Rajeev K. Tyagi

Adapalene loaded solid lipid nanoparticles gel: An effective approach for acne treatment

Salient features such as controlled release, target ability, potential of penetration, improved physical stability, low cost compared to phospholipids, and ease of scaling-up makes solid lipid nanoparticles (SLNs) a viable alternative to liposomes for effective drug delivery. Adapalene (ADA) is a second generation retinoid effective in treating various dermatologic disorders such as Acne vulgaris with a few noticeable dose-mediated side effects. The present study was aimed at developing and characterizing ADA loaded SLNs for effective topical delivery. The formulated SLN system was characterized for particle size, poly dispersity index, entrapment efficiency and drug release properties. The resultant formulation (ADA loaded SLNs incorporated into carbopol hydrogel) was evaluated for in vitro drug release, skin permeation and bio-distribution, rheological behaviour, and texture profile analysis. The SLNs based ADA gel has shown its potential in targeting skin epidermal layer, and reducing systemic penetration. The developed system can avoid systemic uptake of ADA in skin layers, and can localize drug in skin epidermis as confirmed by rat skin model. Our results advocate potential of SLNs as a novel carrier for topical delivery of ADA in topical therapeutic approaches. This study open new avenues for drug delivery which better meets the need of anti-acne research.

Further Improvements of the P. falciparum Humanized Mouse Model

Background It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines. Materials/Methods We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rγ deficiency (NSG mice). Results We demonstrate here the role of aging, of inosine and of the IL-2 receptor γ mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent. Conclusion The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research.

Galactose engineered solid lipid nanoparticles for targeted delivery of doxorubicin

The present investigation reports the preparation, optimization, and characterization of surface engineered solid lipid nanoparticles (SLNs) encapsulated with doxorubicin (DOX). Salient features such as biocompatibility, controlled release, target competency, potential of penetration, improved physical stability, low cost and ease of scaling-up make SLNs viable alternative to liposomes for effective drug delivery. Galactosylation of SLNs instructs some gratifying characteristic, which leads to the evolution of promising delivery vehicles. The impendence of lectin receptors on different cell surfaces makes the galactosylated carriers admirable for targeted delivery of drugs to ameliorate their therapeutic index. Active participation of some lectin receptors in immune responses to antigen overlaid the application of galactosylated carriers in delivery of antigen and immunotherapy for treatment of maladies like cancer. These advantages revealed the promising potential of galactosylated carriers in each perspective of drug delivery. The developed DOX loaded galactosylated SLNs formulation was found to have particle size 239 ± 2.40 nm, PDI 0.307 ± 0.004, entrapment efficiency 72.3 ± 0.9%. Higher cellular uptake, cytotoxicity, and nuclear localization of galactosylated SLNs against A549 cells revealed higher efficiency of the formulation. In a nutshell, the galactosylation strategy with SLNs could be a promising approach in improving the delivery of DOX for cancer therapy.

Surface engineered polymeric nanocarriers mediate the delivery of transferrin–methotrexate conjugates for an improved understanding of brain cancer

The objective of present study was to enhance permeation of bioactive molecules across blood brain barrier (BBB) through polysorbate 80 coated poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with methotrexate-transferrin (Tw-Mtx-Tf-NP) conjugates (Mtx-Tf). The easy trans-BBB migration of developed formulations through endocytosis, and inhibition of P-gp efflux pump present in brain were established by Pluronic F-68 and/or polysorbate 80 (Tween 80/Tw). The over-expression of transferrin (Tf) receptors on cancer cell surface allowed targeted and sustained delivery of Mtx-Tf conjugated to brain cancer cells by receptor mediated endocytosis. The developed formulations showed improved penetration in comparison to non-targeting experimental NP controls. The transportation potential and bio-distribution studies of such nanosized polymeric carriers showing successful migration and trans-BBB passage was carried out by administering FITC labeled drug loaded NPs to albino rats through intravenous route. We have validated anti-tumor efficiency of newly formulated and drug loaded NPs compared to controls in experimentally induced tumor-harboring rat model. The present study suggests greater compatibility, less organ toxicity and higher anti-tumor activity of developed formulations due to their targeting and sustained delivery potential in cancer therapeutic interventions. In conclusion, our findings of targeted and sustained drug delivery potential of NPs for are corroborated with in vitro and in vivo evidence, and formulated novel delivery vehicle shows its value in developing new tools for treating brain cancer.

Mucosal Delivery of Vaccines: Role of Mucoadhesive/Biodegradable Polymers

Majority of infectious microorganism make their gateway to the host through mucosal surfaces, such as gastrointestinal tract, nasal and vaginal tract. Mucosal immune response structured as sIgA can effectively prevent the attachment and invasion of the microorganism from mucosal surface and thereby serves as an efficient tool against infectious disease. There has been an increased demand for the development of novel vaccine that leads to the induction of immune response in systemic circulation as well as at mucosal surfaces against infectious disease. Mucosal delivery of vaccine provides basis for induction of both mucosal as well as systemic immune responses against the infectious organisms. However, a variety of factors such as mucociliary clearance, presence of deteriorating enzymes, pH extremes (GIT), low permeation and metabolic degradation limit the mucosal delivery of vaccine. Numerous strategies have been explored in the meadow of mucosal vaccination for the purpose of efficient antigen delivery through mucosal route(s). Polymeric carrier(s) such as nanoparticles and microparticles loaded with the antigen can serve as the basis for creation of important formulations for improved vaccine. Biodegradable and mucoadhesive polymeric carrier(s) seems to be most promising candidate for mucosal vaccine delivery. Several polymers from natural and synthetic origin, such as polylactide-co-glycolide, chitosan, alginate, carbopol, gelatin etc., have been explored for the efficient mucosal vaccine delivery and significant results have been obtained. This review outlines the polymers used in mucosal vaccine delivery with special reference to mucoadhesive/biodegradable polymers. This article also covers the recent patent granted in the field on polymeric carrier mediated mucosal vaccination.

RNA pulsed dendritic cells: an approach for cancer immunotherapy.

The immunotherapy of cancer is aimed at evoking both branches of immune system to elicite specific immune responses directed against tumor antigens to deal with residual tumor cells upon interaction, and thereby decreases mortality as well as morbidity of cancer patients. As dendritic cells (DCs) are specialized for antigen presentation, and their immunogenicity leads to the induction of antigen specific immune responses, various immunotherapeutic approaches have been designed for using DCs to present tumor-associated antigens to T-lymphocytes. As a part of proposed strategy ex vivo generated DCs might be loaded with antigens and re-infused to the patients and/or they can be used for the ex vivo expansion of anti-tumor lymphocytes. The DCs loaded ex vivo with RNA can be safely administered which proves to be an asset for producing antigen specific immune responses. Furthermore, already conducted studies have prompted clinical trials to be designed to investigate immunological and clinical effects of RNA pulsed DCs administered as an engineered therapeutic vaccine in cancer patients. However, selection of the antigens of interest, methods for introducing TAAgs into MHC class I and II processing pathways, methods for isolation and activation of DCs, and route of administration are the parameters to be considered for designing and conducting clinical trials with engineered DCs. The enhanced RNA transfection efficiency would further improve antigen processing and presentation and T-cell co-stimulation, resulting in the induction of heightened anti-tumor immune responses. Therefore, RNA transfected dendritic cells continue to hold promise for cellular immunotherapy and opens new avenues to devising further strategies for cancer therapeutic interventions.

Analysis of innate defences against Plasmodium falciparum in immunodeficient mice

BackgroundMice with genetic deficiencies in adaptive immunity are used for the grafting of human cells or pathogens, to study human diseases, however, the innate immune responses to xenografts in these mice has received little attention. Using the NOD/SCID Plasmodium falciparum mouse model an analysis of innate defences responsible for the substantial control of P. falciparum which remains in such mice, was performed.MethodsNOD/SCID mice undergoing an immunomodulatory protocol that includes, clodronate-loaded liposomes to deplete macrophages and an anti-polymorphonuclear leukocytes antibody, were grafted with human red blood cells and P. falciparum. The systematic and kinetic analysis of the remaining innate immune responses included the number and phenotype of peripheral blood leukocytes as well as inflammatory cytokines/chemokines released in periphery. The innate responses towards the murine parasite Plasmodium yoelii were used as a control.ResultsResults show that 1) P. falciparum induces a strong inflammation characterized by an increase in circulating leukocytes and the release of inflammatory cytokines; 2) in contrast, the rodent parasite P. yoelii, induces a far more moderate inflammation; 3) human red blood cells and the anti-inflammatory agents employed induce low-grade inflammation; and 4) macrophages seem to bear the most critical function in controlling P. falciparum survival in those mice, whereas polymorphonuclear and NK cells have only a minor role.ConclusionsDespite the use of an immunomodulatory treatment, immunodeficient NOD/SCID mice are still able to mount substantial innate responses that seem to be correlated with parasite clearance. Those results bring new insights on the ability of innate immunity from immunodeficient mice to control xenografts of cells of human origin and human pathogens.

Nanostructured lipid carrier mediates effective delivery of methotrexate to induce apoptosis of rheumatoid arthritis via NF-κB and FOXO1.

Present study was designed to develop novel nano-structured lipid carriers (NLCs) formulated by lipid mixture and chemical permeation enhancer-based hydrogel for an effective transdermal delivery of methotrexate (MTX). The prepared NLCs were optimized with different preparative variables such as particle size <200 nm, poly-dispersity index (PDI) <0.2, and entrapment efficiency ∼85%. The drug incorporated into NLCs-gel base showed excellent spread ability without any grittiness during rheological behavior and texture profile analysis. The in vitro release showed biphasic release pattern with initial fast release of drug (>50%) in 8h followed by sustained release (up to 85%) by the end of 48thh. NLCs showed greater uptake in human hyper-proliferative keratinocyte cell line (HaCaT). NLCs showed increased expression of inflammatory mediators as well asapoptosis in U937 monocytic cells. The greater expression of pro-apoptotic gene Bim regulated by NF-κB-IkB and FOXO1 is supported by fold regulations calculated for various apoptotic and pro-inflammatory biomarkers carried out by RT-PCR. The immunocytochemistry to detect IL-6 expression and immunofluorescence assay suggested that induced apoptosis occurs in experimentally induced in vitro arthritis model treated with NLCs-MTX. We saw reduced inflammation and triggered apoptosis through NF-κB & FOXO1 pathways induced by MTX loaded NLCs in rheumatoid arthritic cells. In addition, formulated NLCs exhibit better skin permeation with higher permeation flux & enhancement ratio as shown by confocal laser scanning microscopy (CLSM). Moreover, histopathological examinations of skin are suggestive of safety potential of NLCs.

Fucose decorated solid-lipid nanocarriers mediate efficient delivery of methotrexate in breast cancer therapeutics.

The present study is designed to engineer fucose anchored methotrexate loaded solid lipid nanoparticles (SLNs) to target breast cancer. The developed nano-carriers were characterized with respect to particle size, PDI, zeta potential, drug loading and entrapment, in-vitro release etc. The characterized formulations were used to comparatively assess cellular uptake, cell-viability, apoptosis, lysosomal membrane permeability, bioavailability, biodistribution, changes in tumor volume and animal survival. The ex-vivo results showed greater cellular uptake and better cytotoxicity at lower IC50 of methotrexate in breast cancer cells. Further, we observed increased programmed cell death (apoptosis) with altered lysosomal membrane permeability and better rate of degradation of lysosomal membrane in-vitro. On the other hand, in-vivo evaluation showed maximum bioavailability and tumor targeting efficiency with minimum secondary drug distribution in various organs with formulated and anchored nano-carrier when compared with free drug. Moreover, sizeable reduction in tumor burden was estimated with fucose decorated SLNs as compared to that seen with free MTX and SLNs-MTX. Fucose decorated SLNs showed promising results to develop therapeutic interventions for breast cancer, and paved a way to explore this promising and novel nano-carrier which enables to address breast cancer.

Vaccination Strategies against Malaria: novel carrier(s) more than a tour de force

The introduction of vaccine technology has facilitated an unprecedented multi-antigen approach to develop an effective vaccine against complex systemic inflammatory pathogens such as Plasmodium spp. that cause severe malaria. The capacity of multi subunit DNA vaccine encoding different stage Plasmodium antigens to induce CD8(+) cytotoxic T lymphocytes and interferon-γ responses in mice, monkeys and humans has been observed. Moreover, genetic vaccination may be capable of eliciting both cell mediated and humoral immune responses. The cytotoxic T cell responses are categorically needed against intracellular hepatic stage and humoral response with antibodies targeted against antigens from all stages of malaria parasite life cycle. Therefore, the key to success for any DNA based vaccine is to design a vector able to serve as a safe and efficient delivery system. This has encouraged the development of non-viral DNA-mediated gene transfer techniques such as liposome, virosomes, microsphere and nanoparticles. Efficient and relatively safe DNA transfection using lipoplexes makes them an appealing alternative to be explored for gene delivery. Also, liposome-entrapped DNA has been shown to enhance the potency of DNA vaccines, possibly by facilitating uptake of the plasmid by antigen-presenting cells (APC). Another recent technology using cationic lipids has been deployed and has generated substantial interest in this approach to gene transfer. In this review we discussed various aspects that could be decisive in the formulation of efficient and stable carrier system(s) for the development of malaria vaccine.