CN102677032B - Method for immobilizing VEGF-carried heparin/polylysine nanoparticles on Ti surface - Google Patents

Abstract

The invention discloses a method for immobilizing heparin/polylysine nanoparticles loaded with VEGF on the surface of Ti. Firstly, by utilizing the property that Hep and PLL can interact electrostatically to form nanoparticles, Hep loaded with VEGF is mixed with PLL to form VEGF-loaded nanoparticles. Then prepare a DM coating on the surface of the Ti material, and use the characteristics of Michael addition and Schiff base reaction between DM and primary amino groups to covalently fix the nanoparticles containing amino groups to the surface of the sample, thereby constructing anticoagulant/promoting endothelial Bifunctional biochemically modified surface. The present invention builds a nanoparticle-modified layer with anticoagulant and re-endothelialization properties on the Ti surface, which significantly improves the blood compatibility and endothelial damage repairing ability of the material.

Description

A method for immobilizing VEGF-loaded heparin/polylysine nanoparticles on Ti surface

Technical field

The invention relates to nanoparticle preparation technology and inorganic material surface modification technology, in particular to a biochemical modification method of artificial organ material titanium surface.

Background technique

Titanium (Ti)-based metal materials have been widely used in the biomedical field due to their good biocompatibility, but for some special applications, such as cardiovascular implant materials for the treatment of cardiovascular diseases, their biocompatibility Sex is far from meeting the clinical requirements. This is mainly due to the poor hemocompatibility of titanium materials, and has no inhibitory effect on vascular intimal hyperplasia and inflammatory reactions.

By biochemically modifying the material surface, endowing the material with good anticoagulant ability and reendothelialization ability is an effective method to improve its biocompatibility. Heparin (Hep) is a common clinical anticoagulant drug, and heparin can bind to a variety of endothelial cell-related biological factors, prolong the half-life of biological factors in vivo, and promote the repair of endothelial damage. In addition, studies have also reported that heparin also has good effects in the prevention and treatment of inflammation. VEGF (vascular endothelial growth factor) is a specific heparin-binding growth factor for vascular endothelial cells, which plays an important role in the proliferation and migration of vascular endothelial cells, as well as the mobilization, homing and differentiation of endothelial progenitor cells.

Dopamine (DM) is a neurotransmitter in organisms, which can self-polymerize under solution conditions. While DM self-polymerizes, part of the catechol groups can form coordination bonds with metals or metal oxides, thereby depositing a poly-DM layer on the surface of the material. The obtained polyDM layer has secondary reactivity, and the o-diquinone group obtained by oxidation can undergo Schiff base reaction or Michael addition reaction with primary amino group (-NH ₂ ) and secondary amino group (-NH).

Hep and polylysine (PLL) can be assembled into nanoparticles through electrostatic interaction, and VEGF can be loaded into the particles by using the interaction between VEGF and heparin to form VEGF-loaded Hep/PLL nanoparticles. The immobilization of the particles on the surface of the material can effectively improve the blood compatibility of the material and promote the repair of endothelial damage. However, there is no relevant report on the immobilization of VEGF-loaded Hep/PLL nanoparticles on the surface of titanium materials.

Contents of the invention

The object of the present invention is to provide a preparation method of Hep/PLL nanoparticles loaded with VEGF and its fixation method on the surface of Ti material, by which the biochemical modification of the surface of Ti material can effectively improve the blood compatibility of the material and cell compatibility.

The technical solution adopted by the present invention to achieve the above object is a method for immobilizing VEGF-loaded heparin/polylysine nanoparticles on the Ti surface, the steps of which are:

A. Sample preparation. Deposit a polydopamine coating on the polished pure Ti surface for use;

B. Preparation of Hep/PLL nanoparticles loaded with VEGF. Add an equal volume of VEGF solution (PBS, pH 7.4) with a concentration of 50-500ng/ml dropwise into a heparin solution (PBS, pH 7.4) with a concentration of 5-20mg/ml, and let stand at 37°C for 1-3h. Then, under room temperature and magnetic stirring conditions, an equal volume of Hep and VEGF mixture was added dropwise to a PLL (MW 150,000-300,000) solution (PBS, pH 7.4) with a concentration of 0.2-1 mg/ml;

C. Nanoparticle immobilization. Soak the Ti sheet with DM deposited in step A in the nanoparticle suspension obtained in step B, react under shaking conditions at 15-50°C for 6-24 hours, rinse with PBS and double distilled water respectively, and obtain the target after drying things.

The reaction process and mechanism of the present invention are mainly divided into two parts. The first part is the preparation of Hep/PLL nanoparticles loaded with VEGF. First, VEGF and Hep are combined through the interaction between Hep and VEGF; secondly, in the PBS system with pH = 7.4, electrostatic interaction can occur after the Hep solution loaded with VEGF is mixed with the positively charged PLL solution, through the intermolecular electrostatic interaction. Combination and molecular entanglement can form nanometer-sized VEGF-loaded Hep/PLL nanoparticles. The second part is the fixation of nanoparticles on the surface of Ti material. First, Ti is soaked in the Tris solution of DM. The catechol groups in DM can form a coordination bond with Ti, and cross-link and polymerize under aerobic conditions, thus forming a firm DM layer on the surface of Ti. The obtained DM polymer layer has secondary reactivity, and the o-diquinyl group obtained by oxidation can undergo Michael addition reaction and Schiff base reaction with the primary amino group (-NH ₂ ) in DM, thereby forming a multilayer polyDM Second, the amino groups on the PLL in the nanoparticles can also undergo Michael addition and Schiff base reactions with the ortho-dikun structure on the surface of the polyDM layer, thereby covalently immobilizing the nanoparticles on the Ti surface.

Compared with prior art, the beneficial effect of the present invention is:

1. Creatively prepared VEGF-loaded Hep/PLL nanoparticles, using the characteristics of the polymer DM layer that can undergo Michael addition and Schiffer base reactions with primary amino groups, the amino-rich nanoparticles can be covalently immobilized on the DM coating covered Ti surface. Through this method, the loading amount of VEGF can be effectively increased, the half-life of VEGF can be prolonged, and the repairing effect of endothelial injury can be enhanced. At the same time, it can also enhance the binding strength of heparin and VEGF to the material, reduce the drug loss rate, and prolong the drug action time.

2. The preparation process and fixation method of nanoparticles are simple and easy to operate, no expensive and complicated equipment is required, the process cost is low, and the effect is remarkable.

3. The fixation of nanoparticles on the surface of the material is carried out by soaking, which can ensure that all parts of the material can be uniformly fixed with nanoparticles, and is also conducive to the realization of various complex cardiovascular implantation devices such as artificial heart valves, vascular stents and other surfaces The nanoparticle immobilization modification has a wide range of applications.

Description of drawings

The method of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

Fig. 1 is a schematic diagram of each step of preparing nanoparticles and immobilizing them on the Ti surface in the method of the present invention.

Figure 2 is a graph showing the particle size distribution of nanoparticles.

Fig. 3 is a scanning electron microscope image of platelet adhesion on the sample surface. (a) Ti; (b) Ti immobilized on nanoparticles.

Figure 4 shows the dynamic release of heparin after the sample is soaked in PBS and the detection of anticoagulant performance on the surface of the sample before and after the release. (a) Hep release amount; (b) Determination result of activated partial thrombin time (APTT) on the sample surface.

Figure 5 is the results of fluorescent staining of endothelial progenitor cells on the sample surface after culture. (a) Ti; (b) Ti immobilized on nanoparticles.

Detailed ways

Embodiment one

Referring to Fig. 1, the first kind of specific embodiment of the present invention is, a kind of method of the heparin/polylysine nanoparticle that carries VEGF on Ti surface immobilization, and its steps are:

A. Sample preparation. Deposit a polydopamine coating on the polished pure Ti surface for use;

B. Preparation of Hep/PLL nanoparticles loaded with VEGF. An equal volume of VEGF solution (PBS, pH 7.4) with a concentration of 500ng/ml was added dropwise to a heparin solution (PBS, pH 7.4) with a concentration of 20mg/ml, and allowed to stand at 37°C for 1h. Then, under room temperature and magnetic stirring conditions, an equal volume of the Hep and VEGF mixture was added dropwise to a PLL (MW 150,000-300,000) solution (PBS, pH 7.4) with a concentration of 1 mg/ml;

C. Nanoparticle immobilization. Soak the Ti sheet deposited with DM in step A in the nanoparticle suspension obtained in step B, react under shaking conditions at 15-50°C for 24 hours, rinse with PBS and double distilled water, and dry to obtain the target product.

Embodiment two

A method for immobilizing VEGF-loaded heparin/polylysine nanoparticles on the Ti surface, the steps of which are:

A. Sample preparation. Deposit a polydopamine coating on the polished pure Ti surface for use;

B. Preparation of Hep/PLL nanoparticles loaded with VEGF. An equal volume of VEGF solution (PBS, pH 7.4) with a concentration of 50 ng/ml was added dropwise to a heparin solution (PBS, pH 7.4) with a concentration of 5 mg/ml, and allowed to stand at 37°C for 2 h. Then, at room temperature and under magnetic stirring conditions, an equal volume of Hep and VEGF mixture was added dropwise to a PLL (MW 150,000-300,000) solution (PBS, pH 7.4) with a concentration of 0.2 mg/ml;

C. Nanoparticle immobilization. The Ti sheet deposited with DM in step A was soaked in the nanoparticle suspension obtained in step B, reacted at 50°C under shaking conditions for 6 hours, rinsed with PBS and double distilled water, and dried to obtain the target product.

Embodiment Three

A method for immobilizing VEGF-loaded heparin/polylysine nanoparticles on the Ti surface, the steps of which are:

A. Sample preparation. Deposit a polydopamine coating on the polished pure Ti surface for use;

B. Preparation of Hep/PLL nanoparticles loaded with VEGF. An equal volume of VEGF solution (PBS, pH 7.4) with a concentration of 200ng/ml was added dropwise to a heparin solution (PBS, pH 7.4) with a concentration of 10mg/ml, and allowed to stand at 37°C for 3h. Then, under room temperature and magnetic stirring conditions, an equal volume of Hep and VEGF mixture was added dropwise to a PLL (MW 150,000-300,000) solution (PBS, pH 7.4) with a concentration of 0.5 mg/ml;

C. Nanoparticle immobilization. The Ti sheet deposited with DM in step A was soaked in the nanoparticle suspension obtained in step B, reacted at 37 °C under shaking conditions for 12 h, rinsed with PBS and double distilled water, and dried to obtain the target product.

Claims (2)

1. the method for the heparin at the fixing year VEGF in Ti surface/poly-lysine nano particle, the steps include:

A. carry the preparation of the Hep/PLL nano particle of VEGF: the VEGF solution equal-volume that is 50-500ng/ml by concentration drops in the heparin solution that concentration is 5-20mg/ml, 37 ℃ of standing 1-3h; Then under room temperature and magnetic agitation condition, Hep and VEGF mixed solution equal-volume are dropped in the PLL solution that concentration is 0.2-1mg/ml, described PLL molecular weight is 150～300KDa;

B. nano particle is fixed: the Ti sheet that will deposit poly-Dopamine HCL is soaked in the nano particle suspension of A step acquisition, and under 15-50 ℃ of oscillating condition, reaction 6-24 hour, use respectively PBS and distilled water rinsing, obtains target compound after drying.

According to claim 1 a kind of on Ti surface the method for the fixing heparin that carries VEGF/poly-lysine nano particle, it is characterized in that: the drying means in described B step is critical point drying, sample, after processed, is placed in the critical point drying instrument and carries out drying.

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