Catechol-functionalized gelatin microsphere and use thereof in preparation of product for inhibiting formation of subcutaneous fluid accumulation and promoting tissue repair

Abstract

The present disclosure relates to the technical field of biomaterials, and provides a catechol-functionalized gelatin microsphere and a use thereof in preparation of a product for inhibiting formation of subcutaneous fluid accumulation and promoting tissue repair. The catechol-functionalized gelatin microsphere designed and prepared in the present disclosure not only has wet tissue adhesion capability, but also has good liquid absorption performance and biodegradability. The results of cell experiments show that abundant interfaces of catechol-functionalized gelatin microspheres can promote cell adhesion and proliferation, and the loose structure of the microspheres can also promote cell migration. The results of chest wall defect repair experiments of New Zealand white rabbits show that the catechol-functionalized gelatin microspheres can not only inhibit formation of subcutaneous fluid accumulation, but also recruit autologous cells in situ and promote proliferation thereof and secretion of extracellular matrix (such as collagen). These results indicate that the catechol-functionalized gelatin microsphere material has clinical application potential of inhibiting the formation of subcutaneous fluid accumulation, and promoting subcutaneous tissue regeneration and wound healing.

Description

Catechol-functionalized gelatin microspheres and their application in the preparation of products for inhibiting subcutaneous fluid formation and promoting tissue repair

[0001] This application claims priority to Chinese Patent Application No. 202411513847.X, filed on October 29, 2024, entitled "Catechol Functionalized Gelatin Microspheres and Their Application in the Preparation of Products for Inhibiting Subcutaneous Fluid Formation and Promoting Tissue Repair", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biomaterials technology, and more specifically, to a catechol-functionalized gelatin microsphere and its application in the preparation of products for inhibiting subcutaneous fluid formation and promoting tissue repair. Background Technology

[0003] Breast cancer is the second leading cause of cancer death among women worldwide, accounting for approximately 31% of all female cancers. Surgical resection is currently the standard treatment. Due to the large area of ​​the free skin flap dissected during breast cancer surgery, a significant cavity is formed, and the superficial skin and subcutaneous tissue cannot adhere tightly, leading to subcutaneous effusion (seroma), which is the most common complication after mastectomy. Subcutaneous effusion can lead to more serious complications; especially in elderly patients and diabetic patients, whose ability to absorb fluid is poor. Recurrent subcutaneous effusion can increase the risk of infection, prolong hospital stays, delay wound healing, flap necrosis, delays in subsequent / adjuvant therapy, and even reoperation. Currently, closed negative pressure wound therapy is the standard operating procedure for postoperative wound care after breast cancer surgery.

[0004] In recent years, with the aging population and the increase in diabetic patients, postoperative care for breast cancer has received increasing attention and importance. Among these, preventing postoperative fluid accumulation and promoting subcutaneous wound healing have become current research hotspots.

[0005] Cellular tissue engineering aims to recruit autologous cells in situ using bioactive scaffold materials to achieve tissue regeneration and repair damaged tissue, and is currently the most promising tissue repair strategy. However, there are few reports on the application of acellular tissue engineering scaffold materials to inhibit post-mastectomy fluid accumulation and simultaneously promote subcutaneous wound healing. Public content

[0006] The purpose of this disclosure includes providing catechol-functionalized gelatin microspheres (ca-CGMSs) and their use in the preparation of products for inhibiting subcutaneous fluid formation and promoting tissue repair, so as to at least alleviate a technical problem existing in the prior art.

[0007] In order to achieve at least one of the above-mentioned objectives of this disclosure, the following technical solution is adopted:

[0008] This disclosure provides a catechin-functionalized gelatin microsphere, which is prepared by the following method:

[0009] (a) A mixed solution of gelatin and catechin-functionalized gelatin was stirred and dispersed in the oil phase, and then cooled in an ice-water bath to prepare physically cross-linked microspheres.

[0010] (b) The physically cross-linked microspheres obtained in step (a) are chemically cross-linked using a chemical cross-linking agent to obtain the catechol-functionalized gelatin microspheres.

[0011] Optionally, the catechin-functionalized gelatin includes gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid, and / or gelatin-3,4-dihydroxybenzoic acid.

[0012] Optionally, the concentration of the mixed solution of gelatin and catechin-functionalized gelatin is 8% to 30% (w / v).

[0013] Optionally, the mass ratio of gelatin to catechin-functionalized gelatin is 1:0.01 to 1:20.

[0014] Optionally, the dry particle size of the catechin-functionalized gelatin microspheres is 50-500 μm.

[0015] Optionally, the solvent of the crosslinking system is a pure organic solvent or a water / organic solvent mixture.

[0016] Optionally, the chemical crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N,N′-dicyclohexylcarbodiimide (DCC), genipin, formaldehyde, and glutaraldehyde.

[0017] This disclosure further provides the use of the above-mentioned catechol-functionalized gelatin microspheres in the preparation of products for inhibiting subcutaneous fluid formation and / or promoting tissue repair.

[0018] Optionally, the catechol-functionalized gelatin microspheres achieve tissue repair by promoting at least one of cell adhesion, proliferation, and migration.

[0019] Optionally, the catechol-functionalized gelatin microspheres achieve tissue repair by inhibiting fluid accumulation or absorbing fluid accumulation.

[0020] Optionally, the tissue repair includes subcutaneous wound healing and tissue repair after breast cancer surgery.

[0021] Compared with the prior art, the beneficial effects of this disclosure include:

[0022] Subcutaneous effusion (seroma) often forms after mastectomy. Poor absorption can lead to slow wound healing, tissue and organ infection, and even life-threatening conditions. This disclosure presents a catechol-functionalized gelatin microsphere material that not only possesses wet tissue adhesion ability but also exhibits good liquid absorption properties and biodegradability. Cell experiments show that the catechol-functionalized gelatin microspheres, due to their large specific surface area, can promote cell adhesion and proliferation, and their loose structure can also promote cell migration. Experiments on the repair of chest wall defects in rabbits show that the catechol-functionalized gelatin microspheres not only have a good ability to inhibit subcutaneous effusion formation but also can recruit autologous cells in situ and promote their proliferation and the secretion of extracellular matrix (such as collagen). These results indicate that the catechol-functionalized gelatin microsphere material has the ability to inhibit subcutaneous effusion formation and promote subcutaneous tissue regeneration and wound healing, demonstrating potential for clinical application. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 shows the synthesis process and 1H NMR characterization of gelatin-3,4-dihydroxyphenylacetic acid containing catechol groups provided in Example 1 of this disclosure.

[0025] Figure 2 shows the synthesis process and 1H NMR characterization of gelatin-dopamine containing catechol groups provided in Example 2 of this disclosure.

[0026] Figure 3 shows the appearance morphology and performance characterization results of ca-CGMSs provided in the experimental examples of this disclosure; where (A) ca-CGMSs powder; (B) scanning electron microscope (SEM) image of ca-CGMSs; (C) bright field micrograph of swollen ca-CGMSs; (DG) particle size distribution of ca-CGMSs; (H) swelling ratio of ca-CGMSs obtained with different crosslinking agent (EDC / NHS) concentrations; and (I) degradation curve of ca-CGMSs.

[0027] Figure 4 shows the results of viability and proliferation detection of L929 cells cultured on ca-CGMSs and fibrin glue (PFS) provided in the experimental examples of this disclosure; wherein, (A) live / dead cell staining of L929 cells in PFS and ca-CGMSs scaffolds obtained in Example 2; (B) live / dead cell staining of L929 cells after 5 days of culture in ca-CGMSs scaffolds obtained in Examples 4, 8 and 11; (C) proliferation of L929 cells in PFS and ca-CGMSs scaffolds obtained in Examples 2, 4 and 11, respectively. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0028] Figure 5 shows the F-actin staining results of L929 cells cultured on ca-CGMSs and PFS provided in this experimental example. The scale bar is 300 μm.

[0029] Figure 6 shows the migration detection results of L929 cells cultured on ca-CGMSs and PFS provided in this experimental example; (A) Crystal violet stained images of L929 cells that have crossed PFS or ca-CGMSs and entered the lower chamber of Transwell, with a scale bar of 300 μm; (B) Statistical analysis of the number of migrating cells. **** indicates p < 0.0001.

[0030] Figure 7 shows the results of constructing the New Zealand rabbit mammary excision model and quantifying the subcutaneous fluid volume provided in this experimental example; (A) actual operation diagram of the New Zealand rabbit mammary excision model; (B) photos of New Zealand rabbits during and after the operation; (C) statistical results of subcutaneous fluid volume, in which the total amount of fluid in each group was statistically analyzed, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0031] Figure 8 shows the statistical results of F-actin staining and DAPI staining of recruited cells 7 and 28 days after subcutaneous tissue defect repair provided in this experimental example; where (A) cell F-actin staining, scale bar is 300 μm; (B) cell nuclear quantitative statistical results; where ** indicates p < 0.01, **** indicates p < 0.0001.

[0032] Figure 9 shows the H&E and Masson staining of newly formed tissue during the subcutaneous tissue defect repair process provided in the experimental example of this disclosure. The scale bar is 300 μm.

[0033] Figure 10 shows Sirius red staining of newly formed tissue during the subcutaneous tissue defect repair process provided in the experimental example of this disclosure. The scale bar is 200 μm.

[0034] Figure 11 shows the immunostaining of type I collagen (Col1) in the newly formed tissue during the subcutaneous tissue defect repair process provided in the experimental example of this disclosure. The scale bar is 300 μm.

[0035] Figure 12 shows the comparison results of (A) gelatin microspheres containing catechol groups and (B) gelatin microspheres without catechol groups in subcutaneous wet tissue adhesion and tissue load-bearing capacity provided in the experimental examples of this disclosure. Detailed Implementation

[0036] Unless otherwise defined herein, the scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any event of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0037] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of this disclosure are generally performed according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature and laboratory procedures and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0038] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply.

[0039] The inventors of this disclosure, considering the enormous clinical application potential of acellular tissue engineering scaffold materials, noted that tissue-inducible scaffold materials have become a hot topic in current tissue engineering research. Gelatin, a natural polymer derived from the denaturation of animal structural collagen, possesses excellent biocompatibility, biodegradability, non-toxicity, and low immunogenicity. Gelatin microspheres are often incorporated into traditional three-dimensional scaffolds as cell or drug carriers. Experiments have shown that catechols can mediate the formation of a series of non-covalent or covalent bonds between materials and biological tissue surfaces, improving their wet tissue adhesion properties; moreover, catechol groups possess antibacterial, anti-inflammatory, and antioxidant functions. However, the application of catechol-functionalized gelatin microspheres in inhibiting subcutaneous fluid formation and promoting subcutaneous tissue repair has not yet been reported.

[0040] Based on this, the first aspect of this disclosure provides catechol-functionalized gelatin microspheres, which are prepared by the following method:

[0041] (a) A mixed solution of gelatin and catechin-functionalized gelatin was stirred and dispersed in the oil phase, and then cooled in an ice-water bath to prepare physically cross-linked microspheres.

[0042] (b) The physically cross-linked microspheres obtained in step (a) are chemically cross-linked using a chemical cross-linking agent to obtain the catechol-functionalized gelatin microspheres.

[0043] This disclosure discloses a catechol-functionalized gelatin microsphere material obtained by introducing catechol groups. This material not only possesses wet tissue adhesion ability but also exhibits good liquid absorption performance and biodegradability. Cell experiments show that the catechol-functionalized gelatin microspheres, due to their ultra-large specific surface area, can promote cell adhesion and proliferation, and their loose structure can also promote cell migration. Experiments on the repair of chest wall defects in rabbits show that the catechol-functionalized gelatin microspheres not only have a good ability to inhibit subcutaneous effusion formation but also can recruit autologous cells in situ and promote their proliferation and the secretion of extracellular matrix (such as collagen). These results indicate that the catechol-functionalized gelatin microsphere material has the ability to inhibit subcutaneous effusion formation and promote subcutaneous tissue regeneration and wound healing, demonstrating potential for clinical application.

[0044] In some optional embodiments, the catechol-functionalized gelatin includes gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid, and / or gelatin-3,4-dihydroxybenzoic acid; gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid, or gelatin-3,4-dihydroxybenzoic acid can be selected separately and mixed with gelatin, or any two or all of gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid, and gelatin-3,4-dihydroxybenzoic acid can be selected and mixed with gelatin-dopamine; when a mixture is selected, the ratio of gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid, and gelatin-3,4-dihydroxybenzoic acid is not limited.

[0045] In some alternative embodiments, the concentration of the mixed solution of gelatin and catechin-functionalized gelatin is 8% to 30% (w / v), for example, but not limited to 8%, 10%, 15%, 20%, 25% or 30%.

[0046] In some optional embodiments, the ratio of gelatin to catechol-functionalized gelatin is 1:0.01 to 1:20, for example, but not limited to 1:0.01, 1:1, 1:5, 1:10, 1:15, or 1:20. When the ratio of gelatin to catechol-functionalized gelatin is within the above range, increasing the amount of catechol-functionalized gelatin can promote the water absorption capacity and tissue adhesion properties of the resulting microspheres. However, if the amount of catechol-functionalized gelatin is too high, it will reduce the cross-linking density of the catechol-functionalized gelatin microspheres and accelerate degradation.

[0047] In some optional embodiments, the dry particle size of the catechol-functionalized gelatin microspheres is 20-550 μm, for example, but not limited to, 20 μm, 30 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, or 550 μm. When the particle size of the catechol-functionalized gelatin microspheres is within the above-mentioned range, the resulting microspheres can not only absorb tissue fluid and adhere to the tissue surface, but also rapidly recruit cells and promote cell proliferation.

[0048] In some optional embodiments, the solvent for the crosslinking system is a pure organic solvent or a water / organic solvent mixture. For example, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, methanol, acetone, etc., can be used as pure organic solvents, or 40% aqueous ethanol solution, 60% aqueous dimethyl sulfoxide solution, 90% aqueous acetone solution, etc., can be used as water / organic solvent mixtures.

[0049] Based on the properties of the catechol-functionalized gelatin microspheres provided in this disclosure, this disclosure also provides their use in the preparation of products for inhibiting subcutaneous fluid formation and / or promoting tissue repair.

[0050] In some alternative embodiments, the catechol-functionalized gelatin microspheres promote tissue repair by facilitating at least one of cell adhesion, proliferation, and migration.

[0051] Optionally, the catechol-functionalized gelatin microspheres promote tissue repair by inhibiting fluid accumulation or absorbing fluid accumulation.

[0052] Subcutaneous fluid accumulation (seroma) often occurs after mastectomy. Poor absorption can lead to slow wound healing, tissue and organ infection, and even endanger life. Utilizing the properties of the catechol-functionalized gelatin microspheres disclosed in this disclosure, subcutaneous wound healing and tissue repair after breast cancer surgery can be effectively promoted.

[0053] The present disclosure will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0054] Example 1

[0055] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, comprising the following steps:

[0056] Step 1: Synthesis of Catechol-functionalized gelatin macromonomers

[0057] Synthesis of 3,4-dihydroxyphenylacetic acid-modified gelatin macromonomer: 2 g of 3,4-dihydroxyphenylacetic acid was dissolved in 200 ml of PBS (pH=7) in a 40°C water bath. 2.0 g of EDC and 1.4 g of NHS were added, and the pH was adjusted to 4-6. After stirring for 30 minutes, 2 g of gelatin was added. After reacting for 36 hours, the precipitate was collected in ethanol, dialyzed in pure water for 3 days, and then lyophilized to obtain gelatin-3,4-dihydroxyphenylacetic acid. (1H NMR spectroscopy) 1 ¹H NMR characterization confirmed the successful synthesis of gelatin-3,4-dihydroxyphenylacetic acid containing catechol groups (Figure 1).

[0058] Step 2: Preparation of physically cross-linked gelatin microspheres

[0059] Catechol-functionalized gelatin microspheres were prepared using a water-in-oil method. Specifically, 1.5 g of gelatin and 1.5 g of catechin-functionalized gelatin were added to 20 ml of double-distilled water and dissolved in an oven at 40°C. The gelatin solution was then poured into 80 ml of a preheated 45°C mixed oil (paraffin oil: edible oil = 3:1) and stirred at 45°C for 30 min using a magnetic stirrer. The reaction mixture was then transferred to an ice / water bath (0°C) and stirred for another 30 min. The precipitate was then washed three times with 1,4-dioxane and acetone, followed by two washes with anhydrous ethanol to obtain physically cross-linked catechin-functionalized gelatin microspheres.

[0060] Step 3: Preparation of ca-CGMSs

[0061] Physically cross-linked catechol-functionalized gelatin microspheres (0.5 g) were placed in 30 mL of PBS / ethanol (3:7 v / v), and EDC (40 mM) and N-hydroxysuccinimide (NHS, 45 mM) were added. The mixture was then shaken for a certain period of time to obtain chemically cross-linked catechol-functionalized gelatin microspheres (ca-CGMSs). The microspheres were washed three times with double-distilled water and dried to obtain a light yellow powder (A in Figure 3), which was then stored at 4°C for later use.

[0062] Example 2

[0063] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 1 in that:

[0064] Step 1: Synthesis of Catechol-functionalized gelatin macromonomers

[0065] Synthesis of dopamine-modified gelatin macromonomer: Gelatin (3g) was dissolved in 200 ml of PBS (pH=7) in a 40℃ water bath. EDC (1.5g) and NHS (1g) were added, and the pH was adjusted to 4-6. After stirring for 30 minutes, dopamine hydrochloride (1.5g) was added. After reacting for 36 hours, the precipitate was collected in industrial ethanol, dialyzed in pure water for 3 days, and then lyophilized to obtain gelatin-dopamine. (1H NMR spectroscopy) 1 ¹H NMR characterization confirmed the successful synthesis of gelatin-dopamine containing catechol groups (Figure 2).

[0066] Example 3

[0067] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0068] In step 3, 88 mM EDC is used instead of 40 mM EDC, and 99 mM NHS is used instead of 45 mM NHS.

[0069] Example 4

[0070] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0071] In step 2, 3g of gelatin and 3g of catechin-functionalized gelatin are added to 20 ml of double-distilled water, so that the concentration of the mixed solution of gelatin and catechin-functionalized gelatin is 30% (w / v).

[0072] Example 5

[0073] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0074] In step 2, 2.5g of gelatin and 2.5g of catechin-functionalized gelatin are added to 20ml of double-distilled water, so that the concentration of the mixed solution of gelatin and catechin-functionalized gelatin is 25% (w / v).

[0075] Example 6

[0076] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0077] In step 2, 2g of gelatin and 2g of catechin-functionalized gelatin are added to 20ml of double-distilled water, so that the concentration of the mixed solution of gelatin and catechin-functionalized gelatin is 20% (w / v).

[0078] Example 7

[0079] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0080] In step 2, 0.8g of gelatin and 0.8g of catechin-functionalized gelatin are added to 20ml of double-distilled water, so that the concentration of the mixed solution of gelatin and catechin-functionalized gelatin is 8% (w / v).

[0081] Example 8

[0082] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0083] In step 2, 2.97g of gelatin and 0.0297g of catechin-functionalized gelatin are added to 20ml of double-distilled water, that is, the mass ratio of gelatin to catechin-functionalized gelatin is 1:0.01.

[0084] Example 9

[0085] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0086] In step 2, 1g of gelatin and 2g of catechin-functionalized gelatin are added to 20ml of double-distilled water, that is, the mass ratio of gelatin to catechin-functionalized gelatin is 1:2.

[0087] Example 10

[0088] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0089] In step 2, 0.273g of gelatin and 2.73g of catechin-functionalized gelatin are added to 20ml of double-distilled water, that is, the mass ratio of gelatin to catechin-functionalized gelatin is 1:10.

[0090] Example 11

[0091] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0092] In step 2, 0.143g of gelatin and 2.857g of catechin-functionalized gelatin are added to 20ml of double-distilled water, that is, the mass ratio of gelatin to catechin-functionalized gelatin is 1:20.

[0093] Example 12

[0094] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0095] In step 2, 0.097g of gelatin and 2.903g of catechin-functionalized gelatin are added to 20ml of double-distilled water, that is, the mass ratio of gelatin to catechin-functionalized gelatin is 1:30.

[0096] Example 13

[0097] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0098] In step 3, DCC is used instead of EDC.

[0099] Example 14

[0100] This embodiment provides a method for preparing catechin-functionalized gelatin microspheres, which differs from Example 2 in that:

[0101] In step 3, use genipin instead of EDC.

[0102] Comparative Example 1

[0103] This comparative example provides a method for preparing gelatin microspheres, which differs from Example 2 in that:

[0104] In step 2, 3g of gelatin is added to 20ml of double-distilled water to prepare catechin-free gelatin microspheres.

[0105] Example 1: Characterization of ca-CGMSs

[0106] Morphological observation: The morphology of ca-CGMSs after gold sputtering was observed by scanning electron microscopy (SEM).

[0107] After the dried ca-CGMSs were sputtered with gold, SEM observation revealed that the ca-CGMSs prepared in Example 2 were round particles with relatively smooth surfaces (B in Figure 3).

[0108] Particle size distribution and swelling rate: The ca-CGMSs prepared in Example 2 were immersed in PBS and their morphology was observed after 48 hours. Similar to SEM observation, they exhibited relatively regular round or elliptical shapes (C in Figure 3). Particle size measurements were performed on the dried ca-CGMSs prepared in Examples 2, 4, 7, and 11 (DG in Figure 3). It was found that the particle size decreased with increasing concentration of the macromolecular mixed solution; for example, the ca-CGMSs prepared in Example 2 had a particle size distribution of 50–350 μm, the ca-CGMSs prepared in Example 4 had a particle size distribution of 20–160 μm, and the ca-CGMSs prepared in Example 7 had a particle size distribution of 50–500 μm. Furthermore, it was found that the ca-CGMSs particle size increased with decreasing proportions of gelatin and catechol-functionalized gelatin in the mixed solution; for example, the ca-CGMSs prepared in Example 11 had a particle size distribution of 50–550 μm.

[0109] After the freshly prepared ca-CGMSs were completely swollen in water, the surface solution was carefully removed, and the wet weight (W) of the ca-CGMSs was obtained by weighing. s After drying, the dry weight (W) is obtained by weighing. d ), using W s / W d The swelling ratio was calculated, and it was found that the crosslinking agent concentration (EDC / NHS: 40 mM / 45 mM or 88 mM / 99 mM) and crosslinking time (6, 12, 24, or 48 hours) of ca-CGMSs had no significant effect on its swelling ratio (H in Figure 3). Subsequently, the effects of the type of catechol-functionalized gelatin, the concentration of the macromolecular mixed solution, and the ratio of gelatin to catechol-functionalized gelatin on the swelling ratio of ca-CGMSs were investigated. Table 1 shows that there was no statistically significant difference in the swelling ratio of ca-CGMSs prepared in Example 1 and Example 2, indicating that the type of catechol-functionalized gelatin, namely gelatin-3,4-dihydroxyphenylacetic acid and gelatin-dopamine, does not significantly affect the swelling ratio of ca-CGMSs. However, increasing the concentration of the macromolecular mixed solution significantly decreased the swelling ratio of ca-CGMSs. Moreover, decreasing the ratio of gelatin to catechol-functionalized gelatin increased the swelling ratio of ca-CGMSs. The swelling rates of the ca-CGMSs prepared in Examples 2, 13 and 14 showed no statistically significant difference, indicating that the commonly used crosslinking agent types (e.g., EDC, DCC, genipin) do not significantly affect the swelling rate of ca-CGMSs at higher concentrations (40 mM).

[0110] Table 1. Effects of catechin-functionalized gelatin type, macromolecular mixed solution concentration, and the ratio of gelatin to catechin-functionalized gelatin on the swelling rate of Ca-CGMSs.

[0111]

[0112] In vitro enzymatic hydrolysis: The ca-CGMSs (~150 mg) prepared in Example 2 were fully swollen in water and then weighed (W). o Place the sample in a 0.001% trypsin solution at 37°C (1 ml, w / v), carefully remove the surface solution at predetermined time points, and weigh (w / v). t ). Using W t / W o × 100% to calculate the remaining degradation rate at different times.

[0113] The results showed that fully swollen ca-CGMSs exhibited good degradability in 0.001% (w / v) trypsin solution at 37°C. The residual degradation rate was almost negatively linearly correlated with degradation time, and ca-CGMSs could be completely degraded in about 5 hours. The biodegradability of ca-CGMSs lays a good foundation for its use as a tissue repair scaffold material (I in Figure 3).

[0114] Experiment Example 2: Cell Experiment

[0115] Cell culture

[0116] 2×10 6 L929 mouse fibroblasts were seeded into 10-cm culture dishes, and 1640 complete culture medium (containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin) was added. The dishes were then placed in a 37 ℃ CO2 (5%) cell culture incubator. When the cells were in the logarithmic growth phase, they were digested with trypsin and counted.

[0117] Cell culture

[0118] Prepare a 1% (w / v) agarose solution and add 200 μL of agarose solution to each well of a 24-well plate, then freeze at 4°C. Resuspend 1 g of the ca-CGMSs prepared in Examples 2, 4, 8, and 11 in 1 mL of cell culture medium and seed 60 μL of ca-CGMS suspension into each well of a 24-well plate. Additionally, add 100 μL of fibrin glue (PFS) to each well of the 24-well plate according to the manufacturer's instructions, and allow it to solidify as a control group. Add 2.5 × 10⁻⁶ ppm of PFS to each well. 5 Cells and 1 mL of complete culture medium.

[0119] Dead / Live Cell Staining

[0120] The material / cell complex was transferred to a 24-well plate. After washing the complex three times with 1640 basal medium, 1 mL of staining solution (1640 basal medium containing 2 μM calcein-AM and 4 μM propidium iodide-PI) was added to each well. The plate was then incubated in a cell culture incubator at 37°C in the dark for 30 min. After soaking twice with 1640 basal medium, the cells were observed under a fluorescence microscope.

[0121] In the detection results, live cells and dead cells exhibited green and red fluorescence, respectively (AB in Figure 4). Staining observation revealed that cells adhered to the ca-CGMSs prepared in Example 2 one day after inoculation, displaying a normal polygonal shape, and the number of dead cells was relatively small. In the PFS group, most cells had adhered and extended, also exhibiting normal morphology, but the number of dead cells was significantly higher than in the ca-CGMSs group. These observations indicate that the ca-CGMSs obtained in Example 2 can provide a suitable microenvironment for the adhesion, extension, and survival of L929 fibroblasts. By day 3 of culture, compared to day 1, the live cell density on the ca-CGMSs obtained in Example 2 increased, and the number of dead cells was lower. Simultaneously, the live cell density on the PFS group also increased, but the number of dead cells remained higher than in the ca-CGMSs group (A in Figure 4). Similarly, compared to day 3, the cell density on both ca-CGMSs and PFS increased again after 5 days of culture, with the number of dead cells in the PFS group still significantly higher than in the ca-CGMSs group. These results indicate that, compared to PFS, the ca-CGMSs obtained in Example 2 not only provide a better microenvironment for the survival of L929 fibroblasts, promoting cell adhesion and extension, but also, due to their unique three-dimensional structure and larger specific surface area, provide more growth space for cell proliferation, thus promoting cell proliferation. Similar to the ca-CGMSs group obtained in Example 2, L929 fibroblasts also exhibited good adhesion and high survival rate in the ca-CGMS scaffolds prepared in Examples 4, 8, and 11 (Figure 4B).

[0122] Cell proliferation detection

[0123] The viability of L929 cells in the material / cell complex was detected using the PrestoBlue kit. PrestoBlue working solution was prepared according to the instructions. At predetermined time points, 1 mL of PrestoBlue working solution was used to replace the cell culture medium, and the cells were incubated at 37 °C in the dark for 30 min. Fluorescence was quantitatively analyzed at 560 / 590 nm (excitation / emission) using a microplate reader.

[0124] Using the quantitative fluorescence intensity of cells in the PFS group on day 1 as a reference (set to 1), the relative cell numbers of each group at different time points were obtained (C in Figure 4). In each group, the cell number showed an increasing trend from day 1 to day 3 and day 5, indicating that PFS, as well as the ca-CGMSs prepared in Examples 2, 4, 8, and 11, can promote cell proliferation. Simultaneously, at each time point, the cell number in the ca-CGMSs groups prepared in Examples 2, 4, 8, and 11 was significantly higher than that in the PFS group. These findings further demonstrate that ca-CGMSs can provide a favorable cell growth microenvironment for L929 fibroblast proliferation. Furthermore, after co-culturing with L929 fibroblasts for 5 days, the cell number in the ca-CGMSs group prepared in Example 4 was significantly higher than that in the ca-CGMSs groups prepared in Examples 2 and 11.

[0125] Phalloid peptide staining

[0126] To investigate cell adhesion to the material, the material / cell complex was stained with phalloidin in this experiment. At the predetermined time, the material / cell complex was transferred to a 1.5 ml centrifuge tube and fixed with 4% paraformaldehyde for 30 min. After washing three times with PBS, cells were permeabilized for 15 min with PBS containing 0.5% Triton X-100 and 3% (w / v) BSA, followed by three washes with PBS. After incubation in phalloidin working solution in the dark for 30 min, the cells were counterstained with DAPI for 30 seconds, washed three times with PBS, and then observed under a fluorescence microscope to examine cell distribution and morphology.

[0127] Experiments (Figure 5) showed that L929 cells expressed F-actin, a cytoskeletal microfilament, on both the ca-CGMSs and PFS obtained in Example 2, promoting their adhesion and extension; cells in both groups exhibited a flattened polygonal shape. Including nuclear staining, this experiment revealed that as the culture time increased to day 3, cells in the PFS group gradually became crowded, a phenomenon unfavorable for further cell proliferation. Cell density in the ca-CGMSs group also gradually increased, but there was still considerable space for cell growth. These observations further indicate that the ca-CGMSs scaffold can provide more growth space for cell adhesion and proliferation. Careful observation revealed that after 5 days of culture, most of the ca-CGMSs were "adheded" together by cells and the extracellular matrix, and the cells maintained their normal polygonal shape; however, cells in the PFS group could only grow on their surface. This example showed that most cells in the PFS group had become rounded, indicating that they might slowly undergo apoptosis. Therefore, compared to the PFS scaffold, ca-CGMSs can provide a better microenvironment for the adhesion, extension, and proliferation of L929 cells.

[0128] Cell migration detection

[0129] The migration of cells in a ca-CGMS scaffold was investigated using Transwell chambers. Transwell chambers were placed in 24-well plates, and either ca-CGMS or PFS were added. After the PFS solidified, L929 cell suspension (5 × 10⁻⁶ cells / well) was added to each chamber. 4 Add 200 μL of cells to the lower chamber, then add 600 μL of complete culture medium and incubate in a 37 °C cell culture incubator containing 5% CO2 for 24 h. Remove the upper chamber, gently wipe away the material and cells inside the lower chamber with a cotton swab, stain with 0.1% crystal violet, and observe under an optical microscope.

[0130] As shown in Figure 6, a large number of cells in the ca-CGMSs group obtained in Example 2 migrated and invaded the lower chamber through the ca-CGMSs scaffold, while no cell migration to the lower chamber was observed in the PFS group, indicating that ca-CGMSs can better promote the migration of L929 cells compared with PFS.

[0131] Experimental Example 3: In vivo experiment

[0132] Establishment and repair of a mastectomy model

[0133] Female New Zealand rabbits, weighing approximately 2 kg, were selected. All animal experimental procedures followed the regulations of the Ethics Committee of the Affiliated Hospital of Qingdao University. Experimental animals were randomly divided into a blank control group (with only a drainage tube) (n=6), a PFS group (n=6), and ca-CGMSs groups obtained in Examples 1, 2, 4, 8, 11, 12, or 14 (n=6). Anesthesia was achieved by intraperitoneal injection of sodium pentobarbital (3%, 40 mg / kg). Rabbits were fixed in a supine position, shaved, and disinfected with povidone-iodine. A midline incision was made from the jugular notch to the xiphoid process using a scalpel and surgical scissors. The right chest flap was raised and separated from the subcutaneous muscle tissue using hemostatic forceps, exposing the edges of the pectoralis major, axilla, clavicle, and latissimus dorsi muscles. The pectoralis major, pectoralis minor, subcutaneous fat, mammary glands, and lymph nodes were removed, resulting in a defect of approximately 5 cm × 3 cm. PFS or ca-CGMSs were implanted in the experimental groups, constructing a mammary gland resection and repair model (Figure 7, A and B). Each group had a drainage tube connected to a negative pressure bulb to collect the accumulated fluid, and the wound was closed using interrupted sutures. Postoperatively, the New Zealand rabbits were allowed to move freely in their cages, and the wound dehiscence and infection status were monitored daily, with the drainage fluid weighed. On 7 and 28 days postoperatively, the New Zealand rabbits were euthanized, and the defect area and surrounding tissues were collected for subsequent histological analysis.

[0134] The experiment showed that the amount of subcutaneous fluid in all groups gradually decreased over time after surgery. On days 1, 2, and 3 post-surgery, the amount of fluid collected in the blank control group was significantly greater than that in the ca-CGMSs and PFS groups prepared in each example (Table 2, Figure 7, C). For example, on day 1, the amount of subcutaneous fluid in the blank control group was 3.6 times that of the ca-CGMSs group obtained in Example 2 and 2.5 times that of the PFS group. By day 5, the volume of fluid collected in all groups had decreased significantly. At each time point, the average amount of fluid in the ca-CGMSs groups obtained in Examples 1, 2, 4, 8, 11, and 14 was less than that in the blank control group, the PFS group, and the ca-CGMSs group obtained in Example 12. Regarding the total amount of fluid collected over 5 days (Sum): 1) the ca-CGMSs groups and PFS groups were significantly lower than the blank control group; 2) the ca-CGMSs groups obtained in Examples 1, 2, 4, 8, 11, and 14 were also lower than the ca-CGMSs group and PFS group obtained in Example 12. In summary, a large amount of fluid will form in the cavity after subcutaneous tissue resection; and compared with the ca-CGMSs group and PFS group obtained in Example 12, the ca-CGMSs obtained in Examples 1, 2, 4, 8, 11 and 14 showed a better ability to inhibit the formation of subcutaneous fluid or absorb the fluid.

[0135] Table 2. Fluid accumulation at different time points and total fluid accumulation over 5 days for different treatment groups

[0136]

[0137] Histological staining

[0138] On postoperative days 7 and 28, full-thickness tissue samples were taken from the tissue defect and surrounding areas. The tissue specimens were fixed in 4% paraformaldehyde solution for 48 hours, then dehydrated, embedded in paraffin, and cut into 5 μm thick sections using a paraffin microtome, which were then attached to anti-detachment slides. After hydration, the tissue sections underwent hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and Sirius red staining. F-actin staining with rhodamine-labeled phalloidin was used to examine cell distribution. For immunofluorescence staining of type I collagen, hydrated sections were incubated in PBS solution containing 3% BSA and 0.1% Triton X-100 for 30 min to prevent nonspecific adsorption, followed by incubation in type I collagen primary antibody solution at room temperature for 60 min, washing twice with PBS (15 min each time), then incubating in 5 μg / mL IgG secondary antibody for 60 min, followed by incubation in 1 μg / mL DAPI solution for 5 min to counterstain cell nuclei, soaking twice in PBS (15 min each time), and observed under a fluorescence microscope (Ti2, NiKon).

[0139] As shown in Figure 8, at each time point, the F-actin and cell (nuclear) densities at the tissue defect site in the ca-CGMSs group obtained in Example 2 were significantly higher than those in the blank control and PFS groups. Seven days post-surgery, the cell (nuclear) density in the repair area of ​​the blank group was significantly lower than that in the PFS and ca-CGMSs groups, with the cell number decreasing in the order of ca-CGMSs group, PFS group, and blank group. The trend of F-actin was consistent with the trend of cell nuclei; and only in the ca-CGMSs group did the cell (F-actin) arrangement show a directional tendency. By day 28, the F-actin and cell (nuclear) densities in both the ca-CGMSs and PFS groups were significantly higher than those on day 7, and the F-actin and cell (nuclear) densities in the ca-CGMSs group were still significantly higher than those in the blank control and PFS groups (Figure 8, B), indicating that compared to the PFS and blank groups, the ca-CGMSs group had more autologous cells migrate to the defect site or promoted in situ cell proliferation. Furthermore, it was found that in the PFS group, F-actin and cells (nuclei) were mostly distributed at the defect edge, with fewer cells distributed inside. However, in the ca-CGMSs group, not only was the density of F-actin and cells (nuclei) higher, but the cells were also almost uniformly distributed throughout the defect site, and the cell arrangement was more directional. These results indicate that ca-CGMSs can promote the migration of more cells to the tissue defect site and provide a favorable cell proliferation microenvironment, which is beneficial to tissue defect repair.

[0140] In this experiment, H&E and Masson staining were performed on each group (Figure 9). Observation of the H&E staining results revealed significant differences in defect repair among the three groups 7 days post-surgery. The extracellular matrix (ECM) in the control group was diffusely distributed, with a significantly lower ECM density than the PFS and ca-CGMSs groups. The staining in the central portion of the PFS group may have originated partly from the PFS itself, and the ECM arrangement in the PFS group was disordered. In contrast, the ECM in the ca-CGMSs group was denser and more directional. Similarly, at 28 days post-surgery, the ECM in the defect repair area of ​​the ca-CGMSs group was denser than that of the PFS and control groups. This indicates that ca-CGMSs can provide a more ideal microenvironment for cell migration to the defect site, adhesion growth, and ECM secretion. Although PFS can adhere to the skin and subcutaneous tissue to some extent and inhibit fluid accumulation, its dense gel structure restricts cell migration to the defect site and also hinders new tissue formation. Through careful observation, this embodiment found that by day 28, the density of the ECM formed at the repaired defect site in the ca-CGMSs group was not significantly different from that of the ECM in the undamaged skin tissue, suggesting the formation of dense connective tissue at the defect site. To further investigate the type of collagen component in the newly formed ECM, Masson staining was performed. The ECM distribution was similar to the results observed with HE staining: at each time point, the ECM density in the ca-CGMSs group was significantly higher than that in the PFS group and the control group. The ECM staining was predominantly blue with a small amount of red, indicating that the newly formed ECM mainly consisted of collagen fibers, with a small amount of muscle fibers, further illustrating that the newly formed tissue was primarily connective tissue.

[0141] To differentiate the types of collagen fibers, the tissue was stained with Sirius red and observed using a polarized light microscope. As shown in Figure 10, the collagen fibers mostly appeared orange-yellow, indicating that the collagen component of the regenerated tissue was mainly type I collagen fibers. At both time points, the number of type I collagen fibers in the control group was significantly less than that in the PFS group, and they showed a scattered and disordered arrangement. The PFS group had less type I collagen fiber deposition compared to the ca-CGMSs group. The collagen fibers in the ca-CGMSs group were more densely arranged than the other two groups, and by day 28, the collagen fibers had filled the entire defect area.

[0142] Finally, immunohistochemistry was used to stain the main ECM component—type I collagen (Col1). As shown in Figure 11, similar to the histochemical staining results, 7 days post-surgery, the blank group had fewer cells in the defect area, and its secreted Col1A1 was lower than the other two groups. The ca-CGMSs group had the highest cell count, and its secreted Col1A1 was significantly higher than the other two groups. At 28 days post-surgery, compared to 7 days post-surgery, the cell count increased in all three groups, but the ca-CGMSs group had the highest Col1A1 density, essentially filling the defect area. These results further demonstrate that the ca-CGMSs scaffold material can not only promote cell proliferation but also promote Col1A1 secretion, thereby promoting in-situ repair of subcutaneous tissue defects.

[0143] As a reference group, this disclosure prepared gelatin microspheres without catechol groups according to Comparative Example 1 and implanted them into subcutaneous defects. As shown in Figure 12, the gelatin microspheres containing catechol groups can bond together well and can support the weight of subcutaneous tissue (Figure 12A). However, although the gelatin microspheres without catechol groups can absorb body fluids and aggregate together, their bonding is not tight and they cannot support the weight of subcutaneous tissue (Figure 12B).

[0144] It should be noted that all quantitative data in this embodiment were obtained through at least three experiments, and the data are presented as mean ± standard deviation (SD). A t-test was used to compare data between two groups. A p-value < 0.05 was considered statistically significant between the two groups. Industrial applicability

[0145] The catechol-functionalized gelatin microspheres designed and prepared in this disclosure have good liquid absorption properties, biocompatibility and biodegradability; they can not only effectively inhibit the formation of subcutaneous fluid, but also recruit autologous cells at tissue defects and promote their proliferation and secretion of extracellular matrix, thereby promoting subcutaneous tissue regeneration and wound healing, and have clinical application potential.

Claims

1. A type of catechin-functionalized gelatin microspheres, characterized in that, It is prepared by the following method: (a) A mixed solution of gelatin and catechin-functionalized gelatin was stirred and dispersed in the oil phase, and then cooled in an ice-water bath to prepare physically cross-linked microspheres. (b) The physically cross-linked microspheres obtained in step (a) are chemically cross-linked using a chemical cross-linking agent to obtain the catechol-functionalized gelatin microspheres.

2. The catechin-functionalized gelatin microspheres according to claim 1, characterized in that, The catechin-functionalized gelatin includes gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid, and / or gelatin-3,4-dihydroxybenzoic acid.

3. The catechin-functionalized gelatin microspheres according to claim 1 or 2, characterized in that, The concentration of the mixed solution of gelatin and catechin-functionalized gelatin is 8%~30% (w / v).

4. The catechin-functionalized gelatin microspheres according to claim 1 or 2, characterized in that, The mass ratio of gelatin to catechin-functionalized gelatin is 1:0.01 to 1:

20.

5. The catechin-functionalized gelatin microspheres according to claim 1 or 2, characterized in that, The dry particle size of the catechin-functionalized gelatin microspheres is 20~550 μm.

6. The catechin-functionalized gelatin microspheres according to claim 1, characterized in that, The chemical crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, N,N′-dicyclohexylcarbodiimide, genipin, formaldehyde, and glutaraldehyde.

7. The use of the catechol-functionalized gelatin microspheres according to any one of claims 1 to 6 in the preparation of products for inhibiting subcutaneous fluid formation and / or promoting tissue repair.

8. The application according to claim 7, characterized in that, The catechin-functionalized gelatin microspheres achieve tissue repair by promoting at least one of cell adhesion, proliferation, and migration.

9. The application according to claim 7, characterized in that, The catechin-functionalized gelatin microspheres promote tissue repair by inhibiting fluid accumulation or absorbing fluid accumulation.

10. The application according to any one of claims 7-9, characterized in that, The tissue repair includes wound healing and tissue regeneration after breast cancer surgery.

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