Use of nanoparticle combining ice-inhibiting polymer and conductive polymer in cell cryopreservation

Abstract

Disclosed is a use of a nanoparticle combining an ice-inhibiting polymer and a conductive polymer as a cell cryopreservative. In the present invention, an ice-inhibiting polymer is used as a dispersion liquid to composite a conductive polymer; the ice-inhibiting polymer can inhibit the growth of ice crystals, while the conductive polymer has efficient photothermal properties, to achieve rapid and uniform warming of cryopreserved cells. Furthermore, using a confocal dish having a large bottom area as a container for cell cryopreservation causes cells to achieve high rates of temperature rise and fall during cryopreservation and recovery processes, thereby ensuring the survival rate of the cells after cryopreservation. The nanoparticle combining the ice-inhibiting polymer and the conductive polymer of the present invention acts as a cryopreservative for cells, is simple and rapid to prepare, has good biocompatibility, can combine with different ice-inhibiting polymers and conductive polymers according to actual conditions, and has good application prospects in the field of cell cryopreservation.

Description

Application of ice-inhibiting polymer composite conductive polymer nanoparticles in cell cryopreservation Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the application of ice-inhibiting polymer composite conductive polymer nanoparticles in cell cryopreservation. Background Art

[0002] Cryopreservation of biological samples, such as cells, tissues, and organs, involves long-term storage in ultra-low temperature environments (-80°C or -196°C). In ultra-low temperature environments, the physical and chemical reactions of cells are greatly restricted, allowing for long-term cryopreservation. Cell cryopreservation technology is crucial for the clinical application of biopharmaceutical products, playing an irreplaceable role in areas such as food security, cell therapy, and assisted reproduction. The development of efficient cryopreservatives is an essential requirement for the advancement of biomedicine.

[0003] During the cooling and warming recovery process, the formation and growth of ice crystals will cause serious and irreversible mechanical damage and osmotic damage to the cells. Rapid cooling that exceeds the critical cooling rate of the solution can cause it to skip the crystallization phase and directly transform into a glassy state during the cooling process. Rapid warming can allow the solution to quickly pass through the freezing temperature range and reduce damage to the cells. Existing cryopreservatives mostly use small molecule dimethyl sulfoxide (DMSO) or glycerol, both of which have high biotoxicity and are difficult to remove from cells. Conventional water bath heating and heating methods make it difficult to achieve rapid and uniform heating of frozen cells, resulting in irreversible damage to the cells. Therefore, lower biotoxicity, better ice inhibition effect, and faster and more uniform heating methods are the development goals of new cell cryopreservatives.

[0004] Invention Disclosure

[0005] To address the problems existing in the prior art, the present invention provides an application of ice-inhibiting polymer composite conductive polymer nanoparticles in cell cryopreservation. The ice-inhibiting polymer composite conductive polymer nanoparticles have low biotoxicity, effectively inhibit the growth of ice crystals, and utilize the photothermal effect of the conductive polymer and the penetrating power of near-infrared lasers to rapidly and uniformly warm frozen cells. Furthermore, using a confocal dish as a container for the cell cryopreservative increases the area of ​​contact between the cryopreservative and the external environment during the warming process, allowing the frozen cells to quickly pass through the freezing temperature range, thereby maintaining cell vitality and stability during the cryo-thaw process.

[0006] In order to achieve the above-mentioned purpose, the technical solutions adopted by the present invention are as follows:

[0007] In a first aspect, the present invention provides the use of ice-inhibiting polymer composite conductive polymer nanoparticles in the preparation of a cell cryopreservative.

[0008] In a second aspect, the present invention provides a cell cryopreservative, the components of which include ice-inhibiting polymer composite conductive polymer nanoparticles and cell basal culture medium.

[0009] Furthermore, in the cell cryopreservative, the concentration of the ice-inhibiting polymer composite conductive polymer nanoparticles is 0.1-100 μg / mL, preferably 50-100 μg / mL.

[0010] Furthermore, the cell basal culture medium does not contain serum or human albumin.

[0011] In a third aspect, the present invention provides a method for cell cryopreservation.

[0012] The cell cryopreservation method comprises the following steps: co-incubating the cell cryopreservation agent and cells to be frozen in a confocal dish, and achieving cell cryopreservation by rapid freezing with liquid nitrogen.

[0013] Furthermore, the ratio of the amount of the cell cryopreservative to the cells to be frozen is: 1 ml of cell cryopreservative is used for 10 6 cells.

[0014] Furthermore, the bottom diameter of the confocal dish may be 35 mm.

[0015] In a fourth aspect, the present invention provides a cryopreserved cell.

[0016] The frozen cells are obtained by freezing using the cell cryopreservation method provided in the third aspect of the present invention.

[0017] In a fifth aspect, the present invention also provides a method for freezing and thawing the above-mentioned frozen cells.

[0018] The method uses near-infrared laser irradiation combined with water bath heating to warm and revive the frozen cells.

[0019] Furthermore, the near-infrared laser has a wavelength of 780-1100 nm and an optical density of 0.5-2 W / cm 2 Near-infrared laser, preferably with a wavelength of 808 nm and an optical density of 1-1.5 W / cm 2 .

[0020] Furthermore, the water bath heating temperature is 36.5-37.5°C.

[0021] The ice-inhibiting polymer composite conductive polymer nanoparticles of the present invention include an ice-inhibiting polymer and a conductive polymer, wherein the ice-inhibiting polymer serves as a dispersion of the conductive polymer;

[0022] Furthermore, different ice-inhibiting polymers have different ice-inhibiting effects, and different conductive polymers have different photothermal effects, and different ice-inhibiting polymer composite conductive polymer nanoparticles are prepared;

[0023] Preferably, the ice-inhibiting polymer is selected from at least one of the following: polyvinyl alcohol (molecular weight 27,000-205,000), polyvinyl pyrrolidone (molecular weight 8,000-580,000), polyethylene glycol (molecular weight 2,000-600,000);

[0024] Preferably, the conductive polymer is selected from at least one of the following: polypyrrole, polyacetylene, polyaniline, polythiophene, and polyphenylene vinylene.

[0025] Furthermore, the ice-inhibiting polymer composite conductive polymer nanoparticles are prepared according to a method comprising the following steps: dissolving the ice-inhibiting polymer in deionized water at 65-85° C., cooling to room temperature, adding an oxidant to the ice-inhibiting polymer solution and stirring; after balancing for 45-90 minutes, adding a monomer for forming a conductive polymer, and reacting at 4-7° C. for 4-6 hours; after the reaction is completed, centrifuging to obtain the ice-inhibiting polymer composite conductive polymer nanoparticles.

[0026] When the conductive polymer is polypyrrole, the oxidant is ferric chloride hexahydrate (FeCl3·6H2O);

[0027] When the conductive polymer is polyaniline, the oxidant is ammonium persulfate.

[0028] For the preparation of PPy@PVA, the ratio of polyvinyl alcohol (PVA), ferric chloride hexahydrate, and pyrrole monomer is: 1.50 g: 1.2434 g: 140 μL.

[0029] The above raw material ratios can be used as a reference for the preparation of other types of ice-inhibiting polymer composite conductive polymer nanoparticles.

[0030] In the above method, the centrifugation conditions are: centrifugation at a rotation speed of 12000 rpm and a temperature of 40° C. for 40 min; the centrifugation is repeated three times.

[0031] The ice-inhibiting polymer composite conductive polymer nanoparticles of the present invention are directly added to achieve cell cryopreservation.

[0032] The cells of the present invention include neural cells, preferably human neuroblastoma cells SH-SY5Y, Schwann cells RSC96, and rat dorsal root ganglion blastoma cells ND7 / 23.

[0033] The present invention has the following beneficial effects:

[0034] The present invention provides a novel cell cryopreservative for cell cryopreservation. The cell cryopreservative is simple and quick to prepare, has low cytotoxicity, can effectively inhibit ice crystal growth at relatively low concentrations, and can also utilize the high absorption rate of conductive polymers in the near-infrared light region I to achieve rapid and uniform heating of frozen cells during thawing. Confocal dishes, as containers for cell cryopreservation, can greatly increase the heating and cooling rates of frozen cells, allowing them to quickly pass through the freezing temperature range and ensure a high recovery rate of cells after freezing and thawing. It has broad application prospects in the field of cell cryopreservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 shows scanning electron micrographs of ice-inhibiting polymer composite conductive polymer nanoparticles after freeze-drying: (a) PPy@PVA-27000; (b) PPy@PVA-67000; (c) PPy@PVA-89000.

[0036] Figure 2 is a graph showing the light absorption performance of ice-inhibiting polymer composite conductive polymer nanoparticles: (a) PPy@PVA-27000; (b) PPy@PVA-67000; (c) PPy@PVA-89000.

[0037] FIG3 is a photothermal conversion performance curve of ice-suppressing polymer composite conductive polymer nanoparticles: (a) concentration ≤ 100 μg / mL; (b) concentration ≥ 100 μg / mL.

[0038] FIG4 is an evaluation of the ice suppression performance of ice suppression polymer composite conductive polymer nanoparticles.

[0039] FIG5 is a cytotoxicity evaluation of ice-inhibiting polymer composite conductive polymer nanoparticles.

[0040] FIG6 shows the cooling rate when different volumes of ice-inhibiting polymer composite conductive polymer nanoparticles are added to the confocal dish.

[0041] Figure 7 shows laser confocal microscopy images of cells taken after the ice-inhibiting polymer composite conductive polymer nanoparticles were used as cell cryopreservation agents: (a) intact RSC96 cells; (b) RSC96 cells frozen without PPy@PVA-67000 solution; (c) RSC96 cells frozen with PPy@PVA-67000 solution.

[0042] Best Mode for Carrying Out the Invention

[0043] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0044] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0045] Example 1

[0046] 1.50 g of polyvinyl alcohol (PVA) with a molecular weight of 67,000 was dissolved in 20 mL of deionized water at 80°C for 30 minutes. After cooling to room temperature, 1.2434 g of ferric chloride hexahydrate (FeCl3·6H2O) was added to the PVA solution with stirring, causing the solution to turn yellow. After equilibration for one hour, 140 μL of pyrrole monomer was added, and the reaction was continued at 5°C for four hours. After the reaction, centrifugation was repeated three times at 12,000 rpm and 40°C for 40 minutes to obtain PPy@PVA-67000. Following the above preparation process, replacing PVA with PVAs of other molecular weights resulted in PVA-composite polypyrrole nanoparticles (PPy@PVA-27000 and PPy@PVA-89000) with molecular weights of 27,000 and 89,000, respectively. Scanning electron micrographs of the resulting particles after freeze-drying are shown in Figure 1. All three materials exhibit spherical shape and uniform particle size.

[0047] Example 2

[0048] The PPy@PVA-27000, PPy@PVA-67000, and PPy@PVA-89000 solutions prepared in Example 1 were diluted with water to concentrations of 5 μg / mL, 15 μg / mL, 25 μg / mL, 35 μg / mL, and 50 μg / mL, respectively. Their light absorption properties were measured using a UV-visible spectrophotometer, as shown in Figure 2. The absorption of all three materials in the near-infrared region I was higher than that at other wavelengths. At the same concentration, the light absorption of the three materials in the near-infrared region I (700-950 nm) was, from highest to lowest, PPy@PVA-27000, PPy@PVA-67000, and PPy@PVA-89000, respectively.

[0049] Example 3

[0050] The photothermal performance of the PPy@PVA-67000 solution prepared in Example 1 was measured using a wavelength of 808 nm and an optical density of 1 W / cm 2A near-infrared laser was used to irradiate 800 μL of water and PPy@PVA-67000 solutions of different concentrations for 10 minutes, and the temperature changes of the irradiated solutions were observed and recorded with thermocouples. As shown in Figure 3a, the net temperature rise of the PPy@PVA-67000 solution with a concentration of 100 μg / mL after 10 minutes of near-infrared laser irradiation can reach 40°C. It was calculated that the photothermal conversion efficiency of the PPy@PVA-67000 solution was 35.86%. As shown in Figure 3b, when the concentration exceeded 100 μg / mL, the photothermal heating effect of the PPy@PVA-67000 solution no longer increased significantly with increasing concentration.

[0051] Example 4

[0052] The PPy@PVA-67000 solution prepared in Example 1 was diluted with water to solution concentrations of 2.5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. A droplet splash experiment was performed to evaluate the inhibitory performance of PPy@PVA-67000 solutions of different concentrations on ice crystal growth. A 10 μL droplet was dropped from a high place to form a thin layer of ice crystals on the silicon wafer below. The silicon wafer with the thin layer of ice crystals was placed in a closed space on a hot and cold stage with an internal temperature of -6°C. The ice crystals were observed using a polarized light microscope and the ice crystal size was counted every 5 minutes for 30 minutes. As shown in Figure 4, as the concentration of PPy@PVA-67000 solution increases, the final size of the ice crystals at 30 minutes continues to decrease. The PPy@PVA-67000 solution with a concentration of 50 μg / mL can effectively inhibit the growth of ice crystals, making the diameter of the ice crystals only 76 μm at 30 minutes.

[0053] Example 5

[0054] The PPy@PVA-67000 solution prepared in Example 1 was used to test its biological toxicity. RSC96 cells were cultured at a density of 3×10 5 / mL concentration was plated in a 96-well plate and incubated until adhered. The PPy@PVA-67000 solution was sterilized in a water bath at 60°C for 6-8 hours and then diluted with the basal culture medium DMEM low glucose (Solarbio, 31600) to a solution concentration of 2.5μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL. 100μL of the above five concentrations of PPy@PVA-67000 solution was added to the 96-well plate to replace the complete culture medium and incubated for 24 hours and 48 hours, respectively. CCK-8 reagent was added to replace the PPy@PVA-67000 solution and incubated for 1 hour. The absorbance at 450nm was detected with a microplate reader and the cell viability was calculated. As shown in Figure 5, in addition to different concentrations of PPy@PVA-67000 solution, four commonly used cell cryopreservation agent components: 10% DMSO, 10% glycerol, 50 mg / mL polyethylene glycol and 20 mg / mL mannitol were taken and diluted with the basic culture medium DMEM low glucose as the control group. The PPy@PVA-67000 solution showed the highest cell survival rate, indicating that the PPy@PVA-67000 solution has the lowest cytotoxicity.

[0055] Example 6

[0056] The PPy@PVA-67000 solution prepared in Example 1 was used to measure the cooling rate in liquid nitrogen using a thermocouple. The four volumes of PPy@PVA-67000 solution, each with a concentration of 50 μg / mL and in volumes of 0.5 mL, 1.0 mL, 1.5 mL, and 2.0 mL, were added to a confocal dish with a bottom diameter of 35 mm. The dish was then placed in an insulated container and liquid nitrogen was added to rapidly cool the solution in the dish. As shown in Figure 6, larger solution volumes resulted in slower cooling rates. The highest cooling rate reached over 100°C / min.

[0057] Example 7

[0058] Take the PPy@PVA-67000 solution prepared in Example 1 and perform cryopreservation on RSC96 cells. First, incubate RSC96 cells on a 35×20mm confocal dish (NEST, 801001) until they adhere to the wall. Add a 50μg / mL PPy@PVA-67000 solution diluted with basal medium DMEM low sugar to the confocal dish to replace the complete medium. Place the confocal dish in an insulated container and add liquid nitrogen to cool it down quickly. When the temperature drops to the lowest and maintains equilibrium, use a wavelength of 808nm and an optical density of 1W / cm 2The cells were irradiated with a near-infrared laser and rewarmed in a water bath (37°C). The cells were then stained with rhodamine phalloidin and DAPI, and images were taken using a laser confocal microscope. The control groups included intact RSC96 cells and RSC96 cells cryopreserved without the PPy@PVA-67000 solution. As shown in Figures 7a and 7b, the cell membrane of RSC96 cells cryopreserved without the PPy@PVA-67000 solution was completely ruptured, and the cytoskeleton was damaged. As shown in Figure 7c, the cell membrane of RSC96 cells cryopreserved with the PPy@PVA-67000 solution was damaged, but the cytoskeleton was preserved.

[0059] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and these variations and improvements fall within the scope of the present invention. The scope of the present invention shall be determined by the appended claims.

[0060] Industrial Applications

[0061] The present invention provides the use of ice-inhibiting polymer composite conductive polymer nanoparticles in cell cryopreservation. The cell cryopreservative is simple and quick to prepare, has low cytotoxicity, can effectively inhibit ice crystal growth at relatively low concentrations, and can also utilize the high absorption rate of the conductive polymer in the near-infrared light zone I to achieve rapid and uniform heating of the frozen cells during thawing. As a container for cell cryopreservation, the confocal dish can greatly increase the heating and cooling rates of the frozen cells, allowing them to quickly pass through the freezing temperature range and ensure a high recovery rate of cells after freezing and thawing. It has broad application prospects in the field of cell cryopreservation.

Claims

1. Application of ice-inhibiting polymer composite conductive polymer nanoparticles in the preparation of cell cryopreservation agents; The ice-suppressing polymer composite conductive polymer nanoparticles include an ice-suppressing polymer and a conductive polymer, wherein: The ice-inhibiting polymer is used as a dispersion liquid of the conductive polymer.

2. The use according to claim 1, characterized in that: The ice-inhibiting polymer is selected from at least one of the following: polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol; Alternatively, the conductive polymer is selected from at least one of the following: polypyrrole, polyacetylene, polyaniline, polythiophene, and polyphenylene vinylene.

3. The use according to claim 1 or 2, characterized in that: The ice-suppressing polymer composite conductive polymer nanoparticles are prepared according to a method comprising the following steps: dissolving the ice-suppressing polymer in deionized water at 65-85° C., adding an oxidant to the ice-suppressing polymer solution and stirring after cooling to room temperature; after balancing for 45-90 minutes, adding a monomer for forming a conductive polymer, and reacting at 4-7° C. for 4-6 hours; after the reaction is completed, centrifuging to obtain the ice-suppressing polymer composite conductive polymer nanoparticles.

4. A cell cryopreservation agent, comprising the ice-suppressing polymer composite conductive polymer nanoparticles according to any one of claims 1 to 3 and a cell basal culture medium.

5. The cell cryopreservation agent according to claim 4, characterized in that: In the cell cryopreservation agent, the concentration of the ice-inhibiting polymer composite conductive polymer nanoparticles is 0.1-100 μg / mL, preferably 50-100 μg / mL; Alternatively, the cell basal culture medium does not contain serum or human albumin.

6. A method for cell cryopreservation, comprising the following steps: co-incubating the cell cryopreservation agent according to claim 4 or 5 with cells to be frozen in a confocal dish, and cryopreserving the cells by rapid freezing with liquid nitrogen.

7. The method according to claim 6, characterized in that: The ratio of the cell cryopreservation agent to the cells to be frozen is: 1 ml of cell cryopreservation is used for 10 6 cells.

8. A cryopreserved cell obtained by cryopreserving the cell using the method of claim 6 or 7.

9. A method for cryo-thawing the frozen cells according to claim 8, comprising irradiating the frozen cells with near-infrared laser combined with water bath heating to thaw the frozen cells.

10. The method according to claim 9, characterized in that: The near-infrared laser has a wavelength of 780-1100 nm and an optical density of 0.5-2 W / cm 2 .

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