Lipid-Based Nanoparticle Functionalization with Coiled-Coil Peptides for In Vitro and In Vivo Drug Delivery

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This article references 63 other publications.

1. 1

   Yang, J.; Bahreman, A.; Daudey, G.; Bussmann, J.; Olsthoorn, R. C. L.; Kros, A. Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes. ACS Cent Sci. 2016, 2 (9), 621– 630,  DOI: 10.1021/acscentsci.6b00172

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   Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes

   Yang, Jian; Bahreman, Azadeh; Daudey, Geert; Bussmann, Jeroen; Olsthoorn, Rene C. L.; Kros, Alexander

   ACS Central Science (2016), 2 (9), 621-630CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)

   Efficient delivery of drugs to living cells is still a major challenge. Currently, most methods rely on the endocytic pathway resulting in low delivery efficiency due to limited endosomal escape and/or degrdn. in lysosomes. Here, we report a new method for direct drug delivery into the cytosol of live cells in vitro and in-vivo utilizing targeted membrane fusion between liposomes and live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of liposomes and cell membranes resp., resulting in targeted membrane fusion with concomitant release of liposome encapsulated cargo including fluorescent dyes and the cytotoxic drug doxorubicin. Using a wide spectrum of endocytosis inhibitors and endosome trackers, we demonstrate that the major site of cargo release is at the plasma membrane. This method thus allows for the quick and efficient delivery of drugs and is expected to have many in-vitro, ex vivo, and in-vivo applications.
2. 2

   Yang, J.; Shimada, Y.; Olsthoorn, R. C. L.; Snaar-Jagalska, B. E.; Spaink, H. P.; Kros, A. Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model. ACS Nano 2016, 10 (8), 7428– 7435,  DOI: 10.1021/acsnano.6b01410

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   Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model

   Yang, Jian; Shimada, Yasuhito; Olsthoorn, Rene C. L.; Snaar-Jagalska, B. Ewa; Spaink, Herman P.; Kros, Alexander

   ACS Nano (2016), 10 (8), 7428-7435CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)

   The complementary coiled coil forming peptides E4 [(EIAALEK)4] and K4 [(KIAALKE)4] are known to trigger liposomal membrane fusion when tethered to lipid vesicles in the form of lipopeptides. In this study, we examd. whether these coiled coil forming peptides can be used for drug delivery applications. First, we prepd. E4 peptide modified liposomes contg. the far-red fluorescent dye TO-PRO-3 iodide (E4-Lipo-TP3) and confirmed that E4-liposomes could deliver TP3 into HeLa cells expressing K4 peptide on the membrane (HeLa-K) under cell culture conditions in a selective manner. Next, we prepd. doxorubicin-contg. E4-liposomes (E4-Lipo-DOX) and confirmed that E4-liposomes could also deliver DOX into HeLa-K cells. Moreover, E4-Lipo-DOX showed enhanced cytotoxicity toward HeLa-K cells compared to free doxorubicin. To prove the suitability of E4/K4 coiled coil formation for in vivo drug delivery, we injected E4-Lipo-TP3 or E4-Lipo-DOX into zebrafish xenografts of HeLa-K. As a result, E4-liposomes delivered TP3 to the implanted HeLa-K cells, and E4-Lipo-DOX could suppress cancer proliferation in the xenograft when compared to nontargeted conditions (i.e., zebrafish xenograft with free DOX injection). These data demonstrate that coiled coil formation enables drug selectivity and efficacy in vivo. It is envisaged that these findings are a step forward toward biorthogonal targeting systems as a tool for clin. drug delivery.
3. 3

   Zeng, Y.; Shen, M.; Singhal, A.; Sevink, G. J. A.; Crone, N.; Boyle, A. L.; Kros, A. Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides. Small 2023, 19, e2301133,  DOI: 10.1002/smll.202301133

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4. 4

   Zeng, Y.; Estapé Senti, M.; Labonia, M. C. I.; Papadopoulou, P.; Brans, M. A. D.; Dokter, I.; Fens, M. H.; van Mil, A.; Sluijter, J. P. G.; Schiffelers, R. M.; Vader, P.; Kros, A. Fusogenic Coiled-Coil Peptides Enhance Lipid Nanoparticle-Mediated mRNA Delivery upon Intramyocardial Administration. ACS Nano 2023, 17 (23), 23466– 23477,  DOI: 10.1021/acsnano.3c05341

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5. 5

   Hald Albertsen, C.; Kulkarni, J. A.; Witzigmann, D.; Lind, M.; Petersson, K.; Simonsen, J. B. The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Deliv Rev. 2022, 188, 114416,  DOI: 10.1016/j.addr.2022.114416

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   The role of lipid components in lipid nanoparticles for vaccines and gene therapy

   Hald Albertsen, Camilla; Kulkarni, Jayesh A.; Witzigmann, Dominik; Lind, Marianne; Petersson, Karsten; Simonsen, Jens B.

   Advanced Drug Delivery Reviews (2022), 188 (), 114416CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)

   A review. Lipid nanoparticles (LNPs) play an important role in mRNA vaccines against COVID-19. In addn., many preclin. and clin. studies, including the siRNA-LNP product, Onpattro, highlight that LNPs unlock the potential of nucleic acid-based therapies and vaccines. To understand what is key to the success of LNPs, we need to understand the role of the building blocks that constitute them. In this Review, we discuss what each lipid component adds to the LNP delivery platform in terms of size, structure, stability, apparent pKa, nucleic acid encapsulation efficiency, cellular uptake, and endosomal escape. To explore this, we present findings from the liposome field as well as from landmark and recent articles in the LNP literature. We also discuss challenges and strategies related to in vitro/in vivo studies of LNPs based on fluorescence readouts, immunogenicity/reactogenicity, and LNP delivery beyond the liver. How these fundamental challenges are pursued, including what lipid components are added and combined, will likely det. the scope of LNP-based gene therapies and vaccines for treating various diseases.
6. 6

   Kang, H.; Rho, S.; Stiles, W. R.; Hu, S.; Baek, Y.; Hwang, D. W.; Kashiwagi, S.; Kim, M. S.; Choi, H. S. Size-Dependent EPR Effect of Polymeric Nanoparticles on Tumor Targeting. Adv. Healthc Mater. 2020, 9 (1), e1901223,  DOI: 10.1002/adhm.201901223

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   Taghipour, Y. D.; Zarebkohan, A.; Salehi, R.; Rahimi, F.; Torchilin, V. P.; Hamblin, M. R.; Seifalian, A. An update on dual targeting strategy for cancer treatment. J. Controlled Release 2022, 349, 67– 96,  DOI: 10.1016/j.jconrel.2022.06.044

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   An update on dual targeting strategy for cancer treatment

   Taghipour, Yasamin Davatgaran; Zarebkohan, Amir; Salehi, Roya; Rahimi, Fariborz; Torchilin, Vladimir P.; Hamblin, Michael R.; Seifalian, Alexander

   Journal of Controlled Release (2022), 349 (), 67-96CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)

   A review. The key issue in the treatment of solid tumors is the lack of efficient strategies for the targeted delivery and accumulation of therapeutic cargoes in the tumor microenvironment (TME). Targeting approaches are designed for more efficient delivery of therapeutic agents to cancer cells while minimizing drug toxicity to normal cells and off-targeting effects, while maximizing the eradication of cancer cells. The highly complicated interrelationship between the physicochem. properties of nanoparticles, and the physiol. and pathol. barriers that are required to cross, dictates the need for the success of targeting strategies. Dual targeting is an approach that uses both purely biol. strategies and physicochem. responsive smart delivery strategies to increase the accumulation of nanoparticles within the TME and improve targeting efficiency towards cancer cells. In both approaches, either one single ligand is used for targeting a single receptor on different cells, or two different ligands for targeting two different receptors on the same or different cells. Smart delivery strategies are able to respond to triggers that are typical of specific disease sites, such as pH, certain specific enzymes, or redox conditions. These strategies are expected to lead to more precise targeting and better accumulation of nano-therapeutics. This review describes the classification and principles of dual targeting approaches and critically reviews the efficiency of dual targeting strategies, and the rationale behind the choice of ligands. We focus on new approaches for smart drug delivery in which synthetic and/or biol. moieties are attached to nanoparticles by TME-specific responsive linkers and advanced camouflaged nanoparticles.
8. 8

   Zhao, Z.; Ukidve, A.; Kim, J.; Mitragotri, S. Targeting Strategies for Tissue-Specific Drug Delivery. Cell 2020, 181 (1), 151– 167,  DOI: 10.1016/j.cell.2020.02.001

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   Targeting strategies for tissue-specific drug delivery

   Zhao, Zongmin; Ukidve, Anvay; Kim, Jayoung; Mitragotri, Samir

   Cell (Cambridge, MA, United States) (2020), 181 (1), 151-167CODEN: CELLB5; ISSN:0092-8674. (Cell Press)

   A review. Off-target effects of systemically administered drugs have been a major hurdle in designing therapies with desired efficacy and acceptable toxicity. Developing targeting strategies to enable site-specific drug delivery holds promise in reducing off-target effects, decreasing unwanted toxicities, and thereby enhancing a drug's therapeutic efficacy. Over the past three decades, a large body of literature has focused on understanding the biol. barriers that hinder tissue-specific drug delivery and strategies to overcome them. These efforts have led to several targeting strategies that modulate drug delivery in both the preclin. and clin. settings, including small mol.-, nucleic acid-, peptide-, antibody-, and cell-based strategies. Here, we discuss key advances and emerging concepts for tissue-specific drug delivery approaches and their clin. translation.
9. 9

   Guidotti, G.; Brambilla, L.; Rossi, D. Cell-Penetrating Peptides: From Basic Research to Clinics. Trends Pharmacol. Sci. 2017, 38 (4), 406– 424,  DOI: 10.1016/j.tips.2017.01.003

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   Cell-Penetrating Peptides: From Basic Research to Clinics

   Guidotti, Giulia; Brambilla, Liliana; Rossi, Daniela

   Trends in Pharmacological Sciences (2017), 38 (4), 406-424CODEN: TPHSDY; ISSN:0165-6147. (Elsevier Ltd.)

   The presence of cell and tissue barriers together with the low biomembrane permeability of various therapeutics often hampers systemic drug distribution; thus, most of the available mols. are of limited therapeutic value. Opportunities to increase medicament concns. in areas that are difficult to access now exist with the advent of cell-penetrating peptides (CPPs), which can transport into the cell a wide variety of biol. active conjugates (cargoes). Numerous preclin. evaluations with CPP-derived therapeutics have provided promising results in various disease models that, in some cases, prompted clin. trials. The outcome of these investigations has thus opened new perspectives for CPP application in the development of unprecedented human therapies that are well tolerated and directed to intracellular targets.
10. 10

    Desale, K.; Kuche, K.; Jain, S. Cell-penetrating peptides (CPPs): an overview of applications for improving the potential of nanotherapeutics. Biomater Sci. 2021, 9 (4), 1153– 1188,  DOI: 10.1039/D0BM01755H

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    Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender, P. A. Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells. J. Am. Chem. Soc. 2004, 126 (31), 9506– 9507,  DOI: 10.1021/ja0482536

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    Role of Membrane Potential and Hydrogen Bonding in the Mechanism of Translocation of Guanidinium-Rich Peptides into Cells

    Rothbard, Jonathan B.; Jessop, Theodore C.; Lewis, Richard S.; Murray, Bryce A.; Wender, Paul A.

    Journal of the American Chemical Society (2004), 126 (31), 9506-9507CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

    The results described herein support a mechanistic hypothesis for how guanidine-rich transporters attached to small cargos (MW ∼<3000) can migrate across the lipid membrane of a cell and directly enter the cytosol. Arginine oligomers are found to partition almost completely into the aq. layer of a water-octanol bilayer. However, when the same partitioning expt. is conducted in the presence of sodium laurate, a representative neg. charged membrane constituent, the arginine oligomer partitions almost completely (>95%) into the octanol layer. In contrast, ornithine oligomers partition almost exclusively into the water layer with and without added sodium laurate. The different partitioning between guanidinium-rich and ammonium-rich oligomers in the presence of sodium laurate is consistent with the ability of the former to form a bidentate hydrogen bonded ion pair. Mono- and dimethylated arginine oligomers, which like ornithine can only efficiently form monodentate hydrogen bonds, were prepd. and found to exhibit poor cellular uptake. Ion pair formation converts a once water-sol. agent to a lipid-sol. agent, thereby reducing the energetic penalty for passage of guanidine-rich transporters through the lipid bilayer. Uptake of guanidine-rich transporters is known to be an energy-dependent process, and this requirement for cellular ATP is now rationalized by the inhibition of guanidine-rich transporter uptake in the presence of agents that reduce the membrane potential. Specifically, incubation of cells in buffers with high potassium ion concns. or pretreatment of cells with gramicidin A reduces the cellular uptake of Fl-aca-arg8-CONH2 by >90%. Furthermore, the reciprocal expt. of hyperpolarizing the cell with valinomycin increased uptake by >1.5 times. In summary, we propose that the water-sol., pos. charged guanidinium headgroups of the transporter form bidentate hydrogen bonds with H-bond acceptor functionality on the cell surface. The resultant ion pair complexes partition into the lipid bilayer and migrate across at a rate related to the membrane potential. The complex dissocs. on the inner leaf of the membrane, and the transporter enters the cytosol. This hypothesis does not preclude uptake by other mechanisms, including endocytosis, which is likely to dominate with large cargos.
12. 12

    Marks, J. R.; Placone, J.; Hristova, K.; Wimley, W. C. Spontaneous membrane-translocating peptides by orthogonal high-throughput screening. J. Am. Chem. Soc. 2011, 133 (23), 8995– 9004,  DOI: 10.1021/ja2017416

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    Spontaneous Membrane-Translocating Peptides by Orthogonal High-Throughput Screening

    Marks, Jessica R.; Placone, Jesse; Hristova, Kalina; Wimley, William C.

    Journal of the American Chemical Society (2011), 133 (23), 8995-9004CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

    Combinatorial peptide chem. and orthogonal high-throughput screening were used to select peptides that spontaneously translocate across synthetic lipid bilayer membranes without permeabilization. A conserved sequence motif was identified that contains several cationic residues in conserved positions in an otherwise hydrophobic sequence. This 9-residue motif rapidly translocates across synthetic multibilayer vesicles and into cells while carrying a large polar dye as a "cargo" moiety. The extraordinary ability of this family of peptides to spontaneously translocate across bilayers without an energy source of any kind is distinctly different from the behavior of the well-known, highly cationic cell-penetrating peptides, such as the HIV tat peptide, which do not translocate across synthetic bilayers, and enter cells mostly by active endocytosis. Peptides that translocate spontaneously across membranes have the potential to transform the field of drug design by enabling the delivery of otherwise membrane-impermeant polar drugs into cells and tissues. Here we describe the chem. tools needed to rapidly identify spontaneous membrane translocating peptides.
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    Borrelli, A.; Tornesello, A. L.; Tornesello, M. L.; Buonaguro, F. M. Cell Penetrating Peptides as Molecular Carriers for Anti-Cancer Agents. Molecules 2018, 23 (2), 295,  DOI: 10.3390/molecules23020295

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    Cell penetrating peptides as molecular carriers for anti-cancer agents

    Borrelli, Antonella; Tornesello, Anna Lucia; Tornesello, Maria Lina; Buonaguro, Franco M.

    Molecules (2018), 23 (2), 295/1-295/28CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)

    Cell membranes with their selective permeability play important functions in the tight control of mol. exchanges between the cytosol and the extracellular environment as the intracellular membranes do within the internal compartments. For this reason the plasma membranes often represent a challenging obstacle to the intracellular delivery of many anti-cancer mols. The active transport of drugs through such barrier often requires specific carriers able to cross the lipid bilayer. Cell penetrating peptides (CPPs) are generally 5-30 amino acids long which, for their ability to cross cell membranes, are widely used to deliver proteins, plasmid DNA, RNA, oligonucleotides, liposomes and anti-cancer drugs inside the cells. In this review, we describe the several types of CPPs, the chem. modifications to improve their cellular uptake, the different mechanisms to cross cell membranes and their biol. properties upon conjugation with specific mols. Special emphasis has been given to those with promising application in cancer therapy.
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    Brooks, H.; Lebleu, B.; Vivès, E. Tat peptide-mediated cellular delivery: back to basics. Adv. Drug Deliv Rev. 2005, 57 (4), 559– 577,  DOI: 10.1016/j.addr.2004.12.001

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    14

    Tat peptide-mediated cellular delivery: back to basics

    Brooks, Hilary; Lebleu, Bernard; Vives, Eric

    Advanced Drug Delivery Reviews (2005), 57 (4), 559-577CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)

    A review. Peptides are emerging as attractive drug delivery tools. The HIV Tat-derived peptide is a small basic peptide that was successfully shown to deliver a large variety of cargoes, from small particles to proteins, peptides and nucleic acids. The 'transduction domain' or region conveying the cell penetrating properties appears to be confined to a small (9 amino acids) stretch of basic amino acids, with the sequence RKKRRQRRR [S. Ruben, A. Perkins, R. Purcell, K. Joung, R. Sia, R. Burghoff, W.A. Haseltine, C.A. Rosen, Structural and functional characterization of human immunodeficiency virus tat protein, J. Virol. 63 (1989) 1-8; S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 664-668; E. Vives, P. Brodin, B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272 (1997) 16010-16017; S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836-5840]. The mechanism by which the Tat peptide adheres to, and crosses, the plasma membrane of cells is currently a topic of heated discussion in the literature, with varied findings being reported. This review aims to bring together some of those findings. Peptide interactions at the cell surface, and possible mechanisms of entry, will be discussed together with the effects of modifying the basic sequence and attaching a cargo.
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    Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (24), 13003– 13008,  DOI: 10.1073/pnas.97.24.13003

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    15

    The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters

    Wender, Paul A.; Mitchell, Dennis J.; Pattabiraman, Kanaka; Pelkey, Erin T.; Steinman, Lawrence; Rothbard, Jonathan B.

    Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (24), 13003-13008CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat49-57 (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved mol. transporters that could deliver agents into cells, a series of analogs of Tat49-57 were prepd. and their cellular uptake into Jurkat cells was detd. by flow cytometry. All truncated and alanine-substituted analogs exhibited diminished cellular uptake, suggesting that the cationic residues of Tat49-57 play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat49-57 in cellular uptake. In contrast, a 9-mer of L-arginine (R9) was 20-fold more efficient than Tat49-57 at cellular uptake as detd. by Michaelis-Menton kinetic anal. The D-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat49-57 play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this anal., we designed and synthesized a class of polyguanidine peptoid derivs. Remarkably, the subset of peptoid analogs contg. a six-methylene space between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat49-57 and even to r9. Overall, a transporter has been developed that is superior to Tat49-57, protease resistant, and more readily and economically prepd.
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    Derossi, D.; Chassaing, G.; Prochiantz, A. Trojan peptides: the penetratin system for intracellular delivery. Trends in Cell Biology 1998, 8 (2), 84– 87,  DOI: 10.1016/S0962-8924(98)80017-2

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    Trojan peptides: the penetratin system for intracellular delivery

    Derossi, Daniele; Chassaing, Gerard; Prochiantz, Alain

    Trends in Cell Biology (1998), 8 (2), 84-87CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Science Ltd.)

    Internalization of exogenous macromols. by live cells provides a powerful approach for studying cellular functions. Understanding the mechanism of transfer from the extracellular milieu to the cytoplasm and nucleus could also contribute to the development of new therapeutic approaches. This article summarizes the unexpected properties of penetratins, a class of peptides with translocating properties and capable of carrying hydrophilic compds. across the plasma membrane. This unique system allows direct targeting of oligopeptides and oligonucleotides to the cytoplasm and nucleus, is non-cell-type specific and highly efficient, and therefore has several applications of potential cell-biol. and clin. interest. Penetratin-1 was a peptide of 16 amino acid residues, corresponding to amino acids 43-58 of the homeodomain (third helix) of the ANTENNAPEDIA protein. Penetratin-1 was internalized into cells and delivered directed to the cytoplasm and cell nucleus, from which it can be retrieved without apparent degrdn. The translocation was not concn. dependent between 10 pM and 100 μM. Internalization of oligonucleotides and peptides by penetratin is discussed.
17. 17

    Deshayes, S.; Heitz, A.; Morris, M. C.; Charnet, P.; Divita, G.; Heitz, F. Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis. Biochemistry 2004, 43 (6), 1449– 1457,  DOI: 10.1021/bi035682s

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    Insight into the Mechanism of Internalization of the Cell-Penetrating Carrier Peptide Pep-1 through Conformational Analysis

    Deshayes, Sebastien; Heitz, Annie; Morris, May C.; Charnet, Pierre; Divita, Gilles; Heitz, Frederic

    Biochemistry (2004), 43 (6), 1449-1457CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    Recently, the authors described a new strategy for the delivery of proteins and peptides into mammalian cells, based on an amphipathic peptide of 21 residues, Pep-1, which was designed on the basis of a protein-interacting domain assocd. with a nuclear localization sequence and sepd. by a linker. This peptide carrier constitutes a powerful tool for the delivery of active proteins or peptides both in cultured cells and in vivo, without requiring any covalent coupling. The authors have examd. the conformational states of Pep-1 in its free form and complexed with a cargo peptide and have investigated their ability to interact with phospholipids and the structural consequences of these interactions. From the conformational point of view, Pep-1 behaves significantly differently from other similarly designed cell-penetrating peptides. CD anal. revealed a transition from a nonstructured to a helical conformation upon increase of the concn. Detn. of the structure by NMR showed that in water, its α-helical domain extends from residues 4-13. CD and FTIR indicate that Pep-1 adopts a helical conformation in the presence of phospholipids. Adsorption measurements performed at the air-water interface are consistent with the helical form. Pep-1 does not undergo conformational changes upon formation of a particle with a cargo peptide. In contrast, the authors observe a partial conformational transition when the complex encounters phospholipids. The authors propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Conformational change of Pep-1 is not assocd. with complexation with its cargo but is induced upon assocn. with the cell membrane.
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    Simeoni, F.; Morris, M. C.; Heitz, F.; Divita, G. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res. 2003, 31 (11), 2717– 2724,  DOI: 10.1093/nar/gkg385

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    Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells

    Simeoni, Federica; Morris, May C.; Heitz, Frederic; Divita, Gilles

    Nucleic Acids Research (2003), 31 (11), 2717-2724CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

    The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localization sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.
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    Ghorai, S. M.; Deep, A.; Magoo, D.; Gupta, C.; Gupta, N. Cell-Penetrating and Targeted Peptides Delivery Systems as Potential Pharmaceutical Carriers for Enhanced Delivery across the Blood-Brain Barrier (BBB). Pharmaceutics 2023, 15 (7), 1999,  DOI: 10.3390/pharmaceutics15071999

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    Cell-Penetrating and Targeted Peptides Delivery Systems as Potential Pharmaceutical Carriers for Enhanced Delivery across the Blood-Brain Barrier (BBB)

    Ghorai, Soma Mondal; Deep, Auroni; Magoo, Devanshi; Gupta, Chetna; Gupta, Nikesh

    Pharmaceutics (2023), 15 (7), 1999CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)

    Among the challenges to the 21st-century health care industry, one that demands special mention is the transport of drugs/active pharmaceutical agents across the blood-brain barrier (BBB). The epithelial-like tight junctions within the brain capillary endothelium hinder the uptake of most pharmaceutical agents. With an aim to understand more deeply the intricacies of cell-penetrating and targeted peptides as a powerful tool for desirable biol. activity, we provide a crit. review of both CPP and homing/targeted peptides as intracellular drug delivery agents, esp. across the blood-brain barrier (BBB). Two main peptides have been discussed to understand intracellular drug delivery; first is the cell-penetrating peptides (CPPs) for the targeted delivery of compds. of interest (primarily peptides and nucleic acids) and second is the family of homing peptides, which specifically targets cells/tissues based on their overexpression of tumor-specific markers and are thus at the heart of cancer research. These small, amphipathic mols. demonstrate specific phys. and chem. modifications aimed at increased ease of cellular internalisation. Because only a limited no. of drug mols. can bypass the blood-brain barrier by free diffusion, it is essential to explore all aspects of CPPs that can be exploited for crossing this barrier. Considering siRNAs that can be designed against any target RNA, marking such mols. with high therapeutic potential, we present a synopsis of the studies on synthetic siRNA-based therapeutics using CPPs and homing peptides drugs that can emerge as potential drug-delivery systems as an upcoming requirement in the world of pharma- and nutraceuticals.
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    Nhàn, N. T. T.; Maidana, D. E.; Yamada, K. H. Ocular Delivery of Therapeutic Agents by Cell-Penetrating Peptides. Cells 2023, 12 (7), 1071,  DOI: 10.3390/cells12071071

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    Koren, E.; Torchilin, V. P. Cell-penetrating peptides: breaking through to the other side. Trends Mol. Med. 2012, 18 (7), 385– 393,  DOI: 10.1016/j.molmed.2012.04.012

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    Cell-penetrating peptides: breaking through to the other side

    Koren, Erez; Torchilin, Vladimir P.

    Trends in Molecular Medicine (2012), 18 (7), 385-393CODEN: TMMRCY; ISSN:1471-4914. (Elsevier Ltd.)

    A review. Cell-penetrating peptides (CPPs) have been previously shown to be powerful transport vector tools for the intracellular delivery of a large variety of cargoes through the cell membrane. Intracellular delivery of plasmid DNA (pDNA), oligonucleotides, small interfering RNAs (siRNAs), proteins and peptides, contrast agents, drugs, as well as various nanoparticulate pharmaceutical carriers (e.g., liposomes, micelles) has been demonstrated both in vitro and in vivo. This review focuses on the peptide-based strategy for intracellular delivery of CPP-modified nanocarriers to deliver small mol. drugs or DNA. In addn., we discuss the rationales for the design of smart' pharmaceutical nanocarriers in which the cell-penetrating properties are hidden until triggered by exposure to appropriate environmental conditions (e.g., a particular pH, temp., or enzyme level), applied local microwave, ultrasound, or radiofrequency radiation.
22. 22

    Reissmann, S.; Filatova, M. P. New generation of cell-penetrating peptides: Functionality and potential clinical application. J. Pept Sci. 2021, 27 (5), e3300,  DOI: 10.1002/psc.3300

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23. 23

    Crone, N. S. A.; van Hilten, N.; van der Ham, A.; Risselada, H. J.; Kros, A.; Boyle, A. L. Azobenzene-Based Amino Acids for the Photocontrol of Coiled-Coil Peptides. Bioconjug Chem. 2023, 34 (2), 345– 357,  DOI: 10.1021/acs.bioconjchem.2c00534

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24. 24

    Daudey, G. A.; Shen, M.; Singhal, A.; van der Est, P.; Sevink, G. J. A.; Boyle, A. L.; Kros, A. Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides. Chem. Sci. 2021, 12 (41), 13782– 13792,  DOI: 10.1039/D0SC06635D

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    Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides

    Daudey, Geert A.; Shen, Mengjie; Singhal, Ankush; van der Est, Patrick; Sevink, G. J. Agur; Boyle, Aimee L.; Kros, Alexander

    Chemical Science (2021), 12 (41), 13782-13792CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)

    Biol. membrane fusion is a highly specific and coordinated process as a multitude of vesicular fusion events proceed simultaneously in a complex environment with minimal off-target delivery. In this study, we develop a liposomal fusion model system with specific recognition using lipidated derivs. of a set of four de novo designed heterodimeric coiled coil (CC) peptide pairs. Content mixing was only obtained between liposomes functionalized with complementary peptides, demonstrating both fusogenic activity of CC peptides and the specificity of this model system. The diverse peptide fusogens revealed important relationships between the fusogenic efficacy and the peptide characteristics. The fusion efficiency increased from 20% to 70% as affinity between complementary peptides decreased, (from KF ≈ 108 to 104 M-1), and fusion efficiency also increased due to more pronounced asym. role-playing of membrane interacting 'K' peptides and homodimer-forming 'E' peptides. Furthermore, a new and highly fusogenic CC pair (E3/P1K) was discovered, providing an orthogonal peptide triad with the fusogenic CC pairs P2E/P2K and P3E/P3K. This E3/P1k pair was revealed, via mol. dynamics simulations, to have a shifted heptad repeat that can accommodate mismatched asparagine residues. These results will have broad implications not only for the fundamental understanding of CC design and how asparagine residues can be accommodated within the hydrophobic core, but also for drug delivery systems by revealing the necessary interplay of efficient peptide fusogens and enabling the targeted delivery of different carrier vesicles at various peptide-functionalized locations.
25. 25

    Versluis, F.; Voskuhl, J.; Vos, J.; Friedrich, H.; Ravoo, B. J.; Bomans, P. H. H.; Stuart, M. C. A.; Sommerdijk, N. A. J. M.; Kros, A. Coiled coil driven membrane fusion between cyclodextrin vesicles and liposomes. Soft Matter 2014, 10 (48), 9746– 9751,  DOI: 10.1039/C4SM01801J

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26. 26

    Crone, N. S. A.; Minnee, D.; Kros, A.; Boyle, A. L. Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning. Int. J. Mol. Sci. 2018, 19 (1), 211,  DOI: 10.3390/ijms19010211

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27. 27

    Mora, N. L.; Boyle, A. L.; van Kolck, B. J.; Rossen, A.; Pokorná, Š.; Koukalová, A.; Šachl, R.; Hof, M.; Kros, A. Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy. Sci. Rep 2020, 10 (1), 3087,  DOI: 10.1038/s41598-020-59926-z

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    27

    Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy

    Mora, Nestor Lopez; Boyle, Aimee L.; van Kolck, Bart Jan; Rossen, Anouk; Pokorna, Sarka; Koukalova, Alena; Sachl, Radek; Hof, Martin; Kros, Alexander

    Scientific Reports (2020), 10 (1), 3087CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)

    Abstr.: We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiol. conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-color time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was obsd. on the lipid bilayer of individual GUVs. In addn. to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biol. systems.
28. 28

    Malle, M. G.; Löffler, P. M. G.; Bohr, S. S.-R.; Sletfjerding, M. B.; Risgaard, N. A.; Jensen, S. B.; Zhang, M.; Hedegård, P.; Vogel, S.; Hatzakis, N. S. Single-particle combinatorial multiplexed liposome fusion mediated by DNA. Nat. Chem. 2022, 14 (5), 558– 565,  DOI: 10.1038/s41557-022-00912-5

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29. 29

    Shen, M.-J.; Olsthoorn, R. C. L.; Zeng, Y.; Bakkum, T.; Kros, A.; Boyle, A. L. Magnetic-Activated Cell Sorting Using Coiled-Coil Peptides: An Alternative Strategy for Isolating Cells with High Efficiency and Specificity. ACS Appl. Mater. Interfaces 2021, 13 (10), 11621– 11630,  DOI: 10.1021/acsami.0c22185

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    29

    Magnetic-Activated Cell Sorting Using Coiled-Coil Peptides: An Alternative Strategy for Isolating Cells with High Efficiency and Specificity

    Shen, Meng-Jie; Olsthoorn, Rene C. L.; Zeng, Ye; Bakkum, Thomas; Kros, Alexander; Boyle, Aimee L.

    ACS Applied Materials & Interfaces (2021), 13 (10), 11621-11630CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)

    Magnetic-activated cell sorting (MACS) is an affinity-based technique used to sep. cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating sepn. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid contg. a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely sepd. from nonlabeled populations. In addn., the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the sepn. of genetically modified cells from their unmodified counterparts.
30. 30

    Kong, L.; Askes, S. H. C.; Bonnet, S.; Kros, A.; Campbell, F. Temporal Control of Membrane Fusion through Photolabile PEGylation of Liposome Membranes. Angew. Chem., Int. Ed. Engl. 2016, 55 (4), 1396– 1400,  DOI: 10.1002/anie.201509673

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31. 31

    Leboux, R. J. T.; Benne, N.; van Os, W. L.; Bussmann, J.; Kros, A.; Jiskoot, W.; Slütter, B. High-affinity antigen association to cationic liposomes via coiled coil-forming peptides induces a strong antigen-specific CD4+ T-cell response. Eur. J. Pharm. Biopharm 2021, 158, 96– 105,  DOI: 10.1016/j.ejpb.2020.11.005

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    31

    High-affinity antigen association to cationic liposomes via coiled coil-forming peptides induces a strong antigen-specific CD4+ T-cell response

    Leboux, R. J. T.; Benne, N.; van Os, W. L.; Bussmann, J.; Kros, A.; Jiskoot, W.; Slutter, B.

    European Journal of Pharmaceutics and Biopharmaceutics (2021), 158 (), 96-105CODEN: EJPBEL; ISSN:0939-6411. (Elsevier B.V.)

    Liposomes are widely investigated as vaccine delivery systems, but antigen loading efficiency can be low. Moreover, adsorbed antigen may rapidly desorb under physiol. conditions. Encapsulation of antigens overcomes the latter problem but results in significant antigen loss during prepn. and purifn. of the liposomes. Here, we propose an alternative attachment method, based on a complementary heterodimeric coiled coil peptide pair pepK and pepE. PepK was conjugated to cholesterol (yielding CPK) and pepE was covalently linked to model antigen OVA323 (yielding pepE-OVA323). CPK was incorporated in the lipid bilayer of cationic liposomes (180 nm in size). Antigen was assocd. more efficiently to functionalized liposomes (Kd 166 nM) than to cationic liposomes (Kd not detectable). In vivo co-localization of antigen and liposomes was strongly increased upon CPK-functionalization (35% -> 80%). CPK-functionalized liposomes induced 5-fold stronger CD4+ T-cell proliferation than non-functionalized liposomes in vitro. Both formulations were able to induce strong CD4+ T-cell expansion in mice, but more IFN-y and IL-10 prodn. was obsd. after immunization with functionalized liposomes. In conclusion, antigen assocn. via coiled coil peptide pair increased co-localization of antigen and liposomes, increased CD4+ T-cell proliferation in vitro and induced a stronger CD4+ T-cell response in vivo.
32. 32

    Zeng, Y.; Shen, M.; Pattipeiluhu, R.; Zhou, X.; Zhang, Y.; Bakkum, T.; Sharp, T. H.; Boyle, A. L.; Kros, A. Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides. Nanoscale 2023, 15, 15026– 15218,  DOI: 10.1039/D3NR02175K

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33. 33

    Shitut, S.; Shen, M.-J.; Claushuis, B.; Derks, R. J. E.; Giera, M.; Rozen, D.; Claessen, D.; Kros, A. Generating Heterokaryotic Cells via Bacterial Cell-Cell Fusion. Microbiol Spectr 2022, 10 (4), e0169322,  DOI: 10.1128/spectrum.01693-22

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34. 34

    Mora, N. L.; Bahreman, A.; Valkenier, H.; Li, H.; Sharp, T. H.; Sheppard, D. N.; Davis, A. P.; Kros, A. Targeted anion transporter delivery by coiled-coil driven membrane fusion. Chem. Sci. 2016, 7 (3), 1768– 1772,  DOI: 10.1039/C5SC04282H

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    34

    Targeted anion transporter delivery by coiled-coil driven membrane fusion

    Mora, Nestor Lopez; Bahreman, Azadeh; Valkenier, Hennie; Li, Hongyu; Sharp, Thomas H.; Sheppard, David N.; Davis, Anthony P.; Kros, Alexander

    Chemical Science (2016), 7 (3), 1768-1772CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)

    Synthetic anion transporters (anionophores) have potential as biomedical research tools and therapeutics. However, the efficient and specific delivery of these highly lipophilic mols. to a target cell membrane is non-trivial. Here, we investigate the delivery of a powerful anionophore to artificial and cell membranes using a coiled-coil-based delivery system inspired by SNARE membrane fusion proteins. Incorporation of complementary lipopeptides into the lipid membranes of liposomes and cell-sized giant unilamellar vesicles (GUVs) facilitated the delivery of a powerful anionophore into GUVs, where its anion transport activity was monitored in real time by fluorescence microscopy. Similar results were achieved using live cells engineered to express a halide-sensitive fluorophore. We conclude that coiled-coil driven membrane fusion is a highly efficient system to deliver anionophores to target cell membranes.
35. 35

    Filipczak, N.; Pan, J.; Yalamarty, S. S. K.; Torchilin, V. P. Recent advancements in liposome technology. Adv. Drug Deliv Rev. 2020, 156, 4– 22,  DOI: 10.1016/j.addr.2020.06.022

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    35

    Recent advancements in liposome technology

    Filipczak, Nina; Pan, Jiayi; Yalamarty, Satya Siva Kishan; Torchilin, Vladimir P.

    Advanced Drug Delivery Reviews (2020), 156 (), 4-22CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)

    A review. The liposomes have continued to be well-recognized as an important nano-sized drug delivery system with attractive properties, such a characteristic bilayer structure assembling the cellular membrane, easy-to-prep. and high bio-compatibility. Extensive effort has been devoted to the development of liposome-based drug delivery systems during the past few decades. Many drug candidates have been encapsulated in liposomes and investigated for reduced toxicity and extended duration of therapeutic effect. The liposomal encapsulation of hydrophilic and hydrophobic small mol. therapeutics as well as other large mol. biologics have been established among different academic and industrial research groups. To date, there has been an increasing no. of FDA-approved liposomal-based therapeutics together with more and more undergoing clin. trials, which involve a wide range of applications in anticancer, antibacterial, and antiviral therapies. In order to meet the continuing demand for new drugs in clinics, more recent advancements have been investigated for optimizing liposomal-based drug delivery system with more reproducible prepn. technique and a broadened application to novel modalities, including nucleic acid therapies, CRISPR/Cas9 therapies and immunotherapies. This review focuses on the recent liposome' prepn. techniques, the excipients of liposomal formulations used in various novel studies and the routes of administration used to deliver liposomes to targeted areas of disease. It aims to update the research in liposomal delivery and highlights future nanotechnol. approaches.
36. 36

    Eygeris, Y.; Gupta, M.; Kim, J.; Sahay, G. Chemistry of Lipid Nanoparticles for RNA Delivery. Acc. Chem. Res. 2022, 55 (1), 2– 12,  DOI: 10.1021/acs.accounts.1c00544

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    36

    Chemistry of Lipid Nanoparticles for RNA Delivery

    Eygeris, Yulia; Gupta, Mohit; Kim, Jeonghwan; Sahay, Gaurav

    Accounts of Chemical Research (2022), 55 (1), 2-12CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)

    A review. Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a homogeneous lipid core. These vesicles are widely used in small-mol. drug and nucleic acid delivery and recently gained much attention because of their remarkable success as a delivery platform for COVID-19 mRNA vaccines. Nonetheless, the utility of transient protein expression induced by mRNA extends far beyond vaccines against infectious diseases-they also hold promise as cancer vaccines, protein replacement therapies, and gene editing components for rare genetic diseases. However, naked mRNA is inherently unstable and prone to rapid degrdn. by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects mRNA from extracellular RNases and assists with intracellular mRNA delivery. We discuss the core features of LNPs for RNA delivery. We focus our attention on LNPs designed to deliver mRNA; however, we also include examples of siRNA-LNP delivery where appropriate to highlight the commonalities and the dissimilarities due to the nucleic acid structure. First, we introduce the concept of LNPs, the advantages and disadvantages of utilizing nucleic acids as therapeutic agents, and the general reasoning behind the mol. makeup of LNPs. We also briefly highlight the most recent clin. successes of LNP-based nucleic acid therapies. Second, we describe the theory and methods of LNP self-assembly. The common idea behind all of the prepn. methods is inducing electrostatic interactions between the nucleic acid and charged lipids and promoting nanoparticle growth via hydrophobic interactions. Third, we break down the LNP compn. with special attention to the fundamental properties and purposes of each component. This includes the identified mol. design criteria, com. sourcing, impact on intracellular trafficking, and contribution to the properties of LNPs. One of the key components of LNPs is ionizable lipids, which initiate electrostatic binding with endosomal membranes and facilitate cytosolic release; however, the roles of other lipid components should not be disregarded, as they are assocd. with stability, clearance, and distribution of LNPs. Fourth, we review the attributes of LNP constructs as a whole that can heavily influence RNA delivery. These attributes are LNP size, charge, internal structure, lipid packing, lipid membrane hydration, stability, and affinity toward biomacromols. We also discuss the specific techniques used to examine these attributes and how they can be adjusted. Finally, we offer our perspective on the future of RNA therapies and some questions that remain in the realm of LNP formulation and optimization.
37. 37

    Lindgren, M.; Hällbrink, M.; Prochiantz, A.; Langel, U. Cell-penetrating peptides. Trends Pharmacol. Sci. 2000, 21 (3), 99– 103,  DOI: 10.1016/S0165-6147(00)01447-4

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    Cell-penetrating peptides

    Lindgren, Maria; Hallbrink, Mattias; Prochiantz, Alain; Langel, Ulo

    Trends in Pharmacological Sciences (2000), 21 (3), 99-103CODEN: TPHSDY; ISSN:0165-6147. (Elsevier Science Ltd.)

    A review with 42 refs. The established view in cellular biol. dictates that the cellular internalization of hydrophilic macromols. can only be achieved through the classical endocytosis pathway. However, in the past five years several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides have been used successfully for the intracellular delivery of macromols. with mol. wts. several times greater than their own. Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques because it is efficient for a range of cell types, can be applied to cells en masse and has a potential therapeutic application.
38. 38

    Liu, X.; Jiang, J.; Ji, Y.; Lu, J.; Chan, R.; Meng, H. Targeted drug delivery using iRGD peptide for solid cancer treatment. Mol. Syst. Des Eng. 2017, 2 (4), 370– 379,  DOI: 10.1039/C7ME00050B

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    Targeted drug delivery using iRGD peptide for solid cancer treatment

    Liu, Xiangsheng; Jiang, Jinhong; Ji, Ying; Lu, Jianqin; Chan, Ryan; Meng, Huan

    Molecular Systems Design & Engineering (2017), 2 (4), 370-379CODEN: MSDEBG; ISSN:2058-9689. (Royal Society of Chemistry)

    Many solid tumor types, such as pancreatic cancer, have a generally poor prognosis, in part because the delivery of a therapeutic regimen is prohibited by pathol. abnormalities that block access to tumor vasculature, leading to poor bioavailability. The recent development of the tumor-penetrating iRGD peptide that is covalently conjugated on the nanocarriers' surface or co-administered with nanocarriers becomes a popular approach for tumor targeting. More importantly, scientists have unlocked an important tumor transcytosis mechanism by which drug-carrying nanoparticles directly access solid tumors (that seems to be independent to leaky vasculature), thereby allowing systemically injected nanocarriers to more abundantly distribute at the tumor site with improved efficacy. In this focused review, we summarize the design and implementation strategy for iRGD-mediated tumor targeting. This includes the working principle of such a peptide and discussion on a patient-specific iRGD effect in vivo, commensurate with the level of key biomarker (i.e. neuropilin-1) expression in tumor vasculature. This highlights the necessity to contemplate the use of a personalized approach when iRGD technol. is used in the clinic.
39. 39

    Morshed, R. A.; Muroski, M. E.; Dai, Q.; Wegscheid, M. L.; Auffinger, B.; Yu, D.; Han, Y.; Zhang, L.; Wu, M.; Cheng, Y.; Lesniak, M. S. Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer. Mol. Pharmaceutics 2016, 13 (6), 1843– 1854,  DOI: 10.1021/acs.molpharmaceut.6b00004

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    39

    Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer

    Morshed, Ramin A.; Muroski, Megan E.; Dai, Qing; Wegscheid, Michelle L.; Auffinger, Brenda; Yu, Dou; Han, Yu; Zhang, Lingjiao; Wu, Meijing; Cheng, Yu; Lesniak, Maciej S.

    Molecular Pharmaceutics (2016), 13 (6), 1843-1854CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)

    As therapies continue to increase the lifespan of patients with breast cancer, the incidence of brain metastases has steadily increased, affecting a significant no. of patients with metastatic disease. However, a major barrier toward treating these lesions is the inability of therapeutics to penetrate into the central nervous system and accumulate within intracranial tumor sites. In this study, we designed a cell-penetrating gold nanoparticle platform to increase drug delivery to brain metastatic breast cancer cells. TAT peptide-modified gold nanoparticles carrying doxorubicin led to improved cytotoxicity toward two brain metastatic breast cancer cell lines with a decrease in the IC50 of at least 80% compared to free drug. I.v. administration of these particles led to extensive accumulation of particles throughout diffuse intracranial metastatic microsatellites with cleaved caspase-3 activity corresponding to tumor foci. Furthermore, intratumoral administration of these particles improved survival in an intracranial MDA-MB-231-Br xenograft mouse model. Our results demonstrate the promising application of gold nanoparticles for improving drug delivery in the context of brain metastatic breast cancer.
40. 40

    Jiang, T.; Zhang, Z.; Zhang, Y.; Lv, H.; Zhou, J.; Li, C.; Hou, L.; Zhang, Q. Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumor-targeted anticancer drug delivery. Biomaterials 2012, 33 (36), 9246– 9258,  DOI: 10.1016/j.biomaterials.2012.09.027

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    40

    Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumor-targeted anticancer drug delivery

    Jiang, Tianyue; Zhang, Zhenhai; Zhang, Yinlong; Lv, Huixia; Zhou, Jianping; Li, Caocao; Hou, Lulu; Zhang, Qiang

    Biomaterials (2012), 33 (36), 9246-9258CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)

    Dual-functional liposomes with pH-responsive cell-penetrating peptide (CPP) and active targeting hyaluronic acid (HA) were fabricated for tumor-targeted drug delivery. A series of synthetic tumor pH-triggered CPPs rich in arginines and histidines were screened by comparing tumor cellular uptake efficiency at pH 6.4 with at pH 7.4, and R6H4 (RRRRRRHHHH) was obtained with the optimal pH-response. To construct R6H4-modified liposomes (R6H4-L), stearyl R6H4 was anchored into liposomes due to hydrophobic interaction. HA was utilized to shield pos. charge of R6H4-L to assemble HA-coated R6H4-L (HA-R6H4-L) by electrostatic effect for protecting the liposomes from the attack of plasma proteins. The rapid degrdn. of HA by hyaluronidase (HAase) was demonstrated by the viscosity and zeta potential detection, allowing the R6H4 exposure of HA-R6H4-L at HAase-rich tumor microenvironment as the protection by HA switches off and cell-penetrating ability of R6H4 turns on. After HAase treatment, paclitaxel-loaded HA-R6H4-L (PTX/HA-R6H4-L) presented a remarkably stronger cytotoxicity toward the hepatic cancer (HepG2) cells at pH 6.4 relative to at pH 7.4, and addnl. coumarin 6-loaded HA-R6H4-L (C6/HA-R6H4-L) showed efficient intracellular trafficking including endosomal/lysosomal escape and cytoplasmic liberation by confocal laser scanning microscopy (CLSM). In vivo imaging suggested the reduced accumulation of near IR dye 15 (NIRD15)-loaded HA-R6H4-L (NIRD/HA-R6H4-L) at the tumor site, when mice were pre-treated with an excess of free HA, indicating the active tumor targeting of HA. Indeed, PTX/HA-R6H4-L had the strongest antitumor efficacy against murine hepatic carcinoma (Heps) tumor xenograft models in vivo. These findings demonstrate the feasibility of using tumor pH-sensitive CPPs and active targeting HA to extend the applications of liposomal nanocarriers to efficient anticancer drug delivery.
41. 41

    Zheng, C.-Y.; Chu, X.-Y.; Gao, C.-Y.; Hu, H.-Y.; He, X.; Chen, X.; Yang, K.; Zhang, D.-L. TAT&RGD Peptide-Modified Naringin-Loaded Lipid Nanoparticles Promote the Osteogenic Differentiation of Human Dental Pulp Stem Cells. Int. J. Nanomedicine 2022, 17, 3269– 3286,  DOI: 10.2147/IJN.S371715

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42. 42

    Sugimoto, Y.; Suga, T.; Umino, M.; Yamayoshi, A.; Mukai, H.; Kawakami, S. Investigation of enhanced intracellular delivery of nanomaterials modified with novel cell-penetrating zwitterionic peptide-lipid derivatives. Drug Deliv 2023, 30 (1), 2191891,  DOI: 10.1080/10717544.2023.2191891

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43. 43

    Carnevale, K. J. F.; Muroski, M. E.; Vakil, P. N.; Foley, M. E.; Laufersky, G.; Kenworthy, R.; Zorio, D. A. R.; Morgan, T. J.; Levenson, C. W.; Strouse, G. F. Selective Uptake Into Drug Resistant Mammalian Cancer by Cell Penetrating Peptide-Mediated Delivery. Bioconjug Chem. 2018, 29 (10), 3273– 3284,  DOI: 10.1021/acs.bioconjchem.8b00429

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    Zhu, Y.; Jiang, Y.; Meng, F.; Deng, C.; Cheng, R.; Zhang, J.; Feijen, J.; Zhong, Z. Highly efficacious and specific anti-glioma chemotherapy by tandem nanomicelles co-functionalized with brain tumor-targeting and cell-penetrating peptides. J. Controlled Release 2018, 278, 1– 8,  DOI: 10.1016/j.jconrel.2018.03.025

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    Highly efficacious and specific anti-glioma chemotherapy by tandem nanomicelles co-functionalized with brain tumor-targeting and cell-penetrating peptides

    Zhu, Yaqin; Jiang, Yu; Meng, Fenghua; Deng, Chao; Cheng, Ru; Zhang, Jian; Feijen, Jan; Zhong, Zhiyuan

    Journal of Controlled Release (2018), 278 (), 1-8CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)

    Glioma is a highly challenging human malignancy as drugs typically exhibit a low blood-brain barrier (BBB) permeability as well as poor glioma selectivity and penetration. Here, we report that tandem nanomicelles co-functionalized with brain tumor-targeting and cell-penetrating peptides, Angiopep-2 and TAT, enable a highly efficacious and specific anti-glioma chemotherapy. Interestingly, tandem nanomicelles with 20mol% Angiopep-2 and 10mol% TAT linked via long and short poly(ethylene glycol)s, resp., while maintaining a high glioma cell selectivity display markedly enhanced BBB permeation, glioma accumulation and penetration, and glioma cell uptake. We further show that docetaxel-loaded tandem nanomicelles have a long blood circulation time in mice and significantly better inhibit orthotopic U87MG human glioma than the corresponding Angiopep-2 single peptide-functionalized control, leading to an improved survival rate with little adverse effects. These tandem nanomicelles uniquely combining brain tumor-targeting and cell-penetrating functions provide a novel and effective strategy for targeted glioma therapy.
45. 45

    Li, J.; Liu, F.; Shao, Q.; Min, Y.; Costa, M.; Yeow, E. K. L.; Xing, B. Enzyme-responsive cell-penetrating peptide conjugated mesoporous silica quantum dot nanocarriers for controlled release of nucleus-targeted drug molecules and real-time intracellular fluorescence imaging of tumor cells. Adv. Healthc Mater. 2014, 3 (8), 1230– 1239,  DOI: 10.1002/adhm.201300613

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    Gabay, M.; Weizman, A.; Zeineh, N.; Kahana, M.; Obeid, F.; Allon, N.; Gavish, M. Liposomal Carrier Conjugated to APP-Derived Peptide for Brain Cancer Treatment. Cell Mol. Neurobiol 2021, 41 (5), 1019– 1029,  DOI: 10.1007/s10571-020-00969-1

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    Kato, N.; Yamada, S.; Suzuki, R.; Iida, Y.; Matsumoto, M.; Fumoto, S.; Arima, H.; Mukai, H.; Kawakami, S. Development of an apolipoprotein E mimetic peptide-lipid conjugate for efficient brain delivery of liposomes. Drug Deliv 2023, 30 (1), 2173333,  DOI: 10.1080/10717544.2023.2173333

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    Wang, L.; Wang, X.; Shen, L.; Alrobaian, M.; Panda, S. K.; Almasmoum, H. A.; Ghaith, M. M.; Almaimani, R. A.; Ibrahim, I. A. A.; Singh, T.; Baothman, A. A.; Choudhry, H.; Beg, S. Paclitaxel and naringenin-loaded solid lipid nanoparticles surface modified with cyclic peptides with improved tumor targeting ability in glioblastoma multiforme. Biomed Pharmacother 2021, 138, 111461,  DOI: 10.1016/j.biopha.2021.111461

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    48

    Paclitaxel and naringenin-loaded solid lipid nanoparticles surface modified with cyclic peptides with improved tumor targeting ability in glioblastoma multiforme

    Wang, Liying; Wang, Xiangbo; Shen, Lina; Alrobaian, Majed; Panda, Sunil K.; Almasmoum, Hussain A.; Ghaith, Mazen M.; Almaimani, Riyad A.; Ibrahim, Ibrahim Abdel Aziz; Singh, Tanuja; Baothman, Abdullah A.; Choudhry, Hani; Beg, Sarwar

    Biomedicine & Pharmacotherapy (2021), 138 (), 111461CODEN: BIPHEX; ISSN:0753-3322. (Elsevier Masson SAS)

    The present work describes the systematic development of paclitaxel and naringenin-loaded solid lipid nanoparticles (SLNs) for the treatment of glioblastoma multiforme (GBM). So far only temozolomide therapy is available for the GBM treatment, which fails by large amt. due to poor brain permeability of the drug and recurrent metastasis of the tumor. Thus, we investigated the drug combination contg. paclitaxel and naringenin for the treatment of GBM, as these drugs have individually demonstrated significant potential for the management of a wide variety of carcinoma. A systematic product development approach was adopted where risk assessment was performed for evaluating the impact of various formulation and process parameters on the quality attributes of the SLNs. I-optimal response surface design was employed for optimization of the dual drug-loaded SLNs prepd. by micro-emulsification method, where Percirol ATO5 and Dynasan 114 were used as the solid lipid and surfactant, while Lutrol F188 was used as the stabilizer. Drug loaded-SLNs were subjected to detailed in vitro and in vivo characterization studies. Cyclic RGD peptide sequence (Arg-Gly-Asp) was added to the formulation to obtain the surface modified SLNs which were also evaluated for the particle size and surface charge. The optimized drug-loaded SLNs exhibited particle size and surface charge of 129 nm and 23 mV, drug entrapment efficiency >80% and drug loading efficiency >7%. In vitro drug release study carried out by micro dialysis bag method indicated more than 70% drug was release obsd. within 8 h time period. In vivo pharmacokinetic evaluation showed significant improvement (p < 0.05) in drug absorption parameters (Cmax and AUC) from the optimized SLNs over the free drug suspension. Cytotoxicity evaluation on U87MG glioma cells indicated SLNs with higher cytotoxicity as compared to that of the free drug suspension (p < 0.05). Evaluation of uptake by florescence measurement indicated superior uptake of SLNs tagged with dye over the plain dye soln. Overall, the dual drug-loaded SLNs showed better chemoprotective effect over the plain drug soln., thus construed superior anticancer activity of the developed nanoformulation in the management of glioblastoma multiforme.
49. 49

    Qin, J.; Xue, L.; Gong, N.; Zhang, H.; Shepherd, S. J.; Haley, R. M.; Swingle, K. L.; Mitchell, M. J. RGD peptide-based lipids for targeted mRNA delivery and gene editing applications. RSC Adv. 2022, 12 (39), 25397– 25404,  DOI: 10.1039/D2RA02771B

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    Figueiredo, P.; Sipponen, M. H.; Lintinen, K.; Correia, A.; Kiriazis, A.; Yli-Kauhaluoma, J.; Österberg, M.; George, A.; Hirvonen, J.; Kostiainen, M. A.; Santos, H. A. Preparation and Characterization of Dentin Phosphophoryn-Derived Peptide-Functionalized Lignin Nanoparticles for Enhanced Cellular Uptake. Small 2019, 15 (24), e1901427,  DOI: 10.1002/smll.201901427

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    Barenholz, Y. Doxil-the first FDA-approved nano-drug: lessons learned. J. Controlled Release 2012, 160 (2), 117– 134,  DOI: 10.1016/j.jconrel.2012.03.020

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    Doxil - The first FDA-approved nano-drug: Lessons learned

    Barenholz, Yechezkel

    Journal of Controlled Release (2012), 160 (2), 117-134CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)

    A review. Doxil, the first FDA-approved nano-drug (1995), is based on three unrelated principles: (i) prolonged drug circulation time and avoidance of the RES due to the use of PEGylated nano-liposomes; (ii) high and stable remote loading of doxorubicin driven by a transmembrane ammonium sulfate gradient, which also allows for drug release at the tumor; and (iii) having the liposome lipid bilayer in a liq. ordered phase composed of the high-Tm (53 °C) phosphatidylcholine, and cholesterol. Due to the EPR effect, Doxil is passively targeted to tumors and its doxorubicin is released and becomes available to tumor cells by as yet unknown means. This review summarizes historical and scientific perspectives of Doxil development and lessons learned from its development and 20 years of its use. It demonstrates the obligatory need for applying an understanding of the cross talk between physicochem., nano-technol., and biol. principles. However, in spite of the large reward, ~ 2 years after Doxil-related patents expired, there is still no FDA-approved generic Doxil available.
52. 52

    Aldughaim, M. S.; Muthana, M.; Alsaffar, F.; Barker, M. D. Specific Targeting of PEGylated Liposomal Doxorubicin (Doxil®) to Tumour Cells Using a Novel TIMP3 Peptide. Molecules 2021, 26 (1), 100,  DOI: 10.3390/molecules26010100

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    Liu, Y.; Ran, R.; Chen, J.; Kuang, Q.; Tang, J.; Mei, L.; Zhang, Q.; Gao, H.; Zhang, Z.; He, Q. Paclitaxel loaded liposomes decorated with a multifunctional tandem peptide for glioma targeting. Biomaterials 2014, 35 (17), 4835– 4847,  DOI: 10.1016/j.biomaterials.2014.02.031

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    Paclitaxel loaded liposomes decorated with a multifunctional tandem peptide for glioma targeting

    Liu, Yayuan; Ran, Rui; Chen, Jiantao; Kuang, Qifang; Tang, Jie; Mei, Ling; Zhang, Qianyu; Gao, Huile; Zhang, Zhirong; He, Qin

    Biomaterials (2014), 35 (17), 4835-4847CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)

    The treatment of glioma is a great challenge because of the existence of the blood-brain barrier (BBB). In order to reduce toxicity to the normal brain tissue and achieve efficient treatment, it is also important for drugs to specifically accumulate in the glioma foci and penetrate into the tumor core after entering into the brain. In this study, a specific ligand cyclic RGD peptide was conjugated to a cell penetrating peptide R8 to develop a multifunctional peptide R8-RGD. R8-RGD increased the cellular uptake of liposomes by 2-fold and nearly 30-fold compared to sep. R8 and RGD resp., and displayed effective penetration of three-dimensional glioma spheroids and BBB model in vitro. In vivo studies showed that R8-RGD-lipo could be efficiently delivered into the brain and selectively accumulated in the glioma foci after systemic administration in C6 glioma bearing mice. When paclitaxel (PTX) was loaded in liposomes, R8-RGD-lipo could induce the strongest inhibition and apoptosis against C6 cells and finally achieved the longest survival in intracranial C6 glioma bearing mice. In conclusion, all the results indicated that the tandem peptide R8-RGD was a promising ligand possessing multi functions including BBB transporting, glioma targeting and tumor penetrating. And R8-RGD-lipo was proved to be a potential anti-glioma drug delivery system.
54. 54

    Uhl, P.; Sauter, M.; Hertlein, T.; Witzigmann, D.; Laffleur, F.; Hofhaus, G.; Fidelj, V.; Tursch, A.; Özbek, S.; Hopke, E.; Haberkorn, U.; Bernkop Schnürch, A.; Ohlsen, K.; Fricker, G.; Mier, W. Overcoming the Mucosal Barrier: Tetraether Lipid Stabilized Liposomal Nanocarriers Decorated with Cell Penetrating Peptides Enable Oral Delivery of Vancomycin. Advanced Therapeutics 2021, 4 (4), 2000247,  DOI: 10.1002/adtp.202000247

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    de Souza Von Zuben, E.; Eloy, J. O.; Araujo, V. H. S.; Gremiao, M. P. D.; Chorilli, M. Insulin-loaded liposomes functionalized with cell-penetrating peptides: influence on drug release and permeation through porcine nasal mucosa. Colloids Surf., A 2021, 622 (1–2), 126624,  DOI: 10.1016/j.colsurfa.2021.126624

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    Hou, X.; Zaks, T.; Langer, R.; Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 2021, 6 (12), 1078– 1094,  DOI: 10.1038/s41578-021-00358-0

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    Lipid nanoparticles for mRNA delivery

    Hou, Xucheng; Zaks, Tal; Langer, Robert; Dong, Yizhou

    Nature Reviews Materials (2021), 6 (12), 1078-1094CODEN: NRMADL; ISSN:2058-8437. (Nature Portfolio)

    A review. MRNA (mRNA) has emerged as a new category of therapeutic agent to prevent and treat various diseases. To function in vivo, mRNA requires safe, effective and stable delivery systems that protect the nucleic acid from degrdn. and that allow cellular uptake and mRNA release. Lipid nanoparticles have successfully entered the clinic for the delivery of mRNA; in particular, lipid nanoparticle-mRNA vaccines are now in clin. use against coronavirus disease 2019 (COVID-19), which marks a milestone for mRNA therapeutics. In this Review, we discuss the design of lipid nanoparticles for mRNA delivery and examine physiol. barriers and possible administration routes for lipid nanoparticle-mRNA systems. We then consider key points for the clin. translation of lipid nanoparticle-mRNA formulations, including good manufg. practice, stability, storage and safety, and highlight preclin. and clin. studies of lipid nanoparticle-mRNA therapeutics for infectious diseases, cancer and genetic disorders. Finally, we give an outlook to future possibilities and remaining challenges for this promising technol.
57. 57

    Herrera-Barrera, M.; Ryals, R. C.; Gautam, M.; Jozic, A.; Landry, M.; Korzun, T.; Gupta, M.; Acosta, C.; Stoddard, J.; Reynaga, R.; Tschetter, W.; Jacomino, N.; Taratula, O.; Sun, C.; Lauer, A. K.; Neuringer, M.; Sahay, G. Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates. Sci. Adv. 2023, 9 (2), eadd4623,  DOI: 10.1126/sciadv.add4623

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58. 58

    Asai, T.; Tsuzuku, T.; Takahashi, S.; Okamoto, A.; Dewa, T.; Nango, M.; Hyodo, K.; Ishihara, H.; Kikuchi, H.; Oku, N. Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery. Biochem. Biophys. Res. Commun. 2014, 444 (4), 599– 604,  DOI: 10.1016/j.bbrc.2014.01.107

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    Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

    Asai, Tomohiro; Tsuzuku, Takuma; Takahashi, Shoya; Okamoto, Ayaka; Dewa, Takehisa; Nango, Mamoru; Hyodo, Kenji; Ishihara, Hiroshi; Kikuchi, Hiroshi; Oku, Naoto

    Biochemical and Biophysical Research Communications (2014), 444 (4), 599-604CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)

    Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepd. for the delivery of small interfering RNA (siRNA) into cells. Lipid derivs. of CPP derived from protamine were newly synthesized and used to prep. CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Anal. of the endocytic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.
59. 59

    Bareford, L. M.; Swaan, P. W. Endocytic mechanisms for targeted drug delivery. Adv. Drug Deliv Rev. 2007, 59 (8), 748– 758,  DOI: 10.1016/j.addr.2007.06.008

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    Endocytic mechanisms for targeted drug delivery

    Bareford, Lisa M.; Swaan, Peter W.

    Advanced Drug Delivery Reviews (2007), 59 (8), 748-758CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)

    A review. Advances in the delivery of targeted drug systems have evolved to enable highly regulated site specific localization to subcellular organelles. Targeting therapeutics to individual intracellular compartments has resulted in benefits to therapies assocd. with these unique organelles. Endocytosis, a mechanism common to all cells in the body, internalizes macromols. and retains them in transport vesicles which traffic along the endolysosomal scaffold. An array of vesicular internalization mechanisms exist, therefore understanding the key players specific to each pathway has allowed researchers to bioengineer macromol. complexes for highly specialized delivery. Membrane specific receptors most frequently enter the cell through endocytosis following the binding of a high affinity ligand. High affinity ligands interact with membrane receptors, internalize in membrane bound vesicles, and traffic through cells in different manners to allow for accumulation in early endosomal fractions or lysosomally assocd. fractions. Although most drug delivery complexes aim to avoid lysosomal degrdn., more recent studies have shown the clin. utility in directed protein delivery to this environment for the enzymic release of therapeutics. Targeting nanomedicine complexes to the endolysosomal pathway has serious potential for improving drug delivery for the treatment of lysosomal storage diseases, cancer, and Alzheimer's disease. Although several issues remain for receptor specific targeting, current work is investigating a synthetic receptor approach for high affinity binding of targeted macromols.
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    Zope, H. R.; Versluis, F.; Ordas, A.; Voskuhl, J.; Spaink, H. P.; Kros, A. In vitro and in vivo supramolecular modification of biomembranes using a lipidated coiled-coil motif. Angew. Chem., Int. Ed. Engl. 2013, 52 (52), 14247– 14251,  DOI: 10.1002/anie.201306033

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    Rabe, M.; Schwieger, C.; Zope, H. R.; Versluis, F.; Kros, A. Membrane interactions of fusogenic coiled-coil peptides: implications for lipopeptide mediated vesicle fusion. Langmuir 2014, 30 (26), 7724– 7735,  DOI: 10.1021/la500987c

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    Membrane Interactions of Fusogenic Coiled-Coil Peptides: Implications for Lipopeptide Mediated Vesicle Fusion

    Rabe, Martin; Schwieger, Christian; Zope, Harshal R.; Versluis, Frank; Kros, Alexander

    Langmuir (2014), 30 (26), 7724-7735CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)

    Fusion of lipid membranes is an important natural process for the intra- and intercellular exchange of mols. However, little is known about the actual fusion mechanism at the mol. level. In this study we examine a system that models the key features of this process. For the mol. recognition between opposing membranes two membrane anchored heterodimer coiled-coil forming peptides called 'E' (EIAALEK)3 and 'K' (KIAALKE)3 were used. Lipid monolayers and IR reflection absorption spectroscopy (IRRAS) revealed the interactions of the peptides 'E', 'K', and their parallel coiled-coil complex 'E/K' with the phospholipid membranes and thereby mimicked the pre- and postfusion states, resp. The peptides adopted α-helical structures and were incorporated into the monolayers with parallel orientation. The strength of binding to the monolayer differed for the peptides and tethering them to the membrane increased the interactions even further. Remarkably, these interactions played a role even in the postfusion state. These findings shed light on important mechanistic details of the membrane fusion process in this model system. Furthermore, their implications will help to improve the rational design of new artificial membrane fusion systems, which have a wide range of potential applications in supramol. chem. and biomedicine.
62. 62

    Paramasivam, P.; Franke, C.; Stöter, M.; Höijer, A.; Bartesaghi, S.; Sabirsh, A.; Lindfors, L.; Arteta, M. Y.; Dahlén, A.; Bak, A.; Andersson, S.; Kalaidzidis, Y.; Bickle, M.; Zerial, M. Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale. J. Cell Biol. 2022, 221, e202110137,  DOI: 10.1083/jcb.202110137

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    62

    Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

    Paramasivam, Prasath; Franke, Christian; Stoeter, Martin; Hoeijer, Andreas; Bartesaghi, Stefano; Sabirsh, Alan; Lindfors, Lennart; Arteta, Marianna Yanez; Dahlen, Anders; Bak, Annette; Andersson, Shalini; Kalaidzidis, Yannis; Bickle, Marc; Zerial, Marino

    Journal of Cell Biology (2022), 221 (2), e202110137CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)

    Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chem. compn. and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resoln. microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quant. parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.
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    Shiba, Y.; Gomibuchi, T.; Seto, T.; Wada, Y.; Ichimura, H.; Tanaka, Y.; Ogasawara, T.; Okada, K.; Shiba, N.; Sakamoto, K.; Ido, D.; Shiina, T.; Ohkura, M.; Nakai, J.; Uno, N.; Kazuki, Y.; Oshimura, M.; Minami, I.; Ikeda, U. Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. Nature 2016, 538 (7625), 388– 391,  DOI: 10.1038/nature19815

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    63

    Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts

    Shiba, Yuji; Gomibuchi, Toshihito; Seto, Tatsuichiro; Wada, Yuko; Ichimura, Hajime; Tanaka, Yuki; Ogasawara, Tatsuki; Okada, Kenji; Shiba, Naoko; Sakamoto, Kengo; Ido, Daisuke; Shiina, Takashi; Ohkura, Masamichi; Nakai, Junichi; Uno, Narumi; Kazuki, Yasuhiro; Oshimura, Mitsuo; Minami, Itsunari; Ikeda, Uichi

    Nature (London, United Kingdom) (2016), 538 (7625), 388-391CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

    Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufg. and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunol. studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 wk with no evidence of immune rejection in monkeys treated with clin. relevant doses of methylprednisolone and tacrolimus, and showed elec. coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Addnl., transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 wk after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.