Gallus gallus domesticus beta-lactoglobulin reabsorption-mediated gambogic acid renal delivery system, synthetic method and application thereof

Abstract

This invention belongs to the field of biomedicine and nanomedicine delivery technology, and relates to a precise renal-targeted delivery system for gambogeylic acid mediated by the β-lactoglobulin reabsorption mechanism, its synthesis method, and applications. The system comprises β-lactoglobulin-PEG-gambogeylic acid nanoparticles, containing β-lactoglobulin as a targeting carrier and gambogeylic acid as the active ingredient, covalently linked by a polyethylene glycol chain. This invention utilizes the suitable molecular size of β-lactoglobulin and its ability to be recognized and reabsorbed by the megalin / cubilin receptor system of renal tubular epithelial cells to construct a nanodelivery system that mimics the physiological process of endogenous protein "filtration-reabsorption." Experiments have shown that this system significantly improves the water solubility and biocompatibility of gambogeylic acid, enabling selective accumulation in the kidneys and efficient capture by renal tubular epithelial cells in vivo. It exhibits superior therapeutic effects in a mouse model of cisplatin- and glycerol-induced acute kidney injury, significantly outperforming the positive control drug NAC.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine delivery technology, and relates to a nanosystem for precise drug delivery by utilizing the physiological reabsorption mechanism of the kidney. Specifically, it relates to a β-lactoglobulin reabsorption mechanism-mediated gambogeylic acid kidney-targeted delivery system, its synthesis method, and its application. Background Technology

[0002] Acute kidney injury (AKI) is a common critical clinical condition characterized by a rapid decline in renal function, accompanied by the accumulation of metabolic waste products, electrolyte disturbances, and acid-base imbalances. Its etiology is complex, including ischemia-reperfusion injury, sepsis, and nephrotoxic drugs (such as cisplatin). Currently, clinical treatment for AKI is mainly limited to supportive therapies (such as renal replacement therapy / dialysis), and there is a lack of specific therapeutic drugs that can effectively promote the recovery of renal structure and function. Therefore, the development of novel, highly effective, low-toxicity drugs that target the kidneys has significant clinical and social value.

[0003] Gambogic acid (GA) is a natural compound isolated from the resin secreted by the Garcinia cambogia plant (Garcinia cambogia). Modern pharmacological studies have shown that, in addition to its broad-spectrum antitumor activity, GA also exhibits significant anti-inflammatory, anti-apoptotic, and anti-oxidative stress effects. Recent studies have indicated that GA can reduce the production of reactive oxygen species (ROS) and has a certain protective effect against renal tubular epithelial cell damage, making it a potential candidate drug for the treatment of acute kidney injury (AKI).

[0004] However, gambogeylic acid faces the following technical bottlenecks in clinical translation and basic research:

[0005] (1) Extremely poor water solubility: Garcinia cambogia is a fat-soluble compound with extremely low solubility in water, resulting in poor bioavailability and limiting its in vivo administration route and therapeutic effect.

[0006] (2) Significant toxic side effects: Due to its lack of selectivity in distribution in the body, gambogeylic acid tends to accumulate in organs such as the liver and kidneys while exerting therapeutic effects, exhibiting significant systemic toxicity (such as hepatotoxicity and vascular irritation), resulting in a narrow therapeutic window and greatly limiting its direct application in the treatment of acute kidney injury.

[0007] (3) Lack of targeting: Traditional administration methods make it difficult for gamboge to specifically accumulate in damaged kidney tissue, resulting in insufficient drug concentration reaching the lesion and difficulty in effectively reversing the progression of AKI.

[0008] To overcome these shortcomings, researchers have attempted to encapsulate gambogeylic acid using nanodelivery systems. However, commonly used synthetic polymer nanocarriers (such as PLGA and liposomes) suffer from problems such as complex preparation processes, relatively insufficient biocompatibility, or potential immunogenicity.

[0009] Utilizing the physiological mechanisms of the kidneys to achieve targeted drug delivery has become a research hotspot in the field of kidney disease treatment in recent years. The kidneys are the main organ for clearing circulating low molecular weight proteins (LMWPs). Studies have shown that proteins with a molecular weight below 30-50 kDa can freely cross the glomerular filtration barrier and enter the primary urine; subsequently, these filtered proteins are efficiently reabsorbed in the proximal tubules of the kidneys—primarily mediated by the megalin / cubilin receptor system on the brush border membrane of the proximal tubular epithelial cells, entering the cells via endocytosis, and finally being degraded in lysosomes. This physiological process of "filtration-reabsorption" provides a unique theoretical basis for targeted drug delivery to the kidneys. Since the 1990s, researchers have explored using low molecular weight proteins such as lysozyme (14 kDa), insulin, and growth hormone as carriers to target small molecule drugs (such as naproxen, captopril, methylprednisolone, and kinase inhibitors) to the kidneys. Studies have shown that after conjugation with LMWP, drugs are primarily taken up by the kidneys, and the parent drug or active metabolites can be released within the kidneys. For example, Franssen et al. reported that after injection of naproxen-lysozyme conjugates, the kidneys were the primary organ of uptake, and their intrarenal distribution was confirmed by radiographic imaging. However, current research mainly focuses on a few proteins such as lysozyme, and often employs chemical conjugation methods (such as covalently binding drugs to proteins via breakable linkers). This approach has drawbacks, including complex linker design, uncertain drug release efficiency, and potential alteration of the protein's native conformation.

[0010] β-Lactoglobulin (β-LG) is the main protein in whey of ruminants (cattle, sheep, etc.), accounting for about 50% of the total whey protein. Its molecular weight is about 18.4 kDa (monomer), and it exists in dimer form (~36.6 kDa) under physiological conditions. β-LG can bind and transport various hydrophobic small molecules (such as retinol, fatty acids, etc.) through non-covalent interactions. Based on its molecular size (~36.6 kDa dimer) falling within the glomerular filtration threshold range, and its potential as a natural protein that can be recognized and reabsorbed by the megalin / cubilin receptor system, β-LG possesses the potential to be an ideal renal targeting carrier: (1) Suitable molecular weight: It can be effectively filtered through the glomerulus without being cleared too quickly; (2) Natural source with excellent biocompatibility: It is biodegradable, has low immunogenicity, and avoids the potential risks of synthesizing high molecular weight carriers; (3) Hydrophobic drug binding ability: It can efficiently encapsulate hydrophobic gambogeylic acid through non-covalent interactions without complex chemical coupling, thus preserving the natural activity of gambogeylic acid; (4) Potential intracellular release mechanism: After entering proximal tubular cells, β-LG can be degraded in lysosomes, thereby achieving intracellular drug release. However, there are currently no reports on using β-LG as a carrier to transport gambogeylic acid and to prepare drugs for the treatment of acute kidney injury.

[0011] In summary, developing a gamwagandhi acid renal targeted delivery system (GA-NPs) mediated by the β-lactoglobulin reabsorption mechanism to achieve solubilization, detoxicity reduction, and enhanced renal targeted delivery capacity of gamwagandhi acid for the treatment of acute kidney injury is of significant research value and application prospects for solving existing technical challenges. Summary of the Invention

[0012] The purpose of this invention is to overcome the technical difficulties of poor water solubility, lack of selectivity in vivo distribution, large toxic side effects, and difficulty in effectively accumulating in renal lesions of gambogeylic acid, and to provide a gambogeylic acid renal delivery system based on β-lactoglobulin reabsorption, its synthesis method, and its application.

[0013] Technical solution

[0014] A renal delivery system for gambogic acid mediated by β-lactoglobulin reabsorption is disclosed. The delivery system comprises β-lactoglobulin-PEG-gambogic acid nanoparticles (GA-NPs), which contain β-lactoglobulin (β-LG) as a targeting carrier and gambogic acid (GA) as an active ingredient. The β-lactoglobulin and gambogic acid are covalently linked by a polyethylene glycol (PEG) chain.

[0015] Preferably, the molecular weight of the polyethylene glycol chain is 2000-5000 Da; more preferably, the polyethylene glycol chain is PEG3400.

[0016] The first objective of this invention is to disclose a method for synthesizing the above-mentioned β-lactoglobulin-PEG-garcinia phosphate nanoparticles (GA-NPs), comprising the following steps:

[0017] S1. Synthesis of intermediate PEG-gamboge acid: Gamboge acid was reacted with activated ester form of carboxyl-polyethylene glycol-amino (COOH-PEG-NH2) in an organic solvent in the presence of a catalyst and under inert gas protection. After the reaction was completed, the intermediate product PEG-gamboge acid was obtained by purification.

[0018] S2. Synthesis of β-lactoglobulin-PEG-gammonic acid nanoparticles (GA-NPs): The carboxyl group at the end of the intermediate product PEG-gammonic acid was activated, and then mixed with β-lactoglobulin in a buffer solution for amidation reaction, so that the amino group on β-lactoglobulin was covalently linked to the activated PEG-gammonic acid, and the target nanoparticles were obtained by purification.

[0019] The activated ester form of carboxyl-polyethylene glycol-amino (COOH-PEG-NH2) described in this invention is a polyethylene glycol with an amino group at one end and a carboxyl group at the other end.

[0020] In a preferred embodiment of the present invention, in step S1, the organic solvent is dichloromethane or N,N-dimethylformamide; and the catalyst is 4-dimethylaminopyridine or an EDCI / NHS system.

[0021] In a preferred embodiment of the present invention, in step S2, the buffer solution is PBS or MES buffer.

[0022] A second objective of this invention is to protect a kidney-targeting fluorescent imaging probe, wherein the probe is the aforementioned β-lactoglobulin-gambogeylic acid nanoparticles labeled with a near-infrared fluorescent dye.

[0023] Preferably, the near-infrared fluorescent dye is ZW800-1.

[0024] The third objective of this invention is to disclose a method for preparing the above-mentioned targeted kidney imaging probe ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1), which involves mixing β-lactoglobulin-PEG-gambogeylic acid nanoparticles GA-NPs and ZW800-1 in activated ester form in a buffer solution, and then purifying the mixture to obtain solid ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1).

[0025] In a preferred embodiment of the present invention, the buffer solution is PBS or MES buffer.

[0026] The activated ester form of ZW800-1 described in this invention is a ZW800-1 active ester near-infrared fluorescent dye prepared by chemical coupling with N-hydroxysuccinimide (NHS) based on the zwitterionic near-infrared fluorophore ZW800-1.

[0027] A fourth object of the present invention is to protect the use of the aforementioned β-lactoglobulin-garcinic acid nanoparticles in the preparation of medicaments for treating acute kidney injury, and in the preparation of reagents for the following purposes:

[0028] (1) Used to prepare reagents for in vitro analysis of cytotoxicity, and to evaluate the killing or protective effect of gamboge on renal cells by measuring cell viability;

[0029] (2) Tracer reagents for preparing colocalization analysis of organelles, which are used to trace the distribution of gambogeylic acid or β-lactoglobulin in living cells and their interaction with organelles such as mitochondria and nucleus by labeling them;

[0030] (3) Used to prepare fluorescent probe reagents for detecting intracellular reactive oxygen species levels, and to evaluate the regulation of oxidative stress by gambogeylic acid by flow cytometry or fluorescence microscopy;

[0031] (4) Used to prepare biochemical analysis reagents for monitoring in vivo renal function, and to evaluate the therapeutic effect of the complex on acute kidney injury by detecting the levels of creatinine and urea nitrogen in serum samples;

[0032] (5) A staining contrast agent for preparing histopathological analysis, wherein the effect of the complex on the improvement of renal tissue pathological morphology is evaluated by H&E staining of renal tissue.

[0033] Beneficial effects

[0034] This invention utilizes the hydrophobic lumen of β-lactoglobulin to encapsulate gambogeylic acid, and through hydrophilic PEG modification, forms nanoparticles with uniform particle size, significantly improving the water solubility of gambogeylic acid. In vitro cell experiments confirmed that these nanoparticles exhibit excellent cell compatibility even at concentrations up to 200 μg / mL, effectively reducing the systemic toxicity risk of gambogeylic acid. This invention is the first to utilize the property that β-lactoglobulin can be specifically recognized by megalin / cubilin receptors on renal tubular epithelial cells to construct an active kidney-targeting nanodelivery system. In vivo imaging experiments demonstrated that these nanoparticles can selectively accumulate and remain in the kidney for a long time, significantly increasing the drug concentration at the lesion site. In two mouse models of AKI induced by cisplatin and glycerol, the treatment group with the nanoparticles of this invention significantly reduced serum creatinine and blood urea nitrogen levels to the normal range, and effectively repaired renal tubular damage and inhibited inflammatory cell infiltration. Its efficacy is significantly better than that of the positive control drug NAC at 20 mg / kg, demonstrating strong nephroprotective effects and therapeutic potential. The probe constructed by coupling with the near-infrared fluorescent dye ZW800-1 enables real-time, non-invasive monitoring of the fate of nanoparticles in vivo. This invention is the first to apply the reabsorption mechanism of β-lactoglobulin to precise drug delivery, providing a novel platform technology for the integrated research of treatment and diagnosis of kidney diseases. Attached Figure Description

[0035] Figure 1 A schematic diagram illustrating the specific synthetic pathway of β-lactoglobulin-PEG-gambogeylic acid and its mechanism of action in treating kidney injury.

[0036] Figure 2 UV absorption and fluorescence spectra of β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs);

[0037] Figure 3 Agarose gel electrophoresis characterization of β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs);

[0038] Figure 4 TEM analysis of β-lactoglobulin-PEG-garnet acid nanoparticles (GA-NPs);

[0039] Figure 5 UV absorption and fluorescence spectra of ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1);

[0040] Figure 6 Agarose gel electrophoresis characterization of ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1);

[0041] Figure 7MTT assay of β-lactoglobulin-PEG-garnet acid nanoparticles (GA-NPs) in HK-2 cells;

[0042] Figure 8 Colocalization imaging of β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs) with organelle dyes;

[0043] Figure 9 ROS analysis of β-lactoglobulin-PEG-garnet acid nanoparticles (GA-NPs) in HK-2 cells;

[0044] Figure 10 In vivo fluorescence imaging was performed in normal mice at different time points after tail vein injection of ZW800-1-β-lactoglobulin-PEG-garcinia tartaric acid nanoparticles (GA-NPs-ZW800-1) and free ZW800-1, respectively.

[0045] Figure 11 The fluorescence time-intensity curves of the kidneys of normal mice were analyzed by tail vein injection of ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1) and free ZW800-1, respectively.

[0046] Figure 12 . Imaging of isolated organs and tissues was performed 8 hours after normal mice were injected with ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1) and free ZW800-1 via tail vein injection, respectively.

[0047] Figure 13 The kidney strength of normal mice was compared 8 hours after injection of ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1) via tail vein injection and free ZW800-1.

[0048] Figure 14 Eight hours after GA-NPs-ZW800-1 was injected via the tail vein, a 40x magnified kidney section was dissected and imaged.

[0049] Figure 15 Serum CRE and BUN levels in the blood of cisplatin-induced AKI mice and glycerol-induced AKI mice in each group (n=5 per group);

[0050] Figure 16 Pathological analysis of kidney sections in cisplatin-induced AKI mice and glycerol-induced AKI mice in different groups. Detailed Implementation

[0051] The present invention will be described in detail below with reference to embodiments to enable those skilled in the art to better understand the present invention, but the present invention is not limited to the following embodiments. Unless otherwise specified, the raw materials and equipment involved in the embodiments are all conventional commercially available products in the art.

[0052] Example 1

[0053] The method for synthesizing β-lactoglobulin-PEG-garcinic acid includes the following steps:

[0054] S1. Synthesis of PEG3400-Galamyric Acid:

[0055] Weigh 1 eq GA (Gatamycin, CAS: 2752-65-0), 1.2 eq EDC·HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, CAS: 25952-53-8), and 1.2 eq NHS (N-hydroxysuccinimide, CAS: 6066-82-6) and add them to 2 mL DMF (N,N-dimethylformamide, CAS: 68-12-2). Add 3 eq TEA (triethylamine, CAS: 121-44-8), stir at room temperature for 5 min, then add 3 eq NH2-PEG3400-COOH, and stir the mixture in a water bath at 37°C for 24 h. After dialysis in water for 24 h, the solution was concentrated by ultrafiltration and passed through a Sephadex LH-20 gel column. The first elution phase solution was collected to obtain a purified intermediate product PEG3400-gambolic acid solution. The solution was then lyophilized to obtain a yellow solid of intermediate product PEG3400-gambolic acid.

[0056] Synthesis of S2.β-lactoglobulin-PEG-garnet acid nanoparticles (GA-NPs):

[0057] Weigh 1 eq PEG3400-Ganamica acid, 10 eq EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, CAS: 25952-53-81), and 5 eq NHS (N-hydroxysuccinimide, CAS: 6066-82-6) and add them to 3 mL of MES (2-morpholinoethanesulfonic acid, CAS: 4432-31-9) buffer at pH 6.0. The mixture is stirred in a water bath at 37°C for 2 h, concentrated by ultrafiltration, and passed through a Sephadex LH-20 gel column. The first eluent is collected and lyophilized to obtain a yellow solid of PEG3400-NHS-Ganamica acid. Weigh 2 eq PEG3400-NHS-Ganamica acid and 1 eq β-LG (β-lactoglobulin, CAS: 9005-43-7) and add them to 3 mL of MES (2-morpholinoethanesulfonic acid, CAS: 4432-31-9) buffer. The mixture was stirred in a water bath at 37°C for 24 hours in MES buffer at pH 8.0. After ultrafiltration and concentration, the mixture was passed through a Sephadex LH-20 gel column. The first elution phase solution was collected and lyophilized to obtain β-lactoglobulin-PEG-garcinia sorbitol nanoparticles (GA-NPs) lyophilized powder.

[0058] like Figure 1 The diagram shows the specific synthesis pathway and kidney injury treatment mechanism of β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs).

[0059] like Figure 2 As shown, the UV-Vis absorption spectroscopy clearly shows that β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs) retain the characteristic absorption peaks of β-LG and GA. Further fluorescence spectroscopy analysis shows that the intrinsic fluorescence of β-LG in β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs) is significantly suppressed, which is more significant than that of free β-LG. This proves the successful synthesis of β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs).

[0060] like Figure 3 As shown, agarose gel electrophoresis revealed that the migration rate of GA-NPs was significantly lower than that of free β-LG, and the significant band shift confirmed successful covalent binding through changes in molecular weight and surface charge. Crucially, when cultured with fetal bovine serum (FBS), GA-NPs maintained their original migration characteristics without the appearance of additional bands or significant smearing, indicating minimal nonspecific binding to serum proteins. This resistance to protein coronation suggests that GA-NPs can effectively evade uptake by the reticuloendothelial system (RES), thereby avoiding rapid clearance from the liver and ensuring the prolonged systemic circulation required for subsequent renal targeting.

[0061] like Figure 4As shown, transmission electron microscopy (TEM) revealed that the prepared GA-NPs nanoparticles possessed a regular morphology and uniform dispersion, with a primary particle size distribution of 5.64 ± 1.44 nm. This particle size is below the glomerular filtration size threshold (≈6 nm), indicating good potential for glomerular filtration, which may facilitate systemic drug delivery and targeted accumulation in renal tissue.

[0062] Example 2

[0063] Synthesis of ZW800-1-β-lactoglobulin-PEG-garnet acid nanoparticles (GA-NPs-ZW800-1)

[0064] The β-lactoglobulin-PEG-gambogeylic acid nanoparticles GA-NPs (1 eq) and ZW800-1-NHS (1 eq) prepared in Example 1 were dissolved in 2 mL PBS, stirred in a 37 °C water bath for 24 h, concentrated by ultrafiltration, passed through a Sephadex LH-20 gel column, and the first eluent was collected to obtain a purified GA-NPs-ZW800-1 solution, which was then freeze-dried to obtain ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1) solid.

[0065] like Figure 5 As shown, the UV-Vis absorption spectroscopy clearly shows that ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1) retain the characteristic absorption peaks of β-LG, GA, and ZW800-1. Further fluorescence spectroscopy analysis shows that the intrinsic fluorescence of ZW800-1 in ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1) is significantly suppressed, which is more significant than that of free ZW800-1. This proves the successful synthesis of ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1).

[0066] like Figure 6 As shown, the agarose gel electrophoresis results further confirmed the target product ZW800-1-β-lactoglobulin-PEG-gambogeylic acid nanoparticles (GA-NPs-ZW800-1).

[0067] Example 3

[0068] Evaluation of the in vitro biological effects of GA-NPs

[0069] 1. Cytotoxicity Assay (MTT Assay): HK-2 cells were seeded at a density of 1 × 10⁴ cells per well in 96-well plates, with 100 μL of cell culture medium per well. After overnight incubation, the cells were treated with fresh cell culture medium containing different concentrations of GA-NPs (0-200 μg / mL), with blank and control groups established. After 24 h of incubation with the drug, cytotoxicity and proliferation were assessed using the MTT assay (3-(4,5-dimethylthiozolium-2-yl)-2,5-diphenyltetrazol bromide). Specifically, the cell culture medium was removed, and 200 μL of fresh medium containing MTT (0.5 mg / mL) was added to each well. The plates were incubated at 37°C for 4 h in a 5% CO₂ incubator. The MTT-containing medium was then removed, and 100 μL of DMSO solution was added to each well. The absorbance intensity was measured at 492 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Results are as follows: Figure 7 The results showed that cell viability was not affected even at concentrations up to 200 μg / mL (P > 0.05), confirming the excellent cell compatibility of GA-NPs and laying a safe therapeutic window for subsequent applications.

[0070] 2. Organelle colocalization and mitochondrial protection: HK-2 cells were seeded on coverslips in 24-well plates and pretreated with GA-NPs before being exposed to H2O2 for 24 h to establish an oxidative stress injury model. Cells were first stained with MitoTracker™ Red CMXRos dye at 37°C for 30 min, followed by Hoechst 33342 staining for 10 min at the same temperature. The coverslips were then fixed with 4% formaldehyde for 10 min, and observed using an FV3000 confocal microscope (Olympus, Japan). Results are as follows: Figure 8 As shown, pretreatment with GA-NPs significantly reduced H2O2-induced mitochondrial damage, indicating that GA-NPs have a protective effect against mitochondrial dysfunction induced by oxidative stress.

[0071] 3. Intracellular Reactive Oxygen Species (ROS) Scavenging Capacity Assay: HK-2 cells were seeded into 24-well plates and grouped as above. After 24 hours of incubation, cells were collected, and intracellular ROS levels and apoptosis were measured using a ROS detection kit or an Annexin V-FITC / PI apoptosis kit. To observe ROS in live cells, HK-2 cells were incubated at 37°C with the DCFH-DA probe for 30 minutes, washed three times with 1×PBS, and then immediately imaged using an Olympus IX73 fluorescence microscope (Olympus, Japan). Results are as follows: Figure 9 As shown, H2O2 treatment triggered a strong surge in ROS production; however, GA-NPs significantly reduced this oxidative stress, demonstrating a strong free radical scavenging ability.

[0072] Example 4

[0073] In vivo evaluation of GA-NPs' renal targeting and verification of its "reabsorption" mechanism

[0074] Based on the studies in Examples 1-3, the ability of GA-NPs to be delivered in vivo to kidney tissue was further evaluated. Images were acquired at different time points (excitation: 710 / 50 nm, emission: 830 / 50 nm, exposure time: 3500 ms). The mice were placed on an imaging bed of a small animal in vivo imaging system (Neoscience FOBI). During imaging, the mice were anesthetized with nasal cones (isoflurane) mounted on the imaging bed. Subsequently, fluorescence images were acquired at different time points under the exact same imaging settings. All post-injection images were taken with the same parameter settings and adjusted to the same maximum values. The temperature of the imaging bed was maintained at 37°C throughout the imaging process.

[0075] like Figure 10-14 As shown, tail vein injection of GA-NPs-ZW800-1 resulted in rapid and preferential accumulation in the kidneys, with a significant fluorescent signal appearing almost immediately in the renal region. Notably, the fluorescence intensity remained stable and did not diminish over 8 hours, indicating that GA-NPs can remain at the lesion site for a long time to achieve a sustained therapeutic effect. In contrast, mice injected with free ZW800-1-NHS exhibited a significantly different distribution pattern, with minimal accumulation in the kidney region and rapid excretion, confirming that it cannot survive independently in kidney tissue. The free ZW800-1-NHS probe had no background interference, verifying its reliability as a fluorescent reporter, capable of accurately tracking the real-time biodistribution and metabolic fate of GA-NPs in vivo. The experimental results also confirmed that binding with β-LG significantly altered the pharmacokinetic profile of the small molecule. Eight hours after injection, major organs (heart, liver, spleen, lung, kidney, and muscle) were collected and subjected to in vitro fluorescence imaging.

[0076] like Figure 12As shown, while no significant fluorescence signal was detected in any organ of the control mice, only a strong signal was observed in the kidneys of mice treated with GA-NPs-ZW800-1. The fluorescence intensity of the experimental group kidneys was approximately 14.7 times that of the control group, consistent with earlier non-invasive imaging results. GA-NPs constructed based on β-LG exhibited good kidney targeting and enhanced in vivo retention in vivo. Fluorescence imaging of kidney sections revealed significant and widespread fluorescence signals in the renal tubular compartments at 40x magnification. This unique localization pattern provides direct histological evidence: GA-NPs were successfully filtered through the glomerulus and captured by renal tubular epithelial cells via reabsorption, thus confirming the precise targeting capability of the β-LG-based system. Combined with the cytoprotective effects observed in vitro, these findings provide a solid experimental basis for the further development of nanodelivery systems for the treatment of kidney diseases. Figure 14 Renal tissue imaging revealed that dark red fluorescence (GA-NPs-ZW800-1) was widely distributed in the renal tubular region and highly overlapped with the tubular structure. This directly demonstrates that GA-NPs, after being filtered by the glomerulus, are efficiently captured by renal tubular epithelial cells through a reabsorption mechanism, providing the most direct experimental evidence for the "reabsorption-mediated" precision delivery strategy of this invention.

[0077] Example 5

[0078] Evaluation of the therapeutic effect of GA-NPs on acute kidney injury

[0079] Establishment and dosing regimen of cisplatin-induced AKI mouse model:

[0080] (1) Animals and Grouping

[0081] Eight-week-old male C57BL / 6 mice, weighing 20-25 g, were selected and acclimatized for one week in an SPF-grade animal facility with free access to food and water and a 12-hour light-dark cycle. The mice were then randomly divided into the following groups (n=5 / group):

[0082] • Blank control group (Sham)

[0083] • Model group (Saline)

[0084] • The complex group of this invention (GA-NPs 5 mg / kg)

[0085] • Positive drug control group (NAC20 mg / kg)

[0086] (2) Model building

[0087] Following existing research methods, a cisplatin-induced acute kidney injury mouse model was established. Except for the blank control group, mice in all other groups were fasted for 12 hours prior to modeling but allowed free access to water, and then received a single intraperitoneal injection of cisplatin (16 mg / kg, freshly prepared with physiological saline). Mice in the blank control group received an equal volume of physiological saline intraperitoneally.

[0088] (3) Dosing regimen

[0089] Mice in the complex group of this invention were intraperitoneally injected with the corresponding dose of β-lactoglobulin-garcinic acid complex every 24 hours for 3 consecutive days before modeling; after modeling on the 4th day, they were injected intraperitoneally with cisplatin and continued to be given the same dose of complex for 3 days (for a total of 3 administrations). Mice in the positive drug control group were intraperitoneally injected with the corresponding dose of NAC every 24 hours for 3 consecutive days before modeling; after modeling on the 4th day, they were injected intraperitoneally with cisplatin and continued to be given the same dose of complex for 3 days (for a total of 3 administrations). Mice in the blank control group and the model group were intraperitoneally injected with the same volume of physiological saline at the same time points.

[0090] (4) Sample collection

[0091] Seventy-two hours after cisplatin injection, mice were weighed, anesthetized with isoflurane inhalation, and blood was collected by enucleation. After standing for 30 minutes, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C to separate the serum, which was then stored at -80°C for later use. Immediately after blood collection, mice were euthanized by cervical dislocation, and both kidneys were removed, rinsed with pre-cooled physiological saline, blotted dry with filter paper, and weighed. The left kidney was fixed in 4% paraformaldehyde for histopathological examination; the right kidney was placed in a cryovial, flash-frozen in liquid nitrogen, and then stored at -80°C for subsequent molecular biological assays.

[0092] Establishment and administration regimen of glycerol-induced AKI mouse model:

[0093] (1) Animal grouping

[0094] Eight-week-old male C57BL / 6 mice, weighing 20-25g, were selected and acclimatized for one week in an SPF-grade animal facility with free access to food and water and a 12-hour light-dark cycle. The mice were then randomly divided into the following groups (n=5 / group):

[0095] • Blank control group (Sham)

[0096] • Model group (Saline)

[0097] • The complex group of this invention (GA-NPs 5mg / kg)

[0098] • Positive drug control group (NAC 20 mg / kg)

[0099] (2) Model building

[0100] A mouse model of glycerol-induced rhabdomyolysis-induced acute kidney injury was established using existing research methods. Except for the blank control group, mice in all other groups had their water intake restricted (but not their food) for 15 hours before modeling, followed by intramuscular injections of 50% glycerol solution (8 mL / kg, 4 mL / kg per hind limb) into both hind limbs. Mice in the blank control group received an equal volume of physiological saline intramuscularly at the same time point.

[0101] (3) Dosing regimen

[0102] Mice in the compound group of this invention were given the same dose of the compound for 3 days (3 times in total) 1 hour after intramuscular injection of glycerol to establish the model. Mice in the positive drug control group were given intraperitoneal injection of NAC 20 mg / kg starting 1 hour after glycerol injection, and continued for 3 days. Mice in the blank control group and the model group were given an equal volume of physiological saline intraperitoneally at the same time points.

[0103] 4. Sample Collection

[0104] 72 hours after glycerol injection, mice were weighed, anesthetized with isoflurane inhalation, and blood was collected by enucleation. After standing for 30 minutes, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C to separate the serum, which was then stored at -80°C for later use. Immediately after blood collection, mice were euthanized by cervical dislocation, and both kidneys were removed, rinsed with pre-cooled physiological saline, blotted dry with filter paper, and weighed. The left kidney was fixed in 4% paraformaldehyde for histopathological examination; the right kidney was placed in a cryovial, flash-frozen in liquid nitrogen, and then stored at -80°C for subsequent molecular biological assays.

[0105] like Figure 15 As shown, GA-NPs treatment significantly reduced elevated levels of CRE and BUN compared to the Saline-treated AKI group. Specifically, in the glycerol-induced model, CRE and BUN levels decreased by 5.4-fold and 4.6-fold, respectively, while in the cisplatin-induced model, CRE decreased by 10.1-fold and BUN by 6.7-fold. After GA-NPs intervention, both models showed that BUN and CRE values ​​were within the normal physiological range. These results confirm a significant recovery of renal function. Notably, a four-fold higher NAC dose (0.2 g / kg) is required to achieve a comparable recovery effect, further emphasizing the superior efficacy of GA-NPs in promoting AKI recovery.

[0106] Example 6

[0107] Renal function index detection and pathological analysis

[0108] Bilateral mouse kidneys were harvested, fixed in formalin, and embedded in paraffin for sectioning. The paraffin sections were then dewaxed and sequentially immersed in xylene I (15 min), xylene II (15 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), 85% ethanol (5 min), and 75% ethanol (5 min), followed by rinsing with distilled water. The slides were then transferred to phosphate-buffered saline (PBS, pH 7.4) and washed three times (5 min each) on a destaining shaker. For DAPI counterstaining, the slides were rinsed three more times (5 min each) with PBS (pH 7.4) on a shaker, briefly filtered, and incubated with DAPI staining solution in the dark at room temperature for 10 min. The slides were then washed three more times (5 min each) with PBS (pH 7.4) on a shaker, briefly drained, fixed with anti-fading mounting medium, and finally observed and imaged under a fluorescence microscope.

[0109] like Figure 16 As shown, histopathological analysis using hemoglobin and eosin (H&E) staining provided direct morphological evidence for kidney protection. Saline-treated AKI mice exhibited severe parenchymal kidney damage, including tubular injury and extensive interstitial inflammatory cell infiltration, consistent with typical AKI pathological findings. In contrast, treatment with GA-NPs almost completely salvaged the kidneys from AKI-induced damage. The kidney morphology of GA-NPs-treated mice was nearly indistinguishable from that of healthy controls, characterized by intact margins, well-developed tubular tissue, and minimal inflammatory cells, highlighting the powerful therapeutic effect of the β-lactoglobulin-PEG-garcinia tinctoria (GA-NPs) targeted nanosystem.

[0110] In summary, this invention successfully constructed a β-lactoglobulin reabsorption-mediated precise renal delivery system for gambogeylic acid (GA-NPs) with excellent water solubility, biocompatibility, and renal targeting. This nanosystem not only solves the water solubility and toxicity issues of gambogeylic acid but also effectively protects renal tubular epithelial cells, scavenge reactive oxygen species, and delivers the drug to the kidneys, demonstrating remarkable therapeutic effects in two AKI mouse models. More importantly, in vitro and in vivo experiments have confirmed for the first time that it can mimic the physiological process of "filtration-reabsorption" of endogenous proteins, achieving precise targeting and efficient treatment of the drug in the renal tubules. Therefore, GA-NPs represent a promising new option for the clinical treatment of AKI.

[0111] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A β-lactoglobulin reabsorption-mediated gambogeylic acid renal delivery system, characterized in that: The delivery system is β-lactoglobulin-PEG-garnet acid nanoparticles, which contain β-lactoglobulin as a targeting carrier and garnet acid as an active ingredient, wherein the β-lactoglobulin and garnet acid are covalently linked by a polyethylene glycol chain.

2. The gambogeylic acid renal delivery system based on β-lactoglobulin reabsorption mediated according to claim 1, characterized in that: The molecular weight of the polyethylene glycol chain is 2000-5000 Da; preferably, the polyethylene glycol chain is PEG3400.

3. The gambogeylic acid renal delivery system based on β-lactoglobulin reabsorption mediated according to claim 1 or 2, characterized in that: The nanoparticles have a particle size of 5-7 nm.

4. A method for synthesizing the gambogeylic acid renal delivery system based on β-lactoglobulin reabsorption mediated by any one of claims 1-3, characterized in that, Includes the following steps: S1. Garcinia cambogia acid and polyethylene glycol with an amino group at one end and a carboxyl group at the other end are subjected to an amidation reaction in an organic solvent in the presence of a catalyst and under inert gas protection. After the reaction is completed, the intermediate product PEG-garcinia cambogia acid is obtained by purification. S2. After activating the carboxyl group at the end of the intermediate product PEG-garcinic acid, it is mixed with β-lactoglobulin in a buffer solution to carry out an amidation reaction, so that the amino group on β-lactoglobulin is covalently linked to the activated PEG-garcinic acid, and the target nanoparticles are obtained by purification.

5. The method for synthesizing the gambogeylic acid renal delivery system based on β-lactoglobulin reabsorption mediated according to claim 4, characterized in that: In step S1, the organic solvent is dichloromethane or N,N-dimethylformamide; the catalyst is 4-dimethylaminopyridine or an EDCI / NHS system; in step S2, the buffer solution is PBS or MES buffer.

6. A kidney-targeting fluorescent imaging probe, characterized in that: The probe is the gambogeylic acid renal delivery system based on β-lactoglobulin reabsorption mediated by any one of claims 1-3, labeled with a near-infrared fluorescent dye.

7. The kidney-targeting fluorescent imaging probe according to claim 6, characterized in that: The near-infrared fluorescent dye is ZW800-1.

8. A method for preparing the fluorescent imaging probe of claim 6 or 7, characterized in that: The delivery system described in any one of claims 1-3 and the activated ester form of ZW800-1 are mixed and reacted in a buffer solution, and then purified to obtain the final product.

9. The use of the β-lactoglobulin reabsorption-mediated gambogeylic acid renal delivery system according to any one of claims 1-3 or the fluorescent imaging probe according to claim 6 or 7 in the preparation of a drug for treating acute kidney injury.

10. The use of the β-lactoglobulin reabsorption-mediated gambogeylic acid renal delivery system according to any one of claims 1-3 in the preparation of reagents for the following purposes: (1) In vitro analysis of renal cell toxicity; (2) Organelle colocalization analysis; (3) Detect the level of reactive oxygen species in cells; (4) Monitor living renal function; (5) Histopathological analysis.

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