CN114522253B - Multifunctional nanoparticle as well as preparation method and application thereof - Google Patents

Multifunctional nanoparticle as well as preparation method and application thereof Download PDF

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CN114522253B
CN114522253B CN202111604923.4A CN202111604923A CN114522253B CN 114522253 B CN114522253 B CN 114522253B CN 202111604923 A CN202111604923 A CN 202111604923A CN 114522253 B CN114522253 B CN 114522253B
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multifunctional nanoparticle
glycolic acid
polylactic acid
mast cell
acid copolymer
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CN114522253A (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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    • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
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    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
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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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    • 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
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    • 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
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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 by the polylactic acid-glycolic acid copolymer or embedded in the polylactic acid-glycolic acid copolymer, the external 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 nanoparticle can be gathered at a thrombus position, can enhance imaging capability of magnetic resonance and a photoacoustic instrument on thrombus, can realize functions of assisting in analyzing thrombus components, identifying morbidity degree and judging early thrombus or late thrombus, can inhibit thrombus formation, and does not increase bleeding risk of patients.

Description

Multifunctional nanoparticle as well as preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to a multifunctional nanoparticle as well as a preparation method and application thereof.
Background
Venous thrombosis is a common disorder in clinical medicine, but traditional imaging technologies such as nuclear magnetism cannot accurately diagnose whether a patient has thrombus, cannot judge whether the patient has thrombus belongs to early thrombus or late thrombus, and cannot analyze the components of thrombus.
In addition, for patients who need to use images to determine whether a thrombotic disorder exists, there is often a high risk of thrombosis, and it is necessary to prevent thrombosis even if it has not yet developed. The drugs for preventing thrombus such as aspirin and clopidogrel which are commonly used clinically at present have a certain effect of preventing thrombus, but have the effects of changing the coagulation-anticoagulation system of patients and increasing the bleeding risk of the patients.
Disclosure of Invention
Based on this, it is necessary to provide a multifunctional nanoparticle capable of enhancing imaging ability, helping to analyze and identify thrombus components and the extent of thrombus onset, and improving thrombus prevention effect, and a preparation method and application thereof.
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 by the polylactic acid-glycolic acid copolymer or embedded in the polylactic acid-glycolic acid copolymer, the external 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 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 in 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 nanoparticle has superparamagnetism; and/or
The average particle diameter of the multifunctional nanoparticle 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 nanoparticle is 0.46+/-0.21 mV; and/or
The encapsulation rate of the mast cell degranulation inhibitor in the polylactic acid-glycolic acid copolymer is more than 60 percent.
The invention also provides a preparation method of the multifunctional nanoparticle in any embodiment, 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 complex emulsion;
adding an aqueous isopropanol solution into the double emulsion, stirring, centrifuging, and discarding the 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 discarding the 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:
firstly dissolving the mast cell degranulation inhibitor and the photosensitizer in dimethyl sulfoxide, and dissolving the polylactic acid-glycolic acid copolymer in dichloromethane;
adding the dissolved mast cell degranulation inhibitor and the photosensitizer to a dichloromethane solution containing the polylactic acid-glycolic acid copolymer;
adding the magnetic resonance contrast agent into the dichloromethane solution.
In one embodiment, the ratio of the charge mass 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).
In one embodiment, 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%.
In one embodiment, during the formation of the first intermediate product, the rotational 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, after EDC, NHS and a first MES buffer solution are added into the first intermediate product, the second intermediate product is placed into a low-temperature shaking table for incubation, the shaking speed is 60 rpm-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the second intermediate product, the rotational speed of centrifugation is 8000-12000 rpm, and the temperature of centrifugation is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticle, after a second MES buffer solution and the targeting ligand protein are added into the second intermediate product, the second intermediate product is placed in a low-temperature shaking table for incubation, the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticle, the rotating speed of centrifugation is 8000-12000 rpm, and the temperature of centrifugation is 3-5 ℃.
In one embodiment, the first MES buffer has a pH of 5 to 6; and/or
The pH value of the second MES buffer solution is 7.5-8.5.
The invention also provides an application of the multifunctional nanoparticle in preparing a thrombus imaging preparation.
The external surface of the multifunctional nanoparticle is connected with the targeting ligand protein which can target fibrin in thrombus, so that the multifunctional nanoparticle can be gathered at a thrombus position, the multifunctional nanoparticle is also loaded with a mast cell degranulation inhibitor, a magnetic resonance contrast agent and a photosensitizer, the magnetic resonance contrast agent and the photosensitizer respectively have magnetic resonance imaging and photoacoustic imaging capabilities, the imaging capabilities of the magnetic resonance and photoacoustic imaging to thrombus can be enhanced, the function of assisting in analyzing thrombus components, identifying the morbidity and judging whether early thrombus or late thrombus can be realized by combining the targeting ligand protein, and the mast cell degranulation inhibitor can inhibit mast cell degranulation in vascular endothelial cells, so that thrombus formation can be inhibited, and the bleeding risk of a patient can not be increased.
Drawings
FIG. 1 is a structural characterization result of the present invention in example 1, comparative example 1 and comparative example 2, wherein a is a transmission electron microscope image of the multifunctional nanoparticle KF/IR780-PLGA-CREKA in comparative example 1; b. c and d are transmission electron microscope images of the multifunctional nanoparticle KF/IR780-PLGA-CREKA in the embodiment 1; e is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 -elemental map analysis of the transmission electron microscope image of CREKA; f is the multifunctional nanoparticle KF/IR780-PLGA-Fe3O in example 1 4 -VSM results of CREKA; g is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 CREKA nanoparticle and multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 Particle size distribution of nanoparticles; h is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 Multifunctional nanoparticle KF/IR780-PLGA-Fe in CREKA and comparative example 2 3 O 4 Is a surface potential of (a); i is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 Multifunctional nanoparticle KF/IR780-PLGA-Fe in CREKA and comparative example 2 3 O 4 Fourier infrared spectra of (a); j is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 Multifunctional nanoparticle KF/IR780-PLGA-Fe in CREKA and comparative example 2 3 O 4 Encapsulation efficiency of KF in (a); k is the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 Multifunctional nanoparticle KF/IR780-PLGA-Fe in CREKA and comparative example 2 3 O 4 Drug release profile of KF in (b).
FIG. 2 shows the results of characterization of the nuclear magnetic resonance imaging ability of examples 1 and 2 and 3, wherein a is the injection of physiological saline in comparative example 3 and the multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 And actual factMultifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 -a CREKA post nuclear magnetic imaging result; b is a nuclear magnetic signal intensity statistical graph corresponding to a; c is T in natural state 2 A linear correlation plot between relaxation rate and fibrin gray scale value; d is the multifunctional nanoparticle KF/IR780-PLGA-Fe in injection example 1 3 O 4 post-CREKA T 2 A linear correlation plot between relaxation rate and fibrin gray scale value; e is T after injection of physiological saline in comparative example 3 2 A linear correlation plot between relaxation rate and fibrin gray scale value; f is injection of multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 Post T 2 A plot of linear correlation between relaxation rate and fibrin grey scale value.
FIG. 3 is a result of characterization of photoacoustic imaging ability of example 1, comparative example 2 and comparative example 3, wherein a is physiological saline injected in comparative example 3, and multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 And multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 -CREKA post photoacoustic imaging results; b is a photoacoustic imaging signal intensity statistical graph corresponding to a.
FIG. 4 is a graph showing the results of characterization of 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 nanoparticle KF/IR780-PLGA-Fe of example 1 were injected 3 O 4 -a CREKA post nuclear magnetic imaging result; b is a nuclear magnetic signal intensity statistical graph corresponding to a.
FIG. 5 shows the results of the characterization of the effects of example 1 and comparative example 3 on coagulation function.
Detailed Description
The multifunctional nanoparticle of the present invention, the preparation method and application thereof are described in further detail below with reference to specific examples. 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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
An embodiment of 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 external surface of the polylactic acid-glycolic acid copolymer is provided with a carboxyl end, and the targeting ligand protein is covalently connected with the carboxyl end outside the polylactic acid-glycolic acid copolymer.
The surface of the multifunctional nanoparticle is connected with the targeting ligand protein which can target fibrin in thrombus, so that the multifunctional nanoparticle can be gathered at the thrombus position, the imaging capability of magnetic resonance and a photoacoustic instrument on thrombus can be enhanced, auxiliary analysis of thrombus components and identification of the morbidity degree can be realized, and 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, no toxicity and degradability, and has good loading effect on functional substances such as mast cell degranulation inhibitors, magnetic resonance contrast agents, photosensitizers and the like by adopting 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 specific example, the mast cell degranulation inhibitor is a drug capable of inhibiting mast cell degranulation in vascular endothelium. In particular, the mast cell degranulation inhibitor may be, but is not limited to being, a mixture of one or more of ketotifen fumarate and nedocromil. The mast cell degranulation inhibitor can inhibit the degranulation of mast cells in vascular endothelium, thereby inhibiting thrombosis, thereby realizing the effect of preventing thrombosis without increasing the risk of bleeding of patients.
In a specific example, the magnetic resonance contrast agent is a contrast agent with superparamagnetism. In particular, the magnetic resonance contrast agent may be, but is not limited to, a mixture of one or more of oleic acid ferroferric oxide and oleic acid manganese dioxide. The tetraoxide and manganese dioxide modified by oleic acid have superparamagnetism and have higher relaxation and sensitivity in magnetic resonance imaging.
In a specific example, the photosensitizer is a dye that enhances photoacoustic imaging capability. Specifically, the photosensitizer may be, but is not limited to, a mixture of one or more of IR780, IR830, and Ce 6. Wherein the chemistry of IR780 is known as 11-chloro-1, 1 '-di-n-propyl-3, 3' -tetramethyl-10, 12-trimethylene indole three-carbocyanine iodide salt, the chemistry of IR830 is known as 2- [ 2-chloro-3- [2- (1, 3-dihydro-1, 3-trimethyl-2H-benzo [ e ] indol-2-ylidene) ethylene ] -1-cyclohexen-1-yl ] vinyl ] -1, 3-trimethyl-1H-benzo [ e ] indol 4-methylbenzenesulfonate, and the chemistry of Ce6 is known as chlorin e6.
In a specific 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 in 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 nanoparticle has 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±0.21mV.
In a specific example, the encapsulation efficiency of the mast cell degranulation inhibitor within the polylactic acid-glycolic acid copolymer is 60% or more. The multifunctional nanoparticle encapsulated mast cell degranulation inhibitor has good effect, can effectively prevent thrombosis and reduce bleeding risk of patients.
An embodiment of the present invention further provides a method for preparing the multifunctional nanoparticle according to 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 steps S111 to S113.
Step S111: firstly, dissolving a mast cell degranulation inhibitor and a photosensitizer in dimethyl sulfoxide, and dissolving a polylactic acid-glycolic acid copolymer in dichloromethane.
Step S112: the dissolved mast cell degranulation inhibitor and the photosensitizer are added into methylene dichloride solution containing polylactic acid-glycolic acid copolymer.
Step S113: a magnetic resonance contrast agent is added to the dichloromethane solution.
Further, the time of the ultrasonic emulsification in the step S110 is 60S-180S. The instrument used for phacoemulsification may be, for example, an ultrasonic sonicator.
Step S120: adding aqueous solution of polyvinyl alcohol into the primary emulsion, and performing ultrasonic emulsification to form a double emulsion.
Further, the concentration of the polyvinyl alcohol aqueous solution is 1% -5%.
Further, the time of the ultrasonic emulsification in the step S120 is 60S-180S.
Step S130: an aqueous isopropanol solution was added to the double emulsion, stirred, and the supernatant was discarded by centrifugation to form a first intermediate product.
Further, the concentration of the isopropanol aqueous solution is 1% -3%.
Further, stirring was carried out until the methylene chloride was completely volatilized.
Further, the rotational speed of centrifugation is 8000 rpm-12000 rpm, the temperature of centrifugation is 3-5 ℃, and the centrifugation time is 8-20 min.
Step S140: EDC, NHS and first MES buffer were added to the first intermediate, incubated, and the supernatant was centrifuged off to form a second intermediate.
Specifically, the incubation refers to the steps of adding EDC, NHS and a first MES buffer solution into a first intermediate product, and then placing 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 ℃.
Further, the pH of the first MES buffer is 5 to 6.
Specifically, the rotational speed of the centrifugation is 8000 rpm-12000 rpm, and the temperature of the centrifugation is 3-5 ℃.
Step S150: and adding a second MES buffer solution and a targeting ligand protein into the second intermediate product, incubating, centrifuging, and discarding the supernatant to form the multifunctional nanoparticle.
Specifically, the incubation refers to that after a second MES buffer solution and a targeting ligand protein are added into a second intermediate product, the second intermediate product is placed in a low-temperature shaking table for incubation, the shaking speed is 60 rpm-100 rpm, and the temperature is 3-5 ℃.
Further, the pH of the second MES buffer is 7.5-8.5.
Specifically, the rotational speed of the centrifugation is 8000 rpm-12000 rpm, and the temperature of the centrifugation is 3-5 ℃.
In a specific example, the charge 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). In the mass ratio range, the polylactic acid-glycolic acid copolymer in the obtained multifunctional nanoparticle is loaded with mast cell degranulation inhibitor, magnetic resonance contrast agent and photosensitizer, and has the best comprehensive effect of being connected with the targeting ligand protein.
An embodiment of the present invention also provides a use of the multifunctional nanoparticle according to any of the above examples for preparing a thrombo-imaging preparation.
The multifunctional nanoparticle can remarkably improve imaging capability of nuclear magnetism and a photoacoustic instrument on thrombus by loading the magnetic resonance contrast agent and the photosensitizer, and the connected targeting ligand protein can target fibrin in the thrombus, so that the multifunctional nanoparticle is aggregated at the thrombus position. Further, the content of fibrin in thrombus is continuously increased along with the enhancement of the morbidity, the more fibrin in the thrombus is in the more advanced stage, the stronger the signals of nuclear magnetism or photoacoustic imaging are, the multifunctional nanoparticle can assist in identifying the morbidity of the thrombus through magnetic resonance or photoacoustic imaging through signal intensity, judging whether the thrombus is early-stage thrombus or late-stage thrombus, and assisting in analyzing thrombus components, and judging whether anticoagulation or thrombolysis is needed. Furthermore, the multifunctional nanoparticle also wraps the mast cell degranulation inhibitor, can prevent thrombosis, does not influence the blood coagulation function, and reduces the bleeding risk.
The following are specific examples. In the following examples, all materials are commercially available unless otherwise specified.
In the following specific examples, polylactic acid-glycolic acid copolymer was purchased from Shandong Dai Biotech Co., ltd, molecular mass 12000Da, abbreviated code PLGA;
the mast cell degranulation inhibitor is ketotifen fumarate, which is purchased from the technical company of the largehead, and abbreviated as KF;
the magnetic resonance contrast agent is ferric oxide oleate, which is purchased from American biological pharmaceutical Co Ltd, and abbreviated as Fe 3 O 4 ；
The photosensitizer is IR780, purchased from the Bacilvish technology Co., ltd, and abbreviated code number is IR780;
the targeting ligand protein is fusion protein purchased from Jiangsu Qiangli biotechnology Co., ltd, abbreviated code CREKA.
Example 1:
step 1: formation of the first intermediate KF/IR780-PLGA-Fe 3 O 4 ：
Dissolving 0.5mg of IR780 and 5mg of ketotifen fumarate in 20 mu l of dimethyl sulfoxide, adding the dissolved IR780, ketotifen fumarate and 50mg of PLGA into 2ml of dichloromethane, adding 200 mu l of oleic acid ferroferric oxide (25 mg/ml), carrying out acoustic shock on the mixture by an ultrasonic acoustic shock instrument for 60s to form colostrum, adding 8ml of PVA solution (with the mass concentration of 4%) into the mixture, carrying out acoustic shock for 60s by the ultrasonic acoustic shock instrument to obtain compound emulsion, adding 10ml of isopropanol (with the mass concentration of 2%), magnetically stirring at room temperature, evaporating the organic solvent dichloromethane, and centrifuging for 10min at the temperature of 4 ℃ at the speed of 10000rpm to obtain a first intermediate product KF/IR780-PLGA-Fe 3 O 4 。
Step 2: forming multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 -CREKA：
To the first intermediate KF/IR780-PLGA-Fe obtained 3 O 4 Adding 0.1mmole EDC, 0.5 mmole NHS and 5ml MES buffer (PH=5.2), placing in a low-temperature shaking table, incubating for 3h at 80rpm and 4 deg.C, centrifuging for 10min at 10000rpm and 4 deg.C, discarding supernatant, adding 0.1mmole MES buffer (PH=8) and 5mg CREKA, placing in a low-temperature shaking table, incubating for 12h at 80rpm and 4 deg.C, centrifuging for 10min at 10000rpm and 4 deg.C, discarding supernatant to obtain multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 -CREKA。
Comparative example 1:
comparative example 1 the preparation procedure was essentially the same as in example 1, with the difference from example 1 that: comparative example 1 without oleic acid ferroferric oxide, the multifunctional nanoparticle formed was KF/IR780-PLGA-CREKA.
Comparative example 2:
comparative example 2 is identical to the preparation step 1 of example 1, with the difference that of example 1: comparative example 2 non-attached targeting ligand protein CREKA, preparation step 2 was not performed, and the multifunctional nanoparticle formed was KF/IR780-PLGA-Fe 3 O 4 。
Comparative example 3:
multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 Replacement of CREKA with physiological saline.
Comparative example 4:
multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 -replacement of CREKA with KF.
As shown in fig. 1, the structures of example 1, comparative example 1 and comparative example 2 were characterized as follows:
a-d show that the multifunctional nanoparticle KF/IR780-PLGA-CREKA in comparative example 1 has a spherical structure, and the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 CREKA is also of spherical structure, and Fe 3 O 4 Successfully loaded on the surface of the multifunctional nanoparticle.
e shows that KF/IR780-PLGA-Fe was synthesized by the multifunctional nanoparticle of example 1 3 O 4 -elemental map analysis of transmission electron microscope images of CREKA, comprising elements of carbon, iron, oxygen and sulphur, the elemental map analysis showing the presence of elemental iron, indicating Fe 3 O 4 Successfully loaded on the surface of the multifunctional nanoparticle.
f shows that KF/IR780-PLGA-Fe was synthesized by the multifunctional nanoparticle of example 1 3 O 4 VSM result analysis of CREK, multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 The CREK nanoparticles have superparamagnetism.
g shows that the multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 The multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 compared with the average particle size 3 O 4 The average particle size of CREKA is 268.87 +/-14.00 nm, the dispersion coefficient in water is 0.06+/-0.02, and the particle size of the multifunctional nanoparticle does not have obvious influence after the targeting ligand protein is connected.
h indicates that the multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 Compared with the surface potential of-17.70+ -0.44 mV, the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 The surface potential of CREKA was 0.46±0.21mV, and negative potential was reduced, indicating successful attachment of the targeting ligand protein to the multifunctional nanoparticle.
i shows that in comparative example 1Multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 Multifunctional nanoparticle KF/IR780-PLGA-Fe in CREKA and comparative example 2 3 O 4 Is characterized by Fourier infrared spectrum of (A), multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 CREKA at 3426.84cm -1 And 1933.96cm -1 Characteristic peaks of (C) indicate KF/IR780-PLGA-Fe 3 O 4 of-COOH in (C) and-NH in CREKA 2 An amide bond is formed by the reaction, which indicates that the targeting ligand protein is successfully connected with the multifunctional nanoparticle.
j indicates that the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 Encapsulation efficiency of KF in CREKA exceeds 60%, and the multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 Compared with the encapsulation efficiency of KF, the encapsulation efficiency of KF does not have obvious difference after the targeting ligand protein is modified.
k indicates the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 The CREKA drug release profile demonstrates successful establishment of KF sustained release system, compared with the multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 3 O 4 There was no statistical difference in drug release profile of KF.
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:
a jugular vein thrombus model was established on SD rats and divided into three groups, and physiological saline in comparative example 3 and multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 were injected respectively 3 O 4 And multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 -CREKA, imaging thrombus with nuclear magnetism.
As shown in fig. 2, the nuclear magnetic imaging results are as follows:
a and b show the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 The CREKA can remarkably improve the imaging capability of nuclear magnetism on venous thrombosis, and the nuclear magnetic signals of late thrombus are stronger than those of early thrombus, so that the fibrin content in the late thrombus is higher. According to the results, the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 was shown 3 O 4 CREKA may help identify whether early or late thrombus.
c-f show that the linear analysis result proves that T is in the natural state 2 There is no linear correlation between the relaxation rate and the fibrin grey value. The multifunctional nanoparticle KF/IR780-PLGA-Fe of example 1 was injected 3 O 4 After CREKA, T 2 There is a good linear correlation between the relaxation rate and the fibrin grey value. Physiological saline in comparative example 3 and multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 were injected 3 O 4 Thereafter, T 2 A nearly nonexistent linear correlation between the relaxation rate and the gray value of fibrin indicates that the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 CREKA may help to analyze how much fibrin component is in the thrombus, helping to guide the individualized treatment of the thrombus.
The photoacoustic imaging ability of example 1, comparative example 2, and comparative example 3 was characterized, specifically, the test method was as follows:
a jugular vein thrombus model was established on SD rats and divided into three groups, and physiological saline in comparative example 3 and multifunctional nanoparticle KF/IR780-PLGA-Fe in comparative example 2 were injected respectively 3 O 4 And multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 CREKA, imaged with a sonographer.
As shown in fig. 3, the imaging results are as follows:
a-b show the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 The CREKA can remarkably improve the imaging capability of the photoacoustic instrument PI mode on venous thrombosis, and the advanced thrombosis has stronger signal than the early thrombosis, so that the fibrin content in the advanced thrombosis is higher.
The thrombus inhibition abilities of example 1, comparative example 3 and comparative example 4 were characterized, and specifically, the test methods were as follows:
SD rats were divided into three groups and respectively injected with physiological saline in comparative example 3, KF in comparative example 4 and multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 After three days, respectively, thrombosis is induced by CREKA, andobserved by nuclear magnetic imaging.
As shown in fig. 4, the thrombus prevention ability test results are as follows:
a-b show that KF/IR780-PLGA-Fe was modified by KF in comparative example 4 and multifunctional nanoparticle KF/IR in example 1 3 O 4 The occurrence rate of thrombus after CREKA treatment is obviously reduced, which shows that the multifunctional nanoparticle KF/IR780-PLGA-Fe in the example 1 3 O 4 CREKA may prevent thrombosis.
The influence of example 1 on the coagulation function was characterized, specifically, the test method was as follows:
SD rats were divided into two groups and physiological saline solution in comparative example 3 and multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 were injected, respectively 3 O 4 Three hours later, clotting routines, including APTT, PT and TT, were examined in two groups of SD rats, respectively.
As shown in fig. 5, the effect of the multifunctional nanoparticle on the coagulation function was tested as follows:
through multifunctional nanoparticle KF/IR780-PLGA-Fe 3 O 4 After CREKA, the clotting functions were not significantly changed, indicating that the multifunctional nanoparticle KF/IR780-PLGA-Fe in example 1 3 O 4 The CREKA does not increase the risk of bleeding.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention should therefore be determined from the appended claims, and the description and drawings may be used to interpret the contents of the claims.

Claims (10)

1. The multifunctional nanoparticle is characterized by 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 by the polylactic acid-glycolic acid copolymer or embedded in the polylactic acid-glycolic acid copolymer, the external surface of the polylactic acid-glycolic acid copolymer is provided with terminal carboxyl groups, and the targeting ligand protein is covalently connected with the terminal carboxyl groups outside the polylactic acid-glycolic acid copolymer; the mast cell degranulation inhibitor is ketotifen fumarate; the targeting ligand protein is fusion protein CREKA.
2. The multifunctional nanoparticle according to claim 1, wherein,
the magnetic resonance contrast agent is one or 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.
3. The multifunctional nanoparticle according to any one of claims 1 to 2, wherein the multifunctional nanoparticle is of 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 nanoparticle has superparamagnetism; and/or
The average particle diameter of the multifunctional nanoparticle 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 nanoparticle is 0.46+/-0.21 mV; and/or
The encapsulation rate of the mast cell degranulation inhibitor in the polylactic acid-glycolic acid copolymer is more than 60 percent.
4. A method for preparing the multifunctional nanoparticle 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 complex emulsion;
adding an aqueous isopropanol solution into the double emulsion, stirring, centrifuging, and discarding the 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 discarding the supernatant to form the multifunctional nanoparticle.
5. The method of preparing a multifunctional nanoparticle according to 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:
firstly dissolving the mast cell degranulation inhibitor and the photosensitizer in dimethyl sulfoxide, and dissolving the polylactic acid-glycolic acid copolymer in dichloromethane;
adding the dissolved mast cell degranulation inhibitor and the photosensitizer to a dichloromethane solution containing the polylactic acid-glycolic acid copolymer;
adding the magnetic resonance contrast agent into the dichloromethane solution.
6. The method of claim 4, wherein the poly (lactic-co-glycolic acid), the mast cell degranulation inhibitor, the magnetic resonance contrast agent, the photosensitizer and the targeting ligand protein are added in a mass ratio of (80-120): (8-12): (8-12): (0.8-1.2): (8-12).
7. The method for preparing the multifunctional nanoparticle according to claim 4, wherein the mass concentration of the aqueous solution of polyvinyl alcohol is 1% -5%; and/or
The mass concentration of the isopropanol water solution is 1-3%.
8. The method of preparing a multifunctional nanoparticle according to any one of claims 4 to 7, wherein in the process of forming the first intermediate product, the rotational 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, after EDC, NHS and a first MES buffer solution are added into the first intermediate product, the second intermediate product is placed into a low-temperature shaking table for incubation, the shaking speed is 60 rpm-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the second intermediate product, the rotational speed of centrifugation is 8000-12000 rpm, and the temperature of centrifugation is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticle, after a second MES buffer solution and the targeting ligand protein are added into the second intermediate product, the second intermediate product is placed in a low-temperature shaking table for incubation, the shaking speed is 60-100 rpm, and the temperature is 3-5 ℃; and/or
In the process of forming the multifunctional nanoparticle, the rotating speed of centrifugation is 8000-12000 rpm, and the temperature of centrifugation is 3-5 ℃.
9. The method of any one of claims 4 to 7, wherein the pH 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 for the preparation of a thrombo-imaging formulation.
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