| Background and Aims:Glioma is the most common and serious brain malignancy in the central nervous system.Clinically,the standard treatment for gliomas is surgical resection with adjuvant radiotherapy and chemotherapy.Temozolomide(TMZ)has become the first-line chemotherapy drug for gliomas due to its wide applicability,low toxicity and high lethality.The resistance of glioma to TMZ is the bottleneck of clinical chemotherapy.Studies have shown that glioma-derived exosomes are related to the formation of their malignant biological behavior,and may be involved in the formation of temozolomide resistance in gliomas.Therefore,inhibiting the secretion of glioma-derived exosomes may have the effect of inhibiting the malignant biological behavior of glioma cells and reversing TMZ resistance.Based on the above research background,this study first evaluated the effect of exosome inhibitor GW4869 on the malignant biological behavior of glioma cells,and further evaluated the reversal of TMZ resistance by exosome inhibitor GW4869 by constructing glioma drugresistant cell lines.Secondly,in order to achieve the purpose of two-drug combination,this study constructed a targeted nano-drug delivery system loaded with both an exosome inhibitor(GW4869)and a chemotherapeutic drug temozolomide(TMZ),which can cross the blood-brain barrier and can successfully deliver drugs to intracranial tumor areas,improve the chemotherapy of brain gliomas to TMZ by reversing the resistance of gliomas to TMZ and inhibit the malignant biological behaviors of gliomas(such as proliferation,metastasis and invasion,etc).This topic evaluates the targeting of the nano-drug delivery system at the cellular and in vivo levels,and evaluates and verifies its combined therapeutic effect at the cellular level,providing a new treatment strategy for glioma.Methods:1.To study the effects of GW4869 on the malignant biological behavior of gliomas: The effects of GW4869 on the migration,invasion and proliferation of gliomas were studied by scratch assay,Transwell assay and plate clone respectively.2.Assess the effect of exosome inhibitor GW4869 in reversing TMZ resistance: use the temozolomide drug concentration increasing method to construct a temozolomide-resistant glioma cell line,and on the basis of the successful construction of its drug-resistant strain,to investigate the ability of GW4869 to reverse TMZ resistance and to explore its mechanism.3.Preparation and characterization of GW4869/TMZ@PM-ApoE: Dual-loaded GW4869 and TMZ drug-loaded micelles were prepared by "thin film hydration method",and then successfully prepared by the special reaction of sulfhydryl and maleamide groups.Drug-loaded micelles modified with targeting peptide ApoE(GW4869/TMZ@PM-ApoE).It was characterized by transmission electron microscope(TEM),dynamic light scattering(DLS),and zeta potential detection,and its stability was investigated.4.Evaluation of the ability of GW4869/TMZ@PM-ApoE to target gliomas in vitro and target therapy in vitro:(1)Western Blot was used to detect the expression of LRP-1 protein on glioma cells(U251),brain microvascular endothelial cells(Bend.3)and primary astrocytes cells;(2)The targeting ability of GW4869/TMZ@PM-ApoE to glioma cells,brain microvascular endothelial cells and primary astrocytes was observed at the cellular level by fluorescence microscopy;(3)To investigate the distribution of GW4869/TMZ@PM-ApoE in vivo,and to evaluate its in vivo targeting ability by Near Infrared Imaging Technology(4)At the cellular level in vitro,the cytotoxicity of GW4869/TMZ@PM-ApoE on glioma cells was detected by Cell Counting Kit-8method.Results:1.GW4869 inhibits the malignant biological behavior of glioma cells.The clone formation rates of GW4869 5μM group and GW4869 10μM group were 40.58±0.55% and 12.66±0.88%,respectively,which was significantly lower than that of the control group,which was 70.99±1.32%.The effect of different concentrations of GW4869 on the invasive ability of glioma cells U251 after 24 hours of treatment was evaluated by Transwell assay.The invasion numbers of cells in GW4869 group were26±4 and 12.6±3.05,which were significantly less than those in control group(148.3±6.5).With the increase of concentration,the number of transmembrane cells gradually decreased.Then we explored the effect of GW4869 treatment on the migration ability of glioma cells U251.The scratch healing rates were 51.39±0.64% and 9.62±0.62%,respectively.Compared with the 78.55±1.28% healing rate of the control group,the migration ability of U251 cells was significantly weakened after GW4869 treatment.2.The TMZ-resistant glioma cell line(U251/TR)constructed in vitro,after the combined treatment of GW4869 and TMZ,the overall apoptotic rate of U251/TR was significantly increased.The total apoptotic rate of each group was: 2.35±0.32%,3.31±0.38%,3.22±0.2% and 16.02±0.44%.The CCK-8 experiment showed similar conclusions.After treatment,the cell survival rates were96.12±0.64%,95.58±1.03% and 77.33±2.93%,respectively.Compared with the control group,the combination of GW4869 and TMZ significantly reduced the cell viability.The survival rate was significantly destructive to drug-resistant cell lines.And q PCR results showed that MGMT expression was significantly down-regulated after GW4869 intervention.The above results were all significantly different(P<0.05).3.The TEM results of the unloaded micelles showed that the polymer micelles self-assembled from the amphiphilic block copolymers were circular,with uniform size distribution and no aggregation.The average hydrated particle size was relatively consistent,and the unsupported micelle solution prepared by the thin-film hydration method had high yield and good morphology.According to the TEM results,the drug-loaded micelles showed no obvious changes in morphology,indicating that the micelle morphology did not change after drug loading.DLS results showed that after loading GW4869 and TMZ and the connecting polypeptide ApoE,the average hydrated particle size slightly increased to 48.65 nm.The polypeptide coupling efficiency was obtained by dividing the amount of ApoE on the surface of the polymer micelle by the amount of ApoE input,which was approximately10.04%.Using HPLC to perform linear regression processing on the concentration by recording the peak area results,the drug loading of TMZ was about 1.53%;the drug loading of GW4869 was about2.073%.4.WB detected high expression of LRP-1 protein on U251 cells and primary cerebral microvascular endothelial cells,but almost no expression of LRP-1 protein on primary glial cells.In evaluating the targeting ability of GW4869/TMZ@PM-ApoE in vitro,GW4869/TMZ@PM-ApoE entered glioma cells U251 faster than GW4869/TMZ@PM group,and GW4869/TMZ@PM-ApoE was more absorbed by the brain Microvascular endothelial cells uptake but not astrocytes,indicating that the prepared GW4869/TMZ@PM-ApoE has better targeting.In the simultaneous in vivo imaging experiment,compared with GW4869/TMZ@PM group,GW4869/TMZ@PM-ApoE can better cross the BBB and achieve aggregation in the tumor area.And at the cell level in vitro,GW4869/TMZ@PM-ApoE produced a significant killing effect: after GW4869/TMZ@PM-ApoE interacted with glioma,the cell survival rate was reduced to 66.34±2.32%,compared with no peptide modification The drug-loaded micelles(83.03±1.42%)had significantly improved lethality,which was also higher than that of GW4869+TMZ without micelles(82.51±0.74%).Conclusion:1.GW4869 has the ability to significantly inhibit the proliferation,growth,invasion and metastasis of glioma cells,especially can effectively reverse the drug resistance of temozolomide-resistant cell line(U251/TR),which can be used for the construction of subsequent nano-drug delivery systems;2.GW4869/TMZ@PM-ApoE was successfully constructed,its particle size is below 100 nm,which can cross the blood-brain barrier,has high stability and biocompatibility,and has less toxic and side effects;3.At the cellular level in vitro and in vivo,GW4869/TMZ@PM-ApoE has superior targeting properties,which can overcome the obstacles of the blood-brain barrier and successfully target the local glioma,improving the efficiency of drug delivery;and in vitro,GW4869/TMZ@PM-ApoE showed high cell killing,suggesting that the combined effect of co-delivery of GW4869 and TMZ enhanced temozolomide sensitivity. |
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