Method for preparing drug-loaded silk protein preparation across blood-brain barrier and application thereof

Abstract

This invention discloses a method for preparing and applying drug-loaded silk protein formulations that cross the blood-brain barrier, relating to the field of biomedical materials technology. To optimize the performance of nanodelivery systems by utilizing silk protein fibers to regulate the self-assembly of bioactive particles and enhance their application value in the treatment of various diseases such as Alzheimer's, the invention specifically includes silk protein nanofibers, functional therapeutic active components, and mannose / rabies virus capsid protein. The silk protein nanofibers self-assemble to form nanoparticles, and the functional therapeutic active components utilize the self-assembly process of the silk protein nanofibers to prepare bioactive silk protein nanocarriers. This invention features a simple and convenient process. The resulting stable drug-loaded silk protein nanodelivery carrier formulation achieves high brain penetration efficiency by binding to different receptors on brain microvascular endothelial cells, providing a novel approach for the design of silk protein nanodelivery carriers.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to the preparation method and application of drug-loaded silk protein nanocarriers that cross the blood-brain barrier. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive impairment and memory loss, severely impacting patients' health and quality of life. It is projected that the number of AD patients worldwide will exceed 150 million by 2050. Given the increasing incidence of AD, its complex pathogenesis, and limited treatment options, related research has become a hot topic in various fields such as clinical medicine, biomaterials, and tissue engineering, and is also an important component of the Healthy China strategy.

[0003] Currently, in research on AD treatment, nanomaterials, due to their advantages such as small size effect and good biocompatibility, can significantly improve the brain-targeting efficiency of drugs. However, facing a wide variety of nanomaterials, how to avoid the problems of traditional nanomaterials such as graphene and zinc oxide, which are not easily degradable and have poor biocompatibility, and how to screen new nanomaterials that are biodegradable, have high efficiency in crossing the blood-brain barrier, and have good effects in preventing and treating AD, is the forefront of this field.

[0004] Among existing nanomaterial systems, silk protein, due to its excellent biocompatibility, low immune response, good modifiability, biodegradability, and rich amino acid composition, can be used as both a matrix material for tissue repair and a carrier for different drugs, to prepare bioactive materials containing a variety of physical, chemical, and biological signals to meet the needs of different tissue repair and regeneration.

[0005] In response to the critical issues of progressively worsening neuroinflammation and microglial dysfunction during the pathogenesis of Alzheimer's disease, existing treatments typically utilize curcumin (Cur) as an inflammatory modulator to alleviate cellular neurotoxicity by improving the intracellular inflammatory microenvironment.

[0006] Simultaneously, epigallocatechin gallate (EGCG) aims to reduce the production and aggregation of β-amyloid protein and restore the migration and phagocytic functions of microglia by alleviating intracellular reactive oxygen species (ROS) levels.

[0007] However, these small molecule inhibitors and drugs have weak ability to penetrate the blood-brain barrier, low efficiency in targeting brain lesions, and low bioavailability, leading to a significant decline in the effectiveness of Alzheimer's disease treatment.

[0008] Therefore, improving the brain-targeted delivery capability of drugs is crucial. Currently, patents report the use of nanosystems to deliver targeted therapeutic drugs and alleviate the pathology of Alzheimer's disease. However, utilizing silk fibroin fibers to regulate the self-assembly of bioactive particles, optimizing the performance of nanodelivery systems, and enhancing their application value in the treatment of Alzheimer's disease remains a gap in this technological field. Therefore, to utilize silk fibroin to improve the therapeutic effect of Alzheimer's disease, this invention proposes a method for preparing and applying a drug-loaded silk fibroin formulation that crosses the blood-brain barrier. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for preparing and applying drug-loaded silk protein formulations that cross the blood-brain barrier.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A drug-loaded silk protein formulation that crosses the blood-brain barrier includes silk protein nanofibers, a functional therapeutic active component, and mannose / rabies virus capsid protein, wherein the silk protein nanofibers form nanoparticles through self-assembly. The functional therapeutic active components are prepared into bioactive silk nanocarriers using the self-assembly process of silk nanofibers. The silk protein nanocarrier and mannose / rabies virus capsid protein are coupled with amino groups and dialyzed to obtain a drug-loaded silk protein nanocarrier formulation that can cross the blood-brain barrier.

[0011] Furthermore: the functional therapeutic active component is composed of a compound system of two natural polyphenol active components with different functions. One component is an anti-inflammatory polyphenol component that mainly inhibits neuroinflammation and repairs nerve cells. The other component is an anti-Aβ antioxidant polyphenolic component that mainly inhibits β-amyloid aggregation and scavenges ROS. The anti-inflammatory polyphenol component is one of the natural polyphenol active components compounded in a system of curcumin, resveratrol, caffeic acid, etc. The anti-Aβ antioxidant polyphenol component is one of the natural polyphenol active components compounded in a system including EGCG, theanine, and proanthocyanidins.

[0012] Furthermore, the concentration of the silk protein nanofiber solution is 1–9 mg / mL.

[0013] Further preferred: the mannose concentration is 0.5–3 mmol / L, and the rabies virus capsid protein peptide concentration is 0.3–3 mmol / L.

[0014] A method for preparing drug-loaded silk protein formulations that cross the blood-brain barrier includes the following steps: S1: The silk fibroin nanofiber solution was diluted with pure water to obtain a solution with a concentration of 1-9 mg / mL. The solution was mixed with an organic solvent, stirred, frozen and thawed, centrifuged, and sonicated to obtain silk fibroin nanoparticles of different sizes. S2: The diluted silk nanofiber solution with a concentration of 1-9 mg / mL was mixed with an organic solvent containing functional therapeutic active components, stirred, frozen-thawed, centrifuged, and sonicated to obtain drug-loaded silk nanoparticles of different sizes. S3: Drug-loaded silk protein nanoparticles were subjected to an amino-coupling reaction with mannose / rabies virus capsid protein peptides to obtain a drug-loaded silk protein nanocarrier that crosses the blood-brain barrier. As a preferred embodiment of the present invention, the preparation method of the silk fibroin nanofiber solution in step S1 includes the following steps: A1: After degumming raw silk in an anhydrous sodium carbonate solution at 100℃ for 60 minutes, remove it and dry it in an oven at 60℃ to obtain dry silk protein fiber; A2: Dry silk protein fibers are treated with 8.0 mol / L... -1 The mixed solution of LiBr and 99% formic acid was dissolved in an oven at 60°C for 4 hours and then placed into a dialysis bag. A3: Dialyze the solution in low-temperature deionized water for 72 h to remove LiBr and formic acid, and centrifuge twice at 4℃ and 9000 rpm for 20 min to remove impurities, to obtain a silk fibroin nanofiber solution. A4: After drying the silk fibroin nanofiber solution in an oven at 60°C, weigh it and calculate the concentration of the silk fibroin nanofiber solution to be 0.1–9 mg / mL. Store it at low temperature for later use.

[0015] As a further embodiment of the present invention: in S1, the stirring speed is 100-700 rpm and the time is 5-60 minutes; The freezing temperature is -70 to -85℃, the freezing time is 24 hours, the centrifugation speed is 12000 rpm, and the centrifugation time is 30 minutes. The ultrasonic treatment power is 50-600W, and the ultrasonic treatment time is 1-10 minutes.

[0016] Based on the aforementioned scheme: in S1, the volume ratio of the organic solvent containing the functional therapeutic active component to the silk nanofiber solution is 1:10–1:1; The organic solvent is composed of one or more solutions of isopropanol, dimethyl sulfoxide, and acetone.

[0017] Based on the aforementioned scheme, the preferred embodiment is as follows: In S3, the amino coupling adopts an EDC / NHS system with a molar ratio of 1:3, a reaction time of 6 hours, and a dialysis time of 24 hours.

[0018] The application of drug-loaded silk protein preparations that cross the blood-brain barrier in the preparation of drugs for treating neurodegenerative diseases, namely Alzheimer's disease.

[0019] The beneficial effects of this invention are as follows: 1. This invention optimizes the interaction between silk nanofibers by controlling the type of organic solvent, the concentration of the silk nanofiber solution, the ultrasonic power and time, and directly obtains nanoparticles of different sizes to meet the small size effect required as a nanodelivery material.

[0020] 2. This invention selects curcumin and epigallocatechin gallate. EGCG is co-assembled with silk fibroin nanofibers to form bioactive nanoparticles. Based on the natural antioxidant, anti-inflammatory, and β-amyloid aggregation inhibitory properties of Curcumin and EGCG, they can better improve the internal environment of dysfunctional microglia in the brain of Alzheimer's disease (AD), reduce the content of β-amyloid aggregates in the AD brain, and thus improve the pathological progression of AD.

[0021] 3. This invention selects mannose and rabies virus capsid protein to modify silk protein nanoparticles. Based on the interaction between mannose and rabies virus capsid protein and specific receptors on brain microvascular endothelial cells, the nanoparticles are endowed with higher brain entry efficiency, further improving the brain bioavailability of Curcumin and EGCG, thereby more effectively improving the pathological process of AD. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the composition of the drug-loaded silk protein formulation that crosses the blood-brain barrier proposed in this invention; Figure 2 This is a schematic flowchart of the preparation method of the drug-loaded silk protein formulation that crosses the blood-brain barrier proposed in this invention; Figure 3 The image shows the morphology of nanoparticles formed by co-assembling silk fibroin nanofiber solution with different organic solvents and functional therapeutic active components. Figure 4 This is a schematic diagram showing the inflammatory regulation and antioxidant properties of different drug-loaded silk protein preparations co-assembled from silk protein nanofiber solutions in isopropanol solution. Figure 5 This is a schematic diagram showing the in vivo imaging results of small animals at different time points after tail vein injection in AD rats, formed by co-assembling different nanoparticles of silk protein nanofiber solution in a Rhodamine Fluorescent Red / isopropanol mixed solution and then modifying them with mannose (Man) or rabies virus capsid protein (RVG29). Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: Drug-loaded silk protein formulations that cross the blood-brain barrier, such as Figure 1-5 As shown, it includes silk protein nanofibers, functional therapeutic active components, and mannose / rabies virus capsid protein, characterized in that the silk protein nanofibers form nanoparticles through self-assembly; The functional therapeutic active components are prepared into bioactive silk nanocarriers using the self-assembly process of silk nanofibers. The silk protein nanocarrier and mannose / rabies virus capsid protein are coupled with amino groups and dialysis to obtain a drug-loaded silk protein nanocarrier formulation that can cross the blood-brain barrier. The drug-loaded silk protein nanocarrier formulation is loaded with a compound system of two natural polyphenol active components with different functions. One component is an anti-inflammatory polyphenol component that mainly inhibits neuroinflammation and repairs nerve cells. The other component is an anti-Aβ antioxidant polyphenolic component that mainly inhibits β-amyloid aggregation and scavenges ROS. The two components work synergistically to form an anti-inflammatory and anti-Aβ aggregation dual pathway, jointly improving the pathological microenvironment of the brain in Alzheimer's disease.

[0025] The anti-inflammatory polyphenol component is one of the natural polyphenol active components compounded in a system of curcumin, resveratrol, caffeic acid, etc. The anti-Aβ antioxidant polyphenol component is one of the natural polyphenol active components compounded in a system including EGCG, theanine, and proanthocyanidins.

[0026] In this embodiment, the functional therapeutic active ingredient is not limited to the addition of curcumin, EGCG and various polyphenols, and can also be a system of other equivalent functional therapeutic active ingredients.

[0027] The concentration of the silk fibroin nanofiber solution is 1–9 mg / mL; the concentration of curcumin is 10–200 μmol / L; and the concentration of epigallocatechin gallate is 0.5–2.5 mmol / L. The mannose concentration is 0.5–3 mmol / L, and the rabies virus capsid protein peptide concentration is 0.3–3 mmol / L.

[0028] A method for preparing drug-loaded silk protein formulations that cross the blood-brain barrier includes the following steps: S1: The silk fibroin nanofiber solution was diluted with pure water to obtain a solution with a concentration of 1-9 mg / mL. The solution was mixed with an organic solvent, stirred, frozen and thawed, centrifuged, and sonicated to obtain silk fibroin nanoparticles of different sizes. S2: The diluted silk nanofiber solution with a concentration of 1-9 mg / mL was mixed with an organic solvent containing functional therapeutic active components, stirred, frozen-thawed, centrifuged, and sonicated to obtain drug-loaded silk nanoparticles of different sizes. S3: Drug-loaded silk protein nanoparticles were subjected to an amino-coupling reaction with mannose / rabies virus capsid protein peptides to obtain a drug-loaded silk protein nanodelivery carrier that crosses the blood-brain barrier.

[0029] In step S1, the preparation method of the silk fibroin nanofiber solution includes the following steps: A1: After degumming raw silk in an anhydrous sodium carbonate solution at 100℃ for 60 minutes, remove it and dry it in an oven at 60℃ to obtain dry silk protein fiber; A2: Dry silk protein fibers are treated with 8.0 mol / L... -1 The mixed solution of LiBr and 99% formic acid was dissolved in an oven at 60°C for 4 hours and then placed into a dialysis bag. A3: Dialyze the solution in low-temperature deionized water for 72 h to remove LiBr and formic acid, and centrifuge twice at 4℃ and 9000 rpm for 20 min to remove impurities, to obtain a silk fibroin nanofiber solution. A4: After drying the silk fibroin nanofiber solution in an oven at 60℃, weigh it and calculate that the concentration of the silk fibroin nanofiber solution is approximately 9 mg / mL. Store it at low temperature for later use.

[0030] In S1, the volume ratio of the organic solvent containing the functional therapeutic active component to the silk nanofiber solution in the silk nanoparticles is 1:10–1:1; the organic solvent is composed of one or more solutions of isopropanol, dimethyl sulfoxide, and acetone. In step S1, the stirring speed is 100-700 rpm and the time is 5-60 minutes; In S1, the freezing temperature is -70 to -85°C, and the freezing time is 24 hours; In S1, the centrifugation speed is 12000 rpm and the centrifugation time is 30 minutes; In S1, the power of the ultrasonic treatment is 50-600W, and the ultrasonic treatment time is 1-10 minutes. In S2, the functional therapeutic active component is composed of one or a mixture of curcumin and epigallocatechin gallate, wherein the concentration of curcumin is 10–200 μmol / L and the concentration of epigallocatechin gallate is 0.5–2.5 mmol / L. In S3, the mannose concentration is 0.5–3 mmol / L, and the rabies virus capsid protein peptide concentration is 0.3–3 mmol / L.

[0031] In this embodiment, a magnetic stirrer is used for mixing, a centrifuge is used for centrifugation, and an ultrasonic device is used for ultrasonication. The magnetic stirrer, centrifuge, and ultrasonic device are respectively a multi-site magnetic stirrer, a low-temperature high-speed centrifuge, and an ultrasonic cell disruptor.

[0032] Example 2: Application of drug-loaded silk protein formulations that cross the blood-brain barrier, such as... Figure 1-5 As shown, the application is used for neurodegenerative diseases such as Alzheimer's disease, to improve the internal environment of dysfunctional microglia in the brain of Alzheimer's patients, reduce the content of β-amyloid protein aggregates in the brain of Alzheimer's patients, and thus improve the pathological process of Alzheimer's disease.

[0033] Experimental Example 1: First, prepare the silk fibroin nanofiber solution according to the preparation method of the silk fibroin nanofiber solution. Then, dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL). Then, mix it at low speed using a magnetic stirrer. Next, the dimethyl sulfoxide solution was added dropwise at a rate of 10 μL per drop to the above-mentioned 2 mg / mL silk nanofiber solution, so that the volume ratio of dimethyl sulfoxide to silk nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0034] Example 2: Prepare silk fibroin nanofiber solution according to the preparation method of silk fibroin nanofiber solution, then dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL), and then mix at low speed using a magnetic stirrer; Next, acetone solution was added dropwise at a rate of 10 μL per drop to the above 2 mg / mL silk nanofiber solution, so that the volume ratio of acetone to silk nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0035] Example 3: Prepare silk fibroin nanofiber solution according to the preparation method of silk fibroin nanofiber solution, then dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL), and then mix at low speed using a magnetic stirrer; Next, the isopropanol solution was added dropwise at a rate of 10 μL per drop to the above-mentioned 2 mg / mL silk fibroin nanofiber solution, so that the volume ratio of isopropanol to silk fibroin nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0036] Example 4: Prepare silk fibroin nanofiber solution according to the preparation method of silk fibroin nanofiber solution, then dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL), and then mix at low speed using a magnetic stirrer; Next, the curcumin / isopropanol solution (curcumin concentration of 200 μmol / L) was added dropwise at a rate of 10 μL to the above-mentioned 2 mg / mL silk nanofiber solution, so that the volume ratio of curcumin / isopropanol solution to silk nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0037] The above-mentioned silk fibroin nanoparticles (500 μg / mL, 10 mL) were reacted with mannose solution (1.5 mM) in EDC / NHS (molar ratio 1:3) solution for 6 hours and dialyzed for 24 hours to obtain mannose-modified drug-loaded silk fibroin nanocarriers, which have good water dispersibility and the ability to cross the blood-brain barrier.

[0038] Example 5: Prepare silk fibroin nanofiber solution according to the preparation method of silk fibroin nanofiber solution, then dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL), and then mix at low speed using a magnetic stirrer; Next, the epigallocatechin gallate / isopropanol solution (epigallocatechin gallate concentration of 2 mmol / L) was uniformly added dropwise at 10 μL per drop to the above 2 mg / mL silk nanofiber solution, so that the volume ratio of the epigallocatechin gallate / isopropanol solution to the silk nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0039] The above-mentioned silk fibroin nanoparticles (500 μg / mL, 10 mL) were reacted with mannose solution (1.5 mM) in EDC / NHS (molar ratio 1:3) solution for 6 hours and dialyzed for 24 hours to obtain mannose-modified drug-loaded silk fibroin nanocarriers, which have good water dispersibility and the ability to cross the blood-brain barrier.

[0040] Example 6: Prepare silk fibroin nanofiber solution according to the preparation method of silk fibroin nanofiber solution, then dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL), and then mix at low speed using a magnetic stirrer; Next, the curcumin / epigallocatechin gallate / isopropanol solution (curcumin concentration of 200 μmol / L; epigallocatechin gallate concentration of 2 mmol / L) was uniformly added dropwise at 10 μL per drop to the above 2 mg / mL silk nanofiber solution, so that the volume ratio of epigallocatechin gallate / isopropanol solution to silk nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0041] The above-mentioned silk fibroin nanoparticles (500 μg / mL, 10 mL) were reacted with mannose solution (1.5 mM) in EDC / NHS (molar ratio 1:3) solution for 6 hours and dialyzed for 24 hours to obtain mannose-modified drug-loaded silk fibroin nanocarriers, which have good water dispersibility and the ability to cross the blood-brain barrier.

[0042] Example 7: A silk fibroin nanofiber solution was prepared according to the preparation method of the silk fibroin nanofiber solution. The silk fibroin nanofiber solution was then diluted with pure water to the required concentration (2 mg / mL, 10 mL), and then mixed at low speed using a magnetic stirrer. Next, the curcumin / epigallocatechin gallate / isopropanol solution (curcumin concentration of 200 μmol / L; epigallocatechin gallate concentration of 2 mmol / L) was uniformly added dropwise at 10 μL per drop to the above 2 mg / mL silk nanofiber solution, so that the volume ratio of epigallocatechin gallate / isopropanol solution to silk nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0043] The above-mentioned silk protein nanoparticles (500 μg / mL, 10 mL) were reacted with rabies virus capsid protein solution (3 mM) in EDC / NHS (molar ratio 1:3) solution for 6 hours and dialyzed for 24 hours to obtain a drug-loaded silk protein nanocarrier modified with rabies virus capsid protein, which gave the drug-loaded silk protein nanocarrier good water dispersibility and the ability to cross the blood-brain barrier.

[0044] Example 8: Prepare silk fibroin nanofiber solution according to the preparation method of silk fibroin nanofiber solution, then dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL), and then mix at low speed using a magnetic stirrer; The next step was to replace curcumin and epigallocatechin gallate with rhodamine fluorescent red dye. A rhodamine fluorescent red dye / isopropanol mixed solution (rhodamine fluorescent red dye concentration of 1 wt%) was uniformly added dropwise at 10 μL per drop to the above 2 mg / mL silk fibroin nanofiber solution, so that the volume ratio of the rhodamine fluorescent red dye / isopropanol mixed solution to the silk fibroin nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0045] The above-mentioned silk fibroin nanoparticles (500 μg / mL, 10 mL) were reacted with mannose solution (1.5 mM) in EDC / NHS (molar ratio 1:3) solution for 6 hours and dialyzed for 24 hours to obtain mannose-modified fluorescent silk fibroin nanocarriers, which gave the drug-loaded silk fibroin nanocarriers good water dispersibility and the ability to cross the blood-brain barrier.

[0046] Example 9: Prepare silk fibroin nanofiber solution according to the preparation method of silk fibroin nanofiber solution, then dilute the silk fibroin nanofiber solution with pure water to the required concentration (2 mg / mL, 10 mL), and then mix at low speed using a magnetic stirrer; The next step was to replace curcumin and epigallocatechin gallate with rhodamine fluorescent red dye. A rhodamine fluorescent red dye / isopropanol mixed solution (rhodamine fluorescent red dye concentration of 1 wt%) was uniformly added dropwise at 10 μL per drop to the above 2 mg / mL silk fibroin nanofiber solution, so that the volume ratio of the rhodamine fluorescent red dye / isopropanol mixed solution to the silk fibroin nanofiber solution in the total system was 1:5. Then, the mixture was stirred at 400 rpm for 30 minutes using an electromagnetic stirrer to obtain a mixed solution. The above-obtained mixed solution was frozen at -80°C for 24 hours, then thawed at room temperature, and then centrifuged twice at 12000 rpm for 30 min each time. The precipitate was then collected and resuspended in pure water. The above resuspended solution was ultrasonically treated with an ultrasonic cell disruptor at 100W for 5 minutes to obtain silk protein nanoparticles, which gave the silk protein nanoparticles good water dispersibility.

[0047] The above-mentioned silk protein nanoparticles (500 μg / mL, 10 mL) were reacted with rabies virus capsid protein solution (3 mM) in EDC / NHS (molar ratio 1:3) solution for 6 hours and dialyzed for 24 hours to obtain a rabies virus capsid protein modified fluorescent silk protein nanodelivery carrier, which gave the drug-loaded silk protein nanodelivery carrier good water dispersibility and the ability to cross the blood-brain barrier.

[0048] Subsequently, the silk protein nanoparticles in Experiments 1-9 were tested and evaluated using morphological characterization and observation methods, inflammation regulation performance detection methods, antioxidant performance calculation and evaluation methods, and blood-brain barrier crossing ability evaluation methods. The morphological characterization observation method includes the following steps: B1: The silk protein nanoparticle dilution solution prepared in Experiments 1-9 was dropped onto the silicon wafer of the electron microscope stage, and then sputtered with gold at 10mA for 90s. B2: The morphology and size of each group of particles were analyzed using cold field scanning electron microscopy at 3 kV.

[0049] Morphological characterization observation such as Figure 3 As shown, Figure 3 The image shows the morphology of nanoparticles formed by co-assembling silk protein nanofiber solutions with different organic solvents and functional therapeutic active components.

[0050] Figure 3 In the image, region (a) represents nanoparticles (SNPs@DMSO) co-assembled from silk fibroin nanofiber solution and dimethyl sulfoxide. (b) Region is composed of nanoparticles (SNPs@Acetone) formed by co-assembly of silk protein nanofiber solution and acetone. (c) Region is composed of nanoparticles (SNPs@IPA) co-assembled from silk fibroin nanofiber solution and isopropanol. (d) is a region of nanoparticles (SNPs@Curcumin / IPA) co-assembled from a silk fibroin nanofiber solution and a curcumin / isopropanol organic solvent mixture. (e) region is nanoparticles (SNPs@EGCG / IPA) formed by co-assembling silk fibroin nanofiber solution with epigallocatechin gallate / isopropanol organic solvent mixture; (f) is a region of nanoparticles (SNPs@Curcumin / EGCG / IPA) co-assembled from a silk fibroin nanofiber solution and a mixture of curcumin / epigallocatechin gallate / isopropanol organic solvent.

[0051] Figure 3 The morphology characterization results showed that silk fibroin nanoparticles could be formed by different organic solvents; among them, the nanoparticles formed by co-assembling isopropanol solution and 2 mg / mL silk fibroin nanofiber solution had more stable and uniform morphology and size.

[0052] Furthermore, the nanoparticles formed by co-assembling a mixture of curcumin / epigallocatechin gallate / isopropanol organic solvent with a 2 mg / mL silk fibroin nanofiber solution exhibit stable and uniform structure and size, making them an ideal choice for nanodelivery carriers in the biomedical field.

[0053] The method for detecting inflammation regulation performance includes the following steps: C1: Mouse microglia (BV-2) stimulated by β-amyloid oligomers were treated with mannose-modified drug-loaded silk protein nanoparticles, and the cell supernatant was collected after 24 hours of co-culture. C2: The anti-inflammatory level of the nanodelivery carrier was assessed using an ELISA kit for detecting inflammation-related factors.

[0054] The method for calculating and evaluating antioxidant performance includes the following steps: D1: The drug-loaded silk protein nanocarriers (2 mg / mL, 100 μL) before and after mannose modification were mixed with DPPH free radicals (200 μM, 1 mL) until homogeneous. D2: After co-incubation at 37℃ for 30 minutes, the absorbance of each group of nanocarrier mixtures was detected by a multifunctional microplate reader, and their in vitro free radical scavenging ability was calculated and evaluated.

[0055] The results of inflammation regulation and antioxidant performance calculations for different drug-loaded silk fibroin nanocarriers co-assembled in isopropanol solution are as follows: Figure 3 As shown: Figure 4 In the figure, (a) and (b) represent the cellular inflammation regulation capacity (tumor necrosis factor-TNF-α, interleukin-6-IL-6) of SNPs@Curcumin / IPA, SNPs@EGCG / IPA, SNPs@Curcumin / EGCG / IPA and single SNPs@IPA vector after mannose modification. Figure 4 (c) represents the in vitro free radical scavenging capacity of SNPs@Curcumin / IPA, SNPs@EGCG / IPA, SNPs@Curcumin / EGCG / IPA, and a single SNPs@IPA vector before and after mannose modification.

[0056] Figure 4 The results in a and b show that, compared with the levels of TNF-α (1182.36 pg / mL) and IL-6 (148.16 pg / mL) in BV-2 cells induced by Aβ protein aggregates, the levels of TNF-α in BV-2 cells treated with the silk protein nanocarrier co-assembled with curcumin and epigallocatechin gallate (EGCG) decreased to 911.58 pg / mL and the level of IL-6 decreased to 70.15 pg / mL. This significantly reduced the levels of pro-inflammatory factors in BV-2 cells, thereby further improving the disordered cellular environment and promoting the transformation of cells to a healthy state. Figure 4 The results in c showed that the DPPH scavenging efficiency of the single filament protein nanoparticles was 7.95%, while that of the drug-loaded filament protein nanocarrier increased to 21.27%. The in vitro DPPH scavenging ability demonstrated the good antioxidant properties of the prepared drug-loaded filament protein nanocarrier, which can improve the intracellular oxidative stress level, promote the restoration of cellular homeostasis, and thus improve the ability of BV-2 to phagocytose β-amyloid protein deposits and improve the pathological process of Alzheimer's disease.

[0057] The method for evaluating the ability to cross the blood-brain barrier includes the following steps: E1: Five-month-old APP / PS1 mice were selected. Fluorescent filament protein nanodelivery carriers modified with mannose and rabies virus capsid protein were administered to the mice via tail vein injection in 200 μL. The distribution of the fluorescent filament protein nanodelivery carriers in vivo was monitored at 1, 3, 5, 8, 12 and 24 hours after injection using a small animal in vivo imaging system. The brain and abdomen were scanned. E2: Evaluation of the blood-brain barrier crossing performance of fluorescent silk protein nanocarriers. Forty-eight hours after tail vein injection, organs (brain, heart, liver, spleen, lung, and kidney) from different parts of mice were removed and subjected to in vitro scanning to analyze the distribution of the fluorescent silk protein nanocarriers within the mice.

[0058] The results of the assessment of ability to cross the blood-brain barrier are as follows Figure 4 As shown, Figure 4 The images show in vivo imaging results of small animals at different time points after tail vein injection in Alzheimer's disease (AD) rats, where different nanoparticles co-assembled from silk fibroin nanofiber solution in a rhodamine fluorescent red / isopropanol mixed solution were modified with mannose or rabies virus capsid protein. Figure 5 Fluorescence imaging of different groups of silk protein nanoparticles in the middle (a) position in the brain of AD mice; (b) is a fluorescence imaging distribution map of different groups of silk protein nanoparticles in the abdomen of AD mice; (c) Fluorescence images of various organs in AD mice 48 hours after tail vein injection of unmodified silk protein nanoparticles; (d) is an in vitro fluorescence image of various organs of AD mice 48 hours after tail vein injection of mannose-modified silk protein nanoparticles.

[0059] The results in the figure demonstrate that the silk protein nanocarrier, after being modified with mannose and rabies virus capsid protein, has a significantly improved efficiency in crossing the blood-brain barrier. This indicates that after the particles are modified with specific ligands targeting brain microvascular endothelial cells, the efficiency of the nanocarrier entering the brain can be significantly improved, which can enhance the bioavailability of AD treatment drugs and improve the efficacy of targeted drugs for AD.

[0060] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A drug-loaded silk protein formulation that crosses the blood-brain barrier, comprising silk protein nanofibers, a functional therapeutically active component, and mannose / rabies virus capsid protein, characterized in that, The silk protein nanofibers form nanoparticles through self-assembly. The functional therapeutic active components are prepared into bioactive silk nanocarriers using the self-assembly process of silk nanofibers. The silk protein nanocarrier and mannose / rabies virus capsid protein are coupled with amino groups and dialyzed to obtain a drug-loaded silk protein nanocarrier formulation that can cross the blood-brain barrier.

2. The drug-loaded silk protein formulation according to claim 1, characterized in that, The functional therapeutic active component is composed of a compound system of two natural polyphenol active components with different functions. One component is an anti-inflammatory polyphenol component that mainly inhibits neuroinflammation and repairs nerve cells. The other component is an anti-Aβ antioxidant polyphenolic component that mainly inhibits β-amyloid aggregation and scavenges ROS. The anti-inflammatory polyphenol component is one of the natural polyphenol active components compounded in a system of curcumin, resveratrol, caffeic acid, etc. The anti-Aβ antioxidant polyphenol component is one of the natural polyphenol active components compounded in a system including EGCG, theanine, and proanthocyanidins.

3. The drug-loaded silk protein formulation according to claim 1, characterized in that, The concentration of the silk protein nanofiber solution is 1–9 mg / mL.

4. The drug-loaded silk protein formulation according to claim 1, characterized in that, The mannose concentration is 0.5–3 mmol / L, and the rabies virus capsid protein peptide concentration is 0.3–3 mmol / L.

5. A method for preparing a drug-loaded silk protein formulation that crosses the blood-brain barrier, characterized in that, Includes the following steps: S1: The silk fibroin nanofiber solution was diluted with pure water to obtain a solution with a concentration of 1-9 mg / mL. The solution was mixed with an organic solvent, stirred, frozen and thawed, centrifuged, and sonicated to obtain silk fibroin nanoparticles of different sizes. S2: The diluted silk nanofiber solution with a concentration of 1-9 mg / mL was mixed with an organic solvent containing functional therapeutic active components, stirred, frozen-thawed, centrifuged, and sonicated to obtain drug-loaded silk nanoparticles of different sizes. S3: Drug-loaded silk protein nanoparticles were subjected to an amino-coupling reaction with mannose / rabies virus capsid protein peptides to obtain a drug-loaded silk protein nanocarrier that crosses the blood-brain barrier.

6. The method for preparing a drug-loaded silk protein formulation that crosses the blood-brain barrier according to claim 5, characterized in that, In step S1, the preparation method of the silk fibroin nanofiber solution includes the following steps: A1: After degumming raw silk in an anhydrous sodium carbonate solution at 100℃ for 60 minutes, remove it and dry it in an oven at 60℃ to obtain dry silk protein fiber; A2: Dry silk protein fibers are treated with 8.0 mol / L... -1 The mixed solution of LiBr and 99% formic acid was dissolved in an oven at 60°C for 4 hours and then placed into a dialysis bag. A3: Dialyze the solution in low-temperature deionized water for 72 h to remove LiBr and formic acid, and centrifuge twice at 4℃ and 9000 rpm for 20 min to remove impurities, to obtain a silk fibroin nanofiber solution. A4: After drying the silk fibroin nanofiber solution in an oven at 60°C, weigh it and calculate the concentration of the silk fibroin nanofiber solution to be 0.1–9 mg / mL. Store it at low temperature for later use.

7. The method for preparing a drug-loaded silk protein formulation that crosses the blood-brain barrier according to claim 5, characterized in that, In step S1, the stirring speed is 100-700 rpm and the time is 5-60 minutes; The freezing temperature is -70 to -85℃, the freezing time is 24 hours, the centrifugation speed is 12000 rpm, and the centrifugation time is 30 minutes. The ultrasonic treatment power is 50-600W, and the ultrasonic treatment time is 1-10 minutes.

8. The method for preparing a drug-loaded silk protein formulation that crosses the blood-brain barrier according to claim 5, characterized in that, In S1, the volume ratio of the organic solvent containing the functional therapeutic active component to the silk nanofiber solution is 1:10–1:

1. The organic solvent is composed of one or more solutions of isopropanol, dimethyl sulfoxide, and acetone.

9. The method for preparing a drug-loaded silk protein formulation that crosses the blood-brain barrier according to claim 5, characterized in that, In S3, the amino coupling adopts an EDC / NHS system with a molar ratio of 1:3, a reaction time of 6 h, and a dialyze time of 24 h.

10. The use of the drug-loaded silk protein formulation that crosses the blood-brain barrier according to any one of claims 1-4 in the preparation of a drug for treating neurodegenerative diseases, characterized in that, The neurodegenerative disease mentioned is Alzheimer's disease.

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