New preservation technologies may allow for organ banking similar to blood and biomaterial banking approaches. Using cryoprotective agents (CPAs), aqueous solutions with organic components such as DMSO, propylene glycol, and added salts and sugars, organs can be used to vitrify and store organs at −140 °C. When needed, these organs can be rewarmed in a rapid and uniform manner if CPAs are supplemented with iron oxide nanoparticles (IONPs) in an applied radiofrequency field. Speed and uniformity of warming are both IONP concentration and CPA suspension dependent. Here we present a coating method of small molecule phosphonate linker (PLink) and biocompatible polymer ( i.e. polyethylene glycol PEG) that tunes stability and increases the maximum allowable concentration of IONPs in CPA suspension. PLink contains a phosphonate 'anchor' for high irreversible binding to iron oxide and a carboxylic acid 'handle' for ligand attachment. PLink-PEG removes and replaces the initial coating layer of commercially available IONPs (EMG1200 (hydrophobic) and EMG308 (hydrophilic) Ferrotec, Inc., increasing colloidal stability and decreasing aggregation in both water and CPAs, (verified with dynamic light scattering) from minutes (uncoated) to up to 6 days. Heating properties of EMG1200, specific absorption rate (SAR), measured using an applied field of 360 kHz and 20 kA m −1 , increased from 20 to 180 W per g Fe with increasing PLink-PEG5000. PEG replacing the initially hydrophobic coating decreased aggregation in water and CPA, consistent with earlier studies on heating performance. Furthermore, although the size is minimized at 0.20 mol PEG per g Fe, heating is not maximized until concentrations above 0.43 mol PEG per g Fe on EMG1200. SAR on hydrophilic EMG308 was preserved at 400 W per g Fe regardless of the amount of PLink added to the core. Herein concentrations of IONP in VS55 (common CPA) significantly above our previous capabilities, sIONP at 10 mg Fe per mL, was reached, 25 mg Fe per mL of 308-PEG5000 and 60 mg Fe per mL of 1200-PEG5000, approaching stock EMG308 in water, 60 mg Fe per mL. Furthermore, at these concentrations cryopreserved Human dermal fibroblast cells were successfully nanowarmed (at applied fields described above), with higher viability as compared to convective rewarming in a water bath and heating rate close to 200 °C min −1 , 2.5 times faster than our current system. Using PLink as the coating method allowed for higher concentrations of IONPs to be successfully suspended in CPA without affecting the heating ability. Additionally, the model ligand, PEG, allowed for increased stability over time in nanowarming experiments.
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Preparation of Scalable Silica‐Coated Iron Oxide Nanoparticles for Nanowarming
Cryopreservation technology allows long‐term banking of biological systems. However, a major challenge to cryopreserving organs remains in the rewarming of large volumes (>3 mL), where mechanical stress and ice formation during convective warming cause severe damage. Nanowarming technology presents a promising solution to rewarm organs rapidly and uniformly via inductive heating of magnetic nanoparticles (IONPs) preloaded by perfusion into the organ vasculature. This use requires the IONPs to be produced at scale, heat quickly, be nontoxic, remain stable in cryoprotective agents (CPAs), and be washed out easily after nanowarming. Nanowarming of cells and blood vessels using a mesoporous silica‐coated iron oxide nanoparticle (msIONP) in VS55, a common CPA, has been previously demonstrated. However, production of msIONPs is a lengthy, multistep process and provides only mg Fe per batch. Here, a new microporous silica‐coated iron oxide nanoparticle (sIONP) that can be produced in as little as 1 d while scaling up to 1.4 g Fe per batch is presented. sIONP high heating, biocompatibility, and stability in VS55 is also verified, and the ability to perfusion load and washout sIONPs from a rat kidney as evidenced by advanced imaging and ICP‐OES is demonstrated.
more » « less- Award ID(s):
- 1642268
- NSF-PAR ID:
- 10457890
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Science
- Volume:
- 7
- Issue:
- 4
- ISSN:
- 2198-3844
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract To extend the preservation of donor hearts beyond the current 4–6 h, this paper explores heart cryopreservation by vitrification—cryogenic storage in a glass‐like state. While organ vitrification is made possible by using cryoprotective agents (CPA) that inhibit ice during cooling, failure occurs during convective rewarming due to slow and non‐uniform rewarming which causes ice crystallization and/or cracking. Here an alternative, “nanowarming”, which uses silica‐coated iron oxide nanoparticles (sIONPs) perfusion loaded through the vasculature is explored, that allows a radiofrequency coil to rewarm the organ quickly and uniformly to avoid convective failures. Nanowarming has been applied to cells and tissues, and a proof of principle study suggests it is possible in the heart, but proper physical and biological characterization especially in organs is still lacking. Here, using a rat heart model, controlled machine perfusion loading and unloading of CPA and sIONPs, cooling to a vitrified state, and fast and uniform nanowarming without crystallization or cracking is demonstrated. Further, nanowarmed hearts maintain histologic appearance and endothelial integrity superior to convective rewarming and indistinguishable from CPA load/unload control hearts while showing some promising organ‐level (electrical) functional activity. This work demonstrates physically successful heart vitrification and nanowarming and that biological outcomes can be expected to improve by reducing or eliminating CPA toxicity during loading and unloading.
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Abstract Vitrification can dramatically increase the storage of viable biomaterials in the cryogenic state for years. Unfortunately, vitrified systems ≥3 mL like large tissues and organs, cannot currently be rewarmed sufficiently rapidly or uniformly by convective approaches to avoid ice crystallization or cracking failures. A new volumetric rewarming technology entitled “nanowarming” addresses this problem by using radiofrequency excited iron oxide nanoparticles to rewarm vitrified systems rapidly and uniformly. Here, for the first time, successful recovery of a rat kidney from the vitrified state using nanowarming, is shown. First, kidneys are perfused via the renal artery with a cryoprotective cocktail (CPA) and silica‐coated iron oxide nanoparticles (sIONPs). After cooling at −40 °C min−1in a controlled rate freezer, microcomputed tomography (µCT) imaging is used to verify the distribution of the sIONPs and the vitrified state of the kidneys. By applying a radiofrequency field to excite the distributed sIONPs, the vitrified kidneys are nanowarmed at a mean rate of 63.7 °C min−1. Experiments and modeling show the avoidance of both ice crystallization and cracking during these processes. Histology and confocal imaging show that nanowarmed kidneys are dramatically better than convective rewarming controls. This work suggests that kidney nanowarming holds tremendous promise for transplantation.
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Abstract In this work, we develop and demonstrate highly stable organic-coated engineered superparamagnetic iron oxide nanoparticles (IONPs), which provide effective osmotic pressure without aggregation, reverse diffusion, or membrane blocking (by nanoparticles) for osmotically driven membrane systems, considering both forward osmosis (FO) and pressure-retarded osmosis (PRO). For this, we synthesized highly water stable, monodisperse 12 nm IONPs with a rational series of water stabilizing surface coatings, including sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and polyethylene glycol (PEG). We then compared the library of surface functionalized IONPs as draw solutes for osmotic pressure-driven membrane processes. As synthesized, surface (organic) coatings are compact, thin, and can have very similar surface charge as the membrane itself, which results in effective osmotic pressure in forward osmosis (FO) mode configuration. To increase the osmotic pressure further, on a per mass basis, we synthesized and demonstrated novel hollow IONPs with identical surface coatings. Finally, water flux was further enhanced for stable particle systems using an oscillating magnetic field, thus physically altering concentration gradients, as a function of particle magnetic properties.
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This study investigates thermomechanical stress in cryopreservation by vitrification of the heart, while exploring the effects of nanowarming-assisted recovery from cryogenic storage. This study expands upon a recently published study, combining experimental investigation and thermal analysis of cryopreservation on a rat heart model. Specifically, this study focuses on scenarios with variable concentrations of silica-coated iron-oxide nanoparticles (sIONPs), while accounting for loading limitations associated with the heart physiology, as well as the properties of cryoprotective agent (CPA) solution and the geometry of the container. Results of this study suggest that variable sIONP concentration based on the heart physiology will elevate mechanical stresses when compared with the mathematically simplified, uniform distribution case. The most dangerous part of rewarming is below glass transition and at the onset of nanowarming past the glass transition temperature on the way for organ recovery from cryogenic storage. Throughout rewarming, regions that rewarm faster, such as the chambers of the heart (higher sIONP concentration), undergo compressive stresses, while the slower rewarming regions, such as the heart myocardium (low sIONP concentration), undergo tension. Being a brittle material, the vitrified organ is expected to fail under tension in lower stresses than in compression. Unfortunately, the location and magnitude of the maximum stress in the investigated cases varied, while general rules were not identified. This investigation demonstrates the need to tailor the thermal protocol of heart cryopreservation on a case-by-case basis, since the location, orientation, magnitude, and instant at which the maximum mechanical stress is found cannot be predicted
a priori . While thermomechanical stress poses a significant risk to organ integrity, careful design of the thermal protocol can be instrumental in reducing the likelihood of structural damage, while taking full advantage of the benefits of nanowarming.
