www.ietdl.org Published in IET Nanobiotechnology Received on 15th May 2013 Revised on 7th October 2013 Accepted on 9th October 2013 doi: 10.1049/iet-nbt.2013.0035
ISSN 1751-8741
Poly(vinyl alcohol)-coated chitosan microparticles act as an effective oral vaccine delivery system for hepatitis B vaccine in rat model Bijaya Shrestha1, Jyoti Prakash Rath1,2 1
Department of Medical Biotechnology, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Chennai, Tamil Nadu 603103, India 2 Department of Chemistry, Gangadhar Meher College, Sambalpur 768004, Odisha, India E-mail: [email protected]
Abstract: The present study focused on the development of an effective oral vaccine delivery system of poly(vinyl alcohol)coated chitosan microparticles-based recombinant hepatitis B vaccine. Chitosan microparticles were prepared by ionotropic gelation technique; they were loaded with recombinant hepatitis B vaccine and coated with poly(vinyl alcohol). The average sizes of the microparticles were measured in the range of 100–410 nm. The optimal loading capacity and loading efficiency were recorded around 3.4 and 74%, respectively. In vitro release study shows that the prepared microparticles release the antigen in a sustained manner. Moreover, the microparticles were resistant to simulated gastric environment and release the antigen in the targeted intestinal milieu. Furthermore, oral immunisation of rats with poly(vinyl alcohol)-coated chitosan hepatitis-B microparticles vaccine shows comparable seroprotective immune response to presently practiced intramuscular vaccination. The results demonstrated that poly(vinyl alcohol)-coated chitosan microparticles have the potential for being used as an oral vaccine delivery system for hepatitis B vaccine and may be a suitable alternative for needle-based vaccination.
1
Introduction
Hepatitis B virus (HBV) infection still remains a global health issue. In its recent bulletin, the World Health Organization has reported about 2 billion infected individuals with the HBV infection [1–3]. Although vaccination is persuaded as the preventive measure against hepatitis B infection and hepatitis B surface antigen-based vaccine is recognised to work effectively, but the potential of vaccination is yet to be fully realised in third world countries [1]. Lack of patient compliance is cited to be the prime reason for low vaccination coverage. Presently practiced lengthy dosing schedule spreading through 6 months is stated to be the main reason for poor compliance rate especially in third world countries [4]. In addition, poor compliance rate is largely influenced by not so user-friendly needle-based vaccination strategy. Moreover, needle stick injury adds to the menace. Approximately 30% of injections for the purpose of vaccination in developing nations are unsafe. There is always a high risk of transmission of blood borne pathogens and it also adds to the waste disposal problem. Hence, there is an urgent need for development of an improved vaccination strategy against hepatitis B. The HBV surface envelope antigen was demonstrated to elicit protective immune response and validated as a candidate antigen (referred to as HBsAg) for successful hepatitis B vaccine development. Alum is primarily used as IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 201–207 doi: 10.1049/iet-nbt.2013.0035
an adjuvant for presently used vaccine formulation. Alum is preferred for its safety, low cost and adjuvanticity for a wide range of antigens. However, the associated limitations, including local reaction and inability to augment cell-mediated immune response are of major concern. In addition, the antibodies generated with alum-adsorbed vaccine fail to reach the mucosal surfaces, the key entry site for HBV. To overcome the issues of conventional vaccine an urgent appeal is to develop a novel vaccine that could facilitate cell-mediated and humoral immunity (both serum and mucosal) and confer complete protection against the pathogen. Oral vaccination is rapidly gaining prominence because of its non-invasive and user-friendly approach that does not require sterile injection by qualified personnel [3]. Moreover, oral vaccination elicits systemic and mucosal immune responses and with the help of an appropriate carrier system, could augment cell-mediated immunity. Nevertheless, the unfriendly environment of the gut, poor transcytosis of the antigen via mucosal epithelium and development of immune tolerance pose a significant challenge for oral vaccination [4, 5]. Nanotechnology could address some of these issues satisfactorily. Among the available options for oral vaccine delivery, the nanocarrierbased delivery systems offer several advantages, including protection of the antigen during transit through mucosal surfaces, and facilitation of antigen uptake by M cells of the mucosal epithelial layer. These delivery systems are easy to 201
& The Institution of Engineering and Technology 2014
www.ietdl.org prepare and characterise, are biocompatible and biodegradable in nature and stabilise the antigen against degradation by gastrointestinal tract (GIT) contents [4–6]. Nanocarriers, which include lipid-based (e.g. liposomes, biolosomes, niosomes and virosomes) and polymeric nanoparticles, are the most sought after carrier adjuvants for oral mucosal vaccine delivery. Their size of a few nanometres mimics the size of pathogenic organisms; therefore they are naturally taken up by M cells and undergo transcytosis to underlying lymphoid tissues. The carrier constructs present several epitopes of the same antigen in a single carrier, which helps in their effective recognition by antigen presenting cell (APC). Several studies have reported the use of many synthetic polymers such as poly(lactic acids), poly(lactic-co-glycolic acid) (PLGA) and poly(ɛ-caprolactone) as drug/vaccine carrier because of their biocompatible, biodegradable and sustained release property [7–9]. Among these, PLGA is predominantly used as oral and mucosal vaccine delivery [10, 11]. However, the relatively tough fabrication method, poor mucoadhesive nature and low immune enhancing ability do not favour it as a polymer of choice for mucosal vaccine delivery. In addition, the negatively charged PLGA will negate the interaction with negatively charged mucosal cell membrane [10–15]. All these limitations speak in favour of the use of chitosan poly(vinyl alcohol) (PVA) combination for oral delivery of hepatitis B vaccine. Chitosan is a biodegradable natural polymer composed of glucosamine (deacetylated unit) and N-acetyl-D-gulcosamine (acetylated unit) linked together by α (1, 4) glycosidic linkage. Chitosan is widely used in medicine and pharmaceutical areas, because of its biodegradability, biocompatibility and less toxic nature [7, 16]. The deacetylated chitosan backbone of glucosamine units has a high density of amine groups, offering strong electrostatic interactions with proteins and genes which carry overall negative charge thus emphasising its role as a putative vaccine adjuvant [7, 17–19]. PVA is a non-digestive coating agent that prevents the drug from digestion [20–22]. In addition, PVA improves the particles adhesion and absorption by intestinal mucosa [19]. PVA is important in many applications such as controlled drug delivery systems, membrane preparation and recycling of polymers [21, 22]. Looking at the prospective of chitosan and PVA for oral vaccination, the present work was designed to develop a PVA-coated chitosan microparticulate oral vaccine delivery system for subunit antigen HBsAg. To meet this objective, chitosan microparticles were synthesised and loaded with HBsAg. The particles were further coated with PVA. Loading capacity, in vitro release was studied and finally in vivo study was conducted in rat model.
2 2.1
Materials and methods Preparation of microparticles
The ionotropic gelation method was followed for the preparation of chitosan microparticles [2, 3]. Different chitosan concentrations, 0.1–0.5% (w/v) were dissolved in 1% acetic acid and pH 5.5 was adjusted. Different concentrations of cross-linking agent (sodium sulphate) 2–10% (w/v) were added dropwise in the solution in stirring condition at room temperature [23]. Opalescent colour was observed and stirring was continued for 60 min. The resulting suspension was centrifuged for 20 min at 202 & The Institution of Engineering and Technology 2014
3000 rpm. The supernatant was discarded and the pellet was re-suspended in distilled water [2, 3, 24]. The mean hydrodynamic diameter of the particles was measured by dynamic light scattering technique by using Zetasizer nano ZS (Malvern, UK). The dynamic light scattering works on the basic principle of light scattering by matter. The striking of light beam on matter induces an oscillating polarisation of electrons of the molecules. This results in secondary sources of light followed by light scattering. The frequency shifts in scattered light are influenced by the size, shape and molecular interaction in the scattering materials. Loading of rHBsAg to chitosan microparticles and further coating with PVA was performed by dissolving variable concentrations of rHBsAg 4–8 μg/ml in 2.5% of sodium sulphate [2]. The solution was added dropwise in chitosan solution in magnetic stirring. It was further stirred for 60 min maintaining 4–8°C [2, 3, 24]. Equal amount of 5% PVA and a few drops of pure glycerol were added to form a strong interaction between chitosan and PVA [21, 22, 25]. It was kept under stirring for 20 min followed by centrifugation for 20 min at 5000 rpm. The supernatant was discarded and the pellet was re-suspended in phosphate-buffered saline (PBS). The microparticles were collected by centrifugation at 20 000 rpm for 10 min and then washed three times with distilled water. The PVA-coated chitosan-rHBsAg microparticles were vacuum freeze-dried for 24 h after pre-freezing of the resultant dispersion at −20°C overnight. 2.2
Characterisation of microparticles
Particle size distribution and mean hydrodynamic diameter of all the microparticles batches were obtained by dynamic light scattering technique using Zetasizer nano ZS. Zeta potential measurements of particles were performed with the same instrument based on the electrophoretic mobility of particles in aqueous suspensions. Fourier transform infrared (FTIR) spectra of various microparticles were recorded in solid mode (Bruker, FTIR Alpha, Germany). Samples were solidified and gently mixed with 300 mg of micronised KBr powder and compressed into discs at a force of 10 kN for 2 min by using a manual tablet pressure. For each spectrum, a 250 scan interferogram was collected with 4 cm−1 resolution in the mid-infrared region at room temperature. The rHBsAg spectrum was obtained in liquid state.
2.3 Evaluation of rHBsAg loading efficiency in chitosan microparticles The loading efficiency of the chitosan microparticles was calculated by an indirect method, quantifying the antigen that remained in solution as described elsewhere [2]. Briefly, the microparticles were separated from the suspension by ultracentrifugation at 10 000 rpm and 4–8°C for 10 min. Supernatant after centrifugation was carefully separated, and the rHBsAg content in the supernatant was analysed with ultraviolet–visible (UV–vis) spectroscopy at 280 nm [5]. A calibration curve was plotted with five different concentrations of rHBsAg. Samples were analysed at each time interval and the rHBsAg loading efficiency in chitosan microparticles was calculated from the curve. The loading efficiency and loading capacity of rHBsAg were IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 201–207 doi: 10.1049/iet-nbt.2013.0035
www.ietdl.org † Group E: rHBsAg (300 μl containing 6 μg of rHBsAg) injected intramuscularly.
calculated by using the following equations [2, 3]. Loading efficiency (%) = Total amount of rHBsAg − free rHBsAg in supernatant/Total amount of rHBsAg × 100 (1) Loading capacity (%) = Total amount of rHBsAg − free rHBsAg/microparticles dryweight × 100 (2) 2.4
Evaluation of in vitro rHBsAg release
In vitro release studies were performed with the goal to investigate the ability of this delivery system to release the associated rHBsAg. PVA-coated chitosan-rHBsAg microparticles were taken in dialysis membrane. Mild agitation was given with magnetic stirring and in vitro rHBsAg release study was conducted for 4 days in simulated gastric and intestinal fluid (pH 2.4 and 6 PBS, respectively) to evaluate rHBsAg release in different GIT targets. One millilitre of the samples was withdrawn at appropriate intervals to measure the optical density (OD) using UV–vis spectrophotometer (UV-1800 SHIMADZU) at 280 nm [26, 27]. After taking the OD, the samples were replaced back every time. The rHBsAg released was calculated using the following equation [2]. Vaccine release % = OD value for release at particular time /OD value of loaded rHBsAg × 100 (3)
2.5
Immunisation studies
The ability of PVA-coated chitosan-rHBsAg microparticles to induce a protective immunity was studied in 4 months old Wister male rats with an average weight of 500 g. The animals were housed in polypropylene cages in standard environmental conditions (20–25°C), fed with standard rodent diet and water. The experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and approved by the Institutional Animal Ethics Committee. Five groups of rats, six in each group were randomly assigned. The animals fed with rHBsAg alone were administered orally with 100 μl of a 7.5% sodium bicarbonate solution immediately before immunisation in order to neutralise the acidic environment of the stomach. The different formulations, corresponding to each treatment group (see Section 2.5.1) were administered with 300 μl volume containing 6 μg of the rHBsAg either intramuscularly or orally, with a prime and a boost dose at 0 and 28 days, respectively [2, 3]. 2.5.1
Treatment groups
† Group A: Control – saline (300 μl) orally fed. † Group B: Blank PVA-coated chitosan microparticles (in 300 μl of PBS saline) orally fed. † Group C: rHBsAg (300 μl with 6 μg of rHBsAg) orally fed. † Group D: PVA-coated rHBsAg (in 300 μl of PBS saline with 6 μg of rHBsAg) loaded chitosan microparticles orally fed. IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 201–207 doi: 10.1049/iet-nbt.2013.0035
Blood samples were withdrawn from the rats retro-orbital at 2 weeks interval beginning on 2 weeks post-immunisation. A micro-capillary tube of 0.7 mm was carefully inserted into the eye cavity and 0.3 ml blood sample was collected from each rat. Serum was separated by centrifugation at 5000 rpm for 20 min. Supernatant was re-suspended in another micro-centrifuge tube and stored at −20°C until further analysis [2, 3]. Serum immunoglobulin G (IgG) endpoint titres for rHBsAg were quantified for each sample by enzyme-linked immunosorbent assay (ELISA). For this antigen (rHBsAg) was diluted to a final concentration of 2 μg/ml per well in 50 mM sodium carbonate, pH 9.6 (coating buffer) [2, 3, 24]. In 96-well flat bottomed microtitre plates, 100 μl of diluted antigen was added in each well and incubated overnight at 4°C. The plates were washed five times with PBS-T (1 × PBS pH 7.4 containing 0.05% of Tween-20), blocked with 1% BSA in PBS-T (200 μl per well) and incubated for 1 h at 37°C [8, 24]. The plates were then washed five times with PBS-T followed by serial dilution of the serum samples with PBS-T. The serum samples were incubated for 2 h at 37°C. The plates were then washed five times with PBS-T. Horseradish peroxidase-conjugated goat anti-rat IgG was diluted 1:2000 in PBS, 50 μl was added in each well and incubated for 1 h at 37°C. The plates were then washed five times with PBS-T. The bound antibodies were revealed by adding 100 μl per well concentration of O-phenylenediamine dihydrochloride 0.5 mg/ml in 10 ml of 1 M citrate buffer pH 5.5 with 10 μl H2O2 in a dark room. The reaction was stopped after 10 min with 50 μl of 3 M HCl to each well [2, 3]. The absorbance was read out at 492 nm in an automatic ELISA reader, and the titres were expressed as mIU/ml, where 1 mIU is the OD mean of the pre-immune serum plus two times the standard deviation (SD). All the serum samples were tested in triplicate. 2.6
Statistical analysis
The difference in antibody (IgG) generated between all the groups in response to different formulations was compared. The quantity of generated antibody among two groups was compared at once. The results were expressed in mean ± SD. For comparison between two groups, analyses were performed with Student’s t-test at 95% confidence limit. A P value
ISSN 1751-8741
Poly(vinyl alcohol)-coated chitosan microparticles act as an effective oral vaccine delivery system for hepatitis B vaccine in rat model Bijaya Shrestha1, Jyoti Prakash Rath1,2 1
Department of Medical Biotechnology, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Kelambakkam, Chennai, Tamil Nadu 603103, India 2 Department of Chemistry, Gangadhar Meher College, Sambalpur 768004, Odisha, India E-mail: [email protected]
Abstract: The present study focused on the development of an effective oral vaccine delivery system of poly(vinyl alcohol)coated chitosan microparticles-based recombinant hepatitis B vaccine. Chitosan microparticles were prepared by ionotropic gelation technique; they were loaded with recombinant hepatitis B vaccine and coated with poly(vinyl alcohol). The average sizes of the microparticles were measured in the range of 100–410 nm. The optimal loading capacity and loading efficiency were recorded around 3.4 and 74%, respectively. In vitro release study shows that the prepared microparticles release the antigen in a sustained manner. Moreover, the microparticles were resistant to simulated gastric environment and release the antigen in the targeted intestinal milieu. Furthermore, oral immunisation of rats with poly(vinyl alcohol)-coated chitosan hepatitis-B microparticles vaccine shows comparable seroprotective immune response to presently practiced intramuscular vaccination. The results demonstrated that poly(vinyl alcohol)-coated chitosan microparticles have the potential for being used as an oral vaccine delivery system for hepatitis B vaccine and may be a suitable alternative for needle-based vaccination.
1
Introduction
Hepatitis B virus (HBV) infection still remains a global health issue. In its recent bulletin, the World Health Organization has reported about 2 billion infected individuals with the HBV infection [1–3]. Although vaccination is persuaded as the preventive measure against hepatitis B infection and hepatitis B surface antigen-based vaccine is recognised to work effectively, but the potential of vaccination is yet to be fully realised in third world countries [1]. Lack of patient compliance is cited to be the prime reason for low vaccination coverage. Presently practiced lengthy dosing schedule spreading through 6 months is stated to be the main reason for poor compliance rate especially in third world countries [4]. In addition, poor compliance rate is largely influenced by not so user-friendly needle-based vaccination strategy. Moreover, needle stick injury adds to the menace. Approximately 30% of injections for the purpose of vaccination in developing nations are unsafe. There is always a high risk of transmission of blood borne pathogens and it also adds to the waste disposal problem. Hence, there is an urgent need for development of an improved vaccination strategy against hepatitis B. The HBV surface envelope antigen was demonstrated to elicit protective immune response and validated as a candidate antigen (referred to as HBsAg) for successful hepatitis B vaccine development. Alum is primarily used as IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 201–207 doi: 10.1049/iet-nbt.2013.0035
an adjuvant for presently used vaccine formulation. Alum is preferred for its safety, low cost and adjuvanticity for a wide range of antigens. However, the associated limitations, including local reaction and inability to augment cell-mediated immune response are of major concern. In addition, the antibodies generated with alum-adsorbed vaccine fail to reach the mucosal surfaces, the key entry site for HBV. To overcome the issues of conventional vaccine an urgent appeal is to develop a novel vaccine that could facilitate cell-mediated and humoral immunity (both serum and mucosal) and confer complete protection against the pathogen. Oral vaccination is rapidly gaining prominence because of its non-invasive and user-friendly approach that does not require sterile injection by qualified personnel [3]. Moreover, oral vaccination elicits systemic and mucosal immune responses and with the help of an appropriate carrier system, could augment cell-mediated immunity. Nevertheless, the unfriendly environment of the gut, poor transcytosis of the antigen via mucosal epithelium and development of immune tolerance pose a significant challenge for oral vaccination [4, 5]. Nanotechnology could address some of these issues satisfactorily. Among the available options for oral vaccine delivery, the nanocarrierbased delivery systems offer several advantages, including protection of the antigen during transit through mucosal surfaces, and facilitation of antigen uptake by M cells of the mucosal epithelial layer. These delivery systems are easy to 201
& The Institution of Engineering and Technology 2014
www.ietdl.org prepare and characterise, are biocompatible and biodegradable in nature and stabilise the antigen against degradation by gastrointestinal tract (GIT) contents [4–6]. Nanocarriers, which include lipid-based (e.g. liposomes, biolosomes, niosomes and virosomes) and polymeric nanoparticles, are the most sought after carrier adjuvants for oral mucosal vaccine delivery. Their size of a few nanometres mimics the size of pathogenic organisms; therefore they are naturally taken up by M cells and undergo transcytosis to underlying lymphoid tissues. The carrier constructs present several epitopes of the same antigen in a single carrier, which helps in their effective recognition by antigen presenting cell (APC). Several studies have reported the use of many synthetic polymers such as poly(lactic acids), poly(lactic-co-glycolic acid) (PLGA) and poly(ɛ-caprolactone) as drug/vaccine carrier because of their biocompatible, biodegradable and sustained release property [7–9]. Among these, PLGA is predominantly used as oral and mucosal vaccine delivery [10, 11]. However, the relatively tough fabrication method, poor mucoadhesive nature and low immune enhancing ability do not favour it as a polymer of choice for mucosal vaccine delivery. In addition, the negatively charged PLGA will negate the interaction with negatively charged mucosal cell membrane [10–15]. All these limitations speak in favour of the use of chitosan poly(vinyl alcohol) (PVA) combination for oral delivery of hepatitis B vaccine. Chitosan is a biodegradable natural polymer composed of glucosamine (deacetylated unit) and N-acetyl-D-gulcosamine (acetylated unit) linked together by α (1, 4) glycosidic linkage. Chitosan is widely used in medicine and pharmaceutical areas, because of its biodegradability, biocompatibility and less toxic nature [7, 16]. The deacetylated chitosan backbone of glucosamine units has a high density of amine groups, offering strong electrostatic interactions with proteins and genes which carry overall negative charge thus emphasising its role as a putative vaccine adjuvant [7, 17–19]. PVA is a non-digestive coating agent that prevents the drug from digestion [20–22]. In addition, PVA improves the particles adhesion and absorption by intestinal mucosa [19]. PVA is important in many applications such as controlled drug delivery systems, membrane preparation and recycling of polymers [21, 22]. Looking at the prospective of chitosan and PVA for oral vaccination, the present work was designed to develop a PVA-coated chitosan microparticulate oral vaccine delivery system for subunit antigen HBsAg. To meet this objective, chitosan microparticles were synthesised and loaded with HBsAg. The particles were further coated with PVA. Loading capacity, in vitro release was studied and finally in vivo study was conducted in rat model.
2 2.1
Materials and methods Preparation of microparticles
The ionotropic gelation method was followed for the preparation of chitosan microparticles [2, 3]. Different chitosan concentrations, 0.1–0.5% (w/v) were dissolved in 1% acetic acid and pH 5.5 was adjusted. Different concentrations of cross-linking agent (sodium sulphate) 2–10% (w/v) were added dropwise in the solution in stirring condition at room temperature [23]. Opalescent colour was observed and stirring was continued for 60 min. The resulting suspension was centrifuged for 20 min at 202 & The Institution of Engineering and Technology 2014
3000 rpm. The supernatant was discarded and the pellet was re-suspended in distilled water [2, 3, 24]. The mean hydrodynamic diameter of the particles was measured by dynamic light scattering technique by using Zetasizer nano ZS (Malvern, UK). The dynamic light scattering works on the basic principle of light scattering by matter. The striking of light beam on matter induces an oscillating polarisation of electrons of the molecules. This results in secondary sources of light followed by light scattering. The frequency shifts in scattered light are influenced by the size, shape and molecular interaction in the scattering materials. Loading of rHBsAg to chitosan microparticles and further coating with PVA was performed by dissolving variable concentrations of rHBsAg 4–8 μg/ml in 2.5% of sodium sulphate [2]. The solution was added dropwise in chitosan solution in magnetic stirring. It was further stirred for 60 min maintaining 4–8°C [2, 3, 24]. Equal amount of 5% PVA and a few drops of pure glycerol were added to form a strong interaction between chitosan and PVA [21, 22, 25]. It was kept under stirring for 20 min followed by centrifugation for 20 min at 5000 rpm. The supernatant was discarded and the pellet was re-suspended in phosphate-buffered saline (PBS). The microparticles were collected by centrifugation at 20 000 rpm for 10 min and then washed three times with distilled water. The PVA-coated chitosan-rHBsAg microparticles were vacuum freeze-dried for 24 h after pre-freezing of the resultant dispersion at −20°C overnight. 2.2
Characterisation of microparticles
Particle size distribution and mean hydrodynamic diameter of all the microparticles batches were obtained by dynamic light scattering technique using Zetasizer nano ZS. Zeta potential measurements of particles were performed with the same instrument based on the electrophoretic mobility of particles in aqueous suspensions. Fourier transform infrared (FTIR) spectra of various microparticles were recorded in solid mode (Bruker, FTIR Alpha, Germany). Samples were solidified and gently mixed with 300 mg of micronised KBr powder and compressed into discs at a force of 10 kN for 2 min by using a manual tablet pressure. For each spectrum, a 250 scan interferogram was collected with 4 cm−1 resolution in the mid-infrared region at room temperature. The rHBsAg spectrum was obtained in liquid state.
2.3 Evaluation of rHBsAg loading efficiency in chitosan microparticles The loading efficiency of the chitosan microparticles was calculated by an indirect method, quantifying the antigen that remained in solution as described elsewhere [2]. Briefly, the microparticles were separated from the suspension by ultracentrifugation at 10 000 rpm and 4–8°C for 10 min. Supernatant after centrifugation was carefully separated, and the rHBsAg content in the supernatant was analysed with ultraviolet–visible (UV–vis) spectroscopy at 280 nm [5]. A calibration curve was plotted with five different concentrations of rHBsAg. Samples were analysed at each time interval and the rHBsAg loading efficiency in chitosan microparticles was calculated from the curve. The loading efficiency and loading capacity of rHBsAg were IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 201–207 doi: 10.1049/iet-nbt.2013.0035
www.ietdl.org † Group E: rHBsAg (300 μl containing 6 μg of rHBsAg) injected intramuscularly.
calculated by using the following equations [2, 3]. Loading efficiency (%) = Total amount of rHBsAg − free rHBsAg in supernatant/Total amount of rHBsAg × 100 (1) Loading capacity (%) = Total amount of rHBsAg − free rHBsAg/microparticles dryweight × 100 (2) 2.4
Evaluation of in vitro rHBsAg release
In vitro release studies were performed with the goal to investigate the ability of this delivery system to release the associated rHBsAg. PVA-coated chitosan-rHBsAg microparticles were taken in dialysis membrane. Mild agitation was given with magnetic stirring and in vitro rHBsAg release study was conducted for 4 days in simulated gastric and intestinal fluid (pH 2.4 and 6 PBS, respectively) to evaluate rHBsAg release in different GIT targets. One millilitre of the samples was withdrawn at appropriate intervals to measure the optical density (OD) using UV–vis spectrophotometer (UV-1800 SHIMADZU) at 280 nm [26, 27]. After taking the OD, the samples were replaced back every time. The rHBsAg released was calculated using the following equation [2]. Vaccine release % = OD value for release at particular time /OD value of loaded rHBsAg × 100 (3)
2.5
Immunisation studies
The ability of PVA-coated chitosan-rHBsAg microparticles to induce a protective immunity was studied in 4 months old Wister male rats with an average weight of 500 g. The animals were housed in polypropylene cages in standard environmental conditions (20–25°C), fed with standard rodent diet and water. The experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and approved by the Institutional Animal Ethics Committee. Five groups of rats, six in each group were randomly assigned. The animals fed with rHBsAg alone were administered orally with 100 μl of a 7.5% sodium bicarbonate solution immediately before immunisation in order to neutralise the acidic environment of the stomach. The different formulations, corresponding to each treatment group (see Section 2.5.1) were administered with 300 μl volume containing 6 μg of the rHBsAg either intramuscularly or orally, with a prime and a boost dose at 0 and 28 days, respectively [2, 3]. 2.5.1
Treatment groups
† Group A: Control – saline (300 μl) orally fed. † Group B: Blank PVA-coated chitosan microparticles (in 300 μl of PBS saline) orally fed. † Group C: rHBsAg (300 μl with 6 μg of rHBsAg) orally fed. † Group D: PVA-coated rHBsAg (in 300 μl of PBS saline with 6 μg of rHBsAg) loaded chitosan microparticles orally fed. IET Nanobiotechnol., 2014, Vol. 8, Iss. 4, pp. 201–207 doi: 10.1049/iet-nbt.2013.0035
Blood samples were withdrawn from the rats retro-orbital at 2 weeks interval beginning on 2 weeks post-immunisation. A micro-capillary tube of 0.7 mm was carefully inserted into the eye cavity and 0.3 ml blood sample was collected from each rat. Serum was separated by centrifugation at 5000 rpm for 20 min. Supernatant was re-suspended in another micro-centrifuge tube and stored at −20°C until further analysis [2, 3]. Serum immunoglobulin G (IgG) endpoint titres for rHBsAg were quantified for each sample by enzyme-linked immunosorbent assay (ELISA). For this antigen (rHBsAg) was diluted to a final concentration of 2 μg/ml per well in 50 mM sodium carbonate, pH 9.6 (coating buffer) [2, 3, 24]. In 96-well flat bottomed microtitre plates, 100 μl of diluted antigen was added in each well and incubated overnight at 4°C. The plates were washed five times with PBS-T (1 × PBS pH 7.4 containing 0.05% of Tween-20), blocked with 1% BSA in PBS-T (200 μl per well) and incubated for 1 h at 37°C [8, 24]. The plates were then washed five times with PBS-T followed by serial dilution of the serum samples with PBS-T. The serum samples were incubated for 2 h at 37°C. The plates were then washed five times with PBS-T. Horseradish peroxidase-conjugated goat anti-rat IgG was diluted 1:2000 in PBS, 50 μl was added in each well and incubated for 1 h at 37°C. The plates were then washed five times with PBS-T. The bound antibodies were revealed by adding 100 μl per well concentration of O-phenylenediamine dihydrochloride 0.5 mg/ml in 10 ml of 1 M citrate buffer pH 5.5 with 10 μl H2O2 in a dark room. The reaction was stopped after 10 min with 50 μl of 3 M HCl to each well [2, 3]. The absorbance was read out at 492 nm in an automatic ELISA reader, and the titres were expressed as mIU/ml, where 1 mIU is the OD mean of the pre-immune serum plus two times the standard deviation (SD). All the serum samples were tested in triplicate. 2.6
Statistical analysis
The difference in antibody (IgG) generated between all the groups in response to different formulations was compared. The quantity of generated antibody among two groups was compared at once. The results were expressed in mean ± SD. For comparison between two groups, analyses were performed with Student’s t-test at 95% confidence limit. A P value
