Spleen-targeted nano-assembly of active ingredients of sanhuang xiyin soup and preparation method and application thereof

Abstract

The application discloses a spleen-targeting nanoassemblies of active ingredients of Sanhuang Xiexin Decoction, a preparation method and application thereof, and belongs to the technical field of biological medicine, and comprises spleen-targeting exosomes and nanoassemblies containing active ingredients of Sanhuang Xiexin Decoction, wherein the nanoassemblies containing active ingredients of Sanhuang Xiexin Decoction are loaded on the spleen-targeting exosomes; and the active ingredients of Sanhuang Xiexin Decoction are three active ingredients corresponding to three traditional Chinese medicines, i.e., rhubarb, Coptis chinensis and Scutellaria baicalensis. The application has the beneficial effects that based on three key active components of Sanhuang Xiexin Decoction, the nano delivery system of the carrier-free pure medicine of three active ingredients of traditional Chinese medicine compound is developed for the first time through a chemical self-assembly technology, support is provided for the development of a new dosage form of the nano preparation of the traditional Chinese medicine compound, and the spleen immune function disorder-mediated diseases have a good targeted treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a spleen-targeting nanoassembly of the active ingredient of Sanhuang Xiexin Decoction, its preparation method, and its application. Background Technology

[0002] Sanhuang Xiexin Decoction is a classic formula in traditional Chinese medicine, originating from the *Synopsis of Prescriptions of the Golden Chamber*. It consists of three bitter herbs: rhubarb, coptis, and scutellaria, and its main functions are clearing heat and detoxifying, and promoting blood circulation and removing blood stasis. Modern pharmacological research has found that it possesses antibacterial, anti-inflammatory, and immunomodulatory activities. Currently, formulas based on Sanhuang Xiexin Decoction (rhubarb:scutellaria:coptis = 2:1:1) are widely used and recognized in the clinical treatment of various diseases. In particular, the main active ingredients extracted and isolated from Sanhuang Xiexin Decoction, inspired by the inherent pharmacological activities of the three herbs themselves, including rhein, berberine, and baicalein, have long been extensively studied for their unique pharmacological activities. However, their clinical application is still limited by issues such as poor solubility, low bioavailability, poor targeting, and toxic side effects of some active molecules.

[0003] The self-assembly nanostrategy of traditional Chinese medicine (TCM) has become a hot topic in nanomedicine in recent years and is developing rapidly. Many active components of TCM, such as amino acids, sugars, nucleoside bases, steroids, and triterpenes, possess self-assembly properties, allowing them to form nanoparticles through various non-covalent forces. Currently, researchers mainly focus on the design and development of two-component self-assembly nanodelivery systems, which not only solve the problem of low bioavailability of conventional TCM active components but also improve the synergistic therapeutic effect of the two components. However, TCM compound prescriptions are one of the most commonly used forms of TCM clinical treatment, and they are complex systems rich in multiple active components. Existing two-component TCM self-assembly nanostrategies cannot fully reflect the multi-component therapeutic advantages and TCM therapeutic connotations of TCM compound prescriptions. Therefore, it is necessary to develop a TCM self-assembly nanostrategy based on three or more key components of traditional TCM compound prescriptions to fully explore and elucidate new dosage forms and pharmacodynamic material bases based on TCM compound prescriptions.

[0004] The spleen, the largest secondary lymphoid organ in the human body, participates extensively in various immune functions and possesses the highest density of antigen-presenting cells and B / T lymphocytes, thus rapidly inducing a stronger immune response and serving as a novel target for immunotherapy strategies. Especially under immune imbalances, spleen-derived immune cells can be rapidly mobilized, proliferated, and released, and can also be quickly recruited to damaged tissues, activating and accelerating the damage process. Designing spleen-targeted nanomedicine delivery systems to effectively treat diseases mediated by spleen immune imbalances is of great significance.

[0005] Chinese patent application CN110357877A discloses an active component of Sanhuang Xiexin Decoction, its extraction method, and its uses. Extracted from Sanhuang Xiexin Decoction, the active component mainly includes berberine, baicalin, berberine, emodin-8-O-β-D-glucoside, wogonin, baicalein, emodin, chrysophanol, and emodin methyl ether. The active component extracted from Sanhuang Xiexin Decoction by this patent exhibits significant blocking effects between nNOS and PSD-95 and anti-stroke activity, showing promise as a new, clearly defined, low-toxicity, and highly effective anti-stroke drug candidate. This patent combines high-surface-area MOF materials, surface molecular imprinting technology with specific adsorption properties, and dispersion solid-phase extraction technology (MIL@MIP-SPE) to efficiently and rapidly prepare the active component, avoiding the non-specificity and inefficiency of traditional separation and extraction methods, and greatly improving the efficiency of research on the material basis of traditional Chinese medicine. However, the patent does not cover how to use exosomes to encapsulate the active ingredients of Sanhuang Xiexin Decoction to prepare nanoassemblies. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to propose a spleen-targeting nano-assembly of the active ingredients of Sanhuang Xiexin Decoction.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The first aspect of this invention proposes a spleen-targeted nanoassembly of the active ingredients of Sanhuang Xiexin Decoction, comprising spleen-targeted cell exosomes and nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction, wherein the nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction are loaded onto spleen-targeted cell exosomes; the spleen-targeted cell exosomes are derived from any one of macrophages, T cells, dendritic cells, and B cells; the active ingredients of Sanhuang Xiexin Decoction are three active ingredients corresponding to the three Chinese medicinal herbs rhubarb, coptis, and scutellaria.

[0009] Preferably, the active ingredient in rhubarb is any one of rhein, chrysophanol, and emodin; the active ingredient in Coptis chinensis is any one of berberine, palmatine, and purslane; and the active ingredient in Scutellaria baicalensis is any one of baicalein, baicalin, and wogonin.

[0010] Preferably, the particle size of the spleen-targeting nanoassembly is 80-180 nm.

[0011] Preferably, the nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction is prepared by the following process: First, the active ingredients of any two of the three Chinese herbs, rhubarb, coptis, and scutellaria, are selected and synthesized into inner layer nanoparticles by electrostatic interaction; second, the active ingredient of the remaining Chinese herb is covalently modified onto the surface of the inner layer nanoparticles by a crosslinking agent that responds to the inflammatory microenvironment, thus obtaining the nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction.

[0012] A second aspect of the present invention provides a method for preparing spleen-targeting nanoassemblies of the above-mentioned active ingredients of Sanhuang Xiexin Decoction, comprising the following steps:

[0013] (1) Preparation of nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction:

[0014] S1: Select any two active ingredients from the three Chinese herbs rhubarb, coptis, and scutellaria, dissolve them in an organic solvent, mix them evenly, adjust the pH of the solution to 5-9, add them dropwise to PBS solution, stir, and then place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 for dialysis. After freeze-drying, the inner layer nanoparticles are obtained.

[0015] S2: Dissolve the inner layer nanoparticles, the active ingredients of the remaining Chinese herbal medicine, EDC, NHS, and the crosslinking agent that responds to the inflammatory microenvironment in a mixed solution of organic solvent and water, stir, and then place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 for dialysis and freeze-drying to obtain a nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction.

[0016] (2) The nano-assemblies containing the active ingredients of Sanhuang Xiexin Decoction are encapsulated by spleen-targeting cell exosomes.

[0017] Note: PBS solution is phosphate buffer solution; EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; NHS is N-hydroxysuccinimide.

[0018] Preferably, the reagent used to adjust the pH of the solution is any one of sodium bicarbonate solution, sodium hydroxide solution, and ammonia solution.

[0019] Preferably, the particle size of the inner layer nanoparticles is 50-150 nm.

[0020] (The inner nanoparticle structure is maintained by π-π interactions, hydrogen bonds, and electrostatic interactions.)

[0021] Preferably, the crosslinking agent for the inflammatory microenvironment response is any one of thioketal, ketethioyl thiocyanate, polysulfide propylene, borate ester, and anthocyanin.

[0022] Preferably, in step S2, the organic solvent is selected from any one or more of dimethyl sulfoxide, polyethylene glycol, propylene glycol, and isopropanol.

[0023] Preferably, the method for coating the spleen-targeting cell exosomes with nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction is any one or more of self-membrane coating, co-extrusion, and ultrasonic fusion.

[0024] Preferably, the preparation method specifically includes the following steps:

[0025] (1) Preparation of nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction:

[0026] S1: Weigh out the active ingredients of rhubarb and coptis in a molar ratio of 10:1 to 1:10, and dissolve them separately in a mixture of 5-20 mL of dimethyl sulfoxide and 100-300 mL of methanol. After mixing, adjust the pH of the solution to 5-9, and add it dropwise to PBS at 10-100℃ and stir for 5-50 minutes. After the reaction is complete, place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 and dialyze for 12-24 hours, and freeze-dry to obtain the inner layer nanoparticles.

[0027] S2: Weigh the above-mentioned inner layer nanoparticles and Scutellaria baicalensis active ingredients in a molar ratio of 6:1 to 1:6, and dissolve them together with 45-135 mg EDC, 27-81 mg NHS and 12-48 mg of crosslinking agent that responds to the inflammatory microenvironment in a mixed solution of dimethyl sulfoxide and water. React at 10-100℃ for 1-5 hours. After the reaction is completed, place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 and dialyze for 12-24 hours. Then freeze-dry to obtain a nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction.

[0028] (2) The nano-assemblies containing the active ingredients of Sanhuang Xiexin Decoction were mixed with spleen-targeting cell exosomes at a particle ratio of 1:1 and ultrasonically broken at 4°C for 5 min. After ultrasonication, spleen-targeting cell exosomes loaded with the three-component nano-assemblies of Sanhuang Xiexin Decoction were obtained.

[0029] A third aspect of the present invention proposes the application of the spleen-targeting nanoassemblies of the above-mentioned active ingredients of Sanhuang Xiexin Decoction in the preparation of spleen-targeting therapeutic drugs.

[0030] The beneficial effects of this invention are as follows:

[0031] 1. This invention provides, for the first time, a three-component assembly strategy based on the active ingredients of Sanhuang Xiexin Decoction, and selects spleen-targeting cell membranes to encapsulate them on their surface, thereby regulating the spleen's immune system to efficiently treat diseases. This method provides a new approach for the development of novel dosage forms of traditional Chinese medicine compound prescriptions.

[0032] 2. This invention provides, for the first time, a three-component assembly strategy based on the active ingredients of Sanhuang Xiexin Decoction, and selects spleen-targeting cell membranes to encapsulate them on their surface, thereby regulating the spleen's immune system to efficiently treat diseases. This method provides a new approach for the development of novel dosage forms of traditional Chinese medicine compound prescriptions. Attached Figure Description

[0033] Figure 1 This is a qualitative analysis diagram of the 1H NMR spectrum prepared in Example 1 of the present invention;

[0034] Figure 2 This is a qualitative analysis diagram of the TK nuclear magnetic resonance hydrogen spectrum prepared in Example 1 of the present invention;

[0035] Figure 3 This is a qualitative analysis diagram of the RBT nuclear magnetic resonance hydrogen spectrum prepared in Example 1 of the present invention;

[0036] Figure 4 These are transmission electron microscope (TEM) images of the RB structure in Example 1 of this invention.

[0037] Figure 5 The particle size distribution of RB prepared in Example 1 of this invention is obtained by dynamic light scattering.

[0038] Figure 6 Transmission electron microscopy (TEM) morphology characterization images of the RBT prepared in Example 1 of the present invention;

[0039] Figure 7 The particle size distribution of the RBT prepared in Example 1 of this invention is obtained by dynamic light scattering.

[0040] Figure 8 Transmission electron microscopy (TEM) morphology characterization images of BVEs prepared in Example 1 of this invention;

[0041] Figure 9 Transmission electron microscopy (TEM) morphology characterization images of the BRBT prepared in Example 1 of the present invention;

[0042] Figure 10 The particle size distribution of BVEs prepared in Example 1 of this invention is obtained by dynamic light scattering.

[0043] Figure 11 This is an evaluation chart of the coating efficiency of BRBT prepared by coating RBT (with FITC dye) with BVEs (with DiL dye) in Example 1 of the present invention.

[0044] Figure 12 The therapeutic effects of different concentrations of BRBT prepared in Example 1 of this invention on RAW264.7 macrophages are shown in the figure.

[0045] Figure 13 The therapeutic effects of different nanoassemblies (RB, RBT, BRBT) prepared in Example 1 of this invention on RAW264.7 macrophages at the same concentration are shown in the figure.

[0046] Figure 14 The spleen targeting ability of different nanoparticles (RB, RBT, BRBT) prepared in Example 1 of the present invention was evaluated by in vivo organ imaging after being inoculated with Cy5 dye.

[0047] Figure 15 The different nanoparticles (RB, RBT, BRBT) prepared in Example 1 of this invention were inoculated with Cy5 dye, and the spleen targeting ability of the materials was evaluated by in vitro organ imaging. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0050] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0051] The thioacetal (TK) used in the following examples was prepared by myself, and the process is as follows: Acetone (9.3 mL, 108 mM), 3-mercaptopropionic acid (3.6 mL, 49 mM), and trifluoroacetic acid (8 mL) were mixed and stirred at 25°C for 5 hours. The mixture was then placed in an ice bath and allowed to stand until crystallization was complete. It was then washed three times with hexane and DI water, and finally dried overnight to obtain the thioacetal.

[0052] Example 1:

[0053] A spleen-targeting nanoassembly of the active ingredients of Sanhuang Xiexin Decoction comprises spleen-targeting cell exosomes and nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction. The nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction are loaded onto the spleen-targeting cell exosomes. The spleen-targeting cell exosomes are derived from B cells. The active ingredients of Sanhuang Xiexin Decoction are three active ingredients corresponding to the three traditional Chinese medicines: rhubarb, coptis, and scutellaria. The active ingredient in rhubarb is rhein, the active ingredient in coptis is berberine, and the active ingredient in scutellaria is baicalin.

[0054] The nano-assemblies containing the active ingredients of Sanhuang Xiexin Decoction are prepared through the following process: First, two active ingredients, rhein and berberine, are selected and synthesized into inner nanoparticles by electrostatic interaction; second, scutellarin is covalently modified onto the surface of the inner nanoparticles using thioacetal, a cross-linking agent that responds to the inflammatory microenvironment, thus obtaining the nano-assemblies containing the active ingredients of Sanhuang Xiexin Decoction.

[0055] The preparation method of the above-mentioned spleen-targeting nanoassemblies specifically includes the following steps:

[0056] First, nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction were prepared:

[0057] (1) Synthesis of a two-component nanoassembly (RB) of rhein (Rhe) and berberine (Ber) was performed as follows: 18 mg of (6.3 × 10⁻⁶) phosphate was weighed... -5 (mol) rhein dissolved in 6 mL of dimethyl sulfoxide solution; weigh (38 mg, 11 × 10) -5 (mol) Berberine was dissolved in 113 mL of methanol solution. The two solutions were mixed thoroughly, and the pH was adjusted to 6 with NaHCO3. The solution was then slowly added dropwise to PBS at 60 °C and stirred for 15 minutes. The mixture was then dialyzed in a 3500 Da dialysis bag for 12 hours. Finally, the mixture was freeze-dried to obtain the rhein-berberine two-component nanoassembly (as inner layer nanoparticles).

[0058] (2) Synthesis of the rhein-berberine-baicalein (Bac) three-component nano-assembly (RBT), the process is as follows: Take the rhein-berberine nanoparticles (9×10⁻⁶) from step (1) respectively. -5 mol), thioacetate (24 mg, 0.1 mM), EDC (15.2 mg, 0.15 mM), NHS (9.2 mg, 0.15 mM), baicalein (9 × 10⁻⁶ mol), thioacetate (24 mg, 0.1 mM), EDC (15.2 mg, 0.15 mM), NHS (9.2 mg, 0.15 mM), ba -5 The extract (mol) was dissolved in a mixed solution of dimethyl sulfoxide and DI water (4 mL each of dimethyl sulfoxide and water), and stirred at 35 °C for 36 hours. The aqueous phase was extracted and collected by adding 20 mL of dichloromethane and 20 mL of sodium bicarbonate. Hydrochloric acid was then added to adjust the pH to acidic, and dichloromethane was added again for extraction to collect the organic phase. The extracted product was then dialyzed in a 3500 Da dialysis bag for 12 hours, and finally freeze-dried to obtain the rhein-berberine-baicalein three-component nanoassembly.

[0059] Secondly, prepare spleen-targeting cell exosomes:

[0060] The procedure is as follows: B cells (Baf3) were placed in high-glucose DMEM culture medium and incubated for 24 hours; the supernatant was discarded, the cells were washed with PBS, and then cultured for 24 hours with serum containing no exosomes. The cell culture supernatant was collected in a centrifuge tube; the collected cell supernatant was centrifuged at 4℃ and 450g for 5 minutes and the supernatant was collected; the collected supernatant was centrifuged at 4℃ and 2000g for 20 minutes and the supernatant was collected; the collected supernatant was centrifuged at 4℃ and 12000g for 60 minutes and the supernatant was discarded, thus obtaining B cell exosomes (BEVs).

[0061] Finally, the three-component nanoassemblies were loaded onto spleen-targeting cell exosomes:

[0062] The process is as follows: The three-component nano-assembly of rhein-berberine-baicalin is mixed with B cell exosomes at a particle ratio of 1:1 and ultrasonically broken at 4℃ for 5 minutes. After ultrasonication, the spleen-targeting nano-assembly of the active ingredients of Sanhuang Xiexin Decoction (BRBT) is obtained.

[0063] Furthermore, the three-component nanoassemblies were labeled with the green fluorescent dye FITC, and the spleen-targeting cell exosomes were labeled with the red fluorescent dye DiL, resulting in fluorescently labeled BRBTs.

[0064] Example 2:

[0065] This embodiment characterizes the structure of the nano-assemblies prepared in Example 1. The specific methods and results are as follows:

[0066] Nanostructure of spleen-targeting nanoassemblies containing active ingredients of Sanhuang Xiexin Decoction

[0067] ①H NMR spectrum of the two-component nanoassembly (RB) of rhein (Rhe)-berberine (Ber)

[0068] 10 mg of RB prepared in Example 1 was dissolved in deuterated dimethyl sulfoxide and then transferred to an NMR tube with an inner diameter of 5 mm. The proton NMR spectrum was determined using NMR spectroscopy. The proton NMR spectrum is shown below. Figure 1 As shown, the successful synthesis of RB was confirmed by assigning each peak in the proton spectrum.

[0069] ②1H NMR spectrum of thioacetate (TK)

[0070] 10 mg of the thioacetate (TK) from Example 1 was dissolved in deuterated chloroform and then transferred to an NMR tube with an inner diameter of 5 mm. The proton NMR spectrum was determined using NMR spectroscopy. The proton NMR spectrum is shown below. Figure 2 As shown, the successful synthesis of TK was confirmed by assigning each peak in the proton spectrum.

[0071] ③ The 1H NMR spectrum of the rhein-berberine-baicalein (Bac) three-component nanoassembly (RBT)

[0072] 20 mg of the RBT prepared in Example 1 was dissolved in deuterated dimethyl sulfoxide and then transferred to an NMR tube with an inner diameter of 5 mm. The proton NMR spectrum was measured using NMR spectroscopy. The proton NMR spectrum is shown below. Figure 3 As shown, the successful synthesis of RBT was confirmed by assigning each peak in the proton spectrum.

[0073] ④ Transmission electron microscopy examination of the two-component nanoassembly of rhein (Rhe) and berberine (Ber)

[0074] The RB two-component nanoassembly prepared in Example 1 was used to prepare a transmission electron microscope (TEM) sample for observation. The results are as follows: Figure 4 As shown. TEM observations revealed that the RB nanoassemblies consisted of uniformly spherical particles with dispersed individual particles. The particle size distribution of RB, measured by dynamic light scattering, is shown below. Figure 5 As shown in the figure, the particle size of the RB two-component nano-assembly is mainly between 50-150 nm.

[0075] ⑤ Transmission electron microscopy examination of the rhein-berberine-baicalein (Bac) three-component nanoassembly (RBT)

[0076] The RBT three-component nanoassembly prepared in Example 1 was used to prepare a transmission electron microscope (TEM) sample for observation. The results are as follows: Figure 6 As shown. TEM observations revealed that the RBT nanoassemblies consisted of uniformly spherical particles with dispersed individual particles. The particle size distribution of RBT, measured by dynamic light scattering, is shown below. Figure 7 As shown in the figure, the particle size of the RBT three-component nanoassembly is mainly distributed in the range of 50-150 nm.

[0077] ⑥ Transmission electron microscopy examination of B cell exosomes (BVEs)

[0078] The BVE exosomes prepared in Example 1 were used to prepare transmission electron microscopy (TEM) samples for observation. The results are as follows: Figure 8 As shown in the image. TEM observations revealed that exosome BVEs appeared as spherical particles.

[0079] ⑦ The spleen-targeting nanoassemblies (BRBTs) containing fluorescently labeled active ingredients of Sanhuang Xiexin Decoction were examined by transmission electron microscopy.

[0080] The BRBT prepared in Example 1 was used to prepare a transmission electron microscope (TEM) sample for observation. The results are as follows: Figure 9 As shown. TEM observations revealed that BRBT exhibits regularly shaped spherical nanoparticles. The particle size distribution of BVEs and BRBT, measured by dynamic light scattering, is shown below. Figure 10 As shown in the figure, the particle size of the BRBT spleen-targeted nanoassemblies is mainly distributed in the range of 80-180 nm.

[0081] ⑧ Evaluation of the fusion efficiency of RBT and BVEs

[0082] The BRBT labeled with fluorescent dye prepared in Example 1 was used to observe the coating of RBT (green fluorescent label) and BVEs (red fluorescent label) using an inverted fluorescence microscope.

[0083] like Figure 11 As shown, BVEs can efficiently coat BRBT with an efficiency of over 80%, further proving the successful preparation of BRBT.

[0084] Example 3:

[0085] This embodiment characterizes the properties of the nanoassemblies prepared in Example 1. The specific methods and results are as follows:

[0086] ① Evaluation of the therapeutic effects of different concentrations of BRBT on RAW264.7 macrophages

[0087] Cell viability was determined using the MTT assay. RAW264.7 macrophages were cultured at a concentration of 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 100 cells / mL in 96-well plates, with a seeding volume of 200 μL per well. Cells were cultured in high-glucose DMEM containing 10% FBS at 37°C in a 5% CO2 incubator. After 24 h of culture, cells were stimulated with 100 ng / mL LPS, and cultured for another 24 h. Then, cells were seeded with different concentrations of BRBT (10... 4 0.5*10 5 10 5 0.5*10 6 10 6 0.5*10 7 10 7 The number of particles / mL was used as the treatment group to assess its therapeutic effect on cells. The BRBT-free group was used as the toxicity group, and the blank cell culture medium group was used as the blank control group. After adding samples, the 96-well plates were placed in an incubator at 37°C and 5% CO2 for 24 hours. After the culture was completed, the supernatant was discarded, and 20 μL of 5 mg / mL MTT solution was added to each well. After culturing for another 4 hours, the supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were then shaken in the dark for 10 minutes, and the OD value at 490 nm was measured using a microplate reader. The experiment was repeated in triplicate.

[0088] The results are as follows Figure 12 As shown in the MTT results, the cell viability in the BRBT group was significantly higher than that in the LPS toxicity group, indicating that BRBT is non-toxic to cells and has good biosafety, especially at a concentration of 0.5*10⁻⁶. 7 Cell viability is maximized when the number of particles per mL is 100.

[0089] ②Evaluation of the therapeutic effects of RB, RBT, and BRBT on RAW264.7 macrophages

[0090] Cell viability was determined using the MTT assay. RAW264.7 macrophages were cultured at a concentration of 1 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of 200 μL / mL in 96-well plates and cultured in high-glucose DMEM containing 10% FBS at 37°C in a 5% CO2 incubator. After 24 h of culture, 100 ng / mL LPS was added to stimulate the cells. After another 24 h of culture, the therapeutic effects of different nanoparticles (RB, RBT, and BRBT) on the cells were assessed. The group without nanoparticles was designated as the toxicity group, and the blank cell culture medium group served as the blank control group. After adding samples, the 96-well plates were placed in a 37°C, 5% CO2 incubator for another 24 h. After the culture was completed, the supernatant was discarded, and 20 μL of 5 mg / mL MTT solution was added to each well. After another 4 h of culture, the supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were then shaken in the dark for 10 min, and the OD value at 490 nm was measured using a microplate reader. The experiment was repeated in triplicate, and the therapeutic effects of RB, RBT, and BRBT at the same concentration were compared.

[0091] The results are as follows Figure 13 As shown in the MTT results, the cell survival rate of the BRBT group reached 92%, and the therapeutic effect was significantly higher than that of other groups, indicating that the BRBT prepared in this invention has the best therapeutic effect.

[0092] The above experimental results indicate that BRBT has no cytotoxicity to inflammatory macrophages and has good biocompatibility, thus it is of great significance for the regulation of macrophages.

[0093] ③ Evaluation of the targeting ability of RB, RBT, and BRBT in the spleen region

[0094] Male C57BL / 6J black mice aged 6-8 weeks were used to construct a PD mouse model by routine intraperitoneal injection of MPTP. Cy5 dye was attached to the RB, RBT, and BRBT prepared in Example 1 by incubation.

[0095] Twelve C57BL / 6J black mice had their backs shaved, and were divided into four groups of three. Each group was then injected intravenously with 200 μL of a nano-formulation prepared in PBS, containing 0.5 x 10⁻⁶ mg / L of the nano-formulation. 7 Solutions containing a certain number of particles per mL (Cy5, RBT-Cy5, BVEs-Cy5, BRBT-Cy5). Mice were anesthetized with a mixture of 2% isoflurane and oxygen at 0h, 1h, 3h, 5h, 8h, and 12h after injection, and the spleen was imaged and analyzed using an in vivo imaging system.

[0096] The results are as follows Figure 14As shown in the in vivo imaging results, 1 hour after tail vein injection, mice in each experimental group exhibited a certain fluorescence intensity in their brains. In the Cy5 group, the fluorescence intensity gradually decreased over time, and the fluorescence essentially disappeared after 12 hours. In the RBT-Cy5 group, the fluorescence intensity gradually decreased over time, and the overall fluorescence intensity was stronger compared to the Cy5-only group, but the fluorescence essentially disappeared after 12 hours. In the BEVs-Cy5 and BRBT-Cy5 groups, the fluorescence intensity gradually increased over time from 0 to 5 hours, and the fluorescence intensity was significantly stronger than other groups. The highest fluorescence intensity was observed in the spleen at 5 hours, indicating good spleen targeting. Furthermore, the combined targeting results of each group suggest that the targeting originates from BVE exosomes.

[0097] In vitro organ imaging in mice: The C57BL / 6J mice were euthanized by cervical dislocation, and their organs (brain, heart, liver, spleen, lung, and kidney) were collected and fixed in paraformaldehyde for 3 hours. Subsequently, imaging analysis of each organ was performed using an in vivo imaging system.

[0098] The results are as follows Figure 15 As shown in the in vivo imaging results, compared with the Cy5 and RBT-Cy5 groups, the BEVs-Cy5 group showed the strongest fluorescence in the spleen, indicating that the two groups of materials have better spleen targeting.

[0099] The above results demonstrate that BEVs and BRBTs have excellent spleen-targeting performance.

[0100] Example 4:

[0101] The difference between this embodiment and Embodiment 1 is that rhein, berberine, and baicalein are replaced with "chrysophanol, palmatine, and baicalin", respectively, while the rest is the same as in Embodiment 1.

[0102] Example 5:

[0103] The difference between this embodiment and Embodiment 1 is that rhein, berberine, and baicalin are replaced with "emodin, rhein alkaloid, and baicalin", respectively, while the rest is the same as in Embodiment 1.

[0104] Example 6:

[0105] The difference between this embodiment and Embodiment 1 is that the preparation steps of the nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction are as follows: first, a two-component nano-assembly of rhein (Rhe) and baicalin (Bac) is prepared, and then a three-component nano-assembly of rhein, baicalin, and berberine (Ber) is prepared. The rest is the same as in Embodiment 1.

[0106] Example 7:

[0107] The difference between this embodiment and Embodiment 1 is that the preparation steps of the nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction are as follows: first, a two-component nano-assembly of baicalin (Bac) and berberine (Ber) is prepared, and then a three-component nano-assembly of baicalin-berberine-rheic acid (Rhe) is prepared. The rest is the same as in Embodiment 1.

[0108] The structures and properties of the spleen-targeting nanoassemblies of the active ingredients of Sanhuang Xiexin Decoction prepared in Examples 4-7 are similar to those in Example 1.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A spleen-targeting nanoassembly of the active ingredients of Sanhuang Xiexin Decoction, characterized in that, The invention comprises spleen-targeting cell exosomes and nanoassemblies containing active ingredients of Sanhuang Xiexin Decoction. The nanoassemblies containing active ingredients of Sanhuang Xiexin Decoction are loaded onto spleen-targeting cell exosomes. The spleen-targeting cell exosomes are derived from any one of macrophages, T cells, dendritic cells, and B cells. The active ingredients of Sanhuang Xiexin Decoction are the three active ingredients corresponding to the three Chinese herbal medicines: rhubarb, coptis, and scutellaria. The particle size of the spleen-targeting nanoassemblies is 80-180 nm. The nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction is prepared by the following process: First, the active ingredients of Coptis chinensis and any one of Rheum palmatum or Scutellaria baicalensis are selected and the inner layer nanoparticles are synthesized by electrostatic interaction; second, the active ingredients of the remaining Chinese medicine are covalently modified onto the surface of the inner layer nanoparticles by a crosslinking agent that responds to the inflammatory microenvironment, thus obtaining the nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction. The active ingredient in rhubarb is any one of rhein, chrysophanol, and emodin; the active ingredient in Coptis chinensis is any one of berberine, palmatine, and purslane; and the active ingredient in Scutellaria baicalensis is any one of baicalein, baicalin, and wogonin.

2. The spleen-targeting nanoassembly of the active ingredients of Sanhuang Xiexin Decoction according to claim 1, characterized in that, The active ingredient in rhubarb is rhein.

3. A method for preparing the spleen-targeting nanoassembly of the active ingredient of Sanhuang Xiexin Decoction according to any one of claims 1-2, comprising the following steps: (1) Preparation of nanoassemblies containing active ingredients of Sanhuang Xiexin Decoction: S1: First, select the active ingredients of Coptis chinensis and any one of the active ingredients of rhubarb or Scutellaria baicalensis, dissolve them in an organic solvent, mix them evenly, adjust the pH of the solution to 5-9, add them dropwise to PBS solution, stir, and then place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 for dialysis, freeze-dry, and the inner layer nanoparticles are obtained. S2: Dissolve the inner layer nanoparticles, the active ingredients of the remaining Chinese herbal medicine, EDC, NHS, and the crosslinking agent that responds to the inflammatory microenvironment in a mixed solution of organic solvent and DI water, stir, and then place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 for dialysis and freeze-drying to obtain a nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction. (2) The nano-assemblies containing the active ingredients of Sanhuang Xiexin Decoction are encapsulated by spleen-targeting cell exosomes.

4. The preparation method according to claim 3, characterized in that, The reagent used to adjust the pH of the solution is any one of sodium bicarbonate solution, sodium hydroxide solution, or ammonia solution.

5. The preparation method according to claim 3, characterized in that, The crosslinking agent for the inflammatory microenvironment response is any one of thioketal, ketethioyl alcohol, polysulfide propylene, or borate ester.

6. The preparation method according to claim 3, characterized in that, The inner layer nanoparticles have a particle size of 50-150 nm.

7. The preparation method according to claim 3, characterized in that, The method for coating the spleen-targeting cell exosomes with nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction is any one or more of self-membrane coating, co-extrusion, and ultrasonic fusion.

8. The preparation method according to claim 7, characterized in that, The method for encapsulating a nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction on a spleen-targeting cell exosome is ultrasonic fusion.

9. The preparation method according to claim 3, characterized in that, The preparation method specifically includes the following steps: (1) Preparation of nanoassemblies containing active ingredients of Sanhuang Xiexin Decoction: S1: Weigh out the active ingredients of rhubarb and coptis in a molar ratio of 10:1 to 1:10, and dissolve them separately in a mixture of 5-20 mL of dimethyl sulfoxide and 100-300 mL of methanol. After mixing, adjust the pH of the solution to 5-9, and add it dropwise to PBS at 10-100 ℃ with constant temperature stirring for 5-50 minutes. After the reaction is complete, place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 and dialyze for 12-24 hours, and freeze-dry to obtain the inner layer nanoparticles. S2: Weigh out the inner layer nanoparticles and Scutellaria baicalensis active ingredients in a molar ratio of 6:1 to 1:6, and dissolve them together with 45-135 mg EDC, 27-81 mg NHS and 12-48 mg of crosslinking agent for inflammatory microenvironment response in a mixed solution of dimethyl sulfoxide and DI water. React at 10-100 °C for 1-5 hours. After the reaction is completed, place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 and dialyze for 12-24 hours. Then freeze-dry to obtain a nano-assembly containing the active ingredients of Sanhuang Xiexin Decoction. (2) The nano-assemblies containing the active ingredients of Sanhuang Xiexin Decoction were mixed with spleen-targeting cell exosomes at a particle ratio of 1:1 and ultrasonically broken at 4°C for 5 min. After ultrasonication, spleen-targeting cell exosomes loaded with the three-component nano-assemblies of Sanhuang Xiexin Decoction were obtained.

10. The spleen-targeting nanoassembly of the active ingredient of Sanhuang Xiexin Decoction as described in any one of claims 1-2, or the spleen-targeting nanoassembly of the active ingredient of Sanhuang Xiexin Decoction prepared by the preparation method described in any one of claims 3-9, in the preparation of spleen-targeting therapeutic drugs.

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