Nano cryopreservation agent with ice suppression, magnetocaloric and photo-thermal effects and preparation method and application of nano cryopreservation agent with ice suppression, magnetocaloric and photo-thermal effects

Abstract

The invention discloses a nano cryopreservation agent with ice suppression, magnetocaloric and photo-thermal effects as well as a preparation method and application thereof, and belongs to the field of biomedicine. The nano cryopreservation agent is obtained by modifying magnetic microparticles with ice-inhibiting macromolecules and photo-thermal conductive macromolecules through ultrasonic and in-situ chemical oxidation polymerization strategies. The nano cryoprotectant has good solution stability and biocompatibility, has excellent ice recrystallization inhibition capability even at low concentration, and can effectively replace and reduce the use of a permeable cryoprotectant. Besides, a nanometer heating technology is developed on the basis of the ice suppression-magnetocaloric-photo-thermal composite nanometer material, and by means of magnetocaloric / photo-thermal synergistic heating, ultra-fast rewarming is achieved within a wide volume range, and the limitation that uniform heating cannot be achieved through traditional convection heating is broken through.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a nano cryopreservation agent with triple effects of ice suppression, magnetic heat and photothermal, as well as a preparation method and application thereof. Background Art

[0002] Cryopreservation involves preserving cells, tissues, organs, and even entire organisms at extremely low temperatures (<-80°C), essentially halting biochemical reactions in biological samples and enabling long-term storage and long-distance transport. Therefore, cryopreservation technology is fundamental for the translation of biopharmaceutical and tissue engineering technologies into clinical applications and is an indispensable component of the biopharmaceutical industry. Vitrification is considered one of the most effective methods, but several challenges remain. First, both the freezing and thawing processes cannot avoid severe physical damage caused by uncontrolled ice crystal nucleation. Second, high concentrations of permeable cryoprotectants (CPAs) can cause irreversible chemical damage to cells and tissues. Furthermore, traditional convection heating cannot achieve uniform heating within the tissue, resulting in not only localized thermal stresses that can cause cracks but also glassy recrystallization (Ostwald ripening), which can produce large amounts of ice crystals and cause severe, irreversible mechanical damage.

[0003] Currently, there are two main strategies for ice suppression during cryopreservation: (1) inhibiting the formation and growth of ice crystals by adding cryopreservatives and adjusting the cooling rate; and (2) controlling the heating rate and changing the heating method to inhibit recrystallization and the generation of local thermal stress during the warming recovery process. However, traditional small molecules (such as glycerol and dimethyl sulfoxide) require high concentrations to achieve the glass transition of biological samples. However, in actual use, high-concentration cryopreservation reagents can cause great damage to cell genetic material and cell function, and are difficult to remove after cryopreservation. Natural antifreeze proteins / glycoproteins developed in recent years have significant thermal hysteresis and immunogenicity, and are expensive. More importantly, existing cryopreservatives cannot effectively inhibit recrystallization and will form more destructive ice crystals inside the sample. At the same time, existing interfacial heating strategies (such as water bath heating) cannot achieve uniform and rapid heating inside the tissue, and microwave heating cannot control the heating process, resulting in overheating and genetic material denaturation. Therefore, the development of new cryoprotectants with strong ice inhibition, rapid and uniform rewarming and low biological toxicity is an inevitable trend in the current development of cryopreservation technology, and has important practical significance in the fields of biomedicine, tissue engineering, agricultural production, etc. Summary of the Invention

[0004] To address these challenges, the present invention has designed a triple-effect ice-inhibiting, magnetocaloric, and photothermal composite (VPF). This composite is synthesized using an organic / inorganic co-assembly strategy, utilizing magnetic microparticles with magnetocaloric effects and conductive polymers with photothermal effects as building blocks. This composite exhibits excellent biocompatibility, maintaining over 90% cell viability and normal physiological function at a high concentration of 10 mg / mL. Furthermore, VPF, driven by hydrogen bonding and adsorption inhibition, exhibits exceptional ice-inhibiting properties, significantly reducing the physical damage caused by ice crystal growth and recrystallization. Its recrystallization inhibition capability significantly surpasses that of other reported similar materials. Importantly, the present invention utilizes nano-warming technology based on a highly penetrating alternating magnetic field and near-infrared light for rewarming. This synergistic magnetocaloric / photothermal warming not only effectively increases the heating rate, allowing frozen samples to rapidly pass through the critical temperature zone, but also offers superior penetration and uniformity compared to convective warming. VPF achieves rapid and uniform rewarming of tissue samples over a wide volume range at a rate exceeding the critical rewarming rate.

[0005] The purpose of the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides an ice-suppressing-magnetocaloric-photothermal triple-effect composite material (VPF), which is prepared by modifying ice-suppressing polymers and photothermal conductive polymers on magnetic microparticles.

[0006] The composite material is in granular form, and the particle diameter is 10-500 nm.

[0007] The magnetic microparticles are selected from at least one of ferroferric oxide, cobalt ferrite, manganese zinc ferrite, and metal alloys, and can be specifically ferroferric oxide; The particle size of the magnetic microparticles may be 1-500 nm; The ice-inhibiting polymer is selected from at least one of polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylamide, polyacrylic acid, antifreeze protein, chitosan, chitin, gelatin, sodium alginate, and hyaluronic acid, and specifically can be polyvinyl pyrrolidone; The photothermal conductive polymer is prepared by in-situ chemical polymerization of conductive polymer monomers through an oxidant. Wherein, the conductive polymer monomer is selected from at least one of pyrrole, aniline, thiophene, acetylene, phenylacetylene, and dopamine, and specifically can be pyrrole; In the ice-suppression-magnetic-thermal-photothermal triple-effect composite material (VPF), the mass ratio of the magnetic microparticles to the ice-suppression polymer and the conductive polymer monomer can be: 1-50:10-500:1-20, specifically 4:18:3.

[0008] In a second aspect, the present invention provides a method for preparing the aforementioned ice suppression-magnetocaloric-photothermal triple-effect composite material (VPF). In the method, the ice suppression-magnetocaloric-photothermal triple-effect composite material (VPF) is synthesized using an organic / inorganic interface co-assembly strategy.

[0009] The preparation method comprises the following steps: The magnetic microparticles and ice-inhibiting polymers are modified and loaded under ultrasonic conditions, and conductive polymer monomers are added and in situ chemically polymerized with an oxidant to obtain an ice-inhibiting polymer / photothermal conductive polymer modified magnetic microparticle composite material, namely, an ice-inhibiting-magnetothermal-photothermal triple-effect composite material (VPF).

[0010] In the method, the magnetic microparticles and the ice-inhibiting polymer are modified and loaded in a solvent, and the solvent can be selected from at least one of water, methanol, ethanol, isopropanol, acetone, butanone, ethyl acetate, butyl acetate, dimethylformamide, dimethylacetamide, ionic liquid, glycerol, ethylene glycol, acetonitrile, sulfuric acid, and phosphoric acid; The concentration of the ice-inhibiting polymer is 10-500 mg / mL; The concentration of the magnetic microparticles is 1-50 mg / mL; The ultrasonic method is one of water bath ultrasonication and probe ultrasonication, or a combination of the two. The ultrasonic time can be 10-500 min and the temperature can be 0-90 ° C. The oxidant may be selected from at least one of ammonium persulfate, potassium persulfate, ferric chloride, and potassium permanganate, and may specifically be ferric chloride; The concentration of the added conductive polymer monomer can be 1-20 mg / mL, and the concentration of the oxidant can be 1-200 mg / mL; The in-situ chemical polymerization is carried out under continuous stirring, and the temperature of the in-situ chemical polymerization can be 0-37 ° C. and the time can be 1-30 h.

[0011] The method further includes: after the reaction is completed, placing the product into a dialysis bag and placing it in deionized water for dialysis, changing the water every 12 hours, and changing it 3-5 times in total; then placing it in a high-speed centrifuge and centrifuging it at a speed of 5000-13000 rpm for 8-40 minutes to obtain an ice-inhibiting polymer / photothermal conductive polymer modified magnetic microparticle composite nanomaterial.

[0012] In a third aspect, the present invention provides the use of the above-mentioned ice-suppression-magnetocaloric-photothermal triple-effect composite material (VPF) as or in the preparation of a cryopreservative.

[0013] The cryopreservative is used for cryopreservation of biological samples, wherein the biological samples are selected from the group consisting of cells, tissues, organs, whole organisms, genetic materials, proteins, and thermosensitive drugs.

[0014] In a fourth aspect, the present invention also provides a method for cryopreservation and thawing of biological samples.

[0015] The biological sample freezing and thawing method comprises the following steps: placing the biological sample to be preserved in a freezing solution containing the above-mentioned ice-inhibiting-magnetic-thermal-photothermal triple-effect composite material (VPF), freezing it by programmed cooling or rapid freezing, and storing it in liquid nitrogen for a long time; when thawing, heating the frozen biological sample under the action of photothermal and / or magnetic heat to thaw and thaw.

[0016] The photothermal and / or magnetothermal effect is achieved by heating by one or a combination of near-infrared laser irradiation (photothermal) and magnetic induction heating (magnetothermal); Furthermore, the wavelength of the near-infrared laser is 780-1100 nm and the optical power density is 0.1-8 W / cm 2 , laser irradiation area is 1-100 cm 2 ; Furthermore, in magnetic induction heating, the current of the alternating magnetic field is 1-50 A, the number of coil turns is 2-10, and the coil diameter is 2-30 cm; Furthermore, the ice suppression-magnetic heat-photothermal triple-effect composite material (VPF) can be used at a concentration of 0.001 mg / mL-50 mg / mL.

[0017] The above method further includes the operation of separating the VPF from the biological sample using a magnetic field after the rewarming is completed to obtain the thawed and revived biological sample.

[0018] The biological sample is selected from the group consisting of: cells, tissues, organs, whole organisms, genetic materials, proteins, and thermosensitive drugs.

[0019] Compared with existing cryopreservation technology, the present invention has the following beneficial effects: The present invention provides a novel cryopreservative that combines the triple effects of ice inhibition, magnetothermal and photothermal. The material has good stability and biocompatibility, and has excellent ice recrystallization inhibition ability even at low concentrations, which can effectively replace and reduce the use of penetrating cryoprotectants. More importantly, the present invention provides a nano-warming technology based on near-infrared laser irradiation and magnetic induction heating. Based on the above-mentioned ice inhibition, magnetothermal and photothermal composite nanomaterials, magnetothermal / photothermal synergistic warming not only achieves ultra-fast rewarming, but also has penetration and uniformity that are difficult to match with traditional convection warming. For biological samples in a wide volume range, nano-warming can achieve rapid and uniform rewarming at a heating rate higher than the critical rewarming rate. In addition, after freezing, the nanomaterial can be quickly separated from the sample by simple magnetic attraction. Therefore, this novel cryopreservative combined with nano-warming technology can not only effectively inhibit the formation and growth of ice crystals during the freezing process, but also quickly and evenly allow frozen samples to pass through the dangerous temperature zone, thereby effectively improving the freezing efficiency. It is applicable to various biological samples such as cells, tissues, and organs, providing a new reference for the development of cryopreservation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Scanning electron microscope image (a), Fourier transform infrared spectrum (b) and X-ray diffraction spectrum (c) of the ice-inhibiting polymer / photothermal conductive polymer modified ferrosoferric oxide composite nanomaterial (PVP / PPy@Fe3O4) prepared in Example 1 of the present invention.

[0021] Figure 2 The photothermal performance of the nano cryopreservative prepared in Example 1 of the present invention includes the photothermal performance of the nano cryopreservative dispersion at 1 W / cm 2 (a), 2 W / cm 2 (b) and 3 W / cm 2 (c) Temperature rise curve under laser power density, (d) Temperature rise curve of 0.3% wt nanocryopreservative dispersion under different light power densities, (e) Temperature change of pure water and nanocryopreservative dispersion after cyclic switching of laser, and (f) Cyclic stability of nanocryopreservative photothermal.

[0022] Figure 3 The magnetocaloric properties of the nanocryopreservative prepared in Example 1 of the present invention, including the heating curves of the nanocryopreservative dispersion under magnetic field currents of 10 A (a), 20 A (b), 30 A (c), and 40 A (d) and the corresponding heating rates (f), and (e) the temperature change of a 0.3% wt nanocryopreservative dispersion under different magnetic field currents.

[0023] Figure 4The magnetothermal / photothermal synergistic heating performance of the nano cryopreservative prepared in Example 1 of the present invention includes (a) photothermal (3 W / cm 2 ), the heating rates of magnetocaloric (30 A) and synergistic heating, (b) the heating curve of 0.3% wt cryopreservative solution thawing from -196 °C, and (c) the heating rates of cryopreservative solution with 0.3% wt, 0.5% wt, and 1% wt nanocryopreservative concentrations thawing from -196 °C.

[0024] Figure 5 The ice inhibition performance of the nano-cryopreservative prepared in Example 1 of the present invention, including an ice crystal image (a) of the nano-cryopreservative dispersion after annealing for 30 minutes and a corresponding ice crystal size change curve (b).

[0025] Figure 6 The biocompatibility of the nano cryopreservative prepared in Example 1 of the present invention, including its cytotoxicity to SH-SY5Y cells (a) and BEAS2B cells (b).

[0026] Figure 7 The application of the nano cryopreservative prepared in Example 1 of the present invention for vitrification and cryopreservation of porcine trachea, including (a) a schematic diagram of the cryopreservation process, (b) a loading method of the tracheal cryoprotectant, (c) a schematic diagram of tracheal stretching, (d) an evaluation of the tracheal stretching curve, and the "Toe region" length (e) and compression curve (f) and compression modulus (g) of the trachea under different vitrification and warming schemes.

[0027] Figure 8 Application of the nanocryopreservative prepared in Example 1 of the present invention for single-cell cryopreservation, including: (a) immediate survival rate of HUVEC, SH-SY5Y, and mesenchymal stem cells after thawing; (b) percentage area and number of HUVEC vascularized tubes; (c) relative osteogenic differentiation rate of mesenchymal stem cells; (d, e) dry weight and differentiated neurite length of SH-SY5Y cells; (f) fluorescence / optical images of HUVEC vascularization, mesenchymal stem cell osteogenesis, and SH-SY5Y cell differentiation.

[0028] Figure 9 Scanning electron microscope image (a), DLS hydrated particle size distribution (b), TGA (c) and DTG (d) curves of the CS / PPy@Fe3O4 ice-inhibiting-magnetocaloric-photothermal triple-effect nano cryopreservation agent prepared in Example 9 of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] Polyvinylpyrrolidone K88-96 (P816208), pyrrole (P915945), and anhydrous ferric chloride (I811935) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Chitosan (M29339-500G) was purchased from Shanghai Myerui Chemical Technology Co., Ltd. Dimethyl sulfoxide (D119415), propylene glycol (P770936), ethylene glycol (E103322), and ferrosoferric oxide (I140494) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. SH-SY5Ys and BEAS-2B cells were purchased from Beyotime Biotech, USA. HUVECs, mesenchymal stem cells, and erythrocytes were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0032] Example 1. Preparation of PVP / PPy@Fe3O4 ice suppression-magnetic heat-photothermal triple-effect nano cryopreservation agent Weigh 0.4 g of ferroferric oxide nanoparticles (particle size 5-20 nm) and 1.8 g of polyvinyl pyrrolidone (molecular weight 1300 kDa) into a conical flask, add 50 mL of deionized water, place it in a water bath ultrasound, and use a mechanical stirrer to continuously stir and react for 2 hours. Then transfer the conical flask to an ice water bath (0-4 ° C), add 0.3 mL of pyrrole monomer after cooling, stir for 10 minutes, and then add 10 mL of oxidant solution containing 2.14 g of anhydrous ferric chloride. React for 10 hours, and keep mechanical stirring during the whole process. After the reaction is completed, transfer the solution to a dialysis bag and place it in deionized water for dialysis. Change the water every 12 hours for three days. Finally, place the obtained solution in a high-speed centrifuge and centrifuge at 7000 rpm for 10 minutes to obtain an ice-inhibiting, magnetic-thermal, and photothermal triple-effect nano-cryopreservative. Scanning electron microscopy shows that the synthesized nanomaterial is a spherical particle with uniform particle size ( Figure 1 Infrared and XRD also show that the ice-suppressing polymer and the conductive polymer are successfully modified on the ferroferric oxide ( Figure 1 b, c).

[0033] Example 2: Photothermal performance of PVP / PPy@Fe3O4 ice suppression-magnetic heat-photothermal triple-effect nano cryopreservation agent The photothermal performance of the nanomaterial prepared in Example 1 was tested. A 1 mL volume of nano-freezing agent aqueous dispersion was irradiated with a near-infrared laser of 808 nm wavelength, and the temperature change of the solution was monitored with an ultra-sensitive thermocouple. Figure 2 The results of the nano-cryopreservative dispersions with different concentrations (0.01%, 0.1%, 0.3%, 0.5%, 1% wt) at 1 W / cm 2 , 2W / cm 2 and 3 W / cm 2 Temperature rising curve under laser power density. Figure 2 Figure d compares the temperature rise data for a 0.3% wt nanocryogenic agent dispersion at different optical power densities. The results show that the temperature rise rate increases with increasing particle concentration and optical power density, reaching a maximum rate of 40 °C / min. Notably, when the particle concentration exceeds 0.1%, the temperature rise rate reaches saturation and remains essentially constant. Figure 2 Figure e records the temperature changes of pure water and nano-cryopreservative dispersion (0.3% wt) after cycling the laser on and off. It can be seen that the nano-cryopreservative dispersion absorbs near-infrared light significantly more than pure water, and the heating rate is greatly accelerated. In addition, Figure 3 Figure f demonstrates the photothermal cyclic stability of the nanocryopreservative.

[0034] Example 3. Magnetothermal properties of PVP / PPy@Fe3O4 ice suppression-magnetothermal-photothermal triple-effect nano cryopreservation agent The magnetocaloric performance of the nanomaterial prepared in Example 1 was tested using a high-frequency magnetic induction device, and the temperature change during the entire magnetocaloric process was monitored using a fluorescent fiber optic thermometer. Figure 3 In Figures ad, the heating curves of aqueous dispersions of nanocryopreservatives with different concentrations (0.1 mg / mL, 1.0 mg / mL, 3.0 mg / mL, 5.0 mg / mL, and 10 mg / mL) were measured under magnetic field currents of 10 A, 20 A, 30 A, and 40 A, respectively, and the dispersion volume was 0.8 mL. Figure 3 Figure e compares the temperature changes of 3.0 mg / mL nano cryopreservative dispersion under different magnetic field currents. Figure 3 The corresponding heating rates are calculated in Figure 1. The data show that the heating rate increases with increasing concentration and magnetic field current, reaching a maximum of 43 °C / min.

[0035] Example 4: Magnetothermal / photothermal synergistic heating performance of PVP / PPy@Fe3O4 ice suppression-magnetothermal-photothermal triple-effect nano cryopreservation agent The photothermal platform and magnetothermal platform of Example 2 and Example 3 were integrated to build a magnetothermal / photothermal synergistic heating integrated platform to test the synergistic heating ability of the nano-cryopreservative. Figure 4 Figure a records the heating rates of the nanocryogenic dispersions at room temperature using photothermal, magnetothermal, and synergistic heating, with a sample concentration of 0.3% wt and an optical power density of 3 W / cm 2 , the magnetic field current is 30 A. It can be seen that the heating rate of the synergistic heating is almost equal to the sum of the heating rates of the individual magnetic heating and photothermal heating, which proves the superiority of the synergistic heating. In addition, Figure 4 Figure b shows the warming curve of the cryopreservative solution containing 0.3% wt nano-cryopreservative recovered from -196 °C. The synergistic heating rate is much higher than the critical heating rate, which can greatly inhibit the recrystallization of ice crystals. Figure 4 Figure c records the heating rate of the cryopreservative solution containing 3.0 mg / mL, 5.0 mg / mL, and 10 mg / mL nanocryopreservative concentrations starting from -196 °C. The maximum heating rate can reach 395 °C / min. The cryopreservative solution was prepared using commercial VS55 vitrification solution as the solvent.

[0036] Example 5: Ice suppression performance of PVP / PPy@Fe3O4 triple-effect nano cryopreservation agent: ice suppression-magnetic heat-photothermal The ice inhibition performance of the nano-cryopreservative prepared in Example 1 was tested. The specific testing procedure was as follows: 10 μL of the solution was dropped onto a liquid nitrogen-precooled silicon wafer at a height of 1.8 m for rapid vitrification. The wafer was then transferred to a -6°C environment for annealing. The size of the ice crystals was recorded every 5 minutes using a polarizing microscope for 30 minutes. Figure 5 Figure a is a picture of ice crystals in aqueous dispersions containing 0, 10, 50, 100, and 200 μg / mL nano cryopreservatives after annealing for 30 min. Figure 5 The corresponding quantitative results are shown in Figure b. It can be seen that the nanocryopreservative almost completely inhibited ice crystal recrystallization at a concentration of 100 µg / mL, with the ice crystal size barely changing within 30 minutes. At a concentration of 200 µg / mL, the recrystallization inhibition rate exceeded 90%.

[0037] Example 6. Biocompatibility of PVP / PPy@Fe3O4 ice suppression-magnetic heat-photothermal triple-effect nano cryopreservation agent The biocompatibility of the nanocryopreservative prepared in Example 1 was tested. Cytotoxicity was determined using the commonly used CCK8 assay. Specifically, the nanocryopreservative was diluted with complete culture medium to different concentrations, up to 10 mg / mL, and incubated for 24 and 48 hours, respectively. Cell viability was then determined using CCK8. Figure 6Figures a and b respectively measured the cytotoxicity of the nanocryopreservative on SH-SY5Y cells and BEAS2B cells. It can be seen that after two days of incubation at the highest concentration of 10 mg / mL, the viability of both cell types can be maintained above 90%, demonstrating the good biocompatibility of the nanocryopreservative.

[0038] Example 7: PVP / PPy@Fe3O4 ice suppression-magnetic heat-photothermal triple-effect nano cryopreservation agent for vitrification of pig trachea The above nano cryopreservation agent and the corresponding nano warming technology were used to cryopreserve pig trachea. The pig trachea was purchased from a local slaughterhouse for slaughter surplus, without intentionally killing the animals. The specific process is as follows ( Figure 7 In a), commonly used VS55 and VS83 were used as vitrification solutions to disperse the nano-cryopreservative prepared in Example 1, and the nano-cryopreservative solutions were prepared at concentrations of 3 mg / mL and 5 mg / mL. Fresh pig trachea purchased from a slaughterhouse was flushed with buffer and then gradient-loaded with vitrification solutions (referring to VS55 and VS83 vitrification solutions, excluding the nano-cryopreservative prepared in Example 1) ( Figure 7 b). After loading the trachea with a 100% concentration of a vitrification solution containing a nano-cryopreservative (VS55 and VS83 vitrification solutions), the trachea was transferred to a vitrification solution containing a nano-cryopreservative. After equilibration at -20°C for 20 minutes, the trachea was transferred to liquid nitrogen for vitrification and long-term storage. During thawing, the vitrified trachea was placed on the magnetothermal / photothermal synergistic heating platform described in Example 4 for heating and rewarming. Magnetothermal heating was performed using a high-frequency magnetic induction heating device with a six-turn coil, a coil diameter of 10 cm, a current of 30 A, and photothermal heating using near-infrared light with a wavelength of 808 nm and a light power density of 3 W / cm. 2 The nano cryopreservative concentrations were 3 mg / mL and 5 mg / mL. The cryopreserved trachea were then evaluated. Figure 7 Figures c and d show the test schematic and evaluation schematic of tracheal tensile properties. Figure 7 The tensile results in Figure e show that the tensile properties of the trachea obtained by nano-heating and reheating are similar to those of the 37°C water bath reheating, and are significantly lower than those of the slow heating (thawed in air), proving that the vascularized fibrous tissue of the trachea is less damaged. In addition, Figure 7 Figures f and g record the compression curves and compression moduli of the trachea under different heating methods. It can be seen that the nano-warming group has the largest compression modulus, second only to the fresh group, proving the superiority of nano-warming technology for vitrification of pig trachea.

[0039] Example 8: PVP / PPy@Fe3O4 ice suppression-magnetic heat-photothermal triple-effect nano cryopreservation agent for single cell cryopreservation The conventional cell cryopreservation protocol was modified using VPF. Specifically, adherent cells at 80% density were digested with 0.25% trypsin and then cryopreserved at 10 7 cells mL -1 Resuspend the cells in culture medium at a density of 1 mL. Add 1 mL of cell suspension to a 1.5 mL centrifuge tube and centrifuge at 250 g for 5 minutes. Prepare 20% FBS and different concentrations of DMSO (5% and 10%) in DEME cryopreservation solution, and add different concentrations of VPF (0, 0.1, 0.5, 1, 2 mg / mL). Resuspend the cells in a centrifuge tube with 1 mL of freezing solution and transfer to a 1.2 mL cryovial. Then, freeze in a refrigerator at 1 °C for 1 min. -1 The cryovials were cooled to -80 °C at a constant cooling rate and, after equilibration for 24 hours, transferred to liquid nitrogen for storage. Based on laser irradiation and alternating magnetic fields, PMH was used to improve the recovery process. The cryovials were placed in a 37 °C water bath and subjected to a near-infrared laser field and high-frequency AMF until the cell suspension was completely thawed. VPF were separated from the cells using a magnet. The cells were centrifuged and resuspended in culture medium for subsequent experiments. Figure 8 Figure a shows the survival rates of HUVEC, SH-SY5Y and MSC cells after freezing. Using 5% DMSO as CPA, the cell survival rates were significantly improved in the presence of VPF, photothermal and magnetic thermal. Figure 8 Middle b records the vascularization area and number of HUVECs after freezing, which are significantly higher than those in the 10% DMSO group and close to those of fresh cells. Figure 8 Figure c records the relative osteogenic differentiation rate of mesenchymal stem cells, which reached 84.6% compared with the fresh group, and its differentiation efficiency was more than twice that of the control group. Figure 8 Figures d and e recorded the dry weight and differentiated axon length of SH-SY5Y cells after cryopreservation, which were significantly higher than those of the control group. Figure 8 Middle (f) shows the fluorescence / optical images of HUVEC angiogenesis, mesenchymal stem cell osteogenesis, and SH-SY5Y cell differentiation.

[0040] Example 9. Preparation of CS / PPy@Fe3O4 ice suppression-magnetic heat-photothermal triple-effect nano cryopreservation agent Weigh 1.0 g of chitosan (molecular weight 150 kDa) into an Erlenmeyer flask and add ferroferric oxide nanoparticles (5-20 nm) at a 1:2, 1:1, and 3:2 ratio of pyrrole mass (0.3 mL). Add 50 mL of deionized water and 0.5 mL of glacial acetic acid. Place the mixture in a water bath for sonication and allow to react for 2 hours with a mechanical stirrer. The flask is then transferred to an ice-water bath (0-4°C). After cooling, 0.3 mL of pyrrole monomer is added. Stir for 10 minutes, followed by 10 mL of an oxidant solution containing 2.14 g of anhydrous ferric chloride. The reaction is continued for 10 hours, with mechanical stirring maintained throughout. Upon completion of the reaction, the solution is transferred to a dialysis bag and dialyzed against deionized water, changing the water every 12 hours for three days. Finally, the resulting solution was placed in a high-speed centrifuge and centrifuged at 7000 rpm for 10 min to obtain the CS / PPy@Fe3O4 ice suppression-magnetic heat-photothermal triple-effect nano cryopreservation agent. Scanning electron microscopy showed that the synthesized nanomaterials were spherical with uniform particle size ( Figure 9 In (a), DLS results demonstrate that the nanoparticles are monodisperse and have a narrow hydrated particle size distribution ( Figure 9 (b) Thermogravimetric (TGA) and DTG results show that the composition ratios of different synthesized CS / PPy@Fe3O4 nanoparticles are different, and the content of Fe3O4 and PPy can be adjusted according to the actual application scenario. ( Figure 9 (c, d).

[0041] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. An ice-suppression-magnetocaloric-photothermal triple-effect composite material, which is prepared by modifying ice-suppression polymers and photothermal conductive polymers on magnetic microparticles.

2. The ice suppression-magnetic thermal-photothermal triple-effect composite material according to claim 1, characterized in that: The magnetic microparticles are selected from at least one of ferroferric oxide, cobalt ferrite, manganese zinc ferrite, and metal alloys; The ice-inhibiting polymer is selected from at least one of polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylamide, polyacrylic acid, antifreeze protein, chitosan, chitin, gelatin, sodium alginate, and hyaluronic acid; The photothermal conductive polymer is prepared by in-situ chemical polymerization of conductive polymer monomers through an oxidant. Wherein, the conductive polymer monomer is selected from at least one of pyrrole, aniline, thiophene, acetylene, phenylacetylene, and dopamine.

3. The ice suppression-magnetic thermal-photothermal triple-effect composite material according to claim 2, characterized in that: In the ice-suppression-magnetic-thermal-photothermal triple-effect composite material, the mass ratios of the magnetic microparticles to the ice-suppression polymer and the conductive polymer monomer are: 1-50:10-500:1-20.

4. A method for preparing the ice-suppressing-magnetocaloric-photothermal triple-effect composite material according to any one of claims 1 to 3, comprising the following steps: modifying and loading magnetic microparticles with ice-suppressing polymers under ultrasonic conditions, adding conductive polymer monomers and performing in-situ chemical polymerization with an oxidant to obtain an ice-suppressing polymer / photothermal conductive polymer modified magnetic microparticle composite material, that is, an ice-suppressing-magnetocaloric-photothermal triple-effect composite material.

5. The method according to claim 4, characterized in that The magnetic microparticles and the ice-inhibiting polymer are modified and loaded in a solvent, wherein the solvent is selected from at least one of water, methanol, ethanol, isopropanol, acetone, butanone, ethyl acetate, butyl acetate, dimethylformamide, dimethylacetamide, ionic liquid, glycerol, ethylene glycol, acetonitrile, sulfuric acid, and phosphoric acid; The concentration of the ice-inhibiting polymer is 10-500 mg / mL; The concentration of the magnetic microparticles is 1-50 mg / mL; The ultrasonic method is water bath ultrasonication, probe ultrasonication, or a combination of the two. The ultrasonic time is 10-500 min and the temperature is 0-90 °C.

6. The method according to claim 4, characterized in that The oxidant is selected from at least one of ammonium persulfate, potassium persulfate, ferric chloride, and potassium permanganate; The concentration of the added conductive polymer monomer is 1-20 mg / mL, and the concentration of the oxidant is 1-200 mg / mL; The in-situ chemical polymerization is carried out under continuous stirring, the temperature of the in-situ chemical polymerization is 0-37 ° C, and the time is 1-30 h.

7. The method according to claim 4, characterized in that The method further comprises: after the reaction is completed, placing the product into a dialysis bag and placing it in deionized water for dialysis, changing the water every 12 hours, and changing it 3-5 times in total; then placing it in a high-speed centrifuge and centrifuging it at a speed of 5000-13000 rpm for 8-40 minutes to obtain an ice-inhibiting polymer / photothermal conductive polymer modified magnetic microparticle composite material; The obtained ice-inhibiting polymer / photothermal conductive polymer modified magnetic microparticle composite material is in granular form with a particle diameter of 10-500 nm.

8. Use of the ice-suppression-magnetic-thermal-photothermal triple-effect composite material according to any one of claims 1 to 3 as or in the preparation of a cryopreservative, wherein the cryopreservative is used for the cryopreservation of biological samples, wherein: The biological sample is selected from the group consisting of: cells, tissues, organs, whole organisms, genetic materials, proteins, and thermosensitive drugs.

9. A method for freezing and thawing biological samples, comprising the following steps: placing the biological sample to be preserved in a freezing solution containing the ice-inhibiting-magnetic-thermal-photothermal triple-effect composite material according to any one of claims 1 to 3, freezing it by programmed cooling or rapid freezing, and storing it in liquid nitrogen for a long term; when thawing, heating the frozen biological sample under the action of photothermal and / or magnetic heat to thaw it.

10. The method according to claim 9, characterized in that The photothermal and / or magnetothermal effect is achieved by heating by one of near-infrared laser irradiation and magnetic induction heating or a combination of both; The wavelength of the near-infrared laser is 780-1100 nm, and the optical power density is 0.1-8 W / cm 2 , laser irradiation area is 1-100 cm 2 ; In magnetic induction heating, the current of the alternating magnetic field is 1-50 A, the number of coil turns is 2-10, and the coil diameter is 2-30 cm; The ice suppression-magnetic heat-photothermal triple-effect composite material is used at a concentration of 0.001 mg / mL-50 mg / mL; The method further includes the operation of separating the ice suppression-magnetic heat-photothermal triple-effect composite material from the biological sample using a magnetic field after the rewarming is completed to obtain a thawed and revived biological sample.

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