Nanoparticular bisphosphonate to selectively target and repolarize liver macrophages for anti-fibrotic treatment

Background and Aims:

Bisphosphonates, e.g. Alendronate (AL), inhibit osteoclasts, a specialized population of macrophages. AL showed an antitumor effect by macrophages (re-)polarization in rat hepatocellular carcinoma [Rogers T et. al., J. Transl. Med. 2011]. However, after intravenous application, bisphosphonates are rapidly excreted by the kidneys. Therefore, we aimed to develop nontoxic nanogel particles (NHP) with covalently linked Alendronate (AL-NHP) that primarily home to the liver.

Method and Results:

NHP with diameters below 100nm were synthesized by controlled-radical polymerization. AL was covalently bound to NHP to generate AL-NHP. In primary murine macrophages, AL-NHP induced a 50% reduction of cell viability at 1 mM AL loading, equal to free AL, while NHP showed no effect. Low concentrations of AL-NHP (˜30µM AL) repolarized M2 polarized macrophages towards anti-tumorous M1 macrophages, increasing their expression of TNF-a and Interferon-g and decreasing CD206 (mannose receptor), as determined on the transcript and protein level via qPCR and FACS analysis. AL-NHP repolarized macrophages more efficiently as equal doses of free AL, while NHP alone had no effect. After intravenous injection in healthy Balb/c mice, more than 80% of near-infrared fluorescence labeled CS800-AL-NHP rapidly accumulated in the liver, whereas CS800 labeled AL was readily cleared via the kidneys. On the cellular level, CS800-AL-NHP were effectively taken up by liver macrophages (> 90%), endothelial cells (80%), hepatocytes (> 90%), portal myofibroblasts (> 90%) and less into T-cells and NKT cells (< 10%), as determined by FACS. In CCl4 fibrotic mice, AL-NHP (˜AL 4 mg/kg or 2 mg/kg) achieved a significant and dose-dependent reduction of collagen (˜60% compared to CCl4 control mice) in the livers as determined by hydroxyproline and morphometrical collagen quantification, while free AL (˜4 mg/kg) was less effective.

Conclusion:

We have designed AL-NHP as biocompatible carriers for the bisphosphonate AL. Nanocarrier-coupled Alendronate was almost exclusively sequestered by the liver and showed promising repolarizing effects on M2-type primary macrophages, shifting their cytokine levels towards a putative anti-fibrotic and anti-tumor M1 phenotype. CS800-AL-NHP were efficiently taken up by liver macrophages, endothelial, fibroblastic and parenchymal cells, but less into T and NKT cells. AL-NHP exhibited in vivo a significant antifibrotic effect in CCl4 liver fibrotic mice.