WO2023165149A1 - 一种荷载中药自组装胶束的"免疫外泌体"纳米粒及其制备方法和应用 - Google Patents

一种荷载中药自组装胶束的"免疫外泌体"纳米粒及其制备方法和应用 Download PDF

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WO2023165149A1
WO2023165149A1 PCT/CN2022/129072 CN2022129072W WO2023165149A1 WO 2023165149 A1 WO2023165149 A1 WO 2023165149A1 CN 2022129072 W CN2022129072 W CN 2022129072W WO 2023165149 A1 WO2023165149 A1 WO 2023165149A1
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traditional chinese
chinese medicine
exosome
self
immune
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French (fr)
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王若宁
崔季维
杜蓥江
曾炜
狄留庆
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南京中医药大学
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of nano preparations, and in particular relates to an "immune exosome" nanoparticle loaded with traditional Chinese medicine self-assembled micelles, a preparation method and application thereof.
  • the chemical components contained in traditional Chinese medicine are complex, including alkaloids, flavonoids, quinones, saponins, etc., which are the material basis for exerting medicinal effects.
  • the diversity of components also necessarily leads to interactions between different components.
  • the self-assembly of organic compound molecules is mainly induced by non-covalent bonds such as hydrogen bonds, van der Waals forces, ⁇ - ⁇ stacking, molecular complexation, electrostatic interactions, and solvation.
  • many components can self-assemble to form nanoparticles.
  • puerarin and baicalin are active ingredients extracted from traditional Chinese medicine Pueraria root and Coptis chinensis, but their solubility and oral bioavailability are poor.
  • Glycyrrhizic acid as the amphiphilic active ingredient in the traditional Chinese medicine licorice, belongs to pentacyclic triterpenoids and consists of one molecule of hydrophobic glycyrrhetinic acid and two molecules of hydrophilic glucuronic acid.
  • Exosomes are extracellular vesicles with a size in the range of 40-160 nm (average 100 nm). Almost all cells are capable of secreting exosomes. Exosomes have a stable phospholipid bilayer structure, and the internal cavity can co-carry a large amount of water-soluble substances, while the hydrophobic region in the middle of the phospholipid bilayer can co-carry hydrophobic substances. This special structure can protect its contents in the cell. Exosomes exist in the external environment for a long time without being degraded or diluted. At the same time, exosomes can also avoid being captured and cleared by the reticuloendothelial system.
  • Exosomes as drug delivery vehicles have the following advantages: 1 prolong the half-life of drugs in the body; 2 not easy to cause immune rejection; 3 can cross the blood-brain barrier; The receptors on the cells bind to achieve targeted drug delivery.
  • serum-derived exosomes are mainly produced by reticulocytes, which are easier to obtain than other cell-derived exosomes, and the acquisition cost is relatively low.
  • transferrin receptors TfR
  • TfR transferrin receptors
  • TfR transferrin receptors
  • TfR transferrin receptors
  • exosome membranes as biomimetic coatings to wrap nanoparticles is also a method that has become popular in recent years.
  • Malignant tumors are one of the major threats to human life and health today.
  • the commonly used clinical treatment methods mainly include surgical resection, drug therapy and radiation therapy.
  • Surgical resection and radiotherapy often only have a good therapeutic effect on local solid tumors.
  • Drug therapy not only runs through surgery and radiotherapy, but is mainly used for the treatment of tumors with a tendency to metastasize.
  • the active ingredients of traditional Chinese medicine have become more and more prominent in the treatment of tumors, such as curcumin, resveratrol, camptothecin, tanshinone IIA, etc.
  • the active ingredients of traditional Chinese medicine have many unique advantages in anti-tumor, such as synergizing efficacy and reducing toxicity, reducing drug resistance of tumor cells, etc.
  • the existence of the BBB prevents most drug molecules from entering the brain. Efficient delivery to sites.
  • Tumor immunotherapy refers to the use of the body's own immune system to activate and kill tumor cells.
  • the FDA has approved several new immunotherapy drugs for various tumor treatments, such as immune checkpoint inhibitors and chimeric antigen receptor T cells.
  • immune adjuvants as non-specific immune enhancers, are mainly used to enhance the body's immune response to antigens or change the type of immune response, and are an important part of many vaccines.
  • CpG-Oligodeoxynucleotide (CpG ODN) as a new type of adjuvant, has been widely concerned and studied because of its powerful immune stimulating effect.
  • CpG ODN has a good application prospect as a vaccine adjuvant in the field of tumor therapy.
  • CpG ODN is a potent Toll-like receptor 9 agonist.
  • CpG ODN can cause the activation of antigen-presenting cells (i.e., macrophages and dendritic cells) in the tumor microenvironment, and the activated dendritic cells migrate to the draining lymph nodes and present tumor antigens to CD8 + T cells, thereby triggering anti-tumor CD8 + T cell-mediated immunity.
  • antigen-presenting cells i.e., macrophages and dendritic cells
  • a single chemotherapeutic drug is difficult to achieve good disease treatment effects, and has relatively high toxicity and side effects, and is prone to drug resistance.
  • people continue to screen and purify effective active ingredients of traditional Chinese medicine.
  • nanocarriers to deliver a single active ingredient of traditional Chinese medicine to the target site of a disease
  • the current construction process of most nano-drug delivery systems is relatively complicated, and more toxic organic reagents will be introduced, the preparation cost is high, and the druggability is not ideal. Therefore, a nano-drug delivery system with simple process, considerable cost and better curative effect needs to be developed urgently.
  • how to efficiently load multiple active ingredients of traditional Chinese medicine and immune adjuvants into exosomes has not been reported before.
  • the purpose of the present invention is an "immune exosome" nanoparticle loaded with self-assembled micelles of traditional Chinese medicine and its preparation method and application.
  • the present invention uses the endogenous exosome membrane to coat the active The components self-assemble nanoparticles, and the exosome membrane is modified with immune adjuvants to achieve multi-drug delivery.
  • the exosome membrane endows it with long-term circulation and targeting capabilities in the body, and plays a role in the synergistic treatment of tumors through chemotherapy and immunotherapy. effect.
  • the nanoparticles are mainly formed by exosome membranes modified by immune adjuvants to coat the self-assembled nanomicelles of the active ingredients of the traditional Chinese medicine. Active Drug Composition.
  • the immune adjuvant is selected from one or more of CpG ODN 1585, CpG ODN 1826, CpG ODN 2395, CpG ODN D-SL03, but not limited to these immune adjuvants;
  • the exosome membrane It is mainly obtained by sonicating exosomes;
  • the exosomes are selected from one or more of exosomes derived from cells, blood, milk and urine, but are not limited to these sources.
  • the amphiphilic active drug of the traditional Chinese medicine is selected from one of the amphiphilic active drugs in the traditional Chinese medicine licorice (glycyrrhizic acid), ginseng (ginsenoside Rh2, ginsenoside Rg3, ginsenoside Rb1, other saponins) or other traditional Chinese medicine.
  • the fat-soluble active drugs of traditional Chinese medicines are selected from the group consisting of traditional Chinese medicine Danshen (tanshinone IIA, cryptotanshinone, dihydrotanshinone, tanshinone I, other tanshinones), turmeric (curcumin), coptis ( Berberine) or one or more of the fat-soluble active ingredients in other traditional Chinese medicines, but not limited to these drugs.
  • the active drug is one of drugs that inhibit tumor cell proliferation, drugs that induce tumor cell apoptosis, drugs that inhibit tumor angiogenesis, and drugs that inhibit tumor cell invasion and migration.
  • drugs that inhibit tumor cell proliferation drugs that inhibit tumor cell proliferation, drugs that induce tumor cell apoptosis, drugs that inhibit tumor angiogenesis, and drugs that inhibit tumor cell invasion and migration.
  • the particle size of the "immune exosome” nanoparticles loaded with self-assembled micelles of traditional Chinese medicine is 140-160nm.
  • the preparation method of the "immune exosome” nanoparticles loaded with self-assembled micelles of traditional Chinese medicines includes using amphiphilic active drugs of traditional Chinese medicines and fat-soluble active drugs of traditional Chinese medicines as raw materials to prepare self-assembled nano-micelles, and exosomes
  • the membrane is coated on the surface of the self-assembled nano micelles, and then the surface of the exosome membrane is modified with an immune adjuvant to obtain.
  • the preparation method of the "immune exosome” nanoparticles loaded with traditional Chinese medicine self-assembled micelles comprises the following steps:
  • step (4) The nanoparticle solution obtained in step (4) is mixed with the solution C obtained in step (5), and incubated;
  • step (1) when the solid mass is mg, the liquid mass is measured in mL, and the mass ratio of the amphiphilic active drug of the traditional Chinese medicine to the fat-soluble active drug of the traditional Chinese medicine is (5-30):1.
  • step (2) the temperature obtained by evaporation under reduced pressure is 37-45° C., the ultrasonic crushing time is 5-15 minutes, and the ultrasonic crushing power is 100-300 W.
  • step (3) the exosomes are extracted and separated by a combination of iodixanol density gradient centrifugation and ultracentrifugation, the time of ultrasonication is 10-30 minutes, and the power of ultrasonication is 100-300W.
  • step (4) the self-assembled nanomicelle is based on the mass of the traditional Chinese medicine amphiphilic active drug therein, and the exosome membrane is based on the protein content therein, and the mass ratio of the self-assembled nanomicelle to the exosome membrane is (1-2): 1, the ultrasonic crushing time is 5-15 minutes, and the ultrasonic crushing power is 100-300W.
  • step (5) in the Tris-HCl buffer solution, the immune adjuvant with the sulfhydryl group is reacted with dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL), To obtain a phospholipid-modified immune adjuvant, that is, to obtain Tris-HCl buffer solution C containing the phospholipid-modified immune adjuvant.
  • DSPE-PEG-MAL dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide
  • the present invention provides the application of the "immune exosome” nanoparticles loaded with traditional Chinese medicine self-assembled micelles in the preparation of drugs for treating tumors or neurodegenerative diseases.
  • it can be dissolved by adding physiological saline, or phosphate buffer, or 5% glucose solution, and administered intravenously, intramuscularly, or orally.
  • physiological saline, or phosphate buffer, or 5% glucose solution When applied, it can be dissolved by adding physiological saline, or phosphate buffer, or 5% glucose solution, and administered intravenously, intramuscularly, or orally.
  • the bioavailability of loaded active drugs has the effect of anti-tumor or treatment of neurodegenerative diseases.
  • the invention prepares the self-assembled nano micelles of active components of traditional Chinese medicine through a thin film dispersion method, fully utilizes the unique properties of the medicine itself, has a very simple method and does not introduce toxic organic reagents, and significantly improves the solubility of fat-soluble active medicines.
  • the exosomes are reorganized by the ultrasonic crushing method to obtain exosome membranes. Then continue to induce the exosome membrane coating on the nanomicelle by sonication, which improves the problem of low drug encapsulation efficiency of the traditional exosome drug loading technology.
  • the present invention reacts the immune adjuvant with sulfhydryl group and dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide to obtain the phospholipid-modified immune adjuvant, and makes the phospholipid-modified immune adjuvant by incubation
  • the adjuvant is embedded in the exosome membrane shell, making full use of the space on the exosome membrane.
  • the present invention utilizes self-assembled nano-micelles of active ingredients of traditional Chinese medicine, exosomes rich in specific proteins and phospholipid-modified immune adjuvants to prepare the obtained "immune exosomes" loaded with self-assembled micelles of traditional Chinese medicine by means of ultrasonic crushing and incubation.
  • Nanoparticles can effectively improve the problems of low drug load of a single carrier, poor biocompatibility, weak targeting penetration, and single therapeutic mechanism. It has the following advantages:
  • Exosomes from natural sources are used to biomimetically coat nanomicelles, and the resulting nanoparticles retain the natural specific proteins on the exosome membrane and are highly endogenous.
  • the particle size of nanoparticles is in the nanoscale range, and it is easy to diffuse from the blood vessel to the outside of the blood vessel; the specific protein on the endogenous exosome membrane enables it to pass through various physiological barriers.
  • the present invention has following advantage:
  • the present invention adopts thin-film dispersion method to prepare self-assembled nano-micelles of active ingredients of traditional Chinese medicine. Biodegradability, biocompatibility and safety of materials.
  • the "immune exosome" nanoparticles loaded with self-assembled micelles of traditional Chinese medicines provided by the present invention can achieve the purpose of combined administration by loading active drugs with different therapeutic mechanisms, and provide a way to achieve multi-drug delivery and multi-channel treatment of diseases. A new design idea.
  • the "immune exosome" nanoparticles loaded with traditional Chinese medicine self-assembled micelles can complete single or multiple anti-tumor active ingredients of traditional Chinese medicine (such as tanshinone IIA, glycyrrhizic acid, glycyrrhetinic acid, curcumin, etc.) or tumor drugs including In vivo co-delivery of active multi-components of traditional Chinese medicine combined with diagnostic and detection reagents.
  • the characteristics of multiple methods provide a model for efficient targeted delivery and multi-method combination therapy for tumors and other neurodegenerative diseases, and has broad application prospects and potential for clinical transformation.
  • Figure 1 is the morphology of the "immune exosome” nanoparticles loaded with traditional Chinese medicine self-assembled micelles in Example 4;
  • Figure 2 shows the expression of characteristic proteins on the surface of "immune exosome” nanoparticles loaded with traditional Chinese medicine self-assembled micelles in Example 5 1.4;
  • Figure 3 is the in vitro hemolytic investigation of the "immune exosome" nanoparticles loaded with traditional Chinese medicine self-assembled micelles in Example 5 1.5;
  • Figure 4 is the investigation of the cellular uptake of "immune exosome" nanoparticles loaded with self-assembled micelles of traditional Chinese medicine in Example 5 1.6;
  • Figure 5 is the cytotoxicity investigation of the "immune exosome" nanoparticles loaded with self-assembled micelles of traditional Chinese medicine in Example 5 1.7;
  • Figure 6 is the observation of apoptosis of "immune exosome” nanoparticles loaded with self-assembled micelles of traditional Chinese medicine in Example 5 1.8;
  • Figure 7 is an investigation of the toxic effect of the "immune exosome" nanoparticles loaded with self-assembled micelles of traditional Chinese medicines on 3D tumor spheres in Example 5 1.9;
  • Fig. 8 is an investigation of the in vitro stimulation of dendritic cell maturation by "immune exosome” nanoparticles loaded with self-assembled micelles of traditional Chinese medicine in Example 5 1.10.
  • Example 1 Preparation process of tanshinone II A (Tan II A)-glycyrrhizic acid (GL) self-assembled nano-micelle (TGM)
  • TanIIA powder Take 1 mg of TanIIA powder and 25 mg of GL powder, dissolve them in 5 mL of absolute ethanol, transfer them to a 100 mL eggplant-shaped bottle, remove the absolute ethanol by rotary evaporation under reduced pressure at 40°C, and form a uniform film on the bottle wall, then add 5 mL of Ultrapure water, slightly oscillated to completely dissolve the drug film in the water. Then the solution was transferred to a 20 mL vial, and ultrasonically crushed in an ice bath for 5 min (power 200 W) to obtain a TGM solution.
  • the mass of fixed TanIIA was 1mg, and the total volume of the solution was 5mL. Further investigate the amount of GL used in the preparation of TGM.
  • the specific investigation prescription is shown in Table 1. Take an appropriate amount of the preparation, dilute it with ultrapure water, and measure the particle size of the preparation with a Malvern laser particle size analyzer. Diameter size, polydispersity index (PDI) and Zeta potential, the results are shown in Table 1.
  • the particle size of TGM is the smallest, which is (111.6 ⁇ 12.1) nm; the PDI is (0.228 ⁇ 0.023), and the particle size distribution is relatively uniform; the Zeta potential is (-22.2 ⁇ 1.5) , so this prescription is selected as the follow-up prescription.
  • Embodiment two the preparation technology of DSPE-PEG-CpG
  • CpG ODN 1826 powder dissolve in Tris-HCl (0.01M, pH7) buffer (100 ⁇ L); take 4.5mg of DSPE-PEG-MAL powder, dissolve in Tris-HCl (0.01M, pH7) buffer (900 ⁇ L )middle. Mix the two solutions evenly, place them in a thermostatic oscillator at room temperature, 300 rpm, and react for 12 hours to obtain a DSPE-PEG-CpG solution.
  • exosome solution Take an appropriate amount of exosome solution, dilute it 10 times with a hypotonic buffer solution (2mM Tris, 1mM MgCl 2 , 1mM KCl) prepared with sterile ultrapure water, and ultrasonically disrupt it for 10 minutes in an ice bath, then use an ultracentrifuge at 40,000rpm , 4°C, centrifuged for 1.5h, and the obtained precipitate was resuspended in PBS solution to obtain an exosome membrane solution, which was stored at 4°C for later use.
  • a hypotonic buffer solution 2mM Tris, 1mM MgCl 2 , 1mM KCl
  • Example 4 Preparation process of "immune exosome" nanoparticles loaded with TGM and modified DSPE-PEG-CpG (CpG-EXO/TGM)
  • Ultrasonic disruption was used to induce exosome membrane coating on TGM.
  • the specific operation was to take 200 ⁇ g of exosome membrane (obtained in Example 3) based on protein content and 200 ⁇ g of TGM (obtained in Example 1) based on the total amount of GL.
  • Mix add PBS to make up the total volume to 360 ⁇ L, and sonicate for 5 minutes under ice bath conditions (power 200W). ), incubated at 37°C for 1.5h, after the incubation, passed through a 0.8 ⁇ m water filter membrane to obtain the "immune exosome" nanoparticles CpG-EXO/TGM loaded with TGM and modified with DSPE-PEG-CpG.
  • Example 2 Take the TGM obtained in Example 1, the EXO/TGM and CpG-EXO/TGM nanoparticles obtained in Example 4, dilute them with ultrapure water, and measure the particle size, PDI and Zeta potential of the nanoparticles with a Malvern laser particle size analyzer. The results are shown in Table 2 , because EXO/TGM and CpG-EXO/TGM have exosome membrane coating, the particle size of TGM increases, and the Zeta potential decreases.
  • TGM was replaced with an equal amount of TanIIA and GL (exosomes before recombination were ultrasonically crushed) to prepare TanIIA-loaded exosomes (EXO/T) and GL-loaded exosomes (EXO/G) and exosomes co-loaded with TanIIA and GL (EXO/T+G).
  • the specific operation is to take 200 ⁇ g of exosomes in terms of protein content (recombinant exosomes obtained in Example 3) and mix them with 8 ⁇ g TanIIA or 200 ⁇ g GL or 8 ⁇ g TanIIA+200 ⁇ g GL, add PBS to make up to 400 ⁇ L, and sonicate in an ice bath After crushing for 5 minutes (with a power of 200W), incubate at a constant temperature of 37° C. for 1.5 hours after sonication.
  • EXO/TGM and CpG-EXO/TGM nanoparticles obtained in Example 4 pass EXO/T, EXO/G, EXO/T+G, EXO/TGM and CpG-EXO/TGM through a 0.8 ⁇ m water filter membrane, add 10 times the amount of chromatographic grade methanol for demulsification, after ultrasonication in a water bath for 10 minutes, centrifuge at 14,000 rpm for 10 minutes, take the supernatant, use high performance liquid chromatography (HPLC) to measure the content of TanIIA and GL in it, and calculate the encapsulation efficiency (EE) and drug loading (Ioading efficiency, IE), the results are shown in Table 3.
  • HPLC high performance liquid chromatography
  • EXO/TGM and CpG-EXO/TGM nanoparticles obtained in Example 4 Take the EXO/TGM and CpG-EXO/TGM nanoparticles obtained in Example 4, and use the BCA protein concentration assay method to measure the total protein content.
  • Exosome membrane, EXO/TGM and CpG-EXO/TGM nanoparticle samples with the same protein concentration were prepared, and the expression of CD63, CD71(TfR), CD81 and ⁇ -actin were detected by Western Blot experiment.
  • the results are shown in Figure 2, the expression of CD63, CD71(TfR), CD81 and ⁇ -actin can be detected in the exosome membrane, EXO/TGM and CpG-EXO/TGM nanoparticles, indicating that EXO/TGM and CpG- EXO/TGM nanoparticles retain the characteristic membrane proteins on the surface of exosomes.
  • GL was dissolved in PBS buffer to prepare a solution with a maximum concentration of 2.5mg/mL.
  • the prepared TGM and CpG-EXO/TGM were diluted with PBS buffer so that the GL concentration in the maximum concentration was 2.5 mg/mL.
  • Three groups of sample solutions were diluted to 5 concentrations, namely 2.5mg/mL, 1.25mg/mL, 0.62mg/mL, 0.31mg/mL, 0.16mg/mL.
  • GL261 cells in the logarithmic growth phase were resuspended in complete medium (2 ⁇ 10 5 cells/mL), inoculated in 12-well plates with 1 mL of cell fluid per well, and cultured overnight at 37°C and 5% CO 2 . After the cells adhered to the wall, remove the medium, add 1mL of DiD-EXO/TGM, CpG-DiD-EXO/TGM and Tf+CpG-DiD-EXO/TGM (DiD concentration is 50 ⁇ M) to the administration wells respectively, and add 1mL to the blank control wells. Add 1 mL of serum-free medium, and incubate at 37°C for 2 hours.
  • GL261 cells in the logarithmic growth phase were resuspended in complete medium (5 ⁇ 10 4 cells/mL), seeded in a 96-well plate with 100 ⁇ L of cell fluid per well, and cultured overnight at 37°C and 5% CO 2 . After the cells adhered to the wall, the medium was aspirated, 100 ⁇ L of serum-free medium was added to the blank control wells, and 100 ⁇ L of serum-free medium containing free TanIIA, free GL, TGM, EXO/TGM and CpG-EXO/TGM were added to the administration wells (the concentration of fixed TanIIA was 2 ⁇ g).
  • GL261 cells in the logarithmic growth phase were resuspended in complete medium (2 ⁇ 10 5 cells/mL), inoculated in 12-well plates, 1 mL per well, and cultured overnight at 37°C and 5% CO 2 . After the cells adhered to the wall, remove the culture medium, add free TanIIA, free GL, TGM, EXO/TGM and CpG-EXO/TGM diluted in serum-free medium (concentration of fixed TanIIA is 4 ⁇ g/mL, concentration of GL is 100 ⁇ g /mL) drug-containing medium, and the cells without drug treatment were used as blank control.
  • a C57BL/6 female mouse was euthanized, the femur and tibia were removed, the muscle on the bone was removed with scissors and tweezers, the bone was soaked in 75% ethanol solution for 5 min, and then washed with PBS 3 times, 3 min each. Cut off the epiphyses at both ends, use a 1mL syringe to absorb PBS to flush out the bone marrow from the bone cavity, blow with a pipette gun to disperse the bone marrow cells, then transfer to a 15mL centrifuge tube, centrifuge at 1500rpm for 5min, and discard the supernatant.
  • BMDCs mouse bone marrow-derived dendritic cells

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Abstract

一种荷载中药自组装胶束的"免疫外泌体"纳米粒及其制备方法和应用，所述纳米粒主要是由免疫佐剂修饰的外泌体膜包覆中药活性成分自组装纳米胶束而形成，所述中药活性成分自组装纳米胶束由中药两亲性活性药物和中药脂溶性活性药物构成。制备工艺简单、条件温和、成本低廉，制得的纳米粒具有高度内源性、高穿透性、天然靶向性、高生物安全性、多组分联合递送、治疗机制互补、治疗手段多元化等优点。

Description

一种荷载中药自组装胶束的“免疫外泌体”纳米粒及其制备方法和应用 技术领域
本发明属于纳米制剂技术领域，具体涉及一种荷载中药自组装胶束的“免疫外泌体”纳米粒及其制备方法和应用。
背景技术
中药中所含的化学成分复杂，包括生物碱类、黄酮类、醌类、皂苷类等，这些是发挥药效的物质基础。成分的多样性也必然会导致不同成分间的相互作用。一般来说，有机化合物分子的自组装主要通过氢键、范德华力、π-π堆积、分子络合、静电作用及溶剂化作用等非共价键诱导形成。在许多种中药汤剂的煎煮过程中，很多成分都能进行自组装形成纳米粒。例如，葛根素和黄芩苷是从中药葛根、黄连中提取所得的活性成分，但其溶解性和口服生物利用度均较差。但在经典方剂葛根芩连汤中，通过甘草进行配伍，葛根素和黄芩苷的溶解性和生物利用度均有增加。甘草酸作为中药甘草中的两亲性活性成分，属于五环三萜类化合物，由一分子疏水性的甘草次酸和两分子亲水性的葡萄糖醛酸组成。可以在水中与脂溶性成分自组装形成纳米胶束，从而使脂溶性成分的水溶性增大。受汤剂中化学成分自组装的启发，很多中药成分也被人为地组装成纳米粒，如黄芩苷-小檗碱、小檗碱-肉桂酸、紫杉醇-甘草酸、吴茱萸碱-甘草酸等。通过中药活性成分间地相互自组装，不仅可以取代某些聚合物纳米载体，实现安全有效地增溶效果，还可以起到协同治疗疾病地作用。
外泌体是一种大小在40-160nm(平均为100nm)范围内的细胞外囊泡。几乎所有细胞都能够分泌外泌体。外泌体具有稳固的磷脂双分子层结构，内部空腔可以协载大量水溶性物质，而磷脂双分子层中间的疏水区可以协载疏水性物质，这种特殊结构能保护其内容物在胞外环境中长期存在而不被降解或稀释，同时，外泌体也能避免被网状内皮系统捕获和清除。外泌体作为药物的递送载体具有以下优势：①延长药物在体内的半衰期；②不易引起机体免疫排斥反应；③可以跨越血脑屏障；④外泌体膜上的某些特异性配体可与细胞上的受体相结合，实现靶向递送药物。例如，血清来源的外泌体主要由网织红细胞产生，相对于其它细胞来源的外泌体更易获得，获取成本也相对较低。而且血清外泌体的膜上有丰富的转铁蛋白受体(Transferrin receptor，TfR)，可以与血液中游离的转铁蛋白(Tf)结合，很多肿瘤细胞的细胞膜上的TfR均过量表达，还有研究证明， 血清外泌体可以通过TfR介导的转胞吞作用穿过血脑屏障(Blood brain barrier,BBB)。目前常用的外泌体直接载药方法包括电穿孔、超声处理、循环冻融、挤出等方法。利用外泌体膜作为仿生涂层包裹纳米颗粒也是几年来逐渐流行的方法。
恶性肿瘤是当今人类生命健康面临的主要威胁之一，目前临床常用的治疗方法主要包括手术切除、药物治疗和放射治疗。手术切除和放射治疗往往只对局部实体瘤有较好的治疗效果，药物治疗不仅贯穿手术和放疗的始终，而且主要用于具有转移倾向的肿瘤的治疗。中药活性成分近年来在肿瘤治疗方面的作用也越来越突出，如姜黄素、白藜芦醇、喜树碱、丹参酮ⅡA等。与大部分合成药物相比，中药活性成分在抗肿瘤中具有许多独特的优势，如增效减毒、降低肿瘤细胞耐药性等。但对于环境中存在特点治疗屏障的肿瘤，如脑胶质瘤，BBB的存在阻碍了绝大部分药物分子进入大脑，为了提高治疗效果，还需借助纳米药物递送技术，实现将中药活性分子向肿瘤部位的高效递送。
肿瘤免疫治疗是指利用人体自身免疫系统激活，杀灭肿瘤细胞。近年来，FDA已批准多个免疫治疗新药应用于各类肿瘤治疗，如免疫检查点抑制剂和嵌合抗原受体T细胞。其中，免疫佐剂作为非特异性免疫增强剂，主要用于增强机体对抗原的免疫应答或改变免疫应答类型，是很多疫苗的重要组成部分。近年来，含CpG基序的寡聚脱氧核苷酸(CpG-Oligodeoxynucleotide，CpG ODN)作为一种新型佐剂，因其强大的免疫刺激作用被广泛关注和研究的。大量动物实验及临床研究证明CpG ODN作为疫苗佐剂在肿瘤治疗领域具有良好的应用前景。CpG ODN是一种强大的Toll样受体9激动剂。CpG ODN可以引起肿瘤微环境中抗原呈递细胞(即巨噬细胞和树突细胞)的活化，激活的树突细胞迁移到引流淋巴结，将肿瘤抗原呈递给CD8 +T细胞，进而引发了抗肿瘤CD8 +T细胞介导的免疫。
目前，单一的化疗药物难以达到良好的疾病治疗效果，并且毒副作用较大、易产生耐药性。人们受中药治疗疾病多靶点、副作用低的启发，不断筛选提纯有效的中药活性成分。虽然利用纳米载体将单一中药活性成分递送到疾病靶部位已有一定的报道，但是递送多种中药活性成分的报道很少。而且，目前大部分纳米药物递送系统的构建工艺较为复杂，且会引入较多毒性较大的有机试剂，制备成本较高，成药性不够理想。因此，工艺简单、成本可观、疗效较佳的纳米药物递送系统亟待开发。此外，如何将多种中药活性成分和免疫佐剂同时高效负载于外泌体中暂未有前人报道。
发明内容
发明目的：针对上述技术问题，本发明目的是一种荷载中药自组装胶束的“免疫外泌体”纳米粒及其制备方法和应用，本发明利用内源性外泌体膜包覆中药活性成分自组装纳米粒，并在外泌体膜上加以免疫佐剂的修饰，实现多药联合递送，通过外泌体膜赋予其体内长循环和靶向能力，发挥通过化学疗法和免疫疗法协同治疗肿瘤的功效。
技术方案：为了达到上述发明目的，本发明所采用的技术方案如下：
所述纳米粒主要是由免疫佐剂修饰的外泌体膜包覆中药活性成分自组装纳米胶束而形成，所述中药活性成分自组装纳米胶束由中药两亲性活性药物和中药脂溶性活性药物构成。
优选的，所述免疫佐剂选自CpG ODN 1585、CpG ODN 1826、CpG ODN 2395、CpG ODN D-SL03中的一种或几种，但不局限于这些免疫佐剂；所述外泌体膜主要是由外泌体进行超声破碎制得；所述外泌体选自细胞来源、血液来源、乳汁来源和尿液来源外泌体中的一种或几种，但不局限于这些来源。
优选的，所述中药两亲性活性药物选自中药甘草(甘草酸)、人参(人参皂苷Rh2、人参皂苷Rg3、人参皂苷Rb1、其他皂苷类)或其他中药中两亲性活性药物中的一种或几种，但不局限于这些药物；所述中药脂溶性活性药物选自中药丹参(丹参酮ⅡA、隐丹参酮、二氢丹参酮、丹参酮Ⅰ、其他丹参酮类)、姜黄(姜黄素)、黄连(小檗碱)或其他中药中脂溶性活性成分中的一种或几种，但不局限于这些药物。
上述活性药物应用于抗肿瘤时，所述活性药物为抑制肿瘤细胞增殖的药物、诱导肿瘤细胞凋亡的药物、抑制肿瘤新生血管生成的药物、抑制肿瘤细胞侵袭和迁移的药物中的一种或几种。
优选的，所述荷载中药自组装胶束的“免疫外泌体”纳米粒的粒径为140-160nm。
所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法，包括以中药两亲性活性药物和中药脂溶性活性药物为原料，制备自组装纳米胶束，将外泌体膜包覆在所述自组装纳米胶束表面，然后用免疫佐剂对外泌体膜表面进行修饰，即得。
优选的，所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法，包括以下步骤：
(1)制备中药两亲性活性药物的有机溶液A，中药脂溶性活性药物的有机溶液B；
(2)将溶液A与溶液B混匀后，减压蒸发除尽有机溶剂，用超纯水重悬药物，随 后在冰浴条件下进行超声破碎，即得自组装纳米胶束；
(3)提取分离获得外泌体，冰浴条件下进行超声破碎，得外泌体膜；
(4)取步骤(2)所得自组装纳米胶束与步骤(3)所得外泌体膜混合，冰浴条件下进行超声破碎，得纳米粒溶液；
(5)在缓冲溶液中制备磷脂修饰的免疫佐剂，得到含磷脂修饰的免疫佐剂的缓冲溶液C；
(6)将步骤(4)所得的纳米粒溶液与步骤(5)所得的溶液C混合，孵育；
(7)过膜去除游离药物，即得。
进一步优选的：
步骤(1)中，当固体质量为mg时，液体质量以mL计，所述中药两亲性活性药物与中药脂溶性活性药物质量比为(5-30)：1。
步骤(2)中，所述减压蒸发得温度为37～45℃，超声破碎时间为5～15min，超声破碎的功率为100-300W。
步骤(3)中，采用碘克沙醇密度梯度离心法和超速离心法相结合的方式提取分离得到外泌体，超声破碎时间为10～30min，超声破碎的功率为100-300W。
步骤(4)中，自组装纳米胶束以其中的中药两亲性活性药物的质量计，外泌体膜以其中的蛋白含量计，所述自组装纳米胶束与外泌体膜的质量比为(1-2)：1，所述超声破碎时间为5～15min，超声破碎的功率为100-300W。
步骤(5)中，在Tris-HCl缓冲溶液中，将连有巯基的免疫佐剂与二棕榈酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺(DSPE-PEG-MAL)进行反应，得磷脂修饰的免疫佐剂，即得含磷脂修饰的免疫佐剂的Tris-HCl缓冲溶液C。
本发明最后提供了所述荷载中药自组装胶束的“免疫外泌体”纳米粒在制备治疗肿瘤药物或神经退行性疾病药物中的应用。应用时，或加生理盐水、或磷酸盐缓冲液、或5％葡萄糖溶液溶解,以静脉注射、或肌肉注射、或口服给药,该纳米粒能够实现中药多活性成分的共递送，显著提高所荷载活性药物的生物利用度,具有抗肿瘤或治疗神经退行性疾病的疗效。
本发明通过薄膜分散法制备了中药活性成分自组装纳米胶束，充分利用了药物本身特有的性质，方法十分简便且不引入毒性较大的有机试剂，显著提高了脂溶性活性药物的溶解性。
本发明通过超声破碎法对外泌体进行重组，获得外泌体膜。然后继续通过超声破碎法诱导外泌体膜包覆在纳米胶束上，改善了传统外泌体载药技术药物包封率低的问题。
本发明通过将连有巯基的免疫佐剂与二棕榈酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺进行反应，得磷脂修饰的免疫佐剂，通过孵育的方法，使磷脂修饰的免疫佐剂嵌合在外泌体膜外壳上，充分利用的外泌体膜上的空间。
本发明利用中药活性成分自组装纳米胶束、富含特异性蛋白的外泌体和磷脂修饰的免疫佐剂，通过超声破碎和孵育的方法制备所得荷载中药自组装胶束的“免疫外泌体”纳米粒，可以有效改善单一载体药物荷载量低、生物相容性差、靶向穿透力弱、治疗机制单一等问题。其具备以下优势：
(1)高度内源性：利用天然来源的外泌体对纳米胶束进行仿生包覆，所得的纳米粒保留了外泌体膜上天然的特异性蛋白，具有高度内源性。
(2)多组分联合递送：通过将中药两亲性活性药物与中药脂溶性活性药物制备为自组装纳米胶束，并荷载于外泌体膜内，能够实现中药活性多组分的联合递送，有利于提高治疗效果。
(3)高穿透性：纳米粒的粒径在纳米级范围内，易从血管内扩散至血管外；内源性外泌体膜上的特异性蛋白使其能够穿过多种生理屏障。
(4)天然靶向性：制备所得荷载中药自组装胶束的“免疫外泌体”纳米粒保留了外泌体膜上天然具有的特异性蛋白，能够与病变细胞表面的相关受体相结合。
(5)高生物安全性：制备所得荷载中药自组装胶束的“免疫外泌体”纳米粒具有良好的生物相容性，可生物降解，毒副作用低。
(6)治疗机制互补：制备所得荷载中药自组装胶束的“免疫外泌体”纳米粒，能够实现联合给药及治疗机制互补。
(7)治疗手段多元化：通过修饰不同的免疫佐剂、荷载不同的药物活性成分或其他诊断检测类成分，从而实现诊断、治疗疾病的能力。
有益效果:与现有技术相比,本发明具有以下优势:
(1)本发明采用薄膜分散法制备了中药活性成分自组装纳米胶束，方法十分简便且不引入毒性较大的有机试剂，显著提高了脂溶性活性药物的溶解性，具有优于人工合成纳米材料的生物可降解性、生物相容性和安全性。
(2)本发明采用超声破碎法和孵育法制备了荷载中药自组装胶束的“免疫外泌体” 纳米粒，方法简便、成本低廉，易于工业化生产。
(3)本发明提供的荷载中药自组装胶束的“免疫外泌体”纳米粒保留了外泌体膜上天然具有的特异性蛋白，能够穿过多种生理屏障并增强病变细胞的摄取。
(4)本发明提供的荷载中药自组装胶束的“免疫外泌体”纳米粒能够通过装载不同治疗机制的活性药物达到联合给药的目的,为实现多药递送、多途径治疗疾病提供一种新的设计思路。
本发明提供的荷载中药自组装胶束的“免疫外泌体”纳米粒可以完成单个或多个中药抗肿瘤活性成分(如丹参酮ⅡA、甘草酸、甘草次酸、姜黄素等)或肿瘤药物包括中药活性多组分与诊断检测试剂联合的体内共递送，该纳米制剂具有高度内源性、高穿透性、天然靶向性、高生物安全性、多组分联合递送、治疗机制互补、治疗手段多元化等特性，为肿瘤及其他神经退行性疾病的高效靶向递送、多手段联合治疗的方法提供了范本，具有广阔的应用前景以及临床转化潜力。
附图说明
图1为实施例四中荷载中药自组装胶束的“免疫外泌体”纳米粒的形态；
图2为实施例五1.4中荷载中药自组装胶束的“免疫外泌体”纳米粒表面特征蛋白的表达情况；
图3为实施例五1.5中荷载中药自组装胶束的“免疫外泌体”纳米粒的体外溶血性考察；
图4为实施例五1.6中荷载中药自组装胶束的“免疫外泌体”纳米粒的细胞摄取考察；
图5为实施例五1.7中荷载中药自组装胶束的“免疫外泌体”纳米粒的细胞毒性考察；
图6为实施例五1.8中荷载中药自组装胶束的“免疫外泌体”纳米粒的细胞凋亡考察；
图7为实施例五1.9中荷载中药自组装胶束的“免疫外泌体”纳米粒对3D肿瘤球的毒性作用考察；
图8为实施例五1.10中荷载中药自组装胶束的“免疫外泌体”纳米粒体外刺激树突状细胞成熟作用考察。
具体实施方式
通过以下实施例进一步对本发明进行进一步阐述。这些实施例完全是例证性的，他们仅用来对本发明进行具体描述，不应当理解为对本发明的限制。下面结合附图及实施 例对发明作进一步描述：
实施例一：丹参酮ⅡA(TanⅡA)-甘草酸(GL)自组装纳米胶束(TGM)的制备工艺
取TanⅡA粉末1mg、GL粉末25mg，溶于5mL无水乙醇中，充分溶解后，转移至100mL茄型瓶中，40℃减压旋转蒸发除去无水乙醇，瓶壁上形成均匀薄膜，随后加入5mL超纯水，轻微振荡使药物薄膜完全溶解于水中。随后将溶液转至20mL西林瓶中，在冰浴条件下超声破碎5min(功率为200W)，即得TGM溶液。
固定TanⅡA的质量为1mg，溶液总体积为5mL，进一步考察TGM制备所用的GL的用量，具体考察处方见表1，取适量的制剂,超纯水稀释,以马尔文激光粒度仪测定制剂的粒径大小、多分散系数(PDI)及Zeta电位，结果见表1。
表1 TGM制备中GL用量考察处方及结果(mean±SD，n＝3)。
Figure PCTCN2022129072-appb-000001
如表1所示，当GL投药量为25mg时，TGM粒径最小，为(111.6±12.1)nm；PDI为(0.228±0.023)，粒径分布较为均匀；Zeta电位为(-22.2±1.5)，因而选择该处方作为后续处方。
实施例二：DSPE-PEG-CpG的制备工艺
取CpG ODN 1826粉末10nmol，溶于Tris-HCl(0.01M，pH7)缓冲液(100μL)中；取DSPE-PEG-MAL粉末4.5mg，溶于Tris-HCl(0.01M，pH7)缓冲液(900μL)中。将两中溶液混匀，至于恒温振荡仪中，室温，300rpm，反应12h，即得DSPE-PEG-CpG溶液。
实施例三：血清来源外泌体的提取及外泌体膜的纯化
取6-8周SD大鼠，腹腔注射2％戊巴比妥钠(50mg/kg)麻醉，随后进行腹主动脉取血获得新鲜血液。全血于常温放置30min，以1600g离心12min后取上清以2000g离心12min，除去血细胞。然后取上清以12000g离心30min进一步去除细胞碎片，取 上清过0.22μm水系滤膜，由此获得血清样品。随后，分别配置50％碘克沙醇溶液、30％碘克沙醇溶液及10％碘克沙醇溶液。每个超速离心管中依次加入4mL 50％碘克沙醇溶液、30％碘克沙醇溶液、10％碘克沙醇溶液及血清样品。27000rpm，4℃，离心2.5h，外泌体密度层位于10％与30％碘克沙醇溶液层之间，吸出外泌体密度层至超速离心管中，加入PBS溶液，40000rpm，4℃，离心1.5h，所得沉淀加PBS溶液重悬，即得外泌体溶液。
取适量外泌体溶液，用无菌超纯水配制的低渗缓冲液(2mM Tris，1mM MgCl 2,1mM KCl)稀释10倍，于冰浴条件下超声破碎10min，随后利用超速离心机，40000rpm，4℃，离心1.5h，所得沉淀加PBS溶液重悬，即得外泌体膜溶液，4℃储存备用。
实施例四：荷载TGM并修饰DSPE-PEG-CpG的“免疫外泌体”纳米粒(CpG-EXO/TGM)的制备工艺
采用超声破碎法诱导外泌体膜包覆于TGM上，具体操作为取以蛋白含量计200μg的外泌体膜(实施例三所得)和以GL总量计200μg的TGM(实施例一所得)混合，加入PBS补足总体积至360μL，在冰浴条件下超声破碎5min(功率为200W)，超声完毕后，得EXO/TGM，加入40μL(0.4nmol)的DSPE-PEG-CpG(实施例二所得)，37℃恒温孵育1.5h，孵育结束后，过0.8μm水系滤膜，即得荷载TGM并修饰DSPE-PEG-CpG的“免疫外泌体”纳米粒CpG-EXO/TGM。
实施例五：荷载中药自组装胶束的“免疫外泌体”纳米粒性质考察
1.1 CpG-EXO/TGM的形态
取实施例四所得CpG-EXO/TGM纳米粒，采用负染法至于透射电镜下观察，结果如图1所示，可见CpG-EXO/TGM粒子形状均为规整的球形，例子之间粘连和聚集的现象较少。
1.2 CpG-EXO/TGM的粒径、PDI及Zeta电位
取实施例一所得TGM、实施例四所得EXO/TGM及CpG-EXO/TGM纳米粒，超纯水稀释，以马尔文激光粒度仪测定纳米粒的粒径、PDI及Zeta电位，结果见表2，由于EXO/TGM和CpG-EXO/TGM具有外泌体膜包覆，粒径TGM有所增大，Zeta电位有所减小。
表2 不同纳米粒的粒径、PDI及电位(mean±SD，n＝3)。
Figure PCTCN2022129072-appb-000002
1.3 CpG-EXO/TGM中TanⅡA和GL的含量
按实施例四方案，将TGM用等量的TanⅡA和GL替换(外泌体使用超声破碎重组前的外泌体)，制备负载TanⅡA的外泌体(EXO/T)、负载GL的外泌体(EXO/G)及共负载TanⅡA和GL的外泌体(EXO/T+G)。具体操作为取以蛋白含量计200μg的外泌体(实施例三所得重组前外泌体)与8μgTanⅡA或200μg GL或8μg TanⅡA+200μg GL混合，加入PBS定容至400μL，在冰浴条件下超声破碎5min(功率为200W)，超声完毕后，于37℃恒温孵育1.5h。并取实施例四所得EXO/TGM和CpG-EXO/TGM纳米粒，将EXO/T、EXO/G、EXO/T+G、EXO/TGM及CpG-EXO/TGM过0.8μm水系滤膜，加10倍量的色谱级甲醇破乳，水浴超声10min后，14000rpm离心10min，取上清，利用高效液相色谱法(HPLC)测定其中TanⅡA和GL的含量，计算包封率(Encapsulation efficiency,EE)及载药量(Ioading efficiency,IE)，结果见表3。
表3 HPLC测定不同制剂中TanⅡA和GL的含量(mean±SD，n＝3)。
Figure PCTCN2022129072-appb-000003
1.4 CpG-EXO/TGM表面特征蛋白的表达
取实施例四所得EXO/TGM及CpG-EXO/TGM纳米粒，采用BCA蛋白浓度测定法测定总蛋白含量。取EXO/TGM或CpG-EXO/TGM溶液与RIPA裂解液等体积混匀，冰 上反应15min，取反应液10μL，加10μLPBS缓冲液混匀，加入200μLBCA工作液，37℃恒温震荡30min，于562nm波长处测定吸光度值，根据标曲计算样品中蛋白浓度。
制备相同蛋白浓度的外泌体膜、EXO/TGM及CpG-EXO/TGM纳米粒样品，通过Western Blot实验，检测CD63、CD71(TfR)、CD81及β-actin的表达情况。结果如图2所示，外泌体膜、EXO/TGM及CpG-EXO/TGM纳米粒中均能检测出CD63、CD71(TfR)、CD81及β-actin的表达，说明EXO/TGM及CpG-EXO/TGM纳米粒保留了外泌体表面的特征膜蛋白。
1.5 CpG-EXO/TGM体外溶血性考察
GL用PBS缓冲液溶解，配制最大浓度为2.5mg/mL的溶液。将制备好的TGM和CpG-EXO/TGM以PBS缓冲液稀释，使最大浓度中GL浓度为2.5mg/mL。三组样品溶液倍比稀释5个浓度，即为2.5mg/mL、1.25mg/mL、0.62mg/mL、0.31mg/mL、0.16mg/mL。
取新鲜C57BL/6小鼠抗凝血液，5000rpm离心6min除去血浆，用生理盐水重悬下层血细胞。重复离心3次，使上层液体不显红色。将血细胞稀释为2％浓度后，分别取0.5mL加入至含药的EP管中，37℃孵育3h。5000rpm离心6min。取上清，加入至96孔板中。540nm检测吸光度。
Figure PCTCN2022129072-appb-000004
As为实验孔吸光度值平均值，Ac为1％Triton孔吸光度平均值，Ab为生理盐水孔吸光度值。
实验结果如图3所示。由图可知，构建CpG-EXO/TGM，大大降低了GL在高浓度下的溶血率，证明了其溶血安全性。
1.6 CpG-EXO/TGM的细胞摄取考察
称取适量DiD以二甲亚砜溶解，配置成10mM的母液，按“实施例四”项下制备EXO/TGM及CpG-EXO/TGM，吸取2μLDiD母液与其混匀，37℃恒温孵育30min，即得DiD-EXO/TGM及CpG-DiD-EXO/TGM；称取适量Tf以PBS缓冲液溶解，配置成4mg/mL的母液，吸取10μL Tf母液与CpG-DiD-EXO/TGM溶液混匀，37℃恒温孵育30min，即得Tf+CpG-DiD-EXO/TGM。
取对数生长期的GL261细胞以完全培养基重悬(2×10 5个/mL)，接种于12孔板中,每孔1mL细胞液，置于37℃，5％CO 2培养过夜。待细胞贴壁后，吸除培养基，给药 孔分别加入DiD-EXO/TGM、CpG-DiD-EXO/TGM及Tf+CpG-DiD-EXO/TGM(DiD浓度为50μM)1mL，空白对照孔加入无血清培养基1mL，37℃培养2h后。PBS缓冲液洗涤细胞2次，将细胞以0.25％胰蛋白酶消化，用0.5mLPBS缓冲液重悬为细胞悬液,放置于流式管中,流式细胞仪进行细胞摄取荧光检测(DiD：Ex/λEm＝648nm/671nm),Kaluza软件进行细胞摄取的结果分析。
结果如图4所示，GL261细胞与DiD-EXO/TGM、CpG-DiD-EXO/TGM及Tf+CpG-DiD-EXO/TGM共孵育2h后，细胞内均有较强的荧光响应，说明3种制剂均能被GL261细胞摄取。Tf+CpG-DiD-EXO/TGM与DiD-EXO/TGM和CpG-DiD-EXO/TGM相比，具有更强的被GL261细胞摄取能力(***p<0.001，**p<0.01)。原因在于Tf介导的胞吞作用提高了细胞对于药物的摄取量。
1.7 CpG-EXO/TGM对细胞的毒性作用考察
取对数生长期的GL261细胞以完全培养基重悬(5×10 4个/mL)，接种于96孔板中,每孔100μL细胞液，置于37℃，5％CO 2培养过夜。待细胞贴壁后，吸除培养基，空白对照孔加入无血清培养基100μL，给药孔分别加入含有游离TanⅡA、游离GL、TGM、EXO/TGM及CpG-EXO/TGM(固定TanⅡA浓度为2μg/mL,GL浓度为50μg/mL)的无血清培养基100μL，置于37℃，5％C0 2培养24h。24h后，取出孔板，吸去每孔药液，每孔加100μL的Calcein AM/EthD-1荧光工作液(10μLEthD-1和2.5μLCalcein AM染液加至5mLPBS中,混匀，现配现用)，室温孵育15min，倒置荧光显微镜下观察各组细胞的存活情况。
结果如图5所示，绿色为活细胞，红色为死细胞，CpG-EXO/TGM组中细胞显红色荧光最多，证明CpG-EXO/TGM组较其他组能够明显抑制GL261细胞的存活。
1.8 CpG-EXO/TGM诱导细胞凋亡作用考察
采用AnnexinV-FITC/PI双染试剂标记，流式细胞仪进行检测。取对数生长期的GL261细胞以完全培养基重悬(2×10 5个/mL)，接种于12孔板中,每孔1mL，置于37℃，5％CO 2培养过夜。待细胞贴壁后，吸除培养基，加入以无血清培养基配制稀释的游离TanⅡA、游离GL、TGM、EXO/TGM及CpG-EXO/TGM(固定TanⅡA浓度为4μg/mL,GL浓度为100μg/mL)的含药培养基，不加药物处理的细胞作为空白对照。37℃，5％CO 2培养24h后，PBS缓冲液洗涤细胞一次，以不含EDTA的0.25％胰酶进行消化，离心弃去上清液,加入结合缓冲液0.5mL重悬细胞，300目尼龙筛网过滤除去细胞团块，加入5 μLAnnexin V-FITC染液和10μLPI染液轻轻混合均匀，室温避光孵育10min，置于流式管中，流式细胞仪进行凋亡检测，Kaluza软件进行结果分析。
结果如图6所示，细胞给予不同药物处理后，细胞凋亡率上升。其中CpG-EXO/TGM处理后细胞凋亡率体现出最强的诱导细胞凋亡的结果，细胞凋亡率为(39.83±3.38)％,显著高于其他药物组处理后的细胞凋亡率(***p<0.001，*p<0.05)。
1.9 CpG-EXO/TGM对3D肿瘤球的毒性作用考察
取对数生长期的U87细胞以完全培养基重悬(10×10 4个/mL)，接种于2％琼脂糖凝胶上，每孔100μL细胞液，置于37℃，5％CO 2培养至细胞成3D球体。U87肿瘤球生成后，取出孔板，吸出瘤球，空白对照孔加入无血清培养基100μL，给药孔分别加入含有游离TanⅡA、游离GL、TGM、EXO/TGM及CpG-EXO/TGM(固定TanⅡA浓度为4μg/mL,GL浓度为100μg/mL)的无血清培养基100μL，置于37℃，5％CO 2培养24h。24h后，取出孔板，吸去每孔药液，每孔加100μL的Calcein AM/EthD-1荧光工作液(10μLEthD-1和2.5μLCalcein AM染液加至5mLPBS中,混匀，现配现用)，室温孵育15min，倒置荧光显微镜下观察各组肿瘤球的染色情况。
结果如图7所示，绿色为活细胞，红色为死细胞，U87肿瘤球给予不同药物处理后，CpG-EXO/TGM组U87肿瘤球中显红色荧光的细胞最多，证明CpG-EXO/TGM组较其他组对体外3D肿瘤球有较强的杀伤作用。
1.10 CpG-EXO/TGM体外刺激树突状细胞成熟作用考察
取一只C57BL/6雌性小鼠执行安乐死，取股骨和胫骨，用剪刀和镊子去除骨头上的肌肉，将骨头浸泡在75％乙醇溶液中5min，随后用PBS冲洗3次，每次3min。剪去两端的骨骺，用1mL注射器吸取PBS从骨腔中冲出骨髓，用移液枪吹打使骨髓细胞分散，随后转移至15mL离心管中，1500rpm离心5min，弃去上清。加入2.5mL红细胞裂解液，裂解4min，加入10mLPBS终止裂解，1500rpm离心5min，弃上清。用含有10％灭活血清的RPMI-1640培养基(含20ng/mL鼠重组GM-CSF和20ng/mL鼠重组IL-4)重悬细胞，并以2×10 5细胞/孔的密度接种于12孔板中，48h后3/4换液，保留半贴壁细胞群，继续培养两天，即得小鼠骨髓来源树突状细胞(BMDCs)。
设置空白对照组、游离TanⅡA、游离GL、游离CpG、TGM、EXO、EXO/TGM、CpG-EXO/TGM组。控制CpG在含药培养基中的浓度为0.5nmol/mL，给药时不吸去原培养基，给药后置于37℃，5％CO 2培养24h。24h后，取出孔板，将每孔中细胞吹打 下来，1000rpm离心5min，弃去上清，用PBS清洗2次，细胞沉淀用500μLPBS重悬，300目尼龙筛网过滤除去细胞团块，加入5μL共刺激分子(CD80和CD86)流式抗体(APC-CD80，PE-CD86)染色，用流式细胞仪分析CD80 ++CD86 +的细胞亚群占比，Kaluza软件进行结果分析。
结果如图8所示，用游离CpG和CpG-EXO/TGM处理的BMDCs中CD80和CD86双阳性的细胞亚群占比比其他组都要多，而且CpG-EXO/TGM组要显著高于游离CpG组(*p<0.05)，证明CpG-EXO/TGM可以显著刺激树突状细胞的成熟，进而促进机体发生抗肿瘤免疫反应。

Claims (10)

  1. 一种荷载中药自组装胶束的“免疫外泌体”纳米粒，其特征在于，所述纳米粒主要是由免疫佐剂修饰的外泌体膜包覆中药活性成分自组装纳米胶束而形成，所述中药活性成分自组装纳米胶束由中药两亲性活性药物和中药脂溶性活性药物构成。
  2. 根据权利要求1所述的荷载中药自组装胶束的“免疫外泌体”纳米粒，其特征在于，所述免疫佐剂选自CpG ODN 1585、CpG ODN 1826、CpG ODN 2395、CpG ODN D-SL03中的一种或几种；所述外泌体膜主要是由外泌体进行超声破碎制得；所述外泌体选自细胞来源、血液来源、乳汁来源和尿液来源外泌体中的一种或几种。
  3. 根据权利要求1所述的荷载中药自组装胶束的“免疫外泌体”纳米粒，其特征在于，所述中药两亲性活性药物选自甘草酸、人参皂苷Rh2、人参皂苷Rg3、人参皂苷Rb1中的一种或几种；所述中药脂溶性活性药物选自紫杉醇、丹参酮ⅡA、隐丹参酮、姜黄素中的一种或几种。
  4. 权利要求1-3任一项所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法，其特征在于，包括以中药两亲性活性药物和中药脂溶性活性药物为原料，制备自组装纳米胶束，将外泌体膜包覆在所述自组装纳米胶束表面，然后用免疫佐剂对外泌体膜表面进行修饰，即得。
  5. 根据权利要求4所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法，其特征在于，包括以下步骤：
    (1)制备中药两亲性活性药物的有机溶液A，中药脂溶性活性药物的有机溶液B；
    (2)将溶液A与溶液B混匀后，减压蒸发除尽有机溶剂，用超纯水重悬药物，随后在冰浴条件下进行超声破碎，即得自组装纳米胶束；
    (3)提取分离获得外泌体，冰浴条件下进行超声破碎，得外泌体膜；
    (4)取步骤(2)所得自组装纳米胶束与步骤(3)所得外泌体膜混合，冰浴条件下进行超声破碎，得纳米粒溶液；
    (5)在缓冲溶液中制备磷脂修饰的免疫佐剂，得到含磷脂修饰的免疫佐剂的缓冲溶液C；
    (6)将步骤(4)所得的纳米粒溶液与步骤(5)所得的溶液C混合，孵育；
    (7)过膜去除游离药物，即得。
  6. 根据权利要求5所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法， 其特征在于，步骤(1)中，当固体质量为mg时，液体质量以mL计，所述中药两亲性活性药物与中药脂溶性活性药物质量比为(5-30)：1。
  7. 根据权利要求5所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法，其特征在于，步骤(2)中，所述减压蒸发得温度为37～45℃，超声破碎时间为5～15min，超声破碎的功率为100-300W。
  8. 根据权利要求5所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法，其特征在于，步骤(3)中，采用碘克沙醇密度梯度离心法和超速离心法相结合的方式提取分离得到外泌体，超声破碎时间为10～30min，超声破碎的功率为100-300W。
  9. 根据权利要求5所述的荷载中药自组装胶束的“免疫外泌体”纳米粒的制备方法，其特征在于，步骤(4)中，自组装纳米胶束以其中的中药两亲性活性药物的质量计，外泌体膜以其中的蛋白含量计，所述自组装纳米胶束与外泌体膜的质量比为(1-2)：1，所述超声破碎时间为5～15min，超声破碎的功率为100-300W。
  10. 权利要求1-3任一项所述的荷载中药自组装胶束的“免疫外泌体”纳米粒在制备抗肿瘤药物或治疗神经退行性疾病药物中的应用。
PCT/CN2022/129072 2022-03-01 2022-11-01 一种荷载中药自组装胶束的"免疫外泌体"纳米粒及其制备方法和应用 WO2023165149A1 (zh)

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