Anti-inflammatory nano drug delivery system containing guanidine structure and preparation method and application thereof

Abstract

The present invention belongs to the technical field of biomedical materials, and specifically relates to an anti-inflammatory nano drug delivery system containing a guanidine structure, a preparation method and an application thereof. The anti-inflammatory nano drug delivery system is based on COF DT B@COF is obtained by loading drugs and wrapping them in red blood cell membranes. DT ‑R, where COF DT It is prepared by high-temperature microwave reaction in a system of mesitylene / 1,4-dioxane / acetic acid using DG and TP as raw materials. The anti-inflammatory nano drug delivery system of the present invention uses covalent organic framework materials as a carrier platform to integrate Bindali into COF DT It protects neurons in the cavity by inhibiting neuroinflammation and neuronal pyroptosis after subarachnoid hemorrhage.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to an anti-inflammatory nano drug delivery system containing a guanidine structure, and a preparation method and application thereof. Background Art

[0002] The main factors for poor prognosis in patients with subarachnoid hemorrhage (SAH) include increased local intracranial pressure leading to cerebral ischemia, cerebral edema, blood-brain barrier disruption, oxidative stress, neuroinflammation, etc., all of which can lead to varying degrees of neuronal damage. Surgical treatment of ruptured arteries, neurocritical care management, and prevention of complications are conventional treatment strategies for SAH patients. However, clinically, these measures are only symptomatic, and there is a lack of effective drugs to intervene in SAH. Many drugs have failed to improve the prognosis of SAH patients in clinical trials due to their short half-life, poor biodistribution specificity, and inability to cross the blood-brain barrier (BBB). Therefore, new therapeutic drugs are urgently needed in the clinic to improve the prognosis of SAH patients.

[0003] Bindarit can exert its anti-inflammatory effect by inhibiting the synthesis of CC motif ligand 2 (CCL2). In addition, Bindarit also has neuroprotective effects. However, the limitations of Bindarit, such as its short half-life, lack of specificity in biodistribution, and difficulty in penetrating the blood-brain barrier, limit the therapeutic effect of Bindarit on SAH.

[0004] Covalent organic framework (COF) is a complex functional system composed of lightweight organic molecular units connected by covalent bonds. It has a highly ordered porous structure, flexible synthesis methods and molecular design, which enables multifunctional integration, delayed and controlled drug release, improved drug solubility, improved bioavailability and reduced drug side effects, which endows COF with important potential application value in drug delivery. In recent years, nanoplatforms based on engineered biomimetic membranes have provided innovative methods for targeted drug delivery, and their excellent biocompatibility and safety have been recognized by many studies. Metformin exhibits anti-inflammatory and antioxidant potential due to its special guanidine structure. The intracranial neuroinflammatory environment after SAH has the pathological characteristics of low pH and excessive reactive oxygen species (ROS) accumulation. Therefore, it is necessary to develop a COF nanosystem that can accumulate in low pH areas and improve the prognosis of SAH. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide an anti-inflammatory nano-drug delivery system containing a guanidine structure, and a preparation method and application thereof. The anti-inflammatory nano-drug delivery system is a covalent organic framework material carrier platform, which can accumulate in SAH injury areas with low pH values, and inhibit post-SAH neuroinflammation and neuronal pyroptosis to protect neuronal cells, improve neuroinflammation and function, thereby significantly improving the therapeutic effect.

[0006] The first object of the present invention is to provide an anti-inflammatory nano drug delivery system containing a guanidine structure, wherein the anti-inflammatory nano drug delivery system is based on COF DT B@COF is obtained by using the drug as a carrier and wrapping the red blood cell membrane on the surface. DT -R; the COF DT It is prepared by high-temperature microwave reaction in a system of mesitylene / 1,4-dioxane / acetic acid using DG (1,3-diaminoguanidine hydrochloride) and TP (1,3,5-triformylphloroglucinol) as raw materials.

[0007] Furthermore, the COF DT It is a covalent organic framework material carrier containing guanidine groups, which has a structure shown in formula (I):

[0008] .

[0009] Furthermore, the drug is Bindarit (B). Specifically, the drug has the following structure:

[0010] .

[0011] The second object of the present invention is to provide a method for preparing an anti-inflammatory nano drug delivery system containing a guanidine structure, which specifically comprises the following steps:

[0012] (1) Preparation of COF DT First, DG and TP were dissolved in a mixed solution of mesitylene / 1,4-dioxane, and a catalyst was added to obtain a reaction solution. The reaction solution was then subjected to three freeze-pump-thaw cycles after ultrasonic treatment for 5 min to 15 min, and then subjected to microwave radiation heating for 20 min to 40 min. Finally, the product was centrifuged, washed, and vacuum dried to obtain COF. DT ;

[0013] (2) Preparation of B@COF DT : First add the drug-containing solution to the COF DT The mixed solution was then reacted on a magnetic stirrer at room temperature for 6 h to 12 h, the product was collected by centrifugation, and dialyzed in PBS for 12 h to 24 h using a 3500 Da dialysis bag, the dialyzate was replaced every 6 h, the dialyzed product was collected by centrifugation, and freeze-dried to obtain the product B@COF DT ;

[0014] (3) Preparation of B@COF DT -R: The red blood cell membrane was first resuspended in deionized water and sonicated for 5 min to 10 min, and large-sized vesicles were obtained using an Avanti micro-extruder. The vesicles were then mixed with B@COF DTAfter mixing and sequentially squeezing back and forth 11 to 21 times using polycarbonate membranes of different small sizes, B@COF with uniform size was obtained. DT -R.

[0015] Specifically, the preparation method of the red blood cell membrane is as follows: whole blood is collected from the eyes of C57BL / 6J mice and stored in an anticoagulant tube; the whole blood is centrifuged at 800g~1000g for 5min~10min at 4°C, the supernatant and attached white blood cells are removed, and then the precipitate is washed with 5 times the volume of 1×PBS until the supernatant becomes colorless, and the deposited red blood cells obtained by hemolysis are resuspended in 10 times the volume of 0.25×PBS solution, and incubated at 4°C for 1h~2h until the solution is well permeabilized and presents a dark red color, and then the red blood cell membrane is centrifuged at 10000g~12000g for 10min~15min at 4°C, and washed with 0.25×PBS solution until the cell membrane is white or light pink, and collected.

[0016] Furthermore, in step (1) of the above technical solution, the molar ratio of DG to TP is 3-2:2-1; the concentration of DG in the reaction solution is 24.6 mg / mL-37 mg / mL; the concentration of TP is 21 mg / mL-42 mg / mL; the catalyst is 6M acetic acid; and the volume ratio of mesitylene, 1,4-dioxane and acetic acid is 2-3:2-3:1.

[0017] Furthermore, in step (1) of the above technical solution, the temperature of the microwave radiation heating treatment is 100°C~120°C; the method for washing the product is: first centrifugally washing the product with tetrahydrofuran (THF), then centrifugally washing the product with acetone, repeating 4 to 6 times, each washing consuming 20mL~25mL of tetrahydrofuran or acetone, and then washing the product with tetrahydrofuran by Soxhlet extraction, consuming 150mL~250mL of tetrahydrofuran.

[0018] Furthermore, in step (2) of the above technical solution, the drug-containing solution is a solution of Bendali dissolved in DMSO (dimethyl sulfoxide); the concentration of Bendali in the mixed solution is 0.6 mg / mL to 1.8 mg / mL; the COF in the mixed solution is 0.6 mg / mL to 1.8 mg / mL; DT The concentration is 1.0mg / mL~3.0mg / mL.

[0019] Furthermore, in step (3) of the above technical solution, the red blood cell membrane and B@COF DTThe volume ratio is 8:1~6:1; the polycarbonate membrane used in the Avanti micro extruder has a large size of 400nm, and the small sizes are 200nm and 100nm respectively. When extruding back and forth, after the Avanti micro extruder is installed and fixed with a polycarbonate membrane of corresponding size (first 200nm and then 100nm), hold one side of the extruder with each hand, extrude back and forth from left to right and then from right to left, and finally collect from the opposite side. The B@COF obtained by this method DT -R is uniform in size and free of contamination.

[0020] Furthermore, in the above technical solution, in each step, the centrifugal speed is 5000rpm~6000rpm, and the time is 5min~10min.

[0021] The third object of the present invention is to provide an application of the above-mentioned guanidine-containing nano drug delivery system in the preparation of drugs for improving neuroinflammation and improving neurological function.

[0022] Advantages compared to the prior art:

[0023] 1. The present invention designs a novel pH-responsive erythrocyte membrane biomimetic guanidine-based covalent organic framework nanocarrier and encapsulates Bindali, aiming to enhance drug delivery across the blood-brain barrier and target the NLRP3 / Caspase-1 / GSDMD axis, thereby inhibiting neuronal pyroptosis. The obtained B@COF DT -R can accumulate in the SAH injury area with low pH value and inhibit neuroinflammation and neuronal pyroptosis after SAH to protect neuronal cells.

[0024] 2. Accepting the B@COF prepared by the present invention DT -R-treated SAH mice showed a significant therapeutic effect with reversed neurological deficits, improved spatial memory and learning abilities, and overall enhanced neurological function scores. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 B@COF of the present invention DT -R preparation flow chart;

[0026] Figure 2 B@COF in Example 1 of the present invention DT - Transmission electron microscopy image of R during self-assembly;

[0027] Figure 3 B@COF in Example 1 of the present invention DT -R particle size distribution during self-assembly;

[0028] Figure 4 B@COF in Example 1 of the present invention DT-R potential diagram during self-assembly;

[0029] Figure 5 B@COF in Example 1 of the present invention DT -Powder X-ray diffraction pattern of R during self-assembly;

[0030] Figure 6 B@COF in Example 1 of the present invention DT - Fourier transform infrared spectra during R self-assembly;

[0031] Figure 7 B@COF in Example 1 of the present invention DT -R Specific surface area test diagram during self-assembly process;

[0032] Figure 8 B@COF in Example 1 of the present invention DT -R drug loading performance evaluation and analysis diagram;

[0033] Fig. 9 B@COF in Example 2 of the present invention DT(Cy5.5) and B@COF DT(Cy5.5) - Fourier transform infrared spectrum of R;

[0034] Fig.10 The in vitro experiment in Example 2 of the present invention verifies B@COF DT -R is enriched in HT22 cell immunofluorescence images;

[0035] Fig.11 This is the in vivo experimental verification of B@COF in Example 2 of the present invention. DT -R can better cross the blood-brain barrier and target SAH brain tissue immunofluorescence images;

[0036] Fig.12 The in vitro experiment in Example 3 of the present invention verifies B@COF DT -R can effectively improve the pyroptosis of HT22 cells induced by heme;

[0037] Fig.13 B@COF in Example 3 of the present invention DT -R treatment and Western Blot analysis of the changes in the expression levels of NLRP3, GSDMD, GSDMD-N, Caspase-1, IL-1β, IL-18 and CCL2 in HT22 cells;

[0038] Fig.14 B@COF in Example 4 of the present invention DT -R improves the brain perfusion of mice after SAH;

[0039] Fig.15B@COF in Example 4 of the present invention DT -R treatment of SAH mice, SAH score, modified Garcia score and brain water content of mice;

[0040] Fig.16 B@COF in Example 4 of the present invention DT -R treatment of SAH mice, distribution of Nissl bodies in the SAH injury site of mouse brain tissue;

[0041] Fig.17 B@COF in Example 4 of the present invention DT -R treatment, Western Blot detection of changes in the expression levels of NLRP3, GSDMD, GSDMD-N, Caspase-1, IL-1β, IL-18 and CCL2 in the brain tissue of SAH model mice, and ELISA detection of IL-1β and IL-18 content in the brain tissue of mice;

[0042] Fig.18 COF in Example 5 of the present invention DT and B@COF DT Biosafety HE staining of the main organs of mice, heart, liver, spleen, lung and kidney;

[0043] Fig.19 COF in Example 5 of the present invention DT and B@COF DT -R hemolysis in vitro experiment. DETAILED DESCRIPTION

[0044] The above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0046] The carrier COF of the present invention DT DG and TP were used as raw materials in a system of mesitylene / 1,4-dioxane / acetic acid by high-temperature microwave reaction; then Binderyne (B) was integrated into COF under stirring at room temperature. DT The surface of the nanostructured carbonyl ion was modified biomimetically with red blood cell membrane to obtain the guanidine-containing nano-drug delivery system B@COF. DT -R. The B@COF DT-R can inhibit neuronal pyroptosis by targeting the NLRP3 / Caspase-1 / GSDMD axis, thus improving neuroinflammation and neurological function. DT The preparation flow chart of -R is as follows Figure 1 shown.

[0047] The present invention is further described in detail below in conjunction with specific embodiments:

[0048] Example 1: B@COF DT -R Preparation Method

[0049] B@COF DT -R preparation method, comprising the following specific steps:

[0050] (1) Preparation of COF DT COF was synthesized by adding DG (37 mg, 0.3 mmol) and TP (42 mg, 0.2 mmol) in a molar ratio of 3:2 to a mixed solution of mesitylene / 1,4-dioxane / 6 M acetic acid (8.4 mL, volume ratio 3:3:1) under microwave heating. DT ; After ultrasonic treatment for 15 minutes, the reaction solution was subjected to three freeze-pump-thaw cycles, the thick-walled pressure bottle was closed, and the product was stirred at 120°C for 20 minutes using a microwave synthesizer and heated by microwave radiation; the product was first centrifuged and washed with THF, and then with acetone, and the washing was repeated three times, the powder was collected, and then washed using THF as a solvent using a standard Soxhlet method to remove any impurities adsorbed in the porous structure; finally, after vacuum drying at 60°C for 24 hours, a brick-red powder of COF was obtained. DT , with a yield of 77.6%.

[0051] (2) Preparation of B@COF DT : 1 mL of DMSO-prepared Bindali (3 mg, 9.25 mM) solution was added to 1 mL of COF DT (5 mg / mL) phosphate buffer solution (pH = 7.4), and reacted on a magnetic stirrer at room temperature for 12 h; the product was collected by centrifugation (5000 rpm, 5 min), and dialyzed in PBS (phosphate buffer) using a 3500 Da dialysis bag for 24 h, with the dialysate replaced every 6 h. The dialyzed product was collected by centrifugation and freeze-dried for 6 h to obtain the product B@COF DT , which is a brick red powder with a yield of 84.1%.

[0052] (3) Preparation of red blood cell membranes: Collect whole blood from the eyes of C57BL / 6J mice and store in anticoagulant tubes; centrifuge the whole blood at 1000 g for 10 min at 4°C, remove the supernatant and attached white blood cells, and then wash the precipitate with 5 volumes of 1× PBS until the supernatant becomes colorless. Resuspend the sedimented red blood cells obtained by hemolysis in 10 volumes of 0.25× PBS solution and incubate at 4°C for 1 h to 2 h until the solution is well permeabilized and appears dark red. Centrifuge the red blood cell membranes at 10,000 g for 15 min at 4°C and wash with 0.25× PBS solution until the cell membrane appears white or light pink. Collect and set aside.

[0053] (4) Preparation of B@COF DT -R: The red blood cell membranes were resuspended in deionized water and sonicated for 5 min (42 kHz, 100 W), and large-sized vesicles (400 nm) were obtained using an Avanti microextruder, which were then mixed with B@COF DT (1 mg / mL) were mixed in a volume ratio of 8:1 and squeezed back and forth 11 times using small-sized polycarbonate membranes (first 200 nm and then 100 nm) to produce B@COF with uniform size. DT -R.

[0054] The B@COF synthesized in the embodiment of the present invention DT -R and its process products were characterized, and the characterization results were as follows Figures 2 to 8 shown.

[0055] Preparation of PBS-dispersed B@COF DT -R dispersion (0.1 mg / mL) was then dropped onto a copper grid to obtain a transmission electron microscopy (TEM) sample. The prepared B@COF DT -R and its process products transmission electron microscope (TEM) Figure 2 shown.

[0056] Among them, the COF prepared in step (1) DT The TEM image shows an ellipsoidal shape (such as Figure 2 As shown in a in FIG), after the drug co-loading process in step (2), a granular texture appears inside the ellipsoid (as shown in FIG. Figure 2 As shown in b in FIG), after the surface modification in step (4), the surface of the nanoparticles becomes rougher and a bubble-like shell appears, and the boundaries become more blurred (as shown in FIG. Figure 2 (as shown in c in the figure).

[0057] Figure 3 and Figure 4 The B@COF prepared in the embodiments of the present invention are DT-R and its process products. Dynamic light scattering measurements show that after steps (2) and (4), B@COF DT -R's hydrodynamic diameter increases significantly ( Figure 3 ); B@COF DT The zeta potential of the cell membrane turned from positive to negative, confirming the successful encapsulation of the cell membrane ( Figure 4 ).

[0058] Figure 5 B@COF prepared in the embodiment of the present invention DT Powder X-ray diffraction (PXRD) patterns of -R and its process products. Figure 5 Shows COF DT Peaks appear at 6.75°, 11.70°, and 13.53°, corresponding to the (100), (110), and (200) planes, respectively, indicating that the COF is reduced by drug loading and surface film modification. DT of crystallinity.

[0059] Figure 6 B@COF prepared in the embodiment of the present invention DT Fourier transform infrared spectroscopy (FT-IR) of -R and its process products. COF was observed in the figure. DT 、B@COF DT and B@COF DT -R FT-IR, it was found that the characteristic C=C and CN stretching vibration peaks appeared at 1593cm -1 and 1347cm -1 .

[0060] Figure 7 B@COF prepared in the embodiment of the present invention DT -R and its process product specific surface area test. The nitrogen adsorption-desorption isotherm data in the figure show COF DT 、B@COF DT and B@COF DT -R's specific surface areas decrease in sequence, which are 841.12m 2 / g, 168.73m 2 / g and 57.05m 2 / g.

[0061] Figure 8 B@COF prepared in the embodiment of the present invention DT -R. Using PBS with different pH levels to simulate different physiological environments, B@COF DT-R was dissolved or dispersed in these pH-adjusted buffer solutions at predetermined concentrations to ensure consistent sample concentrations at each pH condition. At predetermined time points (0, 2, 4, 6, 12, 24, 48 and 96 h), samples were collected and the concentration of Bindali in the samples was determined. Using high performance liquid chromatography (HPLC), the concentration of Bindali at each time point was recorded in order to calculate the release rate and the cumulative release of Bindali. Statistical methods were used to analyze the data and compare the drug release behavior under different pH conditions. From Figure 8 It can be seen that at pH 6.0, B@COF DT -R has a drug release efficiency of up to 66.13%, while in the pH range of 7.0 to 7.4 in normal cells and tissues, the drug release efficiency does not exceed 25.69%, indicating that the nano-drug delivery system has a higher drug release efficiency at low pH.

[0062] Example 2: B@COF DT -R targeted experiments

[0063] The efficacy of SAH treatment drugs depends on their ability to penetrate the blood-brain barrier and accumulate at the site of injury. The coated red blood cell membrane is conducive to the drug penetrating the cell membrane and entering the cell to achieve therapeutic effects. 2-[5-[3-(5-carboxypentyl)-1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benzo[E]indol-2-ylidene]-1,3-pentadienyl]-3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[E]indolium salt (Cyanine5.5, referred to as Cy5.5) is a fluorescent dye used for in vivo imaging. In order to make B@COF DT -R targeting ability and delivery effect visualization, this example prepared B@COF DT(Cy5.5) -R.

[0064] B@COF DT(Cy5.5) -R preparation method is:

[0065] (1) COF was synthesized by adding DG (37 mg, 0.3 mmol) and TP (42 mg, 0.2 mmol) in a molar ratio of 3:2 to a mixed solution of mesitylene / 1,4-dioxane / 6 M acetic acid (8.4 mL, volume ratio 3:3:1) under microwave heating conditions. DT; After ultrasonic treatment for 15 minutes, the reaction solution was subjected to three freeze-pump-thaw cycles, the thick-walled pressure bottle was closed, and the product was stirred at 120°C for 20 minutes using a microwave synthesizer and heated by microwave radiation; the product was first centrifuged and washed with THF, and then with acetone, and the washing was repeated three times, the powder was collected, and then washed using THF as a solvent using a standard Soxhlet method to remove any impurities adsorbed in the porous structure; finally, after vacuum drying at 60°C for 24 hours, a brick-red powder of COF was obtained. DT .

[0066] (2) Add 1 mL of DMSO-prepared solutions of Bindali (3 mg, 9.25 mM) and Cy5.5 (0.05 mg, 0.055 mM) to 1 mL of COF DT The product was collected by centrifugation (5000 rpm, 5 min) in PBS and dialyzed in a 3500 Da dialysis bag for 24 h. The dialyzate was replaced every 6 h. The dialyzed product was collected by centrifugation and freeze-dried for 6 h to obtain the product B@COF. DT(Cy5.5) , which is a black-red powder.

[0067] (3) The erythrocyte membrane (prepared as in Example 1) was resuspended in deionized water and sonicated for 5 min (42 kHz, 100 W). Large-sized vesicles (400 nm) were obtained using an Avanti microextruder and then mixed with B@COF DT(Cy5.5） (1 mg / mL) were mixed in a volume ratio of 8:1 and squeezed back and forth 12 times using small-sized polycarbonate membranes (first 200 nm and then 100 nm) to produce B@COF with uniform size. DT(Cy5.5) -R.

[0068] The B@COF synthesized in the embodiment of the present invention DT(Cy5.5) and B@COF DT(Cy5.5) -R was used for characterization, and the characterization results were as follows Fig. 9 shown.

[0069] Fig. 9 B@COF prepared in the embodiment of the present invention DT(Cy5.5) and B@COF DT(Cy5.5) -R FT-IR characterization data. It can be seen that B@COF DT(Cy5.5) and B@COF DT(Cy5.5) -R all show infrared absorption peaks originating from the sulfonic acid group in Cy5.5. -1 ~1300cm -1 The main characteristic peak in the wave number range is 1060cm -1 and 1180cm-1 , corresponding to the SO and S=O stretching vibrations in the sulfonic acid group. -1 ~1400cm -1 In the wave number range, the main characteristic peak appears at 1250cm -1 and 1350cm -1 , corresponding to the CS and CO stretching vibrations of the sulfonic acid group, respectively. After wrapping the red blood cell membrane, the infrared characteristic peak signal weakened, but there was no obvious shift or disappearance.

[0070] After the cell slides were placed in a six-well plate, 300,000 HT22 cells were added to each well, and 10 μg / mL of Cy5.5 and B@COF DT(Cy5.5) and B@COF DT(Cy5.5) -R co-incubated for 4 hours, first fixed with 4% paraformaldehyde for 30 minutes, rinsed with PBS three times, and then permeabilized with 0.3% Triton X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 30 minutes. After rinsing with PBS, Dapi (4',6-diamidino-2-phenylindole) was used to stain the nucleus, and the slides were sealed and observed under a confocal microscope ( Fig.10 ). The results showed that HT22 cell internalization was significantly enhanced, especially when coated with erythrocyte membrane.

[0071] To further prove the above conclusion, a mouse SAH model was constructed. After treatment with nanomaterials, immunofluorescence analysis of tissue sections in the SAH injury area of ​​the mice also supported this observation, revealing that the B@COF of the present invention was effective in treating SAH. DT(Cy5.5) -R-treated mice showed a significant increase in red fluorescence within the SAH injury area (Fig. Fig.11 (as shown in b in the figure).

[0072] Laser scanning confocal microscopy (CLSM) was used to further analyze the cellular uptake ability of the embodiments of the present invention. The results showed that compared with the unmodified B@COF DT(Cy5.5) In comparison, B@COF DT(Cy5.5 )-R internalization was significantly enhanced, which may be due to the DT(Cy5.5) -R increased bioaffinity. Neuronal cells in vivo and in vitro successfully internalized B@COF DT(Cy5.5) -R is crucial to its therapeutic effect.

[0073] After the SAH model was successfully established in mice, equal amounts of Cy5.5 and B@COF were injected into the peritoneal cavity of SAH mice. DT(Cy5.5) and B@COF DT(Cy5.5)-R, and the mice were killed 3h and 24h after injection, and the brain and major organs such as heart, liver, spleen, lung and kidney were separated and imaged using the IVISLumina XRMS Series III imaging system. The results showed that free Cy5.5 was mainly absorbed by the liver and entered the brain tissue through the blood-brain barrier to a very limited extent; while B@COF DT(Cy5.5) B@COF showed significant enrichment at the SAH injury site. DT(Cy5.5) -R further enhanced their accumulation in SAH brain tissue. And both B@COFDT(Cy5.5) and B@COFDT(Cy5.5)-R were basically metabolized and excreted within 24 hours (e.g. Fig.11 This result shows that B@COF DT(Cy5.5) -R is more conducive to accumulation at the SAH injury site, and B@COF DT(Cy5.5) -R will not remain in major organs for too long, ensuring the necessary biosafety of the drug.

[0074] The above experimental results show that the red blood cell membrane enhances the B@COF DT Cellular internalization enhances the drug's targeting ability and delivery efficiency.

[0075] Example 3: B@COF DT -R in vitro experiments

[0076] 1. In vitro experimental verification of B@COF DT -R inhibits hemin-induced pyroptosis

[0077] In order to evaluate the B@COF prepared in Example 1 DT -R inhibits cell pyroptosis and the release of Bindali molecules in vitro, with the corresponding B, COF DT and B@COF DT Make a comparison.

[0078] After the cell slides were placed in a six-well plate, 300,000 HT22 cells were added to each well. After hemoglobin-induced pyroptosis, 10 μg / mL B and COF were added. DT and B@COF DT -R nanomaterials were co-incubated, and the number and ratio of live and dead cells in each group of cells were determined using a live-dead cell staining (PI) kit; and the amount of LDH released in the cell culture medium of each group was determined using a lactate dehydrogenase (LDH) assay kit. The results showed that B@COF DT -R nanomedicine has better anti-pyroptosis ability in vitro (e.g. Fig.12 a and b in the figure).

[0079] 2. In vitro experimental verification of B@COF DT -R inhibits NLRP3 / GSDMD pyroptosis pathway

[0080] Neuronal pyroptosis is a form of programmed cell death that depends on caspase-1 (cysteine-containing aspartate proteolytic enzyme-1) and is one of the main mechanisms leading to poor prognosis in SAH. The main mechanism of pyroptosis is the assembly of inflammasomes, which are composed of sensors, adapters, and effectors. After SAH occurs, the sensor NLRP3 detects external danger signals within the inflammasome, triggering the activation of caspase-1 by ASC, resulting in the cleavage of GSDMD into its N-terminal fragment GSDMD-N. Subsequently, GSDMD-N binds to the plasma membrane to form a pore that facilitates the passage of ions and molecules. At the same time, active caspase-1 can cleave pro-IL-18 and pro-IL-1β into IL-18 and IL-1β, respectively. IL-18 and IL-1β are released into the extracellular environment through the GSDMD-N pore.

[0081] HT22 cells were treated with 10 μg / mL B and COF DT and B@COF DT -R nanomaterials were co-incubated, 100 μL of RIPA lysis buffer was added to the culture dish, the lysis buffer was collected and the cells were broken using an ultrasonic disruptor, the lysis buffer was centrifuged at 14000 rpm for 15 min in a refrigerated centrifuge to obtain protein, the cell protein was quantified by the BCA method, and 5×SDS buffer was added and boiled to denature the protein. An equal amount of protein was loaded, and the protein was fixed on a PVDF (polyvinylidene fluoride) membrane by electrophoresis and transfer; the antigen epitope was blocked in skim milk, and the corresponding primary antibody dilution was used for antibody incubation, and the corresponding secondary antibody was used for incubation after rinsing, and then developed in ECL luminescent solution (enhanced chemiluminescent solution). When detecting the expression of internal reference and target protein, from Fig.13 It was found that the B@COF of the present invention DT -R can effectively inhibit cell pyroptosis by inhibiting the NLRP3 / GSDMD pyroptosis pathway.

[0082] Example 4: B@COF DT -R in vivo experiments

[0083] 1. In vivo experimental verification of B@COF DT -R therapeutic effect on SAH

[0084] In order to further explore different nanomaterial groups (COF DT 、B@COF DT and B@COF DT -R, prepared by the method of Example 1) for the treatment of SAH, and the LSI system (linear non-shifting system) was used to evaluate the B@COF DTEffects of -R on vascular perfusion of the meningeal system. After the SAH model was successfully established in mice, the mice were immediately treated with 0.05 mg / g of DT 、B@COF DT and B@COF DT The results showed that compared with the sham group, the blood perfusion in the SAH group was reduced on the first day, and B@COF DT -R treatment group showed higher perfusion level than SAH group on day 1, highlighting the B@COF DT -R and COF DT Superior efficacy compared to Fig.14 ). B@COF DT -R treatment results showed that blood flow and lymphatic perfusion increased on the lesion side, contralateral side and sagittal sinus. These findings indicate that B@COF DT -R treatment can inhibit the inflammatory response of neuronal cells and improve the recovery of meningeal fluid perfusion after SAH.

[0085] After the SAH model was successfully established in mice, the dose of 0.05 mg / g (COF DT 、B@COF DT and B@COF DT -R) was administered. After 24 hours, the mice were scored for neurological deficits and then sacrificed, and brain tissues were collected for SAH scoring. SAH score analysis showed no significant differences between the experimental groups (e.g. Fig.15 As shown in a). The modified Garcia score showed that the mice showed obvious neurological dysfunction 24 hours after SAH induction. DT Treatment improved neurological deficits, while B@COF DT and B@COF DT -R group achieved more significant improvement (e.g. Fig.15 b), and B@COF DT -R application can significantly reduce the water content of brain tissue after SAH (such as Fig.15 The Nissl bodies composed of well-developed rough endoplasmic reticulum and free ribosomes reflect neuronal activity. Through Nissl staining, the neurological damage and repair of each group of mice can be better judged. After the SAH model of mice was successfully established, the dose of 0.05 mg / g (COF DT 、B@COF DT and B@COF DT-R) 24 hours after administration, brain tissue was taken out to make frozen sections. After the sections were taken out of the freezing environment, they were placed in a humidified box at room temperature for 3min~5min; 4% paraformaldehyde fully covered the cells, fixed at room temperature for 15min, and washed the cells 3 times with PBS, each time for 5min; Nissl staining solution was used for staining at 56℃ for 10min~15min; 95% alcohol differentiation solution was used for differentiation for a few seconds, and the Nissl bodies were observed under a microscope until they were clear; they were quickly washed with water and allowed to dry slightly; they were dehydrated with anhydrous alcohol for 2min, 2 times, transparentized with xylene for 5min, 2 times, sealed with neutral resin, and then observed under a microscope. Compared with the mice in the SAH group, COF DT The abundance of Nissl bodies was significantly increased ( Fig.16 ). This indicates that COF DT The neuroprotective ability of B@COF DT -R is better than COF DT .

[0086] 2. In vivo experimental verification of B@COF DT -R inhibits pyroptosis of SAH neurons

[0087] Proteins were extracted from the temporal cortex of the area affected by blood stimulation as described above and used for subsequent Western blot analysis. Consistent with the observations at the cellular level, the results showed that COF DT The expression of NLRP3 in brain tissue after SAH was reduced, and the protein levels of caspase-1, GSDMD-N, CCL2, and IL-1β were also reduced, which are genes related to pyroptosis and inflammation (e.g. Fig.17 (a) with COF DT In comparison, B@COF DT and B@COF DT In addition, the cortical tissue was extracted from the ipsilateral hemisphere after brain tissue was taken and homogenized for ELISA experiments. The results showed that IL-1β and IL-18 levels were significantly increased 24 hours after SAH, and B@COF DT -R can significantly and effectively reverse the increase of IL-1β and IL-18 (such as Fig.17 These results show that B@COF DT -R can effectively improve neuronal pyroptosis and inflammation after SAH.

[0088] Example 5: B@COF DT -R biosafety testing

[0089] To further verify the B@COF DT -R biosafety, the mouse SAH model was established with a dose of 0.05 mg / g (COF DTand B@COF DT ) was given for 3 days, and then the mice were killed after being raised for 7 days. The main organs of the mice, heart, liver, spleen, lung and kidney, were removed, fixed with 4% paraformaldehyde and embedded in paraffin. The slices were dewaxed and HE stained. Use hematoxylin staining for 10min~15min; use 0.5%~1.0% hydrochloric acid alcohol for color separation for a few seconds to tens of seconds, and quickly wash with distilled water; blue with light ammonia water, wash with distilled water, and examine with a microscope while differentiating; rinse with running water for 3min to wash away the alkaline water; 1% eosin staining for 5min~10min; quickly wash with distilled water, and dehydrate with alcohol in ascending order, that is, quickly wash with 70%, 80% and 90% alcohol, 95% alcohol for 30s~60s, and examine with a microscope; 100% alcohol for 3min, 2 times; xylene for 5min, 2 times; finally, use neutral gum to seal the slides and observe with a microscope, such as Fig.18 The results showed that the nanoparticles in each group did not cause pathological damage to the main organs. DT -R further weakens the B@COF due to its superior biocompatibility DT pathological toxicity.

[0090] The biosafety of each group of nanomaterials was further verified using a hemolysis experiment. Whole blood was collected from C57BL / 6 mice and centrifuged at 1500g for 3 minutes to separate red blood cells. The red blood cells were then divided into 1.5mL clean EP tubes and treated with different concentrations of nanoparticles, PBS (negative control) and Triton X-100 (positive control). The samples were incubated together in a 37°C constant temperature water bath for 3 hours. Subsequently, the samples were centrifuged at 1500g for 5 minutes, and images were taken. The supernatant in the centrifuge tube was collected, and the absorbance of each group at 570nm was measured using a UV spectrophotometer to further calculate the hemolysis rate. Fig.19 As shown, the results show that COF DT and B@COF DT -R have no obvious hemolytic effect and have high biosafety.

[0091] In summary, the anti-inflammatory nano drug delivery system containing a guanidine structure prepared by the present invention is based on a covalent organic framework material carrier platform containing a guanidine structure. DT The nano drug delivery system has good biosafety and can effectively cross the blood-brain barrier and improve neuroinflammation and neurological function after use.

[0092] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-inflammatory nano drug delivery system containing a guanidine structure, characterized in that: The anti-inflammatory nano drug delivery system is based on COF DT B@COF is obtained by using the drug as a carrier and wrapping the red blood cell membrane on the surface. DT -R; the COF DT It is prepared by high temperature microwave reaction in a system of mesitylene / 1,4-dioxane / acetic acid using DG and TP as raw materials. The COF DT It is a covalent organic framework material carrier containing guanidine groups, which has a structure shown in formula (I): 。 2. The anti-inflammatory nano drug delivery system containing a guanidine structure according to claim 1, characterized in that: The drug is Bindali.

3. The method for preparing an anti-inflammatory nano drug delivery system containing a guanidine structure according to any one of claims 1 to 2, characterized in that: The specific steps include: (1) Preparation of COF DT First, DG and TP were dissolved in a mixed solution of mesitylene / 1,4-dioxane, and a catalyst was added to obtain a reaction solution. The reaction solution was then subjected to three freeze-pump-thaw cycles after ultrasonic treatment for 5 min to 15 min, and then subjected to microwave radiation heating for 20 min to 40 min. Finally, the product was centrifuged, washed, and vacuum dried to obtain COF. DT ; (2) Preparation of B@COF DT : First add the drug-containing solution to the COF DT The mixed solution was then reacted on a magnetic stirrer at room temperature for 6 h to 12 h, the product was collected by centrifugation, and dialyzed in PBS for 12 h to 24 h using a 3500 Da dialysis bag, the dialyzate was replaced every 6 h, the dialyzed product was collected by centrifugation, and freeze-dried to obtain the product B@COF DT ; (3) Preparation of B@COF DT -R: The red blood cell membrane was first resuspended in deionized water and sonicated for 5 min to 10 min, and large-sized vesicles were obtained using an Avanti micro-extruder. The vesicles were then mixed with B@COF DT After mixing and sequentially squeezing back and forth 11 to 21 times using polycarbonate membranes of different small sizes, B@COF with uniform size was obtained. DT -R.

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of DG to TP is 3-2:2-1; the concentration of DG in the reaction solution is 24.6 mg / mL-37 mg / mL; the concentration of TP is 21 mg / mL-42 mg / mL; the catalyst is 6M acetic acid; and the volume ratio of mesitylene, 1,4-dioxane and acetic acid is 2-3:2-3:

1.

5. The preparation method according to claim 3, characterized in that: In step (1), the temperature of the microwave radiation heating treatment is 100°C to 120°C; the method for washing the product is: first centrifugally washing the product with tetrahydrofuran, then centrifugally washing the product with acetone, repeating 4 to 6 times, each washing consumes 20 mL to 25 mL of tetrahydrofuran or acetone, and then using tetrahydrofuran to wash the product by Soxhlet extraction, and the tetrahydrofuran consumed is 150 mL to 250 mL.

6. The preparation method according to claim 3, characterized in that: In step (2), the drug-containing solution is a solution of Bindali dissolved in DMSO; the concentration of Bindali in the mixed solution is 0.6 mg / mL to 1.8 mg / mL; COF DT The concentration is 1.0mg / mL~3.0mg / mL.

7. The preparation method according to claim 3, characterized in that: In step (3), the method for preparing the red blood cell membrane is as follows: whole blood from mouse eyes is collected and stored in an anticoagulant tube, and centrifuged at 800g~1000g for 5min~10min at 4°C, the supernatant and attached white blood cells are removed, and the precipitate is washed with 5 times the volume of 1×PBS until the supernatant becomes colorless, and the deposited red blood cells obtained by hemolysis are resuspended in 10 times the volume of 0.25×PBS solution, and incubated at 4°C for 1h~2h until the solution is well permeabilized and presents a dark red color, and the red blood cell membrane is centrifuged at 10000g~12000g for 10min~15min at 4°C, and washed with 0.25×PBS solution until the cell membrane is white or light pink, and collected.

8. The preparation method according to claim 3, characterized in that: In step (3), the red blood cell membrane and B@COF DT The volume ratio is 8:1~6:1; the polycarbonate membrane used in the Avanti micro extruder has a large size of 400nm and a small size of 200nm and 100nm respectively.

9. Use of the anti-inflammatory nano drug delivery system containing a guanidine structure as described in any one of claims 1 to 2 or the anti-inflammatory nano drug delivery system containing a guanidine structure prepared by the preparation method as described in any one of claims 3 to 8 in the preparation of drugs for improving neuroinflammation and neurological function.

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