Alok Dube

The Effect of pH and Surfactant on the Aggregation Behavior of Chlorin p6: A Fluorescence Spectroscopic Study¶

Abstract Steady state and time-resolved fluorescence properties of chlorin p6, a potential drug for photodynamic therapy, have been investigated as functions of pH. A decrease in pH of the medium has been shown to cause protonation of the ionizable carboxylic acid side chain, leading to an increase in hydrophobicity and consequent aggregation. The aggregates dissociate on further protonation. The dissociation is explained in terms of formation of cations and their mutual repulsion. A synchronous fluorescence spectroscopic study revealed the presence of two anionic forms in equilibrium at physiological pH, with a shift in the equilibrium on slight decrease in the pH. The anionic nature of chlorin p6 in aqueous solutions at physiological pH has been confirmed by complexation with surfactants. The nature of the charge on the headgroups of the surfactants has been found to govern the formation of chlorin–surfactant complexes.

Modulation of macrophage structure and function by low level He–Ne laser irradiation

Studies have shown that He-Ne laser irradiation can affect the biological functions of macrophages. The aim of the present study was to investigate the effect of He-Ne laser irradiation on the various functional parameters of macrophages and look for possible correlations in the effects to understand the mechanisms involved. Mice peritoneal macrophages were irradiated with a He-Ne laser (632.8 nm, approximately 10 W m(-2)) at energy densities ranging from 100 to 600 J m(-2) and the activities of lysozyme and cathepsin, phagocytosis, and cell spreading (markers of cell activation), as well as changes in NAD(P)H autofluorescence, were monitored. He-Ne laser irradiation was observed to lead to significant changes in all the parameters investigated. While lysozyme activity and spreading of the peripheral membrane were found to increase with the irradiation dose over the dose range investigated, the phagocytotic activity of macrophages, the activity of cathepsin, the observed decease in cell membrane fluidity and the observed increase in NAD(P)H level showed a peak at 200 J m(-2). Possible reasons for and the significance of the observed correlations are discussed.

Effect of Helium-Neon Laser Irradiation on Hair Follicle Growth Cycle of Swiss Albino Mice

We report the results of a study carried out to investigate the effect of helium-neon (He-Ne) laser (632.8 nm) irradiation on the hair follicle growth cycle of testosterone-treated and untreated mice. Both histology and optical coherence tomography (OCT) were used for the measurement of hair follicle length and the relative percentage of hair follicles in different growth phases. A positive correlation (R = 0.96) was observed for the lengths of hair follicles measured by both methods. Further, the ratios of the lengths of hair follicles in the anagen and catagen phases obtained by both methods were nearly the same. However, the length of the hair follicles measured by both methods differed by a factor of 1.6, with histology showing smaller lengths. He-Ne laser irradiation (at ∼1 J/cm2) of the skin of both the control and the testosterone-treated mice was observed to lead to a significant increase (p < 0.05) in % anagen, indicating stimulation of hair growth. The study also demonstrates that OCT can be used to monitor the hair follicle growth cycle, and thus hair follicle disorders or treatment efficacy during alopecia.

Effect of poly-L-lysine-chlorin P6-mediated antimicrobial photodynamic treatment on collagen restoration in bacteria-infected wounds.

OBJECTIVE The purpose of this work was to study the effect of poly-L-lysine-conjugated chlorin P6 (pl-cp6)-mediated antimicrobial photodynamic treatment (APDT) on collagen remodeling of murine excisional wounds infected with methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO). BACKGROUND DATA Bacterial infection of wounds leads to compromised collagen remodelling. APDT-induced inactivation of bacteria and bacterial proteases are expected to restore collagen remodeling in wounds. However, published reports on the effect of PDT on wound healing are somewhat contradictory. One of the reasons for these observations could be the random sampling of wound repair outcomes by invasive technques such as histology. METHODS Post-wounding time-dependent changes in collagen restoration were monitored noninvasively using polarization sensitive optical coherence tomography (PSOCT) and compared with histology and hydroxyproline level. Immunoblotting was performed to study matrix metalloproteinase (MMP) level. RESULTS As indicated by retardance measurements from PSOCT images and immunoblotting, bacteria-infected wounds showed slower collagen restoration and higher MMP-8, 9 expression, than did uninfected wounds. In contrast, in infected wounds treated with pl-cp6 and light, retardance was higher (approximately twofold) compared with wounds treated with pl-cp6 alone. These results were consistent with lower MMP-8, 9 level on day 5, more ordered collagen matrix, and higher hydroxyproline content (approximately threefold) on day 18, observed in photodynamically treated wounds, compared with that of untreated infected wounds. CONCLUSIONS APDT expedites healing in bacteria-infected wounds in mice by attenuating collagen degradation and by enhancing epithelialization, hydroxyproline content, and collagen remodelling.

Helium-Neon Laser Preirradiation Induces Protection against UVC Radiation in Wild-Type E. coli Strain K12AB1157

Abstract Kohli, R., Gupta, P.K. and Dube, A. Helium-Neon Laser Preirradiation Induces Protection against UVC Radiation in Wild-Type E. coli Strain K12AB1157. We have observed that preirradiation with a helium-neon laser (632.8 nm) induces protection against UVC radiation in wild-type E. coli strain K12AB1157. The magnitude of protection was found to depend on the helium-neon laser irradiance, exposure time, and period of incubation between helium-neon laser exposure and subsequent UVC irradiation. The optimum values for dose, irradiance and interval between the two exposures were found to be 7 kJ/m2, 100 W/m2 and 1 h, respectively. The possible involvement of singlet oxygen in the helium-neon laser-induced protection is also discussed.

Tumor regression induced by photodynamic treatment with chlorin p6 in hamster cheek pouch model of oral carcinogenesis: Dependence of mode of tumor cell death on the applied drug dose

We investigated tumor regression and the mode of tumor cell death induced by photodynamic treatment (PDT) with chlorin p(6) (Cp(6)) in hamster cheek pouch model of oral squamous cell carcinoma. Cp(6) was administered systemically through intraperitoneal injection and after 4h the tumors were subjected to photodynamic treatment using red light (660±25nm, fluence ∼100J/cm(2)). Tumor response to PDT was monitored by measuring the tumor volume before PDT and 1week after. Results show that smaller tumors (⩽80mm(3)) regressed completely after PDT with Cp(6) dose of 2.0mg/kg body weight and for the bigger tumors (∼180mm(3)) higher dose of Cp(6) (4.0mg/kg) was more effective. Tumors treated with lower Cp(6) dose showed infiltration of immune cells, absence of TUNEL labeling, smeared pattern of DNA fragmentation and no significant increase in caspase-3 activity suggestive of necrotic cell death and inflammation. In tumors treated with higher Cp(6) dose, features characteristic of apoptotic cell death such as extensive TUNEL positive labeling, increase in caspase-3 activity and laddered pattern of DNA fragmentation were observed and there was no infiltration of immune cells. PDT with Cp(6) was also found to lead to expression of matrix metalloprotease-9 (MMP-9) which was greater at lower drug dose PDT as compared to higher drug dose PDT. These results suggest that drug dose plays an important role in determining the mechanism of tumor cell death and effectiveness of PDT.

Photodynamic treatment of oral squamous cell carcinoma in hamster cheek pouch model using chlorin p6-histamine conjugate.

BACKGROUND Over-expression of histamine receptors has been reported in several types of malignancies. Earlier we have successfully demonstrated use of chlorin p6-histamine conjugate (Cp6-his) for improving cellular uptake and photo toxicity of Cp6 in oral cancer cell lines. In the present study, after having confirmed that histamine receptors are over-expressed in tumors of hamster cheek pouch, we investigated the efficacy of Cp6-his for photodynamic treatment (PDT) of tumors in this animal model. METHODS Cp6-his (3mg/kg body weight) was injected intraperitoneally and its accumulation in tumor, surrounding tissue, normal mucosa and abdominal skin was monitored non-invasively by fluorescence spectroscopy. For PDT, tumors at 4h after Cp6-his administration were exposed to red light (660±25nm, 100J/cm(2)). Tumor damage and regression were assessed by histology and tumor volume measurements, respectively. Expression of histamine H2 receptors in tumor and normal mucosa was assessed by immuno-staining. RESULTS The accumulation of Cp6-his was higher in tumors as compared to normal mucosa at 4h after its administration. For Cp6 similar preferential accumulation was observed except that in normal mucosa the accumulation of Cp6 was more as compared to Cp6-his. The clearance of Cp6-his from skin was rapid showing ∼80% decrease within 48h from its peak level at 4h after drug injection. PDT led to extensive cellular damage and tumors of size up to ∼1000mm(3) regressed completely one week after PDT. CONCLUSION Higher tumor selectivity of Cp6-his and complete regression of bigger tumors after PDT suggest that conjugating Cp6 to histamine is a promising approach to improve PDT efficacy.

Fluorescence study of maize irradiated by UVA

Effects of UVA on the growth of maize ( Zea mays L.) plants were investigated by growing them under two different controlled conditions. The weight of plants, length of stem, and length and width of the second leaf of each plant were measured. It was found that the growth of plants was reduced under UVA treatment at an early stage. We observed a clear-cut reduction in the growth of plants. The fluorescence spectra of green maize leaf induced by 337 nm radiation were obtained in each case, consisting of four peak bands at 435 nm (F435), 525 nm (F525), 684 nm (F684) and 740 nm (F740), respectively. The ratios of blue to green bands (F435/F525), blue to red bands (F435/F684) and the chlorophyll fluorescence (F684/F740) ratio were calculated and discussed.

Synthesis and characterization of photodynamic activity of an iodinated Chlorin p6 copper complex

We report the synthesis of a new iodinated Chlorin p6 copper complex (ICp6–Cu) and its efficacy for photodynamic treatment (PDT) of cancer cells. The metal complex is obtained by reacting Chlorin p6 (Cp6) with copper iodide (CuI). The complex formation results in a shift in the Q absorption band of Cp6 from 663 nm to 634 nm and X-ray fluorescence of the complex showed the presence of both copper and iodine. FTIR and EPR spectroscopy suggests that the copper is attached to Cp6 at the two adjacent carboxylic groups. Studies on the photochemical generation of singlet oxygen (1O2) and other reactive oxygen species (ROS) using fluorescence probes revealed that ICp6–Cu acts predominantly through the type I process. PDT of oral cancer cells with ICp6–Cu (10 μM, 3 h) and red light (630 ± 20 nm, ∼12 J cm−2) led to ∼90% phototoxicity. Furthermore, in contrast to Cp6, the phototoxicity induced by ICp6–Cu is not significantly affected under hypoxic conditions.

Chlorin p6 preferentially localizes in endoplasmic reticulum and Golgi apparatus and inhibits Ca2+ release from intracellular store

Subcellular localization of chlorin p6 in human cerebral glioma (U-87MG) cells was studied using laser scanning confocal microscopy. Localization in sub cellular organelles was ascertained by double labeling with specific fluorescent markers of subcellular organelles. The results reveal that chlorin p6 binds to multiple cellular sites but preferential binding sites are endoplasmic reticulum and Golgi apparatus and it does not bind to mitochondria. Significantly the drug localization pattern of proliferating and differentiated cells was notably distinct. In proliferating cells the internalization of drug was faster than in differentiated cells. Localization of chlorin p6 into the cells inhibited Ca(2+) release from endoplasmic reticulum and deregulated cellular Ca(2+) signalling. These results suggest that the fluorescence imaging pattern of chlorin p6 could be useful in identifying the proliferating and differentiated population of cells in tumor tissue.