A hydrogel for protecting biological specimens in pathological preparation and its application

Abstract

The present invention relates to a kind of hydrogel and application thereof for protecting biological specimens in pathological preparation, and belong to the field of medical hydrogel materials. The hydrogel of the present invention includes: the first precursor liquid contains a polymer that can be chemically cross-linked with formaldehyde, and contains a catalyst for catalyzing the chemical cross-linking reaction of formaldehyde, and the second precursor liquid is a formaldehyde solution; the functional group species contained in the polymer structure in the first precursor liquid is one or more of amino, hydroxyl, and sulfhydryl, and the number of functional groups contained in each molecule is greater than or equal to two; the concentration range of the second precursor liquid is 4% 20%. The hydrogel for protecting biological specimens in pathological preparation of the present invention utilizes the formalin soaking step in the specimen processing process to further cross-link liquid glue, so that it is solidified into a protective layer on the surface of biological tissue, with simple operation, the advantage of rapid gelation, and can reduce the damage of various physical and chemical factors to biological samples in conventional pathological preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of medical hydrogel materials, and in particular to a hydrogel for protecting biological specimens in pathological slide preparation, and a preparation method and application thereof. Background Art

[0002] Routine pathological sectioning of biological specimens is an important method for diagnosing diseases and conducting histopathological research. The quality of specimen preparation directly impacts the accuracy of disease diagnosis or research results. For example, in the diagnosis and treatment of oncology, interpretation of pathological sections is the gold standard for determining whether a tumor is benign or malignant, and is one of the primary criteria for distinguishing between various pathological subtypes and early and late stages. Clinicians use pathological results to arrive at a specific diagnosis and treatment plan based on this information. In other words, cancer treatment plans are highly dependent on pathological results, and the appropriateness of treatment plans for the patient's condition is crucial to their prognosis. Therefore, the accuracy of pathological section interpretation is crucial. However, in real-world clinical scenarios, the preparation process for biological specimens involves numerous chemical reagents and processing conditions, which can damage the biological specimens. This can lead to discrepancies between the biological specimens obtained by clinicians and those seen by pathologists. This discrepancy impacts the precise determination of treatment plans for patients and, to a certain extent, reduces their survival expectations. At the current stage, resolving this contradiction mainly relies on repeated communication between clinicians and pathologists and multidisciplinary discussions and collaboration. This approach is undoubtedly inefficient and increases the workload of both parties. Therefore, it becomes extremely critical to eliminate the differences in biological specimens seen by clinicians and pathologists.

[0003] As an emerging polymer material, hydrogels possess excellent biocompatibility, superior mechanical properties, and a vast range of cross-linking modification options. They have found applications in bone and joint repair, hemostasis and coagulation, and sustained drug release in vivo. Therefore, leveraging their excellent biocompatibility makes it possible to apply hydrogels to the preservation of biological specimens. However, most existing hydrogels have strict requirements for gelation conditions, such as temperature, pH, UV wavelength / duration, and enzyme catalysis, making them unsuitable for immediate preservation of biological tissues after ex vivo treatment. Furthermore, excessively acidic or alkaline chemical environments, different enzyme catalysis, and UV light exposure can also damage tissue cells. Existing hydrogels that can adhere to and protect immediately ex vivo biological specimens lack sufficient tissue adhesion and are easily detached. Furthermore, during routine pathology preparation, treatment with multiple chemical reagents, such as formalin solution, ethanol, xylene, paraffin, hematoxylin, and eosin, can significantly damage the hydrogels, with formalin being the most damaging. The standard pathology preparation process requires specimens to be fixed in formalin for 24 hours as soon as possible after removal. This lengthy immersion process places high demands on the hydrogel's mechanical properties and adhesion strength. Therefore, developing a hydrogel that can effectively protect immediately removed biological specimens from the various factors that damage them during conventional pathology preparation has significant clinical application value. Summary of the Invention

[0004] To overcome the above-mentioned deficiencies of the prior art, the present invention aims to provide a hydrogel for protecting biological specimens during pathological section preparation and its application, so as to solve the problem that the existing hydrogel system cannot form good adhesion on biological specimens immediately removed from the body, and overcome the destructive effects of various chemical reagents, mainly formalin solution, used in conventional pathological section preparation.

[0005] A hydrogel for protecting biological specimens in pathological slide preparation comprises a first precursor liquid and a second precursor liquid; the first precursor liquid contains polymers capable of chemically cross-linking with formaldehyde, each of the polymers having two or more functional groups capable of undergoing nucleophilic addition reactions with aldehyde groups, the functional groups being one or more of amino groups, hydroxyl groups, and thiol groups; the second precursor liquid is a formaldehyde solution; when in use, the first precursor liquid is applied to the surface of the biological specimen to be processed, and then the specimen is immersed in the second precursor liquid.

[0006] The polymer capable of chemically cross-linking with formaldehyde in the first precursor solution includes one or more of chitosan, carboxylated chitosan, hydroxyethyl acrylate, chitosan lactate, chitosan quaternary ammonium salt, polyquaternary ammonium salt, polyethyleneimine, polyvinyl alcohol, polyethylene glycol, and polythiol.

[0007] The first precursor liquid also contains a catalyst and a thickener; the catalyst includes acetic acid, dilute hydrochloric acid, and p-toluenesulfonic acid, which are used to catalyze the crosslinking of the first precursor liquid and the second precursor liquid; the thickener includes polyacrylic acid, gelatin, guar gum, agar, carboxymethyl cellulose, bentonite, and diatomaceous earth, which are used to adjust the fluidity of the first precursor liquid.

[0008] The polymer concentration range of the first precursor solution is 1%-50% by mass, the catalyst concentration range is 0-5% by mass, and the thickener concentration range is 0-50% by mass; the formaldehyde concentration range of the second precursor solution is 4%-20% by mass.

[0009] The first precursor solution contains chitosan quaternary ammonium salt and hydroxyethyl acrylate as polymers that react with formaldehyde.

[0010] Photoinitiator I2959 is used as a catalyst, and deionized water is used as a solvent; the second precursor solution includes a 4% neutral formalin solution.

[0011] The first precursor solution contains vinyl alcohol and polyacrylic acid as polymers that react with formaldehyde, glacial acetic acid as a catalyst, and deionized water as a solvent; the second precursor solution includes 4% neutral formalin solution.

[0012] The hydrogel takes formaldehyde to cross-link and solidify the glue layer for 30 seconds to 24 hours. The solidification time is regulated by adjusting the concentration of polymers or catalysts that can chemically cross-link with formaldehyde, or by adding polyacrylic acid, gelatin, guar gum, agar, carboxymethyl cellulose, bentonite, or diatomaceous earth thickeners.

[0013] A method for applying a hydrogel for protecting biological specimens in pathological slide preparation comprises the following steps:

[0014] 1) Dissolve a polymer capable of cross-linking with formaldehyde in deionized water and stir to prepare a first precursor solution;

[0015] 2) Take the formalin solution or formaldehyde solution required for processing biological specimens as the second precursor solution;

[0016] 3) Applying the first precursor liquid to the surface of the biological specimen to be processed and allowing the first precursor liquid to flow smoothly;

[0017] 4) The biological specimen with the surface coated with the first precursor liquid is immersed in the second precursor liquid. The formaldehyde contained in the second precursor liquid cross-links the polymer in the first precursor liquid, thereby solidifying the glue layer.

[0018] In step 1), a polymer capable of chemically cross-linking with formaldehyde is dissolved in deionized water together with a catalyst and a thickener. The time for the formaldehyde cross-linking and curing glue layer is between 30 seconds and 24 hours, which can be regulated by adjusting the concentration of the polymer capable of chemically cross-linking with formaldehyde or the concentration of the catalyst, or by adding polyacrylic acid, gelatin, guar gum, agar, carboxymethyl cellulose, bentonite, or diatomaceous earth thickeners.

[0019] The biological specimens include normal colon tissue, normal rectal tissue, small intestine tissue, stomach tissue, colon tumor tissue, rectal tumor tissue, and stomach tumor tissue.

[0020] Compared with the prior art, the present invention can achieve at least the following beneficial effects:

[0021] 1. The present invention utilizes a nucleophilic addition reaction. The amino, thiol, or hydroxyl groups of the formaldehyde-crosslinked polymer in the first precursor solution react rapidly with the formaldehyde in the second precursor solution under the action of a catalyst. The first precursor solution and the second precursor solution quickly gelate 30 seconds after contact, and are then immersed in a formalin solution for fixation. This solves the problem that biological samples need to be fixed as soon as possible after ex vivo to reduce tissue autolysis.

[0022] 2. The first hydrogel precursor solution of the present invention exists in a sol form. When not in contact with the second precursor solution, the system can remain stable. The fluidity of the system can be adjusted by adjusting the concentration of the polymer, so as to facilitate the formation of a protective layer with a certain thickness on the specimen surface, solving the problem of uniform distribution on the surface of the biological tissue sample.

[0023] 3. The hydrogel of the present invention uses a polymer capable of chemically cross-linking with formaldehyde as the first precursor liquid, which reacts with the formaldehyde molecules contained in the formalin solution during conventional pathology section preparation to form a gel. This transforms the destructive effect of the formalin solution on the hydrogel into a gel-promoting effect, forming an effective protective layer on the surface of the biological sample, thereby solving the problem of damage to the biological sample during conventional pathology section preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1a The hydrogel microstructure of Example 4 was observed using a laser confocal microscope.

[0025] Figure 1b This is the hydrogel microstructure of Comparative Example 1 observed under a laser confocal microscope.

[0026] Figure 2a This is a general picture of the hydrogel described in Example 4 after being coated on the surface of a biological sample.

[0027] Figure 2b This is a general picture of the hydrogel described in Comparative Example 1 after acting on the surface of a biological sample.

[0028] Figure 2c This is a general picture of a biological sample protected by anhydrous gel.

[0029] Figure 3a This is a picture taken under a microscope after the hydrogel described in Example 4 is coated on the surface of a biological sample and then prepared for routine pathological sectioning.

[0030] Figure 3b This is a picture taken under a microscope after the hydrogel described in Comparative Example 1 acts on the surface of a biological sample and is subjected to conventional pathological preparation.

[0031] Figure 3c This is a microscope image of a biological sample without hydrogel protection after routine pathological preparation. DETAILED DESCRIPTION

[0032] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] The present invention discloses a hydrogel for protecting biological specimens in conventional pathological slide preparation, wherein the formalin soaking step in the specimen processing process is utilized to strengthen cross-linking of liquid glue so that it is solidified into a protective layer on the surface of biological tissue. Wherein, the first precursor solution contains a polymer that can be chemically cross-linked with formaldehyde, and contains a catalyst that catalyzes the chemical cross-linking reaction of formaldehyde. When not in contact with the formalin solution, the glue liquid remains stable. The functional group types that can undergo nucleophilic addition reaction with aldehyde groups contained in the polymer structure in the first precursor solution are one or more of amino, hydroxyl, and sulfhydryl groups, and the number of functional groups contained in each molecule is greater than or equal to two. The polymer in the first precursor solution is one or more of chitosan, carboxylated chitosan, chitosan lactate, chitosan quaternary ammonium salt, polyquaternium salt, polyethyleneimine, polyvinyl alcohol, polyethylene glycol, protein, polythiol, etc. The polymer concentration range in the first precursor solution is 1%-50% by mass, the catalyst concentration range is 0-5% by mass, and the thickener concentration range is 0-50% by mass; the formaldehyde concentration range in the second precursor solution is 4%-20% by mass.

[0034] Optionally, a catalyst capable of catalyzing the chemical cross-linking reaction of formaldehyde includes but is not limited to acetic acid, dilute hydrochloric acid, p-toluenesulfonic acid, etc.

[0035] The first precursor liquid is applied on the surface of the tissue to be treated (such as the surface of colon cancer tissue). After the sol is leveled, the second precursor liquid is applied and sprayed onto the surface of the leveled first precursor liquid. It will initially gel in about 2 minutes. The tissue coated with glue is immersed in formalin solution. As the tissue is fixed, formaldehyde further cross-links the glue layer, increases the strength of the gel structure, and forms a strong protective layer on the tissue surface to protect the tissue from damage during the subsequent pathological preparation process.

[0036] Example 1

[0037] Prepare the first precursor solution: Weigh 1g of chitosan lactate, 1g of polyvinyl alcohol 2488, and 10g of deionized water into a glass bottle. Heat the bottle to 70°C on a stirring table and stir for 24 hours to obtain a uniform, transparent sol. Place the sol in a sealed bottle with a lid and store at room temperature.

[0038] Second precursor solution: 4% formaldehyde solution.

[0039] Apply the first precursor liquid to the surface of the biological sample and let it stand for 1 minute. After the sol slowly levels out, spray or apply the second precursor liquid to the surface and let it stand for 1 minute. After initial gelation, immerse the specimen coated with glue in formalin solution. The chitosan lactate in the gel is further cross-linked with the formaldehyde molecules, the gel layer solidifies, and a protective layer is formed on the surface of the specimen.

[0040] Example 2

[0041] Prepare the first precursor solution: Weigh 1g of polyvinyl alcohol 2488, 1g of polyacrylic acid, 0.2g of glacial acetic acid, and 10g of deionized water into a glass bottle. Heat the bottle to 70°C on a stirring table and stir for 24 hours to obtain a uniform, transparent sol. Place the sol in a sealed bottle with a lid and store at room temperature.

[0042] Second precursor solution: 4% formaldehyde solution.

[0043] Apply the first precursor liquid to the surface of the biological sample and let it stand for 1 minute. After the sol slowly levels out, spray or apply the second precursor liquid to the surface and let it stand for 1 minute. After initial gelation, immerse the specimen coated with glue in formalin solution. Under the catalysis of acetic acid, the polyvinyl alcohol 2488 in the gel is further cross-linked with the formaldehyde molecules, the gel layer solidifies, and a protective layer is formed on the surface of the specimen.

[0044] Example 3

[0045] Prepare the first precursor solution: Weigh 1g of bismercapto polyethylene glycol, 1.5g of carboxymethyl cellulose, and 10g of deionized water into a glass bottle. Place the bottle on a stirring table and stir for 24 hours to obtain a uniform, transparent glue. Place the glue in a sealed bottle with a lid and store at room temperature.

[0046] Second precursor solution: 4% formaldehyde solution.

[0047] Apply the first precursor liquid to the surface of the biological sample and let it stand for 1 minute. After the sol slowly levels out, spray or apply the second precursor liquid to the surface and let it stand for 1 minute. After initial gelation, immerse the specimen coated with glue in formalin solution. The dithiol polyethylene glycol in the gel is further cross-linked with the formaldehyde molecules, the gel layer solidifies, and a protective layer is formed on the specimen surface.

[0048] Example 4

[0049] Preparation of the first precursor solution: Weigh 1g of chitosan quaternary ammonium salt and 1.5g of hydroxyethyl acrylate, 0.02g of photoinitiator I2959, and 10g of deionized water, and add them to a glass bottle. Place the glass bottle on a stirring table and stir for 24 hours to obtain a uniform and transparent chitosan quaternary ammonium salt solution. Add the above-mentioned mass of hydroxyethyl acrylate and photoinitiator I2959 to the chitosan quaternary ammonium salt solution and stir evenly on a stirring table. Place the solution in a UV curing box for UV exposure, and under the initiation of photoinitiator I2959, hydroxyethyl acrylate linearly polymerizes. Take out the solution every 30s, observe its viscosity and fluidity, until the viscosity of the solution no longer increases significantly, and obtain a sol. Figure 1a Shown is the hydrogel microstructure observed by laser confocal microscopy.

[0050] Second precursor solution: 4% formaldehyde solution.

[0051] The first precursor liquid is applied to the surface of the biological sample and allowed to stand for 1 minute. After the sol is slowly leveled, the second precursor liquid is sprayed or applied to the surface and allowed to stand for 1 minute. After initial gelation, the specimen coated with glue is immersed in formalin solution. The chitosan quaternary ammonium salt in the gel is further cross-linked with the formaldehyde molecules, the gel layer solidifies, and a protective layer is formed on the surface of the specimen. The performance of the gel applied to the colon cancer specimen is as follows Figure 2a As shown, the biological samples without hydrogel protection are roughly as Figure 2c As shown; after routine pathological preparation, the appearance under the microscope is as follows Figure 3a shown.

[0052] Comparative Example 1

[0053] Powder phase: Weigh 0.1 g of sodium hyaluronate with a molecular weight of 200,000 and 0.1 g of chitosan lactate, put the two powders into a mortar and mix thoroughly for later use.

[0054] Liquid phase: Weigh 0.1 g of glacial acetic acid and 9.9 g of deionized water, add them to a glass bottle, stir and mix them to prepare a uniform solution for later use.

[0055] The powder phase is sprayed on the surface of the biological tissue, and then the liquid phase is sprayed. The powder meets the liquid phase to wet, dissolve, and cross-link, forming a gel layer on the surface of the biological tissue. Figure 1b The following is the hydrogel microstructure observed by laser confocal microscopy after the powder-liquid phase reaction. The gel was applied to the colon cancer specimen as shown in Figure 2b As shown, after routine pathological preparation, the appearance under the microscope is as follows Figure 3b shown.

[0056] Comparative Example 2

[0057] First precursor solution: Weigh 3 g of N,N-dimethylacrylamide, 10 mg of cross-linker N,N-methylenebisacrylamide, 5 g of deionized water, and 0.6 g of tetramethylethylenediamine, add them to a glass bottle and mix well.

[0058] Second precursor solution: Weigh 0.4 g of ammonium persulfate and 9.6 g of deionized water, add them into a glass bottle and mix well.

[0059] The first precursor liquid and the second precursor liquid are sprayed on the surface of the biological tissue, tetramethylethylenediamine and ammonium persulfate synergistically initiate polymerization, and the precursor liquids become gels.

[0060] The hydrogel of Example 4 above is used to protect the serosal cells of colon cancer tissue samples from being damaged and falling off, so as to clearly diagnose the specific depth of tumor infiltration. The obtained colon samples were divided into three groups. Group A used the hydrogel of Example 4, Group B used the gel described in Comparative Example 1, and Group C did not use any gel and the first precursor solution prepared as described in Example 4. The hydrogel of Group A was initially gelled on the surface of the serosal layer of colon cancer tissue after standing for 1 minute, and the hydrogel of Group B was gelled after standing for 5 minutes. The three groups of colon tissue samples were then immersed and fixed in formalin solution, and subsequent routine pathological preparation was performed. The three groups of samples were observed under a microscope, and it was seen that the colon tissue of Group A was well attached to the hydrogel (such as Figure 3a ), the serosal cells were intact and not shed, and the colon tissue in group B was not tightly attached to the hydrogel (e.g. Figure 3b ), hydrogel and serous layer cells were shed to varying degrees, and obvious serous layer cell shed was observed in the colon tissue of group C (e.g. Figure 3c ).

[0061] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that a person skilled in the art would be able to make numerous modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A hydrogel for protecting biological specimens in pathological preparation, characterized in that: include a first precursor solution and a second precursor solution; The first precursor solution contains polymers that can be chemically cross-linked with formaldehyde, each of which has two or more functional groups that can undergo nucleophilic addition reactions with aldehyde groups, and the functional groups are one or more of amino, hydroxyl, and thiol groups; The second precursor liquid is a formaldehyde solution; When in use, the first precursor liquid is applied to the surface of the biological specimen to be processed; then it is immersed in the second precursor liquid; The polymer capable of chemically cross-linking with formaldehyde in the first precursor solution includes one or more of chitosan lactate, chitosan quaternary ammonium salt, and bis-mercapto polyethylene glycol.

2. The hydrogel according to claim 1, wherein The first precursor solution further contains a catalyst and a thickener; The catalyst includes acetic acid, dilute hydrochloric acid, and p-toluenesulfonic acid, which are used to catalyze the cross-linking of the first precursor liquid and the second precursor liquid; The thickener includes polyacrylic acid, gelatin, guar gum, agar, carboxymethyl cellulose, bentonite, and diatomaceous earth, and is used to adjust the fluidity of the first precursor liquid.

3. The hydrogel according to any one of claims 1 to 2, characterized in that The polymer concentration range of the first precursor solution is 1%-50% by mass, the catalyst concentration range is 0-5% by mass, and the thickener concentration range is 0-50% by mass; the formaldehyde concentration range of the second precursor solution is 4%-20% by mass.

4. The hydrogel according to claim 1, wherein The first precursor solution contains chitosan quaternary ammonium salt and hydroxyethyl acrylate as polymers that react with formaldehyde. Photoinitiator I2959 is used as a catalyst, and deionized water is used as a solvent; the second precursor solution includes a 4% neutral formalin solution.

5. The hydrogel according to claim 1, wherein The first precursor solution contains vinyl alcohol and polyacrylic acid as polymers that react with formaldehyde, glacial acetic acid as a catalyst, and deionized water as a solvent; the second precursor solution includes 4% neutral formalin solution.

6. The hydrogel according to claim 1, wherein The time for formaldehyde to cross-link and cure the glue layer is between 30 seconds and 24 hours. The curing time is regulated by adjusting the concentration of polymers or catalysts that can chemically cross-link with formaldehyde, or by adding polyacrylic acid, gelatin, guar gum, agar, carboxymethyl cellulose, bentonite, or diatomaceous earth thickeners.

7. A method for applying the hydrogel according to any one of claims 1-2, characterized in that: The following steps are involved: 1) Dissolve a polymer capable of cross-linking with formaldehyde in deionized water and stir to prepare a first precursor solution; 2) Take the formalin solution or formaldehyde solution required for processing biological specimens as the second precursor solution; 3) Applying the first precursor liquid to the surface of the biological specimen to be processed and allowing the first precursor liquid to flow smoothly; 4) The biological specimen with the surface coated with the first precursor liquid is immersed in the second precursor liquid. The formaldehyde contained in the second precursor liquid cross-links the polymer in the first precursor liquid, thereby solidifying the glue layer.

8. The application method according to claim 7, characterized in that: In step 1), a polymer capable of chemically cross-linking with formaldehyde is dissolved in deionized water together with a catalyst and a thickener. The time for the formaldehyde cross-linking and curing glue layer is between 30 seconds and 24 hours, which can be regulated by adjusting the concentration of the polymer capable of chemically cross-linking with formaldehyde or the concentration of the catalyst, or by adding polyacrylic acid, gelatin, guar gum, agar, carboxymethyl cellulose, bentonite, or diatomaceous earth thickeners.

9. The application method according to claim 8, characterized in that: The biological specimens include normal colon tissue, normal rectal tissue, small intestine tissue, stomach tissue, colon tumor tissue, rectal tumor tissue, and stomach tumor tissue.

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