CN112791238B

CN112791238B - Compound gel for treating bladder ureteral reflux and preparation method thereof

Info

Publication number: CN112791238B
Application number: CN202011527026.3A
Authority: CN
Inventors: 朱剑锋; 金远方
Original assignee: Zhejiang Jingjia Medical Technology Co ltd
Current assignee: Zhejiang Jingjia Medical Technology Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-07-01
Anticipated expiration: 2040-12-22
Also published as: CN112791238A

Abstract

The invention discloses a compound gel for treating vesicoureteral reflux and a preparation method thereof. The preparation method of the compound gel is realized by taking the cross-linked dextran and the hyaluronic acid as raw materials and improving the adsorptivity of the cross-linked dextran and granulating a reaction intermediate.

Description

Compound gel for treating vesicoureteral reflux and preparation method thereof

Technical Field

The invention belongs to the technical field of gel composite materials, and particularly relates to a composite gel for treating bladder ureteral reflux.

Background

Vesicoureteral reflux (VUR) is the reflux of urine back into the ureter and kidney due to congenital insufficiency of the vesicoureteral junction, primary vesicoureteral reflux is common in children. VUR has been reported to occur at a rate of about 1% to 2% in children, whereas up to 20% to 40% of primary VUR in children with urinary tract infections often causes recurrent urinary system infections due to the presence of regurgitation, leading to renal parenchyma damage, delayed development of the kidney, scar kidney formation, progression to reflux nephropathy and ultimately to end stage renal disease.

Antibiotic prophylaxis, injection and surgical treatment are currently the commonly used treatments for vesicoureteral reflux. No endoscopic injection therapy VUR is currently developed in China. The commonly used injection material for the orifice side injection of the ureter under the foreign endoscope is

This product is currently the only injectable material approved by the U.S. Food and Drug Administration (FDA) for treatment VUR.

Is a compound gel consisting of cross-linked sodium hyaluronate and cross-linked dextran (also called cross-linked dextran), and can enlarge local group after injectionAnd (3) weaving, so that the lower ureter keeps a combined state when the bladder is full and contracted, the cross-linked sodium hyaluronate is used as a carrier to convey the cross-linked dextran to an injection site and is gradually absorbed by tissues, and the cross-linked dextran is gradually surrounded by self connective tissues by utilizing the self adsorption performance to form the final filling effect. Since the product is approved to be marketed to the market in 2001 till now, more and more clinical evidence shows that the product is safe and effective, has no side effect and low complication, is an ideal novel injection material, and is injected under an endoscope

The treatment method is the first choice for treating VUR children patients.

In order to inject the compound gel between the mucosa layer and the muscle layer at the lower end of the vesicouretera, the compound gel is usually matched with a cystoscope injection needle for use, the specification of the commonly used injection needle is 23G multiplied by 250mm or 23G multiplied by 350mm, the compound gel has certain viscosity, the length of the injection needle is long, the friction force when the gel passes through is large, and the extrusion force in the injection process is also large, and is usually more than 35N. The pushing force is a very important performance index because the requirement for the injection dosage in the injection process is very accurate, the injection dosage can not be controlled due to the large or uneven pushing force, and the ureteral orifice can be blocked due to excessive injection. The pushing force is generally related to the physical properties (e.g., viscoelasticity, particle size distribution, etc.) of the gel composite and the cystoscope needle used therewith.

Patent CN 108721206 (application No. 201710244912.7) discloses a composition for treating vesicoureteral reflux, stress urinary incontinence and fecal incontinence and a method for preparing the same. The gel comprises 4-6% of cross-linked dextran microspheres, 1-2% of cross-linked sodium hyaluronate and the balance of PBS buffer solution, and the actual form of the composition is that the cross-linked dextran gel microspheres are uniformly dispersed in the cross-linked sodium hyaluronate gel, and the composition is a physical mixing process performed after two polymers are respectively cross-linked. The mixing lacks the adsorption effect of the cross-linked dextran microspheres on the cross-linked hyaluronic acid, the composition has loose form and uneven pushing force in the using process.

Patent CN 105504313 discloses a preparation method and application of hyaluronic acid composite gel. Provides a novel composite gel which is obtained by using multi-parameter combination in multiple time periods to realize the homogenization of a crosslinking system and the preparation of the gel. Specifically, the first section reduces the incidence of crosslinking reaction before the system is not mixed uniformly through low-temperature and high-frequency oscillation, the second section promotes the system to be kept in a homogeneous state during the early crosslinking reaction of the system through high-temperature and high-frequency oscillation, and the third section promotes the formation of gel and reduces the damage of the oscillation to the gel result through high-temperature and low-frequency oscillation. Although the prepared composite gel has good homogeneity, uniform crosslinking and moist heat sterilization resistance, the dextran particles are added into a crosslinking reaction system in an alkaline environment, the whole preparation of the composite gel is a process of physically embedding the dextran particles by hyaluronic acid in a crosslinking process, the dosage of a crosslinking agent is larger, the viscoelastic property of the composite gel is higher, and the extrusion force in the use process is also larger.

Disclosure of Invention

The invention aims to provide a compound gel for treating vesicoureteral reflux and a preparation method thereof. The preparation process of the invention can reduce the usage amount of the cross-linking agent, and the prepared compound gel has lower and stable extrusion force and is suitable for treating the bladder ureteral reflux by endoscopic injection.

In order to realize the purpose, the invention adopts the following technical scheme:

a method for preparing a complex gel for the treatment of vesicoureteral reflux, comprising the steps of:

(1) dissolving hyaluronic acid and cross-linked dextran in neutral aqueous solution containing alcohol, and fully dispersing;

(2) heating the mixture to 35-50 ℃, adding an alkaline solution in which a cross-linking agent is dissolved after the cross-linked dextran is swelled and balanced, and fully dispersing;

(3) crushing the mixture into particles, and standing at a constant temperature of 10-35 ℃ to obtain a cross-linked product;

(4) and (4) washing the cross-linked product obtained in the step (3) for multiple times by using a balanced salt solution to achieve dialysis balance, and preparing the compound gel.

Further, the cross-linked dextran in the step (1) is prepared by cross-linking and polymerizing dextran through ether bonds, and the water absorption capacity is 2.5-20 mL/g.

Furthermore, the cross-linked dextran in the step (1) is microspherical, and the particle size range is 50-500 microns.

Further, the neutral aqueous solution containing alcohol in step (1) is an alcohol solution with four carbon atoms or less, the volume percentage concentration is 3% -10%, and the alcohol solution is preferably an alcohol solution.

Further, the molecular weight of the hyaluronic acid in the step (1) is 1.0 MDa-2.5 MDa.

Further, the crosslinking agent in the step (2) is glycidyl ether. Other difunctional crosslinking agents are also possible.

Further, in the step (3), the mixture is physically crushed into particles having a particle size of less than 5mm to increase the specific surface area of the entire crosslinking reaction system.

The swelling balance is the state that the compound loses fluidity and the crosslinked dextran adsorbs the uncrosslinked hyaluronic acid to the swelling balance.

In order to realize the purpose of the invention, one of the key points is that before the hyaluronic acid is subjected to crosslinking modification, crosslinked dextran is firstly used for adsorbing the hyaluronic acid, and the adsorption is used for increasing the total area of materials in the subsequent whole crosslinking reaction system, improving the crosslinking reaction efficiency and reducing the using amount of a crosslinking agent. The cross-linked dextran is formed by cross-linking dextran through ether bonds, is a high molecular polymer with a porous space network structure, belongs to soft gel, and has strong adsorption capacity due to the fact that a large number of hydroxyl groups are contained on a framework.

Another key point for achieving the object of the present invention is to granulate the mixture of step (2) to increase the specific surface area of the entire crosslinking reaction system and improve the efficiency of the crosslinking reaction. In the conventional preparation method, the whole or massive mixture is usually adopted, so that the contact between the cross-linking agent and the hyaluronic acid is insufficient, the dosage of the cross-linking agent is increased, and a large amount of unreacted cross-linking agent and cross-linking agent with unreacted residual active functional groups are generated, and the residual cross-linking agent causes certain irritation and toxicity to human bodies.

In order to improve the adsorbability of the cross-linked dextran to the hyaluronic acid, the cross-linked dextran is selected to be mixed with the hyaluronic acid in a neutral aqueous solution, so that the hydrolysis of glycosidic bonds in an acidic environment and the degradation of the hyaluronic acid caused by the temperature rise in an alkaline environment are avoided. In order to further improve the adsorption of the cross-linked dextran to hyaluronic acid, alcohol-containing neutral aqueous solution, especially alcohol solution containing less than four carbons, such as ethanol, isopropanol, tert-butanol, etc., is selected.

The temperature rise in step (2) is to further improve the adsorptivity of the cross-linked dextran to reach a state of swelling equilibrium.

The invention also aims to provide the compound gel obtained by the preparation method, wherein the compound gel is injected by a cystoscope injection needle, and the extrusion force of the compound gel is 10N-20N.

The compound gel finally prepared by the invention for treating vesicoureteral reflux has the advantages that:

(1) on the premise of not reducing the viscoelastic property of the compound gel, the usage amount of the cross-linking agent can be reduced, and the biological hazard brought by the residual cross-linking agent is reduced;

(2) the pushing force is within the range of 10N-20N, the pushing resistance is small and smooth, and the endoscope-based vesicopuncture reverse flow injection is suitable for being pushed to a mucous membrane layer under an endoscope and treating vesicoureteral reflux through injection.

Detailed Description

For a better understanding of the present invention, reference will now be made to the following examples and comparative examples.

Example 1 mixing in neutral solution containing alcohol to prepare Complex gel

Dissolving 8g of sodium hyaluronate in 5% ethanol solution, adding cross-linked dextran microsphere particles, fully dispersing at 40 ℃ until the compound loses fluidity, adding a mixed solution of 1% of sodium hydroxide and a cross-linking agent 1, 4-butanediol diglycidyl ether, wherein the mass ratio of the sodium hyaluronate to the cross-linking agent is 200: 1, and uniformly mixing. Crushing the mixture into particles with the diameter less than 5mm by a high-speed shearing machine, standing the mixture for 48 hours at the temperature of 25 ℃, washing the composite by a large amount of phosphate buffer solution for many times, swelling the composite until the composite does not absorb water any more, and sterilizing the composite by high-temperature steam to obtain composite gel.

Example 2 mixing in neutral solution containing alcohol to prepare Complex gel

Dissolving 8g of sodium hyaluronate in 5% ethanol solution, adding cross-linked dextran microsphere particles, fully dispersing at 40 ℃ until the compound loses fluidity, adding a mixed solution of 1% of sodium hydroxide and a cross-linking agent 1, 4-butanediol diglycidyl ether, wherein the mass ratio of the sodium hyaluronate to the cross-linking agent is 250: 1, and uniformly mixing. Crushing the mixture into particles with the diameter less than 5mm by a high-speed shearing machine, standing the mixture for 48 hours at the temperature of 25 ℃, washing the composite by a large amount of phosphate buffer solution for many times, swelling the composite until the composite does not absorb water any more, and sterilizing the composite by high-temperature steam to obtain composite gel.

Example 3 mixing in neutral solution containing alcohol to prepare Complex gel

Dissolving 8g of sodium hyaluronate in 5% ethanol solution, adding cross-linked dextran microsphere particles, fully dispersing at 40 ℃ until the compound loses fluidity, adding a mixed solution of 1% of sodium hydroxide and a cross-linking agent 1, 4-butanediol diglycidyl ether, wherein the mass ratio of the sodium hyaluronate to the cross-linking agent is 180: 1, and uniformly mixing. Crushing the mixture into particles with the diameter less than 5mm by a high-speed shearing machine, standing the mixture for 48 hours at the temperature of 25 ℃, washing the composite by a large amount of phosphate buffer solution for many times, swelling the composite until the composite does not absorb water any more, and sterilizing the composite by high-temperature steam to obtain composite gel.

Example 4 mixing in neutral solution containing alcohol to prepare Complex gel

Dissolving 8g of sodium hyaluronate in 3% isopropanol solution, adding cross-linked dextran microsphere particles, fully dispersing at 40 ℃ until the compound loses fluidity, and adding a mixed solution of 1% of sodium hydroxide and a cross-linking agent 1, 4-butanediol diglycidyl ether, wherein the mass ratio of the sodium hyaluronate: the mass ratio of the cross-linking agent is 200: 1, and the cross-linking agent are uniformly mixed. Crushing the mixture into particles with the diameter less than 5mm by a high-speed shearing machine, standing the mixture for 48 hours at the temperature of 25 ℃, washing the composite by a large amount of phosphate buffer solution for many times, swelling the composite until the composite does not absorb water any more, and sterilizing the composite by high-temperature steam to obtain composite gel.

Comparative example 1 mixing in neutral solution to prepare composite gel

Dissolving 8g of sodium hyaluronate in 1% sodium hydroxide solution, adding dextran microsphere particles, fully dispersing at 40 ℃, adding a mixed solution of 1% sodium hydroxide and a cross-linking agent 1, 4-butanediol diglycidyl ether, wherein the mass ratio of sodium hyaluronate: the mass ratio of the cross-linking agent is 50: 1, and the cross-linking agent are uniformly mixed. Crushing the mixture into particles with the diameter less than 5mm by a high-speed shearing machine, standing the mixture for 48 hours at the temperature of 25 ℃, washing the composite by a large amount of phosphate buffer solution for many times, swelling the composite until the composite does not absorb water any more, and sterilizing the composite by high-temperature steam to obtain composite gel.

Comparative example 2 mixing in alkaline solution to prepare composite gel

Dissolving 8g of sodium hyaluronate in 1% sodium hydroxide solution, adding dextran microsphere particles, fully dispersing at 40 ℃, adding a mixed solution of 1% sodium hydroxide and a cross-linking agent 1, 4-butanediol diglycidyl ether, wherein the mass ratio of sodium hyaluronate: the mass ratio of the cross-linking agent is 20: 1, and the cross-linking agent are uniformly mixed. Crushing the mixture into particles with the diameter less than 5mm by a high-speed shearing machine, standing the mixture for 48 hours at the temperature of 25 ℃, washing the composite by a large amount of phosphate buffer solution for many times, swelling the composite until the composite does not absorb water any more, and sterilizing the composite by high-temperature steam to obtain composite gel.

Example 5

To demonstrate the advantages of the composite gels prepared according to the present invention, the composite gels prepared in the examples and comparative examples were subjected to viscoelasticity and extrusion force tests. The test data are shown in table 1.

The test method is as follows:

viscoelastic properties: the test was carried out at (25. + -. 2). degree.C.at a frequency of 0.01Hz to 100Hz and a shear strain of 1% by means of a rheometer, and the logarithmic graph of the viscous modulus (G ') and the elastic modulus (G') was plotted against the frequency. Values of the viscous modulus and the elastic modulus at a frequency of 0.1Hz are indicated.

Injection and extrusion force: a1.0 mL glass syringe filled with gel is assembled with a cystoscope injection needle (Cook Incorporated) with the specification of 23 Gx 350mm, a small amount of air at the front end of the syringe is discharged, then the syringe is installed on a tension tester, the pushing speed is set to be 12mm/min for measurement, and the maximum pushing force, the minimum pushing force and the average pushing force of a platform area of a pushing force curve are recorded.

TABLE 1 viscoelastic and squeezing force test data for gels of examples

FIG. 1 is a graph of the thrust force of the examples. Table 1 shows that the composite gel prepared by the method of the present invention has the advantages of less cross-linking agent consumption, less extrusion force and smooth extrusion compared with the existing gel. Comparative examples 1-2 are well known in the art and have increased amounts of cross-linking agent in order to obtain a composite gel having similar viscoelastic properties to the composite gel prepared according to the present invention, but the extrusion force becomes large and non-uniform.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A method for preparing a complex gel for the treatment of vesicoureteral reflux, comprising the steps of:

(1) dissolving hyaluronic acid and cross-linked dextran in neutral aqueous solution containing alcohol, and fully dispersing to obtain a mixture; the cross-linked dextran is prepared by cross-linking and polymerizing dextran through ether bonds, and the water absorption capacity is 2.5-20 mL/g;

2. The method according to claim 1, wherein the cross-linked dextran of step (1) is in the shape of a microsphere having a particle size ranging from 50 μm to 500 μm.

3. The method according to claim 1, wherein the neutral aqueous solution containing alcohol in step (1) is an alcoholic solution having four or less carbon atoms and a concentration of 3 to 10% by volume.

4. The method according to claim 3, wherein the neutral aqueous solution containing alcohol is an ethanol solution.

5. The method according to claim 1, wherein the molecular weight of the hyaluronic acid of step (1) is 1.0 to 2.5 MDa.

6. The method according to claim 1, wherein the crosslinking agent in the step (2) is a glycidyl ether.

7. The method according to claim 1, wherein the mixture is physically crushed in the step (3) into granules having a size of less than 5 mm.

8. The complex gel obtained by the preparation method according to any one of claims 1 to 7, wherein the complex gel is injected through a cystoscope injection needle and has a pushing force of 10N to 20N.