Targeted Reversal Of Multidrug Resistance In Ovarian Cancer By Folate Modified Chitosan SiRNA Nanoparticles

Partâ… Preparation and identification of folate-chitosancomplex vectorsObjective: To synthesize folate-chitosan complex vectors and identify the successfulcombination of folate-chitosan complex vectors, determine the folate coupling radio offolate.Methods: 1.Folate-chitosan complex vectors were synthesized using reductive amidation.2.The combined samples were detected in light within the infrared spectrum using theFourier transform infrared (FTIR) spectrometer to determine the folate-chitosan complexvectors.3.Samples were scaned by the UV/Vis spectrophotometer.4.Folate coupling radioswere determined using folate concentration- absorbance standard curve.Results: 1.The purified folate-chitosan complex vectors were obtained and the sampleswere stramineous, exquisite and adqulis fine powder.The solutions were tasteless,stramineous and transparent.2.The results of infrared absorption spectrum showed thereaction key -CO-NH- between the chitosan and folate corresponding to a new bigabsorption peak at 1563^-1cm in the folate-chitosan complex samples.3.The results detectedby the UV/Vis spectrophotometer showed a big ultraviolet absorption peak at 280nm in thefolate-chitosan complexes which was the signal of successful coupling of folate.4.Therewas a good linear relationship between the absorbance of folate and the amount of folate infolate-chitosan complexes from 5.15ug/ml to 30.9μg/ml.The folate tandard curveregression equation was A = 0.0127C + 0.0116, R²=0.9983.There were four folate-chitosan complexes with different folate coupling radio (3%, 7.5%, 11.2% and 17%).Conclusions: We successfully synthesized the folate-chitosan complex vectors usingreductive amidation.By adding different amount of folate and chitosan, we all synthesizedfour folate-chitosan complex vectors with different folate coupling radio at the samereaction condition.The results illustrated that the modification of chitosan can be obtainedby the combination reaction to the active amines of chitosan molecules, and so can changethe physical chemistry function of them.Partâ…¡Peparation of folate modified chitosan siRNA nanoparticles andmeasurement of propertiesObjective: To synthesize folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA)and chitosan PshRNA nanoparticles (CS-PshRNA) and detected their appearance anddiameters.To study the cytotoxity and transfect efficiency to different cell lines andprotective function for DNA of nanoparticles.Methods: 1.Folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA) and chitosanPshRNA nanoparticles (CS-PshRNA) were synthesized using the complex coacervationmethod.2.The appearance and diameters of samples with different folate coupling radiowere detected using transmission electron microscope.3.The protective and packingfunction of different nanoparticles for plasmid DNA was identified by the enzymeprotection experiment.4.MTT method was used to detected the cytotoxity of nanoparticlesto different gynecologic tumor cell lines ((SKOV3,A2780,CAOV3,HeLa and MCF-7).5.The transfection efficiency nanoparticles was examined in different gynecologic cancer cells, such as cervical cancer cell lines HeLa, ovarian cancer cell lines SKOV3 and breastcancer cell lines MCF-7 which were all over-expressing folate receptor.The transfectionwas carried in vitro culture environment.The transfection efficiency of nanoparticles wasanalyzed with flow cytometry and cells were observed by invert fluorescence microscopeand taken photos.Results: 1.Folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA) and chitosanPshRNA nanoparticles (CS-PshRNA) were successfully synthesized using the complexcoacervation method and the nanoparticle solutions were transparent, colorless andtasteless.2.The results of transmission electron microscope showed that the nanoparticleswere found to be near spherical appearance with homogeneous structure and smoothsurface.The particle diameter of chitosan PshRNAnanoparticles was 138.4±0.7nm, and the diameter of 4 kinds of folate modified chitosanPshRNA nanoparticles (folate coupling Radio: 3%,7.5%,11.2%å’Œ17% )were 289.6±0.7nm,78.1±0.3nm,186.6±0.6nm and 212.2±0.5±nm respectively.3.The result of the enzymeprotection experiment declared that chitosan and folate modified chitosan can effectivelycombine and condense the DNA and can protect them from degradation of DNAaseâ… .4.The cototoxity of folate modified chitosan PshRNA and chitosan PshRNA nanoparticles todifferent cells were different.After treatment with chitosan PshRNA nanoparticles, the cellvitality of different cells (MCF-7,A2780,CAOV3,SKOV3 and HeLa) were 87.9±2.4%,91.4±1.0%,42.9±2.1%,102.0±4.0 and 97.4±1.1% respectively compared with the cellsbefore treatment, after treatment with folate modified chitosan PshRNA nanoparticles, the cellvitality were 63.0±2.5%,90.6±1.3%,50.5±0.7%,106.5±1.8% and 99.3±1.6% respectivelycompared with the cells before treatment.5.For MCF-7, SKOV3 cells, the transfectionefficiency were 16.8±1.2% and 24.3±0.7% using folate modified chitosan PshRNAnanoparticles and were significantly increased compared to the transfection efficiency(0.3±0.1% and 0.7±0.1%) using chitosan PshRNA nanoparticles (P<0.05).But for HeLacell, there was no statistics disparity between chitosan PshRNA nanoparticles and folate modified chitosan PshRNA nanoparticles (P>0.05).Conclusions: We successful synthesized the folate modified chitosan siRNA nanoparticelsand chitosan siRNA nanoparticles using the complex coacervation method.The differentfolate coupling radio can affect the diameters of nanoparticles and when the folate couplingradio was 7.5%, the diameter was smallest (78.1±0.3nm).The results illustrated thatchitosan and folate modified chitosan can all effectively combined and condensed the DNAand can protect them from degradation of DNAaseâ… .The transfection efficiency of folatemodified chitosan siRNA nanoparticles were significantly increased compared to thetransfection efficiency of chitosan siRNA nanoparticles, this declared that frommodification of folate, the transfection efficiency of chitosan nanoparticles wassignificantly increased.Partâ…¢Establishment of the drug resistant SKOV3 ovarian cancer cell lineand study of drug resistanceObjective: To induce and culture the multidrug resistant SKOV3 ovarian cancer cell line(SKOV3-ts) using Paclitaxel, and measured the expression of MDR1 gene encodingprotein P-gp in parent cell line SKOV3 and drug resistant cell line SKOV3-ts.To explorethe correlation between the expression of multidrug resistant gene 1 (MDR1) and multidrugresistance (MDR).To supply the cytology foundation for the study of the reversal effectof folate modified chitosan siRNA nanoparticles on ovarian cancer MDR. Methods: 1.The SKOV3-ts cell line was induced and culture by concentration gradientmethod in vitro and paclitaxel was used as inductor.2.IC₅₀ of the cells to paclitaxel andadriamycin in parent cell line SKOV3 and drug resistant cell line SKOV3-ts were measuredby MTT assay.3.Western-blot and laser scanning confocal microscope (LSCM) were usedto detect the expression of P-gp in parent cell line SKOV3 and drug resistant cell lineSKOV3-ts.Results: 1.The SKOV3-ts cell line was successfully established using concentrationgradient method.The cells were bigger than the parent cell line SKOV3.The appearance ofcell was changed from spindle shape to irregular shape and some cells were star like andbranch like patterns.2.The results of western-blot showed that expression level of P-gpwas significant increased in SKOV3-ts cells (1.15±0.02) compared with SKOV3 cells(0.08±0.01) (P<0.05).Photos of LSCM illustrated that there was trace quantity ofP-gp expression in SKOV3 cells.But in SKOV3-ts cell membranes, there was strong P-gpexpression.3.IC₅₀ of SKOV3 and SKOV3-ts cells to paclitaxel were 0.0048±0.0002 and0.3957±0.0075 respectively, there was significant statistics meaning (P<0.05), and IC₅₀of SKOV3 and SKOV3-ts cells to adriamycin were 0.0066±0.0006 and 0.2210±0.0046respectively, there was significant statistics meaning (P<0.05).Conclusions: We successfully established the multidrug resistant SKOV3 ovarian cancercell line SKOV3-ts by concentration gradient method in vitro.The expression of P-gp ofdrug resistant cells SKOV3-ts was significantly higher than parent cell line SKOV3, andthe IC₅₀ of SKOV3-ts cells to paclitaxel and adriamycin were increased compared withSKOV3 cells.This illustrated that there was apparent correlation between over-expressionof MDR1 and MDR of SKOV3-ts cells.So SKOV3-ts cells obtained drug resistance andcan be used as cell model for the study of the reversal effect of folate modified chitosansiRNA nanoparticles on ovarian cancer MDR. Partâ…£Targeted reversal of multidrug resistance in SKOV3-ts cells by folatemodified chitosan siRNA nanoparticlesObjective: To detect the effect of chitosan PshRNA nanoparticles and folate modifiedchitosan PshRNA nanoparticles on the expression of MDR1 in SKOV3 cells.To explorethe changes of drug resistance in SKOV3-ts cells treated by nanoparticles aftermodification of folate.Methods: 1.The folate modified chitosan PshRNA and chitosan PshRNA nanoparticleswere transfected into the SKOV3-ts cells in vitro culture process.(PshRNA was theeukaryonic expression plasmid whicj can express siRNA targeting MDR1 gene, so called chitosansiRNA and chitosan siRNA nanoparticles.2.The expression level of MDR1 mRNA inSKOV3-ts cells were detected by RT-PCR before and after transfection.3.The expressionlevel of P-gp in SKOV3-ts cells were detected by western-blot before and after transfection.4.The IC₅₀ of SKOV3-ts cells to paclitaxel were detected by MTT assay before and aftertransfection.Results: 1.The results of the half quantitative RT-PCR showed that the MDR1 expressionin SKOV3-ts cells transfected by chitosan siRNA and folate modified chitosan siRNAnanoparticles were 0.75±0.01, 0.27±0.01, and there was significant statistic meaning (P<0.05).2.The results of the half quantitative western-blot showed that the P-gpexpression in SKOV3-ts cells transfected by chitosan siRNA and folate modified chitosansiRNA nanoparticles were 0.62±0.01,0.12±0.01, and there was significant statisticmeaning (P<0.05).3.The results of MTT assay showed that the IC₅₀ of SKOV3-ts cells to paclitaxel transfected by chitosan siRNA and folate modified chitosan siRNAnanoparticles were 0.3830±0.0096å’Œ0.0353±0.0006, and there was significant statisticmeaning (P<0.05).Conclusions: Through folate targeted modification, folate modified chitosan siRNAnanoparticles can effectively decreased the expression of MDR1 mRNA and P-gp and canreverse the IC₅₀ of SKOV3-ts cells to paclitaxel.Folate modified chitosan siRNAnanoparticles can reverse the MDR of ovarian cancer mediated by MDR1.