CN115487342A

CN115487342A - Self-developing absorbable embolism microsphere and preparation method thereof

Info

Publication number: CN115487342A
Application number: CN202210986495.4A
Authority: CN
Inventors: 谭奕勋; 杨顶建; 钟明君
Original assignee: Hainan Biomaike Medical Technology Co ltd
Current assignee: Hainan Biomaike Medical Technology Co ltd
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2022-12-20
Anticipated expiration: 2042-08-17
Also published as: CN115487342B

Abstract

The invention provides an auto-developing absorbable embolization microsphere and a preparation method thereof, belonging to the technical field of absorbable embolization microspheres. The invention introduces a developable iodobenzene compound into the gelatin sponge embolism microsphere in a chemical bonding mode, and provides the X-ray-opaque self-development absorbable embolism microsphere. The microsphere has the advantages of convenience for directly observing the position of the embolic agent clinically, judging the embolization endpoint, reducing the risk of ectopic embolization, being biodegradable and absorbable and the like.

Description

Self-developing absorbable embolism microsphere and preparation method thereof

Technical Field

The invention relates to the technical field of absorbable embolism microspheres, in particular to a self-developing absorbable embolism microsphere and a preparation method thereof.

Background

Liver cancer is one of the most common malignant tumors, and most patients are in middle and late stages at the time of diagnosis because of high concealment and unobvious early expression, and are not suitable for surgical resection treatment. For these patients, transcatheter Arterial Chemoembolization (TACE) is the first choice for non-surgical treatment by introducing chemotherapeutics and embolizing agents into tumor vessels via an interventional, super-selective intubation technique to block the blood supply to the tumor and necrotize the tumor under the action of local high-concentration chemotherapeutics. A large amount of clinical data and researches show that TACE can effectively prolong the life cycle of patients with liver cancer in middle and late stages and improve the life quality of the patients.

The particulate solid embolic agents currently used in TACE are classified into absorbable embolic agents, such as gelatin sponges, and non-absorbable embolic agents, such as polyvinyl alcohol, depending on the absorbability of the material. For recurrent tumors and TACE therapies requiring multiple treatments, absorbable embolization agents, after biodegradation, can recanalize blood vessels, leaving access for further treatment, with advantages in a particular clinical setting not available with non-absorbable materials. According to the different physical forms of the embolic agent, the embolic agent can be divided into irregular particle type and regular microsphere type, wherein the microsphere has the characteristics of smooth surface and uniform particle size, so that the vascular compliance of the embolic agent is better. Meanwhile, the microspheres are not easy to gather, and have good elasticity and better conduit trafficability. These advantages have led to the wide clinical acceptance and use of microsphere-based embolic agents.

The conventional solid embolic agent used clinically at present is not developed under X-ray, and the position and the embolization condition of the microsphere are indirectly judged by injecting the mixture under X-ray fluoroscopy after the embolic agent and the contrast agent are mixed. When the flow rate of the mixed solution of the contrast agent and the microspheres is stopped or reflowed, the operator judges that the embolism reaches the endpoint. However, the method has the disadvantages that the final actual position of the embolic agent cannot be accurately judged, and an operator cannot get real-time feedback in the operation, which brings inconvenience to the operation of the operator, affects the accuracy of judging the embolic endpoint, and increases the risk of ectopic embolism at a non-target part. Therefore, in order to enable an operator to directly observe the injection of the solid embolic agent under X-ray fluoroscopy, the convenience of operation is improved, the operator is more confident in pair grasping of embolism, the risk of ectopic embolism in the operation is effectively avoided, a vascular access is provided for subsequent treatment, and the research of the novel self-developing absorbable embolic microsphere has profound significance.

The invention patent with the application number of CN200710026274.8 discloses a biodegradable developing microsphere type vascular embolization material which is composed of a biodegradable material and an X-ray-impermeable developing material, wherein the biodegradable material wraps the developing material to form a particle structure, and the microsphere prepared by the patent has the characteristics of adjustable biodegradation time, good visibility under X-ray, moderate specific gravity, controllable diameter, low cost and the like. The invention patent with application number CN201110068821.5 discloses a developable gelatin sponge suppository for tumor embolization, which consists of three parts of gelatin, a swelling agent and a developing agent in any proportion.

However, the methods for preparing the self-developing absorbable embolization microspheres in the above patents all belong to physical embedding methods, and developing substances do not form chemical bonding with the microspheres, so that although a certain developing effect is achieved, the developing substances embedded in the microspheres can continuously seep out of the microspheres, and the developing effect is difficult to maintain for a long time.

Disclosure of Invention

Because the market lacks the absorbable microsphere of self-development for intervene the embolism art, the art person can't obtain the real-time feedback of embolism microsphere position, is difficult to judge the embolism terminal point and control ectopic embolism, has restricted the use of interveneeing the embolism art to a certain extent, and the absorbable microsphere of self-development that the physics embedding method was prepared simultaneously has the development material to ooze, is difficult to the defect that maintains the development effect for a long time. In view of the above, the present invention provides an autoradiography absorbable embolization microsphere and a preparation method thereof, wherein the invention introduces an X-ray opaque developing substance into a gelatin sponge embolization microsphere through chemical modification, and provides an X-ray opaque autoradiography absorbable embolization microsphere.

The invention provides a preparation method of self-developing absorbable embolism microsphere, which is formed by introducing iodobenzene compound into gelatin for cross-linking polymerization.

Preferably, the gelatin can also be at least one of vegetable gum, microbial gum, seaweed gum and starch. The vegetable gum comprises konjac gum, arabic gum and the like, the microbial gum comprises xanthan gum, gellan gum and the like, and the seaweed gum comprises carrageenan, alginate and the like.

More preferably, the gelatin is gelatin for capsules, the gelatin for capsules is a gelatin which is prepared by selecting the skin, bone, tendon, ligament and other connective tissues of animals such as fresh, healthy, strictly quarantined and non-chemically treated pigs and cattle and the like, is free of fat and protein and is easy to absorb by a human body through complex physicochemical treatment, and the gel strength of the gelatin is more than or equal to 250g Bloom.

Preferably, the iodobenzene compound is any one of 3,5-diiodobenzaldehyde dimethyl acetal, 3,5-diiodobenzoic acid, 2,3,5-triiodophenylacetaldehyde dimethyl acetal, 2,3,5-triiodophenylacetic acid, 2,3,5-triiodobenzoic acid, 2,3,5-triiodobenzaldehyde and 3,4,5-triiodobenzaldehyde.

Preferably, the main chain of the self-developing absorbable embolism microsphere has a structure shown in a general formula 1 or 2, X in the general formula 1 or 2 is a mono-iodine or multi-iodine substituted phenylalkyl functional group, the number of carbon atoms of alkyl in the phenylalkyl functional group is 0-20, the number of iodine atoms of a single benzene ring is 1-5,

general formula 1

General formula 2.

Preferably, the preparation method of the self-developing absorbable embolism microsphere comprises the following steps:

(1) Preparation of gelatin solution:

adding 0.01-2 parts of gelatin into a beaker filled with 0.2-3 parts of water for injection, carrying out constant-temperature water bath, stirring until the gelatin is completely dissolved, carrying out ultrasonic treatment, removing bubbles, and preserving the gelatin in the water bath at constant temperature for later use;

(2) Preparing the self-developing absorbable embolism microsphere by crosslinking iodobenzene compound and gelatin:

adding 1-15 parts of liquid paraffin and 0.006-1 part of span 80 into a reaction kettle at normal temperature, slowly adding the gelatin solution obtained in the step (2), stirring and dispersing for 5min, adding 0.03-3 parts of iodobenzene compound, stirring to completely dissolve the iodobenzene compound, dropwise adding 1-20 parts of formaldehyde solution for crosslinking reaction, keeping the reaction temperature less than or equal to 10 ℃, filtering to obtain microspheres after the reaction is finished, and washing the microspheres with 2% of Tween 80 aqueous solution until the washing water has no oil stains; then the microspheres are soaked and washed by water for injection for at least 1h, and then the microspheres are screened to obtain the finished product of the self-developing absorbable embolism microspheres.

The invention adopts the gelatin with good biocompatibility and low immunogenicity, introduces the developable iodobenzene compound in the chemical crosslinking process, and forms microspheres through crosslinking and curing, and the microspheres can be biodegraded and absorbed, are opaque to X rays and can be developed under medical imaging equipment.

Preferably, the iodo-benzene compound is commercially available or can be prepared by the following specific preparation steps:

reacting 0.2-2 parts of hydrochloric acid solution of anthranilic acid with 0.23-3 parts of iodine chloride hydrochloric acid solution at constant temperature of 30-80 ℃ for 1-4 hours to obtain precipitate, transferring the precipitate into a container, adding glacial acetic acid until the precipitate is weakly acidic, and cooling, filtering and drying to obtain amino-containing iodobenzene carboxylic acid;

mixing 0.5-2 parts of amino-containing iodobenzene carboxylic acid and 1-6 parts of sulfuric acid, slowly adding 0.2-3 parts of sodium nitrite aqueous solution, stirring in an ice bath for reaction for 1-3 hours to obtain a mixture A, blowing air into the mixture A, then carrying out suction filtration to obtain a yellow-green transparent liquid, adding a potassium iodide aqueous solution into the liquid, stirring, standing, heating to remove nitrogen, cooling to room temperature, removing iodine elements, carrying out suction filtration and drying to obtain an orange solid, and recrystallizing a crude product with absolute ethanol to obtain iodobenzene alkyl carboxylic acid;

adding 1-8 parts of borane-tetrahydrofuran solution into a mixed solution of 0.5-2 parts of iodobenzene alkyl carboxylic acid and 0.6-7 parts of anhydrous tetrahydrofuran at 0 ℃ under nitrogen flow, sequentially stirring the mixed solution at 0-5 ℃ for 1-3h, continuously stirring at room temperature for 1-2h to precipitate white solid, sequentially and slowly adding 0.5-6 parts of tetrahydrofuran/water cold mixed solution (13/2) and 2-30 parts of saturated sodium bicarbonate cold water solution to obtain white precipitate, filtering and washing to obtain iodobenzene alkyl alcohol;

under the protection of nitrogen and stirring, 0.02-2 parts of iodobenzene alkyl alcohol is dissolved in 6-70 parts of anhydrous DMSO, then 0.01-3 parts of ethyl acetate solution containing 50% propyl phosphoric anhydride is slowly dropped under the temperature condition of 22-25 ℃, stirring reaction is carried out for 2-5 hours, yellow solution is obtained, deionized water is added, white precipitate is generated, the white precipitate is filtered, the anhydrous DMSO and 50mL of deionized water are used for washing, slurrying, filtering and washing are carried out, vacuum drying is carried out for 15-30 hours at 30-70 ℃, the iodobenzene compound is obtained, and the purity is confirmed by NMR analysis and HPLC.

Preferably, the self-developing absorbable embolization microsphere in step (2) has a particle size in the range of 50-1500 μm.

Preferably, the self-developable absorbable embolization microsphere in step (2) has a developability of 500-10000 HU.

Preferably, the reaction time in the step (2) is 3.5 to 4.5 hours.

Preferably, the stirring speed in the step (2) is 130-170 r/min.

The second purpose of the invention is to provide the self-developing absorbable embolism microsphere prepared by the preparation method.

The self-developing absorbable embolism microsphere prepared by the invention has developing property, larger elastic expansion rate and recovery performance, uniform and controllable granularity and good dispersibility. The time for the self-developing absorbable embolization microspheres to degrade is 14-90 days according to different crosslinking and curing degrees. The CT value of the microsphere can reach 500-10000HU, and the gelatin microsphere can be distinguished from blood (30-451 HU), liver (40-60 HU), brain (20-45 HU) and soft tissue (100-300 HU) due to good developing property, so that the method provides convenience for minimally invasive interventional therapy of tumor diseases.

Compared with the prior art, the invention has the following beneficial effects: the self-developing absorbable embolization microsphere prepared by the invention stably combines the developing substance in the microsphere in a chemical bonding mode, so that the embolization microsphere has absorbability and developability at the same time, the defect that the embolization agent needs to be indirectly positioned by mixing the embolization agent and the contrast agent in the traditional interventional embolization operation is overcome, the clinical direct observation of the position of the embolization agent and the judgment of an embolization endpoint are facilitated, and the risk of ectopic embolization is further reduced. Meanwhile, the self-developing absorbable embolism microsphere produced by the preparation process has good flexibility and elasticity, good vascular compliance, capability of being tightly embedded with vascular walls and capability of providing a passage for secondary treatment after biodegradation and absorption.

Drawings

FIG. 1 is a scanning electron micrograph of an autographic absorbable embolization microsphere prepared according to example 1 of the present invention;

FIG. 2 is a scanning electron micrograph of individual microspheres of the self-developing absorbable embolization microsphere prepared according to example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The test methods or test methods described in the following examples are conventional methods unless otherwise specified; the starting materials and auxiliaries are, unless specified otherwise, either obtained from customary commercial sources or prepared in customary manner.

Example 1:

(1) Synthesis of developable 2,3,5-triiodobenzaldehyde:

the synthesis method comprises the following steps of synthesizing 2-amino-diiodobenzoic acid by using o-aminobenzoic acid, further synthesizing 2,3,5-triiodobenzoic acid, further synthesizing 2,3,5-triiodobenzyl alcohol, and finally synthesizing 2,3,5-triiodobenzaldehyde, and can greatly reduce the cost of raw materials, wherein the synthesis method comprises the following steps:

1) Preparation of 2-amino-diiodobenzoic acid

Adding 33g of hydrochloric acid solution of iodine chloride into 10.3g of hydrochloric acid solution of anthranilic acid under stirring, reacting for 2.5h at 60 ℃ to obtain a precipitate, adding sodium bisulfite solution until the color of the potassium iodide starch test paper is not changed, performing suction filtration and washing, transferring the precipitate into a beaker, adding glacial acetic acid until the precipitate is weakly acidic and a large amount of precipitate is generated, and then sequentially cooling, suction filtration and drying the large amount of precipitate to obtain 66g of off-white solid, wherein the yield is 90.16%, and the melting point is 233-234 ℃;

2) Preparation of 2,3,5-triiodobenzoic acid

Adding 28g of 2-amino-3.5-diiodobenzoic acid and 24ml of sulfuric acid into a 1000ml beaker, cooling in an ice bath, slowly adding 16.3g of sodium nitrite aqueous solution while stirring, stirring for 2 hours, adding a proper amount of crushed ice until the mixture is stirred in the beaker, blowing air into the mixture, then carrying out rapid suction filtration to obtain a yellow-green transparent liquid, adding a proper amount of potassium iodide aqueous solution into the filtrate, stirring, standing, heating to remove nitrogen, cooling to room temperature, removing elemental iodine, carrying out suction filtration, and drying to obtain 36g of orange-yellow solid with the yield of 95%, recrystallizing the crude product with absolute ethyl alcohol to obtain 37g of light-yellow needle-shaped crystals with the yield of 90% and the melting point of 223-224 ℃;

3) Preparation of 2,3,5-triiodobenzyl alcohol

Dropwise adding 26mL of a borane-tetrahydrofuran solution to 61mL of anhydrous tetrahydrofuran in which 30g of 2,3, 5-triiodobenzoic acid was dissolved at 0 ℃ under a dry nitrogen stream, stirring the resulting mixture at 0 ℃ for 1h 15min, then stirring at room temperature for 1h, at the end of the reaction, precipitating a white solid, slowly adding 50mL of a tetrahydrofuran/water (13/2) cold mixed solution, then adding 50mL of a saturated cold aqueous solution of sodium bicarbonate, allowing white precipitation to occur after stirring for 1h, recovering the solid by filtration and washing with water and cold anhydrous ethanol 3 times;

4) Preparation of 2,3,5-triiodobenzaldehyde

In a 50mL three neck round bottom flask equipped with thermometer, nitrogen bubbler and airtight seal 20.6g 2,3, 5-triiodobenzyl alcohol was dissolved in 100mL anhydrous DMSO under nitrogen protection and stirring, then 25mL ethyl acetate solution containing 50% propylphosphoric anhydride was added dropwise over 5min at 22-25 ℃, stirring at room temperature for 4h reaction was completed, the yellow solution was poured into 100mL deionized water under stirring to produce a white precipitate, which was filtered, washed with mother liquor and 50mL deionized water, the filter cake was slurried in 50mL ethyl acetate, filtered and washed again with 50mL water, dried under vacuum at 40 ℃ for 20h to give (7.7 g, yield 75.8%) white solid, structure and purity confirmed by NMR analysis and HPLC.

(2) Preparation of gelatin solution:

adding 4g of gelatin for capsules into a beaker filled with 15g of water for injection, placing the beaker in a constant-temperature water bath kettle, setting the constant-temperature water bath kettle at 70 ℃, stirring the mixture by using a glass rod until the mixture is completely dissolved, then carrying out ultrasonic treatment to enable bubbles to be gathered on the liquid surface, removing the bubbles, and preserving the bubbles in a water bath at 70 ℃ for later use.

(3) 2,3,5-triiodobenzaldehyde and gelatin crosslinking preparation self-developing absorbable embolism microsphere:

adding 36g of liquid paraffin and 0.7g of span 80 into a reaction kettle at the temperature, slowly adding a gelatin solution, stirring and dispersing for 5min, setting the stirring speed to be 130-170 r/m, adding 3.16g of 2,3, 5-triiodobenzaldehyde, stirring to completely dissolve the gelatin solution, dropwise adding 1.36ml of formaldehyde solution to perform crosslinking reaction, continuously stirring for 60min after dropwise adding is finished, switching on a low-temperature cooling liquid circulating water pump to form cold water circulation, cooling a reaction system, keeping the temperature of the reaction system below 10 ℃, keeping the temperature, stirring and reacting for 3.5-4.5 h (including cooling time), filtering to obtain microspheres, washing the microspheres by using a 2% Tween 80 aqueous solution, soaking and washing the microspheres until the washing water has no oil stain, soaking and washing the microspheres by using injection water for at least 1h (the adding amount of the injection water is about 2 times of the weight of the microspheres every time), changing the water every 10min, and then screening to obtain the self-developing absorbable embolism microsphere finished product.

Example 1 the reaction procedure for preparing self-developing absorbable embolization microspheres was as follows:

example 2

2,3,5-triiodobenzoic acid and gelatin crosslinked to prepare self-developing absorbable embolization microspheres:

adding 36g of liquid paraffin and 0.7g of span 80 into a reaction kettle at normal temperature, slowly adding the gelatin solution, stirring and dispersing for 5min, setting the stirring speed to be 130-170 r/m, adding 3.23g of 2,3, 5-triiodobenzoic acid (purchased from Aladdin Co., ltd.), stirring to completely dissolve the gelatin solution, dropwise adding 1.36ml of formaldehyde solution for crosslinking reaction, and continuously stirring for 60min after the addition is finished. And (3) switching on a low-temperature cooling liquid circulating water pump to form cold water circulation, cooling the reaction system, keeping the temperature of the reaction system below 10 ℃, and carrying out heat preservation stirring reaction for 3.5-4.5 h (including cooling time). Filtering to obtain microspheres, washing the microspheres with a 2% Tween 80 aqueous solution, and soaking and washing the microspheres until the washing water has no oil stain; the microspheres were washed with additional water for injection for at least 1 hour (each time water for injection was added in an amount of about 2 times the weight of the microspheres) with water change every 10 minutes. And then screening to obtain a finished microsphere product.

Example 2 the reaction procedure for preparing self-developing absorbable embolization microspheres was as follows:

characterization of the self-developing absorbable embolization microspheres prepared in example 1:

the scanning electron micrographs of the self-developing absorbable embolization microspheres prepared in example 1 are shown in FIGS. 1 and 2.

(1) The dry weight of the absorbable embolic microspheres is measured by removing the filling saline and carrying away the remaining saline with the tissue. The autodevelopable absorbable embolization microspheres were dried under vacuum overnight at 50 ℃ to remove water, thereby obtaining the dry microsphere weight and the solids content (w/w%) of the polymer.

The iodine content (w/w%) in the dry microspheres was measured by elemental analysis according to the Schoniger flask method.

The iodine content in the wet microspheres was calculated as: microsphere solids content (%). The iodine content (% in dry microspheres)

An alternative method of expressing iodine content is to express the iodine content as mg I/mL wet microspheres (wet packed microsphere volume) using the protocol of examples 1 and 2 to achieve an average iodine content of about 150mg I/mL wet microspheres.

(2) And (3) measuring the CT developing capability of the self-developing absorbable embolism microsphere:

the developability of the self-developable absorbable embolization microsphere samples prepared in example 1 was evaluated using an electron computer tomography technique.

Taking 10 parts of samples, adding 25mg of each part of sample into an EP tube containing 1ml of deionized water, scanning by a CT machine at a voltage of 120kV, a current of 350mAs, a layer thickness of 1mm and a screw pitch of 0.6, and measuring a CT value at the same position where microspheres at the bottom of the test tube are uniformly distributed. The average of ten line scans of the self-visualizing absorbable embolization microspheres prepared in example 1 was about 4638HU.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing self-developing absorbable embolism microsphere is characterized in that iodobenzene compound is introduced into gelatin and then cross-linking polymerization is carried out.

2. The method for preparing self-developing absorbable embolization microsphere according to claim 1, wherein the gelatin is at least one of vegetable gum, microbial gum, seaweed gum and starch.

3. The method for preparing the self-developing absorbable embolization microsphere according to claim 2, wherein the gelatin has a gel strength of not less than 250g Bloom.

4. The method for preparing self-developing absorbable embolism microsphere as claimed in claim 1, wherein the iodobenzene compound is 3,5-diiodobenzaldehyde dimethyl acetal, 3,5-diiodobenzoic acid, 2,3,5-triiodophenylacetaldehyde dimethyl acetal, 2,3,5-triiodophenylacetic acid, 2,3,5-triiodobenzoic acid, 2,3,5-triiodobenzaldehyde, 3,4,5-triiodobenzaldehyde.

5. The preparation method of the self-developing absorbable embolization microsphere of claim 1, wherein the self-developing absorbable embolization microsphere has a structure of general formula 1 or 2 on the main chain, X in the general formula 1 or 2 is a mono-or poly-iodo-substituted phenylalkyl functional group, the number of carbon atoms of alkyl in the phenylalkyl functional group is 0-20, the number of iodine atoms in a single benzene ring is 1-5,

6. the method for preparing self-developing absorbable embolism microsphere according to claim 1, which comprises the following steps:

(1) Preparation of gelatin solution:

adding 1-15 parts of liquid paraffin and 0.006-1 part of span 80 into a reaction kettle at normal temperature, slowly adding the gelatin solution obtained in the step (2), stirring and dispersing for 5-60min, adding 0.03-3 parts of iodobenzene compound, stirring to completely dissolve the iodobenzene compound, dropwise adding 1-20 parts of formaldehyde solution for crosslinking reaction, keeping the reaction temperature less than or equal to 10 ℃, filtering to obtain microspheres after the reaction is finished, and washing the microspheres with 0.5-5% of tween 80 aqueous solution until the washing water has no oil stain; then soaking and washing the microsphere for 1-3h by using water for injection, and then screening to obtain the finished product of the self-developing absorbable embolism microsphere.

7. The method for preparing self-developing absorbable embolism microsphere according to claim 6, wherein the particle size of the self-developing absorbable embolism microsphere in the step (2) is 50-1500 μm.

8. The method for preparing self-developing absorbable embolization microspheres according to claim 6, wherein the reaction time in step (2) is 3.5-4.5 h.

9. The method for preparing self-developing absorbable embolism microsphere according to claim 6, wherein the stirring speed in step (2) is 130-170 r/min.

10. The self-developing absorbable embolization microsphere prepared by the preparation method according to any one of claims 1 to 9.