Design,Synthesis And In Vitro Activity Experiments Of GSH-Responsive LND-SS-Pt-TPP/HA-CD Nano Prodrug

Cancer is currently the first or second leading cause of premature death in most countries of the world.As the most populous country in the world,the number of new cancer cases in China far exceeds that in other countries in the world.Therefore,the development of anticancer drugs for the treatment of malignant tumors has been a major global issue.The wide application of prodrugs and nano-drug delivery systems in the field of medicine has made great progress in the treatment of malignant tumors.A prodrug is converted from a virtually pharmacologically inactive molecule to an active parent drug by in vivo enzymatic reactions,chemical reactions,or a combination of the two.Prodrug approaches using vectors have evolved into important tools to overcome different obstacles such as targeting problems.In order to solve the problems of drug resistance and side effects caused by cisplatin in the actual application,a cisplatin prodrug was synthesized and a nano-drug delivery system was constructed to solve the problems of poor targeting and easy drug resistance.The following work will be carried out:In this study,a reduction-stimulus-responsive nano-targeted delivery system of LND-SS-Pt-TPP/HA-CD was designed and synthesized.HA-CD coated prodrug nanoparticles can actively target CD44 on the tumor surface,and then target intracellular mitochondria through triphenyl phosphate groups,thereby delivering the prodrug to mitochondria in tumor cells;disulfide bonds can be selectively degraded and broken by high concentrations of GSH in tumor cells and mitochondria,releasing LND and Pt（IV）,while Pt（IV）is reduced to Pt（II）by the action of GSH and ascorbic acid;LND reduces hexokinase 2 expression,disrupts mitochondria and inhibits glycolysis to block energy supply,while cisplatin causes mitochondrial DNA damage,thus killing tumor cells in a synergistic manner.The inhibition of hexokinase 2expression by LND also has the effect of overcoming cisplatin resistance.The structures of the final synthesized compounds were confirmed by ¹H-NMR,¹³C-NMR and MS analysis.In the study of active targeting preparation and in vitro drug release of nano precursor drugs,the physical and chemical properties of the synthesized carrier and drug loaded nanoparticles were examined and the morphology of the nanoparticles was observed by transmission electron microscopy（TEM）.The results showed that the particle size of the blank carrier was about 119 nm,the PDI was 0.187,and the Zeta potential was about-30 m V;After drug loading,the particle size is about 154nm,the PDI is 0.127,and the Zeta potential is about-33 m V.The decrease of PDI and the increase of absolute zeta potential both proved that the LND-SS-Pt-TPP/HA-CD nano system has good homogeneity and stability.The in vitro release behavior of the nano-loaded drug formulation was investigated by high performance liquid chromatography using a dialysis bag method to simulate the microenvironment of the tumor site in vivo,and the results showed that the nano-loaded drug formulation was completely released only after about 10 h,which proved that the nano-system had a slow release effect.By measuring the degree of fracture reduction of LND-SS-Pt-TPP/HA-CD under different concentrations of GSH,it was demonstrated that the drug could be fracture released under the GSH concentration environment of tumor cells and showed a certain concentration dependence.The reduction products were analyzed by LC-MS,high performance liquid chromatography and divalent platinum probe to resolve the reduction-response drug release mechanism of the disulfide-bridged prodrug,demonstrating that the prodrug was firstly cleaved to LND-SH and TPP-Pt under the action of GSH,and TPP-Pt was further reduced to cisplatin under the action of GSH.The biological activity of the nano-loaded particles of the synthetic drug was investigated in A549 cells and cisplatin-resistant cell line A549.The cell proliferation inhibition ability of LND-SS-Pt-TPP/HA-CD on A549 cells and cisplatin-resistant A549 cells was determined by CCK8 assay with LND,CDDP and LND+CDDP as controls.The results showed that for A549 cells,the IC₅₀was>100μM for LND,6.72μM for CDDP,5.88μM for LND+CDDP,5.14μM for LND-SS-Pt-TPP,and 4.56μM for LND-SS-Pt-TPP/HA-CD;for cisplatin-resistant A549 cells,the IC₅₀was greater than 100μM for LND,56.08μM for CDDP,52.21μM for LND+CDDP,8.23μM for LND-SS-Pt-TPP,and 5.40μM for LND-SS-Pt-TPP/HA-CD.The experimental results indicated that for cisplatin resistant A549 cells,the IC₅₀values of cisplatin,LND+CDDP were about 9-fold higher and the IC₅₀values of LND-SS-Pt-TPP/HA-CD were comparable,demonstrating the ability of LND-SS-Pt-TPP to overcome cisplatin resistance.In vitro cellular uptake experiments by coumarin-6-labeled LND-SS-Pt-TPP/HA-CD nano systems showed a significant increase in cellular uptake of nanoparticles after the use of HA-CD carriers,demonstrating that the nano preparations transported drugs with stronger cellular affinity and targeting.The distribution of platinum in the cytoplasm,nucleus and mitochondria was examined by ICP-MS,and the results showed that the cisplatin-incubated nuclei（N）were around6.24μg/L and mitochondria（M）were around 0.82μg/L.The LP/HA-CD-incubated nuclei（N）were around 2.46μg/L and mitochondria（M）were around 7.62μg/L,and the CDDP M/N 0.13,LP/HA-CD M/N 3.10,the results showed that LND-SS-Pt-TPP/HA-CD had good mitochondrial targeting function.The mitochondrial targeting of the drug was further demonstrated by mitochondrial probe co-localization assay experiments after connecting the TPP moiety.According to the above experiments,this topic combines the advantages of both prodrug systems and nanodrug delivery systems to design and construct a LND-SS-Pt-TPP/HA-CD prodrug nanodrug delivery system that combines GSH,CD44targeting on the cell surface,and mitochondrial targeting within the cell.The system has good antitumor activity,and has outstanding performance in targeting and overcoming cisplatin resistance,laying a foundation for future research in vivo.