CN102677032B - Method for immobilizing VEGF-carried heparin/polylysine nanoparticles on Ti surface - Google Patents
Method for immobilizing VEGF-carried heparin/polylysine nanoparticles on Ti surface Download PDFInfo
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- CN102677032B CN102677032B CN 201210162504 CN201210162504A CN102677032B CN 102677032 B CN102677032 B CN 102677032B CN 201210162504 CN201210162504 CN 201210162504 CN 201210162504 A CN201210162504 A CN 201210162504A CN 102677032 B CN102677032 B CN 102677032B
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Abstract
The invention discloses a method for immobilizing VEGF-carried heparin/polylysine nanoparticles on a Ti surface, which comprises the following steps: by utilizing the characteristic that Hep and PLL can produce static interactions to form nanoparticles, mixing VEGF-carried Hep and PLL to form VEGF-carried nanoparticles; preparing a DM coating on the Ti material surface; and by utilizing the characteristic that the DM and primary amino group can generate Michael addition and Schiff base reaction, immobilizing the amino-containing nanoparticles onto the sample surface in a covalent mode, thereby constructing the biologically modified surface with anticoagulation and endothelium promotion functions. By constructing a nanoparticle modifying layer with anticoagulation and endothelium promotion characteristics on the Ti surface, the invention obviously improves the blood compatibility and endothelium injury restorability of the material.
Description
Affiliated technical field
The present invention relates to nano particle technology of preparing and inorganic material surface modification technology, particularly the bioid method of modifying on artificial organs material titanium surface.
Background technology
Titanium (Ti) Base Metal material has been widely used in biomedical sector because of its good biocompatibility, but the field for some special applications, as the treatment for cardiovascular disorder as cardiovascular embedded material, its biocompatibility also reaches far away clinical requirement.This is mainly because the titanium material blood compatibility is poor, and there is no restraining effect for vascellum endometrial hyperplasia and inflammatory reaction etc.
By material surface is carried out to the bioid modification, give anticoagulation ability that material is good and again the endothelialization ability be to improve the effective ways of its biocompatibility.Heparin (Hep) is a kind of clinical common anticoagulation medicine, and the biotic factor combination that heparin can be relevant to multiple endotheliocyte, extends the biotic factor transformation period in vivo, promotes the endothelial injury reparation.In addition, also studies have reported that heparin also has good effect in the prevention of inflammation and treatment.VEGF (vascular endothelial growth factor) is the specific heparin binding growth factor of a kind of vascular endothelial cell, for the mobilization of the proliferation apoptosis of vascular endothelial cell and endothelial progenitor cells, go back to the nest and all play an important role to the differentiation of endotheliocyte.
Dopamine HCL (DM) is the nerve conduction material in a kind of organism, under solution condition, self-polymeric reaction can occur.When autohemagglutination occurs DM, part catechol group can be combined with metal or metal oxide formation coordination, thereby deposits a strata DM layer at material surface.The poly-DM layer obtained has secondary reaction, neighbour's two a kind of jade groups that its oxidation obtains can with primary amino (NH
2), not alkali reaction or reversal of the Michael addition of west occur in secondary amine (NH).
Hep and poly-lysine (PLL) can be assembled into nano particle by the static interaction, utilize the interaction between VEGF and heparin VEGF can be loaded in particle, form the Hep/PLL nano particle that carries VEGF.This kind of particle, at the fixing blood compatibility that can effectively improve material of material surface, promotes the endothelial injury reparation.And there is no at present the relevant report that the Hep/PLL nano particle that will carry VEGF is fixed in the titanium material surface.
Summary of the invention
The object of the present invention is to provide a kind of year VEGF the Hep/PLL nano particle the preparation method and at the fixing means of Ti material surface, by the method, the Ti material surface is carried out to blood compatibility and the cell compatibility that the bioid modification can effectively improve material.
The present invention realizes that the technical scheme that above purpose adopts is, the method for a kind of heparin at the fixing year VEGF in Ti surface/poly-lysine nano particle, the steps include:
A, sample preparation.In the poly-Dopamine HCL coating of the pure Ti surface deposition of polishing, stand-by;
B, the preparation of carrying the Hep/PLL nano particle of VEGF.The VEGF solution that is 50-500ng/ml by concentration (PBS, pH 7.4) equal-volume drops in the heparin solution that concentration is 5-20mg/ml (PBS, pH 7.4), 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 that concentration is 0.2-1mg/ml (MW 15-30 ten thousand) solution (PBS, pH 7.4);
C, nano particle are fixed.The Ti sheet that deposits DM in the A step is soaked in the nano particle suspension of B step acquisition, under 15-50 ℃ of oscillating condition, reaction 6-24 hour, use respectively PBS and distilled water rinsing, obtains target compound after drying.
Reaction process of the present invention and mechanism mainly are divided into two parts.First part is the preparation of the Hep/PLL nano particle of year VEGF.At first by Hep, with the interaction of VEGF, VEGF is combined with Hep; Secondly in the PBS of pH=7.4 system, the static interaction can occur with being after electropositive PLL solution is mixed in the Hep solution that is loaded with VEGF, by intermolecular electrostatical binding and molecule winding effect, can form the Hep/PLL nano particle that carries VEGF with nano-scale.Second section is nano particle fixing at the Ti material surface.At first Ti is soaked in the Tris solution of DM, the catechol group in DM can form coordination with Ti and be combined, and under aerobic conditions crosslinking polymerization, thereby form firmly DM layer of one deck on Ti surface.The DM polymeric layer obtained has secondary reaction, neighbour's two a kind of jade groups that its oxidation obtains can with DM in primary amino (NH
2) reversal of the Michael addition and Xi Fu alkali reaction occur, thus the poly-DM layer of multilayer formed; Secondly, the amino in nano particle on PLL also can with neighbour's two a kind of jade structure generation Michael addition and Xi Fu alkali reactions on poly-DM layer surface, thereby the nano particle covalency is fixed on to the Ti surface.
Compared with prior art, the invention has the beneficial effects as follows:
One, the creationary Hep/PLL nano particle that carries VEGF of preparing, utilize poly-DM layer can with the characteristic of primary amino generation Michael addition and Xi Fu alkali reaction, can be fixed on the Ti surface that DM applies by being rich in amino nano particle covalency.By this kind of method, can effectively improve the loading capacity of VEGF, extend the transformation period of VEGF, strengthen its endothelial injury repairing effect.Simultaneously also can strengthen the bonding strength of heparin and VEGF and material, reduction drug loss rate, prolong drug action time.
Two, the preparation technology of nano particle and fixing means are all simple to operation, and without expensive complicated equipment, process costs is lower, and effect is remarkable.
Three, nano particle carries out at the fixedly employing immersion way of material surface, can guarantee that the material various piece can fix nano particle uniformly, also be conducive to realize the modification as fixing as the nano particle on the surfaces such as heart valve prosthesis, intravascular stent of various baroque cardiovascular implanting devices, applied widely.
The accompanying drawing explanation
Below in conjunction with drawings and Examples, method of the present invention is described in further detail.
Fig. 1 is that in the inventive method, the nano particle preparation reaches at each fixing step schematic diagram of Ti surface.
Fig. 2 is the nano particle diameter size distribution plot.
The scanning electron microscope (SEM) photograph that Fig. 3 is the sample surfaces platelet adhesion reaction.(a) Ti; (b) the fixing Ti of nano particle.
Fig. 4 is that sample is soaked in the dynamic release situation of postheparin in PBS and discharges front and back sample surfaces anti-freezing Performance Detection.(a) Hep burst size; (b) sample surfaces activated partial thrombin time (APTT) measurement result.
Fig. 5 is fluorescent dye result after the sample surfaces endothelial progenitor cells is cultivated.(a) Ti; (b) the fixing Ti of nano particle.
Embodiment
Embodiment mono-
Referring to Fig. 1, the first embodiment of the present invention is that the method for a kind of heparin at the fixing year VEGF in Ti surface/poly-lysine nano particle, the steps include:
A, sample preparation.In the poly-Dopamine HCL coating of the pure Ti surface deposition of polishing, stand-by;
B, the preparation of carrying the Hep/PLL nano particle of VEGF.The VEGF solution that is 500ng/ml by concentration (PBS, pH 7.4) equal-volume drops in the heparin solution that concentration is 20mg/ml (PBS, pH 7.4), 37 ℃ of standing 1h.Then under room temperature and magnetic agitation condition, Hep and VEGF mixed solution equal-volume are dropped in the PLL that concentration is 1mg/ml (MW 15-30 ten thousand) solution (PBS, pH 7.4);
C, nano particle are fixed.The Ti sheet that deposits DM in the A step is soaked in the nano particle suspension of B step acquisition, under 15-50 ℃ of oscillating condition, reaction is 24 hours, uses respectively PBS and distilled water rinsing, obtains target compound after drying.
Embodiment bis-
The method of a kind of heparin at the fixing year VEGF in Ti surface/poly-lysine nano particle, the steps include:
A, sample preparation.In the poly-Dopamine HCL coating of the pure Ti surface deposition of polishing, stand-by;
B, the preparation of carrying the Hep/PLL nano particle of VEGF.The VEGF solution that is 50ng/ml by concentration (PBS, pH 7.4) equal-volume drops in the heparin solution that concentration is 5mg/ml (PBS, pH 7.4), 37 ℃ of standing 2h.Then under room temperature and magnetic agitation condition, Hep and VEGF mixed solution equal-volume are dropped in the PLL that concentration is 0.2mg/ml (MW 15-30 ten thousand) solution (PBS, pH 7.4);
C, nano particle are fixed.The Ti sheet that deposits DM in the A step is soaked in the nano particle suspension of B step acquisition, under 50 ℃ of oscillating conditions, reaction is 6 hours, uses respectively PBS and distilled water rinsing, obtains target compound after drying.
Embodiment tri-
The method of a kind of heparin at the fixing year VEGF in Ti surface/poly-lysine nano particle, the steps include:
A, sample preparation.In the poly-Dopamine HCL coating of the pure Ti surface deposition of polishing, stand-by;
B, the preparation of carrying the Hep/PLL nano particle of VEGF.The VEGF solution that is 200ng/ml by concentration (PBS, pH 7.4) equal-volume drops in the heparin solution that concentration is 10mg/ml (PBS, pH 7.4), 37 ℃ of standing 3h.Then under room temperature and magnetic agitation condition, Hep and VEGF mixed solution equal-volume are dropped in the PLL that concentration is 0.5mg/ml (MW 15-30 ten thousand) solution (PBS, pH 7.4);
C, nano particle are fixed.The Ti sheet that deposits DM in the A step is soaked in the nano particle suspension of B step acquisition, under 37 ℃ of oscillating conditions, reacts 12h, use respectively PBS and distilled water rinsing, obtain target compound after drying.
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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| CN103736156B (en) * | 2013-10-10 | 2016-01-13 | 西北大学 | A kind of method by poly-Dopamine HCL coating constructing function surface and interface |
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| CN112107733B (en) * | 2020-09-14 | 2024-02-23 | 上海市第六人民医院 | Temperature-sensitive hydrogel-high-dispersion nanoparticle system of injection type platelet lysate loaded and application thereof |
| CN112957543B (en) * | 2021-02-02 | 2022-05-20 | 四川大学华西医院 | Super-hydrophobic multifunctional coating with time-sequential drug release and preparation method thereof |
| CN114887117B (en) * | 2022-04-09 | 2023-10-13 | 中国医科大学附属第一医院 | Drug-loaded nanoparticle and vascular stent for reducing restenosis rate in blood vessel after operation |
| CN115522207B (en) * | 2022-08-30 | 2024-06-04 | 南京医科大学附属口腔医院 | Preparation method for constructing PLL/CPP-ACP self-assembled multilayer film on sandblasted acid etched titanium surface |
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