Preparation and Characterization of Poly-ε-caprolactone Particles for Controlled Insulin Delivery

Journal of Biomedical & Pharmaceutical Engineering 1:1 (2007) 40-44 ISSN: 1793-4532 All Rights Reserved Preparation and Characterization of Poly-ε-caprolactone Particles for Controlled Insulin Delivery A. Mukerjee 1, V.R. Sinha 2, V. Pruthi 1 1 Department of Biotechnology Indian Institute of Technology Roorkee Roorkee, India 2 University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh, 160014, India Corresponding Author: V. PRUTHI; Mailing Address: Department of Biotechnology Indian Institute of Technology Roorkee Roorkee, India 247667; Tel: +91-1332 285530; Fax: +91-1332 273560; Email: vikasfbs@iitr.ernet.in Abstract In recent years, with the aid of newer technology, pharmaceutical and formulation engineering has emerged as a fascinating field with applications in diverse areas such as drug delivery, biomaterials, drug targeting and nanotechnology. In the pharmaceutical industry, novel drug delivery technologies represent a strategic tool for expanding drug markets. This new technology can address issues associated with current pharmaceuticals, by extending product life or by adding to their performance and acceptability, either by increasing efficacy or improving safety and patient compliance. Protein delivery through microspheres and nanospheres is expected to create innovations and play a critical role not only in drug delivery but also in target-specific drug therapy. Microspheres made of biodegradable polymers are preferred because surgical removal of spent device, as in the case of implants, is not required. Furthermore, there is no possibility of toxicological problems, their release rates can be tailored and they degrade in biological fluids to produce biocompatible or non-toxic products in the body, which are removed by normal physiological pathways. Our present work investigates the method for the efficient encapsulation of insulin in poly-ε-caprolactone microspheres and nanospheres using a water-in-oil-in-water double emulsion solvent evaporation method. The microspheres and nanospheres formed were characterized for entrapment efficiency, percent yield, particle size analysis, morphological characteristics and the drug release profiles. Our studies showed the successful formulation of smooth spherical poly-ε-caprolactone microspheres and nanospheres encapsulating insulin, thus highlighting them as potential controlled drug delivery systems. Keywords: microspheres, nanospheres, poly-ε-caprolactone, insulin, drug delivery. Received 25 April 2007; Accepted 11 June 2007 1 INTRODUCTION Major advances in pharma-biotechnology and biochemistry have led to the discovery of a large number of bioactive molecules and vaccines based on peptides, proteins and oligonucleotides but the development of suitable carrier systems for these, remains a major challenge for the pharmaceutical scientist. Carrier technology offers an intelligent approach for drug delivery by entrapping the drug in a carrier particle such as microspheres and nanoparticles, thus protecting them against any enzymatic degradation and can also modulate the release and absorption characteristics. This concept of controlled drug delivery in order to obtain specific release rates or spatial targeting of active ingredients has led to an enormous impact on the health care system. The last two decades in pharmaceutical industry have witnessed an avant-grade interaction among fields of polymer and material science, resulting in development of novel drug delivery systems. Among the biodegradable polymers, poly-εcaprolactone has been receiving increasing attention as matrix materials for controlled release drug formulations. Poly-ε-caprolactone and its derivatives, with a high permeability to many therapeutic drugs and lack of toxicity [1] are well suited for controlled drug delivery. However, despite the rapid progress formulation of these therapeutic molecules, convenient and effective controlled delivery of protein drugs remains a major challenge. Indeed, their large molecular size, their short plasma half-life, their biodegradation by enzymes, and the importance of maintaining a correct 2007 Biomedical & Pharmaceutical Engineering Cluster, Nanyang Technological University 40

conformation all limit their biological activity. Throughout the world, considerable research effort has been devoted to developing appropriate insulin delivery systems to replace or reduce the present injectable insulin dosage regimen [2-6]. A less painful insulin dosage form would be preferred by diabetic patients, leading to better patient compliance. Based on these aspects, the present work investigates various parameters for the efficient formulation of insulin in poly-εcaprolactone (PCL) microspheres and nanospheres using a w/o/w double emulsion solvent evaporation technique. 2 MATERIALS AND METHODS 2.1 Materials Insulin Human Injection USP, 40 IHU/ml, (Eli Lily and Company, USA), Poly-ε-caprolactone (Fluka, USA), Polyvinyl alcohol, Mw 14,000 and Dichloromethane (Sd Fine Chemicals, India), Hydroxypropylmethylcellulose (Himedia, India) and double distilled water were used for all the experiments. 2.2 Preparation of Microspheres Microspheres were prepared by using modified protocols of Sinha et al. [7] and Attivi et al. [8]. For the preparation of PCL microspheres by w/o/w emulsion, first, 3 ml of an aqueous insulin solution (40 IHU/ml) was emulsified in 10 ml of organic solvent (dichloromethane) containing 100mg of the poly-εcaprolactone by stirring at 6000 rpm for 2 minutes, using a mechanical stirrer (RQT-124A, Remi Motors, India). The water-in-oil emulsion, thus formed was thereafter added to 30ml of external aqueous phase (hydroxyl propylmethyl cellulose (HPMC) or polyvinyl alcohol (PVA) solution) and again stirred at 4500 rpm for 150 minutes at 30 C, resulting in the formation of the final water-in-oil-in-water emulsion. To facilitate solvent evaporation, the final emulsion was magnetically stirred for about five hours at 30 C. After complete evaporation of organic solvent, the polymer hardens trapping the encapsulated drug, forming the desired microspheres. The microspheres, thus formed, were isolated by centrifugation. The microspheres were then washed three times with deionized distilled water and dried at room temperature. The dried microspheres were finally weighed to calculate the respective yields. A set of experiments were designed with different concentrations (0.5%, 1.0%, 1.5%each of HPMC and PVA- batches L1-L6) of external aqueous phase with the organic phase being dichloromethane and internal aqueous phase being insulin in water for injecting 40 IHU/ml. For the preparation of nanospheres (batch N1), the above procedure was partially modified using the protocol of Lamprecht et al. [9]. Here, the stirring speed was kept at 12000 rpm for 2 minutes for the primary emulsion step and 5500 rpm for 150 minutes for the secondary emulsion step. The external aqueous phase was kept as 0.5% hydroxyl propylmethyl cellulose (HPMC) solution. 2.3 Encapsulation Efficiency Encapsulation efficiency was determined for each batch of microspheres and nanospheres by analyzing the filtrate of the double emulsion once the microspheres or the nanospheres were removed from it after centrifugation at 5000 rpm for 30 minutes. Micro BCA Protein Estimation Kit (PIERCE ) was used for the estimation [10] and the amount of drug present in the filtrate was calculated from calibration curves of concentration vs absorbance already prepared with known standards of the drug using the same kit. The amount of the drug encapsulated and the Percent Encapsulation in the microspheres is given by: Drug (encapsulated) = Drug (total) Drug (filtrate) % Encapsulation = [Drug (encapsulated) / Drug (total) ] X 100 2.4 Surface Morphology of Microspheres and Nanospheres The surface morphology of the microspheres and nanospheres was investigated using Scanning Electron Microscopy, SEM, (Leo, VP-435, UK). The dry samples of microspheres/ nanospheres were mounted on metal stubs using double sided adhesive tapes. They were then, sputter coated with gold particles under reduced pressure conditions and observed under the scanning electron microscope [11]. The accelerating voltage was kept constant at 15 KV. 2.5 Atomic Force Microscopy of Nanospheres The surface morphology of the nanospheres was investigated using Atomic Force Microscopy (AFM) [9]. The dry samples of nanospheres were mounted on stubs using double sided adhesive tapes and viewed. 2.6 Particles Size Measurements A specific quantity of the microspheres or nanospheres was dispersed in water in an ultrasonic disperser for 1 minute to bring about disaggregation. Particle size of the microspheres/ nanospheres was then measured by laser diffractometry using Laser Particle Size Analyzer. 2.7 In-vitro Release In vitro release of insulin was determined by placing 10mg microspheres, from each batch, in 25mL of release medium (PBS buffer, ph 7.4). The samples were kept in an orbital shaker (SGM-300, Gallenkamp, Sanyo) maintained at 37 C ±5 C stirring at 50 rpm. At specified time intervals, 1 ml aliquot of the release medium was removed and replaced with fresh media. These samples were then centrifuged at 3000 rpm at room temperature. The supernant was then analyzed for drug content using Micro BCA Protein Assay protocol as reported by Katare [12]. Similar protocol was employed for determining the in vitro drug release from the nanospheres. 2007 Biomedical & Pharmaceutical Engineering Cluster, Nanyang Technological University 41

3 RESULTS AND DISCUSSION The choice of a particular method of encapsulation is of enormous importance for the proper entrapment and drug release. In our approach, the double emulsionevaporation process was adopted since it is known to be superior to other incorporation methods in terms of stability of proteins [13]. Table 1: Various parameters of batches L1-L6 and N1 (L1-0.5%HPMC, L2-1.5%HPMC, L3-1.0%HPMC, L4-0.5%PVA, L5-1.5%PVA, L6-1.0%PVA and N1-0.5%HPMC S.No Batch Name 1 L1 2 L2 3 L3 4 L4 5 L5 6 L6 7 N1 % Yield (± SD) 96.1 ± 0.5 89.8 ± 0.9 95.3 ± 0.4 87.8 ± 1.2 82.1 ± 0.9 90.2 ± 0.6 76.5± 0.5 % EE Particle (± SD) Size (µm) 43.7 ± 0.7 30.25 32.4 ± 0.9 43.37 53.0 ± 0.4 28.17 38.2 ± 1.2 35.70 31.8 ± 2.4 39.32 46.3 ± 0.7 30.36 28.3 ± 0.4 0.75 microspheres were found to be comparable as shown in figure 5. A controlled release of insulin is seen over twelve hours after a short initial burst phase. This result is comparable to the results reported by Pe rez [15]. Fitting the release data into the Higuchi equation yielded comparable linearity (figure 6) for all the microsphere and nanosphere batches (batches L1-L6 and N1). Figure 1: PCL microspheres with PVA as external aqueous phase showing pits The effect of formulation variables on yield, encapsulation efficiency, particle size of insulin loaded PCL microspheres and nanospheres are shown in Table 1. The percent encapsulation for PCL microspheres was found to vary within 31.8 to 53%. Dhanaraju et al. [14] showed a similar 46.3 to 57.7% entrapment of levonorgestrol and ethinylestradiol in PCL microspheres formed by w/o/w emulsion technique. A similar result has been reported for hydrophilic drug propranolol hydrochloride in poly-ε-caprolactone microspheres by Pe rez et al. [15]. However, the nanospheres showed a lower insulin entrapment of Figure 2: PCL microspheres with HPMC as external 28.3%. All batches showed formation of spherical aqueous phase showing no pits microspheres with varying smoothness of surface. Though, the microspheres prepared with polyvinyl alcohol were spherical, some showed distinct pits. This is in accordance to the work of Somavarapu [16], who reported pitted microspheres with PVA. However, microspheres prepared with hydroxypropylmethylcellulose as the external aqueous phase were very smooth, spherical and without any pits as shown in figures 1 and 2. The scanning electron microscope and atomic force microscope scans for PCL nanospheres are shown in figures 3 and 4 respectively. The AFM scan in figure 4 reveals the spherical nanoparticles with a relatively monodispersed size. This is in co ordinance with the results showed by Lamprecht [9]. The mean diameter of the microsphere batches was found to lie between 28-43µm. The nanosphere mean diameter was found to be 750nm. The release profiles of all the six batches of Figure 3: SEM scan of PCL nanospheres batch N1 2007 Biomedical & Pharmaceutical Engineering Cluster, Nanyang Technological University 42

Since PCL follows erosion mechanism, the release could be due to erosion of the polymer shell and the subsequent release of insulin to the external media. The nanospheres showed less release of insulin as depicted by the lower line in the release profile graph. 4 CONCLUSION It is therefore concluded that poly-ε-caprolactone microspheres and nanospheres are capable of delivering insulin over prolonged periods, making it a potential candidate for controlled drug delivery. Figure 4: AFM scan of PCL microspheres (noncontact mode) Insulin Release (%) 60 50 40 30 20 10 0 L1 L2 L3 L4 L5 L6 N1 0 2 4 6 8 10 12 Time (h) Figure 5: In-vitro insulin release profile from PCL microspheres and nanospheres. L1-0.5% HPMC, L2-1.5% HPMC, L3-1.0% HPMC, L4-0.5% PVA, L5-1.5% PVA, L6-1.0% PVA and N1-0.5% HPMC (n=3) Insulin Release (%) 70 60 50 40 30 20 10 0 L1 L2 L3 L4 L5 L6 N1 0 0.5 1 1.5 2 2.5 3 3.5 Square Root of Time (h) Figure 6: Higuchi plot of PCL microspheres batch L1- L6 and nanospheres batch N1 REFERENCES [1] Kim SY, Lee YM, Baik DJ and Kang JS. Toxic characteristics of methoxy poly (ethylene glycol)/poly (epsilon-caprolactone) nanospheres; in vitro and in vivo studies in the normal mice. Biomaterials 1985; 24: 55-63 [2] Peppas NA. Devices based on intelligent biopolymers for oral protein delivery. Int. J. Pharm 2004; 277: 11-17 [3] Fossa AC, Goto T, Morishita M and Peppas NA. Development of acrylic-based copolymers for oral insulin delivery. Eur. J. Pharm. Biopharm 2004; 57: 163 169 [4] Kim B and Peppas NA. In vitro release behavior and stability of insulin in complexation hydrogels as oral drug delivery carriers. Int. J. Pharm 2003; 266: 29 37 [5] Nakamura K, Murray RJ, Joseph JI, Peppas NA, Morishita M and Lowman AM. Oral insulin delivery using P(MAA-g-EG) hydrogels: effects of network morphology on insulin delivery characteristics. J. Control. Rel 2004; 95: 589 599 [6] Morishita M, Goto T, Peppas NA, Joseph JI, Torjman MC, Munsick C, Nakamura K, Yamagata T, Takayama K and Lowman AM. Mucosal insulin delivery systems based on complexation polymer hydrogels: effect of particle size on insulin enteral absorption. J. Control. Rel 2004; 97: 115 124 [7] Sinha VR and Trehan A Biodegradable microspheres for protein delivery J. Control. Rel.2003; 90: 261-280 [8] Attivi D, Wehrle P, Ubrich N, Damge C, Hoffman M and Maincent P Formulation of Insulin-Loaded Polymeric Nanoparticles Using Response Surface Methodology Drug Dev. Ind. Pharm. 2005; 31: 179 189 [9] Lamprecht A, Ubrich N, Pe rez MH, Lehr, CM, Hoffman M and Maincent P Influences of process parameters on nanoparticles preparation performed by a double emulsion pressure homogenization technique Int. J. Pharm. 2000; 196: 177 182 [10] Smith PK. Measurement of protein using bicinchonic acid. Anal. Biochem 1985; 150: 76-85. [11] Mukerjee A and Pruthi V. Biodegradable Polymers for Controlled Drug Delivery. Proceedings, 58th Chemical Engineering Congress. New Delhi, Abstract 2005; 2: 308 [12] Katare YK, Muthukumaran T and Panda AK. Influence of particle size, antigen load, dose and additional adjuvant on the immune response from antigen loaded PLA microparticles. Int. J. Pharm. 2005; 301: 149 160 2007 Biomedical & Pharmaceutical Engineering Cluster, Nanyang Technological University 43

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