CN117618635A

CN117618635A - Embolic developing particles loaded with coagulant and preparation method thereof

Info

Publication number: CN117618635A
Application number: CN202311682672.0A
Authority: CN
Inventors: 刘凤永; 田晓梅; 李鑫
Original assignee: Fifth Medical Center of PLA General Hospital
Current assignee: Fifth Medical Center of PLA General Hospital
Priority date: 2023-12-08
Filing date: 2023-12-08
Publication date: 2024-03-01

Abstract

The application relates to the technical field of biological medicines, and particularly discloses an embolism developing particle loaded with a coagulant and a preparation method thereof. The coagulant-loaded embolic developing particles of the present application comprise an ortho-carboxybenzoylated gelatin, a developer, and a coagulant; the preparation method of the o-carboxybenzoylated gelatin comprises the following steps: dissolving gelatin in water to obtain gelatin water solution with concentration of 3-5wt%, and regulating pH of gelatin water solution to 8.5-9.5; then adding phthalic anhydride into the gelatin water solution under the stirring effect, and reacting for 1-2h at 40-50 ℃ to obtain a reaction solution; purifying and freeze-drying to obtain the o-carboxybenzoylated gelatin; the application also provides a preparation method of the embolism developing particles loaded with the coagulant. The application can prepare the embolism developing microsphere with large porosity and good water absorption by utilizing the o-carboxyl benzoyl gelatin, and can obtain the embolism developing particle with high coagulant load by mixing the embolism developing microsphere with the coagulant.

Description

Embolic developing particles loaded with coagulant and preparation method thereof

Technical Field

The application relates to the technical field of biological medicines, in particular to an embolism developing particle loaded with a coagulant and a preparation method thereof.

Background

In the treatment of cancer or vascular hemorrhage, it is important to plug focal blood vessels in time. The embolism is also called embolism treatment, which is to inject the plug into the supply vessel of the lesion organ through the arterial or intravenous catheter in a controlled way, so as to cause the plug to be occluded and interrupt the blood supply, thereby achieving the purposes of controlling bleeding, treating tumor and vascular lesion and eliminating the function of the lesion organ.

Thrombin is a coagulation factor capable of converting soluble fibrinogen in plasma into insoluble fibrin, and in recent years, researchers have obtained a chemical embolic agent by loading thrombin on embolic particles or microspheres, and then injecting it into the blood supply of tumors, and by releasing thrombin at focal blood vessels, an environment of effective concentration of thrombin is locally formed, thereby achieving the effect of double hemostasis of physical embolism and drug coagulation. However, most of the thrombin-loaded embolic particles on the market currently have the problems of poor water absorption, low thrombin loading and the like.

Disclosure of Invention

In order to improve the water absorption and the loading capacity of the thrombin-loaded embolic particles, the application provides a coagulant-loaded embolic developing particle and a preparation method thereof.

In a first aspect, the present application provides a coagulant-loaded embolic developing particle, employing the following technical scheme:

an accelerator-loaded embolic developing particle comprising an o-carboxybenzoylated gelatin, a developer, and an accelerator.

According to the method, o-carboxybenzoylated gelatin is used as a carrier, and a coagulant-loaded embolism developing particle is prepared through loading a developer and a coagulant, the coagulant-loaded embolism developing particle has high water absorption rate and thrombin loading rate, the coagulant-loaded embolism developing particle is injected into a blood vessel through a microcatheter, so that a embolism body can be formed by solidification in blood, the blood vessel can be blocked by the embolism body to cut off blood flow supply, and meanwhile thrombin can form effective auxiliary coagulation locally to manufacture thrombus and achieve the purpose of embolism. In addition, the embolic developing particles loaded with the coagulant contain developing particles, so that the embolic can be clearly developed in operation and early warning can be carried out on ectopic embolism.

In the application, the o-carboxybenzoylated gelatin is obtained by modifying gelatin with phthalic anhydride to perform acylation reaction between phthalic anhydride and amino groups on a gelatin molecular chain. The modification greatly increases the carboxyl content in the gelatin system, and the plug developing particles prepared by utilizing the o-carboxyl benzoyl gelatin can obviously improve the water absorption of the plug developing particles.

Optionally, the preparation method of the o-carboxybenzoylated gelatin comprises the following steps: dissolving gelatin in water to obtain gelatin water solution with concentration of 3-5wt%, and regulating pH of gelatin water solution to 8.5-9.5; then adding phthalic anhydride into the gelatin water solution under the stirring effect, and reacting for 1-2h at 40-50 ℃ to obtain a reaction solution; and purifying and freeze-drying to obtain the o-carboxybenzoylated gelatin.

Optionally, the weight ratio of phthalic anhydride to gelatin is (3-7): 100.

in some embodiments, the weight ratio of phthalic anhydride to gelatin may be (3-5): 100 or (5-7): 100.

in a specific embodiment, the weight ratio of phthalic anhydride to gelatin may be 3: 100. 5:100 or 7:100.

in the application, the weight ratio of phthalic anhydride to gelatin can influence the hydrophilicity and water locking property of the o-carboxybenzoylated gelatin, and experiments prove that the o-carboxybenzoylated gelatin with excellent hydrophilicity and water locking property can be obtained by controlling the weight ratio of phthalic anhydride to gelatin in the range, the prepared embolism developing particles are good in water absorption and coagulant loading, the water absorption capacity can reach 38.2-44.1 times of the self weight of the embolism developing particles, and the thrombin loading capacity can reach 252-482U/100mg.

Optionally, the accelerator-loaded embolic developing particles have a particle size of 70-1300 μm.

In a second aspect, the present application provides a method of preparing a coagulant-loaded embolic developing particle.

A method of preparing coagulant-loaded embolic developing particles, comprising the steps of: preparing gelatin developing solution, emulsifying and crosslinking, and loading coagulant;

preparing gelatin developing solution: dissolving the o-carboxybenzoylated gelatin in water to prepare an o-carboxybenzoylated gelatin solution with the concentration of 5-20wt%; then adding a developer, and uniformly stirring to obtain a gelatin developing solution;

emulsion crosslinking: mixing gelatin developing solution with emulsifier, stirring at 300-450rpm, adding cross-linking agent for cross-linking, cleaning, freeze-drying, and sieving to obtain embolism developing microsphere;

loading coagulant: mixing the embolism developing microsphere with thrombin solution, and freeze-drying to obtain the embolism developing particle loaded with coagulant.

In the preparation method of the embolism developing particles loaded with the coagulant, gelatin developing solution is used as a water phase and an emulsifier is used as an oil phase, the water phase and the oil phase are mixed, the water phase and the oil phase can be subjected to inverse polymerization to form stable suspension, and then a cross-linking agent is added into the suspension, so that stable embolism developing microspheres can be formed. The application finds through experimental investigation that the concentration of the o-carboxybenzoylated gelatin solution and the stirring rate of emulsification are controlled within the above ranges, so that the embolism development microsphere with higher thrombin loading can be obtained.

In some embodiments, the stirring speed may be 300-350rpm, 300-400rpm, 350-450rpm, or 400-450rpm.

In a specific embodiment, the stirring speed may also be 300rpm, 350rpm, 400rpm or 450rpm.

Optionally, the concentration of the phthalylated gelatin solution is 8-15wt%.

In some embodiments, the concentration of the o-carboxybenzoylated gelatin solution may be 5-8wt%, 5-10wt%, 5-15wt%, 8-10wt%, 8-15wt%, or 10-15wt%.

In a specific embodiment, the concentration of the o-carboxybenzoylated gelatin solution may also be 5wt%, 8wt%, 10wt%, or 15wt%.

Optionally, the volume ratio of the gelatin developing solution to the emulsifier is 1: (8-15).

Optionally, the cross-linking agent is a 40-60wt% glutaraldehyde aqueous solution; the crosslinking reaction time is 2-10h.

Optionally, the emulsifier can be paraffin oil or high molecular organic solution; further, the polymer organic acid may be a methylene chloride solution of polylactic acid.

Optionally, the developer is selected from one or more of metal particles, metal oxide particles, metal salts, and iodine-containing developers.

Optionally, the developer is added in an amount of 5-15wt% of the gelatin solution.

Optionally, the thrombin solution has a concentration of 1200-1300U/mL.

Optionally, the usage amount relationship of the embolism developing microsphere and the thrombin solution is as follows: the embolic development microspheres were dissolved in 0.3-0.6mL of thrombin solution per 100g of embolic development microspheres.

In summary, the present application has the following beneficial effects:

1. the method takes the o-carboxybenzoylated gelatin as a carrier, and the o-carboxybenzoylated gelatin is emulsified, crosslinked and loaded with the coagulant, so that the coagulant-loaded embolism developing particle is prepared, and compared with the coagulant-loaded embolism developing particle in the related technology, the coagulant-loaded embolism developing particle has larger porosity and water absorption rate and high thrombin loading rate, and can be widely used for blocking hemorrhagic blood vessels, and embolizing the blood vessels to reduce blood flow/block blood supply for treating diseases such as arteriovenous malformations, aneurysms, venous aneurysms, benign tumors, malignant tumors and the like.

2. The weight ratio of phthalic anhydride to gelatin is controlled to be (3-7): 100, the o-carboxybenzoylated gelatin with excellent hydrophilicity and water locking property can be obtained, the o-carboxybenzoylated gelatin is used for preparing embolism developing particles, the obtained embolism developing particles have good water absorption and coagulant load, the water absorption can reach 38.2-44.1 times of the self weight, and the thrombin load can reach 252-482U/100mg.

3. In the application, by further controlling the concentration of the o-carboxybenzoylated gelatin solution within the range of 8-15wt%, the stirring rotation speed in the emulsification crosslinking step is controlled between 350-450rpm, and the thrombin load of the obtained embolism development particles can reach more than 400U/100mg.

Drawings

FIG. 1 is a topographical view under a microscope of the coagulant-loaded embolic developing particles obtained in example 1.

Detailed Description

The application provides a coagulant-loaded embolic developing particle, the method of making which comprises the steps of:

(1) Preparing gelatin developing solution: dissolving the o-carboxybenzoylated gelatin in purified water to prepare an o-carboxybenzoylated gelatin solution with the concentration of 5-10wt%; then adding 5-15wt% of developer into the gelatin solution, and stirring uniformly to obtain gelatin developing solution.

The preparation method of the o-carboxybenzoylated gelatin comprises the following steps: dissolving gelatin (natural Corii Sus Domestica gelatin) in water to obtain gelatin water solution with concentration of 3-5wt%, and adjusting pH of gelatin water solution to 8.5-9.5; then adding phthalic anhydride into the gelatin water solution under the stirring effect, and reacting for 1-2h at 40-50 ℃ to obtain a reaction solution; and purifying and freeze-drying to obtain the o-carboxybenzoylated gelatin. Wherein, the weight ratio of phthalic anhydride to gelatin is (3-7): 100.

(2) Emulsion crosslinking: gelatin developing solution and paraffin oil were mixed according to 1: (8-13) mixing at a volume ratio, and uniformly stirring at a rotation speed of 300-450 rpm; then dripping glutaraldehyde water solution with the concentration of 40-60wt% into the mixture for crosslinking for 2-10h; after the crosslinking is finished, cleaning the embolism developing microsphere by adopting normal hexane and purified water; and freeze-drying and screening to obtain the embolism developing microsphere.

(3) Loading coagulant: firstly, preparing thrombin solution with the concentration of 1200-1300U/mL; mixing the embolism developing microsphere obtained in the step (2) with thrombin solution, and dissolving each 100g of embolism developing microsphere into 0.3-0.6mL of thrombin solution; and freeze-drying to obtain the embolism developing particles loaded with the coagulant.

In this application, raw materials, reagents, solvents, and the like are all commercially available.

The present application is described in further detail below in connection with preparation examples, examples and performance test.

Preparation examples 1 to 3

Preparation examples 1-3 each provide an orthocarboxybenzoylated gelatin.

The above preparation example is different in that: the weight ratio of phthalic anhydride to gelatin in the process for preparing the carboxybenzoylated gelatin is shown in Table 1 below.

The preparation method of the o-carboxybenzoylated gelatin comprises the following steps: gelatin (natural pigskin gelatin, available from Wuhan polymeric Brilliant biotechnology Co., ltd.) is dissolved in water to prepare gelatin aqueous solution with concentration of 4wt%, and pH of gelatin aqueous solution is adjusted to 8.5; then adding phthalic anhydride into the gelatin water solution under the stirring effect, and reacting for 1h at 40 ℃ to obtain a reaction solution; and purifying and freeze-drying to obtain the o-carboxybenzoylated gelatin.

Comparative preparation examples 1-2

Preparation examples 1-2 each provide an orthocarboxybenzoylated gelatin.

TABLE 1 amounts and weight ratio of phthalic anhydride to gelatin in preparation examples 1-3 and comparative preparation examples 1-2

Examples 1 to 3

Examples 1-3 each provide embolic developing particles loaded with a coagulant.

The above-described embodiments differ in that: in the coagulant-supporting embolic developing particles of examples 1-3, the ortho-carboxybenzoylated gelatin was derived from preparation examples 1-3, respectively.

The preparation method of the coagulant-loaded embolic developing particles provided in examples 1-3 is as follows:

(1) Preparing gelatin developing solution: dissolving the o-carboxybenzoylated gelatin in purified water to prepare an o-carboxybenzoylated gelatin solution with the concentration of 8 wt%; then 10wt% of ferroferric oxide nanoparticles (purchased from the Siam Azimuth biotechnology Co., ltd.) of gelatin solution was added and stirred uniformly to obtain gelatin developing solution.

(2) Emulsion crosslinking: gelatin developing solution and paraffin oil were mixed according to 1:10, and uniformly stirring at a rotating speed of 350 rpm; then, glutaraldehyde aqueous solution with the concentration of 50 weight percent is dripped into the mixture for crosslinking for 5 hours; after the crosslinking is finished, cleaning the embolism developing microsphere by adopting normal hexane and purified water; and freeze-drying and screening to obtain the embolism developing microsphere.

(3) Loading coagulant: firstly preparing a thrombin solution with the concentration of 1250U/mL; then 100mg of the embolism developing microsphere obtained in the step (2) is mixed with 0.4mL of thrombin solution; and freeze-drying to obtain the embolism developing particles loaded with the coagulant.

Examples 4 to 7

Examples 4-7 each provide embolic developing particles loaded with a coagulant.

The above embodiment differs from embodiment 2 in that: (2) In the emulsion crosslinking step, the concentration of the o-carboxybenzoylated gelatin solution is shown in Table 2 below.

TABLE 2 concentration of the o-carboxybenzoylated gelatin solutions in example 2, examples 4-7

Examples	Concentration of the O-carboxybenzoylated gelatin solution (wt%)
		2	8
4	5
		5	10
6	15
		7	20

Examples 8 to 10

Examples 8-10 provide, respectively, embolic developing particles with a loading coagulant.

The above embodiment differs from embodiment 2 in that: (2) The stirring speed in the emulsion crosslinking step is specifically shown in table 3 below.

TABLE 3 stirring speeds for the emulsion crosslinking step in example 2, examples 8-10

Examples	Stirring rotation speed (rpm)
		2	350
8	300
		9	400
10	450

Comparative examples 1 to 2

Comparative examples 1-2 each provide an embolic developing particle carrying a coagulant.

The above comparative example is different from example 2 in that: in the coagulant-supporting embolic developing particles of comparative examples 1-2, the o-carboxybenzoylated gelatin was derived from comparative preparation examples 1-2, respectively.

Comparative example 3

Comparative example 3 provides a coagulant-loaded embolic developing particle.

The above comparative example is different from example 2 in that: the o-carboxybenzoylated gelatin was replaced with gelatin (available from shanxi brocade, open pharmaceutical excipients).

Comparative example 4

Comparative example 4 provides a coagulant-loaded embolic developing particle.

The above comparative example is different from example 2 in that: the o-carboxybenzoylated gelatin was replaced with a methacrylated gelatin (available from markanos technologies limited).

Comparative example 5

Comparative example 5 provides a coagulant-loaded embolic developing particle.

The above comparative example is different from example 2 in that: the o-carboxybenzoylated gelatin is replaced with succinic anhydride modified gelatin.

The preparation method of the succinic anhydride modified gelatin comprises the following steps: dissolving 30g of succinic anhydride in 1L of tetrahydrofuran solvent, suspending 300g of gelatin particles in the solution, adding 0.1g of nicotinamide and 0.1g of polyethylene glycol, controlling the reaction temperature to be 50 ℃, grinding in a colloid mill for 4 hours, and filtering to obtain succinic anhydride modified gelatin.

Performance test

The morphology, thrombin loading amount and in vitro coagulation time of the coagulant-loaded embolic developing particles obtained in examples 1 to 10 and comparative examples 1 to 4 were examined, and the results of the examination are shown in Table 4 below.

(1) The morphology of the coagulant-loaded embolic developing particles was observed under a microscope and the particle size was obtained.

The morphology of the coagulant-loaded embolic developing particles of example 1 under a microscope is shown in FIG. 1, and as can be seen from FIG. 1, the coagulant-loaded embolic developing particles are uniform in size and structure and have a particle size in the range of 70-200. Mu.m.

(2) The porosity detection method comprises the following steps: the porosity of the embolic developing microspheres (unsupported thrombin) was measured using mercury intrusion.

(3) Water absorption multiple: precisely weigh M ₁ 100mg embolic developing microspheres (no thrombin loaded), immersed in a beaker containing 20±1 ℃ water, stirred until the microspheres are completely wetted and all air is expelled, after sufficient water is absorbed; filtering out excessive water in the beaker, weighing the plug developing microsphere after water absorption again, and marking as M ₂ The water absorption multiple A is calculated, and the calculation formula is as follows:

A＝(M ₂ -M ₁ )/M ₁ 。

(4) Thrombin loading: standing the mixed solution of the embolism developing microsphere and thrombin in the step (3) of each example and the comparative example, and performing physical adsorption; then the concentration C of thrombin in the solution was measured at 4 hours after mixing ₁ The thrombin loading Q was calculated as follows:

Q＝(C ₀ -C ₁ )×0.1

TABLE 4 detection results of the coagulant-supporting embolic developing particles of examples 1-10, comparative examples 1-4

As can be seen from the results of examination in Table 4, the plug development microspheres obtained in examples 1 to 10 of the present application had a porosity of 90% or more, and were capable of absorbing water up to 38.2 to 44.1 times their own weight, and 100mg was capable of supporting thrombin 252 to 482U. Whereas the thrombin loading of the embolic developing microspheres obtained with gelatin in comparative example 3, methacrylated gelatin in comparative example 4, and succinic anhydride modified gelatin in comparative example 5 was only 92-166U/100mg. Therefore, the application shows that the plug developing microsphere with higher porosity and water absorption can be prepared by utilizing the o-carboxybenzoylated gelatin, and further the plug developing particle with high thrombin loading can be obtained.

The test results of examples 1-3 and comparative examples 1-2 show that the porosity of the embolism development microsphere is not greatly changed along with the increase of the phthalic anhydride addition amount in the preparation method of the o-carboxybenzoylated gelatin, the water absorption rate is obviously increased and then basically kept unchanged, and the thrombin load is firstly increased and then reduced. The thrombin loading of the embolic developing microspheres obtained in examples 1-3 was 263-406U/100mg, and the thrombin loading of the embolic developing microspheres obtained in comparative examples 1-2 was only 108-150U/100mg. Therefore, the weight ratio of phthalic anhydride to gelatin is controlled to be (3-7): in the range of 100, embolic developing particles with proper porosity, excellent hydrophilicity and high thrombin loading can be obtained.

The results of examples 2 and 4-7 show that as the concentration of the o-carboxybenzoylated gelatin solution increases, the porosity of the embolic developing microspheres gradually decreases, while the water absorption gradually increases, and the thrombin loading tends to increase and then decrease. Further comparison shows that the thrombin loading of the embolic developing microspheres obtained in example 2 and examples 5-6 is > 400U/100mg, while the thrombin loading of the embolic developing microspheres obtained in example 4 and example 7 is < 400U/100mg. Thus, it is demonstrated that the present application further controls the concentration of the o-carboxybenzoylated gelatin solution in the range of 8-15wt% to enable the embolic developing particles to be obtained with high thrombin loading.

The test results of examples 2 and 8-10 show that the porosity of the plug developing microsphere remains substantially unchanged with increasing stirring speed in the emulsification crosslinking step, while the water absorption rate shows a gradual increase trend, and the thrombin loading amount shows a trend of increasing and then decreasing. Especially, the thrombin loading of the embolic developing microspheres obtained in example 2 and examples 9-10 can reach more than 400U/100mg. Therefore, the method has the advantage that the stirring rotation speed in the emulsification and crosslinking step is controlled between 350 and 450rpm, so that the embolic developing particles with higher thrombin loading can be obtained.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A coagulant-loaded embolic developing particle, wherein the coagulant-loaded embolic developing particle comprises an o-carboxybenzoylated gelatin, a developer, and a coagulant.

2. The coagulant-loaded embolic developing particle of claim 1, wherein said o-carboxybenzoylated gelatin is prepared by the process of: dissolving gelatin in water to obtain gelatin water solution with concentration of 3-5wt%, and regulating pH of gelatin water solution to 8.5-9.5; then adding phthalic anhydride into the gelatin water solution under the stirring effect, and reacting for 1-2h at 40-50 ℃ to obtain a reaction solution; and purifying and freeze-drying to obtain the o-carboxybenzoylated gelatin.

3. The accelerator-loaded embolic developing particle of claim 1, wherein the weight ratio of phthalic anhydride to gelatin is (3-7): 100.

4. a coagulant-loaded embolic developing particle according to claim 3, wherein the coagulant-loaded embolic developing particle has a particle size of 70-1300 μm.

5. A method of preparing the coagulant-loaded embolic developing particles according to any of claims 1-4, comprising the steps of: preparing gelatin developing solution, emulsifying and crosslinking, and loading coagulant;

6. The method for preparing coagulant-supported embolic developing particles according to claim 5, wherein the concentration of the o-carboxybenzoylated gelatin solution is 8-15wt%.

7. The method of preparing coagulant-loaded embolic developing particles of claim 5, wherein the volume ratio of the gelatin developing solution to the emulsifier is 1: (8-15).

8. The method for preparing coagulant-supported embolic developing particles according to claim 5, wherein the crosslinking agent is 40-60wt% glutaraldehyde aqueous solution; the crosslinking reaction time is 2-10h.

9. The method for preparing the coagulant-loaded embolic developing particles according to claim 5, wherein the concentration of the thrombin solution is 1200-1300U/mL.

10. The method for preparing the coagulant-loaded embolic developing particles of claim 9, wherein the embolic developing microspheres are used in an amount related to thrombin solution of: the embolic development microspheres were dissolved in 0.3-0.6mL of thrombin solution per 100g of embolic development microspheres.