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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1084-1097 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1087and WatersTM 717 plus autosampler (Millipore,USA). The mobile phase consisted of distilledwater (70%) and acetonitril (30%) withtrifluroacetic acid (0.1%) and 0.6 mmol sodiumsulfate. Chromatography was performed usingC18 reversed phase column (Nova-Pak C18,3.9X150 mm, WatersTM, Ireland) at a flow rateof 1.5 mL/min and the eluent was monitored at220 nm. The concentration of the insulin wasdetermined by integration of the peak area usingthe external standardization method. Allmeasurements were conducted in triplicate.Insulin Integrity: Insulin released fromnanoparticles was characterized using MALDI-TOF MS to assess the molecular mass (20).Nanoparticles were dissolved in acetone, driedat room temperature under vacuum. Thesesamples containing insulin were reconstituted in1 mL of 0.01 N HCl. Reference insulin solutionwas also prepared the same way by dissolvinginto 0.01 NHCl. Aliquots of 2 µL samples werethen mixed with 8 µL of the matrix (α-CHCA) insolution (10 mg/ml in 0.1% TFA and 50%acetonitrile solution) and 2 µL of the mix wasallowed to dry on a plate in a solid spot undervacuum. The plate was then inserted into themass spectrometer.MALDI-TOF MS experiments wereconducted on a MALDI-TOF instrument(Shimadzu, Japan) using a 337-nm nitrogen laser.Spectra were acquired in positive ion linear mode(Acceleration voltage 33 kV).Immunogenicity of insulin: The immunogenicityof insulin was assayed by an ELISA test(Mercodia,Uppsala, Sweden). Accuratelyweighed amounts of nanoparticles were dissolvedinto acetone and centrifuged to precipitate theprotein and the supernatant containing PLGApolymer was discarded. Samples were washedtwice by acetone and centrifuged to separate thepure insulin. Samples were then vacuum driedand re-dissolved in distilled water in theconcentration range of (1–200 mU/L) using themanufacture’s protocol and its relative bioactivitywas calculated by comparison with valuesobtained by BCA analysis of the same aliquots(21).Cell viability assay: MCF-7 cells were growninto 96 well plates in 5,000 cells per well for 24 hat 37 °C, 5% CO2 in DMEM + 5% heatinactivated fetal bovine serum, non-essentialamino acids, and L- glutamine (200 mM). Thecells were serum and insulin starved for another24 h and then incubated with insulin ornanoparticles carrying insulin in concentrationsof 7.5, 10, and 12 µg/mL for 48 hours. Controlwells were treated similarly and PBS of pH 7.4was added instead of insulin. Cell viability wasthen estimated using SRB assay (22).In vivo effects of insulin loaded nanoparticlesAnimals / Conditioning: Male Sprague–Dawley(SD) rats, body weight ranging 160–200 g wereused in the study. The animal protocol wasapproved by the Institutional Animal Care andUse Committee of the University of the Pacific.The animals were grouped in standardpolypropylene cages and maintained undercontrolled room temperature (22±2 °C) andhumidity (55±5% RH) with 12:12h light and darkcycle. All the rats are provided with commerciallyavailable rat normal pellet diet and water adlabium. If the weight of the animals increasesbeyond 250 g, they were discontinued from thestudy.Induction of Diabetes: Diabetes was induced inmale rats (250 ± 30g) by an intra-peritonealinjection of streptozotocin (65 mg/kg) in a 10 mMcitrate buffer at pH 4.5 (23). Streptozotocinsolutions were used within 30 min. Rats wereconsidered diabetic when blood glucose level washigher than 300 mg/dL, a week afterstreptozotocin treatment (24).Mahmoud et al
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1084-1097 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1088Blood glucose levels after subcutaneousadministration of insulin nanoparticles: InsulinLoaded nanoparticles (10 IU/kg) prepared by theabove different methods were injected to feddiabetic rats. Free insulin (10 IU/kg), unloadednanoparticles and PBS (pH 7.4) were alsoadministered in control animals. Samples of 1drop of blood were withdrawn from the tail veinat different time intervals and evaluated for theglucose levels using OneTouch Ultra 2 glucometerbefore injection and 0.5, 1, 1.5, 2, 3, 6, 8, 10, 12,24, 48, and 72 hour post injection. Rats weremaintained fasted during the experiment up to 12h and fed thereafter.Statistical analysis: The mean and the standarderrors were calculated for each group. For groupcomparisons a one-way analysis of variancefollowed by the least significant difference (LSD)as a post-hoc test was applied, using SPSSprogram version 9 software. When P
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(December 7-9, 2011)Venue: Karunya
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