Myeloid-Derived Suppressor Cell Membranebased Multifuncional Nanoparticles For Cancer Theranostics

Background:Cancer,one of the main factors threatening global human health,the latest cancer statistics show that there were approximately 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018.At present,the methods of cancer treatment is still mainly based on surgical resection,adjuvant radiotherapy,chemotherapy and so on.However,traditional treatments have many drawbacks.For example,surgical resection is controversial on the boundary of tumor tissue and difficult to interfere with distant metastasis;Eventhough radiotherapy could kill tumor cell,but it also produces unrecoverable damage for health tissue;Most of the chemotherapy drugs lack specific targeting,which brings great toxic side effects to patients,and the tumors rapidly develop drug resistance due to their strong mutation;In addition,although immunotherapy,as a research hotspot,is currently the star treatment method for cancer,but the clinical patients response rate of mainstream PD-1 and CTLA4 inhibitors is still not high,one of which is due to insufficient amount of infiltrating T cells in the tumor microenvironment,and on the other hand,tumor cells donot exposure immunogenic antigens.Nanomedicine and treatment is a way to treat disease through the unique structure or properties of nanomaterials.With the rapid development of nanotechnology and materials,more and more nanomaterials（Graphene,Black scale,Quantum dots,Aggregation-induced luminescent materials,etc.）have been applied to tumor field,providing a new idea and hope for cancer theranostics.With the development of nanotechnology,biomimetic nanomaterials have received extensive attention in the field of bio-nanomedicine.Compared to general nanomaterials,biomimetic nanomaterials have better biocompatibility and biological functions.At present,the use of encapsulation technology to make biomimetic nanomaterials is one of the most widely used techniques in the field of cancer theranostics.Myeloid-derived suppressor cell（MDSC）is a group of phenotypically heterogeneous cells derived from myeloid progenitor cells and immature myeloid cells,including immature DCs,immature macrophages,and immature granulosa cells.Current research indicates that MDSC can be divided into mononuclear MDSC and polymorphic nuclear MDSC.In humans,the major surface marker molecule of mononuclear MDSC is CD14⁺HLA-DR^-/low;the major surface marker molecule of polymorphonuclear MDSC is CD11b⁺CD14^-CD15⁺or CD11b⁺CD14^-CD66b⁺.The surface marker molecule of MDSC in mice is CD11b⁺Gr-1⁺.There are two types of mononuclear and polymorphic nuclear according to the expression of Ly6G and Ly6C.MDSC is one of the important members of the tumor immune microenvironment.A variety of cytokines produced by the tumor microenvironment can recruit MDSCs to move to the tumor microenvironment.Iron oxide（Fe₃O₄）is a black crystal with magnetic properties,also known as magnetic iron oxide.At present,magnetic Fe₃O₄ nanoparticles（MNP）are usually synthesized by hydrothermal method.Due to its excellent optical and physicochemical properties,Fe₃O₄ nanoparticles have been widely used in the cancer theranostics.In this study,combined with cell membrane-encapsulated nanotechnology,the cell membrane of MDSC sorted from tumor-bearing mice was coating on Fe₃O₄nanoparticles to synthesize MNP@MDSC for cancer theranostics.Part Ⅰ:Preparation and in vitro and in vivo characterization of Fe₃O₄nanoparticles coated with myeloid-derived suppressor cell membrane.Objective:As a multifunctional nanomaterial,Fe₃O₄ nanoparticles have the capabilities of magnetic resonance imaging（MRI）,photothermal,Fenton reaction-induced tumor killing and microenvironment improvement.As a group of cells derived from myeloid progenitor cells and immature myeloid cells,MDSC is able to actively target the tumor site through recruitment of tumor microenvironment.The purpose of this study was to construct a novel multifunctional biomimetic nanomaterial with an active targeting tumor microenvironment function.Methods:Magnetic Fe₃O₄ nanoparticles were synthesized by modified hydrothermal method.MDSC was sorted and purified from tumor-bearing mice by immunomagnetic microbeads,and the cell membrane was extracted by centrifugation.The MDSC membrane-coating magnetic nanoparticles（MNP@MDSC）were synthesized using a mini pusher.Then,the characterization,stability,protein component,and anti-phagocytic ability of MNP@MDSC were analyzed in vitro;the circulation time,biological toxicity and primary organ distribution of MNP@MDSC were detected in vivo.Results:MNP@MDSC was successfully synthesized by the above method.Transmission electron microscopy showed that the cell membrane of MDSC was successfully coated on MNP by detecting the morphology of magnetic iron oxide nanoparticles（MNP）and MNP@MDSC.The Zeta potential test showed that the surface charge of the MNP surface was coated with the MDSC membrane,which further confirmed that the MDSC membrane was successfully wrapped on the surface of the MNP.The results of dynamic light scatterometry showed that the diameter of MNP@MDSC was basically stable in the PBS and FBS during a week.Analysis of the protein components of the MNP@MDSC by SDS-PAGE revealed that MNP@MDSC inherits the surface protein of MDSC membrane.Macrophage phagocytosis experiments show that MNP@MDSC is effective against macrophage phagocytosis compared to MNP.ICR mice experiments showed that the circulation time of MNP@MDSC was significantly improved compared to MNP and MNP@RBC.Blood routine,blood biochemical and histological staining suggest that MNP@MDSC has no significant biological toxicity.Conclusion:The biomimetic MNP@MDSC was successfully constructed.It has significant stability and anti-phagocytic ability in vitro;it has excellent immune escape ability and long circulation time in vivo,and has outstanding biocompatibility,showing no obvious systemic side effects.It can suit the subsequent research for cancer theranostics.Part Ⅱ: Application of MNP@MDSC in Tumor Diagnosis and Its Photothermal EffectObjective: Based on the first part of the synthesized MNP@MDSC.Considering that MNP@MDSC is an integration of MDSC and MNP,we need to test whether MNP@MDSC inherits the targeting tumor microenvironment of MDSC and MRI、 photothermal capacity of MNP.Methods: MNP@MDSC,MNP@RBC and MNP were injected into tumor-bearing mice,and Prussian blue staining and ICP-AES was used to detect whether MNP@MDSC has the ability to actively target the tumor microenvironment;Using small animal magnetic resonance imaging instrument,the T2 relaxation rate of MNP@MDSC was detected to proof whether is similar to MNP.After injection of MNP@MDSC,MNP@RBC and MNP into tumor-bearing mice,the MRI effects and differences of three materials were observed by small animal magnetic resonance imaging instrument;Through laser irradiation,the infrared camera examined the three kinds of nanoparticles in vitro,and then using the xenograft model of malignant melanoma mice to detect the photothermal therapy effects and differences of the three nanoparticles in vivo.Results: The material-targeted experiments of tumor-bearing mice showed that the ability of three nanoparticles to target the tumor microenvironment was: MNP@MDSC>MNP@RBC>MNP;in vitro magnetic resonance immage experiments showed T2 relaxation of MNP@MDSC and MNP was basically similar（R2=86.73）,indicating that the cell membrane coating of MNP@MDSC did not affect MNP’s MRI ability.In vivo,the results showed that the tumor site of tumor-bearing mice injected with MNP@MDSC group was significantly darker than before injection,and the degree of darkening was significantly greater than that of MNP@RBC and MNP treatment groups;in vitro,the photothermal effect showed that the temperature rise curves of the three nanoparticles are basically the same after irradiation by a 808 laser.In vivo,animal experiments showed that the temperature of the tumor site increased in the four groups after 5 minutes of laser irradiation,and the MNP@MDSC injection group increased from 34.4 °C to 54.7 °C after irradiation.The increase degree is highest than the other three groups.Conclusion: MNP@MDSC inherits the ability of MDSC to actively target the tumor microenvironment and the magnetic resonance imaging and photothermal capacity of MNP.And the cell membrane coating of the MNP@MDSC did not interfere with its ability to magnetic resonance imaging and photothermal.Compared to the EPR effect of MNP and the passive targeting ability of MNP@RBC,MNP@MDSC shows excellent ability to actively target the tumor microenvironment,which enriches the system’s lower side effects and lays down a solid foundation for cancer theranostics.Part Ⅲ: The mechnaism research of MNP@MDSC in tumor therapyObjective: Photothermal therapy can cause cysteine protease-dependent apoptosis in tumor cells,and release autoantigen and damage associated molecular patterns（DAMP）to produce immunogenic cell death（ICD）;MNP nanoparticles can induce macrophage differentiation into M1 phenotype in tumor microenvironment.Based on the above two points,we hypothesized that MNP@MDSC induces tumor cell immunogenic cell death by photothermal effect,exposing related antigens（HMGB1 and Calreticulin）;while MNP@MDSC induces macrophage M1 phenotype differentiation in mouse tumor microenvironment.The differentiated M1 phenotype cells further act as antigen presenting cells,further enhancing the anti-tumor immune response,thereby inhibiting tumor growth and reducing tumor metabolic activity.Methods: The expression of M1-phenotype macrophage related genes（i NOS and CD86）and M2-phenotype related genes（Arginase 1 and CD206）were detected by PCR after co-cultured with RAW264.7 and B16/F10 cells in the condition of MNP@MDSC,MNP interference or not;B16/F10 mouse xenograft model was established to detect tumor growth under 808 nm near-infrared laser photothermal therapy after PBS or MNP@MDSC,MNP@RBC and MNP were injection.Flow cytometry was used to detect the number of CD11b⁺F4/80⁺CD86⁺ M1 phenotype macrophages,CD11b⁺F4/80⁺CD206⁺M2 phenotype macrophages,CD4⁺ helper T cells,and CD8⁺ effector T cells.Immunofluorescence（IF）was used to detect the expression of ICD-related proteins（HMGB1 and Calreticulin）,macrophagerelated markers（CD86 and CD206）,effector T cells（CD8）,tumor proliferation and apoptosis related markers（Ki67 and TUNEL）in tumor tissues.Small animal PET was used to detect the changes of tumor metabolism.Results: In vitro,in the condition of MNP@MDSC and MNP promoted the expression of M1 phenotype macrophage associated genes（i NOS and CD86）in RAW264.7 and B16/F10 cell co-culture systems.In contrast,the expression of M2 phenotype macrophages related genes（Arginase 1 and CD206）was inhibited;the results of in vivo anti-tumor experiments showed that the MNP@MDSC photothermal therapy group had better tumor inhibition effect than the other groups（the experimental intervention did not significantly affect the body weight of the mice）.The results of flow cytometry showed that the number of CD11b⁺F4/80⁺CD86⁺ M1 phenotype macrophages increased in mice after MNP@MDSC combined with photothermal therapy in mice tumor microenvironment and the number of CD11b⁺F4/80⁺CD206⁺ M2 phenotype macrophages is reduced.At the same time,the number of CD4⁺ helper T cells and CD8⁺ effector T cells increased in mice;the results of IF suggested that ICD-related proteins（HMGB1 and Calreticulin）are gradually increased in the PBS group,MNP group,alone photothermal treatment group,MNP+photothermal treatment group,MNP@RBC+photothermal treatment group and MNP@MDSC+ photothermal treatment group.At the same time,the expression of apoptosis related protein TUNEL was also enhanced in the above groups.In contrast,the expression of tumor cell proliferation associated protein Ki67 was gradually reduced in the above groups;PET imaging results showed that the metabolic activities of tumor tissues in the three sagittal planes are gradually reduced in the PBS group,MNP group,alone photothermal treatment group,MNP+photothermal treatment group,MNP@RBC+photothermal treatment group and MNP@MDSC+ photothermal treatment group.The SUV（Max）and SUV（Mean）statistics was also gradually reduced in the above groups.Conclusion: MNP@MDSC has the ability to induce macrophage differentiation.In tumor-bearing mice,MNP@MDSC combined with photothermal therapy significantly increased the proportion of M1 macrophages,while the number of M2 macrophages decreased,and photothermal inducing immunogenic cell death（ICD）of tumor cells,which promotes the expression of related proteins.M1 type macrophage and ICD interaction further enhanced the anti-tumor immune response of mice,inhibited the proliferation of tumor cells and promoted the apoptosis of tumor cells;in addition,MNP@MDSC combined with photothermal therapy effectively reduced metabolic activity of tumor cells.In summary,MNP@MDSC combined with photothermal therapy can significantly inhibit tumor growth.As a platform of cancer theranostics,MNP@MDSC has great potential for clinical transformation.