Experimental Studies On Glioblastoma U251 Interfered By Chitosan Nanoparticles Mediated HTERT-siRNA

Malignant tumor is the first cause of death in town and rural inhabitants of China. Furthermore,its morbidity is growing year by year.Malignant gliomas,around 40～50%of intracranial tumors,are the most common malignant tumors of the central nervous system(CNS) clinically.The morbidity of the gliomas is 8～10 per 100,000 persons and rises gradually year by year.Malignant gliomas attacked with high incidence and kill 180,000～600,000 young and middle-aged people every year globally.According to the rank of disease mortality from world health organization(WHO) in 1998,cause of death from malignant gliomas ranked second at age 34 years and younger,and third at age 35 to 54 years.At present,the essential therapeutic tools of malignant gliomas are surgical resection plusing chemotherapy and radiotherapy.However,the gliomas are expelled from the possibility of completed removal and are doomed to recurrence because of their high diffuse infiltrating growth pattern,whilst radio- and chemo-therapy,for low sensitivity and resistance from glioma,can't increase prognosis of glioblastoma patient.Thus,how to improve the clinical treatment effect of glioblastoma should be a major endeavour in modern neuro-oncology.Any new treatment method aimed at glioblastoma is important to improve prognosis.Until now,the best way to treat tumor is to eliminate the tumor,which means, from cellular level,to selectively kill tumor cells as far as possibly.Inhibition of single oncogene is hard to obtain good result for tumor treatment since no all oncogenes and tumorsuppressorgenes take roles during tumorigeness and also because the existence of signal transduction network system make more difficulties to screen key genes related to tumor which is multi-gene and multifactor diseases.RNA interference(RNAi) can inhibit several different genes simultaneously but the inhibition effect doesn't disturb each other,so that RNAi may become a new and powerful potential approach for tumor treatment.RNAi is triggered by double-stranded RNA(dsRNA) and causes degradation of mRNA transcripted by specific tagerting gene in order to suppress expression of targeting protein,and furthermore to provide a novel therapeutics against diseases. Since RNAi technology has been developed from 1990s,it has been widely used in research on gene function,pathway of signal transduction,gene therapy,etc.21- and 23-nucleotide small interfering RNA(siRNA),the mediator of RNAi,is easy to be degraded by RNAse.These exogenous naked nucleotides are difficult to be uptaked by eukaryotic cells.Thus,to avoid the low efficiency of siRNA transfection, it is necessary to select suitable vector for siRNA encapsulating,which can protect siRNA from degradation and help siRNA to transfect cells efficiently.At present, siRNA vectors are still limited in virus,liposome,DNA plasmid,etc.However these vectors are not the best choice for siRNA delivery because they are easy to induce side effect and immune response.Especially to cerebral gliomas,transportation of siRNA into the tumor cells is very difficult because of the existence of the blood-brain barrier(BBB).For this reason,an optimal vector has to be developed for the feasible therapeutics of RNAi. As chitosan has been shown its properties of good biocompatibility,complete biodegradability and anti-tumor activity,in this study,we selected human telomerase reverse transcriptase(hTERT) gene as targeting site for RNAi therapeutics and preparation of chitosan nanoaggregates,in which siRNA was encapsulated to protect them from degradation and to control them for sustained release,to transfect glioblastoma U251 cells in vitro.The results showed that chitosan-siRNA nanoparticles could transfect the U251 cells efficiently to inhibit their growth and proliferation and remarkablely induce their apoptosis,indicating that interference of siRNA targeting hTERT mediated by chitosan nanoparticle was effective on tumor treatment.RNAi might be a new approach for tumor therapeutics.Partâ… :Preparation and characteristic study of chitosan-siRNA nanoparticlesObjective:To explore the characteristics of Chitosan-siRNA nanoparticles prepared by ionic gelation and analyze their phy-chemical properties.Methods:Chitosan-siRNA nanoparticles were prepared by ionic gelation of chitosan with pentasodium tripolyphosphate(TPP) and siRNA targeted human telomerase reverse transcriptase(hTERT).The morphology of the nanoparticle was observed with transmission electron microscope(TEM) and atomic force microscope(AFM). Mean particle diameter and zeta potential of the nanoparticles were measured by Zeta potential/particle analyzer.The loading efficiency of siRNA entrapped into the chitosan nanoparticles and its controlled release ability in vitro was obtained from the determination of free siRNA concentration in the supernatant recovered after particle centrifugation by absorbance measurement at 260 nm.Gel electrophoresis assay was used to detect the siRNA stability in nanoparticles after reaction with foetal bovine serum(FBS).Results:The chitosan-siRNA nanoparticles were successfully prepared,spherical in shape and uniform in size by atomic force microscope(AFM) and transmission electron microscope(TEM).The mean particle diameters were 83.3nm and mean surface potentials were +24.2mV,by measure of Zeta potential/particle analyzer.The loading capacity of the nanoparticles was 95%.Release of siRNA from chitoan-siRNA nanoparticles was slow in vitro,releasing curve was "S" shape and release rate was less than 20%within 24h.After fetal bovine serum(FBS) treatment, Gel electrophoresis assay indicated that chitosan-siRNA nanoparticles were stable in nature and could protect siRNA against RNase degradation.Therefore they were protective for siRNA.Conclusion:Ionic gelation is easy and provides mild condition for the preparation of Chitosan-siRNA nanoparticles which are well-distributed and regular in shape.The prepared particles have high loading efficency and favorable stability.They can obviously delay the release of siRNA and prevent siRNA from degradation in vitro. Such nanoparticles are suitable as genic carrier.Partâ…¡:The experimental study on glioblastoma U251 transfected by chitosan-siRNA in vitroObjective:To investigate cell proliferation and apoptosis of glioblastoma U251 by RNAi with chitosan-siRNA nanoparticle targeted human telomerase reverse transcriptase(hTERT) gene in vitro.Methods:1.Preparation of Chitosan-siRNA nanoparticle targetied hTERT gene(please refer to partâ… )2.Experimental groups:The transfected neurogliocytoma U251 cells were divided into 8 groups:A:blank control group(without transfection reagents or siRNA);B:positive control group(Lipofectamine^TM 2000 transfected siRNA);C: negative control group(3 mmol/L controlled siRNA nanoparticle);D:bare siRNA control group(3 mmol/L siRNA);E:blank CS nanoparticle control group;F,G,H: siRNA nanoparticle groups(1,3,9mmol/L respectively).3.Subculture of U251 cells.4.Neurogliocytoma cells were transfected with chitosan-siRNA U251 at logarithmic growth phase.5.Growth and morphological change of cultured cells were observed under inverted microscope at the 48^th hour after transfection.6.The morphology of the nucleus was observed by Hoechst 33342/propidium iodide(PI) double staining under fluorescence microscope.7.The cell cycle was detected by PI one-step fluorescence staining.8.Apoptosis of U251 cells was assessed by Flow cytometry.9.U251 cells were detected to draw the curve of cellular proliferation and calculate inhibition ratio by means of CCK-8 method.10.Reverse transcription-polymerase chain reaction(RT-PCR) was used to detect the expression changes of hTERT-mRNA.Results:1.Morphologically,cells were shown epithelium-like adherence,fusiform,clear outline,transparent,high diaphaneity,less granulas,growth well and tight structure between cells in the blank control group,negative control group,bare siRNA control group and blank CS nanoparticle group.On the contrasty,in positive control group and siRNA nanoparticle groups cells were shown partly shrink and floating,loosed contact between cells,slow proliferation,more granulas,increasing fragment and poor growth.All of these indicated that cellular proliferation was obviously inhibited in positive control group and siRNA nanoparticle groups(3 and 9 mmol/L).2.The results of Hoechst33342/propidium iodide(PI) double staining under fluorescence microscope revealed that,the normal cells with light blue were mainly showed in the blank control group,negative control group,bare siRNA control group and blank CS nanoparticle group,while the apoptotic cells with dark blue were mainly in the positive control group and siRNA nanoparticle groups(1,3,9 mmol/L).3.Statistical significance of cell cycle in 8 groups was shown at G₀/G₁ phase (F=46.850,P=0.000) and at S phase(F=47.964,P=0.000),respectively,whereas no difference(F=2.532,P=0.059) at G₂/M phase among all groups.Compared with cells in blank control group,negative control group,bare siRNA control group and blank CS nanoparticle group,the cells in positive control group and siRNA nanoparticle groups(1,3,9 mmol/L) were increased at G₀/G₁ phase and decreased at S phase,in which a significant change(P＜0.05) was in the positive control group.4.Apoptosis rates were statistically significant in groups(F=1192.251,P=0.000). There were significant differences between positive control and 1,3,9 mmol/L siRNA nanoparticle groups compared with other groups.There were also significant differences between 1 mmol/L siRNA nanoparticle group and siRNA nanoparticle groups with 3 and 9 mmol/L respectively,but no difference between 3 and 9 mmol/L siRNA nanoparticle groups.5.Absorbance values showed by CCK-8 were statistically significant in groups (F=38.639,P=0.000).There were significant differences between positive control and 3,9 mmol/L siRNA nanoparticle groups compared with other groups,by obvious inhibition of cell proliferation and decrease of absorbance value.6.hTERT-mRNA levels detected by RT-PCR were statistically significant among the groups(F=1218.959,P=0.000).The mRNA levels were obviously decreased in positive control group and siRNA nanoparticle groups,especially in 3 and 9 mmol/L siRNA nanoparticle groups.Conclusion:Chitosan-siRNA effectively inhibits the cells growth and promotes the apoptosis of U251 in vitro.The effect of chitosan-siRNA nanoparticles on growth inhibition of U251 has no dose-effect relations but 3 mmol/L group shows a better result.