| Background:Colorectal cancer is the third most common cancer in the world with increasing incidence and mortality rates globally.Cancer immunotherapy,a novel cancer treatment approach,has recently promoted a transformation in the survival pattern of cancer patients,but it is only effective in a small number of colorectal cancer patients with high microsatellite instability/mismatch repair defects(MSI-H/dMMR).As accumulating evidence has shown,Tumor-associated macrophages(TAMs),as abundant and active infiltrated inflammatory cells in the tumor microenvironment(TME),are the most important components in the tumor immunosuppressive microenvironment,promoting tumor metastasis,and therapy sensitivity for colorectal cancers.In the tumor-promoting microenvironment of colorectal cancer,the inflammatory M2 TAM phenotype accounts for 50%of the tumor cell mass,whereas the immunoprotective M1 TAM phenotype is rare.M2 phenotype macrophages promote tumor cell proliferation by regulating cytokines,chemokines,proteases,and reactive oxygen species.Given their essential roles,TAMs have emerged as promising targets for cancer immunotherapy.Immunotherapeutic strategies to suppress TAMs of the M2 phenotype or to reprogram them against the M1 phenotype of tumors have gained enormous momentum.Current therapies targeting TAMs include inhibition of recruitment,interference with survival,and promotion of M1-type repolarization of TAMs.Among them,the immunotherapy strategy of reprogramming TAMs into M1 type not only retains the innate immunity of macrophages,but also improves the immunosuppressive microenvironment of tumors.It is the focus of current research,and certain progress has been made.Therefore,the key point of this study is to construct and modify dual-mode superparticle platforms to target TAMs and repolarize them to alter the tumor immunosuppressive microenvironment and thus increase the antitumor effect.However,the success of these therapies depends on quantifying the amount and distribution of TAMs in Tmes.Therefore,real-time tracking of the distribution and quantity of TAMs before,during and after immunotherapy is very necessary to improve the efficacy of customized immunotherapy in patients with colorectal cancer.At present,nanomaterials have been widely used in various fields,among which carbon dots(CDs)has received more and more attention.CDs is the most promising candidate in the carbon family,because of its excellent properties such as ultra-small size,high water solubility,low cytotoxicity and inherent photoluminescence,which makes it widely used in the biomedical field.Previous reports on inflammation have shown that carbon dots can repolarize M2-type macrophages,suggesting that carbon dots may be a promising material for targeting TAMs and promoting TAMs repolarization.However,carbon dots rarely stabilize near-infrared luminescence,resulting in poor tissue penetration and limiting their use in vivo imaging.So having stable near-infrared glowing carbon points is crucial for biological imaging.SPIONs is an FDA-approved T2 contrast agent for magnetic resonance MRI and has been widely used to track immune cells such as DC and T cells.Previous studies have found its function beyond labeling,namely,they can induce the transformation of TAMs from M2 to M1 type and inhibit tumor growth.Iron oxide can repolarize M2type TAMs into M1-type Tams,induce Fenton reaction,produce ROS,and promote tumor cell apoptosis.Therefore,iron oxide nanoparticles have the characteristics of superparamagnetism and repolarization and have broad application prospects in clinical practice.The main disadvantages are the inability to distinguish between dead and living cells,the inability of signal holes in MRI to quantitatively report the number of cells,and the dilution of markers of cell replication in vivo.In this study,we developed carbon dots with stable near-infrared luminescence characteristics and constructed a superparticle platform of CDs/Fe3O4 dual-mode imaging based on the near-infrared luminescence characteristics of carbon dots combined with the advantage of T2 negative imaging in magnetic resonance of iron oxide.Targeted imaging was conducted on the CD s/Fe3O4 TAMs with high expression of mannose-receptors on the surface of M2-type Tams.In addition to the real-time quantitative non-invasive tracer of TAMs,the repolarization characteristics of carbon points and iron oxide were utilized to promote the repolarization of TAMs to M1 type,change the immune microenvironment and have anti-tumor therapeutic effect.Aims:The aim of this project is to combine the advantages of carbon points and iron oxide:1 To develop and characterize a stable CDs/Fe3O4 superparticle platform with photomagnetic dual-mode imaging capability.2.In vivo and in vitro experiments were conducted to verify the targeting and in vivo tracer ability of the photomagnetic dualmode nanoparticles to TAMs.3 In vivo and in vitro experiments,it was verified that the photomagnetic dual-mode nanoparticles can improve the tumor immune microenvironment and enhance the anti-tumor effect by inducing TAMs to M1 type repolarization.Methods:1.Preparation and properties determination of photomagnetic dual-mode nanoparticlesIn this project,we use IR-813 p-methylbenzene sulfonate and PEG(PEG400)as raw materials,using solvothermal method,using bottom-up method to prepare CDs with stable near-infrared luminescence.Then DSPE-PEG2000 loaded with D-mannose was used to coat hydrophobic CDs and Fe3O4 with its hydrophilic and hydrophobic supramolecular interaction characteristics to synthesize nanoparticles with good stability and biocompatibility.Then,the prepared DSPE-PEG-Man@Fe3O4-Cds nanoparticles were characterized.Their morphologies were detected by transmission electron microscopy,their particle size and potential were detected by DSL,their fluorescence spectra were measured by spectrophotometer,their T2 negative imaging ability was detected by magnetic resonance imaging,and their T2 mapping was used for quantitative study.The biosafety was determined by CCK8 assay and H&E staining of mouse viscera.2.Targeting of photomagnetic bimodal nanoparticles to TAMsFirstly,the in vitro targeting of TAMs by nanoparticles was tested.Flow cytometry was used to detect the signature cytokines of TAMs and M2-type macrophages,and it was confirmed that the main type of TAMs was M2.Laser confocal microscopy and flow cytometry were used to detect the uptake of nanoparticles by M2 macrophages after addition of targeted and non-targeted nanoparticles and after mannose blocking.Then,the mouse model of subcutaneous transplanted tumor of colorectal cancer and liver metastatic tumor were prepared.After injecting PBS,targeted and non-targeted nanoparticles in the tail vein,fluorescence imaging and magnetic resonance imaging were performed in vivo.3.The photomagnetic dual-mode nanoparticles promote the repolarization of TAMs to M1 typeWe co-cultured the nanoparticles with M2 macrophages,and then tested the repolarization of the nanoparticles from nucleic acid to protein level by qRT-PCR and WB experiments.The changes of ROS levels after co-incubation with various nanoparticles were detected by flow cytometry.Then,the subcutaneous transplanted tumor model of mouse colorectal cancer was prepared and treated in groups after tumor formation.Then,the changes of M1 type and M2 type TAMs in each group after treatment were detected by immunofluorescence method and flow cytometry.4.Photomagnetic dual-mode nanoparticles improve tumor immune microenvironment and anti-tumor effectFirst,macrophages were co-cultured with nanoparticles to prepare conditioned medium,and then CCK8 test was used to detect the cell activity of MC38 cells in mouse colorectal cancer after co-culture with different conditioned medium.Then the tumor bearing mice were treated in groups and the tumor growth was monitored at the same time.After 2 weeks of treatment,the tumor was taken and the number of effector T cells and the apoptosis of tumor cells were detected by immunofluorescence method and Tunel staining method.Results:1.Preparation of photomagnetic dual-mode nanoparticles and determination of their propertiesThe results showed that a kind of nano-biomaterial with good water dispersion,uniform particle size and stable structure was obtained by targeted modification of mannose.Its excellent near-infrared luminescence properties can be used to enhance tissue penetration.Due to its significant negative magnetic resonance T2WI imaging properties,it can provide good spatial resolution and tissue contrast,and it can be quantitatively studied by T2 mapping imaging.The biosafety was verified by in vitro cell experiment and in vivo experiment in mice.2.Targeting of photomagnetic dual-mode nanoparticles to TAMsIn vitro confocal laser microscopy and flow cytometry showed that M2 macrophages were co-incubated with targeted and non-targeted nanoparticles respectively,and M2 macrophages had the strongest uptake capacity of targeted nanoparticles,which was significantly weakened after mannose blocking.Mouse colorectal cancer subcutaneous transplanted tumor model and liver metastatic tumor model were prepared.After injecting different groups of nanoparticles into the tail vein,fluorescence imaging and magnetic resonance imaging were performed in vitro.The results showed that targeted nanoparticles in the two models were mainly taken up by tumor tissues,and the imaging ability of both fluorescence imaging and magnetic resonance imaging was superior to that of non-targeted nanoparticles group.The imaging ability of the latter group was better than that of the PBS control group.3.The photomagnetic dual-mode nanoparticles promote the repolarization of TAMs to M1 typeIn vitro qRT-PCR results showed that the targeted nanoparticles had a repolarization effect similar to that of iron oxide,M1 markers CD86,TNF-α and IL12 were significantly increased,M2 markers CD206,ARG1 and VEGF were significantly decreased.In this experiment,we particularly found that targeted carbon points also promoted the significant increase of M1 markers CD86 and TNF-α,and the significant decrease of M2 markers CD206,ARG1 and VEGF.WB results showed that the expression level of protein STAT1 increased and the expression level of STAT6 and PErk decreased after co-culture with targeted nanoparticles.Flow cytometry was used to detect ROS levels after co-incubation with different groups of nanoparticles,and the results showed that ROS levels in the targeted nanoparticle group were significantly increased.After treatment,tumor immunofluorescence analysis showed that the number of M1 type(CD86+)macrophages increased and M2 type(CD206+)macrophages decreased in the targeted nanoparticle set.Flow cytometry showed that the number of targeted nanoparticle set M1(CD86+)type macrophages increased.4.Opto-magnetic dual-mode nanoparticles improve tumor immune microenvironment and anti-tumor effectThe results of cell experiment in vitro showed that M1 conditioned medium had obvious inhibitory effect on MC38 mouse colorectal cancer cells,and M2 conditioned medium had obvious promoting effect.The conditioned medium induced by targeted nanoparticles had obvious inhibitory effect on MC38 mouse colorectal cancer cells,but the inhibitory effect of non-targeted nanoparticles group was not obvious.In vivo results showed that tumor growth was significantly inhibited after tail vein injection.Immunofluorescence analysis and Tunel analysis of tumor tissues showed that the number of effector T cells(CD3+and CD8+)increased significantly,and the apoptosis of tumor cells was significantly enhanced.Conclusion:1.In this study,a CDs/Fe3O4 superparticle platform for photomagnetic dualmode imaging was successfully constructed.2.The nanoparticles synthesized in this study DSPE-PEG-Man@Fe3O4-Cds can be used to quantitatively and noninvasculatively evaluate TAMs in real time by targeting the action of tumor-associated macrophages and using near-infrared light(NIR)-magnetic resonance(MRI)dual-modal imaging.3.The nanoparticles in this study DSPE-PEG-Man@Fe3O4-Cds significantly enhanced the anti-tumor immune response by changing the polarization spectrum of TAMs in the tumor microenvironment and could recruit effector T cells to improve the tumor immunosuppressive microenvironment. |
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