Method for cryopreserving red blood cells based on cryopreservation protective agent loaded lipid nanoparticles

Abstract

The invention discloses a method for cryopreserving red blood cells based on lipid nanoparticles loaded with a cryopreservation protective agent, and belongs to the field of biological medicines. The method for cryopreserving the red blood cells based on the cryopreservation protective agent loaded lipid nanoparticles comprises the following steps: mixing a cryopreservation protective agent loaded lipid nanoparticle solution, the red blood cells and a cryopreservation protective agent solution to obtain a mixed solution; and controlling the pH value of the mixed solution to be lower than the pKa value of the ionized lipid, incubating at 15-40 DEG C for more than 5 minutes, and freezing in liquid nitrogen. According to the invention, the ionizable lipid nanoparticles are used for loading the cryopreservation protective agent, and the fusion of the lipid nanoparticles and the cell membrane is adjusted by using pH, so that the delivery effect of the cryopreservation protective agent in the red blood cells is effectively improved, and the cryopreservation of the red blood cells is promoted. The method is simple in cryopreservation operation, and can be used for cryopreservation only by mixing at room temperature for a short time; the unfreezing recovery rate of the cryopreserved red blood cells can reach 85%, functions are normal, and clinical use requirements are met; the unfrozen red blood cells can be used for subsequent blood transfusion after being simply cleaned without complicated post-treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for freezing red blood cells based on lipid nanoparticles carrying a cryoprotectant. Background Art

[0002] Red blood cell transfusion is crucial for the vast majority of patients who need to supplement red blood cells and improve oxygen-carrying capacity in clinical practice, such as patients with acute blood loss caused by trauma or surgery, heart, kidney, liver dysfunction, and other blood-related diseases. In order to ensure the supply of red blood cells, a variety of red blood cell preservation methods have been developed. At present, red blood cell products are usually stored in isotonic solutions at 4°C for no more than 42 days, while low-temperature cryopreservation can achieve long-term storage of red blood cells, which helps to stabilize the supply of red blood cells. Cryopreservation is the process of freezing cells at ultra-low temperatures (-65 to -196°C) to maintain their structure and function. This process relies on special cryoprotectants and cryopreservation-resuscitation processes to protect red blood cells from damage due to ice crystal formation and solute changes during freezing and thawing.

[0003] At present, glycerol is mainly used in clinical practice to freeze red blood cells, which can be divided into slow freezing method, using glycerol with a final concentration of 40% (mass / volume) at a temperature below -65°C; or rapid freezing method, using 18% glycerol to freeze at liquid nitrogen temperature (-196°C) and store in liquid nitrogen vapor (-156°C), which can be stored for up to 10 years. In the above method, it is usually only necessary to mix the glycerol preparation with the red blood cells at room temperature and incubate for 30 minutes before freezing. However, although the glycerol method has a long freezing time and good resuscitation effect, the toxicity of glycerol itself will cause side effects such as hemolysis and change of red blood cell morphology during freezing. Before frozen cells are used for transfusion, a long and complicated deglycerolization process is required. This process involves multiple washing steps, which will cause additional hemolysis and morphological changes of red blood cells. In addition, the glycerol remaining in the cells may also cause complications in some patients. The above shortcomings of the glycerol method limit the widespread application of frozen cells and cannot achieve a large supply of high-quality red blood cells in a short period of time. Therefore, it is necessary to develop new red blood cell cryopreservation solutions and cryopreservation methods that have good cryopreservation effects but do not require long-term post-processing, so that patients can better obtain blood.

[0004] Trehalose is a natural cryoprotectant that has attracted much attention for its ability to protect cells and organisms from various stresses, including freezing, drying, heat shock, oxidation, and osmotic stress. Trehalose's polyhydroxy structure enables it to bind to lipid membranes and proteins, thereby replacing lost bound water, reducing ice crystal damage during freezing, and maintaining the water / solute inflow and outflow rate of cells during cryopreservation, thereby maintaining cell morphology. In addition, trehalose is a non-permeable red blood cell cryoprotectant that can avoid expensive and cumbersome deglycerolization procedures, so that cryopreserved red blood cells can be used for transfusion immediately when needed. However, trehalose must be present on both sides of the cell membrane to have the best protective effect, and under normal conditions, red blood cells have no active uptake activity and low membrane permeability. Therefore, trehalose needs to be delivered to red blood cells to achieve a better cryopreservation effect. Currently developed solutions include photothermal, ultrasound, electroosmosis, microinjection, membrane-penetrating peptides, membrane-permeable polymers, biomimetic nanocarriers such as lipid nanoparticles (LNPs), genetic engineering, etc., most of which are still committed to improving the recovery rate of red blood cells after freezing and thawing and improving their practicality.

[0005] So far, due to its excellent biosafety and cell delivery effect, LNP has been widely used in drug and gene delivery. At present, some schemes for delivering trehalose based on LNP have also been developed, including the use of negatively charged phosphatidylserine PS-LNP and red blood cells at 37 ° C for 24 hours to deliver 100 times more trehalose than neutrally charged ones, reaching 15mM, and the survival rate of red blood cell cryopreservation was significantly improved. Another study used LNP containing PS and dipalmitoylphosphatidylcholine (DPPC) to incubate red blood cells at 37 ° C for 4 hours, and its cryopreservation survival rate increased from 29% of trehalose to 64%. The use of high concentrations of LNP (up to 1mM) can increase the concentration of intracellular trehalose to 80mM to a certain extent, effectively improving the cryopreservation survival rate. It can be seen that there are many problems in the above schemes, such as long incubation time of red blood cells and LNP, poor cryopreservation effect or large amount of LNP used.

[0006] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for freezing red blood cells based on lipid nanoparticles carrying cryoprotectants, aiming to solve the problems of the existing method for freezing red blood cells based on LNP delivering trehalose, such as long incubation time between red blood cells and LNP, poor freezing effect or large amount of LNP used.

[0008] The technical solution of the present invention is as follows:

[0009] A method for freezing red blood cells based on lipid nanoparticles carrying cryoprotectants, comprising the steps of:

[0010] A cryoprotectant-loaded lipid nanoparticle solution is provided, wherein the cryoprotectant-loaded lipid nanoparticle solution comprises cryoprotectant-loaded lipid nanoparticles, wherein the cryoprotectant-loaded lipid nanoparticles comprise lipid nanoparticles and a cryoprotectant loaded in the lipid nanoparticles, wherein the lipid nanoparticles are composed of ionizable lipids, wherein the ionizable lipids have good biological safety and a pKa value between 6 and 7, and wherein the cryoprotectant is a cryoprotectant for non-permeable red blood cells;

[0011] Mixing the cryoprotectant-carrying lipid nanoparticle solution, red blood cells and the cryoprotectant solution to obtain a mixed solution;

[0012] The pH of the mixed solution is controlled to be lower than the pKa value of the ionizable lipid, and the mixture is incubated at 15-40° C. for more than 5 minutes. The red blood cell solution obtained after the incubation is frozen in liquid nitrogen.

[0013] Optionally, the concentration of the cryoprotectant-loaded lipid nanoparticles in the cryoprotectant-loaded lipid nanoparticle solution is 0.05-0.5 mg / mL, the concentration of the cryoprotectant solution is above 0.54 M, and the volume ratio of the cryoprotectant-loaded lipid nanoparticle solution to the cryoprotectant solution is 1:10-1:100.

[0014] Optionally, the incubation time is 5 to 60 minutes.

[0015] Optionally, hydrochloric acid, acetic acid or citric acid is used to control the pH of the mixed solution to be below the pKa value of the ionizable lipid.

[0016] Optionally, the cryoprotectant is a macromolecular polymer or a carbohydrate compound.

[0017] Optionally, the carbohydrate compound is one or more of trehalose, sucrose, glucose and mannitol.

[0018] Optionally, the cryoprotectant-carrying lipid nanoparticle solution consists of cryoprotectant-carrying lipid nanoparticles and a cryoprotectant solution;

[0019] The cryoprotectant solution consists of a cryoprotectant and a buffer isotonic with red blood cells.

[0020] Optionally, the method for preparing the cryoprotectant-loaded lipid nanoparticles comprises the steps of:

[0021] preparing a lipid phase solution, wherein the lipid phase solution comprises an ionizable lipid and a solvent;

[0022] preparing an aqueous phase solution, wherein the aqueous phase solution comprises a cryoprotectant and a buffer;

[0023] The lipid phase solution and the aqueous phase solution are mixed at a lipid-water flow rate ratio of 1:2 to 1:10, and the solvent is removed by dialysis or ultrafiltration to obtain lipid nanoparticles loaded with cryoprotectant.

[0024] Optionally, the lipid phase solution includes 1,2-distearoyl-sn-glycero-3-phosphocholine, octadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino)caprylate, cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 and anhydrous ethanol;

[0025] Alternatively, the lipid phase solution includes 1,2-distearoyl-sn-glycero-3-phosphocholine, ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (abbreviated as ALC-0315), cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 and anhydrous ethanol;

[0026] The aqueous phase solution includes trehalose and a buffer;

[0027] The total flow rate of lipid and water is 4.5 mL / min.

[0028] Optionally, the cryoprotectant-loaded lipid nanoparticles are freeze-dried cryoprotectant-loaded lipid nanoparticles, and the cryoprotectant-loaded lipid nanoparticle solution is prepared by dissolving freeze-dried cryoprotectant-loaded lipid nanoparticles in water.

[0029] Beneficial effects: The present invention uses ionizable lipid nanoparticles to load cryoprotectants, and uses pH to adjust the fusion of lipid nanoparticles with cell membranes (pH is used to adjust lipid nanoparticles to be positively charged, while the surface of the red blood cell membrane is negatively charged, and the positive and negative charges interact to promote the adsorption and fusion of lipid nanoparticles with cell membranes), effectively improving the delivery effect of cryoprotectants in red blood cells and promoting the cryopreservation of red blood cells. The present invention shows that short-term incubation at room temperature can achieve a better cryopreservation effect of red blood cells. This condition can be carried out at a temperature of 15 to 40°C, an incubation time of 5 minutes or more, and static or rotating and mixing at different speeds according to specific requirements to achieve a better cryopreservation effect of red blood cells. Compared with the clinical glycerol cryopreservation method, the cryopreservation operation is also simple and easy to obtain, and only needs to be mixed at room temperature for a short time for cryopreservation; the thawing recovery rate of frozen red blood cells can reach 85%, and the function is normal, meeting the clinical use needs; the thawed red blood cells do not need to be similar to the complex post-processing in the glycerol method, and only need to be simply cleaned once for subsequent blood transfusion; the freeze-dried lipid nanoparticles containing cryoprotectants have the same cryopreservation protection effect, which greatly promotes the application scope of the present invention. Therefore, compared with the clinical glycerol cryopreservation method of red blood cells, the present invention is also simple and efficient, and greatly reduces the post-processing steps, which is conducive to the timely and high-quality supply of frozen red blood cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram showing the fusion and cryopreservation effects of LNPs of different particle sizes with red blood cells.

[0031] Figure 2 This is a diagram showing the fusion and cryopreservation effects of LNPs of different compositions with red blood cells.

[0032] Figure 3 ALC-0315 lipid composition LNP is used for red blood cell cryopreservation.

[0033] Figure 4 This is a diagram for optimizing the freezing conditions of LNP and red blood cells.

[0034] Figure 5 Figure 2. Freeze-dried LNP for red blood cell cryopreservation.

[0035] Figure 6 This is a functional chart of red blood cells after cryopreservation and resuscitation.

[0036] Figure 7 This is a scanning electron micrograph of the morphology of red blood cells after cryopreservation and resuscitation. DETAILED DESCRIPTION

[0037] The present invention provides a method for freezing red blood cells based on lipid nanoparticles containing cryoprotectants. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] The embodiment of the present invention provides a method for freezing red blood cells based on lipid nanoparticles carrying cryoprotectants, which comprises the steps of:

[0039] A cryoprotectant-loaded lipid nanoparticle (also referred to as cryoprotectant-loaded LNP) solution is provided, wherein the cryoprotectant-loaded lipid nanoparticle comprises lipid nanoparticles and a cryoprotectant loaded in the lipid nanoparticles, wherein the lipid nanoparticles are composed of ionizable lipids, the ionizable lipids have good biological safety and a pKa between 6 and 7, and the cryoprotectant is a cryoprotectant for non-permeable red blood cells;

[0040] Mixing the cryoprotectant-carrying lipid nanoparticle solution, red blood cells and the cryoprotectant solution to obtain a mixed solution;

[0041] The pH of the mixed solution is controlled to be lower than the pKa value of the ionizable lipid, and the mixture is incubated at 15-40° C. (it can be incubated on a shaker or a horizontal axis mixer) for more than 5 minutes (e.g., 5-60 minutes), and the red blood cell solution obtained after the incubation is frozen in liquid nitrogen.

[0042] In the glycerol cryopreservation method of red blood cells, high concentration of glycerol is used for freezing. Glycerol will penetrate into the red blood cells, resulting in a complex and time-consuming deglycerolization process after freezing to reduce the glycerol concentration in the red blood cells, affecting the red blood cell yield, state and subsequent use. In contrast, the cryoprotectant used in the embodiment of the present invention has good safety, and the cryoprotectant has no cell membrane permeability and cannot enter the red blood cells autonomously; the cryoprotectant in the red blood cells is delivered by lipid nanoparticles, and the concentration is low, which will not cause additional safety risks. Therefore, the red blood cells only need one centrifugal washing after freezing to remove the extracellular high concentration cryoprotectant (such as trehalose), and can be used without complicated post-processing, which greatly improves the convenience, timeliness and safety of the use of frozen red blood cells.

[0043] The embodiment of the present invention uses ionizable LNP to cryopreserve red blood cells, and realizes efficient delivery of cryoprotectant based on pH regulation of LNP and red blood cell membrane fusion, and has good cryoprotectant delivery and red blood cell cryopreserve effect at pH below the pKa value of ionizable lipids. However, the known LNP delivery of trehalose relies on methods such as imitating the lipid ratio of red blood cell membrane, using positively charged lipids, and using high-concentration LNP, and the trehalose delivery efficiency is low or more harmful components are introduced.

[0044] In the embodiment of the present invention, ionizable LNP is used to cryopreserve red blood cells. After the cryoprotectant delivery and red blood cell cryopreservation are achieved at low pH, the incubated or frozen-thawed red blood cells can be resuspended in a physiological environment solution (pH 7.4). The LNP on the red blood cell membrane becomes negatively charged, eliminating the toxicity of the positively charged LNP, so that the red blood cells can be used safely.

[0045] In the embodiment of the present invention, the incubation of LNP containing cryoprotectant and red blood cells can be completed at room temperature, without the need for complicated temperature control equipment and operation, which is consistent with the glycerol method, has good clinical practicality, and is significantly superior to other LNP cryopreservation methods.

[0046] In the embodiment of the present invention, the incubation of the cryoprotectant-loaded LNP and red blood cells at room temperature does not exceed 30 minutes before cryopreservation, which is short and consistent with the glycerol method, and is significantly better than the long incubation requirements in other LNP cryopreservation methods.

[0047] In an embodiment of the present invention, the incubation of the cryoprotectant-loaded LNP and red blood cells can be carried out in a cryoprotectant solution, wherein the cryoprotectant concentration is above 0.54M and has a good cryopreservation effect. Non-permeable cryoprotectants can be delivered to red blood cells using the method shown in the embodiment of the present invention, thereby improving the cryopreservation effect. The cryoprotectant can be a macromolecular polymer or a carbohydrate compound. As an example, the cryoprotectant can be trehalose, and of course trehalose can also be replaced with other cryoprotectants with good biosafety and membrane impermeability, such as sucrose, glucose, mannitol, etc.

[0048] In the embodiment of the present invention, the cryoprotectant-loaded LNP still has a good red blood cell freezing effect after freeze-drying, and the freeze-dried cryoprotectant-loaded LNP has a longer stability, and its re-dissolution only requires the addition of a certain amount of pure water, which greatly facilitates the freezing operation. Preparing the cryoprotectant-loaded LNP as a freeze-dried powder greatly prolongs the stability and shelf life of the material, while being more convenient for storage and transportation, and is also convenient and effective to use, greatly increasing the prospects for clinical application. At present, there is no method of using freeze-dried cryoprotectant-loaded LNP for red blood cell freezing.

[0049] Furthermore, the concentration of the cryoprotectant-loaded lipid nanoparticles in the cryoprotectant-loaded lipid nanoparticle solution is 0.05-0.5 mg / mL, the concentration of the cryoprotectant solution is above 0.54 M, and the volume ratio of the cryoprotectant-loaded lipid nanoparticle solution to the cryoprotectant solution is 1:10-1:100, such as 1:10, 1:20, 1:40, 1:50, 1:70, 1:80, 1:100, etc.

[0050] The embodiments of the present invention rely on the use of ionizable lipids to form LNPs, so that the fusion with the cell membrane can be regulated by pH to improve the delivery of cryoprotectants and the cryopreservation effect of red blood cells. Therefore, other ionizable lipids can be used to synthesize ionizable LNPs for red blood cell cryopreservation as long as they meet the requirements of good biosafety and pKa between 6 and 7.

[0051] Changing the lipid components and composition in LNP can be used for red blood cell cryopreservation as long as pH-mediated LNP charge conversion and cell membrane fusion can be achieved, including but not limited to regulating the molar ratio of each lipid based on the existing components, using new lipids or continuing to add new lipids to obtain five-component, six-component or even more complex LNPs.

[0052] In one embodiment, the lipid nanoparticles include the following ionizable lipid compositions: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), octadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino)caprylate (SM-102), cholesterol (Chol) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-mPEG2000).

[0053] In one embodiment, the lipid nanoparticles include the following ionizable lipid compositions: 1,2-distearoyl-sn-glycero-3-phosphocholine, ALC-0315, cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000.

[0054] In one embodiment, the method for preparing the cryoprotectant-loaded lipid nanoparticles comprises the steps of:

[0055] preparing a lipid phase solution, wherein the lipid phase solution comprises ionizable lipids and a solvent (such as anhydrous ethanol, etc.);

[0056] preparing an aqueous phase solution, wherein the aqueous phase solution comprises a cryoprotectant and a buffer isotonic with red blood cells;

[0057] The lipid phase solution and the aqueous phase solution are mixed at a lipid-water flow rate ratio of 1:2 to 1:10, and the solvent is removed by dialysis or ultrafiltration to obtain lipid nanoparticles loaded with cryoprotectant.

[0058] Among them, the total flow rate of lipid and water is 4.5mL / min.

[0059] In a specific embodiment, the preparation method of the trehalose-loaded lipid nanoparticles (trehalose-loaded LNP) comprises the steps of:

[0060] 1,2-distearoyl-sn-glycero-3-phosphocholine, octadecane-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were mixed in anhydrous ethanol at a molar ratio of 10:50:38:2 to obtain a lipid phase solution; trehalose was dissolved in a buffer isotonic with red blood cells to obtain an aqueous phase solution. A microfluidic device with a herringbone configuration chip was used to prepare trehalose-loaded LNPs at a lipid-water flow rate ratio of 1:2 to 1:10, and ethanol was removed by dialysis or ultrafiltration. A trehalose solution (prepared with a buffer isotonic with trehalose and red blood cells) was used to dilute the trehalose-loaded LNPs to a certain concentration to obtain a trehalose-loaded LNP solution.

[0061] In a specific embodiment, a method for freezing red blood cells using trehalose-loaded LNPs comprises the steps of:

[0062] Fresh human red blood cells and trehalose-loaded LNP solution are mixed in a trehalose solution in a certain ratio, the pH of the solution is controlled below 6.5, and incubated at room temperature (such as 25°C) using a horizontal axis mixer for 5 to 60 minutes. After the incubation, the red blood cell solution is directly frozen in liquid nitrogen.

[0063] When red blood cells are needed, take out the cryopreserved red blood cell freezing solution, thaw quickly at 37-40°C, centrifuge at 1800g for 5 minutes, remove the supernatant, resuspend the red blood cells in isotonic buffer or saline and use them for blood transfusion.

[0064] The present invention is described in detail below through specific embodiments.

[0065] 1. Experimental data

[0066] The main chemicals and reagents were purchased from Merck, Aladdin Reagents, and Xiamen Synobond. Among them, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhB-PE, purchased from Merck Avanti), octadecane-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-mPEG2000) were purchased from Xiamen Synobond, and trehalose and anhydrous ethanol were purchased from Aladdin Reagents. Suspended red blood cells collected from healthy volunteers were provided by Dongguan People's Hospital.

[0067] Main instruments and equipment: microfluidic chip (LNP-B1, Shanghai Pengzan Biotechnology Co., Ltd.), syringe pump (LM2-C, Zhengzhou Lumai Medical Co., Ltd.), dynamic light scattering instrument (NanoTrac Wave II, MicroTrac, USA), multifunctional microplate reader (Synergy H1, BioTek, USA), freeze dryer (LC-10N-60A, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), confocal fluorescence microscope (AX R, Nikon, Japan), flow cytometer (CytoFLEX S, Beckman, USA), scanning electron microscope (SU8010, Hitachi, Japan).

[0068] 2. Detection method

[0069] (1) Particle size and potential of trehalose-loaded LNP

[0070] The purified trehalose-loaded LNP was diluted to 1 mg / mL using 0.01× phosphate buffer (PBS), and the pH of the solution was controlled at 7.4 and 6.5 (adjusted using hydrochloric acid). The particle size and potential were measured using a dynamic light scattering instrument, respectively.

[0071] (2) Trehalose loading rate of trehalose-loaded LNP

[0072] The trehalose content inside LNP was measured using the anthrone colorimetric method. Take 100 μL of the purified trehalose-loaded LNP solution and add 900 μL of trichloroacetic acid solution to extract trehalose. Place it at room temperature for 45 minutes, shake it repeatedly 3 times, and centrifuge it at 8000g for 10 minutes to collect the supernatant. Take 60 μL of the supernatant and add 240 μL of anthrone solution (80% H2SO4 dissolved), heat it at 95°C for 15 minutes, and measure the absorbance at 620 nm. Take a trehalose aqueous solution in the concentration range of 0 to 0.2 mg / mL, and refer to the above method to obtain a standard curve of trehalose concentration and absorbance value. Calculate the trehalose concentration in the trehalose-loaded LNP sample according to the standard curve, and calculate the trehalose loading rate by the following formula (1):

[0073] Trehalose loading rate: LC (%) = mass of trehalose in trehalose-loaded LNP / (mass of trehalose in trehalose-loaded LNP+mass of LNP) × 100 (1)

[0074] (3) LNP hemolysis test

[0075] 100 μL of red blood cells were mixed with 400 μL of deionized water, 0.33×PBS and 0.54M trehalose solution with a concentration of 0.05-0.5 mg / mL trehalose-loaded LNP, respectively, so that the hematocrit was finally maintained at 30%. After the mixture was incubated at 25°C for 1 hour, the supernatant was collected after centrifugation at 1800g for 5 minutes, and the absorbance of the supernatant at a wavelength of 540 nm was measured by a UV spectrophotometer. In the experiment, red blood cells were mixed with deionized water as a positive control for complete hemolysis (A0), red blood cells were mixed with 0.33×PBS as a negative control for almost no hemolysis (A1), and red blood cells were mixed with different concentrations of trehalose-loaded LNP as a sample group (A LNP ), and the red blood cell hemolysis rate was calculated according to the following formula (2):

[0076] Hemolysis rate (%) = (A LNP -A1) / (A0-A1)×100(2)

[0077] (4) Fusion of LNP and red blood cells

[0078] Dissolve trehalose in 0.33×PBS to a concentration of 0.54M to obtain a trehalose cryopreservation solution that is isotonic with red blood cells. When preparing trehalose-loaded LNP, add 0.1% molar ratio of RhB-PE to obtain fluorescent trehalose-loaded LNP, dilute it in 0.54M trehalose cryopreservation solution, the trehalose-loaded LNP concentration is 0.2mg / mL, pH is 7.4 and 6.5, respectively, to obtain LNP trehalose solution. After the red blood cells are purified by centrifugation at 1800g for 5 minutes to remove serum, they are mixed with the above-mentioned LNP trehalose solution, wherein the hematocrit is controlled to be 30%. After incubation at room temperature for 30 minutes, the red blood cells are collected after centrifugation at 1800g for 5 minutes, and the fluorescence (Ex.561nm, Em.591nm) increased by LNP fusion in the red blood cells is measured using a multifunctional microplate detector, and the fusion status of LNP on the red blood cells is observed using a confocal microscope.

[0079] (5) Measurement of trehalose in red blood cells

[0080] 100 μL of red blood cells and 0.2 mg / mL trehalose-loaded LNP were mixed in 0.54 M trehalose solution, pH was controlled at 6.5 and 7.4, hematocrit was 30%, and after incubation at room temperature for 30 minutes, the cells were washed three times with 0.33×PBS at 1800 g for minutes. The red blood cells were collected, lysed with trichloroacetic acid, cell membranes and proteins were precipitated, and placed at room temperature for 45 minutes. After repeated shaking three times, the supernatant was collected by centrifugation at 8000 g for 10 minutes, 60 μL of the supernatant was added to 240 μL of anthrone solution, heated at 95 ° C for 15 minutes, and then the absorbance at 620 nm was measured. The trehalose concentration in the sample was calculated based on the trehalose standard curve, and the trehalose concentration (mM) in red blood cells was obtained by formula (3).

[0081] Trehalose concentration in red blood cells (mM) = M RBC-Tre / (342.3×0.7×V×RBC×HCT)×10 6 (3)

[0082] Among them, M RBC-Tre represents the mass of trehalose in LNP-incubated erythrocytes (g); 342.3 (g / mol) is the molecular weight of trehalose; V (mL) is the volume of erythrocyte suspension; RBC refers to the erythrocyte concentration (×10 12 / L); MCV is the mean corpuscular volume (fL); 0.7 represents the corpuscular volume index because approximately 70% of the cytoplasm of the erythrocyte is in contact with water, while the remainder is filled by the cell membrane.

[0083] (6) Red blood cell cryopreservation survival rate

[0084] 100 μL of blood cells were mixed with 0.2 mg / mL trehalose-loaded LNP in 0.54 M trehalose solution, pH was controlled at 6.5 and 7.4, hematocrit was 30%, and after incubation at room temperature for 30 minutes, the mixture was quickly frozen in liquid nitrogen and kept for at least 2 hours. The cryotube was placed in a 37°C water bath to thaw the sample, then centrifuged at 1800 g for 5 minutes, and the supernatant was collected and the absorbance (A) was measured at a wavelength of 540 nm. LNP ). At the same time, 100 μL of fresh red blood cells were dispersed in 400 μL of deionized water and 0.33×PBS as the positive control for complete hemolysis (A0) and the negative control for non-hemolysis (A1), respectively. The cryopreservation survival rate of red blood cells after thawing was calculated according to the following formula (4):

[0085] Cryopreservation survival rate (%) = (1-(A LNP -A1) / (A0-A1))×100(4)

[0086] (7) Red blood cell function test

[0087] For the above-mentioned frozen and thawed red blood cells, fresh red blood cells were used as controls to analyze the changes in related biological functions, including adenosine triphosphate (ATP) analysis of energy changes in red blood cells, using an ATP kit (C0061M, Beyotime, China); phosphatidylserine (PS) exposure rate to evaluate red blood cell membrane damage, using Annexin V-FITC kit (C1062L, Beyotime, China) for staining and analysis by flow cytometry; 2,3-diphosphoglycerate (2,3-DPG) content to evaluate red blood cell oxygen affinity, using an ELISA kit (YX-E11265, Inotech, China); methemoglobin (MetHb) content to evaluate the oxidation of heme iron, using a related quantitative kit (BC5600, Solebao, China).

[0088] (8) Red blood cell morphology detection

[0089] After freezing, the erythrocytes were thawed and revived at 37°C, washed twice with 0.33×PBS, and fixed with 2.5% glutaraldehyde at 4°C for more than 2 hours. Subsequently, they were washed three times with PBS, dehydrated with a series of ethanol solutions with different volume contents (20%, 40%, 60%, 80%, 90% and 100% (twice), 15 minutes each time) and freeze-dried. Finally, the cell surface was sprayed with gold and imaged with a scanning electron microscope (SU8010, Hitachi, Japan).

[0090] 3. Experimental plan and test results

[0091] (1) Preparation of trehalose-loaded LNP and its effect on red blood cell membrane fusion

[0092] DSPC, SM-102, Chol, DMG-mPEG2000 and RhB-PE were dissolved in anhydrous ethanol at a concentration of 10 mg / mL, sealed and stored at 4°C away from light. According to the molar ratio of DSPC:SM-102:Chol:DMG-mPEG2000=10:50:38:2, 62, 280, 117 and 39 μL of the above lipid ethanol solutions were taken, mixed, and anhydrous ethanol was added to 1 mL to obtain a lipid phase solution with a final concentration of 5 mg / mL. Among them, if fluorescently labeled LNPs need to be prepared, 9 μL of RhB-PE diluted to 1 mg / mL with anhydrous ethanol (equivalent to 0.1% molar ratio of total lipids) is added to the lipid phase solution. At the same time, trehalose was weighed and dissolved in 0.33×PBS to obtain a 184.8 mg / mL (0.54M) aqueous phase solution. The lipid phase solution and the aqueous phase solution were loaded into syringes and placed on the syringe pump, respectively, and the lipid / water flow rate ratio was set at 1:2 to 1:10, with a total flow rate of 4.5 mL / min. The microfluidic chip was a herringbone structure. The effluent was collected to obtain trehalose-loaded LNPs of different particle sizes, and a dialysis bag with a molecular weight cutoff of 3400 Da was used to remove the ethanol in the trehalose-loaded LNP solution, and the dialysis medium was 0.33×PBS. The dialyzed trehalose-loaded LNP solution was loaded into an ultrafiltration tube with a molecular cutoff of 100 kDa, centrifuged at 1500 g for 10 min, the intercepted liquid was collected, and 0.54 M trehalose solution was added to a final volume of 1 mL. According to the concentration-fluorescence intensity standard curve of RhB-PE, the concentration of trehalose-loaded LNP in the obtained trehalose-loaded LNP solution was calculated, and finally 0.54 M trehalose solution was added to adjust the trehalose-loaded LNP concentration to 2 mg / mL. The particle size and potential of trehalose-loaded LNP at pH 7.4 and pH 6.5 were measured using a dynamic light scattering instrument. The results are shown in Table 1 below. It can be seen that as the lipid / water flow rate ratio changes from 1:2 to 1:10, the particle size of trehalose-loaded LNP increases from 137.6 nm to 265.1 nm, that is, when the total flow rate remains unchanged, the faster the lipid phase flow rate, the smaller the particle size; the measured surface potential shows that all trehalose-loaded LNPs are negatively charged at pH 7.4 and become positively charged at pH 6.5; the measured trehalose loading rate of trehalose-loaded LNP increases from 44.6% to about 60% with increasing particle size.

[0093] Table 1

[0094]

[0095]

[0096] The prepared trehalose-loaded LNP solution was stored at 4°C in the dark, and its particle size and trehalose loading rate were measured at regular intervals to determine the storage stability of the trehalose-loaded LNP, and the results are shown in Table 2. Taking the trehalose-loaded LNP with a particle size of 180 nm as an example, it can be seen that within 14 days of storage, its particle size did not change much, and the trehalose loading rate remained unchanged, and there was no trehalose leakage, indicating that the trehalose-loaded LNP stored at 4°C can be used normally for at least 14 days.

[0097] Table 2

[0098]

[0099] The prepared trehalose-loaded LNPs of different particle sizes were diluted to 0.2 mg / mL using 0.54 M trehalose solution. At the same time, the whole blood of healthy people was centrifuged at 1800 g for 5 min to collect red blood cells, and mixed with the trehalose-loaded LNPs diluted with the above trehalose solution. The hematocrit was 30%, and the solution pH was adjusted to 7.4 and 6.5, respectively. After mixing and incubating on a horizontal axis mixer at room temperature for 30 min, the fusion rate of LNP and red blood cells and the survival rate of red blood cells in cryopreservation were measured. The results are as follows Figure 1 shown. Figure 1 A in the middle shows that the fusion of LNP and red blood cells at pH 6.5 is significantly higher than that at pH 7.4, and more red fluorescent LNPs are fused on red blood cells; Figure 1 Middle B shows that at the same concentration, the fusion effect of LNP and red blood cells is not affected by its particle size; Figure 1 Figure C shows the cryopreservation survival rate of red blood cells. At pH 6.5, the cryopreservation survival rate of red blood cells incubated with trehalose-loaded LNPs was significantly higher than that at pH 7.4, and was higher than that incubated with trehalose alone. This result is also consistent with the fusion result of LNP and red blood cells in Figure B, that is, the higher fusion efficiency of LNP and red blood cells causes more trehalose internalization, thereby effectively improving the cryopreservation survival rate of red blood cells.

[0100] In addition, the effect of LNP composition on red blood cell cryopreservation was also detected. The amount of Chol and DMG-mPEG2000 was kept unchanged, and SM-102:DSPC=50:10, 40:20, 30:30, 20:40, 10:50 and 0:60 were adjusted to prepare LNP. The particle size and potential of trehalose-loaded LNP at pH 7.4 and pH 6.5 were measured using a dynamic light scattering instrument. The results are shown in Table 3 below. It can be seen that the particle size of trehalose-loaded LNPs with different compositions is in the range of 200-250nm. All trehalose-loaded LNPs containing SM-102 are negatively charged at pH 7.4 and positively charged at pH 6.5, while trehalose-loaded LNPs without SM-102 have no pH responsiveness. The loading rate of trehalose in each trehalose-loaded LNP is 60%-70%, with little difference. Red blood cells were mixed with these trehalose-loaded LNPs in a 0.54 M trehalose solution, the pH of which was controlled at 7.4 and 6.5, and incubated at room temperature for 30 min. The fusion rate of LNPs with red blood cells and the survival rate of red blood cells in cryopreservation were then measured. Figure 2 shown. Figure 2 Middle A shows the fusion of LNP and red blood cells. It can be seen that the highest fusion effect is achieved when SM-102:DSPC=50:10. When the dosage of SM-102 is reduced, the fusion efficiency of LNP and red blood cells is weakened. Figure 2 Middle B shows the cryopreservation survival rate of red blood cells, and the result is consistent with the fusion result, that is, the LNP with SM-102:DSPC=50:10 has the best cryopreservation protection effect on red blood cells.

[0101] Table 3

[0102]

[0103] SM-102 was replaced with another ionizable lipid ((4-hydroxybutyl) azadiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315, pKa=6.09) and a new trehalose-loaded LNP was synthesized with the same lipid ratio composition. The new LNP was incubated with red blood cells in a 0.54 M trehalose solution at room temperature for 30 minutes with the pH controlled at 6.0 and 7.4. The cryopreservation protective effect of the LNP on red blood cells was measured. The results are as follows: Figure 3 shown. Figure 3 Middle A shows that the particle size of trehalose-loaded LNP based on ALC-0315 is about 155 nm; Figure 3 Figure B shows that its zeta potential changes with pH, ​​which is -17.5 mV at pH 7.4 and changes to +21.5 mV at pH 6.0; Figure 3 Middle C shows that the fusion of ALC-0315-based trehalose-loaded LNP with erythrocytes was significantly improved at pH 6.0; Figure 3Figure D shows that the ALC-0315-based trehalose-loaded LNP has a good red blood cell cryopreservation effect at pH 6.0.

[0104] (2) Optimization of the trehalose-loaded LNP cryopreservation protocol for red blood cells

[0105] The temperature and time of incubation of trehalose-loaded LNP with erythrocytes, the concentration of trehalose-loaded LNP, and the concentration of trehalose in the solution will affect the cryopreservation effect of erythrocytes. After incubating 0.2 mg / mL trehalose-loaded LNP solution with erythrocytes in 0.54 M pH 6.5 trehalose solution at 4, 25, and 37°C for one hour, the cryopreservation effect of erythrocytes was checked. The results are as follows: Figure 4 As shown in Figure A, there is no significant difference in the cryopreservation survival rate of red blood cells at 25°C and 37°C, which is significantly higher than that at 4°C, indicating that incubation at 25°C or room temperature can usually meet the requirements of LNP-mediated trehalose delivery and red blood cell cryopreservation. 0.2 mg / mL trehalose-loaded LNP and red blood cells were incubated in 0.54M pH=6.5 trehalose solution at room temperature for 10, 20, 30, and 60 minutes, and the cryopreservation survival rate of red blood cells was measured. The results are shown in Figure 1. Figure 4 As shown in Figure B, after the red blood cells were incubated with the trehalose-loaded LNP at room temperature for 30 minutes, the cryopreservation survival rate reached a high level and no longer changed with the incubation time, indicating that the 30-minute incubation of LNP is sufficient to meet the cryopreservation requirements of red blood cells. The red blood cells were incubated with 0.05, 0.1, 0.2, and 0.5 mg / mL trehalose-loaded LNP solutions in a 0.54 M pH = 6.5 trehalose solution at room temperature for 30 minutes, and the cryopreservation survival rate and hemolysis of the red blood cells were measured. The cryopreservation results are shown in Figure 2. Figure 4 As shown in C, the cryopreservation survival rate of red blood cells increases with the increase of trehalose-loaded LNP concentration, reaching the highest at 0.2 mg / mL. The higher concentration of 0.5 mg / mL trehalose-loaded LNP actually reduces the survival rate of red blood cells. This is because high concentrations of trehalose-loaded LNP can cause hemolysis of red blood cells. 0.1 mg / mL trehalose-loaded LNP was mixed with red blood cells in 0.18, 0.36, 0.54, 0.72, and 0.90 M trehalose solutions, incubated at room temperature for 30 minutes, and the cryopreservation survival rate of red blood cells was measured. The results are shown in Figure 4 As shown in D, the cryopreservation survival rate of red blood cells increases with the increase of trehalose concentration, reaching a relatively high level at 0.72-0.90 M, indicating that high concentrations of trehalose can be used for cryopreservation.

[0106] (3) Freeze-dried trehalose-loaded LNP for red blood cell cryopreservation

[0107] After ultrafiltration and centrifugation of the prepared trehalose-loaded LNP to remove ethanol, 1 mL of 5 mg / mL trehalose-loaded LNP solution was placed in a glass bottle and pre-frozen in a freeze dryer (pre-freezing temperature -60°C, pre-freezing time 3 hours). Immediately after the pre-freezing is completed, the initial freeze-drying is performed (freeze-drying temperature -60°C, time 12 hours). After the initial drying, secondary drying is performed (freeze-drying temperature 20°C, time 3 hours) to obtain trehalose-loaded LNP freeze-dried powder. 1 mL of ultrapure water was directly added to the freeze-dried powder to dissolve and reconstruct the trehalose-loaded LNP solution, and its particle size and potential were measured using a dynamic light scattering instrument. Referring to the above-mentioned red blood cell freezing method, the freeze-dried and reconstituted trehalose-loaded LNP was used to freeze red blood cells. The results are as follows: Figure 5 shown. Figure 5 Middle A shows that the appearance of trehalose-loaded LNP after freeze-drying and reconstitution is consistent with that of fresh trehalose-loaded LNP; Figure 5 Figures B and C show that the particle size and potential properties of the reconstituted trehalose-loaded LNPs are consistent with those of the freshly prepared product; Figure 5 The results of red blood cell cryopreservation in D showed that freeze-dried trehalose-loaded LNP also had a good red blood cell cryopreservation protection effect.

[0108] (4) Functional test of cryopreserved and revived red blood cells loaded with trehalose LNP

[0109] 0.2 mg / mL trehalose-loaded LNPs were mixed with red blood cells in a 0.72 M trehalose solution (pH 7.4 and pH 6.5), incubated at room temperature for 30 minutes, and then quickly frozen in liquid nitrogen. After ultra-low temperature storage for no less than 2 hours, the frozen red blood cells were taken out and quickly thawed at 37°C, centrifuged at 1800g for 5 minutes, and the red blood cells were collected and suspended in a 0.33× PBS solution. The functional indicators of red blood cells were tested. The results are as follows Figure 6 As shown, Figure 6 A in the figure indicates the ATP content. Figure 6 B in the figure indicates the MetHb content. Figure 6 C in the middle represents 2,3-DPG, Figure 6 D in the middle represents the PS exposure rate of the cell membrane. It can be seen that the above functional indicators of red blood cells frozen with trehalose LNP are not significantly different from those of fresh red blood cells, showing a good cryopreservation protection effect; in contrast, the 2,3-DPG content of red blood cells frozen with trehalose alone is reduced and the PS exposure rate is increased, indicating that the red blood cell membrane is damaged and the oxygen carrying capacity is weakened after freezing.

[0110] The morphology and structure of the frozen and revived red blood cells were also observed using a scanning electron microscope. Figure 7It can be seen that fresh red blood cells have a full shape and smooth edges. In contrast, most of the cells frozen with trehalose alone will be deformed, with twisted edges, similar to jagged edges, and uneven. After adding lipid nanoparticles, this phenomenon is effectively improved. Regardless of pH 6.5 or pH 7.4, the cell morphology remains good.

[0111] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for freezing red blood cells based on lipid nanoparticles carrying cryoprotectants, characterized in that: Includes steps: A cryoprotectant-loaded lipid nanoparticle solution is provided, wherein the cryoprotectant-loaded lipid nanoparticle solution comprises cryoprotectant-loaded lipid nanoparticles, wherein the cryoprotectant-loaded lipid nanoparticles comprise lipid nanoparticles and a cryoprotectant loaded in the lipid nanoparticles, wherein the lipid nanoparticles are composed of ionizable lipids, wherein the ionizable lipids have good biological safety and a pKa value between 6 and 7, and wherein the cryoprotectant is a cryoprotectant for non-permeable red blood cells; Mixing the cryoprotectant-carrying lipid nanoparticle solution, red blood cells and the cryoprotectant solution to obtain a mixed solution; The pH of the mixed solution is controlled to be lower than the pKa value of the ionizable lipid, and the mixture is incubated at 15-40° C. for more than 5 minutes. The red blood cell solution obtained after the incubation is frozen in liquid nitrogen.

2. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 1, characterized in that: The concentration of the cryoprotectant-loaded lipid nanoparticles in the cryoprotectant-loaded lipid nanoparticle solution is 0.05-0.5 mg / mL, the concentration of the cryoprotectant solution is above 0.54 M, and the volume ratio of the cryoprotectant-loaded lipid nanoparticle solution to the cryoprotectant solution is 1:10-1:

100.

3. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 1, characterized in that: The incubation time is 5 to 60 minutes.

4. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 1, characterized in that: The pH of the mixed solution is controlled to be lower than the pKa value of the ionizable lipid using hydrochloric acid, acetic acid or citric acid.

5. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 1, characterized in that: The cryoprotectant is a macromolecular polymer or a sugar compound.

6. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 5, characterized in that: The sugar compound is one or more of trehalose, sucrose, glucose and mannitol.

7. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 1, characterized in that: The cryoprotectant-carrying lipid nanoparticle solution is composed of cryoprotectant-carrying lipid nanoparticles and a cryoprotectant solution; The cryoprotectant solution consists of a cryoprotectant and a buffer isotonic with red blood cells.

8. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 1, characterized in that: The method for preparing the cryoprotectant-loaded lipid nanoparticles comprises the following steps: preparing a lipid phase solution, wherein the lipid phase solution comprises an ionizable lipid and a solvent; preparing an aqueous phase solution, wherein the aqueous phase solution comprises a cryoprotectant and a buffer; The lipid phase solution and the aqueous phase solution are mixed at a lipid-water flow rate ratio of 1:2 to 1:10, and the solvent is removed by dialysis or ultrafiltration to obtain lipid nanoparticles loaded with cryoprotectant.

9. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 8, characterized in that: The lipid phase solution includes 1,2-distearoyl-sn-glycero-3-phosphocholine, octadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 and anhydrous ethanol; Alternatively, the lipid phase solution includes 1,2-distearoyl-sn-glycero-3-phosphocholine, ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 and anhydrous ethanol; The aqueous phase solution includes trehalose and a buffer; The total flow rate of lipid and water is 4.5 mL / min.

10. The method for cryopreservation of red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 1, characterized in that: The cryoprotectant-loaded lipid nanoparticles are freeze-dried cryoprotectant-loaded lipid nanoparticles, and the cryoprotectant-loaded lipid nanoparticle solution is prepared by dissolving freeze-dried cryoprotectant-loaded lipid nanoparticles in water.

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