Inherently Antimicrobial P(MMA- ran-DMAEMA) Copolymers Sensitive to Photodynamic Therapy: A Double Bactericidal Effect for Active Wound Dressing

Abstract

In this work, two compounds belonging to the BODIPY family, and previously investigated for their photosensitizing properties, have been bound to the amino-pendant groups of three random copolymers, with different amounts of methyl methacrylate (MMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) in the backbone. The P(MMA-ran-DMAEMA) copolymers have inherently bactericidal activity, due to the amino groups of DMAEMA and to the quaternized nitrogens bounded to BODIPY. Systems consisting of filter paper discs coated with copolymers conjugated to BODIPY were tested on two model microorganisms, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). On solid medium, irradiation with green light induced an antimicrobial effect, visible as a clear inhibition area around the coated disks. The system based on the copolymer with 43% DMAEMA and circa 0.70 wt/wt% of BODIPY was the most efficient in both bacterial species, and a selectivity for the Gram-positive model was observed, independently of the conjugated BODIPY. A residual antimicrobial activity was also observed after dark incubation, attributed to the inherently bactericidal properties of copolymers.

Keywords: BODIPY; antimicrobial materials; antimicrobial photodynamic therapy (aPDT); photoinactivation; photosensitizers.

Scheme 1

Conjugation of P(MMA-ran-DMAEMA) copolymers with the BODIPYs, A or B. [4,4-difluoro-2,6-diiodio-1,3,5,7-tetramethyl-8-(4-(4-bromobutoxy) phenyl)4-bora-3a,4a-diaza-s-indacene (A); 4,4-difluoro-2,6-diiodo-1,3,5,7-tetramethyl-8-(4-[(8-bromooctoxy] phenyl)4-bora-3a,4a-diaza-s-indacene (B)].

Figure 1

Elution of the copolymer P(MMA-ran-DMAEMA) linked to BODIPY (Table 2, entry 4), through a Sephadex LH 20 column, eluted with methanol. The first peak corresponds to the conjugate copolymer, the second fraction to the free BODIPY.

Figure 2

Representative images of antimicrobial assay on E. coli K12-MG1655. Bacterial cells were inoculated on LB agar and paper disks were placed on solid medium. Disks were prepared with polymers (CoPol1, CoPol2, CoPol3) or polymers with BODIPYs A and B, respectively. After 2 h incubation at 37 °C, petri dishes were dark incubated or irradiated under green light. After overnight incubation at 37 °C, the antimicrobial activity was visible as a clear inhibition growth halo. The experiments were performed three times with independent bacterial cultures.

Figure 3

Representative images of antimicrobial assay on S. aureus ATCC 6538P. Bacterial cells were inoculated on LB agar and paper disks were placed on solid medium. Disks were prepared with polymers (CoPol1, CoPol2, CoPol3) or polymers with BODIPYs A and B, respectively. After 2 h incubation at 37 °C, petri dishes were dark incubated or irradiated under green light. After overnight incubation at 37 °C, the antimicrobial activity was visible as a clear inhibition growth halo. The experiments were performed three times with independent bacterial cultures.

Figure 4

Antimicrobial effect of CoPol1 at decreasing concentrations (15, 10, 5, and 1 mg/mL) on E. coli K12-MG1655 (A) and S. aureus ATCC 6538P (B), respectively. DMSO and ampicillin (x mg/mL) are included as controls.