Preparation method and use of nanoparticles capable of targeting brain injury sites for treating traumatic brain injury

Abstract

This application relates to the field of biomedicine and discloses a method for preparing nanoparticles that can target brain injury sites for the treatment of traumatic brain injury and its use. The nanoparticles are BCA@GP; the preparation method includes the following steps: BSA activation; synthesis of BSA@COA; synthesis of BSA@COA-CaP; synthesis of glycolipid DPG; glucose-modified platelets; and preparation of BCA@GP. Nanoparticles prepared by the method of this application, platelets coupled with glucose can quickly target damaged areas, exert hemostatic and coagulation functions, and release a large amount of Ca after fragmentation. 2+ , amplifying the cascade reaction in the coagulation process, and working with platelets to achieve rapid hemostasis. The loaded COA‑Cl can promote angiogenesis during injury and play a neuroprotective role. At the same time, the rich Ca 2+ Influx into endothelial cells can also promote endothelial cell migration, adhesion, proliferation and angiogenesis.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to a method for preparing nanoparticles that can target brain injury sites for the treatment of traumatic brain injury and its use. Background Art

[0002] Traumatic brain injury (TBI) refers to damage to brain tissue caused by external impacts, shocks, and concussions to the head. It commonly occurs in accidents such as traffic accidents, sports injuries, and violent conflicts, and is a common injury that seriously threatens human life and health. Globally, over 50 million people are threatened by TBI each year, and approximately half of the population may experience one or more TBIs in their lifetime. Its high incidence, mortality, and disability rates have become prominent public health and socioeconomic issues in many countries worldwide. Furthermore, the neurological dysfunction following TBI imposes long-term financial burdens and severe emotional stress on the injured and their families.

[0003] TBI is divided into primary brain injury and secondary brain injury based on the pathophysiological process. Primary injury refers to the damage caused by external mechanical forces immediately after the injury, while secondary injury refers to the pathophysiological process that continues to change from the onset of the injury. Among them, the vascular damage caused by TBI occurs earlier than other injury mechanisms in time, and can cause secondary bleeding, blood-brain barrier damage, brain tissue ischemia and hypoxia, cerebral edema, and neurovascular toxicity, etc., which is considered to be the key mechanism of TBI injury and repair. Therefore, exploring the treatment methods and strategies for vascular repair after craniocerebral trauma is of great significance for improving the prognosis of patients and reducing the mortality and disability rates of TBI patients.

[0004] Furthermore, coagulation disorders caused by TBI can exacerbate intracranial hemorrhage and further increase mortality and morbidity. Improving coagulation function and promoting vascular repair can help improve patient outcomes during TBI treatment. When a blood vessel is damaged, platelets, stimulated by activation factors at the site of injury, begin to aggregate, forming platelet clots that act as primary hemostasis. Platelets then undergo complex changes to produce thrombin, which converts fibrinogen in the adjacent plasma into fibrin. The interwoven fibrin allows the platelet clot to entangle with blood cells, forming a clot. Simultaneously, platelet protrusions extend into the fibrin network. As platelets contract, the clot becomes more solid, more effectively stopping bleeding and exerting a secondary hemostatic effect. Calcium ions, as an essential factor in the coagulation process, can, on the one hand, initiate platelet release reactions, altering platelet shape, facilitating platelet adhesion and aggregation, altering the plasma membrane conformation, and promoting platelet activation. They can also activate contractile proteins in the platelet system, causing platelets to contract and release coagulation factors, promoting blood coagulation and clot contraction. The interaction between calcium ions and platelets plays a crucial role in platelet aggregation and hemostasis; both are essential components of the coagulation process.

[0005] In recent years, with the development of neuroimaging, advancements in treatment concepts, the application of multimodal neurocritical care monitoring, and the improvement and promotion of standardized diagnosis and treatment of traumatic brain injury, the prognosis of TBI patients has improved. Despite this, there is still a lack of recognized effective drugs or methods for the treatment of traumatic brain injury internationally, especially in the field of vascular repair after traumatic brain injury. One of the difficulties lies in the difficulty of drugs to consistently and stably penetrate the blood-brain barrier and reach the site of injury to take effect.

[0006] After TBI, the blood-brain barrier is disrupted, increasing its permeability and facilitating drug delivery into the brain. However, as the barrier recovers, drugs struggle to reach the injury site and exert their therapeutic effects during the subacute phase of TBI and the prolonged recovery period that follows. Nanoparticles hold promise as a solution to this problem. Over the past few decades, the rapid development of nanotechnology has begun to be applied in biological and medical applications. In the field of drug delivery, nanoparticle delivery systems have begun to demonstrate tremendous potential due to their numerous unparalleled advantages. These include, but are not limited to, reduced side effects, prolonged blood circulation, improved drug stability, bioavailability, and targeting efficiency. Therefore, using nanoparticles as carriers to deliver therapeutic drugs may become an effective treatment option for post-traumatic brain injury.

[0007] Currently, there are many common methods of drug delivery to the nervous system in clinical practice. Compared with invasive methods such as stereotactic brain injection and intrathecal injection, peripheral drug delivery has lower technical requirements, less damage, and is safer. However, it also has problems such as wide distribution of the drug during administration and difficulty in achieving effective therapeutic concentrations in specific areas. When designing nanotherapeutic drugs, the inclusion of target recognition substances will enable the nanoparticles to have a high degree of targeted recognition of brain injury sites. This will enable precise targeted treatment after traumatic brain injury through a relatively safe peripheral delivery method.

[0008] With the advancement of nanotechnology and the development of functional nanoparticles, platelets have garnered widespread attention due to their unique physiological properties. Platelets are abundant in human blood, can rapidly respond to sites of vascular damage, target lesions of infection, and are closely associated with tumor metastasis and immunotherapy. Platelets are excellent potential drug carriers, with surface glycoproteins capable of immunomodulation, bioadhesion, and targeted recognition. Furthermore, the numerous primary amine groups on their membranes are suitable for chemical attachment of modifying groups or ligands.

[0009] In terms of promoting angiogenesis, COA-Cl is a new compound synthesized by the Faculty of Pharmacy, Kagawa, Tokushima University of Bunri. It is a small molecule compound that promotes angiogenesis. Its angiogenesis-promoting effect can be summarized as mediating the S1P1 receptor of vascular endothelial cells to promote angiogenesis and promoting VEGF secretion in peripheral cells such as NHDF. It is based on the skeleton of Oxetanocin A, a nucleic acid derivative with antiviral activity isolated from microorganisms. The oxetane ring (C-CC-O cyclic ether) is replaced with a cyclobutane ring (Carbo Oxethane) and Cl ( Figure 1 ), and its angiogenic activity was verified in both cell models and animal models. Summary of the Invention

[0010] Purpose of the invention: To provide a nano-therapeutic drug with targeting ability, easy to penetrate the blood-brain barrier, and exerting pro-coagulant and pro-angiogenic effects, and a preparation method thereof, to solve the difficulties of existing drug treatments.

[0011] To achieve the above objectives and other related objectives, the present invention provides a nanomedicine, which comprises at least a glycosylated engineered platelet membrane, a loaded small molecule compound COA-Cl and its BSA template, and a calcium phosphate ion layer obtained by biomineralization technology.

[0012] In a first aspect, the present application provides a method for preparing nanoparticles that can target brain injury sites for the treatment of traumatic brain injury, wherein the nanoparticles are BCaP-COA-Cl@Glu-PLT;

[0013] The preparation method comprises the following steps:

[0014] Step 1, BSA activation;

[0015] Step 2, synthesis of BSA@COA: taking the activated BSA solution, adding COA-Cl solution for synthesis, and after the synthesis, ultrafiltration, taking the supernatant to obtain BSA@COA;

[0016] Step 3, BSA@COA-CaP synthesis: Take the ultrafiltration supernatant in step 2, slowly add CaCl2 dropwise in multiple times, then continue to slowly add (NH4)2HPO4 dropwise in multiple times, adjust the pH to 10.0, ultrafilter, and take the supernatant to obtain BSA@COA-CaP;

[0017] Step 4, synthesis of glycolipid DSPE-PEG-Glu: dissolve the uncarboxylated PEG lipid copolymer DSPE-PEG-2000-COOH in distilled water, add EDC and stir, then add NHS for activation, continue stirring, add glucosamine, and stir to react to obtain glycolipid DSPE-PEG-Glu;

[0018] Step 5, glucose-modified platelets: platelets are mixed with DSPE-PEG-Glu and incubated with shaking to obtain platelets with glucose modified on the membrane surface;

[0019] Step 6, preparation of BCaP-COA-Cl@Glu-PLT: BSA@COA-CaP was fully and evenly coated with glucose-modified platelets by co-extrusion to obtain BCaP-COA-Cl@Glu-PLT.

[0020] Furthermore, the specific steps of step 1 are as follows: weigh 120 mg BSA, 60 mg sodium dodecyl sulfate, and 4.4 mg dithiothreitol, add them into a 5 mL sample bottle, add 3 mL ultrapure water, fully dissolve them, and place them in an oil bath at 90° C. The oil bath conditions are 180 r for 2 h.

[0021] Furthermore, the specific steps of step 2 are as follows: Synthesis of BSA@COA: Taking a 1 mL system as an example, 10 mg of activated BSA solution with a concentration of 40 mg / mL was taken, 950 μL MES was added, and then 25 μL of 4 mg / mL COA-Cl solution was added. The mixture was synthesized in a 24-well plate at 37 ° C and 800 r / min for 4 hours, followed by washing two to three times with a 100 kd ultrafiltration tube at 4000 r / min for 10 minutes, and the supernatant was taken to obtain BSA@COA.

[0022] Furthermore, the specific steps of step 3 are as follows: take 2 mL of the ultrafiltration supernatant in step 2, place it in a 5 mL glass bottle, set the speed to 520 rpm, slowly add 1000 μL, 2.5 mM CaCl2, add 200 μL each time after 10 min, and then continue to slowly add 1200 μL, 2.5 mM (NH4)2HPO4, add 200 μL each time after 10 min, adjust the pH value of the solution to 10.0 with ammonia water, stir for 30 min, and then use a 15 mL ultrafiltration tube, 4000 r / min, 10 min, ultrafiltration 1 to 2 times, take the supernatant, and obtain BSA@COA-CaP.

[0023] Furthermore, the specific steps of step 4 are as follows: weighing the unterminated carboxylated PEG lipid copolymer DSPE-PEG-2000-COOH, dissolving it in distilled water, adding EDC according to a DSPE-PEG-COOH:EDC:NHS molar ratio of 1:15:15, stirring for 30 minutes, adding NHS to activate the carboxyl group of SPE-PEG-COOH, continuing to stir for 3 hours, and then adding glucosamine at a molar ratio of 35:1 to DSPE-PEG-2000-COOH, stirring at room temperature, and reacting for 24 hours to obtain the glycolipid DSPE-PEG-Glu.

[0024] Furthermore, the specific steps of step 5 are: mixing the platelets with DSPE-PEG-Glu and incubating them in a water bath shaker for 2 hours to obtain platelets with glucose modified on the membrane surface.

[0025] In a second aspect, the present application provides a use of nanoparticles that can target brain injury sites for the treatment of traumatic brain injury, namely, the use of the nanoparticles for rapid hemostasis and coagulation in damaged areas.

[0026] In summary, this application has the following beneficial effects:

[0027] Nanoparticles with a small size (about 70nm in diameter) that can cross the blood-brain barrier were prepared. Platelets coupled with glucose can quickly target the damaged area and exert hemostatic and coagulation functions. After platelet fragmentation, CaP is released. Due to the slightly acidic environment at the site of injury, CaP releases a large amount of Ca 2+ , amplifying the cascade reaction in the coagulation process and cooperating with platelets to achieve rapid hemostasis. The loaded COA-Cl can promote angiogenesis during injury and play a neuroprotective role. 2+ Influx into endothelial cells can also promote endothelial cell migration, adhesion, proliferation and angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Shown is a schematic diagram of the synthesis of BCA@GP of the present invention.

[0029] Figure 2 Shown are the particle size distribution results of nanoparticles (BSA@COA, BSA@COA-CaP, BCA@GP).

[0030] Figure 3 Shown are the potential value results of nanoparticles (BSA@COA, BSA@COA-CaP, BCA@GP).

[0031] Figure 4 Shown are the UV-visible absorbance results of nanoparticles (BSA@COA, BSA@COA-CaP, BCA@GP).

[0032] Figure 5 Shown are the drug loading capacity and encapsulation efficiency of BCA@GP.

[0033] Figure 6 Shown is the 48h drug release curve of drug-loaded particles.

[0034] Figure 7 Shown is the electron microscopy image of BCA@GP nanoparticles.

[0035] Figure 8 Shown is the elemental mapping of BCA@GP nanoparticles. DETAILED DESCRIPTION

[0036] The structure and effects of the present invention are further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0037] The preparation materials are as follows:

[0038]

[0039] The instruments are as follows:

[0040]

[0041] The present invention treats traumatic brain injury, targets the injured area, and promotes coagulation and angiogenesis. Bovine serum albumin (BSA) is used as a template to biomineralize and synthesize CaP, while simultaneously loading the drug COA-Cl. Glucose-modified platelets are then used to coat nanoparticles to obtain BCaP-COA-Cl@Glu-PLT, abbreviated as BCaP@GP, which is used for rapid hemostasis and coagulation in the damaged area.

[0042] The main experimental methods used are self-assembly and chemical cross-linking. These methods utilize the biomacromolecule template effect to nucleate at the organic-inorganic interface in solution through electrostatic forces, chelation, hydrogen bonding, and van der Waals forces. The driving forces are quadrupole-quadrupole, T-shape, π-π stacking, hydrogen bonding, and cation-π interactions.

[0043] Example: Synthesis of BCA@GP

[0044] The preparation method of nanoparticles (BCaP-COA-Cl@Glu-PLT, BCA@GP) for treating traumatic brain injury comprises the following steps:

[0045] Step 1, BSA activation: weigh 120 mg BSA, 60 mg sodium dodecyl sulfate (SDS), and 4.4 mg dithiothreitol (DTT) into a 5 mL sample vial, add 3 mL ultrapure water, dissolve thoroughly, and incubate in a 90°C oil bath at 180 rpm for 2 h.

[0046] Step 2, synthesis of BSA@COA: Taking a 1 mL system as an example, take 25 μL (10 mg) of activated BSA solution (40 mg / mL), add 950 μL MES, and then add 25 μL (4 mg / mL) of COA-Cl solution. Use a 24-well plate to shake at 37°C and 800 rpm for 4 h, then wash twice with a 100 kd ultrafiltration tube at 4000 rpm for 10 min. Take the supernatant to obtain BSA@COA, test the particle size and potential, and use the filtrate for UV quantification (COA-Cl);

[0047] Step 3, BSA@COA-CaP synthesis: Take 2 mL of the ultrafiltration supernatant in step 2, place it in a 5 mL glass bottle, set the speed to 520 rpm, slowly add 1000 μL of CaCl2 (2.5 mM), add 200 μL every 10 min, then continue to slowly add 1200 μL of (NH4)2HPO4 (2.5 mM), add 200 μL every 10 min, adjust the pH value of the solution to 10.0 with ammonia water, stir for 30 min, and then use a 15 mL ultrafiltration tube, 4000 r / min, 10 min, ultrafiltration twice, take the supernatant to obtain BSA@COA-CaP, test the particle size and potential, and use the filtrate for UV quantification (COA-Cl);

[0048] Step 4, synthesis of glycolipid DSPE-PEG-Glu (DPG): weigh the uncarboxylated PEG lipid copolymer DSPE-PEG-2000-COOH, dissolve it in distilled water, add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride): NHS (N-hydroxysuccinimide) at a molar ratio of 1:15:15, stir for 30 minutes, add NHS to activate the carboxyl group of DSPE-PEG-COOH, continue stirring for 3 hours, and then add glucosamine at a molar ratio of 35:1 to DSPE-PEG-2000-COOH. Stir at room temperature and react for 24 hours to obtain the glycolipid DSPE-PEG-Glu;

[0049] Step 5, glucose-modified platelets: platelets were mixed with DSPE-PEG-Glu and incubated in a water bath shaker for 2 h to obtain platelet GP with glucose modified on the membrane surface;

[0050] Step 6, preparation of BCaP-COA-Cl@Glu-PLT (BCA@GP): BSA@COA-CaP was fully and evenly coated with GP by co-extrusion to obtain BCA@GP, and the particle size and potential were tested.

[0051] Detection test

[0052] Test 1: Particle size distribution and potential value

[0053] The particle size distribution and potential value of nanoparticles (BSA@COA, BSA@COA-CaP, BCA@GP) were detected by Malvern laser particle size analyzer. Figure 2 and Figure 3 shown.

[0054] Test 2: UV-Vis absorbance

[0055] The UV-visible absorbance of the nanoparticles (BSA@COA, BSA@COA-CaP, BCA@GP) was tested. The test results were as follows: Figure 4 shown.

[0056] Experiment 3: Drug loading and encapsulation efficiency

[0057] The drug loading and encapsulation efficiency of BCA@GP nanoparticles were tested. Figure 5 shown.

[0058] Test 4: Drug Release

[0059] The drug release performance of BCA@GP nanoparticles was tested, and the results were as follows Figure 6 As shown, it can be seen that BCA@GP nanoparticles can cumulatively release about 70% of the drug in 48 hours.

[0060] Experiment 5: Observation of BCA@GP nanoparticle morphology

[0061] like Figure 7 As shown, the transmission results show that the morphology of BCA@GP composite nanoparticles is spherical and the size is 60-70nm, which is consistent with the particle size measurement results.

[0062] Experiment 6: Elemental mapping of BCA@GP nanoparticles

[0063] The element mapping diagram of BCA@GP is as follows Figure 8 As shown, the mineralized Ca and P elements are evenly dispersed on the shell of the nanoparticles, indicating that a layer of CaP is successfully mineralized on the surface of the nanoparticles.

[0064] The beneficial effect of the present invention is that it prepares nanoparticles with a small size (particle size of about 70nm) that can pass through the blood-brain barrier. The platelets coupled with glucose can quickly target the damaged area and play a role in hemostasis and coagulation. After the platelets are broken, CaP is released. Due to the slightly acidic environment of the damaged area, CaP releases a large amount of Ca 2+ , amplifying the cascade reaction in the coagulation process and cooperating with platelets to achieve rapid hemostasis. The loaded COA-Cl can promote angiogenesis during injury and play a neuroprotective role. 2+ Influx into endothelial cells can also promote endothelial cell migration, adhesion, proliferation and angiogenesis.

[0065] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing nanoparticles that can target brain injury sites for the treatment of traumatic brain injury, characterized in that: The nanoparticles are BCaP-COA-Cl@Glu-PLT; The preparation method comprises the following steps: Step 1, BSA activation; Step 2, synthesis of BSA@COA: taking the activated BSA solution, adding COA-Cl solution for synthesis, and after the synthesis, ultrafiltration, taking the supernatant to obtain BSA@COA; Step 3, BSA@COA-CaP synthesis: Take the ultrafiltration supernatant in step 2, slowly add CaCl2 dropwise in multiple times, then continue to slowly add (NH4)2HPO4 dropwise in multiple times, adjust the pH to 10.0, ultrafilter, and take the supernatant to obtain BSA@COA-CaP; Step 4, synthesis of glycolipid DSPE-PEG-Glu: dissolve the uncarboxylated PEG lipid copolymer DSPE-PEG-2000-COOH in distilled water, add EDC and stir, then add NHS for activation, continue stirring, add glucosamine, and stir to react to obtain glycolipid DSPE-PEG-Glu; Step 5, glucose-modified platelets: platelets are mixed with DSPE-PEG-Glu and incubated with shaking to obtain platelets with glucose modified on the membrane surface; Step 6, preparation of BCaP-COA-Cl@Glu-PLT: BSA@COA-CaP was fully and evenly coated with glucose-modified platelets by co-extrusion to obtain BCaP-COA-Cl@Glu-PLT.

2. The preparation method according to claim 1, characterized in that The specific steps of step 1 are as follows: 120 mg BSA, 60 mg sodium dodecyl sulfate, and 4.4 mg dithiothreitol are weighed and added to a 5 mL sample bottle, 3 mL ultrapure water is added, and the mixture is fully dissolved, and the mixture is placed in an oil bath at 90° C. and the oil bath condition is 180 rpm for 2 h.

3. The preparation method according to claim 1, characterized in that The specific steps of step 2 are as follows: taking a 1 mL system as an example, 10 mg of activated BSA solution with a concentration of 40 mg / mL was taken, 950 μL of MES was added, and then 25 μL of 4 mg / mL COA-Cl solution was added. The mixture was shaken in a 24-well plate at 37°C and 800 r / min for 4 hours, and then washed two to three times with a 100 kd ultrafiltration tube at 4000 r / min for 10 minutes. The supernatant was taken to obtain BSA@COA.

4. The preparation method according to claim 1, characterized in that The specific steps of step 3 are as follows: take 2 mL of the supernatant obtained by ultrafiltration in step 2, place it in a 5 mL glass bottle, set the speed to 520 rpm, slowly add 1000 μL of 2.5 mM CaCl2, add 200 μL every 10 minutes, then continue to slowly add 1200 μL of 2.5 mM (NH4)2HPO4, add 200 μL every 10 minutes, adjust the pH value of the solution to 10.0 with ammonia water, stir for 30 minutes, and then use a 15 mL ultrafiltration tube, 4000 r / min, 10 minutes, ultrafiltration 1 to 2 times, take the supernatant, and obtain BSA@COA-CaP.

5. The preparation method according to claim 1, characterized in that The specific steps of step 4 are as follows: weighing the uncarboxylated PEG lipid copolymer DSPE-PEG-2000-COOH, dissolving it in distilled water, adding EDC at a molar ratio of DSPE-PEG-COOH:EDC:NHS of 1:15:15, stirring for 30 minutes, adding NHS to activate the carboxyl group of SPE-PEG-COOH, continuing to stir for 3 hours, and then adding glucosamine at a molar ratio of 35:1 to DSPE-PEG-2000-COOH, stirring at room temperature, and reacting for 24 hours to obtain the glycolipid DSPE-PEG-Glu.

6. The preparation method according to claim 1, characterized in that The specific steps of step 5 are: mixing the platelets with DSPE-PEG-Glu, shaking and incubating in a water bath shaker for 2 hours to obtain platelets with glucose modified on the membrane surface.

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