Functionalized Silver Nanoparticles as Multifunctional Agents Against Gut Microbiota Imbalance and Inflammation

Abstract

Human pathogenic fungi are the source of various illnesses, including invasive, cutaneous, and mucosal infections. One promising solution is using nanoparticles (NPs) as an antifungal agent. The current study aims to assess the antimicrobial and antifungal effects of drug-loaded silver nanoparticles (AgNPs) with previously reported mebeverine analogue (MA) as a potential drug candidate targeting gut microbiota and inflammation in the gastrointestinal tract. Density Functional Theory (DFT) calculations were conducted to identify possible mechanisms by which AgNPs could prevent microorganisms from growing. In vitro and ex vivo anti-inflammatory, in vitro antimicrobial, ex vivo spasmolytic activities, and in vitro hepatic cell morphology and proliferation of drug-loaded AgNPs were assessed. The drug-loaded AgNPs were considered to have promising antifungal activity against all tested fungal strains, Aspergillus niger, Penicillium chrysogenum, and Fusarium moniliforme, and yeasts, Candida albicans, Saccharomyces cerevisiae, and good antimicrobial activity against Gram-positive and Gram-negative bacterial strains. The results of in vitro and ex vivo determination of anti-inflammatory activity indicated that the drug-loaded AgNPs preserved MA's anti-inflammatory activity and decreased inflammation. A similar effect was observed in spasmolytic activity measurements. Drug-loaded AgNPs also influenced the morphology and proliferation of hepatic cells, indicating a potential for improved gut and liver therapeutic efficacy. Each test was performed in triplicate, and the results were reported as mean values. Based on the results, drug-loaded AgNPs might be a promising antimicrobial agent, maintaining the MA's potential as a spasmolytic and anti-inflammatory agent. Future in vivo and preclinical experiments will contribute to establishing the in vivo properties of drug-loaded AgNPs.

Keywords: DFT; anti-inflammatory activity; antifungal; antimicrobial; silver nanoparticles; spasmolytic.

Scheme 1

Reaction pathway for the synthesis of MA (2).

Figure 1

Structure of MA (2).

Figure 2

Models used in the current work to study the interaction between silver cations (in the form of Ag(H₂O)₄) and the constituents building the outer layer of the microorganism cells [34].

Figure 3

M062X/6-311++G(d,p) optimized structures of the silver-containing complexes of some main constituents building the outer layer of the microorganismic cellular envelope in [A] Gram (+) bacteria; [B] Gram (−) bacteria; [C] Candida albicans; [D] Aspergillus spp. The change in the Gibbs energies in aqueous solution mimicking the intercellular space for yielding these complexes is given in terms of kcal mol⁻¹.

Figure 4

In vitro, the anti-inflammatory activity of (1) and (2) was assessed to prevent albumin denaturation. (n = 3); # p < 0.05 compared to diclofenac; * p < 0.05 compared to acetylsalicylic acid.

Figure 5

Cell cycle distribution of HepG2 cells treated with AgNPs, MA, and AgNPs with MA for 24 h (A) and 72 h (B). The percentage of cells in G₀–G₁ (dark grey), S (light grey), and G₂–M (yellow) phases was determined via flow cytometry and subsequent data evaluation using FlowJo™ software (Version 10, Becton Dickinson, USA). The treatments were applied at concentrations corresponding to the IC50 values determined for each compound and time point: MA—24.69 µg/mL (24 h), 29.39 µg/mL (72 h); AgNPs—6.35 µg/mL (24 h), 17.79 µg/mL (72 h); AgNPs with MA—8.88 µg/mL (24 h), 13.93 µg/mL (72 h).

Figure 6

Representative micrographs of the rat corpus stomach. (A) Control stained for IL-1β, ×400; (B) Control stained for 5HT3, 400×; (C) samples incubated with MA show a very high density of expression for IL-1β in the myenteric plexus (black arrow), 400×; (D) MA showed a moderate level of 5HT3 expression (black arrow), 400×; (E) AgNPs with MA caused weak staining of cells for IL-1β (black arrow), 400×; (F) drug-loaded AgNPs very weakly labeled cells for 5HT3 (black arrow), 400×; scale bar = 50 μm.

Figure 7

Representative isometric tracings illustrating the contractile behavior of gastric SM in response to ACh (10⁻⁶ M) and 5-HT (10⁻⁶ M), and the modulatory effects of compounds (1) and (2) in their background.

Figure 8

Changes in the strength of the contractile response of SM under the influence of compounds (1) and (2) and key neurotransmitters. (A) Effects in the presence of ACh, 5-HT, and atropine (a non-selective muscarinic receptor blocker). (B) Modulation of contractile responses by selective and non-selective antagonists of muscarinic (mAChRs) and nicotinic (nAChRs) ACh receptors. Results are presented as mean ± SEM (n = 6); * p < 0.05 indicates statistical significance.