A biomedical hydrogel and a biological tissue imaging method using the hydrogel

Abstract

This invention belongs to the field of new biomedical materials and relates to a biomedical hydrogel and a biological tissue imaging method using this hydrogel. The invention first involves using a small monomer of propylene oxyalkyl urea ester to penetrate into the tissue at low temperature. By initiating the homopolymerization of this monomer or copolymerization with a crosslinking agent, a polymer hydrogel is rapidly synthesized in situ within the biological tissue, quickly forming a uniformly embedded tissue sample. The embedded sample exhibits good mechanical properties and anti-swelling properties, is easy to cut, and the hydrogel also has good biocompatibility, resulting in excellent fluorescence retention. This makes it suitable for tissue section imaging applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedical imaging materials, specifically relating to a biomedical hydrogel and a biological tissue imaging method using the hydrogel. Background Technology

[0002] Biological tissue embedding materials are widely used in pathological diagnosis and modern biomedical research. Commonly used embedding materials include paraffin, resin, and agarose. However, these materials have drawbacks such as cumbersome preparation processes, unsuitable mechanical properties for cutting, poor biocompatibility, and poor fluorescence retention, limiting their application range. Furthermore, the large molecules do not easily penetrate into the tissue interior, making them merely surface gels that provide external support. Hydrogels, on the other hand, are small-molecule gels that can penetrate into the tissue before polymerization, providing internal support—a homogeneous gel—and exhibiting better biocompatibility compared to traditional embedding materials. Therefore, the preparation of novel high-strength supramolecular hydrogels and the design of simple supramolecular hydrogel preparation processes have significant application value. Supramolecular hydrogels with good mechanical properties and high fluorescence retention facilitate the cutting and preparation of biological tissues for subsequent observation and research. Simple preparation processes are beneficial for the large-scale preparation and widespread application of supramolecular hydrogels, especially for large-sample tissue embedding, where the amount of hydrogel material used is large and quality control requirements are high.

[0003] The desired hydrogels for tissue embedding should have convenient and rapid synthesis and polymerization processes, good mechanical properties, biocompatibility, and fluorescence retention. Most current embedding hydrogel systems form gel structures by forming hydrogen bonds between amide groups and water. However, these systems generally suffer from low hydrogel strength, require high-temperature gel preparation, and the synthesis of these monomers is cumbersome and time-consuming. Furthermore, the next step after tissue embedding involves sectioning, but the mechanical strength of existing amide-based embedding hydrogels is insufficient, and ease of cutting needs to be improved by increasing compressive strength. Additionally, some amide-based embedding hydrogels exhibit excessive swelling, which hinders the maintenance of a stable shape in aqueous solutions, negatively impacting microscopic observation of the samples.

[0004] Therefore, designing a hydrogel with a simple and rapid preparation method, low cost, high purity, good mechanical properties, good biocompatibility, and good fluorescence retention capability for application in the field of biological slide imaging is an urgent problem to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a biomedical hydrogel and a biological tissue imaging method using the hydrogel. By selecting a suitable hydrogel polymerization system and polymerization method, the mechanical strength, fluorescence ability, and section forming speed of the hydrogel are improved, while also enhancing the biological tissue imaging capability.

[0006] The present invention first provides a biomedical hydrogel, which comprises the following components: monomer, crosslinking agent and initiator, wherein the monomer is urea ethyl acrylate or a derivative thereof, and the hydrogel has multiple strong crosslinking points, wherein the strong crosslinking points are intermolecular hydrogen bonds formed by an amino group in one molecule of the monomer and a carbonyl oxygen atom in another molecule.

[0007] According to one embodiment of the present invention, the urea ethyl acrylate or its derivative thereof has the structural formula as shown in formula (a):

[0008]

[0009] Wherein, R1 is H or CH3, and R2 is CH2 or CH2CH2.

[0010] According to one embodiment of the present invention, the initiator is ammonium persulfate or azobisisobutyrazoline hydrochloride, the mass of the initiator is 0.05%-5% of the mass of the monomer, and the crosslinking agent is N,N'-methylenebisacrylamide (MBA) or N,N-methylbisacrylamide (EBA), the mass of the crosslinking agent is 0.01%-10% of the mass of the monomer.

[0011] According to another aspect of the present invention, the present invention also provides a method for biomedical tissue imaging, comprising the following steps:

[0012] S1: Pre-treat biological tissues;

[0013] S2: Mixing monomer, crosslinking agent, initiator and solvent to obtain a mixed solution, wherein the monomer is urea ethyl acrylate or a derivative thereof;

[0014] S3: The pretreated biological tissue is subjected to permeation treatment in the mixed solution;

[0015] S4: The monomer is polymerized in situ in the permeation-treated biological tissue to obtain a hydrogel. The hydrogel has multiple strong cross-linking points, which are intermolecular hydrogen bonds formed by an amino group in one molecule of the monomer and a carbonyl oxygen atom in another molecule.

[0016] S5: Take a section of biological tissue containing the above-mentioned hydrogel and image the section on an imaging system.

[0017] According to one embodiment of the present invention, the biomedical tissue in step S1 is a mouse brain. The pretreatment method is to fix the mouse brain in a fixative for 8-12 hours. After the mouse brain tissue is fixed, it is rinsed multiple times with PBS buffer to remove residual PFA in the mouse brain tissue for later use. The fixative is a 4% PFA solution, and the fixation temperature of the fixative is 4°C.

[0018] According to one embodiment of the present invention, the urea ethyl acrylate or its derivative thereof has the structural formula as shown in formula (a):

[0019]

[0020] Wherein, R1 is H or CH3, and R2 is CH2 or CH2CH2.

[0021] According to one embodiment of the present invention, in step S2, the monomer mass fraction in the mixed solution is 30%-70%, the initiator is ammonium persulfate or azobisisobutyrazoline hydrochloride, the initiator is 0.05%-5% of the monomer mass, and the crosslinking agent is N,N'-methylenebisacrylamide (MBA) or N,N-methylbisacrylamide (EBA), the crosslinking agent is 0.01%-10% of the monomer mass.

[0022] According to one embodiment of the present invention, the permeation treatment temperature in step S3 is 4-10°C, and the permeation treatment time is 6-12h.

[0023] According to one embodiment of the present invention, the reaction temperature of in-situ polymerization in step S4 is 15℃-45℃, and the polymerization time is 1min-24h.

[0024] According to one embodiment of the present invention, the compressive strength of the hydrogel in step S4 is 0.327-0.798 MPa, and the equilibrium swelling ratio in PBS solution is 129%-135%.

[0025] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0026] (1) This invention provides a supramolecular hydrogel polymer, which uses urea ethyl acrylate or its derivatives as the reaction monomer. Compared with traditional embedding hydrogels, it has better swelling ratio, biocompatibility and permeability. After polymerization under the polymerization conditions, it can form strong cross-linking points between hydrogel molecules to provide better mechanical support for imaging sections. It also has a certain fluorescence retention rate. Compared with the monomers used in other embedding hydrogels, the polymer monomers used in this invention are more convenient to synthesize, and a large amount of monomers can be obtained in 5-6 hours of reaction.

[0027] (2) The imaging method of the present invention involves permeating the pretreated hydrogel component into biological tissue and then heating it slightly to reach the hydrogel polymerization temperature. The temperatures required for biological tissue pretreatment, biological tissue permeation treatment, and hydrogel polymerization are not high, which is beneficial for the preservation of biological tissue. Furthermore, the in-situ polymerization method further ensures that the biological tissue is not damaged, resulting in clearer imaging results. In addition, due to the use of a unique hydrogel component, the permeation treatment time can be as low as 6 hours and the polymerization time can be as fast as 1 minute, which speeds up the preparation of biological tissue sections and indirectly speeds up the imaging speed of biological tissue. Attached Figure Description

[0028] Figure 1 This is a diagram of the hydrogel sample synthesized in Example 1 of this invention.

[0029] Figure 2 This is a compressive stress curve of the hydrogel synthesized in Examples 1-5 of the present invention, where the variable is monomer concentration (%) - crosslinking agent concentration (%).

[0030] Figure 3 These are cross-sectional images of the hydrogel synthesized in Example 1 of this invention. a is a cross-section during embedding, and b is a cross-section after embedding a mouse brain.

[0031] Figure 4 This is a swelling curve of the hydrogel synthesized in Example 1 of the present invention in deionized water and PBS buffer, respectively.

[0032] Figure 5 These are images of the hydrogel synthesized in Example 1 of this invention used for fluorescence imaging of mouse brain embedding, wherein (a) is an overall imaging image of a mouse brain slice, (b) is a local imaging image magnified 150 times, and (c) is an image of mouse brain embedded in hydrogel. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other. The following are specific embodiments:

[0034] Preparation of UA monomer

[0035] Two UA monomers were prepared using isocyanate acrylate and ammonia as raw materials. The synthetic routes are shown in formulas (II) and (III). The specific synthetic steps of formula (II) are as follows: A 7M ammonia methanol solution (400 mL, 17 g / mol) was diluted with 200 mL of dichloromethane (84.93 g / mol) in a 2000 mL single-necked round-bottom flask. Isocyanate acrylate (100 g, 708.57 mmol, 141.13 g / mol) diluted with 200 mL of dichloromethane was added under magnetic stirring at 10 °C. The reaction was carried out at 10 °C–45 °C. To maintain low-temperature reaction conditions, liquid nitrogen, alcohol, or ice packs could be used for cooling until all the liquid in the dropper was added. After the raw materials had reacted completely, most of the solvent was removed by rotary evaporation, and the residual solvent was further removed using an oil pump. The mixture was allowed to stand until a white solid precipitated. 400 mL of dichloromethane was added to dissolve the white solid, and then 600 mL of petroleum ether was added under stirring. The white powdery solid was filtered and dried in a 45°C drying oven to constant weight, yielding 100g of the final product, urea ethyl acrylate, with a yield of 97%.

[0036] The successful synthesis of the reaction was verified by proton nuclear magnetic resonance spectroscopy. The characteristic peaks are as follows: 1H NMR (600MHz, DMSO) δ 6.35 (s, 1H), 5.98 (s, 1H), 4.07 (s, 1H), 3.25 (s, 1H), 2.51 (s, 1H), 0.11–0.45 (m, 1H).

[0037]

[0038] Example 1

[0039] This embodiment provides an imaging method, including:

[0040] S1: Mouse brain tissue was pretreated by placing the mouse brain in a 4% PFA (paraformaldehyde fixative) solution at 4°C overnight. After the tissue was fixed, it was rinsed multiple times with PBS to remove residual PFA for later use.

[0041] S2: Mix the monomer, N,N'-methylenebisacrylamide (MBA), and ammonium persulfate (APS) from formula (II) above with deionized water to obtain a mixed solution;

[0042] S3: The pretreated mouse brain tissue is subjected to permeation treatment in the mixed solution at a temperature of 4°C for 12 hours.

[0043] S4: In-situ polymerization of the permeated mouse brain tissue in a mixed solution;

[0044] S5: The polymerized biomedical tissue slices are imaged on an imaging system.

[0045] In this embodiment, the monomer mass fraction in the mixed solution of step S2 is 50%, the initiator mass fraction is 0.75‰, and the crosslinking agent mass fraction is 3%.

[0046] In this embodiment, the operation method of step S3 is as follows: inject the mixture into the prepared EP material sealed mold with a syringe to avoid air bubbles, and keep it at 4°C for 12 hours to prevent polymerization, so that the UA monomer can diffuse fully to support the mouse brain.

[0047] In this embodiment, the operation method of S4 is as follows: An EP material sealed mold is placed at room temperature (20 degrees Celsius) for 1 hour to complete polymerization. Then, the mold is opened and the mouse brain tissue containing the hydrogel is removed, such as... Figure 1 .

[0048] Example 2

[0049] This embodiment provides an imaging method, including:

[0050] S1: Pre-treat mouse brain tissue by placing the mouse brain in a 4% PFA (paraformaldehyde fixative) solution at 4°C overnight. After the tissue is fixed, rinse it several times with PBS to remove residual PFA for later use.

[0051] S2: Combine the monomer, N-methylbisacrylamide (EBA), azobisisobutyrazoline hydrochloride (AIBI) from formula (II) above with deionized water to obtain a mixed solution;

[0052] S3: The pretreated mouse brain tissue is subjected to permeation treatment in the mixed solution at a temperature of 6°C for 10 hours.

[0053] S4: In-situ polymerization of the permeated mouse brain tissue in a mixed solution;

[0054] S5: The polymerized biomedical tissue slices are imaged on an imaging system.

[0055] In this embodiment, the monomer mass fraction, initiator mass fraction, and crosslinking agent mass fraction in the mixed solution in step S2 are 30%, 0.5‰, and 3%, respectively.

[0056] In this embodiment, the polymerization reaction temperature in step S4 is 15°C and the polymerization time is 20h.

[0057] Example 3

[0058] This embodiment provides an imaging method, including:

[0059] S1: Pre-treat mouse brain tissue by placing the mouse brain in a 4% PFA (paraformaldehyde fixative) solution at 4°C overnight. After the tissue is fixed, rinse it several times with PBS to remove residual PFA for later use.

[0060] S2: Mix the monomer, N-methylbisacrylamide (EBA), and ammonium persulfate (APS) from formula (II) above with deionized water to obtain a mixed solution;

[0061] S3: The pretreated mouse brain tissue is subjected to permeation treatment in the mixed solution at a temperature of 8°C for 8 hours.

[0062] S4: In-situ polymerization of the permeated mouse brain tissue in a mixed solution;

[0063] S5: The polymerized biomedical tissue slices are imaged on an imaging system.

[0064] In this embodiment, the monomer mass fraction, initiator mass fraction, and crosslinking agent mass fraction in the mixed solution in step S2 are 50%, 5‰, and 0.01%, respectively.

[0065] In this embodiment, the polymerization reaction temperature in step S4 is 45°C and the polymerization time is 1 min.

[0066] Example 4

[0067] This embodiment provides an imaging method, including:

[0068] S1: Pre-treat mouse brain tissue by placing the mouse brain in a 4% PFA (paraformaldehyde fixative) solution at 4°C overnight. After the tissue is fixed, rinse it several times with PBS to remove residual PFA for later use.

[0069] S2: Mix the monomer, N,N'-methylenebisacrylamide (MBA), azobisisobutyrazoline hydrochloride (AIBI) from formula (III) above with deionized water to obtain a mixed solution;

[0070] S3: The pretreated mouse brain tissue is subjected to permeation treatment in the mixed solution at a temperature of 10°C for 6 hours.

[0071] S4: In-situ polymerization of the permeated mouse brain tissue in a mixed solution;

[0072] S5: The polymerized biomedical tissue slices are imaged on an imaging system.

[0073] In this embodiment, the monomer mass fraction, initiator mass fraction, and crosslinking agent mass fraction in the mixed solution in step S2 are 50%, 1‰, and 10%, respectively.

[0074] In this embodiment, the polymerization reaction temperature in step S4 is 25°C and the polymerization time is 1 hour.

[0075] Example 5

[0076] This embodiment provides an imaging method, including:

[0077] S1: Pre-treat mouse brain tissue by placing the mouse brain in a 4% PFA (paraformaldehyde fixative) solution at 4°C overnight. After the tissue is fixed, rinse it several times with PBS to remove residual PFA for later use.

[0078] S2: Mix the monomer, N,N'-methylenebisacrylamide (MBA), and ammonium persulfate (APS) from formula (III) above with deionized water to obtain a mixed solution;

[0079] S3: The pretreated mouse brain tissue is subjected to permeation treatment in the mixed solution at a temperature of 4°C for 12 hours.

[0080] S4: In-situ polymerization of the permeated mouse brain tissue in a mixed solution;

[0081] S5: The polymerized biomedical tissue slices are imaged on an imaging system.

[0082] In this embodiment, the monomer mass fraction, initiator mass fraction, and crosslinking agent mass fraction in the mixed solution in step S2 are 70%, 0.5‰, and 3%, respectively.

[0083] In this embodiment, the polymerization reaction temperature in step S4 is 35°C and the polymerization time is 24 hours.

[0084] Application Test Example 1

[0085] The hydrogels synthesized in Examples 1-5 were subjected to mechanical property tests. Considering that this invention is mainly used for tissue embedding and cutting, the hydrogels were primarily tested for compression and cutting properties. The polyurea acrylate hydrogels of Examples 1-5 were tested using the following method: using an electronic dynamic and static fatigue tester, the hydrogels were formed into cylinders with a diameter of 12 mm and a height of 8 mm, the compression rate was set to 2 mm / min, and the maximum compressive strain was set to 80%.

[0086] Table 1. Test data on the compressibility of hydrogels prepared in Examples 1-5

[0087] performance Compressive strength (MPa) Example 1 0.798 Example 2 0.583 Example 3 0.350 Example 4 0.327 Example 5 0.638

[0088] In summary, the highest compressive strength of this supramolecular hydrogel reached 0.798 MPa. Figure 2As shown, the Young's modulus of the hydrogel can be calculated to be 4.166 MPa.

[0089] The hydrogel synthesized in Example 1 was subjected to dicing performance testing. Using a vibratory microtome with a dicing speed of 1000 μm / s, hydrogel slices with thicknesses of 1000 μm and 500 μm were cut in a PBS buffer environment. The surfaces were relatively smooth and flat. Figure 3 (a) and Figure 3 As shown in (b).

[0090] Application Test Example 2

[0091] The swelling properties of the hydrogels synthesized in Examples 1-5 were tested using the following method, and the properties are as follows: Figure 4 As shown. The swelling ratio is defined as the ratio of the mass of the gel sample to its original mass at a certain moment. The same hydrogel was cut into two identical pieces and placed in sufficient deionized water and PBS buffer, respectively. The swelling curves are shown below. Figure 4 As shown, in deionized water, constant weight was achieved after 48 hours, with an equilibrium swelling ratio of approximately 120%; in PBS buffer, constant weight was achieved after 36 hours, with an equilibrium swelling ratio of approximately 130%. This indicates that the PUA hydrogels prepared in Examples 1-5 have good biocompatibility and are suitable for embedding biological tissues.

[0092] Table 2. Test data on the swelling properties of the hydrogels prepared in Examples 1-5

[0093] performance PBS equilibrium swelling ratio (%) Example 1 132 Example 2 135 Example 3 134 Example 4 129 Example 5 129

[0094] Application Test Example 3

[0095] A PUA hydrogel polymer solution was prepared according to the preparation conditions described in Example 1. A fluorescent mouse brain was placed in a mold, and the air in the mold was purged using a syringe. The prepared PUA hydrogel polymer solution was then injected into the mold, ensuring it was filled as completely as possible without air bubbles. After embedding, the mold was placed under low temperature conditions and allowed to stand for 12 hours to allow the hydrogel molecules to fully diffuse into the mouse brain. After 12 hours, the mold was removed and allowed to naturally solidify at room temperature. Once solidification was complete, the sample was removed, preserved in PBS solution, and sectioned using a vibratory microtome at a cutting speed of 1000 μm / s to a thickness of 100 μm. The resulting sections were placed on glass slides, and the fluorescent mouse brain was visualized and observed using a confocal microscope. Figure 5 (a) Figure 5 (b) and Figure 5 As shown in (c), the hydrogel maintains a certain fluorescence intensity in PBS solution, with a fluorescence retention rate of 62.20%-72.61%. The imaging effect is relatively clear, and dendrites and axons can be observed clearly after magnification of 150 times.

[0096] Table 3. Fluorescence retention test data of hydrogels prepared in Examples 1-5

[0097] performance Fluorescence retention rate (%) Example 1 72.61 Example 2 72.14 Example 3 65.96 Example 4 62.20 Example 5 71.76

[0098] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of bio-medical tissue imaging, characterized by, The method comprises the following steps: S1: Pretreatment of mouse brain tissue, the mouse brain is placed in a 4% paraformaldehyde fixing solution at 4℃ overnight, after the tissue is fixed, it is rinsed with PBS multiple times to remove residual paraformaldehyde in the tissue for standby; S2: mixing urea methacrylate ethyl ester, N, N'-methylene bisacrylamide, ammonium persulfate and deionized water to obtain a mixed solution; S3: the pretreated mouse brain tissue is subjected to penetration treatment in the mixed solution, the penetration treatment temperature is 4℃, and the treatment time is 12h; S4: the penetrated mouse brain tissue is subjected to in-situ polymerization in the mixed solution at 20 degrees for 1h; S5: the polymerized biomedical tissue is stored in a PBS solution, sliced by using a vibrating microtome, and imaged on an imaging system. In step S2, the mass fraction of urea methacrylate ethyl ester in the mixed solution is 50%, the mass fraction of the initiator is 0.75‰, and the mass fraction of N, N'-methylene bisacrylamide is 3%.

2. The method of claim 1, wherein, In step S4, the compression strength of the hydrogel is 0.798MPa, and the equilibrium swelling ratio in the PBS solution is 132%.

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