Dual Thermo- and pH-Responsive Behavior of Double Zwitterionic Graft Copolymers for Suppression of Protein Aggregation and Protein Release

Abstract

Graft copolymers consisting of two different zwitterionic blocks were synthesized via reversible addition fragmentation chain transfer polymerization. These polymers showed dual properties of thermo- and pH-responsiveness in an aqueous solution. Ultraviolet–visible spectroscopy and dynamic light scattering were employed to study the phase behavior under varying temperatures and pH values. Unlike the phase transition temperatures of other graft copolymers containing nonionic blocks, the phase transition temperature of these polymers was easily tuned by changing the polymer concentration. Owing to the biocompatible and stimuli-responsive nature of the polymers, this system was shown to effectively release proteins (lysozyme) while simultaneously protecting them against denaturation. The positively charged lysozyme was shown to bind with the negatively charged polymer at the physiological pH (pH 7.4). However, it was subsequently released at pH 3, at which the polymer exhibits a positive charge. Protein aggregation studies using a residual enzymatic activity assay, circular dichroism, and a Thioflavin T assay revealed that the secondary structure of the lysozyme was retained even after harsh thermal treatment. The addition of these polymers helped the lysozyme retain its enzymatic activity and suppressed its fibrillation. Both polymers showed excellent protein protection properties, with the negatively charged polymer exhibiting slightly superior protein protection properties to those of the neutral polymer. To the best of the authors’ knowledge, this is the first study to develop a graft copolymer system consisting of two different zwitterionic blocks that shows dual thermo- and pH-responsive properties. The presence of the polyampholyte structure enables these polymers to act as protein release agents, while simultaneously protecting the proteins from severe stress.

Review of the current state of protein aggregation inhibition from a materials chemistry perspective: special focus on polymeric materials

“…Further, we developed a graft polymer where poly-SPB was grafted onto the backbone of carboxylated e-poly-L-lysine (PLL-SA). 432 This was done in a multi-step reaction scheme where PLL-SA was first synthesized by adding succinic anhydride to e-PLL, followed by the controlled substitution of a RAFT agent on PLLSA by acid-amine coupling to obtain the e-PLL-SA-macro RAFT agent. Finally, this macro RAFT agent was used to polymerize the sulfobetaine monomer to obtain the graft polymer (Fig.…”

Advanced Nanomaterials-Assisted Cell Cryopreservation: A Mini Review

“…These graft copolymers showed high protein protection efficiency, with more than 70% of the lysozyme activity being retained even after heating at 90 °C for 30 min. With the multistimuli-responsiveness, these graft copolymers are promising for controlled drug release and protein protection . However, the mentioned stimuli-responsive polymers were only investigated at 4 or 90 °C.…”

“…With the multistimuliresponsiveness, these graft copolymers are promising for controlled drug release and protein protection. 145 However, the mentioned stimuli-responsive polymers were only investigated at 4 or 90 °C. Whether they could be utilized for cryopreservation of cells remains to be further investigated.…”

Double hydrophilic copolymers – synthetic approaches, architectural variety, and current application fields

“…Controlled polymerisation techniques typically provide good control over the side-chain length and Đ . 43,45,48,79,80,82,83 Sun and Cui 43 prepared DHGCs with a hydrophilic poly(2-hydroxy ethyl acrylate) (PHEA) backbone and poly( N -isopropyl acrylamide) (PNIPAAm) or poly(2-(dimethyl amino) ethyl methacrylate) (PDMAEMA) side-chains. They started with a single-electron transfer-living radical polymerization (SET-LRP) initiator-containing monomer (2-hydroxyethyl2-((2-chloropropanoyloxy)methyl)acrylate), which was first polymerised via RAFT (Fig.…”

“…Due to their versatile solution behaviour and efficient interaction with inorganic nanoparticles, 15,79 ions, 15 proteins, 82 or drugs, DHGCs are already found in various applications such as sensing, 15 catalysis, 12,48 suppression of protein aggregation, 82 or cancer treatment. 18 Further utilisation in carrier systems, nanoelectronics, oil recovery, and as dispersants is foreseen.…”