WO2018152989A1 - 一种人参来源的纳米颗粒及其制备和应用 - Google Patents

一种人参来源的纳米颗粒及其制备和应用 Download PDF

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WO2018152989A1
WO2018152989A1 PCT/CN2017/088953 CN2017088953W WO2018152989A1 WO 2018152989 A1 WO2018152989 A1 WO 2018152989A1 CN 2017088953 W CN2017088953 W CN 2017088953W WO 2018152989 A1 WO2018152989 A1 WO 2018152989A1
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macrophages
ginseng
centrifugation
tumor
minutes
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French (fr)
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曹鹏
曹萌
颜怀江
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江苏省中医药研究院
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Priority claimed from CN201710094457.7A external-priority patent/CN106727810A/zh
Priority claimed from CN201710248242.6A external-priority patent/CN106924312B/zh
Application filed by 江苏省中医药研究院 filed Critical 江苏省中医药研究院
Priority to US16/483,021 priority Critical patent/US10925912B2/en
Priority to EP17897934.0A priority patent/EP3563861B1/en
Publication of WO2018152989A1 publication Critical patent/WO2018152989A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
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    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the invention belongs to the field of immunopotentiators, and in particular relates to a nanoparticle of ginseng source and preparation and application thereof.
  • Microvesicles are one type of extracellular Vesicles. Unlike exosomes, microbubbles are mainly nanoscale particles with a diameter of about 100-1000 nm produced by outward germination of the plasma membrane. These nanoparticles often carry a variety of active substances such as proteins, DNA, RNA, etc., and exert various biological activities.
  • An immunopotentiator generally refers to a substance that enhances the immune response of a body when used alone or in combination with an antigen, and is also referred to as an immunomodulator or an immunostimulant.
  • Immune enhancers can act through different pathways, such as enhancing the activity of monocyte-macrophages, enhancing the immunogenicity and stability of antigenic substances, and promoting the synthesis and secretion of antibodies, thereby enhancing the specificity and non-specificity of the organism. Heterosexual immune response.
  • Mononuclear-macrophages are phagocytic cells commonly found in blood, lymph, and all mammalian tissue types. Mononuclear-macrophages are the most malleable cells in the hematopoietic system, and are present in all tissues, and they also have a strong functional diversity. Their main function is to phagocytize aging cells, tumor cells and pathogens in the form of fixed cells or free cells, and activate other immune cells; in the normal development, homeostasis, tissue repair and immune response to pathogens Different effects.
  • monocyte-macrophages play an important role in the body's non-specific immune response and specific immune response, and are widely distributed in vivo, how to regulate the activity of monocyte-macrophages is one of the focuses of immunology research.
  • Traditional granulocyte mononuclear-macrophage colony-stimulating factor can only promote the growth of mononuclear-macrophages, but can not activate TLR and other related immune molecules, thus unable to fully promote the cell-mediated immune response, thereby clearing the located cell.
  • Pathogens and tumor cell components Traditional Chinese medicine immune enhancer has mild drug resistance, low drug resistance, small toxic and side effects, and has the characteristics of two-way adjustment function, and has become a research hotspot of immunopotentiators.
  • Tumor microenvironment refers to tumor cells, stromal cells (including fibroblasts, immune and inflammatory cells, adipocytes, glial cells, smooth muscle cells, and some vascular endothelial cells, etc.) during tumor growth.
  • the local steady-state environment which is combined with extracellular matrix, provides the necessary material basis for the occurrence, development, invasion and metastasis of tumors.
  • the tumor microenvironment has an important support and promotion effect on tumors. How to intervene and improve the tumor microenvironment is the core issue to prevent tumor development and metastasis.
  • TAM Tumor-associated macrophages
  • Macrophage is one of the important immune cells of the body and plays an important role in the host's immune defense and maintaining tissue homeostasis. According to functional characteristics, macrophages are mainly activated into two subtypes: classically activated macrophage and alternative activated macrophage. M1 is regulated by cytokines secreted by Th1, such as interferon- ⁇ (INF- ⁇ ), lipopolysaccharide (LPS) and Toll-like receptor (TLR). It is a pro-inflammatory factor that secretes IL-6, IL-12, IL-23 and tumor necrosis factor- ⁇ (TNF- ⁇ ).
  • INF- ⁇ interferon- ⁇
  • LPS lipopolysaccharide
  • TLR Toll-like receptor
  • M1 macrophages also highly express major histocompatibility complex class I and class II molecules for efficient antigen presentation. Therefore, M1 type macrophages are considered to be cells having killing bacteria and tumor cells and secreting various pro-inflammatory cytokines. M2 macrophages, in contrast to the M1 type, are recognized as cells that promote tumor growth, invasion, and metastasis. The vast majority of TAM belong to M2 macrophages, which are the most important tumor infiltrating leukocytes in humans and mice.
  • TAM Tissue matrix
  • TAM also promotes angiogenesis by releasing angiogenic regulatory enzymes such as matrix metalloprotein 2 (MMP2), MMP-9, MMP-12, and cyclooxygenase 2 (COX2).
  • MMP2 matrix metalloprotein 2
  • MMP-9 MMP-9
  • MMP-12 cyclooxygenase 2
  • Tumor growth delivers nutrients.
  • TAM can release a series of cytokines and growth factors that promote tumor cell infiltration and metastasis, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bEGF). Wait.
  • VEGF vascular endothelial growth factor
  • bEGF basic fibroblast growth factor
  • Ginseng is one of the most important Chinese herbal medicines in China. It is known as the “King of Herbs” and has a very high medicinal value.
  • the nanoparticle-sized microbubbles produced by ginseng carry a variety of active ingredients of ginseng, which may be one of the important ways to exert their effects.
  • the technical problem to be solved by the present invention is to provide a quantifiable production of ginseng-derived nanoparticles (English name: Ginseng derived nano-particles, GDNPs for short), and as a natural immune enhancer and a medicament for treating tumors. use.
  • Nanoparticle microbubbles derived from ginseng contain a variety of bioactive components of ginseng, and thus have a good application prospect as a novel natural immune enhancer.
  • the technical solution adopted by the present invention is:
  • the preparation method of the ginseng-derived nanoparticles comprises the following steps:
  • the precipitate collected by the last centrifugation described in the step (3) is resuspended in a buffer, and then ultracentrifuged once, and the precipitate is collected; the precipitate is resuspended again with a buffer, and then centrifuged at a high speed to collect the supernatant; The supernatant is passed through a sterilizing grade filter to obtain ginseng-derived nanoparticles.
  • the above operation is carried out in an environment of 4 to 25 degrees Celsius.
  • the rotation speed of the low speed screw extrusion is 30 to 60 rpm.
  • the screen is 200 to 2000 mesh, preferably 200 to 1000 mesh.
  • the centrifugal force of the low-speed centrifugation is 100 to 500 ⁇ g, the centrifugation time is 5 to 10 minutes; the centrifugal force of the medium-speed centrifugation is 1000 to 5000 ⁇ g, and the centrifugation time is 10 to 30 minutes; the centrifugal force of the high-speed centrifugation is 8000 to 12000 ⁇ g, the centrifugation time is 30 to 60 minutes; the centrifugal force of the ultracentrifugation is 100,000 to 200,000 ⁇ g, and the centrifugation time is 60 to 120 minutes, the low speed , medium speed, high speed and ultracentrifugation are at least 1 time;
  • the centrifugal force of the low-speed centrifugation is 200-500 ⁇ g; the centrifugal force of the medium-speed centrifugation is 2000-5000 ⁇ g; the centrifugal force of the high-speed centrifugation is 100000 ⁇ 12000 ⁇ g; the centrifugal force of the ultracentrifugation is 100000 ⁇ 120000 ⁇ g.
  • the buffer solution is a phosphate buffer solution
  • the pH of the buffer solution is pH 7.2 to 7.4
  • the centrifugal force of the ultracentrifugation is 100,000 to 200,000 ⁇ g
  • the centrifugation time is 60 to 120 minutes.
  • the centrifugal force of the high-speed centrifugation is 8000 to 12000 ⁇ g, and the centrifugation time is 30 to 60 minutes; and the diameter of the sterilization-grade filter membrane is 0.45 ⁇ m.
  • the ginseng-derived nanoparticles are used for treating diseases caused by various immune functions such as AIDS, active tuberculosis, oral Candida albicans infection, Toxoplasma encephalopathy, Kaposi sarcoma and the like.
  • ginseng-derived nanoparticles in the preparation of a medicament as an immunopotentiator for activating monocytes-macrophages.
  • the method for activating monocytes-macrophages is to promote proliferation of mononuclear-macrophages and formation of colonies or up-regulate immunologically active molecules on the surface of monocyte-macrophages or to promote immunoreactivity of mononuclear-macrophage secretion. Cytokines.
  • the immunologically active molecule is one of Toll-like receptor 2/4 (TLR2/4), leukocyte differentiation antigen 80 (CD80), leukocyte differentiation antigen 86 (CD86), major tissue complex 2 (MHC-II) or A plurality of; the immunologically active cytokine is one or more of tumor necrosis factor alpha (TNF- ⁇ ) and interleukin-6 (IL-6).
  • ginseng-derived nanoparticles in the preparation of a medicament for treating tumors.
  • the method of treating a tumor is to reverse-polarize tumor-associated macrophages.
  • the method for reverse-polarizing tumor-associated macrophages is: down-regulating surface marker molecules of M2 type macrophages and up-regulating surface marker molecules of M1 type macrophages, up-regulating cytokines secreted by M1 type macrophages, and changing tumors The proportion of M1/M2 type macrophages in the microenvironment, thereby improving the tumor microenvironment and killing tumors.
  • the M2 type macrophage-associated surface marker molecule is leukocyte differentiation antigen 206 (CD206); the surface marker molecule associated with the M1 type macrophage is Toll-like receptor 2/4 (TLR2/4), leukocyte differentiation antigen One or more of 80 (CD80), leukocyte differentiation antigen 86 (CD86), and major tissue complex 2 (MHC-II); the M2 type macrophage-associated gene is arginase 1 (Arg-1) And leukocyte differentiation antigen 206, also known as one or more of macrophage mannose receptor (CD206), interleukin 10 (IL-10), chitinase-like molecule (CHI313); M1 macrophage-associated genes are interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), leukocyte differentiation antigen 80 (CD80), chemokine 9 (CXCL9), and chemokine 3 (CCL3).
  • CD206 leukocyte differentiation antigen 206
  • TLR2/4 Toll-like receptor 2
  • inducible nitric oxide synthase iNOS
  • the immunologically active cytokines associated with the M1 type macrophage are tumor necrosis factor alpha (TNF-alpha), interleukin 6 ( One or more of IL-6).
  • a pharmaceutical composition comprising the ginseng-derived nanoparticles.
  • a pharmaceutical formulation comprising a therapeutically effective amount of said ginseng-derived nanoparticles or a pharmaceutically acceptable excipient of said pharmaceutical composition.
  • the ginseng-derived nano-particle material of the invention has wide sources; it can be ginseng, American ginseng, notoginseng, Codonopsis pilosula, Radix Pseudostellariae, Salvia miltiorrhiza, Scrophulariaceae; the preparation method of the invention has the advantages of easy operation, less time consumption and the like.
  • the ginseng-derived nanoparticles of the present invention can effectively induce proliferation and activation of bone marrow-derived mononuclear-macrophages, and simultaneously up-regulate various surface active molecules such as TLR2/4 and CD80, and secrete TNF-a and IL-. 6 and other cytokines. It has a good application prospect in the development of a drug for preparing a natural immune enhancer; the use of the ginseng-derived nanoparticles of the present invention in the preparation of a medicament for treating tumors can effectively make tumor-associated macrophages from M2 which promotes tumor growth.
  • Figure 1 shows the morphology of GDNPs observed by electron microscopy
  • Figure 2 shows the particle size of PDNPs analyzed by Malvern particle size analyzer.
  • the particle size and distribution of ginseng, American ginseng and notoginseng source nanoparticles are 2-1, 2-2 and 2-3, respectively.
  • Figure 3 shows the uptake of GDNPs by mononuclear-macrophages
  • Figure 4 shows the formation of mononuclear-macrophage colonies induced by GDNPs in vitro
  • Figure 5 shows that GDNPs induce mononuclear-macrophage proliferation in vitro
  • Figure 6 shows that GDNPs upregulate a variety of immunologically active molecules on the surface of monocyte-macrophages
  • Figure 7 shows the secretion of cytokines associated with GDNPs stimulating monocyte-macrophage activation
  • Figure 8 shows that GDNPs down-regulate M2 macrophage-associated surface marker molecules in vitro, and up-regulate M1-type surface markers
  • Figure 9 shows that GDNPs downregulate the transcription of M2 macrophage-associated genes in vitro, and up-regulate the transcription of M1-type related genes
  • Figure 10 shows that GDNPs promote M2 type macrophage secretion of M1 type cytokines such as TNF- ⁇ and IL-6 in vitro;
  • Figure 11 is a schematic diagram of in vivo experiments of GDNPs in the treatment of melanoma
  • Figure 12 is a graph showing tumor growth curves during experiments in each group of mice.
  • Figure 13 is the final tumor volume of each group of mice.
  • Figure 14 shows the final tumor weight of each group of mice
  • Figure 15 shows the final tumor size of each group of mice
  • Figure 16 is a graph showing the ratio of macrophages to lymphocytes in tumor tissues of each group of mice.
  • Figure 17 Changes in the number of M1/M2 type macrophages in tumor tissues of each group of mice.
  • GDNPs were 120000 ⁇ g, and after centrifugation for 60 minutes, they were precipitated and compacted. The supernatant was discarded, and the precipitate was fixed with 2.5% (v/v) glutaraldehyde, and processed in a transmission mirror chamber. The morphology of GDNPs was observed on the microscope. . As shown in Fig. 1, under the electron microscope, GDNPs are nano-particles with a membrane structure, and the size is between 150-500 nm, which proves that ginseng-derived nano-scale micro-organisms are effectively recruited.
  • Example 5 Malvern particle size meter for detecting particle size of plant-derived nanoparticles
  • the extracted GDNPs were detected by Malvern particle size analyzer, as shown in Figure 2-1, 2-2, 2-3, and the particle size and distribution of the nanoparticles of ginseng, American ginseng and Panax notoginseng respectively. And the peak homogeneity of the PDNPs we extracted is better.
  • Example 6 GDNPs promote the formation and proliferation of mononuclear-macrophage colonies in vitro
  • BMDM bone marrow-derived mononuclear-macrophages
  • mice 1 C57/BL6 mice were sacrificed by necking, soaked in alcohol for 5 minutes. After fixing the mice, the fur of the front part of the hind limbs of the mice was peeled off the feet. The muscles were cut off and the thighs were cut and placed in a petri dish containing sterile PBS. in.
  • the mouse bone marrow was taken, the red blood cells were lysed, and the cell concentration was adjusted to 1 ⁇ 10 6 /ml, and the medium was DMEM (Gibco) + 10% FBS (Gibco) + 20 ng / ml M-CSF (Peprotech).
  • BMDM was trypsinized, added to a 6-well cell culture plate, 2 x 10 6 cells per well, and 1 ml or 2 ml of medium was added.
  • 1 BMDM was stained by FITC-labeled Anti-mouse F4/80 antibody on the previous day and added to a petri dish with a climbing slide to grow adherently.
  • F4/80 is a specific surface marker molecule of mouse monocyte-macrophages.
  • the mouse BMDM is green-fluorescent by FITC-labeled Anti-mouseF4/80antibody, while PKH26 is a red color.
  • Membrane dyes that bind to membranes on the surface of GDNPs. Under laser confocal microscopy, it was observed that multiple red particles appeared in the mononuclear-macrophage cells, indicating that mononuclear-macrophages can effectively phagocytose GDNPs.
  • the induced BMDM was adjusted to a cell concentration of 5 ⁇ 10 5 /ml, and added to a 96-well cell culture plate at 100 ul per well.
  • the left panel shows the BMDM cultured in the same volume of PBS for 72 hours.
  • the right panel shows the BMDM cultured with GDNPs (20ug/ml) for 72 hours. It can be observed that mononuclear-macrophages are added after the addition of GDNPs. Proliferation is evident and cell clone colonies appear.
  • the results of MTT assay showed that GDNPs could significantly stimulate the proliferation of BMDM. After adding GDNPs, the activity of monocyte-macrophages was significantly higher than that of the control group.
  • Example 7 GDNPs up-regulate CD86, CD80, TLR2/4 and other immunologically active molecules on the surface of monocyte-macrophages
  • GDNPs were added to mouse BMDM induced in vitro, and the supernatant was aspirated after 72 hours.
  • GDNPs up-regulate the expression of surface-associated activating molecules in monocyte-macrophages
  • the culture medium was added to terminate the digestion, and mononuclear-macrophages were collected by centrifugation at 1200 rpm, and the Fc blocking agent was blocked (room temperature, 20 minutes).
  • GDNPs can significantly up-regulate a variety of activating molecules (CD80, CD86, TLR2, TLR4, MHC-II, etc.) on the surface of monocyte-macrophages, thereby activating monocyte-macrophages.
  • activating molecules CD80, CD86, TLR2, TLR4, MHC-II, etc.
  • Example 8 GDNPs promote the secretion of cytokines such as TNF- ⁇ and IL-6 by monocyte-macrophages
  • the capture antibody (anti-mouse TNF- ⁇ or IL-6) was diluted as required by the instructions, and coated in a 96-well plate at 100 ul/well at 4 ° C overnight.
  • the plate was washed 5 times with 4 PBST, and HRP-antimouse-TNF- ⁇ or IL-6 (1:10000) was added, and the mixture was incubated at 37 ° C for 1 hour.
  • the plate was washed 5 times with 5PBST, TMB coloring solution was added, and incubation was carried out for 15 minutes at 37 °C.
  • GDNPs significantly stimulated monocyte-macrophages to secrete cytokines (TNF- ⁇ and IL-6) associated with monocyte-macrophage activation.
  • Example 9 GDNPs down-regulate M2 macrophage surface marker molecules in vitro, and up-regulate M1-type surface marker molecules
  • IL-13 (20 ng/ml) differentiated into M2 type macrophages.
  • the culture medium was added to terminate the digestion, and the macrophages were collected by centrifugation at 1200 rpm, and the Fc blocking agent was blocked (room temperature, 20 minutes).
  • GDNPs can significantly down-regulate the surface marker molecule CD206 of M2 macrophages and up-regulate the surface marker molecules of M1 macrophages (CD80, CD86, TLR2, TLR4, MHC-II, etc.). From the results of surface molecular markers, it was confirmed that GDNPs can polarize M2 type macrophages to M1 type.
  • Example 10 GDNPs downregulate the transcription of M2 macrophage-associated genes in vitro, and up-regulate the transcription of MI-related genes
  • Example 1 Part of the cells in Example 1, adding 1 ml of Trizol, inhaling 1.5 ml of EP tube, pipetting until the liquid clears the cell-free mass, inverting and mixing for 10 times, and allowing to stand at room temperature for 5 minutes.
  • RNA concentration was measured on a microplate reader.
  • RNA concentration adjustment 1 ng - 5 ⁇ g of total RNA was taken as a template.
  • the reaction system is as follows:
  • reaction conditions 42 ° C, 15 minutes ⁇ 85 ° C, 5 seconds ⁇ 4 ° C, ⁇ , after the reaction to obtain cDNA, stored at -20 ° C for use.
  • the M1/M2 macrophage-associated genes IL-6, TNF- ⁇ , CD80, CXCL9, CCL3, iNOS/Arg-1, CD206 were determined by reference to the SYBR Green Realtime PCR Master MIX instructions. Expression of mRNAs such as IL-10 and CHI313.
  • reaction conditions 95 ° C 30 seconds ⁇ PCR cycle (X40 cycle): 95 ° C, 5 seconds; 55 ° C, 10 seconds annealing; 72 ° C, 15 seconds extension ⁇ preparation of melting curve statistical analysis
  • GDNPs can reduce the transcription level of M2 macrophage-associated genes and up-regulate the transcription level of M1 cell-associated genes.
  • the results of transcript levels of M1/M2 related genes confirm that GDNPs make M2 macrophages.
  • the cells are polarized to the M1 type.
  • Example 11 GDNPs promote the secretion of cytokines such as TNF- ⁇ and IL-6 by M2 macrophages in vitro.
  • Orifice plate overnight at 4 °C.
  • the plate was washed 5 times with 4 PBST, and HRP-antimouse-TNF- ⁇ or IL-6 (1:10000) was added, and the mixture was incubated at 37 ° C for 1 hour.
  • the plate was washed 5 times with 5PBST, TMB coloring solution was added, and incubation was carried out for 15 minutes at 37 °C.
  • GDNPs can induce M2 macrophages to secrete M1-associated cytokines (TNF- ⁇ and IL-6). From the perspective of secreted cytokines, GDNPs can make M2 macrophages to M1 polarization.
  • M1-associated cytokines TNF- ⁇ and IL-6
  • Example 12 GDNPs in vivo polarize M2 type macrophages in the tumor microenvironment to M1 type, thereby inhibiting tumor growth
  • mice Male C57/BL6 mice, weighing 18-20 grams, were purchased from the Animal Experimental Center of Yangzhou University. Adaptive growth for 1 week.
  • mice were subcutaneously inoculated subcutaneously into the right melanoma cells-B16F10 (2.5 ⁇ 10 5 cells/only) and observed daily.
  • tumor-bearing model group intraperitoneal injection of PBS
  • intragastric treatment group GDNPs 150ug/only
  • the intraperitoneal injection group (GDNPs 100 ug/only) was treated for 3 days ( Figure 11).

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Abstract

一种人参来源的纳米颗粒及其制备方法和应用。该人参来源的纳米颗粒具有膜结构，粒径为150-500nm，峰值粒径为280-350nm；其通过多次离心、过滤除杂制备。所述人参来源的纳米颗粒可用于制备天然免疫增强剂的药物和天然抗肿瘤的药物。

Description

一种人参来源的纳米颗粒及其制备和应用 技术领域
本发明属于免疫增强剂领域，具体涉及一种人参来源的纳米颗粒及其制备和应用。
背景技术
微泡(Microvesicles)是细胞外囊泡(Extracellular Vesicles)的一种。与外泌体(Exosomes)不同，微泡主要是通过质膜向外出芽产生的直径100-1000nm左右的纳米级颗粒。这些纳米颗粒中往往携带有蛋白、DNA、RNA等多种活性物质，发挥多种生物活性作用。
免疫增强剂通常指的是在单用或与抗原同时使用时可增强机体免疫应答的物质，亦被称为免疫调节剂或免疫刺激剂。免疫增强剂可通过不同的作用途径发挥作用，如增强单核-巨噬细胞的活性、增强抗原物质的免疫原性和稳定性以及促进抗体的合成与分泌等，从而增强机体的特异性和非特异性免疫应答。
单核-巨噬细胞是血液、淋巴和所有哺乳动物组织类型中常见的吞噬细胞。单核-巨噬细胞是造血系统中可塑性最强的一种细胞，所有组织中都有这种细胞，并且其也具有极强的功能多样性。它们的主要功能是以固定细胞或游离细胞的形式对老化细胞、肿瘤细胞以及病原体等进行吞噬作用，并激活其他免疫细胞；在正常发育、体内平衡、组织修复和对病原体的免疫反应中起很多不同作用。
由于单核-巨噬细胞在机体的非特异性免疫应答和特异性免疫应答中皆扮演重要的角色，且在体内分布广泛，如何调节单核-巨噬细胞的活性是免疫学研究的焦点之一。传统的粒细胞单核-巨噬细胞集落刺激因子只能促进单核-巨噬细胞的生长，但不能活化TLR等相关免疫分子，从而不能充分促进细胞介导的免疫应答，进而清除位于系胞内的病原及肿瘤细胞成分。中药免疫增强剂药性温和、耐药性低、毒副作用小，且具有双向调剂功能的特点，日渐成为免疫增强剂的研究热点。
肿瘤微环境(tumor microenvironment)是指在肿瘤生长过程中，由肿瘤细胞、基质细胞(包括成纤维细胞、免疫和炎性细胞、脂肪细胞、胶质细胞、平滑肌细胞，以及一些血管内皮细胞等)和细胞外基质等共同构成的局部稳态环境，为肿瘤的发生、发展、侵袭、转移等提供了必要的物质基础。肿瘤微环境对肿瘤有着重要的支持和促进作用。如何能够干预、改善肿瘤微环境，是防止肿瘤发展、转移的核心问题。
近年来研究表明，肿瘤微环境中生长的巨噬细胞——肿瘤相关巨噬细胞(Tumor-associated macrophages，TAM)是肿瘤微环境的重要组成成分，在肿瘤发展及转移的各个阶段中起关键作用。
巨噬细胞(macrophage，
Figure PCTCN2017088953-appb-000001
)是机体的重要的免疫细胞之一，在宿主的免疫防御及维护组织稳态方面发挥着重要作用。根据功能特征，巨噬细胞主要被活化为2个亚型：经典活化的M1型(classically activated macrophage)及替代活化的M2型(alternatively activated macrophage)。M1型受到Th1分泌的细胞因子，如干扰素-γ(interferon-γ，INF-γ)、细菌脂多糖(lipopolysaccharide，LPS)及Toll样受体(Toll-like receptor，TLR)的调控，其特征是可分泌包括IL-6、IL-12、IL-23及肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)等在内的促炎性因子。此外，M1型巨噬细胞还高表达主要组织相容性复合物(major histocompatibility complex)I类及II类分子进行有效地抗原提呈。因此，M1型巨噬细胞被认为是具有杀伤细菌及肿瘤细胞并可分泌多种促炎性细胞因子的细胞。而M2型巨噬细胞与M1型的作用截然相反，其被认识是一种具有促进肿瘤的生长、侵袭和转移作用的细胞。绝大多数的TAM都属于M2型巨噬细胞，是人和小鼠最主要的肿瘤浸润白细胞。在肿瘤早期侵袭、转移阶段，肿瘤细胞释放趋化因子招募巨噬细胞和其他炎性细胞到达肿瘤周围的基质区域，随后TAM可穿透基底膜，从而使肿瘤细胞逃脱基底膜的束缚到达周围正常组织基质，同时TAM和肿瘤细胞均可刺激血管生成，提高细胞的侵袭性和运动性[11]。TAM还可以通过释放血管生成调节酶类，如基质金属蛋白酶(matrix metalloprotein 2，MMP2)、MMP-9、MMP-12和，环氧合酶2(cyclooxygenase 2，COX2)等，促进血管生成，为肿瘤生长输送养分。此外，TAM还可释放一系列促进肿瘤细胞浸润和转移的细胞因子及生长因子，如血管内皮生长因子(vascular endothelial growth factor，VEGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor，bEGF)等。
基于TAM在肿瘤微环境中的重要作用，如何发挥巨噬细胞的异质性和可塑性，促使TAM向M1型极化，从而改善肿瘤微环境，可能成为肿瘤免疫治疗的重要靶点。
人参是我国最重要的中药材之一，被誉为“百草之王”，有非常高的药用价值。人参所产生的纳米颗粒级微泡携带有多种人参的有效成分，可能是其发挥药效的重要途径之一。
发明内容
本发明要解决的技术问题是，提供一种可量化生产的人参来源的纳米颗粒(英文名：Ginseng derived nano-particles，简称GDNPs)，及其作为天然免疫增强剂和制备治疗肿瘤的药物中的用途。来源于人参的纳米颗粒微泡含有多种人参的生物活性成分，因此作为一种新型的天然免疫增强剂有着良好的应用前景。
为解决上述技术问题，本发明所采用的技术方案是：
一种人参来源的纳米颗粒，具有膜结构，粒径范围为150-500nm，峰值粒径为280～350nm。
所述人参来源的纳米颗粒的制备方法，包括如下步骤：
(1)将新鲜、洗净的人参通过低速螺旋挤压技术榨出原浆；
(2)将步骤(1)中所得原浆通过筛网过滤除杂，收集滤液；
(3)将步骤(2)中得到的滤液依次进行低速、中速、高速和超速离心，每次离心后弃去沉淀，收集上清液进行下一次离心，其中最后一次离心，收集沉淀；
(4)将步骤(3)中所述最后一次离心收集的沉淀用缓冲液重悬后超速离心1次，收集沉淀；所述沉淀用缓冲液再次重悬后高速离心，收集上清液；所述上清液通过除菌级滤膜即得人参来源的纳米颗粒。
优选的，以上操作在4～25摄氏度环境中进行。
步骤(1)中，所述低速螺旋挤压的转速为30～60转/分钟。
步骤(2)中，所述筛网为200～2000目，优选为200～1000目。
步骤(3)中，步骤(3)中，所述低速离心的离心力为100～500×g，离心时间为5～10分钟；所述中速离心的离心力为1000～5000×g，离心时间为10～30分钟；所述高速离心的离心力为8000～12000×g，离心时间为30～60分钟；所述超速离心的离心力为100000～200000×g，离心时间为60～120分钟，所述低速、中速、高速和超速离心的次数为至少1次；
优选的，所述低速离心的离心力为200～500×g；所述中速离心的离心力为2000～5000×g；所述高速离心的离心力为100000～12000×g；所述超速离心的离心力为100000～120000×g。
步骤(4)中，所述缓冲液为磷酸盐缓冲液，所述缓冲液的pH范围为pH7.2～7.4；所述超速离心的离心力为100000～200000×g，离心时间为60～120分钟；所述高速离心的离心力为8000～12000×g，离心时间为30～60分钟；所述除菌级滤膜的孔径为0.45μm。
所述人参来源的纳米颗粒在治疗如艾滋病、活动性肺结核、口腔白色念珠菌感染、弓形虫脑病、卡波济肉瘤等多种免疫功能低下引发的疾病中的应用。
所述人参来源的纳米颗粒在制备作为活化单核-巨噬细胞的免疫增强剂的药物中的应用。
所述活化单核-巨噬细胞的方法为促进单核-巨噬细胞的增殖以及集落的形成或上调单核-巨噬细胞表面的免疫活性分子或促进单核-巨噬细胞分泌的免疫活性细胞因子。
所述免疫活性分子为Toll样受体2/4(TLR2/4)、白细胞分化抗原80(CD80)、白细胞分化抗原86(CD86)、主要组织复合体2(MHC-II)中的一种或多种；所述免疫活性细胞因子为肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)中的一种或多种。
所述人参来源的纳米颗粒在制备治疗肿瘤的药物中的应用。
所述治疗肿瘤的方法为逆极化肿瘤相关的巨噬细胞。
所述逆极化肿瘤相关的巨噬细胞的方法为：下调M2型巨噬细胞的表面标志分子及上调M1型巨噬细胞的表面标志分子，上调M1型巨噬细胞分泌的细胞因子，改变肿瘤微环境中M1/M2型巨噬细胞的比例，从而改善肿瘤微环境，杀伤肿瘤。
所述M2型巨噬细胞相关的表面标志分子为白细胞分化抗原206(CD206)；所述M1型巨噬细胞相关的表面标志分子为Toll样受体2/4(TLR2/4)、白细胞分化抗原80(CD80)、白细胞分化抗原86(CD86)、主要组织复合体2(MHC-II)中的一种或多种；所述M2型巨噬细胞相关基因为精氨酸酶1(Arg-1)、白细胞分化抗原206又称巨噬细胞甘露糖受体(CD206)、白细胞介素10(IL-10)、类几丁质酶3样分子(CHI313)中的一种或多种；所述M1型巨噬细胞相关基因为白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、白细胞分化抗原80(CD80)、趋化因子9(CXCL9)、趋化因子3(CCL3)、诱导型一氧化氮合成酶(iNOS)等中的一种或多种；所述M1型巨噬细胞相关的免疫活性细胞因子为肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)中的一种或多种。
一种药物组合物，包含所述的人参来源的纳米颗粒。
一种药物制剂，包含治疗有效量的所述的人参来源的纳米颗粒或所述的药物组合物的在药学上可接受的赋形剂。
本发明所述的人参来源的纳米颗粒材料来源广泛；可以是人参、西洋参、三七、党参、太子参、丹参、玄参；本发明制备方法，具有易操作、耗时少等优点。
有益效果：本发明的人参来源的纳米颗粒能够有效地诱导骨髓来源的单核-巨噬细胞增殖、活化，同时上调TLR2/4、CD80等多种表面活性分子，并分泌TNF-a、IL-6等细胞因子。在开发用于制备天然免疫增强剂的药物方面有着良好的应用前景；本发明的人参来源的纳米颗粒在制备治疗肿瘤的药物中的应用能够有效地使肿瘤相关巨噬细胞从促进肿瘤生长的M2型向抗肿瘤的M1型极化，同时上调M1型相关的TLR2/4、CD80等多种表面活性分子，并分泌TNF-a、IL-6等细胞因子，从而改善肿瘤微环境，在开发用于制备天然抗肿瘤药物方面有着良好的应用前景。
附图说明
图1为电镜观察GDNPs的形态；
图2为马尔文粒径仪分析的PDNPs的颗粒大小，2-1、2-2、2-3分别为人参、西洋参、三七来源的纳米颗粒的粒径大小及分布；
图3为单核-巨噬细胞对GDNPs的摄取；
图4为GDNPs体外诱导单核-巨噬细胞集落的形成；
图5为GDNPs体外诱导单核-巨噬细胞增殖；
图6为GDNPs上调单核-巨噬细胞表面的多种免疫活性分子；
图7为GDNPs刺激单核-巨噬细胞活化相关的细胞因子的分泌；
图8为GDNPs体外下调M2型巨噬细胞相关表面标志分子，同时上调M1型相关表面标志分子；
图9为GDNPs体外下调M2型巨噬细胞相关基因的转录，同时上调M1型相关基因的转录；
图10为GDNPs体外促使M2型巨噬细胞分泌TNF-α、IL-6等M1型细胞因子；
图11为GDNPs治疗黑色素瘤在体实验示意图；
图12为各组小鼠实验期间肿瘤生长曲线；
图13为各组小鼠肿瘤最终体积；
图14为各组小鼠肿瘤最终重量；
图15为各组小鼠肿瘤最终大小；
图16为各组小鼠肿瘤组织中巨噬细胞占淋巴细胞的比例；
图17各组小鼠肿瘤组织中M1/M2型巨噬细胞数量的变化。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：人参来源的纳米颗粒的制备
(1)将新鲜人参用清水洗净，在20℃环境中通过低速螺旋挤压技术榨出原浆；
(2)将榨出的原浆在20℃环境中经500目筛网过滤除杂，收集滤液；
(3)将收集的滤液先以200×g，离心10分钟，弃沉淀，收集上清；再将上清液以2000×g，离心30分钟，弃沉淀，收集上清；再将上清液以10000×g，离心60分钟，弃沉淀，收集上清；最后将上清液以120000×g，离心60分钟，收集沉淀；
(4)将沉淀用pH7.2的磷酸盐缓冲液重悬后，以120000×g，离心60分钟，收集沉淀；将沉淀再次用pH7.2的磷酸盐缓冲液重悬后，以10000×g，离心60分钟，收集上清液；最后通过孔径为0.45μm的除菌级滤膜后即获得纳米级颗粒。保存于-20℃～-80℃中。
实施例2：西洋参来源的纳米颗粒的制备
(1)将新鲜西洋参用清水洗净，在10℃环境中通过低速螺旋挤压技术榨出原浆；
(2)将榨出的原浆在10℃环境中经200目筛网过滤除杂，收集滤液；
(3)将收集的滤液先以500×g，离心5分钟，弃沉淀，收集上清；再将上清液以 5000×g，离心10分钟，弃沉淀，收集上清；再将上清液以12000×g，离心30分钟，弃沉淀，收集上清；最后将上清液以100000×g，离心90分钟，收集沉淀；
(4)将沉淀用pH7.4的磷酸盐缓冲液重悬后，以100000×g，离心90分钟，收集沉淀；将沉淀再次用pH7.4的磷酸盐缓冲液重悬后，以12000×g，离心30分钟，收集上清液；最后通过孔径为0.45μm的除菌级滤膜后即获得纳米级颗粒。保存于-20℃～-80℃中。
实施例3：三七来源的纳米颗粒的制备
(1)将新鲜三七用清水洗净，在25℃环境中通过低速螺旋挤压技术榨出原浆；
(2)将榨出的原浆在25℃环境中经1000目筛网过滤除杂，收集滤液；
(3)将收集的滤液先以500×g，离心10分钟，弃沉淀，收集上清；再将上清液以5000×g，离心30分钟，弃沉淀，收集上清；再将上清液以12000×g，离心45分钟，弃沉淀，收集上清；最后将上清液以200000×g，离心60分钟，收集沉淀；
(4)将沉淀用pH7.3的磷酸盐缓冲液重悬后，以200000×g，离心60分钟，收集沉淀；将沉淀再次用pH7.3的磷酸盐缓冲液重悬后，以12000×g，离心45分钟，收集上清液；最后通过孔径为0.45μm的除菌级滤膜后即获得纳米级颗粒。保存于-20℃～-80℃中。
实施例4：电镜观察GDNPs的形态
将提取好的GDNPs 120000×g，60分钟超速离心后使其沉淀并压缩紧密，弃上清，沉淀用2.5％(v/v)戊二醛固定，送电镜室进行处理，上镜观察GDNPs的形态。如图1所示：电镜下可见GDNPs为具有膜结构的纳米颗粒，大小在150-500nm之间，证实有效募集了人参来源的纳米级微体。
实施例5：马尔文粒径仪检测植物来源纳米颗粒的粒径
将提取好的GDNPs通过马尔文粒径仪进行粒径检测，如图2-1、2-2、2-3所示，分别为人参、西洋参、三七来源的纳米颗粒的粒径大小及分布，且我们提取的PDNPs的峰值均一性较好。
实施例6：GDNPs体外促进单核-巨噬细胞集落的形成以及增殖
1.骨髓来源的单核-巨噬细胞(Bone marrow derived macrophages，BMDM)的获取
①脱颈处死C57/BL6小鼠，酒精浸泡5分钟，固定小鼠后将小鼠后肢前部的皮毛剥置足部，钝性分离肌肉剪下大腿，放入盛有无菌PBS的培养皿中。
②镊子夹住腿骨，用剪刀剔除其余肌肉，在关节处剪断腿骨。
③用1mL注射器吸取培养皿中的PBS，将针头刺入骨髓腔，反复冲洗骨髓至骨髓变白，将骨髓冲洗液过滤筛网。
④取小鼠骨髓，裂解红细胞，调整细胞浓度至1×106个/ml，培养基：DMEM(Gibco)+10％FBS(Gibco)+20ng/ml M-CSF(Peprotech)。
⑤算第0天取骨髓，第3天，每皿添加10ml含20ng/ml M-CSF的完全培养基。
⑥5天后，每皿添加20ng/ml M-CSF。
⑦7天后，胰酶消化BMDM，加入6孔细胞培养板中，每孔2×106个细胞，加入1ml或2ml培养基。
2.BMDM对GDNPs的摄取
①于前一天通过FITC标记的Anti-mouseF4/80antibody将BMDM染色，并加入带有爬片的培养皿中，使其贴壁生长。
②将100ul GDNPs用稀释液稀释至250ul。
③将1ul PKH26加入250ul稀释液中，作为染料。
④将稀释的GDNPs加入到染料中，快速混合。
⑤25℃孵育的2-5分钟，定时轻轻颠倒离心管保证在25℃充分混匀。
⑥加入等量血清中止染色反应孵育1分钟。
⑦120000×g，60分钟超速离心。
⑧吸弃上清，将染色的GDNPs用100ulPBS重悬。
⑨以20ug/ml的浓度将GDNPs加入培养的BMDM中，培养24小时。
⑩取出爬片，激光共聚焦观察单核-巨噬细胞对GDNPs的吞噬。
如图3.所示：F4/80是小鼠单核-巨噬细胞的特异性表面标志分子，通过FITC标记的Anti-mouseF4/80antibody使小鼠BMDM带有绿色荧光，而PKH26是一种红色的膜染料，能够与GDNPs表面的膜结合。在激光共聚焦显微镜下，可以明显的观察到单核-巨噬细胞胞内出现了多个红色颗粒，表明单核-巨噬细胞能够有效的吞噬GDNPs。
3.GDNPs体外诱导单核-巨噬细胞集落的形成及增殖
①将诱导的BMDM调整至细胞浓度为5×105个/ml，加入96孔细胞培养板，每孔100ul。
②将GDNPs以20ug/ml的终浓度加入各孔中。
③72小时后显微镜观察单核-巨噬细胞集落的生成。
④MTT法检测细胞增殖
如图4.显示：左图为添加同体积PBS培养72小时的BMDM，右图为添加了GDNPs(20ug/ml)培养72小时的BMDM，可以观察到添加了GDNPs之后，单核-巨噬细胞增殖明显且出现细胞克隆集落。如图4.MTT实验结果显示GDNPs能够显著地刺激BMDM增殖，添加了GDNPs之后，单核-巨噬细胞的活力明显高于对照组。
实施例7：GDNPs上调单核-巨噬细胞表面的CD86、CD80、TLR2/4等免疫活性分子
体外诱导的小鼠BMDM添加GDNPs，72小时后吸去上清
1.GDNPs上调单核-巨噬细胞表面相关活化分子的表达
①诱导获得C57/BL6小鼠的BMDM，20ug/ml GDNPs刺激单核-巨噬细胞。
②72小时后，吸去培养上清，PBS清洗一遍后胰酶消化细胞。
③添加培养液终止消化，1200rpm离心收集单核-巨噬细胞，Fc阻断剂封闭(室温，20分钟)。
④分别加入anti-mouse CD80、CD86、TLR2、TLR4、MHC-II等单克隆抗体染色(室温，30分钟)，PBS洗2次。流式细胞仪鉴定上述分子的表达情况。
如图6所示：GDNPs能够显著的上调单核-巨噬细胞表面的多种活化分子(CD80、CD86、TLR2、TLR4、MHC-II等)，从而活化单核-巨噬细胞。
实施例8：GDNPs促进单核-巨噬细胞分泌TNF-α、IL-6等细胞因子
1.实施例3的培养上清中TNF-α、IL-6检测(ELISA法)
①按说明书要求稀释捕获抗体(anti-mouse TNF-α或IL-6)，以100ul/孔量包被于96孔板，4℃过夜。
②用PBST(含0.5％吐温的PBS)洗板3次，每次3分钟。之后用2％的羊血清封闭液封闭，37℃，2小时。
③PBST洗板3次。将培养上清样本及标准品稀释液按顺序加入封闭好的96孔板内，37℃孵育1小时。
④PBST洗板5次，加入HRP-antimouse-TNF-α或IL-6(1∶10000)，37℃孵育1小时。
⑤PBST洗板5次，加入TMB显色液，37℃孵育15分钟。
⑥加入H2SO4终止液，450nm测定OD值。
⑦根据OD值计算TNF-α或IL-6的浓度。
如图7.所示，GDNPs能够显著的刺激单核-巨噬细胞分泌与单核-巨噬细胞活化相关的细胞因子(TNF-α和IL-6)。
实施例9：GDNPs体外下调M2型巨噬细胞表面标志分子，同时上调M1型相关表面标志分子
①通过M-CSF(20ng/ml)诱导获得C57/BL6小鼠的BMDM，添加IL-4(20ng/ml)
和IL-13(20ng/ml)使其分化为M2型巨噬细胞。
②加入GDNPs(20ug/ml)，72小时后，吸去培养上清，PBS清洗一遍后胰酶消化细胞。
③添加培养液终止消化，1200rpm离心收集巨噬细胞，Fc阻断剂封闭(室温，20分钟)。
④分别加入anti-mouse CD206、CD80、CD86、TLR2、TLR4、MHC-II等单克隆抗体染色(室温，30分钟)，PBS洗2次。流式细胞仪鉴定上述分子的表达情况。
如图8所示：GDNPs能够显著的下调M2型巨噬细胞的表面标志分子CD206，同时上调M1型巨噬细胞的表面标志分子(CD80、CD86、TLR2、TLR4、MHC-II等)。从表面分子标志的结果证实GDNPs能够使M2型巨噬细胞向M1型极化。
实施例10：GDNPs体外下调M2型巨噬细胞相关基因的转录，同时上调MI型相关基因的转录
1.巨噬细胞总RNA的提取
①实施例1中的部分细胞，加入1ml Trizol，吸入1.5ml EP管中，吹打至液体澄清无细胞团块，颠倒混匀10下，室温放置5分钟。
②加入200ul氯仿，剧烈震荡15秒，进行RNA抽提，室温放置3分钟。
③4℃，12000RPM，15分钟离心。
④吸取上层水相至新的1.5ml EP管中，加入等体积异丙醇，颠倒混匀，室温放置10分钟。
⑤4℃，12000RPM，10分钟离心。
⑥弃上清，加入预冷的0.5ml的75％乙醇(DEPC水配)洗涤沉淀，混匀后，4℃，7500RPM，5分钟离心。
⑦弃上清，室温下干燥5-10分钟。
⑧加入20-60ul去离子水溶解RNA，吹打混匀，置于56℃水浴锅中10分钟。
⑨快速震荡离心后于酶标仪检测RNA浓度。
2.cDNA的合成
①根据Rever TraAce qPCR RT Kit说明书进行
②根据RNA浓度调整，取1ng-5μg总RNA作为模板。反应体系如下：
Figure PCTCN2017088953-appb-000002
③将以上加入无核酸酶的PCR管中，加入总RNA模板适量体积后，剩余加去离子水补足至20ul，所有操作均在冰上进行。
④反应条件：42℃，15分钟→85℃，5秒→4℃，∞，反应后获得cDNA，-20℃保存备用。
3.荧光定量PCR反应
①以上述cDNA为模板，GAPDH为内参，参照SYBR Green Realtime PCR Master MIX说明书操作测定M1/M2巨噬细胞相关基因IL-6、TNF-α、CD80、CXCL9、CCL3、iNOS/Arg-1、CD206、IL-10、CHI313等的mRNA的表达。
②根据Real-Time PCR试剂盒的说明配制反应液：
Figure PCTCN2017088953-appb-000003
③反应条件：95℃30秒→PCR循环(X40循环)：95℃，5秒；55℃，10秒退火；72℃，15秒延伸→制备熔解曲线统计分析
④以GAPDH作为内参，反应完毕后确认Real-time PCR的扩增曲线和熔解曲线，检测各模板的Ct值，所有样品重复3次，用2-ΔΔCT法计算细胞中mRNA水平的相对表达量。
如图9.所示，GDNPs能够降低M2型巨噬细胞相关基因的转录水平，同时上调M1型细胞相关基因的转录水平，从M1/M2相关基因的转录水平的结果证实GDNPs使M2型巨噬细胞向M1型极化。
实施例11：GDNPs体外促使M2型巨噬细胞分泌TNF-α、IL-6等细胞因子
实施例1的培养上清中TNF-α、IL-6检测(ELISA法)
①按说明书要求稀释捕获抗体(anti-mouse TNF-α或IL-6)，以100ul/孔量包被于96
孔板，4℃过夜。
②用PBST(含0.5％吐温的PBS)洗板3次，每次3分钟。之后用2％的羊血清封闭液封闭，37℃，2小时。
③PBST洗板3次。将培养上清样本及标准品稀释液按顺序加入封闭好的96孔板内，37℃孵育1小时。
④PBST洗板5次，加入HRP-antimouse-TNF-α或IL-6(1∶10000)，37℃孵育1小时。
⑤PBST洗板5次，加入TMB显色液，37℃孵育15分钟。
⑥加入H2SO4终止液，450nm测定OD值。
⑦根据OD值计算TNF-α或IL-6的浓度。
如图10.所示，GDNPs能够促使M2型巨噬细胞分泌M1型相关的细胞因子(TNF-α和IL-6)，从分泌的细胞因子角度证实GDNPs能够使M2型巨噬细胞向M1型极化。
实施例12：GDNPs体内使肿瘤微环境中的M2型巨噬细胞极化为M1型，从而抑制肿瘤的生长
①雄性C57/BL6小鼠，体重18-20克，购于扬州大学动物实验中心。适应性生长1周。
②小鼠右侧腋下皮下接种小鼠黑色素瘤细胞-B16F10(2.5×105个/只)，逐日观察。
③种瘤7天后，小鼠移植瘤大小平均值达到50-120mm3，将荷瘤小鼠随机分为3组：荷瘤模型组(腹腔注射PBS)、灌胃治疗组(GDNPs 150ug/只)、腹腔注射治疗组(GDNPs 100ug/只)，治疗间隔时间为3天(图11)。
④逐日观察各组小鼠生长状态，每2天测小鼠体重及肿瘤体积(肿瘤体积计算＝长×宽2/2)
⑤治疗11天后，测体重后眼眶采血，引颈处死小鼠，剥离肿瘤组织测量体积并称重，其余主要脏器分离后甲醛固定。
⑥取部分小鼠肿瘤组织，胶原酶消化30分钟后研磨，过200目滤网获得肿瘤组织单细胞悬液，Fc阻断剂封闭(室温，20分钟)。
⑦分别加入anti-mouse CD45、CD11b、CD206、CD80等单克隆抗体染色(室温，30分钟)，PBS洗2次。流式细胞仪鉴定上述分子的表达情况。
结果显示：GDNPs的灌胃治疗及腹腔注射治疗皆能抑制小鼠黑色素瘤的生长(图12～15)；通过对各组小鼠肿瘤组织淋巴细胞比例的分析，发现GDNPs的灌胃治疗及腹腔注射治疗组小鼠的肿瘤组织中，巨噬细胞在淋巴细胞中的比例要显著高于荷瘤模型组(图16)；同时，灌胃治疗及腹腔注射治疗组小鼠的肿瘤组织中M1/M2巨噬细胞的比例要显著高于(图17)。这些结果提示我们：GDNPs在体内能够有效地将肿瘤组织中的M2型巨噬细胞向M1型极化，改善肿瘤微环境，抑制肿瘤的生长。

Claims (15)

  1. 一种人参来源的纳米颗粒，其特征在于，具有膜结构，粒径范围为150-500nm，峰值粒径为280～350nm。
  2. 权利要求1所述人参来源的纳米颗粒的制备方法，其特征在于，包括如下步骤：
    (1)将新鲜、洗净的人参通过低速螺旋挤压榨出原浆；
    (2)将步骤(1)中所得原浆通过筛网过滤除杂，收集滤液；
    (3)将步骤(2)中得到的滤液依次进行低速、中速、高速和超速离心，每次离心后弃去沉淀，收集上清液进行下一次离心，其中最后一次离心，收集沉淀；
    (4)将步骤(3)中所述最后一次离心收集的沉淀用缓冲液重悬后超速离心1次，收集沉淀；所述沉淀用缓冲液再次重悬后高速离心，收集上清液；所述上清液通过除菌级滤膜即得人参来源的纳米颗粒。
  3. 根据权利要求2所述的制备方法，其特征在于，所述的人参用西洋参、三七、党参、太子参、丹参或玄参替换。
  4. 根据权利要求2所述的制备方法，其特征在于，步骤(1)中，所述低速螺旋挤压的转速为30～60转/分钟。
  5. 根据权利要求2所述的制备方法，其特征在于，步骤(3)中，所述低速离心的离心力为100～500×g，离心时间为5～10分钟；所述中速离心的离心力为1000～5000×g，离心时间为10～30分钟；所述高速离心的离心力为8000～12000×g，离心时间为30～60分钟；所述超速离心的离心力为100000～200000×g，离心时间为60～120分钟，所述低速、中速、高速和超速离心的次数为至少1次。
  6. 根据权利要求2所述的制备方法，其特征在于，步骤(4)中，所述缓冲液为磷酸盐缓冲液，所述缓冲液的pH范围为pH7.2～7.4；所述超速离心的离心力为100000～200000×g，离心时间为60～120分钟；所述高速离心的离心力为8000～12000×g，离心时间为30～60分钟；所述除菌级滤膜的孔径为0.45μm。
  7. 权利要求1所述人参来源的纳米颗粒在制备治疗如艾滋病、活动性肺结核、口腔白色念珠菌感染、弓形虫脑病、卡波济肉瘤等免疫功能低下疾病的药物中的应用。
  8. 权利要求1所述人参来源的纳米颗粒在制备作为活化单核-巨噬细胞的免疫增强剂的药物中的应用。
  9. 根据权利要求8所述应用，其特征在于，所述活化单核-巨噬细胞的方法为促进单核-巨噬细胞的增殖以及集落的形成或上调单核-巨噬细胞表面的免疫活性分子或促进单核-巨噬细胞分泌的免疫活性细胞因子。
  10. 权利要求1所述人参来源的纳米颗粒在制备治疗肿瘤的药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述治疗肿瘤的方法为逆极化肿 瘤相关的巨噬细胞。
  12. 根据权利要求10所述应用，其特征在于，所述逆极化肿瘤相关的巨噬细胞的方法为：下调M2型巨噬细胞的表面标志分子及上调M1型巨噬细胞的表面标志分子，上调M1型巨噬细胞分泌的细胞因子，改变肿瘤微环境中M1/M2型巨噬细胞的比例，从而改善肿瘤微环境，杀伤肿瘤。
  13. 根据权利要求12所述应用，其特征在于，所述M2型巨噬细胞相关的表面标志分子为白细胞分化抗原206；所述M1型巨噬细胞相关的表面标志分子为Toll样受体2/4、白细胞分化抗原80、白细胞分化抗原86、主要组织相容性复合体2中的一种或多种；所述M2型巨噬细胞相关基因为精氨酸酶1、白细胞分化抗原206、白细胞介素10、类几丁质酶3样分子中的一种或多种；所述M1型巨噬细胞相关基因为白细胞介素6、肿瘤坏死因子α、白细胞分化抗原80、趋化因子9、趋化因子3、诱导型一氧化氮合成酶中一种或多种；所述M1型巨噬细胞相关的免疫活性细胞因子为肿瘤坏死因子α、白细胞介素6中的一种或多种。
  14. 一种药物组合物，包含权利要求1所述的人参来源的纳米颗粒。
  15. 一种药物制剂，包含治疗有效量的如权利要求1所述的人参来源的纳米颗粒或权利要求9所述的药物组合物的在药学上可接受的赋形剂。
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CN116036142A (zh) * 2023-03-06 2023-05-02 上海中医药大学 一种含有人参细胞外囊泡的抗衰老组合物及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102949421A (zh) * 2012-05-17 2013-03-06 包海鹰 纳米化人参的体外抗肿瘤作用
CN106727810A (zh) * 2017-02-22 2017-05-31 江苏省中医药研究院 一种人参来源的纳米颗粒及其制备和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1883556A (zh) * 2005-06-24 2006-12-27 张晴龙 一种康艾纳米注射制剂及其制备方法
CN103479682B (zh) * 2012-06-14 2015-03-11 苏州恒宇生物科技有限公司 一种植物来源活性成分-纳米级膜性囊泡的制备方法
CN103479597B (zh) * 2012-06-14 2015-02-18 苏州恒宇生物科技有限公司 一种葡萄来源活性成分—纳米级膜性囊泡的制备方法及其用途
US10912737B2 (en) * 2016-01-14 2021-02-09 University-Industry Cooperation Group Of Kyung Hee University Nano complex comprising a nano drug delivery matrix; and a ginseng extract or a ginsenoside isolated therefrom
CN105708847B (zh) * 2016-02-02 2019-01-04 成都大学 人参皂苷多组分共载靶向纳米体系的制备方法及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102949421A (zh) * 2012-05-17 2013-03-06 包海鹰 纳米化人参的体外抗肿瘤作用
CN106727810A (zh) * 2017-02-22 2017-05-31 江苏省中医药研究院 一种人参来源的纳米颗粒及其制备和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Separation and purification of extracellular vesicles (Isolation and purification of extracellular vesicles) ", JOURNAL OF DIAGNOSTICS CONCEPTS & PRACTICE, vol. 13, no. 6, 31 December 2014 (2014-12-31), pages 640 - 642, XP009515901, DOI: 10.3969/j.issn.1671-2870.2014.06.022 *
See also references of EP3563861A4 *

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