Nanocrystal nucleus antifreezing agent system for cryopreservation of corneal graft

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a nanocrystal nucleus anti-freezing agent system for low-temperature preservation of a corneal graft. The invention firstly provides a nanocrystal nucleus antifreezing agent which is composed of propylene glycol, ginkgo leaf alcohol extract, trehalose, gold nanoparticles and glycerin, and the nanocrystal nucleus antifreezing agent can inhibit the anti-vitrification of corneal graft, reduce the formation of ice crystals, reduce the damage to cells and tissues to the greatest extent, and improve the anti-vitrification performance of corneal graft. And the survival rate of the corneal graft after cryopreservation is improved. Therefore, the corneal graft is placed in the nanocrystal nucleus anti-freezing agent provided by the technical scheme, and the low-temperature preservation of the corneal graft can be simply and conveniently realized only in an environment of-70 DEG C to-80 DEG C.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a nanocrystalline core antifreeze system for low-temperature preservation of corneal transplants. Background Art

[0002] Cryopreservation is a technique that uses extremely low temperatures to preserve biological samples. It can significantly extend the storage time of organs and tissues, ensuring they remain biologically active until needed. For example, tissues such as corneas, skin, and heart valves can be cryopreserved for long periods at low temperatures, significantly increasing their availability for transplantation.

[0003] There are related studies on organ cryopreservation in the prior art. For example, Chinese Patent CN 102550541 A provides a vitrification cryopreservation solution for long-term organ preservation. This solution is composed of dimethyl sulfoxide, acetamide, glycerol, ethylene glycol, propylene glycol, sucrose, polyethylene glycol, and other ingredients. This solution is beneficial for preserving tissue morphology and structural integrity, completely inhibiting the energy metabolism of tissue cells under freezing conditions, and restoring their biological functions after resuscitation. It can be used for kidney cryopreservation and can also be further applied to the long-term cryopreservation of other organs for scientific research and clinical applications. Another example is Chinese Patent CN 118434843 A, a cryopreservation preparation for corneal endothelial cells and its preparation method, which discloses a method for preserving corneal endothelial cells and / or corneal endothelial-like cells. Specifically, the method comprises freezing the corneal endothelial cells and / or corneal endothelial-like cells in a non-frozen state, wherein the temperature is lowered from the non-frozen temperature to the target freezing temperature, including at least one step of decreasing the temperature at a rate of less than 1°C per minute; and, if necessary, maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state. Although the above schemes achieve organ cryopreservation, they have the disadvantages of complicated procedures or lack of specificity. Therefore, how to provide a nanocrystal core antifreeze system specifically for the cryopreservation of corneal transplants is an urgent problem to be solved in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide a nanocrystalline core antifreeze system for low-temperature preservation of corneal transplants, which has the characteristics of convenient use and high survival rate of corneal transplants.

[0005] The invention provides a nanocrystalline core antifreeze agent, which comprises the following raw materials: propylene glycol, ginkgo biloba leaf alcohol extract, trehalose, gold nanoparticles and glycerol.

[0006] Preferably, the nanocrystalline core comprises the following raw materials in terms of mass percentage: Propylene glycol 60%-65%, ginkgo leaf alcohol extract 13%-15%, trehalose 8%-10%, gold nanoparticles 3%-5% and the balance glycerol.

[0007] Preferably, the particle size of the gold nanoparticles is 50-80 nm.

[0008] Preferably, the preparation method of the ginkgo leaf ethanol extract comprises: Ginkgo biloba leaves are mixed with ethanol and then subjected to ultrasonic extraction to obtain ginkgo biloba leaf alcohol extract; Preferably, the mass volume ratio of the ginkgo leaf to ethanol is 1 g: 6-8 mL; The volume concentration of the ethanol is 70%-75%.

[0009] Preferably, the ultrasonic extraction is performed 2-3 times.

[0010] Preferably, the parameters of the ultrasonic extraction include: time of 30-45 min, power of 175-195 W, and temperature of 35-45°C.

[0011] The present invention provides the use of the nanocrystalline core antifreeze agent described in the above technical solution in the low-temperature preservation of corneal transplants and / or the preparation of low-temperature preservation products for corneal transplants.

[0012] The present invention provides a method for cryopreservation of corneal transplants, comprising the following steps: The corneal transplant is placed in the nanocrystal core antifreeze agent described in the above technical solution and stored at a low temperature of -70°C to -80°C.

[0013] The present invention provides a nanocrystalline core antifreeze system for low-temperature preservation of corneal transplants. The nanocrystalline core antifreeze system comprises the nanocrystalline core antifreeze described in the above technical solution.

[0014] Beneficial effects: The present invention provides a nanocrystalline core antifreeze, comprising the following ingredients: propylene glycol, ginkgo biloba alcohol extract, trehalose, gold nanoparticles, and glycerol. By specifically combining these ingredients, the present invention can achieve vitrification during the cooling process, inhibit devitrification of corneal transplants, reduce ice crystal formation, minimize damage to cells and tissues, and improve the survival rate of corneal transplants after cryopreservation.

[0015] Based on the above technical advantages, the present invention also provides a method for cryopreservation of corneal transplants, which can be achieved simply and conveniently by placing the corneal transplant in the nanocrystalline core antifreeze described in the above technical solution and cryopreserving it in an environment of -70°C to -80°C. DETAILED DESCRIPTION

[0016] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0017] Example 1 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 60%, ginkgo biloba leaf alcohol extract 13%, trehalose 8%, gold nanoparticles 4% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0018] (2) The preparation method of the ginkgo leaf ethanol extract in step (1) is as follows: Ginkgo biloba leaves ( Ginkgo biloba ) was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, subjected to a first ultrasonic extraction at 175 W and 35° C. for 35 min, and filtered for the first time to obtain a first filtrate and a first precipitate; The first precipitate was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, and subjected to a second ultrasonic extraction at 175 W and 35 ° C for 35 min. The second filtrate was obtained by filtering the mixture for the second time. The first filtrate and the second filtrate are combined to obtain the ginkgo leaf ethanol extract.

[0019] Example 2 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 65%, ginkgo biloba leaf alcohol extract 13%, trehalose 9%, gold nanoparticles 3% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0020] (2) The preparation method of the ginkgo leaf ethanol extract in step (1) is as follows: Ginkgo biloba leaves ( Ginkgo biloba ) was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, subjected to a first ultrasonic extraction at 175 W and 35° C. for 35 min, and filtered for the first time to obtain a first filtrate and a first precipitate; The first precipitate was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, and subjected to a second ultrasonic extraction at 175 W and 35 ° C for 35 min. The second filtrate was obtained by filtering the mixture for the second time. The first filtrate and the second filtrate are combined to obtain the ginkgo leaf ethanol extract.

[0021] Example 3 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 60%, ginkgo biloba leaf alcohol extract 13%, trehalose 8%, gold nanoparticles 4% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0022] (2) The preparation method of the ginkgo leaf ethanol extract in step (1) is as follows: Ginkgo biloba leaves ( Ginkgo biloba ) was mixed with 75% ethanol at a mass volume ratio of 1 g:8 mL, subjected to a first ultrasonic extraction at 175 W and 35° C. for 30 min, and filtered for the first time to obtain a first filtrate and a first precipitate; The first precipitate was mixed with 70% ethanol at a mass volume ratio of 1 g:6 mL, and subjected to a second ultrasonic extraction at 175 W and 35 ° C for 30 min. The second filtrate was obtained by filtering the mixture for the second time. The first filtrate and the second filtrate are combined to obtain the ginkgo leaf ethanol extract.

[0023] Comparative Example 1 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 60%, ethylene glycol 13%, trehalose 8%, gold nanoparticles 4% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0024] Comparative Example 2 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 75%, ginkgo biloba leaf alcohol extract 3%, trehalose 8%, gold nanoparticles 4% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0025] (2) The preparation method of the ginkgo leaf ethanol extract in step (1) is as follows: Ginkgo biloba leaves ( Ginkgo biloba ) was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, subjected to a first ultrasonic extraction at 175 W and 35° C. for 35 min, and filtered for the first time to obtain a first filtrate and a first precipitate; The first precipitate was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, and subjected to a second ultrasonic extraction at 175 W and 35 ° C for 35 min. The second filtrate was obtained by filtering the mixture for the second time. The first filtrate and the second filtrate are combined to obtain the ginkgo leaf ethanol extract.

[0026] Comparative Example 3 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 60%, Ginkgo biloba leaf aqueous extract 13%, trehalose 8%, gold nanoparticles 4% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0027] (2) The preparation method of the ginkgo leaf water extract in step (1) is as follows: Ginkgo biloba leaves ( Ginkgo biloba ) was mixed with water at a mass volume ratio of 1 g:8 mL, subjected to a first ultrasonic extraction at 175 W and 35° C. for 35 min, and filtered for the first time to obtain a first filtrate and a first precipitate; The first precipitate was mixed with water at a mass volume ratio of 1 g:8 mL, and subjected to a second ultrasonic extraction at 175 W and 35 ° C for 35 min. The second filtrate was obtained by filtering the mixture for the second time. The first filtrate and the second filtrate were combined to obtain a ginkgo leaf water extract.

[0028] Comparative Example 4 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 60%, ginkgo biloba leaf alcohol extract 13%, trehalose 8%, gold nanoparticles 4% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0029] (2) The preparation method of the ginkgo leaf ethanol extract in step (1) is as follows: Ginkgo biloba leaves ( Ginkgo biloba ) was mixed with 65% ethanol at a mass volume ratio of 1 g:8 mL, subjected to a first ultrasonic extraction at 150 W and 35° C. for 35 min, and filtered for the first time to obtain a first filtrate and a first precipitate; The first precipitate was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, and subjected to a second ultrasonic extraction at 150 W and 35° C. for 35 min. The second filtrate was obtained by filtering the mixture for the second time. The first filtrate and the second filtrate are combined to obtain the ginkgo leaf ethanol extract.

[0030] Comparative Example 5 Provided is a nanocrystalline core antifreeze, comprising the steps of: (1) In terms of mass percentage, the nanocrystalline core includes the following raw materials: Propylene glycol 60%, ginkgo biloba leaf alcohol extract 13%, trehalose 8%, copper nanoparticles 4% and the balance glycerol; The particle size of the gold nanoparticles is 50 nm.

[0031] (2) The preparation method of the ginkgo leaf ethanol extract in step (1) is as follows: Ginkgo biloba leaves ( Ginkgo biloba ) was mixed with 70% ethanol at a mass volume ratio of 1 g:8 mL, subjected to a first ultrasonic extraction at 175 W and 35° C. for 35 min, and filtered for the first time to obtain a first filtrate and a first precipitate; The first precipitate was mixed with 70% ethanol at a mass volume ratio of 1 g: 8 mL, and then subjected to a second ultrasonic extraction at 175 W and 35 ° C for 35 min. The second filtrate was obtained after the second filtration. The first filtrate and the second filtrate are combined to obtain the ginkgo leaf ethanol extract.

[0032] Comparative Example 6 Provided is a nanocrystalline core antifreeze, comprising the steps of: The difference from Example 1 is that the particle size of the gold nanoparticles used in Comparative Example 6 is 150 nm.

[0033] Application Example 1 The nanocrystalline antifreeze agents in Examples 1-3 and Comparative Examples 1-6 were selected as test objects; A number of healthy New Zealand white rabbits weighing 2.5-2.8 kg were selected. Slit lamp examination before the experiment showed that all the rabbits had normal eyes. Rabbits were randomly divided into 9 groups, 15 in each group. The rabbits were sacrificed within 1 hour, and their intact eyeballs were removed and sterilely processed. The sterile eyeballs were processed in a clean bench to obtain rabbit corneal slices, which were quickly placed in corneal bottles at 4°C (one corneal slice per corneal bottle, 10 corneal slices per treatment, repeated three times), with the corneal endothelium facing up. 2 mL of each test object was added to the corneal bottle and stored at -70°C.

[0034] On the 10th, 50th, and 100th day after storage, 10 corneal bottles (i.e., one replicate) were taken from each treatment group. The corneal slices in each treatment were revived, washed, and stained. The activity of the corneal slices in the corneal bottles was counted. The results are shown in Table 1. Among them, active: endothelial cells are neatly arranged and tightly connected under microscopic examination; the combined staining method of alizarin red and trypan blue shows that the cells have red hexagonal outlines, and the blue-stained nuclei are not visible; or the intercellular spaces are slightly enlarged, some cells are still neatly arranged, and most of the cell outlines are round or irregular; Inactive: Endothelial cells are irregularly arranged, with unclear outlines, and nuclei stained with different colors are mostly visible, or cells are missing in patches.

[0035] Table 1 Number of viable corneal transplants in different treatments

[0036] Combined with the data in Table 1, it can be seen that, compared with the comparative example, after 100 days of cryopreservation of corneas using the nanocrystal core antifreeze provided in Examples 1-3, most corneal transplants remained viable. This demonstrates that the technical solution provided by the present invention facilitates the preservation of corneal transplants and achieves a high survival rate.

[0037] In summary, the present invention, through the specific combination of propylene glycol, ginkgo biloba alcohol extract, trehalose, gold nanoparticles and glycerol, can achieve vitrification during the cooling process, inhibit devitrification of corneal transplants, reduce the formation of ice crystals, minimize damage to cells and tissues, and improve the survival rate of corneal transplants after cryopreservation.

[0038] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A nanocrystalline antifreeze agent, characterized in that: The nanocrystalline antifreeze comprises the following raw materials: Propylene glycol, alcoholic extract of ginkgo biloba leaves, trehalose, gold nanoparticles, and glycerol.

2. The nanocrystalline antifreeze agent according to claim 1, characterized in that In terms of mass percentage, the nanocrystalline core comprises the following raw materials: Propylene glycol 60%-65%, ginkgo leaf alcohol extract 13%-15%, trehalose 8%-10%, gold nanoparticles 3%-5% and the balance glycerol.

3. The nanocrystalline antifreeze agent according to claim 1 or 2, characterized in that: The particle size of the gold nanoparticles is 50-80 nm.

4. The nanocrystalline antifreeze agent according to claim 1 or 2, characterized in that: The preparation method of the ginkgo leaf ethanol extract comprises: The ginkgo leaves are mixed with ethanol and then subjected to ultrasonic extraction to obtain the ginkgo leaf alcohol extract.

5. The nanocrystalline antifreeze agent according to claim 4, characterized in that The mass volume ratio of the ginkgo leaf to ethanol is 1 g: 6-8 mL; The volume concentration of the ethanol is 70%-75%.

6. The nanocrystalline antifreeze agent according to claim 4, characterized in that The ultrasonic extraction is performed 2-3 times.

7. The nanocrystalline antifreeze according to claim 4, characterized in that The parameters of the ultrasonic extraction include: time of 30-45 minutes, power of 175-195W, and temperature of 35-45°C.

8. Use of the nanocrystalline core antifreeze according to any one of claims 1 to 7 in the cryopreservation of corneal transplants and / or in the preparation of a corneal transplant cryopreservation product.

9. A method for cryopreservation of corneal transplants, characterized in that: The steps include: The corneal transplant is placed in the nanocrystal core antifreeze according to any one of claims 1 to 7 and stored at a low temperature of -70°C to -80°C.

10. A nanocrystalline core antifreeze system for low-temperature preservation of corneal transplants, characterized in that: The nanocrystalline core antifreeze system comprises: the nanocrystalline core antifreeze according to any one of claims 1 to 7.

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