BCA Reagent was prepared by dissolving about 10 g of bicinchoninic acid, 20 g of sodium carbonate monohydrate, 1.6 g of sodium tartrate, 4 g of sodium hydroxide, and 9.5 g of sodium bicarbonate in water. pH was adjusted with sodium hydroxide to a 11.25. Ultimately volume was made with water to 1 liter.
Copper Sulfate Reagent was prepared by dissolving about 2 g of cupric sulfate in water to a final volume of 50 mL.
Standard Working Reagent (SWR) was prepared by mixing 1 mL of Copper Sulfate Reagent and 50 mL of BCA Reagent.
Preparation of Standard Curve
Various standard dilution of BSA were prepared & 0.4 mL of each Standard Solution was added to 4 mL of the standard working reagent (SWR) , Incubated at 37°C for 30 minutes and were allowed to come to room temperature. The absorbances of standard solutions were made at 562 nm, with Shimadzu UV 1800 spectrophotometer. HBsAg content of bulk antigen was estimated employing standard curve of BSA.
4.1.3. Secondary Structure Investigations by Fourier Transform Infrared Spectroscopy [FTIR]
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Secondary structure of bulk HBsAg was established by performing FTIR.
4.1.4. RP HPLC Analysis of Bulk Antigen (Rajkannan R et al., 2006)
RP HPLC was carried out on a Gracesmart C18 5 micron (250 mm x 4.6mm i.d.) column with mobile phase consisting of 5 mmol potassium dihydrogen phosphate: Methanol (50: 50 v/v) and pH was unadjusted. The flow rate was kept 1.0 mL/min. Detection was carried out at 280 nm.
4.1.5. Evaluation of antigen stability at different temperature
Stability of bulk HBsAg was examined at 4oC & 37oC (RH 65Â± 5%) over a period of one month by RP HPLC technique. Percentage of recovered antigen was determined from relative peak area.
4.1.6. Evaluation of antigen stability at different pH
HBsAg stability was evaluated at pH 4, 6 & 7.4 by RP HPLC. Percentage of recovered antigen was estimated.
4.2. Characterization of polymers by FTIR
FTIR spectroscopy was performed to characterize PLGA, PCL & PLA
4.3. Optimization of Microparticle Preparation Process
The preparation process was optimized initially to produce desired size microparticles of various polymers and blend polymers, suitable for vaccine design. Two kind of microparticles were prepared
Small microparticles (8-12 Âµm) either made of low molecular weight polymer (like PLGA) /or blend PLGA-PCL microparticles/or porous microparticles of PCL suitable for priming of immune response.
Large size microparticles (12-20 Âµm) made of high molecular weight polymer like PLA/or PCL/or PLA-PCL blend. The role of large particles was to sustain release of HBsAg, once priming has been accomplished by smaller particles.
Bovine Serum Albumin (BSA) was used as model antigen for all optimization studies conducted. Particles were prepared by modified reported method (Raghuvanshi et al., 2001). All the particles were produced by double emulsion solvent evaporation process. Briefly 3ml of dichloromethane was taken and desired polymer (1 to 5% w/v) was dissolved in it (Organic Phase, OP). 1 ml of antigen (2 mg/ml, BSA) in PBS (Internal Aqueous Phase, IAP), was added to above solution and was emulsified by sonication (time was varied from 1 to 10 sec for preparing different batches). Resulting primary emulsion (W/O) was added drop wise to 25 ml PVA solution ( 1 to 5 % w/v ) in distilled water (External Aqueous Phase, EAP) under vigorous stirring to get W/O/W emulsion. The stirring was continued for 1 hour & after that the multiple emulsions was transferred on to a magnetic stirrer & was kept for overnight stirring at room temperature to evaporate methylene chloride. The resulting microparticles were collected by centrifugation at 8000 rpm for 15 minutes, washed thrice with distilled water & dried.
In these studies, the effects of the following formulation variables on microparticle size were investigated:
1. Effect of three different polymer concentrations (1, 3, and 5 wt %) in a fixed volume of DCM on particle size (all the other process parameters were kept constant).
2. Once polymer concentration was optimized, three different emulsion stabilizer (PVA) concentrations (1, 3, and 5 wt %) in the external aqueous phase (25 ml) were investigated to observe their effect on particle size, keeping the other parameters constant.
3. Duration of sonication during primary emulsifications (1, 5 & 10 sec) on the particle size of microparticles.
4. At a constant protein/polymer ratio, effect of organic phase volume (0.5, 2 0r 3ml) on final microparticle size was established.
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5. At a constant protein/polymer ratio, effect of internal aqueous phase volume (0.5, 2 0r 3ml) on final microparticle size was ascertained.
4.2. Microencapsulation of Antigen (HBsAg) in Various Polymeric Microparticles
Various polymeric microparticles were prepared using w/o/w solvent evaporation method employing optimized formulas.
HBsAg loaded PLGA particles were fabricated at 3 % w/v polymer concentration & 5% w/v PVA concentration. IAP/OP ratio taken was 1:6 and sonication was performed for 10 sec. Similarly PLGA-PCL blend microparticles & PLGA microparticles encapsulating alum adsorbed HBsAg were also prepared under similar conditions.
HBsAg loaded PLA, blend PCL PLA & PLA alum microparticles were fabricated at 5 % w/v polymer concentration while keeping PVA concentration of 5% w/v. IAP/OP ratio taken was 1:6 and sonication was performed for 10 sec.
PCL particle were fabricated at 4 5 % w/v polymer concentration and other preparation was similar to PLA particle fabrication process. Porous PCL microparticles were prepared at 1: 3 IAP/OP ratio and employing 1 % w/v polymer concentration.
4.2.1. PREPARATION OF PLGA/PCL & PLA/PCL COMPOSITE MICROSPHERES
Composite polymeric microspheres were prepared by water in oil in water (w/o/w) multiple emulsion solvent evaporation method by taking PLGA: PCL (1:1) & PLA/PCL (1:1) respectively in organic phase.
4.3. Characterization of Prepared Microspheres
4.3.1. Determination of Microparticle Size & Surface Morphology
The size of the HBsAg containing particles was measured using particle size analyzer. Small quantity of microparticles was dispersed in phosphate buffer saline containing tween and analyzed for size distribution. The size measurements in optimization studies were carried out using optical microscopy.
Surface morphology of prepared microsphere was investigated using Analytical Scanning Electron Microscope (JEOL JSM 6390A, Japan). Particle diameter was measured from the SEM image using image JÂ® software.
4.3.2. Determination of Antigen Loading Level in the Microparticles
Total loaded protein was predicted using the BCA assay after disruption of the microspheres with 0.2M NaOH/sodium dodecyl sulfate (SDS) solution. Three 5-mL glass vials, each containing 20-mg samples of dry microparticles were taken & 1 mL of 0.2N NaOH/1% sodium dodecyl sulfate (SDS) solution was added to each vial and the vials were placed on a constant shaker at 370C temperature. Parallel samples of blank microparticles (without antigen) were also kept in separate vials to serve as negative controls. 400 Î¼L of the hydrolyzed samples were taken in glass tubes for protein estimation. The BCA reagents were added to all tubes and incubated at 60Â°C for 30 min. The absorbance was measured at Î»max 560 nm. The protein concentration in the microparticle samples were calculated using the BSA standard curve.
The protein loading was calculated as the percent weight of protein per unit weight of polymer. Theoretical loading was calculated as amount of total protein taken for encapsulation per unit weight of polymer. Practical loading is the total protein encapsulated, as determined by protein estimation in unit weight of polymer.
Encapsulation efficiency, defined as the percent of total protein recovered in final particles.
Encapsulation Efficiency = X 100
4.3.3. In Vitro Release of Antigens from the Microparticles
Several vials including 20 mg of microparticles in 2 mL of PBS (pH 7.4) were incubated at 37Â°C on a constant shaking mixer. One vial was withdrawn at day 0, 7, 14, 21, 28, 35 & 42 of the in vitro estimation. The microparticle suspension was centrifuged at 6000g for 15 min and the supernatant was collected. The supernatant was used for protein estimation at various time-points in the release kinetics. Protein release profiles were generated for each type microsphere formulation in terms of cumulative protein release versus time.
4.4. Protein Estimation By BCA Assay
Protein estimation was done using BCA assay. Standard curve was made in the range of 10 Âµg/ml to 100 Âµg/ml of BSA solution and concentration of the unknown sample was calculated with the help of second order equation.
4.5. Immunization Studies
Immunization studies were carried out in BALB/c mice. 40 animals were divided into five groups. Eight animals were taken in each group. Animals were maintained according to the guidelines established by the Institute Animal Ethics Committee of the Bhopal Institute of Technology & Science-Pharmacy, Bhopal. Group I was injected with vaccine formulation 1 (Alum+PLGA+PLA), Group II was injected with vaccine formulation 2 (Alum+PCL(P)+PCL); Group III was injected with vaccine formulation 3 (Alum+PLGA/PCL+PLA/PCL); Group IV was injected with vaccine formulation 4 [ Alum+ PLGA(Al-Hb)+PLA(Al-Hb) ] and Group V was injected with vaccine formulation 5 (Alum Adsorbed Antigen). Before immunization all the formulations (except formulation 5) were reconstituted with alum adsorbed vaccine. Animals were bled at different time interval through retro-orbital plexus and serum anti-HBsAg antibody titers were determined by ELISA. Antibody titers of individual animal (n = 8) were estimated in duplicates and their concentrations (Log10) were presented as geometric mean with standard deviations.
4.5.1. ELISA Protocol
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Anti-HBsAg IgG antibodies in mice sera were estimated as reported earlier. Briefly 10 Âµg of HBsAg in 100 Âµl of PB (50mM, pH 7.4) was coated in each well of 96 well immunoplates. The plates were then washed with PBS-T (1 % v/v tween 20 in PBS) and blocked by adding 300 Âµl solution of blocking buffer to each well (1% bovine serum albumin in PBS-T) and incubating for one hour at 37ËšC. Different dilutions of mice serum in blocking buffer were prepared and 100 Âµl of each solution in duplicates were added into the wells and incubated for 1 h at 37Â°C. After washing thrice with PBS-T, 100 Âµl of goat-anti-mice-HRPO conjugate diluted in PBS-T was added into each well and incubated for 1 h at 37Â°C. Finally, 100 Âµl of O-phenyl diamine (OPD) in citrate-phosphate buffer (pH 4.5) together with hydrogen peroxide was added to each well and incubated for 20 minutes at room temperature. Reaction was stopped by adding 5N H2SO4 (50 Âµl/well) and the absorbance was measured at 492 nm.
4.5.2. Evaluation of Memory Response
For the evaluation of memory response, different groups of mice were administered with 2Âµg soluble HBsAg intramuscularly, 6 months after primary immunization with polymeric microparticles. After this injection, blood samples were collected through retro orbital plexus at day 15, 30 and 60 and serum anti HBsAg IgG concentrations were estimated by ELISA.
4.6. Evaluation of Thermostability of Vaccine Formulations
Vaccine formulation 1, 2, 3 & 5 were exposed to 370C temperature for one week & & thereafter HBsAg was extracted from microparticles by incubating formulations 1, 2 & 3 in PBS with vigorous stirring (800 rpm) for one day. Formulations were centrifuged and supernatants were scrutinized for changes in secondary structure of released HBsAg in amide I & amide II region by FTIR spectroscopy. The changes were compared with alum based vaccine (vaccine Formulation 5).
4.7. Evaluation of Freeze Thaw Stability of Vaccine Formulations
Vaccine formulation 1, 2, 3 & 5 were subjected to 2 cycle of freeze thawing and & thereafter HBsAg was removed from microparticles by incubating formulations 1, 2 & 3 in PBS with vigorous stirring (800 rpm) for one day. Formulations were centrifuged and supernatants were analyzed for the changes in amide I & amide II region of released HBsAg by FTIR spectroscopy. The FTIR spectra were compared with vaccine Formulation 5.
Evaluation of Toxicity & Hypersensitivity
This was done by observing site of injection & performing histopathology.
In vitro biodegradation
The biodegradation test was carried out at 37Â±1Â°C. 10 mg of the microparticles were incubated in pH 7.4 PBS medium. The tube was shaken at customary intervals. On day 14 & 28, the samples were subjected to centrifugation and microparticles were separated from the buffer & desiccated in vacuum at room temperature. The morphology of the samples was studied by scanning electron microscopy (SEM) (Jeol JSM-6400 Electron Microscope).
pH measurements were done by incubating 10 mg microparticles in 2 ml of PBS (pH 7.4) in eppendorf tubes. On the day 7, 14 & 28, sample was centrifuged, the supernatant was collected pH was measured using pH indicator strips.