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Crafting Stable Antibiotic Nanoparticles via Complex Coacervation of Colistin with Block Copolymers
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Cite this: Biomacromolecules 2024, 25, 7, 4267–4280
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https://doi.org/10.1021/acs.biomac.4c00337
Published June 17, 2024

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Abstract

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To combat the ever-growing increase of multidrug-resistant (MDR) bacteria, action must be taken in the development of antibiotic formulations. Colistin, an effective antibiotic, was found to be nephrotoxic and neurotoxic, consequently leading to a ban on its use in the 1980s. A decade later, colistin use was revived and nowadays used as a last-resort treatment against Gram-negative bacterial infections, although highly regulated. If cytotoxicity issues can be resolved, colistin could be an effective option to combat MDR bacteria. Herein, we investigate the complexation of colistin with poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMAA) block copolymers to form complex coacervate core micelles (C3Ms) to ultimately improve colistin use in therapeutics while maintaining its effectiveness. We show that well-defined and stable micelles can be formed in which the cationic colistin and anionic PMAA form the core while PEO forms a protecting shell. The resulting C3Ms are in a kinetically arrested and stable state, yet they can be made reproducibly using an appropriate experimental protocol. By characterization through dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), we found that the best C3M formulation, based on long-term stability and complexation efficiency, is at charge-matching conditions. This nanoparticle formulation was compared to noncomplexed colistin on its antimicrobial properties, enzymatic degradation, serum protein binding, and cytotoxicity. The studies indicate that the antimicrobial properties and cytotoxicity of the colistin-C3Ms were maintained while protein binding was limited, and enzymatic degradation decreased after complexation. Since colistin-C3Ms were found to have an equal effectivity but with increased cargo protection, such nanoparticles are promising components for the antibiotic formulation toolbox.

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1. Introduction

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To address the ongoing rise of multidrug-resistant bacteria, proactive steps are essential for the advancement of antibiotic formulations. (1,2) Over the last few years, antimicrobial peptides (AMPs) have received more attention as the new generation of bactericidal compounds. (2−4) AMPs are naturally occurring peptides, part of the innate immune system in a broad range of organisms. (2) Contrary to conventional antibiotics, the mode of action of AMPs is related to their amphiphilic nature, which results in an affinity toward the cell membrane, creating various disruptive effects. (2,5−7) Their cationic properties create selective affinity toward net negatively charged bacterial membranes over more zwitterionic mammalian cells. (2−4)
One of the oldest classes of AMPs, polymyxins, has been used for decades in clinical practices. (8,9) Polymyxin E, also known as colistin, derived from the Gram-positive Paenibacillus genus, was discovered in 1947. After extensive use in the 50s and 60s, it was found that colistin is nephrotoxic and even mildly neurotoxic. Consequently, its use was banned in the 1980s. (8) However, due to a shortage of bactericidal compounds against Gram-negative bacteria, colistin has seen a partial resurgence and is nowadays used as a last-resort treatment against Gram-negative bacterial infections, administered either ectopically or intravenously. (8,10) Colistin is composed of a fatty acid chain to which a cyclic decapeptide is coupled that contains five l-α-γ-diaminobutyric acid residues. These side groups are the cause of a positive net charge of +5 in physiological conditions. As colistin is cationic, it strongly interacts with anionic lipopolysaccharides (LPS) by electrostatic attraction and cation displacement on the outer membrane of Gram-negative bacteria, after which it efficiently kills the bacteria by penetration of the inner membrane. (11,12) Even though colistin has the highest affinity for LPS, colistin is also able to increase the tubular epithelial cell membrane permeability of mammalian cells, resulting in cell swelling and lysis. (12) This in turn leads to cytotoxic effects in humans. (4,12) It is a general belief that if the cytotoxicity can be reduced, the use of colistin as an antibiotic will be significantly expanded as it is cheap and effective. (13,14)
Several strategies for colistin toxicity reduction have already been assessed over the years that mainly focus on facilitating or encapsulating colistin, aiming to decrease the undesired interactions of colistin causing toxicity. (13,15−21) E.g., conjugation with hyaluronan-chitosan derivatives has been investigated, (15) but also drug delivery systems like colistin loading into liposomes, (13) chelating micelles, (16) aerosolizable particles, (17) nanostructured lipid carriers (NLCs), (18,19) solid lipid nanoparticles (SLNs), (18) poly(lactic-co-glycolic acid) (PLGA) nanoparticles, (20) and complexation into coacervates. (21) In the development of drug delivery methods for colistin, several factors need to be controlled, like solubility, retainability, bioavailability, and functionality. Currently, the main concerns with colistin drug delivery are its stability, chemical alteration, and poor water solubility, leading to low bioavailability. (18,22,23) To resolve these challenges, going deeper into complex coacervation could be a viable strategy as it does not require any chemical modification of the drug and allows for effective encapsulation of water-soluble drugs. (24,25) However, this requires sufficient colloidal stability of the formed complexes. Complexation of colistin could improve the retainability and stability by protecting the drug from degradation and enhancing its therapeutic efficacy in various pharmaceutical formulations. As an additional benefit, colistin effectivity can be improved as well, as most AMPs are known for their low in vivo stability due to protease degradation. (26)
In complex coacervation, microphase separation is induced due to electrostatic attraction and counterion release between oppositely charged polyelectrolytes, thus creating a phase with water-soluble complex coacervates. (27−29) These charged polyelectrolytes are most often grafted, random, or block copolymers but can also be homopolymers, proteins, nutraceuticals, peptides, DNA, (m)RNA, or drugs. (24,27,30) In complex coacervation drug delivery, a charged drug or charge-conjugated drug is complexed with another polyelectrolyte, which often is an ionic polymer or lipid. (27) One of the more recent applications of this methodology is in vaccine technology, in which mRNA is complexed with cationic lipids into lipid nanoparticles. (31−33)
To ensure sufficient colloidal stability and prevent macroscopic phase separation, in many cases, a neutral hydrophilic block is conjugated to one of the charged species. (21,27,33,34) When these charged polyelectrolytes are mixed in favorable charge conditions, the charged polyelectrolytes form a core, and the (neutral) hydrophilic block forms a shell. The shell protects the highly charged core from interaction with other highly charged cores, significantly increasing its stability. (27,28,34) These structures are termed complex coacervate core micelles (C3Ms). (24,27,28,34) Typically, to form C3Ms, block copolymers are used consisting of a charged block and a neutral hydrophilic block. (27) Most often, the neutral block that provides colloidal stability through steric repulsion is poly(ethylene oxide) (PEO), (26,28,35,36) but sometimes other polymer blocks are used, such as poly(vinyl alcohol) (PVA) (37) or poly(acrylamide) (PAAm), (38) to name a few. Previously, other proteins, drugs, and peptides, e.g., (helical) polypeptides, (39,40) doxorubicin, (41) lysozyme, (42,43) myoglobin, (43) bovine serum albumin (BSA), (44) and many others have been successfully complexed into C3Ms. (23,24,27,45,46) In contrast, complex coacervation involving AMPs is limited to a few studies. (21,25,47−51) The AMPs polymyxin B, (48) colistin, (21) temporin-L, (47) KSL-W, (51) P6, (49) and MSI-78 (50) have successfully been complexed into coacervates, with only the latter two into C3Ms, which are generally recognized for their enhanced stability compared to typical coacervates. (27) Răileanu et al. complexed P6 with PEO-b-poly(acrylic acid) (PAA), (49) while Wang et al. prepared C3Ms from the complexation of MSI-78 with methoxyPEO-b-poly(α-glutamic acid) (PGlu). (50) As shown by these examples, in combination with the limited research on the detailed structural investigation of the AMP complexes, the complexation of AMPs is still widely unexplored, even though this structural analysis is potentially highly relevant in the development of new antibiotic formulations.
Herein, we describe the formulation of well-defined spherical and highly stable C3Ms based on the coassembly of colistin with poly(ethylene oxide)-b-poly(methacrylic acid) block copolymers (PEO-b-PMAA) without the use of any additional stabilizing agent, covalently bound species, or any other harmful or harsh chemicals. Our primary motivation was to create a stable and tunable drug delivery system in which colistin is complexed with another component to form C3Ms. Three different PEO-b-PMAA polymers were investigated for complexation with colistin at several charge fractions. We employed P1: PEO45-b-PMAA41, P2: PEO45-b-PMAA81, and P3: PEO114-b-PMAA81 (numbers indicate the degree of polymerization) to achieve nanoparticle complexes with optimal stability and a reproducible structure. The next step comprised the characterization of the properties of the formed C3Ms. We characterized the complex coacervate using scattering techniques: small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) to investigate nanostructure and stability, next to bacterial testing, enzymatic protection, human serum albumin (HSA) binding, and cell toxicity testing. We show that the colistin-C3Ms are highly stable and reproducible, with the highest colistin complexation effectivity found around charge matching. Similar antibiotic activity and cytotoxicity were found between colistin and colistin-C3Ms, while the complexation of colistin was found to improve the protection against enzymatic degradation, while limited protein binding effects were observed. With the increased protection of colistin, while maintaining its activity, colistin-C3Ms could potentially be a new option in the toolbox of antibiotic formulations for intravenous clinical purposes.

2. Experimental Section

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2.1. Complex Coacervate Preparation

PEO-b-PMAA at different block lengths (Polymersource) and colistin sulfate (Sigma Aldrich) were dissolved in 0.05 M TRIS buffer (Sigma Aldrich) (pH = 7.4). Depending on the charge fraction (f+), the samples were diluted separately to two times the desired final concentration before mixing the polymer into colistin solution 1:1 volume-wise. It was made sure that the solution was mixed properly. Final concentrations of either 5.0, 2.5, 1.3, or 1.0 mg/mL were used for analysis and diluted even further when necessary.

2.2. ζ-Potential

ζ-Potential measurements were carried out using the Malvern Zetasizer Nano ZS with Malvern DTS1070 cuvettes. ζ-Potentials were measured between 12 and 20 times for every sample in triplicate at a total concentration of 1.0 mg/mL at 20 °C.

2.3. Dynamic Light Scattering (DLS)

Dynamic light scattering (DLS) experiments were performed using a DLS/SLS instrument from LS Instruments, equipped with a Cobolt high-performance DPSS laser 100 mW (660 nm). The samples were filtered either through 0.22 or 0.45 μm poly(vinylidene difluoride) (PVDF) filters (Millipore) directly into precleaned 2 mm NMR tubes. Complex coacervates were measured at 2.5 or 1.3 mg/mL at 20 °C, including regular checks to avoid multiple scattering and check for concentration-dependent effects. For stability measurements, DLS measurements were taken with one-day intervals in the first week, three-day intervals in the second and third week, and then weekly until three months if no aggregation was observed.

2.4. Small-Angle X-ray Scattering (SAXS)

The small-angle X-ray scattering profiles were measured at 20 °C using the BioSAXS beamline BM29 (52) at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The automated sample changer loaded 50 μL for every sample into a quartz glass capillary of a diameter of 1 mm. Ten scattering frames of 1.0 s each were detected on the Pilatus 3 × 2 M detector, using an energy of 12.5 keV and a sample–detector distance of 2.81 m, measuring a Q-range of 0.007–0.55 Å–1. The background sample (0.05 M TRIS buffer, pH = 7.4) was measured between each sample measurement, and the capillary was cleaned between every measurement. Water was used as a primary standard to scale the data to absolute intensities. Every frame was checked for radiation damage, followed by averaging, buffer subtraction, and binning (from 1000 points to 280), resulting in the final scattering curves that are presented in this paper. Additional SAXS experiments were performed at our in-house Bruker NanoStar instrument (RECX, University of Oslo, Norway). The instrument uses Cu Kα radiation (λ = 1.54 Å) and yields scattering data in the Q range 0.01–0.3 Å–1. According to instrument standard procedures, the scattering intensity was corrected for detector sensitivity, electronic noise, and empty cell scattering, and calibrated to absolute units using water scattering, yielding the macroscopic differential scattering cross-section d∑/dΩ. Then the scattering contribution from the solvent was subtracted.

2.5. SAXS Data Modeling

Fixed partitioning, based on calculated charge matching in the core, was assumed. This significantly reduced the number of fit parameters in the model. With the model, it was possible to get a quantitative analysis of the complex coacervate structures. The fuzzy-surface complex coacervate model with broad interfaces and polydispersity consists of three separate contributions: complex coacervate scattering, free (noncomplexed) polymer and colistin scattering, and an additional structure factor to describe the physical interactions between polyelectrolytes in the core (more elaborate explanation of the model can be found in the Supporting Information). The total scattering function on an absolute scale can be described by eq 1:
I(Q)=φ·fCoa(fclu·S(Q)cluster·ICoa(Q)+(1−fclu)·ICoa(Q)+blob(Q)VCoa)+φ(fPoly·IPoly,free(Q)·fmix+fCol·ICol,free(Q)·(1−fmix))+fCoa·S(Q)internal
(1)
In which φ is the volume fraction, fCoa is the fraction of coacervates, VCoa is the volume of one complex coacervate, fclu is the fraction that is forming clusters, fPoly is the free fraction of polymer, fCol is the free fraction of colistin, and fmix is the molar fraction of polymer in the aqueous phase surrounding the complex coacervates. These fractions are based on the aforementioned assumption that there is stoichiometric charge matching in the core of the complex coacervates.
For the first contribution, the complex coacervates, we used the form factor of a “fuzzy sphere”, i.e., spheres with graded interfaces described by the radius R and width of the interface, σ. Based on the trial fit analysis, we ignored the core–shell nature of the C3Ms since the contrast between the core formed with colistin and PMAA and the PEO shell was too small to allow for a clear separation. Therefore, we described the model of C3Ms with a sphere with a fuzzy interface instead. The form factor for fuzzy spheres (P(Q) = Acore(Q)2) can be described, as follows, and was incorporated in the scattering function for complex coacervates (ICoa(Q)) (53,54) (eqs 2–6).
F0=(Rσ2+1σ)·cos(Q·(R+σ))Q4−3·⁢sin⁡(Q·(R+σ))Q5·σ2+(Rσ2−1σ)·cos⁡(Q·(R−σ))Q4
(2)
F1=−3·sin(Q·(R+σ))+6·sin(Q·R)Q5·σ2−2·R·cos⁡⁡(Q·R)Q4·σ2
(3)
Vn=R33+R·σ26
(4)
Acore(Q)=F0+F1Vn
(5)
Icoa(Q)=Δρaverage2·P2·Vtot2·Acore(Q)2VCoa
(6)
where Icoa(Q) is the scattering contribution from the complex coacervates, Δρaverage is the average scattering length density of the complex coacervates, P is the aggregation number (number of molecules per micelle), and Vtot is the total volume of the blobs. In addition to the form factor, the blob scattering (35,55−59) of the polyelectrolytes in the core of the complex coacervates was considered (eq 7):
blob(Q)=Vtot2·P·Δρaverage2·|fblob|(1+Q2·ξ2)
(7)
where fblob is the fraction of blobs and ξ is the blob correlation length. For the second contribution to the model, for the free component, noncomplexed, scattering of polymer and colistin, the Debye form factor for polymers and polyelectrolytes is used (60) (eq 8).
P(Q)Debye=2(e−Q2·Rg2−1+Q2·Rg2)(Q2·Rg2)2
(8)
In which Rg is the radius of gyration of the polyelectrolyte. The third contribution is the internal structure factor, indicated in Figure 2A. This feature is related to the charge correlations between the blobs of anionic PMAA and cationic colistin (59) and is summarized in eq 9, which was built upon already existing models in the literature. (35,55−60)
S(Q)internal=|C|·e−(Q−Qlocal)2W·Qlocal·2π·2·(W·Qlocal)2
(9)
where C is the fractal scattering, W is the relative width at high Q, and Qlocal is the internal scattering location. In the presence of globular proteins, we approximated the scattering contribution using a prolate/oblate ellipsoid form factor (eqs 10–13). The ellipsoid of revolution has two minor core radii of R and major axis εR, respectively. The form factor can be written as:
r=R(sin2α+ε2cos2α)1/2
(10)
Asph(x)=3[sin(x)−xcos(x)]/x3
(11)
P(Q)Ellipsoid=∫0πAsph(Qr)2sin(α)dα
(12)
The total contribution can then be written as:
I(Q)protein=φp·Mw,p/dp·Δρp2P(Q)Ellipsoid
(13)
where φp is the volume fraction of the globular protein, Mw,p is the molecular weight of the protein, dp is the solution density, and Δρp is scattering length density incorporated by values based on the fitting of the data of pure globular protein samples at three different concentrations. In the case of the addition of another compound next to the complex coacervates, the fitted random chain (Debye equation in eq 8) or ellipsoidal scattering (form factor in eq 12) of these compounds was added to the scattering from the complex coacervate, and then fitted, while the other parameters were kept the same.
For the fit analysis, least-squares fit routines were applied keeping the following parameters free: the total aggregation number (P), the radius of the core including its density distribution, and polydispersity index (Rin, σin, PDI), the free fraction of colistin (fCol), and if necessary, in the presence of cluster formation, the number of complexes in a cluster (Nclu) and fdist the distance between complexes. These parameters mostly describe the low and intermediate Q range. After obtaining a rough fit of low and intermediate Q, to also fit the data well at high Q, the scattering from the blobs themselves was fitted. Moreover, the blob scattering parameters blob fraction (fblob) and blob correlation length (ξ) were fitted. Lastly, to describe the blob charge correlations (internal structure), the relative width high Q (W), the internal scattering location (Qlocal), and fractal scattering (C) were fitted. Finally, all parameters were fitted to the data simultaneously, resulting in the fits presented in this paper.

2.6. Assessing the Effect of Ionic Strength and pH on Colistin-C3Ms

The effect of ionic strength was analyzed in two different setups, the addition of NaCl (Sigma Aldrich) before mixing and after mixing, also known as salt annealing. P1-colistin-C3Ms at f+ = 0.50 were prepared in the same way but with the addition of 0.15 M NaCl in the buffer and mixing using a stopped-flow device, mixing at 6.7 mL/s. Salt annealing was performed by the addition of salt after preparation of the C3Ms at five different concentrations (0.05, 0.10, 0.15, 0.30, and 0.50 M NaCl). For the effect of pH changes, C3Ms were prepared in the same way but in five other pH buffers, citrate buffer at pH = 5.0, maleate buffer at pH = 6.0 and pH = 7.0, and TRIS at pH = 8.0 and pH = 8.7 (all buffers from Sigma Aldrich). All these samples were analyzed using SAXS and modeled accordingly.

2.7. Determination of Critical Micelle Concentration (CMC)

Using an optical tensiometer setup the CMC of C3Ms at f+ = 0.50 was determined. Drops were captured by a CCD camera with a tensiometer setup from Ramé-Hart, inc. The surface tension from the drops was analyzed by using “Drop image”. 100 Frames were captured for each measurement, after which the surface tension was modeled using the Young–Laplace equation and averaged. To determine the CMC, two regions were distinguished between 0.0 and 5.0 mg/mL total concentration. The region of fast surface tension decreases, and the part in which low surface tension decreases is observed. The transition point corresponds to the CMC.

2.8. Antimicrobial Properties of C3Ms and Colistin

To compare the antimicrobial properties of C3Ms to colistin two different methods were tested: disk diffusion assay (DDA) and agar broth dilution testing for MIC50 determination.
In the DDA, the susceptibility of Escherichia coli (DSM613), Serratia indica (NCIMB 8869), Pseudomonas fluorescens (DSM20030), A. vinelandii (DSM2290), and Bacillus subtilis (DSM10) was tested. Sterile filter disks (VWR) were impregnated (using an automatic pipette) with 3.0 μg of colistin from either colistin sulfate or colistin-C3Ms (at f+ = 0.50) in a concentration above the CMC (at 1.0 mg/mL) and dried under sterile air. Bacterial plates were prepared by freshly pouring bacterial medium until it set. Second, a layer of LB medium, that was premixed with 10 μL of bacteria from overnight cultures, was added. Then, the disks were added, and the plates were incubated for 24 h at 37 °C. The sizes of the inhibition zones were measured and averaged. Experiments were performed in triplicate. With the agar broth dilution testing, it was possible to determine the MIC50 values. LB liquid media in test tubes containing equal amounts of E. coli culture (10 μL of bacteria from overnight cultures to 5 mL of media) was mixed with either colistin or colistin-C3Ms with equal colistin for a concentration between 0.0 and 50 μg/mL. The test tubes were incubated for 24 h at 37 °C and their turbidity was measured with a McFarland turbidimeter. The agar broth dilution experiments were done in duplicate. To determine the MIC50 values, curves were averaged and then fitted with a sigmoid function.

2.9. Enzymatic Susceptibility

The enzymatic breakdown susceptibility was analyzed by adding proteinase K (3.4.21.64; Sigma Aldrich), subtilisin (3.4.21.62; Sigma Aldrich), and trypsin (3.4.21.4; Sigma Aldrich) to either colistin (at 3.4 mg/mL) or C3Ms at f+ = 0.50 (total concentration 5.0 mg/mL) at their suppliers’ recommended concentration/ratio for optimized breakdown (molar ratios of enzyme:colistin ≈ 1:130 for proteinase K, ≈ 1:70 for subtilisin and ≈ 1:180 for trypsin). We assessed the difference in susceptibility between noncomplexed colistin and C3Ms. Colistin solution was incubated with enzymes for 24 h at 37 °C, after which polymer solution (kept 24 h at 37 °C) was added while in the other case, premade C3Ms were incubated with enzymes for 24 h at 37 °C. These samples were analyzed using SAXS and modeled, including enzyme scattering (using the Debye form factor since the scattering was too small to use a prolate/oblate ellipsoid form factor, eq 8) to monitor the molecular weight of the complex coacervates after exposure to enzyme degradation.

2.10. HSA Binding Susceptibility

To assess the binding affinity of colistin-C3Ms at f+ = 0.50 with HSA, HSA was added to C3Ms (5.0 mg/mL) at two different molar ratios (1:10 and 1:20) at room temperature (20 °C), and then analyzed using SAXS and modeled including HSA (Sigma Aldrich) (using the prolate/oblate ellipsoid form factor, eq 12) scattering to monitor changes in structure and size of the formed complex coacervates in the presence of HSA.

2.11. Toxicity Assay

To be able to use the colistin-C3Ms, colistin, and PEO-b-PMAA needed for the toxicity assessment, the components were freeze-dried at a total concentration of 1 mg/mL by freezing solutions in liquid nitrogen, after which they were exposed to a vacuum for 24 h. Human embryonic kidney (HEK 293 cells) (ATCC, CRL-1573) were cultured in Dulbecco’s modified Eagle medium (DMEM), low glucose (Sigma Aldrich) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (15140-122; Gibco, Waltham), 1% GlutaMAX (35050; Gibco), and 20% fetal bovine serum (F9665; Sigma Aldrich). Mesenchymal stem cells (MSC, PT-2501; Lonza) were cultured in Mesenchymal Stem Cell Basel Medium (PT-3238; Lonza)) supplemented with MSCGM SingleQuots (PT-4105; Lonza). Human gingival keratinocytes (HGK) (PCS-200-014; ATCC) were cultured in Dermal Cell Basal Medium (PCS-200-030; ATCC) supplemented with Keratinocytes Growth Kit (PCS-200-040; ATCC). Human umbilical cord endothelial cells (HUVEC, 200p-05n; Cell Applications) were cultured in Endothelial Cell Growth Medium (211-500; Cell Applications). The cell types were seeded in 12-well culture-treated plates at a density of 4 × 104 cells/cm2. Upon confluency (set at time point 0), the cells were exposed to cell-specific growth medium containing formulations with a concentration of 1.0 mg/mL total concentration (0.7 mg/mL colistin) of f+ = 0.50 C3Ms or equivalent concentrations of colistin, and polymer as a control. The medium was changed for both control cells and stimulated cells every two days, 24 h before the harvest of the medium to ensure the same window of accumulated cell-secreted factors in the media. Cytotoxicity assays on lactate dehydrogenase (LDH) activity and caspase-3 level were performed at 24, 48, and 72 h and executed in triplicate. LDH activity in the cell culture media was evaluated using a cytotoxicity detection kit (11644793001; Roche). In short, 50 μL of the collected supernatant was mixed with 50 μL of the reaction mixture and incubated in a dark room at room temperature for 30 min. The absorbance was measured at 490 and 600 nm using the BioTek ELx800 Absorbance Microplate Reader. The caspase-3 activity in the cell culture media was evaluated using an enzyme-linked immunosorbent assay (K4221-100; BioVision). A 100 μL sample was added to each well, and the plate was incubated at 37 °C for 1.5 h. After incubation, 100 μL of biotin-detection antibody was added and incubated at 37 °C for 1 h. The plate was washed three times, after which 100 μL of horseradish peroxidase (HRP)-streptavidin conjugate was added, and the samples were incubated at 37 °C for 30 min. The wells plate was washed five times and 90 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was mixed in, after which the plate was incubated at 37 °C for 30 min. After incubation, 50 μL stop solution was added. The absorbance was measured at 450 nm using the BioTek ELx800 Absorbance Microplate Reader. After harvesting, light microscopy at a magnification of 10 times was utilized to visualize morphology changes of different cell types upon treatments.

3. Results and Discussion

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3.1. Charge Dependency and Stability

To analyze the charge dependency of the formation and stability of the colistin complex coacervates, we first investigated the range in which colistin complex coacervates could be formed. To quantify the charge ratios mixing charge fractions were calculated (eq 14), in which a value of 0.50 indicates the charge matching of colistin and PEO-b-PMAA. (27)
f+=1−f−=ZColZCol+ZPoly
(14)
In which ZCol is the number of charges from colistin in the formulation and ZPoly is the number of charges from PEO-b-PMAA in the formulation. For P1, we could prepare colistin complex coacervates for 0.09 ≤ f+ ≤ 0.98 at a total concentration of 5.0 mg/mL. Outside of this region, no complex coacervates were formed, as confirmed by DLS. To investigate the nature of these complex coacervates the ζ-potential was measured, right after mixing and combined with measured stability and sizes by DLS in Figure 1. The stability is based on the number of days after complexation after which aggregation was apparent, as seen in Figure S1 in the Supporting Information.

Figure 1

Figure 1. ζ-Potential measurements (filled black circles) and DLS size measurements right after mixing (open white diamonds), including error bars, combined with the stability of complex coacervates mixing PEO45-b-PMAA41 (P1) and colistin, at a range of charge fractions (f+) of 0.09 ≤ f+ ≤ 0.98 at a formation concentration of 5.0 mg/mL. The stability, measured by follow-up measurements over time with DLS (Figure S1), is indicated by the color coding of the background. 0–1-day(s) stability (red), 2–7-day stability (orange), 8–28-day stability (yellow), and > 28-day stability (green) are separated in the background. To improve visibility by creating sharp color borders a color spacing of 0.02–0.05 was taken horizontally.

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The region of 0.50 ≤ f+ ≤ 0.83 was found to be (size-)stable over a long term, i.e., more than 28 days, as indicated by the green background (Figure 1). The addition of colistin charge does not seem to affect the ζ-potential in the same way the addition of anionic P1-charge does. At f+ < 0.50, the ζ-potential deviates further from zero, resulting in quicker aggregation (from micro to macro phase separation). Therefore lower stability is observed, already visible by the presence of larger sizes at f+ < 0.50, right after mixing. In the stable region, the ζ-potential is closer to zero, while complex coacervates with a size of 35–40 nm are found. This indicates the beneficiality of complex coacervate neutrality in the system at a particular size to achieve the most stable systems. Here, the PEO corona is sufficient to provide colloidal stability via entropic (steric) repulsions between overlapping polymer brushes, like in other C3M formulations. (61,62) It does not hold for f+ > 0.83. Even though the effective charge, the ζ-potential, is close to zero, the systems destabilized quickly (within two days). This is probably due to excess colistin charge, disrupting the homogeneous charge and resulting in the initialization of aggregation after mixing. At these charge fractions, the ionic strength might have exceeded the threshold energy for long-term stability in the systems, causing aggregation, even though the ζ-potential remains at zero when measured. The ζ-potential behavior for a wide range of f+ values differs from complex coacervate system to system. (27) Therefore, the ζ-potential and DLS sizing alone do not give enough information about the charge fractions in the system to conclude on compositional characteristics.
P2-colistin and P3-colistin showed the same trends in stability, with a stable charge fraction region, but showed to have formed complex coacervates in a much smaller window of charge fractions, for P2 0.33 ≤ f+ ≤ 0.83, and for P3 0.50 ≤ f+ ≤ 0.83 (Figures S2 and S3). Additionally, the larger block lengths led to larger colistin complex coacervates at all charge fractions, with the most pronounced size change observed at the long-term stable conditions for all polymers at f+ = 0.50 (Table 1).
Table 1. Hydrodynamic Radii (Rh) of Complex Coacervates from P1, P2, and P3 with Colistin at Long-Term Stable Conditions at f+ = 0.50, Measured by DLS with CONTIN Fits at 120°
polymers used for complexation with colistin at f+ = 0.50Rh (DLS, averaged based on CONTIN fits at 120°, Figure S4) (nm)
P1 (PEO45-b-PMAA41)18 ± 1
P2 (PEO45-b-PMAA81)77 ± 3
P3 (PEO114-b-PMAA81)87 ± 2
The size and shape of nanoparticles used in therapeutics play a crucial role in their bioavailability and biochemical stability once they enter the bloodstream. Therefore, we need to consider the potential threat of spleen and liver accumulation for larger particles (above 100 nm diameter), as well as the renal filtration systems that clear up free unimers from unstable complex coacervate formulations. (63−66) Based on its smallest size and largest stability region, we think P1-colistin complex coacervates would have the most promising therapeutic potential. Hence, they were analyzed further on their charge dependence and compositional characteristics using SAXS. SAXS provides possibilities to analyze and characterize samples in solution at the nanometer scale, allowing resolution of the shape, size, composition, and structure. (67) The SAXS profiles of colistin complex coacervates at f+ = 0.50 (total concentration of 5.0 mg/mL) and the scattering profiles of their separate components are depicted in Figure 2A. In Figure 2B, the colistin complex coacervates in five different charge fractions (total concentration of 5.0 mg/mL), and their fits are shown. We analyzed the SAXS profiles of the complex coacervates using least-squares fit routines with a custom-designed fuzzy-surface complex coacervate model with graded interfaces (see the Experimental Section). The most important parameters fitted to the data were the aggregation number (P), the radius of the core (Rin), the width of the interface of the core (σin), the free fraction of colistin outside of the micelles (fCol) and the polydispersity index of the micelles (PDI). Based on these parameters, the concentration of encapsulated colistin (cColC3M), the total radius of the core (Rtot), molecular weight (Mw), and volume fraction of water in the micelle (fw) could be calculated (calculations are given in the Supporting Information). Noncomplex coacervate structures were analyzed using fit models using either the Debye form factor for polymers and polyelectrolytes (Figure 2A) or the prolate/oblate ellipsoid form factor for globular proteins (Supporting Information).

Figure 2

Figure 2. SAXS profiles of PEO45-b-PMAA41 (orange squares) at 1.6 mg/mL colistin sulfate (blue circles) at 3.4 mg/mL, and colistin complex coacervates at f+ = 0.50 (green triangles (pointing down)) at 5.0 mg/mL total concentration. The lines depict the results of fit analysis using the Debye model for PEO45-b-PMAA41 and colistin sulfate and the fuzzy-surface complex coacervate model for colistin complex coacervates (A). Scattering profiles are depicted of complex coacervates at f+ = 0.17 (orange squares), f+ = 0.33 (blue circles), f+ = 0.50 (green triangles (down)), f+ = 0.67 (gray diamonds), and f+ = 0.83 (pink triangles (up)) at a total concentration of 5.0 mg/mL including fitted curves (B). Scattering patterns in B are fitted using the fuzzy-surface complex coacervate model for the different charge fractions of complex coacervates.

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Generally, in complex coacervation, three phases can be distinguished based on the charge fractions. (27,34) First, the phase in which there is no formation of complex coacervates (single chains), second, the phase in which excess charge causes the formation of either anionic or cationic soluble complex particles (SCPs) and third, the phase where C3Ms are present. To figure out this phase behavior and to understand the colistin-C3M charge dependence, SAXS data was analyzed using the model in the whole range of 0.09 ≤ f+ ≤ 0.98, a larger range than the data shown in Figure 2B. The most relevant fit parameters of the results are summarized in Table 2.
Table 2. Most Relevant (fit) Parameters, Based on Fitting of Different Charge Fractions for C3Ms 0.09 ≤ f+ ≤ 0.98 Using the Fuzzy-Surface Complex Coacervate Modela
f+P·103Rtot (nm)σin (nm)fColcColC3M (mg/mL)Mw (Da)·106fw
0.090.163.80.00.020.80.260.0
0.170.154.10.00.071.40.250.0
0.230.154.20.00.071.80.260.0
0.330.12 ± 0.056.9 ± 0.93.0 ± 0.40.07 ± 0.012.36 ± 0.030.19 ± 0.080.27 ± 0.03
0.441.9 ± 0.615 ± 11.2 ± 0.40.10 ± 0.032.81 ± 0.083 ± 10.59 ± 0.06
0.501.4 ± 0.215.7 ± 0.31.7 ± 0.10.16 ± 0.012.84 ± 0.012.3 ± 0.40.74 ± 0.03
0.551.0 ± 0.115.6 ± 0.11.7 ± 0.10.21 ± 0.012.81 ± 0.041.6 ± 0.10.78 ± 0.02
0.601.2 ± 0.115.8 ± 0.21.9 ± 0.20.38 ± 0.022.34 ± 0.071.9 ± 0.20.80 ± 0.02
0.641.2 ± 0.116.2 ± 0.61.9 ± 0.20.48 ± 0.032.1 ± 0.12.0 ± 0.20.80 ± 0.01
0.671.12 ± 0.0416.2 ± 0.22.0 ± 0.20.54 ± 0.011.86 ± 0.021.9 ± 0.10.78 ± 0.01
0.751.3 ± 0.116.3 ± 0.42.0 ± 0.30.71 ± 0.011.23 ± 0.052.1 ± 0.20.75 ± 0.01
0.831.5 ± 0.616.5 ± 0.32.1 ± 0.30.82 ± 0.010.84 ± 0.012 ± 10.80 ± 0.08
0.911.3 ± 0.217 ± 12 ± 20.91 ± 0.010.45 ± 0.022.2 ± 0.40.80 ± 0.01
0.981.4193.80.980.082.40.72
a

Averages were taken from the fits at three concentrations for f+ ≥ 0.33, while for f+ < 0.33 and f+ > 0.91, only 5.0 mg/mL was possible to be fitted. Since the concentration of colistin is also lower at lower total concentrations, the cColC3M decreases as well, but for comparison, the lower concentrations are scaled (normalized) to 5.0 mg/mL.

The data for charged matched mixtures are shown in Figure 2A and demonstrate the formation of spherical-like micelles upon mixing. While the two individual components display low-intensity scattering with a weak Q–2 dependence at high Q, the scattering curve from the mixed coacervate is much more pronounced with a Guinier plateau at low Q and steep decay at intermediate Q resembling spherical entities. In addition, there is a clear correlation peak at high Q, which indicates an optimal packing distance between the colistin and PMAA chains within the core. This has been found in fully polymeric coacervates and attributed to positional correlations between oppositely charged electrostatic blobs. (59) This feature is the most pronounced feature at f+ = 0.50 and gradually washed out at off-stoichiometric charge balances. For mixtures with f+ < 0.50 (Figure 2B), we also observe upturns at low Q, revealing the formation of micellar clusters. To describe this, a cluster structure factor, based on a fraction of clustered micelles placed at a certain distance from each other, was used (Supporting Information). For 0.33 ≤ f+ ≤ 0.91, fits were obtained at three different concentrations (5.0, 2.5, and 1.3 mg/mL) and could be normalized and averaged because of apparent concentration independence within the experimental error limit (Figure S5, all fits found in Tables S1–S3). Outside of this region, it was not possible to measure at lower concentrations than 5.0 mg/mL because of the increased instability of the system.
The highest concentration of colistin in the complexed fraction (cColC3M) was found at charge-matching conditions, close to f+ = 0.50, and decreasing in both directions further away from charge matching (Table 2). The presence of more anionic charge seems to affect the size and molecular weight of C3Ms, especially in the 0.09 ≤ f+ ≤ 0.33 region, where polymer charges are in excess. The particles formed in this region are smaller and contain fewer molecules, but from DLS we also detect larger aggregates (Figure 1). In theoretical models for complex coacervates, this region is also known as the anionic soluble complex particle (SCP–) region. (27) The border between SCP– and C3Ms is referred to as the critical excess anionic charge (CEAC) point, and in the case of P1-colistin-C3Ms, the CEAC lies between f+ = 0.33 and f+ = 0.44, (27,34) which is clearly shown from the increase of fw. SCPs cannot contain a high volume of water since they are small and negatively charged, whereas C3Ms are stable because of the large volume fraction of water.
C3Ms are present in the 0.44 ≤ f+ ≤ 0.98 region, and even when there is an excess of colistin charge, the aggregation number, size, molecular weight, and water volume fraction of the C3Ms, and the width of the fuzzy surface (σin) remain consistent (confirmed by the density profiles shown in Figure S6). However, the fraction of free colistin exhibits a negative correlation with f+. At higher f+-values, there is a sharp cutoff from C3Ms to no structures (around f+ = 0.98). This is an indication that there is no SCP+ region and consequently, no critical excess cationic charge (CECC) point. The further away from charge matching conditions, we either observe more aggregates and smaller particles (f+ < 0.50) or lower encapsulation efficiency (cColC3M) (f++ > 0.50). These findings are summarized and illustrated in Figure 3.

Figure 3

Figure 3. Graphical illustration of the effect of charge fractions on the formation of complex coacervates. At f+ < 0.09 (I), no complex coacervates are formed, followed by the SCP-phase (II), in which negatively charged small structures are present until the CEAC point between f+ of 0.33 and 0.44. After the CEAC, C3Ms are formed (III), with a decreasing fraction forming micelles the further you go up the scale. From f+ = 0.98 (IV), again, no complex coacervates are formed. Illustrations are not scaled.

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As illustrated in Figure 3, from left to right, four phases can be distinguished. In the first phase (I) where the polymer charge is in excess, the polymer and colistin do not form any complexes but are rather present as single chains. In the second phase (II), we have the negatively charged soluble complex particles that are smaller than C3Ms, in which the polymer charge is in excess. The third phase (III), after the critical excess anionic charge (CEAC) point is crossed, is the C3M phase. The largest number of C3Ms with the lowest free fraction of colistin can be found at charge matching at 16%. At higher f+ values, the number of C3Ms decreases while the free fraction of colistin increases. In the fourth phase (IV), the polymer and colistin do not form complexes, but in this case, the colistin charge is in excess. Considering the focus on stability and toxicity decrease, charge-matching conditions (f+ = 0.50) were taken further into characterization, as this system is stable and has the lowest free fraction of colistin (Figure 3). In addition to these observations, it has to be considered that C3Ms generally are dynamic systems. (27,34,35,68) In the circumstances in which these C3Ms are formed, the micelles are in a kinetically arrested state. However, the metastable C3Ms could be affected by changes in pH, altering the effective charge, addition of ionic strength, or dilution of systems, which are parameters that are especially relevant in biological settings. For many different C3Ms formulations, this behavior is intrinsically different. (27,68) To assess the effect of ionic strength changes and pH changes, we exposed the P1-colistin-C3Ms at f+ = 0.50 to increased ionic strength of physiological levels during mixing (Figure S7, Table S4) and after mixing (“salt annealing”) (Figure S8, Table S5), and mixed P1-colistin-C3Ms at several different pH values between 5.0 and 8.7 (Figure S9, Table S6). We only found a small effect of increasing ionic strength in both mixing and after mixing within physiological ionic strength levels (Figures S7 and S8). Upon gradual addition of 0.30 M of NaCl, the P1-colistin-C3Ms swells (higher fw, while Rtot increased, and P remained the same) and became generally more polydisperse, which was expected from the increased screening of charges. (27) After the addition of 0.30 M NaCl and at 0.50 M NaCl, the C3Ms disintegrate into smaller micelles with lower aggregation numbers. Decreasing the pH caused an increase in polydispersity, as well as a small decrease in size and aggregation, and an increase in the free fraction of colistin (from 16% at pH = 7.4) to 60% at pH 5 and 6. Most likely because of the decreased charge of the PMAA block, which is known to have a pKa between 4 and 5. (69) Less charge in the system from polymer will lead to a decrease in colistin complexation. An increase in pH resulted in the same colistin-C3Ms found at physiological pH (pH = 7.4), with a slightly increased aggregation number and decreased polydispersity.
To further investigate the stability toward dilution, we investigated the (apparent) critical micelle concentration (CMC) with a pendant drop tensiometer (Figure S10). (27,70) Above the CMC, the C3Ms are in an effectively arrested state, while below the CMC, the C3Ms may dissociate into their corresponding compounds. The CMC was found to be around 0.3 mg/mL. SAXS data confirms these findings since around concentrations of 0.3 mg/mL, micelles start dissociating (Figure S11). Since we have established the boundaries of the kinetically arrested state and found the most optimal formulation based on stability, structure, size, and compositional characteristics, we can begin assessing their therapeutic potential. Characterization of f+ = 0.50 colistin-C3Ms encompassing their antimicrobial, enzyme protection, release, serum binding, and toxicity properties was performed.

3.2. Antimicrobial Properties

To compare the antibiotic properties of the C3Ms versus free colistin, first, a disk diffusion assay (DDA) was performed. In the DDA the susceptibility of E. coli, Serratia indica, P. fluorescens, A. vinelandii, and B. subtilis as a control (Figure S12) were tested in the presence of colistin and colistin-C3Ms. (71,72) The best results were obtained for E. coli (Figure 4A) in the conditions that were assessed, since S. indica grew back over the inhibition zones (Figure S12), while P. fluorescens was not visibly affected, and A. vinelandii showed immeasurable small inhibition zones. Second, the MIC50 values (minimum inhibitory concentrations) for both formulations were determined in E. coli culture, to see whether there is a difference in susceptibility based on the introduction of polymer in the bacteria growth medium. (71) To determine the MIC50, the turbidity was measured, with higher turbidity indicating increased bacterial proliferation, after which a sigmoid was fitted to the data (Figure 4B).

Figure 4

Figure 4. Disk diffusion assay (DDA) of colistin and colistin-C3Ms with 3.0 μg of colistin in the E. coli agar medium (A). The inhibition zones were measured, averaged, and compared. MIC50 determination of colistin (orange diamonds) and colistin-C3Ms (blue circles) on E. coli using the agar dilution method (B). The turbidity was plotted against the concentration, which was plotted on a logarithmic scale to improve visibility. The colistin (black line) and colistin-C3Ms curves (black dashed line) were fitted with a sigmoid function from which the MIC50 values were determined, which are plotted in the figure (orange dashed line for colistin and blue dotted line for colistin-C3Ms).

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There were no significant differences between colistin and colistin-C3Ms in the susceptibility of E. coli for both experiments (Figure 4). In the DDA, the sizes of inhibition zones were respectively 1.9 ± 0.1 and 1.8 ± 0.3 mm for colistin and colistin-C3Ms (Figure 4A), while the MIC50 values were 1.5 ± 0.1 and 1.4 ± 0.1 μg/mL respectively (Figure 4B). The MIC50 values measured were in the expected range for colistin, as values between 0.5 and 8.0 μg/mL have been reported before in different assay conditions. (73,74) In the MIC50 determination, the concentrations tested were all below the CMC, meaning no C3Ms were present in the agar samples. Hence, only the effect of the addition of polymer could be assessed, and based on Figure 4B, the polymer did not seem to affect the antimicrobial properties of colistin. Since there are no significant differences between colistin and C3Ms in the DDA, the C3Ms likely disintegrate, releasing the colistin in the process, resulting in no losses in antimicrobial activity. However, another option is that the micelles stay intact and exert their antimicrobial effect as particles, even though this is less likely since C3M systems are susceptible to dilution over the whole agar plate. In addition, Kourmouli et al. (72) found that nanoparticles of similar size containing antibiotics have a significantly decreased diffusivity, and if the antibiotics are not released, no antimicrobial effect could be observed.

3.3. Enzymatic Breakdown Susceptibility

Generally, AMPs, and therefore also colistin, are known for their low in vivo stability due to protease degradation. (26,75) The complexation of colistin could be advantageous if it improves the retainability and stability, by protecting the drug from degradation by enzymes. To assess the enzymatic susceptibility of colistin versus colistin-C3Ms in concentrations above the CMC, two peptidases with a broad specificity were used: proteinase K and subtilisin. Trypsin was also assessed but was found ineffective in breaking down colistin (Figure S13). To visualize the hypothesized effect and experimental design of colistin breakdown for colistin and colistin-C3Ms, an illustration was made (Figure 5A,B). The enzymes were either added to colistin, then incubated, and then complexed with polymer (Figure 5A), or the enzymes were added to the C3Ms and then incubated (Figure 5B). The SAXS patterns are shown in Figure 5C,D and then compared to theoretical, noninteracting (calculated) enzymatic breakdown SAXS patterns. These theoretical SAXS patterns were calculated by simple addition of the scattering of each component separately (polymer, colistin, and the small contribution of enzyme) to resemble 100% enzyme breakdown, called “No C3M protection (A)”. Equivalently, the theoretical scattering curves from C3Ms added to the negligible enzyme scattering (resembling 0% enzyme breakdown) are called “C3M protection (B)” (Figure 5C,D). The data from both methods was analyzed using the fuzzy sphere complex coacervate model and compared to regular C3Ms (Table S7).

Figure 5

Figure 5. Illustrations of hypothesized enzymatic breakdown comparisons (A, enzyme attacks colistin and then undergoes complexation, and (B) enzyme attacks after complexation) and SAXS patterns of enzymatic breakdown of colistin (3.4 mg/mL) versus colistin-C3Ms (total concentration of 5.0 mg/mL) in the presence of proteinase K (C) and subtilisin (D). Theoretical SAXS patterns were calculated based on either the added scattering from C3Ms and enzymes (0% enzyme breakdown, orange squares) or colistin, polymer, and enzyme separately summed up (100% breakdown, blue circles) (C, D). The effect of enzymatic degradation after 24 h at 37 °C was measured by either adding enzyme to colistin, followed by complexation with polymer (illustrated in A, and the graphs in C and D, indicated by green triangles) or addition of enzyme to C3Ms (illustrated in B, and the graphs in C and D, indicated by gray diamonds). The SAXS patterns were fitted using the fuzzy-surface complex coacervate model for complex coacervates.

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From the fits, for proteinase K degradation, the molecular weight of C3Ms changed from Mw = 2.3 ± 0.4 MDa to Mw = 0.13 MDa without C3M protection and Mw = 2.1 MDa with C3M protection. For subtilisin degradation, the decrease in molecular weight of the complex coacervates was more extensive. The molecular weight dropped from Mw = 2.3 ± 0.4 MDa to Mw = 0.042 MDa without C3M protection and to Mw = 1.1 MDa with C3M protection. Therefore, it is apparent that the C3Ms protect colistin from enzymatic breakdown, while in a noncomplexed state, colistin is prone to enzymatic degradation by both proteinase K and subtilisin (Figure 5). Proteinase K can cleave colistin such that the average molecular weight of colistin complex coacervates was reduced by a factor of ≈ 20, while subtilisin breakdown results in a molecular weight reduction of ≈ 50 times. Subsequently, C3Ms seem unaffected by proteinase K, while subtilisin caused a reduction of approximately factor two in molecular weight. In conclusion, C3Ms seem to have a comparable protection effect for both enzymes, compared to noncomplexed colistin: enzymatic breakdown reduction by a factor of ≈20 for its molecular weight. Furthermore, complexation was found to be beneficial to the enzymatic stability and potentially the retainability of colistin. Nevertheless, it must be noted that these studies do not indicate the colistin activity after enzymatic degradation, even though the C3Ms stay intact. However, the retainability of colistin, if improved, can reduce the quantities of colistin needed for treatment, lowering its dose, and reducing the side effects of colistin.

3.4. Human Serum Albumin (HSA) Binding

Next to enzymatic destabilization causing low retainability, (serum) protein binding is another factor that can negatively affect colistin’s in vivo stability. (26,27,62,68) Since colistin-C3Ms would be transported in the bloodstream, it can interact with several different (small) proteins that can bind to colistin or the PEO-b-PMAA and affect the encapsulation effectivity of the colistin-C3Ms. To investigate whether the C3Ms are structurally affected by the presence (of high levels) of serum proteins, HSA was added in two different molar ratios of 1:10 and 1:20 (HSA:colistin) and measured using SAXS (Figure 6). Fits were again performed using the fuzzy-surface complex coacervate model, in which the scattering of HSA was included by adding the form factor of a prolate/oblate ellipsoid to the model.

Figure 6

Figure 6. SAXS patterns of C3Ms (blue circles) and C3Ms with added HSA at a ratio of 1:10 HSA:colistin (green triangles) and 1:20 HSA:colistin (gray diamonds) are presented in (A). To better visualize the effect of HSA addition, we show HSA by themselves (orange squares) and C3Ms by themselves (blue circles) in (B), as well as the scattering patterns in which HSA is combined with C3Ms separated by multiplication of the I(Q) with a factor of 10 for 1:20 HSA:colistin (green triangles) and 100 for 1:10 HSA:colistin (gray diamonds) to increase the visibility of structural changes. The SAXS patterns were fitted using the fuzzy-surface complex coacervate model for complex coacervates with added prolate/oblate ellipsoidal scattering of HSA if present.

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Through the model fits, it was possible to extract the effect of HSA showing only a minor effect on the structure of C3Ms. The total radius was reduced from 15.7 ± 0.3 to 13.8 ± 0.3 nm, while the polydispersity index was essentially the same, 18% and 20% before and after exposure of HSA up to 1:10 (mole ratio). The colistin-C3Ms thus stay intact with a similar aggregation number (P) and molecular weight, while the free fraction of colistin also remained around 16% (Table S8). A possible reason for the size reduction could be the increase of total charged polyelectrolytes in the system, leading to a reduction of water in the core because of osmotic flow. (76,77) This results in a slight condensation of the colistin and polymers in the core. (27,77) Even though the size of the C3Ms was changed significantly, the micelles stayed intact with the same composition, showing promising drug delivery characteristics.

3.5. Toxicity

The level of cytotoxicity indicates the availability of colistin in cellular environments and is an indication of the protection level of the cargo. (28,78) With the quantification of the toxicity level of colistin and colistin-C3Ms, we could find out what cell types are most prone to colistin toxicity, as well as compare how colistin and encapsulated colistin affect the cytotoxicity of living cells. To measure the cytotoxicity of colistin-C3Ms and noncomplexed colistin, four different cell types were investigated that all have relevance to the nephrotoxic properties of colistin. We investigated human embryonic kidney 293 cells (HEK), an immortalized cell line derived from human embryonic kidney cells. Second, we investigated human mesenchymal stem cells (MSC), multipotent cells that can be isolated from several tissues, including the kidney. (79) The third cell type was human gingival keratinocytes (HGK). They play an important role in promoting strong epithelial bonds, which are highly relevant for renal physiology. (80) Lastly, human umbilical cord endothelial cells (HUVEC) were tested, which are generally used as a model for vascularized tissues, like the kidneys. (81) MSC, HGK, and HUVEC were exposed to freeze-dried colistin-C3Ms to facilitate addition to cell cultures (freeze-drying effect: Figure S14) and noncomplexed colistin. To assess the toxicity, the lactate dehydrogenase (LDH) level and caspase-3 level were measured with two colorimetric bioassays (Details in the Experimental Section). LDH is a biomarker for necrosis, (82) whereas caspase-3 is a well-described protease that plays a role in programmed cell death (apoptosis). (83) LDH and caspase-3 were measured after 24, 48, and 72 h of exposure (Figure 7), and light microscopy was used to image the cell morphology (Figure S15).

Figure 7

Figure 7. Cell viability of human embryonic kidney 293 (HEK) cells (A), mesenchymal stem cells (MSCs) (B), human gingival keratinocytes (HGKs) (C), or human umbilical cord endothelial cells (HUVECs) (D) treated with colistin, PEO-b-PMAA, or colistin-C3Ms. Lactate dehydrogenase (LDH) and caspase-3 activity were measured in the corresponding cell culture medium, expressed as fold change of control at 24, 48, and 72 h. Data are presented as the mean effect of 3 parallel cellular experiments for each stimulation at each time point. Significantly different from untreated control cells at each time point at *p < 0.05, **p < 0.01, and ***p < 0.001.

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Based on LDH and caspase-3 measurements, colistin, and colistin-C3Ms were found to have a different effect on the various cell types tested, but in general to be mildly toxic for all cell types (Figure 7). For HEK 293, the caspase-3 levels were up to 3-fold increased on day one and 2-fold increased on day two for both colistin and colistin-C3Ms, while there were no significant differences in LDH activity (Figure 7A). For MSC, the LDH activity was found to be 2-fold increased after 24 h and reduced in the later days because of necrosis-induced dead cell detachment, (82,84) while there was no significant difference in caspase-3 levels after 24 h (Figure 7B). Subsequently, MSC cell death was likely caused by increased necrosis from colistin and colistin-C3M treatments. Colistin, and colistin-C3M both caused necrosis (LDH) and apoptosis (caspase-3) of HGK after 24 h (Figure 7C). Notably, PEO-b-PMAA caused an unexpectedly high spike in LDH level after 48 h as PEO and PMAA are generally found to have low toxicities (85,86) (Figure S15). Lastly, in HUVEC cells, there was no significant effect for any treatment in the first 24 h (Figure 7D). However, after 48 h, a decrease in both LDH and caspase-3 values was observed. These findings are most likely a result of lower cell proliferation or a result in cell arrest of the treated HUVEC cells compared to untreated cells, rather than a toxic effect. This might indicate a higher resistance of this cell type, compared to MSC and HGK.
Generally, for all cell types, we found that the cytotoxicity of colistin-C3M was not lower than that of colistin itself. Most likely this is caused by cell-drug interactions with colistin and C3Ms′ colistin release during the 24 h. Even though no direct reduction of toxicity was found from complexation, it is another indication, next to the antimicrobial effect that the colistin is still active. In any case, more testing needs to be considered on cytotoxicity, in vivo stability, and drug effectivity to validate the viability of colistin-C3Ms for actual in vivo purposes.

4. Conclusions

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In this work, we demonstrated the preparation of PEO-b-PMAA-colistin-C3Ms that are highly stable and reproducible. Scattering techniques (DLS and SAXS) were employed to analyze the structure and stability of colistin complex coacervates at different charge fractions. Using a detailed SAXS model, built up of three scattering contributions: complex coacervate scattering, free component scattering, and an internal structure factor from the polyelectrolyte core, it was possible to quantify the complex coacervates on their structure, molecular weight, size, and composition. Based on the fit analysis the highest therapeutic potential of colistin complex coacervates was found to be at polyelectrolyte charge-matching conditions. C3Ms at this charge fraction exhibited the highest colistin complexation efficiency while being long-term stable. Subsequently, this formulation was assessed on several properties that are important for clinical purposes, like ionic strength effect, pH dependency, CMC, antibiotic properties, (enzymatic) stability, serum protein binding, and toxicity. The C3Ms demonstrated similar antibiotic effectiveness and equivalent toxicity compared to noncomplexed colistin, while the complexation of colistin into C3Ms was found to improve the protection against enzymatic degradation. Moreover, the presence of HSA had a negligible impact on C3Ms, which remained intact upon HSA addition. Comparing C3Ms and noncomplexed colistin, we found a similar antimicrobial/toxic effect, low HSA binding, and increased enzymatic stability. We believe the complexation of colistin in C3Ms could still be a viable strategy to reduce the total toxicity since the retention of colistin in complexed form is improved providing the possibility of reducing the concentration of the administered drug. Consequently, our study suggests that C3Ms incorporating colistin might offer a novel addition to the array of antibiotic formulations suitable for e.g., topical treatments in clinical applications.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.4c00337.

  • Details of the theoretical SAXS data modeling; DLS, including autocorrelation functions, SAXS data showing the concentration dependence of C3Ms, density profiles obtained from SAXS, the effect of ionic strength and pH on C3Ms, CMC determination, additional antimicrobial effects, trypsin breakdown effect, freeze-drying of C3Ms, morphology changes upon cellular treatment, and tables of fit parameter (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Thomas D. Vogelaar - Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway;  Orcidhttps://orcid.org/0000-0002-1653-1477
    • Anne E. Agger - Department of Biomaterials, Institute of Clinical Dentistry, University of Oslo, P.O. Box 1109, Blindern, NO-0317 Oslo, Norway;  Orcidhttps://orcid.org/0000-0002-9180-1297
    • Janne E. Reseland - Department of Biomaterials, Institute of Clinical Dentistry, University of Oslo, P.O. Box 1109, Blindern, NO-0317 Oslo, Norway
    • Dirk Linke - Department of Biosciences, University of Oslo, P.O. Box 1066, Blindern, NO-0316 Oslo, Norway;  Orcidhttps://orcid.org/0000-0003-3150-6752
    • Håvard Jenssen - Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark;  Orcidhttps://orcid.org/0000-0003-0007-0335
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We gratefully acknowledge funding from the Norwegian Research Council (Project Nos. 315666, 294605, and 331752) and the grant from Novo Nordisk Fonden (Project No. 0083282). We acknowledge the assistance of M. Tully and the help from the PSCM lab for the preparation of samples during beamtime at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France). The ESRF is gratefully acknowledged for providing beamtime through the “Norwegian BAG”. We are indebted to the BM29 beamline staff for assistance during the experiments. We acknowledge the use of the Norwegian Centre for X-ray Diffraction, Scattering and Imaging (RECX), supported by the Norwegian Research Council (NRC). In addition, the authors highly acknowledge the help of B. Mathiesen and T. Larsen from the University of Oslo (Norway) in the setup of the antimicrobial property and ζ-potential experiments, respectively. Lastly, we thank C. Cao and L. Ali for the initial experiments in this project.

Abbreviations

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AMP

antimicrobial peptides

C3Ms

complex coacervate core micelles

CEAC

critical excess anionic charge

CECC

critical excess cationic charge

CMC

critical micelle concentration

DDA

disc diffusion assay

DLS

dynamic light scattering

HSA

human serum albumin

MIC

minimum inhibitory concentration

PEO-b-PMAA

poly(ethylene oxide)-b-poly(methacrylic acid)

PDI

polydispersity index

SAXS

small-angle X-ray scattering

SCP

soluble complex particles

References

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This article references 86 other publications.

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    Amidst the ever-rising threat of antibiotics resistance, colistin, a decade-old antibiotic with lingering toxicity concern, is increasingly prescribed to treat many drug-resistant, gram-neg. bacteria. With the aim of improving the safety profile while preserving the antimicrobial activity of colistin, a nanoformulation is herein developed through coacervate complexation with polyanionic peptides. Upon controlled mixing of cationic colistin with polyglutamic acids, formation of liq. coacervates was demonstrated. Subsequent stabilization by DSPE-PEG and homogenization through micro-fluidization of the liq. coacervates yielded nanoparticles 8 nm in diam. In vitro assessment showed that the colistin antimicrobial activity against multiple drug-resistant bacterial strains was retained and, in some cases, enhanced following the nanoparticle assembly. In vivo administration in mice demonstrated improved safety of the colistin nanoparticle, which has a maximal tolerated dose of 12.5 mg/kg compared to 10 mg/kg of free colistin. Upon administration over a 7-day period, colistin nanoparticles also exhibited reduced hepatotoxicity as compared to free colistin. In mouse models of Klebsiella pneumoniae bacteremia and Acinetobacter baumannii pneumonia, treatment with colistin nanoparticles showed equiv. efficacy to free colistin. These results demonstrate coacervation-induced nanoparticle assembly as a promising approach towards improving colistin treatments against bacterial infections. Improving the safety of colistin while retaining its antimicrobial activity has been a highly sought-after objective toward enhancing antibacterial treatments. Herein, we demonstrate formation of stabilized colistin nanocomplexes in the presence of anionic polypeptides and DSPE-PEG stabilizer. The nanocomplexes retain colistins antimicrobial activity while demonstrating improved safety upon in vivo administration. The supramol. nanoparticle assembly of colistin presents a unique approach towards designing antimicrobial nanoparticles.
  22. 22
    Ezike, T. C.; Okpala, U. S.; Onoja, U. L.; Nwike, C. P.; Ezeako, E. C.; Okpara, O. J.; Okoroafor, C. C.; Eze, S. C.; Kalu, O. L.; Odoh, E. C.; Nwadike, U. G.; Ogbodo, J. O.; Umeh, B. U.; Ossai, E. C.; Nwanguma, B. C. Advances in Drug Delivery Systems, Challenges and Future Directions. Heliyon 2023, 9 (6), e17488  DOI: 10.1016/j.heliyon.2023.e17488
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    Ban, E.; Kim, A. Coacervates: Recent Developments as Nanostructure Delivery Platforms for Therapeutic Biomolecules. Int. J. Pharm. 2022, 624, 122058  DOI: 10.1016/j.ijpharm.2022.122058
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    Coacervates: Recent developments as nanostructure delivery platforms for therapeutic biomolecules
    Ban, Eunmi; Kim, Aeri
    International Journal of Pharmaceutics (Amsterdam, Netherlands) (2022), 624 (), 122058CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
    A review. Coacervation is a liq.-liq. phase sepn. that can occur in solns. of macromols. through self-assembly or electrostatic interactions. Recently, coacervates composed of biocompatible macromols. have been actively investigated as nanostructure platforms to encapsulate and deliver biomols. such as proteins, RNAs, and DNAs. One particular advantage of coacervates is that they are derived from aq. solns., unlike other nanoparticle delivery systems that often require org. solvents. In addn., coacervates achieve high loading while maintaining the viability of the cargo material. Here, we review recent developments in the applications of coacervates and their limitations in the delivery of therapeutic biomols. Important factors for coacervation include mol. structures of the polyelectrolytes, mixing ratio, the concn. of polyelectrolytes, and reaction conditions such as ionic strength, pH, and temp. Various compns. of coacervates have been shown to deliver biomols. in vitro and in vivo with encouraging activities. However, major hurdles remain for the systemic route of administration other than topical or local delivery. The scale-up of manufg. methods suitable for preclin. and clin. evaluations remains to be addressed. We conclude with a few research directions to overcome current challenges, which may lead to successful translation into the clinic.
  24. 24
    Blocher, W. C.; Perry, S. L. Complex Coacervate-Based Materials for Biomedicine. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2017, 9 (4), e1442  DOI: 10.1002/wnan.1442
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    Borro, B. C.; Malmsten, M. Complexation between Antimicrobial Peptides and Polyelectrolytes. Adv. Colloid Interface Sci. 2019, 270, 251– 260,  DOI: 10.1016/j.cis.2019.07.001
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    Complexation between antimicrobial peptides and polyelectrolytes
    Borro, Bruno C.; Malmsten, Martin
    Advances in Colloid and Interface Science (2019), 270 (), 251-260CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
    As a result of increasing bacterial resistance against antibiotics, we are facing an emerging health crisis, in which 'simple' infections may no longer be treatable. One class of mols. attracting interest in this context is antimicrobial peptides (AMPs), and considerable research efforts have been directed to identifying selective and potent AMPs. In addn., since in vivo delivery of AMPs is challenging, there is an emerging awareness that successful development of AMP therapeutics can be facilitated by careful design of AMPs delivery systems. In the present overview, we discuss polyelectrolyte complexation as a strategy to deliver AMPs. In doing so, key factors for AMP-polyelectrolyte complexation are illustrated for AMP-polyelectrolyte nanoparticle formation, as well as for AMP incorporation in polyelectrolyte microgels and multilayer structures, and consequences of these for functional performance exemplified.
  26. 26
    Jenssen, H.; Aspmo, S. I. Serum Stability of Peptides. In Peptide-Based Drug Design; Otvos, L., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2008; pp 177– 186.  DOI: 10.1007/978-1-59745-419-3_10 .
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    Voets, I. K.; de Keizer, A.; Cohen Stuart, M. A. Complex Coacervate Core Micelles. Adv. Colloid Interface Sci. 2009, 147–148, 300– 318,  DOI: 10.1016/j.cis.2008.09.012
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    Complex coacervate core micelles
    Voets, Ilja K.; de Keizer, Arie; Cohen Stuart, Martien A.
    Advances in Colloid and Interface Science (2009), 147-148 (), 300-318CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
    A review. In this review we present an overview of the literature on the co-assembly of neutral-ionic block, graft, and random copolymers with oppositely charged species in aq. soln. Oppositely charged species include synthetic (co)polymers of various architectures, biopolymers - such as proteins, enzymes and DNA - multivalent ions, metallic nanoparticles, low mol. wt. surfactants, polyelectrolyte block copolymer micelles, metallo-supramol. polymers, equil. polymers, etc. The resultant structures are termed complex coacervate core/polyion complex/block ionomer complex/interpolyelectrolyte complex micelles (or vesicles); i.e., in short C3Ms (or C3Vs) and PIC, BIC or IPEC micelles (and vesicles). Formation, structure, dynamics, properties, and function will be discussed. We focus on exptl. work; theory and modeling will not be discussed. Recent developments in applications and micelles with heterogeneous coronas are emphasized.
  28. 28
    Magana, J. R.; Sproncken, C. C. M.; Voets, I. K. On Complex Coacervate Core Micelles: Structure-Function Perspectives. Polymers 2020, 12 (9), 1953,  DOI: 10.3390/polym12091953
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    28
    On complex coacervate core micelles: structure-function perspectives
    Magana, Jose Rodrigo; Sproncken, Christian C. M.; Voets, Ilja K.
    Polymers (Basel, Switzerland) (2020), 12 (9), 1953CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
    A review. The co-assembly of ionic-neutral block copolymers with oppositely charged species produces nanometric colloidal complexes, known, among other names, as complex coacervates core micelles (C3Ms). C3Ms are of widespread interest in nanomedicine for controlled delivery and release, while research activity into other application areas, such as gelation, catalysis, nanoparticle synthesis, and sensing, is increasing. In this review, we discuss recent studies on the functional roles that C3Ms can fulfil in these and other fields, focusing on emerging structure-function relations and remaining knowledge gaps.
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    Marciel, A. B.; Srivastava, S.; Ting, J. M.; Tirrell, M. V. SAXS Methods for Investigating Macromolecular and Self-Assembled Polyelectrolyte Complexes. In Methods in Enzymology; Keating, C. D., Ed.; Liquid-Liquid Phase Coexistence and Membraneless Organelles; Academic Press, 2021; Chapter 8, Vol. 646; pp 223– 259.  DOI: 10.1016/bs.mie.2020.09.013 .
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    Rumyantsev, A. M.; Jackson, N. E.; de Pablo, J. J. Polyelectrolyte Complex Coacervates: Recent Developments and New Frontiers. Annu. Rev. Condens. Matter Phys. 2021, 12 (1), 155– 176,  DOI: 10.1146/annurev-conmatphys-042020-113457
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    Uebbing, L.; Ziller, A.; Siewert, C.; Schroer, M. A.; Blanchet, C. E.; Svergun, D. I.; Ramishetti, S.; Peer, D.; Sahin, U.; Haas, H.; Langguth, P. Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-Ray Scattering. Langmuir 2020, 36 (44), 13331– 13341,  DOI: 10.1021/acs.langmuir.0c02446
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    Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering
    Uebbing, Lukas; Ziller, Antje; Siewert, Christian; Schroer, Martin A.; Blanchet, Clement E.; Svergun, Dmitri I.; Ramishetti, Srinivas; Peer, Dan; Sahin, Ugur; Haas, Heinrich; Langguth, Peter
    Langmuir (2020), 36 (44), 13331-13341CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    MRNA (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clin. practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clin. efficacy. In this study, pH-induced changes in internal mol. organization and overall physicochem. characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were detd. for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate detn. of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ detn. of the structural response of the lipoplex systems to changes of the environmental pH similar to that obsd. for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.
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    Grabbe, S.; Haas, H.; Diken, M.; Kranz, L. M.; Langguth, P.; Sahin, U. Translating Nanoparticulate-Personalized Cancer Vaccines into Clinical Applications: Case Study with RNA-Lipoplexes for the Treatment of Melanoma. Nanomedicine 2016, 11 (20), 2723– 2734,  DOI: 10.2217/nnm-2016-0275
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    Translating nanoparticulate-personalized cancer vaccines into clinical applications: case study with RNA-lipoplexes for the treatment of melanoma
    Grabbe, Stephan; Haas, Heinrich; Diken, Mustafa; Kranz, Lena M.; Langguth, Peter; Sahin, Ugur
    Nanomedicine (London, United Kingdom) (2016), 11 (20), 2723-2734CODEN: NLUKAC; ISSN:1743-5889. (Future Medicine Ltd.)
    The development of nucleic acid based vaccines against cancer has gained considerable momentum through the advancement of modern sequencing technologies and on novel RNA-based synthetic drug formats, which can be readily adapted following identification of every patient's tumor-specific mutations. Furthermore, affordable and individual 'on demand' prodn. of molecularly optimized vaccines should allow their application in large groups of patients. This has resulted in the therapeutic concept of an active personalized cancer vaccine, which has been brought into clin. testing. Successful trials have been performed by intranodal administration of sterile isotonic solns. of synthetic RNA vaccines. The second generation of RNA vaccines which is currently being developed encompasses i.v. injectable RNA nanoparticle formulations (lipoplexes), made up from lipid excipients, denoted RNA(LIP). A first product that has made its way from bench to bedside is a therapeutic vaccine for i.v. administration based on a fixed set of four RNA lipoplex drug products, each encoding for one shared tumor antigen (Lipoplex Melanoma RNA Immunotherapy, 'Lipo-MERIT'). This article describes the steps for translating these novel RNA nanomedicines into clin. trials. Graphical Abstr. :.
  33. 33
    Nogueira, S. S.; Schlegel, A.; Maxeiner, K.; Weber, B.; Barz, M.; Schroer, M. A.; Blanchet, C. E.; Svergun, D. I.; Ramishetti, S.; Peer, D.; Langguth, P.; Sahin, U.; Haas, H. Polysarcosine-Functionalized Lipid Nanoparticles for Therapeutic mRNA Delivery. ACS Appl. Nano Mater. 2020, 3 (11), 10634– 10645,  DOI: 10.1021/acsanm.0c01834
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    Polysarcosine-Functionalized Lipid Nanoparticles for Therapeutic mRNA Delivery
    Nogueira, Sara S.; Schlegel, Anne; Maxeiner, Konrad; Weber, Benjamin; Barz, Matthias; Schroer, Martin A.; Blanchet, Clement E.; Svergun, Dmitri I.; Ramishetti, Srinivas; Peer, Dan; Langguth, Peter; Sahin, Ugur; Haas, Heinrich
    ACS Applied Nano Materials (2020), 3 (11), 10634-10645CODEN: AANMF6; ISSN:2574-0970. (American Chemical Society)
    Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for therapeutic application of mRNA were assembled using pSarcosinylated lipids as a tool for particle engineering. Using pSar lipids with different polymeric chain lengths and molar fractions enabled the control of the physicochem. characteristics of the LNPs, such as particle size, morphol., and internal structure. In combination with a suited ionizable lipid, LNPs were assembled, which displayed high RNA transfection potency with an improved safety profile after i.v. injection. Notably, a higher protein secretion with a reduced immunostimulatory response was obsd. when compared to systems based on polyethylene glycol (PEG) lipids. PSarcosinylated nanocarriers showed a lower proinflammatory cytokine secretion and reduced complement activation compared to PEGylated LNPs. In summary, the described pSar-based LNPs enable safe and potent delivery of mRNA, thus signifying an excellent basis for the development of PEG-free RNA therapeutics.
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    Bos, I.; Timmerman, M.; Sprakel, J. FRET-Based Determination of the Exchange Dynamics of Complex Coacervate Core Micelles. Macromolecules 2021, 54 (1), 398– 411,  DOI: 10.1021/acs.macromol.0c02387
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    FRET-Based Determination of the Exchange Dynamics of Complex Coacervate Core Micelles
    Bos, Inge; Timmerman, Marga; Sprakel, Joris
    Macromolecules (Washington, DC, United States) (2021), 54 (1), 398-411CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    Complex coacervate core micelles (C3Ms) are nanoscopic structures formed by charge interactions between oppositely charged macroions and used to encapsulate a wide variety of charged (bio)mols. In most cases, C3Ms are in a dynamic equil. with their surroundings. Understanding the dynamics of mol. exchange reactions is essential as this dets. the rate at which their cargo is exposed to the environment. Here, we study the mol. exchange in C3Ms by making use of Fluorescence resonance energy transfer (FRET) and derive an anal. model to relate the exptl. obsd. increase in FRET efficiency to the underlying macromol. exchange rates. We show that equilibrated C3Ms have a broad distribution of exchange rates. The overall exchange rate can be strongly increased by increasing the salt concn. In contrast, changing the unlabeled homopolymer length does not affect the exchange of the labeled homopolymers and an increase in the micelle concn. only affects the FRET increase rate at low micelle concns. Together, these results suggest that the exchange of these equilibrated C3Ms occurs mainly by expulsion and insertion, where the rate-limiting step is the breaking of ionic bonds to expel the chains from the core. These are important insights to further improve the encapsulation efficiency of C3Ms.
  35. 35
    Amann, M.; Diget, J. S.; Lyngsø, J.; Pedersen, J. S.; Narayanan, T.; Lund, R. Kinetic Pathways for Polyelectrolyte Coacervate Micelle Formation Revealed by Time-Resolved Synchrotron SAXS. Macromolecules 2019, 52 (21), 8227– 8237,  DOI: 10.1021/acs.macromol.9b01072
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    Kinetic Pathways for Polyelectrolyte Coacervate Micelle Formation Revealed by Time-Resolved Synchrotron SAXS
    Amann, Matthias; Diget, Jakob Stensgaard; Lyngsoe, Jeppe; Pedersen, Jan Skov; Narayanan, Theyencheri; Lund, Reidar
    Macromolecules (Washington, DC, United States) (2019), 52 (21), 8227-8237CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    The kinetic pathways for coacervation and micelle formation are still not fully understood. Driven by electrostatic interactions and entropically driven counterion release, complexation of oppositely charged macromols. leads to the formation of micellar nanostructures. Here we study the coacervation process, from initial formation and growth of stable micelles, on a nanometric length scale using time-resolved small-angle X-ray scattering (TR-SAXS). The micellar coacervates are formed through the complexation of anionic polyelectrolyte poly(sodium 4-styrenesulfonate) (PSSS) and cationic block-copolymer poly(ethylene oxide)-block-poly((vinylbenzyl)trimethylammonium chloride) (PEO-b-PVBTA). Mixing the polyelectrolytes in a stoichiometric 1:1 charge ratio resulted in the formation of stable spherical core-shell micellar-like coacervates consisting of a central core of complexed PSSS and PVBTA with a PEO corona. By use of synchrotron SAXS coupled to a stopped-flow mixing app., the whole formation kinetics of coacervates could be followed in situ from a few milliseconds. The results of a detailed data modeling reveal that the formation of these polyelectrolyte coacervates follows a two-step process: (i) first, metastable large-scale aggregates are formed upon a barrier-free complexation immediately after mixing; (ii) subsequently, the clusters undergo charge equilibration upon chain rearrangement and exchange processes yielding micellar-like aggregates with net neutral charge that are pinched off to yield the final stable micelle-like coacervates. While the initial cluster formation is very fast and completed within the dead time of mixing, the subsequent rearrangement becomes significantly slower with increasing mol. wt. of the PVBTA block. Interestingly, the overall kinetic process was essentially concn. independent, indicating that the rearrangement process is mainly accomplished via noncooperative chain rearrangement and chain exchange processes.
  36. 36
    Voets, I. K.; Moll, P. M.; Aqil, A.; Jérôme, C.; Detrembleur, C.; de Waard, P.; de Keizer, A.; Stuart, M. A. C. Temperature Responsive Complex Coacervate Core Micelles With a PEO and PNIPAAm Corona. J. Phys. Chem. B 2008, 112 (35), 10833– 10840,  DOI: 10.1021/jp8014832
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    Temperature Responsive Complex Coacervate Core Micelles With a PEO and PNIPAAm Corona
    Voets, Ilja K.; Moll, Puck M.; Aqil, Abdelhafid; Jerome, Christine; Detrembleur, Christophe; de Waard, Pieter; de Keizer, Arie; Cohen Stuart, Martien A.
    Journal of Physical Chemistry B (2008), 112 (35), 10833-10840CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    In aq. solns. at room temp., poly(N-methyl-2-vinyl pyridinium iodide)-block-poly(ethylene oxide), P2MVP38-b-PEO211 and poly(acrylic acid)-block-poly(isopropylacrylamide), PAA55-b-PNIPAAm88 spontaneously coassemble into micelles, consisting of a mixed P2MVP/PAA polyelectrolyte core and a PEO/PNIPAAm corona. These so-called complex coacervate core micelles (C3Ms), also known as polyion complex (PIC) micelles, block ionomer complexes (BIC), and interpolyelectrolyte complexes (IPEC), respond to changes in soln. pH and ionic strength as their micellization is electrostatically driven. Furthermore, the PNIPAAm segments ensure temp. responsiveness as they exhibit lower crit. soln. temp. (LCST) behavior. Light scattering, two-dimensional 1H NMR nuclear Overhauser effect spectrometry, and cryogenic transmission electron microscopy expts. were carried out to investigate micellar structure and soln. behavior at 1 mM NaNO3, T = 25, and 60 °C, i.e., below and above the LCST of ∼32 °C. At T = 25 °C, C3Ms were obsd. for 7 < pH < 12 and NaNO3 concns. below ∼105 mM. The PEO and PNIPAAm chains appear to be (randomly) mixed within the micellar corona. At T = 60 °C, onion-like complexes are formed, consisting of a PNIPAAm inner core, a mixed P2MVP/PAA complex coacervate shell, and a PEO corona.
  37. 37
    Sproncken, C. C. M.; Surís-Valls, R.; Cingil, H. E.; Detrembleur, C.; Voets, I. K. Complex Coacervate Core Micelles Containing Poly(Vinyl Alcohol) Inhibit Ice Recrystallization. Macromol. Rapid Commun. 2018, 39 (17), e1700814  DOI: 10.1002/marc.201700814
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    Voets, I. K.; van der Burgh, S.; Farago, B.; Fokkink, R.; Kovacevic, D.; Hellweg, T.; de Keizer, A.; Cohen Stuart, M. A. Electrostatically Driven Coassembly of a Diblock Copolymer and an Oppositely Charged Homopolymer in Aqueous Solutions. Macromolecules 2007, 40 (23), 8476– 8482,  DOI: 10.1021/ma071356z
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    Electrostatically Driven Coassembly of a Diblock Copolymer and an Oppositely Charged Homopolymer in Aqueous Solutions
    Voets, Ilja K.; Van der Burgh, Stefan; Farago, Bela; Fokkink, Remco; Kovacevic, Davor; Hellweg, Thomas; De Keizer, Arie; Cohen Stuart, Martien A.
    Macromolecules (Washington, DC, United States) (2007), 40 (23), 8476-8482CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    Electrostatically driven co-assembly of poly(acrylic acid)-block-poly(acrylamide), PAA-b-PAAm, and poly(1-methyl-2-vinylpyridinium iodide), P2MVP, leads to formation of micelles in aq. solns. Light scattering and small angle neutron scattering expts. were performed to study the effect of concn. and length of the corona block (NPAAm = 97, 208, and 417) on micellar characteristics. Small angle neutron scattering curves were analyzed by generalized indirect Fourier transformation and model fitting. All scattering curves could be well described with a combination of a form factor for polydisperse spheres in combination with a hard sphere structure factor for the highest concns. Micellar aggregation nos., shape, and internal structure are relatively independent of concn. for Cp < 23.12 g L-1. The Guinier radius, av. micellar radius, hydrodynamic radius, and polydispersity were found to increase with increasing NPAAm. Micellar mass and aggregation no. were found to decrease with increasing NPAAm.
  39. 39
    Priftis, D.; Leon, L.; Song, Z.; Perry, S. L.; Margossian, K. O.; Tropnikova, A.; Cheng, J.; Tirrell, M. Self-Assembly of α-Helical Polypeptides Driven by Complex Coacervation. Angew. Chem. 2015, 127 (38), 11280– 11284,  DOI: 10.1002/ange.201504861
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    Choi, J.-W.; Heo, T.-Y.; Choi, H.; Choi, S.-H.; Won, J.-I. Co-Assembly Behavior of Oppositely Charged Thermoresponsive Elastin-like Polypeptide Block Copolymers. J. Appl. Polym. Sci. 2022, 139 (38), e52906  DOI: 10.1002/app.52906
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    Lim, C.; Roeck Won, W.; Moon, J.; Sim, T.; Shin, Y.; Chang Kim, J.; Seong Lee, E.; Seok Youn, Y.; Taek Oh, K. Co-Delivery of d -(KLAKLAK) 2 Peptide and Doxorubicin Using a pH-Sensitive Nanocarrier for Synergistic Anticancer Treatment. J. Mater. Chem. B 2019, 7 (27), 4299– 4308,  DOI: 10.1039/C9TB00741E
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    Lindhoud, S.; Voorhaar, L.; de Vries, R.; Schweins, R.; Cohen Stuart, M. A.; Norde, W. Salt-Induced Disintegration of Lysozyme-Containing Polyelectrolyte Complex Micelles. Langmuir 2009, 25 (19), 11425– 11430,  DOI: 10.1021/la901591p
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    Salt-Induced Disintegration of Lysozyme-Containing Polyelectrolyte Complex Micelles
    Lindhoud, Saskia; Voorhaar, Lenny; de Vries, Renko; Schweins, Ralf; Cohen Stuart, Martien A.; Norde, Willem
    Langmuir (2009), 25 (19), 11425-11430CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The salt-induced disintegration of lysozyme-filled polyelectrolyte complex micelles, consisting of pos. charged homopolymers (PDMAEMA150), neg. charged diblock copolymers (PAA42-PAAm417), and lysozyme, was studied with dynamic light scattering (DLS) and small-angle neutron scattering (SANS). These measurements show that, from 0 to 0.2 M NaCl, both the hydrodynamic radius (Rh) and the core radius (Rcore) decrease with increasing salt concn. This suggests that the micellar structures rearrange. Moreover, from ∼0.2 to 0.4 M NaCl the light-scattering intensity is const. In this salt interval, the hydrodynamic radius increases, has a max. at 0.3 M NaCl, and subsequently decreases. This behavior is obsd. in both a lysozyme-contg. system and a system without lysozyme. The SANS measurements on the lysozyme-filled micelles do not show increased intensity or a larger core radius at 0.3 M NaCl. This indicates that from 0.2 to 0.4 M NaCl another structure is formed, consisting of just the diblock copolymer and the homopolymer, because at 0.12 M NaCl the lysozyme-PAA42-PAAm417 complex has disintegrated. One may expect that the driving force for the formation of the complex in this salt range is other than electrostatic.
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    Obermeyer, A. C.; Mills, C.; Dong, X.-H.; Flores, R.; Olsen, B. Complex Coacervation of Supercharged Proteins with Polyelectrolytes. Soft Matter 2016, 12 (15), 3570– 3581,  DOI: 10.1039/C6SM00002A
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    Complex coacervation of supercharged proteins with polyelectrolytes
    Obermeyer, Allie C.; Mills, Carolyn E.; Dong, Xue-Hui; Flores, Romeo J.; Olsen, Bradley D.
    Soft Matter (2016), 12 (15), 3570-3581CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
    Complexation of proteins with polyelectrolytes or block copolymers can lead to phase sepn. to generate a coacervate phase or self-assembly of coacervate core micelles. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase sepd. with strong polycations when the ratio of neg. charged residues to pos. charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of pos. charge in the coacervate phase. The compn. of protein and polymer in the coacervate phase was detd. using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromol. charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temps.
  44. 44
    Xu, A. Y.; Kizilay, E.; Madro, S. P.; Vadenais, J. Z.; McDonald, K. W.; Dubin, P. L. Dilution Induced Coacervation in Polyelectrolyte-Micelle and Polyelectrolyte-Protein Systems. Soft Matter 2018, 14 (12), 2391– 2399,  DOI: 10.1039/C7SM02293J
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    Dilution induced coacervation in polyelectrolyte-micelle and polyelectrolyte-protein systems
    Xu, Amy Y.; Kizilay, Ebru; Madro, Slawomir P.; Vadenais, Justin Z.; McDonald, Kianan W.; Dubin, Paul L.
    Soft Matter (2018), 14 (12), 2391-2399CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
    "Self-suppression", the instability of complex coacervates at high concn., is well-known for polycation-polyanion systems, but the transient nature of those complexes impedes development of a convincing model. The stable polyelectrolyte-micelle complexes of the polycation poly(diallyldimethylammonium chloride) (PDADMAC) with mixed micelles of sodium dodecyl sulfate (SDS)/Triton X-100 (TX100); and the stable complexes of PDADMAC with bovine serum albumin (BSA) can be characterized and identified as coacervate precursors. We observe liq.-liq. phase sepn. upon isoionic diln., a common facet of self-suppression. While complex coacervation usually involves assocn. of near-neutral inter-polymer complexes, diln.-induced coacervation (DIC) proceeds differently: for both systems studied, complex size decreases near the biphasic region: inter-macromol. complexes with hydrodynamic radius Rh ∼ 100 nm dissoc. to intra-polyelectrolyte complexes with Rh ≤ 30 nm. Such small complexes with ≤5 bound micelles are unlikely to be net neutral. In the polyelectrolyte-protein system, complexes are even less likely to be net neutral and the effect of diln. on size is less significant, with complex size diminishing from 50 nm to 35 nm.
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    Marras, A. E.; Ting, J. M.; Stevens, K. C.; Tirrell, M. V. Advances in the Structural Design of Polyelectrolyte Complex Micelles. J. Phys. Chem. B 2021, 125 (26), 7076– 7089,  DOI: 10.1021/acs.jpcb.1c01258
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    Advances in the Structural Design of Polyelectrolyte Complex Micelles
    Marras, Alexander E.; Ting, Jeffrey M.; Stevens, Kaden C.; Tirrell, Matthew V.
    Journal of Physical Chemistry B (2021), 125 (26), 7076-7089CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    A review. Polyelectrolyte complex micelles (PCMs) are a unique class of self-assembled nanoparticles that form with a core of assocd. polycations and polyanions, microphase-sepd. from neutral, hydrophilic coronas in aq. soln. The hydrated nature and structural and chem. versatility make PCMs an attractive system for delivery and for fundamental polymer physics research. By leveraging block copolymer design with controlled self-assembly, fundamental structure-property relationships can be established to tune the size, morphol., and stability of PCMs precisely in pursuit of tailored nanocarriers, ultimately offering storage, protection, transport, and delivery of active ingredients. This perspective highlights recent advances in predictive PCM design, focusing on (1) structure-property relationships to target specific nanoscale dimensions and shapes and (2) characterization of PCM dynamics primarily using time-resolved scattering techniques. We present several vignettes from these two emerging areas of PCM research and discuss key opportunities for PCM design to advance precision medicine.
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    Abbas, M.; Lipiński, W.; Wang, J.; Spruijt, E. Peptide-Based Coacervates as Biomimetic Protocells. Chem. Soc. Rev. 2021, 50 (6), 3690– 3705,  DOI: 10.1039/D0CS00307G
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    Peptide-based coacervates as biomimetic protocells
    Abbas, Manzar; Lipinski, Wojciech P.; Wang, Jiahua; Spruijt, Evan
    Chemical Society Reviews (2021), 50 (6), 3690-3705CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Coacervates are condensed liq.-like droplets formed by liq.-liq. phase sepn. of mols. through multiple weak associative interactions. In recent years it has emerged that not only long polymers, but also short peptides are capable of forming simple and complex coacervates. The coacervate droplets they form act as compartments that sequester and conc. a wide range of solutes, and their spontaneous formation make coacervates attractive protocell models. The main advantage of peptides as building blocks lies in the functional diversity of the amino acid residues, which allows for tailoring of the peptide's phase sepn. propensity, their selectivity in guest mol. uptake and the physicochem. and catalytic properties of the compartments. The aim of this tutorial is to illustrate the recent developments in the field of peptide-based coacervates in a systematic way and to deduce the basic requirements for both simple and complex coacervation of peptides. We a selection of peptide coacervates that illustrates the essentials of phase sepn., the limitations, and the properties that make peptide coacervates biomimetic protocells. Finally, we provide some perspectives of this novel research field in the direction of active droplets, moving away from thermodn. equil.
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    Brancaccio, D.; Pizzo, E.; Cafaro, V.; Notomista, E.; De Lise, F.; Bosso, A.; Gaglione, R.; Merlino, F.; Novellino, E.; Ungaro, F.; Grieco, P.; Malanga, M.; Quaglia, F.; Miro, A.; Carotenuto, A. Antimicrobial Peptide Temporin-L Complexed with Anionic Cyclodextrins Results in a Potent and Safe Agent against Sessile Bacteria. Int. J. Pharm. 2020, 584, 119437  DOI: 10.1016/j.ijpharm.2020.119437
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    Antimicrobial peptide Temporin-L complexed with anionic cyclodextrins results in a potent and safe agent against sessile bacteria
    Brancaccio, Diego; Pizzo, Elio; Cafaro, Valeria; Notomista, Eugenio; De Lise, Federica; Bosso, Andrea; Gaglione, Rosa; Merlino, Francesco; Novellino, Ettore; Ungaro, Francesca; Grieco, Paolo; Malanga, Milo; Quaglia, Fabiana; Miro, Agnese; Carotenuto, Alfonso
    International Journal of Pharmaceutics (Amsterdam, Netherlands) (2020), 584 (), 119437CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
    Concern over antibiotic resistance is growing, and new classes of antibiotics, particularly against Gram-neg. bacteria, are needed. Antimicrobial peptides (AMPs) have been proposed as a new class of clin. useful antimicrobials. Special attention has been devoted to frog-skin temporins. In particular, temporin L (TL) is strongly active against Gram-pos., Gram-neg. bacteria and yeast strains. With the aim of overcoming some of the main drawbacks preventing the widespread clin. use of this peptide, i.e. toxicity and unfavorable pharmacokinetics profile, we designed new formulations combining TL with different types of cyclodextrins (CDs). TL was assocd. to a panel of neutral or neg. charged, monomeric and polymeric CDs. The impact of CDs assocn. on TL soly., as well as the transport through bacterial alginates was assessed. The biocompatibility on human cells together with the antimicrobial and antibiofilm properties of TL/CD systems was explored.
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    Insua, I.; Majok, S.; Peacock, A. F. A.; Krachler, A. M.; Fernandez-Trillo, F. Preparation and Antimicrobial Evaluation of Polyion Complex (PIC) Nanoparticles Loaded with Polymyxin B. Eur. Polym. J. 2017, 87, 478– 486,  DOI: 10.1016/j.eurpolymj.2016.08.023
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    Preparation and antimicrobial evaluation of polyion complex (PIC) nanoparticles loaded with polymyxin B
    Insua, Ignacio; Majok, Sieta; Peacock, Anna F. A.; Krachler, Anne Marie; Fernandez-Trillo, Francisco
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    Here, we describe novel polyion complex (PIC) particles for the delivery of Polymyxin B (Pol-B), an antimicrobial peptide currently used in the clinic as a last resort antibiotic against multidrug-resistant gram-neg. bacteria. A range of conditions for the controlled assembly of Pol-B with poly(styrene sulfonate) (PSS) has been identified which let us prep. stable colloidal PIC particles. This way, PIC particles contg. different Pol-B:PSS ratios have been prepd. and their stability under simulated physiol. conditions (i.e. pH, osmotic pressure and temp.) characterized. Furthermore, preliminary evaluation of the antimicrobial activity of these Pol-B contg. PIC particles has been performed, by monitoring their effect on the growth of Pseudomonas aeruginosa, an opportunistic gram-neg. bacterium.
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    Răileanu, M.; Lonetti, B.; Serpentini, C.-L.; Goudounèche, D.; Gibot, L.; Bacalum, M. Encapsulation of a Cationic Antimicrobial Peptide into Self-Assembled Polyion Complex Nano-Objects Enhances Its Antitumor Properties. J. Mol. Struct. 2022, 1249, 131482  DOI: 10.1016/j.molstruc.2021.131482
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    Wang, C.; Feng, S.; Qie, J.; Wei, X.; Yan, H.; Liu, K. Polyion Complexes of a Cationic Antimicrobial Peptide as a Potential Systemically Administered Antibiotic. Int. J. Pharm. 2019, 554, 284– 291,  DOI: 10.1016/j.ijpharm.2018.11.029
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    Polyion complexes of a cationic antimicrobial peptide as a potential systemically administered antibiotic
    Wang, Chenhong; Feng, Siliang; Qie, Jiankun; Wei, Xiaoli; Yan, Husheng; Liu, Keliang
    International Journal of Pharmaceutics (Amsterdam, Netherlands) (2019), 554 (), 284-291CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
    Antimicrobial peptides (AMPs) are regarded as next-generation antibiotics to replace conventional antibiotics due to their rapid and broad-spectrum antimicrobial properties and far less sensitivity to the development of pathogen resistance. However, they are susceptible to proteolysis in vivo by endogenous or bacterial proteases as well as induce the lysis of red blood cells, which prevent their i.v. applications. In this work, polyion complex (PIC) micelles of the cationic AMP MSI-78 and the anionic copolymer methoxy poly(ethylene glycol)-b-poly(α-glutamic acid) (mPEG-b-PGlu) were prepd. to develop novel antimicrobial agents for potential application in vivo. With an increase in molar ratio of mPEG-b-PGlu to MSI-78, the complexation ability of the PIC micelles increased. FITC-labeled MSI-78 showed a sustained release from the PIC micelles. More importantly, these PIC micelles greatly decreased the hemolytic toxicity of MSI-78 to human red blood cells, without influencing its antimicrobial activity. Thus, this approach could be used as a suitable in vivo delivery method of AMPs in the future.
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    Influence of Shell Thickness and Cross-Link Density on the Structure of Temperature-Sensitive Poly-N-Isopropylacrylamide-Poly-N-Isopropylmethacrylamide Core-Shell Microgels Investigated by Small-Angle Neutron Scattering
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    Langmuir (2006), 22 (1), 459-468CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Swelling properties of doubly temp. sensitive core-shell microgels consisting of two thermosensitive polymers with lower crit. soln. temps. (LCTS) at, resp., 34° in the core and 44° in the shell have been investigated by small-angle neutron scattering (SANS). A core-shell form factor has been employed to evaluate the structure, and the real space particle structure is expressed by radial d. profiles. By this means, the influences of both shell/core mass compn. and shell cross-linker content on the internal structure have been revealed at temps. above, between, and below the LCSTs. Higher shell/core mass ratios lead to an increased expansion of the core at temps. between the LCSTs, whereas a variation of cross-linker in the shell mainly effects the dimensions of the shell. The influence on the core structure was interpreted as resulting from an elastic force developed from the swollen shell. At temps. below the core LCST, the core cannot swell to its native size (i.e., in the absence of a shell), because the max. expanded shell network prohibits further swelling. Thus, depending on temp., the shell either expands or compresses the core.
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    Berndt, I.; Pedersen, J. S.; Richtering, W. Temperature-Sensitive Core–Shell Microgel Particles with Dense Shell. Angew. Chem. 2006, 118 (11), 1769– 1773,  DOI: 10.1002/ange.200503888
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    Structure factors effects in small-angle scattering from block copolymer micelles and star polymers
    Pedersen, Jan Skov
    Journal of Chemical Physics (2001), 114 (6), 2839-2846CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
    Math. expressions for the form factor of a block copolymer micelle model with a spherical core and Gaussian polymer chains attached to the surface presented in J. S. Pedersen and M. C. Gerstenberg [Macromols. 29, 1363 (1996)] were modified to include particle interference effects in scattering expts. in terms of a structure factor. The results are derived assuming that the effective interaction between particles is known, i.e., the structure factor related to the center-center distribution is assumed to be known. The derived expression for the intensity is not a simple product of the form factor and the structure factor, which has the important consequence that the effective structure factor depends on the relative scattering contrast of the core and the corona of polymer chains. The structure factor effects for Gaussian star polymers are described by the same expression for a vanishing core radius. The influences of chain self-avoidance and chain-chain interactions are discussed.
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    Lund, R.; Willner, L.; Stellbrink, J.; Radulescu, A.; Richter, D. Role of Interfacial Tension for the Structure of PEP–PEO Polymeric Micelles. A Combined SANS and Pendant Drop Tensiometry Investigation. Macromolecules 2004, 37 (26), 9984– 9993,  DOI: 10.1021/ma035633n
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    Role of Interfacial Tension for the Structure of PEP-PEO Polymeric Micelles. A Combined SANS and Pendant Drop Tensiometry Investigation
    Lund, Reidar; Willner, Lutz; Stellbrink, Joerg; Radulescu, Aurel; Richter, Dieter
    Macromolecules (2004), 37 (26), 9984-9993CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    We investigated the influence of interfacial tension, γ, on the micellization properties of a highly asym. poly(ethylene-co-propylene)-poly(ethylene oxide) (PEP-PEO) block copolymer in mixed solvents consisting of water and DMF. Both are good solvents for PEO and nonsolvents for PEP but exhibit a large difference in γ with respect to the insol. core block. Micellar characteristics were obtained by small-angle neutron scattering (SANS) and subsequent fitting of a core-shell form factor to the scattering patterns. The curves are perfectly described by a hyperbolic d. profile for the shell, n(r) ∼ r-4/3, indicating a starlike structure of the micelles. The aggregation nos. of the micelles decrease with increasing DMF-water ratio from P = 120 in pure water to nonaggregated chains in pure DMF. Corresponding interfacial tensions were detd. by pendant drop tensiometry using a PEP homopolymer of equal molar mass. A correlation of P with γ reveals a power law dependence, P ∼ γ6/5, in accordance with the scaling prediction of Halperin for starlike micelles. The addn. of DMF leads to a considerable decrease in the micelle radii, which cannot be explained by the decrease in P alone. Measurements of the second virial coeffs., A2, of a PEO homopolymer by SANS reveal clearly reduced values compared to A2 in pure water but still good solvent conditions for PEO in all water/DMF mixts. However, a significant redn. in the radius of gyration was not found. Therefore, it was concluded that the reduced solvent quality has a more pronounced effect for the PEO chain dimensions in the confined geometry of a micellar corona.
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    Makowski, M.; Silva, Í. C.; Pais do Amaral, C.; Gonçalves, S.; Santos, N. C. Advances in Lipid and Metal Nanoparticles for Antimicrobial Peptide Delivery. Pharmaceutics 2019, 11 (11), 588,  DOI: 10.3390/pharmaceutics11110588
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    Antimicrobial peptides (AMPs) have been described as excellent candidates to overcome antibiotic resistance. Frequently, AMPs exhibit a wide therapeutic window, with low cytotoxicity and broad-spectrum antimicrobial activity against a variety of pathogens. In addn., some AMPs are also able to modulate the immune response, decreasing potential harmful effects such as sepsis. Despite these benefits, only a few formulations have successfully reached clinics. A common flaw in the druggability of AMPs is their poor pharmacokinetics, common to several peptide drugs, as they may be degraded by a myriad of proteases inside the organism. The combination of AMPs with carrier nanoparticles to improve delivery may enhance their half-life, decreasing the dosage and thus, reducing prodn. costs and eventual toxicity. Here, we present the most recent advances in lipid and metal nanodevices for AMP delivery, with a special focus on metal nanoparticles and liposome formulations.
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    Eftekhari, A.; Arjmand, A.; Asheghvatan, A.; Švajdlenková, H.; Šauša, O.; Abiyev, H.; Ahmadian, E.; Smutok, O.; Khalilov, R.; Kavetskyy, T.; Cucchiarini, M. The Potential Application of Magnetic Nanoparticles for Liver Fibrosis Theranostics. Front. Chem. 2021, 9, 674786  DOI: 10.3389/fchem.2021.674786
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    A review. Antimicrobial resistance is a leading cause of mortality worldwide. Without newly approved antibiotics and antifungals being brought to the market, resistance is being developed to the ones currently available to clinicians. The reason is the applied evolutionary pressure to bacterial and fungal species due to the wide overuse of common antibiotics and antifungals in clin. practice and agriculture. Biofilms harbor antimicrobial-resistant subpopulations, which make their antimicrobial treatment even more challenging. Nanoparticle-based technologies have recently been shown to successfully overcome antimicrobial resistance in both planktonic and biofilms phenotypes. This results from the combination of novel nanomaterial research and classic antimicrobial therapies which promise to deliver a whole new generation of high-performance active nanocarrier systems. This review discusses the latest developments of promising nanotechnologies with applications against resistant pathogens and evaluates their potential and feasibility for use in novel antimicrobial therapies.
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    A review. The escalation of multidrug-resistant pathogens has created a dire need to develop novel ways of addressing this global therapeutic challenge. Because of their antimicrobial activities, combination of antimicrobial peptides (AMPs) and nanoparticles is a promising tool to kill drug resistant pathogens. In recent years, several studies using AMP-nanoparticle conjugates, esp. metallic nanoparticles, as potential antimicrobial agents against drug resistant pathogens have been published. Amongst these, antimicrobial peptide conjugated gold nanoparticles (AMP-AuNPs) are particularly attractive because of the non-toxic nature of gold and the possibility of fine tuning the AMP-NP conjugation chem. The following review discusses recent developments in the synthesis and antimicrobial activity studies of AMP-AuNPs. Classification of AMPs, their mechanisms of action, methods used for functionalizing AuNPs with AMPs and the antimicrobial activities of the conjugates are discussed.
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    Nolles, A.; Hooiveld, E.; Westphal, A. H.; van Berkel, W. J. H.; Kleijn, J. M.; Borst, J. W. FRET Reveals the Formation and Exchange Dynamics of Protein-Containing Complex Coacervate Core Micelles. Langmuir 2018, 34 (40), 12083– 12092,  DOI: 10.1021/acs.langmuir.8b01272
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    The encapsulation of proteins into complex coacervate core micelles (C3Ms) is of potential interest for a wide range of applications. To address the stability and dynamic properties of these polyelectrolyte complexes, combinations of cyan, yellow, and blue fluorescent proteins were encapsulated with cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Forster resonance energy transfer (FRET) allowed us to det. the kinetics of C3M formation and of protein exchange between C3Ms. Both processes follow first-order kinetics with relaxation times of ±100 s at low ionic strength (I = 2.5 mM). Stability studies revealed that 50% of FRET was lost at I = 20 mM, pointing to the disintegration of the C3Ms. On the basis of exptl. and theor. considerations, the authors propose that C3Ms relax to their final state by assocn. and dissocn. of near-neutral sol. protein-polymer complexes. To obtain protein-contg. C3Ms suitable for applications, it is necessary to improve the rigidity and salt stability of these complexes.
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    In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cytofluorometric and bioluminescent methods are not widely used because they require specified equipment and further evaluation for reproducibility and standardization, even if they can provide rapid results of the antimicrobial agent's effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limitations are reported.
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  1. Thomas D. Vogelaar, Henrik Torjusen, Theyencheri Narayanan, Reidar Lund. The Kinetic Pathways for the Formation of Complex Coacervate Micelles of Antimicrobial Peptides and Block Copolymers. Macromolecules 2025, 58 (1) , 158-168. https://doi.org/10.1021/acs.macromol.4c02077
  2. Thomas D. Vogelaar, Szymon M. Szostak, Reidar Lund. Coacervation in Slow Motion: Kinetics of Complex Micelle Formation Induced by the Hydrolysis of an Antibiotic Prodrug. Molecular Pharmaceutics 2024, 21 (8) , 4157-4168. https://doi.org/10.1021/acs.molpharmaceut.4c00579
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  4. Kaifang Yi, Wenjing Leng, Xiaoyuan Ma, Peiyi Liu, Zibo Li, Dandan He, Li Yuan, Gongzheng Hu, Yajun Zhai. Self-assembly pH-sensitive polyelectrolyte complex co-delivers niclosamide and colistin to overcome colistin resistance in bacterial infections. International Journal of Biological Macromolecules 2025, 306 , 141415. https://doi.org/10.1016/j.ijbiomac.2025.141415
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  • Abstract

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    Figure 1

    Figure 1. ζ-Potential measurements (filled black circles) and DLS size measurements right after mixing (open white diamonds), including error bars, combined with the stability of complex coacervates mixing PEO45-b-PMAA41 (P1) and colistin, at a range of charge fractions (f+) of 0.09 ≤ f+ ≤ 0.98 at a formation concentration of 5.0 mg/mL. The stability, measured by follow-up measurements over time with DLS (Figure S1), is indicated by the color coding of the background. 0–1-day(s) stability (red), 2–7-day stability (orange), 8–28-day stability (yellow), and > 28-day stability (green) are separated in the background. To improve visibility by creating sharp color borders a color spacing of 0.02–0.05 was taken horizontally.

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    Figure 2

    Figure 2. SAXS profiles of PEO45-b-PMAA41 (orange squares) at 1.6 mg/mL colistin sulfate (blue circles) at 3.4 mg/mL, and colistin complex coacervates at f+ = 0.50 (green triangles (pointing down)) at 5.0 mg/mL total concentration. The lines depict the results of fit analysis using the Debye model for PEO45-b-PMAA41 and colistin sulfate and the fuzzy-surface complex coacervate model for colistin complex coacervates (A). Scattering profiles are depicted of complex coacervates at f+ = 0.17 (orange squares), f+ = 0.33 (blue circles), f+ = 0.50 (green triangles (down)), f+ = 0.67 (gray diamonds), and f+ = 0.83 (pink triangles (up)) at a total concentration of 5.0 mg/mL including fitted curves (B). Scattering patterns in B are fitted using the fuzzy-surface complex coacervate model for the different charge fractions of complex coacervates.

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    Figure 3

    Figure 3. Graphical illustration of the effect of charge fractions on the formation of complex coacervates. At f+ < 0.09 (I), no complex coacervates are formed, followed by the SCP-phase (II), in which negatively charged small structures are present until the CEAC point between f+ of 0.33 and 0.44. After the CEAC, C3Ms are formed (III), with a decreasing fraction forming micelles the further you go up the scale. From f+ = 0.98 (IV), again, no complex coacervates are formed. Illustrations are not scaled.

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    Figure 4

    Figure 4. Disk diffusion assay (DDA) of colistin and colistin-C3Ms with 3.0 μg of colistin in the E. coli agar medium (A). The inhibition zones were measured, averaged, and compared. MIC50 determination of colistin (orange diamonds) and colistin-C3Ms (blue circles) on E. coli using the agar dilution method (B). The turbidity was plotted against the concentration, which was plotted on a logarithmic scale to improve visibility. The colistin (black line) and colistin-C3Ms curves (black dashed line) were fitted with a sigmoid function from which the MIC50 values were determined, which are plotted in the figure (orange dashed line for colistin and blue dotted line for colistin-C3Ms).

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    Figure 5

    Figure 5. Illustrations of hypothesized enzymatic breakdown comparisons (A, enzyme attacks colistin and then undergoes complexation, and (B) enzyme attacks after complexation) and SAXS patterns of enzymatic breakdown of colistin (3.4 mg/mL) versus colistin-C3Ms (total concentration of 5.0 mg/mL) in the presence of proteinase K (C) and subtilisin (D). Theoretical SAXS patterns were calculated based on either the added scattering from C3Ms and enzymes (0% enzyme breakdown, orange squares) or colistin, polymer, and enzyme separately summed up (100% breakdown, blue circles) (C, D). The effect of enzymatic degradation after 24 h at 37 °C was measured by either adding enzyme to colistin, followed by complexation with polymer (illustrated in A, and the graphs in C and D, indicated by green triangles) or addition of enzyme to C3Ms (illustrated in B, and the graphs in C and D, indicated by gray diamonds). The SAXS patterns were fitted using the fuzzy-surface complex coacervate model for complex coacervates.

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    Figure 6

    Figure 6. SAXS patterns of C3Ms (blue circles) and C3Ms with added HSA at a ratio of 1:10 HSA:colistin (green triangles) and 1:20 HSA:colistin (gray diamonds) are presented in (A). To better visualize the effect of HSA addition, we show HSA by themselves (orange squares) and C3Ms by themselves (blue circles) in (B), as well as the scattering patterns in which HSA is combined with C3Ms separated by multiplication of the I(Q) with a factor of 10 for 1:20 HSA:colistin (green triangles) and 100 for 1:10 HSA:colistin (gray diamonds) to increase the visibility of structural changes. The SAXS patterns were fitted using the fuzzy-surface complex coacervate model for complex coacervates with added prolate/oblate ellipsoidal scattering of HSA if present.

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    Figure 7

    Figure 7. Cell viability of human embryonic kidney 293 (HEK) cells (A), mesenchymal stem cells (MSCs) (B), human gingival keratinocytes (HGKs) (C), or human umbilical cord endothelial cells (HUVECs) (D) treated with colistin, PEO-b-PMAA, or colistin-C3Ms. Lactate dehydrogenase (LDH) and caspase-3 activity were measured in the corresponding cell culture medium, expressed as fold change of control at 24, 48, and 72 h. Data are presented as the mean effect of 3 parallel cellular experiments for each stimulation at each time point. Significantly different from untreated control cells at each time point at *p < 0.05, **p < 0.01, and ***p < 0.001.

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      Falagas, Matthew E.; Kasiakou, Sofia K.
      Clinical Infectious Diseases (2005), 40 (9), 1333-1341CODEN: CIDIEL; ISSN:1058-4838. (University of Chicago Press)
      A review. The emergence of multidrug-resistant gram-neg. bacteria and the lack of new antibiotics to combat them have led to the revival of polymyxins, an old class of cationic, cyclic polypeptide antibiotics. Polymyxin B and polymyxin E (colistin) are the 2 polymyxins used in clin. practice. Most of the reintroduction of polymyxins during the last few years is related to colistin. The polymyxins are active against selected gram-neg. bacteria, including Acinetobacter species, Pseudomonas aeruginosa, Klebsiella species, and Enterobacter species. These drugs have been used extensively worldwide for decades for local use. However, parenteral use of these drugs was abandoned ∼20 years ago in most countries, except for treatment of patients with cystic fibrosis, because of reports of common and serious nephrotoxicity and neurotoxicity. Recent studies of patients who received i.v. polymyxins for the treatment of serious P. aeruginosa and Acinetobacter baumannii infections of various types, including pneumonia, bacteremia, and urinary tract infections, have led to the conclusion that these antibiotics have acceptable effectiveness and considerably less toxicity than was reported in old studies.
    9. 9
      Trimble, M. J.; Mlynárčik, P.; Kolář, M.; Hancock, R. E. W. Polymyxin: Alternative Mechanisms of Action and Resistance. Cold Spring Harbor Perspect. Med. 2016, 6 (10), a025288  DOI: 10.1101/cshperspect.a025288
      9
      Polymyxin: alternative mechanisms of action and resistance
      Trimble, Michael J.; Mlynarcik, Patrik; Kolar, Milan; Hancock, Robert E. W.
      Cold Spring Harbor Perspectives in Medicine (2016), 6 (10), a025288/1-a025288/23CODEN: CSHPFV; ISSN:2157-1422. (Cold Spring Harbor Laboratory Press)
      Antibiotic resistance among pathogenic bacteria is an ever-increasing issue worldwide. Unfortunately, very little has been achieved in the pharmaceutical industry to combat this problem. This has led researchers and the medical field to revisit past drugs thatwere deemed too toxic for clin. use. In particular, the cyclic cationic peptides polymyxin B and colistin, which are specific for Gram-neg. bacteria, have been used as "last resort" antimicrobials. Before the 1980s, these drugswere known for their renal and neural toxicities; however, new clin. practices and possibly improved manufg. have made them safer to use. Previously suggested to primarily attack the membranes of Gram-neg. bacteria and to not easily select for resistant mutants, recent research exploring resistance and mechanisms of action has provided new perspectives. This review focuses primarily on the proposed alternative mechanisms of action, known resistance mechanisms, and how these support the alternative mechanisms of action.
    10. 10
      Dijkmans, A. C.; Wilms, E. B.; Kamerling, I. M. C.; Birkhoff, W.; Ortiz-Zacarías, N. V.; van Nieuwkoop, C.; Verbrugh, H. A.; Touw, D. J. Colistin: Revival of an Old Polymyxin Antibiotic. Ther. Drug Monit. 2015, 37 (4), 419,  DOI: 10.1097/FTD.0000000000000172
      There is no corresponding record for this reference.
    11. 11
      Liao, F.-H.; Wu, T.-H.; Yao, C.-N.; Kuo, S.-C.; Su, C.-J.; Jeng, U.-S.; Lin, S.-Y. A Supramolecular Trap to Increase the Antibacterial Activity of Colistin. Angew. Chem., Int. Ed. 2020, 59 (4), 1430– 1434,  DOI: 10.1002/anie.201912137
      There is no corresponding record for this reference.
    12. 12
      Ordooei Javan, A.; Shokouhi, S.; Sahraei, Z. A Review on Colistin Nephrotoxicity. Eur. J. Clin. Pharmacol. 2015, 71 (7), 801– 810,  DOI: 10.1007/s00228-015-1865-4
      12
      A review on colistin nephrotoxicity
      Ordooei Javan, Atefeh; Shokouhi, Shervin; Sahraei, Zahra
      European Journal of Clinical Pharmacology (2015), 71 (7), 801-810CODEN: EJCPAS; ISSN:0031-6970. (Springer)
      A review. Purpose: Colistin is an antibiotic that was introduced many years ago and was withdrawn because of its nephrotoxicity. Nowadays, reemergence of this antibiotic for multi-drug resistant Gram-neg. infections, and a new high dosing regimen recommendation increases concern about its nephrotoxicity. This review attempts to give a view on colistin nephrotoxicity, its prevalence esp. in high doses, the mechanism of injury, risk factors, and prevention of this kidney injury. Method: The data collection was done in PubMed, Scopus and Cochrane databases. The keywords for search terms were "colistin", "nephrotoxicity", "toxicity", "renal failure", "high dose", and "risk factor". Randomized clin. trials and prospective or retrospective observational animal and human studies were included. In all, 60 articles have been reviewed. Result and conclusion: Colistin is a nephrotoxic antibiotic; a worldwide increase in nosocomial infections has led to an increase in its usage. Nephrotoxicity is the concerning adverse effect of this drug. The mechanism of nephrotoxicity is via an increase in tubular epithelial cell membrane permeability, which results in cation, anion and water influx leading to cell swelling and cell lysis. There are also some oxidative and inflammatory pathways that seem to be involved in colistin nephrotoxicity. Risk factors of colistin nephrotoxicity can be categorized as dose and duration of colistin therapy, co-administration of other nephrotoxic drugs, and patient-related factors such as age, sex, hypoalbuminemia, hyperbilirubinemia, underlying disease and severity of patient illness.
    13. 13
      Wallace, S. J.; Li, J.; Nation, R. L.; Boyd, B. J. Drug Release from Nanomedicines: Selection of Appropriate Encapsulation and Release Methodology. Drug Delivery Transl. Res. 2012, 2 (4), 284– 292,  DOI: 10.1007/s13346-012-0064-4
      13
      Drug release from nanomedicines: selection of appropriate encapsulation and release methodology
      Wallace, Stephanie J.; Li, Jian; Nation, Roger L.; Boyd, Ben J.
      Drug Delivery and Translational Research (2012), 2 (4), 284-292CODEN: DDTRCY; ISSN:2190-3948. (Springer)
      The characterization of encapsulation efficiency and in vitro drug release from nanoparticle-based formulations often requires the sepn. of nanoparticles from unencapsulated drug. Inefficient sepn. of nanoparticles from the medium in which they are dispersed can lead to inaccurate ests. of encapsulation efficiency and drug release. This study establishes dynamic light scattering as a simple method for substantiation of the effectiveness of the sepn. process. Colistin-loaded liposomes, as an exemplar nanosized delivery particle, were dild. to construct a calibration curve relating the amt. of light scattering to liposome concn. Dynamic light scattering revealed that, in the case of ultracentrifugation and centrifugal ultrafiltration, approx. 2.9% of the total liposomes remained in supernatants or filtrates, resp. In comparison, filtrates obtained using pressure ultrafiltration contained less than 0.002% of the total liposomes from the formulation. Subsequent release studies using dialysis misleadingly implied a slow release of colistin over >48 h. In contrast, pressure ultrafiltration revealed immediate equilibration to the equil. distribution of colistin between the liposome and aq. phases upon diln. Pressure ultrafiltration is therefore recommended as the optimal method of choice for studying release kinetics of drug from nanomedicine carriers.
    14. 14
      Nation, R. L.; Li, J. Colistin in the 21st Century. Curr. Opin. Infect. Dis. 2009, 22 (6), 535,  DOI: 10.1097/QCO.0b013e328332e672
      14
      Colistin in the 21st century
      Nation, Roger L.; Li, Jian
      Current Opinion in Infectious Diseases (2009), 22 (6), 535-543CODEN: COIDE5; ISSN:0951-7375. (Lippincott Williams & Wilkins)
      Purpose of review: Colistin is a 50-yr-old antibiotic that is being used increasingly as a last-line' therapy to treat infections caused by multidrug-resistant Gram-neg. bacteria, when essentially no other options are available. Despite its age, or because of its age, there has been a dearth of knowledge on its pharmacol. and microbiol. properties. This review focuses on recent studies aimed at optimizing the clin. use of this old antibiotic. Recent findings: A no. of factors, including the diversity in the pharmaceutical products available, have hindered the optimal use of colistin. Recent advances in understanding of the pharmacokinetics and pharmacodynamics of colistin, and the emerging knowledge on the relationship between the pharmacokinetics and pharmacodynamics, provide a solid base for optimization of dosage regimens. The potential for nephrotoxicity has been a lingering concern, but recent studies provide useful new information on the incidence, severity and reversibility of this adverse effect. Recent approaches to the use of other antibiotics in combination with colistin hold promise for increased antibacterial efficacy with less potential for emergence of resistance. Summary: Because few, if any, new antibiotics with activity against multidrug-resistant Gram-neg. bacteria will be available within the next several years, it is essential that colistin is used in ways that maximize its antibacterial efficacy and minimize toxicity and development of resistance. Recent developments have improved use of colistin in the 21st century.
    15. 15
      Dubashynskaya, N. V.; Bokatyi, A. N.; Gasilova, E. R.; Dobrodumov, A. V.; Dubrovskii, Y. A.; Knyazeva, E. S.; Nashchekina, Y. A.; Demyanova, E. V.; Skorik, Y. A. Hyaluronan-Colistin Conjugates: Synthesis, Characterization, and Prospects for Medical Applications. Int. J. Biol. Macromol. 2022, 215, 243– 252,  DOI: 10.1016/j.ijbiomac.2022.06.080
      There is no corresponding record for this reference.
    16. 16
      Liao, W.-C.; Wang, C.-H.; Sun, T.-H.; Su, Y.-C.; Chen, C.-H.; Chang, W.-T.; Chen, P.-L.; Shiue, Y.-L. The Antimicrobial Effects of Colistin Encapsulated in Chelating Complex Micelles for the Treatment of Multi-Drug-Resistant Gram-Negative Bacteria: A Pharmacokinetic Study. Antibiotics 2023, 12 (5), 836,  DOI: 10.3390/antibiotics12050836
      There is no corresponding record for this reference.
    17. 17
      Landa, G.; Alejo, T.; Sauzet, T.; Laroche, J.; Sebastian, V.; Tewes, F.; Arruebo, M. Colistin-Loaded Aerosolizable Particles for the Treatment of Bacterial Respiratory Infections. Int. J. Pharm. 2023, 635, 122732  DOI: 10.1016/j.ijpharm.2023.122732
      There is no corresponding record for this reference.
    18. 18
      Vairo, C.; Villar Vidal, M.; Maria Hernandez, R.; Igartua, M.; Villullas, S. Colistin- and Amikacin-Loaded Lipid-Based Drug Delivery Systems for Resistant Gram-Negative Lung and Wound Bacterial Infections. Int. J. Pharm. 2023, 635, 122739  DOI: 10.1016/j.ijpharm.2023.122739
      There is no corresponding record for this reference.
    19. 19
      Sans-Serramitjana, E.; Jorba, M.; Pedraz, J. L.; Vinuesa, T.; Viñas, M. Determination of the Spatiotemporal Dependence of Pseudomonas Aeruginosa Biofilm Viability after Treatment with NLC-Colistin. Int. J. Nanomed. 2017, 12, 4409– 4413,  DOI: 10.2147/IJN.S138763
      There is no corresponding record for this reference.
    20. 20
      Shah, S. R.; Henslee, A. M.; Spicer, P. P.; Yokota, S.; Petrichenko, S.; Allahabadi, S.; Bennett, G. N.; Wong, M. E.; Kasper, F. K.; Mikos, A. G. Effects of Antibiotic Physicochemical Properties on Their Release Kinetics from Biodegradable Polymer Microparticles. Pharm. Res. 2014, 31 (12), 3379– 3389,  DOI: 10.1007/s11095-014-1427-y
      There is no corresponding record for this reference.
    21. 21
      Liu, Y.-H.; Kuo, S.-C.; Yao, B.-Y.; Fang, Z.-S.; Lee, Y.-T.; Chang, Y.-C.; Chen, T.-L.; Hu, C.-M. J. Colistin Nanoparticle Assembly by Coacervate Complexation with Polyanionic Peptides for Treating Drug-Resistant Gram-Negative Bacteria. Acta Biomater. 2018, 82, 133– 142,  DOI: 10.1016/j.actbio.2018.10.013
      21
      Colistin nanoparticle assembly by coacervate complexation with polyanionic peptides for treating drug-resistant gram-negative bacteria
      Liu, Yu-Han; Kuo, Shu-Chen; Yao, Bing-Yu; Fang, Zih-Syun; Lee, Yi-Tzu; Chang, Yuan-Chih; Chen, Te-Li; Hu, Che-Ming Jack
      Acta Biomaterialia (2018), 82 (), 133-142CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
      Amidst the ever-rising threat of antibiotics resistance, colistin, a decade-old antibiotic with lingering toxicity concern, is increasingly prescribed to treat many drug-resistant, gram-neg. bacteria. With the aim of improving the safety profile while preserving the antimicrobial activity of colistin, a nanoformulation is herein developed through coacervate complexation with polyanionic peptides. Upon controlled mixing of cationic colistin with polyglutamic acids, formation of liq. coacervates was demonstrated. Subsequent stabilization by DSPE-PEG and homogenization through micro-fluidization of the liq. coacervates yielded nanoparticles 8 nm in diam. In vitro assessment showed that the colistin antimicrobial activity against multiple drug-resistant bacterial strains was retained and, in some cases, enhanced following the nanoparticle assembly. In vivo administration in mice demonstrated improved safety of the colistin nanoparticle, which has a maximal tolerated dose of 12.5 mg/kg compared to 10 mg/kg of free colistin. Upon administration over a 7-day period, colistin nanoparticles also exhibited reduced hepatotoxicity as compared to free colistin. In mouse models of Klebsiella pneumoniae bacteremia and Acinetobacter baumannii pneumonia, treatment with colistin nanoparticles showed equiv. efficacy to free colistin. These results demonstrate coacervation-induced nanoparticle assembly as a promising approach towards improving colistin treatments against bacterial infections. Improving the safety of colistin while retaining its antimicrobial activity has been a highly sought-after objective toward enhancing antibacterial treatments. Herein, we demonstrate formation of stabilized colistin nanocomplexes in the presence of anionic polypeptides and DSPE-PEG stabilizer. The nanocomplexes retain colistins antimicrobial activity while demonstrating improved safety upon in vivo administration. The supramol. nanoparticle assembly of colistin presents a unique approach towards designing antimicrobial nanoparticles.
    22. 22
      Ezike, T. C.; Okpala, U. S.; Onoja, U. L.; Nwike, C. P.; Ezeako, E. C.; Okpara, O. J.; Okoroafor, C. C.; Eze, S. C.; Kalu, O. L.; Odoh, E. C.; Nwadike, U. G.; Ogbodo, J. O.; Umeh, B. U.; Ossai, E. C.; Nwanguma, B. C. Advances in Drug Delivery Systems, Challenges and Future Directions. Heliyon 2023, 9 (6), e17488  DOI: 10.1016/j.heliyon.2023.e17488
      There is no corresponding record for this reference.
    23. 23
      Ban, E.; Kim, A. Coacervates: Recent Developments as Nanostructure Delivery Platforms for Therapeutic Biomolecules. Int. J. Pharm. 2022, 624, 122058  DOI: 10.1016/j.ijpharm.2022.122058
      23
      Coacervates: Recent developments as nanostructure delivery platforms for therapeutic biomolecules
      Ban, Eunmi; Kim, Aeri
      International Journal of Pharmaceutics (Amsterdam, Netherlands) (2022), 624 (), 122058CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
      A review. Coacervation is a liq.-liq. phase sepn. that can occur in solns. of macromols. through self-assembly or electrostatic interactions. Recently, coacervates composed of biocompatible macromols. have been actively investigated as nanostructure platforms to encapsulate and deliver biomols. such as proteins, RNAs, and DNAs. One particular advantage of coacervates is that they are derived from aq. solns., unlike other nanoparticle delivery systems that often require org. solvents. In addn., coacervates achieve high loading while maintaining the viability of the cargo material. Here, we review recent developments in the applications of coacervates and their limitations in the delivery of therapeutic biomols. Important factors for coacervation include mol. structures of the polyelectrolytes, mixing ratio, the concn. of polyelectrolytes, and reaction conditions such as ionic strength, pH, and temp. Various compns. of coacervates have been shown to deliver biomols. in vitro and in vivo with encouraging activities. However, major hurdles remain for the systemic route of administration other than topical or local delivery. The scale-up of manufg. methods suitable for preclin. and clin. evaluations remains to be addressed. We conclude with a few research directions to overcome current challenges, which may lead to successful translation into the clinic.
    24. 24
      Blocher, W. C.; Perry, S. L. Complex Coacervate-Based Materials for Biomedicine. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2017, 9 (4), e1442  DOI: 10.1002/wnan.1442
      There is no corresponding record for this reference.
    25. 25
      Borro, B. C.; Malmsten, M. Complexation between Antimicrobial Peptides and Polyelectrolytes. Adv. Colloid Interface Sci. 2019, 270, 251– 260,  DOI: 10.1016/j.cis.2019.07.001
      25
      Complexation between antimicrobial peptides and polyelectrolytes
      Borro, Bruno C.; Malmsten, Martin
      Advances in Colloid and Interface Science (2019), 270 (), 251-260CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
      As a result of increasing bacterial resistance against antibiotics, we are facing an emerging health crisis, in which 'simple' infections may no longer be treatable. One class of mols. attracting interest in this context is antimicrobial peptides (AMPs), and considerable research efforts have been directed to identifying selective and potent AMPs. In addn., since in vivo delivery of AMPs is challenging, there is an emerging awareness that successful development of AMP therapeutics can be facilitated by careful design of AMPs delivery systems. In the present overview, we discuss polyelectrolyte complexation as a strategy to deliver AMPs. In doing so, key factors for AMP-polyelectrolyte complexation are illustrated for AMP-polyelectrolyte nanoparticle formation, as well as for AMP incorporation in polyelectrolyte microgels and multilayer structures, and consequences of these for functional performance exemplified.
    26. 26
      Jenssen, H.; Aspmo, S. I. Serum Stability of Peptides. In Peptide-Based Drug Design; Otvos, L., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2008; pp 177– 186.  DOI: 10.1007/978-1-59745-419-3_10 .
      There is no corresponding record for this reference.
    27. 27
      Voets, I. K.; de Keizer, A.; Cohen Stuart, M. A. Complex Coacervate Core Micelles. Adv. Colloid Interface Sci. 2009, 147–148, 300– 318,  DOI: 10.1016/j.cis.2008.09.012
      27
      Complex coacervate core micelles
      Voets, Ilja K.; de Keizer, Arie; Cohen Stuart, Martien A.
      Advances in Colloid and Interface Science (2009), 147-148 (), 300-318CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
      A review. In this review we present an overview of the literature on the co-assembly of neutral-ionic block, graft, and random copolymers with oppositely charged species in aq. soln. Oppositely charged species include synthetic (co)polymers of various architectures, biopolymers - such as proteins, enzymes and DNA - multivalent ions, metallic nanoparticles, low mol. wt. surfactants, polyelectrolyte block copolymer micelles, metallo-supramol. polymers, equil. polymers, etc. The resultant structures are termed complex coacervate core/polyion complex/block ionomer complex/interpolyelectrolyte complex micelles (or vesicles); i.e., in short C3Ms (or C3Vs) and PIC, BIC or IPEC micelles (and vesicles). Formation, structure, dynamics, properties, and function will be discussed. We focus on exptl. work; theory and modeling will not be discussed. Recent developments in applications and micelles with heterogeneous coronas are emphasized.
    28. 28
      Magana, J. R.; Sproncken, C. C. M.; Voets, I. K. On Complex Coacervate Core Micelles: Structure-Function Perspectives. Polymers 2020, 12 (9), 1953,  DOI: 10.3390/polym12091953
      28
      On complex coacervate core micelles: structure-function perspectives
      Magana, Jose Rodrigo; Sproncken, Christian C. M.; Voets, Ilja K.
      Polymers (Basel, Switzerland) (2020), 12 (9), 1953CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
      A review. The co-assembly of ionic-neutral block copolymers with oppositely charged species produces nanometric colloidal complexes, known, among other names, as complex coacervates core micelles (C3Ms). C3Ms are of widespread interest in nanomedicine for controlled delivery and release, while research activity into other application areas, such as gelation, catalysis, nanoparticle synthesis, and sensing, is increasing. In this review, we discuss recent studies on the functional roles that C3Ms can fulfil in these and other fields, focusing on emerging structure-function relations and remaining knowledge gaps.
    29. 29
      Marciel, A. B.; Srivastava, S.; Ting, J. M.; Tirrell, M. V. SAXS Methods for Investigating Macromolecular and Self-Assembled Polyelectrolyte Complexes. In Methods in Enzymology; Keating, C. D., Ed.; Liquid-Liquid Phase Coexistence and Membraneless Organelles; Academic Press, 2021; Chapter 8, Vol. 646; pp 223– 259.  DOI: 10.1016/bs.mie.2020.09.013 .
      There is no corresponding record for this reference.
    30. 30
      Rumyantsev, A. M.; Jackson, N. E.; de Pablo, J. J. Polyelectrolyte Complex Coacervates: Recent Developments and New Frontiers. Annu. Rev. Condens. Matter Phys. 2021, 12 (1), 155– 176,  DOI: 10.1146/annurev-conmatphys-042020-113457
      There is no corresponding record for this reference.
    31. 31
      Uebbing, L.; Ziller, A.; Siewert, C.; Schroer, M. A.; Blanchet, C. E.; Svergun, D. I.; Ramishetti, S.; Peer, D.; Sahin, U.; Haas, H.; Langguth, P. Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-Ray Scattering. Langmuir 2020, 36 (44), 13331– 13341,  DOI: 10.1021/acs.langmuir.0c02446
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      Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering
      Uebbing, Lukas; Ziller, Antje; Siewert, Christian; Schroer, Martin A.; Blanchet, Clement E.; Svergun, Dmitri I.; Ramishetti, Srinivas; Peer, Dan; Sahin, Ugur; Haas, Heinrich; Langguth, Peter
      Langmuir (2020), 36 (44), 13331-13341CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      MRNA (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clin. practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clin. efficacy. In this study, pH-induced changes in internal mol. organization and overall physicochem. characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were detd. for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate detn. of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ detn. of the structural response of the lipoplex systems to changes of the environmental pH similar to that obsd. for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.
    32. 32
      Grabbe, S.; Haas, H.; Diken, M.; Kranz, L. M.; Langguth, P.; Sahin, U. Translating Nanoparticulate-Personalized Cancer Vaccines into Clinical Applications: Case Study with RNA-Lipoplexes for the Treatment of Melanoma. Nanomedicine 2016, 11 (20), 2723– 2734,  DOI: 10.2217/nnm-2016-0275
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      Translating nanoparticulate-personalized cancer vaccines into clinical applications: case study with RNA-lipoplexes for the treatment of melanoma
      Grabbe, Stephan; Haas, Heinrich; Diken, Mustafa; Kranz, Lena M.; Langguth, Peter; Sahin, Ugur
      Nanomedicine (London, United Kingdom) (2016), 11 (20), 2723-2734CODEN: NLUKAC; ISSN:1743-5889. (Future Medicine Ltd.)
      The development of nucleic acid based vaccines against cancer has gained considerable momentum through the advancement of modern sequencing technologies and on novel RNA-based synthetic drug formats, which can be readily adapted following identification of every patient's tumor-specific mutations. Furthermore, affordable and individual 'on demand' prodn. of molecularly optimized vaccines should allow their application in large groups of patients. This has resulted in the therapeutic concept of an active personalized cancer vaccine, which has been brought into clin. testing. Successful trials have been performed by intranodal administration of sterile isotonic solns. of synthetic RNA vaccines. The second generation of RNA vaccines which is currently being developed encompasses i.v. injectable RNA nanoparticle formulations (lipoplexes), made up from lipid excipients, denoted RNA(LIP). A first product that has made its way from bench to bedside is a therapeutic vaccine for i.v. administration based on a fixed set of four RNA lipoplex drug products, each encoding for one shared tumor antigen (Lipoplex Melanoma RNA Immunotherapy, 'Lipo-MERIT'). This article describes the steps for translating these novel RNA nanomedicines into clin. trials. Graphical Abstr. :.
    33. 33
      Nogueira, S. S.; Schlegel, A.; Maxeiner, K.; Weber, B.; Barz, M.; Schroer, M. A.; Blanchet, C. E.; Svergun, D. I.; Ramishetti, S.; Peer, D.; Langguth, P.; Sahin, U.; Haas, H. Polysarcosine-Functionalized Lipid Nanoparticles for Therapeutic mRNA Delivery. ACS Appl. Nano Mater. 2020, 3 (11), 10634– 10645,  DOI: 10.1021/acsanm.0c01834
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      Polysarcosine-Functionalized Lipid Nanoparticles for Therapeutic mRNA Delivery
      Nogueira, Sara S.; Schlegel, Anne; Maxeiner, Konrad; Weber, Benjamin; Barz, Matthias; Schroer, Martin A.; Blanchet, Clement E.; Svergun, Dmitri I.; Ramishetti, Srinivas; Peer, Dan; Langguth, Peter; Sahin, Ugur; Haas, Heinrich
      ACS Applied Nano Materials (2020), 3 (11), 10634-10645CODEN: AANMF6; ISSN:2574-0970. (American Chemical Society)
      Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for therapeutic application of mRNA were assembled using pSarcosinylated lipids as a tool for particle engineering. Using pSar lipids with different polymeric chain lengths and molar fractions enabled the control of the physicochem. characteristics of the LNPs, such as particle size, morphol., and internal structure. In combination with a suited ionizable lipid, LNPs were assembled, which displayed high RNA transfection potency with an improved safety profile after i.v. injection. Notably, a higher protein secretion with a reduced immunostimulatory response was obsd. when compared to systems based on polyethylene glycol (PEG) lipids. PSarcosinylated nanocarriers showed a lower proinflammatory cytokine secretion and reduced complement activation compared to PEGylated LNPs. In summary, the described pSar-based LNPs enable safe and potent delivery of mRNA, thus signifying an excellent basis for the development of PEG-free RNA therapeutics.
    34. 34
      Bos, I.; Timmerman, M.; Sprakel, J. FRET-Based Determination of the Exchange Dynamics of Complex Coacervate Core Micelles. Macromolecules 2021, 54 (1), 398– 411,  DOI: 10.1021/acs.macromol.0c02387
      34
      FRET-Based Determination of the Exchange Dynamics of Complex Coacervate Core Micelles
      Bos, Inge; Timmerman, Marga; Sprakel, Joris
      Macromolecules (Washington, DC, United States) (2021), 54 (1), 398-411CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Complex coacervate core micelles (C3Ms) are nanoscopic structures formed by charge interactions between oppositely charged macroions and used to encapsulate a wide variety of charged (bio)mols. In most cases, C3Ms are in a dynamic equil. with their surroundings. Understanding the dynamics of mol. exchange reactions is essential as this dets. the rate at which their cargo is exposed to the environment. Here, we study the mol. exchange in C3Ms by making use of Fluorescence resonance energy transfer (FRET) and derive an anal. model to relate the exptl. obsd. increase in FRET efficiency to the underlying macromol. exchange rates. We show that equilibrated C3Ms have a broad distribution of exchange rates. The overall exchange rate can be strongly increased by increasing the salt concn. In contrast, changing the unlabeled homopolymer length does not affect the exchange of the labeled homopolymers and an increase in the micelle concn. only affects the FRET increase rate at low micelle concns. Together, these results suggest that the exchange of these equilibrated C3Ms occurs mainly by expulsion and insertion, where the rate-limiting step is the breaking of ionic bonds to expel the chains from the core. These are important insights to further improve the encapsulation efficiency of C3Ms.
    35. 35
      Amann, M.; Diget, J. S.; Lyngsø, J.; Pedersen, J. S.; Narayanan, T.; Lund, R. Kinetic Pathways for Polyelectrolyte Coacervate Micelle Formation Revealed by Time-Resolved Synchrotron SAXS. Macromolecules 2019, 52 (21), 8227– 8237,  DOI: 10.1021/acs.macromol.9b01072
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      Kinetic Pathways for Polyelectrolyte Coacervate Micelle Formation Revealed by Time-Resolved Synchrotron SAXS
      Amann, Matthias; Diget, Jakob Stensgaard; Lyngsoe, Jeppe; Pedersen, Jan Skov; Narayanan, Theyencheri; Lund, Reidar
      Macromolecules (Washington, DC, United States) (2019), 52 (21), 8227-8237CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      The kinetic pathways for coacervation and micelle formation are still not fully understood. Driven by electrostatic interactions and entropically driven counterion release, complexation of oppositely charged macromols. leads to the formation of micellar nanostructures. Here we study the coacervation process, from initial formation and growth of stable micelles, on a nanometric length scale using time-resolved small-angle X-ray scattering (TR-SAXS). The micellar coacervates are formed through the complexation of anionic polyelectrolyte poly(sodium 4-styrenesulfonate) (PSSS) and cationic block-copolymer poly(ethylene oxide)-block-poly((vinylbenzyl)trimethylammonium chloride) (PEO-b-PVBTA). Mixing the polyelectrolytes in a stoichiometric 1:1 charge ratio resulted in the formation of stable spherical core-shell micellar-like coacervates consisting of a central core of complexed PSSS and PVBTA with a PEO corona. By use of synchrotron SAXS coupled to a stopped-flow mixing app., the whole formation kinetics of coacervates could be followed in situ from a few milliseconds. The results of a detailed data modeling reveal that the formation of these polyelectrolyte coacervates follows a two-step process: (i) first, metastable large-scale aggregates are formed upon a barrier-free complexation immediately after mixing; (ii) subsequently, the clusters undergo charge equilibration upon chain rearrangement and exchange processes yielding micellar-like aggregates with net neutral charge that are pinched off to yield the final stable micelle-like coacervates. While the initial cluster formation is very fast and completed within the dead time of mixing, the subsequent rearrangement becomes significantly slower with increasing mol. wt. of the PVBTA block. Interestingly, the overall kinetic process was essentially concn. independent, indicating that the rearrangement process is mainly accomplished via noncooperative chain rearrangement and chain exchange processes.
    36. 36
      Voets, I. K.; Moll, P. M.; Aqil, A.; Jérôme, C.; Detrembleur, C.; de Waard, P.; de Keizer, A.; Stuart, M. A. C. Temperature Responsive Complex Coacervate Core Micelles With a PEO and PNIPAAm Corona. J. Phys. Chem. B 2008, 112 (35), 10833– 10840,  DOI: 10.1021/jp8014832
      36
      Temperature Responsive Complex Coacervate Core Micelles With a PEO and PNIPAAm Corona
      Voets, Ilja K.; Moll, Puck M.; Aqil, Abdelhafid; Jerome, Christine; Detrembleur, Christophe; de Waard, Pieter; de Keizer, Arie; Cohen Stuart, Martien A.
      Journal of Physical Chemistry B (2008), 112 (35), 10833-10840CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      In aq. solns. at room temp., poly(N-methyl-2-vinyl pyridinium iodide)-block-poly(ethylene oxide), P2MVP38-b-PEO211 and poly(acrylic acid)-block-poly(isopropylacrylamide), PAA55-b-PNIPAAm88 spontaneously coassemble into micelles, consisting of a mixed P2MVP/PAA polyelectrolyte core and a PEO/PNIPAAm corona. These so-called complex coacervate core micelles (C3Ms), also known as polyion complex (PIC) micelles, block ionomer complexes (BIC), and interpolyelectrolyte complexes (IPEC), respond to changes in soln. pH and ionic strength as their micellization is electrostatically driven. Furthermore, the PNIPAAm segments ensure temp. responsiveness as they exhibit lower crit. soln. temp. (LCST) behavior. Light scattering, two-dimensional 1H NMR nuclear Overhauser effect spectrometry, and cryogenic transmission electron microscopy expts. were carried out to investigate micellar structure and soln. behavior at 1 mM NaNO3, T = 25, and 60 °C, i.e., below and above the LCST of ∼32 °C. At T = 25 °C, C3Ms were obsd. for 7 < pH < 12 and NaNO3 concns. below ∼105 mM. The PEO and PNIPAAm chains appear to be (randomly) mixed within the micellar corona. At T = 60 °C, onion-like complexes are formed, consisting of a PNIPAAm inner core, a mixed P2MVP/PAA complex coacervate shell, and a PEO corona.
    37. 37
      Sproncken, C. C. M.; Surís-Valls, R.; Cingil, H. E.; Detrembleur, C.; Voets, I. K. Complex Coacervate Core Micelles Containing Poly(Vinyl Alcohol) Inhibit Ice Recrystallization. Macromol. Rapid Commun. 2018, 39 (17), e1700814  DOI: 10.1002/marc.201700814
      There is no corresponding record for this reference.
    38. 38
      Voets, I. K.; van der Burgh, S.; Farago, B.; Fokkink, R.; Kovacevic, D.; Hellweg, T.; de Keizer, A.; Cohen Stuart, M. A. Electrostatically Driven Coassembly of a Diblock Copolymer and an Oppositely Charged Homopolymer in Aqueous Solutions. Macromolecules 2007, 40 (23), 8476– 8482,  DOI: 10.1021/ma071356z
      38
      Electrostatically Driven Coassembly of a Diblock Copolymer and an Oppositely Charged Homopolymer in Aqueous Solutions
      Voets, Ilja K.; Van der Burgh, Stefan; Farago, Bela; Fokkink, Remco; Kovacevic, Davor; Hellweg, Thomas; De Keizer, Arie; Cohen Stuart, Martien A.
      Macromolecules (Washington, DC, United States) (2007), 40 (23), 8476-8482CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Electrostatically driven co-assembly of poly(acrylic acid)-block-poly(acrylamide), PAA-b-PAAm, and poly(1-methyl-2-vinylpyridinium iodide), P2MVP, leads to formation of micelles in aq. solns. Light scattering and small angle neutron scattering expts. were performed to study the effect of concn. and length of the corona block (NPAAm = 97, 208, and 417) on micellar characteristics. Small angle neutron scattering curves were analyzed by generalized indirect Fourier transformation and model fitting. All scattering curves could be well described with a combination of a form factor for polydisperse spheres in combination with a hard sphere structure factor for the highest concns. Micellar aggregation nos., shape, and internal structure are relatively independent of concn. for Cp < 23.12 g L-1. The Guinier radius, av. micellar radius, hydrodynamic radius, and polydispersity were found to increase with increasing NPAAm. Micellar mass and aggregation no. were found to decrease with increasing NPAAm.
    39. 39
      Priftis, D.; Leon, L.; Song, Z.; Perry, S. L.; Margossian, K. O.; Tropnikova, A.; Cheng, J.; Tirrell, M. Self-Assembly of α-Helical Polypeptides Driven by Complex Coacervation. Angew. Chem. 2015, 127 (38), 11280– 11284,  DOI: 10.1002/ange.201504861
      There is no corresponding record for this reference.
    40. 40
      Choi, J.-W.; Heo, T.-Y.; Choi, H.; Choi, S.-H.; Won, J.-I. Co-Assembly Behavior of Oppositely Charged Thermoresponsive Elastin-like Polypeptide Block Copolymers. J. Appl. Polym. Sci. 2022, 139 (38), e52906  DOI: 10.1002/app.52906
      There is no corresponding record for this reference.
    41. 41
      Lim, C.; Roeck Won, W.; Moon, J.; Sim, T.; Shin, Y.; Chang Kim, J.; Seong Lee, E.; Seok Youn, Y.; Taek Oh, K. Co-Delivery of d -(KLAKLAK) 2 Peptide and Doxorubicin Using a pH-Sensitive Nanocarrier for Synergistic Anticancer Treatment. J. Mater. Chem. B 2019, 7 (27), 4299– 4308,  DOI: 10.1039/C9TB00741E
      There is no corresponding record for this reference.
    42. 42
      Lindhoud, S.; Voorhaar, L.; de Vries, R.; Schweins, R.; Cohen Stuart, M. A.; Norde, W. Salt-Induced Disintegration of Lysozyme-Containing Polyelectrolyte Complex Micelles. Langmuir 2009, 25 (19), 11425– 11430,  DOI: 10.1021/la901591p
      42
      Salt-Induced Disintegration of Lysozyme-Containing Polyelectrolyte Complex Micelles
      Lindhoud, Saskia; Voorhaar, Lenny; de Vries, Renko; Schweins, Ralf; Cohen Stuart, Martien A.; Norde, Willem
      Langmuir (2009), 25 (19), 11425-11430CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The salt-induced disintegration of lysozyme-filled polyelectrolyte complex micelles, consisting of pos. charged homopolymers (PDMAEMA150), neg. charged diblock copolymers (PAA42-PAAm417), and lysozyme, was studied with dynamic light scattering (DLS) and small-angle neutron scattering (SANS). These measurements show that, from 0 to 0.2 M NaCl, both the hydrodynamic radius (Rh) and the core radius (Rcore) decrease with increasing salt concn. This suggests that the micellar structures rearrange. Moreover, from ∼0.2 to 0.4 M NaCl the light-scattering intensity is const. In this salt interval, the hydrodynamic radius increases, has a max. at 0.3 M NaCl, and subsequently decreases. This behavior is obsd. in both a lysozyme-contg. system and a system without lysozyme. The SANS measurements on the lysozyme-filled micelles do not show increased intensity or a larger core radius at 0.3 M NaCl. This indicates that from 0.2 to 0.4 M NaCl another structure is formed, consisting of just the diblock copolymer and the homopolymer, because at 0.12 M NaCl the lysozyme-PAA42-PAAm417 complex has disintegrated. One may expect that the driving force for the formation of the complex in this salt range is other than electrostatic.
    43. 43
      Obermeyer, A. C.; Mills, C.; Dong, X.-H.; Flores, R.; Olsen, B. Complex Coacervation of Supercharged Proteins with Polyelectrolytes. Soft Matter 2016, 12 (15), 3570– 3581,  DOI: 10.1039/C6SM00002A
      43
      Complex coacervation of supercharged proteins with polyelectrolytes
      Obermeyer, Allie C.; Mills, Carolyn E.; Dong, Xue-Hui; Flores, Romeo J.; Olsen, Bradley D.
      Soft Matter (2016), 12 (15), 3570-3581CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      Complexation of proteins with polyelectrolytes or block copolymers can lead to phase sepn. to generate a coacervate phase or self-assembly of coacervate core micelles. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase sepd. with strong polycations when the ratio of neg. charged residues to pos. charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of pos. charge in the coacervate phase. The compn. of protein and polymer in the coacervate phase was detd. using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromol. charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temps.
    44. 44
      Xu, A. Y.; Kizilay, E.; Madro, S. P.; Vadenais, J. Z.; McDonald, K. W.; Dubin, P. L. Dilution Induced Coacervation in Polyelectrolyte-Micelle and Polyelectrolyte-Protein Systems. Soft Matter 2018, 14 (12), 2391– 2399,  DOI: 10.1039/C7SM02293J
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      Dilution induced coacervation in polyelectrolyte-micelle and polyelectrolyte-protein systems
      Xu, Amy Y.; Kizilay, Ebru; Madro, Slawomir P.; Vadenais, Justin Z.; McDonald, Kianan W.; Dubin, Paul L.
      Soft Matter (2018), 14 (12), 2391-2399CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
      "Self-suppression", the instability of complex coacervates at high concn., is well-known for polycation-polyanion systems, but the transient nature of those complexes impedes development of a convincing model. The stable polyelectrolyte-micelle complexes of the polycation poly(diallyldimethylammonium chloride) (PDADMAC) with mixed micelles of sodium dodecyl sulfate (SDS)/Triton X-100 (TX100); and the stable complexes of PDADMAC with bovine serum albumin (BSA) can be characterized and identified as coacervate precursors. We observe liq.-liq. phase sepn. upon isoionic diln., a common facet of self-suppression. While complex coacervation usually involves assocn. of near-neutral inter-polymer complexes, diln.-induced coacervation (DIC) proceeds differently: for both systems studied, complex size decreases near the biphasic region: inter-macromol. complexes with hydrodynamic radius Rh ∼ 100 nm dissoc. to intra-polyelectrolyte complexes with Rh ≤ 30 nm. Such small complexes with ≤5 bound micelles are unlikely to be net neutral. In the polyelectrolyte-protein system, complexes are even less likely to be net neutral and the effect of diln. on size is less significant, with complex size diminishing from 50 nm to 35 nm.
    45. 45
      Marras, A. E.; Ting, J. M.; Stevens, K. C.; Tirrell, M. V. Advances in the Structural Design of Polyelectrolyte Complex Micelles. J. Phys. Chem. B 2021, 125 (26), 7076– 7089,  DOI: 10.1021/acs.jpcb.1c01258
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      Advances in the Structural Design of Polyelectrolyte Complex Micelles
      Marras, Alexander E.; Ting, Jeffrey M.; Stevens, Kaden C.; Tirrell, Matthew V.
      Journal of Physical Chemistry B (2021), 125 (26), 7076-7089CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      A review. Polyelectrolyte complex micelles (PCMs) are a unique class of self-assembled nanoparticles that form with a core of assocd. polycations and polyanions, microphase-sepd. from neutral, hydrophilic coronas in aq. soln. The hydrated nature and structural and chem. versatility make PCMs an attractive system for delivery and for fundamental polymer physics research. By leveraging block copolymer design with controlled self-assembly, fundamental structure-property relationships can be established to tune the size, morphol., and stability of PCMs precisely in pursuit of tailored nanocarriers, ultimately offering storage, protection, transport, and delivery of active ingredients. This perspective highlights recent advances in predictive PCM design, focusing on (1) structure-property relationships to target specific nanoscale dimensions and shapes and (2) characterization of PCM dynamics primarily using time-resolved scattering techniques. We present several vignettes from these two emerging areas of PCM research and discuss key opportunities for PCM design to advance precision medicine.
    46. 46
      Abbas, M.; Lipiński, W.; Wang, J.; Spruijt, E. Peptide-Based Coacervates as Biomimetic Protocells. Chem. Soc. Rev. 2021, 50 (6), 3690– 3705,  DOI: 10.1039/D0CS00307G
      46
      Peptide-based coacervates as biomimetic protocells
      Abbas, Manzar; Lipinski, Wojciech P.; Wang, Jiahua; Spruijt, Evan
      Chemical Society Reviews (2021), 50 (6), 3690-3705CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Coacervates are condensed liq.-like droplets formed by liq.-liq. phase sepn. of mols. through multiple weak associative interactions. In recent years it has emerged that not only long polymers, but also short peptides are capable of forming simple and complex coacervates. The coacervate droplets they form act as compartments that sequester and conc. a wide range of solutes, and their spontaneous formation make coacervates attractive protocell models. The main advantage of peptides as building blocks lies in the functional diversity of the amino acid residues, which allows for tailoring of the peptide's phase sepn. propensity, their selectivity in guest mol. uptake and the physicochem. and catalytic properties of the compartments. The aim of this tutorial is to illustrate the recent developments in the field of peptide-based coacervates in a systematic way and to deduce the basic requirements for both simple and complex coacervation of peptides. We a selection of peptide coacervates that illustrates the essentials of phase sepn., the limitations, and the properties that make peptide coacervates biomimetic protocells. Finally, we provide some perspectives of this novel research field in the direction of active droplets, moving away from thermodn. equil.
    47. 47
      Brancaccio, D.; Pizzo, E.; Cafaro, V.; Notomista, E.; De Lise, F.; Bosso, A.; Gaglione, R.; Merlino, F.; Novellino, E.; Ungaro, F.; Grieco, P.; Malanga, M.; Quaglia, F.; Miro, A.; Carotenuto, A. Antimicrobial Peptide Temporin-L Complexed with Anionic Cyclodextrins Results in a Potent and Safe Agent against Sessile Bacteria. Int. J. Pharm. 2020, 584, 119437  DOI: 10.1016/j.ijpharm.2020.119437
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      Antimicrobial peptide Temporin-L complexed with anionic cyclodextrins results in a potent and safe agent against sessile bacteria
      Brancaccio, Diego; Pizzo, Elio; Cafaro, Valeria; Notomista, Eugenio; De Lise, Federica; Bosso, Andrea; Gaglione, Rosa; Merlino, Francesco; Novellino, Ettore; Ungaro, Francesca; Grieco, Paolo; Malanga, Milo; Quaglia, Fabiana; Miro, Agnese; Carotenuto, Alfonso
      International Journal of Pharmaceutics (Amsterdam, Netherlands) (2020), 584 (), 119437CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
      Concern over antibiotic resistance is growing, and new classes of antibiotics, particularly against Gram-neg. bacteria, are needed. Antimicrobial peptides (AMPs) have been proposed as a new class of clin. useful antimicrobials. Special attention has been devoted to frog-skin temporins. In particular, temporin L (TL) is strongly active against Gram-pos., Gram-neg. bacteria and yeast strains. With the aim of overcoming some of the main drawbacks preventing the widespread clin. use of this peptide, i.e. toxicity and unfavorable pharmacokinetics profile, we designed new formulations combining TL with different types of cyclodextrins (CDs). TL was assocd. to a panel of neutral or neg. charged, monomeric and polymeric CDs. The impact of CDs assocn. on TL soly., as well as the transport through bacterial alginates was assessed. The biocompatibility on human cells together with the antimicrobial and antibiofilm properties of TL/CD systems was explored.
    48. 48
      Insua, I.; Majok, S.; Peacock, A. F. A.; Krachler, A. M.; Fernandez-Trillo, F. Preparation and Antimicrobial Evaluation of Polyion Complex (PIC) Nanoparticles Loaded with Polymyxin B. Eur. Polym. J. 2017, 87, 478– 486,  DOI: 10.1016/j.eurpolymj.2016.08.023
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      Preparation and antimicrobial evaluation of polyion complex (PIC) nanoparticles loaded with polymyxin B
      Insua, Ignacio; Majok, Sieta; Peacock, Anna F. A.; Krachler, Anne Marie; Fernandez-Trillo, Francisco
      European Polymer Journal (2017), 87 (), 478-486CODEN: EUPJAG; ISSN:0014-3057. (Elsevier Ltd.)
      Here, we describe novel polyion complex (PIC) particles for the delivery of Polymyxin B (Pol-B), an antimicrobial peptide currently used in the clinic as a last resort antibiotic against multidrug-resistant gram-neg. bacteria. A range of conditions for the controlled assembly of Pol-B with poly(styrene sulfonate) (PSS) has been identified which let us prep. stable colloidal PIC particles. This way, PIC particles contg. different Pol-B:PSS ratios have been prepd. and their stability under simulated physiol. conditions (i.e. pH, osmotic pressure and temp.) characterized. Furthermore, preliminary evaluation of the antimicrobial activity of these Pol-B contg. PIC particles has been performed, by monitoring their effect on the growth of Pseudomonas aeruginosa, an opportunistic gram-neg. bacterium.
    49. 49
      Răileanu, M.; Lonetti, B.; Serpentini, C.-L.; Goudounèche, D.; Gibot, L.; Bacalum, M. Encapsulation of a Cationic Antimicrobial Peptide into Self-Assembled Polyion Complex Nano-Objects Enhances Its Antitumor Properties. J. Mol. Struct. 2022, 1249, 131482  DOI: 10.1016/j.molstruc.2021.131482
      There is no corresponding record for this reference.
    50. 50
      Wang, C.; Feng, S.; Qie, J.; Wei, X.; Yan, H.; Liu, K. Polyion Complexes of a Cationic Antimicrobial Peptide as a Potential Systemically Administered Antibiotic. Int. J. Pharm. 2019, 554, 284– 291,  DOI: 10.1016/j.ijpharm.2018.11.029
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      Polyion complexes of a cationic antimicrobial peptide as a potential systemically administered antibiotic
      Wang, Chenhong; Feng, Siliang; Qie, Jiankun; Wei, Xiaoli; Yan, Husheng; Liu, Keliang
      International Journal of Pharmaceutics (Amsterdam, Netherlands) (2019), 554 (), 284-291CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
      Antimicrobial peptides (AMPs) are regarded as next-generation antibiotics to replace conventional antibiotics due to their rapid and broad-spectrum antimicrobial properties and far less sensitivity to the development of pathogen resistance. However, they are susceptible to proteolysis in vivo by endogenous or bacterial proteases as well as induce the lysis of red blood cells, which prevent their i.v. applications. In this work, polyion complex (PIC) micelles of the cationic AMP MSI-78 and the anionic copolymer methoxy poly(ethylene glycol)-b-poly(α-glutamic acid) (mPEG-b-PGlu) were prepd. to develop novel antimicrobial agents for potential application in vivo. With an increase in molar ratio of mPEG-b-PGlu to MSI-78, the complexation ability of the PIC micelles increased. FITC-labeled MSI-78 showed a sustained release from the PIC micelles. More importantly, these PIC micelles greatly decreased the hemolytic toxicity of MSI-78 to human red blood cells, without influencing its antimicrobial activity. Thus, this approach could be used as a suitable in vivo delivery method of AMPs in the future.
    51. 51
      Niece, K. L.; Vaughan, A. D.; Devore, D. I. Graft Copolymer Polyelectrolyte Complexes for Delivery of Cationic Antimicrobial Peptides. J. Biomed. Mater. Res., Part A 2013, 101 (9), 2548– 2558,  DOI: 10.1002/jbm.a.34555
      There is no corresponding record for this reference.
    52. 52
      Tully, M. D.; Kieffer, J.; Brennich, M. E.; Cohen Aberdam, R.; Florial, J. B.; Hutin, S.; Oscarsson, M.; Beteva, A.; Popov, A.; Moussaoui, D.; Theveneau, P.; Papp, G.; Gigmes, J.; Cipriani, F.; McCarthy, A.; Zubieta, C.; Mueller-Dieckmann, C.; Leonard, G.; Pernot, P. BioSAXS at European Synchrotron Radiation Facility─Extremely Brilliant Source: BM29 with an Upgraded Source, Detector, Robot, Sample Environment, Data Collection and Analysis Software. J. Synchrotron Radiat. 2023, 30 (1), 258– 266,  DOI: 10.1107/S1600577522011286
      There is no corresponding record for this reference.
    53. 53
      Berndt, I.; Pedersen, J. S.; Lindner, P.; Richtering, W. Influence of Shell Thickness and Cross-Link Density on the Structure of Temperature-Sensitive Poly-N-Isopropylacrylamide–Poly-N-Isopropylmethacrylamide Core–Shell Microgels Investigated by Small-Angle Neutron Scattering. Langmuir 2006, 22 (1), 459– 468,  DOI: 10.1021/la052463u
      53
      Influence of Shell Thickness and Cross-Link Density on the Structure of Temperature-Sensitive Poly-N-Isopropylacrylamide-Poly-N-Isopropylmethacrylamide Core-Shell Microgels Investigated by Small-Angle Neutron Scattering
      Berndt, Ingo; Pedersen, Jan Skov; Lindner, Peter; Richtering, Walter
      Langmuir (2006), 22 (1), 459-468CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Swelling properties of doubly temp. sensitive core-shell microgels consisting of two thermosensitive polymers with lower crit. soln. temps. (LCTS) at, resp., 34° in the core and 44° in the shell have been investigated by small-angle neutron scattering (SANS). A core-shell form factor has been employed to evaluate the structure, and the real space particle structure is expressed by radial d. profiles. By this means, the influences of both shell/core mass compn. and shell cross-linker content on the internal structure have been revealed at temps. above, between, and below the LCSTs. Higher shell/core mass ratios lead to an increased expansion of the core at temps. between the LCSTs, whereas a variation of cross-linker in the shell mainly effects the dimensions of the shell. The influence on the core structure was interpreted as resulting from an elastic force developed from the swollen shell. At temps. below the core LCST, the core cannot swell to its native size (i.e., in the absence of a shell), because the max. expanded shell network prohibits further swelling. Thus, depending on temp., the shell either expands or compresses the core.
    54. 54
      Berndt, I.; Pedersen, J. S.; Richtering, W. Temperature-Sensitive Core–Shell Microgel Particles with Dense Shell. Angew. Chem. 2006, 118 (11), 1769– 1773,  DOI: 10.1002/ange.200503888
      There is no corresponding record for this reference.
    55. 55
      Pedersen, J. S.; Svaneborg, C. Scattering from Block Copolymer Micelles. Curr. Opin. Colloid Interface Sci. 2002, 7 (3), 158– 166,  DOI: 10.1016/S1359-0294(02)00044-4
      There is no corresponding record for this reference.
    56. 56
      Lund, R.; Willner, L.; Richter, D. Kinetics of Block Copolymer Micelles Studied by Small-Angle Scattering Methods. In Controlled Polymerization and Polymeric Structures: Flow Microreactor Polymerization, Micelles Kinetics, Polypeptide Ordering, Light Emitting Nanostructures; Abe, A.; Lee, K.-S.; Leibler, L.; Kobayashi, S., Eds.; Advances in Polymer Science; Springer International Publishing: Cham, 2013; pp 51– 158.  DOI: 10.1007/12_2012_204 .
      There is no corresponding record for this reference.
    57. 57
      Pedersen, J. S. Structure Factors Effects in Small-Angle Scattering from Block Copolymer Micelles and Star Polymers. J. Chem. Phys. 2001, 114 (6), 2839– 2846,  DOI: 10.1063/1.1339221
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      Structure factors effects in small-angle scattering from block copolymer micelles and star polymers
      Pedersen, Jan Skov
      Journal of Chemical Physics (2001), 114 (6), 2839-2846CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      Math. expressions for the form factor of a block copolymer micelle model with a spherical core and Gaussian polymer chains attached to the surface presented in J. S. Pedersen and M. C. Gerstenberg [Macromols. 29, 1363 (1996)] were modified to include particle interference effects in scattering expts. in terms of a structure factor. The results are derived assuming that the effective interaction between particles is known, i.e., the structure factor related to the center-center distribution is assumed to be known. The derived expression for the intensity is not a simple product of the form factor and the structure factor, which has the important consequence that the effective structure factor depends on the relative scattering contrast of the core and the corona of polymer chains. The structure factor effects for Gaussian star polymers are described by the same expression for a vanishing core radius. The influences of chain self-avoidance and chain-chain interactions are discussed.
    58. 58
      Lund, R.; Willner, L.; Stellbrink, J.; Radulescu, A.; Richter, D. Role of Interfacial Tension for the Structure of PEP–PEO Polymeric Micelles. A Combined SANS and Pendant Drop Tensiometry Investigation. Macromolecules 2004, 37 (26), 9984– 9993,  DOI: 10.1021/ma035633n
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      Role of Interfacial Tension for the Structure of PEP-PEO Polymeric Micelles. A Combined SANS and Pendant Drop Tensiometry Investigation
      Lund, Reidar; Willner, Lutz; Stellbrink, Joerg; Radulescu, Aurel; Richter, Dieter
      Macromolecules (2004), 37 (26), 9984-9993CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      We investigated the influence of interfacial tension, γ, on the micellization properties of a highly asym. poly(ethylene-co-propylene)-poly(ethylene oxide) (PEP-PEO) block copolymer in mixed solvents consisting of water and DMF. Both are good solvents for PEO and nonsolvents for PEP but exhibit a large difference in γ with respect to the insol. core block. Micellar characteristics were obtained by small-angle neutron scattering (SANS) and subsequent fitting of a core-shell form factor to the scattering patterns. The curves are perfectly described by a hyperbolic d. profile for the shell, n(r) ∼ r-4/3, indicating a starlike structure of the micelles. The aggregation nos. of the micelles decrease with increasing DMF-water ratio from P = 120 in pure water to nonaggregated chains in pure DMF. Corresponding interfacial tensions were detd. by pendant drop tensiometry using a PEP homopolymer of equal molar mass. A correlation of P with γ reveals a power law dependence, P ∼ γ6/5, in accordance with the scaling prediction of Halperin for starlike micelles. The addn. of DMF leads to a considerable decrease in the micelle radii, which cannot be explained by the decrease in P alone. Measurements of the second virial coeffs., A2, of a PEO homopolymer by SANS reveal clearly reduced values compared to A2 in pure water but still good solvent conditions for PEO in all water/DMF mixts. However, a significant redn. in the radius of gyration was not found. Therefore, it was concluded that the reduced solvent quality has a more pronounced effect for the PEO chain dimensions in the confined geometry of a micellar corona.
    59. 59
      Fang, Y. N.; Rumyantsev, A. M.; Neitzel, A. E.; Liang, H.; Heller, W. T.; Nealey, P. F.; Tirrell, M. V.; de Pablo, J. J. Scattering Evidence of Positional Charge Correlations in Polyelectrolyte Complexes. Proc. Natl. Acad. Sci. U.S.A. 2023, 120 (32), e2302151120  DOI: 10.1073/pnas.2302151120
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      Molecular-weight determination by light scattering
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      cf. C.A. 40, 4267.7. The theory of the turbidity method of detg. mol. wt. is presented and the app. used in the measurement is described.
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      Hofs, B.; Brzozowska, A.; de Keizer, A.; Norde, W.; Cohen Stuart, M. A. Reduction of Protein Adsorption to a Solid Surface by a Coating Composed of Polymeric Micelles with a Glass-like Core. J. Colloid Interface Sci. 2008, 325 (2), 309– 315,  DOI: 10.1016/j.jcis.2008.06.006
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      Brzozowska, A. M.; Hofs, B.; de Keizer, A.; Fokkink, R.; Cohen Stuart, M. A.; Norde, W. Reduction of Protein Adsorption on Silica and Polystyrene Surfaces Due to Coating with Complex Coacervate Core Micelles. Colloids Surf., A. 2009, 347 (1), 146– 155,  DOI: 10.1016/j.colsurfa.2009.03.036
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      Makowski, M.; Silva, Í. C.; Pais do Amaral, C.; Gonçalves, S.; Santos, N. C. Advances in Lipid and Metal Nanoparticles for Antimicrobial Peptide Delivery. Pharmaceutics 2019, 11 (11), 588,  DOI: 10.3390/pharmaceutics11110588
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      Advances in lipid and metal nanoparticles for antimicrobial peptide delivery
      Makowski, Marcin; Silva, Itala C.; do Amaral, Constanca Pais; Goncalves, Sonia; Santos, Nuno C.
      Pharmaceutics (2019), 11 (11), 588CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)
      Antimicrobial peptides (AMPs) have been described as excellent candidates to overcome antibiotic resistance. Frequently, AMPs exhibit a wide therapeutic window, with low cytotoxicity and broad-spectrum antimicrobial activity against a variety of pathogens. In addn., some AMPs are also able to modulate the immune response, decreasing potential harmful effects such as sepsis. Despite these benefits, only a few formulations have successfully reached clinics. A common flaw in the druggability of AMPs is their poor pharmacokinetics, common to several peptide drugs, as they may be degraded by a myriad of proteases inside the organism. The combination of AMPs with carrier nanoparticles to improve delivery may enhance their half-life, decreasing the dosage and thus, reducing prodn. costs and eventual toxicity. Here, we present the most recent advances in lipid and metal nanodevices for AMP delivery, with a special focus on metal nanoparticles and liposome formulations.
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      Eftekhari, A.; Arjmand, A.; Asheghvatan, A.; Švajdlenková, H.; Šauša, O.; Abiyev, H.; Ahmadian, E.; Smutok, O.; Khalilov, R.; Kavetskyy, T.; Cucchiarini, M. The Potential Application of Magnetic Nanoparticles for Liver Fibrosis Theranostics. Front. Chem. 2021, 9, 674786  DOI: 10.3389/fchem.2021.674786
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      Weldick, P. J.; Wang, A.; F. Halbus, A.; N. Paunov, V. Emerging Nanotechnologies for Targeting Antimicrobial Resistance. Nanoscale 2022, 14 (11), 4018– 4041,  DOI: 10.1039/D1NR08157H
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      Emerging nanotechnologies for targeting antimicrobial resistance
      Weldick, Paul J.; Wang, Anheng; Halbus, Ahmed F.; Paunov, Vesselin N.
      Nanoscale (2022), 14 (11), 4018-4041CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      A review. Antimicrobial resistance is a leading cause of mortality worldwide. Without newly approved antibiotics and antifungals being brought to the market, resistance is being developed to the ones currently available to clinicians. The reason is the applied evolutionary pressure to bacterial and fungal species due to the wide overuse of common antibiotics and antifungals in clin. practice and agriculture. Biofilms harbor antimicrobial-resistant subpopulations, which make their antimicrobial treatment even more challenging. Nanoparticle-based technologies have recently been shown to successfully overcome antimicrobial resistance in both planktonic and biofilms phenotypes. This results from the combination of novel nanomaterial research and classic antimicrobial therapies which promise to deliver a whole new generation of high-performance active nanocarrier systems. This review discusses the latest developments of promising nanotechnologies with applications against resistant pathogens and evaluates their potential and feasibility for use in novel antimicrobial therapies.
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      Rajchakit, U.; Sarojini, V. Recent Developments in Antimicrobial-Peptide-Conjugated Gold Nanoparticles. Bioconjugate Chem. 2017, 28 (11), 2673– 2686,  DOI: 10.1021/acs.bioconjchem.7b00368
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      Rajchakit, Urawadee; Sarojini, Vijayalekshmi
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      A review. The escalation of multidrug-resistant pathogens has created a dire need to develop novel ways of addressing this global therapeutic challenge. Because of their antimicrobial activities, combination of antimicrobial peptides (AMPs) and nanoparticles is a promising tool to kill drug resistant pathogens. In recent years, several studies using AMP-nanoparticle conjugates, esp. metallic nanoparticles, as potential antimicrobial agents against drug resistant pathogens have been published. Amongst these, antimicrobial peptide conjugated gold nanoparticles (AMP-AuNPs) are particularly attractive because of the non-toxic nature of gold and the possibility of fine tuning the AMP-NP conjugation chem. The following review discusses recent developments in the synthesis and antimicrobial activity studies of AMP-AuNPs. Classification of AMPs, their mechanisms of action, methods used for functionalizing AuNPs with AMPs and the antimicrobial activities of the conjugates are discussed.
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      Da Vela, S.; Svergun, D. I. Methods, Development and Applications of Small-Angle X-Ray Scattering to Characterize Biological Macromolecules in Solution. Curr. Res. Struct. Biol. 2020, 2, 164– 170,  DOI: 10.1016/j.crstbi.2020.08.004
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      Methods, development and applications of small-angle X-ray scattering to characterize biological macromolecules in solution
      Da Vela Stefano; Svergun Dmitri I
      Current research in structural biology (2020), 2 (), 164-170 ISSN:.
      Applications of small-angle X-ray scattering (SAXS) in structural biology are reviewed. A brief introduction of the SAXS basics is followed by the presentation of the structural features of biological macromolecules in solution that can be assessed by SAXS. The approaches are considered allowing one to obtain low resolution three-dimensional (3D) structural models and to describe assembly states and conformations. Metrics and descriptors required for the assessment of model quality are presented and recent biological applications of SAXS are shown.
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      Nolles, A.; Hooiveld, E.; Westphal, A. H.; van Berkel, W. J. H.; Kleijn, J. M.; Borst, J. W. FRET Reveals the Formation and Exchange Dynamics of Protein-Containing Complex Coacervate Core Micelles. Langmuir 2018, 34 (40), 12083– 12092,  DOI: 10.1021/acs.langmuir.8b01272
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      FRET Reveals the Formation and Exchange Dynamics of Protein-Containing Complex Coacervate Core Micelles
      Nolles, Antsje; Hooiveld, Ellard; Westphal, Adrie H.; van Berkel, Willem J. H.; Kleijn, J. Mieke; Borst, Jan Willem
      Langmuir (2018), 34 (40), 12083-12092CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The encapsulation of proteins into complex coacervate core micelles (C3Ms) is of potential interest for a wide range of applications. To address the stability and dynamic properties of these polyelectrolyte complexes, combinations of cyan, yellow, and blue fluorescent proteins were encapsulated with cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Forster resonance energy transfer (FRET) allowed us to det. the kinetics of C3M formation and of protein exchange between C3Ms. Both processes follow first-order kinetics with relaxation times of ±100 s at low ionic strength (I = 2.5 mM). Stability studies revealed that 50% of FRET was lost at I = 20 mM, pointing to the disintegration of the C3Ms. On the basis of exptl. and theor. considerations, the authors propose that C3Ms relax to their final state by assocn. and dissocn. of near-neutral sol. protein-polymer complexes. To obtain protein-contg. C3Ms suitable for applications, it is necessary to improve the rigidity and salt stability of these complexes.
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      Tian, B.; Liu, S.; Lu, W.; Jin, L.; Li, Q.; Shi, Y.; Li, C.; Wang, Z.; Du, Y. Construction of pH-Responsive and up-Conversion Luminescent NaYF4:Yb3+/Er3+@SiO2@PMAA Nanocomposite for Colon Targeted Drug Delivery. Sci. Rep. 2016, 6 (1), 21335  DOI: 10.1038/srep21335
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      In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cytofluorometric and bioluminescent methods are not widely used because they require specified equipment and further evaluation for reproducibility and standardization, even if they can provide rapid results of the antimicrobial agent's effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limitations are reported.
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      Kourmouli, A.; Valenti, M.; van Rijn, E.; Beaumont, H. J. E.; Kalantzi, O.-I.; Schmidt-Ott, A.; Biskos, G. Can Disc Diffusion Susceptibility Tests Assess the Antimicrobial Activity of Engineered Nanoparticles?. J. Nanopart. Res. 2018, 20 (3), 62,  DOI: 10.1007/s11051-018-4152-3
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      El-Andaloussi, S.; Järver, P.; Johansson, H. J.; Langel, Ü. Cargo-Dependent Cytotoxicity and Delivery Efficacy of Cell-Penetrating Peptides: A Comparative Study. Biochem. J. 2007, 407 (2), 285– 292,  DOI: 10.1042/BJ20070507
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      El-Andaloussi, Samir; Jaerver, Peter; Johansson, Henrik J.; Langel, Uelo
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      The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive mols. has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compds. across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore the authors selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. The results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concn., cargo mol. and coupling strategy.
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      Stem cells international (2016), 2016 (), 4798639 ISSN:1687-966X.
      Mesenchymal stem cells form a population of self-renewing, multipotent cells that can be isolated from several tissues. Multiple preclinical studies have demonstrated that the administration of exogenous MSC could prevent renal injury and could promote renal recovery through a series of complex mechanisms, in particular via immunomodulation of the immune system and release of paracrine factors and microvesicles. Due to their therapeutic potentials, MSC are being evaluated as a possible player in treatment of human kidney disease, and an increasing number of clinical trials to assess the safety, feasibility, and efficacy of MSC-based therapy in various kidney diseases have been proposed. In the present review, we will summarize the current knowledge on MSC infusion to treat acute kidney injury, chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, systemic lupus erythematosus, and kidney transplantation. The data obtained from these clinical trials will provide further insight into safety, feasibility, and efficacy of MSC-based therapy in renal pathologies and allow the design of consensus protocol for clinical purpose.
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      A review with 41 refs. Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examd. Thus, caspase-3 is essential for certain processes assocd. with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.
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      Miatmoko, A. Physical Characterization and Biodistribution of Cisplatin Loaded in Surfactant Modified-Hybrid Nanoparticles Using Polyethylene Oxide-b-Polymethacrylic Acid. Adv. Pharm. Bull. 2020, 11 (4), 765– 771,  DOI: 10.34172/apb.2021.086
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.4c00337.

    • Details of the theoretical SAXS data modeling; DLS, including autocorrelation functions, SAXS data showing the concentration dependence of C3Ms, density profiles obtained from SAXS, the effect of ionic strength and pH on C3Ms, CMC determination, additional antimicrobial effects, trypsin breakdown effect, freeze-drying of C3Ms, morphology changes upon cellular treatment, and tables of fit parameter (PDF)


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