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.

Figure 1.

Schematic illustration of the delivery of siRNA and the synthetic strategy for CMM–siRNA. (A) The proposed schematic illustration of receptor-mediated targeted delivery of CMM–siRNA. (B) Strategy for obtaining high-affinity chemically modified mannose–siRNA conjugate (CMM–siRNA). The two approaches used were development of high-affinity mannose ligands and optimization of multivalent branched linkers.

Figure 2.

Synthetic scheme of chemically modified mannose ligands (CMMs) and their binding properties. (A) Synthesis of CMMs using a three-step reaction. (B) SPR results reflecting the inhibition of CD206 binding to the di-mannose surface by modified mannose compounds. Dose-response curves of percent activity were fit using a four-parameter logistic equation with XLfit software (ID Business Solutions, Guilford, UK), and IC₅₀ values were calculated.

Figure 3.

Preparation scheme and physicochemical characterization of CMM–siRNAs. (A) CMM–siRNAs were obtained using a three-step reaction. (B) SPR sensorgrams of the binding of CMM4–siRNA to CD206. (C) Binding affinities of modified mannose–siRNA to C-type lectins. (D) Native PAGE analysis of siRNA and CMM4–siRNAs in 90% mouse serum at 37°C.

Figure 4.

In vitro cellular uptake and gene silencing using CMM–siRNAs. (A) The cellular uptake in human monocyte-derived M1 macrophages. A488-labeled CMM1-siHPRT1-a, CMM4–siHPRT1-a, siHPRT1, and C3N-siHPRT1 were added to each cell type for 6 h. Cellular uptake was analyzed by flow cytometry. (B) Gene silencing of HPRT1 in M1 macrophages. CMM1-siHPRT1-a, CMM4–siHPRT1-a, siHPRT1, C3N-siHPRT1, CMM4-siB2M(h), and CMM4-siCD45 were added to M1 macrophages for 4 days. HPRT1 mRNA expression was determined using quantitative PCR and normalized to that of ACTB mRNA. (C) Gene silencing of B2M in M1 macrophages. CMM1-siB2M(h), CMM4-siB2M(h), siB2M(h), C3N-siB2M(h), CMM4–siHPRT1-a and CMM4-siCD45 were added to M1 macrophages for 4 days. B2M expression was determined using quantitative PCR and normalized to that of GAPDH mRNA.

Figure 5.

Protein downregulation in CD206-positive M1 macrophages. B2M protein downregulation in M1 macrophages, as analyzed by FCM. CMM1-siB2M(h), CMM4-siB2M(h), siB2M(h), C3N-siB2M(h), CMM4–siHPRT1-a and CMM4-siCD45 were added to M1 macrophages for 4 days and analyzed 3 days after medium exchange. The bar chart shows the means ± standard deviations of the geometric mean fluorescence intensity of triplicate experiments.

Figure 6.

In vitro cellular uptake and gene silencing in CD206-positive M2a macrophages, immature DCs, and mature DCs using CMM4–siHPRT1. (A) Cellular uptake in human monocyte-derived M2a macrophages and immature and mature DCs. A488-labeled CMM4–siHPRT1-a and siHPRT1 were added to cells for 6 h. Cellular uptake was analyzed by flow cytometry. (B) Gene silencing activities in M2a macrophages, immature DCs, and mature DCs. CMM4–siHPRT1-a and siHPRT1 were added to cells for 4 days. HPRT1 mRNA expression was determined using quantitative PCR and normalized to that of ACTB mRNA. The data represent means ± standard deviations of triplicate experiments.

Figure 7.

In vivo biodistribution and targeting ability. (A) Blood circulation properties of CMM4–siHPRT1-a, GalNAc4–siHPRT1-a, and siHPRT1 after a single subcutaneous injection (1 mg/kg) into C57BL6/J mice. (B) Biodistributions of siHPRT1, GalNAc4–siHPRT1-a, and CMM4–siHPRT1-a conjugates 24 h after a single subcutaneous injection. After dissociation of the cells from each organ, the amount of siRNA was quantified by stem–loop PCR. The values represent means ± standard deviations of triplicate experiments. (C) In situ hybridization of CMM4–siHPRT1-a in the spleen and liver 24 h after a single subcutaneous injection. Blue signal indicates the localization of modified mannose–siRNA. The boxed areas in the upper panels are magnified in the lower panels. (D) Cell-targeting profiles of A647-labeled CMM4–siHPRT1-a. (E) Cell-targeting profiles of A647-labeled siHPRT1. The population of compounds was determined by FCM using antibodies targeting F4/80 and CD206 markers.

Figure 8.

Gene silencing in CD206-positive macrophages in the spleen, liver, and kidneys of mice treated with CMM4–siHPRT1-a in vivo. (A) Schematic illustration of gene silencing. The gene silencing activities of oligonucleotides on splenic macrophages (B) and hepatic F4/80-positive macrophages (C) and kidney F4/80-positive macrophages (D). Hprt1 mRNA expression was quantified by RT-qPCR and normalized to that of ACTB mRNA.

Figure 9.

Dose-dependency and persistence of gene silencing. (A) Dose-dependent gene silencing of splenic F4/80-positive macrophages. (B) Continuous gene silencing of the target mRNA (Hprt1) with CMM4–siHPRT1-a. CMM4–siHPRT1-a was administered to mice subcutaneously (3 mg/kg, single dose). After 4, 7, and 10 days, the target mRNA in splenic F4/80-positive cells was evaluated using RT-qPCR.

Figure 10.

Effects of the conjugates on gene silencing. Gene silencing in splenic F4/80-positive macrophages was analyzed by RT-qPCR. (A) Four days after subcutaneous administration of CMM4–siRNAs (CMM4-HPRT1-a, CMM4–siHPRT1-b, CMM4–siHPRT1-c) and (B) 10 days after subcutaneous administration of CMM4-siB2M(h) and C3N-siB2M(h).

Figure 11.

Protein downregulation in splenic CD206/F480-positive macrophages. The amount of target protein (B2M protein) in individual animals was quantified by FCM. Values represent means ± standard deviations of the geometric mean fluorescence intensity of triplicate experiments.

Figure 12.

Downregulation in protein levels in mouse kidneys treated with CMM4-siB2M(m) in vivo. (A) The amount of target protein (B2M protein) in individual animals was quantified by FCM. (B) Analysis of downregulation of the target protein (B2M protein) in CD206-high/low populations.