Ayubee Muhammad Salehuddin, Akter Farhana, Ahmed Nadia Tasnim, Kabir Abul Kalam Lutful, Hossain Md Mahboob, Hussain Muhammad Delwar, Kazi Mohsin, Mazid Md Abdul
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka, Bangladesh.
Department of Pharmacy, Northern University Bangladesh, Dhaka, Bangladesh.
PLoS One. 2025 Sep 9;20(9):e0331669. doi: 10.1371/journal.pone.0331669. eCollection 2025.
Antibiotic resistance towards penicillin has been attempted to counter by chemically modifying ampicillin through the conjugation with silver nanoparticles (AgNPs). The current study optimizes the conditions for synthesizing and characterizing AgNP-ampicillin to quantify the conjugation extent, evaluate the antibacterial efficacy, and explore the underlying antibacterial mechanisms.
AgNPs were synthesized from silver nitrate by chemical reduction method, silica-coated with tetraethyl orthosilicate (TEOS) and amine functionalized by (3-aminopropyl) triethoxysilane (APTES), which was then conjugated with ampicillin via the carbodiimide chemistry. UV-visible spectroscopy and DLS were employed to confirm the synthesis of AgNPs, while FT-IR and TGA were used to confirm ampicillin functionalization and conjugation, and SEM and EDX spectroscopy provided morphological insight. Microbial assays were conducted against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa to determine the inhibition zones, MIC, MBC, MPC, MBIC, MBEC, FIC index, and time-dependent efficacy of AgNP-ampicillin. Cytotoxicity was assessed on Vero cells while molecular docking was performed using AutoDock Vina.
The synthesized conjugates demonstrated an approximate conjugation efficiency of 57.7%, with four ampicillin molecules conjugated per AgNP. The AgNP-ampicillin conjugates exhibited enhanced antibacterial activity against the studied clinical isolates compared to AgNPs or ampicillin alone, as evidenced by significant differences in inhibition areas in One-way ANOVA (F=18-25.68, p<0.05), while Tukey's post-hoc analysis suggested synergistic effects. AgNP-ampicillin demonstrated enhanced bacteriostatic and bactericidal activity against both planktonic and biofilm-forming cells with mutant prevention ability, and upto 1.25 times faster bacterial elimination compared to ampicillin and AgNPs alone. Synergistic effects of AgNP-ampicillin were confirmed by an FIC index (≤0.5), and effective protection of ampicillin from β-lactamase degradation was established through molecular docking. Cytotoxicity testing confirmed >95% Vero cell viability, indicating minimal toxicity.
The AgNP-ampicillin conjugates exhibited enhanced antibacterial efficacy, biofilm disruption, and protection against β-lactamase degradation while maintaining low toxicity.
通过将氨苄西林与银纳米颗粒（AgNPs）结合进行化学修饰，以对抗对青霉素的抗生素耐药性。本研究优化了合成和表征AgNP - 氨苄西林的条件，以量化结合程度、评估抗菌效果并探索潜在的抗菌机制。
通过化学还原法由硝酸银合成AgNPs，用正硅酸四乙酯（TEOS）进行二氧化硅包覆，并用（3 - 氨丙基）三乙氧基硅烷（APTES）进行胺功能化，然后通过碳二亚胺化学法将其与氨苄西林结合。采用紫外 - 可见光谱和动态光散射（DLS）来确认AgNPs的合成，同时使用傅里叶变换红外光谱（FT - IR）和热重分析（TGA）来确认氨苄西林的功能化和结合，扫描电子显微镜（SEM）和能谱分析（EDX）提供形态学见解。针对枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌和铜绿假单胞菌进行微生物测定，以确定抑制圈、最低抑菌浓度（MIC）、最低杀菌浓度（MBC）、突变预防浓度（MPC）、最低生物被膜抑制浓度（MBIC）、最低生物被膜消除浓度（MBEC）、联合药敏指数（FIC指数）以及AgNP - 氨苄西林的时间依赖性疗效。在Vero细胞上评估细胞毒性，同时使用AutoDock Vina进行分子对接。
合成的缀合物显示出约为57.7%的结合效率，每个AgNP结合四个氨苄西林分子。与单独的AgNPs或氨苄西林相比，AgNP - 氨苄西林缀合物对所研究的临床分离株表现出增强的抗菌活性，单向方差分析（F = 18 - 25.68，p < 0.05）中抑制区域的显著差异证明了这一点，而Tukey事后分析表明存在协同效应。AgNP - 氨苄西林对浮游细胞和形成生物膜的细胞均表现出增强抑菌和杀菌活性以及突变预防能力，与单独的氨苄西林和AgNPs相比，细菌清除速度快达1.25倍。通过FIC指数（≤0.5）证实了AgNP - 氨苄西林的协同效应，并通过分子对接确定其对氨苄西林具有有效的β - 内酰胺酶降解保护作用。细胞毒性测试证实Vero细胞活力> 95%，表明毒性极小。
AgNP - 氨苄西林缀合物表现出增强的抗菌效果、生物膜破坏作用以及对β - 内酰胺酶降解的保护作用，同时保持低毒性。
