Shalini Asthana

Immunoadjuvant Chemotherapy of Visceral Leishmaniasis in Hamsters Using Amphotericin B-Encapsulated Nanoemulsion Template-Based Chitosan Nanocapsules

ABSTRACT The accessible treatment options for life-threatening neglected visceral leishmaniasis (VL) disease have problems with efficacy, stability, adverse effects, and cost, making treatment a complex issue. Here we formulated nanometric amphotericin B (AmB)-encapsulated chitosan nanocapsules (CNC-AmB) using a polymer deposition technique mediated by nanoemulsion template fabrication. CNC-AmB exhibited good steric stability in vitro, where the chitosan content was found to be efficient at preventing destabilization in the presence of protein and Ca2+. A toxicity study on the model cell line J774A and erythrocytes revealed that CNC-AmB was less toxic than commercialized AmB formulations such as Fungizone and AmBisome. The results of in vitro (macrophage-amastigote system; 50% inhibitory concentration [IC50], 0.19 ± 0.04 μg AmB/ml) and in vivo (Leishmania donovani-infected hamsters; 86.1% ± 2.08% parasite inhibition) experiments in conjunction with effective internalization by macrophages illustrated the efficacy of CNC-AmB at augmenting antileishmanial properties. Quantitative mRNA analysis by real-time PCR (RT-PCR) showed that the improved effect was synergized with the upregulation of tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and inducible nitric oxide synthase and with the downregulation of transforming growth factor β (TGF-β), IL-10, and IL-4. These research findings suggest that a cost-effective CNC-AmB immunoadjuvant chemotherapeutic delivery system could be a viable alternative to the current high-cost commercial lipid-based formulations.

Industrial perspective of gastroretentive drug delivery systems: Physicochemical, biopharmaceutical, technological and regulatory consideration

Introduction: Gastroretentive drug delivery systems (GRDDS) can overcome drawbacks associated with oral drug delivery, by defeating natural physiological principles. Various gastroretentive technologies have been developed in the past, but few of them achieved success on the market. Areas covered: This review is focused on the key concepts required to make a high-quality drug product available in a timely and economical manner. Expert opinion: Pharmacotherapy of various disease states can be amended by drug repurposing through GRDDS. Assessment of the effect of the fed and fasted condition on product performance should be necessary during initial development phases. Dual working technology would be a possible way to overcome drawbacks associated with different GRDDS. Before development of a drug product, the principles of scale up and process validation must be considered to improve the quality and market availability of GRDDS. Knowledge of all regulatory aspects will help to deliver a product to the market within a reasonable timeframe and in a cost-effective manner.

Chitosan-Assisted Immunotherapy for Intervention of Experimental Leishmaniasis via Amphotericin B-Loaded Solid Lipid Nanoparticles

Solid lipid nanoparticles (SLNs) have emerged as an excellent substitute over polymeric nanoparticles and, when incorporated with chitosan which activates the macrophage to impart an immune response, produce excellent results to fight against deleterious diseases like leishmaniasis where its parasite diminishes the immunity of the host to induce resistance. Based upon this hypothesis, chitosan-coated SLNs were developed and loaded with amphotericin B (AmB) for immunoadjuvant chemotherapy of Leishmania infection. Both uncoated and chitosan-coated AmB-loaded SLNs (AmB-SLNs) were fabricated using solvent emulsification and evaporation method. The various processes and formulation parameters involved in AmB-SLN preparation were optimized with respect to particle size and stability of the particles. In vitro hemolytic test credited the formulations to be safe when injected in the veins. The cellular uptake analysis demonstrated that the chitosan-coated AmB-SLN was more efficiently internalized into the J774A.1 cells. The in vitro antileishmanial activity revealed their high potency against Leishmania-infected cells in which chitosan-coated AmB-SLNs were distinguishedly efficacious over commercial formulations (AmBisome and Fungizone). An in vitro cytokine estimation study revealed that chitosan-coated AmB-SLNs activated the macrophages to impart a specific immune response through enhanced production of TNF-α and IL-12 with respect to normal control. Furthermore, cytotoxic studies in macrophages and acute toxicity studies in mice evidenced the better safety profile of developed formulation in comparison to marketed formulations. This study indicates that the AmB-SLNs are a safe and efficacious drug delivery system which promises strong competence in antileishmanial chemotherapy and immunotherapy.

Th-1 biased immunomodulation and synergistic antileishmanial activity of stable cationic lipid–polymer hybrid nanoparticle: Biodistribution and toxicity assessment of encapsulated amphotericin B

To address issues related to Amphotericin B (AmpB) clinical applications, we developed macrophage targeted cationic stearylamine lipid-polymer hybrid nanoparticles (LPNPs) with complementary characteristics of both polymeric nanoparticles and liposomes, for enhancement of therapeutic efficacy and diminishing toxic effect of encapsulated AmpB. The LPNPs (size 198.3 ± 3.52 nm, PDI 0.135 ± 0.03, zeta potential +31.6 ± 1.91 mV) provide core-shell type structure which has the ability to encapsulate amphiphilic AmpB in higher amount (Encapsulation efficiency 96.1 ± 2.01%), sustain drug release and stabilize formulation tremendously. Attenuated erythrocytes and J774A.1 toxicity of LPNPs demonstrated safe applicability for parenteral administration. Elevated macrophage uptake of LPNPs, rapid plasma clearance and higher drug allocation in macrophage abundant liver and spleen illustrated admirable antileishmanial efficacy of AmpB-LPNPs in vitro (IC50, 0.16 ± 0.04 μg AmpB/ml) and in vivo (89.41 ± 3.58% parasite inhibition) against visceral leishmaniasis models. Augmentation in antileishmanial activity due to Th-1 biased immune-alteration mediated by drug-free LPNPs which elevated microbicidal mediators of macrophages. Moreover, minimal distribution to kidney tissues and low level of nephrotoxicity markers (creatinine and BUN) demonstrated the safety profile of AmpB-LPNPs. Conclusively, reliable safety and macrophage directed therapeutic performance of AmpB-LPNPs suggest it as promising alternative to commercial AmpB-formulations for the eradication of intra-macrophage diseases.

Polymeric colloidal particulate systems: intelligent tools for intracellular targeting of antileishmanial cargos

Introduction: Targeted cargo delivery systems can overcome drawbacks associated with antileishmanials delivery, by defeating challenges of physiological barriers. Various colloidal particulate systems have been developed in the past; few of them even achieved success in the market, but still are limited in some ways. Areas covered: This review is focused on the pathobiology of leishmaniasis, interactions of particulate systems with biological environment, targeting strategies along with current conventional and vaccine therapies with special emphasis on polymeric nanotechnology for effective antileishmanial cargo delivery. Expert opinion: The problems concerned with limited accessibility of chemotherapeutic cargos in conventional modes to Leishmania-harboring macrophages, their toxicity, and resistant parasitic strain development can be sorted out through target-specific delivery of cargos. Vaccination is another therapeutic approach employing antigen alone or adjuvant combinations delivered by means of a carrier, and can provide preventive measures against human leishmaniasis (HL). Therefore, there is an urgent need of designing site-specific antileishmanial cargo carriers for safe and effective management of HL. Among various colloidal carriers, polymeric particulate systems hold tremendous potential as an effective delivery tool by providing control over spatial and temporal distribution of cargos after systemic or localized administration along with enhancing their stability profile at a comparatively cost-effective price leading to improved chances of commercial applicability.

Exploitation of lectinized lipo-polymerosome encapsulated Amphotericin B to target macrophages for effective chemotherapy of visceral leishmaniasis.

We have designed lectin functionalized Lipo-polymerosome bearing Amphotericin B (Lec-AmB-L-Psome) for specific internalization via lectin receptors overexpressed on infected macrophages of mononuclear phagocytic system (MPS) for the effective management of intramacrophage diseases such as visceral leishmaniasis. The lipo-polymerosome composed of glycol chitosan-stearic acid copolymer (GC-SA25%) and model lipid cholesterol was surface-functionalized with lectin by the EDC/NHS carbodiimide coupling method. Our designed Lec-AmB-L-Psome showed >2-fold enhanced uptake and significantly higher internalization in macrophages as compared to AmB-L-Psome. Importantly, pharmacokinetic and organ distribution studies illustrate significantly higher accumulation of Lec-AmB-L-Psome in MPS especially in liver, spleen, and lung as compared to AmB-L-Psome, Ambisome, and Fungizone. The IC50 value demonstrated that Lec-AmB-L-Psome has 1.63, 2.23, and 3.43 times higher activity than AmB-L-Psome (p < 0.01), Ambisome (p < 0.05), and Fungizone (p < 0.05), respectively. Additionally, the Lec-AmB-L-Psome showed significantly higher splenic parasite inhibition (78.66 ± 3.08%) compared to Fungizone and Ambisome that caused only 56.54 ± 3.91% (p < 0.05) and 66.46 ± 2.08% (p < 0.05) parasite inhibition, respectively, in Leishmania-infected hamsters. The toxicity profile revealed that Lec-AmB-L-Psome is a safe delivery system with diminished nephrotoxicity which is a limiting factor of Fungizone application. Taken together, these studies suggest that this surface functionalized self-assembled Lec-AmB-L-Psome can introduce a new platform to specifically target macrophages for effective management of intramacrophage diseases.

Perspectives of nanoemulsion assisted oral delivery of docetaxel for improved chemotherapy of cancer

Abstract Context: Nanoemulsions (NE) are one of the robust delivery tools for drugs due to their higher stability and efficacy. Objectives: The purpose of present investigation is to develop stable, effective and safe NE of docetaxel (DTX). Methods: Soybean oil, lecithin, Pluronic F68, PEG 4000 and ethanol were employed as excipients and NEs were prepared by hot homogenization followed by ultra-sonication. NEs were optimized and investigated for different in vitro and in vivo parameters viz. droplet size, poly dispersity index, charge; zeta potential, drug content and in vitro drug release, in vitro cytotoxicity, in vitro cell uptake and acute toxicity. Transmission electron microscopy was performed to study morphology and structure of NEs. Stability studies of the optimized formulation were performed. Results: Droplet size, poly dispersity index, zeta potential, drug content and in vitro drug release were found to be 233.23 ± 4.3 nm, 0.24 ± 0.010, −43.66 ± 1.9 mV, 96.76 ± 1.5%, 96.25 ± 2.1%, respectively. NE F11 exhibited higher cell uptake (2.83 times than control) and strong cytotoxic activity against MCF-7 cancer cells (IC50; 13.55 ± 0.21 µg/mL at 72 h) whereas no toxicity or necrosis was observed with liver and kidney tissues of mice at a dose of 20 mg/kg. Transmission electron microscopy ensured formation of poly-dispersed and spherical droplets in nanometer range. NE F11 (values indicated above) was selected as the optimized formulation based on the aforesaid parameters. Conclusion: Conclusively, stable, effective and safe NE was developed which might be used as an alternative DTX therapy.

Synergistic enhancement of parasiticidal activity of amphotericin B using copaiba oil in nanoemulsified carrier for oral delivery: an approach for non-toxic chemotherapy

The aim of this study was to devise a nanoemulsified carrier system (CopNEC) to improve the oral delivery of amphotericin B (AmB) by increasing its oral bioavailability and synergistically enhance its antileishmanial activity with copaiba oil (Cop).

Targeted chemotherapy of visceral leishmaniasis by lactoferrin-appended amphotericin B-loaded nanoreservoir: in vitro and in vivo studies.

AIM Exploitation of lactoferrin-appended amphotericin B bearing nanoreservoir (LcfPGNP-AmB) for targeted eradication of Leishmania donovani. MATERIALS & METHODS LcfPGNP-AmB was architechtured through ionic adsorption of lactoferrin over core poly (d,l-lactide-co-glycolide) nanoparticles and characterized. Anti-Leishmania activity in visceral leishmaniasis models, immunomodulatory potential, biodistribution and toxicity profile were also assessed. RESULTS LcfPGNP-AmB (size, 196.0 ± 5.28 nm; zeta-potential, +21.7 ± 1.52 mV; encapsulation efficiency, ∼89%) showed reduced toxicity, increased protective proinflammatory mediators expression and down-regulation of disease-promoting cytokines. Biodistribution study illustrated preferential accumulation of LcfPGNP-AmB in liver and spleen. LcfPGNP-AmB showed augmented antileishmanial activity by significantly reducing (∼88%) splenic parasite burden of infected hamsters, compared with commercial-formulations. CONCLUSION Superior efficacy, desired stability and reliable safety of cost-effective LcfPGNP-AmB, suggest its potential for leishmaniasis therapeutics.

Chitosan Coated Hydroxypropyl Methylcellulose- Ethylcellulose Shell Based Gastroretentive Dual Working System to Improve the Bioavailability of Norfloxacin

This investigation was aimed to formulate, evaluate and optimize a gastroretentive dual working system based on mucoadhesion and floating mechanisms to improve the bioavailability of norfloxacin (NFX). Floating microballoons (FMB) were prepared by a non-aqueous emulsification–solvent evaporation method employing hydroxypropyl methylcellulose (HPMC) and ethylcellulose (EC) to develop a core matrix. Furthermore, FMB were coated with chitosan by an ionotropic gelation method to impart mucoadhesive characteristics. Prior to incorporation of NFX into mucoadhesive floating microballoons (MFMB), a solid dispersion (SD) of NFX was prepared using a spray–freeze drying method in order to improve the solubility of NFX. The morphological characteristics of the microballoons were assessed using scanning electron microscopy (SEM) which revealed the spherical shape of the microballoons with a smoother, dense and less porous surface. The developed microballoons were evaluated for various physicochemical parameters such as particle size, surface morphology, entrapment efficiency (EE), in vitro-dissolution, in vitro buoyancy and mucoadhesion. The optimized microballoons were developed with good in vitro-buoyancy coupled with high EE. Microballoons exhibited a zero-order release in simulated gastric fluid (SGF) demonstrating drug release in the range from 64.99 ± 3.26 to 99.94 ± 8.45% after 10 h through various formulations. Chitosan coating over FMB imparted excellent mucoadhesion in rat gut wall and results were also supported by mucin glycoprotein assay. MFMB were able to achieve higher mean plasma concentrations compared to FMB and pure NFX in Wistar rats. Keeping in mind the comprehensible advantages of a developed gastroretentive dual working system over a conventional gastroretentive drug delivery system, it can be concluded that the developed system can be used to target drugs in the gastric cavity.