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.

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.

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.

Modulation of the pH-responsive properties of poly(L-lysine iso-phthalamide) grafted with a poly(ethylene glycol) analogue.

A pH responsive pseudopeptide, poly(L-lysine iso-phthalamide), has been modified with a hydrophilic poly(ethylene glycol) analogue, Jeffamine M-1000 and the effect of grafting ratio on the pH responsive behaviour of the grafted polymers in aqueous solution investigated using fluorescence and 1H NMR spectroscopy. It was demonstrated that at below 35.1 wt% grafting, the modified polymers retained the pH-driven conformational transition of the parent polymer from an expanded structure at high degrees of ionisation to a compact hydrophobically stabilised structure at low degrees of ionisation. The onset of pH response and the pH range over which the conformational transition occurred varied significantly with degree of grafting. At Jeffamine M-1000 ratios in excess of 48.0 wt%, the graft polymer existed in a micellular form over the whole pH studied. Potential applications in drug delivery of both the linear and micellular forms are discussed.

Modulation of cell membrane disruption by pH-responsive pseudo-peptides through grafting with hydrophilic side chains.

The effect of grafting an amphiphilic pseudo-peptide, poly (L-lysine iso-phthalamide), with poly (ethylene glycol) or a hydrophilic poly (ethylene glycol) analogue, Jeffamine M-1000, on the pH-dependent erythrolytic activity and in vitro cytotoxicity have been studied together with the concentration-dependent haemolysis of the polymers with different degrees of grafting. PEGylated polymers showed pH-dependent membrane-disruptive ability similar to the parent poly (L-lysine iso-phthalamide). The polymers showed a better ability to haemolyse the erythrocyte membrane at mildly acidic pHs with increasing degree of PEGylation (up to 17.0 wt.%). Further increasing the degree of PEGylation resulted in a decrease in haemolytic ability. Grafting poly (L-lysine iso-phthalamide) with the lower molecular weight Jeffamine M-1000 had little effect on the haemolytic ability. Finally, the in vitro cytotoxicity of the grafted polymers was assessed by MTT assay, LDH assay and viable cell counts. At pH 7.4, these polymers were well tolerated by a range of mammalian cell lines and grafting reduced the cytotoxicity of polymers. However, at pH 5.5, relative to poly (L-lysine iso-phthalamide), the grafted polymers displayed a better ability to rupture the outer membranes of these 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.

Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process.

A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N''-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.