Anil K. Mishra

The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly(γ-benzyl L-glutamate)-b-hyaluronan polymersomes

We have investigated the intracellular delivery of doxorubicin (DOX) loaded poly(gamma-benzyl L-glutamate)-block-hyaluronan (PBLG-b-HYA) based polymersomes (PolyDOX) in high (MCF-7) and low (U87) CD44 expressing cancer cell models. DOX was successfully loaded into polymersomes using nanoprecipitation method and in vitro drug release pattern were achieved at pH 5.5 and 7.4 up to 10 days. Block copolymer vesicles without loaded DOX were non cytotoxic in both cells at concentration 150-650 microg/mL. Flow cytometry data suggested successful uptake of PolyDOX in cells and high accumulation was found in MCF-7 than U87 cells. Microscopy imagings revealed that in MCF-7 cells PolyDOX was more in cytoplasm and free DOX in nuclei, whereas in U87 cells free DOX was also found in the cytoplasm. Cytotoxicity of the drug was concentration and exposure time dependent. In addition, PolyDOX significantly enhanced reactive oxygen species (ROS) level in both cells. PolyDOX also suppressed growth of breast tumor on female Sprague-Dawley (SD) rats as compared to phosphate buffer saline pH 7.4 (PBS) control group. In addition reduced level of serum enzymes (LDH and CPK) by PolyDOX formulation indicated less cardiotoxicity of DOX after loading in polymersomes. Results suggest that intracellular delivery of PolyDOX was depended on the CD44 expression level in cells due to presence of hyaluronic acid on the surface of polymersomes, and could be used as a self-targeting drug delivery cargo in over-expressed CD44 glycoprotein cells of breast cancer.

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.

In vitro and In vivo Evaluation of Docetaxel Loaded Biodegradable Polymersomes

Formulation of docetaxel (DOC), a hydrophobic anticancer drug, was successfully achieved in poly(gamma-benzyl L-glutamate)-block-hyaluronan polymersomes using a simple and reproducible nanoprecipitation method. The prepared DOC loaded polymersomes (PolyDOC) was stable either in solution or in a lyophilized form, and showed controlled release behaviour over several days. PolyDOC showed high in vitro toxicity after 24 h in MCF-7 and U87 cells compared to free DOC. Biodistribution data demonstrated that (99m)Tc labelled PolyDOC t(1/2) and MRT significantly increased compared to a DOC solution (DS). In addition, PolyDOC uptake in Ehrlich Ascites Tumor (EAT) tumor bearing mice was larger at each time point compared to DS, making such a polymer vesicle formulation an efficient drug nanocarrier for improved DOC cancer therapy.

Targeting Neuropeptide Receptors for Cancer Imaging and Therapy: Perspectives with Bombesin, Neurotensin, and Neuropeptide-Y Receptors

Receptors for some regulatory peptides are highly expressed in tumors. Selective radiolabeled peptides can bind with high affinity and specificity to these receptors and exhibit favorable pharmacologic and pharmacokinetic properties, making them suitable agents for imaging or targeted therapy. The success encountered with radiolabeled somatostatin analogs is probably the first of a long list, as multiple peptide receptors are now recognized as potential targets. This review focuses on 3 neuropeptide receptor systems (bombesin, neurotensin, and neuropeptide-Y) that offer high potential in the field of nuclear oncology. The underlying biology of these peptide/receptor systems, their physiologic and pathologic roles, and their differential distribution in normal and tumoral tissues are described with emphasis on breast, prostate, and lung cancers. Radiolabeled analogs that selectively target these receptors are highlighted.

Proof of concept studies to confirm the delivery of curcumin loaded solid lipid nanoparticles (C-SLNs) to brain

Having achieved a significant bioavailability of curcumin by its incorporation into SLNs (C-SLNs) during pharmacokinetic (32-155 times) and pharmacodynamic (3-4 times) studies, our intent was to proof their targeting to brain. Hence, fluorescent/confocal microscopy, biodistribution and gamma scintigraphy techniques were explored to observe the presence of C-SLNs in the brain. 1h post p.o administration of C-SLNs/free curcumin (C-S) to rats, blood was withdrawn, following which the animals were sacrificed and their harvested brains were frozen at -80°C. The obtained plasma and brain cryosections were observed for fluorescence under fluorescent/confocal microscope. Biodistribution study was performed using (99m)Tc-labeled C-SLNs and C-S in Balb/c mice after p.o. and i.v. administration and % radioactivity/g organ was recorded. Subsequent to this gamma scintigraphs of the New Zealand rabbits following similar treatments were performed. Presence of yellow fluorescent particles in plasma and brain indicated effective delivery of C-SLNs across the gut wall and the BBB. (Blood)AUCoral value for C-SLNs was 8.135 times greater than that for C-S, confirming a prolonged circulation of former. The ratio of blood AUCi.v. C-SLN/C-S in blood is ≤1 while the ratio in brain promisingly indicates 30 times higher preferential distribution of C-SLNs into brain confirming their direct delivery.

Effect of homobifunctional crosslinkers on nucleic acids delivery ability of PEI nanoparticles.

Polyethylenimine (PEI), a widely used cationic polymeric vector with high transfection efficiency, was converted into nanoparticles by introducing ionic and covalent crosslinkers with varying proportion of 1,6-hexanebisphosphate (HP), adipic acid (AA) and 1,4-butane dialdehyde (BA) to obtain a small library of HP-PEI (HPP), AA-PEI (AAP) and BA-PEI (BAP) nanoparticles, respectively. Particles were characterized by spectroscopic technique as well as physicochemical parameters such as size, morphology, surface charge, effect of crosslinking on buffering capacity and DNA binding ability. The entire series of nanoparticles were compared for their cytoxicity and ability to deliver genes in various cell lines. Among various nanoparticles, AAP-3 nanoparticle/DNA complex exhibited higher transfection efficiency (1.5-7.8 folds) than the native PEI (25kDa) and commercially available transfection reagents, such as GenePorter, GenePorter2, Fugene and Superfect, with cell viability >85%. The highest cell viability was observed with BAP nanoparticles (>95%). Importantly, the transfection activity of nanoparticle/DNA complexes was preserved in the presence of serum. Transfection with GFP-siRNA inhibited expression of transfected GFP gene by approximately 81-92%. All nanoparticle types (HPP, AAP and BAP) required a comparable time for entry into cells and subsequent intracellular passage from the cytoplasm to the nucleus. Intravenous delivery of (99)Tc labeled BAP-2/DNA complex to female Balb/c mice revealed the presence of the complex in most of the organs with the highest retention in liver. In conclusion, HPP, AAP and BAP nanoparticles are safe for efficient gene delivery.

The role of ASIC1a in neuroprotection elicited by quercetin in focal cerebral ischemia

One of the major instigators to neuronal cell death and brain damage following cerebral ischemia is calcium dysregulation. The intracellular calcium overload resulting from glutamate excitotoxicity is considered a major determinant for neuronal loss during cerebral ischemia. Moreover, ASIC1a activation due to acidosis also promotes intracellular calcium overload during ischemic insult. Interestingly, ASIC1a was found to be inhibited by some flavonoids which carry an anti-inflammatory property particularly quercetin, which could be exploited in hypoxic conditions like cerebral ischemia. This encourages us to investigate the neuroprotective effect of quercetin besides its possible downstream signaling mechanism in focal cerebral ischemia. The treatment of quercetin 30min before ischemia and 4h after reperfusion shows significant protection from ischemic injury as noticed by reduction in cerebral infarct volume and neurobehavioral deficit. In addition to earlier calcium dependent rise in the levels of nitrite and MDA exhibited marked reduction (P<0.01) in their levels when given quercetin pretreatment in ischemic brain regions. The quercetin treatment also reduced the spectrin break down products (SBDP) caused by ischemic activation of calcium dependent protease calpain. In ex-vivo study, it was also observed that quercetin inhibited the acid mediated intracellular calcium levels in rat brain synaptoneurosomes. These studies suggest the neuroprotective role of quercetin in focal cerebral ischemia by regulation of ASIC1a.

In vitro mechanistic study of cell death and in vivo performance evaluation of RGD grafted PEGylated docetaxel liposomes in breast cancer.

Objectives of the investigations were to prepare RGD grafted docetaxel liposomes (RGD-PEG-LP-DC) using supercritical fluid technology and evaluate it in vitro for cytotoxicity, DNA content analysis, mechanism of cell death, and in vivo for pharmacokinetic and biodistribution studies in BALB/c mice. The RGD-PEG-LP-DCs were found to be most cytotoxic in BT-20 and MDA-MB-231 cell lines. The flowcytometry results shows at 48 hours, 96% G2 phase arrest for RGD-PEG-LP-DC at 5 nM drug concentration. The mode of cell death was found to be mainly by necrosis at low drug equivalent concentration (1 nM) and by apoptosis at high drug equivalent concentration (10 nM). With increase in time and concentration the mode of cell death by apoptosis was found to be increasing. Biodistribution demonstrated that site specific drug distribution, t(1/2), and MRT improved significantly for RGD-PEG-LP-DC. From the studies site specific and sustained intracellular drug delivery from RGD-PEG-LP-DCs may provide promising strategy in enhancing embattled against breast cancer treatment. FROM

Solid Lipid Nanoparticles: Promising Therapeutic Nanocarriers for Drug Delivery

Development of colloidal delivery systems has opened new avenues/frontiers for improving drug delivery. Solid lipid nanoparticles have come up as the latest development in the arena of lipid based colloidal delivery systems after nanoemulsion and liposomes ever since their introduction in the early 1990s. In this review, the authors have made efforts to bring forth the essential and practically relevant aspects of SLNs. This review gives an overview of the preparation methods of solid lipid nanoparticles while mainly focussing on their biological applications including their projected applications in drug delivery. This review critically examines the influential factors governing the formation of SLNs and then discussing in detail the several techniques being utilized for their characterization. This review discusses the drug loading and drug release aspects of SLNs as these are useful biocompatible carriers of lipophilic and to a certain extent hydrophilic drugs. An updated list of drugs encapsulated into various lipids to prepare SLN formulations has been provided. Other relevant aspects pertaining to the clinical use of SLN formulations like their sterilization and storage stability have also been explained. A unique facet of this review is the discussion on the challenging issues of in vivo applications and recent progresses in overcoming these challenges which follows in the end.

Metastatic follicular carcinoma of the thyroid with tumor thrombus in the superior vena cava and right brachiocephalic and internal jugular veins: FDG-PET/CT findings.

Abstract:A 48-year-old woman who had undergone subtotal thyroidectomy with right modified neck dissection underwent F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) for assessing the extent of disease postoperatively (restaging). Biopsy was reported as follicular carcinoma thyroid. A