Beta-cyclodextrin modified gelatin microspheres, deferoxamine-loaded beta-cyclodextrin modified gelatin microspheres, and preparation methods and applications of beta-cyclodextrin modified gelatin microspheres and deferoxamine-loaded beta-cyclodextrin modified gelatin microspheres

Abstract

The invention provides a gelatin microsphere modified by beta-cyclodextrin, a gelatin microsphere modified by deferoxamine loaded beta-cyclodextrin as well as a preparation method and application of the gelatin microsphere, and relates to the technical field of biological materials. Beta-cyclodextrin modified gelatin microspheres with good thermal stability and deferoxamine (DFO) drug loaded beta-cyclodextrin modified gelatin microspheres are successfully designed and prepared by adopting specific materials. Cell experiment results show that the two compounds have good cell compatibility and can promote adhesion, proliferation and migration of cells. The slow release of DFO in the deferoxamine drug loaded beta-cyclodextrin modified gelatin microspheres can promote the vascularization effect of HUVECs cells. The results of rat abdominal wall defect repair experiments show that the microsphere scaffold provided by the invention has obvious advantages in promoting abdominal wall tissue reconstruction, and shows huge clinical application potential.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to beta-cyclodextrin-modified gelatin microspheres, beta-cyclodextrin-modified gelatin microspheres loaded with deferoxamine, and preparation methods and applications thereof. Background Art

[0002] Muscle tissue is indispensable for maintaining normal abdominal wall morphology and function. However, abdominal organ exposure caused by abdominal wall muscle tissue defects due to trauma, tumor resection, severe tissue infection or necrosis, congenital maldevelopment, etc. is a serious clinical problem. Due to the tension of the abdominal wall, abdominal wall defects cannot be sutured directly. Current repair methods include temporary suture technique, hernioplasty, and abdominal wall reconstruction. Temporary suture technique and hernioplasty are mainly used in emergency cases, while abdominal wall reconstruction techniques are used in most routine cases. Abdominal wall reconstruction is an effective method to repair defects using autologous tissue and non-biological material mesh, biological (biomaterials mesh) or composite patch. Autologous tissue repair has a small immune response, but it will cause new wounds, and the source of autologous tissue is limited.

[0003] Currently, the non-degradable patches commonly used in clinical practice include polypropylene mesh (PP)

[13] , polyethylene patch (polypropylene mesh), expanded polytetrafluoroethylene mesh (expanded polytetrafluoroethylene mesh), etc. Non-absorbable patches have high tensile strength, significantly reduce the recurrence rate of the repaired site, and restore the morphological integrity of the abdominal wall. However, non-absorbable meshes are strong and have no biological activity. They usually induce chronic foreign body reactions in tissues, leading to gradual fibrosis and a high risk of postoperative infection, abdominal pain, abdominal wall adhesions, intestinal obstruction, and other serious complications. Therefore, it is difficult to achieve biofunctional abdominal wall defect repair using non-degradable patches.

[0004] Biological patches usually refer to acellular matrix materials, such as small intestinal submucosa (SIS), pericardium (PC), acellular dermal matrix (ADM), etc. Biological patches have the advantages of being absorbable, low immunogenicity, and widely available. In addition, biological patches contain a certain amount of growth factors, such as fibroblast growth factor (FGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), etc., which can promote cell migration, aggregation and proliferation to the defect area, thereby promoting defect repair. Among them, SIS has been applied to the repair of various soft tissue defects. Biological patches have received increasing attention in basic clinical research. However, the rapid degradation rate of simple biological patches limits their clinical application. Their degradation will lead to a sharp decrease in mechanical properties, causing rupture (rupture), tympanites (tympanites) and other complications in the tissue repair area. In addition, some composite mesh materials constructed of non-degradable materials and bio-based materials are also used for abdominal wall defect repair. For example, Parietex composite material (PCO) meshes prevent visceral adhesions by coating a polyester mesh with an anti-adhesion collagen barrier. However, the inherent inflammatory response and lack of microstructure suitable for cell migration and growth of these polyester or PP-based composite meshes may lead to poor wound healing. In addition, the collagen-based barrier will swell and cause deformation of the composite mesh in the moist environment of the abdomen.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a β-cyclodextrin modified gelatin microsphere to at least solve one of the technical problems existing in the prior art.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned β-cyclodextrin modified gelatin microspheres.

[0008] The third object of the present invention is to provide β-cyclodextrin-modified gelatin microspheres loaded with deferoxamine.

[0009] A fourth object of the present invention is to provide a method for preparing the above-mentioned β-cyclodextrin-modified gelatin microspheres loaded with deferoxamine.

[0010] A fifth object of the present invention is to provide the application of the above-mentioned β-cyclodextrin modified gelatin microspheres and the β-cyclodextrin modified gelatin microspheres loaded with deferoxamine.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] The present invention provides a method for preparing β-cyclodextrin modified gelatin microspheres, comprising the following steps:

[0013] (a) stirring and dispersing an aqueous solution of β-cyclodextrin-modified gelatin and gelatin mixed into an oil phase, and cooling the mixture in an ice-water bath to prepare physically cross-linked β-cyclodextrin-modified gelatin microspheres;

[0014] (b) chemically cross-linking the physically cross-linked β-cyclodextrin-modified gelatin microspheres obtained in step (a) using a chemical cross-linking agent to obtain the β-cyclodextrin-modified gelatin microspheres.

[0015] Furthermore, in the step (a), gelatin is added to the carboxymethyl-β-cyclodextrin solution containing a cross-linking agent to obtain the β-cyclodextrin modified gelatin;

[0016] Preferably, the method further comprises a dialysis step, and the β-cyclodextrin-modified gelatin is obtained after dialysis;

[0017] Preferably, the crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide, N,N′-carbonyldiimidazole, and [1-hydroxybenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate.

[0018] Furthermore, the step (a) comprises:

[0019] (a1) dissolving 1 g of carboxymethyl-β-cyclodextrin in 100-500 ml of PBS to obtain a carboxymethyl-β-cyclodextrin solution;

[0020] (a2) adding 0.3-3.2 g of EDC and 0.2-2.5 g of NHS to the carboxymethyl-β-cyclodextrin solution, adjusting the pH to 4-6, to obtain a carboxymethyl-β-cyclodextrin solution containing a cross-linking agent;

[0021] (a3) stirring the carboxymethyl-β-cyclodextrin solution containing the cross-linking agent at 35 to 45° C. for 20 to 40 minutes, and then adding 0.5 to 2.5 g of gelatin;

[0022] (a4) After reacting for 24 to 72 hours, dialyzing in water for 2 to 5 days and then freeze-drying to obtain the β-cyclodextrin-modified gelatin.

[0023] Furthermore, in the step (a), the mass ratio of the β-cyclodextrin-modified gelatin to gelatin is 1:0.5-5;

[0024] Preferably, the volume ratio of the aqueous solution of the β-cyclodextrin-modified gelatin mixed with gelatin to the oil phase is 1:3-5;

[0025] Preferably, the oil phase comprises paraffin oil and edible oil in a volume ratio of 2 to 4:1;

[0026] Preferably, after cooling in an ice-water bath, the step of washing the precipitate is further included, preferably using 1,4-dioxane and acetone to wash the precipitate alternately for 3 times, and then washing it with anhydrous ethanol for 2 times.

[0027] Furthermore, in the step (b), 0.5 g of the physically cross-linked β-cyclodextrin-modified gelatin microspheres are dissolved in 15 to 30 ml of a solvent, and then chemically cross-linked; the solvent includes anhydrous ethanol or a mixed solution of PBS and ethanol;

[0028] Preferably, the chemical cross-linking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, N,N′-dicyclohexylcarbodiimide, N,N′-dicyclohexylcarbodiimide, N,N′-carbonyldiimidazole, and [1-hydroxybenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, genipin, formaldehyde and glutaraldehyde, preferably 0.2-0.3 g EDC and 0.1-0.2 g NHS;

[0029] Preferably, the chemical cross-linking condition is mixing at 0 to 55° C. for 1 to 24 hours;

[0030] Preferably, the method further comprises the steps of washing with water and then drying.

[0031] The present invention also provides beta-cyclodextrin modified gelatin microspheres prepared by the above preparation method.

[0032] The present invention also provides deferoxamine-loaded beta-cyclodextrin-modified gelatin microspheres, in which deferoxamine is loaded.

[0033] The present invention also provides a method for preparing the above-mentioned deferoxamine-loaded β-cyclodextrin-modified gelatin microspheres, which comprises immersing the β-cyclodextrin-modified gelatin microspheres in a deferoxamine solution to obtain the deferoxamine-loaded β-cyclodextrin-modified gelatin microspheres.

[0034] Further, the solvent of the deferoxamine solution includes 60% ethanol aqueous solution;

[0035] Preferably, the soaking comprises soaking the microspheres at 4 degrees for 4 hours, ultrasonicating for 5 minutes, and then soaking the microspheres at 4 degrees for 12 hours;

[0036] Preferably, the method further comprises the step of washing with water twice.

[0037] In addition, the present invention also provides the use of the above-mentioned β-cyclodextrin modified gelatin microspheres and the β-cyclodextrin modified gelatin microspheres loaded with deferoxamine in the preparation of tissue repair products;

[0038] Preferably, the tissue repair comprises abdominal wall tissue defect repair.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention successfully designed and prepared β-cyclodextrin-modified gelatin microspheres (CGMS-β-CD) with good thermal stability and β-cyclodextrin-modified gelatin microspheres loaded with DFO drug and loaded with deferoxamine (DFO@CGMS-β-CD). The results of cell experiments show that CGMS-β-CD and DFO@CGMS-β-CD have good cell compatibility and can promote cell adhesion, proliferation and migration. The introduction of DFO can also promote the angiogenesis of HUVECs cells. The results of the rat abdominal wall defect repair experiment show that compared with PP mesh and DFO@Gel-β-CD, CGMS-β-CD and DFO@CGMS-β-CD have obvious advantages in promoting abdominal wall tissue reconstruction, and loading DFO can promote angiogenesis in new tissues. DFO@CGMS-β-CD shows great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 Characterization of the scaffold material provided for the experimental example of the present invention: (A) SEM image of CGMS-β-CD; (B) Particle size distribution of swollen CGMS-β-CD; (C) H-NMR spectra of CGMS and CGMS-β-CD ( 1 1H NMR).

[0043] Figure 2DFO loading and release detection for CGMS and CGMS-β-CD scaffolds: (A) DFO sustained release kinetics in CGMS and CGMS-β-CD scaffolds; (B) DFO loading amount in 1 g of CGMS or CGMS-β-CD scaffolds.

[0044] Figure 3 Cell activity detection provided for the experimental examples of the present invention: (A) live / dead cell staining, scale bar is 600 μm; (B) quantitative detection of cell proliferation in CGMS-β-CD and DFO@CGMS-β-CD scaffolds.

[0045] Figure 4 This is a crystal violet staining photograph of cells in the Transwell chamber provided in the experimental example of the present invention, with a scale bar of 300 μm.

[0046] Figure 5 The qPCR verification provided for the experimental examples of the present invention shows the expression of vascular markers in the CGMS-β-CD and DFO@CGMS-β-CD scaffolds: (A) the expression of CD31; (B) the expression of VEGF; (C) the expression of FGF2; wherein, * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0047] Figure 6 The construction and repair photos of the rat abdominal wall defect model provided for the experimental examples of the present invention: (A) construction of the abdominal wall defect model; (B) photos of the repair of the abdominal wall defect with different materials at different times.

[0048] Figure 7 H&E staining and Masson staining images of the abdominal wall defect repair area provided in the experimental example of the present invention, with a scale bar of 500 μm.

[0049] Figure 8 This is the Col1 immunohistochemistry provided in the experimental example of the present invention, and the scale bar is 500 μm.

[0050] Fig. 9 This is the VEGF immunohistochemistry provided in the experimental example of the present invention, and the scale bar is 500 μm.

[0051] Fig.10 This is the α-SMA immunohistochemistry provided in the experimental example of the present invention, and the scale bar is 500 μm. DETAILED DESCRIPTION

[0052] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0053] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0054] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In recent years, the application of tissue engineering technology in the repair of abdominal wall defects has attracted more and more attention from researchers, among which the development of scaffold materials is a research hotspot. Ideal tissue-inducing scaffold materials can not only recruit cells in situ, but also support and guide tissue formation. Gelatin microsphere materials have good biocompatibility, biodegradability, low immunogenicity, and can promote cell adhesion and proliferation; these characteristics make them a tissue engineering scaffold with great application potential. Compared with common types of scaffolds (such as porous scaffolds, fiber scaffolds), microspheres have many advantages: for example, 1. Microspheres can adsorb growth factors, drugs or extracellular matrix molecules; 2. Microspheres have a larger specific surface area, which is conducive to cell adhesion and proliferation; 3. The injectability of microspheres facilitates their use. Recently, the inventors found that gelatin microspheres can promote the proliferation of osteoblasts and endothelial cells and the repair of femoral defects. However, the research on gelatin microspheres in the repair of abdominal wall defects has not been reported.

[0056] β-Cyclodextrin (β-CD) has many advantages as a biomaterial. β-CD is a natural cyclodextrin derivative with good biocompatibility and is not prone to cause immune or rejection reactions. It can be used to encapsulate drug molecules to form encapsulation complexes, thereby improving the stability and solubility of drugs and facilitating drug delivery and absorption. It also has good biodegradability and low toxicity.

[0057] Based on this, the first aspect of the present invention provides a method for preparing β-cyclodextrin modified gelatin microspheres, comprising the following steps:

[0058] (a) stirring and dispersing an aqueous solution of β-cyclodextrin-modified gelatin and gelatin mixed into an oil phase, and cooling the mixture in an ice-water bath to prepare physically cross-linked β-cyclodextrin-modified gelatin microspheres;

[0059] (b) chemically cross-linking the physically cross-linked β-cyclodextrin-modified gelatin microspheres obtained in step (a) using a chemical cross-linking agent to obtain the β-cyclodextrin-modified gelatin microspheres.

[0060] The above method is simple in process and easy to operate. The present invention prepares a gelatin-based microsphere material-CGMS-β-CD with a regular circular / elliptical shape and a particle size distribution of 50-300 μm by an oil-in-water emulsification method. Live / dead cell staining and cell proliferation studies show that this new gelatin-based microsphere material-CGMS-β-CD has good cell compatibility, which can not only promote cell adhesion and survival, but also promote cell proliferation; in addition, the DFO@CGMS-β-CD material prepared by introducing DFO also has good cell compatibility; in addition, the transwell experimental results show that compared with the gel-based material (DFO@Gel-β-CD), DFO@CGMS-β-CD can significantly promote cell migration, which will be beneficial for it to recruit autologous cells in vivo and form a material / cell complex to promote tissue regeneration and repair. The results of qPCR test showed that compared with CGMS-β-CD, DFO@CGMS-β-CD could significantly increase the expression of angiogenesis-related factors (such as CD31, VEGF, and FGF2) in HUVECs, indicating that the introduction of DFO will promote the vascularization of new tissues. In the rat abdominal wall defect repair experiment, after 3 months of surgical implantation, we found that the non-degradable PP mesh and the gel-based DFO@Gel-β-CD could not significantly promote the regeneration and repair of abdominal wall tissue. PP mesh cannot be degraded and always occupies the position of tissue defects, forming a physical barrier and inhibiting tissue formation; the new tissue formed in the DFO@Gel-β-CD group is mainly concentrated on the surface, which may be because the gel inhibits the migration of cells into it, and fewer tissue bodies are formed. It is worth noting that compared with PP mesh and DFO@Gel-β-CD, both CGMS-β-CD and DFO@CGMS-β-CD can promote the regeneration of larger / thicker tissues rich in collagen fibers and microvascular tissues; in comparison, the vascularized tissue formation and defect repair in the DFO@CGMS-β-CD implantation group were more ideal, indicating its great clinical application potential in promoting abdominal wall defect repair.

[0061] In some preferred embodiments, step (a) comprises:

[0062] (a1) dissolving 1 g of carboxymethyl-β-cyclodextrin in 100-500 ml of PBS to obtain a carboxymethyl-β-cyclodextrin solution; wherein the amount of PBS used can be, for example, but not limited to, 100, 150 ml, 180 ml, 200 ml, 250 ml, 300 ml, 400 ml or 500 ml.

[0063] (a2) adding 0.3-3.2 g of EDC and 0.2-2.5 g of NHS to the carboxymethyl-β-cyclodextrin solution, adjusting the pH to 4-6, to obtain a carboxymethyl-β-cyclodextrin solution containing a cross-linking agent; wherein the amount of EDC may be, for example, but not limited to 0.3 g, 0.8 g, 1.2 g, 1.8 g, 2.4 g or 3.2 g; the amount of NHS may be, for example, but not limited to 0.2 g, 0.6 g, 1.2 g, 1.8 g, 2 g or 2.5 g; and the pH may be, for example, but not limited to 4, 5 or 6.

[0064] (a3) After mixing the carboxymethyl-β-cyclodextrin solution containing the cross-linking agent at 35-45° C. for 10-40 minutes, 0.5-2.5 g of gelatin is added; wherein the temperature is 35° C., 38° C., 40° C., 42° C. or 45° C.; the mixing time can be, for example, but not limited to, 10 minutes, 20 minutes, 30 minutes, 35 minutes or 40 minutes; the amount of gelatin added can be, for example, but not limited to, 0.5 g, 1 g, 1.5 g, 2 g or 2.5 g.

[0065] (a4) After reacting for 24 to 72 hours, dialyzing in water for 2 to 5 days and then freeze-drying to obtain the β-cyclodextrin modified gelatin; wherein the reaction time can be, for example, but not limited to, 24 hours, 30 hours, 40 hours, 48 ​​hours, 55 hours, 65 hours or 72 hours.

[0066] In some preferred embodiments, in the step (a), the mass ratio of the β-cyclodextrin-modified gelatin to gelatin is 1:0.5-5, for example, but not limited to 1:0.5, 1:1, 1:1.5, 1:2.5 or 1:5;

[0067] In some preferred embodiments, the volume ratio of the aqueous solution of the β-cyclodextrin-modified gelatin mixed with gelatin to the oil phase is 1:3 to 10, for example, but not limited to 1:3, 1:6 or 1:10;

[0068] In some preferred embodiments, the oil phase comprises paraffin oil and edible oil in a volume ratio of 2 to 4:1, for example, it can be, but not limited to, 2:1, 3:1 or 4:1;

[0069] In some preferred embodiments, after cooling in an ice-water bath, a step of washing the precipitate is further included, preferably using 1,4-dioxane and acetone to wash the precipitate alternately for 3 times, and then washing it with anhydrous ethanol for 2 times.

[0070] The physically cross-linked β-cyclodextrin-modified gelatin microspheres are prepared using the above-mentioned specific condition parameters. The preparation process of the physically cross-linked microspheres does not involve chemical cross-linking agents and will not cause agglomeration or agglomeration between the microspheres.

[0071] In some preferred embodiments, in the step (b), each 0.5 g of the physically cross-linked β-cyclodextrin modified gelatin microspheres is dissolved in 15 to 30 ml of solvent, and then chemically cross-linked; for example, it can be, but not limited to, 15 ml, 18 ml, 20 ml, 22 ml, 25 ml, 28 ml or 30 ml. The solvent includes anhydrous ethanol, or a mixed solution of PBS and ethanol, wherein the volume ratio of the two in the mixed solution of PBS and ethanol can be, for example, but not limited to 1:1, 1:2, 1:3, 1:4, 1:10 or 1:20. Using anhydrous ethanol, or a mixed solution of PBS and ethanol as a solvent can not only ensure the stability of the physically cross-linked β-cyclodextrin modified gelatin microspheres, but also facilitate chemical cross-linking at a low swelling rate to form microspheres with good thermal stability.

[0072] In some preferred embodiments, the chemical crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, N,N′-dicyclohexylcarbodiimide, N,N′-dicyclohexylcarbodiimide, N,N′-carbonyldiimidazole, and [1-hydroxybenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, genipin, formaldehyde and glutaraldehyde, preferably 0.2-0.3 g EDC and 0.1-0.2 g NHS.

[0073] In some preferred embodiments, the chemical crosslinking condition is mixing at 0-55°C for 1-24 hours, the crosslinking temperature can be, for example, but not limited to 0°C, 10°C, 20°C, 30°C, 40°C or 55°C, and the mixing time can be, for example, but not limited to 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 20 hours or 24 hours.

[0074] In some preferred embodiments, the step of washing with water and then drying is also included.

[0075] According to a second aspect of the present invention, there is also provided β-cyclodextrin modified gelatin microspheres prepared by the above-mentioned preparation method.

[0076] The β-cyclodextrin-modified gelatin microspheres have good thermal stability, can provide a broad interface and space for cell adhesion and migration, and have good biodegradability. These properties provide guarantees for tissue formation and vascularization.

[0077] According to a third aspect of the present invention, there are provided β-cyclodextrin-modified gelatin microspheres loaded with deferoxamine, wherein deferoxamine is loaded in the above β-cyclodextrin-modified gelatin microspheres.

[0078] Desferrioxamine (DFO) is a commonly used drug approved by the FDA that can promote angiogenesis. It can chelate excess iron ions in the blood and spleen and has long been widely used to treat thalassemia. In recent years, its significant angiogenic function has attracted more and more attention, but achieving its sustained release is the key to promoting microvascular formation. When desferrioxamine is loaded in the above-mentioned β-cyclodextrin-modified gelatin microspheres, its sustained release effect is significant and the ability of tissue repair is stronger.

[0079] According to a fourth aspect of the present invention, a method for preparing the above-mentioned β-cyclodextrin-modified gelatin microspheres loaded with deferoxamine is also provided, wherein the β-cyclodextrin-modified gelatin microspheres are immersed in a deferoxamine solution to obtain the β-cyclodextrin-modified gelatin microspheres loaded with deferoxamine.

[0080] The method has a simple process, and the deferoxamine-loaded β-cyclodextrin-modified gelatin microspheres prepared by the method maintain good appearance, cell adhesion performance, degradability and other properties.

[0081] The solvent of the deferoxamine solution includes 60% ethanol aqueous solution;

[0082] In some preferred embodiments, the soaking includes soaking the microspheres at 4 degrees for 4 hours, ultrasonicating for 5 minutes, and then soaking the microspheres at 4 degrees for 12 hours;

[0083] In some preferred embodiments, the step of washing with water twice is further included.

[0084] In addition, according to the fifth aspect of the present invention, the present invention also provides the use of the above-mentioned β-cyclodextrin modified gelatin microspheres and the β-cyclodextrin modified gelatin microspheres loaded with deferoxamine in the preparation of tissue repair products;

[0085] In some preferred embodiments, the tissue repair comprises abdominal wall tissue repair.

[0086] Since muscle cells are difficult to proliferate, it is very difficult to repair and regenerate abdominal wall muscle tissue defects. The DFO@CGMS-β-CD scaffold has the advantages of large specific surface area and abundant cell growth space, providing a good microenvironment for surrounding cells to migrate to the defect. In addition, its DFO sustained release performance can promote microvascular formation, which is beneficial to the regeneration and repair of abdominal wall muscle tissue.

[0087] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0088] Example 1

[0089] This embodiment provides a β-cyclodextrin modified gelatin microsphere, which is prepared by the following method:

[0090] (1) Preparation of β-cyclodextrin-modified gelatin microspheres (CGMS-β-CD) and gelatin hydrogel (Gel-β-CD): Carboxymethyl-β-cyclodextrin (Carboxymethyl-β-CD, 1 g) was dissolved in 200 mL of PBS (pH = 7), EDC (1 g) and NHS (0.7 g) were added, and the pH of the solution was adjusted to 4-6. After stirring in a 40°C water bath for 30 min, gelatin (1 g) was added. After reacting for 48 h, the mixture was dialyzed in pure water for 3 days and then freeze-dried to obtain gelatin-β-CD.

[0091] (2) CGMS-β-CD was then prepared using the oil-in-water method: 1.5 g of gelatin and 1.5 g of gelatin-β-CD were added to 20 mL of deionized water and dissolved in a 40°C water bath. The gelatin solution was then poured into 80 mL of mixed oil (paraffin oil: edible oil = 3:1) and stirred at 700 rpm on a magnetic stirrer for 30 min. The reaction system was then transferred to an ice-water bath and stirred for another 30 min. The precipitate was then washed alternately with 1,4-dioxane and acetone 3 times, and then washed twice with anhydrous ethanol to obtain physically cross-linked gelatin microspheres (PGMS-β-CD).

[0092] (3) Place 0.5 g of PGMS-β-CD in 20 mL of PBS / ethanol (3:7), add N-hydroxysuccinimide hydrochloride (EDC, 0.22 g) and N-hydroxysuccinimide (NHS, 0.16 g), shake at room temperature for 6 h to obtain CGMS-β-CD, wash three times with deionized water, freeze-dry and store in a refrigerator at 4°C for later use.

[0093] Example 2

[0094] This embodiment provides a β-cyclodextrin modified gelatin microsphere, which is prepared by the following method:

[0095] (1) Preparation of β-cyclodextrin-modified gelatin microspheres (CGMS-β-CD) and gelatin hydrogel (Gel-β-CD): Carboxymethyl-β-cyclodextrin (Carboxymethyl-β-CD, 1 g) was dissolved in 150 mL of PBS (pH = 7), EDC (1.2 g) and NHS (0.5 g) were added, and the pH of the solution was adjusted to 4-6. After stirring in a 45°C water bath for 20 min, gelatin (1.5 g) was added. After reacting for 40 h, the mixture was dialyzed in pure water for 5 days and then freeze-dried to obtain gelatin-β-CD.

[0096] (2) CGMS-β-CD was then prepared using the oil-in-water method: 1.5 g of gelatin and 0.5 g of gelatin-β-CD were added to 20 mL of deionized water and dissolved in a 40°C water bath. The gelatin solution was then poured into 100 mL of mixed oil (paraffin oil: edible oil = 2:1) and stirred at 700 rpm on a magnetic stirrer for 30 min. The reaction system was then transferred to an ice-water bath and stirred for another 30 min. The precipitate was then washed alternately with 1,4-dioxane and acetone 3 times, and then washed twice with anhydrous ethanol to obtain physically cross-linked gelatin microspheres (PGMS-β-CD).

[0097] (3) Place 0.5 g of PGMS-β-CD in 15 mL of PBS / ethanol (3:10), add N-hydroxysuccinimide hydrochloride (EDC, 0.2 g) and N-hydroxysuccinimide (NHS, 0.2 g), shake at room temperature for 5 h to obtain CGMS-β-CD, wash three times with deionized water, freeze-dry and store in a refrigerator at 4 °C for later use.

[0098] Example 3

[0099] This embodiment provides a β-cyclodextrin modified gelatin microsphere, which is prepared by the following method:

[0100] (1) Preparation of β-cyclodextrin-modified gelatin microspheres (CGMS-β-CD) and gelatin hydrogel (Gel-β-CD): Carboxymethyl-β-cyclodextrin (Carboxymethyl-β-CD, 1 g) was dissolved in 300 mL of PBS (pH = 7), EDC (0.8 g) and NHS (1 g) were added, and the pH of the solution was adjusted to 4-6. After stirring in a 35°C water bath for 40 min, gelatin (0.5 g) was added. After reacting for 55 h, the mixture was dialyzed in pure water for 2 days and then freeze-dried to obtain gelatin-β-CD.

[0101] (2) CGMS-β-CD was then prepared using the oil-in-water method: 1.5 g of gelatin and 2.25 g of gelatin-β-CD were added to 20 mL of deionized water and dissolved in a 40°C water bath. The gelatin solution was then poured into 60 mL of mixed oil (paraffin oil: edible oil = 4:1) and stirred at 700 rpm on a magnetic stirrer for 30 min. The reaction system was then transferred to an ice-water bath and stirred for another 30 min. The precipitate was then washed alternately with 1,4-dioxane and acetone 3 times, and then washed twice with anhydrous ethanol to obtain physically cross-linked gelatin microspheres (PGMS-β-CD).

[0102] (3) Place 0.5 g of PGMS-β-CD in 30 mL of PBS / ethanol (3:5), add N-hydroxysuccinimide hydrochloride (EDC, 0.3 g) and N-hydroxysuccinimide (NHS, 0.1 g), shake at room temperature for 8 h to obtain CGMS-β-CD, wash three times with deionized water, freeze-dry and store in a refrigerator at 4 °C for later use.

[0103] Example 4

[0104] This embodiment provides a β-cyclodextrin-modified gelatin microsphere loaded with deferoxamine, which is prepared by the following method:

[0105] Take 1 g of the β-cyclodextrin modified gelatin microspheres provided in Example 1, put them into a 2 ml centrifuge tube, add 1 ml of deferoxamine (DFO) solution (prepared using 60% ethanol aqueous solution); soak the microspheres at 4 degrees for 4 hours, ultrasonicate for 5 minutes, and then soak the microspheres at 4 degrees for 12 hours, wash them twice with double distilled water to obtain a DFO-loaded material (named: CGMS-β-CD@DFO).

[0106] Comparative Example 1

[0107] This comparative example provides a gelatin microsphere loaded with deferoxamine, which is prepared by the following method:

[0108] Take 1g of gelatin microspheres and put them into a 2ml centrifuge tube, add 1ml of DFO solution (prepared using 60% ethanol aqueous solution); soak the microspheres at 4 degrees for 4 hours, ultrasonicate for 5 minutes, then soak the microspheres at 4 degrees for 12 hours, wash twice with double distilled water to obtain a DFO-loaded material (named: CGMS@DFO).

[0109] Experimental Example 1 Microsphere Characterization and Detection

[0110] Scanning electron microscopy: CGMS-β-CD was placed in a freeze dryer and frozen at -20°C overnight to obtain freeze-dried CGMS-β-CD. After spraying with gold, its morphology was observed using a scanning electron microscope.

[0111] H NMR: The dried CGMS-β-CD was completely swollen in heavy water and then subjected to H NMR (1H NMR).

[0112] The results showed that the morphology of CGMS-β-CD was characterized by scanning electron microscopy ( Figure 1 A in the figure, it was found to be round particles with smooth surface; after swelling with PBS, its particle size distribution was 50-300 μm ( Figure 1 In this experiment, the nuclear magnetic resonance spectroscopy ( 1H NMR) studies on the CGMS microspheres without β-CD and CGMS-β-CD ( Figure 1 C), a characteristic peak of β-CD was found at 3.4 ppm, indicating the successful preparation of CGMS-β-CD.

[0113] Experimental Example 2: Study on the release of deferoxamine (DFO)

[0114] Weigh 0.2 g of material (CGMS@DFO provided in Comparative Example 1 or CGMS-β-CD@DFO provided in Example 4) and add it to a 50 ml centrifuge tube, then add 15 ml of 0.1% sodium azide solution; take samples at predetermined time points, take 0.5 ml each time and place them in a 1.5 ml EP tube, and add 0.5 ml of 0.1% NaN to the 50 ml centrifuge tube after sampling. 3 Finally, 100 μl of the culture medium was placed in a 96-well plate and 100 μl of 5 mM ferric chloride (FeCl 3 ) After incubation for 10 min, the OD value was measured at 480 nm on a microplate reader to calculate the amount of DFO released.

[0115] The results are as follows Figure 2 As shown, compared with CGMS, CGMS-β-CD can not only increase the loading amount of DFO, but also achieve the effect of sustained release of DFO.

[0116] Experimental Example 3 Cell culture and planting

[0117] Human umbilical vein endothelial cells (HUVECs) were seeded into 10 cm culture dishes, added with F12K complete medium (F12K medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin), and placed at 37°C, 5% CO 2 Culture in an incubator.

[0118] Prepare 1% agarose solution, add 200 μL of hot agarose solution to each well of a 24-well plate, and freeze in a 4°C refrigerator to solidify. Resuspend 1 g of sterile CGMS-β-CD or DFO@CGMS-β-CD in 1 mL of cell culture medium and inoculate into a 24-well plate, with 60 μL of suspension per well. Add 2.5×10 5 10 HUVECs and 1 mL of complete culture medium were placed at 37°C with 5% CO 2 incubator.

[0119] (1) Dead / live cell staining

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

[0121] (2) Cell proliferation assay

[0122] The viability of HUVEC cells in the material / cell complex was detected using the PrestoBlue kit. According to the instructions, the PrestoBlue working solution was prepared. At the predetermined time point, the cell culture medium was replaced with 1 mL of the PrestoBlue working solution and incubated at 37°C in the dark for 30 minutes. The fluorescence was quantitatively analyzed at 560 / 590 nm (excitation / emission) using an ELISA reader.

[0123] (3) Cell migration assay

[0124] The Transwell chamber was placed in a 24-well plate, and DFO@CGMS-β-CD or DFO@Gel-β-CD was added. HUVEC suspension (5×10 4 cells / 200 μL), and then 600 μL of complete culture medium was added to the lower chamber and placed in a 5% CO 2 The cells were cultured in a 37°C cell culture incubator for 24 h, the upper chamber was taken out, the cells in the upper chamber were carefully wiped with a cotton swab, and the cells were stained with 0.1% crystal violet and observed under an optical microscope.

[0125] The results showed that DFO is a metal cation (such as Fe 3+ ,Al 3+ DFO is a chelating agent of β-cyclodextrin-modified gelatin microspheres and has good antioxidant activity. As a mature stabilizer of hypoxia inducible factor-1α (HIF-1α), DFO can upregulate the level of HIF-1α and related downstream angiogenic factors in cells, thereby accelerating angiogenesis. It has also been reported that DFO has anti-proliferative activity and can induce apoptosis and autophagy in cancer cells. In order to detect the effect of DFO on cell viability in the microenvironment of β-cyclodextrin-modified gelatin microspheres, this experimental example first detected the activity of HUVECs implanted in CGMS-β-CD and DFO@CGMS-β-CD scaffolds by live / dead cell staining (calcein / PI staining). Figure 3A), where live cells show green fluorescence and dead cells show red fluorescence. The survival status of the scaffold-HUVECs was observed after 1, 3, and 7 days of co-culture. In this experimental example, it was found that after the first day of co-culture, a large number of cells survived and adhered to the surface of the scaffold material in both groups; almost no dead cells were observed in the CGMS-β-CD group, but some dead cells were found in the DFO@CGMS-β-CD group. This finding may be attributed to the pro-apoptotic effect of DFO on HUVECs. On the third day, no dead cells were still observed in the CGMS-β-CD group, and no dead cells were found in the DFO@CGMS-β-CD group, indicating that no new cell death occurred, which also indirectly indicated the cell compatibility of the DFO@CGMS-β-CD scaffold. As expected, there were almost no dead cells in both groups on the fifth day. These results show that both CGMS-β-CD and DFO@CGMS-β-CD scaffolds can provide a good microenvironment for cell adhesion and survival, which provides a guarantee for cell behaviors such as cell proliferation and differentiation.

[0126] Subsequently, the proliferation of HUVECs in CGMS-β-CD and DFO@CGMS-β-CD scaffolds was quantitatively detected by Prestoblue method in this experiment ( Figure 3 B). As time goes by, the number of HUVECs in both groups gradually increases with the extension of culture time. At the same time, this experiment found that after 3 and 5 days of culture, the number of cells in the DFO@CGMS-β-CD group was significantly higher than that in the CGMS-β-CD group. This result shows that the DFO@CGMS-β-CD scaffold has good cell compatibility.

[0127] In addition, this experiment used the Transwell chamber assay to detect the migration ability of HUVECs in DFO@CGMS-β-CD and DFO@Gel-β-CD scaffolds. Figure 4 As shown in the figure, after 24 hours of culture, a large number of HUVECs migrated through the DFO@CGMS-β-CD scaffold into the lower layer of the chamber. However, almost no cells migrating through the DFO@Gel-β-CD scaffold were observed in the lower layer of the chamber. This result indicates that compared with DFO@Gel-β-CD, DFO@CGMS-β-CD can provide a better microenvironment for HUVECs migration.

[0128] Experimental Example 4 Real-time fluorescence quantitative PCR (qPCR) to detect the effect of angiogenesis

[0129] In order to study the promoting effect of DFO on angiogenesis, HUVECs were co-cultured with CGMS-β-CD or DFO@CGMS-β-CD for 5 days, and the total RNA in the cells was extracted by Trizol method. The expression of angiogenesis-related genes, including platelet endothelial cell adhesion molecule-1 (CD31), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), was verified by qPCR. Specifically:

[0130] ① RNA extraction

[0131] After the scaffold-HUVECs complex was cultured for a certain period of time, the complex was transferred to a 1.5mL EP tube, 1mL Trizol was added, and then 250μL chloroform was added for every 1mL Trizol. After vigorous shaking for 5-10sec, it was allowed to stand at room temperature for 5min. Centrifuged at 14000g, 4℃ for 20min. Carefully transfer the top layer of liquid to a new 1.5mL EP tube with a pipette, add the same volume of pre-cooled isopropanol and 4μL 5mg / mL glycogen as the supernatant, mix by inversion, and centrifuge at 14000g, 4℃ for 20min. Discard the supernatant, add 1mL pre-cooled 80% ethanol, and centrifuge at 14000g, 4℃ for 20min. Discard the supernatant and air dry for 5-10sec. Add 15-20μL DEPC water to dissolve the precipitate. Measure RNA concentration by Nanodrop.

[0132] ②Reverse transcription

[0133] i. Prepare the following mixture in an RNase free centrifuge tube and mix it by gently pipetting. Incubate at 42℃ for 2 min.

[0134]

[0135] ii. Add directly to the reaction tube in step 1 IIISuperMix plus, mix by gently pipetting.

[0136]

[0137] iii. Reverse transcription standard program settings:

[0138]

[0139] ③ Real-time fluorescence quantitative PCR

[0140] i. Prepare the reaction solution

[0141]

[0142]

[0143] ii. Place in real-time fluorescence quantitative PCR instrument and set the program

[0144]

[0145] The results are as follows Figure 5 As shown: Compared with CGMS-β-CD, the expression levels of CD31, VEGF and FGF2 in the DFO@CGMS-β-CD group were significantly higher, proving that DFO can promote the vascularization of HUVECs in the DFO@CGMS-β-CD scaffold microenvironment.

[0146] Experimental Example 5 Construction and repair of abdominal wall defect model in rats

[0147] Regeneration and repair of abdominal wall defects is a clinical problem. In order to preliminarily evaluate the repair effect of DFO@CGMS-β-CD scaffold on abdominal wall defects, we constructed a rat abdominal wall defect model ( Figure 6 A) in the figure, and different biomaterials, including PP mesh, DFO@Gel-β-CD, CGMS-β-CD and DFO@CGMS-β-CD, were implanted respectively. The animals were euthanized 30 and 90 days after surgery, and the abdominal wall defect and adjacent tissues were harvested.

[0148] The preparation process of DFO@Gel-β-CD group is as follows:

[0149] 1: Transfer the aqueous solution of β-cyclodextrin-modified gelatin and gelatin mixture to a 10-cm diameter cell culture dish, cool it down to obtain physically cross-linked β-cyclodextrin-modified gelatin hydrogel; then perform chemical cross-linking. The specific steps are as follows:

[0150] Add 1.5g gelatin and 1.5g gelatin-β-CD to 20mL deionized water, dissolve evenly in a 40℃ water bath, pour into a mold, solidify in a 4℃ refrigerator, transfer to 20mL PBS / ethanol (3:7), add EDC (0.22g) and NHS (0.16g). Shake horizontally at room temperature for 6h to obtain Gel-β-CD, wash three times with deionized water, and store in a 4℃ refrigerator for later use.

[0151] 2: The steps of loading DFO are similar to those of the microsphere group:

[0152] Place 5 g of Gel-β-CD material in a cell culture dish, add 5 ml of DFO solution (prepared using 60% ethanol aqueous solution); soak the microspheres at 4 degrees for 4 hours, ultrasonicate for 5 minutes, then soak the microspheres at 4 degrees for 12 hours, wash twice with double distilled water to obtain DFO-loaded DFO@Gel-β-CD group material.

[0153] Regenerative repair of abdominal wall defects specifically includes:

[0154] All animal experiments were carried out in accordance with the regulations of the Experimental Animal Management and Animal Welfare Ethics Committee of the School of Medicine of Qingdao University. Male Sprague-Dawley (SD) rats aged 8-10 weeks were used for animal experiments. All animals were housed in the Animal Center of Qingdao University and divided into 4 groups (control group, polypropylene mesh group, DFO@Gel-β-CD group, CGMS-β-CD group and DFO@CGMS-β-CD group) according to the random number table method. The SD rats were fasted and deprived of water the night before surgery. Before surgery, sodium pentobarbital (2%, 30 mg / kg) was injected intraperitoneally to complete anesthesia. After anesthesia, the hair at the surgical site was removed with a depilatory cream. After aseptic treatment of the surgical site with iodine tincture and 75% alcohol cotton balls, the skin was incised on the abdomen of the rat, and the abdominal muscles were cut off to the peritoneal layer to create an abdominal wall injury model (2 cm × 3 cm). Figure 5 As shown in A. The PP patch was placed in the defect and fixed along the edge with sutures; DFO@Gel-β-CD, CGMS-β-CD or DFO@CGMS-β-CD were filled in the defect, covered with Gel-β-CD and fixed along the edge. The skin was then closed with sutures. After euthanasia, the rats in each group were euthanized 30 days and 90 days after surgery, and the whole layer of abdominal wall tissue samples were cut off, fixed in 4% paraformaldehyde, and stored at 4°C.

[0155] like Figure 6As shown in Figure B, at 30 days after surgery, the presence of mesh was clearly observed in the PP mesh group, and the defect was clearly observed in the DFO@Gel-β-CD group; the defect in the CGMS-β-CD group could be distinguished, but tissue repair was clearly observed; the difference between the defect and the surrounding tissue in the DFO@CGMS-β-CD group could no longer be distinguished by the naked eye. At 90 days after surgery, although the surface was covered with a layer of tissue, the presence of mesh was still observed in the defect repair site in the PP mesh group. In the DFO@Gel-β-CD group, it was clearly observed that the abdominal wall defect site was sunken, indicating that its repair was not ideal. The defect in the CGMS-β-CD group was fused with the surrounding tissue, and the defect site could no longer be distinguished by the naked eye; similarly, the defect site in the DFO@CGMS-β-CD group could no longer be distinguished by the naked eye; the defect site in the CGMS-β-CD and DFO@CGMS-β-CD groups needed to be anchored by sutures. These results indicate that CGMS-β-CD and DFO@CGMS-β-CD can promote the regeneration and repair of abdominal wall defects in rats.

[0156] Experimental Example 6 Histological and Immunochemical Analysis

[0157] This experimental example used histological (such as H&E staining, Masson staining) and immunochemical (such as Col1a1, α-SMA, VEGF) staining to examine the repair process of the abdominal wall defect. Specifically:

[0158] The tissue specimens were fixed in 4% paraformaldehyde solution for 48 h, then dehydrated, embedded in paraffin, cut into 5 μm thick sections using a paraffin slicer and attached to anti-shedding slides. After hydration, the tissue sections were stained with hematoxylin and eosin (H&E staining), Masson staining, and immunohistochemical staining of type I collagen, α-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF).

[0159] like Figure 7As shown in the figure, 30 days after surgery, H&E staining results showed that the PP mesh group had the least ECM formation at the defect site, and the average ECM expression gradually increased from DFO@Gel-β-CD, CGMS-β-CD to DFO@CGMS-β-CD groups. Since PP mesh is not degradable, ECM cannot fill the entire defect site. Through careful observation, we can find that the ECM in the DFO@Gel-β-CD group is mainly distributed on the surface. This may be because cells can only adhere to the surface of the hydrogel for growth, and the migration of cells is inhibited before the degradation of the material, so there is not much ECM deposition inside the defect. It is worth noting that the ECM in the CGMS-β-CD and DFO@CGMS-β-CD groups is more evenly distributed in the tissue defect site, and its density is higher. At 90 days, the ECM formed in the PP mesh group is still less, and its density is still the lowest among the four groups; and compared with 30 days, its density has not been significantly improved. The ECM in the DFO@Gel-β-CD group is still mainly concentrated on the outer surface of the material, with less inside the material, which may be attributed to the difficulty of cells migrating to the inside of the hydrogel, and therefore unable to secrete extracellular matrix in situ. Compared with 30 days after surgery, the ECM distribution in the CGMS-β-CD group is more uniform and denser; more capillaries can be seen by careful observation. Similarly, compared with 30 days after surgery, the ECM distribution density in the DFO@CGMS-β-CD group is higher, showing a band-like distribution, and the internal microvessels are rich. These results show that compared with DFO@Gel-β-CD and PP mesh, CGMS-β-CD can provide a better microenvironment for cell proliferation and ECM secretion; in addition, the introduction of DFO (i.e., DFO@CGMS-β-CD) using CGMS-β-CD can also promote angiogenesis and enhance nutrient supply, thereby further promoting cell proliferation and ECM secretion.

[0160] In the PP mesh group, 30 days after surgery, the ECM was mainly blue collagen fibers; purple-red could be seen around the mesh, which might be microvessels, red blood cells and other components. 90 days after surgery, the density of collagen fibers increased slightly, while red-purple microvessels, red blood cells and other components were still distributed around the mesh, and the number also increased, but the overall distribution density of ECM components was still not high. 30 days after surgery, the blue collagen fibers in the DFO@Gel-β-CD group were mainly distributed on the surface, and this observation was similar to the results observed by HE staining. In addition, more microvessels, red blood cells and other components were found in the middle layer in this experimental case, which indicates that there is abundant angiogenesis between the surface of the gel material and the tissue. 90 days after surgery, compared with 30 days, the density of collagen fibers and microvessels and other components in the defect of the DFO@Gel-β-CD group increased. It is worth noting that compared with DFO@Gel-β-CD and PP mesh, the density of collagen fibers formed in the CGMS-β-CD group was higher 30 days after surgery, and they were distributed in a band-like manner, with tiny but abundant microvessels, red blood cells and other components inside. By 90 days after surgery, the density of collagen fibers in the CGMS-β-CD group increased, and a more mature capillary structure was formed. 30 days after surgery, the distribution of collagen fibers, microvessels, red blood cells and other components in the DFO@CGMS-β-CD group was similar to that in the CGMS-β-CD group, and the band-like distribution of collagen fibers was more obvious. 90 days after surgery, the density of the newly formed ECM increased significantly compared to 30 days, and it appeared purple-red, suggesting the formation of muscle fibers. In general, compared with DFO@Gel-β-CD and PP mesh, the newly formed tissues in the CGMS-β-CD and DFO@CGMS-β-CD groups contained more collagen components and abundant microvessels. In addition, the DFO@CGMS-β-CD group generated red-purple and band-like muscle fiber components 90 days after surgery.

[0161] Subsequently, immunohistochemical staining of type I collagen (Col1), the main component of ECM, was performed in this experimental example ( Figure 8). At 30 days after surgery, the positive signals in the PP mesh group and the DFO@Gel-β-CD group were sparse, and the signal distribution in the DFO@Gel-β-CD group was uneven, mainly concentrated in the edge. Compared with the PP mesh group and the DFO@Gel-β-CD group, the expression of Col1 in the CGMS-β-CD and DFO@CGMS-β-CD groups was significantly higher, and showed a band-like distribution. At 90 days, the expression of Col1 in the PP mesh group and the DFO@Gel-β-CD group was still low. The expression of Col1 in the CGMS-β-CD and DFO@CGMS-β-CD groups was higher than that in the PP mesh group and the DFO@Gel-β-CD group. Compared with the CGMS-β-CD group, the expression of Col1 in the DFO@CGMS-β-CD group was higher. These results indicate that compared with PP mesh and hydrogel-based scaffolds (i.e., DFO@Gel-β-CD), gelatin microsphere-based scaffolds can provide a more ideal microenvironment to promote the expression of Col1, and DFO drug has a promoting effect on the secretion of Col1. High expression of Col1 will be beneficial to wound healing and repair of tissue defects.

[0162] Angiogenesis can provide a guarantee for the nutrient supply during the regeneration and repair of the abdominal wall, and promote wound healing and tissue repair. In order to evaluate angiogenesis during the repair of abdominal wall defects, this experimental example stained the samples for angiogenesis markers. VEGF plays a key role in the differentiation, angiogenesis and formation of primitive endothelial cells. Fig. 9 As shown, the VEGF expression levels in the CGMS-β-CD group and DFO@CGMS-β-CD group were significantly higher than those in the PP mesh group and DFO@Gel-β-CD group 30 days after surgery, which will promote angiogenesis and formation; as the results observed in HE staining in this experimental example, the microvessel density in the CGMS-β-CD group and DFO@CGMS-β-CD group was significantly higher than that in the PP mesh group and DFO@Gel-β-CD group 90 days after surgery.

[0163] Next, this experimental example performed immunostaining for the vascular smooth muscle cell marker α-smooth muscle actin (α-SMA) ( Fig.10), further verifying the angiogenesis. As expected, at 30 days after surgery, there was almost no positive signal in the PP mesh group and the DFO@Gel-β-CD group. On the contrary, the CGMS-β-CD group and the DFO@CGMS-β-CD group had obvious positive signals. At 90 days after surgery, the expression density of α-SMA in the CGMS-β-CD group and the DFO@CGMS-β-CD group was significantly higher than that in the PP mesh group and the DFO@Gel-β-CD group. At the same time, this experimental example found that the capillaries formed in the DFO@CGMS-β-CD group had a larger diameter and thicker wall, which may be attributed to the presence of DFO with the ability to promote angiogenesis. The above results show that compared with the non-degradable PP mesh and DFO@Gel-β-CD gel-based scaffold materials, the microsphere scaffold materials, namely CGMS-β-CD and DFO@CGMS-β-CD, have obvious advantages in promoting the repair of abdominal wall tissue defects. In addition, the introduction of DFO can indeed promote the regeneration of abdominal wall tissue and angiogenesis.

[0164] It should be noted that in the experimental examples of the present invention, all quantitative data were tested for no less than three times, and the data were presented as mean ± standard deviation (SD). The data between the two groups were compared using t-test. The data with P < 0.05 were considered statistically significant.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing β-cyclodextrin modified gelatin microspheres, characterized in that: The steps include: (a) stirring and dispersing an aqueous solution of β-cyclodextrin-modified gelatin and gelatin mixed into an oil phase, and cooling the mixture in an ice-water bath to prepare physically cross-linked β-cyclodextrin-modified gelatin microspheres; (b) chemically cross-linking the physically cross-linked β-cyclodextrin-modified gelatin microspheres obtained in step (a) using a chemical cross-linking agent to obtain the β-cyclodextrin-modified gelatin microspheres.

2. The preparation method according to claim 1, characterized in that: In the step (a), gelatin is added to a carboxymethyl-β-cyclodextrin solution containing a cross-linking agent to obtain the β-cyclodextrin-modified gelatin; Preferably, the method further comprises a dialysis step, and the β-cyclodextrin-modified gelatin is obtained after dialysis; Preferably, the crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N,N′-dicyclohexylcarbodiimide, N,N′-carbonyldiimidazole, and [1-hydroxybenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate.

3. The preparation method according to claim 2, characterized in that: The step (a) comprises: (a1) dissolving 1 g of carboxymethyl-β-cyclodextrin in 100-500 ml of PBS to obtain a carboxymethyl-β-cyclodextrin solution; (a2) adding 0.3-3.2 g of EDC and 0.2-2.5 g of NHS to the carboxymethyl-β-cyclodextrin solution, adjusting the pH to 4-6, to obtain a carboxymethyl-β-cyclodextrin solution containing a cross-linking agent; (a3) stirring the carboxymethyl-β-cyclodextrin solution containing the cross-linking agent at 35 to 45° C. for 10 to 40 minutes, and then adding 0.5 to 2.5 g of gelatin; (a4) After reacting for 24 to 72 hours, dialyzing in water for 2 to 5 days and then freeze-drying to obtain the β-cyclodextrin-modified gelatin.

4. The preparation method according to claim 1, characterized in that: In the step (a), the mass ratio of the β-cyclodextrin-modified gelatin to gelatin is 1:0.5-5; Preferably, the volume ratio of the aqueous solution of the β-cyclodextrin-modified gelatin mixed with gelatin to the oil phase is 1:3 to 10; Preferably, the oil phase comprises paraffin oil and edible oil in a volume ratio of 2 to 4:1; Preferably, after cooling in an ice-water bath, the step of washing the precipitate is further included, preferably using 1,4-dioxane and acetone to wash the precipitate alternately for 3 times, and then washing it with anhydrous ethanol for 2 times.

5. The preparation method according to claim 1, characterized in that: In the step (b), 0.5 g of the physically cross-linked β-cyclodextrin-modified gelatin microspheres are dissolved in 15 to 30 ml of a solvent, and then chemically cross-linked; the solvent includes anhydrous ethanol, or a mixed solution of PBS and ethanol; Preferably, the chemical cross-linking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, N,N′-dicyclohexylcarbodiimide, N,N′-dicyclohexylcarbodiimide, N,N′-carbonyldiimidazole, and [1-hydroxybenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, genipin, formaldehyde and glutaraldehyde, preferably 0.2-0.3 g EDC and 0.1-0.2 g NHS; Preferably, the chemical cross-linking condition is mixing at 0 to 55° C. for 1 to 24 hours; Preferably, the method further comprises the steps of washing with water and then drying.

6. β-cyclodextrin modified gelatin microspheres prepared by the preparation method according to any one of claims 1 to 5.

7. β-cyclodextrin-modified gelatin microspheres loaded with deferoxamine, characterized in that: Deferoxamine is loaded in the β-cyclodextrin-modified gelatin microspheres as claimed in claim 6.

8. The method for preparing the deferoxamine-loaded β-cyclodextrin-modified gelatin microspheres according to claim 7, characterized in that: The β-cyclodextrin modified gelatin microspheres are immersed in a deferoxamine solution to obtain the deferoxamine-loaded β-cyclodextrin modified gelatin microspheres.

9. The preparation method according to claim 8, characterized in that: The solvent of the deferoxamine solution includes 60% ethanol aqueous solution; Preferably, the soaking comprises soaking the microspheres at 4 degrees for 4 hours, ultrasonicating for 5 minutes, and then soaking the microspheres at 4 degrees for 12 hours; Preferably, the method further comprises the step of washing with water twice.

10. Use of the β-cyclodextrin modified gelatin microspheres according to claim 6 and the β-cyclodextrin modified gelatin microspheres loaded with deferoxamine according to claim 7 in the preparation of tissue repair products; Preferably, the tissue repair comprises abdominal wall tissue defect repair.

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