Charged Poly(N-isopropylacrylamide) Nanogels for the Stabilization of High Isoelectric Point Proteins

Storage and transportation of protein therapeutics using refrigeration is a costly process; a reliable electrical supply is vital, expensive equipment is needed, and unique transportation is required. Reducing the reliance on the cold chain would enable low-cost transportation and storage of biologics, ultimately improving accessibility of this class of therapeutics to patients in remote locations. Herein, we report on the synthesis of charged poly(N-isopropylacrylamide) nanogels that efficiently adsorb a range of different proteins of varying isoelectric points and molecular weights (e.g., adsorption capacity (Q) = 4.7 ± 0.2 mg/mg at 6 mg/mL initial IgG concentration), provide protection from external environmental factors (i.e., temperature), and subsequently release the proteins in an efficient manner (e.g., 100 ± 1% at 2 mg/mL initial IgG concentration). Both cationic and anionic nanogels were synthesized and selectively chosen based on the ability to form electrostatic interactions with adsorbed proteins (e.g., cationic nanogels adsorb low isoelectric point proteins whereas anionic nanogels adsorb high isoelectric point proteins). The nanogel-protein complex formed upon adsorption increases the stabilization of the protein's tertiary structure, providing protection against denaturation at elevated temperatures (e.g., 84 ± 4% of the protected IgG was stabilized when exposed to 65 °C). The addition of a high molar salt solution (e.g., 40 mM CaCl₂ solution) to protein-laden nanogels disrupts the electrostatic interactions and collapses the nanogel, ultimately releasing the protein. The versatile materials utilized, in addition to the protein loading and release mechanisms described, provide a simple and efficient strategy to protect fragile biologics for their transport to remote areas without necessitating costly storage equipment.