(M1430-07-39) Targeting Resistant Pathogens: The Promise of Vancomycin-Silver Nanoparticle Hybrids Against Vancomycin-Intermediate Staphylococcus Aureus (VISA)

Purpose: The rise in antibiotic resistance poses a critical threat to public health, diminishing the effectiveness of conventional antibiotic therapies. Despite the rise in antibiotic resistance, there has been no considerable progress in developing new antibiotics to combat the superbugs. For decades, the glycopeptide antibiotic vancomycin has been regarded as the ultimate treatment option for severe infections caused by Gram-positive bacteria, often serving as the last line of defense. Over the last two decades, the emergence of vancomycin-resistant bacteria has been alarming because of the high mortality rate. Vancomycin-intermediate Staphylococcus aureus (VISA) remains the pathogen of primary concern, causing difficult-to-treat infections owing to antibiotic resistance to vancomycin. One of the promising ways to combat resistance lies in the utilization of metal nanoparticles for augmenting antibiotic efficacy. Among them, silver nanoparticles (AgNPs) stand out due to their extensively documented broad-spectrum and robust antimicrobial properties, especially against the bacterial biofilms that make them essential against multidrug-resistant pathogens.

Methods: Novel carboxylic acid-capped AgNPs were synthesized employing a chemical reduction method adapted from a recent report.1 Briefly, succinic acid was used as a capping agent in the presence of NaBH₄ as the reducing agent for the AgNO₃ precursor. The synthesized AgNPs were purified by centrifugation, and their formation was confirmed using UV-Vis spectroscopy. For complex preparation, AgNPs concentration was kept constant while varying the vancomycin concentration. Careful monitoring of the complexation reaction by UV-Vis spectra determined the appropriate time and ratio for complete complexation. For coating, we used chitosan solution prepared in 1 % acetic acid at 0.2 % w/v, as mentioned in the literature.² The hydrodynamic radius and zeta potential were measured by dynamic light scattering (DLS). Transmission electron microscopy (TEM) was used to examine the shape. The concentration of nanoparticles was determined by nanoparticle tracking analysis (NTA). The silver and vancomycin content was quantified using inductively coupled plasma mass spectroscopy (ICP-MS) and ultra-performance liquid chromatography (UPLC) systems, respectively. Furthermore, stability testing and biocompatibility studies were conducted. The in vitro antibacterial activity of the formulation was assessed to evaluate aspects of its antibacterial action comprehensively.

Results: Novel succinic acid-capped AgNPs measuring 20 nm with narrow size distribution were synthesized, followed by the formation of a complex with vancomycin and coating with biodegradable chitosan polymer. The hydrodynamic size of AgNPs increased from 23 ± 1 nm to 70 ± 2 nm after formation of the complex and 91 ± 1 nm after coating.  Surface modification of AgNPs with succinic acid resulted in a zeta potential of -20 mV and changed to +30 mV after chitosan coating. The positive charge of coated nanohybrid helps enhance adhesion to bacterial cell walls and vancomycin delivery. The interaction between the vancomycin and AgNPs was investigated using a range of analytical techniques, including UV–vis absorption spectra, FTIR, DLS, TEM, and NTS. Enhanced bactericidal activity was observed at different time points with varying MIC concentrations. The results demonstrate that the nanohybrid is more efficient in killing bacteria compared to using vancomycin alone.

Conclusion: In this study, an efficient AgNPs based vancomycin nanohybrid system was developed to enhance its antibacterial activity against VISA. By leveraging the unique properties of metallic nanoparticles with the therapeutic capabilities of antibiotics, this strategy offers a multifaceted approach to combating resistant bacteria. Metallic nanoparticles (such as silver, copper, gold, and others) possess inherent antimicrobial properties that can augment the efficacy of antibiotics through various mechanisms. However, further research is needed to find out how this combined approach can potentially lower the side effects of antibiotics and facilitate the eradication of resistant strains compared to standard treatments.

References: (1) Irfan, M. I.; Amjad, F.; Abbas, A.; Rehman, M. F. ur; Kanwal, F.; Saeed, M.; Ullah, S.; Lu, C. Novel Carboxylic Acid-Capped Silver Nanoparticles as Antimicrobial and Colorimetric Sensing Agents. Molecules 2022, 27 (11), 3363.
(2) Farhadi, L.; Mohtashami, M.; Saeidi, J.; Azimi-nezhad, M.; Taheri, G.; Khojasteh-Taheri, R.; Rezagholizade-Shirvan, A.; Shamloo, E.; Ghasemi, A. Green Synthesis of Chitosan-Coated Silver Nanoparticle, Characterization, Antimicrobial Activities, and Cytotoxicity Analysis in Cancerous and Normal Cell Lines. J. Inorg. Organomet. Polym. Mater. 2022, 32 (5), 1637–1649.

Figure 1: Characterization of AgNPs and AgNPs-Vancomycin Nanohybrid formulation. (a) UV–visible spectroscopy showing plasmon resonance peak around 398 nm for silver and 280 nm for vancomycin, (b) Zeta potential, (c) TEM image of AgNPs at RT (Scale:100 nm) , (d) Size distribution by DLS


Figure 2: Stability of AgNPs over two-month period assessed by size and polydispersity index (PDI)

Figure 3: Vancomycin-Silver Nanoparticles (AgNPs) Nanohybrid against Vancomycin-intermediate Staphylococcus aureus (VISA) at 1x Minimum Inhibitory concentration (MIC)