Polyfluorocarbon-based Oxygen-carrying Nanovehicles Potentiate Anti-tumor Immune Response

Immunotherapy provides a new effective strategy for the clinical treatment of malignant tumors,but their therapeutic efficacy is closely related to the microenvironment in tumor.The extensive hypoxia in tumor cause robust immunosuppression via a variety of mechanisms,and significantly depressed the antitumor immune responses.In clinic,the photodynamic therapy and radiodynamic therapy can convert the oxygen into reactive oxygen species（ROS）in tumor,which can induce robust immunogenic death（ICD）of cancer cells and boost the anti-tumor immune responses.However,the ROS production was limited by the poor oxygen levels in tumor and the intratumoral delivery of therapeutic agents was hurdled by the complexed tumor stroma barriers,thereby compromising the ICD-inducing activity.Undoubtedly,in view of the hypoxic and immunosuppressive microenvironments in tumor,how to boost the antitumor immunity is emerging as a crucial chanllenge of cancer immunotherapy.On this basis,we herein two oxygen-delivering polyfluorocarbon nanovehicles that respectively load photodynamic Di IC18（5）（Di D）and ROS-sensitive gemcitabine prodrug（GTO）or Di D and halofuginone,aiming to relieve the hypoxic status in tumor and induce robust ICD of cancer cells in combination with photodynamic or radiodynamic therapy,thereby boosting the antitumor immune responses for effective cancer therapy.Firstly,we developed an oxygen-delivering polyfluorocarbon nanovehicle of PF₁₁DG from the amphiphilic polyfluorocarbon material of SMA-F₁₁,amphiphilic polycaprolactone-polyethylene glycol(PCL₅₀₀₀-PEG₅₀₀₀),photodynamic Di D and GTO.PF₁₁DG exhibited good stability in the phosphate buffered saline（PBS,p H 7.4）and complete fetal bovine serum.The PF₁₁DG system displayed better oxygen-carrying capacity with a 2.17-fold improvement versus the counterpart non-oxygen delivering PDG system.Upon laser irradiation,gemcitabine would be rapidly released from the PF₁₁DG system.In 4T1 cancer cells,the PF₁₁DG-mediated photodynamic therapy(PF₁₁DG+L)induced significant cell apoptosis and ICD of cancer cells as well as maturation of DC cells.The PF₁₁DG+L treatment caused significant promotion of DC maturation in lymph nodes,exposure of calreticulin（CRT）in tumor sites.Moreover,in the PF₁₁DG+L treated group,the population of myeloid-derived suppressor cells（MDSC）was reduced 61.1%while that of cytotoxic CD8+T cells and natural killer cells（NK）were respectively enhanced 5.9-and 2.8-fold versus the PBS control.In PAN02 induced tumor model,the PF₁₁DG+L produced a 93.4%inhibition of tumor growth,which was much higher than that of the counterpart PDG+L treatment.Due to the dense stroma barrier in solid tumors and the limited intratumoral delivery efficiency,we further designed a bioinspired oxygen-delivering polyfluorocarbon nanovehicles（M-FDH）with deep tumor penetrating and intratumoral cancer cell-accessing capability,aiming to remodel the stroma barrier with radiodynamic therapy to potentiate the antitumor immune responses.M-FDH was developed by camouflaging the Di D and halofuginone-loaded polymeric micelles with cancer cells membrane.M-FDH had a core-shell structure with an average particle size of about 50 nm and a zeta potential of-6.8±0.9 m V.M-FDH exhibited good stability in PBS（p H 7.4）and fetal bovine serum,and had intriguing ability to dissolve oxygen and produce ROS upon X-ray irradiation.The cellular uptake of M-FDH in 4T1 cells and cancer associated fibroblasts（CAFs）was respectively increased 1.28 and 1.29-fold versus the counterpart PDH without cancer cell membrane modification.Upon X-ray irradiation,M-FDH showed obvious toxicity in 4T1 cells,efficient production of ROS and robust ICD of cancer cells with increased DC maturations.In the 4T1-induced tumor models,M-FDH could preferentially accumulate at tumor sites with deep tumor penetration,notable extravasation from tumor vessels and flexible accessibility to the cancer cell fractions in tumor,thereby causing profound enhancement of tumor oxygenation and reduced expression of hypoxia-inducible factor（HIF-1α）at tumor sites.Upon X-ray irradiation,the M-FDH+X-ray treatment produced a large amount of ROS and robust ICD including CRT exposure and high-mobility group box 1protein（HMGB1）expression in tumor tissues.Concurrently,the M-FDH+X-ray treatment dramatically reduced the frequency of CAFs,depleted the expression of major ECM components of collagen,fibronectin and hyaluronic acid,thereby significantly remodeling the tumor stroma barriers.Compared to the negative control,in the M-FDH+X-ray group,the frequency of MDSC,regulatory T cells（Treg）and M2macrophages were respectively reduced 60.5%,53.4%and 54.1%,while that of CD8⁺T cells,IFN-γpositive CD8⁺T cells and granzyme B-positive CD8⁺T cells was respectively enhanced 2.8-,4.4-and 3.0-fold,thereby strikingly remodel the suppressive tumor microenvironments towards tumoricidal features.Furthermore,the therapeutic benefits of M-FDH-mediated combination with X-ray therapy were evidenced in 4T1 breast cancer models and PANC02 pancreatic cancer models.In summary,we successfully designed two oxygen-delivering polyfluorocarbon nanovehicles of PF₁₁DG and M-FDH,which could strikingly relieve the tumor hypoxic levels and enhance the photodynamic or radiodynamic therapy,thereby inducing robust ICD at tumor sites and eliciting the antitumor immune responses for effective cancer treatment.This study provides an innovative exploration to design the novel oxygen-carrying nanovehicles and represents a plausible strategy to boost the antitumor immunity for cancer immunotherapy.