A brain-targeted nanocarrier and its preparation method and application

Abstract

This invention belongs to the field of drug carrier technology and discloses a brain-targeted nanocarrier, its preparation method, and application. The primary raw materials for preparing the brain-targeted nanocarrier include PLGA-PEG-NHS, D-mannosamine, and a sulfide donor. This brain-targeted nanocarrier utilizes the dual targeting effects of mannose and sulfide to not only target the brain but also effectively improve blood-brain barrier permeability. With its high targeting and high permeability, it can be used as a carrier to effectively deliver therapeutic drugs to the brain to exert therapeutic effects, and has promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug carriers, and in particular relates to a brain-targeted nanocarrier and a preparation method and application thereof. Background Art

[0002] In recent years, the incidence of neurological diseases caused by stressful lifestyles has been increasing. Clinical and basic research is attempting to use nanobiomaterials to deliver drugs for neurological diseases in order to improve the effectiveness of drug treatment. In medicine, nanobiomaterials can not only be used as bionic organs in the human body, but can also be used as drug carriers for drug delivery. As drug carriers, nanobiomaterials have the characteristics of high safety and long circulation, and can be modified with different ligands to give nanobiomaterials specific targeting properties. In terms of economic benefits, nanobiomaterials have simple components, are easy to prepare, and have stable properties, allowing for large-scale production. Although nanobiomaterials have excellent biocompatibility and easy modification, the obstacle of crossing the blood-brain barrier is the first problem that needs to be solved.

[0003] The blood-brain barrier (BBB) ​​is a specialized physiological structure composed of vascular endothelial cells, basement membranes, and neurons. As a crucial barrier protecting the brain, it resists the invasion of foreign substances and protects the central nervous system from harmful substances in the blood. The existence of the BBB significantly blocks the entry of drugs into the brain, limiting their therapeutic effectiveness.

[0004] For drugs to pass through this barrier, they must meet one of the following conditions: 1. Drugs with a molecular weight of 400-500Da and strong lipophilicity can freely pass through the blood-brain barrier; 2. Targeted modified nanocarriers are used to carry drugs to the blood-brain barrier, improving the permeability of the blood-brain barrier and thus increasing the concentration of drugs entering the brain.

[0005] Therefore, the present invention aims to provide a nanocarrier that targets the brain and can improve the permeability of the blood-brain barrier for delivering drugs to treat neurological diseases. Summary of the Invention

[0006] The present invention aims to address at least one of the technical problems existing in the aforementioned prior art. To this end, the present invention provides a brain-targeted nanocarrier, its preparation method, and its application. This brain-targeted nanocarrier utilizes the dual targeting effects of mannose and sulfide, not only targeting the brain but also effectively improving the permeability of the blood-brain barrier. With its high targeting and high permeability, it can be used as a carrier to effectively deliver therapeutic drugs to the brain to exert therapeutic effects, and has promising application prospects.

[0007] The invention provides a brain-targeted nanocarrier, the main preparation raw materials of which include PLGA-PEG-NHS, D-mannosamine and a sulfide donor.

[0008] This invention uses PLGA-PEG-NHS as the carrier material and modifies it with D-mannosamine and a sulfide donor. The resulting nanocarrier not only delivers therapeutic drugs to the brain through the targeting effect of mannose, but also releases hydrogen sulfide molecules through the sulfide donor it carries, thereby opening the blood-brain barrier. The dual targeting effects of mannose and sulfide achieve both high targeting and high penetration, making it an effective delivery vehicle for drugs treating neurological diseases.

[0009] Preferably, the PLGA-PEG-NHS is PLGA 5000 -PEG 5000 -NHS.

[0010] Preferably, the sulfide donor is PEG-DTC.

[0011] The present invention also provides a method for preparing the above-mentioned brain-targeted nanocarrier, comprising the following steps:

[0012] (1) PLGA-PEG-NHS and D-mannosamine were added to a buffer solution for dissolution, and the solution was adjusted to alkaline for reaction to prepare PLGA-PEG-Mannose;

[0013] (2) adding the PLGA-PEG-Mannose and the sulfide donor to a mixed solution of water and an organic solvent to react, and then removing the organic solvent to obtain the brain-targeted nanocarrier.

[0014] Preferably, the buffer solution in step (1) is selected from phosphate buffer.

[0015] Preferably, in step (1), the mass ratio of PLGA-PEG-NHS to D-mannosamine is (15-20):1.

[0016] Preferably, the organic solvent in step (2) is tetrahydrofuran or chloroform.

[0017] Preferably, in step (2), the mass ratio of PLGA-PEG-Mannose to sulfide donor is (3-8):1.

[0018] Preferably, nitrogen is used to remove the organic solvent in step (2).

[0019] Preferably, ultrasound-assisted reaction is used in step (2).

[0020] The present invention also provides the use of the aforementioned brain-targeted nanocarriers in the preparation of products for treating brain diseases. Specifically, the brain disease is a neurological disease. The brain-targeted nanocarriers proposed in the present invention can target the brain and increase the permeability of the blood-brain barrier, enabling targeted brain delivery of therapeutic drugs. They have promising application prospects in the preparation of products for treating brain diseases.

[0021] The present invention also provides a product for treating brain diseases, comprising the above-mentioned brain-targeted nanocarrier and a therapeutic drug.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention innovatively discovered that hydrogen sulfide can improve the permeability of the blood-brain barrier, promoting drug access to brain lesions. Based on this, the present invention leverages the brain-targeting effect of mannose molecules and the blood-brain barrier-opening function of hydrogen sulfide gas molecules to produce brain-targeted nanocarriers with high targeting and high permeability. The efficacy of these nanocarriers has been validated through experiments in cell and animal models.

[0024] Using the brain-targeted nanocarrier as a delivery vehicle for brain therapeutic drugs can effectively promote the entry of therapeutic drugs into the brain to exert therapeutic effects, providing new ideas and new solutions for the treatment of mental illnesses and brain tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 2 shows the construction of the in vitro blood-brain barrier model and the results of the permeability experiment in Example 2; A is the resistance graph of b.End3 cells monitored for 7 days; B is the fluorescence intensity graph of PP@P18, PPM@P18, and S-PPM@P18 passing through b.End3 cells at different time points.

[0026] Figure 2 Figures 1 and 2 show the results of in vivo imaging and organ distribution of healthy mice in Example 3; A shows in vivo imaging of PP@P18, PPM@P18, and S-PPM@P18 at different time points after intravenous administration; B shows the fluorescence intensity of PP@P18, PPM@P18, and S-PPM@P18 in the mouse brain; and C shows in vivo imaging of PP@P18, PPM@P18, and S-PPM@P18 in the isolated brain and organs 4 hours after administration.

[0027] Figure 3 This is a laser confocal image of the mouse brain tissue slice in Example 3, blue is DAPI, and red is the nanomaterial;

[0028] Figure 4The results of constructing the PTSD mouse model in Example 4; A is the movement trajectory of the mouse in the last 5 minutes; B is the statistical chart of the percentage of immobility time in the control group and the PTSD model group, ***P<0.001.

[0029] Figure 5 Figures 2 and 3 show the in vivo imaging and organ distribution results of the PTSD model mice in Example 4; A shows in vivo images taken at different time points after intravenous administration of PPM@P18 and S-PPM@P18; B shows in vivo images of the isolated brain and organs of PPM@P18 and S-PPM@P18 4 hours after administration; and C shows a statistical graph of fluorescence mean values ​​at different time points. DETAILED DESCRIPTION

[0030] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0031] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0032] Example 1: Construction of Nanomicelles PP@P18, PPM@P18, and S-PPM@P18

[0033] Using P18 as a simulated drug delivery system, the following nanomicelles were prepared:

[0034] (1)PP@P18

[0035] a. Prepare 4 ml of deionized water in a glass bottle and add 400 μL of tetrahydrofuran (THF) to obtain a mixed solution 1.

[0036] b. PLGA 5000 -PEG 5000 -NHS and P18 were dissolved in tetrahydrofuran at a mass ratio of 5:1, and tetrahydrofuran was added to 200 μL to obtain mixed solution 2.

[0037] c. Under ultrasonic conditions, mixed solution 2 was added to mixed solution 1. After ultrasonic treatment for 2 min, the organic solvent was removed with nitrogen to obtain nanomicelles PP@P18.

[0038] (2)PPM@P18

[0039] a. 50mg PLGA 5000 -PEG 5000-NHS was dissolved in a pH 7.4 phosphate buffer, followed by the addition of 3 mg of D-Mannosamine (D-mannosamine), and sonication was used to promote dissolution. Triethylamine was used to adjust the pH to approximately 8, and the reaction was allowed to proceed overnight. The reaction solution was collected and dialyzed to remove excess D-Mannosamine. PLGA was then freeze-dried to obtain the product. 5000 -PEG 5000 -Mannose white powder.

[0040] b. Prepare 4 ml of deionized water in a glass bottle and add 400 μL of tetrahydrofuran (THF) to obtain a mixed solution 1.

[0041] c. PLGA 5000 -PEG 5000 -Mannose and P18 were dissolved in tetrahydrofuran at a mass ratio of 5:1, and tetrahydrofuran was added to 200 μL to obtain mixed solution 2.

[0042] d. Under ultrasonic conditions, mixed solution 2 was added to mixed solution 1. After ultrasonic treatment for 2 min, the organic solvent was removed with nitrogen to obtain nanomicelles PPM@P18.

[0043] (3)S-PPM@P18

[0044] a. 50mg PLGA 5000 -PEG 5000 -NHS was dissolved in a pH 7.4 phosphate buffer, followed by the addition of 3 mg of D-Mannosamine, and sonication to promote dissolution. Triethylamine was used to adjust the pH to approximately 8, and the reaction was allowed to proceed overnight. The reaction solution was collected and dialyzed to remove excess D-Mannosamine. PLGA was then freeze-dried to obtain the product. 5000 -PEG 5000 -Mannose white powder.

[0045] b. Prepare 4 ml of deionized water in a glass bottle and add 400 μL of tetrahydrofuran (THF) to obtain a mixed solution 1.

[0046] c. PLGA 5000 -PEG 5000 -Mannose, PEG-DTC and P18 were dissolved in tetrahydrofuran at a mass ratio of 5:1:1, mixed evenly, and tetrahydrofuran was continuously added to 200 μL to obtain mixed solution 2.

[0047] d. Under ultrasonic conditions, mixed solution 2 was added to mixed solution 1. After ultrasonic treatment for 2 min, the organic solvent was removed with nitrogen to obtain nanomicelles S-PPM@P18.

[0048] Among them, PLGA 5000-PEG 5000 -NHS was purchased from Shanghai Maokang Biotechnology Co., Ltd. (model MS5903), P18 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (model S63823), and PEG-DTC was prepared using the method described in an article published in Angewandte Chemie International Edition (A Metal-Phenolic Nanosensitizer Performs Hydrogen Sulfide Reprogrammed Oxygen Metabolism for Cancer Radiotherapy Intensification and Immunogenicity).

[0049] Example 2: Construction of an in vitro blood-brain barrier model and permeability experiment

[0050] Mouse brain microvascular endothelial cells bEnd.3 were selected as an in vitro blood-brain barrier model, with 2×10 5 The cells were seeded in a 12-well Transwell chamber with a pore size of 0.4 μm. The upper chamber volume was 500 μL, and the lower chamber volume was 1 mL. The cells were incubated overnight at 37°C in 5% CO2, with fresh culture medium replaced every other day. The resistance of the bEnd.3 cells was measured using a resistance meter the next day. The in vitro blood-brain barrier model was considered successfully established when the resistance value reached a relatively stable state.

[0051] After the in vitro blood-brain barrier model was successfully established, drug administration began; 50 μg / mL of PP@P18, 50 μg / mL of PPM@P18, and 50 μg / mL of S-PPM@P18 were added to the Transwell chamber, and fresh culture medium was added to the lower layer. The fluorescence intensity of the lower layer was measured using a fluorescence spectrophotometer at 2, 4, and 8 hours, respectively.

[0052] The experimental results are as follows Figure 1 As shown, Figure 1 Middle A shows that the in vitro blood-brain barrier model has been successfully constructed; Figure 1 Figure B shows that when the fluorescence intensity of the lower layer P18 was monitored by a fluorescence spectrophotometer, the three groups of PP@P18, PPM@P18, and S-PPM@P18 all had a time-dependent ability to penetrate the blood-brain barrier; compared with the other groups, S-PPM@P18 showed a significant ability to improve the blood-brain barrier.

[0053] Example 3: Permeability test of healthy mice through the blood-brain barrier

[0054] Healthy 5-week-old male C57 mice were selected as the animal model. Brain hair was removed and 100 μg of PP@P18, 100 μg of PPM@P18, and 100 μg of S-PPM@P18 were administered via the tail vein. In vivo imaging was performed at 2, 4, 6, 8, 12, 24, 48, and 72 hours. Organs were removed and imaged to observe organ distribution. Brain tissue was fixed with 4% paraformaldehyde, dehydrated, and cryosectioned. Fluorescence distribution in brain sections was observed using confocal microscopy.

[0055] The results of in vivo imaging and organ distribution are as follows Figure 2 shown. Figure 2 Figures A and B show that S-PPM@P18 group began to accumulate in the brain 30 minutes after administration and reached the highest level at 4 hours. At 4 hours, the brain and organs were removed and fluorescence was photographed. Figure 2 As shown in Figure C, both PPM@P18 and S-PPM@P18 groups can reach the brain, while S-PPM@P18 group shows stronger penetration. Figure 3 As shown, the results are consistent with the in vivo imaging results.

[0056] Example 4: Construction of a PTSD animal model and permeability experiment

[0057] Eight-week-old male C57 mice were housed in an animal room for one week to familiarize them with the environment and reduce stress. They then underwent electric shock training (placed in a shock box for 5 minutes to acclimate) and two electric shocks (set up a 10-minute program, with the first 5 minutes as an acclimatization phase, without electricity; the last 5 minutes as a stimulation phase, with electricity applied at a current intensity of 0.8 mA every 10 seconds for 10 seconds each, for a total of 15 times). After the electric shocks, a situational fear experiment was performed on the fifth day to select animals for a successful PTSD model.

[0058] like Figure 4 As shown in the figure, the immobility time of mice in the model group was significantly increased, which was statistically different from that in the control group, indicating that the PTSD mouse model was successfully established.

[0059] The brains of established PTSD model mice were shaved, and 100 μg of PP@P18, 100 μg of PPM@P18, and 100 μg of S-PPM@P18 were administered via the tail vein. Live imaging of the animals was performed at 2, 4, 6, 8, 12, 24, 48, and 72 hours. Organ distribution was observed after the time series imaging.

[0060] The results of in vivo imaging and organ distribution are as follows Figure 5As shown, both S-PPM@P18 and PPM@P18 can reach the brain, and S-PPM@P18 shows a stronger advantage in the first 6 hours. Compared with the healthy mice in Example 3, S-PPM@P18 has a longer retention time in PTSD model mice, which can effectively increase the half-life of the drug and prolong the therapeutic effect.

[0061] The embodiments of the present application have been described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present application.

Claims

1. A brain-targeting nanocarrier, characterized in that: The main raw materials for preparing the brain-targeted nanocarrier include PLGA 5000 -PEG 5000 -NHS, D-mannosamine, and PEG-DTC; The method for preparing the brain-targeted nanocarrier comprises the following steps: (1) Add PLGA to the buffer solution 5000 -PEG 5000 -NHS and D-mannosamine were dissolved and adjusted to alkaline for reaction to prepare PLGA-PEG-Mannose; (2) adding the PLGA-PEG-Mannose and PEG-DTC to a mixed solution of water and an organic solvent to react, and then removing the organic solvent to prepare the brain-targeted nanocarrier.

2. The brain-targeting nanocarrier according to claim 1, characterized in that The buffer solution in step (1) is selected from phosphate buffer.

3. The brain-targeting nanocarrier according to claim 1, wherein In the step (1), PLGA 5000 -PEG 5000 The mass ratio of -NHS to D-mannosamine is (15-20):

1.

4. The brain-targeting nanocarrier according to claim 1, wherein The organic solvent in step (2) is tetrahydrofuran or chloroform.

5. The brain-targeting nanocarrier according to claim 1, wherein In the step (2), PLGA 5000 -PEG 5000 -NHS to PEG-DTC mass ratio is (3-8):

1.

6. Use of the brain-targeted nanocarrier according to claim 1 in preparing a product for treating brain diseases.

7. A product for treating brain diseases, characterized in that: The invention comprises the brain-targeting nanocarrier according to claim 1 and a therapeutic drug.

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