The Study On The Anti-glioma Effect Of IRGD Modified Double Drug Loaded Nanoparticles

BackgroundGlioma is the most common type of primary malignant brain tumors,with high morbidity and mortality.The usual therapy involves surgical resection combined with chemotherapy or radiotherapy.However,because of the limitations of surgical resection,drug therapy and the potential recurrence of residual tumor cells,the median survival rate is low and the recurrence rate is high.In addition,the unique physiological barrier of the glioma and the fact that traditional anti-tumor drug molecules cannot distinguish between tumor cells and normal cells,the therapeutic drugs to reach the tumor area is very limited,and it also causes inevitable damage to normal tissues.Previous studies have shown that Nanostructured delivery systems(NDS)are regarded as an ideal drug carrier for their functions of overcoming non-specific phagocytosis,aggregating at tumor sites,binding to tumor cells and entering cells,and controlled drug release.However,due to the role of intracranial physiological barrier,there are few reports about NDS in glioma,and its therapeutic effect on glioma needs to be further studied.At present,chemotherapy combined with immunotherapy has excellent therapeutic effect in a variety of tumors,immune checkpoint inhibitor is one of the most effective immunotherapy methods,but the effect of combined therapy in glioma needs to be further studied.What's more,NDS mediated chemotherapy combined with immunotherapy for glioma has not been reported ever.In order to improve the effective of drug utilization and chemotherapy combined immunotherapy effect,the following properties of tumor targeting drug release carrier needs to be developed,which could simultaneously carry chemotherapy drugs and immunosuppressants,overcome the nonspecific phagocytosis of protective layer and across through the blood brain barrier surrounding the tumor,accumulated in the tumor site and implementation of controlled release within the tumor cells,to prolonged antitumor efficiency.Objective To design and synthesis a new type of drug-loading nanoparticles transmission system,this nanometer system using mesoporous silica nanoparticles as the carrier,has multifunctional including tumor targeting,controlled drug releasing,double drug loading,which could carry anticancer drugs DOX and immune inhibitor 1 MT at the same time and targeted to intracranial glioma area.This new type nanoplatform could kill tumor cells and activate the antitumor immune response,increase the anti-tumor effect of orthotopic glioma model mice.Methods(1)1-methyl-tryptophan(1MT)was prepared by chemical synthesis,and the peptide sequences AD-PEG-KDEVD-1MT and AD-PEG-G-iRGD were synthesized by reference to the standard"Fmoc chemistry"method.Mesoporous silica nanoparticles(MSN)were prepared to synthesize bisulfide functionalized Mesoporous silica nanoparticles modified with acetylene,and then synthesized and connected azide-?-CD.Thus,the reduction response mesoporous silicon drug release control system(DOX@MSN-SS-CD)with cyclodextrin blocking,the reduction response mesoporous silicon drug release control system(DOX@MSN-SS-iRGD)with peptide sequence functionalization and the dual drug loading multi-functional mesoporous silicon drug release control system(DOX@MSN-SS-iRGD&1MT)were prepared.The synthetic peptide was characterized by ~1H NMR and FT-IR.MSN and DOX@MSN-SS-iRGD&1MT were characterized by transmission electron microscopy and scanning electron microscopy.DOX@MSN-SS-CD-iRGD&1MT drug release was detected by fluorescence spectrophotometer and high-performance liquid chromatograph in vitro.MTT assay was performed to detect the killing effect of DOX@MSN-SS-IRGD&1MT on glioma cells in vitro.(2)Glioma GL261 cell with stable luciferase expression was constructed(GL261-luc),GL261-luc orthotopic brain tumor model was constructed in C57/BL6 mic.In vivo bioluminescence technology was used to detect the size of intracranial tumors,and similar fluorescence intensity orthotopic glioma model were screened out.The mice were randomly divided into four groups:intraperitoneal injection of DOX+1MT,DOX@MSN-SS-CD,DOX@MSN-SS-iRGD and DOX@MSN-iRGD&1MT.12h,24h,36h and 48h after injection,blood samples were collected from each mouse and the main organs(heart,liver,spleen,lung,kidney)were completely dissected.Blood routine and biochemical analysis were performed on the blood,and the main organs were analyzed by IVIS system imaging.Meanwhile,ICP-MS assay was performed to detect the silicon content in intracranial glioma tissues of mice 48h after intraperitoneal injection of DOX@MSN-SS-CD,DOX@MSN-SS-iRGDandDOX@MSN-iRGD&1MT.Surfactant assisted tissue removal technique and three-dimensional fluorescence imaging technique were used to detect the intracranial DOX aggregation of mice 36hours after intraperitoneal DOX@MSN-SS-iRGD&1MT injection.(3)CD3~+T cells,CD4~+T cells and CD8~+T cells were detected by flow cytometry in C57/BL6 mouse spleen lymphocytes co-culture with GL261 cells,after 48h after treated PBS,DOX+1MT,DOX@MSN-SS-CD,DOX@MSN-SS-iRGD and DOX@MSN-SS-iRGD&1MT.(4)The orthotopic glioma model of C57/BL6 mice was constructed and randomly divided into 5 groups.In fifth day after surgery,PBS,DOX+1MT,DOX@MSN-SS-CD,DOX@MSN-SS-iRGD and DOX@MSN-iRGD&1MT were intraperitoneal injected,and the injection was once every two days for a total of 5 times.The survival time and body weight of mice were observed and recorded,and the survival curve and body weight curve were drawn.At the same time,In vivo Bioluminescence technology was performed on the fifth day after surgery to observe the tumor size,performed every4 days for a total of 4 times.MRI imaging was performed on each group of mice 20days after tumorigenesis to observe tumor size and followed with the HE stained of the brain tissue sections.(5)Immunohistochemical was used to detect the expressions of CD34 and Ki-67 in intracranial tumor tissues.Meanwhile,immunofluorescence confocal assay was used to detect the expressions of CD8~+T cells,CD86~+DCs and GrzB in tumor tissues.Surfactant assisted tissue clearing technology and three-dimensional fluorescence imaging technology were used to detect the expressions of CD8~+T cells in tumor areas of DOX@MSN-SS-IRGD&1MT treated mice.Flow cytometry was used to detect the proportion of CD3~+T cells,CD4~+T cells,CD8~+T cells and GrzB expression of CD8~+T cells in blood.ELISA was used to detect the secretion of inflammatory factors TNF,IFN?,IL-10 and IL-17 in the glioma region.Western Blot was used to detect the expression of STING,IFN?/?,and p-STAT3,in the glioma region.Results(1)The accuracy of 1MT structure was proved by ~1H NMR,and the chemical structure of AD-PEG8-KDEVD-1MT and AD-PEG8-G-iRGD was proved by mass spectra.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)confirmed the ordered porous structure of MSN and the surface covering connection layer of DOX@MSN-SS-CD-iRGD&1MT.Fourier infrared spectroscopy(FT-IR)was used to detect the surface potential of various synthetic materials.The results of fluorescence spectrophotometer and high-performance liquid chromatograph showed that under the condition of GSH,DOX@MSN-SS-iRGD&1MT drug molecule DOX was rapidly released,and the peptide chain connected to 1MT could be broken and released in the presence of caspase3.The above results show that we have successfully synthesized a newly type combined drug-delivering nano-system,with double drug loading and controlled release function,DOX@MSN-SS-CD-iRGD&1MT,which could kill GL261cells in vitro.(2)D-luciferin detection results showed that luciferase lentivirus transfected GL261cells with an efficiency of more than 90%.Three-dimensional reconstruction results of in vivo bioluminescence technology combined with CT scanning showed that the orthotopic glioma model of C57/BL6 mice was successfully constructed.In vivo bioluminescence technology showed that DOX fluorescence aggregation was observed in the brain of iRGD-modified nanomaterials treated mice 12h after intraperitoneal injection,while the concentration of DOX fluorescence aggregation was low in other organs.The liver of non-iRGD modified group showed strong DOX fluorescence aggregation.Within 36h after intraperitoneal injection,DOX fluorescence intensity increased continuously in the cranial of iRGD-modified nanomaterials treated group,while the DOX fluorescence intensity in the control group showed strongly increased in liver.At 48h,DOX in non-iRGD modified group showed no fluorescence in the heart,liver,spleen,lung and kidney,while a small amount of DOX fluorescence was observed in the intracranial of iRGD modified mice,compared with non-iRGD modified group.The ICP-MS results showed that iRGD-modified nanomaterials had significantly increased the silicon concentration in tumor regions.The above results showed that iRGD modified nanomaterials could carry drugs to the intracranial glioma region,increase the concentration of drugs in glioma and slow down their metabolic rate in intracranial glioma.(3)MTT results showed that DOX@MSN-SS-iRGD&1MT significantly decreased survival rate on glioma GL261 cells.In the co-culture of GL261 and C57/BL6 mice,DOX@MSN-SS-iRGD&1MT,the same to traditional combined administration group,significantly increased the proportion of CD3~+CD8~+T lymphocytes,decreased the proportion of CD3~+CD4~+T lymphocytes,and significantly decreased the ratio of CD3~+CD4~+T lymphocytes to CD3+CD8+T lymphocytes.The above results showed that DOX@MSN-SS-iRGD&1MT could significantly affect the proportion of lymphocytes in the in vitro co-culture cell system and increase the number of cytotoxicity CD3~+CD8~+T lymphocytes.(4)The survival rate analysis results showed that DOX@MSN-SS-iRGD&1MT could significantly prolong the survival rate of the orthotopic glioma model of C57/BL6 mice.In vivo bioluminescence technology showed that the intracranial fluorescence intensity of DOX@MSN-SS-iRGD&1MT treated group had no significant change.The results of MRI and HE staining showed that the volume of intracranial gliomas in DOX@MSN-SS-iRGD&1MT treated group were significantly reduced when compared with the other four groups.The above results showed that DOX@MSN-SS-iRGD&1MT could significantly inhibit the proliferation of tumor cells and enhance the anti-glioma effect of mice in the orthotopic glioma model of C57/BL6 mice.(5)Immunohistochemical results showed that tumor angiogenesis and cell proliferation rate were significantly reduced in the glioma region treated with C57/BL6.Immunofluorescence,surfactant assisted tissue clearing technology and three-dimensional fluorescence imaging showed that significant infiltration of CD8~+T lymphocytes and CD86~+DCs were observed in the glioma area treated with DOX@MSN-SS-iRGD&1MT,at the same time,the expression of cytotoxic factor GrzB was significantly increased.Flow cytometry showed that the proportion of CD3~+CD8~+T lymphocytes in blood and spleen of DOX@MSN-SS-iRGD&1MT treatment group was significantly increased,while the ratio of CD3~+CD4~+T cells to CD3~+CD8~+T cells was significantly decreased.In addition,the expression of GrzB in CD8~+T cells in blood was significantly increased in DOX@MSN-SS-iRGD&1MT treated group.ELISA showed that DOX@MSN-SS-iRGD&1MT could promote the expression of TNF,IFN?and IL-17 while inhibit the expression of IL-10 in the glioma area.WB showed that DOX@MSN-SS-iRGD&1MT could increase the expression of STING and IFN?/?proteins in glioma tissues and decrease the expression of p-STAT3protein.The above results showed that DOX@MSN-SS-iRGD&1MT could not only influence the proliferation of glioma cells,but also activate DCs and promote the proliferation of CD8~+T cells,and further increase the expression of STING,IFN?/?,TNF,IFN?and IL-17,and reduce the expression of inflammatory inhibitor IL-10 and p-STAT3.Conclusions(1)DOX@MSN-SS-iRGD&1MT,a newly type nano-drug delivery system,can carry drugs to the intracranial glioma region,increase the concentration of drugs in glioma and slow down their metabolic rate in intracranial glioma.(2)DOX@MSN-SS-iRGD&1MT can prolong the survival rate of orthotopic glioma model,inhibit the proliferation of glioma cells,while activate DCs and promote the proliferation of CD8~+T cells at the same time,and further increase the expression of cytotoxic factor GrzB,promote the expression of STING,IFN?/?,TNF,IFN?and IL-17,inhibit the expression of IL-10 and p-STAT3.