Study Of Nano-catalytic Particles Loaded On Extracellular Vesicles For The Diagnosis And Treatment Of Hepatocellular Carcinoma

1.Background and objectiveHepatocellular carcinoma（HCC）is the most common pathological type of primary liver cancer and the third leading cause of cancer-related death worldwide.Early diagnosis of HCC is very difficult because it is concealed and progresses rapidly.Most patients lost the opportunity of operation because they were found in the intermediate and advanced stage.For advanced HCC patients,systematic treatment like targeting drugs and precise radiotherapy schemes play an important role in inhibiting tumor progression and improving therapeutic effect.However,the off-target drugs in systematic therapy and the side injury caused by radiation limit their clinical application to some extent.Therefore,in order to meet the clinical needs of managing intermediate and advanced HCC patients,it is urgent to develop new targeting systematic therapy according to the biological characteristics of HCC.As a new chemodynamic therapy（CDT）strategy based on the responsiveness of tumor microenvironment,nano-catalytic drugs have the potential to replace the traditional chemotherapeutic therapy in the tumor microenvironment（TME）.It has been proved that glucose oxidase（GOD）and extremely small iron oxide nanoparticles（ESIONs）can produce cytotoxic hydroxyl radicals through double enzyme like catalytic reaction,which can be used for TME dependent nano-catalytic therapy.However,such treatment also faces many problems,especially on how to avoid the poor treatment effect caused by non-specific injection and off-target,as well as the toxic and side effects similar to traditional chemotherapy due to the cytotoxicity of materials.In addition,the tumor specificity of the nano-catalyst needs to be strengthened,which means the active targeting effect to HCC tumor sites needs to be enhanced.Single cell RNA sequencing technology has been widely used in the study of differential gene expression and immune cell clustering of TME in recent years.At present,the existing research on CDT therapy of tumors lacks the relevant contents of the changes of tumor gene expression and cell clustering of TME,which needs further research and discussion.Since the impact of catalytic nanoparticles may affect many differential genes expression and changes in cell clustering in HCC TME,while CDT may lead to different differentiation directions of immune cells in the tumor ecosystem,single cell sequencing technology is naturally used to study the above mentioned biological changes.It will help us better understand the response of HCC and related immune mechanisms to CDT.2.Methods1)ESIONs were synthesized by a pyrolysis approach and modified by DSPE-PEG and RGD molecules.Extracellular vesicles（EVs）were separated by ultracentrifugation,and catalytic nanoparticles were synthesized by two-step aqueous solution with chemical connection for GOD-ESIONs@EVs（GE@EVs）.2)Transmission electron microscope（TEM）and energy dispersive spectrometer（EDS）were used to analyze the morphology,diffraction,the material characteristics such as uniformity and dispersion in aqueous solution of GE@EVs.Element scanning analysis were carried out to observe the specific element of GE@EVs.3)Particle size potential spectrometer,atomic force microscope,UV-VIS spectrometer,fourier infrared spectrometer,raman spectrometer were used to characterize GE@EVs and its synthetic derivatives and to verify the correct connection of molecules in each synthesis process as well as the special properties of these materials including ESIONs.4)GE@EVs stability experiment:measure the change of particle size under different p H buffer preservation within one week.GE@EVs release experiment:EVs rupture was simulated in ultrasonic environment,and the released GOD concentration was measured.5)Characterization of EVs includes TEM morphology characterization,particle size analysis and at least three EVs specific western bloting.6)The free radicals of GE@EVs were detected by electron spin resonance（ESR）,the Michaelis-Menten kinetic characteristics were measured by 3,3’,5,5’-Tetramethylbenzidine（TMB）,and the reaction rate was calculated.7)DCFH-DA was used to capture hydroxyl radical,and the intracellular endocytosis and reactive oxygen species（ROS）GE@EVs was observed by laser confocal microscope.8)The effect of GE@EVs on the viability of HCC cells in weak acidic environment was tested by CCK-8 experiment,and vitamin C was used for the rescuing experiment.9)Calcein AM/PI staining reagent was used to stain the living and dead state of HCC cells,and the cell state was photographed and analyzed by confocal microscope.After digesting the cells,the proportion and state of apoptosis and necrosis were analyzed by flow cytometry.10)In vitro transwell test was used to judge the invasion of HCC cells according to the number of cells passing through the filtered membrane.11)The ultrastructure of HCC cells after treatment was observed by transmission electron microscope,focusing on the damage of hydroxyl radical produced by catalysis on the mitochondria and other organelles.12)After labeling the relevant nanoparticles with fluorescent dyes Cy5.5,the cell targeting and endocytosis were photographed by laser confocal microscope.13)Drug safety experiment,blood circulation experiment and in vivo metabolism experiment were carried out in mice to judge drug safety and calculate relevant biological metabolism parameters.Then small animal in vivo imaging machine was used to judge the ability of GE@EVs to target HCC tumors.14)The treatment experiment through tail intravenous injection was carried out on tumor bearing nude mice and the related therapeutic effects were recorded.The survival data was also recorded,and the survival analysis was performed as well.The obtained tumor tissues of mice were analyzed by immunohistochemistry.15)Magnetic resonance imaging（MRI）of GE@EVs in mice were verified in MRI T1mode.16)After the treatment of C57 homologous tumor bearing mice,the tumor was digested into single cell suspension.After mixing with barcode beads,a reverse transcriptase library was first constructed,followed by 10×Genomics loading and sequencing.Quality control and preliminary screening of sequencing results were carried out by Cell Ranger software.17)The single cell sequencing results were analyzed such as cluster analysis,cell subgroup difference gene analysis,GSVA analysis,cell cycle analysis,RNA rate analysis and immune cell analysis.18)The patient derived xenograft（PDX）model was constructed and analyzed for GE@EVs standardized treatment to evaluate the efficacy.The tumors after treatment were analyzed by immunohistochemistry.The survival experiment of PDX mice for up to 60days was carried out and the relevant results were analyzed.19)Survival analysis and Cox regression analysis were performed on PDX mice model to determine the relevant risk factors of the PDX mice survival.20)All experimental data and relevant statistical validation were carried out in Graphpad9.0 and Origin 2021 software,and single cell sequencing data were analyzed on R software and R studio platform.P values less than 0.05 were considered statistically significant.3.ResultsThe first part of this study is synthesis and characterization of GE@EVs and its catalytic reaction in vitro.After the successful synthesis of ESIONs by pyrolysis,GE@EVs were synthesized by two-step solution covalent combination.Electron microscopy and atomic force microscopy showed that the morphology was uniform,and the dispersion was good.X-ray diffraction lattice stripes were observed.The characteristic peaks of ESIONs were successfully verified by XRD and XPS.The characterization of EVs verified their origin and the correctness of the extraction methods.Fourier infrared spectroscopy,UV-vis and Raman spectra verified the connection of each modified molecule and quantified the loaded GOD.After the stability test and release test of the carrier was verified for the drug loading performance.ESR spectrum successfully captured the hydroxyl radical catalyzed by GE@EVs with glucose in a weak acidic environment.The Michaelis-Menten kinetic parameters were captured by TMB in weakly acidic environment.The Michaelis-Menten kinetic constant K_mof glucose as substrate is 25.19m M,and the maximum reaction rate V_maxis 5.87*10^-7Ms^-1.The magnetic resonance T1weighted imaging performance of GE@EVs was characterized.The second part is the validation of GE@EVs therapeutic efficacy in Huh7 HCC cells and tumor bearing nude mice.Firstly,DCFH-DA fluorescence verified the intracellular ROS production of GE@EVs.The existence of its cell targeting ability and enhanced endocytosis were verified with Cy5.5.CCK-8 experiment verified cytotoxicity of GE@EVs in weak acidic environment,and vitamin C rescuing experiment proved that the source of cytotoxicity was ROS.Most HCC cells tending to die in vitro was observed by laser confocal with Calcein AM/PI staining.Flow cytometry showed that the proportion of apoptosis and necrosis increased,mainly including acute necrosis cells.Transwell cell migration assay of GE@EVs showed that it has a strong inhibitory effect on the migration of Huh 7 HCC cells.The ultrastructure of cells was observed by electron microscope that GE@EVs destroyed the biological structure of mitochondria and made them swell.At the same time,GE@EVs formed multiple vacuoles in Huh 7 cells,which is difficult to distinguish the morphology of organelles.In vivo safety test and circulating metabolism test of the GE@EVs were verified for biological application.After the targeting ability to HCC tumor site was verified by in vivo fluorescence,the treatment experiments were carried out.GE@EVs did have inhibitory effect in the treatment of HCC solid tumor.Immunohistochemical results showed that proliferative cells decreased and apoptosis increased in the tumor.The magnetic resonance imaging ability of GE@EVs was verified in T1 weighted MRI imaging mode,which could be used to assist in the imaging diagnosis of HCC.The third part is applying single cell sequencing technology for the analysis of HCC transcriptome and microenvironment after GE@EVs treatment.The effective rate of single cell viability was about 90%.After filtration,the gene expression appeared stable and could be used for subsequent analysis.After cluster analysis,the cells were divided into 12different clusters,and their principal component genes were different.On the basis of similar principal components,the similar cells were clustered into the same group.The gene expression of each cell group was different,representing different cell sources.The t SNE chart of dimensionality reduction reflected the effect on HCC cell clustering after CDT.After single R annotation and artificial annotation,each cell cluster was classified again.The differential genes before and after treatment were screened out through gene difference analysis.Enrichment analysis helps to understand the impact of CDT treatment on the related cell functions and signal pathways of HCC.The enrichment results are listed mainly including the inhibition of immune activation of NK cells,the up regulation of genes related to lactose metabolic pathway,down regulation of TGF-βpathway related genes and up regulation of AKT pathway related genes.The efficacy of CDT was further verified by cell cycle analysis and pseudo timing analysis,Through the re-clustering analysis of immune cells,it was found that the proportion of M1 type and TCR macrophages and cytotoxic T cells increased,which revealed that the local immunosuppressive microenvironment of HCC was reversed.The fourth part is the application of GE@EVs in HCC PDX model.The baseline characteristics of patients used to construct PDX model were relatively balanced,and there was no significant difference in their survival analysis.The therapeutic effect of GE@EVs on PDX model was verified,which can significantly inhibit the growth and progression of HCC and prolong the survival time of SCID mice.Cox regression analysis showed that AFP>400μg/L,maximum tumor diameter>5cm,MVI and GE@EVs treatment were the relating factor affecting the survival of mice.The cytotoxic effect was also verified on patient derived cells（PDCs）.The results of immunohistochemistry verified that GE@EVs can inhibit the proliferation and induce apoptosis of HCC in PDX model.4.ConclusionIn this study,a new nano-catalytic particle GE@EVs was successfully designed and synthesized.The physical and chemical properties and catalytic reaction properties of GE@EVs were characterized carefully.Through cell and animal experiments,the effect of HCC targeting therapy based on EVs and RGD molecules was verified,and the efficacy of catalytic therapy in weak acidic environment was confirmed.The results of single cell sequencing reflected the changes of cell clustering and related pathways at the transcriptome level.Finally,the possibility of transformation research of GE@EVs before clinical application was verified by the PDX model,and the influencing factors affecting the survival of PDX mice were analyzed.While revealing therapeutic efficacy and mechanism of GE@EVs,it was also found that the ability of magnetic resonance T1weighted imaging could assist in the diagnosis of HCC.To sum up,this study designed the GE@EVs and verified the potential value in diagnosis and treatment of HCC.The research verified its mechanism at the level of single cell sequencing and also verified the therapeutic effect on the PDX preclinical model.The catalytic diagnosis and treatment system was expected to be used in the clinical practice of HCC.