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Opsonin-Deficient Nucleoproteic Corona Endows UnPEGylated Liposomes with Stealth Properties In Vivo
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  • Francesca Giulimondi
    Francesca Giulimondi
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Elisabetta Vulpis
    Elisabetta Vulpis
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Luca Digiacomo
    Luca Digiacomo
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Maria Valeria Giuli
    Maria Valeria Giuli
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Angelica Mancusi
    Angelica Mancusi
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Anna Laura Capriotti
    Anna Laura Capriotti
    Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
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    Orcidhttps://orcid.org/0000-0003-1017-9625
  • Aldo Laganà
    Aldo Laganà
    Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
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  • Andrea Cerrato
    Andrea Cerrato
    Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
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  • Riccardo Zenezini Chiozzi
    Riccardo Zenezini Chiozzi
    Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Heidelberglaan 8, 3584 CS Utrecht, The Netherlands
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  • Carmine Nicoletti
    Carmine Nicoletti
    Unit of Histology and Medical Embryology, Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Viale A. Scarpa 16, 00161 Rome, Italy
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  • Heinz Amenitsch
    Heinz Amenitsch
    Institute of inorganic Chemistry, Graz University of Technology, Stremayerg 6/IV, 8010 Graz, Austria
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  • Francesco Cardarelli
    Francesco Cardarelli
    NEST, Scuola Normale Superiore, Piazza San Silvestro 12, 56127 Pisa, Italy
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  • Laura Masuelli
    Laura Masuelli
    Department of Experimental Medicine, University of Rome “Sapienza”, Viale Regina Elena 324, 00161 Rome, Italy
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  • Roberto Bei
    Roberto Bei
    Department of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
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  • Isabella Screpanti
    Isabella Screpanti
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Daniela Pozzi
    Daniela Pozzi
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Alessandra Zingoni
    Alessandra Zingoni
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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  • Saula Checquolo*
    Saula Checquolo
    Department of Medico-Surgical Sciences and Biotechnology, Sapienza University of Rome, Corso della Repubblica 79, 04100 Latina, Italy
    *Email: [email protected]
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  • Giulio Caracciolo*
    Giulio Caracciolo
    Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
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    Orcidhttps://orcid.org/0000-0002-8636-4475
Open PDFSupporting Information (2)

ACS Nano

Cite this: ACS Nano 2022, 16, 2, 2088–2100
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https://doi.org/10.1021/acsnano.1c07687
Published January 18, 2022

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Abstract

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For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes’ synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or “proteoDNAsome”, which maintains its synthetic identity in vivo and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles in vivo and their synthetic identity.

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The development of effective and safe gene-delivery systems (GDSs) is the main bottleneck toward successful clinical applications in different fields (e.g., cancer gene therapy, CGT). (1) The GDSs currently at our disposal belong to two distinct groups: viral and nonviral ones. Viral GDSs exploit the intrinsic ability of viruses to transfer their genetic materials into host cells. Unfortunately, however, their clinical application has been hampered by several technical factors, including, among others, complex manipulation procedures, restrictions in size of the target gene, unavoidable host immune responses, and inflammatory toxicities. (2)
Nonviral GDSs, based for instance on the use of polymers, peptides, dendrimers, or liposomes, hold the potential to bypass the limitations of their viral counterparts. Among others, liposomes emerged as a preferred platform owing to their low cost, easy production, and no limitations in gene-payload size. (3)
Still, at least two main requisites must be implemented into an ideal nonviral, liposome-based GDS, which may appear as mutually exclusive: (i) capability to interact with biological membranes, thus favoring endosomal escape and cargo release within the target cells; (4) (ii) stealth properties to prevent, at the same time, clearance by circulating blood cells (e.g., leukocytes). (5)
Requisite (i) is satisfied by cationic liposomes (CLs). CLs show the exquisite advantage of spontaneous interaction with negatively charged, membrane-associated proteoglycans, thus promoting robust cell-uptake of lipoplexes. (6) Unfortunately, concerning requisite (ii), the positive charge of CLs elicits the adsorption of biomolecules from human blood, especially proteins, which in turn induce extensive aggregation and masking of the designed functionality. (2) In particular, it was demonstrated that the peculiar adsorption of opsonins on the CL surface (e.g., IgG complement proteins etc.) stimulates phagocytosis and rapid clearance of CLs from blood circulation. Of note, this effect is not completely prevented by CL-surface functionalization by poly(ethylene glycol) (PEG), as previously thought. (7)
On the other hand, lipoplexes made of anionic (8) or zwitterionic (9) lipids satisfy requisite (ii): they bind low amounts of proteins, leading to longer circulation times and satisfactory clearance profiles. Concerning point (i), however, intrinsic negative charge and consequent poor interaction with cell membranes of these systems are at the basis of their unsatisfactory performances. As result of these limitations, the field of lipoplex-mediated gene delivery has not advanced significantly over the past 2 decades.
To combine both the efficient interaction with cellular membranes and the stealth properties in a single lipoplex, here we propose a change of paradigm in the architecture of the GDS. From one side, DNA forms stable complexes with CLs that have the intrinsic capability to interact with biological membranes of target cells allowing efficient endosomal escape (i.e., requisite “i”); On the other side, DNA is explored, due to its negative charge, as an alternative candidate for the construction of a stealth GDS (i.e., requisite “ii”). In other words, upon exposure to biological fluid, DNA mainly recruits dysopsonins that provide the GDS with stealth properties against capture by immune cells.
Technically, the CLs used in our study were based on a multicomponent CL formulation consisting of DOTAP, DC-Chol, DOPC, and DOPE. This formulation (hereafter referred to as CL1) is a standard choice to maximize endosomal escape, even in hard-to-transfect cells. (10) A second CL variant, hereafter termed CL2, was prepared by substituting DOPC with DOPG: according to previous findings, this change may lead to greater colloidal stability of the particles in serum/plasma but also increases membrane permeability to charged molecules (e.g., DNA), (11) thus leading to superior efficacy. Both CL formulations were incubated with functional plasmid DNA to form stable lipoplexes. As a first step, we thoroughly characterized chemical-physical properties of the lipoplexes. We could identify two distinct lipoplex structures with similar size but opposite zeta-potential: (i) positively charged complexes that contain most of the DNA in the vesicle core are hereinafter referred to as “plain lipoplexes” (PLs); and (ii) negatively charged complexes whose surface is fully decorated with DNA and are therefore termed “DNA-decorated lipoplexes” (DDLs). DDLs and PLs prepared from CL1 are hereafter indicated with DDL1 and PL1, while those prepared from CL2 are denoted with DDL2 and PL2, respectively. Based on previous experience, an adduct must be tested in terms of its ability to attract a protein biomolecular corona (BC) upon interaction with plasma, as BC directly modifies the original features of the adduct and shapes a biological identity that in turn affects its interaction with cells (e.g., immune cells). Of note, exposure of DDLs to low human plasma (HP) concentrations (HP = 5%) promotes formation of an artificial protein corona deficient in typical opsonins (e.g., complement proteins, immunoglobulins) as determined by proteomics experiments. This in turn leads to an unexpected but fundamental result: the absence of opsonins, in fact, can boost the stealth properties imparted by DNA by preventing the DDL2-protein complexes sequestration by blood cells. We explored the ability of these generated “proteoDNAsomes” to evade immune cells both in vitro and in vivo using PEGylated lipoplexes as a reference (Figure 1). We demonstrate the capacity of proteoDNAsomes to avoid capture by immune cells in vitro by using the human monocytic THP-1 and ex vivo leukocyte subpopulations derived from healthy donors. ProteoDNAsomes exhibited a peculiar capacity to evade the immune system in vivo. When administered to C57BL/6 mice, proteoDNAsomes avoided capture by populations of phagocytes in the blood (i.e., monocytes and neutrophils) and in the spleen (i.e., monocytes, neutrophils, and macrophages) (Figure 1) much more efficiently than state-of-the-art PEGylated lipoplexes.

Figure 1

Figure 1. When liposome-DNA complexes (lipoplexes) with a precise synthetic identity are exposed to plasma proteins, they get covered by a protein corona that provides them with a biological identity. (12) The biological identity of lipoplexes ultimately controls their physiological response (e.g., interaction with the immune system). Interaction between lipoplexes and immune cells are tested using (i) human monocytic cell line THP-1 (in vitro), (ii) circulating leukocytes from human donors (ex vivo), and (iii) C57BL/6 mice (in vivo).

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Results

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DNA-Decorated Lipoplexes

First, we performed dynamic light scattering (DLS), microelectrophoresis (ME), and synchrotron small-angle X-ray scattering (SAXS) experiments to explore the hydrodynamic diameter (DH), zeta potential (Zp), and nanostructure of CL1 and CL2. Results are listed in Figure S1 and Table S1. Both formulations were homogeneous, positively charged (Zp > 40 mV), and small in size (DH < 140 nm). As a next step, we prepared lipoplexes by bulk mixing of DNA and CLs. Figure 2 shows the size (black line) and zeta-potential (gray line) of lipoplexes prepared from CL1 (panel a) and CL2 (panel b) as a function of the cationic lipid/DNA weight ratio, ρ. At ρ = 1, lipoplexes were small (DH ≈ 150 nm), and negatively charged (Zp ≈ −40 mV). This value was therefore identified as the proper cationic lipid/DNA weight ratio leading to DDLs in line with the purpose of this study. On the other hand, we selected ρ = 10 as the lipid/DNA weight ratio leading to cationic plain lipoplexes that were used as a control in the following experiments. Comparing the zeta potential distributions of CLs and cationic lipoplexes, we concluded that the surface of PLs was poorly or not at all decorated with DNA molecules. Structures of PLs and DDLs were characterized by synchrotron SAXS. In Figure 2c,d, we compare the SAXS patterns of PLs and DDLs, respectively. In the SAXS patterns, two Bragg peaks were detected at q001 = 0.95 nm–1 and q002 = 0.190 nm–1. They arise from a multilamellar structure (13,14) whose periodicity along the normal to the lipid bilayer, d, is the sum of the membrane thickness (dB) and the thickness of the water layer occupied by DNA molecules (dW): d = dB + dW = 2π/q001 = 6.61 nm. In the SAXS pattern of PL2, another broader Bragg peak was observed at q001 = 0.107 nm–1, corresponding to a lamellar spacing of d = 5.87 nm. According to the literature, this could indicate demixing into lipid-enriched phases. (15)

Figure 2

Figure 2. Characterization of lipoplexes as a function of the cationic lipid/DNA weight ratio. Size (black points) and zeta potential (gray points) of lipoplexes prepared from CL1 (panel a) and CL2 (panel b) as a function of the cationic lipid/DNA weight ratio, ρ. At ρ = 1, lipoplexes were small in size (DH ≈ 150 nm) and negatively charged (zeta potential ≈ −40 mV) (blue and cyan boxes in panels a and b, respectively). This value was therefore identified as the proper cationic lipid/DNA weight ratio leading to DNA-decorated lipoplexes (DDLs) in line with the purpose of this study. On the other hand, we selected ρ = 10 as the lipid/DNA weight ratio leading to cationic plain lipoplexes (PLs, i.e., whose surface is entirely lipidic) that were used as a control in the following experiments (red and gold boxes in panels a and b, respectively). Synchrotron small-angle X-ray scattering (SAXS) patterns of PLs (panel c) and DDLs (panel d). Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold). Vertical green lines indicate the position of the first- and second-order Bragg peaks arising from a multilamellar structure made of alternating lipid bilayers and DNA monolayers. Representative transmission electron microscopy (TEM) images of PLs (panel e) and DDLs (panel f). Both samples are visible nanosized, rounded-shaped vesicles. The presence of DNA filaments on the surface is detectable in DDLs. Bars correspond to 200 nm.

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We also observe that SAXS patterns of DDLs exhibit a stronger diffuse scattering between Bragg peaks. As the solvent scattering background was properly subtracted (see the Materials and Methods), this large interpeak scattering contribution could be due to some excess DNA in solution. This is confirmed by TEM measurements (Figure 2 and Figure S2). The results shown in Figure S2 show that PLs are plain vesicles with a multilamellar inner structure, while DDLs are decorated with DNA protruding from the particle surface. Globally synchrotron SAXS and TEM outcomes suggest that the inner structure of lipoplexes is influenced by the cationic lipid/DNA charge ratio, while it is poorly dependent on the synthetic identity of pristine liposomes.

Lipoplex-Protein Complexes

To promote the formation of lipoplex-protein complexes, we incubated PLs and DDLs in HP for 1-h at increasing protein concentrations. The size of the PL-protein complexes (Figure 3a) increases with increasing HP concentration, whereas the surface charge decreases (Figure 3b). In correspondence to the inversion of the zeta-potential (i.e., 1% < HP < 5%), PL-protein complexes clustered in large aggregates due to their electronic neutrality. Large complexes form at the “isoelectric point”. When this happens, van der Waals interactions overcome the electrostatic repulsion and promote the formation of large neutrally charged assemblies. At higher HP concentrations, the electrostatic repulsion dominates and decreasing-size complexes form (re-entrant condensation).

Figure 3

Figure 3. Characterization of lipoplex-protein complexes as a function of protein concentration. Size (panel a) and zeta potential (panel b) of lipoplex-protein complexes prepared from CL1 and CL2 as a function of human plasma (HP) percentage (%). As PLs are positively charged systems, PL-protein complexes exhibit the typical features of cationic-anionic assemblies, i.e., re-entrant condensation and charge inversion at the isoelectric point. On the other side, the size of the DDL-protein complexes is poorly affected by HP and zeta potential tends to assume similar values. Vertical gray lines indicate the inversion point where PL-protein complexes undergo massive aggregation (panel a) caused by charge neutralization (panel b). (c) Molecular weight (MW) distributions of protein patterns bound to lipoplexes as a function of HP %. While protein patterns of DDL1- and DDL2-protein complexes were almost superimposable up to HP = 20%, those of PL1- and PL2-protein complexes were clearly distinguishable over the protein range especially between 45 and 80 kDa. Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold).

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The evolution profiles of size and zeta potential of PL-protein complexes suggest that anionic plasma proteins act as a molecular glue between distinct cationic lipoplexes and that protein amount is a crucial factor regulating the equilibrium physical-chemical properties of PL-protein complexes. These observations may have relevant implications, as clearance cells remove from the bloodstream objects larger than (roughly) 300 nm. At higher HP percentages, small size PL-protein complexes are observed. These findings agree with previous works obtained using other nanoparticle types. (16) In contrast, the evolution profiles of size and zeta potential of DDL-protein complexes did not show appreciable variation in size and tended to assume similar zeta potentials at HP = 50%. Such an effect, termed “normalization” of the zeta potential has been previously observed for several types of nanomaterials. (17)
Among shaping factors, the protein concentration ratio plays a key role in determining the protein corona composition of NPs. To get an insight into the hard corona characteristics’ evolution, we isolated proteins from lipoplex-protein complexes and used one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate the molecular weight (MW) distributions of the adsorbed coronas (Figure S3). As Figure 3c clearly shows, PL1 and PL2 exhibited marked differences in their protein profiles at each protein concentration. (18) Moreover, both the profiles evolved considerably. According to previous investigations, (19) this indicates that the protein corona of PLs is made of both highly abundant low-affinity and poorly abundant competitive-binding proteins whose enrichment changes with the plasma concentration. Considering that PL1 and PL2 differ only in one of the four lipid species, our results confirm that the protein corona of lipid vesicles is considerably affected by the lipid composition. (20) On the other hand, the corona of DDL1 and DDL2 was quite stable over the range of protein concentrations. This finding suggests that DNA decoration at the particle surface may have a dominant role in determining protein absorption to DDLs irrespective of the underneath lipid surface.

Composition of the Lipoplex-Protein Corona

Given the differences in the evolution of protein profiles as a function of protein concentration, we decided to investigate the composition of the protein corona to identify stealth and biosafe protein coatings for in vivo applications. (21) According to the literature, (19) we characterized protein corona at low (HP = 5%) and high (HP = 50%) protein concentrations, where the largest difference in physical-chemical properties of nanoparticle–protein complexes is typically found. However, as Figure 3a shows, the size of PL-protein complexes at low HP was too large (DH > 500 nm) to be compatible with drug delivery purposes. (22,23) These complexes were therefore excluded from further characterization. Table S2 lists all the plasma proteins identified in the coronas of DDLs and PLs by nano-LC-MS/MS. In Figure 4a–d, we report the relative protein abundance (RPA) of the top-20 proteins. MS/MS results show significant differences in the RPAs of PL1 and PL2. On the other hand, RPA of the top-20 proteins were similar for DDL1 and DDL2. This observation confirms SDS-PAGE findings demonstrating quantitatively that the lipid composition is one of the main factors shaping the protein corona of lipid vesicles, while the DNA layer adsorbed at the lipoplex surface makes the vesicle surface inaccessible. Finally, we observed that protein concentration has a marked effect on protein binding. In detail, APOA1, APOA2, APOC, ACTG1, and HMGB1 were more abundant when DDLs were exposed to low protein concentrations. On the other hand, binding of APOE, HAS, C1QC, C1QB, IGKG, and IGHM was favored by particle incubation at high protein concentrations. Proteins were also categorized according to the biological processes they are involved in. (24) As Figure 4e–j clearly shows, enrichment in proteins involved in complement activation, coagulation, immune response, acute phase, and tissue leakage were appreciably affected by lipoplex characteristics. Experimental evidence supports the idea that decoding the composition of the nanoparticle–protein corona is fundamental to determine the stealth properties of NPs. However, hundreds of proteins are usually associated with NPs in different proportions, and understanding the role played by each protein component in regulating nanoparticle-cell interaction is a very difficult task. Most importantly, the protein corona is a complex “cloud” of biomolecules, and mapping protein location and exposure of functional epitopes is a big challenge. (25−28) A longstanding popular view is that poor enrichment in opsonins is one of the most successful criteria in designing stealth protein coverages. (29,30) Comparing therefore the enrichment in opsonins, we concluded that the protein coating of DDL2 at HP = 5% (total RPA of identified opsonins <3%) has the best stealth properties. A minor abundance of complement proteins and immunoglobulins was detected. We also observed that DDL2 was poorly enriched of fibrinogen that is an endogenous ligand for αMβ2/Mac 1, the integrin receptor that is vital to regulate leukocyte function and trigger innate immunity in vivo. Languino et al. have demonstrated that binding of fibrinogen to vascular cell receptors promotes the adhesion of leukocytes to the endothelium and transendothelial migration, which are the initial events of immune inflammatory responses. (31) More recently, Deng et al. demonstrated that fibrinogen, upon binding to some nanoparticle types undergoes denaturation, activates the integrin receptor Mac-1, and stimulates the NF-κB signaling pathway leading to the release of inflammatory cytokines in THP-1 cells. (32,33)

Figure 4

Figure 4. Bioinformatic classification of corona proteins. (a) Relative protein abundance (RPA) of the 20 most abundant plasma proteins (Top-20) identified in the coronas of DDL1 (blue), PL1 (red), DDL2 (cyan), and PL2 (gold) by quantitative nanoLC–MS/MS at the indicated human plasma (HP) concentrations. Plasma proteins were also grouped according to functional processes as reported in (34) coagulation (e), complement (f), immunoglobulins (g), lipoproteins (e), acute phase (h), lipoproteins (i), and tissue leakage (f). Each value is the average of triplicates ± standard deviation within a single experiment.

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Cellular Uptake by Monocytic THP-1 Cells

As known, nanoparticles interact with the innate immune system, including the complement system and different times of phagocyte populations. These cells display high phagocytic capacity and typically safeguard from foreign materials; however, recognition of nanoparticles as foreign may result in a multilevel immune response against the nanoparticles and eventually lead to a lack of therapeutic efficacy. Hence, we used flow cytometry to analyze the cellular uptake of DDL- and PL-protein complexes by the human monocytic THP-1 cell line, a spontaneously immortalized monocyte-like cell line derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype). (35)
At low protein concentrations (HP = 5%), the percentage of positive cells (Figure 5a) and median fluorescence intensity (Figure 5b) were low for both DDL1 and DDL2. At high protein concentration (HP = 50%), the cellular uptake of PLs was much higher than that of DDLs and dependent on lipid composition with PL1 > PL2. These results would point out the key role of the protein corona of PLs in determining capture by monocytes and consequent removal from the bloodstream in vivo. Second and foremost, the ability of DDLs to escape internalization by THP-1 could result in increased circulation time and consequently boosting the ability to reach therapeutic targets. To test this suggestion, we explored capture by circulating leukocytes in patients’ whole blood.

Figure 5

Figure 5. Sequestration of lipoplex-protein complexes by THP-1 cells in vitro. Cellular uptake of fluorescently labeled lipoplex-protein complexes by human monocyte THP-1 cells via flow cytometry evaluated in terms of percentage of fluorescent cells (a) and median fluorescent intensity (b). Complexes were prepared at low (i.e., HP = 5%) and high (i.e., HP = 50%) protein concentrations. Each value is the average of triplicate samples ± standard deviation within a single experiment. Statistical significance was evaluated by Student’s t test with respect to DDL2 lipoplexes. *p < 0.05, **p < 0.001, no asterisk means not significant. Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold).

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Particle Sequestration by Circulating Leukocytes

Uptake by circulating leukocytes represents part of the immune system which provides efficient surveillance for foreign pathogens. The introduction of nanoparticles via intravenous injection mimics the infection process and might trigger sequestration by leukocytes drastically affecting the particle biodistribution. (5) To validate the trends found in the monocytic THP-1 cell lines, we used flow cytometry to evaluate the sequestration by both circulating monocytes and other distinct leukocyte subpopulations derived from peripheral blood mononuclear cells (PBMCs) of healthy donors (Figure 6). PBMCs have been widely used as an in vitro model to investigate immune system functions. (36) As Figure 6 clearly shows, we observed that (i) leukocytes subpopulations of CD19+ (B lymphocytes) and CD14+ (monocytes) avidly internalized both PLs and DDLs, (ii) for each leukocyte subpopulation and HP percentage, cellular uptake of PLs was higher than that of DDLs and dependent on lipid composition (i.e., PL1 > PL2), (iii) DDLs exhibit a peculiar ability to escape the sequestration by the other subpopulations. In particular, DDL2 at HP = 5% showed the lowest cellular uptake by leukocytes in patients’ whole blood.

Figure 6

Figure 6. Leukocyte uptake of lipoplex-protein complexes. Complexes were prepared at low (5%) and high (50%) human plasma (HP) concentrations. Each value is the average of duplicate samples ± standard deviation within a single experiment. The fluorescence of internalized lipoplex-protein complexes was measured as the percentage of FITC-positive cells by gating on distinct leukocyte subpopulations as indicated. The gating strategy was obtained as explained in ref (37). Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold).

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Keeping in mind that the six tested formulations had, within the limits of the experimental error, the same synthetic identity (i.e., size and zeta potential), we were able to conclude that the differences in uptake by leukocytes were not due to specific internalization mechanisms such as size- (4) or charge-dependent (38) cellular uptake. On the other hand, the considerable differences in the composition of the protein corona led us to conclude that the cellular internalization of lipoplex-protein complexes is largely regulated by receptor-mediated mechanisms. This reiterates the central role of the protein corona in mediating interactions between nanoparticle systems and cells in biological milieu.
Combining the indications arising from (i) physical-chemical characterization of nanoparticles, (ii) protein corona characterization, (iii) cellular uptake in THP-1, and (iv) capture by circulating leukocytes in whole blood, we were prompted to conclude that exposing DDL2 to low protein concentrations (i.e., HP = 5%) leads to the formation of a stealth and biosafe protein coating. Protein-coated DDL2 was therefore identified as the most promising nanocarrier for further in vivo validation.

Capture by Immune Cells In Vivo

In vivo animal experiments were performed to gain insight into the stealth properties of precoated DDL2. Tenzer et al. (34) showed that the protein corona is established quickly (within minutes) and does not substantially change in composition over time. Moreover, Simon et al. (39) have demonstrated that pre-formed protein corona remains stable even after nanoparticles are re-introduced to the plasma. Thus, particle capture by immune cells in vivo could not be biased by changes in corona composition after administration to mice. PEGylated PLs that have long been the gold standard of lipid-mediated in vivo gene delivery (40) were used as a reference. PEG forms hydrogen bonds with water molecules leading to a hydration layer that, together with polymer chains, creates a steric barrier that attenuates protein binding. PEGylation reduces opsonization and macrophage uptake, thereby extending blood circulation times. However, an immune response against PEG hampers the promising role of PEGylation. Many reports have shown that systemic administration of PEGylated PLs induce robust anti-PEG IgM production that results in the accelerated blood clearance (ABC) phenomenon. (41) Consequently, the development and evaluation of therapeutically useful unPEGylated formulations is urgently needed. We administered PEGylated PLs and precoated DDL2 to 8–10 weeks-old female C57BL/6 mice by intravenous venipuncture (i.v.) and evaluated particle capture by the main populations of phagocytes in the blood (i.e., monocytes and neutrophils) and in the spleen (i.e., monocytes, neutrophils, and macrophages). Our findings displayed in Figure 7 show that PEGylated PLs are massively captured by all the phagocyte populations both in the blood and in the spleen (the gating strategy is illustrated in Figure S4). In contrast, the phagocyte uptake of precoated DDL2 appears to be very low both in the blood and the spleen. In this work, we used plasmid DNA, as plasmids are used for several biomedical applications as tools to clone, transfer, and manipulate genes. However, to generalize our results, we prepared a variant of DDL2 using a commercial 20-mer oligonucleotide (in the following text indicated as “DDL2*”, all the details are found in Figure S5 in the Supporting Information). The uptake of precoated DDL2* by populations of phagocytes in the blood and in the spleen is very low (Figure 7). This observation let us to conclude that the DNA size does not affect the stealth properties of “proteoDNAsomes”. The in vivo stealth properties of precoated DDL2 and DDL2* are significant considering that clinically approved PEGylated liposomal formulations cannot completely prevent opsonin adsorption and that the PEG layer can interfere with the interaction of particles with cells in vivo in a rather unpredictable manner depending on the molecular weight and branching of the PEG molecules. (42) For example, Hadjidemetriou et al. investigated the formation of protein coronas on clinically used PEGylated liposomal doxorubicin (DOXIL) nanoparticles following their intravenous administration in CD-1 mice (43) and humans. (44) They found that PEGylated liposomes in vivo become heavily coated with opsonins, and the protein corona has an impact on cellular internalization by immune cells, which helped to understand the poor clinical success of these formulations.

Figure 7

Figure 7. In vivo particle uptake by circulating and resident phagocytes. 8–10 weeks-old female C57BL/6 mice were i.v. injected with three types of lipoplexes, PEGylated plain lipoplexes (PEGylated PL), plasmid DNA-decorated lipoplexes coated with mouse plasma (MP) proteins (DDL2+MP) and 20-mer oligonucleotide-decorated lipoplexes coated with mouse plasma (MP) proteins (DDL2*+MP). In all the groups (n = 6 animals), capture by phagocytes was evaluated in the blood (panel a) and the spleen (panel b) as percentage of FITC-positive cells by flow cytometry gating on distinct populations as indicated. The gating strategy is illustrated in Figure S4. Naïve animals served as controls (n = 9–12). Data are expressed as the mean ± SD. Statistical significance was evaluated by Student’s t test. *p < 0.05, **p < 0.001, no asterisk means not significant.

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As a further investigation, we carried out a biodistribution study by injecting mice with PEGylated PLs, precoated DDL2, and precoated DDL2* and then sacrificing the mice at 1-h after injection. Particle distribution was evaluated by measuring fluorescence in homogenates of the lymphoid (spleen) and the nonlymphoid (liver, lungs, kidneys, and heart) organs. Biodistribution results are reported in Figure 8. Lymphoid organs showed significantly lower levels of precoated DDL2 and precoated DDL2* than those of PEGylated PL. In the lung, accumulation of PEGylated PL was higher than those of precoated DDL2 and DDL2*, but experimental variability limited the statistically significant modifications. The levels of PEGylated PL, precoated DDL2, and precoated DDL2* were low in the kidneys and in the heart and differences among formulations were not statistically significant. In vivo particle uptake by circulating and resident phagocytes and biodistribution of particles demonstrated that precoating DNA-decorated lipoplexes is a powerful strategy to increase systemic circulation.

Figure 8

Figure 8. Biodistribution of lipoplexes in C57BL/6 mice. Distribution of particles in the spleen, liver, lungs, kidneys, and heart of C57BL/6 mice was evaluated by measuring the fluorescence intensity per mg of tissue. Data are expressed as the mean ± SD. Statistical significance was evaluated by Student’s t test. *p < 0.05, **p < 0.001, no asterisk means not significant.

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Many studies have explored the intracellular signaling pathways activated upon the immune response to GDSs. Previous investigations (45) have demonstrated that toll-like receptors (TLRs) are involved in this process and can affect the safety and effectiveness of GDSs. Among them, TLR9 is an important receptor expressed in immune system cells including dendritic cells, macrophages, natural killer cells, and other antigen presenting cells. TLR9 binds DNA that is rich in unmethylated CpG-DNA motifs and triggers signaling cascades that lead to a pro-inflammatory cytokine response. As a last step of our investigation, we asked whether PLs and DDLs could engage TLR9. To this aim, we used a 293-derived reporter cell line engineered to coexpress TLR9 along with an NF-kB-driven luciferase reporter. As shown in Figure S6, exposure to specific TLR9 agonists induced luciferase activity in the reporter cell line, whereas no effect was observed in the presence of distinct lipoplexes, thus suggesting that the DNA sequence is crucial to activate TLR9.

Conclusions

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Due to their positive net charge that promotes association with target cells, CLs have been the preferred template for gene delivery applications so far. Despite these efforts, however, their clinical application is still limited. A major limitation is that, upon exposure to a biological environment, CLs unavoidably elicit the adsorption of biomolecules from human blood, especially proteins, which in turn alter their synthetic identity and influence their final fate and efficacy in the target tissues. A major detrimental effect, for instance, is imposed by spontaneous absorption on the CL surface of opsonins (e.g., IgG, CO3b etc.), which promote cell association, endocytosis, and clearing by macrophages and other phagocytes, thus severely hampering the final efficacy of the gene-delivery adduct. (46,47) In response to these limitations, two main research efforts can be identified in the literature. On one side, researchers developed strategies to abolish protein adsorption. Unfortunately, such nonspecific process cannot be eliminated by grafting polymers at the particle surface (e.g., by PEG). (48,49)
On the other side, researchers attempted to control the composition of the adsorbed proteins to nanoparticles. For instance, the engineering of a preformed protein corona enriched in proteins with null or negligible affinity for the clearance-deputed cellular system became one of the most promising design efforts. Precoating nanoparticles with single protein(s) (e.g., human serum albumin, (50) transferrin, (51) vitronectin) could enable controlled cellular interactions with biological systems. However, when exposed to a physiological environment rich in thousands of plasma proteins (e.g., serum/plasma), it is still possible for the particle to become coated with other proteins by the formation of a double or multiple layer. (52) Alternatively, the idea was to modify the surface of the nanoparticle in such a way that the spontaneously adsorbed protein corona in the physiological environment gets naturally enriched in components of interest, with the undoubted advantage of generating a protein corona that will remain stable in the physiological environment. For instance, some of us designed a cationic lipid/DNA complex that, upon exposure to HP, gets coated with vitronectin-enriched protein corona with the selective ability to target cancer cells expressing high levels of the vitronectin ανβ3 integrin receptor. (53) Unfortunately, this approach, at present, does not allow fine-tuning of the composition of the corona. Also, many adjustments in the synthetic identity and incubation process (e.g., exposure time, protein concentration, temperature) may be necessary to achieve the desired “biological identity” (i.e., size, zeta potential, and protein corona composition). (37)
In this work, we have exploited the electric charge of DNA to generate lipoplexes with a negatively charged surface which, upon exposure to human plasma, gets spontaneously covered with an outer biomolecular layer deficient in opsonins and enriched in dys-opsonins (Figure 9). The result is a peculiar biomolecular corona which can significantly influence the interaction of the whole adduct with immune cells both in vitro and in vivo. To summarize, the final product of the synthetic process is a species of lipoplexes covered by a proteonuclotidic corona, or “proteoDNAsome”, which are endowed with peculiar stealth properties. Our results demonstrate how the synthetic identity of nanocarriers can be engineered by fine-tuning their structural parameters (i.e., biomolecular corona, DNA layer, lipoplex) to shape their biological identity and thus regulate their immune response.

Figure 9

Figure 9. Exposing DNA-decorated lipoplexes to plasma proteins collected form C57BL/6 mice leads to formation of lipoplexes enriched with an opsonin-deficient, dysopsonin-enriched protein corona. The final product of the formulation process is a type of lipoplexes covered by a proteonuclotidic corona, or “proteoDNAsome”, with more pronounced ability to evade capture by immune cells in vivo than PEGylated lipoplexes.

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Material and Methods

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Chemicals

Cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl])-cholesterol (DC-Chol), zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE), and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) were purchased from Avanti Polar Lipids (Alabaster, AL) as previously reported. (7) Lipids were used without additional modification. Human plasma (HP) was purchased from Sigma-Aldrich, Inc. (Merk KGaA, Darmstadt, Germany). Plasmid DNA (pmirGLO plasmid vector) was purchased from Promega Corporation (Madison, WI). Oligonucleotide ODN 2243 was purchased from InvivoGen (Toulouse, France). Cy3-labelled plasmid DNA (Label IT Plasmid Delivery Control Cy3) was purchased from Mirus Bio (Madison, WI). Texas red-DOPE (Life Technologies, Carlsbad, CA) was purchased from Sigma-Aldrich, Inc. (Merk KGaA, Darmstadt, Germany), as previously reported. (54)

Liposome and Lipoplex Preparation

We designed two multicomponent liposomal formulations. The first formulation (CL1) was made of the cationic lipids DOTAP and DC-Chol and the zwitterionic lipids DOPC and DOPE. The second formulation (CL2) was prepared by replacing DOPC with anionic DOPG. Both cationic liposomes were prepared at the desired molar ratio (DOTAP/DC-Chol/DOPC/DOPE = 1:1:1:1 for CL1 and DOTAP/DC-Chol/DOPG/DOPE = 1:1:1:1 for CL2). For in vivo experiments, formulations were labeled with Texas red-DOPE (fluorescent lipid/total lipid molar ratio = 4/1000), as previously reported. (54) Each lipid was dissolved in chloroform, and the solvent was evaporated under a vacuum for 2 h. Lipid films were hydrated to obtain a final lipid concentration of 1 mg/mL with ultrapure water and stored at 4 °C. The obtained liposomal solutions were extruded 20 times through a 0.1 μm polycarbonate carbonate filter with the Avanti Mini-Extruder (Avanti Polar Lipids, Alabaster, AL), as previously reported. (37) Plain and DNA-decorated lipoplexes (PLs and DDLs) were formed by mixing proper amounts of aqueous solutions of liposomes (1 mg/mL) and plasmid DNA (1 mg/mL) or Cy3 (500 ng/μL). The final solutions were left for 20 min at room temperature prior to performing the experiments. For size, zeta-potential, and SDS-PAGE, proteomics non-labelled plasmid DNA was used. For in vitro, ex vivo, and in vivo flow cytometry experiments, Cy3-labeled plasmid DNA was employed.

Lipoplex-Protein Complexes

Lipoplex-protein complexes were prepared by incubating for 1-h proper amounts of lipoplexes in the following plasma concentrations: 1%, 2.5%, 5%, 10%, 20%, and 50%. For DLS and biological experiments, the resulting solution were characterized without further treatment. For SDS-PAGE and proteomics, lipoplexes-protein complexes were isolated by centrifugation for 15 min at 14 000 rpm. The pellets were washed three times with PBS to remove unbound proteins obtaining the “hard corona” (further details can be found in ref (55)).

Size and Zeta Potential Experiments

Size and zeta potential measurements were carried out using a Zetasizer Nano ZS (Malvern, U.K.) at 25 °C. To perform size and zeta-potential experiments, sample solutions were diluted with distilled water (final volume = 1 mL), as previously reported. (37) Results are given as mean ± standard deviation of three replicates.

Synchrotron Small-Angle X-ray Scattering Experiments

SAXS measurements were carried out at the Austrian SAXS beamline at the synchrotron light source of Elettra (Trieste, Italy). SAXS scans were recorded in the q-ranges from 0.05 nm–1 to 5 nm–1 with a resolution of 5 × 10 nm–3. Calibration of detectors was performed with silver behenate powder (d-spacing = 5.838 nm). The sample was held in a 1 mm glass capillary (Hilgenberg, Malsfeld, Germany), and the measurements were executed at room temperature, as previously reported. (37)

Transmission Electron Microscopy Experiments

Samples (10 μL) were dropped on formvar-carbon-coated copper grids (EMS, PA) and allowed to adsorb for 5 min. The resulting film was stained with a 2% uranyl acetate solution for 1 min at room temperature. The excess of staining solution was adsorbed by the filter paper. Grids were air-dried for 1 h before imaging with TEM Morgagni 268D (Philips, The Netherlands), as previously reported. (37)

1D SDS-PAGE Experiments

Lipoplex-protein complexes were resuspended in 40 μL of Laemmli Loading buffer 1× and boiled for 10 min at 100 °C. Each sample was loaded on a gradient polyacrylamide gel stain free (4–20 % TGX precast gels, BioRad) and run at 100 V for about 150 min. Finally, gel images were acquired with a ChemiDoc gel imaging system (Bio-Rad, CA) and processed by means of custom MatLab scripts (MathWorks, Natick, MA), as previously reported. (55)

Proteomics

To perform nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) experiments, lipoplex-protein complex pellets were resuspended in 40 μL of 8 mol L–1 urea and 50 mmol L–1 NH4HCO3 (pH = 7.8). Afterwards, protein solution was treated and prepared for the liquid chromatograph-mass spectrometer through a procedure accurately described elsewhere. (25)

Cell Culture

Human monocyte cell line THP-1 cells were maintained in the RPMI-1640 (Gibco, Carlsbad, CA) medium supplemented with 2 mM l-glutamine, 100 IU/mL penicillin/streptomycin, and 10% fetal bovine serum until use.

Flow Cytometry

To investigate the cellular uptake of lipoplex-protein complexes in the THP-1 cell line, lipoplexes were prepared with Cy3-labeled DNA. THP-1 cells were plated at 500 000 cells mL–1 in 12-well dishes. After the treatment, cells were washed with cold PBS and then run on a FACS Canto (BD Biosciences, San Jose, CA). Cells were gated using forward versus side scatter to exclude debris and then analyzed for the specific emission. The data were analyzed using FlowJo software (FlowJo LLC data analysis software, Ashland, OR), as previously reported. (37) Full experimental details can be found in the supporting Excel file.

Particle Sequestration from Circulating Leukocytes

PBMCs were isolated from peripheral blood of healthy donors by Ficoll–Hypaque gradient centrifugation. Cells were plated at 1 × 106 cells mL–1 and then were incubated with lipoplexes for 1 h at 37 °C. After treatment with PL- and DDL-protein complexes, cells were washed with PBS and then labelled with the following diluted antibodies: anti-CD3/BV510 (catalog no. 564713, dilution 1:50), CD56/BV421 (catalog no. 562751, dilution 1:50), anti-CD4/APC (catalog no. 555349, dilution 1:10), anti-CD14/PerCP (catalog no. 340585, dilution 1:50), anti-CD45/allophycocyanin-H7 (catalog no. 560178, dilution 1:50), and anti-CD19/PE-Cy7 (anti-CD3/BV510 (catalog no. 564713557835, dilution 1:100) all from BD Bioscience. The fluorescence of the internalized liposomes was evaluated by immunofluorescence and FACS analysis using a FACSCanto (BD Biosciences, San Jose, CA) and measured as the percentage of positive cells by gating on distinct leukocyte subpopulations. The data analysis was performed using the FlowJo program, as previously reported. (37) Full experimental details can be found in the supporting Excel file.

In Vivo Animal Experiments

To investigate cellular uptake of nanoparticles in vivo, 200 μL of (i) PEGylated PL, (ii) protein coated-DDL2, and (iii) protein-coated DDL2* were injected by i.v. in 8–10 weeks-old female C57BL/6 mice (Charles River, n = 6 mice per group; (200 μL/each mouse)). The same volume of PBS was injected in the control group. At 1-h after injection, mice were sacrificed followed by cardiac puncture to collect blood, further added to the heparin tubes. Freshly isolated cells from the spleen and blood were prepared and stained, as previously described. (56) Briefly, after lysis of red blood cells, 2 × 106 of splenocytes or 50 μL of peripheral blood mononuclear cells (PBMCs) were washed and resuspended in staining buffer (PBS without Ca2+ Mg2+, BSA 0.5%, EDTA 2 mM, and NaN3 0.025%). After 10 mins of incubation with anti-CD16/32 (clone 24G2), cells were stained with the indicated mAbs (anti-F480/FITC, anti-CD11b/BV421, and anti-GR1/APC) for 25 min at 4 °C. The gating strategy is reported in Figure S4. Samples were analyzed by flow cytometry using a FACSCanto II (BD Biosciences, San Jose, CA), and data analysis was performed using the FlowJo program. For biodistribution analysis, spleen, liver, lungs, kidneys, and heart were collected and kept in PBS on ice. Solid organs were weighed and homogenized, and then PBS was added to each tube to bring it to a specific volume, 2× PBS (weight/volume). Organ homogenates (50 μL) were pipetted into a 96-well plate and then read using a fluorescent plate reader (BD Biosciences). The fluorescence values per mg of tissue were calculated. All animal experiments were approved by local ethic authorities and conducted in accordance with Italian Governing Law (D.lgs 26/2014; Prot. No. 03/2013). Animals were housed in the Institute’s Animal Care Facilities, which meet international standards and were checked regularly by a certified veterinarian responsible for health monitoring, animal welfare supervision, and revision of experimental protocols and procedures. Full experimental details can be found in the supporting Excel file.

NF-kB Luciferase Reporter Assay

TLR-specific activation assays were performed using human embryonic kidney 293 (HEK293) cells expressing luciferase under control of the NF-kB promoter and stably transfected with TLR9 (TLR9-HEK293) or without TLR9 as a negative control (Luc-HEK293) purchased from InvivoGen (Toulouse, France). HEK293-transfected cells were maintained in DMEM supplemented with 4.5 g/L glucose and 10% FBS, 1% penicillin/streptomycin solution (Invitrogen), 5 μg/mL of puromycin (Sigma-Aldrich, St. Louis, MO) and 5 μg/mL of blasticidin (InvivoGen). For the NF-kB luciferase assay, 40 000 cells/well were seeded in 100 μL of complete DMEM without antibiotics in 96-well plates and incubated for 18 h at 37 °C. After this incubation, the medium was removed, cells were washed with PBS, and 200 μL of DMEM without FBS were added. Cells were incubated with suitable amounts of PL1, PL2, DDL1, and DDL2 and left for 5 h. As a positive control, a specific TLR9 agonist was used (i.e., 50 μM ODN2216 from InvivoGen). After incubation, supernatants were aspired from each well, cells were washed with PBS and then were lysed for 15 min at room temperature using 50 μL/well of 1:5 diluted “passive lysis buffer” (Promega, Madison WI). The protein concentration was evaluated by Bio-Rad Protein Assay. In total, 3 μg of total proteins for each sample were diluted in 50 μL of PBS, and 50 μL of luciferase assay substrate (Promega, Madison WI) was added. Emitted light was immediately quantified using a luminometer Glo-Max-Multi Detection System (Promega, Madison WI). Full experimental details can be found in the supporting Excel file.

Statistical Analysis

For all the experiments, results are reported as mean ± standard deviation (error bars in the charts). The number of replicates varied among experiments, as follows: n = 3 for size, zeta-potential, synchrotron SAXS measurements, and proteomics experiments; n = 2 for 1D SDS-PAGE and FACS analysis on the THP-1 cell line. For PBMCs experiments, n = 5. For in vivo experiments, n = 18 mice were sacrificed.

Minimum Information Reporting in Bio–Nano Experimental Literature (MIRIBEL)

The studies conducted herein, including physical-chemical characterization, biological studies, and all the experimental details, conform to the MIRIBEL reporting standard for bio–nano research, and we incorporate a companion checklist of this information as Supplementary Table S3.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.1c07687.

  • Supporting Excel file, scheme of the in vitro and in vivo experiments (XLSX)

  • Figure S1, size, zeta potential, synchrotron SAXS patterns, and the corresponding computed electron density profiles of cationic liposomes CL1 and CL2; Table S1, DLS and synchrotron SAXS parameters for cationic liposomes CL1 and CL2; Figure S2, representative transmission electron microscopy (TEM) images of plain and DNA-decorated lipoplexes; Figure S3, protein corona analysis of plain and DNA-decorated lipoplexes at increasing amounts of human plasma (HP); Table S2, lists of plasma proteins identified in the coronas of DDLs and PLs by nano-LC-MS/MS; Figure S4, gating strategy of mouse phagocyte populations; Figure S5, characterization of CL2/oligonucleotide (ON) lipoplexes as a function of the cationic lipid/ON weight ratio, ρ; Figure S6, luciferase activity in HEK293/Luc reporter cells transfected or not with TLR9 and incubated for 5 h with PL1, PL2, DDL1, DDL2, or a specific TLR9 agonist; and Supplementary Table S3, companion checklist about Minimum Information Reporting in Bio–Nano Experimental Literature (MIRIBEL) (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Saula Checquolo - Department of Medico-Surgical Sciences and Biotechnology, Sapienza University of Rome, Corso della Repubblica 79, 04100 Latina, Italy;  Email: [email protected]
    • Giulio Caracciolo - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy;  Orcidhttps://orcid.org/0000-0002-8636-4475;  Email: [email protected]
  • Authors
    • Francesca Giulimondi - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
    • Elisabetta Vulpis - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
    • Luca Digiacomo - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
    • Maria Valeria Giuli - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
    • Angelica Mancusi - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
    • Anna Laura Capriotti - Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy;  Orcidhttps://orcid.org/0000-0003-1017-9625
    • Aldo Laganà - Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
    • Andrea Cerrato - Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
    • Riccardo Zenezini Chiozzi - Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Heidelberglaan 8, 3584 CS Utrecht, The Netherlands
    • Carmine Nicoletti - Unit of Histology and Medical Embryology, Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Viale A. Scarpa 16, 00161 Rome, Italy
    • Heinz Amenitsch - Institute of inorganic Chemistry, Graz University of Technology, Stremayerg 6/IV, 8010 Graz, Austria
    • Francesco Cardarelli - NEST, Scuola Normale Superiore, Piazza San Silvestro 12, 56127 Pisa, Italy;  Orcidhttps://orcid.org/0000-0003-3049-5940
    • Laura Masuelli - Department of Experimental Medicine, University of Rome “Sapienza”, Viale Regina Elena 324, 00161 Rome, Italy
    • Roberto Bei - Department of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
    • Isabella Screpanti - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
    • Daniela Pozzi - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
    • Alessandra Zingoni - Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy
  • Author Contributions

    F.G. and E.V. contributed equally. Conceptualization was done by I.S., D.P., A.Z., S.C., and G.C.; methodology was done by A.L, A.L.C., A.A., I.S., D.P., H.A., S.C., L.M., R.B., A.M., and G.C.; investigation was done by F.G., E.V., M.V.G., L.D., C.N., A.M., L.M., R.B., R.Z.C.; writing of the original draft preparation was done by F.G., L.D., F.C., and G.C.; writing with reviewing and editing was done by L.D., F.C., D.P., G.C.; supervision was done by A.L., A.L.C., I.S., D.P., A.Z, S.C., G.C., and funding acquisition was done by G.C., D.P, and A.Z. All authors have read and agreed to the published version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

    Data Availability: The data sets generated during and/or analyzed during the current study are available from the corresponding author by reasonable request.


    Code Availability: Custom codes used for SDS-PAGE are available from the corresponding author by request.

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    Torchilin, Vladimir P.
    Nature Reviews Drug Discovery (2005), 4 (2), 145-160CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. Liposomes - microscopic phospholipid bubbles with a bilayered membrane structure - have received a lot of attention during the past 30 years as pharmaceutical carriers of great potential. More recently, many new developments have been seen in the area of liposomal drugs - from clin. approved products to new exptl. applications, with gene delivery and cancer therapy still being the principal areas of interest. For further successful development of this field, promising trends must be identified and exploited, albeit with a clear understanding of the limitations of these approaches.
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  4. 4
    Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. Size-Dependent Internalization Of Particles Via the Pathways of Clathrin-and Caveolae-Mediated Endocytosis. Biochemical journal 2004, 377, 159– 169,  DOI: 10.1042/bj20031253
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    Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis
    Rejman, Joanna; Oberle, Volker; Zuhorn, Inge S.; Hoekstra, Dick
    Biochemical Journal (2004), 377 (1), 159-169CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Non-phagocytic eukaryotic cells can internalize particles <1 μm in size, encompassing pathogens, liposomes for drug delivery or lipoplexes applied in gene delivery. In the present study, we have investigated the effect of particle size on the pathway of entry and subsequent intracellular fate in non-phagocytic B16 cells, using a range of fluorescent latex beads of defined sizes (50-1000 nm). Our data reveal that particles as large as 500 nm were internalized by cells via an energy-dependent process. With an increase in size (50-500 nm), cholesterol depletion increased the efficiency of inhibition of uptake. The processing of the smaller particles was significantly perturbed upon microtubule disruption, while displaying a negligible effect on that of the 500 nm beads. Inhibitor and co-localization studies revealed that the mechanism by which the beads were internalized, and their subsequent intracellular routing, was strongly dependent on particle size. Internalization of microspheres with a diam. <200 nm involved clathrin-coated pits. With increasing size, a shift to a mechanism that relied on caveolae-mediated internalization became apparent, which became the predominant pathway of entry for particles of 500 nm in size. At these conditions, delivery to the lysosomes was no longer apparent. The data indicate that the size itself of (ligand-devoid) particles can det. the pathway of entry. The clathrin-mediated pathway of endocytosis shows an upper size limit for internalization of approx. 200 Nm, and kinetic parameters may det. the almost exclusive internalization of such particles along this pathway rather than via caveolae.
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  5. 5
    Betker, J. L.; Jones, D.; Childs, C. R.; Helm, K. M.; Terrell, K.; Nagel, M. A.; Anchordoquy, T. J. Nanoparticle Uptake by Circulating Leukocytes: a Major Barrier to Tumor Delivery. J. Controlled Release 2018, 286, 85– 93,  DOI: 10.1016/j.jconrel.2018.07.031
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    Nanoparticle uptake by circulating leukocytes: A major barrier to tumor delivery
    Betker, Jamie L.; Jones, Dallas; Childs, Christine R.; Helm, Karen M.; Terrell, Kristina; Nagel, Maria A.; Anchordoquy, Thomas J.
    Journal of Controlled Release (2018), 286 (), 85-93CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    Decades of research into improving drug delivery to tumors has documented uptake of particulate delivery systems by resident macrophages in the lung, liver, and spleen, and correlated short circulation times with reduced tumor accumulation. An implicit assumption in these studies is that nanoparticles present in the blood are available for distribution to the tumor. This study documents significant levels of lipoplex uptake by circulating leukocytes, and its effect on distribution to the tumor and other organs. In agreement with previous studies, PEGylation dramatically extends circulation times and enhances tumor delivery. However, our studies suggest that this relationship is not straightforward, and that particle sequestration by leukocytes can significantly alter biodistribution, esp. with non-PEGylated nanoparticle formulations. We conclude that leukocyte uptake should be considered in biodistribution studies, and that delivery to these circulating cells may present opportunities for treating viral infections and leukemia.
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  6. 6
    Mislick, K. A.; Baldeschwieler, J. D. Evidence for the Role of Proteoglycans in Cation-Mediated Gene Transfer. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 12349– 12354,  DOI: 10.1073/pnas.93.22.12349
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    6
    Evidence for the role of proteoglycans in cation-mediated gene transfer
    Mislick, Kimberly A.; Baldeschwieler, John D.
    Proceedings of the National Academy of Sciences of the United States of America (1996), 93 (22), 12349-12354CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We report evidence that gene complexes, consisting of polycations and plasmid DNA enter cells via binding to membrane-assocd. proteoglycans. Treatment of HeLa cells with sodium chlorate, a potent inhibitor of proteoglycan sulfation, reduced luciferase expression by 69%. Cellular treatment with heparinase and chondroitinase ABC inhibited expression by 78% and 20% with respect to control cells. Transfection was dramatically inhibited by heparin and heparan sulfate and to a smaller extent by chondroitin sulfate B. Transfection of mutant, proteoglycan deficient Chinese hamster ovary cells was 53× lower than of wild-type cells. For each of these assays, the intracellular uptake of DNA at 37°C and the binding of DNA to the cell membrane at 4°C was impaired. Preliminary transfection expts. conducted in mutant and wild-type Chinese hamster ovary cells suggest that transfection by some cationic lipids is also proteoglycan dependent. The variable distribution of proteoglycans among tissues may explain why some cell types are more susceptible to transfection than others.
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  7. 7
    Pozzi, D.; Colapicchioni, V.; Caracciolo, G.; Piovesana, S.; Capriotti, A. L.; Palchetti, S.; De Grossi, S.; Riccioli, A.; Amenitsch, H.; Laganà, A. Effect of Polyethyleneglycol (PEG) Chain Length on the Bio–Nano-Interactions between PEGylated Lipid Nanoparticles and Biological Fluids: from Nanostructure to Uptake in Cancer Cells. Nanoscale 2014, 6, 2782– 2792,  DOI: 10.1039/c3nr05559k
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    7
    Effect of polyethyleneglycol (PEG) chain length on the bio-nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells
    Pozzi, Daniela; Colapicchioni, Valentina; Caracciolo, Giulio; Piovesana, Susy; Capriotti, Anna Laura; Palchetti, Sara; De Grossi, Stefania; Riccioli, Anna; Amenitsch, Heinz; Lagana, Aldo
    Nanoscale (2014), 6 (5), 2782-2792CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    When nanoparticles (NPs) enter a physiol. environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liq. chromatog. coupled with tandem mass spectrometry (nanoLC-MS/MS) anal. allowed us to accurately det. the corona compn. showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry anal. in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.
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  8. 8
    Srinivasan, C.; Burgess, D. J. Optimization and Characterization of Anionic Lipoplexes for Gene Delivery. Journal of controlled release 2009, 136, 62– 70,  DOI: 10.1016/j.jconrel.2009.01.022
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    Optimization and characterization of anionic lipoplexes for gene delivery
    Srinivasan, Charudharshini; Burgess, Diane J.
    Journal of Controlled Release (2009), 136 (1), 62-70CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    Anionic lipoplexes, comprising divalent cations, DNA and anionic liposomes, were optimized for high transfection efficiency and low cytotoxicity. Different molar ratios of anionic to zwitterionic lipid, 1:9 to 1:1 (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), various cations (Ca2+, Mg2+ and Na+) and different anionic lipid/DNA ratios were investigated. The optimized formulation was composed of: anionic/zwitterionic lipid molar ratio 1:4 (DOPG:DOPE); 15-20 mM Ca2+; and 15-20 μg lipid for complexation with 0.8 μg plasmid DNA. Biophys. studies (particle size anal., gel electrophoresis, transmission electron microscopy (TEM), and confocal microscopy) were conducted to characterize the different formulations. TEM revealed structural differences between the complexed and uncomplexed lipoplexes. Gel electrophoresis confirmed the formation of anionic lipoplexes with the amt. of free DNA minimized for the optimized formulation. Confocal imaging showed cellular uptake of the anionic lipoplexes. Most significantly the anionic lipoplex formulation, optimized for the highest transfection efficiency (approx. 78% in the presence of serum) exhibited the highest cell viability approx. 93%, (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) MTT assay. This was compared to Lipofectamine2000 which had a transfection efficiency and cell viability of approx. 68% and 35%, resp. The anionic lipoplex formulation developed here shows promise as a non-viral vector with high transfection efficiency and low cytotoxicity.
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    McManus, J. J.; Rädler, J. O.; Dawson, K. A. Does Calcium Turn a Zwitterionic Lipid Cationic?. J. Phys. Chem. B 2003, 107, 9869– 9875,  DOI: 10.1021/jp034463d
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    Does Calcium Turn a Zwitterionic Lipid Cationic?
    McManus, Jennifer J.; Raedler, Joachim O.; Dawson, Kenneth A.
    Journal of Physical Chemistry B (2003), 107 (36), 9869-9875CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    The structure of a complex formed from DNA, calcium, and DPPC has been studied by small-angle X-ray scattering (SAXS). Previous DSC and temp. scanning ultrasound have shown that in excess lipid two DPPC phases exist, one corresponding to DNA-bound lipid and the other corresponding to lipid not directly involved in complex formation. We have investigated the effect of calcium on both phases within the complex structure. Contrary to the observations in the ternary DPPC/calcium/water system, the binding of calcium to the DPPC membrane in this quaternary system contg. DNA appears to be significantly enhanced. We have estd. the stoichiometry of calcium binding within the complex, and we concluded that the zwitterionic lipid is directly involved in binding to DNA.
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  10. 10
    Palchetti, S.; Pozzi, D.; Marchini, C.; Amici, A.; Andreani, C.; Bartolacci, C.; Digiacomo, L.; Gambini, V.; Cardarelli, F.; Di Rienzo, C.; Peruzzi, G.; Amenitsch, H.; Palermo, R.; Screpanti, I.; Caracciolo, G. Manipulation of Lipoplex Concentration at the Cell Surface Boosts Transfection Efficiency in Hard-to-Transfect Cells. Nanomedicine: Nanotechnology, Biology and Medicine 2017, 13, 681– 691,  DOI: 10.1016/j.nano.2016.08.019
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    10
    Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells
    Palchetti Sara; Pozzi Daniela; Marchini Cristina; Amici Augusto; Andreani Cristina; Bartolacci Caterina; Gambini Valentina; Digiacomo Luca; Cardarelli Francesco; Di Rienzo Carmine; Peruzzi Giovanna; Palermo Rocco; Amenitsch Heinz; Screpanti Isabella; Caracciolo Giulio
    Nanomedicine : nanotechnology, biology, and medicine (2017), 13 (2), 681-691 ISSN:.
    To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results.
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  11. 11
    Pan, J.; Heberle, F. A.; Tristram-Nagle, S.; Szymanski, M.; Koepfinger, M.; Katsaras, J.; Kučerka, N. Molecular Structures of Fluid Phase Phosphatidylglycerol Bilayers As Determined by Small Angle Neutron and X-Ray Scattering. Biochimica et Biophysica Acta (BBA)-Biomembranes 2012, 1818, 2135– 2148,  DOI: 10.1016/j.bbamem.2012.05.007
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    11
    Molecular structures of fluid phase phosphatidylglycerol bilayers as determined by small angle neutron and X-ray scattering
    Pan, Jianjun; Heberle, Frederick A.; Tristram-Nagle, Stephanie; Szymanski, Michelle; Koepfinger, Mary; Katsaras, John; Kucerka, Norbert
    Biochimica et Biophysica Acta, Biomembranes (2012), 1818 (9), 2135-2148CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    We have detd. the mol. structures of commonly used phosphatidylglycerols (PGs) in the commonly accepted biol. relevant fluid phase. This was done by simultaneously analyzing small angle neutron and X-ray scattering data, with the constraint of measured lipid vols. We report the temp. dependence of bilayer parameters obtained using the one-dimensional scattering d. profile model - which was derived from mol. dynamics simulations - including the area per lipid, the overall bilayer thickness, as well as other intrabilayer parameters (e.g., hydrocarbon thickness). Lipid areas are found to be larger than their phosphatidylcholine (PC) counterparts, a result likely due to repulsive electrostatic interactions taking place between the charged PG headgroups even in the presence of sodium counterions. In general, PG and PC bilayers show a similar response to changes in temp. and chain length, but differ in their response to chain unsatn. For example, compared to PC bilayers, the inclusion of a first double bond in PG lipids results in a smaller incremental change to the area per lipid and bilayer thickness. However, the extrapolated lipid area of satd. PG lipids to infinite chain length is found to be similar to that of PCs, an indication of the glycerol-carbonyl backbone's pivotal role in influencing the lipid-water interface.
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  12. 12
    Caracciolo, G. Liposome-Protein Corona in a Physiological Environment: Challenges and Opportunities for Targeted Delivery of Nanomedicines. Nanomedicine: Nanotechnology, Biology, and Medicine 2015, 11, 543– 557,  DOI: 10.1016/j.nano.2014.11.003
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    12
    Liposome-protein corona in a physiological environment: challenges and opportunities for targeted delivery of nanomedicines
    Caracciolo Giulio
    Nanomedicine : nanotechnology, biology, and medicine (2015), 11 (3), 543-57 ISSN:.
    Active targeting that exploits the (over)expression of surface receptors in target cells by ligand incorporation is a central concept in nanomedicine research. Despite unprecedented efforts, no targeted liposome-based therapeutics is commercially available for clinical practice. What is inhibiting the efficient translation of targeted liposome technology from bench to bedside? After introduction in the bloodstream, the lipid surface is immediately modified by the adsorption of a "protein corona" and preserving the surface functionality appears to be challenging. On the other hand, a long-standing corona with receptor-binding sites could associate with the target cell long enough to activate the cell's uptake machinery, triggering liposome endocytosis and intracellular cargo delivery. This opens the intriguing possibility to manipulate the corona composition by liposome design. This review will focus on the emerging field of liposome-protein corona research from basic, descriptive research to readily applicable knowledge and technologies for implementation in drug improvement and development. From the clinical editor: This review is addressing the liposome protein corona research concerning the potential gains in drug improvement and for drug development.
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  13. 13
    Pozzi, D.; Caracciolo, G.; Caminiti, R.; De Sanctis, S. C.; Amenitsch, H.; Marchini, C.; Montani, M.; Amici, A. Toward the Rational Design of Lipid Gene Vectors: Shape Coupling between Lipoplex and Anionic Cellular Lipids Controls the Phase Evolution of Lipoplexes and the Efficiency of DNA Release. ACS Appl. Mater. Interfaces 2009, 1, 2237– 2249,  DOI: 10.1021/am900406b
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    13
    Toward the rational design of lipid gene vectors: Shape coupling between lipoplex and anionic cellular lipids controls the phase evolution of lipoplexes and the efficiency of DNA release
    Pozzi, Daniela; Caracciolo, Giulio; Caminiti, Ruggero; De Sanctis, Sofia Candeloro; Amenitsch, Heinz; Marchini, Cristina; Montani, Maura; Amici, Augusto
    ACS Applied Materials & Interfaces (2009), 1 (10), 2237-2249CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    A viewpoint now emerging is that a crit. factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, the authors found a universal behavior of lipoplex/anionic lipid (AL) mixts.: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely dild. by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, the authors investigated the effect of the membrane charge d. and a general correlation resulted: the higher the membrane charge d. of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release. Lastly, the formation of nonlamellar phases in lipoplex/AL mixts. is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases. Remarkably, also phase transition rates and DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes. These results highlight the phase properties of the carrier lipid/cellular lipid mixts. as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carriers.
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  14. 14
    Pozzi, D.; Marchini, C.; Cardarelli, F.; Rossetta, A.; Colapicchioni, V.; Amici, A.; Montani, M.; Motta, S.; Brocca, P.; Cantu, L.; Caracciolo, G. Mechanistic Understanding of Gene Delivery Mediated by Highly Efficient Multicomponent Envelope-Type Nanoparticle Systems. Mol. Pharmaceutics 2013, 10, 4654– 4665,  DOI: 10.1021/mp400470p
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    14
    Mechanistic Understanding of Gene Delivery Mediated by Highly Efficient Multicomponent Envelope-Type Nanoparticle Systems
    Pozzi, D.; Marchini, C.; Cardarelli, F.; Rossetta, A.; Colapicchioni, V.; Amici, A.; Montani, M.; Motta, S.; Brocca, P.; Cantu, L.; Caracciolo, G.
    Molecular Pharmaceutics (2013), 10 (12), 4654-4665CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and pos. charged, while synchrotron small-angle X-ray scattering (SAXS) and at. force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) expts. indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quant. investigation of cell uptake, intracellular trafficking,endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels obsd. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that ecient transfection requires both the membrane fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.
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    Koltover, I.; Salditt, T.; Safinya, C. Phase Diagram, Stability, and Overcharging of Lamellar Cationic Lipid–DNA Self-Assembled Complexes. Biophys. J. 1999, 77, 915– 924,  DOI: 10.1016/S0006-3495(99)76942-0
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    15
    Phase diagram, stability, and overcharging of lamellar cationic lipid-DNA self-assembled complexes
    Koltover, I.; Salditt, T.; Safinya, C. R.
    Biophysical Journal (1999), 77 (2), 915-924CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Cationic lipid-DNA (CL-DNA) complexes comprise a promising new class of synthetic nonviral gene delivery systems. When pos. charged, they attach to the anionic cell surface and transfer DNA into the cell cytoplasm. A comprehensive x-ray diffraction study is reported of the lamellar CL-DNA self-assemblies as a function of lipid compn. and lipid/DNA ratio, aimed at elucidating the interactions detg. their structure, charge, and thermodn. stability. The driving force for the formation of charge-neutral complexes is the release of DNA and lipid counterions. Neg. charged complexes have a higher DNA packing d. than isoelec. complexes, whereas pos. charged ones have a lower packing d. This indicates that the overcharging of the complex away from its isoelec. point is caused by changes of the bulk structure with absorption of excess DNA or cationic lipid. The degree of overcharging is dependent on the membrane charge d., which is controlled by the ration of neutral to cationic lipid in the bilayers. Importantly, overcharged complexes are obsd. to move toward their isoelec. charge-neutral point at higher concn. of salt co-ions, with pos. overcharged complexes expelling cationic lipid and neg. overcharged complexes expelling DNA. These observations should apply universally to the formation and structure of self-assemblies between oppositely charged macromols.
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    Cukalevski, R.; Ferreira, S. A.; Dunning, C. J.; Berggård, T.; Cedervall, T. IgG and Fibrinogen Driven Nanoparticle Aggregation. Nano Research 2015, 8, 2733– 2743,  DOI: 10.1007/s12274-015-0780-4
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    16
    IgG and fibrinogen driven nanoparticle aggregation
    Cukalevski, Risto; Ferreira, Silvia A.; Dunning, Christopher J.; Berggaard, Tord; Cedervall, Tommy
    Nano Research (2015), 8 (8), 2733-2743CODEN: NRAEB5; ISSN:1998-0000. (Springer GmbH)
    A thorough understanding of how proteins induce nanoparticle (NP) aggregation is crucial when designing in vitro and in vivo assays and interpreting exptl. results. This knowledge is also crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extn. of IgG (IgG) from cow serum results in lower polystyrene NPs aggregation. Moreover, addn. of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concn.; i.e., low protein concns. induced large aggregates, whereas high protein concns. induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, resp. Here, we propose a mechanism behind the protein concn. dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins. [Figure not available: see fulltext.].
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    Walkey, C. D.; Chan, W. C. Understanding and Controlling the Interaction of Nanomaterials with Proteins in a Physiological Environment. Chem. Soc. Rev. 2012, 41, 2780– 2799,  DOI: 10.1039/C1CS15233E
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    Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment
    Walkey, Carl D.; Chan, Warren C. W.
    Chemical Society Reviews (2012), 41 (7), 2780-2799CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Nanomaterials hold promise as multifunctional diagnostic and therapeutic agents. However, the effective application of nanomaterials is hampered by limited understanding and control over their interactions with complex biol. systems. When a nanomaterial enters a physiol. environment, it rapidly adsorbs proteins forming what is known as the protein corona'. The protein corona alters the size and interfacial compn. of a nanomaterial, giving it a biol. identity that is distinct from its synthetic identity. The biol. identity dets. the physiol. response including signalling, kinetics, transport, accumulation, and toxicity. The structure and compn. of the protein corona depends on the synthetic identity of the nanomaterial (size, shape, and compn.), the nature of the physiol. environment (blood, interstitial fluid, cell cytoplasm, etc.), and the duration of exposure. In this crit. review, we discuss the formation of the protein corona, its structure and compn., and its influence on the physiol. response. We also present an adsorbome' of 125 plasma proteins that are known to assoc. with nanomaterials. We further describe how the protein corona is related to the synthetic identity of a nanomaterial, and highlight efforts to control protein-nanomaterial interactions. We conclude by discussing gaps in the understanding of protein-nanomaterial interactions along with strategies to fill them (167 refs.).
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    Caracciolo, G.; Pozzi, D.; Capriotti, A. L.; Cavaliere, C.; Foglia, P.; Amenitsch, H.; Laganà, A. Evolution of the Protein Corona of Lipid Gene Vectors as a Function of Plasma Concentration. Langmuir 2011, 27, 15048– 15053,  DOI: 10.1021/la202912f
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    18
    Evolution of the protein corona of lipid gene vectors as a function of plasma concentration
    Caracciolo, Giulio; Pozzi, Daniela; Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Amenitsch, Heinz; Lagana, Aldo
    Langmuir (2011), 27 (24), 15048-15053CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The concept that the effective unit of interest in the cell-nanomaterial interaction is the particle and its corona of assocd. proteins is emerging. Here we investigate the compositional evolution of the protein corona of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) cationic liposomes (CLs) and DOTAP/DNA lipoplexes over a wide range of plasma concns. (2.5-80%). The compn. of the hard corona of lipoplexes is quite stable, but that of CLs does evolve considerably. We show that the protein corona of CLs is made of both low-affinity and competitive-binding proteins whose relative abundance changes with the plasma concn. This result may have deep biol. implications for the application of lipid-based gene vectors both in vitro and in vivo.
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  19. 19
    Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Baldelli Bombelli, F.; Dawson, K. A. Physical–Chemical Aspects of Protein Corona: Relevance to In Vitro and In Vivo Biological Impacts of Nanoparticles. J. Am. Chem. Soc. 2011, 133, 2525– 2534,  DOI: 10.1021/ja107583h
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    19
    Physical-Chemical Aspects of Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles
    Monopoli, Marco P.; Walczyk, Dorota; Campbell, Abigail; Elia, Giuliano; Lynch, Iseult; Baldelli Bombelli, Francesca; Dawson, Kenneth A.
    Journal of the American Chemical Society (2011), 133 (8), 2525-2534CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    It is now clearly emerging that besides size and shape, the other primary defining element of nanoscale objects in biol. media is their long-lived protein ("hard") corona. This corona may be expressed as a durable, stabilizing coating of the bare surface of nanoparticle (NP) monomers, or it may be reflected in different subpopulations of particle assemblies, each presenting a durable protein coating. Using the approach and concepts of phys. chem., we relate studies on the compn. of the protein corona at different plasma concns. with structural data on the complexes both in situ and free from excess plasma. This enables a high degree of confidence in the meaning of the hard protein corona in a biol. context. Here, we present the protein adsorption for two compositionally different NPs, namely sulfonated polystyrene and silica NPs. NP-protein complexes are characterized by differential centrifugal sedimentation, dynamic light scattering, and zeta-potential both in situ and once isolated from plasma as a function of the protein/NP surface area ratio. We then introduce a semiquant. detn. of their hard corona compn. using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray liq. chromatog. mass spectrometry, which allows us to follow the total binding isotherms for the particles, identifying simultaneously the nature and amt. of the most relevant proteins as a function of the plasma concn. We find that the hard corona can evolve quite significantly as one passes from protein concns. appropriate to in vitro cell studies to those present in in vivo studies, which has deep implications for in vitro-in vivo extrapolations and will require some consideration in the future.
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  20. 20
    Caracciolo, G.; Pozzi, D.; Capriotti, A.; Cavaliere, C.; Piovesana, S.; Amenitsch, H.; Laganà, A. Lipid Composition: a “Key Factor” for the Rational Manipulation of the Liposome–Protein Corona by Liposome Design. Rsc Adv. 2015, 5, 5967– 5975,  DOI: 10.1039/C4RA13335H
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    20
    Lipid composition: a "key factor" for the rational manipulation of the liposome-protein corona by liposome design
    Caracciolo, G.; Pozzi, D.; Capriotti, A. L.; Cavaliere, C.; Piovesana, S.; Amenitsch, H.; Lagana, A.
    RSC Advances (2015), 5 (8), 5967-5975CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    When liposomes are exposed to biol. fluids, a dynamic coating of proteins immediately covers them. Similarly to the aura of plasma surrounding the Sun, plasma proteins are thought of as establishing an aura that surrounds each liposome, hence the phenomenon was dubbed 'protein corona'. This natural functionalization includes proteins engaged from the blood that can interact with receptors (over)expressed on the plasma membrane of target cells, thus targeting the liposomes to their final destination. Exploiting the liposome-protein corona for targeting has the potential to revolutionize the treatment of many disorders and requires a deep understanding of the factors shaping the corona. Following incubation with human plasma (HP), here we manipulated this corona by using six liposomal formulations with systematic changes in lipid compn. The lipids we employed are among the most frequently used lipid species for drug and gene delivery applications in vitro and in vivo. The six liposome-protein coronas were thoroughly characterized by synchrotron small angle X-ray scattering, dynamic light scattering, zeta-potential and nanoliquid-chromatog. tandem mass spectrometry expts. We identified general principles shaping the liposome-protein corona and established clear-cut relationships between lipid species and classes of plasma proteins. This knowledge sets the basis for a rational manipulation of the protein corona for targeted drug delivery by liposome design.
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    Martínez-Negro, M.; González-Rubio, G.; Aicart, E.; Landfester, K.; Guerrero-Martínez, A.; Junquera, E. Insights into Colloidal Nanoparticle-Protein Corona Interactions for Nanomedicine Applications. Adv. Colloid Interface Sci. 2021, 289, 102366,  DOI: 10.1016/j.cis.2021.102366
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    Insights into colloidal nanoparticle-protein corona interactions for nanomedicine applications
    Martinez-Negro, Maria; Gonzalez-Rubio, Guillermo; Aicart, Emilio; Landfester, Katharina; Guerrero-Martinez, Andres; Junquera, Elena
    Advances in Colloid and Interface Science (2021), 289 (), 102366CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
    A review. Nevertheless, the successful implementation of NPs in medicine demands a proper understanding of their interactions with the different proteins found in biol. fluids. Once introduced into the body, NPs are covered by a protein corona (PC) that dets. the biol. behavior of the NPs. The formation of the PC can eventually favor the rapid clearance of the NPs from the body before fulfilling the desired objective or lead to increased cytotoxicity. The PC nature varies as a function of the different repulsive and attractive forces that govern the NP-protein interaction and their colloidal stability. This review focuses on the phenomenon of PC formation on NPs from a physicochem. perspective, aiming to provide a general overview of this crit. process. Main issues related to NP toxicity and clearance from the body as a result of protein adsorption are covered, including the most promising strategies to control PC formation and, thereby, ensure the successful application of NPs in nanomedicine. Colloidal nanoparticles (NPs) have attracted significant attention due to their unique physicochem. properties suitable for diagnosing and treating different human diseases.
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    Danhier, F.; Feron, O.; Préat, V. To Exploit the Tumor Microenvironment: Passive and Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. Journal of controlled release 2010, 148, 135– 146,  DOI: 10.1016/j.jconrel.2010.08.027
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    To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery
    Danhier, Fabienne; Feron, Olivier; Preat, Veronique
    Journal of Controlled Release (2010), 148 (2), 135-146CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    A review. Because of the particular characteristics of the tumor microenvironment and tumor angiogenesis, it is possible to design drug delivery systems that specifically target anti-cancer drugs to tumors. Most of the conventional chemotherapeutic agents have poor pharmacokinetics profiles and are distributed non-specifically in the body leading to systemic toxicity assocd. with serious side effects. Therefore, the development of drug delivery systems able to target the tumor site is becoming a real challenge that is currently addressed. Nanomedicine can reach tumor passively through the leaky vasculature surrounding the tumors by the enhanced permeability and retention effect whereas ligands grafted at the surface of nanocarriers allow active targeting by binding to the receptors overexpressed by cancer cells or angiogenic endothelial cells. This review is divided into 2 parts: the first one describes the tumor microenvironment and the second one focuses on the exploitation and the understanding of these characteristics to design new drug delivery systems targeting the tumor. Delivery of conventional chemotherapeutic anti-cancer drugs is mainly discussed.
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    Sawant, R. R.; Torchilin, V. P. Challenges in Development of Targeted Liposomal Therapeutics. AAPS journal 2012, 14, 303– 315,  DOI: 10.1208/s12248-012-9330-0
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    Challenges in Development of Targeted Liposomal Therapeutics
    Sawant, Rupa R.; Torchilin, Vladimir P.
    AAPS Journal (2012), 14 (2), 303-315CODEN: AJAOB6; ISSN:1550-7416. (Springer)
    A review. Liposomes, phospholipid vesicles with a bilayered membrane structure, have been widely used as pharmaceutical carriers for drugs and genes, in particular for treatment of cancer. To enhance the efficacy of the liposomal drugs, drug-loaded liposomes are targeted to the tumors by means of passive (enhanced permeability and retention mediated) targeting, based on the longevity of liposomes in blood and its accumulation in pathol. sites with compromised vasculature, and active targeting, based on the attachment of specific ligands to the liposomal surface to bind certain antigens on the target cells. Antibody-targeted liposomes loaded with anticancer drugs demonstrate high potential for clin. applications. This review highlights evolution of liposomes for both passive and active targeting and challenges in development of targeted liposomal therapeutics specifically antibody-targeted liposomes.
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    Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nano 2013, 8, 772– 781,  DOI: 10.1038/nnano.2013.181
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    Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology
    Tenzer, Stefan; Docter, Dominic; Kuharev, Joerg; Musyanovych, Anna; Fetz, Verena; Hecht, Rouven; Schlenk, Florian; Fischer, Dagmar; Kiouptsi, Klytaimnistra; Reinhardt, Christoph; Landfester, Katharina; Schild, Hansjoerg; Maskos, Michael; Knauer, Shirley K.; Stauber, Roland H.
    Nature Nanotechnology (2013), 8 (10), 772-781CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    In biol. fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiol. systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biol. relevancy. Here we show that label-free snapshot proteomics can be used to obtain quant. time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 min) and, over time, to change significantly in terms of the amt. of bound protein, but not in compn. Rapid corona formation is found to affect hemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
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    Kelly, P. M.; Åberg, C.; Polo, E.; O’Connell, A.; Cookman, J.; Fallon, J.; Krpetić, Ž.; Dawson, K. A. Mapping Protein Binding Sites on the Biomolecular Corona of Nanoparticles. Nature Nanotechnol. 2015, 10, 472– 479,  DOI: 10.1038/nnano.2015.47
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    25
    Mapping protein binding sites on the biomolecular corona of nanoparticles
    Kelly, Philip M.; Aberg, Christoffer; Polo, Ester; O'Connell, Ann; Cookman, Jennifer; Fallon, Jonathan; Krpetic, Zeljka; Dawson, Kenneth A.
    Nature Nanotechnology (2015), 10 (5), 472-479CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    Nanoparticles in a biol. milieu are known to form a sufficiently long-lived and well-organized 'corona' of biomols. to confer a biol. identity to the particle. Because this nanoparticle-biomol. complex interacts with cells and biol. barriers, potentially engaging with different biol. pathways, it is important to clarify the presentation of functional biomol. motifs at its interface. Here, we demonstrate that by using antibody-labeled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic mol. description of the biol. identity of nanoparticles.
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    Herda, L. M.; Hristov, D. R.; Lo Giudice, M. C.; Polo, E.; Dawson, K. A. Mapping of Molecular Structure of the Nanoscale Surface in Bionanoparticles. J. Am. Chem. Soc. 2017, 139, 111– 114,  DOI: 10.1021/jacs.6b12297
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    Mapping of Molecular Structure of the Nanoscale Surface in Bionanoparticles
    Herda, Luciana M.; Hristov, Delyan R.; Lo Giudice, Maria Cristina; Polo, Ester; Dawson, Kenneth A.
    Journal of the American Chemical Society (2017), 139 (1), 111-114CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Characterizing the orientation of covalently conjugated proteins on nanoparticles, produced for in vitro and in vivo targeting, though an important feature of such a system, has proved challenging. Although extensive physicochem. characterization of targeting nanoparticles can be addressed in detail, relevant biol. characterization of the nanointerface is crucial to select suitable nanomaterials for further in vitro or in vivo expts. The authors adopt a methodol. using antibody fragments (Fab) conjugated to gold nanoparticles (immunogold) to map the available epitopes on a transferrin grafted silica particle (SiO2-PEG8-Tf) as a proxy methodol. to predict nanoparticle biol. function, and therefore cellular receptor engagement. Data from the adopted method suggest that, on av., only ∼3.5% of proteins grafted on the SiO2-PEG8-Tf nanoparticle surface have a favorable orientation for recognition by the cellular receptor.
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    Lara, S.; Alnasser, F.; Polo, E.; Garry, D.; Lo Giudice, M. C.; Hristov, D. R.; Rocks, L.; Salvati, A.; Yan, Y.; Dawson, K. A. Identification of Receptor Binding to the Biomolecular Corona of Nanoparticles. ACS Nano 2017, 11, 1884– 1893,  DOI: 10.1021/acsnano.6b07933
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    Identification of Receptor Binding to the Biomolecular Corona of Nanoparticles
    Lara, Sandra; Alnasser, Fatima; Polo, Ester; Garry, David; Lo Giudice, Maria Cristina; Hristov, Delyan R.; Rocks, Louise; Salvati, Anna; Yan, Yan; Dawson, Kenneth A.
    ACS Nano (2017), 11 (2), 1884-1893CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Biomols. adsorbed on nanoparticles are known to confer a biol. identity to nanoparticles, mediating the interactions with cells and biol. barriers. However, how these mols. are presented on the particle surface in biol. milieu remains unclear. The central aim of this study is to identify key protein recognition motifs and link them to specific cell-receptor interactions. Here, the authors employed an immuno-mapping technique to quantify epitope presentations of two major proteins in the serum corona, low-d. lipoprotein and IgG. Combined with a purpose-built receptor expression system, both proteins present functional motifs to allow simultaneous recognition by low-d. lipoprotein receptor and Fc-gamma receptor I of the corona. The authors' results suggest that the "labeling" of nanoparticles by biomol. adsorption processes allows for multiple pathways in biol. processes in which they may be "mistaken" for endogenous objects, such as lipoproteins, and exogenous ones, such as viral infections.
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    Gianneli, M.; Polo, E.; Lopez, H.; Castagnola, V.; Aastrup, T.; Dawson, K. Label-Free in-Flow Detection of Receptor Recognition Motifs on the Biomolecular Corona of Nanoparticles. Nanoscale 2018, 10, 5474– 5481,  DOI: 10.1039/C7NR07887K
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    Label-free in-flow detection of receptor recognition motifs on the biomolecular corona of nanoparticles
    Gianneli, M.; Polo, E.; Lopez, H.; Castagnola, V.; Aastrup, T.; Dawson, K. A.
    Nanoscale (2018), 10 (12), 5474-5481CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Nanomedicine, nanotargeting and nanotherapeutics have in the last few years faced several difficulties in translating the promising results obtained in vitro to an in vivo scenario. The origin of this discrepancy might be found in the lack of a detailed and realistic characterization of the biol. surface of nanoparticles. Despite the capability to engineer nanomaterials with a great variety and a precise control of the surface functionalization, the targeting capability is lost when the nanoparticles are embedded in complex biol. media, due to the formation of a biol. layer (biomol. corona). This biol. layer represents the ultimate nanoparticle surface, likely to interact with the cell machinery. Therefore, in addn. to traditional nanoparticle characterization techniques, a more insightful investigation of the biomol. corona is needed, including the capability to assess the orientation and functionality of specific key mol. features. Here we present a method for the rapid screening of exposed protein recognition motifs on the surface of nanoparticles exploiting quartz crystal microbalance (QCM). We quantify accessible functional epitopes of transferrin-coated nanoparticles and correlate them to differences in nanoparticle size and functionalization. The target recognition occurs label free in flow, thereby pushing our investigation into a more in vivo-like scenario. Our method is applicable to a wide array of nanoparticles and therefore holds the potential to become an advanced technique for the classification of all kinds of nanobioconstructs based on their biol. external functionality.
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    Moein Moghimi, S.; Patel, H. M. Opsonophagocytosis of Liposomes by Peritoneal Macrophages and Bone Marrow Reticuloendothelial Cells. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1992, 1135, 269– 274,  DOI: 10.1016/0167-4889(92)90230-9
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    Papini, E.; Tavano, R.; Mancin, F. Opsonins and Dysopsonins of Nanoparticles: Facts, Concepts, and Methodological Guidelines. Frontiers in Immunology 2020, 11, 2343,  DOI: 10.3389/fimmu.2020.567365
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    Languino, L. R.; Duperray, A.; Joganic, K. J.; Fornaro, M.; Thornton, G. B.; Altieri, D. C. Regulation of Leukocyte-Endothelium Interaction and Leukocyte Transendothelial Migration by Intercellular Adhesion Molecule 1-Fibrinogen Recognition. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 1505– 1509,  DOI: 10.1073/pnas.92.5.1505
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    Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition
    Languino, Lucia R.; Duperray, Alain; Joganic, Karen J.; Fornaro, Mara; Thornton, George B.; Altieri, Dario C.
    Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (5), 1505-9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesions to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the assocn. of fibrinogen with intercellular adhesion mol. 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen γ-chain sequence N117NQKIVNLKEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.
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    Deng, Z. J.; Liang, M.; Monteiro, M.; Toth, I.; Minchin, R. F. Nanoparticle-Induced Unfolding of Fibrinogen Promotes Mac-1 Receptor Activation and Inflammation. Nat. Nanotechnol 2011, 6, 39– 44,  DOI: 10.1038/nnano.2010.250
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    Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation
    Deng, Zhou J.; Liang, Mingtao; Monteiro, Michael; Toth, Istvan; Minchin, Rodney F.
    Nature Nanotechnology (2011), 6 (1), 39-44CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    The chem. compn., size, shape and surface characteristics of nanoparticles affect the way proteins bind to these particles, and this in turn influences the way in which nanoparticles interact with cells and tissues. Nanomaterials bound with proteins can result in physiol. and pathol. changes, including macrophage uptake, blood coagulation, protein aggregation and complement activation, but the mechanisms that lead to these changes remain poorly understood. Here, we show that neg. charged poly(acrylic acid)-conjugated gold nanoparticles bind to and induce unfolding of fibrinogen, which promotes interaction with the integrin receptor, Mac-1. Activation of this receptor increases the NF-κB signalling pathway, resulting in the release of inflammatory cytokines. However, not all nanoparticles that bind to fibrinogen demonstrated this effect. Our results show that the binding of certain nanoparticles to fibrinogen in plasma offers an alternative mechanism to the more commonly described role of oxidative stress in the inflammatory response to nanomaterials.
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    Deng, Z. J.; Liang, M.; Toth, I.; Monteiro, M. J.; Minchin, R. F. Molecular Interaction of Poly (Acrylic Acid) Gold Nanoparticles with Human Fibrinogen. ACS Nano 2012, 6, 8962– 8969,  DOI: 10.1021/nn3029953
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    Molecular interaction of poly(acrylic acid) gold nanoparticles with human fibrinogen
    Deng, Zhou J.; Liang, Mingtao; Toth, Istvan; Monteiro, Michael J.; Minchin, Rodney F.
    ACS Nano (2012), 6 (10), 8962-8969CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    The binding of fibrinogen to various nanoparticles can result in protein unfolding and exposure of cryptic epitopes that subsequently interact with cell surface receptors. This response is dependent on the size, charge, and concn. of the nanoparticle. Here, the authors examd. the binding kinetics of human fibrinogen to neg. charged poly(acrylic acid)-coated Au-nanoparticles ranging in size from 7 to 22 nm. These particles have previously been shown to elicit an inflammatory response in human cells. The larger nanoparticles bound fibrinogen with increasing affinity and a slower dissocn. rate. Each fibrinogen mol. could accommodate 2 7-nm nanoparticles, but only one when the diam. increased to 10 nm. Nanoparticles of size >12 nm bound multiple fibrinogen mols. in a pos. cooperative manner. However, in the presence of excess nanoparticle, fibrinogen induced the aggregation of the larger particles that could bind more than one protein mol. This was consistent with interparticle bridging by the fibrinogen. Taken together, these results demonstrate that subtle changes in nanoparticle size can influence protein binding both with the surface of the nanoparticle and within the protein corona.
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  34. 34
    Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nature Nanotechnol. 2013, 8, 772– 781,  DOI: 10.1038/nnano.2013.181
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    Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology
    Tenzer, Stefan; Docter, Dominic; Kuharev, Joerg; Musyanovych, Anna; Fetz, Verena; Hecht, Rouven; Schlenk, Florian; Fischer, Dagmar; Kiouptsi, Klytaimnistra; Reinhardt, Christoph; Landfester, Katharina; Schild, Hansjoerg; Maskos, Michael; Knauer, Shirley K.; Stauber, Roland H.
    Nature Nanotechnology (2013), 8 (10), 772-781CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    In biol. fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiol. systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biol. relevancy. Here we show that label-free snapshot proteomics can be used to obtain quant. time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 min) and, over time, to change significantly in terms of the amt. of bound protein, but not in compn. Rapid corona formation is found to affect hemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
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    Tsuchiya, S.; Yamabe, M.; Yamaguchi, Y.; Kobayashi, Y.; Konno, T.; Tada, K. Establishment and Characterization of a Human Acute Monocytic Leukemia Cell Line (THP-1). International journal of cancer 1980, 26, 171– 176,  DOI: 10.1002/ijc.2910260208
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    Establishment and characterization of a human acute monocytic leukemia cell line (THP-1)
    Tsuchiya S; Yamabe M; Yamaguchi Y; Kobayashi Y; Konno T; Tada K
    International journal of cancer (1980), 26 (2), 171-6 ISSN:0020-7136.
    A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia is described. This cell line had Fc and C3b receptors, but no surface or cytoplasmic immunoglobulins. HLA haplotypes of THP-1 were HLA-A2, -A9, -B5, -DRW1 and -DRW2. The monocytic nature of the cell line was characterized by: (1) the presence of alpha-naphthyl butyrate esterase activities which could be inhibited by NaF; (2) lysozyme production; (3) the phagocytosis of latex particles and sensitized sheep erythrocytes; and (4) the ability to restore T-lymphocyte response to Con A. The cells did not possess Epstein-Barr virus-associated nuclear antigen. These results indicate that THP-1 is a leukemia cell line with distinct monocytic markers. During culture, THP-1 maintained these monocytic characteristics for over 14 months.
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    Kleiveland, C.; Verhoeckx, K.; Cotter, P.; López-Expósito, I.; Kleiveland, C.; Lea, T.; Mackie, A. Peripheral Blood Mononuclear Cells. In The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models; Springer: Cham, Switzerland, 2015; pp 161– 167, DOI: 10.1007/978-3-319-16104-4_15 .
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    Giulimondi, F.; Digiacomo, L.; Pozzi, D.; Palchetti, S.; Vulpis, E.; Capriotti, A. L.; Chiozzi, R. Z.; Lagana, A.; Amenitsch, H.; Masuelli, L.; Peruzzi, G.; Mahmoudi, M.; Screpanti, I.; Zingoni, A.; Caracciolo, G. Interplay of Protein Corona and Immune Cells Controls Blood Residency of Liposomes. Nat. Commun. 2019, 10, 3686,  DOI: 10.1038/s41467-019-11642-7
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    Interplay of protein corona and immune cells controls blood residency of liposomes
    Giulimondi, Francesca; Digiacomo, Luca; Pozzi, Daniela; Palchetti, Sara; Vulpis, Elisabetta; Capriotti, Anna Laura; Chiozzi, Riccardo Zenezini; Lagana, Aldo; Amenitsch, Heinz; Masuelli, Laura; Mahmoudi, Morteza; Screpanti, Isabella; Zingoni, Alessandra; Caracciolo, Giulio
    Nature Communications (2019), 10 (1), 3686CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    In vivo liposomes, like other types of nanoparticles, acquire a totally new 'biol. identity' due to the formation of a biomol. coating known as the protein corona that depends on and modifies the liposomes' synthetic identity. The liposome-protein corona is a dynamic interface that regulates the interaction of liposomes with the physiol. environment. Here we show that the biol. identity of liposomes is clearly linked to their sequestration from peripheral blood mononuclear cells (PBMCs) of healthy donors that ultimately leads to removal from the bloodstream. Pre-coating liposomes with an artificial corona made of human plasma proteins drastically reduces capture by circulating leukocytes in whole blood and may be an effective strategy to enable prolonged circulation in vivo. We conclude with a crit. assessment of the key concepts of liposome technol. that need to be reviewed for its definitive clin. translation.
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    Chung, A.; Gao, Q.; Kao, W. J. Macrophage Matrix Metalloproteinase-2/-9 Gene and Protein Expression Following Adhesion to ECM-Derived Multifunctional Matrices Via Integrin Complexation. Biomaterials 2007, 28, 285– 298,  DOI: 10.1016/j.biomaterials.2006.08.038
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    Macrophage matrix metalloproteinase-2/-9 gene and protein expression following adhesion to ECM-derived multifunctional matrices via integrin complexation
    Chung Amy; Gao Qiang; Kao Weiyuan John
    Biomaterials (2007), 28 (2), 285-98 ISSN:0142-9612.
    Macrophages are commonly observed at the biomaterial-tissue interface and interact with the extracellular matrix (ECM) mainly by integrin receptors to play a critical role in ECM turnover by secreting matrix metalloproteinases (MMPs). To investigate beta1 and beta3 containing integrin-mediated adhesion and subsequent MMP-2/-9 protein and gene expression in human blood-derived monocytes, biofunctional peptides immobilized onto flexible polyethylene glycol (PEG) arms were grafted onto a gelatin-based interpenetrating network (IPN). Adherent monocyte density was dramatically greater in the presence of RGD immobilized onto flexible PEG arms of the gelatin-based IPN. Pretreatment of monocytes with either anti-integrin beta1 or beta3 led to a significant decrease in adherent cell density on RGD-PEG-grafted IPNs. MMP-2 and MMP-9 protein and MMP-9 mRNA expression increased in the presence of IPNs initially, independent of ligand identity. Anti-integrin beta1 or beta3 antibody pretreatment of monocytes led to a general decrease in MMP-2/-9 protein expression. These results demonstrate the importance of beta1 and beta3 containing integrins in mediating monocyte adhesion onto RGD immobilized onto flexible PEG arms of the IPN. The results also reveal that MMP-2/-9 protein and gene expression is influenced by the presence of gelatin and not the ligands immobilized on the PEG arms of the IPN.
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    Simon, J.; Müller, L. K.; Kokkinopoulou, M.; Lieberwirth, I.; Morsbach, S.; Landfester, K.; Mailänder, V. Exploiting the Biomolecular Corona: Pre-Coating of Nanoparticles Enables Controlled Cellular Interactions. Nanoscale 2018, 10, 10731– 10739,  DOI: 10.1039/C8NR03331E
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    Exploiting the biomolecular corona: pre-coating of nanoparticles enables controlled cellular interactions
    Simon, Johanna; Mueller, Laura K.; Kokkinopoulou, Maria; Lieberwirth, Ingo; Morsbach, Svenja; Landfester, Katharina; Mailaender, Volker
    Nanoscale (2018), 10 (22), 10731-10739CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Formation of the biomol. corona ultimately dets. the successful application of nanoparticles in vivo. Adsorption of biomols. such as proteins is an inevitable process that takes place instantaneously upon contact with physiol. fluid (e.g. blood). Therefore, strategies are needed to control this process in order to improve the properties of the nanoparticles and to allow targeted drug delivery. Here, we show that the design of the protein corona by a pre-formed protein corona with tailored properties enables targeted cellular interactions. Nanoparticles were pre-coated with Ig depleted plasma to create and design a protein corona that reduces cellular uptake by immune cells. It was proven that a pre-formed protein corona remains stable even after nanoparticles were re-introduced to plasma. This opens up the great potential to exploit protein corona formation, which will significantly influence the development of novel nanomaterials.
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    Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M. PEGylation As a Strategy for Improving Nanoparticle-Based Drug and Gene Delivery. Adv. Drug Delivery Rev. 2016, 99, 28– 51,  DOI: 10.1016/j.addr.2015.09.012
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    PEGylation as a strategy for improving nanoparticle-based drug and gene delivery
    Suk, Jung Soo; Xu, Qingguo; Kim, Namho; Hanes, Justin; Ensign, Laura M.
    Advanced Drug Delivery Reviews (2016), 99 (Part_A), 28-51CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
    Coating the surface of nanoparticles with polyethylene glycol (PEG), or "PEGylation", is a commonly used approach for improving the efficiency of drug and gene delivery to target cells and tissues. Building from the success of PEGylating proteins to improve systemic circulation time and decrease immunogenicity, the impact of PEG coatings on the fate of systemically administered nanoparticle formulations has, and continues to be, widely studied. PEG coatings on nanoparticles shield the surface from aggregation, opsonization, and phagocytosis, prolonging systemic circulation time. Here, we briefly describe the history of the development of PEGylated nanoparticle formulations for systemic administration, including how factors such as PEG mol. wt., PEG surface d., nanoparticle core properties, and repeated administration impact circulation time. A less frequently discussed topic, we then describe how PEG coatings on nanoparticles have also been utilized for overcoming various biol. barriers to efficient drug and gene delivery assocd. with other modes of administration, ranging from gastrointestinal to ocular. Finally, we describe both methods for PEGylating nanoparticles and methods for characterizing PEG surface d., a key factor in the effectiveness of the PEG surface coating for improving drug and gene delivery.
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    Ishida, T.; Ichihara, M.; Wang, X.; Kiwada, H. Spleen Plays an Important Role in the Induction of Accelerated Blood Clearance of PEGylated Liposomes. J. Controlled Release 2006, 115, 243– 250,  DOI: 10.1016/j.jconrel.2006.08.001
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    Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes
    Ishida, Tatsuhiro; Ichihara, Masako; Wang, XinYu; Kiwada, Hiroshi
    Journal of Controlled Release (2006), 115 (3), 243-250CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    It is well known that steric stabilization of the surface of liposomes by a polyethylene glycol (PEG) conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended circulation times of the liposomes (t1/2 ≈ 20 h in rat). Recently, we reported on the "accelerated blood clearance (ABC) phenomenon", causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. We also reported that abundant binding of IgM, secreted into the blood stream after the first dose and, to PEGylated liposomes, plays an essential role in the induction of the ABC phenomenon. Spleen is well known to play a central role in the immune reaction and to produce IgM following a bacterial infection. The aim of the present study was to det. whether spleen contributes to the induction of the ABC phenomenon and to unravel its role in the phenomenon. In rats that were splenectomized (surgical removal of spleen) prior to the first injection of liposomes (0.001 μmol phospholipids/kg), the ABC phenomenon was totally abolished. In these rats serum IgM concns. as well as the amts. of IgM bound to PEGylated liposomes were substantially reduced. Splenectomy attenuated the ABC phenomenon when performed until 3 days post-first injection. Removal of the spleen 4 days post-first injection left the ABC phenomenon unchanged. This finding indicates that the immune reaction in the spleen against the PEGylated liposomes occurs during at least 2-3 days following the first administration and then IgM reactive to PEGylated liposomes is produced. The present study proves that the spleen plays a crit. role in the induction phase of the ABC phenomenon. For effective clin. application, many liposomal drug formulations will require multiple injections. The ABC phenomenon described in this and several preceding papers therefore has important implications for the development and evaluation of therapeutically useful liposomal formulations requiring multiple-dose administration.
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    Hadjidemetriou, M.; Al-Ahmady, Z.; Mazza, M.; Collins, R. F.; Dawson, K.; Kostarelos, K. In Vivo Biomolecule Corona Around Blood-Circulating, Clinically Used and Antibody-Targeted Lipid Bilayer Nanoscale Vesicles. ACS Nano 2015, 9, 8142– 8156,  DOI: 10.1021/acsnano.5b03300
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    In Vivo Biomolecule Corona around Blood-Circulating, Clinically Used and Antibody-Targeted Lipid Bilayer Nanoscale Vesicles
    Hadjidemetriou, Marilena; Al-Ahmady, Zahraa; Mazza, Mariarosa; Collins, Richard F.; Dawson, Kenneth; Kostarelos, Kostas
    ACS Nano (2015), 9 (8), 8142-8156CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    The adsorption of proteins and their layering onto nanoparticle surfaces has been called the "protein corona". This dynamic process of protein adsorption has been extensively studied following in vitro incubation of many different nanoparticles with plasma proteins. However, the formation of protein corona under dynamic, in vivo conditions remains largely unexplored. Extrapolation of in vitro formed protein coronas to predict the fate and possible toxicol. burden from nanoparticles in vivo is of great interest. However, complete lack of such direct comparisons for clin. used nanoparticles makes the study of in vitro and in vivo formed protein coronas of great importance. Our aim was to study the in vivo protein corona formed onto i.v. injected, clin. used liposomes, based on the compn. of the PEGylated liposomal formulation that constitutes the anticancer agent Doxil. The formation of in vivo protein corona was detd. after the recovery of the liposomes from the blood circulation of CD-1 mice 10 min postinjection. In comparison, in vitro protein corona was formed by the incubation of liposomes in CD-1 mouse plasma. In vivo and in vitro formed protein coronas were compared in terms of morphol., compn. and cellular internalization. The protein coronas on bare (non-PEGylated) and monoclonal antibody (IgG) targeted liposomes of the same lipid compn. were also comparatively investigated. A network of linear fibrillary structures constituted the in vitro formed protein corona, whereas the in vivo corona had a different morphol. but did not appear to coat the liposome surface entirely. Even though the total amt. of protein attached on circulating liposomes correlated with that obsd. from in vitro incubations, the variety of mol. species in the in vivo corona were considerably wider. Both in vitro and in vivo formed protein coronas were found to significantly reduce receptor binding and cellular internalization of antibody-conjugated liposomes; however, the in vivo corona formation did not lead to complete ablation of their targeting capability.
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    Hadjidemetriou, M.; Al-Ahmady, Z.; Kostarelos, K. Time-Evolution of In Vivo Protein Corona onto Blood-Circulating PEGylated Liposomal Doxorubicin (DOXIL) Nanoparticles. Nanoscale 2016, 8, 6948– 6957,  DOI: 10.1039/C5NR09158F
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    Time-evolution of in vivo protein corona onto blood-circulating PEGylated liposomal doxorubicin (DOXIL) nanoparticles
    Hadjidemetriou, Marilena; Al-Ahmady, Zahraa; Kostarelos, Kostas
    Nanoscale (2016), 8 (13), 6948-6957CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Nanoparticles (NPs) are instantly modified once injected in the bloodstream because of their interaction with the blood components. The spontaneous coating of NPs by proteins, once in contact with biol. fluids, has been termed the 'protein corona' and it is considered to be a determinant factor for the pharmacol., toxicol. and therapeutic profile of NPs. Protein exposure time is thought to greatly influence the compn. of protein corona, however the dynamics of protein interactions under realistic, in vivo conditions remain unexplored. The aim of this study was to quant. and qual. investigate the time evolution of in vivo protein corona, formed onto blood circulating, clin. used, PEGylated liposomal doxorubicin. Protein adsorption profiles were detd. 10 min, 1 h and 3 h post-injection of liposomes into CD-1 mice. The results demonstrated that a complex protein corona was formed as early as 10 min post-injection. Even though the total amt. of protein adsorbed did not significantly change over time, the fluctuation of protein abundances obsd. indicated highly dynamic protein binding kinetics.
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    Hadjidemetriou, M.; McAdam, S.; Garner, G.; Thackeray, C.; Knight, D.; Smith, D.; Al-Ahmady, Z.; Mazza, M.; Rogan, J.; Clamp, A.; Kostarelos, K. The Human In Vivo Biomolecule Corona onto PEGylated liposomes: a Proof-of-Concept Clinical Study. Adv. Mater. 2019, 31, 1803335,  DOI: 10.1002/adma.201803335
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    Cerullo, V.; Seiler, M. P.; Mane, V.; Brunetti-Pierri, N.; Clarke, C.; Bertin, T. K.; Rodgers, J. R.; Lee, B. Toll-Like Receptor 9 Triggers an Innate Immune Response to Helper-Dependent Adenoviral Vectors. Molecular Therapy 2007, 15, 378– 385,  DOI: 10.1038/sj.mt.6300031
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    Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors
    Cerullo, Vincenzo; Seiler, Michael P.; Mane, Viraj; Brunetti-Pierri, Nicola; Clarke, Christian; Bertin, Terry K.; Rodgers, John R.; Lee, Brendan
    Molecular Therapy (2007), 15 (2), 378-385CODEN: MTOHCK; ISSN:1525-0016. (Nature Publishing Group)
    A major obstacle to the clin. application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying mol. components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, the authors show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after i.v. injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that addnl. innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.
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    Moghimi, S. M.; Patel, H. M. Tissue Specific Opsonins for Phagocytic Cells and Their Different Affinity for Cholesterol-Rich Liposomes. FEBS letters 1988, 233, 143– 147,  DOI: 10.1016/0014-5793(88)81372-3
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    Tissue specific opsonins for phagocytic cells and their different affinity for cholesterol-rich liposomes
    Moghimi, S. Moein; Patel, Harish M.
    FEBS Letters (1988), 233 (1), 143-7CODEN: FEBLAL; ISSN:0014-5793.
    In vivo expts. show that Kupffer cells avidly take up cholesterol-poor but not cholesterol-rich liposomes, whereas splenic phagocytic cells take up preferentially cholesterol-rich rather than cholesterol-poor liposomes in the presence of serum. Apparently, serum contains opsonins specific for hepatic and splenic phagocytic cells and these opsonins have different affinities for cholesterol-rich and cholesterol-poor liposomes.
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    Moghimi, S. M.; Simberg, D.; Skotland, T.; Yaghmur, A.; Hunter, C. The Interplay between Blood Proteins, Complement, and Macrophages on Nanomedicine Performance and Responses. Journal of Pharmacology and Experimental Therapeutics 2019, 370, 581– 592,  DOI: 10.1124/jpet.119.258012
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    The interplay between blood proteins, complement, and macrophages on nanomedicine performance and responses
    Moghimi, S. Moein; Simberg, Dmitri; Skotland, Tore; Yaghmur, Anan; Hunter, A. Christy
    Journal of Pharmacology and Experimental Therapeutics (2019), 370 (3), 581-592CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)
    A review. In the blood, depending on their physicochem. characteristics, nanoparticles attract a wide range of plasma biomols. The majority of blood biomols. bind nonspecifically to nanoparticles. On the other hand, biomols. such as pattern-recognition complement-sensing proteins may recognize some structural determinants of the pristine surface, causing complement activation. Adsorption of nonspecific blood proteins could also recruit natural antibodies and initiate complement activation, and this seems to be a global process with many preclin. and clin. nanomedicines. We discuss these issues, since complement activation has ramifications in nanomedicine stability and pharmacokinetics, as well as in inflammation and disease progression. Some studies have also predicted a role for complement systems in infusion-related reactions, whereas others show a direct role for macrophages and other immune cells independent of complement activation. We comment on these discrepancies and suggest directions for exploring the underlying mechanisms.
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    Verhoef, J.; Anchordoquy, T. Drug Delivery Transl. Res. 2013, 3, 499– 503,  DOI: 10.1007/s13346-013-0176-5
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    Questioning the use of PEGylation for drug delivery
    Verhoef, Johan J. F.; Anchordoquy, Thomas J.
    Drug Delivery and Translational Research (2013), 3 (6), 499-503CODEN: DDTRCY; ISSN:2190-3948. (Springer)
    A review. Polyethylene glycol (PEG) is widely utilized in drug delivery and nanotechnol. due to its reported "stealth" properties and biocompatibility. It is generally thought that PEGylation allows particulate delivery systems and biomaterials to evade the immune system and thereby prolong circulation lifetimes. However, numerous studies over the past decade have demonstrated that PEGylation causes significant redns. in drug delivery, including enhanced serum protein binding, reduced uptake by target cells, and the elicitation of an immune response that facilitates clearance in vivo. This report reviews some of the extensive literature documenting the detrimental effects of PEGylation, and thereby questions the wisdom behind employing this strategy in drug development.
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    Song, J.; Ju, Y.; Amarasena, T. H.; Lin, Z.; Mettu, S.; Zhou, J.; Rahim, M. A.; Ang, C.-S.; Cortez-Jugo, C.; Kent, S. J.; Caruso, F. Influence of Poly (Ethylene Glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle–Immune Cell Interactions in Human Blood. ACS Nano 2021, 15, 10025,  DOI: 10.1021/acsnano.1c01642
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    Influence of Poly(ethylene glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle-Immune Cell Interactions in Human Blood
    Song Jiaying; Ju Yi; Lin Zhixing; Zhou Jiajing; Rahim Md Arifur; Cortez-Jugo Christina; Caruso Frank; Amarasena Thakshila H; Kent Stephen J; Mettu Srinivas; Ang Ching-Seng
    ACS nano (2021), 15 (6), 10025-10038 ISSN:.
    Poly(ethylene glycol) (PEG) is widely used in particle assembly to impart biocompatibility and stealth-like properties in vivo for diverse biomedical applications. Previous studies have examined the effect of PEG molecular weight and PEG coating density on the biological fate of various particles; however, there are few studies that detail the fundamental role of PEG molecular architecture in particle engineering and bio-nano interactions. Herein, we engineered PEG particles using a mesoporous silica (MS) templating method and investigated how the PEG building block architecture impacted the physicochemical properties (e.g., surface chemistry and mechanical characteristics) of the PEG particles and subsequently modulated particle-immune cell interactions in human blood. Varying the PEG architecture from 3-arm to 4-arm, 6-arm, and 8-arm generated PEG particles with a denser, stiffer structure, with increasing elastic modulus from 1.5 to 14.9 kPa, inducing an increasing level of immune cell association (from 15% for 3-arm to 45% for 8-arm) with monocytes. In contrast, the precursor PEG particles with the template intact (MS@PEG) were stiffer and generally displayed higher levels of immune cell association but showed the opposite trend-immune cell association decreased with increasing PEG arm numbers. Proteomics analysis demonstrated that the biomolecular corona that formed on the PEG particles minimally influenced particle-immune cell interactions, whereas the MS@PEG particle-cell interactions correlated with the composition of the corona that was abundant in histidine-rich glycoproteins. Our work highlights the role of PEG architecture in the design of stealth PEG-based particles, thus providing a link between the synthetic nature of particles and their biological behavior in blood.
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    Kristensen, K.; Urquhart, A. J.; Thormann, E.; Andresen, T. L. Binding of Human Serum Albumin to PEGylated Liposomes: Insights into Binding Numbers and Dynamics by Fluorescence Correlation Spectroscopy. Nanoscale 2016, 8, 19726– 19736,  DOI: 10.1039/C6NR05455B
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    Binding of human serum albumin to PEGylated liposomes: insights into binding numbers and dynamics by fluorescence correlation spectroscopy
    Kristensen, Kasper; Urquhart, Andrew J.; Thormann, Esben; Andresen, Thomas L.
    Nanoscale (2016), 8 (47), 19726-19736CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Liposomes for medical applications are often administered by i.v. injection. Once in the bloodstream, the liposomes are covered with a "protein corona", which impacts the behavior and eventual fate of the liposomes. Currently, many aspects of the liposomal protein corona are not well understood. For example, there is generally a lack of knowledge about the liposome binding affinities and dynamics of common types of blood plasma proteins. Fluorescence correlation spectroscopy (FCS) is a powerful exptl. technique that potentially can provide such knowledge. In this study, we have used FCS to investigate the binding of human serum albumin (HSA) to std. types of PEGylated fluid-phase liposomes (consisting of DOPC and DOPE-PEG2k) and PEGylated gel-phase liposomes (consisting of DSPC and DSPE-PEG2k) with various PEG chain surface densities. We detected no significant binding of HSA to the PEGylated fluid-phase liposomes. In contrast, we found that HSA bound tightly to the PEGylated gel-phase liposomes, although only a low no. of HSA mols. could be accommodated per liposome. Overall, we believe that our data provides a useful benchmark for other researchers interested in studying the liposomal protein corona.
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    Jhaveri, A.; Deshpande, P.; Pattni, B.; Torchilin, V. Transferrin-Targeted, Resveratrol-Loaded Liposomes for the Treatment of Glioblastoma. Journal of controlled release 2018, 277, 89– 101,  DOI: 10.1016/j.jconrel.2018.03.006
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    Transferrin-targeted, resveratrol-loaded liposomes for the treatment of glioblastoma
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    Journal of Controlled Release (2018), 277 (), 89-101CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    Glioblastomas (GBMs) are highly aggressive brain tumors with a very grim prognosis even after multi-modal therapeutic regimens. Conventional chemotherapeutic agents frequently lead to drug resistance and result in severe toxicities to non-cancerous tissues. Resveratrol (RES), a natural polyphenol with pleiotropic health benefits, has proven chemopreventive effects in all the stages of cancer including initiation, promotion and progression. However, the poor physico-chem. properties of RES severely limit its use as a free drug. In this study, RES was loaded into PEGylated liposomes (RES-L) to counter its drawbacks as a free drug. Since transferrin receptors (TfRs) are up-regulated in GBM, the liposome surface was modified with transferrin moieties (Tf-RES-L) to make them cancer cell-specific. The liposomal nanomedicines developed in this project were aimed at enhancing the physico-chem. properties of RES and exploiting the passive and active targeting capabilities of liposomes to effectively treat GBM. The RES-L were stable, had a good drug-loading capacity, prolonged drug-release in vitro and were easily scalable. Flow cytometry and confocal microscopy were used to study the assocn. with, and internalization of, Tf-L into U-87 MG cells. The Tf-RES-Ls were significantly more cytotoxic and induced higher levels of apoptosis accompanied by activation of caspases 3/7 in GBM cells when compared to free RES or RES-L. The ability of RES to arrest cells in the S-phase of the cell cycle, and selectively induce prodn. of reactive oxygen species in cancer cells were probably responsible for its cytotoxic effects. The therapeutic efficacy of RES formulations was evaluated in a s.c. xenograft mouse model of GBM. A tumor growth inhibition study and a modified survival study showed that Tf-RES-Ls were more effective than other treatments in their ability to inhibit tumor growth and improve survival in mice. Overall, the liposomal nanomedicines of RES developed in this project exhibited favorable in vitro and in vivo efficacies, which warrant their further investigation for the treatment of GBMs.
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    Moyer, L. S.; Gorin, M. H. Eletectrokinetic Aspects of Surface Chemistry: IX. The Electric Mobilitites of Quartz and Collodion Particles in Mixtures of Horse Serum and Serum Proteins in Relation to the Mechanisms of Film Formation. J. Biol. Chem. 1940, 133, 605– 619,  DOI: 10.1016/S0021-9258(18)73343-7
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    Electrokinetic aspects of surface chemistry. IX. Electric mobilities of quartz and collodion particles in mixtures of horse serum and serum proteins in relation to the mechanism of film formation
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    cf. C. A. 34, 1696.3. The electrophoretic mobilities of quartz and collodion particles were detd. after exposure to mixts. of serum proteins or after they had been coated with one protein and then exposed to another or to serum. There is little tendency for the various constituents used to adsorb on each other, but one protein may replace another at a surface; thus a film of one of the protein components is obtained rather than a mosaic. The nature of the underlying surface influences the adsorption, the more hydrophilic proteins being adsorbed more readily by the more hydrophilic surfaces, and vice versa. Globulin when coated on quartz appears to be either irreversibly adsorbed or the most strongly adsorbed of the three proteins, replacement by albumin not occurring. The tendency to be adsorbed on quartz is roughly in the order albumin A > albumin B = globulin, with the addnl. assumption that globulin can replace the albumins whenever the free energy differences are favorable. On collodion, globulin is more strongly adsorbed than albumin A or B. The biol. significance of these findings is discussed.
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    Caracciolo, G.; Cardarelli, F.; Pozzi, D.; Salomone, F.; Maccari, G.; Bardi, G.; Capriotti, A. L.; Cavaliere, C.; Papi, M.; Laganà, A. Selective Targeting Capability Acquired with a Protein Corona Adsorbed on the Surface of 1,2-Dioleoyl-3-Trimethylammonium Propane/DNA Nanoparticles. ACS Appl. Mater. Interfaces 2013, 5, 13171– 13179,  DOI: 10.1021/am404171h
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    Selective Targeting Capability Acquired with a Protein Corona Adsorbed on the Surface of 1,2-Dioleoyl-3-trimethylammonium Propane/DNA Nanoparticles
    Caracciolo, Giulio; Cardarelli, Francesco; Pozzi, Daniela; Salomone, Fabrizio; Maccari, Giuseppe; Bardi, Giuseppe; Capriotti, Anna Laura; Cavaliere, Chiara; Papi, Massimiliano; Lagana, Aldo
    ACS Applied Materials & Interfaces (2013), 5 (24), 13171-13179CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    A possible turning point in drug delivery has been recently reached: the protein shell, which covers nanocarriers in vivo, can be used for targeting. Here, we show that nanoparticles can acquire a selective targeting capability with a protein corona adsorbed on the surface. We demonstrate that lipid particles made of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and DNA, upon interaction with human plasma components, spontaneously become coated with vitronectin that promotes efficient uptake in cancer cells expressing high levels of the vitronectin ανβ3 integrin receptor.
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    Digiacomo, L.; Cardarelli, F.; Pozzi, D.; Palchetti, S.; Digman, M.; Gratton, E.; Capriotti, A.; Mahmoudi, M.; Caracciolo, G. An Apolipoprotein-Enriched Biomolecular Corona Switches the Cellular Uptake Mechanism and Trafficking Pathway of Lipid Nanoparticles. Nanoscale 2017, 9, 17254– 17262,  DOI: 10.1039/C7NR06437C
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    An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles
    Digiacomo, L.; Cardarelli, F.; Pozzi, D.; Palchetti, S.; Digman, M. A.; Gratton, E.; Capriotti, A. L.; Mahmoudi, M.; Caracciolo, G.
    Nanoscale (2017), 9 (44), 17254-17262CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Following exposure to biol. milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomol. corona (BC) that defines many of their biol. outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biol. fluids, and target cells, resp. By conducting confocal fluorescence microscopy expts. and image correlation analyses, we quant. demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liq. chromatog. tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biol. responses and set the basis for a rational exploitation of the BC for targeted delivery.
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    Digiacomo, L.; Giulimondi, F.; Capriotti, A. L.; Piovesana, S.; Montone, C. M.; Chiozzi, R. Z.; Laganà, A.; Mahmoudi, M.; Pozzi, D.; Caracciolo, G. Optimal Centrifugal Isolating of Liposome–Protein Complexes From Human Plasma. Nanoscale Advances 2021, 3, 3824,  DOI: 10.1039/D1NA00211B
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    Optimal centrifugal isolating of liposome-protein complexes from human plasma
    Digiacomo, Luca; Giulimondi, Francesca; Capriotti, Anna Laura; Piovesana, Susy; Montone, Carmela Maria; Chiozzi, Riccardo Zenezini; Lagana, Aldo; Mahmoudi, Morteza; Pozzi, Daniela; Caracciolo, Giulio
    Nanoscale Advances (2021), 3 (13), 3824-3834CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
    In the past few years, characterization of the protein corona (PC) that forms around liposomal systems has gained increasing interest for the development of novel therapeutic and diagnostic technologies. At the crossroads of fast-moving research fields, the interdisciplinarity of protein corona investigations poses challenges for exptl. design and reporting. Isolation of liposome-protein complexes from biol. fluids has been identified as a fundamental step of the entire workflow of PC characterization but exact specifications for conditions to optimize pelleting remain elusive. In the present work, key factors affecting pptn. of liposome-protein complexes by centrifugation, including time of centrifugation, total sample vol., lipid : protein ratio and contamination from biol. NPs were comprehensively evaluated. Here we show that the total amt. of isolated liposome-protein complexes and the extent of contamination from biol. NPs may vary with influence factors. Our results provide protein corona researchers with precise indications to sep. liposome-protein complexes from protein-rich fluids and include proper controls, thus they are anticipated to catalyze improved consistency of data mining and computational modeling of protein corona compn.
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    Franciosa, G; Diluvio, G; Gaudio, F D.; Giuli, M V; Palermo, R; Grazioli, P; Campese, A F; Talora, C; Bellavia, D; D'Amati, G; Besharat, Z M; Nicoletti, C; Siebel, C W; Choy, L; Rustighi, A; Sal, G D.; Screpanti, I; Checquolo, S Prolyl-Isomerase Pin1 Controls Notch3 Protein Expression and Regulates T-ALL Progression. Oncogene 2016, 35, 4741– 4751,  DOI: 10.1038/onc.2016.5
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    Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression
    Franciosa, G.; Diluvio, G.; Del Gaudio, F.; Giuli, M. V.; Palermo, R.; Grazioli, P.; Campese, A. F.; Talora, C.; Bellavia, D.; D'Amati, G.; Besharat, Z. M.; Nicoletti, C.; Siebel, C. W.; Choy, L.; Rustighi, A.; Sal, G. Del; Screpanti, I.; Checquolo, S.
    Oncogene (2016), 35 (36), 4741-4751CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)
    Deregulated Notch signaling is assocd. with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying mol. mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression anal. of human T-ALL samples we obsd. a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an addnl. target therapy for T-ALL.
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  3. Jingyi Zhu, Limei Xu, Wenxin Wang, Min Xiao, Jian Li, Lushan Wang, Xukai Jiang. Molecular Dynamics Simulations Reveal Octanoylated Hyaluronic Acid Enhances Liposome Stability, Stealth and Targeting. ACS Omega 2024, 9 (31) , 33833-33844. https://doi.org/10.1021/acsomega.4c03526
  4. Francesca Giulimondi, Luca Digiacomo, Serena Renzi, Chiara Cassone, Andrea Pirrottina, Rosa Molfetta, Ilaria Elena Palamà, Gabriele Maiorano, Giuseppe Gigli, Heinz Amenitsch, Daniela Pozzi, Alessandra Zingoni, Giulio Caracciolo. Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles. ACS Applied Bio Materials 2024, 7 (6) , 3746-3757. https://doi.org/10.1021/acsabm.4c00103
  5. Zhenhua He, Shaohua Qu, Li Shang. Perspectives on Protein–Nanoparticle Interactions at the In Vivo Level. Langmuir 2024, 40 (15) , 7781-7790. https://doi.org/10.1021/acs.langmuir.4c00181
  6. Daniela Pozzi, Giulio Caracciolo. Looking Back, Moving Forward: Lipid Nanoparticles as a Promising Frontier in Gene Delivery. ACS Pharmacology & Translational Science 2023, 6 (11) , 1561-1573. https://doi.org/10.1021/acsptsci.3c00185
  7. Cintia Marques, Mohammad Javad Hajipour, Célia Marets, Alexandra Oudot, Reihaneh Safavi-sohi, Mélanie Guillemin, Gerrit Borchard, Olivier Jordan, Lucien Saviot, Lionel Maurizi. Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles. ACS Nano 2023, 17 (13) , 12458-12470. https://doi.org/10.1021/acsnano.3c02041
  8. Jiayu Ren, Nicholas Andrikopoulos, Kelly Velonia, Huayuan Tang, Rong Cai, Feng Ding, Pu Chun Ke, Chunying Chen. Chemical and Biophysical Signatures of the Protein Corona in Nanomedicine. Journal of the American Chemical Society 2022, 144 (21) , 9184-9205. https://doi.org/10.1021/jacs.2c02277
  9. Serena Renzi, Luca Digiacomo, Daniela Pozzi, Erica Quagliarini, Elisabetta Vulpis, Maria Valeria Giuli, Angelica Mancusi, Bianca Natiello, Maria Gemma Pignataro, Gianluca Canettieri, Laura Di Magno, Luca Pesce, Valentina De Lorenzi, Samuele Ghignoli, Luisa Loconte, Carmela Maria Montone, Anna Laura Capriotti, Aldo Laganà, Carmine Nicoletti, Heinz Amenitsch, Marco Rossi, Francesco Mura, Giacomo Parisi, Francesco Cardarelli, Alessandra Zingoni, Saula Checquolo, Giulio Caracciolo. Structuring lipid nanoparticles, DNA, and protein corona into stealth bionanoarchitectures for in vivo gene delivery. Nature Communications 2024, 15 (1) https://doi.org/10.1038/s41467-024-53569-8
  10. Chantalle Moulton, Anna Baroni, Erica Quagliarini, Lucia Leone, Luca Digiacomo, Marta Morotti, Giulio Caracciolo, Maria Vittoria Podda, Ennio Tasciotti. Navigating the nano-bio immune interface: advancements and challenges in CNS nanotherapeutics. Frontiers in Immunology 2024, 15 https://doi.org/10.3389/fimmu.2024.1447567
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  13. Giulio Caracciolo. Artificial protein coronas: directing nanoparticles to targets. Trends in Pharmacological Sciences 2024, 45 (7) , 602-613. https://doi.org/10.1016/j.tips.2024.05.003
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  36. Lishan Cui, Erica Quagliarini, Siyao Xiao, Francesca Giulimondi, Serena Renzi, Luca Digiacomo, Giulio Caracciolo, Junbiao Wang, Augusto Amici, Cristina Marchini, Daniela Pozzi. The protein corona reduces the anticancer effect of graphene oxide in HER-2-positive cancer cells. Nanoscale Advances 2022, 4 (18) , 4009-4015. https://doi.org/10.1039/D2NA00308B
  37. Valentina Palmieri, Giulio Caracciolo. Tuning the immune system by nanoparticle–biomolecular corona. Nanoscale Advances 2022, 4 (16) , 3300-3308. https://doi.org/10.1039/D2NA00290F
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  • Abstract

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    Figure 1

    Figure 1. When liposome-DNA complexes (lipoplexes) with a precise synthetic identity are exposed to plasma proteins, they get covered by a protein corona that provides them with a biological identity. (12) The biological identity of lipoplexes ultimately controls their physiological response (e.g., interaction with the immune system). Interaction between lipoplexes and immune cells are tested using (i) human monocytic cell line THP-1 (in vitro), (ii) circulating leukocytes from human donors (ex vivo), and (iii) C57BL/6 mice (in vivo).

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    Figure 2

    Figure 2. Characterization of lipoplexes as a function of the cationic lipid/DNA weight ratio. Size (black points) and zeta potential (gray points) of lipoplexes prepared from CL1 (panel a) and CL2 (panel b) as a function of the cationic lipid/DNA weight ratio, ρ. At ρ = 1, lipoplexes were small in size (DH ≈ 150 nm) and negatively charged (zeta potential ≈ −40 mV) (blue and cyan boxes in panels a and b, respectively). This value was therefore identified as the proper cationic lipid/DNA weight ratio leading to DNA-decorated lipoplexes (DDLs) in line with the purpose of this study. On the other hand, we selected ρ = 10 as the lipid/DNA weight ratio leading to cationic plain lipoplexes (PLs, i.e., whose surface is entirely lipidic) that were used as a control in the following experiments (red and gold boxes in panels a and b, respectively). Synchrotron small-angle X-ray scattering (SAXS) patterns of PLs (panel c) and DDLs (panel d). Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold). Vertical green lines indicate the position of the first- and second-order Bragg peaks arising from a multilamellar structure made of alternating lipid bilayers and DNA monolayers. Representative transmission electron microscopy (TEM) images of PLs (panel e) and DDLs (panel f). Both samples are visible nanosized, rounded-shaped vesicles. The presence of DNA filaments on the surface is detectable in DDLs. Bars correspond to 200 nm.

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    Figure 3

    Figure 3. Characterization of lipoplex-protein complexes as a function of protein concentration. Size (panel a) and zeta potential (panel b) of lipoplex-protein complexes prepared from CL1 and CL2 as a function of human plasma (HP) percentage (%). As PLs are positively charged systems, PL-protein complexes exhibit the typical features of cationic-anionic assemblies, i.e., re-entrant condensation and charge inversion at the isoelectric point. On the other side, the size of the DDL-protein complexes is poorly affected by HP and zeta potential tends to assume similar values. Vertical gray lines indicate the inversion point where PL-protein complexes undergo massive aggregation (panel a) caused by charge neutralization (panel b). (c) Molecular weight (MW) distributions of protein patterns bound to lipoplexes as a function of HP %. While protein patterns of DDL1- and DDL2-protein complexes were almost superimposable up to HP = 20%, those of PL1- and PL2-protein complexes were clearly distinguishable over the protein range especially between 45 and 80 kDa. Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold).

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    Figure 4

    Figure 4. Bioinformatic classification of corona proteins. (a) Relative protein abundance (RPA) of the 20 most abundant plasma proteins (Top-20) identified in the coronas of DDL1 (blue), PL1 (red), DDL2 (cyan), and PL2 (gold) by quantitative nanoLC–MS/MS at the indicated human plasma (HP) concentrations. Plasma proteins were also grouped according to functional processes as reported in (34) coagulation (e), complement (f), immunoglobulins (g), lipoproteins (e), acute phase (h), lipoproteins (i), and tissue leakage (f). Each value is the average of triplicates ± standard deviation within a single experiment.

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    Figure 5

    Figure 5. Sequestration of lipoplex-protein complexes by THP-1 cells in vitro. Cellular uptake of fluorescently labeled lipoplex-protein complexes by human monocyte THP-1 cells via flow cytometry evaluated in terms of percentage of fluorescent cells (a) and median fluorescent intensity (b). Complexes were prepared at low (i.e., HP = 5%) and high (i.e., HP = 50%) protein concentrations. Each value is the average of triplicate samples ± standard deviation within a single experiment. Statistical significance was evaluated by Student’s t test with respect to DDL2 lipoplexes. *p < 0.05, **p < 0.001, no asterisk means not significant. Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold).

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    Figure 6

    Figure 6. Leukocyte uptake of lipoplex-protein complexes. Complexes were prepared at low (5%) and high (50%) human plasma (HP) concentrations. Each value is the average of duplicate samples ± standard deviation within a single experiment. The fluorescence of internalized lipoplex-protein complexes was measured as the percentage of FITC-positive cells by gating on distinct leukocyte subpopulations as indicated. The gating strategy was obtained as explained in ref (37). Color code: DDL1 (blue), DDL2 (cyan), PL1 (red), PL2 (gold).

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    Figure 7

    Figure 7. In vivo particle uptake by circulating and resident phagocytes. 8–10 weeks-old female C57BL/6 mice were i.v. injected with three types of lipoplexes, PEGylated plain lipoplexes (PEGylated PL), plasmid DNA-decorated lipoplexes coated with mouse plasma (MP) proteins (DDL2+MP) and 20-mer oligonucleotide-decorated lipoplexes coated with mouse plasma (MP) proteins (DDL2*+MP). In all the groups (n = 6 animals), capture by phagocytes was evaluated in the blood (panel a) and the spleen (panel b) as percentage of FITC-positive cells by flow cytometry gating on distinct populations as indicated. The gating strategy is illustrated in Figure S4. Naïve animals served as controls (n = 9–12). Data are expressed as the mean ± SD. Statistical significance was evaluated by Student’s t test. *p < 0.05, **p < 0.001, no asterisk means not significant.

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    Figure 8

    Figure 8. Biodistribution of lipoplexes in C57BL/6 mice. Distribution of particles in the spleen, liver, lungs, kidneys, and heart of C57BL/6 mice was evaluated by measuring the fluorescence intensity per mg of tissue. Data are expressed as the mean ± SD. Statistical significance was evaluated by Student’s t test. *p < 0.05, **p < 0.001, no asterisk means not significant.

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    Figure 9

    Figure 9. Exposing DNA-decorated lipoplexes to plasma proteins collected form C57BL/6 mice leads to formation of lipoplexes enriched with an opsonin-deficient, dysopsonin-enriched protein corona. The final product of the formulation process is a type of lipoplexes covered by a proteonuclotidic corona, or “proteoDNAsome”, with more pronounced ability to evade capture by immune cells in vivo than PEGylated lipoplexes.

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  • References

    This article references 56 other publications.

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      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpt1WlsA%253D%253D&md5=f25804d328c08cbfadeafb90ca7e3554
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      Nanoparticle uptake by circulating leukocytes: A major barrier to tumor delivery
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      Journal of Controlled Release (2018), 286 (), 85-93CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      Decades of research into improving drug delivery to tumors has documented uptake of particulate delivery systems by resident macrophages in the lung, liver, and spleen, and correlated short circulation times with reduced tumor accumulation. An implicit assumption in these studies is that nanoparticles present in the blood are available for distribution to the tumor. This study documents significant levels of lipoplex uptake by circulating leukocytes, and its effect on distribution to the tumor and other organs. In agreement with previous studies, PEGylation dramatically extends circulation times and enhances tumor delivery. However, our studies suggest that this relationship is not straightforward, and that particle sequestration by leukocytes can significantly alter biodistribution, esp. with non-PEGylated nanoparticle formulations. We conclude that leukocyte uptake should be considered in biodistribution studies, and that delivery to these circulating cells may present opportunities for treating viral infections and leukemia.
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      Effect of polyethyleneglycol (PEG) chain length on the bio-nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells
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      Nanoscale (2014), 6 (5), 2782-2792CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      When nanoparticles (NPs) enter a physiol. environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liq. chromatog. coupled with tandem mass spectrometry (nanoLC-MS/MS) anal. allowed us to accurately det. the corona compn. showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry anal. in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.
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      Optimization and characterization of anionic lipoplexes for gene delivery
      Srinivasan, Charudharshini; Burgess, Diane J.
      Journal of Controlled Release (2009), 136 (1), 62-70CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      Anionic lipoplexes, comprising divalent cations, DNA and anionic liposomes, were optimized for high transfection efficiency and low cytotoxicity. Different molar ratios of anionic to zwitterionic lipid, 1:9 to 1:1 (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), various cations (Ca2+, Mg2+ and Na+) and different anionic lipid/DNA ratios were investigated. The optimized formulation was composed of: anionic/zwitterionic lipid molar ratio 1:4 (DOPG:DOPE); 15-20 mM Ca2+; and 15-20 μg lipid for complexation with 0.8 μg plasmid DNA. Biophys. studies (particle size anal., gel electrophoresis, transmission electron microscopy (TEM), and confocal microscopy) were conducted to characterize the different formulations. TEM revealed structural differences between the complexed and uncomplexed lipoplexes. Gel electrophoresis confirmed the formation of anionic lipoplexes with the amt. of free DNA minimized for the optimized formulation. Confocal imaging showed cellular uptake of the anionic lipoplexes. Most significantly the anionic lipoplex formulation, optimized for the highest transfection efficiency (approx. 78% in the presence of serum) exhibited the highest cell viability approx. 93%, (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) MTT assay. This was compared to Lipofectamine2000 which had a transfection efficiency and cell viability of approx. 68% and 35%, resp. The anionic lipoplex formulation developed here shows promise as a non-viral vector with high transfection efficiency and low cytotoxicity.
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      McManus, J. J.; Rädler, J. O.; Dawson, K. A. Does Calcium Turn a Zwitterionic Lipid Cationic?. J. Phys. Chem. B 2003, 107, 9869– 9875,  DOI: 10.1021/jp034463d
      9
      Does Calcium Turn a Zwitterionic Lipid Cationic?
      McManus, Jennifer J.; Raedler, Joachim O.; Dawson, Kenneth A.
      Journal of Physical Chemistry B (2003), 107 (36), 9869-9875CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      The structure of a complex formed from DNA, calcium, and DPPC has been studied by small-angle X-ray scattering (SAXS). Previous DSC and temp. scanning ultrasound have shown that in excess lipid two DPPC phases exist, one corresponding to DNA-bound lipid and the other corresponding to lipid not directly involved in complex formation. We have investigated the effect of calcium on both phases within the complex structure. Contrary to the observations in the ternary DPPC/calcium/water system, the binding of calcium to the DPPC membrane in this quaternary system contg. DNA appears to be significantly enhanced. We have estd. the stoichiometry of calcium binding within the complex, and we concluded that the zwitterionic lipid is directly involved in binding to DNA.
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    10. 10
      Palchetti, S.; Pozzi, D.; Marchini, C.; Amici, A.; Andreani, C.; Bartolacci, C.; Digiacomo, L.; Gambini, V.; Cardarelli, F.; Di Rienzo, C.; Peruzzi, G.; Amenitsch, H.; Palermo, R.; Screpanti, I.; Caracciolo, G. Manipulation of Lipoplex Concentration at the Cell Surface Boosts Transfection Efficiency in Hard-to-Transfect Cells. Nanomedicine: Nanotechnology, Biology and Medicine 2017, 13, 681– 691,  DOI: 10.1016/j.nano.2016.08.019
      10
      Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells
      Palchetti Sara; Pozzi Daniela; Marchini Cristina; Amici Augusto; Andreani Cristina; Bartolacci Caterina; Gambini Valentina; Digiacomo Luca; Cardarelli Francesco; Di Rienzo Carmine; Peruzzi Giovanna; Palermo Rocco; Amenitsch Heinz; Screpanti Isabella; Caracciolo Giulio
      Nanomedicine : nanotechnology, biology, and medicine (2017), 13 (2), 681-691 ISSN:.
      To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results.
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    11. 11
      Pan, J.; Heberle, F. A.; Tristram-Nagle, S.; Szymanski, M.; Koepfinger, M.; Katsaras, J.; Kučerka, N. Molecular Structures of Fluid Phase Phosphatidylglycerol Bilayers As Determined by Small Angle Neutron and X-Ray Scattering. Biochimica et Biophysica Acta (BBA)-Biomembranes 2012, 1818, 2135– 2148,  DOI: 10.1016/j.bbamem.2012.05.007
      11
      Molecular structures of fluid phase phosphatidylglycerol bilayers as determined by small angle neutron and X-ray scattering
      Pan, Jianjun; Heberle, Frederick A.; Tristram-Nagle, Stephanie; Szymanski, Michelle; Koepfinger, Mary; Katsaras, John; Kucerka, Norbert
      Biochimica et Biophysica Acta, Biomembranes (2012), 1818 (9), 2135-2148CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      We have detd. the mol. structures of commonly used phosphatidylglycerols (PGs) in the commonly accepted biol. relevant fluid phase. This was done by simultaneously analyzing small angle neutron and X-ray scattering data, with the constraint of measured lipid vols. We report the temp. dependence of bilayer parameters obtained using the one-dimensional scattering d. profile model - which was derived from mol. dynamics simulations - including the area per lipid, the overall bilayer thickness, as well as other intrabilayer parameters (e.g., hydrocarbon thickness). Lipid areas are found to be larger than their phosphatidylcholine (PC) counterparts, a result likely due to repulsive electrostatic interactions taking place between the charged PG headgroups even in the presence of sodium counterions. In general, PG and PC bilayers show a similar response to changes in temp. and chain length, but differ in their response to chain unsatn. For example, compared to PC bilayers, the inclusion of a first double bond in PG lipids results in a smaller incremental change to the area per lipid and bilayer thickness. However, the extrapolated lipid area of satd. PG lipids to infinite chain length is found to be similar to that of PCs, an indication of the glycerol-carbonyl backbone's pivotal role in influencing the lipid-water interface.
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    12. 12
      Caracciolo, G. Liposome-Protein Corona in a Physiological Environment: Challenges and Opportunities for Targeted Delivery of Nanomedicines. Nanomedicine: Nanotechnology, Biology, and Medicine 2015, 11, 543– 557,  DOI: 10.1016/j.nano.2014.11.003
      12
      Liposome-protein corona in a physiological environment: challenges and opportunities for targeted delivery of nanomedicines
      Caracciolo Giulio
      Nanomedicine : nanotechnology, biology, and medicine (2015), 11 (3), 543-57 ISSN:.
      Active targeting that exploits the (over)expression of surface receptors in target cells by ligand incorporation is a central concept in nanomedicine research. Despite unprecedented efforts, no targeted liposome-based therapeutics is commercially available for clinical practice. What is inhibiting the efficient translation of targeted liposome technology from bench to bedside? After introduction in the bloodstream, the lipid surface is immediately modified by the adsorption of a "protein corona" and preserving the surface functionality appears to be challenging. On the other hand, a long-standing corona with receptor-binding sites could associate with the target cell long enough to activate the cell's uptake machinery, triggering liposome endocytosis and intracellular cargo delivery. This opens the intriguing possibility to manipulate the corona composition by liposome design. This review will focus on the emerging field of liposome-protein corona research from basic, descriptive research to readily applicable knowledge and technologies for implementation in drug improvement and development. From the clinical editor: This review is addressing the liposome protein corona research concerning the potential gains in drug improvement and for drug development.
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    13. 13
      Pozzi, D.; Caracciolo, G.; Caminiti, R.; De Sanctis, S. C.; Amenitsch, H.; Marchini, C.; Montani, M.; Amici, A. Toward the Rational Design of Lipid Gene Vectors: Shape Coupling between Lipoplex and Anionic Cellular Lipids Controls the Phase Evolution of Lipoplexes and the Efficiency of DNA Release. ACS Appl. Mater. Interfaces 2009, 1, 2237– 2249,  DOI: 10.1021/am900406b
      13
      Toward the rational design of lipid gene vectors: Shape coupling between lipoplex and anionic cellular lipids controls the phase evolution of lipoplexes and the efficiency of DNA release
      Pozzi, Daniela; Caracciolo, Giulio; Caminiti, Ruggero; De Sanctis, Sofia Candeloro; Amenitsch, Heinz; Marchini, Cristina; Montani, Maura; Amici, Augusto
      ACS Applied Materials & Interfaces (2009), 1 (10), 2237-2249CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      A viewpoint now emerging is that a crit. factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, the authors found a universal behavior of lipoplex/anionic lipid (AL) mixts.: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely dild. by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, the authors investigated the effect of the membrane charge d. and a general correlation resulted: the higher the membrane charge d. of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release. Lastly, the formation of nonlamellar phases in lipoplex/AL mixts. is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases. Remarkably, also phase transition rates and DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes. These results highlight the phase properties of the carrier lipid/cellular lipid mixts. as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carriers.
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    14. 14
      Pozzi, D.; Marchini, C.; Cardarelli, F.; Rossetta, A.; Colapicchioni, V.; Amici, A.; Montani, M.; Motta, S.; Brocca, P.; Cantu, L.; Caracciolo, G. Mechanistic Understanding of Gene Delivery Mediated by Highly Efficient Multicomponent Envelope-Type Nanoparticle Systems. Mol. Pharmaceutics 2013, 10, 4654– 4665,  DOI: 10.1021/mp400470p
      14
      Mechanistic Understanding of Gene Delivery Mediated by Highly Efficient Multicomponent Envelope-Type Nanoparticle Systems
      Pozzi, D.; Marchini, C.; Cardarelli, F.; Rossetta, A.; Colapicchioni, V.; Amici, A.; Montani, M.; Motta, S.; Brocca, P.; Cantu, L.; Caracciolo, G.
      Molecular Pharmaceutics (2013), 10 (12), 4654-4665CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and pos. charged, while synchrotron small-angle X-ray scattering (SAXS) and at. force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) expts. indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quant. investigation of cell uptake, intracellular trafficking,endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels obsd. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that ecient transfection requires both the membrane fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.
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    15. 15
      Koltover, I.; Salditt, T.; Safinya, C. Phase Diagram, Stability, and Overcharging of Lamellar Cationic Lipid–DNA Self-Assembled Complexes. Biophys. J. 1999, 77, 915– 924,  DOI: 10.1016/S0006-3495(99)76942-0
      15
      Phase diagram, stability, and overcharging of lamellar cationic lipid-DNA self-assembled complexes
      Koltover, I.; Salditt, T.; Safinya, C. R.
      Biophysical Journal (1999), 77 (2), 915-924CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Cationic lipid-DNA (CL-DNA) complexes comprise a promising new class of synthetic nonviral gene delivery systems. When pos. charged, they attach to the anionic cell surface and transfer DNA into the cell cytoplasm. A comprehensive x-ray diffraction study is reported of the lamellar CL-DNA self-assemblies as a function of lipid compn. and lipid/DNA ratio, aimed at elucidating the interactions detg. their structure, charge, and thermodn. stability. The driving force for the formation of charge-neutral complexes is the release of DNA and lipid counterions. Neg. charged complexes have a higher DNA packing d. than isoelec. complexes, whereas pos. charged ones have a lower packing d. This indicates that the overcharging of the complex away from its isoelec. point is caused by changes of the bulk structure with absorption of excess DNA or cationic lipid. The degree of overcharging is dependent on the membrane charge d., which is controlled by the ration of neutral to cationic lipid in the bilayers. Importantly, overcharged complexes are obsd. to move toward their isoelec. charge-neutral point at higher concn. of salt co-ions, with pos. overcharged complexes expelling cationic lipid and neg. overcharged complexes expelling DNA. These observations should apply universally to the formation and structure of self-assemblies between oppositely charged macromols.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXltVyju7w%253D&md5=ef8851417cf746d60ee8bef076e9d8fc
    16. 16
      Cukalevski, R.; Ferreira, S. A.; Dunning, C. J.; Berggård, T.; Cedervall, T. IgG and Fibrinogen Driven Nanoparticle Aggregation. Nano Research 2015, 8, 2733– 2743,  DOI: 10.1007/s12274-015-0780-4
      16
      IgG and fibrinogen driven nanoparticle aggregation
      Cukalevski, Risto; Ferreira, Silvia A.; Dunning, Christopher J.; Berggaard, Tord; Cedervall, Tommy
      Nano Research (2015), 8 (8), 2733-2743CODEN: NRAEB5; ISSN:1998-0000. (Springer GmbH)
      A thorough understanding of how proteins induce nanoparticle (NP) aggregation is crucial when designing in vitro and in vivo assays and interpreting exptl. results. This knowledge is also crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extn. of IgG (IgG) from cow serum results in lower polystyrene NPs aggregation. Moreover, addn. of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concn.; i.e., low protein concns. induced large aggregates, whereas high protein concns. induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, resp. Here, we propose a mechanism behind the protein concn. dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins. [Figure not available: see fulltext.].
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    17. 17
      Walkey, C. D.; Chan, W. C. Understanding and Controlling the Interaction of Nanomaterials with Proteins in a Physiological Environment. Chem. Soc. Rev. 2012, 41, 2780– 2799,  DOI: 10.1039/C1CS15233E
      17
      Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment
      Walkey, Carl D.; Chan, Warren C. W.
      Chemical Society Reviews (2012), 41 (7), 2780-2799CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Nanomaterials hold promise as multifunctional diagnostic and therapeutic agents. However, the effective application of nanomaterials is hampered by limited understanding and control over their interactions with complex biol. systems. When a nanomaterial enters a physiol. environment, it rapidly adsorbs proteins forming what is known as the protein corona'. The protein corona alters the size and interfacial compn. of a nanomaterial, giving it a biol. identity that is distinct from its synthetic identity. The biol. identity dets. the physiol. response including signalling, kinetics, transport, accumulation, and toxicity. The structure and compn. of the protein corona depends on the synthetic identity of the nanomaterial (size, shape, and compn.), the nature of the physiol. environment (blood, interstitial fluid, cell cytoplasm, etc.), and the duration of exposure. In this crit. review, we discuss the formation of the protein corona, its structure and compn., and its influence on the physiol. response. We also present an adsorbome' of 125 plasma proteins that are known to assoc. with nanomaterials. We further describe how the protein corona is related to the synthetic identity of a nanomaterial, and highlight efforts to control protein-nanomaterial interactions. We conclude by discussing gaps in the understanding of protein-nanomaterial interactions along with strategies to fill them (167 refs.).
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    18. 18
      Caracciolo, G.; Pozzi, D.; Capriotti, A. L.; Cavaliere, C.; Foglia, P.; Amenitsch, H.; Laganà, A. Evolution of the Protein Corona of Lipid Gene Vectors as a Function of Plasma Concentration. Langmuir 2011, 27, 15048– 15053,  DOI: 10.1021/la202912f
      18
      Evolution of the protein corona of lipid gene vectors as a function of plasma concentration
      Caracciolo, Giulio; Pozzi, Daniela; Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Amenitsch, Heinz; Lagana, Aldo
      Langmuir (2011), 27 (24), 15048-15053CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The concept that the effective unit of interest in the cell-nanomaterial interaction is the particle and its corona of assocd. proteins is emerging. Here we investigate the compositional evolution of the protein corona of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) cationic liposomes (CLs) and DOTAP/DNA lipoplexes over a wide range of plasma concns. (2.5-80%). The compn. of the hard corona of lipoplexes is quite stable, but that of CLs does evolve considerably. We show that the protein corona of CLs is made of both low-affinity and competitive-binding proteins whose relative abundance changes with the plasma concn. This result may have deep biol. implications for the application of lipid-based gene vectors both in vitro and in vivo.
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    19. 19
      Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Baldelli Bombelli, F.; Dawson, K. A. Physical–Chemical Aspects of Protein Corona: Relevance to In Vitro and In Vivo Biological Impacts of Nanoparticles. J. Am. Chem. Soc. 2011, 133, 2525– 2534,  DOI: 10.1021/ja107583h
      19
      Physical-Chemical Aspects of Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles
      Monopoli, Marco P.; Walczyk, Dorota; Campbell, Abigail; Elia, Giuliano; Lynch, Iseult; Baldelli Bombelli, Francesca; Dawson, Kenneth A.
      Journal of the American Chemical Society (2011), 133 (8), 2525-2534CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      It is now clearly emerging that besides size and shape, the other primary defining element of nanoscale objects in biol. media is their long-lived protein ("hard") corona. This corona may be expressed as a durable, stabilizing coating of the bare surface of nanoparticle (NP) monomers, or it may be reflected in different subpopulations of particle assemblies, each presenting a durable protein coating. Using the approach and concepts of phys. chem., we relate studies on the compn. of the protein corona at different plasma concns. with structural data on the complexes both in situ and free from excess plasma. This enables a high degree of confidence in the meaning of the hard protein corona in a biol. context. Here, we present the protein adsorption for two compositionally different NPs, namely sulfonated polystyrene and silica NPs. NP-protein complexes are characterized by differential centrifugal sedimentation, dynamic light scattering, and zeta-potential both in situ and once isolated from plasma as a function of the protein/NP surface area ratio. We then introduce a semiquant. detn. of their hard corona compn. using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray liq. chromatog. mass spectrometry, which allows us to follow the total binding isotherms for the particles, identifying simultaneously the nature and amt. of the most relevant proteins as a function of the plasma concn. We find that the hard corona can evolve quite significantly as one passes from protein concns. appropriate to in vitro cell studies to those present in in vivo studies, which has deep implications for in vitro-in vivo extrapolations and will require some consideration in the future.
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    20. 20
      Caracciolo, G.; Pozzi, D.; Capriotti, A.; Cavaliere, C.; Piovesana, S.; Amenitsch, H.; Laganà, A. Lipid Composition: a “Key Factor” for the Rational Manipulation of the Liposome–Protein Corona by Liposome Design. Rsc Adv. 2015, 5, 5967– 5975,  DOI: 10.1039/C4RA13335H
      20
      Lipid composition: a "key factor" for the rational manipulation of the liposome-protein corona by liposome design
      Caracciolo, G.; Pozzi, D.; Capriotti, A. L.; Cavaliere, C.; Piovesana, S.; Amenitsch, H.; Lagana, A.
      RSC Advances (2015), 5 (8), 5967-5975CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      When liposomes are exposed to biol. fluids, a dynamic coating of proteins immediately covers them. Similarly to the aura of plasma surrounding the Sun, plasma proteins are thought of as establishing an aura that surrounds each liposome, hence the phenomenon was dubbed 'protein corona'. This natural functionalization includes proteins engaged from the blood that can interact with receptors (over)expressed on the plasma membrane of target cells, thus targeting the liposomes to their final destination. Exploiting the liposome-protein corona for targeting has the potential to revolutionize the treatment of many disorders and requires a deep understanding of the factors shaping the corona. Following incubation with human plasma (HP), here we manipulated this corona by using six liposomal formulations with systematic changes in lipid compn. The lipids we employed are among the most frequently used lipid species for drug and gene delivery applications in vitro and in vivo. The six liposome-protein coronas were thoroughly characterized by synchrotron small angle X-ray scattering, dynamic light scattering, zeta-potential and nanoliquid-chromatog. tandem mass spectrometry expts. We identified general principles shaping the liposome-protein corona and established clear-cut relationships between lipid species and classes of plasma proteins. This knowledge sets the basis for a rational manipulation of the protein corona for targeted drug delivery by liposome design.
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    21. 21
      Martínez-Negro, M.; González-Rubio, G.; Aicart, E.; Landfester, K.; Guerrero-Martínez, A.; Junquera, E. Insights into Colloidal Nanoparticle-Protein Corona Interactions for Nanomedicine Applications. Adv. Colloid Interface Sci. 2021, 289, 102366,  DOI: 10.1016/j.cis.2021.102366
      21
      Insights into colloidal nanoparticle-protein corona interactions for nanomedicine applications
      Martinez-Negro, Maria; Gonzalez-Rubio, Guillermo; Aicart, Emilio; Landfester, Katharina; Guerrero-Martinez, Andres; Junquera, Elena
      Advances in Colloid and Interface Science (2021), 289 (), 102366CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
      A review. Nevertheless, the successful implementation of NPs in medicine demands a proper understanding of their interactions with the different proteins found in biol. fluids. Once introduced into the body, NPs are covered by a protein corona (PC) that dets. the biol. behavior of the NPs. The formation of the PC can eventually favor the rapid clearance of the NPs from the body before fulfilling the desired objective or lead to increased cytotoxicity. The PC nature varies as a function of the different repulsive and attractive forces that govern the NP-protein interaction and their colloidal stability. This review focuses on the phenomenon of PC formation on NPs from a physicochem. perspective, aiming to provide a general overview of this crit. process. Main issues related to NP toxicity and clearance from the body as a result of protein adsorption are covered, including the most promising strategies to control PC formation and, thereby, ensure the successful application of NPs in nanomedicine. Colloidal nanoparticles (NPs) have attracted significant attention due to their unique physicochem. properties suitable for diagnosing and treating different human diseases.
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    22. 22
      Danhier, F.; Feron, O.; Préat, V. To Exploit the Tumor Microenvironment: Passive and Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. Journal of controlled release 2010, 148, 135– 146,  DOI: 10.1016/j.jconrel.2010.08.027
      22
      To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery
      Danhier, Fabienne; Feron, Olivier; Preat, Veronique
      Journal of Controlled Release (2010), 148 (2), 135-146CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      A review. Because of the particular characteristics of the tumor microenvironment and tumor angiogenesis, it is possible to design drug delivery systems that specifically target anti-cancer drugs to tumors. Most of the conventional chemotherapeutic agents have poor pharmacokinetics profiles and are distributed non-specifically in the body leading to systemic toxicity assocd. with serious side effects. Therefore, the development of drug delivery systems able to target the tumor site is becoming a real challenge that is currently addressed. Nanomedicine can reach tumor passively through the leaky vasculature surrounding the tumors by the enhanced permeability and retention effect whereas ligands grafted at the surface of nanocarriers allow active targeting by binding to the receptors overexpressed by cancer cells or angiogenic endothelial cells. This review is divided into 2 parts: the first one describes the tumor microenvironment and the second one focuses on the exploitation and the understanding of these characteristics to design new drug delivery systems targeting the tumor. Delivery of conventional chemotherapeutic anti-cancer drugs is mainly discussed.
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    23. 23
      Sawant, R. R.; Torchilin, V. P. Challenges in Development of Targeted Liposomal Therapeutics. AAPS journal 2012, 14, 303– 315,  DOI: 10.1208/s12248-012-9330-0
      23
      Challenges in Development of Targeted Liposomal Therapeutics
      Sawant, Rupa R.; Torchilin, Vladimir P.
      AAPS Journal (2012), 14 (2), 303-315CODEN: AJAOB6; ISSN:1550-7416. (Springer)
      A review. Liposomes, phospholipid vesicles with a bilayered membrane structure, have been widely used as pharmaceutical carriers for drugs and genes, in particular for treatment of cancer. To enhance the efficacy of the liposomal drugs, drug-loaded liposomes are targeted to the tumors by means of passive (enhanced permeability and retention mediated) targeting, based on the longevity of liposomes in blood and its accumulation in pathol. sites with compromised vasculature, and active targeting, based on the attachment of specific ligands to the liposomal surface to bind certain antigens on the target cells. Antibody-targeted liposomes loaded with anticancer drugs demonstrate high potential for clin. applications. This review highlights evolution of liposomes for both passive and active targeting and challenges in development of targeted liposomal therapeutics specifically antibody-targeted liposomes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xlslant74%253D&md5=e017520ac851c05a4ab607645b15a263
    24. 24
      Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nano 2013, 8, 772– 781,  DOI: 10.1038/nnano.2013.181
      24
      Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology
      Tenzer, Stefan; Docter, Dominic; Kuharev, Joerg; Musyanovych, Anna; Fetz, Verena; Hecht, Rouven; Schlenk, Florian; Fischer, Dagmar; Kiouptsi, Klytaimnistra; Reinhardt, Christoph; Landfester, Katharina; Schild, Hansjoerg; Maskos, Michael; Knauer, Shirley K.; Stauber, Roland H.
      Nature Nanotechnology (2013), 8 (10), 772-781CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      In biol. fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiol. systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biol. relevancy. Here we show that label-free snapshot proteomics can be used to obtain quant. time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 min) and, over time, to change significantly in terms of the amt. of bound protein, but not in compn. Rapid corona formation is found to affect hemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVyqurrL&md5=927a38bd98a5850c71525a7707dc5073
    25. 25
      Kelly, P. M.; Åberg, C.; Polo, E.; O’Connell, A.; Cookman, J.; Fallon, J.; Krpetić, Ž.; Dawson, K. A. Mapping Protein Binding Sites on the Biomolecular Corona of Nanoparticles. Nature Nanotechnol. 2015, 10, 472– 479,  DOI: 10.1038/nnano.2015.47
      25
      Mapping protein binding sites on the biomolecular corona of nanoparticles
      Kelly, Philip M.; Aberg, Christoffer; Polo, Ester; O'Connell, Ann; Cookman, Jennifer; Fallon, Jonathan; Krpetic, Zeljka; Dawson, Kenneth A.
      Nature Nanotechnology (2015), 10 (5), 472-479CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Nanoparticles in a biol. milieu are known to form a sufficiently long-lived and well-organized 'corona' of biomols. to confer a biol. identity to the particle. Because this nanoparticle-biomol. complex interacts with cells and biol. barriers, potentially engaging with different biol. pathways, it is important to clarify the presentation of functional biomol. motifs at its interface. Here, we demonstrate that by using antibody-labeled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic mol. description of the biol. identity of nanoparticles.
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    26. 26
      Herda, L. M.; Hristov, D. R.; Lo Giudice, M. C.; Polo, E.; Dawson, K. A. Mapping of Molecular Structure of the Nanoscale Surface in Bionanoparticles. J. Am. Chem. Soc. 2017, 139, 111– 114,  DOI: 10.1021/jacs.6b12297
      26
      Mapping of Molecular Structure of the Nanoscale Surface in Bionanoparticles
      Herda, Luciana M.; Hristov, Delyan R.; Lo Giudice, Maria Cristina; Polo, Ester; Dawson, Kenneth A.
      Journal of the American Chemical Society (2017), 139 (1), 111-114CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Characterizing the orientation of covalently conjugated proteins on nanoparticles, produced for in vitro and in vivo targeting, though an important feature of such a system, has proved challenging. Although extensive physicochem. characterization of targeting nanoparticles can be addressed in detail, relevant biol. characterization of the nanointerface is crucial to select suitable nanomaterials for further in vitro or in vivo expts. The authors adopt a methodol. using antibody fragments (Fab) conjugated to gold nanoparticles (immunogold) to map the available epitopes on a transferrin grafted silica particle (SiO2-PEG8-Tf) as a proxy methodol. to predict nanoparticle biol. function, and therefore cellular receptor engagement. Data from the adopted method suggest that, on av., only ∼3.5% of proteins grafted on the SiO2-PEG8-Tf nanoparticle surface have a favorable orientation for recognition by the cellular receptor.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFCgs7vL&md5=e168198d8ae5f0ebacabe3ffde2bb533
    27. 27
      Lara, S.; Alnasser, F.; Polo, E.; Garry, D.; Lo Giudice, M. C.; Hristov, D. R.; Rocks, L.; Salvati, A.; Yan, Y.; Dawson, K. A. Identification of Receptor Binding to the Biomolecular Corona of Nanoparticles. ACS Nano 2017, 11, 1884– 1893,  DOI: 10.1021/acsnano.6b07933
      27
      Identification of Receptor Binding to the Biomolecular Corona of Nanoparticles
      Lara, Sandra; Alnasser, Fatima; Polo, Ester; Garry, David; Lo Giudice, Maria Cristina; Hristov, Delyan R.; Rocks, Louise; Salvati, Anna; Yan, Yan; Dawson, Kenneth A.
      ACS Nano (2017), 11 (2), 1884-1893CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Biomols. adsorbed on nanoparticles are known to confer a biol. identity to nanoparticles, mediating the interactions with cells and biol. barriers. However, how these mols. are presented on the particle surface in biol. milieu remains unclear. The central aim of this study is to identify key protein recognition motifs and link them to specific cell-receptor interactions. Here, the authors employed an immuno-mapping technique to quantify epitope presentations of two major proteins in the serum corona, low-d. lipoprotein and IgG. Combined with a purpose-built receptor expression system, both proteins present functional motifs to allow simultaneous recognition by low-d. lipoprotein receptor and Fc-gamma receptor I of the corona. The authors' results suggest that the "labeling" of nanoparticles by biomol. adsorption processes allows for multiple pathways in biol. processes in which they may be "mistaken" for endogenous objects, such as lipoproteins, and exogenous ones, such as viral infections.
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    28. 28
      Gianneli, M.; Polo, E.; Lopez, H.; Castagnola, V.; Aastrup, T.; Dawson, K. Label-Free in-Flow Detection of Receptor Recognition Motifs on the Biomolecular Corona of Nanoparticles. Nanoscale 2018, 10, 5474– 5481,  DOI: 10.1039/C7NR07887K
      28
      Label-free in-flow detection of receptor recognition motifs on the biomolecular corona of nanoparticles
      Gianneli, M.; Polo, E.; Lopez, H.; Castagnola, V.; Aastrup, T.; Dawson, K. A.
      Nanoscale (2018), 10 (12), 5474-5481CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Nanomedicine, nanotargeting and nanotherapeutics have in the last few years faced several difficulties in translating the promising results obtained in vitro to an in vivo scenario. The origin of this discrepancy might be found in the lack of a detailed and realistic characterization of the biol. surface of nanoparticles. Despite the capability to engineer nanomaterials with a great variety and a precise control of the surface functionalization, the targeting capability is lost when the nanoparticles are embedded in complex biol. media, due to the formation of a biol. layer (biomol. corona). This biol. layer represents the ultimate nanoparticle surface, likely to interact with the cell machinery. Therefore, in addn. to traditional nanoparticle characterization techniques, a more insightful investigation of the biomol. corona is needed, including the capability to assess the orientation and functionality of specific key mol. features. Here we present a method for the rapid screening of exposed protein recognition motifs on the surface of nanoparticles exploiting quartz crystal microbalance (QCM). We quantify accessible functional epitopes of transferrin-coated nanoparticles and correlate them to differences in nanoparticle size and functionalization. The target recognition occurs label free in flow, thereby pushing our investigation into a more in vivo-like scenario. Our method is applicable to a wide array of nanoparticles and therefore holds the potential to become an advanced technique for the classification of all kinds of nanobioconstructs based on their biol. external functionality.
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    29. 29
      Moein Moghimi, S.; Patel, H. M. Opsonophagocytosis of Liposomes by Peritoneal Macrophages and Bone Marrow Reticuloendothelial Cells. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1992, 1135, 269– 274,  DOI: 10.1016/0167-4889(92)90230-9
      There is no corresponding record for this reference.
    30. 30
      Papini, E.; Tavano, R.; Mancin, F. Opsonins and Dysopsonins of Nanoparticles: Facts, Concepts, and Methodological Guidelines. Frontiers in Immunology 2020, 11, 2343,  DOI: 10.3389/fimmu.2020.567365
      There is no corresponding record for this reference.
    31. 31
      Languino, L. R.; Duperray, A.; Joganic, K. J.; Fornaro, M.; Thornton, G. B.; Altieri, D. C. Regulation of Leukocyte-Endothelium Interaction and Leukocyte Transendothelial Migration by Intercellular Adhesion Molecule 1-Fibrinogen Recognition. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 1505– 1509,  DOI: 10.1073/pnas.92.5.1505
      31
      Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition
      Languino, Lucia R.; Duperray, Alain; Joganic, Karen J.; Fornaro, Mara; Thornton, George B.; Altieri, Dario C.
      Proceedings of the National Academy of Sciences of the United States of America (1995), 92 (5), 1505-9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesions to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the assocn. of fibrinogen with intercellular adhesion mol. 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen γ-chain sequence N117NQKIVNLKEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.
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    32. 32
      Deng, Z. J.; Liang, M.; Monteiro, M.; Toth, I.; Minchin, R. F. Nanoparticle-Induced Unfolding of Fibrinogen Promotes Mac-1 Receptor Activation and Inflammation. Nat. Nanotechnol 2011, 6, 39– 44,  DOI: 10.1038/nnano.2010.250
      32
      Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation
      Deng, Zhou J.; Liang, Mingtao; Monteiro, Michael; Toth, Istvan; Minchin, Rodney F.
      Nature Nanotechnology (2011), 6 (1), 39-44CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      The chem. compn., size, shape and surface characteristics of nanoparticles affect the way proteins bind to these particles, and this in turn influences the way in which nanoparticles interact with cells and tissues. Nanomaterials bound with proteins can result in physiol. and pathol. changes, including macrophage uptake, blood coagulation, protein aggregation and complement activation, but the mechanisms that lead to these changes remain poorly understood. Here, we show that neg. charged poly(acrylic acid)-conjugated gold nanoparticles bind to and induce unfolding of fibrinogen, which promotes interaction with the integrin receptor, Mac-1. Activation of this receptor increases the NF-κB signalling pathway, resulting in the release of inflammatory cytokines. However, not all nanoparticles that bind to fibrinogen demonstrated this effect. Our results show that the binding of certain nanoparticles to fibrinogen in plasma offers an alternative mechanism to the more commonly described role of oxidative stress in the inflammatory response to nanomaterials.
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    33. 33
      Deng, Z. J.; Liang, M.; Toth, I.; Monteiro, M. J.; Minchin, R. F. Molecular Interaction of Poly (Acrylic Acid) Gold Nanoparticles with Human Fibrinogen. ACS Nano 2012, 6, 8962– 8969,  DOI: 10.1021/nn3029953
      33
      Molecular interaction of poly(acrylic acid) gold nanoparticles with human fibrinogen
      Deng, Zhou J.; Liang, Mingtao; Toth, Istvan; Monteiro, Michael J.; Minchin, Rodney F.
      ACS Nano (2012), 6 (10), 8962-8969CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      The binding of fibrinogen to various nanoparticles can result in protein unfolding and exposure of cryptic epitopes that subsequently interact with cell surface receptors. This response is dependent on the size, charge, and concn. of the nanoparticle. Here, the authors examd. the binding kinetics of human fibrinogen to neg. charged poly(acrylic acid)-coated Au-nanoparticles ranging in size from 7 to 22 nm. These particles have previously been shown to elicit an inflammatory response in human cells. The larger nanoparticles bound fibrinogen with increasing affinity and a slower dissocn. rate. Each fibrinogen mol. could accommodate 2 7-nm nanoparticles, but only one when the diam. increased to 10 nm. Nanoparticles of size >12 nm bound multiple fibrinogen mols. in a pos. cooperative manner. However, in the presence of excess nanoparticle, fibrinogen induced the aggregation of the larger particles that could bind more than one protein mol. This was consistent with interparticle bridging by the fibrinogen. Taken together, these results demonstrate that subtle changes in nanoparticle size can influence protein binding both with the surface of the nanoparticle and within the protein corona.
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    34. 34
      Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nature Nanotechnol. 2013, 8, 772– 781,  DOI: 10.1038/nnano.2013.181
      34
      Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology
      Tenzer, Stefan; Docter, Dominic; Kuharev, Joerg; Musyanovych, Anna; Fetz, Verena; Hecht, Rouven; Schlenk, Florian; Fischer, Dagmar; Kiouptsi, Klytaimnistra; Reinhardt, Christoph; Landfester, Katharina; Schild, Hansjoerg; Maskos, Michael; Knauer, Shirley K.; Stauber, Roland H.
      Nature Nanotechnology (2013), 8 (10), 772-781CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      In biol. fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiol. systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biol. relevancy. Here we show that label-free snapshot proteomics can be used to obtain quant. time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 min) and, over time, to change significantly in terms of the amt. of bound protein, but not in compn. Rapid corona formation is found to affect hemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
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    35. 35
      Tsuchiya, S.; Yamabe, M.; Yamaguchi, Y.; Kobayashi, Y.; Konno, T.; Tada, K. Establishment and Characterization of a Human Acute Monocytic Leukemia Cell Line (THP-1). International journal of cancer 1980, 26, 171– 176,  DOI: 10.1002/ijc.2910260208
      35
      Establishment and characterization of a human acute monocytic leukemia cell line (THP-1)
      Tsuchiya S; Yamabe M; Yamaguchi Y; Kobayashi Y; Konno T; Tada K
      International journal of cancer (1980), 26 (2), 171-6 ISSN:0020-7136.
      A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia is described. This cell line had Fc and C3b receptors, but no surface or cytoplasmic immunoglobulins. HLA haplotypes of THP-1 were HLA-A2, -A9, -B5, -DRW1 and -DRW2. The monocytic nature of the cell line was characterized by: (1) the presence of alpha-naphthyl butyrate esterase activities which could be inhibited by NaF; (2) lysozyme production; (3) the phagocytosis of latex particles and sensitized sheep erythrocytes; and (4) the ability to restore T-lymphocyte response to Con A. The cells did not possess Epstein-Barr virus-associated nuclear antigen. These results indicate that THP-1 is a leukemia cell line with distinct monocytic markers. During culture, THP-1 maintained these monocytic characteristics for over 14 months.
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    36. 36
      Kleiveland, C.; Verhoeckx, K.; Cotter, P.; López-Expósito, I.; Kleiveland, C.; Lea, T.; Mackie, A. Peripheral Blood Mononuclear Cells. In The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models; Springer: Cham, Switzerland, 2015; pp 161– 167, DOI: 10.1007/978-3-319-16104-4_15 .
      There is no corresponding record for this reference.
    37. 37
      Giulimondi, F.; Digiacomo, L.; Pozzi, D.; Palchetti, S.; Vulpis, E.; Capriotti, A. L.; Chiozzi, R. Z.; Lagana, A.; Amenitsch, H.; Masuelli, L.; Peruzzi, G.; Mahmoudi, M.; Screpanti, I.; Zingoni, A.; Caracciolo, G. Interplay of Protein Corona and Immune Cells Controls Blood Residency of Liposomes. Nat. Commun. 2019, 10, 3686,  DOI: 10.1038/s41467-019-11642-7
      37
      Interplay of protein corona and immune cells controls blood residency of liposomes
      Giulimondi, Francesca; Digiacomo, Luca; Pozzi, Daniela; Palchetti, Sara; Vulpis, Elisabetta; Capriotti, Anna Laura; Chiozzi, Riccardo Zenezini; Lagana, Aldo; Amenitsch, Heinz; Masuelli, Laura; Mahmoudi, Morteza; Screpanti, Isabella; Zingoni, Alessandra; Caracciolo, Giulio
      Nature Communications (2019), 10 (1), 3686CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      In vivo liposomes, like other types of nanoparticles, acquire a totally new 'biol. identity' due to the formation of a biomol. coating known as the protein corona that depends on and modifies the liposomes' synthetic identity. The liposome-protein corona is a dynamic interface that regulates the interaction of liposomes with the physiol. environment. Here we show that the biol. identity of liposomes is clearly linked to their sequestration from peripheral blood mononuclear cells (PBMCs) of healthy donors that ultimately leads to removal from the bloodstream. Pre-coating liposomes with an artificial corona made of human plasma proteins drastically reduces capture by circulating leukocytes in whole blood and may be an effective strategy to enable prolonged circulation in vivo. We conclude with a crit. assessment of the key concepts of liposome technol. that need to be reviewed for its definitive clin. translation.
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    38. 38
      Chung, A.; Gao, Q.; Kao, W. J. Macrophage Matrix Metalloproteinase-2/-9 Gene and Protein Expression Following Adhesion to ECM-Derived Multifunctional Matrices Via Integrin Complexation. Biomaterials 2007, 28, 285– 298,  DOI: 10.1016/j.biomaterials.2006.08.038
      38
      Macrophage matrix metalloproteinase-2/-9 gene and protein expression following adhesion to ECM-derived multifunctional matrices via integrin complexation
      Chung Amy; Gao Qiang; Kao Weiyuan John
      Biomaterials (2007), 28 (2), 285-98 ISSN:0142-9612.
      Macrophages are commonly observed at the biomaterial-tissue interface and interact with the extracellular matrix (ECM) mainly by integrin receptors to play a critical role in ECM turnover by secreting matrix metalloproteinases (MMPs). To investigate beta1 and beta3 containing integrin-mediated adhesion and subsequent MMP-2/-9 protein and gene expression in human blood-derived monocytes, biofunctional peptides immobilized onto flexible polyethylene glycol (PEG) arms were grafted onto a gelatin-based interpenetrating network (IPN). Adherent monocyte density was dramatically greater in the presence of RGD immobilized onto flexible PEG arms of the gelatin-based IPN. Pretreatment of monocytes with either anti-integrin beta1 or beta3 led to a significant decrease in adherent cell density on RGD-PEG-grafted IPNs. MMP-2 and MMP-9 protein and MMP-9 mRNA expression increased in the presence of IPNs initially, independent of ligand identity. Anti-integrin beta1 or beta3 antibody pretreatment of monocytes led to a general decrease in MMP-2/-9 protein expression. These results demonstrate the importance of beta1 and beta3 containing integrins in mediating monocyte adhesion onto RGD immobilized onto flexible PEG arms of the IPN. The results also reveal that MMP-2/-9 protein and gene expression is influenced by the presence of gelatin and not the ligands immobilized on the PEG arms of the IPN.
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    39. 39
      Simon, J.; Müller, L. K.; Kokkinopoulou, M.; Lieberwirth, I.; Morsbach, S.; Landfester, K.; Mailänder, V. Exploiting the Biomolecular Corona: Pre-Coating of Nanoparticles Enables Controlled Cellular Interactions. Nanoscale 2018, 10, 10731– 10739,  DOI: 10.1039/C8NR03331E
      39
      Exploiting the biomolecular corona: pre-coating of nanoparticles enables controlled cellular interactions
      Simon, Johanna; Mueller, Laura K.; Kokkinopoulou, Maria; Lieberwirth, Ingo; Morsbach, Svenja; Landfester, Katharina; Mailaender, Volker
      Nanoscale (2018), 10 (22), 10731-10739CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Formation of the biomol. corona ultimately dets. the successful application of nanoparticles in vivo. Adsorption of biomols. such as proteins is an inevitable process that takes place instantaneously upon contact with physiol. fluid (e.g. blood). Therefore, strategies are needed to control this process in order to improve the properties of the nanoparticles and to allow targeted drug delivery. Here, we show that the design of the protein corona by a pre-formed protein corona with tailored properties enables targeted cellular interactions. Nanoparticles were pre-coated with Ig depleted plasma to create and design a protein corona that reduces cellular uptake by immune cells. It was proven that a pre-formed protein corona remains stable even after nanoparticles were re-introduced to plasma. This opens up the great potential to exploit protein corona formation, which will significantly influence the development of novel nanomaterials.
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    40. 40
      Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M. PEGylation As a Strategy for Improving Nanoparticle-Based Drug and Gene Delivery. Adv. Drug Delivery Rev. 2016, 99, 28– 51,  DOI: 10.1016/j.addr.2015.09.012
      40
      PEGylation as a strategy for improving nanoparticle-based drug and gene delivery
      Suk, Jung Soo; Xu, Qingguo; Kim, Namho; Hanes, Justin; Ensign, Laura M.
      Advanced Drug Delivery Reviews (2016), 99 (Part_A), 28-51CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
      Coating the surface of nanoparticles with polyethylene glycol (PEG), or "PEGylation", is a commonly used approach for improving the efficiency of drug and gene delivery to target cells and tissues. Building from the success of PEGylating proteins to improve systemic circulation time and decrease immunogenicity, the impact of PEG coatings on the fate of systemically administered nanoparticle formulations has, and continues to be, widely studied. PEG coatings on nanoparticles shield the surface from aggregation, opsonization, and phagocytosis, prolonging systemic circulation time. Here, we briefly describe the history of the development of PEGylated nanoparticle formulations for systemic administration, including how factors such as PEG mol. wt., PEG surface d., nanoparticle core properties, and repeated administration impact circulation time. A less frequently discussed topic, we then describe how PEG coatings on nanoparticles have also been utilized for overcoming various biol. barriers to efficient drug and gene delivery assocd. with other modes of administration, ranging from gastrointestinal to ocular. Finally, we describe both methods for PEGylating nanoparticles and methods for characterizing PEG surface d., a key factor in the effectiveness of the PEG surface coating for improving drug and gene delivery.
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    41. 41
      Ishida, T.; Ichihara, M.; Wang, X.; Kiwada, H. Spleen Plays an Important Role in the Induction of Accelerated Blood Clearance of PEGylated Liposomes. J. Controlled Release 2006, 115, 243– 250,  DOI: 10.1016/j.jconrel.2006.08.001
      41
      Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes
      Ishida, Tatsuhiro; Ichihara, Masako; Wang, XinYu; Kiwada, Hiroshi
      Journal of Controlled Release (2006), 115 (3), 243-250CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      It is well known that steric stabilization of the surface of liposomes by a polyethylene glycol (PEG) conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended circulation times of the liposomes (t1/2 ≈ 20 h in rat). Recently, we reported on the "accelerated blood clearance (ABC) phenomenon", causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. We also reported that abundant binding of IgM, secreted into the blood stream after the first dose and, to PEGylated liposomes, plays an essential role in the induction of the ABC phenomenon. Spleen is well known to play a central role in the immune reaction and to produce IgM following a bacterial infection. The aim of the present study was to det. whether spleen contributes to the induction of the ABC phenomenon and to unravel its role in the phenomenon. In rats that were splenectomized (surgical removal of spleen) prior to the first injection of liposomes (0.001 μmol phospholipids/kg), the ABC phenomenon was totally abolished. In these rats serum IgM concns. as well as the amts. of IgM bound to PEGylated liposomes were substantially reduced. Splenectomy attenuated the ABC phenomenon when performed until 3 days post-first injection. Removal of the spleen 4 days post-first injection left the ABC phenomenon unchanged. This finding indicates that the immune reaction in the spleen against the PEGylated liposomes occurs during at least 2-3 days following the first administration and then IgM reactive to PEGylated liposomes is produced. The present study proves that the spleen plays a crit. role in the induction phase of the ABC phenomenon. For effective clin. application, many liposomal drug formulations will require multiple injections. The ABC phenomenon described in this and several preceding papers therefore has important implications for the development and evaluation of therapeutically useful liposomal formulations requiring multiple-dose administration.
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    42. 42
      Hadjidemetriou, M.; Al-Ahmady, Z.; Mazza, M.; Collins, R. F.; Dawson, K.; Kostarelos, K. In Vivo Biomolecule Corona Around Blood-Circulating, Clinically Used and Antibody-Targeted Lipid Bilayer Nanoscale Vesicles. ACS Nano 2015, 9, 8142– 8156,  DOI: 10.1021/acsnano.5b03300
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      In Vivo Biomolecule Corona around Blood-Circulating, Clinically Used and Antibody-Targeted Lipid Bilayer Nanoscale Vesicles
      Hadjidemetriou, Marilena; Al-Ahmady, Zahraa; Mazza, Mariarosa; Collins, Richard F.; Dawson, Kenneth; Kostarelos, Kostas
      ACS Nano (2015), 9 (8), 8142-8156CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      The adsorption of proteins and their layering onto nanoparticle surfaces has been called the "protein corona". This dynamic process of protein adsorption has been extensively studied following in vitro incubation of many different nanoparticles with plasma proteins. However, the formation of protein corona under dynamic, in vivo conditions remains largely unexplored. Extrapolation of in vitro formed protein coronas to predict the fate and possible toxicol. burden from nanoparticles in vivo is of great interest. However, complete lack of such direct comparisons for clin. used nanoparticles makes the study of in vitro and in vivo formed protein coronas of great importance. Our aim was to study the in vivo protein corona formed onto i.v. injected, clin. used liposomes, based on the compn. of the PEGylated liposomal formulation that constitutes the anticancer agent Doxil. The formation of in vivo protein corona was detd. after the recovery of the liposomes from the blood circulation of CD-1 mice 10 min postinjection. In comparison, in vitro protein corona was formed by the incubation of liposomes in CD-1 mouse plasma. In vivo and in vitro formed protein coronas were compared in terms of morphol., compn. and cellular internalization. The protein coronas on bare (non-PEGylated) and monoclonal antibody (IgG) targeted liposomes of the same lipid compn. were also comparatively investigated. A network of linear fibrillary structures constituted the in vitro formed protein corona, whereas the in vivo corona had a different morphol. but did not appear to coat the liposome surface entirely. Even though the total amt. of protein attached on circulating liposomes correlated with that obsd. from in vitro incubations, the variety of mol. species in the in vivo corona were considerably wider. Both in vitro and in vivo formed protein coronas were found to significantly reduce receptor binding and cellular internalization of antibody-conjugated liposomes; however, the in vivo corona formation did not lead to complete ablation of their targeting capability.
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    43. 43
      Hadjidemetriou, M.; Al-Ahmady, Z.; Kostarelos, K. Time-Evolution of In Vivo Protein Corona onto Blood-Circulating PEGylated Liposomal Doxorubicin (DOXIL) Nanoparticles. Nanoscale 2016, 8, 6948– 6957,  DOI: 10.1039/C5NR09158F
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      Time-evolution of in vivo protein corona onto blood-circulating PEGylated liposomal doxorubicin (DOXIL) nanoparticles
      Hadjidemetriou, Marilena; Al-Ahmady, Zahraa; Kostarelos, Kostas
      Nanoscale (2016), 8 (13), 6948-6957CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Nanoparticles (NPs) are instantly modified once injected in the bloodstream because of their interaction with the blood components. The spontaneous coating of NPs by proteins, once in contact with biol. fluids, has been termed the 'protein corona' and it is considered to be a determinant factor for the pharmacol., toxicol. and therapeutic profile of NPs. Protein exposure time is thought to greatly influence the compn. of protein corona, however the dynamics of protein interactions under realistic, in vivo conditions remain unexplored. The aim of this study was to quant. and qual. investigate the time evolution of in vivo protein corona, formed onto blood circulating, clin. used, PEGylated liposomal doxorubicin. Protein adsorption profiles were detd. 10 min, 1 h and 3 h post-injection of liposomes into CD-1 mice. The results demonstrated that a complex protein corona was formed as early as 10 min post-injection. Even though the total amt. of protein adsorbed did not significantly change over time, the fluctuation of protein abundances obsd. indicated highly dynamic protein binding kinetics.
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    44. 44
      Hadjidemetriou, M.; McAdam, S.; Garner, G.; Thackeray, C.; Knight, D.; Smith, D.; Al-Ahmady, Z.; Mazza, M.; Rogan, J.; Clamp, A.; Kostarelos, K. The Human In Vivo Biomolecule Corona onto PEGylated liposomes: a Proof-of-Concept Clinical Study. Adv. Mater. 2019, 31, 1803335,  DOI: 10.1002/adma.201803335
      There is no corresponding record for this reference.
    45. 45
      Cerullo, V.; Seiler, M. P.; Mane, V.; Brunetti-Pierri, N.; Clarke, C.; Bertin, T. K.; Rodgers, J. R.; Lee, B. Toll-Like Receptor 9 Triggers an Innate Immune Response to Helper-Dependent Adenoviral Vectors. Molecular Therapy 2007, 15, 378– 385,  DOI: 10.1038/sj.mt.6300031
      45
      Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors
      Cerullo, Vincenzo; Seiler, Michael P.; Mane, Viraj; Brunetti-Pierri, Nicola; Clarke, Christian; Bertin, Terry K.; Rodgers, John R.; Lee, Brendan
      Molecular Therapy (2007), 15 (2), 378-385CODEN: MTOHCK; ISSN:1525-0016. (Nature Publishing Group)
      A major obstacle to the clin. application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying mol. components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, the authors show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after i.v. injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that addnl. innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.
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    46. 46
      Moghimi, S. M.; Patel, H. M. Tissue Specific Opsonins for Phagocytic Cells and Their Different Affinity for Cholesterol-Rich Liposomes. FEBS letters 1988, 233, 143– 147,  DOI: 10.1016/0014-5793(88)81372-3
      46
      Tissue specific opsonins for phagocytic cells and their different affinity for cholesterol-rich liposomes
      Moghimi, S. Moein; Patel, Harish M.
      FEBS Letters (1988), 233 (1), 143-7CODEN: FEBLAL; ISSN:0014-5793.
      In vivo expts. show that Kupffer cells avidly take up cholesterol-poor but not cholesterol-rich liposomes, whereas splenic phagocytic cells take up preferentially cholesterol-rich rather than cholesterol-poor liposomes in the presence of serum. Apparently, serum contains opsonins specific for hepatic and splenic phagocytic cells and these opsonins have different affinities for cholesterol-rich and cholesterol-poor liposomes.
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    47. 47
      Moghimi, S. M.; Simberg, D.; Skotland, T.; Yaghmur, A.; Hunter, C. The Interplay between Blood Proteins, Complement, and Macrophages on Nanomedicine Performance and Responses. Journal of Pharmacology and Experimental Therapeutics 2019, 370, 581– 592,  DOI: 10.1124/jpet.119.258012
      47
      The interplay between blood proteins, complement, and macrophages on nanomedicine performance and responses
      Moghimi, S. Moein; Simberg, Dmitri; Skotland, Tore; Yaghmur, Anan; Hunter, A. Christy
      Journal of Pharmacology and Experimental Therapeutics (2019), 370 (3), 581-592CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)
      A review. In the blood, depending on their physicochem. characteristics, nanoparticles attract a wide range of plasma biomols. The majority of blood biomols. bind nonspecifically to nanoparticles. On the other hand, biomols. such as pattern-recognition complement-sensing proteins may recognize some structural determinants of the pristine surface, causing complement activation. Adsorption of nonspecific blood proteins could also recruit natural antibodies and initiate complement activation, and this seems to be a global process with many preclin. and clin. nanomedicines. We discuss these issues, since complement activation has ramifications in nanomedicine stability and pharmacokinetics, as well as in inflammation and disease progression. Some studies have also predicted a role for complement systems in infusion-related reactions, whereas others show a direct role for macrophages and other immune cells independent of complement activation. We comment on these discrepancies and suggest directions for exploring the underlying mechanisms.
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    48. 48
      Verhoef, J.; Anchordoquy, T. Drug Delivery Transl. Res. 2013, 3, 499– 503,  DOI: 10.1007/s13346-013-0176-5
      48
      Questioning the use of PEGylation for drug delivery
      Verhoef, Johan J. F.; Anchordoquy, Thomas J.
      Drug Delivery and Translational Research (2013), 3 (6), 499-503CODEN: DDTRCY; ISSN:2190-3948. (Springer)
      A review. Polyethylene glycol (PEG) is widely utilized in drug delivery and nanotechnol. due to its reported "stealth" properties and biocompatibility. It is generally thought that PEGylation allows particulate delivery systems and biomaterials to evade the immune system and thereby prolong circulation lifetimes. However, numerous studies over the past decade have demonstrated that PEGylation causes significant redns. in drug delivery, including enhanced serum protein binding, reduced uptake by target cells, and the elicitation of an immune response that facilitates clearance in vivo. This report reviews some of the extensive literature documenting the detrimental effects of PEGylation, and thereby questions the wisdom behind employing this strategy in drug development.
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    49. 49
      Song, J.; Ju, Y.; Amarasena, T. H.; Lin, Z.; Mettu, S.; Zhou, J.; Rahim, M. A.; Ang, C.-S.; Cortez-Jugo, C.; Kent, S. J.; Caruso, F. Influence of Poly (Ethylene Glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle–Immune Cell Interactions in Human Blood. ACS Nano 2021, 15, 10025,  DOI: 10.1021/acsnano.1c01642
      49
      Influence of Poly(ethylene glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle-Immune Cell Interactions in Human Blood
      Song Jiaying; Ju Yi; Lin Zhixing; Zhou Jiajing; Rahim Md Arifur; Cortez-Jugo Christina; Caruso Frank; Amarasena Thakshila H; Kent Stephen J; Mettu Srinivas; Ang Ching-Seng
      ACS nano (2021), 15 (6), 10025-10038 ISSN:.
      Poly(ethylene glycol) (PEG) is widely used in particle assembly to impart biocompatibility and stealth-like properties in vivo for diverse biomedical applications. Previous studies have examined the effect of PEG molecular weight and PEG coating density on the biological fate of various particles; however, there are few studies that detail the fundamental role of PEG molecular architecture in particle engineering and bio-nano interactions. Herein, we engineered PEG particles using a mesoporous silica (MS) templating method and investigated how the PEG building block architecture impacted the physicochemical properties (e.g., surface chemistry and mechanical characteristics) of the PEG particles and subsequently modulated particle-immune cell interactions in human blood. Varying the PEG architecture from 3-arm to 4-arm, 6-arm, and 8-arm generated PEG particles with a denser, stiffer structure, with increasing elastic modulus from 1.5 to 14.9 kPa, inducing an increasing level of immune cell association (from 15% for 3-arm to 45% for 8-arm) with monocytes. In contrast, the precursor PEG particles with the template intact (MS@PEG) were stiffer and generally displayed higher levels of immune cell association but showed the opposite trend-immune cell association decreased with increasing PEG arm numbers. Proteomics analysis demonstrated that the biomolecular corona that formed on the PEG particles minimally influenced particle-immune cell interactions, whereas the MS@PEG particle-cell interactions correlated with the composition of the corona that was abundant in histidine-rich glycoproteins. Our work highlights the role of PEG architecture in the design of stealth PEG-based particles, thus providing a link between the synthetic nature of particles and their biological behavior in blood.
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    50. 50
      Kristensen, K.; Urquhart, A. J.; Thormann, E.; Andresen, T. L. Binding of Human Serum Albumin to PEGylated Liposomes: Insights into Binding Numbers and Dynamics by Fluorescence Correlation Spectroscopy. Nanoscale 2016, 8, 19726– 19736,  DOI: 10.1039/C6NR05455B
      50
      Binding of human serum albumin to PEGylated liposomes: insights into binding numbers and dynamics by fluorescence correlation spectroscopy
      Kristensen, Kasper; Urquhart, Andrew J.; Thormann, Esben; Andresen, Thomas L.
      Nanoscale (2016), 8 (47), 19726-19736CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Liposomes for medical applications are often administered by i.v. injection. Once in the bloodstream, the liposomes are covered with a "protein corona", which impacts the behavior and eventual fate of the liposomes. Currently, many aspects of the liposomal protein corona are not well understood. For example, there is generally a lack of knowledge about the liposome binding affinities and dynamics of common types of blood plasma proteins. Fluorescence correlation spectroscopy (FCS) is a powerful exptl. technique that potentially can provide such knowledge. In this study, we have used FCS to investigate the binding of human serum albumin (HSA) to std. types of PEGylated fluid-phase liposomes (consisting of DOPC and DOPE-PEG2k) and PEGylated gel-phase liposomes (consisting of DSPC and DSPE-PEG2k) with various PEG chain surface densities. We detected no significant binding of HSA to the PEGylated fluid-phase liposomes. In contrast, we found that HSA bound tightly to the PEGylated gel-phase liposomes, although only a low no. of HSA mols. could be accommodated per liposome. Overall, we believe that our data provides a useful benchmark for other researchers interested in studying the liposomal protein corona.
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    51. 51
      Jhaveri, A.; Deshpande, P.; Pattni, B.; Torchilin, V. Transferrin-Targeted, Resveratrol-Loaded Liposomes for the Treatment of Glioblastoma. Journal of controlled release 2018, 277, 89– 101,  DOI: 10.1016/j.jconrel.2018.03.006
      51
      Transferrin-targeted, resveratrol-loaded liposomes for the treatment of glioblastoma
      Jhaveri, Aditi; Deshpande, Pranali; Pattni, Bhushan; Torchilin, Vladimir
      Journal of Controlled Release (2018), 277 (), 89-101CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      Glioblastomas (GBMs) are highly aggressive brain tumors with a very grim prognosis even after multi-modal therapeutic regimens. Conventional chemotherapeutic agents frequently lead to drug resistance and result in severe toxicities to non-cancerous tissues. Resveratrol (RES), a natural polyphenol with pleiotropic health benefits, has proven chemopreventive effects in all the stages of cancer including initiation, promotion and progression. However, the poor physico-chem. properties of RES severely limit its use as a free drug. In this study, RES was loaded into PEGylated liposomes (RES-L) to counter its drawbacks as a free drug. Since transferrin receptors (TfRs) are up-regulated in GBM, the liposome surface was modified with transferrin moieties (Tf-RES-L) to make them cancer cell-specific. The liposomal nanomedicines developed in this project were aimed at enhancing the physico-chem. properties of RES and exploiting the passive and active targeting capabilities of liposomes to effectively treat GBM. The RES-L were stable, had a good drug-loading capacity, prolonged drug-release in vitro and were easily scalable. Flow cytometry and confocal microscopy were used to study the assocn. with, and internalization of, Tf-L into U-87 MG cells. The Tf-RES-Ls were significantly more cytotoxic and induced higher levels of apoptosis accompanied by activation of caspases 3/7 in GBM cells when compared to free RES or RES-L. The ability of RES to arrest cells in the S-phase of the cell cycle, and selectively induce prodn. of reactive oxygen species in cancer cells were probably responsible for its cytotoxic effects. The therapeutic efficacy of RES formulations was evaluated in a s.c. xenograft mouse model of GBM. A tumor growth inhibition study and a modified survival study showed that Tf-RES-Ls were more effective than other treatments in their ability to inhibit tumor growth and improve survival in mice. Overall, the liposomal nanomedicines of RES developed in this project exhibited favorable in vitro and in vivo efficacies, which warrant their further investigation for the treatment of GBMs.
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    52. 52
      Moyer, L. S.; Gorin, M. H. Eletectrokinetic Aspects of Surface Chemistry: IX. The Electric Mobilitites of Quartz and Collodion Particles in Mixtures of Horse Serum and Serum Proteins in Relation to the Mechanisms of Film Formation. J. Biol. Chem. 1940, 133, 605– 619,  DOI: 10.1016/S0021-9258(18)73343-7
      52
      Electrokinetic aspects of surface chemistry. IX. Electric mobilities of quartz and collodion particles in mixtures of horse serum and serum proteins in relation to the mechanism of film formation
      Moyer, Laurence S.; Gorin, Manuel H.
      Journal of Biological Chemistry (1940), 133 (), 605-19CODEN: JBCHA3; ISSN:0021-9258.
      cf. C. A. 34, 1696.3. The electrophoretic mobilities of quartz and collodion particles were detd. after exposure to mixts. of serum proteins or after they had been coated with one protein and then exposed to another or to serum. There is little tendency for the various constituents used to adsorb on each other, but one protein may replace another at a surface; thus a film of one of the protein components is obtained rather than a mosaic. The nature of the underlying surface influences the adsorption, the more hydrophilic proteins being adsorbed more readily by the more hydrophilic surfaces, and vice versa. Globulin when coated on quartz appears to be either irreversibly adsorbed or the most strongly adsorbed of the three proteins, replacement by albumin not occurring. The tendency to be adsorbed on quartz is roughly in the order albumin A > albumin B = globulin, with the addnl. assumption that globulin can replace the albumins whenever the free energy differences are favorable. On collodion, globulin is more strongly adsorbed than albumin A or B. The biol. significance of these findings is discussed.
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    53. 53
      Caracciolo, G.; Cardarelli, F.; Pozzi, D.; Salomone, F.; Maccari, G.; Bardi, G.; Capriotti, A. L.; Cavaliere, C.; Papi, M.; Laganà, A. Selective Targeting Capability Acquired with a Protein Corona Adsorbed on the Surface of 1,2-Dioleoyl-3-Trimethylammonium Propane/DNA Nanoparticles. ACS Appl. Mater. Interfaces 2013, 5, 13171– 13179,  DOI: 10.1021/am404171h
      53
      Selective Targeting Capability Acquired with a Protein Corona Adsorbed on the Surface of 1,2-Dioleoyl-3-trimethylammonium Propane/DNA Nanoparticles
      Caracciolo, Giulio; Cardarelli, Francesco; Pozzi, Daniela; Salomone, Fabrizio; Maccari, Giuseppe; Bardi, Giuseppe; Capriotti, Anna Laura; Cavaliere, Chiara; Papi, Massimiliano; Lagana, Aldo
      ACS Applied Materials & Interfaces (2013), 5 (24), 13171-13179CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      A possible turning point in drug delivery has been recently reached: the protein shell, which covers nanocarriers in vivo, can be used for targeting. Here, we show that nanoparticles can acquire a selective targeting capability with a protein corona adsorbed on the surface. We demonstrate that lipid particles made of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and DNA, upon interaction with human plasma components, spontaneously become coated with vitronectin that promotes efficient uptake in cancer cells expressing high levels of the vitronectin ανβ3 integrin receptor.
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    54. 54
      Digiacomo, L.; Cardarelli, F.; Pozzi, D.; Palchetti, S.; Digman, M.; Gratton, E.; Capriotti, A.; Mahmoudi, M.; Caracciolo, G. An Apolipoprotein-Enriched Biomolecular Corona Switches the Cellular Uptake Mechanism and Trafficking Pathway of Lipid Nanoparticles. Nanoscale 2017, 9, 17254– 17262,  DOI: 10.1039/C7NR06437C
      54
      An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles
      Digiacomo, L.; Cardarelli, F.; Pozzi, D.; Palchetti, S.; Digman, M. A.; Gratton, E.; Capriotti, A. L.; Mahmoudi, M.; Caracciolo, G.
      Nanoscale (2017), 9 (44), 17254-17262CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Following exposure to biol. milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomol. corona (BC) that defines many of their biol. outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biol. fluids, and target cells, resp. By conducting confocal fluorescence microscopy expts. and image correlation analyses, we quant. demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liq. chromatog. tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biol. responses and set the basis for a rational exploitation of the BC for targeted delivery.
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    55. 55
      Digiacomo, L.; Giulimondi, F.; Capriotti, A. L.; Piovesana, S.; Montone, C. M.; Chiozzi, R. Z.; Laganà, A.; Mahmoudi, M.; Pozzi, D.; Caracciolo, G. Optimal Centrifugal Isolating of Liposome–Protein Complexes From Human Plasma. Nanoscale Advances 2021, 3, 3824,  DOI: 10.1039/D1NA00211B
      55
      Optimal centrifugal isolating of liposome-protein complexes from human plasma
      Digiacomo, Luca; Giulimondi, Francesca; Capriotti, Anna Laura; Piovesana, Susy; Montone, Carmela Maria; Chiozzi, Riccardo Zenezini; Lagana, Aldo; Mahmoudi, Morteza; Pozzi, Daniela; Caracciolo, Giulio
      Nanoscale Advances (2021), 3 (13), 3824-3834CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
      In the past few years, characterization of the protein corona (PC) that forms around liposomal systems has gained increasing interest for the development of novel therapeutic and diagnostic technologies. At the crossroads of fast-moving research fields, the interdisciplinarity of protein corona investigations poses challenges for exptl. design and reporting. Isolation of liposome-protein complexes from biol. fluids has been identified as a fundamental step of the entire workflow of PC characterization but exact specifications for conditions to optimize pelleting remain elusive. In the present work, key factors affecting pptn. of liposome-protein complexes by centrifugation, including time of centrifugation, total sample vol., lipid : protein ratio and contamination from biol. NPs were comprehensively evaluated. Here we show that the total amt. of isolated liposome-protein complexes and the extent of contamination from biol. NPs may vary with influence factors. Our results provide protein corona researchers with precise indications to sep. liposome-protein complexes from protein-rich fluids and include proper controls, thus they are anticipated to catalyze improved consistency of data mining and computational modeling of protein corona compn.
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    56. 56
      Franciosa, G; Diluvio, G; Gaudio, F D.; Giuli, M V; Palermo, R; Grazioli, P; Campese, A F; Talora, C; Bellavia, D; D'Amati, G; Besharat, Z M; Nicoletti, C; Siebel, C W; Choy, L; Rustighi, A; Sal, G D.; Screpanti, I; Checquolo, S Prolyl-Isomerase Pin1 Controls Notch3 Protein Expression and Regulates T-ALL Progression. Oncogene 2016, 35, 4741– 4751,  DOI: 10.1038/onc.2016.5
      56
      Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression
      Franciosa, G.; Diluvio, G.; Del Gaudio, F.; Giuli, M. V.; Palermo, R.; Grazioli, P.; Campese, A. F.; Talora, C.; Bellavia, D.; D'Amati, G.; Besharat, Z. M.; Nicoletti, C.; Siebel, C. W.; Choy, L.; Rustighi, A.; Sal, G. Del; Screpanti, I.; Checquolo, S.
      Oncogene (2016), 35 (36), 4741-4751CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)
      Deregulated Notch signaling is assocd. with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying mol. mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression anal. of human T-ALL samples we obsd. a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an addnl. target therapy for T-ALL.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.1c07687.

    • Supporting Excel file, scheme of the in vitro and in vivo experiments (XLSX)

    • Figure S1, size, zeta potential, synchrotron SAXS patterns, and the corresponding computed electron density profiles of cationic liposomes CL1 and CL2; Table S1, DLS and synchrotron SAXS parameters for cationic liposomes CL1 and CL2; Figure S2, representative transmission electron microscopy (TEM) images of plain and DNA-decorated lipoplexes; Figure S3, protein corona analysis of plain and DNA-decorated lipoplexes at increasing amounts of human plasma (HP); Table S2, lists of plasma proteins identified in the coronas of DDLs and PLs by nano-LC-MS/MS; Figure S4, gating strategy of mouse phagocyte populations; Figure S5, characterization of CL2/oligonucleotide (ON) lipoplexes as a function of the cationic lipid/ON weight ratio, ρ; Figure S6, luciferase activity in HEK293/Luc reporter cells transfected or not with TLR9 and incubated for 5 h with PL1, PL2, DDL1, DDL2, or a specific TLR9 agonist; and Supplementary Table S3, companion checklist about Minimum Information Reporting in Bio–Nano Experimental Literature (MIRIBEL) (PDF)


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