Comb-like Pseudopeptides Enable Very Rapid and Efficient Intracellular Trehalose Delivery for Enhanced Cryopreservation of Erythrocytes

Abstract

Cell cryopreservation plays a key role in development of reproducible and cost-effective cell-based therapies. Trehalose accumulated in freezing and desiccation tolerant organisms in nature has been sought as an attractive non-toxic cryoprotectant. Herein, we report a co-incubation method for very rapid and efficient delivery of membrane-impermeable trehalose into ovine erythrocytes through reversible membrane permeabilization using pH-responsive, comb-like pseudopeptides. The pseudopeptidic polymers containing relatively long alkyl side chains were synthesized to mimic membrane-anchoring fusogenic proteins. The intracellular trehalose delivery efficiency was optimized by manipulating the side chain length, degree of substitution and concentration of the pseudopeptides with different hydrophobic alkyl side chains, the pH, temperature and time of incubation, as well as the polymer-to-cell ratio and the concentration of extracellular trehalose.Treatment of erythrocytes with the comb-like pseudopeptides for only 15 min yielded an intracellular trehalose concentration of 177.9 ± 8.6 mM, which resulted in 90.3 ± 0.7% survival after freeze-thaw. The very rapid and efficient delivery was found to be attributed to the reversible, pronounced membrane curvature change as a result of strong membrane insertion of the comb-like pseudopeptides. The pseudopeptides can enable efficient intracellular delivery of not only trehalose for improved cell cryopreservation, but also other membrane-impermeable cargos.

pH-Responsive Amphiphilic Carboxylate Polymers: Design and Potential for Endosomal Escape

“…A similar series of studies, using amphiphilic synthetic pseudopeptides namely poly ( L -lysine isophthalamide) (PLP), also confirm that pH-dependent membrane-permeabilizing capability could be adjusted by grafting amino acids with different hydrophobicity or alkyl chains (Eccleston et al, 2000;Chen et al, 2009Chen et al, , 2017. Increasing the hydrophobicity moieties or changing the polymer structure from linear to branched could increase the interaction with lipid membranes (Wang and Chen, 2017;Chen et al, 2020). Mechanistic insights suggest that phenylalanine modified PLP induced red blood cell membrane thinning of 35-40% normal thickness at endosomal pH, thus facilitating the transport of membrane-impermeable small molecular cargos (Lynch et al, 2011).…”

Facilitating trehalose entry into hRBCs at 4 °C by alkylated ε-poly(l-lysine) for glycerol-free cryopreservation

“…After placing in a water bath at 37 1C for 0.5 h, the samples were centrifuged at 1800g for 5 min and the supernatant was collected for the absorbance measurement at 540 nm on a spectrophotometer (Infinite 200 Pro, Tecan, Switzerland). The percent hemolysis caused by the samples was evaluated using the following eqn (1): 17,21 Hemolysis ð%Þ ¼ OD Sample OD Positive  100…”

“…The collected hRBCs were lysed by deionized water and centrifuged at 12 000g and 4 1C for 5 min, with the supernatant trehalose extraction for subsequent determination. The intracellular trehalose content was calculated according to eqn (2): 17,21 Intracellulartrehalose ðmMÞ¼ c 342:3Â0:7ÂRBCÂMCV Â10 6…”

“…Actually, Lynch and Chen et al ever applied poly(L-lysine isophthalamide) a School of Materials Science and Engineering, and Tianjin Key Laboratory of grafting hydrophobic L-phenyl or decyl groups with pHmediated membrane-disruptive activity on the cell membrane for intracellular delivery of trehalose in sRBCs or hRBCs at 37 1C. [19][20][21] It was noted that most of the previous strategies for intracellular trehalose loading in sRBCs and hRBCs were performed at 37 1C, [17][18][19][20][21] probably because hRBC membranes have two phase transitions at 14 1C and 34 1C, respectively. 22 Above the phase transition temperatures, the membrane fluidity and the motion of trehalose are both accelerated, so that much higher intracellular trehalose can be obtained at 37 1C than that at 4 1C.…”

Formulation Considerations for Autologous T Cell Drug Products

“…Much of this search has focused on the cryopreservation of embryonic stem cells, hematopoietic cell lines, umbilical cord- and bone marrow-derived mesenchymal stem cells [ 93 ]. Examples include polyampholytes such as those based on methacrylates or poly-lysine [ 94 ] and pore-forming amphipathic pH-responsive polymers facilitating the intracellular entry of non-reducing cryoprotectant sugars [ 95 ], such as comb-like pseudopeptides harbouring alkyl side chains that mimic fusogenic proteins [ 96 ]. Despite the burgeoning knowledge of these novel polymers, their introduction as novel excipients in the clinic needs careful consideration of regulatory guidance documents.…”