Construction Of Intestinal Barrier-crossing Nanomotors And Their Application In Oral Treatment Of Orthotopic Colon Cancer

Recent global cancer burden data indicates that colorectal cancer has become one of the most common cancers worldwide,ranking third.In China,both the incidence and mortality rates of colorectal cancer are increasing.According to the latest statistics from the National Cancer Center,it has become the second most prevalent and fourth most deadly cancer among malignant tumors.Therefore,exploring safe and efficient treatment methods for colorectal cancer is of great significance.During the treatment with chemotherapy drugs,drug resistance often occurs,while using immunotherapy drugs,immune escape and immunological tolerance often occur,which can easily result in malignant tumor metastasis.Colon cancer,as a solid tumor,is characterized by complex and abnormal blood vessels,resulting in insufficient oxygen supply.At the same time,tumor cells exhibit vigorous metabolism,which further exacerbates the hypoxic condition.Hence,the therapeutic efficacy of nanomedicine delivery systems has become increasingly significant in addressing tumor hypoxia and chemotherapy resistance.Objectives:1.Exploring strategies to overcome the obstacles of extracellular matrix and mucus in the colon,allowing for greater accumulation of nanomedicines at the site of colon cancer,and investigating the mechanism of nanomotors crossing the intestinal barrier.2.Investigating the use of ultrasound-driven nanomotors to induce iron-mediated cell death in tumor cells,thus eliciting anti-tumor immune response,and exploring the mechanism of ultrasound-driven nanomotor-mediated iron death.3.Exploring the use of orally-administered ultrasound-driven nanomotors to overcome the hypoxic tumor microenvironment,thereby enhancing the sono-dynamic effect against tumor recurrence and metastasis.4.Investigating potential anti-tumor immune mechanisms to prevent recurrence and metastasis of colon cancer in situ by avoiding immune escape and tolerance in tumor immunotherapy.Methods:1.Preparation and characterization of nanomotor(CS-ID@NMs).Extracting regenerated silk protein from cocoon.Preparation of mesoporous manganese dioxide nanomaterials.Surface modification of silks protein material with mesoporous manganese dioxide via solvent evaporation method.By classical amidation reaction,chondroitin sulfate molecules are modified on the surface of silk fibroin to achieve targeting of colon cancer.Characterize the size and zeta potential of the CS-ID@NMs using dynamic light scattering techniques.Analyze the protein secondary structure of the CS-ID@NMs through Fourier Transform Infrared Spectroscopy(FTIR)and X-ray Diffraction(XRD).The traditional dialysis method was used to study the drug release response behavior of nanomotors.2.In vitro anti-tumor experiments of ultrasound-driven CS-ID@NMs.The MTT method was used to detect the anti-tumor activity of ultrasound-driven CS-ID@NMs.The study investigated the targeted phagocytosis and lysosomal escape of colon cancer cells by ultrasound-driven CS-ID@NMs.The release of sonosensitizer from CS-ID@NMs was explored for mitochondrial targeting.The changes in the levels of reactive oxygen species(ROS),glutathione(GSH),glutathione peroxidase 4(GPX4),and lipid peroxidation(LPO)in tumor cells were investigated after treatment with ultrasound-driven CS-ID@NMs.The anti-tumor immune properties of ultrasound-driven CS-ID@NMs and changes in the content of high mobility group protein B1(HMGB1)and calreticulin(CRT)were also studied.The tumor spheroids penetration performance of ultrasound-driven targeted nanomotors was also explored.3.Biological distribution and biosafety evaluation of CS-ID@NMs.An animal fluorescence imaging system was used to capture the fluorescence images of CS-ID@NMs in five organs(heart,liver,spleen,lung and kidney)and the gastrointestinal tract(stomach,duodenum,jejunum,ileum,cecum and colon).Colon cancer-targeted phagocytosis by ultrasound-driven nanomotors in colon tissue was explored by confocal microscopy.Body weight changes and organ index changes after oral administration of different doses of CS-ID@NMs were investigated,and pathological sections of five organs(heart,liver,spleen,lung and kidney)and gastrointestinal tract(stomach,duodenum,jejunum,ileum,cecum and colon)were analyzed,as blood routine results.4.Inhibition and prevention of recurrence and metastasis of orthotopic colorectal cancer by ultrasound-driven CS-ID@NMs.Ultrasound-driven CS-ID@NMs combined with anti-PD-L1(anti-programmed cell death-Ligand 1)to treat orthotopic colon cancer in mice.After the treatment,the size of colon cancer was quantitatively analyzed.Serum was collected for measurement of inflammatory factors(Tumor necrosis factorα,TNF-αand Interferonγ,INF-γ).Hearts,livers,spleens,lungs,and kidneys,as well as colon samples,were collected for Hematoxylin and Eosin(H&E)staining.The colon cancer tissue was stained for cell proliferation(Ki67),apoptosis(Terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling,TUNEL),and immunofluorescence(CD4,CD8,Foxp3,and Hypoxia inducible factor-1,HIF1-α).The changes in the proportion of dendritic cells in the thymus and immune cells in the spleen(CD4~+T cells and CD8~+T cells),as well as the initial memory T cells,pre-effector T cells,central memory T cells,and effector memory T cells in mice treated with ultrasound-driven CS-ID@NMs for orthotopic colorectal cancer were analyzed.The volume and size of the tumor were recorded during the process of treating and preventing metastasis.The collected distal tumor tissues were subjected to H&E staining,cell proliferation(Ki67)staining,apoptosis staining,and immunofluorescence staining(CD4,CD8,and Foxp3).Results:1.By modifying silk protein and chondroitin sulfate on the surface of mesoporous manganese dioxide,a nanomotor with an average particle size of 212.3 nm and surface potential of-27.3 m V was successfully prepared.Additionally,it has good stability in simulated colonic fluid.During the modification process of the mesoporous manganese dioxide surface,CS-ID@NMs formed moreβ-sheet structures compared to free silk protein.Through drug release experiments,it was found that the CS-ID@NMs have various stimuli-responsive drug release characteristics(including p H,GSH,ROS,and ultrasound).2.Ultrasound and modification of targeted chondroitin sulfate molecules both improved the uptake of the nanomotors by tumor cells.The sonosensitizer released from the CS-ID@NMs had mitochondria-targeted performance.Ultrasound improved the nanomotors’lysosomal escape performance.Ultrasound enhanced the ability of CS-ID@NMs to increase the level of ROS,depletion of GSH,and downregulation of GPX4 in tumor cells.Ultrasound-driven CS-ID@NMs promoted the release of HMGB1and the exposure of CRT.Ultrasound promoted the tumor penetration of targeted nanomotors.3.Ultrasound promoted CS-ID@NMs nanomotors to cross the intestinal barrier,and.further enhanced the specific targeting uptake of it in colorectal cancer tissues.No abnormal changes were found in body weight,organ index,pathological sections.There was no damage observed in the major organs(heart,liver,spleen,lungs,and kidneys)and gastrointestinal tissues(stomach,duodenum,jejunum,ileum,cecum,and colon),and no significant hematological changes were noted.4.The combination of ultrasound-driven CS-ID@NMs and anti-PD-L1 treatment could suppress the growth of orthotopic colon cancer.The histological score of colon pathological sections was the lowest,with the highest amount of apoptotic cells and the lowest amount of proliferating cells.Meanwhile,the therapy promoted the greatest maturation of dendritic cells in the thymus and the most proliferation and differentiation of CD8~+T cells in the spleen,with the highest content of CD8 effector memory T cells.Serum levels of inflammatory cytokines(TNF-αand INF-γ)in combined dose group were highest.Immunofluorescence analysis of colon sections revealed that ultrasound-driven CS-ID@NMs in combination with anti-PD-L1 treatment elevated the highest increase and differentiation of CD8~+T cells,the lowest number of immune suppressive cells,and the lowest level of HIF1-αin the colon.The ultrasound-driven CS-ID@NMs combined with anti-PD-L1 therapy effectively prevented the recurrence and metastasis of colon cancer.In the immunofluorescence sections of metastatic tumors,it was found that ultrasound-driven CS-ID@NMs combined with anti-PD-L1therapy promoted the highest amount of apoptosis in distal tumor cells and the lowest amount of proliferating cells.Moreover,the combined therapy improved the highest proliferation and differentiation of CD8~+T cells in the distant tumor tissue,and the lowest number of immunosuppressive cells.Conclusions:In this study,mesoporous manganese oxide-loaded sonosensitizer,which have been modified on the surface with silk protein and chondroitin sulfate,showed tumor microenvironment-responsive drug release performance with multiple stimuli(p H,GSH,ROS,and ultrasound),enabling site-specific and controlled drug release at the tumor site.Under the dual driving force of ultrasound and hydrogen peroxide,CS-ID@NMs can effectively cross the intestinal barrier and specifically target and accumulate in colon cancer cells.Moreover,it can promote dendritic cell maturation and enhance the proliferation and differentiation of CD8~+T cells while reduce the proliferation and differentiation of regulatory T cells,thereby facilitating the proliferation and differentiation of initial memory T cells into pre-effector T cells,central memory T cells,and ultimately into effector memory T cells,enabling the execution of anti-tumor immunotherapy.Ultimately,it can then stimulate the body to produce immunological memory effect,thus effectively inhibiting colon tumors in situ and malignant metastatic tumors.