Amphiphilic Nano-Micelles Of Aptamer-Based DNA For Targeted Drug Delivery

Cancer has become one of the most serious diseases that endanger human life and health for its high morbidity and mortality.Research on targeted drug delivery has shown a bright road for cancer therapy.Excellent drug carriers should have the advantages of precisely targeting ability,sustained release pattern,good biocompatibility and low toxicity.Among many carrier systems,micelles have attracted extensive attention because of their good stability and drug-carrying capacity.Micelles are usually composed of hydrophobic and hydrophilic groups and have core-shell structures.The hydrophobic core dissolves hydrophobic drugs,while the hydrophilic shell acts as a protective shell for drug delivery,preventing premature drug decomposition.DNA is not only the carrier of genetic information,but also a kind of hydrophilic polymer material.Meanwhile,the Watson-Crick base pairing rule provides DNA with unique self-recognition and sequence programmability,which makes it have a broad application prospect in the construction of nanomaterials with exquisite structure and excellent performance.Modification of DNA molecules into micelles could result in various morphologies and novel functions of micelles.In addition,the DNA strands containing functional aptamers enable the micelle system to specifically target cancer cells and perform precise drug delivery.In this dissertation,DNA（hydrophilic molecule）containing specific aptamer was covalently bound to cholesterol（hydrophobic molecule）to form amphiphilic compound.DNA micelle assembled by the above compound was expected to have high solubility of anti-cancer drug molecules and specific target ability for cancer cells,thus improving the effect of drugs for cancer therapy.（1）Cholesterol-AS1411 aptamer-DNA micelle was designed and assembled as a targeted drug delivery system,which was described as Chl-T₈-AS1411 below.In this study,we determined the micelle formation concentration of Chl-T8-AS1411 as 500n M by drug loading method.DLS and AFM tests showed that the micelles were uniformly spherical with average diameter of about 130 nm.The micelles could be stable for more than 7 days under 4℃.Dox could be regularly loaded in the Chl-T₈-AS1411 micelles,with the maximum loading capacity of 94.53%,measured by fluorescence spectrometer.The micelle drug delivery system showed sustained release pattern.AS1411 is a guanine-rich DNA aptamer that specifically recognizes overexpressed nucleolus proteins on the surface and cytoplasm of melanoma cells（B16cells）.The Chl-T₈-AS1411 micelle had a stronger targeting ability to mouse melanoma cells（B16 cells）.Meanwhile,fluorescence microscope images showed clear green fluorescence on the surface of B16 cells,indicating that the Chl-T₈-AS1411 micelle could specifically target B16 cells.The load of Dox micelles system can selectively target B16 cells for drug delivery proved by cell experiment results,which has more obvious killing effect than free doxorubicin.（2）Combination of different therapeutic drugs can effectively improve the treatment effect of cancer.Cholesterol-AS1411 aptamer-DNA micelle（noted as Chl-T₈-Sgc8）was designed as drug delivery system for the combined targeting of anticancer drugs Dox and CPT.Sgc8 aptamer can specifically recognize tyrosine protein kinase 7（PTK7 protein）.Human acute lymphoid leukemia（CCRF-CEM）cells overexpressing this protein were selected as the target cells,and human B lymphoma（Ramos）cells were selected as the negative cells.The formation concentration of Chl-T₈-Sgc8micelles was 1μM,which was measured by fluorescence spectrometer.TEM、DLS and AFM characterizations showed that the size of micelles was about 120 nm,with uniform spherical structure and good stability under 4℃.The loading of the micelles on the two drugs was measured by fluorescence spectrum and ultraviolet spectrophotometer,respectively.As the micelle forming,the maximum emission peak and the maximum absorption peak of the drug molecules exhibit red shift.Thus,Chl-T₈-Sgc8 micelles could co-load the two drugs regularly.The results of flow cytometry and fluorescence microscopy showed that the Chl-T₈-Sgc8 micelles could specifically target CCRF-CEM cells.In addition,the Chl-T₈-Sgc8 micelles could be loaded with Dox and CPT drugs to selectively kill CCRF-CEM cells.The killing effect in the co-loaded system was greatly enhanced compared with the free drug system and the system without aptamer.Chl-T₈-AS1411 and Chl-T₈-Sgc8 micelle systems have good biocompatibility,specific selectivity and high killing rate against B16 cells,which could be used as candidate materials for tumor targeted drug delivery system.