Development And In Vitro And In Vivo Evaluation Of Tumor-targeted MiR155 Delivery System

Triple negative breast cancer（TNBC）is still one of the most aggressive cancers with high mortality and poor prognosis.Tumor infiltrating dendritic cells（TIDCs）and tumor associated macrophages（TAMs）mediate immune responses in the tumor microenvironment.Because it is difficult to treat,constructing targeted nucleic acid delivery system for triple negative breast cancer to enhance immunotherapy,and dual regulation of TIDCs and TAMs may be a potential strategy to enhance tumor immunity and restore immune monitoring.Objective:In the study,we designed and developed a kind of self-assembled micro RNA155（miR155）of p H/ROS dual-responsive nanoparticles（Mi R@PCPm P NPs）,double target TIDCs and TAMs for effective tumor immunotherapy.Methods:The research content consists of carrier synthesis,in vitro and in vivo experiments to evaluate the physical and chemical properties,cell uptake,immune regulation and anti-tumor effect.Mannose was used to connect PEG-CDM-PEI-ox-PCL for carrier synthesis.The blank nanoparticles is composed of acid unstable PEG-CDM-PEI and hydrophobic PCL through reactive oxygen species（ROS）cleavable peroxylate.The copolymer can self-assemble with miR155 through electronic and hydrophobic interaction to form a complex Mi R@PCPm P NPs.The chemical structure of each synthetic part was confirmed by ¹H NMR,the particle size and electric potential of nanoparticles under different p H and H₂O₂ conditions were measured by dynamic light scattering（DLS）,and the morphology of the complex was observed by transmission electron microscopy（TEM）.Agarose gel electrophoresis was used to evaluate the binding of the complexes with different N/P ratios to miR155.In vitro,TIDCs and TAMs cells extracted and sorted from tumor tissue were used for research.The purity of extracted TIDCs and TAMs cells was identified by cell flow cytometry,and the uptake of TIDCs and TAMs cells by nano-composites at different p H values was investigated,and the uptake and lysosomal escape were observed by confocal microscopy（CLSM）.MTT method was used to evaluate the cytotoxicity of each preparation to TAMs and TIDCs cells at different p H values.Cell flow cytometry and ELISA were used to investigate the activity of TIDCs and TAMs cells（CD80,CD86,MHCII;IL-10,IL-12）.In vivo experiments,the distribution of micelles in various organs and tumors was scanned with small animal imaging apparatus,and the distribution of Cy5-miRNA in tumor tissues was observed with CLSM.Set Balb/c female mice as PBS group,Mi R@PCPm P NPS group,Mi R@PCPP NPs group,free miR155 group,NC-Mi R@PCPm P NPs group,the drug was administered every other day,and the tumor volume was weighed and measured regularly.Take the mice in each group who were treated in the early stage,collect the tumor tissue and draining lymph nodes after being killed,and evaluate the immune components（TIDCs（CD11c,CD80,CD86）,TAMs（F4/80,CD206,CD86）,CD8+T cells,Tregs,MDSCs,）by cell flow cytometry on the eleventh day,kill and collect the organs and tumor tissues of each group for HE staining and immunohistochemical analysis,and take pictures of the lung organs of each group.Results:The ¹H NMR spectrum showed that the particle size of the nanoparticles was139.7 nm at p H=6.5,and the surface charge of the nanoparticles increased from+23.5m V to+33.3 m V,indicating that the PEG separation and PEI rotation were caused by CDM fracture under weak acidic conditions.Under the condition of H₂O₂,the particle size increased to 1394nm and the potential increased to+20.7m V.The introduction of H₂O₂led to expansion and irregular appearance,indicating that Mi R@PCPm P NPs decompose under ROS conditions.Under TEM,the nanoparticles were observed to be regular spherical,evenly distributed,and kept intact under weak acid conditions.When the N/P ratio is greater than 3,miR155 and PCPm P NPs are completely complexed,so N/P=4 is selected for subsequent experiments.The purity of TIDCs and TAMs cells extracted in vitro is more than 90%,and cell uptake shows that compared with non-targeted nanoparticles with p H=6.5,Mi R@PCPm P NPs improved the transfection of Cy3-miRNA in TIDCs,indicating the improvement of cell uptake mediated by mannose-mannose receptor（MRs）interaction.Similar results were observed in TAMs,which were consistent with CLSM results.The nanocomposites showed negligible toxicity at the experimental concentration,indicating good biocompatibility and the safety of polymers as carriers.The miR 155released in TIDCs up-regulates the expression of CD80,CD86,MHC II and TAMs,up-regulates the expression of IL-12 and down-regulates the expression of IL-10,indicating that it can promote the maturation of TIDCs and the recovery of the ability of M2 to transform M1 and antigen presentation.In vivo animal imaging experiments show that Mi R@PCPm P NPs showed stronger fluorescence intensity at 48h,indicating that the improvement of surface charge could promote the penetration of nanoparticles into tumor tissue.In mouse experiment Mi R@PCPm P The tumor volume of NPs group was 137.6 mm³,the tumor weight was 0.075 g,and the TGI was 78.2%,indicating that mannose conjugate showed the strongest anti-tumor effect.Mi R@PCPm P NPs group showed a decrease in lung metastatic nodes,indicating that TIDCs and TAMs targeting primary tumors may enhance the anti-tumor immune response against lung metastasis.Cell flow cytometry Mi R@PCPm P NPs group produced higher CD8+T lymphocyte infiltration,decreased the percentage of MDSCs,decreased the proportion of invasive Tregs,decreased the level of IL-10,and increased the level of IL-12,IFN-γThe up-regulation and IL-10reduction indicate that our delivery system can inhibit the immune tumor microenvironment against established breast tumors,and double regulate TIDCs and TAMs.The results of immune response in tumor lymph nodes（TDLNs）show that Mi R@PCPm P NPs group produces higher CD11c+,CD80+,CD86+,IFN-γ,CD8+T cell percentage,indicating Mi R@PCPm P TIDCs activated by NPs can migrate to TNLDs to enhance immune response.Conclusions:The release of intracellular ROS in response to miR155 leads to the activation of TIDCs and the reversion of TAMs,enhances the anti-tumor immune response and double regulates the immunosuppressive tumor microenvironment.Mi R@PCPm P NPs have obvious immunotherapeutic effects in vivo and in vitro,without adverse side effects.To sum up,the dual regulation of TIDCs and TAMs by miR155 complex may be a promising strategy for TNBC immunotherapy,which is worthy of further exploration in other cancers.