CN114163667B

CN114163667B - Cross-linked gel for isolation, preparation method and application

Info

Publication number: CN114163667B
Application number: CN202111486538.4A
Authority: CN
Inventors: 沈延臻
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2024-02-13
Anticipated expiration: 2041-12-07
Also published as: CN114163667A

Abstract

The invention discloses a cross-linked gel for isolation, which is prepared by a method comprising the following steps: adding hyaluronic acid or salt thereof and gelatin into an alkaline solution to obtain a first solution; mixing the first solution with a cross-linking agent to obtain a second solution; and (3) carrying out a crosslinking reaction on the second solution, and swelling a crosslinking reaction product to obtain the crosslinked gel for isolation. The crosslinked gel for isolation has excellent morphological stability and radiation resistance, and is more suitable for radiation therapy isolation protection; the method is convenient to operate, reasonable in reaction time and beneficial to realizing industrial mass production.

Description

Cross-linked gel for isolation, preparation method and application

Technical Field

The invention relates to the technical field of biomedical science, in particular to a crosslinked gel for isolation, a preparation method and application thereof.

Background

Hyaluronic acid, also known as hyaluronic acid, is an acidic mucopolysaccharide. It is an important basic substance for skin care, is a component of human body, has special moisturizing effect, has the weight up to 100 times of the weight of the skin care, is the substance with the best moisturizing property in the nature, and is called ideal natural moisturizing factor. Because of its excellent biocompatibility, it is widely used in various ophthalmic operations such as lens implantation, cornea implantation, and glaucoma-resistant operations. Can also be used for treating arthritis and accelerating wound healing.

Gelatin (Gelatin), which has no fixed structure and relative molecular weight, is degraded by collagen in connective tissue such as animal skin, bone, myomembrane, and muscle charm to become white or yellowish, semitransparent, and slightly glossy flakes or powder; is colorless, odorless, volatile, transparent, hard, amorphous material, and can be dissolved in hot water, and not dissolved in cold water, but can slowly absorb water, expand and soften, and gelatin can absorb water 5-10 times of the weight of gelatin. It is known that gelatin belongs to denatured proteins, has a triple helix structure without bioactivity, is a non-immunogenic substance, and the related literature indicates that injection of gelatin into rabbits, guinea pigs and dogs fails to produce antibodies. Gelatin is one of the very important natural biopolymer materials, and has been widely used in the food, pharmaceutical and chemical industries.

In the radiotherapy process, if the radiation target area is tightly connected with the healthy organ, in order to reduce the damage of radiation to the healthy organ and improve the life quality of a patient after operation, an isolation gel for auxiliary radiotherapy is needed to be used, and a certain volume of space interval is formed between the radiation target area and the healthy organ so as to reduce the radiation dose delivered to the healthy organ during radiotherapy and play an isolation protection role on the tissue of the healthy organ. The barrier gel needs to be stable in the body for a period of time and be fully absorbed by the human body.

The isolation protective gel commonly used in the market at present is polyethylene glycol (PEG) isolation gel, and is often used for assisting the radiotherapy of the prostate cancer. By injecting this product into connective tissue between the fascia of the prostate and the anterior wall of the rectum, a volume of spatial separation is created to reduce the radiation dose delivered to the rectum during radiation therapy of prostate cancer, representing the product as a SpaceOAR spacer gel (augmentx, waltham, MA, USA). The SpaceOAR hydrogel is divided into two bottles, namely a solid powder polyethylene glycol bottle and a tri-lysine diluted solution bottle, and the two bottles are uniformly mixed through a Y-shaped connector when in use, and then a crosslinking reaction occurs immediately after the mixing, so that if an emergency occurs in the injection process, the injection is suspended, equipment can be blocked, a replacement system is required to be prepared, unnecessary pain is brought to a patient, the operation is complex, the requirement is high, and the doctor is also very good in examination.

In summary, in order to simplify the injection procedure of the gel and improve the injection safety, the crosslinking procedure needs to be transferred to the outside of the body, and therefore, it is necessary to prepare a novel gel which can be stably maintained in the body for a period of time and can be completely absorbed by the human body without toxicity. The cohesiveness of the hyaluronic acid gel is strong, so that the gel can keep a certain shape in vivo, but the influence of radioactive rays on the gel can easily degrade the hyaluronic acid gel, so that the in vivo residence time is not long enough; gelatin gels have poor cohesiveness but high radiation resistance, and therefore, a crosslinked hyaluronic acid gelatin gel resistant to radiation degradation is required, and no such products have been reported at present.

Disclosure of Invention

In order to solve the problems, the invention provides a cross-linked gel for isolation, a preparation method and application.

The specific technical scheme of the invention is as follows:

1. a method for preparing a crosslinked gel for isolation, comprising the steps of:

adding hyaluronic acid or salt thereof and gelatin into an alkaline solution to obtain a first solution;

mixing the first solution with a cross-linking agent to obtain a second solution;

and (3) carrying out a crosslinking reaction on the second solution, and swelling a crosslinking reaction product to obtain the crosslinked gel for isolation.

2. The process according to item 1,

the molecular weight of the hyaluronic acid or salt thereof is 1000k-4000kDa, preferably 2000k-4000kDa, and more preferably 3000k-4000kDa.

3. The process according to item 1,

the molecular weight of the gelatin is 20k-500kDa, preferably 100k-500kDa, more preferably 200k-400kDa, and the gelatin is preferably any one of animal gelatin.

4. The process according to item 1,

the cross-linking agent is an esterification cross-linking agent or an etherification cross-linking agent; wherein the esterification crosslinking agent is preferably carbodiimide or bis/polyepoxide, and the etherification crosslinking agent is preferably divinyl sulfone, 1,2,7, 8-diepoxyoctane or 1, 4-butanediol diglycidyl ether;

further preferably, the cross-linking agent comprises 0.5-5wt%, preferably 2-3wt%, of the mass percentage of the sum of the mass of the hyaluronic acid or salt thereof and the gelatin.

5. The process according to item 1,

the mass ratio of the hyaluronic acid or the salt thereof to the gelatin is 0.5-10:1, preferably 5-10:1; preferably, the sum of the contents of hyaluronic acid or a salt thereof and gelatin in the crosslinked gel is 20-50mg/mL.

6. The process according to item 1,

the alkaline solution is selected from one or more of sodium carbonate solution, sodium bicarbonate solution and sodium hydroxide solution;

preferably, the mass percentage concentration of the alkaline solution is 0.1-2wt%;

further preferably, the crosslinking reaction time is from 0.5 to 15 hours, preferably from 8 to 12 hours.

7. A crosslinked gel for insulation, which is prepared by the preparation method of any one of claims 1 to 6.

8. A crosslinked gel for insulation, wherein the crosslinked gel for insulation is prepared by a method comprising the steps of:

9. The crosslinked gel according to item 8,

10. The crosslinked gel according to item 8, wherein,

11. The crosslinked gel according to item 8,

further preferably, the cross-linking agent is used in an amount of 0.5 to 5wt%, preferably 2 to 3wt%, of the sum of the mass of the hyaluronic acid or salt thereof and the gelatin.

12. The crosslinked gel according to item 8,

the mass ratio of the hyaluronic acid or the salt thereof to the gelatin is 0.5-10:1, preferably 5-10:1;

preferably, the sum of the contents of the crosslinked hyaluronic acid or salt thereof and gelatin in the crosslinked gel is 20-50mg/mL.

13. The crosslinked gel according to item 8,

14. A radiation therapy barrier protective agent comprising the crosslinked gel for barrier prepared by the preparation method of any one of claims 1 to 6 or the crosslinked gel for barrier of any one of claims 8 to 13.

15. Use of the crosslinked gel for radiation therapy isolation prepared by the preparation method of any one of claims 1 to 6 or the crosslinked gel for radiation therapy isolation of any one of claims 8 to 13 in the preparation of radiation therapy isolation protection.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention adopts the mixed crosslinking of hyaluronic acid or the salt thereof and gelatin, and both materials have excellent biocompatibility and a large number of active hydroxyl groups, in the crosslinking process, 6-position hydroxyl in the hyaluronic acid or the salt chain thereof and epoxy rings in a crosslinking agent (such as 1, 4-butanediol diglycidyl ether and BDDE) generate nucleophilic reaction to generate stable ether bond, and simultaneously, hydroxyl in the gelatin chain is linked by the crosslinking agent to form crosslinked gel with a semi-interpenetrating network structure. The gel for isolation fully plays the respective excellent characteristics of the two materials, has excellent morphological stability and radiation resistance, and is more suitable for radiation therapy isolation protection; the method is convenient to operate, reasonable in reaction time and beneficial to realizing industrial mass production. The use process is simple and convenient, and the injection process is safer.

Detailed Description

The embodiments described below illustrate the present invention in detail, however, it should be understood that the present invention may be embodied in various forms and should not be limited to the examples set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the invention, but is not intended to limit the scope of the invention, as the description proceeds with reference to the general principles of the description. The scope of the invention is defined by the appended claims.

The term "kDa" refers to 1000Da.

The term "Da" refers to the unit of weight average molecular weight of hyaluronic acid, its salts or gelatin.

The term "mg/mL" refers to the content of the sum of the mass of hyaluronic acid or a salt thereof and gelatin in the crosslinked gel in the gel.

The invention provides a preparation method of cross-linked gel for isolation, which comprises the following steps:

The "cross-linking agent" is also called a bridging agent, which forms a bridge between the polymer molecular chains, becoming an insoluble substance of three-dimensional structure.

The hyaluronic acid or salt thereof may be hyaluronic acid and salts thereof known to those skilled in the art, such as hyaluronic acid, and the hyaluronate is preferably a metal hyaluronate, such as sodium hyaluronate, potassium hyaluronate, etc.

The alkaline solution may be an alkaline solution known to those skilled in the art, such as sodium hydroxide, potassium hydroxide, sodium carbonate solution, etc.;

in some embodiments of the invention, the alkaline solution has a mass percent concentration of 0.1 to 2wt%, for example, may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 09, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0wt% or any range therebetween.

In some embodiments of the invention, the hyaluronic acid or salt thereof has a molecular weight of 1000k-4000kDa, preferably 2000k-4000kDa, further preferably 3000k-4000kDa;

for example, the molecular weight of hyaluronic acid or salt thereof may be 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa, 2600kDa, 2700kDa, 2800kDa, 2900kDa, 3000kDa, 3100kDa, 3200kDa, 3300kDa, 3400kDa, 3500kDa, 3600kDa, 3700kDa, 3800kDa, 3900kDa, 4000kDa, or any range therebetween.

In some embodiments of the invention, the gelatin is any of animal-derived gelatin, preferably porcine-derived gelatin, the molecular weight of the gelatin being 20k-500kDa, preferably 100k-500kDa, further preferably 200k-400kDa;

for example, the molecular weight of the gelatin may be 20kDa, 50kDa, 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, 450kDa, 500kDa or any range therebetween.

In some embodiments of the present invention, the crosslinking agent may use a crosslinking agent well known to those skilled in the art, with the preferred crosslinking agent being an esterified crosslinking agent or an etherified crosslinking agent; wherein the esterified cross-linking agent is preferably a polyol, carbodiimide or di/polyepoxide, and the etherified cross-linking agent is preferably divinyl sulfone, 1,2,7, 8-diepoxyoctane or 1, 4-butanediol diglycidyl ether (BDDE), and more preferably 1, 4-butanediol diglycidyl ether.

In some embodiments of the invention, the cross-linking agent comprises 0.5-5wt%, preferably 2-3wt%, of the sum of the hyaluronic acid or salt thereof and the gelatin mass, e.g., may be 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0wt% or any range therebetween.

In some embodiments of the invention, the mass ratio of the hyaluronic acid or salt thereof to the gelatin is 0.5-10:1, preferably 5-10:1, e.g. may be 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1 or any range therebetween.

In some embodiments of the invention, the crosslinking reaction time is 0.5 to 15 hours, preferably 8 to 12 hours, and may be, for example, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, or any range therebetween.

In some embodiments of the invention, the sum of the contents of cross-linked hyaluronic acid or salt thereof and gelatin in the cross-linked gel is 20-50mg/mL, for example, may be 20, 25, 30, 35, 40, 45, 50mg/mL or any range therebetween.

The invention provides cross-linked hyaluronic acid or salt gel thereof for injection, which is prepared by the preparation method.

The invention provides cross-linked hyaluronic acid or a salt gel thereof for injection, which is prepared by a method comprising the following steps:

In some specific embodiments of the invention, gelatin is dissolved in alkaline solution, sodium hyaluronate is added after complete dissolution, after uniform mixing, a cross-linking agent is added, after uniform stirring, the mixture is put into a water bath kettle for cross-linking reaction, after the cross-linking reaction is finished, a cross-linking reaction product is cut, phosphate buffer solution is used for dialysis and swelling, after swelling to the target weight, the obtained gel block is granulated through a 60-mesh screen to obtain cross-linked gel, and then high-pressure steam sterilization is carried out.

In some embodiments of the invention, the sum of the amounts of cross-linked hyaluronic acid or salt thereof and gelatin in the cross-linked gel is 20-50mg/mL, e.g. may be 20, 25, 30, 35, 40, 45, 50mg/mL or any range therebetween.

The gel absorbs water during dialysis swelling, and the content of the crosslinked gel is related to the weight of the gel after dialysis swelling. The longer the dialysis time, the more water is absorbed, and the lower the sum of the contents of the crosslinked hyaluronic acid or its salt and gelatin is, so the content of the sum of the contents of the crosslinked hyaluronic acid or its salt and gelatin can be controlled by controlling the dialysis time.

The invention provides a radiation therapy isolation protective agent, which comprises the isolation crosslinked gel prepared by the preparation method or the isolation crosslinked gel.

The isolation gel is used for isolating a radiation target tissue from a healthy tissue in radiation therapy, and in the radiation therapy process, if a radiation target region is tightly connected with the healthy organ, in order to reduce the damage of radiation to the healthy organ and improve the postoperative life quality of a patient, the isolation gel for auxiliary radiation therapy is needed to form a certain volume of space interval between the radiation target region and the healthy organ so as to reduce the radiation dose delivered to the healthy organ during radiation therapy and play a role in isolating and protecting the healthy organ tissue. The barrier gel needs to be stable in the body for a period of time and be fully absorbed by the human body.

The invention also provides the cross-linked gel for radiation therapy isolation prepared by the preparation method or the application of the cross-linked gel for radiation therapy isolation in preparation of radiation therapy isolation protection.

The crosslinked gel for isolation provided by the invention has remarkable effect, wherein in a mouse experiment, the feeding amount, the weight, the mental state and the skin condition of a mouse are observed, and the crosslinked gel for isolation provided by the invention can basically ensure that the total feeding amount is not obviously changed, the mental state of the mouse is good and the skin is not damaged. Therefore, the crosslinked gel for isolation provided by the invention has remarkable effect, fully exerts the respective excellent characteristics of the two materials, has excellent morphological stability and radiation resistance, and is more suitable for radiation therapy isolation protection.

The materials used in the test and the test methods are described generally and/or specifically in the examples which follow,% represents wt%, i.e. weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge, wherein Table 1 shows the sources of the raw materials used in the examples.

Table 1 sources of raw materials used in the examples

Example 1

Dissolving 0.4g of gelatin in 10mL of 1wt% sodium hydroxide solution, wherein the molecular weight of gelatin is 200kDa, adding 2g of sodium hyaluronate with the molecular weight of 3000kDa after complete dissolution, wherein the mass ratio of sodium hyaluronate to gelatin is 5:1, adding 48mg of 1, 4-butanediol diglycidyl ether, wherein the using amount of 1, 4-butanediol diglycidyl ether is 2% of the sum of sodium hyaluronate and gelatin, fully stirring uniformly, placing into a water bath kettle for crosslinking reaction for 10h at 30 ℃, cutting a crosslinked reaction product into small blocks after the crosslinking reaction is finished, placing into phosphate buffer solution (1L of phosphate buffer solution: 9g of sodium chloride, 0.22g of disodium hydrogen phosphate, 0.05g of sodium dihydrogen phosphate, and the balance being filled with injection water), granulating the obtained gel blocks through a 60-mesh screen, and obtaining crosslinked gel with the sum of contents of crosslinked sodium hyaluronate and gelatin of 30mg/mL, and sterilizing by high-pressure steam at 121 ℃ for 8min.

Example 2

Example 2 differs from example 1 only in that: the mass ratio of the sodium hyaluronate to the gelatin is 10:1, and the rest conditions are the same.

Example 3

1.6g of gelatin is dissolved in 10mL of 1wt% sodium hydroxide solution, the molecular weight of gelatin is 200kDa, 0.8g of sodium hyaluronate is added after complete dissolution, the molecular weight of sodium hyaluronate is 3000kDa, the mass ratio of sodium hyaluronate to gelatin is 0.5:1, 48mg of 1, 4-butanediol diglycidyl ether is added, the content of 1, 4-butanediol diglycidyl ether is 2% of the sum of the mass of sodium hyaluronate and gelatin, the mixture is fully and uniformly stirred, the mixture is placed in a water bath kettle for crosslinking reaction for 10h at 30 ℃, after the crosslinking reaction is finished, a crosslinked reaction product is cut into small blocks, the small blocks are placed in phosphate buffer solution (1L of phosphate buffer solution: 9g of sodium chloride, 0.22g of disodium hydrogen phosphate, 0.05g of sodium dihydrogen phosphate and the balance of injection water) for dialysis, the obtained gel blocks are granulated through a 60-mesh screen, and then the crosslinked gel with the content sum of crosslinked sodium hyaluronate and gelatin of 30mg/mL is obtained, and the crosslinked gel is sterilized by high-pressure steam at 121 ℃ for 8min.

Example 4

Example 4 differs from example 1 only in that: 12mg of 1, 4-butanediol diglycidyl ether was added, wherein the amount of 1, 4-butanediol diglycidyl ether was 0.5% of the sum of the mass of sodium hyaluronate and gelatin, and the other conditions were the same.

Example 5

Example 5 differs from example 1 only in that: 120mg of 1, 4-butanediol diglycidyl ether was added, wherein the amount of 1, 4-butanediol diglycidyl ether was 3% of the sum of the mass of sodium hyaluronate and gelatin, and the other conditions were the same.

Example 6

Example 6 differs from example 1 only in that: and controlling the sum of the contents of the crosslinked sodium hyaluronate and the gelatin by controlling the dialysis time to obtain the crosslinked gel with the sum of the contents of the crosslinked sodium hyaluronate and the gelatin of 20mg/mL, and the rest conditions are the same.

Example 7

Example 7 differs from example 1 only in that: and controlling the sum of the contents of the crosslinked sodium hyaluronate and the gelatin by controlling the dialysis time to obtain the crosslinked gel with the sum of the contents of the crosslinked sodium hyaluronate and the gelatin of 50mg/mL, and the rest conditions are the same.

Example 8

Example 2 differs from example 1 only in that: the mass ratio of the sodium hyaluronate to the gelatin is 20:1, and the rest conditions are the same.

Example 9

Example 9 differs from example 1 only in that: the mass ratio of the sodium hyaluronate to the gelatin is 0.1:1, and the rest conditions are the same.

Example 10

Example 10 differs from example 1 only in that: 4.8mg of 1, 4-butanediol diglycidyl ether was added, wherein the amount of 1, 4-butanediol diglycidyl ether was 0.2% of the sum of the mass of sodium hyaluronate and gelatin, and the other conditions were the same.

Example 11

Example 11 differs from example 1 only in that: the crosslinked gel with the sum of the contents of the crosslinked sodium hyaluronate and the gelatin being 10mg/mL is obtained, and the other conditions are the same.

Example 12

Example 12 differs from example 1 only in that: the molecular weight of sodium hyaluronate was 2000kDa, the remaining conditions being the same.

Example 13

Example 13 differs from example 1 only in that: the molecular weight of sodium hyaluronate was 3600kDa, the other conditions being the same.

Example 14

Example 14 differs from example 1 only in that: the molecular weight of sodium hyaluronate was 1500kDa, the remaining conditions being the same.

Example 15

Example 15 differs from example 1 only in that: the molecular weight of sodium hyaluronate was 500kDa, the remaining conditions being the same.

Example 16

Example 16 differs from example 1 only in that: the molecular weight of gelatin is 100kDa, the remaining conditions being the same.

Example 17

Example 17 differs from example 1 only in that: the molecular weight of gelatin was 10kDa, the remaining conditions being the same.

Example 18

Example 18 differs from example 1 only in that: the molecular weight of gelatin was 300kDa, the remaining conditions being the same.

Example 19

Example 19 differs from example 1 only in that: the molecular weight of gelatin was 380kDa, the remaining conditions being the same.

Example 20

Example 20 differs from example 1 only in that: 168mg of 1, 4-butanediol diglycidyl ether was added, wherein the amount of 1, 4-butanediol diglycidyl ether was 7% of the sum of the mass of sodium hyaluronate and gelatin, and the other conditions were the same.

Comparative example 1

2.4g of gelatin is dissolved in 10mL of 1wt% sodium hydroxide solution, the molecular weight of gelatin is 200kDa, 48mg of 1, 4-butanediol diglycidyl ether is added, the dosage of 1, 4-butanediol diglycidyl ether is 2% of the sum of the masses of gelatin, the gelatin is fully and uniformly stirred, the gelatin is placed in a water bath kettle for crosslinking reaction at 30 ℃ for 10 hours, after the crosslinking reaction is finished, the crosslinked reaction product is cut into small blocks, the small blocks are placed in phosphate buffer solution (1L of phosphate buffer solution: 9g of sodium chloride, 0.22g of disodium hydrogen phosphate and 0.05g of sodium dihydrogen phosphate, the balance is filled with injection water), the obtained gel blocks are granulated through a 60-mesh screen after dialysis and swelling, and then the crosslinked gel with the content of 30mg/mL is obtained, and is subjected to high-pressure steam sterilization at 121 ℃ for 8min.

Comparative example 2

2.4g of sodium hyaluronate is dissolved in 10mL of 1wt% sodium hydroxide solution, the molecular weight of the sodium hyaluronate is 3000kDa, 48mg of 1, 4-butanediol diglycidyl ether is added, wherein the dosage of the 1, 4-butanediol diglycidyl ether is 2% of the sum of the masses of the sodium hyaluronate, the sodium hyaluronate is fully and uniformly stirred, the sodium hyaluronate is put into a water bath kettle for crosslinking reaction for 10 hours at 30 ℃, after the crosslinking reaction is finished, the crosslinked reaction product is cut into small blocks, the small blocks are put into phosphate buffer solution (1L of phosphate buffer solution: 9g of sodium chloride, 0.22g of disodium hydrogen phosphate, 0.05g of sodium dihydrogen phosphate and the balance of injection water) for dialysis and swelling, and the obtained gel blocks are granulated by a 60-mesh screen to obtain crosslinked gel with the content of 30mg/mL, and then the crosslinked gel is sterilized by high-pressure steam at the temperature of 121 ℃ for 8min.

TABLE 2 raw materials used in examples 1-20 and comparative examples 1-2

Experimental example 1 crosslinked gel vs. particle implant postoperative protection assay

The crosslinked gels obtained in examples 1 to 20 were respectively numbered as gels 1 to 20, and the crosslinked gels obtained in comparative examples 1 to 2 were respectively numbered as gels 21 to 22.

After mice were implanted with radioactive 125I particles, gels 1-22 were examined for their effect in reducing the toxicity of 125I particles implanted into the skin.

Test animals: balb/C mice

Test grouping: 69 mice were randomly divided into 23 groups, 3 groups each, control group, 1 group, 2 group, 3 group, 4 group, 5 group, 6 group, 7 group, 8 group, 9 group, 10 group, 11 group, 12 group, 13 group, 14 group, 15 group, 16 group, 17 group, 18 group, 19 group, 20 group, 21 group, 22 group, gel 1 for 1 group of mice, gel 2 for 2 group of mice, … group and so on, gel 22 for 22 group of mice using the computer random number method. The control group was implanted with 1 empty shell particle (blank) and the other 22 groups were each implanted with 1 particle with 0.6mCi activity 125I. The radioactive particles were implanted while injecting 1.0ml of gel between the skin and the particles, respectively, with the same group number as the test sample number, and the blank group was not injected with the crosslinked gel.

Feeding animals: the animals were housed separately under standard conditions after implantation of the particles.

And (5) observing and indicating: general observations were made on the test animals.

Feed intake (food and water): the total food intake was obtained by subtracting the residual food amount from the daily feed amount. From the preoperative initial observations, post-operative days 5, 10, 30, 90 … were recorded.

Weight of: from the preoperative initial observations, post-operative days 5, 10, 30, 90 … were recorded.

Mental state: subjective evaluation by the same observer was noted as good, general, slightly worse, listless, dead. From the preoperative initial observations, post-operative days 5, 10, 30, 90 … were recorded.

Skin damage: mice were observed contemporaneously for skin lesions such as changes in color, hair loss, ulceration, etc. And skin damage between the different groups.

Residual gel observation: each group of 1 animal was sacrificed at random at 90 and 150 days post-surgery, respectively, and gel residue was observed.

Summary of test results: see table 3.

Table 3 isolation protective Effect test results

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As can be seen from Table 3, by observing and recording the feeding, mental state and skin damage of mice at 5, 10, 30, 90, 150 days after surgery, the combined effect of groups 1 to 20 is better than that of groups 21 and 22, i.e., the isolated crosslinked gels obtained in examples 1 to 20 are superior to those of comparative examples 1 to 2 in terms of isolation, especially the recorded results at 30, 90, 150 days, such as mental state and skin damage. The reason is that in comparative example 1, the barrier crosslinked gel was free of added hyaluronic acid or a salt thereof, and in comparative example 2, no gelatin was added, that is, the barrier crosslinked gel obtained by adding hyaluronic acid or a salt thereof and gelatin simultaneously had a barrier protective effect superior to the case of adding only one of them. The hyaluronic acid or the salt thereof and the gelatin have excellent biocompatibility and a large number of active hydroxyl groups, in the crosslinking process, 6-position hydroxyl in the hyaluronic acid or the salt chain thereof and epoxy rings in a crosslinking agent (such as 1, 4-butanediol diglycidyl ether and BDDE) generate nucleophilic reaction to generate stable ether bonds, and simultaneously, the hydroxyl in the gelatin chain is linked by the crosslinking agent to form crosslinked gel with a semi-interpenetrating network structure, which indicates that the two polymers cooperatively play the effect of isolation protection. In addition, the hyaluronic acid or the salt gel thereof has excellent cohesiveness and gel forming property, can keep stable form under the action of certain external force, is sensitive to irradiation, is easy to degrade after long-term irradiation, has strong irradiation resistance, and can keep stronger form stability after the mixed gel formed by crosslinking the hyaluronic acid or the salt thereof and the gelatin, the gelatin can play a role of a structural node in the irradiation degradation process, and the irradiation resistance of the gel is improved.

Preferably, in examples 1 to 20, the barrier and protection effects of examples 1 to 7 are superior to examples 8 to 11, wherein it can be seen from comparison of examples 1 and 8 that the barrier and protection effects are slightly inferior when the gelatin content is low, because the gelatin content is too low, and an interpenetrating network structure effective for enhancing the crosslinking strength is not formed; as can be seen from comparing example 1 with example 9, when the gelatin content is higher, the isolation protection effect is slightly poor, because the higher gelatin content can cause poor cohesiveness of the gel, the gel is not easy to recover after deformation, and the obvious isolation protection effect cannot be achieved; as can be seen from comparing example 1 with example 10, when the amount of the crosslinking agent is low, the barrier protection effect is slightly poor because the amount of the crosslinking agent is insufficient, the degree of crosslinking is insufficient, and an interpenetrating network structure which effectively enhances the crosslinking strength is not formed; from a comparison of example 1 and example 11, it can be seen that the barrier protection effect is slightly inferior when the gel content is low, because the gel content is too low, the gel is thin, and an effective supporting effect is not achieved.

TABLE 4 summary of residual crosslinked gel results

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The isolation crosslinking gel is used for radiation therapy isolation protection, and is mainly formed by injecting synthetic gel into a gap between a target part and an organ to be protected in a physical interval mode, so that the distance between the two organs is increased, the radiation to the organ to be protected is reduced, and the chronic toxic reaction possibly generated by the protected organ after the radiation therapy is finished is reduced. In order to achieve the goal of stable presence between the target site and the organ to be protected, the isolated crosslinked gel is required to possess a degree of crosslinking stability, radiation resistance and cohesiveness that prevent displacement of the gel during treatment. The gel is generally stable for a certain period of time (typically more than 3 months) and can be completely degraded in vivo after the radiation therapy is completed.

Comparative example 1 was found to leave a lot of gels because comparative example 1 was a pure gelatin crosslinked gel, which was strong in radiation resistance, and was found to leave a lot of gels, but was poor in gelling ability. In comparative example 2, the remaining gel was more, but the protective effect was inferior to that of example 1, because the rays broken part of the molecular chain of hyaluronic acid or its salt, and the gel became looser, and the supporting effect was deteriorated.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

carrying out a crosslinking reaction on the second solution, and swelling a crosslinking reaction product to obtain crosslinked gel for isolation;

wherein the molecular weight of the hyaluronic acid or the salt thereof is 1000k-4000kDa,

the cross-linking agent accounts for 0.5-5wt% of the sum of the hyaluronic acid or salt thereof and the gelatin,

the mass ratio of the hyaluronic acid or the salt thereof to the gelatin is 0.5-10:1.

2. The preparation method according to claim 1, wherein,

the molecular weight of the hyaluronic acid or the salt thereof is 2000k-4000kDa.

3. The preparation method according to claim 1, wherein,

the molecular weight of the hyaluronic acid or the salt thereof is 3000k-4000kDa.

4. The preparation method according to claim 1, wherein,

the molecular weight of the gelatin is 20k-500kDa.

5. The preparation method according to claim 4, wherein,

the molecular weight of the gelatin is 100k-500kDa.

6. The preparation method according to claim 5, wherein,

the molecular weight of the gelatin is 200k-400kDa.

7. The preparation method according to claim 1, wherein,

the gelatin is any one of animal gelatin.

8. The preparation method according to claim 1, wherein,

the cross-linking agent is an esterification cross-linking agent or an etherification cross-linking agent.

9. The preparation method according to claim 8, wherein,

the esterification cross-linking agent is carbodiimide or di/polyepoxide, and the etherification cross-linking agent is divinyl sulfone, 1,2,7, 8-diepoxyoctane or 1, 4-butanediol diglycidyl ether.

10. The preparation method according to claim 1, wherein,

the cross-linking agent accounts for 2-3wt% of the sum of the mass of the hyaluronic acid or salt thereof and the gelatin.

11. The preparation method according to claim 1, wherein,

the mass ratio of the hyaluronic acid or the salt thereof to the gelatin is 5-10:1.

12. The preparation method according to claim 1, wherein,

the sum of the contents of hyaluronic acid or salt thereof and gelatin in the crosslinked gel is 20-50mg/mL.

13. The preparation method according to claim 1, wherein,

the alkaline solution is selected from one or more of sodium carbonate solution, sodium bicarbonate solution and sodium hydroxide solution.

14. The preparation method according to claim 1, wherein,

the mass percentage concentration of the alkaline solution is 0.1-2wt%.

15. The preparation method according to claim 1, wherein,

the crosslinking reaction time is 0.5-15h.

16. The preparation method according to claim 1, wherein,

the crosslinking reaction time is 8-12h.

17. A crosslinked gel for insulation, which is prepared by the preparation method of any one of claims 1 to 16.

18. A radiation therapy shielding agent comprising the crosslinked gel for shielding prepared by the preparation method of any one of claims 1 to 16.

19. Use of the crosslinked gel for isolation prepared by the preparation method of any one of claims 1 to 16 in the preparation of radiation therapy isolation protection.