Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy

Abstract

Here, we report the synthesis and characterization of size-controllable and stimuli-responsive DNA nanohydrogels as effective targeted gene delivery vectors. DNA nanohydrogels were created through a self-assembly process using three kinds of building units, respectively termed Y-shaped monomer A with three sticky ends (YMA), Y-shaped monomer B with one sticky end (YMB), and DNA linker (LK) with two sticky ends. Hybridization at the sticky ends of monomers and LK leads to nanohydrogel formation. DNA nanohydrogels are size-controllable by varying the ratio of YMA to YMB. By incorporating different functional elements, such as aptamers, disulfide linkages, and therapeutic genes into different building units, the synthesized aptamer-based nanohydrogels (Y-gel-Apt) can be used for targeted and stimuli-responsive gene therapy. Y-gel-Apt strongly inhibited cell proliferation and migration in target A549 cells, but not in control cells. By taking advantage of facile modular design and assembly, efficient cellular uptake, and superior biocompatibility, this Y-gel-Apt holds great promise as a candidate for targeted gene or drug delivery and cancer therapy.

Scheme 1. Schematic illustration of Stimuli-Responsive DNA Nanohydrogel Formation

The Y-shaped monomers (YMA and YMB) and linker are designed to crosslink by hybridization of their “sticky end” segments (black lines), leading to nanohydrogel formation.

Figure 1

(a) Analysis by 3% agarose gel electrophoresis. Lane 1 is YMA, lane 2 is YMB, lane 3 is LK, and lane 4 is Y-gel-Apt with molar ratio of 4:1 (YMA/YMB). (b) Size-dependent (in diameter) distribution of hydrogel nanoparticles with different molar ratios of YMA and YMB: (a) 4:1 (NP-1), (b) 3:1 (NP-2), (c) 2:1 (NP-3).

Figure 2

AFM images of YMA (a) and DNA hydrogel nanoparticles with different molar ratios of YMA to YMB: (b) 4:1 (NP-1), (c) 3:1 (NP-2), (d) 2:1 (NP-3).

Figure 3

Flow cytometry histograms to monitor the binding of Y-gel-Apt with (a) A549 cells and (b) HeLa cells. (c) Confocal laser scanning microscopy images of the colocalization of TAMRA-labeled Y-gel-Apt and lysosensor (lysosome marker), indicating the specific internalization of Y-gel-Apt into A549 cells (top) rather than HeLa cells (bottom). Scale bar represents 100 μm, and inset bar represents 25 μm.

Figure 4

Cytotoxicity assay of (a) A549 cells and (b) HeLa cells treated with Y-gel-Apt and Y-gel at different concentrations.

Figure 5

(a) Western blot analysis of MMP-9 protein expression in A549 cells and HeLa cells with Y-gel-Apt and Y-gel treatments. β-Actin serves as a control for equal protein loading. (b) In vitro scratch wound healing assay. Y-gel-Apt inhibits the migration of A549 cells in vitro.