Drug Delivery Systems Based On Camptothecin-grafted Phosphorothioate Dnas And Their Antitumor Applications

Camptothecins are the only clinically approved DNA topoisomerase I inhibitors.However,their extremely low solubility,high toxic and side effects greatly limit their clinical applications.The construction of drug delivery system(DDS)provides a new approach for improving the defect of chemotherapeutic drugs,such as camptothecin,and achieving the better antitumor effect in clinical cancer therapy.Although numerous researches have designed a series of DDS based on polymers,liposomes,inorganic nanoparticles,etc.,these delivery systems usually lack the precise control over drug contents and morphologies.Besides,a drug delivery system is often applied to only one or a class of chemotherapy drugs.These defects make the nanodrugs unable to achieve accurate dosage forms in the clinical transformation and lack of universality,which hinders their large-scale fabrication and clinical application.Furthermore,the biocompatibility and negligible metabolic burden are essential for delivery vehicles.Therefore,construct a biocompatible and universal drug delivery system with precise controlled chemical component and architecture is an urgent demand that needs to be resolved.As an endogenous material,DNA has good biocompatibility and low immunogenicity.The programmable self-assembly based on molecular recognition and base-pairing between DNA strands endows DNA materials with precise and modular structures.Along with the booming development of DNA nanotechnology,DNA materials with designable structure,controllable size and morphology,dynamic response,and functional structural surfaces make them powerful templates for biomedical researches.In order to improve the defects of camptothecin drugs in clinical application,we proposed a new camptothecin(CPT)-conjugated strategy based on DNA backbone modification.Using DNA as drug carriers,a series of precise and controllable DNA drug delivery systems for multiple clinical antitumor treatments were constructed and their antitumor effects in vivo and in vitro were further evaluated.The main research contents and conclusions are described as follows:1.Construction of camptothecin-grafted antisense for combined tumor therapyAntisense oligonucleotides are short sequences of oligonucleotides that can interfere with the expression of related proteins by binding to related mRNAs for gene therapy.For better therapeutic effects,antisenses are usually modified with phosphorothioate groups to resist the degradation of nucleases and improve their pharmacokinetics.Based on the fact that the phosphorothioate groups are good nucleophiles,many functional groups can be conjugated onto the oligonucleotide chains in this way.Therefore,we proposed a new drug-grafted strategy by modifying chemotherapeutic drug,CPT,with electrophilic group and grafting it onto the backbone of the antisense.The genetic therapeutic agent and chemotherapeutic drugs were chemically conjugated to construct a carrier-free drug delivery system.In order to avoid the rapid metabolism of the short antisense strands and improve their tumor accumulations,aptamer sequences were introduced at the 5 'end of the antisenses for tumor target.The chemo and gene-drug co-delivery system realized simple integration of chemotherapeutics and nucleic acids which have dramatic differences in physical-chemical character with controllable drug ratios.The CPT-grafted antisense showed good cell uptake behavior.Based on the cell apoptosis induced by CPT and the down-regulation of anti-apoptotic protein induced by antisense,CPT-grafted chemogene exhibited excellent antitumor effects both in vitro and in vivo.2.Construction of camptothecin-grafted DNA tetrahedron as precise nanomedicine to inhibit the tumor growthThe biocompatibility and well-established chemical modifications make DNA favorable materials in biomedicine.More strikingly,the sequence-directed self-assembly endows their high-precision modularity and designable morphology.Based on the CPT conjugation,the molecular recognition and self-assembly of CPT modified phosphorothioate oligonucleotide were further studied.We speculated that the base-pairing of the complementary strands would not be influenced with a certain amount of CPT loading or CPT-grafted site control,thus a more complex and designable DNA drug delivery system could be constructed.As a proof of concept,we proposed a new strategy to prepare a precise CPT-containing DNA framework with designable morphology and controllable size.In this case,hydrophobic chemotherapeutic agents can be transformed into hydrophilic formulations to enhance the anticancer efficacy,and a general platform for accurate nanodrugs can be built.The hydrophobic drugs were grafted onto DNA via biocleavable linkers,which solved the solubility issue and realized stimuli-responsive drug released behavior.The CPT-containing DNA framework exhibited efficient cellular uptake and enhanced cell cytotoxicity to tumor cells.Furthermore,in an HCT116 tumor-bearing nude mice model,the CPT-grafted DNA tetrahedron showed enhanced accumulation at the tumor site and delayed the tumor progression.The CPT-grafted DNA framework provides a general approach to construct precise nanodrug with defined drug content and architecture.3.Construction of camptothecin-conjugated DNA hydrogel for local chemotherapy to prevent cancer recurrenceTo expand the application scope of DNA-based drug carrier and develop diversified drug delivery system for cancer treatment,we designed a CPT-modified DNA hydrogel for the prevention of tumor recurrence post-surgery.After the oligonucleotides were grafted with drugs,the complementary strands can be self-assembled into CPT-containing Y-shaped motifs The CPT-modified DNA hydrogel was constructed through the well-established“sticky end”associations of each Y-shaped DNA motif.This CPT-grafted DNA hydrogel based on the PS-based drug conjugation and programmable DNA self-assembly provided a long-term antitumor effect and enhanced adjuvant therapeutic effect for the prevention of locoregional recurrence.The injectable feature provided CPT-conjugated DNA hydrogel a convenient and minimally invasive method for drug administration.With enzyme-mediated gradual degradation and disassembly property,this novel CPT modified DNA hydrogel represented a remarkable anti-tumor effect based on the permeation of residual tumor tissue and cellular uptake.Meanwhile,its sustained and GSH-responsive drug release behaviors significantly inhibited cancer recurrence and suppressed tumor regrowth.The CPT-conjugated DNA hydrogel paves a new approach in designing efficient localized DDS for post-surgical adjuvant treatments.