Ashwni Verma

Paclitaxel loaded PEGylated gleceryl monooleate based nanoparticulate carriers in chemotherapy.

A PEGylated drug delivery system of paclitaxel (PTX), based on glyceryl monooleate (GMO) was prepared by optimizing various parameters to explore its potential in anticancer therapy. The prepared system was characterized through polarized light microscopy, TEM, AFM and SAXS to reveal its liquid crystalline nature. As GMO based LCNPs exhibit high hemolytic toxicity and faster release of entrapped drug (66.2 ± 2.5% in 24 h), PEGylation strategy was utilized to increase the hemocompatibility (reduction in hemolysis from 60.3 ± 10.2 to 4.4 ± 1.3%) and control the release of PTX (43.6 ± 3.2% released in 24 h). The cytotoxic potential and cellular uptake was assessed in MCF-7 cell lines. Further, biodistribution studies were carried out in EAT (Ehrlich Ascites tumor) bearing mice using (99m)Tc-(Technetium radionuclide) labeled formulations and an enhanced circulation time and tumor accumulation (14 and 8 times, respectively) were observed with PEGylated carriers over plain ones, at 24 h. Finally, tumor growth inhibition experiment was performed and after 15 days, control group exhibited 15 times enhancement in tumor volume, while plain and PEGylated systems exhibited only 8 and 4 times enhancement, respectively, as compared to initial tumor volume. The results suggest that PEGylation enhances the hemocompatibility and efficacy of GMO based system that may serve as an efficient i.v. delivery vehicle for paclitaxel.

Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone

Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.

Characterization and evaluation of amphotericin B loaded MDP conjugated poly(propylene imine) dendrimers

This paper describes a novel strategy for targeted delivery of amphotericin B (AmB) to macrophages with muramyl dipeptide (MDP) conjugated multimeric poly(propyleneimine) (PPI) dendrimers. Synergistic antiparasitic activity due to immunostimulation by multimeric presentation of MDP on dendrimers was anticipated. MDP conjugated 5.0G PPI (MdPPI) dendrimers were synthesized and characterized. Therapeutic activity and toxicity of dendrimeric formulation of AmB (MdPPIA) were compared with marketed formulations of AmB. Highly significant (P<0.01) reduction in toxicity was observed in hemolytic toxicity and cytotoxicity studies in erythrocytes and J774A.1 macrophage cells, respectively. Formulation MdPPIA showed appreciable macrophage targeting potential and higher or equivalent antiparasitic activity against parasite infected macrophage cell lines and in vivo infection in Balb/c mice. These results suggest the developed MDP conjugated dendrimeric formulation of AmB as a promising immunostimulant targeted drug delivery system and a safer alternative to marketed formulations. From the clinical editor: Parasitic infections remain a significant issue in the clinical setting. The authors in this article studied the use of ligand anchored dendrimeric formulation of Amphotericin B to target infected macrophages and showed reduced toxicity, high anti-leishmanial activity. This may add another treatment option to available formulations in the future.

Investigations into an alternate approach to target mannose receptors on macrophages using 4-sulfated N-acetyl galactosamine more efficiently in comparison with mannose-decorated liposomes: an application in drug delivery

UNLABELLED In this study the potential of 2 different ligands, i.e., palmitoyl mannose (Man-Lip) and 4-SO(4)GalNAc (Sulf-Lip) to target resident macrophages was investigated after surface decoration of Amphotericin B (AmB) loaded liposomes. In the case of Sulf-Lip, the 4-SO(4)GalNAc was adsorbed through electrostatic interaction on cationic liposomes, which was confirmed by change in zeta potential from +48.2 ± 3.7 mV for Lip to +12.2 ± 1.3 mV for Sulf-Lip. The mean particle size of Sulf-Lip and Man-Lip was found to be 139.4 ± 7.4 nm and 147.4 ± 8.6 nm, respectively. Flow cytometric data reveal enhanced uptake of Sulf-Lip in both J774 and RAW cell lines in comparison with the uptake of Man-Lip. Intracellular localization studies indicate that the fluorescence intensity of Sulf-Lip was much higher in comparison with that of Man-Lip and Lip formulations. Sulf-Lip and Man-Lip showed significantly higher localization of AmB at all time points in comparison with Lip (P < 0.05) after intravenous (IV) administration. The studies provide evidence that 4-SO(4)GalNAc possesses a promising feature for targeting resident macrophages and its application in the conditions of leishmaniasis is in the offing. FROM THE CLINICAL EDITOR This in vivo study compares two different ligands to deliver Amphotericin B l(AmB) loaded liposomes to resident macrophages. Targeted approaches showed significantly higher localization of AmB at all time points in comparison to non-targeted liposomes, and future applications in leishmaniasis are already under preparation.

An insight into functionalized calcium based inorganic nanomaterials in biomedicine: Trends and transitions.

Over the recent years the use of biocompatible and biodegradable nanoparticles in biomedicine has become a significant priority. Calcium based ceramic nanoparticles like calcium phosphate (CaP) and calcium carbonate (CaCO3) are therefore considered as attractive carriers as they are naturally present in human body with nanosize range. Their application in tissue engineering and localized controlled delivery of bioactives for bones and teeth is well established now, but recently their use has increased significantly as carrier of bioactives through other routes also. These delivery systems have become most potential alternatives to other commonly used delivery system because of their cost effectiveness, biodegradability, chemical stability, controlled and stimuli responsive behaviour. This review comprehensively covers their characteristic features, method of preparation and applications but the thrust is to focus their recent development, functionalization and use in systemic delivery. On the same platform mineralization of other nanoparticulate delivery system which has widened their application drug delivery will be discussed. The emphasis has been given on their pH dependent properties which make them excellent carriers for tumour targeting and intracellular delivery. Finally this review also attempts to discuss their drawback which limits their clinical utility.

Surface-Engineered Dendrimeric Nanoconjugates for Macrophage-Targeted Delivery of Amphotericin B: Formulation Development and In Vitro and In Vivo Evaluation

ABSTRACT The present study aimed to develop an optimized dendrimeric delivery system for amphotericin B (AmB). Fifth-generation (5.0G) poly(propylene imine) (PPI) dendrimers were synthesized, conjugated with mannose, and characterized by use of various analytical techniques, including Fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (1H-NMR) spectroscopic analysis, and atomic force microscopy (AFM). Mannose-conjugated 5.0G PPI (MPPI) dendrimers were loaded with AmB and evaluated for drug loading efficiency, in vitro drug release profile, stability, hemolytic toxicity to human erythrocytes, cytotoxicity to and cell uptake by J774A.1 macrophage cells, antiparasitic activity against intracellular Leishmania donovani amastigotes, in vivo pharmacokinetic and biodistribution profiles, drug localization index, toxicity, and antileishmanial activity. AFM showed the nanometric size of the MPPI dendrimers, with a nearly globular architecture. The conjugate showed a good entrapment efficiency for AmB, along with pH-sensitive drug release. Highly significant reductions in toxicity toward human erythrocytes and macrophage cells, without compromising the antiparasitic activity of AmB, were observed. The dendrimeric formulation of AmB showed a significant enhancement of the parasiticidal activity of AmB toward intramacrophagic L. donovani amastigotes. In the in vitro cell uptake studies, the formulation showed selectivity toward macrophages, with significant intracellular uptake. Further pharmacokinetic and organ distribution studies elucidated the controlled delivery behavior of the formulation. The drug localization index was found to increase significantly in macrophage-rich organs. In vivo studies showed a biocompatible behavior of MPPIA, with negligible toxicity even at higher doses, and promising antileishmanial activity. From the results, we concluded that surface-engineered dendrimers may serve as optimized delivery vehicles for AmB with enhanced activity and low or negligible toxicity.

New Fluoranthene FLUN-550 as a Fluorescent Probe for Selective Staining and Quantification of Intracellular Lipid Droplets

A new class of live cell permeant, nontoxic fluoranthene-based fluorescent probe (FLUN-550) having a high Stokes shift in aqueous medium has been discovered. It showed selective staining of lipid droplets (LDs, dynamic cytoplasmic organelles) at a low concentration without background noise in in vitro live cell imaging of 3T3-L1 preadipocytes, J774 macrophages, MCF7 breast cancer cells, and single-celled, parasitic protozoa Leishmania donovani promastigotes and in vivo nonparasitic soil nematode C. elegans.

Emerging trend in nano-engineered polyelectrolyte-based surrogate carriers for delivery of bioactives.

Importance of the field: In recent decades a new colloidal drug delivery system based on layer-by-layer (LbL) technology has emerged, which offers promising means of delivering bioactive agents, specifically biological macromolecules including peptides and DNA. Nano-engineered capsules specifically fabricated from biocompatible and biodegradable polyelectrolytes (PEs) can provide a better option for encapsulation of cells thereby protecting cells from immunological molecules in the body, and their selective permeability can ensure the survival of encapsulated cells. Areas covered in this review: This review encompasses a strategic approach to fabricate nano-engineered microcapsules through meticulous selection of polyelectrolytes and core materials based on LbL technology. The content of the article provides evidence for its wide array of applications in medical therapeutics, as indicated by the quantity of research and patents in this area. Recent developments and approaches for tuning drug release, biocompatibility and cellular interaction are discussed thoroughly. What the reader will gain: This review aims to provide an overview on the development of LbL capsules with specific orientation towards drug and macromolecular delivery and its integration with other drug delivery systems, such as liposomes. Take home message: Selection of PEs for the fabrication of LbL microcapsules has a profound effect on stability, drug release, biocompatibility and encapsulation efficacy. The release can be easily modulated by varying different physicochemical as well as physiological conditions. Scale-up approaches for the fabrication of LbL microcapsules by means of automation must be considered to improve the possibility of application of LbL microcapsules on a large scale.

Vitamin B12 functionalized layer by layer calcium phosphate nanoparticles: A mucoadhesive and pH responsive carrier for improved oral delivery of insulin.

UNLABELLED The present study investigates the potential of layer by layer coated calcium phosphate nanoparticles - for oral delivery of insulin where Vitamin B12 grafted chitosan and sodium alginate have been used as cationic and anionic polyelectrolyte respectively. The major emphasis has been given on the role Vitamin B12 conjugated chitosan as cationic polyelectrolyte (VitB12-Chi) in the delivery system. VitB12-Chi conjugate was prepared by carbodiimide reaction. The formulated VirB12-Chi-CPNPs were tested for in vitro and in vivo efficacy studies carried out in Caco-2 monolayers and diabetic rats. VitB12-Chi-CPNPs with particle size <250nm and zeta potential+32.56(±2.34) exhibited pH responsive insulin release at simulated gastric fluid and simulated intestinal fluid. Fluorescence microscopy and flow cytometry studies revealed higher uptake of VitB12-Chi-CPNPs in Caco-2 monolayer in comparison to Chi-CPNPs. Further reduction in TEER supported paracellular transport of insulin because of opening of tight epithelial junctions. In vivo intestinal uptake of FITC tagged Vit-B12-Chi-CPNPs from different intestinal segments supported paracellular and receptor mediated uptake of VitB12-Chi-CPNPs. Plasma insulin and blood glucose levels were measured in diabetic rats and showed about four fold increases in insulin bioavailability and sustained hypoglycemic effects up to 12h of administration with VitB12-Chi-CPNPs in comparison to Chi-CPNPs. Results of the study revealed the potential of layer by layer nanoparticles for oral insulin delivery. The study also specifically highlighted the role of VitB12 as a pH sensitive and targeting ligand which significantly participated in enhancing insulin oral bioavailability. STATEMENT OF SIGNIFICANCE Oral delivery of insulin is always the most desirable approach for diabetic patients however its also the most challenging in respect to formulation development due to harsh gastrointestinal conditions. Several groups have been working from decades for oral delivery of insulin. However the beauty of this prototype formulation is that it exhibits the pH responsive behavior in natural condition of gastrointestinal tract. It resists the release of insulin at gastric condition however stimulate the release at intestinal conditions. Apart from pH responsive behavior it utilizes multiple pathways to improve the overall bioavailability of insulin including paracellular transport and receptor mediated endocytosis.

Development of stabilized Paclitaxel nanocrystals: In-vitro and in-vivo efficacy studies.

OBJECTIVE The aim of the study was to develop stable Paclitaxel nanocrystals (PTX/NCs) for enhanced oral delivery of Paclitaxel (PTX) by circumventing its difficult solubilization properties and rapid metabolism. METHODS Preparation of nanocrystals (NCs) was carried out using high pressure homogenizer (Microfluidizer™) without using any organic solvent. Effect of various process and formulation parameters on development and stability of nanocrystals (NCs) were investigated. Particle characteristics, stability studies, in-vitro cellular studies and oral pharmacokinetics in male Wistar rats were examined. RESULTS It was found that different stabilizer used had different effect on size reduction and stability. Surfactants (Tween 80) and low molecular weight synthetic polymer sodium poly styrene sulfonate (PSS) found more suitable and efficient compared to high molecular weight polymers glycol chitosan (GC) and sodium alginate (SA). In-vitro cytotoxicity and cell cycle arrest studies on MCF7 and MDA-MB breast cancer cell lines revealed that PTX/NCs retained the activity even after processing at high pressure and also NCs were more potent and efficacious than PTX solution. The oral in-vivo pharmacokinetic studies demonstrated that PTX/NCs exhibit significant increase in AUC0-t, Cmax, MRT and decrease in Tmax, compared to plain PTX crystals. The increase in AUC was almost 9-10 fold compared to plain PTX crystals. CONCLUSION Altogether study showed that PTX/NC can be a clinically relevant drug delivery system for oral chemotherapy as it can remarkably increases the pharmacological effect by increasing oral bioavailability.