Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity

Cryopreservation enables long-term preservation of cells at ultralow temperatures. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)­ethyl methacrylate (DMAEMA) and methacrylic acid­(MAA) (poly­(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepared two zwitterionic polymers, poly­(sulfobetaine) (SPB) and poly­(carboxymethyl betaine) (CMB), and compared their efficiency with poly­(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the molecular mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Additionally, cryoprotective polymers, especially their hydrophobic derivatives, inhibit the recrystallization of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency

Surface-Selective Control of Cell Orientation on Cyanobacterial Liquid Crystalline Gels

Liquid crystalline hydrogels (LCGs) with layer structures and oriented pores were created using sacran which is a cyanobacterial heteropolysaccharide possessing functional sulfate, carboxylate, and amide groups in common with glycosaminoglycan. The LCG biocompatibility with L929 mouse fibroblasts was confirmed under the appropriate conditions. Enhanced growth and proliferation of L929 cells without exhibiting any toxicity were confirmed. The water contact angle and protein adsorption ability on the LCG were well-controlled by the cross-linking degree. Additionally, fibroblasts were finely oriented on the LCG side face where layer edges made a striped morphology on its surface, whereas the flat top faces of the LCG did not induce any specific cell orientation

Surface-Selective Control of Cell Orientation on Cyanobacterial Liquid Crystalline Gels

Liquid crystalline hydrogels (LCGs) with layer structures and oriented pores were created using sacran which is a cyanobacterial heteropolysaccharide possessing functional sulfate, carboxylate, and amide groups in common with glycosaminoglycan. The LCG biocompatibility with L929 mouse fibroblasts was confirmed under the appropriate conditions. Enhanced growth and proliferation of L929 cells without exhibiting any toxicity were confirmed. The water contact angle and protein adsorption ability on the LCG were well-controlled by the cross-linking degree. Additionally, fibroblasts were finely oriented on the LCG side face where layer edges made a striped morphology on its surface, whereas the flat top faces of the LCG did not induce any specific cell orientation

Design of Highly Selective Zn-Coordinated Polyampholyte for Cancer Treatment and Inhibition of Tumor Metastasis

Developing anticancer agents with negligible cytotoxicity against normal cells while mitigating multidrug resistance and metastasis is challenging. Previously reported cationic polymers have effectively eradicated cancers but are clinically unsuitable due to their limited selectivity. Herein, a series of poly(l-lysine)- and nicotinic acid-based polymers were synthesized using varying amounts of dodecylsuccinic anhydride. Zn-coordinating polymers concealed their cationic charge and enhanced selectivity. These Zn-bound polymers were highly effective against liver and colon cancer cells (HepG2 and Colon 26, respectively) and prevented cancer cell migration. They also displayed potent anticancer activity against drug-resistant cell lines (COR-L23/R): their cationic structure facilitated cancer cell membrane disruption. Compared to these polymers, doxorubicin was less selective and less efficacious against drug-resistant cell lines and was unable to prevent cell migration. These polymers are potential cancer treatment agents, offering a promising solution for mitigating drug resistance and tumor metastasis and representing a novel approach to designing cancer therapeutics

pH-Responsive Polyion Complex Vesicle with Polyphosphobetaine Shells

When a bioactive molecule is taken into cells by endocytosis, it is sometimes unable to escape from the lysosomes, resulting in inefficient drug release. We prepared pH-responsive polyion complex (PIC) vesicles that collapse under acidic conditions such as those inside a lysosome. Furthermore, under acidic conditions, cationic polymer was released from the PIC vesicles to break the lysosome membranes. Diblock copolymers (P₂₀M₁₆₇ and P₂₀A₁₉₀) consisting of water-soluble zwitterionic poly­(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block and cationic or anionic blocks were synthesized via reversible addition–fragmentation chain transfer (RAFT) radical polymerization. Poly­(3-(methacrylamidopropyl) trimethylammonium chloride) (PMAPTAC) and poly­(sodium 6-acrylamidohexanoate) (PAaH) were used as the cationic and anionic blocks, respectively. The pendant hexanoate groups in the PAaH block are ionized in basic water and in phosphate buffered saline (PBS), while the hexanoate groups are protonated in acidic water. In basic water, PIC vesicles were formed from a charge neutralized mixture of oppositely charged diblock copolymers. At the interface of PIC vesicle and water exists biocompatible PMPC shells. Under acidic conditions, the PIC vesicles collapsed, because the charge balance shifted due to protonation of the PAaH block. After collapse of the PIC vesicles, P₂₀A₁₉₀ formed micelles composed of protonated PAaH core and PMPC shells, while P₂₀M₁₆₇ was released as unimers. PIC vesicles can encapsulate hydrophilic nonionic guest molecules into their hollow core. Under acidic conditions, the PIC vesicles can release the guest molecules and P₂₀M₁₆₇. The cationic P₂₀M₁₆₇ can break the lysosome membrane to efficiently release the guest molecules from the lysosomes to the cytoplasm

The permeability of PN mouse and pig embryos to COOH-PLL.

<p>The embryos were exposed to FITC-labeled COOH-PLL (5% (w/v)) for 5 min. After exposure, embryos with or without washing were examined under a laser scanning microscope. Scale bars denote 50 um.</p

The kinetics of COOH-PLL in mouse oocytes.

<p>Mouse oocytes were exposed to PB1(-) supplement with FITC-labeled COOH-PLL (5% (w/v)). Green: FITC-labeled COOH-PLL.</p

The effect of COOH-PLL concentration on the number of cells in a blastocyst.

<p>The effect of COOH-PLL concentration on the number of cells in a blastocyst.</p

The effects of COOH-PLL on survival, fertility and developmental ability of vitrified mouse oocytes after IVF.

<p>Data are shown as means ± S.E.M. Different superscripts denote a significant difference (P<0.05). Numbers of oocytes used in each group were described under the treatment group.</p

<i>In vivo</i> development of porcine embryos vitrified with P20.

<p><i>In vivo</i> development of porcine embryos vitrified with P20.</p