Targeted delivery to macrophages and dendritic cells by chemically modified mannose ligand-conjugated siRNA

Abstract Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose–siRNA (CMM–siRNA) conjugates to macrophages and dendritic cells (DCs). CMM–siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.

This PDF file includes:

General information of chemical synthesis
Reagents and solvents obtained from commercial suppliers were used without purification or drying unless otherwise noted. Methyl α-D-mannopyranoside was purchased from FUJIFILM Wako Pure Chemical Corporation. Compound 1 was prepared as described in the literature (1). Compound 15 was prepared as described in the literature (2)

General procedure 1: Condensation using alkylamine
To a 0.1 M stirred solution of compound 1 (1 eq) in DMF, we added N, N-diisopropylethylamine (DIEA, 6 eq) and alkylamine (2.5 eq) at room temperature, and the reaction mixture was stirred at the same temperature for 48 h. The reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give the crude compound. The crude compound was purified by reverse-phase column chromatography using a grace instrument, eluted in 10% MeCN/water (0.1% formic acid), and concentrated under reduced pressure to afford compounds 2a-f.

General procedure 2: Reduction using triphenylphosphine
To a 0.05 M stirred solution of compounds 2a-f (1 eq) in THF/H2O (100:1), we added triphenylphosphine (Ph3P, 1.2 eq) at room temperature, and the reaction mixture was stirred at the same temperature for 20 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give a residue. The residue was washed with n-pentane to give a crude compound. The crude compound was purified by preparative HPLC to afford compounds 3a-f.

General procedure 3: Deprotection using sodium methoxide
Compounds 3a-f (1 eq) were dissolved in 2 M methylamine in MeOH to yield a 0.05 M solution at room temperature, and the reaction mixture was stirred at the same temperature for 16 h. The reaction was monitored by LC-MS. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give a crude compound. The crude compound was purified by preparative HPLC to afford compounds 4a-f.  To a solution of crude dimethyl 5-hydroxyisophthalate (5, 100 mg, 476 μmol) in THF (5 ml) was added benzyl (2-hydroxyethyl) carbamate (139 mg, 714 μmol) and triphenyl phosphine (476 mg, 1.43 mmol), followed by the addition of bis(2-methoxyethyl) azodicarboxylate (DMEAD, 167 mg, 714 μmol). After stirring overnight at room temperature, ethyl acetate and water were added to the reaction mixture.
The aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried with MgSO4 and concentrated in vacuo. The residue was separated by silica gel chromatography, and compound 6 was obtained as a white solid (73.0 mg, 40%).
The aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried with MgSO4 and concentrated in vacuo. Compound 7 was obtained (3.08 g, 87%) and subjected to esterification with pentafluorophenyl trifluoroacetate (PFP-TFA) without further purification.  To a cooled solution of compound 9 (89.7 mg, 29.0 μmol) in DCM (8 mL) was added trifluoro acetic acid (2 mL). After stirring for 3 h at room temperature, the reaction mixture was concentrated in vacuo.
Diethyl ether was added to the residue, and the white precipitate was isolated as the deprotected compound (72.4 mg, 87%). Compound 10 was subjected to amidation with compound 4a without further purification.