Liver-targeted exosome polydopamine silicon nano drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a liver-targeted exosome polydopamine silicon nano drug delivery system as well as a preparation method and application thereof, and relates to the field of inflammatory microenvironment targeted controlled-release drug delivery. The system is composed of exosome, active oxygen response bond modified mesoporous silica, PDA and an anti-inflammatory drug, the water solubility of natural medicinal compounds such as TB can be remarkably improved, the bioavailability can be improved, the effects of in-vivo and in-vitro specific targeted inflammation sites and efficient liver protection and hepatitis treatment can be achieved, the biotoxicity of TB is remarkably reduced, and the application prospect is broad. The compound is expected to be applied to safe and efficient acute liver injury treatment and prevention of natural medicinal compounds such as TB, so that a nanotechnology delivery strategy is provided for clinical application of other natural product monomers; the invention has important fundamental research significance and pre-clinical application value in the fields of accurate treatment, prevention and health care of acute hepatitis and acute liver injury.

Description

Technical Field

[0001] The present invention relates to the field of controlled-release delivery of drugs targeted to inflammatory microenvironments, and in particular to a liver-targeted exosome polydopamine silicon nano-drug delivery system and a preparation method and application thereof. Background Art

[0002] Acute liver injury (ALI) is a disease characterized by massive liver cell death and liver dysfunction. Liver transplantation is currently a common treatment for ALI. However, this technology is limited by conditions such as transplant rejection and shortage of donor organs. Currently, anti-inflammatory therapy in the early stage of ALI has become a potential alternative treatment for ALI.

[0003] Tokinolide B (TB) is isolated from the rhizomes of the traditional Chinese medicine plants Angelica sinensis and Chuanxiong, and belongs to the dimeric phthalide class. Its chemical name is: (3'Z)-(3S,8R,3A'S,6'R)-3,3A':8,6'-biligustilide, and its molecular formula is C 24 H 28 O4, molecular weight is 380.48.

[0004] Traditional Chinese medicines of the Umbelliferae family, such as Angelica sinensis and Chuanxiong, are often used in the treatment of inflammatory response syndromes such as gynecological inflammation and cardiovascular diseases. At the same time, TB, as a bioactive dimer phthalide compound isolated from Chuanxiong and Angelica sinensis, has a good in vitro anti-inflammatory effect.

[0005] However, TB has the same problems as the current natural phthalide compounds, such as wide distribution in the body, large first-pass effect, and low bioavailability. In addition, the phthalide dimer compound TB has the problem of poor water solubility, which restricts its further research and application. Therefore, it is necessary to improve the above shortcomings of TB through new drug dosage forms.

[0006] The present invention innovatively designs a silicon nano drug delivery system modified by exosomes (Exo) coated with polydopamine (PDA) for loading the natural drug compound TB, which is a new multifunctional drug delivery system with good water dispersibility, long in vivo circulation time, good inflammation targeting and controllable drug release performance. The present invention is based on the dual anti-inflammatory effects of PDA and TB and is used as a therapeutic and preventive drug for ALI disease. The designed exosome polydopamine mesoporous silica nanocarrier system has significant significance in delivering natural anti-inflammatory active ingredients of traditional Chinese medicine including natural anti-inflammatory active monomers such as TB for anti-inflammatory treatment of ALI, and is expected to improve the anti-inflammatory ability of the loaded drug in inflammation treatment, and achieve high efficiency and low toxicity, with a wide range of application prospects. Summary of the invention

[0007] The purpose of the present invention is to solve the above-mentioned problems of low solubility, low bioavailability and poor stability of TB in water in the prior art, and to provide a liver-targeted exosome polydopamine silicon nano-drug delivery system and its preparation method and application.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme:

[0009] A method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system comprises the following steps:

[0010] 1) First, the silica is amino-modified, then carboxyl-modified, and then the drug and the modified silica are mixed and shaken for co-incubation, and finally washed to obtain drug-loaded mesoporous silica;

[0011] 2) covalently linking the drug-loaded mesoporous silica obtained in step 1) to polydopamine via a reactive oxygen species (ROS) responsive bond to obtain PDA-modified drug-loaded mesoporous silica;

[0012] 3) The exosomes are coated onto the outside of the PDA-modified drug-loaded mesoporous silica to obtain the exosome polydopamine silica nano-drug delivery system.

[0013] The pore size of the mesoporous silica is 8-12 nm.

[0014] The drug is an anti-inflammatory natural medicinal compound, including at least one of oriental angelica lactone B, triptolide, and triptolide.

[0015] The active oxygen responsive bond includes at least one of a diselenide bond (-SESE-), a disulfide bond (-SS-), and a thioketal bond (TK).

[0016] The exosomes include at least one of macrophage-derived exosomes, hepatitis cell-derived exosomes, and liver cancer cell-derived exosomes.

[0017] In step 2), the surface carboxyl groups of the drug-loaded mesoporous silica are activated with EDC and NHS, and then a selenocystamine dihydrochloride solution is added, stirred in the dark, and washed to obtain diselenide bond-modified drug-loaded mesoporous silica.

[0018] In step 2), dopamine hydrochloride is added to the drug-loaded mesoporous silica modified with a diselenide bond in a slightly alkaline environment, stirred in the dark, and washed to obtain the PDA-modified drug-loaded mesoporous silica.

[0019] In step 3), after the exosomes are mixed and incubated with the PDA-modified drug-loaded mesoporous silica, the exosome polydopamine silica nano-drug delivery system is obtained by physical means (such as extrusion, ultrasound, etc.).

[0020] A liver-targeted exosome polydopamine silicon nano-drug delivery system is prepared by the above-mentioned preparation method, and comprises exosomes, mesoporous silica nano-carriers modified with active oxygen responsive bonds, polydopamine and anti-inflammatory natural medicinal compounds.

[0021] The application of the liver-targeted exosome polydopamine silicon nano-drug delivery system is used to prepare drugs for treating and / or preventing hepatitis, acute liver injury, and liver cancer caused by hepatitis.

[0022] The present invention uses mesoporous silica nanoparticles (MSNs) as a carrier, which has a regular internal structure and a large pore size, which is conducive to the entry of different types of drug molecules and a wider drug loading space. At the same time, it has rich surface chemical groups, which makes it easier to modify the surface of biomacromolecules or targeting ligands. Polydopamine, as a self-polymerization form of dopamine, has the advantages of extraordinary adhesion, excellent biocompatibility, mild synthesis requirements, etc. The PDA rich in reducing functional groups in the present invention can be used as a free radical scavenger to eliminate the reactive oxygen species (ROS) generated during the inflammatory response. Exosomes (Exo) are a type of lipid bilayer membrane vesicles with a size of about 30 to 150 nm formed by plasma membrane internalization, which can come from most living cells and be released into the extracellular medium. Exos have low immunogenicity and good tolerance in body fluids. They can be stably present in body fluids such as blood, urine, amniotic fluid, breast milk and ascites. Exo can transport the encapsulated contents through the plasma membrane to a specific intracellular location in a target-specific manner. The present invention will utilize the inherent homing ability of macrophage-derived Exo to target inflammatory sites.

[0023] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0024] The exosome polydopamine silicon nano-drug delivery system provided by the present invention is composed of mesoporous silica nanoparticles, polydopamine, macrophage exosomes and drug TB. Compared with amorphous silicon materials such as liposomes and micelles, mesoporous silica nanomaterials have the advantages of excellent biocompatibility, good biosafety, large specific surface area and pore volume, uniform and controllable mesopore size, excellent stability and high drug loading capacity.

[0025] The present invention can greatly solve the problems of poor water solubility and low bioavailability of TB, and provide a new dosage form for clinical use of TB.

[0026] The present invention can make TB and PDA produce synergistic effect in anti-inflammatory aspect, and provide a new scheme for the application of active ingredients in traditional Chinese medicine in anti-inflammatory aspect.

[0027] The final particle size of the nanomaterial of the present invention is less than 100 nm, and will not form drug administration embolism. It can be suitable for intravenous injection or intraperitoneal administration of patients, thereby providing multiple possibilities for the use of TB drugs.

[0028] The present invention can increase the biocompatibility of the carrier and reduce the toxicity of the drug due to the modification of the PDA coating on the carrier surface, and PDA can also participate in anti-inflammation by removing excessive ROS. At the same time, the nano drug-carrying particles provided by the present invention can release drugs in a controlled manner through dual modification of diselenide bonds and PDA. PDA has the property of pH controllability, and diselenide bonds have ROS responsiveness. Therefore, in the acidic environment of the inflammation site, the diselenide bonds break and the PDA depolymerizes, which can realize the dual drug release of hydrogen peroxide and acid response, thereby achieving the purpose of safe and efficient slow and controlled release of anti-inflammatory drugs.

[0029] The present invention can increase the inflammation targeting ability of the carrier due to the modification of the surface macrophage exosomes, reduce the systemic toxic side effects caused by the non-specific and extensive distribution of the drug, and reduce the dosage of the drug.

[0030] The present invention provides a new idea for the design of nano-preparations for other similar Chinese medicine ingredients. The exosome polydopamine silicon nano-drug delivery system of the present invention is used in drugs for the treatment and prevention of diseases related to hepatitis, acute liver injury, liver cancer caused by hepatitis, etc.

[0031] In summary, the present invention improves the physicochemical properties and bioavailability of TB through the exosome polydopamine silicon nano-drug delivery system, while enabling PDA and TB to synergistically exert anti-inflammatory effects. Further, through the modification of macrophage exosomes, the nanocarrier is endowed with inflammation targeting ability, thereby broadening the application research of functionalized modified mesoporous silica nanocarrier systems in anti-inflammatory treatment, and providing a new strategy for drug dosage form design for the application of effective natural medicinal monomers in traditional Chinese medicine in the treatment and prevention of inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a transmission electron microscopy image of the mesoporous silica nanoparticles (MSNs) prepared in Example 1.

[0033] Figure 2 This is the Fourier infrared spectrum of the nanoparticles during the preparation process of Example 1.

[0034] Figure 3 This is a transmission electron micrograph of the exosomes from macrophage RAW264.7 cells extracted in Example 1.

[0035] Figure 4 This is the transmission electron microscopy image of Exo-M-SESE-PDA@TB prepared in Example 1.

[0036] Figure 5This is the in vitro ROS and pH responsive release diagram of the exosome polydopamine silicon nanodrug delivery system in Example 2.

[0037] Figure 6 This is a diagram of the in vitro targeting analysis of Exo-M-SESE-PDA on liver cancer cells with an inflammatory microenvironment in Example 3.

[0038] Figure 7 This is the fluorescence distribution diagram of the main in vitro organs (including liver) 16 hours after the injection of Exo-M-SESE-PDA in mice with acute liver injury in Example 4; HE is the heart, LI is the liver, SP is the spleen, LU is the lung, and KI is the kidney.

[0039] Figure 8 This is a diagram showing the anti-inflammatory effect of Exo-M-SESE-PDA@TB on inflammatory cells in vitro in Example 5.

[0040] Fig. 9 This is a diagram showing the therapeutic effect of Exo-M-SESE-PDA@TB on acute liver injury in mice in Example 6.

[0041] Fig.10 This is a diagram showing the protective effect of Exo-M-SESE-PDA@TB on mouse hepatitis in vivo in Example 7. DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0043] The preparation method of the present invention comprises the following steps:

[0044] 1) First, prepare amino-modified MSNs (M-NH2);

[0045] 2) The surface amino group of M-NH2 is carboxylated with succinic anhydride, and TEOA is added and stirred at room temperature in the dark, and washed to obtain a carboxy-modified MSNs derivative (M-COOH); the mass ratio of M-NH2:succinic anhydride:TEOA can be (0.8-1.2):1:1;

[0046] 3) TB and M-COOH dissolved in a solvent are mixed in a certain ratio, shaken and incubated in a mixer at room temperature, and then washed to obtain drug-loaded nanoparticles M-COOH@TB; in step 3), the mass ratio of TB:M-COOH can be 1:5-10;

[0047] 4) After the surface carboxyl groups of M-COOH@TB were activated with EDC / NHS, selenocystamine dihydrochloride was added and stirred at room temperature in the dark, and then washed to obtain the diselenide bond-modified MSNs drug-loaded derivative (M-SESE-NH2@TB); the mass ratio of M-COOH@TB:EDC:NHS:selenocystamine dihydrochloride can be (38-42):20:(4-6):40;

[0048] 5) In a slightly alkaline environment, dopamine hydrochloride is added to M-SESE-NH2@TB, stirred at room temperature in the dark, and washed to obtain a PDA-modified MSNs drug-loaded derivative (M-SESE-PDA@TB); the mass ratio of PDA:M-SESE-NH2@TB can be 1-2:1; the slightly alkaline environment can be pH 8.0-9.0;

[0049] 6) Collect the supernatant of RAW264.7 cells and extract exosomes (Exo) by ultra-high-speed centrifugation;

[0050] 7) Under low temperature conditions, the Exo obtained in step 6) and the M-SESE-PDA@TB in step 5) are extruded multiple times to obtain exosome-coated MSNs drug-loaded nanoderivatives (Exo-M-SESE-PDA@TB); the volume ratio of the M-SESE-NH2@TB to Exo can be (1-3):1.

[0051] Specific examples are given below.

[0052] Example 1

[0053] Preparation of exosome polydopamine silicon nanodrug delivery system.

[0054] 1) Using hexadecyltrimethylammonium chloride (CTAC) as a template, triethanolamine (TEOA) as an alkali source, and tetraethyl orthosilicate (TEOS) as a silicon source, 0.4 g CTAC and 0.18 g TEOA were uniformly dissolved in 80 mL ultrapure water, and placed on a magnetic stirrer, stirred at a constant temperature for 1 h, and then 1.5 mL TEOS was added dropwise, and the reaction continued for 2 h. After the reaction was completed, mesoporous silica nanoparticles MSNs (M-OH) were obtained by washing.

[0055] 2) The M-OH obtained in step 1) was dissolved in 50 mL of anhydrous ethanol (concentration of 1 mg / mL), stirred and refluxed at 90°C for 4 h, 300 μL of 3-aminopropyltriethoxysilane (APTES) was added to partially aminize the surface hydroxyl groups, and the mixture was stirred and refluxed at constant temperature for 24 h. The amino-modified MSNs (M-NH2) were obtained by washing.

[0056] 3) Take 40 mg of M-NH2 obtained in step 2), suspend it in a round-bottom flask containing 15 mL of ultrapure water, and stir it on a magnetic stirrer; weigh 400 mg of succinic anhydride (SA), dissolve SA evenly in 5 mL of N,N-dimethylformamide (DMF), and slowly add it dropwise to the round-bottom flask; weigh 400 mg of TEOA, slowly add it dropwise to the round-bottom flask, continue to stir overnight in the dark, and wash to obtain M-COOH.

[0057] 4) TB dissolved in a solvent and the M-COOH obtained in step 3) were added to the tube at a mass ratio of 1:8, and incubated in a mixer at room temperature for 24 hours, followed by washing to obtain drug-loaded nanoparticles M-COOH@TB.

[0058] 5) Weigh 4 mg of M-COOH or M-COOH@TB obtained in step 3) or step 4) and suspend it in a beaker containing 1 mL of ultrapure water, and place it on a magnetic stirrer for stirring; weigh 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and add it to the beaker and stir in the dark for 5 min; after the timing is over, weigh 5 mg of N-hydroxysuccinimide (NHS) and add it to the beaker, and then immediately drop 42 mg of selenocystamine hydrochloride solution dissolved in 7 mL of ultrapure water and stir overnight in the dark; after the reaction is completed, wash to obtain M-SESE-NH2 or M-SESE-NH2@TB.

[0059] 6) Weigh 2 mg of M-SESE-NH2 or M-SESE-NH2@TB obtained in step 5) and suspend it in a beaker containing 5 mL of Tris-HCl at pH 8.5, and place it on a magnetic stirrer for stirring at room temperature; take 2 mg of dopamine hydrochloride (DA) and dissolve it in 1 mL of Tris-HCl at pH 8.5, add it dropwise into the beaker in the dark, and then continue to stir at room temperature in the dark for 3 h; after the reaction is completed, wash it to obtain the PDA-modified MSNs derivative (M-SESE-PDA or M-SESE-PDA@TB).

[0060] 7) Collect the supernatant of RAW264.7 cells and extract exosomes (Exo) by ultra-high-speed centrifugation.

[0061] 8) At 4°C, 1 mg of Exo and 5 mg of M-SESE-PDA or M-SESE-PDA@TB in step 6) were suspended in 2 mL of cold PBS, mixed evenly and incubated at 4°C for 30 min, and then the mixture was repeatedly extruded through 800 nm and 400 nm polycarbonate membranes 15 times using a manual extruder. After ultrasonication for 2 min, the exosome-modified PDA silica nanoparticles or drug delivery system (Exo-M-SESE-PDA or Exo-M-SESE-PDA@TB) was obtained by centrifugation.

[0062] The transmission electron microscopy (TEM) image of the mesoporous silica nanoparticles prepared in step 1) of Example 1 is shown in Figure 1 It can be seen that a mild and simple preparation process can be used to synthesize spherical mesoporous silica nanoparticles with a particle diameter of about 60 nm. The particles have a branched mesoporous structure with a pore size of 8-12 nm.

[0063] The Fourier transform infrared spectrum of the nanoparticles during the preparation process of Example 1 is shown in Figure 2 All nanoparticles 3443 cm -1 attributed to Si-OH at 1091 cm -1 and 966 cm -1 The peaks at 1528 cm-1 are attributed to the stretching and rocking vibrations of Si-O-Si, indicating that the SiO2 structural network has been successfully synthesized. -1 The NH stretching vibration peak appeared at 1730 cm -1 When the carboxyl group forms a diselenide bond modified by an amide bond with selenocystamine hydrochloride, the peak at 1730 cm -1 The C=O peak at 1636 cm -1 and 1560 cm -1 The amide peak at 1636 cm -1 and 1560 cm -1 The absorption peak nearby weakened, which was due to the coverage of amide groups by PDA, further proving the successful modification of PDA.

[0064] The transmission electron microscopy (TEM) image of the exosomes extracted in step 7) of Example 1 is shown in Figure 3 It can be seen that the extracted exosomes present a typical cup-and-saucer structure, with thick and smooth edges, a concave middle, and a stable morphology. Its double-layer membrane structure is clear and the size is 50-80 nm, which proves the successful extraction of exosomes.

[0065] The transmission electron microscopy (TEM) image of Exo-M-SESE-PDA@TB prepared in step 8) of Example 1 is shown in Figure 4 It can be seen that the particle size of the exosomes increased to about 90 nm after being coated with PDA silicon nanoparticles. At the same time, compared with the adjacent uncoated PDA silicon nanocarriers, the outer exosome layer also became rounder and brighter, indicating the successful coating of the exosomes on the PDA silicon nanocarriers.

[0066] Example 2

[0067] Analysis of in vitro responsive release ability of exosome polydopamine silicon nanodrug delivery system.

[0068] PBS solutions of pH 7.4, pH 5.0, 10 mM H2O2+pH 7.4, and 10 mM H2O2+pH 5.0 were prepared as release media. At the same time, 5% Tween-80 was added to the release media to increase the solubility of TB. 5 mg of Exo-M-SESE-PDA@TB sample was weighed and evenly dispersed into 400 μL of release medium by ultrasonic dispersion, and then added to a sterile dialysis bag and tied tightly; the dialysis bag was placed in a 4 mL EP tube, and 3 mL of release medium was added; then the EP tube was placed in a constant temperature mixer at 37°C and shaken at 250 rpm. 200 μL of dialysate was taken out at different time intervals (0, 2, 4, 6, 8, 12, 24, 48, 72h) and added to the new release medium of the same volume. Finally, the absorbance of all the dialysates taken out was measured at 288 nm, and the drug release rate was calculated.

[0069] The in vitro responsive release curve of the exosome polydopamine silicon nanoparticle drug delivery system in Example 2 is shown in Figure 5Under neutral (pH 7.4) and normal ROS (0 mM H2O2) conditions, only about 12% of TB was released within 72 h; under neutral and high ROS (10 mM H2O2) conditions, the TB release rate increased, and about 37% of TB was released within 72 h; under acidic (pH 5.0) and normal ROS conditions, about 53% of TB was released within 72 h; and under acidic and high ROS conditions simulating inflammation sites, about 71% of TB was released within 72 h. The increase in TB release under acidic conditions may be due to the acidic responsiveness of the modified PDA. In a neutral environment, the PDA layer can maintain structural integrity and reduce drug release, while under acidic conditions, the PDA layer depolymerizes and increases drug release. The increased release of TB under high ROS conditions may be due to the cleavage of the modified ROS-responsive diselenide bond in Exo-M-SESE-PDA@TB, which allows the drug to further leak from the pores of mesoporous silica, increasing the release of the drug. The above results prove that Exo-M-SESE-PDA@TB has ROS and pH-responsive release capabilities.

[0070] Example 3

[0071] Analysis of the targeting ability of exosome polydopamine silica nanodrug delivery system to inflammatory hepatocytes in vitro.

[0072] Place 5*5 mm sterile glass slides in a 24-well plate and add 3×10 4 A number of HepG2 cells were plated. FITC with a final concentration of 2 μg / mL, FITC-loaded nanoparticles M-SESE-PDA@FITC and Exo-M-SESE-PDA@FITC were prepared. When the cell density reached 70%, the samples were added in reverse chronological order (2 h, 4 h) by changing the cell medium and incubated in the dark. After the incubation, the culture medium was discarded, the cells were washed three times with sterile PBS, and Mitotracker Deepred with a final concentration of 75 nM was added and incubated for another 30 min. The old solution was discarded, the cells were washed three times with sterile PBS, and 4% paraformaldehyde was added and incubated for 30 min. The old solution was discarded, the cells were washed twice with sterile PBS, and the slides were sealed with sealing oil containing DAPI. Confocal photography was performed after the sealing oil dried.

[0073] Inflammatory hepatocyte targeting in Example 3 is shown in Figure 6Cancer cells usually have an inflammatory microenvironment. HepG2 liver cancer cells were used to simulate liver cells in an in vitro inflammatory microenvironment to compare their targeted uptake ability. The FITC-labeled carriers were observed by laser confocal microscopy to investigate their uptake and distribution. At incubation times of 2 h and 4 h, no obvious FITC entry into the cells was observed, indicating that HepG2 cells had a low uptake of FITC alone. After loading with nanocarriers, cell uptake of the carriers was observed in the M-SESE-PDA@FITC and Exo-M-SESE-PDA@FITC groups at 2 h. At 4 h, the fluorescence of FITC in the M-SESE-PDA@FITC and Exo-M-SESE-PDA@FITC groups was significantly enhanced, and the green light of FITC in the Exo-M-SESE-PDA@FITC group overlapped more with the red fluorescence of mitochondria in the cytoplasm, indicating that the uptake of nanoparticles by HepG2 cells was time-consuming and the coating of Exo enhanced the uptake of carriers by liver cells in an inflammatory microenvironment.

[0074] Example 4

[0075] Analysis of the in vivo inflammatory liver targeting ability of exosome polydopamine silicon nanodrug delivery system.

[0076] Acute liver injury model was established by intraperitoneal injection of 8 mg / kg LPS into female ICR mice weighing about 20 g. Ten minutes later, Cy7, M-COOH@Cy7, M-SESE-PDA@Cy7 and Exo-M-SESE-PDA@Cy7 were injected through the tail vein (the injection volume was calculated based on the Cy7 content, and the Cy7 dosage was 0.5 mg / kg). Sixteen hours after the injection, the main isolated organs (heart, liver, spleen, lung, and kidney) of the mice were collected for small animal fluorescence imaging analysis.

[0077] The results of mouse isolated organ targeted analysis in Example 4 are shown in Figure 7 Compared with the normal group (Saline) mice, Exo-M-SESE-PDA@Cy7 was more enriched in the liver of the inflammatory group (LPS) mice, proving that it has good responsiveness to the inflammatory microenvironment. Compared with the free Cy7 treatment group, the fluorescence signals of M-COOH@Cy7, M-SESE-PDA@Cy7 and Exo-M-SESE-PDA@Cy7 in the liver organ were enhanced in turn, showing that the final exosome polydopamine silicon nano-drug delivery system Exo-M-SESE-PDA@Cy7 has enhanced targeting and retention capabilities in the inflamed liver.

[0078] Example 5

[0079] In vitro anti-inflammatory effect of Exo-M-SESE-PDA@TB on inflammatory cells in vitro.

[0080] TB, M-SESE-PDA@TB, Exo-M-SESE-PDA, and Exo-M-SESE-PDA@TB samples were prepared with culture medium at a final concentration of 15 μM. RAW264.7 cells were cultured, and LPS at a final concentration of 1 μg / mL was added to construct an inflammatory model, and the culture was continued for 12 h. The drug sample was added for pretreatment 2 h before the addition of LPS, and then incubated with LPS for 12 h; 50 μL of supernatant was aspirated into a new 96-well plate, and the NO detection kit was used for determination, and the absorbance at 560 nm was detected by an ELISA instrument. The model group (LPS group) was taken as 100%, and the NO release rate of the control group (not treated with LPS and drugs) and the sample group was calculated.

[0081] The in vitro anti-inflammatory (NO inhibition) graph of Example 5 is shown in Figure 8 After LPS modeling, the level of inflammatory mediator NO increased significantly, while TB pretreatment for two hours could reduce the NO level to 59.26% of the LPS group, and Exo-M-SESE-PDA@TB could further reduce the NO level to 35.22% of the LPS group. Therefore, the anti-inflammatory effect of TB can be increased by the loading of the carrier, and the PDA in the carrier can play a synergistic anti-inflammatory role and reduce the production of inflammatory mediator NO and pro-inflammatory factors.

[0082] Example 6

[0083] Anti-inflammatory effect of Exo-M-SESE-PDA@TB on mice with acute liver injury.

[0084] The acute liver injury mouse model was established by intraperitoneal injection of 8 mg / kg LPS, and 0.6 mg / mL TB saline solution (corresponding to the dosage of 6 mg / mL) was prepared with 3% DMSO and 5% Tween-80 as cosolvents. M-COOH@TB, M-SESE-PDA@TB, and Exo-M-SESE-PDA@TB with the corresponding TB concentration were prepared with saline, and ultrasonic dispersion was performed to make them uniform. The normal group and the inflammatory group were first injected with saline through the tail vein, and the drug-treated group was injected with TB (6 mg / mL), M-COOH@TB (TB 6 mg / mL), M-SESE-PDA@TB (TB 6 mg / mL), and Exo-M-SESE-PDA@TB (TB 6 mg / mL) through the tail vein, respectively. 6 hours later, the normal group was intraperitoneally injected with saline, and the inflammatory group (LPS) and the drug-treated group were intraperitoneally injected with LPS to establish the acute liver injury model. After 12 hours, the mice were killed by cervical dislocation after blood was taken from the eyeballs, and the main organs were removed and kept for later use. The collected blood was placed at room temperature for 2 h, centrifuged at 4°C, 3000 rpm for 10 min, the supernatant was retained, and the supernatant was centrifuged at 4°C, 3000 rpm for 5 min, the serum was collected, and stored in aliquots at 4°C. The ALT and AST levels in the serum were detected by a blood biochemical analyzer.

[0085] In vivo anti-inflammatory treatment of Example 6 (taking liver function indicators ALT and AST as an example) is shown in FIG. Fig. 9 After intraperitoneal injection of exogenous irritant LPS, AST and ALT levels in the LPS group increased to about 4 and 3.7 times that of the normal group mice (Saline), respectively, proving the success of ALI mouse modeling; after tail vein injection of 3 mg / kg of free TB, AST and ALT levels were suppressed, indicating that liver damage was alleviated. When the TB dose was increased to 6 mg / kg, AST and ALT levels further decreased. Therefore, 6 mg / kg may be a better dose of TB for the treatment of ALI; after tail vein injection of 3 mg / kg of M-COOH@TB, AST and ALT levels decreased, but were not much different from those of the free TB group; after tail vein injection of 3 mg / kg of M-SESE-PDA@TB, AST and ALT levels showed a downward trend compared with the M-COOH@TB group, indicating that the modification of PDA can enhance the anti-inflammatory ability of the drug carrier, thereby inhibiting ALI; after tail vein injection of 3 After adding 6 mg / kg of Exo-M-SESE-PDA@TB, the levels of AST and ALT were further reduced compared with the M-SESE-PDA@TB group, and showed a certain dose dependence. The Exo-M-SESE-PDA@TB at a dose of 6 mg / kg had the best effect in treating ALI.

[0086] Example 7

[0087] The preventive and protective effect of Exo-M-SESE-PDA@TB on liver damage in mice in vivo.

[0088] The acute liver injury mouse model was established by intraperitoneal injection of 8 mg / kg LPS, and 0.6 mg / mL TB saline solution (corresponding to the dosage of 6 mg / mL) was prepared with 3% DMSO and 5% Tween-80 as cosolvents. M-COOH@TB, M-SESE-PDA@TB, and Exo-M-SESE-PDA@TB with the corresponding TB concentration were prepared with saline, and ultrasonic dispersion was performed to make them uniform. The normal group and the inflammatory group were first injected with saline through the tail vein, and the drug-treated group was injected with TB (6 mg / mL), M-COOH@TB (TB 6 mg / mL), M-SESE-PDA@TB (TB 6 mg / mL), and Exo-M-SESE-PDA@TB (TB 6 mg / mL) through the tail vein, respectively. 6 hours later, the normal group was intraperitoneally injected with saline, and the inflammatory group (LPS) and the drug-treated group were intraperitoneally injected with LPS to establish the acute liver injury model. After 12 hours, the mice were killed by cervical dislocation after blood was taken from the eyeballs, and the main organs were removed and kept for later use. The collected blood was placed at room temperature for 2 h, centrifuged at 4°C, 3000 rpm for 10 min, the supernatant was retained, and the supernatant was centrifuged at 4°C, 3000 rpm for 5 min, the serum was collected, and stored in aliquots at 4°C. The levels of TNF-α and IL-6 in serum were detected by the Elisa kit according to the instructions of the kit.

[0089] The results of the in vivo protective effect of Exo-M-SESE-PDA@TB on liver damage in mice in Example 7 (taking inflammatory factors TNF-α and IL-6 as examples) are shown in the figure below: Fig.10 The levels of inflammatory factors TNF-α and IL-6 in serum were detected by Elisa kits. After pretreatment and protection by Exo-M-SESE-PDA@TB, the levels of inflammatory factors TNF-α and IL-6 in the inflammation model group were reduced from 696 pg / mL and 608 pg / mL to 332 pg / mL and 209 pg / mL, respectively. The inflammation caused by LPS was inhibited. Exo-M-SESE-PDA@TB effectively prevented and protected the acute inflammation caused by ALI.

[0090] The drug of the present invention includes active monomers extracted from natural medicinal plants, such as TB. The nanocomposite carrier is based on mesoporous silica, with diselenide bonds and PDA modified on the surface, and macrophage exosomes coated on the outside. The dual drug-loaded composite nanoparticles obtained by the above method have good solution dispersibility, controllable responsive release ability, and strong targeting of inflammatory liver, so as to deliver natural active monomers and target inflammatory liver for synergistic treatment, thereby broadening the therapeutic application of poorly soluble natural active monomers in liver inflammatory diseases.

Claims

1. A method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system, characterized in that: The following steps are involved: 1) The mesoporous silica is first amino-modified, then carboxyl-modified, and then the drug and the modified silica are mixed and shaken for co-incubation, and finally washed to obtain the drug-loaded mesoporous silica; 2) covalently linking the drug-loaded mesoporous silica obtained in step 1) to polydopamine via an active oxygen responsive bond to obtain PDA-modified drug-loaded mesoporous silica; 3) The exosomes are coated onto the outside of the PDA-modified drug-loaded mesoporous silica to obtain the exosome polydopamine silica nano-drug delivery system.

2. The method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system according to claim 1, characterized in that: The pore size of the mesoporous silica is 8-12 nm.

3. The method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system according to claim 1, characterized in that: The drug includes at least one of oriental angelica lactone B, triptolide, and triptolide.

4. The method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system according to claim 1, characterized in that: The active oxygen responsive bond includes at least one of a diselenide bond, a disulfide bond, and a thioketal bond.

5. The method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system according to claim 1, characterized in that: The exosomes include at least one of macrophage-derived exosomes, hepatitis cell-derived exosomes, and liver cancer cell-derived exosomes.

6. The method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system according to claim 1, characterized in that: In step 2), the surface carboxyl groups of the drug-loaded mesoporous silica are activated with EDC and NHS, and then a selenocystamine dihydrochloride solution is added, stirred in the dark, and washed to obtain diselenide bond-modified drug-loaded mesoporous silica.

7. The method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system according to claim 6, characterized in that: In step 2), dopamine hydrochloride is added to the drug-loaded mesoporous silica modified with a diselenide bond in a slightly alkaline environment, stirred in the dark, and washed to obtain the PDA-modified drug-loaded mesoporous silica.

8. The method for preparing a liver-targeted exosome polydopamine silicon nanoparticle drug delivery system according to claim 1, characterized in that: In step 3), after the exosomes are mixed and incubated with the PDA-modified drug-loaded mesoporous silica, the exosome polydopamine silica nano-drug delivery system is obtained by physical means.

9. A liver-targeted exosome polydopamine silicon nanoparticle drug delivery system, characterized in that: Prepared by the preparation method described in claims 1 to 8.

10. The use of a liver-targeted exosome polydopamine silicon nano-drug delivery system according to claim 9, characterized in that: Used for preparing drugs for treating and / or preventing hepatitis, acute liver damage, and liver cancer caused by hepatitis.

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