Chitosan Inhibits the Rehabilitation of Damaged Microbes Induced by Photodynamic Inactivation.

Previously, we showed that chitosan could augment the biocidal efficacy mediated by photodynamic treatment against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. In this study, we showed that the antimicrobial action of chitosan in augmenting photodynamic inactivation (PDI) is related to the increase in cell surface destruction. The microbial cell surfaces exhibit severe irregular shapes after PDI in the presence of chitosan as demonstrated by transmitted electron microscopy. Furthermore, increases in the concentration or incubation time of chitosan significantly reduced the amounts of photosensitizer toluidine blue O required, indicating that chitosan could be an augmenting agent used in conjunction with PDI against S. aureus, P. aeruginosa, and C. albicans. A prolonged lag phase was found in microbial cells that survived to PDI, in which chitosan acted to completely eradicate the cells. Once the exponential log stage and cell rebuild began, their cellular functions from PDI-induced damage returned and the increased cytotoxic effect of chitosan disappeared. Together, our results suggest that chitosan can prevent the rehabilitation of PDI-surviving microbial cells, leading to increased biocidal efficacy.

Assessment of Photodynamic Inactivation against Periodontal Bacteria Mediated by a Chitosan Hydrogel in a 3D Gingival Model.

Chitosan hydrogels containing hydroxypropyl methylcellulose (HPMC) and toluidine blue O were prepared and assessed for their mucoadhesive property and antimicrobial efficacy of photodynamic inactivation (PDI). Increased HPMC content in the hydrogels resulted in increased mucoadhesiveness. Furthermore, we developed a simple In Vitro 3D gingival model resembling the oral periodontal pocket to culture the biofilms of Staphylococcus aureus (S. aureus), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Porphyromonas gingivalis (P. gingivalis). The PDI efficacy of chitosan hydrogel was examined against periodontal biofilms cultured in this 3D gingival model. We found that the PDI effectiveness was limited due to leaving some of the innermost bacteria alive at the non-illuminated site. Using this 3D gingival model, we further optimized PDI procedures with various adjustments of light energy and irradiation sites. The PDI efficacy of the chitosan hydrogel against periodontal biofilms can significantly improve via four sides of irradiation. In conclusion, this study not only showed the clinical applicability of this chitosan hydrogel but also the importance of the light irradiation pattern in performing PDI for periodontal disease.

Optimization and Evaluation of a Chitosan/Hydroxypropyl Methylcellulose Hydrogel Containing Toluidine Blue O for Antimicrobial Photodynamic Inactivation.

Photodynamic inactivation (PDI) combined with chitosan has been shown as a promising antimicrobial approach. The purpose of this study was to develop a chitosan hydrogel containing hydroxypropyl methylcellulose (HPMC), chitosan and toluidine blue O (TBO) to improve the bactericidal efficacy for topical application in clinics. The PDI efficacy of hydrogel was examined in vitro against the biofilms of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). Confocal scanning laser microscopy (CSLM) was performed to investigate the penetration level of TBO into viable S. aureus biofilms. Incorporation of HMPC could increase the physicochemical properties of chitosan hydrogel including the hardness, viscosity as well as bioadhesion; however, higher HMPC concentration also resulted in reduced antimicrobial effect. CSLM analysis further demonstrated that higher HPMC concentration constrained TBO diffusion into the biofilm. The incubation of biofilm and hydrogel was further performed at an angle of 90 degrees. After light irradiation, compared to the mixture of TBO and chitosan, the hydrogel treated sample showed increased PDI efficacy indicated that incorporation of HPMC did improve antimicrobial effect. Finally, the bactericidal efficacy could be significantly augmented by prolonged retention of hydrogel in the biofilm as well as in the animal model of rat skin burn wounds after light irradiation.

5-Aminolevulinic acid induced photodynamic inactivation on Staphylococcus aureus and Pseudomonas aeruginosa.

The aim of the present study was to develop a simple and fast screening technique to directly evaluate the bactericidal effects of 5-aminolevulinic acid (ALA)-mediated photodynamic inactivation (PDI) and to determine the optimal antibacterial conditions of ALA concentrations and the total dosage of light in vitro. The effects of PDI on Staphylococcus aureus and Pseudomonas aeruginosa in the presence of various concentrations of ALA (1.0 mM, 2.5 mM, 5.0 mM, 10.0 mM) were examined. All bacterial strains were exponentially grown in the culture medium at room temperature in the dark for 60 minutes and subsequently irradiated with 630 ± 5 nm using a light-emitting diode (LED) red light device for accumulating the light doses up to 216 J/cm2. Both bacterial species were susceptible to the ALA-induced PDI. Photosensitization using 1.0 mM ALA with 162 J/cm2 light dose was able to completely reduce the viable counts of S. aureus. A significant decrease in the bacterial viabilities was observed for P. aeruginosa, where 5.0 mM ALA was photosensitized by accumulating the light dose of 162 J/cm2. We demonstrated that the use of microplate-based assays-by measuring the apparent optical density of bacterial colonies at 595 nm-was able to provide a simple and reliable approach for quickly choosing the parameters of ALA-mediated PDI in the cell suspensions.

The use of Chitosan to enhance photodynamic inactivation against Candida albicans and its drug-resistant clinical isolates.

Drug-resistant Candida infection is a major health concern among immunocompromised patients. Antimicrobial photodynamic inactivation (PDI) was introduced as an alternative treatment for local infections. Although Candida (C.) has demonstrated susceptibility to PDI, high doses of photosensitizer (PS) and light energy are required, which may be harmful to eukaryotic human cells. This study explores the capacity of chitosan, a polycationic biopolymer, to increase the efficacy of PDI against C. albicans, as well as fluconazole-resistant clinical isolates in planktonic or biofilm states. Chitosan was shown to effectively augment the effect of PDI mediated by toluidine blue O (TBO) against C. albicans that were incubated with chitosan for 30 min following PDI. Chitosan at concentrations as low as 0.25% eradicated C. albicans; however, without PDI treatment, chitosan alone did not demonstrate significant antimicrobial activity within the 30 min of incubation. These results suggest that chitosan only augmented the fungicidal effect after the cells had been damaged by PDI. Increasing the dosage of chitosan or prolonging the incubation time allowed a reduction in the PDI condition required to completely eradicate C. albicans. These results clearly indicate that combining chitosan with PDI is a promising antimicrobial approach to treat infectious diseases.

Liposome-encapsulated photosensitizers against bacteria.

Photodynamic therapy (PDT), utilizing photosensitizers and light, has received considerable interests for its potential to treat microbial infections. The advantages of antimicrobial PDT include a broad spectrum of action, efficient killing against wild-type as well as drug-resistant pathogens. Therefore, antimicrobial PDT could be valuable to rapidly reduce the microbial burden during the management of local infections, especially for the antibiotic resistance. A variety of photosensitizers have been examined its efficacy against pathogens. To increase the efficacy of photosensitizers, various drug delivery systems have been developed. Among these carrier systems, liposomes showed their PDT efficacy and safety in delivering photosensitizers. This review is focused on the application of liposomes mediated photodynamic inactivation of bacteria along with the discussion of few of recent patents.

Chitosan nanoparticles for antimicrobial photodynamic inactivation: characterization and in vitro investigation.

The growing resistance to antibiotics has rendered antimicrobial photodynamic inactivation (PDI) an attractive alternative treatment modality for infectious diseases. Chitosan (CS) was shown to further potentiate the PDI effect of photosensitizers and was therefore used in this study to investigate its ability to potentiate the activity of erythrosine (ER) against bacteria and yeast. CS nanoparticles loaded with ER were prepared by ionic gelation method and tested for their PDI efficacy on planktonic cells and biofilms of Streptococcus mutans, Pseudomonas aeruginosa and Candida albicans. The nanoparticles were characterized for their size, polydispersity index and zeta potential. No toxicity was observed when planktonic cells and biofilms were treated with the nanoparticles in the dark. However, when the cells were exposed to light irradiation after treatment with free ER or ER/CS nanoparticles, a significant phototoxicity was observed. The antimicrobial activity of ER/CS nanoparticles was significantly higher than ER in free form. The particle size and incubation time of the nanoparticles also appeared to be important factors affecting their PDI activity against S. mutans and C. albicans.