Tumor Microenvironment Responsive Self-assembled Nanomedicines For Cancer Chemotherapy And Immunotherapy

By comprehensive consideration of in vivo transport of CNs,complexity of nano-systems and characteristics of cancer,we constructed three kinds of self-assembled nano-prodrugs for cancer chemotherapy and immunotherapy using paclitaxel,doxorubicin and cisplatin respectively.We first developed a tumor redox-heterogeneity responsive prodrug platform with self-induced ROS amplification property for facilitating rapid drug release,and overcoming MDR and lung metastasis.The prodrug can self-assemble into polymer micelles（PMs）with high drug loading content（³0%）,good physiological stability,prolonged systemic circulation and enhanced tumor distribution.The prodrug PMs can respond to either GSH or ROS,resulting in rapid release of paclitaxel and D-α-tocopheryl polyethylene glycol 1000 succinate（TPGS）.TPGS would stimulate tumor-specific ROS signal amplification,which in turn improve the drug release rate,so the cycle.This effect built a positive feedback loop of ROS generation,maintaining a high level of intracellular ROS necessary for rapid and complete drug release.On one hand,the elevated ROS can induce mitochondria mediated apoptosis through the classical ROS/Cyt C/caspase-9/3 pathway.On the other hand,the increased ROS can inhibit inherent and acquired drug resistance by altering expression of Bcl-2 protein family and by reducing mitochondria membrane potential and ATP level in cancer cells,respectively.As a result,the prodrug PMs showed enhanced efficacy for inhibiting tumor growth in S180 sarcoma tumor model and in drug-resistant tumor model MCF-7/ADR and preventing lung metastasis in 4T1 in situ breast cancer model.We then exploited a sequentially responsive and structure-transformable nanoparticle assembled from 2-（Nap）-FFK_TPA-DOXAGLDDRGD,for comprehensively improving the‘CAPIR cascade’and eventually enhancing therapeutic efficacy.This chimeric peptide was composed of four modules including（i）targeting ligand（Arg-Gly-Asp,RGD）which can specifically recognize integrinαvβ3 that abundantly expressed on tumor endothelial cells as well as on several types of tumors;（ii）2-（Nap）-FFK motif that was inclined to form stable fibrous structures through intermolecular hydrogen bonds;（iii）Ala-Gly-Leu-Asp-Asp（AGLDD）served as a matrix metalloproteinase-9（MMP9）responsive linker which would be selectively cleaved by the overexpressed MMP9 in tumor microenvironment;（iv）DOX was coupled to peptide via a pH-responsive hydrazone bond with 4-formylbenzoic acid（TPA）as a linker that was rapidly broken under intracellular weakly acidic condition.The chimeric peptide can self-assemble into spherical nanoparticles（RGD-sNPs）at physiological condition with relatively small size of 45.7±5.4 nm.By combination of enhanced permeability and retention（EPR）effect and RGD-integrinαvβ3 medicated active-targeting mechanism,RGD-sNPs can be efficiently accumulated at tumor site.Subsequently,the gathered nanoparticles would be transformed into rod-like short nanofibers（S-NFs）with mean diameter of 18±1.6 nm and length of 225.7±10.2 nm,triggered by overexpressed MMP9 in tumor microenvironment.Owing to suitable diameter and length of S-NFs,it showed significant advantages of deep tissue penetration as determined by experiments of in vitro multicellular tumor spheroids and in vivo tumor tissue penetration,extended drug retention（¹5.1%ID/g at 24 h and even³.7%ID/g at 96 h）and 2-fold enhanced cellular uptake.Under intracellular weakly acidic environment,the pH-sensitive hydrazone bond was rapidly cleaved accompanied with active DOX release for subsequent trigger of cytotoxicity and apoptosis.Meanwhile,the remaining peptide was further converted to long fibers（length>5μm）with cytotoxicity,thereby exerting a synergetic antineoplastic effect by caspase-3 apoptotic pathway and promiscuously interaction with microtubules.As a result,RGD-sNPs exhibited superior therapeutic efficacy on hepatoma tumor model and double increased median survival period to 55 days,and minimal side effects.This is the first report that fusing structural transformation with synergistic antitumor effect of chimeric peptide nanoparticles for comprehensively improving the‘CAPIR cascade’of nanomedicines and eventually enhancing their therapeutic efficacy.Tri-negative breast cancer（TNBC）is a kind of breast cancer with high risk of metastasis and poor prognosis.Immunohistochemical results show that its estrogen receptor（ER）,progesterone receptor（PR）and humor epidermal growth factor receptor 2（Her-2）are all negative.There is no specific treatment guideline for TNBC,which is still treated with conventional chemotherapy for breast cancer.Pt,as a first-line drug for TNBC,can significantly improve the survival of patients,but it has strong toxic side effects such as nephrotoxicity,ototoxicity and neurotoxicity.In addition,the MDR of Pt is also an important clinical problem.By combination of preliminary researches,we constructed a Pt prodrug conjugate based on Pt,adjudin（ADD）and（2-Nap）-FFKPLGVRGGGGG.The prodrug can also self-assemble into nanoparticles（2-NPs）,which processed a series of similar property with RGD-sNPs,including long circulation,high tumor targeting ability,deep tumor penetration,prolonged drug retention and so on.ADD,as a male contraceptive,can enhance ROS production,thus induce mitochondria mediated apoptosis through the classical ROS/Cyt C/caspase-9/3 pathway,which is beneficial for improvement of antitumor efficacy and inhibition of MDR.2-NPs can effectively alleviate the nephrotoxicity and gastrointestinal toxicity of Pt,and process significantly enhanced antitumor efficacy in a dose-dependent manner during the administration in 4T1 orthotopic tumor model.Subsequently,we studied the associated mechanisms of 2-NPs on improved efficacy.The results indicated 2-NPs can induce immunogenic cell death（ICD）by way of endoplasmic reticulum stress and autophagy,which facilitated calreticulin（CRT）migration to cell membrane,and secretion of high mobility group protein-1（HMGB-1）and ATP.ATP,as a kind of chemokine,can promote the recruitment of dendritic cells（DCs）into tumor environment.CRT can specifically bind with CD91 receptor on the surface of DCs,so that promote the engulfment of tumor antigens.The binding of HMGB-1with toll like receptor 4 will advance the antigen presentation to T cells.The ICD effect induced by 2-NPs is beneficial to the improvement of tumor suppressive microenvironment,such as the enhancement of CD8⁺T cells and IFN-gamma,the down-regulation of PD-L1 expression and FOXP3⁺Treg cells.To further improve the ICD effect,WKYMVm（W-peptide）was introduced into the skeleton of peptide,and another Pt prodrug（2-Nap）-FFK_Pt-2TPA-ADDGGGPLGVRG-WKYMVm-mPEG₁₀₀₀ was developed.W-peptide is a formyl peptide receptor-1（FPR-1）agonist,which can activate FPR-1receptor on the surface of DCs,make DCs more likely to approach dying tumor cells,and establish stable contacts with corpses,which is conducive to the subsequent steps of ICD.Taken together,this kind of Pt prodrug-enabled activation and enhancement of ICD cascade can effectively inhibit the growth and lung metastasis of tumors and prolong the survival time of tumor-bearing mice.