Amphipathic pH-responsive polymers have shown to increase the permeability of cell membranes to trehalose hence improving the cryopreservation of mammalian cells. However, the trafficking of both the polymer and trehalose across the cell membrane has not yet been thoroughly analysed. The objective of this study was to investigate the effect on cryopreservation of the trafficking of the disaccharide trehalose along PP-50, an amphipathic polymer, through an osteosarcoma cell line (SAOS-2). Confocal microscopy analysis confirmed the presence of intracellular labelled trehalose only when incubated in the presence of PP-50. Further analysis confirmed that both trehalose and PP-50 localised in the cytoplasm, accumulated mainly in the perinuclear area. Quantitative analysis of the colocalisation between trehalose and PP-50 showed Pearson and Manders coefficients of 0.862 ± 0.008 and 0.766 ± 0.033, respectively, suggesting a high degree of intracellular colocalisation between these molecules. Cryopreserved cells pre-incubated with trehalose and PP-50 showed increased cryosurvival when compared with cells pre-incubated in the absence of the polymer. PP-50 showed to be directly involved in the uptake of trehalose, a critical characteristic towards use in cryopreservation and biomedical applications.
Biopolymers have become important drug delivery systems for therapeutic molecules by enhancing their accessibility and efficacy intracellularly. However, the transport of these drugs across the cell membrane and their release into the cytosol remain a challenge. The trafficking of poly (l-lysine iso-phthalamide) grafted with phenylalanine (PP-50) was investigated using an osteosarcoma cell line (SAOS-2). Colocalisation of this amphipathic biopolymer with endocytosis tracers, such as transferrin and lactosylceramide, suggested that PP-50 is partially internalised by both clathrin and caveolin-mediated endocytosis. Macropinocytosis was also investigated, but a smaller correlation was found between this mechanism and PP-50 transport. A significant decrease in polymer-mediated calcein uptake was found when cells were pre-incubated with endocytosis inhibitors, suggesting also the use of a combination of mechanisms for cell internalisation. In addition, PP-50 colocalisation with endosome and lysosome pathway markers showed that the polymer was able to escape the endolysosomal compartment before maturation. This is a critical characteristic of a biopolymer towards use as drug delivery systems and biomedical applications.
The use of amphipathic pH-responsive polymers to transport sugars across cell membranes has been shown to improve the cryopreservation of mammalian cells. However, the effect of these polymers on cell viability and morphology has not yet been thoroughly analysed. In this study, the objective was to investigate the functional and structural effects of an amphipathic polymer, PP-50, upon an osteosarcoma cell line (SAOS-2). Cellular growth curves confirmed similar doubling times in PP-50 treated cells and untreated cells. PP-50 concentrations (10-2000 µg/ml) were well tolerated by cells after 2, 24 and 48 hours of incubation, as measured by mitochondrial enzyme activity and lactate dehydrogenase release. Analysis by flow cytometry demonstrated that PP-50 did not induce any additional apoptosis or necrosis in polymer treated cells compared to untreated cells. Phase contrast, confocal and transmission electron microscopy analysis of PP-50 treated cells revealed no signs of morphological changes, with cells maintaining their nucleus size and membrane integrity after treatment. PP-50 was shown to have no negative cellular effects, which is a critical characteristic of polymers towards use in cryopreservation and biomedical applications. In addition, the methodology and protocols established in this work provided a robust and comprehensive analysis of PP-50's cellular effects, thus making them well suited for determining these effects in mammalian cells exposed to other polymers intended for biological applications.
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