This study used poly(L-lysine)20 (s-PLL20) and a sheet-like, hydrophobic polypeptide (poly(L-phenylalanine)5 (PPhe5), poly(L-alanine) 5 (PAla5), poly(L-valine)5 (PVal5) or poly(L-leucine) 5 (PLeu5)) to synthesize a star-shaped copolypeptide. The effect on the hydrogelation of those block copolopeptides based on polypeptide topology and composition was studied. It was found that except PAla, the remaining segments could form hydrogel well in an aqueous environment. It demonstrated that the hydrophobic block and the arm number affected the mechanical properties of hydrogels and hydrogelation, dictated by the balance, the interactions and the bonding between polypeptide segments. In this study, the readily available alcohols containing different numbers of −OH groups were used as star-shaped initiators to carry out ring-opening polymerization (ROP) of the lysine segment, and the nucleophilic functional group at the end of the first block was used for the second-stage polymerization to obtain a variety of the composition of diblock polymerization. A rheometer was used to test the strength properties of hydrogel under different operating conditions. It is found that these hydrogels have excellent recovery properties, and the viscoelastic properties are related to the arm number and composition of polymers. CD spectrometer and FT-IR spectroscopy were used to confirm the existence of secondary structure, and the content of secondary structure is analyzed by software. These polymers have the ability to form β-shift/β-turn structure to aid in gelation. Moreover, the star-shaped topology can affect the hydrogelation of those polypeptides due to the ability serving as interacting depots between hydrophobic block of branching chains. Comparing to the 3-armd block copolypeptides, the hydrogelation ability of 6-armed ones was better and s-PLL20-b-PPhe5 was the best among them because PPhe segment possessed the benzyl groups which could form the additional π-π interactions. This study highlighted that polypeptide composition and topology could be additional parameters to manipulate polypeptide hydrogelation. Additionally, SEM, XRD, and SAXS analyses were employed to characterize the morphology, macromolecular, and packing morphology.

[1] Appel, E. A.; del Barrio, J.; Loh, X. J.; Scherman, O. A., Supramolecular polymeric hydrogels. Chem. Soc. Rev. 2012, 41 (18), 6195-6214.
[2] Anderson, A. J.; Culver, H. R.; Bryant, S. J.; Bowman, C. N., Viscoelastic and thermoreversible networks crosslinked by non-covalent interactions between “clickable” nucleic acid oligomers and DNA. Polym. Chem. 2020, 11 (17), 2959-2968.
[3] Strandman, S.; Zhu, X., Self-healing supramolecular hydrogels based on reversible physical interactions. Gels 2016, 2 (2), 16.
[4] Barrett-Catton, E.; Ross, M. L.; Asuri, P., Multifunctional Hydrogel Nanocomposites for Biomedical Applications. Polymers 2021, 13 (6), 856.
[5] Pham, T.-N.; Su, C.-F.; Huang, C.-C.; Jan, J.-S., Biomimetic hydrogels based on L-Dopa conjugated gelatin as pH-responsive drug carriers and antimicrobial agents. Colloids Surf. B. Biointerfaces 2020, 196 111316.
[6] Hogan, K. J.; Mikos, A. G., Biodegradable thermoresponsive polymers: Applications in drug delivery and tissue engineering. Polymer 2020, 211 123063.
[7] Gao, Q.; Zhang, C.; Wang, M.; Wu, Y.; Gao, C.; Zhu, P., Injectable pH-responsive poly (γ-glutamic acid)-silica hybrid hydrogels with high mechanical strength, conductivity and cytocompatibility for biomedical applications. Polymer 2020, 197 122489.
[8] Ravichandran, R.; Sundarrajan, S.; Venugopal, J. R.; Mukherjee, S.; Ramakrishna, S., Advances in polymeric systems for tissue engineering and biomedical applications. Macromol. Biosci. 2012, 12 (3), 286-311.
[9] Li, Y.; Rodrigues, J.; Tomás, H., Injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications. Chem. Soc. Rev. 2012, 41 (6), 2193-2221.
[10] Pham, T.-N.; Jiang, Y.-S.; Su, C.-F.; Jan, J.-S., In situ formation of silver nanoparticles-contained gelatin-PEG-dopamine hydrogels via enzymatic cross-linking reaction for improved antibacterial activities. Int. J. Biol. Macromol. 2020, 146 1050-1059.
[11] Chen, Y.-F.; Chen, G.-Y.; Chang, C.-H.; Su, Y.-C.; Chen, Y.-C.; Jiang, Y.-s.; Jan, J.-S., TRAIL encapsulated to polypeptide-crosslinked nanogel exhibits increased anti-inflammatory activities in Klebsiella pneumoniae-induced sepsis treatment. Mater. Sci. Eng. C. 2019, 102 85-95.
[12] Hsu, F.-M.; Hu, M.-H.; Jiang, Y.-S.; Lin, B.-Y.; Hu, J.-J.; Jan, J.-S., Antibacterial polypeptide/heparin composite hydrogels carrying growth factor for wound healing. Mater. Sci. Eng. C. 2020, 112 110923.
[13] Sun, Y.; Wollenberg, A. L.; O’Shea, T. M.; Cui, Y.; Zhou, Z. H.; Sofroniew, M. V.; Deming, T. J., Conformation-directed formation of self-healing diblock copolypeptide hydrogels via polyion complexation. J. Am. Chem. Soc. 2017, 139 (42), 15114-15121.
[14] Huang, J.; Hastings, C. L.; Duffy, G. P.; Kelly, H. M.; Raeburn, J.; Adams, D. J.; Heise, A., Supramolecular hydrogels with reverse thermal gelation properties from (oligo) tyrosine containing block copolymers. Biomacromolecules 2013, 14 (1), 200-206.
[15] O’Brien, S.; Brannigan, R. P.; Ibanez, R.; Wu, B.; O’Dwyer, J.; O’Brien, F. J.; Cryan, S.-A.; Heise, A., Biocompatible polypeptide-based interpenetrating network (IPN) hydrogels with enhanced mechanical properties. J. Mater. Chem. B 2020, 8 (34), 7785-7791.
[16] Hou, S.-S.; Fan, N.-S.; Tseng, Y.-C.; Jan, J.-S., Self-Assembly and Hydrogelation of Coil–Sheet Poly (l-lysine)-block-poly (l-threonine) Block Copolypeptides. Macromolecules 2018, 51 (20), 8054-8063.
[17] Gaspard, J.; Silas, J. A.; Shantz, D. F.; Jan, J.-S., Supramolecular assembly of lysine-b-glycine block copolypeptides at different solution conditions. Supramol. Chem. 2010, 22 (3), 178-185.
[18] Huang, Y.-C.; Arham, M.; Jan, J.-S., Alkyl chain grafted poly (l-lysine): self-assembly and biomedical application as carriers. Soft Matter 2011, 7 (8), 3975-3983.
[19] Zhang, Y.-X.; Chen, Y.-F.; Shen, X.-Y.; Hu, J.-J.; Jan, J.-S., Reduction-and pH-Sensitive lipoic acid-modified Poly (l-lysine) and polypeptide/silica hybrid hydrogels/nanogels. Polymer 2016, 86 32-41.
[20] Cheng, J.; Deming, T. J., Synthesis of polypeptides by ring-opening polymerization of α-amino acid N-carboxyanhydrides. Peptide-based materials 2011, 1-26.
[21] Aoi, K.; Tsutsumiuchi, K.; Okada, M., Glycopeptide Synthesis by an. alpha.-Amino Acid N-Carboxyanhydride (NCA) Method: Ring-Opening Polymerization of a Sugar-Substituted NCA. Macromolecules 1994, 27 (3), 875-877.
[22] Jiang, J.; Zhang, X.; Fan, Z.; Du, J., Ring-Opening Polymerization of N-Carboxyanhydride-Induced Self-Assembly for Fabricating Biodegradable Polymer Vesicles. ACS Macro Lett. 2019, 8 (10), 1216-1221.
[23] Zhou, P.; Dai, X.-G.; Kong, J.; Ling, J., Synthesis of Well-defined Poly (tetrahydrofuran)-b-Poly (a-amino acid) s via Cationic Ring-opening Polymerization (ROP) of Tetrahydrofuran and Nucleophilic ROP of N-thiocarboxyanhydrides. Chin. J. Polym. Sci. 2021, 1-7.
[24] Marzano, M.; Falanga, A. P.; Marasco, D.; Borbone, N.; D’Errico, S.; Piccialli, G.; Roviello, G. N.; Oliviero, G., Evaluation of an analogue of the marine ε-PLL peptide as a ligand of G-quadruplex DNA structures. Mar. Drugs 2020, 18 (1), 49.
[25] Deming, T. J., Synthesis of side-chain modified polypeptides. Chem. Rev. 2016, 116 (3), 786-808.
[26] Hou, S.-S.; Hsu, Y.-Y.; Lin, J.-H.; Jan, J.-S., Alkyl-poly (L-threonine)/cyclodextrin supramolecular hydrogels with different molecular assemblies and gel properties. ACS Macro Lett. 2016, 5 (11), 1201-1205.
[27] Hsiao, L.-W.; Lai, Y.-D.; Lai, J.-T.; Hsu, C.-C.; Wang, N.-Y.; Steven, S.-S. W.; Jan, J.-S., Cross-linked polypeptide-based gel particles by emulsion for efficient protein encapsulation. Polymer 2017, 115 261-272.
[28] Shen, X.-Y.; Tang, C.-C.; Jan, J.-S., Synthesis and hydrogelation of star-shaped poly (L-lysine) polypeptides modified with different functional groups. Polymer 2018, 151 108-116.
[29] Shen, Y.; Zhang, S.; Wan, Y.; Fu, W.; Li, Z., Hydrogels assembled from star-shaped polypeptides with a dendrimer as the core. Soft Matter 2015, 11 (15), 2945-2951.
[30] Murphy, R.; Borase, T.; Payne, C.; O'Dwyer, J.; Cryan, S.-A.; Heise, A., Hydrogels from amphiphilic star block copolypeptides. RSC Adv. 2016, 6 (28), 23370-23376.
[31] Tang, C.-C.; Zhang, S.-H.; My Phan, T. H.; Tseng, Y.-C.; Jan, J.-S., Block length and topology affect self-assembly and gelation of poly(L-lysine)-block-poly(S-benzyl-l-cysteine) block copolypeptides. Polymer 2021, 123891.
[32] Chen, Y.-F.; Lai, Y.-D.; Chang, C.-H.; Tsai, Y.-C.; Tang, C.-C.; Jan, J.-S., Star-shaped polypeptides exhibit potent antibacterial activities. Nanoscale 2019, 11 (24), 11696-11708.
[33] Breedveld, V.; Nowak, A. P.; Sato, J.; Deming, T. J.; Pine, D. J., Rheology of block copolypeptide solutions: hydrogels with tunable properties. Macromolecules 2004, 37 (10), 3943-3953.
[34] Zhang, D.; Qi, D.; Wang, J.; Yu, S.; He, C.; Deng, M., Effects of ethyl-L-glutamated and phenylalanine ratio/sequence on the secondary structure and gelation properties of their PEGylated copolymers. Polymer 2020, 191 122276.
[35] Thornton, P. D.; Billah, S. M. R.; Cameron, N. R., Enzyme‐Degradable Self‐Assembled Hydrogels From Polyalanine‐Modified Poly (ethylene glycol) Star Polymers. Macromol. Rapid Commun. 2013, 34 (3), 257-262.
[36] Garrett, R.; Grisham, C. M., Biochemistry, Cengage Learning. Inc., Boston 2008,
[37] Schulz, G. E.; Schirmer, R. H., Patterns of folding and association of polypeptide chains. In Principles of Protein Structure, Springer: 1979; pp 66-107.
[38] Merrifield, R. B., Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85 (14), 2149-2154.
[39] Löfblom, J., Bacterial display in combinatorial protein engineering. Biotechnology journal 2011, 6 (9), 1115-1129.
[40] Yazawa, K.; Numata, K., Recent advances in chemoenzymatic peptide syntheses. Molecules 2014, 19 (9), 13755-13774.
[41] Deming, T. J., Polypeptide and polypeptide hybrid copolymer synthesis via NCA polymerization. Peptide hybrid polymers 2006, 1-18.
[42] Deming, T. J., Facile synthesis of block copolypeptides of defined architecture. Nature 1997, 390 (6658), 386-389.
[43] Pahovnik, D.; Hadjichristidis, N., Ring-Opening Polymerization of N-Carboxyanhydrides for Preparation of Polypeptides and Polypeptide-Based Hybrid Materials with Various Molecular Architectures. In Anionic Polymerization, Springer: 2015; pp 307-337.
[44] Deng, C.; Wu, J.; Cheng, R.; Meng, F.; Klok, H.-A.; Zhong, Z., Functional polypeptide and hybrid materials: Precision synthesis via α-amino acid N-carboxyanhydride polymerization and emerging biomedical applications. Prog. Polym. Sci. 2014, 39 (2), 330-364.
[45] Daly, W. H.; Poché, D., The preparation of N-carboxyanhydrides of α-amino acids using bis (trichloromethyl) carbonate. Tetrahedron Lett. 1988, 29 (46), 5859-5862.
[46] Mansky, P.; Liu, Y.; Huang, E.; Russell, T.; Hawker, C., Controlling polymer-surface interactions with random copolymer brushes. Science 1997, 275 (5305), 1458-1460.
[47] Jarosz, T.; Gebka, K.; Stolarczyk, A., Recent advances in conjugated graft copolymers: Approaches and applications. Molecules 2019, 24 (16), 3019.
[48] Arandia, I.; Meabe, L.; Aranburu, N.; Sardon, H.; Mecerreyes, D.; Müller, A. J., Influence of chemical structures on isodimorphic behavior of three different copolycarbonate random copolymer series. Macromolecules 2020, 53 (2), 669-681.
[49] Pérez-Camargo, R. A.; Arandia, I.; Safari, M.; Cavallo, D.; Lotti, N.; Soccio, M.; Müller, A. J., Crystallization of isodimorphic aliphatic random copolyesters: Pseudo-eutectic behavior and double-crystalline materials. Eur. Polym. J. 2018, 101 233-247.
[50] Singh, R. P.; Nayak, B. R.; Biswal, D. R.; Tripathy, T.; Banik, K., Biobased polymeric flocculants for industrial effluent treatment. Mater. Res. Innovations 2003, 7 (5), 331-340.
[51] Mishra, S.; Sinha, S.; Dey, K. P.; Sen, G., Synthesis, characterization and applications of polymethylmethacrylate grafted psyllium as flocculant. Carbohydr. Polym. 2014, 99 462-468.
[52] Ding, J.; Zhuang, X.; Xiao, C.; Cheng, Y.; Zhao, L.; He, C.; Tang, Z.; Chen, X., Preparation of photo-cross-linked pH-responsive polypeptide nanogels as potential carriers for controlled drug delivery. J. Mater. Chem. 2011, 21 (30), 11383-11391.
[53] Kumar, D.; Pandey, J.; Raj, V.; Kumar, P., A review on the modification of polysaccharide through graft copolymerization for various potential applications. The open medicinal chemistry journal 2017, 11 109.
[54] Huang, C.-J.; Chang, F.-C., Polypeptide diblock copolymers: syntheses and properties of poly (N-isopropylacrylamide)-b-polylysine. Macromolecules 2008, 41 (19), 7041-7052.
[55] Shim, W. S.; Yoo, J. S.; Bae, Y. H.; Lee, D. S., Novel injectable pH and temperature sensitive block copolymer hydrogel. Biomacromolecules 2005, 6 (6), 2930-2934.
[56] He, C.; Kim, S. W.; Lee, D. S., In situ gelling stimuli-sensitive block copolymer hydrogels for drug delivery. J. Controlled Release 2008, 127 (3), 189-207.
[57] Kissel, T.; Li, Y.; Unger, F., ABA-triblock copolymers from biodegradable polyester A-blocks and hydrophilic poly (ethylene oxide) B-blocks as a candidate for in situ forming hydrogel delivery systems for proteins. Adv. Drug Del. Rev. 2002, 54 (1), 99-134.
[58] Hamley, I. W., Developments in block copolymer science and technology. John Wiley & Sons, (2004).
[59] Murphy, R. D.; Cryan, S.-A.; Heise, A., Disulphide crosslinked star block copolypeptide hydrogels: influence of block sequence order on hydrogel properties. Polym. Chem. 2018, 9 (28), 3908-3916.
[60] Van Bemmelen, J., Das hydrogel und das krystallinische hydrat des kupferoxyds. Zeitschrift für anorganische Chemie 1894, 5 (1), 466-483.
[61] Hoffman, A. S., Hydrogels for biomedical applications. Adv. Drug Del. Rev. 2012, 64 18-23.
[62] Abandansari, H. S.; Aghaghafari, E.; Nabid, M. R.; Niknejad, H., Preparation of injectable and thermoresponsive hydrogel based on penta-block copolymer with improved sol stability and mechanical properties. Polymer 2013, 54 (4), 1329-1340.
[63] Sawamura, K.; Ikeda, T.; Nagae, M.; Okamoto, S.-i.; Mikami, Y.; Hase, H.; Ikoma, K.; Yamada, T.; Sakamoto, H.; Matsuda, K.-i., Characterization of in vivo effects of platelet-rich plasma and biodegradable gelatin hydrogel microspheres on degenerated intervertebral discs. Tissue Engineering Part A 2009, 15 (12), 3719-3727.
[64] Hamedi, H.; Moradi, S.; Hudson, S. M.; Tonelli, A. E., Chitosan based hydrogels and their applications for drug delivery in wound dressings: A review. Carbohydr. Polym. 2018, 199 445-460.
[65] Pupkaite, J.; Rosenquist, J.; Hilborn, J. n.; Samanta, A., Injectable Shape-Holding Collagen Hydrogel for Cell Encapsulation and Delivery Cross-linked Using Thiol-Michael Addition Click Reaction. Biomacromolecules 2019, 20 (9), 3475-3484.
[66] Lee, J. H., Injectable hydrogels delivering therapeutic agents for disease treatment and tissue engineering. Biomaterials research 2018, 22 (1), 1-14.
[67] Yu, F.; Cao, X.; Li, Y.; Zeng, L.; Yuan, B.; Chen, X., An injectable hyaluronic acid/PEG hydrogel for cartilage tissue engineering formed by integrating enzymatic crosslinking and Diels–Alder “click chemistry”. Polym. Chem. 2013, 5 (3), 1082-1090.
[68] Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J., Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles. Nature 2002, 417 (6887), 424-428.
[69] Li, Z.; Wang, F.; Roy, S.; Sen, C. K.; Guan, J., Injectable, highly flexible, and thermosensitive hydrogels capable of delivering superoxide dismutase. Biomacromolecules 2009, 10 (12), 3306-3316.
[70] Buwalda, S. J.; Boere, K. W.; Dijkstra, P. J.; Feijen, J.; Vermonden, T.; Hennink, W. E., Hydrogels in a historical perspective: From simple networks to smart materials. J. Controlled Release 2014, 190 254-273.
[71] Hirokawa, Y.; Okamoto, T.; Kimishima, K.; Jinnai, H.; Koizumi, S.; Aizawa, K.; Hashimoto, T., Sponge-like heterogeneous gels: hierarchical structures in poly (N-isopropylacrylamide) chemical gels as observed by combined scattering and confocal microscopy method. Macromolecules 2008, 41 (21), 8210-8219.
[72] Zhang, J.-T.; Bhat, R.; Jandt, K. D., Temperature-sensitive PVA/PNIPAAm semi-IPN hydrogels with enhanced responsive properties. Acta Biomater. 2009, 5 (1), 488-497.
[73] Hua, J.; Li, Z.; Xia, W.; Yang, N.; Gong, J.; Zhang, J.; Qiao, C., Preparation and properties of EDC/NHS mediated crosslinking poly (gamma-glutamic acid)/epsilon-polylysine hydrogels. Mater. Sci. Eng. C. 2016, 61 879-892.
[74] Rizwan, M.; Yahya, R.; Hassan, A.; Yar, M.; Azzahari, A. D.; Selvanathan, V.; Sonsudin, F.; Abouloula, C. N., pH sensitive hydrogels in drug delivery: Brief history, properties, swelling, and release mechanism, material selection and applications. Polymers 2017, 9 (4), 137.
[75] Suzuki, M.; Hayakawa, Y.; Hanabusa, K., Thixotropic supramolecular gel based on L-lysine derivatives. Gels 2015, 1 (1), 81-93.
[76] Wu, D.; Xie, X.; Kadi, A. A.; Zhang, Y., Photosensitive peptide hydrogels as smart materials for applications. Chin. Chem. Lett. 2018, 29 (7), 1098-1104.
[77] Kim, M. S.; Dayananda, K.; Choi, E. K.; Park, H. J.; Kim, J. S.; Lee, D. S., Synthesis and characterization of poly (l-glutamic acid)-block-poly (l-phenylalanine). Polymer 2009, 50 (10), 2252-2257.
[78] Kousar, A.; Liu, J.; Mehwish, N.; Wang, F.; Dang-i, A.; Feng, C., pH-Regulated supramolecular chirality of phenylalanine-based hydrogels. Mater. Today. Chem. 2019, 11 217-224.
[79] Triftaridou, A. I.; Chécot, F.; Iliopoulos, I., Poly (N, N‐dimethylacrylamide)‐block‐Poly (L‐lysine) Hybrid Block Copolymers: Synthesis and Aqueous Solution Characterization. Macromol. Chem. Phys. 2010, 211 (7), 768-777.
[80] Cheng, Y.; He, C.; Xiao, C.; Ding, J.; Zhuang, X.; Huang, Y.; Chen, X., Decisive role of hydrophobic side groups of polypeptides in thermosensitive gelation. Biomacromolecules 2012, 13 (7), 2053-2059.
[81] Zhang, S.; Fu, W.; Li, Z., Supramolecular hydrogels assembled from nonionic poly (ethylene glycol)-b-polypeptide diblocks containing OEGylated poly-L-glutamate. Polym. Chem. 2014, 5 (10), 3346-3351.
[82] Chen, B.-Y.; Huang, Y.-C.; Jan, J.-S., Molecular assembly of alkyl chain-grafted poly (L-lysine) tuned by backbone chain length and grafted alkyl chain. RSC Adv. 2015, 5 (29), 22783-22791.
[83] Chen, B.-Y.; Huang, Y.-F.; Huang, Y.-C.; Wen, T.-C.; Jan, J.-S., Alkyl chain-grafted poly (L-lysine) vesicles with tunable molecular assembly and membrane permeability. ACS Macro Lett. 2014, 3 (3), 220-223.
[84] Chen, Y.-F.; Shiau, A.-L.; Chang, S.-J.; Fan, N.-S.; Wang, C.-T.; Wu, C.-L.; Jan, J.-S., One-dimensional poly (L-lysine)-block-poly (L-threonine) assemblies exhibit potent anticancer activity by enhancing membranolysis. Acta Biomater. 2017, 55 283-295.
[85] Holowka, E. P.; Pochan, D. J.; Deming, T. J., Charged polypeptide vesicles with controllable diameter. J. Am. Chem. Soc. 2005, 127 (35), 12423-12428.
[86] Hoffman, R. L., Explanations for the cause of shear thickening in concentrated colloidal suspensions. J. Rheol. 1998, 42 (1), 111-123.
[87] Chu, B., Laser light scattering: basic principles and practice. Courier Corporation, (2007).
[88] Tsai, Y.-L.; Tseng, Y.-C.; Chen, Y.-M.; Wen, T.-C.; Jan, J.-S., Zwitterionic polypeptides bearing carboxybetaine and sulfobetaine: synthesis, self-assembly, and their interactions with proteins. Polym. Chem. 2018, 9 (10), 1178-1189.