M2 macrophage specific gene silencing in the liver using third generation mannose coated, siRNA-loaded nanohydrogel particles

Background and aims:

Efficient in vivo transport of active siRNA to specific liver cells remains challenging. First and second generation cationic nanohydrogel particles (NHP) are excellent oligonucleotide carriers [Kaps L et al, Adv. Healthcare Mat. 2015; J. Contr. Rel. 2016]. We designed a third generation of NHP where mannose residues are covalently linked to the surface (Man-NHP), enabling targeting of CD206 positive M2 macrophages, which are implicated in HCC and liver fibrosis progression.

Method and Results:

Man-NHP composed of block copolymers (pentafluorophenyl methacrylate and tri-(ethylene glycol) methyl ether methacrylate), with diameters around 40nm and loaded with control siRNA (scsiRNA) up to 400 nM did not show cytotoxicity for murine 3T3 fibroblasts, Raw macrophages, SVEC endothelial cells or HepG2 human hepatoma cells. Man-NHP labelled with FITC showed a preferential binding to in vitro polarized CD206 high M2 compared to CD206 low non-polarized M0 or M1-polarized bone-marrow derived macrophages (BMDM), as determined by FACS analysis. Man-NHP were shielded from unspecific interactions compared to non-mannose coated NHP, which exhibited high binding to macrophages regardless of their phenotype. Man-NHP loaded with siRNA to CSFR-1, an M2 macrophage specific target, yielded a robust knockdown in M2 polarized BMDM after 48h of incubation.

After intravenous injection in mice with CCl₄-induced liver fibrosis, near infrared (NIR, CW800-dye) labeled Man-NHP loaded with Cy5-labelled scsiRNA distributed preferentially to the liver (> 80%), as determined by in vivo and ex vivo NIR imaging. Ex vivo colocalization studies of liver single cell suspension and confocal fluorescence microscopy of cryo liver sections obtained from Man-NHP-NIR/Cy5-scsiRNA treated fibrotic mice revealed that Man-NHP-NIR/Cy5-scsiRNA complexes were preferentially taken up in CD206+ M2 macrophages in contrast to other (non)-parenchymal liver cells.

Conclusion:

Third generation NHP were derivatized with surface mannose residues for effective M2 macrophage targeting via the mannose receptor CD206 in vitro and in vivo. Man-NHP are a promising tool to specifically target therapeutic siRNA to fibrosis and HCC promoting M2 macrophages.