full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1084-1097 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1093was considered an appropriate means fordetection of potential changes of the antigenicactivity. It can confirm that the receptor bindingepitopes on insulin were maintained afterencapsulation (16). This could be true wheneverthe antiginicity is close to 100 %, however, in thisstudy, reduction in antiginicity as tested by ELISAwas not detrimental on receptor binding epitopes.Results showed that nanoparticles prepared byESE method had 100% immunogenicity, whereas,nanoparticles prepared by MM method showedonly 28.18% insulin immunogenicity based onELISA testing. As shown in Table 2, the presenceof minute amounts of methoxy PEG 350 afterpurification of nanoparticles was responsible forthe reduced immunogenicity, while Tween-20 didnot show any such effect. It is commonly knownthat PEGs could interfere with antibody antigeninteraction. This effect is presumably the resultof reduced exposure of hydrophobic parts of theprotein in the presence of PEG (41). Low PEGresidues on the nanoparticle surface, being morehydrophilic, decreased their opsonization, andreduced immune system recognition (42). Thisimplied that the organic solvent/water interfaces,surfactants (PVA and Tween 20) andhomogenization steps used in the preparation ofnanoparticles did not alter insulin integrity.However, low immunogenicity did not result lowerbioactivity obtained from cell viability assay.The insulin present in the supplementedserum free medium seemed to be responsible forthe enhanced cell growth (43). Insulin haspotentiated lysophosphatidic acid to stimulateMCF-7cell cycle progression and DNA synthesis(44). Hence, cell viability assay performed toelucidate the insulin bioactivity on growth of MCF-7 cells was studied using nanoparticles preparedby ESE and novel MM methods. Higher cellviability was evident from nanoparticles whencompared to standard insulin solution at the sameconcentrations. The intimate contact ofnanoparticles to cells increased the concentrationof insulin closure to cellular membranes enhancingcell viability. Since, the nanoparticles preparedby MM method exhibited good bioactivity whilereducing the immunogenicity, this method ofnanoparticle preparation seems to haveadvantages.The biological effects of insulinencapsulated into nanoparticles were also provedby the in vivo studies. Nanoparticles preparationmethods that involved organic solvents andsurfactants showed prolonged reduction in bloodglucose levels of diabetes induced rats, furthersuggesting that the biological activity of insulin isnot affected by preparation methods. The bloodglucose profiles of insulin nanoparticles showed4 phases as shown in Figure 7. In the first phasea rapid reduction in blood glucose levels wasobserved that peaked at 3 hours, which correlatedwith insulin absorbed from the burst effect in vitrorelease media. In the second phase, blood glucoselevels have increased as the absorbed insulinrapidly cleared from the rats between 3rd and6th hours. The third plateau phase after 12 hourscould be due to slow release of insulin fromnanoparticles. A fourth phase of blood glucoselevels recovery back to 100% and continued atthat level at 48 and 72 hours revealed completeclearance of insulin from the rats. The reducedblood levels observed between 12 and 24 hoursmight from the insulin release due to thedegradation of the polymer (19). On the otherhand, insulin solution showed only first 2 phasesshown by nanoparticles. This data clearly showedthe unique advantages of nanoparticulate deliverysystems, which include offering protection fromdegradation by enzymes in the body (44). A similarstudy showed lowered blood glucose levels indiabetic rats over 24 hours followingintraperitoneal injection of PLGA nanoparicles(45). In that study, blood glucose level showed aminimum of 10 mmol/L at 1 hour after nanoparticleMahmoud et al