CN119678911A

CN119678911A - A method for cryopreservation of red blood cells based on lipid nanoparticles carrying cryoprotectants

Info

Publication number: CN119678911A
Application number: CN202411905100.9A
Authority: CN
Inventors: 魏裕双; 元冰; 李向阳
Original assignee: Songshan Lake Materials Laboratory
Current assignee: Songshan Lake Materials Laboratory
Priority date: 2024-12-23
Filing date: 2024-12-23
Publication date: 2025-03-25

Abstract

The invention discloses a method for cryopreserving erythrocytes based on lipid nanoparticles carrying a cryopreservation protective agent, and belongs to the field of biological medicines. The method for freezing and storing the red blood cells based on the lipid nanoparticles carrying the freezing and storing protective agent comprises the steps of mixing the lipid nanoparticle solution carrying the freezing and storing protective agent, the red blood cells and the freezing and storing protective agent solution to obtain a mixed solution, controlling the pH value of the mixed solution to be lower than the pKa value of the ionizable lipid, incubating the mixed solution for more than 5 minutes at 15-40 ℃, and then freezing the mixed solution in liquid nitrogen. The invention utilizes the ionized lipid nano particles to load the cryopreservation protective agent, utilizes the pH to regulate the fusion of the lipid nano particles and cell membranes, effectively improves the delivery effect of the cryopreservation protective agent in erythrocytes, and promotes the cryopreservation of erythrocytes. The invention has simple freezing operation, can be used for freezing only by mixing at room temperature for a short time, has the thawing recovery rate of the frozen erythrocytes up to 85 percent, has normal functions and meets the clinical use requirement, and the thawed erythrocytes can be used for subsequent blood transfusion without complex post-treatment and simple cleaning.

Description

Method for cryopreserving erythrocytes based on lipid nanoparticles carrying cryopreservation protectant

Technical Field

The invention relates to the field of biological medicine, in particular to a method for freezing and storing erythrocytes based on lipid nano particles carrying a freezing and storing protective agent.

Background

Erythrocyte infusion is critical for most patients who need to be supplemented with erythrocytes and have an increased capacity to carry oxygen, such as those suffering from acute blood loss caused by trauma or surgery, cardiac, renal, hepatic insufficiency, and other blood-related diseases. In order to ensure the supply of erythrocytes, various methods of preserving erythrocytes have been developed. Currently, red blood cell products are typically stored in isotonic solutions at 4 ℃ for no more than 42 days, whereas cryopreservation can allow long-term storage of red blood cells, helping to stabilize the red blood cell supply. Cryopreservation is to freeze cells at ultra-low temperatures (-65 to-196 ℃) to maintain their structure and function, which relies on special cryopreservation protectants and cryopreservation-resuscitation procedures to protect erythrocytes from damage due to ice crystal formation and solute changes during the freezing and thawing process.

Currently, glycerol is used for freezing erythrocytes mainly in clinic, and is classified into a slow freezing method, wherein glycerol with a final concentration of 40% (mass/volume) is stored at a temperature below-65 ℃, or a fast freezing method, wherein 18% glycerol is frozen at a liquid nitrogen temperature (-196 ℃) and stored in liquid nitrogen vapor (-156 ℃) for 10 years. In the above method, the glycerol preparation is usually only incubated with the erythrocytes for 30 minutes at room temperature for freezing. However, although glycerol has long freezing time and good resuscitation effect, glycerol itself has toxicity, and can cause side effects such as hemolysis and change of erythrocyte morphology during freezing. The long and complex deglyceridization process required before the frozen cell transfusion can be used, and this process involves multiple washing steps, which can lead to additional hemolysis and morphological changes of the red blood cells. In addition, glycerol remaining in the cells may also cause complications in some patients. The above drawbacks of the glycerol method limit the wide application of frozen cells and do not allow for a large supply of high quality erythrocytes in a short time. Therefore, development of a new cryopreservation solution and a cryopreservation method for erythrocytes is needed, which have good cryopreservation effect and do not require long-time post-treatment, so that patients can obtain blood better.

Trehalose is a natural cryoprotectant that has been of great interest because of its ability to protect cells and organisms from a variety of stresses, including freezing, drying, heat shock, oxidative and osmotic stresses. The polyhydroxy structure of trehalose enables the trehalose to combine with lipid membranes and proteins, thereby replacing the lost combined water, reducing the damage of ice crystals in the freezing process, simultaneously maintaining the water/solute in and out rate of cells in the freezing process, and maintaining the cell morphology. In addition, trehalose is an impermeable erythrocyte cryoprotectant, avoiding an expensive and cumbersome deglyceridization procedure, so that cryopreserved erythrocytes can be immediately used for transfusion when needed. However, trehalose must be present on both sides of the cell membrane to have the best protective effect, whereas under normal conditions, erythrocytes have no active uptake activity and the membrane permeability is low. Therefore, trehalose needs to be delivered into erythrocytes to achieve a better cryopreservation effect, and currently developed schemes include photo-thermal, ultrasound, electro-osmotic, microinjection, membrane-penetrating peptides, membrane-penetrating polymers, biomimetic nano-carriers such as Lipid Nanoparticles (LNP), genetic engineering, etc., wherein most of the methods are still working to increase the recovery rate of erythrocytes after freeze thawing and improve their practicality.

To date, LNP has been widely used in drug and gene delivery due to excellent biosafety and cell delivery effects. Some protocols for the delivery of trehalose based on LNP have also been developed, including the use of negatively charged phosphatidylserine PS-LNP with 100-fold higher trehalose than neutral charge delivery to 15mM after 24 hours incubation at 37℃with significantly improved erythrocyte cryopreservation survival. Another study increased the cryopreservation viability from 29% to 64% of trehalose after 4 hours incubation with erythrocytes at 37 ℃ using LNP containing PS and dipalmitoyl lecithin (DPPC). The LNP with high concentration (up to 1 mM) can improve the concentration of intracellular trehalose to 80mM to a certain extent, thereby effectively improving the survival rate of freezing. It can be seen that the above scheme has many problems such as long incubation time of erythrocytes and LNP, poor cryopreservation effect or large LNP usage.

Accordingly, the prior art is still in need of improvement and development.

Disclosure of Invention

In view of the shortcomings of the prior art, the invention aims to provide a method for cryopreserving erythrocytes based on lipid nanoparticles carrying a cryopreservation protective agent, and aims to solve the problems of long incubation time of erythrocytes and LNP, poor cryopreservation effect, large LNP usage amount and the like in the existing method for delivering trehalose to cryopreserve erythrocytes based on LNP.

The technical scheme of the invention is as follows:

A method for cryopreserving erythrocytes based on lipid nanoparticles carrying a cryopreservation protectant, comprising the steps of:

Providing a lipid nanoparticle solution carrying a cryopreservation protective agent, wherein the lipid nanoparticle solution carrying the cryopreservation protective agent comprises lipid nanoparticles carrying the cryopreservation protective agent, the lipid nanoparticles carrying the cryopreservation protective agent comprise lipid nanoparticles and the cryopreservation protective agent loaded in the lipid nanoparticles, the lipid nanoparticles are composed of ionizable lipids, the ionizable lipids are good in biological safety, the pKa is between 6 and 7, and the cryopreservation protective agent is a cryopreservation protective agent of non-permeable erythrocytes;

Mixing the lipid nanoparticle solution carrying the cryopreservation protective agent, the red blood cells and the 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 ionizable lipid, incubating for more than 5 minutes at 15-40 ℃, and placing the erythrocyte solution obtained after incubation in liquid nitrogen for freezing.

Optionally, the concentration of the freeze-carried protective agent lipid nanoparticle in the freeze-carried protective agent lipid nanoparticle solution is 0.05-0.5 mg/mL, the concentration of the freeze-carried protective agent solution is above 0.54M, and the volume ratio of the freeze-carried protective agent lipid nanoparticle solution to the freeze-carried protective agent solution is 1:10-1:100.

Optionally, the incubation time is 5-60 min.

Optionally, the pH of the mixture is controlled below the pKa value of the ionizable lipid using hydrochloric acid, acetic acid, or citric acid.

Optionally, the cryopreservation protective agent is a macromolecular polymer or a saccharide compound.

Optionally, the saccharide compound is one or more of trehalose, sucrose, glucose and mannitol.

Optionally, the cryoprotectant-loaded lipid nanoparticle solution consists of cryoprotectant-loaded lipid nanoparticles and a cryoprotectant solution;

the cryopreservation protective agent solution consists of a cryopreservation protective agent and a buffer solution isotonic with red blood cells.

Optionally, the preparation method of the lipid nanoparticle carrying the cryopreservation protective agent comprises the following steps:

preparing a lipid phase solution comprising an ionizable lipid and a solvent;

Preparing an aqueous phase solution, wherein the aqueous phase solution comprises a freezing protecting agent and a buffer solution;

and mixing the lipid phase solution and the aqueous phase solution according to a lipid water flow ratio of 1:2-1:10, and removing the solvent by dialysis or ultrafiltration to obtain the lipid nanoparticle carrying the cryopreservation protective agent.

Alternatively, the lipid phase solution comprises 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, octadeca-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate, cholesterol, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, and absolute ethanol;

Or the lipid phase solution comprises 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, ((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 absolute ethanol;

the aqueous phase solution comprises trehalose and buffer solution;

the total flow rate of lipid water was 4.5mL/min.

Optionally, the freeze-drying and freeze-preserving protective agent-carrying lipid nanoparticle is a freeze-drying and freeze-preserving protective agent-carrying lipid nanoparticle, and the freeze-drying and freeze-preserving protective agent-carrying lipid nanoparticle solution is prepared by dissolving the freeze-drying and freeze-preserving protective agent-carrying lipid nanoparticle in water.

The invention has the beneficial effects that the ionized lipid nanoparticle is used for loading the cryopreservation protective agent, the pH is used for regulating the fusion of the lipid nanoparticle and the cell membrane (the pH is used for regulating the lipid nanoparticle to be positive charge, the surface of the erythrocyte membrane is negative charge, and the adsorption and fusion of the lipid nanoparticle and the cell membrane are promoted by the interaction of positive and negative charges), so that the delivery effect of the cryopreservation protective agent in the erythrocyte is effectively improved, and the cryopreservation of the erythrocyte is promoted. The invention shows that the incubation can achieve better erythrocyte cryopreservation effect in a short time at room temperature, the incubation time can be 5min or more at the temperature of 15-40 ℃ according to specific requirements, and the incubation can be carried out by standing or rotating and mixing at different speeds so as to achieve better erythrocyte cryopreservation effect. Compared with a glycerol freezing method used clinically, the method has the advantages that freezing operation is simple and easy to obtain, freezing can be achieved only by mixing at room temperature for a short time, the thawing recovery rate of frozen erythrocytes can reach 85%, the functions are normal, clinical use requirements are met, the thawed erythrocytes do not need complex post-treatment similar to the glycerol method, can be used for subsequent blood transfusion by only needing one-time simple cleaning, and the lyophilized lipid nanoparticle carrying the freezing protective agent has the same freezing protective effect, so that the application range of the method is greatly promoted. Therefore, compared with the clinical glycerol cryopreservation method of the erythrocytes, the method is simple, convenient and efficient, greatly reduces post-treatment steps, and is favorable for timely and high-quality supply of the cryopreserved erythrocytes.

Drawings

FIG. 1 is a graph showing the effects of fusion and cryopreservation of LNP and erythrocytes of different particle sizes.

FIG. 2 is a graph showing the effects of fusion and cryopreservation of LNP and erythrocytes of different compositions.

FIG. 3 is a graph of LNP for erythrocyte cryopreservation using ALC-0315 lipid composition.

FIG. 4 is a graph of LNP and erythrocyte cryopreservation condition optimization.

Fig. 5 is a freeze-dried LNP for erythrocyte cryopreservation.

FIG. 6 is a functional diagram of erythrocytes after cryopreservation and resuscitation.

Fig. 7 is a scanning electron microscope image of the morphology of erythrocytes after cryopreservation and resuscitation.

Detailed Description

The invention provides a method for cryopreserving erythrocytes based on lipid nanoparticles carrying a cryopreservation protective agent, which is used for making the purposes, technical schemes and effects of the invention clearer and more definite, and is further described in detail below. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The embodiment of the invention provides a method for cryopreserving erythrocytes based on lipid nanoparticles carrying a cryopreservation protective agent, which comprises the following steps:

Providing a lipid nanoparticle (also called as a lipid-based cryoprotectant LNP for short) solution of a lipid-based cryoprotectant, wherein the lipid nanoparticle comprises a lipid nanoparticle and a cryoprotectant loaded in the lipid nanoparticle, the lipid nanoparticle is composed of ionizable lipid, the biosafety of the ionizable lipid is good, the pKa is between 6 and 7, and the cryoprotectant is a cryoprotectant of non-permeable erythrocytes;

and controlling the pH value of the mixed solution to be lower than the pKa value of the ionizable lipid, incubating (which can be placed on a shaking table or a transverse axis mixer for incubation) at 15-40 ℃ for more than 5min (for example, 5-60 min), and placing the erythrocyte solution obtained after the incubation is finished in liquid nitrogen for freezing.

The high-concentration glycerol freezing method is used for freezing and storing the red blood cells, and the glycerol can permeate into the red blood cells, so that a complex and time-consuming deglyceridization process exists after the freezing and storing to reduce the concentration of the glycerol in the red blood cells, and the yield, the state and the subsequent use of the red blood cells are affected. Compared with the method, the cryopreservation protective agent used in the embodiment of the invention has good safety, the cryopreservation protective agent has no cell membrane permeability and can not enter erythrocytes independently, and the cryopreservation protective agent in erythrocytes is delivered by lipid nanoparticles, so that the concentration is low and no extra safety risk is caused, therefore, the extracellular high-concentration cryopreservation protective agent (such as trehalose) can be removed by centrifugal cleaning for only once after the erythrocytes are frozen, the cryopreservation protective agent can be used without complex post-treatment, and the convenience, timeliness and safety of using the cryopreserved erythrocytes are greatly improved.

The embodiment of the invention uses the ionized LNP cryopreserved erythrocyte, realizes the high-efficiency delivery of the cryopreservation protective agent based on the fusion of the LNP and erythrocyte membrane by regulating and controlling the pH, and has good cryopreservation protective agent delivery and erythrocyte cryopreservation effects below the pKa value of the ionized lipid. Whereas the known LNP delivery of trehalose relies on methods of red cell membrane lipid profiling, the use of positively charged lipids, the use of high concentrations of LNP, etc., trehalose delivery is inefficient or more harmful components are introduced.

According to the embodiment of the invention, the ionized LNP frozen erythrocytes are used, after the delivery of the frozen protective agent and the frozen erythrocytes are realized at low pH, the erythrocytes after incubation or frozen-thawed can be resuspended in a physiological environment solution (pH 7.4), LNP on erythrocyte membranes becomes negative charge, and the toxicity of the positively charged LNP is eliminated, so that the erythrocytes can be safely used.

In the embodiment of the invention, incubation of the LNP carrying the cryopreservation protective agent and the erythrocytes can be completed at room temperature, complicated temperature control equipment and operation are not needed, and the LNP carrying cryopreservation protective agent is consistent with a glycerol method, has good clinical practicability and is obviously superior to other LNP cryopreservation methods.

In the embodiment of the invention, the incubation of LNP carrying the cryopreservation protective agent and erythrocytes can be used for cryopreservation at room temperature for not more than 30 minutes, the time is short, and the LNP carrying the cryopreservation protective agent is consistent with a glycerol method and is obviously superior to the long-time incubation requirements in other LNP cryopreservation methods.

In the embodiment of the invention, incubation of the LNP carrying the cryopreservation protective agent and the erythrocytes can be performed in a cryopreservation protective agent solution, wherein the concentration of the cryopreservation protective agent is above 0.54M, and the LNP carrying the cryopreservation protective agent has good cryopreservation effect. The impermeable cryopreservation protectant can be delivered into erythrocytes using the methods of the embodiments of the invention, thereby improving the cryopreservation effect. The cryoprotectant may be a macromolecular polymer or a saccharide compound. For example, the cryoprotectant may be trehalose, but of course, trehalose may be replaced with other cryoprotectants with good biosafety and impermeable membranes, such as sucrose, glucose, mannitol, and the like.

In the embodiment of the invention, the freeze-dried LNP carrying the freeze-dried protective agent still has good erythrocyte freeze-drying effect, while the freeze-dried LNP carrying the freeze-dried protective agent has longer stability, and the re-dissolution of the LNP carrying the freeze-dried protective agent only needs to be added with a certain amount of pure water, thereby greatly facilitating the freeze-drying operation. The freeze-dried protective agent LNP is prepared into the freeze-dried powder, so that the stability and the preservation time of the material are greatly prolonged, the storage and the transportation are more convenient, the use is convenient and effective, and the clinical application prospect is greatly improved. There is currently no method known for cryopreserving erythrocytes using the lyophilized cryoprotectant LNP.

Further, the concentration of the lipid nanoparticle carrying the cryopreservation protective agent in the lipid nanoparticle carrying the cryopreservation protective agent solution is 0.05-0.5 mg/mL, the concentration of the lipid nanoparticle carrying the cryopreservation protective agent solution is more than 0.54M, and the volume ratio of the lipid nanoparticle carrying the cryopreservation protective agent to the lipid nanoparticle carrying the cryopreservation protective agent solution is 1:10-1:100, such as 1:10, 1:20, 1:40, 1:50, 1:70, 1:80, 1:100 and the like.

The embodiment of the invention relies on the use of ionizable lipid to form LNP, so that the delivery of the cryopreservation protective agent and the cryopreservation effect of erythrocytes can be improved by regulating and controlling the fusion of the LNP and cell membranes. Therefore, other ionizable lipids can be used for synthesizing the ionizable LNP for cryopreservation of erythrocytes as long as the requirements of good biosafety and pKa between 6 and 7 are met.

The lipid composition and composition of LNP can be changed, so long as pH-mediated LNP charge transformation and cell membrane fusion can be realized, and the LNP can be used for erythrocyte cryopreservation, including but not limited to regulating the molar ratio of each lipid based on the existing components, and five-component, six-component or even more complex LNP can be obtained by using new lipids or continuously adding new lipids.

In one embodiment, the lipid nanoparticle comprises an ionizable lipid composition of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), octadecan-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate (SM-102), cholesterol (Chol), and 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-mPEG 2000).

In one embodiment, the lipid nanoparticle comprises an ionizable lipid profile of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, ALC-0315, cholesterol, and 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000.

In one embodiment, the method for preparing the lipid nanoparticle carrying the cryopreservation protective agent comprises the following steps:

preparing a lipid phase solution comprising an ionizable lipid and a solvent (e.g., absolute ethanol, etc.);

Preparing an aqueous phase solution, wherein the aqueous phase solution comprises a cryopreservation protective agent and a buffer solution isotonic with red blood cells;

Wherein, the total flow rate of the fat water is 4.5mL/min.

In a specific embodiment, the method for preparing the trehalose-loaded lipid nanoparticle (trehalose-loaded LNP) comprises the steps of:

1, 2-distearoyl-sn-glycero-3-phosphorylcholine, octadecane-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) caprylate, cholesterol and 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were mixed in absolute ethanol according to a molar ratio of 10:50:38:2 to give a lipid phase solution, and trehalose was dissolved in a buffer isotonic with erythrocytes to give an aqueous phase solution. And preparing the trehalose-carrying LNP by using a microfluidic device with a herringbone configuration chip at a lipid-water flow ratio of 1:2-1:10, and removing ethanol by using dialysis or ultrafiltration. And diluting the trehalose-carrying LNP to a determined concentration by using a trehalose solution (prepared from a buffer solution with trehalose and red blood cells being isotonic), thus obtaining the trehalose-carrying LNP solution.

In a specific embodiment, a method for cryopreserving erythrocytes using trehalose-loaded LNP comprises the steps of:

mixing fresh human blood erythrocytes and a trehalose-loaded LNP solution into the trehalose solution according to a certain proportion, controlling the pH of the solution to be below 6.5, and incubating at room temperature (such as 25 ℃) for 5-60 min by using a transverse axis mixer. After the incubation, the red blood cell solution was frozen directly in liquid nitrogen.

When the erythrocytes are needed to be used, taking out the cryopreserved erythrocyte solution, rapidly thawing at 37-40 ℃, centrifuging for 5 minutes at 1800g, removing the supernatant, and re-suspending the erythrocytes in isotonic buffer solution or normal saline to be used for blood transfusion.

The present invention will be described in detail with reference to the following examples.

1. Experimental data

The main chemicals and reagents were purchased from merck, ala Ding Shiji and mansion, inc. Wherein 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), cholesterol (Chol) and 1, 2-dipalmitol-sn-glycero-3-phosphoethanolamine-N- (lissamine rhodamine B sulfonyl) (RhB-PE, available from the merck company 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-mPEG 2000) were purchased from the Xiamen Saunopong company, and trehalose and absolute ethanol were purchased from the Aba Ding Shiji company. Suspension red blood cells collected from healthy volunteers were supplied by Dongguan city people hospital.

The main instrument devices are microfluidic chip (LNP-B1, shanghai Pengpan Biotechnology Co., ltd.), syringe pump (LM 2-C, zheng Zhoulu Mich., ltd.), dynamic light scattering instrument (NanoTrac Wave II, USA, microTrac), multifunctional microplate detector (Synergy H1, USA, bioTek), freeze dryer (LC-10N-60A, shanghai Libang West Instrument Co., ltd.), confocal fluorescence microscope (AX R, japan, nikon), flow cytometer (CytoFLEX S, beckman, USA), scanning electron microscope (SU 8010, japan, hitachi).

2. Detection method

(1) Trehalose-carrying LNP particle size and potential

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

(2) Trehalose loading rate of trehalose-loaded LNP

The internal trehalose content of LNP was measured using anthrone colorimetry. 100 mu L of the purified trehalose-carrying LNP solution is added with 900 mu L of trichloroacetic acid solution to extract trehalose, the mixture is placed for 45 minutes at room temperature, and 8000g of the mixture is centrifuged for 10 minutes after repeated shaking for 3 times, and the supernatant is collected. 60. Mu.L of the supernatant was added to 240. Mu.L of an anthrone solution (80% dissolved in H ₂SO₄), and after heating at 95℃for 15min, the absorbance at 620nm was measured. And taking a trehalose aqueous solution within the concentration range of 0-0.2 mg/mL, and obtaining a standard curve of trehalose concentration and absorbance by referring to the method. The trehalose concentration in the trehalose-loaded LNP samples was calculated from the standard curve and the trehalose loading rate was calculated by the following formula (1):

Trehalose loading ratio LC (%) =trehalose mass in trehalose-carrying LNP/(trehalose mass in trehalose-carrying lnp+lnp mass) ×100 (1)

(3) LNP hemolysis test

100. Mu.L of red blood cells are respectively mixed with 400. Mu.L of deionized water, 0.33 XPBS and 0.54M trehalose solution with the concentration of 0.05-0.5 mg/mL trehalose-carrying LNP, so that the hematocrit is finally kept at 30%. After incubating the mixture at 25℃for 1 hour, 1800g was centrifuged for 5 minutes, and the supernatant was collected and absorbance of the supernatant at 540nm was measured by an ultraviolet spectrophotometer. In the experiment, red blood cells are mixed with deionized water to form a positive control (A ₀) which is completely hemolyzed, red blood cells are mixed with 0.33 XPBS to form a negative control (A ₁) which is basically not hemolyzed, red blood cells are mixed with trehalose-carrying LNP with different concentrations to form a sample group (A _LNP), and the hemolysis rate of the red blood cells is calculated according to the following formula (2):

Hemolysis ratio (%) = (a _LNP-A₁)/(A₀-A₁) ×100 (2)

(4) LNP and erythrocyte fusion

Trehalose was dissolved in 0.33×pbs to a concentration of 0.54M to obtain a trehalose cryopreservation solution isotonic with erythrocytes. When preparing the trehalose-carrying LNP, the RhB-PE label with the molar ratio of 0.1% is doped to obtain the fluorescence trehalose-carrying LNP, the fluorescence trehalose-carrying LNP is diluted in 0.54M trehalose cryopreservation solution, the concentration of the trehalose-carrying LNP is 0.2mg/mL, and the pH is 7.4 and 6.5 respectively, so as to obtain the LNP trehalose solution. After removal of serum purification from erythrocytes by centrifugation at 1800g for 5 min, they were mixed with the above-mentioned LNP trehalose solution, wherein the hematocrit was controlled to 30%. After 30 minutes incubation at room temperature, 1800g was centrifuged for 5 minutes and the erythrocytes were collected, and the increased fluorescence (ex.561 nm, em.591 nm) of LNP fusion in the erythrocytes was measured using a multi-functional microplate detector while observing the fusion status of LNP on the erythrocytes using a confocal microscope.

(5) Measurement of trehalose in erythrocytes

Mu.L of erythrocytes were mixed with 0.2mg/mL trehalose-loaded LNP in 0.54M trehalose solution, pH was controlled to 6.5 and 7.4, hematocrit 30%, incubated for 30min at room temperature, and after incubation the cells were washed 3 times with 0.33 XPBS at 1800g centrifugation min. Red blood cells are collected, lysed with trichloroacetic acid, cell membranes and proteins are precipitated, the cells are placed at room temperature for 45 minutes, after repeated shaking for 3 times, 8000g of centrifugation is carried out for 10 minutes, the supernatant is collected, 60 mu L of supernatant is added into 240 mu L of anthrone solution, absorbance at 620nm wavelength is measured after heating at 95 ℃ for 15 minutes, trehalose concentration in a sample is calculated according to a trehalose standard curve, and the intracellular trehalose concentration (mM) is obtained through a formula (3).

Erythrocyte trehalose concentration (mM) =m _RBC-Tre/(342.3×0.7×V×RBC×HCT)×10⁶ (3)

Where M _RBC-Tre represents the mass (g) of trehalose in the LNP incubated erythrocytes measured, 342.3 (g/mol) is the molecular weight of trehalose, V (mL) is the volume of the erythrocyte suspension, RBC is the erythrocyte concentration (×10 ¹²/L), MCV is the mean erythrocyte volume (fL), 0.7 represents the erythrocyte volume index, since about 70% of cytoplasmic erythrocytes can be contacted with water, while the residues are filled by the cell membrane.

(6) Erythrocyte cryopreservation survival rate

Mu.L of blood cells were mixed with 0.2mg/mL trehalose-loaded LNP in 0.54M trehalose solution, pH was controlled to 6.5 and 7.4, hematocrit 30%, incubated for 30 minutes at room temperature, and the mixture was rapidly frozen in liquid nitrogen and kept for at least 2 hours or more. The frozen tube was placed in a 37 ℃ water bath to thaw the sample, after which centrifugation at 1800g for 5 minutes, the supernatant was collected and absorbance was measured at 540nm (a _LNP). Meanwhile, 100. Mu.L of fresh erythrocytes were dispersed in 400. Mu.L of deionized water and 0.33 XPBS as a total hemolytic positive control (A ₀) and a non-hemolytic negative control (A ₁), respectively. The frozen survival rate of erythrocytes after thawing was calculated according to the following formula (4):

survival in frozen state (%) = (1- (a _LNP-A₁)/(A₀-A₁)) ×100 (4)

(7) Erythrocyte function test

For the cryopreserved and recovered erythrocytes, the relevant biological function changes were analyzed using fresh erythrocytes as a control, including Adenosine Triphosphate (ATP) analysis of intracellular energy changes, ATP kit (C0061M, bi yun, china) analysis, phosphatidylserine (PS) exposure rate to assess erythrocyte membrane damage, annexin V-FITC kit (C1062L, bi yun, china) staining, and analysis by flow cytometry, 2, 3-diphosphoglycerate (2, 3-DPG) content to assess erythrocyte oxygen affinity, ELISA kit (YX-E11265, enoki, china) to assess heme iron oxidation, and methemoglobin (hb) content to assess heme iron oxidation, using relevant quantification kit (BC 5600, labao, china).

(8) Erythrocyte morphology detection

The erythrocytes after freezing were thawed and resuscitated at 37 ℃, washed 2 times with 0.33×pbs, and fixed with 2.5% glutaraldehyde for 2 hours or more at 4 ℃. The cells were then washed 3 times with PBS, dehydrated with a series of ethanol solutions of different volume contents (20%, 40%,60%,80%,90% and 100% (twice for 15min each) and freeze-dried. Finally, the cell surface was gold sprayed and imaged with a scanning electron microscope (SU 8010, hitachi, japan).

3. Experimental protocol and test results

(1) Preparation of trehalose-loaded LNP and erythrocyte membrane fusion effect

DSPC, SM-102, chol, DMG-mPEG2000 and RhB-PE were dissolved in absolute ethanol at a concentration of 10mg/mL, respectively, and stored in a sealed dark place at 4 ℃. According to the molar ratio of DSPC to SM-102 to Chol to DMG-mPEG 2000=10:50:38:2, respectively taking 62, 280, 117 and 39 mu L of the lipid ethanol solution in sequence, mixing, and adding absolute ethanol to 1mL to obtain the lipid phase solution with the final concentration of 5 mg/mL. Wherein, if it is desired to prepare a fluorescent-labeled LNP, 9. Mu.L of RhB-PE diluted to 1mg/mL with absolute ethanol (equivalent to 0.1% mole ratio of total lipid) is additionally added to the lipid phase solution. Meanwhile, trehalose was weighed and dissolved in 0.33 XPBS to obtain 184.8mg/mL (0.54M) aqueous solution. And respectively loading the lipid phase solution and the aqueous phase solution into an injector, arranging the injector in an injection pump, and setting the ratio of lipid to water flow to be 1:2-1:10, wherein the total flow rate is 4.5mL/min, and the microfluidic chip is of a herringbone structure. Collecting effluent liquid to obtain trehalose-carrying LNP with different particle sizes, removing ethanol in the trehalose-carrying LNP solution by using a dialysis bag with molecular weight cut-off of 3400Da, and dialyzing with 0.33 XPBS. The dialyzed trehalose-loaded LNP solution was loaded into a ultrafiltration tube having a molecular cutoff of 100kDa, centrifuged at 1500g for 10min, the cutoff was collected, and 0.54M trehalose solution was added to a final volume of 1mL. According to the concentration-fluorescence intensity standard curve of RhB-PE, calculating the concentration of the trehalose-carrying LNP in the obtained trehalose-carrying LNP solution, and finally adding 0.54M trehalose solution to adjust the concentration of the trehalose-carrying LNP to 2mg/mL. The particle size and potential of the trehalose-carrying LNP at pH 7.4 and pH 6.5 were measured using a dynamic light scattering instrument, and the results are shown in Table 1 below, and it can be seen that as the lipid/water flow ratio was changed from 1:2 to 1:10, the particle size of the trehalose-carrying LNP was increased from 137.6nm to 265.1nm, i.e., as the total flow rate was unchanged, the lipid phase flow rate was faster, the particle size was smaller, the surface potential was measured to show that all the trehalose-carrying LNP was negatively charged at pH 7.4 and positively charged at pH 6.5, and the trehalose loading rate of the trehalose-carrying LNP was increased from 44.6% to about 60% as the particle size was increased.

TABLE 1

The prepared trehalose-loaded LNP solution was stored at 4 ℃ in a dark place, and the particle size and the trehalose loading rate were measured at intervals, and the storage stability of the trehalose-loaded LNP was found, and the results are shown in table 2 below. Taking the trehalose-carrying LNP with the particle size of 180nm as an example, the particle size is not changed greatly in 14 days of storage, the trehalose loading rate is kept unchanged, and no trehalose leakage exists, so that the trehalose-carrying LNP stored at the temperature of 4 ℃ can be normally used in at least 14 days.

TABLE 2

The prepared trehalose-carrying LNP with different particle sizes was diluted to 0.2mg/mL with 0.54M trehalose solution. Meanwhile, the obtained whole blood of healthy people is centrifuged for 5min at 1800g to collect red blood cells, and the red blood cells are mixed with the trehalose-loaded LNP diluted by the trehalose solution, the hematocrit is 30%, and the pH of the solution is respectively regulated to 7.4 and 6.5. After incubation for 30min at room temperature on a horizontal axis mixer, the LNP fusion rate with erythrocytes and the erythrocyte cryopreservation survival rate were measured and the results are shown in fig. 1. In FIG. 1, A shows that the fusion of LNP and erythrocytes is obviously higher than the performance of LNP and erythrocytes at pH 7.4 at pH 6.5, more red fluorescent LNP is fused on erythrocytes, in FIG. 1, B shows that the fusion effect of LNP and erythrocytes is not influenced by the particle size of the erythrocytes at the same concentration, in FIG. 1, C shows that the cryopreservation survival rate of erythrocytes is obviously higher than that of erythrocytes incubated with trehalose at pH 7.4 at pH 6.5, and is higher than that of erythrocytes incubated with trehalose alone, and the result also corresponds to the fusion result of LNP and erythrocytes in FIG. B, namely, the higher fusion efficiency of LNP and erythrocytes causes more trehalose internalization, thereby effectively improving the cryopreservation survival rate of erythrocytes.

In addition, the effect of LNP composition on erythrocyte cryopreservation was also examined. The particle size and potential of trehalose-loaded LNP at pH7.4 and pH 6.5 were measured using a dynamic light scatterometer with the amounts of Chol and DMG-mPEG2000 unchanged, adjusting SM-102:dspc=50:10, 40:20, 30:30, 20:40, 10:50, and 0:60 to prepare LNP, and the results are shown in table 3 below. It can be seen that the particle size of the downloaded trehalose LNP with different compositions is in the range of 200-250 nm, all the trehalose-carrying LNPs containing SM-102 show negative electricity at pH7.4 and positive electricity at pH 6.5, while the trehalose-carrying LNP without SM-102 has no pH responsiveness. The loading rate of trehalose in each trehalose-carrying LNP is 60% -70% and the difference is not great. Erythrocytes were mixed with these trehalose-loaded LNPs in 0.54M trehalose solution, pH controlled at 7.4 and 6.5, incubated at room temperature for 30min, after which the LNP fusion with erythrocytes and the cryopreservation survival of erythrocytes were measured and the results are shown in fig. 2. In fig. 2a shows the fusion of LNP with erythrocytes, it can be seen that the LNP has the highest fusion effect at SM-102:dspc=50:10, the use amount of SM-102 is reduced, the fusion efficiency of LNP with erythrocytes is reduced, and in fig. 2B shows the cryopreservation survival rate of erythrocytes, the result is consistent with the fusion result, i.e. the LNP of SM-102:dspc=50:10 has the best cryopreservation protection effect of erythrocytes.

TABLE 3 Table 3

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 the same lipid formulation was synthesized to give a new trehalose-loaded LNP, which was incubated with erythrocytes in 0.54M trehalose solution for 30 min at room temperature, pH was controlled at 6.0 and 7.4, and the cryopreservation protection effect on erythrocytes was measured, and the results are shown in fig. 3. FIG. 3A shows that the particle size of the trehalose-carrying LNP based on ALC-0315 is about 155nm, FIG. 3B shows that the zeta potential of the trehalose-carrying LNP varies with pH and is-17.5 mV at pH 7.4 and is converted into +21.5mV at pH 6.0, FIG. 3C shows that the fusion of the trehalose-carrying LNP based on ALC-0315 and erythrocytes is significantly improved at pH 6.0, and FIG. 3D shows that the trehalose-carrying LNP based on ALC-0315 has good cryopreservation effect at pH 6.0.

(2) Optimization of trehalose-loaded LNP cryopreserved erythrocyte protocol

The temperature, time and concentration of the trehalose-carrying LNP and the erythrocytes are used for incubating, and the concentration of the trehalose-carrying LNP and the concentration of the trehalose in the solution can influence the cryopreservation effect of the erythrocytes. After 0.2mg/mL of trehalose-loaded LNP solution and erythrocytes were incubated in 0.54M trehalose solution at pH 6.5 for one hour at 4, 25 and 37 ℃, the effect of cryopreservation of erythrocytes was examined, and as shown in FIG. 4A, the viability of erythrocytes at 25℃and 37℃was not significantly different, and was significantly higher than that of erythrocytes at 4℃indicating that LNP-mediated trehalose delivery and erythrocyte cryopreservation requirements could be met by ordinary 25℃or room temperature incubation. The viability of the erythrocytes was measured by incubating 0.2mg/mL trehalose-loaded LNP with erythrocytes in a trehalose solution at 0.54M ph=6.5 for 10, 20, 30, 60 minutes at room temperature, and as shown in fig. 4B, after incubation of erythrocytes with trehalose-loaded LNP for 30 minutes at room temperature, the viability reached a higher level and no longer changed with incubation time, indicating that the LNP incubation for 30 minutes was sufficient to meet the cryopreservation requirements of erythrocytes. The cryopreservation viability and hemolysis of erythrocytes were measured by incubating erythrocytes with 0.05, 0.1, 0.2, 0.5mg/mL of trehalose-loaded LNP solution in 0.54M ph=6.5 trehalose solution for 30 minutes at room temperature, and the cryopreservation results are shown in fig. 4C, wherein the cryopreservation viability of erythrocytes increases with increasing concentration of trehalose-loaded LNP, to the maximum at 0.2mg/mL, and the higher concentration of 0.5mg/mL of trehalose-loaded LNP conversely decreases the viability of erythrocytes, because high concentration of trehalose-loaded LNP causes hemolysis of erythrocytes. Mixing 0.1mg/mL of trehalose-loaded LNP with red blood cells in 0.18, 0.36, 0.54, 0.72, 0.90M trehalose solution, incubating at room temperature for 30 minutes, and measuring the cryopreservation survival rate of the red blood cells, as shown in FIG. 4D, shows that the cryopreservation survival rate of the red blood cells increases with increasing trehalose concentration, and reaches a relatively high level at 0.72-0.90M, which indicates that high concentration of trehalose can be used for cryopreservation.

(3) Freeze-dried trehalose-loaded LNP for cryopreservation of erythrocytes

After removing ethanol by ultrafiltration and centrifugation of the prepared trehalose-carrying LNP, 1mL of 5mg/mL of the trehalose-carrying LNP solution is placed in a glass bottle, and is placed in a freeze dryer for pre-freezing (pre-freezing temperature-60 ℃ C., pre-freezing time is 3 h), primary freeze drying (freeze drying temperature-60 ℃ C., time is 12 h) is immediately carried out after the pre-freezing is finished, and secondary drying (freeze drying temperature is 20 ℃ C., time is 3 h) is carried out after the primary drying, so that the trehalose-carrying LNP freeze-dried powder is obtained. 1mL of ultrapure water was directly added to the lyophilized powder to dissolve and reconstruct the trehalose-carrying LNP solution, and the particle size and potential thereof were measured using a dynamic light scattering instrument. The results of cryopreserving erythrocytes using freeze-dried reconstituted trehalose-loaded LNP with reference to the above-described erythrocyte cryopreservation method are shown in fig. 5. The freeze-dried and re-dissolved trehalose-carrying LNP is consistent with fresh trehalose-carrying LNP in appearance in FIG. 5, the particle size and potential properties of the re-dissolved trehalose-carrying LNP in FIG. 5 are consistent with those of a newly prepared product, and the freeze-dried trehalose-carrying LNP in FIG. 5 has good erythrocyte freezing protection effect.

(4) Trehalose-loaded LNP cryopreservation resuscitated erythrocyte function test

0.2Mg/mL of trehalose-loaded LNP was mixed with red blood cells in 0.72M trehalose solution (pH 7.4 and pH 6.5), and after incubation for 30 min at room temperature, liquid nitrogen flash frozen. After the cryopreservation for at least 2 hours, taking out the cryopreserved red blood cells, rapidly thawing at 37 ℃, centrifuging for 5 minutes at 1800g, collecting the red blood cells, suspending the red blood cells in a 0.33 XPBS solution, and detecting each functional index of the red blood cells, wherein the results are shown in a graph in FIG. 6, A in FIG. 6 shows the ATP content, B in FIG. 6 shows the MetHb content, C in FIG. 6 shows the 2,3-DPG, and D in FIG. 6 shows the PS exposure rate of the cell membranes. Compared with the red blood cells frozen by singly trehalose, the red blood cells frozen by the trehalose have reduced 2,3-DPG content and increased PS exposure rate, which indicates that the red blood cell membrane is damaged and the oxygen carrying capacity is reduced after the freezing.

The morphology of the cryopreserved and recovered erythrocytes was also observed by scanning electron microscopy, and the results are shown in fig. 7. Compared with the fresh red blood cells, the cells frozen by the trehalose have mostly deformed, distorted edges, saw-tooth-like and uneven edges, and the phenomenon is effectively improved after the lipid nano particles are added, and the cell morphology is kept good no matter the pH value is 6.5 or the pH value is 7.4.

It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Claims

1. A method for freezing red blood cells based on lipid nanoparticles carrying cryoprotectants, characterized in that it comprises the steps of:

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 cryoprotectant lipid nanoparticles according to claim 1 is characterized in that the concentration of the cryoprotectant lipid nanoparticles in the cryoprotectant 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 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, wherein 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 below 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, wherein the cryoprotectant is a macromolecular polymer or a carbohydrate compound.

6. The method for freezing red blood cells using lipid nanoparticles carrying cryoprotectants according to claim 5, wherein the carbohydrate 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, wherein the lipid nanoparticle solution carrying cryoprotectants is composed of lipid nanoparticles carrying cryoprotectants 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 containing cryoprotectants according to claim 1, wherein the preparation method of the lipid nanoparticles containing cryoprotectants comprises the steps of:

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, wherein the lipid phase solution comprises 1,2-distearoyl-sn-glycero-3-phosphocholine, octadecane-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)caprylate, 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 freezing red blood cells using cryoprotectant-loaded lipid nanoparticles 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.