Design And Biological Evaluation Of Multi-drug Delivery Systems With Accurate Tunability Of Drug Ratios For Synergistic Drug Combination Cancer Targeted Therapy

A Cancer Journal for Clinicians（CA）,the most influential academic journal,released the latest global cancer statistical data.In 2020,there were about 19.29 million new cancer cases and 9.96 million deaths with a rising incidence and mortality in the past few decades.Cancer has become the number one killer threatening human health.Cancer is a group of diseases that are comprised of following unique qualities:uncontrolled cell division,replicative immortality,counterattacking apoptosis,unobstructed reproduction,stimulating angiogenesis,activating invasion and metastasis.Conventional mono-chemotherapy is a common and indispensable treatment of various cancers.However,almost all clinical first-line anticancer chemotherapeutic agents showed dose-dependent,non-specific cytotoxicity and tumor cells may exhibit multidrug resistance（MDR）to different antitumor drugs due to the physiological complexity and heterogeneity of cancer,which are the main obstacles to achieve the expected therapeutic effect.In view of the low pace and long time periods of new drug development,the combination of two or more currently available drugs with non-overlapping systemic toxicities and different therapeutic mechanisms is recognized as the cornerstone of cancer therapy.Remaining optimal drug-to-drug ratio to the tumor site is critical for achieving synergistic drug combination effect.Smart drug delivery systems（DDSs）for cancer treatment represent an innovative and promising strategy,which enhances the cellular uptake of drugs and selectivity through passive and active targeting accumulation,to overcome the various limitations of traditional chemotherapy drugs.This study focuses on a cascade responsive prodrug nano-platform to precisely deliver multiple drug combination for efficient personalized cancer therapy.1.In this work,redox-sensitive amphiphilic block polymers poly（N-（2-hydroxy）propylmethacrylamide）-b-poly（D,L-lactide）with tumor-targeting peptide（termed as PDLLA-ss-PHPMA-i RGD）was rationally designed and synthesized through substitution,ring-opening polymerization（ROP）,atom transfer radical Polymerization（ATRP）,and click chemistry.Subsequently,dimeric prodrug CPT-ss-PDLLA-ss-CPT,PTX-ss-PDLLA-ss-PTX and DOX-ss-PDLLA-ss-DOX based on three chemotherapeutics camptothecin（CPT）,paclitaxel（PTX）and doxorubicin（DOX）were obtained by chemical modification.We engineered nano-medicine using a microfluidics-based mixer by combining rationally designed polymeric prodrugs of three commercial chemotherapeutics and a cascade-responsive block copolymer in parallel,presented the identical nano-property with similar size of～100 nm and narrow dispersity of～0.15,showing excellent controllability and malleability;the nano-medicine possessed ratiometric drug loading and synchronized drug release due to the high degree of consistency in the drug release mechanism of the three prodrug designs.On the other hand,to overcome the low delivery efficiency and limited tumor penetration of nanoparticles to achieve effective tumor treatment,i RGD was employed to endow active targeting property by conjugating with redox-sensitive amphiphilic polymer.Compared with i RGD-free nanocarriers,nanoparticles modified with i RGD definitely enhanced the cellular uptake and penetrated into deeper areas of the multicellular 3D tumor spheres.In subcutaneous 4T1-tumor mice for in vivo distribution characterization,the concentration of active targeting nanoparticles accumulated at the tumor site was much higher than that of the free group and the nanocarrier group without i RGD modification.With the capability of ratiometric co-delivery and synchronized release of multiple drugs,multi-drug-loaded nanoparticles significantly enhances synergistic efficacy against breast tumor growth and metastatic liver cancers with reduced side effects.The combination of three synergistic drugs co-encapsulated in a single carrier to achieve drug synergistic inhibition effects at low dosage had been proved by in vitro and in vivo experiments.It is of great significance to enhance and improve the therapeutic effect of malignant tumors that are clinically resistant to monotherapy and to achieve personalized combination therapy.2.In view of the multi-drug resistance of cancer cells caused by the drug efflux transporter P-glycoprotein（P-gp）on the cell membrane and the inspiration brought by the work of the previous chapter,TPGS and VE that had been proved to inhibit P-gp activity were selected to redesign the amphiphilic polymer m PEG₁₁₄-b-P（MA-ss-VE）_nand the prodrug CPT-ss-VE,DOX-ss-VE,PTX-ss-VE based on CPT,DOX and PTX were rationally designed and synthesized.Hence,stimuli-responsive disulfide bonds were introduced into the polymer repeating unit aims to improve sensitivity of the polymer response to GSH by adjusting the repeating unit of the response segment.The inhibition of P-gp protein activity and triple synergistic drug delivery overcome multidrug resistance in cancer cells simultaneously.This strategy offers a potential solution to broaden the range of compounds that can be modified and to enable modular design of nanocarrier-based multi-drug combinations for cancer therapies,or other diseases.The main obstacles current synergistic drug combinations have to overcome is how to keep all component drugs at the fixed ratio from quantitative loading in vitro to synchronized release in vivo,which is essential to achieve synergistic effect.Considering that three free drugs DOX,PTX and CPT can’t be directly co-encapsulated in the same carrier due to their different chemical properties,these drugs can be rationally designed to be prodrug by chemical reactions.Novel stimuli-responsive amphiphilic polymer based on tumor microenvironment was designed as carrier of co-encapsulation of synergistic drug combinations.The in vitro and in vivo data had proved that as-designed strategy can well unify the physical properties of various parent drugs,which allowed the quantitative loading of prodrug combinations,the accurate control of the drug ratios and the synchronized release of loaded drugs,which is capable of precise tunability of drug ratios to obtain an optimal ratio for combination therapy of different cancer types.Improved synergistic therapeutic effects of triple-drug nanoparticles for combinational cancer therapy has been demonstrated by various experiments,providing a promising platform for precisely personalized therapy,which will be the inevitable direction of cancer treatment.This strategy shows a potential platform for extending drug bank and tailoring therapeutic regimen for each individual patient with cancers and potentially other diseases.