CAVITY-CONTAINING OLIGOMERS AND POLYMERS EFFECTIVE IN

CRY OPRE SERVATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 62/586,904, filed November 16, 2017, and U.S. Provisional Application No. 62/587,482, filed November 17, 2017, the disclosures of which are herein incorporated by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Phase I Small Business Innovation Research Award No. 1622240, awarded by the National Science Foundation. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Cryoprotective agents (CPAs) are critical additives that improve the post-thaw viability of cryopreserved biological systems from cells to large and complex tissues/organs by preventing ice crystal nucleation and growth. Membrane permeable CPAs also prevent osmotic shrinkage of the cells and reduce the volume of available water by penetrating and equilibrating across the cell membrane. All known CPAs exhibit various levels of cytotoxicity at effective concentration, which may be decreased by reducing the CPA loading temperature and exposure time. However, most CPAs become effectively impermeable at sub-zero temperatures. This is largely associated with the dysfunction of transmembrane protein channels at low temperature that serve as mass transport gates. As can be seen in FIG. 1, transmembrane protein channels function normally at physiological temperature (L); however, during the CPA loading process, transmembrane protein channels will become unable to function at low temperature leading to impermeability of the cell membrane leading to lethal cell shrinkage (M). Longer loading times lead to greater cell shrinkage (R) and cell membrane damage. [0004] An unbalanced gradient of CPA and salt across the cell membrane leads to detrimental cell shrinkage as water effluxes out of the cell. This could be detrimental to the fate of large, complex solid tissues/organs by damaging cell-cell and cell-matrix junctions. As shown in FIG. 2, cell shrinkage is more detrimental to large complex tissues/organs due to cell-cell and cell- matrix interactions.

[0005] As such, there is a need in the art for a novel cellular delivery system that can effectively facilitate the intracellular delivery and transmembrane equilibration of CPAs at sub 0 °C and cryogenic temperatures. The present disclosure satisfies this need and provides other advantages as well.

BRIEF SUMMARY OF THE INVENTION

[0006] In brief, the present invention relates to in part the following: (1) use of certain cavity- containing oligomers and polymers to enhance cryopreservation of cells, tissues, and organs, (2) incorporating an advanced cryoprotectant solution, and (3) a novel method of cryoprotectant cellular or intracellular delivery.

[0007] Firstly, the present invention uses size- and function-tunable, cavity-containing oligomers and polymers to aid in cryopreservation by serving as synthetic membrane channels that remain open and capable of molecular transport at subzero temperature to effectively deliver CPAs across the cell membrane as in FIG. 3. Relatedly, this aspect also pertains to the delivery of therapeutic agents and diagnostic agents to cells, tissues or organs. In particular, the present invention relates, in part, to compositions and methods that use a class of compositions such as oligomers and polymers that automatically fold into helices with large (i.e., 8 A to 50 A) tubular cavities. The interior of the helical composition features amide-0 atoms, which make the tubular cavities hydrophilic. The internal diameters of the helices are adjustable by combining meta- and para-di substituted benzene rings or by using larger aromatic rings, such as derivatives of naphthalene and anthracene. As nanotubes, these helices are useful as artificial pore-forming agents which are readily functionalized.

[0008] In part, the present invention relates to the use of cavity-containing oligomers and polymers to allow a significant decrease in both the CPA exposure time and loading/unloading temperature during freezing. The supramolecular assembly feature, or other engineered environmentally sensitive stimuli, allows organic nanopores to seal off at or above physiological temperature, which offers minimum interference of membrane integrity and low toxicity.

[0009] In some embodiments, the oligomers and polymers disclosed and discussed in US Patent No. 6,495,680 Bl (Gong; herein incorporated by reference in its entirety for all purposes) find utility in cryopreservation and drug or therapeutic agent delivery compositions and methods of the present invention.

[0010] In one aspect, provided herein is a composition for cryopreservation of a population of cells, a biological tissue, or an organ. In some embodiments, the composition comprises: (a) a cryoprotectant solution comprising a compound selected from the group consisting of an ionic species, a penetrating cryoprotectant, a non-penetrating cryoprotectant, an antioxidant, a cell membrane stabilizing compound, a channel-forming compound, an alcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinylpyrrolidone, polyvinyl alcohol, hyaluronan, formamide, a natural or synthetic hydrogel, and a combination thereof; and (b) an oligomer or polymer comprising a backbone that comprises a structure according to Formula (I):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CFb, -CH2CH2OCH3, -CFhCFhCXCFhCFhC^zCFb, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C 1-12 alkylamino, -OH, -SH, -NH2, -NHY, -N ⁺ R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein n is an integer having a value of 4 to 100.

[0011] In some embodiments, n has a value of 6 to 64. In some embodiments, n has a value of 6 to 16. In some embodiments, n is 6, 8, 10, or 12.

[0012] In some embodiments, the backbone of the oligomer or polymer comprises a plurality of aromatic substituents linked by at least one amide group, wherein the backbone is curved due at least in part to intramolecular hydrogen bonds that rigidify the amide linkage of each amide group to each aromatic substituent and at least in part to an interaction between the aromatic substituents, whereby the curved backbone is stabilized, and wherein the backbone comprises a structure according to Formula (II):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH2OCH3, -CFhCFhCXCFhCFhC^zCFb, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C 1-12 alkylamino, -OH, -SH, -NH2, -NH3 ⁺ , -N ⁺ Rri, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein m is an integer having a value of 2 to 50. In some embodiments, m has a value of 2 to 26. In some embodiments, m has a value of 2 to 8. [0013] In some embodiments, the backbone of the oligomer or polymer comprises a structure according to Formula (III):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CFb, -CH2CH2OCH3, -CFhCFhCXCFhCFhC^zCFb, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C 1-12 alkylamino, -OH, -SH, -NH2, -NHV, -N ⁺ Rri, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; and wherein z is an integer having a value of 1 to 10.

[0014] In some embodiments, the backbone of the oligomer or polymer comprises a structure according to Formula (IV):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH2OCH3, -CH2CH20(CH2CH20) _Z CH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C 1-12 alkylamino, -OH, -SH, -NH2, -NH ₃ ⁺ , -N ⁺ Rri, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein m is an integer having a value of 2 to 50. In some embodiments, m has a value of 2 to 26. In some embodiments, m has a value of 2 to 8.

[0015] In some embodiments, the aryl group is selected from the group consisting of phenyl, benzyl, napthyl, alkyl, and an aryl group with a polar terminal functional end. In some embodiments, the polar terminal functional end is selected from the group consisting of -OH, - COOH, -NH ₂ , -NH ₃ ⁺ , -N ⁺ R'3, -CH2CH2C6H5, CH2C6H4-P-OH, -CH2COOCH3, -CH2CONH2, - CH2CH2CONH2, and -CH2COOCH2-(3-indolyol), wherein R’ can be any alkyl or aryl group. In some embodiments, at least one instance of R is an oxy ether or thioether. In some embodiments, at least one instance of R comprises the structure.

[0016] In some embodiments, the backbone of the oligomer or polymer comprises a structure according to Formula (VII): (vii),

wherein X and Y are independently selected from the group consisting of H, optionally substituted Ci-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C 1-12 alkylamino, -OH, -SH, -NH2, -NHY, -NkRri, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; and wherein m is an integer having a value of 2 to 50. In some embodiments, m has a value of 2 to 26. In some embodiments, m is 3, 4, 5, 6, 7, or 8.

[0017] In another aspect, provided herein is a composition for cryopreservation of a population of cells, a biological tissue, or an organ. In some embodiments, the composition comprises: (a) a cryoprotectant solution comprising a compound selected from the group consisting of an ionic species, a penetrating cryoprotectant, a non-penetrating cryoprotectant, an antioxidant, a cell membrane stabilizing compound, a channel-forming compound, an alcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinylpyrrolidone, polyvinyl alcohol, and a combination thereof; and (b) an oligomer or polymer comprising a backbone that comprises a plurality of aromatic substituents linked by at least one amide group, wherein the backbone is curved due at least in part to intramolecular hydrogen bonds that rigidify the amide linkage of each amide group to each aromatic substituent and at least in part to an interaction between the aromatic substituents, whereby the curved backbone is stabilized, and wherein the backbone comprises a structure according to Formula (V):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH2OCH3, -CH2CH20(CH2CH20) _Z CH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C 1-12 alkylamino, -OH, -SH, -NH2, -NH ₃ ⁺ , -N ⁺ R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein m is an integer having a value of 2 to 50. In some embodiments, m has a value of 2 to 26. In some embodiments, m has a value of 2 to 8.

[0018] In some embodiments, the backbone of the oligomer or polymer comprises a structure according to Formula (VI):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH2OCH3, -CH2CH20(CH2CH20) _Z CH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C 1-12 alkylamino, -OH, -SH, -NH2, -NH ₃ ⁺ , -N ⁺ R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; and wherein z is an integer having a value of 1 to 10.

[0019] In some embodiments, the aryl group is selected from the group consisting of phenyl, benzyl, napthyl, alkyl, and an aryl group with a polar terminal functional end. In some embodiments, the polar terminal functional end is selected from the group consisting of -OH, - COOH, -NH2, -NH ₃ ⁺ , -N ⁺ R'3, -CH2CH2C6H5, CH2C6H4-P-OH, -CH2COOCH3, -CH2CONH2, - CH2CH2CONH2, and -CH2COOCH2-(3-indolyol). In some embodiments, at least one instance of R is an oxyether or thioether. In some embodiments, at least one instance of R comprises the structure:

[0020] In some embodiments, the oligomer or polymer comprises the structure:

[0021] In some embodiments, the alcohol is selected from the group consisting of propylene glycol, ethylene glycol, glycerol, methanol, butylene glycol, adonitol, ethanol, trimethylene glycol, diethylene glycol, polyethylene oxide, erythritol, sorbitol, xythyritol, polypropylene glycol, 2- methyl-2,4-pentanediol (MPD), mannitol, inositol, dithioritol, 1, 2-propanediol, and a combination thereof. In some embodiments, the sugar is a monosaccharide, disaccharide, or polysaccharide. In some embodiments, the oligomer or polymer is present at a concentration of about 100 nM to about 1,000 mM.

[0022] In another aspect, provided herein is a method for cryopreserving a population of cells with improved cell viability. In some embodiments, the method comprises contacting the population of cells with a cryopreservation composition of the present invention to produce a cryopreserved population of cells. In some embodiments, the method further comprises cooling the population of cells to a temperature of from about 0 °C to about -200 °C. In some embodiments, the population of cells is cooled for a time period of at least about 3 hours.

[0023] In some embodiments, the population of cells comprises a tissue of any kind or an organ of any kind. In some embodiments, the tissue or organ is a bioengineered tissue of any kind or bioengineered organ of any kind. In some embodiments, the population of cells is selected from the group consisting of primary cells, heart cells, liver cells, lung cells, kidney cells, pancreatic cells, gastric cells, intestinal cells, muscle cells, skin cells, neural cells, blood cells, immune cells, fibroblasts, genitourinary cells, bone cells, stem cells, sperm cells, oocytes, embryonic cells, epithelial cells, endothelial cells, and a combination thereof. In some instances, the cell are mammalian cells. In particular instances, the cells are human cells.

[0024] In another aspect, provided herein is a method for cry opreserving a biological tissue with improved biological tissue viability. In some embodiments, the method comprises: (a) contacting the biological tissue with a cryopreservation composition of the present invention; and (b) cooling the biological tissue to a temperature of from about 0 °C to about -200 °C over a time period of at least about 3 hours to produce a cryopreserved biological tissue. In some embodiments, the tissue is selected from the group consisting of heart tissue, liver tissue, lung tissue, kidney tissue, gall bladder tissue, reproductive system tissue, pancreatic tissue, gastric tissue, intestinal tissue, muscle tissue, skin tissue, neural tissue, blood, genitourinary tissue, bone, epithelial tissue, endothelial tissue, corneal tissue, heart valve tissue, and a combination thereof. In some embodiments, the tissue is a bioengineered tissue of any kind.

[0025] In another aspect, provided herein is a method for cry opreserving an organ with improved organ viability. In some embodiments, the method comprises: (a) contacting the organ with a cryopreservation composition of the present invention; and (b) cooling the organ to a temperature of from about 0 °C to about -200 °C over a time period of at least about 3 hours to produce a cryopreserved organ. In some embodiments, the organ is selected from the group consisting of heart, liver, lung, kidney, a reproductive organ, pancreas, stomach, gall bladder, intestine, muscle, skin, bone, and a combination thereof. In some embodiments, the organ is a bioengineered organ of any kind.

[0026] In another aspect, provided herein is a method for cryopreserving a population of cells, a tissue, or an organ with improved cell, tissue, or organ viability, the method comprising: (a) cooling the population of cells, tissue, or organ to a temperature of about 4 °C; and (b) contacting the population of cells, tissue, or organ with a cryopreservation composition of the present invention at the temperature of about 4 °C.

[0027] In another aspect, provided herein is a method for delivering a therapeutic agent or diagnostic agent into a cell, a population of cells, a tissue, or an organ, the method comprising contacting the cell, population of cells, tissue, or organ with the therapeutic agent or diagnostic agent and a cryopreservation composition of the present invention. In some embodiments, the therapeutic agent or diagnostic agent is selected from the group consisting of a drug, a small molecule, a nutrient, an imaging agent, and a combination thereof. In some embodiments, the imaging agent is selected from the group consisting of a radioactive tracer, a fluorescent tracer, and a combination thereof. In some embodiments, the therapeutic agent or diagnostic agent is contacted with the cell, population of cells, tissue, or organ after cooling the cell, population of cells, tissue, or organ to a temperature of about 4 °C or lower. In some instances, the cell, population of cells, tissue, or organ is cooled to a temperature of about 4 °C.

[0028] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows that (left) transmembrane protein channels function normally at physiological temperature but are (middle) impaired at low temperature during CPA loading process, leading to impermeability of the cell membrane to detrimental cell shrinkage. The graph on the right shows that longer loading times lead to great cell shrinkage. [0030] FIG. 2 shows that cell shrinkage is more detrimental to large complex tissues/organs due to cell-cell and cell-matrix interactions.

[0031] FIG. 3 shows an illustration of a partial element of the present invention.

[0032] FIG. 4 shows a schematic diagram showing that the macrocycle (labeled“a”) stacks into tubular assemblies.

[0033] FIGS. 5A-5C show schematic diagrams depicting routes of syntheses of monomers for type I oligomers and polymers using derivatives of 2,4-dihydroxy-5-nitrobenzoic acid.

[0034] FIGS. 6A-6C show schematic diagrams depicting routes of syntheses for type I oligomers and polymers.

[0035] FIGS. 7 A and 7B show schematic diagrams depicting routes of syntheses for dimers for syntheses of type II oligomers and polymers.

[0036] FIGS. 8A and 8B show schematic diagrams depicting routes of syntheses of type II oligomers.

[0037] FIG. 9 shows fast and reliable CPA loading via incorporation of cavity-containing oligomers and polymers, resulting in 50% less time required to reach effective intracellular concentration compared to current methods with high osmotic pressure. This leads to much less cellular injury.

[0038] FIG. 10 shows the results of a cell toxicity study performed on K562 cells in which cavity-containing oligomer and polymer Compounds 1 or 2 was added to the cell culture media. Since both Compound 1 and 2 solutions contained 1% DMSO, a sample in which no cavity- containing oligomer or polymer compound was present, but contained 1% DMSO, was used to serve as a control. Serial dilutions were performed in order to test different concentrations of Compounds 1 and 2. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively (i.e., the value of m in Formula (II) is 8 and 16, respectively).

[0039] FIG. 11 shows the results of a cryopreservation assay performed on K562 cells, comparing serial dilutions of cavity-containing oligomer and polymer Compounds 1 and 2 with both solutions containing 1% DMSO in DMEM with 40% FBS to a solution containing 1% DMSO alone with 40% FBS in DMEM. An additional condition using PBS as the freezing medium served as a control. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively (i.e., the value of m in Formula (II) is 8 and 16, respectively).

[0040] FIG. 12 shows the results of a cryopreservation assay performed on K562 cells, comparing serial dilutions of cavity-containing oligomer and polymer Compounds 1 and 2 with both solutions containing 3% glycerol and 1% DMSO with 40% FBS in DMEM to a solution containing 3% glycerol and 1% DMSO with 40% FBS in DMEM without Compound 1 or 2. A condition with Compound 1 alone in DMEM acted as a control to demonstrate it is not an ice- interacting compound. A solution containing DMEM and 40% FBS served as another control. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively (i.e., the value of m in Formula (II) is 8 and 16, respectively).

[0041] FIG. 13 shows the results of a cryopreservation assay performed on K562 cells, comparing a solution of 3% glycerol and 1% DMSO with serial dilutions of cavity-containing oligomer and polymer Compounds 1 and 2 with 40% FBS in DMEM to a 3% glycerol and 1% DMSO solution with 40% FBS in DMEM. A solution containing 3% glycerol with 40% FBS in DMEM and a solution only containing PBS served as another control. The images directly below the condition represent the confocal microscopy images of the condition using calcein AM to indicate cell viability. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively (i.e., the value of m in Formula (II) is 8 and 16, respectively).

[0042] FIG. 14 shows the results of a cryopreservation assay performed on K562 cells, comparing a solution of 3% sorbitol and 1% DMSO with serial dilutions of cavity-containing oligomer and polymer Compounds 1 and 2 with 40% FBS in DMEM to a 3% sorbitol and 1% DMSO solution with 40% FBS in DMEM. A condition with Compound 1 alone in DMEM acted as a control to demonstrate it is not an ice-interacting compound. A solution containing 3% sorbitol with 40% FBS in DMEM served as another control. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively (i.e., the value of m in Formula (II) is 8 and 16, respectively).

[0043] FIG. 15 shows the results of a cryopreservation assay performed on K562 cells, comparing solutions containing 64 mM of cavity-containing oligomer and polymer Compounds 1, 3, and 4 mixed with either 2% DMSO or 0.6% DMSO. Other formulas are included for comparison containing 40% FBS in DMEM and either 2% DMSO or 0.6% DMSO. A condition with DMEM served as a control. Compounds 1, 3, and 4 are cavity-containing oligomers which vary in residue length. Compounds 1, 3, and 4 comprise the structure of Formula (II) and have 8, 6, and 10 residues, respectively (i.e., the value of m in Formula (II) is 8, 6, and 10, respectively).

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0044] The banking of cells and tissues at low temperatures using cryopreservation is critical for many biological products and applications, but remains a significant problem that has yet to allow the successful full recovery of viable therapeutic cells, tissues, or organs. Cryopreservation offers a revolutionizing opportunity to achieve long-term biobanking. In nature, species including microorganisms, plants, insects, fish, and amphibians can survive at ultralow temperature with controlled ice growth to form stable glasses in which the intracellular medium contains large amounts of saccharides (particularly glucose, trehalose and sucrose) and glycols to protect from chilling injury. Inspired by nature, small molecule cryoprotective agents (CPAs) have been widely applied in cryopreservation by biologists. However, a successful preservation and restoration of a medically relevant organ has not occurred. In mammalian cells, intracellular CPA concentration is limited due to cellular regulation, thus outsourcing is required to transport 1.0-2.0 M CPAs across cell membrane during cryopreservation. This remains a significant challenge.

[0045] Biological ions and molecular channels play many functions that are vital to the survival of cells and organisms. At low ( e.g ., sub-zero) temperatures, the effectiveness of most CPAs is impeded due to the fact that biological channels (e.g., glucose transporters (GLUTs) and aquaporins) typically become dysfunctional and can no longer serve as gates for transporting CPAs. Long loading time and disruptive localized pressure is often required to reach effective concentration of intracellular CPAs. As a circumstance, the cell can experience detrimental shrinkage caused by dehydration, which leads to apoptosis after thawing. This is more lethal for large complex solid organs/tissues compared to single cells, as the functional junctions between cells are permanently damaged. For instance, the upload of CPAs to rabbit kidney during vitrification can take up to 3 hours, as the loading rate significantly decreases while temperature drops, which surges toxicity level and post-thaw death rate.

[0046] Eroglu and Toner et. al. (Nat. Biotechnol. 2000 Feb; l8(2): l63-7) demonstrated that intracellular delivery of 0.2 M trehalose significantly improved post-thaw cell viability when transported through a genetically engineered variant of the pore forming toxin, a-hemolysin. The large lumen of a-hemolysin (14Ά) can allow sufficient transport of large molecules like trehalose, but replaces adverse CPA cytotoxicity with cytotoxicity caused by a lack of selective transport and large pore size, especially at physiological temperature. A rational blockage strategy was critical to reduce toxicity and the genetically engineered pore forming protein was blocked with the addition of Zn ²⁺ ion for 18 hours.

[0047] Overcoming the deficiencies of natural protein channels by developing synthetic channels capable of mimicking natural systems has attracted the interest of many chemists over the last several decades. These channels provide significant advantages such as synthetic efficiency and structure diversity to engineer various functions such as responsiveness and selective transport. Many systems focused on selective ion transport have been developed.

[0048] For instance, a variety of rigid macrocyclic molecules have been discovered, many of which were subsequently found to stack into nanotubular assemblies that contain cylindrical inner pores of different size and properties ranging from 5 to 29 A. These pore-containing tubular stacks promoted ion conductivity and selectivity across the cell membrane. For example, aromatic oligoamide macrocycles disclosed in US Patent No. 6,495,680 B 1 were found to stack into tubular structures that transported ions across lipid bilayers in very large conductance. It was found that macrocycles rapidly increased proton flux across lipid bilayers. The formation of transmembrane channels with a cavity size of 8.5 A was confirmed by measured values of single channel conductance (800-900 pS) that are much larger than those of typical synthetic or natural ion channels. The observed values were similar to those of large protein nanopores. These observations are consistent with the tubular assembly shown in FIG. 4.

[0049] Cavity-containing oligomers and polymers can undergo tubular stacking into self- assembling nanotubes with rational design. The inner pores of these nanotubes not only mediate efficient and selective molecular and ion transport, but also allow structural and functional fine- tuning. These previous studies have laid a solid foundation for constructing channel-forming molecules that facilitate the transport and uptake of CPAs.

II. Abbreviations and Definitions

[0050] The abbreviations used herein are conventional, unless otherwise defined.

[0051] The terms“a,”“an,” or“the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells and reference to“the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0052] The term“about” as used herein to modify a numerical value indicates a defined range around that value. If“X” were the value,“about X” would indicate a value from 0.9X to 1.1X, and more preferably, a value from 0.95X to 1.05X. Any reference to“about X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus,“about X” is intended to teach and provide written description support for a claim limitation of, e.g .,“0.98X.”

[0053] The term“alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C1-2, C1-3, Ci-4, Ci-5, C1-6, Ci-7, Ci-8, Ci-9, Ci-io, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. For example, Ci-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 30 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or un substituted. Alkyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0054] The term“alkenyl” refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, l-butenyl, 2-butenyl, isobutenyl, butadienyl, l-pentenyl, 2-pentenyl, isopentenyl, l,3-pentadienyl, l,4-pentadienyl, l-hexenyl, 2-hexenyl, 3-hexenyl, l,3-hexadienyl, l,4-hexadienyl, l,5-hexadienyl, 2,4-hexadienyl, or l,3,5-hexatrienyl. Alkenyl groups can be substituted or unsubstituted. Alkenyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0055] The term“alkynyl” refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, l-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, l-pentynyl, 2-pentynyl, isopentynyl,

1.3-pentadiynyl, l,4-pentadiynyl, l-hexynyl, 2-hexynyl, 3-hexynyl, l,3-hexadiynyl,

1.4-hexadiynyl, l,5-hexadiynyl, 2,4-hexadiynyl, or l,3,5-hexatriynyl. Alkynyl groups can be substituted or unsubstituted. Alkynyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0056] The term“alkylene” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene group. For instance, a straight chain alkylene can be the bivalent radical of -(CH2)n-, where n is any number of suitable carbon atoms. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene. Alkylene groups can be substituted or unsubstituted. Alkylene groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0057] The term“alkenylene” refers to an alkenyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkenylene can be linked to the same atom or different atoms of the alkenylene. Alkenylene groups include, but are not limited to, ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene and hexenylene. Alkenylene groups can be substituted or unsubstituted. Alkenylene groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0058] The term“alkynylene” refers to an alkynyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkynylene can be linked to the same atom or different atoms of the alkynylene. Alkynylene groups include, but are not limited to, ethynylene, propynylene, isopropynylene, butynylene, sec-butynylene, pentynylene and hexynylene. Alkynylene groups can be substituted or unsubstituted. Alkynylene groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0059] The term“halogen” or“halo” refers to fluorine, chlorine, bromine and iodine.

[0060] The term“amine” or“amino” refers to an -N(R)2 group where the R groups can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, among others. The R groups can be the same or different. The amino groups can be primary (each R is hydrogen), secondary (one R is hydrogen) or tertiary (each R is other than hydrogen). The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0061] The term“hydroxyl” or“hydroxy” refers to an -OH group. The hydroxyl can be at any suitable carbon atom.

[0062] The term“thiol” refers to an -SH group. The thiol group can be at any suitable carbon atom.

[0063] The term“oxo” refers to a double bonded O group (=0, -C(O)-). The oxo group can be at any suitable carbon atom. [0064] The term“thioxo” refers to a double bonded S group (=S). The thioxo group can be at any suitable carbon atom.

[0065] The term“nitro” refers to a -NO2 group. The nitro group can be at any suitable carbon atom.

[0066] The term“carboxy” refers to a carboxylic acid group of the formula -C(0)OH or -CO2H.

[0067] The term“cycloalkyl” refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C3-6, C ₄ -6, C5-6, C3-8, C ₄ -8, C5-8, C6-8, C3-9, C3-10, C3-11, and C3-12. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbomane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1, 4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1, 5-isomers), norbornene, and norbornadiene. When cycloalkyl is a saturated monocyclic C3-8 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C3-6 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. Cycloalkyl groups can be optionally substituted with one or more moieties selected from alkyl, alkenyl, alkynyl, halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, thiol, nitro, oxo, thioxo, and cyano. For example, cycloalkyl groups can be substituted with C1-6 alkyl or oxo (=0), among many others.

[0068] The term“heterocycloalkyl” refers to a saturated ring system having from 3 to 12 ring members and from 1 to 4 heteroatoms of N, O, or S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si, and P. The heteroatoms can also be oxidized to form moieties including, but not limited to, -S(O)- and -S(0)2-. Heterocycloalkyl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms can be included in the heterocycloalkyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. The heterocycloalkyl group can include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4- isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. The heterocycloalkyl groups can also be fused to aromatic or non-aromatic ring systems to form members including, but not limited to, indoline. Heterocycloalkyl groups can be unsubstituted or substituted. Heterocycloalkyl groups can be optionally substituted with one or more moieties selected from alkyl, alkenyl, alkynyl, halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. For example, heterocycloalkyl groups can be substituted with Ci- ₆ alkyl or oxo (=0), among many others.

[0069] The heterocycloalkyl groups can be linked via any position on the ring. For example, aziridine can be 1- or 2-aziridine, azetidine can be 1- or 2- azetidine, pyrrolidine can be 1-, 2-, or 3 -pyrrolidine, piperidine can be 1-, 2-, 3-, or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or 4- pyrazolidine, imidazolidine can be 1-, 2-, 3-, or 4-imidazolidine, piperazine can be 1-, 2-, 3-, or 4- piperazine, tetrahydrofuran can be 1- or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4-, or 5- oxazolidine, isoxazolidine can be 2-, 3-, 4-, or 5 -isoxazolidine, thiazolidine can be 2-, 3-, 4-, or 5- thiazolidine, isothiazolidine can be 2-, 3-, 4-, or 5- isothiazolidine, and morpholine can be 2-, 3-, or 4-morpholine.

[0070] When heterocycloalkyl includes 3 to 8 ring members and 1 to 3 heteroatoms, representative members include, but are not limited to, pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene, thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, morpholine, thiomorpholine, dioxane, and dithiane. Heterocycloalkyl can also form a ring having 5 to 6 ring members and 1 to 2 heteroatoms, with representative members including, but not limited to, pyrrolidine, piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, and morpholine. [0071] The term“aryl” refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be substituted or unsubstituted. Aryl groups can be optionally substituted with one or more moieties selected from alkyl, alkenyl, alkynyl, halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0072] The term“heteroaryl” refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, O or S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si, and P. The heteroatoms can also be oxidized to form moieties including, but not limited to, -S(O)- and -S(0)2-. Heteroaryl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms can be included in the heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups can have from 5 to 8 ring members and from 1 to 4 heteroatoms, or from 5 to 8 ring members and from 1 to 3 heteroatoms, or from 5 to 6 ring members and from 1 to 4 heteroatoms, or from 5 to 6 ring members and from 1 to 3 heteroatoms. The heteroaryl group can include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3- , 1,2,4- and 1,3, 5 -isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. The heteroaryl groups can also be fused to aromatic ring systems, such as a phenyl ring, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyri dines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups can be substituted or un substituted. Heteroaryl groups can be optionally substituted with one or more moieties selected from alkyl, alkenyl, alkynyl, halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0073] The heteroaryl groups can be linked via any position on the ring. For example, pyrrole includes 1-, 2-, and 3 -pyrrole, pyridine includes 2-, 3-, and 4-pyridine, imidazole includes 1-, 2-, 4-, and 5-imidazole, pyrazole includes 1-, 3-, 4-, and 5-pyrazole, triazole includes 1-, 4-, and 5- triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes 2-, 4-, 5-, and 6- pyrimidine, pyridazine includes 3- and 4-pyridazine, l,2,3-triazine includes 4- and 5-triazine, l,2,4-triazine includes 3-, 5-, and 6-triazine, 1,3, 5-triazine includes 2-triazine, thiophene includes 2- and 3- thiophene, furan includes 2- and 3-furan, thiazole includes 2-, 4-, and 5-thiazole, isothiazole includes 3-, 4-, and 5-isothiazole, oxazole includes 2-, 4-, and 5-oxazole, isoxazole includes 3-, 4- , and 5-isoxazole, indole includes 1-, 2-, and 3-indole, isoindole includes 1- and 2-isoindole, quinoline includes 2-, 3-, and 4-quinoline, isoquinoline includes 1-, 3-, and 4-isoquinoline, quinazoline includes 2- and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline, benzothiophene includes 2- and 3 -benzothiophene, and benzofuran includes 2- and 3-benzofuran.

[0074] Some heteroaryl groups include those having from 5 to 10 ring members and from 1 to 3 ring atoms including N, O, or S, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3, 5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include those having from 5 to 8 ring members and from 1 to 3 heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3, 5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Some other heteroaryl groups include those having from 9 to 12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, benzofuran and bipyridine. Still other heteroaryl groups include those having from 5 to 6 ring members and from 1 to 2 ring atoms including N, O, or S, such as pyrrole, pyridine, imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. [0075] The term“(cycloalkyl)alkyl” refers to a radical having an alkyl component and a cycloalkyl component, where the alkyl component links the cycloalkyl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the cycloalkyl component and to the point of attachment. The alkyl component can include any number of carbons, such as Ci-6, C1-2, C1-3, C 1-4, C 1-5, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. The cycloalkyl component is as defined within. Exemplary (cycloalkyl)alkyl groups include, but are not limited to, methyl-cyclopropyl, methyl-cyclobutyl, methyl-cyclopentyl, and methyl-cyclohexyl.

[0076] The term“(heterocycloalkyl)alkyl” refers to a radical having an alkyl component and a heterocycloalkyl component, where the alkyl component links the heterocycloalkyl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the heterocycloalkyl component and to the point of attachment. The alkyl component can include any number of carbons, such as Co-6, C1-2, C1-3, C1-4, Ci-5, Ci-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. The heterocycloalkyl component is as defined above. (Heterocycloalkyl)alkyl groups can be substituted or unsubstituted.

[0077] The term “arylalkyl” refers to a radical having an alkyl component and an aryl component, where the alkyl component links the aryl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the aryl component and to the point of attachment. The alkyl component can include any number of carbons, such as Co-6, C 1-2, C1-3, C 1-4, C1-5, Ci-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. The aryl component is as defined above. Examples of arylalkyl groups include, but are not limited to, benzyl and ethyl -benzene. Arylalkyl groups can be substituted or unsubstituted.

[0078] The term“heteroarylalkyl” refers to a radical having an alkyl component and a heteroaryl component, where the alkyl component links the heteroaryl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the heteroaryl component and to the point of attachment. The alkyl component can include any number of carbons, such as Co-6, C1-2, C1-3, C 1-4, C1-5, Ci-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C ₄ - ₆ and C5-6. The heteroaryl component is as defined within. Heteroarylalkyl groups can be substituted or unsubstituted.

[0079] The term“carboxyalkyl” refers to a carboxy group linked to an alkyl, as described above, and generally having the formula -C1-12 alkyl-C(0)0H. Any suitable alkyl chain is useful. Carboxyalkyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0080] The term“acyl” refers to an alkyl that contains an oxo substituted carbon at the point of attachment ( -C(O) -C1-12 alkyl ). Any suitable alkyl chain can be used. Acyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

[0081] The term“hydroxyalkyl” refers to an alkyl group, as defined above, where at least one of the hydrogen atoms is replaced with a hydroxy group. As for the alkyl group, hydroxyalkyl groups can have any suitable number of carbon atoms, such as C1-6. Exemplary hydroxyalkyl groups include, but are not limited to, hydroxy-methyl, hydroxyethyl (where the hydroxy is in the 1- or 2-position), hydroxypropyl (where the hydroxy is in the 1-, 2- or 3-position), hydroxybutyl (where the hydroxy is in the 1-, 2-, 3- or 4-position), hydroxypentyl (where the hydroxy is in the 1-, 2-, 3-, 4- or 5-position), hydroxyhexyl (where the hydroxy is in the 1-, 2-, 3-, 4-, 5- or 6-position), 1, 2-dihydroxy ethyl, and the like. Hydroxyalkyl groups can be optionally substituted with one or more moieties selected from halo, thiol, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. One of skill in the art will appreciate that other hydroxyalkyl groups are useful in the present invention.

[0082] The term“alkoxy” refers to an alkyl group having at least one bridging oxygen atom. The bridging oxygen atom can be anywhere within the alkyl chain (alkyl-O-alkyl) or the bridging oxygen atom can connect the alkyl group to the point of attachment (alkyl-O-). In some embodiments, the bridging oxygen atom is not present as a terminal hydroxy group (i.e., -OH). In some instances, the alkoxy contains 1, 2, 3, 4, or 5 bridging oxygen atoms. As for alkyl groups, alkoxy groups can have any suitable number of carbon atoms, such as C 1-2, C1-4, and C 1-6. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, methyloxy-ethyloxy-ethyl (C1-O-C2-O-C2- ), etc. One example of an alkoxy group is polyethylene glycol (PEG) wherein the polyethylene glycol chain can include between 2 to 20 ethylene glycol monomers. Alkoxy groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, thiol, alkylamino, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. Alkoxy groups can be substituted or unsubstituted.

[0083] The term“alkylamino” refers to an alkyl group as defined within, having one or more amino groups. The amino groups can be primary, secondary or tertiary. Alkylamino groups useful in the present invention include, but are not limited to, ethyl amine, propyl amine, isopropyl amine, ethylene diamine, and ethanolamine. The amino group can link the alkylamino to the point of attachment with the rest of the compound, be at any position of the alkyl group, or link together at least two carbon atoms of the alkyl group. Alkylamino groups can be optionally substituted with one or more moieties selected from halo, hydroxy, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. One of skill in the art will appreciate that other alkylaminos are useful in the present invention.

[0084] The term“alkylthio” refers to an alkyl group as defined within, having one or more thiol groups. Alkylthio groups useful in the present invention include, but are not limited to, ethyl thiol, propyl thiol, and isopropyl thiol. The thiol group can link the alkylthio to the point of attachment with the rest of the compound, be at any position of the alkyl group, or link together at least two carbon atoms of the alkyl group. Alkylthio groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. One of skill in the art will appreciate that other alkylthio are useful in the present invention.

[0085] The term“oxy ethyl” refers to a divalent radical having the formula -OCH2CH2-.

[0086] The term“wavy line” signifies the point of attachment of a substituent to the remainder of a molecule. When the wavy line is not depicted as being specifically appended to a specific ring atom, the point of attachment can be to any suitable atom of the substituent. For example, the wavy line in the following structure:

is intended to include, as the point of attachment, any of the substitutable atoms.

[0087] The term“bioengineered tissue” refers to one or more synthetically created cells, tissues, or organs created for the purposes of regenerative medicine. In some embodiments, bioengineered tissue refers to cells, tissues, or organs that were developed in the laboratory. In some embodiments, bioengineered tissues refers to laboratory derived heart, liver, lung, kidney, pancreas, intestine, thymus, cornea, stem cells ( e.g ., human pluripotent stem cells, hematopoietic stem cells), lymphocytes, granulocytes, immune system cells, bone cells, primary cells, organoids, embryonic cells, genitourinary cells (e.g., sperm cells, oocytes, corpus cavernosum cells (e.g, smooth muscle corpus cavernosum cells, epithelial corpus cavernosum cells), urinary bladder cells, urethral cells, ureter cells, kidney cells, testicular cells), blood platelets, nerve cells, or a combination thereof.

[0088] The terms“vitrify” and“vitrification” mean the transformation of a substance into a glass (i.e., a non-crystalline amorphous solid). In the context of water, vitrification refers to the transformation of water into a glass without the formation of ice crystals, as opposed to ordinary freezing, which results in ice crystal formation. Vitrification is often achieved through very rapid cooling and/or the introduction of agents that suppress ice crystal formation. On the other hand, “devitrify” and“devitrification” refer to the process of crystallization in a previously crystal-free (amorphous) glass. In the context of water ice, devitrification can mean the formation of ice crystals as the previously non-crystalline amorphous solid undergoes melting.

III. Detailed Description of the Embodiments A. Novel cavity-containing oligomers and polymers

[0089] In one aspect, the present invention relates to the use of nanotube compositions which comprise the rigidification of an amide group flanked by two aromatic rings. According to one aspect of the invention, the rigidification is accomplished by introducing intramolecular H-bonds. An oligomer having amide-liked benzene rings forms a curved backbone when the amide linkers are meta to each other. The existence of intramolecular H-bonds provides the curved conformation to the oligomer backbone. The use of the cavity-containing oligomers and polymers described herein are useful, for example, in improving or increasing cryopreservation efficacy and/or improving or increasing efficacy of delivering therapeutic or diagnostic agents to cells.

[0090] In some embodiments, cavity-containing oligomers and polymers used in compositions and methods of the present invention comprise a plurality of aromatic substituents linked by at least one amide group, wherein the oligomer or polymer has a curved backbone. In some embodiments, the curvature of the backbone is due at least in part to intramolecular hydrogen bonds that rigidify the amide linkage of each amide group to each aromatic substituent. In some embodiments, the curvature of the backbone is due at least in part to an interaction between the aromatic substituents. In some embodiments, the curved backbone is stabilized. In some embodiments, the oligomer or polymer comprises a plurality of aromatic substituents.

[0091] In some embodiments, the cavity-containing oligomer or polymer backbone comprises a structure according to Formula (I):

[0092] In some embodiments, the cavity-containing oligomer or polymer backbone comprises a structure according to Formula (II):

[0093] In some embodiments, the cavity-containing oligomer or polymer backbone comprises a structure according to Formula (III):

[0094] In some embodiments, the cavity-containing oligomer or polymer backbone comprises a structure according to Formula (IV):

[0095] In some embodiments, the cavity-containing oligomer or polymer backbone comprises a structure according to Formula (V):

[0096] In some embodiments, the cavity-containing oligomer or polymer backbone comprises a structure according to Formula (VI):

[0097] Each instance of R may be independently selected. In some embodiments, R is selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is ( e.g ., in general) chiral, -CEE, -CH2CH2OCH3, - CFFCFFCXCFhCFhCrizCFF, aaryl group (e.g., phenyl, benzyl, napthyl, alkyl or an aryl group with a polar terminal functional end (e.g, -OH, -COOH, -NH2, -NHG, -N ⁺ R'3, -CH2CH2C ₆ H5, CH2C ₆ H4-P-OH, -CH2COOCH ₃ , -CH2CONH2, -CH2CH2CONH2, -CH ₂ COOCH ₂ -(3-indolyol))). In some embodiments, z is an integer having a value between 1 and 10 ( e.g 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In varying embodiments, each instance of R may be the same or each instance of R may be different. In some embodiments, each instance of R may be the same or different from unit to unit.

[0098] In some embodiments, the oligomer or polymer side group R is selected from the group of monomers set forth in Table 1. A person of skill in the art will recognize that the bounds of this invention are not limited to the monomers listed in Table 1, and that any useful alkyl or aryl group can be used as a side group.

[0099] In some embodiments, the cavity-containing oligomer or polymer contains capping groups X and Y that are independently selected. X and Y may be the same, or they may be different. In some embodiments, X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted C1-12 acyl, optionally substituted C1-12 alkylamino, -OH, -SH, -NH2, -NHY, -N ⁺ R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond. R’ can be any alkyl or aryl group.

[0100] In some embodiments, the oligomer or polymer terminal group X is selected from the group of monomers set forth in Table 2. A person of skill in the art will recognize that the bounds of this invention are not limited to the monomers listed in Table 2, and that any useful terminal group can be used as a terminal group, including any amino acid.

[0101] In some embodiments, the oligomer or polymer terminal group Y is selected from the group of monomers set forth in Table 3. A person of skill in the art will recognize that the bounds of this invention are not limited to the monomers listed in Table 3, and that any useful terminal group can be used as a terminal group, including any amino acid.

[0102] In some embodiments, the cavity-containing oligomer or polymer contains a capping group that is an amide-linked amino acid.

[0103] In some embodiments, the cavity-containing oligomer or polymer contains a side-chain R that is selected from the set in Table 4. A person of skill in the art will recognize that the bounds of this invention are not limited to the monomers listed in Table 4, and that any useful terminal group can be used as a terminal group, including any amino acid.

[0104] In some embodiments, the cavity-containing oligomer or polymer contains a monomer unit possessing at least one side chain bearing an oxyether or thioether ( i.e R is an oxyether or thioether). In some embodiments, the cavity-containing oligomer or polymer contains a monomer unit possessing at least one side chain of linear and branched chain ether groups.

[0105] In some embodiments, the cavity-containing oligomer or polymer contains a monomer unit possessing at least one side chain R that comprises the structure:

[0106] In some embodiments, the cavity-containing oligomer or polymer backbone comprises a structure according to Formula (VII):

(VII).

[0107] In some embodiments, the cavity-containing oligomer or polymer comprises the following structure:

[0108] In some embodiments, the sequence length of the cavity-containing oligomer or polymer (e.g, n in Formula (I)) is between 4 and 128. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is between 4 and 100. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is between 4 and 64. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is between 4 and 52. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is between 4 and 32. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is between 4 and 16. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is between 4 and 8. In some embodiments, the sequence length of the cavity-containing oligomer or polymer can be from about 8 to about 64, from about 16 to about 100, from about 28 to about 48, from about 32 to about 44, or from about 36 to about 40. In some embodiments, the sequence length of the cavity-containing oligomer or polymer can be from about 16 to about 100, from about 16 to about 90, from about 16 to about 80, from about 16 to about 70, from about 16 to about 60, from about 20 to about 50, from about 20 to about 40, or from about 20 to about 30. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is from about 6 to about 16. In some embodiments, the sequence length of the cavity-containing oligomer or polymer is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or more. In some embodiments, the sequence length of the cavity- containing oligomer or polymer is 6, 8, 10, or 12.

[0109] In some embodiments, the oligomers and polymers ranging in length from about 4 monomeric subunits to about 128 monomeric subunits are particularly useful in the present invention.

[0110] In some embodiments, the oligomers and polymers ranging in length from about 4 monomeric subunits to about 64 monomeric subunits are particularly useful in the present invention. [0111] In some embodiments, the oligomers and polymers ranging in length from about 4 monomeric subunits to about 32 monomeric subunits are more preferred and useful in the present invention.

[0112] In some embodiments, the oligomers and polymers ranging in length from about 4 monomeric subunits to about 16 monomeric subunits are more preferred and useful in the present invention.

[0113] In some embodiments, the oligomers and polymers ranging in length from about 4 monomeric subunits to about 8 monomeric subunits are most preferred and useful in the present invention.

[0114] In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is between 2 and 64. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is between 2 and 50. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is between 2 and 26. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is between 2 and 32. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is between 2 and 16. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is between 2 and 8. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is between 2 and 4. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII) is from about 4 to about 32, from about 8 to about 50, from about 14 to about 24, from about 16 to about 22, or from about 18 to about 20. In some embodiments, value of m in Formula (II), (IV), (V), or (VII) can be from about 8 to about 50, from about 8 to about 45, from about 8 to about 40, from about 8 to about 35, from about 8 to about 30, from about 10 to about 25, from about 10 to about 20, from about 10 to about 15, or from about 3 to about 8. In some embodiments, the value of m in Formula (II), (IV), (V), or (VII)is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or more. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9. 10. 11. 12. 13. 14. 15. or 16.

[0115] In some embodiments, the concentration of the cavity-containing oligomer or polymer ( e.g present in a composition, formulation, or product such as a cryoprotectant solution or antifreeze solution) is between about 1 nM and about 100 mM. In some embodiments, the concentration of the cavity-containing oligomer or polymer is between about 100 nM and about 1,000 mM. In certain embodiments, the concentration of the cavity-containing oligomer or polymer is between about 1 nM and about 250 nM, between about 250 nM and about 500 nM, between about 500 nM and about 750 nM, between about 750 nM and about 1 mM, between about 1 pM and about 5 pM, between about 5 pM and about 25 pM, between about 25 pM and about 50 pM, between about 50 pM and about 100 pM, between about 100 pM and about 250 pM, between about 250 pM and about 500 pM, between about 500 pM and about 750 pM, between about 750 pM and about 1 mM, between about 1 mM and about 10 mM, between about 10 mM and about 50 mM, between about 50 mM and about 100 mM. In other embodiments, the concentration of the cavity-containing oligomer or polymer is about 100 nM, about 1 pM, about 10 pM, about 100 pM, about 1 mM, about 10 mM, or about 100 mM. In certain embodiments, the concentration of cavity-containing oligomer or polymer is between about 100 nM and about 1,000 mM. In particular embodiments, the concentration of the cavity-containing oligomer or polymer is between about 1 and 200 mM ( e.g ., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM). In particular embodiments, the concentration of the cavity-containing oligomer or polymer is between about 1 and 1000 nM (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM). In particular embodiments, the concentration of the cavity-containing oligomer or polymer is about 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 pM.

Methods of Synthesis

[0116] FIG. 5A shows the synthesis of 2,4-dihydroxy-5-nitrobenzoic acid from the commercially available 2,4-dihydroxybenzoic acid. The acid is then esterified and alkylated in high yields. The alkylation reaction is quite general, since methyl, ally, iso-butyl, octyl, and decyl bromides all have resulted in corresponding products with high yields (>95%).

[0117] Alternative steps to the synthetic method shown in FIG. 5A include:

[0118] From 1 to 2: other general methods for nitrating aromatic rings, such as concentrated HNO3 alone, in H2SO4 at 0 °C, in acetic anhydride, and NaNCh in trifluoroacetic acid, are effective for this conversion.

[0119] From 2 to 3: this esterification step can also be effected by using acidic catalysts such as HC1, tosylsulfonic acid, BFy ether, and other strong acids. [0120] From 3 to 4: this alkylation step can also be carried out with alkyl halides and alkyl iodide under the same conditions. Preparation of m-nitrobenzoic acids were prepared before as reference US Patent No. 6495,680 Bl (Gong), herein incorporated by reference in its entirety for all purposes.

[0121] FIG. 5B shows the synthesis of the trifluoroacetyl-protected acid chloride type I monomer 7 from 5. The nitro-group is reduced and trifluoroacetylated. The benzoic acid derivative is then converted to the acid chloride using oxalyl chloride.

[0122] FIG. 5C shows the synthesis of the /er/-butoxy carbonyl -protected aniline type I monomer 9 from 5. The benzoic acid derivative is first protected using oxalyl chloride then potassium tert- butoxide. The nitro-group is then reduced.

[0123] Preparation of 7 and 9 were prepared before as reference US 6495,680 B 1 (Gong).

[0124] FIG. 6A shows the synthesis of type I dimer 10. Synthesis is carried out in the presence of base in high yield.

[0125] Preparation of 10 was previously reported (Yuan U; Sanford, A. R.; Feng, W.; Zhang, A.; Zhu, T; Zeng, FL; Yamato, K.; Li, M.; Ferguson, L; Gong, B. (2005) J. Org. Chem., 70, 10660; herein incorporated by reference in its entirety for all purposes).

[0126] FIG. 6B shows the synthesis of type I tetramer 13. The /er/-butoxy carbonyl of 13 is deprotected with trifluoroacetic acid to form 11. Separately, the trifluoroacetyl group of 13 is deprotected with sodium hydroxide to form 12. The tetramer is then formed by combining 11 and 12 in the presence of base, albeit in lower yields.

[0127] Preparation of 13 was previously reported (Yuan L.; Sanford, A. R.; Feng, W.; Zhang, A.; Zhu, J.; Zeng, H.; Yamato, K.; Li, M.; Ferguson, J.; Gong, B. (2005) J. Org. Chem., 70, 10660).

[0128] FIG. 6C shows the general coupling of two type I n-mers to form a type I 2n-mer. The same coupling strategy to synthesize 13 is employed.

[0129] FIG. 7A shows the synthesis of type II monomer 17 from 4,6-dihydroxyisophthalic acid. [0130] Preparation of 17 was reported before US 6495,680 Bl (Gong). [0131] FIG. 7B shows the synthesis of type II monomer 19 and type II dimer 21.

[0132] Preparation of 19 was reported before (Hamuro, Y.; Geib, S.J.; Hamilton, A.D. (1997) I. Am. Chem. Soc., 1997, 119, 10587, herein incorporated by reference in its entirety for ail purposes). Preparation of 21 was reported before (US 6495,680 B1 (Gong).

[0133] FIG. 8 A shows the synthesis of a type II tetramer 24 from the dimerization of 21.

[0134] Preparation of 24 was prepared before as reference US 6495,680 B 1 (Gong).

[0135] FIG. 8B shows the general coupling of two type II n-mers to form a type II 2n-mer. The same coupling strategy to synthesize 24 is employed.

[0136] The structures of synthesized building blocks, intermediates and oligomers are characterized by standard techniques such as 'H- and ¹³ C-NMR, mass spectrometry and elemental analysis. The solid-state structures of these oligomers are determined by X-ray crystallography. The crystal structures provide detailed and conclusive three-dimensional structural information, including bond lengths, bond angles and the proposed folding patterns, which guides further the design and structural modification of the oligomer.

[0137] Further methods are described in Zhao Y, Connor AL, Sobiech TA, Gong B. Effects of Oligomer Length, Solvents, and Temperature on the Self-Association of Aromatic Oligoamide Foldamers. Org. Lett. 2018 Sep 7;20(l7):5486-5489. doi: l0. l02l/acs.orglett.8b02438. Epub 2018 Aug 17, incorporated herein by reference in its entirety for all purposes.

[0138] It is understood that these methods described are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

B. Incorporating cavity-containing oligomers and polymers into cryoprotectant

solutions for enhanced efficacy of cryopreservation

[0139] The present invention is based in part on the unexpected discovery that incorporating a low concentration of one or more cavity-containing oligomers or polymers greatly enhances the efficacy of cryoprotectant compositions and solutions. Cavity-containing oligomers and polymers can undergo tubular stacking into self-assembling nanotubes by design. The inner pores of these nanotubes not only mediate efficient and selective molecular and ion transport, but also allow structural and functional fine-tuning. Previous studies have laid a solid foundation for constructing channel-forming molecules that facilitate the transport and uptake of CPAs. The present invention is related to the cavity-containing oligomers and polymers facilitating the intracellular delivery of cryoprotectant solution. In some aspects, the present invention relates to compositions for cryopreservation that comprise a cavity-containing oligomer or polymer described herein and a cryoprotectant solution described herein. In particular embodiments, a composition for cryopreservation of the present invention comprises two or more different cavity-containing oligomers or polymers described herein.

[0140] The term“cryoprotectant solution” refers to a solution that is used to reduce or prevent freezing damage caused by ice crystal formation. In some embodiments, the cryoprotectant solution protects a biological sample from freezing damage. In some embodiments, the cryoprotectant solution protects a biological sample from ice crystal formation. In some embodiments, the cryoprotectant solution preserves a biological sample for an amount of time longer than if the biological sample were not exposed to reduced temperatures.

[0141] In other embodiments, the cryoprotectant solution comprises a compound selected from the group consisting of an ionic species, a penetrating cryoprotectant, a non-penetrating cryoprotectant, an antioxidant, a cell membrane stabilizing compound, a channel-forming compound, an alcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, hyaluronan, formamide, a natural or synthetic hydrogel, and a combination thereof. In particular embodiments, the penetrating cryoprotectant penetrates the cell membrane and reduces the intracellular water concentration, thereby reducing the amount of ice formed at any temperature. In other particular embodiments, the non-penetrating cryoprotectant induces changes in colloidal osmotic pressure and modifies cell membrane associations with extracellular water by induced ionic interaction.

[0142] In some embodiments, the cavity-containing oligomer or polymer facilitates the cellular delivery of a cryoprotectant solution comprising a compound selected from the group consisting of an ionic species, a penetrating cryoprotectant, a non-penetrating cryoprotectant, an antioxidant, a cell membrane stabilizing compound, an alcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropylene glycol (PPG), Ficoll ^® , polyvinylpyrrolidone, polyvinyl alcohol, hyaluronan, formamide, a natural or synthetic hydrogel, or any combination thereof.

[0143] In some embodiments, the cavity-containing oligomer or polymer facilitates the cellular delivery of a cryoprotectant solution comprising an alcohol selected from the group consisting of propylene glycol, ethylene glycol, glycerol, methanol, butylene glycol, adonitol, ethanol, trimethylene glycol, diethylene glycol, polyethylene oxide, erythritol, sorbitol, xythyritol, polypropylene glycol, 2-methyl-2,4-pentanediol (MPD), mannitol, inositol, dithioritol, 1 ,2- propanediol, or any combination thereof.

[0144] In some embodiments, the cavity-containing oligomer or polymer facilitates the cellular delivery of a cryoprotectant solution comprising a sugar that is selected from the group consisting of a monosaccharide, a disaccharide, a polysaccharide, or any combination thereof. In some instances, the sugar is a monosaccharide selected from the group consisting of sorbitol, glucose, galactose, arabinose, fructose, xylose, mannose, 3-O-Methyl-D-glucopyranose, or any combination thereof. In other instances, the sugar is a disaccharide selected from the group consisting of sucrose, trehalose, lactose, maltose, or any combination thereof. In still other instances, the sugar is a polysaccharide selected from the group consisting of raffmose, dextran, or any combination thereof.

[0145] In some embodiments, the cavity-containing oligomer or polymer facilitates the cellular delivery of a cryoprotectant solution further comprising a PEG or PPG that has an average molecular weight less than about 3,000 g/mol, less than about 2,000 g/mol, or less than about 1,000 g/mol. In particular instances, the PEG or PPG has an average molecular weight between about 200 and 400 g/mol.

[0146] In some embodiments, the cavity-containing oligomer or polymer facilitates the cellular delivery of a cryoprotectant solution comprising a protein selected from the group consisting of bovine serum albumin, human serum albumin, gelatin, or any combination thereof. In some embodiments, the cryoprotectant solution comprises a natural or synthetic hydrogel that comprises alginate, gelatin, chitosan, hyaluronic acid, or any combination thereof. In some embodiments, the cryoprotectant solution comprises a nonionic surfactant selected from the group consisting of polyoxyethylene lauryl ether, polysorbate 80, or any combination thereof.

[0147] Incorporation of functional supramolecular assemblies based on cavity-containing oligomers or polymers to enhance membrane permeability of CPAs could lead to a revolutionary solution to long-term cryopreservation of large or complex tissues and organs.

[0148] The present invention incorporates the cavity-containing oligomer or polymer to significantly reduce toxicity and cell injury (FIG. 9) that is caused by, for example, osmotic shrinkage caused by CPAs and salt during both the cooling and rewarming processes via (1) reducing CPA loading and unloading time, (2) enabling rapid CPA loading and unloading at the wider temperature range instead of the conventional 4 °C loading and unloading due to toxicity, and (3) greatly reduce the CPA concentration that is required to cryopreserve a biological system, therefore reducing cytotoxicity related to CPA and improve the viability of the cells/tissues/organs. Moreover, versatile functional cavity-containing oligomers or polymers of diverse sizes and properties can be prepared by modifying inner cavities to allow selective CPA transport while preventing ion exchange. This method is effective when CPA loading in tissues using the“liquidus tracking” or step-wise methods where increasingly concentrated solutions of CPA are loaded in the tissue/organ at progressively decreasing temperatures.

[0149] Cell-based cryopreservation assays have shown significant improvement of cell viability with the presence of small molecule penetrating and non-penetrating cryoprotectant agents at 3- 10X reduced concentration, which includes, but is not meant to limit, DMSO, glycerol and sorbitol.

Details of cryopreservation compositions

[0150] In some aspects, the present invention provides a composition for cryopreservation. In some embodiments, the composition for cryopreservation comprises a cavity-containing oligomer and/or polymer described herein and a cryoprotectant solution comprising a compound selected from the group consisting of an ionic species, a penetrating cryoprotectant, a non-penetrating cryoprotectant, an antioxidant, a cell membrane stabilizing compound, a channel-forming compound, an alcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropylene glycol (PPG), Ficoll®, polyvinylpyrrolidone, polyvinyl alcohol, hyaluronan, formamide, a natural or synthetic hydrogel, and a combination thereof. In particular embodiments, the penetrating cryoprotectant penetrates the cell membrane and reduces the intracellular water concentration, thereby reducing the amount of ice formed at any temperature. In other particular embodiments, the non-penetrating cryoprotectant induces changes in colloidal osmotic pressure and modifies cell membrane associations with extracellular water by induced ionic interaction.

[0151] In some instances, the cryoprotectant solution comprises an alcohol that is selected from the group consisting of propylene glycol, ethylene glycol, glycerol, methanol, butylene glycol, adonitol, ethanol, trimethylene glycol, diethylene glycol, polyethylene oxide.

[0152] In other instances, the cryoprotectant solution comprises PEG or a plurality of different PEG compounds. In some instances, at least one of the PEG compounds has an average molecular weight less than about 3,000 g/mol ( e.g ., less than about 3,000, 2,950, 2,900, 2,850, 2,800, 2,750, 2,700, 2,650, 2,600, 2,550, 2,500, 2,450, 2,400, 2,350, 2,300, 2,250, 2,000, 1,550, 15,00, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100 g/mol). In particular instances, at least one of the PEG compounds has an average molecular weight between about 200 g/mol and 400 g/mol (e.g., about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 g/mol). In some instances, the cryoprotectant solution comprises PEG or a plurality of PEG compounds selected from the group consisting of PEG 200, PEG 300, PEG 400, and a combination thereof.

[0153] In other instances, the cryoprotectant solution comprises PPG or a plurality of different PPG compounds. In some instances, at least of the PPG compounds has an average molecular weight less than about 3,000 g/mol (e.g, less than about 3,000, 2,950, 2,900, 2,850, 2,800, 2,750, 2,700, 2,650, 2,600, 2,550, 2,500, 2,450, 2,400, 2,350, 2,300, 2,250, 2,000, 1,550, 15,00, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100 g/mol). In particular instances, at least one of the PPG compounds has an average molecular weight between about 200 g/mol and 400 g/mol (e.g, about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 g/mol). In some instances, the cryoprotectant solution comprises PPG or a plurality of PPG compounds selected from the group consisting of PEG 200, PEG 300, PEG 400, and a combination thereof.

[0154] In other instances, the cryoprotectant solution comprises a protein that is selected from the group consisting of egg albumin, bovine serum albumin, human serum albumin gelatin, and a combination thereof. In still other instances, the cryoprotectant solution comprises a natural or synthetic hydrogel, wherein the natural or synthetic hydrogel comprises chitosan, hyaluronic acid, or a combination thereof. In yet other instances, the cryoprotectant solution comprises a nonionic surfactant selected from the group consisting of polyoxyethylene lauryl ether, polysorbate 80, and a combination thereof.

[0155] Non-limiting examples of various properties of the cryoprotectant solution such as effective concentration, viscosity, water solubility, and/or membrane permeability can be assessed using a model cell or tissue, including, but not limited to, stem cells, liver tissue or hepatocytes, kidney, intestine, heart, pancreas, genitourinary cells ( e.g ., sperm cells, oocytes, corpus cavemosum cells (e.g., smooth muscle corpus cavemosum, epithelial corpus cavernosum cells), urinary bladder cells, urethral cells, ureter cells, kidney cells, testicular cells), bone marrow, organoids, and other biological tissues for cryopreservation.

[0156] In some embodiments, the concentration of cavity-containing oligomers and polymers in the cryopreservation composition is between 100 nM and about 1,000 mM. In some embodiments, the concentration of the cavity-containing oligomers and polymers in the cryopreservation composition is between about 100 nM and about 250 nM, between about 250 nM and about 500 nM, between about 500 nM and about 750 nM, between about 750 nM and about 1 mM, between about 1 pM and about 5 pM, between about 5 pM and about 25 pM, between about 25 pM and about 50 pM, between about 50 pM and about 100 pM, between about 100 pM and about 250 pM, between about 250 pM and about 500 pM, between about 500 pM and about 750 pM, between about 750 pM and about 1 mM, between about 1 mM and about 10 mM, between about 10 mM and about 50 mM, between about 50 mM and about 100 mM, between about 100 mM and about 250 mM, between about 250 mM and about 500 mM, between about 500 mM and about 750 mM, or between about 750 mM and about 1,000 mM. In some embodiments, the concentration of the cavity-containing oligomers and polymers in the cryopreservation composition is about 100 nM, about 1 mM, about 10 mM, about 100 mM, about 1 mM, about 10 mM, about 100 mM, or about 1,000 mM. In particular embodiments, the concentration of the cavity-containing oligomers and polymers in the cryopreservation composition is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM. In certain embodiments, the concentration of cavity-containing oligomer or polymer in the cryopreservation composition is between about 100 nM and about 1,000 mM. In particular embodiments, the concentration of the cavity-containing oligomer or polymer in the cryopreservation composition is between about 1 and 200 mM ( e.g ., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM). In particular embodiments, the concentration of the cavity-containing oligomer or polymer in the cryopreservation composition is between about 1 and 1000 nM (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM).

[0157] In some embodiments, the cryopreservation composition comprises a cavity-containing oligomer and/or polymer described herein.

[0158] In some aspects, a method for cryopreserving a biological sample is provided. In some embodiments, the biological sample possesses cellular composition. In some embodiments, the biological sample comprises a tissue. In other embodiments, the biological sample comprises an organ. The tissue or organ may be of any kind. In still other embodiments, the biological sample comprises a cell. In particular embodiments, the biological sample comprises one or more tissues, one or more organs, one or more cells or a combination thereof. In some embodiments, the method comprises contacting the biological sample with a cavity-containing oligomer or polymer described herein, a cavity-containing oligomer or polymer described herein, a cryoprotectant solution described herein, or a combination thereof. In some embodiments, the method comprises contacting the biological sample with a composition for cryopreservation of the present invention. In some instances, when a combination of compositions or solutions is used, contacting the biological sample with the compositions or solutions can be accomplished in multiple steps. As a non-limiting example, a biological sample can first be contacted with a cavity-containing oligomer or polymer described herein, and then at a later point the biological sample can be contacted with a cryoprotectant solution described herein. As another non-limiting example, the biological sample can first be contacted with the cryoprotectant solution described herein, and then at a later point be contacted with the cavity-containing oligomer or polymer described herein. [0159] In some embodiments, the tissue is a bioengineered tissue or organ. The bioengineered tissue or organ can be of any kind. In some instances, the biological sample is selected from the group consisting of heart, liver, lung, kidney, pancreas, intestine, thymus, cornea, nerve cells, blood platelets, genitourinary cells ( e.g ., sperm cells, oocytes, corpus cavernosum cells (e.g, smooth muscle corpus covemosum, epithelial corpus cavernosum cells), urinary bladder cells, urethral cells, ureter cells, kidney cells, testicular cells), embryonic cells, stem cells, bone cells, and a combination thereof.

[0160] In some embodiments, the biological sample comprises a cell or a population of cells selected from the group consisting of primary cells, heart cells, liver cells, lung cells, kidney cells, pancreatic cells, gastric cells, intestinal cells, muscle cells, skin cells, neural cells, blood cells, immune cells, fibroblasts, genitourinary cells, bone cells, stem cells, sperm cells, oocytes, embryonic cells, epithelial cells, endothelial cells, and a combination thereof. In some instances, the cells are mammalian (e.g, human) cells.

[0161] In some embodiments, the biological sample comprises a tissue selected from the group consisting of heart tissue, liver tissue, lung tissue, kidney tissue, gall bladder tissue, reproductive system tissue, pancreatic tissue, gastric tissue, intestinal tissue, muscle tissue, skin tissue, neural tissue, blood, genitourinary tissue, bone, epithelial tissue, endothelial tissue, corneal tissue, heart valve tissue, and a combination thereof. In some instances, the tissue is mammalian (e.g, human) tissue.

[0162] In some embodiments, the biological sample comprises an organ selected from the group consisting of heart, liver, lung, kidney, a reproductive organ, pancreas, stomach, gall bladder, intestine, muscle, skin, bone, and a combination thereof. In some instances, the organ is a mammalian (e.g, human) organ.

[0163] Cryoprotection of biological samples is useful for any number of purpose. Non-limiting examples include organoid preservation, stem cell preservation (e.g, hematopoietic stem cells, embryonic stem (ES) cells, pluripotent stem cells (PSCs), and induced pluripotent stem cells (iPSCs)), preservation of adult cells and cell lines (e.g, lymphocytes, granulocytes, immune system cells, bone cells), preservation of embryos, sperm, and oocytes, tissue preservation, and organ preservation. Preservation of tissues, organs, and other biological samples and structures is especially useful, for example, in the field of organ transplantation. Other useful applications of the present invention to biological sample cryoprotection will readily be known to one of skill in the art.

[0164] In yet other aspects, provided herein is a method for preserving one or more biological macromolecules. In some embodiments, the method comprises comprising the one or more biological macromolecules with a composition for cryopreservation of the present invention. Said biological macromolecules can be naturally or unnaturally occurring. Non-limiting examples of biological macromolecules that are suitable for cryoprotection by compositions and methods of the present invention include nucleic acids ( e.g ., DNA, RNA), amino acids, proteins, peptides, lipids, and composite structures (e.g., liposomes). In some embodiments, the method comprises contacting the biological macromolecule with the cavity-containing oligomers or polymers described herein, a cryoprotectant solution described herein, or a combination thereof. In some instances, the biological macromolecule is an isolated protein. In particular instances, the isolated protein is a protease protein. In some instances, when a combination of compositions or solutions is used, contacting the one or more biological macromolecules with the compositions or solutions can be accomplished in multiple steps. As a non-limiting example, the one or more biological macromolecules can first be contacted with the cavity-containing oligomers or polymers herein, and then at a later point the biological sample can be contacted with a cryoprotection solution described herein. As another non-limiting example, the biological sample can be contacted with a cryoprotection solution described herein, and then at a later point the biological sample can be contacted with the cavity-containing oligomers and polymers described herein.

[0165] Cryoprotection of biological macromolecules using compositions and methods of the present invention is useful for any number of purposes. Non-limiting examples of such purposes include the preservation of DNA (e.g, genomic DNA) and RNA samples, the preservation of stem cell growth factors, and the preservation of antibodies. Other useful purposes and applications appropriate for compositions and methods of the present invention will be readily known by one of skill in the art.

[0166] Biological samples and macromolecules that are suitable for cryoprotection according to the compositions and methods of the present invention can come from any biological kingdom (e.g., Animalia (including, but not limited to, humans and livestock animals), Plantae, Fungi (including, but not limited to, mushrooms), Protista, Archaea/Archaeabacteria, and B acteri a/Eub acted a) .

C. Novel delivery method of cavity-containing oligomers and polymers in combination with CPAs

[0167] In some aspects, methods for cry opreserving or supercooling a biological sample (e.g, a cell, a population of cells, a tissue, and/or an organ) or a macromolecule are provided herein. In some embodiments, the method comprises contacting the biological sample or macromolecule with a cavity-containing oligomer or polymer described herein and a cryoprotectant solution provided herein. In some embodiments, the method comprises contacting the biological sample or macromolecule with a composition for cryopreservation provided herein (i.e., comprising a cavity-containing oligomer or polymer provided herein and a cryoprotectant solution provided herein). In other embodiments, the cavity-containing oligomers or polymers are directly combined and mixed together in a whole cryoprotectant solution, and delivered into a targeted biological system. In some embodiments, the biological sample or macromoelcule is contacted with the cavity-containing oligomer or polymer and the cryoprotectant solution separately (e.g, the cavity- containing oligomer or polymer and the cryoprotectant solution are present in different compositions (e.g, solutions)).

[0168] A method for cry opreserving a biological sample is provided by the present invention by a novel CPA loading and uploading method using synthetic cavity-containing oligomers or polymers. In some embodiments, the cavity-containing oligomers and polymers are firstly delivered into a target system at certain temperature (about 37 °C to about 1 °C) for certain incubation time to precondition the cells, followed by CPA loading at the same or lower hypothermic temperature. A high influx rate of CPAs through the cavity/pore can be maintained during cooling as a function of the concentration gradient across cell membrane, thereby reducing the required time to reach effective cryopreservation concentrations. Upon rewarming, these oligomers and polymers will facilitate rapid removal of CPAs to reduce exposure time and the consequent toxic side effects. At or above physiological temperature, the nanopores will seal off, and be washed out from the system resulting in low toxicity. [0169] As a non-limiting example, compositions and methods of the present invention are useful for cryopreservation during freezing protocols (e.g, 0 °C to -196 °C). Freezing protocols are typically performed at a controlled rate (sometimes referred to as slow freezing) during at least part of the temperature reduction. For example, a biological sample (e.g, a cell, population of cells, tissue, or organ) or macromolecule can be contacted with composition for cryopreservation, cavity-containing oligomer or polymer, and/or cryoprotectant solution described herein, and the temperature can be reduced at a controlled rate until a desired temperature is reached (e.g, between -80 °C and - 180 °C), and then the sample or macromolecule can be flash frozen (e.g. , by immersing the sample or macromolecule in liquid nitrogen or placing the sample or macromolecule above liquid nitrogen). The composition for cryopreservation can be contacted with the sample or macromolecule being cryopreserved at any point during the protocol, as long as it is before the formation of ice crystals that damage the sample or macromolecule being preserved.

[0170] As another non-limiting example, compositions and methods of the present invention are useful for cryogenic freezing protocols (e.g., -90 °C to -196 °C). For example, a biological sample (e.g, cell, population of cells, tissue, or organ) or macromolecule can be contacted with a composition for cryopreservation, cavity-containing oligomer or polymer, and/or cryoprotectant solution described herein, then plunged into liquid nitrogen or a stream of liquid nitrogen vapor in order to quickly freeze the sample without formation of ice crystals. No ice lattice forms and so the water within the sample or macromolecule has an amorphous or glass-like state. Therefore, damaging ice is not formed.

[0171] The compositions and methods described herein are suitable for use in any number of cryopreservation protocols. As a non-limiting example, compositions and methods of the present invention are useful for cryopreservation during supercooling to high sub-zero temperatures (e.g. 0 °C to -20 °C). In the field of organ transplantation, organs are typically cooled on ice (e.g. 0 °C to -20 °C), which limits the transplantation window to about ten hours. By using ex vivo machine perfusion with cryoprotectants containing standard small molecule CPAs, it has been possible to preserver organs for up to 96 hours at a temperature of -6 °C. While it is desirable to further reduce the cryopreservation temperature below -6 °C, which would extend the possible cryopreservation time, it has not been possible to do so because the high concentrations of standard CPAs necessary, resulting in irreversible organ damage owing to CPA-related toxicity. For more information, see, e.g. Uygun K, et. al. Nat. Protoc. l0(3):484-94 (2015). Employing ex vivo perfusion methods or otherwise contacting biological samples (e.g, organs and tissues) or macromolecules with compositions for cryopreservation, cavity-containing oligomers or polymers, and/or cryoprotectant solutions described herein is useful for supercooling to high sub-zero temperatures, allowing cryopreservation for longer periods of time and at lower temperatures than is currently feasible. Other suitable applications of the present invention to high sub-zero temperature supercooling will readily be known to one of skill in the art.

[0172] In some embodiments, the biological sample (e.g, cell, population of cells, tissue, and/or organ) or macromolecule is cooled to a temperature of about 0 °C to about -20 °C (e.g, at about 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, or -20 °C). In other embodiments, the biological sample (e.g, cell, population of cells, tissue, and/or organ) or macromolecule is cooled to a temperature of about -20 °C to about -40 °C (e.g, at about -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, or -40 °C). In certain embodiments, the biological sample or macromolecule is cooled to a temperature of about -20 °C. In certain other embodiments, the biological sample or macromolecule is cooled to a temperature of about -40 °C to about -200 °C (e.g, at about -40, -45, -50, -55, -60, -65, -70, -75, - 80, -85, -90, -95, -100, -105, -110, -115, -120, -125, -130, -135, -140, -145, -150, -155, -160, -165, -170, -175, -180, -185, -190, -195, or -200 °C).

[0173] In some embodiments, the biological sample (e.g, cell, population of cells, tissue, and/or organ) or macromolecule is cooled to a temperature of temperature within about 0 °C to about - 200 °C (e.g, about -196 °C, within about -10 °C to about -190 °C, within about -20 °C to about - 180 °C, within about -30 °C to about -170 °C, within about -40 °C to about -160 °C, within about -50 °C to about -150 °C, within about -60 °C to about -140 °C, within about -70 °C to about -140 °C, within about -80 °C to about -130 °C, within about -90 °C to about -120 °C, or within about - 100 °C to about -110 °C.

[0174] In some embodiments, the composition for cryopreservation reduces or inhibits ice crystal formation at a temperature within about 0 °C to about -20 °C (e.g, at about 0, -1, -2, -3, - 4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, or -20 °C). In other embodiments, the composition for cryopreservation reduces or inhibits ice crystal formation at a temperature within about -20 °C to about -40 °C ( e.g ., at about -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, - 30, -31, -32, -33, -34, -35, -36, -37, -38, -39, or -40 °C). In certain embodiments, composition for cryopreservation reduces or inhibits ice crystal formation at about -20 °C. In certain other embodiments, the composition for cryopreservation reduces or inhibits ice crystal formation at a temperature within about -40 °C to about -200 °C (e.g., at about -40, -45, -50, -55, -60, -65, -70, - 75, -80, -85, -90, -95, -100, -105, -110, -115, -120, -125, -130, -135, -140, -145, -150, -155, -160, -165, -170, -175, -180, -185, -190, -195, or -200 °C).

[0175] In some embodiments, the composition for cryopreservation reduces or inhibits ice crystal formation at a temperature within about 0 °C to about -200 °C, within about -10 °C to about -190 °C, within about -20 °C to about -180 °C, within about -30 °C to about -170 °C, within about -40 °C to about -160 °C, within about -50 °C to about -150 °C, within about -60 °C to about -140 °C, within about -70 °C to about -140 °C, within about -80 °C to about -130 °C, within about -90 °C to about -120 °C, or within about -100 °C to about -110 °C.

[0176] In some embodiments, the biological sample (e.g, cell, population of cells, tissue, and/or organ) or macromolecule is cooled for a period of time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or longer. In some embodiments, the biological sample or macromolecule is cooled for a period of time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or longer.

[0177] In some embodiments, a method of cryopreservation provided herein further comprises warming the biological sample (e.g, cell, population of cells, tissue, and/or organ) or macromolecule. In some embodiments, the biological sample or macromolecule is warmed to a temperature of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 °C, or higher.

[0178] In some embodiments, the biological sample (e.g, cell, population of cells, tissue, and/or organ) or macromolecule has increased survival, viability or function after being warmed to a temperature of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 °C, or higher. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75,% 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of a population of cells survive after being warmed. [0179] In a related aspect, provided herein is a method for cryopreserving a population of cells, a tissue, or an organ with improved cell, tissue, or organ viability, the method comprising: (a) cooling the population of cells, tissue, or organ to a temperature of about 4 °C; and (b) contacting the population of cells, tissue, or organ with a cryopreservation composition provided herein at the temperature of about 4 °C.

[0180] In some embodiments, improved cell viability comprises enhanced proliferation of the population of cryopreserved (e.g, supercooled) cells that survive after warming compared to a control population of cells. In some embodiments, the control population of cells has not been contacted with the cryopreservation composition. In some embodiments, the number of cells in the population of cryopreserved cells at about 3, 4, 5, 6, 7, 8, 9, or 10 days after warming is at least about 1-, l . l-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2- fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or lO-fold greater than the number of cells in the control population of supercooled cells.

[0181] One of skill in the art will readily appreciate that the concentrations and compositions of cavity-containing oligomers or polymers and/or cryoprotectant solutions described herein can be modified depending on the particular cavity-containing oligomer or polymer employed, cryoprotectant employed, biological sample, and/or macromolecule being cryopreserved and the particular cryopreservation protocol being employed.

Cell-based Viability Assay Post Cryopreservation

[0182] A post-thaw viability assay with living cells can be performed to assess the cryoprotective activity of assisted CPA uptake from a composition for cryopreservation, cavity- containing oligomer and/or polymer, and/or cryoprotectant solution. Cells are seeded at high density (e.g, 50% confluence) and allowed to adhere and divide for, e.g, 24 hours.

[0183] The cells seeded in suitable containers are treated with a composition for cryopreservation, cavity-containing oligomer and/or polymer, and/or cryoprotectant solution at different concentrations, e.g, in 3 replicates. The plate is frozen by placing it bottom down on an aluminum shelf in the -80 °C freezer. After the desired time period, the plates are warmed by placing them bottom down on an aluminum surface in the cell culture incubator while cells adhere and recover for 24 hours.

[0184] Cell viability is tested using the alamarBlue® Cell Viability Assay Protocol provided by Thermo Fisher Scientific, Inc. Briefly, alamarBlue® is the trade name of resazurin (7-Hydroxy- 3H-phenoxazin-3-one lO-oxide) which is a non-toxic cell permeable compound that is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufm, a compound that is red in color and highly fluorescent. Viable cells continuously convert resazurin to resorufm, increasing the overall fluorescent and color of the media surrounding the cells. Non- viable cells do not convert the resazurin to resorufm; thus the overall fluorescence and color of the media surrounding the cells is an indication of the relative amount of viable cells in the sample. In a standard experiment, cells and the composition for cryopreservation, cavity-containing oligomer and/or polymer and/or cryoprotectant solution is mixed in any suitable container. The mixture is then cooled to the desired sub 0 °C temperature and held for the desired amount of time. Cells are then returned to ambient temperatures and the alamarBlue® reagent is added, incubated, and measured following the Thermo Fisher protocol. Typically, direct readout of cell viability is determined by measuring the relative fluorescence of the samples at the wavelengths XE -560 nm/lE m -590 nm.

[0185] Other times, cell viability is tested using the MTT assay for total cellular metabolic activity per well. The MTT assay is a colorimetric cell viability and proliferation assays that relies upon the reduction of yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide) to the insoluble formazan, which is purple in color. Tetrazolium dye reduction is dependent on NAD(P)H-dependent oxidoreductase enzymes, primarily located in the cytosolic compartment of metabolically active cells. The MTT assay is commercially available. In a standard experiment, cells and the composition for cryopreservation, cavity- containing oligomer and/or polymer, and/or cryoprotectant solution is mixed in any suitable container. The mixture is then cooled to the desired sub 0 °C temperature and held for a desired amount of time. Cells are then returned to ambient temperatures and the MTT reagent is added, incubated, and measured following the ATCC or Sigma-Aldrich protocol. Typically, absorbance of converted dye is measured at a wavelength of 570 nm with a background subtraction at 630- 690 nm. [0186] A third, more sensitive, two-color dual-parameter cell viability assay can be performed using promising solutions. Calcein AM is a cell-permeant and non-fluorescent compound that is widely used for determining cell viability and can be used at nM concentrations. In live cells, it is hydrolyzed by intracellular esterases into the strongly green fluorescent anion calcein. Fluorescent calcein is well-retained in the cytoplasm of live cells. Cells that die via the necrosis pathway, in contrast to those with apoptosis, rapidly lose membrane integrity and do not retain calcein (le _c at 494 nm/lEhi at 517 nm), and so can instead be stained by red-fluorescent ethidium homodimer- 1 (le _c at 528 nm/lEhi at 617 nm). After the freeze-thaw process, samples are incubated in a stain solution containing calcein AM and ethidium homodimer-l (1 :4) for 30 minutes at r.t. Samples are then imaged, e.g., via a fluorescence microscope. The sample loading stage can be programed to scan 96/384-well plates. A parallel comparison of images of each sample provides direct insight to identify the most potent cryoprotectant compounds and formulas.

[0187] In some embodiments, cell viability is tested using the LIVE/DEAD® Viability/Cytotoxicity Kit, for mammal cells provided by Thermo Fisher Scientific, Inc. This kit uses two indicator molecules: calcein AM and Ethidium homodimer-l (EthD-l). Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluore scent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells (le _c -495 nm / le _c -515 nm). Conversely, EthD- 1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (le _c -495 nm / ¾Em -635 nm). Notably, EthD-l is excluded by the intact plasma membrane of live cells, so the determination of live and dead cells is easily distinguishable. Calcein and EthD-l can be viewed simultaneously with a conventional fluorescein longpass filter. Alternatively, the fluorescence from these dyes may also be observed separately; calcein can be viewed with a standard fluorescein bandpass filter, and EthD-l can be viewed with filters for propidium iodide or Texas Red® dye. In a standard experiment, cells and the cryopreservation composition are mixed in any suitable container. The mixture is then cooled to the desired sub 0 °C temperature, held at that temperature for the desired amount of time, and then returned to ambient temperatures. Subsequent steps involving the addition of the calcein AM and EthD-l reagents and measuring the assay results are performed as described in the Thermo Fisher protocol. Typically, direct readout of cell viability is determined by measuring the relative fluorescence at the above indicated wavelengths for both reagents.

Methods for Delivering Therapeutic and Diagnostic Agents

[0188] Cavity-containing oligomers and polymers and cryopreservation compositions of the present invention are also useful for delivering compounds such as therapeutic agents and diagnostic agents to cells, populations of cells, tissues, and organs (i.e., the nanopore can be used as a delivery vehicle). In some embodiments, a method for delivering a therapeutic agent or diagnostic agent to a cell, a population of cells, a tissue, or an organ comprises contacting the cell, population of cells, tissue, and/or organ with the therapeutic or imaging agent and the cavity- containing oligomer or polymer, or cryopreservation composition.

[0189] Suitable therapeutic and diagnostic agents include, but are not limited to, drug, small molecules, nutrients, imaging agents, and combinations thereof. The imaging agent can be, for example, a radioactive tracer, a fluorescent tracer, or a combination thereof.

[0190] In some embodiments, the method further comprises comprising cooling the cell, population of cells, tissue, or organ ( e.g ., after the cell, population of cells, tissue, or organ has been contacted with the therapeutic or diagnostic agent) to a temperature of about 4 °C or lower (e.g., about 4, 3, 2, 1, 0, -1 -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -

19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -

40, -41, -42, -43, -44, -45, -46, -47, -48, -49, -50, -51, -52, -53, -54, -55, -56, -57, -58, -59, -60, -

61, -62, -63, -64, -65, -66, -67, -68, -69, -70, -71, -72, -73, -74, -75, -76, -77, -78, -79, -80, -81, -

82, -83, -84, -85, -86, -87, -88, -89, -90, -91, -92, -93, -94, -95, -96, -97, -98, -99, -100, -101, -102, -103, -104, -105, -106, -107, -108, -109, -110, -111, -112, -113, -114, -115, -116, -117, -118, -119,

-120, -121, -122, -123, -124, -125, -126, -127, -128, -129, -130, -131, -132, -133, -134, -135, -136,

-137, -138, -139, -140, -141, -142, -143, -144, -145, -146, -147, -148, -149, -150, -151, -152, -153,

-154, -155, -156, -157, -158, -159, -160, -161, -162, -163, -164, -165, -166, -167, -168, -169, -170,

-171, -172, -173, -174, -175, -176, -177, -178, -179, -180, -181, -182, -183, -184, -185, -186, -187,

-188, -189, -190, -191, -192, -193, -194, -195, -196, -197, -198, -199, or -200 °C). In some embodiments, the cell, population of cells, tissue, or organ is cooled to a temperature of about 4 °C.

[0191] In addition, compositions of the present invention can be used to provide arrays of nanopores that can be used, for example, in methods of purification or separation (e.g. of molecules (e.g, biological molecules) and/or ions).

IV. Examples

[0192] The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1. Cavity-containing oligomers and polymers (Compound 1 and 2) exhibit no cellular toxicity over 24-hour incubation with K562 cells.

[0193] This example shows that cavity-containing oligomer and polymer compounds have no cell toxicity. In particular, Compound 1 achieved a superior cryopreservation, reducing the necessary amount of CPAs.

Cytotoxicity assays

[0194] In order to demonstrate the safety of the cavity-containing oligomer and polymer composition, a cell based cytotoxicity assay was developed utilizing the K562 cell line, a human cell line derived from chronic myelogenous leukemia. The solutions contained culture media, buffers and a polymer (Compound 1 or Compound 2) for a final concentration of 10% FBS and 1% DMSO in DMEM. Serial dilutions were performed to obtain solutions containing various concentrations of Compound 1 and Compound 2 (128 mM, 32 pM, 8 pM, 2 pM, 0.5 pM, 0.125 M). Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively (i.e., the value of m in Formula (II) is 8 and 16, respectively).

[0195] For these experiments, cells were seeded at a density of 50,000 cells, exposed to the solution containing Compound 1 or Compound 2 and placed in a 37 °C incubator for 24 h. The compound cytotoxicity was assessed via Alamar blue assay. [0196] As can be seen in FIG. 10, as analyzed by Alamar blue at 24 h, the toxicity of Compound 1 and 2 did not significantly deviate from the one of culture media (DMEM +10% FBS) with 1% DMSO. Notably Compound 1 and Compound 2 did not show toxicity also at the highest concentration examined.

Example 2. Addition of cavity-containing oligomers and polymers (Compound 1) improve K562 cell post-thaw survival compared to K562 cells cryopreserved by cryoprotectant solution alone consisting of 1% DMSO, 10% FBS and DMEM.

[0197] DMSO is the most widely used CPA for cell and tissue cryopreservation, but it is often used at a concentrations that can result in significant cytotoxicity ( e.g ., 10% for routine cell cryopreservation). At 1% concentration, DMSO shows limited cytotoxicity. However it is not effective to successfully cryopreserve cells even with presence of FBS. It is demonstrated that Compound 1 can facilitate the delivery of 1% DMSO even at low concentrations and help the survival of cells that have been stored at -80 °C. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively ( i.e the value of m in Formula (II) is 8 and 16, respectively).

[0198] This example serves to show cavity-containing oligomers and polymers achieved superior cryopreservation when used in the presence of DMSO as a cryoprotectant at low concentrations.

Cryopreservation assays

[0199] Cryopreservation efficacy of a cryoprotectant solution containing 1% DMSO and 40% FBS in DMEM in the presence of various concentrations of Compound 1 and Compound 2 was evaluated by post-thaw cell viability assay. Each freezing solution was prepared at 64 mM, 16 pM, 4 pM, 1 pM, 0.25 pM, or 0.0625 pM compound concentrations with final concentrations of 1% DMSO and 40% FBS in DMEM.

[0200] K562 cells were grown in culture. lxlO ⁶ cells were pelleted and resuspended in 200 pl of one of the sample solutions. Vials with the resuspended cells were prechilled for 10 min at 4 °C and then stored at -80 °C for a period of 3 days or 7 days. After this period samples were rewarmed in a 37 °C water bath for 5 min. The cells were immediately diluted 1 : 10 with Phenol red-free media and plated in a 96 well plate at a density of 50,000 cells per well. After 24 h recovery cells were stained with Alamar blue following vendor instruction and cell viability measured using a fluorescence plate reader.

[0201] In a first set of experiments, each freezing solution was prepared at 64 mM, 16 pM, 4 pM, 1 pM, 0.25 pM, or 0.0625 pM compound concentrations with final concentrations of 1% DMSO and 40% FBS in DMEM. Controls included one without polymer but with 1% DMSO and 40% FBS in DMEM (shown as 1% DMSO). A negative control of cells frozen in PBS was also added. Results are shown in FIG. 11.

[0202] The addition of Compound 1 greatly improved cell survival in comparison to control cryoprotectant solutions and demonstrated the ability of increasing the effectiveness of DMSO based cryoprotection by about 9 times at the highest concentration of 64 pM cavity-containing oligomer.

Example 3. Addition of cavity-containing oligomers and polymers improve K562 cell cryopreservation after 3 days post-thaw survival compared to K562 cells cryopreserved by cryoprotectant solution alone, which consists of 3% Glycerol, 10% FBS and DMEM.

[0203] This example shows that cavity-containing oligomers and polymers achieved superior cryopreservation when used in the presence of glycerol as a cryoprotectant at low concentrations.

Cryopreservation assays

[0204] Cryopreservation efficacy of a cryoprotectant solution that contained 3% glycerol and 40% FBS in DMEM in the presence of various concentration of Compound 1 and Compound 2 have been evaluated by post-thaw cell viability assay. The results demonstrate that Compound 1 likely facilitates the delivery of glycerol even at low concentrations to improve the survival of cells that have been stored at -80 °C. Each freezing solution was prepared at 64 pM, 16 pM, 4 pM, 1 pM, 0.25 pM, or 0.0625 pM compound concentrations with final concentrations of 40% FBS in DMEM. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively (i.e., the value of m in Formula (II) is 8 and 16, respectively).

[0205] K562 cells were grown in culture. lxlO ⁶ cells were pelleted and resuspended in 200 pl of one of the sample solutions. Vials with the resuspended cells were prechilled 10 min at 4 °C and then stored at -80 °C for a period of 3 days or 7 days. After this period samples were rewarmed in a 37 °C water bath for 5 min. The cells were immediately diluted 1 : 10 with Phenol red-free media and plated in a 96-well plate at a density of 50,000 cells per well. After a 24-hour recovery cells were stained with Alamar blue following vendor instruction and cell viability measured using a fluorescence plate reader.

[0206] A second set of experiments was performed to evaluate the cryopreservation potential of formulation containing different concentrations of Compound 1 and Compound 2 (64 mM, 16 mM, 4 pM) plus a mixture of 3% glycerol, 40% FBS and DMEM. Controls included one solution with polymer only without glycerol, one solution without polymer but with glycerol, and one solution without glycerol and polymers.

[0207] As shown in FIG. 12, 24 hours post-thawing after a 3 -day storage at -80 °C, the viability of the cells improved as much as two times using Compound 1 and 2 in combination with glycerol. The best result was achieved at the highest concentration of Compound 1. Further, the lack of cryopreservation performance for Compound 1 alone demonstrated that Compound 1 was not an ice-interacting compound by itself, but instead facilitated the performance of CPAs.

Example 4. Addition of cavity-containing oligomers and polymers improve K562 cells cryopreserved for 7 days post-thaw survival compared to K562 cells cryopreserved by cryoprotectant solution alone, which consists of 3% Glycerol, 10% FBS and DMEM.

[0208] This example shows that cavity-containing oligomers and polymers achieved superior cryopreservation when used in the presence of glycerol as a cryoprotectant at low concentrations during extended 7 day storage at -80 °C.

Cryopreservation assays

[0209] Glycerol is a known cryoprotectant, but is often used at very high concentrations. To reduce the cryoprotectant performance of the freezing media and allow the performance of the cavity-containing oligomers and polymers to be more apparent, preliminary cryopreservation assays were performed on test solutions with a low amount of glycerol in the presence or absence of Compound 1 and 2. Results demonstrated that Compound 1 facilitated the delivery of glycerol even at low concentrations and helped the survival of cells that had been stored at -80 °C. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively ( i.e the value of m in Formula (II) is 8 and 16, respectively). [0210] Each freezing solution was prepared at 64 mM, 16 pM, 4 pM, 1 pM, 0.25 pM, or 0.0625 pM compound concentrations with final concentrations of 3% glycerol and 40% FBS in DMEM.

[0211] K562 cells were grown in culture. lxlO ⁶ cells were pelleted and resuspended in 200 pl of one of the sample solutions. Vials with the resuspended cells were prechilled for 10 min at 4 °C and then stored at -80 °C for a period of 3 days or 7 days. After this period samples were rewarmed in a 37 °C water bath for 5 min. The cells were immediately diluted 1 : 10 with Phenol red-free media and plated in a 96-well plate at a density of 50,000 cells per well. After a 24-hour recovery cells were stained with Alamar blue following vendor instruction and cell viability measured using a fluorescence plate reader.

[0212] An additional set of experiments was performed to evaluate the cryopreservation potential of cryoprotectant solutions containing different concentrations of Compound 1 and Compound 2 (64 pM, 16 pM, 4 pM) plus a mixture of 3% glycerol, 40% FBS and DMEM. Controls included one solution with cavity-containing oligomer alone without glycerol, one solution without cavity-containing oligomer but with glycerol, and one without glycerol or cavity- containing oligomer.

[0213] As shown in FIG. 13, 24 hours post-thawing after a 7 day storage at -80 °C the viability of the cells improved as much as seven times using Compound 1 in combination with glycerol. The best result was achieved at the highest concentration of Compound 1.

[0214] Confocal microscopic live cell imaging support improved cell viability with Compound 1 and glycerol, as shown in FIG. 13. In FIG. 13, bright spots indicate viable cells, wherein the higher number of brighter spots indicates higher cell viability. As shown in FIG. 14, the 64 pM, 16 pM, and 4 pM concentrations of Compound 1 led to a greater number of viable cells per well.

[0215] This data confirms the previous 3-day storage result for longer term 7-day storage.

Example 5. Addition of cavity-containing oligomers and polymers improve K562 cell 7 days post-thaw survival compared to K562 cells cryopreserved by cryoprotectant solution alone, which consists of 3% Sorbitol, 10% FBS and DMEM.

[0216] This example shows that cavity-containing oligomers and polymers achieved superior cryopreservation when used in the presence of sorbitol as a cryoprotectant at low concentrations. Cryopreservation assays

[0217] Sorbitol is a known cryoprotectant, but it is often used at very high concentrations. To reduce the cryoprotectant performance of the freezing media and allow the performance of the cavity-containing oligomers and polymers to be more apparent, preliminary cryopreservation assays were performed to test solutions with a low amount of sorbitol in the presence or absence of Compound 1 and 2. We demonstrated that Compound 1 facilitated the delivery of sorbitol, even at low concentrations, and increased the survival of cells that have been stored at -80 °C. Compounds 1 and 2 comprise the structure of Formula (II) and have 8 and 16 residues, respectively ( i.e the value of m in Formula (II) is 8 and 16, respectively).

[0218] Each freezing solution was prepared at 64 mM, 16 pM, 4 pM, 1 pM, 0.25 pM, or 0.0625 pM compound concentrations with final concentrations of 1% DMSO and 40% FBS in DMEM.

[0219] K562 cells were grown in culture. lxlO ⁶ cells were pelleted and resuspended in 200 pl of one of the sample solutions. Vials with the resuspended cells were prechilled 10 min at 4 °C and then stored at -80 °C for a period of 3 days or 7 days. After this period samples were rewarmed in a 37 °C water bath for 5 min. The cells were immediately diluted 1 : 10 with Phenol red-free media and plated in a 96-well plate at a density of 50,000 cells per well. After a 24-hour recovery cells were stained with Alamar blue following vendor instruction and cell viability measured using a fluorescence plate reader.

[0220] Another similar experiment was performed adding to the solution 3% sorbitol in the presence or absence of Compound 1 at different concentrations (64 pM, 16 pM, 4 pM). While the solution with sorbitol alone and the solution with the compound alone did not increase the cell survival, a combination of the two greatly improved the viability of the cells in FIG. 14.

[0221] In addition, Compounds 3 and 4 were shown to be effective for cryopreservation (FIG. 15). Compounds 3 and 4 comprise the structure of Formula (II) and have 6 and 10 residues, respectively {i.e., the value of m in Formula (II) is 6 and 10, respectively).

[0222] The present invention includes cavity-containing oligomers and polymers that do not appear as ice-interacting compounds and that can transport DMSO, glycerol and sorbitol intracellularly to assist cell survival after cryopreservation. These polymers can achieve superior cryopreservation and reduce the necessary amount of CPAs, thus reducing also the cell toxicity often associated with CPAs.

[0223] It is understood that the examples and embodiments described are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes.

V. Exemplary Embodiments

[0224] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

1. A composition for cryopreservation of a population of cells, a biological tissue, or an organ, the composition comprising:

(a) a cryoprotectant solution comprising a compound selected from the group consisting of an ionic species, a penetrating cryoprotectant, a non-penetrating cryoprotectant, an antioxidant, a cell membrane stabilizing compound, a channel-forming compound, an alcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinylpyrrolidone, polyvinyl alcohol, hyaluronan, formamide, a natural or synthetic hydrogel, and a combination thereof; and

(b) an oligomer or polymer comprising a backbone that comprises a structure according to Formula

(I):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH20CH3, -CH2CH20(CH2CH20)zCH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted Cl 12 acyl, optionally substituted C 1-12 alkylamino, OH, SH, NH2, -NH3+, -N+R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein n is an integer having a value of 4 to 100.

2. The composition of embodiment 1, wherein n has a value of 6 to 64.

3. The composition of embodiment 1 or 2, wherein n has a value of 6 to 16.

4. The composition of any one of embodiments 1 to 3, wherein n is 6, 8, 10, or 12.

5. The composition of any one of embodiments 1 to 4, wherein the backbone of the oligomer or polymer comprises a plurality of aromatic substituents linked by at least one amide group, wherein the backbone is curved due at least in part to intramolecular hydrogen bonds that rigidify the amide linkage of each amide group to each aromatic substituent and at least in part to an interaction between the aromatic substituents, whereby the curved backbone is stabilized, and wherein the backbone comprises a structure according to Formula (II):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH20CH3, -CH2CH20(CH2CH20)zCH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted Cl 12 acyl, optionally substituted C 1-12 alkylamino, OH, SH, NH2, -NH3+, -N+R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein m is an integer having a value of 2 to 50.

6. The composition of embodiment 5, wherein m has a value of 2 to 26.

7. The composition of embodiment 5 or 6, wherein m has a value of 2 to 8.

8. The composition of any one of embodiments 1 to 7, wherein the backbone of the oligomer or polymer comprises a structure according to Formula (III):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH20CH3, -CH2CH20(CH2CH20)zCH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted Cl 12 acyl, optionally substituted C 1-12 alkylamino, OH, SH, NH2, -NH3+, -N+R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; and wherein z is an integer having a value of 1 to 10.

9. The composition of any one of embodiments 1 to 7, wherein the backbone of the oligomer or polymer comprises a structure according to Formula (IV):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH20CH3, -CH2CH20(CH2CH20)zCH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted Cl 12 acyl, optionally substituted C 1-12 alkylamino, OH, SH, NH2, -NH3+, -N+R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein m is an integer having a value of 2 to 50.

10. The composition of embodiment 9, wherein m has a value of 2 to 26.

11. The composition of embodiment 9 or 10, wherein m has a value of 2 to 8.

12. The composition of embodiment 1, 5, 8, or 9, wherein the aryl group is selected from the group consisting of phenyl, benzyl, napthyl, alkyl, and an aryl group with a polar terminal functional end.

13. The composition of embodiment 12, wherein the polar terminal functional end is selected from the group consisting of -OH, -COOH, -NH2, -NH3+, -N+R'3, -CH2CH2C6H5, CH2C6H4- p-OH, -CH2COOCH3, -CH2CONH2, -CH2CH2CONH2, and -CH2COOCH2-(3-indolyol), wherein R’ can be any alkyl or aryl group. 14. The composition of any one of embodiments 1 to 13, wherein at least one instance of R is an oxy ether or thioether.

15. The composition of embodiment 14, wherein at least one instance of R comprises the structure:

16. The composition of embodiment 15, wherein he oligomer or polymer backbone comprises a structure according to Formula (VII):

(VII), wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted Cl 12 acyl, optionally substituted C 1-12 alkylamino, OH, SH, NH2, -NH3+, -N+R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; and wherein m is an integer having a value of 2 to 50.

17. The composition of embodiment 16, wherein m has a value of 2 to 26.

18. The composition of embodiment 16 or 17, wherein m is 3, 4, 5, 6, 7, or 8.

19. A composition for cryopreservation of a population of cells, a biological tissue, or an organ, the composition comprising:

(a) a cryoprotectant solution comprising a compound selected from the group consisting of an ionic species, a penetrating cryoprotectant, a non-penetrating cryoprotectant, an antioxidant, a cell membrane stabilizing compound, a channel-forming compound, an alcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinylpyrrolidone, polyvinyl alcohol, and a combination thereof; and

(b) an oligomer or polymer comprising a backbone that comprises a plurality of aromatic substituents linked by at least one amide group, wherein the backbone is curved due at least in part to intramolecular hydrogen bonds that rigidify the amide linkage of each amide group to each aromatic substituent and at least in part to an interaction between the aromatic substituents, whereby the curved backbone is stabilized, and wherein the backbone comprises a structure according to Formula (V):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH20CH3, -CH2CH20(CH2CH20)zCH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted Cl 12 acyl, optionally substituted C 1-12 alkylamino, OH, SH, NH2, -NH3+, -N+R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; wherein z is an integer having a value of 1 to 10; and wherein m is an integer having a value of 2 to 50.

20. The composition of embodiment 19, wherein m has a value of 2 to 26.

21. The composition of embodiment 19 or 20, wherein m has a value of 2 to 8.

22. The composition of any one of embodiments 19 to 21, wherein the backbone of the oligomer or polymer comprises a structure according to Formula (VI):

wherein each instance of R is independently selected from the group consisting of a linear chain alkyl group, a branched chain alkyl group, a linear chain ether group, a branched chain ether group, a linear chain thioether group, a branched chain thioether group, a group that is in general chiral, -CH3, -CH2CH20CH3, -CH2CH20(CH2CH20)zCH3, and an aryl group; wherein X and Y are independently selected from the group consisting of H, optionally substituted C1-12 alkyl, optionally substituted Cl 12 acyl, optionally substituted C 1-12 alkylamino, OH, SH, NH2, -NH3+, -N+R’3, carboxy, tert-butyl ester, trifluoroacetamide, optionally substituted C1-12 hydroxyalkyl, optionally substituted C1-12 alkylamino, optionally substituted C2-12 alkylthio, optionally substituted C1-12 carboxyalkyl, and halogen, or alternatively X and Y are taken together to form a covalent bond; wherein R’ can be any alkyl or aryl group; and wherein z is an integer having a value of 1 to 10.

23. The composition of embodiment 19 or 22, wherein the aryl group is selected from the group consisting of phenyl, benzyl, napthyl, alkyl, and an aryl group with a polar terminal functional end.

24. The composition of embodiment 23, wherein the polar terminal functional end is selected from the group consisting of -OH, -COOH, -NH2, -NH3+, -N+R'3, -CH2CH2C6H5, CH2C6H4- p-OH, -CH2COOCH3, -CH2CONH2, -CH2CH2CONH2, and -CH2COOCH2-(3-indolyol).

25. The composition of any one of embodiments 19 to 24, wherein at least one instance of R is an oxy ether or thioether.

26. The composition of embodiment 25, wherein at least one instance of R comprises the structure:

27. The composition of embodiment 26 the oligomer or polymer comprises the structure:

28. The composition of any one of embodiments 1 to 27, wherein the alcohol is selected from the group consisting of propylene glycol, ethylene glycol, glycerol, methanol, butylene glycol, adonitol, ethanol, trimethylene glycol, diethylene glycol, polyethylene oxide, erythritol, sorbitol, xythyritol, polypropylene glycol, 2-methyl-2,4-pentanediol (MPD), mannitol, inositol, dithioritol, 1, 2-propanediol, and a combination thereof.

29. The composition of any one of embodiments 1 to 27, wherein the sugar is a monosaccharide, disaccharide, or polysaccharide.

30. The composition of any one of embodiments 1 to 29, wherein the oligomer or polymer is present at a concentration of about 100 nM to about 1,000 mM.

31. A method for cryopreserving a population of cells with improved cell viability, the method comprising contacting the population of cells with the cryopreservation composition of any one of embodiments 1 to 30 to produce a cryopreserved population of cells. 32. The method of embodiment 31, wherein the method further comprises cooling the population of cells to a temperature of from about 0 °C to about -200 °C.

33. The method of embodiment 32, wherein the population of cells is cooled for a time period of at least about 3 hours.

34. The method of any one of embodiments 31 to 33, wherein the population of cells comprises a tissue of any kind or an organ of any kind.

35. The method of embodiment 34, wherein the tissue or organ is a bioengineered tissue of any kind or organ of any kind.

36. The method of any one of embodiments 31 to 35, wherein the population of cells is selected from the group consisting of primary cells, heart cells, liver cells, lung cells, kidney cells, pancreatic cells, gastric cells, intestinal cells, muscle cells, skin cells, neural cells, blood cells, immune cells, fibroblasts, genitourinary cells, bone cells, stem cells, sperm cells, oocytes, embryonic cells, epithelial cells, endothelial cells, and a combination thereof.

37. The method of embodiment 36, wherein the cells are mammalian cells.

38. The method of embodiment 37, wherein the mammalian cells are human cells.

39. A method for cry opreserving a biological tissue with improved biological tissue viability, the method comprising:

(a) contacting the biological tissue with the cryopreservation composition of any one of embodiments 1 to 30; and

(b) cooling the biological tissue to a temperature of from about 0 °C to about -200 °C over a time period of at least about 3 hours to produce a cryopreserved biological tissue.

40. The method of embodiment 39, wherein the tissue is selected from the group consisting of heart tissue, liver tissue, lung tissue, kidney tissue, gall bladder tissue, reproductive system tissue, pancreatic tissue, gastric tissue, intestinal tissue, muscle tissue, skin tissue, neural tissue, blood, genitourinary tissue, bone, epithelial tissue, endothelial tissue, corneal tissue, heart valve tissue, and a combination thereof.

41. The method of embodiment 39, wherein the tissue is a bioengineered tissue of any kind.

42. A method for cryopreserving an organ with improved organ viability, the method comprising:

(a) contacting the organ with the cryopreservation composition of any one of embodiments 1 to 30; and

(b) cooling the organ to a temperature of from about 0 °C to about -200 °C over a time period of at least about 3 hours to produce a cryopreserved organ.

43. The method of embodiment 42, wherein the organ is selected from the group consisting of heart, liver, lung, kidney, a reproductive organ, pancreas, stomach, gall bladder, intestine, muscle, skin, bone, and a combination thereof.

44. The method of embodiment 42, wherein the organ is a bioengineered organ of any kind.

45. A method for cry opreserving a population of cells, a tissue, or an organ with improved cell, tissue, or organ viability, the method comprising:

(a) cooling the population of cells, tissue, or organ to a temperature of about 4 °C; and

(b) contacting the population of cells, tissue, or organ with the cryopreservation composition of any one of embodiments 1 to 30 at the temperature of about 4 °C.

46. A method for delivering a therapeutic agent or diagnostic agent into a cell, a population of cells, a tissue, or an organ, the method comprising contacting the cell, population of cells, tissue, or organ with the therapeutic agent or diagnostic agent and the cryopreservation composition of any one of embodiments 1 to 30.

47. The method of embodiment 46, wherein the therapeutic agent or diagnostic agent is selected from the group consisting of a drug, a small molecule, a nutrient, an imaging agent, and a combination thereof. 48. The method of embodiment 47, wherein the imaging agent is selected from the group consisting of a radioactive tracer, a fluorescent tracer, and a combination thereof.

49. The method of any one of embodiments 46 to 48, wherein the therapeutic agent or diagnostic agent is contacted with the cell, population of cells, tissue, or organ after cooling the cell, population of cells, tissue, or organ to a temperature of about 4 °C or lower.

50. The method of embodiment 49, wherein the cell, population of cells, tissue, or organ is cooled to a temperature of about 4 °C.