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

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.

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.

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

BACKGROUND AND PURPOSE: 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). EXPERIMENTAL APPROACH: The AmB encapsulated NEC (CopNEC-AmB) comprised of Cop, d-α-tocopheryl polyethylene glycol 1000 succinate and phosphatidylcholine was prepared by high-pressure homogenization method. Stability study of CopNEC-AmB was carried out in simulated gastric fluid and simulated intestinal fluid. The CopNEC-AmB and plain AmB were compared as regards their in vitro antileishmanial activity, pharmacokinetics, organ distribution and toxicity. KEY RESULTS: The optimal CopNEC-AmB had a small globule size, low polydispersity index, high ζ potential and encapsulation efficiency. The high resolution transmission electron microscopy illustrated spherical particle geometry with homogeny in their sizes. The optimal CopNEC-AmB was found to be stable in gastrointestinal fluids showing insignificant changes in globule size and encapsulation efficiency. The AUC0-48 value of CopNEC-AmB in rats was significantly improved showing 7.2-fold higher oral bioavailability than free drug. The in vitro antileishmanial activity of CopNEC-AmB was significantly higher than that of the free drug as Cop synergistically enhanced the antileishmanial effect of AmB by causing drastic changes in the morphology of Leishmania parasite and rupturing its plasma membrane. The CopNEC-AmB showed significantly less haemolytic toxicity and cytotoxicity and did not change the histopathology of kidney tissues as compared with AmB alone. CONCLUSIONS AND IMPLICATIONS: This prototype CopNEC formulation showed improved bioavailability and had a non-toxic synergistic effect on the antileishmanial activity of AmB.

Overexpressed Macrophage Mannose Receptor Targeted Nanocapsules- Mediated Cargo Delivery Approach for Eradication of Resident Parasite: In Vitro and In Vivo Studies.

PURPOSE: Since, Leishmania protozoans are obligate intracellular parasites of macrophages, an immunopotentiating macrophage-specific Amphotericin B (AB) delivery system would be ideally appropriate to increase its superiority for leishmaniasis treatment and to eliminate undesirable toxicity. Herein, we report AB entrapped mannose grafted chitosan nanocapsules (MnosCNc-AB) that results in effective treatment of visceral leishmaniasis, while also enhancing L. donovani specific T-cell immune responses in infected host. METHODS: MnosCNc-AB were prepared via synthesized mannosylated chitosan deposition on interface of oil/water nanoemulsion intermediate and were characterized. J774A.1 macrophage uptake potential, antileishmanial activity and immunomodulatory profile were evaluated in hamster. Tissue localization, biodistribution and toxicity profile were also investigated. RESULTS: MnosCNc-AB had nanometric size (197.8 ± 8.84 nm), unimodal distribution (0.115 ± 0.04), positive zeta potential (+31.7 ± 1.03 mV) and 97.5 ± 1.13% cargo encapsulation efficiency. Superior macrophage internalization of mannosylated chitosan nanocapsules compared to unmodified chitosan nanocapsules was observed by fluorescence-based assessment, further confirmed by rapid blood clearance and, greater localization and higher accumulation in macrophage rich liver and spleen. While, MnosCNc-AB mediated cargo distribution to kidney decreased. Augmented in vitro antileishmanial activity and in vivo pro-inflammatory mediator's expression were observed with MnosCNc-AB, led to significant reduction (∼90%) in splenic parasite burden. CONCLUSIONS: Results demonstrated that mannose ligand grafted chitosan nanocapsules could improve selective delivery of AB into macrophages via interactions with overexpressed mannose receptors thus reduce undesirable toxicity. Study provides evidence for MnosCNc-AB potential to leishmaniasis therapeutics and presents valuable therapeutic strategies for combating chronic macrophage-resident microbial infections.

Antigen presenting cells targeting and stimulation potential of lipoteichoic acid functionalized lipo-polymerosome: a chemo-immunotherapeutic approach against intracellular infectious disease.

Antigen presenting cells (APC) are well-recognized therapeutic targets for intracellular infectious diseases, including visceral leishmaniasis. These targets have raised concerns regarding their potential for drug delivery due to overexpression of a variety of receptors for pathogen associated molecular pathways after infection. Since, lipoteichoic acid (LTA), a surface glycolipid of Gram-positive bacteria responsible for recognition of bacteria by APC receptors that also regulate their activation for pro-inflammatory cytokine secretion, provides additive and significant protection against parasite. Here, we report the nanoarchitechture of APC focused LTA functionalized amphotericin B encapsulated lipo-polymerosome (LTA-AmB-L-Psome) delivery system mediated by self-assembly of synthesized glycol chitosan-stearic acid copolymer (GC-SA) and cholesterol lipid, which can activate and target the chemotherapeutic agents to Leishmania parasite resident APC. Greater J774A and RAW264.7 macrophage internalization of FITC tagged LTA-AmB-L-Psome compared to core AmB-L-Psome was observed by FACSCalibur cytometer assessment. This was further confirmed by higher accumulation in macrophage rich liver, lung and spleen during biodistribution study. The LTA-AmB-L-Psome overcame encapsulated drug toxicity and significantly increased parasite growth inhibition beyond commercial AmB treatment in both in vitro (macrophage-amastigote system; IC50, 0.082 ± 0.009 µg/mL) and in vivo (Leishmania donovani infected hamsters; 89.25 ± 6.44% parasite inhibition) models. Moreover, LTA-AmB-L-Psome stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase and nitric oxide with down-regulation of disease susceptible cytokines, like transforming growth factor-ß (TGF-ß), IL-10, and IL-4. These data demonstrate the potential use of LTA-functionalized lipo-polymerosome as a biocompatible lucrative nanotherapeutic platform for overcoming toxicity and improving drug efficacy along with induction of robust APC immune responses for effective therapeutics of intracellular diseases.

Self assembled ionically sodium alginate cross-linked amphotericin B encapsulated glycol chitosan stearate nanoparticles: applicability in better chemotherapy and non-toxic delivery in visceral leishmaniasis.

OBJECTIVES: To investigate the applicability, localization, biodistribution and toxicity of self assembled ionically sodium alginate cross-linked AmB loaded glycol chitosan stearate nanoparticles for effective management of visceral leishmaniasis. METHODS: Here, we fabricated Amphotericin B (AmB) encapsulated sodium alginate-glycol chitosan stearate nanoparticles (AmB-SA-GCS-NP) using strong electrostatic interaction between oppositely charged polymer and copolymer by ionotropic complexation method. The tagged FAmB-SA-GCS-NP was compared with tagged FAmB for in vitro macrophagic uptake in J774A macrophages and in vivo localization in liver, spleen, lung and kidney tissues. The AmB-SA-GCS-NP and plain AmB were compared for in vitro and in vivo antileishmanial activity, pharmacokinetics, organ distribution and toxicity profiling. RESULTS: The morphology of SA-GCS-NP revealed as nanocrystal (size, 196.3 ± 17.2 nm; PDI, 0.216 ± 0.078; zeta potential, (-) 32.4 ± 5.1 mV) by field emission scanning electron microscopy and high resolution transmission electron microscopy. The macrophage uptake and in vivo tissue localization studies shows tagged FAmB-SA-GCS-NP has significantly higher (~1.7) uptake compared to tagged FAmB. The biodistribution study of AmB-SA-GCS-NP showed more localized distribution towards Leishmania infected organs i.e. spleen and liver while lesser towards kidney. The in vitro (IC50, 0.128 ± 0.024 µg AmB/ml) and in vivo (parasite inhibition, 70.21 ± 3.46%) results of AmB-SA-GCS-NP illustrated significantly higher (P < 0.05) efficacy over plain AmB. The monomeric form of AmB within SA-GCS-NP, observed by UV-visible spectroscopy, favored very less in vitro and in vivo toxicities compared to plain AmB. CONCLUSION: The molecular organization, toxicity studies, desired localization and biodistribution of cost effective AmB-SA-GCS-NP was found to be highly effective and can be proved as practical delivery platform for better management of leishmaniasis.

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.

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.

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.

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.

Immunoadjuvant chemotherapy of visceral leishmaniasis in hamsters using amphotericin B-encapsulated nanoemulsion template-based chitosan nanocapsules.

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 Ca(2+). 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 [IC(50)], 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 ß (TGF-ß), 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.