Stimuli-responsive Polymeric Nanomedicine For Modulating Tumor Immune Microenvironment

Tumor,especially malignant tumor,remains a major threat to global public health problems.The traditional chemopeutics mainly target tumor cells,resulting in limited therapeutic effect and strong side effects.In fact,tumor tissue is not just a collection of tumor cells,which contains medium tumor cells,matrix cells,immune cells,and various cytokines.Matrix cells,immune cells and various cytokines make up the tumor microenvironment,which affects the tumor progress and the therapeutic effect of chemopeutics.The crosstalk between tumor and immune cells a central scenario in the tumor microenvironment(TME),which is complex and variable.The predominant effect of tumor cells on immune cells is establishing an immunosuppressive context by adjusting the immunophenotype of the immune cells into immunosuppressive type through immunonegative regulatory molecules such as cyclooxygenase-2(COX-2)/prostaglandin E2(PGE2).Immunosuppressive cells in turn promote tumor growth,metastasis,and evading immune surveillance.However,under certain conditions,such as the existence of a large amount of reactive oxygen(ROS)action,tumor cells are induced death and release tumor antigen,resulting in boosted tumor cell specific antitumor immunity.In this paper,we designed stimuli responsive polymeric nanomedicine for improving the tumor immunosuppressive microenvironment and boosting specific antitumor immunity based on the features of the tumor microenvironment,and hoped that these strategies can provide new solutions for future tumor treatment.The main contents and conclusions are as follows:(1)Dexamethasone(DEX)can inhibit the expression of COX-2 mRNA,resulting in reduced production of COX-2 and its downstream product PGE2.Based on the redox content of TME,we modified poly(ethylene glycol)-b-poly(L-lysine)(mPEG-b-PLL)with 3,3’-dithiodipropionic acid,and then directly conjugated DEX onto the scaffold materials to obtain glutathione responsive polypeptide-DEX congujate(L-SS-DEX),with a drug content of 17.1%.We also prepared a non-sensitive polypeptide-DEX congujate(L-SA-DEX)as controland,and its drug content was 13.2%.The hydrodynamics radii(Rh)of L-SS-DEX and L-SA-DEX self-assembled nano-micelles were 33 nm and 38 nm,respectively.TEM images proved the uniform sizes and morphologies of the two nano-micelles.In vitro drug release experiment proved that L-SS-DEX could release more DEX in the condition of high GSH concentration and low pH.In contrast,the contents of DEX release from L-SA-DEX were all below 10%in all conditions.The half-time of blood circulation of L-SS-DEX was about 8.9 h,which was about 2.5 times to that of free DEX.And the concentration of L-SS-DEX in tumor tissues was 9.7 times that of free DEX 48 h post intravenous injection.Besides,L-SS-DEX achieved 86%tumor suppression rate(TSR)in CT26 model,which was much more than that of free DEX(49%TSR)and L-SA-DEX(41%TSR).More than that,L-SS-DEX showed much stronger ability in modulating tumor microenvironment compared to free DEX and L-SA-DEX,characterized as more inflatration of CD8+T cells and M1 macrophages,and reduced infiltration of MDSCs and Tregs.After the treatment of L-SS-DEX,the content of COX-2 in tumor tissues was decreased to 50%of that in PBS group,and the content of PGE2 was also significantly reduced.(2)Aspirin is a kind of nonsteroidal anti-inflammatory drugs,which has been proved to inhibit the activity of COX-2,reduce the production of PGE2,neutralize tumor-promoting inflammation,increase the inflatration of CD8+T cell.Based on the high level of ROS in tumor tissues,we synthesized a ROS-responsive aspirin polymeric prodrug(P3C-Asp)through Passerini three components reaction of aspirin with 4-formylbenzeneboronic acid pinacol ester and 5-isocyanopent-1-yne,followed by azide-alkyne Huisgen cycloaddition "click" reaction between aspirin prodrug with dextran,the drug loading of P3C-Asp was 10%.In the in vitro drug release experiment,an increased drug release was observed in the existence of hydrogen peroxide(H2O2).We further found more P3C-Asp accumulated in tumor tissues compared with that of free aspirin 24 h post injection.In the therapeutic experiment,the treatment of P3C-Asp achieved 60%TSR,while free aspirin only results in 25%TSR.Moreover,after the treatment of P3C-Asp,about 1.9%of CD8+T cells infiltrated in tumor tissues,which was much higher than taht after the treatment of free aspirin(0.6%).Once P3C-Asp combined with aPD-1,the CT26 subcutaneous tumors were completely eliminated.In contrast,there was no significant difference between Aspirin+αPD-1 and aPD-1.(3)Immunogenic death(ICD)has drawn extensive attention due to its can effectiveness in activating tumor specific antitumor immunity.The generation of large amounts of ROS in tumor cells is an important inducer for ICD.We designed a tumor specific enhanced oxidative stress polymer conjugate(TSEOP)for inducing ICD.The TSEOP is prepared by Passerini reaction between cinnamaldehyde(CA),4-formylbenzeneboronic acid pinacol ester and 5-isocyanopent-1-yne,followed by azide-alkyne click reaction with poly(L-glutamic acid)-graft-poly(ethylene glycol)monomethyl ether(PLG-g-mPEG).The number average molecular weight(Mn)of TSEOP was 81.6 k Da with poly dispersion index of 1.69.The Rh of TSEOP self-assembled nano-micelles was about 42 nm.After incubation in PBS with pH 6.8 containing 100 mM H2O2 for 24 hours,the self-assembled nano-micelles of TSEOP dissociates and showed irregular morphology.Under tumor stimuli condition,CA and quinone methide(QM)were quickly generated from TSEOP.CA could consume intracellular thiol,resulting in over-produced ROS in the mitochondria.The benzene boric acid group could generate QM in the presence of H2O2 and consume GSH.The combination of CA and QM cooperatively triggered the accumulation of ROS,oxidative stress and ICD in tumor cells.More importingly,TSEOP was more cytotoxic to tumor cells(CT26 mouse colorectal cancer cells)than to normal cells(3T3 mouse fibroblasts).Single TSEOP treatment could eradicate both CT26 colon tumor and 4T1 tripal negative breast tumors.The vaccination and rechallenge experiments further proved TSEOP coud boost tumor specific antitumor immunity.Through the investigation of this dissertation,we hope to utlize stimuli-responsive polymers to modelate TME and boost antitumor immunity by regulating the crosstalk between tumor and immune cells,which should be benefit for handling current problem in cancer therapy and prrvide an important guidance for designing nano therapeutics.