Research On Macrophage Membrane Biomimetic Nanoparticles In The Immunotherapy Of Renal Cancer

ObjectivesRenal cell carcinoma is one of the tumors with abundant immune cell infiltration.However,renal cell carcinoma cells can evade immune surveillance and develop immune tolerance in the process of progression.An important factor is tumor associated macrophages in the immune microenvironment,which coexists with tumor cells and are polarized into M2 macrophages by the tumor immune microenvironment to promote tumor proliferation or metastasis.In the past decade,tumor immunotherapy has achieved rapid development,focusing on improving the activation of immune effector cells in the tumor environment,but the overall treatment efficiency is still limited,so new methods need to be explored.Nanomaterials are substances with a diameter of 1nm-100nm.Different nanomaterials have different physical and biological effects.Some nanomaterials also have tumor targeting and anti-tumor properties.Nanoparticles are loaded with antitumor drugs and coated with biofilm to form biomimetic nanoparticles,which can not only avoid immune phagocytosis and prolong blood circulation time,but also affect tumor progression by releasing specific drugs.Therefore,they show great potential in the field of antitumor therapy.In this study,we provide a macrophage membrane biomimetic nanoparticle with PLGA as the core and coated with M2macrophage membrane.Further,M2 macrophage specific targeting peptide was modified on the outer membrane,and a TLR7/TLR8 agonist(R848)was loaded into M2macrophage membrane biomimetic nanoparticles to observe its remodeling function on the immune microenvironment of renal cell carcinoma and activate anti-tumor immune effect.At the same time,the biosafety performance of the biomimetic nanoparticles was judged by histological staining and blood biochemical detection,which provided a basis for clinical transformation.Methods1.UALCAN and GEPIA databases analyzed the tumor microenvironment landscape of renal clear cell carcinoma,and analyzed the infiltration of M1macrophages and M2 macrophages in the tumor microenvironment,as well as CD8~+T cells and their secreted cytokines and exhaustion indicators.The phenotypic characteristics of macrophages and CD8~+T cells in the tumor environment were verified by immunofluorescence double labeling and q RT-PCR.In addition,we obtained tumor tissues in the constructed orthotopic renal cell carcinoma mouse model,and performed immunofluorescence double labeling and q RT-PCR to verify the above results again.2.We cultured mouse macrophage cell line Raw264.7 and induced M1polarization with LPS(100ng/ml)and IFN-γ(20ng/ml),while induced M2polarization with IL-4(20ng/ml).The phenotype of polarized cells were identified using flow cytometry and q RT-PCR.The polarized macrophage membranes were extracted by hypotonic ultrasonic lysis and high-speed centrifugation.PLGA nanoparticles and the loading of R848 drugs or ICG were prepared by magnetic stirring and centrifugation.The extracted macrophage membranes were encapsulated on the surface of nanoparticles using a physical extruder,and the macrophage membrane biomimetic nanoparticles were finally formed.Transmission electron microscopy and scanning electron microscopy were used to observe the morphology of the prepared biomimetic nanoparticles.The overall surface charge and particle size distribution of macrophage membrane nanoparticles were analyzed by dynamic light scattering.3.After macrophage induced polarization,we detected the expression of CSF-1R,IL-10R,and TGF-βR using q RT-PCR and flow cytometry.We next prepared different biomimetic nanoparticles and detected CSF-1R,IL-10R,TGF-βR by Western blot(WB).We incubated different biomimetic nanoparticles with cytokine standards(concentration of about 1000pg/ml)for30 minutes,followed by high-speed centrifugation.ELISA was used to detect the concentration of cytokines contained in the upper sample to determine the absorption rate of cytokines by nanoparticles.In addition,we re-cultured M0macrophages in the culture medium treated with different nanoparticles and analyzed the changes in surface markers of macrophages using flow cytometry.4.Mouse kidney cancer cell line(RANCA)was cultured and a fluorescent reporter gene was transfected by lentivirus.The successfully transfected RANCA cells were subsequentlycultured and subcutaneously injected into the right hindquarter of a Balb/c mouse at a density of 5×10~7/ml(200μl).When the tumor grew to a certain volume,it was cut into small pieces of 1mm~3and transferred to the right renal capsule of a new Balb/c mouse.Different membrane types of nanoparticles carrying ICG were injected into the tail vein of mice with the same tumor burden to determine the distribution of nanoparticles in the mouse’s body at different times after injection to predict the tumor targeting ability of nanoparticles.At the same time,the expression of chemokines in mouse tumors and chemokine receptors in polarized macrophages were analyzed by q RT-PCR.In order to improve the targeting performance,we further modified M2 macrophage targeting peptide(M2pep)with M2 macrophage membrane biomimetic nanoparticles,and observed its targeting potential through small animal live imaging system.5.Live imaging of small animals was performed 10 days after the establishment of the orthotopic renal cancer model.The successfully constructed mouse models with the same tumor burden were randomly divided into 5 groups.Nanoparticles were injected through the tail vein of mouse every other days.The 5 groups were injected with NC(PBS),PLGA,PLGA@M2PLGA@M2pep,PLGA+R848@M2pep.Small animal live imaging technology was used to dynamically monitor the growth of tumors in each group,and the anti-tumor effect of biomimetic nanoparticles was observed.After treatment,mouse and tumors were dissected,and cytokines in tumor tissues of each group were detected by q RT-PCR and ELISA.The phenotypic changes of macrophages and CD8~+T cells in tumors of each group were further analyzed by immunofluorescence double label staining.6.Finally,HE staining evaluated the effects of nanoparticles in each group on important organs and tissues during the treatment.The in vivo toxicity and side effects of biomimetic nanoparticles were judged by analyzing the growth and weight changes of mouse during the injection of biomimetic nanoparticles,as well as the detection of liver and kidney functions,myocardial enzymes and other biochemical indicators after treatment.Results1.We obtained from the UALCAN and GEPIA databases that CD8A and monocyte macrophages(CD68 and CD14)accounted for a larger proportion in renal clear cell carcinoma compared with normal renal tissue,but the secretion of IFN-γand IL-2 in the microenvironment were less,while the exhaustion markers of CD8~+T cells were elevated(PDCD-1,CTLA-4,HAVCR-2,LAG-3,TIGIT,etc.),indicating that CD8~+T cells were in a state of functional exhaustion.In the analysis of macrophage phenotype,the expression of M1macrophage markers,such as NOS2,CSF-2,IL-1b,and IL-6,showed no significant differences,but the expression of M2 macrophage markers,such as CD163,TGF-β,CSF-1,and VEGFA were increased,indicating that M2macrophages are the main macrophages in the tumor microenvironment of renal cancer.Subsequently,we performed immunofluorescence double labeling and q RT-PCR analysis in 12 clinical human advanced renal cell carcinoma tissue specimens.The results showed that CD8~+T cells were in a state of depletion,while macrophages were dominated by M2 macrophages.Similar conclusions were obtained in the orthotopic model of mouse renal cancer.2.We successfully prepared macrophage membrane biomimetic nanoparticles.First,q RT-PCR and flow cytometry identification showed that mouse macrophage cell line RAW264.7 successfully induced polarization into M1 and M2 macrophages in different conditioned media.Then,the polarized macrophage membranes were extracted by hypotonic lysis and ultrasonic dispersion method,mixed with the prepared PLGA nanoparticles,and then the macrophage biomimetic nanoparticles were prepared by physical co-extrusion.The particle size of biomimetic nanoparticles is about 150-200nm according to scanning electron microscopy.Dynamic light scattering analysis showed that the surface of nanoparticles were negatively charged.3.After macrophage induced polarization,the expression of CSF-1R,IL-10R,TGF-βR in M2 macrophages were more than that of M1 and M0 macrophages.WB analysis of the outer layer protein of the prepared macrophage membrane biomimetic nanoparticles also showed that CSF-1R,IL-10R and TGF-βR were highly expressed in M2 macrophages.In the experiment of biomimetic nanoparticles absorbing inflammatory factors,ELISA results showed that M1macrophage biomimetic nanoparticles neutralized more inflammatory cytokines,such as IL-6,IL-1b,TNF-α,While M2 macrophage biomimetic nanoparticles neutralized more inflammatory suppressors,such as CSF-1,IL-10,TGF-β.4.In order to study the distribution of biomimetic nanoparticles in vivo,we first successfully constructed an orthotopic mouse model of renal cancer carrying a luciferase reporter gene.On the 10th day after the operation of orthotopic inoculation of tumor in the kidney,a significant renal tumor pattern could be observed,and when it reached about 20 days,the kidney was almost infiltrated by tumor cells.The prepared PBS and nanoparticles(PLGA、PLGA@M0、PLGA@M1、PLGA@M2)were injected into mice with the same tumor burden from the tail vein of mice.According to small animal live imaging detection,macrophage biomimetic nanoparticles were gradually enriched in kidney tumors after a few hours,PLGA@M1 and PLGA@M2 all has tumor targeting ability,but it was observed that PLGA@M2 nanoparticles have better targeting performance.QRT-PCR detection revealed that a variety of chemokines were highly expressed in tumors,and after macrophage polarization,the expression of multiple chemokine receptors in M1 and M2macrophages increased.In addition,small animal live imaging results showed that the tumor targeting of PLGA@M2pep group was better than that of PLGA@M2 Group.Through immunofluorescence staining,we found that the distribution of nanoparticles in tumor tissues was significantly higher than that in normal kidney tissues.The above results confirmed that the M2 macrophage biomimetic nanoparticles had good targeting properties,which can be further enhanced when M2pep is modified.5.To verify the inhibitory effect of M2 macrophages biomimetic nanoparticles on renal cell carcinoma,we randomly divided the mouse model of renal cell carcinoma into five groups(NC,PLGA@,PLGA@M2,PLGA@M2pep,PLGA+R848@M2pep),and nanoparticles were injected through tail vein of mouse every two days.Through the small animal live imaging system and pathological examination,we found that the PLGA+R848@M2pep group had a strongest inhibitory effect on tumors,followed by the PLGA@M2pep group and PLGA@M2 group.Through small animal CT,we also proved that the PLGA+R848@M2pep group had significant inhibitory effects on tumors and protected the kidney to reduce the invasion of tumor.The results of q RT-PCR and ELISA showed that the expression of CSF-1,IL-10,and TGF-βwas significantly reduced in the PLGA+R848@M2pep group.Immunofluorescence staining of tumor tissues revealed that after treatment with PLGA+R848@M2pep,the degree of neovascularization and the proliferation of cells were significantly reduced,apoptosis was increased,and the proportion of exhausted T cells was reduced.Further analysis revealed that the proportion of M1 macrophages(CD68 and NOS2)was significantly increased,while that of M2 macrophages(CD68 and CD163)was relatively reduced.Above results demonstrated that PLGA+R848@M2pep effectively reversed the polarization direction of macrophages to M1 macrophages,thus restoring the activity of T cells and increasing their anti-tumor effect.6.HE staining showed that the tissue morphology of important organs such as heart,liver,spleen,lung,kidney and so on in mice remained basically intact without abnormal damage.During the drug treatment,the body weight of mice in each group was not significantly different,and the biochemical indexes such as liver and kidney function and myocardial enzyme spectrum were basically normal,indicating that the biomimetic nanoparticle is a safe and reliable biological agent with less toxic and side effects.ConclusionsWe successfully constructed macrophage membrane biomimetic nanoparticles.By adjusting the appropriate particle size,the nanoparticles were more likely to be enriched in kidney tumors.After being biomimeticized with M2 macrophage membranes,the nanoparticles could be captured by inflammatory chemokines in the tumor microenvironment under the action of chemokine receptors on the surface of M2 macrophage membranes.Due to the high expression of CSF-1R,TGF-βR,IL-10R and other receptors on the surface of M2 macrophage membranes,the nanoparticles could neutralized the inhibitory cytokines such as CSF-1,TGF-β,IL-10 in the tumor environment and reshaped the tumor microenvironment,increased the proportion of M1/M2 macrophages in the tumor microenvironment and improved the activity of CD8~+T cells.In order to enhance the targeting and therapeutic properties,we modified the surface of nanoparticles with M2 macrophage targeting peptide and loaded TLR7/TLR8agonist(R848).The results showed that in the orthotopic model mice of renal cell carcinoma,M2 macrophage biomimetic nanoparticles effectively targeted renal cell carcinoma and adsorbed inflammatory suppressors such as CSF-1、TGF-β、IL-10 through their highly expressed receptors on the surface.With the increase of repolarization from M2 macrophages to M1 macrophages,the proportion of exhausted T cells gradually decreased,while the proportion of cytotoxic T cells gradually increased,leading to better inhibition and killing effects on tumors.In conclusion,we provided a targeted,therapeutic and safe anti-tumor way through M2 macrophage membrane nanoparticles.This study developed a new strategy for remodeling the tumor microenvironment to enhance cancer immunotherapy,which can not only be translated to the clinic,but also be combined with various anti-tumor drugs,so it has broad application prospect.