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

Abstract

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.

Intracellular Delivery of Trehalose for Cell Banking

“…Synthetic polymers have been investigated as facilitators of trehalose into cytoplasm by interacting with the cell membrane and temporarily increasing permeability [39][40][41][42][43][44] . Amphipathic polymers with weakly ionizable carboxyl acid side groups and hydrophobic side chains have garnered interest because of their high affinity for lipid membranes and ability to elevate membrane permeability by mimicking viral peptides as well as promote endosomal escape 62,63 .…”

“…blood to a nucleated cell line derived from human osteosarcoma (SAOS-2) 40,42 . PP50 was able to safely and effectively load trehalose into the nucleated human cell line.…”

Advanced Biotechnology for Cell Cryopreservation

“…Chemical delivery methods utilize cell membrane perforating agents like synthetic polymers [42,43], α-hemolysin [44][45][46], and nanoparticles [47][48][49] to achieve intracellular delivery of trehalose. These chemical materials usually involve a complex preparation process.…”

“…Trehalose has been used as sole CPA in combination with these chemical delivery methods to successfully cryopreserve RBCs, fibroblasts, keratinocytes, and human mesenchymal stem cells (hMSCs). Mercado et al [43] designed and synthesized a series of biomimetic derivatives of PLP polymer to facilitate intracellular delivery of trehalose. Particularly, co-incubation of PP50 (composed of PLP grafted with l-phenylalanine) with erythrocytes and trehalose suspension increased the intracellular concentration of trehalose to 123 ± 16 mmol/L.…”

A Versatile Polymer‐Based Platform for Intracellular Delivery of Macromolecules

“…PP50, one of the polymers from the PP‐family, is obtained by grafting PLP with l ‐phenylalanine at a stoichiometric ratio of 50 mol% relative to the pendant carboxylic acid groups . PP50 has been previously demonstrated to deliver small‐molecule trehalose through simple mixing to human erythrocytes and osteosarcoma cells at a mildly acidic extracellular pH for cryopreservation applications, while causing minimal cytotoxicity . In this paper, we explore the possibility of greatly expanding the scope of using PP50 as a bio‐inspired, next generation delivery agent, compatible with many model and functional macromolecular payloads and enabling cytoplasmic delivery to a wide range of cell types.…”