γ-Glutamyl Transpeptidase Stimuli-responsive Blocked Polymer Prodrug-derived For Glioma Treatment

Objective:Glioma is the primary malignant tumor that commonly appeared in central nervous system of adult,and with a very poor prognosis.At present,the treatment of glioma is basically based on surgery,supplemented by radiotherapy and chemotherapy,but the overall treatment effect is not satisfying.Therefore,it is very necessary to discover new treatment methods for glioma in order to find more effective treatment measures.The target delivery of anti-tumor drugs to tumor sites by drug delivery system is one of the most important direction of researches in this field.γ-Glutamyl transpeptidase（GGT）is an enzyme that is highly expressed in glioma and the blood tumor barrier,but with relatively low expression in normal tissues of living bodies.In this study,we planned to design a blocked drug delivery system for GGT targeting response,which could concentrate at the glioma site through targeted GGT response and then release anti-tumor drug,so as to reduce the side effects of the carried drug and increase it’s anti-tumor efficiency.Materials and Methods:In this study,the GGT responsive amphiphilic block polymers were synthesized firstly through reversible addition-fragmentation chain transfer,and then loaded with podophyllotoxin（PPT）to form the GGT responsive amphiphilic block polymers PPT delivery system（p BEAGA-b-p PPT,GGTPPT）.Next,we studied the physical and chemical characteristics of the drug delivery system.Dynamic Light Scattering（DLS）,Scanning Electron Microscope（SEM）,¹H Nuclear Magnetic Resonance（¹H NMR）,¹³C Nuclear Magnetic Resonance(¹³C NMR),High resolution mass spectrometry（HR-MS）and High Performance Liquid Chromatography（HPLC）were used for the studies.After the physical and chemical characterization of the drug delivery system,we studied the expression level of GGT in glioma cell lines by Western blot（WB）firstly.Then,we used confocal laser scanning microscope（CLSM）and flow cytometry for the study of cellular uptake of the drug delivery system.The penetration of the drug delivery system in glioma multicellular tumor spheriod（MTS）was also studied through CLSM.Cell counting kit-8（CCK8）was then used for experiment of the cell survival rate of glioma that co-incubated with GGTPPT.At in vivo experiments parts,we firstly studied the expression of GGT in glioma tumor tissues through WB.The distribution of the GGTPPT at glioma was then studied using in vivo fluorescence.The anti-glioma efficacy of the GGTPPT was studied through mouse orthotopic glioma model,and in vivo fluorescence system and 7T magnetic resonance imaging（MRI）were used for the glioma monitoring.Hematoxylin-Eosinstaining（HE）staining and immunohistochemical staining were used to study the changes of main organs and tumor tissues in the mouse model after administration of GGTPPT.In the the therapeutic mechanism study part,we firstly used bright field imaging of microscope for study of the morphological changes of glioma cells after co-incubated with GGTPPT.Then CLSM,structure illumination microscopy（SIM）was used to observe the microtubule changes of glioma cells.The survival situation of glioma cells after administration of GGTPPT was studied by dead and living cell fluorescent staining.The change of cell cycle of glioma after co-incubation with GGTPPT was studied by flow cytometry.Then the apoptosis of glioma cells after co-incubation with GGTPPT was also studied through flow cytometry.Finally,the expression level changes of proteins in apoptosis pathway in glioma affected by GGTPPT were studied by WB.Results:In this study,a GGT specific responsive amphiphilic block polymer drug delivery system was successfully designed and synthesized.At the beginning of this study,the drug delivery system was synthesized step by step according to the designed synthesis route,and then the synthesis products of each step were characterized.After the completion of synthesis,the physical and chemical characterization of the GGTPPT drug delivery system was also carried out.The results of the characterization of the intermediate products showed that the structure of all products in the synthesis process was consistent with the designed chemical structure.In the characterization results of GGTPPT drug delivery system,the particle size of GGTPPT drug delivery system was 87.15±5.04 nm and the basic zeta potential was-3.50±0.69 m V measured by DLS.Stability test showed that the particle size of GGTPPT drug delivery system was basically stable at about 85 nm within 48 hours in PBS solution.The results of the charge reversal experiments showed that the GGTPPT delivery system could achieve charge reversal in the microenvironment with high GGT level in vitro,and could maintain electronegative in the microenvironment of low GGT and without GGT.The responsive drug release of GGTPPT drug delivery system was detected by HPLC.The experiment results showed that GGTPPT drug delivery system be able to release Podophyllotoxin（PPT）in vitro with high concentrations of Glutathione（GSH）,and barely no PPT released at phosphate buffer saline（PBS）.In cell experiment,we first verified that the expression level of GGT in U87glioma cells was higher than that of astrocytes（P<0.05）.Subsequently,it was observed through CLSM that GGTPPT drug delivery system labeled with Cy5 could be successfully ingested by U87 glioma cells,and the semi-quantitative analysis results of flow cytometry showed that the uptake of GGTPPT drug delivery system by glioma cells was positively correlated with the co-incubation time.GGTPPT could also be successfully ingested by U87 glioma cell MTS,and GGTPPT could successfully penetrated into MTS,and the amount of infiltration was positively correlated with incubation time.CCK8 cytotoxicity experiment results showed that the half maximal inhibitory concentration（IC50）of free PPT on U87 glioma cell is about 25 n M,and the IC50 of GGTPPT drug delivery system increased to 550 n M of relative concentration of PPT.The results suggested that GGTPPT could successfully release the antineoplastic drugs PPT,but for the need of longer time to enter glioma cells and need time for drug release also,the IC50 of GGTPPT is higher compared to free PPT.In vivo experiments,this study successfully established the nude mouse U87glioma model firstly,and then detected the expression level of GGT in glioma tissues and normal brain tissues of the mouse model by WB.The results showed that the expression level of GGT in glioma tissue was higher than that in normal brain tissue,and the semi-quantitative analysis showed that the expression level of GGT in tumor tissue was about 4 times compared to normal brain tissue.In the treatment study,the distribution of GGTPPT at tumor site was detected through living fluorescence.The results showed that GGTPPT could selective gathered at glioma tumor site,and could persisted for a quite long time.Specifically,GGTPPT drug delivery system concentration level at glioma site reached the top level at about 12 h after drug administration.In addition,fluorescence imaging of U87luc glioma tissue from nude mice orthotopic glioma model also showed that GGTPPT could be highly aggregated at the glioma site.Fluorescence imaging results showed that both free PPT and GGTPPT drug delivery system could inhibit the growth of glioma in nude mice.But the inhibition effect of GGTPPT drug delivery system is better,and the glioma treated with free PPT was observed with rapid growth of tumor size upto PBS treated group at about 21 days after first time of dosing.What’s more,half of the mice in PPT treated group died after the third time dosing,which caused by the high system toxicity of free PPT.In the treatment experiment,the T2-weighted images of7T MRI showed that the glioma growth characteristics were consistent with the results of fluorescence imaging semi-quantitative analysis that the GGTPPT drug delivery system had a better glioma inhibition effect.HE staining of the main organs and tumor tissues showed no significant difference among groups.However,in the HE staining of glioma tumor tissue,large area of necrosis was observed around vessels in free PPT group,but there was no obvious change of the glioma cells away from vessels.In the HE staining of tumor tissues of GGTPPT drug delivery system group,it was observed that the cell nucleus density of glioma cells was relatively low,and this change was also observed in the tumor tissues that were far from blood vessels,indicating that the drug delivery system could successfully penetrate into the tumor tissues and then inhibit glioma growth.Immunohistochemical staining of glioma tissues showed that the expression of Ki67 in GGTPPT group was relatively low compared to the expression of Ki67 in other groups,indicating that the proliferation activity of glioma in the drug delivery system group was significantly inhibited.The results of terminal-deoxynucleoitidyl transferase mediated nick end labeling（TUNEL）fluorescence staining showed that there was more apoptosis fluorescence signals in GGTPPT group compared with other groups,indicating a higher proportion of apoptosis in glioma at the drug delivery system group.In the study of the therapeutic mechanism,the bright field images of U87 cells that treated with drugs showed that the morphology of U87 glioma cells changed significantly in free PPT group and GGTPPT group.Glioma cell of the two groups were both become the shape elliptic or irregular from of fusiform.The CLSM and SIM images of fluorescent dyed microtubules showed that microtubules of U87glioma cell in both free PPT group and GGTPPT group presented the state of aggregation,while the microtubules of control group presented a continuous parallel linear form,indicating that free PPT and GGTPPT could both anti-depolymerize microtubules in glioma cells.Then,the dead and living cell fluorescent staining of monolayer U87 glioma cells showed that the glioma cells in both free PPT group and the GGTPPT group have a higher percent of death cells.At the same time,the dead and living cell fluorescent staining of MTS showed that GGTPPT group have more glioma cell death in MTS compared to free PPT group.This result was consistent with the MTS penetration experiment results above,indicating GGTPPT could successfully penetrate MTS and then play the role of anti-glioma.Flow cytometry detection of glioma cell cycle results showed that the cell cycle of glioma cells in free PPT group and GGTPPT group were both blocked at G2/M phase.The following flow cytometry apoptosis detection results show that both free PPT group and GGTPPT group could induce apoptosis of U87 glioma cells,and the proportion of early apoptosis cell were both about 25%of all tested glioma cells,indicating that the drug delivery system and free PPT can both depress glioma cells through inducing apoptosis.Conclusion:The GGTPPT drug delivery system designed and synthesized in this study can achieve targeted aggregation in U87 glioma site and enhance penetration into solid U87 tumor through GGT responsive charge reversal in GGT highly expressed glioma site.GGTPPT drug delivery system can be retained at the tumor site for a long time.At the same time,it could contiguously release anti-tumor drug PPT through response to the high GSH level microenvironment in glioma,by which exert anti-glioma effect and significantly reduces the systemic toxicity of PPT.The expression level of GGT in glioma tissue of nude mouse glioma model was significantly higher than that in normal brain tissue.The anti-glioma mechanism of GGTPPT drug delivery system is basically the same as that of free PPT,both of which can inhibit the microtubule depolymerization of glioma cells in the mitotic.The proliferation of glioma is arrested at G2/M phase,and the anti-glioma effect is produced by inducing the apoptosis of glioma cells.To sum up,this study successfully constructed the GGT responsive amphiphilic block polymer drug delivery system and did the systemic glioma therapy research.The results proved that the drug delivery system can give the drug that cannot be used routinely in clinical the ability of targeting release at glioma site.The system could reduce the system toxicity of carried drug,and improve the effect of anti-glioma at the same time,thus achieving a good therapeutic effect in orthotopic glioma model.Therefore,the GGT-responsive amphiphilic block polymer drug delivery system is expected to become an effective anti-glioma drug delivery system,which has a promising research prospect and is worthy of further study.