Poly Lactic-co-Glycolic Acid (PLGA) Loaded with a Squaraine Dye as Photosensitizer for Antimicrobial Photodynamic Therapy

Abstract

Antimicrobial Photodynamic Therapy (aPDT) is an innovative and promising method for combating infections, reducing the risk of antimicrobial resistance compared to traditional antibiotics. Squaraine (SQ) dyes can be considered promising photosensitizers (PSs) but are generally hydrophobic molecules that can self-aggregate under physiological conditions. To overcome these drawbacks, a possible solution is to incorporate SQs inside nanoparticles (NPs). The present work deals with the design and development of innovative nanophotosensitizers based on poly lactic-co-glycolic acid (PLGA) NPs incorporating a brominated squaraine (BrSQ) with potential application in aPDT. Two designs of experiments (DoEs) based on the single emulsion and nanoprecipitation methods were set up to investigate how different variables (type of solvent, solvent ratio, concentration of PLGA, stabilizer and dye, sonication power and time) can affect the size, zeta (ζ)-potential, yield, entrapment efficiency, and drug loading capacity of the SQ-PLGA NPs. SQ-PLGA NPs were characterized by NTA, FE-SEM, and UV-Vis spectroscopy and the ability to produce reactive oxygen species (ROS) was evaluated, proving that ROS generation ability is preserved in SQ-PLGA. In vitro antimicrobial activity against Gram-positive bacteria in planktonic state using Staphylococcus aureus was conducted in different conditions and pH to evaluate the potential of these nanophotosensitizers for aPDT in the local treatment of infections.

Keywords: Antimicrobial Photodynamic Therapy; Gram-positive bacteria; PLGA nanoparticles; designs of experiments; squaraine dyes.

Figure 1

BrSQ structure (a); Schematic set-up of antibacterial PDT protocol (b).

Figure 4

Significant coefficients (green bars) and corresponding confidence intervals (black lines) for the EE (left) and yield (right) models, computed by MODDE software, for the single emulsion method (DoE-2).

Figure 2

Synthetic workflow for the BrSQ-loaded PLGA nanoparticles preparation via the (a) nanoprecipitation (DoE-1) technique and (b) single emulsion method (DoE-2).

Figure 3

Significant coefficients (green bars) and corresponding confidence interval (black lines) for the NPs size (left) and yield (right) models computed for DoE-1 by MODDE software.

Figure 5

FE-SEM images of (a) PLGA, (b) BrSQ-PLGA, and (c) nano-tracking analysis of PLGA and BrSQ-PLGA.

Figure 6

(a) UV-Vis spectra of BrSQ dissolved in different percentages of DMSO and PBS, (b) comparison of the UV-Vis spectra of free BrSQ in 100% DMSO, 100% PBS, and BrSQ-PLGA NPs in 100% PBS. The concentration of BrSQ was kept constant for all the measurements.

Figure 7

Decay of the absorption band of DPBF at 418 nm as a function of the irradiation time in the presence of free BrSQ and BrSQ-loaded PLGA NPs. Data are reported as an average of 3 independent experiments and error bars represent the standard deviation.

Figure 8

In vitro antimicrobial effect of different concentrations of BrSQ, PLGA, and BrSQ-PLGA on S. aureus in the dark and after irradiation (640 nm LED, fluence 6.2 J/cm², irradiance 7 mW/cm² for 15 min). For dye-loaded PLGA NPs, the concentrations refer to dyes incorporated into PLGA (from 100 nM to 1 µM) in PBS buffer, pH 7.4. Data are reported as an average of 3 independent experiments. p values: * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001.

Figure 9

In vitro antimicrobial effect of different concentrations of BrSQ, PLGA, and BrSQ-PLGA on S. aureus in the dark and after irradiation (640 nm LED, fluence 6.2 J/cm², irradiance 7 mW/cm² for 15 min). For dye-loaded PLGA NPs, the concentrations refer to dyes incorporated into PLGA (from 2 µM to 5 µM) in PBS buffer, pH 7.4. Data are reported as an average of 3 independent experiments. p values: * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001.

Figure 10

In vitro antimicrobial effect of different concentrations of BrSQ, PLGA, and BrSQ-PLGA on S. aureus in the dark and after irradiation (640 nm LED, fluence 15.7 J/cm², irradiance 17.4 mW/cm² for 15 min). For dye-loaded PLGA NPs, the concentrations refer to dyes incorporated into PLGA (from 2 µM to 5 µM) in PBS buffer, pH 7.4. Data are reported as an average of 3 independent experiments. p values: * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001.

Figure 11

In vitro antimicrobial effect of different concentrations of BrSQ, PLGA, and BrSQ-PLGA on S. aureus in the dark and after irradiation (640 nm LED, fluence 15.7 J/cm², irradiance 17.4 mW/cm² for 15 min). For dye-loaded PLGA NPs, the concentrations refer to dyes incorporated into PLGA (from 2 µM to 5 µM) in PBS buffer, pH 5.5. Data are reported as an average of 3 independent experiments. p values: * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001.