Brain-targeted nano drug delivery system, preparation method and application

Abstract

The invention belongs to the technical field of biological medicine, and discloses a brain-targeted nano drug delivery system, a preparation method and application. According to the invention, dipentaerythritol-polycaprolactone-histidine (DPE-PCL-HIS) is used as a basic drug carrier, and drug curcumin (Cur) molecules are entrapped in a physical embedding manner; and modifying bovine serum albumin (BSA) grafted with a bifunctional intermediate connection compound 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) on a precoating layer, and finally connecting an apolipoprotein E mimic peptide as a brain-targeting ligand, so as to construct the brain-targeting bionic protein crown nano drug delivery system, wherein the bovine serum albumin (BSA) is grafted with the bifunctional intermediate connection compound 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC). According to the prepared brain-targeted nano drug delivery system, apolipoprotein E mimic peptide [RLLRKRLK (PEG2000) RLGWC] is used as a ligand and enters the brain under the transendocytosis action of the apolipoprotein E mimic peptide [RLLRKRLK (PEG2000) RLGWC] and a blood-brain barrier (BBB) surface receptor, the blood-brain barrier permeability of a drug is increased, the brain targeting property and bioavailability of drug-loaded nanoparticles are improved, and the brain-targeted nano drug delivery system has wide application prospects in the aspect of treating brain diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a brain-targeted nano drug delivery system, a preparation method and a use thereof. Background Art

[0002] Effective treatment of brain diseases is a major challenge in clinical practice. The existence of the blood-brain barrier (BBB) ​​hinders the passage of most drugs. Any complex nanocarrier will interact with proteins to form a protein corona during the passage through biological fluids, thus affecting the targeting. However, most studies have shown that protein-corona modified nanodrug delivery systems exhibit good tumor targeting and low clearance effects in the treatment of brain diseases. Albumin as a protein corona modified nanoparticle has the effect of reducing plasma protein adsorption and complement activation (C3b, C4b), and is therefore widely used in the design of nanodrug delivery systems.

[0003] Curcumin is a natural phenolic antioxidant extracted from ginger plants. It has good anti-inflammatory and anti-cancer properties and good biosafety. However, due to its poor water solubility, stability, and low in vivo bioavailability, its clinical application is limited (Chen Rong, Yan Ye, He Zixuan, et al. Research progress on strategies and technologies to improve the efficacy of curcumin [J]. Chinese Herbal Medicine, 2024, 55(15): 5315-5330.); Studies have shown that curcumin can significantly inhibit the occurrence and development of gliomas. It can achieve anti-tumor effects through a variety of molecular mechanisms and has great potential in the treatment of gliomas (Fan Yang, Zhang Xi'an, Liu Zhanhui. Research progress on curcumin in the treatment of gliomas [J]. Progress in Clinical Medicine, 2024, 14(6): 1030-1036.).

[0004] Studies have shown that apolipoprotein E is one of the main apolipoproteins in the central nervous system and is involved in the pathophysiology of neurodegenerative diseases. When acting as a ligand, it has a high affinity for LDL and LRP1 receptors on the surface of the blood-brain barrier. Therefore, apolipoprotein E can mediate the efficient binding of drug molecules or nanocarriers to the blood-brain barrier. However, due to its large molecular weight, apolipoprotein E is not easy to cross the BBB, so its application is limited. Currently, researchers have developed many apolipoprotein E small molecule mimetic peptides, which can better simulate natural apolipoprotein E, maintain its binding ability to receptors such as LDL and LRP1 by retaining the specific lipid binding domain of apolipoprotein E, and minimize the molecular weight of the mimetic peptide to give it an advantage in BBB penetration. It is becoming a potential treatment for central nervous system diseases. Summary of the invention

[0005] The purpose of the present invention is to improve the defects of traditional nano drug delivery systems in brain targeting applications and to optimize the deficiencies in the targeting research strategy of biomimetic protein crown nanoparticles, and to provide a brain targeting nano drug delivery system, preparation method and use.

[0006] In order to achieve the above objectives, the present invention adopts the following technical scheme: As a first aspect, the present invention provides a brain-targeted nano drug delivery system, comprising a brain-targeted peptide, a protein, a bifunctional intermediate connecting compound, a polymer material, and an anti-tumor drug, wherein the molar ratio of the brain-targeted peptide to the protein is 50:1 to 500:1; the molar ratio of the bifunctional intermediate connecting compound to the protein is 2:1 to 20:1; the mass ratio of the protein to the polymer material is 1:1 to 10:1; the mass ratio of the polymer material to the anti-tumor drug is 5:1 to 25:1.

[0007] Furthermore, the high molecular polymer material is dipentaerythritol-polycaprolactone-histidine, and the molecular weight is 8000-12000.

[0008] Furthermore, the brain targeting peptide is an apolipoprotein E mimetic peptide, the sequence of which is [RLLRKRLK(PEG2000)RLGWC] and the molecular weight is 3698.13.

[0009] Furthermore, the protein is bovine serum albumin, and the anti-tumor drug is curcumin with a purity of 98%.

[0010] Furthermore, the bifunctional intermediate connecting compound is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt, which is used to connect the protein and the brain targeting peptide.

[0011] As a second aspect, the present invention also provides a method for preparing the above-mentioned brain-targeted nano drug delivery system, comprising the following steps:

[0012] (1) reacting dipentaerythritol-polycaprolactone and Boc-histidine at a molar ratio of 1:15 to 1:60 under nitrogen protection at room temperature with stirring for 24 hours to allow the hydroxyl group at the end of dipentaerythritol-polycaprolactone to undergo an esterification reaction with the carboxyl group of Boc-histidine to obtain a first reaction solution;

[0013] (2) adding trifluoroacetic acid in an ice bath, wherein the volume of trifluoroacetic acid is 40-60% of the total volume of the first reaction solution, and reacting at room temperature for 2-3 hours with stirring to remove the tert-butyloxycarbonyl group of the Boc-histidine protecting group;

[0014] (3) washing the first reaction liquid with a saturated sodium bicarbonate aqueous solution, extracting the dichloromethane oil phase layer, and then extracting and washing with dichloromethane 2-3 times, adding anhydrous sodium sulfate to remove water, filtering and dropping the filtrate into ice ether for precipitation, suction filtering, and freeze-drying at -60°C to constant weight to obtain a high molecular polymer material dipentaerythritol-polycaprolactone-histidine;

[0015] (4) Dissolving the freeze-dried dipentaerythritol-polycaprolactone-histidine and curcumin in acetone at a mass ratio of 5:1 to 25:1, adding the solution to a vitamin E polyethylene glycol succinate aqueous solution containing PBS at pH = 7.4, stirring at room temperature for 30 minutes to 1 hour, and vacuum drying until the acetone is removed to obtain a drug-loaded nanoparticle Cur / DPE-PCL-HIS NPs solution; the concentration of the vitamin E polyethylene glycol succinate aqueous solution is 0.5 mg / ml to 2 mg / ml, the added volume is 30% to 70% of the total volume of the drug-loaded nanoparticle Cur / DPE-PCL-HIS NPs solution, and the volume of the pH = 7.4 PBS is 35% to 70% of the total volume of the vitamin E polyethylene glycol succinate aqueous solution;

[0016] (5) dissolving bovine serum albumin and sulfoSMCC in a molar ratio of 1:2 to 1:20 in PBS at pH 7.4, stirring at room temperature for 5 to 6 hours to allow the amino groups on the surface of BSA to react specifically with the active ester structure of sulfoSMCC, thereby obtaining a second reaction solution;

[0017] (6) adding the second reaction solution into a dialysis bag and dialyzing for more than 24 hours, and freeze-drying at -60°C to obtain BSA-sulfoSMCC;

[0018] (7) The Cur / DPE-PCL-HIS NPs solution and BSA-sulfoSMCC are mixed at a mass ratio of 1:1 to 1:10 and incubated for 4 to 6 hours, the mixed solution after incubation is transferred to an ultrafiltration tube, centrifuged at a speed of 3000×g / min for 10 min / time, and washed and resuspended with PBS to obtain a biomimetic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSA NPs solution;

[0019] (8) The bionic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSANPs solution and the apolipoprotein E mimetic peptide are mixed at a molar ratio of 1:50 to 1:500 and incubated for 4 to 6 hours. The mixed solution after incubation is transferred to an ultrafiltration tube, centrifuged at a speed of 3000×g / min for 10 min / time, and washed and resuspended with PBS to obtain the apolipoprotein E mimetic peptide-modified brain-targeted bionic protein corona nanodrug delivery system Cur / DPE-PCL-HIS / BSA-Peptide NPs, wherein the molar ratio of the bionic albumin corona nanoparticle solution to the apolipoprotein E mimetic peptide is the ratio of BSA-sulfoSMCC bound to the apolipoprotein E mimetic peptide on the surface of the bionic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSA NPs.

[0020] Furthermore, before dipentaerythritol-polycaprolactone and Boc-histidine are stirred and reacted at room temperature under nitrogen protection conditions, the method also includes: pre-dissolving Boc-histidine in dichloromethane, adding 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N-diisopropylethylamine catalyst, and activating at room temperature for 2 to 3 hours; wherein the ratio of Boc-histidine to dichloromethane is 1 mol / L, and the molar ratio of Boc-histidine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N-diisopropylethylamine catalyst is 6:6:7:20.

[0021] Preferably, the molecular weight cut-off of the dialysis bag is 8000-14000.

[0022] Furthermore, it also includes: after obtaining the bionic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSA NPs solution, determining the BSA-sulfoSMCC content in the bionic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSA NPs solution by the BCA method.

[0023] As a third aspect, the present invention also provides a use of the above-mentioned brain-targeted nano-drug delivery system in the preparation of drugs for treating brain diseases.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The present invention is reasonably designed and constructs a novel biomimetic protein crown coated nanoparticle, which is based on dipentaerythritol-polycaprolactone-histidine (DPE-PCL-HIS) as a basic drug carrier, encapsulates drug curcumin (Cur) molecules in a physical embedding manner, and then pre-coats and modifies bovine serum albumin connected with a bifunctional intermediate connecting compound 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), and finally connects an apolipoprotein E mimetic peptide as a brain targeting ligand, thereby solving the problem that traditional nano drug delivery systems are easily cleared in vivo. Most importantly, the nanoparticle has good brain targeting and has great potential in the treatment of brain diseases. This brain-targeted nano-drug delivery system improves the problems of curcumin (Cur), such as poor water solubility, low bioavailability and poor blood-brain barrier permeability. The modification of the albumin corona on the surface of the nanoparticles improves the biocompatibility of the nanoparticles. At the same time, this brain-targeted nano-drug delivery system uses the apolipoprotein E-mimicking peptide on the surface of the biomimetic protein corona nanoparticles to bind to the low-density lipoprotein receptor on the surface of the blood-brain barrier and penetrate the BBB through transcytosis, thereby improving the ability of the nanocarrier to cross the BBB and the bioavailability of the drug, and reducing the toxic side effects of the drug.

[0026] 2. The brain-targeted nano-drug delivery system prepared by the present invention has good biocompatibility and prolonged in vivo circulation time. It has been proven to have lower cytotoxicity and lower macrophage clearance effect in vitro. Fluorescence can still be observed in brain tissue and major organ tissues 48 hours after administration via rat tail vein injection.

[0027] 3. The brain-targeted nano drug delivery system prepared by the present invention can be used for intravenous injection, and can selectively enrich the drug at the brain tumor site to achieve a better drug effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the synthetic reaction formula of brain-targeted nano drug delivery system;

[0029] Figure 2 It is the infrared spectra of the synthesized high molecular polymer nanocarrier materials DPE-PCL and DPE-PCL-HIS;

[0030] Figure 3 The figures are hydrogen NMR spectra of synthesized high molecular polymer nanocarrier materials DPE-PCL and DPE-PCL-HIS; wherein (A) is the hydrogen NMR spectrum of synthesized DPE-PCL, (B) is the hydrogen NMR spectrum of synthesized DPE-PCL-HIS, (C) is the hydrogen NMR spectrum of part of DPE-PCL in Figure (A), and (D) is the hydrogen NMR spectrum of part of DPE-PCL-HIS in Figure (B);

[0031] Figure 4 (A) is a high performance liquid chromatogram of BSA-sulfoSMCC, and (B) is a high performance liquid chromatogram of BSA-sulfoSMCC-ApoE Peptide obtained by the reaction of BSA-sulfoSMCC and apolipoprotein E mimetic peptide;

[0032] Figure 5 (A) and (B) are the particle size distribution and Zeta potential distribution of the prepared nanodrug nanoparticles Cur / DPE-PCL-HIS, Cur / DPE-PCL-HIS / BSA, and Cur / DPE-PCL-HIS / BSA-Peptide NPs, respectively;

[0033] Figure 6 (A), (B), and (C) are transmission electron micrographs of the prepared nanodrug nanoparticles Cur / DPE-PCL-HIS, Cur / DPE-PCL-HIS / BSA, and Cur / DPE-PCL-HIS / BSA-Peptide NPs, respectively;

[0034] Figure 7 It is the in vitro release curve of the brain-targeted nano drug delivery system under the conditions of pH = 7.4 and pH = 5.6;

[0035] Figure 8 Free DIR, DIR / DPE-PCL-HIS nanoparticles, DIR / DPE-PCL-HIS / BSA nanoparticles, and DIR / DPE-PCL-HIS / BSA-Peptide nanoparticles were injected into the tail vein of SD rats with brain glioma, and the fluorescence distribution of each treatment group and the fluorescence distribution of the brain of each group of SD rats after sacrifice were measured by small animal living imaging. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0037] The invention adopts the independently synthesized high molecular polymer material dipentaerythritol-polycaprolactone-histidine (DPE-PCL-HIS) as the drug carrier. The end group modification of histidine (HIS) makes the nanomaterial have a certain pH responsiveness, which is beneficial to the release of the drug. In addition, after the drug is encapsulated by the physical embedding method, it is positively charged, which is beneficial to the adsorption of bovine serum albumin (BSA) on its surface, which not only improves the biocompatibility of the nanoparticles, but also reduces the toxicity of the nanomaterial. Finally, the surface of bovine serum albumin (BSA) is pre-modified. The modified 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfoSMCC) is linked to the modified apolipoprotein E mimetic peptide on its surface as a brain-targeting ligand. The apolipoprotein E mimetic peptide can better replace apolipoprotein E to bind to the BBB surface receptor and then enter the brain through transcytosis. Its molecular weight is much smaller than that of apolipoprotein E and is conducive to penetrating the BBB to reach the brain. Therefore, the constructed brain-targeted nano-drug delivery system has good biocompatibility, BBB targeting and penetration.

[0038] The present invention provides a preparation method and application of a brain-targeted nano drug delivery system, which is mainly composed of a brain-targeted peptide, a protein, a bifunctional intermediate connecting compound, a high molecular polymer material, and an anti-tumor drug. The molar ratio of the brain-targeted peptide to the protein incubation is 50:1-500:1; the molar ratio of the bifunctional intermediate connecting compound to the protein connection is 2:1-20:1; the mass ratio of the protein to the high molecular polymer material incubation is 1:1-10:1; and the mass ratio of the high molecular polymer material to the anti-tumor drug is 5:1-25:1.

[0039] The high molecular polymer material described in the present invention is dipentaerythritol-polycaprolactone-histidine (DPE-PCL-HIS); the brain targeting peptide is an apolipoprotein E mimetic peptide, whose sequence is [RLLRKRLK(PEG2000)RLGWC] and whose molecular weight is 3698.13; the protein corona is composed of bovine serum albumin (BSA); the bifunctional intermediate connecting compound is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfoSMCC), which can connect the protein corona and the brain targeting peptide; the anti-tumor drug is curcumin (Cur) with a purity of 98%.

[0040] Among them, the brain-targeted nanodrug delivery system is a nanoparticle with a particle size of 80 to 200 nm, a particle size distribution PDI of 0.12 to 0.19, and a potential of -3.12 to -6.14 mv.

[0041] The present invention provides a method for preparing a brain-targeted nano drug delivery system, which comprises the following steps:

[0042] (1) Boc-histidine (Boc-HIS) was pre-dissolved in dichloromethane, and 1-hydroxybenzotriazole (HOBT), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N,N-diisopropylethylamine (DIPEA) catalyst were added, and activated at room temperature for 2 to 3 hours, wherein the ratio of Boc-histidine to dichloromethane was 1 mol / L, and the molar ratio of Boc-histidine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N-diisopropylethylamine catalyst was 6:6:7:20. Then, dipentaerythritol-polycaprolactone (DPE-PCL) and Boc-HIS were stirred at room temperature for 24 hours under nitrogen protection conditions at a molar ratio of 1:15 to 1:60, so that the hydroxyl group at the end of DPE-PCL and the carboxyl group of Boc-HIS were esterified to obtain a first reaction solution;

[0043] (2) adding trifluoroacetic acid (TFA) to the first reaction solution under ice bath, stirring at room temperature for 2 to 3 hours to remove the Boc-histidine protecting group tert-butyloxycarbonyl (Boc group);

[0044] (3) The reaction solution was washed with a saturated sodium bicarbonate (NaHCO3) aqueous solution, the dichloromethane oil phase layer was extracted with a separatory funnel, and then extracted and washed with dichloromethane 2-3 times, anhydrous sodium sulfate (NaSO4) was added to remove water, and the filtrate was dropped into 250 ml of ice ether for precipitation after filtration, suction filtered, and freeze-dried at -60°C to obtain a high molecular polymer material dipentaerythritol-polycaprolactone-histidine (DPE-PCL-HIS);

[0045] (4) The DPE-PCL-HIS and Cur obtained after freeze-drying were dissolved in acetone at a mass ratio of 5:1 to 25:1, added to a vitamin E polyethylene glycol succinate (TPGS) aqueous solution containing phosphate buffer (PBS) at pH = 7.4, stirred at room temperature for 30 minutes to 1 hour, and placed in a vacuum drying oven for volatilization to remove acetone, thereby obtaining a drug-loaded nanoparticle (Cur / DPE-PCL-HISNPs) solution;

[0046] (5) dissolving bovine serum albumin (BSA) and sulfoSMCC in a molar ratio of 1:2 to 1:20 in phosphate buffered saline (PBS) at pH 7.4, stirring at room temperature for 5 to 6 hours to allow the amino groups on the surface of BSA to react specifically with the active ester structure of sulfoSMCC, thereby obtaining a second reaction solution;

[0047] (6) collecting the reaction solution of bovine serum albumin (BSA) and sulfoSMCC (second reaction solution), adding it to a dialysis bag for dialysis for more than 24 hours, and freeze-drying it at -60°C to obtain BSA-sulfoSMCC;

[0048] (7) The Cur / DPE-PCL-HIS NPs solution prepared in (4) and the BSA-sulfoSMCC prepared in (6) were mixed at a mass ratio of 1:1 to 1:10 and incubated for 4 to 6 hours, the mixed solution after incubation was transferred to an ultrafiltration tube (MW 100 kDa), centrifuged at 3000 × g / min for 10 min / time, and washed and resuspended with PBS to obtain a biomimetic albumin corona nanoparticle (Cur / DPE-PCL-HIS / BSA NPs) solution, and the BSA-sulfoSMCC content in the biomimetic albumin corona nanoparticle (Cur / DPE-PCL-HIS / BSANPs) solution was determined;

[0049] (8) The bionic albumin corona nanoparticle solution prepared in (7) was mixed with apolipoprotein E mimetic peptide at a molar ratio of 1:50 to 1:500 and incubated for 4 to 6 hours. The mixed solution after incubation was transferred to an ultrafiltration tube (MW 100 kDa), centrifuged at 3000 × g / min for 10 min / time, and washed and resuspended with PBS to obtain apolipoprotein E mimetic peptide-modified brain-targeted bionic protein corona nanodrug delivery system (Cur / DPE-PCL-HIS / BSA-Peptide NPs).

[0050] Wherein, the volume of trifluoroacetic acid (TFA) added in step (2) is 40-60% of the total volume of the reaction solution.

[0051] Wherein, the concentration of the vitamin E polyethylene glycol succinate (TPGS) aqueous solution described in step (4) is 0.5 mg / ml to 2 mg / ml, the added volume is 30% to 70% of the total volume of the drug-loaded nanoparticles (Cur / DPE-PCL-HIS NPs) solution, and the volume of the added pH=7.4 phosphate buffer (PBS) is 35% to 70% of the total volume of the polyethylene glycol succinate (TPGS) aqueous solution.

[0052] Wherein, the molecular weight cut-off of the dialysis bag used in step (6) is 8000-14000.

[0053] The drug-loaded nanoparticles (Cur / DPE-PCL-HIS NPs) described in step (7) are combined with bovine serum albumin (BSA) through electrostatic forces, van der Waals forces, hydrogen bonds and other interaction forces, and the BSA-sulfoSMCC content is determined by the BCA method.

[0054] In which, the apolipoprotein E mimetic peptide in step (8) is linked to the surface of biomimetic albumin corona nanoparticles (Cur / DPE-PCL-HIS / BSA NPs) through sulfoSMCC modified on the surface of bovine serum albumin (BSA), the molar ratio of the biomimetic albumin corona nanoparticle solution to the apolipoprotein E mimetic peptide is the ratio of BSA-sulfoSMCC bound to the surface of biomimetic albumin corona nanoparticles (Cur / DPE-PCL-HIS / BSA NPs) to the apolipoprotein E mimetic peptide, and the method for linking the apolipoprotein E mimetic peptide and sulfoSMCC is preferably to react and connect the thiol group of the apolipoprotein E mimetic peptide with the maleimide group at one end of the sulfoSMCC.

[0055] The brain-targeted nano-drug delivery system of the present invention and the system without modified brain-targeted head group were loaded with fluorescent dye DIR and injected into the tail vein of SD rats. The in vivo distribution characteristics of the nano-drug delivery system were investigated using a small animal in vivo imaging device. The observation results showed that the accumulation of the brain-targeted peptide-modified group in the brain was significantly higher than that of the non-targeted group. The results indicate that apolipoprotein E mimetic peptide can efficiently mediate the entry of the brain-targeted nano-drug delivery system into the brain.

[0056] The brain-targeted nano drug delivery system constructed by the present invention is suitable for targeting brain glioma cells.

[0057] Example 1

[0058] Synthetic polymer material DPE-PCL-HIS

[0059] 30mmol Boc-histidine (Boc-HIS) was pre-dissolved in 30ml dichloromethane, and 30mmol 1-hydroxybenzotriazole (HOBT), 35mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 100mmol N,N-diisopropylethylamine (DIPEA) catalyst were added, and activated at room temperature for 2-3 hours. Then, 0.5mmol dipentaerythritol-polycaprolactone (DPE-PCL) synthesized in advance was added, and the reaction was stirred at room temperature for 24 hours under nitrogen protection to obtain a reaction solution containing DPE-PCL-HIS product.

[0060] Example 2

[0061] Purification of polymer materials DPE-PCL-HIS

[0062] The reaction solution containing DPE-PCL-HIS product was washed with 200 ml saturated sodium bicarbonate solution (pH 7.0-8.0), the lower oil phase was extracted, and then extracted and washed 2-3 times with an appropriate amount of dichloromethane, an appropriate amount of anhydrous sodium sulfate solid was added to absorb water, filtered, and the filtrate was precipitated in 250 ml of ice ether, filtered, and the obtained product was collected, freeze-dried at -60 ° C to constant weight, and analyzed by nuclear magnetic resonance hydrogen spectrum. 1 The structure of the compound was analyzed by H-NMR and Fourier transform infrared spectroscopy FT-IR. Figure 2 and Figure 3 A, B, C, D.

[0063] Example 3

[0064] Preparation of Cur / DPE-PCL-HIS nanoparticles

[0065] Take 5 mg DPE-PCL-HIS and 0.25 mg curcumin, dissolve them in 1 ml acetone, and slowly add them dropwise to a mixed aqueous solution containing 350 μl PBS buffer with pH = 7.4 and 1 ml TPGS with a concentration of 1 mg / ml. Stir at 500 rpm for 30 min at room temperature, vacuum dry to remove acetone, and dilute with purified water to obtain a Cur / DPE-PCL-HIS nanoparticle solution with a concentration of 2.5 mg / ml.

[0066] Example 4

[0067] Preparation of Cur / DPE-PCL-HIS nanoparticles

[0068] Take 5 mg DPE-PCL-HIS and 0.5 mg curcumin, dissolve them in 1 ml acetone, slowly add them to a mixed aqueous solution containing 350 μl PBS buffer with pH = 7.4 and 1 ml TPGS with a concentration of 1 mg / ml, stir at room temperature at 500 rpm for 30 min, vacuum dry to remove acetone, dilute with purified water to obtain a Cur / DPE-PCL-HIS nanoparticle solution with a concentration of 2.5 mg / ml, place it in a Malvern nanometer and use the software ZetaSizer to analyze the particle size distribution and potential distribution of the nanoparticles. Each sample was measured three times and the average value was taken to obtain the Cur / DPE-PCL-HIS NPs particle size of 77.92 ± 0.204 nm, the potential distribution of 8.90 ± 0.550, and the particle size distribution PDI of 0.149 ± 0.004. The results are as follows Figure 5 shown.

[0069] Example 5

[0070] Preparation of Cur / DPE-PCL-HIS nanoparticles

[0071] Take 5 mg DPE-PCL-HIS and 1 mg curcumin, dissolve them in 1 ml acetone, and slowly add them dropwise to a mixed aqueous solution containing 350 μl PBS buffer with a pH of 7.4 and 1 ml TPGS with a concentration of 1 mg / ml. Stir at 500 rpm for 30 min at room temperature, vacuum dry to remove acetone, and dilute with purified water to obtain a Cur / DPE-PCL-HIS nanoparticle solution with a concentration of 2.5 mg / ml.

[0072] Example 6

[0073] Preparation of BSA-sulfoSMCC

[0074] Take 10g BSA and 328.6mg sulfoSMCC, dissolve them in 50ml PBS solution with pH=7.4, react for 5h at room temperature with stirring, transfer to a dialysis bag with a molecular cutoff of 8000-14000, dialyze with purified water for more than 24h, and freeze-dry at -60℃ to obtain BSA-sulfoSMCC. The BSA-sulfoSMCC reaction was verified by HPLC. The results are as follows Figure 4 As shown in A, BSA-sulfoSMCC was successfully synthesized.

[0075] Example 7

[0076] Take 2 ml of Cur / DPE-PCL-HIS nanoparticle solution with a concentration of 2.5 mg / ml, add 10 mg of bovine serum albumin (BSA-SulfoSMCC), and dilute the nanoparticle solution to 5 ml with PBS buffer at pH = 7.4, incubate at 37°C, 100 rpm for 4 hours, add the mixed solution to an ultrafiltration centrifuge tube (MW = 100 kDa), centrifuge at 3000 × g / min for 10 min / time, wash and resuspend with PBS to obtain biomimetic albumin crown nanoparticles (Cur / DPE-PCL-HIS / BSA NPs) solution, place it in a Malvern nanometer for detection and use the software ZetaSizer to analyze the particle size distribution and potential distribution of the nanoparticles. Each sample is measured three times, and the average value is taken to obtain Cur / DPE-PCL-HIS / BSA The particle size of NPs was 133.3 ± 0.313 nm, the potential distribution was -5.88 ± 2.545, the particle size distribution PDI was 0.150 ± 0.047, the drug loading was 3.75% to 10.2%, and the encapsulation efficiency was 45.4% to 97.5%. The results are shown in Figure 5 shown.

[0077] Example 8

[0078] Take 2 ml of Cur / DPE-PCL-HIS nanoparticle solution with a concentration of 2.5 mg / ml, add 5 mg bovine serum albumin (BSA-SulfoSMCC), and dilute the nanoparticle solution to 5 ml with PBS buffer at pH = 7.4. Incubate at 37°C and 100 rpm for 4 hours, add the mixed solution to an ultrafiltration centrifuge tube (MW = 100 kDa), centrifuge at 3000 × g / min for 10 min / time, wash with PBS and resuspend to obtain a biomimetic albumin crown nanoparticle (Cur / DPE-PCL-HIS / BSA NPs) solution.

[0079] Example 9

[0080] Take 2 ml of Cur / DPE-PCL-HIS nanoparticle solution with a concentration of 2.5 mg / ml, add 25 mg bovine serum albumin (BSA-SulfoSMCC), and dilute the nanoparticle solution to 5 ml with PBS buffer at pH = 7.4. Incubate at 37°C and 100 rpm for 4 hours, add the mixed solution to an ultrafiltration centrifuge tube (MW = 100 kDa), centrifuge at 3000 × g / min for 10 min / time, wash with PBS and resuspend to obtain a biomimetic albumin corona nanoparticle (Cur / DPE-PCL-HIS / BSA NPs) solution.

[0081] Example 10

[0082] Take a bionic albumin corona nanoparticle solution containing 100 mg Cur / DPE-PCL-HIS / BSA NPs, add 200 mg bovine serum albumin (BSA-SulfoSMCC), and dilute the nanoparticle solution to 20 ml with PBS buffer at pH = 7.4. Incubate at 37°C, 100 rpm for 4 to 8 hours with shaking. Put the incubation solution into a centrifuge tube and centrifuge at 10000 r / min for more than 20 min. After removing the supernatant, wash the precipitate three times with 5 ml PBS each time, and then resuspend the nanoparticles to 2 ml with an appropriate amount of PBS buffer (PH = 7.4). The BCA method was used to measure the BSA-SulfoSMCC contained in the 100 mg Cur / DPE-PCL-HIS / BSA NPs solution to be approximately 50 to 65 μg.

[0083] Embodiment 11

[0084] Verification of the reaction between BSA-sulfoSMCC and apolipoprotein E mimetic peptide

[0085] Take 10 mg of freeze-dried BSA-sulfoSMCC and 27.84 mg of apolipoprotein E mimetic peptide, dissolve them in PBS solution with pH = 7.4, shake and incubate for 4 to 6 hours at 37°C and 100 rpm, transfer the reaction solution to a dialysis bag with a molecular cutoff of 8000 to 14000, dialyze with purified water for more than 48 hours, and verify the BSA-sulfoSMCC reaction by HPLC. The results are as follows: Figure 4 As shown in B, BSA-sulfoSMCC reacted successfully with the apolipoprotein E mimetic peptide.

[0086] Example 12

[0087] Take a biomimetic albumin corona nanoparticle solution containing 5 mg Cur / DPE-PCL-HIS / BSA NPs, add 220-275 μg apolipoprotein E mimetic peptide, and dilute the nanoparticle solution to 5-7 ml with PBS buffer at pH = 7.4. Incubate the reaction at 37°C and 100 rpm for 4-6 hours. Add the reaction solution to an ultrafiltration centrifuge tube (MW = 100 kDa), centrifuge at 3000 × g / min for 10 min / time, wash 3 times with PBS and resuspend, and obtain a brain-targeted biomimetic protein corona nanodrug delivery system modified with apolipoprotein E mimetic peptide (Cur / DPE-PCL-HIS / BSA -Peptide NPs solution). Place it in a Malvern nanometer for detection and use the software ZetaSizer to analyze the particle size distribution and potential distribution of the nanoparticles. Repeat the measurement three times for each sample, take the average value, and obtain Cur / DPE-PCL-HIS / BSA -Peptide The particle size of NPs was 146.5 ± 0.147 nm, the potential distribution was -6.14 ± 1.018, and the particle size distribution PDI was 0.193 ± 0.010. Figure 5 shown.

[0088] Example 13

[0089] The Cur / DPE-PCL-HIS, Cur / DPE-PCL-HIS / BSA, and Cur / DPE-PCL-HIS / BSA-Peptide NPs solutions prepared above were diluted with purified water, and one drop of each was placed on a 50-mesh copper grid for staining. The samples were observed and photographed under a transmission electron microscope at an operating voltage of 120 kV. The results are shown in FIG. Figure 6 As shown in A, B, and C.

[0090] Embodiment 14

[0091] The in vitro drug release kinetics of Cur / DPE-PCL-HIS / BSA-Peptide NPs were studied by dialysis method, and the Cur content was determined by ultraviolet spectrophotometry (UV). A certain amount of Cur, Cur / DPE-PCL-HIS NPs, and Cur / DPE-PCL-HIS / BSA-Peptide NPs were dissolved or diluted in 0.01M PH=7.4 and PH=5.6 phosphate buffer (PBS), and then loaded into dialysis bags with a molecular weight cutoff MD=8000 and placed in 200 ml of 0.01M PBS containing 0.6% Tween-80. The samples were collected at 2 to 192 hours in phosphate buffer solution at pH 7.4 and pH 5.6 and then released at 37°C and 100 rpm. The samples were then added with an equal volume of fresh release medium. The samples were collected three times at each time point. The absorbance values ​​were determined. The cumulative release rate was calculated according to the cumulative release formula and the release curve was plotted. Figure 7 shown.

[0092] Embodiment 15

[0093] The in vitro cell uptake experiment of the brain-targeted nano-drug delivery system Cur / DPE-PCL-HIS / BSA-Peptide NPs was conducted using C6 cells as a model. 5 The cells were inoculated with different nanoparticle groups at different densities in laser confocal dish, and then cultured and adhered to the wall. After the drug-containing medium was removed, they were washed with PBS for 3 to 5 times. After being fixed with paraformaldehyde, the cells were observed under a microscope. The results of flow cytometry also showed that Cur / DPE-PCL-HIS / BSA-Peptide NPs were easily taken up by C6 cells.

[0094] Example 16

[0095] Evaluation of the in vivo distribution of the nano-drug delivery system: The nano-drug delivery system was labeled with DIR as a fluorescent probe and its brain targeting was detected. The SD rats with brain glioma weighing 250 g ± 30 g and 7 days after modeling were randomly divided into four groups, namely, free DIR group, DIR / DPE-PCL-HIS NPs group, DIR / DPE-PCL-HIS / BSA NPs group, and DIR / DPE-PCL-HIS / BSA-Peptide NPs group; the above nanoparticle solutions were prepared with PBS to 0.15 mg / ml (calculated based on the DIR concentration) and injected into the tail vein at a dose of 0.15 mg / kg; after the end of the administration, the brain targeting fluorescence was detected using a small animal in vivo imager at 4h, 24h and 48h respectively. It can be seen that the fluorescence of the DIR / DPE-PCL-HIS / BSA-Peptide NPs group was stronger than that of the other drug-treated groups. The brain fluorescence distribution results obtained by dissection are shown as follows Figure 8 shown.

[0096] The above embodiments are only used to illustrate the present invention but are not limited thereto. It should be understood that there may be various modifications or replacements without departing from the scope of the present invention.

Claims

1. A brain-targeted nano drug delivery system, characterized in that: The invention comprises a brain targeting peptide, a protein, a bifunctional intermediate connecting compound, a high molecular polymer material and an anti-tumor drug. The molar ratio of the brain targeting peptide to the protein is 50:1 to 500:1; the molar ratio of the bifunctional intermediate connecting compound to the protein is 2:1 to 20:1; the mass ratio of the protein to the high molecular polymer material is 1:1 to 10:1; and the mass ratio of the high molecular polymer material to the anti-tumor drug is 5:1 to 25:

1.

2. The brain-targeted nano drug delivery system according to claim 1, characterized in that: The high molecular polymer material is dipentaerythritol-polycaprolactone-histidine, and the molecular weight is 8000-12000.

3. The brain-targeted nano drug delivery system according to claim 1, characterized in that: The brain targeting peptide is an apolipoprotein E mimetic peptide, the sequence of which is [RLLRKRLK(PEG2000)RLGWC] and the molecular weight is 3698.

13.

4. The brain-targeted nano drug delivery system according to claim 1, characterized in that: The protein is bovine serum albumin, and the anti-tumor drug is curcumin with a purity of 98%.

5. The brain-targeted nano-drug delivery system according to claim 1, characterized in that: The bifunctional intermediate connecting compound is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt, which is used to connect the protein and the brain targeting peptide.

6. A method for preparing the brain-targeted nano drug delivery system according to any one of claims 1 to 5, characterized in that: The steps include: (1) reacting dipentaerythritol-polycaprolactone and Boc-histidine at a molar ratio of 1:15 to 1:60 under nitrogen protection at room temperature with stirring for 24 hours to allow the hydroxyl group at the end of dipentaerythritol-polycaprolactone to undergo an esterification reaction with the carboxyl group of Boc-histidine to obtain a first reaction solution; (2) adding trifluoroacetic acid in an ice bath, wherein the volume of trifluoroacetic acid is 40-60% of the total volume of the first reaction solution, and reacting at room temperature for 2-3 hours with stirring to remove the tert-butyloxycarbonyl group of the Boc-histidine protecting group; (3) washing the first reaction liquid with a saturated sodium bicarbonate aqueous solution, extracting the dichloromethane oil phase layer, and then extracting and washing with dichloromethane 2-3 times, adding anhydrous sodium sulfate to remove water, filtering and dropping the filtrate into ice ether for precipitation, suction filtering, and freeze-drying at -60°C to constant weight to obtain a high molecular polymer material dipentaerythritol-polycaprolactone-histidine; (4) Dissolving the freeze-dried dipentaerythritol-polycaprolactone-histidine and curcumin in acetone at a mass ratio of 5:1 to 25:1, adding the solution to a vitamin E polyethylene glycol succinate aqueous solution containing PBS at pH = 7.4, stirring at room temperature for 30 minutes to 1 hour, and vacuum drying until the acetone is removed to obtain a drug-loaded nanoparticle Cur / DPE-PCL-HIS NPs solution; the concentration of the vitamin E polyethylene glycol succinate aqueous solution is 0.5 mg / ml to 2 mg / ml, the added volume is 30% to 70% of the total volume of the drug-loaded nanoparticle Cur / DPE-PCL-HIS NPs solution, and the volume of the pH = 7.4 PBS is 35% to 70% of the total volume of the vitamin E polyethylene glycol succinate aqueous solution; (5) dissolving bovine serum albumin and sulfoSMCC in a molar ratio of 1:2 to 1:20 in PBS at pH 7.4, stirring at room temperature for 5 to 6 hours to allow the amino groups on the surface of BSA to react specifically with the active ester structure of sulfoSMCC, thereby obtaining a second reaction solution; (6) adding the second reaction solution into a dialysis bag and dialyzing for more than 24 hours, and freeze-drying at -60°C to obtain BSA-sulfoSMCC; (7) The Cur / DPE-PCL-HIS NPs solution and BSA-sulfoSMCC are mixed at a mass ratio of 1:1 to 1:10 and incubated for 4 to 6 hours, the mixed solution after incubation is transferred to an ultrafiltration tube, centrifuged at a speed of 3000×g / min for 10 min / time, and washed and resuspended with PBS to obtain a biomimetic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSANPs solution; (8) The bionic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSA NPs solution and the apolipoprotein E mimetic peptide are mixed at a molar ratio of 1:50 to 1:500 and incubated for 4 to 6 hours. The mixed solution after incubation is transferred to an ultrafiltration tube, centrifuged at a speed of 3000×g / min for 10 min / time, and washed and resuspended with PBS to obtain the apolipoprotein E mimetic peptide-modified brain-targeted bionic protein corona nanodrug delivery system Cur / DPE-PCL-HIS / BSA-Peptide NPs, wherein the molar ratio of the bionic albumin corona nanoparticle solution to the apolipoprotein E mimetic peptide is the ratio of BSA-sulfoSMCC bound to the apolipoprotein E mimetic peptide on the surface of the bionic albumin corona nanoparticle Cur / DPE-PCL-HIS / BSANPs.

7. The method for preparing the brain-targeted nano drug delivery system according to claim 6, characterized in that: Before dipentaerythritol-polycaprolactone and Boc-histidine are stirred at room temperature for reaction under nitrogen protection conditions, the method also includes: pre-dissolving Boc-histidine in dichloromethane, adding 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N-diisopropylethylamine catalyst, and activating at room temperature for 2 to 3 hours; wherein the ratio of Boc-histidine to dichloromethane is 1 mol / L, and the molar ratio of Boc-histidine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N-diisopropylethylamine catalyst is 6:6:7:

20.

8. The method for preparing the brain-targeted nano drug delivery system according to claim 6, characterized in that: The molecular weight cut-off of the dialysis bag is 8000-14000.

9. The method for preparing the brain-targeted nano drug delivery system according to claim 6, characterized in that: Also includes: After obtaining the biomimetic albumin corona nanoparticles Cur / DPE-PCL-HIS / BSANPs solution, the BSA-sulfoSMCC content in the biomimetic albumin corona nanoparticles Cur / DPE-PCL-HIS / BSANPs solution was determined by the BCA method.

10. Use of the brain-targeted nano drug delivery system according to any one of claims 1 to 5 in the preparation of drugs for treating brain diseases.

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