Preparation And Therapy Research Of Telomerase Antisense Oligonucleotide Nanoparticles On Malignant Glioma

SummaryGlioma is the most common primary intracranial tumors and growth is very rapid.The ways of treatment are usually operation and radiotherapy and chemotherapy and immunotherapy at present.Due to the invasive ability of tumor cell,the glioblastoma infiltrativly grows into the normal brain tissue;so the operation of total resection is very difficult and the recurrence rate is high after the operation. The prognosis of most of patients can not be improved,so the effective treatment method become an exploration of neurosurgery problem.With the development of oncomolecularbiology,it has be confirmed that telomere and telomerase are basement of cell division and multiplication.The telomere is the compound of DNA series and protein in eucaryotic cell chromosome ends.Under normal circumstances,it becomes gradually short with the each round of cell division.When it decurtates to borderline length,the cells cease division and age and decease.So the length of telomere is the molecular marker of cell reproductive and life.Telomerase is the special retroviridase composed of hTERT and hTR and hTPI.With the autospecific formwork of hTR,it can catalyze the duplication of telomere repetitive sequence and prevent telomere shortening and maintain cell proliferation activity by way of reverse transcription. Abnomal reactivation of telomerase is the basic reason of glioblastoma infinite and restraining apoptosis.As the telomerase is the most generally and specificity tumor marker,it's closely related to genesis and development and prognosis of the malignant tumor.The telomerase as a target,it is a new strategy that the telomerase inhibitor in coordination with conventional methods treat brain tumors.There have been many ways to inhibit telomerase at present,but the antisense technology is the most outstanding.The antisense oligodeoxynucleotides(ASODN) by the antisense technology is a kind of fragment of oligonucleotide man-made composition and expressed vector expression of oligonucleotide.The unique nucleotide sequence determines that it can complement with the basic group of target DAN and target RNA.and multi-channelly block the expression of the target gene.A considerable amount of research shows that ASODN injection in vitro and intratumor can significantly inhibit the malignancy accrementition of neurogliocytoma,and promote apoptosis,and increase the chemotherapeutics sensitivity of tumor cells.ASODN has been the potential vitality in treating neurogliocytoma.However with the polyanion of ASODN and the negative charge of cells envelope and the degradation of endonuclease in transport processing,we should search a kind of carrier to assist ASODN.Nanoparticle(NP) as a novel drug delivery system generally refers to the particle size in the 1-1000 nm particles.As a giant molecule material,drugs can be dissolved and adsorbed and wrapped in the material.NPs possess biological advantages of dimension effection and surface effecttion and easy absorption.Currently the nano-technology generally applicate in the fields of the targetion administration and the giant molecule material.To increase the transfection of tumour cells and the stability of durgs,ASODN was adsorbed in the surface of NPs with domestic and overseas ways.But the effect in vivo is not ideal.So we demand a new kind of carrier to parcel and protect ASODN.Objective of the researchIn research,optimizing the giant molecule materials—butyleyanoacrylate(B C A) as the carrier of NPs and applying interfacial polymerization,we parcel and freeze and dry NPs by optimizing technology.We investigate the stability of NPs and observe the inhibitive impact on C6 neurogliocytoma.Pramary methods,contents and conclusionsPartâ… :Optimize technology of preparaing ASODN in NP.In this experiment,BCA was the biodegradable polymer materials and the carrier;the solution of ASODN in NP was prepared by the interfacial polymerization. According to the test results of the single factor,we optimized ASODN concentration, pH and Tween-80 as three main factors and choosed three levels in each factor.The orthogonal design principle by orthogonal design table L₉(3⁴) was employed.The best process parameters were 5.0×10^-6 mol/L density of ASODN in NP,8.0pH and 1.25g Twween-80.Partâ…¡:The study of property,purification and freeze-drying.The solution of ASODN in NP was uniformity and translucence disperse system of a little opalescence.The particle size and the distribution of ASODN in NP were determined by photo correlation spectroscopy(PCS).The encapsulation efficiency (EE) and the drug loading(DE) were examined by high performance liquid chromatography(HPLC).The property of ASODN in NP:Dn=94.9±6.2 nm, EE=96.67±1.45%,DL=10.1±0.35%,PI=0.05±0.012,Zeta potential=-37.2 3.11mv.The surface of NPs was the uniform particle size and smooth observed in transmission electron microscopy(TEM).It showed that the purification of ASODN in NPs was freezingly centrifuged at 4℃,20000 rpm for 30 min and washed for three times.The freeze-dry protector was lactase of 5%and freeze-dried samples was smooth roundness.Partâ…¢:The stability of ASODN in NPThe stability of ASODN in NP freeze-dried powder was investigated by acceleration testing in the temperature of 40±2℃and the humidity of 75±5%for 3 months.The samples were conducted every month.:the appearance colour,PH,Dn, DL,and redispersion etc.As the control,the stability of ASODN in NP solution was also studied at the room temperature for three months and checked for the appearance colour,Dn,DL,PH and so on.The results of the acceleration testing manifested that the appearance colour,Dn,DL,and pH had no significant variation,while the results of ASODN in NP solutions in the room temperature stability testing showed the Dn became bigger.DL and EE became smaller as time went by.It meaned that lyophilizing ASODN in NP solutions into freeze-dry powder can markedly increase the stability of ASODN in NP.ASODN in NP,ASODN-NP and FREE ASODN were prepared as described The different preparations were mixed with the fetal calf serum at 37℃.After inactivating serum enzymes,we obtained ASODN by incubation with NaOH(4M).ASODN was assayed by polyacrylamide-7M urea sequencinggel(PAGE),EBS staining.The experimental results:There were bright straps in ASODN in NP and ASODN-NP samples.FREE ASODN had been completely degraded,so there was not bright strap in FREE ASODN sample.ASODN in NP can effectively protect ASODN.Such protection was superior to ASODN-NP.Partâ…£:Growth suppressive effect of ASODN in NP on C6 glioma cells.Grouping:A culture solution control;B BCA-NP;C FREE ASODN;D ASODN-NP;E ASODN in NP.1.Flow cytometry(FCM) detected cell cycle:C6 cells of exponential phase of growth had serially subcultivated for 24h in 24 orifices.Experimental groups had trnsfected C6 cells for 48h.Prepare FCM samples and analyze cell cycle.FCM results: compared with the control,there were obviously change except BCA-NP in the cell cycles after transfection.There were significantly difference(P＜0.01) in ASODN in NP.The cells amounts of Go/G1 phase significantly increased.DNA synthesis(S phase) cells proportionally reducted.ASODN in NP group also showed significantly differences(P＜0.01)in compared with ASODN-NP.ASODN in NP can effectively block C6 cells in G1 phase to S phase,inhibit cell proliferation,and promote apoptosis.2.CCK-8 kit detected relative growth rate of cells.After passage by trypsin digestion,low-speed centrifugation,collection and cells count,the cell density was adjusted to 5×10⁷/L.C6 cells were inoculated in the 96-well culture plate and cultivated in the banjo of containing CO₂.The cell growth was observed in the inverted mieroscop.There were different concentrations of each group dilute solution (final concentration of ASODN:5.0,10.0,15.0,20.0,25.0,30.0μmol/L,respectively) added into the start-transfected cells,the blank transfected as the control group. ASODN in NP and ASODN-NP were equivalent in the amount of ASODN and the control group was only added into the culture medium.After 24 h,we added CCK-8 10μl to each hole and continued to cultivate for 4 h.The absorbance(A) was determined at 450 nm.We accorded to the following formula to calculate the relative growth rate(RGR%),RGR%= A value of the experimental group / A value of the control group×100%.The results of the C6 glioma cell growth inhibition showed that when the final concentration of ASODN in NP was 5-10μmol/L,C6 cells began to be inhibited. The effection of inhibition was better than Free ASODN and ASODN-NP.When the relative final concentration of ASODN was 10-15μmol/L,ASODN in NP group demonstrated strong inhibitory effectction.Proliferation activity reduced with the increase of the ASODN concentration.C6 cells proliferation dose-dependent was significantly better than other groups.ASODN in NP can be more effectively play on the inhibitory effection on glioma cells.