Antibacterial photodynamic activity of photosensitizer-embedded alginate-pectin-carboxymethyl cellulose composite biopolymer films.

Antimicrobial photodynamic therapy (APDT) is a promising approach for treatment of wounds infected with antibiotic-resistant bacteria. In this approach, delivery of appropriate concentration of photosensitizer (PS) at the infected site is a critical step; it is therefore essential that PS need to be administered at the infected site in a suitable formulation. Here, we report preparation of PS-embedded composite biopolymer films and their photobactericidal properties against methicillin-resistant Staphylococcus aureus (MRSA) and biocompatibility. Sodium alginate (SA), pectin (PC), and carboxymethyl cellulose (CMC) were used for preparing films containing chlorin p6 (Cp6, anionic PS) or methylene blue (MB, cationic PS). Films containing 1% CMC (15 mm diameter; 110 ± 09 µm thickness) showed ~ 55% light transmission in 500 to 750 nm region and high swelling rate as indicated by ~ 38% increase in diameter within 1 h. Absorption spectroscopic studies of PS-embedded films revealed that while Cp6 existed mainly in monomeric state, MB existed in both dimeric and monomeric forms. MRSA incubated with the film for 1 h displayed substantial uptake of Cp6 and MB as indicated by the presence of Cp6 fluorescence and MB staining in cells under the microscope. Furthermore, photodynamic treatment (660 nm, 10 J/cm2) of MRSA with Cp6 embedded in film or free Cp6 resulted in ~ 3 log reduction in colony-forming units (cfu), whereas decrease in cfu was less (~ 1 log) for MB-embedded film than for free MB (~ 6 logs). Studies on human keratinocyte (HaCaT) cells showed that there was no significant change in the viability of cells when they were incubated with solubilized films (plain) for 24 h or subjected to treatment with PS-containing films followed by PDT. These results suggest that films are biocompatible and have potential application in photodynamic treatment of MRSA-infected wounds.

A study of normal foot abduction across various age groups in children.

INTRODUCTION: The relapse rate after Ponseti method of correction has reduced in recent years which is attributable to the better appreciation of the need to achieve the correct degree of abduction. Ponseti recommended clinical 'overcorrection' of the foot to 700 of abduction. However, no scientific basis for this figure was found in literature. As the indications of Ponseti method extend to older children, we conducted a study to find out the amount of foot abduction to be achieved before applying a foot abduction brace in various age groups. METHODS: The normal 197 feet of children up to eight years of age were considered for study. The measurements included foot-bimalleolar angle in neutral and maximum possible abduction, thigh foot angle and leg foot angle. RESULTS AND DISCUSSION: The foot bimalleolar angle in neutral had a mean of 82.6 degrees and in maximum abduction a mean of 99.0 degrees. The mean leg foot angle was 66.4 degrees and the mean thigh foot angle was 60.5 degrees. It was found that these variables do not change with age. From the study we concluded that achieved abduction should be about 60-70 degrees before applying foot abduction brace in all children till age eight years with clubfeet treated with Ponseti technique, keeping the leg foot angle or the thigh foot angle as a guideline. This is against the common perception of keeping the abduction at 70 degrees for infants and reducing the abduction to 30 to 40 degrees for older children. Both the leg foot angle and thigh foot angles are reliable indicators of correction.

Photo-biomodulatory response of low-power laser irradiation on burn tissue repair in mice.

The present work reports the photo-biomodulatory effect of red (632.8 nm) and near infrared (785 and 830 nm) lasers on burn injury in Swiss albino mice. Animals were induced with a 15-mm full thickness burn injury and irradiated with various fluences (1, 2, 3, 4, and 6 J/cm2) of each laser wavelength under study having a constant fluence rate (8.49 mW/cm2). The size of the injury following treatment was monitored by capturing the wound images at regular time intervals until complete healing. Morphometric assessment indicated that the group treated with 3-J/cm2 fluence of 830 nm had a profound effect on healing as compared to untreated controls and various fluences of other wavelengths under study. Histopathological assessment of wound repair on treatment with an optimum fluence (3 J/cm2) of 830 nm performed on days 2, 6, 12, and 18 post-wounding resulted in enhanced wound repair with migration of fibroblasts, deposition of collagen, and neovascularization as compared to untreated controls. The findings of the present study have clearly demonstrated that a single exposure of 3-J/cm2 fluence at 830-nm enhanced burn wound healing progression in mice, which is equivalent to 5 % povidone iodine treatment (reference standard), applied on a daily basis till complete healing.

Topical antimicrobial photodynamic therapy improves angiogenesis in wounds of diabetic mice.

We report the results of our investigations on the effect of antimicrobial photodynamic therapy (APDT) on angiogenesis in wounds of diabetic mice. For this, measurements were made on levels of nitric oxide (NO), vascular endothelial growth factor-A (VEGF-A), and markers of proinflammatory stress (phosphorylated nuclear factor kappa B and p(38) mitogen-activated protein kinase) on day 3 post-wounding. For uninfected and infected wounds, the levels of NO, VEGF-A were lower and the levels of phospho-NF-kB-p65, phospho-p(38)MAPK were higher in diabetic mice compared with that in nondiabetic mice. For infected wounds, multiple APDT (fluence ~60 J/cm(2)) led to increase in NO, VEGF-A levels and a decrease in the phospho-NF-kB-p65, phospho-p(38)MAPK. Further, compared with aminoguanidine, and silver nitrate, multiple APDT was observed to result in a much improved proangiogenic response.

Enhancement of phototoxicity of curcumin in human oral cancer cells using silica nanoparticles as delivery vehicle.

We report results on the use of organically modified silica nanoparticles (SiNp) as a vehicle for the delivery of curcumin in human oral cancer cells for improvement of uptake and phototoxicity. Nanoformulated drug (curcumin-SiNp complex) was prepared by postloading curcumin in SiNp, and the complex was soluble in aqueous solution. Cellular uptake studied by fluorescence microscopy and spectroscopy showed that curcumin accumulation was higher when cells were incubated with curcumin-SiNp complex as against free curcumin. Studies carried out on incubation time-dependent cytotoxicity, inhibition of NF-κB activity, suppression of NF-κB-regulated proteins involved in invasion (MMP-9), angiogenesis (VEGF), and inflammation (TNF-α) showed that curcumin-SiNp leads to significant improvement over free curcumin in dark as well as on exposure to light.

Topical photodynamic treatment with poly-L-lysine-chlorin p6 conjugate improves wound healing by reducing hyperinflammatory response in Pseudomonas aeruginosa-infected wounds of mice.

We report the results of our investigations on the effect of antimicrobial photodynamic treatment (APDT) with poly-lysine-conjugated chlorin p6 (pl-cp6) on proinflammatory cytokine expression and wound healing in a murine excisional wound model infected with Pseudomonas aeruginosa. Treatment of infected wounds with pl-cp6 and light doses of 60 and 120 J/cm(2) reduced the bacterial load by ~1.5 and 2.0 log, respectively, after 24 h. The treated wounds healed ~5 days earlier as compared to untreated control and wound closure was not dependent on light dose. Interestingly, at 96 h post-treatment, drug-treated wounds irradiated at 60 J/cm(2) showed considerable reduction of proinflammatory cytokines IL-6 (approximately five times) and TNF-α (approximately four times) compared to untreated control. Further, exposure of culture supernatants to similar light dose and pl-cp6 concentration under in vitro conditions reduced the protease activity by ~50 % as compared to the untreated control, suggesting inactivation of extracellular virulent factors. Additionally, histological analysis of treated infected wounds showed complete reepithelialization, ordered collagen fibers, and considerable decrease in inflammatory cell infiltration compared to untreated wounds. These results imply that pl-cp6-mediated PDT reduces hyperinflammatory response of infected wounds, leading to acceleration of wound healing.

Noninvasive imaging of ethanol-induced developmental defects in zebrafish embryos using optical coherence tomography.

In this article, we report the use of optical coherence tomography for noninvasive cross-sectional real-time imaging of ethanol-induced developmental defects in zebrafish embryos larvae. For ethanol concentration of over 300 mM, developmental defects of eye (shrinkage and retinal abnormalities), malformation of the notochord and ataxia arising due to the toxic effects of ethanol were observed in OCT images from 3 days post fertilization onwards. The results suggest that OCT could be a valuable tool for noninvasive assessment of birth defects in small animal systems.

Optimizing the Efficacy of Learning Objectives through Pretests.

Learning objectives (LOs) are statements that typically precede a study session and describe the knowledge students should obtain by the end of the session. Despite their widespread use, limited research has investigated the effect of LOs on learning. In three laboratory experiments, we examined the extent to which LOs improve retention of information. Participants in each experiment read five passages on a neuroscience topic and took a final test that measured how well they retained the information. Presenting LOs before each corresponding passage increased performance on the final test compared with not presenting LOs (experiment 1). Actively presenting LOs increased their pedagogical value: Performance on the final test was highest when participants answered multiple-choice pretest questions compared with when they read traditional LO statements or statements that included target facts (experiment 2). Interestingly, when feedback was provided on pretest responses, performance on the final test decreased, regardless of whether the pretest format was multiple choice or short answer (experiment 3). Together, these findings suggest that, compared with the passive presentation of LO statements, pretesting (especially without feedback) is a more active method that optimizes learning.

Wound healing activity of curcumin conjugated to hyaluronic acid: in vitro and in vivo evaluation.

Curcumin is a promising wound healing agent but its clinical application is limited due to hydrophobicity and lack of stability. In this article, we report the results of a study on wound healing efficacy of curcumin conjugated to hyaluronic acid (HA) which is a natural polysaccharide known to influence the healing process. Studies on proliferation, antioxidant activity and scratch wound healing carried out in human keratinocyte cells revealed that HA-conjugated curcumin treatment enhanced cell proliferation, decreased oxidative damage induced by H2O2 and also improved migration of cells in scratch wounds as compared to treatment with native curcumin. HA conjugated curcumin exhibited bactericidal activity in dark and phototoxicity when irradiated with blue light against antibiotic resistant bacteria. Furthermore, wound healing efficacy studied in diabetic mice demonstrated that topical application of the conjugate on wounds led to better healing as compared to treatment with HA-free curcumin and HA alone. These results suggest that HA conjugation is a promising formulation of curcumin for enhancing its healing efficacy.

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6.

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.

Modulation of inflammatory response of wounds by antimicrobial photodynamic therapy.

BACKGROUND AND AIMS: Management of infections caused by Pseudomonas aeruginosa is becoming difficult due to the rapid emergence of multi-antibiotic resistant strains. Antimicrobial photodynamic therapy (APDT) has a lot of potential as an alternative approach for inactivation of antibiotic resistant bacteria. In this study we report results of our investigations on the effect of poly-L-lysine conjugate of chlorine p6 (pl-cp6) mediated APDT on the healing of P.aeruginosa infected wounds and the role of Nuclear Factor kappa B (NF-kB) induced inflammatory response in this process. MATERIALS AND METHOD: Excisional wounds created in Swiss albino mice were infected with ∼10(7) colony forming units of P.aeruginosa. Mice with wounds were divided into three groups: 1) Uninfected, 2) Infected, untreated control (no light, no pl-cp6), 3) Infected, APDT. After 24 h of infection (day 1 post wounding), the wounds were subjected to APDT [pl-cp6 applied topically and exposed to red light (660 ± 25 nm) fluence of ∼ 60 J/cm(2)]. Subsequent to APDT, on day 2 and 5 post wounding (p.w), measurements were made on biochemical parameters of inflammation [toll like receptor-4 (TLR-4), NF-kB, Inteleukin (IL)-[1α, IL-ß, and IL-2)] and cell proliferation [(fibroblast growth factor-2 (FGF-2), alkaline phosphatase (ALP)]. RESULTS: In comparison with untreated control, while expression of TLR-4, NF-kB (p105 and p50), and proinflammatory interleukins (IL-1α, IL-1ß,IL-2) were reduced in the infected wounds subjected to APDT, the levels of FGF-2 and ALP increased, on day 5 p.w. CONCLUSION: The measurements made on the inflammatory markers and cell proliferation markers suggest that APDT reduces inflammation caused by P.aeruginosa and promotes cell proliferation in wounds.

Cytotoxicity of curcumin silica nanoparticle complexes conjugated with hyaluronic acid on colon cancer cells.

We report results of our investigations on the cytotoxic efficacy of Organically modified silica nanoparticle (SiNp)-curcumin complex conjugated with hyaluronic acid (HA) (HA-SiNp-cur) and HA free SiNp-cur complex in human colon carcinoma (colo-205) cells. Curcumin was loaded in SiNp and resulting complexes were conjugated with HA, which has a strong affinity for cancer cells expressing CD44. After conjugation with HA, the average size of the SiNp-cur nanoparticles increased from 45 nm to 70 nm, and zeta potential changed to -33 mV from -26 mV. Compared to free curcumin and SiNp-cur, curcumin in HA-SiNp was more stable. The uptake and cytotoxicity of curcumin delivered through HA-SiNp-cur was significantly higher in monolayer and spheroids as compared to free curcumin and HA free SiNp-cur. Concomitantly, HA-SiNp-cur complex treatment resulted in higher inhibition of growth and migration of cells in spheroids. Further, incubation of colo-205 cancer cells with an excess of HA impaired the uptake of HA-SiNp-cur confirming the involvement of receptor mediated endocytosis in the uptake of HA conjugated nanocomplex. Time dependent increase in the fluorescence of curcumin observed in the release media when HA-SiNp-cur was incubated with hyaluronidase suggests involvement of enzyme in release of curcumin from nanoparticle.

Photodynamic inactivation of antibiotic resistant strain of Pseudomonas aeruginosa by porphyrins induced by delta-aminolaevulinic acid.

BACKGROUND & OBJECTIVES: The widespread occurrence of antibiotic resistant strains of Pseudomonas aeruginosa in hospitals is a matter of growing concern. We report the results of a study on photodynamic inactivation of antibiotic resistant strain of P. aeruginosa by delta-aminolaevulinic acid (ALA). METHODS: Exponentially growing P. aeruginosa cells were incubated in growth medium with ALA for various durations. Subsequently, the cells were washed and resuspended in phosphate buffered saline (PBS). These cells were incubated with different concentrations of glutathione (GSH) in PBS for 15 min. Porphyrins synthesized with and without GSH were detected by fluorescence spectroscopy. The ALA treated cells were irradiated with light at 405 nm with and without subsequent incubation in GSH. Cell survival was measured by colony forming ability. RESULTS: Incubation of cells in growth medium with ALA led to increased synthesis of protoporphyrins in cells which saturated beyond 4 h. The level of protoporphyrin synthesis increased significantly when ALA treated cells were subsequently incubated with GSH in PBS for 15 min. Irradiation of cells incubated with ALA alone led to weak inactivation. However, substantial cell death was observed in ALA treated cells irradiated in the presence of 15 mM GSH. INTERPRETATION & CONCLUSION: Photodynamic inactivation of P. aeruginosa by ALA induced porphyrins can be enhanced if ALA treated cells are further incubated with GSH and irradiated using 405 nm light. These findings may be useful for inactivation of antibiotic resistant strains of P. aeruginosa causing burn and wound infections in hospitalized patients.

Photodynamic action of merocyanine 540 on carcinoma of cervix cells.

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.

Extra cellular pH influences uptake and photodynamic action of pyropheophorbide-a entrapped in folate receptor targeted organically modified silica nanoparticle.

BACKGROUND: Photodynamic efficacy of pyropheophorbide-a (PPa) is limited due to poor aqueous solubility. In the present study, organically modified silica nanoparticles (ORMOSIL) entrapping PPa and its folate receptor targeted conjugate (FR-Np-PPa) were prepared and the effect of pH on uptake and photodynamic action of plain and FR-Np-PPa in squamous cell carcinoma (Nt-8e) cells and adenocarcinoma of breast (MCF-7) cells were studied. METHODS: Nanoformulations of PPa were characterized by absorption and fluorescence spectroscopy. Dynamic light scattering was used for size measurements. The uptake of the two nanoformulations by cells incubated in media of pH 6.5 and 7.4 was studied by confocal fluorescence microscopy and spectrofluoremetrically. Phototoxicity of PPa was studied by MTT assay. RESULTS: In MCF-7 and Nt-8e cells, while the uptake of PPa was observed to increase with a decrease in pH of the incubation media for folate receptor targeted Np, uptake of Np-PPa was not influenced by a change in the pH of the media. Inhibition in the uptake of PPa in presence of free folic acid for cells incubated in a medium of pH 6.5 with targeted nanoparticles was higher compared to physiological pH. Consistent with uptake studies in both the cell lines phototoxicity of PPa delivered through FR-Np-PPa was higher in medium of pH 6.5 as compared to physiological pH and phototoxicity induced by NP-PPa was independent of the pH of medium. CONCLUSION: Acidic pH enhances the photodynamic efficacy of FR-targeted nanoformulated PPa.

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.

Viral markers in patients with hemophagocytosis: a prospective study in a tertiary care hospital.

BACKGROUND: Hemophagocytic syndrome (HPS) is a rare clinicopathological condition characterized by the activation of macrophages with prominent hemophagocytosis in bone marrow and other reticulo-endothelial systems. HPS can be familial or secondary to infections including viruses. AIM: To study the viral markers in patients with HPS. MATERIALS AND METHODS: Serum samples of patients with HPS and control group were screened for anti EBV VCA IgM, and IgG, anti-Parvo B19 IgM, and anti-CMV IgM antibodies using commercially available ELISA kits and CMV and ParvoB19 DNA by polymerase chain reaction (PCR). RESULTS AND DISCUSSION: The present prospective study reports the profile of viral markers in HPS cases from north India. Among the 14 HPS cases 43% (6/14) were positive for at least one viral marker tested, of which EBV was found to be the most prevalent (3/6: 50%) followed by parvovirus B19(2/6: 33%) and cytomegalovirus (1/6: 17%). Mortality was noted in 33% of virus associated HPS patients. Our study highlights the higher association of Epstein-Barr virus (EBV) with HPS as compared to other viruses along with higher rate of mortality in both parvovirus B 19 and EBV associated HPS.

Atomic force microscopic study on morphological alterations induced by photodynamic action of Toluidine Blue O in Staphylococcus aureus and Escherichia coli.

Topographical alterations induced by Toluidine Blue O (TBO) mediated photodynamic treatment in Staphylococcus aureus and Escherichia coli was studied using atomic force microscopy (AFM). The AFM images showed distinct differences in the effect of photodynamic treatment on the morphology of S. aureus and E. coli. In S. aureus, photodynamic treatment with TBO resulted in light dose dependent increase in surface bleb formation suggesting breakage in the contact between the cell wall and the membrane with no significant change in the cell dimensions. Photosensitization of E. coli, resulted in surface indentations, significant reduction in the mean cell height, and flattening of bacteria as compared to the bacteria treated with the photosensitizers in the dark. These results indicate damage to the bacterial membrane and reduction of cell volume due to the loss of cytoplasmic materials. Leakage of intracellular contents measured using absorption spectrophotometry was higher and occurred faster in E. coli as compared to S. aureus and correlated with the morphological alterations. The results suggest that with AFM imaging it is possible to distinguish the membranolytic action of TBO in Gram-positive and Gram-negative bacteria.

Toluidine blue-mediated photodynamic effects on staphylococcal biofilms.

Staphylococci are important causes of nosocomial and medical-device-related infections. Their virulence is attributed to the elaboration of biofilms that protect the organisms from immune system clearance and to increased resistance to phagocytosis and antibiotics. Photodynamic treatment (PDT) has been proposed as an alternative approach for the inactivation of bacteria in biofilms. In this study, we have investigated the effect of the photodynamic action of toluidine blue O (TBO) on the viability and structure of biofilms of Staphylococcus epidermidis and of a methicillin-resistant Staphylococcus aureus strain. Significant inactivation of cells was observed when staphylococcal biofilms were exposed to TBO and laser simultaneously. The effect was found to be light dose dependent. Confocal laser scanning microscopic study suggested damage to bacterial cell membranes in photodynamically treated biofilms. In addition, scanning electron microscopy provided direct evidence for the disruption of biofilm structure and a decrease in cell numbers in photodynamically treated biofilms. Furthermore, the treatment of biofilms with tetrasodium EDTA followed by PDT enhanced the photodynamic efficacy of TBO in S. epidermidis, but not in S. aureus, biofilms. The results suggest that photodynamic treatment may be a useful approach for the inactivation of staphylococcal biofilms adhering to solid surfaces of medical implants.

Generation of ROS in cells on exposure to CW and pulsed near-infrared laser tweezers.

We report the results of a study on generation of reactive oxygen species (ROS) and changes in the membrane potential of mitochondria of carcinoma of cervix (HeLa) and Chinese hamster ovary (CHO) cells following exposure to continuous wave (cw) or pulsed Nd: YAG laser (1064 nm). For a given laser irradiation, the generation of ROS and induced changes in the membrane potential of mitochondria were more pronounced for HeLa cells as compared to CHO cells. However, in both the cells the laser dose required to elicit a given change was much lower with pulsed laser exposure compared to that required with a cw laser exposure. This suggests involvement of photothermal effects in the laser irradiation induced changes. Mechanistic studies using quenchers for ROS suggest that laser irradiation leads to generation of hydroxyl radicals.