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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1084-1097 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1089Insulin release (%)PLGA 50:50 (EtAc:ESE)PLGA 50:50 (DCM:ESE)PLGA 50:50 (MPEG:MM)Insulin release (%)pH 1.2pH 5.6pH 7.4Time (hr)Fig. 2. Insulin release (%) from nanoparticles prepared bydifferent methods in phosphate buffer pH 7.4 at 37 0 C Datarepresented as the mean+SD (n=3)84% entrapment efficiency (EE) as shown inTable 1. Of the different methods of preparation,MM method resulted in lower EE%. However,there was no significant difference in Insulin EE%compared to nanoparticles prepared by ESEmethods (p>0.05). All nanoparticles prepared bydifferent methods showed negative zeta potentialat pH 7.4.Insulin Release from nanoparticles preparedby different methods : The release of insulinfrom nanoparticles prepared by different methodswas found to be biphasic (Fig. 2). The initial rapidrelease (burst effect) was high, where, 52%, 52.4,and 54.2% of the entrapped insulin was releasedfrom nanoparticles prepared by ESE (DCM),MM, ESE (ET-AC), respectively. This burstrelease in the first 5 minutes was followed byabout 8% release of insulin after 30 minutes incase of ESE (DCM) and MM methods. On theother hand, about 16% of insulin was releasedfrom nanoparticles of ESE (ET-AC) methodfollowing the burst release. After the first 30minutes, very slow release of minute amounts ofinsulin was observed over 24 hours.To use nanoparticles via the oral route, itis important to test the release of insulin atdifferent pH range. Insulin release fromnanoparticles of ESE (DCM) method wasselected for the test as they show no significantdifferences in release compared to MM method(P>0.05). The release of insulin was significantlyFig. 3. Effect of release medium pH on insulin release fromPLGA (50:50) nanoparticles prepared using ESE (DCM)method. Data represented as the mean+ SD. (n=3).affected by the external pH. Complete releaseof insulin occurred in 5 minutes at pH 1.2 asshown in Figure 3. However, the initial burstrelease was only 8% at pH 5.6 and 52% at pH7.4. After 24 hours, only 42.5% of insulin wasFig. 4. MALDI-TOF Mass spectra of standrd insulin (aand e), insulin extracted from nanoparticles prepared usingMM (b), ESE (ET-AC) (c), and ESE (DCM) (d) methods.Insulin concentration used was 0.2 mg/ml.Insulin immunogenicity (%)Time (hrs)Mass/ChargeESE (DCM) ESE (ET-AC) MMFig. 5. Immunogenicity of insulin, determined as the ratioof insulin detected by ELISA test to insulin detected byBCA protein assay kit.Mahmoud et al
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1084-1097 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1090found released at pH 5.6, whereas 63.5% and96% of insulin was released in pH 7.4 and 1.2media, respectively.Effect of Method of preparation on InsulinIntegrity and Bioactivity : The integrity of insulinduring the preparation of nanoparticles (organicsolvent, homogenization, surfactants, freeze dryingsteps), was evaluated by extracting insulin fromnanoparticles and analyzing the molecular weightby MALDI-TOF Mass Spectrometry. The massspectra showed only a molecular peak at m/z2866.4 (doubly charged, molecular weight ofbovine insulin is 5733.5 Da) for samples preparedby both methods as well as the insulin solution(Fig. 4). This indicated that insulin retained itsintegrity and insulin was not degraded in thepreparation process in all the methods evaluated.The immunogenicity of insulin afterencapsulation into nanoparticles by differentmethods was evaluated using ELISA test. Thedata showed that the antigen activity of insulinremained particularly unaffected by DCMTable 2: Effect of tween 20 concentration onimmunogenicty of insulin. Data represented as themean+SD. (n=3).Tween 20Concentration (%) 0 1 2 4Antigenicity (%) 23.11+0.02 29.51+0.72 27.61+0.16 28.18+13.38(102.46% + 19% ), or ET-AC (97.63%+ 18.18%)in ESE method. However, only 28.18+13.38%insulin antigenicity was retained fromnanoparticles prepared by MM method (Fig. 5).Tween-20 and MPEG-350 were used inthe preparation of insulin nanoparticles using MMmethod. To differentiate which of these twosurfactants reduced antigenicity of insulin, ELISAassays were conducted using nanoparticlesprepared at different Tween-20 concentrations.The antigenicity of insulin remained constant inthe Tween-20 range of 1%-4% while it isFig. 6. Cell Viability (%) in the presence of insulin solutionand insulin nanoparticles. Data presented as the mean+SD.(n=3).significantly reduced (P
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(December 7-9, 2011)Venue: Karunya
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