Biocompatible DNA/gold nanoparticle complex with a protective calcium phosphate (CaP) coating was prepared by incubating DNA/gold nanoparticle complex coated by hyaluronic acid in SBF (simulated body fluid) with a Ca concentration above 2 mM. The CaP-coated DNA complex was revealed to have high compatibility with cells and resistance against enzymatic degradation. By immersion in acetate buffer (pH 4.5), the CaP capsule released the contained DNA complex. This CaP capsule including a DNA complex is promising as a sustained-release system of DNA complexes for gene therapy.
Gene therapy has been proposed as a novel strategy for the treatment of refractory disease. However, direct injection of naked DNA coding a therapeutic gene generally fails to exhibit a satisfactory therapeutic effect [
Recently, drug delivery systems composed of inorganic nanoparticles, such as silica nanoparticles [
However, DNA/gold particle complexes are generally unstable in plasma because of their positive surface charge [
In this study, we developed novel DNA/gold nanoparticle complexes with protective calcium phosphate (CaP) coating. The effects of the CaP coating on the protection against degradation by DNase and suppression of adverse interactions with cells were investigated.
Chloroauric acid (HAuCl4), sodium borohydride (NaBH4), and 2-aminoethanethiol were purchased from Wako Pure Chemical Industries, Ltd. Hyaluronic acid sodium salt (from a microorganism) and YOYO-1 iodide were obtained from Nacalai Tesque, Inc, and Invitrogen Corp, respectively. GFP-coding plasmid DNA (pDNA) with cytomegalovirus promoter was obtained from Clontech Laboratories, Inc. It was amplified in
A solution of 0.01% HAuCl4 (2 mL) was reduced using 1
HA aqueous solution (0.5
A suspension of pDNA/AET/Au/HA with 1.5 SBF was mixed with an equal volume of 30% NaCl solution and incubated for 24 hours at 37°C to dissociate the DNA complex. The DNA complex was then diluted with pure water to adjust DNA concentration ([DNA] = 2
The sizes of Au nanoparticles and DNA/AET/Au/HA complex encapsulated in calcium phosphate were measured by a dynamic light scattering method (DLS) with a particle analyzer (Malvern Zetasizer Nano ZS). DNA/AET/Au complex or DNA/AET/Au/HA complex suspension was diluted with water to 1 mL, and
DNA/AET/Au/HA encapsulated by calcium phosphate was dropped onto adhesive carbon tape and vacuum-dried overnight. The surface was evaluated by SEM-EDS (JSM-7600F, JEOL Ltd., Japan) operated at 5 kV.
Cytotoxicity of DNA/AET/Au/HA encapsulated by calcium phosphate was evaluated by WST-1 assay as follows: MLC-6 cells, an osteoclast-like cell line derived from a mouse, were seeded onto 24-well plates at
Plasmid DNA was fluorescently labeled with YOYO-1 at a YOYO-1/nucleotide ratio of 0.1. DNA/AET/Au/HA complex was then made of the fluorescent DNA and mixed with 1.5 SBF to be encapsulated by calcium phosphate (final Ca = 2.6 mM). It was added to the cells (1.65
The protective effect against the enzymatic degradation of DNA by encapsulation with calcium phosphate was evaluated using Hind III (Takara Bio Inc.) as follows: Hind III (0.5 unit) was added to the DNA/AET/Au/HA encapsulated by calcium phosphate suspension (DNA = 190 ng) in accordance with the instructions for the reagent. The degradation of DNA was evaluated by agarose gel electrophoresis ([agarose gel] = 1%).
Significant differences between two independent groups were examined by Student's
Small gold nanoparticles were readily obtained by reduction of HAuCl4 by NaBH4. As shown in Figure
Size distribution profile of the gold nanoparticles.
DNA/AET/Au complex was then encapsulated in a CaP membrane using SBF. An SBF has a similar inorganic ion concentration to that of human blood plasma and is supersaturated against hydroxyapatite (Ca ion = 2.5 mM). In this study, 1.5 times concentrated SBF (Ca ion = 3.8 mM, pH 7.25) was used to deposit apatite onto the DNA/gold complex surfaces. An apatite layer is known to be formed on bioactive materials with phosphoric acid or carboxylic acid groups [
Various amounts of HA were added to DNA/AET/Au complex (DNA : AET = 1 : 7.2 in weight), and
DNA/AET/Au complex coated by HA (DNA : AET : HA = 1 : 7.2 : 23.3 in weight) was added to the SBF, and deposition of CaP layer on the surface of DNA/AET/Au/HA complex was attempted. DNA complex suspension was added to 1.5 times concentrated SBF at a final Ca concentration of 1.4, 2.0, 2.6, or 3.1 mM. To examine the deposition of CaP, dissociation behavior of the DNA complex in a concentrated NaCl solution was evaluated. DNA complex immersed in SBF with 1.4 mM Ca ion concentration was dissociated by concentrated NaCl solution and showed bands at similar positions to those of DNA complex without SBF. On the other hand, the DNA complexes immersed in SBF with more than 2.0 mM Ca did not show bands from dissociated DNA molecules. This indicates that CaP could be deposited onto a surface of DNA complex coated by HA by immersion in SBF with more than 2.0 mM Ca and form a stable encapsulated complex (Figure
Agarose gel electrophoresis profile of the DNA complexes treated in SBF with various Ca concentrations. Complexes were electrophoresed after dissociation in 15% NaCl. The lowest line represents the result with a DNA complex treated in SBF with [Ca] = 2.6 mM and degraded in an acetate buffer.
Degradation of the CaP capsule in an acidic solution was then examined. An equal volume of pH 4.5 acetate buffer was added to the suspension of DNA/AET/Au/HA complex encapsulated in CaP, which was prepared with 2.6 mM Ca. After stirring at 37°C for 24 h, 30% NaCl solution was added. When it was electrophoresed, a clear band of the dissociated DNA molecule was observed (Figure
In SEM-EDS images, differences in surface morphology and composition were observed (Figure
SEM-EDS analysis of (a) DNA/AET/Au/HA complex; (b) DNA/AET/Au/HA complex encapsulated in calcium phosphate (prepared in SBF with [Ca] = 2.6 mM).
Size distribution profile of the DNA/AET/Au/HA complex encapsulated in calcium phosphate (prepared in SBF with Ca = 2.6 mM).
Figure
Cytotoxicity of Au nanoparticle and the DNA complexes with or without calcium phosphate envelope.
Plasmid DNA was fluorescently labeled by YOYO-1, complexed with gold, and then packaged by CaP. When they were incubated with MLC-6 cells, the cells became luminescent, while the cells treated with naked DNA/YOYO-1 complex did not show the fluorescence (Figure
Cellular uptake of fluorescence-labeled naked DNA and its complex with Au encapsulated with CaP.
Enzymatic degradation behavior of the DNA molecule was evaluated by incubation with Hind III followed by agarose gel electrophoresis. DNA molecule in the DNA/AET/Au/HA complex without CaP was degraded by the enzyme and showed bands of degradation products. A smeared band was observed, unlike for the naked DNA (Figure
Agarose gel electrophoresis profile of the DNA complex encapsulated in CaP after degradation by Hind III.
CaP-encapsulated DNA/gold nanoparticle has high biocompatibility and resistance against enzymatic degradation and also the releasing property by cellular degradation. It is expected to be a safe and durable nonviral system for gene therapy.
DNA/gold complex was efficiently included in a CaP capsule by coating the complex with hyaluronic acid followed by immersion in SBF with a Ca concentration above 2 mM. Biocompatibility and resistance against enzymatic degradation were apparently enhanced by the encapsulation with CaP. Incubation of the CaP capsule including DNA complex in an acidic acetate buffer invited the release of DNA complex from the capsule. This shows the high potential of the CaP capsule as an injectable slow-release device, which would release the contained DNA complex by degradation by osteoclasts.
The authors thank Professor K. Yoshikawa (Kyoto University) for his help with the