Peptide-modified Liposome Co-loading And Therapeutic Studies Of Honokiol And Copper Disulfiram Targeting The Glioma Microenvironment

Research purposesGlioma,especially Glioblastoma(GBM),is one of the most invasive tumors.Although people use multiple modes of treatment intervention,the median survival of GBM patients could not be significantly improved.The main reason is the invasive growth of tumors,the traditional treatments such as surgery and radiotherapy and chemotherapy have a low cure rate,which is easy to induce tumor recurrence.The recent study has found that in brain tumor,the tumor immune microenvironment(TIME)has undergone tremendous changes,resulting in poor prognosis.Among the TIME,macrophages play an important non-specific immune cell,which would be recruited by tumor microenvironment factors and have strong plasticity.Therefore,immunotherapies targeting macrophages in glioblastoma would be a new treatment to improve the survival rate of patients with glioma.Since the effective treatment for GBM is dismal,our study using the chemotherapeutic drugs:the combination of disulfiram/copper complex(DSF/Cu)and honokiol(HNK)coloaded via liposomes,through receptor-mediated strategies for targeted delivery to brain tumors.It inhibited the tumor growth as well as targeted at tumor-associated macrophages and regulated the tumor immune microenvironment.And then we preliminary investigated its possible molecular mechanism,promising to provide a theoretical basis for future clinical applications.Methods1.The functional material CDX-PEG-DSPE was prepared,and the CDX polypeptide modified PEGylated liposome was co-loaded with HNK and DSF/Cu.The purification and characterization have been carried out,and the experimental verification of particle size,zeta potential,transmission electron microscopy(TEM),encapsulation efficiency(EE%),drug loading and in vitro stability was carried out.2.The in vitro activity of CDX polypeptide-modified liposomes was further studied.The uptake of liposomes on glioma U87 and macrophages was qualitatively and quantitatively determined,and the blood-brain barrier was simulated in vitro.The ability of liposomes to cross the blood-brain barrier is verified.In addition,3D tumor spheres were used to simulate solid tumors to verify the permeability of this liposome to the interior of the tumor.3.To explore the anti-tumor effect and mechanism of CDX-LIPO liposome,in order to detect the apoptosis cell of tumor,we used cytotoxicity and flow cytometry,and to induce intracellular reactive oxygen species and hypoxia by kit and Western Blot.The protein expression such as factor and autophagy levels and its related pathways were studied.4.At the animal level,the biopsy distribution and pharmacodynamics of liposomes were further investigated in situ tumor model mice bearing a glioma model,and the distribution in vivo was compared after tail vein administration.At the same time,the biosafety of the liposome was examined by measuring the organ coefficient,H&E pathology and other experiments.5.Establish an animal model of glioma.Observe the ability of CDX-LIPO liposome to inhibit tumor growth by observing the survival curve and body weight of mice.Flow cytometry methods and immunofluorescence were used to investigate in the tumor tissue,and to explore the changes of metabolic level and the mechanism of regulating the tumorassociated macrophages and immune cells.ResultsThe liposome modified by CDX polypeptide prepared by our research has uniform morphology and uniform particle size distribution,good stability and no burst effect.It can be effectively taken up by glioma cells and significantly increase the ability of the drug delivery system to penetrate the BBB and accumulate in the tumor site,thereby significantly improving the therapeutic effect of the tumor.The results showed that modification of CDXLIPO not only can enhance the glioma treatment effects,but also could effectively regulate the tumor microenvironment.The number of M2 macrophages was down-regulated,and the infiltration of M1 macrophages increased accordingly.Moreover,the number of NK cells also increased correspondingly,while the infiltration of MDSC cells was significantly downregulated.Moreover,the expression of IL-6 in tumor tissues was also significantly downregulated,with the expression of TNF-? was increased.It is revealed the mechanism of CDX-LIPO on tumor cells and macrophages is mainly related to the induction of autophagy and ICD.Through the activation of Akt/mTOR pathway,it can change energy metabolism and inhibit glycolysis.And then to restore the immune function of the whole body and promote the apoptosis of tumor cells.ConclusionThe study has successfully designed liposomes can regulate the polarization of tumorassociated macrophages and improve the tumor immune microenvironment,inhibiting tumor growth and progression.