Formulation and Physical Characterization of Bio-Degradable Chitosan-Poloxamer Gel Base for Local Drug Delivery

Objective: Thermo-modulated in-situ hydrogel (TSHG) are formulated routinely utilizing poloxamer for extended drug release. However physical properties of such formulations may have some flaws, which can be rectified using a combination of polymers with better physical properties such as chitosan. The purpose of the present study was to fabricate biodegradable chitosan-poloxamer-based in-situ drug delivery systems and assessment of their physical properties. Methods: The present chitosan-poloxamer gel base was formulated using a two-stage method. Initially, chitosan gel was prepared by dissolving 1% w/w chitosan in glacial acetic acid. The poloxamer gel was prepared using “cold method”. The final chitosan-poloxamer gel base was prepared by mixing equal amounts of both solutions and evaluated for physical and mechanical properties. Result and Discussion: The DSC thermogram demonstrated no obvious interactions among ingredients or micellization temperature. The gelation temperature of the gel was between 27 and 330C. The pH was 7 with slight clarity. The viscosity of the gel ranged from 15.14 to 41.19 pa.s. The gel was syringable between 4-300C and biodegradable under physiological conditions. The mean particle size of the gel under SEM was found in the range of 300-554 nm. Conclusion: After the evaluation of the formulation, it can be concluded that all the ingredients in the gel showed good compatibility with each other, which could form a stable and homogeneous gel with favorable mechanical and physical properties. Keywords: chitosan, drug delivery system, hydrogels, poloxame

Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani

Quantitative proteomic profiling of the promastigotes and the intracellular amastigotes of Leishmania donovani isolates identifies novel proteins having a role in leishmania differentiation and intracellular survival

Protozoan parasites of the genus Leishmania are important human pathogens that cycle between an extracellular promastigote stage residing in the sandflies and an intracellular amastigote stage colonizing the phagolysosomal compartment of the mammalian macrophages. Here, we used the isobaric tagging method to quantify the global proteomic differences between the promastigotes and the intracellular amastigotes of three different Leishmania donovani clones derived from the THP-1 human macrophage cell line. We identified a substantial number of differentially modulated proteins involved in nutrient acquisition and energy metabolism, cell motility and cytoskeleton, transport, cell signaling and stress response. Proteins involved in vesicular trafficking and endocytosis like the rab7 GTP binding protein, GTP-binding proteins of the Ras superfamily and developmentally regulated GTP-binding protein 1 revealed enhanced expression and also a putative dynein heavy chain protein was found to be up-regulated in the amastigotes and it probably has a role in cargo transport inside the vesicles. Significantly, in the amastigotes the expression of a protein involved in glucose transport was increased eight to fifteen-fold, whereas concentrations of several proteins associated with cell motility and cytoskeleton were reduced. Thus, the quantitative proteomic analysis of L. donovani isolates sheds light on some novel proteins that may have a role in Leishmania differentiation and intracellular survival

Leishmania donovani encodes a functional enzyme involved in vitamin C biosynthesis: Arabino-1,4-lactone oxidase

Plants and most animals can synthesize ascorbate (vitamin C) for their own requirements, but humans have lost this ability during evolution. The last step in the biosynthesis of l-ascorbic acid involves the conversion of an aldonolactone substrate to ascorbate (or analogues), reactions catalyzed by a family of flavoprotein aldonolactone oxidase/dehydrogenases. We report cloning, molecular characterization, localization and functional importance of arabinonolactone oxidase (LdALO), an enzyme from L. donovani, a protozoan parasite that causes visceral leishmaniasis. L. donovani arabinonolactone oxidase gene is 1509-bp and encodes a putative 502-amino acid protein with a molecular mass of 57-kDa. A 57-kDa protein was obtained by heterologous expression of LdALO in Escherichia coli. Recombinant arabinonolactone oxidase (LdALO) obeys Michaelis–Menten kinetics utilizing d-arabinono-γ-lactone as a substrate, a property characteristic of the yeast enzyme. Activity towards the mammalian substrate, l-gulono-γ-lactone, could not be detected. The inhibitor study profile suggested the essentiality of cysteine residues for the activity of this enzyme. LdALO displayed glycosomal localization as in other kinetoplastids. Overexpression of LdALO in L. donovani resulted in better ability of survival of the parasite within the host in comparison to the vector transfectants. d-Arabinono-γ-lactone oxidase required for synthesizing ascorbate in Leishmania could be considered as a therapeutically exploitable target

Differential expression of proteins in antimony-susceptible and -resistant isolates of Leishmania donovani

Visceral Leishmaniasis (VL) is a parasitic disease caused by the protozoan parasite Leishmania donovani. Resistance to pentavalent antimonials (SbV), the mainstay therapy for leishmaniasis is now a major concern, due to emergence of drug resistance. Hence, understanding the underlying mechanism of resistance to antimonials is required. Here we used quantitative mass spectrometery to identify global proteome differences between antimony-susceptible/-resistant isolates. We detected modification of expression of proteins involved in the key metabolic pathways. Comparative proteomic analysis indicated increase in glycolysis in the antimony-resistant isolates. Elevated expression of stress related proteins implicated in oxidative stress was observed in the resistant parasites. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival of the parasite in the resistant isolates. The identified parasite proteins could serve as surrogate markers for resistance or susceptibility and would also help in understanding the underlying mechanism of resistance to antimonials

Plasmenylethanolamine synthesis in Leishmania major

Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT-null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI-anchored surface molecules. EPT-null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors

In vitro toxicity assessment of chitosan oligosaccharide coated iron oxide nanoparticles

Iron oxide nanoparticles (INPs) have potential biological, biomedical and environmental applications. These applications require surface modification of the iron oxide nanoparticles, which makes it non-toxic, biocompatible, stable and non-agglomerative in natural and biological surroundings. In the present study, iron oxide nanoparticles (INPs) and chitosan oligosaccharide coated iron oxide nanoparticles (CSO-INPs) were synthesized to evaluate the effect of surface coating on the stability and toxicity of nanoparticles. Comparative in vitro cytotoxicity of nanoparticles was evaluated in HeLa (human cervix carcinoma), A549 (human lung carcinoma) and Hek293 (human embryonic kidney) cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay along with flow cytometry study for cell viability, membrane integrity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production. Morphological alteration in nanoparticles treated cells was analyzed by Acridine orange/ethidium bromide double staining and electron microscopy. Synthesized nanoparticles were found to be spherical in shape, well dispersed and stable at various pH values, making them suitable for biomedical and environmental applications. The present study also indicates that the chitosan oligosaccharide coating on iron oxide nanoparticles results in the decrease in cellular damage and moderate ROS production, thereby, significantly decreasing the cytotoxic impact of bare iron oxide nanoparticles

ATRT_manuscript_model_prediction_figure

This data is exported from Code Ocean resease capsule v1.0 . It has all the code, data and instructions to reproduce the model prediction results presented in the Manuscript</p