Brain-targeted delivery system as well as preparation method and application thereof

Abstract

The invention provides a brain-targeted delivery system. The brain-targeted delivery system comprises mesoporous Prussian blue nano-enzyme serving as a drug carrier and an erythrocyte membrane wrapping the surface of the drug carrier, the erythrocyte membrane is modified by rabies virus glycoprotein polypeptide. The invention further provides a medicine which further comprises an active substance loaded on the medicine carrier, and the active substance comprises at least one of L-arginine and butylphthalide. When the brain-targeted delivery system is used for synergistically treating cerebral arterial thrombosis, the immune escape ability is remarkably enhanced, the brain-targeted efficiency is improved by more than 10 times compared with that of unmodified nanoparticles, and the retention time in the brain is more than or equal to 24 hours.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and nanotechnology, and specifically relates to a multifunctional nanodelivery system for the synergistic treatment of ischemic stroke (CIS). Background Technology

[0002] Ischemic stroke (CIS) is one of the leading causes of disability and death worldwide, and its pathological mechanisms involve multiple factors such as oxidative stress, inflammatory cascades, and vascular dysfunction. Although clinical methods such as intravenous thrombolysis and mechanical thrombectomy can partially restore cerebral blood flow, the strict time window limitation (usually <4.5 hours) and reperfusion injury severely restrict the treatment effect. In recent years, neuroprotective drugs targeting single pathological mechanisms (such as free radical scavenging or anti-inflammation) have shown potential in animal models, but have generally failed in clinical trials. The main reasons include: ① Blood-brain barrier (BBB) ​​limitation: Even if the BBB is partially open during acute ischemia, its dynamic repair characteristics make it difficult to continuously deliver drugs to the lesion area; ② Insufficient multi-target regulation: Post-CIS neurological injury involves the synergistic effects of oxidative stress (ROS accumulation), inflammation (microglial activation), and vascular stenosis (endothelial dysfunction), and single-pathway intervention is difficult to comprehensively block the pathological process; ③ Defects in drug delivery systems: Traditional nanocarriers (such as liposomes and polymer micelles) are easily cleared by the reticuloendothelial system (RES) and lack the ability to actively target ischemic areas, resulting in low bioavailability.

[0003] While existing nanoparticle-based drug delivery systems can partially improve drug solubility and circulation time, they still have significant limitations: ① Immunogenicity issues: Synthetic materials (such as PLGA and PEG) may trigger complement activation or accelerate blood clearance; ② Single-function design: Most systems focus only on drug encapsulation or passive targeting (EPR effect), and cannot simultaneously integrate antioxidant, anti-inflammatory, and vascular repair functions; ③ Controllable release challenges: The dynamic changes in the microenvironment of ischemic areas (such as pH gradient and ROS level) require drug release to be spatiotemporally matched with the pathological process, while traditional carriers lack environmental responsiveness.

[0004] Against this backdrop, biomimetic nanotechnology and multi-mechanism synergistic therapeutic strategies have become a research hotspot. For example, erythrocyte membrane camouflage can evade immune recognition through a "self-labeling" mechanism, prolonging the circulating half-life; mesoporous Prussian blue nanozymes (HMPBs), due to their superoxide dismutase (SOD) and catalase (CAT)-like activities, are used for ROS scavenging; and targeted peptides (such as RVGs) can penetrate the BBB via receptor-mediated transport. However, current technologies have not yet effectively integrated these advantages, especially lacking a multifunctional platform capable of simultaneously regulating oxidative stress, inflammatory responses, and vascular function. Summary of the Invention

[0005] This invention develops a nanodelivery system that combines highly efficient targeting, multi-mechanism synergy, and controlled release, representing a key direction for overcoming the bottlenecks in CIS treatment. By integrating erythrocyte membrane biomimetic technology, free radical scavenging nanozymes, and multi-drug synergistic loading, this invention aims to address the problems of low delivery efficiency and limited treatment mechanisms in existing technologies, providing an innovative treatment solution for CIS.

[0006] In a first aspect of this application, a brain-targeted delivery system is provided, comprising: a mesoporous Prussian blue nanozyme as a drug carrier, and a red blood cell membrane encapsulated on the surface of the drug carrier; the red blood cell membrane is modified with rabies virus glycoprotein polypeptide.

[0007] Optionally, the mesoporous Prussian blue nanozyme has a hollow mesoporous structure and a specific surface area of ​​130-140 m². 2 / g, with an average pore size of 18-22nm, and exhibits catalytic activity for SOD, CAT, and peroxidase (POD).

[0008] Optionally, the preparation method of the mesoporous Prussian blue nanozyme includes the following steps: a1: Dissolve K3[Fe(CN)6] and PVP in hydrochloric acid solution and stir until the solution turns yellow and transparent; a2: Heat the solution obtained in step a1 in a water bath, centrifuge, wash, and dry the precipitate to obtain Prussian blue nanozyme; a3: Mix Prussian blue nanozyme and PVP in hydrochloric acid solution, stir, and heat at 120-140℃ for 3-5 hours to obtain mesoporous Prussian blue nanozyme.

[0009] Optionally, the concentration of hydrochloric acid in step a1 is 0.005–0.015 M; the concentration of hydrochloric acid in step a3 is 0.8–1.2 M.

[0010] In a second aspect of this application, this application provides the application of the brain-targeted delivery system described in the first aspect of this invention in the delivery of drugs for ischemic stroke.

[0011] Optionally, the drug is composed of L-arginine and butylphthalide.

[0012] In a third aspect of this application, this application provides a drug for a brain-targeted delivery system as described in the first aspect of the present invention, comprising: a mesoporous Prussian blue nanozyme as a drug carrier, an active drug substance loaded on the drug carrier, and a red blood cell membrane encapsulated on the surface of the drug carrier; wherein the red blood cell membrane is modified with a rabies virus glycoprotein polypeptide, and the active substance comprises at least one of L-arginine and butylphthalide.

[0013] Optionally, the active substance is composed of L-arginine and butylphthalide.

[0014] Optionally, the loading amounts of L-arginine and butylphthalide on the drug carrier are: The encapsulation efficiency of L-arginine is 40-45%, and the drug loading is 20-22%. Butylphthalide has an encapsulation efficiency of 55-60% and a drug loading of 2.5-3%.

[0015] Optionally, the drug may also contain pharmaceutically acceptable excipients.

[0016] In a fourth aspect, this application provides a method for preparing a drug for the brain-targeted delivery system described in the third aspect of the invention, comprising the following steps: S1: Co-loaded drug: L-arginine and butylphthalide were dissolved and added dropwise to the mesoporous Prussian blue nanozyme dispersion. After stirring and reacting, the mixture was centrifuged and freeze-dried. S2: Red blood cell membrane encapsulation: Red blood cell membranes are extracted, mixed with mesoporous Prussian blue nanozyme / L-arginine-butylphthalide, and then extruded by ultrasonication. S3: Rabies virus glycoprotein peptide modification: DSPE-PEG2000-rabies virus glycoprotein peptide was added to the product of step S2 and incubated, followed by dialysis purification.

[0017] Optionally, in step S1, the solvent for L-arginine is PBS, and the solvent for butylphthalide is ethanol.

[0018] Further, optionally, the drug loading pH in step S1 is 7.4.

[0019] Optionally, the red blood cell membrane is derived from mouse red blood cell membranes.

[0020] Furthermore, optionally, the ultrasonic temperature in step S2 is 4°C, and the extrusion process is performed on ice.

[0021] Furthermore, optionally, the dialysis temperature in step S3 is 4°C.

[0022] In a fifth aspect of this application, this application provides the use of the brain-targeted delivery system described in the first aspect of this invention and / or the brain-targeted delivery system described in the third aspect of this invention in the preparation of a drug for the prevention or treatment of ischemic stroke.

[0023] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention provides a brain-targeted delivery system, comprising: a mesoporous Prussian blue nanozyme as a drug carrier, and a red blood cell membrane encapsulated on the surface of the drug carrier; the red blood cell membrane is modified with rabies virus glycoprotein peptides. This delivery system can scavenge free radicals through the catalytic activity of SOD and CAT of HMPB; evade immune recognition by "self-labeling" with CD47 using RCM camouflage, thus prolonging blood circulation time; and achieve active targeting by specifically binding to BBB and neuronal acetylcholine receptors (nAChR) via RVG. In vivo, this brain-targeted delivery system exhibits: significantly enhanced immune escape capability, brain targeting efficiency more than 10 times higher than unmodified nanoparticles, and brain retention time ≥24 hours.

[0024] 2. This application provides an application of the above-mentioned brain-targeted delivery system in the delivery of drugs for ischemic stroke. The drug can achieve vasodilation and ROS clearance by releasing NO through L-arginine in the acidic environment (pH=5.0) of the ischemic area, and improve cerebral microcirculation and promote collateral blood flow reconstruction in the ischemic area by using butylphthalide.

[0025] 3. This application also provides a drug for the above-mentioned brain-targeted delivery system, comprising: a mesoporous Prussian blue nanozyme as a drug carrier, an active drug substance loaded on the drug carrier, and a red blood cell membrane encapsulated on the surface of the drug carrier; the red blood cell membrane is modified with rabies virus glycoprotein, and the active substance includes at least one of L-arginine and butylphthalide. This drug can achieve vasodilation and ROS clearance through NO release via L-arginine in the acidic environment (pH=5.0) of the ischemic area, and improve cerebral microcirculation and promote collateral blood flow reconstruction in the ischemic area via butylphthalide.

[0026] 4. This invention provides the application of the above-described brain-targeted delivery system and / or the above-described drug in the preparation of drugs for the prevention or treatment of ischemic stroke. The brain-targeted delivery system penetrates the BBB to treat CIS. Attached Figure Description

[0027] Figure 1 This is a process flow diagram for preparing RVG-M@HMPB / L-Arg-NBP in Example 1 of this application; Figure 2 The basic particle sizes of HMPB, HMPB / L-Arg-NBP, M@HMPB / L-Arg-NBP, and RVG-M@HMPB / L-Arg-NBP in Example 1 are shown below. Figure 3 The potentials of HMPB, HMPB / L-Arg-NBP, M@HMPB / L-Arg-NBP, and RVG-M@HMPB / L-Arg-NBP in Example 1; Figure 4The TEM images of HMPB and HMPB / L-Arg-NBP in Example 1 are shown, with a scale bar of 200 nm. in, Figure 4 (A) is a TEM image of HMPB in Example 1; Figure 4 (B) is a TEM electron microscope image of the HMPB / L-Arg-NBP of this application; Figure 5 The images shown are TEM images of M@HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP from Example 1; where, Figure 5 (A) is a TEM image of M@HMPB / L-Arg-NBP in Example 1; Figure 5 (B) is a TEM image of RVG-M@HMPB / L-Arg-NBP in Example 1; Figure 6 The isothermal adsorption-desorption curve of HMPB in Example 1; Figure 7 The X-ray diffraction pattern of HMPB in Example 1; Figure 8 Fourier transform infrared spectra of HMPB and HMPB / L-Arg-NBP in Example 1; Figure 9 The ultraviolet spectra of PB, HMPB, Arg, HMPB / Arg-NBP, and NBP in Example 1 are shown. Figure 10 WB images of RCM and RVG-M@HMPB / L-Arg-NBP in Example 1; Figure 11 The particle size stability of RVG-M@HMPB / L-Arg-NBP in PBS solution at 4°C in Example 2; Figure 12 Release curves of HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP in Example 3 under neutral (pH=7.4) and acidic (pH=5.0) conditions; in, Figure 12 (A) shows the L-Arg release curves of HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP in Example 3 under neutral (pH=7.4) and acidic (pH=5.0) conditions; Figure 12(B) shows the NBP release curves of HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP in Example 3 under neutral (pH=7.4) and acidic (pH=5.0) conditions; Figure 13 The following data are presented in Example 4: SOD catalytic activity, CAT catalytic activity, POD catalytic activity, and NO generation at different concentrations of RVG-M@HMPB / L-Arg-NBP. in, Figure 13 (A) shows the SOD catalytic activity of RVG-M@HMPB / L-Arg-NBP at different concentrations in Example 4; Figure 13 (B) shows the CAT catalytic activity of RVG-M@HMPB / L-Arg-NBP at different concentrations in Example 4; Figure 13 (C) shows the POD catalytic activity of RVG-M@HMPB / L-Arg-NBP at different concentrations in Example 4; Figure 13 (D) represents the NO generation at different times under different concentrations of RVG-M@HMPB / L-Arg-NBP in Example 4; Figure 14 The results of the cytotoxicity experiment of RVG-M@HMPB / L-Arg-NBP in Example 5 are the results of the cytotoxicity experiment (n=3) after different concentrations of RVG-M@HMPB / L-Arg-NBP were incubated with bEnd.3 cells or PC12 cells for 24 h. Figure 15 The uptake of different materials by RAW264.7 in Example 6 (scale bar: 20 μm); Figure 16 The fluorescence images of the lower cavity PC12 after incubation for 4 h with Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in Example 7 are shown (scale bar: 20 μm). Figure 17 Fluorescence imaging and corresponding quantitative analysis of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in MCAO mice in Example 8; fluorescence imaging and corresponding quantitative analysis of major ex vivo organs 24 h after administration of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 (n=3); in, Figure 17(A) Fluorescence imaging of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in MCAO mice in Example 8; Figure 17 (B) Fluorescence imaging of major ex vivo organs 24 hours after administration of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in Example 8; Figure 17 (C) shows the fluorescence quantification of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in MCAO mice in Example 8; Figure 17 (D) The fluorescence quantification of the heart, liver, spleen, lungs and kidneys after 24 hours of administration of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5 and RVG-M@HMPB / Cy5.5 in Example 8; Figure 17 (E) The corresponding fluorescence quantification (n=3) of the brain fluorescence imaging after 24 hours of administration of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in Example 8. Detailed Implementation

[0028] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to the application, and these equivalent forms also fall within the protection scope of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the application.

[0029] the term: In this application, terms such as "further" and "even further" are used to describe the purpose and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0030] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0031] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0032] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0033] In this application, unless otherwise specified, the terms "size", "particle size", and "diameter" generally refer to average values.

[0034] Example 1 The preparation method of mesoporous Prussian blue nanoparticles encapsulated in erythrocyte membranes and co-loaded with L-arginine (L-Arg) and butylphthalide (NBP) (M@HMPB / L-Arg-NBP) includes the following steps: Synthesis of mesoporous Prussian blue nanozyme (HMPB): HMPB was synthesized via a hydrothermal method. First, K3[Fe(CN)6] (132 mg) and PVP (3 g) were dissolved in 40 mL of 0.01 M hydrochloric acid solution and stirred at room temperature for 1 h until the solution turned yellow and transparent. The solution was then transferred to a three-necked flask and heated in a water bath at 80 °C for 24 h. The precipitate was dried under vacuum after frequent centrifugation and washing with deionized water and ethanol to obtain Prussian blue nanozyme (PB). Then, PB (20 mg) and PVP (100 mg) were mixed in 20 mL of 1 M hydrochloric acid solution. After stirring for one hour, the solution was transferred to a reaction vessel with a polytetrafluoroethylene inner wall and heated at 140 °C for 4 h to obtain mesoporous Prussian blue nanozyme (HMPB). During this process, the PVP layer protects the outer shell from acidic H2O. + The erosion effect of ions forms a mesoporous structure.

[0035] Drug loading: 10 mg HMPB was dispersed in 10 mL of ultrapure water. NBP dissolved in ethanol (1 mg / mL, 1 mL) and L-Arg dissolved in PBS (10 mg / mL, 1 mL) were added dropwise to the HMPB solution, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 10 min, washed three times, and lyophilized to obtain HMPB / L-Arg-NBP, which was then stored at -20 °C.

[0036] Prepare L-Arg standard solutions (1, 2, 5, 10, 15, 20 μg / mL) using PBS. Turn on the UV spectrophotometer and preheat the device for 10-15 minutes. First, zero the instrument. Add pure PBS to the photocell, set the detection wavelength to 210 nm, and adjust to the zero point. Then process the sample: Take 200 μL of RVG-M@HMPB / L-Arg-NBP nanoparticles, add 800 μL of methanol, and sonicate to break the emulsion for 30 min using an ultrasonic cleaner. After breaking the emulsion, centrifuge the sample at 12000 rpm for 20 min, and add PBS to 2 mL of the supernatant. Transfer the sample to the photocell and record the absorbance value. After completing the test, clean the photocell with pure solvent and turn off the instrument. Prepare NBP standard solutions (10, 15, 20, 30, 60, 90 μg / mL) using ethanol. The sample breaking process is the same as above, and the detection wavelength is set to 274 nm. The absorbance of L-Arg and NBP was measured using a UV spectrophotometer, and standard curves were plotted: the standard curve for L-Arg was A = 0.0028C + 0.0149(R). 2 =0.9992); the standard curve for NBP is A = 0.0177C - 0.0556(R). 2 =0.9953).

[0037] Table 1. Drug loading and encapsulation efficiency of the two drugs in HMPB / L-Arg-NBP As shown in Table 1, based on the standard curve, the encapsulation efficiency of L-Arg is 42.8% and the drug loading is 21.4%; the encapsulation efficiency of NBP is 55.4% and the drug loading is 2.8%.

[0038] Red blood cell membrane (RCM) wraps around: Red blood cell membranes were extracted using a hypotonic centrifugation method. Whole blood from C57 / BL6 mice was collected in heparinized sodium centrifuge tubes and centrifuged at 2500 rpm for 5 min at 4°C. The supernatant plasma was removed, leaving dark red packed red blood cells. The lower red blood cells were washed three times with 1×PBS (1:3) and centrifuged at low speed to obtain the packed red blood cells. The red blood cells were then hypotonic in 0.25×PBS (1:40) on ice for 40–60 min and centrifuged at 13000 rpm for 15 min at 4°C. The lower blood layer was washed multiple times with 0.25×PBS until the supernatant was colorless, yielding pink red blood cell membranes. These membranes were resuspended in 1×PBS and stored at 4°C. The obtained 10 mg RCM was mixed with 1 mL HMPB / L-Arg-NBP solution (10 mg / mL), and the mixture was sonicated in a water bath for 10 min. The mixture was then extruded repeatedly about 10 times under polycarbonate membranes with pore sizes of 400 nm and 200 nm using a liposome extruder to obtain biomimetic nanoparticles M@HMPB / L-Arg-NBP solution, which was then stored at 4 °C.

[0039] Rabies virus glycoprotein (RVG) modification: First, 5 mg of DSPE-PEG2000-RVG29 powder was added to M@HMPB / L-Arg-NBP solution (0.5 mg / mL, 2 mL), and incubated overnight at 4°C. Then, the resulting solution was dialyzed in deionized water for 12 h using a dialysis bag (MWCO = 10 kDa). This yielded the RVG-M@HMPB / L-Arg-NBP solution.

[0040] Take HMPB, HMPB / L-Arg-NBP, M@HMPB / L-Arg-NBP, and RVG-M@HMPB / L-Arg-NBP, dilute them with deionized water, pour the diluted solutions into cuvettes and potentiometers, and use a Malvern particle size and potential analyzer to determine the average particle size, PDI, and Zeta potential of the nanoparticles.

[0041] The results are as follows Figure 2 , Figure 3 As shown, the hydrated particle sizes of HMPB, HMPB / L-Arg-NBP, M@HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP, as detected by a laser diffractometer (NANO ZS90), were 170.6 nm, 184.4 nm, 210 nm and 243.9 nm, respectively, and their potentials were -13.61 mV, -17.6 mV, -17 mV and -9.4 mV, respectively.

[0042] Deionized water was added to HMPB and HMPB / L-Arg-NBP to obtain nanoparticle dispersions of HMPB and HMPB / L-Arg-NBP. 10 μL of each HMPB / L-Arg-NBP nanoparticle dispersion was dropped onto a copper grid with a carbon support film and allowed to dry naturally at room temperature. The morphology of the nanoparticles was observed and photographed under a transmission electron microscope.

[0043] The results are as follows Figure 4 As shown in the figure, HMPB and HMPB / L-Arg-NBP exhibit a cubic morphology based on electron microscopy, with a particle size of approximately 100 nm.

[0044] Take 10 μL of M@HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP nanoparticle dispersions respectively, drop them onto a copper grid with a carbon support film, and let them air dry at room temperature. Observe the morphology of the nanoparticles under a transmission electron microscope and take pictures.

[0045] The results are as follows Figure 5 As shown, M@HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP exhibit a typical core-shell bilayer structure, confirming that the erythrocyte membrane has been successfully deposited on the surface of HMPB / L-Arg-NBP.

[0046] To prove that the synthesized HMPB has a mesoporous surface and a hollow core, 100 mg of HMPB solid powder was obtained by freeze-drying. After drying at 700 °C, the surface pore size distribution was tested using a fully automated surface area and porosity analyzer (BET, Micromeritics ASAP 2460, USA).

[0047] The results are as follows Figure 6 As shown, the specific surface area of ​​HMPB is 135.095 m². 2 / g, with an average pore size of 20.167nm.

[0048] 10 mg of HMPB solid powder was obtained by freeze-drying. The HMPB sample was subjected to wide-angle testing using BRUKER (D8 Advance, Germany), and specific X-ray diffraction peaks were obtained.

[0049] The results are as follows Figure 7 As shown, the diffraction peaks of HMPB correspond to crystal planes (200), (220), (400), (420), (422), (440), (600), and (620), respectively, which are in perfect agreement with the standard card (73-0687) of the face-centered cubic Prussian blue (PB) lattice (space group Fm3m).

[0050] Take 2 mg each of HMPB and HMPB / L-Arg-NBP solid powder and place them in the sample cell of an FTIR spectrophotometer (Thermo Fisher Scientific, USA). The wavenumber starts from 499 cm⁻¹. -1 Scan to 4000cm -1 The resulting spectrum showed a specific infrared absorption peak for HMPB.

[0051] The results are as follows Figure 8 As shown, HMPB / L-Arg-NBP at 2090 cm⁻¹ -1 and 1670cm -1 Characteristic absorption peaks appeared at 2090 cm⁻¹. -1 The peak at 1670 cm⁻¹ belongs to the -CN- stretching vibration of the Fe-CN-Fe bond in HMPB, while the peak at 1670 cm⁻¹ belongs to the -CN- stretching vibration of the Fe-CN-Fe bond. -1 The peak at that point corresponds to the C=O stretching vibration of the polyvinylpyrrolidone (PVP) amide unit. These results further confirm the chemical structural integrity of HMPB / L-Arg-NBP.

[0052] Take 2 mL of HMPB, NBP, L-Arg, and HMPB / L-Arg-NBP solution (0.05 mg / mL) and add them sequentially to the sample cell of the UV-Vis spectrophotometer. Set the detection wavelength to 200 nm to 1000 nm and detect the characteristic UV absorption peaks of each component.

[0053] The results are as follows Figure 9 As shown, PB, HMPB, and HMPB / L-Arg-NBP all exhibited a distinct Prussian blue characteristic absorption peak near 710 nm. Meanwhile, HMPB / L-Arg-NBP showed characteristic absorption peaks for L-Arg and NBP near 210 nm and 274 nm, respectively, further confirming the success of the dual-drug loading.

[0054] The extracted erythrocyte membranes and biomimetic nanoparticles RVG-M@HMPB / L-Arg-NBP were placed in centrifuge tubes, and 200 μL of RIPA (strong) lysis buffer containing 1% protease inhibitor was added. The mixture was then repeatedly pipetted and thoroughly mixed, and lysed on ice for 1 h. After centrifugation at 4℃ and 13000 rpm for 10 min, the precipitate was discarded. The erythrocyte membrane protein content in each sample was measured using the BCA method. The samples were then loaded for SDS-PAGE gel electrophoresis, and the resulting gel was transferred to a polyvinylidene fluoride (PVDF) membrane for Western blotting analysis. Through a series of steps including membrane transfer, skim milk blocking, incubation with primary antibody, incubation with secondary antibody, and color development, the expression of CD47 protein on the biomimetic nanoparticles was obtained.

[0055] The results are as follows Figure 10As shown, a band of CD47 protein from the erythrocyte membrane was observed in the band of RVG-M@HMPB / L-Arg-NBP, verifying that the erythrocyte membrane was successfully coated on the surface of HMPB / L-Arg-NBP.

[0056] Example 2 The preliminary stability of RVG-M@HMPB / L-Arg-NBP prepared in Example 1 was investigated.

[0057] Specifically, the prepared RVG-M@HMPB / L-Arg-NBP solution was stored at 4℃, and its particle size and PDI were measured at different times (0, 1, 3, 5, 7 days). The preliminary stability of the nanoparticles within one week was observed, and the data were recorded and a trend graph was plotted.

[0058] The results are as follows Figure 11 As shown, the particle size of RVG-M@HMPB / L-Arg-NBP remained essentially unchanged over 7 days at 4°C, demonstrating the stability of the nanodelivery system.

[0059] Example 3 Based on the HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP prepared in Example 1, their in vitro drug release was tested.

[0060] Specifically, to investigate the release of L-Arg and NBP under different pH conditions, the samples were divided into four groups: ① HMPB / L-Arg-NBP (pH=5.0), ② HMPB / L-Arg-NBP (pH=7.4), ③ RVG-M@HMPB / L-Arg-NBP (pH=5.0), and ④ RVG-M@HMPB / L-Arg-NBP (pH=7.4). To investigate the release efficiency of L-Arg and NBP, 1 mL of HMPB / L-Arg-NBP (2 mg / mL) solution sealed in a dialysis bag was immersed in 20 mL of PBS buffer (pH=7.4) or 20 mL of PBS buffer (pH=5.0). The RVG-M@HMPB / L-Arg-NBP (2 mg / mL) solution underwent the same treatment. Every 12 hours, 2 mL of dialysate was removed, and fresh PBS solution was added to bring the volume back to the original level. The amount of L-Arg and NBP released was monitored using a UV spectrophotometer.

[0061] The results are as follows Figure 12 As shown, Figure 12 (A) shows the L-Arg release curves of HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP in Example 3 under neutral (pH=7.4) and acidic (pH=5.0) conditions. Figure 12(A) It can be seen that both HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP exhibit rapid cleavage characteristics under acidic conditions (pH=5.0). Specifically, under pH=5.0 conditions, the cumulative release of L-Arg reached 97.3% and 81.3%, respectively, while under neutral conditions (pH=7.4), the release of L-Arg was 69.6% and 51.7%, respectively. Figure 12 (B) shows the NBP release curves of HMPB / L-Arg-NBP and RVG-M@HMPB / L-Arg-NBP in Example 3 under neutral (pH=7.4) and acidic (pH=5.0) conditions. Figure 12 (B) It can be seen that, similarly, the cumulative release of NBP at pH=5.0 was 94.3% and 78.5%, respectively, while at pH=7.4, the release was 56.3% and 39.3%, respectively.

[0062] Example 4 Based on the RVG-M@HMPB / L-Arg-NBP prepared in Example 1, its reactive oxygen species scavenging performance and NO release were tested.

[0063] Specifically, the SOD activity of RVG-M@HMPB / L-Arg-NBP was detected using the WST-8 Total Superoxide Dismutase Assay Kit. 20 μL of HMPB was mixed with 200 μL of WST-8 working solution and 20 μL of xanthine oxidase solution. After 30 min, the absorbance was measured, and the SOD catalytic activity was determined at 450 nm. The CAT activity of RVG-M@HMPB / L-Arg-NBP was measured using a catalase assay kit. Hydrogen peroxide is catalyzed by CAT enzyme to produce oxygen and water. The chromogenic substrate can be oxidized by the remaining hydrogen peroxide. This produces a red product with a maximum absorption wavelength of 520 nm, which can be used to calculate the catalytic activity. POD activity was measured at 650 nm using the substrate (3,5,3',5'-tetramethylbenzidine, TMB) in an HAc-NaAc buffer solution containing H2O2. L-Arg can generate NO under the action of H2O2. The NO release from RVG-M@HMPB / L-Arg-NBP nanoparticles (200 μL) was plotted as a function of reaction time in the presence and absence of H2O2 (10 mM). The NO content generated during the reaction was detected using a NO content detection kit (Griess method) (Beyotime, Shanghai).

[0064] The results are as follows Figure 13 As shown, by Figure 13 It can be seen that, Figure 13(A) shows the SOD catalytic activity of RVG-M@HMPB / L-Arg-NBP at different concentrations in Example 4; Figure 13 A. The effect of RVG-M@HMPB / L-Arg-NBP on ·O2 was evaluated using a superoxide dismutase (SOD) activity assay kit. - Its clearing ability. (By) Figure 13 (A) With the increase of RVG-M@HMPB / L-Arg-NBP concentration, its SOD-like activity was significantly enhanced, showing an effect on ·O2. - The inhibition rate of the generated product was significantly improved. Figure 13 (B) shows the CAT catalytic activity at different concentrations of RVG-M@HMPB / L-Arg-NBP in Example 4; Figure 13 (B) It can be seen that RVG-M@HMPB / L-Arg-NBP consumes H2O2 significantly in a concentration-dependent manner, confirming that it has good CAT-like activity. Figure 13 (C) shows the POD catalytic activity of RVG-M@HMPB / L-Arg-NBP at different concentrations in Example 4; Figure 13 (C) shows that as the concentration of RVG-M@HMPB / L-Arg-NBP increases, the absorbance of the TMB oxidation product gradually increases, indicating that it has significant POD-like activity. Figure 13 (D) represents the NO generation at different times under different concentrations of RVG-M@HMPB / L-Arg-NBP in Example 4; Figure 13 The ability of RVG-M@HMPB / L-Arg-NBP to release NO was evaluated using the Griess reagent method. Figure 13 (D) It can be seen that the NO release level gradually increases over time, indicating that RVG-M@HMPB / L-Arg-NBP can efficiently release NO through H2O2 activation, further highlighting its application potential in the treatment of ischemic stroke.

[0065] Example 5 Based on the RVG-M@HMPB / L-Arg-NBP prepared in Example 1, its cytotoxicity was detected by a cytotoxicity assay.

[0066] Specifically, the CCK8 assay was used to detect the cytotoxicity of RVG-M@HMPB / L-Arg-NBP against PC12 and bEnd.3 cells. Both cell types were seeded into 96-well plates at 5000 cells per well and cultured in 200 μL of medium. Then, RVG-M@HMPB / L-Arg-NBP (0, 10, 20, 50, 100, 200 μg / mL) was added. After 24 h of incubation, CCK8 was added, and incubation was continued for another 2 h. Absorbance was measured at 450 nm, and cell viability was calculated.

[0067] The results are as follows Figure 14 As shown, when the concentration of RVG-M@HMPB / L-Arg-NBP is ≤200μg / mL, the survival rate of bEnd.3 cells and PC12 cells is maintained above 90%, indicating that the material has no obvious cytotoxicity and good biocompatibility within this concentration range.

[0068] Example 6 Based on RVG-M@HMPB / L-Arg-NBP prepared in Example 1, its immune evasion ability was determined by macrophage uptake assay after Cy5.5 fluorescent labeling.

[0069] Specifically, the immune evasion ability of RVG-M@HMPB / L-Arg-NBP was determined using a macrophage uptake assay. Log-grown RAW264.7 cells (1×10⁻⁶) were used. 4 (Number of cells) were seeded into 12-well plates and cultured for approximately 2 hours. Floating cells were washed away with PBS, and the adherent macrophages were cultured overnight. After treating the cells with cell culture medium containing LPS (1 μg / mL) for 12 hours, 200 μL of cell culture medium containing Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, or RVG-M@HMPB / Cy5.5 was added, and the cells were cultured for 4 hours. Cell uptake was then observed using a fluorescence microscope.

[0070] The results are as follows Figure 15 As shown, compared with the free Cy5.5 and HMPB / Cy5.5 groups, the accumulation of RVG-M@HMPB / L-Arg-NBP encapsulated in erythrocyte membranes in macrophages was significantly reduced, indicating that it has an effective immune escape capability.

[0071] Example 7 Based on RVG-M@HMPB / L-Arg-NBP prepared in Example 1, its in vitro blood-brain barrier penetration ability was determined by the Transwell method after fluorescent labeling.

[0072] Specifically, the in vitro blood-brain barrier penetration ability of RVG-M@HMPB / L-Arg-NBP was determined using the Transwell assay. Simply put, bEnd.3 cells were loaded at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded per well into a 24-well trans-well upper chamber with a pore size of 1 μm and cultured for 1 week. The culture medium was then changed every two days. After treating the cells with LPS (1 μg / mL) for 12 hours, 200 μL of Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, or RVG-M@HMPB / Cy5.5 was added to the upper chamber, transferred to the lower chamber containing PC12, and incubated for another 4 hours. Fluorescence imaging of the PC12 cells cultured in the lower chamber was then performed.

[0073] The results are as follows Figure 16 As shown, the free Cy5.5 group, HMPB / Cy5.5 group, and M@HMPB / Cy5.5 group exhibited only weak fluorescence signals, while the fluorescence intensity of RVG29-modified nanoparticles (RVG-M@HMPB / L-Arg-NBP) in the inferior vena cava was significantly enhanced, increasing by 10.8 times compared to the free Cy5.5 group (P<0.001). This result confirms that modification with RVG29 peptide can significantly enhance the ability of nanoparticles to penetrate the BBB, thereby improving the targeted delivery efficiency of drugs in the central nervous system. In vivo fluorescence imaging analysis showed that the RVG29-modified nanoparticles (RVG-M@HMPB / L-Arg-NBP) exhibited a significant advantage in brain-targeted delivery compared to the free Cy5.5, HMPB / Cy5.5, and M@HMPB / Cy5.5 groups (P<0.0001).

[0074] Example 8 Based on RVG-M@HMPB / L-Arg-NBP prepared in Example 1, its in vivo targeting ability was studied after fluorescent labeling.

[0075] Specifically, a mouse model of MCAO was first established. One hour after perfusion, mice were intravenously injected via the tail vein with free Cy5.5 and Cy5.5-labeled HMPB, HMPB / L-Arg-NBP, M@HMPB / L-Arg-NBP, and RVG-M@HMPB / L-Arg-NBP. At 4 and 24 hours, mice were imaged using an in vivo imaging system (IVIS) to assess targeting and biodistribution. Quantitative analysis was performed using LivingImage software.

[0076] The results are as follows Figure 17 As shown, Figure 17 (A) Fluorescence imaging of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in MCAO mice in Example 8; Figure 17 (C) shows the corresponding fluorescence quantification of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in MCAO mice as shown in Example 8; Figure 17 As shown in (A) and (C), the fluorescence signal in the brain of mice in the RVG-M@HMPB / L-Arg-NBP group continued to increase over time and maintained a high intensity even after 24 hours. Figure 17 (B) Fluorescence imaging of major ex vivo organs 24 hours after administration of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in Example 8; Figure 17 (D) The fluorescence quantification of the heart, liver, spleen, lungs and kidneys after 24 hours of administration of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5 and RVG-M@HMPB / Cy5.5 in Example 8; Figure 17 (E) Quantitative fluorescence imaging of the brain 24 hours after administration of Free Cy5.5, HMPB / Cy5.5, M@HMPB / Cy5.5, and RVG-M@HMPB / Cy5.5 in Example 8, corresponding to the corresponding fluorescence quantification (n=3). Figure 17 As shown in (B), (D), and (E), fluorescence analysis of ex vivo tissues revealed that the distribution of free Cy5.5, HMPB@Cy5.5, and M@HMPB@Cy5.5 in the liver was stronger than that of the RVG-M@HMPB@Cy5.5 group. Furthermore, the brain fluorescence of the RVG-M@HMPB@Cy5.5 group was higher than that of the free Cy5.5, HMPB@Cy5.5, and M@HMPB@Cy5.5 groups. This long-lasting retention effect can be attributed to a dual modification strategy: active targeting mediated by the erythrocyte membrane and the RVG29 peptide.

[0077] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A brain-targeted delivery system, characterized in that, include: The mesoporous Prussian blue nanozyme serves as a drug carrier, and the red blood cell membrane is encapsulated on the surface of the drug carrier; the red blood cell membrane is modified with rabies virus glycoprotein polypeptide.

2. The brain-targeting delivery system according to claim 1, characterized in that, The mesoporous Prussian blue nanozyme has a hollow mesoporous structure and a specific surface area of ​​130-140 m². 2 / g, with an average pore size of 18-22nm.

3. The brain-targeting delivery system according to claim 1, characterized in that, The preparation method of the mesoporous Prussian blue nanozyme includes the following steps: a1: Dissolve K3[Fe(CN)6] and PVP in hydrochloric acid solution and stir until the solution turns yellow and transparent; a2: The solution obtained in step a1 is heated in a water bath, centrifuged, washed, and the precipitate is dried to obtain Prussian blue nanozyme; a3: Mix Prussian blue nanozyme and PVP in hydrochloric acid solution, stir, and heat at 120-140℃ for 3-5 hours to obtain mesoporous Prussian blue nanozyme.

4. The brain-targeting delivery system according to claim 3, characterized in that, The concentration of hydrochloric acid in step a1 is 0.005-0.015M; the concentration of hydrochloric acid in step a3 is 0.8-1.2M.

5. The use of the brain-targeted delivery system according to any one of claims 1-4 in the delivery of drugs for ischemic stroke.

6. A medicament based on the brain-targeting delivery system according to any one of claims 1-4, characterized in that, include: The drug carrier consists of a mesoporous Prussian blue nanozyme, an active drug loaded on the drug carrier, and a red blood cell membrane encapsulated on the surface of the drug carrier; the red blood cell membrane is modified with rabies virus glycoprotein polypeptide, and the active substance includes at least one of L-arginine and butylphthalide.

7. The drug according to claim 6, characterized in that, The active substance is composed of L-arginine and butylphthalide.

8. The drug according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. A method for preparing the drug according to claim 6 or 7, characterized in that, Includes the following steps: S1: Co-loaded drug: L-arginine and butylphthalide were dissolved and added dropwise to the mesoporous Prussian blue nanozyme dispersion. After stirring and reacting, the mixture was centrifuged and freeze-dried to obtain mesoporous Prussian blue nanozyme / L-arginine-butylphthalide. S2: Red blood cell membrane encapsulation: Red blood cell membranes are extracted, mixed with mesoporous Prussian blue nanozyme / L-arginine-butylphthalide, and then extruded by ultrasonication. S3: Rabies virus glycoprotein peptide modification: DSPE-PEG2000-rabies virus glycoprotein peptide was added to the product obtained in step S3 and incubated, then purified by dialysis to obtain the drug.

10. The use of a brain-targeted delivery system according to any one of claims 1-4 and / or the medicament according to claims 6-8 in the preparation of a medicament for the prevention or treatment of ischemic stroke.

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