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

Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK.
Journal of Controlled Release (Impact Factor: 7.71). 12/2005; 108(1):63-72. DOI: 10.1016/j.jconrel.2005.07.011
Source: PubMed


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.

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Available from: Rongjun Chen, Dec 08, 2014
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    • "The pH-mediated aqueous solution behaviour of the polymers with different degrees of PEGylation has been investigated by conventional fluorescence characterisation at concentrations up to 1.0 mg/ml using pyrene as a probe [23], and is further substantiated in this paper at elevated polymer concentrations by dynamic light scattering. pH-dependent cell membrane lysis of the polymers with PEG grafting levels below 23.4 wt% has been demonstrated using a haemolysis model and their in vitro cytotoxicity has been evaluated using MTT and LDH assays [24]. Here their kinetic properties and mechanisms of membrane disruption are presented, together with the effect of levels of PEGylation above 25.6 wt% on the membrane lytic activity. "
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    ABSTRACT: 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.
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    ABSTRACT: The pH mediated intra-molecular association and inter-molecular aggregation of a range of amphiphilic poly(l-lysine iso-phthalamide) polymers have been investigated in aqueous solution over a range of pH values and concentrations. The desired functionality of these novel bioresponsive amphiphilic polymers was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Incorporation of low levels of bis-functional Cy3 (poly-Cy3) and/or Cy5 dye (poly-Cy3/5 or poly-Cy5) co-monomers in the responsive polymer backbone allowed detailed probing of the pH mediated hydrophobic association using a combination of optical spectroscopic techniques. Both steady-state fluorescence spectroscopy and fluorescence lifetime measurements of poly-Cy3 revealed a conformational transition at pH 4.5. Thus, below a critical pH the polymer collapsed into a compact globular structure (hypercoil) bringing the fluorophore molecules into close proximity with one another. This resulted in a dramatic reduction in fluorescence intensity and fluorescent lifetime in the single fluorophore systems (poly-Cy3) accompanied by a red shift in the maximum emission wavelength. Observed redshifts in the emission maxima and enhancements of fluorescent lifetimes with increasing polymer concentration suggested the formation of polymer aggregates. Fluorescence resonance energy transfer (FRET) was measured in mixtures of single fluorophore containing poly-Cy3 (donor) and poly-Cy5 (acceptor) and dual fluorophore containing poly-Cy3 (donor)/Cy5 (acceptor) in an effort to distinguish between intra-molecular versus inter-molecular association. The relevance of the results with respect to potential in vivo applications (drug delivery and biodiagnostics) is discussed.
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