A micro-hydrogel encapsulating platelets, a lyophilized powder and a preparation method, a use method and an application thereof

Abstract

The application belongs to the technical field of biomedical engineering, and particularly relates to a micro-hydrogel wrapping platelets, a freeze-dried powder and a preparation method, use method and application thereof. The micro-hydrogel wrapping platelets, the micro-hydrogel is in a core-shell or multi-chamber structure, and the platelets are wrapped inside; the micro-hydrogel is formed by emulsifying a dispersed phase solution in a continuous phase solution and spontaneously physically gelling, and the platelets are wrapped inside; the micro-hydrogel can reduce the damage to the platelets caused by the freeze-drying process and rehydration, and the platelets can be stored for a long time after freeze-drying; when used, the micro-hydrogel can be dissociated by only rehydrating, centrifuging and heating, without the need of adding an additional decrosslinking agent, and the operation is convenient. The micro-hydrogel wrapping platelets has higher expression of hemostasis-related glycoprotein and lower de-sialylation degree after freeze-drying, rehydration and dissociation, and is expected to have better hemostasis ability and longer in-vivo circulation time; and is expected to greatly extend the storage time of platelets and alleviate the shortage of platelets.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering, and specifically relates to a micro-hydrogel wrapped with platelets, a freeze-dried powder and a preparation method, use method and application thereof. BACKGROUND

[0002] Platelets are cell fragments produced by megakaryocytes and are the key components of hemostasis in blood, playing a key role in hemostasis. Resting platelets are disc-shaped, with a diameter of 2-3 μm, and the normal concentration in the human body is (100-300) × 10 9 / L. When a wound occurs, collagen in the extracellular matrix will be exposed. At this time, GP Ibα (also known as CD42b) on the surface of the platelets will bind with von Willebrand factor (vWF) and adhere to the collagen through vWF as a bridge, achieving the effect of initial hemostasis. Subsequently, collagen contacts GP VI on the surface of the platelets, stimulating platelet activation, causing the platelets to release agonists such as ADP and thrombin. These agonists can activate free platelets in the blood, causing morphological changes and extending pseudopod-like tentacles. The activated platelets have a larger specific surface area, and the surface GP IIb-IIIa (also known as CD41 / 61) will connect with fibrin to form a clot. In addition, platelets can also link and aggregate through CD42b and vWF. Activated platelets will further release more agonists, triggering a coagulation cascade to achieve hemostasis.

[0003] Platelets are in great demand in clinical practice. Emergencies such as car accidents, surgical bleeding, and delivery bleeding require immediate platelet transfusion for hemostasis. In addition, platelet transfusion is an effective method for treating thrombocytopenia. Patients with thrombocytopenia have a blood platelet concentration of less than 100 × 10 9 / L, and after capillary bleeding in the body, hemostasis cannot be achieved in time, and blood stasis will deposit on the surface of the skin to form purpura. As the number of platelets decreases, bleeding worsens, and patients with very few platelets may experience massive gastrointestinal bleeding or even life-threatening intracranial bleeding without injury. The "Chinese Expert Consensus on Diagnosis and Treatment of Adult Thrombocytopenia" states: "Platelet count <20 × 10 9 / L with bleeding or other internal bleeding, platelet transfusion is an effective treatment measure."

[0004] Short shelf life is one of the main reasons for the shortage of platelets. The standard storage method for apheresis platelets is 25 °C oscillation storage, with a shelf life of only 3-5 days. In contrast, 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 amount of platelets being discarded. The short shelf life of room temperature oscillation stored platelets results in a large amount of platelet waste, which cannot meet the demand for platelets for patients with trauma and platelet deficiency.

[0005] Researchers have tried various methods to extend the shelf life of platelets, but none have been successful.

[0006] As early as last century, researchers tried to store platelets at 4 °C in the refrigerator, and it was once the mainstream method. Refrigeration can reduce bacterial growth and inhibit protein degradation, theoretically extending the shelf life of platelets. However, during long-term storage at 4 °C, the rewarming process can cause platelets to desialylation, and desialylated platelets can expose galactose, leading to rapid clearance in the liver, resulting in a significant decrease in platelet circulation time in the body, rapid clearance in the body, and a significant decrease in hemostatic effect. If it is short-term refrigeration at 0 °C, the platelet surface will expose β-GlcNAc, which will then be cleared by 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 been withdrawn from clinical use.

[0007] Some researchers have also tried to freeze platelets. However, frozen platelets are also rapidly cleared in the body, 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 claim to have invented "no-wash" platelet freezing technology: after thawing, platelets do not need to be washed to remove DMSO before being infused into the body. However, frozen platelets may also undergo desialylation and be rapidly cleared, with a recovery rate in the body of only about half that of fresh platelets. In addition, the toxicity of DMSO is a potential risk of platelet freezing. Moreover, freezing relies on -80 °C freezers, which are not conducive to transportation and cannot meet the demand for platelets in war-torn areas and remote areas.

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

[0010] Freeze-drying involves three steps: freezing, primary drying, and secondary drying. The water-containing sample is first frozen into ice, then kept at a low temperature, where reduced pressure is applied to allow the ice to sublime directly into gas. This step, known as primary drying, removes most of the free water. Maintaining low pressure while slowly increasing the temperature further removes bound water, a process known as secondary drying. The freeze-dried sample loses over 90% of its water content and can be stored long-term at room temperature or 4°C. When needed, it can be rehydrated by adding the appropriate solution. During the freeze-drying process, the sample undergoes a dramatic phase transition, which can cause irreversible damage. To maintain the activity of the freeze-dried sample, trehalose is often added as a preservative. Trehalose, through its "water replacement" effect, mitigates the aggregation and inactivation of proteins and lipids in environments such as high temperature, extreme cold, and aridity. Currently, bioactive samples such as proteins, exosomes, and mRNA vaccines have been freeze-dried for long-term storage using 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 were used in the pretreatment solution, and plasma was used as a protective agent in the freeze-drying protective solution. Although the appearance of the platelets after freeze-drying was acceptable and the cytokine concentration was maintained, the platelet aggregation ability decreased after freeze-drying. In addition, for freeze-dried platelets, no attention was paid to the expression of platelet surface glycoproteins related to hemostasis ability, and the degree of surface desialylation related to the circulation time in the body. In CN202010377816.1, platelets were incubated in a solution containing trehalose, and then the platelets were fixed with glutaraldehyde and ethanol, and then freeze-dried. Although the expression of platelet surface glycoproteins was maintained, the fixed platelets lost their activity and could not 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 the platelet membrane, 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 platelet surface 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 short, there is currently no universally recognized solution to the platelet storage problem. While there is a huge clinical demand for platelets, a large number of platelets are discarded. Therefore, there is an urgent need for improved methods to extend the storage period of platelets. Summary of the Invention

[0013] To meet the clinical use requirements, the 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 infusion. In addition, the flux of freeze-dried platelets should be large to meet the use requirements of patients with massive hemorrhage and patients with thrombocytopenia.

[0014] In the current research on freeze-dried platelets, various protective agents are added for direct freeze-drying, including trehalose, mannitol, albumin, and poly-sucrose. Although these protective agents can partially alleviate the damage during freeze-drying, they are difficult to provide sufficient protection; the current freeze-dried platelets generally have the problems of decreased hemostatic ability (such as a large decrease in the expression of hemostasis-related glycoprotein CD42b) and reduced circulation time (de-sialylation leading to high expression of galactose).

[0015] During freeze-drying, platelets will undergo three serious damages. First, in the freezing stage, the generated sharp ice crystals will pierce the cell membrane and other structures of the platelets, causing mechanical damage; second, in the drying stage, the escape of water will cause irreversible aggregation of the lipid and protein of the platelet membrane, losing activity; finally, in the rehydration stage, during the transition from solid to liquid, the platelets directly contact water molecules, which will cause a sharp change in osmotic pressure, leading to osmotic shock. In the face of these damages, trehalose may not be able to provide sufficient protection (see Fig. (a) of Figure 1 The decrease in the hemostatic ability and in vivo circulation time of freeze-dried platelets is mainly due to the damage caused by freeze-drying and rehydration.

[0016] To solve the above technical problems in the prior art, the present application provides a micro-hydrogel for wrapping platelets, a freeze-dried powder and a preparation method, use method and application thereof. The micro-hydrogel for wrapping platelets provided by the present application wraps platelets inside the micro-hydrogel, which can reduce the damage to platelets caused by freeze-drying and rehydration. The micro-hydrogel for wrapping platelets can be stored for a long time after freeze-drying, prolonging the effective period of platelets. When needed, the platelets can be dissociated from the micro-hydrogel by rehydration-centrifugation-heating without the need for additional addition of a decrosslinking agent, which is convenient to operate. Compared with the current method of directly freeze-drying platelets, the micro-hydrogel for wrapping platelets provided by the present application has higher surface glycoprotein expression and higher aggregation ability after freeze-drying preservation and rehydration dissociation, which can enable the platelets to maintain normal hemostatic ability and inhibit the de-sialylation process, and can circulate in the body for a longer time. Through the freeze-drying preservation method of the micro-hydrogel for wrapping platelets provided by the present application, it is expected to greatly prolong the storage time of platelets and alleviate the shortage of platelets.

[0017] The use of micro-hydrogel to encapsulate platelets can reduce the damage during the freeze-drying process. During the freezing process, the macromolecules that constitute the micro-hydrogel 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, the freeze-drying protectant such as trehalose can alleviate the aggregation of proteins and lipids through the "water replacement" effect. Finally, during the rehydration process, the external hydrogel will first contact the water and then slowly release the water to the internal platelets, thereby reducing the osmotic shock (see Figure 1 Therefore, the micro-hydrogel encapsulated platelets combined with freeze-drying is expected to become an ideal method for storing platelets.

[0018] In a first aspect, the present application provides a micro-hydrogel encapsulating platelets, wherein the micro-hydrogel is in 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 some embodiments, the water-soluble polymer component is selected from dextran, gelatin, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, hyaluronic acid or a salt thereof (such as a metal salt of hyaluronic acid, which can be sodium hyaluronate), collagen, chitosan, sodium alginate, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol.

[0020] In some 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 of dextran and sodium hyaluronate. Specifically, the mass ratio of gelatin to dextran and / or sodium hyaluronate is (1-1000): 1, preferably (2-500): 1, more preferably (4-100): 1, (5-50): 1, (10-30): 1, such as 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 some embodiments, the micro-hydrogel is spherical or spheroidal, with a diameter of 1-1000 μm, preferably 5-150 μm.

[0022] In some embodiments, the micro-hydrogel further contains one or more of trehalose, sucrose, glucose, and maltose in the interior, preferably the micro-hydrogel further contains trehalose in the interior. The trehalose, sucrose, glucose, and maltose in the interior of the micro-hydrogel can synergistically play a freeze-drying protective role for the platelets during freeze-drying.

[0023] In a second aspect, the present application provides a method for preparing a micro-hydrogel encapsulating platelets, comprising the following steps: after emulsifying a dispersed phase solution containing platelets in a continuous phase solution, the micro-hydrogel is formed by spontaneous physical gelation, 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; wherein the water-soluble polymer components A and the water-soluble polymer components B are immiscible with each other.

[0024] The present application forms a stable emulsion structure by emulsifying the dispersed phase solution containing water-soluble polymer components A in the continuous phase, because the dispersed phase solution and the continuous phase solution are immiscible with each other, the interactions between the components are different, and through the phase separation-water transfer principle, the liquid-liquid phase separation occurs spontaneously, and the water-soluble polymer components A spontaneously physically gelate to form micro-hydrogels. After adding platelets in the dispersed phase solution, the platelets can be encapsulated inside the micro-hydrogels. The micro-hydrogel encapsulating platelets of the first aspect can be prepared by the preparation method described in the present application.

[0025] In some embodiments, the platelets are pre-treated and incubated platelets. The pre-treatment and incubation refers to adding an incubation solution to the platelet precipitate separated from blood, resuspending, heating and incubating, and collecting by centrifugation; preferably, the amount of the incubation solution is 1-100% of the volume of the original blood, preferably 10% of the volume of the original blood. 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 (ethylene glycol-bis (2-aminoethanol)- N,N,N',N'- 1-10 mM PGE1 (prostaglandin E1); preferably the composition of the hatching solution is 50 mM trehalose, 100 mM sodium chloride, 10 mM potassium chloride, 10 mM imidazole, 10 mM EGTA, 1 mM 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 10-200 rpm, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200 rpm, and the incubation time is 1-10 h, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 h. Preferably, the incubation temperature is 35-38 °C, 30-60 rpm, and the incubation time is 2-5 h. The conditions for centrifugal collection are a centrifugal force of 100-1000 g, such as 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g. The duration of the centrifugation is 3-30 min, such as 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 force of 500-1000 g and a duration of 10-15 min.

[0026] In certain embodiments, the platelet pellet is isolated from blood by adding PGE1 to 1-10 μΜ (preferably 1 μΜ) to inhibit platelet activation in the platelet rich plasma after removal of most of the red blood cells, white blood cells, and centrifugation to obtain the platelet pellet. The centrifugation is at 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 is for 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 centrifugation is at 500-1000 g for 10-15 min.

[0027] In certain embodiments, the platelets (preferably pre-processed incubated platelets) are resuspended using a dispersing phase, the concentration of platelets in the dispersing phase solution is 0.1-10 x 10 9 / mL. For example, 0.1 x 10 9 / mL, 0.2 x 10 9 / mL, 0.3 x 10 9 / mL, 0.4 x 10 9 / mL, 0.5 x 10 9 / mL, 0.6 x 10 9 / mL, 0.7 x 10 9 / mL, 0.8 x 10 9 / mL, 0.9 x 10 9 / mL, 1.0 x 10 9 / mL, 1.1 x 10 9 / mL, 1.2 x 10 9 / mL, 1.3 x 10 9 / mL, 1.4 x 10 9 / mL, 1.5 x 10 9 / mL, 1.6 x 10 9 / mL, 1.7 x 10 9 / mL, 1.8 x 10 9 / mL, 1.9 x 10 9 / mL, 2.0 x 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-co-glycolic acid, and polyethylene glycol. In certain embodiments, water-soluble polymer component A is selected from gelatin, dextran, and 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 both of dextran and sodium hyaluronate, and the continuous phase solution is an aqueous solution containing one or both of polyethylene glycol and polyethylene glycol.

[0029] In certain embodiments, the total concentration of the one or more water-soluble polymer components A in the dispersed 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 the one or more water-soluble polymer components A is 2-15% (w / v). The total concentration of the 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 the 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 dextran and / or sodium hyaluronate), preferably an aqueous solution containing 2-10% (w / v) gelatin (such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), and 0.1-0.5% (w / v) dextran and / or sodium hyaluronate (such as 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% (such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%) (w / v) polyethylene glycol and / or polyvinyl alcohol.

[0031] In certain embodiments, the solvent in the dispersed phase solution is pure water. In certain embodiments, the solvent in the dispersed phase solution is water, and one or more osmotic pressure adjusting agents are added to adjust the osmotic pressure, for example, the osmotic pressure adjusting agent is selected from the group consisting of HEPES (4- hydroxyethylpiperazineethanesulfonic acid), Tricine (tris(hydroxymethyl)methylamino acid), sodium chloride, mannitol, and the like. Preferably, the osmotic pressure adjusting agent is mannitol. In certain embodiments, the total concentration of the osmotic pressure adjusting agent 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 adjusting agent in the dispersed phase solution is 1-8% (w / v). More preferably, the solvent in the dispersed phase solution is water and contains 3-5% mannitol.

[0032] In certain embodiments, when the continuous phase solution is an aqueous phase, the solvent is one or more of a lyoprotectant solution, pure water, or a buffer solution, for example, one or more of 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 TIS buffer. Preferably, when the continuous phase solution is an aqueous phase, the solvent is a lyoprotectant solution. The total concentration of the solutes in the buffer solution is 1-1000 mM.

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

[0034] In certain embodiments, when the continuous phase solution is an oil phase, the oil-based component is selected from the group consisting of n-hexadecane, iso-hexadecane, mineral oil, stearyl alkane, isostearyl alkane, squalane, decane, dodecane, dimethicone, paraffin oil, olive oil, coconut oil, grape seed oil, apricot kernel oil, soybean oil, isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl linoleate, dodecanol benzoate, isostearyl isostearate, fatty acid lactylate, 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 components is 80-90%.

[0035] In certain embodiments, when the continuous phase solution is an oil phase, the surfactant is selected from the group consisting of Span 80, Span 60, polysorbate 20, polysorbate 60, polysorbate 80, cocoglycoside, lauryl glucoside, glyceryl stearate, dimethicone alcohol, sodium lauryl sulfate, sodium lauroyl glutamate, sodium methyl cocoyl taurate, stearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, cocamidopropyl betaine, lauramidopropyl 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 and the continuous phase solution is 1:1-1000, preferably 1:1-100, for example 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 and the continuous phase solution is 1:20-50.

[0037] In some embodiments, the dispersed phase solution is an aqueous solution containing 5.5% (w / v) gelatin, 0.25% (w / v) dextran, 3-5% mannitol; the continuous phase solution is a lyoprotectant 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, 3-5% mannitol; the continuous phase solution is a lyoprotectant 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, 3-5% mannitol; the continuous phase solution is a lyoprotectant solution containing 4.5% (w / v) polyethylene glycol.

[0038] In some embodiments, the emulsification method is not particularly limited as long as it can achieve sufficient contact and mixing of the dispersed phase and the continuous phase, and the emulsification method includes, but is not limited to, one or more of vortexing, shaking, pipette pipetting, electrospraying, membrane emulsification, and micro-channel extrusion.

[0039] In a third aspect, the present application provides a lyophilized powder of platelet- wrapped microhydrogel, which is obtained by freeze-drying the platelet-wrapped microhydrogel of the first aspect or the platelet-wrapped microhydrogel prepared according to the preparation method of the second aspect. Preferably, freeze-drying is performed in a lyoprotectant solution.

[0040] In a fourth aspect, the present application provides a preparation method of a lyophilized powder of platelet-wrapped microhydrogel, which comprises the step of freeze-drying the platelet-wrapped microhydrogel of the first aspect or the platelet-wrapped microhydrogel prepared according to the preparation method of the second aspect. Preferably, freeze-drying is performed in a lyoprotectant solution.

[0041] In some embodiments, when preparing the platelet-wrapped microhydrogel, the solvent of the continuous phase solution is a lyoprotectant solution, and the microhydrogel is directly freeze-dried after emulsification.

[0042] In some embodiments, when the solvent of the continuous phase solution is not the lyoprotectant or the continuous phase solution is oil-based in the preparation of the microhydrogel encapsulating platelets, the microhydrogel encapsulating platelets is collected by centrifugation after emulsification to form the microhydrogel, and then resuspended in the lyoprotectant 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 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 collection condition is that the centrifugal speed is 500-1000 g, and the centrifugal time is 10-15 min. The volume of the lyoprotectant used for resuspension is preferably 1 / 2-2 times the volume before centrifugal collection, more preferably equal to the volume before centrifugal collection.

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

[0044] In certain embodiments, the platelet-coated microhydrogels are transferred into a lyophilization container for lyophilization, preferably, further comprising a step of nitrogen protection after lyophilization, sealing (e.g. stoppering, aluminizing), vacuumizing. The lyophilization container includes, but is not limited to, a Schlenk flask, 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 (PE tube), a polypropylene tube (PP tube), a glass tube, an aluminum tube, a polyester tube (PET tube). The volume of the lyophilization container used is 0.1-30 mL, for example, 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, 27 mL, 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 shelf, dry ice, liquid nitrogen.

[0046] In certain embodiments, the temperature of the lyophilizer shelf is set to -50 °C -10 °C during primary drying. 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 duration of primary drying is 3-30 h. 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, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h.

[0047] In certain embodiments, the secondary drying is performed at an elevated temperature. The rate of temperature increase is 0.1-50°C / min, such as 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, 33°C / min, 34°C / min, 35°C / min, 36°C / min, 37°C / min, 38°C / min, 39°C / min, 40°C / min, 41°C / min, 42°C / min, 43°C / min, 44°C / min, 45°C / min, 46°C / min, 47°C / min, 48°C / min, 49°C / min, 50°C / min. The secondary drying temperature is 10-35°C, such as 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 is performed for 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 platelet- wrapped microhydrogel lyophilized powder of the third aspect, which comprises the steps of: rehydrating, centrifuging to collect the platelet-wrapped microhydrogel, heating to dissociate, and centrifuging to collect the harvested platelets. The method is suitable for the platelet-wrapped microhydrogel lyophilized powder of the third aspect or the platelet-wrapped microhydrogel lyophilized powder prepared according to the fourth aspect.

[0049] In some embodiments, the rehydration refers to resuspension of the platelet-encapsulated microhydrogel lyophilized powder in one or more of pure water, plasma, or a buffer (e.g., phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, carbonate buffer, triethanolamine buffer, Tricine solution, Tris buffer, Tyrode's solution). The total concentration of solutes in the buffer is 1-1000 mM. The volume of the solution used for rehydration is 1 / 2-2 times the volume before lyophilization. Preferably, the volume of the solution used for rehydration is based on the volume equivalent to that before lyophilization. More preferably, the platelet-encapsulated microhydrogel lyophilized powder is rehydrated using pure water.

[0050] In some embodiments, the microhydrogel after lyophilization-rehydration is spherical or spheroidal, with a diameter of 1-1000 μm, preferably 5-150 μm.

[0051] In some embodiments, the microhydrogel after lyophilization-rehydration is a core-shell structure or a multi-chamber structure.

[0052] In some embodiments, the heat dissociation refers to heat incubation of the resuspended platelet-encapsulated microhydrogel in one or more of pure water, plasma, or a buffer (e.g., phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, carbonate buffer, triethanolamine buffer, Tricine solution, Tris buffer, Tyrode's solution). The total concentration of solutes in the buffer is 1-1000 mM. Preferably, the platelet-encapsulated microhydrogel is resuspended in 1-20 mM Tyrode's solution for heat incubation. The volume of the solution used for incubation is 1 / 2-2 times the volume before lyophilization. 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 condition is 10-200 rpm, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200 rpm, and the incubation time is 1 min-1 h, for example, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60 min. Preferably, the incubation temperature is 35-38 °C, 30-60 rpm, and the incubation time is 2-5 min.

[0053] In some embodiments, PGE1 is added to 1-10 µM after heat incubation, preferably, PGE1 is added to 1 µM after heat incubation, and the harvested platelets are collected by centrifugation.

[0054] The conditions of the two-step centrifugation (centrifugation of the micro-hydrogel encapsulating platelets in the method of use of the fifth aspect, centrifugation of the harvested platelets) are that the centrifugation 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 conditions of the centrifugation are that the centrifugation speed is 500-1000 g, and the centrifugation time is 10-15 min.

[0055] In some embodiments, after the centrifugation of the harvested platelets, the platelets can be resuspended in one or more of pure water, plasma, or 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) for subsequent use. The total concentration of the solutes in the buffer is 1-1000 mM. Preferably, after the centrifugation of the harvested platelets, the platelets can be 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 the sixth aspect, the present application provides a method for lyophilization, storage and / or transportation and use of platelets, which comprises the following steps: preparing the micro-hydrogel encapsulating platelets according to the method of the second aspect, preparing the lyophilized powder according to the method of the fourth aspect, storage and / or transportation, and obtaining the harvested platelets according to the method of use of the fifth aspect. Preferably, the storage or transportation is carried out at room temperature, 4°C, -20°C or -80°C.

[0057] In the seventh aspect, the present application provides the use of the micro-hydrogel encapsulating platelets of the first aspect, the micro-hydrogel encapsulating platelets prepared according to the method of the second aspect, the lyophilized powder of the micro-hydrogel encapsulating platelets of the third aspect, or the lyophilized powder of the micro-hydrogel encapsulating platelets prepared according to the method of the fourth aspect in the preparation of a biomedical product.

[0058] Compared with the prior art, the present application has the following beneficial effects:

[0059] 1) The micro-hydrogel for wrapping platelets provided in the present application, platelets are wrapped inside the micro-hydrogel, which can reduce the damage of the freeze-drying process and the rehydration process to platelets. Compared with the existing direct freeze-drying method, the platelets wrapped in the micro-hydrogel have higher glycoprotein expression, higher aggregation ability, and lower desialidation degree after freeze-drying and rehydration dissociation, which is expected to have better hemostatic ability and in vivo circulation time.

[0060] 2) The preparation method of the micro-hydrogel for wrapping platelets provided in the present application is to emulsify the dispersed phase solution in the continuous phase solution, and form a micro-hydrogel by spontaneous physical gelation through liquid-liquid phase separation-water transfer principle. After adding platelets in the dispersed phase, the platelets can be wrapped inside the micro-hydrogel. The freeze-dried powder of the micro-hydrogel for wrapping platelets can be dissociated by rehydration, centrifugation and heating, and the wrapped platelets can be released without adding additional crosslinking agent, which is simple and convenient. Therefore, the preparation and freeze-drying method of the micro-hydrogel for wrapping platelets provided in the present application can further scale up the production to meet the clinical needs.

[0061] 3) The components of the micro-hydrogel for wrapping platelets provided in the present application (such as dextran, gelatin, hyaluronic acid and its salt, polyethylene glycol, polyvinyl alcohol, etc.) are all pharmacopoeia components, which are safe, non-toxic and low in price.

[0062] 4) The micro-hydrogel for wrapping platelets, freeze-dried powder and preparation method thereof provided in the present application can greatly extend the storage time of platelets and alleviate the shortage of platelets. BRIEF DESCRIPTION OF DRAWINGS

[0063] The above and / or additional aspects and advantages of the present application will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:

[0064] Figure 1 (a) Damage of platelets in the freeze-drying process and (b) protective effect of the micro-hydrogel.

[0065] Figure 2 It is a microscope observation diagram of the micro-hydrogel for wrapping platelets (left) and the dissociated free platelets (right) in Example 2. Among them, the platelets are labeled with FITC anti-human CD41 / CD61.

[0066] Figure 3 It is a preparation flowchart of the micro-hydrogel for wrapping platelets in Example 3.

[0067] Figure 4 It is a microscope observation diagram of the micro-hydrogel for wrapping platelets before and after freeze-drying in Example 3. Among them, the platelets before freeze-drying are labeled with FITC anti-human CD41 / CD61.

[0068] Figure 5 Size distribution of the platelet-encapsulated microhydrogels before and after lyophilization in Example 3.

[0069] Figure 6 Photographs of the platelet-encapsulated microhydrogel lyophilized powder (Encap-Lyo, left) and the platelets directly lyophilized (Lyo, right) in Example 3.

[0070] Figure 7 Graphs of the expression of hemostatic-related glycoproteins CD41 / 61 and CD42b of the platelet-encapsulated microhydrogel lyophilized platelets (Encap-Lyo), the platelets directly lyophilized (Lyo), and the fresh platelets (Fresh) detected by flow cytometry in Example 4.

[0071] Figure 8 Graphs of the degree of desialylation of the platelet-encapsulated microhydrogel lyophilized platelets (Encap-Lyo), the platelets directly lyophilized (Lyo), and the fresh platelets (Fresh) detected by flow cytometry in Example 4.

[0072] Figure 9 Graphs of the in vitro aggregation ability of the platelet-encapsulated microhydrogel lyophilized platelets (Encap-Lyo), the platelets directly lyophilized (Lyo), and the fresh platelets (Fresh) in Example 4.

[0073] Figure 10 Graphs of the expression of hemostatic-related glycoproteins CD41 / 61 and CD42b of the platelet-encapsulated microhydrogel lyophilized platelets (Encap-Lyo), the platelets directly lyophilized (Lyo), and the fresh platelets (Fresh) detected by flow cytometry after storage in Example 5.

[0074] Figure 11 Graphs of the degree of desialylation of the platelet-encapsulated microhydrogel lyophilized platelets (Encap-Lyo), the platelets directly lyophilized (Lyo), and the fresh platelets (Fresh) detected by flow cytometry after storage in Example 5. DETAILED DESCRIPTION

[0075] The specific embodiments of the present application will be described in detail below. It should be understood that the detailed description and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, and are not by way of limitation.

[0076] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges are generally provided as approximations. Unless otherwise stated, the endpoints of the ranges are not to be understood as being singular, but are merely to be understood as approximations. It is also understood that the endpoints of the ranges can be appropriately adjusted to take into account the possibility of an exact value.

[0077] Features described or illustrated as part of one embodiment or certain embodiments can be used in other embodiments or certain other embodiments, to produce still further embodiments.

[0078] Before describing the present application in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which is defined solely by the claims. In order to more fully understand the present application described herein, the following terms are defined as follows. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0079] Definitions

[0080] Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0081] Unless specifically stated otherwise, the terms "including," "comprising," and "having" and the like, as used herein, are understood to be open-ended and to mean "including but not limited to."

[0082] The term "microhydrogel" refers to a micrometer-sized gel composed of water-soluble polymer materials, with a three-dimensional network structure, capable of absorbing and retaining a large amount of water while maintaining structural stability, with a size typically between 1 micrometer and several hundred micrometers. Its excellent water absorption and environmental responsiveness make it sensitive to external stimuli such as pH, temperature, light, ionic strength, etc., capable of adjusting its own swelling and drug release. Due to its good biocompatibility, microhydrogels are widely used in drug delivery, tissue engineering and regenerative medicine, especially suitable for soft tissue repair, biosensors and intelligent drug delivery systems, with great potential for application.

[0083] The term "encapsulation" refers to the process by which microhydrogel materials encapsulate biologically active substances such as drugs, proteins, cells, etc. This encapsulation technique encapsulates these substances in the three-dimensional network structure of the hydrogel, forming a protective barrier that allows them to be released slowly or isolated under specific conditions, thereby providing protection.

[0084] The term "dispersed phase" refers to one phase in a multiphase system, usually a small particle or droplet of liquid, solid or gas, which is dispersed in another substance (i.e. the continuous phase) in a discontinuous manner. In an emulsion, suspension or colloidal solution, the size of the dispersed phase can range from nanometers to micrometers. The dispersed phase particles are usually introduced into the system by external energy (e.g. shaking, stirring or ultrasonication), and are in a thermodynamically unstable state, often with a tendency to aggregate or coagulate. Without sufficient stabilizing mechanisms (e.g. emulsifiers or stabilizers), the dispersed phase particles will gradually form larger particles through collision and coalescence, leading to instability of the system. The specific morphology of the dispersed phase in an 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).

[0085] The term "continuous phase" refers to the continuously distributed matrix in a multiphase system, which is the substance acting as a solvent or matrix relative to the dispersed phase, and is able to uniformly surround and distribute the particles of the dispersed phase. In an emulsion or colloidal solution, the continuous phase occupies the majority of the volume of the system and is the medium in which the dispersed phase exists. The properties of the continuous phase have important influences 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 selection and physical properties (e.g. viscosity, surface tension) of the continuous phase can significantly affect the stability of the dispersed phase.

[0086] The term "emulsification" refers to the process of mixing two immiscible liquids by shaking, mechanical stirring, ultrasonication, homogenization or other physical methods, so that one liquid (the dispersed phase) is dispersed in the form of droplets in the other liquid (the continuous phase) 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 uniformly distributed in the continuous phase. Due to the high interfacial energy, this dispersed state is thermodynamically unstable, so emulsions usually require the addition of emulsifiers to reduce interfacial tension and prevent droplet aggregation and separation. Emulsifiers are molecules with both hydrophilic and lipophilic properties (surfactants) that will adsorb on the interface between the dispersed and continuous phases, forming a stable interfacial film to prevent droplet fusion. Common emulsifiers include surfactants, proteins, polymers, etc.

[0087] The term "liquid-liquid phase separation" refers to the separation of two originally mixed liquids into two immiscible liquid phases in a system due to energy release or changes in external conditions. This phenomenon often occurs when emulsions become unstable, such as when emulsifiers fail, temperature changes, or external forces are disturbed. 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 eventually forming two immiscible liquid layers. For example, oil and water originally form an emulsion under emulsification conditions, but when emulsification is destroyed, they separate into an oil layer and a water layer, which is a typical liquid-liquid phase separation phenomenon. The process of liquid-liquid phase separation is related to the phase diagram of thermodynamics, and the system may exist in multiple phases under certain conditions, while under other conditions it may exhibit a single phase of uniform mixing. Liquid-liquid phase separation is widely used in chemical industry and biology (such as intracellular phase separation phenomenon).

[0088] The term "physical gelation" refers to the process by which one or more substances change from a liquid state to a solid or semi-solid state 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, allowing the substance to remain solid or have elasticity.

[0089] The term "gelatin" refers to a natural high molecular weight protein obtained by hydrolysis of animal collagen, mainly derived from the skin, bones and connective tissue of animals such as cattle, pigs and fish. It has good solubility in water and can form a gel when cooled, with excellent gelation properties. The hydration of gelatin allows it to thicken, gel, and retain water in hydrogels, making it widely used in biomedical, hydrogel drug delivery systems, tissue engineering, wound dressings and other fields. Gelatin not only has good biocompatibility and biodegradability, but also can be compounded with other high molecular weight materials to adjust the physical properties and mechanical strength of hydrogels, becoming an important component in the pharmaceutical, food, cosmetics and other industries.

[0090] The term "dextran" refers to a high molecular weight polysaccharide composed of glucose molecules connected by α-1,6-glycosidic bonds, with a branched structure, widely found in some bacteria (such as Proteus and Lactobacillus). The molecular chain of dextran can be linear or highly branched, with a large molecular weight, and generally has good water solubility and hydrophilicity. In the field of biomedicine, dextran is often used as a biocompatible material as a blood volume expander, drug carrier and stabilizer, especially in plasma substitutes, drug delivery systems and biological macromolecular carriers. It also has biodegradability and good immunocompatibility, so it is often used in drug delivery, tissue engineering and medical diagnosis.

[0091] 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 and animal body. It is composed of alternating glucosamine and glucuronic acid, has strong moisturizing ability, and can absorb and hold a large amount of water. Sodium hyaluronate has a wide range of applications in medicine and cosmetics, as a moisturizer, lubricant, and drug carrier, for skin care, anti-aging, joint treatment, and other aspects. It is often used as an injection filler to help facial cosmetic or relieve arthritis, and also plays an important role in ophthalmic surgery, eye drops, and wound 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, due to its good biocompatibility and biodegradability, sodium hyaluronate is also widely used in drug delivery systems and other medical fields.

[0092] The term "polyethylene glycol" (PEG) refers to a class of synthetic high molecular compounds formed by the polymerization of ethylene glycol or oxirane. According to different polymerization degrees, PEG can have different molecular weights, usually ranging from several hundred to several million daltons, and 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, widely used in medicine, cosmetics, food, industry, and biotechnology fields. In the medical 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.

[0093] The term "polyvinyl alcohol" (PVA) is a high molecular compound made from vinyl acetate through alcoholysis reaction and polymerization, with good hydrophilicity and water solubility. Its chemical structure contains multiple alcohol groups (-OH), which endow it with 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, and the common molecular weight range is from several thousand to several million daltons. Due to its good biocompatibility, polyvinyl alcohol is widely used in medicine, industry, cosmetics, and environmental protection fields. 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 adhesives, coatings, textiles, and paper processing; in cosmetics, polyvinyl alcohol is used as an emulsifier, thickener, and moisturizer,

[0094] The term "Tyrode's buffer" refers to a buffered solution used in biological and biochemical experiments, typically to maintain a stable pH and ionic environment required for cells or tissues during experiments. Tyrode's solution is composed of various salts and other components designed to mimic physiological conditions in the body, particularly widely used in cardiac physiology and cell biology research. A standard Tyrode's solution typically 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 examples is 4.6 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 25 mM Na2HPO4, 5 mM HEPES, 10 mM glucose.

[0095] Example 1 Collection of platelets

[0096] Blood was collected from volunteers who passed the clinical ethics. In the blood, the original volume was added 1 / 3 of hydroxyethyl starch, and after 30 minutes of sedimentation, 50g centrifugation for 10 minutes, 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, and the obtained supernatant was platelet-rich plasma.

[0097] PGE1 was added to the platelet-rich plasma to 1 μM, and centrifuged at 1000g for 12 minutes, and the supernatant was platelet-poor plasma, and the lower layer was platelet sediment. The platelet-poor plasma was collected and stored at -20°C for standby.

[0098] Example 2 Encapsulation and dissociation of platelets

[0099] A mixed solution of 5.5% gelatin (Sigma-Aldrich 73865), 0.25% dextran (Macron D872024), and 3% mannitol (Solabio M8140) was prepared as the dispersed phase solution using water as the solvent. A freeze-drying protective solution was used as the solvent, and 5% PEG was added as the continuous phase solution. The freeze-drying protective solution consisted of 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. The platelet sediment was resuspended with 500 μL of the dispersed phase solution, and then blown and mixed uniformly. The concentration of the platelets was adjusted to 2 x 10 8 / mL by adding the dispersed phase solution. 0.5 μl of FITC anti-human CD41 / CD61 (Biolegend, 359810) was added, mixed, and incubated in the dark for 20 min.

[0100] Take 50 μL of the dispersed phase solution after adding platelets, emulsify by blowing several times in 1 mL of the continuous phase solution. Place in a 24-well plate and observe under a microscope. If Figure 2 As shown on the left, the dispersed phase solution and the continuous phase solution are emulsified to form microhydrogels, most of the platelets are wrapped inside the microhydrogels, showing green fluorescence, and the microhydrogels are in a core-shell structure.

[0101] Centrifuge the platelet-wrapped microhydrogels 500 g for 5 minutes, remove the supernatant, resuspend with an equal volume of Tyrode's solution, and heat to 37°C to dissociate the microhydrogels. Place in a 24-well plate and observe under a microscope. If Figure 2 As shown on the right, the microhydrogels can be seen to dissociate after heating, and the platelets are released.

[0102] Example 3 Preparation of platelet-wrapped microhydrogel lyophilized powder

[0103] Platelet incubation

[0104] Add 1 / 10 of the original volume of incubation solution to the platelet pellet for resuspension. The incubation solution composition is: 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, 37°C, 40 rpm, and incubate for 4 hours.

[0105] Platelet wrapping

[0106] Different component microhydrogel formulations:

[0107] Microhydrogel 1: Dextran-Gelatin-PEG

[0108] Continuous phase: 4.5% PEG (Solvay P8280) dissolved in lyophilization protection solution

[0109] Dispersed phase: aqueous solution, 5.5% gelatin, 0.25% dextran, 3% mannitol

[0110] Microhydrogel 2: Dextran-Gelatin-PVA

[0111] Continuous phase: 15% PVA (Sigma-Aldrich P8136) dissolved in lyophilization protection solution

[0112] Dispersed phase: aqueous solution, 5.5% gelatin, 0.25% dextran, 3% mannitol

[0113] Microhydrogel 3: Sodium Hyaluronate-Gelatin-PEG (SH-Gelatin-PEG)

[0114] Continuous phase: 4.5% PEG dissolved in freeze-dried protective solution;

[0115] Dispersed phase: aqueous solution, 5.33% gelatin, 0.27% sodium hyaluronate (Source Leaf Biotechnology S24592), 3% mannitol.

[0116] The above percentages are all by mass.

[0117] The components of the freeze-drying 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.

[0118] Prepare the dispersed phase solution and the continuous phase solution according to the above recipe.

[0119] After the platelet incubation time is over, the platelets are removed from the incubator and centrifuged at 1000 g for 12 minutes. The supernatant is removed and the platelets are resuspended in the dispersed phase solution to adjust the platelet concentration to 2×10 8 / mL, filter through a 40μm filter. Add 0.5μl FITCanti-human CD41 / CD61 (Biolegend, 359810), mix well, and incubate in the dark for 20 minutes.

[0120] Take 50 μL of the dispersed phase solution after adding platelets and emulsify it in 1 mL of the continuous phase solution to obtain platelet micro-hydrogels with platelets wrapped inside the micro-hydrogels. Place it in 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 platelets are encapsulated inside the micro-hydrogels, showing green fluorescence. Dextran-Gelatin-PEG and Dextran-Gelatin-PVA micro-hydrogels have a core-shell structure, while SH-Gelatin-PEG micro-hydrogels have a multi-compartment structure. The size of the micro-hydrogels is as follows: Figure 5 as shown (Lyo stands for freeze-dried).

[0121] The sizes of the micro-hydrogels in each group 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).

[0122] Lyophilization of platelet-encapsulated microhydrogels

[0123] An equal amount of freeze-drying protection solution was directly added to an equal amount of platelets and resuspended to serve as the direct freeze-drying control group.

[0124] The platelet-encapsulated microhydrogel or the directly resuspended platelets were transferred to a test tube with 1 mL volume, and pre-frozen at -80°C for 10 hours.

[0125] The pre-frozen test tubes were transferred to a freeze dryer, and the plate temperature was set at -5°C and the vacuum degree was set at 1 pa. The first drying was performed for 10 hours.

[0126] The temperature was raised to 25°C at a rate of 0.5°C / min, and the second drying was performed for 10 hours to obtain the freeze-dried powder.

[0127] As shown in FIG. 1, the platelets encapsulated in the microhydrogel after freeze-drying were more plump than the directly resuspended platelets. Figure 6 Figure 6 As shown in FIG. 1, the platelets encapsulated in the microhydrogel after freeze-drying were more plump than the directly resuspended platelets. Figure 6

[0128] The platelet-encapsulated microhydrogel or the directly resuspended platelets were transferred to a test tube with 1 mL volume, and pre-frozen at -80°C for 10 hours. Figure 4 Figure 5 As shown in FIG. 1, the platelets encapsulated in the microhydrogel after freeze-drying were more plump than the directly resuspended platelets.

[0129] The size of each group of microhydrogels after freeze-drying was as follows: Dextran-Gelatin-PEG (12.45 μm-99.88 μm); Dextran-Gelatin-PVA (9.78 μm-98.96 μm); and SH-Gelatin-PEG (4.68 μm-55.95 μm).

[0130] Example 4: Functional detection of platelet-encapsulated microhydrogel freeze-dried powder

[0131] Rehydration and dissociation of platelets

[0132] The platelet-encapsulated microhydrogel or the directly resuspended platelets were transferred to a test tube with 1 mL volume, and pre-frozen at -80°C for 10 hours.

[0133] For the platelet-encapsulated microhydrogel freeze-dried powder, the ddH2O resuspension was centrifuged at 500 g for 5 minutes, and the supernatant was removed. The precipitate was resuspended in the same volume of Tyrode's solution, and incubated at 37°C for 5 minutes to release the platelets.

[0134] Flow cytometry detection of platelet surface glycoprotein content and de-sialylation degree

[0135] ​​​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 7 / mL.

[0136] 500 μL of platelet suspension was placed in a 1.5 mL centrifuge tube. 0.5 μL of Brilliant Violet 650™ anti-human CD42b (Biolegend, 303926, specifically binding to glycoprotein CD42b), 0.5 μL of PE / Cyanine7 anti-human CD41 / CD61 (Biolegend, 359812, specifically binding to glycoprotein CD41 / 61), and 0.2 μL of FITC-RCA-I (Vector, FL-1081-1, specifically binding to galactose molecules exposed after desialylation) were added, respectively. The platelets were incubated in the dark for 20 min.

[0137] After incubation, the cells were filtered through a 40 μm filter and placed in a flow cytometer. The surface glycoprotein content and desialylation degree of each group were detected using a flow cytometer (BD LSRFortessaSORP).

[0138] like Figure 7 As shown in the data, compared with fresh platelets (Fresh), the CD41 / 61 and CD42b double-positive ratio of platelets in the direct freeze-dried (Lyo) group decreased significantly, reaching only 65.00±7.99%; the CD41 / 61 and CD42b double-positive ratio of platelets in the microhydrogel freeze-dried platelet-encapsulated (Encap-Lyo) group was maintained at 90.10±5.90%, which is very close to the 93.97±5.56% of fresh platelets.

[0139] like Figure 8 As shown, the galactose expression level of fresh platelets (a molecule exposed after desialylation) is set at 1. Directly freeze-dried (Lyo) platelets undergo severe desialylation, with a relative increase in galactose expression to 2.71±0.75. Desialylation is significantly inhibited in platelets freeze-dried with microhydrogels (Encap-Lyo), with a relative increase in galactose expression to 1.32±0.32, which is not significantly different from the fresh group.

[0140] Platelet aggregation assay in vitro

[0141] 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.

[0142] Take 300 μL platelet suspension into a special test tube, add the rotor, incubate at 37°C for 5 min. Put into the aggregometer, adjust zero. Add thrombin to 2 U / mL, observe the aggregation.

[0143] As shown in Figure 9 , the platelet aggregation ability of direct lyophilization (Lyo) is significantly weakened, only 65.33 ± 4.16%, and the platelet aggregation ability of micro-hydrogel-encapsulated platelets lyophilized (Encap-Lyo) is 95.67 ± 7.51%, which is not significantly different from the aggregation ability of fresh platelets (Fresh) 85.67 ± 2.08%. The results of platelet aggregometer further prove the effect of micro-hydrogel encapsulation, and can be verified by flow cytometry results.

[0144] The test results show that the platelets encapsulated in the micro-hydrogel lyophilized and rehydrated have higher glycoprotein expression, higher aggregation ability, and lower desialidation degree.

[0145] Example 5 Long-term storage of micro-hydrogel-encapsulated platelet lyophilized powder

[0146] After preparing the platelet lyophilized powder (micro-hydrogel-encapsulated platelet Dextran-Gelatin-PEG lyophilized powder / direct lyophilized powder) according to Example 3, fill nitrogen in situ in the lyophilizer, add an aluminum cover, and store at room temperature (25°C) in a vacuum sealed state. After one month, detect the surface glycoprotein content and desialidation degree of the lyophilized platelets according to the method in Example 4.

[0147] Reserve fresh platelets of the same batch, and detect the surface glycoprotein content and desialidation degree of the fresh platelets according to the method in Example 4.

[0148] As shown in Figure 10 , compared with fresh platelets (Fresh), the proportion of CD41 / 61 and CD42b double positive platelets in the direct lyophilization (Lyo) group after one month of storage decreased significantly, only 51.3%; the proportion of CD41 / 61 and CD42b double positive platelets in the micro-hydrogel-encapsulated platelet lyophilized (Encap-Lyo) group was maintained at 83.2%, which was close to 98.6% of fresh platelets.

[0149] As shown in Figure 11The expression of galactose (a molecule exposed after desialylation) of fresh platelets was set as 1. The platelets of direct lyophilization (Lyo) suffered severe desialylation, and the expression of galactose was relatively increased to 1.57. The degree of desialylation of the platelets of the micro-hydrogel-encapsulated lyophilized (Encap-Lyo) group was significantly inhibited, and the expression of galactose was relatively increased to 1.09, close to that of the fresh group (Fresh).

[0150] For the functional detection of platelets, the micro-hydrogel-encapsulated lyophilized powder provided by the application can be stored for a long time, and the rehydrated and dissociated platelets after storage have higher glycoprotein expression and inhibit desialylation.

[0151] The effects of the embodiments of the application show that the platelets released after rehydration and dissociation of the platelet-encapsulated micro-hydrogel lyophilized powder have higher hemostasis-related glycoprotein expression and lower desialylation, and are expected to have better hemostasis ability and longer in vivo circulation time. Through the platelet-encapsulated micro-hydrogel, lyophilized powder and the preparation method thereof provided by the application, the storage time of platelets can be greatly prolonged, and the shortage of platelets can be alleviated.

[0152] The scope of protection of the application is not limited to the above-mentioned embodiments. Obviously, those skilled in the art can make various modifications and changes to the application without departing from the scope and spirit of the application. If these modifications and changes belong to the scope of the claims of the application and its equivalent technologies, the intention of the application also includes these modifications and changes.

[0153] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, various possible combinations are not described again in the application.

[0154] In addition, any combination of various different embodiments of the application can be made as long as it does not deviate from the idea of the application, and it should also be considered as disclosed in the application.

Claims

1. A micro-hydrogel encapsulating platelets, characterized in that: The micro-hydrogel has a core-shell or multi-compartment structure, with platelets encapsulated inside the micro-hydrogel. The micro-hydrogel is formed by spontaneous physical gelation after a dispersed phase solution containing platelets is emulsified in a continuous phase solution. The dispersed phase solution is an aqueous solution containing gelatin and one or two selected from dextran, hyaluronic acid or their salts, and containing one or more osmotic pressure regulators; the continuous phase solution is an aqueous solution containing one or two of polyethylene glycol and polyvinyl alcohol.

2. The platelet-wrapped micro-hydrogel according to claim 1, characterized in that: The dispersed phase solution is an aqueous solution containing gelatin and one or two selected from dextran and sodium hyaluronate.

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

1.

4. The platelet-encapsulating micro-hydrogel according to claim 3, characterized in that: The mass ratio of gelatin, dextran and / or sodium hyaluronate is (10-30):

1.

5. The platelet-encapsulating micro-hydrogel according to any one of claims 1 to 4, characterized in that: The micro-hydrogel is spherical or quasi-spherical, with a diameter of 1-1000 μm.

6. The platelet-encapsulating micro-hydrogel according to any one of claims 1 to 4, characterized in that: The micro-hydrogel further contains one or more of trehalose, sucrose, glucose and maltose.

7. A method for preparing a micro-hydrogel for encapsulating platelets, characterized in that: It includes the following steps: After the dispersed phase solution containing platelets is emulsified in the continuous phase solution, it spontaneously physically gels to form micro-hydrogels, and the platelets are encapsulated inside the micro-hydrogels; The dispersed phase solution is an aqueous solution containing gelatin and one or two selected from dextran, hyaluronic acid or their salts, and contains one or more osmotic pressure regulators; the continuous phase solution contains an aqueous solution of one or two selected from polyethylene glycol and polyvinyl alcohol.

8. The preparation method according to claim 7, characterized in that The platelets are pre-treated and incubated platelets.

9. The preparation method according to claim 8, characterized in that The pretreatment incubation refers to adding incubation solution to platelet sediment separated from blood, resuspending, heating and incubating, and centrifuging and collecting.

10. The preparation method according to claim 9, wherein The amount of the incubation solution used 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.

11. The preparation method according to any one of claims 7 to 10, characterized in that: The concentration of platelets in the dispersed phase solution is 0.1-10×10 9 / mL.

12. The preparation method according to claim 7, characterized in that The dispersed phase solution is an aqueous solution containing gelatin and one or two selected from dextran and sodium hyaluronate.

13. The preparation method according to claim 12, 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.

14. The preparation method according to claim 7, 12 or 13, 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.

15. The preparation method according to claim 14, characterized in that The dispersed phase solution contained 3-5% w / v mannitol.

16. The preparation method according to claim 7, 12 or 13, characterized in that: The solvent of the continuous phase solution is one or more of freeze-dried protective solution, pure water, or buffer solution.

17. The preparation method according to claim 16, 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.

18. The preparation method according to claim 16, characterized in that The freeze-drying protective solution contains one or more of trehalose, sucrose, glucose, maltose, glycerol, and mannitol, and optionally, one or more of magnesium chloride, potassium chloride, sodium chloride, HEPES, and hydroxypropyl-β-cyclodextrin.

19. The preparation method according to claim 18, wherein 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.

20. The preparation method according to claim 7, characterized in that The volume ratio of the dispersed phase solution to the continuous phase solution is 1:1-100.

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

22. The preparation method according to claim 7, 18, 19, 20 or 21, 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% w / v 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% w / v 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% w / v mannitol; the continuous phase solution is a freeze-drying protective solution containing 4.5% w / v polyethylene glycol.

23. A micro-hydrogel encapsulating platelets, characterized in that: The invention is prepared according to the preparation method according to any one of claims 7 to 22.

24. A micro-hydrogel freeze-dried powder encapsulating platelets, characterized in that: The micro-hydrogel is obtained by freeze-drying the platelet-wrapped micro-hydrogel according to any one of claims 1 to 6 or the platelet-wrapped micro-hydrogel according to claim 23.

25. The platelet micro-hydrogel freeze-dried powder according to claim 24, characterized in that: Freeze-dry in lyophilization solution.

26. A method for preparing platelet-encapsulating micro-hydrogel freeze-dried powder, 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 6 or the platelet-wrapped micro-hydrogel according to claim 23.

27. The preparation method according to claim 26, characterized in that Freeze-dry in lyophilization solution.

28. The preparation method according to claim 26, 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 the micro-hydrogels and then freeze-dried directly; when the solvent of the continuous phase solution is not a freeze-drying protective solution, the micro-hydrogels encapsulating the platelets are collected by centrifugation after emulsification to form the micro-hydrogels, and then resuspended in a freeze-drying protective solution for freeze-drying.

29. The preparation method according to any one of claims 26 to 28, characterized in that: The freeze-drying process includes three steps: pre-freezing, primary drying and secondary drying.

30. The preparation method according to any one of claims 26 to 28, characterized in that: The platelet-wrapped microhydrogel is transferred to 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 process further includes nitrogen protection, sealing, and vacuuming steps after freeze drying.

31. The preparation method according to claim 30, characterized in that The platelet-wrapped micro-hydrogel is transferred to a vial for freeze-drying. Optionally, the method further comprises nitrogen protection, sealing, and vacuuming steps after freeze-drying.

32. A method for processing the platelet-encapsulating micro-hydrogel freeze-dried powder according to any one of claims 24-25, or the platelet-encapsulating micro-hydrogel freeze-dried powder obtained by the preparation method according to any one of claims 26-31, 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.

33. The processing method according to claim 32, characterized in that The rehydration refers to resuspending the platelet-wrapped micro-hydrogel freeze-dried powder 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 to 2 times the volume before freeze-drying.

34. The processing method according to claim 32, characterized in that The heat dissociation refers to resuspending the platelet-wrapped microhydrogel 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.

35. The processing method according to claim 34, characterized in that After heating and incubation, PGE1 was added to a concentration of 1-10 μM and the platelets were harvested by centrifugation.

36. The processing method according to claim 32, 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 to 2 times the volume before lyophilization.

37. The treatment method according to claim 33, 34 or 36, 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.

38. A method for freeze-drying, storing and / or transporting and processing platelets, characterized in that: The method comprises the following steps: preparing a micro-hydrogel encapsulating platelets according to the preparation method described in any one of claims 7 to 22, preparing a freeze-dried powder of the micro-hydrogel encapsulating platelets according to the method described in any one of claims 26 to 31, storing or transporting the platelets, and obtaining harvested platelets according to the processing method described in any one of claims 32 to 37.

39. The method for freeze-drying, storing and / or transporting and processing platelets according to claim 38, characterized in that: Store or transport at room temperature, 4°C, -20°C, or -80°C.

40. Use of the platelet-encapsulating microhydrogel according to any one of claims 1 to 6, the platelet-encapsulating microhydrogel according to claim 23, the platelet-encapsulating microhydrogel freeze-dried powder according to any one of claims 24 to 25, or the platelet-encapsulating microhydrogel freeze-dried powder prepared according to the preparation method of any one of claims 26 to 31 in the preparation of biomedical products.

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