Assessment of Chitosan-Based Hydrogel and Photodynamic Inactivation against Propionibacterium acnes

Abstract

Chitosan (CH) is a biopolymer that exhibits a number of interesting properties such as anti-inflammatory and antibacterial activity and is also a promising platform for the incorporation of photosensitizing agents. This study aimed to evaluate the efficacy of antimicrobial activity of chitosan hydrogel formulation alone and in combination with the methylene blue (MB) associated with antimicrobial photodynamic therapy (aPDT) against planktonic and biofilm phase of Propionibacterium acnes. Suspensions were sensitized with 12.5, 25.0, 37.5, 50.0 μg/mL of MB for 10 min and biofilms to 75, 100 and 150 μg/mL for 30 min then exposed to red light (660 nm) at 90 J/cm² and 150 J/cm² respectively. After treatments, survival fractions were calculated by counting the number of colony-forming units. The lethal effect of aPDT associated with CH hydrogel in planktonic phase was achieved with 12.5 µg/mL MB and 1.9 log₁₀ biofilm reduction using 75 µg/mL MB. Rheological studies showed that formulations exhibited pseudoplastic non-Newtonian behavior without thixotropy. Bioadhesion test evidenced that the formulations are highly adhesive to skin and the incorporation of MB did not influence the bioadhesive force of the formulations.

Keywords: Propionibacterium acnes; chitosan hydrogel; methylene blue; photodynamic therapy.

Figure 1

Flow rheograms of: (A) HG1, HG2 and HG3 at room temperature (25.0 ± 0.5 °C); (B) HG1, HG2 and HG3 at skin temperature (32.0 ± 0.5 °C); (C) HG1-MB12.5 and HG1-MB75 at room temperature (25.0 ± 0.5 °C) and (D) HG1-MB12.5 and HG1-MB75 at skin temperature (32.0 ± 0.5 °C). Notes: The flow properties were performed using a controlled shear rate procedure ranging from 0.01 to 100 s⁻¹ (or ascendent curve—filled symbols) and back (or descendant curve—empty symbols). Standard deviations have been omitted for clarity; however, in all cases, the coefficient of variation of triplicate analyses was less than 10%.

Figure 2

Frequency sweep profile of (A) HG1, HG2 and HG3 at room temperature (25.0 ± 0.5 °C); (B) HG1, HG2 and HG3 at skin temperature (32.0 ± 0.5 °C); (C) HG1-MB12.5 and HG1-MB75 at room temperature (25.0 ± 0.5 °C) and (D) HG1-MB12.5 and HG1-MB75 at skin temperature (32.0 ± 0.5 °C). Notes: The storage modulus G’ are the filled symbols and the loss modulus G” are the empty symbols.The SDs have been omitted for clarity; however, in all cases, the coefficients of variation of the triplicate analyses were less than 10%.

Figure 3

The peak of bioadhesion of the formulations HG1, HG1-MB12.5 and HG1-MB75. Each value represents the mean ± standard deviation of four replicates. The data were collected at 32 ± 0.5 °C.

Figure 4

Bacterial suspension incubated with the chitosan hydrogel at 0.25% for thirty and sixty minutes. Columns represent the average of three independent assays and bars represent the standard deviation. The asterisks indicatewhere there is a significant difference in comparison with the groups treated and the group no treated (NT) (one-way ANOVA with Tukey’s post-hoc). ** p < 0.01.

Figure 5

P. acnes biofilm incubated with the chitosan hydrogel at 0.25% for thirty and sixty minutes. Columns represent the average of three independent assays and bars represent the standard deviation. The asterisks indicate the statistical difference between the groups and the group no treated (NT). (One-way ANOVA with Tukey’s post-hoc). * p < 0.05.

Figure 6

aPDT mediated by MB in solution and MB concentrations incorporated into 0.25 chitosan hydrogel over the standard suspension of P. acnes. Columns represent the average of three independent assays and bars represent the standard deviation. The asterisks indicate where there is a statistical difference in comparison with the groups treated and the group no treated (NT); (two-way ANOVA with post-test Bonferroni), *** p < 0.001.

Figure 7

aPDT mediated by MB in solution and MB concentrations incorporated into 0.25 chitosan hydrogel over biofilm of P. acnes. The columns represent the average of three independent assays and bars represent the standard deviation. The asterisks represent the statistical difference between the groups treated and the group no treated (NT)(two-way ANOVA with post-test Bonferroni).** p < 0.01; *** p < 0.001.

Figure 8

Absorption spectrum of 0.25% chitosan hydrogel with methylene blue.