Compositions – Frost-preventing – ice-thawing – thermostatic – thermophoric,...
Reexamination Certificate
2000-11-30
2003-09-09
Green, Anthony J. (Department: 1755)
Compositions
Frost-preventing, ice-thawing, thermostatic, thermophoric,...
C047S002000, C106S013000, C252S071000, C252S073000, C252S077000, C435S001100, C435S001300, C435S260000
Reexamination Certificate
active
06616858
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods of inhibiting ice formation using polyglycerol and related molecules.
BACKGROUND OF THE INVENTION
Preventing the freezing of water, and solutions that contain water, is a problem of substantial environmental, agricultural, industrial, and biomedical interest. Ice on walkways, roads and aircraft wings constitutes an environmental hazard to transportation. Ice formation on and inside plants causes expensive damage to crops and gardens. Freezing of antifreeze solutions, pipeline contents, paints, wet concrete and other aqueous solutions subjected to cold temperatures are issues of concern for industry. Avoiding ice formation during cold storage of proteins, viruses, cells, tissues, and organs is an important problem in cryobiology.
Below a critical temperature (the equilibrium freezing point), the crystallization of water into ice becomes thermodynamically favored. The equilibrium freezing point of water can be lowered by adding solutes that lower the vapor pressure of water such that it becomes equivalent to the vapor pressure of ice only at a lower temperature. This classical means of freezing point depression is termed “colligative” freezing point depression, and is approximately independent of the nature of the added solute, the effect being proportional instead to the mole fraction of the added solute regardless of its nature. Colligative freezing point depression is the physical basis on which essentially all currently used antifreeze agents (such as glycols and salts) operate. The disadvantage of colligative freezing point depression is that large quantities of solutes (10% or more) are required to lower the freezing point by even a few degrees Celsius.
Beyond colligative freezing point depression, there are two independent means of lowering the practical freezing point of water. The first is to inactivate heterogeneous nucleating agents, and the second is to inhibit growth of small ice crystals despite cooling to below the equilibrium freezing point.
Pure water freezes spontaneously (homogeneous nucleation) at just above −40° C. when ice is not previously nucleated by impurities in the water known generically as heterogeneous nucleating agents. Biogenic heterogeneous nucleating agents are often simply called ice nucleating agents (INAs). Biogenic INAs have apparently evolved to reduce or eliminate supercooling in a variety of contexts, but minerals and organic nucleators also exist. Even highly purified laboratory grade water retains significant nucleation tendency. If INAs can be removed or inhibited, water and water solutions can be supercooled to temperatures many degrees below the freezing point without actually freezing.
Cold-hardy plants, insects, and fish have evolved antifreeze proteins that selectively adsorb onto the surface of ice or INAs, thereby preventing water molecules from coming into contact with surfaces that trigger ice growth (Devries, A. L., and Wohlschlag, D. E. “Freezing resistance in some Antarctic fishes” Science 163, pp. 1074-1075, 1969). Antifreeze proteins thus act as non-colligative antifreeze agents, and very small concentrations (less than 1%) are able to suppress the temperature at which ice forms, in some cases by several degrees. Soon after the original discovery of antifreeze proteins, it was speculated that “many polymeric molecules (not just proteins) ought to be able to inhibit nucleation (of ice) in this way” (Klotz, I. M. in “The Frozen Cell” pp. 5-26. J. & A. Churchill, London, 1970). These speculations opened the door to the possibility that inexpensive synthetic compounds might be found with non-colligative antifreeze activity.
In 1983, Caple et al (“Polymeric Inhibition of Ice Nuclei Active Sites” Cryo-Letters 4, pp. 51-58, 1983 and U.S. Pat. No. 4,484,409) reported significant enhancement of water supercooling tendency by adding small quantities of methyl acrylate-co-vinyl pyrrolidone polymer or methyl methacrylate-co-vinyl pyrrolidone polymer. While showing proof of concept, these observations were limited in a number of important respects. First, these copolymers were not tested for toxicity and may be toxic. Second, release of these polymers into the environment, or their inclusion in foods, may not be permissible. Third, the polymers required substantial hydrophobicity for effectiveness, which limits their utility in water solutions. Fourth, the nature of these polymers and of the methods for their synthesis may make them too expensive for practical use. Finally, their performance was not fully characterized, and may be limited in a variety of ways. In any case, no commercial use of Caple's polymers has appeared in the 16 years since their publication, implying unreported deficiencies of these polymers for practical ice antinucleation applications. Similarly, the Japanese investigators Watanabe et al. showed that they could reduce nucleation by silver iodide using an NMR assay method by reacting proteins with hydrophobic aliphatic chains of varying lengths (US Patent Application, recently lapsed). But this method appeared to require the resulting modified proteins to form micelles in order to gain the antinucleation activity reported, a factor that will limit the accessibility of the antinucleators to nucleating bodies in general, and that may prevent the invention from being used in organ perfusion applications wherein the micelles may not penetrate through capillaries into the interstitial space. In any case, no industrial use of their invention is known, and the US rights to their invention recently lapsed due to non-payment of maintenance fees by the assignee, implying a lack of utility. A variety of other antinucleation substances has been described, but these are generally either chemically reactive substances that destroy ice nucleators and would be expected to also damage vital biomolecules present in cells or the environment, or are complicated organic chains that may have unacceptable toxicity and chemical reactivity and that tend to be hydrophobic and otherwise difficult or problematic to use.
In 1995, Fahy (“Novel Ice-Controlling Molecules and Their Applications” International Patent Application PCT/U.S.96/04284, Publication # WO 96/30459, 1996, superceded by PCT application PCT/U.S.98/20834, Publication # WO 99/18169, published on Apr. 15, 1999) proposed creating synthetic ice interface dopants (“ice blockers”) specifically designed to bind to the basal plane and prism faces of ice crystals (and ice nucleators). Molecules were to be designed by spacing polar groups at intervals corresponding to the lattice spacing of water molecules on the crystal faces of ice. Numerous specific molecules and polymers were proposed, and data were presented showing reduction of ice crystal growth rates by 6% w/v 1,3-cis-cyclohexanediol and augmentation of the thermal hysteresis effect of fish antifreeze glycoprotein by 1,3,5-cis,cis-cyclohexanetriol, but the latter effect was said to be impossible to utilize due to the pro-nucleating effect of 1,3,5-cis,cis-cyclohexanediol. Also, no data were shown indicating thermal hysteresis augmentation by any other agent, nor confirming any ice-bonding effect of 1,3-cis-cyclohexanediol or any other proposed agent.
Claims were presented for several specific polymers as agents for inhibiting ice crystal growth, but none of these polymers was shown to inhibit ice crystal growth, and none anticipated either the polyvinylacetate/polyvinylalcohol (PVA) antinucleating copolymers of Wowk (U.S. patent application Ser. No. 09/400,791) or the novel antinucleating species disclosed herein.
At sufficiently high concentrations (typically 50% or more), conventional colligative antifreeze agents can prevent ice formation completely, allowing aqueous solutions to be cooled to arbitrarily low temperatures without freezing. In the field of cryobiology, this is the basis of cryopreservation by vitrification (Fahy, G. M. et al “Vitrification as an approach to cryopreservation” Cryobiology 21, pp. 407-426, 1984). However the utility of
Fahy Greg
Wowk Brian
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