Yuvraj Singh

Engineered Nanocrystal Technology: in-vivo fate, targeting and applications in drug delivery

Formulation of nanocrystals is a robust approach which can improve delivery of poorly water soluble drugs, a challenge pharmaceutical industry has been facing since long. Large scale production of nanocrystals is done by techniques like precipitation, media milling and, high pressure homogenization. Application of appropriate stabilizers along with drying accords long term stability and commercial viability to nanocrystals. These can be administered through oral, parenteral, pulmonary, dermal and ocular routes showing their high therapeutic applicability. They serve to target drug molecules in specific regions through size manipulation and surface modification. This review dwells upon the in-vivo fate and varying applications in addition to the facets of drug nanocrystals stated above.

Targeting of gastrointestinal tract for amended delivery of protein/peptide therapeutics: Strategies and industrial perspectives

Delivery of proteins/peptides to the gastrointestinal (GI) tract via peroral/oral route involves tremendous challenges due to unfavorable environmental conditions like harsh pH, presence of proteolytic enzymes and absorption barriers. Detailed research is being conducted at the academic and industrial levels to diminish these troubles and various products are under clinical trials. Several approaches have been established to optimize oral delivery of proteins and peptides and can be broadly categorized into chemical and physical strategies. Chemical strategies include site specific mutagenesis, proteinylation, glycosylation, PEGylation and prodrug approaches, whereas physical strategies comprise formulation based approaches including application of absorption enhancers and metabolism modifiers along with delivering them via colloidal carrier systems such as nanoparticles, liposomes, microparticles, and micro- and nano-emulsions. This review stands to accomplish the diverse aspects of oral delivery of proteins/peptides and summarizes the key concepts involved in targeting the biodrugs to specific sites of the GI tract such as the intestine and colon. Furthermore some light has also been shed on the current industrial practices followed in developing oral formulations of such bioactives.

Nanoemulsion: Concepts, development and applications in drug delivery

ABSTRACT Nanoemulsions are biphasic dispersion of two immiscible liquids: either water in oil (W/O) or oil in water (O/W) droplets stabilized by an amphiphilic surfactant. These come across as ultrafine dispersions whose differential drug loading; viscoelastic as well as visual properties can cater to a wide range of functionalities including drug delivery. However there is still relatively narrow insight regarding development, manufacturing, fabrication and manipulation of nanoemulsions which primarily stems from the fact that conventional aspects of emulsion formation and stabilization only partially apply to nanoemulsions. This general deficiency sets up the premise for current review. We attempt to explore varying intricacies, excipients, manufacturing techniques and their underlying principles, production conditions, structural dynamics, prevalent destabilization mechanisms, and drug delivery applications of nanoemulsions to spike interest of those contemplating a foray in this field.

Immunotherapeutic vitamin E nanoemulsion synergies the antiproliferative activity of paclitaxel in breast cancer cells via modulating Th1 and Th2 immune response.

Paclitaxel (PTX) is used as first line treatment for metastatic breast cancer but the relief comes at a heavy cost in terms of accompanying adverse effects. The pharmaceutical credentials of PTX are further dampened by the intrinsically low aqueous solubility. In order to sideline such insidious tendencies, PTX was incorporated in a vitamin E nanoemulsion using high pressure homogenization. The encapsulation efficiency of PTX in nanoemulsion was 97.81±2.7% and a sustained drug release profile was obtained. PTX loaded nanoemulsion exhibited higher cytotoxicity in breast cancer cell line (MCF-7) when compared to free PTX and marketed formulation (Taxol). Cell cycle arrest study depicted that MCF-7 cells treated with PTX loaded nanoemulsion showed high arrest in G2-M phase. Moreover blank nanoemulsion induced additional apoptosis in breast cancer cells through G1-S arrest by disrupting mitochondrial membrane potential. Cytokine estimation study in macrophages showed that both PTX loaded nanoemulsion and blank nanoemulsion enhanced secretion of IL-12 and downregulated secretion of IL-4 and IL-10. Results suggest that inclusion of vitamin E in nanoemulsion opened multiple complementary molecular effects which not only magnified the principle antiproliferative activity of PTX but also independently showcased potential in restoring the proactive nature of the breast cancer slackened chronic immune response. In-vivo anticancer activity showed significantly improved efficacy of PTX loaded nanoemlsion compare to Taxol and free PTX. The list of plausible advantages of PTX nanoemulsification was further substantiated by acceptable haemolytic potential, reduced in-vivo toxicity and conveniently modified pharmacokinetic profile in which the AUC and MRT were extended considerably. Overall, there were strong evidences that developed formulation can serve as a viable alternative to currently available PTX options.

Bridging small interfering RNA with giant therapeutic outcomes using nanometric liposomes

The scope of RNAi based therapeutics is unquestionable. However, if we dissect the current trend of clinical trials for afore mentioned drug class, some stark trends appear: 1) naked siRNA only exerts influence in topical mode whilst systemic delivery requires a carrier and 2) even after two decades of extensive efforts, not even a single siRNA containing product is commercially available. It was therefore felt that a perspective simplifying the unique intricacies of working with a merger of siRNA and liposomes from a pharmaceutical viewpoint could draw the attention of a wider array of interested researchers. We begin from the beginning and attempt to conduit the gap between theoretical logic and experimental/actual constraints. This, in turn could stimulate the next generation of investigators, gearing them to tackle the conundrum, which is siRNA delivery.

Targeting tumor associated macrophages (TAMs) via nanocarriers

&NA; Recruitment of inflammatory cells to tumor has been well documented, with the most frequent inhabitants being macrophages termed as tumor associated macrophages, (TAMs). Their presence was thought to be an evidence of immune system initiating a fight response towards the tumor, i.e. immune surveillance. This is the case too initially, when TAMs majorly exhibit an M1 phenotype, but their continued presence in tumor microenvironment brings about their polarization to M2 phenotype, which not only participate in continued sustenance of existing tumor but also open up deleterious avenues for further progression and metastasis of cancer. Current perspective is built around this very premise and focuses specifically on TAMs and how they are being targeted by researchers working in annals of nanomedicine. To do so, we dwell into tumor microenvironment and focus on nanotechnology based drug delivery aspects which have either been already or can be potentially employed in the future to target tumor associated macrophages for improved immunoadjuvant therapy of cancer. Graphical abstract Figure. No caption available.

Enhanced apoptotic and anticancer potential of paclitaxel loaded biodegradable nanoparticles based on chitosan

Taxanes have established and proven effectivity against different types of cancers; in particular breast cancers. However, the high hemolytic toxicity and hydrophobic nature of paclitaxel and docetaxel have always posed challenges to achieve safe and effective delivery. Use of bio-degradable materials with an added advantage of nanotechnology could possibly improve the condition so as to achieve better and safe delivery. In the present study paclitaxel loaded chitosan nanoparticles were formulated and optimized using simple w/o nanoemulsion technique. The observed average size, pdi, zeta potential, entrapment efficiency and drug loading for the optimized paclitaxel loaded chitosan nanoparticle formulation (PTX-CS-NP-10) was 226.7±0.70nm, 0.345±0.039, 37.4±0.77mV, 79.24±2.95% and 11.57±0.81%; respectively. Nanoparticles were characterized further for size by Transmission Electron Microscopy (TEM). In vitro release studies exhibited sustained release pattern and more than 60% release was observed within 24h. Enhanced in vitro anticancer activity was observed as a result of MTT assay against triple negative MDA-MB-231 breast cancer cell lines. The observed IC50 values obtained for PTX-CS-NP-10 was 9.36±1.13μM and was almost 1.6 folds (p<0.05) less than the pure drug. Similarly, PTX-CS-NP-10 were extremely biocompatible and safe as observed for haemolytic toxicity which was almost 4 folds less (p<0.05) than the naïve drug. Anticancer activity was further evaluated using flow cytometry for apoptosis. Cell apoptosis study revealed that PTX-CS-NP-10 treatment resulted into enhanced (almost double) late cell apoptosis than naïve paclitaxel. Hence the developed nanoparticulate formulation not only reduced the overall toxicity but also resulted into improved anticancer efficacy of paclitaxel. It can be concluded that a robust, stable and comparatively safe nanoformulation of paclitaxel was developed, characterized and evaluated.

Subcutaneously Administered Ultrafine PLGA Nanoparticles Containing Doxycycline Hydrochloride Target Lymphatic Filarial Parasites.

Systemic chemotherapeutic targeting of filarial parasites is unfocused due to their deep seated location in lymphatic vessels. This warrants a prolonged dosing regimen in high doses for an anthelmintic like doxycycline hydrochloride (DOX). In order to provide an alternative, we have constructed ultrafine PLGA nanoparticles of DOX (DPNPs), so as to exploit the peculiarity of lymphatic vasculature underneath the subcutaneous layer of skin, which preferentially allows entry of only 10-100 nm sized particles. DPNPs were constructed using a novel solvent diffusion method aided by probe sonication, which resulted in an average size 95.43 ± 0.8 nm as per DLS, PDI 0.168 ± 0.03, zeta potential -7.38 ± 0.32, entrapment efficiency 75.58 ± 1.94%, and refrigerator stability of 7 days with respect to size in the optimized batch. TEM further substantiated the spherical shape of DPNPs along with their actual nonhydrated size as being well below 100 nm. FTIR analysis of DOX, dummy nanoparticles, and freeze-dried DPNPs revealed that the formulation step did not induce prominent changes in the chemical nature of DOX. The drug release was significantly altered (p < 0.05) with 64.6 ± 1.67% release in 48 h from DPNPs and was dictated by Fickian diffusion. Pharmacokinetic studies in Wistar rats further revealed that DPNPs caused a 16-fold prolongation in attainment of plasma Tmax and a 2-fold extension of elimination half-life (28.569 ± 1.27 h) at a dose of 5 mg/kg when compared to native drug (DOX solution) of the same strength. Contrastingly the trend was reversed in regional lymph nodes where Cmax for DPNPs (820 ± 84 ng/mg) was 4-fold greater, and lymphatic Tmax was attained in one-fourth of what was required for DOX solution. This size based preferential lymphatic targeting resulted in significantly greater in vivo antifilarial activity of DPNPs when compared to DOX solution as gauged by several parameters in Brugia malayi infected Mastomys coucha. Interestingly, the magnification in efficacy was obtained despite equivalent in vitro antifilarial activity of DOX solution and DPNPs against B. malayi worms.

Nanosized complexation assemblies housed inside reverse micelles churn out monocytic delivery cores for bendamustine hydrochloride

Objective We explore a plausible method of targeting bendamustine hydrochloride (BM) to circulatory monocytes by exploiting their intrinsic endocytic/phagocytic capability. Methods We do so by complexation of sodium alginate and chitosan inside dioctyl sulfo succinate sodium (AOT) reverse micelles to form bendamustine hydrochloride loaded nanoparticles (CANPs). Dynamic light scattering, electrophoretic mobility and UV spectroscopy were used to detail intra‐micellar complexation dynamics and to prove that drug was co‐captured during interaction of carbohydrate polymers. A fluorescent conjugate of drug (RBM) was used to trace its intracellular fate after its loading into nanoparticles. Results CANPs were sized below 150 nm, had 75% drug entrapment and negative zeta potential (−30 mV). Confocal microscopy demonstrated that developed chitosan alginate nanoparticles had the unique capability to carry BM specifically to its site of action. Quantitative and mechanism based cell uptake studies revealed that monocytes had voracious capacity to internalize CANPs via simultaneous scavenger receptor based endocytic and phagocytic mechanism. Comparative in vitro pharmacokinetic studies revealed obtainment of significantly greater intracellular drug levels when cells were treated with CANPs. This caused reduction in IC50 (22.5 ± 2.1 &mgr;g/mL), enhancement in G2M cell cycle arrest, greater intracellular reactive oxygen species generation, and increased apopotic potential of bendamustine hydrochloride in THP‐1 cells. Conclusion Selective monocytic targeting of bendamustine hydrochloride using carbohydrate constructs can prove advantageous in case of leukemic disorders displaying overabundance of such cells. Graphical abstract No caption available.

Macrophage-targeted chitosan anchored PLGA nanoparticles bearing doxorubicin and amphotericin B against visceral leishmaniasis

Novel chitosan-coated nanoparticles with a high payload of amphotericin B (AmB) and doxorubicin (Dox) were formulated employing a nanoprecipitation technique and evaluated for antileishmanial activity against Leishmania donovani. FTIR, DSC and TG-DTA analysis ensured the physicochemical compatibility of drugs and polymers. The chitosan-coated optimized nanoparticle formulation resulted in a mean particle size; 374.4 ± 4.8 nm, PDI; 0.227 ± 0.035 and zeta potential; (+) 32.9 ± 1.10 mV. The entrapment efficiency was determined to be 70.2 ± 4.76 and 93.86 ± 2.61% for AmB and Dox respectively. An in vitro drug release study demonstrated the release of 27.29 and 36.93% AmB and Dox, respectively after 24 h from chitosan-coated PLGA nanoparticles which is slower than the release obtained from uncoated PLGA nanoparticles of AmB and Dox (32.82 and 57.93% AmB and Dox respectively after 24 h). Stability studies confirmed no remarkable alterations in the physicochemical properties of nanoparticles. Cs-PLGA-ABDx was less hemotoxic (22.87 ± 0.487%) than PLGA-ABDx (36.71 ± 2.08%) and the ABDx suspension (97.04 ± 5.01%) at 42.78 μg mL−1 AmB and 80 μg mL−1 Dox. Cell uptake investigation showed the mean florescence intensity of chitosan-coated PLGA-FITC was 2.02 fold higher than uncoated PLGA-FITC nanoparticles. The cytotoxicity in J774A.1 cells revealed Cs-PLGA-ABDx was less cytotoxic compared to the ABDx suspension and PLGA-ABDx, whereas the IC50 of Cs-PLGA-ABDx against infected macrophages was significantly (p < 0.05) lower than PLGA-ABDx indicating the effectiveness of Cs-PLGA-ABDx. No significant increase in the biomedical markers AST, BUN and PC was observed in Cs-PLGA-ABDx treated groups at 1 and 3 mg kg−1 dose. These experimental findings put forward Cs-PLGA-ABDx to be a suitable alternative in the management of visceral leishmaniasis.