Figure 2: SEM photomicrograph of pure drug (A), RL PO (B) and RS PO (C) microspheres.

Drug Content and Percent Entrapment Efficiency (% EE):

An amount of 25mg of the samples was weighed and dispersed in 10 ml of 0.01 N HCl. It was sonicated for 10 min, the samples were centrifuged at 2000 rpm for 10 min. Supernatant was diluted with suitable quantity of 0.01N HCl and analyzed through UV-Visible Spectrophotometer (Shimadzu UV-1700, Tokyo, Japan) at 281nm.

Dissolution Studies:

A Lab India Dissolution test apparatus type II (Paddle) at rotation speed of 100 rpm was used for the study. Dissolution of the samples was carried out on an equivalent of 300 mg of RFB using 0.01 N HCl as dissolution media. The volume and temperature of the dissolution media were 900 ml and 37 ± 0.2 ⁰C, respectively. After fixed time interval 5 ml of samples were withdrawn (sink was maintained) and assayed through ultraviolet absorbance measurement at 281 nm with UV-Visible Spectrophotometer (Shimadzu UV-1700, Tokyo, Japan) by an analytically validated method (r² = 0.9995). To increase the reliability of the observations, the dissolution studies were performed in triplicate.

Scanning Electron Microscopy (SEM):

Morphological evaluation of the freeze dried samples was performed by JSM-6400 scanning electron microscope (JEOL, Tokyo, Japan). Samples were fixed on aluminum stubs with conductive double sided adhesive tape and coated with the gold by sputter coater at 50 mA for 50 s.

Table 1: Different release models applied to the dissolution data of the RL and RS microspheres.

Batch

Zero Order

First Order

Hixon Crowell

Korsmeyer Peppas

Higuchi Plot

0.9421

0.6511

0.8090

0.85448

0.9961

K= 0.036

0.9035

0.5812

0.7449

0.7332

0.9823

K= 0.035

Table 2: Flow properties of the drug, RL and RS microspheres.

Parameter	Pure Drug	RS	RL
Bulk Density (σ_b) (g/ml)	0.270	0.334	0.326
Tapped density (σ_t) (g/ml)	0.415	0.431	0.428
Carr’s Index (%)	34.94	22.50	23.83
Hausner ratio	1.54	1.29	1.31

Powder X-Ray Diffraction (PXRD):

Crystallinity of the drug and the samples were determined using the Philips Analytical XRD (Model: PW 3710, Holland), with copper target. The conditions were: 40 kV voltage; 30 mA current; at room temperature. The samples were loaded on to the difffractometer and scanned over a range of 2θ values form 5 to 60 ⁰ at a scan rate of 0.02 ⁰/min.

Differential Scanning Calorimentry (DSC):

Thermal properties of the drug and the samples were analyzed by DSC (TA Instruments, USA, Model: SDT 2960). The samples were heated in a hermetically sealed aluminum pans. Heat runs for each sample were set from 30 to 350 ⁰C at a heating rate of 10 ⁰C/min, using nitrogen as blanket gas.

Photo-stability Studies:

Photo-stability study of the drug and the samples was performed in a chamber containing UV lamp, 30W, emitting radiations at 254 nm. The drug and the samples (equivalent to 10 mg of the drug, n=3) were kept in a petri dish. The samples were collected after 10, 20, 30 days and analyzed for drug content.

Stability studies:

RL and RS microspheres were passed through accelerated stability studies. The samples (each equivalent to 5mg of drug, n=3) were kept for stability studies at 40 ± 2 ⁰C and 75 ± 5 % RH for a period of 3 months in environmental test chamber (HMG India, Mumbai, India). The samples were kept in glass vials sealed with rubber plugs. After 30, 60 and 90 days, the samples were taken out and analyzed for drug content

Moisture Uptake Study:

A weighed quantity of drug and samples (each 10 mg) was placed in a crucible at accelerated condition of temperature and humidity, 40 ± 2 ⁰C and 75% ± 5 % RH respectively in environmental test chamber (HMG India, Mumbai, India). The gain in weight of the drug and samples was determined.

Flow Properties:

Flow properties of the drug and microspheres were studied by determining the bulk density (σ_b), tap density (σ_t), Carr’s Index and Hausner ratio.

Carr’s Index = [(σ_t – σ_b)/ σ_t] x 100

Hausner ratio = (σ_t)/ (σ_b)

Figure 3: XRD pattern of pure drug (A), RL PO (B) and RS PO (C) microspheres.

RESULTS:

The polymeric microspheres of RFB were prepared by emulsion solvent diffusion method, utilizing eudragit RL PO and RS PO polymers. The process was carried out at constant stirring rate of 2000 rpm using baffles which has enhanced the processing efficiency. The rate of addition of the organic phase to the stabilizer solution has a direct effect on size of the microspheres. The organic phase of the drug and the polymers were added at different rates with the help of a syringe using 16# gauge needle. It was noticed that at slower rate of addition (12 ml of organic phase in 3 min) uniformity in the size and shape of the microspheres increased. The drug content of the RS and RL microspheres was found to be 43.16 ± 1.21 and 45.48 ± 0.94 % respectively. However, the entrapment efficiency of the RS and RL microspheres was 86.32 ± 2.42 and 90.96 ± 1.88 % respectively.

Dissolution studies of the drug and samples were carried out in 0.01N HCl. Figure 1 depicts the release rate of the drug from the microspheres. The untreated drug has released 99.47% of the drug at the end of 2 hrs, while the microspheres have sustained the release upto 12 hrs. RL and RS microspheres released 98.04 % and 93.76% of the drug respectively, at the end of 12 hrs. The mechanism of drug release was studied by applying different dissolution models. Higuchi plot has explained the release of the drug from the microspheres (For RS, r² = 0.9961; for RL, r² = 0.9823) (Table 1).

Figure 4: DSC thermo grams of pure drug (A), RL PO (B) and RS PO (C) microspheres.

Spherical shape of the microspheres was confirmed by the SEM images whereas the drug was having smooth surface with discrete shape (Fig. 2). The untreated drug and the prepared microspheres were checked for its crystallinity and change in enthalpy by performing the X-RD and DSC studies. XRD pattern of the untreated drug and microspheres did not show any characteristic peak denoting its amorphous nature (Fig. 3). Figure 4 predicts the results for DSC studies, showing an endothermic peak at 122.45⁰C for untreated drug, while there is a shift in this endothermic peak at 107.68⁰C for RL microspheres and this peak was diminished in case of the RS microspheres indicating complete entrapment of the drug in the polymer matrix.

Microspheres were passed through photo-stability study, drug content of the RL and RS microspheres were found to be, 98.74 ± 0.11 and 99.12 ± 0.06, after 10 days, 97.87 ± 0.14 and 98.66 ± 0.08, after 20 days, 96.93 ± 0.21and 97.45 ± 0.19, after 30 days. The results of the stability study indicated no change in the physical properties. After accelerated stability studies, drug content of the RL and RS microspheres were observed to be, 99.41 ± 0.07 and 99.71 ± 0.04, after 30 days, 98.43 ± 0.04 and 98.51 ± 0.05, after 60 days, 97.88 ± 0.05 and 97.90 ± 0.03, after 90 days. In moisture uptake study, no change in the weight of the microspheres was observed.

The flow properties of the drug were studied by bulk and tap density determination. The Carr’s index and Hausner ratio were calculated from the bulk and the tap density. Table 2 depicts the values of flow properties of the drug and the microspheres. The bulk and tap density of the untreated drug were observed to be 0.270 and 0.415 g/ml respectively (Table 2). The values of the bulk (σ_b) and tap (σ_t) density for the RL microspheres were discovered to be 0.326 and 0.428 g/ml respectively, whereas for RS microspheres these values were 0.334 and 0.421 g/ml respectively. The Carr’s Index (compressibility index) and the Hausner ratio were determined from the bulk and the tapped density. The Carr’s index and Hausner ratio for the untreated drug was observed to be 34.94 % and 1.54 respectively indicating poor flow. The Carr’s index and Hausner ratio for RL microspheres were observed to be 23.83 % and 1.31 respectively, while for RS microspheres it was 22.50 % and 1.29 respectively (Table 2).

DISSCUSSION:

The microspheres of RFB were prepared by emulsion solvent diffusion. Methylene chloride and ethanol were mixed together as organic phase. Ethanol is water miscible solvent, hence it is expected to diffuse into the large phase and small spherical globules of methylene chloride (water immiscible solvent) form, aftermath organic phase will evaporate with formation of micro-sized spherical particles. PVA a mostly used surfactant was used as a stabilizer to stabilize these formed particles. Baffles used in the process increases the turbulent flow; avoid foam and vortex formation. Addition rate of the organic solvent has high impact on the size uniformity; it may be due to the rate of diffusion of the organic phase into the aqueous phase. Total 12 ml of the organic phase was added at 0, 1 and 3 min. At slower rate of addition (12 ml in 3 min) the organic phase got sufficient time to diffuse uniformly throughout the aqueous phase whereas at faster rate of addition of the solvent, it may not get sufficient time to diffuse uniformly throughout the large phase. Entrapment efficiency (RS = 86.32 ± 2.42 % and RL = 90.96 ± 1.88 %) of the drug in microspheres was quite good and very less amount of the drug was wasted while preparation.

The dissolution studies predicted more than 90% of the drug release from microspheres at the end of 12 hrs (Fig. 1). Eudragit is chemically polymethacrylates. Eudragit RL and RS refers to the ammonio-methacrylate copolymers synthesized from acrylic acid and methaycrylic acid esters with RL and RS having 10 and 5 % of functional quaternary ammonium groups respectively. These polymers are mostly used for sustained release. Ammonium groups are present as salt, which increases the solubility and pH independent permeability of the drugs (8). Initially bust release was observed from the RL microspheres, it may be due to the adsorption of drug on the surface of the microspheres prepared. Interestingly among the applied release kinetic determinant models, the Higuchi models resulted in highest regression (For RS, r² = 0.9961; for RL, r² = 0.9823). Although the diffusion and dissolution are the mechanisms operating for the dosage forms one of them is dominating. The values of r were highest when evaluating the diffusion methodology suggesting that the mechanism of release was through wetting and diffusion.

Morphological characteristics of the drug and the prepared microspheres were examined by SEM (Fig. 2). The untreated drug was observed with large particles with

smooth surface and discrete shape. In contrast the prepared microspheres are spherical in shape. As compared to the RS, RL microspheres were with rough porous surface support the fast release of the drug from the RL microspheres. The untreated drug did not show any peak in its XRD spectra, dictating its amorphous nature (Fig. 3). Similarly, RL and RS microspheres did not shown any peak in their XRD spectra, denoting retention of the amorphous nature of the drug in microspheres. The DSC results for the untreated drug showed an endothermic peak at 122.45 ⁰C correspondences to its melting (Fig. 4). In comparison, for RL an endothermic peak was observed at 107.68 corresponding to its melting point. For RS microspheres no endothermic peak was observed in the heating range indicating complete entrapment of the drug in the microspheres.

Increased stability of the drug in the microspheres was discovered from the accelerated and photo-stability studies; due to the entrapment of the drug in the polymer matrix. In photo-stability study slight decrease in the drug content may be due to the presence of the drug on the surface of the microspheres. The moisture uptake study was done to confirm the non-hygroscopic nature of the microspheres. No change in the weight of the microspheres indicated its non-hygroscopic nature. Pure drug showed 34.94 % value of carr’s index indicating very poor flow (9), whereas for RL and RS microspheres the values were 23.83 and 22.50 % (Table 2) respectively dictating improved flow properties which may be due to the presence of the polymer matrix and the spherical shape of the microspheres.

CONCLUSION:

From the above discussion it is concluded that the dissolution of the drug from microspheres was sustained up to 12 hrs with improved Photo and accelerated stability of the drug. Further optimization of the processes will allow its implementation on large scale.

ACKNOWLEDGEMENTS:

We are thankful to Lupin Ltd, Aurangabad for providing gift sample of RFB, Alembic, Vadodara for providing excipients and Shivaji University, Kolhapur for getting facilities to perform XRD and DSC.

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