Dual-load and dual-target nano material for treating acute kidney injury as well as preparation method and application of dual-load and dual-target nano material
By constructing dual-load and dual-targeted nanomaterials, the adverse reactions of traditional means and the lack of fundamental treatment in the treatment of acute renal injury are solved, and the co-delivery of drugs and layered responses are achieved to achieve efficient repair of renal injury.
Patent Information
- Application Number
- CN202510094994.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-06-03
AI Technical Summary
The prior art cannot effectively treat acute renal injury. Traditional treatment methods are prone to trigger adverse reactions in humans and lack fundamental treatment capabilities, which cannot reverse the damage to renal tissue.
Dual-loaded and dual-targeted nanomaterials were constructed through nanoprecipitation and coupling methods to achieve co-delivery of the two drugs and intracellular layered response capabilities. The specific steps include using PLGA-PEG amphiphilic block copolymer as assembly motifs, preparing TP@MNPs, and modifying drugs and antibodies through amide reactions to form dual-loaded and dual-targeted nanomaterials.
The stratified response and graded drug release of drugs in cells is achieved, and the synergy between the two drugs is fully utilized, the effective dose of each drug is reduced, and the effective repair of the lesions is achieved, and the renal precision targeting is achieved.
Smart Images
Figure CN120078788A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of pharmaceutical materials, and particularly relates to a dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury, and a preparation method and application thereof. Background Art
[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function within a short period of time caused by various etiologies. It is manifested as a rapid impairment of renal function in a short time, accompanied by a high hospitalization rate and mortality rate. At present, the main treatment methods for AKI are to use free drugs such as diuretics, antibiotics, and vasodilators to improve the hemodynamics or local inflammation of patients. However, this traditional treatment method is prone to cause adverse reactions in the human body and lacks fundamental treatment ability, and cannot reverse the damage of renal tissue.
[0003] The nano-drug delivery system (NDDS) is a new type of nano-drug developed based on nanomaterials. It can increase the solubility of traditional drugs, change the in vivo distribution of traditional drugs, and improve the targeting of traditional drugs, thereby improving the therapeutic effect and reducing the incidence of adverse reactions. Therefore, it has become the research focus of efficient therapies for various diseases. NDDS can achieve in vivo transportation across biological barriers through multiple mechanisms and finally localize in the target organ or cell. In the targeting process of NDDS, the targeting method combining passive targeting and active targeting can optimize the drug delivery at the lesion site and minimize the non-target drug distribution.
[0004] The multi-drug co-delivery nanocarrier is a combined treatment method that applies a synergistic drug delivery strategy and synergistically improves the therapeutic effect by leveraging the advantages of different therapies. This method can achieve efficient repair of the lesion while reducing the drug dose.
[0005] At present, there is no multi-drug co-delivery system for the efficient treatment of acute kidney injury. Summary of the Invention
[0006] Aiming at the above-mentioned shortcomings and deficiencies of the prior art, the present invention aims to provide a dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury, and a preparation method and application thereof; the present invention constructs a nanomaterial with long-range dual targeting, two-drug co-delivery, and intracellular hierarchical response capabilities through the nanoprecipitation method and the coupling method.
[0007] The object of the present invention is achieved by the following technical solutions:
[0008] The present invention provides a preparation method for a dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury, comprising the following steps:
[0009] (1) Under stirring conditions, a solution of triptolide and PLGA-PEG in an organic solvent was dropped into an aqueous solution of Poloxamer 188, and the organic solvent was evaporated by stirring, followed by centrifugation to obtain TP@MNPs;
[0010] (2) After the carboxyl group of captopril was activated, it reacted with NH 2 -PEG-NH 2 to obtain Cap-PEG-NH 2 ;
[0011] (3) After the carboxyl group of the GFLG polypeptide was activated, it reacted with Cap-PEG-NH 2 to obtain Cap-PEG-GFLG;
[0012] (4) After the carboxyl group of the Nephrin antibody was activated, it reacted with Cap-PEG-GFLG to obtain Cap-Nephrin;
[0013] (5) After the carboxyl group of TP@MNPs was activated, it reacted with Cap-Nephrin to obtain a dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury.
[0014] Furthermore, the organic solvent in step (1) is acetonitrile.
[0015] Furthermore, the preparation method of PLGA-PEG in step (1) includes the following steps:
[0016] Polylactic acid-glycolic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and polyethylene glycol reacted in anhydrous chloroform, and were precipitated and centrifuged to obtain PLGA-PEG.
[0017] Preferably, the precipitation and centrifugation is to add an ether / methanol mixture for precipitation and then centrifugal separation;
[0018] More preferably, the volume ratio of ether to methanol in the ether / methanol mixture is 1:1 - 1:3.
[0019] Furthermore, the carboxyl group activation of captopril in step (2) is obtained by the activation reaction of captopril with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution;
[0020] Preferably, the activation reaction time is 7 - 11 h, and the buffer solution is phosphate buffer solution.
[0021] Furthermore, the carboxyl group activation of the GFLG polypeptide in step (3) is obtained by the activation reaction of the GFLG polypeptide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution;
[0022] Preferably, the activation reaction time is 7 - 11 h, the buffer solution is phosphate buffer solution, and the pH of the buffer solution is 6.4 - 8.4;
[0023] Furthermore, the reaction time in step (3) is 10 - 14 h.
[0024] Furthermore, the carboxyl activation of Nephrin antibody in step (4) is obtained by the activation reaction of Nephrin antibody with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution;
[0025] Preferably, the activation reaction time is 1 - 5 h, the buffer solution is phosphate buffer solution, and the pH of the buffer solution is 4.8 - 6.8;
[0026] Furthermore, the reaction temperature in step (4) is room temperature and the time is 10 - 14 h;
[0027] Furthermore, the carboxyl activation of TP@MNPs in step (5) is obtained by the activation reaction of TP@MNPs with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution;
[0028] Preferably, the activation reaction time is 13 - 17 min, and the buffer solution is phosphate buffer solution;
[0029] Furthermore, the reaction in step (5) is carried out in a buffer solution, the pH of the buffer solution is 5 - 9; the reaction temperature is room temperature and the time is 1 - 4 h.
[0030] The present invention provides a dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury prepared by the above preparation method.
[0031] The present invention provides an application of the above dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury in the preparation of drugs for the treatment of acute kidney injury.
[0032] The present invention uses PLGA-PEG amphiphilic block copolymer as an assembly unit, and prepares TP@MNPs by the method of nanoprecipitation. Based on the hydrophobic interaction between the PLGA segment in PLGA-PEG and TP, TP is encapsulated inside the obtained MNPs. Cap is a hydrophilic drug with active -COOH, so it can rely on the -COOH in the drug and NH 2 -PEG-NH 2 in -NH 2The amide reaction was used to modify it on PEG to obtain a PEG-drug conjugate. Since the GFLG polypeptide has a structure with active -COOH at both ends, it can also be grafted to the PEG-drug conjugate through the formation of an amide bond to form a GFLG-PEG-drug conjugate. Finally, the above conjugate can be modified onto an antibody (i.e., Nephrin) to obtain an antibody-drug conjugate (Cap-Nephrin). Cap-Nephrin continued to react with TP@MNPs to obtain a dual-loaded and dual-targeted nanomaterial (Dualnanocarrier) for the treatment of acute kidney injury.
[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0034] (1) The dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury prepared by the present invention can achieve the co-delivery of two drugs and enable hierarchical response and graded drug release in the intracellular environment, giving full play to the synergistic effect of the two loaded drugs, while reducing the effective dose of each drug, so that the drug dose is safe and efficient repair of the lesion can be achieved.
[0035] (2) The dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury prepared by the present invention has renal precise targeting. Description of the Drawings
[0036] Figure 1 It is a scanning electron microscope (SEM) image of the dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury prepared in Example 1.
[0037] Figure 2 It is a scanning electron microscope (SEM) image of TP@MNPs prepared in Example 2.
[0038] Figure 3 It is a scanning electron microscope (SEM) image of MNPs-Cap prepared in Example 3.
[0039] Figure 4 It is a line graph of the encapsulation rate and drug loading rate of TP and Cap in the dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury in Example 4.
[0040] Figure 5 It is a fluorescence imaging map of 4-μm thick frozen sections of the kidney 24 h after intravenous injection of Cy7-labeled TP@MNPs and Cy7-labeled dual-loaded and dual-targeted nanomaterials (Dual nanocarriers) in Example 5.
[0041] Figure 6 It is a uACR expression map of PAN model mice intervened with different materials in Example 6.
[0042] Figure 7 Expression diagrams of TC, TG, and ALB in PAN model mice intervened with different materials in Example 6. Detailed implementation manners
[0043] The following further illustrates the specific implementation of the present invention in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. Reagents or instruments without indicating the manufacturer are regarded as conventional products that can be obtained through commercial purchase.
[0044] Example 1
[0045] A preparation method of a dual-loaded and dual-targeted nanomaterial for the treatment of acute kidney injury, comprising the following steps:
[0046] First, PLGA (polylactic acid-glycolic acid), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and PEG (polyethylene glycol) are mixed and stirred in anhydrous chloroform; after reacting for 12 h, 15 mL of an ether / methanol mixture (v / v 1:1, 0 °C) is added to the aforementioned mixture to precipitate the product, and the lower-layer precipitate is collected by centrifugation (10,000 rpm, 15 min). Subsequently, the precipitate is dissolved in acetonitrile and then an ether-methanol mixture is added to re-precipitate the product, and the product is collected by centrifugation 3 times (10,000 rpm, 15 min). Then, the product is placed in a vacuum drying oven (35 °C), and after 2 h, the product (PLGA-PEG) is collected at the wall of the centrifuge tube and stored in a -20 °C refrigerator.
[0047] TP (triptolide) is dissolved in the acetonitrile solution of PLGA-PEG, and under stirring conditions, it is slowly added dropwise (0.5 mL / min) to the aqueous solution of Poloxamer 188; after waiting for the evaporation of acetonitrile, TP@MNPs are collected by centrifugation (6,600 rpm, 10 min).
[0048] Dilute the Nephrin antibody at a ratio of 1:100 and sonicate it for 10 min to disperse it in 5 mL of phosphate buffer (PB, 10 mM, pH 5.8); subsequently, add EDC (50 mg / mL) and NHS (5 mg / mL) thereto, and stir at room temperature for 3 h to activate the carboxyl groups on its surface. Resuspend the obtained Nephrin-NHS in PB (5 mL, 10 mM, pH 7.4). Then dilute the GFLG polypeptide at a ratio of 1:50, disperse it in PBS (pH 7.4), and add EDC (50 mg / mL) and NHS (5 mg / mL) thereto for reaction for 9 h. Subsequently, add EDC (50 mg / mL) and NHS (5 mg / mL) to the Cap solution for reaction for 9 h. Add 5 mL of NH 2 -PEG-NH 2 (10 mM), and use N 2 Dry the obtained sample, add the obtained concentrated product to the dispersed solution of the activated GFLG polypeptide, react on a shaker for 12 h, and use N again 2 Dry the obtained sample, add the concentrated product to the Nephrin-NHS dispersed solution, sonicate the mixture for 10 min. Finally, place the aforementioned mixture under stirring at room temperature for reaction overnight (12 h), and then collect the prepared Cap-Nephrin by centrifugation (10000 rpm, 10 min, 4 °C), and wash the sample three times with ultrapure water (5 mL). Subsequently, lyophilize the obtained Cap-Nephrin and store it at -20 °C.
[0049] First, dissolve TP@MNPs in PB (5 mL, 10 mM, pH 6.0). Prepare an activation solution for use by adding 100 μL of 200 mM EDC and NHS (5 mg / mL) to PB (5 mL, 10 mM, pH 7.4); mix TP@MNPs with the aforementioned prepared activation solution and stir for 15 min, and then use a Zeba TM desalting spin column to remove the excess EDC and EDC by-products. Adjust the pH value of the activated TP@MNPs solution to 7.0, add 100 μL of Cap-Nephrin, react at room temperature for 2 h, subsequently collect the product, and centrifuge and wash the product three times with PBS (pH 7.4) to obtain a dual-loaded, dual-targeted nanomaterial (Dual nanocarrier) for the treatment of acute kidney injury, and store it at -20 °C.
[0050] Example 2
[0051] A preparation method of a nanomaterial TP@MNPs encapsulating triptolide, comprising the following steps:
[0052] First, PLGA (polylactic-co-glycolic acid), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and PEG (polyethylene glycol) were mixed and stirred in anhydrous chloroform; after reacting for 12 h, 15 mL of an ether / methanol mixture (v / v 1:1, 0 °C) was added to the aforementioned mixture to precipitate the product. The lower-layer precipitate was collected by centrifugation (10,000 rpm, 15 min). Subsequently, the precipitate was dissolved in acetonitrile and then an ether-methanol mixed solution was added to reprecipitate the product. The product was collected by centrifugation 3 times (10,000 rpm, 15 min). Then, the product was placed in a vacuum drying oven (35 °C). After 2 h, the product (PLGA-PEG) was collected at the wall of the centrifuge tube and stored in a -20 °C refrigerator.
[0053] TP (triptolide) was dissolved in the acetonitrile solution of PLGA-PEG. Under stirring conditions, it was slowly added dropwise (0.5 mL / min) to an aqueous solution of Poloxamer 188; after waiting for the acetonitrile to evaporate, TP@MNPs were collected by centrifugation (6600 rpm, 10 min).
[0054] Example 3
[0055] A preparation method of a nanomaterial MNPs-Cap that only encapsulates captopril, comprising the following steps:
[0056] First, PLGA (polylactic-co-glycolic acid), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and PEG (polyethylene glycol) were mixed and stirred in anhydrous chloroform; after reacting for 12 h, 15 mL of an ether / methanol mixture (v / v 1:1, 0 °C) was added to the aforementioned mixture to precipitate the product. The lower-layer precipitate was collected by centrifugation (10,000 rpm, 15 min). Subsequently, the precipitate was dissolved in acetonitrile and then an ether-methanol mixed solution was added to reprecipitate the product. The product was collected by centrifugation 3 times (10,000 rpm, 15 min). Then, the product was placed in a vacuum drying oven (35 °C). After 2 h, the product (PLGA-PEG) was collected at the wall of the centrifuge tube and stored in a -20 °C refrigerator.
[0057] Under stirring conditions, the acetonitrile solution of PLGA-PEG was slowly added dropwise (0.5 mL / min) to an aqueous solution of Poloxamer 188. After waiting for the acetonitrile to evaporate, MNPs were collected by centrifugation (6600 rpm, 10 min) and washed 3 times with water.
[0058] Dilute the Nephrin antibody at a ratio of 1:100 and sonicate it for 10 min to disperse it in 5 mL of phosphate buffer (PB, 10 mM, pH 5.8); subsequently, add EDC (50 mg / mL) and NHS (5 mg / mL) thereto and stir at room temperature for 3 h to activate the carboxyl groups on its surface, and resuspend the obtained Nephrin-NHS in PB (5 mL, 10 mM, pH 7.4). Then dilute the GFLG polypeptide at a ratio of 1:50, disperse it in PBS (pH 7.4), and add EDC (50 mg / mL) and NHS (5 mg / mL) thereto and react for 9 h. Subsequently, add EDC (50 mg / mL) and NHS (5 mg / mL) to the Cap solution and react for 9 h, and add 5 mL of NH 2 -PEG-NH 2 (10 mM), and use N 2 Dry the obtained sample, add the obtained concentrated product to the dispersed solution of the activated GFLG polypeptide, react on a shaker for 12 h, and use N again 2 Dry the obtained sample, add the concentrated product to the Nephrin-NHS dispersed solution, sonicate the mixture for 10 min, and finally, place the aforementioned mixture at room temperature and stir and react overnight (12 h), then collect the prepared Cap-Nephrin by centrifugation (10000 rpm, 10 min, 4 °C), and wash the sample three times with ultrapure water (5 mL). Subsequently, lyophilize the obtained Cap-Nephrin and store it at -20 °C.
[0059] First, dissolve the MNPs in PB (5 mL, 10 mM, pH 6.0). Prepare an activation solution for use by adding 100 μL of 200 mM EDC and NHS (5 mg / mL) to PB (5 mL, 10 mM, pH 7.4); mix the MNPs with the prepared activation solution above and stir for 15 min, then use a Zeba TM desalting spin column to remove the excess EDC and EDC by-products. Adjust the pH value of the activated MNPs solution to 7.0, add 100 μL of Cap-Nephrin, react at room temperature for 2 h, then collect the product, and centrifuge and wash the product three times with PBS (pH 7.4) to obtain MNPs-Cap.
[0060] Characterize the dual-loaded and dual-targeted nanomaterials prepared in Examples 1-3; TP@MNPs; MNPs-Cap; the results are as follows:
[0061] Use a scanning electron microscope (SEM) to analyze the surface structure of the nanoparticles of the products obtained in Examples 1-3, and the results are as Figures 1-3As shown, it indicates that before modifying the antibody, the MNPs nanoparticles exhibit a smooth spherical structure on the surface, while after modifying the antibody, the surface of the nanoparticles becomes significantly rougher, indicating the successful loading of Cap-Nephrin on the surface of MNPs.
[0062] Example 4
[0063] Investigate the encapsulation efficiency and drug loading of TP and Cap in the dual-loaded and dual-targeted nanomaterials for the treatment of acute kidney injury:
[0064] As Figure 4 shown, when the concentration of Cap is fixed, the EE (encapsulation efficiency) of TP decreases with the increase in the concentration of TP. This is because the proportion of TP not encapsulated in MNPs increases with the increase in the added amount of TP. When the added amount of TP is 1 mg / mL, the encapsulation rate of the dual-loaded and dual-targeted nanomaterials for TP is as high as 83%. When the added concentration of TP is 10 mg / mL, more than 30% of TP can still be encapsulated into MNPs. In addition, with the increase in the concentration of TP, the drug loading efficiency (DLE) of MNPs for TP also increases. Combining with the encapsulation efficiency of TP, MNPs can achieve a drug loading of ∼60%, which is beneficial to reducing the effective dose of TP with greater toxicity in the dual-loaded and dual-targeted nanomaterials, thereby weakening its biological toxicity and potential side effects in vivo. When the concentration of TP is constant, the changing trends of the EE and DLE of Cap with the increase in Cap are similar to those of TP. When the added concentration of Cap is 0.1 mg / mL, the highest EE of Cap can reach ∼35%; when the added amount of Cap is 1.2 mg / mL, its DLE drops to ∼25%. Combining with the encapsulation efficiency of Cap, its drug loading can reach 15%, only one-fourth of that of MNPs. This is determined by its loading position in the dual-loaded and dual-targeted nanomaterials because it is exposed on the outside of the dual-loaded and dual-targeted nanomaterials, and it is difficult for the nanoparticles to achieve effective encapsulation of it. However, due to the specific recognition of the GFLG polypeptide in Cap-Nephrin by cathepsin B, Cap will not produce drug off-target phenomena during the blood circulation of the dual-loaded and dual-targeted nanomaterials. Furthermore, the dual-loaded and dual-targeted nanomaterials can deliver an effective dose of Cap to the targeted site. It is beneficial to reduce the effective drug amount of Cap. Therefore, the encapsulation efficiency and drug loading of the dual-loaded and dual-targeted nanomaterials for the treatment of acute kidney injury drugs for TP and Cap can meet the requirements of this experiment to fully exert the synergistic effect of the two loaded drugs while reducing the effective dose of each drug.
[0065] Example 5
[0066] Cy7-labeled TP@MNPs and Cy7-labeled dual-loaded, dual-targeted nanomaterials were prepared separately and injected into mice via the tail vein. Kidneys of the mice were selected 24 h after administration for frozen kidney section, immunofluorescence staining and imaging studies. Figure 5 It is an immunofluorescence staining imaging diagram of frozen kidney sections of mice. Among them, the blue fluorescence is the nuclear fluorescence stained by DAPI, and the red fluorescence is Cy7 in the dual-loaded, dual-targeted nanomaterials or TP@MNPs. From Figure 5 As can be seen from a in Figure 5 , in the mice injected with the dual-loaded, dual-targeted nanomaterials via the tail vein, there is partial overlap between the blue fluorescence of the glomerulus and the red fluorescence of the materials, which fully demonstrates that the dual-loaded, dual-targeted nanomaterials can be enriched in the glomerular region. Since the glomerular filtration barrier of the model mice is damaged, that is, podocytes are shed or fused, the damage of the glomerular filtration barrier will lead to the passage of large-sized substances. Based on this, the dual-loaded, dual-targeted nanomaterials can reach the slit diaphragm space of podocytes through the fenestrated diaphragm and the basement membrane. Through the specific recognition of Nephrin antibody and Nephrin protein expressed in the slit diaphragm space, the dual-loaded, dual-targeted nanomaterials can be located and stay in the podocyte region for a long time. From
[0067] Example 6
[0068] Blood and urine of PAN mice were examined for biochemical indicators to evaluate the therapeutic effect of the dual-loaded, dual-targeted nanomaterials. The biochemical indicators include albumin (ALB), total cholesterol (TC) and triglyceride (TG). At the same time, urine protein and creatinine were detected, and the ratio of urine protein to creatinine (uACR) was calculated.
[0069] Figure 6It is a graph showing the changes in uACR of mice in different groups. uACR can well evaluate the impairment of renal function. As can be seen from the graph, after intervention by injecting puromycin through the tail vein, compared with the NS group (i.e., the normal mouse group), the uACR of mice in the PAN group showed a significant increase 5 - 14 days after puromycin intervention. This result indicates that puromycin has damaged the glomerular filtration membrane of mice, that is, it has caused the destruction of the glomerular filtration barrier and the production of proteinuria. At the same time, this also proves that the PAN model mice were successfully constructed. Subsequently, TP@MNPs, MNPs-Cap, and dual-loaded, dual-targeted nanomaterials (Dualnanocarrier) were used to intervene and treat the PAN model mice. As Figure 6 shown by the purple curve (TP@MNPs) in the figure, in the mouse group intervened with TP@MNPs (TP loading amount is 0.01 mg / mL), the uACR value remained at a relatively high level within 5 - 14 days after intervention. This indicates that the glomerular filtration rate is still in a state of loss, and the damaged glomerular filtration barrier has not been repaired. This is because this nanomaterial does not carry Cap-Nephrin, so there is no Nephrin antibody on its surface that can recognize Nephrin in the slit diaphragm of podocytes. This means that it is very difficult for this nanomaterial to achieve long-term localization in the glomerulus, and thus it cannot repair the damage of the glomerular filtration barrier in PAN model mice. In addition, the TP loading amount of TP@MNPs (0.01 mg / mL) is much lower than the effective drug dose of TP (3 mg / mL), so it is very difficult for TP@MNPs to achieve effective treatment. As Figure 6 shown by the blue curve (MNPs-Cap), for the mice intervened with MNPs-Cap, the uACR index began to slightly decrease after the 10th day, but still could not return to the normal level at 21 days. This is because the presence of Nephrin antibody enables MNPs-Cap to target the glomerulus for a relatively long time, but because there is only one drug and the loading amount of Cap (0.005 mg / mL) is much lower than the effective dose of Cap (5 mg / mL), and there is a lack of synergistic effect between the two drugs, so the therapeutic effect of MNPs-Cap is very limited. However, for the mouse group intervened with dual-loaded, dual-targeted nanomaterials (where the loading amount of TP is 0.01 mg / mL, which is an ineffective drug dose, and the effective drug dose of TP is 3 mg / mL; the loading amount of Cap is 0.005 mg / mL, which is an ineffective drug dose, and the effective drug dose of Cap is 5 mg / mL), the uACR index decreased to a level close to that of normal mice at 21 days after intervention ( Figure 6 green curve). This indicates that the dual-loaded, dual-targeted nanomaterials have a good repair effect on glomerular damage and the destruction of the glomerular filtration barrier, and this repair effect is achieved by loading ineffective drug doses of single-loaded drug nanoparticles.
[0070] In the PAN model mice intervened by TP@MNPs, MNPs-Cap, and dual-loaded, dual-targeted nanomaterials, the biochemical indices of ALB, TC, and TG changed within 21 days. As can be seen from Figure 7 it, in the mice intervened by the dual-loaded, dual-targeted nanomaterials, after 21 days, all their biochemical indices could be restored to levels close to those of the normal mouse group, indicating that the kidney and body functions of the PAN model mice had been effectively improved. In the PAN model mice intervened by TP@MNPs and MNPs-Cap, their various biochemical indices recovered to some extent but could not be restored to the normal level.
Claims
1. A method for preparing a dual-loaded, dual-targeted nanomaterial for the treatment of acute kidney injury, characterized in that: The steps include: (1) Under stirring conditions, the organic solvent solution of triptolide and PLGA-PEG is added dropwise to the aqueous solution of Poloxamer 188, the organic solvent is evaporated by stirring, and TP@MNPs are obtained by centrifugation; (2) After the carboxyl group of captopril is activated, it reacts with NH2-PEG-NH2 to obtain Cap-PEG-NH2; (3) After the carboxyl group of GFLG polypeptide is activated, it reacts with Cap-PEG-NH2 to obtain Cap-PEG-GFLG; (4) After the carboxyl group of the nephrin antibody is activated, it reacts with Cap-PEG-GFLG to obtain Cap-Nephrin; (5) After carboxyl group activation, TP@MNPs reacted with Cap-Nephrin to obtain dual-loaded, dual-targeted nanomaterials for the treatment of acute kidney injury.
2. The preparation method according to claim 1, characterized in that: In step (1), the organic solvent is acetonitrile.
3. The preparation method according to claim 1, characterized in that: The preparation method of PLGA-PEG in step (1) comprises the following steps: Polylactic acid-glycolic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and polyethylene glycol are reacted in anhydrous chloroform, and the precipitation is centrifuged to obtain PLGA-PEG.
4. The preparation method according to claim 3, characterized in that: The precipitation centrifugation is to add ether / methanol mixture to precipitate and then centrifuge; The volume ratio of ether to methanol in the ether / methanol mixture is 1:1-1:
3.
5. The preparation method according to claim 1, characterized in that: The carboxyl activation of captopril in step (2) is obtained by an activation reaction of captopril with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution; the activation reaction time is 7 to 11 hours, and the buffer solution is a phosphate buffer solution.
6. The preparation method according to claim 1, characterized in that: In step (3), the carboxyl group activation of the GFLG polypeptide is obtained by an activation reaction of the GFLG polypeptide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution; the activation reaction time is 7 to 11 hours, the buffer solution is a phosphate buffer solution, and the pH of the buffer solution is 6.4 to 8.4; The reaction time in step (3) is 10 to 14 hours.
7. The preparation method according to claim 1, characterized in that: In step (4), the carboxyl group activation of the nephrin antibody is obtained by an activation reaction of the nephrin antibody with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a buffer solution; the activation reaction time is 1 to 5 hours, the buffer solution is a phosphate buffer solution, and the pH of the buffer solution is 4.8 to 6.8; The reaction temperature in step (4) is room temperature and the reaction time is 10 to 14 hours.
8. The preparation method according to claim 1, characterized in that: In step (5), the carboxyl activation of TP@MNPs is obtained by an activation reaction of TP@MNPs with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution; the activation reaction time is 13 to 17 minutes, and the buffer solution is a phosphate buffer solution; The reaction in step (5) is carried out in a buffer solution having a pH of 5 to 9; the reaction temperature is room temperature and the reaction time is 1 to 4 hours.
9. A dual-loaded, dual-targeted nanomaterial for the treatment of acute kidney injury prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the dual-loaded, dual-targeted nanomaterial for treating acute kidney injury according to claim 9 in preparing a drug for treating acute kidney injury.