A medical hydrogel containing ultrasonically detectable bubble microspheres and its preparation method

Abstract

This invention relates to the field of pharmaceutical preparation technology, specifically to a medical hydrogel containing ultrasonically detectable bubble microspheres and its preparation method. The hydrogel is formed by in-situ crosslinking of a polyethylene glycol precursor solution and a polyamine crosslinking agent solution containing bubble microspheres. The polyethylene glycol precursor solution consists of a multi-arm polyethylene glycol derivative and buffer solution A. Without affecting the gelling properties of the hydrogel, the provided medical hydrogel is clearly visible under ultrasound, enabling real-time observation of the hydrogel within the human body, and it can be completely degraded without residue.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, specifically to a medical hydrogel containing ultrasonically detectable bubble microspheres and its preparation method. Background Technology

[0002] Hydrogels are soft materials containing a large amount of water, obtained by cross-linking hydrophilic polymers. They possess excellent physicochemical properties and biological characteristics, including strong conformability, biodegradability, and good biocompatibility. Therefore, hydrogel products have attracted much attention in the biomedical field, including applications in drug delivery, isolation and protection, and vascular embolization.

[0003] However, hydrogels cannot be visualized by conventional ultrasound imaging after implantation, making it difficult for researchers to conveniently and effectively observe their location, shape, and degradation within the body. Although numerous studies have been conducted on improving the imaging properties of hydrogels, very few methods are currently effective. The main approach is to introduce materials with densities different from water to achieve ultrasound visualization, such as introducing water-insoluble imaging particles into the hydrogel. Chinese patent application (publication number CN116077744A) discloses an absorbable self-illuminating hydrogel, its preparation method, and its applications. The imaging function of the hydrogel originates from inorganic nanoparticles generated by the in-situ binding of anions and salt ions in the buffer solution component. However, this method has several problems, such as poor imaging effect, a time requirement for visualization after implantation, and changes in hydrogel properties. Pre-adding imaging particles to the hydrogel can give it better imaging performance; however, these particles generally have a high density, settle quickly, and their effectiveness is unstable. The most controversial aspect is that water-insoluble imaging particles, after hydrogel degradation, can remain in the body for a long time and may migrate, potentially leading to foreign body reactions and thrombosis. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a medical hydrogel containing ultrasonically detectable bubble microspheres and its preparation method. Without affecting the gelling properties of the hydrogel, the provided medical hydrogel is clearly visible under ultrasound, enabling real-time observation of the hydrogel within the human body, and it can be completely degraded without residue.

[0005] One aspect of this invention provides a medical hydrogel containing ultrasonically detectable bubble microspheres, which is formed by in-situ crosslinking of a polyethylene glycol precursor solution and a polyamine crosslinking agent solution containing bubble microspheres. The polyethylene glycol precursor solution is composed of a multi-arm polyethylene glycol derivative and buffer solution A.

[0006] As a preferred technical solution, the multi-arm polyethylene glycol derivative is selected from at least one of 3-arm polyethylene glycol derivatives, 4-arm polyethylene glycol derivatives, 6-arm polyethylene glycol derivatives, and 8-arm polyethylene glycol derivatives.

[0007] Preferably, the multi-arm polyethylene glycol derivative is an aldehyde-terminated multi-arm polyethylene glycol derivative, wherein the aldehyde group of the aldehyde-terminated multi-arm polyethylene glycol derivative is linked to the multi-arm polyethylene glycol through an ether bond, amide bond, urethane bond, imine bond or urea bond.

[0008] Preferably, the number-average molecular weight of the multi-arm polyethylene glycol derivative is 10 kDa to 20 kDa.

[0009] Preferably, the multi-arm polyethylene glycol derivative is a 4-arm polyethylene glycol with amide bonds and benzaldehyde groups at the end, and a number average molecular weight of 10 kDa.

[0010] As a preferred technical solution, the mass fraction of the multi-arm polyethylene glycol derivative in the polyethylene glycol precursor solution is 10-24%, preferably 10-20%.

[0011] As a preferred technical solution, the raw materials for preparing the polyamine crosslinking agent solution containing bubble microspheres include at least a polyamine polymer, serum albumin, and buffer solution B.

[0012] As a preferred technical solution, the polyamino polymer is composed of one or more of trilysine, polyethyleneimine, and polylysine, preferably a combination of polyethyleneimine and polylysine.

[0013] As a preferred technical solution, the mass fraction of the polyamine polymer in the polyamine crosslinking agent solution containing bubble microspheres is 9-17%, preferably 9.128-16.13%. Preferably, the concentration ratio of polyethyleneimine to polylysine is (4-7):(5-11).

[0014] The medical hydrogel provided by this invention is prepared by cross-linking and curing four-arm polyethylene glycol (PEG) with benzaldehyde-terminated amide bonds linked by amide groups with a number average molecular weight of 10 kDa with polyethyleneimine and polylysine. Further, the mass fraction of the multi-arm PEG derivative in the PEG precursor solution is controlled to be 10-24%. After mixing with a multi-amino polymer solution containing microspheres and containing 9-17% polyamino polymer, gelation occurs in a short time, ensuring an average gelation time of less than 10 seconds. Simultaneously, the cured hydrogel exhibits good stability and a low swelling rate. However, a low mass fraction of the polyamino polymer results in an excessively long gelation time, which cannot meet the requirements of practical applications.

[0015] As a preferred technical solution, the mass fraction of serum albumin in the polyamino crosslinking agent solution containing bubble microspheres is 10-30%, preferably 10-20%. The serum albumin is preferably bovine serum albumin.

[0016] To address current technological limitations, this invention prepares a multi-amino crosslinking agent solution containing bovine serum albumin (BSA) bubble microspheres by adding BSA to a crosslinking system. This solution is then mixed with a polyethylene glycol (PEG) precursor solution and cured via two-component in-situ crosslinking to form an ultrasound-detectable medical hydrogel. The low density of the gas allows the hydrogel to be clearly visualized under ultrasound. Furthermore, the inventors discovered that by controlling the mass fraction of BSA in the multi-amino crosslinking agent solution containing bubble microspheres to 10-20%, the hydrogel's gelling properties are maintained, it is completely degraded without residue, and its stability is enhanced. The inventors believe the reason for this is that the amino groups in the BSA shell of the bubble microspheres can chemically bond with multi-arm PEG derivatives, allowing the bubble microspheres to be stably loaded into the hydrogel through long-term chemical bonding and gradually released as the hydrogel degrades. This ensures the stability of the hydrogel's ultrasound imaging and its synchronization with the hydrogel's degradation, facilitating real-time observation of the hydrogel in the human body. Excessive BSA addition accelerates the degradation rate, while insufficient BSA addition affects the imaging performance.

[0017] As a preferred technical solution, the buffer solution A and buffer solution B are phosphate buffer or borax buffer with pH = 4 to 10; preferably, the buffer solution A is phosphate buffer with pH = 5.6 and the buffer solution B is borax buffer with pH = 9.2.

[0018] Another aspect of the present invention provides a medical hydrogel containing ultrasonically detectable bubble microspheres and a method for preparing the same, comprising at least the following steps:

[0019] (1) Dissolve the multi-arm polyethylene glycol derivative in buffer solution A to prepare a polyethylene glycol precursor solution;

[0020] (2) The polyethylene glycol precursor solution and the polyamino crosslinking agent solution containing bubble microspheres are mixed and injected by a dual syringe at a volume ratio.

[0021] Preferably, the preparation method of the polyamine crosslinking agent solution containing bubble microspheres is as follows: dissolve the polyamine polymer in buffer solution B to prepare a polyamine crosslinking agent solution base solution; control the temperature and dissolve serum albumin in the polyamine crosslinking agent solution base solution; after complete dissolution, simultaneously perform ultrasonic treatment and nitrogen gas treatment on the solution; immediately place it in a constant temperature water bath for low temperature treatment, then take it out and restore it to 20-30℃ to obtain the polyamine crosslinking agent solution containing bubble microspheres.

[0022] A further preferred embodiment of the preparation method of the medical hydrogel containing ultrasonically detectable bubble microspheres specifically includes the following steps:

[0023] S1: Dissolve the multi-arm polyethylene glycol derivative in phosphate buffer solution at pH 5.6 to prepare a polyethylene glycol precursor solution;

[0024] S2: Dissolve the polyamine polymer in a borax buffer solution with pH=9.2 to prepare a polyamine crosslinking agent solution base solution;

[0025] S3: Control the temperature, dissolve bovine serum albumin in the base solution of the polyamine crosslinking agent, and after complete dissolution, simultaneously perform ultrasonic treatment and nitrogen gas treatment on the solution. After the treatment, immediately place it in a constant temperature water bath for low temperature treatment, and then take it out and restore it to 20-30℃ to obtain a polyamine crosslinking agent solution containing bovine serum albumin bubble microspheres.

[0026] S4: The polyethylene glycol precursor solution and the polyamine crosslinking agent solution containing bubble microspheres are mixed and injected by a dual syringe at the specified volume ratio.

[0027] As a preferred technical solution, the temperature in step S3 is controlled at 55-65℃.

[0028] As a preferred technical solution, the ultrasonic treatment and nitrogen gas treatment in step S3 take 10-30 seconds and are performed 1-5 times.

[0029] As a preferred technical solution, the low-temperature treatment in step S3 is performed at a temperature of -5 to 0°C for a time of 40 to 60 minutes.

[0030] As a preferred technical solution, the volume ratio of the polyethylene glycol precursor solution to the polyamine crosslinking agent solution containing bubble microspheres is 1:1.

[0031] This invention, through a specific process, creatively introduces bovine serum albumin microspheres into a two-component gel system, solving many problems existing in water-insoluble imaging particle imaging systems. This makes the provided ultrasonic imaging hydrogel material a promising new material for the fields of medicine and tissue engineering, applicable to implantable medical devices, permanent embolization, artificial tissue scaffolds, etc., enabling clear ultrasonic imaging observation during and after implantation.

[0032] The third aspect of the present invention provides an application of a medical hydrogel containing ultrasound-detectable bubble microspheres, which can be used in implantable medical devices, permanent embolization, and artificial tissue scaffolds.

[0033] Beneficial effects

[0034] 1. This invention provides a medical hydrogel containing ultrasonically detectable bubble microspheres. Without affecting the gelling properties of the hydrogel, the provided medical hydrogel can be clearly visualized under ultrasound, enabling real-time observation of the hydrogel in the human body, and it can be completely degraded without residue.

[0035] 2. In this invention, by controlling the mass fraction of multi-arm polyethylene glycol derivatives in the polyethylene glycol precursor solution to be 10-24%, and mixing it with a multi-amino crosslinking agent solution with a mass fraction of 9-17% of the multi-amino polymer, gelation occurs in a short time. This ensures that the average gelation time is less than 10 seconds, while the cured hydrogel exhibits good stability and low swelling rate.

[0036] 3. This invention prepares a multi-amino crosslinking agent solution containing bovine serum albumin bubble microspheres by adding bovine serum albumin to a crosslinking system. After mixing with a polyethylene glycol precursor solution, the solution is cured by two-component in-situ crosslinking to form an ultrasound-detectable medical hydrogel. The low density of the gas allows the hydrogel to be clearly visible under ultrasound.

[0037] 4. This invention controls the mass fraction of serum albumin in the polyamino crosslinking agent solution to 10-20%, thereby achieving both the effect of not affecting the gelling properties of the hydrogel, complete degradation without residue, and also contributing to the stability of the hydrogel.

[0038] 5. This invention, through a specific process, creatively introduces bovine serum albumin bubble microspheres into a two-component gel system, solving many problems existing in the current water-insoluble imaging particle imaging system. This makes the provided ultrasonic imaging hydrogel material a promising new material for the fields of medicine and tissue engineering, applicable to implantable medical devices, permanent embolization, artificial tissue scaffolds, etc., enabling clear ultrasonic imaging observation during and after implantation. Attached Figure Description

[0039] Figure 1 This is a comparison chart showing the swelling rate changes over time for Examples 3, 1, 2, and 4 of the present invention and the control example.

[0040] Figure 2 This is a comparison chart showing the swelling rate changes over time for Examples 3, 5, and 6 of the present invention and the control example.

[0041] Figure 3 This is a comparison chart showing the swelling rate changes over time for Examples 3, 9, and 10 of the present invention and the control example.

[0042] Figure 4 The figures show the shape characterization of bovine serum albumin bubble microspheres under optical fiber microscopes at 10x and 20x magnification for Examples 3 and 9 of the present invention. In the figures, a is Example 3 (10x magnification), b is Example 3 (20x magnification), c is Example 9 (10x magnification), and d is Example 9 (20x magnification).

[0043] Figure 5The images show the ultrasound imaging effects of Embodiments 3 and 9 of the present invention and the control example, where the red circles indicate the implanted materials. In the images, a is the ultrasound imaging effect of the control example, b is the ultrasound imaging effect of Embodiment 3, and c is the ultrasound imaging effect of Embodiment 9. Detailed Implementation

[0044] Example 1

[0045] In one aspect, Embodiment 1 of the present invention provides a medical hydrogel containing ultrasonically detectable bubble microspheres, which is formed by in-situ crosslinking of a polyethylene glycol precursor solution and a polyamine crosslinking agent solution containing bubble microspheres. The polyethylene glycol precursor solution is composed of a multi-arm polyethylene glycol derivative and buffer solution A.

[0046] The multi-arm polyethylene glycol derivative is a 4-arm polyethylene glycol with amide bonds and benzaldehyde groups at the end, and has a number average molecular weight of 10 kDa (purchased from Beijing Jiankai Technology Co., Ltd.).

[0047] The mass fraction of the multi-arm polyethylene glycol derivative in the polyethylene glycol precursor solution is 10%.

[0048] The raw materials for preparing the polyamine crosslinking agent solution containing bubble microspheres include polyamine polymer, serum albumin, and buffer B solution.

[0049] The polyamine crosslinking agent solution containing bubble microspheres has a mass fraction of 16.13% for polyamine polymers, which are a combination of polylysine and polyethyleneimine, with a concentration ratio of 6.078:10.052 for polyethyleneimine and polylysine.

[0050] The mass fraction of serum albumin in the polyamino crosslinking agent solution containing bubble microspheres is 20%. The serum albumin is bovine serum albumin.

[0051] Buffer A is a phosphate buffer solution with pH = 5.6; buffer B is a borax buffer solution with pH = 9.2.

[0052] Example 1 of the present invention provides a method for preparing a medical hydrogel containing ultrasonically detectable bubble microspheres, specifically including the following steps:

[0053] S1: Dissolve the polyethylene glycol derivative in 1 mL of buffer solution A to prepare a polyethylene glycol precursor solution;

[0054] S2: Dissolve the polyamine polymer in 1 mL of buffer solution B to prepare the polyamine crosslinking agent solution base solution;

[0055] S3: Control the temperature, dissolve bovine serum albumin in the base solution of the polyamine crosslinking agent, and after complete dissolution, simultaneously perform ultrasonic treatment and nitrogen gas treatment on the solution. After the treatment, immediately place it in a constant temperature water bath for low temperature treatment, and then take it out and restore it to 25°C to obtain a polyamine crosslinking agent solution containing bovine serum albumin bubble microspheres.

[0056] S4: The polyethylene glycol precursor solution and the polyamine crosslinking agent solution are mixed and injected by a dual syringe at the specified volume ratio.

[0057] In step S3, the temperature is controlled at 60°C; the ultrasonic treatment and nitrogen purging treatment in step S3 last for 15 seconds, and are performed once each.

[0058] In step S3, the low-temperature treatment is performed at -5°C for 45 minutes.

[0059] The volume ratio of the polyethylene glycol precursor solution to the polyamino crosslinking agent solution containing bovine serum albumin microspheres is 1:1.

[0060] Example 2

[0061] Example 2 of the present invention provides a medical hydrogel containing ultrasonically imaging bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of the multi-arm polyethylene glycol derivative in the polyethylene glycol precursor solution is 13%.

[0062] Example 3

[0063] Example 3 of the present invention provides a medical hydrogel containing ultrasonically imaging bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of the multi-arm polyethylene glycol derivative in the polyethylene glycol precursor solution is 20%.

[0064] Example 4

[0065] Example 4 of the present invention provides a medical hydrogel containing ultrasonically imaging bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of the multi-arm polyethylene glycol derivative in the polyethylene glycol precursor solution is 24%.

[0066] Example 5

[0067] Example 5 of the present invention provides a medical hydrogel containing ultrasonically imitative bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of the polyamine polymer in the polyamine crosslinking agent solution containing bubble microspheres is 13.153%, and the polyamine polymer is a combination of polylysine and polyethyleneimine, and the concentration ratio of polyethyleneimine to polylysine is 5.539:7.614.

[0068] Example 6

[0069] Example 6 of the present invention provides a medical hydrogel containing ultrasonically imitative bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of the polyamine polymer in the polyamine crosslinking agent solution containing bubble microspheres is 9.128%, and the polyamine polymer is a combination of polylysine and polyethyleneimine, and the concentration ratio of polyethyleneimine to polylysine is 4.052:5.076.

[0070] Example 7

[0071] Example 7 of the present invention provides a medical hydrogel containing ultrasonically imitative bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of the polyamine polymer in the polyamine crosslinking agent solution containing bubble microspheres is 8.065%, the polyamine polymer is a combination of polylysine and polyethyleneimine, and the concentration ratio of polyethyleneimine to polylysine is 3.039:5.026.

[0072] Example 8

[0073] Example 8 of the present invention provides a medical hydrogel containing ultrasonically imitative bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of the polyamine polymer in the polyamine crosslinking agent solution containing bubble microspheres is 6.515%, the polyamine polymer is a combination of polylysine and polyethyleneimine, and the concentration ratio of polyethyleneimine to polylysine is 2.076:4.439.

[0074] Example 9

[0075] Example 9 of the present invention provides a medical hydrogel containing ultrasonically detectable bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of serum albumin in the polyamino crosslinking agent solution containing bubble microspheres is 10%.

[0076] Example 10

[0077] Example 10 of the present invention provides a medical hydrogel containing ultrasonically imaging bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of serum albumin in the polyamino crosslinking agent solution containing bubble microspheres is 30%.

[0078] Example 11

[0079] Example 11 of the present invention provides a medical hydrogel containing ultrasonically detectable bubble microspheres and its preparation method. The specific implementation method is the same as that of Example 1, except that the mass fraction of serum albumin in the polyamino crosslinking agent solution containing bubble microspheres is 40%.

[0080] Comparison Example

[0081] The comparative example of the present invention provides a medical hydrogel containing ultrasonically imitative bubble microspheres and its preparation method. The specific implementation method is the same as that in Example 1, except that the mass fraction of serum albumin in the polyamino crosslinking agent solution containing bubble microspheres is 0%.

[0082] Performance testing methods

[0083] 1. Gel Formation Time: Take 0.6 mL and 2.5 mL vials of the polyethylene glycol precursor solution and the polyamine crosslinking agent solution containing bubble microspheres from the examples and control examples, respectively, and place them in a water bath at 37±0.5℃. After standing for 5 minutes, use a pipette to take 200 μL of the polyethylene glycol precursor solution into a 2.5 mL vial. Then, use a different pipette tip to take 200 μL of the polyamine crosslinking agent solution containing bubble microspheres and add it to the vial containing the polyethylene glycol precursor solution. Vibrate your wrist at a frequency of 3 times / s for 3 seconds, and then repeatedly tilt the vial at a frequency of 1 time / s until the liquid stops flowing. Start timing at the beginning of adding the polyethylene glycol precursor solution and stop timing when the liquid stops flowing. The timing duration is the gel formation time. Measure three groups of samples in parallel and take the average value. The results are recorded in Table 1.

[0084] Table 1

[0085]

[0086] 2. Degradation Performance: The hydrogels provided in the examples and control examples, excluding Examples 7, 8, and 11, were subjected to accelerated aging experiments (in vitro degradation simulation experiments, incubated in a 60°C constant temperature incubator). The time for complete degradation of the hydrogels was recorded, and the results are shown in Table 2. The data in Table 2 demonstrate that the complete degradation time of the provided hydrogel products varies from 7 to 14 days (60°C, 7 days is approximately equal to 37°C, 34 days; 60°C, 9 days is approximately equal to 37°C, 44 days; 60°C, 11 days is approximately equal to 37°C, 54 days; 60°C, 14 days is approximately equal to 37°C, 69 days), which can meet the needs of various clinical application scenarios for different degradation times.

[0087] Table 2

[0088]

[0089]

[0090] 3. Hydrogel swelling rate: The hydrogel swelling rate was calculated based on the hydrogel degradation performance data provided in the examples and control examples. The results are shown in [link to relevant documentation]. Figure 1 , Figure 2 , Figure 3 The comparison shows that the products provided in Examples 3 and 9 have better hydrogel stability and lower hydrogel swelling rate.

[0091] 4. The hydrogels provided in Examples 3 and 9 were observed under an optical microscope to determine the shape of the bovine serum albumin microspheres. The results are shown in [reference needed]. Figure 4 .

[0092] 5. The hydrogels provided in Examples 3 and 9 and the control example were implanted subcutaneously into rats and subjected to ultrasound imaging. The imaging results under ultrasound are shown in [reference needed]. Figure 5 .

Claims

1. A medical hydrogel containing ultrasonically detectable bubble microspheres, characterized in that, This product is formed by in-situ crosslinking of a polyethylene glycol (PEG) precursor solution and a polyamine crosslinking agent solution containing bubble-filled microspheres. The PEG precursor solution consists of a multi-arm PEG derivative and buffer solution A. The polyamine crosslinking agent solution containing bubble-filled microspheres is prepared from at least a polyamine polymer, bovine serum albumin, and buffer solution B. The multi-arm PEG derivative is a four-arm PEG derivative. Specifically, it is an aldehyde-terminated multi-arm PEG derivative, where the aldehyde group is linked to the multi-arm PEG via an amide bond. The number-average molecular weight of the multi-arm PEG derivative is 10 kDa ~ 20 kDa. kDa; the mass fraction of the multi-arm polyethylene glycol derivative in the polyethylene glycol precursor solution is 10-24%; the mass fraction of the polyamine polymer in the polyamine crosslinking agent solution containing bubble microspheres is 9-17%; the mass fraction of bovine serum albumin in the polyamine crosslinking agent solution containing bubble microspheres is 10-20%; the polyamine polymer is a composition of polyethyleneimine and polylysine, and the concentration ratio of polyethyleneimine to polylysine is (4-7):(5-11).

2. The medical hydrogel containing ultrasonically detectable bubble microspheres according to claim 1, characterized in that, The buffer solution A and buffer solution B are phosphate buffer or borax buffer with pH=4~10.

3. A method for preparing a medical hydrogel containing ultrasonically detectable bubble microspheres according to any one of claims 1-2, characterized in that, At least the following steps are included: (1) Dissolve the multi-arm polyethylene glycol derivative in buffer solution A to prepare a polyethylene glycol precursor solution; (2) The polyethylene glycol precursor solution and the polyamine crosslinking agent solution containing bubble microspheres are mixed and injected by a dual syringe at a volume ratio.

4. The method for preparing a medical hydrogel containing ultrasonically detectable bubble microspheres according to claim 3, characterized in that, The preparation method of the polyamine crosslinking agent solution containing bubble microspheres is as follows: dissolve the polyamine polymer in buffer solution B to prepare a polyamine crosslinking agent solution base solution; control the temperature and dissolve bovine serum albumin in the polyamine crosslinking agent solution base solution. After complete dissolution, simultaneously perform ultrasonic treatment and nitrogen gas treatment on the solution. After the treatment, immediately place it in a constant temperature water bath for low temperature treatment. Then remove it and restore it to 20-30℃ to obtain the polyamine crosslinking agent solution containing bubble microspheres.

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