Enhanced photodynamic therapy and effective elimination of cancer stem cells using surfactant-polymer nanoparticles.

Photodynamic therapy is a potentially curative treatment for various types of cancer. It involves energy transfer from an excited photosensitizer to surrounding oxygen molecules to produce cytotoxic singlet oxygen species, a process termed as type II reaction. The efficiency of photodynamic therapy is greatly reduced because of the reduced levels of oxygen, often found in tumor microenvironments that also house cancer stem cells, a subpopulation of tumor cells that are characterized by enhanced tumorigenicity and resistance to conventional therapies. We show here that encapsulation of a photosensitizer, methylene blue, in alginate-Aerosol OT nanoparticles leads to an increased production of reactive oxygen species (ROS) under both normoxic and hypoxic conditions. ROS generation was found to depend on the interaction of the cationic photosensitizer with the anionic alginate polymer. Dye-polymer interaction was characterized by formation of methylene blue dimers, potentially enabling electron transfer and a type I photochemical reaction that is less sensitive to environmental oxygen concentration. We also find that nanoparticle encapsulated methylene blue has the capacity to eliminate cancer stem cells under hypoxic conditions, an important goal of current cancer therapy.

Reduction of Endotracheal Tube Biofilms Using Antimicrobial Photodynamic Therapy.

BACKGROUND: Ventilator-associated pneumonia (VAP) is reported to occur in 12 to 25% of patients who require mechanical ventilation with a mortality rate of 24 to 71%. The endotracheal (ET) tube has long been recognized as a major factor in the development of VAP since biofilm harbored within the ET tube become dislodged during mechanical ventilation and have direct access to the lungs. The objective of this study was to demonstrate the safety and effectiveness of a non-invasive antimicrobial photodynamic therapy (aPDT) treatment method of eradicating antibiotic resistant biofilms from ET tubes in an in vitro model. METHODS: Antibiotic resistant polymicrobial biofilms of Pseudomonas aerugenosa and MRSA were grown in ET tubes and treated, under standard ventilator conditions, with a methylene blue (MB) photosensitizer and 664nm non-thermal activating light. Cultures of the lumen of the ET tube were obtained before and after light treatment to determine efficacy of biofilm reduction. RESULTS: The in vitro ET tube biofilm study demonstrated that aPDT reduced the ET tube polymicrobial biofilm by >99.9% (p<0.05%) after a single treatment. CONCLUSIONS: MB aPDT can effectively treat polymicrobial antibiotic resistant biofilms in an ET tube.

Antimicrobial photodynamic therapy treatment of chronic recurrent sinusitis biofilms.

BACKGROUND: Chronic recurrent sinusitis (CRS) is an inflammatory disease of the facial sinuses and nasal passages that is defined as lasting longer than 12 weeks or occurring more than 4 times per year with symptoms usually lasting more than 20 days. The National Institute for Health Statistics estimates that CRS is one of the most common chronic conditions in the United States, affecting an estimated 37 million Americans. The potential etiologies of CRS include bacteria, viruses, allergies, fungi, superantigens, and microbial biofilms. In clinical practice there is a significant subpopulation of patients with CRS who remain resistant to cure despite rigorous treatment regimens including surgery, allergy therapy, and prolonged antibiotic therapy. The reason for treatment failure is thought to be related to the destruction of the sinus mucociliary defense by the chronic sinus infection resulting in the development of secondary antibiotic-resistant microbial colonization of the sinuses and biofilm formation. Antimicrobial photodynamic therapy (aPDT) is a nonantibiotic broad-spectrum antimicrobial treatment that has been demonstrated to eradicate antibiotic-resistant bacteria and biofilms. The objective of this study was to demonstrate the effectiveness of a noninvasive aPDT treatment method of eradicating antibiotic resistant biofilms/microorganisms known to cause CRS in an in vitro model. METHODS: Antibiotic-resistant planktonic bacteria and fungi and polymicrobial biofilms of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) were grown on silastic sheets and treated with a methylene blue photosensitizer and 670 nm non-thermal-activating light. Cultures of the planktonic microorganisms and biofilms were obtained before and after light treatment to determine efficacy of planktonic bacteria and biofilm reduction. RESULTS: The in vitro CRS planktonic microorganism and biofilm study demonstrated that aPDT reduced the CRS polymicrobial biofilm by >99.9% after a single treatment. CONCLUSION: aPDT can effectively treat CRS polymicrobial antibiotic-resistant bacteria, fungi, and biofilms in vivo. Human clinical studies are currently planned to assess the safety and efficacy of this treatment for CRS.

Effect of Ca+ on the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria and the dye affinity for lipopolysaccharides.

BACKGROUND AND OBJECTIVES: Methylene blue (MB) and toluidine blue (TB) form metachromatic complexes with lipopolysaccharides (LPS). The greater photobactericidal efficacy of TB may be explained by its affinity for LPS. This study aims to elucidate the difference in photobactericidal efficacies between the dyes using Ca(2+) as a competitor for dye-binding sites on the bacterial outer membrane. STUDY DESIGN/MATERIALS AND METHODS: Fixed dye concentration solutions with gram-negative bacteria and increasing concentrations of CaCl(2) were exposed to red laser light. Bacterial survival and spectrophotometry were used to describe the effect of Ca(2+) on dye interaction with bacteria and LPS. RESULTS: MB-mediated photokilling was inhibited more significantly than that of TB. CaCl(2) inhibited dye photobleaching and suppressed the metachromatic reaction between the dyes and LPS, in particular TB. CONCLUSIONS: CaCl(2) inhibits bacterial photokilling by binding with LPS, as well as other anionic polymers including outer membrane proteins. LPS is chiefly involved in TB-mediated photokilling, whereas outer membrane proteins probably are more involved in MB-mediated photokilling.

The interaction of lipopolysaccharides with phenothiazine dyes.

BACKGROUND AND OBJECTIVES: The difference in the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria may result from their primary reaction with lipopolysaccharides (LPS) of the outer bacterial membrane. The aim of the present study was to compare the reactivity of these dyes with LPS extracted from different gram-negative bacteria. STUDY DESIGN/MATERIALS AND METHODS: The interactions of methylene blue and toluidine blue with LPS from Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), and Serratia marcescens (S. marcescens) were studied spectrophotometrically in 0.45% saline. The dyes were used at the concentration of 10 microM. The concentrations of LPS ranged from 5-100 microg/ml. RESULTS: Methylene blue and toluidine blue enter into a metachromatic reaction with the LPS resulting the in generation of dimers of methylene blue and higher aggregates of toluidine blue. The more significant hypochromic and hypsochromic effects in the reaction of the latter with LPS indicate a greater metachromatic efficacy of toluidine blue than methylene blue. The equilibrium constants of the metachromatic complex between toluidine blue and different LPS were calculated. The spectrophotometric titration of LPS with the dyes was used to estimate the equivalent weight of LPS. CONCLUSIONS: Toluidine blue interacts with LPS more significantly than methylene blue in vitro. This may be one of the main factors determining its greater photobactericidal efficacy against gram-negative bacteria.