WO2009113070A1 - Freeze-free method for storage of polypeptides - Google Patents

Abstract

ABSTRACT A method of preserving an activity of an isolated polypeptide is disclosed. The method comprises storing the polypeptide for at least 6 hours at a temperature greater than 0 °C with a liquid composition having a liquid and nanostructures, each of the nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state, thereby preserving the activity of the polypeptide. Freeze-free compositions of isolated polypeptides are also disclosed.

Description

FREEZE-FREE METHOD FOR STORAGE OF POLYPEPTIDES

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of storing polypeptides.

The use of polypeptides in industrial and research products and processes requires that these biomolecules remain active over a variety of temperatures. Whereas the required basic techniques such as isolation and purification of proteins are mostly established, the most difficult aspect of the use of these biomolecules resides in maintenance for the envisioned application, of the desired native, or, active molecular properties. Particularly, in the storage and transport of these biomolecules, losses in activity often have to be taken into account, whereby the success of later applications is put in risk. Commercially obtainable protein preparations therefore for the most part comprise compounds whose presence can minimize the activity loss during storage or transport. Furthermore, storage and transport is mostly carried out under low temperatures, which is costly, and for particular proteins may be undesirable because of molecular changes.

One area where the stabilization of polypeptides at room temperature is of particular relevance is the stabilization of enzymatic activity in liquid formulations. For example, a pre-formulated liquid enzyme concentrate may sometimes be stored for weeks or months before eventually being blended into a final product (e.g., a personal care product, such as a hand cream; or a cleaning product, such as a liquid detergent). Similarly, formulated liquid products containing enzymes may sit in storage for lengthy periods of time before use, as well. For a variety of reasons, the activity of enzymes in liquid formulations typically decreases over time.

Another area where the stabilization of polypeptides at room temperature is of relevance is in the field of molecular biology. Enzymes used for molecular cloning such as restriction enzymes, polymerases, ligases and endonucleases are particularly unstable and are subject to conformational and other changes that may occur during improper storage. In addition, these enzymes are often stored in the presence of Mg²⁺ which serves to contaminate and inactivate the enzyme preparations. One well known prior art method of storage of enzymes is freeze-drying. In this method, an aqueous solution of the polypeptide in a conventional storage buffer and in the presence of a cryoprotectant is first frozen, typically to -40 °C to -50°C. Water is then removed from the biological solution and the residual material becomes more concentrated until the material crystallizes. Ice is then removed by sublimation under vacuum. When the last traces of residual moisture are removed, a dry crystalline powder remains. An active polypeptide may be reconstituted from this powder.

Unfortunately, exposure of a freeze-dried product to ambient temperatures can result in significant enzyme activity loss. Additionally, some enzymes are often not completely freeze-stable. Further, the freeze-drying cycle may take several days and the activity of the reconstituted enzyme may not be very reproducible.

The "freeze/thaw" storage method involves mixing the enzyme with a cryoprotectant, freezing and storing, usually below -50 °C and sometimes in liquid nitrogen. The enzyme is then thawed immediately before use. Some enzymes will not survive a freezing and thawing cycle. Further, this technique can be expensive (especially with respect to transportation to customers).

Another technique involves storing an enzyme solution at -20 °C. This type of refrigerated storage for enzymes usually involves the addition of the cryoprotection additive glycerol to depress the freezing point and avoid freezing the enzyme. Thus for example, restriction enzymes are usually stored in 50 % glycerol.

However, as well as being costly, this type of storage poses a significant problem for some enzymes e.g. restriction enzymes, since the presence of glycerol in a restriction enzyme reaction can lead to what is known as specificity relaxation (such as "star activity"). In this regard, when restriction enzyme digestions are performed in the presence of glycerol, the specificity of the enzyme for a particular restriction site becomes relaxed and DNA is no longer cleaved only at that restriction site. The lack of enzyme specificity may lead to confusion as to which DNA fragments are the result of true restriction enzyme cleavage. It is generally understood that a glycerol concentration of greater than 5 % can create a significant possibility of star activity in an enzymatic digest. High enzyme concentration in the DNA cleavage reaction is also known to contribute to star activity, even in the absence of glycerol. To date, the art has often sought to minimize specificity relaxation by using lower than desired concentrations of enzyme and/or longer than desired incubation times.

SUMMARY OF THE INVENTION According to an aspect of some embodiments of the present invention there is provided a method of preserving an activity of an isolated polypeptide, the method comprising storing the polypeptide for at least 6 hours at a temperature greater than 0 °C with a liquid composition having a liquid and nanostructures, each of the nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state, thereby preserving the activity of the polypeptide.

According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising a polypeptide and a liquid composition having a liquid and nanostructures, each of the nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state, the composition not comprising a cryoprotectant.

According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising a polynucleotide-modifying- enzyme, a sugar and a liquid composition having a liquid and nanostructures, each of the nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state.

According to some embodiments of the invention, the activity is an enzymatic activity.

According to some embodiments of the invention, the activity is a binding activity.

According to some embodiments of the invention, the polypeptide is selected from the group consisting of an enzyme, an antibody and a structural protein. According to some embodiments of the invention, the polypeptide is an enzyme. According to some embodiments of the invention, the enzyme is a restriction enzyme.

According to some embodiments of the invention, the enzyme is a polymerase enzyme. According to some embodiments of the invention, the temperature is between about 25 °C and 45 °C.

According to some embodiments of the invention, at least a portion of thefluid molecules are identical to molecule of the liquid.

According to some embodiments of the invention, at least a portion of the fluid molecules are in a gaseous state.

According to some embodiments of the invention, a concentration of the nanostructures is lower than 10²⁰ nanostructures per liter.

According to some embodiments of the invention, the nanostructures are capable of forming clusters of the nanostructures. According to some embodiments of the invention, the nanostructures are capable of maintaining long range interaction thereamongst.

According to some embodiments of the invention, the liquid composition comprises a buffering capacity greater than a buffering capacity of water.

According to some embodiments of the invention, the liquid composition is capable of altering polarization of light.

According to some embodiments of the invention, the composition further comprises sugar.

According to some embodiments of the invention, the the cryoprotectant is glycerol. According to some embodiments of the invention, the polynucleotide-modifying- enzyme is a restriction enzyme.

According to some embodiments of the invention, the polynucleotide-modifying- enzyme is a polymerase enzyme.

According to some embodiments of the invention, the sugar is sucrose or ficoll. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings: FIG. 1 is a photograph of an agarose gel displaying amplification products of Neo water™ and double distilled water (DDW)-diluted Taq polymerase boiled over time. Taq polymerase (either Peqlab (a) or Biolab (b) originated brands) was diluted in Neowater™ or DDW and incubated at 95 °C for the indicated times.

FIG. 2 is a graph illustrating the effect of DDW vs. Neowater™ on Taq's functionality over time at room temperature (RT). Taq polymerase was diluted 1 :50 in DDW or Neowater™ followed by incubation at RT. Upon completion of incubation period (1-7 days), samples were placed at -20 °C until the end of the experiment. At the end of the experiment, samples were thawed (including non-treated (NT) samples which were kept at -20 °C for the whole length of the experiment) and were used in qPCR reactions as detailed in the Materials and Methods. FIG. 3 is a graph of amplification plots of Neowater™ and DDW-based FF qPCR mix following heat incubation. FF qPCR mix was prepared using either DDW or Neowater™ and an aliquot was removed (triplicate wise; plots showing average) for the indicated incubation period at 40 °C followed by freezing at -20 ⁰C until the end of the experiment. At the end of the experiment, a master base mix containing template, primers, dye and DDW was prepared and added to the samples followed by qPCR. FIG. 4A is a bar graph illustrating the effect of time on Freeze-free (FF) PCR batches. The activities of the PCR mixes were tested on the 22/11/07.

FIG. 4B is a graph comparing the Ct-value ratio of 150407-Cargo to Invitrogen's mix and 150407-4°C to Invitrogen's mix. FIG. 5 is a photograph of a Gel electrophoresis of pUC19 plasmid restricted with BgII following incubation at 37 °C and 47 °C for 5 minutes. Legend: Lane 1, Uncut pUC19; Lane 2, duplicate of BgII diluted in DDW and incubated at 37 ⁰C for 5 minutes; Lane 3, duplicate of BgII diluted in Neowater™ and incubated at 37 °C for 5 minutes; Lane 4, duplicate of BgII diluted in DDW and incubated at 47 ⁰C for 5 minutes; Lane 5, duplicate of BgII diluted in Neowater™ and incubated at 47 °C for 5 minutes.

*- The 1568bp fragment is not visible in the gel due to the fact that it merged with the uncut super-coiled DNA.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of storing polypeptides and compositions for same.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

In order to retain protein activity over an extended period of time, typically proteins are frozen either in the presence or absence of cryoprotectants. However, this process is costly, especially in terms of transport and storage and in the case of particular proteins may be undesirable because of molecular changes.

Whilst reducing the present invention to practice, the present inventors have uncovered that compositions comprising nanostructures (such as described in U.S. Pat.

Appl. Nos. 60/545,955 and 10/865,955, and International Patent Application,

Publication No. WO2005/079153) may be used to preserve the activity of a protein at room temperature. As illustrated hereinbelow and in the Examples section which follows the present inventors have demonstrated that nanostructures and liquid preserved the activity of a polymerase enzyme at room temperature over prolonged periods of time.

Thus, according to one aspect of the present invention, there is provided a method of preserving an activity of an isolated polypeptide, the method comprising storing the polypeptide for at least 6 hours at a temperature above 0 °C with a liquid composition having a liquid and nanostructures, each of the nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state, thereby preserving the activity of the polypeptide.

As used herein, the phrase "preserving an activity of an isolated polypeptide" refers to maintaining the polypeptide in a state such that an activity thereof is at least 50 %, more preferably at least 60 %, more preferably at least 70 %, more preferably at least 80 % and even more preferably at least 90 % of its original activity prior to addition of the liquid composition of the present invention.

According to one embodiment the polypeptide activity is a functional activity. It will be appreciated that the composition of the present invention is capable of preserving the activity of a polypeptide, regardless of the activity itself and as such all polypeptide activities are contemplated by the present invention. Non-limiting examples of polypeptide activities include, enzymatic activities, binding activities and/or structural activities.

As used herein, the phrase "enzymatic activity" refers to the ability of a polypeptide to catalyze a reaction. Enzymes are proteins produced by all living organisms that mediate, cause and/or promote reactions that change a chemical into another type of chemical without themselves being altered or destroyed. Exemplary enzyme classes include oxidoreductases (ECl); transferases (EC2); hydrolases (EC3); lyases (EC4); isomerases (EC5); and ligases (EC6).

The phrase "binding activity", as used herein, refers to the ability of a polypeptide to bind to another macromolecule (e.g. DNA, RNA, protein, carbohydrate, lipid and a combination of same) either specifically (i.e. by recognizing a specific structure or sequence) or non-specifically. The phrase "structural activity" as used herein, refers to an ability of a polypeptide to give structure or support. Typically polypeptides that comprise structural activities are fibrous polypeptides including, but not limited to keratin, actin, calbindin, myosin, elastin, microtubule, tensin, collagen, silk and insect fibers. It will be appreciated that a particular protein may comprise more than one activity - i.e. it may be capable of binding a macromolecule and act as an enzyme in a biological reaction. Thus, the present invention, contemplates preserving at least one activity of an isolated polypeptide, although it also contemplates preserving all activities thereof. As used herein, the phrase "isolated polypeptide" refers to a polypeptide that has been removed from its natural environment. The polypeptide may be further purified following its removal. Thus, according to one embodiment, the polypeptide is substantially free from other macromolecules which are present in its natural environment. According to still another embodiment, the polypeptide is a recombinant polypeptide.

The present invention contemplates preserving the activity of all polypeptides including for example enzymes, antibodies and structural proteins.

According to one embodiment, the enzyme to be stabilized is one which is typically used in the field of molecular biology (i.e. a polynucleotide modifying enzyme). Such enzymes are widely commercially available, such as from New England Biolabs, Promega and Fermentas.

Thus, according to an embodiment of this aspect of the present invention, the enzyme is a restriction enzyme (i.e. a restriction endonuclease). Examples of restriction enzymes that may be stabilized according to the method of the present invention include Aatll, Acc65I, Accl, Acil, AcII, Acul, Afel, AfIII, AfIIII, Agel, Ahdl, AIeI, AIuI, AIwI, AIwNI, Apal, ApaLI, ApeKI, Apol, Ascl, Asel, AsiSI, Aval, Avail, Avrll, Bael, BamHI, Bael, BamHI, Banl, Banll, Bbsl, BbvCI, Bbvl, Bed, Bcgl, BciVI, BcII, Bfal, BmAI, BfuCI, BgII, BgIII, BIpI, Bmel580I, BmgBI, Bmrl, Bmtl, Bpml, BpulOI, BpuEI, BsaAI, BsaBI, BsaHI, Bsal, BsaJI, BsaWI, BsaXI, BseRI, BseYI, Bsgl, BsiEI, BsiHKAI, BsiWI, BsII, BsmAI, BsmBI, BsmFI, Bsml, BsoBI, Bsp 12861, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, Bsrl, BssHII, BssKI, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, Btgl, BtgZI, BtsCI, Btsl, Ca8I, CIaI, CspCI, CviAII, CviKI-1, CviOI, Ddel, Dpnl, DpnII, Dral, Dralll, Drdl, Eael, EagI, Earl, Ecil, EcoNI, DcoO109I, EcoP151, EcoRI, EcoRV, Fatl Faul, Fnu4HI, Fokl Fsel, Fspl, Haell, Haelll, Hgal, Hhal, Hindi, Hindlll, Hinfl, HinPlI, Hpal, Hpall, HphI, Hpyl66II, Hpyl88I, Hpyl88III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, KasI, Mbol, MboII, Mfel, MIuI, MIyI, Mmel, MnII, Mscl, Msel, MsII, MspAlI, Mspl, Mwol, Nael, Narl, Neil, Ncol, Ndel, NgoMIV, NheI, Nhel-HF^™, NIaIII, NIaIV, NmeAIII, NotI, Nrul, Nsil, Nspl, Pad, PaeR7I, Pcil, PfIFI, PfIMI, Phol, PIeI, Pmel, PmII, PpuMI, PshAI, Psil, PspGI, PspOMI, PspXI, Pstl, Pvul, PvuII, PvuII-HF™, Rsal, RsrII, Sad, SacII, Sail, Sall-HF™, Sapl, Sau3AI, Sau96I, Sbfl, Seal, Scal-HF™, ScrFI, SexAI, SfaNI, Sfd, Sfil, Sfol, SgrAI, Smal, SmII, SnaBI, Spel, SphI, Sphl-HF^™, Sspl, Stul, StyD4I, Styl, Swal, Taq^αl, Tfil, TIiI, Tsel, Tsp45I, Tsp509I, TspMI, TspRI, Tthl 1 II, Xbal, Xctnl, Xhol, Xmal, Xmnl, Zral.

According to another embodiment, the enzyme is a homing endonuclease, such as for example I-Ceul, I-Scel, PI-PspI or PI-SeI. According to another embodiment, the enzyme is a nicking endonuclease, such as for example Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, NtBstNBI and Nt.CviPII.

According to still another embodiment, the enzyme is a polymerase, including but not limited to a thermophilic DNA polymerase, a mesophilic DNA polymerase, a reverse transcriptase and an RNA polymerase.

Other enzymes involved in DNA and RNA modifying are also contemplated for stabilization, such as for example, DNA ligases, RNA ligases, RNases, nucleases, methyltransferase, phosphatases, sulfurylases and recombinases.

As mentioned, the present invention also contemplates preserving the activity (i.e. binding activity) of antibodies.

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

The antibodies may also be attached to a detectable moiety. In addition, the antibodies may be monoclonal or polyclonal.

Antibodies are widely commercially available, such as from Santa Cruz, Sigma,

Abeam and Enco. Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane,

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

According to one embodiment, the antibodies are those that are used for research purposes, such as for example flow cytometry, immunoprecipitations, Western blots, immunofluorescent, immunohistochemical staining procedures and also those used for diagnostic and imaging procedures.

Exemplary antibodies that may be stabilized according to the method of the present invention, include, but are not limited to those that recognize mammalian and non-mammalian cellular targets including tumor suppressors, cell cycle regulators, kinases, phophatases, signaling intermediates, structural proteins, transcription factors, lymphocyte signaling molecules, synthesis and degradation machinery proteins and cell adhesion and trafficking proteins

As mentioned, the method of the present invention is effected by contacting the polypeptide with the composition of the present invention. The contacting may be effected during the isolation process or following the isolation process.

If the polypeptide is already situated in a liquid composition, the present invention contemplates substituting at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, and some say at least 99 % thereof with the liquid composition of the present invention. Methods of substituting one liquid for another are well known in the art, such as by evaporation of the first liquid and subsequent addition of the second or by dialysis. Typically, the composition of the present invention is left in contact with the polypeptide during the entire storage period. The storage period may be any length of time, for example 6 hours, 24 hours, 1 week, 1 month, 2, months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. The length of time for which the polypeptides may be stored typically depends on the storage temperature. Thus, for example a polypeptide which is stored at 0 °C or 4 ⁰C may be stored for a longer period of time without effecting its activity in the composition of the present invention than if it were stored at 25 °C for example. According to one embodiment, the polypeptides are stored at room temperature - i.e. between about 25 ⁰C and 45 °C, or between about 25 ⁰C and 36 °C. As exemplified for a polymerase enzyme, storage in the composition of the present invention preserved the activity of the enzyme for seven months at room temperature.

As mentioned, the composition of the present invention comprises a liquid and nanostructures, each of the nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state,

As used herein the term "nanostructure" refers to a structure on the sub- micrometer scale which includes one or more particles, each being on the nanometer or sub-nanometer scale and commonly abbreviated "nanoparticle". The distance between different elements (e.g., nanoparticles, molecules) of the structure can be of order of several tens of picometers or less, in which case the nanostructure is referred to as a "continuous nanostructure", or between several hundreds of picometers to several hundreds of nanometers, in which the nanostructure is referred to as a "discontinuous nanostructure". Thus, the nanostructure of the present embodiments can comprise a nanoparticle, an arrangement of nanoparticles, or any arrangement of one or more nanoparticles and one or more molecules.

The liquid of the above-described composition is preferably an aquatic liquid e.g., water. According to one preferred embodiment of this aspect of the present invention the nanostructures of the liquid composition comprise a core material of a nanometer size enveloped by ordered fluid molecules, which are in a steady physical state with the core material and with each other. Such a liquid composition is described in U.S. Pat. Appl. Nos. 60/545,955 and 10/865,955 and International Pat. Appl. Publication No.

WO2005/079153 to the present inventor, the contents of which are incorporated herein by reference.

Examples of such core materials include, without being limited to, a ferroelectric material, a ferromagnetic material and a piezoelectric material. A ferroelectric material is a material that maintains, over some temperature range, a permanent electric polarization that can be reversed or reoriented by the application of an electric field. A ferromagnetic material is a material that maintains permanent magnetization, which is reversible by applying a magnetic field. Preferably, the nanostructures retains the ferroelectric or ferromagnetic properties of the core material, thereby incorporating a particular feature in which macro scale physical properties are brought into a nanoscale environment.

The core material may also have a crystalline structure.

As used herein, the phrase "ordered fluid molecules" refers to an organized arrangement of fluid molecules which are interrelated, e.g., having correlations thereamongst. For example, instantaneous displacement of one fluid molecule can be correlated with instantaneous displacement of one or more other fluid molecules enveloping the core material.

As used herein, the phrase "steady physical state" is referred to a situation in which objects or molecules are bound by any potential having at least a local minimum. Representative examples, for such a potential include, without limitation, Van der Waals potential, Yukawa potential, Lenard-Jones potential and the like. Other forms of potentials are also contemplated.

Preferably, the ordered fluid molecules of the envelope are identical to the liquid molecules of the liquid composition. The fluid molecules of the envelope may comprise an additional fluid which is not identical to the liquid molecules of the liquid composition and as such the envelope may comprise a heterogeneous fluid composition. Due to the formation of the envelope of ordered fluid molecules, the nanostructures of the present embodiment preferably have a specific gravity that is lower than or equal to the specific gravity of the liquid.

The fluid molecules may be either in a liquid state or in a gaseous state or a mixture of the two.

A preferred concentration of the nanostrucutures is below 10²⁰ nanostructures per liter and more preferably below 10¹⁵ nanostructures per liter. Preferably a nanostructure in the liquid is capable of clustering with at least one additional nanostructure due to attractive electrostatic forces between them. Preferably, even when the distance between the nanostructures prevents cluster formation (about 0.5-10 μm), the nanostructures are capable of maintaining long-range interactions.

Production of the nanostructures according to this aspect of the present invention may be carried out using a "top-down" process. The process comprises the following method steps, in which a powder (e.g., a mineral, a ceramic powder, a glass powder, a metal powder, or a synthetic polymer) is heated, to a sufficiently high temperature, preferably more than about 700 ⁰C. Examples of solid powders which are contemplated include, but are not limited to, BaTiO₃, WO₃ and Ba₂F₉O₁₂. Surprisingly, the present inventors have shown that hydroxyapetite (HA) may also be heated to produce the liquid composition of the present invention. Hydroxyapatite is specifically preferred as it is characterized by intoxocicty and is generally FDA approved for human therapy.

It will be appreciated that many hydroxyapatite powders are available from a variety of manufacturers such as Sigma Aldrich and Clarion Pharmaceuticals (e.g. Catalogue No. 1306-06-5).

The heated powder is then immersed in a cold liquid, (water), below its density anomaly temperature, e.g., 3 °C or 2 °C. Simultaneously, the cold liquid and the powder are irradiated by electromagnetic RF radiation, preferably above 500 MHz, 750 MHz or more, which may be either continuous wave RF radiation or modulated RF radiation.

It has been demonstrated by the present inventor that during the production process described above, some of the large agglomerates of the source powder disintegrate and some of the individual particles of the source powder alter their shape and become spherical nanostructures. It is postulated [Katsir et ai, "The Effect of rf-

Irradiation on Electrochemical Deposition and Its Stabilization by Nanoparticle Doping", Journal of The Electrochemical Society, 154(4) D249-D259, 2007] that during the production process, nanobubbles are generated by the radiofrequency treatment and cavitation is generated due to the injection of hot particles into water below the anomaly temperature. Since the water is kept below the anomaly temperature, the hot particles cause local heating that in turn leads to a local reduction of the specific volume of the heated location that in turn causes under pressure in other locations. It is postulated that during the process and a time interval of a few hours or less following the process, the water goes through a self-organization process that includes an exchange of gases with the external atmosphere and selective absorption of the surrounding electromagnetic radiation. It is further postulated that the self-organization process leads to the formation of the stable structured distribution composed of the nanobubbles and the nanostructures.

The liquid composition of the present embodiments is characterized by a non- vanishing circular dichroism signal. Circular dichroism is an optical phenomenon that results when a substance interacts with plane polarized light at a specific wavelength. Circular dichroism occurs when the interaction characteristics of one polarized-light component with the substance differ from the interaction characteristics of another polarized-light component with the substance. For example, an absorption band can be either negative or positive depending on the differential absorption of the right and left circularly polarized components for the substance.

It is recognized that non-vanishing circular dichroism signal of the liquid composition indicates that the liquid composition is an optically active medium. Thus, the liquid composition of the present embodiments can alter the polarization of light while interacting therewith. The present inventor postulates that the optical activity of the liquid composition of the present embodiments is a result of the long-range order which is manifested by the aforementioned formation of stable structured distribution of nanobubbles and nanostructures.

Since the nanostructures of the present invention are capable of preserving the activity of a polypeptide at room temperature, polypeptides comprised in same do not have to be frozen. As such, the present invention contemplates compositions of matter comprising polypeptides and the nanostructures of the present invention, the compositions not comprising cryoprotectants. Such freeze-free compositions may be stored for extended periods of time, as detailed herein above.

The present inventors have shown that a composition comprising the nanostructures of the present invention together with a sugar is particularly effective at enhancing preservation of polypeptides at room temperature. Accordingly the present inventors also contemplate compositions of matter comprising polypeptides (e.g. polynucleotide modifying enzymes), the nanostructures of the present invention and sugars (e.g. sucrose or ficoll). An exemplary concentration of sucrose or ficoll is about

2.5 M. Exemplary cryoprotectants include, but are not limited to glycerol, hydroxyethyl starch (HES) ethylene glycol and DMSO.

The polypeptide compositions of the present invention may be packaged in containers that are suitable for storage at room temperature and not those used for storing at freezing temperatures. The packaging may comprise labels indicating that the polypeptides may be stored above 0 ⁰C, or at room temperature and for a predetermined period of time.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of and "consisting essentially of.

The phrase "consisting essentially of means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or

"at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate some embodiments of the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes MII Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorpotaed by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Compositions comprising nanoparticles protect Taq Polymerase from heat stress

The aim of this experiment was to show that (a) Neowater™ (hereafter, NW) can protect Taq polymerase from heat stress. Two commercial Taq polymerase enzymes (Peq-lab and Bio-lab) were checked in a PCR reaction to determine their activities in DDW and NW.

MATERIALS AND METHODS

The Peq-lab samples were prepared as follows:

(a) 20.4 μl of either NW or DDW.

(b) 0.1 μl Taq polymerase (Peq-lab, Taq DNA polymerase, 5 U/ μl) The Bio-lab samples were prepared as follows:

(a) 18.9 μl of either Neo water™ or distilled water (Reverse Osmosis= RO).

(b) 0.1 μl Taq polymerase (Bio-lab, Taq polymerase, 5 U/ μl)

Three samples were made and placed in a PCR machine at constant temperature of 95 °C. Incubation times were: 60, 75 and 90 minutes. Following boiling of the Taq polymerase the following components were added to the reaction tube of the Peq-lab Taq polymerase samples:

2.5 μl 1OX reaction buffer Y (Peq-lab) 0.5 μl dNTPs 1OmM (Bio-lab) 1 μl primer GAPDH mix 10 pmol/ μl 0.5 μl genomic DNA 35 μg/ μl

Five samples were made of each brand of Taq polymerase and placed in a PCR machine at constant temperature of 95 °C. Incubation times were: 60, 75, 90 120 and 150 minutes.

After boiling the taq enzyme the following components were added to the reaction tube of the Biolab Taq polymerase samples: 2.5 μl TAQ 1OX buffer Mg- free (Bio-lab) 1.5 μl MgC12 25 mM (Bio-lab) 0.5 μl dNTPs 1OmM (Bio-lab) 1 μl primer GAPDH mix (10 pmol/ μl) 0.5 μl genomic DNA (35 μg/ μl)

For each treatment (Neowater™ or DDW) a positive and negative control were made (Positive control contained not treated Taq polymerase; Negative control contained not treated Taq polymerase and without DNA template in the reaction).

Samples were placed in a PCR machine with the following conditions: 1. 94 °C - 2'

2. 94 ⁰C - 30"

3. 60 ⁰C - 30" 4. 72 ⁰C - 30" (cycle back to step #2) x30 times (total cycles)

5. 72 °C - 10'

RESULTS As illustrated in Figure 1, Taq polymerase prediluted in NW maintains activity even after 90 minutes at 95 °C. DDW, on the other hand, lost activity after 60 minutes. This result implies that NW might enhance/protect the Taq polymerase enzyme and other enzymatic reactions which are heat dependent or heat sensitive.

EXAMPLE 2

Compositions comprising nanoparticles protect Taq Polymerase at room temperature

The aim of this experiment was to show that NW can stabilize Taq polymerase at room temperature (RT) for prolonged period of times. MATERIALS AND METHODS Taq polymerase (Biolab) was diluted from stock concentration (5U/μl, 50 % glycerol as stated from the manufacturer) to a final concentration of 0.5 U/5μl (1:50 dilution factor): 2μl of Biolab Taq polymerase was mixed into 98 μl of DDW (referred to herein as Working stock). The working stock was left on the bench for the whole period of the experiment during which, each day 5μl of the working stock was removed to strips of PCR tubes (0.2 ml volume) in triplicates (for NW and DDW dilutions, total of 6 samples) and tubes were immediately put in -20 °C. Non-treated samples were prepared and frozen at the same dilutions as the Taq in DDW/NW. At the end of the experiment, all samples were thawed on an ice block and were subjected to a standard qPCR reaction utilizing SYBR green (SG) as a detection dye on an Mx3000p QPCR system (Stratagene) (including not treated samples (NT) that were thawed only prior to performing the PCR amplification itself). Dissociation curves were performed at the end of the experiment so as to verify the authenticity of the products amplified (exclusion of primer-dimer formation).

To the 5μl of treated samples a master mix was prepared as follows: 1. Biolab xlO buffer (supplied with the Taq)

2. Mg²⁺ (25 mM)

3. dNTPs (10 mM each) 4. SG - diluted 1 :20,000 from x 10,000 solution

5. Plasmid DNA harboring the gene to be amplified, PDX (10 pg)

6. Primers set adequate for the amplification of the above gene (10 pmol)

7. DDW up to 20 μl of volume) 15μl of master mix was added to each sample, after which samples were placed into a Real-Time Thermal Cycler type Mx3000P (Stratagene) and a standard program was applied:

Amplification stage 1. 95 ⁰C - 2' 2. 95 °C - 30"

3. 57 ⁰C - 30"

4. 72 ⁰C - 30" (cycle back to step #2) x39 times

Dissociation curve stage 1. 95 °C - r 2. 57 °C - 30" Then incremental temperature increase to 95 °C

3. 95 ⁰C - 30"

RESULTS

As can be seen from Figure 2, Taq diluted in Neowater™ exhibited prolonged activity in comparison to the DDW-diluted Taq samples, which lost its full activity after 4 days. The Neowater™ kept the activity for 7 days and began showing reducing activity after 8 days.

EXAMPLE 3 Freeze Free - A novel PCR/qPCR mix incorporating Neowater™ which displays stability at 40 ⁰C over time

The aim of this experiment was to show that a PCR/qPCR Neowater™-based formulation, termed Freeze Free (hereafter, FF), displays stability at 40 °C for a period of two weeks. MATERIALS AND METHODS

FF qPCR mix was prepared according to the following protocol as DDW and Neowater™ based mix (x4 concentrated): KCl-based Buffer Mg²⁺

Sugar-based stabilizer dNTPs Biolab Taq Polymerase

DDW/ Neowater™

Five microliters of each mix were aliquoted (triplicate-wise) into 0.2 ml PCR tubes and immediately sealed and incubated at 40 ⁰C for the indicated time points.

Each day a triplicate of DDW and Neowater™ was removed from 40 ⁰C and frozen at - 20 ⁰C until the end of the experiment. At the end of the experiment a base mix was prepared as follows:

Human Genomic DNA Beta Actin-specific primers set SYBR Green dye DDW

Fifteen microliters of the base mix were aliquoted into each sample and samples were placed into a Real-Time Thermal Cycler type Mx3000P (Stratagene) and a standard program was applied:

Amplification stage 1. 95 ⁰C - 2'

2. 95 °C - 30"

3. 55 °C - 30"

4. 72 ⁰C - 30" (cycle back to step #2) x39 times

Dissociation curve stage 1. 95 ⁰C - 1'

2. 55 °C - 30" Then incremental temperature increase to 95 °C

3. 95 °C - 30"

RESULTS As illustrated in Figure 3, after 1 week at 40 ⁰C both Neowater™ and DDW based FF qPCR mix exhibit similar behavior. This similarity diminishes after an additional week, in which a gap of 21 cycles separates is evident in favor of Neowater ^M

(equivalent of 2²¹ more template than DDW).

EXAMPLE 4 Freeze Free - Stability at room temperature (RT) for prolonged periods of time

The aim of this experiment was to evaluate freeze-free's (FF's) activity over prolong periods of time at RT.

MATERIALS AND METHODS

FF qPCR mix was prepared according to the following protocol as NW/DDW- based mix (x4 concentrated):

KCl-based Buffer

Mg²⁺

Sugar-based stabilizer (sucrose 2.5 M) dNTPs Biolab Taq Polymerase

DDW/NW

At the indicated time points, a triplicate of the FF mix was removed from the stock and was tested in qPCR (triplicate-wise).

Several FF qPCR and non FF mix were prepared as follows: (1) 1 1-06-07 - FF qPCR mix prepared at 1 1/06/07

(2) 15-04-07 - Cargo - FF qPCR prepared 15/04/07, flown back and forth in the passenger seat in the Cargo bay, kept for approximately one month at a warehouse and subsequently stored at room temperature.

(3) 15-04-07 - 4 °C - FF qPCR prepared 15/04/07, (same as Cargo) and stored at 4°C.

(4) KCL-P - KCl-based FF containing the PDX primer set.

(5) NH4-P - (NH₄)₂SO4-based FF containing the PDX primer set.

(6) - KCl-based FF

(7) NH4 - (NH₄)₂SO4-based FF (8) Invitrogen - Invitrogen's Platinum® SYBR® Green qPCR SuperMix-UDG

P/N 56750 - served as a Positive control

A base mix was prepared as follows: (a) Plasmid DNA containing the human PDX gene

(b) Human PDX-specific primers set

(c) SYBR Green dye

(d) DDW Twenty microliters of FF mix were mixed with 20 μl of DDW followed by aliqouting in triplicates. Base mix was prepared and added to the different mixes (in accordance with the specific mix) and samples were placed into a Real-Time Thermal Cycler type Mx3000P (Stratagene) and a standard program was applied:

Amplification stage 1. 95 ⁰C - 2'

2. 95 °C - 30"

3. 57 °C - 30"

4. 72 ⁰C - 30" (cycle back to step #2) x39 times

Dissociation curve stage 1. 95 °C - r

2. 57 °C - 30" Then incremental temperature increase to 95 ⁰C 3. 95 °C - 30"

RESULTS As illustrated in Figures 4A-B, the FF formulation, based on Neowater™ technology retained activity even after seven months at room temperature.

EXAMPLE 5 Stability of restriction enzymes with Neowater™ The aim of the experiments is to evaluate whether Neowater™ protects/stabilizes restriction enzymes when incubated under heat conditions in the presence of Neowater™ as compared to DDW. In this experiment, BgII was tested for it's ability to double digest pUC19 plasmid at positions 251 and 1819 following heat incubation (generating two fragments, 1568 bp and 1 1 18 bp in length) MATERIALS AND METHODS

1. 4 μl of BgII restriction enzyme (New England Biolabs, lOu/μl ) were diluted in 36 μl of Neowater™ and DDW (MiIiQ) (to reach a concentration of lu/μl). 2. Enzyme suspension (both Neowater™ and DDW) were further diluted to reach a working concentration of lu/36μl which was then aliquoted to duplicate PCR tube and incubated at 37 °C for 5 minutes or 47 ⁰C for five minutes; Control samples were left at 4 °C till the end of the incubation period. 3. Upon completion of incubation period, samples were added with a mix of pUC19 (500 ng/μl) and the appropriate buffer (Buffer 3, New England Biolabs).

4. Following pipetation, samples were put back in a thermal cycler heat block and were incubated at 37 °C for 90 minutes; In addition, a fresh restriction reaction mix was prepared as a positive control. Samples were then loaded on a 1% agarose gel and were visualized via UV table.

RESULTS

The results of the restriction analysis are illustrated in Figure 5. As can be seen, Neowater™ can maintain activity of restriction enzymes, in this case BgII, even after heat incubation at 37 ⁰C and 47 ⁰C.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of preserving an activity of an isolated polypeptide, the method comprising storing the polypeptide for at least 6 hours at a temperature greater than 0 °C with a liquid composition having a liquid and nanostructures, each of said nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, said core material and said envelope of ordered fluid molecules being in a steady physical state, thereby preserving the activity of the polypeptide.

2. The method of claim 1, wherein the activity is an enzymatic activity.

3. The method of claim 1, wherein the activity is a binding activity.

4. The method of claim 1, wherein the polypeptide is selected from the group consisting of an enzyme, an antibody and a structural protein.

5. The method of claim 4, wherein the polypeptide is an enzyme.

6. The method of claim 5, wherein said enzyme is a restriction enzyme.

7. The method of claim 5, wherein said enzyme is a polymerase enzyme.

8. The method of claim 1, wherein said temperature is between about 25 °C and 45 °C.

9. The method of claim 1, wherein at least a portion of said fluid molecules are identical to molecule of said liquid.

10. The method of claim 1, wherein said at least a portion of said fluid molecules are in a gaseous state.

11. The method of claim 1, wherein a concentration of said nanostructures is lower than 10²⁰ nanostructures per liter.

12. The method of claim 1, wherein said nanostructures are capable of forming clusters of said nanostructures.

13. The method of claim 1, wherein said nanostructures are capable of maintaining long range interaction thereamongst.

14. The method of claim 1, wherein said liquid composition comprises a buffering capacity greater than a buffering capacity of water.

15. The method of claim 1, wherein said liquid composition is capable of altering polarization of light.

16. A composition of matter comprising a polypeptide and a liquid composition having a liquid and nanostructures, each of said nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, said core material and said envelope of ordered fluid molecules being in a steady physical state, the composition not comprising a cryoprotectant.

17. The composition of matter of claim 16, further comprising sugar.

18. The composition of matter of claim 16, wherein the polypeptide is selected from the group consisting of an enzyme, an antibody and a structural protein.

19. The composition of matter of claim 16, wherein the polypeptide is an enzyme.

20. The composition of matter of claim 19, wherein said enzyme is a restriction enzyme.

21. The composition of matter of claim 19, wherein said enzyme is a polymerase enzyme.

22. The composition of matter of claim 16, wherein at least a portion of said fluid molecules are identical to molecule of said liquid.

23. The composition of matter of claim 16, wherein said at least a portion of said fluid molecules are in a gaseous state.

24. The composition of matter of claim 16, wherein a concentration of said nanostructures is lower than 10²⁰ nanostructures per liter.

25. The composition of matter of claim 16, wherein said nanostructures are capable of forming clusters of said nanostructures.

26. The composition of matter of claim 16, wherein said nanostructures are capable of maintaining long range interaction thereamongst.

27. The composition of matter of claim 16, wherein said liquid composition comprises a buffering capacity greater than a buffering capacity of water.

28. The composition of matter of claim 16, wherein said liquid composition is capable of altering polarization of light.

29. The composition of matter of claim 16, wherein the cryoprotectant is glycerol.

30. A composition of matter comprising a polynucleotide-modifying- enzyme, a sugar and a liquid composition having a liquid and nanostructures, each of said nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, said core material and said envelope of ordered fluid molecules being in a steady physical state.

31. The composition of matter of claim 30, wherein said polynucleotide- modifying-enzyme is a restriction enzyme.

32. The composition of matter of claim 30, wherein said polynucleotide- modifying-enzyme is a polymerase enzyme.

33. The composition of matter of claim 30, wherein at least a portion of said fluid molecules are identical to molecule of said liquid.

34. The composition of matter of claim 30, wherein said at least a portion of said fluid molecules are in a gaseous state.

35. The composition of matter of claim 30, wherein a concentration of said nanostructures is lower than 10²⁰ nanostructures per liter.

36. The composition of matter of claim 30, wherein said nanostructures are capable of forming clusters of said nanostructures.

37. The composition of matter of claim 30, wherein said nanostructures are capable of maintaining long range interaction thereamongst.

38. The composition of matter of claim 30, wherein said liquid composition comprises a buffering capacity greater than a buffering capacity of water.

39. The composition of matter of claim 30, wherein said liquid composition is capable of altering polarization of light.

40. The composition of matter of claim 30, wherein said sugar is sucrose or ficoll.