Mark E. Eccleston

Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells

Frequency domain fluorescence lifetime imaging microscopy (FLIM) has been used in combination with laser scanning confocal microscopy to study the cellular uptake behavior of the antitumor drug doxorubicin (DOX) and micellar-encapsulated DOX (PLyAd-DOX). The endocytosis uptake process of PLyAd-DOX was monitored over 72 hours using confocal microscopy, with a maximum fluorescence recorded at incubation periods around 24 hours. The micellar structure was not found to release the encapsulated DOX during the time course of imaging. FLIM revealed single lifetime distributions of PLyAd-DOX during accumulation in the cytoplasm. The free DOX in contrast was observed both in the cytoplasm and the nuclear domain of the cell, showing bimodal lifetime distributions. There was a marked dependence of the measured free-DOX lifetime on concentration within the cell, in contrast to reference experiments in aqueous solution, where no such dependence was found. The results suggest the formation of macromolecular structures inside the living cells.

The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pH-responsive pseudo-peptides

pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly(l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL(-1), the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly(l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications.

pH-responsive pseudo-peptides for cell membrane disruption

We describe pseudo-peptides obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various hydrophobic dicarboxylic acid moieties. In aqueous solution, when the carboxylic acid groups are charged, the polymers dissolve. When they are fully neutralised the hydrophobic moieties cause the polymer to precipitate. The pH range over which reversible precipitation occurs can be adjusted by changing the intramolecular hydrophilic/hydrophobic balance, by using a carboxylic acid moiety with a different pK(a) value or by changing the apparent pK(a) value of the polymer through chemical modifications of the backbone. These bio-degradable materials are well tolerated by a range of mammalian cell lines at physiological pH but display an ability to associate with the outer membranes of these cells, which they rupture to varying degrees at pH 5.5. Relative to the degree of lysis displayed by poly(L-lysine iso-phthalamide), lysis was reduced by partial esterification and increased by replacing the aromatic iso-phthaloyl moiety with a long chain aliphatic dodecyl moiety. Similar behaviour was observed for the pH-dependent rupture of human erythrocytes, where poly(L-lysine dodecanamide) displayed enhanced cell lysis at pH values <7.0 relative to poly(L-lysine iso-phthalamide).

Aqueous solution behaviour and membrane disruptive activity of pH-responsive PEGylated pseudo-peptides and their intracellular distribution

The effect of PEGylation on the aqueous solution properties and cell membrane disruptive activity of a pH-responsive pseudo-peptide, poly(l-lysine iso-phthalamide), has been investigated by dynamic light scattering, haemolysis and lactate dehydrogenase (LDH) assays. Intracellular trafficking of the polymers has been examined using confocal and fluorescence microscopy. With increasing degree of PEGylation, the modified polymers can form stabilised compact structures with reduced mean hydrodynamic diameters. Poly(l-lysine iso-phthalamide) with a low degree of PEGylation (17.4 wt%) retained pH-dependent solution behaviour and showed enhanced kinetic membrane disruptive activity compared to the parent polymer. It facilitated trafficking of endocytosed materials into the cytoplasm of HeLa cells. At levels of PEGylation in excess of 25.6 wt%, the modified polymers displayed a single particle size distribution unresponsive to pH, as well as a decrease in cell membrane lytic ability. The mechanism involved in membrane destabilisation was also investigated, and the potential applications of these modified polymers in drug delivery were discussed.

Synthesis and pH-responsive properties of pseudo-peptides containing hydrophobic amino acid grafts

Pseudo-peptidic polymers have been synthesised by grafting L-valine (PV), L-leucine (PL) and L-phenylalanine (PP) onto the pendant carboxylic acid moieties of a pH-responsive polyamide, poly(L-lysine isophthalamide). The pH-responsive aqueous solution properties of PV-75, PL-75 and PP-75 with a stoichiometric degree of substitution of 75 mol% have been compared with those of the parent poly(L-lysine isophthalamide) using UV-visible and fluorescence spectroscopy. At low concentrations (≤0.1 mg mL−1), the grafted polymers displayed pH-dependent conformation. The pH at the onset of hydrophobic association (pHh) and the pH range over which association occurred varied significantly between the different amino acid grafts. The pHh values of PV-75, PL-75 and PP-75 at 0.025 mg mL−1 were 6.2, 7.0 and 7.2, respectively. Increasing concentration enhanced intermolecular aggregation. A bis-functional Cy5 derivative, incorporated within the backbones of poly(L-lysine isophthalamide) (polyCy5) and PP-75 (PDP-75), was demonstrated to act as a fluorescence reporter on the state of polymer conformation and aggregation. The intracellular trafficking of PDP-75, examined by confocal microscopy, indicates potential applications of the grafted polymers in drug delivery and medical imaging.

Effect of L-Leucine Graft Content on Aqueous Solution Behavior and Membrane-Lytic Activity of a pH-Responsive Pseudopeptide

A series of pH-responsive polymers have been synthesized by grafting L-leucine onto the pendant carboxylic acid groups of the linear pseudopeptide, poly(L-lysine iso-phthalamide). The effect of the degree of grafting on aqueous solution properties, cell membrane-disruptive activity, and in vitro cytotoxicity was examined by UV-visible and fluorescence spectroscopy, hemolysis, alamar blue staining, and propidium iodide fluorescence assays. Modification of poly(L-lysine iso-phthalamide) with < or =23.6 mol % L-leucine caused a marginal effect on the pH-mediated hydrophobic association and hemolytic activity. Increasing the degree of grafting from 31.9 to 61.2 mol % resulted in polymers with progressively enhanced hydrophobic association and cell membrane disruption, thus confirming that the pH responsiveness and the extent of hydrophobic association and membrane disruption of the polymers can be modulated by varying the degree of grafting with hydrophobic amino acids. The pH responses were demonstrated to be concentration-dependent. At certain degrees of leucine grafting, the polymers were nonmembrane-lytic at physiological pH but mediated considerable membrane lysis at endosomal pH values (5.0-6.8), a feature critical for potential drug delivery applications.

The effect of high intensity focussed ultrasound (HIFU) on pH responsive PEGylated micelles

The effect of HIFU on pH responsive PEGylated micelles was investigated. Micelles can be used as drug carrier vehicles reducing unwanted drug toxicity. HIFU is able to release drugs from the circulating micelles, as well as improving intracellular uptake of both micelle‐encapsulated and free drugs non‐invasively. Large molecules generally enter cells by endocytosis. Endosomes gradually become acidic and fuse with enzyme containing lysosomes degrading the endosomal contents and preventing them from reaching their intracellular target. Using pH responsive polymers enables endosomes to be distrupted, releasing their contents into the cytoplasm before degradation occurs. Addition of poly(ethylene glycol), referred to as PEGylation, prolongs circulatory half‐life and reduces degradation within the bloodstream. HIFU did enable release of encapsulated molecules from the modified micelles, and the micelles were taken up by H69 human carcinoma cells in vitro. Further work will investigate optimization of the micel...