Studies on narrow bandwidth light effects on cellular cultures and animal models

Photodynamic therapy (PDT) is a clinically approved modality for the treatment of cancer and macular degeneration. Currently this modality is also being explored for the treatment of other diseases such as superficial bacterial infections and restenosis of arteries.  PDT utilizes light and oxygen in combination with a photosensitizing drug to achieve cellular destruction through production of cytotoxic reactive oxygen species.  At LBAID, RRCAT studies have been carried out to explore the effect of photodynamic treatment on cancer cells and bacteria.  Chlorin p6, (Cp6) a chlorophyll derivative prepared in-house has shown promising results in cancer cells and hamster cheek pouch model of oral cancer. These studies showed that pH influences the hydrophobicity of the drug and thus the cellular uptake and the photodynamic efficacy of the drug [1]. Cp6 accumulated preferentially in tumor, cleared rapidly from the skin and resulted in complete tumor regression subsequent to PDT with red light [1]. The cell death mode after PDT is of interest since it influences the response of the immune system and therefore the effectiveness of the treatment. Our Studies revealed that PDT with Cp6 led to cell death predominantly by apoptosis at physiological pH and necrosis was more common when the pH was lowered to slightly acidic [2].  In hamster cheek pouch model, Cp6 given intraperitoneal (1.5 mg/kg body weight) showed good tumor selectivity if the size of tumors was smaller than 5 mm, led to complete tumor damage subsequent to PDT with red light and excreted rapidly from normal tissue [3]. For larger tumor sizes accumulation of the drug was poor for systemically administered drug. Here topical application at 1.0 mg/kg body weight gave better results. The current interest of this activity is to improve the delivery and photodynamic efficacy of chlorin p6 in tumor cells by conjugating it with molecules that can attach to cancer cells via surface receptors. In addition, use of nanoparticles has also been investigated. 

Results of studies on the antimicrobial effects of photodynamic treatment showed that photosensitization with phenothiazinium dyes induced not only lethal effect but also some favorable responses at sub lethal doses e.g. decrease in virulence of Pseudomonas aeruginosa [4]. Photodynamic treatment was also found to be effective for inactivation of antibiotic resistant Staphylococcus biofilms [5]. Since the interaction of photosensitizer with the cell envelop plays a critical role in photoinactivation of bacteria, studies on cell surface alterations induced by toludine blue (TBO) mediated photodynamic treatment on a Gram-positive bacterium, Staphylococcus aureus and a Gram-negative bacterium, Escherichia coli were carried out using Atomic force microscopy [6].  Results of this study showed bleb formation in S. aureus, suggesting breakage in the contact between the cell wall and the membrane. In E. coli, surface indentations, flattening of bacteria suggesting damage to the bacterial membrane and loss of cytoplasmic materials was more obvious. To explore the antimicrobial potential of PDT for control of wound infection, superficial and excision skin wound infection model in mice has been established. Use of Polarization-sensitive Optical coherence tomography (PS- OCT,) and real time OCT for monitoring the morphological changes associated with healing of uninfected and bacteria infected wounds has been demonstrated [7].  One of the main concern in the use of PDT as antimicrobial modality is the inability of some good photosensitizers to cross the complex cell envelop of Gram negative bacteria. To overcome such problems, photosensitizer conjugated to cationic molecules such as polylysin show considerable promise.  Studies have also been carried out using Cp6 conjugated to polylysin and significant improving in photodynamic killing of bacteria both in vitro and in vivo has been obtained.

1. Mrinalini Sharma, Alok Dube, Harsha Bansal, Pradeep Kumar Gupta, Effect of pH on uptake and photodynamic action of chlorin p6 on human colon and breast adenocarcinoma cell lines. Photochem. Photobiol. Sci. 3, 231-235 (2004).
   
   
2. M. Sharma, K. Sahu, A. Dube, H. Bansal, P.K. Gupta, Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6, Journal of Photochemistry Photobiology. B, 81,107-113, (2005).
   
   
3. Alok Dube, Sulbha Sharma, and P K Gupta, evaluation of chlorin p6 for photodynamic treatment of squamous cell carcinoma in hamster cheek pouch model. oral oncology 42, 76-81 (2006).
   
   
4. Mrinalini Sharma, Harsha Bansal, Pradeep Kumar Gupta, Virulence of Pseudomonas aeruginosa Cells Surviving Photodynamic Treatment with Toluidine Blue. Current Microbiology 50 277–280 (2005).
   
   
5. Mrinalini Sharma,Livia Visai, Francesca Bragheri, Ilaria Cristiani, Pradeep Kumar Gupta,Pietro Speziale, Toluidine Blue-Mediated Photodynamic Effects on Staphylococcal Biofilms, Antimicrobial agents and chemotherapy 52, 299–305 (2008).
   
   
6. K. Sahu, H. Bansal, C. Mukharjee, M. Sharma, P.K. Gupta, Atomic force microscopic study on morphological alterations induced by photodynamic action of toluidine blue in Staphylococcus aureus and Escherichia coli. J. Photochem. Photobiol. B, 96, 9-16, (2009).
   
   
7. K. Sahu, Y. Verma, M. Sharma, K. D. Rao and P. K. Gupta, Noninvasive assessment of healing of bacteria infected and uninfected wounds using optical coherence tomography, Skin Research and Technology In press.