Mrinalini Sharma

Toluidine Blue-Mediated Photodynamic Effects on Staphylococcal Biofilms

ABSTRACT 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.

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.

Controlled induction, enhancement, and guidance of neuronal growth cones by use of line optical tweezers

We report an optical tweezers based approach for efficient and controlled manipulation of neuronal growth cones. The approach exploits asymmetric transverse gradient force created in a line optical tweezers to transport actin monomers in the desired growth direction. With this approach induction of artificial growth cones from the neuronal cell body and enhancement of the growth rate of the natural growth cones have been achieved. The use of this approach to bring two growth cones into close proximity for establishing a neuronal connection is also discussed.

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.

Use of common path phase sensitive spectral domain optical coherence tomography for refractive index measurements

We report the use of a common path phase sensitive spectral domain optical coherence tomography setup for the measurement of the refractive index (RI) of a biomimetic material (glucose solution in water having intralipid as the scattering medium) and a single biological cell (keratinocyte). The RI of glucose solutions could be measured with a precision of ∼0.00015, which corresponds to a precision of ∼2 nm in the optical path length measurement in our setup. The precision obtained in the measurement of the RI of a single keratinocyte cell was ∼0.0004.

Virulence of Pseudomonas aeruginosa Cells Surviving Photodynamic Treatment with Toluidine Blue

We report results of a study carried out to investigate photodynamic effect of Touildine blue (TBO) on virulent factors secreted by Pseudomonas aeruginosa. Viability of MCF-7 cells incubated with culture supernatants of bacteria photosensitized with TBO (experimental) was found to be higher than that for MCF-7 cells incubated with culture supernatants of bacterial cells treated either with TBO in dark (control II) or not receiving any treatment (neither TBO nor light (control I)). Furthermore, whereas MCF-7 cells incubated with supernatants of bacteria, control I and II, showed significant morphological alterations, no such changes were observed in MCF-7 cells incubated with supernatant of experimental cells. The activities of phospholipase C and proteases, virulent factors of P. aeruginosa, were also found to decrease on photosensitization. These results suggest that the potency of virulent factors is reduced in cells surviving photodynamic treatment with TBO and this may have implication in treatment of infections caused by P. aeruginosa.

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.

Non-invasive assessment of healing of bacteria infected and uninfected wounds using optical coherence tomography

Background/purpose: Bacterial infection is one of the main predisposing factors for the delay in wound healing. To facilitate a timely decision for correct therapy, it is important to accurately monitor the morphological changes in the infected wounds using noninvasive tools. In the present study, we have explored the use of optical coherence tomography (OCT) for monitoring the healing of superficial wounds infected with Staphylococcus aureus in mice under in vitro and in vivo conditions and studied the changes in collagen birefringence in the infected wounds.

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.

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.