Temperature-responsive bulk hydrogels for encapsulating biological products, lyophilized powders, methods of making, methods of treatment, and uses thereof

Abstract

The application belongs to the technical field of biomedical engineering, and particularly relates to a temperature-responsive block hydrogel wrapping biological products, a freeze-dried powder and a preparation method, a processing method and application thereof. The application mixes crowding macromolecules such as gelatin and dextran with biological products, and the gelatin is converted into a block hydrogel by liquid-liquid phase separation and physical gelation under the promotion of the crowding macromolecules at low temperature, so as to wrap the biological products inside. The block hydrogel can reduce the damage caused by the freeze-drying process and rehydration to the biological products, and the biological products can be stored for a long time after freeze-drying. When used, the biological products can be released by rehydration and temperature rise, without the need of adding an additional decrosslinking agent, and the operation is convenient. When the method is used for platelets and PRP, the platelets after freeze-drying storage and rehydration release can effectively maintain the expression of hemostasis-related glycoproteins, and significantly inhibit desialylation, and show good hemostasis ability and longer in-vivo circulation time; and the method is expected to greatly extend the storage time of platelets, and alleviate the shortage of platelets.

Description

Technical Field

[0001] This application belongs to the field of biomedical engineering technology, specifically relating to a temperature-responsive block hydrogel for encapsulating biological products, a lyophilized powder, and its preparation, processing and application methods. Background Technology

[0002] Platelets, as an indispensable hemostatic component of blood, play a crucial role in clinical emergency care and treatment. Whether dealing with acute bleeding caused by severe trauma, surgery, or childbirth, or treating thrombocytopenia caused by decreased platelet production or excessive platelet destruction, platelet transfusion is the most direct and effective treatment option. Clinical data shows that when the platelet count is below 100 × 10⁻⁶, platelet transfusion is crucial. 9 At a count of 20 × 10⁹ / L, the risk of microvascular bleeding increases significantly, manifesting as purpura; as the count further decreases, the bleeding risk increases exponentially. Once it falls below 20 × 10⁹ / L, the risk becomes even greater. 9 Patients with thrombocytopenia ( / L) are highly susceptible to massive gastrointestinal bleeding or fatal spontaneous intracranial hemorrhage. The "Expert Consensus on the Diagnosis and Treatment of Thrombocytopenia in Chinese Adults" clearly states that platelet transfusion is a necessary and effective intervention for such patients with bleeding risks.

[0003] However, the contradiction between high clinical demand and severe resource shortage is becoming increasingly acute. Taking Shanghai as an example, the annual platelet shortage is as high as 30,000 therapeutic units (approximately 7.5 × 10⁻⁶). 15 The platelet count (per unit of platelets) is over 37% shortfall. The core constraint causing this predicament lies in the extremely short shelf life of platelets. Compared to red blood cells, which can be stored at 4°C for 42 days and plasma, which can be stored at -20°C for over a year, apheresis platelets are currently mainly stored at room temperature (around 22-25°C) with agitation, and their shelf life is only 3-5 days. These stringent storage conditions result in a persistently high platelet waste rate globally, around 20% in most countries and as high as 30% in places like New Zealand. Therefore, developing a new technology that can effectively extend the shelf life of platelets has become a common problem urgently needing to be solved in the field of hematology.

[0004] Researchers have attempted to refrigerate platelets, but refrigeration causes desialylation. Desialylated platelets expose galactose, leading to rapid clearance from the liver and a significant decrease in circulation time. This rapid clearance weakens hemostatic effects. Some researchers have also tried cryopreserving platelets. However, cryopreserved platelets are also rapidly cleared from the body, indicating that desialylation may also occur. Furthermore, dimethyl sulfoxide (DMSO) used in cryopreservation has cytotoxic properties. If it remains in the formulation and enters the human body, it may cause cytotoxicity, immune responses, or other adverse biological effects, posing a potential safety hazard.

[0005] Freeze-drying is an advanced method for preserving biological products. Compared to refrigeration and cryopreservation, freeze-dried biological products can be transported at room temperature and stored for extended periods. Currently, although freeze-dried platelets can still maintain some function, some issues remain.

[0006] To replace toxic chemical reagents, many patents (such as US5827741A and its family of patents, as well as several improved patents based on this approach) use a combination of human serum albumin (HSA) and trehalose as a freeze-drying protectant. The theoretical basis is that high concentrations of albumin form an amorphous glassy matrix, providing external physical support for the platelet membrane during drying. However, this does not verify the in vivo function of freeze-dried platelets. Although albumin can improve the appearance of the freeze-dried cake, its macromolecular characteristics prevent it from entering platelet cells and resisting the sublimation stress of water within the cells during drying. Furthermore, albumin itself is derived from blood products, presenting an extremely high cost barrier. US patent US9682104B2 abandons the preservation of intact platelet cells, instead repeatedly freezing and thawing platelets before freeze-drying. Although this platelet lysate freeze-dried powder is rich in growth factors such as PDGF and TGF-β, showing excellent performance in in vitro wound healing and cell culture, it completely loses its complete cellular structure. This means it lacks the core hemostatic ability of platelets. Therefore, these products are essentially biological excipients and cannot replace platelet transfusions to treat thrombocytopenia or systemic bleeding.

[0007] Chinese patent CN202510018685.0 describes the preparation of bulk hydrogels through liquid-liquid phase separation and physical gelation, encapsulating platelets within the bulk hydrogels. After freeze-drying, the glycoproteins such as CD41 and CD42b of the platelets are maintained, and their aggregation ability remains essentially unchanged. Based on the aforementioned research, this application further improves the preparation of the hydrogel, providing an improved method to extend the preservation time of platelets. Summary of the Invention

[0008] To meet clinical needs, lyophilized platelets should possess good hemostatic ability and long circulation time. All reagents used in the lyophilization process should also be non-toxic to avoid risks after transfusion. Furthermore, the throughput of lyophilized platelets should be high to meet the needs of patients with massive bleeding and thrombocytopenia.

[0009] Current research on freeze-dried platelets mainly involves direct freeze-drying after the addition of various protective agents, including trehalose, mannitol, albumin, and sucrose. While these protective agents can partially alleviate damage during the freeze-drying process, they are insufficient to provide adequate protection. Currently, freeze-dried platelets commonly exhibit decreased hemostatic ability (e.g., a significant decrease in the expression of the hemostasis-related glycoprotein CD42b) and reduced circulation time (due to desialylation leading to high galactose expression). The decrease in platelet hemostatic ability and in vivo circulation time after freeze-drying is primarily due to damage caused during the freeze-drying and rehydration processes.

[0010] To address the aforementioned technical problems in the prior art, this application provides a bulk hydrogel encapsulating biological products, a lyophilized powder, and its preparation, processing, and applications. This application mixes crowded macromolecules such as gelatin and dextran with biological products such as platelets and PRP. Under low-temperature induction, the gelatin undergoes liquid-liquid phase separation and physical gelation promoted by the crowded macromolecules, ultimately transforming into a bulk hydrogel that encapsulates the biological product. During lyophilization, the bulk hydrogel can mitigate damage during the process, and the encapsulated lyophilized biological product can be stored at room temperature for extended periods, prolonging its shelf life. When needed, the bulk hydrogel only requires dissociation, rehydration, and heating (e.g., to 37°C), causing the gel to revert to a solution state and release the biological product, without the need for additional decrosslinking agents.

[0011] The block-shaped hydrogel encapsulation system of this application effectively maintains the expression levels of key membrane proteins (CD41 / CD61, CD42b) of platelets without affecting their structural integrity through physical embedding and interfacial protection mechanisms, thus preserving their biological hemostatic function. Simultaneously, this system significantly inhibits desialylation of glycoprotein terminals, slowing the rate of platelet clearance in vivo and thereby prolonging their circulating half-life. This technology has been validated in lyophilized platelet samples from various sources (including human and mouse), demonstrating good functional retention and cross-species applicability.

[0012] In a first aspect, this application provides a block hydrogel encapsulating a biological product, which is formed by liquid-liquid phase separation and physical gelation of an aqueous solution containing gelatin, crowding macromolecules and the biological product, wherein the crowding macromolecules are selected from one or more of dextran and its derivatives, polyvinyl alcohol, hyaluronic acid and its salts (such as sodium hyaluronate), and polyethylene glycol.

[0013] In some embodiments, the biological product may be platelets, platelet-rich plasma (PRP), etc., and the biological product may be derived from humans or animals, such as mammals (mice, rabbits, monkeys, etc.). In addition to being rich in platelets, the platelet-rich plasma also contains various growth factors and proteins found in blood. During the formation of the block hydrogel, these growth factors and proteins are also encapsulated inside the hydrogel, reducing their damage during the freeze-drying process.

[0014] In some embodiments, the mass ratio of gelatin to crowding macromolecules in the aqueous solution is 1:0.05-0.5, such as 1:0.05, 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, or 1:0.5.

[0015] In some embodiments, the aqueous solution is an aqueous solution containing gelatin, dextran, and biological products. Specifically, the mass ratio of gelatin to dextran is 1:0.1-0.4, preferably 1:0.2-0.3, and more preferably 1:0.25.

[0016] In some embodiments, the water content of the bulk hydrogel is 40%-99%; preferably, the water content of the bulk hydrogel is 60%-95%.

[0017] In some embodiments, the bulk hydrogel further contains one or more of trehalose, sucrose, glucose, maltose, mannitol, lactose, and sorbitol. Preferably, the bulk hydrogel also contains trehalose. The trehalose, sucrose, glucose, maltose, mannitol, lactose, and sorbitol within the bulk hydrogel can synergistically provide freeze-drying protection for biological products (such as platelets and PRP) during freeze-drying.

[0018] In some embodiments, the aqueous solution undergoes liquid-liquid phase separation and physical gelation at -5 to 10°C (e.g., -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C), preferably at -1 to 5°C, and more preferably at 0 to 1°C.

[0019] In a second aspect, this application provides a method for preparing the block hydrogel containing the biological product described in the first aspect, comprising the following steps: forming a block hydrogel by liquid-liquid phase separation and physical gelation of an aqueous solution containing gelatin, crowding macromolecules and the biological product; wherein the crowding macromolecules are selected from one or more of dextran and its derivatives, polyvinyl alcohol, hyaluronic acid and its salts (such as sodium hyaluronate), and polyethylene glycol.

[0020] In some embodiments, the biological product may be platelets or PRP, and the platelets or PRP in the aqueous solution are pretreated and incubated platelets or PRP. Conventional pretreatment and incubation methods in the art can be used, such as incubating platelets or PRP in a solution containing sugars (e.g., trehalose), optionally selected from one or more of sodium chloride, potassium chloride, imidazole, EGTA, PGE1, and EDTA. The pretreatment and incubation refers to adding an incubation solution containing sugars (e.g., trehalose), optionally selected from one or more of sodium chloride, potassium chloride, imidazole, EGTA, PGE1, and EDTA, to the platelet precipitate separated from blood, resuspending it, and then incubating. After incubation, the platelets can be collected by centrifugation. The amount of incubation solution added to the platelets is 1-100% of the original blood volume, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100%, preferably 10% of the original blood volume. The pretreatment incubation involves adding sugars (such as trehalose) to the PRP separated from the blood, optionally selected from one or more of sodium chloride, potassium chloride, imidazole, EGTA, PGE1, and EDTA, and then incubating the mixture. The incubation solution contains 1-100 mM trehalose, 1-1000 mM sodium chloride, 1-100 mM potassium chloride, 1-100 mM imidazole, and 1-100 mM EGTA (ethylene glycol-bis(2-aminoethyl ether)- N,N,N′,N′1-10 μM trehalose, 100 mM sodium chloride, 10 mM potassium chloride, 10 mM imidazole, 10 mM EGTA, and 1 μM PGE1; preferably, the incubation solution contains no other components besides water, platelets, or PRP. The incubation temperature is 20-40℃, for example, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, and 40℃. The incubation conditions are 10-200 rpm, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, and 200 rpm, and the incubation time is 1-10 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 hours. Preferably, the incubation temperature is 35-38℃, the incubation speed is 30-60 rpm, and the incubation time is 2-5 hours. The centrifugation collection conditions are a centrifugation speed of 100-1000 g, such as 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, and 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, and 30 min. Preferably, the centrifugation collection conditions are a centrifugation speed of 500-1000 g and a centrifugation time of 10-15 min.

[0021] In some embodiments, platelet precipitation is obtained by conventional methods in the art, such as obtaining platelet precipitation from blood through the following steps: adding PGE1 to 1-10 μM (preferably 1 μM) to PRP (from which most red blood cells and white blood cells have been removed) to inhibit platelet activation, followed by centrifugation to obtain platelet precipitation. 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, or 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, and 30 min. Preferably, the centrifugation speed is 500-1000 g and the centrifugation time is 10-15 min.

[0022] In some embodiments, the biological product is PRP, which is prepared by removing red blood cells and white blood cells from the blood (e.g., by centrifugation) and collecting the supernatant plasma. For the preparation of PRP, the preferred centrifugation speed is 100-300g (e.g., 200g), the centrifugation time is 10-20min (e.g., 15min), and after centrifugation, about 2 / 3 of the supernatant plasma volume is collected to avoid aspirating the white film layer.

[0023] In some embodiments, to further increase the platelet concentration in the PRP, the plasma obtained from the initial separation can be concentrated by secondary centrifugation before optional pretreatment incubation. Specific steps include: centrifuging the plasma obtained from the initial separation a second time (preferably at a centrifugation speed of 1000-2000g, such as 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000g, for a centrifugation time of 5-15 min, such as 5, 10, or 15 min), causing platelets to settle or accumulate at the bottom of the tube; removing approximately 2 / 3-4 / 5 of the upper volume of anemic platelet plasma (PPP); and resuspending the platelets at the bottom using the remaining plasma, thereby obtaining a smaller volume of concentrated PRP with a higher platelet concentration.

[0024] In some embodiments, the biological product is platelets or PRP, and platelets (preferably pre-treated incubated platelets) are resuspended in an aqueous solution containing gelatin and crowding macromolecules; or an aqueous solution is obtained by mixing a concentrated hydrogel matrix stock solution (containing a high concentration of gelatin and crowding macromolecules) with PRP (preferably pre-treated incubated PRP); followed by liquid-liquid phase separation and physical gelation. The platelet concentration in the aqueous solution is 0.1-10 × 10⁻⁶. 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 × 109 / 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 platelet concentration in the aqueous solution is 0.1-5 × 10⁹ / mL. 9 / mL. More preferably, the platelet concentration in the aqueous solution is 0.5-1 ×10⁻¹⁰. 9 / mL.

[0025] In some embodiments, the concentration of gelatin and crowding macromolecules in the concentrated hydrogel matrix stock solution is 2-10 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 times, preferably 3-5 times, for example 4 times) the concentration of gelatin in the final aqueous solution. During mixing, the concentrated hydrogel matrix stock solution is mixed with PRP at a volume ratio of 1:1-1:9 (preferably 1:2-1:5, for example 1:3). This concentrated formulation strategy can ensure that the concentration of gelatin and crowded macromolecules in the final aqueous solution remains within the threshold range required for physical gelation (e.g., 0.1-15% (w / v), such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 % w / v; preferably 1-5% (w / v), more preferably 2-3% (w / v)) while introducing a high proportion of bioproducts (such as PRP).

[0026] In some embodiments, the mass ratio of gelatin to crowding macromolecules in the aqueous solution is 1:0.05-0.5, such as 1:0.05, 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, or 1:0.5.

[0027] In some embodiments, the aqueous solution is an aqueous solution containing gelatin, dextran, and biological products. Specifically, the mass ratio of gelatin to dextran is 1:0.1-0.4, preferably 1:0.2-0.3, and more preferably 1:0.25.

[0028] In some embodiments, the aqueous solution contains 0.1-15% (w / v) gelatin, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15% w / v; preferably 1-5% (w / v) gelatin, more preferably 2-3% (w / v) gelatin.

[0029] In some embodiments, the medium of the aqueous phase solution is a lyophilization protective base solution, preferably a 0.5-2 × lyophilization protective base solution (i.e., the concentration of each component is 0.5-2 times the concentration in a commonly used 1 × lyophilization protective base solution), such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 × lyophilization protective base solutions; more preferably a 1-1.5 × lyophilization protective base solution.

[0030] In some embodiments, the lyophilization protective base solution is a conventionally used lyophilization protective base solution in the art, which may contain one or more of trehalose, sucrose, glucose, maltose, mannitol, and glycerol. Preferably, the lyophilization protective base solution contains trehalose. The lyophilization protective base solution may also contain one or more of magnesium chloride, potassium chloride, sodium chloride, HEPES, and hydroxypropyl-β-cyclodextrin. The concentrations of each component in the 1× lyophilization protective base solution are 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, and 1000mM. In some embodiments, the 1× lyophilization protective base 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; preferably, the 1× lyophilization protective base solution is composed 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.

[0031] In some embodiments, the aqueous solution is physically gelled at -5 to 10°C (e.g., -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5°C), preferably at -1 to 5°C, and more preferably at 0 to 1°C.

[0032] In a third aspect, this application provides a freeze-dried block hydrogel powder encapsulating biological products, which is obtained by freeze-drying the block hydrogel encapsulating biological products described in the first aspect, or the block hydrogel encapsulating biological products prepared according to the preparation method described in the second aspect.

[0033] In a fourth aspect, this application provides a method for preparing a block hydrogel freeze-dried powder encapsulating biological products, comprising the following steps: freeze-drying the block hydrogel encapsulating biological products described in the first aspect, or the block hydrogel encapsulating biological products prepared according to the preparation method described in the second aspect.

[0034] In some embodiments, when preparing bulk hydrogels that encapsulate biological products, if the medium of the aqueous phase solution is a freeze-drying protective base solution, the bulk hydrogel is directly freeze-dried after it is formed.

[0035] In some embodiments, freeze-drying is performed using methods conventional in the art. The freeze-drying process includes three steps: pre-freezing, primary drying, and secondary drying. The block-shaped hydrogel freeze-dried powder encapsulating biological products can be stored or transported at room temperature (15-35°C), 4°C, -20°C, or -80°C; it can be stored at room temperature for extended periods.

[0036] In some embodiments, freeze-drying is performed using containers conventional in the art. For example, an aqueous solution containing gelatin, crowded macromolecules, and biological products is directly subjected to liquid-liquid phase separation and physical gelation in a freeze-drying container to form a bulk hydrogel, followed by freeze-drying; or the bulk hydrogel encapsulating the biological product is transferred to a freeze-drying container for freeze-drying. Optionally, the freeze-drying process also includes nitrogen protection, sealing (e.g., stoppering, adding an aluminum cap), and vacuuming steps.

[0037] In some embodiments, the freeze-drying container serves as a mold defining the shape and size of the bulk hydrogel, wherein the bulk hydrogel has a size of 1 mm to 1000 mm in at least one dimension in three-dimensional space; preferably, the bulk hydrogel has a size of 1 mm to 100 mm in at least one dimension in three-dimensional space.

[0038] In some embodiments, the freeze-drying container includes, but is not limited to, one or more of the following: 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, polyester tubes (PET tubes), and freeze-drying trays. The volume of the freeze-drying container used is 0.1-1000mL, 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, 2mL, 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, 16mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1000 mL.

[0039] In some 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, freeze dryer plates, dry ice, and liquid nitrogen.

[0040] In some embodiments, the freeze dryer plate temperature is set to -50℃ to -10℃ during a single drying cycle. Examples include -50℃, -49℃, -48℃, -47℃, -46℃, -45℃, -44℃, -43℃, -42℃, -41℃, -40℃, -39℃, -38℃, -37℃, -36℃, -35℃, -34℃, -33℃, -32℃, -31℃, -30℃, -29℃, -28℃, -27℃, -26℃, -25℃, -24℃, -23℃, -22℃, -21℃, -20℃, -19℃, -18℃, -17℃, -16℃, -15℃, -14℃, -13℃, -12℃, -11℃, and -10℃. The drying time for a single cycle is 3-30 hours. 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.

[0041] In some embodiments, the plates need to be heated during secondary drying. The heating rate is 0.1-50℃ / min, for example 0.1℃ / min, 0.2℃ / min, 0.3℃ / min, 0.4℃ / min, 0.5℃ / min, 0.6℃ / min, 0.7℃ / min, 0.8℃ / min, 0.9℃ / min, 1.0℃ / min, 2℃ / min, 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, 2 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℃, for example, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, and 35℃. The secondary drying time is 3-30 hours, for example, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, and 30 hours.

[0042] In a fifth aspect, this application provides a method for processing a block hydrogel freeze-dried powder encapsulating biological products as described in the third aspect, or a block hydrogel freeze-dried powder encapsulating biological products prepared according to the preparation method described in the fourth aspect, comprising the following steps: rehydration, dissociation (including but not limited to heating incubation, enzymatic hydrolysis, chemical degradation, etc.), and centrifugation to collect the harvested biological products.

[0043] In some embodiments, the heating and 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, or 40°C. The incubation time is 1 min to 1 h, such as 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, or 60 min. Preferably, the heating and incubation temperature is 35-38°C, and the incubation time is 3-6 min.

[0044] In some embodiments, the processing method includes the following steps: rehydrating the block-shaped hydrogel lyophilized powder encapsulating the biological product with pure water or a suitable solution, heating and incubating to dissociate the biological product, and centrifuging to collect the harvested biological product; optionally, resuspending it in a suitable solution for subsequent use. The volume of the rehydration solution is 1 / 10 to 5 times the volume before lyophilization, such as 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times, preferably 1 / 2 to 2 times, and more preferably, the volume of the rehydration solution is substantially equal to the volume before lyophilization. After centrifugation to collect the harvested biological products, the volume of the solution used for resuspension is 1 / 10 to 5 times the volume before freeze-drying, such as 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times, preferably 1 / 2 to 2 times, and more preferably, the volume of the solution used for resuspension is substantially equal to the volume before freeze-drying.

[0045] In some embodiments, the biological product is platelets or PRP. The block-shaped hydrogel lyophilized powder encapsulating platelets or PRP is rehydrated by resuspending it in pure water, plasma, or one or more buffer solutions (such as phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, carbonate buffer, triethanolamine buffer, Tricine solution, Tris buffer, Tyrode's solution). The total concentration of solute in the buffer solution is 1-1000 mM. More preferably, the block-shaped hydrogel lyophilized powder encapsulating platelets or PRP is rehydrated by resuspending it in pure water.

[0046] In some embodiments, the biological product is platelets or PRP. After heating and incubation to dissociate the platelets, PGE1 is added to 1-10 µM. Preferably, PGE1 is added to 1 µM after heating and incubation, and the harvested biological product is obtained by centrifugation.

[0047] In some embodiments, the biological product is platelets or PRP. After centrifugation to collect the harvested biological product, it is resuspended in pure water, plasma, or one or more buffer solutions (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 solute concentration in the buffer solution is 1-1000 mM. Preferably, after centrifugation to collect the harvested biological product, it is resuspended in Tyrode's solution or plasma for subsequent use. More preferably, after centrifugation to collect the harvested biological product, it is resuspended in platelet-rich plasma for subsequent use.

[0048] The centrifugation collection conditions are as follows: centrifugation speed of 100-1000g, for example, 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, and 1000 g; and centrifugation time of 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, and 30 min. Preferably, the centrifugation collection conditions are: centrifugation speed of 500-1000g and centrifugation time of 10-15 min.

[0049] In a sixth aspect, this application provides a method for freeze-drying, storing, and / or transporting and processing a biological product, comprising the following steps: preparing a block-shaped hydrogel encapsulating the biological product 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 the product; and obtaining the harvested biological product according to the processing method described in the fifth aspect. Preferably, storage or transportation is carried out at room temperature, 4°C, -20°C, or -80°C.

[0050] In a seventh aspect, this application provides the use of the bulk hydrogel containing the biological product of the first aspect, the bulk hydrogel containing the biological product prepared in the second aspect, the lyophilized powder of the bulk hydrogel containing the biological product of the third aspect, or the lyophilized powder of the bulk hydrogel containing the biological product prepared in the fourth aspect in the preparation of biomedical products.

[0051] Compared with the prior art, the beneficial effects of this application are as follows:

[0052] 1. The block hydrogel encapsulation technology proposed in this application can reduce damage during the freeze-drying process. It can be used not only to encapsulate platelets or PRP and freeze-dry them, but also to encapsulate and freeze-dry other biological products. It can protect biological products from damage during the freeze-drying process and maintain their normal function.

[0053] 2. The bulk hydrogel proposed in this application possesses good biocompatibility and convertibility. The components of the hydrogel in this application (gelatin and dextran) are all reagents permitted for use in the pharmacopoeia, inexpensive, safe, and non-toxic.

[0054] 3. The block hydrogel provided in this application has temperature-responsive release properties. During the preparation process, gelatin will undergo liquid-liquid separation and physical gelation under low temperature induction. Under the promotion of crowded macromolecules, block hydrogel can be formed to encapsulate biological products such as platelets and PRP. After freeze-drying, the encapsulated biological products can be released simply by adjusting the temperature. No additional decrosslinking agent is required, making the operation simple.

[0055] 4. Compared with direct lyophilization, platelets encapsulated in block hydrogels and then lyophilized with PRP exhibit normal glycoprotein expression and inhibit desialylation, resulting in better hemostatic ability and longer in vivo circulation time. Furthermore, it demonstrates good protective and functional preservation effects in platelets from different species (such as human and mouse), exhibiting good cross-species adaptability.

[0056] 5. Compared with microhydrogel encapsulation freeze-drying, this application uses block hydrogel, which makes the preparation and operation process simpler and facilitates large-scale production; the encapsulation rate is higher, which improves the utilization rate of effective ingredients.

[0057] The method of freeze-drying biological products by encapsulating them in block hydrogels as described in this application allows for long-term storage of biological products at room temperature, which is expected to significantly extend the storage time of biological products, especially platelets, and alleviate the current platelet shortage. Attached Figure Description

[0058] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 The temperature response solution-gel change diagram of the hydrogel for encapsulating platelets provided in Example 1.

[0059] The top, middle, and bottom images show incubation at room temperature (22℃), 0℃ for 3 minutes, and 0℃ for 5 minutes, respectively.

[0060] From left to right: Gelatin-PEG hydrogel, Gelatin-SH hydrogel, Gelatin-Dextran hydrogel, Gelatin-PVA hydrogel, and pure gelatin.

[0061] Figure 2 Morphological images of the gelatin-PEG hydrogel, gelatin-SH hydrogel, gelatin-Dextran hydrogel, gelatin-PVA hydrogel and pure gelatin after freeze-drying, which are platelet-encapsulating gelatin provided in Example 1.

[0062] Figure 3 The graph shows the water content of the platelet-encapsulating gelatin-PEG hydrogel, gelatin-SH hydrogel, gelatin-Dextran hydrogel, gelatin-PVA hydrogel, and pure gelatin provided in Example 1.

[0063] Figure 4 The images show the state and dimensions of the Gelatin-Dextran hydrogel provided in Example 2 before (left) gelation, after (middle) gelation, and in different freeze-drying containers (right).

[0064] Figure 5 The image shows the expression levels of hemostasis-related glycoproteins CD41 / 61 and CD42b in bulk-Lyo, directly freeze-dried human platelets (Lyo), and fresh blood platelets (Fresh) encapsulated in the block-shaped Gelatin-Dextran hydrogel provided in Example 3.

[0065] Figure 6 The diagram shows the degree of desalting acidification of lyophilized human platelets (Bulk-Lyo), directly lyophilized human platelets (Lyo), and fresh human platelets (Fresh) encapsulated in the block-shaped Gelatin-Dextran hydrogel provided in Example 3.

[0066] Figure 7 The diagram shows the in vitro aggregation capacity of the block-shaped Gelatin-Dextran hydrogel-encapsulated lyophilized human platelets (Bulk-Lyo), directly lyophilized human platelets (Lyo), and fresh human platelets (Fresh) provided in Example 4.

[0067] Figure 8 The diagram shows the degree of desialylation of lyophilized mouse platelets (Bulk-Lyo), directly lyophilized mouse platelets (Lyo), and fresh mouse platelets (Fresh) encapsulated in the block-shaped Gelatin-Dextran hydrogel provided in Example 5.

[0068] Figure 9 The diagram shows the in vitro aggregation capacity of the block-shaped Gelatin-Dextran hydrogel-encapsulated lyophilized mouse platelets (Bulk-Lyo), directly lyophilized mouse platelets (Lyo), and fresh mouse platelets (Fresh) provided in Example 5.

[0069] Figure 10The in vivo circulation time diagrams (n=4) of lyophilized mouse platelets (Bulk-Lyo), directly lyophilized mouse platelets (Lyo), and fresh mouse platelets (Fresh) encapsulated in the block-shaped Gelatin-Dextran hydrogel provided in Example 6.

[0070] Figure 11 This is a comparison chart of the encapsulation efficiency of the bulk Gelatin-Dextran hydrogel and the micro hydrogel provided in Example 7.

[0071] Figure 12 A comparison of hemostasis time between the bulk Gelatin-Dextran hydrogel (Bulk-Lyo) and the micro-hydrogel (Micro-Lyo) provided in Example 7.

[0072] Figure 13 Comparison of CD41 / 61 and CD42b expression in human lyophilized PRP (Bulk-PRP) and fresh platelets provided in Example 8.

[0073] Figure 14 is a comparison of the expression levels of hemostasis-related glycoproteins CD41 / 61 and CD42b in lyophilized human platelets encapsulated in block-shaped Gelatin-Dextran hydrogel provided in Example 9 after 30 days of storage at room temperature (25°C) (Bulk-Lyo-30d) with those in fresh human platelets. Detailed Implementation

[0074] The specific embodiments of this application are described in detail below. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this application.

[0075] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0076] Features described or illustrated as part of one or more embodiments may be used in another or more embodiments to produce further embodiments.

[0077] Before describing this application in detail, it should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of this application, which is defined solely by the appended claims. For a more complete understanding of the 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 meaning as understood by one of ordinary skill in the art to which this application pertains.

[0078] definition Unless otherwise specified, the following terms used in this application shall have the following definitions.

[0079] Unless otherwise specified, the terms “comprising,” “including,” and “containing,” as well as similar expressions, shall be interpreted in an open and inclusive sense as “including but not limited to.”

[0080] The term "bulk hydrogel" refers to a polymer material with a continuous three-dimensional network structure on a macroscopic scale. Its characteristic feature is that the polymer chains form a single, monolithic, and physically continuous phase structure in space through physical or chemical cross-linking. The geometry of the bulk hydrogel is defined by its preparation container, mold, or in-situ formed tissue cavity, and its minimum macroscopic dimension is typically not less than 1 mm. Unlike the micropores contained within it, this "bulk" property emphasizes its non-particulate and monolithic nature in macroscopic mechanical properties and physical morphology. It should be clearly understood that the "bulk hydrogel" described in this invention is fundamentally different from aggregates formed by the stacking of "hydrogel microspheres" or "microgels." Bulk hydrogels consist of a continuous polymer network and do not possess physical interfaces or interparticle voids formed by particle stacking; while hydrogel microspheres exhibit discrete polydisperse or monodisperse phases, with diameters typically between 1 μm and 1000 μm. Furthermore, the stress transfer of bulk hydrogels is based on a complete molecular network framework, exhibiting significant structural integrity; while the mechanical strength of microsphere aggregates is limited by interparticle contact forces, electrostatic attraction, or auxiliary adhesives. Finally, bulk hydrogels are typically obtained through bulk polymerization or overall cross-linking, rather than through processes that produce discrete particles, such as emulsification, microfluidics, or spray drying.

[0081] The term "encapsulation" refers to the process of encapsulating bioactive substances (such as drugs, proteins, and cells) in bulk hydrogel materials. This encapsulation technology forms a protective barrier by encapsulating these substances within the three-dimensional network structure of the hydrogel, allowing them to be slowly released or isolated under specific conditions, thereby providing protection.

[0082] 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. Instead, it relies on non-covalent interactions between molecules (such as hydrogen bonds and van der Waals forces) to form a three-dimensional network structure, which keeps the substance solid or elastic.

[0083] The term "gelatin" refers to a natural high-molecular-weight protein obtained by hydrolyzing animal collagen. It primarily originates from the skin, bones, and connective tissues of animals such as cattle, pigs, and fish, as well as from recombinant gelatin or collagen-like peptides / proteins with controllable sequences prepared using synthetic biology methods. It exhibits excellent solubility in water and forms a gel upon cooling, demonstrating superior gelation properties. The hydration properties of gelatin enable it to act as a thickener, gelling agent, and moisture retainer in hydrogels, thus finding wide applications in biomedicine, hydrogel drug delivery systems, tissue engineering, and wound dressings. Gelatin not only possesses good biocompatibility and biodegradability but can also be compounded with other polymers to adjust the physical properties and mechanical strength of hydrogels, making it an important component in the pharmaceutical, food, and cosmetic industries.

[0084] The term "dextran" refers to a high-molecular-weight polysaccharide composed of glucose molecules linked by α-1,6-glycosidic bonds. It has a branched structure and is widely found in some bacteria (such as Proteus and Lactobacillus). Dextran molecules can be linear or highly branched, have a large molecular weight, and typically exhibit good water solubility and hydrophilicity. In the biomedical field, dextran is a biocompatible material commonly used as a blood volume expander, drug carrier, and stabilizer, particularly in plasma substitutes, drug delivery systems, and biomolecular carriers. It also possesses biodegradability and good immunocompatibility, thus frequently used in drug delivery, tissue engineering, and medical diagnostics.

[0085] The term "sodium hyaluronate" (SH) refers to a water-soluble salt compound formed by the combination of hyaluronic acid and its sodium salt. It is widely found in the connective tissue, skin, eyes, and synovial fluid of the human and animal bodies. Composed of alternating glucosamine and glucuronic acid, it possesses strong moisturizing capabilities, absorbing and retaining large amounts of water. Sodium hyaluronate has extensive applications in the medical and cosmetic fields, serving as a moisturizer, lubricant, and drug carrier in skin care, anti-aging, and joint treatment. It is commonly used in injectable fillers to aid facial aesthetics or alleviate arthritis, and also plays an important role in ophthalmic surgery, eye drops, and wound healing. In cosmetics, sodium hyaluronate is used as a moisturizing ingredient to improve skin hydration, reduce wrinkles and fine lines, and maintain skin elasticity. Furthermore, due to its excellent biocompatibility and biodegradability, sodium hyaluronate is also widely used in medical fields such as drug delivery systems.

[0086] The term "polyethylene glycol" (PEG) refers to a class of synthetic polymers formed by the polymerization of ethylene glycol or ethylene oxide. Depending on the degree of polymerization, PEG can have different molecular weights, typically ranging from several 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, widely used in pharmaceuticals, cosmetics, food, industry, and biotechnology. In the pharmaceutical field, PEG is commonly used as a drug carrier, sustained-release agent, solvent, solubilizer, and drug modifier to improve drug solubility, stability, and bioavailability, thereby enhancing therapeutic efficacy.

[0087] The term "polyvinyl alcohol" (PVA) refers to a high molecular weight compound produced by the alcoholysis and polymerization of vinyl acetate. It possesses excellent hydrophilicity and water solubility. Its chemical structure contains multiple alcohol groups (-OH), endowing it with superior film-forming properties, solubility, colloidal properties, and adhesiveness. The molecular weight and water solubility of PVA can be controlled by adjusting the degree of polymerization and alcoholysis, with common molecular weights ranging from several thousand to several million Daltons. Due to its good biocompatibility, PVA is widely used in medicine, industry, cosmetics, and environmental protection. In the pharmaceutical 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; and in cosmetics, PVA is used as an emulsifier, thickener, and humectant.

[0088] The term "Tyrode's buffer" refers to a buffer solution used in biological and biochemical experiments, typically to maintain a stable pH and ionic environment for cells or tissues during experiments. Tyrode's buffer consists of various salts and other components designed to mimic physiological conditions in vivo, and is widely used, particularly in cardiac physiology and cell biology research. Standard Tyrode's buffer usually contains ions such as sodium, potassium, calcium, chloride, and hydrocarbonate, as well as glucose. The Tyrode's buffer used in this study contained sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, HEPES, and glucose. The specific composition of the Tyrode's buffer used in the examples was 4.6 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 25 mM Na2HPO4, 5 mM HEPES, and 10 mM glucose.

[0089] The term "lyophilization protective base solution" typically refers to a lyophilization protective base solution used in the art consisting 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 2x lyophilization protective base solution used in this application example has each component concentration twice that described above.

[0090] Platelet precipitate: Platelet precipitate can be obtained using conventional platelet collection methods in the art. The platelet precipitate used in this embodiment was prepared using the following method: Blood was collected from ethically approved volunteers. Hydroxyethyl starch (1 / 3 of the original volume) was added to the blood, and after settling for 30 minutes, the mixture was centrifuged at 50g for 10 minutes. The supernatant was collected, and the red blood cells at the bottom of the tube were discarded. The supernatant was centrifuged at 400g for 15 minutes, and the white blood cells at the bottom of the tube were discarded. The supernatant was centrifuged again at 400g for 15 minutes to further remove the white blood cells at the bottom. The resulting supernatant was platelet-rich plasma. PGE1 to 1 μM was added to the platelet-rich plasma, and the mixture was centrifuged at 1000g 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 later use.

[0091] This application embodiment can use pretreated incubated platelets. The specific pretreatment steps are as follows: resuspend the platelet precipitate in 1 / 10 of its original volume of incubation solution. The incubation solution consists of: 50 mM trehalose, 100 mM sodium chloride, 10 mM potassium chloride, 10 mM imidazole, 10 mM EGTA, and 1 μM PGE1. Transfer to a constant temperature incubator and incubate at 37°C and 40 rpm for 4 hours. After the platelet incubation time is completed, remove the platelets from the incubator and centrifuge at 1000 g for 12 minutes. Remove the supernatant to obtain the platelet precipitate.

[0092] The freeze-drying procedure used in this embodiment is as follows: pre-freeze at -80℃ for 10 hours, transfer the pre-frozen vials to a freeze dryer, set the plate temperature to -5℃ and the vacuum degree to 1 Pa. Dry for 10 hours initially. Then, heat to 25℃ at a rate of 0.5℃ / min and dry for a second time for 10 hours to obtain the freeze-dried powder.

[0093] Example 1: Preparation of temperature-responsive block hydrogels encapsulating platelets and study on their freeze-drying properties 1.1 Experimental Materials and Solution Preparation Gelatin was selected as the gelling matrix, and polyethylene glycol (PEG), sodium hyaluronate (SH), dextran, and polyvinyl alcohol (PVA) were added as crowding macromolecules or synergistic components.

[0094] The preparation methods for each solution are as follows: 8% Gelatin solution: Weigh out gelatin powder and dissolve it in deionized water. Heat and stir in a 65°C water bath until completely dissolved.

[0095] 20% PVA solution: Weigh PVA into deionized water and dissolve it by heating in a high-temperature autoclave (about 121°C) to ensure full dispersion.

[0096] 10% Dextran solution, 10% PEG solution: Weigh Dextran or PEG into deionized water and stir to dissolve at room temperature.

[0097] 0.5% SH solution: Weigh SH into deionized water and dissolve by stirring at room temperature in the dark.

[0098] 1.2 Preparation and Encapsulation Process of Temperature-Response Bulk Hydrogels Component mixing: Using 2 times the lyophilized protective base solution as the solvent, add the above solution to make the final concentration of gelatin in the system 2% (w / v). The other auxiliary components are set as follows: 0.5% PEG, 0.15% SH, 0.5% Dextran, and 0.5% PVA, or no other auxiliary component is added.

[0099] Platelet loading: Resuspend the platelet precipitate using the above mixed solution and adjust the final platelet concentration to 5 × 10⁻⁶. 8 / mL.

[0100] Weighing and Packaging: Weigh the empty vial (W0), fill the vial with the platelet-containing mixed solution, and record the total weight (W1).

[0101] 1.3 Temperature response properties and freeze-drying characterization Solution state (22℃): At room temperature, all components in the inverted vial exhibit a clear fluid solution state (e.g., Figure 1 (As shown in the upper layer).

[0102] Gelation induction (0℃): The vials were incubated at 0℃. Experiments showed that under low-temperature induction, gelatin molecules in different formulations underwent physical cross-linking to form blocky hydrogels. Among them, the Gelatin-Dextran component exhibited the best gel strength within 3-5 minutes of incubation, and the vials did not flow when inverted (e.g., [missing information]). Figure 1 (As shown in the middle and lower layers).

[0103] Freeze-drying process: The vials are transported to a freeze dryer via dry ice for programmed freeze-drying.

[0104] Dry powder morphology: After freeze-drying, it was observed that the freeze-dried cakes formed by all components had relatively intact structures, loose texture, and no obvious collapse or shrinkage (e.g., Figure 2 (As shown).

[0105] 1.4 Moisture Content Calculation and Analysis Remove the lyophilized vial and weigh it, recording the weight as W2. The formula for calculating water content is as follows: Water weight = W1 - W2 Total weight of contents = W1 - W0 Moisture content calculation: Moisture content (%) = (W1 - W2) / (W1 - W0) * 100% Experimental results are as follows Figure 3 As shown, by comparing different formulations, it was found that the Gelatin-Dextran system formed the most stable physical framework while maintaining a low water content. This indicates that Dextran, as a crowded macromolecule, can effectively assist gelatin in expelling excess water during freeze-drying, forming an amorphous glassy matrix that is conducive to the long-term preservation of platelets.

[0106] Example 2: Effect of different container sizes on the gelling properties of bulk hydrogels To verify the gelation stability of the temperature-responsive bulk hydrogel at different scales and the feasibility of industrial-scale production, this experiment used various sizes of encapsulation containers for testing.

[0107] 2.1 Experimental Methods The platelet-containing mixture was prepared according to the preferred Gelatin-Dextran formulation in Example 1. Three different sizes of vials (10 mL, 30 mL, and 100 mL) were used as loading containers.

[0108] The platelet-containing mixture was injected into the containers described above, and the liquid flow was recorded at room temperature (22°C). All containers were then incubated at 0°C for 5 minutes.

[0109] 2.2 Experimental Results and Analysis Experimental results are as follows Figure 4 As shown.

[0110] At room temperature: At 22°C, the system maintains good liquid flowability in both small 10 mL and large 100 mL containers, facilitating large-scale dispensing and automated filling.

[0111] Low-temperature gel formation: At 0°C, the liquids in all three container sizes underwent a phase change simultaneously, forming a structurally complete blocky hydrogel. Even in a large-volume container of 100 mL, the hydrogel maintained sufficient mechanical strength to support its own weight without collapsing or flowing.

[0112] The above results demonstrate that the temperature-responsive bulk hydrogel preparation method provided by this invention has extremely high tolerance for container size. This "size-independent" gelation characteristic proves that this technology is not only suitable for the preparation of small laboratory samples, but also for large-scale, industrial platelet freeze-drying production processes, and has extremely strong clinical translational value.

[0113] Example 3: Verification of Functional Protein Maintenance and Desialylation Inhibition in Lyophilized Platelets 3.1 Sample rehydration and pretreatment Sample grouping: The experiment was divided into fresh platelet group (Fresh), direct freeze-dried platelet group (Lyo), and block-shaped Gelatin-Dextran hydrogel-encapsulated freeze-dried platelet group provided in Example 1 (Bulk-Lyo).

[0114] Directly lyophilized platelets group: An equal volume of lyophilization protection base solution was added directly to an equal volume of platelets for resuspension, followed by lyophilization.

[0115] Rehydration and release: 1 mL of double-distilled water (ddH2O, the same volume as the solution before lyophilization) was added to all lyophilized vials for rehydration. The rehydrated solution was then transferred to 1.5 mL centrifuge tubes and incubated in a 37°C water bath for 5 minutes to fully liquefy the block hydrogel and release platelets using the temperature response characteristics.

[0116] Washing and resuspending: PGE1 was added to both the lyophilized and fresh samples to a final concentration of 1 µM to inhibit abnormal platelet activation, and the samples were centrifuged at 1200g for 12 minutes. The supernatant was discarded, and the platelets were resuspended in 1 mL of physiological saline.

[0117] Filtration: Pass 200 µL of the resuspension through a 40 µm filter to remove any possible minute impurities.

[0118] 3.2 Flow cytometry detection and concentration analysis Antibody labeling: Specific fluorescent antibodies were added to the platelet samples after the above treatment and incubated in the dark for 20 minutes. The antibody components were as follows: 0.2 µL PE-Cy7 labeled CD41 antibody (platelet marker); 0.2 µL BV650 labeled CD42b antibody (a key glycoprotein for hemostasis); 0.1 µL FITC labeled RCA-I phytohemagglutinin (used to detect surface-exposed galactose and assess the degree of desialylation).

[0119] Detection and statistics: The above indicators were detected by flow cytometry, and the platelet recovery concentration of each group was simultaneously determined by a complete blood count instrument.

[0120] 3.3 Analysis of Experimental Results Expression of hemostasis-related glycoproteins: such as Figure 5 As shown, flow cytometry results revealed that CD42b expression in the directly lyophilized group (Lyo) was significantly lower than that in the fresh group. However, platelets encapsulated in the bulk hydrogel of this invention (Bulk-Lyo) maintained CD41 / CD61 and CD42b expression levels to a great extent, approaching the levels of fresh platelets. This demonstrates that the bulk hydrogel system can effectively protect platelet membrane proteins from freeze-drying stress damage.

[0121] Assessment of the degree of desialylation: such as Figure 6 As shown, increased RCA-I binding strength indicates platelet desialylation. Figure 6 As shown, the direct freeze-dried group (Lyo) exhibited a significant rightward shift of the RCA-I binding peak, indicating substantial exposure of surface galactose. In contrast, the RCA-I binding level in the Bulk-Lyo group was significantly lower than that in the direct freeze-dried group, effectively inhibiting the desialylation reaction.

[0122] The above results indicate that this application, through the protective mechanism of bulk hydrogel, not only maintains the expression of the platelet hemostatic core protein CD42b, but also significantly inhibits the desialylation process that leads to the rapid clearance of platelets in vivo, providing a biological basis for prolonging the in vivo circulation time of freeze-dried platelets.

[0123] Example 4: Validation of the in vitro aggregation function of lyophilized platelets 4.1 Experimental Materials and Sample Preparation Experimental reagents: Thrombin, prostaglandin E1 (PGE1), platelet-poor plasma (PPP), platelet-rich plasma (PRP), phosphate-buffered saline (PBS).

[0124] Sample grouping: The experiment was divided into fresh platelet group (Fresh), direct freeze-dried platelet group (Lyo), and block-shaped Gelatin-Dextran hydrogel-encapsulated freeze-dried platelet group provided in Example 1 (Bulk-Lyo).

[0125] Directly lyophilized platelets group: An equal volume of lyophilization protection base solution was added directly to an equal volume of platelets for resuspension, followed by lyophilization.

[0126] Rehydration and Pretreatment: The lyophilized platelets were rehydrated and released according to the method in Example 3. PGE1 was added to both the lyophilized and fresh samples to a final concentration of 1 µM, and the samples were centrifuged at 1200g for 12 minutes. The supernatant was discarded, and the samples were resuspended in platelet-poor plasma (PPP). The final platelet concentration was adjusted to 2 × 10⁻⁶ using a complete blood count machine. 8 / mL.

[0127] 4.2 Aggregation Experiment Procedure Sample incubation: Take 300 µL of the resuspended platelet sample and put it into a special test tube for platelet aggregator, and put in the matching rotor, and incubate at 37°C for 5 minutes.

[0128] Instrument zeroing: Place the test tube in the concentration instrument detection position, set the rotation speed to 600 rpm, and press the Baseline button to zero the baseline.

[0129] Induction of aggregation: Thrombin was added to the test tube as an inducing agent to achieve a final concentration of 2 U / mL.

[0130] Data recording: Observe and record the platelet aggregation curve in real time, and record the change in the aggregation percentage within 10 minutes.

[0131] 4.3 Analysis of Experimental Results Experimental results are as follows Figure 7 As shown.

[0132] Aggregation curve characteristics: After the addition of the inducing agent, the aggregation curve of the direct freeze-dried group (Lyo) fluctuated slightly, and the maximum aggregation rate was significantly lower than that of the fresh group, indicating that its core hemostatic function was severely damaged.

[0133] Evaluation of protective effect: The bulk-Lyo hydrogel encapsulation group provided in this application showed good aggregation response, and the slope of its aggregation curve and the maximum aggregation percentage were highly consistent with the trend of the fresh group.

[0134] The above results demonstrate that, when platelets are encapsulated and protected by the temperature-responsive block hydrogel prepared in this invention, they can still maintain good in vitro aggregation activity after undergoing freeze-drying and rehydration processes. Under the induction of thrombin, platelets can rapidly aggregate, thus possessing the functional basis for performing clinical hemostasis tasks.

[0135] Example 5: Validation of the functional stability of mouse platelets under the protection of bulk hydrogel To further verify the universality of the temperature-responsive block hydrogel provided in this application in platelet protection of different species, this experiment used mouse platelets (mPLTs) as the research object to investigate their protein expression and aggregation function after freeze-drying and rehydration.

[0136] 5.1 Treatment and rehydration of mouse platelets Sample preparation: Following the preferred formulation and method in Example 1, block hydrogels encapsulating mouse platelets were prepared and lyophilized (Bulk-Lyo) in 10ml vials with a volume of 1ml. A fresh mouse platelet group (Fresh) and a direct lyophilized group (Lyo) were also prepared.

[0137] Directly lyophilized platelets group: An equal volume of lyophilization protection base solution was added directly to an equal volume of platelets for resuspension, followed by lyophilization.

[0138] Rehydration and release: 1 mL of double-distilled water (ddH2O, the same volume as the solution before lyophilization) was added to all lyophilized vials for rehydration. The rehydrated solution was then transferred to 1.5 mL centrifuge tubes and incubated in a 37°C water bath for 5 minutes. The temperature response characteristics were used to completely liquefy the block hydrogel and release mouse platelets.

[0139] Purification: PGE1 was added to both the lyophilized and fresh samples to a final concentration of 1 µM to inhibit abnormal platelet activation. The samples were centrifuged at 1200g for 12 minutes, the supernatant was discarded, and the samples were resuspended in 200 µL of physiological saline and filtered through a 40 µm filter.

[0140] 5.2 Flow cytometry detection Experimental method: 0.1 µL of FITC-labeled RCA-I phytohemagglutinin was added to the platelet samples of each group of mice after treatment, and the samples were incubated in the dark for 20 minutes before flow cytometry detection.

[0141] Results analysis: Figure 8 The results showed that the direct freeze-dried group (Lyo) exhibited a strong RCA-I binding signal, indicating that a large number of galactose residues were exposed on the platelet surface, resulting in severe desialylation. In contrast, the RCA-I fluorescence intensity of the Bulk-Lyo group was similar to that of the Fresh group, but significantly lower than that of the Lyo group. This demonstrates that the bulk hydrogel system can effectively inhibit the desialylation reaction of mouse platelets in cross-species applications.

[0142] 5.3 In vitro platelet aggregation experiment in mice Experimental Procedure: Platelets from the lyophilized group were rehydrated and released according to method 5.1. PGE1 was added to both the lyophilized and fresh groups to a final concentration of 1 µM, and the samples were centrifuged at 1200g for 12 minutes. The supernatant was discarded, and the samples were resuspended in platelet-rich plasma (PPP). The final platelet concentration was adjusted to 2 × 10⁻⁶ using a complete blood count (CBC) system. 8 / mL. Take 300 µL of the resuspended sample and place it into a special test tube for platelet aggregator, and put in the matching rotor. Incubate at 37°C for 5 minutes.

[0143] Instrument zeroing: Place the test tube in the concentration instrument detection position, set the rotation speed to 600 rpm, and press the Baseline button to zero the baseline.

[0144] Results analysis: such as Figure 9 As shown, the platelet aggregation curves of the Bulk-Lyo group were consistent with those of the Fresh group, with the maximum aggregation percentage far exceeding that of the Lyo group, demonstrating that its hemostatic activity was well preserved.

[0145] The above results demonstrate that the temperature-responsive block hydrogel encapsulation scheme proposed in this application has excellent protective effects on platelets from different sources (including human and mouse), showcasing the broad applicability of this technology in the biomedical field.

[0146] Example 6: Circulatory kinetics evaluation of lyophilized platelets in vivo 6.1 Experimental Materials and Sample Preparation Blood collection and anticoagulation: Blood was collected from the heart of healthy mice and treated with an anticoagulant.

[0147] Platelet-rich plasma (PRP) preparation: Dilute whole blood with an equal volume of physiological saline, and add prostaglandin E1 (PGE1) to a final concentration of 1 µM. Centrifuge at 230g for 9 minutes and collect the supernatant.

[0148] Sample reconstitution and centrifugation: Following the method in Example 5, 1 mL of double-distilled water (ddH2O) was added to the bulk hydrogel encapsulation group (Bulk) and the direct freeze-drying group (Lyo), and incubated at 37°C for 5 minutes. After the hydrogel liquefies, mouse platelets are completely released.

[0149] Purification: PGE1 was added to both the lyophilized and fresh samples to a final concentration of 1 µM to inhibit abnormal platelet activation. The samples were centrifuged at 1200g for 12 minutes and the supernatant was discarded.

[0150] 6.2 Fluorescent Labeling and Competitive Tracing Resuspension and labeling: Platelets from each group were resuspended using 1 mL of Tyrode's solution.

[0151] Dye selection and labeling: To distinguish different groups within the same receptor, three different wavelengths of cell-tracking dyes were added to the three groups of samples. Specifically: Fresh group: the first fluorescent dye (green, such as succinimide ester derivatives) was added; Bulk-Lyo group: the second fluorescent dye (far-infrared, such as long-chain cyanide dyes) was added; Directly lyophilized group (Lyo): the third fluorescent dye (blue, such as coumarin derivatives) was added.

[0152] Incubation and washing: Incubate at 37°C in the dark for 30 minutes. Then wash twice with Tyrode's solution containing 1% bovine serum albumin (BSA) to thoroughly remove excess free dye.

[0153] 6.3 Sample Transfusion and In Vivo Monitoring Volume adjustment and mixing: Adjust the platelet concentration of each group to be consistent (each group contains approximately 1×10⁻⁶). 8 (1 platelet) and mix them in equal volumes.

[0154] Infusion and sampling: 200 µL of the mixture was infused into recipient mice via the tail vein, and this was repeated for 4 groups. Sampling was performed at multiple pre-defined time points after infusion.

[0155] Results Analysis: The proportions of fluorescently labeled platelets of different colors in circulating blood were detected using flow cytometry. The experimental results are as follows: Figure 10 As shown, the directly freeze-dried group (Lyo) is rapidly eliminated after entering the body; while the bulk hydrogel group (Bulk-Lyo) provided by the present invention has a circulation trajectory in the body that closely matches that of the fresh group (Fresh), significantly prolonging the circulation time.

[0156] Example 7: Performance Comparison of Bulk and Microhydrogel Encapsulation Technologies 7.1 Sample Preparation The present invention group (Bulk): According to the method shown in Example 1, platelets are encapsulated in situ using a gelatin and dextran system under low temperature conditions.

[0157] Prior art group (Micro): Using the method disclosed in CN202510018685.0, microhydrogels are prepared to encapsulate platelets through liquid-liquid phase separation and physical gelation processes.

[0158] Control group: included fresh platelet group (Fresh) and blank saline group (PBS).

[0159] 7.2 Encapsulation Efficiency Comparison Experiment Experimental method: Under the same initial platelet input, the number of platelets successfully loaded into the hydrogel matrix in both groups after preparation was counted.

[0160] Experimental results: such as Figure 11 As shown, the encapsulation efficiency of the micro hydrogel group is only about 25%, while the encapsulation efficiency of the bulk hydrogel group of the present invention is as high as 90% or more.

[0161] Technical Advantages Analysis: The encapsulation efficiency of the bulk hydrogel group and the microhydrogel group showed a statistically significant difference (p = 0.0002). This is because the microhydrogel preparation process involves complex phase separation and centrifugal washing, resulting in significant platelet loss; while the bulk hydrogel directly captures platelets through in-situ physical phase transition with almost no loss, significantly improving the utilization rate of the effective components.

[0162] 7.3 Comparative Experiment on In Vivo Hemostatic Efficacy Experimental methods: Using a mouse tail artery bleeding model, under the premise of ensuring that the original amount of platelets in the two groups of samples is equal, the effect of platelet samples after rehydration transfusion (Bulk-Lyo and Micro-Lyo) on hemostasis time was observed.

[0163] Experimental results: such as Figure 12 As shown, the average hemostasis time in the Bulk-Lyo group (approximately 300 seconds) was significantly shorter than that in the Micro-Lyo group (approximately 1000 seconds).

[0164] Technical Advantages Analysis: Statistical results show that the hemostatic efficacy of the Bulk-Lyo group is superior to that of the Micro-Lyo group (p = 0.0056), and there is no significant difference compared with the Fresh group (fresh platelets). This result demonstrates that, under the same production input cost, the block-shaped hydrogel encapsulating platelets of this application not only has higher production efficiency (higher encapsulation rate) but also exhibits stronger hemostatic efficacy in clinical applications.

[0165] Example 8: Preparation of PRP freeze-drying protection system and freeze-drying treatment method 8.1 Preparation of lyophilized PRP First, the core stock solution was prepared: 3.42 g of trehalose was dissolved in ultrapure water and brought to a final volume of 10 mL. After filtration and sterilization, a 1 M trehalose stock solution was prepared. Simultaneously, under a salt-free environment, 0.4 g of gelatin and 0.2 g of hydroxypropyl-β-cyclodextrin were added to approximately 2.5 mL of ultrapure water and heated in a 60°C water bath with shaking until completely transparent. Then, 1.0 mL of 10% (w / w) dextran (Dextran 500) stock solution was added and brought to a final volume of 5 mL to obtain the "4X" composite hydrogel matrix stock solution, which was then stored at a constant temperature of 37°C in a water bath for later use. Next, PRP was prepared using a single centrifugation method. Whole blood was centrifuged at 200 g for 15 min at room temperature, with the centrifuge set to soft brake mode to maintain stable blood cell levels. After centrifugation, only the upper two-thirds of the pale yellow plasma layer was aspirated, strictly avoiding the aspiration of the middle white membrane layer to ensure no leukocyte contamination.

[0166] During the loading and gelation stage, 0.3 µL of PGE1 stock solution and 150 µL of the aforementioned 1 M trehalose stock solution were added sequentially to 3 mL of the prepared PRP to achieve an initial trehalose concentration of approximately 47.6 mM. The mixture was then incubated at 37°C for 4 hours to achieve intracellular loading of trehalose. After loading, 3.15 mL of the mixture was removed and 1.05 mL of "4X" hydrogel matrix stock solution at 37°C was added. The mixture was then gently pipetted to ensure thorough mixing. At this point, the trehalose concentration was reduced to approximately 35.7 mM, and the overall osmotic pressure was maintained at approximately 270 mOsm / L. Finally, the mixture was aliquoted while still warm into lyophilization vials (1 mL / vial) for pre-freezing and vacuum lyophilization.

[0167] 8.2 Characterization of lyophilized PRP Rehydration and release: 1 mL of double-distilled water (ddH2O) was added to all lyophilized vials for rehydration. The rehydrated solution was then transferred to 1.5 mL centrifuge tubes and incubated in a 37°C water bath for 5 minutes to utilize the temperature response characteristics to completely liquefy the block hydrogel and release platelets.

[0168] Washing and resuspending: PGE1 was added to both the lyophilized and fresh samples to a final concentration of 1 µM to inhibit abnormal platelet activation, and the samples were centrifuged at 1200g for 12 minutes. The supernatant was discarded, and the platelets were resuspended in 1 mL of physiological saline.

[0169] Filtration: Pass 200 µL of the resuspension through a 40 µm filter to remove any possible minute impurities.

[0170] Antibody labeling: Add specific fluorescent antibodies to the platelet samples after the above treatment and incubate in the dark for 20 minutes. The antibody components are as follows: 0.2 µL PE-Cy7 labeled CD41 antibody (platelet marker); 0.2 µL BV650 labeled CD42b antibody (key glycoprotein for hemostasis).

[0171] Detection and statistics: The above indicators were detected by flow cytometry, and the platelet recovery concentration of each group was simultaneously determined by a complete blood count instrument.

[0172] 8.3 Analysis of Experimental Results like Figure 13 As shown in the flow cytometry results, the expression levels of CD41 / CD61 and CD42b in the PRP encapsulated by the bulk hydrogel of this invention were maintained to a great extent, approaching the levels of fresh platelets. This demonstrates that the bulk hydrogel system can effectively protect platelet membrane proteins from freeze-drying stress damage.

[0173] Example 9: Study on the long-term room temperature stability of lyophilized platelets encapsulated in block hydrogel 9.1 Sample Preparation and Storage Following the preferred formulation and preparation method provided in Example 1, multiple groups of block hydrogels encapsulating human platelets were prepared and subjected to programmed lyophilization. The resulting lyophilized powder samples were placed in sealed containers and stored at room temperature (22-25°C) for one month. A fresh platelet group was set up as a control group.

[0174] 9.2 Sample Rehydration and Treatment One month after storage, the lyophilized powder samples from the Bulk Lyo group were retrieved. 1 mL of double-distilled water (ddH2O) was added to each vial for rehydration. The rehydrated solution was then transferred to a 1.5 mL centrifuge tube and incubated at 37°C for 5 minutes to allow the temperature-responsive hydrogel to completely liquefy and release platelets. PGE1 was added to both the rehydrated and fresh platelet samples to a final concentration of 1 µM to inhibit abnormal platelet activation. The samples were centrifuged at 1200g for 12 minutes, the supernatant was discarded, and the suspension was resuspended in physiological saline and filtered through a 40 µm filter.

[0175] 9.3 Flow cytometry detection Add specific fluorescent antibodies to the platelet samples after the above treatment and incubate in the dark for 20 minutes. The detection indicators include: 0.2 µL of PE-Cy7 labeled CD41 antibody and 0.2 µL of BV650 labeled CD42b antibody.

[0176] 9.4 Analysis of Experimental Results like Figure 14 Flow cytometry results showed that after one month of storage at room temperature, the expression levels of surface markers CD41 / CD61 and CD42b in the Bulk Lyo group were not statistically significantly different from those in fresh platelets. This result demonstrates that the block-shaped hydrogel encapsulation system provided in this application can provide excellent room-temperature storage protection for lyophilized platelets, effectively preventing membrane protein degradation or structural damage during long-term storage, and significantly extending the clinical shelf life of the product.

[0177] This application proposes a method for encapsulating and lyophilizing biological products using temperature-responsive bulk hydrogels. The bulk hydrogels enable large-scale encapsulation and release of biological products such as platelets and PRPs through temperature response, eliminating the need for additional decrosslinking agents and simplifying the process. After lyophilization, room temperature storage, and dissociation, platelets and PRPs encapsulated in the bulk hydrogels maintain normal surface glycoprotein expression and inhibit desialylation, thus preserving the normal hemostatic capacity of platelets and extending their in vivo circulation time. Compared to microhydrogel encapsulation technology, the bulk hydrogels provided in this application offer higher encapsulation efficiency, are simpler to operate, and exhibit higher in vivo hemostatic efficacy after encapsulation. The method for encapsulating platelets and PRPs with bulk hydrogels in this application is expected to significantly extend platelet storage time and alleviate the current platelet shortage.

[0178] The scope of protection of this application is not limited to the embodiments described above. Obviously, those skilled in the art can make various modifications and variations to this application without departing from the scope and spirit of the invention. If such modifications and variations fall within the scope of the claims of this application and their equivalents, then the intent of this application also includes such modifications and variations.

[0179] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

[0180] Furthermore, various different implementations of this application can be combined in any way, as long as they do not violate the spirit of this application, they should also be regarded as the content disclosed in this application.

Claims

1. A block-shaped hydrogel encapsulating biological products, characterized in that, It is formed by liquid-liquid phase separation and physical gelation of an aqueous solution containing gelatin, crowding macromolecules and biological products, wherein the crowding macromolecules are selected from one or more of dextran and its derivatives, polyvinyl alcohol, hyaluronic acid and its salts, and polyethylene glycol.

2. The block hydrogel encapsulating biological products according to claim 1, characterized in that, The biological product is platelet-rich plasma (PRP) of humans or animals.

3. The block hydrogel encapsulating biological products according to claim 1 or 2, characterized in that, The mass ratio of gelatin to crowded macromolecules in the aqueous solution is 1:0.05-0.5; Preferably, the aqueous solution is an aqueous solution containing gelatin, dextran, and biological products, wherein the mass ratio of gelatin to dextran is 1:0.1-0.4; More preferably, the mass ratio of gelatin to dextran is 1:0.2-0.

3.

4. The block hydrogel encapsulating biological products according to any one of claims 1-3, characterized in that, The block hydrogel has a water content of 40%-99%; and / or, the block hydrogel also contains one or more of trehalose, sucrose, glucose, maltose, mannitol, lactose, and sorbitol. Preferably, the water content of the block hydrogel is 60%-95%.

5. The block hydrogel encapsulating biological products according to any one of claims 1-4, characterized in that, The aqueous solution undergoes liquid-liquid phase separation and physical gelation at -5 to 10°C, preferably at -1 to 5°C.

6. A method for preparing a block hydrogel encapsulating a biological product as described in any one of claims 1-5, characterized in that, Includes the following steps: A block hydrogel is formed by liquid-liquid phase separation and physical gelation of an aqueous solution containing gelatin, crowding macromolecules and biological products; the crowding macromolecules are selected from one or more of dextran and its derivatives, polyvinyl alcohol, hyaluronic acid and its salts, and polyethylene glycol.

7. The preparation method according to claim 6, characterized in that, The biological product is platelets or PRP, and the platelets or PRP in the aqueous solution are pretreated and incubated platelets or PRP. Preferably, platelets or PRP are incubated in a solution containing sugars, optionally selected from one or more of sodium chloride, potassium chloride, imidazole, EGTA, PGE1, and EDTA. More preferably, an incubation solution containing trehalose, optionally selected from one or more of sodium chloride, potassium chloride, imidazole, EGTA, PGE1, and EDTA, is added to the platelet precipitate separated from blood, resuspended, and incubated; optionally, after incubation, the platelets are collected by centrifugation. Alternatively, trehalose, optionally selected from one or more of sodium chloride, potassium chloride, imidazole, EGTA, PGE1, and EDTA, can be added to PRP separated from blood and then incubated.

8. The preparation method according to claim 6 or 7, characterized in that, The biological product is platelets or PRP. Platelets are resuspended in an aqueous solution containing gelatin and crowding macromolecules; or an aqueous solution is obtained by mixing a concentrated hydrogel matrix stock solution containing gelatin and crowding macromolecules with PRP; followed by liquid-liquid phase separation and physical gelation; the platelet concentration in the aqueous solution is 0.1-10 × 10⁻⁶. 9 / mL; Preferably, the concentrated hydrogel matrix stock solution is mixed with PRP at a volume ratio of 1:1 to 1:9; the platelet concentration in the aqueous solution is 0.1-5 × 10⁻⁶. 9 / mL; More preferably, the concentrated hydrogel matrix stock solution is mixed with PRP at a volume ratio of 1:2 to 1:

5.

9. The preparation method according to any one of claims 6-8, characterized in that, The aqueous solution contains 0.1-15% w / v gelatin, preferably 1-5% w / v gelatin, and more preferably 2-3% w / v gelatin.

10. The preparation method according to any one of claims 6-9, characterized in that, The medium of the aqueous solution is a lyophilization protective base solution; preferably, the lyophilization protective base solution is a 0.5-2 × lyophilization protective base solution; more preferably, the lyophilization protective base solution is a 1-1.5 × lyophilization protective base solution.

11. The preparation method according to claim 10, characterized in that, The freeze-drying protective base solution contains one or more of trehalose, sucrose, glucose, maltose, glycerol, and mannitol, and optionally, contains one or more of magnesium chloride, potassium chloride, sodium chloride, HEPES, and hydroxypropyl-β-cyclodextrin. Preferably, the 1× lyophilized protective base solution contains 1-100mM trehalose, 1-10mM magnesium chloride, 1-10mM potassium chloride, 1-1000mM sodium chloride, 1-100mM HEPES, and 1-100mM hydroxypropyl-β-cyclodextrin.

12. A block-shaped hydrogel lyophilized powder encapsulating biological products, characterized in that, It is obtained by freeze-drying the block hydrogel containing biological products as described in any one of claims 1-5, or the block hydrogel containing biological products prepared by any one of claims 6-11.

13. A method for preparing a block-shaped hydrogel lyophilized powder encapsulating biological products, characterized in that, It includes the following steps: freeze-drying the block hydrogel containing the biological product as described in any one of claims 1-5, or the block hydrogel containing the biological product prepared by the preparation method according to any one of claims 6-11.

14. The preparation method according to claim 13, characterized in that, The freeze-drying process includes three steps: pre-freezing, primary drying, and secondary drying.

15. The preparation method according to claim 13 or 14, characterized in that, The aqueous solution containing gelatin, crowded macromolecules and biological products is directly subjected to liquid-liquid phase separation and physical gelation in a freeze-drying container to form a block hydrogel, which is then freeze-dried; or the block hydrogel containing biological products is transferred to a freeze-drying container for freeze-drying; optionally, it also includes nitrogen protection, sealing and vacuuming steps after freeze-drying. Preferably, the freeze-drying container serves as a mold defining the shape and size of the block hydrogel, wherein the block hydrogel has a size of 1 mm to 1000 mm in at least one dimension in three-dimensional space; and / or, the freeze-drying container is one or more of the following: vials, aluminum foil bags, composite film bags, vacuum packaging bags, modified atmosphere packaging bags, polyester film bags, glass bottles, plastic bottles, polyethylene tubes, polypropylene tubes, glass tubes, aluminum tubes, polyester tubes, and freeze-drying trays; More preferably, the bulk hydrogel has a size of 1 mm to 100 mm in at least one dimension in three-dimensional space.

16. A method for processing the block hydrogel lyophilized powder encapsulating biological products as described in claim 12, or the block hydrogel lyophilized powder encapsulating biological products prepared by any of the preparation methods according to claims 13-15, characterized in that, Includes the following steps: The harvested biological products are obtained by rehydration, dissociation, and centrifugation; preferably, dissociation is achieved by heating and incubation.

17. The processing method according to claim 16, characterized in that, The heating and incubation temperature is 20-40℃, and the incubation time is 1 min-1 h; preferably, the heating and incubation temperature is 35-38℃, and the incubation time is 3-6 min.

18. The processing method according to claim 16 or 17, characterized in that, The process includes the following steps: resuspending the block-shaped hydrogel lyophilized powder containing the biological product in pure water or a suitable solution for rehydration; heating and incubating to dissociate the biological product; and centrifuging to collect the harvested biological product; optionally, resuspending it in pure water or a suitable solution for subsequent use. Preferably, the volume of the solution used for rehydration is 1 / 10-5 times the volume before freeze-drying; after centrifugation to collect the harvested biological products, the volume of the solution used for resuspension is 1 / 10-5 times the volume before freeze-drying. More preferably, the volume of the solution used for rehydration is 1 / 2 to 2 times the volume before lyophilization; the volume of the solution used for resuspension is 1 / 2 to 2 times the volume before lyophilization.

19. The processing method according to claim 18, characterized in that, The lyophilized block hydrogel powder containing platelets or PRP is resuspended in one or more of pure water, plasma, or buffer solution, heated to dissociate the biological product, PGE1 to 1-10 µM is added, and the harvested biological product is collected by centrifugation; optionally, it is resuspended in one or more of pure water, plasma, or buffer solution for subsequent use. Preferably, the lyophilized block hydrogel powder encapsulating platelets or PRP is resuspended in pure water, centrifuged to collect the harvested biological product, and then resuspended in anemic platelet plasma for subsequent use.

20. A method for freeze-drying, storing, and / or transporting and processing a biological product, characterized in that, The process includes the following steps: preparing a block hydrogel encapsulating a biological product according to any one of the preparation methods described in claims 6-11; preparing a freeze-dried powder of the block hydrogel encapsulating the biological product according to any one of the methods described in claims 13-15; storing or transporting the product; and obtaining the harvested biological product according to any one of the processing methods described in claims 16-19.

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

22. The use of the bulk hydrogel encapsulating biological products according to any one of claims 1-5, the bulk hydrogel encapsulating biological products prepared according to any one of claims 6-11, the lyophilized powder of the bulk hydrogel encapsulating biological products according to claim 12, or the lyophilized powder of the bulk hydrogel encapsulating biological products prepared according to any one of claims 13-15 in the preparation of biomedical products.

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