CN115177741A - Metal-organic composite nano-drug and preparation method and application thereof - Google Patents

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

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CN115177741A
CN115177741A CN202110360364.0A CN202110360364A CN115177741A CN 115177741 A CN115177741 A CN 115177741A CN 202110360364 A CN202110360364 A CN 202110360364A CN 115177741 A CN115177741 A CN 115177741A
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nano
drug
nanoparticles
particles
cofeb
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宋玉军
赵枭雄
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University of Science and Technology Beijing USTB
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University of Science and Technology Beijing USTB
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Priority to PCT/CN2022/083976 priority patent/WO2022206815A1/en
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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

Abstract

The invention discloses a metal-organic composite nano-drug, which comprises heterostructure metal-based nano-particles and a drug component compound, wherein the heterostructure metal-based nano-particles are sequentially treated by a first surface modifier and a cross-linking agent, the drug component compound is treated by a second surface modifier, the nano-particles are coupled with the drug component compound through the cross-linking agent, the drug component compound is ginsenoside, and the nano-particles are Au @ CoFeB. The invention also discloses the preparation and application of the nano-drug. The nano-drug provided by the invention can well inhibit the development of liver cancer, and has the in vitro and living body molecular image tracing functions.

Description

Metal-organic composite nano-drug and preparation method and application thereof
Technical Field
The invention relates to the technical field of nano-drugs, in particular to a metal-organic composite nano-drug and a preparation method and application thereof.
Background
Nanoparticulates have controllable size, large mass ratio and unique physicochemical properties and are therefore useful at the molecular level for monitoring, control, diagnosis and treatment of biological systems and for tissue repair (Tekade, R.; maheshwari, R.; soni, N.; tekade, M.; chougle, M., nanotechnology-Based applications for Targeting and Delivery of Drugs and genes. Elsevier, amsterdam,2017, pp 3-61.). In recent years, the main methods for synthesizing nano-drugs have focused on the following aspects: 1) Diagnosis with consistent results is achieved by increasing the sensitivity and comprehensiveness of the analytical method (Alhareth, k.; sancy, l.; tsapis, n.; mignet, N., how shoulded we plate the future of nano-medicine for cancer diagnosis and therapy International Journal of pHarmaceutics 2017,532, (2), 657-659); 2) Drug delivery with biologically active agents (Peng, f.; zhang, w.; qiu, F., self-assembling Peptides in Current Nanomedicine, versatile Nanomaterials for Drug delivery. Current-real Medicinal Chemistry 2019,26, (1), 1-26.); 3) Tissue engineering and implants have been used to overcome limitations associated with vascular grafts (Hu, e; baylindrir-Buchhalter, I.; goebel, U.S., nanomedicine, biofab-location, tissue Engineering and Much More-Advanced Healthcare Materials Welcomes 2019.Advanced Healthcare Materials-properties 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, polymeric nanoparticles, iron oxides, metallic nanoparticles, dendrimers, liposomes, and the like. Thus, nano-drugs improve and extend the pharmacokinetics, solubility, and stability of a range of drug molecules that have been widely used in a variety of biomedical applications, including specific drug delivery, therapy, imaging, and diagnostics. However, the use of nanomedicines still has numerous unclear effects on human health and characteristics of various biological barriers to overcome, including stability, surface modification and functionalization, multimodal function, effective drug delivery, and balance and side effects between the two (Cencini, E.; sicuranza, A.; fabbri, A.; ferrigno, I.; rigacci, L.; cox, M.C.; raspadori, D.; bocchia, M., study of gene polymorph Hisms precursors of gene efficacy and toxicity in tissues with index-n-hod-hodgkin and mantle cell lysate, 8978), 89223-231).
Nanohybrids composed of noble metals and Magnetic components are expected to find wide application in fields 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 uniformity of Ni @ Ag core shell nonwovens with cellular catalytic efficiency 8978 z 8978, (38), 9; medical Imaging (S.D.; gwenin, V.V.; gwenin, C.D., magnetic Nanoparticles for use in medicine, and filtration, 14, detection of diseases (application) and filtration). In addition, appropriate 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 changes make certain mixed nanoparticles attractive features for diagnosis, imaging, drug delivery, therapy and even synthetic vaccine development and thus can be used as effective chemotherapeutic drugs for a variety of diseases (Chen, H.; liu F.; lei, Z.; ma, L.; wang, Z.; fe2O3@ Au core @ Shell Nanopane Nanocompositites as therapeutic Agents for Bioimaging and Chemo-pHothermal Synergistic therapy for RSC Advances 2015,5, (103), 84980-84987.). Thus, the controlled preparation and functionalization of multimodal nanomedicines extends their interdisciplinary applications to basic research and clinical applications (e.g., ultrasensitive bioprobes, highly potent nanomedicines and nanoenzymes, ultrasensitive biomedical molecular imaging and diagnosis of certain diseases, etc.).
The existing nano-drugs are either prepared by simply nano-crystallizing the original drugs, especially for organic or polymer drugs, nano-crystallizing improves the dispersibility of the drugs, but other properties, especially the curative effect is improved a little, but the toxic and side effects become larger, and the simple nano-crystallized drugs do not have the quantum effect and the surface activation effect which play an important role in the curative effect of metal-based nano-materials under the nano-scale. Pure inorganic nano-drugs such as inorganic non-metals like silicon dioxide and the like basically serve as carriers, and pure metals such as gold and the like can be good carriers for escape and metastasis of cancer cells, and have great side effects. In addition, the nanoparticles, whether inorganic non-based, organic polymer based or metal based, have large particle sizes at present, and each component is basically larger than 10nm, so that after entering the human body, the nanoparticles are difficult to be removed by epidermal network cells, renal tubules and the like of each organ or tissue of the human body, and have potential hazards of permanently retaining the human body. For inorganic non-metallic and metallic nanoparticles, the component size is preferably less than 6nm, but there is currently no method for mass production of such nanoparticles, which is very expensive at one time and unacceptable to patients for a while. The organic polymer nano-drugs, if decomposed continuously, have the damage and canceration effects of the decomposition products (especially the generated active functional groups) on normal tissues and organs. In addition, for the research of the mechanism of the curative effect, the nano-drug with the tracing image function which can be closely contrasted with the living body in vitro is needed, and the nano-drug is basically marked by a fluorescent agent which has short service life and is easy to be photosensitized and decomposed at present. Especially, for organic nano-drugs, it is almost impossible to conduct stable molecular imaging studies on their pharmacokinetics and pharmacodynamics mechanisms for a long time. Therefore, nano-drugs with both in vitro and in vivo molecular imaging tracing functions are urgently needed.
Disclosure of Invention
In order to solve the problems of the prior art, it is an object of the present invention to provide a metal-organic composite nano-drug comprising nanoparticles treated with a first surface modifier and an activator in this order and a drug ingredient compound treated with a second surface modifier; the nanoparticles are coupled to the drug ingredient compound via a cross-linking agent, wherein the drug ingredient compound is ginsenoside and the nanoparticles are au @ cofeb.
The nano-drug according to the present invention, wherein the first surface modifier is selected from at least one of 3-aminopropyltrimethoxysiloxane, vinyltrimethoxysilane, a phosphate bis-titanate coupling agent and diisopropoxydidiacetylacetone titanate; 3-aminopropyltrimethoxysilane is preferred.
According to the nano-drug, the second surface modifier is selected from at least one of 3-aminopropyltrimethoxy siloxane, 3-mercaptopropyl-triethoxy silane coupling agent, heptadecafluorodecyltrimethyloxy silane and isopropyl tri (dioctyl pyrophosphato acyloxy) titanate; preferably at least one of 3-aminopropyltrimethoxy siloxane, 3-mercaptopropyl-triethoxysilane coupling agent and isopropyltris (dioctylpyrophosphate) titanate.
The nano-drug according to the present invention, wherein the cross-linking agent is selected from at least one of suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (N-hydroxysuccinimide succinate), polyethylene glycol disuccinimide succinate, succinimidyl-succinate poly, ethylene glycol succinimidyl succinate and aziridine cross-linking agent XR-100; preferably at least one of suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (N-hydroxysuccinimide succinate), polyethylene glycol disuccinimide succinate, polyethylene glycol succinimidyl succinate and aziridine crosslinking agent XR-100.
The nano-drug according to the present invention, in a specific embodiment, is prepared by a method comprising the steps of:
(1) Preparing Au @ CoFeB nanoparticles;
(2) Surface modification of nanoparticles:
a) Adding the nano-particles in the step (1) into a first organic solution containing a first surface modifier for stirring,
b) Carrying out ultrasonic cleaning, centrifuging and drying on the first organic solvent solution added with the nano-particles in the step a) to obtain surface-modified nano-particles;
(3) Coupling and activating of the nanoparticles:
a) Dispersing the surface modified nano-particles obtained in the step (2) and the cross-linking agent into a second organic solvent respectively, stirring and then incubating,
b) Centrifuging and drying the solution added with the surface modified nano-particles and the cross-linking agent in the step a) to obtain activated nano-particles;
(4) pH regulation of nanoparticles:
dispersing the activated nano particles obtained in the step (3) into a buffer solution to adjust the pH value, and then carrying out centrifugal-ultrasonic cleaning to obtain surface modified and activated nano particles;
(5) Surface modification of ginsenoside:
a) Adding the ginsenoside into a third organic solution containing a second surface modifier and stirring;
b) Ultrasonically cleaning, centrifuging and drying a third organic solvent solution added with ginsenoside in a) to obtain surface-modified ginsenoside;
(6) Coupling of nanoparticles and ginsenoside:
a) Incubating the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) in a fourth organic solvent;
b) Centrifuging, ultrasonically cleaning, centrifuging and drying the fourth organic solvent solution added with the surface modified and activated nano-particles and the surface modified ginsenoside in the a) to obtain the composite nano-medicament.
The nano-drug is characterized in that the nano-particles are of a core-shell structure, and the metal inner core of the core-shell structure is Au of 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 the size of 250-350nm, which is constructed by coupling ultra-small nano-drug units with the size of 6-7.2 nm; the kinetic radius of the nanoparticles is about 100-200nm;
the surface of the nano particle is provided with +7-12mV; the surface of the nano-drug is provided with plus potential of 25-30 mV.
The nano-drug according to the present invention, wherein the first organic solvent is at least one selected from the group consisting of benzene, toluene, p-xylene and m-xylene; preferably at least one of toluene, p-xylene and m-xylene.
The second organic solvent is at least one selected from the group consisting of p-dimethyl sulfoxide, N-methylformamide, N-methyl-2-pyrrolidone, xylene and o-xylene; at least one of dimethyl sulfoxide, N-methylformamide and N-methyl-2-pyrrolidone is preferable.
The third organic solvent is at least one selected from p-xylene, o-xylene and toluene; toluene is preferred.
The fourth organic solvent is at least one selected from dimethyl sulfoxide, N-methyl pyrrolidone, N-ethyl acetamide and N-methyl formamide; preferably at least one of dimethylsulfoxide, N-methylpyrrolidone and N-ethylacetamide.
The buffer used in step (4) according to the nano-drug of the present invention may be based on a buffer known to those skilled in the art, and for example, the buffer may be phosphate PBS buffer, citric acid-Na 2 HPO 4 At least one of buffer, trizma buffer, fetal bovine serum FBS buffer, sodium carbonate-sodium bicarbonate buffer, and sodium acetate-acetic acid buffer, preferably phosphate buffer, fetal bovine serum FBS buffer, and citric acid-Na 2 HPO 4 At least one of the buffers is more preferably phosphate PBS buffer.
Another object of the present invention is to provide a method for preparing a composite nano-drug, comprising the steps of:
(1) Preparing Au @ CoFeB nanoparticles;
(2) Surface modification of nanoparticles:
a) Adding the nano-particles in the step (1) into a first organic solution containing a first surface modifier for stirring,
b) Carrying out ultrasonic cleaning, centrifuging and drying on the first organic solvent solution added with the nano-particles in the step a) to obtain surface-modified nano-particles;
(3) Coupling and activating of the nanoparticles:
a) Respectively dispersing the surface modified nano-particles obtained in the step (2) and a cross-linking agent into a second organic solvent, stirring and then incubating,
b) Centrifuging and drying the solution added with the surface modified nano-particles and the cross-linking agent in the step a) to obtain activated nano-particles;
(4) pH regulation of nanoparticles:
dispersing the activated nano particles obtained in the step (3) into a buffer solution to adjust the pH value, and then carrying out centrifugal-ultrasonic cleaning to obtain surface modified and activated nano particles;
(5) Surface modification of ginsenoside:
a) Adding ginsenoside into the third organic solution containing the second surface modifier and stirring;
b) Ultrasonically cleaning, centrifuging and drying a third organic solvent solution added with ginsenoside in a) to obtain surface-modified ginsenoside;
(6) Coupling of nanoparticles and ginsenoside:
a) Incubating the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) in a fourth organic solvent;
b) Centrifuging, ultrasonically cleaning, centrifuging and drying the fourth organic solvent solution added with the surface modified and activated nano-particles and the surface modified ginsenoside in the a) to obtain the composite nano-medicament.
As a preferred scheme, in the step (1), the nano-particles of Au @ CoFeB are prepared by a microfluidic method, a hydrothermal method, a magnetron sputtering method and an electrodeposition method; more preferably, microfluidic methods.
Specifically, the microfluidic method is a continuous flow reaction process constructed by using a micrometer scale (sub millimeter) to control the processes of mixing reactants, reaction nucleation, nanoparticle or drug growth and growth termination in the synthesis of nano materials or drugs in a reaction volume of μ L to pL or less. Compared with the traditional kettle type reactor, the method has the advantages of accurate design and regulation of kinetic parameters of different stages of reaction, rapid material and energy exchange, uniform mixing and reaction and capability of parallel amplification operation. The tank-free reactor has the characteristics of inevitable amplification effect, environmental friendliness, safety, minimized waste and capability of fully utilizing the high specific surface area effect of the micro-channel to regulate and control reaction products. The method for preparing the nano particles by the microfluidic method can refer to the prior art, and is not described in detail herein.
As a preferable mode, in the step (2) -a), the stirring time is preferably 20 to 28 hours; in steps (2) -b), the preferred conditions for centrifugation include: the centrifugal speed is 10000-20000rpm; the centrifugation time is 5-40 minutes; preferably 12000-16000rpm; the centrifugation time is 10-30 minutes.
As a preferable mode, in the step (3) -a), the stirring time is preferably 1 to 3 hours; in steps (3) -b), the preferred conditions for centrifugation include; the centrifugal speed is 10000-20000rpm; the centrifugation time is 5-15 minutes.
As a preferable scheme, in the step (4), the pH value is adjusted to 7-7.8; preferred conditions for centrifugation include; the centrifugal speed is 10000-14000rpm; the centrifugation time is 5-15 minutes.
As a preferable mode, in the step (5) -a), the stirring time is preferably 20 to 28 hours; preferred conditions for centrifugation include; the centrifugal speed is 10000-14000rpm; the centrifugation time is 5-15 minutes.
As a preferable mode, in the step (6) -a), the incubation time is preferably 1.5 to 2.0 hours; in the step (6) -a), the fourth organic solvent is preferably dimethyl sulfoxide.
The invention also aims to provide the application of the composite nano-medicament in preparing the medicament for treating liver cancer.
The application of the invention, wherein the concentration of Au @ CoFeB in the composite nano-drug is 0.00001-100000 mu g/mL.
The composite nano-drug provided by the invention can fully utilize the curative effect generated by the activity and quantum effect of multi-mode and surface multi-layer atomic layers of inorganic, especially metal-based nano-particles, the curative effect of organic drugs and the protective effect on metal inner layers and living organisms to construct the inorganic-organic composite nano-drug, fully exert the synergistic effect of the organic drugs and the inorganic nano-particles, obtain the original nano-drug with high curative effect, low or no toxic or side effect, and simultaneously has the functions of in vitro and living body molecular image tracing.
Drawings
FIG. 1 is a schematic diagram of a microfluidic device for synthesizing nanoparticles.
FIG. 2A is a wide-angle transmission electron micrograph of Au @ CoFeB-Rg3 nano-drug prepared by the process of example 1, wherein the upper right of the micrograph is a size distribution diagram, and the lower part of the micrograph is an enlarged view of a single particle image. FIGS. 2B-8 are XRD spectra of nanoparticles Au @ CoFeB prepared using the process of example 1. FIGS. 2B-9 are XRD spectra of nanoparticles Au @ CoFeB-Rg3 prepared using the process of example 1 and FIGS. 2C-10 are XPS spectra of nanoparticles Au @ CoFeB prepared using the process of example 1. FIG. 2C-11 is the XPS spectrum of the nano-drug Au @ CoFeB-Rg 3. FIG. 2D-12 is the FT-IR spectrum of nanoparticle Au @ CoFeB. FIG. 2D-13 is the FT-IR spectrogram of the nano-drug Au @ CoFeB-Rg 3. FIGS. 2E-14 are hydrodynamic diameter profiles of nanoparticles Au @ CoFeB prepared using the process of example 1. FIGS. 2E-15 are hydrodynamic diameter profiles of the NanoTagen Au @ CoFeB-Rg3 prepared using the process of example 1. FIGS. 2F-16 are surface Zeta potentials for nanoparticles Au @ CoFeB prepared using the process of example 1. FIG. 2F-17 is the surface Zeta potential of the nano-drug Au @ CoFeB-Rg3 prepared using the process of example 1.
FIGS. 3A-18 are surface plasmon scattering light patterns of Au @ CoFeB nanoparticles prepared using the process of example 2 and photographed by dark field microscopy. FIGS. 3A-19 are Au @ CoFeB nanoparticle surface plasmon scattering spectra characterized by a dark field microspectrometer prepared using the process of example 2. FIGS. 3B-20 are surface plasmon scattering light patterns of Au @ CoFeB-Rg3 nano-drugs photographed by dark field microscope prepared using the process of example 2. 3B-21 are surface plasmon scattering spectra of Au @ CoFeB-Rg3 nano-drugs characterized by dark field microspectrometers prepared using the process of example 2. FIG. 3C is a magnetic resonance image (upper image) and magnetic resonance relaxation rate (T.sub.2 WI concentration (0. Mu.g/mL, 7.5. Mu.g/mL, 15.1. Mu.g/mL, 31.3. Mu.g/mL, 93.5. Mu.g/mL, 187.0. Mu.g/mL)) dependent magnetic resonance image (upper image) and magnetic resonance relaxation rate (T.sub.0. Mu.g/mL) of Au @ CoFeB nanoparticles prepared using the procedure of example 2 2 -1 Unit is s -1 ) Concentration dependence curve of (a). FIG. 3D is the T2WI concentrations (0. Mu.g/mL, 4.1. Mu.g/mL, 12.4. Mu.g/mL, B/G) of Au @ CoFeB-Rg3 nano-drugs prepared using the procedure of example 2,37.3. Mu.g/mL, 112.0. Mu.g/mL) dependent magnetic resonance image (upper image) and magnetic resonance relaxation rate (T 2 -1 Unit is s -1 ) Concentration dependence curve of (a). FIG. 3E is a concentration-dependent curve of Au @ CoFe (B) -Rg3 nano-drug concentration (400. Mu.g/mL, 800. Mu.g/mL, 1100. Mu.g/mL, 1500. Mu.g/mL, 4000. Mu.g/mL) dependent computer-assisted tomography (CT) images (upper image in figure) and three-point averaged signal (HU) prepared using the process of example 2.
FIG. 4A is the 24 hour survival rate of Jurkat-CT (4A-22), 3T3 (4A-23), K562-CT (4A-24) and HEP-G2/C3A (4A-25) cells with concentration dependence of Au @ CoFeB nanoparticles prepared using the process of example 1. FIG. 4B is the 24-hour survival rate of cells with Jurkat-CT (4B-26), 3T3 (4B-27), K562-CT (4B-28) and HEP-G2/C3A (4B-29) dependent on the concentration of Au @ CoFeB-Rg3 nano-drug prepared using the process of example 1.
FIG. 5A is the cell proliferation survival rate of Jurkat-CT (5A-30), 3T3 (5A-31), K562-CT (5A-32) and HEP-G2/C3A (5A-33) cells dependent on the concentration of Au @ CoFeB nanoparticles prepared using the process of example 3. FIG. 5B is the cell proliferation survival rate of Jurkat-CT (5B-34), 3T3 (5B-35), K562-CT (5B-36) and HEP-G2/C3A (5B-37) with concentration dependence of Au @ CoFeB-Rg3 nano-drug prepared by the process of example 3.
FIG. 6 Au @ CoFeB nanoparticles and Au @ CoFeB-Rg3 nano-drug prepared using the process of example 4, PBS buffer solution and other nano-drugs (Fe @ Fe) 3 O 4 -Rg3、FePt@Fe 3 O 4 -Rg 3) at two concentrations (diagonal columns, 95-190 μ g/mL; mesh column, 474-947 ug/mL) for human chronic myelogenous leukemia cell K562 cancer cell, and the survival rate of the cells incubated for 24 hours.
FIG. 7A is an image of liver tumors after sacrifice of mice after different dosing regimens in vivo animal experiments using Au @ CoFeB-Rg3 and Au @ CoFeB nanoparticles anti-liver cancer efficacy prepared by the process of example 5. FIG. 7B is a fluorescence plot of liver tumor size at different stages of mice under different dosing regimens via bioluminescence labeling using in vivo animal experiments of anti-liver cancer efficacy of Au @ CoFeB-Rg3 and Au @ CoFeB nanoparticles prepared by the process of example 5. FIG. 7C is the absolute (upper fold) and relative weight change (lower fold) of mice in different groups within 21 days in the in vivo animal experiments using the anti-hepatoma efficacy of Au @ CoFeB (7C-40) and Au @ CoFeB-Rg3 (7C-41) and control saline (7C-38) and Rg3 (7C-39) prepared by the process of example 5. FIG. 7D is a graph showing the quantitative values of the bioluminescence intensity at the tumor site after different drug treatments in the in vivo animal experiments of anti-liver cancer efficacy of Au @ CoFeB nanoparticles (7D-44) and Au @ CoFeB-Rg3 (7D-45) prepared by the process of example 5 and normal saline (7D-42) and Rg3 (7D-43) of the control group.
Description of the reference numerals
1. A first syringe pump; 2. a second syringe pump; 3. a first microchannel tube; 4. a second microchannel tube; 5. a Y-shaped reaction material liquid mixer; 6. a third microchannel tube; 7. a collector.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Synthesis of nanoparticles and Nanopharmaceuticals
Example 1:
(1) Preparation of au @ cofeb nanoparticles:
as shown in FIG. 1, 0.42g of polyvinylpyrrolidone (PVP, K-30, weight average molecular weight 30,000), 0.07g (0.35 mmol) of FeCl 2 ·4H 2 O and 0.0833g (0.35 mmol) CoCl 2 ·6H 2 O was dissolved in 50ml of N-methyl-2-pyrrolidone (NMP) under nitrogen to form a metal salt solution. Then, 0.4g (10.5 mmol) of NaBH was added under nitrogen protection 4 Dissolved in 50ml of NMP to form a reducing solution.
Next, a microfluidic synthesis was performed at 120 ℃ under nitrogen using the procedure shown in fig. 1: 50ml of the PVP-bearing metal salt solution and 50ml of the reducing solution were introduced into each syringe separately, and the syringes were fixed to the first syringe pump 1 and the second syringe pump 2. Then the reaction mixture is led into a Y-shaped reaction material liquid mixer 5 through a first micro-flow channel pipe 3 and a second micro-flow channel pipe 4 to complete the reduction reaction, and the flow rates of a first injection pump 1 and a second injection 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 the nanoparticles. CoFeB nanoparticles were formed according to the following reaction scheme. When the reaction is completed, the obtained fresh nanoparticle dispersed solution is collected in the collector 7. The solution was then pelleted using a centrifuge at 15,000rpm for 30 minutes and the top supernatant was decanted. The obtained particles were washed twice with NMP to give pre-synthesized CoFeB nanoparticles.
Figure BDA0003005294960000101
2.5ml (0.36 mmol) of HAuCl 4 The solution was dissolved in 50ml of NMP. The microfluidic synthesis was then performed at room temperature under nitrogen protection using the apparatus shown in figure 1: 50ml of diluted HAuCl were injected using a first syringe pump 1 and a second syringe pump 2, respectively 4 The solution and 50ml of pre-synthesized CoFeB nanoparticle NMP solution are pumped into a first micro-flow channel pipe 3 and a second micro-flow channel pipe 4 and enter a Y-shaped reaction material liquid mixer 5 together to complete reduction reaction and rapid nucleation, and the flow rates of the first injection pump 1 and the second injection pump 2 are both 3ml/min. In this step, 0.2g of NaBH is added 4 Dissolved in 20ml of NMP and placed in the collector 7 beforehand. When the reaction was completed, the collector 7 was thoroughly shaken and left to stand for 30 minutes to complete the displacement, reduction and surface rearrangement of the CoFeB nanoparticles. 2ml of ethanol was then added to the collector to disrupt the equilibrium of the solution. The precipitated nanoparticles were re-dissolved in the same volume of NMP. The washing process was repeated twice to remove most of the surfactant. The final black slurry in the bottle was finally dried under vacuum to a black powder and stored in a desiccator for future use.
(2) Surface modification of nanoparticles:
10mg of the Au @ CoFeB nanoparticles prepared in step (1) were first dissolved in 50ml of an anhydrous toluene solution containing 1wt% of 3-Aminopropyltrimethoxysiloxane (APTMS). Then, the mixed solution was stirred at room temperature for 24 hours. After the completion of the stirring, the mixture was centrifuged at 12000rpm for 10 minutes using a centrifuge. Subsequently, the top supernatant was decanted and the precipitated nanoparticles were washed once with ethanol. And finally obtaining the Au @ CoFeB-APTMS nano-particles.
(3) Coupling and activating of the nanoparticles:
first, 5mg of prepared Au @ CoFeB-APTMS nanoparticles were dissolved in 5ml of dimethyl sulfoxide. Then, 5mg 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 12,000rpm for 10 minutes. Then, the supernatant at the top is poured out, and the finally precipitated nanoparticles in the bottle are dried under vacuum, so that the Au @ CoFeB-APTMS-DSS nanoparticles can be obtained.
(4) pH regulation of nanoparticles:
the dried sample was dissolved in 10mL of phosphate buffer solution (pH = 7.4) and the pH of the nanoparticles was adjusted. After centrifugation at 12,000rpm for 10 minutes, the final slurry in the bottle was washed with deionized water and dried under vacuum.
(5) Surface modification of ginsenoside:
200mg of ginsenoside Rg3 was first dissolved in 50mL of anhydrous toluene solution containing 1w% of APTMS. After stirring at ambient temperature for 24 hours, the mixed solution was centrifuged at 1200rpm for 10 minutes. Then, the precipitated slurry was washed 3 times with ethanol to obtain modified ginsenoside Rg3.
(6) Coupling of nanoparticles and ginsenoside:
after drying in vacuum, 5mg of surface modified ginsenoside Rg3 and 5mg of surface modified and activated Au @ CoFeB nanoparticles were dispersed in 5ml of dimethyl sulfoxide. Incubate at room temperature for 2 hours to achieve coupling between au @ cofeb nanoparticles and ginsenoside Rg3. The solution was then centrifuged and washed twice with deionized water. The precipitated slurry was then dried in vacuum to obtain Au @ CoFeB-Rg3 nano-drug.
Examples 2-5 all utilized the process and apparatus of example 1 to prepare nanoparticles, but varied some of the starting materials, starting material amounts, and reaction conditions, as shown in table 1.
TABLE 1 summary of the process parameters of examples 2-5
Figure BDA0003005294960000111
Figure BDA0003005294960000121
Figure BDA0003005294960000131
Figure BDA0003005294960000141
The microstructures and multimodal imaging properties of typical nanoparticles and nanomedicines prepared by the method of example 1 were characterized. According to a high-resolution TEM image (figure 2A) and an XRD spectrogram (figure 2B), before and after the nanoparticles are coupled with the drug, the metal core still keeps the core-shell structure of the Au and CoFeB alloy shell layer with the surface-centered cubic crystal structure; XPS spectra (fig. 2c, 10, 11) show that the nanopharmaceutical contains Au, co, fe, B, si (Ti), C, N (P) and O elements, where Si is mainly derived from the surface modification and cross-linking agent of the nanoparticles, C, N (P) and O are derived from organic pharmaceuticals and coupling agents, such as Rg3 and suberic acid (N-hydroxysuccinimide ester), and boron (B) is derived from the reducing agent used, sodium borohydride; when a phosphate bis-titanate coupling agent is used, si of the drug becomes Ti, and an element P is added. The FT-IR spectrogram (figure 2D) confirms that the method is used for obtaining the metal-organic compound nano-drug Au @ CoFeB-Rg3 (figure 2D; combining TEM image (fig. 2A) and hydrodynamic diameter (fig. 2E) detection results, it is shown that the overall structure of the nanoparticle and the nano-drug after the drug is coupled into the nano-drug (fig. 2e 15) is indeed a nano-drug aggregate with a size of about 300nm constructed by coupling together ultra-small nano-drug units of about 6.6 nm; the kinetic radius of the pure metal nanoparticles (fig. 2e 14) is about 150nm, mainly due to the CoFeB magnetic material of the shell layer of the inorganic part of the nano-drug, the magnetic dipole effect generated by the magnetic material has the function of gathering the nano-drug together, the nano-drug can also exist as aggregates in aqueous solution, the aggregates smaller than 1 micron are very beneficial to improving the retention time of the drug in vivo and the efficiency of transporting the drug to a focus, and the slightly acidic cell microenvironment at the focus can be rapidly dissociated into single drugs to improve the permeability to cells and tissues of the focus; the Zeta potential (fig. 2F) characterization result shows that the surfaces of the nanoparticles (fig. 2f: the medicine utilization rate is low and is less than 1.0 percent on average.
Fig. 3 is a multi-modal imaging functional characterization of nanoparticles and nano-drugs prepared by example 2. As shown in fig. 3A, for the magnetic heterostructure nanoparticles containing noble metal components, which have strong surface plasmon resonance (LSPR) scattering light characteristics, although they are only around 5nm, since they can scatter light with optical size of 400-600 nm, single nanoparticle images can be observed by optical microscope (fig. 3A. After the surface is modified and coupled with the drug, although the dielectric constant of the surface is changed, the LSPR scattering light intensity is still kept strong (figures 3B-20), and the image tracing can still be carried out by using a dark field microscope and a surface plasma spectrometer. Dark field microscopic LSPR optical images of the prepared Au @ CoFe (3A-18) and the Au @ CoFe-Rg3 nano-drug (3B-20) coupled with Rg3 and LSPR spectra (3B-21) of one particle (3A-19) and the drug are conjugated through the drug, and the scattered light of the whole particle is red-shifted due to the change of the surface dielectric constant.
FIG. 3C is a self-selection of pure Au @ CoFeB nanoparticles prepared by example 2 under T2WI experimental conditions-the echo sequence is 33ms for echo (TE) duration and 2500ms for pulse repetition time interval (TR) -it has very good T2WI enhancement effect, its relaxation rate (T2 WI enhancement effect) 2 -1 ，s -1 ) And the concentration of the nano particles have a good linear relation. FIG. 3D is the magnetic resonance imaging result of the synthesized pure Au @ CoFeB-Rg3 nano-drug under the T2WI experimental conditions with the echo sequence (TE) time of 33ms and the pulse repetition time interval (TR) of 2500ms, which has the good T2WI enhancement effect and the relaxation rate (T2 WI) as the nano-particles 2 -1 ，s -1 ) Has good linear relation with the concentration of the nano particles, and simultaneously shows that the relaxation rate of the nano medicament is higher than that of the nano particles, which indicates that the metal-organic composite is favorable for further enhancing the molecular image enhancement effect of the nano medicament.
FIG. 3E top image is the concentration dependence of the computer assisted tomography (CT) image and the three-point averaged signal (Henschel units: HU) at different concentrations of Au @ CoFeB-Rg3 nano-drug (400. Mu.g/mL, 800. Mu.g/mL, 1100. Mu.g/mL, 1500. Mu.g/mL, 4000. Mu.g/mL) (curves in the figure). Therefore, the method has excellent CT image effect; meanwhile, in a large concentration range, the CT value (HU is a measuring unit for measuring the density of a certain local tissue or organ of a human body, namely air is-1000, and compact bones are 1000) and the concentration have an excellent linear relation, and the clinical research on pathology and pharmacology can be carried out on line at high time-space resolution by a molecular image tracer method.
Application examples
Antitumor effects of nano-drugs Au @ CoFeB and Au @ CoFeB-Rg3 and pharmacological research thereof
Application example 1
The anti-tumor effect of the synthesized nano-drug and the toxic and side effect on normal cells are researched by taking a human chronic myelogenous leukemia K562 cell line and a liver cancer cell line (HEP-G2/C3A) as a cancer cell pathological model and taking a human epithelial fibroblast line 3T3 and a human immune cell Jurkat T cell line as a normal healthy cell model through a cell biology method.
Phosphate PBS buffer solutions for cell culture with different concentrations of Au @ CoFeB and Au @ CoFeB-Rg3 were prepared and passed through BD LSRFortessa TM The cell analyzer (BD biosciences) detects the 24-hour survival rate of various cells after the cells are incubated for 24 hours at different concentrations of Au @ CoFeB and Au @ CoFeB-Rg3 by using a light scattering method.
FIG. 4A shows the effect of Au @ CoFeB (concentration from 0.00001. Mu.g/mL to 500. Mu.g/mL) and Au @ CoFeB-Rg3 (concentration from 0.00001. Mu.g/mL to 1000. Mu.g/mL) prepared in example 1 on toxicity of normal cells (3 T3. The 24-hour cytotoxicity detection shows that (4A and 4B), the toxicity of the Au @ CoFeB-Rg3 nano-drug (figure 4B) prepared in example 1 on K562 cells (4B-28) and HEP-G2/C3A (4B-29) cells is obviously higher than that of Au @ CoFeB (4A, 24 and 25) and is also higher than the lethality of pure Rg3 on hepatoma cells and blood cancer cells, and the obvious synergistic antitumor effect between the heterostructure metal nanoparticles and the organic drug is demonstrated. The 24-hour toxicity of Au @ CoFeB on immune cells and blood cancer cells is obviously lower than that on 3T3 and liver cancer cells, which indicates that Au @ CoFeB has higher toxicity on cells capable of forming solid tissues than free suspension cells, and meanwhile, au @ CoFeB is shown to be at low concentration (9.5 mu g/mL). When the concentration of Au @ CoFeB is lower than 250 mu g/mL, the cytotoxicity is very low, and the concentration can be used as an optical biomolecule nano probe. When the concentration of Au @ CoFeB-Rg3 is 95 mug/mL, the lethal rate to K562 cells is strong; no more than this concentration was significantly toxic to other cells (cell survival > 70%). When the concentration of Au @ CoFeB-Rg3 exceeds 200 mug/mL, the lethality rate of the Au @ CoFeB-Rg3 to liver cancer cells and blood cancer cells is greatly improved; while this concentration is also significantly toxic to Jurkat cells, the toxicity to 3T3 is significantly less than for cancer cells.
Application example 2
The cell biology method takes a human chronic myelogenous leukemia K562 cell line and a liver cancer cell line (HEP-G2/C3A) as a cancer cell pathology model, and takes a human epithelial fibroblast line 3T3 and a human immune cell Jurkat T cell line as positiveThe normal healthy cell model researches the antitumor effect of the synthesized nano-drug and the toxic and side effect of normal cells. PBS buffers for cell culture were prepared at different concentrations of Au @ CoFeB and Au @ CoFeB-Rg3, and the buffer solutions were passed through BD LSRFortessa TM Cell analysis (BD biosciences available from BD biosciences Inc.) the 24-hour survival rate of each cell was measured by light scattering method after incubation for 24 hours at different concentrations of Au @ CoFeB and Au @ CoFeB-Rg 3.
The concentrations of Au @ CoFeB prepared in example 1 were 2500. Mu.g/mL, 10000. Mu.g/mL, 100000. Mu.g/mL, respectively, were used this time; the concentrations of Au @ CoFeB-Rg3 were 2500. Mu.g/mL, 20000. Mu.g/mL, 100000. Mu.g/mL. The toxicity of the compounds on leukemia cells K562 and liver cancer cells HEP-G2/C3A is detected, the cell survival rate in 24 hours is zero, and 100 percent of the compounds are killed.
Application example 3
Cell proliferation rates of K562, hepG2/C3A, and Jurkat cells at different concentrations of nanoparticles and nano-drugs prepared by example 3 were measured by cell titer-glo fluorescent cell viability assay (Promega, G7570) based on ATP cell viability assay using 96-well plates. Wherein the buffer solution is 10% FBS buffer solution, and the concentrations of Au @ CoFeB in the buffer solution are selected to be 0.00001 μ g/ml,9.5 μ g/ml,47.0 μ g/ml,95.0 μ g/ml and 474.0 μ g/ml; au @ CoFe-Rg3 was selected to be 0.00001. Mu.g/ml, 19. Mu.g/ml, 95.0. Mu.g/ml, 190.0. Mu.g/ml and 950.0. Mu.g/ml. Together in a cell culture incubator (3% CO) 2 And, after 4 days of incubation in 310K) the proliferation was examined.
The effect of nanoparticles and nano-drugs on cell proliferation indicates: when the concentration of Au @ CoFeB exceeds 50 mug/mL (figure 5A) and the concentration of Au @ CoFeB-Rg3 exceeds 100 mug/mL (figure 5B), the nano-drug has obvious inhibition effect on cancer cell proliferation but has no obvious toxicity on normal cell 3T3 (5A-31, 5B-35) no matter the nano-drug is a solid tumor liver cancer cell (HEP-G2/C3A: 5A-33, 5B-37) or a leukemia cancer cell (K562: 5A-32, 5B-36) which can flow freely; particularly, when the concentration of Au @ CoFeB-Rg3 reaches 950 [ mu ] g/mL, cancer cells almost all die after proliferation culture for 4 days. The proliferation inhibition effect research shows that Au @ CoFeB has obvious inhibition effect on the proliferation of cancer cells, and particularly, when the concentration reaches 474 mu g/mL, the proliferation of the cancer cells is basically and completely inhibited. One particular phenomenon is that when low concentrations of Au @ CoFeB are used (e.g., 19. Mu.g/mL), it can increase the proliferation rate of immune cells Jurkat (5A-30), but the nano-drugs have certain toxicity to it (5B-34), suggesting how to properly design the size, composition and structure of the nanoparticles, which can have the effect of increasing the activity of immune cells, and the nanoparticles of specific concentrations and components can be used in combination with immunotherapy to improve the cancer treatment effect in the future.
Application example 4
The cell proliferation rates of K562, hepG2/C3A, and Jurkat cells at different concentrations of the various nano-drugs prepared by example 3 were measured by cell titer-glo fluorescent cell viability assay (Promega, G7570) based on ATP cell viability assay using 96-well plates. The concentration of Au @ CoFeB used at this time is 2500 mu g/mL, 10000 and 100000 mu g/mL respectively; the concentrations of Au @ CoFeB-Rg3 were 5000. Mu.g/mL, 20000. Mu.g/mL, and 100000. Mu.g/mL. Together in a cell culture incubator (3% CO) 2 And, after 4 days of incubation in 310K) the proliferation was examined. Experimental results show that when the concentration of Au @ CoFeB exceeds 2500 mu g/mL and the concentration of Au @ CoFeB-Rg3 exceeds 5000 mu g/mL, the blood cancer cell K562 and the solid tumor liver cancer HepG2/C3A cell all die. While Jurkat cells and 3T3 cells still have 30% and more than 20% of the survival rate of 4-day proliferation culture respectively. The medicine has high lethality to cancer cells and certain toxicity to normal cells at high concentration, so the dosage of the medicine needs to be controlled in clinical application.
Application example 5
The Au @ CoFeB nano-particles and the Au @ CoFeB-Rg3 nano-drugs prepared by the process of the embodiment 4 and other nano-drugs (Fe @ Fe) are considered 3 O 4 -Rg3、FePt@Fe 3 O 4 -Rg 3) at two concentrations) of human chronic myelogenous leukemia cells K562 cancer cells incubated for 24 hours. As shown in fig. 6: a diagonal column with the drug concentration of 95-190 mug/mL; the concentration of the drug is 474-947 mug/mL. The result shows that the killing rate of the Au @ CoFeB-Rg3 nano-drug to the leukemia cells is lower than that of the Au @ CoFeB-Rg3 nano-drug and the CoFeB-Rg3 nano-drugThe concentration (95-190 mug/mL) is improved by nearly 7 times; at high concentrations (474-947. Mu.g/mL) the increase was 40-fold. While Au @ CoFeB nano-particles are higher than Fe @ Fe at high concentration 3 O 4 -Rg3 and FePt @ Fe 3 O 4 the-Rg 3 nano-drug is respectively improved by 27 times and 23 times; at low concentration than Fe @ Fe respectively 3 O 4 -Rg3 and FePt @ Fe 3 O 4 the-Rg 3 nano-drug is improved by 1.4 times and 1.5 times.
Application example 6
Nude mice were used and an orthotopic liver cancer model was established in their liver by dimethylnitrosamine. Administration was started after approximately 4 weeks of tumor growth, every two days with about 70mg/kg each, and was discontinued after 5 consecutive days, and the mice were then reared, weighed every 7 days, and the relative size of the tumor was determined by fluorescence analysis of the tumor site. Four groups were co-divided, one group was a group using physiological saline as a control, and the other three groups were a pure Rg3 group, a pure au @ cofeb prepared by example 5, and a nano-drug au @ cofeb-Rg3 group, respectively. By day 21, all mice were sacrificed and dissected, and the liver and its tumors were dissected and subjected to various biochemical and pathological section analyses. FIG. 7A is an image of the tumor of the last mouse under treatment with different drugs and controls, FIG. 7B is a fluorescence image of the tumor size of the mice characterized by fluorescence at different time points, and FIG. 7C is the absolute (upper fold line) and relative weight change (lower fold line) measured for different groups of mice at different time points after dosing (saline: 38 Rg3. FIG. 7D is a graph that measures tumor size at different time nodes after dosing for different groups based on fluorescence values (saline: 42, rg3: 44 Au @ CoFeB-Rg3: 45. It can be seen that the tumor of the simple Rg3 group (43) is slightly smaller than that of the control group (42), while the tumor of Au @ CoFeB (44) is slightly smaller than that of the control group, which indicates that Au @ CoFeB has strong anti-liver cancer effect; the strongest inhibition effect on mouse tumor is a group of nano-drugs compounded by Au @ CoFeB and Rg3, and it can be seen that after the administration, the nano-drugs have more and more obvious inhibition effect on the development of liver cancer along with the administration and the time lapse after the administration (45).

Claims (10)

1. A metal-organic composite nano-drug comprises heterostructure metal-based nanoparticles treated by a first surface modifier and a cross-linking agent in sequence and a drug component compound treated by a second surface modifier, wherein the nanoparticles are coupled with the drug component compound through the cross-linking agent, and the drug component compound is ginsenoside, and the nanoparticles are Au @ CoFeB.
2. The nano-drug of claim 1, wherein the first surface modifier is selected from at least one of 3-aminopropyltrimethoxysiloxane, vinyltrimethoxysilane, a phosphate bis-titanate coupling agent, and diisopropoxydidiacetone titanate; 3-aminopropyltrimethoxysiloxane is preferred.
3. The nano-drug of claim 1, wherein the cross-linking agent is selected from at least one of suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (N-hydroxysuccinimide succinate), polyethylene glycol disuccinimidyl succinate, succinimidyl-succinate, polyethylene glycol succinimidyl-succinate, and aziridine cross-linking agent XR-100; preferably at least one of suberic acid (N-hydroxysuccinimide ester), ethylene glycol bis (N-hydroxysuccinimide succinate), polyethylene glycol disuccinimide succinate, polyethylene glycol succinimidyl succinate and aziridine crosslinking agent XR-100.
4. The nano-drug of claim 1, wherein the second surface modifier is selected from at least one of 3-aminopropyltrimethoxy siloxane, 3-mercaptopropyl-triethoxysilane coupling agent, heptadecafluorodecyltrimethoxysilane, and isopropyltris (dioctylpyrophosphate) titanate; preferably at least one of 3-aminopropyltrimethoxy siloxane, 3-mercaptopropyl-triethoxysilane coupling agent and isopropyltris (dioctylpyrophosphate) titanate.
5. The nano-drug of claim 1, wherein the nano-drug is prepared using a method comprising the steps of:
(1) Preparing Au @ CoFeB nanoparticles;
(2) Surface modification of nanoparticles:
a) Adding the nanoparticles in the step (1) into a first organic solution containing a first surface modifier, stirring,
b) Carrying out ultrasonic cleaning, centrifuging and drying on the first organic solvent solution added with the nanoparticles in the step a) to obtain surface-modified nanoparticles;
(3) Coupling and activating of the nanoparticles:
a) Dispersing the surface modified nano-particles obtained in the step (2) and the cross-linking agent into a second organic solvent respectively, stirring and then incubating,
b) Centrifuging and drying the solution added with the surface modified nano-particles and the cross-linking agent in the step a) to obtain activated nano-particles;
(4) pH regulation of nanoparticles:
dispersing the activated nano particles obtained in the step (3) into a buffer solution to adjust the pH value, and then carrying out centrifugal-ultrasonic cleaning to obtain surface modified and activated nano particles;
(5) Surface modification of ginsenoside:
a) Adding ginsenoside into the third organic solution containing the second surface modifier, stirring,
b) Ultrasonically cleaning, centrifuging and drying a third organic solvent solution added with ginsenoside in a) to obtain surface-modified ginsenoside;
(6) Coupling of nanoparticles and ginsenoside:
a) Incubating the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) in a fourth organic solvent;
b) And (b) carrying out centrifugal-ultrasonic cleaning-centrifugal drying treatment on the fourth organic solvent solution added with the surface modified and activated nano-particles and the surface modified ginsenoside in the step a) to obtain the composite nano-medicament.
6. The nano-drug according to claim 5, wherein the Au @ CoFeB nano-particles in step (1) are prepared by a microfluidic method, a hydrothermal method, a magnetron sputtering method or an electrodeposition method.
7. The nano-drug according to claim 5, wherein the nano-particle is of a core-shell structure, and the metal core of the core-shell structure is Au of 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 the size of 250-350nm, which is constructed by coupling ultra-small nano-drug units with the size of 6-7.2 nm; the kinetic radius of the nanoparticles is about 100-200nm;
the surface of the nano particle is provided with +7-12mV; the surface of the nano-drug is provided with plus potential of 25-30 mV.
8. A process for the preparation of a nano-drug according to any one of claims 1 to 7, comprising the steps of:
(1) Preparing Au @ CoFeB nanoparticles;
(2) Surface modification of nanoparticles:
a) Adding the nanoparticles in the step (1) into a first organic solution containing a first surface modifier, stirring,
b) Carrying out ultrasonic cleaning, centrifuging and drying on the first organic solvent solution added with the nano-particles in the step a) to obtain surface-modified nano-particles;
(3) Coupling and activating of the nanoparticles:
a) Dispersing the surface modified nano-particles obtained in the step (2) and the cross-linking agent into a second organic solvent respectively, stirring and then incubating,
b) Centrifuging and drying the solution added with the surface modified nano-particles and the cross-linking agent in the step a) to obtain activated nano-particles;
(4) pH regulation of nanoparticles:
dispersing the activated nano particles obtained in the step (3) into a buffer solution to adjust the pH value, and then carrying out centrifugal-ultrasonic cleaning to obtain surface modified and activated nano particles;
(5) Surface modification of ginsenoside:
a) Adding ginsenoside into the third organic solution containing the second surface modifier and stirring;
b) Ultrasonically cleaning, centrifuging and drying a third organic solvent solution added with ginsenoside in a) to obtain surface-modified ginsenoside;
(6) Coupling of nanoparticles and ginsenoside:
a) Incubating the surface-modified and activated nanoparticles obtained in step (3) and the surface-modified ginsenosides obtained in step (5) in a fourth organic solvent;
b) Centrifuging, ultrasonically cleaning, centrifuging and drying the fourth organic solvent solution added with the surface modified and activated nano-particles and the surface modified ginsenoside in the a) to obtain the composite nano-medicament.
9. Use of the nano-drug of any one of claims 1 to 7 in the preparation of a medicament for treating liver cancer.
10. Use according to claim 9, wherein the concentration of au @ cofeb in the nano-drug is 0.00001-5000 μ g/mL.
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