Preparation and Characterisation of Alginate Coated Chitosan Microspheres for  Bacterial  Vaccines.


Kalaivani M*, Manju Jose Pulikottil, Nisha Mary BM, Sakthivel R and Rajasekaran A

KMCH College of Pharmacy, Kalapatti Road, Coimbatore-641 048, Tamil Nadu, India.

*Corresponding Author E-mail:



Tetanus toxoids vaccine is widely used to prevent tetanus. As it has a short biological half-life, a long acting tetanus toxoids formulation is desirable to improve patient compliance. The chitosan microspheres were prepared by an emulsion polymerization method using glutaraldehyde as the crosslinking agent. All microspheres were spherical and smooth with the mean particle size in the range of 30–38 μm. Drug release from the chitosan microspheres displayed a biphasic pattern characterized by an initial fast release, followed by a slower release. The released amount was decreased with an increase in the glutaraldehyde concentration. The time required for complete degradation of microspheres was increased from 144 to 264 h when the glutaraldehyde concentration increased from 0.1 to 0.7 ml. Biodistribution studies indicated that the degree of uptake by the M-cells of the peyer’s patches in the gut was higher than that of the other organs. All these results demonstrated that tetanus toxoids loaded chitosan microspheres can be used for passive M-cell targeting.


KEYWORDS: Chitosan microsphere; Tetanus toxoid; mucosal immunization; Controlled-release



Sustained release of macromolecular drugs from polymeric matrices has received increasing attention in recent years. In view of the fact that many of the future drugs are going to be of recombinant DNA origin. Vaccines that are required to be given in multiple divided doses are not efficacious if only one dose is given without boosting. In developing countries, the drop-out rates from individuals receiving the first dose, but a not successive dose is high. In order to be effective, most vaccines require two or three booster doses after primary immunization. Therefore, the conversion of multiple dose vaccines into single dose vaccines may  represent an important advance because recombinant proteins are expensive. It should also lead to improved vaccination coverage as well as reduced costs1. Among the various tentatives for improving the administration of proteins, the microencapsulation into biodegradable polymers represents a practical and promising approach. The biodegradable and biocompatible polymer, chitosan are the primary candidates for the development of single dose vaccines, because they have been used in humans as suture material and as controlled release delivery systems for peptide drugs2,3. Microspheres (<10 µm) promote their entry into lymph nodes and provide a high level concentration of antigen over an extended time-period.


It also promotes the interaction of encapsulated antigen with antigen presenting cells (APCs) such as macrophages, dendritic cells (DCs)4.Various therapeutic proteins have been formulated using chitosan microspheres and some are also undergoing clinical trials5,6,7. Though microspheres have high potential for the controlled release of macromolecules, there are some difficulties, which explain to a certain extent, the limited number of formulations in the market. These drawbacks include; low encapsulation efficiency, protein inactivation during the encapsulation process and difficulties for controlling the release of the active protein8,9,10.The prospect of stabilizing antigens and administering primary and secondary doses in a “single shot” by formulating vaccines in these polymers is a promising development of vaccination in both developed and developing countries11. In the case that the chitosan  particles  are not very porous, the antigen will be preferentially adsorbed to the particle surface. This can cause stability problems because processes like desorption or the attack of the antigens by enzymes or acidic substances from the body fluids may occur. These obstacles may be overcome by coating those particles with an acid resistant polymer, like sodium alginate. Sodium alginate has been used for preparing nanoparticles12,13, microspheres14,15,16,17,microcapsules18 and beads17, for oral delivery. In particularly, the use of alginate microparticles as an antigen delivery system has been described in several publications and there are some indications that they are able to induce a mucosal and systemic immune response in a variety of animal species by both oral and intranasal administration19,20. Over the last years, sodium alginate has also been used as a coating material for cells with some advantages. It seems that the coating acts as a barrier to microbial contamination, and thus improved survival prospects of the coated cells21. In another study, the coating is performed to protect donor mammalian cells against antibodies and cytotoxic cells of the host immune system, allowing the transplantation of cells in the absence of immunosuppression22. In the present study the TT is loaded with adsorption method and loaded chitosan microspheres is coated with sodium alginate is formulated to increase the immune response .In view to reduce the cost, chitosan can be used to replace the expensive polymer. Advances in this line will be illustrated using a model protein oval bumin (OVA) and the antigen tetanus toxoid (TT). Tetanus toxoid represents an ideal protein that would substantially benefit from the microencapsulation technologies. Moreover, because of its easy availability and characteristics, TT appears as a suitable antigen to attempt alternative approaches to the existing vaccines.




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, Tetanus toxoid (MW 150 kDa) having concentration 1250 LfU/ml and standard tetanus anti-toxin were obtained from Pasteur Institute of India, Coonoor, India. All other chemicals and reagents were of analytical grade and purchased from commercial vendors.


Preparation of Chitosan Microsphere:

Chitosan microspheres are prepared by using cross-linking agent glutaraldehyde saturated toluene23. Where 4 ml of 1% chitosan gel was dissolved in 1ml of 0.01N HCl aqueous solution and stirred well for 5mins. Then 50mL of Toluene containing 10% volume of span-80 is added to the aqueous phase with continuous stirring, for one hour using magnetic stirring, which resulted in water-in-oil type of emulsion. After formulation of the emulsion 10mL  of 50% GST containing 10% span-80 is added to the emulsion drop by drop with continuous stirring and the stirring was continued for one and half hour. After one and  half hour the microspheres suspension obtained are centrifuged at 2000rpm for 5mins and sediment (microspheres) was washed with toluene thrice and with acetone thrice. Then microspheres are dried at room temperature and collected as dry free flowing powder. Five concentrations of chitosan are used for preparing microspheres, stable particles are selected and it is used for coating with alginate.


Loading of Tetanus Toxoid onto Chitosan Microspheres By Adsorption Method:

The dried and swelled microspheres were collected. To the dry microspheres 4ml of plain TT (600 Lf) was added and kept in an incubator at 37şC for 20 hours  in shaker system.  After incubation the adsorbed TT microspheres were centrifuged at 10,000rpm for 20 min. The pellets were washed with methanol and acetone to remove excess of TT and stored in a refrigerator23.


Preparation of alginate coated Microsphere:

Alginate coated microspheres were obtained by mixing equal volumes of the loaded microspheres suspension and a buffer phosphate solution of sodium alginate (1% w/v) under magnetic stirring. The agitation was maintained for 20min. The suspension was then centrifuged for 10 min at 1600rpm and the supernatant is discarded24.


In Vitro Evaluation Studies:

Determination of particle size distribution:

The particle size distribution of microsphere 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 gold 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 TT 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 The 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 a vail 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. Placebo microspheres (without antigen) containing alginate are used as control.


Estimation of Tetanus Toxoid by Lowry’s Method:

Concenteration of 20, 40, 60, 80mg of standard protein solution was pipetted out into a series of test tubes and the volume of  each test tube was made upto 1mL with distilled water and in one test tube 1ml of distilled water without any standard protein served as blank. Volume of sample was taken and made upto 10mL with distilled water, a volume of 5mL alkaline solution was added to all test tube. Mix well and allow to stand for 10min. Then a volume of 0.5mL of Folin’s reagent was added to all test tubes. Mix well and incubated for 30min. Blue colour developed is measured at 660nm in spectrophotometer against the reagents (blank). A standard graph was plotted by taking concentration of protein on X-axis and optical density on Y-axis. From which amount of protein in the sample was calculated.



Characterization of the microsphere.

Preparation of the vaccine delivery system:

Chitosan, a natural polymer was selected for developing mucosal delivery system for vaccines, because of it’s mucoadhsive property, biodegradability and non-antigenic property Microspheres were formulated using different concentration 1, 1.5, 2, 2.5, 3% of chitosan and characterized for its size. At the concentration 1% of chitosan maximum number of uniform sized microparticles were obtained and it was in the range of 30-40 µm (Figure 1)


Figure 1.Showing size of Microspheres in size range of 30-40µm at different concentration of Chitosan


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 tetanus toxoid in chitosan microspheres was evaluated. The encapsulation efficiency of tetanus toxoid in chitosan microspheres was found to be 48% (Table 1).


Table.1: Encapsulation efficiency of tetanus toxoid loaded chitosan microparticles


Encapsulation efficiency

Mean in %

















Release studies

The TT releases from microspheres with a low burst release of 42% in 24 hrs. This was followed by a slow and sustained released profile. After 3 days, 64% TT was release (Table 2). These results clearly indicate that release of TT from microsphere was controlled.

Table.2: In vitro release of Tetanus toxoid from microspheres at different time intervals

S. No

Time interval in days

In vitro release of drug concentration in µg/mL





















This work describes a chitosan microsphere used for mucosal vaccine delivery system, consisting of chitosan microsphere  that are prepared by cross-linking with glutaraldehyde. The chitosan microsphere are loaded under very mild conditions with a tetanus toxoid antigen, which was negatively charged in the buffer systems. In order to increase the stability of the loaded chitosan microsphere at physiological conditions, a coating process with sodium alginate was developed and the antigen released from the coated microsphere was reduced in comparison to the uncoated chitosan microsphere. In vivo studies are under way to show the efficacy of these systems for mucosal vaccination.



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Received on 10.11.2009                             Modified on 21.01.2009

Accepted on 23.02.2010                            © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 503-506