Biomimetic DNA nanoballs for oligonucleotide delivery
Introduction
Recently, functional oligonucleotides have been studied as a major class of nucleic acid-based therapeutics [1]. Antisense oligonucleotides (ASOs) and micro RNA are examples of oligonucleotides with therapeutic potentials. However, these oligonucleotides suffer from drawbacks, such as instability against nucleases and limited cellular uptake owing to their high negative charges. To overcome these drawbacks, researchers delivered oligonucleotides using cationic lipid or polymer-based delivery systems. Cationic nanoparticles have previously been used to deliver single-stranded oligonucleotides based on charge–charge loading to cationic liposomes [2], [3] and polymer-based nanoparticles [4], [5]. However, the cationic nanoparticle-associated cytotoxicity [6] requires the development of other carriers not cationic as well as biomimetic for delivery of oligonucleotides.
Rolling circle amplification (RCA) is an enzymatic process that produces a single-stranded DNA (ssDNA) from a circular template. The resulting ssDNA is composed of complementary-sequence repeats of the circular template. RCA technology was originally used for DNA detection in genomics; however, additional applications using RCA have since been reported [7]. Recently, RCA was applied to construct DNA nanostructures and DNA hydrogels [8], [9], [10], [11].
In this study, we used RCA-amplified DNA to produce an oligonucleotide delivery system. As model oligonucleotides, we chose two ASO oligonucleotides. To load ASO to the delivery system, we used sequence-specific hybridization rather than cationic nanoparticle-based charge–charge interaction. The template for RCA was designed to have complementary sequences for two different ASOs and to produce poly-binding sites for ASOs upon RCA amplification. Exploiting the DNA condensation mechanisms of viruses, we employed cationic Mu peptides derived from the adenovirus core complex to condense ASO-hybridized RCA products (ARP) and produce DNA nanoballs. The surface of ASO-loaded DNA nanoballs was coated with hyaluronic acid (HA), a ligand for CD44 receptors overexpressed by tumor cells [12], for tumor-targeted delivery.
Here, we demonstrated that DNA nanoballs with ASO-complementary sequences could provide sequence-specific loading of dual ASOs, and coating with HA promoted CD44 receptor-mediated delivery of ASOs to tumor cells. Importantly, ASOs delivered by HA-coated DNA nanoballs silenced the expression of their target mRNAs of ASO, exerting potent anticancer effects in vitro and in vivo.
Section snippets
Construction of ASO-hybridized DNA nanoballs
ASO-hybridized DNA nanoballs were constructed by (1) amplifying ssDNA containing ASO-binding sites by RCA, (2) hybridizing with ASO, and (3) condensing with cationic Mu peptides. In the first step, an RCA template for complementary binding with ASOs was circularized using primers. Briefly, 0.5 μM of 5′-phosphorylated linear ssDNA template and primer (Macrogen Inc., Daejeon, Republic of Korea) were annealed in hybridization buffer (10 mM Tris–HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0) and mixed with
Sequence-specific hybridization of dual ASOs onto DNA nanoballs
The sequences of scrambled and dual ASO-complementary RCA templates are illustrated in Fig. 1A and B, respectively. ARP was formed by hybridization of Dz13 and OGX-427 (Fig. 1C). Loading amounts of Dz13 and OGX-427 onto RCA products depended on the sequences of RCA templates. As compared to RCA products of scrambled template, the RCA products of dual ASO-complementary RCA templates provided 8.1- and 6.7-fold higher loading amounts for Dz13 and OGX-427, respectively (Fig. 1D). ARP was then
Discussion
Here, we demonstrated that the adenovirus-derived Mu peptide played an essential role in condensing ARP to form MA nanoballs, and surface coating with HA promoted CD44 receptor-mediated delivery of HMA nanoballs to tumor cells. Importantly, ASOs delivered by HMA nanoballs silenced the expression of their target mRNAs of ASO, exerting potent anticancer effects in vitro and in vivo upon co-treatment with Dox.
The goal of this study was to test the delivery of therapeutic oligonucleotides via
Conclusions
In this study, biomimetic HMA nanoballs were developed for in vitro and in vivo delivery of functional oligonucleotides. RCA was used to amplify ASO-binding sequences, and dual ASOs were loaded onto HMA nanoballs through sequence-specific complementary hybridization. Soft, ball-shaped, ASO-loaded RCA products were condensed to nanoballs upon biomimetic condensation with adenovirus core complex-derived Mu peptide. Surface coating of MA nanoballs with HA facilitated the delivery of ASOs to cells
Acknowledgment
This work was supported by research grants from the Ministry of Science, ICT and Future Planning (NRF-2014K2A2A4001156, NRF-2015R1A2A1A01005674), and from the Ministry of Trade, Industry & Energy (Technology Innovation Program, Grant No. 10050648), Republic of Korea.
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Both authors contributed equally to this work.