INTRODUCTION:

Sustained release of macromolecular drugs from polymeric matrices has received increasing attention in recent years. Vaccines that are required to be given in multiple divided doses are not efficacious if only one dose is given without boosting. 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. Among the various tentative for improving the administration of proteins, the microencapsulation into biodegradable polymers represents a practical and promising approach.¹

The most widely used delivery form of protein and peptide drugs is as injectables². The microspheres vaccines delivery system based on biodegradable polylactide (PLA) and polylactide-co-glycolide(PLGA) has been extensively investigated due to the many advantages of the controlled-release delivery system.^3,4

PLA and PLGA are biodegradable and biocompatible polymers, which are nonimmunogenic and have a long history of safe use in humans as sutures and as controlled delivery systems.⁵

The potential applicability of biodegradable microspheres as drugs, peptides, proteins and antigen delivery systems is based not only on the protection from acidic and proteolytic degradation, but also on the fact that the controlled release profile could be adjusted by polymer degradation. Especially release microspheres using biodegradables such polylactide (PLA), poly (DL-lactide-co-glycolide) (PLGA)^6,7 and poly-DL-lactide-co-poly-(ethylene glycol) (PELA)^8,9have been investigated and satisfactory results have been obtained.

Biodegradable polymers, especially in the form of injectable microparticles, have been investigated extensively for their capability of releasing therapeutically useful proteins in controlled way^10,11,12. Microparticulate protein release systems, such as microspheres, microcapsules, as well as nanospheres, are generally designed to achieve either sustained or pulsed release over a prolonged period of time, typically weeks or months.¹³To mimic conventional repetitive vaccination schedules with a single injection, pulsed release is aimed at for vaccine components, such as bacterial toxoids^14-19 and viral antigens^20,21. It has the advantage that it can be freeze dried and therefore avoid the need for cold storage.²²Tetanus toxoid is a 150-kDa protein that has been safely used in human for several decades as aluminium-adsorbed vaccine, requiring three injections, at 0, 3, and 6-12 months, to induce protective immunity. Tetanus toxoid was used as (model) antigen for microencapsulation.²³

Figure No.1 Effect of change in concentration of cross-linking agent on loading (TT adsorbed on to PLA microspheres)

MATERIALS AND METHODS: Materials:

Poly lactic acid was purchased from Sigma Aldrich, Germany (FLUKA). Tetanus Toxoid and Tetanus Antisera was obtained from Biological E. Ltd., Hyderabad, India. Dichloro methane was obtained Sd Fine Chem. Ltd., Mumbai, India. Toluene was obtained from Nice Chemicals (P) Ltd., Cochin, India. Acetone was obtained from LOBA Chemical, Mumbai, India. Linseed oil, Cotton seed oil and Castor oil were obtained from Scientific Lab. Chemical, Madras, India. All the reagents used were of analytical grade.

Methods:

Formulation of microspheres was done using emulsion cross-linking technique using different variables to establish the parameters necessary to obtain ideal batches of Tetanus toxoid. By using the above technique Tetanus Toxoid was loaded into the microspheres by the following two methods:

1. Adsorption

2. Encapsulation.

I. Preformulation studies:

Formulation of microspheres was done, using different variables to establish the parameters necessary to obtain ideal batches of TT adsorbed and encapsulated microspheres.

A. Variables:

1. Cross-linking agent concentration:

Effect of cross-linking agent concentration was studied by preparing 5 batches of microspheres containing 2ml of 2%, 3%, 4%, 5%, 6% GST (Gluteraldehyde Saturated Toluene) respectively. Other variables were kept constant.

2. Polymer concentration:

Effect of polymer concentration was studied by preparing 5 batches of microspheres using 3ml. of 0.2, 0.5, 1, 2, 3% polymer. Other variables were kept constant.

3. Stirrer Speed:

Effect of stirrer speed was studied by preparing 5 batches of microspheres with 1000, 1200, 1500, 1700, 2000 rpm as stirrer speed. Other variables were kept constant.

Figure No.2 Shows the effect of change in concentration of PLA on loading (T.T adsorbed onto PLA microspheres)

4. Dispersion medium:

Effect of dispersion medium was studied by preparing three batches of microspheres using Linseed oil, cotton seed oil and castor oil. Other variables were kept constant.

5. Washing solvent:

The effect of washing solvents was studied by preparing 3 batches of microspheres using Acetone, dichloromethane, chloroform (after the initial toluene wash). Other variables were kept constant.

The resulting microspheres of the various formulations were accessed for size uniformity, shape, surface characteristics and percentage yield.

B. Formulation of TT Adsorbed on to PLA Microspheres²⁴:

PLA microspheres were prepared by emulsion cross linking technique.

i. Preparation of PLA solution:

A solution of 1% PLA was prepared in dichloromethane this was stirred using remi stirrer at 3000rpm for one hour.

ii. Formation of empty PLA microspheres:

3ml of the 1% PLA solution was added into the dispersion medium containing 12.5ml of Linseed oil, 12.5ml of Toluene and 1% of Tween 80. The mixture was stirred for half an hour at 1700rpm. Next, the 2ml of 5% gluteraldehyde saturated toluene was introduced drop wise into the beaker as a cross-linking agent. The stirring was continued for 4 hours, and finally an emulsion was formed.

5% of gluteraldehyde saturated toluene solution was prepared by mixing equal volume of 5% gluteraldehyde and toluene for 10 minutes using a cyclone mixer The mixture was allowed to separate and the upper toluene layer was used as the cross linking agent.

Figure No.3 Effect of change in concentration of cross-linking agent on loading (TT encapsulated PLA microspheres)

iii. Washing of emulsion:

The emulsion was centrifuged at 5,000 rpm for 10 minutes. The pellet formed to separate the microspheres from the dispersion medium was resuspended in Toluene. Again it was centrifuged at 5,000 rpm for 10 minutes. This was repeated 5 times with toluene and 3 times with acetone to remove residual oil. After washing, the microspheres obtained were dried in a vacuum desiccator. Many batches of microspheres thus obtained were pooled and stored under refrigeration.

Figure No.4 Shows the effect of change in concentration of PLA on loading (TT encapsulated in PLA microspheres)

iv. Swelling of microspheres:

From the pooled microspheres 200mg were weighed and suspended in 20ml of sterile water and kept 24 hours for swelling. After swelling, the microspheres were centrifuged at 10,000rpm for 10 minutes. The swelled microspheres thus obtained were washed twice with acetone. The powder was formed, and 2ml of plain Tetanus toxoid was added into the powder and it was kept in an incubator at 37^oC for 20 hours for adsorption. After incubation, the adsorbed microspheres were centrifuged at 10,000 rpm for 20 minutes. The pellet was formed and it was washed with methanol and acetone to remove water. Many batches of Tetanus toxoid adsorbed PLA microspheres thus obtained were pooled and stored in refrigerator.

C. Formulation of TT Encapsulated in PLA Microspheres²⁴:

Emulsion cross-linking technique was used to encapsulate PLA microspheres with Tetanus toxoid.

i. Formation of PLA solution:

A solution of 1% PLA was prepared in Dichloromethane and stirred using remi stirrer at 3000rpm for one hour.

ii. Formation of TT encapsulated Tetanus microspheres:

3ml of the 1% PLA solution was mixed with 2ml of TT in a cyclone mixer for 10 minutes. The above solution was dispersed in the dispersion medium containing 12.5ml of linseed oil, 12.5ml of toluene and 1% of Tween 80. The stirring was continued for half an hour. To this 2ml of 5% Gluteraldehyde saturated toluene solution was added and the stirring was continued at 1700 rpm for 5hours.

Table No.1 Table shows In vitro release of TT in terms of Limes flocculation, from the batch of TT adsorbed onto PLA microspheres

Time (hrs)	Amount of plain T.T. released (Lf/ml)	Percentage release (%)	Cumulative percentage release
0	0	0.00	0.00
2	0	0.00	9.00
6	5	15.00	15.01
12	11	33.00	34.51
24	13	39.00	42.32
36	16	48.00	51.92
48	21	63.01	67.83
60	22	66.01	72.33
72	25	75.01	81.63
84	26	78.01	85.53
96	28	84.01	91.84

iii. Washing of emulsion:

The above formed emulsion was centrifuged at 5000 rpm for 10 minutes for removing the oil layer. The pellet was resuspended in toluene and centrifuged at 5000 rpm for 10 minutes. The same procedure was continued 5 times with toluene and 3 times with acetone. After the washing procedure, the microspheres were dried in a vacuum desicator. Many batches of microspheres thus obtained were pooled and stored in refrigerator.

Figure No.5 In vitro release of TT in terms of Limes flocculation, from the batch of TT adsorbed onto PLA microspheres

vi. Determination of Average Diameter of Microspheres:

Determination of average diameter of PLA microspheres was carried out by optical microscopy in which stage micrometer was employed. Minute quantities of microspheres were spread on a clean glass slide and average sizes of 400 microspheres were determined in each batch.

II. Estimation of TT Incorporated into PLA Microspheres (By Flocculation method)²⁴:

Limes Flocculation means the tube which contains the optimum concentration of toxin and antitoxin flocculates first and the corresponding unit of the antitoxin is taken as the Lf value of the toxin.

A. Estimation of TT Adsorbed onto PLA microspheres:

Weighed quantity (10mg) was digested in 10ml of 15% sodium citrate solution and it was kept in an incubator at 37^oC for 24 hours. After the incubation, the microspheres were separated by centrifugation. The supernatant containing the vaccine protein was taken and the limes flocculation was estimated by flocculation test.

Procedure:

To each of a series of flocculation tubes containing 1ml of sample solution, various volumes of (0.05, 0.1, 0.15, 0.2, 0.25) ml of standard antitoxin were added and the volume was made upto 2ml using normal saline solution. The contents in the tube were mixed properly and kept in the flocculation chamber at 45 to 50^oC and observed for the most rapidly flocculation mixture.

B. Estimation of TT Encapsulated in PLA microspheres:

Weighed quantity (10mg) was digested in 10ml of normal saline solution, then this solution was immediately washed. To the pellet, 10ml of 1M NaCl was added and kept in refrigerator for 2 days. After that, the microspheres were separated by centrifugation. The supernatant containing the vaccine protein was taken and the potency was estimated by Limes flocculation test procedure.

To each of a series of flocculation tubes containing 1ml of sample solution, various volumes of (0.01, 0.02, 0.03, 0.04, 0.05) ml of standard antitoxin were added and the volume was made up to 2ml using normal saline solution. The contents in the tube were mixed properly and kept in the flocculation chamber at 45 to 50^oC and observed for the most rapidly flocculating mixture.

III. In vitro Dissolution Study:

(In vitro dissolution study for TT adsorbed and encapsulated in PLA microspheres)

50 mg of TT adsorbed and encapsulated in microspheres were taken in a two separate 250ml conical flask. To each, 50ml of phosphate buffer (pH 7.4) was added; the flask was agitated at 90-95 revolution per minute in an incubator maintained at 37^oC. From this 5ml sample were withdrawn at various time interval, freshly prepared 5ml of phosphate buffer (pH 7.4) being replaced immediately. This was continued for 60 days. The collected samples were centrifuged and supernatant solution was analyzed in terms of Limes flocculation.

Table No.2 Table shows In vitro of TT in terms of Lime flocculation from the batch of PLA microspheres containing encapsulated TT

Time (Days)	Amount of plain T.T. released(Lf/ml)	Percentage release (%)	Cumulative percentage release
0	0	0.00	0.00
0.02	0	0.00	3.00
0.08	0	0.00	0.00
0.16	0	0.00	0.00
0.5	4	12.00	12.00
1	6	18.00	19.21
2	9	27.00	28.81
6	10	30.00	32.71
10	12	36.00	39.02
16	14	42.00	45.62
20	15	45.00	49.22
24	17	51.01	55.52
30	20	60.01	65.13
35	21	63.01	69.03
40	23	69.01	75.33
45	25	75.01	81.93
50	27	81.01	88.54
60	28	84.01	92.14

IV. Surface analysis of microspheres:

The surface analysis of the microspheres was examined by the Scanning Electron Microscope (SEM). The photographs were taken with a Scanning Electron Microscope (SEM, Hitachi, Tokyo, Japan).

RESULTS AND DISCUSSION:

I.A. Pre Formulation Studies:

To achieve the ideal microsphere formulations for both TT adsorbed and encapsulated., the systematic preformulation studies were carried out. The variables in the study were:

i. Cross linking agent concentration

ii. Polymer concentration

iii. Stirrer speed

iv. Dispersion medium composition

v. Washing solvents

The effect of each parameter was accessed in terms of uniformity in size, shape and yield of microspheres.

i. Effect of cross linking agent concentration:

Various batches of microspheres were obtained using different concentrations of the rigidising agent. It was found that the yield of microspheres was high but the formulations corresponding to 2%, 3%, 4%, 6% Gluteraldehyde, the microspheres were highly irregular, clumping of polymer was seen and the presence of Gluteraldehyde crystals was also noted. The 5% of gluteraldehyde gave highly uniform sized, spherical, smooth surfaced microspheres and the percentage yield of microspheres of the ideal combination was 89.4%.

ii. Effect of polymer concentration:

The various batches of microspheres were obtained with different concentrations of polymer were used in this study. It was found that the yield of microspheres was high but in the case of 0.2%, 0.5%, 2%, 3%, the microspheres show clumping, irregular with many gluteraldehyde crystals. The 1% polymer, gave highly spherical and smooth surface and the percentage yield of the ideal formulation was 92.3%. The size of microspheres were seen to be directly proportional to polymer concentration.

Figure No.6 In vitro of TT in terms of Lime flocculation from the batch of PLA microspheres containing encapsulated TT

iii. Effect of stirrer speed:

Various stirrer speeds of 1000, 1200, 1500, 1700, 2000 rpm were used to find out the optimum speed necessary to obtain microspheres. From this it was found 1700rpm was the speed at which homogenous mixing of polymer in the dispersion medium took place and the microspheres were uniform sized and spherical in shape and the percentage yield of the ideal formulation was 90.2%.. The higher stirrer speeds (2000 rpm) were difficult to work with due to excessive splashing and in the case of lower stirrer speeds (1200, 1500rpm) failed to convert the polymer into microspheres and showed clumping.

iv. Effect of dispersion medium composition:

Different vegetable oils were used along with toluene in varying proportions in order to obtain the right viscosity of the dispersion medium. Cotton seed oil and castor oil were left out of further study because of excessive loss of the reaction mixture from the container during stirring. The percentage yield was very less. The mixture of linseed oil and toluene was found suitable under the given conditions of agitation and yielded microspheres that were highly uniform and spherical and percent yield of ideal formulation is 94.86%.

v. Effect of washing solvents:

Formulation of TT loaded microspheres requires TT to come into contact with various solvents. Washing of the microspheres was carried out using dichloromethane and toluene. Toluene rendered the microspheres discrete but dichloromethane fail to do so and coalescing of microspheres was observed. To obtain dry-free flowing microspheres acetone was used as the final washing solvent.

vi. Determination of average diameter of microspheres:

Size distribution analysis was carried out by counting 400 microspheres from each of the above batches and the average size and standard deviation was calculated as per the procedure discussed under materials and methods. It was seen that there was proportional increase in microspheres size with an increase in Gluteraldehyde concentration, polymer concentration and inversely proportional with stirrer speed. In the case of varying Gluteraldehyde concentrations, the microsphere size of ideal batch (5%) was found to be 160±10 and the 3% and 4% of Gluteraldehyde shows the size was less but the standard deviation was high. Varying the polymer concentration, stirrer speed and dispersion medium composition, the microsphere size of the ideal batches were 140±5, 140±15, 162.5±7.5 respectively. Standard deviations of the other concentrations of the polymer, stirrer speed and dispersion medium composition were very high, as well as size.

B. Formulation:

From the preformulation studies, the ideal parameters which gave discrete microspheres having uniform size, shape, which gave a high yield of microspheres and showed good loading efficiency were chosen. For both TT adsorbed and encapsulated in PLA microspheres, the 1% of polymer and 5% of Gluteraldehyde as rigidising agent stirred in a dispersion medium of equal volumes of toluene and linseed oil at 1700 rpm.

After harvesting the microspheres, viz. separating the microspheres from the reaction mixture by centrifugation washing with toluene and acetone, the product was a free-flowing, cream coloured powder.

II. In vitro dissolution study:

In vitro dissolution studies were carried out on both batches for a period of 96 hours and 60 days in a dissolution apparatus at a temperature of 37^oC and stirring speed 95 rpm. The samples were analysed by Lf testing. It was found that in the case of the TT adsorbed batch, TT started to release on the 6^thhours itself. Results were shown in Table No.1 Fig. No. 5.

From the plots of cumulative % release Vs. Time in hours, the release of TT from TT adsorbed PLA microspheres was steady upto the 96 hours and the cumulative percentage release at this point being 91.84%.

From the plots of cumulative % release Vs. No. of days, the release of TT from TT encapsulated PLA microspheres was steady and follows first order upto the 60^th day and the cumulative percentage release at this point being 92.14%.

In the case of TT encapsulated microspheres, it showed different release profile and the TT started to release only on the 0.5 day and thereafter showed a pulsed release upto the 60^th day, with a cumulative percentage release of 92.14% of the vaccine. After this, a plateau was seen. Results were shown in Table No.2 Fig. No. 6.

CONCLUSION:

The main objective of the study was to develop a single contact vaccine using controlled release systems and to improve the efficacy of currently available vaccines.

In the past years PLA has been studied extensively by various scientists and has been found to be an excellent polymer approved for human use and is incorporated into controlled release microspheres which can provide continuous release of Tetanus toxoid over a period of several months.

The PLA microspheres were prepared by emulsion cross-linking method. Preformulation studies were done during the formulation of PLA microspheres to obtain ideal concentration of polymer (1%) and cross-linking agent (5%). Size distribution analysis was also studied. From size distribution analysis, uniformity of size, shape, percentage yield and TT loading efficiency, the ideal batches of PLA microspheres were chosen for in-vitro studies.

In-vitro dissolution studies were performed. In the case of TT adsorbed onto PLA microspheres, the release follows more or less a first order kinetics but in the case of TT encapsulated PLA microspheres there was a delayed initial release which thereafter showed a pulsed release upto the 60^th day. The release was estimated by Lf testing.

In both release studies, a high percentage of the loaded TT was released. At the end of the study, TT adsorbed onto PLA microspheres showed approximately 91.84% release of the vaccine, whereas the TT encapsulated in PLA microspheres showed approximately 92.14% release.

The Scanning Electron Micrograph shows that TT loaded PLA microspheres were spherical in shape and having smooth surface.

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A Saravanakumar*, Sunita Minz, Madhulika Pradhan, Pavani Sure, Atul N Chandu, Umesh Mishra, K Kamalakannan and T Sivakumar.

*Corresponding Author E-mail:saravanan_biotech@yahoo.com

A Saravanakumar*, Sunita Minz, Madhulika Pradhan, Pavani Sure, Atul N Chandu, Umesh Mishra, K Kamalakannan and T Sivakumar.

*Corresponding Author E-mail: saravanan_biotech@yahoo.com

*Corresponding Author E-mail:saravanan_biotech@yahoo.com