Probe and medicine for targeting kidney, and preparation method and application of probe and medicine

Abstract

The invention relates to the technical field of biological medicine, in particular to a kidney targeting probe, a medicine, a preparation method and application thereof. According to the application of the protein provided by the invention in preparation of the kidney-targeted product, the protein can be reabsorbed by a healthy kidney; the proteins have high renal tubule targeting, can be accurately positioned to renal tubule epithelial cells, and can be used as a delivery carrier, a loaded dye to prepare a probe or a loaded drug to prepare a drug, so that the accuracy and effectiveness of detection and treatment are ensured. And the detection of the renal tubule injury is successfully realized by loading a fluorescent dye, the process is rapid and intuitive, and the efficiency and accuracy of kidney disease diagnosis are greatly improved. By loading the medicine, the therapeutic medicine is directly conveyed to the renal tubular epithelial cells, so that the effective treatment of kidney diseases is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to a probe, a drug, a preparation method and uses thereof that target the kidney. Background Art

[0002] As the core functional unit of the kidney, the renal tubule and the renal corpuscle (composed of the glomerulus and the renal capsule) together form the basic functional and structural unit of the kidney - the nephron. This complex epithelial tissue structure undertakes multiple physiological functions, including selectively reabsorbing the components of the primary urine, actively secreting metabolites, finely regulating the body fluid electrolyte balance, and maintaining the acid-base homeostasis and other important physiological processes. In the field of clinical nephrology, renal tubule-related diseases have an important clinical status, and among them, the damage of renal tubular epithelial cells often leads to serious clinical consequences.

[0003] Taking Acute Kidney Injury (AKI) as an example, the main pathological mechanism of this critical illness involves the abnormal response of renal tubular epithelial cells to ischemia / hypoxia or nephrotoxic substances. Despite the continuous progress of modern medicine, the in-hospital mortality rate of AKI patients still remains above 20%, which is closely related to the limitations of current diagnostic techniques. The current clinical diagnosis mainly relies on two criteria: the progressive increase in serum creatinine (Scr) within 48 hours and the decrease in urine output. However, such biochemical indicators not only have limited sensitivity, but their significant time lag is more likely to delay the best treatment opportunity.

[0004] In response to the above diagnostic dilemma, some scholars have attempted to use the tracer clearance method to measure the Glomerular Filtration Rate (GFR) to improve the detection efficiency of AKI. A fluorescently labeled substance that is filtered through the glomerulus and has less interaction with the renal tubule is used to determine the filtration ability of the glomerulus, and the change in GFR is monitored in real time to judge whether renal function damage has occurred. Although this method does improve the detection timeliness, from a pathophysiological perspective, there are fundamental defects: the core injury target of AKI is located in the renal tubule rather than the glomerulus. Typical histopathological changes include the formation of casts blocked by apoptosis / necrosis of renal tubular epithelial cells, and the back-leakage of luminal fluid caused by changes in cell polarity. And based on the detection indicators of glomerular filtration function, abnormalities can only appear when renal tubular damage involves the interstitium and causes changes in glomerular hemodynamics. At this time, it often has entered the stage of injury progression. Obviously, this detection mode (that is, first there is renal tubular damage, and then it affects glomerular function) fundamentally determines the lag of existing detection means. For kidney diseases with renal tubular damage in particular, timely detection and treatment have very important clinical significance. Summary of the Invention

[0005] Therefore, the first technical problem to be solved by the present invention is to provide the use of a protein in the preparation of a product targeting the kidney, where the protein is a protein that can be reabsorbed by a healthy kidney. Using this protein, a product targeting the kidney, a detection probe, or a drug can be prepared. These probes and drugs can specifically target the kidney, especially the renal tubules, thereby enabling early and accurate detection and treatment of kidney diseases.

[0006] The second technical problem to be solved by the present invention is to provide a probe targeting the kidney, its preparation method, and its application. Here, the detection of kidney diseases such as renal tubule injury and kidney tumors is achieved through the probe targeting the kidney. The probe targeting the kidney has good renal tubule targeting ability and can rapidly diagnose renal tubule diseases through imaging or signal strength, opening up a new method for the diagnosis of kidney diseases.

[0007] The third technical problem to be solved by the present invention is to provide a drug targeting the kidney, its preparation method, and its application. The treatment of kidney diseases such as renal tubule injury is achieved through the drug targeting the kidney. The drug targeting the kidney has good renal tubule targeting ability, can effectively deliver relevant therapeutic drugs to renal tubular epithelial cells, has high application value, and opens up a new method for the development of the treatment of kidney diseases.

[0008] For this reason, the present invention provides the following technical solutions:

[0009] An embodiment of the present invention provides the use of a protein in the preparation of a product targeting the kidney, where the protein is a protein that can be reabsorbed by a healthy kidney. The protein can be absorbed by the renal tubules.

[0010] In some embodiments, the product targeting the kidney includes, but is not limited to, probes and drugs. They all use a protein that can be reabsorbed by a healthy kidney as the core component. These proteins have high renal tubule targeting properties, can accurately locate to renal tubular epithelial cells, and can be used as delivery carriers to load dyes to prepare probes or load drugs to make drugs, thereby ensuring the accuracy and effectiveness of detection and treatment. In some embodiments, through loading fluorescent dyes, the fluorescence imaging detection of renal tubule injury has been successfully achieved. This process is rapid and intuitive, greatly improving the efficiency and accuracy of the diagnosis of kidney diseases. In some embodiments, through loading drugs, therapeutic drugs are directly delivered to renal tubular epithelial cells, achieving effective treatment of kidney diseases.

[0011] In some embodiments, the protein that can be reabsorbed by a healthy kidney is a protein with a molecular weight less than 70KD. The protein can be filtered by the glomerulus and then reabsorbed by healthy renal tubular epithelial cells.

[0012] In a preferred embodiment, the protein comprises at least one of β-lactoglobulin, α-lactalbumin or β2-microglobulin.

[0013] In an embodiment of the present invention, a probe targeting the kidney is provided, comprising: a carrier: comprising the protein described above; a signal substance; and the signal substance is modified on the carrier.

[0014] In the above embodiment, since the signal substance, such as a dye, is small relative to the protein, it has little effect on the metabolism of the protein in vivo, and the kidney targeting property of the protein can be ensured. The probe targeting the kidney can target and accumulate in the area of normal kidney tissue and maintain a high signal for a long time. When the kidney is unhealthy, such as in kidney disease AKI where the renal tubular epithelial cells are damaged, the ability of the probe targeting the kidney to be taken up by the damaged renal tubular epithelial cells decreases, and the retention time in the kidney tissue is short, resulting in a decrease in the accumulation of the probe targeting the kidney in the kidney tissue area and a weakening of the signal. Therefore, the signal strength of normal and unhealthy kidneys can be compared to detect kidney-related diseases. Further, by comparing the fluorescence signal intensity of urine, since in unhealthy kidneys such as kidney disease AKI, the ability of the probe targeting the kidney to be taken up by the damaged renal tubular epithelial cells decreases, more probes are excreted through urine. Therefore, more signal substances can be detected in urine, that is, compared with normal kidneys, the signal in the urine excreted by unhealthy kidneys is significantly enhanced to detect kidney-related diseases.

[0015] In some embodiments, the signal substance is a substance capable of generating a signal commonly used in the fields of disease detection and imaging, such as a fluorescent dye, a radioactive isotope or a magnetic resonance imaging contrast agent, etc. The selection of these signal substances can be determined according to specific detection requirements and imaging techniques. After being excited by light of a specific wavelength, the fluorescent dye can emit fluorescence of a specific wavelength to achieve visual detection; the radioactive isotope can be imaged by the rays generated by radioactive decay; and the magnetic resonance imaging contrast agent can change the signal intensity of tissues in magnetic resonance imaging and improve the imaging contrast. By selecting a suitable signal substance, sensitive and accurate detection of kidney diseases can be achieved. In a preferred embodiment, the signal substance comprises a dye, the dye comprises a fluorescent dye; the fluorescent dye comprises a fluorescent dye in the ultraviolet-visible region, a far-infrared region dye or a near-infrared region dye; the fluorescent dye comprises at least one of a fluorescein-based dye, a rhodamine-based dye, a cyanine-based dye or a quantum dot dye. Further, the dye is a fluorescent dye, and further still, the dye includes but is not limited to at least one of ZW800-1, FITC, Cy5.

[0016] In some embodiments, the signaling substance is modified on the carrier, and the modification method is a common method in the art, such as by chemical bonding, physical adsorption, or encapsulation. Chemical bonding can connect the signaling substance to the carrier through covalent bonds, ionic bonds, or coordination bonds; physical adsorption utilizes the physical interaction force between the signaling substance and the carrier, such as van der Waals force, to attach the signaling substance to the surface of the carrier; the encapsulation method is to encapsulate the signaling substance inside or on the surface of the carrier. The selection of these modification methods can be determined according to the properties of the signaling substance, the characteristics of the carrier, and specific application requirements to ensure that the signaling substance can stably and effectively bind to the carrier and play a detection or imaging role at the target site. In a preferred embodiment, when the signaling substance is a dye, the dye can be selected to be covalently modified on the carrier, and the bonding sites of the dye and the carrier are the amino or mercapto groups of the carrier. Further, the NHS esters modified on ZW800-1, FITC, and Cy5 are bonded to the amino group of the carrier.

[0017] An embodiment of the present invention provides a method for preparing the probe targeting the kidney, comprising the following steps:

[0018] (1) Modify the signaling substance;

[0019] (2) Modify the signaling substance obtained in step (1) on the carrier.

[0020] In some embodiments, the signaling substance is a substance capable of generating signals commonly used in the fields of disease detection and imaging, such as fluorescent dyes, radioactive isotopes, or magnetic resonance imaging contrast agents. The selection of these signaling substances can be determined according to specific detection requirements and imaging techniques. Fluorescent dyes can emit fluorescence of a specific wavelength after being excited by light of a specific wavelength, thereby realizing visual detection; radioactive isotopes can be imaged through the rays generated by radioactive decay; magnetic resonance imaging contrast agents can change the signal intensity of tissues in magnetic resonance imaging and improve imaging contrast. By selecting a suitable signaling substance, sensitive and accurate detection of kidney diseases can be achieved. In a preferred embodiment, the signaling substance includes dyes, and the dyes include fluorescent dyes; the fluorescent dyes include fluorescent dyes in the ultraviolet-visible region, far-infrared region, or near-infrared region; the fluorescent dyes include at least one of fluorescein dyes, rhodamine dyes, cyanine dyes, or quantum dot dyes; further, the dyes include but are not limited to at least one of ZW800-1, FITC, and Cy5.

[0021] In some embodiments, the signal substance is modified in a manner common in the art by modifying appropriate functional groups to enhance its binding ability and stability to the carrier. For example, when the signal substance is a fluorescent dye, NHS esters can be introduced to enable it to form a covalent bond with the amino group on the carrier, or maleimide can be introduced to form a covalent bond with the sulfhydryl group on the carrier through a covalent bond. The introduction of these functional groups not only improves the binding efficiency of the signal substance to the carrier but also helps to maintain the stability of the signal substance on the carrier, preventing it from being rapidly degraded or excreted in the body, thus ensuring that the probe plays a lasting detection or imaging role at the target site. In addition, factors such as the optical properties, water solubility, and biocompatibility of the signal substance can be considered during the modification process to further optimize the performance of the probe.

[0022] In some embodiments, the signal substance is modified on the carrier, and the modification method is a common method in the art, such as by chemical bonding, physical adsorption, or encapsulation. Chemical bonding can connect the signal substance to the carrier through covalent bonds, ionic bonds, or coordination bonds; physical adsorption utilizes the physical interaction force between the signal substance and the carrier, such as van der Waals forces, to attach the signal substance to the surface of the carrier; the encapsulation method is to encapsulate the signal substance inside or on the surface of the carrier. The selection of these modification methods can be determined according to the properties of the signal substance, the characteristics of the carrier, and specific application requirements to ensure that the signal substance can stably and effectively bind to the carrier and play a detection or imaging role at the target site. In a preferred embodiment, the dye can be selected to be covalently modified on the carrier, and the bonding site between the dye and the carrier is the amino group or sulfhydryl group of the carrier. Further, the NHS esters modified on ZW800-1, FITC, and Cy5 form covalent bonds with the amino group of the carrier.

[0023] In a preferred embodiment, when the signal substance is a dye, in step (1), the dye is either carboxyl-modified or not carboxyl-modified, and is either NHS-modified or not NHS-modified. Further, the step of NHS-modifying the carboxyl-modified dye includes dissolving the carboxyl-modified dye in an organic solvent and then adding dipyrrolidino(N-succinimidyloxy)carbon hexafluorophosphate for reaction. Further, the molar ratio of the carboxyl-modified dye to dipyrrolidino(N-succinimidyloxy)carbon hexafluorophosphate is 1:1 - 1:10, and the molar ratio can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, and more preferably 1:2.

[0024] In a preferred embodiment, when the signaling substance is a dye, in step (2), the dye obtained in step (1) is mixed with a carrier and stirred at 100 - 1000 rpm (more preferably 300 rpm) in the dark at 0 - 37 °C for 0.5 - 48 h. Further, the temperature in the dark can be any value among 0, 5, 10, 15, 20, 25, 30, 37 °C or a range value between any two values, and the stirring time can be any value among 0.5, 1, 2, 5, 10, 15, 20, 24, 30, 35, 40, 48 h or a range value between any two values, more preferably 24 h. Further, the molar ratio of the modified dye obtained in step (1) to the carrier is 10:1 to 1:10. For example, the molar ratio of the modified dye to the carrier can be any value among 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:8, 1:10 or a range value between any two values.

[0025] The embodiments of the present invention provide the use of the kidney-targeted probe or the kidney-targeted probe prepared by the preparation method described above in any one of the following:

[0026] (1) Use in kidney-targeted imaging;

[0027] (2) Use in the preparation of products for detecting kidney diseases.

[0028] In the above embodiments, since the carrier in the kidney-targeted probe can be reabsorbed by healthy kidneys and the signaling substance is smaller than the protein molecule, the influence of the signaling substance on the reabsorption of the protein by healthy kidneys is small. Therefore, the kidney-targeted probe can be reabsorbed by healthy kidneys (tubular absorption), has a long residence time in the kidney tissue area, and realizes imaging of healthy kidneys. Similarly, since the kidneys with kidney diseases cannot normally uptake the protein and have a short residence time in the kidney tissue area, resulting in fewer probes in the kidney tissue area and a decrease in signal intensity. By comparing with the signal intensity of healthy kidneys, those with a weakened signal intensity have kidney diseases, thus realizing the detection of kidney diseases.

[0029] In a preferred embodiment, it includes the use in any one of the following:

[0030] (1) Use in tubular-targeted imaging;

[0031] (2) Use in the preparation of products for detecting kidney injury or kidney tumors;

[0032] The products include reagents and kits.

[0033] In some embodiments, the reagent may comprise the kidney-targeting probe described above, as well as necessary components such as buffers and stabilizers for imaging or detecting the kidney in vitro or in vivo. The kit may then comprise the reagent described above, as well as instructions, tools, etc. for operating the reagent to facilitate the detection of kidney diseases by users. Through the products described above, rapid and accurate detection of kidney diseases can be achieved, providing strong support for the diagnosis and treatment of kidney diseases.

[0034] Embodiments of the present invention provide a kidney-targeting drug, comprising:

[0035] A carrier: comprising the protein described above;

[0036] A drug;

[0037] The drug is modified on the carrier.

[0038] In the above embodiments, since the protein can be a protein that can be reabsorbed by the kidney, the protein can be used as a carrier to deliver drugs. In early-stage damaged renal tubular cells, some renal tubular cells necrose and exfoliate, and some renal tubular cells can uptake the corresponding protein (a protein that can be reabsorbed by a healthy kidney). When the protein carrying the drug passes through the kidney tissue, it is phagocytosed by the remaining renal tubular epithelial cells that still have the function of uptaking the protein, and then slowly metabolized through the renal tubules, thereby slowing down its clearance through the kidney, prolonging its residence time in the body, and increasing the therapeutic effect of the drug on kidney diseases.

[0039] In some embodiments, the drug comprises drugs for antioxidative stress injury, including but not limited to drugs for treating kidney injury caused by oxidative stress, such as gambogic acid, resveratrol, glutathione, etc. These drugs are delivered to the kidney tissue through the carrier, i.e., the protein that can be reabsorbed by the kidney, effectively alleviating kidney injury caused by oxidative stress. Gambogic acid has pharmacological effects such as inhibiting cell proliferation and inducing apoptosis, and can reduce oxidative stress injury of kidney cells; resveratrol is a potent antioxidant that can scavenge free radicals and protect kidney cells from oxidative damage; glutathione is an important antioxidant in the body that can maintain the redox balance of kidney cells. Through the kidney-targeting drug described above, precise treatment of kidney diseases can be achieved, improving the therapeutic effect and reducing drug side effects.

[0040] In some embodiments, the drug is modified on the carrier, and the modification methods are common in the art, such as by chemical bonding, physical adsorption, or encapsulation. Chemical bonding can connect the signaling substance to the carrier through covalent bonds, ionic bonds, or coordination bonds; physical adsorption utilizes the physical interaction between the signaling substance and the carrier, such as van der Waals forces, to attach the drug to the surface of the carrier; the encapsulation method is to encapsulate the drug inside or on the surface of the carrier. The selection of these modification methods can be determined according to the properties of the drug, the characteristics of the carrier, and specific application requirements to ensure that the drug can stably and effectively bind to the carrier and exert corresponding therapeutic effects at the target site. In a preferred embodiment, when the drug is gambogic acid, the gambogic acid can be covalently modified on the carrier. Further, the gambogic acid can be covalently modified on the carrier through a linker. The linker can be polyethylene glycol, and the polyethylene glycol includes amino functional groups and carboxyl functional groups. The amino functional group (primary amine or secondary amine) is amide-linked to the carboxyl group of the gambogic acid, and the carboxyl functional group of the polyethylene glycol is NHS-modified and then covalently bonded to the amino group on the carrier.

[0041] An embodiment of the present invention provides a method for preparing the drug targeting the kidney, comprising the following step: modifying the drug on the carrier.

[0042] In some embodiments, a drug such as gambogic acid can be directly modified on the carrier in the common ways as described above in the art.

[0043] In a preferred embodiment, functional groups can be connected between the drug, the carrier, or both, such as to increase solubility to improve the bioavailability of the drug or to improve its stability in vivo. For example, when the drug is gambogic acid, some functional groups can be introduced to enhance its solubility in water, which is beneficial to the transport and absorption of the drug in vivo. At the same time, these functional groups can also serve as a connecting bridge to firmly bind the drug to the carrier through chemical bonding to ensure that the drug does not fall off or become inactivated before reaching the target site. In addition, the introduction of functional groups can also perform targeted modification of the drug as needed, such as introducing specific ligands or antibodies to achieve precise recognition and binding of the drug to kidney cells.

[0044] In a preferred embodiment, to increase the water solubility of gambogic acid, PEG is connected to gambogic acid and then to the carrier. PEG serves as a linker between gambogic acid and the carrier, improving solubility on the one hand and firmly binding the drug to the carrier on the other hand to ensure that the drug does not fall off or become inactivated before reaching the target site. The method includes the following steps:

[0045] (1) Modify gambogic acid with polyethylene glycol containing at least 1 amino functional group and at least 1 carboxyl functional group through the amino functional group to obtain Compound 1;

[0046] (2) Perform NHS modification on the carboxyl terminus of polyethylene glycol in Compound 1 obtained in step (1) to obtain Compound 2;

[0047] (3) Mix Compound 2 obtained in step (2) with a carrier and carry out a reaction.

[0048] In some embodiments, the molecular weight of the polyethylene glycol is ≥ 3000 Da, and a preferred embodiment is 3400 Da.

[0049] In some embodiments, in step (1), the molar ratio of polyethylene glycol containing at least 1 amino functional group and at least 1 carboxyl functional group to gambogic acid is 1:1 - 10:1. The molar ratio can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and a preferred embodiment is 3:1;

[0050] In some embodiments, in step (1), dissolve gambogic acid in an organic solvent, then add polyethylene glycol containing at least 1 amino functional group and at least 1 carboxyl functional group, and react at 0 - 37 °C for 0.5 - 48 h; further, the temperature in the dark can be any value among 0, 5, 10, 15, 20, 25, 30, 37 °C or the range value between any two values, and the stirring time can be any value among 0.5, 1, 2, 5, 10, 15, 20, 24, 30, 35, 40, 48 h or the range value between any two values, and more preferably 24 h.

[0051] In some embodiments, in step (2), dissolve Compound 1, then add N - hydroxysuccinimide and mix. The molar ratio of the two is 1:1 - 1:10. The molar ratio can be any value among 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or the range value between any two values, and more preferably 1:5. The reaction conditions are to react at 0 - 37 °C for 0.5 - 2 h; the temperature can be any value among 0, 5, 10, 15, 20, 25, 30, 37 °C or the range value between any two values, and the reaction time can be any value among 0.5, 1, 1.5, 2 h or the range value between any two values.

[0052] In some embodiments, in step (3), the compound 2 obtained in step (3) is mixed with a carrier and dissolved. The molar ratio of compound 2 to the carrier is 10:1 - 1:10. The molar ratio can be any one value among 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:2, 8:3, 7:4, 6:5, 5:6, 4:7, 3:8, 2:9 or a range value between any two values. More preferably, it is 2:1. The reaction conditions are to react at 0 - 37°C for 0.5 - 48 h. The reaction temperature can be any one value among 0, 5, 10, 15, 20, 25, 30, 37°C or a range value between any two values. The reaction time can be any one value among 0.5, 1, 2, 5, 10, 15, 20, 24, 30, 35, 40, 48 h or a range value between any two values. More preferably, it is 24 h.

[0053] The embodiments of the present invention provide the use of the described kidney-targeting drug or the kidney-targeting drug prepared by the described preparation method in the preparation of a drug for targeted prevention or treatment of kidney diseases.

[0054] In some embodiments, the kidney diseases include tubular injury.

[0055] The technical solution of the present invention has the following advantages:

[0056] 1. The use of the protein provided by the present invention in the preparation of a kidney-targeting product, where the protein is a protein that can be reabsorbed by a healthy kidney; these proteins have high tubular targeting properties, can accurately locate to renal tubular epithelial cells, and can be used as delivery carriers to load dyes to prepare probes or load drugs to make drugs, thereby ensuring the accuracy and effectiveness of detection and treatment. For example, by loading a fluorescent dye, the detection of tubular injury has been successfully achieved. This process is rapid and intuitive, greatly improving the efficiency and accuracy of the diagnosis of kidney diseases. By loading drugs, therapeutic drugs are directly delivered to renal tubular epithelial cells, achieving effective treatment of kidney diseases.

[0057] 2. The use of the protein provided by the present invention in the preparation of a kidney-targeting product, where the protein includes at least one of β-lactoglobulin, α-lactalbumin, or β2-microglobulin; the present invention has found through research that the above-mentioned proteins can have a long retention time in normal kidneys, can target renal tubular epithelial cells, and can be taken up by renal tubular epithelial cells. Therefore, the above-mentioned proteins can be used as carriers to deliver drugs or signaling substances to prepare kidney-targeting products, such as drugs or probes, for the treatment or detection of kidney diseases.

[0058] 3. The kidney-targeting probe provided by the present invention includes: a carrier: including the protein described above; a dye; the signal substance is modified on the carrier; signal substances such as ZW800-1 and most small molecule dyes can pass through the glomerular filtration, and then quickly pass through the renal tubule lumen, and then are excreted from the body through urine. For the protein modified with the selected signal substance, it can be phagocytosed by renal tubular epithelial cells and then slowly metabolized through the renal tubules, thereby slowing down its clearance through the kidneys, prolonging its residence time in the body, increasing its targeting effect on the renal tubules, prolonging its aggregation in the renal tubules, and enabling long-term renal tubular imaging in the kidney region. In kidney diseases such as kidney injury, some renal tubular epithelial cells are necrotic or shed. Compared with normal kidney tissues, the number of renal tubular epithelial cells capable of taking up the protein is reduced. Therefore, the uptake amounts of the probe by normal kidney tissues and kidney tissues with kidney diseases are different. Compared with normal kidney tissues, the signal intensity in kidney diseases such as kidney injury or tumor areas is significantly weakened, while the signal intensity in the excreted urine is the opposite. It can be used for kidney injury detection or kidney tumor imaging by comparing the signal intensities, observing the boundary between the tumor and kidney tissues, and providing assistance for fluorescence-guided tumor resection surgery.

[0059] 4. The kidney-targeting drug provided by the present invention includes: a carrier: including the protein described above; a drug; the drug is modified on the carrier; most small molecule drugs can pass through the glomerular filtration, and then quickly pass through the renal tubule lumen, and then are excreted from the body through urine. For early damaged renal tubular epithelial cells, some renal tubular epithelial cells are necrotic and shed, and some renal tubular epithelial cells can take up the corresponding protein (protein that can be reabsorbed by healthy kidneys). The protein carrying the drug can be phagocytosed by renal tubular epithelial cells and then slowly metabolized through the renal tubules, thereby slowing down its clearance through the kidneys, prolonging its residence time in the body, increasing its targeting effect on the renal tubules, prolonging its aggregation time in the renal tubules. After the protein carrying the drug is taken up, it can rescue these cells and play a therapeutic role, achieving the treatment of kidney diseases such as kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0061] Figure 1 It is the gel electrophoresis test results of each probe in Experimental Example 1 of the present invention;

[0062] Figure 2It is the UV-visible absorption spectra of each probe in Experimental Example 2 of the present invention;

[0063] Figure 3 It is the fluorescence imaging of each group of mice injected with ZW800-1-LG, ZW800-1-MG or ZW800-1-LA within 0 - 30 min in Experimental Example 3 of the present invention;

[0064] Figure 4 It is the fluorescence imaging of each group of mice injected with ZW800-1-LG, ZW800-1-MG or ZW800-1-LA within 1 h - 7 d in Experimental Example 3 of the present invention;

[0065] Figure 5 It is the fluorescence imaging of the main organs dissected at 30 minutes and 7 days after injection of ZW800-1-LG, ZW800-1-MG or ZW800-1-LA in each group of mice in Experimental Example 3 of the present invention;

[0066] Figure 6 It is the fluorescence imaging of the main organs dissected 30 minutes after injection of Cy5-LG or FITC-LG in each group of mice in Experimental Example 4 of the present invention;

[0067] Figure 7 It is the fluorescence imaging results of each group of mice injected with ZW800-1-LG within 10 min - 8 h in Experimental Example 5 of the present invention;

[0068] Figure 8 It is the results of the fluorescence intensity of the kidneys 10 minutes after injection of ZW800-1-LG in each group of mice in Experimental Example 5 of the present invention;

[0069] Figure 9 It is the fluorescence imaging and the results of the fluorescence intensity of the kidneys 10 minutes after injection of ZW800-1-MG in each group of mice in Experimental Example 5 of the present invention;

[0070] Figure 10 It is the fluorescence imaging and the results of the fluorescence intensity of the kidneys 10 minutes after injection of ZW800-1-LA in each group of mice in Experimental Example 5 of the present invention;

[0071] Figure 11 It is the results of the fluorescence intensity of urine 30 minutes after injection of ZW800-1-LG in each group of mice in Experimental Example 5 of the present invention;

[0072] Figure 12 It is the fluorescence imaging results of each group of mice injected with ZW800-1-LG within 10 min - 8 h in Experimental Example 6 of the present invention;

[0073] Figure 13 It is the results of the fluorescence intensity of the kidneys 10 minutes after injection of ZW800-1-LG in each group of mice in Experimental Example 6 of the present invention;

[0074] Figure 14 They are the fluorescence imaging results of the main organs dissected 20 min after injection of ZW800-1-LG in each group of mice in Experimental Example 7 of the present invention;

[0075] Figure 15 They are the co-localization results of cells after treatment with ZW800-1-LG in Experimental Example 8 of the present invention;

[0076] Figure 16 They are the fluorescence observation results of sections prepared from kidney tissues taken 20 min after injection of ZW800-1-LG in mice in Experimental Example 9 of the present invention;

[0077] Figure 17 They are the detection results of kidney tumors by ZW800-1-LG in Experimental Example 10 of the present invention;

[0078] Figure 18 They are the gel electrophoresis results and absorption spectrum detection results of GA-PEG-β-LG in Experimental Example 11 of the present invention; the left figure is the gel electrophoresis result, and the right figure is the absorption spectrum detection result;

[0079] Figure 19 They are the gel electrophoresis results and absorption spectrum detection results of GA-PEG-MG in Experimental Example 11 of the present invention; the left figure is the gel electrophoresis result, and the right figure is the absorption spectrum detection result;

[0080] Figure 20 They are the gel electrophoresis results and absorption spectrum detection results of GA-PEG-LA in Experimental Example 11 of the present invention; the left figure is the gel electrophoresis result, and the right figure is the absorption spectrum detection result;

[0081] Figure 21 They are the microscopic observation results of HK-2 cells after treatment with GA-PEG-β-LG in Experimental Example 12 of the present invention;

[0082] Figure 22 They are the results of GA-PEG-β-LG in treating AKI in Experimental Example 13 of the present invention;

[0083] Figure 23 They are the design principles and effect schematic diagrams of each probe targeting renal tubular cells in the present invention. Detailed implementation manners

[0084] The following embodiments are provided to better further understand the present invention. They are not limited to the described optimal implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0085] For those not specifying specific experimental procedures or conditions in the examples, operations or conditions of conventional experimental procedures described in the literature in this field can be followed. For reagents or instruments without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0086] ZW800-1, FITC, and Cy5 involved in the following examples are all common commercially available products.

[0087] β-Lactoglobulin was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0088] α-Lactalbumin was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0089] β2-Microglobulin was purchased from Nanjing Lideng Medical Technology Co., Ltd.

[0090] Example 1 ZW800-1-LG

[0091] This example provides a preparation method for a kidney-targeted probe ZW800-1-LG, which includes the following steps:

[0092] 1) Activate ZW800-1 modified with -COOH to form N-hydroxysuccinimide active ester, namely ZW800-1-NHS. Specifically, weigh ZW800-1 modified with -COOH (1 mmol, see: Angew. Chem. Int. Ed., 50: 6258-6263) and add it to 50 mL of DMSO (dimethyl sulfoxide, CAS: 67-68-5), add TEA (triethylamine, CAS: 121-44-8) to adjust the pH to 10-11, weigh (2 mmol) HSPyU (dipyrrolidino(N-succinimidyloxy)carbon hexafluorophosphate, CAS: 207683-26-9) and add it to the ZW800-1 solution modified with -COOH, and stir for 30 minutes. After the reaction is completed, slowly drop the reaction solution into a mixed solution of 500 mL of acetone (CAS: 67-64-1): ethanol (CAS: 64-17-5) = 1:1 (v:v) for recrystallization, collect the precipitate, dry it, and obtain ZW800-1-NHS powder, which is stored in the dark at -20 °C.

[0093] 2) Weigh ZW800-1-NHS and dissolve it in PBS to prepare a 0.9 mmol / L ZW800-1-NHS solution. Weigh β-lactoglobulin (LG) and add it to PBS to prepare a 0.45 mmol / L β-lactoglobulin solution. Mix the above two solutions in equal volumes and stir at 37 °C in the dark at 300 rpm for 24 h. Then ultrafilter and concentrate, and purify through a Sephadex LH-20 gel column using water as the eluent. Collect the water elution phase solution to obtain the ZW800-1-LG solution. It can also be further made into a freeze-dried powder and stored in the dark at -20 °C.

[0094] Example 2 ZW800-1-LA

[0095] The difference between this example and Example 1 is that β-lactoglobulin is replaced with α-lactalbumin (LA) in equimolar amounts.

[0096] Example 3 ZW800-1-MG

[0097] The difference between this example and Example 1 is that β-lactoglobulin is replaced with β2-microglobulin (MG) in equimolar amounts.

[0098] Example 4 FITC-NHS-LG

[0099] The difference between this example and Example 1 is that ZW800-1 is replaced with FITC in equimolar amounts.

[0100] Example 5 Cy5-NHS-LG

[0101] The difference between this example and Example 1 is that ZW800-1 is replaced with Cy5 in equimolar amounts.

[0102] Example 6 GA-PEG-LG

[0103] This example provides a preparation method of a drug targeting the kidney, including the following steps:

[0104] First, modify gambogic acid (GA) with polyethylene glycol (PEG) to form GA-PEG, which can improve the water solubility of gambogic acid while providing a chemical bond connection. Then, activate the other end of polyethylene glycol and react with β-lactoglobulin (LG) to form GA-PEG-LG. The specific steps are as follows:

[0105] 1) Weigh (5 mg, 1 eq) GA (gambogic acid, CAS: 2752-65-0), (10.22 mg, 3 eq) COMU ((ethyl 2-oximino-cyanoacetate)-N,N-dimethyl-morpholinourea hexafluorophosphate, CAS: 1075198-30-9) and add them to 2 mL of DMF (N,N-dimethylformamide, CAS: 68-12-2). Then add (3.324 μL, 3 eq) TEA (triethylamine, CAS: 121-44-8), stir at room temperature for five minutes, and then add (81.11 mg, 3 eq) NH2-PEG 3400 -COOH. Stir and react at 37 °C in a water bath for 24 h. After dialysis in water for 24 h, further purify and concentrate by ultrafiltration, and then use water as the eluent to purify through a Sephadex LH-20 gel column. Collect the water elution phase solution to obtain a purified GA-PEG solution. Then prepare the solution into a freeze-dried powder to obtain a yellow solid of GA-PEG.

[0106] 2) Weigh (32.45 mg, 1 eq) GA-PEG, (18.29 mg, 10 eq) EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, CAS: 25952-53-81), (5.49 mg, 5 eq) NHS (N-hydroxysuccinimide, CAS: 6066-82-6) and add them to 3 mL of MES (2-morpholinoethanesulfonic acid) buffer solution with pH = 6.0. Stir and react at 37 °C in a water bath for 2 h. Further concentrate by ultrafiltration, and then use water as the eluent to purify through a Sephadex LH-20 gel column. Collect the water elution phase solution, and after freeze-drying, obtain a yellow solid of GA-PEG-NHS.

[0107] 3) Weigh (81.11 mg, 2 eq) GA-PEG-NHS, (219.47 mg, 1 eq) β-LG (β-lactoglobulin, CAS: 9005-43-7) and add them to 3 mL of MES (2-morpholinoethanesulfonic acid) buffer solution with pH = 8.0. Stir and react at 37 °C in a water bath for 24 h. Further concentrate by ultrafiltration, and then use water as the eluent to purify through a Sephadex LH-20 gel column. Collect the water elution phase solution, and after freeze-drying, obtain a freeze-dried powder of GA-PEG-LG. Store at -4 °C.

[0108] Example 7 GA-PEG-LA

[0109] The difference between this example and Example 6 is that β-lactoglobulin is replaced with α-lactalbumin (LA) in equimolar amounts.

[0110] Example 8 GA-PEG-MG

[0111] The difference between this example and Example 6 is that β-lactoglobulin is replaced by β2-microglobulin (MG) in equimolar amounts.

[0112] Experimental Example 1 Gel Electrophoresis Test

[0113] Gel electrophoresis was used to test and prove the successful modification of proteins by dyes in the probes prepared in Examples 1-5. The specific operations are as follows:

[0114] Each time, it was divided into an experimental group (solutions prepared after purification of the dye-modified proteins, namely: ZW800-1-LG, ZW800-1-MG, ZW800-1-LA, FITC-LG, Cy5-LG, Step 2) and a control group (prepared from the dyes used to modify the proteins, namely: ZW800-1-NHS, FITC-NHS or Cy5-NHS, Step 1), diluted to the same concentration as the experimental group). 2% agarose gel electrophoresis was used to analyze the dye-modified proteins and the corresponding fluorescent dyes. The method was as follows: 20 μL of the sample to be tested was added to the sample wells of the gel plate respectively, and the wire was connected for electrophoresis. When it moved 1-2 cm from the front edge, the electrophoresis was stopped. Fluorescent images were taken at 730 nm / 825 nm through the FOBI imaging system.

[0115] The finally obtained gel electrophoresis results are as Figure 1 shown. As can be seen from the figure, the modified proteins can emit fluorescence through the corresponding fluorescent channels, and their displacements in 2% agarose are different from those of the dyes before the reaction. Since the charges of ZW800-1-NHS, FITC-NHS or Cy5-NHS change after coupling with proteins, their displacement positions after electrification are different from those of the individual dyes, which proves the successful modification of proteins by dyes in Examples 1-5.

[0116] Experimental Example 2 Spectral Property Test of Probes

[0117] The spectral properties of the probes prepared in Examples 1-5 were characterized respectively. Specifically as follows: Using a UV-2550 spectrometer, the ultraviolet-visible absorption spectra of proteins (β-lactoglobulin (LG), β2-microglobulin (MG), α-lactalbumin (LA)) and dye-modified proteins (ZW800-1-LG, ZW800-1-MG, ZW800-1-LA, FITC-LG and Cy5-LG) were tested respectively. The method was as follows: The lyophilized powders of the above-mentioned tested proteins and dye-modified proteins were added to water respectively to prepare solutions with a concentration of 0.2 mg / mL, and a UV-2550 spectrometer was used to test the ultraviolet-visible absorption spectra in the spectral range of 200 nm - 800 nm.

[0118] The results are as Figure 2As shown, the probes ZW800-1-LG, ZW800-1-MG, ZW800-1-LA, FITC-LG, and Cy5-LG simultaneously have the absorption peak of proteins at 280 nm and the absorption peaks of the corresponding dyes (the absorption peak of ZW800-1 is around 780 nm, the absorption peak of FITC is around 490 nm, and the absorption peak of Cy5 is around 650 nm). The above indicates the successful modification of the dyes to the proteins, and the probes prepared in Examples 1-5 are successful.

[0119] Experimental Example 3 In Vivo Imaging of ZW800-1-LG, ZW800-1-MG, and ZW800-1-LA

[0120] This experimental example investigated that the probes prepared in Examples 1-3 specifically target the kidneys.

[0121] Using an in vivo fluorescence imaging system, the method of fluorescence photography was adopted to determine the distribution of each protein labeled with the near-infrared fluorescent dye ZW800-1 (specifically including: ZW800-1-LG, ZW800-1-MG, and ZW800-1-LA) in mice after intravenous injection. The specific operation is as follows:

[0122] Healthy Balb / c mice (6-8 weeks old, weighing about 20 g, female gender, 3 mice corresponding to each probe) were anesthetized by the animal anesthesia system SWD, and ZW800-1-LG, ZW800-1-MG, and ZW800-1-LA were respectively injected through the tail vein (the concentration of the fluorescent dye ZW800-1 in the injection solution was 4.93 μmol / L for all, and the injection volume was 125 μL for all). Then, at different time points, fluorescence imaging was performed on the mice. The imaging and photographing conditions were: excited by an 808 nm laser, and photographed with a FOBI near-infrared camera for Balb / c female mice through a 900 nm long-pass filter FELH0900. As Figure 3 shown, within 5 minutes after injection, fluorescence was detected in the kidney region of the mice injected with each probe. As Figure 4 shown, the fluorescence in the kidney region basically disappeared one week (7 days) after injection of ZW800-1-LG, ZW800-1-MG, or ZW800-1-LA.

[0123] Furthermore, the mice injected with ZW800-1-LG, ZW800-1-MG, and ZW800-1-LA were dissected at 30 minutes and 7 days respectively, and fluorescence imaging was performed on the main organs (including: heart, liver, spleen, lung, kidney). The imaging and photographing conditions were: excited by an 808 nm laser, and photographed with a FOBI near-infrared camera for Balb / c female mice through a 900 nm long-pass filter FELH0900. The results are as Figure 5As shown, consistent with the in vivo imaging results, 30 minutes after injecting each probe, the fluorescence was mainly concentrated in the kidney region; after 7 days, the fluorescence intensity in each organ was weak. These results demonstrated that both ZW800-1-LG, ZW800-1-MG, and ZW800-1-LA could rapidly and specifically target the kidney.

[0124] Experimental Example 4 Fluorescence Imaging of Cy5-LG and FITC-LG

[0125] This experimental example investigated the specific targeting of the probes prepared in Examples 4 - 5 to the kidney.

[0126] Due to the tissue penetration depth limitations of Cy5 (excitation wavelength: 640 nm; emission wavelength: 670 nm) and FITC (excitation wavelength: 490 nm; emission wavelength: 520 nm), the kidney targeting of Cy5-LG and FITC-LG was determined by detecting the fluorescence distribution of Cy5-LG and FITC-LG in each major tissue and organ after dissection. The specific operation was as follows: Cy5-LG and FITC-LG (the dye concentration in the injection solution was 4.93 μmol / L for both, and the injection volume was 125 μL for both) were respectively injected into healthy Balb / c mice (6 - 8 weeks old, weighing about 20 g, female, 3 mice for each probe) via the tail vein. 30 minutes after injection, the mice were dissected, and the major organs (including: heart, liver, spleen, lung, kidney) were taken for fluorescence imaging. The imaging conditions were 625 nm excitation and 680 nm emission for Cy5; 470 nm excitation and 540 nm emission for FITC.

[0127] The results were as Figure 6 shown, and the main fluorescence aggregation sites of Cy5-LG and FITC-LG were both the kidney. This result indicated that Cy5-LG and FITC-LG rapidly and specifically targeted the kidney, further demonstrating that different dye modifications did not affect the targeting of the probe, and dyes with different wavelengths could be selected according to needs.

[0128] Experimental Example 5 Detection of Kidney Injury

[0129] This experimental example investigated the effect of the probes prepared in Examples 1 - 5 on detecting kidney injury.

[0130] The FOBI in vivo imaging system was used to observe the distribution and metabolism of the probe in vivo.

[0131] Method for establishing an AKI mouse model: Healthy Balb / c female mice (6 - 8 weeks old, weighing about 20 g, female) were deprived of water 15 h in advance, and a 50% (v / v) glycerol solution (8 mL / kg) was injected intramuscularly once to establish an AKI mouse model.

[0132] The specific steps for each probe are as follows: Weigh the lyophilized powder of the probe and dissolve it in PBS to prepare a solution with a concentration of 1 mg / mL. Divide 6 Balb / c mice (6 - 8 weeks old, weighing about 20 g, female) with the hair removed from the imaging area into 2 groups, with 3 mice in each group (healthy mouse group, glycerol-induced acute kidney injury mouse group). Among them, for the glycerol-induced acute kidney injury mouse group, a glycerol AKI model was established. 2 hours after injecting the glycerol solution, the mice in each group were anesthetized by the animal anesthesia system SWD. The 2 groups of mice were respectively injected with the 1 mg / mL probe solution via the tail vein, and the injection volume was 125 μL / mouse. Then, fluorescence imaging was performed on the mice at different time points. Imaging and photographing conditions: Excited by an 808 nm laser, and photographed with a FOBI near-infrared camera for Balb / c female mice through a 900 nm long-pass filter FELH0900.

[0133] After injecting ZW800-1-LG via the tail vein, the results were as Figure 7 and Figure 8 shown. A large amount of ZW800-1-β-LG could be observed to accumulate in the kidney tissue area of normal mice within 10 minutes. The fluorescence signal of ZW800-1-β-LG in the kidney remained at a high fluorescence intensity for a long time after intravenous injection. By comparing with the fluorescence imaging of glycerol-modeled AKI mice, it could be seen that the fluorescence intensity of the AKI mice's kidneys was significantly weaker. This was because when the renal tubular epithelial cells were damaged in AKI mice, the uptake ability of this probe by the damaged cells decreased, resulting in a lower fluorescence intensity in the damaged kidneys. Therefore, the probe could be used to detect AKI.

[0134] After injecting ZW800-1-LA via the tail vein, the results were as Figure 9 shown; after injecting ZW800-1-MG via the tail vein, the results were as Figure 10 shown. Similar to ZW800-1-LG, the fluorescence intensity of the kidneys of AKI mice was lower than that of normal mice. This result indicated that the uptake amounts of the three kidney-targeting proteins verified in Experimental Example 3 - lactoglobulin (LG), lactalbumin (LA), and microglobulin (MG) - were all lower under kidney injury conditions, resulting in a weaker fluorescence intensity in the damaged kidneys. Further, the schematic diagrams of the design principles and effects of each probe targeting renal tubular cells of the present invention are as Figure 23 shown.

[0135] Furthermore, AKI was rapidly detected through urine:

[0136] Method for establishing an AKI mouse model: Healthy Balb / c female mice (6 - 8 weeks old, weighing about 20 g, female) were deprived of water 15 h in advance, and an AKI mouse model was established by intramuscular injection of 50% (v / v) glycerol (8 mL / kg) once.

[0137] The specific steps for each probe are as follows: Weigh the freeze-dried powder of the probe and make a solution with a concentration of 1 mg / mL using PBS. Divide 6 Balb / c mice (6 - 8 weeks old, weighing about 20 g, female) with the hair removed from the imaging area into 2 groups, with 3 mice in each group (healthy mouse group, glycerol-induced acute kidney injury mouse group). Among them, the glycerol-induced acute kidney injury mouse group was subjected to glycerol AKI modeling. 2 hours after injecting the glycerol solution, the mice in each group were anesthetized by the animal anesthesia system SWD. The two groups of mice were respectively injected with a 1 mg / mL probe solution via the tail vein, with an injection volume of 125 μL / mouse. 30 minutes after injection, the urine of the mice was collected through the bladder and the total amount of fluorescent substances in the urine was analyzed.

[0138] The results are as Figure 11 shown. More fluorescence was detected in the urine of the acute kidney injury mice. This is because due to renal tubular injury, the uptake of ZW800-1-LG by renal tubular epithelial cells decreased, and more ZW800-1-LG was excreted through urine. Therefore, AKI can be detected by comparing the fluorescence intensity in the urine. Further, the design principle and effect schematic diagram of each probe targeting renal tubular cells of the present invention are as Figure 23 shown.

[0139] Experimental Example 6

[0140] This experimental example investigated the renal injury detection effect of the probe prepared in Example 1 on different AKI mouse models.

[0141] Method for establishing an AKI mouse model: Healthy Balb / c female mice (6 - 8 weeks old, weighing about 20 g, female) were deprived of water 15 h in advance, and an AKI mouse model was established by intraperitoneal injection of a physiological saline solution (15 mg / kg) of 1 mg / mL cisplatin once.

[0142] The specific steps of the probe are as follows: Weigh the freeze-dried powder of the probe and dissolve it in PBS to make a solution with a concentration of 1 mg / mL. Divide 6 Balb / c mice (6 - 8 weeks old, weighing about 20 g, female) with the hair removed from the imaging area into 2 groups, with 3 mice in each group (healthy mouse group, cisplatin-induced acute kidney injury mouse group). One group was intraperitoneally injected with a physiological saline solution of 1 mg / mL cisplatin (15 mg / kg) to establish an AKI mouse model; the other group was the control group with physiological saline. After 3 days, the mice with the hair removed from the imaging area were anesthetized by the animal anesthesia system SWD. Both groups of mice were respectively injected with a 1 mg / mL ZW800-1-β-LG solution via the tail vein, and the injection volume was 125 μL per mouse. Then, fluorescence imaging was performed on the mice at different time points. Imaging and photographing conditions: Excited by an 808 nm laser, and photographed with a FOBI near-infrared camera for Balb / c female mice through a 900 nm long-pass filter FELH0900.

[0143] The results are as Figure 12 and Figure 13 shown. At 10 minutes after injecting ZW800-1-LG, it was also possible to immediately observe that cisplatin-induced AKI mice had lower renal fluorescence. This indicates that ZW800-1-LG has good application prospects in the immediate detection of kidney injury.

[0144] Experimental Example 7

[0145] In this experimental example, the mice were dissected, and fluorescence imaging was performed on the main organs (including: heart, liver, spleen, lung, kidney) to investigate the kidney injury detection effect of each probe.

[0146] Performed according to Experimental Example 5, the mice 20 min after injecting the probe were dissected, and the organ tissues of the heart, liver, spleen, lung, and kidney were taken for near-infrared fluorescence ex vivo imaging. The imaging conditions were: Excited by an 808 nm laser, and photographed with a FOBI near-infrared camera for Balb / c female mice through a 900 nm long-pass filter FELH0900.

[0147] The results are as Figure 14 shown. From the organ fluorescence imaging diagrams 20 min after injecting ZW800-1-LG via the tail vein, it can be seen that obvious fluorescence signals can be observed in the kidneys of both normal mice and kidney injury mice, while the fluorescence signals of other organ tissues are relatively low. This further indicates that ZW800-1-LG can selectively light up the kidney area and maintain a strong fluorescence signal in the kidney area for a relatively long time. And the fluorescence of the kidneys of kidney injury mice is significantly weaker than that of normal healthy mice. By comparing the bar graphs of the fluorescence intensities of the kidneys of normal mice and kidney injury mice, it can also be seen that the fluorescence intensity of the kidneys of normal mice is higher than that of kidney injury mice.

[0148] Experimental Example 8 Cell Co-localization Test

[0149] This experimental example investigated the phagocytosis of the probe prepared in Example 1 by renal tubular epithelial cells.

[0150] The probe was operated as follows: The probe (final concentration of 10 μg / mL) was added to the culture medium of HK2 cells (cell density of about 50%) (the culture medium was RPMI1640) and incubated for 2 h. At the same time, Lyso Tracker Green (operated according to the instructions) and Hoechst 33342 (operated according to the instructions) were used to stain lysosomes and cell nuclei. The distribution of the probe in the cells was observed.

[0151] Through Figure 15 fluorescence imaging, ZW800-1-LG could be effectively taken up by HK2 cells and was mainly distributed in the cytoplasm. The above indicates that the said probe can be taken up by normal renal tubular epithelial cells.

[0152] Experimental Example 9 Kidney Distribution

[0153] This experimental example investigated the uptake of the probe prepared in Example 1 by the kidney.

[0154] The probe was operated as follows: 125 μL of the probe, which was a 1 mg / mL ZW800-1-β-LG solution, was injected into healthy Balb / c mice (6-8 weeks old, weighing about 20 g, female) through the tail vein. After 20 min, the kidneys were immediately fixed in 10% neutral buffered formalin, and then standard dehydration and paraffin embedding were carried out. The embedded tissue was cut into 4-μm sections. The final sections were observed under an Olympus IX 73 fluorescence microscope.

[0155] The results were as Figure 16 shown. By injecting the said probe through the tail vein, ZW800-1-LG was mainly taken up by renal tubular epithelial cells.

[0156] Experimental Example 10 Kidney Tumor Detection

[0157] This experimental example investigated the effect of the probe prepared in Example 1 on detecting kidney tumors.

[0158] nude mice (6-8 weeks old, weighing about 20 g, female) were taken and an in-situ renal tumor model was established on the right kidney using the ACHN cell line. The specific method was as follows: Cells in the logarithmic growth phase were digested with 0.25% trypsin and resuspended. After cell viability determination and cell counting, the density of viable cells was adjusted to 5×10 7cells / mL. The nude mice were anesthetized by intraperitoneal injection of 3.5% chloral hydrate solution, then fixed and disinfected. The skin and peritoneum were incised along the midline of the abdomen to expose the right kidney. A sterile syringe was used to aspirate the cell suspension with adjusted cell viability and concentration, and the needle was inserted from the lower pole of the kidney through the renal parenchyma until under the capsule of the upper pole of the kidney. The sign of successful injection was the swelling of the renal parenchyma and the whitening of the renal capsule color. The injection dose was 0.01 mL. After suturing the skin, the mice were placed under SPF conditions and continued to be raised.

[0159] The operation of each probe is as follows: One hour after injecting 125 μL of 1 mg / mL probe through the tail vein, fluorescence imaging of the mouse kidney was performed. Imaging and photographing conditions: Excited by an 808 nm laser, and photographed with a FOBI near-infrared camera for Balb / c female mice through a 900 nm long-pass filter FELH0900.

[0160] The results are as Figure 17 shown. There are obvious differences in fluorescence intensity between the tumor on the right kidney and the normal tissue of the kidney. The fluorescence on the tumor at the edge of the kidney is significantly weaker, indicating that the probe can be taken up by renal tubular epithelial cells in the normal kidney but not by cancerous tumor cells. Therefore, the distinction between normal kidney tissue and tumor is initially judged by the general outline of the photographed kidney. In addition, it is also applicable to intraoperative imaging to help distinguish the tumor boundary.

[0161] Experimental Example 11 Gel Electrophoresis and Absorption Spectrum Characterization

[0162] The drugs prepared in Examples 6-8 were subjected to gel-electrophoresis testing and absorption spectrum characterization. The specific operations are as follows:

[0163] Experimental group: GA-PEG-LG (or GA-PEG-LA, GA-PEG-MG) powder was added to 20 μL of phosphate buffer solution (PBS) to prepare a test sample with a concentration of 0.02 mM, and 5 μL of Coomassie Brilliant Blue (CBB) 250 staining solution was added for mixed staining. The CBB 250 staining solution contains 10% (v / v) CBB and 90% (v / v) PBS.

[0164] Control group: β-LG (or LA, or MG) powder was added to 20 μL of PBS buffer solution to prepare test samples with a concentration of 0.02 mmol / L respectively, and 5 μL of CBB 250 staining solution was added for mixed staining.

[0165] Two samples were analyzed by 2% agarose gel electrophoresis. The results are as Figure 18 shown in the left figure in the middle. Both GA-PEG-LG and β-LG migrated towards the positive electrode, but GA-PEG-LG carried less negative charge. In comparison, GA-PEG-LG migrated more slowly towards the positive electrode. Similarly, in Figure 19As shown in the left middle figure, both GA-PEG-MG and MG move towards the positive electrode. However, GA-PEG-MG carries less negative charge. In comparison, GA-PEG-MG moves towards the positive electrode more slowly. In Figure 20 As shown in the left middle figure, both GA-PEG-LA and LA move towards the positive electrode. However, GA-PEG-LA carries less negative charge. In comparison, GA-PEG-LA moves towards the positive electrode more slowly.

[0166] The absorption spectral properties of the prepared GA-PEG-β-LG, GA-PEG-LA, and GA-PEG-MG solutions were characterized. The results are as Figures 18 - 20 shown in the right middle figure. GA-PEG-β-LG has characteristic absorption peaks of both β-LG and GA. GA-PEG-LA has characteristic absorption peaks of both LA and GA. GA-PEG-MG has characteristic absorption peaks of both MG and GA.

[0167] The above shows that in the present invention, gambogic acid was successfully loaded onto three proteins, and GA-PEG-β-LG, GA-PEG-LA, and GA-PEG-MG were successfully prepared.

[0168] Experimental Example 12

[0169] The drugs prepared in Examples 6-8 were subjected to a cell co-localization test to verify that GA-PEG-LG can protect HK-2 (human proximal tubular epithelial cells) from H2O2-induced oxidative stress. GA-PEG-β-LG, GA-PEG-LA, or GA-PEG-MG (final incubation concentration 10 μg / mL) dissolved in PBS and H2O2 (final incubation concentration 350 μM) were incubated into HK-2 cells (cell density approximately 50%). At the same time, Mitochondria (operated according to the instructions) and Hoechst 33342 (operated according to the instructions) were used to stain the mitochondria and nuclei, and then microscopic observation was carried out. The results are as Figure 21 shown. It can be clearly seen that H2O2 induces mitochondrial fragmentation in HK-2 cells, while pretreatment with GA-PEG-β-LG (10 μg / mL) can effectively reduce the oxidative stress damage of the cells.

[0170] Experimental Example 13

[0171] This experimental example investigated the effects of the drugs prepared in Examples 6-8 on kidney injury.

[0172] Each drug was operated as follows: 10 normal C57BL / 6 mice (aged 6 - 8 weeks, weighing about 20 g, female) were taken. After blood collection, an AKI model with glycerol injury was established in the same way as in Experimental Example 5. Subsequently, the mice were randomly divided into two groups and injected with GA-PEG-LG, GA-PEG-LA or GA-PEG-MG (concentration 40 mg / mL) and PBS respectively, with an injection volume of 125 μL. After 24 hours, blood was collected again for the tests of CRE (serum creatinine) and BUN (blood urea nitrogen). The above CRE and BUN were detected using conventional commercially available kits.

[0173] The results are as Figure 22 shown. The levels of renal function indexes CRE and BUN in the GA-PEG-LG treatment group were much lower than those in the PBS group and there was no significant difference from those before injury. This shows that GA-PEG-β-LG can significantly improve renal injury and restore renal function, having good application prospects. In early renal injury, some cells necrosis and exfoliate, but some adjacent cells can still perform corresponding functions. In the treatment of renal injury in this experiment, for the early injured renal tubular epithelial cells, some renal tubular epithelial cells necrosis and exfoliate, and some renal tubular cells can uptake the corresponding proteins (proteins that can be reabsorbed by healthy kidneys). After the protein drugs (GA-PEG-LG, GA-PEG-LA or GA-PEG-MG) carrying antioxidant drugs are uptaken, these cells can be rescued, ROS can be eliminated, and these cells can be prevented from oxidative damage, achieving the purpose of treatment.

[0174] Obviously, the above embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. Use of a protein in the preparation of a product targeting the kidney, characterized in that, The protein is a protein that can be reabsorbed by a healthy kidney.

2. The use according to claim 1, characterized in that, The protein includes at least one of β-lactoglobulin, α-lactalbumin or β2-microglobulin.

3. A kidney-targeting probe, characterized in that, Comprising: Carrier: including the protein described in any one of claims 1-2; Signal substance; The signal substance is modified on the carrier.

4. The kidney-targeting probe according to claim 3, wherein The signal substance includes a dye; the dye includes a fluorescent dye; the fluorescent dye includes an ultraviolet-visible region fluorescent dye, a far-infrared region dye or a near-infrared region dye; the fluorescent dye includes at least one of fluorescein dyes, rhodamine dyes, cyanine dyes or quantum dot dyes; And / or, the signal substance is modified on the carrier by chemical bonding, physical adsorption or coating.

5. The kidney-targeting probe according to claim 4, wherein, The dye includes at least one of ZW800-1, FITC, Cy5; And / or, the dye is covalently modified on the carrier; the bonding site of the dye and the carrier is the amino group or sulfhydryl group of the carrier; optionally, the NHS ester modified on ZW800-1, FITC, Cy5 is bonded to the amino group of the carrier.

6. A method for preparing a kidney-targeting probe according to any one of claims 3-5, characterized in that, Comprising the following steps: (1) Modify the signal substance; (2) Modify the signal substance obtained in step (1) on the carrier.

7. The method for preparing the kidney-targeting probe according to claim 6, characterized in that, When the signal substance is a dye, in step (1), the dye is carboxyl-modified or not carboxyl-modified, and then NHS-modified or not NHS-modified; And / or, when the signal substance is a dye, the modified dye in step (1) is covalently modified on the carrier.

8. Use of a kidney-targeting probe as described in any one of claims 3-5 or a kidney-targeting probe prepared by the preparation method described in any one of claims 6-7 in any of the following: (1) Use in kidney-targeted imaging; (2) Use in the preparation of products for detecting kidney diseases.

9. The use according to claim 8, wherein, Comprising use in any of the following: (1) Use in tubular-targeted fluorescence imaging; (2) Use in the preparation of products for detecting kidney injury or kidney tumors; The product includes a reagent or a kit.

10. A drug targeting the kidney, characterized in that, Comprising: Carrier: including the protein described in any one of claims 1-2; Drug; The drug is modified on the carrier.

11. The kidney-targeted drug according to claim 10, wherein The drug includes an antioxidant stress injury drug; And / or, the drug is modified on the carrier by chemical bonding, physical adsorption or coating.

12. The kidney-targeted drug according to claim 11, wherein The drug includes gambogic acid; And / or, the drug is covalently modified on the carrier.

13. A method for preparing a kidney-targeted drug according to any one of claims 10-12, characterized in that, Comprising: Modify the drug on the carrier.

14. The preparation method of the kidney-targeted drug according to claim 13, wherein, When the drug is selected as gambogic acid, it includes: (1) Modify gambogic acid with polyethylene glycol containing at least one amino functional group and at least one carboxyl functional group through the amino functional group to obtain compound 1; (2) Perform NHS modification on the carboxyl end of polyethylene glycol in the compound 1 obtained in step (1) to obtain compound 2; (3) Mix the compound 2 obtained in step (2) with the carrier and carry out a reaction.

15. The preparation method of the kidney-targeted drug according to claim 14, wherein Comprising: In step (1), the molecular weight range of polyethylene glycol is ≥3000Da; And / or, in the step (1), the molar ratio of polyethylene glycol containing at least 1 amino functional group and at least 1 carboxyl functional group to gambogic acid is 1:1 - 10:1; And / or, in the step (1), gambogic acid is dissolved in an organic solvent, and then polyethylene glycol containing at least 1 amino functional group and at least 1 carboxyl functional group is added, and the reaction is carried out at 0 - 37 °C for 0.5 - 48 h; And / or, in the step (2), compound 1 is dissolved, and then N-hydroxysuccinimide is added and mixed, and the molar ratio of the two is 1:1 - 1:10, and the reaction condition is to react at 0 - 37 °C for 0.5 - 2 h; And / or, in the step (3), the compound 2 obtained in the step (3) is mixed with the carrier and dissolved, and the molar ratio of compound 2 to the carrier is 10:1 - 10:1, and the reaction condition is to react at 0 - 37 °C for 0.5 - 48 h.

16. Use of a drug targeting the kidney according to any one of claims 10 - 12 or a drug targeting the kidney prepared by the preparation method according to any one of claims 13 - 15 in the preparation of a drug for targeted prevention or treatment of kidney diseases.

17. The use according to claim 16, characterized in that, The kidney diseases include renal tubular injury.

Images