Artificial Exosomes Mediated The Epigenetic Reprogramming Of Tumor-associated Macrophages To Remodel The Tumor Microenvironment

BackgroundCancer is a serious threat to human health.Immunotherapy is a promising cancer therapy,which recovers anti-tumor immune response by reactivating immune cells.However,immunotherapy often exhibits unsatisfactory therapeutic effect because of the suppressive tumor microenvironment induced by a large number of tumor-associated macrophage(TAM).TAM polarizes into M1 type and M2 type.M1 TAM kills tumor cells,stimulates anti-tumor response,and inhibits tumor cell invasion and metastasis.However,TAM generally manifests as M2 type in tumor tissues,which promotes tumor growth,angiogenesis,invasion,and immunosuppression.Phosphoinositide-3-kinaseγ(PI3Kγ)is a key molecule switch that controls TAM polarization.The M2 TAM can be reprogramed into M1 TAM when the PI3Kγis inhibited,exhibiting anti-tumor effect.However,there is a lack of selective inhibitor of PI3Kγ.CRISPR interference(CRISPRi)is a powerful tool for inhibiting gene expression.Under the guidance of small guide RNA(sg RNA),d Cas9 protein(dead Cas9)specifically binds to the gene(PI3Kγ)and inhibit gene transcription.However,CRISPRi system is negative charged and has disadvantages such as high molecular weight,easy degradation and immunogenicity.Deliver CRISPRi system safely and effectively into cells in vivo is a difficult problem.ObjectiveIn this study,a type of artificial exosomes was constructed to deliver CRISPRi system(d Cas9-KRAB-sg PI3Kγencoding plasmid).The artificial exosomes were designed to target into the nuclear of M2 TAM efficiently and epigenetic inhibited PI3Kγselectively.The M2 TAM would be reprogramed into M1 type,accompanying with suppressive tumor microenvironment remodeling with the aim of cancer treatment.Methods1.Construction and characterization of artificial exosomes.A HEK 293T cell line was constructed to express TAM targeted peptide(CRVLRSGSC,CRV)on exosome membrane through CRISPR/Cas9.Thus,exosomes that overexpressed CRV(CRV-EM)were obtained sustainably.The nuclear localization signal peptide(NLS)was conjugated to polyβ-amino ester(PBAE)to form NLS-PBAE.NLS-PBAE loaded d Cas9-KRAB-sg PI3Kγencoding plasmid through electrostatic adsorption to prapare NPC.Finally,CRV-EM coated NPC to construct CENPC.The particle size,zeta potential and gel retardation of NPC and CENPC were determined to optimize the weight ratio.The morphologies of NPC and CENPC were observed by transmission electron microscopy,and the stabilities of NPC and CENPC was detected.Coomassie blue staining was used to verify the proteins of CENPC.2.Bio-viability of CENPC in vitro.The cellular uptake of CENPC by M2macrophages was investigated through confocal laser microscopy and flow cytometry.Cytotoxicity of CENPC was determined by CCK-8.The expression levels of PI3Kγ,iNOS and CD206 in M2 macrophages treated with CENPC were detected by WB to detect the reprogram efficiency.Lewis lung cancer cells(LLC)were treated with spent medium from different macrophages to explore the inhibition of reprogrammed macrophages.Cell viability was analyzed by Live/Dead staining,apoptosis and scratch.3.Distribution and antitumor effect of CENPC in vivo.Small animal imaging and photoacoustic imaging were performed to observe the distribution of CENPC in tumor-bearing mice.Tumor-bearing mice were randomly divided into serval groups and treated with different nanoparticles and CENPC to evaluate the tumor inhibition.HE staining was used to detect the toxicity of CENPC to other organs.The amount changes of TAM,T cells and myeloid-derived suppressor cells were detected by immunofluorescence staining and flow cytometry.Results1.NPC and CENPC were spherical nanoparticles with particle sizes of 104.3 nm and 131.2 nm,respectively.NPC was able to complex p DNA,and released it in the acidic environment in a short time.CRV-EM tightly coated NPC and improved stability.Coomassie blue staining indicated that CENPC retained most of the proteins of exosome membranes.2.The uptake of CENPC was time-dependent and dose-dependent.CENPC facilitated lysosomes escaped,showing good nuclear deliver capability.CENPC significantly reduced the expressions of PI3Kγand CD206(M2 TAM marker),increased the levels of iNOS(M1 TAM marker)in M2 macrophages.Reprogrammed TAM secreted TNFαand IL6,inducing LLC apoptosis and migration inhibition.3.CENPC targeted to tumor in tumor-bearing mice and retained for a long time.Compared with other groups,CENPC significantly inhibited tumor growth.After CENPC treatment,the number of M2 TAM decreased significantly in tumor microenvironment,while the number of M1 TAM increased.Alongside,CD4~+and CD8~+T cells increased while myeloid-derived suppressor cells decreased.ConclusionIn conclusion,we have successfully fabricated a type of artificial exosome(CENPC).CENPC effectively loaded CRISPRi plasmid and targeted to TAM.CENPC reprogramed the TAM to M1 in situ,awakening the“hot”tumor-immunity and inhibited tumor growth significantly.Artificial exosomes showed great potential in tumor immunotherapy,providing a new strategy for tumor therapy.