Microcrystal sustained release preparation and preparation method thereof
The microcrystalline sustained-release formulation with edaravone rod-shaped microcrystalline double-shell structure solves the problems of drug difficulty in accurately reaching the target tissue and uncontrollable release rate in the treatment of inner ear diseases, and achieves long-term sustained release and biocompatible therapeutic effects for inner ear diseases.
Patent Information
- Application Number
- CN202511271866.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-08
- Publication Date
- 2025-12-26
AI Technical Summary
In current treatments for inner ear diseases, local drug delivery is inefficient, drugs are difficult to accurately reach the target tissue, the release rate is uncontrollable, biocompatibility is poor, and traditional preparations have weak adhesion to the middle ear mucosa, resulting in poor treatment effects and potential complications.
Using edaravone rod-shaped microcrystals as the core, the double-shell structure consists of an inner layer of polyethylene glycol-polylysine copolymer and an outer layer of hyaluronic acid modified with CD44 targeting peptide. The microcrystal sustained-release formulation is formed through cross-linking and curing to ensure drug targeting and sustained release.
It significantly improves the binding efficiency of drugs to inner ear target cells, prolongs the drug residence time in the inner ear, reduces the need for repeated injections, reduces the risk of inflammation, and achieves long-lasting release and biocompatibility.
Smart Images
Figure CN121197103A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of sustained-release formulation technology, specifically to a microcrystalline sustained-release formulation and its preparation method. Background Technology
[0002] The clinical treatment of inner ear diseases has long faced the problem of low efficiency in local drug delivery. Existing systemic drug delivery methods are limited by the blood-labyrinthine barrier, making it difficult for drugs to reach the target tissues of the inner ear, resulting in limited efficacy and a high risk of systemic side effects. Traditional local drug delivery (such as intratympanic injection of ordinary solutions) requires frequent injections to maintain efficacy because the drugs have a short retention time in the middle ear cavity and are easily lost through the Eustachian tube, which not only increases patient suffering but may also cause complications such as middle ear infections.
[0003] To address these issues, existing technologies attempt to prolong drug retention time using carriers such as gels and polymer microspheres. However, these technologies still suffer from drawbacks, including insufficient targeting, difficulty in precisely reaching the round window membrane and inner ear hair cells, uncontrollable release rates, initial burst release leading to excessively high local concentrations or insufficient release later, and poor biocompatibility. Furthermore, most formulations are spherical, exhibiting weak adhesion to the middle ear mucosa and round window membrane, further reducing drug delivery efficiency. Therefore, developing an inner ear local delivery system that combines active targeting, long-lasting sustained release, and excellent biocompatibility is crucial for improving the treatment outcomes of inner ear diseases. Summary of the Invention
[0004] This invention addresses the technical problems existing in the prior art by providing a microcrystalline sustained-release formulation and its preparation method.
[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a microcrystalline sustained-release formulation and its preparation method, comprising the following steps: the sustained-release formulation comprises a core composed of edaravone rod-shaped microcrystals, and has 2-4 shells composed of bilayers, wherein the shells comprise at least one polyethylene glycol-polylysine copolymer inner layer and at least one hyaluronic acid outer layer modified with CD44 targeting peptide.
[0006] In a preferred embodiment, the edaravone rod-shaped microcrystals have an apparent shape of cylindrical rods, with a length of 15-30 μm and a diameter of 2-5 μm.
[0007] In a preferred embodiment, the total size of the sustained-release formulation is 18-35 μm.
[0008] This invention also provides a method for preparing a microcrystalline sustained-release formulation, comprising: S101. Dissolve edaravone active pharmaceutical ingredient to prepare a solvent-free system; S102, edaravone DMSO solution was added dropwise to a non-solvent system for crystal induction and growth; S103, the suspension of induction and growth was purified and dried, and modified with polyethylene glycol-polylysine copolymer inner layer and hyaluronic acid outer layer with CD44 targeting peptide; S104, after cross-linking and curing, the sustained-release formulation is formed.
[0009] In a preferred embodiment, the pre-prepared non-solvent system for dissolving edaravone active pharmaceutical ingredient includes: Edaravone active pharmaceutical ingredient was dissolved in DMSO at 37°C, 200-500 rpm, and 30-60 min with stirring. The concentration of the edaravone DMSO solution was controlled at 9-10 mg / mL. Furthermore, heating and stirring were used to accelerate the dissolution of the active pharmaceutical ingredient, ensuring a homogeneous solution and avoiding undissolved particles. The 9-10 mg / mL concentration provided a suitable supersaturation for subsequent crystallization; too high a concentration could lead to agglomeration, while too low a concentration would result in irregular crystal forms. Tween 80 at a final concentration of 0.05-0.1 g / L was added to ultrapure water, and a non-solvent solution was formed under magnetic stirring at 37°C and 400-800 rpm. Furthermore, Tween 80 forms a molecular film in the non-solvent, reducing the interfacial tension between DMSO and water and providing a stable interface for microcrystal growth; the 37°C temperature is consistent with the subsequent crystallization temperature, avoiding the influence of temperature fluctuations on the crystal form.
[0010] In a preferred embodiment, the edaravone DMSO solution is added dropwise to a non-solvent system for crystal induction and growth, including: Add edaravone DMSO solution dropwise to the non-solvent solution at a rate of 0.8 ml / min using a constant flow pump, while maintaining magnetic stirring at 400-800 rpm. After the addition is complete, continue stirring for 3-6 hours until a white suspension is formed.
[0011] Furthermore, a slow drop rate of 0.8 mL / min controls the diffusion rate of DMSO, avoiding excessive local supersaturation that could lead to microcrystal aggregation; moderate shear force is generated to induce edaravone molecules to grow axially and form rod-shaped structures; stirring for 3-6 hours ensures complete crystallization and reduces the proportion of amorphous particles.
[0012] In a preferred embodiment, the induction and growth suspension is purified and dried, and then modified with a polyethylene glycol-polylysine copolymer inner layer and a hyaluronic acid outer layer containing a CD44-targeting peptide, including: The suspension was transferred to centrifuge tubes and centrifuged at 8000-10000 rpm for 15-40 min at 4°C. The supernatant was discarded, and the precipitate was washed with ultrapure water at least 3 times, adding ultrapure water each time. After vortexing and resuspending, the precipitate was centrifuged again and finally freeze-dried at -50°C and 0.1-0.5 mbar for 24-48 h to obtain white, fluffy edaravone rod-shaped microcrystalline powder.
[0013] Furthermore, centrifugation at 4°C avoids the dissolution of microcrystals due to temperature rise, while centrifugation at 8000-10000 rpm ensures complete precipitation of microcrystals; washing with ultrapure water removes residual DMSO and free Tween 80, reducing the risk of toxicity; and freeze-drying avoids damage to the crystal form by high temperature, maintaining the integrity of the rod-shaped structure.
[0014] After obtaining edaravone rod-shaped microcrystalline powder, the morphology was observed by scanning electron microscopy, the size was measured by laser particle size analyzer, and the crystal form was verified by X-ray diffraction. The rod-shaped structure was intact, with a length of 15-30 μm and a diameter of 2-5 μm. The XRD characteristic peaks were consistent with type A edaravone, with no crystal form transformation and a dispersion of ≤15%.
[0015] In a preferred embodiment, it further includes: Weigh out edaravone rod-shaped microcrystals, add them to a polyethylene glycol-polylysine copolymer solution, and place them on a shaker at 150-300 rpm for 20-30 minutes at room temperature to form solution one; Centrifuge at 8000-10000 rpm for 15-30 min at 4℃, discard the supernatant, and wash the precipitate with PBS at least twice to obtain microparticles a; In the above process, the polyethylene glycol-polylysine copolymer solution is adsorbed onto the microcrystal surface due to the positively charged amino groups and the weakly negatively charged electrostatic effect of the Tween 80 residue. To ensure sufficient adsorption, centrifugation and washing are used to remove unbound free polyethylene glycol-polylysine copolymer, exposing polyethylene glycol on the surface, reducing van der Waals forces between microcrystals, improving dispersibility, and preventing agglomeration during the modification process. Resuspend particle a in hyaluronic acid solution containing CD44 targeting peptide, shake on a shaker at 150-300 rpm for 15-30 min at room temperature to form solution two, centrifuge at 8000-10000 rpm for 10-15 min at 4℃, discard the supernatant, and wash the precipitate at least twice with PBS to obtain particle b. Furthermore, the carboxyl group of hyaluronic acid containing CD44 targeting peptides carries a negative charge and combines with the positively charged amino group of polyethylene glycol-polylysine copolymer through electrostatic interaction to form a stable bilayer. The CD44 targeting peptides are exposed on the surface, providing recognition sites for subsequent active targeting of inner ear hair cells. Repeat the operation at least twice to obtain particle c.
[0016] Preferably, the three bilayers have moderate thickness, with a total shell thickness of about 1-2 μm, which ensures the target peptide density while avoiding excessively large formulation size, with the total size controlled at 18-35 μm; and the multilayer structure enhances shell stability, preventing the target molecule from falling off during delivery.
[0017] In a preferred embodiment, the method further includes: the cross-linked and cured sustained-release formulation molding process, which includes: resuspending the microparticles c in 10 mL of 1% genipin aqueous solution to make the microparticle concentration 5 mg / mL, and letting it stand at room temperature in the dark for 2 hours, during which time it is gently vortexed once every 30 minutes. Genipin undergoes a condensation reaction with the hydroxyl groups of HA and the amino groups of PLL to form a covalent cross-linked network, which enhances the mechanical strength of the shell. A 2-hour reaction time ensures sufficient cross-linking; light protection is necessary to prevent genipin from oxidizing and becoming ineffective. A particle concentration of 5 mg / mL ensures sufficient contact between the crosslinking agent and the shell material; Centrifuge at 4000-8000 rpm for 15 min at 4℃, discard the supernatant, wash the precipitate three times with ultrapure water to remove residual genipin, and freeze-dry under vacuum for 12-24 h to obtain a white powdery microcrystalline sustained-release formulation.
[0018] The beneficial effects of this invention are: the shell-modified CD44-targeting peptide can specifically recognize CD44 receptors on the surface of inner ear hair cells, significantly improving the binding efficiency of the formulation to the round window membrane and target cells, reducing the diffusion of the drug to non-target tissues, and increasing the local drug concentration. The multi-layered shell structure, combined with the curing effect of the natural cross-linking agent genipin, can regulate the drug release rate, avoid the problems of burst release or rapid clearance of traditional formulations, prolong the effective duration of drug action in the inner ear, and reduce the need for repeated injections. The shell materials are polyethylene glycol-polylysine copolymer and hyaluronic acid, both of which have good biocompatibility. The cross-linking agent is genipin of natural origin, avoiding the potential toxicity of chemically synthesized cross-linking agents and reducing the risk of inflammation in the middle and inner ear tissues. Attached Figure Description
[0019] Figure 1 This is a flowchart of the present invention; Figure 2 This is a schematic diagram of the release rate curves of the experimental group and the control group in the example. Detailed Implementation
[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0021] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the stated features. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.
[0022] In the description of this application, the term "for example" is used to mean "used as an example, illustration, or description." Any embodiment described as "for example" in this application is not necessarily to be construed as being more preferred or advantageous than other embodiments. The following description is provided to enable any person skilled in the art to make and use the invention. Details are set forth in the following description for purposes of explanation. It should be understood that those skilled in the art will recognize that the invention can be made without using these specific details. In other instances, well-known structures and processes will not be described in detail to avoid obscuring the description of the invention with unnecessary detail. Therefore, the invention is not intended to be limited to the embodiments shown, but is consistent with the broadest scope of the principles and features disclosed in this application.
[0023] This embodiment provides: a microcrystalline sustained-release formulation, the sustained-release formulation comprising a core composed of edaravone rod-shaped microcrystals, having 2-4 bilayer shells, the shells comprising at least one polyethylene glycol-polylysine copolymer inner layer, and at least one hyaluronic acid outer layer modified with CD44 targeting peptide.
[0024] Edaravone rod-shaped microcrystals have an apparent cylindrical rod shape, with a length of 15-30 μm and a diameter of 2-5 μm.
[0025] The total size of the sustained-release formulation is 18-35 μm.
[0026] This invention also provides a method for preparing a microcrystalline sustained-release formulation, comprising: S101. Dissolve edaravone active pharmaceutical ingredient to prepare a solvent-free system; include: Edaravone active pharmaceutical ingredient was dissolved in DMSO at 37°C, 200-500 rpm, and 30-60 min with stirring. The concentration of the edaravone DMSO solution was controlled at 9-10 mg / mL. Furthermore, heating and stirring were used to accelerate the dissolution of the active pharmaceutical ingredient, ensuring a homogeneous solution and avoiding undissolved particles. The 9-10 mg / mL concentration provided a suitable supersaturation for subsequent crystallization; too high a concentration could lead to agglomeration, while too low a concentration would result in irregular crystal forms. Tween 80 at a final concentration of 0.05-0.1 g / L was added to ultrapure water, and a non-solvent solution was formed under magnetic stirring at 37°C and 400-800 rpm. Furthermore, Tween 80 forms a molecular film in the non-solvent, reducing the interfacial tension between DMSO and water and providing a stable interface for microcrystal growth; the 37°C temperature is consistent with the subsequent crystallization temperature, avoiding the influence of temperature fluctuations on the crystal form.
[0027] S102, edaravone DMSO solution was added dropwise to a non-solvent system for crystal induction and growth; Add edaravone DMSO solution dropwise to the non-solvent solution at a rate of 0.8 ml / min using a constant flow pump, while maintaining magnetic stirring at 400-800 rpm. After the addition is complete, continue stirring for 3-6 hours until a white suspension is formed.
[0028] Furthermore, a slow drop rate of 0.8 mL / min controls the diffusion rate of DMSO, avoiding excessive local supersaturation that could lead to microcrystal aggregation; moderate shear force is generated to induce edaravone molecules to grow axially and form rod-shaped structures; stirring for 3-6 hours ensures complete crystallization and reduces the proportion of amorphous particles.
[0029] S103, the suspension of induction and growth was purified and dried, and modified with polyethylene glycol-polylysine copolymer inner layer and hyaluronic acid outer layer with CD44 targeting peptide; The suspension was transferred to centrifuge tubes and centrifuged at 8000-10000 rpm for 15-40 min at 4°C. The supernatant was discarded, and the precipitate was washed with ultrapure water at least 3 times, adding ultrapure water each time. After vortexing and resuspending, the precipitate was centrifuged again and finally freeze-dried at -50°C and 0.1-0.5 mbar for 24-48 h to obtain white, fluffy edaravone rod-shaped microcrystalline powder.
[0030] Furthermore, centrifugation at 4°C avoids the dissolution of microcrystals due to temperature rise, while centrifugation at 8000-10000 rpm ensures complete precipitation of microcrystals; washing with ultrapure water removes residual DMSO and free Tween 80, reducing the risk of toxicity; and freeze-drying avoids damage to the crystal form by high temperature, maintaining the integrity of the rod-shaped structure.
[0031] After obtaining edaravone rod-shaped microcrystalline powder, the morphology was observed by scanning electron microscopy, the size was measured by laser particle size analyzer, and the crystal form was verified by X-ray diffraction. The rod-shaped structure was intact, with a length of 15-30 μm and a diameter of 2-5 μm. The XRD characteristic peaks were consistent with type A edaravone, with no crystal form transformation and a dispersion of ≤15%.
[0032] Weigh out edaravone rod-shaped microcrystals, add them to a polyethylene glycol-polylysine copolymer solution, and place them on a shaker at 150-300 rpm for 20-30 minutes at room temperature to form solution one; Centrifuge at 8000-10000 rpm for 15-30 min at 4℃, discard the supernatant, and wash the precipitate with PBS at least twice to obtain microparticles a; In the above process, the polyethylene glycol-polylysine copolymer solution is adsorbed onto the microcrystal surface due to the positively charged amino groups and the weakly negatively charged electrostatic effect of the Tween 80 residue. To ensure sufficient adsorption, centrifugation and washing are used to remove unbound free polyethylene glycol-polylysine copolymer, exposing polyethylene glycol on the surface, reducing van der Waals forces between microcrystals, improving dispersibility, and preventing agglomeration during the modification process. Resuspend particle a in hyaluronic acid solution containing CD44 targeting peptide, shake on a shaker at 150-300 rpm for 15-30 min at room temperature to form solution two, centrifuge at 8000-10000 rpm for 10-15 min at 4℃, discard the supernatant, and wash the precipitate at least twice with PBS to obtain particle b. Furthermore, the carboxyl group of hyaluronic acid containing CD44 targeting peptides carries a negative charge and combines with the positively charged amino group of polyethylene glycol-polylysine copolymer through electrostatic interaction to form a stable bilayer. The CD44 targeting peptides are exposed on the surface, providing recognition sites for subsequent active targeting of inner ear hair cells. Repeat the operation at least twice to obtain particle c.
[0033] Preferably, the three bilayers have moderate thickness, with a total shell thickness of about 1-2 μm, which ensures the target peptide density while avoiding excessively large formulation size, with the total size controlled at 18-35 μm; and the multilayer structure enhances shell stability, preventing the target molecule from falling off during delivery.
[0034] S104, after cross-linking and curing, the sustained-release formulation is formed.
[0035] Particle c was resuspended in 10 mL of 1% genipin aqueous solution to make the particle concentration 5 mg / mL, and allowed to stand at room temperature in the dark for 2 h, with gentle vortexing once every 30 min during the period; Genipin undergoes a condensation reaction with the hydroxyl groups of HA and the amino groups of PLL to form a covalent cross-linked network, which enhances the mechanical strength of the shell. A 2-hour reaction time ensures sufficient cross-linking; light protection is necessary to prevent genipin from oxidizing and becoming ineffective. A particle concentration of 5 mg / mL ensures sufficient contact between the crosslinking agent and the shell material; Centrifuge at 4000-8000 rpm for 15 min at 4℃, discard the supernatant, wash the precipitate three times with ultrapure water to remove residual genipin, and freeze-dry under vacuum for 12-24 h to obtain a white powdery microcrystalline sustained-release formulation.
[0036] Example 1: This example uses a controlled experiment to verify the targeting, sustained-release effect, inner ear retention time, and neuroprotective efficiency of the microcrystalline sustained-release formulation. The control system includes unmodified microcrystals, a formulation without a targeting shell, and a commercially available common injectable formulation.
[0037] The experimental group used a targeted sustained-release formulation, administered via intratympanic injection of 0.1 mL. Control group 1 used a non-targeting shell formulation, consisting only of the inner PEG-PLL layer, with 3 bilayers and a size of 18-35μm. The administration method was the same as that of the experimental group. The control group 2 used unmodified rod-shaped microcrystals without a shell, with a size of 15-30 μm, and the administration method was the same as that of the experimental group. The blank control group used physiological saline, the same as the experimental group.
[0038] Regarding experimental methods: All formulations were labeled with the fluorescent dye Cy5.5 (labeling rate ≥95%, without affecting the formulation structure).
[0039] Guinea pigs in each group were anesthetized and injected into the tympanic cavity. They were sacrificed 1 hour later, the auditory bullae were separated, and the round window membrane tissue was taken.
[0040] The detection methods are as follows: observation of fluorescence distribution on the surface of the circular window membrane using laser confocal microscopy, and quantitative detection of the proportion of fluorescent positive particles in the circular window membrane tissue using flow cytometry.
[0041] The results are shown in the table below:
[0042] This demonstrates that the targeted sustained-release formulation significantly enhances the binding efficiency with the circular window membrane through the CD44 targeting peptide, validating the advantages of active targeting.
[0043] Example 2: Based on Example 1, control group 3 used commercially available edaravone injection, solution type, without sustained-release function, and the administration method was the same as the experimental group. Control group 4 used a single-shell formulation, one bilayer, PEG-PLL+HA-CD44p, and the administration method was the same as the experimental group.
[0044] Perilymph was collected at 1 h, 6 h, 24 h, 72 h, and 168 h after drug administration. The drug concentration was determined by high performance liquid chromatography, and the area under the curve and mean residence time were calculated.
[0045] The results are shown in the table below:
[0046] In summary, the MRT in the experimental group was 8.3 times that of control group 3 and 1.8 times that of control group 4, verifying the long-term sustained-release advantage of the multi-layer shell.
[0047] Example 3, based on Example 1, uses edaravone as the active ingredient, with the same edaravone content as the experimental group. It is unmodified, spherical in appearance, and has no directional structure. It contacts the middle ear mucosa and round window membrane via point-to-surface contact, with a diameter controlled at 18-35 μm. Five control groups were used. The administration method was the same as the experimental group. At 24h, 72h, and 168h after administration, the middle ear cavity was separated, and the residual amount of the formulation was observed using scanning electron microscopy, showing the percentage of the initial administered formulation in the middle ear cavity.
[0048] The results are shown in the table below:
[0049] In summary, the rod-shaped structure significantly prolongs the retention time by increasing the mechanical adhesion to the middle ear mucosa, thus validating the advantages of shape optimization.
[0050] Example 4: Based on Example 1, this example uses a targeted sustained-release formulation injected into the tympanic cavity 24 hours after noise exposure as the experimental group, six control groups are injected into the tympanic cavity 24 hours after noise exposure, and the model control group is injected with physiological saline 24 hours after noise exposure.
[0051] The ABR threshold at 4 kHz was measured 7 days and 14 days after drug administration, and cochlear sections were stained with HE to count the survival rate of outer hair cells in the basement membrane.
[0052] The results are shown in the table below:
[0053] This demonstrates that targeted sustained-release formulations, through long-term targeted delivery, significantly improve hearing recovery and hair cell protection in noise-induced hearing loss, validating their advantages in clinical application.
[0054] It should be noted that the descriptions of each embodiment in the above embodiments have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.
[0055] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.
[0056] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A microcrystalline sustained-release formulation, characterized in that, The sustained-release formulation comprises a core composed of edaravone rod-shaped microcrystals, and two to four bilayer shells, each shell comprising at least one inner layer of polyethylene glycol-polylysine copolymer and at least one outer layer of hyaluronic acid modified with a CD44-targeting peptide.
2. The microcrystalline sustained-release formulation and its preparation method according to claim 1, characterized in that, The edaravone rod-shaped microcrystals have an apparent shape of cylindrical rods, with a length of 15-30 μm and a diameter of 2-5 μm.
3. The microcrystalline sustained-release formulation and its preparation method according to claim 2, characterized in that, The total size of the sustained-release formulation is 18-35 μm.
4. A method for preparing a microcrystalline sustained-release formulation, characterized in that, The method includes: S101. Dissolve edaravone active pharmaceutical ingredient to prepare a solvent-free system; S102, edaravone DMSO solution was added dropwise to a non-solvent system for crystal induction and growth; S103, the suspension of induction and growth was purified and dried, and modified with polyethylene glycol-polylysine copolymer inner layer and hyaluronic acid outer layer with CD44 targeting peptide; S104, after cross-linking and curing, the sustained-release formulation is formed.
5. The method for preparing a microcrystalline sustained-release formulation according to claim 4, characterized in that, The pre-prepared non-solvent system for dissolving edaravone active pharmaceutical ingredient includes: Edaravone active pharmaceutical ingredient was dissolved in DMSO at 37℃, 200-500 rpm, and 30-60 min with stirring. The concentration of the edaravone DMSO solution was controlled to be 9-10 mg / mL. A non-solvent solution was formed by adding Tween 80 to ultrapure water at a final concentration of 0.05-0.1 g / L and stirring with a magnetic stirrer at 37°C and 400-800 rpm.
6. The method for preparing a microcrystalline sustained-release formulation according to claim 4, characterized in that, The edaravone DMSO solution is added dropwise to a non-solvent system for crystal induction and growth, including: Add edaravone DMSO solution dropwise to the non-solvent solution at a rate of 0.8 ml / min using a constant flow pump, while maintaining magnetic stirring at 400-800 rpm. After the addition is complete, continue stirring for 3-6 hours until a white suspension is formed.
7. The method for preparing a microcrystalline sustained-release formulation according to claim 4, characterized in that, The suspension used for induction and growth is purified and dried, and then modified with a polyethylene glycol-polylysine copolymer inner layer and a hyaluronic acid outer layer containing a CD44-targeting peptide, including: The suspension was transferred to centrifuge tubes and centrifuged at 8000-10000 rpm for 15-40 min at 4°C. The supernatant was discarded, and the precipitate was washed with ultrapure water at least 3 times, adding ultrapure water each time. After vortexing and resuspending, the precipitate was centrifuged again and finally freeze-dried at -50°C and 0.1-0.5 mbar for 24-48 h to obtain white, fluffy edaravone rod-shaped microcrystalline powder.
8. The method for preparing a microcrystalline sustained-release formulation according to claim 7, characterized in that, Also includes: Weigh out edaravone rod-shaped microcrystals, add them to a polyethylene glycol-polylysine copolymer solution, and place them on a shaker at 150-300 rpm for 20-30 minutes at room temperature to form solution one; Centrifuge the solution at 8000-10000 rpm for 15-30 min at 4℃, discard the supernatant, and wash the precipitate with PBS at least twice to obtain microparticles a; Resuspend particle a in hyaluronic acid solution containing CD44 targeting peptide, shake on a shaker at 150-300 rpm for 15-30 min at room temperature to form solution two. Centrifuge solution two at 8000-10000 rpm for 10-15 min at 4℃, discard the supernatant, and wash the precipitate at least twice with PBS to obtain particle b. Repeat the operation at least twice to obtain particle c.
9. The method for preparing a microcrystalline sustained-release formulation according to claim 4, characterized in that, The cross-linked and cured sustained-release formulation is formed by: resuspending the microparticles c in 10 mL of 1% genipin aqueous solution to make the microparticle concentration 5 mg / mL, and letting it stand at room temperature in the dark for 2 hours, during which time it is gently vortexed once every 30 minutes. Centrifuge at 4000-8000 rpm for 15 min at 4℃, discard the supernatant, wash the precipitate three times with ultrapure water to remove residual genipin, and freeze-dry under vacuum for 12-24 h to obtain a white powdery microcrystalline sustained-release formulation.