Poly(l-alanine-co-l-lysine)-g-Trehalose as a Biomimetic Cryoprotectant for Stem Cells

Abstract

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.

Gold Nanoparticle-Tethered Peptide Nanofibrils for Monitoring Ice Recrystallization Inhibition: Implications for Cryopreservation

“…Peptides consisting of short amino acid sequences that can be synthesized through simple synthetic routes are good candidates that can directly introduce an ice-binding motif by controlling self-assembling structures involving a specific secondary conformation. − Short peptides containing N-terminal aromatic protecting groups such as pyrene, naphthalene, and fluorenylmethoxycarbonyl (Fmoc) are well known to form highly ordered self-assembled fibrillar structures through intermolecular interactions such as hydrogen bonding and hydrophobic interactions, along with enhanced π–π stacking between building peptides. , Such fibrils are hierarchically formed from peptides to secondary structures and then to fibrils, providing a nanoscaffold for inducing cryopreservation ability by regularly arranging ice-binding moieties . The cryopreservation capability that is determined by the ice shape and growth control, freezing point depression in a non-colligative fashion (thermal hysteresis (TH)), and ice recrystallization inhibition (IRI) can be improved when rationally designed peptide agents bind to a certain ice crystal plane. − For example, cryodamage of the cell can be reduced if the peptide is well-adhered to the ice crystals to maintain a non-pointed shape during freezing and thawing. , In addition to the design of peptide agents, the correlative analysis elucidating the AFP–ice interaction mechanism should also be developed because multiscale tools are required to observe the AFP–ice interface at the atomic level, ice-binding of peptide agents at the nanoscale, and the shape of ice crystals at the microscale.…”

“…Peptides consisting of short amino acid sequences that can be synthesized through simple synthetic routes are good candidates that can directly introduce an ice-binding motif by controlling self-assembling structures involving a specific secondary conformation. 18 thalene, and fluorenylmethoxycarbonyl (Fmoc) are well known to form highly ordered self-assembled fibrillar structures through intermolecular interactions such as hydrogen bonding and hydrophobic interactions, along with enhanced π−π stacking between building peptides. 21,22 Such fibrils are hierarchically formed from peptides to secondary structures and then to fibrils, providing a nanoscaffold for inducing cryopreservation ability by regularly arranging ice-binding moieties.…”

Sulfonium-Containing Glycopolypeptides Tethering Trehalose for Protein Stabilization

“…26−28 Recently, poly(L-alanine-co-L-lysine)-g-trehalose was also prepared as a biomimetic cryoprotectant for stem cells. 29 However, so far, there are few reports on trehalose-containing glycopolypeptides for protein preservation. Considering the preciousness of bioactive proteins, reasonable strategies of protection are crucial for multiple stimuli.…”

“…Inspired by glycoproteins, biomimetic glycopolymers with carbohydrates conjugating to the polymeric backbone could realize important physiological functions . Alternatively, glycopolypeptides could be used for protection of biological units due to their well-defined structures, which were dominant in morphological control compared with natural biomacromolecules. , In our previous work, ε-poly­( l -lysine)­s with carboxylated trehalose pendant were adopted to protect red blood cells from cryo-injury, exhibiting excellent performance of membrane stabilization. − Recently, poly­( l -alanine- co - l -lysine)- g -trehalose was also prepared as a biomimetic cryoprotectant for stem cells . However, so far, there are few reports on trehalose-containing glycopolypeptides for protein preservation.…”

“…Herein, a series of cationic glycopolypeptides tethering trehalose with well-defined and fully trehalose-glycosylated structures are synthesized, without hydroxyl protection of trehalose. ,, A detailed synthesis and structural characterizations are given in the Supporting Information (Figure S1∼S23). As shown in Scheme , positively charged polypeptides, poly­(ethylene glycol)- b -( l -methionine- g -glycidyl propargyl ether)­s (PEG 45 - b -(Met- g -GP) x , PMG x ), were first prepared by using amine-initiated ring-opening polymerization (ROP) of α-methionine- N -carboxyanhydride (Met-NCA), and followed by alkylation of sulfur atoms with glycidyl propargyl ether.…”