WO2022206815A1 - Metal-organic composite nano-drug, preparation method therefor, and application thereof - Google Patents

Metal-organic composite nano-drug, preparation method therefor, and application thereof Download PDF

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WO2022206815A1
WO2022206815A1 PCT/CN2022/083976 CN2022083976W WO2022206815A1 WO 2022206815 A1 WO2022206815 A1 WO 2022206815A1 CN 2022083976 W CN2022083976 W CN 2022083976W WO 2022206815 A1 WO2022206815 A1 WO 2022206815A1
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nanoparticles
modified
cofeb
nanomedicine
ginsenosides
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French (fr)
Chinese (zh)
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宋玉军
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北京科技大学
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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
    • A61K47/69—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
    • A61K47/6921—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 particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K47/52—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 inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to the technical field of nano-medicine, in particular to a metal-organic composite nano-medicine and a preparation method and application thereof.
  • Nanomedicines have controllable size, large mass ratio, and unique physicochemical properties, so they can be used at the molecular level for monitoring, control, diagnosis and therapy of biological systems, and tissue repair (Tekade, R.; Maheshwari, R.; Soni, N.; Tekade, M.; Chougule, M., Nanotechnology-Based Approaches for Targeting and Delivery of Drugs and Genes. Elsevier, Amsterdam, 2017, pp 3-61.).
  • nanomedicines improve and extend the pharmacokinetics, solubility, and stability of a range of drug molecules that have been used in a wide variety of biomedical applications, including specific drug delivery, therapy, imaging, and diagnostics.
  • the use of nanomedicines is still characterized by numerous unclear effects on human health and various biological barriers to overcome, including stability, surface modification and functionalization, multimodal functionality, efficient drug delivery, and both.
  • nanohybrids composed of noble metals and magnetic components are expected to be used in energy conversion including energy conversion (Wang, S.; Niu, S.Y.; Li, H.S.; Lam, K.K.; Wang , Z.R.; Du, P.Y.; Leung, C.W.; Qu, S.X., Synthesis and controlled morpHology of Ni@Ag core shell nanowires with excellent catalytic efficiency and recyclability. Nanotechnology 2019,30,(38),9.), Medical Imaging (S.D. Gwenin, V.V.; Gwenin, C.D., Magnetic Functional-ized Nanoparticles for Biomedical, Drug Delivery and Imaging Applications.
  • controllable preparation and functionalization of multimodal nanomedicines extend their interdisciplinary applications to basic research and clinical applications (e.g., ultrasensitive bioprobes, highly efficient nanomedicines and nanozymes, ultrasensitive biomedical molecular imaging and certain diagnosis of certain diseases, etc.).
  • the current nano-drugs are either simple nano-drugs of the original drugs, especially for some organic or macromolecular drugs.
  • nano-drugs In addition to improving their dispersibility, nano-drugs have little improvement in other properties, especially the curative effect, while the toxic and side effects will change. Even larger, these simply nanosized drugs do not have quantum effects and surface activation effects that play an important role in therapeutic efficacy at the nanoscale like metal-based nanomaterials.
  • Pure inorganic nano-drugs, such as inorganic non-metals, like silica, etc. basically act as carriers, while pure metals, such as gold, may be a good carrier for cancer cells to escape and metastasize. big side effects.
  • the current particle size is very large, and each component is basically larger than 10nm. Epidermal network cells and renal tubules are removed, and there is a potential hazard of permanent retention in the human body.
  • the size of their components is preferably less than 6nm, but there is basically no mass production method for such nanomaterials, and the one-time price is very expensive, which is temporarily unacceptable to patients.
  • the organic polymer nano-drugs are continuously decomposed, there will also be damage to normal tissues and organs and carcinogenesis of the decomposed products (especially the generated active functional groups).
  • nano-drugs with tracer imaging function that can be closely compared in vitro and in vivo are required.
  • it basically depends on the fluorescent agent labeling with short lifespan and easy photosensitization and decomposition.
  • organic nano-drugs it is basically impossible to conduct molecular imaging studies stably for a long time to study their pharmacokinetics and pharmacodynamic mechanisms. Therefore, there is an urgent need for nanomedicines that can simultaneously trace in vitro and in vivo molecular imaging.
  • one object of the present invention is to provide a metal-organic composite nanomedicine, comprising nanoparticles treated with a first surface modifier and an activator in turn and a second surface modifier.
  • the treated pharmaceutical ingredient compound; the nanoparticle is coupled with the pharmaceutical ingredient compound through a cross-linking agent, wherein the pharmaceutical ingredient compound is ginsenoside, and the nanoparticle is Au@CoFeB.
  • the first surface modifier is selected from 3-aminopropyltrimethoxysiloxane, vinyltrimethoxysilane, phosphate bis-titanate coupling agent and diisopropyl At least one of oxydiacetylacetonate titanate; preferably 3-aminopropyltrimethoxysiloxane.
  • the second surface modifier is selected from the group consisting of 3-aminopropyltrimethoxysiloxane, 3-mercaptopropyl-triethoxysilane coupling agent, heptadecafluorodecyl triglyceride At least one of methyloxysilane and isopropyl tris (dioctyl pyrophosphate acyloxy) titanate; preferably 3-aminopropyl trimethoxy siloxane, 3-mercaptopropyl-tri At least one of an ethoxysilane coupling agent and isopropyl tris(dioctyl pyrophosphate acyloxy) titanate.
  • the crosslinking agent is selected from suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (succinate N-hydroxysuccinimide ester), polyethylene glycol At least one of alcohol disuccinimidyl succinate, succinyl-imide succinate poly, ethylene glycol succinyl-imide succinate and aziridine crosslinking agent XR-100; preferably Suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (N-hydroxysuccinimide ester of succinate), polyethylene glycol disuccinimidyl succinate, polyethylene glycol At least one of succinyl-imide succinate and aziridine crosslinker XR-100.
  • the nanomedicine is prepared by a method comprising the following steps:
  • step (3) a) putting the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) into a fourth organic solvent for incubation;
  • the metal core of the core-shell structure is Au with a face-centered cubic crystal structure
  • the shell layer of the core-shell structure is CoFeB with a face-centered cubic crystal structure
  • the overall structure of the nano-drug is a nano-drug aggregate with a size of 250-350 nm constructed by coupling together ultra-small nano-drug units of 6-7.2 nm; the dynamic radius of the nano-particle is about 100-200 nm;
  • the nanoparticle surface has +7-12mV; the nanomedicine surface has a positive potential of +25-30mV.
  • the first organic solvent is selected from at least one of benzene, toluene, para-xylene and meta-xylene; preferably at least one of toluene, para-xylene and meta-xylene.
  • the second organic solvent is selected from at least one of p-dimethyl sulfoxide, N-methylformamide, N-methyl-2-pyrrolidone, xylene and o-xylene; preferably dimethyl sulfoxide, N -At least one of methylformamide and N-methyl-2-pyrrolidone.
  • the third organic solvent is selected from at least one of para-xylene, ortho-xylene and toluene; preferably toluene.
  • the fourth organic solvent is selected from at least one of dimethyl sulfoxide, N-methylpyrrolidone, N-ethylacetamide and N-methylformamide; preferably dimethylsulfoxide, N-methylpyrrolidone and N -At least one of ethylacetamide.
  • the buffer used in step (4) can be a buffer known to those skilled in the art, for example, the buffer can be phosphate PBS buffer, citric acid-Na 2 HPO 4 buffer , Trizma buffer, fetal bovine serum FBS buffer, sodium carbonate-sodium bicarbonate buffer, and at least one of sodium acetate-acetate buffer, preferably phosphate buffer, fetal bovine serum FBS buffer and citric acid -At least one of Na 2 HPO 4 buffers, more preferably phosphate PBS buffers.
  • the buffer can be phosphate PBS buffer, citric acid-Na 2 HPO 4 buffer , Trizma buffer, fetal bovine serum FBS buffer, sodium carbonate-sodium bicarbonate buffer, and at least one of sodium acetate-acetate buffer, preferably phosphate buffer, fetal bovine serum FBS buffer and citric acid -At least one of Na 2 HPO 4 buffers, more preferably phosphate PBS buffers.
  • Another object of the present invention is to provide a method for preparing a composite nanomedicine, comprising the following steps:
  • step (3) a) putting the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) into a fourth organic solvent for incubation;
  • the nanoparticles of Au@CoFeB are prepared by microfluidic method, hydrothermal method, magnetron sputtering method, and electrodeposition method; more preferably, microfluidic method is used.
  • the microfluidic method is a continuous flow reaction process constructed at a micrometer scale (sub-millimeter), and the mixing, reaction nucleation, nanoparticle or drug growth and growth termination of the reactants during the synthesis of nanomaterials or drugs Processes are controlled in reaction volumes from ⁇ L to pL or even smaller.
  • the method has the advantages of precise design and regulation of kinetic parameters in different stages of the reaction, rapid material and energy exchange, uniform mixing and reaction, and parallel scale-up operations.
  • the tankless reactor has the unavoidable amplification effect, environmental friendliness, safety and waste minimization, and can make full use of the high specific surface area effect of the microfluidic channel to control the reaction products.
  • the stirring time is preferably 20-28 hours; in step (2)-b), the preferred centrifugal conditions include: the centrifugal rotation speed is 10000-20000rpm; the centrifugal time is 5 -40 minutes; preferably 12000-16000 rpm; centrifugation time 10-30 minutes.
  • the stirring time is preferably 1-3 hours; in step (3)-b), the preferred centrifugation conditions include; the centrifugal speed is 10000-20000rpm; the centrifugal time is 5 -15 minutes.
  • step (4) the pH value is adjusted to 7-7.8; the preferred centrifugation conditions include; the centrifugation speed is 10000-14000rpm; and the centrifugation time is 5-15 minutes.
  • the stirring time is preferably 20-28 hours; the preferred centrifugation conditions include; the centrifugal speed is 10000-14000rpm; the centrifugation time is 5-15 minutes.
  • step (6)-a) the incubation time is preferably 1.5-2.0 hours; in step (6)-a), the fourth organic solvent is preferably dimethyl sulfoxide.
  • Another object of the present invention is to provide the application of the composite nanomedicine of the present invention in the preparation of a medicine for treating liver cancer.
  • the concentration of Au@CoFeB in the composite nanomedicine is 0.00001-100000 ⁇ g/mL.
  • the composite nanomedicine provided by the invention can make full use of the multimodality of inorganic, especially metal-based nanoparticle, the activity of several atomic layers on the surface and the curative effect produced by quantum effect, as well as the curative effect of organic medicine and the effect on metal inner layer and living organisms
  • Protective effect, construct inorganic-organic composite nano-drugs give full play to the synergistic effect of organic drugs-inorganic nanoparticles, and obtain original nano-drugs with high efficacy, low or no toxic side effects, and the drugs have both in vitro and in vivo molecular imaging display. tracking function.
  • Figure 1 Schematic diagram of the microfluidic device structure for the synthesis of nanoparticles.
  • Figure 2A is a wide-angle transmission electron microscope photograph of the Au@CoFeB-Rg3 nanomedicine prepared by the process of Example 1.
  • the upper right of the figure is the size distribution map, and the lower part is an enlarged image of a single particle image.
  • 2B-8 are XRD spectra of nanoparticle Au@CoFeB prepared using the process of Example 1.
  • Figures 2B-9 are XRD patterns of nanoparticles Au@CoFeB-Rg3 prepared by the process of Example 1.
  • Figures 2C-10 are XPS spectra of nanoparticles Au@CoFeB prepared by the process of Example 1.
  • Figure 2C-11 is the XPS spectrum of the nanodrug Au@CoFeB-Rg3.
  • Figure 2D-12 is the FT-IR spectrum of nanoparticle Au@CoFeB.
  • Figure 2D-13 is the FT-IR spectrum of the nanodrug Au@CoFeB-Rg3.
  • 2E-14 are hydrodynamic diameter distribution diagrams of nanoparticles Au@CoFeB prepared using the process of Example 1.
  • 2E-15 are the hydrodynamic diameter distribution diagrams of the nanomedicine Au@CoFeB-Rg3 prepared by the process of Example 1.
  • Figures 2F-16 are the surface Zeta potentials of Au@CoFeB nanoparticles prepared using the process of Example 1.
  • 2F-17 is the surface Zeta potential of the nano-drug Au@CoFeB-Rg3 prepared by the process of Example 1.
  • 3A-18 are surface plasmon scattered light patterns of Au@CoFeB nanoparticles prepared using the process of Example 2, photographed by dark field microscopy.
  • 3A-19 are surface plasmon scattering light spectra of Au@CoFeB nanoparticles prepared using the process of Example 2 characterized by a dark-field optical spectrometer.
  • 3B-20 are the surface plasmon scattering light patterns of the Au@CoFeB-Rg3 nanomedicine prepared by the process of Example 2 and photographed by a dark field microscope.
  • 3B-21 are surface plasmon scattering optical spectra of Au@CoFeB-Rg3 nanomedicines prepared by the process of Example 2 and characterized by dark-field microspectroscopy.
  • Figure 3C is the T2WI concentration (0 ⁇ g/mL, 7.5 ⁇ g/mL, 15.1 ⁇ g/mL, 31.3 ⁇ g/mL, 93.5 ⁇ g/mL, 187.0 ⁇ g/mL) dependence of Au@CoFeB nanoparticles prepared by the process of Example 2
  • the magnetic resonance image (upper image of the figure) and the concentration-dependent curve of the magnetic resonance relaxation rate (T 2 ⁇ 1 , in s ⁇ 1 ).
  • Figure 3D shows the T2WI concentration (0 ⁇ g/mL, 4.1 ⁇ g/mL, 12.4 ⁇ g/mL, 37.3 ⁇ g/mL, 112.0 ⁇ g/mL)-dependent magnetic resonance of the Au@CoFeB-Rg3 nanomedicine prepared by the process of Example 2 Image (upper image of panel) and concentration-dependent curve of magnetic resonance relaxation rate (T 2 -1 in s -1 ).
  • Figure 3E is the computer-aided X-ray tomography of the Au@CoFe(B)-Rg3 nanomedicine prepared by the process of Example 2 depending on the concentration (400 ⁇ g/mL, 800 ⁇ g/mL, 1100 ⁇ g/mL, 1500 ⁇ g/mL, 4000 ⁇ g/mL) Concentration-dependent curves of scan (CT) images (upper image of the figure) and three-point averaged signal (HU).
  • CT Concentration-dependent curves of scan
  • Figure 4A shows the concentration-dependent Au@CoFeB nanoparticles prepared using the process of Example 1 for Jurkat-CT (4A-22), 3T3 (4A-23), K562-CT (4A-24) and HEP-G2/C3A (4A -25) 24 hour cell viability.
  • Figure 4B shows the concentration-dependent Jurkat-CT (4B-26), 3T3 (4B-27), K562-CT (4B-28) and HEP-G2/C3A of Au@CoFeB-Rg3 nanomedicines prepared using the process of Example 1 (4B-29) 24-hour cell viability.
  • Figure 5A is the concentration-dependent Au@CoFeB nanoparticles prepared using the process of Example 3 for Jurkat-CT (5A-30), 3T3 (5A-31), K562-CT (5A-32) and HEP-G2/C3A (5A -33) Cell proliferation survival rate.
  • Figure 5B shows the concentration-dependent Jurkat-CT (5B-34), 3T3 (5B-35), K562-CT (5B-36) and HEP-G2/C3A of Au@CoFeB-Rg3 nanomedicines prepared using the process of Example 3 (5B-37) Cell proliferation survival.
  • Fig. 6 Prepared Au@CoFeB nanoparticles and Au@CoFeB-Rg3 nanomedicines and PBS buffer and other nanomedicines (Fe@ Fe3O4 - Rg3, FePt@ Fe3O4 -Rg3 prepared by the process of Example 4 ) ) at two concentrations (twill bars, 95-190 ⁇ g/mL; grid bars, 474-947 ⁇ g/mL) on human chronic myeloid leukemia cells K562 cancer cells incubated for 24 hours of survival experimental results.
  • Figure 7A is an image of liver tumor after mice were sacrificed after different drug regimens in the in vivo animal experiment of the anti-liver cancer efficacy of Au@CoFeB-Rg3 and Au@CoFeB nanoparticles prepared by the process of Example 5.
  • FIG. 7B is a fluorescence image of the size of liver tumors labeled by biofluorescence at different stages in the in vivo animal experiment of the anti-liver cancer efficacy of Au@CoFeB-Rg3 and Au@CoFeB nanoparticles prepared by the process of Example 5. .
  • Figure 7C shows the anti-liver cancer efficacy of Au@CoFeB (7C-40) and Au@CoFeB-Rg3 (7C-41) prepared by the process of Example 5, and normal saline (7C-38) and Rg3 (7C-39) in the control group Absolute (upper line) and relative body weight changes (lower line) of different groups of mice during 21 days in live animal experiments.
  • Figure 7D shows the Au@CoFeB nanoparticles (7D-44) and Au@CoFeB-Rg3 (7D-45) prepared by the process of Example 5 and the control group with normal saline (7D-42) and Rg3 (7D-43) against liver cancer Quantitative values of bioluminescence intensity at tumor sites after treatment with different drugs in vivo animal experiments.
  • microfluidic synthesis was performed at 120 °C under nitrogen protection using the procedure shown in Fig. 1: 50 ml of metal salt solution with PVP and 50 ml of reduction solution were introduced into each syringe, respectively, and the syringes were fixed in the first syringe pump 1 and the second syringe pump 2. Then it is introduced into the Y-shaped reaction material-liquid mixer 5 through the first microfluidic pipe 3 and the second microfluidic pipe 4 to complete the reduction reaction, and the flow rates of the first syringe pump 1 and the second syringe pump 2 are both 3ml/min . Next, the solution enters the third microchannel tube 6 to complete the rapid nucleation and complete the growth of nanoparticles.
  • CoFeB nanoparticles were formed according to the following reaction scheme.
  • the reaction was completed, the obtained fresh nanoparticle dispersion solution was collected in the collector 7 . Then, the solution was pelleted using a centrifuge at 15,000 rpm for 30 minutes, and the top supernatant was decanted. The obtained particles were washed twice with NMP to obtain pre-synthesized CoFeB nanoparticles.
  • HAuCl 4 solution 2.5 ml (0.36 mmol) of the HAuCl 4 solution was dissolved in 50 ml of NMP.
  • the microfluidic synthesis was then performed at room temperature under nitrogen protection using the apparatus shown in Figure 1: 50 ml of the diluted HAuCl solution and 50 ml of NMP of pre-synthesized CoFeB nanoparticles were mixed using the first syringe pump 1 and the second syringe pump 2, respectively .
  • the solution is pumped into the first microfluidic pipe 3 and the second microfluidic pipe 4, and enters the Y-shaped reaction material-liquid mixer 5 together to complete the reduction reaction and rapid nucleation.
  • the first syringe pump 1 and the second syringe pump 2 The flow rates were all 3ml/min.
  • 0.2 g of NaBH 4 was dissolved in 20 ml of NMP and placed in the collector 7 in advance.
  • the collector 7 was shaken thoroughly and left to stand for 30 min to complete the displacement, reduction and surface rearrangement of the CoFeB nanoparticles.
  • 2 ml of ethanol was added to the collector to disrupt the equilibrium of the solution.
  • the precipitated nanoparticles were redissolved in the same volume of NMP. Repeat the cleaning process twice to remove most of the surfactant.
  • the final black slurry in the bottle was finally dried under vacuum to a black powder and kept in a desiccator for future use.
  • Au@CoFeB nanoparticles prepared in step (1) were dissolved into 50 ml of anhydrous toluene solution containing 1 wt% 3-aminopropyltrimethoxysiloxane (APTMS). Then, the mixed solution was stirred at room temperature for 24 hours. After stirring was completed, centrifugation was performed at 12,000 rpm for 10 minutes using a centrifuge. Subsequently, the top supernatant was decanted and the precipitated nanoparticles were washed once with ethanol. Finally, Au@CoFeB-APTMS nanoparticles were obtained.
  • APIMS 3-aminopropyltrimethoxysiloxane
  • ginsenoside Rg3 200 mg was dissolved in 50 mL of anhydrous toluene solution containing 1 w% APTMS. After stirring for 24 hours at ambient temperature, the mixed solution was centrifuged at 1200 rpm for 10 minutes. Then, the precipitated slurry was washed three times with ethanol to obtain modified ginsenoside Rg3.
  • Example 2-5 the process and device of Example 1 were used to prepare nanoparticles, but some of the raw materials, the amount of raw materials and the reaction conditions were changed, as shown in Table 1.
  • microstructure and multimodal imaging properties of typical nanoparticles and nanomedicines prepared by Example 1 were characterized. According to the high-resolution TEM images (Fig. 2A) and XRD patterns (Fig. 2B), before and after the nanoparticles were coupled with the drug, the metal core of the nanoparticles still maintained the core of Au with a face-centered cubic crystal structure and the CoFeB alloy shell with a face-centered cubic crystal structure. Core-shell structure; XPS spectra (Fig.
  • the nanomedicine contains Au, Co, Fe, B, Si(Ti), C, N(P) and O elements, in which Si mainly comes from nanoparticles surface modification and crosslinking agents, C, N(P) and O are derived from organic drugs and coupling agents such as Rg3 and suberic acid (N-hydroxysuccinimide ester), while boron (B) is derived from
  • the reducing agent used is sodium borohydride; when a phosphate bis-titanate coupling agent is used, the Si of the drug will become Ti, and an element P will be added at the same time.
  • the FT-IR spectrum Fig.
  • the cellular internalization process plays a key role in drug utilization.
  • FIG. 3 is a multimodal imaging functional characterization of nanoparticles and nanomedicines prepared by Example 2.
  • FIG. 3A for the magnetic heterostructure nanoparticles containing noble metal components, they have strong surface plasmon resonance (LSPR) scattering light characteristics. Although they are only about 5 nm, they have 400- With an optical size of 600 nanometers, images of individual nanoparticles can still be observed by optical microscopy (Fig. 3A: 18, 20) and can be characterized by surface plasmon spectroscopy for the optical scattering properties of individual nanoparticles (Fig. 3A: 19, 21) ). After surface modification and drug coupling, although the dielectric constant of the surface has changed, the LSPR scattered light intensity remains strong (Fig.
  • LSPR surface plasmon resonance
  • Figure 3C is the magnetic resonance imaging of the pure Au@CoFeB nanoparticles prepared by Example 2 under T2WI experimental conditions - the echo sequence is the echo (TE) duration is 33ms, and the pulse repetition time interval (TR) is 2500ms. As a result, it has a good T2WI enhancement effect, and its relaxation rate (T 2 -1 , s -1 ) has a good linear relationship with the nanoparticle concentration.
  • Figure 3D is the MRI results of the synthesized pure Au@CoFeB-Rg3 nanomedicines under T2WI experimental conditions - echo sequence with echo (TE) time is 33ms, pulse repetition time interval (TR) is 2500ms, and nanoparticles Similarly, it has a good T2WI enhancement effect, and its relaxation rate (T 2 -1 , s -1 ) has a good linear relationship with the concentration of nanoparticles. higher, indicating that the metal-organic composite is beneficial to further enhance its molecular image enhancement effect.
  • TE echo sequence with echo
  • TR pulse repetition time interval
  • Fig. 3E The upper image of Fig. 3E is the computer-aided tomography (CT) image and three Concentration-dependent curve of the point-averaged signal (Hinz units: HU) (curve in the figure). It can be seen that it has excellent CT imaging effect; at the same time, in a large concentration range, its CT value (HU is a measurement unit for measuring the density of a certain local tissue or organ in the human body: air is -1000, dense bone is 1000 ) and concentration have an excellent linear relationship, which can be used to study pathology and pharmacology online with high spatial and temporal resolution by molecular imaging tracing method.
  • CT computer-aided tomography
  • the human chronic myeloid leukemia K562 cell line and the hepatoma cell line were used as the pathological models of cancer cells, and the human epithelial fibroblast cell line 3T3 and the human immune cell JurkatT cell line were used as normal healthy cells by cell biology methods. model, the antitumor effects and toxic side effects on normal cells of the synthesized nanomedicines were studied.
  • Phosphate PBS buffers for cell culture with different concentrations of Au@CoFeB and Au@CoFeB-Rg3 were prepared, and the BD LSRFortessa TM cell analyzer (BD biosciences) used light scattering to detect various cells in different Au@CoFeB and Au@CoFeB - 24-hour viability of various cells after 24-hour incubation at Rg3 concentration.
  • Figure 4A shows the effects of Au@CoFeB (concentration from 0.00001 ⁇ g/mL to 500 ⁇ g/mL) and Au@CoFeB-Rg3 (concentration from 0.00001 ⁇ g/mL to 1000 ⁇ g/mL) prepared in Example 1 on normal cells (3T3: 4A-23, 4B-27), T cells (Jurkat: 4A-22, 4B-26), leukemia cells K562 (4A-24, 4B-28) and solid tumor hepatoma cells (HEP-G2/C3A) (4A- 25, 4B-29) effects of toxicity.
  • the 24-hour cytotoxicity assay showed (4A and 4B) that the Au@CoFeB-Rg3 nanomedicine (Fig.
  • Example 4B prepared in Example 1 was toxic to K562 cells (4B-28) and HEP-G2/C3A (4B-29) cells Significantly higher than that of Au@CoFeB (4A: 24, 25), and also higher than the lethality of pure Rg3 on liver cancer cells and blood cancer cells, indicating that there is an obvious synergistic anti-tumor effect between heterostructured metal nanoparticles and organic drugs .
  • the concentration of Au@CoFeB is lower than 250 ⁇ g/mL, the toxicity to the investigated cells is very low, indicating that this concentration can be used as an optical biomolecular nanoprobe.
  • the concentration of Au@CoFeB-Rg3 is 95 ⁇ g/mL, it has a strong lethality to K562 cells; it has no obvious toxicity to other cells (cell survival>70%) when the concentration does not exceed this concentration.
  • the concentration of Au@CoFeB-Rg3 exceeds 200 ⁇ g/mL, its lethality to liver cancer cells and blood cancer cells is greatly increased; although this concentration is also significantly toxic to Jurkat cells, the toxicity to 3T3 is significantly smaller than that of cancer cells.
  • the human chronic myeloid leukemia K562 cell line and the hepatoma cell line were used as the pathological models of cancer cells, and the human epithelial fibroblast cell line 3T3 and the human immune cell JurkatT cell line were used as normal healthy cells by cell biology methods. model, the antitumor effects and toxic side effects on normal cells of the synthesized nanomedicines were studied.
  • PBS buffers for cell culture with different concentrations of Au@CoFeB and Au@CoFeB-Rg3 were prepared, and a BD LSRFortessa TM cell analyzer (purchased from BD biosciences, BD biosciences) was used to detect the presence of various cells in different Au@CoFeB by light scattering method. and Au@CoFeB-Rg3 concentrations, the 24-hour viability of various cells after 24-hour incubation.
  • the concentrations of Au@CoFeB prepared in Example 1 were 2500 ⁇ g/mL, 10000 ⁇ g/mL, and 100000 ⁇ g/mL, respectively; the concentrations of Au@CoFeB-Rg3 were 2500 ⁇ g/mL, 20000 ⁇ g/mL, and 100000 ⁇ g/mL. Detecting their toxicity to leukemia cells K562 cells and hepatoma cells HEP-G2/C3A, the 24-hour cell viability was zero and 100% was killed.
  • the buffer was 10% fetal bovine serum FBS buffer, and the concentrations of Au@CoFeB in the buffer were selected as 0.00001 ⁇ g/ml, 9.5 ⁇ g/ml, 47.0 ⁇ g/ml, 95.0 ⁇ g/ml and 474.0 ⁇ g/ml; Au @CoFe-Rg3 was selected as 0.00001 ⁇ g/ml, 19 ⁇ g/ml, 95.0 ⁇ g/ml, 190.0 ⁇ g/ml and 950.0 ⁇ g/ml.
  • the proliferation was detected after 4 days of incubation in a cell incubator (3% CO 2 , 310K) together.
  • the cell proliferation rate of the cells was detected.
  • the buffer is 10% fetal bovine serum FBS buffer.
  • concentrations of Au@CoFeB used this time are 2500 ⁇ g/mL, 10000, 100000 ⁇ g/mL; Au@CoFeB-Rg3 concentrations are 5000 ⁇ g/mL, 20000 ⁇ g/mL, 100000 ⁇ g /mL.
  • the proliferation was detected after 4 days of incubation in a cell incubator (3% CO 2 , 310K) together.
  • the Au@CoFeB nanoparticles and Au@CoFeB-Rg3 nanomedicines and other nanomedicines (Fe@Fe 3 O 4 -Rg3, FePt@Fe 3 O 4 -Rg3) prepared by the process of Example 4 were investigated at two concentrations ) on the survival rate of human chronic myeloid leukemia cells K562 cancer cells incubated for 24 hours.
  • the drug concentration is 95-190 ⁇ g/mL
  • grid columns the drug concentration is 474-947 ⁇ g/mL.
  • An orthotopic liver cancer model was established in the liver of nude mice by dimethylnitrosamine.
  • the drug is given at about 70 mg/kg every two days, and the drug is stopped after 5 consecutive times. Then the mice are raised, and the mice are weighed every 7 days and analyzed by fluorescence at the tumor. The relative size of the tumor was measured.
  • There are four groups in total one is the normal saline group as a control, the other three groups are the pure Rg3 group, the pure Au@CoFeB prepared by Example 5 and the nano-drug Au@CoFeB-Rg3 group. After day 21, all mice were sacrificed and dissected, and the liver and its tumor were dissected and analyzed for various biochemical and pathological sections.
  • Figure 7A is the tumor image of the final mice treated with different drugs and control groups
  • Figure 7B is the fluorescence image of the tumor size of the mice characterized by fluorescence method at different time points
  • Figure 7C is the measured tumor size of different groups of mice. Absolute (upper line) and relative body weight change (lower line) at different time points after drug administration (saline: 38; Rg3: 39; Au@CoFe: 40; Au@CoFeB-Rg3: 41).
  • Figure 7D shows the tumor size of different groups at different time points after administration according to the fluorescence value (saline: 42; Rg3: 43; Au@CoFe: 44; Au@CoFeB-Rg3: 45).
  • the tumor of the pure Rg3 group (43) is slightly smaller than that of the control group (42), while the tumor of Au@CoFeB (44) is much smaller than that of the control group, indicating that Au@CoFeB itself has a strong anti-tumor effect.
  • Hepatocellular carcinoma effect the strongest inhibitory effect on mice tumor is the group of nano-drugs compounded by Au@CoFeB and Rg3.

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Abstract

Disclosed in the present invention is a metal-organic composite nano-drug, comprising a heterostructural metal-based nanoparticle treated by a first surface modifier and a cross-linking agent in sequence, and a pharmaceutical ingredient compound treated by a second surface modifier. The nanoparticle is coupled with the pharmaceutical ingredient compound by means of the cross-linking agent, the pharmaceutical ingredient compound is ginsenoside, and the nanoparticle is Au@CoFeB. Further disclosed in the present invention are preparation and application of the nano-drug. The nano-drug provided by the present invention can well inhibit the development of liver cancer, and has an in-vitro and in-vivo molecular image tracing function.

Description

一种金属-有机复合纳米药物及其制备方法和应用A metal-organic composite nanomedicine and its preparation method and application 技术领域technical field
本发明涉及纳米药物技术领域，具体涉及一种金属-有机复合纳米药物及其制备方法和应用。The invention relates to the technical field of nano-medicine, in particular to a metal-organic composite nano-medicine and a preparation method and application thereof.
背景技术Background technique
纳米药物具有尺寸可控、大的质量比和独特的物理化学特性，因此可在分子水平上用于生物***的监测、控制、诊断和治疗以及组织修复(Tekade,R.；Maheshwari,R.；Soni,N.；Tekade,M.；Chougule,M.,Nanotechnology-Based Approaches for Targeting and Delivery of Drugs and Genes.Elsevier,Amsterdam,2017,pp 3-61.)。近年来，合成纳米药物的主要方法集中在以下几个方面：1)通过提高分析方法的敏感性和综合性来获得连贯结果的诊断(Alhareth,K.；Sancey,L.；Tsapis,N.；Mignet,N.,How should we plan the future of nanomedicine for cancer diagnosis and therapy？International Journal of pHarmaceutics 2017,532,(2),657-659)；2)携带生物活性剂的药物输送(Peng,F.；Zhang,W.；Qiu,F.,Self-assembling Peptides in Current Nanomedicine:Versatile Nanomaterials for Drug Delivery.Cur-rent Medicinal Chemistry 2019,26,(1)，1-26.)；3)通过组织工程和植入物以克服与血管移植物有关的局限性(Hu,E.；Bayindir-Buchhalter,I.；Goebel,U.,Nanomedicine,Biofab-rication,Tissue Engineering and Much More-Advanced Healthcare Materials Welcomes2019.Advanced Healthcare Mate-rials 2019,8,(1),1801576.)；和4)生物利用度提高和医疗设备。此外，已经使用了广泛的纳米材料来制备纳米药物，包括纳米悬浮液、聚合物纳米颗粒、氧化铁、金属纳米颗粒、树枝状聚合物和脂质体等。因此，纳米药物改善并扩展了一系列药物分子的药代动力学、溶解性和稳定性，这些分子已广泛用于各种生物医学应用中，包括特定的药物输送、治疗、成像和诊断。然而，纳米药物的使用仍然具有对人类健康的众多不清楚的影响以及需要克服的各种生物障碍的特征，包括稳定性、表面修饰和功能化、多模式功能，有效的药物递送以及两者之间的平衡和副作用(Cencini,E.；Sicuranza,A.；Fabbri,A.；Ferrigno,I.；Rigacci,L.；Cox,M.C.；Raspadori,D.；Bocchia,M.,Study of gene polymorpHisms as predictors of treatment efficacy and toxicity in patients with indo-lent non‐hodgkin lympHomas and mantle cell lympHoma receiv-ing bendamustine and rituximab.British Journal of Haematology 2019,184,(2),223-231.)。Nanomedicines have controllable size, large mass ratio, and unique physicochemical properties, so they can be used at the molecular level for monitoring, control, diagnosis and therapy of biological systems, and tissue repair (Tekade, R.; Maheshwari, R.; Soni, N.; Tekade, M.; Chougule, M., Nanotechnology-Based Approaches for Targeting and Delivery of Drugs and Genes. Elsevier, Amsterdam, 2017, pp 3-61.). In recent years, the main methods for synthesizing nanomedicines have focused on the following aspects: 1) Diagnosis to obtain coherent results by improving the sensitivity and comprehensiveness of analytical methods (Alhareth, K.; Sancey, L.; Tsapis, N.; Mignet, N., How should we plan the future of nanomedicine for cancer diagnosis and therapy? International Journal of pHarmaceutics 2017, 532, (2), 657-659); 2) Drug delivery with bioactive agents (Peng, F. ; Zhang, W.; Qiu, F., Self-assembling Peptides in Current Nanomedicine: Versatile Nanomaterials for Drug Delivery. Cur-rent Medicinal Chemistry 2019, 26, (1), 1-26.); 3) By tissue engineering and Implants to overcome limitations associated with vascular grafts (Hu, E.; Bayindir-Buchhalter, I.; Goebel, U., Nanomedicine, Biofab-rication, Tissue Engineering and Much More-Advanced Healthcare Materials Welcomes 2019. Advanced Healthcare Mate-rials 2019, 8, (1), 1801576.); and 4) Bioavailability enhancement and medical devices. In addition, a wide range of nanomaterials have been used to prepare nanomedicines, including nanosuspensions, polymer nanoparticles, iron oxides, metal nanoparticles, dendrimers, and liposomes, among others. As a result, nanomedicines improve and extend the pharmacokinetics, solubility, and stability of a range of drug molecules that have been used in a wide variety of biomedical applications, including specific drug delivery, therapy, imaging, and diagnostics. However, the use of nanomedicines is still characterized by numerous unclear effects on human health and various biological barriers to overcome, including stability, surface modification and functionalization, multimodal functionality, efficient drug delivery, and both. The balance between and side effects (Cencini, E.; Sicuranza, A.; Fabbri, A.; Ferrigno, I.; Rigacci, L.; Cox, M.C.; Raspadori, D.; Bocchia, M., Study of gene polymorpHisms as predictors of treatment efficacy and toxicity in patients with indo-lent non‐hodgkin lympHomas and mantle cell lympHoma receiv-ing bendamustine and rituximab.British Journal of Haematology 2019,184,(2),223-231.).
由贵金属和磁性成分组成的纳米杂化物由于其独特的理化性质、高稳定性和优异的应用前景，有望在包括能量转换(Wang,S.；Niu,S.Y.；Li,H.S.；Lam,K.K.；Wang,Z.R.；Du,P.Y.；Leung,C.W.；Qu,S.X.,Synthesis and controlled morpHology of Ni@Ag core shell nanowires with excellent catalytic efficiency and recyclability.Nanotechnology 2019,30,(38),9.)、医学成像(S.D.；Gwenin,V.V.；Gwenin,C.D.,Magnetic Functional-ized Nanoparticles for Biomedical,Drug Delivery and Imaging Applications.Nanoscale Research Letters.2019,14,(1),188.)和疾病检测在内的广泛领域中应用。此外，对这些纳米颗粒的表面进行适当的修饰和/或与某些药物或有机成分的结合可导致宽广的治疗窗和临床应用，而对健康细胞和/或组织的副作用则没有或明显减少。这些改变使某些混合纳米颗粒具有诊断，成像，药物输送，治疗甚至合成疫苗开发等吸引人的特征，因而可作为多种疾病的有效化学治疗药物(Chen,H.；Liu,F.；Lei,Z.；Ma,L.；Wang,Z.,Fe2O3@Au Core@Shell Nanoparticle-GrapHene Nanocomposites as Theranostic Agents for Bioimaging and Chemo-pHotothermal Synergistic Therapy.RSC Advances 2015,5,(103),84980-84987.)。因此，多模式纳米药物的可控制备和功能化将其跨学科应用扩展到基础研究和临床应用(例如，超灵敏生物探针，高效纳米药物和纳米酶，超灵敏的生物医学分子成像和某些疾病的诊断等)。Due to their unique physicochemical properties, high stability and excellent application prospects, nanohybrids composed of noble metals and magnetic components are expected to be used in energy conversion including energy conversion (Wang, S.; Niu, S.Y.; Li, H.S.; Lam, K.K.; Wang , Z.R.; Du, P.Y.; Leung, C.W.; Qu, S.X., Synthesis and controlled morpHology of Ni@Ag core shell nanowires with excellent catalytic efficiency and recyclability. Nanotechnology 2019,30,(38),9.), Medical Imaging (S.D. Gwenin, V.V.; Gwenin, C.D., Magnetic Functional-ized Nanoparticles for Biomedical, Drug Delivery and Imaging Applications. Nanoscale Research Letters. 2019, 14, (1), 188.) and a wide range of applications including disease detection. Furthermore, proper modification of the surface of these nanoparticles and/or incorporation of certain drugs or organic components can lead to a broad therapeutic window and clinical application with no or significantly reduced side effects on healthy cells and/or tissues. These alterations enable some hybrid nanoparticles to have attractive features for diagnosis, imaging, drug delivery, therapy, and even the development of synthetic vaccines, thus serving as effective chemotherapeutic drugs for a variety of diseases (Chen, H.; Liu, F.; Lei , Z.; Ma, L.; Wang, Z., Fe2O3@Au Core@Shell Nanoparticle-GrapHene Nanocomposites as Theranostic Agents for Bioimaging and Chemo-pHotothermal Synergistic Therapy.RSC Advances 2015,5,(103),84980-84987. ). Therefore, the controllable preparation and functionalization of multimodal nanomedicines extend their interdisciplinary applications to basic research and clinical applications (e.g., ultrasensitive bioprobes, highly efficient nanomedicines and nanozymes, ultrasensitive biomedical molecular imaging and certain diagnosis of certain diseases, etc.).
现在的纳米药物要么是将原有药物简单纳米化，特别是对以一些有机物或高分子类药物，纳米化除了提高其分散性外，其他性能，尤其是疗效提高微乎其微，而毒副作用反而会变得更大，这些简单纳米化的药物不存在像金属基纳米材料在纳米尺度下的对疗效起重要作用的量子效应和表面活化效应。而纯的无机纳米药物，如无机非金属类，像二氧化硅等，其基本充当载体作用，而对纯金属，如金等可能还是癌细胞逃逸转移的很好的运载工具，同时也具有很大的副作用。另外，无论是无机非类、有机聚合物类还是金属类的纳米颗粒，目前的粒径都很大，各个组份基本都大于10nm以上，其进入人体后，很难被人体各器官或组织的表皮网络细胞以及肾小管等给清除掉，存在永久滞留人体的潜在危害。对于无机非金属和金属类纳米颗粒，其组成部分尺寸最好都小于6nm，但目前基本没有此类纳米材料的可量产方法，一次价格非常昂贵，病人一时无法接受。而有机聚合物类纳米药物，如果持续分解，还存在分解物(特别是产生的活性官能团)对正常组织和器官的损害以及癌变作用。另外，对于疗效的机理研究，需要能够进行离体和活体密切对照的具有示踪影像功能的纳米药物，目前基本全靠具有寿命短、易光敏化分解的荧光剂标记。特别是对有机类纳米药物，基本无法长时间稳定地进行分子影像学研究其药代动力学和药效机理。因此迫切需要能够同时具有离体和活体分子影像示踪功能的纳米药物。The current nano-drugs are either simple nano-drugs of the original drugs, especially for some organic or macromolecular drugs. In addition to improving their dispersibility, nano-drugs have little improvement in other properties, especially the curative effect, while the toxic and side effects will change. Even larger, these simply nanosized drugs do not have quantum effects and surface activation effects that play an important role in therapeutic efficacy at the nanoscale like metal-based nanomaterials. Pure inorganic nano-drugs, such as inorganic non-metals, like silica, etc., basically act as carriers, while pure metals, such as gold, may be a good carrier for cancer cells to escape and metastasize. big side effects. In addition, whether it is inorganic non-type, organic polymer or metal nanoparticles, the current particle size is very large, and each component is basically larger than 10nm. Epidermal network cells and renal tubules are removed, and there is a potential hazard of permanent retention in the human body. For inorganic non-metallic and metal-based nanoparticles, the size of their components is preferably less than 6nm, but there is basically no mass production method for such nanomaterials, and the one-time price is very expensive, which is temporarily unacceptable to patients. On the other hand, if the organic polymer nano-drugs are continuously decomposed, there will also be damage to normal tissues and organs and carcinogenesis of the decomposed products (especially the generated active functional groups). In addition, for the mechanism research of curative effect, nano-drugs with tracer imaging function that can be closely compared in vitro and in vivo are required. At present, it basically depends on the fluorescent agent labeling with short lifespan and easy photosensitization and decomposition. Especially for organic nano-drugs, it is basically impossible to conduct molecular imaging studies stably for a long time to study their pharmacokinetics and pharmacodynamic mechanisms. Therefore, there is an urgent need for nanomedicines that can simultaneously trace in vitro and in vivo molecular imaging.
发明内容SUMMARY OF THE INVENTION
为解决现有技术中存在的问题，本发明的一个目的在于提供一种金属-有机复合纳米药物，包含依次经第一表面改性剂和活化剂处理的纳米颗粒以及经第二表面改性剂处理的药物成分化合物；所述纳米颗粒通过交联剂与所述药物成分化合物偶联，其中所述药物成分化合物为人参皂苷，所述纳米颗粒为Au@CoFeB。In order to solve the problems existing in the prior art, one object of the present invention is to provide a metal-organic composite nanomedicine, comprising nanoparticles treated with a first surface modifier and an activator in turn and a second surface modifier. The treated pharmaceutical ingredient compound; the nanoparticle is coupled with the pharmaceutical ingredient compound through a cross-linking agent, wherein the pharmaceutical ingredient compound is ginsenoside, and the nanoparticle is Au@CoFeB.
根据本发明的纳米药物，其中所述第一表面改性剂选自3-氨丙基三甲氧基硅氧烷、乙烯基三甲氧基硅烷、磷酸酯双钛酸酯偶联剂和二异丙氧基二乙酰丙酮钛酸酯中的至少一种；优选为3-氨丙基三甲氧基硅氧烷。According to the nanomedicine of the present invention, wherein the first surface modifier is selected from 3-aminopropyltrimethoxysiloxane, vinyltrimethoxysilane, phosphate bis-titanate coupling agent and diisopropyl At least one of oxydiacetylacetonate titanate; preferably 3-aminopropyltrimethoxysiloxane.
根据本发明的纳米药物，所述第二表面改性剂选自3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂、十七氟癸基三甲基氧基硅烷和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种；优选为3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种。According to the nanomedicine of the present invention, the second surface modifier is selected from the group consisting of 3-aminopropyltrimethoxysiloxane, 3-mercaptopropyl-triethoxysilane coupling agent, heptadecafluorodecyl triglyceride At least one of methyloxysilane and isopropyl tris (dioctyl pyrophosphate acyloxy) titanate; preferably 3-aminopropyl trimethoxy siloxane, 3-mercaptopropyl-tri At least one of an ethoxysilane coupling agent and isopropyl tris(dioctyl pyrophosphate acyloxy) titanate.
根据本发明的纳米药物，其中所述交联剂选自辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、琥珀酰-亚胺丁二酸酯聚、乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种；优选为辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、聚乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种。According to the nanomedicine of the present invention, wherein the crosslinking agent is selected from suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (succinate N-hydroxysuccinimide ester), polyethylene glycol At least one of alcohol disuccinimidyl succinate, succinyl-imide succinate poly, ethylene glycol succinyl-imide succinate and aziridine crosslinking agent XR-100; preferably Suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (N-hydroxysuccinimide ester of succinate), polyethylene glycol disuccinimidyl succinate, polyethylene glycol At least one of succinyl-imide succinate and aziridine crosslinker XR-100.
根据本发明的纳米药物，在一种具体实施方案中，所述纳米药物采用包含以下步骤的方法制备：According to the nanomedicine of the present invention, in a specific embodiment, the nanomedicine is prepared by a method comprising the following steps:
(1)制备Au@CoFeB纳米颗粒；(1) Preparation of Au@CoFeB nanoparticles;
(2)纳米颗粒的表面改性：(2) Surface modification of nanoparticles:
a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌，a) adding the nanoparticles in (1) to the first organic solution containing the first surface modifier and stirring,
b)将a)中的加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥，得到表面改性的纳米颗粒；b) ultrasonically cleaning, centrifuging and drying the first organic solvent solution with nanoparticles in a) to obtain surface-modified nanoparticles;
(3)纳米颗粒的偶联活化：(3) Coupling activation of nanoparticles:
a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中，搅拌后进行孵化，a) dispersing the surface-modified nanoparticles and cross-linking agent obtained in (2) into the second organic solvent, respectively, and incubating after stirring,
b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥，得到活化后的纳米颗粒；b) centrifuging and drying the solution added with the surface-modified nanoparticles and cross-linking agent in a) to obtain activated nanoparticles;
(4)纳米颗粒的pH调控：(4) pH regulation of nanoparticles:
将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值，随后经离心-超声清洗，得到经表面改性和活化的纳米颗粒；Dispersing the activated nanoparticles obtained in (3) into a buffer to adjust the pH value, and then performing centrifugation-ultrasonic cleaning to obtain surface-modified and activated nanoparticles;
(5)人参皂苷的表面改性：(5) Surface modification of ginsenosides:
a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌；a) adding ginsenoside to the third organic solution containing the second surface modifier and stirring;
b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥，得到表面改性的人参皂苷；b) ultrasonically cleaning, centrifuging and drying the third organic solvent solution added with ginsenosides in a) to obtain surface-modified ginsenosides;
(6)纳米颗粒和人参皂苷的偶联：(6) Coupling of nanoparticles and ginsenosides:
a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化；a) putting the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) into a fourth organic solvent for incubation;
b)将a)中的加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理，得到复合纳米药物。b) The fourth organic solvent solution in a) with the surface-modified and activated nanoparticles and the surface-modified ginsenosides is subjected to centrifugation-ultrasonic cleaning-centrifugation and then drying to obtain a composite nanomedicine.
根据本发明的纳米药物，其中所述纳米颗粒为核壳结构，核壳结构的金属内核为面心立方晶体结构的Au；核壳结构的壳层为面心立方晶体结构的CoFeB；According to the nanomedicine of the present invention, wherein the nanoparticle has a core-shell structure, the metal core of the core-shell structure is Au with a face-centered cubic crystal structure; the shell layer of the core-shell structure is CoFeB with a face-centered cubic crystal structure;
所述纳米药物的整体结构为6-7.2nm的超小纳米药物单元偶联在一起构建的尺寸为250-350nm的纳米药物聚集体；纳米颗粒的动力学半径约100-200nm；The overall structure of the nano-drug is a nano-drug aggregate with a size of 250-350 nm constructed by coupling together ultra-small nano-drug units of 6-7.2 nm; the dynamic radius of the nano-particle is about 100-200 nm;
所述纳米颗粒表面带有+7-12mV；所述纳米药物表面带有+25-30mV的正电势。The nanoparticle surface has +7-12mV; the nanomedicine surface has a positive potential of +25-30mV.
根据本发明的纳米药物，其中第一有机溶剂选自苯、甲苯、对二甲苯和间二甲苯中的至少一种；优选为甲苯、对二甲苯和间二甲苯中的至少一种。According to the nanomedicine of the present invention, wherein the first organic solvent is selected from at least one of benzene, toluene, para-xylene and meta-xylene; preferably at least one of toluene, para-xylene and meta-xylene.
第二有机溶剂选自对二甲基亚砜、N-甲基甲酰胺、N-甲基-2-吡咯烷酮、二甲苯和邻二甲苯中的至少一种；优选为二甲基亚砜、N-甲基甲酰胺和N-甲基-2-吡咯烷酮中的至少一种。The second organic solvent is selected from at least one of p-dimethyl sulfoxide, N-methylformamide, N-methyl-2-pyrrolidone, xylene and o-xylene; preferably dimethyl sulfoxide, N -At least one of methylformamide and N-methyl-2-pyrrolidone.
第三有机溶剂选自对二甲苯、邻二甲苯和甲苯中的至少一种；优选为甲苯。The third organic solvent is selected from at least one of para-xylene, ortho-xylene and toluene; preferably toluene.
第四有机溶剂选自二甲亚砜、N-甲基吡咯烷酮、N-乙基乙酰胺和N-甲基甲酰胺中的至少一种；优选为二甲亚砜、N-甲基吡咯烷酮和N-乙基乙酰胺中的至少一种。The fourth organic solvent is selected from at least one of dimethyl sulfoxide, N-methylpyrrolidone, N-ethylacetamide and N-methylformamide; preferably dimethylsulfoxide, N-methylpyrrolidone and N -At least one of ethylacetamide.
根据本发明的纳米药物，步骤(4)中所用缓冲液可以为基于本领域技术人员所公知的缓冲液，例如所述缓冲液可以为磷酸盐PBS缓冲液、柠檬酸–Na 2HPO 4缓冲液、Trizma缓冲液、胎牛血清FBS缓冲液、碳酸钠-碳酸氢钠缓冲溶液、和醋酸钠-醋酸缓冲液中的至少一种，优选为磷酸盐缓冲液、胎牛血清FBS缓冲液和柠檬酸-Na 2HPO 4缓冲液中的至少一种，进一步优选为磷酸盐PBS缓冲液。 According to the nanomedicine of the present invention, the buffer used in step (4) can be a buffer known to those skilled in the art, for example, the buffer can be phosphate PBS buffer, citric acid-Na 2 HPO 4 buffer , Trizma buffer, fetal bovine serum FBS buffer, sodium carbonate-sodium bicarbonate buffer, and at least one of sodium acetate-acetate buffer, preferably phosphate buffer, fetal bovine serum FBS buffer and citric acid -At least one of Na 2 HPO 4 buffers, more preferably phosphate PBS buffers.
本发明的另一个目的在于提供一种复合纳米药物的制备方法，包括以下步骤：Another object of the present invention is to provide a method for preparing a composite nanomedicine, comprising the following steps:
(1)制备Au@CoFeB纳米颗粒；(1) Preparation of Au@CoFeB nanoparticles;
(2)纳米颗粒的表面改性：(2) Surface modification of nanoparticles:
a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌，a) adding the nanoparticles in (1) to the first organic solution containing the first surface modifier and stirring,
b)将a)中的加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥，得到表面改性的纳米颗粒；b) ultrasonically cleaning, centrifuging and drying the first organic solvent solution with nanoparticles in a) to obtain surface-modified nanoparticles;
(3)纳米颗粒的偶联活化：(3) Coupling activation of nanoparticles:
a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中，搅拌后进行孵化，a) dispersing the surface-modified nanoparticles and cross-linking agent obtained in (2) into the second organic solvent, respectively, and incubating after stirring,
b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥，得到活化后的纳米颗粒；b) centrifuging and drying the solution added with the surface-modified nanoparticles and cross-linking agent in a) to obtain activated nanoparticles;
(4)纳米颗粒的pH调控：(4) pH regulation of nanoparticles:
将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值，随后经离心-超声清洗，得到经表面改性和活化的纳米颗粒；Dispersing the activated nanoparticles obtained in (3) into a buffer to adjust the pH value, and then performing centrifugation-ultrasonic cleaning to obtain surface-modified and activated nanoparticles;
(5)人参皂苷的表面改性：(5) Surface modification of ginsenosides:
a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌；a) adding ginsenoside to the third organic solution containing the second surface modifier and stirring;
b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥，得到表面改性的人参皂苷；b) ultrasonically cleaning, centrifuging and drying the third organic solvent solution added with ginsenosides in a) to obtain surface-modified ginsenosides;
(6)纳米颗粒和人参皂苷的偶联：(6) Coupling of nanoparticles and ginsenosides:
a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化；a) putting the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) into a fourth organic solvent for incubation;
b)将a)中的加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理，得到复合纳米药物。b) The fourth organic solvent solution in a) with the surface-modified and activated nanoparticles and the surface-modified ginsenosides is subjected to centrifugation-ultrasonic cleaning-centrifugation and then drying to obtain a composite nanomedicine.
作为一种优选方案，步骤(1)中，Au@CoFeB的纳米颗粒采用微流控法、水热法、磁控溅射法、电沉积法制备；进一步优选为微流控法。As a preferred solution, in step (1), the nanoparticles of Au@CoFeB are prepared by microfluidic method, hydrothermal method, magnetron sputtering method, and electrodeposition method; more preferably, microfluidic method is used.
具体而言，所述微流控法就是使用微米尺度(亚毫米)构建的连续流反应工艺，将合成纳米材料或药物时的反应物的混合、反应成核、纳米颗粒或药物生长和生长终止过程控制在μL到pL甚至更小的反应体积内。该方法和传统的釜式反应器相比，具有精确设计和调控反应不同阶段的动力学参数、快速的物质和能量交换、混合和反应均匀、可以平行放大作业。无釜式反应器不可避免的放大效应、环境友好、安全和废物最小化、可充分利用微流道的高比表面积效应对反应产物进行调控的特点。微流控法制备纳米颗粒的方法可以 参考现有技术，在此不再赘述。Specifically, the microfluidic method is a continuous flow reaction process constructed at a micrometer scale (sub-millimeter), and the mixing, reaction nucleation, nanoparticle or drug growth and growth termination of the reactants during the synthesis of nanomaterials or drugs Processes are controlled in reaction volumes from μL to pL or even smaller. Compared with the traditional tank reactor, the method has the advantages of precise design and regulation of kinetic parameters in different stages of the reaction, rapid material and energy exchange, uniform mixing and reaction, and parallel scale-up operations. The tankless reactor has the unavoidable amplification effect, environmental friendliness, safety and waste minimization, and can make full use of the high specific surface area effect of the microfluidic channel to control the reaction products. For the method of preparing nanoparticles by microfluidic method, reference may be made to the prior art, which will not be repeated here.
作为一种优选方案，步骤(2)-a)中,搅拌时间优选为20-28小时；步骤(2)-b)中,优选离心的条件包括：离心转速为10000-20000rpm；离心时间为5-40分钟；优选为12000-16000rpm；离心时间为10-30分钟。As a preferred solution, in step (2)-a), the stirring time is preferably 20-28 hours; in step (2)-b), the preferred centrifugal conditions include: the centrifugal rotation speed is 10000-20000rpm; the centrifugal time is 5 -40 minutes; preferably 12000-16000 rpm; centrifugation time 10-30 minutes.
作为一种优选方案，步骤(3)-a)中,搅拌时间优选为1-3小时；步骤(3)-b)中,优选离心的条件包括；离心转速为10000-20000rpm；离心时间为5-15分钟。As a preferred solution, in step (3)-a), the stirring time is preferably 1-3 hours; in step (3)-b), the preferred centrifugation conditions include; the centrifugal speed is 10000-20000rpm; the centrifugal time is 5 -15 minutes.
作为一种优选方案，步骤(4)中,将pH值调节为7-7.8；优选离心的条件包括；离心转速为10000-14000rpm；离心时间为5-15分钟。As a preferred solution, in step (4), the pH value is adjusted to 7-7.8; the preferred centrifugation conditions include; the centrifugation speed is 10000-14000rpm; and the centrifugation time is 5-15 minutes.
作为一种优选方案，步骤(5)-a)中,搅拌时间优选为20-28小时；优选离心的条件包括；离心转速为10000-14000rpm；离心时间为5-15分钟。As a preferred solution, in step (5)-a), the stirring time is preferably 20-28 hours; the preferred centrifugation conditions include; the centrifugal speed is 10000-14000rpm; the centrifugation time is 5-15 minutes.
作为一种优选方案，步骤(6)-a)中,孵化时间优选为1.5-2.0小时；步骤(6)-a)中，所述第四有机溶剂优选为二甲基亚砜。As a preferred solution, in step (6)-a), the incubation time is preferably 1.5-2.0 hours; in step (6)-a), the fourth organic solvent is preferably dimethyl sulfoxide.
本发明的另一个目的在于提供本发明的复合纳米药物在制备治疗肝癌药物中的应用。Another object of the present invention is to provide the application of the composite nanomedicine of the present invention in the preparation of a medicine for treating liver cancer.
根据本发明的应用，其中复合纳米药物中的Au@CoFeB的浓度为0.00001-100000μg/mL。According to the application of the present invention, the concentration of Au@CoFeB in the composite nanomedicine is 0.00001-100000 μg/mL.
本发明提供的复合纳米药物可以充分利用无机，特别是金属基纳米颗粒的多模态和表面数层原子层的活性和量子效应产生的疗效，以及有机药物的疗效和对金属内层及生命有机体的保护作用，构建无机-有机复合纳米药物，充分发挥有机药物-无机纳米颗粒的协同效果，获得高疗效、低或无毒副作用的原创型纳米药物，该药物同时具有离体和活体分子影像示踪功能。The composite nanomedicine provided by the invention can make full use of the multimodality of inorganic, especially metal-based nanoparticle, the activity of several atomic layers on the surface and the curative effect produced by quantum effect, as well as the curative effect of organic medicine and the effect on metal inner layer and living organisms Protective effect, construct inorganic-organic composite nano-drugs, give full play to the synergistic effect of organic drugs-inorganic nanoparticles, and obtain original nano-drugs with high efficacy, low or no toxic side effects, and the drugs have both in vitro and in vivo molecular imaging display. tracking function.
附图说明Description of drawings
图1.合成纳米颗粒的微流控装置结构示意图。Figure 1. Schematic diagram of the microfluidic device structure for the synthesis of nanoparticles.
图2A是使用实施例1工艺制备的Au@CoFeB-Rg3纳米药物的广角透射电镜照片，图中右上是尺寸分布图，其下是单个颗粒图像的放大图。图2B-8是是使用实施例1工艺制备的纳米颗粒Au@CoFeB的XRD谱。图2B-9是是使用实施例1工艺制备的纳米颗粒Au@CoFeB-Rg3的XRD谱.图2C-10是是使用实施例1工艺制备的纳米颗粒Au@CoFeB的XPS谱图。图2C-11是纳米药物Au@CoFeB-Rg3的XPS谱图。图2D-12是纳米颗粒Au@CoFeB的FT-IR谱图。图2D-13是纳米药物Au@CoFeB-Rg3的FT-IR谱图。图2E-14是是使用实施例1工艺制备的纳米颗粒Au@CoFeB的水动力学直径分布图。图2E-15是是使用实施例1工艺制备的纳米药物Au@CoFeB-Rg3的水动力学直径分布图。图2F-16是使 用实施例1工艺制备的纳米颗粒Au@CoFeB的表面Zeta电势。图2F-17是是使用实施例1工艺制备的纳米药物Au@CoFeB-Rg3的表面Zeta电势。Figure 2A is a wide-angle transmission electron microscope photograph of the Au@CoFeB-Rg3 nanomedicine prepared by the process of Example 1. The upper right of the figure is the size distribution map, and the lower part is an enlarged image of a single particle image. 2B-8 are XRD spectra of nanoparticle Au@CoFeB prepared using the process of Example 1. Figures 2B-9 are XRD patterns of nanoparticles Au@CoFeB-Rg3 prepared by the process of Example 1. Figures 2C-10 are XPS spectra of nanoparticles Au@CoFeB prepared by the process of Example 1. Figure 2C-11 is the XPS spectrum of the nanodrug Au@CoFeB-Rg3. Figure 2D-12 is the FT-IR spectrum of nanoparticle Au@CoFeB. Figure 2D-13 is the FT-IR spectrum of the nanodrug Au@CoFeB-Rg3. 2E-14 are hydrodynamic diameter distribution diagrams of nanoparticles Au@CoFeB prepared using the process of Example 1. 2E-15 are the hydrodynamic diameter distribution diagrams of the nanomedicine Au@CoFeB-Rg3 prepared by the process of Example 1. Figures 2F-16 are the surface Zeta potentials of Au@CoFeB nanoparticles prepared using the process of Example 1. 2F-17 is the surface Zeta potential of the nano-drug Au@CoFeB-Rg3 prepared by the process of Example 1.
图3A-18是使用实施例2工艺制备的由暗场显微镜拍摄的Au@CoFeB纳米颗粒的表面等离子体散射光图形。图3A-19是使用实施例2工艺制备的由暗场显微光波谱仪表征的Au@CoFeB纳米颗粒表面等离子体散射光波谱。图3B-20是使用实施例2工艺制备的由暗场显微镜拍摄的Au@CoFeB-Rg3纳米药物的表面等离子体散射光图形。图3B-21是使用实施例2工艺制备的由暗场显微光波谱仪表征的Au@CoFeB-Rg3纳米药物的表面等离子体散射光波谱。图3C是使用实施例2工艺制备的Au@CoFeB纳米颗粒的T2WI浓度(0μg/mL、7.5μg/mL、15.1μg/mL、31.3μg/mL、93.5μg/mL、187.0μg/mL)依赖性的磁共振图像(图上部图像)和磁共振弛豫速率(T 2 -1，单位为s -1)的浓度依赖性曲线。图3D是使用实施例2工艺制备的Au@CoFeB-Rg3纳米药物的T2WI浓度(0μg/mL、4.1μg/mL、12.4μg/mL、37.3μg/mL、112.0μg/mL)依赖性的磁共振图像(图上部图像)和磁共振弛豫速率(T 2 -1，单位为s -1)的浓度依赖性曲线。图3E是使用实施例2工艺制备的Au@CoFe(B)-Rg3纳米药物浓度(400μg/mL、800μg/mL、1100μg/mL、1500μg/mL、4000μg/mL)依赖性的计算机辅助X光断层扫描(CT)图像(图上部图像)和三点平均的信号(HU)的浓度依赖曲线。 3A-18 are surface plasmon scattered light patterns of Au@CoFeB nanoparticles prepared using the process of Example 2, photographed by dark field microscopy. 3A-19 are surface plasmon scattering light spectra of Au@CoFeB nanoparticles prepared using the process of Example 2 characterized by a dark-field optical spectrometer. 3B-20 are the surface plasmon scattering light patterns of the Au@CoFeB-Rg3 nanomedicine prepared by the process of Example 2 and photographed by a dark field microscope. 3B-21 are surface plasmon scattering optical spectra of Au@CoFeB-Rg3 nanomedicines prepared by the process of Example 2 and characterized by dark-field microspectroscopy. Figure 3C is the T2WI concentration (0 μg/mL, 7.5 μg/mL, 15.1 μg/mL, 31.3 μg/mL, 93.5 μg/mL, 187.0 μg/mL) dependence of Au@CoFeB nanoparticles prepared by the process of Example 2 The magnetic resonance image (upper image of the figure) and the concentration-dependent curve of the magnetic resonance relaxation rate (T 2 −1 , in s −1 ). Figure 3D shows the T2WI concentration (0 μg/mL, 4.1 μg/mL, 12.4 μg/mL, 37.3 μg/mL, 112.0 μg/mL)-dependent magnetic resonance of the Au@CoFeB-Rg3 nanomedicine prepared by the process of Example 2 Image (upper image of panel) and concentration-dependent curve of magnetic resonance relaxation rate (T 2 -1 in s -1 ). Figure 3E is the computer-aided X-ray tomography of the Au@CoFe(B)-Rg3 nanomedicine prepared by the process of Example 2 depending on the concentration (400 μg/mL, 800 μg/mL, 1100 μg/mL, 1500 μg/mL, 4000 μg/mL) Concentration-dependent curves of scan (CT) images (upper image of the figure) and three-point averaged signal (HU).
图4A是使用实施例1工艺制备的Au@CoFeB纳米颗粒浓度依赖的Jurkat-CT(4A-22)、3T3(4A-23)、K562-CT(4A-24)和HEP-G2/C3A(4A-25)细胞24小时存活率。图4B是使用实施例1工艺制备的Au@CoFeB-Rg3纳米药物浓度依赖的Jurkat-CT(4B-26)、3T3(4B-27)、K562-CT(4B-28)和HEP-G2/C3A(4B-29)细胞24小时存活率。Figure 4A shows the concentration-dependent Au@CoFeB nanoparticles prepared using the process of Example 1 for Jurkat-CT (4A-22), 3T3 (4A-23), K562-CT (4A-24) and HEP-G2/C3A (4A -25) 24 hour cell viability. Figure 4B shows the concentration-dependent Jurkat-CT (4B-26), 3T3 (4B-27), K562-CT (4B-28) and HEP-G2/C3A of Au@CoFeB-Rg3 nanomedicines prepared using the process of Example 1 (4B-29) 24-hour cell viability.
图5A是使用实施例3工艺制备的Au@CoFeB纳米颗粒浓度依赖的Jurkat-CT(5A-30)、3T3(5A-31)、K562-CT(5A-32)和HEP-G2/C3A(5A-33)细胞增殖存活率。图5B是使用实施例3工艺制备的Au@CoFeB-Rg3纳米药物浓度依赖的Jurkat-CT(5B-34)、3T3(5B-35)、K562-CT(5B-36)和HEP-G2/C3A(5B-37)细胞增殖存活率。Figure 5A is the concentration-dependent Au@CoFeB nanoparticles prepared using the process of Example 3 for Jurkat-CT (5A-30), 3T3 (5A-31), K562-CT (5A-32) and HEP-G2/C3A (5A -33) Cell proliferation survival rate. Figure 5B shows the concentration-dependent Jurkat-CT (5B-34), 3T3 (5B-35), K562-CT (5B-36) and HEP-G2/C3A of Au@CoFeB-Rg3 nanomedicines prepared using the process of Example 3 (5B-37) Cell proliferation survival.
图6使用实施例4工艺制备的制备的Au@CoFeB纳米颗粒和Au@CoFeB-Rg3纳米药物和PBS缓冲液以及其它纳米药物(Fe@Fe 3O 4-Rg3、FePt@Fe 3O 4-Rg3)在两种浓度下(斜纹柱，95-190μg/mL；网格柱，474-947μg/mL)对人慢性髓系白血病细胞K562癌细胞孵化24小时的存活率实验结果。 Fig. 6 Prepared Au@CoFeB nanoparticles and Au@CoFeB-Rg3 nanomedicines and PBS buffer and other nanomedicines (Fe@ Fe3O4 - Rg3, FePt@ Fe3O4 -Rg3 prepared by the process of Example 4 ) ) at two concentrations (twill bars, 95-190 μg/mL; grid bars, 474-947 μg/mL) on human chronic myeloid leukemia cells K562 cancer cells incubated for 24 hours of survival experimental results.
图7A是使用实施例5工艺制备的Au@CoFeB-Rg3和Au@CoFeB纳米颗粒抗肝癌疗效的活体动物实验在不同用药方案后将小鼠牺牲后的肝部肿瘤图像。图7B是使用实施例5 工艺制备的Au@CoFeB-Rg3和Au@CoFeB纳米颗粒抗肝癌疗效的活体动物实验在不同用药方案的小鼠在不同阶段处通过生物荧光标记的肝部肿瘤大小荧光图。图7C是使用实施例5工艺制备的Au@CoFeB(7C-40)和Au@CoFeB-Rg3(7C-41)和对照组生理盐水(7C-38)及Rg3(7C-39)抗肝癌疗效的活体动物实验在21天内不同组小鼠的绝对(上部折线)和相对体重变化(下部折线)。图7D是使用实施例5工艺制备的Au@CoFeB纳米颗粒(7D-44)和Au@CoFeB-Rg3(7D-45)和对照组生理盐水(7D-42)及Rg3(7D-43)抗肝癌疗效的活体动物实验在不同药物处理后的肿瘤部位生物荧光强度的定量值。Figure 7A is an image of liver tumor after mice were sacrificed after different drug regimens in the in vivo animal experiment of the anti-liver cancer efficacy of Au@CoFeB-Rg3 and Au@CoFeB nanoparticles prepared by the process of Example 5. FIG. 7B is a fluorescence image of the size of liver tumors labeled by biofluorescence at different stages in the in vivo animal experiment of the anti-liver cancer efficacy of Au@CoFeB-Rg3 and Au@CoFeB nanoparticles prepared by the process of Example 5. . Figure 7C shows the anti-liver cancer efficacy of Au@CoFeB (7C-40) and Au@CoFeB-Rg3 (7C-41) prepared by the process of Example 5, and normal saline (7C-38) and Rg3 (7C-39) in the control group Absolute (upper line) and relative body weight changes (lower line) of different groups of mice during 21 days in live animal experiments. Figure 7D shows the Au@CoFeB nanoparticles (7D-44) and Au@CoFeB-Rg3 (7D-45) prepared by the process of Example 5 and the control group with normal saline (7D-42) and Rg3 (7D-43) against liver cancer Quantitative values of bioluminescence intensity at tumor sites after treatment with different drugs in vivo animal experiments.
附图标记说明Description of reference numerals
1、第一注射泵；2、第二注射泵；3、第一微流道管；4、第二微流道管；5、Y字形反应物料液混合器；6、第三微流道管；7、收集器。1. The first syringe pump; 2. The second syringe pump; 3. The first microfluidic tube; 4. The second microfluidic tube; 5. The Y-shaped reaction material-liquid mixer; 6. The third microfluidic tube 7. Collector.
具体实施方式Detailed ways
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.
纳米颗粒和纳米药物的合成Synthesis of Nanoparticles and Nanomedicines
实施例1：Example 1:
(1)制备Au@CoFeB纳米颗粒：(1) Preparation of Au@CoFeB nanoparticles:
如图1所示，将0.42g聚乙烯吡咯烷酮(PVP，K-30，重均分子量30,000)，0.07g(0.35mmol)的FeCl 2·4H 2O和0.0833g(0.35mmol)的CoCl 2·6H 2O在氮气保护下溶解在50毫升N-甲基-2-吡咯烷酮(NMP)中，形成金属盐溶液。接着，在氮气保护下将0.4g(10.5mmol)的NaBH 4溶解在50ml的NMP中以形成还原溶液。 As shown in Figure 1, 0.42 g of polyvinylpyrrolidone (PVP, K-30, weight average molecular weight 30,000), 0.07 g (0.35 mmol) of FeCl 2 ·4H 2 O and 0.0833 g (0.35 mmol) of CoCl 2 ·6H 2 O was dissolved in 50 mL of N-methyl-2-pyrrolidone (NMP) under nitrogen to form a metal salt solution. Next, 0.4 g (10.5 mmol) of NaBH 4 was dissolved in 50 ml of NMP under nitrogen protection to form a reducing solution.
接下来，使用图1所示过程在氮气保护下于120℃进行微流体合成：将50ml带PVP的金属盐溶液和50ml还原溶液分别引入每个注射器中，并将注射器固定在第一注射泵1和第二注射泵2上。随后将其通过第一微流道管3和第二微流道管4引入Y字形反应物料液混合器5完成还原反应，第一注射泵1和第二注射泵2的流速均为3ml/min。接下来，溶液进入第三微通道管6以完成快速成核并完成纳米颗粒的生长。根据以下反应路线形成CoFeB纳米颗粒。当反应完成时，将获得的新鲜纳米颗粒分散溶液收集在收集器7中。 然后，使用离心机以15,000rpm的速度沉淀溶液30分钟，并倾析出顶部上清液。使用NMP将获得的颗粒洗涤两次，得到预合成的CoFeB纳米颗粒。Next, microfluidic synthesis was performed at 120 °C under nitrogen protection using the procedure shown in Fig. 1: 50 ml of metal salt solution with PVP and 50 ml of reduction solution were introduced into each syringe, respectively, and the syringes were fixed in the first syringe pump 1 and the second syringe pump 2. Then it is introduced into the Y-shaped reaction material-liquid mixer 5 through the first microfluidic pipe 3 and the second microfluidic pipe 4 to complete the reduction reaction, and the flow rates of the first syringe pump 1 and the second syringe pump 2 are both 3ml/min . Next, the solution enters the third microchannel tube 6 to complete the rapid nucleation and complete the growth of nanoparticles. CoFeB nanoparticles were formed according to the following reaction scheme. When the reaction was completed, the obtained fresh nanoparticle dispersion solution was collected in the collector 7 . Then, the solution was pelleted using a centrifuge at 15,000 rpm for 30 minutes, and the top supernatant was decanted. The obtained particles were washed twice with NMP to obtain pre-synthesized CoFeB nanoparticles.
Figure PCTCN2022083976-appb-000001
Figure PCTCN2022083976-appb-000001
将2.5ml(0.36mmol)的HAuCl 4溶液溶解在50ml的NMP中。然后使用图1所示装置在氮气保护条件下在室温下进行微流体合成：分别使用第一注射泵1和第二注射泵2将50ml稀释的HAuCl 4溶液和50ml预合成的CoFeB纳米颗粒的NMP溶液泵送入第一微流道管3和第二微流道管4，一起进入Y字形反应物料液混合器5完成还原反应和快速成核，第一注射泵1和第二注射泵2的流速均为3ml/min。在该步骤中，将0.2g的NaBH 4溶解在20ml的NMP中，并预先放入收集器7中。当反应完成时，将收集器7彻底摇动并静置30分钟，以完成CoFeB纳米颗粒的置换，还原和表面重排。随后向收集器中加入2ml乙醇以破坏溶液的平衡。沉淀的纳米颗粒重新溶解在相同体积的NMP中。重复清洗过程两次，以去除大部分表面活性剂。最后将瓶中的最终黑色浆料在真空条件下干燥成黑色粉末，并保存在干燥器中以备将来使用。 2.5 ml (0.36 mmol) of the HAuCl 4 solution was dissolved in 50 ml of NMP. The microfluidic synthesis was then performed at room temperature under nitrogen protection using the apparatus shown in Figure 1: 50 ml of the diluted HAuCl solution and 50 ml of NMP of pre-synthesized CoFeB nanoparticles were mixed using the first syringe pump 1 and the second syringe pump 2, respectively . The solution is pumped into the first microfluidic pipe 3 and the second microfluidic pipe 4, and enters the Y-shaped reaction material-liquid mixer 5 together to complete the reduction reaction and rapid nucleation. The first syringe pump 1 and the second syringe pump 2 The flow rates were all 3ml/min. In this step, 0.2 g of NaBH 4 was dissolved in 20 ml of NMP and placed in the collector 7 in advance. When the reaction was completed, the collector 7 was shaken thoroughly and left to stand for 30 min to complete the displacement, reduction and surface rearrangement of the CoFeB nanoparticles. Then 2 ml of ethanol was added to the collector to disrupt the equilibrium of the solution. The precipitated nanoparticles were redissolved in the same volume of NMP. Repeat the cleaning process twice to remove most of the surfactant. The final black slurry in the bottle was finally dried under vacuum to a black powder and kept in a desiccator for future use.
(2)纳米颗粒的表面改性：(2) Surface modification of nanoparticles:
首先将10mg步骤(1)制备的Au@CoFeB纳米颗粒溶解到50ml含有1wt％3-氨丙基三甲氧基硅氧烷(APTMS)无水甲苯溶液中。然后，将混合溶液在室温下搅拌24小时。搅拌完成后，使用离心机以12000rpm的速度沉离心10分钟。随后，倾析出顶部上清液，并将沉淀的纳米颗粒用乙醇洗涤一次。最后获得Au@CoFeB-APTMS纳米颗粒。First, 10 mg of Au@CoFeB nanoparticles prepared in step (1) were dissolved into 50 ml of anhydrous toluene solution containing 1 wt% 3-aminopropyltrimethoxysiloxane (APTMS). Then, the mixed solution was stirred at room temperature for 24 hours. After stirring was completed, centrifugation was performed at 12,000 rpm for 10 minutes using a centrifuge. Subsequently, the top supernatant was decanted and the precipitated nanoparticles were washed once with ethanol. Finally, Au@CoFeB-APTMS nanoparticles were obtained.
(3)纳米颗粒的偶联活化：(3) Coupling activation of nanoparticles:
首先，将5mg制备的Au@CoFeB-APTMS纳米颗粒溶解在5ml的二甲基亚砜中。然后，将5mg辛二酸(N-羟基琥珀酰亚胺酯)偶联剂溶液缓慢加入到溶液中。在室温下孵育1小时以上后，将混合溶液以12,000rpm的速度离心10分钟。然后倒出顶部上清液，并将瓶中最终沉淀的纳米颗粒在真空下干燥，即可以得到Au@CoFeB-APTMS-DSS纳米颗粒。First, 5 mg of the prepared Au@CoFeB-APTMS nanoparticles were dissolved in 5 ml of dimethyl sulfoxide. Then, 5 mg of suberic acid (N-hydroxysuccinimide ester) coupling agent solution was slowly added to the solution. After incubation at room temperature for more than 1 hour, the mixed solution was centrifuged at 12,000 rpm for 10 minutes. Then, the top supernatant was poured out, and the finally precipitated nanoparticles in the bottle were dried under vacuum to obtain Au@CoFeB-APTMS-DSS nanoparticles.
(4)纳米颗粒的pH调控：(4) pH regulation of nanoparticles:
将干燥后的样品溶于10mL磷酸盐缓冲溶液(pH＝7.4)中，调节纳米颗粒的pH值。然后以12,000rpm的速度离心10分钟后，将瓶中的最终浆液用去离子水洗涤并在真空下干燥。The dried samples were dissolved in 10 mL of phosphate buffer solution (pH=7.4) to adjust the pH of the nanoparticles. Then after centrifugation at 12,000 rpm for 10 minutes, the final slurry in the bottle was washed with deionized water and dried under vacuum.
(5)人参皂苷的表面改性：(5) Surface modification of ginsenosides:
首先将200mg人参皂苷Rg3溶解在50mL含有1w％APTMS的无水甲苯溶液中。在环境温度下搅拌24小时后，将混合溶液以1200rpm的速度离心10分钟。然后，将沉淀的浆液用乙醇洗涤3次，获得改性后的人参皂苷Rg3。First, 200 mg of ginsenoside Rg3 was dissolved in 50 mL of anhydrous toluene solution containing 1 w% APTMS. After stirring for 24 hours at ambient temperature, the mixed solution was centrifuged at 1200 rpm for 10 minutes. Then, the precipitated slurry was washed three times with ethanol to obtain modified ginsenoside Rg3.
(6)纳米颗粒和人参皂苷的偶联：(6) Coupling of nanoparticles and ginsenosides:
在真空中干燥后，将5mg表面改性后的人参皂苷Rg3和5mg经过表面改性和活化的Au@CoFeB纳米颗粒分散在5ml的二甲基亚砜中。在室温下孵育2小时以实现Au@CoFeB纳米颗粒与人参皂苷Rg3之间的偶联。随后将溶液离心并用去离子水洗涤两次。随后将沉淀的浆液在真空中干燥，获得Au@CoFeB-Rg3纳米药物。After drying in vacuum, 5 mg of surface-modified ginsenoside Rg3 and 5 mg of surface-modified and activated Au@CoFeB nanoparticles were dispersed in 5 ml of dimethyl sulfoxide. Incubate for 2 h at room temperature to achieve the coupling between Au@CoFeB nanoparticles and ginsenoside Rg3. The solution was then centrifuged and washed twice with deionized water. The precipitated slurry was subsequently dried in vacuum to obtain Au@CoFeB-Rg3 nanomedicines.
实施例2-5都利用了实施例1的工艺和装置制备纳米颗粒，但改变了部分的原料、原料用量以及反应条件，如表1所示。In Examples 2-5, the process and device of Example 1 were used to prepare nanoparticles, but some of the raw materials, the amount of raw materials and the reaction conditions were changed, as shown in Table 1.
表1实施例2-5中各项工艺参数一览表List of various process parameters in the embodiment 2-5 of table 1
Figure PCTCN2022083976-appb-000002
Figure PCTCN2022083976-appb-000002
Figure PCTCN2022083976-appb-000003
Figure PCTCN2022083976-appb-000003
Figure PCTCN2022083976-appb-000004
Figure PCTCN2022083976-appb-000004
对通过实施例1中制备的典型的纳米颗粒和纳米药物的微观结构以及多模成像特性进行表征。根据高分辨TEM图像(图2A)和XRD谱图(图2B)，纳米颗粒偶联药物前后，其金属内核仍保持核心为面心立方晶体的Au和面心立方晶体结构的CoFeB合金壳层的核壳结构；XPS谱图(图2C：10、11)则表明该纳米药物含有Au、Co、Fe、B、Si(Ti)、C、N(P)和O元素，其中Si主要来自纳米颗粒的表面改性和交联剂，C、N(P)和O来自有机的药物和偶联剂，如Rg3和辛二酸(N-羟基琥珀酰亚胺酯)，而硼(B)则来自使用的还原剂硼氢化钠；当使用磷酸酯双钛酸酯类偶联剂时，药物的Si会变成Ti，同时增 加一个元素P。FT-IR谱图(图2D)则证实了使用该方法的确获得了由异质结构金属Au@CoFeB(图2D：12)和药物Rg3共轭构建的金属-有机复合化的纳米药物Au@CoFeB-Rg3(图2D：13)；结合TEM图像(图2A)和水动力学直径(图2E)检测结果则表明纳米颗粒和药物偶联成纳米药物后的纳米药物(图2E：15)整体结构的确是由约6.6nm的超小纳米药物单元偶联在一起构建的尺寸约300nm的纳米药物聚集体；而单纯的金属纳米颗粒(图2E：14)的动力学半径约150nm，主要是由于该纳米药物的无机部分的壳层CoFeB磁性材料，其产生的磁偶极作用具有将其聚集在一起的功能，在水溶液中也会以聚集体存在，这些小于1微米的聚集体非常有利于提高药物在体内停留时间和输运到病灶的效率，而在病灶处微酸性的细胞微环境，则又很快解离成单个的药物，提高对病灶细胞及组织内的渗透力；而Zeta电势(图2F)表征结果表明，该类纳米颗粒(图2F：16)及纳米药物(图2F：17)表面分别带有+10mV和+27mV的正电势，这些正电势特别有利于提高药物递送速率和提高药物利用率起关键作用的细胞内化过程，药物的这个特点和药物以聚集体形式的集团输运模式一起，共同提高药物进入细胞内的比率，即药物的利用率，克服纳米药物在癌症治疗上的关键难题：药物利用率低下，平均不到1.0％。The microstructure and multimodal imaging properties of typical nanoparticles and nanomedicines prepared by Example 1 were characterized. According to the high-resolution TEM images (Fig. 2A) and XRD patterns (Fig. 2B), before and after the nanoparticles were coupled with the drug, the metal core of the nanoparticles still maintained the core of Au with a face-centered cubic crystal structure and the CoFeB alloy shell with a face-centered cubic crystal structure. Core-shell structure; XPS spectra (Fig. 2C: 10, 11) show that the nanomedicine contains Au, Co, Fe, B, Si(Ti), C, N(P) and O elements, in which Si mainly comes from nanoparticles surface modification and crosslinking agents, C, N(P) and O are derived from organic drugs and coupling agents such as Rg3 and suberic acid (N-hydroxysuccinimide ester), while boron (B) is derived from The reducing agent used is sodium borohydride; when a phosphate bis-titanate coupling agent is used, the Si of the drug will become Ti, and an element P will be added at the same time. The FT-IR spectrum (Fig. 2D) confirmed that the metal-organic composite nano-drug Au@CoFeB constructed by the heterostructure metal Au@CoFeB (Fig. 2D: 12) and drug Rg3 conjugated was indeed obtained using this method. -Rg3 (Fig. 2D: 13); combined with TEM image (Fig. 2A) and hydrodynamic diameter (Fig. 2E) detection results show that the nanoparticle and drug are coupled into nanodrugs (Fig. 2E: 15) overall structure It is indeed a nano-drug aggregate with a size of about 300 nm constructed by coupling together ultra-small nano-drug units of about 6.6 nm; while the kinetic radius of pure metal nanoparticles (Fig. 2E: 14) is about 150 nm, mainly due to the The CoFeB magnetic material in the shell layer of the inorganic part of the nanomedicine, the magnetic dipole produced by it has the function of gathering it together, and it also exists as aggregates in the aqueous solution. These aggregates smaller than 1 micron are very beneficial to improve the drug. The residence time in the body and the efficiency of transport to the lesion, while the slightly acidic cell microenvironment at the lesion is quickly dissociated into a single drug, which improves the penetration force into the lesion cells and tissues; while the Zeta potential (Fig. 2F) characterization results show that the surfaces of the nanoparticles (Fig. 2F: 16) and nano-drugs (Fig. 2F: 17) have positive potentials of +10 mV and +27 mV, respectively, which are particularly beneficial to improve the drug delivery rate and increase the The cellular internalization process plays a key role in drug utilization. This feature of drugs, together with the group transport mode of drugs in the form of aggregates, together improve the rate of drug entry into cells, that is, drug utilization, and overcome the role of nano-drugs in cancer treatment. The key problem on the list: The drug utilization rate is low, on average less than 1.0%.
图3是对通过实施例2制备的纳米颗粒和纳米药物的多模态影像功能表征。如图3A所示，对于含有贵金属组份的磁性异质结构纳米颗粒，其具有很强的表面等离子体共振(LSPR)散射光特性，虽然其只有5nm左右，但由于其可以散射光具有400-600纳米的光学尺寸，仍然可以通过光学显微镜观测到单个纳米颗粒图像(图3A：18、20)，并可以用表面等离子体波谱仪对单个纳米颗粒的光学散射特性表征(图3A：19、21)。而经过表面改性并偶联上药物后，虽然表面的介电常数有所改变，但其LSPR散射光强度仍然保持很强(图3B-20)，仍可以使用暗场显微镜和表面等离子体波谱仪对其成像示踪。制备的Au@CoFe(3A-18)和偶联有Rg3的Au@CoFe-Rg3纳米药物(3B-20)的暗场显微LSPR光学图像和其中一个颗粒(3A-19)和药物的LSPR波谱(3B-21)，经过药物共轭后，由于表面介电常数改变，整体颗粒的散射光发生了红移。FIG. 3 is a multimodal imaging functional characterization of nanoparticles and nanomedicines prepared by Example 2. FIG. As shown in Figure 3A, for the magnetic heterostructure nanoparticles containing noble metal components, they have strong surface plasmon resonance (LSPR) scattering light characteristics. Although they are only about 5 nm, they have 400- With an optical size of 600 nanometers, images of individual nanoparticles can still be observed by optical microscopy (Fig. 3A: 18, 20) and can be characterized by surface plasmon spectroscopy for the optical scattering properties of individual nanoparticles (Fig. 3A: 19, 21) ). After surface modification and drug coupling, although the dielectric constant of the surface has changed, the LSPR scattered light intensity remains strong (Fig. 3B-20), and dark-field microscopy and surface plasmon spectroscopy can still be used. image tracer. Dark-field microscopy LSPR optical images of as-prepared Au@CoFe (3A-18) and Rg3-conjugated Au@CoFe-Rg3 nanomedicine (3B-20) and LSPR spectra of one of the particles (3A-19) and the drug (3B-21), after drug conjugation, the scattered light of the bulk particle undergoes a red shift due to the change of the surface permittivity.
图3C是通过实施例2制备的纯Au@CoFeB纳米颗粒在T2WI实验条件的自选-回波序列为回波(TE)持续时间是33ms、脉冲重复时间间隔(TR)是2500ms下的磁共振成像结果，其具有很好的T2WI增强效果，其弛豫速率(T 2 -1，s -1)和纳米颗粒浓度具有很好的线性关系。图3D是合成的纯Au@CoFeB-Rg3纳米药物在T2WI实验条件的自选-回声序列为回声(TE)时间是33ms、脉冲重复时间间隔(TR)是2500ms下的磁共振成像结果，和纳米颗粒一样，其具有很好的T2WI增强效果，其弛豫速率(T 2 -1，s -1)和纳米颗粒浓度 具有很好的线性关系，同时可以看出，纳米药物的弛豫速率比纳米颗粒更高，说明金属-有机复合有利于进一步增强其分子影像增强效果。 Figure 3C is the magnetic resonance imaging of the pure Au@CoFeB nanoparticles prepared by Example 2 under T2WI experimental conditions - the echo sequence is the echo (TE) duration is 33ms, and the pulse repetition time interval (TR) is 2500ms. As a result, it has a good T2WI enhancement effect, and its relaxation rate (T 2 -1 , s -1 ) has a good linear relationship with the nanoparticle concentration. Figure 3D is the MRI results of the synthesized pure Au@CoFeB-Rg3 nanomedicines under T2WI experimental conditions - echo sequence with echo (TE) time is 33ms, pulse repetition time interval (TR) is 2500ms, and nanoparticles Similarly, it has a good T2WI enhancement effect, and its relaxation rate (T 2 -1 , s -1 ) has a good linear relationship with the concentration of nanoparticles. higher, indicating that the metal-organic composite is beneficial to further enhance its molecular image enhancement effect.
图3E上部图像是是Au@CoFeB-Rg3纳米药物不同浓度下(400μg/mL、800μg/mL、1100μg/mL、1500μg/mL、4000μg/mL)的计算机辅助X光断层扫描(CT)图像和三点平均的信号(亨氏单位：HU)的浓度依赖曲线(图中曲线)。可见，其具有很优异的CT影像效果；同时在很大浓度范围内，其CT值(HU是测定人体某一局部组织或器官密度大小的一种计量单位：空气为-1000，致密骨为1000)和浓度具有优异的线性关系，可以在临床中通过分子影像示踪法高时空分辨在线研究病理和药理。The upper image of Fig. 3E is the computer-aided tomography (CT) image and three Concentration-dependent curve of the point-averaged signal (Hinz units: HU) (curve in the figure). It can be seen that it has excellent CT imaging effect; at the same time, in a large concentration range, its CT value (HU is a measurement unit for measuring the density of a certain local tissue or organ in the human body: air is -1000, dense bone is 1000 ) and concentration have an excellent linear relationship, which can be used to study pathology and pharmacology online with high spatial and temporal resolution by molecular imaging tracing method.
应用实施例Application Example
纳米药物Au@CoFeB和Au@CoFeB-Rg3抗肿瘤效果及其药理研究Antitumor effects and pharmacological studies of nano-drugs Au@CoFeB and Au@CoFeB-Rg3
应用实施例1Application Example 1
通过细胞生物学方法以人慢性髓性白血病K562细胞系和肝癌细胞系(HEP-G2/C3A)为癌细胞病理模型、以人上皮成纤细胞系3T3和人免疫细胞JurkatT细胞系为正常健康细胞模型，对合成的纳米药物的抗肿瘤效果和对正常细胞的毒副作用进行了研究。The human chronic myeloid leukemia K562 cell line and the hepatoma cell line (HEP-G2/C3A) were used as the pathological models of cancer cells, and the human epithelial fibroblast cell line 3T3 and the human immune cell JurkatT cell line were used as normal healthy cells by cell biology methods. model, the antitumor effects and toxic side effects on normal cells of the synthesized nanomedicines were studied.
配制不同Au@CoFeB和Au@CoFeB-Rg3浓度的细胞培养用磷酸盐PBS缓冲液，通过BD LSRFortessa TM细胞分析仪(BD biosciences)使用光散射法检测各种细胞在不同Au@CoFeB和Au@CoFeB-Rg3浓度下，孵育24小后，各种细胞的24小时存活率。 Phosphate PBS buffers for cell culture with different concentrations of Au@CoFeB and Au@CoFeB-Rg3 were prepared, and the BD LSRFortessa TM cell analyzer (BD biosciences) used light scattering to detect various cells in different Au@CoFeB and Au@CoFeB - 24-hour viability of various cells after 24-hour incubation at Rg3 concentration.
如图4A所示为实施例1制备的Au@CoFeB(浓度从0.00001μg/mL到500μg/mL)和Au@CoFeB-Rg3(浓度从0.00001μg/mL到1000μg/mL)对正常细胞(3T3：4A-23、4B-27)、T细胞(Jurkat：4A-22、4B-26)、白血病细胞K562(4A-24、4B-28)和实体瘤肝癌细胞(HEP-G2/C3A)(4A-25、4B-29)毒性的影响。24小时细胞毒性检测表明(4A和4B)，实施例1制备的Au@CoFeB-Rg3纳米药物(图4B)对K562细胞(4B-28)和HEP-G2/C3A(4B-29)细胞的毒性明显高于Au@CoFeB(4A：24、25)，也都高于纯Rg3对肝癌细胞和血癌细胞的致死率，说明异质结构金属纳米颗粒和有机的药物之间有明显的协同抗肿瘤效果。Au@CoFeB对免疫细胞和血癌细胞的24小时毒性明显低于对3T3和肝癌细胞，说明Au@CoFeB对可以成实体组织的细胞比自由悬浮细胞具有高毒性，同时可以看出，Au@CoFeB在低浓度(9.5μg/mL)。当Au@CoFeB浓度低于250μg/mL时，对所考察细胞毒性很低，说明该浓度下可以作为光学生物分子纳米探针使用。当Au@CoFeB-Rg3浓度为95μg/mL，对K562细胞已具有强致死率；不超过该浓度对其他细胞无明显毒性(细胞 存活>70％)。当Au@CoFeB-Rg3浓度超过200μg/mL，其对肝癌细胞和血癌细胞致死率大幅度提高；虽然该浓度对Jurkat细胞也有明显毒性，但对3T3的毒性明显比对癌细胞的小很多。Figure 4A shows the effects of Au@CoFeB (concentration from 0.00001 μg/mL to 500 μg/mL) and Au@CoFeB-Rg3 (concentration from 0.00001 μg/mL to 1000 μg/mL) prepared in Example 1 on normal cells (3T3: 4A-23, 4B-27), T cells (Jurkat: 4A-22, 4B-26), leukemia cells K562 (4A-24, 4B-28) and solid tumor hepatoma cells (HEP-G2/C3A) (4A- 25, 4B-29) effects of toxicity. The 24-hour cytotoxicity assay showed (4A and 4B) that the Au@CoFeB-Rg3 nanomedicine (Fig. 4B) prepared in Example 1 was toxic to K562 cells (4B-28) and HEP-G2/C3A (4B-29) cells Significantly higher than that of Au@CoFeB (4A: 24, 25), and also higher than the lethality of pure Rg3 on liver cancer cells and blood cancer cells, indicating that there is an obvious synergistic anti-tumor effect between heterostructured metal nanoparticles and organic drugs . The 24-hour toxicity of Au@CoFeB to immune cells and blood cancer cells was significantly lower than that to 3T3 and liver cancer cells, indicating that Au@CoFeB was more toxic to cells that could form solid tissues than free suspension cells. Low concentration (9.5 μg/mL). When the concentration of Au@CoFeB is lower than 250 μg/mL, the toxicity to the investigated cells is very low, indicating that this concentration can be used as an optical biomolecular nanoprobe. When the concentration of Au@CoFeB-Rg3 is 95μg/mL, it has a strong lethality to K562 cells; it has no obvious toxicity to other cells (cell survival>70%) when the concentration does not exceed this concentration. When the concentration of Au@CoFeB-Rg3 exceeds 200 μg/mL, its lethality to liver cancer cells and blood cancer cells is greatly increased; although this concentration is also significantly toxic to Jurkat cells, the toxicity to 3T3 is significantly smaller than that of cancer cells.
应用实施例2Application Example 2
通过细胞生物学方法以人慢性髓性白血病K562细胞系和肝癌细胞系(HEP-G2/C3A)为癌细胞病理模型、以人上皮成纤细胞系3T3和人免疫细胞JurkatT细胞系为正常健康细胞模型，对合成的纳米药物的抗肿瘤效果和对正常细胞的毒副作用进行了研究。配制不同Au@CoFeB和Au@CoFeB-Rg3浓度的细胞培养用PBS缓冲液，通过BD LSRFortessa TM细胞分析仪(BD biosciences购自BD biosciences公司，)使用光散射法检测各种细胞在不同Au@CoFeB和Au@CoFeB-Rg3浓度下，孵育24小后，各种细胞的24小时存活率。 The human chronic myeloid leukemia K562 cell line and the hepatoma cell line (HEP-G2/C3A) were used as the pathological models of cancer cells, and the human epithelial fibroblast cell line 3T3 and the human immune cell JurkatT cell line were used as normal healthy cells by cell biology methods. model, the antitumor effects and toxic side effects on normal cells of the synthesized nanomedicines were studied. PBS buffers for cell culture with different concentrations of Au@CoFeB and Au@CoFeB-Rg3 were prepared, and a BD LSRFortessa TM cell analyzer (purchased from BD biosciences, BD biosciences) was used to detect the presence of various cells in different Au@CoFeB by light scattering method. and Au@CoFeB-Rg3 concentrations, the 24-hour viability of various cells after 24-hour incubation.
这次使用实施例1制备的Au@CoFeB浓度分别是2500μg/mL、10000μg/mL、100000μg/mL；Au@CoFeB-Rg3浓度为2500μg/mL、20000μg/mL、100000μg/mL。检测它们对白血病细胞K562细胞和肝癌细胞HEP-G2/C3A的毒性，其24小时的细胞存活率均为零，100％被杀死。The concentrations of Au@CoFeB prepared in Example 1 were 2500 μg/mL, 10000 μg/mL, and 100000 μg/mL, respectively; the concentrations of Au@CoFeB-Rg3 were 2500 μg/mL, 20000 μg/mL, and 100000 μg/mL. Detecting their toxicity to leukemia cells K562 cells and hepatoma cells HEP-G2/C3A, the 24-hour cell viability was zero and 100% was killed.
应用实施例3Application Example 3
使用96孔板通过基于ATP细胞活力检测的细胞效价-glo荧光细胞活力测定仪(Promega,G7570)对通过实施例3制备的不同浓度下纳米颗粒和纳米药物的K562,HepG2/C3A,和Jurkat细胞的细胞增殖率进行检测。其中缓冲液使用10％的胎牛血清FBS缓冲液，缓冲液中Au@CoFeB的浓度选取为0.00001μg/ml,9.5μg/ml,47.0μg/ml,95.0μg/ml和474.0μg/ml；Au@CoFe-Rg3选取为0.00001μg/ml,19μg/ml,95.0μg/ml,190.0μg/ml和950.0μg/ml。一起在细胞培养箱(3％CO 2，310K)中培育4天后检测其增值。 K562, HepG2/C3A, and Jurkat of nanoparticles and nanomedicines prepared by Example 3 at different concentrations were measured by ATP-based cell viability assay-glo fluorescence cell viability assay (Promega, G7570) using a 96-well plate The cell proliferation rate of the cells was detected. The buffer was 10% fetal bovine serum FBS buffer, and the concentrations of Au@CoFeB in the buffer were selected as 0.00001μg/ml, 9.5μg/ml, 47.0μg/ml, 95.0μg/ml and 474.0μg/ml; Au @CoFe-Rg3 was selected as 0.00001 μg/ml, 19 μg/ml, 95.0 μg/ml, 190.0 μg/ml and 950.0 μg/ml. The proliferation was detected after 4 days of incubation in a cell incubator (3% CO 2 , 310K) together.
纳米颗粒和纳米药物对细胞增殖的效果表明：当Au@CoFeB浓度超过50μg/mL(图5A)、Au@CoFeB-Rg3浓度超过100μg/mL(图5B)后，不论是实体瘤肝癌细胞(HEP-G2/C3A：5A-33、5B-37)还是可以自由流动的白血病癌细胞(K562：5A-32、5B-36)，纳米药物对癌细胞增殖具有明显抑制作用，但对正常细胞3T3(5A-31、5B-35)无明显毒性；特别是当Au@CoFeB-Rg3浓度达到950μg/mL时，增殖培养4天后，癌细胞基本全部死亡。该增殖抑制效果研究表明，Au@CoFeB自身对癌细胞的增殖具有明显抑制作用，特别是当浓度达到474μg/mL时，癌细胞增殖基本全部被抑制。一个特别的现象是，当使用低的 Au@CoFeB浓度(如19μg/mL)时，其可以提高免疫细胞Jurkat的增殖速率(5A-30)，但是纳米药物对其有一定毒性(5B-34)，说明如何适当设计好纳米颗粒的尺寸、组成和结构，它们可以具有提高免疫细胞活性的作用，将来特定浓度和成分的纳米颗粒可以和免疫疗法联合使用，提高癌症治疗效果。The effects of nanoparticles and nanomedicines on cell proliferation showed that when the concentration of Au@CoFeB exceeded 50 μg/mL (Fig. 5A) and the concentration of Au@CoFeB-Rg3 exceeded 100 μg/mL (Fig. 5B), no matter the solid tumor hepatoma cells (HEP) -G2/C3A: 5A-33, 5B-37) are still free-flowing leukemia cancer cells (K562: 5A-32, 5B-36), nanomedicine has obvious inhibitory effect on the proliferation of cancer cells, but on normal cells 3T3 ( 5A-31, 5B-35) had no obvious toxicity; especially when the concentration of Au@CoFeB-Rg3 reached 950 μg/mL, after 4 days of proliferation and culture, almost all cancer cells died. The study on the inhibitory effect of proliferation showed that Au@CoFeB itself had a significant inhibitory effect on the proliferation of cancer cells, especially when the concentration reached 474 μg/mL, the proliferation of cancer cells was basically completely inhibited. A particular phenomenon is that when a low concentration of Au@CoFeB (such as 19 μg/mL) is used, it can increase the proliferation rate of immune cell Jurkat (5A-30), but the nanomedicine has some toxicity to it (5B-34) , to explain how to properly design the size, composition and structure of nanoparticles, which can improve the activity of immune cells. In the future, nanoparticles of specific concentration and composition can be used in combination with immunotherapy to improve the effect of cancer treatment.
应用实施例4Application Example 4
使用96孔板通过基于ATP细胞活力检测的细胞效价-glo荧光细胞活力测定仪(Promega,G7570)对通过实施例3制备的不同纳米药物在其不同浓度下的K562,HepG2/C3A,和Jurkat细胞的细胞增殖率进行检测。其中缓冲液使用10％的胎牛血清FBS缓冲液.这次使用的Au@CoFeB浓度分别是2500μg/mL、10000、100000μg/mL；Au@CoFeB-Rg3浓度为5000μg/mL、20000μg/mL、100000μg/mL。一起在细胞培养箱(3％CO 2，310K)中培育4天后检测其增值。实验结果表明，当Au@CoFeB浓度超过2500μg/mL、Au@CoFeB-Rg3浓度超过5000μg/mL后，血癌细胞K562和实体瘤肝癌HepG2/C3A细胞均全部死亡。而Jurkat细胞和3T3细胞仍然分别有30％和20％以上的4天增值培养存活率。说明，高浓度下，该类药物对癌细胞具有高致死性，而对正常细胞也有一定毒性，因此，临床应用必须控制用药量。 K562, HepG2/C3A, and Jurkat at different concentrations of the different nanomedicines prepared by Example 3 were measured by ATP-based cell viability assay-glo fluorescence cell viability assay (Promega, G7570) using a 96-well plate The cell proliferation rate of the cells was detected. The buffer is 10% fetal bovine serum FBS buffer. The concentrations of Au@CoFeB used this time are 2500μg/mL, 10000, 100000μg/mL; Au@CoFeB-Rg3 concentrations are 5000μg/mL, 20000μg/mL, 100000μg /mL. The proliferation was detected after 4 days of incubation in a cell incubator (3% CO 2 , 310K) together. The experimental results showed that when the concentration of Au@CoFeB exceeded 2500 μg/mL and the concentration of Au@CoFeB-Rg3 exceeded 5000 μg/mL, the blood cancer cell K562 and the solid tumor hepatoma HepG2/C3A cells all died. While Jurkat cells and 3T3 cells still had 4-day proliferation culture survival rates of over 30% and 20%, respectively. It is indicated that at high concentrations, this type of drug is highly lethal to cancer cells, but also has certain toxicity to normal cells. Therefore, the dosage must be controlled in clinical application.
应用实施例5Application Example 5
考察了使用实施例4工艺制备的Au@CoFeB纳米颗粒和Au@CoFeB-Rg3纳米药物与其它纳米药物(Fe@Fe 3O 4-Rg3、FePt@Fe 3O 4-Rg3)在两种浓度下)对人慢性髓系白血病细胞K562癌细胞孵化24小时的存活率实验结果。如图6所示：斜纹柱，药物浓度为95-190μg/mL；网格柱，药物浓度为474-947μg/mL。结果表明，Au@CoFeB-Rg3纳米药物对该类白血病细胞的杀死率比前两者在低浓度下(95-190μg/mL)均提高了近7倍；在高浓度下(474-947μg/mL)提高了40多倍。而Au@CoFeB纳米颗粒在高浓度下则比Fe@Fe 3O 4-Rg3和FePt@Fe 3O 4-Rg3纳米药物也分别提高了27倍和23倍；在低浓度下分别比Fe@Fe 3O 4-Rg3和FePt@Fe 3O 4-Rg3纳米药物提高1.4倍和1.5倍。 The Au@CoFeB nanoparticles and Au@CoFeB-Rg3 nanomedicines and other nanomedicines (Fe@Fe 3 O 4 -Rg3, FePt@Fe 3 O 4 -Rg3) prepared by the process of Example 4 were investigated at two concentrations ) on the survival rate of human chronic myeloid leukemia cells K562 cancer cells incubated for 24 hours. As shown in Figure 6: diagonal bars, the drug concentration is 95-190 μg/mL; grid columns, the drug concentration is 474-947 μg/mL. The results showed that the killing rate of Au@CoFeB-Rg3 nanomedicine on leukemia cells was nearly 7 times higher than that of the former two at low concentrations (95-190 μg/mL); at high concentrations (474-947 μg/mL) mL) increased by more than 40 times. And Au@CoFeB nanoparticles at high concentrations are 27 times and 23 times better than Fe@Fe 3 O 4 -Rg3 and FePt@Fe 3 O 4 -Rg3 nanomedicines, respectively; 3 O 4 -Rg3 and FePt@Fe 3 O 4 -Rg3 nanomedicines were improved by 1.4 times and 1.5 times.
应用实施例6Application Example 6
使用裸鼠并通过二甲基亚硝胺在其肝部建立原位肝癌模型。当肿瘤长大约4周后开始给药，每两天给一次，每次约70mg/kg，连续5次后停药，然后饲养老鼠，每7天对老鼠 称体重，并通过肿瘤处的荧光分析检测肿瘤相对大小。共分四组，一组为使用生理盐水组作为对照，另三组分别为纯Rg3组、通过实施例5制备的纯Au@CoFeB和纳米药物Au@CoFeB-Rg3组。到第21天后，将所有老鼠牺牲掉并进行解剖，对肝部及其肿瘤进行解剖并作各种生化和病理切片分析。图7A是不同药物和对照组处理下的最后的小鼠的肿瘤图像，图7B是不同时间点处通过荧光法表征的小鼠肿瘤大小的荧光图像，图7C是测定的不同组小鼠在给药后不同时间点的绝对(上部折线)和相对体重变化(下部折线)(生理盐水：38；Rg3：39；Au@CoFe：40；Au@CoFeB-Rg3：41)。图7D是根据荧光值测定不同组在给药后不同时间节点处的肿瘤大小(生理盐水：42；Rg3：43；Au@CoFe：44；Au@CoFeB-Rg3：45)。可以看出，单纯Rg3组(43)比对照组(42)的肿瘤稍微小一点，而Au@CoFeB(44)则比对照组的肿瘤稍小很多，说明Au@CoFeB自身就具有很强的抗肝癌效果；对小鼠肿瘤抑制作用最强的是经过Au@CoFeB和Rg3复合成纳米药物的一组，同时可以看出给药后，随着用药及用药后时间推移，纳米药物对肝癌的发展的抑制作用越来越明显(45)。An orthotopic liver cancer model was established in the liver of nude mice by dimethylnitrosamine. When the tumor grows for about 4 weeks, the drug is given at about 70 mg/kg every two days, and the drug is stopped after 5 consecutive times. Then the mice are raised, and the mice are weighed every 7 days and analyzed by fluorescence at the tumor. The relative size of the tumor was measured. There are four groups in total, one is the normal saline group as a control, the other three groups are the pure Rg3 group, the pure Au@CoFeB prepared by Example 5 and the nano-drug Au@CoFeB-Rg3 group. After day 21, all mice were sacrificed and dissected, and the liver and its tumor were dissected and analyzed for various biochemical and pathological sections. Figure 7A is the tumor image of the final mice treated with different drugs and control groups, Figure 7B is the fluorescence image of the tumor size of the mice characterized by fluorescence method at different time points, and Figure 7C is the measured tumor size of different groups of mice. Absolute (upper line) and relative body weight change (lower line) at different time points after drug administration (saline: 38; Rg3: 39; Au@CoFe: 40; Au@CoFeB-Rg3: 41). Figure 7D shows the tumor size of different groups at different time points after administration according to the fluorescence value (saline: 42; Rg3: 43; Au@CoFe: 44; Au@CoFeB-Rg3: 45). It can be seen that the tumor of the pure Rg3 group (43) is slightly smaller than that of the control group (42), while the tumor of Au@CoFeB (44) is much smaller than that of the control group, indicating that Au@CoFeB itself has a strong anti-tumor effect. Hepatocellular carcinoma effect; the strongest inhibitory effect on mice tumor is the group of nano-drugs compounded by Au@CoFeB and Rg3. At the same time, it can be seen that after administration, the development of nano-drugs on hepatocellular carcinoma with the passage of time after administration and administration The inhibitory effect is increasingly evident (45).

Claims (10)

  1. 一种金属-有机复合纳米药物，包含依次经第一表面改性剂和交联剂处理的异质结构金属基纳米颗粒以及经第二表面改性剂处理的药物成分化合物，所述纳米颗粒通过交联剂与所述药物成分化合物偶联，其特征在于，所述药物成分化合物为人参皂苷，所述纳米颗粒为Au@CoFeB。A metal-organic composite nanomedicine, comprising heterostructured metal-based nanoparticles treated with a first surface modifier and a cross-linking agent in turn, and a drug component compound treated with a second surface modifier, the nanoparticles passing through The cross-linking agent is coupled with the pharmaceutical ingredient compound, wherein the pharmaceutical ingredient compound is ginsenoside, and the nanoparticle is Au@CoFeB.
  2. 根据权利要求1所述的纳米药物，其中所述第一表面改性剂选自3-氨丙基三甲氧基硅氧烷、乙烯基三甲氧基硅烷、磷酸酯双钛酸酯偶联剂和二异丙氧基二乙酰丙酮钛酸酯中的至少一种；优选为3-氨丙基三甲氧基硅氧烷。The nanomedicine of claim 1, wherein the first surface modifier is selected from the group consisting of 3-aminopropyltrimethoxysiloxane, vinyltrimethoxysilane, phosphate bis-titanate coupling agent and At least one of diisopropoxydiacetylacetonate titanate; preferably 3-aminopropyltrimethoxysiloxane.
  3. 根据权利要求1所述的纳米药物，其中所述交联剂选自辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、琥珀酰-亚胺丁二酸酯、聚乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种；优选为辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、聚乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种。The nanomedicine according to claim 1, wherein the cross-linking agent is selected from the group consisting of suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (succinate N-hydroxysuccinimide ester), At least one of polyethylene glycol disuccinimidyl succinate, succinyl-imide succinate, polyethylene glycol succinyl-imide succinate and aziridine crosslinking agent XR-100 species; preferably suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (N-hydroxysuccinimide succinimide ester), polyethylene glycol disuccinimidyl succinate, polyethylene glycol At least one of ethylene glycol succinyl-imide succinate and aziridine crosslinking agent XR-100.
  4. 根据权利要求1所述的纳米药物，其中所述第二表面改性剂选自3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂、十七氟癸基三甲基氧基硅烷和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种；优选为3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种。The nanomedicine according to claim 1, wherein the second surface modifier is selected from the group consisting of 3-aminopropyltrimethoxysiloxane, 3-mercaptopropyl-triethoxysilane coupling agent, seventeen seventeen At least one of fluorodecyl trimethyloxysilane and isopropyl tris (dioctyl pyrophosphate acyloxy) titanate; preferably 3-aminopropyl trimethoxy siloxane, 3-mercapto At least one of propyl-triethoxysilane coupling agent and isopropyl tris(dioctyl pyrophosphate acyloxy) titanate.
  5. 根据权利要求1所述的纳米药物，其中所述纳米药物采用包含以下步骤的方法制备：The nanomedicine according to claim 1, wherein the nanomedicine is prepared by a method comprising the following steps:
    (1)制备Au@CoFeB纳米颗粒；(1) Preparation of Au@CoFeB nanoparticles;
    (2)纳米颗粒的表面改性：(2) Surface modification of nanoparticles:
    a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌，a) adding the nanoparticles in (1) to the first organic solution containing the first surface modifier and stirring,
    b)将a)中加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥，得到表面改性的纳米颗粒；b) ultrasonically cleaning, centrifuging and drying the first organic solvent solution added with nanoparticles in a) to obtain surface-modified nanoparticles;
    (3)纳米颗粒的偶联活化：(3) Coupling activation of nanoparticles:
    a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中，搅拌后进行孵化，a) dispersing the surface-modified nanoparticles and cross-linking agent obtained in (2) into the second organic solvent, respectively, and incubating after stirring,
    b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥，得到活化后的纳米颗粒；b) centrifuging and drying the solution added with the surface-modified nanoparticles and cross-linking agent in a) to obtain activated nanoparticles;
    (4)纳米颗粒的pH调控：(4) pH regulation of nanoparticles:
    将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值，随后经离心-超声清洗，得到经表面改性和活化的纳米颗粒；Dispersing the activated nanoparticles obtained in (3) into a buffer to adjust the pH value, and then performing centrifugation-ultrasonic cleaning to obtain surface-modified and activated nanoparticles;
    (5)人参皂苷的表面改性：(5) Surface modification of ginsenosides:
    a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌，a) adding ginsenoside to the third organic solution containing the second surface modifier and stirring,
    b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥，得到表面改性的人参皂苷；b) ultrasonically cleaning, centrifuging and drying the third organic solvent solution added with ginsenosides in a) to obtain surface-modified ginsenosides;
    (6)纳米颗粒和人参皂苷的偶联：(6) Coupling of nanoparticles and ginsenosides:
    a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化；a) putting the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) into a fourth organic solvent for incubation;
    b)将a)中加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理，得到复合纳米药物。b) The fourth organic solvent solution in a) with the surface-modified and activated nanoparticles and the surface-modified ginsenosides is subjected to centrifugation-ultrasonic cleaning-centrifugation and then drying to obtain a composite nanomedicine.
  6. 根据权利要求5所述的纳米药物，其中步骤(1)中的Au@CoFeB纳米颗粒采用微流控法、水热法、磁控溅射法或电沉积法制备。The nanomedicine according to claim 5, wherein the Au@CoFeB nanoparticles in step (1) are prepared by a microfluidic method, a hydrothermal method, a magnetron sputtering method or an electrodeposition method.
  7. 根据权利要求5所述的纳米药物，其中所述纳米颗粒为核壳结构，核壳结构的金属内核为面心立方晶体结构的Au；核壳结构的壳层为面心立方晶体结构的CoFeB；The nanomedicine according to claim 5, wherein the nanoparticle has a core-shell structure, and the metal core of the core-shell structure is Au with a face-centered cubic crystal structure; the shell layer of the core-shell structure is CoFeB with a face-centered cubic crystal structure;
    所述纳米药物的整体结构为6-7.2nm的超小纳米药物单元偶联在一起构建的尺寸为250-350nm的纳米药物聚集体；纳米颗粒的动力学半径约100-200nm；The overall structure of the nano-drug is a nano-drug aggregate with a size of 250-350 nm constructed by coupling together ultra-small nano-drug units of 6-7.2 nm; the dynamic radius of the nano-particle is about 100-200 nm;
    所述纳米颗粒表面带有+7-12mV；所述纳米药物表面带有+25-30mV的正电势。The nanoparticle surface has +7-12mV; the nanomedicine surface has a positive potential of +25-30mV.
  8. 权利要求1-7中任一项纳米药物的制备方法，包括以下步骤：The preparation method of any one of nano-medicine in claim 1-7, comprises the following steps:
    (1)制备Au@CoFeB纳米颗粒；(1) Preparation of Au@CoFeB nanoparticles;
    (2)纳米颗粒的表面改性：(2) Surface modification of nanoparticles:
    a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌，a) adding the nanoparticles in (1) to the first organic solution containing the first surface modifier and stirring,
    b)将a)中的加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥， 得到表面改性的纳米颗粒；b) ultrasonically cleaning, centrifuging and drying the first organic solvent solution with nanoparticles added in a) to obtain surface-modified nanoparticles;
    (3)纳米颗粒的偶联活化：(3) Coupling activation of nanoparticles:
    a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中，搅拌后进行孵化，a) dispersing the surface-modified nanoparticles and cross-linking agent obtained in (2) into the second organic solvent, respectively, and incubating after stirring,
    b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥，得到活化后的纳米颗粒；b) centrifuging and drying the solution added with the surface-modified nanoparticles and cross-linking agent in a) to obtain activated nanoparticles;
    (4)纳米颗粒的pH调控：(4) pH regulation of nanoparticles:
    将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值，随后经离心-超声清洗，得到经表面改性和活化的纳米颗粒；Dispersing the activated nanoparticles obtained in (3) into a buffer to adjust the pH value, and then performing centrifugation-ultrasonic cleaning to obtain surface-modified and activated nanoparticles;
    (5)人参皂苷的表面改性：(5) Surface modification of ginsenosides:
    a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌；a) adding ginsenoside to the third organic solution containing the second surface modifier and stirring;
    b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥，得到表面改性的人参皂苷；b) ultrasonically cleaning, centrifuging and drying the third organic solvent solution added with ginsenosides in a) to obtain surface-modified ginsenosides;
    (6)纳米颗粒和人参皂苷的偶联：(6) Coupling of nanoparticles and ginsenosides:
    a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化；a) putting the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) into a fourth organic solvent for incubation;
    b)将a)中的加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理，得到复合纳米药物。b) The fourth organic solvent solution in a) with the surface-modified and activated nanoparticles and the surface-modified ginsenosides is subjected to centrifugation-ultrasonic cleaning-centrifugation and then drying to obtain a composite nanomedicine.
  9. 权利要求1-7中任一项所述的纳米药物在制备治疗肝癌药物中的应用。Application of the nanomedicine according to any one of claims 1 to 7 in the preparation of a medicine for treating liver cancer.
  10. 根据权利要求9中的应用，其中所述纳米药物中的Au@CoFeB的浓度为0.00001-5000μg/mL。The application according to claim 9, wherein the concentration of Au@CoFeB in the nanomedicine is 0.00001-5000 μg/mL.
PCT/CN2022/083976 2021-04-02 2022-03-30 Metal-organic composite nano-drug, preparation method therefor, and application thereof WO2022206815A1 (en)

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