A nanoguan-based drug system, a preparation method and application thereof

Abstract

The application discloses a nano guanido drug system and a preparation method and application thereof, and relates to the technical field of medicine, in particular to a GCOF DHM @CAQK platform, can efficiently deliver drugs to a TBI injury site, can accumulate in a TBI brain injury area, can inhibit neuroinflammation, oxidative stress and protect neuron cells after TBI, can precisely intervene in secondary injury of TBI, and provides a new strategy for improving the prognosis of patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a nano-guanidine-based drug system, its preparation method, and its application. Background Technology

[0002] Traumatic brain injury (TBI) refers to severe damage to the structure and function of brain tissue caused by external force. The causes of TBI are diverse, with traffic accidents, falls, violence, and sports injuries being the most common. The pathological process of TBI is complex, divided into primary mechanical injury and secondary injury. Secondary injury occurs within hours to days after the primary injury, and its mechanisms involve excitatory neurotoxicity, oxidative stress, calcium overload, inflammatory response, neuronal death, blood-brain barrier disruption, increased intracranial pressure, and cerebral edema, which can further aggravate the injury. Although secondary injury is an interventional target and a core target for TBI treatment, clinical practice still focuses on symptomatic treatment, lacking effective drugs to intervene in secondary brain injury caused by TBI. Most candidate drugs have failed to effectively improve the prognosis of TBI patients in clinical trials due to their short half-life, poor biospecificity, and low efficiency in crossing the blood-brain barrier (BBB). Therefore, there is an urgent clinical need to develop new therapeutic drugs to optimize the outcome of TBI patients and reduce the social and family burden they bring.

[0003] Dihydromyricetin (DHM), a natural compound with definite neuroprotective activity, exerts its neuroprotective effect by inhibiting inflammasome-mediated neuroinflammatory responses and blocking neuronal death. It holds promise for alleviating secondary brain injury following total brain injury (TBI) by targeting related pathological processes. However, limitations such as DHM's short half-life, lack of specificity in biodistribution, and low BBB penetration efficiency restrict its therapeutic efficacy against TBI. Therefore, developing a drug that can target and deliver DHM directly to the site of TBI injury is of great significance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a nano-guanidine-based drug system, its preparation method, and its applications. This nano-guanidine-based drug system is a covalent organic framework material carrier platform that can efficiently deliver drugs to the site of TBI injury. It can accumulate in the TBI brain injury area and inhibit neuroinflammation and oxidative stress after TBI, while protecting neurons. It can precisely intervene in secondary damage caused by TBI, providing a new strategy for improving patient prognosis.

[0005] The purpose of this invention is to provide a nano-guanidine-based drug system, wherein the nano-guanidine-based drug system is a GCOF obtained by loading the drug DHM onto a GCOF carrier and then modifying the surface with CAQK. DHM @CAQK; The GCOF is a guanidine-containing carrier with a structure as shown in formula (I):

[0006] .

[0007] Covalent organic frameworks (COFs) are functional systems formed by the covalent connection of lightweight organic building blocks. Characterized by their highly ordered porous structure and synthetic flexibility, they can optimize drug release, solubility, and bioavailability, demonstrating significant potential in drug delivery. CAQK peptides are functional peptides that target damaged brain tissue, achieving targeted enrichment by binding to chondroitin sulfate proteoglycan highly expressed in the damaged area. This invention constructs a targeted delivery system for TBI-damaged areas by loading the drug DHM into the COF cavity and modifying the COF nanomaterial surface with CAQK peptides. This system combines the drug delivery advantages of COFs with the targeting properties of CAQKs, efficiently delivering DHM to the TBI lesion site. Simultaneously, utilizing the unique guanidine structure, it possesses anti-inflammatory and antioxidant potential, enabling precise intervention in secondary TBI damage and providing a new strategy for improving patient prognosis.

[0008] Furthermore, the DHM has a structure as shown in formula (II):

[0009] .

[0010] Furthermore, the CAQK is a targeting peptide with the peptide sequence Cys-Ala-Gln-Lys.

[0011] This invention also provides a method for preparing a nano-guanidine drug system, comprising the following steps:

[0012] (1) Synthesis of GCOF: The monomers triaminoguanidine hydrochloride (TG) and trialdehyde phloroglucinol (TP) were dissolved in a mixed solvent, and a catalyst was added to prepare a reaction solution. The reaction solution was subjected to ultrasonic treatment for 5 min-30 min and 2-5 cycles of freezing-vacuuming-thawing. After the reaction was completed, the reaction solution was centrifuged, washed with organic solvent, and vacuum dried to obtain GCOF.

[0013] (2) Drug loading: The GCOF obtained in step (1) was dispersed in phosphate buffer, and a pre-dissolved DHM solution was added. The mixture was stirred at room temperature for 6-24 hours. After the reaction, the mixture was centrifuged, the solid was collected, washed, and then dialyzed in a dialysis bag for 12-48 hours. The dialysis product was collected by centrifugation and freeze-dried to obtain the drug-loaded complex GCOF. DHM ;

[0014] (3) Targeted peptide modification: The drug-loaded complex GCOF obtained in step (2) is modified. DHMThe drug was dispersed in phosphate buffer, and a pre-prepared target peptide CAQK solution was added. After sonication for 2-15 minutes, the mixture was stirred at room temperature for 6-24 hours. After the reaction, the mixture was centrifuged, the solid was collected, washed, and dialyzed in a dialysis bag for 12-36 hours. The dialysis product was collected by centrifugation and freeze-dried to obtain the covalent organic framework drug-loaded target complex GCOF. DHM @CAQK.

[0015] Further, in step (1), the molar ratio of the monomer TG to TP is 1-1.5:1-2; the mixed solution is a mixed solution of mesitylene and 1,4-dioxane in a volume ratio of 1-2.5:1-2; the catalyst is acetic acid with a concentration of 3M-9M, and the amount added is 1%-15% of the total volume of the reaction solution.

[0016] Furthermore, in step (1), the reaction temperature in the microwave reactor is 100℃-150℃; the vacuum drying temperature is 50℃-80℃, and the drying time is 6h-24h; the washing organic solvent is one or more of methanol, acetone, and tetrahydrofuran, and the washing is carried out in sequence.

[0017] Furthermore, in step (2), the mass ratio of DHM to GCOF is 1:5-15; the DHM solution is obtained by dissolving DHM in dimethyl sulfoxide, ethanol or a mixture of the two solvents, and the final amount used is no more than 5% of the volume fraction in the loaded reaction system.

[0018] Furthermore, in step (3), CAQK and GCOF DHM The mass ratio is 1:30-50; the target peptide CAQK solution is obtained by dissolving CAQK in phosphate buffer or water.

[0019] Furthermore, in steps (2) and (3), the pH of the phosphate buffer is 6.8-7.4; the molecular weight cutoff of the dialysis bag is 3500 Da; and the dialysis solution is changed every 4-8 hours during dialysis.

[0020] This invention also provides the application of the above-mentioned nanoguanidine drug system in the preparation of drugs that improve BBB function and cerebral edema, reduce neuroinflammation, and protect neurological function.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. This invention designs a novel guanidine-based covalent organic framework (GCOF) nanocarrier and constructs GCOF nanocarriers. DHMThe @CAQK system, by integrating DHM into the COF cavity and modifying the COF nanomaterial surface with CAQK short peptides, constructs a targeted delivery platform for TBI-damaged areas. This platform can deliver DHM drugs directly to the TBI injury site, effectively penetrating the BBB and improving neuroinflammation and neurological function, significantly enhancing treatment efficacy. This nano-guanidine-based drug system can precisely intervene in secondary TBI damage and can be applied to the preparation of drugs that improve BBB function and cerebral edema, reduce neuroinflammation, and protect neurological function, providing a new strategy for improving the prognosis of TBI patients.

[0023] 2. Mouse experiments showed that the GCOF designed in this invention... DHM @CAQK treatment reversed neurological deficits in TBI mice, improved spatial memory and learning abilities, enhanced overall neurological function scores, restored BBB function, reduced inflammation and edema, and demonstrated high biocompatibility. Attached Figure Description

[0024] Figure 1 GCOF is an embodiment of the present invention. DHM Scanning electron microscope (SEM) images (scale bar: 1 μm) and transmission electron microscope (TEM) images (scale bar: 0.2 μm) of @CAQK during its self-assembly process.

[0025] Figure 2 GCOF is an embodiment of the present invention. DHM Powder X-ray diffraction pattern during the self-assembly process of @CAQK;

[0026] Figure 3 GCOF is an embodiment of the present invention. DHM Fourier transform infrared spectrum of @CAQK during self-assembly process;

[0027] Figure 4 GCOF is an embodiment of the present invention. DHM Specific surface area test diagram during the CAQK self-assembly process;

[0028] Figure 5 To verify GCOF in in vitro experiments in this embodiment of the invention DHM @CAQK can better cross the blood-brain barrier and target damaged HT22 neurons (immunofluorescence map);

[0029] Figure 6 To verify GCOF in vivo in this embodiment of the invention DHM @CAQK can better target damaged brain tissue in small animal organ imaging images by crossing the blood-brain barrier;

[0030] Figure 7 To verify GCOF in in vitro experiments in this embodiment of the invention DHM @CAQK can effectively improve serum deprivation-induced HT22 cell death (Figure);

[0031] Figure 8 GCOF is an embodiment of the present invention. DHM Image showing Evans blue staining of brain tissue in TBI mice after treatment with @CAQK;

[0032] Figure 9 GCOF is an embodiment of the present invention. DHM Statistics on brain water content in TBI mice after treatment with @CAQK;

[0033] Figure 10 GCOF is an embodiment of the present invention. DHM Immunohistochemical images of GFAP and Iba-1 in brain tissue of TBI mice after treatment with @CAQK;

[0034] Figure 11 GCOF is an embodiment of the present invention. DHM Distribution of Nissl bodies in brain tissue of mice after @CAQK treatment of TBI mice;

[0035] Figure 12 GCOF is an embodiment of the present invention. DHM Representative route maps and statistical analysis charts of learning and memory abilities of mice in each group after @CAQK treatment of TBI mice;

[0036] Figure 13 GCOF is an embodiment of the present invention. DHM and GCOF DHM @CAQK measured IC in HT22 cells 50 data;

[0037] Figure 14 DHM and GCOF are embodiments of the present invention. DHM and GCOF DHM HE staining image of the biosafety of @CAQK on mouse heart, liver, spleen, lungs and kidneys. Detailed Implementation

[0038] To facilitate understanding of the present invention, a more complete description will be given below with reference to various embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0039] 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 invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the formulations involved in the following examples are all commercially available products and can be purchased from the market.

[0041] The carrier GCOF of the nano-guanidine drug system of this invention was prepared by TG and TP; then DHM was integrated into the GCOF cavity under stirring at room temperature; finally, its surface was targeted modified with CAQK to obtain the GCOF. DHM @CAQK can precisely target areas of neuronal damage, and has the effect of improving neuroinflammation and neurological function.

[0042] The present invention will be further described in detail below with reference to specific embodiments:

[0043] Example 1: GCOF DHM @CAQK preparation method

[0044] GCOF DHM The preparation method of @CAQK includes the following specific steps:

[0045] (1) Synthesis of GCOF

[0046] The monomers triaminoguanidine hydrochloride (TG) and trialdehyde phloroglucinol (TP) in a molar ratio of 1:1 were dissolved in a mixed solvent of mesitylene and 1,4-dioxane in a volume ratio of 1:1. 6M acetic acid, at 5% by volume of the total reaction solution, was added as a catalyst to form the reaction solution. The reaction solution was sonicated for 15 min, followed by three cycles of freezing-evacuation-thawing to remove oxygen. The sealed reaction tube was placed in a microwave reactor and reacted at 120 °C for 30 min. After the reaction, the mixture was cooled to room temperature and centrifuged at 12,000 rpm for 10 min to collect the yellow solid. The solid was washed three times each with methanol, acetone, and tetrahydrofuran. The washed product was then vacuum dried at 60 °C for 12 h to obtain a yellow GCOF powder.

[0047] (2) Drug loading (preparation of GCOF) DHM )

[0048] Take 10 mg of GCOF synthesized in step (1), disperse it in 5 mL of phosphate-buffered saline (PBS) at pH 7.4, and sonicate for 15 min to obtain a homogeneous dispersion with a concentration of 2 mg / mL; separately, dissolve 1 mg of drug DHM in 50 μL of anhydrous dimethyl sulfoxide (DMSO), and then slowly add it to the GCOF dispersion at a mass ratio of DHM to GCOF of 1:10. Mix the mixture and stir magnetically at room temperature in the dark for 12 h. After the reaction is complete, centrifuge at 12,000 rpm for 10 min to collect the solid and wash it twice with PBS. Redisperse the product in PBS, transfer it to a 3500 Da dialysis bag, and dialyze it in 1 L of PBS for 24 h, changing the dialysate every 6 h. After dialysis, centrifuge to collect the solid, freeze-dry it, and obtain the drug-loaded complex GCOF. DHM .

[0049] (3) Targeted peptide modification (preparation of GCOF) DHM @CAQK)

[0050] Take 10 mg of the GCOF obtained in step (2) DHM The 2 mg / mL CAQK was dispersed in 5 mL of PBS (pH 7.4) and sonicated for 5 min to obtain a dispersion. 0.2 mg of the targeting peptide CAQK was dissolved in 1 mL of PBS to obtain a 0.2 mg / mL peptide solution. The CAQK was then reacted with GCOF... DHM The peptide solution was added to the material dispersion at a mass ratio of 1:50, and the mixture was sonicated for 5 min to ensure homogeneity. The mixture was then stirred and reacted at room temperature in the dark for 12 h. After the reaction, the solid was collected by centrifugation at 12,000 rpm for 10 min and washed twice with PBS. The product was redispersed in PBS and transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed in 1 L of PBS at 4 °C in the dark for 24 h, with the dialysis buffer changed every 6 h. After dialysis, the solid was collected by centrifugation and freeze-dried to obtain the final product GCOF. DHM @CAQK.

[0051] The GCOF synthesized in this embodiment DHM @CAQK and its process products were characterized.

[0052] Preparation of PBS-dispersed GCOF DHM The CAQK dispersion (concentration 0.5 mg / mL) was then dropped onto a silicon wafer and dried at room temperature to obtain a scanning electron microscope (SEM) sample. The solution was then diluted 10 times and dropped onto a copper grid to obtain a transmission electron microscope (TEM) sample. The GCOF prepared in this example... DHM SEM and TEM images of @CAQK and its process products are shown below. Figure 1As shown in the figure. The SEM image of the GCOF prepared in step (1) shows that it has a regular, chrysanthemum-like microstructure, as... Figure 1 As shown in A; the TEM image shows that GCOF is composed of interwoven filamentous petal-like units, such as... Figure 1 The illustration of A in the figure shows that after the drug co-loading process in step (2), the SEM image shows GCOF. DHM A morphological change occurs, forming a network structure, such as Figure 1 As shown in B; TEM images further confirm the existence of an interwoven network, such as... Figure 1 The illustration of B in the figure shows that after the surface modification treatment in step (3), GCOF DHM @CAQK exhibits a typical sea urchin-like morphology, with its surface covered by dense needle-like protrusions, such as... Figure 1 C and its illustration are shown in the figure.

[0053] Figure 2 GCOF prepared in this embodiment DHM Powder X-ray diffraction (PXRD) patterns of @CAQK and its process products are shown. Peaks are observed at 4.75°, 8.14°, and 26.67°, corresponding to the (100), (200), and (002) planes, respectively. The crystallinity of GCOF was reduced by drug loading and surface-targeting peptide modification.

[0054] Figure 3 GCOF prepared in this embodiment DHM Fourier transform infrared (FT-IR) spectra of @CAQK and its process products. GCOF and GCOF2 are observed in the figure. DHM and GCOF DHM @CAQK's FT-IR revealed that the characteristic C=N stretching vibration peaks all appeared at 1634 cm⁻¹. -1 .

[0055] Figure 4 GCOF prepared in this embodiment DHM Specific surface area of ​​@CAQK and its process products. The nitrogen adsorption-desorption isotherm data in the figure show GCOF and GCOF. DHM and GCOF DHM The specific surface area of ​​@CAQK decreases sequentially, reaching 380.52 m². 2 / g、273.40m 2 / g and 50.49m 2 / g.

[0056] Example 2: GCOF DHM @CAQK Targeted Experiment

[0057] The efficacy of TBI treatment drugs depends on their ability to penetrate the blood-brain barrier and accumulate at the site of injury. GCOF DHM @CAQK can directly target the damaged area and enter cells to achieve a therapeutic effect. Cyanine 5.5 (Cy5.5 for short) is a fluorescent dye used for in vivo imaging, used to enable GCOF... DHM The targeting capability and delivery performance of @CAQK were visualized; GCOF was prepared in this embodiment. DHM(Cy5.5) @CAQK.

[0058] 1. Fluorescent labeling (preparation of GCOF) DHM(Cy5.5) )

[0059] Take 10 mg of GCOF obtained in step 2 of Example 1 DHM The sample was dispersed in 5 mL of pH 8.5 to prepare a dispersion with a concentration of 2 mg / mL; 0.02 mg of the near-infrared fluorescent dye Cy5.5-NHS ester was dissolved in 20 μL of anhydrous DMSO. Under light-protected conditions, Cy5.5-NHS ester was reacted with GCOF... DHM The dye solution was added dropwise to GCOF at a mass ratio of 1:500. DHM In a dispersion, the reaction was carried out at room temperature under argon protection with stirring for 6 hours in the dark. After the reaction was completed, the solid was collected by centrifugation at 12,000 rpm for 10 min, and washed repeatedly with PBS by centrifugation 4-5 times until the supernatant showed no fluorescence signal in the near-infrared region, yielding the fluorescently labeled intermediate GCOF. DHM(Cy5.5) .

[0060] 2. Targeted peptide modification (preparation of GCOF) DHM(Cy5.5) @CAQK)

[0061] Take 10 mg of the GCOF obtained in step 1 DHM(Cy5.5) The 2 mg / mL CAQK was dispersed in 5 mL of PBS (pH 7.4) and sonicated for 5 min to obtain a dispersion. 0.2 mg of the targeting peptide CAQK was dissolved in 1 mL of PBS to obtain a 0.2 mg / mL peptide solution. The CAQK was then reacted with GCOF... DHM(Cy5.5) The peptide solution was added to the material dispersion at a mass ratio of 1:50, and the mixture was sonicated for 5 min to ensure homogeneity. The mixture was then stirred and reacted at room temperature in the dark for 12 h. After the reaction, the solid was collected by centrifugation at 12,000 rpm for 10 min and washed twice with PBS. The product was redispersed in PBS and transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed in 1 L of PBS at 4 °C in the dark for 24 h, with the dialysis buffer changed every 6 h. After dialysis, the solid was collected by centrifugation and freeze-dried to obtain the final product GCOF. DHM(Cy5.5) @CAQK.

[0062] For Cy5.5, GCOF DHM(Cy5.5) GCOF DHM(Cy5.5) @CAQK conducted in vitro experimental verification.

[0063] An in vitro blood-brain barrier model was established using the Transwell system. The chambers of this system were lined with a monolayer of brain microvascular endothelial cells (bEnd.3). After seeding cell spreaders into six-well plates, 300,000 HT22 cells were added to each well. Cells were cultured in medium containing 10% fetal bovine serum and serum-deprived medium, respectively, and cultured with 10 μg / mL GCOF. DHM(Cy5.5) and GCOF DHM(Cy5.5) Cells were co-incubated with CAQK for 6 h, using a serum deprivation method to simulate the TBI neuronal injury environment in vitro. They were first fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, then permeabilized with 0.3% Triton-X for 30 min, washed with PBS again, and stained with DAPI. After mounting, the slides were observed under a confocal microscope. Figure 5 As shown, the results indicate that serum-deprived HT22 cell internalization was significantly enhanced with the addition of CAQK peptide.

[0064] After successful TBI modeling in mice, Cy5.5 and GCOF were injected intraperitoneally. DHM(Cy5.5) and GCOF DHM(Cy5.5) @CAQK. After euthanizing the mice, the brain and major organs, including the heart, liver, spleen, lungs, and kidneys, were separated and imaged using the IVIS Lumina XRMS Series III imaging system. (See attached image.) Figure 6 As shown, the results indicate that GCOF DHM(Cy5.5) @CAQK is more likely to accumulate at the site of TBI injury, and GCOF DHM(Cy5.5) @CAQK does not remain in major organs for too long, ensuring the drug's necessary biosafety.

[0065] Example 3: GCOF DHM @CAQK in vitro experiments

[0066] 1. GCOF DHM In vitro evaluation of the anti-ROS, anti-inflammatory and neuroprotective effects of @CAQK

[0067] To evaluate the GCOF prepared in Example 1 DHM @CAQK's in vitro anti-ROS, anti-inflammatory, and neuroprotective effects, with DHM and GCOF DHM Make a comparison.

[0068] After seeding cell spreaders into six-well plates, 300,000 HT22 cells were added to each well. Cells were then incubated in DMEM medium containing 10% fetal bovine serum and serum-free medium at 37°C with 5% CO2 for 24 hours. The cells were then reacted with 10 μg / mL DHM and GCOF. DHM and GCOF DHM @CAQK nanomaterials were co-incubated, and the number and ratio of live and dead cells in each group were determined using a live-dead cell staining (PI) kit. Cells were cultured using a serum deprivation method to simulate the neuronal injury microenvironment caused by traumatic brain injury in vitro. Cell viability was detected using a calcein acetoxymethyl ester / propidium iodide (AM / PI) double staining method, with AM and PI labeling live and dead cells, respectively. Figure 7 As shown, the results indicate that GCOF DHM @CAQK nanomedicines have better ability to protect neurons in vitro.

[0069] Example 4: GCOF DHM @CAQK in vivo experiments

[0070] 1. In vivo experimental verification of GCOF DHM @CAQK's treatment effect on TBI

[0071] Evaluation studies on blood-brain barrier (BBB) ​​repair, cerebral blood flow improvement, acute inflammation suppression, and short-term neuroprotection should focus on the acute phase of traumatic brain injury (TBI). This phase is characterized by severe BBB disruption, cerebral edema, inflammatory cytokine outbreaks, microglia activation, and rapid neuronal damage. A mouse TBI model was constructed using a free-fall impact device. Mice were anesthetized with isoflurane and fixed to the experimental platform. After routine disinfection, a longitudinal scalp incision was made along the midline of the head to expose the skull. A bone window with a diameter of approximately 3 mm was prepared using a cranial drill on the right side of the midline (aligned with the coronal suture). A 60 g weight was dropped from a height of 20 cm to impact the target site, creating a local lesion with a diameter of approximately 2 mm. After suturing the scalp incision, the mice were placed in cages for routine resuscitation. The sham-operated group mice underwent the same procedures as the model group except for the absence of the free-fall impact. Treatment interventions (DHM, GCOF) were also performed. DHM and GCOF DHM @CAQK) was administered once daily at a dose of 0.05 mg / g on days 0, 1, and 2 after injury to maximize the intervention effect during the critical therapeutic window, thereby evaluating the role of nanomedicine in blood-brain barrier repair and inhibition of neuroinflammatory cascades.

[0072] The integrity of the brain brain (BBB) ​​in each treatment group was preferentially assessed using the Evans blue exudation method: 4% Evans blue dye was intravenously injected into mice at a dose of 2.5 mL / kg, followed by a 3-hour circulation cycle to ensure sufficient dye distribution. Mice were then deeply anesthetized, and 20 mL of physiological saline was perfused through the heart to remove residual dye from the blood vessels. After perfusion, brain tissue was precisely separated and imaged using a digital camera to visually observe the dye exudation. The injured cerebral hemisphere was separated and weighed, and then homogenized using a tissue homogenizer. 300 μL of the brain homogenate sample was added to 700 μL of acetone and incubated at room temperature for 24 h. After centrifugation, the absorbance was measured at 620 nm using a spectrophotometer. Finally, the Evans blue content was calculated according to the standard curve. The results are as follows: Figure 8 As shown.

[0073] To assess the severity of cerebral edema in each group of mice, brain water content was measured using the wet weight method. After establishing the TBI mouse model, treatment interventions (DHM, GCOF) were administered. DHM and GCOF DHM (@CAQK) The drug was administered once daily on days 0, 1, and 2 post-injury. On day 3, mice were sacrificed, and their brains were collected, divided into left and right hemispheres, with the cerebellum and brainstem included in the wet weight range. The samples were then dehydrated at 105°C for 24 hours to obtain dry weight. The formula for calculating the percentage of water content in the brain is: (wet weight - dry weight) / wet weight × 100%. Figure 9 The results showed GCOF DHM The application of @CAQK can significantly reduce the water content of brain tissue after TBI.

[0074] After successfully establishing the mouse TBI model, a dose of 0.05 mg / g (DHM, GCOF) was administered. DHM and GCOF DHM @CAQK) was administered once daily on days 0, 1, and 2 after injury. On the third day, mouse brain tissue was removed under anesthesia, dehydrated, embedded, and prepared into paraffin sections. After dewaxing, antigen retrieval was performed using citrate buffer at high temperature, followed by cooling and washing with PBS. The sections were then blocked with 3% BSA at room temperature for 30 min. Primary antibodies GFAP (astrocytocyte marker) and Iba-1 (microglia marker) were added, and the sections were incubated overnight at 4°C. The sections were then warmed again the next day and washed with PBS. Secondary antibodies labeled with fluorescence or HRP were added, and the sections were incubated at room temperature for 1 h, followed by washing with PBS. HRP-labeled secondary antibodies were developed using DAB, and hematoxylin was used to counterstain cell nuclei. Fluorescently labeled secondary antibodies were directly mounted. Images were observed and acquired under an optical microscope to analyze the number and expression intensity of positive cells. Figure 10 The results showed GCOF DHM @CAQK can significantly improve neuroinflammation mediated by astrocytes and microglia.

[0075] Nissl staining can effectively assess the neurological function impairment and repair status in each group of mice. After successful establishment of the mouse TBI model, a dose of 0.05 mg / g (DHM, GCOF) was administered. DHM and GCOF DHM @CAQK) was administered once daily on days 0, 1, and 2 after injury. On the third day, mouse brain tissue was removed after anesthesia. The fixed brain tissue was dehydrated, embedded, and prepared into paraffin sections. After dewaxing, Nissl stain was added, and the sections were incubated at room temperature for 10-20 minutes. The sections were rapidly differentiated with graded ethanol until the background was clear, and the differentiation was terminated by rinsing with distilled water. The sections were then dehydrated with high-concentration ethanol and cleared with xylene. The sections were mounted and observed under a light microscope. Nissl bodies appeared deep blue or purple, which can be used to assess neuronal morphology and number. Figure 11 As shown, compared with the TBI group mice, GCOF DHM It significantly increased the abundance of Nissl bodies, and GCOF DHM @CAQK is more effective than GCOF. DHM .

[0076] On day 15 after preparing the TBI animal model, a Moss water maze experiment was conducted with the water temperature set at 24°C. For the first 5 days, mice were randomly placed in a quadrant of the water. These mice were allowed to swim freely until they found a resting platform hidden 1 cm below the surface, and the time (t) and swimming distance (s) were recorded. Each mouse had 300 seconds to swim; mice that could not find the platform before the end of the time limit were guided to the platform and remained there for 30 seconds. On day 7, the platform was removed, and a quadrant was randomly selected so that the mice could find the platform's original location. Each mouse swam for 300 seconds, and the time taken to reach the platform's original location and the time taken to traverse the quadrant were recorded. All data during the test were recorded and analyzed using a video tracking system. Each mouse underwent a swimming test before the experiment; mice that could not swim were excluded, and each group consisted of 5 mice. Figure 12 As shown, the experimental results indicate that GCOF DHM @CAQK significantly enhanced the recovery of neurological function in mice after TBI and improved their motor, learning, and memory functions.

[0077] Example 5: GCOF DHM @CAQK's biosafety testing

[0078] To further verify GCOF DHM @CAQK's biosafety has been assessed through in vitro and in vivo biosafety testing experiments.

[0079] HT22 cells were taken, resuspended in complete culture medium, and the cell concentration was adjusted to 5 × 10⁻⁶. 3Cells were seeded at a density of 100 μL per well in a 96-well plate and incubated for 24 h in a constant temperature incubator to allow the cells to adhere and grow. The target nanomedicine (GCOF) was then introduced. DHM and GCOF DHM @CAQK) A series of concentration gradients were prepared by diluting the drug in complete culture medium, with three replicates per group; a blank control group (complete culture medium only, no cells) and a negative control group (cells + complete culture medium, no drug) were also set up. The original culture medium in the 96-well plate was discarded, and 100 μL of the corresponding concentration of drug culture medium was added to each well of the drug group. An equal volume of complete culture medium was added to the negative control group and the blank control group. The plates were incubated for 24 h. After incubation, a mixture of 10 μL of CCK-8 reagent and 90 μL of complete culture medium was added to each well. The plate was gently shaken to mix thoroughly and incubated in an incubator for 1 h. The optical density (OD) value of each well was measured at 450 nm using a microplate reader. The OD value of the blank control group was used to subtract background interference. The dose-response curve was fitted using GraphPad Prism software with the logarithm of drug concentration as the x-axis and cell viability as the y-axis to calculate the half-maximal inhibitory concentration (IC50) of the drug for HT22 cells. 50 ) value. Result as follows Figure 13 As shown, the experimentally measured GCOF DHM IC50 in HT22 cells 50 The concentration was 67.84 mg / L, and the GCOF was... DHM @CAQK in HT22 cells IC 50 It was 91.63 mg / L.

[0080] To further verify GCOF DHM The biocompatibility of @CAQK was assessed by constructing a mouse TBI model and administering it at a dose of 0.05 mg / g (DHM, GCOF). DHM and GCOF DHM @CAQK) After 3 days of drug administration, mice were sacrificed after 7 days of feeding. Major organs (heart, liver, spleen, lungs, kidneys) were removed, fixed in 4% paraformaldehyde, embedded in paraffin, dewaxed, and stained with hematoxylin and eosin (HE). Hematoxylin staining was performed for 10-15 minutes; separation was achieved with 0.5%-1.0% hydrochloric acid-alcohol for several seconds to tens of seconds, followed by rapid washing with distilled water; blueing with dilute ammonia, washing with distilled water, and microscopic examination during differentiation; rinsing with running water for 3 minutes to remove alkaline water; staining with 1% eosin for 5-10 minutes; rapid washing with distilled water, followed by stepwise dehydration with alcohol (70%, 80%, and 90% alcohol), then 30-60 seconds with 95% alcohol, followed by microscopic examination; 100% alcohol for 3 minutes, twice; xylene for 5 minutes, twice; finally, mounting with neutral resin and microscopic observation. Figure 14 As shown, the results indicate that none of the nanoparticles caused pathological damage to major organs, demonstrating high biosafety.

[0081] In summary, this invention prepares a nano-guanidine-based drug system by integrating DHM into the COF cavity and modifying the COF nanomaterial surface with a short CAQK peptide. This system can target the TBI-damaged area and effectively cross the BBB to improve neuroinflammation and neurological function, exhibiting good therapeutic effects and high biosafety. It can be applied to the preparation of drugs that improve BBB function and cerebral edema, reduce neuroinflammation, and protect neurological function, providing a new strategy for improving the prognosis of TBI patients.

Claims

1. A nano-guanidine-based drug system, characterized in that, The nano-guanidine drug system is a GCOF obtained by loading the drug DHM onto a GCOF carrier and then modifying the surface with CAQK. DHM @CAQK; The GCOF is a guanidine-containing carrier with a structure as shown in formula (I): ； The DHM has a structure as shown in formula (II): ； The CAQK is a targeting peptide with the peptide sequence Cys-Ala-Gln-Lys.

2. The method for preparing the nano-guanidine drug system according to claim 1, characterized in that, Includes the following steps: (1) Synthesis of GCOF: The monomers triaminoguanidine hydrochloride and trialdehyde phloroglucinol were dissolved in a mixed solvent, and a catalyst was added to prepare a reaction solution. The reaction solution was subjected to ultrasonic treatment for 5 min-30 min and 2-5 cycles of freezing-vacuuming-thawing. After the reaction was completed, GCOF was obtained by centrifugation, washing with organic solvent and vacuum drying. (2) Drug loading: The GCOF obtained in step (1) was dispersed in phosphate buffer, and a pre-dissolved DHM solution was added. The mixture was stirred at room temperature for 6-24 hours. After the reaction, the solid was collected by centrifugation, washed, and then dialyzed in a dialysis bag for 12-48 hours. The dialysis product was collected by centrifugation and freeze-dried to obtain the drug-loaded complex GCOF. DHM ; (3) Targeted peptide modification: The drug-loaded complex GCOF obtained in step (2) is modified. DHM Dispersed in phosphate buffer, a pre-prepared target peptide CAQK solution was added, and the mixture was sonicated for 2-15 minutes. The mixture was then stirred at room temperature for 6-24 hours. After the reaction, the solid was centrifuged, collected, washed, and dialyzed in a dialysis bag for 12-36 hours. The dialysis product was collected by centrifugation and freeze-dried to obtain GCOF. DHM @CAQK.

3. The method for preparing the nano-guanidine drug system according to claim 2, characterized in that, In step (1), the molar ratio of the monomer triaminoguanidine hydrochloride and trialdehyde phloroglucinol is 1-1.5:1-2; the mixed solvent is a mixture of mesitylene and 1,4-dioxane in a volume ratio of 1-2.5:1-2; the catalyst is acetic acid with a concentration of 3M-9M, and the amount added is 1%-15% of the total volume of the reaction solution.

4. The method for preparing the nano-guanidine drug system according to claim 2, characterized in that, In step (1), the reaction temperature in the microwave reactor is 100℃-150℃; the vacuum drying temperature is 50℃-80℃, and the drying time is 6h-24h; the organic solvent used for washing is one or more of methanol, acetone, and tetrahydrofuran.

5. The method for preparing the nano-guanidine drug system according to claim 2, characterized in that, In step (2), the mass ratio of DHM to GCOF is 1:5-15; the DHM solution is obtained by dissolving DHM in dimethyl sulfoxide, ethanol or a mixture of the two solvents, and the final amount used is no more than 5% of the volume fraction in the loaded reaction system.

6. The method for preparing the nano-guanidine drug system according to claim 2, characterized in that, In step (3), CAQK and GCOF DHM The mass ratio is 1:30-50; the target peptide CAQK solution is obtained by dissolving CAQK in phosphate buffer or water.

7. The method for preparing the nano-guanidine drug system according to claim 2, characterized in that, In steps (2) and (3), the pH of the phosphate buffer is 6.8-7.4; the molecular weight cutoff of the dialysis bag is 3500 Da; and the dialysis solution is changed every 4-8 hours during dialysis.

8. The use of the nanoguanidine drug system as described in claim 1 or the nanoguanidine drug system prepared by the preparation method as described in any one of claims 2-7 in the preparation of drugs for treating traumatic brain injury.

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