Department of Chemistry , Indian Institute of Technology Ropar , Rupnagar 140001 , Punjab , India.
Medicinal Chemistry Division , CSIR-Indian Institute of Integrative Medicine , Canal Road , Jammu Tawi 180001 , Jammu and Kashmir , India.
Biomacromolecules. 2018 Mar 12;19(3):782-792. doi: 10.1021/acs.biomac.7b01582. Epub 2018 Feb 14.
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-γ-L-Phe-PEA (NH007) and Boc-L-Phe-γ-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.
细菌感染对人类构成严重威胁，因此人们非常有兴趣设计和开发使用超短肽的自组装肽凝胶，将其应用于抗菌领域。含有天然氨基酸的肽凝胶在对抗蛋白酶方面稳定性较差。因此，需要设计和开发稳定性更高的肽凝胶来对抗蛋白酶。在本工作中，我们报告了 Boc-D-Phe-γ-L-Phe-PEA（NH007）和 Boc-L-Phe-γ-L-Phe-PEA（NH009）的合成和表征，以提高其对蛋白酶的稳定性。这两种二肽都被发现可以在水-DMSO（3-5%w/v）中自组装成凝胶，FTIR 和 CD 的自组装过程研究表明 NH007 凝胶中形成了反平行β-折叠片层和无规卷曲，NH009 凝胶中形成了无规或无序构象。流变学研究表明两种凝胶都具有粘弹性特征；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''）。此外，当在 0.1 和 30%（200 s 每次）的不同应变下进行六次连续循环时，两种凝胶都表现出自修复特性。将肽凝胶与蛋白酶 K、胃蛋白酶和糜蛋白酶的模拟混合酶孵育，并使用反相高效液相色谱法（RP HPLC）监测稳定性。在 24 和 36 小时内，NH007（3%，w/v）的降解率分别为 23%和 40%，NH009（3%，w/v）的降解率分别为 77%和 94%。因此，含有 D-Phe 的α/γ 混合肽凝胶比使用 L-Phe 制成的凝胶具有更高的稳定性。在α/γ 混合肽中使用 D-残基显著提高了肽对蛋白酶的稳定性，因为本工作中报告的稳定性数据可能是同类中最好的。两种肽凝胶都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）具有广谱抗菌活性。特别是铜绿假单胞菌和金黄色葡萄球菌已知会产生耐药性。NH007（3%，w/v）显示出 65%的抑制作用，而 NH009（3%，w/v）显示出 78%的抑制作用，对铜绿假单胞菌具有很强的活性。使用 SEM、高分辨率 TEM 和细菌死活检测的机制研究表明，细菌被捕获在凝胶网络中，随后与细胞膜成分相互作用并发生裂解。细胞活力（MTT 测定）和毒性（LDH 测定）研究表明，两种凝胶对 NIH 3T3 小鼠胚胎成纤维细胞（哺乳动物）均无毒性。MTT 测定显示细胞活力超过 85%，LDH 测定显示细胞毒性不超过 15%，即使在较高浓度（5%，w/v）和较长暴露时间（48 小时）下也是如此。总的来说，研究表明，源自α/γ 混合肽的凝胶在预防生物材料相关感染方面具有潜在的应用前景。
