CN115487342B

CN115487342B - Self-developing absorbable embolism microsphere and preparation method thereof

Info

Publication number: CN115487342B
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: 2023-11-24
Anticipated expiration: 2042-08-17
Also published as: CN115487342A

Abstract

The invention provides an self-developing absorbable embolism microsphere and a preparation method thereof, belonging to the technical field of absorbable embolism microsphere. The invention introduces a developable iodinated benzene compound into gelatin sponge embolism microsphere by chemical bonding, and provides an X-ray-opaque self-developing absorbable embolism microsphere. The microsphere provided by the invention has the advantages of facilitating the clinical direct observation of the embolic agent position, judging the embolic end point, reducing the risk of ectopic embolism, 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 microsphere, in particular to an self-developing absorbable embolism microsphere and a preparation method thereof.

Background

Liver cancer is one of the most common malignant tumors, and is not obvious in early stage due to high concealment, so that most patients are middle and late stages in diagnosis, and are not suitable for surgical excision treatment. For such patients, transcatheter Arterial Chemoembolization (TACE) is the method of choice for non-operative treatment by injecting chemotherapeutics and embolizing agents into tumor vessels via an interventional super-selective catheterization technique, blocking tumor blood supply, and causing tumor necrosis under the action of local high concentrations of chemotherapeutics. A large amount of clinical data and researches show that TACE can effectively prolong the life cycle of middle and late liver cancer patients and improve the life quality of the patients.

The particulate solid embolic agents currently in use in TACE can be classified into absorbable embolic agents, such as gelatin sponges, and non-absorbable embolic agents, such as polyvinyl alcohol, according to the absorbability of the material. For recurrent tumors and TACE therapies requiring multiple treatments, absorbable embolization agents, after biodegradation, the blood vessels can be recaptured, leaving a pathway for retreatment, with the advantage of not being possessed by non-absorbable materials in specific clinical settings. 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 microsphere is better. Meanwhile, the microspheres are not easy to aggregate, have good elasticity and have better passage property. These advantages have led to the wide acceptance and use of microspherical embolic agents in the clinic.

At present, the conventional solid embolic agent used clinically is not developed under X-rays, and the embolic agent and the contrast agent are mixed and then injected under X-ray perspective to indirectly judge the positions of the microspheres and the embolic condition. When the flow rate of the mixed solution of contrast agent and microspheres stagnates or returns, the operator judges that the embolism reaches the end point. However, the method has the defects that the final actual position of the embolic agent cannot be accurately judged, and an operator cannot obtain real-time feedback in the operation, so that inconvenience is brought to the operation of the operator, the accuracy of the embolic end point judgment is affected, and the risk of ectopic embolism of a non-target part is increased. Therefore, in order to enable operators to directly observe the injection of the solid embolic agent under X-ray perspective, the operation convenience is improved, the paired control of the embolism is more confident, the risk of abnormal embolism in the operation is effectively avoided, simultaneously, a vascular passageway is provided for the subsequent treatment, and the novel self-developing absorbable embolic microsphere is researched to have profound significance.

The invention patent with the application number of CN200710026274.8 discloses a biodegradable developing microsphere vascular embolism material, which consists of a biodegradable material and an X-ray opaque 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 the application number of CN201110068821.5 discloses a developable gelatin sponge embolic agent for tumor embolism, which consists of gelatin, an expanding agent and a developing agent in any proportion.

However, the methods for preparing self-developing absorbable embolism microsphere in the patent belong to physical embedding methods, and the developing substance does not form a combined bond with the microsphere, so that the microsphere has a certain developing effect, but the developing substance embedded in the microsphere can continuously ooze out of the microsphere, and the developing effect is difficult to maintain for a long time.

Disclosure of Invention

Because of the lack of self-developing absorbable microspheres in the market, operators cannot obtain real-time feedback of the positions of the embolic microspheres in interventional embolization, and are difficult to judge the embolic end point and monitor ectopic embolization, the use of interventional embolization is limited to a certain extent, and meanwhile, the self-developing absorbable microspheres prepared by a physical embedding method have the defects that developing substances exude, and the developing effect is difficult to maintain for a long time. In view of this, the present invention provides an self-developing absorbable embolic microsphere and a method of preparing the same, which introduces an X-ray opaque developing substance into a gelatin sponge embolic microsphere by chemical modification, and provides an X-ray opaque self-developing absorbable embolic microsphere.

The invention provides a preparation method of self-developing absorbable embolism microsphere, which is prepared by introducing an 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 konjak gum, acacia 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.

Further preferably, the gelatin is gelatin for capsules, wherein the gelatin for capsules is fresh, healthy, strictly quarantined and not subjected to any chemical treatment, and is made of connective tissues such as skin, bones, tendons, ligaments and the like of pigs, cattle and the like, and is prepared by complex physicochemical treatment, and the gelatin for capsules has no fat, high protein and easy absorption by human body, and has the gel strength of more than or equal to 250g Bloom.

Preferably, the iodobenzene compound is specifically 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 and 2,3, 5-triiodobenzaldehyde, 3,4, 5-triiodobenzaldehyde.

Preferably, the self-developing absorbable embolism microsphere has a structure shown in a general formula 1 or 2 on the main chain, 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 fully dissolved, carrying out ultrasonic treatment, removing bubbles, and preserving the gelatin in the water bath at constant temperature for later use;

(2) Self-developing absorbable embolic microspheres prepared by crosslinking an 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 iodo benzene compound, stirring to completely dissolve the iodo benzene compound, then dripping 1-20 parts of formaldehyde solution for crosslinking reaction, keeping the reaction temperature to be less than or equal to 10 ℃, filtering to obtain microspheres after the reaction is completed, and washing the microspheres with 2% Tween 80 aqueous solution until washing water has no oil marks; and washing the microspheres with water for injection for at least 1h, and then sieving to obtain the finished product of the self-developing absorbable embolism microsphere.

The invention adopts gelatin with good biocompatibility and low immunogenicity, introduces developable iodo-benzene compound in the chemical crosslinking process, and forms microspheres by crosslinking and curing, the microspheres can be biologically degraded and absorbed, and can be developed under medical imaging equipment without X-rays.

Preferably, the iodo benzene compound can be commercially purchased or prepared as follows, and the specific preparation steps of the iodo benzene compound are as follows:

reacting 0.2-2 parts of hydrochloric acid solution of anthranilic acid with 0.23-3 parts of iodine chloride hydrochloric acid solution at a constant temperature of 30-80 ℃ for 1-4 hours to obtain a 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 iodo benzene carboxylic acid;

mixing 0.5-2 parts of iodic benzene carboxylic acid containing amino and 1-6 parts of sulfuric acid, slowly adding 0.2-3 parts of sodium nitrite aqueous solution, stirring in an ice bath, reacting for 1-3 hours to obtain a mixture A, blowing air into the mixture A, performing suction filtration to obtain a yellowish green transparent liquid, adding potassium iodide aqueous solution into the liquid, stirring, standing, heating to remove nitrogen, cooling to room temperature, removing iodine element, performing suction filtration, drying to obtain an orange yellow solid, and recrystallizing a crude product with absolute ethyl alcohol to obtain iodic benzene 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 under the condition of 0 ℃ and nitrogen flow, stirring the mixed solution for 1-3 hours at the temperature of 0-5 ℃ in sequence, continuing stirring for 1-2 hours at room temperature to precipitate white solid, 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 in sequence 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% of propyl phosphoric anhydride is slowly dripped at the temperature of 22-25 ℃ and stirred for 2-5 hours to obtain yellow solution, deionized water is added to generate white precipitate, the white precipitate is filtered, washed with the anhydrous DMSO and 50mL of deionized water, pulped, filtered and washed, and vacuum-dried for 15-30 hours at the temperature of 30-70 ℃ to obtain the iodobenzene compound, and the purity is confirmed by NMR analysis and HPLC.

Preferably, the particle size of the self-developing resorbable embolic microspheres in step (2) is in the range of 50-1500 μm.

Preferably, the self-developing resorbable embolic microspheres in step (2) have a developability of 500-10000 HU.

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

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

It is a second object of the present invention to provide self-developing absorbable embolic microspheres prepared by the preparation method.

The self-developing absorbable embolism microsphere prepared by the invention has the advantages of developing property, larger elastic expansion rate and recovery property, uniform and controllable granularity and good dispersibility. The degradation time of the self-developing absorbable embolism microsphere is 14-90 days according to the different crosslinking and curing degrees. The CT value of the microsphere can reach 500-10000HU, and the good developing property can lead the gelatin microsphere to be distinguished from blood (30-451 HU), liver (40-60 HU), brain (20-45 HU) and soft tissue (100-300 HU), thereby providing 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 embolism microsphere prepared by the invention stably combines the developing substance into the microsphere in a chemical bonding mode, so that the embolism microsphere has absorbability and developability, the defect that the embolism agent and the contrast agent are required to be mixed in the traditional interventional embolism operation so as to indirectly position the embolism agent is overcome, the position of the embolism agent is conveniently and directly observed clinically, the end point of the embolism is judged, and the risk of ectopic embolism is further reduced. Meanwhile, the self-developing absorbable embolism microsphere produced by the preparation process has good elasticity and elasticity, has good vascular compliance, can be tightly embedded into the vascular wall, and provides a passage for retreatment after biodegradation and absorption.

Drawings

FIG. 1 is a scanning electron microscope spectrum of self-developing absorbable embolism microsphere prepared in example 1 of the present invention;

FIG. 2 is a scanning electron microscope spectrum of a single microsphere of the self-developing absorbable embolism microsphere prepared in 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 will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the invention, are within the scope of the invention.

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

Example 1:

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

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

1) Preparation of 2-amino-diiodobenzoic acid

Adding 33g of iodine chloride into 10.3g of hydrochloric acid solution of anthranilic acid under stirring, reacting for 2.5 hours at 60 ℃ to obtain a precipitate, adding sodium bisulphite solution until the starch potassium iodide test paper does not change color, carrying out suction filtration and washing, transferring the precipitate into a beaker, adding glacial acetic acid until the solution is weak acid, generating a large amount of precipitate, and then sequentially cooling, carrying out suction filtration and drying a 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 into the beaker, stirring, blowing air into the mixture, then rapidly carrying out suction filtration to obtain a yellow green transparent liquid, adding a proper amount of potassium iodide aqueous solution into filtrate, standing and heating after stirring to remove nitrogen, cooling to room temperature, removing elemental iodine, carrying out suction filtration, drying to obtain 36g of orange yellow solid, recrystallizing the crude product with absolute ethyl alcohol to obtain 37g of light yellow needle-like crystals, wherein the yield is 90%, and the melting point is 223-224 ℃;

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

26mL of borane-tetrahydrofuran solution was added dropwise to 61mL of anhydrous tetrahydrofuran dissolved with 30g of 2,3, 5-triiodobenzoic acid at 0℃under dry nitrogen flow, the resulting mixture was stirred for 1h 15min at 0℃and then for 1h at room temperature, at the end of the reaction a white solid precipitated, 50mL of a cold mixed solution of tetrahydrofuran/water (13/2) was slowly added, then 50mL of a saturated sodium hydrogencarbonate cold water solution was added, white precipitation occurred after stirring for 1h, the solid was recovered by filtration and washed 3 times with water and cold anhydrous ethanol;

4) Preparation of 2,3, 5-triiodobenzaldehyde

In a 50mL three-necked round bottom flask equipped with a thermometer, nitrogen bubbler and airtight seal, 20.6g of 2,3, 5-triiodobenzyl alcohol was added and dissolved in 100mL anhydrous DMSO under nitrogen protection and stirring, then 25mL ethyl acetate solution containing 50% of propylphosphoric anhydride was added dropwise over 5min at 22-25℃and stirring for 4h reaction was completed, the yellow solution was poured into 100mL deionized water under stirring to give 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, and dried in vacuo at 40℃for 20h to give (7.7 g, yield 75.8%) as a white solid, whose structure and purity were 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 into a constant-temperature water bath, setting the temperature of the constant-temperature water bath to 70 ℃, stirring the mixture with a glass rod until the mixture is fully dissolved, then performing ultrasonic treatment to ensure that bubbles are gathered on the liquid surface, fishing out the bubbles, and preserving the bubbles in the water bath at 70 ℃ for later use.

(3) Self-developing absorbable embolic microspheres prepared by crosslinking 2,3, 5-triiodobenzaldehyde with gelatin:

adding 36g of liquid paraffin and 0.7g of span 80 into a reaction kettle at a temperature, slowly adding gelatin solution, stirring and dispersing for 5min, setting stirring speed to be 130-170 r/m, adding 3.16g of 2,3, 5-triiodobenzaldehyde, stirring to completely dissolve the gelatin solution, dripping 1.36ml of formaldehyde solution for crosslinking reaction, continuing stirring for 60min after dripping is finished, connecting 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 and stirring for 3.5-4.5 h (including cooling time), filtering to obtain microspheres, washing the microspheres with 2% Tween 80 aqueous solution, soaking the microspheres until the washing water is free of oil marks, soaking the microspheres with water for injection for at least 1h (the adding amount of water for injection is about 2 times the weight of the microspheres), changing the water every 10min, and sieving to obtain the finished product of the self-developing absorbable embolism microspheres.

Example 1 the reaction procedure for preparing self-developing resorbable embolic microspheres is as follows:

example 2

Self-developing absorbable embolic microspheres prepared by crosslinking 2,3, 5-triiodobenzoic acid with gelatin:

36g of liquid paraffin and 0.7g of span 80 are added into a reaction kettle at normal temperature, a gelatin solution is slowly added, stirring and dispersing are carried out for 5min, the stirring speed is set to be 130-170 r/m, 3.23g of 2,3, 5-triiodobenzoic acid (purchased from Aladin Co., ltd) is added, after stirring to completely dissolve, 1.36ml of formaldehyde solution is dripped for crosslinking reaction, and stirring is continued for 60min after the adding is completed. And (3) a low-temperature cooling liquid circulating water pump is connected to form cold water circulation to cool the reaction system, the reaction system is kept below 10 ℃, and the reaction is carried out for 3.5-4.5 hours (including cooling time) by heat preservation and stirring. Filtering to obtain microspheres, washing the microspheres with 2% Tween 80 water solution until the washing water has no oil trace; the microspheres were then rinsed with water for injection for at least 1 hour (with each injection being added at about 2 times the weight of the microspheres) with water being changed every about 10 minutes. And then sieving to obtain the microsphere finished product.

Example 2 the reaction procedure for preparing self-developing resorbable embolic microspheres is as follows:

characterization of self-developable resorbable embolic microspheres prepared in example 1:

the scanning electron microscope spectra of the self-developing absorbable embolism microsphere prepared in example 1 are shown in fig. 1 and fig. 2.

(1) The self-imaging can absorb the dry weight of the embolic microspheres, as measured by removing the filled saline and taking the remaining saline away with the tissue. The self-developable resorbable embolic microspheres were dried in vacuo at 50 ℃ overnight to remove water, thereby obtaining dry microsphere weight and solids content of polymer (w/w%).

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 (%) iodine content (%)

An alternative method of expressing iodine content is mg I/mL wet microsphere (wet filled microsphere volume), using the protocols of examples 1 and 2, an average iodine content of about 150mg I/mL wet microsphere is achieved.

(2) CT developability determination of self-developable absorbable embolic microspheres:

an electron computer tomography technique was used to evaluate the developability of the self-developable absorbable embolic microsphere samples prepared in example 1.

10 samples are taken, 25mg of each sample is added into an EP tube containing 1ml of deionized water, the sample is scanned under a CT machine at the voltage of 120kV, the current of 350mAs, the thickness of the layer of the sample is 1mm and the screw pitch is 0.6, and the CT value is measured at the same position of uniform microsphere distribution at the bottom of the test tube. The average of ten line scans of the self-developable resorbable embolic microspheres prepared in example 1 was about 4638HU.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The preparation method of the self-developing absorbable embolism microsphere is characterized by comprising the following steps of:

(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 (1), stirring and dispersing for 5-60 min, adding 0.03-3 parts of iodo benzene compound, stirring to completely dissolve the gelatin solution, dripping 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, washing the microspheres with 0.5-5% Tween 80 aqueous solution until no oil marks exist in washing water, washing the microspheres with water for injection for 1-3h, and sieving to obtain a finished product of self-developing absorbable embolism microsphere;

the gelatin is gelatin for capsules, and the gel strength of the gelatin is more than or equal to 250gBloom;

the iodo benzene compound is specifically any one of 2,3, 5-triiodo benzoic acid, 2,3, 5-triiodo benzaldehyde or 3,4, 5-triiodo benzaldehyde;

the self-developing absorbable embolism microsphere has a structure shown in a general formula 1 or 2 on a main chain, wherein X in the general formula 1 or 2 is a multi-iodine substituted phenylalkyl functional group, the number of carbon atoms of alkyl in the phenylalkyl functional group is 1, the number of iodine atoms of a single benzene ring is 3,

；

general formula 1

；

General formula 2.

2. The method of preparing self-developing absorbable embolic microspheres of claim 1, wherein the particle size of the self-developing absorbable embolic microspheres in step (2) is in the range of 50-1500 μm.

3. The method for preparing self-developing absorbable embolism microsphere according to claim 1, wherein the reaction time in the step (2) is 3.5-4.5 h.

4. The method for preparing self-developing absorbable embolism microsphere according to claim 1, wherein the stirring speed in the step (2) is 130-170 r/min.

5. An self-developing absorbable embolism microsphere prepared by the preparation method of any one of claims 1-4.