A nanocapsule for brain delivery system, preparation method and application thereof

Abstract

The present invention relates to a nanocapsule for a brain delivery system, a preparation method and application thereof, and belongs to the technical field of nanomedicine. The present invention uses 2-methacryloyloxyethyl phosphorylcholine and N-(3-aminopropyl) methacrylamide as monomers, N,N'-methylenebisacrylamide as a crosslinker, and a nanomaterial formed by free radical polymerization. Bovine serum albumin is used as a model protein to wrap inside the nanomaterial to form a nanocapsule, and naloxone molecules are modified on the surface of the nanocapsule to specifically bind to brain opioid receptors to form a new type of protein drug nanocarrier that efficiently crosses the blood-brain barrier mediated by receptors.

Description

Technical Field

[0001] The invention relates to a nano capsule for a brain delivery system, a preparation method and application thereof, and belongs to the technical field of nano medicines. Background Art

[0002] The blood-brain barrier (BBB) ​​is a physiological barrier that protects the brain from blood toxins and infectious substances. It is mainly composed of endothelial cells, pericytes and astrocytes. If it is not actively transported, the blood-brain barrier can block 98% of molecules from passing through. Therefore, the blood-brain barrier has become an insurmountable obstacle for brain drug delivery.

[0003] In recent years, scientists have proposed various strategies to circumvent the blood-brain barrier. For example, brain targeting mediated by adsorption can be achieved because the negatively charged BBB can interact with positively charged drug delivery systems on the basis of electrostatic effects, thereby promoting drug passage through the blood-brain barrier. However, poor selectivity is an inherent disadvantage of adsorption-mediated targeting because most biological membranes are negatively charged. For this reason, adsorption-mediated targeting is rarely used for systemic drug delivery. In addition, compounds or nanoparticles can be allowed to diffuse directly into the brain by temporarily opening the BBB. Although the BBB can be temporarily opened by several physical or chemical methods, these methods all cause certain damage to the BBB. Summary of the invention

[0004] The purpose of the present invention is to address the defects of the prior art and to propose a nanocapsule for a brain delivery system, and a preparation method and application thereof.

[0005] The present invention solves the technical problem through the following technical solutions: The present invention first provides a nanomaterial for a brain delivery system, which is composed of nanocapsules modified with naloxone hydrochloride derivatives. The naloxone hydrochloride derivatives have free carboxyl groups. The nanocapsules are formed by 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl) methacrylamide, N,N'-methylenebisacrylamide, ammonium persulfate, tetramethylethylenediamine, and bovine serum albumin as a model protein.

[0006] Endogenous opioid peptides are mainly β-endorphin (β-EP), which is mostly distributed in the hypothalamus, pituitary gland, cerebral cortex, brainstem, diaphragm and cerebrospinal fluid. β-EP in the blood is mainly released by the pituitary gland, and it plays an important regulatory role in analgesia, endocrine activity, neuropsychiatric and respiratory circulatory systems. Naloxone hydrochloride, trade name Narcan, is a derivative of hydroxydihydromorphone. It is a pure antagonist of opioid receptors without agonist activity. Its affinity for opioid receptors is greater than that of morphine and enkephalin, and it can competitively block and replace the binding of opioid substances to receptors. Naloxone has a high lipid solubility and can be quickly distributed throughout the body, especially in the brain, heart, lungs, and kidneys. 5 minutes after administration, its distribution in the brain is 4-6 times higher than that in plasma.

[0007] Therefore, based on the property of naloxone that can easily cross the BBB, it is combined with nanocapsules that can improve drug stability to form a new type of protein drug nanocarrier that can efficiently cross the BBB mediated by opioid receptors. The application of this new nanocarrier will provide a new solution for cross-BBB brain drug delivery.

[0008] The present invention further provides a method for preparing the material, specifically a method for preparing a nanomaterial for a brain delivery system, comprising the following steps:

[0009] The first step is to modify the hydroxyl group on the benzene ring of the naloxone molecule to produce a free carboxyl group;

[0010] The second step is to prepare nanomaterials by free radical polymerization using 2-methacryloyloxyethyl phosphorylcholine and N-(3-aminopropyl) methacrylamide as monomers, N,N'-methylenebisacrylamide as a crosslinker, ammonium persulfate and tetramethylethylenediamine as initiators, and bovine serum albumin as a model protein to form nanocapsules;

[0011] The third step is to couple the compound from the first step with the nanocapsules from the second step to obtain nanocapsules with naloxone as the surface modification.

[0012] In the first step of the above method, the modified step is naloxone Figure 1 (A) reacts with tert-butyl bromoacetate to obtain the intermediate compound naloxone-tert-butyl acetate, i.e., compound 1 Figure 1 (B); Use trifluoroacetic acid to remove the tert-butyl group of compound 1 to obtain a free carboxyl group, namely compound 2 Figure 1 (C). The reaction is as follows:

[0013] In the second step, 2-methacryloyloxyethyl phosphorylcholine: N-(3-aminopropyl) methacrylamide: N,N'-methylenebisacrylamide: ammonium persulfate: tetramethylethylenediamine: bovine serum albumin = 10000:10:5000:300:10:1, 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl) methacrylamide, N,N'-methylenebisacrylamide, tetramethylethylenediamine, bovine serum albumin, and ammonium persulfate are added to the centrifuge tube in proportion in this order, reacted on a shaker overnight, and purified by a hydrophobic column to remove free protein to obtain nanocapsules.

[0014] In the third step, the naloxone-modified compound 2 is first dissolved in 1×PBS, the pH is adjusted to 5-6 using a dilute HCL solution, 1.2 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and after about 1 minute, the pH is adjusted to about 8-9 using a dilute NaOH solution, and 1.2 equivalents of N-hydroxysuccinimide are added, and the nanocapsules are added at a ratio of nanocapsules: compound 2 = 1:100 for 2-3 minutes. The concentration of the nanocapsules is based on the concentration of the protein they encapsulate.

[0015] The present invention further provides the use of nanomaterials for brain delivery systems, including the use of nanomaterials in the preparation of brain-targeted drugs.

[0016] The application also includes the use of nanomaterials in the preparation of drugs for delivering macromolecular therapeutic drugs to sites of brain diseases.

[0017] The present invention uses 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3-aminopropyl) methacrylamide (APM) as monomers, N,N'-methylenebisacrylamide (BIS) as a cross-linking agent, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) as initiators, and utilizes a nanomaterial formed by free radical polymerization. Bovine serum albumin (BSA) is used as a model protein to wrap the nanomaterial inside to form a nanocapsule, and naloxone molecules are modified on the surface of the nanocapsule to specifically bind to brain opioid receptors to form a novel protein drug nanocarrier that efficiently crosses the blood-brain barrier mediated by receptors.

[0018] Through research, it was found that it can effectively transport large molecular proteins to the brain. Compared with natural BSA, after loading into the nanocapsules modified with naloxone, the amount of BSA accumulated in the brain was 8 times higher. At the same time, compared with the previously studied nBSA-PMPC nanocarriers with brain targeting function, whether it is administered through the tail vein, eye drops, or nose drops, the nBSA-PMPC-Naloxone nanoparticles modified with naloxone have a better brain targeting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Naloxone modification steps.

[0020] Figure 2 H-NMR spectrum of compound B.

[0021] Figure 3 H-NMR spectrum of compound C.

[0022] Figure 4 MALDI-TOF MS spectrum of BSA.

[0023] Figure 5 MALDI-TOF MS spectrum of BSA-Naloxone.

[0024] Figure 6 DLS measurement results of particle size of nBSA-PMPC-Naloxone and BSA.

[0025] Figure 7 TEM of nBSA-PMPC-Naloxone.

[0026] Figure 8 Zeta potential of nBSA-PMPC-Naloxone and BSA

[0027] Fig. 9 Fluorescence imaging of mouse brain tissue after administration of nBSA-PMPC and nBSA-PMPC-Naloxone via the tail vein.

[0028] Fig.10 Quantification of ROI in fluorescent imaging of mouse brain tissue after administration of nBSA-PMPC and nBSA-PMPC-Naloxone via tail vein.

[0029] Fig.11 Fluorescence intensity of mouse brain tissue trituration solution after administration of nBSA-PMPC and nBSA-PMPC-Naloxone via tail vein.

[0030] Fig.12 Fluorescence imaging of mouse brain tissue after ocular administration of nBSA-PMPC and nBSA-PMPC-Naloxone.

[0031] Fig.13 Quantification of ROIs by fluorescent imaging of mouse brain tissue after ocular administration of nBSA-PMPC and nBSA-PMPC-Naloxone.

[0032] Fig.14 Fluorescence intensity of mouse brain tissue trituration solution after ocular administration of nBSA-PMPC and nBSA-PMPC-Naloxone.

[0033] Fig.15 Fluorescence imaging of mouse brain tissue after nasal administration of nBSA-PMPC and nBSA-PMPC-Naloxone.

[0034] Fig.16 Quantification of ROI in fluorescent imaging of mouse brain tissue after intranasal administration of nBSA-PMPC and nBSA-PMPC-Naloxone.

[0035] Fig.17 Fluorescence intensity of mouse brain tissue trituration solution after nasal administration of nBSA-PMPC and nBSA-PMPC-Naloxone. DETAILED DESCRIPTION

[0036] The technical solutions of the embodiments of the present invention will be described clearly and completely below. Example

[0037] 1. Modification of Naloxone

[0038] Experimental steps:

[0039] (1) Figure 1 A (800 mg, 2.198 nmol) and 5 eq of potassium carbonate (1518.8 mg) were added to 10 ml of acetonitrile and stirred at 50 degrees for 30 min. Then 2 eq of tert-butyl bromoacetate was added and the temperature was raised to 90 degrees. The reaction time was monitored by TLC and was about 2 hours. The reaction solution was filtered and the obtained liquid phase was rotary evaporated to obtain a yellow oily liquid which was purified by silica gel column chromatography (mobile phase dichloromethane: methanol = 20:1). H-NMR of compound B is shown in Figure 2 .

[0040] (2) Figure 1 Add 2 ml of trifluoroacetic acid to B (100 mg\0.226 nmol) and stir at room temperature for 1 h. After the reaction is completed by TLC detection, add 5 ml of chloroform three times each time and evaporate by rotary evaporation. Then add 5 ml of ether twice each time and evaporate by rotary evaporation. Add about 1 ml of ethanol to the obtained compound to completely dissolve it. Then add ether about 50 times the volume of ethanol, and a white solid precipitates. The white solid obtained by filtration is Figure 1 Compound C. H-NMR of compound C is shown in Figure 3 .

[0041] 2. Synthesis of Nanocapsules

[0042] BSA was dissolved in 1×PBS, and then MPC, APM, and BIS (10000:10:5000:300:10:1, n / n, MPC:APM: BIS: BSA) were added to the solution, and then APS (500:1, n / n, APS: BSA) and TEMED (1000:1, n / n, TEMED: BSA, pH=7.0) were added to initiate free radical polymerization and reacted at room temperature for 2 h. nBSA-PMPC was obtained. Purification and separation by hydrophobic column

[0043] 3. Conjugation of naloxone and nanocapsules

[0044] After compound C was dissolved in 1×PBS, the pH was adjusted to about 5-6 with dilute HCL solution. After adding the corresponding amount of EDC, the pH was adjusted to about 8-9 with dilute NaOH, and NHS was added. Finally, nBSA-PMPC (100:1.2:1.2:1, n / n, compound C: EDC: NHS: nBSA-PMPC) was added, and the reaction was shaken for 12 hours, and nBSA-PMPC-Naloxone was removed by dialysis.

[0045] 4. Characterization of Nanocapsules

[0046] 1 mL of the nanocapsules dialyzed in the PB solution was taken and subjected to a dynamic light scattering test using a Malvern Zetasizer Nano ZSE instrument to obtain the zeta potential of the nanocapsules.

[0047] 5. Cy7 fluorescent labeling

[0048] (1) Dissolve 1 mg sulfo-Cy7-NHS in 200 μl DMSO to a final concentration of 5 mg / ml in a dark environment. Wrap the mixture in aluminum foil and freeze it in a -20°C refrigerator in a dark environment until ready for use.

[0049] (2) nBSA-PMPC was adjusted to pH 8 with NaOH solution, and ulfo-Cy7-NHS was added to the system at a molar ratio of 1:3. The system was reacted at room temperature in the dark for 4 h.

[0050] (3) Perform dialysis in the dark at room temperature, changing the dialysis solution every 4 hours for 12 hours. 1. Specific implementation methods

[0052] 6. Tissue Imaging

[0053] (1) Tail vein injection: 8 mice were divided into two groups, 4 mice in each group, the experimental group (nBSA-PMPC-Naloxone) and the control group (nBSA-PMPC). Each mouse was given 25.5 mg / kg. After 10 hours, the mice were perfused by heart and the brain tissue was taken for fluorescence imaging.

[0054] (2) Nasal administration: Eight mice were divided into two groups with four mice in each group, namely the experimental group (nBSA-PMPC-Naloxone) and the control group (nBSA-PMPC). 50 μl of the sample was dripped into the nasal cavity of the mice using a pipette. Each mouse was given 6.4 mg / kg of the drug. After 10 hours, the mice were perfused by heart and the brain tissue was taken for fluorescence imaging.

[0055] (3) Ocular administration: Eight mice were divided into two groups with four mice in each group, namely the experimental group (nBSA-PMPC-Naloxone) and the control group (nBSA-PMPC). A total of 50 μl of the sample was dripped into both eyes of the mice using a pipette. Each mouse was given 1.28 mg / kg of the drug. After 10 hours, the mice were perfused by heart and the brain tissues were taken for fluorescence imaging.

[0056] 7. Fluorescence Assay

[0057] The fluorescence intensity of brain tissue was further quantitatively studied using a fluorescent microplate reader to determine the degree of aggregation of nBSA-PMPC-Naloxone-Cy7 in the brain.

[0058] (1) Place the removed brain tissue in a 4 ml centrifuge tube at a volume of 100 g / ml with 1× PBS.

[0059] (2) High-throughput tissue grinder, 50 Hz, 10 s. After three grindings, centrifuge in a high-speed centrifuge and collect the supernatant solution.

[0060] (3) Add 100 μl to each well of a black 96-well plate.

[0061] (4) Determination by fluorescence microplate reader.

[0062] 8. Experimental Results

[0063] go through Figure 1 The reaction of the step was analyzed by NMR results ( Figure 2-Figure 3 ) proved that Naloxone modification was successful. Figure 4-Figure 5 ) proved that Naloxone and BSA were successfully coupled through the condensation reaction between carboxyl and amino groups. DLS ( Figure 6 ) found that the particle size of nBSA-PMPC was about 30 nm, TEM ( Figure 7) imaging further confirmed this point. This particle size is obviously larger than that of natural BSA (<10 nm), and the change of zeta potential on the surface of the binding protein ( Figure 8 ) All of these indicate the successful synthesis of nBSA-PMPC.

[0064] In order to verify the ability of nBSA-PMPC-Naloxone to cross the BBB and deliver to the brain, we first observed the delivery of nBSA-PMPC-Naloxone in the rat brain by administering it through the rat tail vein. Figure 9-10 ) Results We found that the protein aggregation in the brain of mice treated with nBSA-PMPC was significantly lower than that of mice treated with nBSA-PMPC-Naloxone. The fluorescence intensity of brain tissue homogenate ( Fig.11 ) also further proved this conclusion. Figure 12-14 ) and intranasal administration in mice ( Figure 15-17 ), we also observed similar results. The protein aggregation in the brain of mice administered with nBSA-PMPC was significantly lower than that of mice administered with nBSA-PMPC-Naloxone. These results show that the nanocapsules modified with naloxone can significantly improve the ability of proteins to cross the BBB and accumulate in the brain in various routes of administration. They are combined with nanocapsules that can improve drug stability to form a new type of protein drug nanocarrier that efficiently crosses the BBB mediated by opioid receptors. Its application will provide a new solution for cross-BBB brain drug delivery.

[0065] In addition to the above implementations, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A nanomaterial for a brain delivery system, comprising nanocapsules modified with naloxone hydrochloride, wherein the preparation method comprises the following steps: The first step is to modify the hydroxyl group on the benzene ring of the naloxone molecule to produce a free carboxyl group; The second step is to prepare nanomaterials by free radical polymerization using 2-methacryloyloxyethyl phosphorylcholine and N-(3-aminopropyl) methacrylamide as monomers, N,N'-methylenebisacrylamide as a crosslinker, ammonium persulfate and tetramethylethylenediamine as initiators, and form nanocapsules using bovine serum albumin as a model protein; the third step is to couple the compound in the first step with the nanocapsule in the second step to obtain nanocapsules with naloxone as the surface modification; in the first step, the modification step is to react naloxone with tert-butyl bromoacetate to obtain the intermediate compound naloxone-ethyl The tert-butyl ester of the acid is compound B; the tert-butyl group of compound B is removed with trifluoroacetic acid to obtain compound C; in the third step, the naloxone-modified compound C is first dissolved in 1×PBS, the pH is adjusted to 5-6 with a dilute HCL solution, 1.2 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and after 1 minute, the pH is adjusted to about 8-9 with a dilute NaOH solution, and 1.2 equivalents of N-hydroxysuccinimide are added. After 2-3 minutes, the nanocapsules are added at a ratio of nanocapsules: compound C = 1:

100.

2. The nanomaterial for brain delivery system according to claim 1, characterized in that: The concentration of the nanocapsules is based on the concentration of the protein they encapsulate.

3. Use of the nanomaterial of the brain delivery system according to claim 1 in the preparation of brain-targeted drugs.

4. The use of the nanomaterial for brain delivery system according to claim 3, characterized in that: Includes the use of nanomaterials in the preparation of drugs for delivering macromolecular therapeutic drugs to sites of brain disease.

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