Bacterial infections pose a serious threat to mankind, and there is immense interest in the design and development of self-assembled peptide gels using ultrashort peptides for antibacterial applications. The peptide gels containing natural amino acids suffer from poor stability against proteolytic enzymes. Therefore, there is a need to design and develop peptide gels with improved stability against proteolytic enzymes. In the present work, we report the synthesis and characterization of α/γ hybrid peptides Boc-D-Phe-γ⁴-L-Phe-PEA (NH007) and Boc-L-Phe-γ⁴-L-Phe-PEA (NH009) to improve the proteolytic stability. Both of the dipeptides were found to self-assemble into gels in aqueous DMSO (3–5% w/v), and the self-assembly process was studied using FTIR and CD, which indicated antiparallel β-sheet formation with random coils in NH007 gels and random or unordered conformation in NH009. The rheological studies indicated viscoelastic characteristics for both gels; the storage modulus (G′) for NH007 and NH009 gels (3% w/v) was estimated as 0.2 and 0.5 MPa, higher than the loss modulus (G′′). Also, both gels demonstrated self-healing characteristics for six consecutive cycles when subjected to varying strains of 0.1 and 30% (200 s each). The peptide gels were incubated with a mocktail of proteolytic enzymes, proteinase K, pepsin, and chymotrypsin, and stability was monitored using RP HPLC. Up to 23 and 40% degradation was observed for NH007 (3%, w/v) in 24 and 36 h, and 77 and 94% degradation was observed for NH009 (3%, w/v), within the same period. Thus α/γ hybrid peptide gels containing D-Phe exhibited higher stability than gels fabricated using L-Phe. The use of D-residue in α/γ hybrid peptide significantly enhanced the stability of peptides against proteolytic enzymes, as the stability data reported in this work are possibly the best in class. Both peptide gels exhibited broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The Pseudomonas aeruginosa and Staphylococcus aureus, in particular, are known to develop resistance. The NH007 (3%, w/v) demonstrated 65% inhibition, whereas NH009 (3%, w/v) showed 78% inhibition, with potent activity against Pseudomonas aeruginosa. Mechanistic studies, using SEM, HR-TEM, and bacterial live–dead assay, indicated entrapment of bacteria in gel networks, followed by interaction with cell membrane components and lysis. Cell viability (MTT assay) and toxicity (LDH assay) studies showed that both gels are not toxic to NIH 3T3 mouse embryonic fibroblast cells (mammalian). MTT assay showed >85% cell viability, and LDH assay exhibited not more than 15% cytotoxicity, even at higher concentrations (5%, w/v) and prolonged exposures (48 h). Overall, studies indicate the potential application of gels developed from the α/γ hybrid peptides in preventing biomaterial-related infections.

中文翻译：

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蛋白水解稳定的自组装αγ-杂合肽凝胶的广谱抗菌活性

细菌感染对人类构成了严重威胁，并且人们对使用和设计用于抗菌应用的超短肽的自组装肽凝胶的设计和开发产生了浓厚的兴趣。含有天然氨基酸的肽凝胶对蛋白水解酶的稳定性差。因此，需要设计和开发对蛋白水解酶具有改善的稳定性的肽凝胶。在目前的工作中，我们报告的合成和表征α/γ杂合肽的Boc-d-PHE-γ ⁴ -L-PHE-PEA（NH007）和Boc-L-PHE-γ ⁴ -L-PHE-PEA（NH009）以提高蛋白水解稳定性。发现这两种二肽都可以在DMSO水溶液中（3-5％w / v）自组装成凝胶，并且使用FTIR和CD研究了自组装过程，这表明在NH007中反平行的β-折叠形成并带有无规卷曲凝胶和NH009中的随机或无序构象。流变学研究表明两种凝胶都具有粘弹性。NH007和NH009凝胶（3％w / v）的储能模量（G '）估计为0.2和0.5 MPa，高于损耗模量（G''）。同样，两种凝胶在经受0.1％和30％（每个200 s）的变化应变时，在六个连续的循环中均表现出自愈特性。将肽凝胶与蛋白水解酶，蛋白酶K，胃蛋白酶和胰凝乳蛋白酶的混合物一起孵育，并使用RP HPLC监测稳定性。在24和36小时内，观察到NH007的降解高达23％和40％（3％，w / v），而NH009观察到77和94％的降解（3％，w / v），在同一时期内。因此，与使用L-Phe制备的凝胶相比，含有D-Phe的α/γ杂合肽凝胶显示出更高的稳定性。在α/γ杂合肽中使用D残基可显着增强肽对蛋白水解酶的稳定性，因为这项工作中报道的稳定性数据可能是同类中最好的。两种肽凝胶均显示出对革兰氏阴性和革兰氏阳性细菌（如大肠杆菌，铜绿假单胞菌，枯草芽孢杆菌和金黄色葡萄球菌）的广谱抗菌活性。该绿脓杆菌和金黄色葡萄球菌，特别是被称为发展性。该NH007（3％，w / v）（3％，w / v）表现出65％的抑制作用，而NH009（3％，w / v）表现出78％的抑制作用，对铜绿假单胞菌具有有效活性。使用SEM，HR-TEM和细菌活死分析进行的机理研究表明，细菌被截留在凝胶网络中，随后与细胞膜成分相互作用并裂解。细胞活力（MTT测定）和毒性（LDH测定）研究表明，两种凝胶均对NIH 3T3小鼠胚胎成纤维细胞（哺乳动物）无毒。MTT分析显示> 85％的细胞活力，而LDH分析显示出不超过15％的细胞毒性，即使在更高的浓度（5％，w / v）和长时间暴露（48小时）下也是如此。总体而言，研究表明，由α/γ杂合肽开发的凝胶在预防生物材料相关感染中的潜在应用。