阅读说明：本技术 一种固定化金属离子-磁性脂质体及其制备方法和应用 (Immobilized metal ion-magnetic liposome and preparation method and application thereof ) 是由 伍善广 陆苑 冯学珍 蒙华琳 陆玉婷 韦啟球 廖娜 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种固定化金属离子-磁性脂质体及其制备方法和应用。本发明的金属离子-磁性脂质体由磷脂、N-十六烷基亚氨基二乙酸包裹磁性纳米四氧化三铁,组成具有磷脂双层膜结构的大分子脂质体后,并在其表面负载、固定化过渡金属离子。与传统磁性脂质体制备方法不同,本发明采用原位反应法制备磁性脂质体,主要方法为将FeCl<Sub>3</Sub>·6H<Sub>2</Sub>O和FeCl<Sub>2</Sub>·4H<Sub>2</Sub>O溶液在一定温度、高速搅拌、氮气保护下,通过滴加氨水进行反应,然后加入磷脂-乙醇溶液获得磁性脂质体,再偶联过渡金属离子。本发明的固定化金属离子-磁性脂质体具备仿细胞膜亲和吸附及金属离子亲和吸附双重功能,制备方法简单,稳定性好。(The invention discloses an immobilized metal ion-magnetic liposome and a preparation method and application thereof. The metal ion-magnetic liposome is formed by wrapping magnetic nano ferroferric oxide with phospholipid and N-hexadecyl iminodiacetic acid, and then the macromolecular liposome with a phospholipid double-layer membrane structure is loaded and immobilized with transition metal ions on the surface. Different from the traditional preparation method of the magnetic liposome, the invention adopts an in-situ reaction method to prepare the magnetic liposome, and the main method is to use FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 The O solution reacts by dripping ammonia water at a certain temperature under the protection of nitrogen gas and high-speed stirring, and then phospholipid-ethanol is addedThe solution is used to obtain magnetic liposome and then coupled with transition metal ions. The immobilized metal ion-magnetic liposome has the dual functions of imitating cell membrane affinity adsorption and metal ion affinity adsorption, and has simple preparation method and good stability.)

1. An immobilized metal ion-magnetic liposome, which is characterized in that: after the macromolecular liposome with the phospholipid double-layer membrane structure is formed by wrapping magnetic nano ferroferric oxide with phospholipid and N-alkyl iminodiacetic acid, transition metal ions are loaded and immobilized on the surface of the macromolecular liposome.

2. The immobilized metal ion-magnetic liposome of claim 1, wherein: the phospholipid content of the immobilized metal ion-magnetic liposome is 0-300 mu g/mg, and the transition metal ion content is 0-100 mu mol/g.

3. The immobilized metal ion-magnetic liposome of claim 1, wherein: the phospholipid is selected from one or more of lecithin, soybean phospholipid, distearoyl phosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylethanolamine.

4. The immobilized metal ion-magnetic liposome of claim 1, wherein: the alkyl group of the N-alkyl iminodiacetic acid is C12-C18.

5. the process for producing an immobilized metal ion-magnetic liposome according to any one of claims 1 to 4, wherein: preparing magnetic liposome by in-situ reaction method, and adding FeCl₃·6H₂O and FeCl₂·4H₂Under the protection of nitrogen and high-speed stirring, dropwise adding ammonia water to the O solution for reaction, and then adding a phospholipid-ethanol solution to obtain a magnetic liposome; re-fusion of N-alkyliminodiacetic acid, azolinking transition metal ions to obtain the immobilized metal ion-magnetic liposome.

6. The method for preparing immobilized metal ion-magnetic liposome of claim 5, comprising the following steps:

(1) With FeCl₃·6H₂O and FeCl₂·4H₂O is used as raw material to prepare magnetic Fe by chemical coprecipitation method₃O₄A nanoparticle; adding phospholipid-ethanol solution for reaction to obtain magnetic liposome for later use;

(2) Magnetic liposome coupling transition metal ion:

Weighing 0.5-15g of N-alkyl iminodiacetic acid, dissolving with 50-100mL of 0.005mol/L sodium hydroxide solution to obtain N-alkyl iminodiacetic acid solution, weighing 0.5-2.5g of magnetic liposome, adding into the N-alkyl iminodiacetic acid solution, and magnetically stirring at 50-55 ℃ for 80-90 min; taking out the magnetic liposome under the action of an external magnetic field, and washing the magnetic liposome by deionized water; putting the cleaned magnetic liposome into a centrifuge tube, adding 50-200mL of 0.005-0.2mol/L NiSO₄Shaking the solution at room temperature for 50-70min, separating the magnetic liposome under the action of external magnetic field, and washing with deionized water to obtain immobilized metal ion-magnetic liposome.

7. The method for preparing immobilized metal ion-magnetic liposome of claim 6, wherein the specific operation steps of step (1) are as follows:

1.3661g of FeCl were weighed₃·6H₂O and 0.5334g FeCl₂·4H₂Dissolving O in 100ml deionized water, stirring, placing in 30 deg.C constant temperature water bath, adding dropwise NH under nitrogen protection and strong mechanical stirring₃·H₂O, adjusting the pH value of the solution to 8.5, reacting for 30min, adding 50-70 mL0.5-1.5% (w/v) phospholipid-ethanol solution to generate a large amount of black particles, heating to 90 ℃, dropwise adding 1mol/L NaOH, adjusting the pH value to 12, stirring strongly for 30min, and after the reaction is finished, applying an external magnetic fieldAnd separating the prepared black particles, and washing with deionized water to obtain the magnetic liposome.

8. The use of the immobilized metal ion-magnetic liposome prepared by the preparation method of claim 5, wherein: the immobilized metal ion-magnetic liposome is applied to biomagnetic separation of active protein.

Technical Field

The invention relates to the technical field of liposome, in particular to immobilized metal ion-magnetic liposome and a preparation method and application thereof.

Background

In the 21 st century, with the rapid development of life science, people are more and more concerned about the rapid and efficient separation of biomacromolecules by using new technologies. The biomagnetic separation technology is a new separation means developed on the basis of the traditional magnetic separation technology, is a high-efficiency separation technology taking organisms such as cells, bacteria, nucleic acid, protein and the like as application objects, and has the advantages of high efficiency, rapidness, simplicity and the like; the characteristic that a magnetic or magnetic marked organism performs directional motion under the action of an external magnetic field is utilized to realize extraction, enrichment, separation and purification of a target organism.

At present, the immobilized metal ion affinity technology is easy to prepare, has low cost, and is widely applied to the separation of active proteins, such as histidine-tagged recombinant polypeptides and recombinant proteins, phosphorylated proteins and cytochromes. The liposome is a phospholipid bilayer structure similar to a biological membrane formed by dispersing phospholipid in water and automatically arranging the phospholipid under certain conditions. The magnetic particle surface coated with a layer of liposome can be used as a simulated biological membrane functional particle and can be applied to biological magnetic separation. The traditional biological magnetic separation technology of active protein needs to carry out complex pretreatment operations such as filtration, centrifugation and the like on sample liquid, and the separation efficiency is low.

Disclosure of Invention

The invention aims to provide an immobilized metal ion-magnetic liposome, which is formed by wrapping magnetic nano ferroferric oxide with phospholipid and N-alkyl iminodiacetic acid to form a macromolecular liposome with a phospholipid double-layer membrane structure, and then loading and immobilizing transition metal ions on the surface of the macromolecular liposome.

Furthermore, the content of the phospholipid of the immobilized metal ion-magnetic liposome is 0-300 mu g/mg, and the content of the transition metal ion is 0-100 mu mol/g.

Further, the phospholipid is selected from one or more of lecithin, soybean phospholipid, distearoyl phosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylethanolamine.

Further, the alkyl group of the N-alkyl iminodiacetic acid is C12-C18.

the N-alkyl iminodiacetic acid can be obtained by a preparation method of the N-alkyl iminodiacetic acid applied by the applicant in patent CN 201711466648.8.

The second purpose of the invention is to provide a preparation method of the immobilized metal ion-magnetic liposome, which comprises the following steps: preparing magnetic liposome by in-situ reaction method, and adding FeCl₃·6H₂O and FeCl₂·4H₂Under the protection of nitrogen and high-speed stirring, dropwise adding ammonia water to the O solution for reaction, and then adding a phospholipid-ethanol solution to obtain a magnetic liposome; then fusing N-alkyl iminodiacetic acid and coupling transition metal ions to obtain the immobilized metal ion-magnetic liposome.

Further, the preparation method of the immobilized metal ion-magnetic liposome specifically comprises the following steps:

(1) With FeCl₃·6H₂O and FeCl₂·4H₂O is used as raw material to prepare magnetic Fe by chemical coprecipitation method₃O₄A nanoparticle; adding phospholipid-ethanol solution for reaction to obtain magnetic liposome for later use;

(2) Magnetic liposome coupling transition metal ion:

Weighing 0.5-15g of N-alkyl iminodiacetic acid, and dissolving with 50-100mL of 0.005mol/L sodium hydroxide solution to obtain N-alkyl iminodiacetic acid solutionweighing 0.5-2.5g of magnetic liposome, adding into N-alkyl imino diacetic acid solution, and magnetically stirring at 50-55 deg.C for 80-90 min; under the action of an external magnetic field, taking out the magnetic liposome, and washing the magnetic liposome by using a large amount of deionized water; putting the cleaned magnetic liposome into a centrifuge tube, adding 50-200mL of 0.005-0.2mol/L NiSO₄Shaking the solution at room temperature for 50-70min, separating the magnetic liposome under the action of an external magnetic field, and washing with a large amount of deionized water to obtain the immobilized metal ion-magnetic liposome.

Further, the specific operation steps of the step (1) are as follows:

1.3661g of FeCl were weighed₃·6H₂O and 0.5334g FeCl₂·4H₂Dissolving O in 100ml deionized water, stirring, placing in 30 deg.C constant temperature water bath, adding dropwise NH under nitrogen protection and strong mechanical stirring₃·H₂And O, adjusting the pH value of the solution to 8.5, reacting for 30min, adding 50-70 mL0.5-1.5% (w/v) phospholipid-ethanol solution, generating a large amount of black particles in the solution, heating to 90 ℃, dropwise adding 1mol/L NaOH, adjusting the pH value to 12, stirring strongly for 30min, separating the prepared black particles under the action of an external magnetic field after the reaction is finished, and washing with a large amount of deionized water to obtain the magnetic liposome.

The immobilized metal ion-magnetic liposome is formed by coupling transition metal ions on the surface of the magnetic liposome, and has the dual functions of imitating cell membrane affinity adsorption and metal ion affinity adsorption.

The third purpose of the invention is to provide the application of the immobilized metal ion-magnetic liposome, in particular to the biomagnetic separation of active protein.

The invention has the following advantages and technical effects:

the immobilized metal ion-magnetic liposome of the invention is loaded and immobilized with transition metal ions on the surface of the magnetic liposome, so the immobilized metal ion-magnetic liposome can be called as the immobilized metal ion-magnetic liposome. The immobilized metal ion-magnetic liposome is a biomagnetic separation material developed by combining liposome, magnetic separation and immobilized metal ion affinity technology, and has more excellent characteristics. The immobilized metal ion-magnetic liposome has the dual functions of imitating cell membrane affinity adsorption and metal ion affinity adsorption, and has good stability. The preparation method has the advantages of easily available raw materials, simple method, mild conditions and low cost, can successfully prepare the immobilized metal ion-magnetic liposome, and is easy for scale-up production.

The invention combines a plurality of technologies such as liposome, magnetic separation and immobilized metal ion affinity to obtain the magnetic liposome with high stability and high selectivity and the surface coupled with transition metal ions. The immobilized metal ion-magnetic liposome is applied to biomagnetic separation of active protein, complex pretreatment operations such as filtration, centrifugation and the like are not needed to be carried out on a sample liquid, only the immobilized metal ion-magnetic liposome is directly put into a mixed solution containing target protein, the target protein is tightly combined with the immobilized metal ion-magnetic liposome, and then the separation is rapidly carried out by utilizing the auxiliary action of an external magnetic field, so that the separation efficiency is greatly improved.

Drawings

FIG. 1 is an electron micrograph of immobilized metal ion-magnetic liposomes prepared in example 1 (X60000);

FIG. 2 is a distribution diagram of particle size of the immobilized metal ion-magnetic liposome prepared in example 1.

Detailed Description

the invention is further described with reference to the following figures and specific examples.