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The main objective of this study was to prepare Hepatitis B surface antigen (HBsAg) loaded poly(lactic-co-glycolic acid) (PLGA), Trimethyl chitosan (TMC) as well as TMC-coated PLGA nanoparticles and compare their efficacy as nasal vaccine. The developed formulations were characterized for size, zeta potential, entrapment efficiency, mucin adsorption ability, Dentritic cells interaction, in vitro and in vivo studies. PLGA nanoparticles demonstrated negative zeta potential whereas TMC and PLGA–TMC nanoparticles showed higher positive zeta potential. Results indicated that TMC and PLGA–TMC nanoparticles demonstrated substantially higher mucin adsorption when compared to PLGA nanoparticles. The nanoparticles were nontoxic to isolated nasal epithelium. Immunogenicity increased as a function of particle size upon nasal administration. HBsAg encapsulated in PLGA–TMC particles elicited a significantly higher secretory (IgA) immune response compared to that encapsulated in PLGA and TMC particles. Similar to in vivo immune response data, fluorescent-labelled nanoparticles of smaller size are taken up more efficiently by rat alveolar macrophages compared to those of larger size. Results indicated that alum based HBsAg induced strong humoral but less mucosal immunity. However, PLGA–TMC nanoparticles induced stronger immune response at both of the fronts as compared to generated by PLGA or TMC nanoparticles. Present study demonstrates that PLGA–TMC nanoparticles with specific size range can be a better carrier adjuvant for nasal subunit vaccines.

Stable polymeric microspheres capable of controlled release of tetanus toxoid (TT) for periods ranging from days to over months were developed. TT was stabilized, encapsulated in microspheres prepared from poly(d,l)-lactide-co-glycolide (PLGA) and chitosan by using protein stabilizer (trehalose) and its immune response was compared. The influence of co-encapsulated protein stabilizer on tetanus toxoid’s stability and release from the microspheres was studied. The protein stabilizer (trehalose) prevented structural losses and aggregation of microencapsulated TT. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polymer, we also co-incorporated into the polymer an antacid, (Mg(OH)2), which neutralized the acidity during degradation of the polymer and also prevented TT structural losses and aggregation. The in vitro release experiments with PLGA and chitosan microspheres were performed and the release of TT was increased up to 80–90%. The antigen integrity was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie brilliant blue staining. The SDS-PAGE analysis confirmed that antigen integrity was not affected by the encapsulation procedure. In addition, the immunogenicity of PLGA and chitosan microspheres based single dose vaccine was evaluated in guinea pigs and compared with multiple doses of alum adsorbed TT. Results indicated that a single injection of PLGA and chitosan microspheres containing TT could maintain the antibody response at a level comparable to the booster injections of conventional alum adsorbed vaccines. The both PLGA and chitosan based stable vaccine formulations produced an equal immune response. Hence chitosan can be used to replace the expensive polymer PLGA. This approach should have potential application in the field of vaccine delivery. © 2005 Elsevier B.V. All rights reserved.

The purpose of this study was to develop a stable single-dose vaccine based on recombinant hepatitis B surface antigen (HBsAg) in poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres, in which HBsAg was stabilized by a protein stabilizer (trehalose) and an antacid (Mg(OH)2). The microspheres were prepared by the double emulsion method and characterized by scanning electron microscopy. To neutralize the acids liberated by the biodegradable lactic/glycolic acid based polymer, we co-incorporated into the polymer an antacid, Mg(OH)2, which neutralized the acidity during degradation of the polymer and also prevented HBsAg structural losses and aggregation. The antigen integrity after encapsulation was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining, isoelectric focusing and Western blotting techniques, which confirmed that antigen remained intact after encapsulation. In-vitro release experiments were performed in phosphate-buffered saline (pH 7.4) and the release of antigen was found to be improved by the protein stabilizer (trehalose). In stability studies, performed at 37°C, the microspheres were found to be stable for 16 days. The immunogenicity of stable microsphere formulations bearing HBsAg was compared with the conventional alum-absorbed HBsAg vaccine in a guinea-pig model. The antibody titre indicated that a single injection of stabilized HBsAg-PLGA microspheres produced a better immune response than two injections of alum-formulated HBsAg vaccine. The findings suggest that recombinant HBsAg can be stabilized by use of a protein stabilizer and antacid during entrapment, and this stabilized preparation can be useful for antigen delivery.

Controlled porosity osmotic pumps (CPOPs) are devoid of delivery orifice to release core contents and essentially possess pore-forming agent(s) in coating composition. When the pump comes in contact with aqueous media, pore-forming agent(s) generate pores through which core contents are delivered. Diltiazem hydrochloride (DLTZ) is a freely water-soluble drug and the release rates of DLTZ are higher from oral osmotic pumps including CPOPs, in which the drug release is controlled by concentration of pore-forming agents. The effect of appropriate concentration of hydroxypropyl methyl cellulose and sodium carboxy methyl cellulose mixture on the release of DLTZ from CPOPs was studied. In vitro drug release profiles were compared with that of different marketed controlled release formulations and statistically analysed to examine the suitability of CPOP for twice or once daily administration. Dissolution models were applied to drug release data in order to establish the mechanism of drug release and kinetics. Drug release from the CPOPs was effectively modified with the concentration of pore-forming agent in membrane and concentration of hydrophilic polymers in the core. CPOPs showed minimum 65% of consistent DLTZ release at 16 h. Statistical analysis confirmed that with an increase in the amount of hydrophilic polymers release rate decreased. Drug release from the systems follows Hixson-Crowell cube root model and mechanism of release follow non-Fickian diffusion.

Surface-modified DL-lactide/glycolide copolymer (PLGA) microspheres with chitosan (CS) were developed for nasal immunization using recombinant Hepatitis B (HBsAg) surface protein for the induction of humoral, cellular and mucosal immunity. Modified PLGA microspheres were characterized in vitro for their size, shape, entrapment efficiency and zeta potential. The nasal clearance rate was evaluated by gamma scintigraphy in rabbits. The antigen integrity, in vitro release and its stability at 37 degrees C were also evaluated. The designed cationic microspheres possessed 27.2 mV zeta potential and an average size less than 10 microm with antigen loading efficiency of 80+/-5%. However, zeta potential of unmodified PLGA microspheres was measured to be negative (-8.7 mV). The modified PLGA microspheres showed the lowest nasal clearance rate when compared with unmodified PLGA microspheres and lactose powder. The antigen integrity was retained intact in encapsulated form as well as on release. The immune-stimulating activity was studied by measuring anti-HBsAg titre, secretory IgA level in serum, vaginal, nasal and salivary secretions (mucosal secretions) and cytokine level (interleukin-2 (IL-2) and interferon-gamma (IFN-gamma)) in spleen homogenates following nasal administration of modified PLGA microspheres in Balb/c mice and compared with alum-HBsAg vaccine injected subcutaneously. The serum anti-HBsAg titre obtained after nasal administration of modified PLGA microspheres was comparable with titre recorded after alum-HBsAg was administered subcutaneously. Moreover, alum-HBsAg vaccine did not elicit sIgA in mucosal secretions as it was induced and measured in the case of nasal administration of modified PLGA microspheres. Similarly, there was no cellular response (cytokine level) in case of alum-HBsAg vaccine. Modified PLGA microspheres (cationic microspheres) thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. These data demonstrate high potential of modified PLGA microspheres for their use as a carrier adjuvant for nasal subunit vaccines.

The present study was aimed at preparation, characterization, and performance evaluation of amphotericin B (Amp B)-loaded aerosolized liposomes for their selective presentation to lungs (alveolar macrophages), that being the densest site of Aspergillosis infection. Egg phosphatidylcholine (PC)- and cholesterol (Chol)-based liposomes were modified by coating them with alveolar macrophage-specific ligands (O-palmitoyl mannan, OPM, and O-polmitoyl pullulan, OPP). The prepared formulations were characterized in vitro for vesicle morphology, mean vesicle size, vesicle size distribution and percent drug entrapment. Pressurized packed systems based on preformed liposomal formulations in chlorofluorocarbon aerosol propellants were prepared. In vitro airways penetration efficiency of the liposomal aerosols was determined by percent dose reaching the peripheral airways, it was recorded 1.4-1.6 times lower as compared to plain drug solution-based aerosol. In vivo tissue distribution studies on albino rats suggested the preferential accumulation of OPM- and OPP-coated formulations in the lung macrophages. Higher lung drug concentration was recorded in case of ligand-anchored liposomal aerosols as compared to plain drug solution and plain liposome-based aerosols. The drug was estimated in the lung in high concentration even after 24 h. The drug-localization index calculated after 6 h was nearly 1.42-, 4.47-, and 4.16-fold, respectively, for plain, OPM-, and OPP-coated liposomal aerosols as compared to plain drug solution-based aerosols. These results suggest that the ligand anchored liposomal aerosols are not only effective in rapid attainment of high-drug concentration in lungs with high population of alveolar macrophages but also maintain the same over prolonged period of time. The significance of targeting potential of the developed systems was established.

An oral osmotic system which can deliver theophylline and salbutamol sulphate simultaneously for extended period of time was developed and characterized in a view to reduce the problems associated with the multidrug therapy of asthma. Simple controlled porosity osmotic pump contained both drugs (in freely soluble form) did not provide satisfactory extended release of theophylline. A modified two-layered, push-pull osmotic system was developed by using the basic designs of various oral osmotic pumps, such as controlled porosity osmotic pump (CPOP), elementary osmotic pump (EOP) and push-pull osmotic pump (PPOP). Scanning electron microscopy of cellulose acetate coating membrane after dissolution revealed that 25% (w/w) of sorbitol can be used as an optimized concentration of pore forming agent with 25% (w/w) of plasticizer, which was kept constant. Formulations were initially developed for theophylline and the release was optimized by using two different soluble forms of theophylline with varying amount of hydrophilic polymer mixture in upper layer and polyethylene oxide (expandable hydrogel) in lower layer. Further, the release of salbutamol sulphate was optimized by keeping the drug in upper or lower layer or both layers. In vitro release studies showed satisfactory controlled release profiles of both drugs. The release profiles of both drug statistically compared with respective marketed controlled release formulations. An optimized system was selected to study the effect of concentration of pore forming agent and orifice diameter on the release of both drugs.