Spleen-targeted nano assembly of Sanhuang heart-fire purging decoction active ingredients as well as preparation method and application of spleen-targeted nano assembly

Abstract

The invention discloses a spleen-targeted nano assembly of an active component of Sanhuang heart-fire purging decoction as well as a preparation method and application of the spleen-targeted nano assembly, and belongs to the technical field of biological medicines. The spleen-targeted nano assembly comprises a spleen-targeted cell exosome and a nano assembly containing the active component of the Sanhuang heart-fire purging decoction, the nano assembly containing the active ingredients of the Sanhuang heart-fire purging decoction is loaded on the spleen-targeted cell exosome; the active ingredients of the Sanhuang heart-fire purging decoction comprise three active ingredients corresponding to three traditional Chinese medicines, namely rheum officinale, coptis chinensis and scutellaria baicalensis. The invention has the following beneficial effects: on the basis of three key active components of the Sanhuang heart-fire-purging decoction, a nano delivery system of a carrier-free pure drug of three active components of a traditional Chinese medicine compound is developed for the first time through a chemical self-assembly technology, and support is provided for development of a new dosage form of a traditional Chinese medicine compound nano preparation; and the compound has a good targeted therapeutic effect on diseases mediated by spleen immune dysfunction.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a spleen-targeted nano-assembly of the active ingredients of Sanhuang Xiexin Decoction, and a preparation method and application thereof. Background Art

[0002] Sanhuang Xiexin Decoction is a classic prescription in clinical Chinese medicine. It comes from the Synopsis of the Golden Chamber. It is composed of three bitter herbs: rhubarb, coptis chinensis, and scutellaria baicalensis. Its main effects are clearing away heat and detoxifying, promoting blood circulation and removing blood stasis. Modern pharmacological studies have found that it has antibacterial, anti-inflammatory and immunomodulatory activities. At present, the "Xiexin Decoction" prescription based on Sanhuang Xiexin Decoction (rhubarb: scutellaria baicalensis: coptis chinensis = 2:1:1) has been widely used and recognized in the clinical treatment of various diseases. In particular, the main active ingredients extracted and separated from Sanhuang Xiexin Decoction are inspired by the pharmacodynamic activities of the three traditional Chinese medicines themselves. Rhein, berberine and baicalein, as typical active ingredients, have also been widely studied for their unique pharmacological activities for a long time. However, due to the poor solubility, low bioavailability, poor targeting, toxic side effects and other problems of some active molecules, their clinical application is still limited.

[0003] As one of the hot topics of nanomedicine in recent years, the self-assembly nano-strategy of traditional Chinese medicine is developing rapidly. Many active ingredients of traditional Chinese medicine, such as amino acids, sugars, nucleoside bases, steroids, triterpenes, etc., have self-assembly properties, allowing them to form nanoparticles through various non-covalent forces. At present, researchers mainly focus on the design and development of self-assembly nano-delivery systems based on two components, which not only solves the low bioavailability of active ingredients of conventional traditional Chinese medicine, but also improves the synergistic therapeutic effect of the two components. However, traditional Chinese medicine compound is one of the most commonly used forms of traditional Chinese medicine in clinical treatment, which is a complex system rich in multiple active ingredients. The existing two-component traditional Chinese medicine self-assembly nano-strategy is difficult to reflect the multi-component therapeutic advantages of traditional Chinese medicine compound and the connotation of traditional Chinese medicine treatment. Therefore, it is necessary to develop a traditional Chinese medicine self-assembly nano-strategy based on the key three or more components of traditional Chinese medicine compound, in order to fully explore and explain the new dosage form and efficacy material basis based on traditional Chinese medicine compound.

[0004] The spleen is the largest secondary lymphoid organ in the human body. It is widely involved in various immune functions of the body and has the highest density of antigen presenting cells and B / T lymphocytes, which can quickly induce stronger immune responses and can be used as a new target for immunotherapy strategies. Especially under the condition of immune imbalance, spleen-derived immune cells can not only be quickly mobilized, proliferated and released, but also be quickly recruited to damaged tissues to activate and accelerate the damage process. For diseases mediated by splenic immune imbalance, it is of great significance to design spleen-targeted nanodrug delivery systems to efficiently treat diseases by regulating the splenic immune system.

[0005] The Chinese patent application document with publication number CN110357877A discloses an active component of Sanhuang Xiexin Decoction and its extraction method and use. The components extracted from Sanhuang Xiexin Decoction mainly include coptisine, baicalin, berberine, emodin-8-O-β-D-glucoside, wogonin, baicalin, emodin, chrysophanol and emodin methyl ether. The active component of Sanhuang Xiexin Decoction extracted in this patent has obvious blocking effect on the coupling between nNOS and PSD-95 and anti-stroke effect, and is expected to become a new candidate drug for anti-stroke with clear ingredients, low toxicity and high efficiency; this patent combines MOFs materials with high specific surface area, surface molecular imprinting technology with specific adsorption and dispersed solid phase extraction technology (MIL@MIP-SPE), and efficiently and quickly prepares active components, avoiding the nonspecificity and inefficiency of traditional separation and extraction, and greatly improving the efficiency of basic research on Chinese medicine substances. However, the patent does not involve how to use exosomes to encapsulate and load the active ingredients of Sanhuang Xiexin Decoction to prepare nanoassemblies. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to propose a spleen-targeted nanoassembly of the active ingredients of Sanhuang Xiexin Decoction.

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

[0008] The first aspect of the present invention proposes a spleen-targeted nanoassembly of the active ingredients of Sanhuang Xiexin Decoction, including 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 on 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 of rhubarb, coptis chinensis, and scutellaria baicalensis.

[0009] Preferably, the active ingredient in the rhubarb is any one of rhein, chrysophanol and rhein, the active ingredient in the coptis chinensis is any one of berberine, bamipine and jatrorrhizine, and the active ingredient in the 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 nanoassembly 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 medicinal herbs, rhubarb, coptis chinensis, and scutellaria baicalensis, are selected and synthesized into inner layer nanoparticles by electrostatic action; secondly, the active ingredient of the remaining Chinese medicinal herb is covalently modified on the surface of the inner layer nanoparticles by a cross-linking agent that responds to the inflammatory microenvironment, thereby obtaining a nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction.

[0012] The second aspect of the present invention provides a method for preparing the spleen-targeted nanoassembly of the active ingredients of the 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 of the three Chinese medicines of rhubarb, coptis chinensis and scutellaria baicalensis, dissolve them in an organic solvent, mix them evenly, adjust the pH of the solution to 5-9, add them dropwise to a PBS solution, stir, and then dialyze the solution in a dialysis bag with a molecular weight cutoff of less than 10,000, and freeze-dry to obtain inner layer nanoparticles;

[0015] S2: The inner layer nanoparticles, the active ingredient of the remaining Chinese medicine, EDC, NHS and the cross-linking agent for inflammatory microenvironment response are dissolved in a mixed solution of an organic solvent and water, stirred, and then the solution is dialyzed in a dialysis bag with a molecular weight cutoff of less than 10,000, and freeze-dried to obtain a nanoassembly containing the active ingredient of Sanhuang Xiexin Decoction;

[0016] (2) The nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction is coated on the spleen-targeted cell exosomes to obtain the nanoassembly.

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

[0018] Preferably, the reagent for adjusting the pH of the solution is any one of a sodium bicarbonate solution, a sodium hydroxide solution, and an 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 cross-linking agent responsive to the inflammatory microenvironment is any one of thioketal, ketalthiool, polypropylene sulfide, borate, 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 of coating the nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction on spleen-targeted cell exosomes is any one or more of self-membrane coating, co-extrusion, and ultrasonic fusion.

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

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

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

[0027] S2: Weigh the inner layer nanoparticles and the active ingredient of Scutellaria baicalensis 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 a cross-linking agent responsive to an inflammatory microenvironment in a mixed solution of dimethyl sulfoxide and water, react at 10-100° C. for 1-5 hours, and after the reaction, dialyze the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 for 12-24 hours, and freeze-dry to obtain a nanoassembly containing the active ingredient of Sanhuang Xiexin Decoction;

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

[0029] The third aspect of the present invention proposes the use of the spleen-targeted nanoassembly of the active ingredients of the Sanhuang Xiexin Decoction in the preparation of spleen-targeted therapeutic drugs.

[0030] The beneficial effects of the present invention are:

[0031] 1. The present invention provides for the first time a three-component assembly strategy based on the active ingredients of Sanhuang Xiexin Decoction, and selects spleen-targeted cell membranes to wrap on its surface, thereby regulating the spleen's immune system to efficiently treat diseases. This method provides a new idea for the development of new dosage forms of traditional Chinese medicine compounds.

[0032] 2. The present invention provides for the first time a three-component assembly strategy based on the active ingredients of Sanhuang Xiexin Decoction, and selects spleen-targeted cell membranes to wrap on its surface, thereby regulating the spleen's immune system to efficiently treat diseases. This method provides a new idea for the development of new dosage forms of traditional Chinese medicine compounds. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0035] Figure 3 This is a qualitative analysis diagram of the H NMR spectrum of RBT prepared in Example 1 of the present invention;

[0036] Figure 4 This is a transmission electron microscope (TEM) morphology characterization picture of Example 1RB of the present invention;

[0037] Figure 5 The particle size distribution diagram of RB prepared in Example 1 of the present invention measured by dynamic light scattering;

[0038] Figure 6 This is a transmission electron microscope (TEM) morphology characterization picture of the RBT prepared in Example 1 of the present invention;

[0039] Figure 7 The particle size distribution diagram of RBT prepared in Example 1 of the present invention measured by dynamic light scattering;

[0040] Figure 8 This is a transmission electron microscope (TEM) morphology characterization picture of the BVEs prepared in Example 1 of the present invention;

[0041] Fig. 9 This is a transmission electron microscope (TEM) morphology characterization picture of the BRBT prepared in Example 1 of the present invention;

[0042] Fig.10 The particle size distribution of BVEs and BRBT prepared in Example 1 of the present invention measured by dynamic light scattering;

[0043] Fig.11 This is a diagram for evaluating the coating efficiency of BRBT obtained by coating RBT (connected with FITC dye) with BVEs (connected with DiL dye) in Example 1 of the present invention;

[0044] Fig.12 This is a diagram showing the therapeutic effects of different concentrations of BRBT prepared in Example 1 of the present invention on macrophage RAW264.7;

[0045] Fig.13 This is a diagram showing the therapeutic effects of different nanoassemblies (RB, RBT, BRBT) prepared in Example 1 of the present invention on macrophages RAW264.7 at the same concentration;

[0046] Fig.14 Different nanoparticles (RB, RBT, BRBT) prepared in Example 1 of the present invention are connected with Cy5 dye, and the spleen targeting ability of the materials is evaluated by in vivo organ imaging;

[0047] Fig.15 Different nanoparticles (RB, RBT, BRBT) prepared in Example 1 of the present invention were coated with Cy5 dye, and the spleen targeting ability of the materials was evaluated by in vitro organ imaging. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0050] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0051] The thioketal (TK) used in the following examples was prepared by myself, and the process was 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 placed in an ice bath and allowed to stand until crystallization was complete. The thioketal was then washed with hexane and DI water for 3 times, and finally dried overnight to obtain the thioketal.

[0052] Embodiment 1:

[0053] A spleen-targeted nanoassembly of the active ingredients of Sanhuang Xiexin Decoction includes 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 on spleen-targeted cell exosomes; the spleen-targeted cell exosomes are derived from B cells; the active ingredients of Sanhuang Xiexin Decoction are three active ingredients corresponding to the three Chinese medicines of rhubarb, coptis chinensis, and scutellaria baicalensis. The active ingredient in rhubarb is rhein, the active ingredient in coptis chinensis is berberine, and the active ingredient in scutellaria baicalensis is baicalein.

[0054] Among them, the nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction was prepared by the following process: first, two active ingredients, rhein and berberine, were selected and the inner layer nanoparticles were synthesized by electrostatic action; secondly, the cross-linking agent thioketal that responds to the inflammatory microenvironment was used to covalently modify baicalin on the surface of the inner layer nanoparticles, thereby obtaining the nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction.

[0055] The preparation method of the spleen-targeted nanoassembly specifically comprises the following steps:

[0056] First, a nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction was prepared:

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

[0058] (2) Synthesis of rhein-berberine-baicalein (Bac) three-component nanoassembly (RBT) was carried out as follows: the rhein-berberine nanoparticles (9×10 -5 mol), thioketal (24 mg, 0.1 mM), EDC (15.2 mg, 0.15 mM), NHS (9.2 mg, 0.15 mM), baicalein (9 × 10 -5 mol) was dissolved in a mixed solution of dimethyl sulfoxide and DI water (4 ml of dimethyl sulfoxide and water), and stirred at 35°C for 36 hours. 20 mL of dichloromethane and 20 mL of sodium bicarbonate were added to extract and collect the aqueous phase. Hydrochloric acid was then added to adjust the pH to acidic, and dichloromethane was added to extract and collect the organic phase. The extracted product was then placed in a 3500Da dialysis bag for 12 hours, and finally freeze-dried to obtain the three-component nanoassembly of rhein-berberine-baicalein.

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

[0060] The process is as follows: B cells (Baf3) are placed in high-glucose DMEM culture medium and incubated for 24 hours; the supernatant is discarded, the cells are washed with PBS, and then exosome-free serum is added for 24 hours, and the cell culture supernatant solution is collected in a centrifuge tube; the collected cell supernatant is centrifuged at 4°C and 450g for 5 minutes, and the supernatant is collected; the collected supernatant is centrifuged at 4°C and 2000g for 20 minutes, and the supernatant is collected; the collected supernatant is centrifuged at 4°C and 12000g for 60 minutes, and the supernatant is discarded to obtain B cell exosomes (BEVs).

[0061] Finally, the three-component nanoassembly was loaded into spleen-targeted cell exosomes:

[0062] The process is as follows: the three-component nanoassembly of rhein-berberine-baicalein is mixed with B cell exosomes in a particle number of 1:1, and ultrasonically disrupted at 4°C for 5 minutes. After the ultrasonication, the spleen-targeted nanoassembly (BRBT) of the active ingredient of Sanhuang Xiexin Decoction is obtained.

[0063] Furthermore, the three-component nanoassembly was labeled with the green fluorescent dye FITC, and the spleen-targeted cell exosomes were labeled with the red fluorescent dye DiL to obtain fluorescent dye-labeled BRBT.

[0064] Embodiment 2:

[0065] This example performs structural characterization on the nanoassembly prepared in Example 1, and the specific method and results are as follows:

[0066] Nanostructure investigation of spleen-targeted nanoassemblies of active ingredients from Sanhuang Xiexin Decoction

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

[0068] 10 mg of RB prepared in Example 1 was dissolved in deuterated dimethyl sulfoxide and then transferred to a nuclear magnetic resonance tube with an inner diameter of 5 mm. The hydrogen spectrum was measured by nuclear magnetic resonance spectroscopy. The hydrogen spectrum was as follows: Figure 1 As shown, the successful synthesis of RB was confirmed by assigning the peaks in the hydrogen spectrum.

[0069] ②H NMR spectrum of thioketal (TK)

[0070] 10 mg of the thioketal (TK) prepared in Example 1 was dissolved in deuterated chloroform and then transferred to a nuclear magnetic resonance tube with an inner diameter of 5 mm. The hydrogen spectrum was measured by nuclear magnetic resonance spectroscopy. Figure 2 As shown, the successful synthesis of TK was confirmed by assigning the peaks in the hydrogen spectrum.

[0071] ③NMR spectroscopy of rhein-berberine-baicalein (Bac) three-component nanoassembly (RBT)

[0072] 20 mg of RBT prepared in Example 1 was dissolved in deuterated dimethyl sulfoxide and then transferred to a nuclear magnetic resonance tube with an inner diameter of 5 mm. The hydrogen spectrum was measured by nuclear magnetic resonance spectroscopy. The hydrogen spectrum was as follows: Figure 3 As shown, the successful synthesis of RBT was confirmed by assigning the peaks in the hydrogen spectrum.

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

[0074] The RB two-component nanoassembly prepared in Example 1 was used to prepare a transmission electron microscope (TEM) sample and observe it. Figure 4 As shown in Figure 2. According to TEM observation, RB nanoassemblies are uniform spherical particles and are individually dispersed. The particle size distribution of RB measured by dynamic light scattering is shown in Figure 2. Figure 5 As shown in the figure, it can be seen that the particle size of the RB two-component nanoassembly is mainly between 50-150nm.

[0075] ⑤ Transmission electron microscopy study of 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 and observe it. Figure 6 As shown in Figure 2. According to TEM observation, RBT nanoassemblies are uniform spherical particles and are individually dispersed. The particle size distribution of RBT measured by dynamic light scattering is shown in Figure 2. Figure 7 As shown, it can be seen from the figure that the particle size of the RBT three-component nanoassembly is mainly distributed in 50-150nm.

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

[0078] The BVEs exosomes prepared in Example 1 were used to prepare transmission electron microscope (TEM) samples and observe them. Figure 8 As shown in Figure 2, according to TEM observation, exosome BVEs appeared as spherical particles.

[0079] ⑦ Transmission electron microscopy examination of the spleen-targeted nanoassembly (BRBT) of the fluorescently labeled active ingredients of Sanhuang Xiexin Decoction

[0080] The BRBT prepared in Example 1 was used to prepare a transmission electron microscope (TEM) sample and observe it. Fig. 9 As shown in Figure 2. According to TEM observation, BRBT is a spherical nanoparticle with regular morphology. The particle size distribution of BVEs and BRBT measured by dynamic light scattering is shown in Figure 2. Fig.10 As shown, it can be seen from the figure that the particle size of BRBT spleen-targeted nanoassemblies is mainly distributed in the range of 80-180 nm.

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

[0082] Take the BRBT labeled with fluorescent dye prepared in Example 1 and observe the coating of RBT (green fluorescent label) and BVEs (red fluorescent label) by fluorescence inverted microscope.

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

[0084] Embodiment 3:

[0085] This example characterizes the performance of the nanoassembly prepared in Example 1, and the specific method and results are as follows:

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

[0087] The cell viability was determined by MTT assay. RAW264.7 macrophages were cultured at 1×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, with a volume of 200 μL per well, and cultured in a 37°C, 5% CO2 incubator with high-glucose DMEM containing 10% FBS. After 24 hours of culture, 100 ng / mL LPS was added to stimulate the cells. After another 24 hours of culture, 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 detect its therapeutic effect on cells, the group without BRBT was used as the toxicity group, and the blank cell culture medium group was used as the blank control group. After adding the sample, the 96-well plate was placed in an incubator at 37°C and 5% CO2 for further 24 hours. After the culture was completed, the supernatant was discarded, and 20μL of 5mg / mL MTT solution was added to each well. After continuing to culture for 4 hours, the supernatant was discarded, and 150μL DMSO was added to each well. After being placed on a shaker in the dark for 10 minutes, the OD value at a wavelength of 490nm was measured with an enzyme marker. The experiment was repeated three times in parallel.

[0088] The results are as follows Fig.12 As shown in the MTT results, the cell activity in the BRBT group was greater than that in the LPS toxicity group, indicating that BRBT was non-toxic to cells and had good biosafety. 7 When the number of particles / mL is less than 100, the cell activity is maximum.

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

[0090] The cell viability was determined by MTT assay. RAW264.7 macrophages were cultured at 1×10 5The cells were inoculated at a density of 100 μL / mL in a 96-well plate, with a volume of 200 μL per well. The cells were cultured in a high-glucose DMEM cell culture medium containing 10% FBS at 37°C and 5% CO2 incubator. After 24 hours of culture, 100 ng / mL LPS was added to stimulate the cells. After 24 hours of culture, different nanoparticles RB, RBT, and BRBT were used as treatment groups to detect their therapeutic effects on cells. The group without nanoparticles was used as a toxicity group, and the blank cell culture medium group was used as a blank control group. After adding the sample, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. After the culture was completed, the supernatant was discarded, 20 μL, 5 mg / mL MTT solution was added to each well, and after 4 hours of culture, the supernatant was discarded, 150 μL DMSO was added to each well, and the cells were placed on a shaker in the dark for 10 minutes. The OD value at a wavelength of 490 nm was measured with an enzyme marker. The experiment was repeated three times in parallel, and the therapeutic effects of RB, RBT, and BRBT at the same concentration were compared.

[0091] The results are as follows Fig.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 by the present invention has the best therapeutic effect.

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

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

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

[0095] Twelve C57BL / 6J mice were depilated from their backs and divided into four groups of three mice each. Each group was injected with 200 μL of the nanoparticle preparation prepared with PBS containing 0.5*10 7 The 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 Fig.14As shown. According to the results of in vivo imaging, 1 hour after tail vein injection, the brains of mice in each experimental group had a certain fluorescence intensity. In the Cy5 group, the fluorescence intensity gradually weakened with time, and the fluorescence basically disappeared at 12 hours. In the RBT-Cy5 group, the fluorescence gradually weakened with time, and the overall fluorescence was stronger than that of the simple Cy5 group, but the fluorescence basically disappeared at 12 hours. In the BEVs-Cy5 and BRBT-Cy5 groups, the fluorescence intensity gradually increased with time within 0-5 hours, and the fluorescence intensity was significantly stronger than that of other groups. The fluorescence intensity in the spleen reached the highest at 5 hours, indicating that it has good spleen targeting, and the comprehensive targeting results of each group show that its targeting comes from BVEs exosomes.

[0097] Mouse in vitro organ imaging: The C57BL / 6J black mice were killed by cervical dislocation, and their organs, including brain, heart, liver, spleen, lung, and kidney, were taken 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 Fig.15 According to the results of in vivo imaging, the fluorescence of BEVs-Cy5 and BRBT-Cy5 groups was the strongest in the spleen compared with Cy5 and RBT-Cy5 groups, indicating that the two groups of materials have good spleen targeting.

[0099] The above results indicate that BEVs and BRBT have excellent spleen targeting performance.

[0100] Embodiment 4:

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

[0102] Embodiment 5:

[0103] The difference between this embodiment and embodiment 1 is that rhein, berberine and baicalein are replaced by "rhein, jatrorrhizine and wogonin" respectively, and the rest is the same as embodiment 1.

[0104] Embodiment 6:

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

[0106] Embodiment 7:

[0107] The difference between this embodiment and embodiment 1 is that the preparation steps of the nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction are: first prepare a two-component nanoassembly of baicalein (Bac) and berberine (Ber), and then prepare a three-component nanoassembly of baicalein-berberine-rhein (Rhe), and the rest are the same as embodiment 1.

[0108] The structures and properties of the spleen-targeted 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, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A spleen-targeted nanoassembly of the active ingredient of Sanhuang Xiexin Decoction, characterized in that: It includes 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 on 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 of rhubarb, coptis chinensis, and scutellaria baicalensis.

2. The spleen-targeted nanoassembly of the active ingredient of Sanhuang Xiexin Decoction according to claim 1, characterized in that: The active ingredient in the rhubarb is any one of rhein, chrysophanol and rhein; the active ingredient in the coptis chinensis is any one of berberine, bamipine and jatrorrhizine; the active ingredient in the scutellaria baicalensis is any one of baicalein, baicalin and wogonin.

3. The spleen-targeted nanoassembly of the active ingredient of Sanhuang Xiexin Decoction according to claim 1, characterized in that: The particle size of the spleen targeting nanoassembly is 80-180 nm.

4. The spleen-targeted nanoassembly of the active ingredient of Sanhuang Xiexin Decoction according to claim 1, characterized in that: The nanoassembly 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 medicinal herbs, rhubarb, coptis chinensis, and scutellaria baicalensis, are selected and synthesized into inner layer nanoparticles by electrostatic action; secondly, the active ingredient of the remaining Chinese medicinal herb is covalently modified on the surface of the inner layer nanoparticles by a cross-linking agent that responds to the inflammatory microenvironment, thereby obtaining the nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction.

5. The method for preparing the spleen-targeted nanoassembly of the active ingredient of Sanhuang Xiexin Decoction according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Preparation of nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction: S1: Select any two active ingredients of the three Chinese medicines of rhubarb, coptis chinensis and scutellaria baicalensis, dissolve them in an organic solvent, mix them evenly, adjust the pH of the solution to 5-9, add them dropwise to a PBS solution, stir, and then dialyze the solution in a dialysis bag with a molecular weight cutoff of less than 10,000, and freeze-dry to obtain inner layer nanoparticles; S2: The inner layer nanoparticles, the active ingredient of the remaining Chinese medicine, EDC, NHS and the cross-linking agent for inflammatory microenvironment response are dissolved in a mixed solution of an organic solvent and DI water, stirred, and then the solution is dialyzed in a dialysis bag with a molecular weight cutoff of less than 10,000, and freeze-dried to obtain a nanoassembly containing the active ingredient of Sanhuang Xiexin Decoction; (2) The nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction is coated on the spleen-targeted cell exosomes to obtain the nanoassembly.

6. The preparation method according to claim 5, characterized in that: The reagent for adjusting the pH of the solution is any one of sodium bicarbonate solution, sodium hydroxide solution, and ammonia solution; the cross-linking agent for inflammatory microenvironment response is any one of thioketal, ketalthiool, polypropylene sulfide, borate, and anthocyanin.

7. The preparation method according to claim 5, characterized in that: The particle size of the inner layer nanoparticles is 50-150nm.

8. The preparation method according to claim 5, characterized in that: The method for coating the nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction on spleen-targeted cell exosomes is any one or more of self-membrane coating, co-extrusion, and ultrasonic fusion.

9. The preparation method according to claim 5, characterized in that: The preparation method specifically comprises the following steps: (1) Preparation of nanoassemblies containing the active ingredients of Sanhuang Xiexin Decoction: S1: Weigh the active ingredients of rhubarb and coptis chinensis in a molar ratio of 10:1 to 1:10, dissolve them in a mixture of 5-20 mL of dimethyl sulfoxide and 100-300 mL of methanol, adjust the pH of the solution to 5-9 after mixing, add it dropwise to PBS at 10-100°C and stir at a constant temperature for 5-50 minutes; after the reaction is completed, place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 for 12-24 hours, and freeze-dry to obtain inner layer nanoparticles; S2: Weigh the inner layer nanoparticles and the active ingredient of Scutellaria baicalensis 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 inflammatory microenvironment response in a mixed solution of dimethyl sulfoxide and DI water, react at 10-100°C for 1-5 hours, and after the reaction, place the solution in a dialysis bag with a molecular weight cutoff of less than 10,000 for 12-24 hours, and freeze-dry to obtain a nanoassembly containing the active ingredient of Sanhuang Xiexin Decoction; (2) The nanoassembly containing the active ingredients of Sanhuang Xiexin Decoction and spleen-targeted cell exosomes were mixed in a particle number of 1:1 and ultrasonically disrupted at 4°C for 5 min. After the ultrasonication, spleen-targeted cell exosomes of the three-component nanoassembly loaded with the active ingredients of Sanhuang Xiexin Decoction were obtained.

10. Use of the spleen-targeted nanoassembly of the active ingredient of Sanhuang Xiexin Decoction according to any one of claims 1 to 4 or the spleen-targeted nanoassembly of the active ingredient of Sanhuang Xiexin Decoction prepared by the preparation method according to any one of claims 5 to 9 in the preparation of spleen-targeted therapeutic drugs.

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