An antibacterial carboxylated collagen-based corneal repair material and a preparation method thereof

Abstract

The present application belongs to the technical field of tissue engineering keratoprosthesis, and particularly relates to an antibacterial carboxylated collagen-based keratoprosthesis repair material. The present application utilizes citric acid to perform carboxylated modification on a collagen membrane, increases carboxyl functional groups on the surface of the collagen membrane which can produce amide bonding reaction with aminoglycoside antibiotics, and then increases the amount of aminoglycoside antibiotics loaded on the surface through chemical crosslinking, so as to prepare a high-efficiency antibacterial carboxylated collagen-based membrane. The present application utilizes 1-ethyl-3-(3-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as crosslinking agents to graft aminoglycoside antibiotics to the surface of the carboxylated collagen membrane, so as to realize high-efficiency antibacterial functionalization of the collagen-based biomaterial on the basis of not changing the good physical and chemical properties and excellent biocompatibility of the collagen-based membrane.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering artificial cornea technology, and specifically relates to an antibacterial carboxylated collagen-based corneal repair material and its preparation method. Background Technology

[0002] A significant clinical problem affecting millions of blind people is corneal disease, and corneal transplantation is one of the effective treatments for corneal blindness. However, the severe lack of high-quality allogeneic corneal tissue limits its clinical application. Therefore, researchers both domestically and internationally have focused on developing corneal tissue alternatives using natural biomaterials. Among these, collagen, as the main load-bearing component of connective tissue, possesses excellent physicochemical properties and biocompatibility. Therefore, it has been extensively studied as a scaffold material for corneal tissue engineering.

[0003] While collagen-based scaffolds offer numerous advantages in replacing pathological corneal tissue, their application often leads to intraoperative or postoperative bacterial infections, sometimes even causing transplantation or repair failure. Currently, to reduce the risk of bacterial infection from collagen-based biomaterials, researchers both domestically and internationally are attempting to enhance their antibacterial properties through physical or chemical methods, such as adding antibiotics, antibacterial inorganic nanoparticles, and polycationic antibacterial agents. However, achieving highly efficient antibacterial functionalization without significantly affecting the excellent optical properties, superior biocompatibility, and low immunogenicity of collagen-based corneal repair materials is crucial for improving their clinical application. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a novel, highly efficient antibacterial carboxylated collagen-based corneal repair material and its preparation method. This study utilizes citric acid to carboxylate and modify collagen membranes, increasing the number of carboxyl functional groups on their surface capable of forming amide bonds with aminoglycoside antibiotics. Further, chemical crosslinking is used to increase the amount of aminoglycoside antibiotics loaded on the surface, thereby preparing a highly efficient antibacterial carboxylated collagen-based film. Aminoglycoside antibiotics, by inhibiting bacterial protein synthesis, have effective antibacterial effects against most Gram-positive and Gram-negative aerobic bacteria. In this study, 1-ethyl-3-(3-3-dimethylaminopropyl)carbodiimide (EDC) and n-hydroxysuccinimide (NHS) are used as crosslinking agents to graft aminoglycoside antibiotics onto the surface of the carboxylated collagen membrane. Through this method, we aim to achieve highly efficient antibacterial functionalization of collagen-based biomaterials without altering the good physicochemical properties and excellent biocompatibility of the collagen-based film.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A highly effective antibacterial carboxylated collagen-based corneal repair material includes the following steps:

[0007] (1) Prepare a collagen solution, using carbodiimide and N-hydroxysuccinyl as crosslinking agents and catalysts respectively, stir thoroughly to produce a crosslinking reaction, pour the crosslinked collagen solution into a mold, and air dry it to form a collagen film (Col);

[0008] (2) Prepare a citric acid solution and use citric acid molecules to chemically modify the surface of the collagen membrane obtained in step (1) to obtain a carboxylated collagen membrane;

[0009] (3) Prepare an aminoglycoside antibiotic solution, immerse the carboxylated collagen membrane obtained in step (2) in the aminoglycoside antibiotic solution, and add carbodiimide and N-hydroxysuccinyl as crosslinking agent and catalyst respectively, so that the aminoglycoside antibiotic molecules and carboxylated collagen molecules undergo chemical crosslinking to form amide bonds, and obtain an aminoglycoside antibiotic-carboxylated collagen membrane crosslinking system;

[0010] (4) The aminoglycoside antibiotic-carboxylated collagen membrane crosslinking system obtained in step (3) is placed in a mold and air-dried to form a film, thus forming a novel antibacterial carboxylated collagen corneal repair material containing aminoglycoside antibiotics.

[0011] Further, in step (1), the collagen solution has a mass concentration of 7~10 mg / mL, and the collagen in the collagen solution is bovine Achilles tendon type I collagen.

[0012] Further, in step (1), the mass ratio of collagen, carbodiimide, and N-hydroxysuccinyl is (6.0~6.5):(1~1.2):(1~1.2).

[0013] Further, in the citric acid solution described in step (2), the mass ratio of citric acid to collagen is 1-1.5:1; and the mass concentration of the citric acid solution is 2 mg / mL.

[0014] Furthermore, the aminoglycoside antibiotics mentioned in step (3) typically include streptomycin, gentamicin, tobramycin, spectinomycin, amikacin, netilmicin, etilmicin, isopamicin, etc.; the mass concentration of the aminoglycoside antibiotic solution is 15 mg / mL.

[0015] Furthermore, in step (3), the mass ratio of carboxylated collagen, carbodiimide, and N-hydroxysuccinyl is (6.0~6.5):(1~1.2):(1~1.2).

[0016] In this study, addressing the limitation of traditional collagen-based antibacterial corneal materials due to the limited antibacterial components, we successfully developed a novel, highly efficient antibacterial carboxylated collagen-based corneal repair material. This material enhances the antibacterial properties of the collagen-based material without altering its excellent physicochemical properties and biocompatibility. Citric acid, as an edible acid, possesses good biocompatibility. After modification with citric acid, the collagen-based film still retains its good physicochemical properties and excellent biocompatibility. Clearly, the chemical modification of collagen with citric acid increases the number of carboxyl groups on the collagen surface, thereby increasing the number of aminoglycoside antibiotic molecules grafted onto the Col membrane surface via amide bonding, thus achieving highly efficient antibacterial functionalization of the collagen-based material. Preliminary results indicate that this novel, highly efficient antibacterial carboxylated collagen-based film may have potential applications in corneal tissue engineering. Attached Figure Description

[0017] Figure 1 The transmittance of the gentamicin-containing antibacterial carboxylated collagen film prepared in Example 2 of this invention;

[0018] Figure 2 Morphology of the gentamicin-containing antibacterial carboxylated collagen cornea prepared in Example 2 of this invention;

[0019] Figure 3 Microscopic images (left) and fluorescence staining images (right) of human corneal epithelial cells cultured with gentamicin-containing antibacterial carboxylated collagen cornea for 48 hours, prepared as in Example 2 of this invention.

[0020] Figure 4 The carboxylated collagen membrane loaded with tobramycin prepared in Example 5 of the present invention (right figure), and the anti-Escherichia coli performance of the collagen membrane loaded with tobramycin (middle figure) and the collagen membrane (left figure) are compared.

[0021] Figure 5 The carboxylated collagen membrane loaded with tobramycin prepared in Example 5 of this invention (right figure) is compared with the anti-Staphylococcus aureus performance of the collagen membrane loaded with tobramycin (middle figure) and the collagen membrane (left figure). Implementation

[0022] To better understand the present invention, the following description is based on embodiments, but the scope of protection of the present invention is not limited to the scope represented by the embodiments mentioned.

[0023] In the specific embodiments of the present invention, the collagen used is bovine Achilles tendon type I collagen, purchased from HSBC Biotechnology Co., Ltd.; citric acid is purchased from Aladdin® (Shanghai, China); EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are both purchased from Aladdin® (Shanghai, China).

[0024] Example 1

[0025] A novel, highly effective antibacterial, carboxylated collagen-based corneal repair material containing gentamicin was prepared using collagen, aminoglycoside antibiotics, and citric acid as raw materials. The preparation steps of this highly effective collagen-based antibacterial material are as follows:

[0026] (1) Dissolve 3.5g of collagen in 0.1mol / mL HCl and dilute to 500mL to prepare a collagen solution with a concentration of 7mg / mL. Then, add collagen to the collagen solution according to the mass of the collagen in the solution. Col :m EDC :m NHS Add 583 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 583 mg of n-hydroxysuccinimide (NHS) in a ratio of 6:1:1, stir thoroughly for 5 hours to allow cross-linking reaction to occur, and dispense the cross-linked collagen solution into 35 mL molds and air dry to form a film (Col).

[0027] (2) Prepare 130 mL of 2 mg / mL citric acid solution;

[0028] (3) Immerse 0.2508g of the Col film dried in step (1) into the citric acid solution in step (2) for full absorption, and then air dry to form a film (ColCA);

[0029] (4) Prepare 130 mL of 15 mg / mL gentamicin (GM) solution;

[0030] (5) At room temperature, according to m ColCA+ m GM :m EDC :m NHS EDC and NHS were added to the gentamicin solution obtained in step (4) at a mass ratio of 6:1:1, and the ColCA film was stirred and reacted for 5 hours.

[0031] (6) The ColCA-GM film obtained by crosslinking in step (5) is rinsed three times with deionized water and air-dried to form a film, thus obtaining the ColCA-GM film. The water absorption rate and tensile strength of the film are tested, and the results are shown in Table 1. The gentamicin drug loading of the film is tested, and the results are shown in Table 2.

[0032] Example 2

[0033] The difference between this embodiment and Example 1 is that 4g of collagen was dissolved in 0.1mol / mL HCl and diluted to 500mL to prepare 500mL of a collagen solution with a concentration of 8mg / mL. The remaining procedures were the same as in Example 1. The water absorption rate and tensile strength of the film were tested, and the results are shown in Table 1. The gentamicin drug loading of the film was tested, and the results are shown in Table 2.

[0034] The transmittance of the novel gentamicin-containing, highly effective antibacterial carboxylated collagen-based film prepared in Example 2 was evaluated. From... Figure 1 As can be seen, the transparency of the ColCA-GM film differs slightly from that of the Col film, with the former being slightly higher. With increasing wavelength, the transmittance of the film increases to its maximum value (above 90%). The transmittance of both films tends to remain constant in the visible light range.

[0035] Figure 2 Morphological image of the novel gentamicin-containing antibacterial carboxylated collagen-based cornea prepared in Example 2: As can be seen from the figure, the film is transparent, with a smooth and uniform surface and no obvious phase separation.

[0036] Figure 3 Microscopic images (left) and fluorescence-stained images (right) of human corneal epithelial cells cultured on the novel gentamicin-containing antibacterial carboxylated collagen-based cornea prepared in Example 2 after 48 hours: After 48 hours of seeding on the material surface, the cells completely covered the entire surface of the ColCA-GM membrane. To observe the proliferation and adhesion of HCECs on the cell membrane surface, we used immunofluorescence staining to observe cell growth. Blue fluorescence was used to observe the cell nucleus, and red fluorescence was used to observe the cytoskeleton. The blue fluorescence appeared circular, and the red fluorescence appeared spindle-shaped, indicating that the cells had adhered to the cell membrane surface and were growing well.

[0037] Example 3

[0038] The difference between this embodiment and Example 1 is that 4.5g was dissolved in 0.1mol / mL HCl and the solution was diluted to 500mL to prepare a collagen solution with a concentration of 9mg / mL. The remaining procedures were the same as in Example 1. The water absorption rate and tensile strength of the film were tested, and the results are shown in Table 1. The gentamicin drug loading of the film was tested, and the results are shown in Table 2.

[0039] The test methods for water absorption and tensile strength are as follows:

[0040] (1) The specific method for testing water absorption rate is as follows: Cut the dried sample into a square film of 20 mm × 20 mm, weigh the film using a balance and record the weight as Mo. Place the sample film in PBS (10 mmol / L, pH=7.4) solution, and after 24 h, quickly absorb the surface moisture with filter paper. Weigh and record the sample mass as Mw. Perform five parallel experiments for each sample and calculate the average value. The water absorption rate of the sample is calculated according to formula (Ⅰ): X=(Mw-Mo) / Mw×100% (Formula Ⅰ)

[0041] Where Mo is the weight of the dry dressing (g); Mw is the weight of the film after water absorption (g); and X is the saturated water absorption rate of the film (%).

[0042] (2) Tensile strength: The test method was conducted according to the pharmaceutical industry standard YY / T 0471.4-2004, using an electronic tensile testing machine to test the tensile strength of the film. The specific steps are as follows: The sample was cut into a long strip with a length of 90 mm and a width of 25 mm. The thickness was measured with vernier calipers and recorded. Tensile testing was performed under constant temperature and humidity conditions (temperature 25℃, relative humidity 70%). The clamping distance of the sample was 50 mm, and the tensile rate was 300 mm / min. The test procedure was set according to the test method, and the test was conducted. Five sets of valid data were collected for each test, and the average value was taken. The tensile strength (TS) of the dressing was calculated according to the following formula: TS=F max / (L×W) (Formula II)

[0043] Where TS is tensile strength (MPa), F max L is the maximum tensile force (N) that the sample can withstand when it breaks, L is the thickness of the dressing (mm), and W is the width of the dressing (mm).

[0044] Table 1. Water absorption rate and tensile strength of the films prepared in Examples 1-3

[0045]

[0046] Table 1 shows that the saturated water absorption rate of the antibacterial carboxylated collagen-based materials prepared in this invention is all above 90%, exhibiting excellent water absorption performance. This high water absorption rate is mainly due to the presence of many hydrophilic groups such as amino and carboxyl groups in the molecules of collagen, gentamicin, and citric acid. Considering the application of collagen-based materials in the cornea, good water absorption can provide a suitable environment for cell growth and adhesion to its surface. Furthermore, compared with Col films and ColCA films, the tensile strength of the antibacterial carboxylated collagen-based films prepared in this invention is all above 9 MPa, demonstrating improved mechanical properties. This is attributed to the modification of citric acid molecules and the introduction of gentamicin molecules, which strengthens the intermolecular interactions and increases the steric hindrance effect of molecular motion, thereby enhancing its tensile strength.

[0047] The gentamicin drug loading of the membrane was tested, and the results are shown in Table 2. The test methods for the gentamicin drug loading of the membrane are as follows:

[0048] The sample film was placed in a 15 mg / mL gentamicin solution, according to m Col +m GM :m EDC :m NHS EDC and NHS were added to a 15 mg / mL gentamicin solution in a 6:1:1 ratio, and 3 ml of this solution was taken as mother liquor 1. The mixture was then stirred for 5 hours to allow for complete cross-linking, and 3 ml of this solution was taken as liquid 2 after cross-linking. Finally, the absorbance of mother liquor 1 and liquid 2 was measured using a UV spectrophotometer (X1 and X2, respectively), and the gentamicin loading in the film was calculated. The formula for drug loading is as follows:

[0049] X = (X1 - X2) / X1 × 100% (Formula III)

[0050] Where X1 is the absorbance value of mother liquor 1; X2 is the absorbance value of liquid 2; and X is the drug loading rate of the film (%).

[0051] Table 2 Gentamicin drug loading of films prepared in Examples 1-3

[0052]

[0053] Table 2 shows that the gentamicin drug loading of the carboxylated collagen-based material prepared in this invention is significantly higher than that of the control group. This is mainly because the carboxylation modification of the collagen membrane by citric acid increases the number of carboxyl functional groups on the surface of the collagen membrane that can react with gentamicin, thereby increasing the gentamicin drug loading of the material and improving its antibacterial properties.

[0054] Example 4

[0055] A novel, highly effective antibacterial carboxylated collagen-based corneal repair material containing tobramycin was prepared using collagen, tobramycin, and citric acid as raw materials. The preparation steps of this highly effective collagen-based antibacterial material are as follows:

[0056] (1) Prepare 500 mL of collagen solution with a concentration of 8 mg / mL. Then add the collagen solution according to m Col :m EDC :m NHS 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and n-hydroxysuccinimide (NHS) were added in a ratio of 6:1:1. The mixture was stirred thoroughly for 5 hours to induce a cross-linking reaction. The cross-linked collagen solution was then dispensed into 35mL molds and air-dried to form a film (Col).

[0057] (2) Prepare 130 mL of 2 mg / mL citric acid solution;

[0058] (3) Immerse the Col film dried in step (1) in the 2 mg / ml citric acid solution in step (2) until fully absorbed, and then air dry to form a film (ColCA).

[0059] (4) Prepare 130 mL of a 15 mg / mL tobramycin solution;

[0060] (5) At room temperature, according to m ColCA +m Tob :m EDC :m NHS EDC and NHS were added to the tobramycin solution obtained in step (4) in a ratio of 6:1:1, and the ColCA film was stirred and reacted for 5 hours. The film was then rinsed three times with deionized water and air-dried to form a film, thus obtaining the ColCA-Tob film.

[0061] (6) At room temperature, the Col membrane from step (1) is directly loaded with Tob, according to m Col +m Tob :m EDC :m NHS EDC and NHS were added to the tobramycin solution obtained in step (4) in a ratio of 6:1:1, and the Col film was stirred and reacted for 5 hours. The film was then rinsed three times with deionized water and air-dried to form a film, thus obtaining the Col-Tob film.

[0062] Figure 4 The carboxylated collagen membrane loaded with tobramycin prepared for this embodiment (right figure), the collagen membrane loaded with tobramycin (middle figure), and the anti-Escherichia coli performance of the collagen membrane (left figure) are compared: As can be seen from the figures, compared with the collagen membrane, the antibacterial performance of the collagen-based film loaded with tobramycin after citric acid carboxylation modification is significantly improved.

[0063] Figure 5 The carboxylated collagen membrane loaded with tobramycin prepared for this embodiment (right figure), the collagen membrane loaded with tobramycin (middle figure), and the collagen membrane (left figure) are compared in terms of their anti-Staphylococcus aureus performance: As can be seen from the figures, compared with the collagen membrane, the antibacterial performance of the collagen-based film loaded with tobramycin after citric acid carboxylation modification is significantly improved.

Claims

1. A method for preparing an antibacterial carboxylated collagen-based corneal repair material, characterized in that, Includes the following steps: (1) Prepare a collagen solution with a concentration of 7~10mg / mL, using carbodiimide and N-hydroxysuccinyl as crosslinking agents and catalysts respectively. The mass ratio of collagen, carbodiimide and N-hydroxysuccinyl is 6.0~6.5:1~1.2:1~1.

2. Stir thoroughly to induce a crosslinking reaction, pour the crosslinked collagen solution into a mold, and air dry it to form a collagen film; (2) Prepare a citric acid solution with a concentration of 2 mg / mL, and use citric acid molecules to chemically modify the surface of the collagen membrane obtained in step (1) to obtain a carboxylated collagen membrane; (3) Prepare an aminoglycoside antibiotic solution. Soak the carboxylated collagen membrane obtained in step (2) in the aminoglycoside antibiotic solution, and add carbodiimide and N-hydroxysuccinyl as crosslinking agent and catalyst, respectively, so that the aminoglycoside antibiotic molecules and carboxylated collagen molecules undergo chemical crosslinking and form amide bonds to obtain an aminoglycoside antibiotic-carboxylated collagen membrane crosslinking system; the concentration of the aminoglycoside antibiotic solution is 15 mg / mL; the mass ratio of carboxylated collagen membrane, carbodiimide and N-hydroxysuccinyl is 6.0~6.5:1~1.2:1~1.2; (4) The aminoglycoside antibiotic-carboxylated collagen membrane crosslinking system obtained in step (3) is placed in a mold and air-dried to form a film, thereby obtaining an antibacterial carboxylated collagen-based corneal repair material containing aminoglycoside antibiotics; The aminoglycoside antibiotics are selected from one or more of streptomycin, gentamicin, tobramycin, spectinomycin, amikacin, netilmicin, etilmicin, and isapamicin.

2. The method for preparing the antibacterial carboxylated collagen-based corneal repair material according to claim 1, characterized in that, The collagen is bovine Achilles tendon type I collagen.

3. The method for preparing the antibacterial carboxylated collagen-based corneal repair material according to claim 1, characterized in that, In step (2), the mass ratio of citric acid to collagen membrane is 1-1.5:

1.

4. An antibacterial carboxylated collagen-based corneal repair material, characterized in that, It is prepared by the method described in any one of claims 1-3.

Images