In-vivo trapping agent for diquat molecules as well as preparation method and application of in-vivo trapping agent

Abstract

The invention belongs to the technical field of biology, and particularly relates to an in-vivo trapping agent for diquat molecules and a preparation method and application thereof.The in-vivo trapping agent O-TCP (at) M is obtained by synthesizing carboxymethyl alpha-cyclodextrin, constructing rotaxane, preparing a crosslinked polyethyleneimine encapsulated rotaxane compound and finally using macrophages for loading. The carboxymethyl alpha-cyclodextrin highly matched with the aquacide molecular structure is utilized to realize accurate recognition and combination of toxin molecules, and the TK-PEI dendrimer with positive electricity is used for wrapping, so that the structural stability and ROS responsiveness are enhanced. The in-vivo accurate capture and inflammation regulation effects are achieved, poisoning symptoms are relieved, and kidney functions are protected and tissue damage is reduced by accurately capturing and neutralizing toxins.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an in vivo capture agent for diquat molecules and a preparation method and application thereof. Background Art

[0002] Diquat, with its molecular structure of a hydrophobic center and a hydrophilic end, resembles a dumbbell with positive charges at both ends. Once diquat enters the body, it exists in a cationic form, does not bind to macromolecules in the body, and rapidly accumulates in the kidneys, causing persistent damage. It causes mitochondrial dysfunction by reducing mitochondrial membrane potential and produces a large amount of cellular reactive oxygen species (ROS). In addition, under physiological conditions, diquat can also directly react with oxygen to produce ROS, and its ability to induce ROS production is 10 to 40 times that of paraquat, directly damaging cellular proteins, lipids, nucleic acids, and other biomacromolecules, and may also trigger inflammatory responses and disrupt cell signaling pathways, ultimately leading to tissue damage, especially kidney damage. The main clinical manifestations include damage to renal tubular epithelial cells, significant increases in blood creatinine and urea nitrogen, ataxia caused by central nervous system toxicity, and the main cause of death is renal failure.

[0003] At present, intervention options for poisoning, including diquat, are still limited to symptomatic treatment, including diuresis, mechanical ventilation, and blood purification according to current standard guidelines. However, it cannot be ignored that toxins usually continue to accumulate in a certain organ, and the toxin concentration in the blood is usually not high, resulting in inefficient clearance through the blood. The final elimination of toxins in the body mainly depends on the patient's own toxin metabolism and excretion ability. However, patients usually have liver and kidney dysfunction, which further reduces the toxin processing ability, forming a vicious cycle after poisoning. Even if a capture system for a certain toxin is found under in vitro conditions by chance, the direct application of the capture agent to the body will be limited by the distribution of the capture agent itself in the body (the distribution of the capture agent in the body is often inconsistent with the toxin, and the capture agent in the body may also be quickly cleared, further reducing its availability), and the capture agent is difficult to use as an in vivo detoxifier. For example, it is known that the carboxyl-modified pillar aromatic hydrocarbon WP6 has strong paraquat capture and ROS reduction capabilities at the in vitro and cellular levels, but it faces challenges in in vivo application. To date, there is no specific toxin capture strategy for the target organ of poisoning in clinical settings or even in the field of basic research. All of this greatly reduces the possibility of emergency intervention. Therefore, there is an urgent need to develop specific antidotes for toxins, as well as effective strategies to optimize the body distribution of antidotes and enable them to act on demand.

[0004] The complexity and abundance of substances in the body can easily render antidotes, especially those that capture via supramolecular recognition, inactive before reaching the target organ due to competition with other freely diffusing analogs. Utilizing in situ supramolecular replacement and in situ activation strategies may help resolve this dilemma. Specifically, the active compartment of the antidote should be temporarily occupied by a substance, rendering it unresponsive to other analogs in vivo, and then, when faced with the specific microenvironment of the lesion site where action is required, the temporary binding complex will dissociate according to the designed stimuli-responsive dissociation, restoring the compartment with the ability to capture toxins.

[0005] The inflammatory response in the body after poisoning is the body's natural mechanism to fight against foreign invasion. During this period, macrophages are attracted to the site of inflammation through a series of complex signal transduction and cell migration processes. This actually provides a directional solution strategy for transporting antidotes to the site where toxins accumulate.

[0006] In clinical practice, many types of acute poisoning have specific antidotes (e.g., neutralization by antibodies in antivenom, and anticholinergics for organophosphate poisoning), but there are still some widely and heavily used substances (such as paraquat and diquat) that do not have effective, specific, and consistent antidotes. There are two fundamental reasons why these substances do not have antidotes: first, they are distributed too quickly after entering the body and concentrate in target organs, making it difficult to eliminate them through means such as hemodialysis or gastric lavage; second, these molecules have a very unique structure, usually with a strong positive charge. If an antidote with a strongly negatively charged structure is matched to it, it will be eliminated by the body too early.

[0007] Macrophages play a key role in the innate immune system. They rapidly respond to pathogens and subsequently promote an anti-inflammatory phenotype to limit tissue damage and promote repair. Macrophage engineering is an effective strategy that exploits the natural law of chemotaxis to achieve efficient delivery of substances in the body. Itaconate is a key anti-inflammatory metabolite that is most abundant in macrophages, reaching 5 mM in mouse bone marrow-derived macrophages after LPS stimulation. Itaconate is essential for the activation of the anti-inflammatory transcription factor Nrf2 in mouse and human macrophages: it alkylates cysteine ​​residues on the protein KEAP1, thereby enhancing the expression of antioxidant and anti-inflammatory genes by Nrf2. Through Nrf2, itaconate limits inflammation and modulates the effects of type I interferons. Structurally, itaconate is anionic and hydrophilic, which makes it difficult to cross cell membranes; it is endogenously produced by IRG1 in the mitochondrial matrix and acts on Nrf2 in the cytoplasm. 4-OI is an esterified prodrug of itaconate that can cross cell membranes and be hydrolyzed to itaconate in the cytoplasm or act as a direct substitute for itaconate. In addition, 4-OI has a stronger ability to upregulate Nrf2 under ROS conditions. Reports indicate that the highest concentration of exogenous 4-OI treatment that does not impair cell activity can reach 45 pmol / mg of cell protein.

[0008] The mechanism of diquat poisoning is that its special molecular structure causes cellular reactive oxygen species (ROS) damage, and the main target organ is the kidney, leading to acute kidney injury. There are currently no specific guidelines for the treatment of diquat poisoning, nor are there specific in vivo capture antidotes specifically for diquat. Therefore, intervention strategies are usually derived from methods for treating sepsis. Therefore, the problems that need to be solved urgently are: 1) Find or construct an in vivo capture antidote specifically for diquat; 2) Deliver the antidote to the target organ (i.e., the kidney) to accurately capture the toxin and reduce its ability to produce ROS; 3) Reduce the damage caused and protect kidney function. Summary of the invention

[0009] In order to overcome the deficiencies in the prior art, the present invention provides an in vivo capture agent for diquat molecules and a preparation method and application thereof. The technical solution of the present invention is as follows: A method for preparing an in vivo capture agent for diquat molecules comprises the following steps: (1) Synthesis of carboxymethyl α-cyclodextrin (CCD): α-cyclodextrin was added to a sodium hydroxide aqueous solution under stirring to obtain solution A; chloroacetic acid was dissolved in water to obtain a chloroacetic acid solution; solution A was heated to 60°C, and the chloroacetic acid solution was added dropwise to solution A at a controlled rate, and the reaction was continued at 60°C for 3 hours after the addition was completed; after returning to room temperature, the solution was neutralized with hydrochloric acid to a pH of 7, and dialyzed in pure water with a MW1K dialysis bag for 24 hours, and freeze-dried to obtain CCD; (2) Construction of rotaxane: CCD and chain polyethylene glycol 5K (PEG 5K) were directly dispersed in distilled water, vortexed and ultrasonically treated at room temperature, and then heated to 60°C for insulation. The rotaxane suspension CCD@PEG was obtained by centrifugal ultrafiltration. (3) Preparation of cross-linked polyethyleneimine encapsulated rotaxane complex (TC@PEG): cross-linking polyethyleneimine (MW 1.2K) with reactive oxygen species (ROS)-responsive thioketal (sulfonic acid-NHS-TK-sulfonic acid-NHS) was performed, and TK-PEI was obtained after purification by dialysis; TK-PEI was added to the rotaxane suspension CCD@PEG obtained in step (2), stirred at 25°C, and subjected to rapid centrifugal ultrafiltration to obtain cross-linked polyethyleneimine encapsulated rotaxane complex TC@PEG; (4) Macrophage loading capture agent: Mouse macrophage Raw264.7 cell line was cultured in a cell culture dish (density: 1 million per dish), the cell culture medium was removed, and the cells were washed with serum-free medium. TC@PEG was introduced into the cells. After incubation for 2 h, the cells were washed with PBS buffer to obtain the TP@M intermediate state. Then, 4-OI (octyl itaconate) was added and dissolved with DMSO. The cells were incubated for 0.5 h and washed with PBS buffer to obtain the final in vivo capture agent O-TCP@M.

[0010] Preferably, in step (1), the concentration of sodium hydroxide is 1.5 M, the concentration of α-cyclodextrin is 0.05 g / mL, and the concentration of chloroacetic acid is 0.1 g / mL.

[0011] Preferably, the time for adding the chloroacetic acid solution in step (1) is 1 hour.

[0012] Preferably, in step (2), the mass ratio of CCD to PEG 5K is 3:1, the ultrasonic treatment time is 30 minutes, and the insulation time is 3 to 8 days.

[0013] Preferably, in step (3), the mass ratio of polyethyleneimine to thioketal is 5:1.

[0014] Preferably, in step (3), the mass volume ratio of TK-PEI to CCD@PEG suspension is 1:1, mg / mL.

[0015] Preferably, in step (3), the stirring speed is 100 rpm and the stirring time is 30 minutes.

[0016] Preferably, in step (4), the ratio of TC@PEG, 4-OI to cells is 10 mg: 1 mg: 10 mL.

[0017] The present invention also includes the use of the in vivo capture agent O-TCP@M in the preparation of a drug for intervening in poisoning caused by diquat and reducing kidney damage.

[0018] In the present invention, carboxymethyl-α-cyclodextrin is used as a specific antidote for diquat, which is strung on a PEG axis to form a rotaxane structure, and a low molecular weight PEI cross-linked by TK bonds is coated on the periphery by electrostatic attraction to achieve further stability and ROS-responsive dissociation to form a special nanofiber. This nanofiber can be taken up by macrophages and further treated with 4-OI (4-OI can activate Nrf2 by alkylating macrophage KEAP1, greatly enhancing its antioxidant and anti-inflammatory abilities), completing a bionic antidote delivery system.

[0019] The present invention firstly designs a special in vivo capture agent precisely according to the structural characteristics and toxicity principles of the toxin, temporarily masks the molecular recognition ability of the in vivo capture agent, and integrates it into a bionic system based on chemotactic cells through modular design; then, with the help of natural chemotactic behavior in the body, the entire bionic system chemotacts to the lesion site, releases and activates the in vivo capture agent in the specific microenvironment of the lesion site, captures the toxin, and reduces the damage of the toxin to the body.

[0020] Sugammadex® is a neuromuscular blocker approved by the FDA in 2015. The researchers matched the hydrophobic cavity of γ-cyclodextrin to the structure of rocuronium bromide (API) and modified the periphery of γ-cyclodextrin with negatively charged 3-mercaptopropionic acid to attract the positive charge of rocuronium bromide, resulting in a 10 ^7 M ^-1 The host-guest complex constant. Through the principle of molecular design, the present invention initially designed, synthesized and screened six cyclodextrin hosts (non-toxic and degradable cyclic polysaccharides), and performed simulation calculations. We found that the cavity size of α-cyclodextrin is most suitable for forming a complex with diquat, and the carboxymethyl group is the best choice for interacting with the charge of diquat. Through molecular design, dynamic simulation and reasonable screening, the present invention finally selected carboxymethyl α-cyclodextrin (CCD) as an antidote specifically for diquat.

[0021] Of course, the premise for CCD to exert its detoxification effect is that it exists in the inflamed area (mainly the kidney, because the main cause of death caused by diquat poisoning is renal failure caused by ROS-induced kidney damage). Diquat can cause human renal epithelial cells (HEK293) to produce significant ROS accumulation (such as Figure 2 ), while CCD-encapsulated diquat significantly reduced its redox potential and ROS production capacity through precise molecular recognition (as shown in Figure 2). Therefore, the next specific task is to successfully achieve the effective and precise enrichment of CCD in the inflammatory injury area of ​​the kidney. In situ molecular recognition and supramolecular self-assembly in vivo are crucial in the detoxification process, which temporarily occupies the CCD compartment and prevents early interactions with non-target molecules circulating in the blood (especially cholesterol, which has a significant binding affinity to CCD); at the same time, blocking the signals that have already been generated and developed, adopting a dual approach to achieve detoxification and reduce inflammatory responses.

[0022] Rotaxanes are able to string together multiple cyclic molecules using axes and are considered suitable for integrating "dispersed" host molecules into a whole, making them suitable as delivery packages in vivo. Based on the cyclic properties of CCD, we used PEG5K to string CCDs together to form an integrated "molecular necklace". Subsequently, we coated the highly negatively charged "molecular necklace" with positively charged and ROS-cleavable TK-PEI dendrimers, which were stacked together to form nanofibers. Due to the occupation of the cavity, the CCD on PEG temporarily lost the ability to recognize diquat; in the presence of ROS, local ROS would cleave and strip TK-PEI, allowing the CCD to fall off from PEG and restore the CCD's ability to recognize and capture diquat.

[0023] In the present invention, under in vitro culture conditions, macrophages can internalize nanofibers, and the macrophages that have internalized nanofibers are further incubated with 4-OI, and finally a bionic toxin capture system is rationally constructed. After following the cells to the target site, the ROS-rich inflammatory environment triggers the release of CCD from PEG, making the chamber accessible and releasing 4-OI. The system can target the kidneys of poisoned mice, achieve the regulation of renal inflammation, ROS-specific release of antidotes, and ultimately reduce the renal inflammatory response and ROS damage in poisoned mice, and improve the survival rate of poisoned mice.

[0024] Technical features and beneficial effects of the present invention: (1) The present invention uses carboxymethyl α-cyclodextrin (CCD) which is highly compatible with the molecular structure of diquat to achieve accurate recognition and binding of toxin molecules. Using PEG as the axis, multiple CCD molecules are connected in series to form a "molecular necklace" and wrapped with positively charged TK-PEI dendrimers, which enhances the stability of the structure and ROS responsiveness.

[0025] (2) The present invention utilizes the natural chemotaxis of macrophages to effectively transport the detoxification system to the inflammatory area, especially the kidney, and increase the concentration and activity of the detoxifier in the target organ. In the inflammatory microenvironment, the release of CCD is achieved through ROS-induced TK-PEI cleavage, restoring its ability to capture diquat, thereby achieving accurate removal of toxins at the lesion site. By loading 4-OI, the Nrf2 signaling pathway is activated, the antioxidant and anti-inflammatory capabilities of macrophages are enhanced, and it helps to reduce the inflammatory response caused by poisoning. The safety of the detoxification system was verified by in vitro and in vivo experiments, and no obvious damage to other organs was found, showing good biocompatibility.

[0026] (3) The present invention achieves precise in vivo capture and inflammation regulation effects, which not only relieves the symptoms of poisoning, but also protects kidney function, reduces tissue damage, and improves the survival rate of poisoned animals by precisely capturing and neutralizing toxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 For CCD thin layer chromatography (TLC) analysis and 1 H NMR further characterized; among them, A) Structural formula of carboxymethyl α-cyclodextrin (CCD); B) Precise, unique and stable molecular recognition guided by inappropriate charge attraction, hydrophobic interaction and size matching between host and guest molecules; C) 2D NMR ROESY spectrum of diquat@CCD in aqueous solution; D) 2D NMR ROESY spectrum of single component CCD and diquat / CCD in aqueous solution 1 H NMR chemical shift changes; E) Job curve for determining the host-guest ratio in supramolecular recognition; F) As the CCD ratio increases, diquat 1 H NMR peak deformation; G) After adding common interfering ions in vivo, the diquat / CCD pair 1 Deformation of H NMR peaks; H) Determination of the spatial compatibility of CCD and diquat from different angles using Gaussian simulation; Figure 2 is the change of ROS production with time and concentration dependence; A) The mechanism by which CCD reduces the ability of diquat to produce reactive oxygen species (ROS) through charge neutralization; B) Different electrochemical voltammetric curves of diquat and diquat@CCD obtained in aqueous phase; C) Concentration dependence of diquat on ROS production in HEK293 cells measured by flow cytometry and D) corresponding quantitative results; E) Time dependence of diquat on ROS production in HEK293 cells measured by flow cytometry and F) corresponding quantitative results; G) ELISA (5×10 ^6HEK293 cells) Quantitative results of measuring the time-dependent ROS production of diquat; H) Effects of different ratios of CCD on reducing ROS levels in HEK293 cells after 1 hour of intoxication, and I) corresponding quantitative results; J) ROS levels produced after treating HEK293 cells with different ratios of CCD pre-encapsulated with diquat; K) Effects of different ratios of CCD on reducing ROS levels in HEK293 cells after 12 hours of intoxication, and L) corresponding quantitative results; M) Activity trend of HEK293 cells over time in the presence of different concentrations of diquat; N) Activity of HEK293 cells after treatment with 2 equivalents of CCD for 1 hour and 12 hours; O) Activity of HEK293 cells at increasing concentrations of CCD after 48 hours of exposure to diquat; P) ALT, Q) AST, R) ALB, S) ALP, T) BUN and U) CREA is a liver and kidney index in mice injected with diquat and diquat@CCD (2 equivalents) 3 days later; Figure 3 The experimental results of the ROS response process and the incubation time and phagocytic efficiency of macrophages with TC@PEG-Cy5; A) The continuous process of PEG inserting CCD into the rotaxane structure and then being wrapped into nanofibers by TK-PEI; B) The compatibility of PEG axis and CCD shuttle on the spatial scale under molecular simulation; C) 2D NMR ROESY spectrum of CCD@PEG in aqueous solution; D) After adding CCD (30 equivalents) to the aqueous PEG solution, 1 The intensity of each peak in H NMR changes with time, and E) the corresponding quantitative results (taking the CCD peak at 3.65 ppm as an example); F) the intensity of the single CCD peak and CCD@PEG in aqueous solution 1H NMR chemical shift changes; G) C / H / N / S components of intermediates and final products determined by elemental analysis, and the average precise composition ratio of TC@PEG was calculated; H) Microscopic morphology of TC@PEG aqueous solution observed by SEM after vacuum drying and gold coating; I~J) Specific length and diameter of TC@PEG determined by AFM; K) ROS-induced dissociation of TK-PEI and detachment of CCD from PEG axis for diquat capture in in situ supramolecular replacement model; L) TK-PEI (1 equivalent) captures diquat over time in a dialysis bag (1K MW) in the presence or absence of ROS; M) Different ratios of TK-PEI (1 equivalent and 10 equivalents) capture diquat in poisoned HEK293 cells over time in a dialysis bag (1K MW) in the presence or absence of ROS; Microscopic morphology of TC@PEG aqueous solution at different time points of ROS treatment under TEM observation: N) 0 hour, O) 1 h and P) 2 h; Q) Hemolysis of different formulations; R) Schematic diagram of the construction process of the final capture system: macrophage extraction process, TC@PEG endocytosis (Trojan horse strategy into TCP@M), and the final step of loading 4-OI into O-TCP@M; S) Concentration dependence of the effect of the capture system on the viability of HEK293 cells; T) Macrophage image of endocytic nanofibers observed using bioprojection movies; U) Incubation time dependence of macrophage endocytosis of nanofibers; V) Incubation dose dependence of macrophage endocytosis of nanofibers; Figure 4 This is the distribution test process in mice; A) Distribution of TC@PEG and O-TCP@M labeled with Cy5 in major organs (in vitro organs: heart, liver, spleen, lung, kidney) at 4, 12 and 24 hours; B) Semi-quantitative results at 4 hours; C) Semi-quantitative results at 12 hours; D) Semi-quantitative results at 24 hours; E) Semi-quantitative results of kidney accumulation; F) Distribution of TC@PEG and O-TCP@M labeled with Cy5 in kidney sections after accumulation and their co-localization with inflammatory sites at different time points, and G) Local magnified images, and H) Corresponding semi-quantitative results, where arrows indicate non-specific co-localized signals; Figure 5 The application process of diquat in vivo; wherein, A) The establishment process of the animal poisoning model, the timetable of intervention treatment, and the arrangement of subsequent efficacy evaluation follow the reality and standards of clinical treatment; efficacy evaluation, including B) survival rate, C) body weight change, and D) total water intake of each group of animals after animal poisoning and treatment; E) spleen index after 15 days of treatment, and behavioral evaluation results (including F) face wiping times, G) standing times, H) crossing times, and I) moving time); liver and kidney function indicators after 15 days of treatment (including J) BUN, K) CREA, L) UA, M) ALT, N) AST, O) total bilirubin T-BIL, P) albumin ALB, Q) alkaline phosphatase ALP, R) γ-glutamyl transpeptidase GGT, and S) total bile acid TBA); T) kidney tissue pathological sections after 15 days of treatment, and U) Caspase-3 and V) Ki-67 staining results. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is described in detail below through specific implementation methods. It should be understood that the following specific implementation methods are only exemplary, and any changes or modifications should be within the scope of protection of the rights of the present invention as long as they do not deviate from the technical solution design of the present invention.

[0029] Example 1 Preparation process of in vivo capture agent (1) Synthetic CCD A three-necked round-bottom flask was charged with 100 ml of deionized water, and 43 g of sodium hydroxide and 65 g of α-cyclodextrin were added under stirring. The mixture was stirred for 30 minutes to ensure complete dissolution. A chloroacetic acid solution was prepared by dissolving 50 g of chloroacetic acid (7.92 equivalents) in 50 ml of water with stirring. The reaction flask was heated to 60°C, and then the chloroacetic acid solution was added dropwise over 1 h. After the addition was completed, the reaction was continued at 60°C for an additional 3 hours. After returning to room temperature, the solution was neutralized with hydrochloric acid to pH = 7, dialyzed in pure water with a MW1K dialysis bag for 24 h, and freeze-dried to obtain CCD. Samples were taken for thin layer chromatography (TLC) analysis, and the CCD was obtained by 1 H NMR was used for further characterization. Figure 1 .in Figure 1 A describes the specific chemical structure of CCD, Figure 1 B simulates the molecular recognition and capture process of diquat molecules by CCD; Figure 1 C is the two-dimensional NMR spectrum of the CCD molecule after complexing with diquat in aqueous solution, which confirms the obvious inclusion result of CCD and diquat. Figure 1 D characterizes the chemical shift data of each H atom in the one-dimensional nuclear magnetic hydrogen spectrum before and after the inclusion of CCD and diquat. It can be clearly seen that the H-5 on the CCD has the largest shift, which proves the successful recombination of CCD and diquat.

[0030] (2) Construction of rotaxane CCD (100 equivalents) and PEG 5K (1 equivalent) were directly dispersed in distilled water (10 ml), vortexed and sonicated at room temperature for 30 min, then heated to 60 °C and maintained at this temperature for 8 days. The resulting suspension was then treated with rapid centrifugal ultrafiltration to remove unpenetrated CCD to obtain the rotaxane suspension CCD@PEG.

[0031] The simulation diagram of the continuous process of PEG inserting CCD into the rotaxane structure and then being wrapped into nanofibers by TK-PEI is shown in Figure 2. Figure 3 As shown in A; the compatibility simulation diagram of the PEG axis and the CCD shuttle on the spatial scale under molecular simulation is shown in Figure 3 B; 2D NMR ROESY spectrum of CCD@PEG in aqueous solution is shown in Figure 3 C; for detailed test results, see Figure 3 A~3F. Figure 3 A describes the sequential process of PEG penetrating CCD into the rotaxane structure and then being wrapped into nanofibers by TK-PEI; Figure 3 B) The compatibility of the PEG axis and the CCD shuttle on a spatial scale was demonstrated by molecular simulation, demonstrating its theoretical feasibility. Figure 3 C depicts the 2D NMR ROESY spectrum of CCD@PEG in aqueous solution. The HH correlation results confirm that PEG can be well strung into the CCD cavity. Figure 3 In D, CCD (30 equivalents) was added to the aqueous PEG solution. 1 The intensity of each peak in the H NMR changes with time, and Figure 3 E) The corresponding quantitative results (taking the peak of CCD at 3.65 ppm as an example) confirm that CCD can have a good complex effect with PEG; Figure 3 F lists the single CCD peak and CCD@PEG in aqueous solution 1 The change of H NMR chemical shift proves the good intercalation complex between CCD and PEG to form nanowires.

[0032] (3) Preparation of TC@PEG complex Polyethylene glycol amine was cross-linked with reactive oxygen species (ROS)-responsive thioketal, with a mass ratio of polyethyleneimine to thioketal of 5:1, and then TK-PEI was purified by dialysis. TK-PEI was added to the CCD@PEG suspension, with a mass volume ratio of TK-PEI to CCD@PEG suspension of 1:1, mg / mL, and stirred at 100 rpm for 30 minutes at 25°C to promote coating and encapsulation. The suspension was then treated again with rapid centrifugal ultrafiltration to remove unreacted TK-PEI to obtain the TC@PEG complex. After the TC@PEG aqueous solution was vacuum dried and gold-plated, the microscopic morphology observed by SEM was as follows Figure 3 H, for detailed test results and morphological characterization, see Figure 3 G~3J.

[0033] (4) Macrophage-loaded capture agent Mouse macrophage Raw264.7 cell line (density: 1 million per dish) was cultured in a cell culture dish, the cell culture medium was removed, and the cells were washed with serum-free medium. TC@PEG was introduced into the cells and the incubation period began. After 2 hours of incubation, the cells were washed with PBS buffer to remove the uninternalized TC@PEG to obtain the TP@M intermediate state; then 4-OI (octyl itaconate) was added and DMSO was used to assist in dissolution. The cells were incubated for 0.5 hours. The ratio of TC@PEG, 4-OI to cells was 10 mg: 1 mg: 10 mL; the cells were washed with PBS buffer to remove the uninternalized 4-OI to obtain the final in vivo capture agent O-TCP@M.

[0034] When testing the loading efficiency of macrophages, TC@PEG was replaced with TC@PEG-Cy5, and the rest of the steps were similar. A microplate reader was used to determine the incubation time and phagocytic efficiency of macrophages with TC@PEG-Cy5. A microplate reader was used to measure the fluorescence intensity of Cy5 at specific excitation and emission wavelengths, which confirmed that TC@PEG-Cy5 can be well internalized by macrophages. For specific concentration-dependent test results and macrophage internalization process, please refer to Figure 3 T~3V.

[0035] Test Example 1 Effect of CCD on reducing toxicity of diquat In aqueous solution, the redox potential of diquat before and after CCD encapsulation (same concentration, 1 mM) was tested by electrochemical workstation-cyclic voltammetry, and the curves were recorded and compared respectively; the time dependence of diquat on ROS production of HEK293 cells was measured by flow cytometry; and the time-dependent ROS production of diquat was measured by microplate reader. HEK293 cells were cultured to 80%-90% density. The cells were divided into experimental group and control group. Different concentrations of diquat were added to the experimental group, and the control group was treated accordingly. For flow cytometer measurement, DCFH-DA was used as a probe, and a staining solution of appropriate concentration was prepared. The cells were incubated with the staining solution for 20-30 minutes, and the mixture was mixed every 3-5 minutes. For microplate reader measurement, DCFH-DA was also used, and similar operations were performed. The cells were digested with trypsin, and the cell suspension was collected after centrifugation. The cells were resuspended in PBS and tested on the machine. The excitation wavelength of the flow cytometer was set to 488nm and the emission wavelength was set to 525nm. The treated cell suspension was returned to the 96-well plate. The corresponding excitation and emission wavelengths were set on the microplate reader to read the fluorescence intensity. Data at different time points were collected to analyze the changes in ROS production over time and in concentration dependence. For details on the mechanism and experimental results, see Figure 2 .in Figure 2 A describes the mechanism by which CCD reduces the ability of diquat to produce reactive oxygen species (ROS) through a charge neutralization mechanism; Figure 2 B. Electrochemical voltammetry curves revealed that the mechanism of reduced toxicity of diquat@CCD is the change of redox potential; Figure 2 C~2D Flow cytometry revealed the concentration dependence of diquat on ROS production in HEK293 cells; Figure 2 E~2F used flow cytometry to reveal the time dependence of diquat on ROS production in HEK293 cells; Figure 2 G was analyzed by microplate reader (5 × 10 ^6 HEK293 cells) revealed that diquat produced ROS in a time-dependent manner; Figure 2 H~2I shows the effect of different ratios of CCD in reducing ROS levels in HEK293 cells after 1 h of intoxication; Figure 2 J described that the treatment of HEK293 cells with CCD pre-encapsulated with diquat at different ratios could significantly reduce the level of ROS produced; Figure 2 K~2L expressed the effect of different ratios of CCD in significantly reducing ROS levels in HEK293 cells after 12 hours of poisoning; Figure 2 M reveals the survival rate of HEK293 cells in the presence of different concentrations of diquat; Figure 2 N describes the increased survival of HEK293 cells after 1 and 12 h of treatment with 2 equivalents of CCD; Figure 2O depicts the increase in HEK293 cell viability with CCD ratio at increasing concentrations after 48 h exposure to diquat; Figure 2 P~2U respectively describe the liver and kidney indicators of mice injected with diquat and diquat@CCD (2 equivalents) at the in vivo level, including ALT, AST, ALB, ALP, BUN and CREA. The above experimental cases and experimental results all show that CCD can significantly reduce the toxicity of diquat by reducing the production of ROS.

[0036] Test Example 2 ROS response process A 5 μg / mL diquat solution was prepared in a phosphate buffered saline (PBS) solution (10 mM) at a pH of 7.4, and different dialysis conditions were added, including 1 equivalent and 10 equivalents of CCD, 10 equivalents of CCD rotaxane, and 10 equivalents of CCD rotaxane with continuous supplementation of hydrogen peroxide. Diquat was determined by UV-visible spectrometry. The changes in UV absorption outside the dialysis bag (200-400 nm, at room temperature) were monitored; the UV absorption value at the maximum absorption wavelength (311 nm) was plotted as a curve. For detailed experimental results and microscopic morphological changes, see Figure 3 K~3P. Figure 3 K depicts the mechanism of ROS-induced TK-PEI dissociation and CCD shedding from the PEG shaft for diquat capture in the in situ supramolecular displacement model; Figure 3 L depicts the effect of TK-PEI (1 equivalent) capturing diquat over time in a dialysis bag (1K MW) in the presence or absence of ROS. Figure 3 M represents the capture of diquat in poisoned HEK293 cells by different ratios of TK-PEI (1 equivalent and 10 equivalent) in a dialysis bag (1K MW) over time in the presence or absence of ROS; The microscopic morphology of TC@PEG aqueous solution at different time points of ROS treatment under TEM observation: Figure 3 M describes the effect of different ratios of TK-PEI (1 equivalent and 10 equivalents) in capturing diquat in poisoned HEK293 cells over time in a dialysis bag (1K MW) in the presence or absence of ROS; The microscopic morphology of TC@PEG aqueous solution at different time points of ROS treatment under TEM observation: Figure 3 N: 0 hours, Figure 3 O: 1 hour and Figure 3 P: 2 hours. The above results all indicate that the constructed rotaxane system can activate and release active CCD in the presence of ROS, achieving effective capture of diquat.

[0037] Test Example 3 In vivo distribution study process A diquat solution was prepared in 0.9% saline and an acute poisoning model was established at a dose of 30 mg / kg body weight. Treatment began one day after poisoning, and the in vivo distribution was observed at 24 hours, 12 hours, and 4 hours. Each mouse was injected with 1 million macrophages loaded with TC@PEG-Cy5 and TC@PEG-Cy5 alone before normalizing the fluorescence intensity. The in vivo distribution was observed using IVIS. At multiple time points after intravenous injection, the mice were imaged by IVIS with an exposure time set to 15 seconds. Prior to imaging of ex vivo organs, the mice were anesthetized, perfused with saline, and then killed. Major organs and tumors were removed from the mice at multiple time points, rinsed with saline, and in vitro images were obtained using IVIS. For detailed experimental results, see Figure 4 A. The experimental results show that the entire carrier capture system can be effectively enriched in the inflammatory damaged kidney area.

[0038] The distribution pattern is described by semi-quantitatively measuring the fluorescence intensity within the region of interest (ROI). Figure 4 B~4E. For tissue observation, mice were anesthetized and perfused with saline or 4% formaldehyde solution before being sacrificed. Organs were removed, fixed in 4% formaldehyde for 12 hours, embedded in OCT compound at -80°C, and cut into 20-micron-thick sections using a freezing microtome. The sections were then stained with DAPI and 8-OHG and visualized under a fluorescence microscope. For specific experimental images and corresponding semi-quantitative results, see Figure 4 F~4H. The experimental results showed that the entire carrier capture system had obvious co-localization with the site of renal inflammatory damage.

[0039] Test Example 4 Application process of diquat in vivo After administration of high and low doses of diquat (30 mg / kg body weight), different treatment strategies were applied starting 3 days after intoxication. During this period, survival curves, body weight changes, and total water intake were monitored. Behavioral tests were performed on days 4 and 15, strictly following animal ethics guidelines (see the detailed experimental procedures for details). Figure 5 A. ). On day 25, all rodents were euthanized for animal welfare reasons. In addition, a new group was established to evaluate the extent of kidney damage using immunohistochemistry and measure the spleen index on day 15 after treatment. Blood samples were collected to test liver and kidney function indicators. The safety assessment method was as follows: After administration of therapeutic doses of different dosage forms, mice were euthanized. Sections of major organs (i.e., heart, liver, spleen, lungs, and kidneys) were prepared and stained with hematoxylin and eosin. The stained sections were then observed and captured using an inverted fluorescence microscope (Leica, Wetzlar, Germany). The survival rate (see for details) was Figure 5 B), weight changes (see Figure 5 C), and the total water intake of each group of animals after poisoning and treatment (see Figure 5 D); spleen index after 15 days of treatment (see Figure 5 E), and behavioral assessment results, including the number of face wipes (see Figure 5 F), number of standing times (see Figure 5 G), the number of times you cross the grid (see Figure 5 H), and movement time (see Figure 5 I)); liver and kidney function indicators after 15 days of treatment (including BUN (see Figure 5 J), CREA (see Figure 5 K), UA (see Figure 5 L), ALT (see Figure 5 M), AST (see Figure 5 N), total bilirubin T-BIL (see Figure 5 O), albumin ALB (see Figure 5 P), alkaline phosphatase ALP (see Figure 5 Q), γ-glutamyl transpeptidase GGT (see Figure 5 R), and total bile acid TBA (see Figure 5 S)); kidney tissue pathological section after 15 days of treatment (see Figure 5 T), Caspase-3 (see Figure 5 U) and Ki-67 (see Figure 5 The experimental results show that the entire carrier capture system can effectively capture diquat in vivo and reduce damage to the liver and kidneys.

Claims

1. A method for preparing an in vivo capture agent for diquat molecules, characterized in that: The preparation method comprises the following steps: (1) Synthesis of carboxymethyl α-cyclodextrin (CCD): α-cyclodextrin was added to a sodium hydroxide aqueous solution under stirring to obtain solution A; chloroacetic acid was dissolved in water to obtain a chloroacetic acid solution; solution A was heated to 60°C, and the chloroacetic acid solution was added dropwise to solution A at a controlled rate, and the reaction was continued at 60°C for 3 hours after the addition was completed; after returning to room temperature, the solution was neutralized with hydrochloric acid to a pH of 7, and dialyzed in pure water with a MW1K dialysis bag for 24 hours, and freeze-dried to obtain CCD; (2) Construction of rotaxane: CCD and chain polyethylene glycol 5K (PEG 5K) were directly dispersed in distilled water, vortexed and ultrasonically treated at room temperature, and then heated to 60°C for insulation. The rotaxane suspension CCD@PEG was obtained by centrifugal ultrafiltration. (3) Preparation of cross-linked polyethyleneimine encapsulated rotaxane complex (TC@PEG): cross-linking polyethyleneimine (MW 1.2K) with reactive oxygen species (ROS)-responsive thioketal (sulfonic acid-NHS-TK-sulfonic acid-NHS) was performed, and TK-PEI was obtained after purification by dialysis; TK-PEI was added to the rotaxane suspension CCD@PEG obtained in step (2), stirred at 25°C, and subjected to rapid centrifugal ultrafiltration to obtain cross-linked polyethyleneimine encapsulated rotaxane complex TC@PEG; (4) Macrophage loading capture agent: Mouse macrophage Raw264.7 cell line was cultured in a cell culture dish (density: 1 million per dish), the cell culture medium was removed, and the cells were washed with serum-free medium. TC@PEG was introduced into the cells. After incubation for 2 h, the cells were washed with PBS buffer to obtain the TP@M intermediate state. Then, 4-OI (octyl itaconate) was added and dissolved with DMSO. The cells were incubated for 0.5 h and washed with PBS buffer to obtain the final in vivo capture agent O-TCP@M.

2. The preparation method according to claim 1, characterized in that In the step (1), the concentration of sodium hydroxide is 1.5 M, the concentration of α-cyclodextrin is 0.05 g / mL, and the concentration of chloroacetic acid is 0.1 g / mL.

3. The preparation method according to claim 1, characterized in that: The time for adding the chloroacetic acid solution in step (1) is 1 hour.

4. The preparation method according to claim 1, characterized in that In the step (2), the mass ratio of CCD to PEG 5K is 3:

1.

5. The preparation method according to claim 1, characterized in that: In the step (2), the ultrasonic treatment time is 30 minutes and the insulation time is 3 to 8 days.

6. The preparation method according to claim 1, characterized in that: In the step (3), the mass ratio of polyethyleneimine to thioketal is 5:1; the mass volume ratio of TK-PEI to CCD@PEG suspension is 1:1, mg / mL.

7. The preparation method according to claim 1, characterized in that In the step (3), the stirring speed is 100 rpm and the stirring time is 30 minutes.

8. The preparation method according to claim 1, characterized in that: In the step (4), the ratio of TC@PEG, 4-OI and cells is 10 mg: 1 mg: 10 mL.

9. The in vivo capture agent O-TCP@M obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the in vivo capture agent O-TCP@M as claimed in claim 9 in the preparation of a drug for intervening in poisoning caused by diquat and reducing kidney damage.

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