Precise control over the size and shape of ice crystals is a key factor to consider in designing antifreezing and cryoprotecting molecules for cryopreservation of cells. Here, we report that a poly(ethylene glycol)−poly(L-alanine) (PEG−PA) block copolymer exhibits excellent cryoprotecting properties for stem cells and antifreezing properties for water. As the molecular weight of PA increased from 500, 760, and 1750 Da (P1, P2, and P3) at the same PEG molecular weight of 5000 Da, the β-sheet content decreased and α-helix content increased. Comparing P2 (PEG−PA; 5000−760) and P4 (PEG−PA: 1000−750), β-sheets increased as the PEG block length decreased. The critical micelle concentration of the PEG−PA block copolymers was in a range of 0.5−3.0 mg/mL and was proportional to the hydrophobicity of the PEG−PA block copolymers. The P1, P2, and P3 self-assembled into spherical micelles, whereas P4 formed micelles with cylindrical morphology. The difference in the block copolymer structure affected ice recrystallization inhibition (IRI) activity and cryopreservation of cells. IRI activity was assayed via mean largest grain size (MLGS), and interactions between polymers and ice crystal surfaces were studied by dynamic ice-shaping studies. The MLGS decreased to 58 → 53 → 45 → 35 → 23% of that of PBS, as the polymer (PEG−PA 5000−500) concentration increased from 0.0 (PBS; control) → 1.0 → 5.0 → 10 → 30 → 50 mg/mL. The MLGS of PEG 5k solutions (negative control) decreased to 74 → 71 → 64 → 44 → 37% of that of PBS in the same concentration range. P3 and P4 with a longer hydrophobic PA block developed elongated ice crystals at above 30 mg/mL. The dynamic ice-shaping study exhibited that ice crystals became needle-shaped, as the hydrophobicity of the polymer increased as in P2−P4. The cell recovery in the P1 system after cryopreservation at −196 °C for 7 days was 87% of that of the dimethyl sulfoxide (DMSO) 10% system (positive control). The cell recovery was 48% for the P2 system and drastically decreased to less than 30% of that of the DMSO 10% system in the P3, P4, PEG 5k, PEG 1k, PVA 80H, and PVA 100H systems. Current studies suggest that IRI activity, round ice crystal shaping, and membrane stabilization activity of P1 cooperatively provide excellent cell recovery among the candidate systems. Recovered stem cells exhibited excellent proliferation and multilineage differentiation into osteocytes, chondrocytes, and adipocytes. To conclude, the PEG−PA (5000−500) block copolymer is suggested to be a promising antifreezing cryoprotectant for stem cells.

The control of ice recrystallization is very important in cryo-technological fields such as the food industry, biopharmaceuticals, and cell storage. Ice recrystallization inhibition (IRI) compounds are therefore designed to limit the growth of ice crystals, decrease the crystal size, and control the crystal shape. To improve the IRI activity of cryo-systems, various synthetic polymers such as biomimetic polypeptides from polar fish, facially amphiphilic polymers, polyampholytes, poly­(vinyl alcohol) derivatives, and block copolymers with hydrophilic–hydrophobic balance have been developed. Except for graphene oxide, poly­(vinyl alcohol) has thus far exhibited the best performance among these polymers. Herein, poly­(l-alanine-co-l-lysine) (PAK) was shown to exhibit a similar IRI activity to that of poly­(vinyl alcohol). Moreover, in contrast to the needle-shaped ice crystals generated by the aqueous PVA solution, the PAK solution was shown to generate cubic-to-spherical shaped ice crystals. Furthermore, neither poly­(l-alanine-co-l-aspartic acid) (PAD) nor poly­(ethylene glycol) (PEG) with a similar molecular weight provided any significant IRI activity. Examination by FTIR and circular dichroism spectroscopies indicated that the PAK forms α-helices, whereas the PAD forms random coils in water. Further, a dynamic ice shaping study suggested that PAK strongly interacts with ice crystals, whereas PAD and PEG only weakly interact. These results suggest that PAK is an important compound with superior IRI activity and that this activity is dependent upon the functional groups and secondary structure of the polypeptides.

Poly(L-alanine-co-L-lysine)-graft-trehalose (PAK T ) was synthesized as a natural antifreezing glycopolypeptide (AFGP)-mimicking cryoprotectant for cryopreservation of mesenchymal stem cells (MSCs). FTIR and circular dichroism spectra indicated that the content of the α-helical structure of PAK decreased after conjugation with trehalose. Two protocols were investigated in cryopreservation of MSCs to prove the significance of the intracellularly delivered PAK T . In protocol I, MSCs were cryopreserved at −196 °C for 7 days by a slow-cooling procedure in the presence of both PAK T and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAK T solution, followed by cryopreservation at −196 °C in the presence of free trehalose for 7 days by the slow-cooling procedure. Polymer and trehalose concentrations were varied by 0.0−1.0 and 0.0−15.0 wt %, respectively. Cell recovery was significantly improved by protocol II with preincubation of the cells in the PAK T solution. The recovered cells from protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, and adipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Ice recrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly delivered PEG-PAK T was the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosis and caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAK T . 1 H NMR and FTIR spectra suggested that the intracellular PEG-PAK T s interact with water and stabilize the cells during cryopreservation.

Recently, poly(L-alanine-co-L-lysine) (PAK) was reported to exhibit an ice recrystallization inhibition (IRI) activity comparable to that of poly(vinyl alcohol), which has the highest IRI activity among synthetic polymers (ACS Macro Lett. 2021, 10, 1436−1442. As a continuation of the study, we synthesized a series of PAK copolymers to investigate the structure−property relationship of PAK on the IRI activity and its mechanism by varying the (1) composition of the L-alanine/L-lysine (A/K) ratio among 0/100, 50/50, 60/40, and 70/30 and (2) molecular weight of PAK among 4, 9, and 15 kDa at a fixed 50/50 ratio of A/K. As the hydrophobic L-alanine composition of PAK increased from 0 to 70%, the mean largest grain size (MLGS) relative to phosphatebuffered saline decreased from 74 to 11% at 1.0 mg/mL. As the molecular weight of PAK increased from 4 to 15 kDa, the MLGS decreased from 32 to 15% at 1.0 mg/mL. The ice nucleation temperature decreased as the hydrophobic L-alanine composition and the molecular weight of PAK increased. The thermal hysteresis (TH) of PAK was about 0.05 °C at 1.0 mg/mL and increased to 0.2 °C as the molecular weight increased from 4 to 15 kDa. The ice crystal size of ice cream showed a similar trend to IRI activity. To understand the underlying chemistry of the IRI effect of PAK, circular dichroism spectroscopy, a dynamic ice shaping study, and Fourier-transform infrared spectroscopy were carried out. These studies suggest that the α-helicity, hydrophobic/hydrophilic balance, molecular weight of PAK, and extent of interactions between polymers and ice crystals are related to the IRI activity, which extends to ice nucleation inhibition, TH of ice formation, ice shaping, and even the ice crystal size of ice cream.

Hydrogels are a three-dimensional network material with a high equilibrium water content where chemical, physical, or biomolecular crosslinking systems have been used for the network formation. In this study, we report a thermosensitive cytogel of lactobionic acid/butanoic acid-conjugated poly(ε-L-lysine) (PKLC4). The thermogelation of the aqueous PKLC4 solution (3.5 wt %) was induced by partial dehydration accompanying a random coil-to-β-sheet transition of the polymer. During the sol-to-gel transition, the modulus increased from <0.05 Pa at <10 °C to 1300−1360 Pa at 37 °C. When HepG2 cells were incorporated into the PKLC4 solution, the gel modulus at 37 °C increased to 2300−2670 Pa. Moreover, the gel modulus was significantly affected by the cell type, population of the HepG2 cells, and live/dead states of the HepG2 cells. The cells proliferate better in the biointeractive PKLC4 thermogel than in the bioinert PEG-PA thermogel. To conclude, by combining thermosensitivity and specific binding of the receptor to the substrate, the hydrogel attained a high modulus without delay in gel time. This study provides new insights into hydrogel preparation in that substrate−receptor binding can be utilized as a crosslinking system to control the hydrogel modulus as well as a design principle for three-dimensional cache that improves cytocompatibility for cells.