Amit K. Goyal

M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B

The transcytotic capability and expression of distinct carbohydrate receptors on the intestinal M-cells render it a potential portal for the targeted oral vaccine delivery. PLGA nanoparticles loaded with HBsAg were developed and antigen was stabilized by co-encapsulation of trehalose and Mg(OH)2. Additionally, Ulex europaeus 1 (UEA-1) lectin was anchored to the nanoparticles to target them to M-cells of the peyes patches. The developed systems was characterized for shape, size, polydispersity index and loading efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitro determination of lectin activity and specificity. The targeting potential of the lectinized nanoparticles were determined by Confocal Laser Scanning Microscopy (CLSM) using dual staining technique. The immune stimulating potential was determined by measuring the anti-HBsAg titre in the serum of Balb/c mice orally immunized with various lectinized formulations and immune response was compared with the alum-HBsAg given intramuscularly. Induction of the mucosal immunity was assessed by estimating secretary IgA (sIgA) level in the salivary, intestinal and vaginal secretion. Additionally, cytokines (interleukin-2; IL-2 and interferon-γ; IFN-γ) level in the spleen homogenates was also determined. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of protein stabilizers. The lectinized nanoparticles have demonstrated approximately 4-fold increase in the degree of interaction with the BSM as compared to plain nanoparticles and sugar specificity of the lectinized nanoparticles was also maintained. CLSM showed that lectinized nanoparticles were predominantly associated to M-cells. The serum anti-HBsAg titre obtained after oral immunization with HBsAg loaded stabilized lectinized nanoparticles was comparable with the titre recorded after alum-HBsAg given intramuscularly. The stabilized UEA-1 coupled nanopartilces exhibited enhanced immune response as compared to stabilized non-lectinized nanoparticles. Furthermore, the stabilized lectinized nanoparticles elicited sIgA in the mucosal secretion and IL-2 and IFN-γ in the spleen homogenates. These stabilized lectinized nanoparticles could be a promising carrier-adjuvant for the targeted oral-mucosal immunization.

Effect of lipid core material on characteristics of solid lipid nanoparticles designed for oral lymphatic delivery.

Solid lipid nanoparticles (SLNs) are essentially composed of triglyceride(s) that orient to form a polar core with polar heads oriented toward the aqueous phase, resembling chylomicrons. The composition of such SLNs may alter the course of drug absorption predominantly to and through lymphatic route and regions, presumably following a transcellular path of lipid absorption, especially by enterocytes and polar epithelial cells of the intestine. SLNs were prepared using stearic acid, glycerol monostearate, tristearin, and Compritol 888 ATO by solvent diffusion method using demineralized double-distilled water as the dispersion medium. The SLNs were characterized for shape, size, zeta potential, and percentage drug content and its release. The characterization of SLNs suggests that Compritol 888 ATO-based nanoparticles were heterogeneous with better drug-loading and release characteristics as compared with the other formulations. The selected products were studied for in vivo absorption and hence bioavailability by measure of area under the blood plasma curve plotted as a function of time. Periodic lymphatic concentration of drug following oral administration of respective formulations was also determined by mesenteric duct cannulation and collection of samples. The comparative study conducted on methotrexate (MTX)-bearing SLNs revealed that the formulation based on Compritol 888 ATO could noticeably improve the oral bioavailability of MTX, presumably following SLNs constituting lipid digestion and co-absorption through lymphatic transport and route.

Microfold-cell targeted surface engineered polymeric nanoparticles for oral immunization

Present work was envisaged to develop novel M-cell targeted polymeric particles that are capable of protecting the antigen from harsh gastric conditions. Ulex europaeus agglutinin (UEA-1) lectin was anchored for selective delivery of antigen to gut-associated lymphoid tissue (GALT). In the present investigation, chitosan nanoparticles were prepared by ionic gelation followed by antigen (bovine serum albumin, BSA) adsorption. Developed nanoparticles were further coated by UEA-1 lectin conjugated alginate and characterized for size, shape, zeta-potential, entrapment efficiency, and in vitro release. The immunological response of the developed system were performed in Balb/c mice and compared with aluminium hydroxide gel-based conventional vaccine. Results indicated that immunization with UEA-1 lectin conjugated alginate-coated particles induces efficient systemic as well as mucosal immune responses against BSA compared to other formulations. Aluminium-based vaccine dominated throughout the study, while failed in case of mucosal antibody. Additionally, IgG1 and IgG2a isotypes were determined to confirm the TH1/TH2 mixed immune response. The developed formulation exhibited superior systemic response along with dominating mucosal immunity. These data demonstrate the potential of UEA-alginate-coated nanoparticles as effective delivery system via oral route.

Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.

Biomaterial-based scaffolds – current status and future directions

Introduction: Biomaterial-based scaffold formulations (three-dimensional Porous matrix, nano-fibre mesh, hydrogels and microspheres) are the major components that are used to deliver the bioactive molecules into the body organs through different routes for an effective treatment of various diseases. Areas covered: Various fabrication techniques such as freeze-drying, polymerisation, spray drying, gas foaming, supercritical fluid technology, etc., are successfully used for fabrication of scaffold formulations. Due to their unique characteristics, these formulations are widely used against various diseases such as tuberculosis, bone defects, cartilage repair, skin diseases, cardiovascular diseases, periodontal diseases, wound dressing, etc. Expert opinion: The study of biomaterial-based scaffold formulations is exhilarating with novel approaches to drug/cell/gene delivery being developed all the time. At present, there is a huge extent of research being performed worldwide on all aspects of tissue engineering/drug or gene delivery. In the future, the main focus will be on the development of more patient compliant, sustained and controlled delivery systems against various diseases by modification of polymers, manufacturing technologies as well as carrier systems.

Surface modified liposomes for nasal delivery of DNA vaccine

The aim of the present work was to investigate the potential utility of glycol chitosan coated liposomes as nasal vaccine delivery vehicle for eliciting viral specific humoral mucosal and cellular immune responses. Plasmid pRc/CMV-HBs(S) encapsulated liposomes were prepared by dehydration-rehydration method and subsequently coated with glycol chitosan by simple incubation method. Liposomes were then characterized for their size, surface charge, entrapment efficiency, and ability to protect encapsulated DNA against nuclease digestion and for their mucoadhesiveness. The liposomes were then administered to mice in order to study their feasibility as nasal vaccine carriers. The developed liposomes possessed +9.8 mV zeta potential and an average vesicle size less than 1 microm and entrapment efficiency of approximately 53%. Following intranasal administration, glycol chitosan coated liposomes elicited humoral mucosal and cellular immune responses that were significant as compared to naked DNA justifying the potential advantage of mucosal vaccination in the production of local antibodies at the sites where pathogens enters the body.

Synthesis, characterization and evaluation of novel triblock copolymer based nanoparticles for vaccine delivery against hepatitis B

Poly lactic acid (PLA) is one of widely used biodegradable polymer in vaccine delivery. However, the use is restricted due to hydrophobic nature and generation of acidic microenvironment upon its degradation, rendering it unfavorable to the encapsulated antigen. In the present study we have synthesized PEG derivatized block copolymers of PLA for development of nanoparticles encapsulating HBsAg for mucosal vaccination against hepatitis B. The copolymers of compositions AB, ABA and BAB (PLA as A-block and PEG as B-block) were synthesized and characterized by 1H NMR spectroscopy and gel permeation chromatography. Nanoparticles were characterized to determine the effect of copolymer. Among all, BAB produced nanoparticles of smallest size and lowest zeta potential, suggesting highest PEG density on their surface. The in vitro release experiments were performed in PBS (pH7.4). SDS-PAGE analysis confirmed the structural stability and integrity of the released antigen. Results were compared for immunogenicity with plain PLA nanoparticles and conventional alum-HBsAg based vaccine. BAB nanoparticles produced better humoral response as compared to other polymeric nanoparticles. The extent of humoral response obtained in single dose of BAB nanoparticles was comparable to the response produced by alum based vaccine (which received a booster dose). Block copolymeric nanoparticles also produced better sIgA level at all local and distal mucosal sites as compare of PLA nanoparticles, where alum based formulation failed to give any considerable response. Additionally, IgG1 and IgG2a isotype were determined to confirm the T(H)1/T(H)2 mixed immune response. These data demonstrate the potential of BAB nanoparticles as mucosal vaccine delivery system capable of eliciting high and prolonged immune response.

Development and characterization of nano-fiber patch for the treatment of glaucoma.

In the present work polymeric nano-fiber patches was developed for the effective treatment of glaucoma using timolol maleate and dorzolamide hydrochloride as model drugs. The nano-fibers were prepared by electrospinning technique and were characterized on the basis of fiber diameter, morphology, entrapment efficiency, mucoadhesive strength, and drug release behavior, etc. Final formulations were inserted in the cul-de-sac of glaucoma induced rabbits and the efficacy of the formulation was evaluated. The results clearly indicated the potential of the developed formulation for occur drug delivery. There was a significant fall in the intraocular pressure compared to commercial eye drops.

Evaluation of mucoadhesive PLGA microparticles for nasal immunization.

In this study, hepatitis B surface antigen (HBsAg) loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were prepared and coated with chitosan and trimethyl chitosan (TMC) to evaluate the effect of coating material for nasal vaccine delivery. The developed formulations were characterized for size, zeta potential, entrapment efficiency, and mucin adsorption ability. Plain PLGA microparticles demonstrated negative zeta potential. However, coated microparticles showed higher positive zeta potential. Results indicated that TMC microparticles demonstrated substantially higher mucin adsorption when compared to chitosan-coated microparticles and plain PLGA microparticles. The coated and uncoated microparticles showed deposition in nasal-associated lymphoid tissue under fluorescence microscopy. The coated and uncoated microparticles were then administered intranasally to mice. Immune-adjuvant effect was determined on the basis of specific antibody titer observed in serum and secretions using enzyme-linked immunosorbent assay. It was observed that coated particles showed a markedly increased anti-HBsAg titer as compared to plain PLGA microparticles, but the results were more pronounced with the TMC-coated PLGA microparticles.

Oral immunization against hepatitis B using bile salt stabilized vesicles (bilosomes)

PURPOSE Most of the presently available vaccines including hepatitis B vaccines administered through parenteral route, fail to induce a mucosal antibody response. Therefore, oral immunization appears to be an effective and attractive alternative to parenteral immunization. However, the problem of degradation of antigen in the harsh and hostile environment of the gastrointestinal tract consequently requires larger doses and more frequent dosing of antigen. Furthermore, much larger doses can induce antigen tolerance. Therefore the purpose of the present study was firstly to overcome these problems by the use of bile salt stabilized vesicles (bilosomes) and HBsAg as the model antigen,which could provide both protection to the antigen as well as enable transmucosal uptake and subsequent immunization. Another purpose of this study was to determine the dose that could produce serum antibody titres against hepatitis B via the oral route compared to those following intramuscular immunization. METHODS In the present study bilosomes containing recombinant hepatitis B surface antigen were prepared by a lipid cast film method. HBsAg loaded bilosomeswere characterized in vitro for their shape, size, percent antigen entrapment and stability. Fluorescence microscopy was carried out to confirm the uptake of bilosomes by gut associated lymphoid tissues (GALT). The in vivo part of the study comprised estimation of anti-HBsAg IgG response in serum and anti-HBsAg sIgA in various body secretions using specific ELISA techniques following oral immunization with low dose loaded bilosomes (B1, 10 microg), intermediate dose loaded bilosomes (B2, 20 microg) and high dose loaded bilosomes (B3, 50 microg) in BALB/c mice. RESULTS Fluorescence microscopy suggested that there was an increase in fluorescence intensity following the uptake of bilosomes entrapped FITC-BSA in gut associated lymphoid tissues. The high dose HBsAg bilosomes (B3, 50 microg) produced comparable anti-HBsAg IgG levels in serum to those observed in the case of intramuscular administration of alum adsorbed HBsAg (10 microg). In addition, the bilosomal preparations elicited measurable sIgA in mucosal secretions, where the highest responses were observed with high dose HBsAg bilosomes (B3,50 microg) and as expected, intramuscular administered alum adsorbed HBsAg (10 microg) failed to elicit such responses. CONCLUSIONS HBsAg loaded bilosomes produced both systemic as well as mucosal antibody responses upon oral administration. Furthermore, bilosomes with a five times higher dose upon oral administration produced comparable serum antibody titres to those obtained after intramuscular immunization without the induction of systemic tolerance.