Chitosan nanoparticles enhance the efficiency of methylene blue-mediated antimicrobial photodynamic inactivation of bacterial biofilms: An in vitro study

Highlights

• Significant phototoxicity of MB+CSNPs on S. aureus and P. aeruginosa biofilms compared to MB alone.

• 84.9% survival of fibroblast cells after phototoxicity assessment using MB+CSNPs.

• Great potential of MB+CSNPs as a dual-action synergistic mixture to fight biofilm.

Abstract

Background

Biodegradable chitosan nanoparticles (CSNPs) with an intrinsic antimicrobial activity may be a good choice to improve the effectiveness of antimicrobial photodynamic inactivation (APDI). The aim of this study was to investigate the effect of CSNPs on the efficiency of methylene blue (MB)-mediated APDI of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. We also assessed the phototoxicity of MB + CSNPs towards human fibroblasts.

Methods

CSNPs were prepared using ionic gelation method and characterized by dynamic light scattering (DLS) and field-emission scanning electron microscope (FESEM). Biofilms were developed in a 96-well polystyrene plate for 24 h. In vitro phototoxic effect of MB + CSNPs (at final concentrations of 50 μM MB) at fluence of 22.93 J/cm²) on biofilms were studied. Appropriate controls were included. Also, in vitro cytotoxicity and phototoxicity of the above mixture was assessed on human dermal fibroblasts.

Results

DLS and FESEM measurements confirmed the nanometric size of the prepared CSNPs. APDI mediated by the mixture of MB and CSNPs showed significant anti-biofilm photoinactivation (P < 0.001, >3 and >2 log₁₀ CFU reduction in S. aureus and P. aeruginosa biofilms, respectively) while MB-induced APDI led to approximately <1 log₁₀ CFU reduction. At the same experimental conditions, only 25.1% of the fibroblasts were photoinactivated by MB + CSNPs.

Conclusion

Our findings showed that CSNPs enhanced the efficacy of MB-APDI; it may be due to the disruption of biofilm structure by polycationic CSNPs and subsequently deeper and higher penetration of MB into the biofilms.

Introduction

Biofilms as microbial communities are the most ancient multicellular life forms that in 1980s their pathogenic role during the infection process was recognized [1]. A biofilm is an aggregate of microbial cells embedded in a self-produced matrix composed of different extracellular polymeric substances (EPS), including exopolysaccharides, proteins, nucleic acids and lipids [2], [3]. According to the National Institutes of Health, biofilms have been estimated to account for 80% of human infections [4].

Microbial biofilms have been found to be involved in a variety of challenging clinical conditions, especially in chronic and medical device-related infections [5]. Biofilm-associated infections mainly include chronic wound infection, chronic otitis media, chronic osteomyelitis, chronic prostatitis, endocarditis, cystic fibrosis-associated lung infection, recurrent urinary tract infection, dental caries and periodontitis [1], [2].

Antimicrobial Photodynamic Inactivation (APDI) or Antimicrobial Photodynamic therapy (APDT) may emerge as one of the promising alternative treatments to combat both biofilm and antimicrobial-related resistance due to its rapid effectiveness, broad spectrum of action, and the efficient inactivation of antibiotic-resistant strains [6].

Photodynamic therapy (PDT) is based on localized administration of a non-toxic light-sensitive drug/compound (photosensitizer) followed by exposure to light of an appropriate wavelength to produce singlet oxygen or other reactive oxygen species that are able to kill target cells via oxidative stress to cell membranes and other cellular components [7].

Usually, photosensitizers with positive charge are more efficient than their neutral and negative charged analogues for anti-biofilm PDT due to their high affinity to negatively charged bacterial cell wall and EPS-molecules of biofilms [8], [9].

At the present time, phenothiazinium salts, such as methylene blue (MB), are used clinically for antimicrobial therapy [6]. MB is an attractive candidate for APDI because of its favorable properties such as its high quantum yield of ¹O₂, hydrophilicity, low molecular weight, low mammalian cell toxicity and cationic form at physiological pH [6], [10]. However, biofilm susceptibility toward APDI can be affected by some specific protective factors such as EPS and efflux pumps [11].

The decreased penetration of the photosensitizer into the inner regions of the biofilm structure caused by extracellular matrix is one of the main causes of the low susceptibility of biofilm to APDI. In the other words, the effective penetration of photosensitizer into the biofilm structure can be a critical parameter for the performance of APDI. Nanoparticles with ability to disrupting the three-dimensional organization of biofilm can be a promising choice for this goal [12].

Chitosan nanoparticles (CSNPs) are known to possess broad spectrum-antibacterial activity mainly because of their polycationic charge [13]. It has been proved that CSNPs can disrupt biofilm structure (12, 14), so, they can be assessed as a potential choice to enhance the effectiveness of APDI.

Methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant (MDR) Pseudomonas aeruginosa are particularly problematic in chronic wound infection due to their antibiotic resistance and ability to form biofilm [15]. So, the main purpose of this work was to investigate whether CSNPs can enhance the efficiency of MB-mediated APDI of MRSA and MDR P. aeruginosa biofilms.