CN114522253A - Multifunctional nanoparticles and preparation method and application thereof - Google Patents

Multifunctional nanoparticles and preparation method and application thereof Download PDF

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CN114522253A
CN114522253A CN202111604923.4A CN202111604923A CN114522253A CN 114522253 A CN114522253 A CN 114522253A CN 202111604923 A CN202111604923 A CN 202111604923A CN 114522253 A CN114522253 A CN 114522253A
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multifunctional
glycolic acid
mast cell
magnetic resonance
polylactic acid
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CN114522253B (en
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黄连弟
钟毅欣
伍宏耘
胡柳
廖钰琨
郭大静
冉海涛
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Second Affiliated Hospital of Chongqing Medical University
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Second Affiliated Hospital of Chongqing Medical University
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    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4535—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom, e.g. pizotifen
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    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
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    • 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
    • A61K47/6931—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 the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—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 the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
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    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
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    • A61K49/14—Peptides, e.g. proteins
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    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1857—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA
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Abstract

The invention relates to a multifunctional nanoparticle, which comprises a polylactic acid-glycolic acid copolymer, a mast cell degranulation inhibitor, a magnetic resonance contrast agent, a photosensitizer and a targeting ligand protein, wherein the mast cell degranulation inhibitor, the magnetic resonance contrast agent and the photosensitizer are coated in or embedded in the polylactic acid-glycolic acid copolymer, the outer surface of the polylactic acid-glycolic acid copolymer is provided with a terminal carboxyl group, and the targeting ligand protein is covalently connected with the terminal carboxyl group outside the polylactic acid-glycolic acid copolymer. The multifunctional nanoparticles can be gathered at the thrombus part, the imaging capability of the magnetic resonance and photoacoustic instruments on thrombus can be enhanced, the functions of analyzing thrombus components in an auxiliary manner, identifying the degree of disease attack and judging whether the thrombus is early thrombus or late thrombus can be realized, the thrombus formation can be inhibited, and the bleeding risk of patients can not be increased.

Description

Multifunctional nanoparticles and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a multifunctional nanoparticle and a preparation method and application thereof.
Background
Venous thrombosis is a common disease in clinical medicine, but the traditional imaging technology such as nuclear magnetism cannot accurately diagnose whether a patient suffers from thrombosis, cannot judge whether the suffered thrombosis belongs to early thrombosis or late thrombosis, and cannot analyze components of the thrombosis.
In addition, for patients who need to use images to determine whether they have thrombotic disorders, there is often a high risk of thrombus formation, and it is necessary to prevent thrombus formation even if it has not yet occurred. Although clinically common thrombus-preventing medicines such as aspirin and clopidogrel have a certain thrombus-preventing effect, the blood coagulation-anticoagulation system of a patient is changed, and the bleeding risk of the patient is increased.
Disclosure of Invention
Therefore, a multifunctional nanoparticle which can enhance imaging capability, help to analyze and identify thrombus components and thrombus incidence degree in an auxiliary manner and improve thrombus prevention effect, and a preparation method and application thereof are needed.
The invention provides a multifunctional nanoparticle, which comprises a polylactic acid-glycolic acid copolymer, a mast cell degranulation inhibitor, a magnetic resonance contrast agent, a photosensitizer and a targeting ligand protein, wherein the mast cell degranulation inhibitor, the magnetic resonance contrast agent and the photosensitizer are coated in or embedded in the polylactic acid-glycolic acid copolymer, the outer surface of the polylactic acid-glycolic acid copolymer is provided with a terminal carboxyl group, and the targeting ligand protein is covalently connected with the terminal carboxyl group outside the polylactic acid-glycolic acid copolymer.
In one embodiment, the mast cell degranulation inhibitor is a mixture of one or more of ketotifen fumarate and nedocromil; and/or
The magnetic resonance contrast agent is one or a mixture of more of oleic acid ferroferric oxide and oleic acid manganese dioxide; and/or
The photosensitizer is a mixture of one or more of IR780, IR830 and Ce 6; and/or
The targeting ligand protein is fibrin.
In one embodiment, the multifunctional nanoparticle is a spherical structure, and the mast cell degranulation inhibitor, the magnetic resonance contrast agent and the photosensitizer are coated inside the polylactic acid-glycolic acid copolymer; and/or
The multifunctional nanoparticles have superparamagnetism; and/or
The average grain diameter of the multifunctional nanoparticles is 268.87 +/-14.00 nm, and the dispersion coefficient in water is 0.06 +/-0.02; and/or
The average surface potential of the multifunctional nanoparticles is 0.46 +/-0.21 mV; and/or
The mast cell degranulation inhibitor has an encapsulation rate of 60% or more in the polylactic acid-glycolic acid copolymer.
The invention also provides a preparation method of the multifunctional nanoparticle, which comprises the following steps:
mixing the polylactic acid-glycolic acid copolymer, the mast cell degranulation inhibitor, the magnetic resonance contrast agent and the photosensitizer in a solvent, and performing ultrasonic emulsification to form a primary emulsion;
adding a polyvinyl alcohol aqueous solution into the primary emulsion, and performing ultrasonic emulsification to form a double emulsion;
adding an isopropanol aqueous solution into the double emulsion, stirring, centrifuging and removing a supernatant to form a first intermediate product;
adding EDC, NHS and a first MES buffer solution into the first intermediate product, incubating, centrifuging and discarding the supernatant to form a second intermediate product;
and adding a second MES buffer solution and the targeting ligand protein into the second intermediate product, incubating, centrifuging and removing a supernatant to form the multifunctional nanoparticle.
In one embodiment, the step of mixing the polylactic acid-glycolic acid copolymer, the mast cell degranulation inhibitor, the magnetic resonance contrast agent, and the photosensitizer in a solvent further comprises:
dissolving the mast cell degranulation inhibitor and the photosensitizer in dimethyl sulfoxide, and dissolving the polylactic acid-glycolic acid copolymer in dichloromethane;
adding the mast cell degranulation inhibitor and the photosensitizer after dissolution to a dichloromethane solution containing the polylactic acid-glycolic acid copolymer;
adding the magnetic resonance contrast agent to the dichloromethane solution.
In one embodiment, the polylactic acid-glycolic acid copolymer, the mast cell degranulation inhibitor, the magnetic resonance contrast agent, the photosensitizer and the targeting ligand protein are fed in a mass ratio of (80-120): (8-12): (8-12): (0.8-1.2): (8-12).
In one embodiment, the mass concentration of the polyvinyl alcohol aqueous solution is 1-5%; and/or
The mass concentration of the isopropanol aqueous solution is 1-3%.
In one embodiment, the rotating speed of the centrifugation is 8000 rpm-12000 rpm, and the temperature of the centrifugation is 3-5 ℃ in the process of forming the first intermediate product; and/or
In the process of forming the second intermediate product, the incubation is to add EDC, NHS and a first MES buffer solution into the first intermediate product, and then place the first intermediate product into a low-temperature shaker for incubation, wherein the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the second intermediate product, the centrifugal rotating speed is 8000-12000 rpm, and the centrifugal temperature is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticles, after a second MES buffer solution and the targeted ligand protein are added into the second intermediate product, the mixture is placed into a low-temperature shaking table for incubation, wherein the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticles, the centrifugal rotation speed is 8000 rpm-12000 rpm, and the centrifugal temperature is 3-5 ℃.
In one embodiment, the pH value of the first MES buffer is 5-6; and/or
The pH value of the second MES buffer solution is 7.5-8.5.
The invention also provides application of the multifunctional nanoparticles in any embodiment in preparation of a thrombus imaging preparation.
The external surface of the multifunctional nanoparticle is connected with the targeted ligand protein capable of targeting fibrin in thrombus, the multifunctional nanoparticle can be gathered at the thrombus part, a mast cell degranulation inhibitor, a magnetic resonance contrast agent and a photosensitizer are also loaded in the multifunctional nanoparticle, the magnetic resonance contrast agent and the photosensitizer have magnetic resonance imaging and photoacoustic imaging capabilities respectively, the imaging capability of magnetic resonance and a photoacoustic instrument on the thrombus can be enhanced, the function of combining the targeted ligand protein and targeting fibrin can be used for realizing the auxiliary analysis of thrombus components, the disease incidence degree can be identified, the function of judging whether early thrombus or late thrombus is realized, the mast cell degranulation inhibitor can inhibit the mast cell degranulation in vascular endothelial cells, the thrombus formation can be inhibited, and the bleeding risk of patients can not be increased.
Drawings
FIG. 1 is a structural characterization result of example 1, comparative example 1 and comparative example 2 of the present invention, wherein a is a transmission electron microscope image of the multifunctional nanoparticles KF/IR780-PLGA-CREKA in comparative example 1; b. c and d are transmission electron microscope images of the multifunctional nanoparticles KF/IR780-PLGA-CREKA in the embodiment 1; e is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 13O4-elemental map analysis of transmission electron microscopy images of CREKA; f is the multifunctional nanoparticle KF/IR780-PLGA-Fe3O in example 14VSM results for CREKA; g is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 13O4-CREKA nanoparticles and multifunctional nanoparticles KF/IR780-PLGA-Fe in comparative example 23O4The particle size distribution of the nanoparticles; h is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 13O4-CREKA and KF/IR780-PLGA-Fe as the multifunctional nanoparticle in comparative example 23O4The surface potential of (a); i is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 13O4-CREKA and KF/IR780-PLGA-Fe as the multifunctional nanoparticle in comparative example 23O4(ii) a Fourier infrared spectrum; j is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 13O4-CREKA and KF/IR780-PLGA-Fe as the multifunctional nanoparticle in comparative example 23O4The encapsulation efficiency of KF in (1); k is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 13O4-CREKA and KF/IR780-PLGA-Fe as the multifunctional nanoparticle in comparative example 23O4The drug release profile of KF in (1).
FIG. 2 shows the results of the nuclear magnetic imaging performance characterization of example 1 and comparative example 2 and comparative example 3, wherein a is the physiological saline solution injected in comparative example 3, and the multifunctional nanoparticles KF/IR in comparative example 2780-PLGA-Fe3O4And the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4-post CREKA nuclear magnetic imaging results; b is a nuclear magnetic signal intensity statistical chart corresponding to a; c is T in natural state2A linear correlation relationship graph between the relaxation rate and the fibrin grey value; d is the multifunctional nanoparticle KF/IR780-PLGA-Fe in injection example 13O4-post CREKA T2A linear correlation relationship graph between the relaxation rate and the fibrin grey value; e is T after injection of physiological saline in comparative example 32A linear correlation relationship graph between the relaxation rate and the fibrin grey value; f is the multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative injection example 23O4Last T2And (3) a linear correlation graph between the relaxation rate and the fibrin grey value.
FIG. 3 shows the photoacoustic imaging capability characterization results of example 1, comparative example 2 and comparative example 3, wherein a is the physiological saline solution injected in comparative example 3, and the multifunctional nanoparticles KF/IR780-PLGA-Fe injected in comparative example 23O4And the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4-post CREKA photoacoustic imaging results; b is a photoacoustic imaging signal intensity statistical graph corresponding to the a.
FIG. 4 is a result of characterizing thrombus preventing abilities of example 1, comparative example 3 and comparative example 4, in which a physiological saline solution of comparative example 3, KF of comparative example 4 and multifunctional nanoparticles KF/IR780-PLGA-Fe of example 1 were injected3O4-post CREKA nuclear magnetic imaging results; b is a nuclear magnetic signal intensity statistical chart corresponding to the a.
FIG. 5 is a graph showing the results of characterizing the influence of example 1 and comparative example 3 on blood coagulation function.
Detailed Description
The multifunctional nanoparticle of the present invention, the preparation method thereof, and the application thereof are further described in detail with reference to the following embodiments. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
An embodiment of the present invention provides a multifunctional nanoparticle, including a polylactic acid-glycolic acid copolymer, a mast cell degranulation inhibitor, a magnetic resonance contrast agent, a photosensitizer, and a targeting ligand protein, where the mast cell degranulation inhibitor, the magnetic resonance contrast agent, and the photosensitizer are coated with or embedded in the polylactic acid-glycolic acid copolymer, the outer surface of the polylactic acid-glycolic acid copolymer has a terminal carboxyl group, and the targeting ligand protein is covalently linked to the terminal carboxyl group outside the polylactic acid-glycolic acid copolymer.
The surface of the multifunctional nanoparticle is connected with the targeting ligand protein capable of targeting fibrin in thrombus, so that the multifunctional nanoparticle can be gathered at the thrombus part, the imaging capability of magnetic resonance and a photoacoustic instrument on the thrombus can be enhanced, the thrombus components can be analyzed in an auxiliary manner, the morbidity degree can be identified, and the thrombus formation can be inhibited.
In a specific example, the polylactic acid-glycolic acid copolymer has a molecular weight of 10000Da or more. The polylactic acid-glycolic acid copolymer has good biocompatibility, is nontoxic and degradable, and has good loading effect on functional substances such as mast cell degranulation inhibitors, magnetic resonance contrast agents, photosensitizers and the like by taking the polylactic acid-glycolic acid copolymer with the molecular weight of more than 10000Da as a carrier of the multifunctional nanoparticles. Further, the molecular weight of the polylactic acid-glycolic acid copolymer is 12000Da or more.
In a particular example, the mast cell degranulation inhibitor is a drug capable of inhibiting mast cell degranulation in the vascular endothelium. In particular, the mast cell degranulation inhibitor may be, but is not limited to, a mixture of one or more of ketotifen fumarate and nedocromil. The mast cell degranulation inhibitor can inhibit the degranulation of the mast cells in vascular endothelium so as to inhibit the formation of thrombus, thereby realizing the effect of preventing thrombus without increasing the bleeding risk of patients.
In a specific example, the magnetic resonance contrast agent is a contrast agent with superparamagnetic properties. Specifically, the magnetic resonance contrast agent may be, but is not limited to, a mixture of one or more of ferroferric oxide oleate and manganese dioxide oleate. The trimaran tetroxide and manganese dioxide modified by oleic acid have superparamagnetism and higher relaxation and sensitivity in magnetic resonance imaging.
In one particular example, the photosensitizer is a dye that enhances photoacoustic imaging capabilities. Specifically, the photosensitizer may be, but is not limited to, a mixture of one or more of IR780, IR830, and Ce 6. Wherein the chemical formula of IR780 is 11-chloro-1, 1' -di-n-propyl-3, 3,3',3' -tetramethyl-10, 12-trimethyleneindole tricarbocyanine iodide, the chemical formula of IR830 is 2- [2- [ 2-chloro-3- [2- (1, 3-dihydro-1, 1, 3-trimethyl-2H-benzo [ e ] indol-2-ylidene) ethylene ] -1-cyclohexen-1-yl ] vinyl ] -1,1, 3-trimethyl-1H-benzo [ e ] indol-4-methylbenzenesulfonate, and the chemical formula of Ce6 is chlorin e 6.
In one particular example, the targeting ligand protein has specificity for targeting fibrin in a thrombus. In particular, the targeting ligand protein may be, but is not limited to, a fusion protein.
In a specific example, the multifunctional nanoparticle is a spherical structure, and the mast cell degranulation inhibitor, the magnetic resonance contrast agent and the photosensitizer are coated inside the polylactic acid-glycolic acid copolymer.
In a specific example, the multifunctional nanoparticle has superparamagnetism, which is beneficial to improving the magnetic resonance imaging capability of the multifunctional nanoparticle.
In a specific example, the multifunctional nanoparticles have an average particle size of 268.87 ± 14.00nm and a dispersion coefficient in water of 0.06 ± 0.02.
In a specific example, the average surface potential of the multifunctional nanoparticle is 0.46 ± 0.21 mV.
In a specific example, the mast cell degranulation inhibitor has an encapsulation rate of 60% or more in the polylactic acid-glycolic acid copolymer. The multifunctional nanoparticle has a good effect of encapsulating the mast cell degranulation inhibitor, can effectively prevent thrombosis and reduce the bleeding risk of patients.
An embodiment of the present invention further provides a method for preparing multifunctional nanoparticles as in any one of the above examples, including the following steps S110 to S150.
Step S110: mixing polylactic acid-glycolic acid copolymer, mast cell degranulation inhibitor, magnetic resonance contrast agent and photosensitizer in solvent, and ultrasonic emulsifying to form primary emulsion.
Specifically, the step of mixing the polylactic acid-glycolic acid copolymer, the mast cell degranulation inhibitor, the magnetic resonance contrast agent, and the photosensitizer in the solvent in step S110 may be further subdivided into step S111 to step S113.
Step S111: the mast cell degranulation inhibitor and photosensitizer are dissolved in dimethyl sulfoxide, and the polylactic acid-glycolic acid copolymer is dissolved in dichloromethane.
Step S112: adding the dissolved mast cell degranulation inhibitor and photosensitizer into dichloromethane solution containing polylactic acid-glycolic acid copolymer.
Step S113: the magnetic resonance contrast agent is added to the dichloromethane solution.
Further, the time of the ultrasonic emulsification in the step S110 is 60S to 180S. The apparatus used for phacoemulsification may be, for example, an ultrasonic sonicator.
Step S120: adding polyvinyl alcohol water solution into the primary emulsion, and performing ultrasonic emulsification to form a double emulsion.
Furthermore, the concentration of the polyvinyl alcohol aqueous solution is 1 to 5 percent.
Further, the time of the ultrasonic emulsification in step S120 is 60S to 180S.
Step S130: an aqueous isopropanol solution was added to the double emulsion, stirred, centrifuged and the supernatant discarded to form a first intermediate.
Further, the concentration of the isopropanol aqueous solution is 1-3%.
Further, the mixture was stirred until methylene chloride was completely volatilized.
Furthermore, the rotating speed of the centrifugation is 8000rpm to 12000rpm, the temperature of the centrifugation is 3 ℃ to 5 ℃, and the centrifugation time is 8min to 20 min.
Step S140: adding EDC, NHS and a first MES buffer solution into the first intermediate product, incubating, centrifuging and discarding the supernatant to form a second intermediate product.
Specifically, the incubation is to add EDC, NHS and a first MES buffer solution into the first intermediate product, and then place the mixture into a low-temperature shaker for incubation, wherein the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃.
Further, the pH value of the first MES buffer solution is 5-6.
Specifically, the centrifugal rotating speed is 8000-12000 rpm, and the centrifugal temperature is 3-5 ℃.
Step S150: and adding a second MES buffer solution and the target ligand protein into the second intermediate product, incubating, centrifuging and removing the supernatant to form the multifunctional nanoparticle.
Specifically, the incubation is to add a second MES buffer solution and a targeting ligand protein into a second intermediate product, and then place the second intermediate product in a low-temperature shaking table for incubation, wherein the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃.
Further, the pH value of the second MES buffer solution is 7.5-8.5.
Specifically, the rotating speed of the centrifugation is 8000-12000 rpm, and the temperature of the centrifugation is 3-5 ℃.
In a specific example, the feeding mass ratio of the polylactic acid-glycolic acid copolymer, the mast cell degranulation inhibitor, the magnetic resonance contrast agent, the photosensitizer and the targeting ligand protein is (80-120): (8-12): (8-12): (0.8-1.2): (8-12). Within the mass ratio range, the polylactic acid-glycolic acid copolymer in the obtained multifunctional nanoparticles loads mast cell degranulation inhibitors, magnetic resonance contrast agents and photosensitizers, and has the best comprehensive effect of being connected with targeted ligand proteins.
An embodiment of the present invention further provides an application of the multifunctional nanoparticle as in any one of the above examples in preparing a thrombus imaging preparation.
The multifunctional nanoparticles can obviously improve the imaging capability of a nuclear magnetic resonance and photoacoustic instrument on thrombus by loading a magnetic resonance contrast agent and a photosensitizer, and the connected targeting ligand protein can target fibrin in the thrombus, so that the multifunctional nanoparticles are aggregated at the thrombus part. Further, the content of fibrin in the thrombus is continuously improved along with the enhancement of the incidence of disease degree, the more fibrin is in the thrombus of later period, the stronger the signal of nuclear magnetism or photoacoustic imaging, the multifunctional nanoparticles can assist in identifying the incidence of disease degree of the thrombus through magnetic resonance or photoacoustic imaging, judge whether the thrombus is in early period or later period through signal strength, and assist in analyzing thrombus components, judge whether only need to carry out anticoagulation or need to carry out thrombolysis. Furthermore, the multifunctional nanoparticles also wrap the mast cell degranulation inhibitor, so that the formation of thrombus can be prevented, the blood coagulation function cannot be influenced, and the bleeding risk is reduced.
The following are specific examples. In the following examples, all the starting materials are commercially available unless otherwise specified.
In the following specific examples, the polylactic acid-glycolic acid copolymer is available from Shandong Dai Ting Bio-technology Ltd, molecular mass 12000Da, abbreviated as PLGA;
the mast cell degranulation inhibitor is ketotifen fumarate, available from carbofuran technologies ltd, abbreviated by KF;
the magnetic resonance contrast agent is ferroferric oxide oleate, which is purchased from American biopharmaceutical limited company of America and has the abbreviation Fe3O4；
The photosensitizer is IR780, available from Bailingwei science and technology Limited, and abbreviated as IR 780;
the targeting ligand protein is a fusion protein, purchased from Jiangsu Qiangyao Biotechnology limited company, and abbreviated as CREKA.
Example 1:
step 1: forming a first intermediate KF/IR780-PLGA-Fe3O4：
Dissolving 0.5mg of IR780 and 5mg of ketotifen fumarate in 20 mul of dimethyl sulfoxide, adding the dissolved IR780, ketotifen fumarate and 50mg of PLGA into 2ml of dichloromethane, adding 200 mul of ferroferric oleate (25mg/ml), sonicating the mixture for 60s by using an ultrasonic sonicator to form colostrum, adding 8ml of PVA solution (mass concentration is 4%) into the colostrum by using an ultrasonic sonicator to obtain multiple emulsions, adding 10ml of isopropanol (mass concentration is 2%), magnetically stirring and evaporating the dichloromethane serving as an organic solvent at room temperature, and centrifuging for 10min under the conditions of the rotating speed of 10000rpm and the temperature of 4 ℃ to obtain a first intermediate product KF/IR780-PLGA-Fe3O4。
Step 2: forming multifunctional nano-particles KF/IR780-PLGA-Fe3O4-CREKA：
To the first intermediate KF/IR780-PLGA-Fe obtained3O4Adding 0.1mmol EDC, 0.5mmol NHS and 5ml MES buffer (PH is 5.2), placing in a low temperature shaking table at 80rpm and 4 ℃ for incubation for 3h, centrifuging at 10000rpm and 4 ℃ for 10min, discarding the supernatant, adding 0.1mmol MES buffer (PH is 8) and 5mg CREKA, placing in a low temperature shaking table at 80rpm and 4 ℃ for incubation for 12h, centrifuging at 10000rpm and 4 ℃ for 10min, discarding the supernatant to obtain the multifunctional nanoparticle KF/IR780-PLGA-Fe3O4-CREKA。
Comparative example 1:
comparative example 1 was prepared substantially the same as example 1 except that: comparative example 1 no oleic acid ferroferric oxide was added and the multifunctional nanoparticles formed were KF/IR 780-PLGA-CREKA.
Comparative example 2:
comparative example 2 is the same as preparation step 1 of example 1, except that: comparative example 2 is not connected with the targeted ligand protein CREKA, the preparation step 2 is not carried out, and the formed multifunctional nanoparticle is KF/IR780-PLGA-Fe3O4。
Comparative example 3:
the multifunctional nano-particle KF/IR780-PLGA-Fe3O4-CREKA was replaced with saline.
Comparative example 4:
the multifunctional nano particles KF/IR780-PLGA-Fe3O4-CREKA was replaced by KF.
As shown in fig. 1, the structures of example 1, comparative example 1 and comparative example 2 were characterized as follows:
a to d show that the multifunctional nanoparticles KF/IR780-PLGA-CREKA in the comparative example 1 are spherical structures, and the multifunctional nanoparticles KF/IR780-PLGA-Fe in the example 13O4CREKA is also a spherical structure and Fe3O4Successfully loaded on the surface of the multifunctional nanoparticle.
e shows that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4Elemental mapping analysis of transmission electron microscopy images of CREKA, including elements of carbon, iron, oxygen and sulfur, showing iron in the elemental mapping analysis, indicating Fe3O4Successfully loaded on the surface of the multifunctional nano-particle.
f shows that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4VSM result analysis of-CREK shows that the multifunctional nano-particle KF/IR780-PLGA-Fe3O4the-CREK nanoparticles have superparamagnetism.
g shows that the multifunctional nano-particle KF/IR780-PLGA-Fe in the comparative example 23O4Compared with the average particle size of the particles, the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4The average grain diameter of the-CREKA is 268.87 +/-14.00 nm, the dispersion coefficient in water is 0.06 +/-0.02, and after the multifunctional nanoparticle is connected with the targeting ligand protein, the grain diameter of the multifunctional nanoparticle can not be obviously influenced.
h shows that the multifunctional nanoparticles KF/IR780-PLGA-Fe in comparative example 23O4The surface potential of the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 1 is-17.70 + -0.44 mV compared with that of the multifunctional nanoparticles KF/IR780-PLGA-Fe3O4The surface potential of the CREKA is 0.46 +/-0.21 mV, and the negative potential is reduced, which indicates that the targeting ligand protein is successfully connected with the multifunctional nanoparticle.
i shows that the multifunctional nanoparticles KF/IR780-PLGA-Fe in comparative example 13O4-CREKA and the multifunctional nano-particle KF/IR780-PLGA-Fe in the comparative example 23O4The multifunctional nanoparticles KF/IR780-PLGA-Fe3O4CREKA at 3426.84cm-1And 1933.96cm-1The characteristic peak of (A) indicates KF/IR780-PLGA-Fe3O4of-COOH and of-NH in CREKA2The reaction forms amido bond, which shows that the target ligand protein is successfully connected with the multifunctional nano-particle.
j indicates that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4The encapsulation rate of KF in-CREKA exceeds 60 percent, compared with the multifunctional nano-particle KF/IR780-PLGA-Fe in comparative example 23O4Compared with the encapsulation efficiency of KF in (1), the encapsulation efficiency of KF is not obviously different after the KF is modified by the targeting ligand protein.
k indicates that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4The successful establishment of a KF slow-release system is proved by a CREKA drug release curve, and compared with the multifunctional nano-particle KF/IR780-PLGA-Fe in the comparative example 23O4The drug release curves of KF in (a) were compared, and there was no statistical difference.
The nuclear magnetic imaging capabilities of example 1 and comparative example 2 and comparative example 3 were characterized, specifically, the test methods were as follows:
jugular vein thrombosis models are established on SD rats and divided into three groups, and the physiological saline in the comparative example 3 and the multifunctional nanoparticles KF/IR780-PLGA-Fe in the comparative example 2 are respectively injected3O4And the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4CREKA, imaging of thrombi with nuclear magnetic resonance.
As shown in fig. 2, the results of the magnetic resonance imaging are as follows:
a and b show that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4The CREKA can obviously improve the imaging capability of nuclear magnetism on venous thrombosis, and the nuclear magnetic signal of late thrombosis is stronger than that of early thrombosis, which indicates that the fibrin content in the late thrombosis is more. Based on the above results, it is shown that the multi-function in example 1KF/IR780-PLGA-Fe nanoparticles3O4CREKA can help to identify whether a thrombus is early or late.
c-f show that the results of the linear analysis confirm T in the natural state2There is no linear correlation between the relaxation rate and the fibrin grayscale value. The multifunctional nanoparticles KF/IR780-PLGA-Fe in example 1 were injected3O4after-CREKA, T2There is a good linear correlation between the relaxation rate and the grey value of fibrin. The physiological saline in the comparative example 3 and the multifunctional nanoparticle KF/IR780-PLGA-Fe in the comparative example 2 were injected3O4Then, T2Almost nonexistent linear correlation relationship between relaxation rate and fibrin gray value shows that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4CREKA can help to analyze how much fibrin component is in the thrombus and help to guide individualized treatment of thrombus.
The photoacoustic imaging capabilities of example 1, comparative example 2, and comparative example 3 were characterized, specifically, the test methods were as follows:
jugular vein thrombosis models are established on SD rats and divided into three groups, and the physiological saline in the comparative example 3 and the multifunctional nanoparticles KF/IR780-PLGA-Fe in the comparative example 2 are respectively injected3O4And the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4CREKA, imaging with a photoacoustic instrument.
As shown in fig. 3, the imaging results are as follows:
a to b show that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4The CREKA can obviously improve the imaging capacity of the vein thrombus in a PI mode of a photoacoustic instrument, and the late thrombus has stronger signal than the early thrombus, which indicates that the fibrin content in the late thrombus is more.
The thrombus-inhibiting abilities of example 1, comparative example 3, and comparative example 4 were characterized, specifically, the test methods were as follows:
SD rats were divided into three groups, and physiological saline in comparative example 3, KF in comparative example 4, and multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 were injected respectively3O4-CREKA，After three days, thrombosis was induced separately and observed by nuclear magnetic imaging.
As shown in fig. 4, the thrombus prevention ability test results were as follows:
a to b show that KF in comparative example 4 and KF/IR780-PLGA-Fe, which is a multifunctional nanoparticle in example 13O4The incidence of thrombosis after CREKA treatment was significantly reduced, indicating that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4CREKA can prevent thrombosis.
Example 1 was characterized with respect to the effect of coagulation function, specifically the test method was as follows:
SD rats were divided into two groups, and physiological saline in comparative example 3 and multifunctional nanoparticles KF/IR780-PLGA-Fe in example 1 were injected respectively3O4CREKA, three hours later, two groups of SD rats were tested for clotting routine, including APTT, PT and TT, respectively.
As shown in fig. 5, the test results of the effect of the multifunctional nanoparticles on the blood coagulation function are as follows:
by multifunctional nano-particles KF/IR780-PLGA-Fe3O4After the action of-CREKA, the blood coagulation function is not obviously changed, which indicates that the multifunctional nanoparticles KF/IR780-PLGA-Fe in example 13O4CREKA does not increase the risk of bleeding.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims, and the description and drawings can be used for explaining the contents of the claims.

Claims (10)

1. A multifunctional nanoparticle comprising a polylactic acid-glycolic acid copolymer, a mast cell degranulation inhibitor, a magnetic resonance contrast agent, a photosensitizer, and a targeting ligand protein, wherein the mast cell degranulation inhibitor, the magnetic resonance contrast agent, and the photosensitizer are coated with or embedded in the polylactic acid-glycolic acid copolymer, the polylactic acid-glycolic acid copolymer has terminal carboxyl groups on the outer surface, and the targeting ligand protein is covalently linked to the terminal carboxyl groups on the outer portion of the polylactic acid-glycolic acid copolymer.
2. The multifunctional nanoparticle according to claim 1, wherein the mast cell degranulation inhibitor is a mixture of one or more of ketotifen fumarate and nedocromil; and/or
The magnetic resonance contrast agent is one or a mixture of more of oleic acid ferroferric oxide and oleic acid manganese dioxide; and/or
The photosensitizer is a mixture of one or more of IR780, IR830 and Ce 6; and/or
The targeting ligand protein is a fusion protein.
3. The multifunctional nanoparticle according to any one of claims 1 to 2, wherein the multifunctional nanoparticle is a spherical structure, and the mast cell degranulation inhibitor, the magnetic resonance contrast agent and the photosensitizer are coated inside the polylactic acid-glycolic acid copolymer; and/or
The multifunctional nanoparticles have superparamagnetism; and/or
The average grain diameter of the multifunctional nanoparticles is 268.87 +/-14.00 nm, and the dispersion coefficient in water is 0.06 +/-0.02; and/or
The average surface potential of the multifunctional nanoparticles is 0.46 +/-0.21 mV; and/or
The mast cell degranulation inhibitor has an encapsulation rate of 60% or more in the polylactic acid-glycolic acid copolymer.
4. A method for preparing multifunctional nanoparticles according to any one of claims 1 to 3, comprising the steps of:
mixing the polylactic acid-glycolic acid copolymer, the mast cell degranulation inhibitor, the magnetic resonance contrast agent and the photosensitizer in a solvent, and performing ultrasonic emulsification to form a primary emulsion;
adding a polyvinyl alcohol aqueous solution into the primary emulsion, and performing ultrasonic emulsification to form a double emulsion;
adding an isopropanol aqueous solution into the double emulsion, stirring, centrifuging and removing a supernatant to form a first intermediate product;
adding EDC, NHS and a first MES buffer solution into the first intermediate product, incubating, centrifuging and discarding a supernatant to form a second intermediate product;
and adding a second MES buffer solution and the targeting ligand protein into the second intermediate product, incubating, centrifuging and removing a supernatant to form the multifunctional nanoparticle.
5. The method of claim 4, wherein the step of mixing the poly (lactic-co-glycolic acid), the mast cell degranulation inhibitor, the magnetic resonance contrast agent, and the photosensitizer in a solvent further comprises:
dissolving the mast cell degranulation inhibitor and the photosensitizer in dimethyl sulfoxide, and dissolving the polylactic acid-glycolic acid copolymer in dichloromethane;
adding the mast cell degranulation inhibitor and the photosensitizer after dissolution to a dichloromethane solution containing the polylactic acid-glycolic acid copolymer;
adding the magnetic resonance contrast agent to the dichloromethane solution.
6. The preparation method of the multifunctional nanoparticle according to claim 4, wherein the feeding mass ratio of the polylactic acid-glycolic acid copolymer, the mast cell degranulation inhibitor, the magnetic resonance contrast agent, the photosensitizer and the targeting ligand protein is (80-120): (8-12): (8-12): (0.8-1.2): (8-12).
7. The preparation method of the multifunctional nanoparticles according to claim 4, characterized in that the mass concentration of the polyvinyl alcohol aqueous solution is 1-5%; and/or
The mass concentration of the isopropanol water solution is 1-3%.
8. The preparation method of the multifunctional nanoparticles according to any one of claims 4 to 7, wherein in the process of forming the first intermediate product, the rotation speed of centrifugation is 8000rpm to 12000rpm, and the temperature of centrifugation is 3 ℃ to 5 ℃; and/or
In the process of forming the second intermediate product, the incubation is to add EDC, NHS and a first MES buffer solution into the first intermediate product, and then place the first intermediate product into a low-temperature shaking table for incubation, wherein the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the second intermediate product, the centrifugal rotating speed is 8000 rpm-12000 rpm, and the centrifugal temperature is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticles, after a second MES buffer solution and the targeted ligand protein are added into the second intermediate product, the mixture is placed into a low-temperature shaking table for incubation, wherein the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticles, the centrifugal rotating speed is 8000-12000 rpm, and the centrifugal temperature is 3-5 ℃.
9. The preparation method of the multifunctional nanoparticles according to any one of claims 4 to 7, wherein the pH value of the first MES buffer is 5 to 6; and/or
The pH value of the second MES buffer solution is 7.5-8.5.
10. Use of the multifunctional nanoparticle of any one of claims 1 to 3 in the preparation of a thrombus imaging preparation.
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