Advancing Rewarming for Cryopreservation Through Scalable Polymer Coating of Iron Oxide Nanoparticles

The short time limit for preserving donated organs after collection leads to organ shortages, morbidity and mortality of transplant recipients. We have successfully rewarmed vitrified, cryopreserved (-140 °C – indefinite storage) biological samples, using radiofrequency (RF) excited iron oxide nanoparticles (IONPs) in cryoprotective agents (CPAs).¹ CPAs are aqueous (high concentration) solutions of salts, sugars, and organics such as DMSO and propylene glycol. We previously demonstrated that silica-shell coated IONPs form stable colloidal suspension in CPA and that their production is scalable. Unfortunately, these IONPs have limited saturation concentration.² Herein, we present a new small molecule phosphonate linker (PLink) biocompatible polymer (i.e. polyethylene glycol PEG) for coating IONPs that 1) is stable in CPAs, 2) improves saturation concentration, and 3) is inexpensive for scale-up (&gt; 1g per batch).<br/>PLink enables ligand attachment to commercial IONPs (Ferrotec Inc.) such as EMG-1200 (hydrophobic) and EMG-308 (hydrophilic) by displacing the initial coating. PLink contains a phosphonate anchoring moiety that has high affinity for iron oxide and a carboxyl chemical handle for ligand attachment. PLinked-PEG increases colloidal stability and decreases aggregation in water and CPAs from minutes (uncoated) up to 6 days. The heating properties of EMG-1200, specific absorption rate (SAR), increased from 20 to 150 W/g Fe, which we attribute to decreased aggregation as the PLink-PEG5000 replaced the initial hydrophobic coating. Heating is also preserved when the stability of the colloidal suspension is unaffected; as is the case in replacing EMG308s hydrophilic ligand with hydrophilic PEG. We successfully rewarmed cryopreserved HDF cells in CPA by tuning the concentration of coated IONPs, 1200PLink-PEG from 20-60 mg Fe/mL, and 308PLink-PEG from 8-25 mg Fe/mL and achieved higher viability then convective rewarming in a water bath.<br/>Designing proper PLink-PEG coating with appropriate molecular weight and concentration leads to the formation of stable colloidal suspensions of IONPs in CPAs while preserving their heating ability. The simple coating method is both inexpensive and scalable, as needed for future manufacturing of PEG coatings for bulk cryopreservation and other biomedical IONP applications. In future experiments, PLink IONPs will be tested at higher saturation Fe concentration in CPAs, potentially increasing the heating rates and improving cryopreservation outcomes.<br/>1. Manuchehrabadi, N.; Gao, Z.; Zhang, J.; Ring, H. L.; Shao, Q.; Liu, F.; McDermott, M.; Fok, A.; Rabin, Y.; Brockbank, K. G. M.; Garwood, M.; Haynes, C. L.; Bischof, J. C., Improved tissue cryopreservation using inductive heating of magnetic nanoparticles. <i>Science translational medicine </i><b>2017,</b> <i>9</i> (379).<br/>2. Gao, Z.; Ring, H. L.; Sharma, A.; Namsrai, B.; Tran, N.; Finger, E. B.; Garwood, M.; Haynes, C. L.; Bischof, J. C., Preparation of Scalable Silica-Coated Iron Oxide Nanoparticles for Nanowarming. <i>Advanced Science </i><b>2020,</b> <i>7</i> (4), 1901624.