Chondroitin Sulfate-Coated Heteroduplex-Molecular Spherical Nucleic Acids

Abstract

Molecular Spherical Nucleic Acids (MSNAs) are atomically uniform dendritic nanostructures and potential delivery vehicles for oligonucleotides. The radial formulation combined with covalent conjugation may hide the oligonucleotide content and simultaneously enhance the role of appropriate conjugate groups on the outer sphere. The conjugate halo may be modulated to affect the delivery properties of the MSNAs. In the present study, [60]fullerene-based molecular spherical nucleic acids, consisting of a 2'-deoxyribonucleotide and a ribonucleotide sequence, were used as hybridization-mediated carriers ("DNA and RNA-carriers") for an antisense oligonucleotide, suppressing Tau protein, (i. e. Tau-ASO) and its conjugates with chondroitin sulfate tetrasaccharides (CS) with different sulfation patterns. The impact of the MSNA carriers, CS-moieties on the conjugates and the CS-decorations on the MSNAs on cellular uptake and - activity (Tau-suppression) of the Tau-ASO was studied with hippocampal neurons in vitro. The formation and stability of these heteroduplex ASO-MSNAs were evaluated by UV melting profile analysis, polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and size exclusion chromatography equipped with a multi angle light scattering detector (SEC-MALS). The cellular uptake and - activity were studied by confocal microscopy and Western blot analysis, respectively.

Keywords: Chondroitin sulfates; Drug Delivery; Heteroduplex ASOs; Molecular Spherical Nucleic Acids; Nanoparticles.

Figure 1

Illustration of the MSNA structures in the study. MSNA1 and MSNA2 are hybridized with ON5–8 to yield MSNA3–10 (cf. Scheme 1). Filled sugar symbols refer to sulfated N‐acetylgalactosamine units.

Scheme 1

A) Structures of the azidopropyl‐modified CS tetrasaccharides. B) Synthesis of AF488‐labeled oligonucleotide conjugates ON5–8. The oligonucleotides are consisted of phosphorothioate backbone. Underlined letters represent 2′‐O‐(2‐methoxyethyl) ribonucleotides, the rest are 2′‐deoxyribonucleotides. Underlined C is 5‐methylated. Reagents and conditions: (i) 3 equivalents of azidopropyl‐modified CS tetrasaccharides 1–3, H₂O, overnight at room temperature. (ii) 20 equivalents of AF488 NHS ester, 0.1 M sodium borate (pH 8.4), H₂O:DMSO (9 : 1, v/v), overnight at room temperature

Scheme 2

Synthesis and hybridization‐mediated assembly of MSNAs. Letters in bold represent ribonucleotides Conditions: i) ON9 or ON10 (0.3 equivalents/4), DMSO:H₂O (9 : 1, v/v), overnight at room temperature; ii) ON9 or ON10 (1.2 or 1.5 equivalents / azide arm of 4), 1.5 M or 0.75 M NaCl (aq), 3 days at room temperature.

Figure 2

PAGE analysis of MSNAs. The ladder is only a reference and not suitable for determining the size of MSNAs. For conditions, see experimental section. (Note: With the given conditions, the excess oligonucleotide is eluted outside of the gel electropherogram)

Figure 3

SEC‐MALS analysis of MSNAs. For conditions, see experimental section

Figure 4

Uptake of ON5–8 and MSNA3–10 in neurons. A–B) Maximum intensity projections of five Z‐sections of 0.4 μm each through nuclei of hippocampal neurons treated at 7 days in culture with 120 nM ON5–8 and 10 nM MSNA3–10 for 10 days are shown (the effective ASO concentration of 120 nM in each case). Signal for the conjugates is shown in cyan while nuclei are highlighted in magenta. Scale=10 μm. C) Mean somatic intensity of AF488 signal relative to ON5 from 3–6 cells +/− S.E.M for each conjugate is shown. The signal intensity refers to intact molecules (fluorescence quenching on MSNAs considered). D) Total Tau levels relative to actin and normalized to untreated cells analyzed by western blotting of Hippocampal neurons treated with indicated ASO conjugates and MSNAs as in A–B. Mean data +/− S.E.M from 3 repeats is shown. Significance was determined using Student's t‐test. *p<0.05; **p<0.01; ***p<0.001. (E) Representative images for treatment in D are shown.