Comparative assessment of efficacy and safety potential of multifarious lipid based Tacrolimus loaded nanoformulations.

The present work reports the development, optimization and characterization of novel lipid based nanoformulations viz., Liquid crystalline nanoparticles (LCNP), solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) and liposomes loaded with Tacrolimus (Tac) for topical delivery. Different nanoformulations were developed after screening lipids and suitable surfactants depending upon emulsification ability. The various nanoformulations were then optimized (to achieve higher entrapment efficacy, lower particle size, PDI and zeta potential), characterized and loaded into gel. The gels loaded with nanoformulations were also characterized depending upon rheology and viscosity. The gels were analyzed for in vitro drug release, HaCaT cell lines studies and skin permeation studies. The in vivo efficacy studies were carried out using mouse tail model and skin irritation studies using Draize patch test and measurement of TEWL. The developed nanoformulations showed optimum particle size (<200 nm), polydispersity index (PDI < 0.3), zeta potential (≥-10 mV) and higher entrapment efficiency (>85%). The nanoformulations showed higher penetration of Tac into skin. Tac-LCNP, Tac-SLN, Tac-NLC and Tac-liposomes loaded gels showed 14, 11.5, 12.5 and 3.7 folds increment in dermal bioavailability respectively, in comparison to free Tac loaded gel and 2.5, 2 and ∼2 folds augmentation in dermal bioavailability respectively as compared to Tacroz™ Forte. In case of Tac-liposomes, the dermal bioavailability was lower as compared with the marketed formulation, Tacroz™ Forte. Despite, the increased bioavailability into the skin, the developed nanoformulations showed no significant skin irritation. The above mentioned nanoformulations were able to achieve greater penetration of Tac into the skin as compared to marketed ointment of Tac, Tacroz™ Forte.

Immunostimulatory effect of tetanus toxoid loaded chitosan nanoparticles following microneedles assisted immunization.

The present study investigated potential of tetanus toxoid loaded chitosan nanoparticles (TT-Ch-NPs) following bare topical and microneedles assisted immunization. The TT-Ch-NPs were prepared by ionotropic gelation method using poly(sodium-4-styrene sulfonate) (PSS) as crosslinking agent which exhibited ~208 nm size and ~99% entrapment efficiency. The manufacturing process did not have any detrimental effect on integrity and conformation of antigen. The in vitro analysis demonstrated higher skin penetration following microneedles assisted immunization. In vivo immunization studies exhibited that TT-Ch-NPs delivered through microneedles induced comparable IgG and IgG1 titer, yet higher IgG2a titer than commercial TT vaccine. Similarly, microneedles assisted administration of TT-Ch-NPs generated higher Th1 cytokines, albeit no significant alteration in Th2 cytokines levels than commercial TT vaccine. In conclusion, microneedles assisted administration of TT-Ch-NPs especially via hollow microneedles (HMN) could be considered as best preferred route for immunization due to induction of more balanced Th1/Th2 biased immune response. From the Clinical Editor: The use of skin as a route for vaccination has been a clinically important topic for some time. In this article, the authors investigated the efficacy of both solid microneedles and hollow microneedles as methods for topical delivery of tetanus toixoid. The positive finding in the experiments could provide a better method for vaccination in the clinical setting in the future.

Trilateral '3P' Mechanics of Stabilized Layersomes Technology for Efficient Oral Immunization.

The main objective of the present work is to investigate the potential of polyelectrolyte stabilized layersomes as vaccine adjuvants for oral immunization. Herein, bovine serum albumin (BSA) loaded vesicular nanoformulations were prepared via thin film hydration method. The effect of various explanatory variables on responses were examined by a 6-factor, 3-level Box-Behnken design using JMP statistical software. An improved stability of vesicular nanoformulations in simulated biological media revealed that alternate layer-by-layer coating using polyacrylic acid and polyallylamine hydrochloride improved the robustness of vesicular structure. The formulation was lyophilized to improve long-term storage stability using trehalose as cryoprotectant. As evident from in process stability analysis, the chemicals, process and lyophilization conditions had no detrimental effect on the conformational stability and integrity of antigen. Mathematical modelling and DDsolver analysis confirmed that the release profile of vesicular nanoformulations altered from Baker-Lonsdale to Higuchi fashion, following integration of layer-by-layer coating. Confocal microscopy and spectrofluorometric quantification demonstrated a selective, dose and time dependent accumulation of nanoformulations in antigen presenting cells. As evident from in vivo immunization study, layersomes produced higher humoral (mean IgG in serum), mucosal (mean sIgA titre in biological fluids) and cellular immune response (IL-2 and IFN-y level) compared to free antigen. The observed effects might be attributed to improved protection, permeation and presentation of antigen using layersomes. In conclusion, polyelectrolyte stabilized vesicular nanoformulations contribute promising toolbox for mass immunization using human/veterinary vaccine or other protein/peptides delivery especially by oral route.

Downstream Processing, Formulation Development and Antithrombotic Evaluation of Microbial Nattokinase.

The present research work describes the downstreaming of nattokinase (NK) produced by Bacillus subtilis under solid state fermentation; and the role of efficient oral formulation of purified NK in the management of thrombotic disorders. Molecular weight of purified NK was estimated to be 28 kDa with specific activity of 504.4 FU/mg. Acid stable nattokinase loaded chitosan nanoparticles (sNLCN) were fabricated for oral delivery of this enzyme. Box-Behnken design (BBD) was employed to investigate and validate the effect of process (independent) variables on the quality attributes (dependent variables) of nanoparticles. The integrity, conformational stability and preservation of fibrinolytic activity of NK (in both free and sNLCN forms) were established by SDS-PAGE, CD analysis and in vitro clot lytic examination, respectively. A 'tail thrombosis model' demonstrated significant decrease in frequency of thrombosis in Wistar rats upon peroral administration of sNLCN in comparison with negative control and free NK group. Furthermore, coagulation analysis, namely the measurement of prothrombin and activated partial thromboplastin time illustrated that sNLCN showed significantly (p < 0.001) higher anti-thrombotic potential in comparison to the free NK. Further, sNLCN showed anti-thrombotic profile similar to warfarin. This study signifies the potential of sNLCN in oral delivery of NK for the management of thrombotic disorders.

Tetanus toxoid-loaded layer-by-layer nanoassemblies for efficient systemic, mucosal, and cellular immunostimulatory response following oral administration.

The present study reports the tetanus toxoid (TT)-loaded layer-by-layer nanoassemblies (layersomes) with enhanced protection, permeation, and presentation for comprehensive oral immunization. The stable and lyophilized TT-loaded layersomes were prepared by a thin-film hydration method followed by alternate layer-by-layer coating of an electrolyte. The developed system was assessed for in vitro stability of antigen and formulation, cellular uptake, ex vivo intestinal uptake, and immunostimulatory response using a suitable experimental protocol. Layersomes improved the stability in simulated biological media as well as protected the integrity/conformation and native 3D structure of TT as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorescence spectroscopy, respectively. The cell culture studies demonstrated a 3.8-fold higher permeation of layersomes in Caco-2 cells and an 8.5-fold higher uptake by antigen-presenting cells (RAW 264.7). The TT-loaded layersomes elicited a complete immunostimulatory profile consisting of higher systemic (serum IgG titer), mucosal (sIgA titer), and cellular (interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels) immune response after peroral administration in mice. The modified TT inhibition assay further confirmed the elicitation of complete protective levels of anti-TT antibody (>0.1 IU/mL) by layersomes. In conclusion, the proposed strategy is expected to contribute significantly in the field of stable liposome technology for mass immunization through the oral route.

Development of dual toxoid-loaded layersomes for complete immunostimulatory response following peroral administration.

AIM: Present study reports the development of divalent vaccine with enhanced protection, permeation and presentation following peroral immunization. MATERIALS & METHODS: Layersomes were prepared by layer-by-layer tuning of polyelectrolytes on liposomes template. The developed system was evaluated for in vitro stability of antigen and layersomes, cell-based assays and immunization experiments in mice. RESULTS: Layersomes exhibited enhanced stability in simulated biological fluids, still preserving the integrity, biological activity and conformational stability of toxoids. Layersomes also exhibited complete and protective (>0.1 IU/ml) immunostimulatory response include serum IgG titer, mucosal sIgA titer and cytokines (IL-2 and IFN-γ) levels following peroral administration. CONCLUSION: The positive findings of proposed strategy are expected to contribute significantly in the field of stable liposomes technology and peroral immunization.

Divalent toxoids loaded stable chitosan-glucomannan nanoassemblies for efficient systemic, mucosal and cellular immunostimulatory response following oral administration.

The present study reports dual tetanus and diphtheria toxoids loaded stable chitosan-glucomannan nanoassemblies (sCh-GM-NAs) formulated using tandem ionic gelation technique for oral mucosal immunization. The stable, lyophilized sCh-GM-NAs exhibited ~152 nm particle size and ~85% EE of both the toxoids. The lyophilized sCh-GM-NAs displayed excellent stability in biomimetic media and preserved chemical, conformation and biological stability of encapsulated toxoids. The higher intracellular APCs uptake of sCh-GM-NAs was concentration and time dependent which may be attributed to the receptor mediated endocytosis via mannose and glucose receptor. The higher Caco-2 uptake of sCh-GM-NAs was further confirmed by ex vivo intestinal uptake studies. The in vivo evaluation revealed that sCh-GM-NAs posed significantly (p<0.001) higher humoral, mucosal and cellular immune response than other counterparts by eliciting complete protective levels of anti-TT and anti-DT (~0.1 IU/mL) antibodies. Importantly, commercial 'Dual antigen' vaccine administered through oral or intramuscular route was unable to elicit all type of immune response. Conclusively, sCh-GM-NAs could be considered as promising vaccine adjuvant for oral mucosal immunization.

Tetanus toxoids loaded glucomannosylated chitosan based nanohoming vaccine adjuvant with improved oral stability and immunostimulatory response.

PURPOSE: The present report embarks on rational designing of stable and functionalized chitosan nanoparticles for oral mucosal immunization. METHODS: Stable glucomannosylated sCh-GM-NPs were prepared by tandem cross linking method followed by lyophilization. The in vitro stability of antigen and formulation, cellular uptake and immunostimulatory response were assessed by suitable experimental protocol. RESULTS: Stability testing ensured the chemical and conformation permanency of encapsulated TT as well as robustness of sCh-GM-NPs in simulated biological media. The antigen release from sCh-GM-NPs followed initial burst followed by controlled Weibull's type of release profile. The higher intracellular uptake of sCh-GM-NPs in Raw 264.7 and Caco-2 was concentration and time dependent which mainly attributed to Clathrin and receptor mediated endocytosis via mannose and glucose receptor. The in vivo evaluation in animals revealed that sCh-GM-NPs posed significantly (p < 0.001) higher humoral, mucosal and cellular immune response than other counterparts. More importantly, commercial TT vaccine administered through oral or intramuscular route was unable to elicit all type of immune response. CONCLUSION: The sCh-GM-NPs could be considered as promising vaccine adjuvant for oral tetanus immunization. Additionally, this technology expected to benefit the design and development of stable peroral formulation for administration of protein, peptides and variety of other antigens.

Solid lipid nanoparticles-loaded topical gel containing combination drugs: an approach to offset psoriasis.

AIM: The primary aim of present work was to develop effective combination drug therapy for topical treatment of psoriasis. METHODS: Betamethasone dipropionate and calcipotriol loaded solid lipid nanoparticles (CT-BD-SLNs) were prepared by hot melt high shear homogenization technique, which were then incorporated in Carbopol gel matrix. The anti-psoriatic potential was tested by sequential in vitro (skin permeation and dermal distribution, anti-proliferative effect in HaCaT cells) and in vivo (Draize patch irritation, transepidermal water loss (TEWL) and anti-psoriatic mouse tail studies) experiments. RESULTS: A negligible amount in receptor compartment, yet confined distribution of drugs to epidermal and dermal region of skin was observed in case of SLNs, which is essential for safe and effective anti-psoriatic therapy. Draize patch test and TEWL demonstrated negligible skin irritation and better skin tolerability of SLNs. The in vitro HaCaT cell line study demonstrated that SLNs delayed the abrupt growth of keratinocytes, while in vivo mouse tail model showed that SLNs gel significantly decreased the epidermal thickness and increased melanocyte count in comparison to commercial Daivobet® ointment. CONCLUSIONS: The developed SLNs gel is expected to be potential strategies for treatment of psoriasis and other topical diseases.

Oral mucosal immunization using glucomannosylated bilosomes.

The present study embarks on the feasibility of GM-bilosomes as a rationally designed vehicle for oral mucosal immunization. Bilosomes containing BSA as a model antigen were found to have vesicle size of 157 +/- 3 nm, PDI of 0.287 +/- 0.045, zeta potential of -21.8 +/- 2.01 mV and entrapment efficiency of 71.3 +/- 4.3%. Bilosomal formulations were freeze dried and entrapped BSA in freeze dried formulations was found to retain its structural and conformational stability as evident by SDS-PAGE and CD analysis. The GM-bilosomes were also found stable in different simulated biological fluids and bile salt solutions of different concentrations. In-vitro drug release revealed that GM-bilosomes were able to sustain drug release up to 24 h. In-vitro cell uptake in RAW 264.7 macrophage cells demonstrated significantly higher uptake of GM-bilosomes in comparison with bilosomes and free antigen. Intestinal uptake studies on excised rat intestinal sections further demonstrated higher uptake of vesicular systems throughout the intestinal region in comparison with free antigen. Significantly higher (p < 0.05) systemic immune response (serum IgG level) was observed in case of GM-bilosomes in comparison with bilosomes and alum adsorbed BSA (BSA-AL) following oral administration. The immune response observed in case of GM-bilosomes was comparable to BSA-AL administered through im route without any significant difference (p > 0.05). More importantly, GM-bilosomes were found capable of inducing mucosal immune response as well as cell mediated immune response which was not induced by im BSA-AL. In conclusion, GM-bilosomes could be considered as promising carrier and adjuvant system for oral mucosal immunization and productively exploited for oral delivery of other candidate antigens.

Development of stabilized glucomannosylated chitosan nanoparticles using tandem crosslinking method for oral vaccine delivery.

AIM: The aim of this study was to develop a novel platform technology, comprising of stable glucomannosylated chitosan nanoparticles, for oral immunization. MATERIALS & METHODS: Chitosan nanoparticles were stabilized by tandem crosslinking using tripolyphosphate followed by glutaraldehyde. Process and formulation variables were optimized using a 'Box-Behnken' design. The in vitro and in vivo performances were established in RAW 264.7 and BALB/c mice, respectively. RESULTS: The lyophilized formulation was exceptionally stable in simulated biological media and the enclosed antigen was conformationally stable. The mechanistic understanding of glucomannosylated chitosan nanoparticles in RAW 264.7 revealed transcellular uptake via both mannose and glucose transporter-mediated endocytosis. Glucomannan modification resulted in significantly higher systemic (serum IgG titer), mucosal (secretory IgA) and cell-mediated (IL-2 and IFN-γ) immune responses in comparison with nonmodified chitosan nanoparticles. CONCLUSION: The present strategy is expected to contribute some novel tools for the oral delivery of numerous biomacromolecules.

Improved stability and immunological potential of tetanus toxoid containing surface engineered bilosomes following oral administration.

The present study was designed with the objective to investigate the stability and potential of glucomannan-modified bilosomes (GM-bilosomes) in eliciting immune response following oral administration. GM-bilosomes exhibited desired quality attributes simultaneously maintaining the chemical and conformation stability of the tetanus toxoid (TT) entrapped in to freeze dried formulations. The GM-bilosomes exhibited excellent stability in different simulated biological fluids and sustained release profile up to 24 h. GM-bilosomes elicited significantly higher (P<0.05) systemic immune response (serum IgG level) as compared to bilosomes, niosomes and alum adsorbed TT administered through oral route. More importantly, GM-bilosomes were found capable of inducing mucosal immune response, i.e. sIgA titre in salivary and intestinal secretions as well as cell mediated immune response (IL-2 and IFN-γ levels in spleen homogenate) which was not induced by i.m. TT, the conventional route of immunization. Conclusively, GM-bilosomes could be considered as a promising carrier and adjuvant system for oral mucosal immunization. FROM THE CLINICAL EDITOR: This team reports on the development and effects of a glucomannan-modified bilosome as an oral vaccine vector, using tetanus toxoid in the experiments. These GM-bilosomes not only elicited significantly higher systemic immune response as compared to bilosomes, niosomes and alum adsorbed orally administered TT, but also demonstrated mucosal immune response induction as well as cell mediated immune responses, which were not induced by the conventional route of immunization.

Improved stability and antidiabetic potential of insulin containing folic acid functionalized polymer stabilized multilayered liposomes following oral administration.

The present study reports the folic acid (FA) functionalized insulin loaded stable liposomes with improved bioavailability following oral administration. Liposomes were stabilized by alternating coating of negatively charged poly(acrylic acid) (PAA) and positively charged poly(allyl amine) hydrochloride (PAH) over liposomes. Furthermore, folic acid was appended as targeting ligand by synthesizing folic acid-poly(allyl amine) hydrochloride conjugate. The insulin entrapped within the freeze-dried formulation was found stable both chemically as well as conformationally and developed formulation exhibited excellent stability in simulated biological fluids. Caco-2 cell and ex vivo intestinal uptake studies revealed higher uptake of folic acid functionalized layersomes in comparison with their plain counterparts. In vivo pharmacodynamic and pharmacokinetic studies further revealed almost double hypoglycemia and approximately 20% relative bioavailability in comparison with subcutaneously administered standard insulin solution. Overall the proposed strategy is expected to contribute significantly in the field of designing ligand-anchored, polyelectrolyte-based stable systems in drug delivery.

Solid lipid nanoparticles: an oral bioavailability enhancer vehicle.

INTRODUCTION: The therapeutic efficacy of perorally administered drugs is often obscured by their poor oral bioavailability (BA) and low metabolic stability in the gastrointestinal tract (GIT). Solid lipid nanoparticles (SLNs) have emerged as potential BA enhancer vehicles for various Class II, III and IV drug molecules. AREA COVERED: This review examines the recent advancements in SLN technology, with regards to oral drug delivery. The discussion critically examines the effect of various key constituents on SLN absorption and their applications in oral drug delivery. The relationship between the complexity of absorption (and various factors involved during absorption, including particle size), stability and the self-emulsifying ability of the lipids used has been explored. EXPERT OPINION: The protective effect of SLNs, coupled with their sustained/controlled release properties, prevents drugs/macromolecules from premature degradation and improves their stability in the GIT. An extensive literature survey reveals that direct peroral administration of SLNs improves the BA of drugs by 2- to 25-fold. Overall, the ease of large-scale production, avoidance of organic solvents and improvement of oral BA make SLNs a potential BA enhancer vehicle for various Class II, III and IV drugs.