INTRODUCTION:

If microparticulate drug delivery systems are used for intestinal drug delivery, the associated compound should be released upon arrival at the epithelium. Only particles smaller than about 100 nm have shown to be taken up by Caco-2 cells¹. Larger microparticles end up in the Peyer’s patches after uptake by the M-cells ^{2, 3} for vaccine delivery, the lymphoid tissue should be targeted. Access to mucosal lymphoid tissue is provided by antigen sampling cells These M-cells are located in between the epithelial cells and take up antigens and microparticles smaller than 10 mm ^{4, 5}. Particulate drug carrier systems administered to mucosal surfaces may protect the drug from degradation during the passage through the gastrointestinal tract, enhance the uptake by the epithelium, and act as a controlled release system resulting in prolonged blood concentrations. The oral route is well accepted and easily allows the vaccination of large populations. However, the acidic environment of the stomach and the presence of enzymes make the oral delivery of vaccines a challenge where is difficult to achieve high and reproducible effects.

In order to solve these difficulties, a considerable number of polymeric microparticulate systems are under investigation to deliver vaccines to the intestine while protecting them from adverse conditions that could affect their bioactivity⁶.

Chitosan microparticulate carrier systems were reviewed. Chitosan microparticles for colonic delivery of prednisolone were prepared by a precipitation/coacervation technique using chitosan and sodium sulfate as precipitant agent⁷. Chitosan microparticles as an oral and intranasal vaccine delivery system were already used in our group showing promising. Capabilities^{8, 9, 10}. In these studies, the vaccine was loaded by a mild and simple but effective adsorption method. By this method, deleterious preparation conditions, like elevated temperatures, high shear rates or the presence of organic solvents were avoided. This method has also been described by other groups that reported good adsorption capacities for different substances^11,
12. Therefore, the goal of this study was to prepare and fully characterize alginate microspheres as antigen delivery system.

MATERIALS AND METHODS:

Materials:

Chitosan was purchased from Primex BioChemicals AS. According to the provider’s specifications, the degree of deacetylation is 95% (titration method) and the viscosity is 8 cP (1% solutions in 1% acetic acid). Low viscosity sodium alginate, Bovine serum albumin (BSA) (fraction V), were provided by Sigma. All other chemicals and reagents were of analytical grade and purchased from commercial vendors.

Preparation of chitosan microsphere:

Chitosan microspheres were prepared by the precipitation/ coacervation method described previously⁷. Shortly, chitosan was dissolved at a concentration of 0.20% (w/v) in a solution with 2 %( v/v) of acetic acid and 1% (w/v) of Tween® 80. The formation of the particles was achieved after the addition of 3.5 ml of sodium sulfate solution (10%, w/v) to 200 ml of the chitosan solution. The addition was made at a rate of 1 ml/min under mild agitation (<50 rpm) and continuous sonication (vibracell sonicator; 600-W model). Sonication was maintained for additional 15 min and the agitation for 60 min at room temperature. The suspension was centrifuged for 30 min at 3500 rpm (2800×g) and the supernatant was discarded. The particles were re-suspended twice in Millipore water, centrifuged again for 30 min and the supernatants were discarded. The particles were frozen in liquid nitrogen and freeze-dried overnight using a Christ freeze-dryer. The dry powder was kept frozen until further use.

Loading of bovine serum albumin onto chitosan microspheres by adsorption method:

The first step of the loading procedure was the suspension of the freeze-dried particles in a phosphate buffer (pH 7.4) placed in an ultrasound bath for 30 min in order to disaggregate the particles. The loading was done by incubating a solution of ovalbumin with chitosan particles under mild agitation at room temperature¹³.

In vitro evaluation studies:

Determination of particle size distribution:

The particle size distribution of microspheres was done by optical microscope and self scaled scanning electron microscope. The sample for SEM was prepared by sprinkling the micrisphere powder on a double adhesive tape that stuck to an aluminium stab was then coated with platinum to a thickness of about 300A using a sputter coater. The samples were then randomly scanned and photographs were taken.

Determination of the encapsulation efficiency:

This method involves alkaline hydrolysis of the microspheres and determination of the BSA recovered. Ten mg of microspheres were shaken overnight with 10mL of 5% (W/V) SDS in 0.1 M sodium hydroxide solution (NaOH : SDS). Following centrifugation, the protein content was determined by the Lowry method.

In vitro release of antigens from chitosan microspheres:

The release of antigen from the chitosan microspheres are studied in PBS (pH 7.4) containing 0.02% (W/V) Tween-80 (PBST). Several vials containing 10mg of microspheres and 5mL of PBST were incubated at 37°C on a constant shaking mixer .The content of avail is withdrawn for estimation of released antigen on days 1 ,3 , 7 , 14 ,28 and 35. The micro spheres suspension is centrifuged at 8000rpm for 10min the supernatant was collected and used for protein estimation by Lowry method.

^{RESULTS AND DISCUSSION:}

Formulation of chitosan microsphere:

^{Chitosan, a natural polymer was selected for developing
mucosal delivery system for vaccines, because of its mucoadhsive property,
biodegradability and non-antigenic property. Microspheres were formulated using
different concentration 1, 1.5, 2.0, 2.5, 3.0% of chitosan and characterized
for its size [14]. At the concentration 1.0% of chitosan maximum numbers of
uniform sized microparticles were obtained and it was in the range of 1-10 µm.
The results were showed in Fig no: 1.}

^{Fig. 1. Showing size of
Microspheres in size range of 1-10 µm at different concentration of Chitosan}

Characterization of microspheres

Morphology.

Chitosan microspheres were prepared by the precipitation/ coacervation method. The surface of these microspheres as observed under scanning electron microscopy (SEM) (jsm-6360) Fig no: 2 , 3, was free from any pores. The actual size of individual microspheres was in the range of 1-10^{µm and few microspheres were measured of size up to 50 µm. Microspheres
that are less than 10 µm in size are reportedly taken up by the APCS (macrophages
and dendritic cells) and thus considered to be most suitable for optimum
adjuvant effect 15,16. A 1.0% (w/v) solution of chitosan was selected, which
was suitable for making small microspheres (10 µm).}

Fig. 2. Scanning electron microscopy photograph of BSA loaded chitosan microshere

^{Encapsulation Efficiency:}

^{Incorporation of therapeutic agent into chitosan
microspheres can be influenced by factors such as method of preparation, drug,
drug and polymer binding. In this study encapsulation of BSA in chitosan
microspheres was evaluated. The encapsulation efficiency of BSA in chitosan
microspheres was found to be 61.2% are summarized in Table no:1.}

Fig. 3. Scanning electron microscopy photograph of BSA loaded single chitosan microshere

^{Table 1; Encapsulation efficiency of BSA loaded
chitosan microparticles}

^Sample

^{Encapsulation
efficiency}

^{Mean
in %}

⁴

⁵

^69%

^53%

^64%

^59%

^61%

^61.2%

Release studies:

^{The model protein releases from chitosan microspheres with
a low release of 29.5% in 24 hrs. This was followed by a slow and sustained
release profile. After 3 days, 36.5% model protein was released (Fig no: 5).
these results clearly indicate that release of model protein from chitosan
microsphere was controlled.}

^{Fig. 4. Invitro release of model
protein loaded chitosan microsphere}

^CONCLUSION:

Chitosan being natural biocompatible polysaccharide has been widely used in controlled drug delivery and it may provide non toxic carrier for successful mucosal delivery of BSA. This study demonstrated that spherical and non aggregated chitosan microspheres was in the range of 1-10^{µm and few microspheres were measured of size up
to 50 µm}can be prepared by the cross-linking with sodium sulphate method. High encapsulation efficiency of BSA (61.2%) was achieved. The release of BSA from chitosan microspheres was quite fast, particularly in PBS. However, it was possible to delay the release of BSA. Therefore, chitosan microspheres appear, technologically, a promising delivery system for antigen.

ACKNOWLEDGEMENTS:

We express our sincere thanks to the Dr. Nalla G. Palaniswami, Chairman and Dr.Thavamani D Palaniswami, Managing trustee, of Kovai Medical Center Research and Educational Trust, Coimbatore. For giving the facilities and encouragement to carry out this work.

REFERENCES:

1. Desai MP, Labhasetwar V, Walker E, Levy RJ, Amidon GL. The mechanism of uptake of biodegradable microparticles in Caco-2 cells is dependent on size. Pharm Res 1997; 14:1568–1573.

2. Jani PU, McCarthy DE, Florence AT. Nanosphere and microsphere uptake via Peyer’s patches: observation of the rate of uptake after a single oral dose. Int J Pharm 1992; 86:239–246.

3. Smith MW, Thomas NW, Jenkins NW, Miller PG, Cremaschi NGA, Porta C. Selective transport of microparticles across Peyer’s patches follicle associated M-cells from mice and rats. Exp Phys 1995; 80:735–743.

4. Eldridge JH, Hammond CJ, Meulbroek JA, Staas JK, Gilley RM, Rice TR. Controlled vaccine release in the gut associated lymphoid tissues; orally administered biodegradable micro spheres target the Peyer’s patches. J Control Release 1990; 11:205–214.

5. Lydyard P, Gross C. The lymphoid system, In: Immunology, Vol 5, Mosby: London; 1998; 31–42.

6. Singh M, O’Hagan D. The preparation and characterization of polymeric antigen delivery systems for oral administration. Adv Drug Deliv Rev 1998;34: 285–304.

7. Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs. J Control Release 1996; 39: 17–25.

8. Van der Lubben IM, Konings FA, Borchard G, Verhoef JC, Junginger HE. In vivo uptake of chitosan microparticles by murine Peyer’s patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry. J Drug Target 2001a; 9: 39–47.

9. Van der Lubben IM, Verhoef JC, van Aelst AC, Borchard G, Junginger HE. Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine Peyer’s patches. Biomaterials 2001b; 22:687–694.

10. Bivas-Benita M, Laloup M, Versteyhe S, Dewit J, De Braekeleer J, Jongert E, Borchard G. Generation of Toxoplasma gondii GRA1 protein and DNA vaccine loaded chitosan particles: preparation, characterization, and preliminary in vivo studies. Int J Pharm 2003; 266:17–27.

11. Mi FL, Shyu SS, Chen CT, Schoung JY. Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release. Biomaterials 1999; 20:1603–1612.

12. Hejazi R, Amiji M, Stomach-specific anti-H, pylori therapy I. Preparation and characterization of tetracyline-loaded chitosan microspheres. Int J Pharm 2002; 235: 87–94.

13. Olga Borges, Gerrit Borchard, Coos Verhoef J. Adriano de Sousa, Hans E Junginger, Preparation of coated nanoparticles for a new mucosal vaccine delivery system, International Journal of Pharmaceutics. 2005; 299: 155–166.

14. Jaganathan KS, Raob YUB, Paramjit Singh, Prabakaran D, Swati Gupta, Anubhav Jain and Suresh P Vyasa. Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(d,l-lactic-co-glycolic acid) versus chitosan microspheres. International Journal of Pharmaceutics 2005; 294: 23–32.

15. Eldridge JH, Staas JK, Meulbroek JA, Tice TR and Gilley RM. Biodegradable and biocompatible poly(dl-lactideco-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies. Infect Immun 1991; 59: 2978–2986.

16. O'Hagan DT, MacKichan ML and Singh M. Recent developments in adjuvants for vaccines against infectious diseases. Biomol Eng 2001; 18: 69-85.

Received on 26.07.2011 Modified on 03.08.2011

Research J. Pharm. and Tech. 4(10): Oct. 2011; Page 1630-1632