| Glioma is the most common primary intracranial tumor of the central nervous system,also known as glioma,accounting for 51.4%of all primary brain tumors,and its 5-year overall survival rate does not exceed 35%.From 2000 to 2015,the malignant tumor mortality rate of glioma in my country ranked tenth,slightly higher than the global mortality rate of glioma(2.5%),and since 2000,the prevalence rate has shown an upward trend.However,almost all patients with glioma will continue to develop the disease after receiving first-line chemotherapy for 7 to 10 months.The primary method of clinical treatment of glioma is still surgical resection.However,because gliomas mostly grow in important parts of the brain and show diffuse and infiltrating growth,the location of the tumor is difficult to distinguish,so the operation is difficult and risky.It is difficult to be cured by surgical resection.The existence of blood brain barrier(BBB)and(brain blood tumor barrier,BBTB),as well as the high degree of tumor infiltration of normal brain tissue,have brought great difficulties to the diagnosis and treatment of glioma.Current research mainly focuses on molecular targeted therapy,immunotherapy,gene therapy and new drug delivery technologies.However,so far,the results of single-agent therapy have been disappointing,and combination therapy can achieve a broader and long-lasting anti-tumor effect.Therefore,the immunotherapy of glioma has gradually attracted attention of researchers,combined with traditional chemotherapy to explore strategies for prolonging the overall survival after surgical resection combined with chemotherapy.Lipoprotein from human body is a kind of supramolecular nanostructure that plays an important role in cell function,lipid metabolism and disease.In the lipoprotein family,the size of high-density lipoprotein(HDL)composed of apolipoprotein apoA-I and phospholipids is only about 10 nm.The small particle size and endogenous nature of HDL make it particularly suitable for the construction of nanocarrier platforms targeting inflammatory diseases and tumor diseases.Lipoprotein-like nanocarriers are nanocarriers artificially assembled in vitro based on the structure of lipoproteins using lipids,apolipoproteins(apo A,C,E)and cholesterol.It has the characteristics of high biocompatibility,small size,BBB targeting and multi-mode drug delivery,and avoids blood-borne contamination of native lipoproteins.It is an ideal delivery vehicle for glioma treatment drugs.Arsenic trioxide(ATO)is the main component of the traditional Chinese medicine mineral medicine arsenic.It was recorded in the "Compendium of Materia Medica" in the Ming Dynasty to be used for "destroying carbuncle and septic flesh".Recent studies have found that it is also good in the treatment of solid tumors such as glioma.ATO can reduce the proliferation and recurrence of glioblastoma by down-regulating the Notch pathway;combined with the p53 mutant,the p53 mutant has thermal stability and transcriptional activity,and the mutant p53 is reactivated to inhibit tumors.However,ATO’s low targeting,rapid renal clearance,short half-life,and differences in biodistribution lead to serious side effects on normal tissues.The narrow therapeutic window limits the application of ATO in the treatment of glioma.Melittin(Mel)is the main component of traditional Chinese medicine bee venom.It can interact with cell membranes and destroy the stability of the phospholipid bilayer,thereby killing tumor cells.As a natural cationic host defense peptide,recent studies have found that it can also cause an immune response characterized by local inflammation,induce a significant increase in the number of CD4+T cells in the body,and exert an immune effect to treat tumors.However,the hydrophilic C-terminal of Mel has a cationic charge,which makes it have significant hemolytic side effects.Transition metal Mn2+is a T1 contrast agent.On the one hand,Mn2+can enhance MRI signals for brain imaging;on the other hand,the precipitate formed by the complexation of Mn2+with ATO has the ability to respond to release in the acidic microenvironment of tumors,and can achieve Integration of diagnosis and treatment of glioma.This project design uses ATO with anti-glioma effect to complex with MRI imaging agent Mn2+to generate MnAs co-precipitation,load it on lipoprotein-like nanocarriers,and combine with Mel with immunotherapeutic effect on the surface to construct a kind of glioma MelLNPs/MnAs is a biomimetic nano-delivery system that integrates functions with targeted,realtime MRI imaging and multiple treatment mechanisms.This project investigated the reaction conditions and concentration of hydrophobic MnAs coprecipitation.By examining the affinity of different phospholipids to DMPC,the dosage of different phospholipids,the particle size,particle size distribution coefficient and Zeta potential were used as evaluation indicators to determine the type and dosage of phospholipids..The stability of Mel-LNPs/MnAs was further investigated.Finally,characterize and evaluate the morphology and properties of Mel-LNPs/MnAs,including transmission electron microscopy(TEM),energy dispersive X-ray spectroscopy(EDX)analysis,determination of content and encapsulation efficiency,in vitro pH responsive release,and in vitro MRI imaging Ability and hemolysis in vitro.The hydrophobic fluorescent dye coumarin 6 was used to replace the MnAs core,and label the preparations to investigate the uptake of fluorescent dyes in different preparation carriers by GL261 cells;use Sulfo-Cy5-E SE fluorescent probes to analyze α-Mel Mark to investigate the position of α-Mel in tumor cells.The LIVE/DEAD Viability/Cytotoxicity Kit was used to stain the GL261 cells after administration,and the MTT method was used to investigate the toxic effects of free drugs and different preparations on GL261 cells.An in vitro 3D tumor spheroid model was established to investigate the tumor sphere permeability of different preparations.The live/dead cell staining of the tumor spheroids after administration was performed using the LIVE/DEAD Viability/Cytotoxicity Kit to investigate the effects of free drugs and different preparations on tumor spheres.The near-infrared dye DiR was used to replace the MnAs core to label the preparations to investigate the crossover of different preparations in vivo The ability of BBB to target the brain;a mouse orthotopic glioma model was established to investigate the glioma targeting ability of different DiR-labeled preparations in vivo.Through the in vivo pharmacokinetic study,the As content in rat plasma was detected by ICPMS,and the pharmacokinetic parameters were calculated by DAS 3.0 software to investigate the pharmacokinetics of Mel-LNPs/MnAs in rats.Features provide a reference for the further application of Mel-LNPs/MnAs.Use the near-infrared dye DiR to replace the MnAs core and label the preparations to investigate the ability of different preparations to cross the BBB and target the brain in vivo;establish a mouse orthotopic glioma model to investigate the different DiR-labeled preparations in vivo Glioma targeting ability.An experimental animal model of GL261 intracranial glioma was established,and the MRI imaging capabilities of different preparations in glioma in situ were investigated.Free ATO,free ATO+Mel,Lipos/MnAs,LNPs/MnAs and Mel-LNPs/MnAs inhibits the growth of glioma.The anti-tumor mechanism of Mel-LNPs/MnAs was investigated by immunohistochemistry,immune factors and immune cell analysis in the spleen,and evaluated by blood routine,serum biochemical indicators,and H&E staining of main organs.In vivo safety of Mel-LNPs/MnAs. |
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