Platelet-wrapped micro-hydrogel, freeze-dried powder, and preparation method, use method and application of freeze-dried powder

Abstract

The invention belongs to the technical field of biomedical engineering, and particularly relates to micro-hydrogel and freeze-dried powder for wrapping platelets as well as a preparation method, a use method and application of the micro-hydrogel and the freeze-dried powder. According to the micro-hydrogel wrapping the blood platelets, the micro-hydrogel is of a core-shell or multi-cavity structure, and the blood platelets are wrapped in the micro-hydrogel; the preparation method comprises the following steps: emulsifying a dispersed phase solution in a continuous phase solution, and spontaneously and physically gelatinizing to form micro-hydrogel to wrap platelets; the micro-hydrogel can reduce the damage to platelets caused by rehydration in the freeze-drying process, and can be stored for a long time after being freeze-dried; during use, dissociation can be achieved only through rehydration, centrifugation and heating, no extra decrosslinking agent needs to be added, and operation is convenient and fast. The blood platelets obtained after freeze-drying preservation and rehydration dissociation of the micro-hydrogel wrapping the blood platelets have higher hemostasis related glycoprotein expression and lower sialylation removal degree, and are expected to show better hemostasis capacity and longer in-vivo circulation time; the platelet storage time is expected to be greatly prolonged, and the platelet shortage situation is relieved.

Description

Technical Field

[0001] The present application belongs to the field of biomedical engineering technology, and specifically relates to a micro-hydrogel for encapsulating platelets, a freeze-dried powder, and a preparation method, a use method, and an application thereof. Background Art

[0002] Platelets are cell fragments produced by megakaryocytes. They are the key components of hemostasis in the blood and play a key role in hemostasis. Resting platelets are disc-shaped with a diameter of 2-3 μm. The concentration in a normal human body is (100-300)×10 9 / L. When a wound occurs, collagen in the extracellular matrix is ​​exposed. At this time, GPⅠbα (also known as CD42b) on the surface of platelets will bind to von Willebrand factor (vWF) and adhere to collagen with vWF as a bridge, which has the effect of initial hemostasis. Subsequently, collagen contacts GPVI on the surface of platelets, stimulating platelet activation and causing platelets to release agonists such as ADP and thrombin. These agonists can activate free platelets in the blood, change their morphology, and extend pseudopodia-like tentacles. Activated platelets have a larger specific surface area, and surface GPⅡb-Ⅲa (also known as CD41 / 61) will connect with fibrin and aggregate to form clots. In addition, platelets can also aggregate through CD42b and vWF. Activated platelets will further release more agonists, causing a coagulation cascade reaction to achieve the effect of hemostasis.

[0003] There is a huge demand for platelets in clinical practice. Emergency situations such as car accidents, surgical bleeding, and childbirth bleeding require immediate platelet transfusion to stop bleeding. In addition, platelet transfusion is an effective method for treating thrombocytopenia. The platelet concentration in patients with thrombocytopenia is less than 100×10 9 / L, if the capillary bleeding in the body cannot be stopped in time, the blood will be deposited on the skin surface to form purpura. As the number of platelets decreases, the bleeding worsens. Patients with very few platelets may suffer from massive blood loss in the digestive tract, or life-threatening bleeding may occur even if there is no injury in the brain. The "Chinese Expert Consensus on the Diagnosis and Treatment of Thrombocytopenia in Adults" states: "A platelet count <20×10 9 / L is accompanied by bleeding or other internal bleeding, transfusion of single-donor platelets is an effective treatment measure. "

[0004] Short shelf life is one of the main reasons for platelet shortage. The standard storage method for single-donor platelets is 25°C shaking storage, and the shelf life is only 3-5 days. In comparison, red blood cells can be stored at 4°C for 42 days, and plasma can be stored at -20°C for more than a year. Among various blood components, platelets have the shortest shelf life, which leads to a large number of platelets being discarded. The short shelf life of platelets stored at room temperature with shaking leads to a large amount of platelets being wasted, which cannot fully supply the platelet needs of patients with severe traumatic bleeding and thrombocytopenia.

[0005] Researchers have tried various methods to extend the shelf life of platelets, but have been unsuccessful.

[0006] As early as the last century, researchers tried to store platelets at 4°C, which once became the mainstream method. Refrigeration can reduce bacterial growth, inhibit protein degradation, and theoretically extend the shelf life of platelets. However, when stored at 4°C for a long time, the rewarming process will cause the platelets to be desialylated. Desialylated platelets will expose galactose, leading to rapid clearance in the liver, which is manifested as a significant decrease in the circulation time of platelets in the body and rapid clearance in the body, resulting in a significant weakening of the hemostatic effect. If refrigerated at 0°C for a short period of time, β-acetylglucosamine (β-GlcNAc) will be exposed on the surface of platelets and subsequently cleared by liver macrophages (Kupffer cells). Both of these situations will significantly reduce the circulation time and recovery rate of platelets. Since the beginning of this century, refrigerated platelets have gradually withdrawn from clinical use.

[0007] Some researchers have also tried freezing platelets. However, frozen platelets are also rapidly cleared in vivo, indicating that desialylation may also occur. In addition, the toxicity of DMSO used in freezing is also a potential risk.

[0008] By adding 6% DMSO to platelets, platelets can be stored at -80°C for two years. Some researchers also claim to have invented a "no-wash" platelet cryopreservation technology: after thawing, platelets can be transfused into the body without washing to remove DMSO. However, frozen platelets may also undergo desialylation, resulting in rapid clearance, and their recovery rate in the body is only about half of that of fresh platelets. In addition, the toxicity of DMSO is a potential risk of platelet cryopreservation. Moreover, cryopreservation relies on -80°C refrigerators, which is not conducive to transportation and cannot supply the platelet needs in war zones and remote areas in a timely manner.

[0009] Freeze drying (lyophilization) is an advanced method for preserving biological products. Compared with refrigeration and freezing, freeze-dried biological products can be transported and stored for a long time at room temperature. At present, although platelets can still maintain some functions after freeze drying, there are still some problems.

[0010] Freeze drying is divided into three steps: freezing, primary drying and secondary drying. The water-containing sample is first frozen into ice, then kept at a low temperature, and the ice is directly sublimated into gas by decompression. This step is the primary drying, which can remove most of the free water. By maintaining low pressure and slowly heating up, the bound water can be further removed, which is the secondary drying process. The freeze-dried sample has lost more than 90% of its water content and can be stored for a long time at room temperature or 4°C. When needed, the corresponding solution can be added to rehydrate. During the freeze-drying process, the sample will undergo a drastic phase change, which will cause irreversible damage to the sample. In order to maintain the activity of the freeze-dried sample, trehalose is usually added as a protective agent. Trehalose can reduce the aggregation and inactivation of proteins and lipids in high temperature, high cold, dry and other environments through the "water substitution" effect. At present, biologically active samples such as proteins, exosomes, and mRNA vaccines have been stored for a long time through freeze drying under the action of trehalose.

[0011] Some researchers have tried to preserve platelets by freeze drying, but the effect is limited. For example, in CN201510111401.9, trehalose and reversible platelet activation inhibitor PGE1 are used in the pretreatment solution, and plasma is used as a protective agent in the freeze-drying protective solution. Although the appearance of platelets after freeze drying is acceptable and the cytokine concentration is maintained, the aggregation ability of platelets after freeze drying decreases; in addition, for freeze-dried platelets, the expression of platelet surface glycoproteins related to hemostasis ability and the degree of surface desialylation related to the circulation time in the body are not paid attention to. In CN202010377816.1, platelets are incubated in a solution containing trehalose, and then the platelets are fixed with glutaraldehyde and ethanol, and then freeze-dried. Although the expression of platelet surface glycoproteins is maintained, the fixed platelets lose their activity and cannot circulate in the body to function. In US11529587B2, trehalose is used as a protective agent, polysucrose 400 is used as a stabilizer, ethanol is used to increase the permeability of platelet membranes, and heat treatment is performed after freeze-drying to remove pathogens; glycoproteins such as CD41 on the surface of freeze-dried platelets are maintained, and functions such as thrombin generation and thrombus formation remain basically unchanged. However, a paper published by the applicant later showed that the expression of CD42b on the surface of platelets was greatly reduced after freeze-drying, and the circulation time in the body was also greatly reduced, which may lead to a decrease in the hemostatic ability of freeze-dried platelets in the body.

[0012] In summary, there is currently no recognized method to solve the platelet storage problem. On the one hand, there is a huge demand for platelets in clinical practice, and on the other hand, a large number of platelets are discarded. Therefore, there is an urgent need for an improved method to extend the storage period of platelets. Summary of the invention

[0013] In order to meet the needs of clinical use, freeze-dried platelets should have good hemostatic ability and in vivo circulation time. The various reagents used in the freeze-drying process should also be non-toxic to avoid risks after transfusion. In addition, the flux of freeze-dried platelets should be large to meet the needs of patients with severe bleeding and thrombocytopenia.

[0014] At present, the research on freeze-dried platelets mainly focuses on direct freeze-drying after adding various protective agents, including trehalose, mannitol, albumin, polysucrose, etc. Although these protective agents can partially alleviate the damage during the freeze-drying process, it is difficult to provide sufficient protection; currently, freeze-dried platelets generally have problems such as decreased hemostatic ability (such as a significant decrease in the expression of hemostasis-related glycoprotein CD42b) and decreased circulation time (desialylation leads to high expression of galactose).

[0015] During the freeze-drying process, platelets will experience three serious injuries. First, during the freezing stage, the sharp ice crystals generated will pierce the platelet cell membrane and other structures, causing mechanical damage; second, during the drying stage, the escape of water will cause the lipids and proteins in the platelet membrane to aggregate irreversibly and lose their activity; finally, during the rehydration stage, during the transition from solid to liquid, the platelets will directly contact water molecules and the osmotic pressure will change dramatically, leading to osmotic shock. In the face of these injuries, trehalose may not provide sufficient protection (see Figure 1 The decrease in the hemostatic ability and in vivo circulation time of freeze-dried platelets is mainly due to the damage caused during the freeze-drying and rehydration process.

[0016] In order to solve the above-mentioned technical problems existing in the prior art, the present application provides a micro-hydrogel, freeze-dried powder and its preparation method, use method and application for encapsulating platelets. The micro-hydrogel encapsulating platelets provided by the present application, the platelets are encapsulated inside the micro-hydrogel, and the micro-hydrogel can reduce the damage caused to the platelets by the freeze-drying process and rehydration. The micro-hydrogel encapsulating platelets can be stored for a long time after freeze-drying, extending the shelf life of platelets. When needed, the platelets can be dissociated from the micro-hydrogel by rehydration-centrifugation-heating, without the need to add an additional cross-linking agent, and the operation is convenient. Compared with the current method of directly freeze-drying platelets, the platelets encapsulating platelets provided by the present application are freeze-dried and stored, and the platelets after rehydration and dissociation have higher surface glycoprotein expression and higher aggregation ability, which can keep the platelets in normal hemostasis ability, and can inhibit the desialylation process, and can circulate in the body for a longer time. Through the method of freeze-drying and storage of the micro-hydrogel encapsulating platelets provided by the present application, it is expected to greatly extend the storage time of platelets and alleviate the current situation of platelet shortage.

[0017] Using micro-hydrogels to wrap platelets can reduce damage during the freeze-drying process. During the freezing process, the macromolecules that make up the micro-hydrogels will form a dense network, thereby inhibiting the growth of ice crystals and reducing the mechanical damage caused by ice crystals. In addition, during the drying process, freeze-drying protectants such as trehalose can reduce the aggregation of proteins and lipids through the "water replacement" effect. Finally, during the rehydration process, the external hydrogel will first contact water, and then slowly release water to the internal platelets to reduce osmotic shock (see Figure 1 (b)). Therefore, micro-hydrogel encapsulation of platelets combined with freeze-drying is expected to be an ideal method for storing platelets.

[0018] In a first aspect, the present application provides a micro-hydrogel encapsulating platelets, wherein the micro-hydrogel has a core-shell or multi-chamber structure, and the platelets are encapsulated inside the micro-hydrogel; the micro-hydrogel is formed by physical gelation of one or more water-soluble polymer components.

[0019] In certain embodiments, the water-soluble polymer component is selected from dextran, gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, hyaluronic acid or its salt (such as hyaluronic acid metal salt, which can be sodium hyaluronate), collagen, chitosan, sodium alginate, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polyethylene glycol.

[0020] In certain embodiments, the water-soluble polymer component is selected from gelatin, dextran, and sodium hyaluronate. Preferably, the micro-hydrogel is formed by physical gelation of gelatin and one or two selected from dextran and sodium hyaluronate. Specifically, the mass ratio of gelatin: dextran and / or sodium hyaluronate is (1-1000):1, preferably (2-500):1, more preferably (4-100):1, (5-50):1, (10-30):1, for example 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1.

[0021] In certain embodiments, the micro-hydrogel is spherical or quasi-spherical, with a diameter of 1-1000 μm, preferably 5-150 μm.

[0022] In certain embodiments, the micro-hydrogel further contains one or more of trehalose, sucrose, glucose, and maltose, preferably the micro-hydrogel further contains trehalose. The trehalose, sucrose, glucose, and maltose in the micro-hydrogel can cooperate with the micro-hydrogel to protect platelets from freeze-drying during freeze-drying.

[0023] In a second aspect, the present application provides a method for preparing a micro-hydrogel encapsulating platelets, which comprises the following steps: emulsifying a dispersed phase solution containing platelets in a continuous phase solution, spontaneously physically gelling to form a micro-hydrogel, and encapsulating the platelets inside the micro-hydrogel; wherein the dispersed phase solution is an aqueous solution containing one or more water-soluble polymer components A; the continuous phase solution is an aqueous phase or an oil phase, wherein the aqueous phase is an aqueous solution containing one or more water-soluble polymer components B, and the oil phase contains one or more oil-based components, and one or more surfactants; wherein the water-soluble polymer component A and the water-soluble polymer component B are immiscible with each other.

[0024] The present application forms a stable emulsion structure by emulsifying a dispersed phase solution containing a water-soluble polymer component A in a continuous phase. Because the dispersed phase solution and the continuous phase solution are immiscible and the components interact differently, spontaneous liquid-liquid phase separation occurs through the phase separation-water transfer principle, and the water-soluble polymer component A spontaneously physically gels to form a micro-hydrogel. After platelets are added to the dispersed phase solution, the platelets can be encapsulated inside the micro-hydrogel. The micro-hydrogel encapsulating platelets described in the first aspect can be prepared by the preparation method described in the present application.

[0025] In certain embodiments, the platelets are platelets that have been pre-treated and incubated. The pre-treatment and incubation refers to adding an incubation solution to the platelet precipitate separated from the blood, resuspending, heating and incubating, and centrifuging and collecting; preferably, the amount of the incubation solution is 1-100% of the original blood volume, preferably 10% of the original blood volume. The incubation solution contains 1-100mM trehalose, 1-1000mM sodium chloride, 1-100mM potassium chloride, 1-100mM imidazole, 1-100mM EGTA (ethylene glycol-bis(2-aminoethyl ether)- N,N,N′,N′-tetraacetic acid), 1-10μM PGE1 (prostaglandin E1); preferably, the composition of the incubation solution is 50mM trehalose, 100mM sodium chloride, 10mM potassium chloride, 10mM imidazole, 10mM EGTA, 1μM PGE1. The incubation temperature is 20-40°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. The incubation conditions are 10-200rpm, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200rpm, and the incubation time is 1-10h, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10h. Preferably, the incubation temperature is 35-38°C, 30-60rpm, and the incubation time is 2-5h. The centrifugal collection condition is that the centrifugal speed is 100-1000g, such as 100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g. The centrifugation time is 3-30 min, for example 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min. Preferably, the centrifugal collection condition is that the centrifugal speed is 500-1000g and the centrifugal time is 10-15 min.

[0026] In certain embodiments, platelet precipitates are obtained by separating blood through the following steps, PGE1 is added to 1-10 μM (preferably 1 μM) in platelet-rich plasma from which most red blood cells and white blood cells are removed to inhibit platelet activation, and platelet precipitates are obtained by centrifugation. The centrifugal speed is 100-1000 g, for example, 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g. The centrifugal time is 3-30 min, for example, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min. Preferably, the centrifugal speed is 500-1000 g and the centrifugal time is 10-15 min.

[0027] In certain embodiments, the platelets (preferably pre-treated platelets) are resuspended in a dispersed phase, and the concentration of platelets in the dispersed phase solution is 0.1-10×10 9 / mL. For example, 0.1 × 10 9 / mL, 0.2 × 10 9 / mL, 0.3 × 10 9 / mL, 0.4 × 10 9 / mL, 0.5 × 10 9 / mL, 0.6 × 10 9 / mL, 0.7 × 10 9 / mL, 0.8 × 10 9 / mL, 0.9 × 10 9 / mL, 1.0 × 10 9 / mL, 1.1 × 10 9 / mL, 1.2 × 10 9 / mL, 1.3 × 10 9 / mL, 1.4 × 10 9 / mL, 1.5 × 10 9 / mL, 1.6 × 10 9 / mL, 1.7 × 10 9 / mL, 1.8 × 10 9 / mL, 1.9 × 10 9 / mL, 2.0 × 10 9 / mL, 2.1 × 10 9 / mL, 2.2 × 10 9 / mL, 2.3 × 10 9 / mL, 2.4 × 10 9 / mL, 2.5 × 10 9 / mL, 2.6 × 10 9 / mL, 2.7 × 10 9 / mL, 2.8 × 10 9 / mL, 2.9 × 10 9 / mL, 3.0 × 10 9 / mL, 3.1 × 10 9 / mL, 3.2 × 10 9 / mL, 3.3 × 10 9 / mL, 3.4 × 10 9 / mL, 3.5 × 10 9 / mL, 3.6 × 10 9 / mL, 3.7 × 10 9 / mL, 3.8 × 10 9 / mL, 3.9 × 10 9 / mL, 4.0 × 10 9 / mL, 5.0 × 10 9 / mL, 6.0 × 10 9 / mL, 7.0 × 10 9 / mL, 8.0 × 10 9 / mL, 9.0 × 10 9 / mL, 10.0 × 10 9 / mL. Preferably, the concentration of platelets in the dispersed phase solution is 0.1-1×10 9 / mL. More preferably, the concentration of platelets in the dispersed phase solution is 0.2×10 9 / mL.

[0028] In certain embodiments, water-soluble polymer component A and water-soluble polymer component B are independently selected from dextran, gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, hyaluronic acid and its salts (such as sodium hyaluronate), collagen, chitosan, sodium alginate, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polyethylene glycol. In certain embodiments, water-soluble polymer component A is selected from gelatin, dextran, sodium hyaluronate, and water-soluble polymer component B is selected from polyethylene glycol and polyvinyl alcohol. Preferably, the dispersed phase solution is an aqueous solution containing gelatin and one or two selected from dextran and sodium hyaluronate, and the continuous phase solution is an aqueous solution containing one or two of polyethylene glycol and polyethylene glycol.

[0029] In certain embodiments, in the dispersed phase solution, the total concentration of one or more water-soluble polymer components A is 0.1-20% (w / v). For example, 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 5.5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. Preferably, the total concentration of one or more water-soluble polymer components A is 2-15% (w / v). The total concentration of one or more water-soluble polymer components B in the continuous phase solution is 0.1-20% (w / v). For example, 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 5.5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. Preferably, the total concentration of one or more water-soluble polymer components B is 2-15% (w / v).

[0030] In certain embodiments, the dispersed phase solution is an aqueous solution containing 1-15% (w / v) gelatin, and 0.05-1% (w / v) dextran and / or sodium hyaluronate (referring to the total concentration of glucose and / or sodium hyaluronate), preferably an aqueous solution containing 2-10% (w / v) gelatin (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), and 0.1-0.5% (w / v) dextran and / or sodium hyaluronate (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%). The continuous phase solution is an aqueous solution containing 1-20% (w / v) polyethylene glycol and / or polyvinyl alcohol (referring to the total concentration of polyethylene glycol and / or polyvinyl alcohol), preferably an aqueous solution containing 2-15% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%) (w / v) polyethylene glycol and / or polyvinyl alcohol.

[0031] In some embodiments, the solvent in the dispersed phase solution is pure water. In some embodiments, the solvent in the dispersed phase solution is water, and one or more osmotic pressure regulators need to be added to adjust the osmotic pressure, for example, the osmotic pressure regulator is selected from HEPES (4-hydroxyethylpiperazineethanesulfonic acid), Tricine (tris(hydroxymethyl)methylglycine), sodium chloride, mannitol, etc. Preferably, the osmotic pressure regulator is mannitol. In some embodiments, the total concentration of the osmotic pressure regulator in the dispersed phase solution is 0.1-10% (w / v), for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. Preferably, the total concentration of the osmotic pressure regulator in the dispersed phase solution is 1-8% (w / v). More preferably, the solvent of the dispersed phase solution is water and contains 3-5% mannitol.

[0032] In certain embodiments, when the continuous phase solution is an aqueous phase, its solvent is a freeze-drying protective solution, pure water, or one or more of a buffer, such as a phosphate buffer, an acetate buffer, a citrate buffer, a HEPES buffer, a carbonate buffer, a triethanolamine buffer, a Tricine solution, a Tris buffer, and a Tyrode's solution. Preferably, when the continuous phase solution is an aqueous phase, its solvent is a freeze-drying protective solution. The total concentration of the solute in the buffer is 1-1000 mM.

[0033] In certain embodiments, the freeze-drying protective solution contains one or more of trehalose, sucrose, glucose, maltose, mannitol, and glycerol. Preferably, the freeze-drying protective solution contains trehalose. The freeze-drying protective solution also contains one or more of magnesium chloride, potassium chloride, sodium chloride, HEPES, and hydroxypropyl-β-cyclodextrin. The concentration of each component in the lyophilized protective solution is 1mM-1000mM, for example, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 150mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM. In certain embodiments, the lyophilization protective solution contains 1-100mM trehalose, 1-10mM magnesium chloride, 1-10mM potassium chloride, 1-1000mM sodium chloride, 1-100 mM HEPES, and 1-100mM hydroxypropyl-β-cyclodextrin; preferably, the composition of the lyophilization protective solution is 30mM trehalose, 1mM magnesium chloride, 4.8mM potassium chloride, 142.5mM sodium chloride, 9.5mM HEPES, and 7mM hydroxypropyl-β-cyclodextrin.

[0034] In certain embodiments, when the continuous phase solution is an oil phase, the oil-based component is selected from n-hexadecane, isohexadecane, mineral oil, stearane, isostearane, squalane, decane, dodecane, dimethicone, paraffin oil, olive oil, coconut oil, grape seed oil, almond oil, soybean oil, isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl linoleate, lauryl benzoate, isostearyl isostearate, fatty acid lactate, decyl oleate, octyl palmitate. The total concentration of the one or more oil-based components is 70%-99.9% (w / v). For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%. Preferably, the total concentration of the oil-based component is 80%-90%.

[0035] In certain embodiments, when the continuous phase solution is an oil phase, the surfactant is selected from Span 80, Span 60, polysorbate 20, polysorbate 60, polysorbate 80, cocoyl glucoside, lauryl glucoside, glyceryl stearate, dimethiconol, sodium lauryl sulfate, sodium lauroyl glutamate, sodium methyl cocoyl taurate, stearyl trimethyl ammonium chloride, dioctadecyl dimethyl ammonium chloride, cocamidopropyl betaine, and lauroamidopropyl betaine. The total concentration of the surfactant is 0.1-30% (w / v). For example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%. Preferably, the total concentration of the surfactant is 1-10% (w / v).

[0036] In certain embodiments, the volume ratio of the dispersed phase solution to the continuous phase solution is 1:1-1000, preferably 1:1-100, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100. Preferably, the volume ratio of the dispersed phase solution to the continuous phase solution is 1:20-50.

[0037] In certain embodiments, the dispersed phase solution is an aqueous solution containing 5.5% (w / v) gelatin, 0.25% (w / v) dextran, and 3-5% mannitol; the continuous phase solution is a freeze-drying protective solution containing 4.5% (w / v) polyethylene glycol. The dispersed phase solution is an aqueous solution containing 5.5% (w / v) gelatin, 0.25% (w / v) dextran, and 3-5% mannitol; the continuous phase solution is a freeze-drying protective solution containing 15% (w / v) polyvinyl alcohol. The dispersed phase solution is an aqueous solution containing 5.33% (w / v) gelatin, 0.27% (w / v) sodium hyaluronate, and 3-5% mannitol; the continuous phase solution is a freeze-drying protective solution containing 4.5% (w / v) polyethylene glycol.

[0038] In certain embodiments, there is no particular limitation on the emulsification method, as long as sufficient contact and sufficient mixing of the dispersed phase and the continuous phase can be achieved. The emulsification method includes, but is not limited to, one or more of vortexing, shaking, pipette blowing, electrospraying, membrane emulsification, and microfluidic extrusion.

[0039] In a third aspect, the present application provides a platelet-encapsulating micro-hydrogel freeze-dried powder, which is obtained by freeze-drying the platelet-encapsulating micro-hydrogel described in the first aspect or the platelet-encapsulating micro-hydrogel prepared according to the preparation method described in the second aspect. Preferably, freeze-drying is performed in a freeze-drying protective solution.

[0040] In a fourth aspect, the present application provides a method for preparing a platelet-encapsulating micro-hydrogel freeze-dried powder, which comprises the following steps: freeze-drying the platelet-encapsulating micro-hydrogel described in the first aspect or the platelet-encapsulating micro-hydrogel prepared according to the preparation method described in the second aspect. Preferably, freeze-drying is performed in a freeze-drying protective solution.

[0041] In certain embodiments, when preparing micro-hydrogels encapsulating platelets, if the solvent of the continuous phase solution is a freeze-drying protective solution, freeze-drying is directly performed after emulsification to form micro-hydrogels.

[0042] In certain embodiments, when preparing micro-hydrogels encapsulating platelets, if the solvent of the continuous phase solution is not a freeze-drying protective solution or the continuous phase solution is oil-based, the micro-hydrogels encapsulating platelets are collected by centrifugation after emulsification to form micro-hydrogels, and then resuspended in a freeze-drying protective solution for freeze drying. The centrifugal collection condition is that the centrifugal speed is 100-1000 g, for example, 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g. The centrifugation time is 3-30 min, for example, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min. Preferably, the centrifugal collection condition is a centrifugal speed of 500-1000g and a centrifugal time of 10-15 min. The volume of the lyophilization protection solution used during resuspension is preferably 1 / 2-2 times the volume before centrifugal collection, and more preferably equal to the volume before centrifugal collection.

[0043] In certain embodiments, the freeze drying comprises three steps: pre-freezing, primary drying, and secondary drying. The micro-hydrogel freeze-dried powder encapsulating platelets can be stored or transported at room temperature (15-35°C), 4°C, -20°C, or -80°C; and can be stored for a long time at room temperature.

[0044] In certain embodiments, the micro-hydrogel encapsulating platelets is transferred to a freeze-drying container for freeze drying, preferably, further comprising nitrogen protection, sealing (such as stoppering, adding aluminum cap), and vacuuming steps after freeze drying. The freeze-drying container includes, but is not limited to, vials, aluminum foil bags, composite film bags, vacuum packaging bags, modified atmosphere packaging bags, polyester film bags, glass bottles, plastic bottles, polyethylene tubes (PE tubes), polypropylene tubes (PP tubes), glass tubes, aluminum tubes, and polyester tubes (PET tubes). The volume of the lyophilization container used is 0.1-30mL, such as 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27mL, 28 mL, 29 mL, 30 mL.

[0045] In certain embodiments, the pre-freezing medium includes, but is not limited to, a 4°C refrigerator, a -20°C refrigerator, a -30°C refrigerator, a -80°C refrigerator, a lyophilizer plate, dry ice, and liquid nitrogen.

[0046] In certain embodiments, during the primary drying, the freeze dryer plate temperature is set to -50°C to -10°C. For example, -50°C, -49°C, -48°C, -47°C, -46°C, -45°C, -44°C, -43°C, -42°C, -41°C, -40°C, -39°C, -38°C, -37°C, -36°C, -35°C, -34°C, -33°C, -32°C, -31°C, -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C. The primary drying time is 3-30h. For example, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25h, 26 h, 27 h, 28 h, 29 h, 30 h.

[0047] In certain embodiments, the board layer needs to be heated during secondary drying. The heating rate is 0.1-50°C / min, for example, 0.1°C / min, 0.2°C / min, 0.3°C / min, 0.4°C / min, 0.5°C / min, 0.6°C / min, 0.7°C / min, 0.8°C / min, 0.9°C / min, 1.0°C / min, 2°C / min, 3°C / min, 4°C / min, 5°C / min, 6°C / min, 7°C / min, 8°C / min, 9°C / min, 10°C / min, 11°C / min, 12°C / min, 13°C / min, 14°C / min, 15°C / min, 16°C / min, 17°C / min, 18°C / min, 19°C / min, 20°C / min, 21°C / min, 22°C / min, 23°C / min, 24°C / min, 25°C / min, 26°C / min, 27°C / min, 28°C / min, 29°C / min, 30°C / min, 31°C / min, 32°C / min 0℃ / min, 21℃ / min, 22℃ / min, 23℃ / min, 24℃ / min, 25℃ / min, 26℃ / min, 27℃ / min, 28℃ / min, 29℃ / min, 30℃ / min, 31℃ / min, 32℃ / min, 33℃ / min, 34℃ / min, 35℃ / min, 36℃ / min, 37℃ / min, 38℃ / min, 39℃ / min, 40℃ / min, 41℃ / min, 42℃ / min, 43℃ / min, 44℃ / min, 45℃ / min, 46℃ / min, 47℃ / min, 48℃ / min, 49℃ / min, 50℃ / min. The secondary drying temperature is 10-35°C, for example, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C. The secondary drying time is 3-30h. For example, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h.

[0048] In a fifth aspect, the present application provides a method for using the micro-hydrogel freeze-dried powder of platelets encapsulated in the third aspect, which comprises the following steps: rehydration, centrifugal collection of the micro-hydrogel encapsulated in the platelets, heating dissociation, and centrifugal collection to obtain harvested platelets. The method is applicable to the micro-hydrogel freeze-dried powder of platelets encapsulated in the third aspect or the micro-hydrogel freeze-dried powder of platelets encapsulated prepared according to the preparation method of the fourth aspect.

[0049] In certain embodiments, the rehydration refers to resuspending the micro-hydrogel freeze-dried powder encapsulating platelets using pure water, plasma, or one or more of a buffer (such as a phosphate buffer, an acetate buffer, a citrate buffer, a HEPES buffer, a carbonate buffer, a triethanolamine buffer, a Tricine solution, a Tris buffer, a Tyrode's solution). The total concentration of the solute in the buffer is 1-1000 mM. The volume of the solution used for rehydration is 1 / 2-2 times the volume before freeze-drying. Preferably, the volume of the solution used for rehydration is based on the volume equal to the volume before freeze-drying. More preferably, the micro-hydrogel freeze-dried powder encapsulating platelets is rehydrated using pure water.

[0050] In certain embodiments, after freeze-drying and rehydration, the micro-hydrogel is spherical or quasi-spherical, with a diameter of 1-1000 μm, preferably 5-150 μm.

[0051] In certain embodiments, the micro-hydrogel after freeze-drying-rehydration has a core-shell structure or a multi-chamber structure.

[0052] In certain embodiments, the heat dissociation refers to enriching the micro-hydrogel encapsulating platelets and then resuspending it in pure water, plasma, or one or more of buffers (such as phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, carbonate buffer, triethanolamine buffer, Tricine solution, Tris buffer, Tyrode's solution) for heating and incubation. The total concentration of the solute in the buffer is 1-1000mM. Preferably, the micro-hydrogel encapsulating platelets is resuspended in 1-20mM Tyrode's solution for heating and incubation. The volume of the solution used for incubation is 1 / 2-2 times the volume before freeze-drying. The incubation temperature is 20-40°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. The incubation conditions are 10-200rpm, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200rpm, and the incubation time is 1min-1h, such as 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60min. Preferably, the incubation temperature is 35-38°C, 30-60rpm, and the incubation time is 2-5min.

[0053] In certain embodiments, PGE1 is added to 1-10 µM after the heating and incubation. Preferably, PGE1 is added to 1 µM after the heating and incubation, and the platelets are harvested by centrifugation.

[0054] The conditions of the two-step centrifugal collection (centrifugal collection of microhydrogels encapsulating platelets and centrifugal collection of harvested platelets in the method of use of the fifth aspect) are that the centrifugal speed is 100-1000 g, for example, 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g. The centrifugal time is 3-30 min, for example, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min. Preferably, the conditions for centrifugal collection are a centrifugal speed of 500-1000 g and a centrifugal time of 10-15 min.

[0055] In certain embodiments, after the harvested platelets are collected by centrifugation, they are resuspended in pure water, plasma, or one or more buffers (such as phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, carbonate buffer, triethanolamine buffer, Tricine solution, Tris buffer, Tyrode's solution) for subsequent use. The total concentration of the solute in the buffer is 1-1000mM. Preferably, after the harvested platelets are collected by centrifugation, they are resuspended in Tyrode's solution or plasma for subsequent use. The volume of the solution used for resuspension is 1 / 2-2 times the volume before lyophilization.

[0056] In a sixth aspect, the present application provides a freeze-drying, storage and / or transportation and use method of platelets, which comprises the following steps: preparing a micro-hydrogel encapsulating platelets according to the method described in the second aspect, preparing a freeze-dried powder according to the method described in the fourth aspect, storing and / or transporting, and obtaining harvested platelets according to the use method described in the fifth aspect. Preferably, the storage or transportation is performed at room temperature, 4°C, -20°C or -80°C.

[0057] In the seventh aspect, the present application provides the use of the platelet-encapsulating microhydrogel of the first aspect, the platelet-encapsulating microhydrogel prepared by the second aspect, the platelet-encapsulating microhydrogel lyophilized powder of the third aspect, or the platelet-encapsulating microhydrogel lyophilized powder prepared by the fourth aspect in the preparation of biomedical products.

[0058] Compared with the prior art, the beneficial effects of this application are: 1) The micro-hydrogel encapsulating platelets provided in the present application, in which platelets are encapsulated inside the micro-hydrogel, can reduce the damage to platelets during the freeze-drying process and the rehydration process. Compared with the existing direct freeze-drying method, the platelets encapsulated in the micro-hydrogel after freeze-drying, rehydration and dissociation have higher glycoprotein expression, higher aggregation ability, and lower degree of desialylation, and are expected to show better hemostatic ability and in vivo circulation time.

[0059] 2) The method for preparing the platelet-encapsulating micro-hydrogel provided in the present application is: emulsifying the dispersed phase solution in the continuous phase solution, and forming the micro-hydrogel through spontaneous physical gelation according to the liquid-liquid phase separation-water transfer principle. After adding platelets to the dispersed phase, the platelets can be encapsulated inside the micro-hydrogel. The platelet-encapsulating micro-hydrogel freeze-dried powder can be dissociated by rehydration, centrifugal heating, and the encapsulated platelets can be released without the need to add additional cross-linking agents, and the operation is simple and convenient. Therefore, the preparation and freeze-drying methods of the platelet-encapsulating micro-hydrogel provided in the present application can be further scaled up for production to meet clinical needs.

[0060] 3) The components of the micro-hydrogel for encapsulating platelets provided in this application (such as dextran, gelatin, hyaluronic acid and its salts, polyethylene glycol, polyvinyl alcohol, etc.) are all pharmacopoeia components, which are safe, non-toxic and inexpensive.

[0061] 4) The platelet-encapsulating micro-hydrogel, freeze-dried powder and preparation method thereof provided in this application can significantly extend the storage time of platelets and alleviate the current situation of platelet shortage. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which: Figure 1 Damage to platelets during freeze-drying (a) and the protective effect of micro-hydrogel (b).

[0063] Figure 2 The microscopic observation pictures of the micro-hydrogel encapsulating platelets (left) and the free platelets after dissociation (right) in Example 2. The platelets were labeled with FITC anti-human CD41 / CD61.

[0064] Figure 3 Schematic diagram of the preparation process of the platelet-encapsulating micro-hydrogel lyophilized powder in Example 3.

[0065] Figure 4 The microscopic observation pictures of the micro-hydrogel encapsulating platelets before and after freeze-drying in Example 3. The platelets before freeze-drying were labeled with FITC anti-human CD41 / CD61.

[0066] Figure 5 This is a statistical diagram of the size of the micro-hydrogel encapsulating platelets in Example 3 before and after freeze-drying.

[0067] Figure 6 Actual pictures of the platelet-encapsulated microhydrogel freeze-dried powder (Encap-Lyo, left) and directly freeze-dried platelets (Lyo, right) in Example 3.

[0068] Figure 7 This is a graph showing the expression of hemostasis-related glycoproteins CD41 / 61 and CD42b in platelet-encapsulated micro-hydrogel freeze-dried platelets (Encap-Lyo), directly freeze-dried platelets (Lyo) and fresh platelets (Fresh) detected by flow cytometry in Example 4.

[0069] Figure 8 This is a graph showing the degree of desialylation of platelets encapsulated in micro-hydrogel freeze-dried platelets (Encap-Lyo), directly freeze-dried platelets (Lyo) and fresh platelets (Fresh) detected by flow cytometry in Example 4.

[0070] Fig. 9 This is a graph showing the in vitro aggregation ability of platelets encapsulated in micro-hydrogel (Encap-Lyo), directly freeze-dried platelets (Lyo) and fresh platelets (Fresh) in Example 4.

[0071] Fig.10 This is a graph showing the expression of hemostasis-related glycoproteins CD41 / 61 and CD42b in platelet-encapsulated microhydrogel freeze-dried platelets (Encap-Lyo), directly freeze-dried platelets (Lyo) and fresh platelets (Fresh) after storage detected by flow cytometry in Example 5.

[0072] Fig.11 This is a graph showing the degree of desialylation of platelet-encapsulated micro-hydrogel freeze-dried platelets (Encap-Lyo), directly freeze-dried platelets (Lyo) and fresh platelets (Fresh) after storage detected by flow cytometry in Example 5. DETAILED DESCRIPTION

[0073] The specific implementation of the present application is described in detail below. It should be understood that the specific implementation described here is only used to illustrate and explain the present application, and is not used to limit the present application.

[0074] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0075] Features illustrated or described as part of one embodiment or certain embodiments can be used on another embodiment or certain other embodiments to yield a still further embodiment.

[0076] Before describing the present application in detail, it should be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present application, which is limited only by the appended claims. In order to more fully understand the present application described herein, the following terms are used, and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by a person of ordinary skill in the art to which the present application belongs.

[0077] definition Unless otherwise specified, the following terms used in this application have the following definitions.

[0078] Unless otherwise stated, the terms "comprising," "including," and "containing" and similar expressions should be interpreted in this specification and claims in an open and inclusive sense as "including but not limited to."

[0079] The term "micro-hydrogel" refers to a micron-sized gel composed of water-soluble polymer materials, with a three-dimensional network structure that can absorb and retain a large amount of water while maintaining structural stability. The size is usually between 1 micron and several hundred microns. Its excellent water absorption and environmental responsiveness make it sensitive to external stimuli (such as pH, temperature, light, ionic strength, etc.), and it can regulate its own swelling and drug release. Due to its good biocompatibility, micro-hydrogels are widely used in drug delivery, tissue engineering and regenerative medicine, and are particularly suitable for soft tissue repair, biosensors and smart drug delivery systems, with great potential application value.

[0080] The term "encapsulation" refers to the process of encapsulating bioactive substances (such as drugs, proteins, cells, etc.) with micro-hydrogel materials. This encapsulation technology encapsulates these substances in the three-dimensional network structure of the hydrogel to form a protective barrier, allowing them to be slowly released or isolated under specific conditions, thereby playing a protective role.

[0081] The term "dispersed phase" refers to one phase in a multiphase system, usually small particles or droplets of liquid, solid or gas, which are dispersed in another substance (i.e., continuous phase) in a discontinuous manner. In emulsions, suspensions or colloidal solutions, the size of the dispersed phase can range from nanometers to micrometers. Dispersed phase particles are usually introduced into the system by external energy (such as vibration, stirring or ultrasound), are in a thermodynamically unstable state, and often tend to aggregate or agglomerate. If there is no sufficient stabilizing mechanism (such as emulsifiers or stabilizers), the particles of the dispersed phase will gradually form larger particles through collision and merger, resulting in an unstable system. The specific form of the dispersed phase in the emulsion can be oil droplets dispersed in water (O / W emulsion), water droplets dispersed in oil (W / O emulsion), or water droplets dispersed in water (W / W emulsion).

[0082] The term "continuous phase" refers to the continuously distributed matrix in a multiphase system. It is a substance that acts as a solvent or matrix, relative to the dispersed phase, and can evenly surround and distribute the particles of the dispersed phase. In an emulsion or colloidal solution, the continuous phase occupies the majority of the system's volume and is the medium in which the dispersed phase exists. The characteristics of the continuous phase have an important influence on the overall properties of the system, such as rheology, stability, and mass transfer performance. In O / W or W / W emulsions, water is the continuous phase; in W / O emulsions, oil is the continuous phase. The choice and physical properties of the continuous phase (such as viscosity, surface tension, etc.) can significantly affect the stability of the dispersed phase.

[0083] The term "emulsification" refers to the process of mixing two immiscible liquids through shaking, mechanical stirring, ultrasound, homogenizer or other physical methods, so that one liquid (dispersed phase) is dispersed in the other liquid (continuous phase) in the form of droplets to form an emulsion. The essence of emulsification is to overcome the interfacial tension between the two liquids so that the dispersed phase droplets remain evenly distributed in the continuous phase. Due to the high interfacial energy, this dispersed state is thermodynamically unstable, so the emulsified system usually requires the addition of emulsifiers to reduce the interfacial tension and prevent the droplets from aggregating and separating. Emulsifiers are molecules (surfactants) with both hydrophilic and lipophilic properties. They will adsorb on the interface between the dispersed phase and the continuous phase to form a stable interfacial film to prevent the fusion of droplets. Common emulsifiers include surfactants, proteins, polymers, etc.

[0084] The term "liquid-liquid phase separation" refers to the separation of two originally mixed liquids into two immiscible liquid phases in a system through energy release or changes in external conditions. This phenomenon usually occurs when the emulsified system is unstable, such as emulsifier failure, temperature change, external force disturbance, etc. The essence of liquid-liquid phase separation is that the system tends to reduce free energy, driving the dispersed phase to separate from the continuous phase, and finally forming two immiscible liquid phase layers. For example, oil and water originally form an emulsion under emulsification conditions, but when the emulsification is broken, they will separate into an oil layer and a water layer, which is a typical liquid-liquid phase separation phenomenon. The liquid-liquid phase separation process is related to the thermodynamic phase diagram. The system may have multiple mutually separated phases under certain conditions, while it may show a single-phase uniform mixture under other conditions. Liquid-liquid phase separation is widely used in chemical industry and biology (such as intracellular phase separation phenomenon).

[0085] The term "physical gelation" refers to the process in which one or more substances change from liquid to solid or semi-solid under physical action. Unlike chemical gelation, physical gelation does not involve chemical reactions or the use of cross-linking agents, but relies on non-covalent interactions between molecules (such as hydrogen bonds, van der Waals forces, etc.) to form a three-dimensional network structure, which keeps the substance solid or elastic.

[0086] The term "gelatin" refers to a natural high-molecular protein obtained by hydrolyzing animal collagen, mainly from the skin, bones and connective tissues of cattle, pigs, fish and other animals. It has good solubility in water and can form gel when cooled, with excellent gelation properties. The hydration of gelatin enables it to play the role of thickening, gelling and retaining moisture in hydrogels, so it is widely used in biomedicine, hydrogel drug delivery systems, tissue engineering, wound dressings and other fields. Gelatin not only has good biocompatibility and biodegradability, but can also be compounded with other polymer materials to adjust the physical properties and mechanical strength of hydrogels, becoming an important ingredient in the pharmaceutical, food, cosmetics and other industries.

[0087] The term "dextran" refers to a high molecular weight polysaccharide composed of glucose molecules connected by α-1,6-glycosidic bonds. It has a branched structure and is widely found in some bacteria (such as Proteus and lactic acid bacteria). The molecular chain of dextran can be linear or highly branched, with a large molecular weight and usually good water solubility and hydrophilicity. In the biomedical field, dextran, as a biocompatible material, is often used as a blood volume expander, drug carrier and stabilizer, especially in plasma substitutes, drug delivery systems and biomacromolecule carriers. It also has biodegradability and good immunocompatibility, so it is often used in drug delivery, tissue engineering and medical diagnosis.

[0088] The term "sodium hyaluronate" (SH) refers to a water-soluble salt compound formed by the combination of hyaluronic acid and sodium salt, which is widely present in the connective tissue, skin, eyes, joint fluid and other parts of the human body and animals. It is composed of alternating glucosamine and glucuronic acid, has a strong moisturizing ability, and can absorb and retain a large amount of water. Sodium hyaluronate is widely used in the fields of medicine and beauty. It is used as a moisturizer, lubricant and drug carrier for skin care, anti-aging, joint treatment and other aspects. It is often used in injectable fillers to help facial beauty or relieve arthritis, and also plays an important role in ophthalmic surgery, eye drops and trauma repair. In cosmetics, sodium hyaluronate is used as a moisturizing ingredient to improve skin hydration, reduce wrinkles and fine lines, and maintain skin elasticity. In addition, sodium hyaluronate is also widely used in medical fields such as drug delivery systems due to its good biocompatibility and biodegradability.

[0089] The term "polyethylene glycol" (PEG) refers to a class of synthetic polymer compounds formed by polymerization of ethylene glycol or ethylene oxide. Depending on the degree of polymerization, PEG can have different molecular weights, usually ranging from a few hundred to several million Daltons. Common molecular weight ranges include: low molecular weight (200-600 Da, liquid state), medium molecular weight (600-6000 Da, paste or semi-solid), and high molecular weight (>6000 Da, solid). PEG is a non-toxic, highly hydrophilic, chemically stable and biocompatible polymer material that is widely used in medicine, cosmetics, food, industry and biotechnology. In the pharmaceutical field, PEG is often used as a drug carrier, sustained-release agent, solvent, solubilizer and drug modification to improve the solubility, stability and bioavailability of drugs and enhance the therapeutic effect of drugs.

[0090] The term "polyvinyl alcohol" (PVA) is a polymer compound made from vinyl acetate through alcoholysis and polymerization. It has good hydrophilicity and water solubility. Its chemical structure contains multiple alcohol groups (-OH), which give it excellent film-forming, solubility, colloid and adhesion properties. The molecular weight and water solubility of polyvinyl alcohol can be controlled by adjusting the degree of polymerization and alcoholysis. The common molecular weight ranges from thousands to millions of Daltons. Due to its good biocompatibility, polyvinyl alcohol is widely used in medicine, industry, cosmetics and environmental protection. In the medical field, PVA is used in drug delivery systems, artificial tears, wound dressings and biomaterials; in the industrial field, it is widely used in glues, coatings, textiles and paper processing; in cosmetics, polyvinyl alcohol is used as an emulsifier, thickener and moisturizer. The term "Tyrode's buffer" refers to a buffer solution used in biological and biochemical experiments, usually used to maintain a stable pH and ion environment required by cells or tissues during the experiment. Tyrode's solution is composed of a variety of salts and other ingredients, designed to simulate physiological conditions in vivo, and is widely used in cardiac physiology and cell biology research. Standard Tyrode's solution usually contains sodium, potassium, calcium, chloride, bicarbonate ions and glucose. The Tyrode's solution used in this study contains sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, HEPES and glucose. The specific composition of the Tyrode's solution used in the example is 4.6mM NaCl, 5.4mM KCl, 1mM MgCl2, 25mM Na2HPO4, 5mM HEPES, and 10mM glucose.

[0091] Example 1 Collection of platelets Blood was collected from volunteers who passed the clinical ethics. One-third of the original volume of hydroxyethyl starch was added to the blood. After settling for 30 minutes, the blood was centrifuged at 50g for 10 minutes, and the supernatant was taken, and the red blood cells at the bottom of the test tube were discarded. The supernatant was centrifuged at 400g for 15 minutes, and the supernatant was taken, and the white blood cells at the bottom of the test tube were discarded. The supernatant was centrifuged at 400g for 15 minutes again to further remove the white blood cells at the bottom. The resulting supernatant was platelet-rich plasma.

[0092] PGE1 was added to the platelet-rich plasma to 1 μM, and the mixture was centrifuged at 1000 g for 12 minutes. The supernatant was platelet-poor plasma, and the lower layer was platelet precipitate. The platelet-poor plasma was collected and stored at -20°C for future use.

[0093] Example 2 Platelet encapsulation and dissociation Using water as solvent, prepare a mixed solution of 5.5% gelatin (Sigma-Aldrich 73865), 0.25% dextran (McLean D872024), and 3% mannitol (Solebo M8140) as the dispersed phase solution. Using freeze-dried protective solution as solvent, add 5% PEG as the continuous phase solution. The freeze-dried protective solution consists of 30mM trehalose, 1mM magnesium chloride, 4.8mM potassium chloride, 142.5mM sodium chloride, 9.5mM HEPES, and 7mM hydroxypropyl-β-cyclodextrin. Resuspend the platelet precipitate with 500µL of dispersed phase solution, blow evenly, and continue to add dispersed phase solution to adjust the platelet concentration to 2×10 8 / mL. Add 0.5µl FITC anti-humanCD41 / CD61 (Biolegend, 359810), mix well, and incubate for 20min in the dark.

[0094] Take 50 µL of the dispersed phase solution after adding platelets, blow it several times in 1 mL of the continuous phase solution to emulsify it. Place it in a 24-well plate and observe it under a microscope. Figure 2 As shown on the left, the dispersed phase solution and the continuous phase solution are emulsified to form a micro-hydrogel. Most of the platelets are encapsulated inside the micro-hydrogel, showing green fluorescence, and the micro-hydrogel has a core-shell structure.

[0095] The platelet-encapsulated micro-hydrogel was centrifuged at 500 g for 5 minutes, the supernatant was removed, and the micro-hydrogel was resuspended with an equal volume of Tyrode's solution and heated to 37°C to dissociate the micro-hydrogel. The micro-hydrogel was placed in a 24-well plate and observed under a microscope. Figure 2 As shown on the right, upon heating, the micro-hydrogel can be seen to dissociate and the platelets are released.

[0096] Example 3 Preparation of platelet-encapsulating microhydrogel freeze-dried powder platelet incubation Add 1 / 10 of the original volume of incubation solution to the platelet pellet and resuspend. The incubation solution contains: 50 mM trehalose, 100 mM sodium chloride, 10 mM potassium chloride, 10 mM imidazole, 10 mM EGTA, 1 μM PGE1. Transfer to a constant temperature incubator and incubate at 37°C, 40 rpm for 4 hours.

[0097] Platelet packaging Micro hydrogel formula with different components: Microhydrogel 1: Dextran-Gelatin-PEG Continuous phase: 4.5% PEG (Solabo P8280) dissolved in freeze-dried protective solution; Dispersed phase: aqueous solution, 5.5% gelatin, 0.25% dextran, 3% mannitol.

[0098] Micro-hydrogel 2: Dextran-Gelatin-PVA Continuous phase: 15% PVA (Sigma-Aldrich P8136) dissolved in lyophilized protective solution Dispersed phase: aqueous solution, 5.5% gelatin, 0.25% dextran, 3% mannitol.

[0099] Micro-hydrogel 3: Sodium hyaluronate-gelatin-PEG (SH-Gelatin-PEG) Continuous phase: 4.5% PEG dissolved in freeze-dried protective solution; Dispersed phase: aqueous solution, 5.33% gelatin, 0.27% sodium hyaluronate (Yuanye Biotechnology S24592), 3% mannitol.

[0100] The above percentages are all mass percentages.

[0101] The components of the freeze-dried protective solution are: 30 mM trehalose, 1 mM magnesium chloride, 4.8 mM potassium chloride, 142.5 mM sodium chloride, 9.5 mM HEPES and 7 mM hydroxypropyl-β-cyclodextrin.

[0102] The dispersed phase solution and the continuous phase solution were prepared according to the above recipes.

[0103] After the platelet incubation time is over, the platelets are taken out of the incubator and centrifuged at 1000 g for 12 minutes. The supernatant is removed and the platelets are resuspended in the dispersed phase solution. The platelet concentration is adjusted to 2×10 8 / mL, filter through a 40μm filter. Add 0.5μl FITCanti-human CD41 / CD61 (Biolegend, 359810), mix well, and incubate for 20 minutes in the dark.

[0104] Take 50 μL of the dispersed phase solution after adding platelets and blow it into 1 mL of the continuous phase solution to emulsify it, and obtain the platelet micro-hydrogel with platelets wrapped inside the micro-hydrogel. Put it into a 24-well plate and observe it under a microscope. Figure 4 As shown above, the dispersed phase solution and the continuous phase solution are emulsified to form micro-hydrogels. Most of the platelets are wrapped inside the micro-hydrogels, showing green fluorescence. Dextran-Gelatin-PEG and Dextran-Gelatin-PVA micro-hydrogels have a core-shell structure, and SH-Gelatin-PEG micro-hydrogels have a multi-chamber structure. The size of the micro-hydrogels is as shown in Figure 5 As shown (Lyo stands for freeze-dried).

[0105] The sizes of each group of micro-hydrogels before freeze-drying were: Dextran-Gelatin-PEG (9.71μm-77.80μm); Dextran-Gelatin-PVA (15.69μm-116.00μm); SH-Gelatin-PEG (13.50μm-127.92μm).

[0106] Lyophilization of platelet-encapsulated microhydrogels An equal amount of freeze-dried protective solution was directly added to an equal amount of platelets and resuspended to serve as a direct freeze-dried control group.

[0107] Transfer the platelet-encapsulated micro-hydrogel or directly resuspended platelets into vials, each with a volume of 1 mL, and pre-freeze at -80°C for 10 hours.

[0108] Transfer the pre-frozen vials to the freeze dryer, set the plate temperature to -5°C, and the vacuum to 1 Pa. Dry for 10 hours.

[0109] The temperature was raised to 25°C at a rate of 0.5°C / min and the freeze-dried powder was obtained after secondary drying for 10 hours.

[0110] like Figure 6 As shown, the platelets (such as Figure 6 Left, the Dextran-Gelatin-PEG encapsulated platelets are more plump, while the platelets directly resuspended after freeze-drying (such as Figure 6 Right) A collapse occurred.

[0111] Add the same volume of ddH2O to the platelet freeze-dried powder prepared by the above method, blow and mix evenly. Place in a 24-well plate and observe under a microscope. Figure 4 As shown below, it can be seen that the micro-hydrogel wrapped with platelets after freeze-drying can maintain the original structure before freeze-drying. The size of the micro-hydrogel after freeze-drying is as follows: Figure 5 shown.

[0112] The sizes of each group of micro-hydrogels after freeze-drying were: Dextran-Gelatin-PEG (12.45μm-99.88μm); Dextran-Gelatin-PVA (9.78μm-98.96μm); SH-Gelatin-PEG (4.68μm-55.95μm).

[0113] Example 4 Functional testing of platelet-encapsulated microhydrogel freeze-dried powder Platelet rehydration and dissociation The same volume of ddH2O was added to the platelet freeze-dried powder prepared in Example 3 (Dextran-Gelatin-PEG micro-hydrogel freeze-dried powder encapsulating platelets / direct freeze-dried powder), and the mixture was pipetted and mixed evenly.

[0114] For the platelet-encapsulated micro-hydrogel freeze-dried powder, the ddH2O resuspension was centrifuged at 500 g for 5 minutes, the supernatant was removed, the same volume of Tyrode's solution was added to resuspend the precipitate, incubated at 37°C for 5 minutes, and the released platelets were harvested.

[0115] Flow cytometry to detect the content of platelet surface glycoprotein and the degree of desialylation PGE1 was added to the platelet-encapsulated micro-hydrogel freeze-dried group, the direct freeze-dried group, and fresh platelets to a concentration of 1 μM and centrifuged at 1000 g for 12 minutes. The supernatant was removed and the platelet concentration was adjusted to 5×10 with Tyrode's solution. 7 / mL.

[0116] Take 500 μL of platelet suspension in a 1.5 mL centrifuge tube, add 0.5 μL Brilliant Violet 650™ anti-human CD42b (Biolegend, 303926, specifically binds to glycoprotein CD42b), 0.5 μL PE / Cyanine7 anti-human CD41 / CD61 (Biolegend, 359812, specifically binds to glycoprotein CD41 / 61), and 0.2 μL FITC-RCA-I (Vector, FL-1081-1, specifically binds to galactose molecules exposed after desialylation), and incubate in the dark for 20 minutes.

[0117] After incubation, the cells were filtered through a 40 μm filter and placed in a flow tube. The surface glycoprotein content and desialylation degree of each group were detected in a flow cytometer (BD LSRFortessaSORP).

[0118] like Figure 7 As shown, compared with fresh platelets (Fresh), the double-positive ratio of CD41 / 61 and CD42b of platelets in the direct freeze-dried (Lyo) group was significantly reduced to only 65.00±7.99%; the double-positive ratio of CD41 / 61 and CD42b of platelets in the microhydrogel freeze-dried (Encap-Lyo) group encapsulating platelets was maintained at 90.10±5.90%, which is very close to the 93.97±5.56% of fresh platelets.

[0119] like Figure 8 As shown, the expression of galactose (a molecule exposed after desialylation) in fresh platelets was set to 1. Directly freeze-dried (Lyo) platelets were severely desialylated, and the galactose expression level increased to 2.71±0.75. The desialylation degree of platelets in the microhydrogel freeze-dried (Encap-Lyo) group encapsulating platelets was significantly inhibited, and its galactose expression level increased to 1.32±0.32, which was not significantly different from the fresh group (Fresh).

[0120] Platelet aggregation assay in vitro PGE1 was added to the platelet-encapsulated micro-hydrogel freeze-dried group, the direct freeze-dried group, and fresh platelets to a concentration of 1 μM and centrifuged at 1000 g for 12 minutes. The supernatant was removed and the platelet concentration was adjusted to 2×10 with platelet-poor plasma. 8 / mL.

[0121] Take 300 µL of platelet suspension and put it into a special test tube. Add a rotor and incubate at 37°C for 5 minutes. Put it into the aggregation instrument and adjust to zero. Add thrombin to 2 U / mL and observe the aggregation.

[0122] like Fig. 9 As shown in the results, the aggregation ability of platelets in the direct freeze-dried (Lyo) group was significantly weakened, only 65.33±4.16%, and the aggregation ability of platelets in the micro-hydrogel freeze-dried (Encap-Lyo) group encapsulating platelets was 95.67±7.51%, which was not significantly different from the aggregation ability of fresh platelets (Fresh) of 85.67±2.08%. The results of the platelet aggregation instrument further proved the effect of micro-hydrogel encapsulation and could be mutually verified with the results of flow cytometry.

[0123] The test results showed that the platelets encapsulated by the microhydrogel freeze-dried, rehydrated and dissociated platelets had higher glycoprotein expression, higher aggregation ability, and lower degree of desialylation.

[0124] Example 5 Long-term storage of platelet-encapsulated microhydrogel lyophilized powder After preparing platelet freeze-dried powder (Dextran-Gelatin-PEG micro-hydrogel freeze-dried powder / direct freeze-dried powder encapsulating platelets) according to Example 3, the freeze-dryer was filled with nitrogen in situ, covered with an aluminum cap, and vacuum-sealed and stored at room temperature (25° C.). One month later, the surface glycoprotein content and desialylation degree of the freeze-dried platelets were detected according to the method in Example 4.

[0125] Fresh platelets from the same batch were reserved, and the surface glycoprotein content and desialylation degree of the fresh platelets were detected according to the method in Example 4.

[0126] like Fig.10 As shown, compared with fresh platelets (Fresh), the CD41 / 61 and CD42b double-positive ratio of platelets in the direct freeze-dried (Lyo) group decreased significantly after one month of storage, to only 51.3%; the CD41 / 61 and CD42b double-positive ratio of platelets in the microhydrogel freeze-dried (Encap-Lyo) group encapsulating platelets was maintained at 83.2%, which is close to 98.6% of fresh platelets.

[0127] like Fig.11 As shown, the expression of galactose (a molecule exposed after desialylation) in fresh platelets is set to 1. Directly freeze-dried (Lyo) platelets suffer from severe desialylation, and the galactose expression level is relatively increased to 1.57. The desialylation degree of platelets in the microhydrogel freeze-dried (Encap-Lyo) group encapsulating platelets is significantly inhibited, and its galactose expression level is relatively increased to 1.09, which is close to the fresh group (Fresh).

[0128] Functional testing of platelets shows that the microhydrogel freeze-dried powder encapsulating platelets provided in the present application can be stored for a long time, and the platelets after rehydration and dissociation after storage have higher glycoprotein expression and inhibit desialylation.

[0129] The effects of the examples of the present application show that the platelets encapsulated in the micro-hydrogel have a higher expression of hemostasis-related glycoproteins and a lower degree of desialylation after rehydration and dissociation, and are expected to show better hemostasis ability and longer in vivo circulation time. The micro-hydrogel, lyophilized powder and preparation method of platelets encapsulated in the present application can significantly extend the storage time of platelets and alleviate the current situation of platelet shortage.

[0130] The protection scope of the present application is not limited to the above-mentioned embodiments. Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the scope and spirit of the present invention. If these changes and modifications fall within the scope of the claims of the present application and their equivalents, the intention of the present application also includes these changes and modifications.

[0131] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0132] In addition, the various implementation modes of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed in the present application.

Claims

1. A micro-hydrogel encapsulating platelets, characterized in that: The micro-hydrogel has a core-shell or multi-chamber structure, and the platelets are wrapped inside the micro-hydrogel; the micro-hydrogel is formed by physical gelation of one or more water-soluble polymer components.

2. The platelet-encapsulating micro-hydrogel according to claim 1, characterized in that: The water-soluble polymer component is selected from dextran, gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, hyaluronic acid or its salt, collagen, chitosan, sodium alginate, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polyethylene glycol.

3. The platelet-encapsulating micro-hydrogel according to claim 2, characterized in that: The micro hydrogel is formed by physical gelation of gelatin and one or two selected from dextran and sodium hyaluronate.

4. The platelet-encapsulating micro-hydrogel according to claim 3, characterized in that: The mass ratio of gelatin, dextran and / or sodium hyaluronate is (2-500):

1.

5. The platelet-encapsulating micro-hydrogel according to claim 4, characterized in that: The mass ratio of gelatin, dextran and / or sodium hyaluronate is (10-30):

1.

6. The platelet-encapsulating micro-hydrogel according to any one of claims 1 to 5, characterized in that: The micro-hydrogel is spherical or quasi-spherical, with a diameter of 1-1000 μm.

7. The platelet-encapsulating micro-hydrogel according to any one of claims 1 to 5, characterized in that: The micro-hydrogel also contains one or more of trehalose, sucrose, glucose and maltose.

8. A method for preparing a micro-hydrogel encapsulating platelets, characterized in that: It includes the following steps: After emulsifying a dispersed phase solution containing platelets in a continuous phase solution, it undergoes spontaneous physical gelation to form a micro-hydrogel, and the platelets are encapsulated inside the micro-hydrogel; wherein the dispersed phase solution is an aqueous solution containing one or more water-soluble polymer components A; the continuous phase solution is an aqueous phase or an oil phase, wherein the aqueous phase is an aqueous solution containing one or more water-soluble polymer components B, and the oil phase contains one or more oil-based components and one or more surfactants; The water-soluble polymer component A and the water-soluble polymer component B are insoluble in each other.

9. The preparation method according to claim 8, characterized in that: The platelets are pre-treated and incubated platelets.

10. The preparation method according to claim 9, characterized in that: The pretreatment incubation refers to adding the incubation solution to the platelet precipitate separated from the blood, resuspending, heating and incubating, and centrifuging and collecting.

11. The preparation method according to claim 10, characterized in that The amount of the incubation solution is 1-100% of the original blood volume, and the incubation solution contains 1-100 mM trehalose, 1-1000 mM sodium chloride, 1-100 mM potassium chloride, 1-100 mM imidazole, 1-100 mM EGTA, and 1-10 μM PGE1.

12. The preparation method according to any one of claims 8 to 11, characterized in that: The concentration of platelets in the dispersed phase solution is 0.1-10×10 9 / mL.

13. The preparation method according to claim 8, characterized in that: The water-soluble polymer component A and the water-soluble polymer component B are independently selected from dextran, gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, hyaluronic acid and its salts, collagen, chitosan, sodium alginate, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polyethylene glycol.

14. The preparation method according to claim 13, characterized in that: The dispersed phase solution is an aqueous solution containing gelatin and one or two selected from dextran and sodium hyaluronate, and the continuous phase solution contains polyethylene glycol and an aqueous solution of one or two selected from polyethylene glycol.

15. The preparation method according to claim 8, 13 or 14, characterized in that: In the dispersed phase solution, the total concentration of one or more water-soluble polymer components A is 0.1-20% (w / v); and the total concentration of one or more polymer components B in the continuous phase solution is 0.1-20% (w / v).

16. The preparation method according to claim 15, characterized in that: The dispersed phase solution is an aqueous solution containing 2-10% (w / v) gelatin and 0.1-0.5% (w / v) dextran and / or sodium hyaluronate; the continuous phase solution is an aqueous solution containing 2-15% (w / v) polyethylene glycol and / or polyvinyl alcohol.

17. The preparation method according to claim 8, 13 or 14, characterized in that: The solvent of the dispersed phase solution is pure water, or the solvent in the dispersed phase solution is water, and one or more osmotic pressure regulators are added.

18. The preparation method according to claim 17, characterized in that: The osmotic pressure regulator is selected from HEPES, Tricine, sodium chloride, and mannitol; wherein the total concentration of the osmotic pressure regulator is 0.1-10% (w / v).

19. The preparation method according to claim 18, characterized in that: The solvent of the dispersed phase solution is water and contains 3-5% mannitol.

20. The preparation method according to claim 8, 13 or 14, characterized in that: The continuous phase solution is an aqueous phase, and its solvent is one or more of a freeze-drying protective solution, pure water, or a buffer solution.

21. The preparation method according to claim 20, characterized in that: The buffer is selected from phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, carbonate buffer, triethanolamine buffer, Tricine solution, Tris buffer, and Tyrode's solution; The total concentration of solute in the buffer is 1-1000 mM.

22. The preparation method according to claim 20, characterized in that: The freeze-drying protective solution contains one or more of trehalose, sucrose, glucose, maltose, glycerol, and mannitol, and optionally, contains one or more selected from magnesium chloride, potassium chloride, sodium chloride, HEPES, and hydroxypropyl-β-cyclodextrin.

23. The preparation method according to claim 22, characterized in that: The freeze-dried protective solution contains 1-100 mM trehalose, 1-10 mM magnesium chloride, 1-10 mM potassium chloride, 1-1000 mM sodium chloride, 1-100 mM HEPES, and 1-100 mM hydroxypropyl-β-cyclodextrin.

24. The preparation method according to claims 8 and 13, characterized in that: The continuous phase solution is an oil phase, and the oil-based component is selected from n-hexadecane, isohexadecane, mineral oil, stearane, isostearane, squalane, decane, dodecane, dimethicone, paraffin oil, olive oil, coconut oil, grape seed oil, almond oil, soybean oil, isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl linoleate, lauryl benzoate, isostearyl isostearate, fatty acid lactate, decyl oleate, and octyl palmitate; the surfactant is selected from Span 80, Span 60, polysorbate 20, polysorbate 60, polysorbate 80, coconut glucoside, lauryl glucoside, glyceryl stearate, dimethiconol, sodium lauryl sulfate, sodium lauroyl glutamate, sodium methyl cocoyl taurate, stearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, cocamidopropyl betaine, and lauroamidopropyl betaine.

25. The preparation method according to claim 24, characterized in that The total concentration of the one or more oil-based components is 70-99.9% (w / v); the total concentration of the surfactant is 0.1-30% (w / v).

26. The preparation method according to claim 8, characterized in that: The volume ratio of the dispersed phase solution to the continuous phase solution is 1:1-100.

27. The preparation method according to claim 26, characterized in that: The volume ratio of the dispersed phase solution to the continuous phase solution is 1:20-50.

28. The preparation method according to claim 8, 22, 23, 26 or 27, characterized in that: The dispersed phase solution is an aqueous solution containing 5.5% (w / v) gelatin, 0.25% (w / v) dextran, and 3-5% mannitol; the continuous phase solution is a freeze-drying protective solution containing 4.5% (w / v) polyethylene glycol; or the dispersed phase solution is an aqueous solution containing 5.5% (w / v) gelatin, 0.25% (w / v) dextran, and 3-5% mannitol; the continuous phase solution is a freeze-drying protective solution containing 15% (w / v) polyvinyl alcohol; or the dispersed phase solution is an aqueous solution containing 5.33% (w / v) gelatin, 0.27% (w / v) sodium hyaluronate, and 3-5% mannitol; the continuous phase solution is a freeze-drying protective solution containing 4.5% (w / v) polyethylene glycol.

29. A micro-hydrogel freeze-dried powder encapsulating platelets, characterized in that: The micro-hydrogel is obtained by freeze-drying the micro-hydrogel encapsulating platelets as described in any one of claims 1 to 7 or the micro-hydrogel encapsulating platelets obtained by the preparation method as described in any one of claims 8 to 28.

30. The platelet micro hydrogel freeze-dried powder according to claim 29, characterized in that: Freeze-dry in lyophilization solution.

31. A method for preparing a micro-hydrogel freeze-dried powder encapsulating platelets, characterized in that: The method comprises the following steps of freeze-drying the platelet-wrapped micro-hydrogel according to any one of claims 1 to 7 or the platelet-wrapped micro-hydrogel prepared by the preparation method according to any one of claims 8 to 28.

32. The preparation method according to claim 31, characterized in that: Freeze-dry in lyophilization solution.

33. The preparation method according to claim 31, characterized in that: When preparing micro-hydrogels encapsulating platelets, when the solvent of the continuous phase solution is a freeze-drying protective solution, the micro-hydrogels are emulsified to form micro-hydrogels and then freeze-dried directly; when the solvent of the continuous phase solution is not a freeze-drying protective solution or the continuous phase solution is oil-based, the micro-hydrogels encapsulating platelets are collected by centrifugation after emulsification to form micro-hydrogels, and then resuspended in a freeze-drying protective solution for freeze-drying.

34. The preparation method according to any one of claims 31 to 33, characterized in that: The freeze drying comprises three steps: pre-freezing, primary drying and secondary drying.

35. The preparation method according to any one of claims 31 to 33, characterized in that: The platelet-wrapped micro-hydrogel is transferred to a vial, an aluminum foil bag, a composite film bag, a vacuum packaging bag, a modified atmosphere packaging bag, a polyester film bag, a glass bottle, a plastic bottle, a polyethylene tube, a polypropylene tube, a glass tube, an aluminum tube, or a polyester tube for freeze drying. Optionally, the freeze drying process also includes nitrogen protection, sealing, and vacuuming steps.

36. A method for using the platelet-encapsulating micro-hydrogel freeze-dried powder according to any one of claims 29 to 30, or the platelet-encapsulating micro-hydrogel freeze-dried powder obtained by the preparation method according to any one of claims 31 to 35, characterized in that: The following steps are involved: The platelets are harvested by rehydration, centrifugation to collect the micro-hydrogel encapsulating the platelets, heating to dissociate, and centrifugation to collect.

37. The method of use according to claim 36, characterized in that: The rehydration refers to resuspending the micro-hydrogel freeze-dried powder encapsulating the platelets using one or more of pure water, plasma, or buffer; the volume of one or more of the pure water, plasma, or buffer is 1 / 2-2 times the volume before freeze-drying.

38. The method of use according to claim 36, characterized in that: The heat dissociation refers to resuspending the micro-hydrogel encapsulating platelets in one or more of pure water, plasma, or buffer solution and heating and incubating; the volume of one or more of the pure water, plasma, or buffer solution is 1 / 2-2 times the volume before freeze-drying.

39. The method of use according to claim 38, characterized in that: After heating and incubation, PGE1 was added to 1-10 µM and the platelets were harvested by centrifugation.

40. The method of use according to claim 36, characterized in that: After the harvested platelets are collected by centrifugation, they are resuspended in one or more of pure water, plasma, or buffer for subsequent use; the volume of one or more of the pure water, plasma, or buffer is 1 / 2-2 times the volume before lyophilization.

41. The method of use according to claims 37, 38 and 40, characterized in that: The buffer is selected from phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, carbonate buffer, triethanolamine buffer, Tricine solution, Tris buffer, and Tyrode's solution; the total concentration of the solute in the buffer is 1-1000 mM.

42. A method for freeze-drying, storing and / or transporting and using platelets, characterized in that: The method comprises the following steps: preparing micro-hydrogel encapsulating platelets according to the preparation method described in any one of claims 8 to 28, preparing lyophilized micro-hydrogel encapsulating platelets according to the method described in any one of claims 31 to 35, storing or transporting the micro-hydrogel, and obtaining harvested platelets according to the use method described in any one of claims 36 to 41.

43. The method for freeze-drying, storing and / or transporting and using platelets according to claim 42, characterized in that: Store or transport at room temperature, 4°C, -20°C, or -80°C.

44. Use of the platelet-encapsulating micro-hydrogel according to any one of claims 1-7, the platelet-encapsulating micro-hydrogel prepared according to the preparation method according to any one of claims 8-28, the platelet-encapsulating micro-hydrogel lyophilized powder according to any one of claims 29-30, or the platelet-encapsulating micro-hydrogel lyophilized powder prepared according to the preparation method according to any one of claims 31-35 in the preparation of biomedical products.

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