Construction And Anti Temozolomide-resistant Glioma Activity Of Brain-targted Co-delivery System Of SiPD-L1 And Temozolomide

Glioma is the most common malignant tumor in the central nervous system.At present,the standardized regimen for clinical treatment of glioma is surgical resection combined with radiotherapy,followed by three courses of chemotherapy with temozolomide(TMZ).However,it is easy for glioma to develop TMZ resistance and the failure of chemotherapy.At present,the median survival time is 14.6 months for glioma patientsRecent research has found that the resistance of glioma to TMZ is not only related to the gene expression of glioma cells itself,but also closely related to tumor microenvironment.Our previous study showed that the ligand of programmed cell death-1(PD-1)receptor(PD-L1)was significantly overexpressed in TMZ-resistant glioma cells.It is reported that high expression of PD-L1 leads to continuous activation of PD-1/PD-L1 signal pathway,which causes tumor cells to evade immune surveillance and being killed Finally,tumor growth is promoted,and the resistance of tumor to TMZ is also aggravated When the protein expression of PD-L1 in TMZ-resistant glioma cells was inhibited by using PD-L1-specific siRNA(siPD-L1),the expression of TMZ-resistant related protein 06-methylguanine-DNA methyltransferase(MGMT)was also significantly decreased in TMZ-resistant glioma cells,and the inhibitive effect of TMZ on TMZ-resistant glioma cells was obviously enhanced.These results suggest that silencing the protein expression of PD-L1 in TMZ-resistant glioma cell by using siPD-L1 can not only inhibit the PD-1/PD-L1 signal pathway in tumor microenvironment but also enhance the killing effect of body immune system on glioma cells.Moreover,it can also increase the sensitivity of TMZ-resistant glioma to TMZ by decreasing the protein expression of MGMT and enhance the toxicity of TMZ to TMZ-resistant glioma cells.Finally,the growth of TMZ-resistant glioma is inhibited by dually,and the therapeutic effect of TMZ on TMZ-resistant glioma is greatly improved.However,siPD-L1 is unstable in the blood circulation.It is easy for siPD-L1 to be degraded by the RNA enzyme in the body and siPD-L1 can be quickly eliminated by kidney.In addition,many biological barriers in the body,such as the blood-brain barrier(BBB),can hinder the transport of siPD-L1.Thus,ensure the stability of siPD-L1 in serum the accumulation of siPD-L1 in glioma is greatly limited.How to make siPD-L1 across the BBB and efficiently enter the cytoplasm of TMZ-resistant glioma cells to silence the PD-L1 expression is a key point in using siPD-L1 to treat TMZ-resistant gliomaObjective:The siPD-L1 and TMZ co-delivery system based on lipid polymer nanoparticle with core shell structure was prepared to increase the stability of siPD-L1 in the blood circulation.It also enhanced the penetration of nanoparticle across BBB and the accumulation of nanoparticle in glioma cells via glucose transporter on the brain capillary endothelial cells and glioma cells.The siPD-L1 and TMZ were released in TMZ-resistant glioma cells,and the protein expression of PD-L1 and MGMT was decreased.Finally,the growth of TMZ-resistant glioma was inhibited by enhancing the killing effect of body immune system on glioma and improving the sensitivity of TMZ-resistant tumor to TMZ via decreasing the protein expression of MGMTMethods:(1)The polyethyleneimine(PEI,the molecular weight was 600,1200 and 1800)was grafted onto polyaspartic acid(PASP)via disulfide bond to form PASP-g-PEI cationic polymer to condense siPD-L1.2-Deoxyglucose(Glu)was connected with 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol by amide bond to form DSPE-PEG-Glu.DSPE-PEG-Glu was used as brain and glioma cells target material DSPE-PEG-Glu and PASP-g-PEI1soo were identified by proton nuclear magnetic resonance spectra(1H NMR)(2)The complex of PASP-g-PEI and siPD-L1 was used as the core,and the lipid membrane consisted of DSPE-PEG-Glu,cholesterol and 2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE)was used as the shell.The siPLKl and TMZ co-loaded nanoparticle TMZ/siPD-L1@GLPN(PASP-g-PEI1800)with core shell structure was prepared by the thin film dispersion method.The N/P ratio was optimized,and the most suitable PASP-g-PEI was selected out to condense siPD-L1.The particle size,zeta potential,drug loading,environmental response characteristics and stability of nanoparticle were investigated(3)The toxicity of blank nanoparticle scrambled siRNA?PEI25K,scrambled siRNA@PASP-g-PEI1200,scrambled siRNA@PASP-g-PEI1800 and scrambled siRNA@GLPN(PASP-g-PEI1800)was evaluated by using MTT assay,hemolysis test and animal experiment(scrambled siRNA is a negative control for siPD-L1)(4)Eight different siPD-L1 sequences were designed,and the siPD-L1 sequence with the strongest silencing effect on PD-L1 in C6 glioma cells was screened out by western blot(5)The silencing effect of TMZ/siPD-L1@GLPN(PASP-g-PEI,800)on PD-L1 in C6 cells and C6/TR cells was detected by western blot.The effects of TMZ/siPD-L1@GLPN(PASP-g-PEI,800)on the proliferation,migration and invasion in C6 cells and C6/TR cells were investigated by MTT experiment,colony formation experiment,migration experiment and invasion experiment respectively.The effect of TMZ/siPD-L1@GLPN(PASP-g-PEI1800)on protein expression of MGMT in C6/TR cells was investigated by using western blot(6)The cellular uptake and intracellular distribution of siPD-L1@GLPN(PASP-g-PEI1800)in C6 cells and C6/TR cells were investigated by confocal laser scanning microscope(CLSM).The dissociation of scrambled siRNA from PASP-g-PEI in C6/TR cells was also studied by using CLSM(7)An in vitro BBB model was established.The ability of scrambled siRNA@GLPN(PASP-g-PEI1800)to penetrate BBB in vitro and the uptake of scrambled siRNA@GLPN(PASP-g-PEI1800)by bEnd3 cells and C6/TR cells in BBB model were investigated by using fluorescence spectrophotometer,flow cytometer and CLSM.The penetrability of scrambled siRNA@GLPN(PASP-g-PEI1800)in the three-dimensional(3D)tumor spheroids after it penetrated BBB was also studied by using CLSM(8)Luciferase-labeled C6 cells and Luciferase-labeled C6/TR cells were injected into the brain of ICR mice to reproduce in situ glioma model.One week after inoculation of tumor cells,TMZ/siPD-L1@GLPN(PASP-g-PEI1800)was injected to tumor-bearing mice via tail vein once every three days and for consecutive 4 times.The growth of glioma was observed by the Caliper IVIS Lumina ? in vivo image system.28 days after the last administration of nanoparticle,the effects of TMZ/siPD-L1@GLPN(PASP-g-PEI1800)on the expression of PD-L1 and MGMT in glioma tissue were investigated by western blot The number of effector T cells and regulatory T cells in brain tissue of tumor-bearing mice was detected by flow cytometry.The survival time of tumor-bearing mice was recorded(9)The biodistribution of TMZ/siPD-L1@GLPN(PASP-g-PEI18800)in organs and tumor tissue was detected by the Caliper IVIS Lumina ? in vivo image system after TMZ/siPD-L1@GLPN(PASP-g-PEI1800)was injected via the tail vein of the tumor-bearing mice,and the biodistribution of TMZ/siPD-L1@GLPN(PASP-g-PEI1800)in glioma and normal brain tissue was evaluated with fluorescence microscopy.Moreover,the biodistribution of TMZ in organs and tumor tissue was detected by high performance liquid chromatography(HPLC)after the injection of TMZ/siPD-L1@GLPN(PASP-g-PEI1800)via the tail vein of the miceResults:(1)The results of 1H NMR analysis showed that the synthesized compounds were the objective productions.PASP-g-PEI1800 showed the similar compression effect on siPD-L1 as PEI2sK did.Moreover,siPD-L1 could dissociate from PASP-g-PEI1800 in the presence of glutathione.The average diameter and zeta potential of TMZ/siPD-L1@GLPN(PASP-g-PEI1800)were 92 nm and-33 mV,respectively.The drug loading of TMZ in TMZ/siPD-L1@GLPN(PASP-g-PEI1800)was 5.23%.The result of SEM revealed that the TMZ/siPD-L1@GLPN(PASP-g-PEI1800)showed spherical shape Besides,TMZ/siPD-L1@GLPN(PASP-g-PEI1800)could improve the stability of siPD-L1 in serum(2)Compared with scrambled siRNA@PEI25K and scrambled siRNA@PASP-g-PEI1800,scrambled siRNA@GLPN(PASP-g-PEI1800)exhibited the lower toxicity on C6 cells,erythrocyte and mice.Besides,scrambled siRNA@PASP-g-PEI1800 showed lower toxicity than scrambled siRNA?PEI25K(3)The cd274-mus-362 of siPD-L1 sequence exhibited the strongest silence effect on PD-L1 protein expression in C6 cells(4)The protein expression of PD-L1 and MGMT markedly increased in C6/TR cells in comparsion with that in C6 cells.TMZ/siPD-L1@GLPN(PASP-g-PEI1800)significantly decreased the protein expression of PD-L1 in C6 cells and C6/TR cells TMZ/siPD-L1@GLPN(PASP-g-PEI1800)significantly inhibited the proliferation,colony formation,migration and invasion in C6 cells and C6/TR cells as compared with TMZ/siPD-L1@LPN(PASP-g-PEI1800)(without 2-deoxyglucose modification)and free TMZ.In addition,the protein expression of MGMT in C6/TR cells obviously decreased after the treatment of TMZ/siPD-L1@GLPN(PASP-g-PEI1800)(5)The cellular uptake of scrambled siPD-L1@GLPN(PASP-g-PEI1800)in C6 cells and C6/TR cells significantly increased as compared with scrambled siPD-L1@LPN(PASP-g-PEI1800)(without 2-deoxyglucose modification).Phlorizin obviously attenuate the cellular uptake of siPD-L1@GLPN(PASP-g-PEI1800)in C6 cells and C6/TR cells,indicating that the cellular uptake of siPD-L1@GLPN(PASP-g-PEI1800)in C6 cells and C6/TR cells was mediated by glucose transporter.After being uptaken by C6/TR cells,a large amount of siPD-L1@GLPN(PASP-g-PEI)was distributed in the cytoplasm,and little amount of siPD-L1@GLPN(PASP-g-PEI1800)was localized in the lysosome.siPD-L1 could dissociate from PASP-g-PEI1800 effectively in C6/TR cell cytoplasm(6)The penetration efficiency of scrambled siRNA@GLPN(PASP-g-PEI1800)across BBB was higher than that of scrambled siRNA@LPN(PASP-g-PEI1800).The accumulation of scrambled siRNA@GLPN(PASP-g-PEI1800)in bEnd3 cells and C6/TR cells was significantly higher than siPD-L1@LPN(PASP-g-PEI1800).Besides,scrambled siRNA@GLPN(PASP-g-PEI1800)penetrated into deeper area of the three-dimensional tumor spheroids of C6/TR cells after it crossed over BBB.The above results indicated that 2-deoxyglucose modification increased the transport efficiency of scrambled siRNA@GLPN(PASP-g-PEI1800)across BBB,and scrambled siRNA@GLPN(PASP-g-PEI1800)exhibited the strong penetrability to glioma tissue(7)TMZ/siPD-L1@GLPN(PASP-g-PEI1800)significantly inhibited the growth of tumor in C6 cells planted glioma-bearing mice.The survival time of C6 cells glioma bearing mice was prolonged after the treatment of TMZ/siPD-L1@GLPN(PASP-g-PEI,800),and 53.3%of C6 cells planted glioma-bearing mice was survival at day 100 after inoculation of tumor cells.Compared with TMZ/siPD-L1@LPN(PASP-g-PEI1800),siPD-L1@GLPN(PASP-g-PEI1800)and free TMZ,TMZ/siPD-L1@GLPN(PASP-g-PEI1800)showed the strongest inhibitive effect on tumor growth in C6 cells planted glioma-bearing mice.Moreover,in C6/TR cells planted glioma-bearing mice,free TMZ showed no inhibitive effect on tumor growth,while TMZ/siPD-L1@GLPN(PASP-g-PEI1800)significantly inhibited the tumor growth and markedly prolonged survival time of C6/TR cells planted glioma-bearing mice TMZ/siPD-L1@GLPN(PASP-g-PEI1800)obviouly decreased the protein expression of PD-L1 and MGMT in glioma tissue.After the treatment of TMZ/siPD-L1@GLPN(PASP-g-PEI1800),the number of effector T cells increased while the number of regulatory T cells decreased in brain tissue in C6 cells and C6/TR cells planted glioma-bearing mice(8)The accumulation of TMZ/siPD-L1@GLPN(PASP-g-PEI,800)in brain significantly increased in comparsion with siPD-L1@LPN(PASP-g-PEI.)after they were injected via the tail vein of the tumor-bearing mice.The distribution of TMZ/siPD-L1@GLPN(PASP-g-PEI1800)in glioma tissue was obviously higher than that in normal brain tissue.Compared with TMZ/siPD-L1@LPN(PASP-g-PEI1800),TMZ/siPD-L1@GLPN(PASP-g-PEI1800)delivered a large amount of TMZ to the mice brain tissueConclusions:TMZ/siPD-L1@GLPN(PASP-g-PEI1800)effectively condensed siPD-L1 and improved the stability of siPD-L1 in serum.TMZ/siPD-L1@GLPN(PASP-g-PEI1800)could be uptaken by C6 cells and C6/TR cells and release free siPD-L1 in cytoplasm TMZ/siPD-L1@GLPN(PASP-g-PEI1800)penetrated the BBB and accumulated in brain tissue with high efficiency.TMZ/siPD-L1@GLPN(PASP-g-PEI1800)not noly enhanced the activity of body immune system to inhibit the tumor growth by blocking the PD-1/PD-L1 signal pathway in glioma microenvironment but also increased the sensitivity and cytotoxicity of TMZ to TMZ-resistant glioma cells through decreasing the protein expression of MGMT.Finally,the growth of TMZ-resistant glioma was significantly inhibited by body immune system and TMZ.