The naturally occurring substances like alginates, found in brown algae are the main attraction for the development of controlled release drug delivery systems. Alginates can be considered as block polymers, which mainly consist of mannuronic acid (M), mannuronic- guluronic (MG) blocks and is known to be non toxic when taken orally and also have a protective effect on the mucous membranes.^9-12 Also the dried alginate beads have property of reswelling and thus they can act as controlled release system.¹³ Microspheres have attracted the main interest because they are promising systems to fulfill the requirements of controlled release and drug targeting.¹⁴

Mucoadhesive microspheres have been proved to be the excellent carriers in the design of drug delivery systems to prolong the residence time of the dosage form at the site of application or absorption and to facilitate intimate contact of the dosage form with the underlying.¹⁵

Nicotine bitartrate dihydrate is a diacidic salt of free base of nicotine with tartaric acid. It undergoes first pass metabolism upto ~ 70% when administered orally.⁴ Hence the present research work was designed with an aim to develop novel mucoadhesive microspheres to explore the buccal administration of nicotine bitartrate dihydrate which may help to increase the bioavailability.

2. MATERIALS AND METHODS:

2.1 Materials:

Nicotine bitartrate dihydrate was obtained as a generous gift from Alchem International, Pvt. Ltd., Gujarat. Sodium alginate &. Span 80 were obtained from S.d.fine chemicals, Mumbai. Calcium chloride was obtained from Qualigens, Mumbai. Other reagents and chemicals of analytical grade were used.

2.2 Methods:

2.2.1 Preparation of mucoadhesive microspheres:¹⁶

The mucoadhesive microspheres were prepared by using the ionic cross linking technique. The polymer (3.5%w/v) was dispersed in water and stirred under the overhead stirrer for 2 minutes to obtain a homogenously viscous dispersion. In case of the drug loaded batches drug was dissolved in polymer solution. The liquid paraffin was used as an external phase in this process was taken in another beaker and Span 80 (2%w/v) used as an emulsifier was added to external phase. The pre emulsion was formed by addition of the polymer solution to the external phase by continuous stirring under the overhead stirrer. The stirring rate was varied from 1000- 1500 rpm for the period of 1hour. Further, the solution of cross linking agent (5%w/v) was added drop wise to the pre emulsion with continuous stirring and kept for 10 minutes for sufficient curing of the microspheres. Thus the microspheres formed were then filtered using the vacuum filtration method and collected after washing with isopropyl alcohol solution and air dried.

2.2.2 Effect of variables:

To study the effect of variables on percentage yield different batches were prepared using 3² factorial design. Drug: polymer ratio and polymer: cross linking agent ratio was selected as two independent variables. Effect of these variables on percentage yield is shown in fig. 1. Amount of Span 80 was kept constant. Values of all variables and batch codes are as shown in table1.

Fig: 1 Response surface graph for effect of drug: polymer ratio and polymer: cross linking agent ratio on % yield.

Table 1: Experimental design with coded levels of variables and actual values

Batch code	Factor combination	Drug: Polymer ratio	Polymer: cross linking agent ratio (%w/v)	% yield
B1	(-1,-1)	1:1	1:1	61.36
B2	(-1,0)	1:1	1:1.5	64.06
B3	(-1,1)	1:1	1:2	39.31
B4	(0,-1)	1:2	1:1	72.38
B5	(0,0)	1:2	1:1.5	96.32
B6	(0,1)	1:2	1:2	21.59
B7	(1,-1)	2:1	1:1	56.38
B8	(1,0)	2:1	1:1.5	59.48
B9	(1,1)	2:1	1:2	29.78

3. Evaluation of drug loaded microspheres:

The batches (B1-B9) were employed for the further physicochemical evaluation, which can be explained as below:

3.1. Percent yield:¹⁷

The percentage of production yield (w/w) was calculated from the weight of dried microspheres (W₁) recovered from each of 3 batches and the sum of the initial dry weight of starting solid materials (W₂= Wt. of sodium alginate + Wt. of Cross linking agent) as the following equation:

% of production yield = W₁/ W₂× 100

The results are shown in table 1 and fig1.

3.2. Particle size determination:

The size and size distribution of the microspheres was analyzed using optical image analyzer. The particles were counted in different field, average particle size & particle size distribution was recorded. The results are given in table 2.

3.3. Angle of Repose:

The angle of repose gives an indication of the flow ability of the substance. It is the measure of inter particle friction. It was measured by the fixed funnel method. Funnel was adjusted such that the stem of the funnel lies 2 cm above the horizontal surface. The weighed amount of microspheres was allowed to flow from the funnel under the gravitational force till the tip of the pile just touched the tip of the funnel, so the height of the pile was taken as 2 cm. Drawing a boundary along the circumference of the pile and taking the average of six diameters determined the diameter of the pile.

Table 2: Evaluation of selected batches of mucoadhesive microspheres

Sr. No.

Test

Particle size(µm)

126

185

100

Angle of Repose(θ)

32.39

±0.2

30.78

±0.1

30.54

±0.3

31.23

±0.5

30.53

±0.2

31.22

±0.4

28.34

±0.4

32.33

±0.1

31.46

±0.5

Bulk Density (g/cc)

0.660

±0.02

0.645

±0.04

0.574

±0.04

0.651

±0.02

0.634

±0.02

0.578

±0.05

0.559

±0.05

0.567

±0.04

0.584

±0.5

Tapped Density (g/cc)

0.675

±0.08

0.669

±0.06

0.569

±0.06

0.679

±0.06

0.654

±0.05

0.554

±0.5

0.560

±0.08

0.580

±0.06

0.572

±0.5

Drug Content (%)

95.67

±0.2

99.04

±0.3

101.56

±0.3

99.38

±0.11

102.14

±0.2

99.55

±0.5

99.98

±0.4

101.56

±0.3

99.61

±0.5

Swelling index

1.019

1.037

1.033

1.143

1.167

0.970

0.971

1.033

0.969

Muco- adhesion time (hrs)

6.3

±0.2

6.5

±0.5

6.12

±0.5

8.0

±0.8

8.4

±0.5

6.1

±0.5

6.12

±0.5

7.0

±0.2

6.1

±0.5

The angle of repose was recorded for all the batches (B1-B9). The experiment was done in triplicate. The results are given in table 2.

3.4. Bulk Density:^18,19

A known weight of microspheres (2.5g) was allowed to flow in a fine stream into a graduated cylinder of a mechanical tapping device. The measuring cylinder was tapped until no further change in the volume was observed (100 taps). The final volume after tapping was noted (V_b). The experiment was done in triplicate.

Untapped bulk density:

It was obtained as weight of microspheres divided by the initial untapped volume (V_u).

δ_u =M / V_u

Tapped bulk density:

It was obtained as weight of microspheres divided by the final tapped volume.

δ_u = M / V_b

The results are enlisted in table 2.

3.5. Drug content:

The accurately weighed quantity of microspheres (50 mg) were taken in 10 ml of 0.1N HCl and allowed to vortex under the magnetic stirrer for 15 minutes, to allow the complete rupture of the microspheres and the complete release of the drug. The drug solubilized in the solvent was then filtered through the Whatmann filter paper no. 20 and the filtrate was used for the determination of drug content. The drug content was estimated by UV spectrophotometric analysis at λmax 259 nm against the filtrate obtained from the blank microspheres processed in the same manner. The results are enlisted in table 2.

3.6. Swelling index:²⁰

Swelling of microspheres was determined by soaking 0.5 ml of microsphere bed in 5 ml phosphate buffer pH 6.8 in 10 ml measuring cylinder. Volume of microspheres was determined after 12 hrs. Swelling index was calculated by using following formula:

Swelling index= Volume after 12 hrs/ original volume

The results are shown in table 2.

3.7. Mucoadhesion time:²¹

A freshly cut piece (5 cm long) of porcine buccal mucosa was obtained from a local slaughter house within 1 hour of killing the animal. It was cleaned by washing with isotonic saline solution. An accurate weight of microspheres was placed on mucosal surface, which was attached over a polyethylene plate that fixed in an angle of 40° relative to the horizontal plane, and pH 6.8 phosphate buffer warmed at 37°C was peristaltically pumped at a rate of 5ml/min over the tissue. The duration for complete washing of microspheres from the porcine buccal mucosa was recorded and averaged from 3 determinations. The results are enlisted in table 2.

3.8. In vitro drug release study:²²

Microspheres (equivalent to drug dose 6.15mg) were filled in dialysis membrane and tied on both sides with the help of a thread. The dialysis bag in turn was tied to the basket of the rotating paddle. The US Pharmacopeia XXIII rotating paddle method was used to study drug release from the microspheres; 200 mL of phosphate buffer (pH 6.8) was used as the dissolution medium, at 37.0 ± 0.5°C, and a rotation speed of 50 rpm was used. Samples of 5ml were withdrawn at predetermined time intervals of 15 mins, 30 mins, 1, 2, 3, 4, 5, 6, 7and 8 hours and replaced with the fresh medium. The samples were filtered through 0.2 µm Whatmann filter paper (Whatmann, Brentford, UK) and analyzed after appropriate dilution by UV Spectrophotometer at 259 nm. The results are shown in fig 4.

From the results of the above characterization tests the batch B5 of the drug-loaded microspheres were found to be optimized batch, which was used for the further characterization.

3.9 Kinetic modeling:²³

Further to explore the kinetic behavior, in vitro release were further fitted into the following Korsmeyer and Peppas equation: M_T/ M_∞= K tⁿ , where M_T and M_∞ are the amounts of drug released at time t and the overall amount released, respectively, K is the release rate constant and n is the release exponent indicative of release mechanism. The values of n, R and K are enlisted in the table 3.

Table 3: Results of Korsmeyer- Peppas Equation Treatment of in vitro drug release data

Batch Code	K	N	R	R²
B1	0.177	0.602	0.9808	0.9620
B2	0.176	0.601	0.9869	0.974
B3	0.174	0.604	0.9852	0.970
B4	0.413	0.420	0.9832	0.965
B5	0.399	0.439	0.9853	0.971
B6	0.409	0.421	0.9839	0.968
B7	0.369	0.545	0.9929	0.986
B8	0.364	0.554	0.9934	0.987
B9	0.365	0.548	0.9944	0.9888

3.10. Characterization of the optimized batch (B5) of drug loaded microspheres:

3.10.1. Appearance:

The surface morphology of the alginate microspheres was investigated using scanning electron microscope (Joel, JSM-6360, Japan). They were mounted onto the SEM sample stub using double-sided sticking tape and coated with platinum film. Scanning Electron photographs were taken at an accelerating voltage of 15 kV, chamber pressure of 0.6 mm Hg and 100X magnification shown in fig.3.

3.10.2. Particle size determination:

The size and size distribution of the microspheres was analyzed using optical image analyzer. The particles were counted in different field & average particle size & particle size distribution was calculated as shown in table 3 and fig. 2.

Fig. 2 SEM Photograph of optimized mucoadhesive microspheres (Batch B5)

3.10.3. Differential Scanning Calorimetry (DSC):

DSC is one of the most widely used calorimetric technique for qualitative and quantitative determinations of physiochemical properties of drug. DSC measures the heat capacity (CP) of the system as a function of temperature. The melting endotherm of nicotine bitartrate dihydrate, drug loaded batch was performed using Mettler-Toledo DSC 821 instrument. The sample was heated at a constant tare of 10⁰C/min over a temperature range of 30 to 300⁰C. DSC thermo analytical curve for nicotine bitartrate dihydrate and optimized drug loaded microspheres of (B5) are given in fig no. 3 and 4 respectively.

Fig. 3 DSC thermogram of pure drug (nicotine bitartrate dihydrate)

Fig. 4 DSC thermogram of drug loaded microspheres

Fig. 5 In vitro drug release profiles of selected batches of drug loaded microspheres

Fig. 6 Graph of correlation between in vitro drug release and ex vivo drug permeation studies

3.10.4. Ex vivo drug permeation studies:²⁴

Preparation of porcine buccal tissue: The mucosal membrane was excised by removing the underlying connective and adipose tissue of freshly slaughtered pig and was equilibrated at 37 + 1.0 °C for 30 mins in phosphate buffer pH 7.4. The microspheres equivalent to drug dose (6.15mg) were weighed accurately and placed on the porcine mucosa for the in vitro drug permeation studies by using the apparatus of modified Franz diffusion cell (fig.5.8.4.1). The receptor compartment was filled with phosphate buffer pH 7.4 (20 ml) and donor compartment was filled with the simulated saliva pH 6.8 (4 ml). The temperature was maintained at 37 ± 0.5 ⁰C. 2 ml sample was withdrawn from the receptor compartment at various time points upto 10 hours and replaced by fresh medium. The samples were filtered using Whatmann filter paper no. 20. Each sample was analyzed by using UV spectrophotometer at λ _max259 nm. The ex-vivo release profiles and correlation between in vitro drug release and ex vivo drug permeation studies are given in fig.6.

3.10.6. Stability studies:^25,26

Stability of the optimized batch (B5) was carried out as per ICH guidelines at 4-8⁰C, 25⁰C ± 2⁰C / 60% ± 5% RH, 30⁰C ± 2⁰C / 65% ± 5% RH & 40⁰C ± 2⁰C / 75% ± 5% RH for 3 months. Effects of temperature and RH on the physical and chemical attributes like appearance, color, odor and drug content during the stability period were studied.

4. RESULTS AND DISCUSSION:

The batches of the drug loaded microspheres were first evaluated for the percentage yield. As shown in fig. 1 and table: 1, it was observed that the percentage yield was found to be increased with the increase in the concentration of the polymer in the drug: polymer ratio B5, B1, B4, and decrease with concentration of the cross linking agent in the polymer: cross linking agent ratio B6, B9, B3, B7, B4. The batch (B5) with the drug: polymer ratio 1:2 and polymer: cross linking agent ratio 1:1.5 gave maximum yield (96.32%) of microspheres. This may be attributed to the optimum concentration of the cross linking agent for the cross linking of the polymer residues and optimum concentration of the polymer for the engulfment of the drug. The batches B3, B6 and B9 showed inefficient yield of 39.31, 21.59, and 29.78% respectively.

Particle size determination:

The particle size of the microspheres was determined using the optical image analyzer. As shown in the table 2 the particle size of the microspheres was found to be in the range 84-105 μm. The increase in the particle size of the microspheres can be attributed to the more amount of the sodium alginate residues for cross linking with the divalent cations of the calcium ions, whereas the increase in the cross liking agent led to the decrease in the particle size due to excessive curing of the polymer.

Angle of repose:

As shown in the table 2 the angle of repose of all the selected formulation was found to be in the range 28° - 33°. The values were found to be below 35° and hence according to the standards of the angle of repose in the I.P. the microspheres indicated good flow properties.

Bulk density and tapped density:

The term density refers to a measure used to describe packing of particles. It is weight per volume of the substances expressed in gm/cm³. As shown in table 2 the bulk density and tapped density were found to be in the range 0.559± 0.05 - 0.660 ±0.02 and 0.560 ±0.08 - 0.669 ±0.06. The observations were found to be satisfactory in case of all the batches to give good flow properties. Thus it indicated that microspheres were found to be bulkier in nature.

Swellability:

Swellability is an indicative parameter for rapid availability of drug solution for diffusion with greater flux. It was found that the swelling index decreases with the increase in the concentration of the cross linking agent which may be attributed to the increase in the rigidity of the microspheres and vice versa was the case with increase in the polymer concentration which may be due to increase in the hydrophilicity of the increased polymer concentration. All the batches showed a significant index of swelling between a range of 0.950- 1.170 (table 2).

Drug content (%):

The drug content of the formulations (B1-B9) was found to be in compliance range between 95- 105%w/w (table 2).

Mucoadhesion time:

Mucoadhesion is an important factor as it imparts the advantage of increase in the contact time of the drug with the site of absorption. As shown in table 2 it was observed that the mucoadhesion time increased with increase in the concentration of the polymer relative to drug concentration and decreased with the relative increase in the concentration of the cross linking agent of the polymer concentration in the formulations. The batch B5 with drug: polymer ratio 1:2 and polymer: cross linking agent ratio 1:1.5 was found to have maximum mucoadhesion time of 8 hours.

In vitro drug release study:

The drug profiles of the selected batches for optimization are shown in the fig.4. Formulations of batches B1 and B2 showed entire drug release in 8 hrs, while the formulations of batches B4 and B5 showed the complete drug release in 10 hours. In case of batches B7 and B8 rapid drug release was found within 6 hours. It indicates that as the polymer concentration increases the time required for the complete release of the drug which may be attributed to the increase in the path that the drug has to traverse during the diffusion from the microspheres.

The graph (fig.4) reveals that the drug release followed a biphasic pattern of drug release is characteristic of matrix diffusion kinetics.

Kinetic modeling:

Further to explore the kinetic behavior, in vitro release were further fitted into the following Korsmeyer and Peppas equation:

M_T/ M_∞= K tⁿ

Where, M_T/ M_∞ is the fraction of drug released after time t, K is a kinetic constant, and n is a release exponent that characterizes the drug transport and was in the range of 0.420- 0.602, indicating the Fickian drug diffusion (table 3).

Thus, from the above observations it can be concluded that batch B5 is the optimized batch with maximum production yield of 98.32%, significant mucoadhesion time of 8.4±0.5 hrs and expected controlled drug release for 8 hours. For further studies of the drug permeation through the buccal mucosa the ex vivo drug permeation studies were conducted.

Characterization of the optimized batch (B5) of drug loaded microspheres:

Appearance:

Scanning electron microscopy revealed that alginate microspheres in the optimized batch were discrete and spherical in shape with a smooth outer surface (fig.2). The average diameter of the microspheres was found to be 105 μm.

Differential Scanning Calorimetry (DSC) studies:

DSC is very useful in the investigation of the thermal properties of the microspheres, providing both the qualitative and quantitative information about the physicochemical state of drug inside the microspheres. There is no detectable endotherm if the drug is present in a molecular dispersion or solid solution state in the polymer microspheres loaded with the drug. DSC thermogram of pure drug showed a sharp peak in the endotherm at 89.10⁰C (fig.3) and where as that of drug loaded microspheres showed a shifted peak of endotherm at 124⁰C (fig.4). The shift in the peak reveals the engulfment of the drug in the microspheres.

Ex vivo drug permeation studies:

The ex vivo permeation studies were conducted on the optimized batch (B5) of the mucoadhesive microspheres in order to evaluate the in vivo performance of the drug permeation through the buccal mucosa. The graph (fig. 6) indicates that 88.91% of the drug was found to be permeated through the buccal mucosa in 8 hours.

The correlation between in vitro drug release (%) and ex vivo permeation studies was found to be positive with the correlation coefficient (R²) of 0.9960 (fig.6).

Stability studies:

Stability of the optimized batch (B5) of mucoadhesive microspheres was carried out for 3 months at various temperatures as per ICH guidelines. The drug content of the microspheres was same throughout the stability studies period. Whereas the colour of the microspheres showed an significant change from yellowish to brown at the end of 3 months of the stability studies period, which may be due to the degradation of the polymer. The microspheres were found to be unstable at the accelerated temperatures.

CONCLUSION:

The results of our study clearly indicate that mucoadhesive microspheres can be a good pass way for the first pass metabolism of nicotine bitartrate dihydrate. However, more extensive pharmacokinetic and pharmacodynamic studies are needed to be done.

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Received on 13.12.2009

Accepted on 10.02.2010

Research Journal of Pharmaceutical Dosage Forms and Technology. 2(1): Jan. –Feb. 2010, 90-95