Apoptotic Cell Membrane-Inspired Polymer for Immunosuppression

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Apoptotic Cell Membrane-Inspired Polymer for Immunosuppression Yasuhiro Nakagawa,†,‡ Atsuhiro Saitou,∥ Takao Aoyagi,⊥ Masanobu Naito,‡,# and Mitsuhiro Ebara*,†,‡,§ †

International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1, Namiki, Tsukuba, Ibaraki, Japan ‡ Materials and Science Engineering, Graduate School of Pure and Applied Science, University of Tsukuba, Ibaraki, Japan § Department of Materials Science and Technology, Graduate School of Industrial Science and Technology, Tokyo University of Science, Tokyo, Japan ∥ Development of Medical Innovation, Osaka University Hospital, Osaka, Japan ⊥ Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo, Japan # Research Center for Strategic Materials, National Institute for Materials Science (NIMS), Ibaraki, Japan S Supporting Information *

ABSTRACT: Apoptotic cell death serves important roles in homeostasis by eliminating dangerous, damaged, or unnecessary cells without causing an inflammatory response by externalizing phosphatidylserine to the outer leaflet in the phospholipid bilayer. In this study, we newly designed apoptotic cell membrane-inspired monomer and polymer which have the phosphoryl serine group as the antiinflammatory functional moiety and demonstrate here for the first time that administration of an apoptotic cell membrane-inspired phosphorylserine polymer can protect murine macrophages (RAW 264.7) from lipopolysaccharideinduced inflammation. Interestingly, statistical copolymers with phosphorylcholine monomer that mimicked more precisely the apoptotic cell membrane result in more effective suppression of macrophage activation. This study provides new insights into the rational design of effective polymeric materials for anti-inflammatory therapies.

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no synthetic polymer materials research based on antiinflammatory therapy using the apoptotic immunosuppressive mechanism. In this work, we designed an apoptotic cell membrane-inspired polymer and demonstrated its postinflammatory effects on macrophages (Figure 1). First, we synthesized methacryloyloxyethyl phosphorylserine (MPS) using 2-hydroxyethyl methacrylate, N-α-(t-butoxycarbonyl)-L-serine t-butyl ester, and tert-butyl tetraisopropylphosphorodiamidite by the phosphoramidite method commonly used in solid phase syntheses of DNA (Scheme S1a in the Supporting Information).22 The advantage of using this reaction is its high selectivity under mild conditions when forming a phosphate ester between two hydroxyl groups (yield >78%).23 Next, poly(MPS) was obtained by free radical polymerization and then deprotection and oxidation of the PtdSer using tert-butyl peroxide and trifluoroacetic acid (Scheme S2b in the Supporting Information). The molecular weight and molecular weight dispersity were estimated to be

nflammatory responses are necessary to maintain homeostasis in our bodies; however, it has been known that impaired immunity leads to serious illnesses such as autoimmune diseases and post inflammatory injuries.1 Over the past few decades, many researchers have shown an interest in immunotolerance systems to treat these serious diseases.2−6 Numerous anti-inflammatory drugs such as steroidal,7 nonsteroidal,8 and molecular targeting drugs9 have been developed; however, these methods have low therapeutic efficacies. In recent years, apoptotic cells have attracted attention for use in immunosuppressive therapy as they have been shown to induce immunotolerance on inflammatory activated regions.10,11 For example, Voll et al. reported that apoptosis induced lymphocytes to increase secretion of anti-inflammatory cytokines and decrease secretion of pro-inflammatory cytokines.12 It has been revealed that apoptotic cells modify the morphology of an immune cell such as macrophage to an immunosuppressive phenotype via externalized phosphatidylserine (PtdSer) on their surface.13,14 Hence, many immunosuppressive treatment studies focus on using PtdSer/PtdCho (PtdCho; phosphatidylcholine) liposomes.15−17 These studies are important as they show potential for new immunotherapy materials to treat autoimmune diseases;18−21 however, there is © XXXX American Chemical Society

Received: August 9, 2017 Accepted: August 30, 2017

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DOI: 10.1021/acsmacrolett.7b00592 ACS Macro Lett. 2017, 6, 1020−1024

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Figure 1. Design of an apoptotic cell membrane-mimetic polymer for anti-inflammatory therapies.

Figure 2. (a) Optical microscope images of RAW 264.7 macrophages cultured in the presence of LPS and poly(MPS). (b) Pseudopodia ratios of RAW 264.7 macrophages cultured in the presence of LPS and poly(MPS). Effects of poly(MPS) (c) and poly(MPC) (d) on the NF-κB expression in RAW macrophages determined by SEAP reporter gene assay.

25.8k and 2.54 by GPC (Table S1). The cytotoxicity of the obtained poly(MPS) was evaluated by an Alamar blue assay on RAW264.7 and HeLa cells. High relative cell viabilities at all concentrations (up to 50 mM) were observed, indicating that poly(MPS) is biocompatible (Figure S4 in the Supporting Information).

Optical microscope images show the effect of poly(MPS) on morphological changes in macrophages, as amoeboid cell extensions in bacterial lipopolysaccharide (LPS) stimulated conditions are inhibited in poly(MPS)-treated groups (Figure 2a). Despite the amoeboid extension, the ratio of LPS added condition was reached around 80%, and poly(MPS) addition significantly inhibited the amoeboid extension ratio to below 1021

DOI: 10.1021/acsmacrolett.7b00592 ACS Macro Lett. 2017, 6, 1020−1024

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Figure 3. (a) Confocal microscope images of the RAW macrophages cultured for 24 h in the presence of rhodamine-modified poly(MPS) (TRITCpoly(MPS)). Cellular nuclear is stained by hoechst. (b) Dose-dependent distribution of poly(MPS) in the RAW macrophages (c) Incubation timedependent immunosuppressive effect of poly(MPS) against the RAW macrophages.

(MPC) had no effect on the expression of NF-κB (Figure 2d). This result indicates that although poly(MPC) does not cause any inflammation it also does not stimulate immunosuppression. This observation indicates that poly(MPS) has a potential to protect macrophages from LPS-induced inflammation. In addition, we compared the immunosuppressive effects using poly(MPS)s with different molecular weight because molecular weight is another important factor which affects recognition from macrophages. Figure S6 shows that there are no significant differences between two samples. This result indicates that the concentration of the PS group is more important for macrophage recognition than molecular weight. To investigate how macrophages recognize poly(MPS), the localization of rhodamine-modified poly(MPS) (TRITC-poly(MPS)) on RAW 264.7 cells was studied. In general, PtdSer moieties are known to be opsonized by several kinds of proteins (e.g., milk fat globule-EGF factor 8 protein (MFG-E8), growth arrest-specific 6 (Gas6), and thrombospondin) and recognized by immune cells via a PtdSer receptor such as proto-oncogene tyrosine-protein kinase MER (MERTK).13,31,32 However, the mechanisms of immunosuppression and phagocytosis of apoptotic cells are not fully understood despite many discussions in molecular biological hypotheses. Confocal microscopy images in Figure 3a show a homogeneous distribution of the poly(MPS) in the cytoplasm. The amount of absorbed poly(MPS) in the macrophages was quantified using an Alexa 488 modified poly(MPS). Several concentrations of fluorescence-modified poly(MPS) (0.1−50 mM in medium) were incubated with the macrophages for 24 h, and after washing twice with PBS, the fluorescent strength in each well was measured by a fluorescence plate reader. Figure 3b shows a linear relationship between the added and absorbed amount of poly(MPS) in the macrophages up to 40 mM. After 40 mM, no extra poly(MPS) is absorbed, indicating the maximum uptake of poly(MPS) by macrophages. In addition, comparing absorbed poly(MPS) and the suppression of NF-κB activity (Figure1c, and Figure 2b, and Figure S8), we found that 50 pg or more of poly(MPS) within each macrophage is necessary to inhibit inflammatory behavior.

20% (Figure 2b). The geometric shape of macrophages is correlated to their phenotype. For example, Porcheray et al. reported that the expression of amoeboid differentiation in RAW 264.7 is remarkable upon the addition of macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage (GM)-CSF which are involved in inflammatory activation and differentiation of macrophages and dendritic cells.24−27 In the case of this experiment, despite LPS addition induced amoeboid extension that indicates inflammatory activation to RAW macrophages as CSF addition, poly(MPS) addition suppressed that inflammatory amoeboid extension. Next, we evaluated poly(MPS) dose-dependency on immune suppressive behaviors in LPS -stimulated RAW264.7 cell lines. In this investigation, we used RAW264.7 macrophages with a chromosomal integration of a secreted alkaline phosphatase (SEAP) reporter construct inducible by nuclear factor-kappa B (NF-κB). NF-κB is a positive regulator of inflammation and an important indicator for inflammatory activity on immune cells.28 We quantify the expression amount of NF-κB on the macrophages to investigate immunosuppressive effect of obtained poly(MPS). Several concentrations of poly(MPS) (0.1−50 mM in medium) were incubated with macrophages for 24 h, and 1 μg of LPS was added. After incubation, SEAP concentrations in the culture supernatant were measured by absorption spectroscopy (λ = 655 nm). Despite that few expressions of NF-κB were observed at the no LPS addition condition, a significant increase of NF-κB expression was observed in the LPS addition sample (Figure S5 in the Supporting Information). Using this result as a standard, we plotted poly(MPS) dose-dependent NF-κB expression and confirmed the effective immunosuppression as poly(MPS) dose-dependent suppression of NF-κB was observed over 10 mM (Figure 2c). Additionally, we have examined the immunosuppressive behavior of poly-2-methacryloyloxyethyl phosphorylcholine (poly(MPC)). Poly(MPC) pioneered by Ishihara is one of the most extensively studied biomimetic polymers.29,30 MPC has often been used in biomaterials due to its exceptional biocompatibility, versatility, mechanical stability, and mechanical strength. Despite this, we found that poly1022

DOI: 10.1021/acsmacrolett.7b00592 ACS Macro Lett. 2017, 6, 1020−1024

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Figure 4. Comparison of the immunosuppressive effects of apoptotic cell-inspired polymer in different polymeric conformation: negative control (white bar), positive control (black bar), homo poly(MPS) (blue), the mixture of homo poly(MPS) and homo poly(MPC) (pink), and a statistical copolymer of poly(MPS-st-MPC) (green).

macrophages as an immunosuppressive agent for the first time. The exposure time, dosage, and composition of MPS copolymers with MPC are key factors for ideal immunosuppressive effects. The findings in this study are expected to open new doors in developing advanced immunosuppressive agents and materials for anti-inflammatory therapies.

Furthermore, we investigated the time-dependent immunosuppression induction of poly(MPS) by incubating poly(MPS)treated macrophages for 0, 1, 3, 6, and 12 h. Figure 3c shows NF-κB expression was inhibited after 3 h incubating. This finding shows that there is a minimum time for immunosuppression. This amount of time may be related to a potential immunosuppression mechanism. The Myeloid differentiation primary response gene 88 protein (MyD88) is an adaptor protein for TLR4 that is essential for responses to a broad range of pro-inflammatory stimuli.33 Furthermore, administering apoptotic cells and PtdSer liposomes to RAW macrophages induces ubiquitination to the MyD88 protein.34,35 We hypothesized that the result of time dependency of poly(MPS) immunosuppression is due to this invalidation mechanism of TLR4-related signals by degradation of the TLR adaptor protein MyD88. Finally, we designed a copolymer of MPS with MPC to mimic the apoptotic cell membrane as nearly 90% of its phospholipids are neutral, such as phosphatidylcholine and sphingomyelin. This means real apoptotic cells expose less than 10% of phosphatidylserine on their neutral phospholipid surfaces. Therefore, we synthesized a statistical copolymer of MPS and MPC (poly(MPS-st-MPC)) with a copolymerizing ratio of MPS:MPC = 1:9, similar to apoptotic cell membranes. To evaluate its immunomodulatory efficacy, we compared the immunosuppression effects among poly(MPS), a mixture of poly(MPS) and poly(MPC), and poly(MPS-st-MPC) on LPSstimulated macrophages by an SEAP reporter gene assay. The administered amount of each polymer was standardized to 5 mM phosphoryl serine units (Figure 4). No significant difference in NF-κB expression was observed in poly(MPS) (blue) and the mixture of poly(MPS) and poly(MPC) (pink) treated samples. However, the macrophages with poly(MPS-stMPC) (green) showed a significant decrease in NF-κB compared to other polymers. PtdSer in apoptotic cells is recognized by binding to receptors via opsonization with proteins such as Gas6. The anionic charge and threedimensional structure of the PtdSer headgroup are crucial components of PtdSer recognition by macrophages. Due to this, we hypothesize that the statistical copolymer of MPS and MPC showed excellent immunosuppressive efficiency and ideal opsonization due to its balanced surface electrical charge, similar to apoptotic membranes. In summary, apoptotic cell membrane-inspired polymers have been successfully synthesized and applied to LPS-activated

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00592. Materials and methods, synthesis of MPS, synthesis of poly(MPS), poly(MPC), and poly(MPS-st-MPC), characteristic data of poly(MPS), poly(MPC), and poly(MPS-st-MPC), NMR spectrum of MPS, mass spectrum of MPS, NMR spectra of poly(MPS) and intermediates, cell toxicity test of MPS on RAW 264.7 and HeLa, evaluation of NF-κB expression on RAW264.7 w/or w/o LPS, standard curve of quantification of distribution of Alexa-poly(MPS) and its microscope image, and distribution amount dependency of immunosuppression efficacy of poly(MPS) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yasuhiro Nakagawa: 0000-0002-2152-3519 Masanobu Naito: 0000-0001-7198-819X Mitsuhiro Ebara: 0000-0002-7906-0350 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to express their gratitude to the Grantsin-Aid for Scientific Research (C) (16K01402) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. The authors are grateful to Prof. Allan S. Hoffman (University of Washington) for continued and valuable discussion. This study was supported by NIMS Molecule & Material Synthesis Platform in “Nanotechnology Platform Project” operated by the MEXT, Japan. 1023

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(20) Wilson, J. T.; Keller, S.; Manganiello, M. J.; Cheng, C.; Lee, C.C.; Opara, C.; Convertine, A.; Stayton, P. S. pH-Responsive Nanoparticle Vaccines for Dual-Delivery of Antigens and Immunostimulatory Oligonucleotides. ACS Nano 2013, 7, 3912−3925. (21) Das, D.; Gerboth, D.; Postma, A.; Srinivasan, S.; Kern, H.; Chen, J.; Ratner, D. M.; Stayton, P. S.; Convertine, A. J. Synthesis of zwitterionic, hydrophobic, and amphiphilic polymers via RAFT polymerization induced self-assembly (PISA) in acetic acid. Polym. Chem. 2016, 7, 6133−6143. (22) King, R. B.; Sadanani, N. D. Dialkylaminophosphines. J. Chem. Soc., Chem. Commun. 1984, 955−956. (23) Guanti, G.; Banfi, L.; Basso, A.; Bondanza, L.; Guglieri, G.; Powles, K.; Riva, R. Optimized synthesis of phosphatidylserine. Amino Acids 2010, 39, 367−373. (24) Porcheray, F.; Viaud, S.; Rimaniol, A. C.; Léone, C.; Samah, B.; Dereuddre-Bosquet, N.; Dormont, D.; Gras, G. Macrophage activation switching: an asset for the resolution of inflammation. Clin. Exp. Immunol. 2005, 142, 481−489. (25) Ushach, I.; Zlotnik, A. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage. J. Leukocyte Biol. 2016, 100, 481−489. (26) Thorens, B.; Mermod, J.-J.; Vassalli, P. Phagocytosis and inflammatory stimuli induce GM-CSF mRNA in macrophages through posttranscriptional regulation. Cell 1987, 48, 671−679. (27) Cates, E. C.; Fattouh, R.; Wattie, J.; Inman, M. D.; Goncharova, S.; Coyle, A. J.; Gutierrez-Ramos, J.-C.; Jordana, M. Intranasal Exposure of Mice to House Dust Mite Elicits Allergic Airway Inflammation via a GM-CSF-Mediated Mechanism. J. Immunol. 2004, 173, 6384−6392. (28) Gilmore, T. D. Introduction to NF-[kappa]B: players, pathways, perspectives. Oncogene 2006, 25, 6680−6684. (29) Ishihara, K.; Aragaki, R.; Ueda, T.; Watenabe, A.; Nakabayashi, N. Reduced thrombogenicity of polymers having phospholipid polar groups. J. Biomed. Mater. Res. 1990, 24, 1069−1077. (30) Jawanda, M.; Lai, B. F. L.; Kizhakkedathu, J. N.; Ishihara, K.; Narain, R. Linear and hyperbranched phosphorylcholine based homopolymers for blood biocompatibility. Polym. Chem. 2013, 4, 3140−3146. (31) Wu, Y.; Tibrewal, N.; Birge, R. B. Phosphatidylserine recognition by phagocytes: a view to a kill. Trends Cell Biol. 2006, 16, 189−197. (32) Graham, D. K.; DeRyckere, D.; Davies, K. D.; Earp, H. S. The TAM family: phosphatidylserine-sensing receptor tyrosine kinases gone awry in cancer. Nat. Rev. Cancer 2014, 14, 769−785. (33) Fitzgerald, K. A.; Palsson-McDermott, E. M.; Bowie, A. G.; Jefferies, C. A.; Mansell, A. S.; Brady, G.; Brint, E.; Dunne, A.; Gray, P.; Harte, M. T.; McMurray, D.; Smith, D. E.; Sims, J. E.; Bird, T. A.; O’Neill, L. A. J. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 2001, 413, 78−83. (34) Huynh, M.-L. N.; Fadok, V. A.; Henson, P. M. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation. J. Clin. Invest. 2002, 109, 41−50. (35) DaMatta, R. A.; Seabra, S. H.; Deolindo, P.; Arnholdt, A. C. V.; Manhães, L.; Goldenberg, S.; de Souza, W. Trypanosoma cruzi exposes phosphatidylserine as an evasion mechanism. FEMS Microbiol. Lett. 2007, 266, 29−33.

REFERENCES

(1) Tabas, I.; Glass, C. K. Anti-Inflammatory Therapy in Chronic Disease: Challenges and Opportunities. Science 2013, 339, 166−172. (2) Libby, P.; Ridker, P. M.; Maseri, A. Inflammation and Atherosclerosis. Circulation 2002, 105, 1135−1143. (3) Huang, J.; Upadhyay, U. M.; Tamargo, R. J. Inflammation in stroke and focal cerebral ischemia. Surgical Neurology 2006, 66, 232− 245. (4) Nian, M.; Lee, P.; Khaper, N.; Liu, P. Inflammatory Cytokines and Postmyocardial Infarction Remodeling. Circ. Res. 2004, 94, 1543− 1553. (5) Austin, J. W.; Kang, C. E.; Baumann, M. D.; DiDiodato, L.; Satkunendrarajah, K.; Wilson, J. R.; Stanisz, G. J.; Shoichet, M. S.; Fehlings, M. G. The effects of intrathecal injection of a hyaluronanbased hydrogel on inflammation, scarring and neurobehavioural outcomes in a rat model of severe spinal cord injury associated with arachnoiditis. Biomaterials 2012, 33, 4555−4564. (6) Chonpathompikunlert, P.; Yoshitomi, T.; Vong, L. B.; Imaizumi, N.; Ozaki, Y.; Nagasaki, Y. Recovery of Cognitive Dysfunction via Orally Administered Redox-Polymer Nanotherapeutics in SAMP8Mice. PLoS One 2015, 10, e0126013. (7) Bunim, J. J.; Black, R. L.; Lutwak, L.; Peterson, R. E.; Whedon, G. D. Studies on dexamethasone, a new synthetic steroid, in rheumatoid arthritisa preliminary report. Arthritis Rheum. 1958, 1, 313−331. (8) Gøtzsche, P. C. Methodology and overt and hidden bias in reports of 196 double-blind trials of nonsteroidal antiinflammatory drugs in rheumatoid arthritis. Controlled Clin. Trials 1989, 10, 31−56. (9) Abdel-Razzak, Z.; Loyer, P.; Fautrel, A.; Gautier, J. C.; Corcos, L.; Turlin, B.; Beaune, P.; Guillouzo, A. Cytokines down-regulate expression of major cytochrome P-450 enzymes in adult human hepatocytes in primary culture. Mol. Pharmacol. 1993, 44, 707−715. (10) Ren, Y.; Xie, Y.; Jiang, G.; Fan, J.; Yeung, J.; Li, W.; Tam, P. K. H.; Savill, J. Apoptotic Cells Protect Mice against LipopolysaccharideInduced Shock. J. Immunol. 2008, 180, 4978−4985. (11) Thum, T.; Bauersachs, J.; Poole-Wilson, P. A.; Volk, H.-D.; Anker, S. D. The Dying Stem Cell Hypothesis. J. Am. Coll. Cardiol. 2005, 46, 1799−1802. (12) Voll, R. E.; Herrmann, M.; Roth, E. A.; Stach, C.; Kalden, J. R.; Girkontaite, I. Immunosuppressive effects of apoptotic cells. Nature 1997, 390, 350−351. (13) Birge, R. B.; Boeltz, S.; Kumar, S.; Carlson, J.; Wanderley, J.; Calianese, D.; Barcinski, M.; Brekken, R. A.; Huang, X.; Hutchins, J. T.; Freimark, B.; Empig, C.; Mercer, J.; Schroit, A. J.; Schett, G.; Herrmann, M. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ. 2016, 23, 962−978. (14) Fadok, V. A.; Bratton, D. L.; Frasch, S. C.; Warner, M. L.; Henson, P. M. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ. 1998, 5, 551−562. (15) Harel-Adar, T.; Mordechai, T. B.; Amsalem, Y.; Feinberg, M. S.; Leor, J.; Cohen, S. Modulation of cardiac macrophages by phosphatidylserine-presenting liposomes improves infarct repair. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1827−1832. (16) Hashioka, S.; Han, Y.-H.; Fujii, S.; Kato, T.; Monji, A.; Utsumi, H.; Sawada, M.; Nakanishi, H.; Kanba, S. Phosphatidylserine and phosphatidylcholine-containing liposomes inhibit amyloid β and interferon-γ-induced microglial activation. Free Radical Biol. Med. 2007, 42, 945−954. (17) Wu, Z.; Ma, H. M.; Kukita, T.; Nakanishi, Y.; Nakanishi, H. Phosphatidylserine-Containing Liposomes Inhibit the Differentiation of Osteoclasts and Trabecular Bone Loss. J. Immunol. 2010, 184, 3191−3201. (18) Uthaman, S.; Lee, S. J.; Cherukula, K.; Cho, C.-S.; Park, I.-K. Polysaccharide-Coated Magnetic Nanoparticles for Imaging and Gene Therapy. BioMed Res. Int. 2015, 2015, 14. (19) Cheng, Y.; Yumul, R. C.; Pun, S. H. Virus-Inspired Polymer for Efficient In Vitro and In Vivo Gene Delivery. Angew. Chem., Int. Ed. 2016, 55, 12013−12017. 1024

DOI: 10.1021/acsmacrolett.7b00592 ACS Macro Lett. 2017, 6, 1020−1024