CN112972754B - Visual fucoidin embolism microsphere and preparation method thereof - Google Patents

Abstract

A visual fucoidin embolism microsphere and a preparation method thereof belong to the field of embolism microspheres. The embolism microsphere adopts fucoidin as a main component, and has the function of magnetic resonance imaging through the functional modification of the fucoidin. The embolic agent has low cost and can replace the prior expensive embolic microsphere products at home and abroad.

Description

Visual fucoidin embolism microsphere and preparation method thereof

Technical Field

The application relates to the field of embolism microspheres, in particular to a visualized fucoidin embolism microsphere and a preparation method thereof.

Background

Transcatheter Arterial embolization (TACE) has become the preferred treatment for non-surgical removal of tumors. The principle is as follows: under the assistance of imaging equipment, a doctor injects an embolic agent into blood vessels of a focus part through a microcatheter to plug the blood vessels of the focus and cut off the nutrition supply of the focus part, so that the tumor is atrophied and necrotized, and finally the treatment is realized.

As a key to success or failure in TACE treatment, the choice of embolic agent is critical.

Because the current clinical embolic agent does not have an imaging function, the embolization effect cannot be evaluated, and in addition, the embolization microsphere product has high technical threshold, few categories and small selectivity, so that the price of the embolization microsphere product is high, and the hospitalization cost of a patient is greatly increased.

Therefore, it is significant to develop an embolization microsphere product which can replace the current clinical use.

Disclosure of Invention

The application provides a visual fucoidin embolism microsphere and a preparation method thereof, which are used for replacing the existing embolism products.

The application is realized as follows:

in a first aspect, the present application provides the use of fucoidan for the preparation of an embolic agent.

In some examples of the present application, the embolic agent described above is magnetic resonance imageable; optionally, the embolic agent is magnetic resonance imageable by the fucoidan loading of the contrast agent; alternatively, the fucoidan is present in the embolic agent as a pharmaceutical carrier; or water is a poor solvent for the embolic agent, allowing the embolic agent to be stable in water; optionally, the embolic agent is stabilized in water by the fucoidan-loaded polymer.

In some examples of the application, the contrast agent comprises a longitudinal relaxation contrast agent or a transverse relaxation contrast agent; optionally, the contrast agent is ferroferric oxide; optionally, the ferroferric oxide surface is modified with carboxyl for combining with hydroxyl on the fucoidin surface. And/or the polymer is prepared by the polymerization reaction of acrylic acid loaded on the surface of the fucoidin and 2-acrylamido 2-methylpropanesulfonic acid, and the medicine is adsorbed by sulfonic acid groups on the surface of the fucoidin.

In a second aspect, examples of the present application provide a microsphere comprising: a particle comprising fucoidan having a polymer formed on the surface thereof by polymerization.

Optionally, the polymer is formed with a contrast material loaded on the fucoidan through a chemical bond; optionally, the polymer is hydrophobic, further optionally, the polymer is hydrophobic and has a residue of a sulfonic acid group.

In some examples of the present application, the microspheres include one or more of the following: a first definition, the size of the particles is between 70 and 1000 μm; and/or the particles in the microspheres have a size distribution deviation between-5% and 5%; the contrast material in the second definition, microspheres, includes gadopentetate meglumine or ferroferric oxide; optionally, in the microsphere, the content of ferroferric oxide used as a transverse relaxation contrast agent is 5 to 18 wt%; a third definition, the polymer is crosslinked from a first reactant and a second reactant; alternatively, the first reactant comprises acrylic acid and the second reactant comprises 2-acrylamido 2-methylpropanesulfonic acid; fourthly, the 10-minute adsorption rate of the particles to the small molecule drug can reach more than 92 percent, and optionally, the small molecule drug is adriamycin; fifth, the particles have elasticity.

In a third aspect, examples of the present application provide an injectable comprising the aforementioned microspheres.

In some examples of the present application, the injectable article comprises: pharmaceutically acceptable adjuvants; alternatively, the injectable may comprise: a load adsorbed to the microsphere as a carrier, wherein the load comprises an anti-tumor agent, optionally, the anti-tumor agent comprises doxorubicin or epidoxorubicin; alternatively, the injectable is an embolic agent and is in the form of microspheres dispersed in water to form water; alternatively, the injectable is a slow release formulation and includes an envelope for containing and gradually releasing the microspheres.

In a fourth aspect, examples of the present application provide a method of preparing an embolic agent comprising providing an intermediate by functionally modifying fucoidan, the intermediate having fucoidan and being modified with an unsaturated bond and a contrast agent; the unsaturated bond of the intermediate is polymerized.

In some examples of the present application, a method of polymerizing unsaturated bonds of an intermediate comprises: forming an emulsion from the intermediate and the polymerization raw materials, and initiating by an initiator to generate crosslinking; alternatively, the emulsion is obtained by membrane emulsification of the intermediate and the polymerization starting material.

In a fifth aspect, examples of the present application provide a method of preparing an embolic agent. The preparation method comprises the following steps:

the method comprises the following steps: preparation of functionalized fucoidan

Dissolving fucoidin in alkaline aqueous solution to prepare fucoidin aqueous solution; then adding acrylic acid and carboxylated ferroferric oxide nano-particles; heating and stirring to obtain an unsaturated bond modified magnetic fucoidin aqueous solution, precipitating by using an organic reagent, and then washing and drying to obtain functionalized magnetic fucoidin;

step two: formulating the dispersed and continuous phases

Dissolving functionalized magnetic fucoidin in water to obtain a magnetic fucoidin water solution, and uniformly dispersing 2-acrylamide-based 2-methylpropanesulfonic acid in the magnetic fucoidin water solution to obtain a dispersion phase; mixing vegetable oil with emulsifier to obtain continuous phase;

step three: preparation of embolic agent

Injecting the dispersed phase into the continuous phase in a flowing state by pressurizing the dispersed phase through pores of the porous membrane by inert gas to obtain a mixed phase in an emulsion state; adding an initiator into the mixed phase to polymerize the acrylic acid and the 2-acrylamido 2-methylpropanesulfonic acid; the microspheres are isolated from the polymerization system and dispersed in water.

In a sixth aspect, examples of the present application provide a method of preparing an embolic agent. The preparation method comprises the following steps:

dissolving fucoidin solid in alkaline aqueous solution with the pH value of 7.8-9.0, and then adding carboxyl modified ferroferric oxide nano particles and acrylic acid into the aqueous solution; heating and stirring, using ethanol as a precipitator, and performing reversed-phase precipitation, washing and vacuum drying to obtain a functionalized modified magnetic fucoidin intermediate;

dissolving the magnetic fucoidin intermediate in water at 60-80 deg.C under stirring to obtain fucoidin water solution; uniformly dispersing 2-acrylamido 2-methylpropanesulfonic acid into a fucoidin water solution under the condition of stirring at room temperature to obtain a mixed solution, wherein the content of the fucoidin intermediate in the mixed solution is 5 wt% to 20 wt%, and the content of the 2-acrylamido 2-methylpropanesulfonic acid in the mixed solution is 4 wt% to 15 wt%;

taking the mixed solution as a dispersed phase, and taking a vegetable oil solution containing 1 to 5 weight percent of emulsifier as a continuous phase; pouring the dispersed phase into a dispersion tank, adding the continuous phase into a beaker, and stirring the solution of the continuous phase at the stirring speed of 300-800rpm to keep the continuous phase in fluidity;

pressurizing the dispersed phase by nitrogen to make the dispersed phase enter the continuous phase through membranes with different pore sizes; then taking out the continuous phase, adding an initiator into the continuous phase, and stirring for 6-12h at the temperature of 50-75 ℃ and the speed of 300-800rpm to ensure that the 2-acrylamide-based 2-methanesulfonic acid and the magnetic fucoidan modified with unsaturated double bonds have polymerization reaction; then filtering, removing oil phase, washing with ethanol and water in sequence, and dispersing the obtained microspheres in water.

In the implementation process, the visualized fucoidin embolism microsphere provided by the embodiment of the application can be used for medical imaging, can carry out medicine loading efficiently, and is simple in process and easy to implement, so that large-scale production is facilitated.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments or the prior art of the present application, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a T2 magnetic resonance image of magnetic fucoidan embolization microspheres in rabbit liver tumor;

FIG. 2 shows optical microscope pictures of fucoidan embolic microspheres having dimensions of 100 μm (a), 300 μm (b), 500 μm (c), 700 μm (d), respectively;

fig. 3 (a) is a scanning electron microscope image of the internal structure of the magnetic fucoidan embolic microsphere, which shows that the magnetic fucoidan embolic microsphere has a reticular pore structure; (b) high-power scanning electron microscope pictures show that ferroferric oxide nano particles are doped in the framework of the embolism microsphere;

in fig. 4, (a) a photograph before applying pressure to the magnetic fucoidan embolization microspheres, (b) a photograph after applying 20g of pressure to the magnetic fucoidan embolization microspheres, the compressible deformation of the microspheres was measured to be 80%, and (c) a photograph after removing the pressure, the fucoidan embolization microspheres recovered the spherical shape.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following detailed description is made for a visualized fucoidan embolic microsphere and a preparation method thereof according to the embodiment of the present application:

the common embolic agents at present comprise polyvinyl alcohol particles, sodium alginate microspheres, EMG embolic particle balls, gelatin sponge particles, drug-loaded microspheres and the like. However, as far as the present inventors know, none of the TACE microsphere products currently on the market can be imaged, and it is difficult to evaluate the embolization effect after operation. Therefore, there is a need to improve upon existing embolic solutions. Based on such practical needs, the inventors propose a visualized embolization microsphere product with imaging function in the present application.

And is different from the above-mentioned conventional embolization agent scheme. In the present application, Fucoidan (Fucoidan), a natural high molecular compound, was selected.

Fucoidin is a water-soluble heteropolysaccharide sulfate, and contains L-fucose and sulfate radical as main components, galactose, arabinose, mannose, xylose, glucuronic acid, etc. In the prior art, fucoidin is mostly used for preparing substances such as food, beverage, beer, health care products and the like. Also, in these applications, the fucoidan is typically present in a separate form in the product as a composition (e.g., an emulsion). For example, it is mechanically mixed with other Chinese medicinal materials. In the application, fucoidin is applied to the embolism microsphere, so that the application field of the embolism microsphere is greatly expanded.

The selection of fucoidan in the present application is based on the consideration that:

the fucoidin has good biocompatibility and multiple biological functions. Meanwhile, because the molecular structure of the fucoidin contains a large amount of sulfate groups (the sulfate groups have strong electronegativity), the fucoidin has very strong drug adsorption activity, so that antitumor drugs (such as adriamycin and epiadriamycin) can be efficiently and quickly adsorbed.

In addition, fucoidan itself has anticancer function. The anticancer effect is mainly embodied in three aspects:

(1) Inhibiting cell division, and inducing cancer cell apoptosis.

(2) Inhibiting tumor angiogenesis and inhibiting cancer cell metastasis.

(3) Enhancing immunity and activating immune system.

Therefore, based on the above thought, the inventor prepares an embolization microsphere product (which can enter peripheral blood vessels such as capillary vessels to block blood supply and realize physical embolization) with uniform size, fucoidan as a base material and good biocompatibility through research. In addition, the embolism microsphere product also has the loading performance of active drugs and the visualization effect. At the same time, the product is a material with good water dispersibility, thereby being beneficial to the use in an aqueous environment.

Since pure fucoidan is readily soluble in water, especially hot water. Therefore, direct use thereof will cause it to dissolve in water, thereby failing to exert the embolizing effect. In view of this, the present application modifies its molecular structure so that it changes from a water-soluble substance to a water-insoluble substance and thus can exist stably in water. For example, the surface of the material is modified with groups to form a hydrophobic surface group structure. In the present application, the polymer backbone is formed by polymerization (e.g., crosslinking), thereby significantly improving its stable presence in water.

Further, in view of the drug loading requirements of fucoidan, it is desirable that the embolic microspheres are adsorptive of active drugs (e.g., small molecule drugs) and greater amounts of adsorption may provide better benefits. However, as described above, surface structure modification thereof may cause groups on the surface of fucoidan to be "blocked", which may cause a decrease in drug loading.

Therefore, in combination with the above means for stabilizing fucoidan in water, in the present example, the hydrophobic surface group structure (e.g., polymer backbone) is introduced, and at the same time, a group capable of supporting a drug is introduced, so that the present example has the characteristics of drug loading and stable existence in water.

Still further, other performance improvements may be made in addition to this, as desired. For example, in some examples of the present application, contrast agents are introduced into the embolic balls based on the need for location awareness of the embolic balls, thereby enabling visual adaptation of the new embolic balls. Wherein, the visualization means that: the localization can be performed by imaging means, such as magnetic resonance imaging. In the present application, contrast agents that can be localized by imaging means of magnetic resonance imaging, such as ferroferric oxide (T2 contrast agent), are introduced into the embolic ball, or may be replaced by other contrast agents (such as T1 contrast agent gadopentetate meglumine, etc.).

Thus, in some instances, the resulting embolic microsphere is a visible, drug-loaded, and water-stable product. An example of a visualization thereof can be disclosed in fig. 1.

Microscopically, the surface of the embolization microspheres obtained in the examples of the present application has a porous structure, so that loading of the drug (rapid loading, and high loading rate) can also be achieved. For example, in some cases, it was verified that the adsorption rate of the embolization microspheres to doxorubicin (chemotherapeutic drug) could reach more than 92% in 10 minutes. The adsorption rate (v) — (m1-m2)/m1 × 100%. Where m1 is the mass of drug added and m2 is the mass of drug that was not adsorbed after 10 minutes.

In addition, the embolization microspheres also have particle size uniformity through the choice of process. For example, by selective membrane emulsification, embolizing microspheres with a small particle size distribution (high uniformity of particle size of individual microspheres) can be obtained. In some examples, the particle size of the embolization microspheres may be controlled to be between 70 microns and 1000 microns, and may illustratively be 75 μm, 100 μm, 300 μm, 500 μm, or 700 μm. In some optimized schemes, the size distribution can reach +/-5%.

The morphology of different sizes of embolization microspheres and the uniformity of their size may be disclosed in fig. 2. As can be seen from fig. 2, the embolization microspheres have a spherical morphology. In use, it is delivered to the lesion through a catheter, and such a topography can allow for good flow through the catheter so that clogging of the catheter does not occur.

The microstructure of the embolization microspheres may be shown in fig. 3. As shown in fig. 3, the ferroferric oxide nano-particle has a network-like porous structure, and the surface of the network skeleton is provided with the ferroferric oxide nano-particle.

The embolization microspheres also have good dispersibility. This helps to disperse it in water, thereby facilitating transport and thus easier access to the location where embolization is desired. Meanwhile, the embolism microsphere is elastic, so that the embolism microsphere is more convenient to transport in a human body blood vessel or other channels. Such as a vessel collapsing at a portion of the site to reduce closure, or a vessel bending to cause a decrease in the smoothness of the inner wall. The embolization may be performed by "passing" these regions "through the site of the lesion due to the elasticity of the embolization microspheres. For example, due to its elasticity, it can pass through the embolic catheter smoothly, avoiding clogging the catheter. Through test calculation, the compression deformation ratio of the magnetic fucoidin embolism microsphere is determined to be up to 80%. The compression is shown in fig. 4, which is capable of returning to its original shape after removal of the squeezing force at a relatively high compression ratio, as shown in fig. 4.

In some examples of the application, ferroferric oxide, acrylic acid and 2-acrylamido 2-methylpropanesulfonic acid are respectively selected as modifying agents to modify fucoidan. And the ferroferric oxide, the acrylic acid and the 2-acrylamido 2-methylpropanesulfonic acid are bonded (combined) on the fucoidan in a chemical bond mode, so that the method is different from simple physical mixing.

Wherein, ferroferric oxide (Fe)₃O₄For example, the loading may be between 5 wt% and 18 wt%) can impart visualization properties to the embolization microspheres. If the loading amount is too low, the imaging effect is influenced; accordingly, if the loading is too high, the amount of modification of the acrylic acid and thus the subsequent crosslinking is affected.

In the present application, the ferroferric oxide is surface-modified with carboxyl groups (commercially available products may be selected). Therefore, the carboxyl-modified ferroferric oxide can be bonded (bonded) to fucoidan by reacting the carboxyl of the ferroferric oxide with the hydroxyl in the molecular structure of the fucoidan.

The acrylic acid can be used as anchor point or graft structure, so as to transfer the 2-acrylamide 2-methyl propane sulfonic acid on the fucoidan structure. Specifically, carboxyl groups in the molecular structure of acrylic acid react with hydroxyl groups in the molecular structure of fucoidan. And unsaturated bonds (double bonds) in the molecular structure of the 2-acrylamido 2-methylpropanesulfonic acid and unsaturated bonds (double bonds) in the molecular structure of the acrylic acid can be polymerized through addition reaction to form a polymer skeleton. The polymer skeleton also has exposed sulfonic acid groups introduced by 2-acrylamide-based 2-methylpropanesulfonic acid, so that the polymer skeleton can also play a role in adsorbing drugs (mainly drugs with positive groups) (when the embolic microspheres loaded with drugs are prepared, the drugs and the embolic microspheres can be mixed, and the drugs can be adsorbed by electrostatic action to realize drug loading). Thus, 2-acrylamido 2-methylpropanesulfonic acid can be bonded to fucoidan through acrylic acid. Meanwhile, as the fucoidan is also bonded with ferroferric oxide, the ferroferric oxide is randomly dispersed in the polymer skeleton in the embolism microsphere.

In order to facilitate the implementation of the protocol of the present application by the person skilled in the art, the following description will be given with reference to some examples of the preparation method thereof.

In this method, the preparation of microspheres is mainly carried out by a membrane emulsion method, and polymerization is effected in the process. Specifically, the method in the example mainly comprises three steps, and decibels are: (1) preparing unsaturated bond modified fucoidin macromolecules; (2) preparing a dispersed phase and a continuous phase by a membrane emulsification method; (3) the dispersed phase and the continuous phase react to obtain the embolism microsphere. Obviously, in the foregoing steps, the two steps of step (1) and step (2) may be performed simultaneously, or may be performed sequentially in other orders, and are not performed in the foregoing order. In other words, the preparation method proposed in the examples of the present application is a material preparation step, and a step of performing a polymerization reaction using the prepared material to form the embolic microsphere.

The preparation method of the unsaturated bond modified fucoidin macromolecule comprises the following steps:

fucoidan is dissolved in an alkaline solution (to prevent corrosion of subsequently used ferroferric oxide) in an amount of, for example, 10 to 30 wt% to obtain a first solution. Wherein the pH value of the alkaline solution can be approximately controlled between 7.8 and 9.0. The aqueous alkaline solution may be, for example, a soluble carbonate, an aqueous phosphate solution, an aqueous acetate solution. Alternatively, soluble salts include, but are not limited to, any one or more of potassium carbonate, sodium bicarbonate, sodium phosphate, sodium hydrogen phosphate and sodium acetate.

Then, adding acrylic acid and carboxyl modified ferroferric oxide nanoparticles into the first solution. Wherein, the mass ratio of the acrylic acid to the fucoidin is 1:3 to 1:1, and the dosage of the ferroferric oxide can be 5 to 18 weight percent. Heating is then carried out to raise the temperature (for example to 55-75 ℃). The magnetic fucoidan aqueous solution modified by the unsaturated bond can be obtained by continuously stirring overnight (under the conditions of, for example, a speed of 300-500rpm for 8-12 hours).

This may be followed by purification to separate the acrylic acid and ferroferric oxide bound fucoidan. For example, ethanol is used as a precipitant, and reversed-phase precipitation is performed. In other words, the obtained fucoidan modified with unsaturated bonds and having magnetism is water-soluble, but it is not alcohol-soluble for ethanol. Meanwhile, water and ethanol can be completely miscible, so that the fucoidan modified by unsaturated bonds and having magnetism is separated out from the solution system and is precipitated. The precipitate is then washed (e.g., with water to remove inorganic salts or other water insoluble impurities; or, alternatively, with ethanol to remove unreacted acrylic acid by dissolution) and dried under vacuum (e.g., rotary evaporation) to obtain the functionally modified magnetic fucoidan intermediate.

The preparation method of dispersed phase and continuous phase in the film emulsion method.

The membrane emulsion method can effectively control the uniformity of the particle size. It has better dispersing effect than mechanical dispersing method. In principle, the membrane emulsion method (membrane emulsification method) is a method in which a dispersed phase (fluid, mainly liquid) is pressurized to permeate a porous structure (which may be a porous plate or a porous membrane, for example, an SPG membrane) having a desired or designed uniform or non-uniform pore size to form a fine liquid and disperse the fine liquid in a continuous phase to prepare an emulsion.

First, the dispersed phase is prepared as follows:

the functionalized and modified magnetic fucoidan intermediate obtained as described above is dispersed in water, and then dissolved by heating (e.g., 60 to 80 ℃) with stirring, thereby obtaining an aqueous fucoidan solution (wherein the content of the functionalized and modified magnetic fucoidan intermediate is 5 to 20 wt%). Then adding 2-acrylamido 2-methylpropanesulfonic acid to the fucoidan aqueous solution. Then stirring and fully mixing the mixture at room temperature to obtain a dispersed phase mixed solution. Wherein the content of the 2-acrylamido 2-methylpropanesulfonic acid is from 4 to 15%.

Secondly, the preparation method of the continuous phase is as follows: the emulsifier is dispersed in the vegetable oil to form an emulsified oil phase. Wherein the content of the emulsifier is 1 to 5 wt%. The emulsifier component may be one or both of an anionic surfactant or a nonionic surfactant. Alternatively, anionic surfactants include, but are not limited to: carboxylates, sulfates and sulfonates. Or in other examples, the emulsifier may be selected from sodium stearate, sodium dodecyl sulfate or sodium dodecyl benzene sulfonate. Alternatively, nonionic surfactants include, but are not limited to: tween 20, Tween 60, Tween 80, F127 or P123, and the like.

The method for preparing the emulsion by the membrane emulsion method comprises the following steps:

pouring the magnetic fucoidin and a dispersed phase (9-35 percent of the total weight of the magnetic fucoidin and the dispersed phase) with the content of 2-acrylamido 2-methylpropanesulfonic acid of 4-15 weight percent into a dispersion tank; the continuous phase was added to the beaker and agitated at a stirring speed of 300-800rpm to maintain fluidity. Pressurizing the dispersed phase with certain nitrogen (other inert or non-oxidizing atmosphere, such as argon) pressure (e.g., 0.01MPa-0.1MPa) to make it enter the continuous phase through membranes with different pore sizes, standing for a proper time, and taking out the emulsified mixed solution of the dispersed phase and the continuous phase.

The method for preparing the embolism microsphere through polymerization reaction comprises the following steps:

an initiator is added to the prepared emulsified mixture, and then polymerization is simultaneously carried out by heating (e.g., 50-75 ℃) under stirring (conditions such as: speed 300-. The initiator may be, for example, an oxidation-reduction species such as ammonium sulfate and sodium bisulfite. Alternatively, AIBN/azobisisobutyronitrile may be used as the initiator. In the polymerization reaction, mainly acrylic acid and 2-acrylamido-2-methylsulfonic acid copolymerize to form a copolymer (e.g., a crosslinked network). Wherein the polymerization reaction is polymerization between acrylic acid which is a main form forming a cross-linked network (acrylic acid-modified fucoidan) and polymerization of acrylic acid and 2-acrylamido-2-methylsulfonic acid for modifying sulfonic acid groups.

The polymerization reaction is followed by isolation and purification. For example, the polymerization reaction system is filtered to remove the oil phase therefrom. Liquid substances (including water and an oil phase) in the reaction system are filtered, and the obtained solid is washed by ethanol (such as three times of washing) and water (such as once of washing), so that the microspheres are obtained. Dispersing the obtained microspheres in water to obtain a visualized fucoidin embolism microsphere product which stably exists in the water and has uniform size.

By implementing the visualized fucoidan embolic microsphere and the preparation method thereof in the examples of the application, the following effects can be obtained through proper selection and adjustment of the method:

(1) the current marketed embolization microsphere products cannot be imaged, and the embolization effect after TACE treatment is difficult to evaluate. The visualized fucoidan product prepared by the method can be used for evaluating the embolism effect of the microsphere through magnetic resonance imaging after operation.

(2) The fucoidin material selected by the application has good biocompatibility and an anti-tumor effect, and is an ideal embolism microsphere material.

(3) The embolism microsphere products on the market at present are few in types and expensive in price. The preparation method takes fucoidin as a base material, and the prepared novel visual drug-loaded embolism microsphere product is expected to replace the embolism microsphere product used clinically, so that the medical cost of a patient is reduced.

(4) The fucoidin embolism microsphere product prepared by the method has the characteristics of uniform size (the particle size range can be regulated and controlled in the range of 100-1000 microns), good dispersibility, larger elasticity and rapid and efficient drug loading, thereby filling the blank of the fucoidin embolism microsphere.

(5) The membrane emulsification method can accurately control the size of the microspheres, is simple to operate, low in preparation cost and capable of realizing large-scale production.

The present application is described in further detail with reference to examples below.

Example 1

The preparation method of the visual fucoidin embolism microsphere by the membrane emulsification method comprises the following steps:

the method comprises the following steps: preparation of functionalized fucoidan macromolecule:

dissolving 50g of fucoidin solid in 150ml of sodium carbonate aqueous solution with the pH value of 8.0 to prepare fucoidin aqueous solution with the mass concentration of 25%; then adding 10ml of acrylic acid and 0.2g of carboxyl modified ferroferric oxide nano particles into the system, raising the temperature of the system to 65 ℃, and stirring at 400rpm for 10 hours to obtain unsaturated bond and ferroferric oxide modified fucoidin; and finally, using ethanol as a precipitator, and performing reversed-phase precipitation, washing and vacuum drying to obtain the functionalized magnetic fucoidin intermediate.

Step two: preparation of dispersed phase by membrane emulsification method:

taking 10g of the fucoidin modified by the unsaturated bond in the step one, dissolving the fucoidin modified by the unsaturated bond in 30ml of water, stirring at 70 ℃, and uniformly stirring at 300rpm to obtain a fucoidin water solution with the mass fraction of 25%; adding 1g of 2-acrylamide 2-methylpropanesulfonic acid into the system, and uniformly stirring at room temperature to obtain a disperse phase mixed solution for a membrane emulsification method.

Step three: preparing the embolism microsphere:

adding 1g of F127 into 50ml of vegetable oil, and uniformly mixing to obtain a continuous phase; and pouring the dispersed phase obtained in the second step into a dispersion tank, and stirring the continuous phase solution at a stirring speed of 300rpm to keep the fluidity of the continuous phase.

Pressurizing the dispersed phase with 0.05MPa of nitrogen pressure, allowing it to enter the continuous phase through a membrane with a pore size of 100 microns, and taking out the continuous phase after 30 min.

Adding 0.5g of azobisisobutyronitrile into the continuous phase, stirring for 6h at 60 ℃ and 500rpm, then filtering, removing the oil phase, and washing with ethanol and water to obtain the visualized fucoidan embolism microsphere product with the size of 150 microns.

Example 2

The preparation method of the visual fucoidin embolism microsphere by the membrane emulsification method comprises the following steps:

the method comprises the following steps: preparation of functionalized fucoidan macromolecule:

Dissolving 50g of fucoidin solid in 150ml of sodium carbonate water solution with pH value of 8.0 to prepare fucoidin water solution with mass concentration of 25%; 10ml of acrylic acid and 0.6g of carboxyl-modified ferroferric oxide nanoparticles are then added to the system.

Raising the temperature of the system to 65 ℃, stirring at 400rpm for 10h to obtain fucoidin aqueous solution modified by unsaturated bonds; and finally, using ethanol as a precipitator, and performing reversed-phase precipitation, washing and vacuum drying to obtain the functionalized magnetic fucoidin intermediate.

Step two: preparation of dispersed phase by film emulsification method

Taking 10g of the fucoidin modified by the unsaturated bond in the step one, dissolving the fucoidin modified by the unsaturated bond in 30ml of water, stirring at 70 ℃, and uniformly stirring at 300rpm to obtain a fucoidin water solution with the mass fraction of 25%; 1g of 2-acrylamide-based 2-methylpropanesulfonic acid is added into the system, and the mixture is uniformly stirred at room temperature to obtain a dispersed phase mixed solution for a membrane emulsification method.

Step three: preparing the embolism microsphere:

adding 1g of F127 into 50ml of vegetable oil, and uniformly mixing to obtain a continuous phase;

and (4) pouring the dispersed phase in the step two into a dispersing tank, and stirring the continuous phase solution at a stirring speed of 300rpm to keep the fluidity of the continuous phase.

The dispersed phase was pressurized with nitrogen pressure of 0.05MPa to pass through a membrane with a pore size of 250 microns into the continuous phase. And (3) taking out the continuous phase after 30min, adding an initiator into the continuous phase, stirring at 60 ℃ and 500rpm for 6h, then filtering, removing an oil phase, and washing with ethanol and water to obtain a visualized fucoidan embolism microsphere product with the size of 300 microns.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (2)

1. A method of preparing a medically imageable embolic agent, the method comprising:

the method comprises the following steps: preparing functionalized fucoidan;

dissolving fucoidin in alkaline water solution to prepare fucoidin water solution; then adding acrylic acid and carboxylated ferroferric oxide nano-particles; heating and stirring to obtain an unsaturated bond modified magnetic fucoidin aqueous solution, precipitating by using an organic reagent, and then washing and drying to obtain functionalized magnetic fucoidin;

Step two: preparing a dispersed phase and a continuous phase;

dissolving the functionalized magnetic fucoidin in water to obtain a magnetic fucoidin water solution, and uniformly dispersing 2-acrylamide 2-methylpropanesulfonic acid in the magnetic fucoidin water solution to obtain a dispersion phase; mixing vegetable oil with emulsifier to obtain continuous phase;

step three: preparing an embolic agent;

injecting the dispersed phase into the continuous phase in a flowing state by pressurizing the dispersed phase through pores of a porous membrane by an inert gas to obtain a mixed phase in an emulsion state; adding an initiator into the mixed phase to polymerize acrylic acid and 2-acrylamido 2-methylpropanesulfonic acid; the microspheres are isolated from the polymerization system and dispersed in water.

2. A method of preparing a medically imageable embolic agent, the method comprising:

dissolving fucoidin solid in alkaline aqueous solution with the pH value of 7.8-9.0, and adding carboxyl modified ferroferric oxide nano particles and acrylic acid into the solution; heating and stirring, and then using ethanol as a precipitator, and performing reversed-phase precipitation, washing and vacuum drying to obtain a functionalized modified magnetic fucoidin intermediate;

dissolving the magnetic fucoidin intermediate in water at the temperature of 60-80 ℃ by stirring to obtain fucoidin water solution; uniformly dispersing 2-acrylamido 2-methylpropanesulfonic acid into the fucoidin water solution under the condition of stirring at room temperature to obtain a mixed solution, wherein the content of the fucoidin intermediate in the mixed solution is 5 wt% to 20 wt%, and the content of the 2-acrylamido 2-methylpropanesulfonic acid in the mixed solution is 4 wt% to 15 wt%;

Taking the mixed solution as a disperse phase, and taking a vegetable oil solution containing 1-5 wt% of an emulsifier as a continuous phase; pouring the dispersed phase into a dispersion tank, adding the continuous phase into a beaker, and stirring the continuous phase solution at the stirring speed of 300-800rpm to keep the continuous phase in fluidity;

pressurizing the dispersed phase by nitrogen to enable the dispersed phase to enter the continuous phase through membranes with different pore sizes; then taking out the continuous phase, adding an initiator into the continuous phase, and stirring for 6-12h at the temperature of 50-75 ℃ and the speed of 300-800rpm to ensure that the 2-acrylamide-based 2-methanesulfonic acid and the magnetic fucoidan modified with unsaturated double bonds have polymerization reaction; then filtering, removing oil phase, washing with ethanol and water in sequence, and dispersing the obtained microspheres in water.

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