Synthesis and biological characterisation of HPMA copolymer-mannose conjugates designed for intracellular delivery of anti-leishmanial compounds to macrophages

摘要

is the second largest parasitic killer in the world (after malaria) with 59,000 deaths annually. The parasite resides in macrophages, within a compartment called the parasitophorous vacuole, but many anti-leishmanial drugs are toxic and poorly effective due to the low concentrations attained in this compartment. Therefore, the aim of this study was to establish the basis for design of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates that would target the parasitophorous vacuole, with potential to deliver improved treatments for leishmaniasis. Mannose was selected as a ligand for macrophage targeting and amphotericin B as a model drug. First, in vitro macrophage models (RAW 264.7 and THP-1 cells) were established that displayed mannose receptor expression and uptake of mannosylated macromolecular ligands. A library of HPMA copolymer-mannose conjugates containing Oregon Green (OG) were then synthesised to define the optimal mannose loading needed for targeting. To define the fate of internalised conjugates a density gradient subcellular fractionation method was developed for THP-1 cells. Finally HPMA copolymer-amphotericin B mannose conjugates were synthesised and their cell uptake, intracellular fate and preliminary haemolytic and cytoxicity profiles established. HPMA copolymer-OG-Man conjugates with a mannose loading 4 mol % showed significantly higher uptake by THP-1 cells. The subcellular fractionation and confocal fluorescence microscopy confirmed time-dependent trafficking of such conjugates to late endosomes/lysosomes. Release of free OG (as a drug model) from the biodegradable polymer-OG linker was seen. HPMA copolymer-amphotericin B-mannose conjugates were taken up 5-fold faster than the control and accumulated in the late endosome/lysosomal compartment. HPMA copolymer-amphotericin B-mannose conjugates displayed reduced haemolysis and cytotoxicity against the THP-1 cells. This study has established methods to investigate the intracellular trafficking of polymer-drug conjugates, and has demonstrated the potential of mannose-targeted HPMA copolymer conjugates for effective targeting of anti-leishmanial drugs to macrophages.