The Effect Of Double-loaded Nanomicelles On Glioblastoma Cell In Vitro

BackgroundGlioblastoma(GBM)is a common intracranial malignancy that originates in neuroepithelial cells.At present,the main treatment is surgical resection combined with radiotherapy and chemotherapy,but the prognosis is still poor,and the survival rate is still low.One of the main reasons is that the tumor is infiltrating and most of them are in important parts of the brain.The surgery can not be excised cleanly,on the other hand,it is difficult for the chemotherapy drugs to cross the blood-brain barrier to reach the tumor site and the single drug treatment effect is poor.Therefore,new treatment strategies urgently need to be studied,and the combined use of two or more drugs and targeted delivery to the tumor site has become a hot topic in tumor treatment strategies.Curcumin(CUR)is a yellow polyphenolic natural active substance extracted from the plant turmeric.Studies have shown that CUR has a variety of pharmacological effects such as sensitization,anti-inflammation,anti-cancer.The anti-cancer effect has been obtained in animal experiments,but poor water solubility limited its application.Doxorubicin(DOX)is commonly used in clinical broad-spectrum anti-tumor drugs,but there is a short window of treatment,strong resistance,cardiac toxicity and other issues.Studies have confirmed that the combined use of DOX and other drugs can increase its anti-tumor effect and slow drug resistance.With the advent of nanomedicine,the relationship between the physical and chemical properties of nanostructures and the ability of drugs to effectively deliver to tumor cells has been elucidated.Because of its ability to increase drug utilization,nanopharmaceutical systems increase drug solubility and prolong drug circulation time.The advantages of achieving tumor targeting and drug co-delivery are of concern.The use of nano drug-loaded compounds has become a potential strategy for the diagnosis and treatment of glioblastoma.Studies have shown that the co-delivery of different drugs through nanoparticles(NPs)can effectively improve drug efficacy and achieve drug synergy,which has a good application prospects.In recent years,it has been reported in the literature that the inclusion of chemotherapeutic drugs into NPs can significantly enhance the anti-glioblastoma effect,but the effect of different types of nanoparticles and chemotherapeutic drugs on different tumors is somewhat different,and an excellent preparation scheme is available.The appropriate treatment strategy is still in urgent need of study.In this paper,the self-assembly of CUR and DOX encapsulated by polyethylene glycol(PEG)was used to prepare the nano-micelles PEG/DOX/CUR with DOX and CUR,and the synergistic effect of DOX and CUR on the antiglioblastoma cells and the mechanism of PEG/DOX/CUR entry into glioblastoma cells were discussed.ObjectiveTo study the effect of PEG/DOX/CUR on glioblastoma cells in vitro and the synergistic effect of DOX and CUR,and to explore the mechanism of PEG/DOX/CUR uptake.Methods1.Preparation and characterization of PEG/DOX/CUR.According to the single factor investigation method,the optimal preparation method of PEG/DOX/CUR was selected.PEG/DOX/CUR was characterized by scanning electron microscope,particle size potential scanner,ultraviolet spectrophotometer,fluorescence spectrophotometer and NMR spectrometer.The release efficiency of DOX and CUR from PEG/DOX/CUR was determined by using dialysis bag to simulate the release environment in vivo,and the release efficiency of PEG/DOX/CUR in acidic environment was studied by adjusting the release environment of DOX and CUR.2.To study the synergistic effect of DOX and CUR and the effect of PEG/DOX/CUR on glioblastoma cells.The cell survival rate was measured by cytotoxicity test.The 50% Inhibiting concentration(Inhibiting concentration,IC50)and the combined drug index(Combination index,CI)were calculated by the formula.The apoptosis-promoting effect of PEG/DOX/CUR was detected by double staining of calcein(Calcein,CA)/propidium iodide(Propidium iodide,PI),and the ability of PEG/DOX/CUR to inhibit cell migration was studied by scratch test.3.To study the mechanism of PEG/DOX/CUR entry.The cellular internalization of PEG/DOX/CUR was observed by confocal microscope,and the cell uptake efficiency was analyzed by flow cytometry.Cell transmembrane transport inhibitors were used to study the mechanism of PEG/DOX/CUR entry into cells.Results1.Successfully constructed double-loaded nanomicelles PEG/DOX/CUR.The double-loaded nanomicelles PEG/DOX/CUR was successfully constructed with a concentration of 0.5 mg/mL,and the ratio of DOX: CUR: PEG was 1:1:7.The minimum particle size and average particle diameter of PEG/DOX/CUR were constructed using membrane hydration.About 60 nm,the polydispersity index(Polydispersity index,PDI)is less than 0.2,the zeta potential is about-47 mV,the encapsulation efficiency of DOX and CUR is more than 90%,the drug loading is about 8%,and the stability of PEG/DOX/CUR is good.The double loaded micelle PEG/DOX/CUR has a slow drug release effect.In vitro release results showed that DOX and CUR showed slow release in PEG/DOX/CUR,and DOX and CUR released faster in acid environment.2.The double loaded micelles PEG/DOX/CUR have anti-glioblastoma effect.The results of cytotoxicity test showed that DOX and CUR have a synergistic effect.The blank NPs have no obvious toxicity to cells.PEG/DOX/CUR can effectively promote apoptosis and inhibit cell migration.Different concentrations of PEG/DOX/CUR are synergistic.Inhibits the effects of glioblastoma cells.3.The micellar PEG/DOX/CUR mainly entered the cells through the caveolin-mediated endocytic pathway.The results of cell uptake experiments showed that PEG/DOX/CUR could effectively enter the cells with time and energy dependence.The endocytic pathway experiment results showed that PEG/DOX/CUR mainly enters cells through the caveolin-mediated endocytic pathway.ConclusionPEG/DOX/CUR was successfully constructed,with small particle size,good stability and slow release characteristics.PEG/DOX/CUR has anti-glioblastoma cell effects and DOX and CUR have synergistic effect,shows that double-loaded nanomicelles PEG/DOX/CUR enter cells mainly through endocytic pathways.The pathway for PEG/DOX/CUR entry into cells was primarily the caveolin-mediated endocytic pathway.