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

Visceral leishmaniasis 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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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

Visceral leishmaniasis 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

Impact of polymer tacticity on the physico-chemical behaviour of polymers proposed as therapeutics

Although water-soluble polymers are finding increasing use as polymer therapeutics, there has been little consideration of the effect of polymer stereochemistry on their physico-chemical and biological properties. The aim of this study was to investigate these properties using polymethacrylic acids (PMAs) of similar molecular weights with a difference in syndiotacticity of about 20% of rr triad content. Experiments to characterize the solution behaviour were conducted at pHs encountered during the transport, endocytic uptake and intracellular trafficking (7.4–3.0). These showed that with increasing rr triads, the polymer become less hydrophobic, a stronger acid, displayed a locally ordered solution conformation at pH 5.5, and interacted more strongly with dodecyl trimethylammonium bromide (DTAB) micelles. Preliminary cytotoxicity experiments using B16F10 melanoma cells showed lower toxicity in the concentration range of 1–100 μg/mL with increased rr triads. These observations indicate that the higher content of rr triads could drive a chain organization that minimize the influence of negative charges and so underline the importance of further, systematic studies to investigate the effect of tacticity on the behaviour of polymers in respect of their pharmacokinetics, toxicity and efficacy

The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery

Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy

Heat shock protein-based therapy as a potential candidate for treating the sphingolipidoses.

Lysosomal storage diseases (LSDs) often manifest with severe systemic and central nervous system (CNS) symptoms. The existing treatment options are limited and have no or only modest efficacy against neurological manifestations of disease. We demonstrate that recombinant human heat shock protein 70 (HSP70) improves the binding of several sphingolipid-degrading enzymes to their essential cofactor bis(monoacyl)glycerophosphate in vitro. HSP70 treatment reversed lysosomal pathology in primary fibroblasts from 14 patients with eight different LSDs. HSP70 penetrated effectively into murine tissues including the CNS and inhibited glycosphingolipid accumulation in murine models of Fabry disease (Gla(-/-)), Sandhoff disease (Hexb(-/-)), and Niemann-Pick disease type C (Npc1(-/-)) and attenuated a wide spectrum of disease-associated neurological symptoms in Hexb(-/-) and Npc1(-/-) mice. Oral administration of arimoclomol, a small-molecule coinducer of HSPs that is currently in clinical trials for Niemann-Pick disease type C (NPC), recapitulated the effects of recombinant human HSP70, suggesting that heat shock protein-based therapies merit clinical evaluation for treating LSDs

GM1 ganglioside-independent intoxication by Cholera toxin

<div><p>Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (Le^X) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the Le^X glycan <i>in vitro</i> when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to Le^X. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.</p></div

CT induced ion secretion can be inhibited by pretreating the tissue with AAL and PNA.

<p>Human jejunal mucosae were pre-incubated with or without AAL or PNA at the indicated concentrations, mounted in an Ussing chamber and exposed to CT. <b>(A)</b> Dot plot showing percent difference in I_ep to control tissue for jejunal mucosae over time. Each dot represents a mean of 4–7 donors (each treatment for each donor was tested in duplicates) with SEM error bars. Significance was calculated using a two-way-ANOVA with Tukey correction (compared to the CT). * represent CT to CT+AAL comparison and † represent CT to CT+PNA comparison (**** = p<0.0001 and ** = p<0.01). <b>(B)</b> Dot plot showing percent of start I_ep for jejunal mucosae at 180 min. AAL, PNA-treated or untreated tissue were treated with forskolin (or forskolin analog NKH477) bilaterally at 200 min. CT treated tissue were treated with bumetanide at 200 min. Each dot represents a mean of 2–3 donors (each treatment for each donor was tested in duplicates). Error bars show SEM.</p

Le^X blocks binding of CTB to human granulocytes but not murine leukocytes.

<p><b>(A-B and D-E)</b> Histograms from flow cytometry analyses of CTB-, G33D- and OVA-binding to granulocytes in human peripheral blood. CTB was pretreated or not with titrated amounts of <b>(A)</b> Le^X-os, <b>(B)</b> GM1-os, <b>(D)</b> os-HSA (not titrated) and <b>(E)</b> Le^X-os and GM1-os. Graphs show the percent of gMFI of CTB binding to the cells where 100% represents CTB staining with no blocking oligosaccharide. <b>(C)</b> Histograms from flow cytometry analyses of CTB-, G33D- and OVA-binding to CD3+ T cells gated from murine splenocytes. CTB was pretreated or not with the indicated os or os-HSA. <b>(A-B)</b> n = 3, <b>(C)</b> One representative out of three independent experiments, <b>(D)</b> n = 8, <b>(E)</b> n = 4–9. Error bars show SD. Each dot represents one donor and significance was calculated using a one-way-ANOVA with Tukey correction compared to CTB without block if not indicated otherwise with bars (**** = p<0,0001, *** = p<0,005, ** = p<0,01).</p