The Self-assembly And Application Of Drug-drug Conjugate

Nanotechnology has been widely used in cancer treatment.However,the widely and large amount use of,high diversity and complexity of excipients in traditional nanomedicine has triggered many problems,such as low drug loading content,immunogenicity and toxicity concerns,sophisticated nanoparticle preparation method,inferior repeatability and controllability,which could hardly meet the needs of large-scale production and clinical translation of nanomedicine.In the past few years,self-assembled nanomedicine has attracted attentions from researchers by taking advantages of the self-assembly properties of drugs.It can greatly reduce the usage and avoid the safety concerns of carriers,improve the encapsulation efficiency and drug loading content,simplify the preparation method and cut down the complexity and instability of the nanomedicine.Self-assembled has shown great promise in promoting large-scale production and clinical application of nanodrug.Herein,we synthesized two kinds of drug-drug conjugate,doxorubicin-dichloroacetic acid?DOX-DCA?and a series of platinum?IV?-adjudin?Pt?IV?-ADD?prodrugs,and developed the corresponding self-assembled nanomedicines.The nanomedicines were designed to reduce the side effects of DOX and resistance of cisplatin,respectively.Meanwhile,they also can improve the therapeutic effects and avoid the drawback of traditional nanomedicine.The structure of target compounds were validated by ¹H NMR,¹³C NMR,MS or HPLC.The self-assembly property of drug-drug conjugates were evaluated and the particle size,zeta potential,morphology,stability,encapsulation rate and drug loading content of the corresponding nanoparticles were also analyzed.The in vitro cell experiments of self-assembled nanomedicines were carried out,including cell uptake,intracellular pharmacokinetics,MTT assay or apoptosis study.The in vivo antitumor effects were further evaluated on hepatic carcinoma H22,melanoma B16F10 or triple negative breast cancer MDA-MB-231tumor model.The in vivo drug distribution and safety study of DOX-DCA NPs were also investigated.DOX-DCA conjugate was first synthesized with high purity.It can self-assemble into nanoparticles with small amount of PEGylated lipid DSPE-PEG,leading to high drug loading?71.8%,w/w?and diminished excipient associated concerns.The nanoparticles possessed an average particle size of 55.8 nm and negative zeta potential of-28.6±1.1 mV.After uptake by B16F10 cells,the nanoparticles can gradually accumulate into cell nucleus and be dissociated into free drug DOX under acid condition or in the presence of esterase.The nanoparticles also showed good tumor targeting capability in murine melanoma model,comparable antitumor efficacy with DOX and enhanced tumor killing effect by increasing the dosage.Most importantly,the nanoparticles exhibited invisible systemic toxicity with a high maximum tolerated dosage of 75 mg DOX equiv./kg,which was 15-fold higher than that of free DOX.This work provides a promising strategy to simplify the drug preparation process,increase drug loading content,reduce systemic toxicity as well as enhance antitumor efficiency of DOX.A small library of Pt?IV?-ADD prodrugs were successfully synthesized,including ADD monosubstituted prodrug with different carbon chain length(C₂-Pt-ADD,C₄-Pt-ADD,C₆-Pt-ADD,C₈-Pt-ADD and C₁₂-Pt-ADD)and ADD disubstituted prodrug?Pt-2ADD?.All the Pt?IV?-ADD prodrugs can self-assemble into nanoparticles.DSPE-PEG was further introduced to improve the stability and in vivo circulation of NPs.The particle size of Pt?IV?-ADD@DSPE-PEG NPs varied from 60 nm to 140 nm and increased with the augment of carbon chain.They possessed uniform morphology,negative zeta potential,very high drug encapsulation efficiency?78.8%-92.6%?and drug loading content?84%-86%?.There were big difference in the stability of those nanoparticles.C₂-Pt-ADD@DSPE-PEG NPs,C₈-Pt-ADD@DSPE-PEG NPs,C₁₂-Pt-ADD@DSPE-PEG NPs and Pt-2ADD@DSPE-PEG NPs showed extremely good stability in PBS for over 270 d,while C4-Pt-ADD@DSPE-PEG NPs and C6-Pt-ADD@DSPE-PEG NPs can only last for 48 h and 24 h,respectively.It may be caused by the rearrangement of secondary structure in C₄-Pt-ADD@DSPE-PEG NPs and C₆-Pt-ADD@DSPE-PEG NPs.In order to evaluate to antitumor effect of Pt?IV?-ADD prodrugs and relative nanoformulations,cell viability were evaluated on drug resistant cancer cells?MDA-MB-231?and drug sensitive cancer cells?hepatocellular carcinoma HepG2 and non-small cell lung cancer A549?.The cell killing capacity of each compound and the corresponding nanoparticles were demonstrated as follows,C₄-Pt-ADD>C₆-Pt-ADD>C₂-Pt-ADD>C₈-Pt-ADD>C₁₂-Pt-ADD>Pt-2ADD.The good antitumor effect of C₄-Pt-ADD and C₆-Pt-ADD may have a relationship with their structure rearrangement.In drug sensitive cancer cells A549 and HepG2,the anticancer effects of nanomedicine was increased up to 5-and 7-fold compared to cisplatin,while in resistant cancer cells MDA-MB-231,the anticancer effects of nanomedicine was increased up to 266.4-fold,which can greatly reduce the resistance of cisplatin.C₂-Pt-ADD@DSPE-PEG NPs could suppress tumor growth almost completely in MDA-MB-231 tumor model.It also showed good safety without any body weight loss.In summary,two kinds of self-assembled nanomedicines based on drug-drug conjugate were successfully developed in this project.It can greatly reduce the side effects of DOX and overcome the resistance of cisplatin.The nanoparticles can be very simple in fabrication with high drug loading content,which can greatly reduce the use of carrier and overcome the many problems caused by carriers.This work may provide a promising strategy for large-scale production and clinical translation of nanodrug delivery system.