Modulation of cell membrane disruption by pH-responsive pseudo-peptides through grafting with hydrophilic side chains.
ABSTRACT 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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ABSTRACT: PURPOSE: In breast cancer, a significant decrease in riboflavin (RF) serum levels and increase in RF carrier protein occurs, indicating a potential role of RF in disease progression. To evaluate RF's ability to serve as a targeting agent, mitomycin C (MMC)-conjugated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and targeted to the RF internalization pathway in human breast cancer cells. METHODS: Competitive uptake studies were used to determine specificity of RF-targeted conjugates, and an MTT assay established the IC50 for the conjugates. Endocytic mechanisms were investigated by confocal microscopy. RESULTS: Studies revealed a high-affinity endocytic mechanism for RF-specific internalization of fluorescently-labeled conjugates in both MCF-7 and SKBR-3 cells, whereas folic acid-mediated endocytosis showed high specificity only in SKBR-3 cells. MMC internalization was significantly higher following nontargeted and RF-targeted MMC-conjugate administration compared to that of free MMC. Cytotoxic analysis illustrated potent IC50 values for RF-targeted MMC conjugates similar to free MMC. Maximum nuclear accumulation of MMC resulted from lysosomal release from RF-targeted and nontargeted MMC-conjugates following 6 h incubations, unlike that of free MMC seen within 10 min. CONCLUSION: Targeting polymer-MMC conjugates to the RF internalization pathway in breast cancer cells enabled an increase in MMC uptake and nuclear localization, resulting in potent cytotoxic activity.Pharmaceutical Research 04/2013; · 4.74 Impact Factor
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ABSTRACT: Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications.Acta biomaterialia 03/2011; 7(7):2846-56. · 5.09 Impact Factor
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ABSTRACT: The efficient cytoplasmic delivery of therapeutic agents is especially important for the induction of a maximal therapeutic effect. Previously, we reported that 200-nm-sized nanoparticles composed of hydrophobically-modified poly(gamma-glutamic acid) (gamma-PGA) showed great potential as protein carriers. Moreover, protein-encapsulated hydrophobic gamma-PGA (gamma-hPGA) nanoparticles efficiently delivered loaded proteins from the endosomes to the cytoplasm in dendritic cells, but the mechanism of the nanoparticle translocation into the cytoplasm remains to be elucidated. In this study, we examined how polymer composition, hydrophobic modification, size, conformation and surface properties of the amphiphilic nanoparticles are related to functional membrane-disruptive activities. To evaluate their potential applications as membrane-disruptive nanoparticles, the nanoparticles were characterized with respect to their hemolytic activity against erythrocytes as a function of pH. The pH-dependent conformation changes of the nanoparticles were studied by Fourier transform infrared (FT-IR) spectroscopy. The gamma-hPGA nanoparticles showed hemolytic activity with decreasing pH from 7 to 5.5, and were membrane-inactive at physiological pH. This activity was dependent on the hydrophobicity of gamma-PGA. The mechanism responsible for the pH-dependent hemolysis by the nanoparticles involved a conformational change of gamma-hPGA and corresponding increase in the surface hydrophobicity. We conclude that gamma-hPGA nanoparticles have significant potential as membrane-disruptive carriers. These results have important implications for the design of endosome-disruptive nanoparticles as drug, protein and DNA delivery systems.Journal of Biomaterials Science Polymer Edition 01/2010; 21(3):315-28. · 1.70 Impact Factor