Polypeptide-based cryoprotectant as well as preparation method and application thereof

Abstract

The invention belongs to the field of cryoprotectants, and particularly relates to a polypeptide-based cryoprotectant as well as a preparation method and application thereof. The polypeptide-based cryopreservation protective agent comprises an anti-freezing agent, a polypeptide ice resistance agent and magnetic nanoparticles, the anti-freezing agent comprises betaine, trehalose and equilibrium liquid. In order to solve the problems that an existing vitrification cryopreservation protective agent is high in concentration (larger than 6 M) and large in toxicity, a series of polypeptides with good solubility are designed and synthesized, and a polypeptide-based low-concentration vitrification cryopreservation protective agent formula is developed and used for cryopreservation of blood vessels.

Description

Technical Field

[0001] The present invention belongs to the field of cryopreservation agents, and in particular relates to a polypeptide-based cryopreservation protective agent and a preparation method and application thereof. Background Art

[0002] Coronary artery disease has become a major cause of morbidity and mortality in developed countries. According to statistics, the global mortality rate from cardiovascular disease is expected to increase from 17.5 million in 2012 to 22.2 million in 2030. The current gold standard for treating cardiovascular disease is autologous vascular transplantation, but due to the scarcity of autologous vascular donors and the potential damage to donors during surgery, allogeneic vascular transplantation has become an indispensable option. However, the preservation of allogeneic blood vessels faces significant challenges. Donor blood vessels usually need to be stored at 4°C, but even at this low temperature, the metabolic activity of cells still remains at about 10% of the normal level. This residual metabolic activity will lead to the production of a large amount of metabolic waste, which will cause extensive initial damage to the vascular endothelium, platelets, and leukocyte adhesion. This damage can cause endothelial denudation and dysfunction, thereby affecting the long-term function of blood vessels. Therefore, the development and research of effective long-term preservation techniques are crucial to improving the clinical application of allogeneic vascular transplantation.

[0003] Cryopreservation technology achieves long-term preservation of tissues and avoids deterioration by slowing down or even suspending physiological metabolism. Traditional cryopreservation methods usually use slow cooling (cooling rate is about 1℃ / min) to deep low temperature to achieve tissue preservation. At the same time, low concentrations of protective agents are added to inhibit the excessive growth of ice crystals, thereby protecting cells and tissue structures. However, this method inevitably leads to ice crystal damage, which causes irreversible damage to cells and tissues.

[0004] In contrast, vitrification technology avoids the formation of ice crystals by forming amorphous ice, thus avoiding ice crystal damage. Currently, vitrification is considered the most promising method for successful cryopreservation of organs. However, it usually takes up to 10 7 The cooling rate is ℃ / min, which is unrealistic for the cryopreservation of tissues and organs.

[0005] Therefore, it is usually necessary to add high concentrations of cryoprotectants (CPAs) to reduce the cooling rate required to achieve vitrification. For example, high concentrations of CPAs (such as 6.4M dimethyl sulfoxide (DMSO), 6.5M glycerol (Gly), 6.0M DP6, and 8.4M VS55) can effectively inhibit ice crystal formation, but also bring new challenges. Traditional cryoprotectants such as DMSO and glycerol may cause greater toxicity and difficulty in elution, and the use of high concentrations of CPAs will aggravate these problems. Therefore, the development of low-concentration vitrification cryoprotectants with good biosafety has become a key direction of current research. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a polypeptide-based cryoprotectant and a preparation method and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A polypeptide-based cryoprotectant (vitrification cryoprotectant) comprises an antifreeze agent, a polypeptide ice inhibitor and magnetic nanoparticles; the antifreeze agent comprises betaine, trehalose and a balancing solution.

[0009] The mass concentration of betaine in the polypeptide-based cryoprotectant is ≥40%; the mass concentration of trehalose in the polypeptide-based cryoprotectant is ≥5%; the total molar concentration of betaine, trehalose and polypeptide ice inhibitor is ≥3.6 mol / L; preferably 3.6 mol / L.

[0010] The polypeptide ice inhibitor is polyglutamic acid (PG lu), which accounts for 0.5% by weight in the cryoprotectant; the degree of polymerization of the polyglutamic acid (PG lu) is 5-15, preferably 5.

[0011] The balancing solution is one of PBS buffer, DPBS buffer, physiological saline or Euro-Colli ns preservation solution.

[0012] The magnetic nanoparticles include magnetic spheres and biocompatible materials wrapped on the surface of the magnetic spheres; preferably, the size is 10nm to 1000nm, the concentration is 5-40mg / mL, and preferably, the concentration is 20mg / mL;

[0013] Preferably, the magnetic balls are Fe3O4 balls or FeO balls; and the biocompatible materials are gum arabic or polysaccharides.

[0014] The magnetic nanoparticles are prepared by: 1) using a co-precipitation method to prepare magnetic spheres; 2) adding the magnetic spheres into an aqueous solution of a biocompatible material.

[0015] The present invention also includes a method for preparing the polypeptide-based cryoprotectant, comprising the following steps: adding betaine, trehalose and polypeptide ice inhibitor into a balance solution to prepare a vitrification cryoprotectant of a certain concentration, and adding magnetic nanoparticles therein to obtain the vitrified cryoprotectant.

[0016] The present invention also includes an application of the polypeptide-based cryoprotectant to vitrification of tissues or organs. Preferably, the organ is a blood vessel.

[0017] The method includes the following steps: 1) loading of vitrification solution: placing the blood vessel in a polypeptide-based cryoprotectant for equilibrium; 2) setting of vitrification program: cooling the cryotube containing the blood vessel using a programmable temperature controller after loading; 3) magnetic thermal rewarming: after the cryopreservation is completed, the cryotube containing the blood vessel is quickly placed in a magnetic induction coil for magnetic thermal rewarming, and taken out after the solution temperature rises to 0°C. 4) elution program: diluting the vitrification cryoprotectant with a gradient PBS solution, and using a magnet to absorb the nanoparticles.

[0018] In step 1), gradient concentration balance is adopted; preferably, the mixture is placed in 1.2M, 2.4M, and 3.6M polypeptide-based cryoprotectants for balance; preferably, the molar concentration of the gradient dilution in step 4) is 2.4M, 1.2M, and 0.6M;

[0019] The cooling program of step 2) is: after equilibration at 4°C for 10 minutes, cool to -120°C at a rate of 30°C / min, anneal for 20 minutes, cool to -150°C, and finally place in a liquid nitrogen tank for cryopreservation.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) In order to solve the problem of high toxicity of vitrification cryoprotectants at high concentrations, the present invention develops a cryoprotectant formula of natural zwitterionic betaine, trehalose, and polypeptide ice inhibitors, and uses magnetic nanoparticles with good biocompatibility to increase the rewarming rate, and finally prepares a vitrification cryoprotectant formula with a concentration as low as 3.6M.

[0022] Specifically, betaine is a small molecule with good biocompatibility and permeability. It has a strong ability to bind to water molecules and can effectively inhibit the formation of ice nuclei. Trehalose is a polyhydroxy sugar that forms hydration by forming hydrogen bonds with water molecules, which is conducive to vitrification. Polyglutamic acid polypeptide is more dispersed and evenly distributed due to its surface hydrophilicity and hydrophobicity, and has good solubility in betaine-based cryopreservation protection. It disrupts the distribution of water molecules in the vitrification solution by forming a large number of hydrogen bonds with water molecules, thereby reducing the concentration of the vitrification protective agent. Magnetic nanoparticles have high magnetothermal conversion characteristics and can achieve uniform and rapid heating under magnetic field conditions.

[0023] 2) The present invention measures the vitrification ability of the vitrification cryoprotectant, and designs and comprehensively optimizes the vitrification cryopreservation method based on the vitrification ability. The vitrification cryopreservation method avoids tissue osmotic pressure damage by gradient loading / unloading of the vitrification cryoprotectant, and reduces thermal stress during the cryopreservation process by gradient cooling and annealing treatment, thereby ultimately achieving ice-free vitrification cryopreservation.

[0024] 3) The present invention utilizes the above-designed low-concentration vitrification cryopreservation protection strategy to perform deep cryopreservation on pig blood vessels, so that after 2 months of cryopreservation, the blood vessels have intact tissue structure, normal fiber and glycosaminoglycan content in the matrix, stable biomechanical properties, and good responsive contraction and relaxation capabilities.

[0025] In summary, in order to solve the problems of high concentration (>6M) and high toxicity of existing vitrification cryopreservation protective agents, the present invention designed and synthesized a series of polypeptides with good solubility, and developed a betaine-based low-concentration vitrification cryopreservation protective agent formula for the cryopreservation of porcine blood vessels.

[0026] In order to reduce the freezing temperature and thermal stress distribution of large-sized blood vessels, we synthesized magnetic nanoparticles with good biocompatibility, and used magnetothermal thawing technology to evenly thaw the frozen blood vessels, further reducing the concentration of the vitrification cryoprotectant to 3.6M, which is 40% lower than the commonly used vitrification cryoprotectant VS55. Finally, the preservation effect of the frozen pig blood vessels was evaluated, and the pig blood vessels maintained good tissue integrity, fiber structure integrity and biomechanical properties after 2 months of freezing. The new vitrification cryoprotectant and its cryopreservation method used in the present invention not only provide new possibilities for the long-term preservation of large-sized biological tissues, but also provide strong technical support for the source of donors for allogeneic vascular transplantation in future clinical applications, and are expected to be applied to a wider range of biological tissue and organ preservation fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of a method for applying a low-concentration vitrification cryoprotectant of the present invention to blood vessel cryopreservation;

[0028] Figure 2 This is a graph showing the particle size distribution of GA-MNPs in Example 1 of the present invention;

[0029] Figure 3 The graph is a graph showing the heating curve and rate results of nanoparticles of different concentrations in Example 1 of the present invention;

[0030] Figure 4 The temperature curve results of the protective agent with different concentrations in Example 1 of the present invention are shown;

[0031] Figure 5 This is a graph showing the temperature curve of the BT protective agent after adding different polypeptides in Example 1 of the present invention;

[0032] Figure 6 This is a graph showing the temperature curve results of polyglutamic acid cryoprotectants with different polymerization degrees in Example 1 of the present invention;

[0033] Figure 7 This is a graph showing the test results of the critical cooling rate and critical heating rate of the vitrification cryoprotectant in Example 2 of the present invention;

[0034] Figure 8 The temperature curve and rewarming rate result diagram of the vitrification cryoprotectant in a 50 mL system in Example 2 of the present invention;

[0035] Fig. 9 The graph is a result of the penetration ability, loading and unloading procedures of the vitrification cryoprotectant in Example 3 of the present invention;

[0036] Fig.10 This is a graph showing the staining and fiber content results of blood vessels after vitrification in Example 3 of the present invention;

[0037] Fig.11 This is a graph showing the biomechanical properties of blood vessels after vitrification in Example 3 of the present invention;

[0038] Fig.12 The corresponding results of contraction and relaxation of blood vessels after vitrification in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

[0040] Example 1: Preparation and concentration selection of nanoparticles and vitrification cryoprotectants

[0041] Preparation of magnetic nanoparticle solution:

[0042] 1) Fe3O4 nanoparticles were prepared by coprecipitation method. Specifically, 4.9 g FeCl3·6H2O was dissolved in deionized water under nitrogen protection, 2.1 g FeCl2·4H2O was added and stirred to dissolve, 8 mL of concentrated ammonia was slowly added dropwise, and then heated to 70°C for 1 hour, and then washed repeatedly with ethanol and deionized water for 3 times.

[0043] 2) Preparation of Fe3O4 magnetic nanoparticles coated with gum arabic: 0.6 g of Fe3O4 nanoparticles was added to 300 g of 10% gum arabic solution, mechanically stirred for 20 minutes and then washed with deionized water three times to obtain Fe3O4 magnetic nanoparticles coated with gum arabic (GA-MNPs). Figure 2 The particle size shown is approximately 100 nm.

[0044] The Fe3O4 magnetic nanoparticles coated with gum arabic were dispersed in PBS solution and prepared into 5 mg / mL ( Figure 3 Nanowarming-1), 10 mg / mL (Nanowarming-2), 20 mg / mL (Nanowarming-3), 30 mg / mL (Nanowarming-4) solutions in a.

[0045] like Figure 3 As shown, the above solution is added to a centrifuge tube, cooled by liquid nitrogen, and then placed in a magnetic induction coil for heating. At the same time, a temperature sensor is used to measure the temperature change of the solution. The rewarming rate of the solution increases with the increase of the concentration of the nanoparticles. When the concentration of the nanoparticles is 20 mg / mL, the rewarming rate is 14.2°C / s, which is 446% higher than that of convection rewarming (37°C water bath heating). Considering the content of the nanoparticles and the rewarming rate, 20 mg / mL is selected as the use concentration of the nanoparticles.

[0046] Preparation of cryoprotectant: dissolve betaine, trehalose and polypeptide ice blocker in PBS buffer in sequence, add the magnetic nanoparticles after they are evenly dissolved, and make the concentration of the nanoparticles 20 mg / mL;

[0047] Dissolve 600 mg of betaine in PBS solution (60B), dissolve 300 mg, 400 mg, 450 mg of betaine and 50 mg of trehalose in PBS buffer solution in sequence, with a total solution mass of 1 g, to prepare 30B+5T, 40B+5T, and 45B+5T solutions, respectively; dissolve 400 mg of betaine, 50 mg of trehalose and 5 mg of polyglutamic acid in 545 mg of PBS buffer solution in sequence to prepare 40B+5T+0.5PG lu (3.6M) solution.

[0048] like Figure 4 As shown, the above solution is added to a centrifuge tube, cooled by liquid nitrogen, and then placed in a magnetic induction coil for heating. At the same time, the temperature change of the solution is measured by a temperature sensor. It is found that when the concentration of pure betaine reaches 60%, vitrification can be successfully achieved, while pure trehalose has a low solubility and cannot be successfully vitrified.

[0049] When betaine and trehalose are mixed, the temperature rise curve shows an inflection point when the solution concentration is low (such as 40B+5T), indicating the formation of ice crystals. When the concentration increases to 45% betaine and 5% trehalose (45B+5T), the solution can be successfully vitrified, indicating that the lowest concentration is the combination of 45% betaine and 5% trehalose.

[0050] When 0.5% polyglutamic acid is added, the concentration of the vitrification solution can be reduced to a combination of 40% betaine and 5% trehalose (40B+5T+0.5PG lu).

[0051] In order to study the effect of other water-soluble peptides on reducing the glass transition concentration of the system, 5 mg of polylysine (PLys), polyaspartic acid (PAsn), polyserine (PSer) and polyproline (PPro) were added in sequence with 40B+5T (BT) as the basic protective agent. Figure 5 As shown, the above solution was added to a centrifuge tube, cooled by liquid nitrogen, and then placed in a magnetic induction coil for heating. At the same time, the temperature change of the solution was measured by a temperature sensor. It was found that only the BT+PG lu solution had a smooth curve in the rewarming stage and no devitrification behavior occurred.

[0052] In order to explore the effect of the polymerization degree of polyglutamic acid on the vitrification ability of the system, 40B+5T (BT) was used as the basic protective agent, and 5 mg of polyglutamic acid with a polymerization degree of 5, 10, 15, and 20 were added in sequence to prepare BT+PG l u5, BT+PG lu 10 、BT+PG lu 15 and BT+PG lu 20 Solution. Figure 6 As shown, the above solution was added to a centrifuge tube and cooled by liquid nitrogen, and then placed in a magnetic induction coil for heating. At the same time, the temperature change of the solution was measured by a temperature sensor. It was found that when the degree of polymerization concentration was between 5 and 15, the BT solution could be vitrified. Considering the cost of synthesis, a polyglutamic acid solution with a degree of polymerization of 5 was selected, and finally a vitrified solution (BTP) with a minimum concentration of 3.6 M (total molar concentration) was obtained.

[0053] Example 2: Cryopreservation and thawing program setting of the vitrified cryoprotectant

[0054] In order to set a suitable cooling and heating program, the critical cooling rate and critical heating rate of the low-concentration vitrification cryoprotectant were studied. The heat flow of the low-concentration vitrification cryoprotectant at different cooling rates and heating rates was analyzed by differential scanning calorimetry. The cooling rates were 0.2℃ / min, 1℃ / min, 5℃ / min, 10℃ / min, 20℃ / min and 40℃ / min, respectively. The ice content (q) and cooling rate curve were drawn by fitting the heat flow peak area in the DSC curve, which conformed to the Boutron curve model. The heating rates were 5℃ / min, 10℃ / min, 20℃ / min, 40℃ / min and 80℃ / min, respectively. The relationship between Tm-Td and heating rate was drawn by analyzing the melting point (Tm) and devitrification temperature (Td) in the DSC curve, which conformed to the linear relationship.

[0055] like Figure 7 As shown, according to the Boutron method, when the ice content is less than 0.2%, it is considered that the solution can be vitrified, and the cooling rate at this time is the critical cooling rate. The critical cooling rate of the above-mentioned vitrification cryoprotectant is 11.02°C / min; for the heating stage, it is considered that when the ice content is less than 0.5%, the devitrification behavior can be ignored, and the critical heating rate of the above-mentioned vitrification cryoprotectant is 202.24°C / min.

[0056] In order to alleviate the thermal stress problem caused by the large thermal gradient caused by rapid cooling during the cryopreservation process, a cooling program greater than the critical cooling rate of the protective agent was selected. The optimized cooling rate was 30℃ / min. When the solution was cooled to -120℃, it was kept for 20 minutes for annealing, and then continued to cool to -150℃ / min, and finally frozen in liquid nitrogen.

[0057] The low-concentration vitrification cryoprotectant was placed in a 50 mL centrifuge tube and cooled using the above cooling program. The temperature change after the volume expansion was then tested using magnetic thermal rewarming. Figure 8 It was found that after the volume was expanded to 50 mL, the solution could still be successfully vitrified, and the heating rate was 262.4℃ / min, which exceeded its critical heating rate.

[0058] Example 3: Vitrification of blood vessels

[0059] The blood vessel was immersed in the low concentration vitrification cryopreservation protective agent in Example 1, and the penetration ability of the protective agent was tested. Fig. 9 As shown, when the equilibrium time is 20 min, the loading concentration of the protective agent exceeds 90%, so 20 min is selected as the protective agent gradient loading period.

[0060] The vitrification cryopreservation protection strategy for blood vessels using the low-concentration vitrification cryopreservation protective agent in Example 1 is as follows: the blood vessels are placed in vitrification cryopreservation protective agents with concentrations of 1.2M, 2.4M, and 3.6M, respectively, and balanced for 20 minutes respectively; after loading, the cryopreservation tube containing the blood vessel is cooled using a program temperature controller, after balancing at 4°C for 10 minutes, it is cooled to -120°C at a rate of 30°C / min, annealed for 20 minutes, cooled to -150°C, and finally placed in a liquid nitrogen tank for cryopreservation. After the cryopreservation is completed, the cryopreservation tube containing the blood vessel is quickly placed in a magnetic induction coil for magnetic thermal rewarming, and taken out after the solution temperature rises to 0°C.

[0061] Comparative Example 1: Cryopreservation of blood vessels in PBS solution: The blood vessels were placed in a cryotube containing 30 mL of PBS solution, and after cryopreservation using the above-mentioned cryopreservation procedure, the blood vessels were thawed by convection heating. Specifically, the cryotube containing the blood vessels was placed in a 37°C water bath for heating.

[0062] Comparative Example 2: Cryopreservation of blood vessels using the commonly used vitrification cryopreservation protection VS55: VS55 is composed of 3.1M DMSO, 3.1M formamide and 2.2M 1,2-propylene glycol. The blood vessels were placed in the VS55 solution, cryopreserved using the above freezing procedure, and then thawed by convection heating.

[0063] Thawing steps of blood vessels with vitrification cryoprotectant in Example 1:

[0064] The cryotubes containing blood vessels were rewarmed and transferred to solutions with concentrations of 2.4M, 1.2M, and 0.6M (total molar concentration) in sequence for equilibration for 20 minutes respectively, and then washed with PBS solution to obtain the blood vessels.

[0065] Detection and analysis of the vitrification protection results of blood vessels.

[0066] 1. Perform the following functional tests on blood vessels after vitrification and storage for 1 week and 2 months and then thawing

[0067] (1) Histomorphological detection: After cutting the blood vessel samples obtained in Example 3, they were processed by hematoxylin-eosin (H&E) and collagen-elastic fiber composite (EVG) staining methods according to the instructions of the kit. These staining techniques are used to analyze the density and morphological changes of vascular tissue after cryopreservation to evaluate the integrity of its tissue structure.

[0068] (2) Fiber and glycosaminoglycan content detection: The frozen blood vessels were analyzed by using the hydroxyproline (HYP) kit, hydroxylysylpyridinoline (HP) kit, lysylpyridinoline (LP) kit, and glycosaminoglycan (GAGs) kit. According to the method in the kit instructions, the contents of hydroxyproline, hydroxylysylpyridinoline, lysylpyridinoline, and glycosaminoglycan in the blood vessel samples were measured to evaluate the changes in the content of collagen fibers and glycosaminoglycans.

[0069] (3) Biomechanical properties testing: The biomechanical properties of blood vessels were measured using a universal testing machine. First, the blood vessel samples were cut using a dumbbell cutter. The gauge length was set to 1 cm, and the blood vessel samples were clamped in the fixture of the testing instrument. Then, a uniaxial tensile test was performed at a rate of 10 mm / min until the blood vessel sample broke. The elongation at break was determined by analyzing the tensile stress-strain curve, and the Young's modulus was obtained by calculating the slope of the linear region of the curve.

[0070] (4) Assessment of contractile tension: The isometric contraction and relaxation responses of the blood vessels were assessed by adding different agonists and antagonists to the organ bathing fluid, and the measurement data were recorded using a universal testing machine. A preload tension of 0.01 N was first applied to the blood vessel sample and allowed to stabilize at the baseline. Norepinephrine (doses ranging from 10 -8 M to 10 -4 M) to induce vasoconstriction. To assess the vascular relaxation response, first use 10 -5 The blood vessels were pre-contracted to a stable level with 10 M norepinephrine. -4 M sodium nitroprusside and 10 -4 The endothelium-dependent relaxation (EDR) and non-endothelium-dependent relaxation (non-EDR) responses of the blood vessels were evaluated by acetylcholine (ACh).

[0071] 2. Results Analysis

[0072] (1) Fig.10 The results of H&E staining, EVG staining and fiber content after blood vessel cryopreservation are shown, indicating that the structural morphology of blood vessel tissue after one week of vitrification using the low-concentration formula was less changed than that of fresh tissue, and was significantly better than that using the conventional vitrification cryoprotectant VS55. In contrast, the blood vessel samples in the convection heating group showed obvious fiber breakage, cell nucleus leakage and increased tissue gaps, and the content of hydroxylysylpyridinoline and lysylpyridinoline increased, indicating that the triple helix chain of collagen was more cross-linked, resulting in collagen denaturation, which may be due to the formation of ice crystals. Even if the cryopreservation time was extended to 2 months, the samples using this formula still retained 92.1% of tissue density and retained similar proportions of collagen and glycosaminoglycans, showing excellent cryopreservation effect.

[0073] (2) Fig.11 The results of biomechanical properties of blood vessels after cryopreservation show that after 1 week or 2 months of vitrification using this BTP, the elongation at break and Young's modulus of blood vessels are still not significantly different from those of the fresh group. However, after 2 months of cryopreservation using the commonly used vitrification cryopreservation protective agent VS55, the elongation at break was significantly reduced, and the Young's modulus was significantly increased, indicating that the hardness of the blood vessels increased and the elasticity decreased significantly.

[0074] (3) Fig.12 The results showed that the contraction and relaxation ability of blood vessels after cryopreservation increased with the increase of norepinephrine concentration. The contraction response of the control group was significantly reduced due to the damage of ice crystals to smooth muscle cells. The blood vessels treated with BTP vitrification cryopreservation protectant can still maintain good contraction ability even after being frozen for 2 months. In addition, we used non-endothelial smooth muscle relaxant sodium nitroprusside and endothelial smooth muscle relaxant acetylcholine to evaluate the vascular relaxation ability. The results showed that the maximum vascular relaxation ability of the BTP group after 2 months of cryopreservation reached 87% and 79% of the fresh group, respectively, showing high sensitivity and functional retention.

[0075] In summary, in order to solve the problems of high concentration and high toxicity of existing vitrification cryoprotectants, we have developed a new biocompatible low-concentration vitrification cryoprotectant (CPA) formula for cryopreservation of blood vessels. Based on the excellent hydration of polyglutamic acid, the formula can effectively disrupt water molecules to form ice crystals, thereby successfully reducing the concentration of the vitrification protectant to 3.6M. In order to improve the rewarming efficiency and avoid devitrification, we designed a magnetic nanoparticle (GA-MNPs) with good biocompatibility. The nanoparticles not only increase the heating rate, but also effectively reduce the thermal stress during the heating process, thereby significantly improving the rewarming effect of the blood vessels. Using this vitrification cryopreservation strategy, we successfully cryopreserved the blood vessels for 2 months, and the structural integrity, biomechanical properties and good contraction and relaxation ability of the blood vessels were retained after rewarming. For the first time, the present invention uses a peptide to achieve a vitrification cryoprotectant with a concentration as low as 3.6M and applies it to the vitrification preservation of blood vessels. This low-concentration vitrification preservation strategy is expected to be further optimized to achieve the cryopreservation of a wider range of biological tissues and organs, and promote research and clinical applications in related fields.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide-based cryoprotectant, characterized in that: It comprises antifreeze agent, polypeptide ice inhibitor and magnetic nanoparticles; the antifreeze agent comprises betaine, trehalose and balance solution.

2. The polypeptide-based cryoprotectant according to claim 1, characterized in that: The mass concentration of betaine in the polypeptide-based cryoprotectant is ≥40%, preferably 40%; the mass concentration of trehalose in the polypeptide-based cryoprotectant is ≥5%, preferably 5%; the total molar concentration of betaine, trehalose and polypeptide ice inhibitor is ≥3.6 mol / L, preferably 3.6 mol / L.

3. The polypeptide-based cryoprotectant according to claim 1, characterized in that: The polypeptide ice inhibitor is polyglutamic acid (PGlu), which accounts for 0.5% by mass in the cryoprotectant; the polymerization degree of the polyglutamic acid (PGlu) is 5-15.

4. The polypeptide-based cryoprotectant according to claim 1, characterized in that: The balancing solution is one of PBS buffer, DPBS buffer, physiological saline or Euro-Collins preservation solution.

5. The polypeptide-based cryoprotectant according to claim 1, characterized in that: The magnetic nanoparticles include magnetic spheres and biocompatible materials wrapped on the surface of the magnetic spheres; preferably, the size is 10nm to 1000nm, the concentration is 5-40mg / mL, and preferably, the concentration is 20mg / mL; Preferably, the magnetic balls are Fe3O4 balls or FeO balls; and the biocompatible materials are gum arabic or polysaccharides.

6. The polypeptide-based cryoprotectant according to claim 5, characterized in that: The magnetic nanoparticles are prepared by: 1) using a co-precipitation method to prepare magnetic spheres; 2) adding the magnetic spheres into an aqueous solution of a biocompatible material.

7. A method for preparing the polypeptide-based cryoprotectant according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: adding betaine, trehalose and polypeptide ice inhibitor into a balance solution to prepare a vitrification cryopreservation protective agent of a certain concentration, and adding magnetic nanoparticles therein to obtain the vitrification protective agent.

8. Use of the polypeptide-based cryoprotectant according to any one of claims 1 to 6, characterized in that: Applicable to vitrification of tissues or organs, preferably, the organ is a blood vessel.

9. The use of the polypeptide-based cryoprotectant according to claim 8, characterized in that: The method comprises the following steps: 1) loading of vitrification solution: placing the blood vessel in a polypeptide-based cryoprotectant for equilibrium; 2) setting of vitrification cryoprotectant program: after loading, cooling the cryotube containing the blood vessel using a programmable temperature controller; 3) magnetic thermal rewarming: after the cryoprotectant is completed, quickly placing the cryotube containing the blood vessel in a magnetic induction coil for magnetic thermal rewarming, and taking it out after the solution temperature rises to 0°C; 4) elution program: gradiently diluting the vitrification cryoprotectant with a PBS solution, and using a magnet to absorb the nanoparticles.

10. The use of the polypeptide-based cryoprotectant according to claim 9, characterized in that: The cooling program of step 2) is: after equilibration at 4°C for 10 minutes, cool to -120°C at a rate of 30°C / min, anneal for 20 minutes, cool to -150°C, and finally place in a liquid nitrogen tank for cryopreservation.

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