Tanmoy Bera

One pot synthesis of gold nanoparticles and application in chemotherapy of wild and resistant type visceral leishmaniasis

Gold nanoparticles (Aunp) through biogenetic processes have induced enormous interest for lower toxicity and precise applications. A rapid, one pot synthesis for uniformly sized gold nanoparticles was developed using polyphenolic compound quercetin. Reduction process was followed at low temperatures in a simple bath type sonicator. Nanoparticle plasmon response was recorded at 540 nm and the average size in TEM was observed at 15.07 nm. Detailed X-ray diffraction (XRD) observations proved fcc crystalline structure of metallic gold and the Fourier transform infrared (FTIR) analysis has confirmed nanoparticles conjugation with quercetin. Leishmaniasis, is a neglected tropical disease (NTD) classified by the World Health Organization (WHO). The leishmanial parasite multiply in host macrophages and most strains have developed drug resistance to available chemotherapeutics. Drug delivery is therefore a major problem in macrophage specific leishmanial parasite infections. New quercetin conjugated gold nanoparticles (QAunp) were successfully evaluated for the first time against leishmanial macrophage infections. Antileishmanial efficiency of QAunp was established against wild type (IC50 15±3), sodium stibogluconate resistant strain (IC50 40±8) and the paramomycin resistant (IC50 30±6) strains. Macrophage uptake of QAunp was complete within an hour as observed in TEM experiments.

Andrographolide nanoparticles in leishmaniasis: characterization and in vitro evaluations.

Andrographolide (AG) is a diterpenoid lactone isolated from the leaves of Andrographis paniculata. AG is a potent and low-toxicity antileishmanial agent. Chemotherapy applications of AG are, however, seriously constrained because of poor bioavailability, short plasma half-life, and inappropriate tissue localization. Nanoparticulation of AG was therefore envisaged as a possible solution. AG nanoparticles (AGnp) loaded in 50:50 poly(DL-lactide-co-glycolic acid) were prepared for delivery into the monocyte–macrophage cells infested with the amastigote form of leishmanial parasite for evaluation in the chemotherapy of leishmaniasis. Particle characteristics of AGnp were optimized by proportionate application of a stabilizer, polyvinyl alcohol (PVA). Physicochemical characterization of AGnp by photon correlation spectroscopy exhibited an average particle size of 173 nm and zeta potential of −34.8 mV. Atomic force microscopy visualization revealed spherical nanoparticles with a smooth surface. Antileishmanial activity was found to be significant for the nanoparticle preparation with 4% PVA (IC50 34 μM) in about one-fourth of the dosage of the pure compound AG (IC50 160 μM). AGnp therefore have significant potential to target the infested macrophage cells and prove valuable in chemotherapy of neglected tropical diseases such as leishmaniasis.

In vitro susceptibilities of wild and drug resistant leishmania donovani amastigote stages to andrographolide nanoparticle: role of vitamin E derivative TPGS for nanoparticle efficacy.

Visceral leishmaniasis (VL) is a chronic protozoan infection in humans associated with significant global morbidity and mortality. There is an urgent need to develop drugs and strategy that will improve therapeutic response for effective clinical treatment of drug resistant VL. To address this need, andrographolide (AG) nanoparticles were designed with P-gp efflux inhibitor vitamin E TPGS (D-α-tocopheryl polyethyleneglycol 1000 succinate) for sensitivity against drug resistant Leishmania strains. AG loaded PLGA (50∶50) nanoparticles (AGnps) stabilized by vitamin E TPGS were prepared for delivery into macrophage cells infested with sensitive and drug resistant amastigotes of Leishmania parasites. Physico-chemical characterization of AGnps by photon correlation spectroscopy exhibited an average particle size of 179.6 nm, polydispersity index of 0.245 and zeta potential of −37.6 mV. Atomic force microscopy and transmission electron microscopy visualization revealed spherical nanoparticles with smooth surfaces. AGnps displayed sustained AG release up to 288 hours as well as minimal particle aggregation and drug loss even after three months study period. Antileishmanial activity as revealed from selectivity index in wild-type strain was found to be significant for AGnp with TPGS in about one-tenth of the dosage of the free AG and one-third of the dosage of the AGnp without TPGS. Similar observations were also found in case of in vitro generated drug resistant and field isolated resistant strains of Leishmania. Cytotoxicity of AGnp with and without TPGS was significantly less than standard antileishmanial chemotherapeutics like amphotericin B, paromomycin or sodium stibogluconate. Macrophage uptake of AGnps was almost complete within one hour as evident from fluorescent microscopy studies. Thus, based on these observations, it can be concluded that the low-selectivity of AG in in vitro generated drug resistant and field isolated resistant strains was improved in case of AG nanomedicines designed with vitamin E TPGS.

Immuno-modulation effect of sulphated polysaccharide (porphyran) from Porphyra vietnamensis.

Our investigation explores the immuno-efficiency of sulphated polysaccharides enriched Porphyra vietnamenis. Isolated polysaccharide fraction (17.1-25.8%) was characterized by FTIR and NMR spectroscopy which showed the presence of typical linear backbone structure called as porphyran. Oral administration of porphyran (200-500 mg/kg) evoked a significant (P ≤ 0.05) increase in weight of the thymus, spleen and lymphoid organ cellularity. The total leucocyte and lymphocyte count was increased significantly (P<0.005). The increase in the percent neutrophil adhesion to nylon fibres as well as a dose-dependent increase in antibody titre values was observed. A decreased response to DTH reaction induced by SRBC was recorded. A potential phagocytic response was seen and significant changes were observed in the formation of formazone crystals. It also prevented myelosuppression in cyclophosphamide drug treated rats. The results indicated that P. vietnamenis possesses potential immunomodulatory activity and has therapeutic potential for the prevention of autoimmune diseases.

The γ-guanidinobutyramide pathway of l-arginine catabolism in Leishmania donovani promastigotes

L-Arginine stimulates the respiration of Leishmania donovani to rates comparable to those observed with D-glucose. gamma-Guanidinobutyramide, CO2, urea and succinate have been identified as products of L-arginine catabolism by the cell-free extract. The reactions involved in CO2 and urea formation require aerobic conditions. An enzymatic reaction that converts radiolabelled L-arginine to gamma-guanidinobutyramide occurs in cell-free extracts. The enzyme catalyzes a reaction in which O2 consumption and CO2 production are equimolar. The O2 uptake and CO2 production are stimulated by Mg2+, Mn2+, FMN, pyridoxal phosphate, and inhibited by hydroxylamine and NaBH4. L-Arginine decarboxyoxidase is suggested as the trivial name for this enzyme. The enzyme has maximum activity at pH 6.7, and its Km for L-arginine is 3.8 mM. L-Arginine decarboxyoxidase initiates the catabolism of L-arginine (pH less than or equal to 7) in this species, and is followed by the other enzymes of gamma-guanidinobutyramide pathway. Assay procedures have been devised to assay the multiple enzymes of this pathway.

Submicron-size biodegradable polymer-based didanosine particles for treating HIV at early stage: an in vitro study

Human immunodeficiency viruses (HIV) hide themselves in macrophages at the early stage of infection. Delivering drug in a sustained manner from polymeric nanoparticles in those cells could control the disease effectively. The study was intended to develop poly(d,l-lactic-co-glycolic acid)-based nanoparticles containing didanosine and to observe their uptake by macrophages in vitro. Various physicochemical evaluations related to nanoparticles, such as drug–excipient interaction, surface morphology, particle size, zeta potential, polydispersity index, drug loading, in vitro drug release and nanoparticle-uptake by macrophages in vitro were determined. Homogenising speeds and drug–polymer ratio varied drug loading and polydispersity index of nanoparticles, providing sustained drug release. Dimethyl sulphoxide/polyethylene glycol improved drug loading predominantly. Nanoparticle-uptake by macrophages was concentration dependent. Experimental nanoparticles successfully transported didanosine to macrophages in vitro, suggesting reduction of dose, thus minimising toxicity and side effects. Developed nanoparticle may control HIV infection effectively at an early stage.

Stage specific developmental changes in the mitochondrial and surface membrane associated redox systems of Leishmania donovani promastigote and amastigote.

Energy metabolism of Leishmania donovani parasite has been investigated under conditions imitating intralysosomal-like environment in the host organism. Trans-plasma membrane electron transport and oxygen uptake were inhibited progressively when promastigote cells were exposed to pH 5.5 and 37°C. A special feature of the respiratory chain in amastigote was the absence of complex I, II, and IV. When L. donovani was grown at pH 5.5 and 37°C, the acid excretory product succinate was increased in comparison to cells grown at pH 7.5 and 24°C. The findings of this study showed that the amastigote form catabolized fatty acid to excrete succinic acid when oxidative phosphorylation was impaired. Amastigote mitochondria failed to generate membrane potential by oxidizable substrates. On the other hand, the amastigote cell showed absorbance change of safranine O when fatty acid was the oxidizable substrate. The safranine signal was completely reversed by valinomycin, carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone, malonate, and oxaloacetate. Our data suggest that the generation of metabolic energy from succinate/H+ efflux will contribute to energy requiring process of amastigote significantly. On the basis of these results, we conclude that due to absence of oxidative phosphorylation in amastigotes, energy linked functions in amastigotes might occur through fumarate reduction leading to ΔpH generation by succinate excretion.

In Vitro susceptibilities of wild and drug resistant Leishmania donovani amastigotes to piperolactam A loaded hydroxypropyl-β-cyclodextrin nanoparticles.

Leishmaniasis is an epidemic in various countries, and the parasite Leishmania donovani is developing resistance against available drugs. In the present study the antileishmanial action of piperolactam A (PL), isolated after bioactivity guided fractionation from root extracts of Piper betle was accentuated in detail. Activity potentiation was achieved via cyclodextrin complexation. Crude hydro-ethanolic extract (PB) and three fractions obtained from PB and fabricated PL-hydroxypropyl-β-cyclodextrin (HPBCD) nanoparticles were evaluated for antileishmanial activity. Tests were performed against L. donovani wild-type, sodium stibogluconate, paromomycin and field isolated (GE1) resistant strains in axenic amastigote and amastigote in macrophage models. PL-HPBCD complex was characterized and FITC loaded HPBCD nanoparticles were assessed for macrophage internalization in confocal microscopic studies. Isolated and purified PL from most potent, alkaloid rich ethyl acetate fraction of PB showed high level of antileishmanial activities in wild-type (IC50=36 μM), sodium stibogluconate resistant (IC50=103 μM), paromomycin resistant (IC50=91 μM) and field isolated resistant (IC50=72 μM) strains together with cytotoxicity (CC50=900 μM) in mouse peritoneal macrophage cells. Inclusion of PL in HPBCD nanoparticles resulted in 10-fold and 4-10-fold increase in selectivity indexes (CC50/IC50) for wild-type and drug resistant strains, respectively. Drug-carrier interactions were clearly visualized in FT-IR studies. Complete incorporation of PL in HPBCD cavity was ascertained in DSC and XRD analyses. 180nm size stable nanospheres showed macrophage internalization within 1h of incubation. Piperolactam A (PL), a representative of the inchoate skeleton of aristolactam chassis might be the source of safe and affordable antileishmanial agents for the cure of deadly Leishmania infections.

Significance of Algal Polymer in Designing Amphotericin B Nanoparticles

Development of oral amphotericin B (AmB) loaded nanoparticles (NPs) demands a novel technique which reduces its toxicity and other associated problems. Packing of AmB in between two oppositely charged ions by polyelectrolyte complexation technique proved to be a successful strategy. We have developed a novel carrier system in form of polyelectrolyte complex of AmB by using chitosan (CS) and porphyran (POR) as two oppositely charged polymers with TPP as a crosslinking agent. Initially POR was isolated from Porphyra vietnamensis followed by the fact that its alkali induced safe reduction in molecular weight was achieved. Formulation was optimized using three-factor three-level (33) central composite design. High concentration of POR in NPs was confirmed by sulfated polysaccharide (SP) assay. Degradation and dissolution studies suggested the stability of NPs over wide pH range. Hemolytic toxicity data suggested the safety of prepared formulation. In vivo and in vitro antifungal activity demonstrated the high antifungal potential of optimized formulation when compared with standard drug and marketed formulations. Throughout the study TPP addition did not cause any significant changes. Therefore, these experimental oral NPs may represent an interesting carrier system for the delivery of AmB.

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and FoF1-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and FoF1-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by FoF1-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.