CN113041363B - Magnetic hollow protein microsphere and preparation method and application thereof - Google Patents
Magnetic hollow protein microsphere and preparation method and application thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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- A—HUMAN NECESSITIES
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- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
- A61K9/5078—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings with drug-free core
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5094—Microcapsules containing magnetic carrier material, e.g. ferrite for drug targeting
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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Abstract
The invention discloses a magnetic hollow protein microsphere and a preparation method and application thereof. The magnetic hollow protein microsphere comprises an outer layer structure, an intermediate layer structure and a cavity structure, wherein the cavity structure is arranged in the intermediate layer structure, and the outer layer structure is arranged on the outer surface of the intermediate layer structure; the outer layer structure is a cross-linked protein membrane with a stimulus response characteristic, the middle layer structure is a drug-loaded layer loaded with a magnetic material and a drug, the cavity structure is formed by filling stable gas, and the particle size of the magnetic hollow protein microsphere is 2-10 mu m. The invention takes the hollow protein microspheres as a carrier, and utilizes an emulsion method to entrap magnetic materials and drugs so as to form a diagnosis and treatment integrated carrier with image diagnosis and targeted treatment, can be used for diagnosis and targeted treatment of atherosclerosis, and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of drug delivery systems, in particular to a magnetic hollow protein microsphere and a preparation method and application thereof.
Background
Atherosclerosis is an inflammatory disease characterized primarily by lipid deposition, mononuclear-macrophage infiltration, and plaque formation. The atherosclerotic plaque can make the wall of the artery continuously thicken and lose elasticity, and as the plaque is continuously enlarged, the atherosclerotic plaque can cause the stenosis of the lumen, which is an important pathological basis for causing ischemic cerebrovascular diseases. Plaque vulnerability is closely related to ischemic events, vulnerable plaque rupture, and subsequent formation of a thrombus or ulcer. Thrombus or related fragments fall off to form embolism under the impact of high-speed blood flow, and the embolism is an important reason for ischemic stroke of a patient. Therefore, accurate assessment of plaque vulnerability is of great significance in the prevention and treatment of atherosclerosis and the prevention of ischemic stroke events.
With the rapid development of material science, the carrier only used for drug delivery cannot meet the requirements of people on accurate treatment of diseases, so that the diagnosis and treatment integrated carrier with clinical diagnosis and treatment functions is widely concerned by researchers. The diagnosis and treatment integrated carrier can timely adjust the treatment scheme by monitoring the drug carrier and the disease tissues in real time, enhance the high efficiency and accuracy of treatment and realize accurate treatment. Based on this, it is very important to design and prepare a diagnosis and treatment integrated platform with accurate diagnosis and targeted therapy. Chinese patent CN109498592A discloses a hollow protein sustained-release microsphere and a preparation method thereof, although the elastic shell of the microsphere can realize force buffering, strong pressure resistance, difficult rupture in the delivery process and targeted drug delivery, the particle size of the microsphere is large, so that vascular administration for atherosclerosis diagnosis or treatment cannot be performed, and diagnosis and treatment integration of diagnosis and targeted treatment cannot be realized.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a magnetic hollow protein microsphere.
The second purpose of the invention is to provide a preparation method of the magnetic hollow protein microsphere.
The third purpose of the invention is to provide the application of the magnetic hollow protein microsphere.
The above object of the present invention is achieved by the following technical solutions:
the magnetic hollow protein microsphere comprises an outer layer structure, an intermediate layer structure and a cavity structure, wherein the cavity structure is arranged in the intermediate layer structure, and the outer layer structure is arranged on the outer surface of the intermediate layer structure; the outer layer structure is a cross-linked protein film with a stimulus response characteristic, the middle layer structure is a drug-loaded layer loaded with a magnetic material and a drug, and the cavity structure is formed by filling stable gas; the particle size of the magnetic hollow protein microsphere is 2-10 mu m.
The magnetic hollow protein microsphere takes the hollow protein microsphere as a carrier, a diagnosis and treatment integrated carrier with image diagnosis and targeted treatment can be formed by loading a magnetic material and a medicament in a medicament-carrying layer, the magnetic hollow protein microsphere has good magnetic targeting and contrast functions, can release the medicament under external stimulation, has the particle size of 2-10 mu m, can be administered through blood vessels, and is used for diagnosis and targeted treatment of diseases such as atherosclerosis and the like.
Preferably, the magnetic material is Fe3O4Nano particles,Fe2O3One or more of nanoparticles or metal doped ferrite nanomaterials.
Further preferably, the metal-doped ferrite nanomaterial is nano cobalt ferrite.
Preferably, the drug is one or more of a small molecule drug, a large molecule drug or a nano-drug. Such as the drug paclitaxel.
The invention also provides a preparation method of any one of the magnetic hollow protein microspheres, which comprises the following steps:
s1, adding a magnetic material and a medicine into a 10-20% protein solution, and uniformly mixing;
s2, adding an organic solvent containing a surfactant into the mixed solution obtained in the step S1, and uniformly mixing; the volume ratio of the protein solution to the organic solution containing the surfactant is 1: 3-8;
s3, continuously introducing gas into the mixed liquid obtained in the step S2, and homogenizing and stirring to form emulsion; the homogenizing and stirring speed is 18.0-20.0 Krpm;
s4, dropwise adding a cross-linking agent solution into the emulsion obtained in the step S3 to form a stable magnetic hollow protein microsphere solution; the concentration of the cross-linking agent is 20-40%;
s5, centrifuging the magnetic hollow protein microsphere solution prepared in the step S4, and removing the upper layer liquid to obtain the magnetic hollow protein microspheres loaded with the magnetic material and the medicine.
The invention takes protein as a material, utilizes an emulsion method to entrap a magnetic material and a micromolecule medicine in protein microspheres, further solidifies by a chemical crosslinking method, and successfully prepares the magnetic hollow protein microspheres loaded with the magnetic material and the medicine with the particle size of less than 10 mu m by controlling the concentration of a protein solution, the proportion of a water phase and an oil phase, the homogenizing and stirring speed and the concentration of a crosslinking agent. The magnetic hollow protein microspheres have good magnetic targeting and contrast functions, can release drugs under external stimulation, show good atherosclerotic plaque detection effect in vitro experiments, and can be used as an atherosclerosis diagnosis and treatment integrated platform.
Preferably, the concentration of the protein solution in the step S1 is 20%.
Preferably, the final concentration of the magnetic material and the drug in step S1 is 1-5 mg/mL (preferably 1 mg/mL); the mass ratio of the magnetic material to the medicine is 1: 1.
preferably, the surfactant in step S2 is span 80.
Further preferably, the volume ratio of the protein solution to the organic solution containing the surfactant in step S2 is 1: 6.
preferably, the gas introduced in step S3 is a biocompatible gas.
Further preferably, the gas is sulfur hexafluoride gas.
Preferably, the homogenizing and stirring speed in step S3 is 18.0 Krpm.
Preferably, the crosslinking agent in step S4 is terephthaloyl chloride.
Further preferably, the concentration of the cross-linking agent in step S4 is 25%.
Preferably, the centrifugation speed in step S5 is 0.5Krpm to 2.0Krpm (preferably 1.0 Krpm).
The magnetic hollow protein microsphere has good magnetic targeting and contrast functions, can release medicines under external stimulation, shows good atherosclerotic plaque detection effect in vitro experiments, and can be used as an atherosclerosis diagnosis and treatment integrated platform. Therefore, the invention also provides the application of any one of the magnetic hollow protein microspheres in preparing a target diagnosis product for atherosclerotic plaques.
Compared with the prior art, the invention has the following beneficial effects:
the magnetic hollow protein microsphere provided by the invention takes the hollow protein microsphere as a carrier, can form a diagnosis and treatment integrated carrier with image diagnosis and targeted therapy by encapsulating a magnetic material and a medicament, has a particle size of 2-10 mu m, can release the medicament under external stimulation, can be used for diagnosis and targeted therapy of atherosclerosis, and has a wide application prospect.
Drawings
Fig. 1 is a schematic structural diagram of the magnetic hollow protein microsphere of the present invention.
FIG. 2 is an optical microscope photograph of the hollow protein microspheres prepared in example 2.
FIG. 3 is an optical microscope photograph of the hollow protein microspheres prepared in example 3.
FIG. 4 is an optical microscope photograph of the hollow protein microspheres prepared in example 4.
FIG. 5 is an optical microscope photograph of the hollow protein microspheres prepared in example 5.
FIG. 6 is an optical microscope photograph of the magnetic hollow protein microspheres prepared in example 6.
FIG. 7 shows the result of the magnetic response characteristics of the hollow protein magnetic microspheres.
FIG. 8 shows the results of the magnetic hollow protein microspheres prepared in example 6 for the in vitro ultrasound imaging detection of atherosclerotic plaques.
FIG. 9 is the ultrasonic stimulated drug release curve of the magnetic hollow protein microsphere prepared in example 6.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1
As shown in fig. 1, the magnetic hollow protein microsphere comprises an outer layer structure 1, an intermediate layer structure 2 and a cavity structure 3, wherein the cavity structure 3 is arranged in the intermediate layer structure, and the outer surface of the intermediate layer structure 2 is provided with the outer layer structure 1; the outer layer structure 1 is a cross-linked protein film with a stimulus response characteristic, the middle layer structure 2 is a drug-loaded layer 2 loaded with a magnetic material and a drug, and the cavity structure 3 is formed by filling stable gas. The cross-linked protein membrane with the stimulus response characteristic is formed by the cross-linking reaction of bovine serum albumin and a cross-linking agentThe housing structure of (1); the magnetic material is Fe3O4Nanoparticles, the drug being paclitaxel; the stable gas is sulfur hexafluoride gas.
Example 2
Respectively adding 20 wt%, 30 wt% and 40 wt% BSA solutions into a 10% (v/v) span 80 toluene solution, wherein the volume ratio of the BSA solution to the span 80 toluene solution is 1: 10. homogenizing and stirring under the condition of continuously introducing sulfur hexafluoride gas, wherein the stirring speed is 10.0Krpm, and then slowly dropwise adding a cross-linking agent terephthaloyl chloride solution, wherein the concentration of the cross-linking agent is 25%, so that the stable hollow protein microspheres are formed. Centrifuging under the condition of 1.0Krpm, and removing the upper layer solution to obtain the hollow protein microspheres. FIG. 2 is an optical microscope image of hollow protein microspheres. (A) It was shown that the average particle size of the fibroin microspheres was 16.8 μm at a BSA concentration of 20 wt%; (B) it was shown that the average particle size of the fibroin microspheres was 25.3 μm at a BSA concentration of 30 wt%; (C) it was shown that the average particle size of the fibroin microspheres was 35.7 μm at a BSA concentration of 40 wt%. The BSA concentration of 20 wt% was chosen for subsequent experiments.
Example 3
Adding 20 wt% BSA solution into 10% (v/v) span 80 toluene solution, wherein the volume ratio of the BSA solution to the span 80 toluene solution is 1: 10. homogenizing and stirring under the condition of continuously introducing sulfur hexafluoride gas, wherein the stirring speed is 10.0Krpm, and then slowly dropwise adding a cross-linking agent terephthaloyl chloride solution, wherein the concentrations of the cross-linking agents are respectively 25% and 50%, so that the stable hollow protein microspheres are formed. Centrifuging under the condition of 1.0Krpm, and removing the upper layer solution to obtain the hollow protein microspheres. FIG. 3 is an optical microscope photograph of hollow protein microspheres. (A) It was shown that the average particle size of the fibroin microspheres was 16.8 μm at a crosslinker concentration of 25 wt%; (B) it was shown that the average particle size of the fibroin microspheres was 20.5 μm at a crosslinker concentration of 50 wt%. The crosslinker concentration was chosen to be 25% for subsequent testing.
Example 4
Adding 20% BSA solution into 10% (v/v) span 80 toluene solution, wherein the volume ratio of the BSA solution to the span 80 toluene solution is 1: 10. homogenizing and stirring under the condition of continuously introducing sulfur hexafluoride gas, wherein the stirring speed is 4.0Krpm, 10.0Krpm and 18.0Krpm respectively, and then slowly dropwise adding a cross-linking agent terephthaloyl chloride solution, wherein the concentration of the cross-linking agent is 25%, so that the stable hollow protein microspheres are formed. Centrifuging under the condition of 1.0Krpm, and removing the upper layer solution to obtain the hollow protein microspheres. Fig. 4 is an optical microscope image of the hollow protein microspheres. (A) It was shown that the average particle size of the hollow protein microspheres was 28.2 μm at a stirring speed of 4.0 Krpm; (B) it was shown that the average particle size of the hollow protein microspheres was 16.8 μm at a stirring speed of 10.0 Krpm; (C) it was shown that the average particle size of the hollow protein microspheres was 12.5 μm at a stirring speed of 18.0 Krpm. The subsequent experiments were carried out with a homogeneous stirring speed of 18.0 Krpm.
Example 5
Adding 20% BSA solution into 10% (v/v) span 80 toluene solution, wherein the volume ratio of the BSA solution to the span 80 toluene solution is 1: 10 and 1: 6. homogenizing and stirring under the condition of continuously introducing sulfur hexafluoride gas, wherein the stirring speed is 18.0Krpm, and then slowly dropwise adding a cross-linking agent terephthaloyl chloride solution, wherein the concentration of the cross-linking agent is respectively 25%, so that stable hollow protein microspheres are formed. Centrifuging under the condition of 1.0Krpm, and removing the upper layer solution to obtain the hollow protein microspheres. Fig. 5 is an optical microscope image of hollow protein microspheres. (A) The volume ratio of the BSA solution to the span 80 toluene solution was shown to be 1: when 10, the average grain diameter of the cardioglobulin microspheres is 12.5 mu m; (B) the volume ratio of the BSA solution to the span 80 toluene solution was shown to be 1: the average particle size of the fibroin microspheres at 6 hours was 7.0. mu.m. The volume ratio of the selectin solution to the span 80 toluene solution is 1: 6 subsequent tests were performed.
Example 6
Carrying magnetic material Fe3O4Preparing magnetic hollow protein microspheres of nanoparticles and paclitaxel: adding a magnetic material and a medicament (in a mass ratio of 1:1) with a final concentration of 1mg/mL into a BSA solution with a concentration of 20%, uniformly stirring, and continuously adding a toluene solution containing span 80 (10% (v/v)), wherein the volume ratio of the BSA solution to the span 80 toluene solution is 1: 6. homogenizing and stirring under the condition of continuously introducing sulfur hexafluoride gas, wherein the stirring speed is 18.0Krpm, then slowly dropwise adding a cross-linking agent paraphthaloyl chloride solution, wherein the concentration of the cross-linking agent is 25%, so as to form stable magneticHollow protein microspheres; centrifuging under the condition of 1.0Krpm, and removing the upper layer solution to obtain the magnetic hollow protein microspheres. FIG. 6 is an optical microscopic image of the magnetic hollow protein microsphere, showing that the magnetic hollow protein microsphere has a regular spherical structure with a particle size of less than 10 μm.
And (3) testing and analyzing the hollow protein magnetic microsphere powder with different magnetic particle contents by using a magnetic performance measuring system at 37 ℃, recording saturated magnetization data when an external magnetic field is 0-10000 Oe. As a result, as shown in FIG. 7, the hollow protein magnetic microsphere has a magnetic response characteristic.
Example 7
Carrying magnetic material Fe2O3Preparing magnetic hollow protein microspheres of nanoparticles and paclitaxel: adding a magnetic material and a medicament (in a mass ratio of 1:1) with a final concentration of 1mg/mL into a BSA solution with a concentration of 10%, uniformly stirring, and continuously adding a toluene solution containing span 80 (8% (v/v)), wherein the volume ratio of the BSA solution to the span 80 toluene solution is 1: 8. homogenizing and stirring under the condition of continuously introducing sulfur hexafluoride gas, wherein the stirring speed is 20.0Krpm, and then slowly dropwise adding a cross-linking agent paraphthaloyl chloride solution, wherein the concentration of the cross-linking agent is 20%, so that stable magnetic hollow protein microspheres are formed; centrifuging under the condition of 1.0Krpm, and removing the upper layer solution to obtain the magnetic hollow protein microspheres. The results show that the magnetic hollow protein microspheres have a regular spherical structure and the particle size is less than 10 mu m.
Example 8
The preparation of the magnetic hollow protein microsphere loaded with the magnetic nano material cobalt ferrite and the drug paclitaxel comprises the following steps: adding a magnetic material and a medicament (in a mass ratio of 1:1) with a final concentration of 1mg/mL into a BSA solution with a concentration of 15%, uniformly stirring, and continuously adding a toluene solution containing span 80 (12% (v/v)), wherein the volume ratio of the BSA solution to the span 80 toluene solution is 1: 4. homogenizing and stirring under the condition of continuously introducing sulfur hexafluoride gas, wherein the stirring speed is 19.0Krpm, and then slowly dropwise adding a cross-linking agent paraphthaloyl chloride solution, wherein the concentration of the cross-linking agent is 40%, so that stable magnetic hollow protein microspheres are formed; centrifuging under the condition of 1.0Krpm, and removing the upper layer solution to obtain the magnetic hollow protein microspheres. The results show that the magnetic hollow protein microspheres have a regular spherical structure and the particle size is less than 10 mu m.
Example 9
In vitro detection of atherosclerotic plaques: a1% w/v agarose solution was poured into a container and embedded in a silicone tube with gel fragments immobilized inside to simulate atherosclerotic plaques, and a prosthesis simulating vascular plaques was formed after the hydrogel was solidified. The channel of the simulated blood vessel was filled with the magnetic hollow protein microsphere solution of example 6, and then visualized using a portable ultrasound imaging device. The result is shown in fig. 8, after the magnetic hollow protein microsphere solution is introduced, the plaque prosthesis presents high ultrasonic signals, and the magnetic hollow protein microsphere can detect atherosclerotic plaques through ultrasonic contrast.
Example 10
The ultrasound-mediated magnetic hollow protein microsphere releases the medicine: 0.5 g of paclitaxel-loaded magnetic hollow protein microspheres (example 6) were weighed into a centrifuge tube and dissolved in 1mL of ultrapure water, followed by application of ultrasound (40KHz, 3.6W/cm)2) Stimulate drug release and the supernatant is taken at set time points for testing. The results are shown in fig. 9, and the magnetic hollow protein microspheres can release the drug by ultrasonic stimulation.
Claims (8)
1. The magnetic hollow protein microsphere is characterized by comprising an outer layer structure, an intermediate layer structure and a cavity structure; the cavity structure is arranged in the middle layer structure, an outer layer structure is arranged on the outer surface of the middle layer structure, the outer layer structure is a cross-linked protein film with a stimulus response characteristic, the middle layer structure is a drug-loaded layer loaded with a magnetic material and a drug, and the cavity structure is formed by filling stable gas; the particle size of the magnetic hollow protein microsphere is 2-10 mu m; the preparation method of the magnetic hollow protein microspheres comprises the following steps:
s1, taking a protein solution with the concentration of 10-20%, adding a magnetic material and a medicine, and uniformly mixing;
s2, adding an organic solvent containing 8-12% (v/v) of a surfactant into the mixed solution obtained in the step S1, and uniformly mixing; the volume ratio of the protein solution to the organic solution containing the surfactant is 1: 3-8;
s3, continuously introducing gas into the mixed liquid obtained in the step S2, and homogenizing and stirring to form emulsion; the homogenizing and stirring speed is 18.0-20.0 Krmp;
s4, dropwise adding a cross-linking agent solution into the emulsion obtained in the step S3 to form a stable magnetic hollow protein microsphere solution; the concentration of the cross-linking agent is 20-40%;
s5, centrifuging the magnetic hollow protein microsphere solution prepared in the step S4, and removing the upper layer liquid to obtain the magnetic hollow protein microspheres loaded with the magnetic material and the medicine.
2. The magnetic hollow protein microsphere of claim 1, wherein the magnetic material is Fe3O4Nanoparticles, Fe2O3One or more of nanoparticles or metal doped ferrite nanomaterials.
3. The magnetic hollow protein microsphere according to claim 1, wherein the drug is one or more of a small molecule drug, a large molecule drug or a nano-drug.
4. The magnetic hollow protein microsphere of claim 1, wherein the final concentration of the magnetic material and the drug in step S1 is 1-5 mg/mL.
5. The magnetic hollow protein microsphere of claim 1, wherein the surfactant in step S2 is span 80.
6. The magnetic hollow protein microsphere of claim 1, wherein the gas in step S3 is sulfur hexafluoride gas.
7. The magnetic hollow protein microsphere of claim 1, wherein the cross-linking agent in step S4 is terephthaloyl chloride.
8. Use of the magnetic hollow protein microsphere of any one of claims 1 to 7 in the preparation of an atherosclerotic plaque targeted diagnostic product.
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| CN110201194A (en) * | 2019-07-23 | 2019-09-06 | 山东百多安医疗器械有限公司 | A kind of load medicine contrast microbubbles and preparation method thereof for treating atherosclerotic plaque |
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| CN109498592A (en) * | 2017-09-15 | 2019-03-22 | 中山大学 | A kind of hollow albumen sustained-release micro-spheres and preparation method thereof |
| CN110201194A (en) * | 2019-07-23 | 2019-09-06 | 山东百多安医疗器械有限公司 | A kind of load medicine contrast microbubbles and preparation method thereof for treating atherosclerotic plaque |
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