An adaptive drug delivery system for ischemic stroke, its preparation method and application

Abstract

This invention discloses an adaptive drug delivery system for ischemic stroke, its preparation method, and its application. MMP-2 responsive peptide-drug dimers are loaded onto the surface of mesoporous polydopamine for intervention or treatment of cerebral ischemia-reperfusion injury. The system allows for targeted drug delivery based on individual differences in disease progression among patients, precisely promoting brain injury repair. This invention applies MMP-2 responsive peptides to the treatment of ischemic stroke, using mesoporous polydopamine as a drug carrier, providing reactive sites and loading space for efficient drug loading. The chemical reaction between catechol on the polydopamine surface and the thiol groups on the MMP-2 responsive peptides loads the peptide-drug dimers, thereby achieving responsive drug release on the carrier surface. Simultaneously, this invention utilizes the ability of polydopamine to scavenge reactive oxygen species (ROS), rapidly clearing local ROS and reducing acute damage caused by ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to an adaptive drug delivery system for ischemic stroke, its preparation method, and its application. Background Technology

[0002] Ischemic stroke is a clinical syndrome characterized by brain tissue necrosis and neurological dysfunction caused by insufficient blood supply to the brain. It is characterized by five highs: high incidence, high recurrence rate, high disability rate, high mortality rate, and high economic burden. After thrombosis, reperfusion, such as intravenous thrombolysis or arterial thrombectomy, is the primary method to restore blood flow and ensure energy supply to the brain. Timely reperfusion is the most effective way to restore blood supply to the ischemic lesion; however, the increased oxygen content resulting from reperfusion after ischemia can induce a series of neuroinflammatory cascades. It is important to note that the occurrence and development of neuroinflammatory disease are spatiotemporally specific. That is, in the early stages of inflammation, immune cells, including microglia, are recruited / activated, initiating an immune response to the injury; however, as inflammation progresses, the environment in the injured area gradually deteriorates, inducing neuronal apoptosis and causing irreversible pathological damage. The individualized progression of neuroinflammatory disease varies greatly among patients, with the transition time ranging from several hours to several days; even within the same patient, the time of inflammatory progression in different regions of the stroke brain varies significantly.

[0003] Minocycline is a drug that can regulate the progression of neuroinflammation in the brain. It has been shown to induce the transformation of immune cells from M1 pro-inflammatory to M2 anti-inflammatory types through the NF-κB pathway, reducing secondary damage caused by inflammation and promoting brain repair. As mentioned earlier, the progression of the disease varies greatly among different patients. If minocycline is administered too early, the drug will be rapidly metabolized; if it is administered too late, inflammatory damage has already occurred, rendering the intervention ineffective. Continuous, high-dose treatment with minocycline can easily lead to increased drug toxicity. Therefore, there is an urgent need for an intervention program that is adaptive to different stroke patients and different disease progressions to achieve enhanced treatment efficacy and reduced toxicity.

[0004] In recent years, the emergence of nanomedicine delivery systems has provided new avenues for the treatment of ischemic stroke due to their unique small size effect and functionalizability. However, past technological breakthroughs have mainly focused on how to rapidly scavenge reactive oxygen species (ROS) using nanomedicine delivery systems, or on using ROS as a response mechanism to release drugs. This type of research only focuses on the acute injury phase after ischemia-reperfusion and rarely addresses the regulation of inflammatory progression. Therefore, how to treat ischemic brain injury according to the pathological characteristics at different time points is a significant technical challenge in the field. Based on these challenges, developing a safe, effective, and precise interventional treatment to achieve adaptive therapy based on individual patient differences is of great significance for both basic research and clinical application of drug therapy. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of existing technologies by providing a method for preparing an adaptive drug delivery system for ischemic stroke. The nano-drug delivery system synthesized by this method can respond to matrix metalloproteinase-2 (MMP-2) secreted by microglia, promptly regulating the inflammatory environment after the deterioration of neuroinflammation and converting pro-inflammatory immune cells into anti-inflammatory ones; simultaneously, the carrier material itself has a highly efficient ROS binding capacity, capable of scavenging reactive oxygen species caused by reoxygenation.

[0006] The nano-drug delivery system involved in this invention is prepared stepwise using a process, and the specific technical solution is as follows:

[0007] Step (1): Preparation of mesoporous polydopamine nanomaterials

[0008] Solution A was obtained by dissolving Pluronic F127 block copolymer in ethanol, and solution B was obtained by dissolving dopamine hydrochloride in water. Solution A and solution B were mixed, and 0.3-1.0% (v / v) of 1,3,5-trimethylbenzene was added and mixed thoroughly. The pH was adjusted to 8-11 and the reaction was continued for 30-180 minutes to obtain mesoporous polydopamine nanomaterials.

[0009] As a preferred embodiment, the mass-to-volume ratio of Prönnick F127 block copolymer to ethanol is 50 mg: 1.5 mL.

[0010] Preferably, the mass-to-volume ratio of dopamine hydrochloride to water is 15 mg: 1.5 mL.

[0011] Preferably, the mesoporous polydopamine nanomaterial has a particle size of 120-150 nm and a pore size of 20-40 nm.

[0012] As a preferred method, a certain amount of alkaline solution is added when adjusting the pH.

[0013] As a preferred embodiment, the mass ratio of Pluronic F127 block copolymer to dopamine hydrochloride is 50:15.

[0014] Step (2): Conjugation of MMP-2 responsive peptides to drug molecules and preparation of drug delivery systems

[0015] The MMP-2 responsive peptide was mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide. After reacting for a certain period of time, the drug was added to continue the reaction, thus synthesizing the MMP-2 responsive peptide-drug dimer.

[0016] Preferably, the drug is 9-aminominocycline.

[0017] Preferably, the MMP-2 responsive peptide sequence includes a -GPLGIAGQ- sequence, with short chain peptides consisting of no less than 3 serine residues linked to each end of the sequence, and one end of the sequence having a thiol group.

[0018] More preferably, the MMP-2 responsive peptide sequence is CSSSGPLGIAGQSSS, where the -GPLGIAGQ- sequence is the MMP-2 enzyme-responsive sequence, and the flanking -SSS- sequences are used to enhance the peptide's water solubility. Simultaneously, the cysteine ​​(C) residue at one end of the peptide contains a thiol group for linking to the carrier. Before adding the drug, the pH of the reaction solution is 4-7; after adding the drug, the pH of the reaction solution is 7-10. After synthesis, the peptide can be purified by dialysis and lyophilization.

[0019] Preferably, the molar ratio of the MMP-2 responsive peptide to the drug is 1:1.

[0020] Step (3): Mesoporous polydopamine-loaded MMP-2 responsive peptide-drug dimer

[0021] In an aqueous environment, mesoporous polydopamine nanomaterials were mixed with MMP-2 responsive peptide-drug dimers, and the mixture was stirred and reacted in an aerobic environment to load the MMP-2 responsive peptide-drug dimers onto the surface of the mesoporous polydopamine. After synthesis, the mixture was purified by centrifugation and lyophilization. The mass ratio of MMP-2 responsive peptide-drug dimers to mesoporous polydopamine was greater than 1:1 to maximize the drug loading of the mesoporous polydopamine.

[0022] Preferably, the pH of the reaction solution is 7-10.

[0023] The second objective of this invention is to provide an adaptive ischemic stroke drug delivery system prepared using the above method, which employs a drug carrier that precisely regulates the progression of neuroinflammatory disease after reperfusion injury. This system can precisely release anti-stroke drugs and regulate the neuroinflammatory process for different stages of the disease.

[0024] An adaptive drug delivery system for ischemic stroke is made of mesoporous polydopamine nanomaterials and MMP-2 responsive peptide-drug dimers; the MMP-2 responsive peptide contains a -GPLGIAGQ- sequence, and one end of the sequence is a thiol group; wherein each end of the -GPLGIAGQ- sequence is connected to a short chain peptide consisting of at least 3 serine residues.

[0025] Preferably, the molar ratio of MMP-2 responsive peptide to drug in the MMP-2 responsive peptide-drug dimer is 1:1.

[0026] A third objective of this invention is to provide the application of the aforementioned drug delivery system in the preparation of drugs for the intervention or treatment of cerebral ischemia-reperfusion injury. The aforementioned drug delivery system is suitable for different patients and can adaptively regulate the neuroinflammatory process and promote brain injury repair.

[0027] The specific beneficial effects of this invention are as follows:

[0028] 1) This invention proposes to apply MMP-2 responsive peptides to the field of ischemic stroke technology, utilizing their responsiveness to solve the problem of drug differences between different individuals and achieve adaptive treatment.

[0029] 2) To address the varying microglial polarization patterns among individual patients and at different disease stages within the same patient, this invention employs mesoporous polydopamine as the main structure and MMP-2 responsive peptides as the drug release module to assemble a responsive nanoparticle drug delivery system. This system can responsively release drugs based on differences in polarization time, resolving the issue of different dosing times and sites of action among different patients.

[0030] 3) This invention proposes to construct a drug delivery system using mesoporous polydopamine as the main structure and introducing MMP-2 enzyme-responsive peptide as an intermediate chain to load the drug. This drug delivery system utilizes the Schiff base reaction between catechol on the surface of polydopamine and the thiol group on the MMP-2 responsive peptide to load the peptide-drug dimer, thereby realizing the responsive release of the drug on the surface of the carrier.

[0031] Here, mesoporous polydopamine serves as both a drug carrier, providing reactive sites and loading space for efficient drug loading, and also utilizes the property of polydopamine itself to scavenge ROS, rapidly removing local ROS.

[0032] 4) This invention uses mesoporous polydopamine with nanoscale size and large specific surface area as a carrier. The increased specific surface area improves the ROS scavenging efficiency of polydopamine itself, and the structural features of its 20-40nm mesoporous channels improve the drug loading capacity.

[0033] 5) This invention uses MMP-2 enzyme-responsive peptides with good water solubility and thiol groups at the ends as drug linking molecules, which can improve the water solubility of drugs, prevent aggregation, and achieve drug loading in a one-step process in a mild aqueous system.

[0034] 6) This invention addresses the differences in the development of neuroinflammatory processes after cerebral ischemia-reperfusion injury, achieving responsive and precise regulation of the intracranial inflammatory environment, avoiding excessive drug use, and combining the ROS scavenging properties of the carrier material itself to achieve highly efficient treatment of ischemia-reperfusion injury.

[0035] In summary, this invention utilizes mesoporous polydopamine and MMP-2 enzyme-responsive peptides to construct an adaptive anti-stroke drug delivery system. The synthesis is simple, the design concept is clear, and the resulting nano-drug delivery system has well-defined structural components and functional properties. Further studies in vitro at the cellular level and in animal models validate this invention, providing an adaptive ischemic stroke drug delivery system that releases drugs in response to individualized differences in inflammation progression, precisely regulating the intracranial inflammatory environment, and is expected to have good therapeutic effects on different patients. Attached Figure Description

[0036] Figure 1 Scanning electron microscope image of the mesoporous polydopamine nanoparticles prepared in Example 1.

[0037] Figure 2 The nitrogen adsorption-desorption curve and pore size analysis diagram of the mesoporous polydopamine prepared in Example 1 are shown.

[0038] Figure 3 Thermogravimetric analysis curves of mesoporous polydopamine (mPDA), drug molecule (Mino), and drug-loaded mesoporous polydopamine nanoparticles (mPDA-Pep-Mino) in Example 1 are shown.

[0039] Figure 4 The image shows the enzyme response drug release curves of the nano-drug delivery system in Example 1 under different stimulating factors (MMP-2, pH, H2O2).

[0040] Figure 5 This is a graph showing the ROS scavenging ability of mesoporous polydopamine in nerve cells in Example 1. PDA refers to non-mesoporous polydopamine nanoparticles with the same particle size and concentration as mPDA.

[0041] Figure 6 This study analyzes the anti-acute injury effect of the nano-drug delivery system in Example 2 in a mouse model of middle cerebral artery occlusion. After different treatments, A) staining of the infarcted area in mouse brain tissue; B) analysis of infarct volume in mouse brain tissue; and C) analysis of behavioral neurological scores in mice.

[0042] Figure 7This study analyzes the pro-chronic repair effect of the nano-drug delivery system in Example 2 in a mouse model of middle cerebral artery occlusion. A) Survival curves of mice at 7 days after different treatments; B) Behavioral neurological scores of mice at 3 and 7 days. In the figure, a represents Saline, b represents T-mPDA, c represents Mino, and d represents T-mPDA-Pep-Mino.

[0043] Figure 8 The results show the effects of different administration methods on the regulation of intracranial inflammatory processes in Example 3. Detailed Implementation

[0044] As mentioned above, in view of the shortcomings of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention, which is mainly based on at least the following:

[0045] 1) This invention uses mesoporous polydopamine nanoparticles as the carrier body to prepare a nano-drug delivery system by coupling MMP-2 enzyme-responsive peptide-drug dimer. Selecting an appropriate surface modification ratio is crucial for simultaneously achieving precise regulation of the intracranial inflammatory environment by releasing drugs in response to the MMP-2 enzyme.

[0046] 2) The MMP-2 enzyme-responsive peptide is coupled with an amino-containing drug molecule, and then the residual thiol group at the other end reacts with catechol on the surface of mesoporous polydopamine to achieve drug loading.

[0047] 3) Mesoporous polydopamine carriers exhibit highly efficient ROS scavenging activity in vivo. This activity can treat and promote the recovery of local lesions caused by various inflammations, including those of the central nervous system and peripheral systems. The preparation of nanoparticles with suitable size and pore dimensions is crucial for the carrier's ability to enter ischemic areas, scavenge ROS, and load drugs.

[0048] 4) By sequentially reacting mesoporous polydopamine, drugs, and MMP-2 enzyme-responsive peptides, the resulting drug delivery system can specifically respond to the pathological characteristics during the transformation of the inflammatory environment in the brain, and further explore the ROS scavenging effect of the carrier material. After loading drugs, the nano-drug delivery system prepared in this invention can target the microenvironmental characteristics of neuroinflammation at different stages of development after ischemia-reperfusion injury, overcome individual differences among patients, and achieve responsive regulation of the neuroinflammation process to achieve the goal of precision treatment.

[0049] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0050] The present invention provides a method for preparing an adaptive drug delivery system for ischemic stroke, the specific implementation of which is as follows:

[0051] 1) Preparation of mesoporous polydopamine with a particle size of 120-150 nm and a pore size of 20-40 nm

[0052] 50 mg of Pranic F127 polymer was dissolved in 1.5 mL of ethanol to obtain solution A; 15 mg of dopamine hydrochloride was dissolved in 1.5 mL of water to obtain solution B. After mixing solutions A and B, 9-30 μL of 1,3,5-trimethylbenzene was added, and the mixture was sonicated with a probe until it changed from turbid to clear. Then, while maintaining rapid stirring, 90 mL of concentrated ammonia was added to induce spontaneous polymerization of dopamine. After 90 min of open reaction, the mixture was centrifuged at high speed (10,000 rpm, 10 min) to remove unreacted material, and the precipitate was washed multiple times with a mixture of ethanol and water to obtain mesoporous polydopamine nanoparticles for later use.

[0053] 2) Preparation of MMP-2 enzyme-responsive peptides and drug dimers

[0054] 20 mg of MMP-2 enzyme-responsive peptide was weighed and dissolved in 2 mL of 0.1 M MES buffer (pH = 5.2). 21.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide were added. After stirring for 15 min, the pH of the solution was adjusted to approximately 8.5 with 2 M Na₂CO₃. 29.2 mg of 9-aminominocycline was added, and the reaction was continued for 24 hours. After the reaction was complete, the product was dialyzed in aqueous solution for 48 hours and then further lyophilized for 24 hours for purification.

[0055] 3) Drug load

[0056] Weigh 20 mg of the lyophilized peptide-drug dimer and dissolve it in 5 mL of weakly alkaline aqueous solution at a mass ratio of 1:1 with mesoporous polydopamine. Stir the solution over an open container for 24 hours. After the reaction, remove the unloaded dimer by centrifugation and washing with water multiple times to obtain the purified drug-loaded nanodelivery system.

[0057] Example 1:

[0058] 1) Preparation of mesoporous polydopamine with a particle size of 120 nm and a pore size of 20 nm

[0059] 50 mg of Pranic F127 polymer was dissolved in 1.5 mL of ethanol to prepare solution A; 15 mg of dopamine hydrochloride was dissolved in 1.5 mL of water to prepare solution B. Solutions A and B were mixed, and 15 μL of 1,3,5-trimethylbenzene was added. The mixture was then sonicated using a probe until it changed from turbid to clear. Then, while maintaining rapid stirring, 90 mL of concentrated ammonia was added to induce spontaneous polymerization of dopamine. After 90 min of open reaction, the mixture was centrifuged at high speed (10,000 rpm, 10 min) to remove unreacted material. The precipitate was washed multiple times with a mixture of ethanol and water to obtain mesoporous polydopamine nanoparticles for later use.

[0060] Scanning electron microscope images of mesoporous polydopamine nanoparticles are shown below. Figure 1 Nitrogen adsorption-desorption curves and material pore size analysis are shown in [reference needed]. Figure 2 .

[0061] 2) Preparation of MMP-2 enzyme-responsive peptides and drug dimers

[0062] 20 mg of MMP-2 enzyme-responsive peptide was weighed and dissolved in 2 mL of 0.1 M MES buffer (pH = 5.2). 21.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide were added. After stirring for 15 min, the pH of the solution was adjusted to approximately 8.5 with 2 M Na₂CO₃. 29.2 mg of 9-aminominocycline was added, and the reaction was continued for 24 hours. After the reaction was complete, the product was dialyzed in aqueous solution for 48 hours and then further lyophilized for 24 hours for purification.

[0063] 3) Drug load

[0064] Weigh 20 mg of the lyophilized peptide-drug dimer and dissolve it in 5 mL of weakly alkaline aqueous solution at a mass ratio of 1:1 with mesoporous polydopamine. Stir the solution over an open container for 24 hours. After the reaction, remove the unloaded dimer by centrifugation and washing with water multiple times to obtain the purified drug-loaded nanodelivery system.

[0065] Thermogravimetric analysis curves of mesoporous polydopamine (mPDA), drug molecule (Mino), and drug-loaded mesoporous polydopamine nanoparticles (mPDA-Pep-Mino) are shown in the figure. Figure 3 .

[0066] 4) Nanoparticle drug delivery system responds to MMP-2 enzyme to release drugs

[0067] The nano-drug delivery system was co-incubated with 0.1 μg / mL and 1 μg / mL MMP-2 enzyme for specific times (1, 2, 3, 4, 8, 12, 24, 32, 40, and 48 hours), respectively. After centrifugation, the supernatant was collected, and the released drug content was determined by high-performance liquid chromatography (HPLC). This confirmed that the nano-drug delivery system was sensitive to MMP-2 enzyme and could respond and release drug compared with the blank control group. Simultaneously, the effects of other potential interfering factors (weakly acidic environment, H2O2) on drug release from the delivery system were determined.

[0068] The MMP-2 enzyme-responsive drug release curve of the nano-drug delivery system is shown in the figure. Figure 4 .

[0069] 5) Nanoparticle drug delivery systems scavenge cellular free radicals

[0070] SH-SY5Y neural cells were selected as a cell model, and ROS increase was induced in the neural cells using the ROSup kit. After co-incubating the neural cells with a 10 μg / mL nano-drug delivery system for 24 hours, the changes in cell fluorescence intensity were observed using a fluorescence microscope and compared with the control group. This confirmed that the nano-drug delivery system could effectively scavenge abnormally elevated free radicals in neural cells. The results are as follows: Figure 5 .

[0071] Example 2:

[0072] 1) Preparation of mesoporous polydopamine with a particle size of 150 nm and a pore size of 30 nm

[0073] 50 mg of Pranic F127 polymer was dissolved in 1.5 mL of ethanol to prepare solution A; 15 mg of dopamine hydrochloride was dissolved in 1.5 mL of water to prepare solution B. After mixing solutions A and B, 30 μL of 1,3,5-trimethylbenzene was added, and the mixture was sonicated with a probe until it changed from turbid to clear. Then, while maintaining rapid stirring, 90 mL of concentrated ammonia was added to induce spontaneous polymerization of dopamine. After 90 min of open reaction, the mixture was centrifuged at high speed (10,000 rpm, 10 min) to remove unreacted material, and the precipitate was washed multiple times with a mixture of ethanol and water to obtain mesoporous polydopamine nanoparticles for later use.

[0074] 2) Preparation of MMP-2 enzyme-responsive peptides and drug dimers

[0075] 20 mg of MMP-2 enzyme-responsive peptide was weighed and dissolved in 2 mL of 0.1 M MES buffer (pH = 5.2). 21.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide were added. After stirring for 15 min, the pH of the solution was adjusted to approximately 8.5 with 2 M Na₂CO₃. 29.2 mg of 9-aminominocycline was added, and the reaction was continued for 24 hours. After the reaction was complete, the product was dialyzed in aqueous solution for 48 hours and then further lyophilized for 24 hours for purification.

[0076] 3) Drug load

[0077] Weigh 20 mg of the lyophilized peptide-drug dimer and dissolve it in 5 mL of weakly alkaline aqueous solution at a mass ratio of 1:1 with mesoporous polydopamine. Stir the solution over an open container for 24 hours. After the reaction, remove the unloaded dimer by centrifugation and washing with water multiple times to obtain the purified drug-loaded nanodelivery system.

[0078] 4) Brain-targeted modification of nanodelivery systems

[0079] First, a polyethylene glycol-modified brain-targeting peptide was prepared. 10 mg of thiol-modified polyethylene glycol N-hydroxysuccinimide (HS-PEG-NHS ester) and 66 mg of the brain-targeting peptide RAP-12 (EAKIEKHNHYQK) were dissolved in 2 mL of 0.2 M Na₂CO₃ solution. After stirring for 24 hours, the mixture was purified by dialysis to obtain a thiol-terminated polyethylene glycol-modified brain-targeting peptide. 20 mg of this peptide was added to 2 mL of a 4 mg / mL nanoparticle drug delivery system solution, and the pH was adjusted to weakly alkaline with Na₂CO₃. After reacting for 24 hours, unreacted material was removed by centrifugation to obtain the brain-targeting modified nanoparticle drug delivery system.

[0080] 5) The role of nano-drug delivery systems in repairing ischemia-reperfusion injury

[0081] A mouse model of middle cerebral artery occlusion was established. The injured animals were treated with a nanomedicine delivery system. Twenty-four hours later, the brain injury area was stained and neurological function was assessed to clarify the role of the nanomedicine delivery system in reducing acute reperfusion. Results are as follows: Figure 6 The survival status of mice was monitored for 7 consecutive days, and neurological function scores were assessed on days 3 and 7. This clarified the role of the nanodelivery system in promoting long-term brain repair after injury. The results are as follows: Figure 7 .

[0082] Example 3:

[0083] 1) Preparation of mesoporous polydopamine with a particle size of 150 nm and a pore size of 30 nm

[0084] 50 mg of Pranic F127 polymer was dissolved in 1.5 mL of ethanol to prepare solution A; 15 mg of dopamine hydrochloride was dissolved in 1.5 mL of water to prepare solution B. After mixing solutions A and B, 30 μL of 1,3,5-trimethylbenzene was added, and the mixture was sonicated with a probe until it changed from turbid to clear. Then, while maintaining rapid stirring, 90 mL of concentrated ammonia was added to induce spontaneous polymerization of dopamine. After 90 min of open reaction, the mixture was centrifuged at high speed (10,000 rpm, 10 min) to remove unreacted material, and the precipitate was washed multiple times with a mixture of ethanol and water to obtain mesoporous polydopamine nanoparticles for later use.

[0085] 2) Preparation of MMP-2 enzyme-responsive peptides and drug dimers

[0086] 20 mg of MMP-2 enzyme-responsive peptide was weighed and dissolved in 2 mL of 0.1 M MES buffer (pH = 5.2). 21.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide were added. After stirring for 15 min, the pH of the solution was adjusted to approximately 8.5 with 2 M Na₂CO₃. 29.2 mg of 9-aminominocycline was added, and the reaction was continued for 24 hours. After the reaction was complete, the product was dialyzed in aqueous solution for 48 hours and then further lyophilized for 24 hours for purification.

[0087] 3) Drug load

[0088] Weigh 20 mg of the lyophilized peptide-drug dimer and dissolve it in 5 mL of weakly alkaline aqueous solution at a mass ratio of 1:1 with mesoporous polydopamine. Stir the solution over an open container for 24 hours. After the reaction, remove the unloaded dimer by centrifugation and washing with water multiple times to obtain the purified drug-loaded nanodelivery system.

[0089] 4) Brain-targeted modification of nanodelivery systems

[0090] First, a polyethylene glycol-modified brain-targeting peptide was prepared. 10 mg of thiol-modified polyethylene glycol N-hydroxysuccinimide (HS-PEG-NHS ester) and 66 mg of the brain-targeting peptide RAP-12 (EAKIEKHNHYQK) were dissolved in 2 mL of 0.2 M Na₂CO₃ solution. After stirring for 24 hours, the mixture was purified by dialysis to obtain a thiol-terminated polyethylene glycol-modified brain-targeting peptide. 20 mg of this peptide was added to 2 mL of a 4 mg / mL nanoparticle drug delivery system solution, and the pH was adjusted to weakly alkaline with Na₂CO₃. After reacting for 24 hours, unreacted material was removed by centrifugation to obtain the brain-targeting modified nanoparticle drug delivery system.

[0091] 5) Therapeutic effect analysis of adaptive nanodelivery systems and combined drug delivery methods

[0092] A mouse model of middle cerebral artery occlusion was constructed. The animals were randomly divided into 3 groups and treated as follows.

[0093] Group 1 (Day 0 dosing group): Animals were immediately treated with empty mesoporous polydopamine and minocycline after reperfusion;

[0094] Group 2 (Day 3 dosing group): Animals were given empty mesoporous polydopamine immediately after reperfusion, followed by minocycline treatment on Day 3;

[0095] Group 3 (Nanodelivery System Group): Animals were treated with the nanodelivery system immediately after reperfusion.

[0096] Seven days after treatment, the animals were sacrificed and mouse brain tissue was extracted. Immunofluorescence staining was performed on the ischemic areas to clarify the inflammatory environment in the brains of mice in different groups. The results are as follows: Figure 8 As shown, DAPI represents nuclear staining; CD86 represents M1 pro-inflammatory microglia; and CD206 represents M2 anti-inflammatory microglia.

[0097] Depend on Figure 8 It was found that the ischemic areas of the brain tissue of mice in the day 0 and day 3 drug administration groups still contained a large number of M1 pro-inflammatory microglia. Due to individual differences, administering the drug too early or too late would not achieve an adaptive therapeutic effect and would not transform the microglia into M2 anti-inflammatory microglia. In contrast, the number of M1 pro-inflammatory microglia in the third group (nano-drug delivery system group) was significantly reduced compared to the first two groups, and the microglia had already transformed into M2 anti-inflammatory microglia, achieving a better adaptive therapeutic effect without the need for continuous, high-dose administration.

Claims

1. A method for preparing an adaptive drug delivery system for ischemic stroke, characterized in that, The preparation method includes the following steps: Step (1): Preparation of mesoporous polydopamine nanomaterials Solution A was obtained by dissolving Pluronic F127 block copolymer in ethanol and solution B was obtained by dissolving dopamine hydrochloride in water. Solution A and solution B were mixed and 0.3-1.0% (v / v) of 1,3,5-trimethylbenzene was added and mixed thoroughly. The pH was adjusted to 8-11 and the reaction was continued for 30-180 minutes to obtain mesoporous polydopamine nanomaterials. Step (2): Conjugation of MMP-2 responsive peptides to drug molecules and preparation of drug delivery systems The MMP-2 responsive peptide was mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide. After reacting for a certain period of time, the drug was added and the reaction continued to synthesize the MMP-2 responsive peptide-drug dimer. The MMP-2 responsive peptide contained a -GPLGIAGQ- sequence, one end of which was a thiol group. Each end of the -GPLGIAGQ- sequence was connected to a short-chain peptide consisting of at least three serine residues. The drug was 9-aminominocycline. Step (3): Mesoporous polydopamine-loaded MMP-2 responsive peptide-drug dimer In an aqueous environment, mesoporous polydopamine nanomaterials were mixed with MMP-2 responsive peptide-drug dimers and stirred in an aerobic environment to load the MMP-2 responsive peptide-drug dimers onto the surface of the mesoporous polydopamine. After loading, the mixture was purified by centrifugation and lyophilization. The mass ratio of MMP-2 responsive peptide-drug dimers to mesoporous polydopamine was greater than 1:

1.

2. The method for preparing an adaptive drug delivery system for ischemic stroke according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of Prönnick F127 block copolymer to ethanol is 50 mg: 1.5 mL.

3. The method for preparing an adaptive drug delivery system for ischemic stroke according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of dopamine hydrochloride to water is 15 mg: 1.5 mL.

4. The method for preparing an adaptive drug delivery system for ischemic stroke according to claim 1, characterized in that, The mesoporous polydopamine nanomaterials obtained in step (1) have a particle size of 120-150 nm and a pore size of 20-40 nm.

5. The method for preparing an adaptive drug delivery system for ischemic stroke according to claim 1, characterized in that, Step (1) The mass ratio of Pronnic F127 block copolymer to dopamine hydrochloride is 50:

15.

6. The method for preparing an adaptive drug delivery system for ischemic stroke according to claim 1, characterized in that, In step (2), the molar ratio of the MMP-2 responsive peptide to the drug is 1:

1.

7. The method for preparing an adaptive drug delivery system for ischemic stroke according to claim 1, characterized in that, Step (3) The pH of the reaction solution is 7-10.

8. An adaptive drug delivery system for ischemic stroke, prepared by the method described in any one of claims 1-7.

9. The use of the drug delivery system of claim 8 in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

Images