Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

Sharp, Duncan M.C.; Picken, Andrew; Morris, Timothy; Hewitt, Christopher J.; Coopman, Karen; Slater, Nigel K.H.

doi:10.1016/j.cryobiol.2013.09.002

Cited by 30 publications

(41 citation statements)

References 36 publications

(39 reference statements)

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“…Interestingly, such high amounts of intracellular trehalose didn’t appear to adversely impact the viability and function of the hepatocytes. Although the presence of intracellular trehalose is expected to increase intracellular osmolality and induce osmotic swelling, several cell types have been observed to tolerate the osmotic impact of various levels of intracellular trehalose [ 26 , 29 , 36 , 37 ]. A byproduct of 6-O-Ac-Tre hydrolysis is acetic acid which can result in a significant reduction in intracellular pH.…”

Section: Discussionmentioning

confidence: 99%

Engineered Trehalose Permeable to Mammalian Cells

et al. 2015

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Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

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Section: Discussionmentioning

confidence: 99%

Engineered Trehalose Permeable to Mammalian Cells

et al. 2015

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“…Recent studies have shown the impact of PP-50, a biopolymer consisting of poly(L-lysine iso-phthalamide) grafted with the hydrophobic amino acid L-phenylalanine, on increased post-thaw viabilities of cryopreserved red blood cells and osteosarcoma cells [26,27].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Increased cryosurvival of osteosarcoma cells using an amphipathic pH-responsive polymer for trehalose uptake

Mercado

Slater

2016

Cryobiology

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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.

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“…PP-50, an amphipathic polymer, has shown recently to be able to transport hydrophilic molecules into cells and to be biocompatible [15,16]. Despite its successful use in cryopreservation, the trafficking of PP-50 through cell membranes has not been previously investigated in depth.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

The intracellular fate of an amphipathic pH-responsive polymer: Key characteristics towards drug delivery

Mercado

Orellana-Tavra

Chen

et al. 2016

Materials Science and Engineering: C

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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.

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Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

Cited by 30 publications

References 36 publications

Engineered Trehalose Permeable to Mammalian Cells

Engineered Trehalose Permeable to Mammalian Cells

Increased cryosurvival of osteosarcoma cells using an amphipathic pH-responsive polymer for trehalose uptake

The intracellular fate of an amphipathic pH-responsive polymer: Key characteristics towards drug delivery

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