Evaluation of the potential of Delta-aminolevulinic acid for simultaneous detection of bioburden and anti-microbial photodynamic therapy of MRSA infected wounds in Swiss albino mice.

BACKGROUND: The dramatic increase of drug-resistant bacteria necessitates urgent development of platforms to simultaneously detect and inactivate bacteria causing wound infections, but are confronted with various challenges. Delta amino levulinic acid (ALA) induced protoporphyrin IX (PpIX) can be a promising modality for simultaneous bioburden diagnostics and therapeutics. Herein, we report utility of ALA induced protoporphyrin (PpIX) based simultaneous bioburden detection, photoinactivation and therapeutic outcome assessment in methicillin resistant Staphylococcus aureus (MRSA) infected wounds of mice. METHODS: MRSA infected wounds treated with 10% ALA were imaged with help of a blue LED (∼405 nm) based, USB powered, hand held device integrated with a modular graphic user interface (GUI). Effect of ALA application time, bacteria load, post bacteria application time points on wound fluorescence studied. PpIX fluorescence observed after excitation with blue LEDs was used to detect bioburden, start red light mediated antimicrobial photodynamic therapy (aPDT), determine aPDT effectiveness and assess selectivity of the approach. RESULTS: ALA-PpIX fluorescence of wound bed discriminates infected from uninfected wounds and detects clinically relevant load. While wound fluorescence pattern changes as a function of ALA incubation and post infection time, intra-wound inhomogeneity in fluorescence correlates with the Gram staining data on presence of biofilms foci. Lack of red fluorescence from wound granulation tissue treated with ALA suggests selectivity of the approach. Further, significant reduction (∼50%) in red fluorescence, quantified using the GUI, relates well with bacteria load reduction observed post topical aPDT. CONCLUSION: The potential of ALA induced PpIX for simultaneous detection of bioburden, photodynamic inactivation and "florescence-guided aPDT assessment" is demonstrated in MRSA infected wounds of mice.

In vitro photoinactivation effectiveness of a portable LED device aimed for intranasal photodisinfection and a photosensitizer formulation comprising methylene blue and potassium iodide against bacterial, fungal, and viral respiratory pathogens.

Antimicrobial photodynamic therapy (aPDT) can be a viable option for management of intranasal infections. However, there are light delivery, fluence, and photosensitizer-related challenges. We report in vitro effectiveness of an easily fabricated, low-cost, portable, LED device and a formulation comprising methylene blue (MB) and potassium iodide (KI) for photoinactivation of pathogens of the nasal cavity, namely, methicillin-resistant Staphylococcus aureus, antibiotic-resistant Klebsiella pneumoniae, multi-antibiotic-resistant Pseudomonas aeruginosa, Candida spp., and SARS-CoV-2.In a 96-well plate, microbial suspensions incubated with 0.005% MB alone or MB and KI formulation were exposed to different red light (~ 660 ± 25 nm) fluence using the LED device fitted to each well. Survival loss in bacteria and fungi was quantified using colony-forming unit assay, and SARS-CoV-2 photodamage was assessed by RT-PCR.The results suggest that KI addition to MB leads to KI concentration-dependent potentiation (up to ~ 5 log10) of photoinactivation in bacteria and fungi. aPDT in the presence of 25 or 50 mM KI shows the following photoinactivation trend; Gm + ve bacteria > Gm - ve bacteria > fungi > virus. aPDT in the presence of 100 mM KI, using 3- or 5-min red light exposure, results in complete eradication of bacteria or fungi, respectively. For SARS-CoV-2, aPDT using MB-KI leads to a ~ 6.5 increase in cycle threshold value.The results demonstrate the photoinactivation effectiveness of the device and MB-KI formulation, which may be helpful in designing of an optimized protocol for future intranasal photoinactivation studies in clinical settings.

Azadirachta indica (AI)leaf extract coated ZnO-AInanocore-shell particles for enhanced antibacterial activity against methicillin-resistantStaphylococcus aureus(MRSA).

With the rise in microbial resistance to traditional antibiotics and disinfectants, there is a pressing need for the development of novel and effective antibacterial agents. Two major approaches being adopted worldwide to overcome antimicrobial resistance are the use of plant leaf extracts and metallic nanoparticles (NPs). However, there are no reports on the antibacterial potential of NPs coated with plant extracts, which may lead to novel ways of treating infections. This study presents an innovative approach to engineer antibacterial NPs by leveraging the inherent antibacterial properties of zinc oxide NPs (ZnO NPs) in combination withAzadirachta indica(AI) leaf extract, resulting in enhanced antibacterial efficacy. ZnO NPs were synthesised by the precipitation method and subsequently coated withAIleaf extract to produce ZnO-AInanocore-shell structures. The structural and morphological characteristics of the bare and leaf extract coated ZnO NPs were analysed by x-ray diffraction and field emission scanning electron microscopy, respectively. The presence of anAIleaf extract coating on ZnO NPs and subsequent formation of ZnO-AInanocore-shell structures was verified through Fourier transform infrared spectroscopy and photoluminescence techniques. The antibacterial efficacy of both ZnO NPs and ZnO-AInanocore-shell particles was evaluated against methicillin-resistantStaphylococcus aureususing a zone of inhibition assay. The results showed an NP concentration-dependent increase in the diameter of the inhibition zone, with ZnO-AInanocore-shell particles exhibiting superior antibacterial properties, owing to the combined effect of ZnO NPs and the poly phenols present inAIleaf extract. These findings suggest that ZnO-AInanocore-shell structures hold promise for the development of novel antibacterial creams and hydrogels for various biomedical applications.

Evaluation of antimicrobial photodynamic action of a pluronic and pectin based film loaded with methylene blue against methicillin resistantStaphylococcus aureus.

Antimicrobial wound dressings play a crucial role in treatment of wound infections. However, existing commercial options fall short due to antibiotic resistance and the limited spectrum of activity of newly emerging antimicrobials against bacteria that are frequently encountered in wound infections. Antimicrobial photodynamic therapy (aPDT) is very promising alternative therapeutic approach against antibiotic resistant microbes such as methicillin resistantStaphylococcus aureus (MRSA). However, delivery of the photosensitizer (PS) homogeneously to the wound site is a challenge. Though polymeric wound dressings based on synthetic and biopolymers are being explored for aPDT, there is paucity of data regarding theirin vivoefficacy. Moreover, there are no studies on use of PS loaded, pluoronic (PL) and pectin (PC) based films for aPDT. We report development of a polymeric film for potential use in aPDT. The film was prepared using PL and PC via solvent casting approach and impregnated with methylene blue (MB) for photodynamic inactivation of MRSAin vitroandin vivo. Atomic force microscopic imaging of the films yielded vivid pictures of surface topography, with rough surfaces, pores, and furrows. The PL:PC ratio (2:3) was optimized that would result in an intact film but exhibit rapid release of MB in time scale suitable for aPDT. The film showed good antibacterial activity against planktonic suspension, biofilm of MRSA upon exposure to red light. Investigations on MRSA infected excisional wounds of mice reveal that topical application of MB loaded film for 30 min followed by red light exposure for 5 min (fluence; ∼30 J cm-2) or 10 min (fluence; ∼60 J cm-2) reduces ∼80% or ∼92% of bioburden, respectively. Importantly, the film elicits no significant cytotoxicity against keratinocytes and human adipose derived mesenchymal stem cells. Taken together, our data demonstrate that PS-loaded PL-PC based films are a promising new tool for treatment of MRSA infected wounds.

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.

Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments.

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MɸCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MɸCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

Low-level laser therapy as an adjunct to conventional therapy in the treatment of diabetic foot ulcers.

Foot ulcers are serious complications of diabetes mellitus (DM) and are known to be resistant to conventional treatment. This study was conducted to evaluate the efficacy of low-level laser therapy (LLLT) for the treatment of diabetic foot ulcers in a tertiary care centre (Department of Surgery, Mahatma Gandhi Memorial Medical College and Maharaja Yashwantrao Hospital, A.B. Road, Indore). A total of 30 patients with type 2 DM having Meggitt-Wagner grade I foot ulcers of more than 6 weeks duration with negative culture were studied. Patients were randomized into two groups of 15 each. Patients in study group received LLLT (660 ± 20 nm, 3 J/cm2) along with conventional therapy and those in control group were treated with conventional therapy alone. The primary outcome measure was the absolute and relative wound size reduction at 2 weeks compared to the baseline parameter. Percentage ulcer area reduction was 37 ± 9% in the LLLT group and 15 ± 5.4% in the control group (p < 0.001). For ∼75% of wounds of the treatment group, wound area reduction of 30-50% was observed. In contrast, for the control group, ∼80% of wounds showed a wound area reduction of <20% on day 15. Further, the wounds with initial wound area 1000-2000 mm2 seems to have better final outcome than the groups with larger areas. The treated groups showed higher amount of granulation than the control group. The results suggest that LLLT is beneficial as an adjunct to conventional therapy in the treatment of diabetic foot ulcers.

Human ex-vivo oral tissue imaging using spectral domain polarization sensitive optical coherence tomography.

We report the use of spectral domain polarization sensitive optical coherence tomography for ex-vivo imaging of human oral mandibular tissue samples. Our results show that compared to the changes observed in the epithelium thickness and the decay constant of A-scan intensity profile, a much larger degree of change was observed in the phase retardation for tissue sites progressing from normal to the malignant state. These results suggest that monitoring of tissue retardance can help in better differentiation of normal and cancerous oral tissue sites.

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.

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.

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.

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.

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.

He-Ne laser (632.8 nm) pre-irradiation gives protection against DNA damage induced by a near-infrared trapping beam.

We report the results of a study carried out to investigate the effect of He-Ne laser (632.8 nm) pre-irradiation on DNA damage induced by continuous wave 1064 nm trapping beam exposure in MCF-7 cells. A significant decrease in % tail DNA (p < 0.05) was observed in MCF-7 cells pre-exposed to He-Ne laser beam. The dependence of the induced protection against 1064 nm trapping beam irradiation induced DNA damage on the time interval between the two irradiations as well as the He-Ne laser pre-irradiation parameters is presented.

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.