A method for improving the freeze resistance of metal-organic frameworks and its application in the cryopreservation of red blood cells

Abstract

This invention provides a method for improving the cryoprotective properties of metal-organic frameworks (MOFs) and their application in erythrocyte cryoprotection. By preparing highly stable MOF materials, the method addresses the issue of low crystallinity in MOF materials prepared via hydrothermal reactions, avoiding structural damage caused by phosphonic acids to low-crystallinity MOFs. This facilitates the maintenance of effective interfacial hydrogen bond matching in phosphonic acid-modified MOF materials, thus preserving high cryoprotective capacity. The MOF materials prepared by the method described in this invention, as erythrocyte cryoprotectants, exhibit a higher erythrocyte recovery rate (over 98%) compared to existing MOF materials, solving the problem of insufficient cryoprotective effect of MOF materials and promoting their further clinical application.

Description

Technical Field

[0001] This invention relates to a method for improving the cryoprotective properties of metal-organic frameworks and the application of the improved metal-organic frameworks as cryoprotectants in the cryoprotection of red blood cells. Background Technology

[0002] Cryopreservation of biological samples requires the addition of cryoprotectants to reduce damage during freezing. However, traditional permeable organic cryoprotectants such as DMSO have certain toxicity and require long washing times after thawing, which cannot meet the needs of blood in emergency situations. Therefore, the development of novel cryoprotectants with high biocompatibility and strong antifreeze capabilities to replace traditional permeable cryoprotectants such as glycerol and DMSO is of great practical significance. Metal-organic frameworks (MOFs) have periodically arranged surface structures that can match the ice crystal interface, thereby inhibiting ice crystal formation and growth. They are a new type of erythrocyte cryoprotectant with excellent antifreeze and anti-crystallization capabilities discovered in recent years. However, the final erythrocyte recovery rate when using existing MOFs as cryoprotectants is too low, which cannot meet the actual needs of clinical use. Surface modification of the MOF interface structure (such as phosphonic acid surface modification) can increase the surface hydrogen bond density, enhance the antifreeze and anti-crystallization properties of MOFs, and thus improve the final cryopreservation efficiency of biological samples. However, due to the high affinity of phosphonic acids for metal sites, high stability must be achieved before phosphonic acid surface modification of metal-organic frameworks to avoid damage to the MOF structure. Improving the antifreeze properties of metal-organic frameworks will help promote the practical application of metal-organic frameworks in the field of cryoprotection. Summary of the Invention

[0003] The primary objective of this invention is to overcome the shortcomings of the prior art by introducing phosphoric acid through the rational design of the metal-organic framework interface structure to improve the anti-ice and antifreeze properties of the metal-organic framework, by increasing the crystallinity of the metal-organic framework to improve its stability and thus avoid the destruction of the MOF structure by phosphonic acid, and by improving the final red blood cell cryoprotection recovery rate when the existing metal-organic framework is used as a red blood cell cryoprotectant, thereby providing a phosphonic acid surface-modified metal-organic framework.

[0004] A second objective of this invention is to provide a method for preparing the above-mentioned phosphonic acid surface-modified metal-organic framework.

[0005] A third objective of this invention is to provide applications of phosphonic acid surface-modified metal-organic frameworks.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] A phosphonic acid-modified metal-organic framework is prepared by surface modification of a highly crystalline metal-organic framework with phosphonic acid.

[0008] The method for preparing the highly crystalline metal-organic framework includes the following steps:

[0009] (1) Take organic ligand 2, ultrasonically disperse it in an alkaline solution, add dilute acid solution to adjust the pH of the solution to 6-8, and add organic solvent to obtain H2O / organic solvent solution of organic ligand 2;

[0010] (2) Take metal-organic framework A and add it to the prepared organic ligand 2 H2O / organic solvent solution, disperse it by ultrasonication, react, centrifuge, wash, dry and collect the solids;

[0011] (3) Add the solid collected in (2) to the prepared H2O / organic solvent solution of organic ligand 2, disperse by ultrasonication, react, centrifuge, wash, and dry to obtain a highly crystalline metal-organic framework.

[0012] The organic ligand 2 mentioned in step (1) is an organic ligand with hydrogen-rich matching sites;

[0013] The metal-organic framework A mentioned in step (2) includes at least one of UiO-66, UiO-67 and UiO-68.

[0014] Further, the phosphonic acid includes N-(phosphonomethyl)glycine, 3-phosphono-L-alanine, dialanine, phosphonopyruvate, tripropylphosphonic acid, hydroxyethylidene diphosphonic acid, 2-hydroxyphosphonoacetic acid, methylphosphonic acid, phenylphosphonic acid, phosphonoacetic acid, phosphonoacetaldehyde, ethylphosphonic acid, vinylphosphonic acid, 2-aminoethylphosphonic acid, (2-chloroacetyl)phosphonic acid, 2-amino-4-[hydroxy(methyl)phosphono]butanoic acid ammonium, 2-hydroxyethylphosphonic acid, (2-amino-1-hydroxyethyl)phosphonic acid, etc. At least one of the following: phosphonic acid, diethylenetriaminepentamylidenephosphonic acid, ethylenediaminetetramylidenephosphonic acid, aminotrimylidenephosphonic acid, zoledronic acid, phosphonocarboxylic acid, (1R,2S)-1,2-epoxypropylphosphonic acid, 2-hydroxyethyl-[(E)-14-iodotetradecane-13-ene-3,5-diyneoxy]phosphonic acid, L-arginino-L-2-amino-5-phosphoryl-3Z-pentenoic acid; further comprising at least one of 2-aminoethylphosphonic acid, vinylphosphonic acid, and 2-hydroxyethylphosphonic acid.

[0015] Further, the organic ligand 2 mentioned in step (1) includes 2-hydroxyterephthalic acid, 2,3-dihydroxyterephthalic acid, 2,5-dihydroxyterephthalic acid, 2,6-dihydroxyterephthalic acid, 2,3-diaminoterephthalic acid, 2,5-diaminoterephthalic acid, 4,4'-dihydroxy-3,3'-biphenyl dicarboxylic acid, 2,5-dihydroxybiphenyl dicarboxylic acid, and 2',5'-dihydroxy-[1,1':4 [1,1”-terphenyl]-4,4”-dicarboxylic acid, 2-amino-4,4'-biphenyl dicarboxylic acid, 4,4'-diaminobiphenyl-2,2'-dicarboxylic acid; further comprising at least one of 2-hydroxyterephthalic acid, 2,5-diaminoterephthalic acid, 2,5-dihydroxybiphenyl dicarboxylic acid and 2',5'-dihydroxy-[1,1':4',1”-terphenyl]-4,4”-dicarboxylic acid.

[0016] Further, the alkaline solution mentioned in step (1) includes at least one of NaOH solution, Na2CO3 solution, NaHCO3 solution, KOH solution, K2CO3 solution and KHCO3 solution; and even more specifically, at least one of NaOH solution and KOH solution.

[0017] Further, the molar ratio of the organic ligand 2 and the alkali in the alkaline solution mentioned in step (1) is:

[0018] Further, the dilute acid solution mentioned in step (1) includes at least one of dilute sulfuric acid solution, dilute nitric acid solution, dilute hydrochloric acid solution and dilute phosphoric acid solution; and even more specifically, dilute hydrochloric acid solution.

[0019] Furthermore, the pH adjustment of the solution in step (1) is to adjust the pH of the conditional solution to 7 to 7.4.

[0020] Further, the organic solvent mentioned in step (1) includes at least one of N,N-dimethylformamide (DMF) and N,N-diethylformamide (DEF).

[0021] Further, the metal-organic framework A described in step (2) includes at least one of UiO-66(Zr), UiO-67(Zr), UiO-68(Zr), UiO-66(Hf), UiO-67(Hf) and UiO-68(Hf).

[0022] Furthermore, the organic ligand 1 of the metal-organic framework A described in step (2) includes at least one of terephthalic acid, biphenyl dicarboxylic acid, and terphenyl dicarboxylic acid.

[0023] Furthermore, the preparation of the metal-organic framework A described in step (2) includes the following steps:

[0024] S1. Weigh the metal compound and organic ligand 1, and sonicate them separately in a solvent to prepare metal compound solution and organic ligand 1 solution respectively. After thorough sonication, mix the metal compound solution and organic ligand 1 solution, then add crystal regulator, continue sonication to mix thoroughly, perform hydrothermal reaction, centrifuge, wash with washing solution, and dry to obtain metal-organic framework 1.

[0025] Furthermore, the metal compound described in step S1 includes at least one of zirconium propoxide, zirconium tetrachloride, zirconium dichloride, hafnium propoxide, hafnium tetrachloride, and hafnium dichloride.

[0026] Furthermore, the organic ligand 1 described in step S1 includes at least one of terephthalic acid, biphenyl dicarboxylic acid, and terphenyl dicarboxylic acid.

[0027] Furthermore, the solvent in step S1 includes at least one of water, N,N-dimethylformamide (DMF), and N,N-diethylformamide (DEF).

[0028] Furthermore, the crystal regulator described in step S1 includes at least one of formic acid, acetic acid, benzoic acid, hydrochloric acid, and trifluoroacetic acid; and even further includes acetic acid.

[0029] Furthermore, the molar ratio of the metal compound, organic ligand 1, and crystal regulator mentioned in step S1 is 0.5-2:0.5-2:20-100; and even further, it is 0.1:0.1:5.

[0030] Furthermore, the hydrothermal reaction conditions described in step S1 are 80–120°C for 12–36 hours.

[0031] Furthermore, the washing described in step S1 is as follows: washing with DMF and anhydrous methanol in sequence.

[0032] Furthermore, the molar ratio of the metal-organic framework A and the organic ligand 2 in step (2) is 1:1 to 10.

[0033] Furthermore, the reaction described in step (2) is carried out at 80-120°C for 12-72 hours; even further, it is carried out at 80-100°C for 48 hours.

[0034] Further, the washing described in step (2) is: washing with DMF and anhydrous methanol in sequence.

[0035] The preparation of the phosphonic acid surface-modified metal-organic framework includes the following steps:

[0036] A highly crystalline metal-organic framework solution and a phosphonic acid solution were stirred and reacted, then centrifuged, washed, and dried to obtain a phosphonic acid-modified metal-organic framework.

[0037] Furthermore, the molar ratio of the highly crystalline metal-organic framework to phosphonic acid is 1:1 to 100; even further, it is 1:20.

[0038] Furthermore, the solvent for the highly crystalline metal-organic framework solution includes at least one of ethanol, methanol, acetone, and deionized water.

[0039] Furthermore, the solvent for the phosphonic acid solution includes at least one of ethanol, methanol, acetone, and deionized water.

[0040] Furthermore, the washing liquid used for washing includes at least one of ethanol, methanol, acetone and deionized water; even further, the washing liquid used for washing is water.

[0041] The above-mentioned application of phosphonic acid surface-modified metal-organic frameworks in cryopreservation of red blood cells.

[0042] The ratio of the phosphonic acid surface-modified metal-organic framework to cryopreserved red blood cells is 2–4000 mg: 2 × 10⁻⁶ mg. 9 indivual.

[0043] The concentration of the phosphonic acid surface-modified metal-organic framework used for cryopreservation of red blood cells is 1–10 mg / mL; more specifically, it is 10 mg / mL.

[0044] The above-mentioned application of phosphonic acid surface-modified metal-organic frameworks in the preparation of erythrocyte cryopreservation products.

[0045] Compared with the prior art, the present invention has the following advantages and effects:

[0046] (1) The red blood cell cryoprotectant prepared by the method of the present invention has a higher red blood cell liquid nitrogen cryoprotection recovery rate (over 98%) compared with the existing metal-organic framework, which solves the problem of insufficient cryoprotection effect of metal-organic framework and is conducive to further promoting the practical application of metal-organic framework in clinical practice.

[0047] (2) The method of improving the antifreeze performance of the metal-organic framework of the present invention can improve the crystallinity of the same metal-organic framework prepared by hydrothermal reaction, avoid the structural damage caused by phosphonic acid to low crystallinity metal-organic framework, and help the phosphonic acid surface-modified metal-organic framework maintain effective antifreeze ability. Attached Figure Description

[0048] Figure 1 This is a transmission electron microscopy (TEM) image of the red blood cell cryoprotectant from Example 1.

[0049] Figure 2 This is a transmission electron microscopy (TEM) image of the cryoprotectant for red blood cells in Comparative Example 1.

[0050] Figure 3 The image shows the optical microscope results of erythrocytes after thawing in the 20 mg / mL erythrocyte cryoprotectant group in Example 1. Detailed Implementation

[0051] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0052] Example 1:

[0053] A method for preparing a highly stable phosphonic acid surface-modified metal-organic framework as a cryoprotectant for erythrocytes includes the following steps:

[0054] (1) Weigh 4 mmol of sodium hydroxide and dissolve it in 2 mL of ultrapure water to prepare a sodium hydroxide solution with a mass fraction of 8%. Weigh 0.5 mmol of 2,5-diaminoterephthalic acid and dissolve it in the solution. Add dilute hydrochloric acid solution to adjust the pH of the solution to 7.4. Add 0.5 mL of DMF to obtain an H2O / DMF solution of 2,5-diaminoterephthalic acid.

[0055] (2) Weigh 0.1 mmol of metal-organic framework UiO-66 and add it to the H2O / DMF solution of 2,5-diaminoterephthalic acid prepared in step (1). Disperse by sonication and react hydrothermally at 100°C for 36 hours. Centrifuge and wash with N,N-dimethylformamide and anhydrous methanol in sequence. Dry and collect the solid. Add the collected solid product to the newly prepared H2O / DMF solution of 2,5-diaminoterephthalic acid (prepared in the same way as step (1)). Disperse by sonication and react hydrothermally at 100°C for 36 hours. Centrifuge and wash with N,N-dimethylformamide and anhydrous methanol in sequence. Dry to obtain highly crystalline metal-organic framework UiO-66-(NH2)2.

[0056] (3) Prepare equal volumes of 0.04 mol / L metal-organic framework UiO-66-(NH2)2 and 0.80 mol / L 2-aminoethylphosphonic acid aqueous solutions, stir the reaction, centrifuge, wash with ultrapure water, and dry to obtain highly stable phosphonic acid surface-modified metal-organic framework UiO-66-(NH2)2, and observe its morphology using transmission electron microscopy.

[0057] The metal-organic framework UiO-66 was prepared by the following method:

[0058] Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol terephthalic acid, and sonicate them separately in DMF to prepare equal volumes of zirconium tetrachloride solution and terephthalic acid solution. After thorough sonication, mix the two solutions, then add 5.0 mmol acetic acid and continue to sonicate thoroughly. The mixture is then subjected to hydrothermal reaction at 100 °C for 24 hours. After centrifugation, the mixture is washed successively with DMF and anhydrous methanol and dried to obtain metal-organic framework UiO-66.

[0059] A method for cryopreserving red blood cells using the aforementioned highly stable phosphonic acid surface-modified metal-organic framework as a red blood cell cryoprotectant:

[0060] Using 1×PBS buffer as solvent, highly stable phosphonic acid surface-modified metal-organic framework UiO-66-(NH2)2 dispersions with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL, and 20 mg / mL were prepared as erythrocyte cryoprotectants.

[0061] 1×10 7 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0062] Example 2:

[0063] A method for preparing a highly stable phosphonic acid surface-modified metal-organic framework as a cryoprotectant for erythrocytes includes the following steps:

[0064] (1) Weigh 4 mmol of sodium hydroxide and dissolve it in 2 mL of ultrapure water to prepare a sodium hydroxide solution with a mass fraction of 8%. Weigh 0.6 mmol of 2,5-dihydroxybiphenylcarboxylic acid and dissolve it in the solution. Add dilute hydrochloric acid solution to adjust the pH of the solution to 7.4. Add 0.5 mL of DMF to obtain an H2O / DMF solution of 2,5-dihydroxybiphenylcarboxylic acid.

[0065] (2) Weigh 0.1 mmol of metal-organic framework UiO-67 and add it to the H2O / DMF solution of 2,5-dihydroxybiphenylcarboxylic acid prepared in step (1). Disperse by sonication and react hydrothermally at 90°C for 48 hours. Centrifuge and wash with N,N-dimethylformamide and anhydrous methanol in sequence. Dry and collect the solid. Add the collected solid to the newly prepared H2O / DMF solution of 2,5-dihydroxybiphenylcarboxylic acid (prepared in the same way as step (1)). Disperse by sonication and react hydrothermally at 90°C for 48 hours. Centrifuge and wash with N,N-dimethylformamide and anhydrous methanol in sequence. Dry and collect the solid to obtain highly crystalline metal-organic framework UiO-67-OH.

[0066] (3) Prepare equal volumes of aqueous solutions of 0.04 mol / L metal-organic framework UiO-67-OH and 0.80 mol / L vinylphosphonic acid, stir the reaction, centrifuge, wash with ultrapure water, and dry to obtain highly stable phosphonic acid surface-modified metal-organic framework UiO-67-OH.

[0067] The metal-organic framework UiO-67 was prepared by the following method:

[0068] Weigh 0.1 mmol zirconium dichloride and 0.1 mmol biphenyl dicarboxylic acid, and sonicate them separately in DMF to prepare equal volumes of zirconium dichloride solution and biphenyl dicarboxylic acid solution. After thorough sonication, mix the two solutions, then add 5.0 mmol acetic acid and continue to sonicate thoroughly. The mixture is then subjected to hydrothermal reaction at 120 °C for 24 hours. After centrifugation, the mixture is washed successively with DMF and anhydrous methanol and dried to obtain metal-organic framework UiO-67.

[0069] Using 1×PBS buffer as solvent, highly stable phosphonic acid surface-modified metal-organic framework UiO-67-OH dispersions with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL, and 20 mg / mL were prepared as erythrocyte cryoprotectants.

[0070] 2×10 9 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0071] Example 3:

[0072] A method for preparing a highly stable phosphonic acid surface-modified metal-organic framework as a cryoprotectant for erythrocytes includes the following steps:

[0073] (1) Weigh 4 mmol of sodium hydroxide and dissolve it in 2 mL of ultrapure water to prepare a potassium hydroxide solution with a mass fraction of 8%. Weigh 0.8 mmol of 2',5'-dihydroxy-[1,1':4',1”-terphenyl]-4,4”-dicarboxylic acid and dissolve it in the solution. Add dilute hydrochloric acid solution to adjust the pH of the solution to 7.4. Add 0.5 mL of DMF to obtain a H2O / DMF solution of 2',5'-dihydroxy-[1,1':4',1”-terphenyl]-4,4”-dicarboxylic acid.

[0074] (2) Weigh 0.1 mol of metal-organic framework UiO-68 and add it to the H2O / DMF solution of 2',5'-dihydroxy-[1,1':4',1”-terphenyl]-4,4”-dicarboxylic acid prepared in step (1). Disperse the mixture by sonication and react it hydrothermally at 90°C for 48 hours. Centrifuge the mixture and wash it with N,N-dimethylformamide and anhydrous methanol in sequence. Dry the mixture and collect the solid. Add the collected solid to the newly prepared H2O / DMF solution of 2',5'-dihydroxy-[1,1':4',1”-terphenyl]-4,4”-dicarboxylic acid (prepared in the same way as step (1)). Disperse the mixture by sonication and react it hydrothermally at 90°C for 48 hours. Centrifuge the mixture and wash it with N,N-dimethylformamide and anhydrous methanol in sequence. Dry the mixture and collect the solid to obtain highly crystalline metal-organic framework UiO-68-OH.

[0075] (3) Prepare equal volumes of aqueous solutions of 0.04 mol / L metal-organic framework UiO-68-OH and 0.80 mol / L 2-hydroxyethyl phosphoric acid, stir the reaction, centrifuge, wash with ultrapure water, and dry to obtain highly stable phosphonic acid surface-modified metal-organic framework UiO-68-OH.

[0076] The metal-organic framework UiO-68 was prepared by the following method:

[0077] Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol 4,4'-terphenyl dicarboxylic acid, and sonicate them separately in DMF to prepare equal volumes of zirconium tetrachloride solution and 4,4'-terphenyl dicarboxylic acid solution. After thorough sonication, mix the two solutions, then add 5.0 mmol acetic acid and continue to sonicate thoroughly. The mixture is then subjected to hydrothermal reaction at 120 °C for 24 hours. After centrifugation, the mixture is washed successively with DMF and anhydrous methanol and dried to obtain metal-organic framework UiO-68.

[0078] Using 1×PBS buffer as solvent, highly stable phosphonic acid surface-modified metal-organic framework UiO-68-OH dispersions with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL, and 20 mg / mL were prepared as erythrocyte cryoprotectants.

[0079] 2×10 9 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0080] Example 4:

[0081] A method for preparing a highly stable phosphonic acid surface-modified metal-organic framework as a cryoprotectant for erythrocytes includes the following steps:

[0082] (1) Weigh 4 mmol of sodium hydroxide and dissolve it in 2 mL of ultrapure water to prepare a potassium hydroxide solution with a mass fraction of 8%. Weigh 0.5 mmol of 2-hydroxyterephthalic acid and dissolve it in the solution. Add dilute hydrochloric acid solution to adjust the pH of the solution to 7.4. Add 0.5 mL of DMF to obtain an H2O / DMF solution of 2-hydroxyterephthalic acid.

[0083] (2) Weigh 0.1 mmol of metal-organic framework UiO-66 and add it to the 2-hydroxyterephthalic acid H2O / DMF solution prepared in step (1). Disperse by sonication, react hydrothermally at 90°C for 48 hours, centrifuge, wash with N,N-dimethylformamide and anhydrous methanol in sequence, dry, collect the solid, and add the collected solid to the freshly prepared 2-hydroxyterephthalic acid H2O / DMF solution (prepared in the same way as step (1)), disperse by sonication, react hydrothermally at 90°C for 48 hours, centrifuge, wash with N,N-dimethylformamide and anhydrous methanol in sequence, dry, and collect the solid to obtain highly crystalline metal-organic framework UiO-66-OH.

[0084] (3) Prepare equal volumes of aqueous solutions of 0.04 mol / L metal-organic framework UiO-66-OH and 0.80 mol / L 2-hydroxyethyl phosphoric acid, stir the reaction, centrifuge, wash with ultrapure water, and dry to obtain highly stable phosphonic acid surface-modified metal-organic framework UiO-66-OH.

[0085] The metal-organic framework UiO-66 was prepared by the following method:

[0086] Weigh 0.1 mmol zirconium dichloride and 0.1 mmol terephthalic acid, and sonicate them separately in DMF to prepare equal volumes of zirconium dichloride solution and terephthalic acid solution. After thorough sonication, mix the two solutions, then add 5.0 mmol acetic acid and continue to sonicate thoroughly. The mixture is then subjected to hydrothermal reaction at 120 °C for 24 hours. After centrifugation, the mixture is washed successively with DMF and anhydrous methanol and dried to obtain metal-organic framework UiO-66.

[0087] Using 1×PBS buffer as solvent, highly stable phosphonic acid surface-modified metal-organic framework UiO-66-OH dispersions with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL, and 20 mg / mL were prepared as erythrocyte cryoprotectants.

[0088] 2×10 9Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0089] Comparative Example 1:

[0090] A method for preparing a low-crystallinity phosphonic acid surface-modified metal-organic framework as a cryoprotectant for erythrocytes includes the following steps:

[0091] (1) Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol 2,5-diaminoterephthalic acid, and disperse them separately in DMF by ultrasonication to prepare equal volumes of zirconium tetrachloride solution and 2,5-diaminoterephthalic acid. After thorough ultrasonication, mix the two solutions, then add 5.0 mmol acetic acid and continue to ultrasonicate thoroughly. The mixture is then subjected to hydrothermal reaction at 120°C for 24 hours. After centrifugation, the mixture is washed with DMF and anhydrous methanol in sequence and dried to obtain metal-organic framework UiO-68-(NH2)2.

[0092] (2) Prepare equal volumes of 0.04 mol / L metal-organic framework material UiO-66-(NH2)2 and 0.80 mol / L methylphosphonic acid aqueous solutions, react under suitable conditions, centrifuge, wash with ultrapure water, and dry to obtain low crystallinity phosphonic acid surface-modified metal-organic framework material UiO-66-(NH2)2, and observe its morphology using transmission electron microscopy.

[0093] (3) Using 1×PBS buffer as solvent, prepare dispersions of low-crystallinity metal-organic framework material UiO-68-(NH2)2 with phosphonic acid surface modification at concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL and 20 mg / mL, as erythrocyte cryoprotectants.

[0094] 1×10 9 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0095] Comparative Example 2:

[0096] A method for preparing a low-crystallinity phosphonic acid surface-modified metal-organic framework as a cryoprotectant for erythrocytes includes the following steps:

[0097] (1) Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol 2,5-dihydroxybiphenylcarboxylic acid, and disperse them separately in DMF by ultrasonication to prepare equal volumes of zirconium tetrachloride solution and 2,5-dihydroxybiphenylcarboxylic acid. After thorough ultrasonication, mix the two solutions, then add 5.0 mmol acetic acid and continue to ultrasonicate thoroughly. The mixture is then subjected to hydrothermal reaction at 120°C for 24 hours. After centrifugation, the mixture is washed with DMF and anhydrous methanol in sequence and dried to obtain the metal-organic framework UiO-67-OH.

[0098] (2) Prepare equal volumes of 0.04 mol / L metal-organic framework material UiO-67-OH and 0.80 mol / L methylphosphonic acid aqueous solutions, react under suitable conditions, centrifuge, wash with washing solution, and dry to obtain low crystallinity phosphonic acid surface-modified metal-organic framework material UiO-67-OH, and observe its morphology with transmission electron microscopy.

[0099] (3) Using 1×PBS buffer as solvent, prepare dispersions of low-crystallinity metal-organic framework material UiO-67-OH with phosphonic acid surface modification at concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL and 20 mg / mL, as erythrocyte cryoprotectants.

[0100] 1×10 9 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0101] Comparative Example 3:

[0102] A method for preparing a low-crystallinity phosphonic acid surface-modified metal-organic framework material as a cryoprotectant for erythrocytes includes the following steps:

[0103] (1) Weigh 0.1 mmol of zirconium oxychloride and 0.1 mmol of 2',5'-dihydroxy-[1,1':4',1”-terphenyl]-4,4”-dicarboxylic acid, and ultrasonically disperse them in DMF to prepare equal volumes of zirconium oxychloride solution and 4,4'-terphenyl dicarboxylic acid solution. After thorough ultrasonication, mix the two solutions, then add 5.0 mmol of trifluoroacetic acid and continue to ultrasonically mix thoroughly. The mixture is then subjected to hydrothermal reaction at 120℃ for 24 hours. After centrifugation, the mixture is washed with DMF and anhydrous methanol in sequence and dried to obtain metal-organic framework material UiO-68-OH.

[0104] (2) Prepare equal volumes of aqueous solutions of 0.04 mol / L metal-organic framework material UiO-68-OH and 0.80 mol / L ethyl phosphoric acid, react under suitable conditions, centrifuge, wash with ultrapure water, and dry to obtain low crystallinity phosphonic acid surface-modified metal-organic framework material UiO-68-OH.

[0105] (3) Using 1×PBS buffer as solvent, prepare dispersions of low-crystallinity metal-organic framework material UiO-68-OH with phosphonic acid surface modification at concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL and 20 mg / mL, as erythrocyte cryoprotectants.

[0106] 5×10 8 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0107] Comparative Example 4:

[0108] A method for preparing low-crystallinity metal-organic framework materials as erythrocyte cryoprotectants via a simple hydrothermal reaction includes the following steps:

[0109] (1) Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol 2,5-diaminoterephthalic acid, and disperse them separately in DMF by ultrasonication to prepare equal volumes of zirconium tetrachloride solution and 2,5-diaminoterephthalic acid. After thorough ultrasonication, mix the two solutions, then add 5.0 mmol acetic acid and continue to ultrasonicate thoroughly. The mixture is then subjected to hydrothermal reaction at 120°C for 24 hours. After centrifugation, the mixture is washed with DMF and anhydrous methanol in sequence and dried to obtain metal-organic framework material UiO-68-(NH2)2.

[0110] (2) Using 1×PBS buffer as solvent, prepare metal-organic framework material UiO-68-(NH2)2 dispersions with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL and 20 mg / mL for use as erythrocyte cryoprotectants.

[0111] 2×10 9 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, and 20 mg / mL red blood cell cryoprotectants at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0112] Comparative Example 5: A method for preparing metal-organic framework materials lacking hydrogen bond matching sites as erythrocyte cryoprotectants, comprising the following steps:

[0113] (1) Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol terephthalic acid, and disperse them separately in DMF by ultrasonication to prepare equal volumes of zirconium tetrachloride solution and terephthalic acid solution. After thorough ultrasonication, mix the two solutions, then add 5.0 mmol acetic acid, continue ultrasonication to mix thoroughly, and react hydrothermally at 100°C for 24 hours. After centrifugation, wash with DMF and anhydrous methanol in sequence, and dry to obtain metal-organic framework UiO-66.

[0114] (2) Prepare equal volumes of 0.04 mol / L metal-organic framework UiO-66 and 0.80 mol / L 3-phosphonyl-L-alanine aqueous solutions, stir the reaction, centrifuge, wash with ultrapure water, and dry to obtain phosphonic acid surface-modified metal-organic framework UiO-66.

[0115] (3) Using 1×PBS buffer as solvent, prepare dispersions of phosphonic acid surface-modified metal-organic framework material UiO-66 with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL and 20 mg / mL as erythrocyte cryoprotectants.

[0116] 5×10 7 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0117] Comparative Example 6: A method for preparing metal-organic framework materials lacking hydrogen bond matching sites as erythrocyte cryoprotectants, comprising the following steps:

[0118] (1) Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol biphenyl dicarboxylic acid, and disperse them separately in DMF by ultrasonication to prepare equal volumes of zirconium tetrachloride solution and biphenyl dicarboxylic acid solution. After thorough ultrasonication, mix the two solutions, then add 5.0 mmol acetic acid, continue ultrasonication to mix thoroughly, and react hydrothermally at 100℃ for 24 hours. After centrifugation, wash with DMF and anhydrous methanol in sequence, and dry to obtain metal-organic framework UiO-67.

[0119] (2) Prepare equal volumes of aqueous solutions of 0.04 mol / L metal-organic framework UiO-67 and 0.80 mol / L methylphosphonic acid, stir the reaction, centrifuge, wash with ultrapure water, and dry to obtain phosphonic acid surface-modified metal-organic framework UiO-67.

[0120] (3) Using 1×PBS buffer as solvent, prepare dispersions of phosphonic acid surface-modified metal-organic framework material UiO-67 with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL and 20 mg / mL as erythrocyte cryoprotectants.

[0121] 5×10 8 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0122] Comparative Example 7: A method for preparing metal-organic framework materials lacking hydrogen bond matching sites as erythrocyte cryoprotectants, comprising the following steps:

[0123] (1) Weigh 0.1 mmol zirconium tetrachloride and 0.1 mmol triphenyl dicarboxylic acid, and disperse them separately in DMF by ultrasonication to prepare equal volumes of zirconium tetrachloride solution and triphenyl dicarboxylic acid solution. After thorough ultrasonication, mix the two solutions, then add 5.0 mmol acetic acid, continue ultrasonication to mix thoroughly, and react hydrothermally at 100°C for 24 hours. After centrifugation, wash with DMF and anhydrous methanol in sequence, and dry to obtain metal-organic framework UiO-68.

[0124] (2) Prepare equal volumes of aqueous solutions of 0.04 mol / L metal-organic framework UiO-68 and 0.80 mol / L ethylphosphonic acid, stir the reaction, centrifuge, wash with ultrapure water, and dry to obtain phosphonic acid surface-modified metal-organic framework UiO-68.

[0125] (3) Using 1×PBS buffer as solvent, prepare dispersions of phosphonic acid surface-modified metal-organic framework material UiO-68 with concentrations of 2 mg / mL, 4 mg / mL, 10 mg / mL and 20 mg / mL, as erythrocyte cryoprotectants.

[0126] 1×10 9 Red blood cells per mL were mixed with 2 mg / mL, 4 mg / mL, 10 mg / mL, or 20 mg / mL red blood cell cryoprotectant at a 1:1 volume ratio, incubated at 4°C for 10 minutes, frozen in liquid nitrogen for 24 hours, and then rapidly thawed in a 37°C water bath to obtain thawed red blood cells.

[0127] Performance testing

[0128] Transmission electron microscopy observation of the morphology of the cryoprotectant for red blood cells in Example 1 (results are shown in Figure 1). Figure 1 As shown), the morphology of the cryoprotectant for red blood cells in Comparative Example 1 (results are shown in Figure 1). Figure 2(as shown);

[0129] from Figure 1 , Figure 2 It can be seen that: the phosphonic acid surface-modified metal-organic framework material in Example 1 has high crystallinity and can maintain a good morphology, while the phosphonic acid surface-modified metal-organic framework material in Comparative Example 2 is fragmented and has an irregular morphology. The framework of the metal-organic framework material has been destroyed. This shows that the method of improving the antifreeze performance of metal-organic framework materials of the present invention can avoid the structural damage caused by phosphonic acid to metal-organic framework materials and is conducive to maintaining the effective antifreeze ability of metal-organic framework materials.

[0130] The recovery rate of red blood cells in the mixture of cryoprotectant and red blood cells after thawing in Examples 1-4 and Comparative Examples 1-7 was determined.

[0131] The mixtures of thawed red blood cell cryoprotectant and red blood cells from Examples 1-4 and Comparative Examples 1-7 were centrifuged at 2300 rpm for 8 minutes, and the absorbance A of the supernatant was measured using an ELISA reader. 540 .

[0132] The recovery rate R of red blood cells in the mixture of thawed red blood cell cryoprotectant and red blood cells in Examples 1-4 and Comparative Examples 1-7 was calculated according to formula (1). 冻存 (%)

[0133]

[0134] The negative control was a red blood cell sample that was not treated and was thoroughly mixed with 1×PBS buffer at a volume ratio of 1:1 and stored at 4°C.

[0135] The positive control was a red blood cell sample that was not treated and was thoroughly mixed with 1×PBS buffer at a volume ratio of 1:1, sonicated thoroughly, and then stored in liquid nitrogen. After thawing, the sample was completely hemolyzed.

[0136] The test samples were: the mixture of thawed erythrocyte cryoprotectant and erythrocytes in Examples 1-4, and the erythrocyte samples in the mixture of thawed erythrocyte cryoprotectant and erythrocytes in Comparative Examples 1-7.

[0137] The recovery rates of erythrocytes from the thawed mixtures of cryoprotectant and erythrocytes in Examples 1-4 and Comparative Examples 1-7 are shown in Table 1 below. Figure 3 As shown.

[0138] Table 1: Recovery rate of thawed red blood cells in Examples 1-4 and Comparative Examples 1-7

[0139]

[0140] As can be seen from Table 1, the highly stable phosphonic acid surface-modified metal-organic framework materials in Examples 1 to 4 all have high erythrocyte cryoprotectant effects.

[0141] Example 1: Highly stable phosphonic acid surface-modified metal-organic framework material as a cryoprotectant for erythrocytes. The recovery rate of erythrocytes in the mixture of cryoprotectant and erythrocytes after thawing is higher than that in Comparative Example 1: Low crystallinity phosphonic acid surface-modified metal-organic framework material as a cryoprotectant for erythrocytes. The recovery rate of erythrocytes in the mixture of cryoprotectant and erythrocytes after thawing is higher than that in Comparative Example 4: Metal-organic framework material prepared by simple hydrothermal reaction as a cryoprotectant for erythrocytes. The recovery rate of erythrocytes in the mixture of cryoprotectant and erythrocytes after thawing is higher than that in Comparative Example 4.

[0142] Example 2: The recovery rate of red blood cells in the mixture of red blood cell cryoprotectant and red blood cells after thawing of highly stable phosphonic acid surface-modified metal-organic framework material as a red blood cell cryoprotectant was higher than that in Comparative Example 2: The recovery rate of red blood cells in the mixture of cryoprotectant and red blood cells after thawing of low crystallinity phosphonic acid surface-modified metal-organic framework material as a red blood cell cryoprotectant was higher than that in Comparative Example 2.

[0143] Example 3: The recovery rate of red blood cells in the mixture of red blood cell cryoprotectant and red blood cells after thawing of the highly stable phosphonic acid surface-modified metal-organic framework material as a red blood cell cryoprotectant was higher than that in Comparative Example 3: The recovery rate of red blood cells in the mixture of cryoprotectant and red blood cells after thawing of the low crystallinity ....

[0144] Comparative Examples 5-7 show that metal-organic framework materials lacking hydrogen bond matching sites in their ligands have low cryoprotective effects on erythrocytes. This indicates that the method of improving the antifreeze properties of metal-organic framework materials in this invention can significantly improve the recovery rate of erythrocytes after cryopreservation, and can achieve a high cryoprotective recovery rate of up to 98% for cryopreserved erythrocytes, which is comparable to the cryoprotective effect of commercially available glycerol-containing erythrocytes.

[0145] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of phosphonic acid surface-modified metal-organic frameworks in cryopreserved erythrocytes, characterized in that, The phosphonic acid surface-modified metal-organic framework is prepared by surface modification of a highly crystalline metal-organic framework with phosphonic acid. The method for preparing the highly crystalline metal-organic framework includes the following steps: (1) Take organic ligand 2, sonicate it in an alkaline solution, add dilute acid solution to adjust the pH of the solution to 6-8, and add organic solvent to obtain a mixed solution of organic ligand 2; (2) Take metal-organic framework A and add it to the prepared mixed solution of organic ligand 2, disperse it by ultrasonication, react it, centrifuge it, wash it, dry it, and collect the solid. (3) Add the solid collected in (2) to the prepared mixed solution of organic ligand 2, disperse by ultrasonication, react, centrifuge, wash, and dry to obtain a highly crystalline metal-organic framework. The organic ligand 2 mentioned in step (1) is an organic ligand with hydrogen-rich matching sites; The metal-organic framework A mentioned in step (2) includes at least one of UiO-66, UiO-67 and UiO-68.

2. The application according to claim 1, characterized in that, The phosphonic acids mentioned include N-(phosphonomethyl)glycine, 3-phosphono-L-alanine, dialamidophosphonic acid, phosphonopyruvate, tripropylphosphonic acid, hydroxyethylidene diphosphonic acid, 2-hydroxyphosphonoacetic acid, methylphosphonic acid, phenylphosphonic acid, phosphonoacetic acid, phosphonoacetaldehyde, ethylphosphonic acid, vinylphosphonic acid, 2-aminoethylphosphonic acid, (2-chloroacetyl)phosphonic acid, 2-amino-4-[hydroxy(methyl)phosphono]butanoic acid ammonium, 2-hydroxyethylphosphonic acid, etc. At least one of the following: phosphonic acid, (2-amino-1-hydroxyethyl)phosphonic acid, diethylenetriaminepentamylidenephosphonic acid, ethylenediaminetetramylidenephosphonic acid, aminotrimylidenephosphonic acid, zoledronic acid, phosphonocarboxylic acid, (1R,2S)-1,2-epoxypropylphosphonic acid, 2-hydroxyethyl-[(E)-14-iodotetradecane-13-en-3,5-diacetyloxy]phosphonic acid, and L-arginino-L-2-amino-5-phosphoryl-3Z-pentenoic acid.

3. The application according to claim 1, characterized in that, The organic ligand 2 mentioned in step (1) includes at least one of 2-hydroxyterephthalic acid, 2,3-dihydroxyterephthalic acid, 2,5-dihydroxyterephthalic acid, 2,6-dihydroxyterephthalic acid, 2,3-diaminoterephthalic acid, 2,5-diaminoterephthalic acid, 4,4'-dihydroxy-3,3'-biphenyldicarboxylic acid, 2,5-dihydroxybiphenyldicarboxylic acid, 2',5'-dihydroxy-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid, 2-amino-4,4'-biphenyldicarboxylic acid, and 4,4'-diaminobiphenyl-2,2'-dicarboxylic acid.

4. The application according to claim 1, characterized in that, The metal-organic framework A mentioned in step (2) includes at least one of UiO-66-Zr, UiO-67-Zr, UiO-68-Zr, UiO-66-Hf, UiO-67-Hf and UiO-68-Hf; The organic ligand 1 of the metal-organic framework A mentioned in step (2) includes at least one of terephthalic acid, biphenyl dicarboxylic acid, and terphenyl dicarboxylic acid.

5. The application according to claim 4, characterized in that, The preparation of the metal-organic framework A described in step (2) includes the following steps: S1. Weigh the metal compound and organic ligand 1, and sonicate them separately in a solvent to prepare metal compound solution and organic ligand 1 solution respectively. After thorough sonication, mix the metal compound solution and organic ligand 1 solution, then add a crystal regulator, continue sonication to mix thoroughly, perform hydrothermal reaction, centrifuge, wash with washing solution, and dry to obtain metal-organic framework A.

6. The application according to claim 5, characterized in that, The metal compound mentioned in step S1 includes at least one of zirconium propoxide, zirconium tetrachloride, zirconium dichloride, hafnium propoxide, hafnium tetrachloride, and hafnium dichloride; The organic ligand 1 mentioned in step S1 includes at least one of terephthalic acid, biphenyl dicarboxylic acid, and triphenyl dicarboxylic acid; The solvent mentioned in step S1 includes at least one of water, N,N-dimethylformamide, and N,N-diethylformamide; The crystal regulator mentioned in step S1 includes at least one of formic acid, acetic acid, benzoic acid, hydrochloric acid, and trifluoroacetic acid; The molar ratio of the metal compound, organic ligand 1, and crystal regulator mentioned in step S1 is 0.5–2:0.5–2:20–100; The hydrothermal reaction conditions described in step S1 are 80–120°C for 12–36 hours.

7. The application according to claim 1, characterized in that, The molar ratio of the metal-organic framework A and the organic ligand 2 in step (2) is 1:1 to 10; The reaction described in step (2) is carried out at 80-120°C for 12-72 hours.

8. The application according to any one of claims 1 to 7, characterized in that, The method for preparing the phosphonic acid surface-modified metal-organic framework includes the following steps: A highly crystalline metal-organic framework solution and a phosphonic acid solution were stirred and reacted, then centrifuged, washed, and dried to obtain a phosphonic acid-modified metal-organic framework. The molar ratio of the highly crystalline metal-organic framework to phosphonic acid is 1:1 to 100.

9. The application according to any one of claims 1 to 7, characterized in that, The concentration of the phosphonic acid surface-modified metal-organic framework used for cryopreservation of red blood cells is 1–10 mg / mL.

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