CN101757643B - Emulsion based on stabilizing agent, preparation method and application thereof - Google Patents

Abstract

A stabilizer-based emulsion in the field of biomedical technology and its preparation method and application, the stabilizer is prepared by polylactic acid and iron halide by chemical co-precipitation; or further using 1-(3 dimethylaminopropyl) 3 ethyl Carbodiimide and polyethylenimine are modified to obtain stabilizers; emulsions are prepared by mixing stabilizers with water and oil phases, and emulsions are applied to gene carriers, magnetic resonance imaging contrast agents, or contrast agents for ultrasound diagnosis . The stabilizer prepared by the present invention has good hydrophilicity, lipophilicity and magnetism, and has a certain positive charge after modification; the prepared emulsion has magnetism and positive charge, and has the ability to become a magnetic gene carrier and magnetic resonance imaging. Potential for contrast agents. The emulsion prepared by passing sulfur hexafluoride also has the potential to be a contrast agent for ultrasonic diagnosis.

Description

Emulsion based on stabilizer and its preparation method and use

technical field

The invention relates to an emulsion in the technical field of biomedicine and its preparation method and application, in particular to a stabilizer-based emulsion and its preparation method and application.

Background technique

An emulsion is a heterogeneous mixture of droplets of one liquid phase dispersed in another completely incompatible liquid phase. The mutual incompatibility of the two liquid phases causes the two to generate surface tension where they contact each other. When one liquid phase is dispersed in the other liquid phase in the form of droplets, the contact area between the two increases significantly. , leading to an increase in the chemical potential energy, and the droplets will spontaneously collide and fuse to make up for the lost free energy. Therefore, the emulsion is a thermodynamically unstable system. The preparation of a stable emulsion needs to create a layer of kinetic energy between the droplets. Physical barriers to prevent their accumulation. The use of surfactants lies in its amphiphilicity, which can stabilize between two incompatible phases and reduce surface tension, thereby reducing the free energy lost in the process of emulsion formation. However, its amphiphilicity also allows it to be adsorbed on the surface of any solid medium, significantly affecting the wettability of the latter, which limits the application range of emulsions to a certain extent, such as the surfactant used in cosmetics. There is some irritation to the skin.

In recent years, microparticles are considered to be able to replace surfactants to prepare stable emulsions. This kind of emulsion prepared with solid particles as a stabilizer is called Pickering emulsion to commemorate his discoverer Mr. SUPickering. Researches and reports on Pickering emulsions have emerged in an endless stream in recent years, involving various aspects such as preparation and characterization analysis. Pickering emulsions are also used in production and life, such as adhesives, sealants and waterproof coatings, in cosmetics such as shampoos and conditioners, etc. The solid particles used to stabilize the Pickering emulsion must also meet certain conditions, and their shape, size and hydrophobicity all have varying degrees of influence on the stability of the emulsion. For example, the contact angle between particles and droplets is one of the characteristics of hydrophobicity. The smaller the contact angle, the easier it is for the particles to be wetted by droplets, the less likely it is to prevent the aggregation between droplets, and the more unstable the emulsion formed. Only partially hydrophobic particles, that is to say, particles whose contact angle with the droplet is close to 90 degrees, can be wetted by the two incompatible liquid phases respectively, so as to better bind to the surface of the droplet and prevent its aggregation, which stabilizes the emulsion better. The particles commonly used to prepare Pickering emulsions include silicates, aluminates, titanates, silicon aluminum, metal oxides, carbon black, nitrides, cadmium selenide, etc., and most of them are based on SiO ₂ constructed particles.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, provide a kind of emulsion based on stabilizer and its preparation method and application, can prepare the stabilizer with better hydrophilic lipophilicity, and have positive charge and magnetism, utilize The double emulsion prepared by the modified stabilizer has magnetism and positive charge, and can be used as a magnetic gene carrier or a contrast agent for magnetic resonance imaging.

The present invention is achieved through the following technical solutions,

In the first aspect, the present invention relates to a stabilizer, which can be prepared by the following method: take a high molecular polymer and iron halide and prepare magnetic nanoparticles by chemical co-precipitation method, that is, the stabilizer;

The preparation method specifically refers to: dissolving _FeCl3 and _FeCl2 in degassed water to prepare an iron salt solution; dissolving a high molecular polymer in degassed water to obtain a solution, mixing the solution with an iron salt solution to obtain a mixed solution; place the mixed solution in an ice-water bath, and carry out the following operations while stirring under the protection of nitrogen: add the aqueous solution of NaOH drop by drop, stop the titration when the pH value is 10.5 to 11, and continue the ice bath; put the solution under the protection of nitrogen oil bath, centrifuge, take the supernatant, and dry to obtain the stabilizer.

In a second aspect, the present invention relates to a modification method of the above-mentioned stabilizer: get the MES of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the stabilizer and polyethyleneimine After the solution is mixed and stirred, it is dried by dialysis to obtain a positively charged stabilizer.

In a third aspect, the present invention relates to a stabilizer-based emulsion comprising: an aqueous phase, an oil phase and said stabilizer.

In the fourth aspect, the present invention relates to an emulsion based on a positively charged stabilizer. The emulsion is a double emulsion with a water-in-oil-in-water structure, and its components are: a water phase, an oil phase and the positively charged stabilizer.

In a fifth aspect, the present invention relates to a method for preparing the above-mentioned emulsion based on a positively charged stabilizer, comprising the steps of:

Step 1, using ultrasonic method to prepare colostrum with oil-in-water structure;

Step 2, using the oil-in-water colostrum and a positively charged stabilizer to prepare a double emulsion with an oil-in-water structure, ie, an emulsion.

The preparation described in step 2 refers to: carrying out the reaction under continuous ultrasonic environment.

In the sixth aspect, the present invention relates to the use of the above-mentioned emulsion, which is used to prepare a gene carrier or a magnetic resonance imaging contrast agent.

In a seventh aspect, the present invention relates to another method for preparing an emulsion based on a positively charged stabilizer, comprising the steps of:

Step 1, using ultrasonic method to prepare colostrum with oil-in-water structure;

Step 2, using the oil-in-water structure of the colostrum and the positively charged stabilizer, under the condition of continuously feeding sulfur hexafluoride, preparing the double emulsion of the water-in-oil-in-water structure, that is, the emulsion.

The preparation described in step 2 refers to: carrying out the reaction under continuous ultrasonic environment;

In an eighth aspect, the present invention relates to the use of the above-mentioned emulsion, which is used to prepare a contrast agent for ultrasonic diagnosis.

Compared with the prior art, the present invention has the following beneficial effects: the stabilizer prepared by the present invention is a solid particle, which has better hydrophilicity and lipophilicity after compound modification with PEI, and has positive charge and magnetism; The emulsion prepared by using the modified stabilizer of the present invention has magnetism and positive charge, and can be used as a magnetic gene carrier or a contrast agent for magnetic resonance imaging; at the same time, during the process of preparing the emulsion, sulfur hexafluoride gas is passed while ultrasonic, The emulsion containing sulfur hexafluoride can be obtained, and this emulsion can be used as a new contrast agent for ultrasonic imaging diagnosis.

Description of drawings

Fig. 1 is the appearance diagram of W/O colostrum and W/O/W double milk;

Fig. 2 is the result figure of W/O colostrum and W/O/W double emulsion observed under a 40 times optical microscope;

Fig. 3 is the agarose gel electrophoresis figure after W/O/W double milk is combined with DNA;

Figure 4 is the comparison result of transfection of COS-7 cells carried by W/O/W double milk carrying pGL3-control plasmid;

Fig. 5 is the in vitro magnetic resonance imaging (T2 weighted phase) photo of W/O/W double emulsion and stabilizer;

Figure 6 is the morphological diagram of W/O/W double emulsion under TEM;

Figure 7 is the in vitro imaging results of W/O/W double milk under the ultrasonic diagnostic instrument.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

Example 1

The experimental materials involved in this embodiment are as follows:

Ferrous chloride, ferric chloride, and sodium hydroxide were all analytically pure, purchased from China Pharmaceutical Group Shanghai Chemical Reagent Company;

Polylactic acid (PLA), purchased from Shanghai Bybo Biotechnology Co., Ltd.;

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was purchased from Sigma;

Polyethyleneimine (PEI) 25000 is purchased from Sigma company;

soybean oil, commercially available;

Sulfur hexafluoride (SF ₆ ) gas was purchased from CNNC Honghua Special Gas Co., Ltd.;

African green monkey kidney cell line COS-7 cells were purchased from ATCC;

RPMI-1640 medium and serum were purchased from GIBICO;

The luciferase reporter plasmid pGL3-control was purchased from Promega, USA;

The PCS plasmid is disclosed in the publication specification of Chinese invention patent application CN101095951;

The luciferase detection kit was purchased from Promega, USA;

BCA protein quantification kit was purchased from Shanghai Karma Biotechnology Company;

The stabilizer preparation method of the present embodiment is as follows:

Weigh 730mg FeCl ₃ and 360mg FeCl _{2 and} dissolve in 5ml degassed water to obtain iron salt solution; then take 2.8g PLA and dissolve it in 2ml degassed water to obtain a solution, mix this solution with iron salt solution to obtain a mixed solution Place the mixed solution in an ice-water bath (2-4°C), and carry out the following operations while stirring under nitrogen protection: add an aqueous solution of NaOH (5M) drop by drop, and monitor the pH value online with a pH meter. When the pH value is 10.5- Stop the titration at 11:00, and continue the ice bath for 30 minutes; under the protection of nitrogen, heat the solution in an oil bath at 85°C for 2 hours to mature the particles; after the reaction is completed, centrifuge at 9000rpm and 4°C for 10 minutes to remove the large precipitated particles and keep the supernatant. Centrifuge again under the same conditions for 10 minutes, further remove the large particle precipitate, retain the supernatant, and obtain a magnetic particle solution of about 10ml; further, the magnetic nanoparticle solid (ie, MP-PLA magnetic particle) can be obtained by dialysis and drying, that is, the stabilizer.

The emulsion preparation method of the present embodiment is as follows:

Ultrasonic preparation of W/O emulsion using the above stabilizer: Take the solution obtained by dialysis (i.e. the stock solution of PLA magnetic particles), dilute it 5 times, take 100ul to mix with 900ul soybean oil, 25°C, 30% ultrasonic intensity, ultrasonic 7 times , every time 5S on, 6S off, get W/O emulsion.

Example 2

The present embodiment adopts the same materials and steps as in Example 1 to prepare a stabilizer, and the method for preparing an emulsion based on the stabilizer specifically includes the following steps:

Ultrasonic preparation of O/W emulsion using the above stabilizer: Take the solution obtained by dialysis (i.e. the stock solution of PLA magnetic particles), dilute it 5 times, take 900ul to mix with 100ul soybean oil, 25 ° C, 30% ultrasonic intensity, ultrasonic 7 times , every time 5S on, 6S off, get O/W emulsion.

Example 3

The present embodiment adopts the material identical with embodiment 1 to prepare positively charged stabilizer, and this preparation method specifically comprises the following steps:

Step 1, taking polylactic acid, FeCl ₃ and FeCl ₂ , and preparing magnetic nanoparticles by chemical co-precipitation method;

Weigh 730mg FeCl ₃ and 360mg FeCl _{2 and} dissolve in 5ml degassed water to obtain iron salt solution; then take 2.8g PLA and dissolve it in 2ml degassed water to obtain a solution, mix this solution with iron salt solution to obtain a mixed solution Place the mixed solution in an ice-water bath (2-4°C), and carry out the following operations while stirring under nitrogen protection: add an aqueous solution of NaOH (5M) drop by drop, and monitor the pH value online with a pH meter. When the pH value is 10.5- Stop the titration at 11:00, and continue the ice bath for 30 minutes; under the protection of nitrogen, heat the solution in an oil bath at 85°C for 2 hours to mature the particles; after the reaction is completed, centrifuge at 9000rpm and 4°C for 10 minutes to remove the large precipitated particles and keep the supernatant. Centrifuge again under the same conditions for 10 minutes, further remove the large particle precipitate, keep the supernatant, and obtain about 10ml of magnetic particle solution; further, the magnetic nanoparticle solid can be obtained by drying;

Step 2, take 1-(3 dimethylaminopropyl) 3 ethyl carbodiimide, magnetic nanoparticles, MES solution of polyethyleneimine, mix, stir to make it react, dialyze, dry to obtain a stabilizer;

Dissolve PEI25000 in the MES solution (0.05M MES, 0.5M NaCl) of pH 6.0, so that the final concentration of PEI25000 is 0.1667g/ml to obtain the MES solution of PEI; take 400ul of the magnetic particle solution obtained in step 1, and 240ul of PEI MES solution, 80ul EDC, mixed, stirred and reacted at room temperature for 2 hours, then placed the solution in a dialysis bag with a molecular weight of 10,0000 and dialyzed for 4 hours to remove free PEI and EDC to obtain MP-PLA-EDC-PEI magnetic particles The stock solution was dried to obtain a stabilizer.

The emulsion preparation method of the present embodiment comprises the steps:

Step 1, using the above-mentioned positively charged stabilizer and ultrasonic method to prepare W/O colostrum: take the solution obtained by dialysis (that is, the stock solution of MP PLA EDC PEI magnetic particles), dilute it 5 times, take 100ul and mix it with 900ul soybean oil , 25°C, 30% ultrasonic intensity, ultrasonic 7 times, each time 5S on, 6S off, to get W/O colostrum;

Step 2, using the above-mentioned positively charged stabilizer and W/O colostrum to ultrasonically prepare W/O/W double emulsion, that is, emulsion: take the solution obtained by dialysis (that is, the stock solution of MP-PLA-EDC-PEI magnetic particles), Mix 900 ul of the 6-fold diluted solution with 100 ul of W/O colostrum, then 25°C, 30% ultrasonic intensity, and 6 times of ultrasonication to obtain a relatively uniform double emulsion, ie an emulsion. The composition of the emulsion is as follows: by volume, the ratio of the water phase to the oil phase is 91:9, and the concentration of the stabilizer in the water phase is 0.032 g/ml.

Implementation Effect

(1) Characterization analysis of magnetic particles before and after connection with PEI

Take the magnetic particles (MP-PLA) and the stabilizer (MP-PLA-EDC-PEI), respectively take an equal volume of deionized water ddH ₂ O to dilute, and then measure the Zeta potential and particle size. The results are shown in Table 1, from which it can be seen that , compared with MP-PLA, the particle size of MP-PLA-EDC-PEI becomes larger, and the surface potential also changes from negative to positive, indicating that PEI has been connected to the surface of MP-PLA, because it reacts with some carboxyl groups on PLA, so The negative charge on the surface of the magnetic particles is reduced, resulting in particle aggregation and particle size increase; at the same time, because PEI is connected to the surface of MP-PLA, the surface of MP-PLA-EDC-PEI has many amino groups, so the surface potential is positive.

Table 1

MP-PLA MP-PLA-EDC-PEI W/O/Wmulti-emulsion particle size 34nm 1.59um 140.4nm Zetapotential -34.1mv 40.8mv 55.5mv

(2) Investigation of the appearance and stability of the emulsion

The appearance of colostrum and double milk was observed with ordinary camera and optical microscope. The results of ordinary cameras are shown in Figure 1. A is the oil phase and water phase before the formation of colostrum, B is W/O colostrum, and C is W/O/W double emulsion; then the colostrum is diluted 5 times with PBS respectively, and the double The milk was diluted 40 times and observed under a 40 times optical microscope. The results are shown in Figure 2. A is the diluted colostrum, and B is the diluted double milk. It can be seen that the formed colostrum and double milk are comparable in appearance. Uniformity, the shape of emulsion droplets observed under a light microscope is relatively stable, the particle size distribution of emulsion droplets in double emulsion is more uniform, and it can be seen that many large emulsion droplets are surrounded by small emulsion droplets, indicating that W/O/W double emulsion has indeed been formed , and further proved that in the absence of any surfactant, only MP-PLA-EDC-PEI magnetic particles can be used as a stabilizer to form a relatively stable double emulsion. The double emulsion is stable for 48 hours at room temperature, and then gradually divides into two layers, but the boundary between the upper and lower layers is still very blurred. It is still a double emulsion, but the proportion of the upper oil phase is large, and the proportion of the lower water phase is large; dilute it After standing still for 36 hours at 100 times, uniformly dispersed emulsion droplets can still be seen under an optical microscope. The above evidence shows that the stability of the double emulsion prepared by using the stabilizer of this example is better.

(3) DNA-binding ability of W/O/W double emulsion in vitro

Dilute the double emulsion 10 times and place it in a 10,0000 dialysis bag, dialyze in PBS for 4 hours, then take 2ul of the double emulsion after dialysis and dilute it 5 times and mix it with 0.5, 1, 1.5, 2, 2.5, 3 respectively , 3.5, and 4ul of 1.25ug/ul PCS plasmids were mixed, allowed to stand at room temperature for 10 minutes, and then loaded for electrophoresis, and 1ul of the PCS plasmid was used as a control. 0.8% agarose gel, 80V, electrophoresis for 20 minutes, and then observed and photographed under the gel imaging system to find the optimal ratio of double emulsion and DNA binding. The results of agarose gel electrophoresis are shown in Figure 3. Bands 1 to 9 represent the electrophoresis lanes after complexing 2ul double emulsion with 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4ul of 1.25ug/ul PCS plasmid, respectively. It can be seen that there are almost no bands in the first 4 lanes, while DNA bands appear in the rest of the lanes, which shows that 2ul double milk can complex 2ul PCS plasmid.

(4) In vitro gene transfection of W/O/W double milk

According to the density of 2*10 ⁴ cells/well, COS-7 cells were uniformly inoculated in 24-well plate, after 16 hours of culture, replaced with serum-free 1640 medium for 4 hours, and then in the same serum-free 1640 medium In each hole, add different volumes (0.1, 0.2, 0.3, 0.4ul) of emulsion and 3ul 0.4ug/ul of the pGL3-control plasmid for transfection, and 0.024ul of 0.1667g/ml PEI is also mixed with 3ul of 0.4 The ug/ul pGL3-control plasmid was compounded as a positive control; 4 parallel wells for each concentration were transfected for 3.5 hours. After removing the supernatant, replace it with a medium containing 10% serum and continue culturing for 24 hours, then remove the culture medium, wash the cells twice with PBS, and then lyse the cells with lysis solution, 130ul lysis solution/well, 37°C, lysis for 0.5 hours , and then take 10ul of the lysate and mix it with 10ul of Luciferace substrate mixture, and detect it under the illuminance meter. The results are shown in Figure 4. From the transfection results, the W/O/W double emulsion prepared with MP-PLA-EDC-PEI magnetic particles as a stabilizer can carry exogenous genes for in vitro transfection of cos-7 cells. And has a certain transfection efficiency. Among the four different emulsion concentrations, the transfection effect of 0.2ul is the best, indicating that the emulsion also has certain toxicity, and the best transfection effect can only be achieved by finding a suitable N/P.

(5) In vitro magnetic resonance (MRI) imaging of W/O/W double emulsion

The stock solution of the stabilizer and the stock solution of the W/O/W double emulsion were taken respectively, and were appropriately double-diluted with deionized water to obtain a series of dilutions of the stabilizer and W/O/W double emulsion containing different iron concentrations. The MRI signal intensity (T2 weighted phase) of the above samples was then detected with a 3T MRI scanner (TrioTim) from Siemens. The parameters used are: 20°C, 3T, spin-ethosequence, TR is 8000ms, and TE is 19ms. The specific results are shown in Figure 5. A is stabilizer MP-PLA-EDC-PEI, B is W/O/W double emulsion. It can be seen that within a certain range, for a given iron concentration, the W/O/W double emulsion shows a relatively darker image compared with the stabilizer MP-PLA-EDC-PEI, that is to say W/O/W double emulsion can increase the contrast of the image relatively stronger, and has the potential to become a contrast agent for magnetic resonance (MRI) imaging.

(6) Preparation of W/O/W double emulsion containing SF ₆

Prepare W/O colostrum according to the method in step 1 of the emulsion preparation method, then dilute the stock solution of MP-PLA-EDC-PEI magnetic particles 6 times, take 450ul as the outer water phase, and 50ul W/O colostrum as the inner water After the two are mixed together, the SF ₆ is passed continuously with ultrasound (100W) for 2 minutes and 5 minutes respectively, and two kinds of double emulsions containing gas are obtained. Observe under TEM (transmission electron microscope) after diluting 50 times, compare the influence of different aeration times on the shape of the formed double milk droplets, and use the hospital's ultrasonic detector to detect whether there is gas in the double milk droplets and the gas content. How many.

Figure 6 is the morphological diagram of W/O/W double emulsion under TEM; it can be seen that the emulsion droplets formed by short aeration time (2min) are generally very small under TEM, with a particle size of about 100-200nm, and the emulsion droplets Lack of three-dimensionality, see Figure 6A and Figure 6B; while the double emulsion formed by long ventilation time (5min) is observed under TEM. The feeling is very strong, see Figure 6C, Figure 6D. Through the comparison of the two double emulsions, it is proved that SF ₆ gas is contained in the emulsion droplets, which makes the emulsion droplets larger, and the center of the emulsion droplets is brighter and the periphery is darker. It acts as a stabilizer on the outer surface of the emulsion droplet. Ultrasonic detector detection, using CPS MSK mode, 14.0MHz frequency. The double milk formed by ventilation for 2 minutes was observed in the in vitro ultrasonic mode. There were relatively few bubbles at the beginning, and the developing effect gradually weakened under continuous ultrasound conditions, which can last for about 6 minutes, as shown in Figure 7A and Figure 7B; while the double milk formed by ventilation for 5 minutes was in the In the external ultrasonic mode, the bubbles are relatively dense at the beginning, and the developing effect can be maintained for about 10 minutes under continuous ultrasonic conditions, as shown in Figure 7C and Figure 7D, which shows that SF ₆ gas is wrapped in the emulsion droplets of double milk, And the formed double emulsion has the potential to be a contrast agent for ultrasonic diagnosis.

Claims (11)

1. magnetic nano-particle is as the application of stabilizing agent in preparation Emulsion, and the preparation method of described magnetic nano-particle is: get high molecular polymer and iron halide and adopt chemical coprecipitation to prepare;

Wherein, describedly get high molecular polymer and iron halide and adopt chemical coprecipitation preparation specifically to refer to: with FeCl ₃And FeCl ₂Be dissolved in the de aerated water preparation iron salt solutions; Get high molecular polymer and be dissolved in the de aerated water, get solution, this solution is mixed with iron salt solutions, get mixed solution; Mixed solution is placed ice-water bath, under nitrogen protection, proceed as follows while stirring: dropwise drip the aqueous solution of Na0H, when pH value is 10.5～11, stop titration, continue ice bath; With the solution oil bath, centrifugal under nitrogen protection, get supernatant, drying gets stabilizing agent.

2. Emulsion, this Emulsion is water in oil emulsion or oil in water emulsion, it is characterized in that, the component of described Emulsion is: water, oil phase and stabilizing agent, the preparation method of described stabilizing agent is: get high molecular polymer and iron halide and adopt chemical coprecipitation to prepare magnetic nano-particle, be i.e. stabilizing agent; Wherein, describedly get high molecular polymer and iron halide and adopt chemical coprecipitation preparation specifically to refer to: with FeCl ₃And FeCl ₂Be dissolved in the de aerated water preparation iron salt solutions; Get high molecular polymer and be dissolved in the de aerated water, get solution, this solution is mixed with iron salt solutions, get mixed solution; Mixed solution is placed ice-water bath, under nitrogen protection, proceed as follows while stirring: dropwise drip the aqueous solution of NaOH, when pH value is 10.5～11, stop titration, continue ice bath; With the solution oil bath, centrifugal under nitrogen protection, get supernatant, drying gets stabilizing agent.

3. the purposes of an Emulsion according to claim 2 is characterized in that, with this Emulsion for the preparation of magnetic resonance imaging contrast.

4. a positively charged stabilizing agent is characterized in that, gets the 1-(3-dimethylamino-propyl) after the MES solution of-3 ethyl carbodiimide, magnetic nano-particle and polymine mixes and stir, through the dialysis dried, get the positively charged stabilizing agent; The preparation method of described magnetic nano-particle is: get high molecular polymer and iron halide and adopt chemical coprecipitation to prepare; Wherein, describedly get high molecular polymer and iron halide and adopt chemical coprecipitation preparation specifically to refer to: with FeCl ₃And FeCl ₂Be dissolved in the de aerated water preparation iron salt solutions; Get high molecular polymer and be dissolved in the de aerated water, get solution, this solution is mixed with iron salt solutions, get mixed solution; Mixed solution is placed ice-water bath, under nitrogen protection, proceed as follows while stirring: dropwise drip the aqueous solution of NaOH, when pH value is 10.5～11, stop titration, continue ice bath; With the solution oil bath, centrifugal under nitrogen protection, get supernatant, drying gets stabilizing agent.

5. an Emulsion is characterized in that, this Emulsion is the emulsion of W/O/W structure, and its component is: water, oil phase and positively charged stabilizing agent according to claim 4.

6. the preparation method of Emulsion according to claim 5 is characterized in that, comprises the steps:

Step 1 adopts ultrasonic method to prepare the colostrum of oil-in-water structure;

Step 2 is utilized the colostrum of oil-in-water structure and the emulsion that the positively charged stabilizing agent prepares the W/O/W structure, i.e. Emulsion.

7. the preparation method of Emulsion according to claim 6 is characterized in that, the preparation described in the step 2 refers to: react under lasting ultrasonic environment.

8. the purposes of an Emulsion according to claim 5 is characterized in that, with this Emulsion for the preparation of genophore.

9. an Emulsion that utilizes positively charged stabilizing agent claimed in claim 4 to be synthesized is characterized in that, this Emulsion is the emulsion of W/O/W structure, and its component is: water, oil phase, described positively charged stabilizing agent and sulfur hexafluoride.

10. the preparation method of an Emulsion according to claim 9 is characterized in that, comprises the steps:

Step 1 adopts ultrasonic method to prepare the colostrum of oil-in-water structure;

Step 2 is utilized colostrum and the positively charged stabilizing agent of oil-in-water structure, at the emulsion that continues to pass into preparation W/O/W structure under the condition of sulfur hexafluoride, i.e. Emulsion.

11. the purposes of an Emulsion according to claim 9 is characterized in that, with this Emulsion for the preparation of the ultrasonic diagnosis contrast agent.

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