Developable hyaluronic acid microspherical embolic agent, preparation method and use thereof

Abstract

A developable hyaluronic acid microspherical embolic agent includes a hyaluronic acid and an X-ray opaque contrast material; wherein the hyaluronic acid is a modified hyaluronic acid, wherein a modifying method for preparing the modified hyaluronic acid is cross-linking, grafting, esterification, or recombination, and is preferably cross-linking.

Claims

1. A method of preparing a developable hyaluronic acid microspherical embolic agent, wherein the method comprises the following steps: 1) preparing a hyaluronic acid solution having a concentration ranging from 0.1 g/mL to 0.3 g/mL, wherein the solution contains an iodine compound; 2) adding the hyaluronic acid solution resulted from step 1) to a liquid paraffin containing an emulsifying agent, and emulsifying with a shearing machine at a speed ranging from 500 rpm to 2000 rmp, to produce a water-in-oil emulsion comprising hyaluronic acid microspheres embedded with the iodine compound; 3) adding 0.2-2 vol% of a cross-linking agent to the emulsion resulted from step 2), stirring the emulsion at room temperature for 4-6 hours for cross-linking reaction, keeping the emulsion still after the reaction is finished to form an oil phase layer and an aqueous phase layer, removing the oil phase layer, then washing the aqueous phase layer orderly with an acid solvent and a water soluble organic solvent, and vacuum drying the aqueous phase layer to obtain hyaluronic acid microspheres containing the iodine compound, wherein the microspheres are the hyaluronic acid microspherical embolic agents developable under an X-ray machine.

2. The method of preparing the developable hyaluronic acid microspherical embolic agent according to claim 1, wherein: the hyaluronic acid is sodium hyaluronate produced by bacterial fermentation and has a molecular weight ranging from 0.3 million to 3 million dalton; the cross-linking agent is one selected from the group consisting of epoxides, divinyl sulphone, 1,4-butanediol diglycidyl ether, and diglycidyl ether; and the emulsifying agent is sorbitan oleate, or polyoxyethylene (20) sorbitan monooleate, or one or more from the combinations of sorbitan oleate and polyoxyethylene (20) sorbitan monooleate.

3. The method of claim 1, wherein the washing with the acid solvent in step 3) lowers the pH value of the aqueous phase layer to about 4.0 to 5.0.

4. The method of claim 1, wherein the aqueous phase layer after being washed with the acid solvent, is washed in turn with ethyl acetate and absolute ethanol.

5. The method of claim 1, wherein the iodine compound is one selected from the group consisting of sodium iodide, N,N.sup.40-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-[(2-methoxyacetyl)amino ]-N-methylbenzene-1,3 -carboxamide (iopromide), N,N-bis(2,3 -dihydroxypropyl)-5 -[N-(2,3 -dihydroxypropyl) acetamino]-2,4,6-triiodoisophthalamide (iohexol), (S)-N,N-bis[2-hydroxy-1-(hydroxymethy)ethyl]-5-[N-[(2-hydroxy-1-oxopropyl)amino ]-2,4,6-triiodo-1,3-benzenedicarboxamide(iopamidol), and 5,5-((2-hydroxy-1,3-propane)bis (acetylimino))bis(N,N-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide (iodixanol).

6. The method of claim 4, wherein the acid solvent is acetic acid.

7. The method of claim 4, wherein the water soluble organic solvent is one selected from the group consisting of ethyl acetate, absolute ethanol, and acetone.

8. The method of claim 4, wherein 1) the hyaluronic acid is sodium hyaluronate produced by bacterial fermentation and has a molecular weight ranging from 0.3 million to 3 million Dalton; 2) the iodine compound is iohexol; 3) the emulsifying agent is sorbitan oleate; 4) the cross-linking agent is 1,4-butanediol diglycidyl ether; 5) the acid solvent is acetic acid; and 6) the water soluble organic solvent is ethyl acetate and absolute ethanol.

9. A method of preparing a developable hyaluronic acid microspherical embolic agent, wherein the method comprises the following steps: 1) preparing a hyaluronic acid in sodium hydroxide solution having a concentration of about 0.1 g/mL, wherein the solution contains 0.07 g/mL of iohexol; 2) adding the hyaluronic acid solution resulted from step 1) to a liquid paraffin containing about 200-205g of liquid paraffin and about 3.0 to 4.0g sorbitan oleate, and emulsifying with a shearing machine at a speed ranging from 500 rpm to 2000 rmp, to produce a water-in-oil emulsion comprising hyaluronic acid microspheres embedded with iohexol; 3) adding about 1% of 1,4-butanediol diglycidyl ether to the emulsion resulted from step 2), stirring the emulsion at room temperature for 4-6 hours for cross-linking reaction, keeping the emulsion still after the reaction is finished to form an oil phase layer and an aqueous phase layer, removing the oil phase layer, then washing the aqueous phase layer orderly with an acetic acid and in turn with ethyl acetate and absolute ethanol, and vacuum drying the aqueous phase layer to obtain hyaluronic acid microspheres containing iohexol, wherein the microspheres are hyaluronic acid microspherical embolic agents developable under an X-ray machine.

10. The method of claim 9, wherein the hyaluronic acid is sodium hyaluronate produced by bacterial fermentation and has a molecular weight ranging from 0.3 million to 3 million Dalton.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A and FIG. 1B show development effects of the product prepared according to example 1 (FIG. 1A) and a positive control (FIG. 1B) under X-ray radiation.

(2) FIG. 2 is a view of the product prepared according to example 1 under a scanning electron microscope, in which microspheres have uniform shapes and smooth surfaces; and

(3) FIG. 3A and FIG. 3B show a comparison of the product prepared according to example 1 before (FIG. 3A) and after (FIG. 3B) the product is compressed to deform.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) Examples will be provided below to explain the present disclosure in more detail, but the present disclosure is not limited thereto.

Example 1: Preparation of Hyaluronic Acid Microspherical Embolic Agent Containing Iohexol

(5) A hyaluronic acid solution having a concentration of 0.1 g/mL was prepared by using 8.04 g of solid power of sodium hyaluronate and 0.5% of sodium hydroxide solution. The hyaluronic acid solution contained 0.07 g/mL of iohexol. The hyaluronic acid solution was added to an oil phase containing 200.71 g of liquid paraffin and 3.41 g of Span 80. The resultant mixed phase was emulsified and dispersed for 10 minutes by using a shearing machine at 1000 rpm, to obtain a water-in-oil hyaluronic acid microspheres emulsion. 1% of 1,4-butanediol diglycidyl ether was added to the emulsion and the resultant mixture was stirred for 4 hours at room temperature for cross-linking reaction. After the stirring was finished, the mixture was kept still overnight to leave the microspheres to settle. The oil phase at the upper layer was poured away, and then acetic acid was added to adjust the pH value of the remaining mixture to be in a range of from 4.0 to 5.0. After that, the remaining mixture was washed in turn with ethyl acetate and absolute ethanol to remove the residual organic phase, and then dried in vacuum to obtain sodium hyaluronate microspheres containing the iodine compound. At last, the microspheres were sieved and sterilized to obtain a developable hyaluronic acid microspherical embolic agent.

Example 2: Preparation of Hyaluronic Acid Microspherical Embolic Agent Containing Iohexol

(6) A hyaluronic acid solution having a concentration of 0.1 g/mL was prepared by using 8.02 g of solid power of sodium hyaluronate and 0.5% of sodium hydroxide solution. The hyaluronic acid solution contained 0.07 g/mL of iohexol. The hyaluronic acid solution was added to an oil phase containing 201.01 g of liquid paraffin and 3.45 g of Span 80. The resultant mixed phase was emulsified and dispersed for 10 minutes by using a shearing machine at 1000 rpm, to obtain a water-in-oil hyaluronic acid microspheres emulsion. 1% of 1,4-butanediol diglycidyl ether was then added to the emulsion and the resultant mixture was stirred for 4 hours at room temperature for cross-linking reaction. After the stirring was finished, the mixture was kept still overnight to leave the microspheres to settle. The oil phase at the upper layer was poured away, and the remaining mixture was washed in turn with ethyl acetate and absolute ethanol to remove the residual organic phase, and then vacuum dried to obtain sodium hyaluronate microspheres containing the iodine compound. At last, the microspheres were sieved and sterilized to obtain a developable hyaluronic acid microspherical embolic agent.

(7) Measurement of Content of Developing Agent in Hyaluronic Acid Microspherical Embolic Agent

(8) Dried microspheres prepared according to the above examples were respectively taken and dissolved in normal saline. After the microspheres were well swollen, the content of the developing agent in the microspheres was measured. The content of iohexol was measured by ultraviolet spectrophotometry.

(9) The content of iohexol in the microspheres prepared in example 1 was 26%.

(10) The content of iohexol in the microspheres prepared in example 2 was 18%.

(11) Test of Development Effect of Hyaluronic Acid Microspherical Embolic Agent

(12) The hyaluronic acid microspherical embolic agent prepared in example 1 was taken to mix with 1 mL of normal saline. 1 mL of iohexol was measured out separately to be used as a positive control. Development test was carried out under an X-ray machine.

Comparative Example 1: Preparation of Cross-Linked Hyaluronic Acid Microspherical Embolic Agent Free of Developing Material

(13) A hyaluronic acid solution having a concentration of 0.1 g/mL was prepared by using 8.14 g of solid power of sodium hyaluronate and 0.5% of sodium hydroxide solution. The solution was added to an oil phase containing 200.98 g of liquid paraffin and 3.42 g of Span 80. The resultant mixed phase was emulsified and dispersed for 10 minutes by using a shearing machine at 1000 rpm, to obtain a water-in-oil hyaluronic acid microspheres emulsion. 1% of 1,4-butanediol diglycidyl ether was then added to the emulsion and the resultant mixture was stirred for 4 hours at room temperature for cross-linking reaction. After the stirring was finished, the mixture was kept still overnight to leave the microspheres to settle. The oil phase at the upper layer was poured away, and then the remaining mixture was washed in turn with ethyl acetate and absolute ethanol to remove the residual organic phase. The resulted mixture was then vacuum dried to obtain sodium hyaluronate microspheres containing the iodine compound. At last, the microspheres were sieved and sterilized to obtain a developable hyaluronic acid microspherical embolic agent.

(14) FIG. 1A shows the development effects of the product prepared according to example 1 under X-ray radiation, and FIG. 1B shows the development effects of a positive control (product in comparative example 1).

Test Example 1: Property Test of Hyaluronic Acid Microspherical Embolic Agent Embedded with a Developing Agent

(15) Samples prepared in the examples and the comparative example were subjected to tests on compression deformation property, swelling degree, and content of sodium hyaluronate, to assess whether or not the samples embedded with the developing agent still retained their original properties. The compression deformation property represents the elasticity of the microspheres. Good compression deformation property allows the embolic microspheres to deform effectively in order to pass through an injector and a microcatheter and then return to their original states. Swelling degree represents the cross-linking degree of the microspheres, which is manifested in the degradability of the microspheres in vivo. The content of sodium hyaluronate represents the effective component of the embolic microspheres, and it was determined by carbazole method.

(16) As shown in FIG. 2, the product prepared in example 1 includes microspheres having uniform shapes and smooth surfaces. FIGS. 3A and 3B shows a comparison of the product prepared according to example 1 before (FIG. 3A) and after (FIG. 3B) the product is compressed to deform.

(17) TABLE-US-00001 Comparative Property Test Example 1 Example 2 Example 1 Compression 62.4% 44.2% 59.7% Deformation Swelling Degree 96 68 90 Content of Sodium 12.5 mg/mL 16.2 mg/mL 13.4 mg/mL Hyaluronate

(18) A comparison between the determined contents of the developing agents of example 1 and example 2 shows that, with other preparation procedures being the same, according to the method of the present disclosure, the use of an acid solvent to wash the microspheres obtained after the cross-linking reaction to decrease the pH value of the environment can lead to microspheres with a higher content of the developing agent and a better embedding efficiency, as compared to directly using a water soluble organic solvent to wash the microspheres obtained after the cross-linking reaction.

(19) A comparison between example 1 and comparative example 1 shows that, with the feed ratio and process parameters being the same, a procedure of embedding a developing agent material was added in example 1, but the results of the property tests of the microsherical embolic agents prepared in example 1 and comparative example 1 are basically the same.

(20) The above examples are only specific examples of the present disclosure, and features of the present disclosure are not limited thereto. Any variations or modifications in the field of the present disclosure made by any person skilled in the art shall fall within the protection scope of the present disclosure.