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.

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.

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.

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders.

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.

Circular PVT1: an oncogenic non-coding RNA with emerging clinical importance.

The importance of circular RNAs (circRNAs) in pathological processes like cancer is evident. Among the circRNAs, recent studies have brought circPVT1 under focus as the most potent oncogenic non-coding RNA. Recent studies on various aspects of circPVT1, including its biogenesis, molecular alteration and its probable role in oncogenesis, have been conducted for research and clinical interest. In this review, a first attempt has been made to summarise the available data on circPVT1 from PubMed and other relevant databases with special emphasis on its role in development, progression and prognosis of various malignant conditions. CircPVT1 is derived from the same genetic locus encoding for long non-coding RNA lncPVT1; however, existing literature suggested circPVT1 and lncPVT1 are transcripted independently by different promoters. The interaction between circRNA and microRNA has been highlighted in majority of the few malignancies in which circPVT1 was studied. Besides its importance in diagnostic and prognostic procedures, circPVT1 seemed to have huge therapeutic potential as evident from differential drug response of cancer cell line as well as primary tumors depending on expression level of the candidate. circPVT1 in cancer therapeutics might be promising as a biomarker to make the existing treatment protocol more effective and also as potential target for designing novel therapeutic intervention.