A Multifunctional\nDNA Nanoassembly for Cancer Cell\nDetection and Combined Gene–Chemotherapy Therapy

Although DNAzyme is a promising gene therapy agent, low\ncellular\nuptake efficiency, poor biological stability, and the unsatisfactory\neffect of monotherapy limit its development. Herein, a multifunctional\nDNA nanoassembly (RCA product-aptamer-DNAzyme, RAD) was constructed\nfor cancer cell detection and targeted delivery of doxorubicin (DOX)\nand DNAzyme. Briefly, the rolling circle amplification (RCA) product\nwas employed as a scaffold, and each repeated sequence was designed\nto combine with three single-stranded DNA (ssDNA), which carried the\naptamer AS1411 sequence, fluorescent group, and DNAzyme sequence,\nrespectively. Up to 40 groups of ssDNA can be assembled into an RCA\nproduct, resulting in a high affinity for cancer cells and stronger\nfluorescent signals. Due to the high binding affinity, RAD displayed\nhigh sensitivity for the detection of HepG-2 cells (the limit of detection\nwas 200 cells/mL). In addition, with the formation of the double helix\nstructure, each RAD could load up to 200 DOX molecules. Subsequently,\nRAD could efficiently and selectively deliver DOX and DNAzyme into\ncancer cells through the multivalent interaction between the aptamers\nand membrane nucleolin. Then, the released DNAzyme could recognize\nand cleave survivin mRNA under the action of Mg<sup>2+</sup>, leading\nto the apoptosis of HepG-2 cells for gene therapy, while DOX inserted\ninto intracellular DNA to inhibit cell proliferation, realizing chemotherapy.\nAccording to the results, RAD-DOX displayed enhanced therapeutic effects\ncompared with individual gene therapy or chemotherapy, and RAD could\nprotect membrane nucleolin-negative cells from the effects of DOX.\nOverall, given the enhanced serum stability, high drug-loading capacity,\nand excellent selective cellular uptake ability of RAD, this strategy\nshows great potential in the field of cancer therapy.