Construction Of Bionic Nano-delivery System Based On Multi-mode Collaborative Therapy And Research On Targeted Therapy For Malignant Brain Tumor

Objective Glioblastoma multiform(GBM)is one of the most aggressive primary malignant brain tumors in the central nervous system(CNS),with a five-year survival rate of less than 10%and the median survival was only 15 months.Currently,FDA-approved treatments include surgical resection,chemotherapy,and radiation.The aggressive nature of glioblastoma,combined with the fact that it is located in the brain,makes it difficult for surgery to completely remove the diseased tissue.The existence of the blood-brain barrier(BBB)limits the efficacy of chemotherapy and radiotherapy,preventing most drugs from reaching their targets.Chemodynamic therapy(CDT)is a strategy to mediate the production of reactive oxygen species(ROS),which converts H2O2 into highly cytotoxic hydroxyl radicals through Fentontype reactions(·OH)to induce tumor cell apoptosis,bringing a new dawn to tumor therapy.However,most of the related nanoformulations of CDT are based on Metal-Organic Framework(MOF)materials,which are inevitably neurotoxic,coupled with high concentrations of glutathione in the unique tumor microenvironment(TME)Peptide(glutathione,GSH),insufficient hydrogen peroxide concentration,and other problems limit its efficacy.Based on this,this project preliminarily envisages enhancing the concentration of H2O2 in the tumor area and depleting GSH.Glucose oxidase(Glucose oxidase,Gox)is an important enzyme catalyst,which catalyzes the formation of gluconic acid and H2O2 from glucose,O2,and H2O.This catalytic reaction solves the problem of insufficient H2O2 concentration at the tumor site.In addition,this process also consumes O2 and glucose,resulting in hypoxia in the tumor region.Tumor cells typically use glycolysis to fuel themselves,even in the presence of O2,known as the Warburg effect.According to the Warburg effect,tumor cells require more glucose to function.Gox consumes glucose and cuts off the energy source of tumor cells to achieve cancer starvation therapy(CST).However,Gox is easily cleared in vivo and its low enzymatic activity hinders its application.How to efficiently penetrate the BBB,maintain Gox activity while reducing neurotoxicity,and relieve hypoxia in the tumor area remains to be explored.Innate immunity plays an important role in the body’s defense.As an important pathway in innate immunity,STING(stimulator of interferon genes)can be activated by cyclic GMPAMP Synthase(cGAS)cyclic GAMP synthase through double-stranded DNA,or by other small molecule agonists under specific conditions Activated,resulting in the release of a variety of inflammatory-related cytokines such as interferon(IFN),which triggers an innate immune response.STING-related immune responses in tumor cells can inhibit tumor development.Mn2+,as an essential trace element for the body,also plays an important role in tumor immunotherapy.Mn2+ can bind and activate the function of cGAS even in the absence of dsDNA,enhancing the binding affinity of cGAS-STING.And Mn2+is also the initiator of the Fenton-like reaction,which catalyzes the conversion of H2O2 into ·OH.At the same time,it has peroxidase-like activity and H2O2 to generate O2,which relieves hypoxia in the tumor area.Polydopamine(PDA)is the product of dopamine oxidative self-polymerization.It is similar to natural mussels and has a catechol-like structure.It can be wrapped in almost"Nanocoating" is formed on the surface of any object.Good biocompatibility makes it widely used in biomedicine,nanomaterials,and other fields.In addition,the GSH-depleting properties and acid response of PDA suggest that it can be applied to deliver glucose oxidase to enhance the efficiency of CDT,reduce the occurrence of side effects,and facilitate the synergistic treatment of Cancer Starvation therapy and Chemodynamic therapy.Cell membrane-based smart biomimetic nano-drug delivery strategies are one of the recent research hotspots.Zhang ’s research group first proposed to use red blood cell membrane to wrap the surface of nanoparticles to escape the clearance of phagocytic cells in vivo and prolong the circulation time in vivo.Cell membrane encapsulation has natural advantages.For example,cancer cell membrane encapsulated nanocarriers can achieve homologous targeting for precise delivery.Melanoma has the characteristics of brain metastasis,so the B16F10 cell membrane has the advantage of innately crossing the blood-brain barrier.We used the B16F10 cell membrane to encapsulate the nanozyme to penetrate the blood-brain barrier so that the GMPC could efficiently accumulate at the tumor site.In conclusion,we designed a melanoma cell membrane biomimetic co-loaded with glucose oxidase,Mn2+,and polydopamine.The multifunctional GMPC utilizes the B16F10 cell membrane to span the BBB and is enriched in the tumor microenvironment with a high concentration of GSH.Under the action of GSH and slightly acidic pH,the polydopamine in the GMPC disintegrates and releases Gox and Mn2+.Gox consumes glucose and produces H2O2,and Mn2+ mediates a Fenton-like reaction to produce·OH and induce apoptosis.In addition,Mn2+ and H2O2 generate oxygen to improve hypoxia while activating the STING pathway,inhibiting tumor development in multiple modes and multiple ways.Methods 1.Using Gox as a template,electrostatic adsorption,and Michael’s addition reaction were used to co-load Mn2+and polydopamine,and the B16F10 cell membrane was biomimetic to prepare GMPC.2.The hydrated particle size and Zeta unit of the obtained GMPC were measured by Malvern laser particle size analyzer,the morphology was observed by transmission scanning electron microscope,and the elemental composition was analyzed by X-ray photoelectron spectroscopy.3.Measure the pH value of GMPC in glucose solution by pH meter to verify that it catalyzes the generation of gluconic acid.4.The ability of GMPC to generate H2O2 and·OH in vitro and deplete GSH was investigated by using a full-wavelength scanning UV-Vis spectrophotometer.5.CCK-8 kit to determine GMPC for tumor cytotoxicity and biocompatibility.6.The apoptosis rate of each group was detected by Annexin V-PI kit by flow cytometry,and the ROS generation in the treatment group was observed by an inverted fluorescence microscope.7.Western Blot method was used to detect the expression levels of STING,HIF-α,and Caspase-3 after GMPC treatment.8.Calreticulin(CRT)translocation,high mobility group protein B1(HMGB1)release,mitochondrial membrane potential decrease,and DNA oxidative damage were determined by an inverted fluorescence microscope and laser confocal microscope.9.Determination of ROS-related indicators by multi-function microplate reader:intracellular H2O2 level,GSH content,lipid-oxidized malondialdehyde(MDA)level,total antioxidant capacity(T-AOC),and superoxide dismutase(SOD)activity10.The GL261 glioma orthotopic tumor-bearing mouse model was established,and GMPC was injected into the tail vein.The small animal in vivo imaging system tracked the distribution of GMPC at different times and quantitatively analyzed the fluorescence intensity of isolated organs.11.Tail vein injection of GMPC was used to treat tumor-bearing mice.Survival and body weight changes were detected during the treatment.Intravital imaging analyzes changes in fluorescence intensity at brain sites.At the same time,immunohistochemical staining was performed on tissue sections.Results 1.The biomimetic nano-enzyme GMPC was prepared.The hydrated particle size measured by the Malvern laser particle size analyzer was about 186 nm.The Zeta potential was-25.1 mv,which was lower than the GM potential.Transmission scanning electron microscopy showed that its morphology was spherical and the core-shell structure was obvious,the surface is covered with a layer of the cell membrane.X-ray photoelectron spectroscopy analysis showed that it is composed of C,N,O,Mn,and P elements.2.The results of the pH meter showed that GMPC and glucose solution co-incubated,and the pH of the solution showed a decreasing trend.3.The UV absorption spectrum showed that the absorbance value of the GMPC group increased at 420 nm and there were three broad absorption peaks at 600-900 nm,indicating the generation of H2O2.The decrease in absorbance at 660 nm indicates that methylene blue(MB)degrades the generated ·OH.The absorbance at 660 nm of the GMPC group was lower than that of the GM group,indicating that GMPC could consume GSH,increase the generation level of ·OH,and avoid being cleared by GSH.4.The CCK-8 experiment showed that GMPC had a higher inhibitory ability on tumor cells than GM under the same conditions,and the addition of L-ascorbic acid increased the cell viability,indicating that the anti-tumor effect was derived from the induced oxidative stress.The safety investigation of normal cells shows that it has good biocompatibility.5.The ROS detection experiment showed that GMPC could induce the generation of reactive oxygen species(the proportion of reactive oxygen species accounted for 43.5%),and the apoptosis experiment also showed that the GMPC group induced the highest apoptosis rate of tumor cells.6.GMPC induces the up-regulation of STING protein expression,indicating that the encapsulated Mn2+can activate the STING pathway.In addition,Caspase-3 and HIF-α of tumor cells were up-regulated and down-regulated after GMPC treatment,which indicated that GMPC could induce apoptosis and improve the hypoxia of tumor cells.7.GMPC induces oxidative stress in tumor cells,CRT translocation,HMGB1 release,mitochondrial membrane potential decrease,and DNA damage.8.After GMPC treatment of cells,a series of indicators related to ROS changed:intracellular H2O2 content increased,GSH level decreased,MDA content increased,T-AOC decreased,SOD activity increased.9.The brain targeting ability was investigated using in situ tumor-bearing mice.The results of in vivo imaging showed that GMPC could aggregate to the tumor site in the brain after treatment.The imaging results of in vitro organs also demonstrated that GMPC has a good ability to target.Brain targeting ability.10.The in vivo experiments showed that the GMPC has a good tumor suppressing ability,the survival period was significantly prolonged,and the bodyweight of the mice in the GMPC treatment group did not change much.In vivo imaging experiments showed that after GMPC treatment,the tumor had lower fluorescence intensity compared with other groups,and the determination of biochemical blood indicators revealed that it had excellent biocompatibility.Conclusions 1.GMPC has pH responsiveness,GSH depletion,and glucose sensitivity,and releases and encapsulates various components in TME.In vitro experiments confirmed that it can generate·OH,deplete GSH to increase the efficiency of Fenton-like reaction,and at the same time generate O2 to relieve hypoxia.2.GMPC induces tumor cell apoptosis and activates intracellular oxidative stress through the generation of·OH,causing tumor cell CRT translocation,HMGB1 release,DNA oxidative damage,changes in ROS-related indicators,and mitochondrial membrane potential decrease.GMPC consumes glucose,cutting off the energy supply to cancer cells.In addition,Mn2+also played a role in activating the STING pathway,inducing the upregulation of the STING protein.3.GMPC has the ability to target brain tumors,and the brain still has a strong fluorescence signal 60 hours after tail vein injection.The imaging results of isolated organs also confirmed its ability to effectively focus on brain organs.4.The survival time of mice in the GMPC treatment group was significantly prolonged without significant changes in body weight.It is safe,non-toxic,and has good biocompatibility,and the three-way synergy is expected to become a new strategy for brain tumor treatment.