Nanoparticle with anti-inflammatory and autophagy activation functions, preparation method of nanoparticle and application of nanoparticle in preparation of medicine for treating acute kidney injury

Abstract

The invention relates to the technical field of nano-drugs, in particular to nanoparticles with anti-inflammatory and autophagy activation functions, a preparation method of the nanoparticles and application of the nanoparticles to preparation of drugs for treating acute kidney injury. The invention relates to a nanoparticle with anti-inflammatory and autophagy activation functions. The nanoparticle comprises bovine serum albumin as a scaffold; dihydromyricetin is adsorbed on the bovine serum albumin; and the dihydromyricetin is chelated with ferric ions. According to the technical scheme, the ultra-small nanoparticles are successfully constructed and synthesized, ROS and RNS can be effectively removed, autophagy is promoted, and inflammation is regulated. The nanoparticles are quickly accumulated in the kidney, high clearance rate and excellent biological safety are shown, and renal tubule injury is relieved. According to the technical scheme, the technical problems of chemical instability, low solubility, poor membrane permeability and relatively poor bioavailability of natural flavonoid compounds for treating acute kidney injury in the prior art can be solved, and the natural flavonoid compounds have ideal application prospects in the aspects of adjusting inflammation, remolding an oxidative stress environment, activating autophagy capacity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a nanoparticle with anti-inflammatory and autophagy activating functions, a preparation method thereof, and an application of the nanoparticle in preparing a medicine for treating acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is a common and serious complication of critically ill patients, characterized by inflammatory tubular damage and potentially reversible decline in renal function. Epidemiological studies have shown that AKI affects 10-15% of hospitalized patients and up to 50% of patients in intensive care units. Clinically, AKI is characterized by a sudden decline in renal function, manifested as a decrease in glomerular filtration rate and increased levels of nitrogenous waste in the blood. Effective early management of AKI includes avoiding the use of nephrotoxic drugs, providing supportive care, and implementing strategies to prevent and deal with complications. As there are currently no approved drug therapies with comprehensive, synergistic, and multifaceted benefits for the management of AKI, the morbidity and mortality of AKI remain high, highlighting the urgent need for effective treatment options.

[0003] During acute kidney injury, the formation of an oxidative stress microenvironment is marked by the abnormal overproduction of reactive oxygen species (ROS). These ROS interact with biomolecules to cause oxidative stress, acute kidney injury, and renal dysfunction. The imbalance between reactive species and antioxidant defense triggers apoptosis, necrosis, and renal dysfunction. Current therapeutic strategies mainly target the scavenging of ROS and reactive nitrogen species (RNS), such as superoxide radicals, hydroxyl radicals, and nitrogen radicals. In addition, excessive ROS production can lead to nuclear and mitochondrial damage and is a key factor in activating inflammasomes such as NLRP3 and leading to apoptosis. A large number of studies have shown that scavenging ROS can restore the homeostasis of the oxidative stress microenvironment, reduce inflammation, and inhibit apoptosis. However, ROS-induced damage to organelles such as mitochondria and nuclei persists, and ROS scavengers alone are usually insufficient to quickly and completely repair and restore the homeostasis of the cellular environment.

[0004] Autophagy is a tightly regulated process that is essential for maintaining basic metabolic functions. Autophagy helps to remove damaged or harmful components, promotes catabolism and recycling, and thus maintains intracellular dynamic homeostasis. Under normal conditions, all types of cells exhibit low levels of autophagy. Previous studies have shown that multiple signaling pathways, such as Sirt1 (Sirtuin 1), PI3K (Phosphoinositide 3-kinase), and MAPK (Mitogen-Activated Protein kinase), can activate the autophagy pathway. Enhanced cellular autophagy can not only degrade oxidatively damaged proteins, but also remove organelles that produce ROS, such as mitochondria and peroxisomes, thereby alleviating oxidative stress and preventing ROS-mediated cell damage. Therefore, autophagy can be effectively used to remove ROS, alleviate various forms of cell damage, and maintain homeostasis. However, there are currently only a few clinical drugs that alleviate AKI-induced damage by simultaneously promoting ROS clearance and enhancing autophagy.

[0005] Natural flavonoids, including dihydromyricetin (DMY), quercetin, kaempferol, and naringenin, have a range of beneficial physiological and biochemical effects. Among them, DMY is well known for its powerful antioxidant and anti-inflammatory properties, which helps reduce the incidence of neurological and cardiovascular diseases and other free radical-related inflammatory conditions. Notably, DMY may stimulate cellular autophagy through the Sirt1-LC3 pathway, thereby maintaining cellular homeostasis. Despite the potential benefits of these natural flavonoids, their clinical application is severely limited due to their chemical instability, low solubility, poor membrane permeability, and poor bioavailability. How to construct an effective nanodrug delivery system loaded with natural flavonoids to give full play to the antioxidant, anti-inflammatory, and autophagy-promoting effects of natural flavonoids, including DMY, is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] The present invention aims to provide a nanoparticle with anti-inflammatory and autophagy activation functions to solve the technical problems of the prior art that natural flavonoid compounds used to treat acute kidney injury are chemically unstable, low solubility, poor membrane permeability and poor bioavailability.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A nanoparticle with anti-inflammatory and autophagy activating functions, comprising bovine serum albumin as a scaffold; dihydromyricetin adsorbed on the bovine serum albumin; and dihydromyricetin chelated with trivalent iron ions.

[0009] Furthermore, the trivalent iron ions are provided by ferric chloride hexahydrate.

[0010] Furthermore, the mass ratio of bovine serum albumin, dihydromyricetin and ferric chloride hexahydrate is 5-10:30-50:10-15.

[0011] Furthermore, its particle size was 6.92±2.57 nm and its surface potential was -6.3 mV.

[0012] The technical scheme also provides a method for preparing nanoparticles with anti-inflammatory and autophagy activation functions, wherein a dihydromyricetin solution is added dropwise to a bovine serum albumin solution under stirring conditions; after the reaction, a ferric chloride solution is added dropwise; and after stirring, culture and ultrafiltration, DF@BSA nanoparticles are obtained.

[0013] Furthermore, the concentration of the bovine serum albumin solution is 0.5-1 mg / mL; the concentration of the dihydromyricetin solution is 30-50 mg / mL, and the solvent is dimethyl sulfoxide; and the concentration of the ferric chloride solution is 2-15 mg / mL.

[0014] Further, the dihydromyricetin solution was added dropwise to the bovine serum albumin solution, and the reaction time was 60 min.

[0015] Further, after the ferric chloride solution is added dropwise, the stirring culture time is 12-14 hours and the rotation speed is 800-1000 rpm.

[0016] The technical solution also provides an application of nanoparticles with anti-inflammatory and autophagy activating functions in the preparation of drugs for treating acute kidney injury.

[0017] The principle of this technical solution is:

[0018] This technical scheme successfully constructed and synthesized ultrasmall DF@BSA nanoparticles (nanozymes), which showed good ability to mimic superoxide dismutase (SOD), catalase (CAT), and HORAC activities. These nanozymes effectively scavenged ROS and RNS and showed significant protective effects in vitro, including promoting autophagy and regulating inflammation. In vivo, DF@BSA nanozymes accumulated rapidly in the kidneys, showing high clearance and excellent biosafety. These nanozymes enhanced renal function, reduced tubular damage, and promoted autophagy. Overall, DF@BSA's ability to regulate inflammation, reshape the oxidative stress environment, and activate autophagy makes it an excellent candidate for anti-inflammatory therapy.

[0019] More specifically, DF@BSA efficiently removes reactive oxygen species in vivo and in vitro through nano-dihydromyricetin (DMY), and promotes autophagy to maintain cell homeostasis. In addition, through the phagocytosis of renal tubular megalin, BSA can effectively target the kidneys and stay in the kidneys. Because of its synthesized ultra-small diameter nanozymes, they can be effectively cleared in the kidneys without causing major organ metabolic toxicity. Due to its strong chelating ability, trivalent iron ions can self-assemble into nanoparticles with natural flavonoids. This method not only improves the water solubility and safety of the compound, but also solves key problems and improves the bioavailability of natural flavonoids. This scheme successfully synthesized ultra-small Fe 3+ -coordinated (DMY) nanoparticles. Bovine serum albumin (BSA) was used as the nanozyme scaffold, which has good biocompatibility (safety) and water solubility. The poorly water-soluble DMY was successfully adsorbed onto the BSA surface, promoting the dispersion of DMY in the BSA aqueous solution. DMY chelated with trivalent iron ions to achieve self-assembly and form BSA-encapsulated Fe 3+ -DMY nanoparticles DF@BSA. DF@BSA nanozymes are able to reshape the oxidative stress microenvironment by scavenging ROS, inhibiting apoptosis, and activating autophagy to promote cell repair. On the one hand, it can be observed that DF@BSA inhibits the activation of NLRP3 inflammasomes in a dose-dependent manner, which is associated with reduced ASC spot formation, reduced caspase-1 cleavage, and reduced levels of various inflammatory factors. On the other hand, DF@BSA increases the autophagic activity of inflammatory cells by upregulating the expression of Sirt1, enhancing the expression of LC3, and increasing the formation of autophagosomes. In addition, DF@BSA nanozymes upregulate the anti-apoptotic protein b lymphocytoma-2 (Bcl-2) and inhibit the apoptosis-related protein Bcl-2-associated protein (Bax). The nanoparticles of this scheme provide a new therapeutic strategy for acute kidney injury, namely, the use of multifunctional nanoparticles to regulate the oxidative stress microenvironment and induce autophagy. This scheme can enhance the potential of next-generation nanomedicines for the treatment of oxidative stress-related diseases and pave the way for the treatment of AKI.

[0020] The beneficial effects of this technical solution are:

[0021] In current clinical practice, the guiding principles for the management of early AKI are to avoid the use of nephrotoxic drugs, provide supportive care, and prevent and manage complications. However, there is currently no approved drug therapy that can provide comprehensive, synergistic, and multifaceted improvements in the treatment of AKI. This study provides an effective treatment option. The effectiveness of DF@BSA nanozymes in treating AKI by scavenging excess ROS and promoting intracellular autophagy indicates that as an easily accessible natural antioxidant drug, dihydromyricetin has the potential to be a natural antioxidant nanoplatform for AKI and other ROS-related diseases. This technical solution uses dihydromyricetin as a technical strategy for treating AKI by combining the two mechanisms of scavenging ROS and promoting cellular autophagy, and achieves ideal therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscope image of DF@BSA of Example 1 of the present invention (scale: 100 nm).

[0023] Figure 2 This is the particle size distribution image of DF@BSA in Example 1 of the present invention.

[0024] Figure 3 This is the potential distribution image of DF@BSA in Example 1 of the present invention.

[0025] Figure 4 This is the iron ion XPS image of DF@BSA in Example 1 of the present invention.

[0026] Figure 5 This is the Fourier infrared image of DF@BSA of Example 1 of the present invention.

[0027] Figure 6 This is an image of the experimental results of the superoxide scavenging ability of DF@BSA in Example 1 of the present invention.

[0028] Figure 7 This is an image of the experimental results of the hydroxyl radical scavenging ability of DF@BSA in Example 1 of the present invention.

[0029] Figure 8 This is an image of the experimental results of the nitrogen radical scavenging ability of DF@BSA in Example 1 of the present invention.

[0030] Fig. 9 This is an image of the experimental results of the oxygen production capacity of DF@BSA in Example 1 of the present invention.

[0031] Fig.10The experimental results of the in vitro anti-inflammatory and repairing ability of DF@BSA of Example 2 of the present invention are shown in Figure 2 (A, B: cell viability of RAW 264.7 and HK-2 cells after incubation with DF@BSA, data are expressed as mean ± SD, n = 3; C: cell viability of HK-2 cells after being stimulated by H2O2 and treated with different doses of DF@BSA, data are expressed as mean ± SD, n = 3; D: apoptosis rate of HK-2 cells after being stimulated by H2O2 and treated with different concentrations of DF@BSA; E: CLSM analysis of ROS after HK-2 cells were stimulated by H2O2 and treated with different concentrations of DF@BSA, scale bar: 50μm; F: CLSM: changes in mitochondrial potential after H2O2 stimulation and treatment with different doses of DF@BSA, scale bar: 50μm; G, J, I: IL-1β, TNF-α and IL-6 levels secreted by HK-2 cells after being activated by H2O2 and treated with different doses of DF@BSA, data are expressed as mean ± SD, n = 3; all statistical significance was analyzed by analysis of variance, *: p < 0.05, **: p < 0.01, ***; p < 0.001, ns: not significant).

[0032] Fig.11 These are images of experimental results of the ability of DF@BSA of Example 3 of the present invention to activate autophagy and promote in vitro repair (A: transmission electron microscopy shows autophagosomes in HK-2 cells after different treatments; B, C: immunofluorescence staining of DHE in HK-2 cells after different treatments and corresponding quantitative analysis; scale bar: 50 μm; data are expressed as mean ± SD, n = 3; D, E: mRNA of Beclin-1 and Sirt1 after different treatments; data are expressed as mean ± SD, n = 3; all statistical significances were analyzed by variance analysis, *: p < 0.05, **: p < 0.01, ***; p < 0.001, ns: no significant significance).

[0033] Fig.12 The experimental results of in vivo biosafety and biodistribution of DF@BSA in Example 4 of the present invention are as follows (A: HE staining of major organs by DF@BSA at different concentrations, scale bar: 50 μm; BE: blood biochemical indices of mice after intravenous injection of DF@BSA, data are expressed as mean ± SD, n = 3; F: in vivo living images of mice at different time intervals after injection of Cy5-labeled DF@BSA; G, H: in vitro FL images of major organs (heart, liver, spleen, lung, kidney) and corresponding quantitative FL analysis of kidney at 2h and 4h, data are expressed as mean ± SD, n = 3; all statistical significances were analyzed by variance analysis, *: p < 0.05, **: p < 0.01, ***; p < 0.001, ns: no significant significance).

[0034] Fig.13 The experimental results of evaluating the therapeutic effect of DF@BSA on AKI mice in Example 5 of the present invention are as follows (A: Schematic diagram of the experimental process for exploring the therapeutic effect of DF@BSA on AKI mice; B: Hematoxylin / eosin staining of renal tissue, scale bar: 100 μm; C: Periodic acid-Schiff staining of renal tissue, scale bar: 100 μm; D, E: levels of urea nitrogen and creatinine in serum under different conditions; F: Representative TUNEL staining of renal sections after different treatments, scale bar: 50 μm; G, H, I: IL-1β, IL-6 and TNF-α levels of HK-2 cells after different treatments, data are expressed as mean ± SD, n = 3); all statistical significances were analyzed by variance analysis, *: p < 0.05, **: p < 0.01, **; p < 0.001, ns: no significant significance).

[0035] Fig.14 These are experimental results images of the in vivo anti-inflammatory and autophagy activation mechanisms of DF@BSA of Example 6 of the present invention (A, B: immunofluorescence staining of DHE in kidney sections after different treatments and corresponding quantitative analysis, scale bar: 50 μm, data are shown as mean ± SD, n = 3; CE: immunohistochemical staining of BAX and BCL in kidney sections after different treatments and corresponding quantitative analysis, scale bar: 100 μm, data are shown as mean ± SD, n = 3; F, G: immunofluorescence staining of LC3 in kidney sections after different treatments and corresponding quantitative analysis, scale bar: 50 μm, data are shown as mean ± SD, n = 3; all statistical significances were analyzed by variance analysis, *: p < 0.05, **: p < 0.01, ***; p < 0.001, ns: no significant significance).

[0036] Fig.15 The biocompatibility (safety) evaluation results of the nanoparticles of Comparative Example 2 of the present invention (using ethanol as the solvent).

[0037] Fig.16 This is the particle size distribution diagram of the nanoparticles of Comparative Example 2 of the present invention (first adding trivalent iron ions and then adding DMY).

[0038] Fig.17 This is a projection electron micrograph of the nanoparticles of Comparative Example 2 of the present invention (stirring for 0.5 h and 12 h after adding trivalent iron ions).

[0039] Fig.18 This is the appearance of the nanoparticles of Comparative Example 2 of the present invention after ultrafiltration centrifugation (using different filter membranes). DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0041] Example 1: Preparation of Nanoparticles

[0042] 10 mg BSA (bovine serum albumin) was dissolved in 10 mL sterile water, and then 50 mg DMY (dihydromyricetin, dissolved in 1 mL dimethyl sulfoxide) was added dropwise (about 10 drops / min) under stirring (about 1000 rpm). After stirring at about 1000 rpm for 60 min, 10 mg FeCl3·6H2O (ferric chloride hexahydrate) dissolved in 1 mL sterile water was added dropwise, and cultured overnight (about 12 h) under stirring conditions of 1000 rpm. After culture, the product was subjected to conventional ultrafiltration centrifugation (MVCO 100KD) three times (the speed was controlled between 2000-3000 rpm, each time for about 10 min), and the product supernatant was collected to obtain DF@BSA nanoparticles (nanozymes).

[0043] In actual operation, the following methods can be used to achieve the expected effect: 5-10 mg BSA (bovine serum albumin) is dissolved in 10 mL sterile water, and then 30-50 mg DMY (dihydromyricetin, dissolved in 1-2 mL dimethyl sulfoxide) is added at a rate of 10-15 drops / min under stirring at 800-1000 rpm. After 60-90 min, 10-15 mg FeCl3·6H2O (ferric chloride hexahydrate) dissolved in 1-5 mL sterile water is added dropwise, and cultured for 12-14 h under stirring conditions of 800-1000 rpm. After culture, ultrafiltration centrifugation (MVCO 100KD) at 2000-3000 rpm for 3-5 times, the product supernatant is collected, and DF@BSA nanoparticles (nanozymes) are obtained. Ultrafiltration centrifugation is a conventional method in the prior art. The key to affecting the effect lies in the pore size of the ultrafiltration membrane. The ultrafiltration speed and time need to be set conventionally.

[0044] The obtained DF@BSA was tested, and the transmission electron microscopy image was as follows: Figure 1 Particle size distribution and potential distribution are shown in Figure 2 , Figure 3 The average particle size of the nanoparticles is 6.92±2.57nm, and the average potential is -6.3mV. Figure 4 As shown, the Fourier infrared image is Figure 5 The polydispersity index (PDI) of the nanoparticles was 0.225.

[0045] The superoxide scavenging ability, hydroxyl radical scavenging ability, nitrogen radical scavenging ability and oxygen production ability of the nanoparticles were tested. Figure 6 , Figure 7 , Figure 8 and Fig. 9 DF@BSA has an ideal ability to scavenge ROS and RNS, as well as a certain oxygen production capacity, which can restore the homeostasis of the oxidative stress microenvironment.

[0046] Example 2: In vitro anti-inflammatory and repair abilities of DF@BSA

[0047] In order to evaluate the biocompatibility (safety) of DF@BSA nanoparticles, we incubated RAW264.7 (mouse macrophages) and HK-2 (human renal tubular epithelial cells) with different concentrations of DF@BSA for 24 h (the cell dosage was conventionally used in the prior art, about 5 × 10 6 Even at a high concentration of 400 μg / mL, cell viability remained above 85%, indicating that DF@BSA has good biocompatibility ( Fig.10 A, B). Interestingly, treatment with 400 μg / mL DF@BSA significantly increased the viability of HK-2 cells compared with the other nanoparticle groups, indicating that DF@BSA has a mild stimulatory effect on HK-2 cell growth.

[0048] To further evaluate the anti-inflammatory and anti-apoptotic effects of DF@BSA in vitro, we used CCK-8 assay to assess its protective effect on HK-cells against H2O2-induced injury (H2O2 250 μM was used to induce injury and then treated for 4 h). Fig.10 As shown in (C), DF@BSA significantly increased the viability of H2O2-stimulated cells and reduced cytotoxicity in a dose-dependent manner.

[0049] In order to study the effect of DF@BSA on cell apoptosis, we performed Annexin V-FITC / PI cell apoptosis assay. The experimental method is the conventional method of the prior art: Annexin V-FITC and PI dyes were diluted with basal culture medium (1:1000), added to the well plate, incubated at 37°C for 30-60 minutes, and their values ​​were measured. Flow cytometry results showed that the cell apoptosis rate in the H2O2 treatment group was as high as 89.10%. As the DF@BSA concentration increased from 50μg / mL to 400μg / mL, the cell apoptosis rate decreased from 49.70% to 12.94% ( Fig.10 D).

[0050] In addition, we used confocal laser scanning microscopy to observe the ROS scavenging and anti-inflammatory abilities of DF@BSA. The experimental method was the conventional method in the prior art: DCFH-DA dye was diluted with basal culture medium (1:1000), added to the dish, incubated at 37°C for 30 minutes, and observed under a microscope. Fig.10 As shown in E, after incubation with H2O2 and ROS staining with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), the fluorescence intensity of the DF@BSA group decreased from 80% to 20% compared with the control group, indicating that DF@BSA can significantly remove intracellular ROS and regulate the oxidative stress microenvironment.

[0051] To further evaluate the protective effect of DF@BSA against H2O2-induced mitochondrial damage, we used JC-1 dye to monitor MMP changes. The experimental method is the conventional method of the prior art: JC-1 dye was diluted with basal culture medium (1:200), added to the dish, incubated at 37°C for 20 minutes, and observed under a microscope. When the cell membrane potential decreases, the JC-1 dye changes from a red fluorescent aggregated state to a green fluorescent dispersed state. Confocal laser scanning microscopy results showed that compared with the control group, cells treated with DF@BSA showed an increase in green fluorescence signals, while the higher the concentration of nanoparticles, the weaker the red fluorescence signal, indicating that the JC-1 dye changed from an aggregated state to a dispersed state ( Fig.10 F). This indicates that DF@BSA promotes the recovery of mitochondrial function by inhibiting toxic ROS.

[0052] When stimulated by substances, HK-2 cells enter a proinflammatory state and secrete proinflammatory cytokines, such as IL-6, TNF-α, and IL-1β, which play a vital role in the occurrence and development of AKI. To investigate the anti-inflammatory properties of DF@BSA in vitro, we measured the levels of inflammatory cytokines in HK-2 cells. ELISA analysis confirmed that H2O2 stimulation significantly increased the levels of IL-1β, IL-6, and TNF-α ( Fig.10 GI), which is consistent with previous reports. In contrast, DF@BSA significantly suppressed the levels of these inflammatory cytokines. These results suggest that DF@BSA can alleviate inflammatory responses.

[0053] In summary, DF@BSA nanoparticles can regulate the stress microenvironment by scavenging intracellular ROS, promoting mitochondrial recovery, and inhibiting the expression of inflammatory cytokines, thereby inhibiting cell apoptosis.

[0054] Example 3: Ability of DF@BSA to activate autophagy and promote in vitro repair

[0055] To explore the ability of DF@BSA nanozymes to activate autophagy and promote cell repair in vitro, we conducted a series of experiments. Excessive ROS mainly causes cellular oxidative damage, leading to apoptosis, necrosis, and impaired mitochondrial function. Based on the above experiments, we hypothesized that DF@BSA triggers mitophagy.

[0056] To test this hypothesis, HK-2 cells (1.4×10 7 ) and prepared as biological electron microscopy samples. Fig.11 As shown in A, TEM showed significant changes in mitochondrial morphology. In untreated cells, mitochondria appeared normal with smooth membranes, intact cristae, and few autophagosomes. However, in HK-2 cells treated with H2O2, we observed swollen mitochondria with severe structural damage and the formation of some autophagosomes. Notably, DF@BSA treatment significantly alleviated these changes, reducing mitochondrial swelling and increasing the number of double-membrane autophagosomes, especially perimitochondrial autophagosomes (mitosomes) as well as autophagolysosomes.

[0057] To further confirm these observations, we performed fluorescent staining to assess mitophagy. 6 The cells were stimulated with 250 μm H2O2 for 4 h and then cultured with different concentrations of DF@BSA (50, 100, 200, 400 μg / ml) for 12 h before testing. The results showed that the autophagy-related fluorescence increased significantly in a concentration-dependent manner after DF@BSA treatment ( Fig.11 B, C). This indicates that DF@BSA effectively reduced H2O2-induced ROS accumulation and promoted the repair of damaged cells.

[0058] In addition, we examined whether DF@BSA could promote autophagy through the Sirt1 pathway (drug treatment method was the same as above, and the concentration of DF@BSA was 50 and 400ug / ml). qPCR analysis showed that Sirt1 and Beclin-1, key markers of autophagy, were significantly upregulated in cells treated with DF@BSA ( Fig.11 D, E). These results strongly support the view that DF@BSA activates mitophagy by upregulating Sirt1 and Beclin-1, thereby helping to repair cellular damage. These results strongly support the view that DF@BSA activates mitophagy by upregulating Sirt1 and Beclin-1, thereby helping to repair cellular damage.

[0059] Example 4: In vivo biosafety and biodistribution of DF@BSA

[0060] To evaluate the biosafety of DF@BSA, we administered DF@BSA nanozymes (100, 200, 400 μg / mL) in mice by intravenous injection. Blood samples and major organs (heart, liver, spleen, lungs, and kidneys) were collected 24 h after injection. We observed that the renal tissues in the DF@BSA-treated group did not show any significant damage compared to the control group, even at higher doses, indicating the absence of renal toxicity. In addition, H&E staining of other major organs showed that DF@BSA administration had no adverse side effects ( Fig.12 A). In addition, the renal and liver functions as well as hematological parameters in the DF@BSA-treated group remained within the normal range, similar to those observed in the control group ( Fig.12 BE).

[0061] To further evaluate the biosafety, we performed a hemolytic test using mouse erythrocytes, and no hemolytic activity was found within the injection concentration range. In addition, the survival rate of mice injected with different concentrations of nanozymes showed no statistically significant difference compared with the control group. These results indicate that DF@BSA nanozymes have excellent in vivo biocompatibility and low toxicity, supporting their potential for clinical application.

[0062] To further investigate the biodistribution of DF@BSA, we monitored the real-time distribution of intravenously injected nanoparticles in mice by in vivo fluorescence imaging. Fig.12 As shown in Figure F, the fluorescence intensity of the mouse abdomen reached a peak at 2h and decreased significantly at 24h, indicating that the nanoparticles were effectively metabolized by the organs within 24h. The in vitro fluorescence intensity of organs at different time points showed that Cy5-labeled DF@BSA mainly accumulated in the liver and kidneys, and only weak fluorescence was observed in the heart, spleen, and lungs. It is worth noting that the fluorescence intensity of the kidney at 2h and 4h was 4.1 times and 3.1 times that of the liver, respectively ( Fig.12 G, H). This indicates that the ultrasmall hydrodynamic diameter of DF@BSA (≈6.92 nm) enables them to pass the kidney filtration threshold of 6-8 nm and effectively target the kidney. After 24 h, almost no fluorescence was detected in the isolated organ, further confirming that the nanoparticles were effectively metabolized by the organ. In summary, DF@BSA nanozymes mainly target the kidney, are effectively metabolized by the organ, and exhibit good biosafety.

[0063] Example 5: Evaluation of therapeutic effects on AKI mice

[0064] In order to evaluate the therapeutic effect of DF@BSA as a potential treatment for AKI, a glycerol-induced AKI mouse model was used. The model was established by inducing rhabdomyolysis and hemolysis through water deprivation, followed by intramuscular injection of 50% glycerol, resulting in renal oxidative stress-related damage and subsequent renal dysfunction. DF@BSA (10 mL / kg, 2 mg / ml; for example, based on an average of 20 g per mouse, 0.2 ml, 400 μg of the drug needs to be injected; this amount is a safe dose for mice) was administered by intravenous injection 4 h after modeling for therapeutic evaluation ( Fig.13 A). Before modeling, mice were deprived of water but allowed to consume food for 16 h, followed by intramuscular injection of 50% glycerol / saline mixture. 4 h later, the successful AKI model was treated with intravenous DF@BSA (10 mL / kg), and the effect was evaluated 24 h later. For comparison with other ROS scavengers, N-acetylcysteine ​​(NAC), a widely recognized small molecule ROS inhibitor, was introduced as a positive control at a concentration of 7 mL / kg (5 mM). Histological analysis using hematoxylin-eosin staining and periodic acid-Schiff (PAS) staining showed that DF@BSA significantly alleviated renal tubular damage in AKI mice ( Fig.13 B, C). Since oxidative stress marked by high ROS levels is a key factor in the progression of AKI, traditional renal function markers such as creatinine (CRE) and blood urea nitrogen (BUN) also showed significant decreases after DF@BSA treatment ( Fig.13 D, E), indicating its effectiveness in alleviating renal injury. In addition, kidney dissection and TUNEL staining further confirmed the protective effect of DF@BSA on renal cell injury ( Fig.13 F). To investigate the in vivo antioxidant capacity of DF@BSA and its effect on inflammatory cytokines, serum IL-1β, IL-6, and TNF-α levels were measured using ELISA. As expected, the AKI control group showed a significant upregulation of IL-1β, IL-6, and TNF-α, while these levels were significantly decreased in the DF@BSA-treated group ( Fig.13 GI). These findings suggest that DF@BSA effectively alleviates oxidative stress and inflammatory responses, thereby promoting renal repair and showing great promise as a therapeutic agent for AKI.

[0065] Example 6: In vivo anti-inflammatory and autophagy activation mechanisms of DF@BSA

[0066] We further investigated whether DF@BSA could effectively remove ROS, especially superoxide, produced in the damaged kidneys of AKI mice. As expected, glycerol-induced renal cells produced a large amount of superoxide, resulting in a strong red fluorescence signal in the dihydroethidium (DHE)-stained sections of the AKI model group ( Fig.14A). In contrast, DF@BSA treatment significantly reduced superoxide levels (red fluorescence) by approximately 50%, almost reaching the level observed in the control group. In the NAC-treated group, DHE fluorescence intensity decreased by 28.7% compared with the model group, indicating partial relief of oxidative stress ( Fig.14 B). These results indicate that DF@BSA has a stronger ability to eliminate ROS in mouse kidneys than NAC. Bax is a pro-apoptotic protein that is mainly located on the outer membrane of mitochondria. As a single-chain membrane protein, Bax plays a vital role in regulating apoptosis. The activation and aggregation of Bax can lead to changes in mitochondrial membrane permeability and release apoptotic molecules such as cytochrome c from mitochondria. On the other hand, Bcl-2 is an anti-apoptotic protein that is mainly present on the outer membrane of mitochondria and other organelles such as the endoplasmic reticulum. The main function of Bcl-2 is to inhibit Bax activity, thereby preventing changes in mitochondrial membrane permeability and cell apoptosis. The Bcl-2 / BAX ratio reflects the degree of cell anti-apoptotic ability and plays an important role in maintaining cell survival. Immunohistochemical analysis showed that Bax expression was significantly increased and Bcl-2 expression was decreased in the renal tissue of the model group compared with the normal control group. In contrast, the DF@BSA-treated group showed a dose-dependent decrease in Bax expression and an increase in Bcl-2 expression, indicating that DF@BSA has a strong anti-apoptotic ability ( Fig.14 CE).

[0067] In addition, immunofluorescence staining was used to analyze the classic autophagy marker LC-3 ( Fig.14 F). Compared with NAC, DF@BSA not only exhibited excellent ROS scavenging ability in the kidney, but also enhanced autophagy expression, promoted mitophagy, improved mitochondrial function, and maintained intracellular homeostasis ( Fig.14 G). These findings suggest that nanozymes exert their therapeutic effects mainly by scavenging ROS, inhibiting the expression of inflammatory proteins, regulating the oxidative stress microenvironment, and promoting the autophagy pathway, thereby effectively alleviating AKI damage and promoting recovery.

[0068] Comparative Example 1

[0069] This technical solution is the first to use BSA as a scaffold to load DMY. There is no report in the prior art on how to effectively load DMY onto BSA, ensure the loading amount of DMY and give full play to its excellent performance. The inventor first tried the conventional hydrothermal method to synthesize nanoparticles. However, BSA will denature into a viscous flocculent in a hydrothermal environment, causing changes in physical properties and making it impossible to effectively load DMY. Therefore, the inventor abandoned the hydrothermal method commonly used in the prior art and explored a new preparation method.

[0070] Since heating will cause the shape of BSA to change, the inventors then tried to prepare nanoparticles at room temperature. However, the loading effect of DMY on BSA is limited, and it is necessary to improve the efficiency of preparing nanoparticles by adding ligands. The inventors tried to add PEG (polyethylene glycol) and PVP (polyvinyl pyrrolidone) as ligands to improve the dispersibility of nanoparticles and the loading amount of DMY.

[0071] More specifically, on the basis of Example 1, the step of dropping FeCl3·6H2O was omitted, and after mixing BSA and DMY, the mixture was directly stirred overnight. The polydispersity index (PDI.) of the nanoparticles was about 0.514. Compared with the nanoparticles prepared in Example 1 (PDI. 0.225), the dispersibility is very unsatisfactory. Generally speaking, PDI.>0.5 indicates that the particle size distribution in the system is wide, the particle size is not uniform, and there is a large degree of polydispersity. The nanoparticles obtained in Example 1 have a PDI. of 0.225 and a very narrow particle size distribution, indicating that the particle size is uniform and the distribution is very concentrated, further ensuring the quality uniformity of the nanoparticles.

[0072] On the basis of the technical solution of Example 1, 10 mg of FeCl3·6H2O dissolved in 1 mL of sterile water was replaced by 10% (mass volume ratio) of PEG with a molecular weight of about 6000, and the obtained nanoparticles had a polydispersity index (PDI.) of 0.482. In addition, on the basis of the technical solution of Example 1, 10 mg of FeCl3·6H2O dissolved in 1 mL of sterile water was replaced by 10% (mass volume ratio) of PVP with a molecular weight of about 30,000, and the obtained nanoparticles had a polydispersity index (PDI.) of 0.539. It can be seen that the particle size uniformity of the nanoparticles of this scheme cannot be improved by using ligands at will, and the use of PEG and PVP as ligands is not ideal for improving the dispersibility of nanoparticles.

[0073] In summary, compared with Example 1, the replacement of the ligand resulted in poor dispersibility of the nanoparticles and a lower loading amount of DMY, indicating that the present solution uses trivalent iron as a ligand to achieve unexpected technical effects in improving the performance of the nanoparticles.

[0074] Comparative Example 2

[0075] (1) Solvent type

[0076] In this scheme, water is used as the solvent for BSA and trivalent iron ions, and DMSO is used as the solvent for DMY, which is very critical to ensure the good biosafety of the nanoparticles. Based on Example 1, the solvents for BSA, trivalent iron ions, and DMY are all replaced with an equal amount of ethanol, and the obtained nanoparticles are subjected to the following steps: Fig.10 The biosafety experiment shown in A, the results are as follows Fig.15 As shown. And Fig.10 Compared with A, the biosafety of nanoparticles is very unsatisfactory, so the selection of solvent is very critical to the quality of the nanoparticles of this technical solution.

[0077] The solubility of DMY in water is low, and the dissolution rate is gradually improved as the heating temperature rises. After being replaced by first / ethanol and DMSO solvents, the solubility of DMY is improved. However, if the solvents of all substances adopt first / ethanol and DMSO, the biosafety of nanoparticles will be unsatisfactory. Therefore, this scheme adopts the method of dissolving DMY in DMSO at a higher concentration, and then diluting it into the aqueous solution of BSA, which not only ensures the effective load of DMY on nanoparticles, but also ensures the biosafety of nanoparticles. Those skilled in the art will generally believe that the whole system for preparing nanoparticles needs to adopt a system with a higher solubility for DMY (both all solvents adopt organic solvents such as first / ethanol and DMSO, and water cannot be used), so as to ensure the load of DMY on nanoparticles. However, this technical scheme has been found through attempts that it is only necessary to prepare a DMSO solution of DMY at a high concentration, and then disperse it in the aqueous solution of BSA to ensure the load of DMY, which has not been reported in the prior art, and is the first time that the inventor has discovered and adopted it, overcoming the defects that the drug loading in nanoparticles and the biosafety of nanoparticles are difficult to meet at the same time.

[0078] (2) Raw material mixing order

[0079] The mixing order of DMY, BSA and trivalent iron ions will have a certain influence on the formation of nanoparticles. For example, the production process is slightly changed based on Example 1:

[0080] 10 mg BSA (bovine serum albumin) was dissolved in 10 mL sterile water, and then 10 mg FeCl3·6H2O (ferric chloride hexahydrate) dissolved in 1 mL sterile water was added dropwise under stirring, and then 50 mg DMY (dihydromyricetin, dissolved in 1 mL dimethyl sulfoxide) was added dropwise under stirring, and cultured overnight under stirring conditions of 1000 rpm. After culture, the product supernatant was collected by ultrafiltration centrifugation three times, and the particle size of the obtained nanoparticles was detected, such as Fig.16As shown. Because of the filtering (6-8nm) characteristics of the kidney, we want to make the nanomaterials able to pass through the glomerular filtration. The particle size of the nanoparticles obtained according to the method is relatively large (average particle size is close to 100nm), which does not meet the application requirements. The nanoparticles obtained according to the method of implementation 1 have a particle size of 6.92±2.57nm, which meets the application requirements. It can be seen that the mixing order of DMY, BSA and ferric ions is very critical to the particle size of the nanoparticles. The inventor further analyzed the reason that this may be because DMY is first added to mix evenly with BSA, and then ferric ions are added, so that DMY is chelated with it, further stabilizing DMY therein, and controlling the particle size of the nanoparticles at a relatively small level.

[0081] (3) Concentration of BSA and ferric ions

[0082] The concentration of BSA and ferric iron has a more obvious influence on the performance of nanoparticles. For example, on the basis of Example 1, the manufacturing process is slightly changed, and the concentration of BSA and ferric iron is mainly adjusted. As the BSA concentration is reduced from 5mg / ml to 0.5mg / ml, and the ferric iron concentration is increased from 10mM / L to 500mM / L, its particle diameter gradually decreases. The above-mentioned BSA and ferric iron affect the law of nanoparticle particle diameter. The inventor first discovered that considering the loading capacity of BSA to DMY and the biological toxicity of ferric iron, on the basis of ensuring drug loading rate and biosafety, suitable concentration, i.e. the material dosage of Example 1, is selected to ensure that the particle diameter of nanoparticles can pass through the renal tubules.

[0083] (4) Overnight stirring time

[0084] After adding trivalent iron ions, the material was stirred overnight. The inventors also investigated the effect of stirring time on the quality of the nanoparticles, and took characterization images of the nanoparticles at stirring times of 0.5, 1, 2, 4, 6, 12, and 24 h (see Fig.17 ), it was found that the statistical difference between the characterization images of stirring for 12 and 24 h was not obvious, so the stirring time was selected as overnight (12 h).

[0085] (5) Ultrafiltration centrifugation

[0086] In the process of filtering the obtained nanoparticles, three methods were used: dialysis, differential centrifugation and ultrafiltration centrifugation. It was found that dialysis would cause the materials to agglomerate together, and the dispersion of the nanoparticles was significantly reduced. In the process of differential centrifugation, although BSA would be centrifuged off, the content of BSA and DMY in the remaining solution would be reduced. In ultracentrifugation, 100KD and 30KD centrifuge tubes were used for centrifugation. It was found that the substances could not be separated in the 30KD centrifuge tube, but obvious substance separation could be observed in the 100KD centrifuge tube (see Fig.18, the left is 100KD, the right is 30KD), so the selected centrifuge tube filtration size is 100KD.

[0087] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A nanoparticle with anti-inflammatory and autophagy activating functions, characterized in that: The invention comprises bovine serum albumin as a scaffold; dihydromyricetin is adsorbed on the bovine serum albumin; and dihydromyricetin is chelated with trivalent iron ions.

2. The nanoparticles with anti-inflammatory and autophagy activating functions according to claim 1, characterized in that: The ferric iron ions are provided by ferric chloride hexahydrate.

3. The nanoparticles having anti-inflammatory and autophagy activating functions according to claim 2, characterized in that: The mass ratio of bovine serum albumin, dihydromyricetin and ferric chloride hexahydrate is 5-10:30-50:10-15.

4. The nanoparticles with anti-inflammatory and autophagy activating functions according to claim 3, characterized in that: Its particle size is 6.92±2.57nm and its surface potential is -6.3mV.

5. A method for preparing nanoparticles with anti-inflammatory and autophagy activating functions according to any one of claims 1 to 4, characterized in that: Under stirring conditions, the dihydromyricetin solution was added dropwise to the bovine serum albumin solution; After the reaction, ferric chloride solution was added dropwise; after stirring, culture and ultrafiltration, DF@BSA nanoparticles were obtained.

6. The method for preparing nanoparticles having anti-inflammatory and autophagy activating functions according to claim 5, characterized in that: The concentration of the bovine serum albumin solution is 0.5-1 mg / mL; the concentration of the dihydromyricetin solution is 30-50 mg / mL, and the solvent is dimethyl sulfoxide; and the concentration of the ferric chloride solution is 2-15 mg / mL.

7. The method for preparing nanoparticles having anti-inflammatory and autophagy activating functions according to claim 6, characterized in that: The dihydromyricetin solution was added dropwise to the bovine serum albumin solution, and the reaction time was 60-90 minutes.

8. The method for preparing nanoparticles having anti-inflammatory and autophagy activating functions according to claim 7, characterized in that: After the ferric chloride solution is added dropwise, the stirring culture time is 12-14 hours at a rotation speed of 800-1000 rpm.

9. Use of a nanoparticle having anti-inflammatory and autophagy activating functions according to any one of claims 1 to 4 in the preparation of a drug for treating acute kidney injury.

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