Method for improving blood brain barrier permeability

Abstract

The invention discloses a black phosphorus-methylene blue nano composition for improving blood brain permeability and a synthesis method thereof. The synthesis method comprises the following steps: step 1, mixing black phosphorus nanosheets with polyethylene glycol for reaction; step 2, carrying out centrifugal cleaning on the reaction liquid in the step 1, and collecting a lower-layer reactant; step 3, adding aminated superparamagnetic nanoparticles and methylene blue into the lower reactant obtained in the step 2 for reaction; and 4, carrying out centrifugal cleaning on the reaction liquid obtained in the step 3, and collecting a lower-layer reactant. The invention also discloses the black phosphorus-methylene blue nano composition synthesized by the synthesis method, the drug loading rate of methylene blue in the composition exceeds 60%, and the composition has good biological safety, is non-toxic to cells and mice, is very simple and convenient in preparation process, and has a wide application prospect in the aspect of improving the blood-brain barrier permeability.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a method for improving the permeability of the blood-brain barrier. Background Technology

[0002] The blood-brain barrier (BBB) ​​is formed by a single, tightly sealed layer of endothelial cells along the vascular tree. It regulates the transport of molecules into and out of the central nervous system (CNS), strictly controlling the chemical composition of the neuronal environment necessary for normal neuronal function and preventing toxic plasma components, blood cells, and pathogens from entering the brain. However, this tight structure also exhibits low paracellular permeability, significantly reducing the ability of therapeutic drugs to reach target brain regions, thus hindering effective treatment. Some studies have used viral vectors and target ligands to facilitate intracerebral drug delivery; however, these strategies typically require specific pathological conditions and high doses. Furthermore, the post-binding drug release behavior is often unknown, potentially leading to side effects. Therefore, there is an urgent need to develop more effective, minimally invasive techniques that are not limited by disease progression. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the present invention aims to provide a synthetic method for improving the permeability of the blood-brain barrier.

[0004] The first aspect of this invention provides a method for synthesizing a black phosphorus-methylene blue nanocomposite that enhances blood-brain barrier permeability, comprising the following steps: (1) Mix black phosphorus nanosheets with polyethylene glycol and react them; (2) Centrifuge and wash the reaction solution in (1) and collect the lower layer of reactants; (3) Add amino-modified superparamagnetic nanoparticles and methylene blue to the lower reactant obtained in (2) and react; (4) Centrifuge and wash the reaction solution obtained in (3) and collect the lower layer of reaction product. Preferably, in the present invention, the molecular weight of polyethylene glycol in step one of the synthesis method of the black phosphorus-methylene blue nanocomposite for improving blood-brain barrier permeability is 2000.

[0005] In this invention, in step (3) of the method for synthesizing a black phosphorus-methylene blue nanocomposite to improve blood-brain barrier permeability, the volume molar ratio of aminated superparamagnetic nanoparticles and methylene blue is 1-2:1-2.

[0006] Preferably, in step (3) of the synthesis method of the black phosphorus-methylene blue nanocomposite for improving blood-brain barrier permeability described in this invention, the volume molar ratio of aminated superparamagnetic nanoparticles to methylene blue is 1:1.

[0007] In this invention, the method for synthesizing a black phosphorus-methylene blue nanocomposite that improves blood-brain barrier permeability, wherein in step (1), the black phosphorus nanosheet dispersant needs to be sonicated for 20-40 minutes before the reaction.

[0008] In this invention, the method for synthesizing a black phosphorus-methylene blue nanocomposite that improves blood-brain barrier permeability includes step (1) of the reaction being magnetically stirred in a water bath at 4°C for 8 to 12 hours.

[0009] In this invention, the method for synthesizing a black phosphorus-methylene blue nanocomposite that improves blood-brain barrier permeability, wherein the reaction time of step (3) is 8 to 12 hours.

[0010] In this invention, the method for synthesizing a black phosphorus-methylene blue nanocomposite that improves blood-brain barrier permeability, wherein the centrifugation conditions in step (2) are: temperature 4°C, rotation speed 9000~10000r / min, centrifugation for 10 minutes each time.

[0011] In this invention, the method for synthesizing a black phosphorus-methylene blue nanocomposite that improves blood-brain barrier permeability includes step (4) centrifugation conditions of 4°C, 10000-12000 r / min, and 10 minutes per centrifugation.

[0012] The second aspect of this invention proposes a method for improving the permeability of the blood-brain barrier using the above-mentioned black phosphorus-methylene blue nanocomposite, and for determining the content of the black phosphorus-methylene blue nanocomposite in brain tissue solution using in vivo fluorescence imaging.

[0013] In this invention, the method of improving blood-brain barrier permeability using the aforementioned black phosphorus-methylene blue nanocomposite was employed to develop ir680-labeled-BP-PEG-SPION@MB, which was then injected into mice via the tail vein. Its distribution in the brain and fluorescence signal intensity were compared. Female C57 mice were randomly divided into two groups (n = 6-8 per group): (i) ir680-labeled-BP-PEG-SPION@MB, and (ii) ir680-labeled-BP-PEG-SPION@MB + NIR irradiation. All mice were intravenously injected with a nano-inhibitor solution (BP: 3 mg / kg). -1 ), 1 hour later, use 1.0 W·cm -2 Mice in groups (ii) and (iii) were irradiated with near-infrared lasers. During this period, the local temperature of the head was continuously monitored using a thermal infrared imager at 41–43 °C for 5 min. After 24 h, the mouse brains were extracted for in vivo fluorescence imaging.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention does not require complicated experimental conditions. Based on the photothermal properties of black phosphorus nanosheets (BPNSs), it can significantly improve the permeability of the blood-brain barrier and improve drug-brain permeability. It is simple to operate, highly reproducible, and easy to apply in clinical treatment of Alzheimer's disease. Attached Figure Description

[0015] Figure 1 The results are obtained from the Zeta potentiometer analysis of BP (-30.47mV), SPION (24.44mV), BP-PEG (-33.91mV), and BP-PEG-SPION@MB (-11.19mV) in Example 1.

[0016] Figure 2 The transmission electron microscopy (TEM) analysis results of BP, BP-PEG and BP-PEG-SPION@MB in Example 1 show that the average lateral size of the pure black phosphorus nanosheets (BP) is about 130 nm (scale bar: 20 nm), and the size of the synthesized nanocomposites BP-PEG and BP-PEG-SPION@MB is about 200 nm (scale bar: 200 nm).

[0017] Figure 3 The results are energy-dispersive X-ray spectroscopy (EDS) analysis of BP, SPION, and the final reaction product in Example 1. The results show that BP and SPION successfully combined with MB to form BP-PEG-SPION@MB.

[0018] Figure 4 The results are the FTIR Fourier transform infrared spectra of each substance in Example 1.

[0019] Figure 5 The UV-Vis spectroscopic analysis results of MB, SPION, BP and the final reaction product BP-PEG-SPION@MB in Example 1.

[0020] Figure 6 This is a schematic diagram of the BBB in vitro model in Example 3.

[0021] Figure 7 This is a comparison of the in vitro blood-brain barrier permeability of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB(NPs) under 808nm NIR irradiation in Example 3.

[0022] Figure 8 The results show the cell viability after treatment with different concentrations of BP-PEG-SPION@MB in Example 4.

[0023] Figure 9This is a schematic diagram illustrating brain temperature monitoring in mice under different treatment groups in Example 5. Black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB(NPs).

[0024] Figure 10 The images show the EB staining results of mouse brains in different treatment groups in Example 5. Black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB(NPs)

[0025] Figure 11 This is a comparison of in vitro brain fluorescence imaging results in mice from different treatment groups in Example 6. Black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB(NPs)

[0026] Figure 12 This is a comparison of the total fluorescence signal results in the in vitro brains of mice in different treatment groups in Example 6. Black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB(NPs)

[0027] Figure 13 This is a comparison of the average fluorescence signal in the brains of mice in different treatment groups in Example 6. Black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB(NPs)

[0028] Figure 14 The results of hemolysis after treatment with different concentrations of BP-PEG-SPION@MB in Example 7 are shown. Detailed Implementation

[0029] Example 1

[0030] This embodiment proposes a method for synthesizing a black phosphorus-methylene blue nanocomposite to improve blood-brain barrier permeability, specifically including the following steps: Step 1: Take 2 mL of black phosphorus nanosheet dispersant (BPNSs, 1 mg / mL) and sonicate for 30 minutes. Then, weigh 20 mg of polyethylene glycol 2000 (HO(CH2CH2O)nH) and dissolve it in 2 mL of distilled water (dH2O). Slowly add the sonicated black phosphorus nanosheet dispersant and place the mixture in a 4°C water bath and stir magnetically overnight. Step 2: Place the mixture from Step 1 into a high-speed centrifuge and centrifuge at 4°C and 10,000 rpm for 10 minutes. After centrifugation, wash and repeat the centrifugation three times. Collect the supernatant and aliquot the lower reaction mixture and store it at 4°C in the dark. Step 3: Slowly add aminated superparamagnetic nanoparticles and methylene blue (MB) to the lower layer reactant obtained in Step 2. After complete dissolution, incubate in a shaker at room temperature for 8-12 hours. The molar volume ratio of aminated superparamagnetic nanoparticles to methylene blue (MB) is 1:1. Step 4: Place the mixture from Step 3 in a high-speed centrifuge and centrifuge at 4°C and 12000 r / min for 10 minutes. After centrifugation, wash and repeat the centrifugation three times. Collect the supernatant and quantitatively aliquot the lower layer of reactants and store them at 4°C in the dark. Step 5: As shown in Table 1, the encapsulation efficiency was calculated by detecting the MB content in the supernatant using a UV-Vis spectrometer. The results showed that after the reaction with aminated superparamagnetic nanoparticles, the encapsulation efficiency of BP-PEG-SPION@MB reached 63.49%. Table 1 Encapsulation efficiency calculation for Example 1 Results were obtained by Zeta potentiometer (see results). Figure 1 As shown), transmission electron microscopy (TEM) results are as follows. Figure 2 As shown), energy-dispersive X-ray spectrometer (results as shown) Figure 3 (as shown), FTIR Fourier transform infrared spectrometer (results as shown) Figure 4 As shown), ultraviolet spectrophotometer (results as shown) Figure 5 As shown in the figure, the immobilization of each component in BP-PEG-SPION@MB was detected. The results all showed that after the addition of aminated superparamagnetic nanoparticles, MB and SPION could be successfully bound and solidified with BP. In this embodiment, after modifying the polyethylene glycol by electrostatic adsorption, the oxidation of black phosphorus is effectively reduced. After adding aminated superparamagnetic nanoparticles, the specific surface area effect and magnetic effect are utilized to easily embed biopolymers on the crystal surface, which become the binding bridge between BP and MB, and efficiently solidify BP and MB together.

[0031] Example 2

[0032] This embodiment presents a method for determining the MB concentration in BP-PEG-SPION@MB solution using a UV spectrophotometer, specifically including the following steps: Step 1: Plot the standard curve of MB UV absorbance: Dissolve 1 mg MB completely in dH2O and dilute to different concentrations (1, 2, 2.5, 3, 4, 5, 6, 10 μg / mL). Then measure the absorbance at the characteristic wavelength of MB 664 nm using a UV-Vis spectrometer to obtain the concentration-absorbance standard curve of MB. Step 2: Measure and calculate the MB concentration in BP-PEG-SPION@MB. Quantify the BP-PEG-SPION@MB supernatant collected in Step 4 of Example 2 to 20 mL, then dilute it 100 times with dH2O. Measure the absorbance at a wavelength of 664 nm using a UV-Vis spectrometer, and calculate the MB concentration based on the absorbance value. The results are shown in Table 1.

[0033] Example 3

[0034] This embodiment proposes an in vitro experimental method for verifying the effect of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB on improving blood-brain barrier permeability, which specifically includes the following steps: Step 1: Bend.3 cells (5 × 10⁵ cells per well) were seeded into the upper cavity of a Transwell model plate to form a dense monolayer. The cells were then analyzed using a TEER instrument (…). Merck, Germany) measured the transepithelial resistance (TEER). When it reached 200 Ω·cm... 2 At that time, the same concentration of black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB was added to the upper cavity of each well. Step 2: Cell monolayers are heated under an 808nm laser at a rate of 0.6W·cm⁻¹. -1 Irradiation was performed for 30 minutes. Simultaneously, an unirradiated cell monolayer served as a control. The concentration of BP-PEG-SPION@MB in the lower chamber was measured using a UV spectrophotometer. Step 3: Calculate using the transport ratio calculation formula of the BBB model. The results of this embodiment are shown below. Figure 6 and Figure 7 During NIR irradiation, BP-PEG-SPION@MB from the upper chamber migrates to the lower chamber. The quantity of BP-PEG-SPION@MB is measured to determine the permeability of the blood-brain barrier. Figure 6 ).according to Figure 7 The results showed that under NIR irradiation, the proportion of BP-PEG-SPION@MB transferred to the lower cavum increased from 23.7% to 32.5%, indicating that BP-PEG-SPION@MB has a strong photothermal effect on improving blood-brain barrier permeability. Therefore, the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB can improve the permeability of the blood-brain barrier model in vitro.

[0035] Example 4

[0036] This embodiment illustrates an in vitro method for verifying the biosafety of BP-PEG-SPION@MB, which specifically includes the following steps: 100 μL of logarithmic growth phase 293 cell suspension was seeded into 96-well plates, approximately 5000 cells / well. After 24 h of cell culture, different amounts of BP-PEG-SPION@MB were added to achieve final BP-PEG-SPION@MB concentrations of 1, 5, 10, 20, 40, 60, and 100 μg / ml. After 12 hours of incubation, 10 μL of CCK8 solution was added to each well. Blank wells were prepared with equal volumes of cell culture medium, BP-PEG-SPION@MB, and CCK8 solution, but without cells. Two hours after adding CCK8 solution, the absorbance at 450 nm was measured using a microplate reader for each well. The results are as follows Figure 8 As shown, after incubation with different concentrations of BP-PEG-SPION@MB for 12 hours, the viability of 293 cells remained above 0.95, indicating that BP-PEG-SPION@MB has good biocompatibility.

[0037] Example 5

[0038] This embodiment illustrates an experimental method for in vivo verification of the effect of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB on improving blood-brain barrier permeability, specifically including the following steps: Female C57 mice were randomly assigned to groups (n = 6–8 per group): (i) BP-PEG-SPION@MB, (ii) NIR irradiation, and (iii) BP-PEG-SPION@MB + NIR irradiation. All mice were intravenously injected with Evans Blue (EB) solution (100 μL, 2 wt%) dispersed in physiological saline (EB, SHBN1051, Sigma, UAS). Groups (i) and (iii) were intravenously injected with a black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB solution (BP: 3 mg·kg⁻¹). -1 ), 1 hour later, use 1.0 W·cm -2 Mice in groups (ii) and (iii) were irradiated with near-infrared lasers. During this period, the local temperature of the head was continuously monitored using a thermal infrared imager and kept at 41–43 °C for 5 min. The mouse brains were extracted and photographed 24 h later. The results are as follows Figure 9 , Figure 10 As shown, compared with other groups, due to the strong photothermal effect of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB, the brain temperature of mice injected with BP-PEG-SPION@MB increased more significantly under near-infrared irradiation than that of mice treated with near-infrared irradiation alone. Figure 9Maintaining brain temperature at 41–43°C facilitated the crossing of the blood-brain barrier by the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB. After 24 hours, we observed the staining of EB in the brains of mice injected with the blue black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB.

[0039] Example 6

[0040] This embodiment illustrates an experimental method for in vivo and in vitro verification of the effect of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB on improving blood-brain barrier permeability, specifically including the following steps: To further investigate the effect of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB on improving blood-brain barrier permeability, we developed ir680-labeled-BP-PEG-SPION@MB and injected it into mice via the tail vein, comparing its distribution and fluorescence signal intensity in the brain. Female C57 mice were randomly divided into two groups (n = 6-8 per group): (i) ir680-labeled-BP-PEG-SPION@MB, and (ii) ir680-labeled-BP-PEG-SPION@MB + NIR irradiation. All mice were intravenously injected with a nano-inhibitor solution (BP: 3 mg·kg⁻¹). One hour later, mice in groups (ii) and (iii) were irradiated with a 1.0 W·cm⁻² near-infrared laser. During this period, the local head temperature was continuously monitored using a thermal infrared imager and maintained at 41–43 °C for 5 min. After 24 hours, mouse brains were extracted for in vivo fluorescence imaging. The results are as follows Figure 11 , Figure 12 , Figure 13 As shown, the brain fluorescence signal of ir680-1 abeled-BP-PEG-SPION@MB in mice was more pronounced after near-infrared irradiation treatment. Figure 11 This indicates an increase in the total amount of nanomedicine entering the brain; furthermore, the total drug amount was 11.4% higher than that of the control group that did not receive laser light irradiation. Figure 12 The average drug concentration in the brain was 67.3% higher than that in the control group. Figure 13 ).

[0041] Example 7

[0042] This embodiment illustrates an in vivo experimental method for verifying the biosafety of BP-PEG-SPION@MB, which specifically includes the following steps: Blood was collected from the orbital cavity (500 μL). The blood was centrifuged at 3,500 rpm for 5 min, the precipitate was collected, and red blood cells were extracted. The cells were then washed five times with PBS (3,500 rpm, 5 min) and resuspended in 6 mL PBS. The diluted red blood cell suspension was then mixed thoroughly with saline solutions containing different concentrations of BP-PEG-SPION@MB and incubated at room temperature for 3 h. After centrifugation, photographs were taken, and the absorbance of the supernatant at 541 nm was measured using UV-Vis. Red blood cells from the saline-treated group served as a positive control, and red blood cells from the deionized water-treated group served as a negative control. The hemolysis rate was calculated using the following formula: The results are as follows Figure 14 As shown, no abnormal hemolysis was observed after incubation with different concentrations of BP-PEG-SPION@MB, indicating that BP-PEG-SPION@MB has good in vivo biocompatibility. Definitions of abbreviations and key terms Tau: Tau protein is a microtubule-associated protein that is widely distributed in nerve cells of the nervous system and is an important component used to stabilize microtubules, which serve as the neuronal cytoskeleton. AD: Alzheimer's disease. MB: Methylene Blue. BP: Black phosphorus, a black semiconductor crystal with a metallic luster, has a density of 2.70 g / cm³. 3 It has a hardness of 2 and its crystal lattice is composed of two atomic layers, each of which is composed of tortuous chains of phosphorus atoms. PEG: Polyethylene glycol. BP-PEG-SPION@MB: Black phosphorus polyethylene glycol superparamagnetic iron oxide methylene blue composition. BPNSs: Black phosphorus nano-dispersants. SPION: Aminated iron oxide magnetic nanoparticles. dH2O: Distilled water. 293 cells: HEK293 cells, also known as human embryonic kidney cells 293, are a cell line derived from human embryonic kidney cells. They are characterized by high transfection efficiency and ease of culture. PMSF: Methylsulfonyl fluoride, is an irreversible serine protease inhibitor used in various research and laboratory processes. Double antibiotics: Double antibiotic culture medium is a type of culture medium in which the double antibiotics are a mixture of penicillin and streptomycin. ROS: Reactive oxygen species are oxygen-containing chemically reactive substances, including peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha oxygen. FBS: Fetal bovine serum. DMEM: A culture medium containing various amino acids and glucose. PBS: Phosphate Buffer Solution, is one of the most widely used buffer solutions in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl and KCl. It is generally used as a solvent to dissolve and protect reagents. Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. Application of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB in the preparation of reagents for measuring blood-brain barrier permeability.

2. The application of the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB as described in claim 1 in the preparation of reagents for measuring blood-brain barrier permeability, wherein the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB is composed of black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB particles with the following particle sizes: Particle size range 150–200 nm, average particle size 175 ± 15 nm, by weight.

3. The application of the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB as described in claim 2 in the preparation of a reagent for measuring blood-brain barrier permeability, wherein the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB is prepared into a solution with water, and the solution concentration is 20-200ug / ml.

4. The preparation method of the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB as described in any one of claims 1 to 3, using polyethylene glycol 2000 (H(OCH) 2 CH 2 ) n The product is prepared by reacting OH with black phosphorus nanosheet dispersant (BPNSs), aminated superparamagnetic nanoparticles, and methylene blue (MB), characterized in that: Based on volume molar ratio, polyethylene glycol 2000(H(OCH) 2 CH 2 ) n The volume molar ratio of OH to black phosphorus nanosheet dispersant (BPNSs), aminated superparamagnetic nanoparticles, and methylene blue (MB) was 10:1:10:10 to obtain a black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB solution. After filtration, the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB solution was centrifuged at 10,000-120,000 rpm at 4°C, the supernatant was discarded, and the solution was dissolved in 100 ml of double-distilled water and centrifuged at 10,000-120,000 rpm at 4°C. This process was repeated three times, and finally, the volume was adjusted to 100 ml with double-distilled water.

5. The preparation method of the black phosphorus-methylene blue nanocomposite BP-PEG-SPION@MB as described in claims 1-3, wherein the reaction temperature is 4°C and the reaction time is 8-12 hours.

6. A method for measuring the blood-brain barrier permeability of the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB according to any one of claims 1-3, wherein the concentration of the black phosphorus-methylene blue nanocomposition BP-PEG-SPION@MB in brain tissue solution is determined by in vivo fluorescence imaging.

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