Assembly of Bleomycin Saccharide-Decorated Spherical Nucleic Acids

Abstract

Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AON_ARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AON_ARV7 conjugates were hybridized with complementary strands of a C₆₀-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AON_ARV7 conjugates (120 nM solutions) and of the corresponding glyco-decorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.

Scheme 1. Synthesis of Azide-Modified Carbamoyl Mannose and Bleomycin Disaccharide Precursors

Reagents and conditions: (i) Ph₂SO, Tf₂O, 2,4,6-tri-tert-butyl pyrimidine, 2-(2-azidoethoxy)ethanol, CH₂Cl₂, −60 °C to room temperature (rt); (ii) 0.4 N NH₃ in tetrahydrofuran (THF), CH₂Cl₂; (iii) 2-(2-azidoethoxy)ethanol, TMSOTf, 0 °C.

Scheme 2. Synthesis of Trivalent Clusters of Carbamoyl Mannose and Bleomycin Disaccharide

(A) Crude reversed-phase high-performance liquid chromatography (RP-HPLC) profiles of compounds 7 and 8. (B) Crude RP-HPLC profiles of compounds 10α and 10β. Reagents and conditions: (i) sodium ascorbate, CuSO₄, dioxane/H₂O, overnight at 55 °C; (ii) N(Me)hydroxylamine hydrochloride, NaHCO₃, dimethylformamide (DMF), 1 h at room temperature; RP-HPLC conditions: an analytical C-18 column (250 × 4.6 mm, 5 μm), detection at λ = 260 nm, flow rate: 1.0 mL min^–1, a gradient elution from 25 to 100% MeCN in 0.01 mol L^–1 aqueous triethylammonium acetate over 30 min.

Scheme 3. Synthesis of AF488-Labeled Glycocluster–Oligonucleotide Conjugates ON5–7

The oligonucleotides are 2′-OMe-ribonucleotide phosphorothioates. (A) Crude RP-HPLC profile of ON2. (B) RP-HPLC profile of purified ON7. Reagents and conditions: (i) 7N NH₃ in MeOH, 2 days at rt; (ii) ON1 + 11, 12α, or 12β (2 equiv), H₂O, overnight at rt; (iii) AF488 N-hydroxysuccinimide ester (20 equiv in dimethyl sulfoxide, DMSO), 0.1 M sodium borate (aq, pH 8.5), overnight at room temperature. RP-HPLC conditions: (A) an analytical C-18 column (250 × 4.6 mm, 5 μm), detection at λ = 260 nm, gradient elution (0–25 min) from 0 to 50% MeCN in 0.1 M aqueous triethylammonium acetate, flow rate 1.0 mL min^–1; (B) same as A, except gradient elution (0–25 min) from 5 to 95% MeCN in 0.1 M aqueous triethylammonium acetate.

Scheme 4. SNA Synthesis and Hybridization of AF488-Labeled Glycocluster–Oligonucleotide Conjugates ON5–ON7 with Complementary SNA1

(A) Crude RP-HPLC profile of SNA1. (B) RP-HPLC profile of purified SNA1. (C) Electrospray ionization mass spectrometry (MS-ESI) of SNA1 (a spectrometer equipped with a hybrid quadrupole orbitrap and nano-ESI ionization). (D) Polyacrylamide gel electrophoresis (PAGE) of SNAs 1–5. Conditions: (i) ON9 (0.3 equiv/compound 15), DMSO/H₂O 9:1 v/v, overnight at room temperature; (ii) ON9 (1.2 equiv/azide arm of 16), 1.5 M NaCl (aq), 3 days at room temperature; (A and B) An analytical RP-HPLC column Phenomenex, Aeris 3.6 μm WIDEPORE XB-C18 200 Å, 150 × 4.6 mm, linear gradient from 5 to 45% MeCN in 50 mmol L^–1 triethylammonium acetate over 30 min, a flow rate of 1.0 mL min^–1, detection at 260 nm; (D) for conditions, see the Experimental Section.

Figure 1

Uptake of AF488-labeled carbamoyl mannose conjugate ON5 and SNA2 by PC3 cells (Note: the scale bar is 20 μm). The cells were incubated (37 °C with 5% CO₂) with 120 nM oligonucleotide and 10 nM SNA concentrations or in PBS (controls) for 4 h. After incubation, the cells were washed three times with PBS and fixed using 4% paraformaldehyde solution (diluted in PBS). The intracellular delivery of oligonucleotides and SNAs was quantified via wide-field microscopy on a Nikon Eclipse Ti2-E microscope using Alexa 488 (475 nm) and 4′,6-diamidino-2-phenylindole (DAPI) (395 nm) channels and normalized based on the intensity of the untreated control cell.