Macrophage-specific RNA interference targeting via "click", mannosylated polymeric micelles

Abstract

Macrophages represent an important therapeutic target, because their activity has been implicated in the progression of debilitating diseases such as cancer and atherosclerosis. In this work, we designed and characterized pH-responsive polymeric micelles that were mannosylated using "click" chemistry to achieve CD206 (mannose receptor)-targeted siRNA delivery. CD206 is primarily expressed on macrophages and dendritic cells and upregulated in tumor-associated macrophages, a potentially useful target for cancer therapy. The mannosylated nanoparticles improved the delivery of siRNA into primary macrophages by 4-fold relative to the delivery of a nontargeted version of the same carrier (p < 0.01). Further, treatment for 24 h with the mannose-targeted siRNA carriers achieved 87 ± 10% knockdown of a model gene in primary macrophages, a cell type that is typically difficult to transfect. Finally, these nanoparticles were also avidly recognized and internalized by human macrophages and facilitated the delivery of 13-fold more siRNA into these cells than into model breast cancer cell lines. We anticipate that these mannose receptor-targeted, endosomolytic siRNA delivery nanoparticles will become an enabling technology for targeting macrophage activity in various diseases, especially those in which CD206 is upregulated in macrophages present within the pathologic site. This work also establishes a generalizable platform that could be applied for "click" functionalization with other targeting ligands to direct siRNA delivery.

FIGURE 1. Smart Polymeric Nanoparticles for Mannose Receptor-Targed Cytosolic Delivery of siRNA

Schematic representation of the triblock copolymers and formulation into multi-functional nanoscale siRNA delivery vehicles. The blocks include (red) a pH-responsive block that is capable of disrupting endosomes at low pH, (blue) a cationic block for condensation of nucleic acids, and (green) an azide-displaying block for conjugation of targeting motifs (purple) via ‘click’ chemistry.

FIGURE 2. Morphologic and Functional Characterization of Micelles composed of Diblock Copolymers and Mannosylated Triblock Copolymers

(A–B) Uranyl acetate-counterstained transmission electron micrographs of (A) micelles of diblock copolymers (See 2 in Scheme 1), which had an average diameter of 13.0 ± 6.1 nm (n = 367). (B) ManNPs had an average diameter of 9.7 ± 6.2 nm (n = 415). Scale bars = 50 nm. (C) Gel retardation assay of siRNA-loaded ManNPs confirmed increased complexation of siRNA with increasing NH⁺:PO⁻ ratios. Free FAM-labeled siRNA (S) appears as a control. The cationic ManNPs migrate in the opposite direction from the siRNA (gray arrowhead), consistent with their opposing charges. (D) Protection of siRNA from degradation by RNases. Micelle/siRNA complexes were incubated with RNase cocktails. RNase-mediated degradation of siRNA was characterized by a hyperchromic effect at 260 nm, and within 10–15 min, all siRNA in each sample has completely degraded as signified by asymptotic behavior of the results. siRNA and micelle/siRNA complexes which were left in buffer without RNases did not exhibit this hyperchromic effect. (E–F) Dynamic light scattering was used to analyze the (E) hydrodynamic diameters and (F) ζ-potentials of the polymeric micelles following complexation with siRNA. (G) Both polymers exhibit pH- and concentration-dependent hemolysis, with minimal disruption of erythrocyte phospholipid membranes at physiologic pH, but increased disruption at pH ranges mimicking endosomal pH (pH < 6.5). Error bars represent standard deviation of 4 replicates.

FIGURE 3. ManNPs are Cytocompatible and Selectively Enhance siRNA Delivery into Immortalized Human Macrophages

(A) Cytotoxicity assay of immortalized THP-1 macrophages, treated with ManNPs complexed with siRNA at various N:P ratios. Error bars represent standard deviation from 3 experiments (*,** p < 0.01; n = 3). (B) Representative flow cytometry histograms of THP-1, MDA-MB-231, or MDA-MB-468 cells at 0 (red), 1 (blue), or 4 h (orange) after transfection with FAM-siRNA loaded into diblock micelles (left column) or ManNPs (right column). (C) Mean fluorescence intensities of all of the groups in (B) have been quantified and shown. Error bars represent standard deviation of n = 3 experiments. ManNPs enhanced siRNA delivery to macrophages up to 26-fold over two model breast cancer cell lines, and 3-fold in macrophages relative to untargeted diblock carriers, as measured via flow cytometry. (*p < 0.01 vs. all other treatment groups at 4 h timepoint).

FIGURE 4. CD206-dependent siRNA Delivery to Primary Macrophages using ManNPs

(A) Following 4h of transfection with FAM-siRNA (green; free or complexed into nanoparticles), BMDMs were fixed, nuclei stained with DAPI (blue), and imaged via confocal microscopy. (Scale bars = 50 µm for Serum-free; 20 µm for + Serum). Mannosylation of the polymeric vehicles enhanced their internalization by BMDMs. This could be competitively inhibited through co-administration of the ManNPs with D-mannose. A similar trend was observed when transfections were performed under serum conditions. FAM brightness and contrast were enhanced equally for all samples within the ‘+ Serum’ condition, but unaltered for the ‘Serum-Free’ condition. For the ‘Serum-free condition’, brightness & contrast were also enhanced in the DAPI channel to account for small differences in staining between samples. (B) Flow cytometry quantification of FAM-siRNA delivery into BMDMs via ManNPs (blue) relative to untargeted nanoparticles (orange) or free siRNA without vehicle (black) within 4 h of administration under serum-free conditions. Quantification of mean fluorescence intensity in each treatment group is in Supplementary Figure S6.

FIGURE 5. Kinetics of ManNP-Mediated siRNA Delivery into Primary Macrophages

BMDMs were transfected with FAM-siRNA (green; complexed into ManNPs) for 1–4 h prior to being fixed, stained with DAPI (blue), and imaged via confocal microscopy. (Scale bars = 50 µm). As a comparison, BMDMs treated with non-fluorescent, scrambled siRNA (also complexed into ManNPs) are also shown. Brightness & contrast were enhanced in the DAPI channel to account for small differences in staining between samples. All settings were identical for FAM imaging. Punctate green signal is observed within 1–2 h of administration, suggesting internalization of siRNA into vesicles. At 4h, more green fluorescence has accumulated and the staining pattern is more diffuse, consistent with endosomal escape of the siRNA into the cytosol.

FIGURE 6. ManNPs Mediate Knockdown of PPIB Expression in BMDMs

siRNA-mediated knockdown of PPIB expression using different transfection vehicles relative to controls. qRT-PCR confirmed ManNPs carrying anti-PPIB siRNA (ManNPs/PPIB) mediated 87 ± 10% decrease in target gene expression following 24 h of treatment, relative to non-transfected (NT) cells. Data was normalized to expression of the housekeeping gene GAPDH as an internal control. Error bars represent standard deviation of 3 independent experiments (*p < 0.05 by one-way ANOVA; **Not statistically significant). Some BMDMs were also transfected with ManNPs carrying scrambled siRNA (ManNPs/SCR) as an additional negative control.

Scheme 1. RAFT Polymerizations

Synthetic scheme for RAFT polymerization of triblock copolymers composed of blocks of 2-azidoethyl methacrylate (AzEMA), 2-dimethylaminoethyl methacrylate (DMAEMA), and the DMAEMA-co-BMA-co-PAA terpolymer (butyl methacrylate = BMA; 2-propylacrylic acid = PAA).