Biocompatible infinite-coordination-polymer nanoparticle-nucleic-acid conjugates for antisense gene regulation

Abstract

Herein, we report the synthesis of DNA-functionalized infinite-coordination-polymer (ICP) nanoparticles as biocompatible gene-regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3-hydroxy-4-pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe(III) to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe(III)-HOPO ICP particles by copper-free click chemistry afforded colloidally stable nucleic-acid nanoconstructs. The DNA-ICP particles, when cross-linked through sequence-specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA-ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.

Keywords: antisense gene regulation; coordination polymers; gene knockdown; iron; nanoparticles.

Figure 1

Characterization of DNA-ICP particles. AFM image of (a) Bare ICP particles drop-cast and dried on mica. b) DNA-functionalized ICP particles drop-cast and dried on mica. c) DLS histograms comparing size distributions of bare and DNA-functionalized ICPs. d) Cooperative melting of ICP-DNA aggregates.

Figure 2

UV-Vis analysis of DNA-ICP particles. a) Comparison of bare ICP particles with DNA-ICP particles showing the DNA absorbance at 260 nm. Inset: determination of LMCT ε₄₆₀. b) pH dependence of LMCT absorbance. The red-shift of λ_max with decreasing pH is indicative of complex dissociation (see inset).

Figure 3

Cellular uptake and gene knockdown. Confocal microscopy image of HeLa cells treated with (a) Cy5-ssDNA and (b) DNA-ICP particles (100 nM DNA in each case). Hoechst stain denotes the nucleus in blue while the Cy5 dye attached to the DNA is red. c) Fluorescence intensity of Cy5 dye quantified by flow cytometry. d) Naked-eye visualization of DNA-ICPs taken up in pelleted SKOV-3 ovarian cancer cells e) Expression of HER2 protein in SKOV-3 cells treated with non-targeting DNA-ICPs, HER2 targeting ssDNA + Lipofectamine (25 nM DNA basis), and HER2 targeting DNA-ICPS. Starred bars (**) indicate knockdown was significant (p<0.05) as determined by unpaired student's T test.

Scheme 1

Synthesis and assembly of ICP particles and their cellular uptake. a) Synthetic scheme for bis-3,4-HOPO azide (4). b) Assembly of ICP particles from Fe(NO₃)₃ and compound 4, followed by conjugation with DNA via a Cu free ‘Click’ reaction. c) Scheme depicting the cellular uptake of ICP-DNA conjugates. Reaction conditions: i) maltol, n-propanol, reflux, 16 h. ii) maltol, ethoxyethanol, 64 h, reflux. iii) 4-azido-butan-1-amine, HATU, diisopropylethylamine, DMSO, RT, 4 h. iv) Fe(NO₃)₃·9H₂O, NaOH (aq.), RT, 10 min. v) Dibenzocyclooctyne-DNA, 0.5M NaCl, RT, 16 h