Revolutionizing Antimicrobial Biomaterials: Integrating an Enzyme Degradation-Resistant Sequence into Self-Assembled Nanosystems to Overcome Stability Limitations of Peptide-Based Drugs

Abstract

Incorporating enzyme-resistant peptide sequences into self-assembled nanosystems is a promising strategy to enhance the stability and versatility of peptide-based antibacterial drugs, aiming to replace ineffective antibiotics. By combining newly designed enzymatic-resistant sequences with synthetically derived compounds bearing single, double, triple, or quadruple aromatic rings. A series of nanoscale antimicrobial self-assembled short peptides for the purpose of combating bacterial infections are generated. Nap* (Nap–^DNal–Nal–Dab–Dab–NH₂, where Nap represents the 1-naphthylacetyl group) possesses the greatest clinical potential (GM_SI = 23.96) among the peptides in this series. At high concentrations in an aqueous environment, Nap* spontaneously generates nanofibers to capture bacteria and prevent their evasion, exhibiting broad-spectrum antimicrobial effects and exceptional biocompatibility. In the presence of physiological salt ions and serum, the antimicrobial agent exhibits strong effectiveness and retains impressive resistance even when exposed to high levels of proteases (trypsin, chymotrypsin, pepsin). Nap* exhibits negligible in vivo toxicity and effectively alleviates systemic bacterial infections in mice. Mechanistically, Nap* initially captures bacteria and induces bacterial cell death primarily through membrane dissolution, achieved by multiple synergistic mechanisms. In summary, these advances have the potential to greatly expedite the clinical evolution of nanomaterials based on short peptides combined with naphthyl groups and foster the development of peptides integrated with self-assembled systems in this domain.

Keywords

Naphthyl / Nanofibers / Antimicrobial peptide / Self-assembled / Protease resistance / Antibacterial mechanism

References

[1] Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini C, Kahlmeter G, Kluytmans J, Carmeli Y, Ouellette M, Outterson K, Patel J, Cavaleri M, Cox EM, Houchens CR, Grayson ML, Hansen P, Singh N, Theuretzbacher U, Magrini N. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis, 2018, 18: 318-327,

[2] Magana M, Pushpanathan M, Santos AL, Leanse L, Fernandez M, Ioannidis A, Giulianotti MA, Apidianakis Y, Bradfute S, Ferguson AL, Cherkasov A, Seleem MN, Pinilla C, de la Fuente-Nunez C, Lazaridis T, Dai T, Houghten RA, Hancock REW, Tegos GP. The value of antimicrobial peptides in the age of resistance. Lancet Infect Dis, 2020, 20: e216-e230,

[3] Gao N, Wang JJ, Fang CY, Bai PF, Sun Y, Wu WP, Shan AS. Combating bacterial infections with host defense peptides: shifting focus from bacteria to host immunity. Drug Resist Updat, 2024, 72: 101030,

[4] Li WL, Xiao XM, Qi YC, Lin XH, Hu HQ, Shi MQ, Jiang WA, Liu LQ, Chen K, Wang K, Liu RH, Zhou M. Host-defense-peptide-mimicking β-peptide polymer acting as a dual-modal antibacterial agent by interfering quorum sensing and killing individual bacteria simultaneously. Research, 2023, 6: 0051,

[5] Shan JY, Che JY, Song CH, Zhao YJ. Emerging antibacterial nanozymes for wound healing. Smart Med, 2023,

[6] Abadehie FS, Dehkordi AH, Zafari M, Bagheri M, Yousefiasl S, Pourmotabed S, Mahmoodnia L, Validi M, Ashrafizadeh M, Zare EN, Rabiee N, Makvandi P, Sharifi E. Lawsone-encapsulated chitosan/polyethylene oxide nanofibrous mat as a potential antibacterial biobased wound dressing. Eng Regen, 2021,

[7] Fang YX, Zhu YH, Li L, Lai ZH, Dong N, Shan AS. Biomaterial-interrelated bacterial sweeper: simplified self-assembled octapeptides with double-layered trp zipper induces membrane destabilization and bacterial apoptosis-like death. Small Methods, 2021, 5: e2101304,

[8] Wang CS, Shao CX, Fang YX, Wang JJ, Dong N, Shan AS. Binding loop of sunflower trypsin inhibitor 1 serves as a design motif for proteolysis-resistant antimicrobial peptides. Acta Biomater, 2021, 124: 254-269,

[9] Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, Cao W, Wang YH, Su B, Nestle FO, Zal T, Mellman I, Schröder JM, Liu YJ, Gilliet M. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature, 2007, 449: 564-569,

[10] Chu H, Pazgier M, Jung G, Nuccio SP, Castillo PA, de Jong MF, Winter MG, Winter SE, Wehkamp J, Shen B, Salzman NH, Underwood MA, Tsolis RM, Young GM, Lu W, Lehrer RI, Bäumler AJ, Bevins CL. Human α-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets. Science, 2012, 337: 477-481,

[11] Yu WK, Wang JJ, Wang ZH, Li LX, Li WY, Song J, Zhang SS, Shan AS. PEGylation of the antimicrobial peptide PG-1: a link between propensity for nanostructuring and capacity of the antitrypsin hydrolytic ability. J Med Chem, 2021, 64: 10469-10481,

[12] Yu WK, Sun Y, Li WY, Guo X, Liu XS, Wu WP, Yu WQ, Wang JJ, Shan AS. Self-assembly of antimicrobial peptide-based micelles breaks the limitation of trypsin. ACS Appl Mater Interfaces, 2023, 15: 494-510,

[13] Lai ZH, Yuan XJ, Chen HY, Zhu YH, Dong N, Shan AS. Strategies employed in the design of antimicrobial peptides with enhanced proteolytic stability. Biotechnol Adv, 2022, 59: 107962,

[14] Li GY, Lai ZH, Shan AS. Advances of antimicrobial peptide-based biomaterials for the treatment of bacterial infections. Adv Sci, 2023, 10: e2206602,

[15] Di YP, Lin Q, Chen C, Montelaro RC, Doi Y, Deslouches B. Enhanced therapeutic index of an antimicrobial peptide in mice by increasing safety and activity against multidrug-resistant bacteria. Sci Adv, 2020, 6: eaay6817,

[16] Pan M, Lu C, Zheng MC, Zhou W, Song FL, Chen WD, Yao F, Liu DJ, Cai JF. Unnatural amino-acid-based star-shaped poly(l-Ornithine)s as emerging long-term and biofilm-disrupting antimicrobial peptides to treat pseudomonas aeruginosa-infected burn wounds. Adv Healthc Mater, 2020, 9: e2000647,

[17] Wang JJ, Song J, Yang ZY, He SQ, Yang Y, Feng XJ, Dou XJ, Shan AS. Antimicrobial peptides with high proteolytic resistance for combating gram-negative bacteria. J Med Chem, 2019, 62: 2286-2304,

[18] He SQ, Yang ZY, Li XF, Wu H, Zhang LC, Shan AS, Wang JJ. Boosting stability and therapeutic potential of proteolysis-resistant antimicrobial peptides by end-tagging β-naphthylalanine. Acta Biomater, 2023, 164: 175-194,

[19] He SQ, Yang ZY, Li XF, Zhang LC, Wang JJ, Shan AS. Optimized proteolytic resistance motif (DabW)-based U1-2WD: a membrane-induced self-aggregating peptide to trigger bacterial agglutination and death. Acta Biomater, 2022, 153: 540-556,

[20] Zhu YJ, Shao CX, Li GY, Lai ZH, Tan P, Jian Q, Cheng BJ, Shan AS. Rational avoidance of protease cleavage sites and symmetrical end-tagging significantly enhances the stability and therapeutic potential of antimicrobial peptides. J Med Chem, 2020, 63: 9421-9435,

[21] Shao CX, Jian Q, Li BW, Zhu YJ, Yu WK, Li ZY, Shan AS. Ultrashort all-hydrocarbon stapled α-helix amphiphile as a potent and stable antimicrobial compound. J Med Chem, 2023, 66: 11414-11427,

[22] Cai XM, Dong J, Liu J, Zheng HZ, Kaweeteerawat C, Wang FJ, Ji ZX, Li RB. Multi-hierarchical profiling the structure-activity relationships of engineered nanomaterials at nano-bio interfaces. Nat Commun, 2018, 9: 4416,

[23] Cruz MA, Bohinc D, Andraska EA, Alvikas J, Raghunathan S, Masters NA, van Kleef ND, Bane KL, Hart K, Medrow K, Sun M, Liu H, Haldeman S, Banerjee A, Lessieur EM, Hageman K, Gandhi A, de la Fuente M, Nieman MT, Kern TS, Maas C, de Maat S, Neeves KB, Neal MD, Gupta AS, Stavrou EX. Nanomedicine platform for targeting activated neutrophils and neutrophil-platelet complexes using an α(1)-antitrypsin-derived peptide motif. Nat Nanotechnol, 2022, 17: 1004-1014,

[24] Herrera-Barrera M, Ryals RC, 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 AK, Neuringer M, Sahay G. Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates. Sci Adv, 2023, 9: eadd4623,

[25] An HW, Mamuti M, Wang XF, Yao HD, Wang MD, Zhao LN, Li LL. Rationally designed modular drug delivery platform based on intracellular peptide self-assembly. Exploration, 2021,

[26] Feng XF, Hou XL, Cui CJ, Sun SB, Sadik S, Wu SH, Zhou F. Mechanical and antibacterial properties of tannic acid-encapsulated carboxymethyl chitosan/polyvinyl alcohol hydrogels. Eng Regen, 2021,

[27] Chen Y, Yuan ZC, Sun WY, Shafiq M, Zhu J, Chen JF, Tang H, Hu L, Lin WK, Zeng YX, Wang L, Zhang L, She YL, Zheng H, Zhao GF, Xie D, Mo XM, Chen C. Vascular endothelial growth factor-recruiting nanofiber bandages promote multifunctional skin regeneration via improved angiogenesis and immunomodulation. Adv Fiber Mater, 2023, 5: 327-348,

[28] Wang ZJ, Hu WK, Wang W, Xiao Y, Chen Y, Wang XH. Antibacterial electrospun nanofibrous materials for wound healing. Adv Fiber Mater, 2023, 5: 107-129,

[29] Lai ZH, Jian Q, Li GY, Shao CX, Zhu YJ, Yuan XJ, Chen HY, Shan AS. Self-assembling peptide dendron nanoparticles with high stability and a multimodal antimicrobial mechanism of action. ACS Nano, 2021, 15: 15824-15840,

[30] Li P, Zhou C, Rayatpisheh S, Ye K, Poon YF, Hammond PT, Duan HW, Chan-Park MB. Cationic peptidopolysaccharides show excellent broad-spectrum antimicrobial activities and high selectivity. Adv Mater, 2012, 24: 4130-4137,

[31] Wang C, Hong TT, Cui PF, Wang JH, Xia J. Antimicrobial peptides towards clinical application: delivery and formulation. Adv Drug Deliv Rev, 2021, 175: 113818,

[32] Fang YX, Li L, Sui MR, Jiang QZ, Dong N, Shan AS, Jiang JG. Protein transduction system based on tryptophan-zipper against intracellular infections via inhibiting ferroptosis of macrophages. ACS Nano, 2023, 17: 12247-12265,

[33] Chen WY, Chang HY, Lu JK, Huang YC, Harroun SG, Tseng YT, Li YJ, Huang CC, Chang HT. Self-assembly of antimicrobial peptides on gold nanodots: against multidrug-resistant bacteria and wound-healing application. Adv Funct Mater, 2015, 25: 7189-7199,

[34] Ong ZY, Gao SJ, Yang YY. Short synthetic β-sheet forming peptide amphiphiles as broad spectrum antimicrobials with antibiofilm and endotoxin neutralizing capabilities. Adv Funct Mater, 2013, 23: 3682-3692,

[35] Stern D, Cui HG. Crafting polymeric and peptidic hydrogels for improved wound healing. Adv Healthc Mater, 2019, 8: e1900104,

[36] Craik DJ, Fairlie DP, Liras S, Price D. The future of peptide-based drugs. Chem Biol Drug Des, 2013, 81: 136-147,

[37] Shao CX, Zhu YJ, Jian Q, Lai ZH, Tan P, Li GY, Shan AS. Cross-strand interaction, central bending, and sequence pattern act as biomodulators of simplified β-hairpin antimicrobial amphiphiles. Small, 2021, 17: e2003899,

[38] Kim W, Zou GJ, Hari TPA, Wilt IK, Zhu WP, Galle N, Faizi HA, Hendricks GL, Tori K, Pan W, Huang XW, Steele AD, Csatary EE, Dekarske MM, Rosen JL, Ribeiro NQ, Lee K, Port J, Fuchs BB, Vlahovska PM, Wuest WM, Gao HJ, Ausubel FM, Mylonakis E. A selective membrane-targeting repurposed antibiotic with activity against persistent methicillin-resistant Staphylococcus aureus. Proc Natl Acad Sci USA, 2019, 116: 16529-16534,

[39] Yang ZY, He SQ, Wei YX, Li XF, Shan AS, Wang JJ. Antimicrobial peptides in combination with citronellal efficiently kills multidrug resistance bacteria. Phytomedicine, 2023, 120: 155070,

[40] Lai ZH, Tan P, Zhu YJ, Shao CX, Shan AS, Li L. Highly stabilized α-helical coiled coils kill gram-negative bacteria by multicomplementary mechanisms under acidic condition. ACS Appl Mater Interfaces, 2019, 11: 22113-22128,

[41] Tan P, Lai ZH, Jian Q, Shao CX, Zhu YJ, Li GY, Shan AS. Design of heptad repeat amphiphiles based on database filtering and structure-function relationships to combat drug-resistant fungi and biofilms. ACS Appl Mater Interfaces, 2020, 12: 2129-2144,

[42] Bai YS, Meng QW, Wang C, Ma KD, Li JB, Li JP, Shan AS. Gut Microbiota mediates Lactobacillus rhamnosus GG alleviation of deoxynivalenol-induced anorexia. J Agric Food Chem, 2023, 71: 8164-8181,

[43] Li JZ, Li QK, Wu QH, Gao N, Wang ZH, Yang Y, Shan AS. Exopolysaccharides of lactobacillus rhamnosus GG ameliorate salmonella typhimurium-induced intestinal inflammation via the TLR4/NF-κB/MAPK pathway. J Anim Sci Biotechnol, 2023, 14: 23,

[44] Song MR, Liu Y, Huang XY, Ding SY, Wang Y, Shen JZ, Zhu K. A broad-spectrum antibiotic adjuvant reverses multidrug-resistant gram-negative pathogens. Nat Microbiol, 2020, 5: 1040-1050,

[45] Wang YC, Kadiyala U, Qu ZB, Elvati P, Altheim C, Kotov NA, Violi A, VanEpps JS. Anti-biofilm activity of graphene quantum dots via self-assembly with bacterial amyloid proteins. ACS Nano, 2019, 13: 4278-4289,

[46] Chen TT, Lyu YF, Tan MS, Yang CY, Li Y, Shao CX, Zhu YJ, Shan AS. Fabrication of supramolecular antibacterial nanofibers with membrane-disruptive mechanism. J Med Chem, 2022, 65: 888,

[47] Glossop HD, de Zoysa GH, Hemar Y, Cardoso P, Wang K, Lu J, Valéry C, Sarojini V. Battacin-inspired ultrashort peptides: nanostructure analysis and antimicrobial activity. Biomacromol, 2019, 20: 2515-2529,

[48] Kurnaz LB, Luo YY, Yang XM, Alabresm A, Leighton R, Kumar R, Hwang J, Decho AW, Nagarkatti P, Nagarkatti M, Tang CB. Facial amphiphilicity index correlating chemical structures with antimicrobial efficacy. Bioact Mater, 2023, 20: 519-527

[49] Li QK, Li JZ, Yu WK, Wang ZH, Li JW, Feng XJ, Wang JJ, Shan AS. De novo design of a pH-triggered self-assembled β-hairpin nanopeptide with the dual biological functions for antibacterial and entrapment. J Nanobiotechnology, 2021, 19: 183,

[50] Singh P, Brar SK, Bajaj M, Narang N, Mithu VS, Katare OP, Wangoo N, Sharma RK. Self-assembly of aromatic α-amino acids into amyloid inspired nano/micro scaled architects. Mater Sci Eng C Mater Biol Appl, 2017, 72: 590-600,

[51] Ge CL, Zhu JL, Ye H, Wei YS, Lei YH, Zhou RX, Song ZY, Yin LC. Rational construction of protein-mimetic nano-switch systems based on secondary structure transitions of synthetic polypeptides. J Am Chem Soc, 2023, 145: 11206-11214,

[52] Fielden SDP, Derry MJ, Miller AJ, Topham PD, O'Reilly RK. Triggered polymersome fusion. J Am Chem Soc, 2023, 145: 5824-5833,

[53] Nandi N, Gayen K, Ghosh S, Bhunia D, Kirkham S, Sen SK, Ghosh S, Hamley IW, Banerjee A. Amphiphilic peptide-based supramolecular, noncytotoxic, stimuli-responsive hydrogels with antibacterial activity. Biomacromol, 2017, 18: 3621-3629,

[54] Lombardi L, Shi YJ, Falanga A, Galdiero E, de Alteriis E, Franci G, Chourpa I, Azevedo HS, Galdiero S. Enhancing the potency of antimicrobial peptides through molecular engineering and self-assembly. Biomacromol, 2019, 20: 1362-1374,

[55] Dou XJ, Zhu X, Wang JJ, Dong N, Shan AS. Novel design of heptad amphiphiles to enhance cell selectivity, salt resistance, antibiofilm properties and their membrane-disruptive mechanism. J Med Chem, 2017, 60: 2257-2270,

[56] Zhao W, Róg T, Gurtovenko AA, Vattulainen I, Karttunen M. Role of phosphatidylglycerols in the stability of bacterial membranes. Biochimie, 2008, 90: 930-938,

[57] Cartron ML, England SR, Chiriac AI, Josten M, Turner R, Rauter Y, Hurd A, Sahl HG, Jones S, Foster SJ. Bactericidal activity of the human skin fatty acid cis-6-hexadecanoic acid on Staphylococcus aureus. Antimicrob Agents Chemother, 2014, 58: 3599-3609,

[58] Saeloh D, Tipmanee V, Jim KK, Dekker MP, Bitter W, Voravuthikunchai SP, Wenzel M, Hamoen LW. The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity. PLoS Pathog, 2018, 14: e1006876,

[59] Parasassi T, Di Stefano M, Loiero M, Ravagnan G, Gratton E. Influence of cholesterol on phospholipid bilayers phase domains as detected by laurdan fluorescence. Biophys J, 1994, 66: 120-132,

[60] Gong HN, Hu XZ, Liao MR, Fa K, Ciumac D, Clifton LA, Sani MA, King SM, Maestro A, Separovic F, Waigh TA, Xu H, McBain AJ, Lu JR. Structural disruptions of the outer membranes of gram-negative bacteria by rationally designed amphiphilic antimicrobial peptides. ACS Appl Mater Interfaces, 2021, 13: 16062-16074,

[61] Epand RM, Walker C, Epand RF, Magarvey NA. Molecular mechanisms of membrane targeting antibiotics. Biochim Biophys Acta, 2016, 1858: 980-987,

[62] Jin JC, Wu XJ, Xu J, Wang BB, Jiang FL, Liu Y. Ultrasmall silver nanoclusters: highly efficient antibacterial activity and their mechanisms. Biomater Sci, 2017, 5: 247-257,

[63] Lam SJ, O'Brien-Simpson NM, Pantarat N, Sulistio A, Wong EH, Chen YY, Lenzo JC, Holden JA, Blencowe A, Reynolds EC, Qiao GG. Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers. Nat Microbiol, 2016, 1: 16162,

[64] Engelberg Y, Landau M. The Human LL-37(17–29) antimicrobial peptide reveals a functional supramolecular structure. Nat Commun, 2020, 11: 3894,

[65] Fan Y, Li XD, He PP, Hu XX, Zhang K, Fan JQ, Yang PP, Zheng HY, Tian W, Chen ZM, Ji L, Wang H, Wang L. A biomimetic peptide recognizes and traps bacteria in vivo as human defensin-6. Sci Adv, 2020, 6: eaaz4767,

[66] Cao JW, Xu R, Geng Y, Xu SW, Guo MY. Exposure to polystyrene microplastics triggers lung injury via targeting toll-like receptor 2 and activation of the NF-κB signal in mice. Environ Pollut, 2023, 320: 121068,

[67] Wang FH, Zhang QR, Cui J, Bao BW, Deng X, Liu L, Guo MY. Polystyrene microplastics induce endoplasmic reticulum stress, apoptosis and inflammation by disrupting the gut microbiota in carp intestines. Environ Pollut, 2023, 323: 121233,

[68] Cai L, Zhu XY, Ruan HJ, Yang J, Wei W, Wu Y, Zhou LZ, Jiang HJ, Ji MH, Chen J. Curcumin-stabilized silver nanoparticles encapsulated in biocompatible electrospun nanofibrous scaffold for sustained eradication of drug-resistant bacteria. J Hazard Mater, 2023, 452: 131290,

[69] Sun TT, Liu XS, Su YZ, Wang ZH, Cheng BJ, Dong N, Wang JJ, Shan AS. The efficacy of anti-proteolytic peptide R7I in intestinal inflammation, function, microbiota, and metabolites by multi-omics analysis in murine bacterial enteritis. Bioeng Transl Med, 2023, 8: e10446,

[70] Zheng KY, Setyawati MI, Lim TP, Leong DT, Xie JP. Antimicrobial cluster bombs: silver nanoclusters packed with daptomycin. ACS Nano, 2016, 10: 7934-7942,

Funding

National Natural Science Foundation of China(32002215); Heilongjiang Provincial Collaborative Innovation Achievement Project for Universities - Incubation Project(LJGXCG2022-022); Heilongjiang Touyan Innovation Team Program(54600112); Natural Science Foundation of Heilongjiang Province(YQ2022C015)