Biodegradable DNA-Brush Block Copolymer Spherical Nucleic Acids Enable Transfection Agent-Free Intracellular Gene Regulation

Abstract

By grafting multiple DNA strands onto one terminus of a polyester chain, a DNA-brush block copolymer that can assemble into micelle structure is constructed. These micelle spherical nucleic acids have a density of nucleic acids that is substantively higher than linear DNA block copolymer structures, which makes them effective cellular transfection and intracellular gene regulation agents.

Keywords: DNA-brush block copolymers; cellular uptake; gene regulation; micelles; spherical nucleic acids.

Figure 1

Characterization of as-synthesized polycaprolactone-based micelle-SNAs. (a) 1% agarose gel electrophoresis; nucleic acids were stained with ethidium bromide; (b) A typical DLS measurement of micelle-SNAs derived from (top) linear and (bottom) brush block copolymer structures; (c) Melting transition behaviour for micelle-SNAs hybridized to complementary 15 nm AuNP-SNAs; (black trace) micelle-SNAs made from linear structures and (red trace) micelles made from the brush architecture; (d) Cryogenic TEM images of micelle-SNAs derived from brush block copolymer. Both DBBC-SNA and LDBC-SNA nanoparticles remain intact under cryogenic conditions and are relatively uniform in size with diameters ~ 30-50 nm.

Figure 2

Cellular uptake of micelle-SNAs. (a-e) Fluorescence micrograph of HeLa cells incubated with different forms of nucleic acids at a total DNA concentration of 1 μM for 16 h. DNA strands were labelled at the 5’-end with fluorescein and the dye molecules are located at the outside terminus of the micelle-SNA structure. a) negative control, cells without DNA incubation; b) single stranded fluorescein-labelled DNA (Fluo-DNA); c) positive control, single stranded Fluo-DNA transfected with Lipofectamine® 2000; d) micelle-SNAs derived from linear block copolymer structures; e) micelle-SNAs derived from brush block copolymer structures. In the fluorescence images, micelle-SNAs assembled from the brush block copolymer show significantly higher uptake compared to those derived from the linear analog or component single stranded DNA; (f) Fluorescence-activated cell sorting (FACS) analysis of the cells when incubated with different forms of nucleic acids. FACS data also confirm the brush block copolymer based micelle-SNA has higher cell uptake efficiency than that of the linear block copolymer based micelle-SNA. Single stranded DNA and DNA transfected by conventional Lipofectamine® 2000 were used as controls.

Figure 3

Confocal microscopy of fluorescein-labelled DBBC-SNA and immunofluorescence staining of organelle markers (red, labelled by Alexa Fluor 663). Biomarkers are Rab9 (for late endosomes) and LAMP-1 (for lysosomes). Note that most DBBC-SNAs colocalize with late endosomes during the incubation in HeLa cells. There is no significant colocalization of DBBC-SNAs with lysosomes, which is consistent with the behaviour of AuNP-SNAs.

Figure 4

Gene regulation by DBBC-based micelle-SNAs. (a) Fluorescence micrograph of C166 mouse endothelial cells that highly express the EGFP protein before DBBC-based micelle-SNA treatment. (b) Fluorescence micrograph of C166 mouse endothelial cells after DBBC-SNA treatment. The green fluorescence was significantly suppressed due to the EGFP gene knockdown. The micelle-SNAs were equipped with anti-EGFP sequence and the cells were cultured for another 2 days after SNA transfection. (c) Western blotting of EGFP expression in C166 cells after treatment with anti-EGFP DBBC-based micelle-SNAs and single stranded DNA samples under various total DNA concentrations. (d) Western blotting of EGFP expression in C166 cells after treatment with anti-EGFP DBBC-SNAs and control samples under total DNA concentrations of 2 μM. The positive control sample was antisense DNA transfected by conventional Lipofectamine® 2000. Actin was used as an internal reference.

Figure 5

The pH-dependent degradation of DBBC-based micelle-SNAs. Samples were incubated in 50 mM MES buffer (pH 5.5), 50 mM HEPES buffer (pH 6.0), or 1X PBS (pH 7.4). Agarose gel electrophoresis was used to monitor the degradation process. a) Degradation of DBBC-based micelle-SNAs after 24 hour incubation under different pH buffer conditions. Note that a significant amount of single stranded DNA can be observed in the gel image; b) the mobility and shape of the DBBC-based SNA bands over incubation time under different buffer conditions. In the first 24 hours, the SNA bands look relatively sharp. However, as the incubation time increases, the bands become smeared and diffuse, indicating the gradual degradation of the entire micellar structures. Note that the micelles held at lower pH (5.5) suffer considerably more degradation after 72 hours.

Figure 6

Cellular toxicity of DBBC-based micelle-SNAs analyzed by a standard MTT assay. Cells treated with DBBC-SNAs (red), LDBC-SNAs (blue), and single stranded DNA (black) at the same total DNA concentration (2 μM). Viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570 nm, and cell viability was quantified by normalization of the absorbance at 570 nm to non-treated cells. Error bar are standard deviation of absorbance at 570 nm from 3 independent wells of cells in a 96-well plate.

Scheme 1

Schematic showing the synthesis of DNA grafted block copolymer-based micelle SNAs. (a) The synthesis of the linear DNA-b-PEO-b-PCL block copolymer and the corresponding formation of micelle-SNAs (LDBC-SNAs). (b) The synthesis of the brush DNA-g-PCL-b-PCL block copolymer and the formation of micelle SNAs (DBBC-SNAs) with a higher surface density of nucleic acids.