Drug loading system based on hollow mesoporous polydopamine nanoparticles as well as preparation process and application of drug loading system

Abstract

The invention discloses a drug loading system based on hollow mesoporous polydopamine nanoparticles as well as a preparation process and application of the drug loading system. The nanoparticle provided by the invention comprises a hollow polydopamine layer, a pharmaceutically acceptable component is coated in the hollow structure of the polydopamine layer, and hyaluronic acid for sealing the component in the hollow structure of the polydopamine layer is loaded outside the polydopamine layer. Through the integrated design of active targeting, intelligent drug release and multi-effect synergy, the defects that in an existing AKI treatment strategy, targeting is poor, release is uncontrollable, and the effect is single are systematically overcome, and a brand new efficient treatment strategy is provided for AKI.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a drug delivery system based on hollow mesoporous polydopamine nanoparticles, its preparation process, and its application. Background Technology

[0002] Acute kidney injury (AKI) and the resulting nephritis are common and critical clinical conditions, with their core pathological mechanisms involving excessive inflammatory responses and oxidative stress. Flavonoids, such as those extracted from okra flowers, have shown great potential in treating AKI due to their potent anti-inflammatory and antioxidant activities. However, these drugs generally suffer from poor water solubility, poor chemical stability, low oral bioavailability, and a lack of targeted delivery to specific lesions in vivo. This makes it difficult to achieve and maintain effective therapeutic concentrations at the site of kidney lesions via conventional administration routes, limiting efficacy and potentially causing unnecessary side effects due to systemic distribution.

[0003] Nanomedicine delivery systems can increase drug stability, offering the possibility of improving drug properties. However, existing conventional nanocarriers (such as conventional liposomes and polymer nanoparticles) still have significant limitations in the treatment of kidney diseases: First, they mainly rely on the high permeability and retention effect of solid tissues to achieve passive targeting, which has limited efficiency and low specificity; second, the drug release behavior of most systems lacks intelligent regulation, and the release rate is slow or uncontrollable, making it difficult to meet the urgent need for rapid and sufficient drug delivery to the lesion in diseases such as AKI.

[0004] For example, polydopamine (PDA) nanoparticles have good drug loading capacity, but they suffer from leakage during drug delivery due to open pores; furthermore, their targeting is insufficient, resulting in poor drug accumulation at the lesion site. Current technologies can achieve nanoparticle targeting through the specific binding of hyaluronic acid (HA) to the CD44 receptor, i.e., further loading HA onto the surface of PDA nanoparticles. However, the current blocking efficiency and stability of HA are insufficient, which may lead to reduced drug loading or premature release.

[0005] CN112274651A discloses a polydopamine nanocarrier delivery system for targeting and activating CD44 molecules, used for diagnosing vulnerable plaques or diseases associated with vulnerable plaques. The polydopamine nanocarrier is a dopamine nanocarrier and / or a dopamine-tannic acid copolymer nanocarrier, the surface of which is partially modified with a targeting ligand that specifically binds to activated CD44 molecules. However, this nanocarrier delivery system requires approximately 80 hours to fully release the drug at pH 5.0, resulting in a slow onset of action and failing to meet the treatment needs of acute AKI-related diseases.

[0006] Therefore, developing a novel nanodelivery system that combines active targeting capability with intelligent rapid drug release characteristics is of great significance for the treatment of kidney diseases. Summary of the Invention

[0007] To overcome the above technical problems, this invention provides a drug delivery system based on hollow mesoporous polydopamine nanoparticles. Hyaluronic acid is used to seal the pores of the nanoparticles and target the binding of the CD44 receptor, achieving efficient accumulation and precise release of the drug at the renal lesion site. This significantly improves therapeutic efficacy, reduces systemic toxicity, and provides an efficient and safe innovative solution for the treatment of AKI. To achieve the above objectives, the technical solution provided by this invention is as follows: In a first aspect, the present invention provides a drug delivery system based on hollow mesoporous polydopamine nanoparticles, the nanoparticles comprising a hollow polydopamine layer, wherein the hollow structure of the polydopamine layer is coated with a pharmaceutically acceptable component, and the exterior of the polydopamine layer is loaded with hyaluronic acid that seals the component within the hollow structure of the polydopamine layer.

[0008] The polydopamine layer of this invention is polymerized from dopamine and is in the form of a hollow sphere. The hollow interior contains pharmaceutically acceptable components. The hollow sphere shell itself has a microporous structure, and hyaluronic acid loaded on the outside of the polydopamine layer seals the microporous structure, achieving drug loading and encapsulation with a suitable renal targeted drug release time. In some embodiments, the pharmaceutically acceptable components have a therapeutic effect on kidney disease; the drug delivery system is used to bind to CD44 receptors in the kidney to achieve targeted drug release; the absolute value of the Zeta potential of the drug delivery system is less than 5, preferably less than 3, and more preferably less than 1.

[0009] In some embodiments, the particle size of the drug delivery system is between 150-350 nm, preferably between 180-300 nm, and more preferably between 200-250 nm.

[0010] In some embodiments, the component having therapeutic effects on nephropathy is a poorly soluble nephropathy drug component.

[0011] The poorly soluble components mentioned in this invention refer to components that are slightly soluble, sparingly soluble, very slightly soluble, or almost insoluble or insoluble, as defined in Part I of the 2025 edition of the Chinese Pharmacopoeia.

[0012] In some embodiments, the component is a natural flavonoid component; preferably, the natural flavonoid component is selected from flavonoids, flavonols, dihydroflavonoids, dihydroflavonols, anthocyanins, flavan-3,4-diols, bisbenzopyrones, chalcones, and biflavonoids; furthermore, the natural flavonoid component includes one or more of hyperoside, rutin, isoquercitrin, hibifolin-8-O-β-D-glucuronide, quercetin-3'-O-glucoside, hibifolin-3'-O-glucoside, myricetin-3-O-glucoside, quercetin-3-O-sophoroside, hibifolin, myricetin, and quercetin.

[0013] In some embodiments, the component is okra flower extract; Furthermore, the total flavonoid content of the okra flower extract is 5% or more, further 8% or more, further 55% or more, and even further 68% or more; Furthermore, the okra flower extract contains 12%-41% hyperoside, 0.1%-1.6% rutin, 6%-18% isoquercitrin, 12.5%-22.5% gossypol-8-O-β-D-glucuronide, 1.5%-5.5% myricetin, 9.5%-19.5% quercetin-3'-O-glucosidase, and 0.5%-8.5% quercetin. Furthermore, the okra flower extract contains hyperoside at a content of 12.54%-40.71%, rutin at a content of 0.14%-1.55%, isoquercitrin at a content of 6.46%-17.81%, gossypol-8-O-β-D-glucuronide at a content of 12.68%-22.17%, myricetin at a content of 1.92%-5.2%, quercetin-3'-O-glucosidide at a content of 9.9%-19.37%, and quercetin at a content of 0.59%-8.07%. Furthermore, the okra flower extract contains hyperoside 13.67%-40.71%, rutin 0.15%-1.55%, isoquercitrin 8.93%-17.81%, gossypol-8-O-β-D-glucuronide 13.07%-22.17%, myricetin 2.6%-5.2%, quercetin-3'-O-glucosidide 9.9%-19.37%, and quercetin 0.93%-8.07%. or, The extract of the okra flower contains hyperoside 13.67%-19.8%, rutin 0.14%-0.55%, isoquercitrin 12.56%-16.83%, gossypol-8-O-β-D-glucuronide 13.12%-22.01%, myricetin 3.27%-4.65%, quercetin-3'-O-glucosidide 13.46%-19.06%, and quercetin 3.7%-7.75%. Furthermore, the okra flower extract contains 13.8% hyperoside, 0.14% rutin, 13.63% isoquercitrin, 13.84% gossypol-8-O-β-D-glucuronide, 4.33% myricetin, 15.63% quercetin-3'-O-glucosidide, and 7.52% quercetin. or, The extract of Okra flower contains hyperoside at a content of 6.4 wt% to 19.0 wt%, isoquercitrin at a content of 4.4 wt% to 14.0 wt%, and total flavonoids at a content of 50 wt% to 80 wt%.

[0014] In some embodiments, the okra flower extract is prepared by a method comprising the following steps: (1) Extract the flowers or medicinal parts of the yellow hibiscus with ethanol to obtain an extract; (2) The extract is concentrated and then extracted to obtain the extract solution; (3) After removing the solvent from the extract, it was eluted with macroporous resin to obtain the extract of Hibiscus syriacus flower; Alternatively, it can be prepared by a method including the following steps: (1) Extract the flowers or medicinal parts of the yellow hibiscus with ethanol to obtain an extract; (22) Add clarifying agent to the extract, treat with water bath, and filter to remove supernatant; (33) The supernatant was eluted with polyamide resin to obtain okra flower extract; Alternatively, it can be prepared by a method including the following steps: (1) The flowers of the yellow hibiscus were extracted with ethanol to obtain an extract; (23) After removing the solvent from the extract, it was eluted with macroporous resin to obtain the extract of okra flower.

[0015] In some implementations, in step (1), the amount of ethanol used is 10-25 times that of the okra flower, and the ethanol is a 60-95% ethanol solution; In step (2), the extractant used for extraction is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-to-liquid ratio of extraction is 0.8-4:1, and the number of extraction stages is 1-5. In steps (3) and (23), the macroporous resin is of type D101, HPD100 or AB-8, and the elution process of the macroporous resin is as follows: the macroporous resin diameter-to-height ratio is 1:4-9, the sample concentration is 0.10-0.30 g crude drug / mL, the sample volume is 4-12 BV, the sample is loaded at a flow rate of 1-4 BV / h, impurities are removed by 4-8 BV of pure water and 1-5 BV of 3-15% ethanol at a flow rate of 0.5-4 BV / h, and elution is performed by 2-8 BV of 50-80% ethanol at a flow rate of 1-5 BV / h. In step (22), the water bath temperature is 50-70℃ and the water bath time is 30-90min; In step (33), the resin diameter-to-height ratio is 1:4-9, the loading solution concentration is 0.10-0.60 g crude drug / mL, the loading solution volume is 4-12 BV, and elution is performed with 4-8 BV of pure water and 4-8 BV of 60-95% ethanol.

[0016] In some embodiments, the preparation of the okra flower extract includes the following steps: (201) The flowers of the yellow hibiscus were crushed into coarse powder and extracted by percolation with 18 times the amount of 80% ethanol to obtain the yellow hibiscus flower extract; (202) The extract of okra flower was deethanoled, diluted with water, and continuously countercurrently extracted with ethyl acetate. The material-to-liquid ratio was 2.5:1 and the extraction stage was 4 stages. The solvent was recovered from the extract under reduced pressure at 40°C. (203) The macroporous resin was treated with D101 macroporous resin. The macroporous resin treatment process was as follows: the macroporous resin diameter-to-height ratio was 1:5, the sample concentration was 0.15 g crude drug / mL, the sample volume was 3 BV, the sample was loaded at a flow rate of 1.5 BV / h, and impurities were removed by 6 BV pure water and 3 BV 10% ethanol at a flow rate of 2 BV / h. The eluent was obtained by elution with 4 BV 60% ethanol at a flow rate of 1.5 BV / h. The eluent was then subjected to vacuum recovery at 60℃ to obtain the extract of Hibiscus syriacus flower.

[0017] In some embodiments, the okra flower extract is prepared by the following method: okra flowers are extracted with 70%–90% ethanol 1–2 times, each time for 1–3 hours, filtered, and the filtrate is concentrated under reduced pressure at 60°C to a relative density of 1.13–1.20. The concentrate is allowed to stand for 30–40 hours to remove oil, and water is added to a relative density of 1.10–1.12. The concentrate is then refrigerated at -2–5°C for 20–40 hours, filtered, and the filtrate is passed through a macroporous adsorption resin chromatography column. Three column volumes are eluted with water, and three to five column volumes each are eluted with 15%–25%, 30%–40%, and 60%–70% ethanol. The ethanol eluent is concentrated under reduced pressure to obtain the okra flower extract. In some embodiments, the okra flower extract is prepared by the following method: okra flowers are extracted once with 80% ethanol for 2 hours, the extracts are combined, filtered, and the filtrate is concentrated to a relative density of 1.13-1.20 at 60°C. An appropriate amount of water is added and the mixture is allowed to precipitate for 30-40 hours. The filtrate is then filtered, passed through a macroporous adsorption resin column, and eluted with water for 3 column volumes, with 10% and 30% ethanol each for 3 column volumes, and with 70% ethanol for 1 column volume. The eluent is concentrated, dried, and pulverized to obtain the total flavonoid extract of okra flowers. In some embodiments, the okra flower extract is prepared by the following method: okra flowers are extracted by reflux with 85%–95% ethanol 1–3 times, each time for 1–2 hours, filtered, the filtrates are combined and the ethanol is recovered, the filtrate is concentrated to a specific gravity of 1.20–1.35, the concentrate is allowed to stand at 0°C–4°C for 24–48 hours, the oil layer of the refrigerated liquid is removed, the pH is adjusted to 6.0–7.0, concentrated, and then rapidly dried in a thin layer or by vacuum belt drying to obtain the okra flower extract.

[0018] In a second aspect, the present invention provides a method for preparing the drug delivery system described in the first aspect, comprising the following steps: (Z1) Hollow mesoporous polydopamine nanoparticles, denoted as HMPDA, were prepared by the template method; (Z2) A pharmaceutically acceptable component is encapsulated in the HMPDA obtained in step 1, denoted as Fla@HMPDA; (Z3) The Fla@HMPDA obtained in step (Z2) is loaded with dopamine hydrochloride and hyaluronic acid, and is denoted as Fla@HMPDA@HA.

[0019] In some implementations, the specific preparation steps of the template method in step (Z1) are as follows: prepare F-127 dispersion; add tetramethylbenzidine (TMB) to F-127 dispersion to obtain a first mixed system; then add Tris and dopamine hydrochloride to the first mixed system, stir, and separate the solid and liquid to obtain HMPDA.

[0020] In some embodiments, in step (Z1), the concentration of the F-127 dispersion is 3-10 mg / mL, preferably 4-8 mg / mL, more preferably 6 mg / mL; the solvent for preparing the F-127 dispersion is an aqueous ethanol solution, which is a mixture of deionized water and ethanol, wherein the volume ratio of deionized water to ethanol is 0.5-1.5:0.5-1.5, preferably 1:1; the volume ratio of the F-127 dispersion to TMB is 5-150:1, preferably 50-120:1, more preferably 100:1; the Tris is added in the form of a Tris aqueous solution, the concentration of which is 20-100 mg / mL, preferably 30-60 mg / mL, more preferably 45 mg / mL; the dopamine hydrochloride is added in the form of an aqueous dopamine hydrochloride solution, the concentration of which is 5-10 mg / mL, preferably 7-8 mg / mL. mg / mL, more preferably 7.5 mg / mL; the volume ratio of the first mixing system, Tris aqueous solution and dopamine hydrochloride aqueous solution is 10-40:0.5-1.5:0.5:1.5, preferably 20-30:0.8-1.2:0.8-1.2, more preferably 27.25:1:1.

[0021] In some embodiments, in step (Z1), the stirring reaction time is 10-30 h, preferably 15-25 h, more preferably 18-20 h; the stirring reaction temperature is 0-50 °C, preferably 10-40 °C, more preferably room temperature.

[0022] In some implementations, in step (Z1), solid-liquid separation is preferably carried out by centrifugation or filtration. More preferably, a washing solution is added during the separation process. The washing solution used for centrifugal washing is a mixed solution of acetone and ethanol with a volume ratio of 0.5-5:0.5-5, preferably 1-2:1-2, and more preferably 1:2.

[0023] In some implementations, the specific preparation steps of step (Z2) are as follows: the hollow mesoporous polydopamine nanoparticles obtained in step (Z1) are dispersed in a saturated aqueous solution of flavonoid components, stirred to obtain a mixture, and then separated into solid and liquid components to obtain Fla@HMPDA.

[0024] In some implementations, in step (Z2), the stirring time is 5-25 h, preferably 10-20 h, and more preferably 13-18 h.

[0025] In some implementations, the specific preparation steps of step (Z3) are as follows: add dopamine hydrochloride and hyaluronic acid to the Fla@HMPDA prepared in step (Z2), stir, and separate the solid and liquid to obtain Fla@HMPDA@HA.

[0026] In some embodiments, in step (Z3), the mass ratio of added dopamine hydrochloride to hyaluronic acid is 1-5:1-5, preferably 1-2:1-3, more preferably 1-2:1; the stirring time is 0.5-3h, preferably 1-2h, more preferably 1.5h.

[0027] In some implementations, the solid-liquid separation involved in steps (Z2) and (Z3) is the same as that in step (Z1).

[0028] The present invention provides a method for preparing a drug delivery system for hollow mesoporous polydopamine nanoparticles as described in the first and second aspects, comprising the following steps: (1) F-127 was added to a mixed solution of deionized water and ethanol in a volume ratio of 0.5-1.5:0.5-1.5 to obtain an F-127 dispersion; tetramethylbenzidine was added to the F-127 dispersion to obtain a first mixed system; Tris aqueous solution and dopamine hydrochloride aqueous solution were added to the first mixed system, stirred for 10-30 h, and solid-liquid separation was performed to obtain HMPDA; wherein, the volume ratio of F-127 dispersion to TMB was 5-150:1; the concentration of the Tris aqueous solution was 20-100 mg / mL; the concentration of the dopamine hydrochloride aqueous solution was 5-10 mg / mL; the volume ratio of the first mixed system, Tris aqueous solution and dopamine hydrochloride aqueous solution was 10-40:0.5-1.5:0.5:1.5; (2) Disperse the hollow mesoporous polydopamine nanoparticles obtained in step (Z1) in a dispersion containing a component with the effect of treating kidney disease, stir for 5-25 h to obtain a mixture, separate the solid and liquid to obtain Fla@HMPDA; (3) Add dopamine hydrochloride and hyaluronic acid to the mixture prepared in step (Z2), stir for 0.5-3h, separate the solid and liquid to obtain Fla@HMPDA@HA, wherein the mass ratio of added dopamine hydrochloride and hyaluronic acid is 1-5:1-5.

[0029] Fourthly, the present invention provides a drug delivery system based on hollow mesoporous polydopamine nanoparticles prepared by the preparation method described in the third aspect above.

[0030] Fifthly, the present invention provides the application of the aforementioned drug delivery system based on hollow mesoporous polydopamine nanoparticles in the preparation of drugs for treating kidney diseases. Preferably, the kidney disease is chronic kidney disease, chronic nephritis, diabetic nephropathy, diabetic nephropathy or nephritis with renal fibrosis, lupus nephritis, contrast agent-induced kidney injury, membranous glomerulonephritis, postoperative acute kidney injury, or IgA nephropathy.

[0031] In some implementations, kidney disease is defined as acute kidney injury.

[0032] In this invention, the drug delivery system can target the kidneys.

[0033] In this invention, the relevant terms are explained as follows: In this invention, the term "drug delivery system" refers to a carrier that encapsulates a drug, specifically hollow mesoporous polydopamine nanoparticles that encapsulate poorly soluble drugs (such as flavonoids).

[0034] The term "hollow" refers to a structure that is hollow inside.

[0035] In this invention, the term "HMPDA" refers to hollow mesoporous polydopamine nanoparticles, specifically nanoscale particles that are polymerized from dopamine and are in the form of hollow spheres.

[0036] In this invention, the term "template method" refers to the process of using a material with a certain three-dimensional structure as a template, and depositing the atoms or ions of the substance into the pores or surface of the template through physical, chemical or biological methods, and then removing the template to obtain the desired nanostructured material. Specifically, in this invention, polydopamine is filled into a template formed by polymerization of F127 to ultimately form a polydopamine nanocarrier.

[0037] In this invention, the term "Fla" ​​refers to a component with therapeutic effects on kidney disease, specifically a flavonoid component. The term "HA" in this invention refers to hyaluronic acid, with the molecular formula (C...). 14 H 21 NO 11 )n is a homogeneous, repeating linear glycosaminoglycan polymerized from disaccharide units of N-acetylglucosamine and glucuronic acid.

[0038] In this invention, the term "@" refers to a core-shell structure, where one substance is encased within another substance. For example, Fla@HMPDA means that Fla is encased within HMPDA, and so on.

[0039] The structural formula of the flavonoid component involved in this invention is as follows:

[0040] Hypericin, Rutin, Isoquercetin

[0041] Hibiscus 8-O-β-D-glucuronide (hibifolin) and Quercetin 3'-O-glucan

[0042] Cosmin-3'-O-glucoside and Myricetin-3-O-glucoside

[0043] Quercetin-3-O-sophoroside Gossypol

[0044] Myricetin Quercetin Compared with the prior art, the present invention has the following beneficial effects: First, this invention achieves precise renal-targeted drug delivery. It utilizes hollow mesoporous polydopamine nanoparticles coated with pharmaceutically acceptable components (especially those with nephrotic therapeutic effects, such as flavonoids), and then modifies their surface with hyaluronic acid. This allows the nanoparticles to specifically recognize and bind to CD44 receptors, which are highly expressed at the site of acute kidney injury (primarily located on the surface of damaged renal tubular epithelial cells and infiltrating inflammatory cells). This "lock-and-key" recognition mechanism enables the drug-loaded nanoparticles to actively accumulate in the renal lesion area from the systemic bloodstream, significantly increasing the drug concentration in the diseased tissue. This addresses the key problem of poor efficacy of current nephrotic drugs due to a lack of targeting. Furthermore, the precise renal-targeted delivery improves bioavailability, effectively treating AKI while reducing the total dosage and avoiding potential adverse reactions caused by high-dose systemic exposure.

[0045] Secondly, the drug delivery system of this invention possesses rapid and intelligent response release characteristics suitable for kidney targeting. This invention constructs a core-shell structured nanosystem that, based on the sensitivity of hyaluronic acid and the polydopamine carrier to a weakly acidic environment, maintains structural stability and slow drug release in normal blood and physiological environments (pH≈7.4). When the nanoparticles are targeted to the inflamed area of ​​the kidney (pH≈5.0-6.5) or taken up by cells into acidic lysosomes, the carrier structure undergoes a responsive change, resulting in rapid drug release. This characteristic of "stable in normal circulation and rapid release at the lesion site" ensures drug stability during delivery and meets the clinical need for rapid drug onset in acute kidney injury.

[0046] Third, this invention leverages the advantages of multi-mechanism synergistic therapy. The drug delivery system provided by this invention is not only a delivery tool but also a therapeutic system, with its therapeutic principles manifested in several aspects: 1) The released flavonoid components can directly neutralize excess reactive oxygen species at the site of injury, reducing oxidative stress; 2) It can effectively inhibit the expression of core inflammatory factors such as TNF-α, IL-6, and IL-1β, blocking cytokine storms; 3) It can regulate the transformation of local macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory and repair-promoting M2 phenotype, improving the immune microenvironment; 4) By protecting mitochondrial function, it inhibits renal tubular epithelial cell apoptosis. These mechanisms are interconnected and synergistic, collectively forming a three-dimensional and highly efficient therapeutic network for acute kidney injury.

[0047] In summary, this invention, through its integrated design of "active targeting, intelligent drug release, and multi-effect synergy," systematically overcomes the shortcomings of existing treatment strategies, such as poor targeting, uncontrollable release, and single-effect, providing a novel and highly efficient treatment strategy for acute kidney injury. Attached Figure Description

[0048] Figure 1 The average particle size diagram (Fig. a) and zeta potential diagram (Fig. b) are for HMPDA, Fla@HMPDA, and Fla@HMPDA@HA. Figure 2 Transmission electron microscopy (TEM) images of HMPDA, Fla@HMPDA, and Fla@HMPDA@HA; Figure 3 Comparison of infrared spectra of HMPDA, Fla, HA, physical mixture of HMPDA and Fla at a mass ratio of 7:3, Fla@HMPDA, and Fla@HMPDA@HA; Figure 4 Comparison of particle size, zeta potential, and PDI of Fla@HMPDA@HA at 25℃ and 37℃; Figure 5 The pH-responsive drug release curves for Fla@HMPDA@HA are shown. Figure 6 This is a comparison of the in vitro antioxidant effects of Fla@HMPDA@HA; Figure 7 Figure a shows the quantitative analysis of the proportion of M2 macrophages induced in vitro by Fla@HMPDA@HA (Figure a) and the representative flow cytometry diagrams of M2 polarization of macrophages in each group (Figure b). Figure 8 Figure showing the in vitro anti-inflammatory effect of Fla@HMPDA@HA on NRK-52E cells; Figure 9 Figure showing the in vitro anti-inflammatory effect of Fla@HMPDA@HA on iBMDM cells; Figure 10 This is a diagram illustrating the pharmacodynamics of nanoparticles in vivo against nephritis. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are not intended to limit the invention, but only to illustrate it. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. The following exemplarily describes the preparation method of flavonoid component A used in the following embodiments of the present invention, but the present invention is not limited thereto; all preparation methods of flavonoid components mentioned in the present invention are applicable. 25 kg of dried okra corollas were pulverized into coarse powder and extracted by percolation with 18 times the amount of 70% ethanol to obtain okra flower extract. The ethanol was removed from the okra flower extract, diluted with water, and then continuously extracted countercurrently with ethyl acetate at a solid-liquid ratio (extractant:extract) of 2.5:1, with four extraction stages. After solvent recovery under reduced pressure at 40℃, the extract was treated with D101 macroporous resin. The macroporous resin treatment process was as follows: macroporous resin diameter-to-height ratio of 1:5, sample concentration of 0.15 g crude drug / mL, sample volume of 3 BV, and a flow rate of 1.5 BV / h, using 6 BV of pure water and 3 BV of... Impurities were removed by 10% ethanol at a flow rate of 2 BV / h, followed by elution with 4 BV of 60% ethanol at a flow rate of 1.5 BV / h to obtain the eluent. The eluent was then subjected to reduced pressure at 60°C to recover the ethanol, yielding the total solids of *Abelmoschus mandshurica* flowers. Seven specific components were added and adjusted (each component's content did not exceed 20% of the total weight) to achieve the contents shown in Table 1 below (the content of each component was determined using the UPLC method reported in "Determination of Seven Components in *Abelmoschus mandshurica* by a Single Test and Multiple Evaluation Method" (Journal of Pharmaceutical Analysis, 2013, Vol. 12, pp. 2082-2087)). This yielded *Abelmoschus mandshurica* flower extract (component A). The quercetin-3-O-robinin content was 0.20-0.50%.

[0050] The extraction material-to-liquid ratio (i.e., extractant: extractant) refers to the volume ratio.

[0051] Table 1

[0052] Flavonoid component B: Hibiscus flowers were extracted by reflux with 85%–95% ethanol 1–3 times, 1–2 hours each time, filtered, the filtrates were combined and the ethanol was recovered, the filtrate was concentrated to a specific gravity of 1.20–1.35, the concentrate was allowed to stand at 0℃–4℃ for 24–48 hours, the oil layer of the refrigerated liquid was removed, the pH was adjusted to 6.0–7.0, concentrated, and vacuum dried to obtain Hibiscus flower extract (component B).

[0053] Unless otherwise specified, the preparation process of each drug delivery system in the following examples was carried out under stirring (stirring speed of 5000-30000 rpm) and ultrasonic (ultrasonic frequency of 10-50 kHz and ultrasonic power of 100-500 W). However, the present invention is not limited to this, and any operation method that can make the reaction system uniformly dispersed can be used instead. Example 1

[0054] Preparation of HMPDA F-127 was added to a mixed solution of deionized water and ethanol in a volume ratio of 1:1 and dispersed evenly to obtain an F-127 dispersion with a concentration of 6 mg / mL. TMB was added to the F-127 dispersion (volume ratio of F-127 dispersion to TMB was 100:1), and after 2 hours, a first mixed system was obtained. Then, a Tris aqueous solution with a concentration of 45 mg / mL and a dopamine hydrochloride aqueous solution with a concentration of 7.5 mg / mL were added to the first mixed system (volume ratio of the first mixed system to the Tris aqueous solution to the dopamine hydrochloride aqueous solution was 27.25:1:1), and the mixture was stirred for 18 hours to obtain a mixed solution. The liquid was collected and centrifuged twice (washing solution was acetone and ethanol in a volume ratio of 1:2, centrifugation conditions were 13000 rpm, 10 min). Hollow mesoporous polydopamine nanoparticles with an average particle size of 150 nm were collected and designated as HMPDA, and stored under cold storage.

[0055] Preparation of Fla@HMPDA The HMPDA obtained in the previous step was fully dispersed in a saturated aqueous solution of okra extract (component A), and stirred at 11,000 rpm for 15 hours to obtain a mixture. The mixture was then centrifuged and washed (under the same conditions as in the HMPDA preparation step), and the solid was collected and denoted as Fla@HMPDA.

[0056] Preparation of Fla@HMPDA@HA Repeat the steps before centrifugation and washing in the preparation of Fla@HMPDA. Add dopamine hydrochloride and hyaluronic acid (mass ratio of dopamine hydrochloride to hyaluronic acid is 2:1) to the prepared mixture. Stir at 11,000 rpm for 1.5 h, then centrifuge and wash once (under the same conditions as in the preparation of HMPDA). Collect the precipitate and denote it as Fla@HMPDA@HA. Example 2

[0057] Preparation of HMPDA F-127 was added to a mixed solution of deionized water and ethanol in a volume ratio of 1:1.2 and dispersed evenly to obtain an F-127 dispersion with a concentration of 8 mg / mL. TMB was added to the F-127 dispersion (volume ratio of F-127 dispersion to TMB was 80:1), and after 2 hours, a first mixed system was obtained. Then, a Tris aqueous solution with a concentration of 30 mg / mL and a dopamine hydrochloride aqueous solution with a concentration of 7 mg / mL were added to the first mixed system (volume ratio of the first mixed system to the Tris aqueous solution to the dopamine hydrochloride aqueous solution was 30:1:1.2), and the mixture was stirred for 20 hours to obtain a mixed solution. The liquid was collected and centrifuged twice (washing solution was acetone and ethanol in a volume ratio of 1:1, centrifugation conditions were 13000 rpm, 10 min). Hollow mesoporous polydopamine nanoparticles with an average particle size of 150 nm were collected and designated as HMPDA, and stored under cold storage.

[0058] Preparation of Fla@HMPDA The HMPDA obtained in the previous step was fully dispersed in a saturated aqueous solution of okra extract (component A), and stirred at 11,000 rpm for 13 hours to obtain a mixture. The mixture was then centrifuged and washed (under the same conditions as in the HMPDA preparation step), and the solid was collected and denoted as Fla@HMPDA.

[0059] Preparation of Fla@HMPDA@HA Repeat the steps before centrifugation and washing in the preparation of Fla@HMPDA. Add dopamine hydrochloride and hyaluronic acid (mass ratio of dopamine hydrochloride to hyaluronic acid is 1:1) to the prepared mixture. Stir at 11,000 rpm for 1 h, then centrifuge and wash once (under the same conditions as in the preparation of HMPDA). Collect the precipitate and denote it as Fla@HMPDA@HA. Example 3

[0060] Preparation of HMPDA F-127 was added to a mixed solution of deionized water and ethanol in a volume ratio of 1:1 and dispersed evenly to obtain an F-127 dispersion with a concentration of 4 mg / mL. TMB was added to the F-127 dispersion (volume ratio of F-127 dispersion to TMB was 120:1), and after 2 hours, a first mixed system was obtained. Then, a Tris aqueous solution with a concentration of 60 mg / mL and an aqueous solution of dopamine hydrochloride with a concentration of 8 mg / mL were added to the first mixed system (volume ratio of the first mixed system to the Tris aqueous solution to the dopamine hydrochloride aqueous solution was 27.25:1:1.2), and the mixture was stirred for 23 hours to obtain a mixed solution. The liquid was collected and centrifuged twice (washing solution was acetone and ethanol in a volume ratio of 1:1, centrifugation conditions were 13000 rpm, 10 min). Hollow mesoporous polydopamine nanoparticles with an average particle size of 150 nm were collected and designated as HMPDA, and stored under cold storage.

[0061] Preparation of Fla@HMPDA The HMPDA obtained in the previous step was fully dispersed in a saturated aqueous solution of okra extract (component A), and stirred at 11,000 rpm for 18 hours to obtain a mixture. The mixture was then centrifuged and washed (under the same conditions as in the HMPDA preparation step), and the solid was collected and denoted as Fla@HMPDA.

[0062] Preparation of Fla@HMPDA@HA Repeat the steps before centrifugation and washing in the preparation of Fla@HMPDA. Add dopamine hydrochloride and hyaluronic acid (mass ratio of dopamine hydrochloride to hyaluronic acid is 1:2.5) to the prepared mixture. Stir at 11,000 rpm for 2 hours, then centrifuge and wash once (under the same conditions as in the preparation of HMPDA). Collect the precipitate and denote it as Fla@HMPDA@HA. Example 4

[0063] The preparation method is basically the same as in Example 3, except that the okra extract (component A) is replaced with an equal amount of quercetin glycoside. Example 5

[0064] The preparation method is basically the same as in Example 3, except that the okra extract (component A) is replaced with an equal amount of hyperoside. Example 6

[0065] The preparation method is basically the same as in Example 3, except that the okra extract (component A) is replaced with an equal amount of okra extract (component B).

[0066] Experimental Examples: Investigation of the Properties of the Nanoparticles of the Present Invention 1. Particle size and potential The HMPDA, Fla@HMPDA, and Fla@HMPDA@HA prepared in Example 1 were diluted with deionized water until they were transparent. Their average particle size and Zeta potential were measured using a dynamic light scattering instrument at 37°C. The results are as follows: Figure 1 As shown in the figure, the Fla@HMPDA@HA nanoparticles prepared in this invention have a particle size of approximately 232 nm, which brings clear advantages to their targeted renal therapy. First, this size facilitates the nanoparticles' passage through blood vessels at the site of renal inflammation, thereby effectively accumulating in the lesion area and increasing local drug concentration. Second, this size provides ample space for the hollow structure, enabling it to carry more drug molecules and significantly improving the efficiency of single-dose administration. Furthermore, this particle size falls within the range easily taken up by cells, allowing the nanoparticles to efficiently enter target cells such as renal tubular epithelial cells, thereby directly exerting antioxidant and anti-inflammatory therapeutic effects. By combining surface hyaluronic acid with targeted modification, the nanoparticles can be further guided to precisely reach the kidneys, reducing retention in non-target organs, ultimately achieving safer and more efficient targeted therapy.

[0067] The particle size and potential of the drug delivery systems prepared in Examples 2-6 were measured. The particle size range was 150-350 nm and the absolute value of the Zeta potential was less than 5.

[0068] 2. TEM The microstructures of HMPDA, Fla@HMPDA, and Fla@HMPDA@HA prepared in Example 1 were observed using transmission electron microscopy, such as... Figure 2 As shown, HMPDA has a hollow structure, while Fla@HMPDA and Fla@HMPDA@HA are spherical with no clearly visible hollow structure. The drug delivery systems of Examples 2-6 have similar microstructures.

[0069] Infrared spectroscopy was performed on six samples, including the prepared HMPDA, Fla@HMPDA, Fla@HMPDA@HA, and a physical mixture of raw materials Fla, HA, HMPDA and Fla in a mass ratio of 7:3. The results are as follows: Figure 3 As shown, compared with free flavonoids (Fla), the characteristic absorption peak intensity of the carbonyl group (C=O) at 1650 cm⁻¹ in the drug-loaded nanoparticles prepared in this invention is significantly weakened and broadened. This spectroscopic behavior proves that the flavonoid molecules are not simply physically adsorbed, but are successfully encapsulated within the mesoporous structure of HMPDA.

[0070] Infrared spectroscopy was performed on the drug delivery systems prepared in Examples 2-6, and all results indicated that the flavonoid components were successfully encapsulated within the mesoporous structure of HMPDA.

[0071] 3. Stability assessment of Fla@HMPDA@HA nanoparticles The Fla@HMPDA@HA nanoparticles prepared in Example 1 were tested by immersing them in PBS solution at pH 7.4 for 72 h at 25 °C and 37 °C, respectively. The particle size, potential, and PDI (polydispersity index) stability of the nanoparticles at the two temperatures were compared. The results are as follows: Figure 4 As shown, the particle size of Fla@HMPDA@HA nanoparticles varies at different temperatures. Figure 4 -a), Potential ( Figure 4 -b), PDI ( Figure 4 -c) It is basically stable, indicating that it has good physical stability under in vitro conditions.

[0072] The stability of the drug delivery systems prepared in Examples 2-6 was investigated, and all showed good physical stability under in vitro conditions.

[0073] 4. Drug loading rate test The drug loading rate of Fla@HMPDA@HA prepared in Examples 1-6 was tested by dialysis under acidic conditions. The specific operating steps are as follows: First, accurately weigh 5.0 mg of lyophilized Fla@HMPDA@HA nanoparticle powder and disperse it in 1.0 mL of deionized water. Sonicate the powder to ensure uniform dispersion. Then, transfer the dispersion to a dialysis bag with a molecular weight cutoff of 3000 Da and clamp both ends. Completely immerse the dialysis bag in a beaker containing 20 mL of a pH 3.6 acetate-sodium acetate buffer solution.

[0074] The release system was placed in a constant-temperature shaker (37℃, 100 rpm) and shaken for 24 hours in the dark. After release, all dialysate was collected, and its absorbance was measured using a UV-Vis spectrophotometer. The drug concentration in the release medium was calculated based on a pre-established flavonoid standard curve, and the total mass of released drug was then determined. The drug loading rate (DL%) was calculated using the following formula: DL (%) = M Fla / M total ×100%, where M Fla M represents the total mass of drug actually released. total The mass of Fla@HMPDA@HA nanoparticle powder.

[0075] Table 2. Drug loading rates of Fla@HMPDA@HA nanoparticles prepared in Examples 1-6

[0076] As shown in Table 2 above, the Fla@HMPDA@HA nanoparticles prepared by this invention have a high drug loading rate of about 30%.

[0077] 5. pH responsiveness study of Fla@HMPDA@HA The release behavior of Fla@HMPDA@HA nanoparticles prepared in Example 1 under different pH conditions was evaluated using dialysis (molecular weight cutoff 3000 Da): 1 mL of Fla@HMPDA@HA nanoparticle suspension (concentration 2.3 mg / mL) was placed in a dialysis bag and immersed in 29 mL of PBS (pH 7.4), sodium acetate buffer (pH 5), and acetate buffer (pH 3.6), respectively, and shaken at 37°C (100 rpm) for 72 hours. Samples were taken periodically, and the flavonoid content was determined by UV spectroscopy. The cumulative release rate was calculated, and the results are as follows: Figure 5 As shown, the drug release rate is higher at pH 5 (the acidic environment of the renal tubular inflammation site). This indicates that the nanoparticles of the present invention have the characteristic of accelerated release in an acidic environment, enabling them to effectively release drugs at the site of renal tubular inflammation and enhance the local therapeutic effect.

[0078] The drug delivery systems prepared in Examples 2-6 were all subjected to pH response tests, and they had the same pH response as those in Example 1.

[0079] 6. In vitro antioxidant (ROS scavenging) capacity assessment DPPH scavenging activity assay: Add the test sample to 2 mL of 0.2 mM DPPH solution, mix well, and incubate in the dark at 37°C for 30 min. Then, take a small amount of the test sample and measure and record the ultraviolet spectrum in the 500–600 nm band. The results are shown in the figure. Figure 6 (a).

[0080] Superoxide dismutase (SOD) activity assay: A test solution was prepared using 100 μL of 1.2 mmol / L riboflavin, 400 μL of 0.1 mol / L ethylenediaminetetraacetic acid (EDTA), 150 μL of nitrotetrazole chloride (NBT), and 5.8 mL of purified water. The sample was added to the test solution, shaken to mix, and reacted at 37°C for 5 min. The reaction was then initiated by irradiation with a 27-watt lamp for 2 min. Excess superoxide ions react with NBT to produce a blue product. A small amount of the sample was taken and the UV absorption spectrum in the 450–650 nm band was measured using a UV scanner to determine the amount of uneliminated superoxide ions. The results are shown in [Figure number missing]. Figure 6 (b).

[0081] Hydroxyl radical scavenging activity assay: 9 mM FeSO4 and 4 mM H2O2 were reacted for 10 min; the sample to be tested was then added to scavenge hydroxyl radicals. Finally, 9 mM salicylic acid was added and reacted with the hydroxyl radicals for 5 min to generate 2,3-dihydroxybenzoic acid. A small amount of the sample was taken and the UV absorption spectrum in the 500–600 nm band was measured using a UV scanner. The results are shown below. Figure 6 (c).

[0082] Detection of catalase-like activity: Prepare a 10 mM H2O2 stock solution. Add the sample to be tested to a 96-well plate, then add the H2O2 stock solution to the 96-well plate and shake to mix. After incubation for 5 min, add the H2O2 detection solution to the wells, mix well, and incubate in the dark for 30 min. Scan the absorbance in the 400-700 nm wavelength range using a microplate reader. The results are shown in the figure. Figure 6 (d).

[0083] It can be seen that the Fla@HMPDA@HA of the present invention exhibits the strongest antioxidant activity in four different in vitro antioxidant experiments, and its performance is superior to Fla, HMPDA and Fla@HPMDA alone.

[0084] The in vitro antioxidant capacity of the drug delivery systems prepared in Examples 2-6 was investigated, and they showed similar in vitro antioxidant capacity to those in Example 1.

[0085] 7. Examination of macrophage differentiation capacity in vitro Test method: iBMDM cells were prepared at a concentration of 1×10⁻⁶. 6Cells were seeded at a density of 5 μg / mL in 6-well plates, and LPS (lipopolysaccharide) was added to each well to a concentration of 5 μg / mL (an equal volume of PBS was added to the control group). After 24 h, the supernatant was aspirated, and fresh culture medium was added, followed by the test samples (free Fla, HMPDA, Fla@HMPDA, and Fla@HMPDA@HA) to achieve a final drug concentration of 30 μg / mL for each group. After incubation for 24 h, each well was gently washed three times with PBS to remove free and loosely bound nanoparticles, and collected by centrifugation at 1000 rpm for 5 min. Cells were then resuspended in PBS containing 0.1 mL of antibody (containing 1 μL FTIC anti-CD11b antibody, 0.5 μL PE / Cy7 anti-F4 / 80 antibody, and 0.5 μL PE anti-CD86 antibody) and stained for 30 min for extracellular staining. After staining, all samples underwent intracellular immunostaining for CD206. Cells were collected by centrifugation and resuspended in 0.1 mL PBS containing 0.5 μL of APC anti-CD206 antibody for intracellular staining. Finally, stained cells were collected and resuspended in 0.2 mL PBS for further analysis using a NovoCyte 2060R flow cytometer (ACEA, San Diego, CA92121, USA). Untreated cells served as a blank control group. To avoid error, three samples were used for each group. Results are as follows: Figure 7 As shown.

[0086] It can be seen that the phenotypic analysis data from flow cytometry consistently and strongly demonstrate that Fla@HMPDA@HA can effectively differentiate LPS-induced M1 macrophages into M2 macrophages in vitro, and the conversion of macrophages from M1 to M2 is a core step in inflammation resolution and tissue repair.

[0087] The drug delivery systems prepared in Examples 2-6 were all tested for their ability to induce macrophage differentiation in vitro, and they showed similar ability to induce macrophage differentiation in vitro as in Example 1.

[0088] 8. In vitro anti-inflammatory capacity assessment (1) NRK-52E cells were seeded at a density of 5*10⁴ cells in 24-well plates and cultured. After 24 h, the supernatant was aspirated, serum-free medium was added, and H₂O₂ was added to bring the final concentration to 500 μM. The cells were then incubated for 2 h. After modeling, the medium was aspirated, washed three times with PBS, and then fresh medium was added. The test samples (free Fla, HMPDA, Fla@HMPDA, and Fla@HMPDA@HA) were added to bring the final concentration of each drug to 30 μg / mL. After co-incubation for 24 h, the cells were centrifuged at 3000 rpm for 10 min and the supernatant was collected. The levels of TNF-α, IL-6, IL-1β, and IL-10 were measured using an ELISA kit. The results are as follows: Figure 8As shown.

[0089] As can be seen, in the H2O2-induced NRK-52E cell inflammation model, compared with the control group, H2O2 treatment significantly increased the secretion levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α, and decreased the expression of anti-inflammatory factor IL-10, indicating that the inflammation model was successfully constructed. After intervention with different test samples, it can be seen that free flavonoids Fla and HMPDA both reduced the levels of pro-inflammatory factors to some extent, while Fla@HMPDA and Fla@HMPDA@HA further enhanced the inhibitory effect on inflammatory factors. Among them, Fla@HMPDA@HA showed the most significant effect in reducing the levels of IL-6, IL-1β, and TNF-α, while significantly upregulating the secretion of IL-10, suggesting that it has superior in vitro anti-inflammatory activity.

[0090] (2) iBMDM cells at 5*10 4 The samples were seeded at high density in 24-well plates; 5 μg / mL of LPS was added to each well (an equal volume of PBS was added to the blank control group). After 24 h, the supernatant was aspirated, fresh culture medium was added, and the drugs (free Fla, HMPDA, Fla@HMPDA, and Fla@HMPDA@HA) were added to each well to achieve a final drug concentration of 30 μg / mL for each group. After co-incubation for 24 h, the samples were centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The levels of TNF-α, IL-6, IL-1β, and IL-10 were measured using an ELISA kit. The results are as follows: Figure 9 As shown.

[0091] As can be seen, in the LPS-induced iBMDM cell inflammation model, compared with the blank control group, LPS stimulation significantly induced high expression of IL-6, IL-1β, and TNF-α, and inhibited IL-10 secretion. Different drug-treated groups could alleviate the LPS-induced inflammatory response to varying degrees. Among them, Fla@HMPDA@HA showed the most significant inhibitory effect on pro-inflammatory factors and effectively restored IL-10 expression levels, demonstrating superior anti-inflammatory effects compared to free flavonoids and uncoated drug-loaded MPDA.

[0092] In summary, Fla@HMPDA@HA exhibited significant anti-inflammatory effects in both in vitro inflammation models, effectively inhibiting the release of pro-inflammatory factors and promoting the secretion of anti-inflammatory factors. Its anti-inflammatory effect was superior to that of free drugs and uncoated drug delivery systems, indicating that HA modification and nanodelivery systems have significant advantages in improving the in vitro anti-inflammatory efficacy of drugs.

[0093] 9. Pharmacodynamic studies of its anti-nephritis effects in vivo After one week of acclimatization, mice were intraperitoneally injected with 10 mg / kg LPS to induce nephritis. One hour after the intraperitoneal injection of LPS, the drug was administered via tail vein injection. Three groups were established: a normal control group (receiving an equal volume of physiological saline), a model control group (LPS + physiological saline), and a nanoparticle therapy intervention group (LPS + different nanoparticles). All mice were sacrificed 24 hours after the first administration, and blood samples were collected. Serum was separated by centrifugation, and renal function indicators, serum urea nitrogen (BUN) and creatinine (Cr) levels, were measured. Bilateral kidneys were dissected and processed as follows: a portion of kidney tissue was homogenized, and the levels of inflammatory factors TNF-α, IL-1β, IL-6, and the anti-inflammatory factor IL-10 were detected by ELISA; another portion of kidney tissue was fixed in 4% paraformaldehyde, sectioned, and subjected to H&E staining to observe histopathological changes and Masson's trichrome staining to assess collagen deposition. The results are as follows. Figure 10 As shown.

[0094] As can be seen, all three indicators increased significantly after LPS treatment, indicating that inflammatory stimulation successfully induced significant renal tissue damage. Compared with the control group, the LPS group showed a significant increase in fibrin casts, and the renal tubular injury score and overall score were also significantly higher (P < 0.05 or P < 0.01).

[0095] In different intervention groups, HMPDA alone did not improve LPS-induced kidney injury. The fibrin cast formation and renal tubular injury scores were similar to those in the LPS group, suggesting that naked HMPDA does not have a renal protective effect.

[0096] In contrast, the Fla@HMPDA@HA group showed the most significant nephroprotective effect. This group exhibited significant decreases in all three indicators, with less fibrin cast formation. These results suggest that the Fla@HMPDA@HA nanoparticle form can enhance the accumulation and intensity of Fla in renal tissue, improving its bioavailability and therapeutic efficacy.

[0097] In summary, Fla@HMPDA@HA can effectively alleviate LPS-induced kidney injury.

[0098] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A drug delivery system based on hollow mesoporous polydopamine nanoparticles, characterized in that, The nanoparticles comprise a hollow polydopamine layer, the hollow structure of which encapsulates a pharmaceutically acceptable component, and the exterior of the polydopamine layer is loaded with hyaluronic acid that seals the component within the hollow structure of the polydopamine layer.

2. A method for preparing the drug delivery system according to claim 1, characterized in that, Includes the following steps: (Z1) Hollow mesoporous polydopamine nanoparticles, denoted as HMPDA, were prepared by the template method; (Z2) A pharmaceutically acceptable component is encapsulated in the HMPDA obtained in step (1), denoted as Fla@HMPDA; (Z3) The Fla@HMPDA obtained in step (2) is loaded with dopamine hydrochloride and hyaluronic acid, and is denoted as Fla@HMPDA@HA.

3. The drug delivery system according to claim 1 or the preparation method according to claim 2, characterized in that, The pharmaceutically acceptable component has a therapeutic effect on kidney disease; the drug delivery system is used to bind to CD44 receptors in the kidney to achieve targeted drug release; furthermore, the absolute value of the Zeta potential of the drug delivery system is less than 5, preferably less than 3, and more preferably less than 1.

4. The drug delivery system according to claim 1 or the preparation method according to claim 2, characterized in that, The particle size of the drug delivery system is between 150-350 nm, preferably between 180-300 nm, and more preferably between 200-250 nm.

5. The preparation method according to claim 2, characterized in that, The preparation steps of step (Z1) are as follows: prepare F-127 dispersion; add tetramethylbenzidine to F-127 dispersion to obtain the first mixed system; then add Tris and dopamine hydrochloride to the first mixed system, stir, and separate the solid and liquid to obtain HMPDA; Further, the concentration of the F-127 dispersion is 3-10 mg / mL, preferably 4-8 mg / mL, more preferably 6 mg / mL; the solvent for preparing the F-127 dispersion is an aqueous ethanol solution, which is a mixture of deionized water and ethanol, wherein the volume ratio of deionized water to ethanol is 0.5-1.5:0.5-1.5, preferably 1:1; the volume ratio of the F-127 dispersion to TMB is 5-150:1, preferably 50-120:1, more preferably 100:1; the Tris is added in the form of a Tris aqueous solution, the concentration of which is 20-100 mg / mL, preferably 30-60 mg / mL, more preferably 45 mg / mL; the dopamine hydrochloride is added in the form of an aqueous dopamine hydrochloride solution, the concentration of which is 5-10 mg / mL, preferably 7-8 mg / mL. mg / mL, more preferably 7.5 mg / mL; the volume ratio of the first mixing system, Tris aqueous solution, and dopamine hydrochloride aqueous solution is 10-40:0.5-1.5:0.5:1.5, preferably 20-30: 0.8-1.2:0.8-1.2, more preferably 27.25:1:1; In step (Z1), the stirring reaction time is 10-30 h, preferably 15-25 h, more preferably 18-20 h; solid-liquid separation is preferably carried out by centrifugation or filtration, more preferably, a washing solution is added during the separation process, and the washing solution used for centrifugal washing is a mixed solution of acetone and ethanol, with a volume ratio of 0.5-5:0.5-5, preferably 1-2:1-2, more preferably 1:

2.

6. The preparation method according to claim 2, characterized in that, The preparation steps of step (Z2) are as follows: the hollow mesoporous polydopamine nanoparticles obtained in step (Z1) are dispersed in the dispersion of the components, stirred to obtain a mixture, and then separated into solid and liquid components to obtain Fla@HMPDA. The preparation steps of step (Z3) are as follows: add dopamine hydrochloride and hyaluronic acid to the mixture obtained in step (Z2), stir, and separate the solid and liquid to obtain Fla@HMPDA@HA.

7. The preparation method according to claim 6, characterized in that, In step (Z2), the dispersion of the component is a saturated aqueous solution containing the component; the stirring time is 5-25 h, preferably 10-20 h, and more preferably 13-18 h; In step (Z3), the mass ratio of added dopamine hydrochloride to hyaluronic acid is 1-5:1-5, preferably 1-2:1-3, and more preferably 1-2:1; the stirring time is 0.5-3h, preferably 1-2h, and more preferably 1.5h.

8. The drug delivery system according to claim 1 or the preparation method according to any one of claims 2-7, characterized in that, The component is a natural flavonoid component; preferably, the natural flavonoid component is selected from flavonoids, flavonols, dihydroflavonoids, dihydroflavonols, anthocyanins, flavan-3,4-diols, bisbenzopyrones, chalcones, and biflavonoids; furthermore, the natural flavonoid component includes one or more of hyperoside, rutin, isoquercitrin, gossypol-8-O-β-D-glucuronide, quercetin-3'-O-glucoside, gossypol-3'-O-glucoside, myricetin-3-O-glucoside, quercetin-3-O-sophoroside, gossypol, myricetin, and quercetin.

9. The drug delivery system according to claim 1 or the preparation method according to any one of claims 2-7, characterized in that, The component is okra flower extract; Furthermore, the total flavonoid content of the okra flower extract is 5% or more, further 8% or more, further 55% or more, and even further 68% or more; Furthermore, the okra flower extract contains 12%-41% hyperoside, 0.1%-1.6% rutin, 6%-18% isoquercitrin, 12.5%-22.5% gossypol-8-O-β-D-glucuronide, 1.5%-5.5% myricetin, 9.5%-19.5% quercetin-3'-O-glucosidase, and 0.5%-8.5% quercetin. Furthermore, the okra flower extract contains hyperoside at a content of 12.54%-40.71%, rutin at a content of 0.14%-1.55%, isoquercitrin at a content of 6.46%-17.81%, gossypol-8-O-β-D-glucuronide at a content of 12.68%-22.17%, myricetin at a content of 1.92%-5.2%, quercetin-3'-O-glucosidide at a content of 9.9%-19.37%, and quercetin at a content of 0.59%-8.07%. Furthermore, the okra flower extract contains hyperoside 13.67%-40.71%, rutin 0.15%-1.55%, isoquercitrin 8.93%-17.81%, gossypol-8-O-β-D-glucuronide 13.07%-22.17%, myricetin 2.6%-5.2%, quercetin-3'-O-glucosidide 9.9%-19.37%, and quercetin 0.93%-8.07%. or, The extract of the okra flower contains hyperoside 13.67%-19.8%, rutin 0.14%-0.55%, isoquercitrin 12.56%-16.83%, gossypol-8-O-β-D-glucuronide 13.12%-22.01%, myricetin 3.27%-4.65%, quercetin-3'-O-glucosidide 13.46%-19.06%, and quercetin 3.7%-7.75%. Furthermore, the okra flower extract contains 13.8% hyperoside, 0.14% rutin, 13.63% isoquercitrin, 13.84% gossypol-8-O-β-D-glucuronide, 4.33% myricetin, 15.63% quercetin-3'-O-glucosidide, and 7.52% quercetin. or, The extract of Okra flower contains hyperoside at a content of 6.4 wt% to 19.0 wt%, isoquercitrin at a content of 4.4 wt% to 14.0 wt%, and total flavonoids at a content of 50 wt% to 80 wt%.

10. The drug delivery system according to claim 9, characterized in that, The okra flower extract was prepared by a method comprising the following steps: (1) Extract the flowers or medicinal parts of the yellow hibiscus with ethanol to obtain an extract; (2) The extract is concentrated and then extracted to obtain the extract solution; (3) After removing the solvent from the extract, it was eluted with macroporous resin to obtain the extract of Hibiscus syriacus flower; Alternatively, it can be prepared by a method including the following steps: (1) Extract the flowers or medicinal parts of the yellow hibiscus with ethanol to obtain an extract; (22) Add clarifying agent to the extract, treat with water bath, and filter to remove supernatant; (33) The supernatant was eluted with polyamide resin to obtain okra flower extract; Alternatively, it can be prepared by a method including the following steps: (1) The flowers of the yellow hibiscus were extracted with ethanol to obtain an extract; (23) After removing the solvent from the extract, it was eluted with macroporous resin to obtain the extract of okra flower.

11. The drug delivery system according to claim 10, characterized in that, In step (1), the amount of ethanol used is 10-25 times that of the okra flower, and the ethanol is a 60-95% ethanol solution. In step (2), the extractant used for extraction is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-to-liquid ratio of extraction is 0.8-4:1, and the number of extraction stages is 1-5. In steps (3) and (23), the macroporous resin is of type D101, HPD100 or AB-8, and the elution process of the macroporous resin is as follows: the macroporous resin diameter-to-height ratio is 1:4-9, the sample concentration is 0.10-0.30 g crude drug / mL, the sample volume is 4-12 BV, the sample is loaded at a flow rate of 1-4 BV / h, impurities are removed by 4-8 BV of pure water and 1-5 BV of 3-15% ethanol at a flow rate of 0.5-4 BV / h, and elution is performed by 2-8 BV of 50-80% ethanol at a flow rate of 1-5 BV / h. In step (22), the water bath temperature is 50-70℃ and the water bath time is 30-90min; In step (33), the resin diameter-to-height ratio is 1:4-9, the loading solution concentration is 0.10-0.60 g crude drug / mL, the loading solution volume is 4-12 BV, and elution is performed with 4-8 BV of pure water and 4-8 BV of 60-95% ethanol.

12. The drug delivery system according to claim 9, characterized in that, The preparation of the okra flower extract includes the following steps: (201) The flowers of the yellow hibiscus were crushed into coarse powder and extracted by percolation with 18 times the amount of 80% ethanol to obtain the yellow hibiscus flower extract; (202) The extract of okra flower was deethanoled, diluted with water, and continuously countercurrently extracted with ethyl acetate. The material-to-liquid ratio was 2.5:1 and the extraction stage was 4 stages. The solvent was recovered from the extract under reduced pressure at 40°C. (203) The macroporous resin was treated with D101 macroporous resin. The treatment process of macroporous resin was as follows: the diameter-to-height ratio of macroporous resin was 1:5, the concentration of the loading solution was 0.15 g crude drug / mL, the loading solution volume was 3 BV, the loading rate was 1.5 BV / h, the impurities were removed by 6 BV pure water and 3 BV 10% ethanol at a flow rate of 2 BV / h, the eluent was obtained by elution with 4 BV 60% ethanol at a flow rate of 1.5 BV / h, and then the eluent was subjected to vacuum recovery at 60℃ to obtain the extract of Hibiscus syriacus flower. Alternatively, the okra flower extract is prepared by the following method: okra flowers are extracted with 70%–90% ethanol 1–2 times, each time for 1–3 hours, filtered, and the filtrate is concentrated under reduced pressure at 60°C to a relative density of 1.13–1.

20. The concentrate is allowed to stand for 30–40 hours to remove oil, and water is added to a relative density of 1.10–1.

12. The concentrate is then refrigerated at -2–5°C for 20–40 hours, filtered, and the filtrate is passed through a macroporous adsorption resin chromatography column. Three column volumes are eluted with water, and three to five column volumes each are eluted with 15%–25%, 30%–40%, and 60%–70% ethanol. The ethanol eluent is concentrated under reduced pressure to obtain the okra flower extract. Alternatively, the extract of Hibiscus syriacus flowers can be prepared by the following method: Hibiscus syriacus flowers are extracted once with 80% ethanol for 2 hours, the extracts are combined, filtered, and the filtrate is concentrated to a relative density of 1.13-1.20 at 60°C. An appropriate amount of water is added and the mixture is allowed to precipitate for 30-40 hours. The filtrate is then filtered and passed through a macroporous adsorption resin column. The column is eluted with water for 3 column volumes, with 10% and 30% ethanol for 3 column volumes each, and with 70% ethanol for 1 column volume. The eluent is concentrated, dried, and pulverized to obtain the total flavonoid extract of Hibiscus syriacus flowers. Alternatively, the okra flower extract can be prepared by the following method: okra flowers are extracted by reflux with 85%–95% ethanol 1–3 times, each time for 1–2 hours, filtered, the filtrates are combined and the ethanol is recovered, the filtrate is concentrated to a specific gravity of 1.20–1.35, the concentrate is allowed to stand at 0℃–4℃ for 24–48 hours, the oil layer of the refrigerated liquid is removed, the pH is adjusted to 6.0–7.0, concentrated, and then rapidly dried in a thin layer or by vacuum belt drying to obtain the okra flower extract.

13. A method for preparing the drug delivery system according to any one of claims 1-12, characterized in that, Includes the following steps: (1) F-127 was added to a mixed solution of deionized water and ethanol in a volume ratio of 0.5-1.5:0.5-1.5 to obtain an F-127 dispersion; tetramethylbenzidine was added to the F-127 dispersion to obtain a first mixed system; Tris aqueous solution and dopamine hydrochloride aqueous solution were added to the first mixed system, stirred for 10-30 h, and solid-liquid separation was performed to obtain HMPDA; wherein, the volume ratio of F-127 dispersion to TMB was 5-150:1; the concentration of the Tris aqueous solution was 20-100 mg / mL; the concentration of the dopamine hydrochloride aqueous solution was 5-10 mg / mL; the volume ratio of the first mixed system, Tris aqueous solution and dopamine hydrochloride aqueous solution was 10-40:0.5-1.5:0.5:1.5; (2) Disperse the hollow mesoporous polydopamine nanoparticles obtained in step (Z1) in a dispersion containing the components, stir for 5-25 h to obtain a mixture, and then separate the solid and liquid to obtain Fla@HMPDA; (3) Add dopamine hydrochloride and hyaluronic acid to the mixture prepared in step (Z2), stir for 0.5-3h, separate the solid and liquid to obtain Fla@HMPDA@HA, wherein the mass ratio of added dopamine hydrochloride and hyaluronic acid is 1-5:1-5.

14. The use of the drug delivery system according to any one of claims 1-13 in the preparation of a drug for treating kidney disease, preferably, the kidney disease is chronic kidney disease, chronic nephritis, diabetic nephropathy, diabetic nephropathy or nephritis with renal fibrosis, lupus nephritis, contrast agent-induced kidney injury, membranous glomerulonephritis, postoperative acute kidney injury, or IgA nephropathy.

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