A nano-carrier crossing blood-brain barrier and improving brain delivery efficiency and preparation and application thereof

Abstract

The application belongs to the technical field of medicine, and discloses a nano-carrier capable of penetrating through the blood-brain barrier and improving brain delivery efficiency, and a preparation and application thereof. The preparation method comprises the following steps: 1) dopamine hydrochloride is deposited on the surface of nanoparticles through an oxidative self-polymerization reaction to obtain polydopamine modified particles; and 2) glucose functionalized polyethylene glycol and end-capped polyethylene glycol are reacted with the polydopamine modified particles to obtain the nano-carrier. The polydopamine coating is used to modify the nanoparticles, and the glucose functionalized polyethylene glycol is used to secondarily modify the polydopamine coating, so that the ability of the nanoparticles to penetrate through the BBB and enter brain tissues and brain gliomas is enhanced. The targeting effect of the glucose functionalized PEG and the synergistic effect of the PDA coating in enhancing cell uptake are combined in the application, so that the brain and brain tumor delivery capacity of the nano-carrier is enhanced. The nano-carrier is used for loading drugs.

Description

Technical Field

[0001] This invention belongs to the technical field of drug carriers, specifically relating to a nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency, as well as its preparation and application. Background Technology

[0002] Central nervous system (CNS) diseases are a serious threat to human health, and their incidence and mortality rates are rising with an aging population. The key to effective treatment of CNS diseases lies in delivering drugs to target sites in the brain. The brain is a vital organ, but also very vulnerable, thus protected by numerous physiological barriers, among which the blood-brain barrier (BBB) ​​is the primary barrier controlling substance exchange. While protecting the brain, the BBB also prevents 98% of small molecule drugs and almost all large molecule drugs (such as peptides, gene therapies, and protein drugs) from entering the brain. Therefore, the treatment of CNS diseases is severely limited by the BBB. Nanoparticle-mediated brain drug delivery is considered a promising, multifunctional brain delivery system. For decades, various carriers, such as viral vectors, exosomes, and various nanoparticle formulations, have been developed to enhance drug delivery to the brain. Among these, nanocarriers (such as liposomes, polymers, and inorganic nanoparticles) have shown promise in delivering a variety of drugs to the central nervous system. However, the preparation of nanoparticles for brain delivery typically requires complex modifications to enable them to penetrate the brain's blood-brain barrier (BBB). Furthermore, strategies that solely target BBB endothelial cell surface receptors are inefficient and yield unsatisfactory results.

[0003] This invention constructs a simple, efficient nanocarrier capable of crossing the blood-brain barrier and improving intrabrain delivery efficiency. This nanocarrier can enhance the delivery of drugs to the brain. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency, as well as a method for its preparation. The method of the present invention is simple and efficient, and the prepared nanocarrier can achieve intrabrain delivery. The present invention uses a PDA coating with enhanced cellular uptake to modify nanoparticles of different sizes and materials, and further modifies the polydopamine coating with glucose-functionalized PEG. The modified nanoparticles can penetrate the blood-brain barrier (BBB) ​​and enter brain tissue and gliomas. In this system, the PDA coating enhances cellular uptake of the nanocarrier while providing the possibility of modifying it with materials of various properties. Glucose-functionalized PEG provides endothelial cell targeting of the nanocarrier to the BBB and also improves the stability of the PDA-modified nanoparticles. Similar to the synergistic effect of attachment factors and receptor-promoted endocytosis in viruses, the targeting effect of glucose-functionalized PEG and the synergistic effect of the PDA coating enhancing cellular uptake enhance the delivery capability of the nanocarrier to the brain and brain tumors.

[0005] Another object of the present invention is to provide applications of the aforementioned nanocarriers. The nanoparticle carriers of the present invention are used to load drugs for treating brain diseases.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for preparing a nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency includes the following steps:

[0008] 1) Dopamine hydrochloride is deposited on the surface of nanoparticles via an oxidative self-polymerization reaction to obtain polydopamine-modified particles;

[0009] 2) Glucose-functionalized polyethylene glycol, end-capped polyethylene glycol, and polydopamine-modified particles are reacted to obtain nanocarriers; the end-capped polyethylene glycol is methoxy polyethylene glycolamine or methoxy polyethylene glycol-mercapto.

[0010] The molar ratio of glucose-functionalized polyethylene glycol to capped polyethylene glycol is 1:(0.5-10), preferably 1:(2-5).

[0011] The molecular weight of polyethylene glycol in end-capped polyethylene glycol is 1000-6000.

[0012] In step 2), when the nanoparticles are gold nanoparticles, the molar ratio of glucose-functionalized polyethylene glycol to polydopamine-modified particles is 1:(0.5-10)*10. -3 When the nanoparticles are silica nanoparticles (SN) or PLGA-PEG-PLGA nanoparticles, the mass ratio of (glucose-functionalized polyethylene glycol + end-capped polyethylene glycol) to polydopamine-modified particles is (1-5):1.

[0013] Step 1) involves mixing nanoparticles with dopamine hydrochloride in a Tris solution and then reacting the mixture with ultrasound to obtain nanoparticles with PDA surface modification.

[0014] The nanoparticles are gold nanoparticles (AuNP), silica nanoparticles (SN), PLGA-PEG-PLGA nanoparticles, etc., with a particle size of 10-150 nm.

[0015] The Tris solution has a final concentration of 5–15 mM and a pH of 8–12 in the system. The dopamine hydrochloride has a final concentration of 0.1–0.5 mg / mL in the system. When the nanoparticles are gold nanoparticles or silica nanoparticles (SN), the final concentration of the nanoparticles in the system is 0.1–1 nM. When the nanoparticles are PLGA-PEG-PLGA nanoparticles, the final concentration of the nanoparticles in the system is 0.1–1 mg / mL.

[0016] The duration of the ultrasonic response is 1 to 12 hours.

[0017] The thickness of the polydopamine coating is 5-20 nm.

[0018] In step 2), the glucose-functionalized polyethylene glycol is modified with thiol or amino groups adjacent to the second, fifth, or sixth carbon of glucose. The structural formulas are as follows: as well as

[0019] The reaction conditions described in step 2) are 20–35°C for 20–28 h.

[0020] In step 2), a Tris solution is added to the reaction process, with a final concentration of 5–15 mM in the system.

[0021] After the reaction in step 2) is completed, purification is performed; the purification method includes, but is not limited to, high-speed centrifugation, dialysis or ultrafiltration.

[0022] The nanoparticles described in step 1) are prepared by the following method:

[0023] When the nanoparticles are AuNP, citric acid-stabilized gold nanoparticles AuNP are obtained by reacting tetrachloroauric acid solution with sodium citrate solution at 95–100 °C for 15–45 minutes. Then, methoxylated polyethylene glycol mercaptoyl groups are used. The resulting AuNP reacts with the obtained AuNP to give polyethylene glycol-stabilized AuNP.

[0024] The tetrachloroauric acid solution is a 1% (w / w) aqueous solution of tetrachloroauric acid trihydrate.

[0025] The sodium citrate solution is a 1% (w / w) aqueous solution of sodium citrate hydrate.

[0026] The volume ratio of the tetrachloroauric acid solution to the sodium citrate solution is 1:(1-3).

[0027] The M-PEG-SH has a molecular weight of 1000-5000, and the reaction conditions are ultrasonic treatment for 1.5-2.5 hours.

[0028] When the nanoparticles are SN, they are obtained by stirring a silicon source in ammonia water.

[0029] The silicon source is tetraethoxysilane.

[0030] The volume ratio of the silicon source to ammonia is 200:1 to 10, and the concentration of ammonia is 5 wt%. The silicon source and ammonia are dissolved in water to react, and the volume ratio of ammonia to water is (1 to 10) μL:20 mL.

[0031] The reaction temperature is 30–35°C, and the reaction time is 20–28 hours.

[0032] When the nanoparticles are PLGA-PEG-PLGA micelles, PLGA-PEG-PLGA (with a ratio of 1 to 5: 1: 1 to 5 of the three blocks) is dissolved in an organic solvent, and then slowly dripped into water under ice bath conditions. After stirring to evaporate the organic solvent, the nanoparticles are obtained.

[0033] The PLGA block in the PLGA-PEG-PLGA has a molecular weight of 1000-5000, and the PEG block has a molecular weight of 1000-5000.

[0034] The organic solvent is acetone, and the volume ratio of acetone to water is 1:(5-30).

[0035] Taking the modification of polyethylene glycol thiol or amino groups with the hydroxyl group adjacent to the sixth carbon of glucose as an example, the amino or thiol-terminated glucose-functionalized polyethylene glycol (Glu-PEG-NH2, Glu-PEG-SH) is prepared by the following method: 3,5-O-benzylene-1,2-O-isopropylidene-α-D-furanose glucose (BIG)-functionalized polyethylene glycol (BIG-PEG-NH2, BIG-PEG-SH) with amino or thiol-terminated 3,5-O-benzylene-1,2-O-isopropylidene-α-D-furanose (BIG) is reacted with trifluoroacetic acid.

[0036] The structures of BIG-PEG-NH2 and BIG-PEG-SH are respectively

[0037] The structures of Glu-PEG-NH2 and Glu-PEG-SH are as follows:

[0038] The molecular weight of the glucose-functionalized polyethylene glycol with amino or thiol-terminated groups is 2000-5000.

[0039] The reaction time is 1 to 2 hours, and the reaction temperature is 20 to 35°C.

[0040] The amino or thiol-terminated BIG-functionalized polyethylene glycol is obtained by the following method:

[0041] When BIG-functionalized polyethylene glycol is amino-terminated (BIG-PEG-NH2), it is obtained by reacting ammonia with methanesulfonated glucose polyethylene glycol (BIG-PEG-OMs).

[0042] The ammonia concentration is 20-35%. The reaction time is 24-72 hours. The reaction temperature is 20-35°C. The mass ratio of ammonia to BIG-PEG-Oms is 20-40:1.

[0043] The BIG-PEG-OMs are made from methanesulfonyl chloride and BIG-PEG-OH (structural formula: The reaction is obtained.

[0044] The molar ratio of methanesulfonyl chloride to BIG-PEG-OH is (1-10):1. The reaction solvent is anhydrous tetrahydrofuran, and the catalyst is triethylamine.

[0045] When BIG-functionalized polyethylene glycol is thiol-terminated (BIG-PEG-SH) (structural formula is...) The product is obtained by reacting thioacetic acid-esterified BIG-functionalized polyethylene glycol (BIG-PEG-SAc) with hydrazine hydrate. The mass ratio of BIG-PEG-Sac to hydrazine hydrate is 1:5 to 20.

[0046] The reaction solvent is methanol, and the reaction temperature is 20–35°C. The reaction time is 12–24 hours.

[0047] The BIG-PEG-SAc is obtained by reacting p-toluenesulfonated BIG-functionalized polyethylene glycol (BIG-PEG-OTs) with potassium thioacetate.

[0048] The mass ratio of BIG-PEG-OTs to potassium thioacetate is 1:(5-10).

[0049] The reaction solvent is N,N-dimethylformamide, the reaction temperature is 70–100°C, and the reaction time is 12–24 hours.

[0050] The BIG-PEG-OTs are made from p-toluenesulfonyl chloride and BIG-PEG-OH (structural formula: The reaction yields the product.

[0051] The mass ratio of BIG-PEG-OH to p-toluenesulfonyl chloride is 1:(5-15).

[0052] The reaction solvent is dichloromethane, and the reaction temperature is 35–50°C.

[0053] The BIG-PEG-OH uses 3,5-O-benzyl-1,2-O-isopropylidene-α-D-furanose (BIG) (structural formula: It is obtained by ring-opening polymerization of ethylene oxide (EO) as an initiator.

[0054] The reaction solvent is tetrahydrofuran, the reaction temperature is room temperature, and the reaction time is 20–28 hours.

[0055] The BIG used is 1,2-O-isopropylidene-α-D-furanose (MIG) (structural formula: It is obtained by reacting benzaldehyde with a catalyst.

[0056] The molar ratio of MIG to benzaldehyde is 1:(5-10), the reaction time is 3-12 hours, and the catalyst is ZnCl2.

[0057] The equation for preparing hydroxylated, amino, and thiol-modified glucose-functionalized polyethylene glycol according to this invention is as follows:

[0058]

[0059] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0060] This invention not only prepares a nanoplatform that can effectively penetrate the blood-brain barrier and enter the brain parenchyma under simple conditions, but also realizes the intrabrain delivery of nanomedicine carriers of different sizes and materials, which has great application potential in the treatment and diagnosis of brain diseases.

[0061] The coating of this invention can endow nanomedicine carriers with the ability to enter the brain, and the method is simple, fast, highly repeatable, and compatible with different nanocarriers. Attached Figure Description

[0062] Figure 1 This is the hydrogen NMR spectrum of BIG;

[0063] Figure 2 The 1H NMR spectrum of BIG-PEG-OH;

[0064] Figure 3 The hydrogen NMR spectrum of BIG-PEG-OMs;

[0065] Figure 4 The 1H NMR spectrum of BIG-PEG-NH2;

[0066] Figure 5 Particle size distribution of gold nanoparticles determined by dynamic light scattering; (a) AuNP; (b) PEG-AuNP; (c) PDA@AuNP; (d) PPDA@AuNP; (e) GPDA@AuNP;

[0067] Figure 6 Electron transmission microscopy images of gold nanoparticles; (a) AuNP; (b) PEG-AuNP; (c) PDA@AuNP; (d) PPDA@AuNP; (e) GPDA@AuNP;

[0068] Figure 7 The figure shows the results of cellular validation of glucose and PDA modification enhancing AuNP uptake at the cellular level.

[0069] Figure 8 Figure showing the results of validating the BBB penetration efficiency of AuNP enhanced by glucose and PDA modification in an in vitro BBB model;

[0070] Figure 9 Figures showing the qualitative and quantitative results of glucose and PDA-modified AuNP accumulation in the brain in vivo; (A) in vitro imaging, (B) semi-quantitative analysis results of in vitro imaging software, and (C) quantitative results of Au content detected by ICP-MS.

[0071] Figure 10 The results of GPDA@SN penetrating the BBB and entering brain tissue in vivo are shown in the figure; (A) in vitro imaging of brain tissue 24 hours after tail vein administration, (B) semi-quantitative analysis results of in vitro imaging software and (C) quantitative results of SN content detected by ICP-MS are shown in the figure.

[0072] Figure 11 This image shows the cumulative in vivo imaging results of brain tumors to validate the effects of glucose and PDA-modified PLGA-PEG-PLGA nanoparticles. Detailed Implementation

[0073] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0074] Example 1: Glu-PEG 5k Preparation of -NH2

[0075] Glu-PEG 5k-NH2 is prepared by using BIG as an initiator through epoxy ring-opening polymerization and amination.

[0076] Preparation of BIG:

[0077] 500 mg of MIG (1,2-O-isopropylidene-α-D-furanose) and 620 mg of anhydrous zinc chloride were weighed and mixed with 2 mL of benzaldehyde at room temperature and stirred for 6 hours at room temperature. The benzaldehyde was then dried under vacuum, diluted with 10 mL of ethyl acetate, washed three times with pure water, and dried over anhydrous magnesium sulfate. The mixture was filtered and the organic solvent was evaporated by rotary evaporation. Finally, BIG (white powder) was recrystallized in n-hexane in 85% yield. The 1H NMR spectrum of BIG is shown below. Figure 1 As shown.

[0078] BIG structure:

[0079]

[0080] Preparation of BIG-PEG-OH:

[0081] Ethylene oxide (EO) gas was passed into a flask containing NaH in an ice bath. After collecting 6 mL of EO, the EO flow was stopped, and the mixture was stirred in an ice bath for three hours to obtain dry EO. Simultaneously, 200 mg of dry BIG, a catalytic amount of triethylboron, and phosphazene base were weighed and dissolved in anhydrous THF (3 mL). Then, 6 mL of dry EO was added to the mixture, and the mixture was stirred in an ice bath for 10 minutes, followed by stirring at room temperature for 24 hours. Finally, BIG-PEG-OH (white solid) was precipitated by cold diethyl ether with a yield of 90%. The NMR spectrum of BIG-PEG-OH is shown below. Figure 2 As shown.

[0082] The structure of BIG-PEG-OH:

[0083]

[0084] Preparation of BIG-PEG-OMs:

[0085] BIG-PEG-OH (300 mg) and triethylamine (63 μL) were dissolved in THF (4 mL) and slowly added to a THF solution containing methanesulfonyl chloride (29 μL) in an ice bath. After returning to room temperature, the mixture was stirred for 6 hours. After evaporating the solvent, BIG-PEG-OMs (white solid) were obtained by precipitation with ice-cold diethyl ether, with a yield of 90%. The NMR spectra of BIG-PEG-OMs are shown below. Figure 3 As shown.

[0086] Structure of BIG-PEG-OMs:

[0087]

[0088] Preparation of BIG-PEG-NH2:

[0089] BIG-PEG-OMs (270 mg) were dissolved in 30 wt% ammonia solution (10 mL), stirred at room temperature for 48 hours, and after evaporation of the solvent, BIG-PEG-NH2 was obtained by precipitation with cold diethyl ether, with a yield of 85%. The NMR spectrum of BIG-PEG-NH2 is shown below. Figure 4 As shown.

[0090] The structure of BIG-PEG-NH2:

[0091]

[0092] Preparation of Glu-PEG-NH2:

[0093] BIG-PEG-NH2 (100 mg) was dissolved in a solution of trifluoroacetic acid and water at a volume ratio of 4:1. The mixture was stirred at room temperature for one hour. The trifluoroacetic acid was removed by rotary evaporation. After the solvent was dried, Glu-PEG-NH2 was obtained by cold ether precipitation with a yield of 90%.

[0094] The structure of Glu-PEG-NH2:

[0095]

[0096] Example 2: Preparation of glucose and polydopamine modified gold particles, silica nanoparticles, and PLGA-PEG-PLGA nanoparticles.

[0097] PDA@AuNP is prepared by oxidative self-polymerization of dopamine hydrochloride in an alkaline solution and deposition onto the surface of PEG-AuNP.

[0098] Preparation of AuNP (15nm):

[0099] Take 300 μL of freshly prepared 1 wt% tetrachloroauric acid trihydrate aqueous solution and add it to 30 mL of ultrapure water. Stir and heat to 100 °C. Then, add 900 μL of freshly prepared 1 wt% trisodium citrate solution and stir vigorously at 100 °C for 30 minutes to obtain AuNP. The dynamic light scattering (DLS) particle size results of AuNP are as follows. Figure 5 As shown in Figure a, the electron transmission microscopy (TEM) imaging results are as follows: Figure 6 As shown in a.

[0100] Preparation of PEG@AuNP:

[0101] Take 15 mL of LAuNP (1 nM) and add 15 mL of M-PEG-SH (structural formula: (1 μM), sonicated for 2 hours, and centrifuged (20000g, 30 minutes) to remove unreacted M-PEG-SH, yielding PEG@AuNP. The DLS particle size results for PEG@AuNP are shown below. Figure 5 As shown in b, the TEM imaging results are as follows: Figure 6 As shown in b.

[0102] Preparation of PDA@AuNP:

[0103] 13.5 mL of PEG@AuNP (1 nM) was added to 13.5 mL of dopamine hydrochloride solution (0.5 mg / mL), followed by 3 mL of Tris solution (100 mM). The mixture was sonicated for 2 hours. Subsequently, centrifugation (20000 g, 30 min) was performed to remove unreacted dopamine hydrochloride, yielding PDA@AuNP. The DLS particle size results for PDA@AuNP are shown below. Figure 5 As shown in c, the TEM imaging results are as follows: Figure 6 As shown in c.

[0104] Preparation of GPDA@AuNP and PPDA@AuNP:

[0105] Take 15 mL of PDA@AuNP (1 nM) and add 15 mL of Glu-PEG-NH2 and M-PEG-NH2 (molar ratio 1:3) or M-PEG-NH2 (structural formula: (1 μM), stirred at room temperature for 24 h, centrifuged (20000 g, 30 min) to remove Glu-PEG-NH2 or M-PEG-NH2 that had been reacted, to obtain GPDA@AuNP and PPDA@AuNP. The DLS particle size results of GPDA@AuNP and PPDA@AuNP are as follows: Figure 5 As shown in d and 5e, the TEM imaging results are as follows: Figure 6 As shown in d, 6e.

[0106] Preparation of silica nanoparticles (SN):

[0107] 200 μL of tetraethoxysilane was dissolved in 6 μL of 5 wt% ammonia in 20 mL of water. The reaction was carried out at 35 °C for 24 hours. The mixture was centrifuged three times (10000 g, 10 min), the precipitate was collected, resuspended in ethanol, and stored at 4 °C.

[0108] Preparation of PDA@SN:

[0109] First, take 15 mL of SN (65 nm, 1 nM) and centrifuge three times (10000 g, 15 min) to remove ethanol. Resuspend in 25 mL of ultrapure water, add 2 mL of dopamine hydrochloride solution (3.75 mg / mL) and 3 mL of Tris solution (100 mM), sonicate for 2 h, centrifuge three times (10000 g, 15 min) to collect the precipitate, resuspend the precipitate in ultrapure water to obtain PDA@SN, and store at 4 °C.

[0110] Preparation of PEG-SN:

[0111] First, take 15 mL of SN (65 nm, 1 nM), add 2 mL of silane polyethylene glycol (10 μM), reflux at 50 °C for 24 h, collect the precipitate, resuspend the precipitate with ultrapure water to obtain PEG-SN, and store it at 4 °C.

[0112] Preparation of GPDA@SN and PPDA@SN:

[0113] To prepare PEG- or glucose-functionalized PEG-modified PDA@SN (PPDA@SN and GPDA@SN), the following steps were performed: 10 mL of PDA@SN (concentration 1 nM) was taken, and RB-PEG was added respectively. 2k -NH2 (Rhodamine B-PEG amino) and M-PEG 5k -NH2 (molar ratio 1:3) or RB-PEG 2k -NH2, Glu-PEG 5k -NH2,M-PEG 5k -NH2 (molar ratio 1:1:3), concentration 10 strands / nm 2 The PEG solution was then added. 1 mL of Tris buffer (100 mM) was added to each mixture, and the mixture was stirred at room temperature for 24 hours. Subsequently, each reaction mixture was centrifuged three times (12000 g, 15 min) using a high-speed centrifuge to remove unreacted PEG and Tris buffer, and the precipitate was collected. The precipitate was resuspended in ultrapure water to obtain PPDA@SN and GPDA@SN, which were then stored separately at 4°C.

[0114] Preparation of PPDA@P and GPDA@P nanoparticles:

[0115] Dissolve 10 mg of PLGA-PEG-PLGA and 1 mg of DiR in 2 mL of acetone. Slowly add the mixture dropwise to 20 mL of pure water and stir overnight at room temperature to evaporate the acetone. Dialyze for 48 h to obtain PLGA-PEG-PLGA nanocarriers (Polymer). Dilute to 25 mL with ultrapure water, add 2 mL of dopamine hydrochloride solution (3.75 mg / mL) and 3 mL of Tris solution (100 mM), sonicate for 2 h, centrifuge three times (10000 g, 15 min), collect the precipitate, resuspend the precipitate in ultrapure water to obtain PDA@P, and store at 4 °C.

[0116] To prepare PEG or glucose-functionalized PEG-modified PDA@P, the following steps were performed: 10 mL of PDA@P (concentration 0.5 mg / mL) was taken, and 1 mL of RB-PEG (concentration 10 mg / mL) was added respectively. 2k -NH2 and M-PEG 5k -NH2 (molar ratio 1:3) or RB-PEG 2k -NH2, Glu-PEG 5k -NH2,M-PEG 5k A PEG solution with an -NH2 ratio of 1:1:3 was prepared. 1 mL of 100 mM Tris buffer was added to each mixture, and the mixture was stirred at room temperature for 24 hours. Subsequently, each reaction mixture was centrifuged three times (12000 g, 15 min) to remove unreacted PEG and Tris buffer, and the precipitate was collected. The precipitate was resuspended in ultrapure water and its morphology, particle size, surface potential, and other properties were characterized using dynamic light scattering and transmission electron microscopy to obtain PPDA@P and GPDA@P, which were stored at 4°C.

[0117] Example 3: Cellular-level verification of glucose and PDA modification enhancing cellular uptake of AuNP.

[0118] bEnd.3(1×10) 5 Cells were added to 24-well plates and cultured overnight in DMEM complete medium. After complete cell adhesion, 0.5 nM GPDA@AuNP, PPDA@AuNP, and PEG-AuNP (gold core diameter 35 nm) were added as control groups and co-incubated with bEnd.3 cells for 12 hours. Cells were then washed three times with PBS, digested with 0.25% trypsin to collect and count the cells, and finally digested with aqua regia. The results were analyzed by ICP-MS. The experimental results are shown below. Figure 7 As shown in the figure (PPDA@AS corresponds to PPDA@AuNP, and GPDA@AS corresponds to GPDA@AuNP). Figure 7The content of nanoparticles in bEnd.3 cells of each group was detected by ICP (n=3).

[0119] Conclusion: Compared with AuNP modified only by PEG (PEG-AuNP), PDA modification significantly enhanced the uptake of nanoparticles by bEnd.3 cells. The number of nanoparticles per cell in the PPDA@AuNP group increased from 2.6 × 10⁻⁶ to 2.6 × 10⁻⁶ compared to the PEG-AuNP group. 4 Increased to 7.8×10 4 Furthermore, glucose modification further enhanced the uptake of nanoparticles by bEnd.3 cells; compared to the PPDA@AuNP group, the number of particles per cell in the GPDA@AuNP group increased from 7.8 × 10⁻⁶ to 10⁻⁶. 4 Increased to 11.2 × 10 4 .

[0120] Example 4: In vitro BBB model verification of glucose and PDA modification to enhance AuNP's BBB penetration ability.

[0121] bEnd.3(5×10) 4 The cells were added to Transwell chambers (each well) and cultured in DMEM complete medium. After seven days, serum-free DMEM medium was added to the chambers to starve the cells for 30 minutes. Then, 12.5 μg / mL of PEG-AuNP, PPDA@AuNP, and GPDA@AuNP were added and the cells were co-incubated for 12 hours. The culture medium in the lower chamber was collected, digested with aqua regia, and the gold concentration in the lower chamber was detected by ICP-MS. The experimental results are shown below. Figure 8 As shown in the figure (PPDA@AS corresponds to PPDA@AuNP, and GPDA@AS corresponds to GPDA@AuNP). Figure 8 The gold content in the lower chamber of the Transwell after penetrating a dense monolayer of bEnd.3 cells 12 hours after administration was measured by ICP, and the percentage of gold in the total dose was calculated to obtain the transport efficiency of each group of nanoparticles penetrating the in vitro BBB.

[0122] Conclusion: Compared to AuNP modified with PEG alone (PEG-AuNP), PDA modification significantly enhanced the ability of nanoparticles to penetrate the in vitro BBB. The transport efficiency of PPDA@AuNP increased from 0.1% to 1.6%, a 16-fold increase, compared to PEG-AuNP. Furthermore, glucose modification further enhanced the in vitro BBB penetration ability of the nanoparticles; the transport efficiency of the GPDA@AuNP group increased from 1.6% to 2.0% compared to the PPDA@AuNP group.

[0123] Example 5: In vivo verification of glucose and PDA modification enhancing the BBB penetration ability of AuNP and SN.

[0124] In this experiment, different types and concentrations of nanocarriers were injected into 6-week-old Balb / c mice via tail vein injection. After 24 hours, in vitro imaging and ICP-MS were used to qualitatively and quantitatively detect the content of GPDA@AuNP and GPDA@SN in brain tissue. Specifically: for AuNP, PEG-AuNP, PPDA@AuNP, and GPDA@AuNP with a total surface area equivalent to 25 μg of AuNP with a diameter of 35 nm were injected; for SN, PEG-SN, PPDA@SN, and GPDA@SN with a silicon content of 30 μg were injected. Mice were sacrificed 24 hours later, and the fluorescence intensity of brain tissue was detected using in vitro fluorescence imaging, followed by ICP-MS detection of the gold or silicon content in the brain tissue.

[0125] Conclusion: For AuNP, strong accumulation of PPDA@AuNP and GPDA@AuNP was observed in the brain in ex vivo imaging, while PEG-AuNP was almost invisible. Further semi-quantitative and quantitative analyses showed that 24 hours after intravenous injection of the nanocarrier, the fluorescence intensity and percentage of injected dose reaching brain tissue of GPDA@AuNP were 3.2 and 5.4 times that of PEG-AuNP, respectively. The difference between the semi-quantitative and quantitative results may be due to tissue autofluorescence and fluorescence quenching; therefore, the quantitative results will be used as the standard in the following analysis. ICP-MS quantitative results showed that the percentage of injected dose reaching brain tissue of GPDA@AuNP was 1.5 times that of PPDA@AuNP. These results indicate that, under in vivo conditions, a ligand-cooperative strategy mimicking viruses can significantly improve the efficiency of AuNP entry into brain tissue.

[0126] For SN, strong accumulation of PPDA@SN and GPDA@SN in the brain was observed in ex vivo imaging, while only weak fluorescence was observed in PEG-SN. Further semi-quantitative and quantitative analyses showed that 24 hours after intravenous injection of the nanocarrier, the fluorescence intensity of GPDA@SN in the brain and the percentage of injected dose reaching the brain tissue were 1.7 and 2.9 times that of PEG-SN, respectively. The difference between the semi-quantitative and quantitative results may be due to tissue autofluorescence; therefore, the quantitative results will be used as the standard in the following analysis. ICP-MS quantitative results showed that the percentage of injected dose reaching the brain tissue of GPDA@AuNP was 1.3 times that of PPDA@AuNP. These results indicate that, under in vivo conditions, a ligand-cooperative strategy mimicking viruses can significantly improve the efficiency of SN entry into brain tissue.

[0127] Figure 9Figures showing the qualitative and quantitative results of glucose and PDA-modified AuNP accumulation in the brain in vivo; (A) in vitro imaging, (B) semi-quantitative analysis results of in vitro imaging software, and (C) quantitative results of Au content detected by ICP-MS.

[0128] Figure 10 The results of GPDA@SN penetrating the BBB and entering brain tissue in vivo are shown in the figure; (A) in vitro imaging of brain tissue 24 hours after tail vein administration, (B) semi-quantitative analysis results of in vitro imaging software, and (C) quantitative results of SN content detected by ICP-MS are shown in the figure.

[0129] Example 6: In vivo validation of the PDA blood-brain barrier platform for delivery of PLGA-PEG-PLGA to brain tumors.

[0130] Six-week-old female C57BL / 6 mice, weighing between 18 and 22 g, were selected. The mice were fixed on a stereotactic apparatus. Under aseptic conditions, the scalp was incised along the midline with a scalpel to expose the skull. A small hole was drilled in the right side of the brain (coordinates: anterior horn -2 mm, midline +2 mm, depth -3 mm), and 5 μL of solution containing 10... 5 A suspension of GL261-Luc cells was slowly injected into the subcortical layer of the cerebral cortex at a depth of 3 mm. The wound was sutured and disinfected, and the recovery of the mice was observed. Bioluminescent signals in the mouse head were detected at regular intervals using an in vivo imaging system. To detect the content and distribution of GPDA@P in brain tissue, this experiment injected it into mice with an established glioma model via the tail vein, and observed it using both in vivo and ex vivo imaging. Specifically, 100 μg of Polymer (PLGA-PEG-PLGA), PPDA@P, and GPDA@P nanocarriers were injected via the tail vein. Fluorescence signals in the mouse head region were observed using a fluorescence in vivo imaging system at 4, 12, and 24 hours, and fluorescence intensity was analyzed using software. Mice were sacrificed at 24 hours, and the fluorescence intensity of the brain tissue was detected using ex vivo fluorescence imaging, and the fluorescence intensity was analyzed using software.

[0131] Conclusion: GPDA@P exhibited the highest fluorescence signal, and the fluorescence signals of both PPDA@P and GPDA@P remained stable over 24 hours. However, while polymer particles could enter brain tissue through the gap between the BBTB and cells within 4 hours, the fluorescence signal significantly decreased after 24 hours. This may be because untaken polymer particles or released DIR had been expelled from the brain tissue via cerebrospinal fluid.

[0132] Figure 11 This image shows the cumulative in vivo imaging results of brain tumors to validate the effects of glucose and PDA-modified PLGA-PEG-PLGA nanoparticles.

Claims

1. A method for preparing a nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency, characterized in that: Includes the following steps: 1) Dopamine hydrochloride is deposited on the surface of nanoparticles via an oxidative self-polymerization reaction to obtain polydopamine-modified particles; 2) Glucose-functionalized polyethylene glycol, end-capped polyethylene glycol, and polydopamine-modified particles were reacted to obtain nanocarriers; The nanoparticles are one or more of gold nanoparticles, silica nanoparticles, and PLGA-PEG-PLGA nanoparticles. The end-capped polyethylene glycol mentioned in step 2) is methoxy polyethylene glycol amine or methoxy polyethylene glycol-mercapto; The glucose-functionalized polyethylene glycol is , , , , , One or more.

2. The method for preparing the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 1, characterized in that: The molar ratio of glucose-functionalized polyethylene glycol to end-capped polyethylene glycol is 1:(0.5~10). The molecular weight of polyethylene glycol in end-capped polyethylene glycol is 1000~6000; In step 2), when the nanoparticles are gold nanoparticles, the molar ratio of glucose-functionalized polyethylene glycol to polydopamine-modified particles is 1:(0.5~10)*10. -3 When the nanoparticles are silica nanoparticles or PLGA-PEG-PLGA nanoparticles, the mass ratio of (glucose-functionalized polyethylene glycol + capped polyethylene glycol) to polydopamine-modified particles is (1~5):

1. The thickness of the polydopamine coating is 5~20 nm.

3. The method for preparing the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 2, characterized in that: The molar ratio of glucose-functionalized polyethylene glycol to end-capped polyethylene glycol is 1:(2~5).

4. The method for preparing the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 1, characterized in that: The nanoparticles have a particle size of 10–150 nm; Step 1) involves mixing nanoparticles with dopamine hydrochloride in a Tris solution and then reacting the mixture with ultrasound to obtain nanoparticles with PDA surface modification.

5. The method for preparing the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 4, characterized in that: In step 1), the final concentration of the Tris solution in the system is 5–15 mM, and the pH is 8–12; the final concentration of the dopamine hydrochloride in the system is 0.1–0.5 mg / mL. The duration of the ultrasonic response is 1 to 12 hours.

6. The method for preparing the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 1, characterized in that: The reaction conditions described in step 2) are 20~35℃ for 20~28h; In step 2), a Tris solution is added to the reaction process, with a final concentration of 5–15 mM in the system. After the reaction in step 2) is completed, purification is performed; the purification method is high-speed centrifugation, dialysis or ultrafiltration.

7. The method for preparing the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 1, characterized in that: The nanoparticles described in step 1) are prepared by the following method: When the nanoparticles are AuNP, a tetrachloroauric acid solution and a sodium citrate solution are reacted at 95–100 °C for 15–45 minutes to obtain citric acid-stabilized gold nanoparticles AuNP. Then, methoxy polyethylene glycol mercapto groups are reacted with the obtained AuNP to obtain polyethylene glycol-stabilized AuNP. When the nanoparticles are PLGA-PEG-PLGA micelles, PLGA-PEG-PLGA is dissolved in an organic solvent, then slowly dripped into water under ice bath conditions. After stirring to evaporate the organic solvent, the nanoparticles are obtained.

8. The method for preparing the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 7, characterized in that: The tetrachloroauric acid solution is a 1 wt% aqueous solution of tetrachloroauric acid trihydrate; the sodium citrate solution is a 1 wt% aqueous solution of sodium citrate hydrate; the volume ratio of the tetrachloroauric acid solution to the sodium citrate solution is 1:(1-3); the molecular weight of the methoxy polyethylene glycol mercapto group is 1000-5000; and the reaction conditions are ultrasonic treatment for 1.5-2.5 hours. The PLGA block in the PLGA-PEG-PLGA has a molecular weight of 1000-5000, and the PEG block has a molecular weight of 1000-5000; the organic solvent is acetone, and the volume ratio of acetone to water is 1:(5-30).

9. A nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency, obtained by the preparation method according to any one of claims 1 to 8.

10. The application of the nanocarrier that crosses the blood-brain barrier and improves brain delivery efficiency according to claim 9, characterized in that: The nanocarriers that cross the blood-brain barrier and improve brain delivery efficiency are used to prepare drugs for treating brain diseases.

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