阅读说明：本技术 一种两亲性化合物用于制备血液红细胞冷冻保护剂的应用 (Application of amphiphilic compound in preparation of blood erythrocyte cryoprotectant ) 是由 霍炳臣 金晟琳 郭存悦 王健君 于 2020-04-09 设计创作，主要内容包括：本发明提供了一种两亲性化合物用于制备血液红细胞冷冻保护剂的应用。本发明的两亲性化合物由葡萄糖酸内酯和丝氨酸反应制备,所述化合物具有两亲性,可有效抑制水溶液中的冰晶生长和重结晶,用于血液红细胞的冷冻保存具有优异的细胞恢复率,无需加入有机类抗冻成分,成分简单,可保持良好的生物相容性,具有广泛的应用前景。(The invention provides an application of an amphiphilic compound in preparation of a blood erythrocyte cryoprotectant. The amphiphilic compound is prepared by reacting gluconolactone and serine, has amphipathy, can effectively inhibit the growth and recrystallization of ice crystals in aqueous solution, has excellent cell recovery rate when being used for freezing and storing blood erythrocytes, does not need to add organic anti-freezing components, has simple components, can keep good biocompatibility, and has wide application prospect.)

1. Use of a compound that inhibits ice crystal growth in an aqueous solution as an antifreeze agent, the compound being of formula (I):

2. the use of claim 1, wherein the compound of formula (I) is prepared by reacting gluconolactone with serine;

preferably, the gluconolactone is reacted with serine in an organic solvent, for example methanol;

preferably, the reaction temperature of the gluconolactone and the serine is 30-70 ℃, the reaction time is 24-48 h, and the molar ratio of the feed rate to the gluconolactone to the serine is 1: (1-5).

3. The application of the compound shown in the formula (I) in preparing a reagent for cryopreservation of red blood cells,

4. an erythrocyte cryopreservation reagent, which comprises a compound shown as a formula (I);

preferably, the erythrocyte cryopreservation reagent comprises a compound shown as a formula (I) and a buffer solution;

5. the erythrocyte cryopreservation reagent of claim 4, which is free of organic compounds.

6. The cryopreservation reagent for red blood cells according to claim 5, consisting of the compound represented by formula (I) and a buffer.

7. The erythrocyte cryopreservation reagent of any one of claims 4 to 6, wherein the buffer is selected from any one of PBS buffer, DPBS buffer, hepes-buffered HTF buffer or other cell culture buffer;

preferably, the PBS buffer has a pH of 7.0-7.4.

8. A method of cryopreservation of red blood cells comprising: preparing a solution containing the compound shown in the formula (I), mixing the suspension of the red blood cells with the prepared solution to prepare a frozen red blood cell solution, freezing and storing by liquid nitrogen,

9. the method for cryopreservation of red blood cells according to claim 8, wherein the molar concentration of the compound in the frozen red blood cell solution is 18-520mM, preferably 100-400mM, such as 360 mM.

10. The method for cryopreservation of red blood cells according to claim 8 or 9, wherein the solution containing the compound represented by formula (I) is a solution obtained by dissolving the compound represented by formula (I) in a buffer,

preferably, the buffer is selected from any one of PBS buffer, DPBS buffer, hepes-buffered HTF buffer or other cell culture buffer;

preferably, the PBS buffer has a pH of 7.0-7.4.

Technical Field

The invention belongs to the field of cell cryopreservation, and particularly relates to an application of an amphiphilic compound in preparation of a blood erythrocyte cryoprotectant.

Background

Cryopreservation is a technique of extremely reducing or even stopping the metabolism of cells, tissues or organs at an ultralow temperature to prolong the physiological activity of tissue cells. Such as: whole blood can be preserved for about 30 days at 2-6 deg.C, hematopoietic stem cells can be preserved for one year in-80 deg.C ultra-low temperature refrigerator, and can be preserved for a long time in-196 deg.C liquid nitrogen. The shape and genetic stability of the preserved object are ensured by freezing preservation, and the method has very important scientific and market values. Therefore, cryopreservation has been widely used in the fields of biomedical research, food science, agricultural breeding, and the like. However, in this process, the crystallization of ice and its recrystallization are one of the major causes of cell damage and death. In order to avoid the risks associated with ice crystallization, high concentrations of organic solvents such as dimethyl sulfoxide (DMSO), Ethylene Glycol (EG), and glycerol are often used extensively to achieve vitrification and ice-free freezing of cryopreservation media during cryopreservation.

The red blood cells can be stored at 2-6 deg.C for 30 days, or at-196 deg.C for a long period. Blood transfusion is a life-saving means for trauma patients with leukemia, hemolytic anemia and severe blood loss. In cryopreservation of blood cells, recrystallization of ice is a major cause of cell damage. Traditional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol, while providing better survival rates after cryopreservation recovery, use of residual organic solvents poses a risk of hemolysis in the infused patient. Glycerol is widely used for cryopreservation of red blood cells at present, but the residual amount of glycerol in blood vessels during blood transfusion can reach 1% by using glycerol as a cryoprotectant, and the residual amount is enough to cause serious side effects on patients receiving blood transfusion. Therefore, prior to clinical use, glycerol must be removed from the red blood cells. However, the removal of glycerol from red blood cells is a time-consuming process and requires special equipment, and therefore the development of new cryoprotectants for red blood cells has a tremendous incentive for the application of red blood cells.

Disclosure of Invention

The invention aims to provide an application of a small molecular compound for cryopreservation of red blood cells, and an organic solvent is not required. The micromolecule compound has good water solubility and amphipathy (ice-philic property and hydrophilcity), and can effectively inhibit the recrystallization of ice in the rewarming process, thereby avoiding the damage of cells caused by the over-fast growth of the ice crystals. The invention further provides a cryopreservation reagent for cryopreservation of blood cells.

The invention provides an application of an amphiphilic compound as an antifreeze, wherein the antifreeze inhibits the growth of ice crystals in an aqueous solution, and the compound is shown as a formula (I):

according to the invention, the amphiphilic compound shown in the formula (I) can be prepared by reacting glucolactone with serine. In one embodiment, the gluconolactone is prepared by reaction with serine in an organic solvent, for example methanol. In a specific embodiment, the reaction temperature of the gluconolactone and the serine is 30-70 ℃, the reaction time is 24-48 h, the molar ratio of the feed rate to the feed rate is 1: (1-5).

In another embodiment, the amphiphilic compound of formula (I) is a gluconolactone and serine synthesized by solid phase synthesis comprising: swelling resin, linking an amino acid protected by amino group to the swelled resin, deprotection, adding saccharide compound (such as glucolactone), condensation reaction, cutting, purifying and the like.

The invention further provides an application of the amphiphilic compound shown in the formula (I) in preparing a reagent for cryopreservation of red blood cells.

According to the invention, the erythrocyte cryopreservation reagent comprises an amphiphilic compound shown as a formula (I). In one embodiment, the erythrocyte cryopreservation reagent comprises an amphiphilic compound represented by formula (I) and a buffer.

The invention provides a reagent for cryopreservation of red blood cells, which comprises an amphiphilic compound shown as a formula (I). In one embodiment, the erythrocyte cryopreservation reagent comprises an amphiphilic compound represented by formula (I) and a buffer.

According to a preferred embodiment of the present invention, the cryopreservation reagent does not contain an organic solvent, including but not limited to Dimethylformamide (DMF), ethylene glycol, propylene glycol, glycerol, and the like.

According to a preferred embodiment of the present invention, the cryopreservation reagent is composed of an amphiphilic compound represented by formula (I) and a buffer.

The invention also provides a cryopreservation method of the red blood cells, which comprises the following steps: preparing a solution containing the amphiphilic compound shown in the formula (I), mixing the suspension of the red blood cells with the prepared solution to prepare a frozen red blood cell solution, and freezing and storing the frozen red blood cell solution by liquid nitrogen.

According to the invention, the molar concentration of the amphipathic compound in the blood cell cryopreservation solution is 18-520mM, preferably 100-400mM, such as 360 mM.

According to the invention, the solution comprising the amphiphilic compound is a solution of the compound dissolved in a buffer.

In the present invention, the buffer may be selected from any one of cell culture buffers known in the art, such as PBS buffer, DPBS buffer, hepes-buffered HTF buffer, or other cell culture buffers, preferably PBS buffer. In one embodiment, the PBS buffer has a pH of 7.0 to 7.4.

In the present invention, the erythrocytes are derived from animal blood, e.g., blood of a warm-blooded mammal, including but not limited to humans, non-human primates, livestock (e.g., cows, sheep, pigs), other mammals (e.g., dogs, cats). The red blood cells can be isolated from blood by methods known in the art for clinical use.

In the present invention, "amphiphilic" means having hydrophilicity and ice-affinity. The hydrophilicity is the non-covalent interaction with water molecules, such as hydrogen bond with water, Van der Waals interaction, electrostatic interaction, hydrophobic interaction or pi-pi interaction; by ice-philic is meant a material that is non-covalent with ice, e.g., capable of forming hydrogen bonds with ice, van der waals interactions, electrostatic interactions, hydrophobic interactions, or pi-pi interactions, etc.

Has the advantages that:

the compound has a polyhydroxy structure, good hydrophilicity and higher IRI activity (figure 4), and can effectively inhibit the recrystallization of ice crystals in the rewarming process in the cryopreservation process, thereby having good effect on cryopreservation of red blood cells. The blood cells can reach 40 to 60 percent of cell recovery rate after being frozen and preserved in the freezing and preserving reagent of the invention without adding organic solvent. The erythrocyte preservation material provided by the invention is simple to prepare, rich in raw materials, good in cell compatibility and good in cryopreservation effect.

Drawings

FIG. 1 shows nuclear magnetic hydrogen spectra (upper) and carbon spectra (lower) of the compounds G to S prepared in example 1 (solvent: heavy water);

FIG. 2 is an optical image of the pure PBS buffer and G-S PBS buffer of example 1 to inhibit recrystallization;

FIG. 3 is the average largest ice crystal size ratio of PBS (%) in PBS buffer solutions of different concentrations of G-S in example 1;

FIG. 4 shows the recovery rate of cell cryopreservation of pure PBS buffer and G-S PBS buffer described in example 3.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The materials are commercially available from the open literature unless otherwise specified.

EXAMPLE 1 preparation of the Compound of formula (I)

Dissolving 1mmol of gluconolactone in 30mL of methanol, fully stirring and dissolving, then adding 10mmol of serine, stirring and reacting for 48h at 60 ℃, then cooling the reaction solution for 5h in ice water bath, filtering, and carrying out rotary evaporation on the filtrate to obtain the product.

The nuclear magnetic hydrogen spectrum and carbon spectrum (AVANCE II 400M NMR) of the obtained product are shown in figure 1, and the prepared product can be determined to have the structure shown in formula (I) according to figure 1 and is marked as a compound G-S.

The resulting product was subjected to IRI activity analysis to evaluate its ice inhibition performance. IRI activity assays were performed by the Splat-Cooling method. The experimental setup used to study IRI activity was a nikon polarized optical microscope (AZ100) and a Linkman (LTS420) cold stage. All samples were dissolved in PBS solution at the desired concentration. 10 μ L of the sample solution was dropped from 1.5 m onto a cold stage previously cooled to-60 ℃. The droplets instantaneously coalesce into a thin layer of ice. The cold plate was then raised to-6 ℃ at a ramp rate of 10 ℃/min. The frozen sample was then annealed at this temperature for 30 minutes. The ice crystals were then photographed by a camera on a microscope and the images were processed using Image J software, the results are shown in figure 2. The largest 25 ice crystals were selected from each photograph and the length of the largest axis was counted. This procedure was repeated three times, the average was calculated and the average value was compared to the average value of the maximum ice crystal size in the PBS buffer solution, i.e., the average maximum ice crystal size ratio PBS (%). The average maximum ice crystal size ratio PBS (%) in the solutions of the compounds G-S at different concentrations is shown in FIG. 3. As can be seen from fig. 3, the average maximum ice crystal size fraction PBS (%) of the G-S solution was around 20-55%, indicating that compound G-S significantly reduced the ice crystal growth size in PBS buffer, significantly inhibiting ice crystal growth in solution. When the concentration of the G-S compound in the solution is 360mM, the G-S compound is minimum, and the average maximum ice crystal size ratio PBS (%) is only about 20-25%, so that the excellent ice inhibition effect is obtained.

EXAMPLE 2 preparation of cryopreservation reagents

The product obtained in example 1 was dissolved in water, sufficiently stirred to dissolve, and then freeze-dried. The dried product was dissolved in PBS buffer (NaCl (136.9mmol L)^-1),KCl(2.7mmol L^-1),Na₂HPO₄(10.0mmol L^-1)，KH₂PO₄(2.0mmol L^-1) pH7.4) for use.

EXAMPLE 3 sheep Red blood cell cryopreservation experiment

The cells used in this experiment were sheep blood cells. Selecting 5ml of blood cells, rotating at 4000r/min, centrifuging for 5min, and centrifuging and washing for a plurality of times until the suspension is clear. The obtained blood cells were stored in a refrigerator at 4 ℃ for further use. 50. mu.L of the prepared blood cells were collected, and the same volume of G-S solution in which the molar concentration of compound G-S was 720mM was added. The final cryopreservation solution containing cells was obtained with a concentration of compound G-S of 360 mM. Then, the frozen cells are frozen and preserved in liquid nitrogen for 2 hours, the frozen cells are rewarming at the water bath temperature of 45 ℃, and then the cell recovery rate is tested, and in fig. 4, the cell recovery rate of the frozen cells preserved by pure PBS buffer solution is basically 0%, while the cell recovery rate of the frozen blood cells of the frozen preservation reagent prepared in the embodiment 2 can reach more than 50% and even 60%, and an organic solvent is not required to be added, so that a good freezing preservation effect is obtained.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.