Naresh Mittapelly

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.

Therapeutic interventions in sepsis: current and anticipated pharmacological agents

Sepsis is a clinical syndrome characterized by a multisystem response to a pathogenic assault due to underlying infection that involves a combination of interconnected biochemical, cellular and organ–organ interactive networks. After the withdrawal of recombinant human‐activated protein C (rAPC), researchers and physicians have continued to search for new therapeutic approaches and targets against sepsis, effective in both hypo‐ and hyperinflammatory states. Currently, statins are being evaluated as a viable option in clinical trials. Many agents that have shown favourable results in experimental sepsis are not clinically effective or have not been clinically evaluated. Apart from developing new therapeutic molecules, there is great scope for for developing a variety of drug delivery strategies, such as nanoparticulate carriers and phospholipid‐based systems. These nanoparticulate carriers neutralize intracorporeal LPS as well as deliver therapeutic agents to targeted tissues and subcellular locations. Here, we review and critically discuss the present status and new experimental and clinical approaches for therapeutic intervention in sepsis.

Investigating the role of Pluronic-g-Cationic polyelectrolyte as functional stabilizer for nanocrystals: Impact on Paclitaxel oral bioavailability and tumor growth.

UNLABELLED Paclitaxel (PTX) is a potent anticancer drug which suffers limitations of extremely low oral bioavailability due to low solubility, rapid metabolism and efflux by P-gp transporters. The main objective of this study was to overcome the limitation of PTX by designing delivery systems that can enhance the absorption using multiple pathways. A novel Pluronic-grafted chitosan (Pl-g-CH) copolymer was developed and employed as a functional stabilizer for nanocrystals (NCs) and hypothesized that it would improve PTX absorption by several mechanisms and pathways. Pl-g-CH was synthesized and characterized using (1)H NMR and then used as a stabilizer during nanocrystal development. To establish our proof of concept the optimized formulation having a particle size 192.7 ± 9.2 nm and zeta potential (+) 38.8 ± 3.12 mV was studied extensively on in vitro Caco-2 model. It was observed that nanocrystals rendered higher PTX accumulation inside the cell than Taxol™. P-gp inhibitory potential of Pl-g-CH was proved by flow cytometry and fluorescence microscopy where the much enhanced fluorescence intensity of Rhodamine 123 (Rho-123, P-gp substrate) was observed in the presence of Pl-g-CH. In addition, a significant decrease in Trans Epithelial Electrical Resistance (TEER) of Caco-2 cell monolayers was observed with nanocrystals as well as with Taxol™ (in the presence of free Pl-g-CH compared to only Taxol™). This supports the role of the stabilizer in reversible opening of tight junctions between cells which can allow paracellular transport of drug. The in vivo results were in complete corroboration with in vitro results. Nanocrystals resulted in much enhanced absorption with 12.6-fold improvement in relative bioavailability to that of Taxol™. Concomitantly efficacy data in B16 F10 murine melanoma model also showed a significant reduction in tumor growth with nanocrystals compared to Taxol™ and control. Based on the results it can be suggested that nanocrystals with functional stabilizers can be a promising approach for the oral delivery of anticancer drugs which are P-gp substrates STATEMENT OF SIGNIFICANCE Nanocrystals are currently one of the most explored novel drug delivery systems especially for oral delivery of drugs because of ease in synthesis and high drug loading. But their use is still limited for oral delivery of anticancer drugs which are P-gp substrates. This particular study aims at widening the scope of nanocrystals by using a functional stabilizer which participates in enhancing the oral absorption of anticancer drugs and controlling the tumor growth.

Dual functioning microspheres embedded crosslinked gelatin cryogels for therapeutic intervention in osteomyelitis and associated bone loss.

In the present research,we simultaneously addressed the condition of osteomyelitis and osteoporosis by developing a gelatin based chemically cross linked cryogel system embedded with CaCO3 microspheres and ciprofloxacin hydrochloride was incorporated in both the microspheres and the 3D matrix of cryogel. The fabricated cryogel was characterized for the swelling ratio, swelling kinetics, porosity, pore volume, compression strength and in vitro rate of degradation which were found to be dependent on the concentration of gelatin, duration of freezing and number of freeze-thaw cycles. The sustained release of drug was obtained up to 21days after the initial burst, and the concentration was maintained above the MIC for the entire duration of the study. The in vitro antibacterial study in Staphylococcus aureus and Escherichia coli exhibited 33mm, 30mm, 28mm, 27mm and 43mm, 37mm, 37mm, and 36mm zone of inhibition respectively at day 1, 3, 5 and 7. The cell viability, number of cells in the growth phase and alkaline phosphatase levels were found to be significantly higher in rat osteoblasts cultured in cryogel as compared to 2D surface. All these results demonstrate the propitious potential of this microsphere incorporated, ciprofloxacin-loaded, industrially scalable cryogel system for therapeutic intervention in osteoporosis and associated osteomyelitis.

Investigation of salt formation between memantine and pamoic acid: Its exploitation in nanocrystalline form as long acting injection.

In the present work, we prepared memantine-pamoic acid (MEM-PAM) salt by counter ion exchange in the aqueous phase to reduce the water solubility of MEM hydrochloride (native form) to make it suitable for long acting injection. The ratio of MEM to PAM in salt formation was optimized to maximize the loading efficiency and complexation efficiency. The 2:1 molar ratio of MEM to PAM salt form displayed nearly 95% complexation efficiency and 50% drug loading. The solubility was decreased by a ∼1250 folds. Thermo Gravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), and Powder X-ray Diffraction Analysis (PXRD) studies revealed the formation of new solid phase. Additionally, Nuclear Magnetic Resonance (NMR) spectroscopy confirmed the anhydrous nature of the salt form. Through Fourier transformation infrared spectroscopy (FT-IR) we identified the molecular interactions. Further, the microcrystals of the salt were transformed into nanocrystals (NCs) using high pressure homogenization. The particle size distribution and atomic force microscopy confirmed the monodispersed and spherical shape of the NCs. The in vitro dissolution studies were performed under sink condition in phosphate buffer saline pH 6.8. The results of MTT assay in murine fibroblast 3T3 cell line show that the NCs were less cytotoxic and more tolerable than plain MEM HCl. The in vivo performance of NCs administered as i.m. injection at three different doses in female Sprague-Dawley rats showed that the plasma levels lasted till the 24th day of the study. The pharmacokinetic parameters AUC0-∞ and Cmax increased linearly with increasing dose. Therefore, the results suggest that injectable NCs could represent a therapeutic alternative for the treatment of AD.

Co-delivery of hesperetin enhanced bicalutamide induced apoptosis by exploiting mitochondrial membrane potential via polymeric nanoparticles in a PC-3 cell line

In this research, we reported the co-delivery of anti-androgen drug Bicalutamide (BCT) with Hesperetin (HSP) in chitosan (CS) coated polycaprolactone (PCL) nanoparticles (NPs) to increase their therapeutic efficacy against an androgen independent prostate cancer cell line. The PCL-BCT-HSP-CS NPs were prepared by anti-solvent precipitation followed by ionic gelation method, and showed narrow particle size distribution, high encapsulation efficiency (about 90%) and sustained drug release behavior for both drugs. During the preparation of NPs; HSP crystallinity was retained, whereas polymorphic transition of BCT from form I to form II was observed by thermogravimetric analysis. Additionally, DPPH radical scavenging assay confirmed the structural integrity of HSP in the NPs. The kinetic solubility of BCT and HSP in the NPs was increased by 61.66 and 6.75 fold, respectively, as compared to the free drugs. Further, the in vitro therapeutic efficacy of co-loaded PCL-BCT-HSP-CS NPs was compared with free BCT, HSP and their combination (BCT plus HSP) in androgen independent PC-3 cell lines. Interestingly, co-loaded NPs exhibited higher in vitro cytotoxicity by G1-S phase cell cycle arrest and apoptosis at equivalent concentrations. This superior activity may be attributed to enhanced mitochondrial membrane potential loss by co-loaded NPs. Cell uptake study showed significantly higher (P < 0.05) uptake of NPs by cancer cells. Additionally, in vivo pharmacokinetics of NPs were explored in SD male rats and revealed higher AUC0–t and Cmax of BCT (1.46 and 1.42 fold) and HSP (4.16 and 3.79 fold) than an aqueous suspension when administered orally at 20 mg kg−1. We demonstrated that co-delivery with HSP via polymeric NPs might have better therapeutic potential for in vitro management of androgen independent prostate cancer.

Validation of RP-HPLC Method for Simultaneous Quantification of Bicalutamide and Hesperetin in Polycaprolactone-Bicalutamide-Hesperetin-Chitosan Nanoparticles

Bicalutamide is a non-steroidal anti-androgen drug used for the treatment of androgen-dependent prostate cancer. Hesperetin is a natural bioflavonoid that can be used in combination with bicalutamide to improve efficacy and decrease tolerance. The aim of the present work was to develop and validate a simple, sensitive, rapid reverse phase-high performance liquid chromatographic method for simultaneous estimation of bicalutamide and hesperetin. The validation parameters such as specificity, linearity, precision and accuracy, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. Chromatographic separation was achieved on Lichrocart(®) CN column (250 × 4 mm, 5 µm, MERCK) with isocratic elution. The retention times and detection wavelength, for hesperetin and bicalutamide were 4.28 min, 288 nm and 5.90 min, 270 nm respectively. The intra-day and inter-day assay precision and accuracy were found to be <2% over linearity of 50-2000 ng/mL with R(2) 0.999. LOD and LOQ, of bicalutamide and hesperetin was 14.70, 44.57 ng/mL and 16.11, 48.84 ng/mL, respectively. The method was successfully applied for encapsulation efficiency and drug release studies from bicalutamide and hesperetin loaded nanoparticles.

Dried and free flowing granules of Spinacia oleracea accelerate bone regeneration and alleviate postmenopausal osteoporosis.

Objective: The aim of this study was to demonstrate the efficacy of extract derived from Spinacia oleracea extract (SOE) in reversing bone loss induced by ovariectomy and bone healing properties in a drill-hole fracture model in rats. Methods: SOE was administered orally for 12 weeks in adult ovariectomized Sprague Dawley rats after inducing osteopenic condition. Bone micro-architecture, expressions of osteogenic and resorptive gene markers, biomechanical strength, new bone formation, and bone turnover markers were studied. Uterine histomorphometry was used to assess estrogenicity. Bone regeneration potential of SOE was assessed in a drill-hole fracture model. Fracture healing was assessed by calcein intensity and micro-CT analysis of callus at fracture region. Results: SOE prevented ovariectomy-induced bone loss as evident from 122% increase in bone volume/tissue volume (BV/TV) and 29% decline in Tb.Sp in femoral trabecular micro-architecture. This was corroborated by the more than twofold stimulation in the expression of osteogenic genes runt-related transcription factor 2, osterix, osteocalcin, bone morphogenetic protein 2, collagen-1. Furthermore in the fracture healing model, we observed a 25% increase in BV/TV and enhancement in calcein intensity at the fractured site. The extract when converted into dried deliverable Spinaceae oleracea granule (SOG) form accelerated bone regeneration at fracture site, which was more efficient as evident by a 39% increase in BV/TV. Transforming SOE into dried granules facilitated prolonged systemic availability, thus providing enhanced activity for a period of 14 days. Conclusions: SOE treatment effectively prevents ovariectomy-induced bone loss and stimulated fracture healing in adult rats. The dried granular form of the extract of Spinaceae oleracea was effective in fracture healing at the same dose.

P-gp modulatory acetyl-11-keto-β-boswellic acid based nanoemulsified carrier system for augmented oral chemotherapy of docetaxel☆

In spite of being a very potent and promising drug against many types of cancer, docetaxel suffers the disadvantage of low solubility and poor bioavailability rendering it unsuitable for oral administration. Also, the available marketed formulation for intravenous administration has its inherent drawbacks owing to the presence of polysorbate 80. Here, we exploited the anticancer and P-gp inhibitory potential of naturally occurring frankincense oil to fabricate a stable docetaxel loaded nanoemulsified carrier system for oral delivery. The nanoemulsion possessing desirable particle size (122±12nm), polydispersity (0.086±0.007) and zeta potential (-29.8±2.1mV) was stable against all type of physical stresses and simulated physiological conditions tested. The formulation showed higher uptake in Caco-2 cells and inhibited P-gp transporter significantly (P<0.05). In MDA-MB-231 cells, it showed less IC50, arrested cells in G2-M phase and exhibited higher degree of apoptosis than marketed formulation Taxotere®. The 182.58±4.16% increment in relative oral bioavailability led to higher in vivo anti-proliferative activity manifesting 19% more inhibition than Taxotere®. Conclusively, it is revealed that the developed nanoemulsion will be a propitious approach towards alternative docetaxel therapy.

Multifunctional Glycoconjugate Assisted Nanocrystalline Drug Delivery for Tumor Targeting and Permeabilization of Lysosomal-Mitochondrial Membrane

Nanotechnology has emerged as the most successful strategy for targeting drug payloads to tumors with the potential to overcome the problems of low concentration at the target site, nonspecific distribution, and untoward toxicities. Here, we synthesized a novel polymeric conjugate comprising chondroitin sulfate A and polyethylene glycol using carbodiimide chemistry. We further employed this glycoconjugate possessing the propensity to provide stability, stealth effects, and tumor targeting via CD44 receptors, all in one, to develop a nanocrystalline system of docetaxel (DTX@CSA-NCs) with size < 200 nm, negative zeta potential, and 98% drug content. Taking advantage of the enhanced permeability and retention effect coupled with receptor mediated endocytosis, the DTX@CSA-NCs cross the peripheral tumor barrier and penetrate deeper into the cells of tumor mass. In MDA-MB-231 cells, this enhanced cellular uptake was observed to exhibit a higher degree of cytotoxicity and arrest in the G2 phase in a time dependent fashion. Acting via a mitochondrial-lysosomotropic pathway, DTX@CSA-NCs disrupted the membrane potential and integrity and outperformed the clinically used formulation. Upon intravenous administration, the DTX@CSA-NCs showed better pharmacokinetic profile and excellent 4T1 induced tumor inhibition with significantly less off target toxicity. Thus, this glycoconjugate stabilized nanocrystalline formulation has the potential to take nano-oncology a step forward.