In vivo capture agent for diquat molecules, preparation method and application thereof

Abstract

This invention belongs to the field of biotechnology and specifically relates to an in vivo capture agent for diquat molecules, as well as its preparation method and application. The invention synthesizes carboxymethyl α-cyclodextrin, constructs a rotaxane, prepares a cross-linked polyethyleneimine-encapsulated rotaxane complex, and finally uses macrophages for loading to obtain the in vivo capture agent O-TCP@M. The invention utilizes carboxymethyl α-cyclodextrin, which is highly structurally compatible with diquat, to achieve precise recognition and binding of toxin molecules. The positively charged TK-PEI dendrimers are then used for encapsulation, enhancing structural stability and ROS responsiveness. This invention achieves precise in vivo capture and inflammation regulation, alleviating poisoning symptoms while also protecting kidney function and reducing tissue damage by precisely capturing and neutralizing toxins.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an in vivo capture agent for diquat molecules, a preparation method and an 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 generating large amounts of cellular reactive oxygen species (ROS). In addition, under physiological conditions, diquat can also directly react with oxygen to produce ROS, with an induction capacity 10 to 40 times that of paraquat, directly damaging cellular proteins, lipids, nucleic acids, and other biomacromolecules. It 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, a significant increase in blood creatinine and urea nitrogen, ataxia caused by central nervous system toxicity, and renal failure as the main cause of death.

[0003] Currently, intervention options for poisonings, including diquat, are limited to symptomatic treatment, including diuresis, mechanical ventilation, and blood purification according to current standard guidelines. However, it is important to note that toxins often accumulate persistently in specific organs, and blood concentrations are often low, resulting in inefficient blood clearance. Ultimately, elimination of toxins from the body relies primarily on the patient's own ability to metabolize and excrete them. However, patients often suffer from liver and kidney dysfunction, further reducing toxin processing capacity and creating a vicious cycle of poisoning. Even if a toxin-capturing system is discovered in vitro, direct application in vivo is limited by the capture agent's inherent distribution (the capture agent's distribution in vivo is often inconsistent with the toxin, and the capture agent may also be rapidly cleared in the body, further reducing its availability), making it difficult for the capture agent to serve as an in vivo detoxifier. For example, the carboxyl-modified pillararene WP6 is known to have potent paraquat-capturing and ROS-reducing capabilities in vitro and in cells, but its in vivo application faces challenges. To date, no specific toxin-capturing strategy targeting the target organ of poisoning has been developed in the clinical setting, or even in basic research. All of this significantly reduces the potential for urgent 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 target organs due to competition with freely diffusing analogs. Utilizing in situ supramolecular displacement and activation strategies may help address this dilemma. Specifically, the active compartment of the antidote should be temporarily occupied by a substance, rendering it unresponsive to other analogs in vivo. Then, when faced with the specific microenvironment of the lesion site where action is required, the temporary binding complex dissociates according to a designed stimuli-responsive dissociation, restoring the compartment's ability to capture the toxin.

[0005] The inflammatory response that occurs after poisoning is the body's natural mechanism for combating foreign invasion. During this process, macrophages are attracted to the site of inflammation through a complex series of signaling and cell migration processes. This actually provides a targeted solution strategy for the delivery of antidotes to the site of toxin accumulation.

[0006] In clinical practice, specific antidotes are available for many types of acute poisoning (for example, neutralizing antibodies in antivenom and anticholinergics for organophosphate poisoning). However, some widely and heavily used substances, such as paraquat and diquat, lack effective, specific, and consistent antidotes. There are two fundamental reasons why these substances lack antidotes: first, they are rapidly distributed after entering the body and concentrate in target organs, making their elimination difficult through methods such as hemodialysis or gastric lavage. Second, these molecules have very unique structures, often with a strong positive charge. If an antidote with a strongly negatively charged structure is matched to them, the body will clear them prematurely.

[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, a key anti-inflammatory metabolite, is most abundant in macrophages, reaching levels as high as 5 mM in mouse bone marrow-derived macrophages after LPS stimulation. Itaconate is essential for activation of the anti-inflammatory transcription factor Nrf2 in both mouse and human macrophages: it alkylates cysteine ​​residues on the protein KEAP1, thereby enhancing Nrf2's expression of antioxidant and anti-inflammatory genes. Through Nrf2, itaconate limits inflammation and modulates the effects of type I interferons. Structurally, itaconate is anionic and hydrophilic, making 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 it can act as a direct replacement for itaconate. Furthermore, 4-OI has a stronger ability to upregulate Nrf2 under ROS conditions. Reports indicate that the highest concentration of exogenous 4-OI treatment without compromising cellular activity can reach 45 pmol / mg of cellular protein.

[0008] The mechanism of diquat poisoning is that its unique molecular structure triggers cellular reactive oxygen species (ROS) damage, primarily targeting the kidneys, leading to acute kidney injury. Currently, there are no specific guidelines for the management of diquat poisoning, nor are there specific in vivo capture antidotes specifically targeting diquat. Therefore, intervention strategies are often derived from methods used to treat sepsis. Therefore, urgent challenges remain: 1) identifying or constructing an in vivo capture antidote specifically targeting diquat; 2) delivering the antidote to the target organ (i.e., the kidneys) to accurately capture the toxin and reduce its ability to generate ROS; and 3) minimizing the resulting damage and protecting renal 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, a preparation method, and an application thereof. The technical solutions of the present invention are as follows:

[0010] A method for preparing an in vivo capture agent for diquat molecules comprises the following steps:

[0011] (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;

[0012] (2) Construction of rotaxane: CCD and chain polyethylene glycol 5K (PEG 5K) were directly dispersed in distilled water, vortex-mixed and ultrasonically treated at room temperature, and then heated to 60°C for insulation. The rotaxane suspension CCD@PEG was obtained by centrifugal ultrafiltration.

[0013] (3) Preparation of cross-linked polyethyleneimine encapsulated rotaxane complex (TC@PEG): Polyethyleneimine (MW 1.2K) was cross-linked with reactive oxygen species (ROS)-responsive thioketal (sulfonic acid-NHS-TK-sulfonic acid-NHS), 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;

[0014] (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, incubated for 2 h, and then washed with PBS buffer to obtain the TP@M intermediate state; then 4-OI (octyl itaconate) was added, DMSO-assisted dissolution was performed, incubated for 0.5 h, and washed with PBS buffer to obtain the final in vivo capture agent O-TCP@M.

[0015] 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.

[0016] Preferably, the time for adding the chloroacetic acid solution in step (1) is 1 hour.

[0017] 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.

[0018] Preferably, in step (3), the mass ratio of polyethyleneimine to thioketal is 5:1.

[0019] Preferably, in step (3), the mass volume ratio of TK-PEI to CCD@PEG suspension is 1:1, mg / mL.

[0020] Preferably, in step (3), the stirring speed is 100 rpm and the stirring time is 30 minutes.

[0021] Preferably, in step (4), the ratio of TC@PEG, 4-OI to cells is 10 mg:1 mg:10 mL.

[0022] 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.

[0023] In this invention, carboxymethyl-α-cyclodextrin (CMD) is used as a specific antidote for diquat. It is strung onto a PEG axle to form a rotaxane structure. The structure is then coated with a low-molecular-weight polyethylene ether (PEI) cross-linked by TK bonds via electrostatic attraction for further stabilization and ROS-responsive dissociation, resulting in a specialized nanofiber. These nanofibers can be taken up by macrophages and further treated with 4-OI (4-OI activates Nrf2 by alkylating macrophage KEAP1, significantly enhancing its antioxidant and anti-inflammatory properties), completing a biomimetic antidote delivery system.

[0024] The present invention first precisely designs a dedicated in vivo capture agent based on 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 chemotaxis 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 to the body caused by the toxin.

[0025] Sugammadex® is a neuromuscular blocker approved by the FDA in 2015. 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 host-guest complex constant. Using molecular design principles, the present inventors initially designed, synthesized, and screened six cyclodextrin hosts (non-toxic and biodegradable cyclic polysaccharides) and conducted simulations. They found that the cavity size of α-cyclodextrin was optimal for complex formation with diquat, and that the carboxymethyl group was the best candidate for charge interaction with diquat. Through molecular design, dynamic simulations, and rational screening, the present inventors ultimately selected carboxymethyl α-cyclodextrin (CCD) as a specific antidote for diquat.

[0026] 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 2Therefore, the next specific task is to successfully achieve the efficient and precise enrichment of CCDs in areas of renal inflammatory damage. In situ molecular recognition and supramolecular self-assembly in vivo are crucial for the detoxification process. This temporarily occupies the CCD compartment, preventing early interactions with non-target molecules circulating in the blood (particularly cholesterol, which has a significant binding affinity for CCDs). Simultaneously, it blocks signals that have already been generated and developed, employing a dual approach to achieve detoxification and reduce inflammatory responses.

[0027] Rotaxanes, which can string together multiple cyclic molecules using an axis, are considered suitable for integrating "dispersed" host molecules into a single entity, making them suitable for in vivo delivery packages. Taking advantage of the cyclic nature of CCDs, we used PEG5K to string the CCDs together to form an integrated "molecular necklace." Subsequently, we coated the highly negatively charged "molecular necklace" with positively charged ROS-cleavable TK-PEI dendrimers, which were stacked together to form nanofibers. Due to the occupied cavity, the CCDs on the PEG temporarily lost their ability to recognize diquat. However, in the presence of ROS, local ROS cleaved and stripped the TK-PEI, allowing the CCDs to fall off the PEG and restore their ability to recognize and capture diquat.

[0028] Under in vitro culture conditions, the present invention allows macrophages to internalize the nanofibers. These internalized macrophages are then incubated with 4-OI, ultimately creating a rationally designed biomimetic toxin capture system. Once the cells reach the target site, the ROS-rich inflammatory environment triggers the release of the CCD from the PEG, making the cavity accessible and releasing 4-OI. This system can target the kidneys of poisoned mice, modulating renal inflammation and specifically releasing an antidote to ROS, ultimately reducing renal inflammation and ROS damage in the poisoned mice and improving their survival rate.

[0029] The technical features and beneficial effects of the present invention are as follows:

[0030] (1) This invention utilizes carboxymethyl α-cyclodextrin (CCD), which has a highly compatible molecular structure with diquat, to achieve precise recognition and binding of toxin molecules. Using PEG as the core, multiple CCD molecules are connected in series to form a "molecular necklace" and then encapsulated with positively charged TK-PEI dendrimers, enhancing structural stability and ROS responsiveness.

[0031] (2) The present invention utilizes the natural chemotaxis of macrophages to effectively transport the detoxification system to the inflammatory area, especially the kidney, thereby increasing 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 precise clearance 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 alleviate 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.

[0032] (3) The present invention achieves precise in vivo capture and inflammation regulation effects, which not only alleviates 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

[0033] Figure 1 For CCD thin layer chromatography (TLC) analysis and 1 H NMR further characterized; among them,

[0034] 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 Distortion of the H NMR peak; G) After adding common interfering ions in vivo, the diquat / CCD pair 1 H NMR peak deformation; H) Gaussian simulation was used to determine the steric compatibility between CCD and diquat at different angles;

[0035] Figure 2 is the change of ROS production with time and concentration dependence;

[0036] A) The mechanism by which CCD reduces the ability of diquat to generate reactive oxygen species (ROS) through charge neutralization; B) Different electrochemical voltammetry curves of diquat and diquat@CCD 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) Electrochemical voltammetry (ELISA) (5×10 ^6 (H) Quantitative results of measuring the time-dependent ROS production of diquat in HEK293 cells; 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 generated after treatment of 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) Viability trend of HEK293 cells over time in the presence of different concentrations of diquat; N) Viability of HEK293 cells after treatment with 2 equivalents of CCD for 1 hour and 12 hours; O) Viability 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 3 days after injection of diquat and diquat@CCD (2 equivalents);

[0037] Figure 3 The experimental results of the ROS response process and the incubation time and phagocytic efficiency of macrophages with TC@PEG-Cy5 are shown in Figure 2.

[0038] A) The continuous process of PEG penetrating CCD into the rotaxane structure and then being wrapped into nanofibers by TK-PEI; B) The compatibility of the 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 CCD peak alone and CCD@PEG in aqueous solution. 1H NMR chemical shift changes; G) C / H / N / S compositions of the intermediates and final products determined by elemental analysis, and the average precise composition ratio of TC@PEG was calculated; H) SEM micromorphology of TC@PEG aqueous solution after vacuum drying and gold coating; I-J) Specific length and diameter of TC@PEG determined by atomic force microscope (AFM); K) ROS-induced dissociation of TK-PEI and detachment of CCD from the PEG axis for diquat capture in an in situ supramolecular displacement model; L) TK-PEI (1 equivalent) captures diquat over time in a dialysis bag (1K MW) in the presence or absence of ROS; M) TK-PEI (1 equivalent and 10 equivalents) captures diquat in poisoned HEK293 cells over time in a dialysis bag (1K MW) in the presence or absence of ROS; TEM micromorphology of TC@PEG aqueous solution at different time points after ROS treatment: N) 0 hour, O) 1 hour and P) 2 hours; Q) Hemolysis of different formulations; R) Schematic diagram of the final capture system construction process: 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 HEK293 cell viability; T) Image of macrophages endocytosing nanofibers using bioprojection movies; U) Incubation time dependence of macrophage endocytosis of nanofibers; V) Incubation dose dependence of macrophage endocytosis of nanofibers;

[0039] Figure 4 This is the distribution test process in mice;

[0040] A) Distribution of Cy5-labeled TC@PEG and O-TCP@M 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 Cy5-labeled TC@PEG and O-TCP@M in kidney sections after accumulation and their colocalization with inflammatory sites at different time points, G) Local magnified images, and H) Corresponding semi-quantitative results, where arrows indicate nonspecific colocalized signals;

[0041] Figure 5 For the application process of diquat in vivo; wherein,

[0042] A) The establishment process of the animal poisoning model, the timeline of intervention treatment, and the subsequent arrangement of efficacy evaluation follow the realities and standards of clinical treatment; efficacy evaluation, including B) survival rate, C) body weight change, and D) total water consumption of animals in each group during poisoning and after treatment; E) spleen index and behavioral assessment results after 15 days of treatment (including F) number of face wipes, G) number of standing times, H) number of grid jumps, and I) movement 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 pathology sections after 15 days of treatment, and U) Caspase-3 and V) Ki-67 staining results. DETAILED DESCRIPTION

[0043] 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 that do not depart from the technical solution design of the present invention should be within the scope of protection of the rights of the present invention.

[0044] Example 1

[0045] Preparation process of in vivo capture agent

[0046] (1) Synthetic CCD

[0047] 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 hour. After the addition was complete, 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 hours, 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 1D 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.

[0048] (2) Construction of rotaxane

[0049] CCD (100 equivalents) and PEG 5K (1 equivalent) were directly dispersed in distilled water (10 ml), vortex-mixed, and sonicated at room temperature for 30 minutes. The resulting suspension was 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, yielding the rotaxane suspension CCD@PEG.

[0050] 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 the figure. Figure 3 As shown in A; the compatibility simulation diagram of the PEG axis and 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 subsequently being wrapped into nanofibers by TK-PEI; Figure 3 B) The compatibility of the PEG axis and the CCD shuttle on a spatial scale is demonstrated by molecular simulations, 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 inserted 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 H NMR changes with time, and Figure 3 E) The corresponding quantitative results (taking the CCD peak at 3.65 ppm as an example) confirm that CCD can form a good complex with PEG; Figure 3 F lists the peaks of single CCD and CCD@PEG in aqueous solution. 1 The change in H NMR chemical shift confirmed the good intercalation complexation between CCD and PEG to form nanowires.

[0051] (3) Preparation of TC@PEG complex

[0052] 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 micromorphology observed by SEM was as follows Figure 3 H, for detailed test results and morphological characterization, see Figure 3 G~3J.

[0053] (4) Macrophage-loaded capture agent

[0054] 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 and an incubation period was started. After 2 hours of incubation, the cells were washed with PBS buffer to remove uninternalized TC@PEG, obtaining the TP@M intermediate state. 4-OI (octyl itaconate) was then added and dissolved with the aid of DMSO. 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 uninternalized 4-OI, obtaining the final in vivo capture agent O-TCP@M.

[0055] When testing the macrophage loading efficiency, TC@PEG was replaced with TC@PEG-Cy5, and the remaining 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 detailed concentration-dependent test results and macrophage internalization process, please refer to Figure 3 T~3V.

[0056] Test Example 1

[0057] Effect of CCD on reducing the toxicity of diquat

[0058] In aqueous solution, the redox potential of diquat (at the same concentration, 1 mM) was measured using an electrochemical workstation-based cyclic voltammetry method before and after CCD coating. The curves were recorded and compared. The time-dependent effect of diquat on ROS production in HEK293 cells was measured using flow cytometry and a microplate reader. HEK293 cells were cultured to 80%-90% confluence. The cells were divided into experimental and control groups. The experimental groups were treated with varying concentrations of diquat, while the control group received the corresponding treatments. For flow cytometric measurements, DCFH-DA was used as the probe. A staining solution of appropriate concentration was prepared and the cells were incubated with the staining solution for 20-30 minutes, mixing by inversion every 3-5 minutes. For microplate reader measurements, DCFH-DA was also used, and similar procedures were followed. Cells were trypsinized, and the cell suspension was collected after centrifugation. The cells were resuspended in PBS and analyzed. The flow cytometer was set to an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The treated cell suspension was returned to the 96-well plate. The corresponding excitation and emission wavelengths were set on the microplate reader and the fluorescence intensity was read. Data at different time points were collected to analyze the changes in ROS production over time and in a concentration-dependent manner. 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 used flow cytometry to reveal 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 expresses the effect of different ratios of CCD in significantly reducing ROS levels in HEK293 cells after 12 hours of intoxication; Figure 2 M reveals the survival rate of HEK293 cells in the presence of different concentrations of diquat; Figure 2 N describes the enhanced viability of HEK293 cells treated with 2 equivalents of CCD for 1 and 12 h; Figure 2O depicts the increase in HEK293 cell viability following 48 h of diquat exposure with increasing CCD ratio at increasing concentrations; Figure 2 P~2U respectively describe the liver and kidney parameters, including ALT, AST, ALB, ALP, BUN, and CREA, in mice three days after injection with diquat and diquat@CCD (2 equivalents). These experimental examples and results demonstrate that CCD significantly reduces diquat toxicity by reducing ROS production.

[0059] Test Example 2

[0060] ROS response process

[0061] 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 replenishment 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. Detailed experimental results and micromorphological changes are detailed in [1]. 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 equivalents) 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.

[0062] Test Example 3

[0063] In vivo distribution study process

[0064] 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. Before imaging the organs in vitro, the mice were anesthetized, perfused with saline, and then killed. At multiple time points, the main organs and tumors were removed from the mice, rinsed with saline, and in vitro images were obtained using IVIS. For detailed experimental results, see Figure 4 A. Experimental results show that the entire carrier capture system can be effectively enriched in the inflammatory damaged kidney area.

[0065] The distribution pattern was 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-μm-thick sections using a freezing microtome. The sections were then stained with DAPI and 8-OHG and visualized under a fluorescence microscope. For detailed 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.

[0066] Test Example 4

[0067] The application process of diquat in vivo

[0068] After administration of high and low doses of diquat (30 mg / kg body weight), different treatment strategies were implemented starting 3 days after intoxication. During this period, survival curves, body weight changes, and total water intake were monitored. Behavioral testing was performed on days 4 and 15, strictly adhering to animal ethics guidelines (detailed experimental procedures are available in the literature). Figure 5A. ). 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 is 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 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 carboxymethyl α-cyclodextrin CCD; (2) Construction of rotaxane: CCD and chain polyethylene glycol 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): polyethyleneimine with active oxygen responsive sulfonic acid-NHS-TK-sulfonic acid-NHS cross-linking reaction, wherein the polyethyleneimine has a molecular weight of 1200 and is purified by dialysis to obtain TK-PEI; TK-PEI is 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-loaded capture agent: Mouse macrophage Raw264.7 cell line was cultured in a cell culture dish at a density of 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, incubated for 2 h, and then washed with PBS buffer to obtain the TP@M intermediate state; then octyl itaconate was added, DMSO-assisted dissolution was performed, 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, wherein 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.

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 step (2), the mass ratio of CCD to polyethylene glycol 5K is 3:

1.

5. The preparation method according to claim 1, characterized in that In 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 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, wherein In 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 step (4), the ratio of TC@PEG, octyl itaconate, 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 according to claim 9 in the preparation of a drug for intervening in poisoning caused by diquat and reducing kidney damage.

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