Construction Of HER2-targeted DNA Nanocarriers Incorporating Polymeric 5-fluorodeoxyuridine And Their Antitumor Applications

Nucleoside analogues,as a kind of traditional chemotherapy drugs,often appear in many standard chemotherapy schemes for malignant tumors.However,there are still many shortcomings that limit their clinical application,such as poor tumor targeting and blood stability,serious side effects and unsatisfactory therapeutic effect.In order to solve the above problems,the nanoscale drug delivery systems(DDS)enable nucleoside analogues to achieve sustained-release in the blood,extend its half-life,and improve the concentration of drugs in the tumor site and reduce their toxic and side effects by the active or passive targeted delivery.Up to now,a variety of DDS,which composed of synthetic organic or inorganic nanomaterials,have been developed and achieved good therapeutic effects,but the comprehensive and detailed analysis of safety profiles and metabolism pathways of nanomaterials in vivo must be performed before their introduction to further studies and clinical applications.In addition,due to the good water solubility of nucleoside analogues,the drug loading of nanocarriers cannot be accurately controlled,and the repeatability is poor,which hinders their industrial production and clinical application.Thus,how to achieve the active targeted delivery of nucleoside antitumor drugs,obtain accurate drug loading and safe and controllable nanocarriers are important issues that need to be solved urgently.In addition,targeting can play an important role in improving the efficiency of cellular uptake of chemotherapeutic drugs,enhancing their therapeutic effects,and reducing their toxic and side effects.Thus,in our study,based on the structure similarity to normal nucleosides,5-fluorodeoxyuridine(FUdR)was selected to be integrated into the DNA strands by solid-phase synthesis technology and three DNA nanodrugs were designed and constructed for targeted therapy of HER2-positive breast cancer and gastric cancer.Meanwhile,a preliminary analysis of nano-drugs' targeted binding,biodegradation and cancer suppression mechanism was carried out.The specific research contents are as follows:(1)A new affibody-FUdR-DNA tetrahedron nanostructure(affi-F/TDNs)was first designed and constructed to improve the targeting of FUdR,and reduce its toxic and side effects.FUdR was integrated into four 41-mer DNA strands by solid-phase DNA synthesis.Affibody molecule was attached to the end of one of the DNA strands to enhance targeted uptake of the drug molecule.Then,DNA nanodrugs containing 40 FUdR molecules were selfassembled,and the drug loading rate was 19.6%.In vitro test results showed that affi-F/TDNs had high selectivity and inhibitory effect on breast cancer BT474 cells overexpressing HER2,while it had low toxicity in MCF-7 cells with low HER2 expression.In vivo anti-tumor results suggested that affi-F/TDNs had good stability in blood,achieved accumulation in tumor areas,and exhibited significant anti-tumor efficacy.Therefore,affibody-DNA tetrahedron,as a simple and effective active targeted delivery nanocarrier,provides a new way for the transportation of nucleoside antitumor drugs.(2)In order to achieve the targeted co-delivery of nucleoside analogues and anthracycline chemotherapeutics,and to improve their therapeutic effect,an affibody modified DNA-AuNPs(affi-F/AuNPs)integrated with 5-fluorodeoxyuridine(FUdR)was successfully constructed for co-loading FUdR and doxorubicin(Dox).Each new dual-drug-containing DNA-AuNPs(Dox@affi-F/AuNPs)had about 30 affi-DNA hybrid strands with polymeric FUdR,in which about 800 FUdRs were integrated by DNA solid phase synthesis and more than 200 Dox molecules were encapsulated via noncovalent interactions.Compared with the simple mixture of FUdR and Dox,Dox@affi-F/AuNPs exhibited higher inhibitory effect on HER2-overexpressing breast cancer cells and better synergistic antitumor activity due to affibodymediated endocytosis.The related mechanistic studies proved that Dox@affi-F/AuNPs achieved a remarkable combined antitumor activity of Dox and FUdR by promoting more cells to enter apoptosis pathway.Our work provides a novel nanomedicine platform for targeted codelivery of nucleoside analogs and other chemotherapeutic drugs acting on DNA to achieve synergistic treatment of HER2-positive tumors.(3)Combination therapy is emerging as an important strategy for cancer treatment with decreased side effects and improved treatment effect.However,chemotherapeutic drugs with different solubility characteristics are not easy to realize co-delivery in traditional drug delivery systems.Here,the affibody modified G-quadruplex DNA micelles integrated with polymeric 5-fluorodeoxyuridine(FUdR)(affi-F/GQs)were designed as the new drug carriers to solve this problem.The affi-F/GQs exhibited excellent stability and targeting in a series of tests.By encapsulating curcumin(Cur)into their hydrophobic core,the affi-F/GQs co-loaded with FUdR and Cur(Cur@affi-F/GQs)were obtained,and the drug loading rate of FUdR and Cur was 21.1% and 5.5%,respectively.Compared with the physical combination of FUdR and Cur,Cur@affi-F/GQs showed higher cytotoxicity and greater synergistic effect on HER2 overexpressing gastric cancer N87 cells.Moreover,the anticancer mechanism studies revealed that Cur@affi-F/GQs enhanced the production and activity of apoptosis-associated proteins in the apoptotic pathway induced by FUdR.This study presents a promising candidate for targeted drug delivery system,and provides a novel approach for co-delivery of drugs with significant difference in solubility.