Preparation process of freeze-dried platelet aggregation activation reagent

Abstract

The invention discloses a preparation process of a freeze-dried platelet aggregation activation reagent, and relates to the technical field of medical reagent preparation, and the preparation process comprises the following steps: (1) preparing trehalose grafted chitosan; (2) preparing phenylboronic acid-trehalose-chitosan; (3) preparing a multifunctional freeze-drying protection carrier; (4) preparing a supramolecular complex binding solution; and (5) sub-packaging the supramolecular complex binding liquid, and performing programmed freeze-drying to obtain the freeze-dried platelet aggregation activation reagent. According to the invention, an environmental response network is constructed to anchor the conformation of the active substance, and a long chain with an antioxidant group and a programmed freeze-drying technology are cooperated, so that free radicals are removed, a porous structure is maintained, and the redissolution efficiency, the biological activity and the long-term storage stability of the reagent are improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical reagent preparation technology, specifically to a preparation process for a lyophilized platelet aggregation activating reagent. Background Technology

[0002] In the preparation and storage of lyophilized platelet aggregation activators, traditional physical mixture-type protective agents often face the problem of efficacy failure when dealing with freeze-drying stress. During freeze-drying, single physical mixture systems are prone to phase separation from the active substances, preventing the protective agent from effectively constraining the core protein or reagent. This phase separation makes it impossible for the reagent's structure to be fixed during dehydration, and during the subsequent ice crystal growth stage, it can easily cause irreversible mechanical damage to the spatial conformation of the platelet aggregation activator. Under long-term storage and freeze-drying-reconstitution conditions, the system is susceptible to continuous attack by free radicals, triggering oxidative degradation reactions of the active components.

[0003] The combined effect of these complex factors leads to negative phenomena in the practical application of existing lyophilized reagents, such as abnormally long reconstitution time, low microporosity, and poor retention of biological activity, making it difficult to meet the high standards of long-term stable storage requirements. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a preparation process for a lyophilized platelet aggregation activating agent, comprising the following steps: (1) Disperse water-soluble low molecular weight chitosan and trehalose-6-aldehyde (mass ratio 1:1-1:5) in a first buffer solution with pH 5.0-5.5, add reducing agent and reduce amination at 20-35℃ for 24-48 hours, and dialysis to purify trehalose-grafted chitosan. (2) Dissolve 4-carboxyphenylboronic acid in a second buffer solution at pH 6.0, add an activator to activate it, and obtain an activated solution; the activator is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and the molar ratio of 4-carboxyphenylboronic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1-3):(1-3); add it dropwise to a buffer solution at pH 7.0-8.5 containing the product of step (1), with the mass ratio of 4-carboxyphenylboronic acid to the product being 1:2-1:10, react at 20-35℃ for 12-24 hours, and dialysis to purify phenylboronic acid-trehalose-chitosan; (3) Polyethylene glycol N-hydroxysuccinimide ester with 3,5-di-tert-butyl-4-hydroxyphenyl end group modified was added to a third buffer solution at pH 7.5 containing the product of step (2) for amidation coupling. The mass ratio of the ester to the product of step (2) was 1:1-1:5. The reaction was carried out at 20-35℃ for 12-24 hours, dialyzed and freeze-dried to form a multifunctional lyophilized protective carrier. (4) Dissolve the platelet aggregation activating reagent in the fourth buffer solution at pH 7.8-8.5, add the carrier from step (3) and incubate in the dark to obtain a supramolecular complex binding solution; (5) After the liquid obtained in step (4) is dispensed, it is subjected to programmed freeze-drying to obtain freeze-dried platelet aggregation activator.

[0005] Preferably, in step (1), the degree of deacetylation of the water-soluble low molecular weight chitosan is greater than 90%, and the molecular weight is 5000 Da; the first buffer is an acetate-sodium acetate buffer with pH 5.0-5.5; and the reducing agent is sodium cyanoborohydride.

[0006] Preferably, in step (2), the second buffer is a MES buffer with pH 6.0; the activator is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0007] Preferably, in step (3), the number average molecular weight of polyethylene glycol N-hydroxysuccinimide ester with 3,5-di-tert-butyl-4-hydroxyphenyl end group modified is 2000-5000; the third buffer is PBS buffer with pH 7.5.

[0008] Preferably, in step (4), the platelet aggregation activating agent is selected from one of adenosine diphosphate, collagen, arachidonic acid or thrombin; the fourth buffer is HEPES buffer or Tris-HCl buffer with pH 7.8-8.5.

[0009] Preferably, in step (4), the temperature for incubation under light-protected conditions is 4℃-8℃, and the incubation time is 1-2 hours; the final mass concentration of the multifunctional freeze-dried protective carrier in the supramolecular complex binding solution is 20mg / mL.

[0010] Preferably, in step (5), the programmed freeze drying includes a pre-freezing stage, a primary drying stage, and a secondary drying stage: The pre-freezing process involves cooling the temperature at a rate of 1-2℃ / min down to -45℃ to -50℃ and holding it at that temperature for 2-4 hours. The operation of the first drying stage is as follows: reduce the vacuum degree to below 10 Pa, raise the shelf temperature to -15°C to -5°C at a heating rate of 0.5°C / min, and keep it at that temperature for 10-15 hours; The secondary drying stage is operated as follows: maintain the vacuum degree below 5Pa, raise the shelf temperature to 25℃, keep it warm for 4-6 hours, and then seal it under vacuum.

[0011] Compared with the prior art, the present invention has the following beneficial effects: Through the binding between a specific polymer copolymer carrier and a platelet aggregation activator, a stable structural constraint is formed under dehydration conditions. This can anchor the spatial conformation of the active substance during the freeze-drying stage, thereby preventing the spatial structure of the active substance from collapsing or degrading. During reconstitution, it can automatically dissociate according to changes in the system environment, ensuring that the biological activity of the reagent is completely preserved. A long-chain structure with high flexibility and steric hindrance was introduced into the carrier, and it carries specific antioxidant groups. This structure can interfere with the regular growth of ice crystals during freezing, maintain the uniform porous microstructure of the freeze-dried powder, improve the dissolution efficiency during reagent reconstitution, and continuously remove free radicals generated in the system. This breaks the cascade mechanism of oxidative degradation and improves the anti-aging ability of the product during long-term storage. By combining programmed freeze-drying technology, a variable temperature mechanism is used to induce the slow growth and sublimation of ice crystals. This process utilizes the thermal hysteresis effect to release the stress within the system, avoiding mechanical damage to the porous network framework caused by rapid temperature changes. This drying mechanism and the polymer carrier have a good synergistic effect, ensuring the uniformity of the finished product's appearance and the integrity of its internal structure, and improving the reconstitution efficiency and overall quality of the finished product. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 This is a flowchart of the preparation process of the present invention. Detailed Implementation

[0013] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0014] Please see Figure 1This invention provides a preparation process for a lyophilized platelet aggregation activating agent. Trehalose-6-aldehyde used in this invention can be obtained commercially. The polyethylene glycol N-hydroxysuccinimide ester with 3,5-di-tert-butyl-4-hydroxyphenyl end groups used in this invention can be obtained commercially or custom-made, or prepared by condensing terminal amino polyethylene glycol with 3,5-di-tert-butyl-4-hydroxybenzoic acid, followed by activation of the end groups with succinic anhydride and N-hydroxysuccinimide. Before detailing each embodiment, the present invention establishes a dynamic correlation model between freeze-drying dehydration stress and the spatial conformation of the multifunctional freeze-drying protective carrier in order to determine the optimal control nodes for each reaction stage. The multifunctional freeze-drying protective carrier of the present invention is a polymer copolymer that integrates a water substitution mechanism, a dynamic covalent crosslinking mechanism, and an antioxidant pore-forming mechanism; multiple functional groups are covalently grafted onto the same polymer backbone through chemical reaction; it aims to solve the technical problem that a single physical mixture system is prone to phase separation from active substances under freeze-drying stress, and to generate structural stability through the synergistic constraint of spatial conformation; Furthermore, the phenylboronic acid group in the phenylboronic acid-trehalose-chitosan of this invention is defined as a responsive, dynamically covalently bound group; for activating reagents with cis-diol structures such as ribose rings, based on the principle of phenylboronic acid-cis-diol complexation reaction: ; By utilizing the dynamic covalent borate ester bond formed with platelet aggregation activators in a weakly alkaline environment, or by using the steric hindrance effect provided by the long polymer chain and dense network to deeply encapsulate hydrophobic activators lacking this covalent binding structure, the structure can be fixed during the freeze-drying process. At the same time, by utilizing the physiological pH and competitive free sugar environment of the blood sample, the borate ester bond can be dissociated during reconstitution, forming an environmentally responsive release mechanism.

[0015] Example 1: This embodiment provides a specific implementation method for the preparation process of a lyophilized platelet aggregation activating reagent; the specific preparation process is as follows: Water-soluble low molecular weight chitosan with a deacetylation degree of 91% and a molecular weight of 5000 Da was dispersed in an acetate-sodium acetate buffer solution at pH 5.0 with trehalose-6-aldehyde. In this embodiment, trehalose-6-aldehyde is defined as a monooxidation product in which the primary hydroxyl group at the C6 position of one of the two glucose units in the trehalose molecule is selectively oxidized to an aldehyde group, while the other glucose unit retains its original structure. Sodium cyanoborohydride was added to carry out a reductive amination reaction. The reaction temperature was set at 20°C and the reaction time was 24 hours. After the reaction, trehalose-grafted chitosan was obtained by dialysis purification. In this step, the suitable acidic environment helps the chitosan molecular chain to fully unfold, so that trehalose-6-aldehyde can be efficiently grafted to the amino site, and a rich water-substituted hydrogen bond network is constructed in situ. 4-Carboxyphenylboronic acid was dissolved in MES buffer at pH 6.0. A mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide was added to activate the carboxyl group, obtaining an activation solution. The molar ratio of 4-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was 1:1:1. This activation solution was then added dropwise to a pH 7.0 buffer containing trehalose-grafted chitosan for reaction. The mass ratio of 4-carboxyphenylboronic acid to trehalose-grafted chitosan was 1:2. The reaction temperature was 20°C, and the reaction time was 12 hours. After the reaction, the phenylboronic acid-trehalose-chitosan was purified by dialysis. The phenylboronic acid group introduced in this reaction stage serves as a responsive dynamic covalent bonding group, aiming to utilize it to form a dynamic covalent borate ester bond with a platelet aggregation activator under a weakly alkaline environment, thereby achieving structural fixation during the lyophilization dehydration process. A polyethylene glycol N-hydroxysuccinimide ester with a molecular weight of 2000 and end-group modified with 3,5-di-tert-butyl-4-hydroxyphenyl was added to a PBS buffer containing phenylboronic acid, trehalose, and chitosan at pH 7.5 for amidation coupling reaction at a mass ratio of 1:1. The reaction temperature was 20°C, and the reaction time was 12 hours. After the reaction, the mixture was purified by dialysis and freeze-dried to obtain a multifunctional lyophilized protective carrier. The introduction of this modifying group not only endowed the long polymer chain with flexible steric hindrance, but its hindered phenolic structure could also effectively remove steric hindrance from the system. The free radicals within the body exert an antioxidant mechanism; after obtaining the multifunctional lyophilized protective carrier, a platelet aggregation activating agent selected from adenosine diphosphate was dissolved in HEPES buffer at pH 7.8 to obtain a reagent solution, and the multifunctional lyophilized protective carrier was added to it. The solution was incubated at 4°C for 1 hour under light-protected conditions, with a final carrier concentration of 20 mg / mL, to obtain a supramolecular complex binding solution; during this period, the weakly alkaline environment triggered a specific complexation between phenylboronic acid and the cis-diol structure on the ribose ring of adenosine diphosphate, forming a stable supramolecular complex; The supramolecular complex binding liquid was dispensed and then subjected to programmed freeze-drying. In the pre-freezing stage, the temperature was lowered to -45°C at a rate of 1°C / min and held for 2 hours. In the first drying stage, the vacuum was lowered to 9 Pa, and the shelf temperature was raised to -15°C at a rate of 0.5°C / min and held for 10 hours. In the second drying stage, the vacuum was maintained at 4 Pa, the shelf temperature was raised to 25°C, and held for 4 hours. After the process, the container was sealed under vacuum. Programmed freeze-drying utilizes the thermal hysteresis effect to induce slow growth and sublimation of ice crystals, preventing mechanical damage caused by rapid temperature changes. In this embodiment, the parameter values ​​are taken as the lower limit, mainly to verify the basic performance under low temperature and low proportion modification conditions; the trehalose fragment with a low grafting rate in the multifunctional freeze-dried protective carrier can still form a moderate polymer network structure by combining with the phenylboronic acid group; the low cooling rate used in the pre-freezing stage makes the ice crystal growth relatively slow, which verifies that the hindered phenol-modified polyethylene glycol long chain can still interfere with the regular growth of ice crystals at the basic content, reduce the mechanical damage of ice crystals to the spatial conformation of platelet aggregation activator, and demonstrate the robustness of the basic formulation.

[0016] Example 2: This embodiment provides a specific implementation method for the preparation process of a lyophilized platelet aggregation activating reagent; the specific preparation process is as follows: Water-soluble low molecular weight chitosan with a degree of deacetylation of 95% and a molecular weight of 5000 Da was dispersed in an acetate-sodium acetate buffer solution at pH 5.2 at a mass ratio of 1:3. A reductive amination reaction was then carried out with sodium cyanoborohydride at a reaction temperature of 28°C for 36 hours. After the reaction, the chitosan was purified by dialysis. In this step, the appropriate grafting ratio ensured that the polymer backbone had balanced hydrophilic nodes. 4-Carboxyphenylboronic acid was dissolved in MES buffer at pH 6.0, and the carboxyl groups were activated with an activator to obtain an activated solution. The molar ratio of 4-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was 1:2:2. This activated solution was added dropwise to a pH 7.5 buffer containing trehalose-grafted chitosan for reaction. The mass ratio of 4-carboxyphenylboronic acid to trehalose-grafted chitosan was 1:6. The reaction temperature was 28℃, and the reaction time was 18 hours. After the reaction, the phenylboronic acid-trehalose-chitosan was obtained by dialysis purification. During this process, the number of covalently bound phenylboronic acid groups was moderate, providing sufficient binding sites for subsequent dynamic crosslinking. A polyethylene glycol N-hydroxysuccinimide ester with a molecular weight of 3500 and 3,5-di-tert-butyl-4-hydroxyphenyl end groups was added to a PBS buffer containing phenylboronic acid, trehalose, and chitosan at pH 7.5 for amidation coupling reaction at a mass ratio of 1:3. The reaction temperature was 28°C, and the reaction time was 18 hours. After the reaction, the mixture was purified by dialysis and freeze-dried to obtain a multifunctional lyophilized protective vector. After obtaining the multifunctional lyophilized protective vector, a platelet aggregation activating agent selected from collagen was dissolved in Tris-HCl buffer at pH 8.0 to obtain a reagent solution. The multifunctional lyophilized protective vector was added to the solution, and the mixture was incubated at 6°C for 1.5 hours under light-protected conditions. The final mass concentration of the vector was 20 mg / mL, resulting in a supramolecular complex binding solution. The supramolecular complex binding liquid was dispensed and then subjected to programmed freeze-drying. In the pre-freezing stage, the temperature was lowered to -48°C at a rate of 1.5°C / min and held for 3 hours. In the first drying stage, the vacuum was lowered to 8 Pa and the shelf temperature was raised to -10°C at a rate of 0.5°C / min and held for 12 hours. In the second drying stage, the vacuum was maintained at 3 Pa and the shelf temperature was raised to 25°C and held for 5 hours. After the process was completed, the container was sealed under vacuum. In this embodiment, the parameter values ​​are taken as the median values ​​under normal operating conditions; the appropriate ratio of trehalose-6-aldehyde and 4-carboxyphenylboronic acid provides a balanced hydrogen bond network and covalent binding sites; the hindered phenolic groups fully exert their scavenging effect on free radicals in the system; the supramolecular complex binding solution exhibits stable sublimation and desorption characteristics in the primary and secondary drying stages; and the lyophilized platelet aggregation activator exhibits a uniform porous microstructure, demonstrating the excellent synergistic effect of the proportion of each component in the preparation process and the lyophilization procedure.

[0017] Example 3: This embodiment provides a specific implementation method for the preparation process of a lyophilized platelet aggregation activating reagent; the specific preparation process is as follows: Water-soluble low molecular weight chitosan with a deacetylation degree of 99% and a molecular weight of 5000 Da was dispersed in an acetate-sodium acetate buffer at pH 5.5 with trehalose-6-aldehyde, defined as a specific isomer in which the C6 hydroxyl group of a single glucose residue in the trehalose disaccharide backbone is converted into an aldehyde group. Sodium cyanoborohydride was added for a reductive amination reaction at a reaction temperature of 35°C for 48 hours. After the reaction, the trehalose-grafted chitosan was obtained by dialysis purification. In this step, the extremely high degree of deacetylation combined with the high proportion of trehalose monoaldehyde is intended to maximize the density of water-substituted groups on the polymer backbone. 4-Carboxyphenylboronic acid was dissolved in MES buffer at pH 6.0, and the carboxyl groups were activated by adding an activator to obtain an activated solution. The molar ratio of 4-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was 1:3:3. The activated solution was added dropwise to a pH 8.5 buffer containing trehalose-grafted chitosan for reaction. The mass ratio of 4-carboxyphenylboronic acid to trehalose-grafted chitosan was 1:10. The reaction temperature was 35℃ and the reaction time was 24 hours. After the reaction, the phenylboronic acid-trehalose-chitosan was obtained by dialysis purification. A polyethylene glycol N-hydroxysuccinimide ester with a molecular weight of 5000 and 3,5-di-tert-butyl-4-hydroxyphenyl end groups was added to a PBS buffer containing phenylboronic acid, trehalose, and chitosan at pH 7.5 for amidation coupling reaction at a mass ratio of 1:5. The reaction temperature was 35℃ and the reaction time was 24 hours. After the reaction, the mixture was purified by dialysis and freeze-dried to obtain a multifunctional lyophilized protective carrier. After obtaining the multifunctional lyophilized protective carrier, a platelet aggregation activating agent selected from arachidonic acid was dissolved in HEPES buffer at pH 8.5 to obtain a reagent solution. The multifunctional lyophilized protective carrier was added to the solution and incubated at 8℃ for 2 hours under light-protected conditions. The final mass concentration of the carrier was 20 mg / mL, resulting in a supramolecular complex binding solution. The supramolecular complex binding liquid was dispensed and then subjected to programmed freeze drying. In the pre-freezing stage, the temperature was lowered to -50°C at a rate of 2°C / min and held for 4 hours. In the first drying stage, the vacuum was reduced to 7 Pa and the shelf temperature was increased to -5°C at a rate of 0.5°C / min and held for 15 hours. In the second drying stage, the vacuum was maintained at 2 Pa and the shelf temperature was increased to 25°C and held for 6 hours. After the process was completed, the container was sealed under vacuum. In this embodiment, the parameter values ​​are taken as upper limits to verify the performance under high grafting rate and high temperature conditions. The high-density trehalose grafted chitosan endows the system with a rich polyhydroxy structure, maintaining the absolute stability of the water substitution mechanism under dehydration. The high molecular weight polyethylene glycol long chain increases steric hindrance, deeply embedding and protecting the hydrophobic platelet aggregation activator. This design demonstrates the excellent structural stability of the multifunctional freeze-dried protective carrier in the face of extreme oxidation risks and dehydration stress environments.

[0018] Example 4: This embodiment provides a specific implementation method for the preparation process of a lyophilized platelet aggregation activating reagent; the specific preparation process is as follows: Water-soluble low molecular weight chitosan with a degree of deacetylation of 93% and a molecular weight of 5000 Da was dispersed with trehalose-6-aldehyde at a mass ratio of 1:2 in an acetate-sodium acetate buffer solution at pH 5.1. Sodium cyanoborohydride was added to carry out a reducing amination reaction. The reaction temperature was set at 24℃ and the reaction time was 30 hours. After the reaction was completed, trehalose-grafted chitosan was obtained by dialysis purification. 4-Carboxyphenylboronic acid was dissolved in MES buffer at pH 6.0, and the carboxyl groups were activated with an activator to obtain an activated solution. The molar ratio of 4-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was 1:1.5:1.5. The activated solution was added dropwise to a pH 7.2 buffer containing trehalose-grafted chitosan for reaction. The mass ratio of 4-carboxyphenylboronic acid to trehalose-grafted chitosan was 1:4. The reaction temperature was 24℃, and the reaction time was 15 hours. After the reaction, the phenylboronic acid-trehalose-chitosan was obtained by dialysis purification. A polyethylene glycol N-hydroxysuccinimide ester with a molecular weight of 2500 and 3,5-di-tert-butyl-4-hydroxyphenyl end groups was added to a PBS buffer containing phenylboronic acid, trehalose, and chitosan at pH 7.5 for amidation coupling reaction at a mass ratio of 1:2. The reaction temperature was 24°C and the reaction time was 15 hours. After the reaction, the mixture was purified by dialysis and freeze-dried to obtain a multifunctional lyophilized protective vector. After obtaining the multifunctional lyophilized protective vector, a platelet aggregation activating agent selected from thrombin was dissolved in Tris-HCl buffer at pH 7.9 to obtain a reagent solution. The multifunctional lyophilized protective vector was added to the solution, and the mixture was incubated at 5°C for 1.2 hours under light-protected conditions. The final mass concentration of the vector was 20 mg / mL, resulting in a supramolecular complex binding solution. The supramolecular complex binding liquid was dispensed and then subjected to programmed freeze-drying. In the pre-freezing stage, the temperature was lowered to -46°C at a rate of 1.2°C / min and held for 2.5 hours. In the first drying stage, the vacuum was reduced to 8 Pa and the shelf temperature was increased to -12°C at a rate of 0.5°C / min and held for 11 hours. In the second drying stage, the vacuum was maintained at 4 Pa ​​and the shelf temperature was increased to 25°C and held for 4.5 hours. After the process was completed, the container was sealed under vacuum. In this embodiment, a combination of parameters with relatively low values ​​was used, and thrombin was selected as the platelet aggregation activator. The grafting reaction of water-soluble low molecular weight chitosan with each functional group was carried out under mild conditions, and the resulting multifunctional freeze-dried protective carrier had a suitable spatial network structure. The cooling rate of the pre-freezing stage and the heating rate of the primary drying stage were precisely matched, so that the ice crystal size and sublimation channel distribution were reasonable, which helped to maintain the protein spatial conformation of thrombin and greatly reduced the degradation of activity.

[0019] Example 5: This embodiment provides a specific implementation method for the preparation process of a lyophilized platelet aggregation activating reagent; the specific preparation process is as follows: Water-soluble low molecular weight chitosan with a degree of deacetylation of 97% and a molecular weight of 5000 Da was dispersed with trehalose-6-aldehyde at a mass ratio of 1:4 in an acetate-sodium acetate buffer solution at pH 5.4. Sodium cyanoborohydride was added to carry out a reducing amination reaction. The reaction temperature was set at 32℃ and the reaction time was 42 hours. After the reaction was completed, trehalose-grafted chitosan was obtained by dialysis purification. 4-Carboxyphenylboronic acid was dissolved in MES buffer at pH 6.0, and the carboxyl groups were activated by adding an activator to obtain an activated solution. The molar ratio of 4-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide was 1:2.5:2.5. The activated solution was added dropwise to a pH 8.0 buffer containing trehalose-grafted chitosan for reaction. The mass ratio of 4-carboxyphenylboronic acid to trehalose-grafted chitosan was 1:8. The reaction temperature was 32℃, and the reaction time was 21 hours. After the reaction, the phenylboronic acid-trehalose-chitosan was obtained by dialysis purification. Polyethylene glycol N-hydroxysuccinimide ester with a molecular weight of 4000 and terminally modified with 3,5-di-tert-butyl-4-hydroxyphenyl was added to a PBS buffer containing phenylboronic acid, trehalose, and chitosan at pH 7.5 for amidation coupling reaction at a mass ratio of 1:4. The reaction temperature was 32℃ and the reaction time was 21 hours. After the reaction, the mixture was purified by dialysis and freeze-dried to obtain a multifunctional lyophilized protective vector. After obtaining the multifunctional lyophilized protective vector, a platelet aggregation activating agent selected from adenosine diphosphate was dissolved in HEPES buffer at pH 8.2 to obtain a reagent solution. The multifunctional lyophilized protective vector was added to the solution and incubated at 7℃ for 1.8 hours under light-protected conditions. The final mass concentration of the vector was 20 mg / mL, and a supramolecular complex binding solution was obtained. The supramolecular complex binding liquid was dispensed and then subjected to programmed freeze drying. In the pre-freezing stage, the temperature was lowered to -49°C at a rate of 1.8°C / min and held for 3.5 hours. In the first drying stage, the vacuum was lowered to 8 Pa and the shelf temperature was raised to -8°C at a rate of 0.5°C / min and held for 14 hours. In the second drying stage, the vacuum was maintained at 3 Pa and the shelf temperature was raised to 25°C and held for 5.5 hours. After the process was completed, the container was sealed under vacuum. In this embodiment, a combination of parameters with relatively high values ​​was used; the higher reaction temperature and longer reaction time significantly improved the grafting conversion rate of phenylboronic acid-trehalose-chitosan; the ribocyclic structure of the platelet aggregation activator reacted fully with 4-carboxyphenylboronic acid to form a dense dynamic covalent system; the deep low-temperature pre-freezing and long-term vacuum drying in the programmed freeze-drying process ensured that the bound water was fully removed, which greatly improved the hydrolysis resistance of the freeze-dried platelet aggregation activator during long-term storage.

[0020] Comparative Example 1: This comparative example provides a conventional lyophilization reagent preparation process, the parameters of which are consistent with those of Example 2. The difference is that: instead of synthesizing a multifunctional lyophilization protective carrier, 5% trehalose and 1% bovine serum albumin are directly added to the reagent solution as physical mixing protective agents. The other reagent types and programmed lyophilization process parameters are the same as those of Example 2. This comparative example serves as a blank control for conventional methods and is used to verify the technical effect of the polymer copolymer protective agent of this invention in preventing phase separation.

[0021] Comparative Example 2: This comparative example provides a lyophilization reagent preparation process with parameters consistent with Example 2. The difference is that in step 2, 4-carboxyphenylboronic acid and activator are not added, the synthesis step of phenylboronic acid-trehalose-chitosan is skipped, and the trehalose-grafted chitosan obtained in step 1 is directly used for the reaction in step 3. This comparative example lacks a responsive dynamic covalent binding mechanism, which is used to verify the necessity of phenylboronic acid groups locking the spatial conformation of active substances under lyophilization dehydration stress.

[0022] Comparative Example 3: This comparative example provides a lyophilization reagent preparation process with parameters consistent with those of Example 2. The difference is that in step 3, polyethylene glycol N-hydroxysuccinimide ester with 3,5-di-tert-butyl-4-hydroxyphenyl end-group modification was not added for the reaction. This comparative example lacks the antioxidant and pore-forming mechanisms and is used to verify the necessity of hindered phenolic long chains in scavenging free radicals and interfering with the regular growth of ice crystals.

[0023] Comparative Example 4: This comparative example provides a lyophilized reagent preparation process with parameters consistent with Example 2. The difference is that in step 5, instead of using a programmed freeze-drying process, the supramolecular complex binding liquid is dispensed and directly placed in an ultra-low temperature freezer at -80°C for 2 hours, and then transferred to a conventional freeze dryer for continuous drying at a constant temperature of 25°C and a vacuum of 10 Pa for 24 hours. This comparative example is used to verify the necessity of controlling the pre-freezing cooling rate and the multi-stage heating mechanism in programmed freeze-drying to prevent mechanical damage to ice crystals and structural collapse.

[0024] Verification experiment: To further demonstrate the synergistic effect of the multifunctional freeze-dried protective carrier and the programmed freeze-drying process of the present invention, a verification experiment was set up to test the performance of the freeze-dried platelet aggregation activating reagents prepared in Examples 1-5 and Comparative Examples 1-4. At the same time, in order to verify the critical point effect and necessity of the final mass concentration of the multifunctional freeze-dried protective carrier in step (4) being preferably 20 mg / mL, Comparative Examples 5 and 6 were set up in addition. The preparation process parameters of Comparative Example 5 were consistent with those of Example 2, except that the final mass concentration of the multifunctional lyophilized protective carrier in step (4) was 10 mg / mL. The preparation process parameters of Comparative Example 6 were consistent with those of Example 2, except that the final mass concentration of the multifunctional lyophilized protective carrier in step (4) was 30 mg / mL. The test aimed to reveal the key role of dynamic covalent crosslinking mechanism and antioxidant pore-forming mechanism in resolution efficiency and long-term stability, and to verify the scientific validity of the fixed concentration parameters.

[0025] Test method description: Reconstitution time and porosity determination: Samples from each group were taken, and a standard volume of purified water was added. The time required from the addition of water to the complete dissolution of the lyophilized powder into a clear solution was recorded. The micropores of the lyophilized powder were determined using a mercury porosimeter to characterize the structural integrity. Bioactivity retention determination: The effective concentration of platelet aggregation activator in the reconstituted solution was determined by high performance liquid chromatography, and the retention rate relative to the initial concentration before lyophilization was calculated. Accelerated aging activity retention rate determination: Each group of samples was sealed and placed in a constant temperature and humidity chamber at 37℃ and 75% relative humidity for 30 days for accelerated aging. Subsequently, the same high performance liquid chromatography method was used to determine its bioactivity retention rate. All tests were independently repeated 3 times, and the average value of the data was taken.

[0026] Specific testing process: During the testing process, the ambient temperature was strictly controlled at 25±1℃. When performing the reconstitution test, the reconstitution solvent was injected at a constant speed along the tube wall using a pipette to avoid interference from mechanical vibration. In the liquid chromatography detection of activity retention rate, a C18 reversed-phase column was used. The mobile phase was adaptively adjusted according to different platelet aggregation activating reagents, such as adenosine diphosphate or thrombin. The detection wavelength was set to the maximum absorption peak of the corresponding substance to ensure the accuracy and reproducibility of quantitative analysis.

[0027] Table 1 Performance test data of Examples 1-5 and Comparative Examples 1-6 Test Group Reconstitution time (s) Porosity (%) Bioactivity retention rate (%) Activity retention rate (%) after 30 days of accelerated aging at 37℃ Example 1 6.5 82.1 89.5 84.3 Example 2 2.5 91.2 98.2 96.8 Example 3 5.8 84.6 91.4 86.7 Example 4 3.0 90.1 97.1 94.3 Example 5 2.6 90.8 98.0 96.2 Comparative Example 1 42.5 65.3 82.4 55.6 Comparative Example 2 15.3 78.6 86.7 68.2 Comparative Example 3 28.6 72.4 94.5 72.4 Comparative Example 4 35.2 68.9 80.1 61.3 Comparative Example 5 18.4 76.5 85.2 70.4 Comparative Example 6 22.1 71.8 88.6 74.5 As can be seen from the test data in Table 1, the preparation process of the freeze-dried platelet aggregation activating reagent of the present invention has significantly improved the reconstitution efficiency and long-term stability. Significant effects of dynamic covalent crosslinking and spatial network embedding: Comparing Example 2 and Comparative Example 2, it can be seen that after introducing phenylboronic acid groups, the bioactivity retention rate increased from 86.7% to 98.2%, and the accelerated aging activity retention rate jumped from 68.2% to 96.8%. This indicates that for reagents containing cis-diol structures, the dynamic covalent borate ester bond formed by phenylboronic acid and platelet aggregation activating reagents plays a strong structural anchoring role under freeze-drying dehydration stress. For activating reagents such as arachidonic acid that do not have this covalent binding site, a dense physical embedding network is formed through the modified polymer skeleton. Both can effectively prevent the spatial conformational collapse and degradation of active substances. Significant impact of process parameter range and determination of optimal range: Comparison of Examples 1, 2 and 3 shows that Example 2 is significantly better than Examples 1 and 3 in terms of reconstitution time and bioactivity retention rate; This indicates that there is a significant parabolic performance change within the parameter range such as raw material ratio. A moderate grafting ratio can provide the best hydrogen bond network and dynamic crosslinking sites, while values ​​that are too low or too high will lead to a decrease in protective performance or excessive steric hindrance. Furthermore, the final mass concentration of the multifunctional lyophilized protective carrier in step (4) was fixed at 20 mg / mL. Based on the data from Comparative Examples 5 and 6, it can be seen that when the concentration is below 20 mg / mL, the reconstitution time is extended to 18.4 s, and the accelerated aging activity retention rate drops to 70.4%, indicating that low concentration cannot achieve complete encapsulation and effective protection of the activating reagent; when the concentration is above 20 mg / mL, the reconstitution time is extended to 22.1 s, and the porosity drops to 71.8%, indicating that high concentration easily causes polymer chain entanglement and phase separation, hindering the ice crystal sublimation channel; this verifies the protective effect of 20 mg / mL as the critical saturation concentration, so it is used as a fixed parameter to ensure the best implementation effect; Synergistic effect of antioxidant and pore-forming mechanism: Compared with Comparative Example 3, the absence of hindered phenol-modified polyethylene glycol long chains led to an increase in resolution time from 2.5s to 28.6s and a decrease in porosity from 91.2% to 72.4%. This confirms that the introduction of flexible long chains not only increases steric hindrance to maintain the porous network structure, but its hindered phenol groups also effectively scavenge free radicals during the aging process, interrupting the cascade mechanism of oxidative degradation, thereby ensuring the long-term stability of the reagent. Anti-collapse function of programmed freeze-drying: Compared with Comparative Example 4, the porosity of the sample dried by non-programmed rapid freeze-drying was significantly reduced to 68.9%, and the reconstitution time was significantly longer. This shows that programmed freeze-drying with gradient temperature utilizes the thermal hysteresis effect to effectively release internal stress, prevent the rapid growth of ice crystals from mechanically tearing the polymer microcapsule network, and solve the problem of kinetic mismatch. This invention solves the technical defect of phase separation in physical mixing systems by dual coupling of multifunctional freeze-drying protective carrier and programmed freeze-drying, and achieves the expected technical indicators.

[0028] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A preparation process for a lyophilized platelet aggregation activating agent, characterized in that, Includes the following steps: (1) Disperse water-soluble low molecular weight chitosan and trehalose-6-aldehyde (mass ratio 1:1-1:5) in a first buffer solution with pH 5.0-5.5, add reducing agent and reduce amination at 20-35℃ for 24-48 hours, and dialysis to purify trehalose-grafted chitosan. (2) Dissolve 4-carboxyphenylboronic acid in a second buffer solution at pH 6.0, add an activator to activate it, and obtain an activated solution; the activator is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and the molar ratio of 4-carboxyphenylboronic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1-3):(1-3); add it dropwise to a buffer solution at pH 7.0-8.5 containing the product of step (1), with the mass ratio of 4-carboxyphenylboronic acid to the product being 1:2-1:10, react at 20-35℃ for 12-24 hours, and dialysis to purify phenylboronic acid-trehalose-chitosan; (3) Polyethylene glycol N-hydroxysuccinimide ester with 3,5-di-tert-butyl-4-hydroxyphenyl end group modified was added to a third buffer solution at pH 7.5 containing the product of step (2) for amidation coupling. The mass ratio of the ester to the product of step (2) was 1:1-1:

5. The reaction was carried out at 20-35℃ for 12-24 hours, dialyzed and freeze-dried to form a multifunctional lyophilized protective carrier. (4) Dissolve the platelet aggregation activating reagent in the fourth buffer solution at pH 7.8-8.5, add the carrier from step (3) and incubate in the dark to obtain a supramolecular complex binding solution; (5) After the liquid obtained in step (4) is dispensed, it is subjected to programmed freeze-drying to obtain freeze-dried platelet aggregation activator.

2. The preparation process of the lyophilized platelet aggregation activating reagent according to claim 1, characterized in that, In step (1), the degree of deacetylation of the water-soluble low molecular weight chitosan is greater than 90%, and the molecular weight is 5000 Da; the first buffer is an acetate-sodium acetate buffer with a pH of 5.0-5.5; and the reducing agent is sodium cyanoborohydride.

3. The preparation process of the lyophilized platelet aggregation activating reagent according to claim 1, characterized in that, In step (2), the second buffer is a pH 6.0 MES buffer; the activator is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

4. The preparation process of the lyophilized platelet aggregation activating reagent according to claim 1, characterized in that, In step (3), the number average molecular weight of polyethylene glycol N-hydroxysuccinimide ester with 3,5-di-tert-butyl-4-hydroxyphenyl end groups is 2000-5000; the third buffer is PBS buffer at pH 7.

5.

5. The preparation process of the lyophilized platelet aggregation activating reagent according to claim 1, characterized in that, In step (4), the platelet aggregation activating agent is selected from one of adenosine diphosphate, collagen, arachidonic acid or thrombin; the fourth buffer is HEPES buffer or Tris-HCl buffer with pH 7.8-8.

5.

6. The preparation process of a lyophilized platelet aggregation activating reagent according to claim 1 or 5, characterized in that, In step (4), the incubation temperature under light-protected conditions is 4℃-8℃, and the incubation time is 1-2 hours; the final mass concentration of the multifunctional freeze-dried protective carrier in the supramolecular complex binding solution is 20mg / mL.

7. The preparation process of the lyophilized platelet aggregation activating reagent according to claim 1, characterized in that, In step (5), the programmed freeze drying includes a pre-freezing stage, a primary drying stage, and a secondary drying stage: The pre-freezing process involves cooling the temperature at a rate of 1-2℃ / min down to -45℃ to -50℃ and holding it at that temperature for 2-4 hours. The operation of the first drying stage is as follows: reduce the vacuum degree to below 10 Pa, raise the shelf temperature to -15°C to -5°C at a heating rate of 0.5°C / min, and keep it at that temperature for 10-15 hours; The secondary drying stage is operated as follows: maintain the vacuum degree below 5Pa, raise the shelf temperature to 25℃, keep it warm for 4-6 hours, and then seal it under vacuum.

Images