Antimicrobial and anti-inflammatory activity of chitosan-alginate nanoparticles: a targeted therapy for cutaneous pathogens

Abstract

Advances in nanotechnology have demonstrated potential application of nanoparticles (NPs) for effective and targeted drug delivery. Here we investigated the antimicrobial and immunological properties and the feasibility of using NPs to deliver antimicrobial agents to treat a cutaneous pathogen. NPs synthesized with chitosan and alginate demonstrated a direct antimicrobial activity in vitro against Propionibacterium acnes, the bacterium linked to the pathogenesis of acne. By electron microscopy (EM) imaging, chitosan-alginate NPs were found to induce the disruption of the P. acnes cell membrane, providing a mechanism for the bactericidal effect. The chitosan-alginate NPs also exhibited anti-inflammatory properties as they inhibited P. acnes-induced inflammatory cytokine production in human monocytes and keratinocytes. Furthermore, benzoyl peroxide (BP), a commonly used antiacne drug, was effectively encapsulated in the chitosan-alginate NPs and demonstrated superior antimicrobial activity against P. acnes compared with BP alone while demonstrating less toxicity to eukaryotic cells. Together, these data suggest the potential utility of topical delivery of chitosan-alginate NP-encapsulated drug therapy for the treatment of dermatologic conditions with infectious and inflammatory components.

Figure 1. Synthesis of chitosan-alginate nanoparticles

Nanoparticles were synthesized using chitosan and alginate. The structure was analyzed using (a) TEM (bar = 50 nm) and analytical sizing perform using DLS.

Figure 2. Antimicrobial effects of chitosan-alginate nanoparticles

Various concentrations (1%, .5%, .2%, .1%) of chitosan-alginate NPs were incubated with P. acnes for 4 h and tested for antimicrobial activity using CFU assay (mean CFU/ml) and compared to chitosan and alginate as controls. These data are derived from eight independent experiments ± SEM (p-values: † ≤ 0.005, ‡ ≤ 0.001).

Figure 3. Anti-inflammatory effect of chitosan-alginate nanoparticles

Chitosan-alginate NPs at various concentrations (6.5, 25 and 50 percent of stock, respectively) were incubated with primary human monocytes or HaCaT cells which were subsequently stimulated with P. acnes. (a) IL-12p40 and (c) IL-6 levels were determined by ELISA expressed by mean cytokine concentration (pg/ml) and are representative of four independent experiments + SEM (p-values: ** ≤ 0.01). (b,d) Cells were collected and evaluated for cell viability using a MTT (b) and MTS (d) assay. Data is expressed by mean percentage as compared to untreated cells and is composite of four independent experiments ± SEM (b. pvalues: * ≤ 0.05, † ≤ 0.005, ‡ ≤ 0.001; d. p-values: * ≤ 0.05, ** ≤ 0.01, ≤ 0.0001).

Figure 4. Synergistic activity of benzoyl peroxide encapsulated chitosan-alginate nanoparticles

(a) Benzoyl peroxide encapsulated into chitosan-alginate NPs ([BP 0.1%] NP) were incubated with P. acnes for 4 h and tested for antimicrobial activity using CFU assay (mean CFU/ml) and compared to chitosan-alginate NPs, BP 0.1%, and alginate as a negative control. These data are composite of eight distinct experiments ± SEM (p-values: ** ≤ 0.01, † ≤ 0.005, ‡ ≤ 0.001). (b) Primary human monocytes were stimulated with BP 0.1%, [BP 0.1%] NP, and Na₂Cr₂O₇ as a positive control for toxicity, and evaluated for cell viability using MTT assay. Data is expressed by mean percentage as compared to untreated cells and is composite of three independent experiments ± SEM (p-value: ‡ ≤ 0.001).

Figure 5. Effects of chitosan-alginate NP, benzoyl peroxide and NP encapsulated benzoyl peroxide on the cell structure of P. acnes.

SEM (bar = 1 µm), low power TEM (bar = 1 µm) and high power TEM (bars = 100 nm) demonstrated the effect of chitosan-alginate NP, benzoyl peroxide alone (BP 0.1%) and NP encapsulated BP ([BP 0.1%] NP) on P. acnes after 4 h of incubation with the bacteria. EM studies represent data from at least three different experiments.