Dual-Driven Nanomotors Enable Tumor Penetration And Hypoxia Alleviation For Calcium Overload-Photo-Immunotherapy Against Colorectal Cancer

The tumor microenvironment of colorectal cancer has three typical characteristics:low penetration,low oxygen and immunosuppression,which greatly limit the therapeutic effect of colorectal cancer.The development of a multifunctional drug with the advantages of relieving hypoxia,immunosuppressive microenvironment and improving tissue permeability is of great significance for the treatment of colon cancer.Here,we report calcium overload phototherapy with mitochondria-targeting drug N770-NHS（N770）modified mesoporous silica（MSN）nanoparticles（called CaO₂-N770@MSN）based on delivery of CaO₂ to release oxygen to alleviate hypoxia symptoms.The results showed that the drug loading of CaO₂-N770@MSN was14.0wt%,which could meet the therapeutic dose.Under near-infrared（NIR）irradiation,CaO₂-N770@MSN can penetrate dense mucus and accumulate in tumor tissues with the aid of oxygen generation by CaO₂.Meanwhile,calcium ions(Ca²⁺)released by CaO₂-N770@MSN exhibit excellent antitumor efficiency by targeting mitochondrial membrane destruction.Calcium overload combined with phototherapy has good tumor sensitivity and biosafety.It also regulates intestinal microbes and,most importantly,causes immunogenic cell death（ICD）in tumor cells.Combined with PD-L1 checkpoint blockers,tumor growth is further inhibited and gradually eradicated while relieving the immunosuppressive microenvironment.In conclusion,we anticipate that CaO₂-N770@MSN,together with enhanced calcium overload phototherapy,opens new frontiers for anti-hypoxic colon cancer therapy,improving sensitivity and safety.In this paper,the preparation and characterization of nanoparticles were designed,the antitumor effect of nanoparticles was evaluated in vitro,the antitumor effect of nanoparticles was evaluated in vivo,and the antitumor effect of nanoparticles combined with immunotherapy was evaluated.The specific experiment contents are as follows:1:Preparation and characterization of nanoparticles.Through the high drug loading of mesoporous silica nanoparticles,N770 and CaO₂ were loaded,so that the nanoparticles could not only alleviate the hypoxia status at the tumor site,but also have good photodynamic and photothermal effects,and the two elements present a complementary state,making the anti-tumor effect greater than any one element alone.A series of experiments verified that the nanoparticles were successfully synthesized and had good oxygen generation ability,excellent photothermal and photodynamic effects.In addition,CaO₂ components can be driven by the O₂ bubble to enter the colorectal tumor tissue through the mucus layer infiltration through the nano motor（CaO₂-N770@MSNs）.The thermal therapy generated by near-infrared irradiation can further improve its accumulation in the deep tissue of the tumor,and the produced oxygen also successfully solves the hypoxia status of the tumor site,thereby enhancing the effect of light therapy.2:In vitro antitumor activity of nanoparticles.In order to successfully reach the tumor site,nanoparticles need to penetrate the mucus layer and the tumor tissue,and finally reach the inside of the tumor cells.In this study,the excellent mucus penetration and tumor penetration of CaO₂-N770@MSNs were demonstrated,and the phototherapy facilitated the nanoparticles to enter the tumor cells and escape from lysosomal clearance.Then,the anti-tumor ability of the nanoparticles was verified by MTT,live-death staining and cell apoptosis.The changes of intracellular reactive oxygen species（ROS）content and mitochondrial membrane potential were detected to determine whether the cells had ICD.3:Anti-tumor effect of nanoparticles in vivo.A mouse model of orthotopic colon cancer was established,and the drug was administered to the mice by enema,assisted by light therapy.The experimental results show that calcium overload combined with phototherapy has a high therapeutic efficiency on orthotopic colorectal tumors,and alleviates the immunosuppressive microenvironment.At the same time,the microbial diversity of the intestinal tract of mice was evaluated and analyzed to determine the effect of treatment on the microbial abundance of the intestinal tract of mice.Treatment effectively increased the abundance of beneficial microorganisms（such as Lactobacillaceae and Lachnospiraceae）and reduced the abundance of harmful microorganisms（such as Bacteroidaceae and Muribaculaceae）.4.Combination therapy of nanoparticles with phototherapy and immunotherapy.The mice with orthotopic colon cancer were constructed,and the colon was administered with drugs to assist phototherapy.On this basis,αPD-L1 was injected into the tail vein for immunotherapy.The body weight of the mice was monitored during the treatment period.After the treatment,subcutaneous tumors were implanted under the skin of the mice,and the growth of subcutaneous tumors was observed for more than one week.Through the detection of a series of indicators in mice,it can be obtained that the combined treatment for the removal of the tumor in situ and the inhibition of the growth of the subcutaneous tumor,showing a better immune activation and the release of tumor site immunosuppression.In summary,by constructing nanoparticles that can produce oxygen by themselves to relieve hypoxic distress in tumor sites and have good phototherapy effect,combined with immunotherapy in treatment,a good therapeutic effect can be achieved in inhibiting tumor growth and eliminating tumor.And can regulate the microbial diversity in the gut,and then treat colon cancer from a variety of nanoparticles.