FORMULATION AND EVALUATION OF BSA LOADED PLGA MICROPARTICLES

FORMULATION AND EVALUATION OF BSA LOADED PLGA MICROPARTICLES

Santosh Voruganti*, J. Surya Chandra Padman

Department of Pharmaceutics, Mother Teresa College of Pharmacy, Ghatkesar, Hyderabad, Andhra Pradesh, India

ABSTRACT: Protein drug delivery has emerged to be an important area of research in the field of novel drug delivery technology. The objective of the study was to prepare poly (D, L-lactide-co-glycolide) (PLGA) microspheres containing bovine serum albumin (BSA) as a  model  drug  and  to  evaluate  the  various  physicochemical  characteristics  of  the formulations, namely morphology, particle size, FTIR, DSC, BSA encapsulation  efficiency  and   in-vitro  BSA  release profile. BSA-loaded microspheres were  prepared  by  double  emulsion  solvent  evaporation  method  with  different  BSA: PLGA  ratios  and  at  different  speeds  of  homogenization  keeping  the  amount  of  BSA constant in all the formulations. Out of those 1:10 was selected as a optimized (drug: polymer) for BSA loaded PLGA microspheres, there after internal parameters like volume of inner aqueous phase(2ml), volume of DCM(10ml), concentration of polymer (9.09%),speed of homogenization, were selected as a optimized formulation parameters. Accelerated  stability  testing  was  performed  with  the  optimized formulations  for  a  period  of  eight  weeks. The  mean  particle  size  and  encapsulation efficiency  of  the  microspheres  were  found  to  decrease  as  the  speed  of  homogenization increased.  And  the  same  were  found  to  increase  simultaneously  with  increase  in  the amount of polymer. The in vitro release study showed a slow and steady release pattern of BSA. Accelerated stability studies indicated that formulations here stable during the period of study. Thus, a sustained release formulation of protein loaded PLGA microspheres was developed

Microspheres,Bovine Serum Albumin, PLGA 50:50, Double emulsion solvent evaporation, SEM, DSC, Homogenization

INTRODUCTION: Microspheres have played a vital role in the development of controlled/sustained release drug delivery systems. Microspheres have been of particular interest from the pharmaceutical point of view providing the possibility to achieve sustained and controlled drug release ¹. In a very short time, since their emergence, the field of controlled delivery of proteins has grown immensely. Because of their relatively large size, they have low transdermal bio-availabilities. Oral bioavailability is generally poor since they are poorly absorbed and easily degraded by proteolytic enzymes in the gastrointestinal tract ocular and nasal delivery is also unfavorable due to degradation by enzymes present in tissues and nasal mucosa, thus parenteral delivery is currently most demanding and suitable for delivery of such molecules. In systemic delivery  of proteins, biodegradable microspheres  as parenteral depot formulation  occupy an important place because of  several  aspects like  protection of sensitive proteins from degradation, prolonged or modified release, pulsatile release patterns. The main objective in developing controlled release  protein injectables  is avoidance of regular invasive doses which in turn provide patient  compliance, comfort as well as control over blood levels. BSA  is a water-soluble protein, which is chosen for loading in to  microspheres, development of microspheres  containing  BSA successfully  gives a way for  further development of other proteicious drugs, which have similar properties of BSA ². Among the various tentative for improving the administration of proteins, the microencapsulations into biodegradable polymers ³ represent a practical and promising approach. On the other hand, biodegradable microspheres can be injected ³ subcutaneously or intramuscularly and release the entrapped proteins for extended periods of time ⁴.

TABLE 1: COMMERCIAL BIODEGRADABLE DRUG PRODUCTS

MATERIALS AND METHODS: Materials: BSA was obtained from Celon Laboratories (Hyderabad), PLGA was obtained fromEvonik Roehm Gmbh (Germany), Poly vinyl alcohol obtained from S.D. Fine chemicals (Mumbai). All solvents were HPLC grade and were obtained from Merck chemicals, Mumbai.

Preparation of BSA Microspheres: Double emulsion (W/O/W) solvent evaporation method was employed in the preparation of BSA loaded PLGA microspheres. This method for preparation of microsphere was reported to overcome the problem of low encapsulation efficiency of water soluble drug prepared by conventional water/oil emulsion solvent evaporation method. Polymer [Poly (L-lactic glycolic acid) (PLGA)] is dissolved in organic phase DCM (Dichloro methane).

In this organic phase, aqueous drug solution is emulsified using high speed homogenizer (IKA) operating around 10000 rpm for about 5 minutes to prepare water¹ /oil (w¹/o) primary emulsion. This primary emulsion is added to external aqueous phase containing surfactant (poly vinyl alcohol is used to prepare w¹/o/w² emulsion) at homogenizer speed around 8000 rpm for 3 minutes and then shifted to mechanical stirrer which was stirred at 1000 rpm for 1 hour at 2-8^oC then next 2 hrs at room temperature to permit evaporation of DCM.

The microspheres obtained is collected by centrifugation, filtration and then dried. Formed microspheres are evaluated ⁵.

Evaluation of Microspheres:

Percentage yield: The prepared microspheres were collected and weighted. The actual weight of obtained microspheres divided by the total amount of all material that was used for the preparation of the microspheres multiplied by 100 gives the % yield of microspheres (equation) ⁷:

% Yield = Actual weight of product/ Total weight of excipients and drug × 100

Drug entrapment efficiency: The amount of drug entrapped was estimated by dissolving the 100mg of microspheres in DCM and water in 3:1 ratio ,under vigorous shaking for 1hr, the resultant solution is centrifuged, both layers were separated, cytarabine was soluble in water but not in DCM. The drug content in aqueous solution was analyzed Spectrophoto-metrically by using UV-Vis spectrophotometer at 272.7nm with further dilutions against appropriate blank. The amount of the drug entrapped in the microspheres was calculated using the formula ⁶:

Encapsulation efficiency=

Actual weight of drug in sample x 100

Theoretical weight of drug in sample

Scanning Electron Microscopy: Microspheres were observed and photographed with scanning electron microscopy (SEM) (Using Hitachi-S-3700N). Scanning electron microscopy was carried out to study the morphological characteristics of cytarabine PLA microspheres. The samples for the SEM analysis were prepared by sprinkling the microspheres on one side of adhesive stub. Then the microspheres were coated with gold (100A°) before microscopy. Finally the morphology of the microspheres was observed with the scanning electron microscopy ⁷.

Particle Size Analysis: Determination of average particle size of cytarabine microspheres was very important character. It was carried out by using Malvern instruments, Startech Labs Pvt. Ltd.

In-vitro Drug Release: An in vitro release method using a regenerated cellulose membrane dialysis apparatus (Float-a-Lyzer) was suitable for studying in vitro release of cytarabine-loaded biodegradable microspheres. Microspheres suspension containing known amount of drug was placed in Float-a-Lyzer. The Float-a-Lyzer was placed in beaker containing 50ml of PBS (pH 7.4), maintained at 37^oC and stirred with the help of a magnetic stirrer. Aliquots (2ml) of release medium were withdrawn at different time intervals and the sample was replaced with fresh PBS (pH 7.4) to maintain constant volume and sink conditions. The samples analyzed for drug content by UV-Vis Spectrophotometer at 272.7nm. After every one week the complete medium was withdrawn   and replaced by fresh medium to avoid saturation of the medium.

In-vitro Drug Release Kinetic Study: In order to describe the kinetics of the release process of drug in the different formulations, zero order (Qt = Q0+K0t), First order (lnQt = lnQ0+K1t), Higuchi KHt1/2) and Korsmeyer- Peppas (Qt/Q8= Ktn) models were fitted to the dissolution data of all formulations using linear regression analysis. A value of n=0.5indicates case-I (Fickian) diffusion or square root of time kinetics, 0.5<n<1anomalous (non-Fickian) diffusion, n=1 Case-II transport and n>1 Super Case-II transport ⁸.

Stability studies: To assess the physical and chemical stability of the microspheres, stability studies were conducted for 2 months under various storage conditions mentioned in ICH guidelines. The sample containing optimized formulation were placed in vials and stored at 40±2^oC/75±5% RH. After 60 days the formulations were checked for physical appearance and drug content.

TABLE 2: THE FORMULATION COMPOSITION AND RATIOS OF BSA MICROSPHERES

RESULTS AND DISCUSSION:

Standard calibration curve of BSA in UV Spectro photometer: The UV absorbance’s of BSAstandard solution in the range of 10-50 µg/ml of drug in buffer, pH 7.4 showed linearity at λ max 279nm. The linearity was plotted for absorbance against concentration with R2 value 0.9991 and with the slope equation y=0.0006x-0.0033. The absorbance values and standard curve shown in Figure 1.

FIGURE 1: STANDARD GRAPH OF BSA IN PHOSPHATE BUFFER OF pH 7.4

Preformulation Studies:

Compatibility Studies:The compatibility between the drug and the selected polymer and other excipients was evaluated using FTIR peak matching method. There was no appearance or disappearance of peaks in the drug-polymer mixture, which confirmed the absence of any chemical interaction between the drug, polymer and other excipients. The results are shown in Table 3 and Figure 2, 3, 4.

FIGURE 2:   FTIR OF BSA

FIGURE 3: FTIR OF PLGA

FIGURE 4: FTIR OF BSA WITH PLGA FORMULATION

TABLE 3: INTERPRETATION OF FTIR SPECTRUMS OF BSA, PLGA, BSA WITH PLGA.

Formulation optimization: The Microspheres were prepared by double emulsion technique using homogenizer (IKA). Formulations was optimized for in vitro release profile , particle size and entrapment efficiency. The drug polymer ratio was 1:10 for optimized formulation, PVA concentration was 0.5%, aqueous phase volume was 2ml and DCM volume was 10ml. The formulation containing BSA kept at constant strength was prepared with different ratios of DCM, poly l-lactic acid, PVA, water and all other parameters like temperature and rpm were optimized.

Evaluation of Microspheres:

Percentage yield and Entrapment efficiency of BSA microspheres: The percentage yield and encapsulation efficiency were determined for all the formulations from F1to F8 it was in the ranges from, percentage yield (58.3% - 69.9%) and encapsulation efficiency (75.4%-80.1%).

Among those compositions, 3 Formulations are selected as optimized batches for further evaluation based on in vitro dissolution profile and entrapment efficiency.

TABLE 4: PERCENTAGE YIELD AND ENTRAPMENT EFFICIENCY OF BSA MICROSPHERES

Scanning Electron Microscopy: SEM micrographs and typical surface morphology of the microspheres are given in Fig. 5-6 for F5, F6 formulations. It was observed that microspheres were spherical with smooth surface.

FIGURE 5: SEM PHOTOGRAPHY OF MICROSPHERES FOR F5 FORMULATION

FIGURE 6:  SEM PHOTOGRAPHY OF MICROSPHERES FOR F6 FORMULATION

Particle Size Distribution: The particle size distribution was analyzed for F5, F6, F7 formulations of BSA microspheres by wet method.  The  particle  size  was  optimum  in  F5 Formulation, When compared to F6 and F7, The results were shown in Table 5 and Figure  7, 8, 9.

TABLE 5:  PARTICLE SIZE DISTRIBUTION OF BSA MICROSPHERES

FIGURE 7: PARTICLE SIZE DISTRIBUTION OF BSA MICROSPHERES FOR F5  FORMULATION

FIGURE 8: PARTICLE SIZE DISTRIBUTION OF BSA MICROSPHERES FOR F6 FORMULATION

FIGURE 9: PARTICLE SIZE DISTRIBUTION OF BSA MICROSPHERES FOR F7 FORMULATION

In-vitro cumulative % Drug Release Profile: The in vitro dissolution profile of prepared formulations was determined by modified dissolution apparatus method. The dissolution was carried out for a period of 30 days in 7.4 pH phosphate buffer. The cumulative percent release of F1-F8 formulations at various time intervals was calculated and tabulated in Table 6 for F5 formulation 86.8% drug release was achieved on 30th day.

TABLE 6:   IN VITRO CUMULATIVE % DRUG RELEASE DATA OF BSA MICROSPHERES

FIGURE 10 : COMPARISON OF CUMULATIVE % DRUG RELEASE OF ALL FORMULATIONS

Kinetic Profiles: The release kinetics of F5, F6, F7 formulations was studied. All formulations follow Zero order release kinetics and follow non- fickian diffusion when it applied to the Korsmeyer-Peppa’s Model for mechanism of drug release. F5 formulation has better kinetic results when compared to F6 and F7 formulations. The results are shown in Figure  11-14.

FIGURE 11:  COMPARISION OF ZERO ORDER RELEASE STUDIES FOR OPTIMIZED FORMULATIONS F5, F6, F7.

FIGURE 12:  COMPARISION OF FIRST  ORDER RELEASE STUDIES FOR OPTIMIZED FORMULATIONS F5, F6, F7

FIGURE 13: COMPARISION OF  HIGUCHI’S MODEL RELEASE STUDIES FOR OPTIMIZED  FORMULATIONS   F5, F6, F7.

FIGURE 14:  COMPARISON OF KORSEMEYER- PEPPAS MODEL RELEASE STUDIES FOR OPTIMIZED     FORMULATIONS    F5, F6, F7

TABLE 7: REGRESSION COEFFICIENT (R²) VALUES OF DIFFERENT KINETIC MODELS AND DIFFUSION EXPONENT (n) OF PEPPAS MODEL FOR BSA MICROSPHERES

 Stability test: The stability of the BSA microspheres was evaluated after storage at 40^oC/75%%RH and room temperature for 60 days. The assays of the samples were determined as a function of the storage time. The microspheres stored at 40^oC were found to be stable for duration of 60 daysdays (table 8).

TABLE 8: STABILITY TEST DATA FOR OPTIMIZED FORMULATIONS F5, F6, F7

Differential Scanning Calorimetry (DSC): In order to confirm the physical state of BSA in microspheres, DSC of BSA, PLGA, physical mixture of BSA and polymer, BSA microspheres formulations were carried out and were shown in graph .The DSC trace of BSA showed a sharp endothermic peak 68.6^oC. the physical mixture of BSA and polymer showed same thermal behavior 69.1^oC, as the individual component, indicating that there was no interaction between drug and polymer in solid state.

The reported melting point range of BSA is between 68^oC-70^oC, thus indicating there is no change of BSA in pure state, physical mixture of drug and polymer. . The absence of endothermic peak of the BSA at 68.6 ^oC in the DSC of the BSA microspheres suggests that the BSA existed in an amorphous or disordered crystalline phase as a molecular dispersion in polymeric matrix¹⁰.

FIGURE 15: DSC PEAK OF BSA.

FIGURE 16:  DSC PEAK OF POLYMER PLGA 50:50

FIGURE 17: DSC PEAK OF PHYSICAL MIXTURE

FIGURE 18: DSC PEAK OF FORMULATION

CONCLUSION: The conclusions drawn from the present investigation are given below:

• Preformulation studies like solubility, melting point, and uv analysis of BSA complied with IP standards.
• The FTIR spectra revealed that, there was no interaction between polymer and BSA , All the excipients used were compatible with the BSA.
• Surface smoothness of the BSA microspheres was increased by increasing the polymer concentration, which was confirmed by SEM.
• As the drug to polymer ratio was increased, the mean particle size of BSA microspheres was also increased. The BSA microspheres with normal frequency distribution were obtained.
• Entrapment efficiency increase with increase in the polymer concentration From the results it can be concluded that as the drug to polymer ratio decrease, initial burst release will increases.
• The co-efficient of determination indicated that the release data was best fitted with Zero order kinetics. Higuchi equation explains the diffusion controlled release mechanism. The diffusion exponent ‘n’ values of Korsemeyer-Peppas model was found to be less than 0.45 for the BSA microspheres indicating Fickian diffusion of drug from microspheres.
• Accelerated stability studies indicating that BSA microspheres are stable on storage.
• The DSC data indicates that there is no inter action between drug and polymer and it also indicates that BSA still present in lattice structure in physical mixture where as it was completely amorphous in microspheres.
• From the study, it is evident that promising sustained release microspheres of BSA may be developed by double emulsion solvent evaporation method by using polymers like PLGA.

 ACKNOWLEDGEMENTS: The authors acknowledge Celon Laboratories, Hyderabad, India, for gift sample of BSA, PLGA. The authors also thank Aravind. G, Manager (FRD), Celon Labs, Mahesh Kumar, Summit Shah, Senior Research Associate (FRD), Celon Labs, Hyderabad.

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   How to cite this article:

   Voruganti S, Padman JSC: Formulation and Evaluation of BSA Loaded PLGA Microparticles. Int J Pharm Sci Res. 2013; 4(3); 1013-1021.

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