Synergistic Nanocomposites of Different Antibiotics Coupled with Green Synthesized Chitosan-Based Silver Nanoparticles: Characterization, Antibacterial, in vivo Toxicological and Biodistribution Studies

Abstract

Purpose: The present study reports chitosan functionalized green synthesized CS-AgNPs, conjugated with amoxicillin (AMX), cefixime (CEF), and levofloxacin (LVX) for safe and enhanced antibacterial activity.

Methods: The CS-AgNPs and conjugates CS-AgNPs+AMX CS-AgNPs+CEF, and CS-AgNPs+LVX were characterized by UV-Vis, FTIR, SEM, TEM, EDX spectroscopy. The size distribution and zeta potential were measured using the dynamic light scattering (DLS) technique. The interaction between CS-AgNPs and antibiotic molecules was also investigated using UV-Vis spectroscopy at the concentrations of 5, 50, 500, and 5000 µM for each antibiotic. Antibacterial activity and synergism were assessed by the Fractional Inhibitory Concentration (FIC) index. The mechanism for synergistic activity was investigated by the detection of hydroxyl species based on the chemiluminescence of luminol. The biocompatibility index (BI) was calculated from IC₅₀ using the HeLa cell line. In vivo toxicity and tissue distribution of silver ions were evaluated on Sprague Dawley rats. Physical interactions of antibiotics and significant (P<0.05) antibacterial activity were observed after loading on CS-AgNPs surfaces.

Results: The spherical shape nanocomposites of CS-AgNPs with different antibiotics were prepared with mean size ranges of 80-120 nm. IC₅₀ of antibiotics-conjugated CS-AgNPs decreased compared to CS-AgNPs. The biocompatibility (BI) index showed that antibiotics-conjugated CS-AgNPs have high antibacterial potential and low toxicity. Highly significant (P<0.005) increase in the generation of hydroxyl species indicated the radical scavenging mechanism for synergistic activity of CS-AgNPs after combined with different antibiotics. Biochemical analysis and histopathological examinations confirmed low toxicity with minor hepatotoxicity at higher doses. After oral administration, extensive distribution of Ag ion was observed in spleen and liver.

Conclusion: The study demonstrates positive attributes of antibiotics-conjugated CS-AgNPs, as a promising antibacterial agent with low toxicity.

Keywords: antibiotic resistance; chitosan functionalized silver nanoparticles; in vivo toxicity; synergistic antibacterial activity; tissue distribution.

Figure 1

Antibacterial activities of CS-AgNP with amoxicillin against (A) Escherichia coli; (B) Klebsiella pneumoniae; (C) Staphylococcus aureus; (D) Salmonella typhi; (E) Pseudomonas aeruginosa.

Figure 2

Antibacterial activities of CS-AgNP with cefixime against (A) Escherichia coli; (B) Klebsiella pneumoniae; (C) Staphylococcus aureus; (D) Salmonella typhi; (E) Pseudomonas aeruginosa.

Figure 3

Antibacterial activities of CS-AgNP with levofloxacin against (A) Escherichia coli; (B) Klebsiella pneumoniae; (C) Staphylococcus aureus; (D) Salmonella typhi; (E) Pseudomonas aeruginosa.

Figure 4

Fold increase in inhibition zones of antibiotics after conjugation with CS-AgNPs against tested isolates. All experiments were performed in triplicates and reported as mean ± SD. *p ≤ 0.05 significant as compared to control (considered as no increase in activity), **p ≤ 0.005 highly significant as compared to control.

Figure 5

CS-AgNPs-associated generation of hydroxyl radicals in the presence of different antibacterial agent. *The chemiluminescence of luminol (stage 1) in the presence of CS-AgNPs (stage 2) antibiotic (stage 3) CS-AgNPs combined with different antibiotics (stage 4). **RLU, relative luminescence units; AMX, amoxicillin; CEF, cefixime; LVX, levofloxacin. All experiments were performed in triplicates and reported as mean. *p ≤ 0.05 significant as compared to control (luminol), **p ≤ 0.005 highly significant as compared to control.

Figure 6

% viability of HeLa cells against AgNPs, CS-AgNPs alone, and combination with different antibiotics while IC₅₀ of each agent are given in Table 4. All experiments were performed in triplicates and reported as mean.

Figure 7

Cytotoxic activity of combined antibacterial agents (CS-AgNPs and antibiotics) against HeLa cell line. Both antimicrobials were used at concentrations of their FIC. 1 = (4 µg/mL of CS-AgNPs+64 µg/mL AMX or 32 µg/mL CEF or 1 µg/mL LVX); 2 = (8 µg/mL of CS-AgNPs+32 µg/mL AMX or 4 µg/mL of CS-AgNPs+8 µg/mL CEF or 8 µg/mL of CS-AgNPs+4 µg/mL LVX); 3 = (4 µg/mL of CS-AgNPs+16 µg/mL AMX or CEF or 4 µg/mL LVX); 4 = (4 µg/mL of CS-AgNPs+16 µg/mL AMX or 2 µg/mL CEF or 2 µg/mL of CS-AgNPs+0.0625 µg/mL LVX); 5 = (4 µg/mL of CS-AgNPs+32 µg/mL AMX or CEF or 128 µg/mL LVX). AMX, amoxicillin; CEF, cefixime; LVX, levofloxacin.

Figure 8

Histopathological examinations of heart, kidney and liver of male rats after administration of CS-AgNPs at different doses using the 400× magnification. Arrows in figures shows the minor tissue inflammation at higher doses of CS-AgNPs.

Figure 9

Histopathological examinations of heart, kidney and liver of female rats after administration of CS-AgNPs at different doses using the 400× magnification. Arrows in figures shows the minor tissue inflammation at higher doses of CS-AgNPs.

Figure 10

Silver concentrations (ng/g tissue wet weight) in different organs of both male and female after administration of CS-AgNPs. All experiments were performed in triplicates and reported as mean. *p ≤ 0.05 significant as compared to control (considered as zero concentration of silver ion), **p ≤ 0.005 highly significant as compared to control.