Design And Preparation Of Organelle-targeted Intelligent Responsive ApDC

Aptamers are one kind of promising targeting ligands for constructing targeted antitumor drugs due to their high specificity,low immunogenicity,modifiable chemical structures and various biological targets.Aptamer-Drug Conjugate(ApDC),which is formed by conjugating aptamer and drug with a linker,can efficiently deliver drugs into tumor cells,thereby improving the therapeutic effect of drugs and decreasing the side effect.Recently,in order to further enhance the specificity of drugs,many works have focused on the precise delivery of drugs into specific organelles,which could enhance the drug potency with less drug dosage and thus improve the side effect.In this paper,we combined two strategies of cell specific-targeting and organelle specific-targeting with aptamers and organelle-located small molecular ligands,and achieved targeted delivery and controlled release for precise tumor treatment.The details can be listed as follows:(1)Metabolic abnormalities,such as the up-regulation of important enzymes and small biological molecules,are highly related to the occurrence,development,escape and resistance of tumors,which are also potential markers for cancer diagnosis and treatment.Here,five reactive tumor biomarkers,i.e.,carboxylesterase,cytochrome P4502J2 enzyme,proline aminopeptidase,reactive oxygen species and glutathione,were screened by analyzing the data of genes,proteins and metabolites in the Cancer Cell Line Database,involving more than 1000 cells of 30 different tumor cell lines.The analysis data covering the distribution maps and expression levels of different markers in different organs and cells provide the design of tumor microenvironment responsive linkers with theoretical support and design ideas.(2)Subsequently,we designed and synthesized nine responsive and cleavable linkers with similar structures and reactivities,which can respond to UV light,H2O2,hypochlorous acid,acetylcholinesterase,cytochrome P4502J2 enzyme,esterase,nitroreductase,phosphatase,and azo reductase.The linkers,which are flexible in structure and fast in assembly,provide a binding site for the access of various chemotherapeutic drugs.The alkynyl at the tail of the linkers can be covalently coupled with a biological targeting ligand through click chemistry,providing a general molecular library for the design of stimuli-responsive drug conjugates.Besides,we optimized the synthetic route to prepare the cleavable linkers in a simple and efficient way,with high yields of more than 60%.Thus,we developed a robust way to synthesize stimuli-responsive linkers,which could conjugate various drugs and targeting ligands.Especially,we further expanded our system to design linkers responsive to other reactive triggers.(3)Furthermore,we used cleavable linkers as bridges,to conjugate chemical drugs(exatecan)and the organelle-targeting ligands(e.g.,those targeting the endoplasmic reticulum,lysosomal and mitochondrial).In addition,we covalently coupled these drug blocks with aptamers via click chemistry to construct dual-targeting ApDC(dt-ApDC).dt-ApDC,which could specially recognize the c-Met receptors overexpressed on tumor cells,was internalized into cells via receptor-mediated endocytosis.dt-ApDC would escape from the endosome and accumulate on the specific organelle for controlled drug release.According to the results of flow cytometry,ApDC drugs could specifically bind to He La and A549 cells,and the modification of small prodrugs significantly enhanced the targeting binding ability of aptamers.As shown by cell localization experiments,ApDC were successfully internalized into cancer cells and located at the designated organelles,thus achieving precise targeted drug delivery at organelle level.In vitro drug release experiments and cell viability experiments demonstrated that,the specific recognition and high binding of aptamers to target cells and the response rupturing ability of cleavable linkers are of great significance for improving the anti-tumor cell proliferation ability of ApDC drugs.In this work,we developed a novel molecular library of cleavable linkers,and engaged to conjugate aptamer,organelle-targeted ligand,and chemical drugs.Through targeted recognition by aptamer and targeted location by small ligand,we realized targeted delivery and controlled drug release to induce precise tumor cell death.In short,our molecular library has provided a new way to design smart medicine and biomaterials,and will promote the development of cancer treatment and drug delivery.