Use of cellulose-based materials as biological cryoprotective agents

Abstract

The application discloses application of a cellulose-based material as a biological cryoprotectant and relates to the technical field of materials. The cellulose-based material with an average polymerization degree ranging from 2 to 1000 and a surface charge is used as the biological cryoprotectant, so that the cellulose-based material can be combined on an ice crystal surface, the growth of the crystal surface is inhibited, and the movement of water between the crystal boundaries is inhibited, so that the effect of modifying the ice crystal morphology, inhibiting the growth of the ice crystal and inhibiting recrystallization is achieved, the high-efficiency antifreezing effect is achieved at a low concentration, and the cellulose-based material has great potential in the fields of cryopreservation of cells, vaccines and organs.

Description

Technical Field

[0001] This invention relates to the field of materials technology, and in particular to the application of cellulose-based materials as biological cryoprotectants. Background Technology

[0002] Cryopreservation technology significantly slows down or stops intracellular chemical and biological reactions by lowering temperatures, enabling long-term preservation of organisms. However, in extreme low-temperature environments, multiple stresses such as ice crystal formation, osmotic pressure changes, and temperature stress can lead to cell damage. Effectively inhibiting ice crystal growth and recrystallization is key to improving cryopreservation performance. Currently, the most common method is to add osmotic cryoprotectants, such as dimethyl sulfoxide and glycerol, to reduce ice formation. However, these often exhibit biotoxicity and require high concentrations to achieve the desired effect. Further research has revealed that antifreeze proteins (AFPs) can modify ice crystal morphology, significantly inhibiting ice crystal growth and recrystallization, and possessing cryopreservation activity. However, the preparation of antifreeze proteins (AFPs) is complex, costly, and unstable, limiting their application in industrial fields.

[0003] To overcome the above problems, some artificially synthesized biomimetic materials for AFPs have been prepared, including some nanomaterials and polymers such as graphene oxide and polyvinyl alcohol, but they still have problems such as poor biocompatibility. Summary of the Invention

[0004] The purpose of this invention is to provide the application of cellulose-based materials as biological cryoprotectants to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] Cellulose is a widely distributed substance in nature. It is abundant, renewable, and has the characteristics of low cost, biodegradability, and good biocompatibility. It is widely used in fields such as biology, food, and medicine.

[0007] This invention provides an application of a cellulose-based material as a biological cryoprotectant, wherein the cellulose-based material has an average degree of polymerization ranging from 2 to 1000 and is surface-charged.

[0008] Furthermore, the cellulose-based material includes cellulose, 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) modified cellulose, phosphorylated cellulose, sulfated cellulose, carboxymethylated cellulose, tetramethylpiperidine oxide (TEMPO) oxidized cellulose, nitrocellulose, citric acid modified cellulose, acrylic acid modified cellulose, or pyridine modified cellulose.

[0009] Furthermore, during application, the concentration of cellulose-based material in the biological cryoprotectant is 0.1-100 mg / ml, more preferably 1-15 mg / ml.

[0010] The present invention also provides a biological cryoprotectant comprising a cellulose-based material; the cellulose-based material having an average degree of polymerization ranging from 2 to 1000 and a surface charge.

[0011] Furthermore, the aforementioned biological cryoprotectants also include dispersants.

[0012] Further, the dispersant comprises one or more of the following: pure water, phosphate buffer (PBS), citrate buffer, borate buffer, 0.9% sodium chloride solution, glycine buffer, tris(hydroxymethyl)aminomethane buffer, 2-(N-methyl)-diaminoethane sulfonic acid buffer, N-hydroxyethyl-1,3-propanediamine-N,N,N'-triacetic acid buffer, triacetate buffer, piperazine sulfonic acid buffer, N-(tris(hydroxymethyl)-glycine) buffer, or 3-(N-methyl)-1-propanesulfonic acid buffer.

[0013] Furthermore, the aforementioned biological cryoprotectants also include additives.

[0014] Furthermore, the additives include one or more of the following: dimethyl sulfoxide, glycerol, mannitol, D-mannitol, gelatin, glucose, trehalose, sucrose, propylene glycol, ethylene glycol, polyethylene glycol, glycine, bovine serum albumin (BSA), magnesium sulfate, L-cysteine, or zinc sulfate.

[0015] This invention discovers that using specific types of cellulose-based materials as cryogenic agents can modify ice crystal morphology, inhibit ice crystal growth, and inhibit recrystallization, thereby increasing the survival rate of cells after cryopreservation by more than 2 times and effectively improving the activity of vaccines and organ status after cryopreservation.

[0016] This invention reveals that, compared with commonly used organic antifreeze agents, cellulose-based antifreeze agents are simple, rapid, and economical to prepare, require low concentrations, can use different cellulose materials as needed, and have good biocompatibility and biosafety.

[0017] The present invention discloses the following technical effects:

[0018] This invention uses a specific type of cellulose-based material as a biological cryoprotectant, which can bind to the surface of ice crystals, inhibiting the growth of the crystal surface and the movement of water between crystal boundaries, thereby achieving the effects of modifying the morphology of ice crystals, inhibiting ice crystal growth and recrystallization, and achieving a highly efficient antifreeze effect at low concentrations.

[0019] When the cellulose-based materials of the present invention are applied to biological cryoprotectants, different average degrees of polymerization, different surface charges, and different chemical groups can be used to modify cellulose materials as needed to achieve different antifreeze effects, which has great potential in the fields of cryopreservation of cells, vaccines, and organs. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a flowchart illustrating the preparation process of a typical cellulose-based antifreeze agent according to the present invention.

[0022] Figure 2 The images show the morphology of a single ice crystal before and after modification with cellulose-based materials as described in Examples 1 (Precipitate B), 3, 4, 5, 6, 1 (Comparative Example), and 2 (Comparative Example).

[0023] Figure 3 Comparison of growth rate curves of cellulose-based antifreeze aqueous dispersion and pure water system under different supercooling conditions in Examples 3, 4 and 5 (10 mg / ml).

[0024] Figure 4 The image shows the recrystallization morphology of ice crystals in Examples 1-5 at a concentration of 10 mg / ml (a) and the quantitative determination results of the recrystallized crystal size (b).

[0025] Figure 5 The graph shows the cell viability of HeLa cells after being stored at -80℃ and -196℃ for one day using different cell cryopreservation solutions and then thawed.

[0026] Figure 6 The average fluorescence intensity of the mRNA-LNP vaccine after cryopreservation at 4 / -20℃ with + / -10 mg / ml cellulose-based cryoprotectant.

[0027] Figure 7 Scanned images of mouse kidney sections stained with H&E(ac) and terminal deoxynucleotidyl transferase dUTP nick end marker (TUNEL)(df) after freezing for 3 days in water, UW solution and UW solution containing 10 mg / mL MCOs at -10°C. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1

[0034] Preparation of cellulose-based antifreeze agents:

[0035] 1) Add 20g of α-cellulose to a beaker containing 380g of 83% phosphoric acid while stirring. Dissolve the solution at 60°C for 5 minutes, stirring with a glass rod. Repeat this process several times until the solution becomes clear and transparent.

[0036] 2) Hydrolyze the cellulose in a 60℃ oven for 8 hours to obtain a hydrolyzed cellulose phosphoric acid solution;

[0037] 3) The hydrolyzed cellulose phosphoric acid solution was mixed with an equal mass of water, stored at 4°C for 24 hours, and then centrifuged to obtain precipitate A. The supernatant was mixed with three times the volume of isopropanol, stored at 4°C for 24 hours, and then centrifuged to obtain precipitate B.

[0038] 4) After washing precipitates A and B to neutral, freeze-dry them. Their average degrees of polymerization are 18 and 6, respectively, and their Zeta potentials are -12.4 mV and -16.2 mV, respectively. Resuspend precipitates A and B in buffer solution respectively. After mixing, the concentration of both is 10 mg / ml, which can be used as an antifreeze agent.

[0039] Example 2

[0040] Preparation of cellulose-based antifreeze agents:

[0041] 1) Add 20g of α-cellulose to a beaker containing 200g of 96% sulfuric acid while stirring. This process is carried out in an ice bath.

[0042] 2) Hydrolyze the cellulose in an oven at 40℃ for 2 hours to obtain a hydrolyzed cellulose sulfuric acid solution;

[0043] 3) The hydrolyzed cellulose sulfuric acid solution was mixed with an equal mass of water, stored at 4°C for 24 hours, and then centrifuged to obtain precipitate A. The supernatant was mixed with three times the volume of isopropanol, stored at 4°C for 24 hours, and then centrifuged to obtain precipitate B.

[0044] 4) Wash precipitates A and B until neutral and freeze-dry them. Their average degrees of polymerization are 18 and 6, respectively, and their Zeta potentials are -20.1 mV and -17.6 mV, respectively. Resuspend precipitates A and B in buffer solution respectively. After mixing, the concentration of both is 10 mg / ml, which can be used as an antifreeze agent.

[0045] Example 3

[0046] Preparation of cellulose-based antifreeze agents:

[0047] 1) Add 11g of 77% NaOH aqueous solution to a beaker containing 1g of cellulose precipitate A (precipitate A in Example 1) with an average degree of polymerization of 18, and stir at room temperature for 30 minutes.

[0048] 2) Add 2.81 g of 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) and react for 5 hours in a water bath at 65°C;

[0049] 3) Wash with water until neutral, freeze dry to obtain the product with a Zeta potential of 11.7 mV, resuspend it in buffer, and the concentration after resuspending is 10 mg / ml, which can be used as an antifreeze (labeled as EPTMAC modified DP18).

[0050] Example 4

[0051] Preparation of cellulose-based antifreeze agents:

[0052] 1) Disperse 1g of cellulose precipitate A (precipitate A in Example 1) with an average degree of polymerization of 18 in a beaker containing 100ml of water, add 0.016g of tetramethylpiperidine oxide (TEMPO) and 0.1g of NaBr, and stir until TEMPO is completely dissolved.

[0053] 2) Add 15 mL of NaClO solution, and adjust the pH of the solution to 10 with HCl while stirring at 150 rpm;

[0054] 3) As the reaction proceeds, the pH of the system continuously decreases. Under stirring at 150 rpm, the pH is maintained at 10 with 0.5 M NaOH. The reaction is carried out for 1 hour, and then a small amount of ethanol is added to terminate the reaction.

[0055] 4) Wash the product four times by centrifugation with ethanol, dry it at 60°C to obtain the product, the zeta potential is -27.5mV, resuspend it in buffer, the concentration after resuspending is 10mg / ml, and it can be used as an antifreeze (labeled as Tempo oxidized DP18).

[0056] Example 5

[0057] Preparation of cellulose-based antifreeze agents:

[0058] 1) Add 15 mL of isopropanol and 1.5 mL of 20% NaOH aqueous solution to a capped glass bottle containing 0.5 g of cellulose precipitate A (precipitate A in Example 1) with an average degree of polymerization of 18, and stir magnetically at room temperature for 1 hour.

[0059] 2) Add 0.46g of chloroacetic acid, seal with parafilm membrane, and react at 55℃ for 3 hours;

[0060] 3) Add 30 mL of ethanol and neutralize with acetic acid until neutral;

[0061] 4) After adding 3 times the volume of isopropanol, the precipitated solid was washed 4 times by centrifugation with ethanol, dried at 60°C to obtain the product with a Zeta potential of -21.4mV. The product was resuspended in buffer solution to a concentration of 10mg / ml, which can then be used as an antifreeze agent (labeled as carboxymethylated DP18).

[0062] Example 6

[0063] Preparation of cellulose-based antifreeze agents:

[0064] High molecular weight carboxymethyl cellulose (average degree of polymerization 600, Zeta potential -59.25mV) is resuspended in buffer solution to a concentration of 10mg / ml, which can then be used as an antifreeze.

[0065] Comparative Example 1

[0066] Cellulose nanocrystals (CNC) (purchased from Tianjin Wood Elf Biotechnology Co., Ltd., model: TOCNF-PL10) were resuspended in buffer solution. After resuspending, the concentration of cellulose nanocrystals was 10 mg / ml. They could not modify the morphology of ice crystals and had no antifreeze activity.

[0067] Comparative Example 2

[0068] TEMPO oxidized cellulose nanofibers (Tempo-CNF) (purchased from Tianjin Wood Elf Biotechnology Co., Ltd., model: SCN-PS150) were resuspended in buffer solution. After resuspending, the concentration of Tempo-CNF was 10 mg / ml. It could not modify the morphology of ice crystals and had no antifreeze activity.

[0069] Example 1: Evaluation of antifreeze activity

[0070] Nanoliters of cellulose-based antifreeze were injected into the silicone oil in the sample holder using a microsyringe equipped with a capillary tube. The solution was then rapidly cooled to freeze the droplet, followed by slow heating to allow it to melt gradually. When only a small ice crystal remained, the temperature was adjusted to maintain this state for 20 seconds, preventing further growth and melting. This temperature was recorded as the melting point (Tm). The temperature was then lowered to a target temperature (Tf), which is the supercooling ΔT = Tm - Tf. The entire process was observed and recorded using a high-speed camera, monitoring the ice crystal morphology and growth rate. The more pronounced the non-circular morphology of the ice crystal and the slower its growth rate, the stronger the antifreeze activity.

[0071] Figure 2 The images show the morphology of a single ice crystal before and after modification with cellulose-based materials from Example 1 (precipitate B), Example 3, Example 4, Example 5, Example 6, Comparative Example 1, and Comparative Example 2. This demonstrates that the prepared cellulose-based antifreeze agent can be adsorbed onto the surface of ice crystals, thereby modifying the morphology of the ice crystals. In contrast, nanocellulose crystals (CNC) and TEMPO oxidized cellulose nanofibers cannot modify the morphology of ice crystals and have no antifreeze activity. Figure 3 The graphs show the growth rate curves of the aqueous dispersion and pure water system of cellulose-based antifreeze in Examples 3, 4 and 5 at different supercooling degrees, demonstrating that the addition of cellulose-based antifreeze can reduce the ice crystal growth rate.

[0072] Example 2: Evaluation of Ice Crystal Recrystallization Inhibition Activity (IRI)

[0073] A square glass slide was fixed on a cold stage, which was pre-cooled to -60°C. 10 μL of cellulose-based antifreeze (10 mg / mL) was dropped onto the slide from a height of 1.4 meters, and the temperature was rapidly raised to -10°C, then annealed at -10°C for 30 minutes. The ice crystal size was then observed and photographed using an optical microscope equipped with a digital camera (OLYMPUS, model BX63). Image processing was performed using ImageJ software, and the ten largest ice crystal sizes (longest length on any axis) were measured from each photograph. The experiment was repeated three times, and the average value was calculated to obtain the mean maximum crystal size (MLGS). A smaller MLGS indicates a stronger ability to inhibit recrystallization and higher IRI activity.

[0074] Figure 4 The images show the recrystallization morphology of ice crystals and the quantitative determination results of the recrystallized crystal size of the cellulose-based antifreeze agent in Examples 1-5 at a concentration of 10 mg / ml. Compared with the recrystallization morphology of pure water, it was found that adding a low concentration of cellulose-based antifreeze agent to water can significantly reduce the ice crystal size and has high IRI activity.

[0075] Example 3: Evaluation of cell viability after cryopreservation and thawing

[0076] Figure 5 The image shows the cell viability of HeLa cells after being stored at -80℃ and -196℃ for one day and then thawed using different cell cryopreservation solutions. Compared with the traditional cell cryopreservation solution (90% FBS + 10% DMSO), the cell survival rate was doubled after adding 10 mg / ml of cellulose-based antifreeze.

[0077] Example 4: Activity evaluation of mRNA-LNP vaccine after cryopreservation

[0078] Figure 6 The average fluorescence intensity of the mRNA-LNP vaccine after cryopreservation at 4 / -20℃ with a cellulose-based cryoprotectant of + / - 10 mg / ml is shown. Compared with unfrozen and -20℃ mRNA-LNP vaccines, the addition of 10 mg / ml cellulose-based cryoprotectant effectively improved vaccine activity.

[0079] Example 5: Evaluation of tissue condition after organ cryopreservation and thawing

[0080] Figure 7 Scanned images of mouse kidney sections stained with H&E(ac) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (df) after freezing for 3 days in water, UW solution, and UW solution containing 10 mg / mL MCOs at -10°C. Compared with water and commercially available UW solutions, the addition of 10 mg / mL cellulose-based cryoprotectant significantly reduced tissue cracks and the number of dead cells.

[0081] This invention uses a specific type of cellulose-based material as a biological cryoprotectant, which has good biocompatibility and can achieve good cryoprotection effect, and can be applied to the field of cell cryopreservation; at the same time, the method of regulating the cellulose-based material in this invention is simple and rapid.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of cellulose-based materials as biological cryoprotectants, characterized in that, The cellulose-based material has an average degree of polymerization ranging from 2 to 18 and carries a surface charge; The cellulose-based material includes one or more of the following: cellulose, 2,3-epoxypropyltrimethylammonium chloride modified cellulose, phosphorylated cellulose, sulfated cellulose, carboxymethylated cellulose, tetramethylpiperidine oxide oxidized cellulose, nitrocellulose, citric acid modified cellulose, acrylic acid modified cellulose, or pyridine modified cellulose. The concentration of the cellulose-based material in the biological cryoprotectant is 0.1-100 mg / ml.

2. A biological cryoprotectant, characterized in that, It includes cellulose-based materials, dispersants, and additives; the cellulose-based materials have an average degree of polymerization ranging from 2 to 18 and are surface-charged; The dispersant comprises one or more of the following: pure water, phosphate buffer, citrate buffer, borate buffer, 0.9% sodium chloride solution, glycine buffer, tris(hydroxymethyl)aminomethane buffer, 2-(N-methyl)-diaminoethane sulfonic acid buffer, N-hydroxyethyl-1,3-propanediamine-N,N,N'-triacetic acid buffer, triacetate buffer, piperazine sulfonic acid buffer, N-(tris(hydroxymethyl)-glycine) buffer, or 3-(N-methyl)-1-propanesulfonic acid buffer. The additives include one or more of the following: dimethyl sulfoxide, glycerol, mannitol, gelatin, glucose, trehalose, sucrose, propylene glycol, ethylene glycol, polyethylene glycol, glycine, bovine serum albumin, magnesium sulfate, L-cysteine, or zinc sulfate.

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