CN112472875A

CN112472875A - Heparinized magnetic nanoparticles and preparation method and application thereof

Info

Publication number: CN112472875A
Application number: CN201910865484.9A
Authority: CN
Inventors: 刘文广; 吴元昊
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-09-12
Filing date: 2019-09-12
Publication date: 2021-03-12

Abstract

The invention discloses heparinized magnetic nanoparticles with an anticoagulant function and a preparation method and application thereof. The magnetic nano-particles have high specific surface area, can increase the contact area of heparin and blood, can uniformly spread the surface of a base material under the action of a magnetic field, have good biocompatibility, and have simple and easy preparation method.

Description

Heparinized magnetic nanoparticles and preparation method and application thereof

Technical Field

The invention relates to heparinized magnetic nanoparticles (FeNPs-HepDA) and a preparation method and application thereof, in particular to core-shell mechanism nanoparticles formed by electrostatic interaction of polylysine modified ferric oxide magnetic nanoparticles (FeNPs) and dopamine modified heparin (HepDA), wherein the nanoparticles have superparamagnetism, magnetic response ordered arrangement and good biocompatibility.

Background

By nanomaterials are meant, in a broad sense, materials that have at least one dimension in three dimensions in the nanoscale range or that are composed of them as elementary units. Among the nanomaterials, magnetic nanomaterials are receiving wide attention from the material science community due to their special magnetic properties, such as superparamagnetism, high coercivity, low curie temperature, high magnetic susceptibility, and the like. At present, the material is widely applied to the fields of magnetofluid, environmental protection, data storage, catalysis, biomedicine and the like, and is a functional material with wide application. The research of the magnetic nano material began in the 70 s of the 20 th century, and scientists at university of paris in france in 1988 discovered the giant magnetoresistance effect when researching the multilayer film of the Fe/Cr nano structure, further promoting the development of the magnetic nano material. Therefore, exploring and synthesizing the magnetic nano material with special performance has a vital significance for promoting the development of nano science and technology, and can generate a profound influence on the development of the human society.

Different from macroscopic bulk materials, the nano magnetic material has some special physical and chemical properties, such as excellent characteristics of superparamagnetism, small-size effect, surface effect, quantum tunneling effect and the like, and by utilizing the properties, a plurality of new materials which cannot be achieved by the performance of the bulk materials can be manufactured. Surface modification of magnetic nanoparticles is commonly used for the synthesis of functionalized magnetic nanoparticles as one of the most common chemical methods. The iron-based nanoparticles are used as the most common magnetic nano-materials, and organic ligands are modified on the surfaces of the iron-based magnetic nanoparticles through specific chemical reactions such as silanization coupling reaction, complexation reaction, esterification reaction and the like, so that the magnetic nano-composite materials with different surface functional group modifications are prepared. Dung et al prepared chitosan-modified Fe by suspension crosslinking method with glutaraldehyde as crosslinking agent₃O₄Magnetic nanoparticles (Dung, D.T.K.; Hai, T.H.; Phuc, L.H.et al.preparation and Characterizat)ion of Magnetic Nanoparticles with Chitosan coating. journal of Physics: Conference Series 2009,187,012036.). Habibi adds cellulose solution with certain concentration modified by amino to Fe by using dip coating method₃O₄Magnetic nano-composite is prepared in aqueous solution of magnetic nano-particles, and the composite can be used as functionalized biological material in the fields of drug delivery, tumor treatment, enzyme engineering and the like (Habibi, N., Functional Biocompatible Magnetite-Cellulose Nanocomposite Networks: chromatography by Source Transformed induced Spectroscopy, X-Ray Powder Diffraction and Field Emission Scanning Electron Microscopy analysis. Spectrochem. acta. A. mol. Biomol. Spectrosc.2015,136, 1450-1453.).

The blood compatibility of biomedical implant materials is a primary consideration in clinical applications, and since the surface of the material is in direct contact with blood and the surface chemistry of the material determines the anticoagulation property, the surface modification of the material is the most commonly used means to improve the blood compatibility. Heparin is used as the most widely clinical anticoagulant drug with anticoagulation and thrombosis inhibition, a large number of carboxyl functional groups are enriched on the surface of the anticoagulant drug, the heparin can be fixed by a method of forming nano particles through electrostatic interaction, and the anticoagulant drug is applied to drug release.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides heparinized magnetic nanoparticles and a preparation method and application thereof.

The technical purpose of the invention is realized by the following technical scheme.

The invention relates to heparinized magnetic nanoparticles and a preparation method and application thereof, which are carried out according to the following steps:

(1) preparing dopamine modified heparin (HepDA) by taking Dopamine (DA) and heparin (Hep) as raw materials and utilizing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to perform amidation reaction;

(2) dissolving the dopamine modified heparin (HepDA) prepared in the step (1) and polylysine modified ferric oxide nanoparticles (FeNPs) in a Tris buffer solution, and stirring at room temperature for reaction so as to compound the polylysine modified ferric oxide magnetic nanoparticles with positive charges and the dopamine modified heparin with negative charges into the nanoparticles with the core-shell structure by utilizing electrostatic interaction.

And (3) the pH value of the Tris buffer solution in the step (2) is 8.5, and the concentration is 5 mg/mL.

Dissolving the dopamine modified heparin (HepDA) and the polylysine modified ferric oxide nanoparticles (FeNPs) in a Tris buffer solution, and stirring at room temperature for reaction for 2-4 h.

And (3) after the reaction in the step (2) is finished, centrifuging the reaction solution for 20-30min at the rotation speed of 4000-6000rpm, and then drying the lower-layer precipitate in vacuum to obtain the nano particles with the core-shell structure.

The heparinized magnetic nanoparticles (FeNPs-HepDA) are applied to preparation of anticoagulant materials.

The application of dopamine in dispersing and fixing heparinized magnetic nanoparticles is characterized in that the heparinized magnetic nanoparticles take polylysine modified ferric oxide nanoparticles as an inner core, and dopamine modified heparin is loaded on the surface of the magnetic nanoparticles and effectively dispersed and fixed on the surface of a dopamine modified substrate under the control of a magnetic field.

The invention has the beneficial effects that: the invention provides a core-shell structure nano particle which takes polylysine modified ferric oxide magnetic nano particles (FeNPs) as an inner core and loads dopamine modified heparin (HepDA) on the surface, and the nano particle with the core-shell structure not only shows some inherent properties of the magnetic nano particle such as magnetic response arrangement, but also has an anticoagulation function and good biocompatibility, and can be effectively dispersed and fixed on the surface of a dopamine modified substrate under the control of a magnetic field.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of HepDA of the present invention.

Fig. 2 is a fourier infrared spectrum of HepDA of the present invention.

Fig. 3 is an ultraviolet-visible light absorption spectrum of HepDA of the present invention.

FIG. 4 is a graph showing the distribution of the particle size of the nanoparticles of the present invention.

FIG. 5 is a graph showing the change in particle size of the nanoparticles of the present invention.

FIG. 6 is a graph of zeta-point variation for nanoparticles of the present invention.

FIG. 7 is a thermogravimetric analysis of the nanoparticles of the present invention.

FIG. 8 is a Scanning Electron Microscope (SEM) image of nanoparticles of the present invention.

FIG. 9 is a cytotoxicity plot of nanoparticles of the invention.

FIG. 10 is a graph of Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) for nanoparticles of the invention.

FIG. 11 is an Atomic Force Microscope (AFM) image of a nanoparticle of the present invention.

Detailed Description

The following is a further description of the invention and is not intended to limit the scope of the invention.

Dopamine modified heparin (HepDA) was prepared according to the method of reference (young, i.; Kang, s.m.; Byun, y.; Lee, h., Enhancement of the blood compatibility of poly (urethane) substrettes by muscle-immobilized adjuvant coating, bioconjugate. chem.2011,22,1264-9): firstly, dissolving 1g of heparin in 100mL of MES buffer solution with pH 5.5, then adding 5mM N-hydroxysuccinimide (NHS) and 10mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), stirring uniformly, and then stirring the mixed solution at room temperature for 1h to activate carboxyl groups on the heparin; after stirring, 0.3g of dopamine is dissolved in MES buffer solution and added into the reaction solution, and stirred vigorously at room temperature for reaction overnight; after the reaction is finished, dialyzing in a dialysis bag with the molecular weight cutoff of 3500Da for 24h to remove unreacted molecules, and freeze-drying to obtain the product, namely the dopamine modified heparin (HepDA).

The hydrogen nuclear magnetic resonance spectroscopy detection of HepDA prepared above showed that d ═ 2.8ppm (methyl) corresponds to the characteristic peak of methylene group on dopamine, as seen in fig. 1, thus confirming the successful grafting of dopamine to heparin.

Infrared spectrum detection is carried out on the HepDA prepared above, and as can be seen from figure 2, the characteristic peak is as follows ν ═ 3436cm^-1(s,NH),2924cm-1(m,NH),1557cm^-1(vs, C ═ O), gray area 1557cm in the figure^-1The appearance of characteristic peaks represents the production of amide bonds, thus demonstrating that dopamine is covalently bound to heparin through amidation, i.e. dopamine is successfully grafted to heparin.

After the products are obtained according to the steps, the dopamine, heparin and dopamine modified heparin are respectively subjected to an ultraviolet and visible light absorption test, the absorbance and the absorption peak are tested at the speed of 1nn/s in the wavelength range of 200-1000nm, as can be seen from figure 3, the dopamine and heparin respectively have characteristic absorption peaks at 280nm and 258nm, when dopamine is covalently grafted on the heparin, a new absorption peak appears at 263nm, and the grafting rate of the dopamine can be obtained through the intensities of different absorption peaks.

Dissolving the dopamine modified heparin (HepDA) prepared in the step into 10mM Tris buffer solution with the pH value of 8.5, wherein the concentration is 5mg/mL, mixing polylysine modified ferric oxide nanoparticle (FeNPs) solution with the solution, stirring for 2h at room temperature, then centrifuging at 5000rpm for 20min, and drying in vacuum to obtain the core-shell structured nanoparticle (FeNPs-HepDA).

And (3) carrying out dynamic light scattering and Zeta point position analysis on the polylysine modified ferric oxide nanoparticles before and after modification, and detecting the particle size and potential change before and after modification.

In fig. 4, a is a dynamic light scattering particle size distribution diagram of polylysine-modified iron trioxide nanoparticles (FeNPs) before modification, and b is a dynamic light scattering particle size distribution diagram of polylysine-modified iron trioxide nanoparticles (FeNPs-HepDA) after modification. As can be seen from fig. 4, the unmodified FeNPs showed a narrow distribution, a particle size of 59.9 ± 7.1nm, and after HepDA was loaded on the surfaces of the FeNPs by electrostatic interaction, the particle size increased, and the distribution became wide, a particle size of 162 ± 10.3nm, thereby confirming the formation of the outer coating thereof.

In order to confirm that dopamine grafted heparin (HepDA) can be further reacted, unmodified heparin was used as a negative control group, and heparin was loaded on the surfaces of FeNPs by the same procedure as described above to obtain FeNPs-Hep, which was subjected to dynamic light scattering. As can be seen from FIG. 5, the particle size of heparin-modified FeNPs (FeNPs-Hep) can only reach 106 + -10.3 nm, which is smaller than that of FeNPs-HepDA, mainly because the thickness of the coating can be significantly increased due to further oxidative self-polymerization reaction after heparin grafts with dopamine.

As can be seen from FIG. 6, polylysine-modified FeNPs are positively charged at a potential of 31.8. + -. 2.7mV, whereas heparin-and dopamine-modified heparin-loaded FeNPs-Hep and FeNPs-HepDA have potentials of-35.5. + -. 3.3mV and-27.1. + -. 2.6mV, respectively. Heparin has a large number of carboxyl groups, so the surface of the heparin presents negative charges, and dopamine modified heparin occupies partial carboxyl groups to form amide bonds, so the potential of FeNPs-HepDA is lower than that of FeNPs-Hep, and the successful synthesis of a core-shell structure is laterally proved.

Thermogravimetric analysis is used for detecting the thermogravimetric change of the polylysine modified ferric oxide nanoparticles before and after modification, and the content of HepDA loaded on the surface of the FeNPs can be obtained through the thermogravimetric analysis in the process of 25-700 ℃, and the content of HepDA can be seen from figure 7 to be 9.7%.

The surface morphology and structure of the nanoparticles of the invention were examined using the following methods. In order to visually see the surface morphology and the structure of the nano particles prepared by the method, different nano particles are dispersed in an ethanol solution, the mixture is ultrasonically stirred uniformly, 20L of the solution is dripped on a silicon wafer cleaned in advance, the silicon wafer is dried for 12 hours at room temperature and then dried for 2 hours in vacuum, and after gold spraying treatment is carried out on the surface of the silicon wafer, the observation is carried out through a Scanning Electron Microscope (SEM). In FIG. 8, a is an SEM photograph of polylysine-modified iron trioxide nanoparticles (FeNPs) before modification, and b is an SEM photograph of polylysine-modified iron trioxide nanoparticles (FeNPs-HepDA) after modification. As can be seen from fig. 8, the particle size of the polylysine-modified iron sesquioxide nanoparticles (FeNPs) is significantly increased after being modified by HepDA, and the surface of the polylysine-modified iron sesquioxide nanoparticles forms a core-shell structure.

As can be seen from FIG. 9, the cellular activity of FeNPs-HepDA reached more than 80%, demonstrating an effect.

The Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) reflect primarily intrinsic and extrinsic coagulation system conditions. As can be seen from fig. 10, both the heparin-modified nanoparticles and pure heparin have good anticoagulant performance, wherein the APTT test value exceeds 120s, while the APTT value of the normal blank group is only 18 s; and the PT value is basically within the normal fluctuation range compared with the blank group, so that whether the anticoagulation effect is changed or the intrinsic coagulation behavior can be proved.

In FIG. 11, a is an AFM photograph of a polytetrafluoroethylene substrate (PTFE), b is an AFM photograph of a polytetrafluoroethylene substrate modified with heparin composite nanoparticles (FeNPs-HepDA) under a magnetic field condition, c is an AFM photograph of a polytetrafluoroethylene substrate modified with heparin composite nanoparticles (FeNPs-HepDA) under a non-magnetic field condition, and d is a water contact angle of the polytetrafluoroethylene substrate (PTFE) and modified Polydopamine (PDA) and heparin composite nanoparticles (FeNPs-HepDA), respectively. As can be seen from fig. 11, the pure polytetrafluoroethylene substrate (PTFE) has a relatively smooth surface, and a rough surface is formed by modifying the heparin composite nanoparticles, so that the nanoparticles can be uniformly spread on the PTFE surface under the magnetic field condition, the surface roughness is uniform, and the nanoparticles partially agglomerate and have irregular surface morphology when not controlled by the magnetic field; according to the water contact angle characterization, PTFE shows a hydrophobic property, the surface water contact angle is 100.2 degrees, the contact angle is 67.9 degrees after the dopamine structure is modified, the surface becomes hydrophilic, the contact angle is 37.1 degrees after the nanoparticles are modified on the surface, and the surface hydrophilic property is changed to be beneficial to the spreading of the nanoparticles.

The preparation of the heparinized magnetic nanoparticles can be realized by adjusting the process parameters according to the content of the invention, and the performance of the heparinized magnetic nanoparticles is basically consistent with that of the embodiment of the invention.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

Claims

1. A heparinized magnetic nanoparticle characterized by: prepared by the following steps:

(1) dopamine and heparin are used as raw materials, and dopamine modified heparin is prepared by amidation reaction of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;

(2) dissolving the dopamine-modified heparin and polylysine-modified ferric oxide nanoparticles prepared in the step (1) into a Tris buffer solution, and stirring at room temperature for reaction so as to compound the polylysine-modified ferric oxide nanoparticles with positive charges and the dopamine-modified heparin with negative charges into core-shell structured nanoparticles by utilizing electrostatic interaction.

2. The heparinized magnetic nanoparticle of claim 1, wherein: and (3) the pH value of the Tris buffer solution in the step (2) is 8.5, and the concentration is 5 mg/mL.

3. The heparinized magnetic nanoparticle of claim 1, wherein: dissolving the dopamine-modified heparin and the polylysine-modified ferric oxide nanoparticles in the step (2) into a Tris buffer solution, and stirring at room temperature for reaction for 2-4 h.

4. The heparinized magnetic nanoparticle of claim 1, wherein: and (3) after the reaction in the step (2) is finished, centrifuging the reaction solution for 20-30min at the rotation speed of 4000-6000rpm, and then drying the lower-layer precipitate in vacuum to obtain the nano particles with the core-shell structure.

5. A preparation method of heparinized magnetic nanoparticles is characterized by comprising the following steps: the method comprises the following steps:

6. The method of claim 5, wherein the heparinized magnetic nanoparticles are prepared by: and (3) the pH value of the Tris buffer solution in the step (2) is 8.5, and the concentration is 5 mg/mL.

7. The method of claim 5, wherein the heparinized magnetic nanoparticles are prepared by: dissolving the dopamine-modified heparin and the polylysine-modified ferric oxide nanoparticles in the step (2) into a Tris buffer solution, and stirring at room temperature for reaction for 2-4 h.

8. The method of claim 5, wherein the heparinized magnetic nanoparticles are prepared by: and (3) after the reaction in the step (2) is finished, centrifuging the reaction solution for 20-30min at the rotation speed of 4000-6000rpm, and then drying the lower-layer precipitate in vacuum to obtain the nano particles with the core-shell structure.

9. Use of heparinized magnetic nanoparticles according to any one of claims 1 to 4 for the preparation of anticoagulant materials.

10. The application of dopamine in dispersing and fixing heparinized magnetic nanoparticles is characterized in that: the heparinized magnetic nanoparticles take polylysine-modified ferric oxide nanoparticles as an inner core, and dopamine-modified heparin is loaded on the surface of the magnetic nanoparticles and effectively dispersed and fixed on the surface of a dopamine-modified substrate under the control of a magnetic field.