Drug-loaded Micelles Combined With A2AR Inhibitors For Enhancing The Chemo-immunotherapy Of Breast Cancer

Among the cancer statistics in 2020,female breast cancer has become the number one cancer in the world.At present,cancer immunotherapy,different from traditional cancer therapy,has been given a promising treatment that aims to activate self-adaptive immune response to specifically kill cancer cells.Both the effective presentation of antigens and the activation of dendritic cells（DCs）are essential to adaptive immune response.On the one hand,the classic chemotherapy drug doxorubicin（DOX）can induce immunogenic cell death（ICD）,promote the exposure of calreticulin（CRT）on the surface of cancer cells and the secretion of high mobility group protein（HMGB1）,thereby recruiting DCs and promoting DCs phagocytic cancer cell-related antigens;on the other hand,DCs are in a state of immunosuppression without the participation of activation signals.As an adjuvant,Resimod（R848）is a Toll-like Receptor 7/8（TLR7/8）agonist stimulating the TLR7 and TLR8 receptors of DCs to induce the maturation and activation of DCs.Besides,in the tumor microenvironment（TME）,there is a highly expressed molecule-adenosine（ADO）.ADO activates the adenosine pathway by binding to the A2AR receptor（a type of G protein-coupled receptor）on the surface of most lymphocytes（such as regulatory T cells（Tregs）,NK cells,and T cells）,changing the metabolic level of these cells.However,the current clinical response rate of immunotherapy drugs is not high,and it is accompanied by side effects such as drug off-target.With the rise of nanomedicine technology,these problems can be solved perfectly,and the efficacy of anti-cancer drugs can be further improved.Based on this,this paper designed a kind of arginine-glycine-aspartic acid-polyphenylene glycol-ss-polycaprolactone micelles（RGD-PEG-ss-PCL,RPsP）,loading two hydrophobic drugs DOX and R848 into it to prepare D/R@RPsP.The RGD on the surface of D/R@RPsP actively targets the integrinα_Vβ₃ on the surface of 4T1cells to increase the concentration of drugs in the tumor area.In response to the high concentration of glutathione（GSH）in tumor cells,the redox reaction polymer chain breaks,and the contained drug molecules are directly released.Furthermore,the A2AR receptor inhibitor SCH58261 competitively binds to the A2AR receptor in the presence of adenosine,inhibits the proliferation of Tregs,promotes the anti-tumor effect of T cells and NK cells,and improves the immunosuppression of the tumor microenvironment into immunity the effect of promotion.Therefore,we propose that the combination of intravenous（i.v.）injection of D/R@RPsP and intraperitoneal（i.p.）injection of the A2AR receptor inhibitor SCH58261 can synergistically produce a highly effective and low-toxic cancer immunotherapy effect.First,through chemical synthesis and high-performance liquid chromatography（HPLC）,liquid chromatography-mass spectrometry（LC-MS）,1H-NMR,and other detection methods,the basic materials RGD,alcohol carboxylation(HOOC-PEG₂₀₀₀-COOH),tert-butyl carbonate monoprotected NH₂（Boc-ss-NH₂）,tert-butyl carbonate monoprotected polycaprolactone（Boc-ss-PCL）,monocarboxylate Polyethylene glycol-ss-polycaprolactone(HOOC-PEG₂₀₀₀-ss-PCL),monomethoxy ether polyethylene glycol-ss-polycaprolactone(m PEG₂₀₀₀-ss-PCL)and RGD-PEG₂₀₀₀-ss-PCL were successfully synthesized.D/R@RPsP were prepared by solvent volatilization,and their hydration particle size,polydispersity coefficient（PDI）,and potential（Zeta）were measured by dynamic light scattering nanoparticle size analyzer（DLS）.The true morphology of the micelles was observed by transmission electron microscopy（TEM）,the critical micelle concentration was tested by fluorescence spectrophotometer and the stability of the micelles was examined by DLS,and the drug loading and encapsulation efficiency of the drug-loaded micelles were evaluated by UV spectrophotometer.From the analysis of the results,it was concluded that the micelle D/R@RPsP were around nanoparticle with uniform dispersion,uniform size,excellent stability,and high loading efficiency.By analyzing the change of the particle size of RPsP at different GSH concentrations and the comparison of ¹H-NMR results,it fully showed that RPsP had a great reduction response.The drug release of D/R@RPsP at different GSH concentrations was also consistent with it.Then,through cell death staining and the Alamar Blue method,it was proved that the micelle RPsP had excellent cytocompatibility.Based on ensuring safety in vitro,the IC₅₀ of drug-loaded micelles,the active targeting performance,the strength of inducing ICD,and the maturation and activation of DCs were investigated.The analysis of the results showed that the IC₅₀ of D/R@RPsP was 3.72±0.13μg/mL,which was 1.2 times higher than the IC₅₀ of D/R@RPsP without targeting of 4.39±0.12μg/mL,and the induced ICD was also the highest,the fluorescence of CRT expression was the strongest,the secretion level of HGMB1 reached 13.2±2.2μg/L,and the degree of mediated DCs maturity also reached 92.6±0.8%.Finally,the anti-tumor activity in vivo was investigated.The optimal group D/R@RPsP+SCH58261 was analyzed by normal organ H&E staining and blood routine results analysis,indicating that it had a systemic safety effect.And its tumor suppression rate was the highest among all groups,89.9±11.5%,and the complete regression of breast cancer in one mouse was observed on the 6th day and the 8th day,indicating that the treatment effect was the best.Besides,the optimal group D/R@RPsP+SCH58261 CRT in terms of the degree of induction of ICD and the activation and maturation of DCs was as high as 69.9%by flow cytometry analysis and the amount of HMGB1 measured with the ELISA kit was also the highest,which was 239.9±26.8μg/L,and the maturity of DCs was up to 57.2±1.1%.Further study the influence of A2AR antagonism in the tumor microenvironment on the adenosinergic pathway.The results show that D/R@RPsP synergistic SCH58261 could reduce the expression of A2AR,promote the proliferation of CD8⁺T cells and NK cells,and inhibit the activity of Tregs.Finally,a combination of chemo-immune therapy to improve the adenosinergic pathway in the tumor microenvironment is realized.