Electrostatic spinning fiber membrane, preparation method and application

Abstract

According to the electrostatic spinning fiber membrane and the preparation method thereof provided by the invention, the electrostatic spinning fiber membrane selects poly (L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan as materials, and the toughness of the poly (L-lactide-co-caprolactone) and the strength of polyurethane are utilized to improve the mechanical properties of the electrostatic spinning fiber; meanwhile, due to the addition of carboxymethyl chitosan, the hydrophilicity of the electrospun fibrous membrane is improved, the electrospun fibrous membrane is more beneficial to cell adhesion, and the electrospun fibrous membrane has a fibrous structure similar to an extracellular matrix, the hydrophilicity is improved, the mechanical property is enhanced, and the electrospun fibrous membrane has a proper swelling ratio and good biocompatibility and has a good application prospect in the field of blood contact biomedical materials.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to an electrospun fiber membrane, a preparation method and applications thereof. Background Art

[0002] Traditional blood-contact materials have the following limitations: First, intrinsic toxicity: Fibrinogen adsorbed on the surface of the material activates Toll-like receptors on macrophages, releasing pro-inflammatory factors, triggering an inflammatory cascade and causing cell damage. Second, traditional materials suffer from excessive surface roughness and poor mechanical stability, making them unable to maintain surface hydrophilicity over the long term. Furthermore, their uneven nanostructure accelerates fibrinogen adsorption and platelet adhesion, leading to thrombosis.

[0003] By regulating the parameters of the polymer solution (such as viscosity, conductivity, etc.) and process conditions (voltage, receiving distance, etc.), electrospinning technology can prepare a fiber network structure with multi-scale characteristics (single filament diameter range of 50nm~5μm, porosity of 70~95%). Its topological morphology is highly biomimetic of the collagen fiber arrangement pattern of the natural extracellular matrix (ECM), thereby significantly improving the efficiency of cell adhesion. Based on the composite spinning system of synthetic polymers and natural polymers, the mechanical properties and degradation rate of the scaffold can be accurately matched to meet the needs of different clinical scenarios. At the same time, by optimizing the motion mode of the receiving device (such as adjusting the linear speed of the rotating drum and the translation speed of the spinneret), the fibers can be arranged in a direction to form a specific orientation structure, and its pore size distribution forms a multi-scale coupling effect with the pore topological characteristics of the natural ECM.

[0004] Polyurethane (PU) is a semicrystalline polymer with a structure consisting of alternating hard and soft segments. The functional groups of polycaprolactone (PCL) are similar to those of PU. Poly(l-lactic acid)-co-poly(ε-caprolactone), PLCL, is a random copolymer of lactic acid and caprolactone. It has a similar structure to PCL and exhibits excellent biocompatibility, making it a promising candidate for use as a tissue engineering scaffold in applications such as blood vessels, tendons, and cartilage. Carboxymethyl chitosan (CMCS) is a water-soluble derivative of chitosan with functionalizable chemical groups (-NH2 and -COOH), antioxidant properties, and antimicrobial properties.

[0005] At present, there is no similar report on the preparation of electrospun fiber membranes for blood-contact biomedical materials based on the advantages of the above materials. Summary of the Invention

[0006] In view of this, it is necessary to provide a preparation method and application of a biocompatible electrospun fiber membrane to address the defects of the existing technology.

[0007] To solve the above problems, this application adopts the following technical solutions:

[0008] One of the purposes of this application is to provide a method for preparing an electrospun fiber membrane, comprising the following steps:

[0009] dissolving poly(L-lactide-co-caprolactone), polyurethane, and carboxymethyl chitosan in an organic solvent at a mass ratio of 1:(1-9):(0.25-1) to obtain a polymer solution;

[0010] electrospinning the polymer solution to obtain an electrospun membrane;

[0011] The electrospun membrane is dried and then subjected to steam cross-linking treatment;

[0012] The electrospun membrane after steam cross-linking treatment is sterilized to obtain the electrospun fiber membrane.

[0013] In some embodiments, in the step of dissolving poly(L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan in an organic solvent in a mass ratio of 1:(1-9):(0.25-1) to obtain a polymer solution, the number average molecular weight of the poly(L-lactide-co-caprolactone) is 10-200 kDa, the molar ratio of lactide to caprolactone is 25:75-70:30, the density of the polyurethane is 1.1-1.3 g / mL, and the degree of carboxylation of the carboxymethyl chitosan is 87%-90%.

[0014] In some embodiments, the organic solvent includes a mixed solvent of chloroform and N,N-dimethylformamide, and the volume ratio of chloroform to N,N-dimethylformamide is (4-8):1.

[0015] The concentration of the polymer solution is 100-200 mg / mL.

[0016] In some embodiments, in the step of electrospinning the polymer solution to obtain an electrospun membrane, the electrospinning conditions are as follows: the stainless steel needle is 18-20G, the syringe capacity is 5-20mL, the injection rate is 0.5-1.5mL / h, the electrospinning voltage is 10-15kV, the ambient temperature is 30-35°C, the ambient humidity is 25%-30%, the receiving distance is 15-20cm, the electrospinning time is 8-12h, and the thickness of the electrospun fiber membrane is 10-70μm.

[0017] In some embodiments, the step of steam cross-linking the electrospun membrane after drying specifically includes the following steps:

[0018] The electrospun membrane was vacuum dried for more than 96 hours to remove the organic solvent remaining on the surface;

[0019] The dried electrospun membrane is cross-linked with glutaraldehyde vapor at room temperature for 1 to 3 hours.

[0020] In some embodiments, the step of sterilizing the electrospun membrane after steam cross-linking treatment to obtain the electrospun fiber membrane specifically includes the following steps:

[0021] The electrospun membrane after steam cross-linking treatment is maintained in a disinfectant for 30 to 60 minutes, and then vacuum dried for 12 to 48 hours to obtain the electrospun fiber membrane, and the disinfectant includes a glycine solution.

[0022] The second purpose of this application is to provide an electrospun fiber membrane prepared by the preparation method.

[0023] In some embodiments, the tensile strength of the electrospun fiber membrane can reach above 5 MPa, the thickness is 10-70 μm, and the electrospun fiber membrane is composed of fibers of 1-2 μm.

[0024] The third purpose of this application is to provide an application of the electrospun fiber membrane in blood-contact biomedical materials.

[0025] This application adopts the above technical solution, and its beneficial effects are as follows:

[0026] The present application provides an electrospun fiber membrane and a preparation method thereof. The electrospun fiber membrane uses poly(L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan as materials, and utilizes the toughness of poly(L-lactide-co-caprolactone) and the strength of polyurethane to improve the mechanical properties of the electrospun fiber. At the same time, the addition of carboxymethyl chitosan improves the hydrophilicity of the electrospun fiber membrane, making it more conducive to cell adhesion. It has a fiber structure similar to that of the extracellular matrix, with improved hydrophilicity and enhanced mechanical properties, and has a suitable swelling rate and good biocompatibility, and has good application prospects in the field of blood-contact biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0028] Figure 1This is a flow chart of the steps of the method for preparing the electrospun fiber membrane provided in Example 1 of the present application.

[0029] Figure 2 This is the infrared spectrum of the PU-PLCL-CMCS electrospun nanofibers provided in Example 1 of the present application.

[0030] Figure 3 The CCK-8 method provided in Example 1 of the present application was used to measure the cell activity (c) and OD value (d) corresponding to cell proliferation of four groups of electrospun fiber scaffolds after 1 day (a), 4 days (b), and 7 days.

[0031] Figure 4 This is a scanning electron microscope image of the attachment and morphology of cells 48 hours after the incubation provided in Example 1 of the present application. DETAILED DESCRIPTION

[0032] The following describes in detail embodiments of the present application, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0033] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on this application.

[0034] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0035] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0036] See also Figure 1 , is a flow chart of the steps of the method for preparing the electrospun fiber membrane provided in an embodiment of the present application, comprising the following steps:

[0037] Step S110: dissolving poly(L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan in an organic solvent at a mass ratio of 1:(1-9):(0.25-1) to obtain a polymer solution.

[0038] In this embodiment, the number average molecular weight of the poly(L-lactide-co-caprolactone) is 200 kDa, the molar ratio of lactide to caprolactone is 70:30, the density of the polyurethane is 1.18 g / mL, and the degree of carboxylation of the carboxymethyl chitosan is 87% to 90%.

[0039] In this embodiment, the organic solvent includes a mixed solvent of chloroform and N,N-dimethylformamide, and the volume ratio of chloroform to N,N-dimethylformamide is (4-8):1.

[0040] In this embodiment, the concentration of the polymer solution is 100-200 mg / mL.

[0041] As can be understood, since polyurethane (PU) is a semicrystalline polymer with a structure of alternating hard and soft segments, electrospun nanofibers prepared from PU exhibit excellent biocompatibility and high mechanical and viscoelastic properties. Polycaprolactone (PCL) has functional groups similar to those of PU and can serve as an ideal reinforcing agent for PU electrospun membranes. Poly(L-lactic acid)-co-poly(ε-caprolactone) (PLCL), formed by the random copolymerization of lactic acid and caprolactone, has a similar structure to PCL and good biocompatibility, and has been used as a tissue engineering scaffold for applications such as blood vessels, tendons, and cartilage. Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan, possesses functionalizable chemical groups (-NH2 and -COOH), antioxidant and antimicrobial properties, promoting wound healing and promoting collagen secretion. Thus, PLCL, CMCS and PU are mixed and prepared into electrospun membranes which can be applied in the field of biomedical materials.

[0042] Step S120: electrospinning the polymer solution to obtain an electrospun membrane.

[0043] In this embodiment, in the step of electrospinning the polymer solution to obtain an electrospun membrane, the electrospinning conditions are as follows: the stainless steel needle is 18-20G, the syringe capacity is 5mL, the injection rate is 0.5-1.5mL / h, the electrospinning voltage is 10-15kV, the ambient temperature is 30-35°C, the ambient humidity is 25%-30%, the receiving distance is 15-20cm, the electrospinning time is 8-12h, and the thickness of the electrospun fiber membrane is 10-70μm.

[0044] Step S130: drying the electrospun membrane and then performing a steam cross-linking treatment.

[0045] In this embodiment, the step of steam cross-linking the electrospun membrane after drying specifically includes the following steps: placing the electrospun membrane obtained in step S120 in a vacuum drying oven and drying it for more than 96 hours to remove the organic solvent remaining on the surface; then taking out the dried electrospun membrane, cutting it into the same size with scissors, placing it on the partition in the middle of the dryer, adding 10% glutaraldehyde solution at the bottom, the volume of glutaraldehyde is 10 mL, the mass ratio is 50%, and steam cross-linking is performed at room temperature for 3 hours.

[0046] Step S140: sterilizing the electrospun membrane after the steam cross-linking treatment to obtain the electrospun fiber membrane.

[0047] In this embodiment, the cross-linked material in step S130 is immersed in a disinfectant for 30 minutes and then placed in a vacuum drying oven for drying for 12 hours to obtain a biomedical material suitable for blood contact. The disinfectant includes a glycine solution.

[0048] The electrospun fiber membrane provided in the present application has a fiber structure similar to that of the extracellular matrix, has improved hydrophilicity, enhanced mechanical properties, and has a suitable swelling rate and good biocompatibility.

[0049] In this embodiment, the tensile strength of the electrospun fiber membrane can reach above 5 MPa, the thickness is 10-70 μm, and the electrospun fiber membrane is composed of fibers with a thickness of 1-2 μm.

[0050] The electrospun fiber membrane and preparation method provided in the above embodiments of the present application use poly(L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan as materials, and utilize the toughness of poly(L-lactide-co-caprolactone) and the strength of polyurethane to improve the mechanical properties of the electrospun fiber; at the same time, the addition of carboxymethyl chitosan improves the hydrophilicity of the electrospun fiber membrane, making it more conducive to cell adhesion, and has a fiber structure similar to the extracellular matrix, with improved hydrophilicity and enhanced mechanical properties, and has a suitable swelling rate and good biocompatibility, and has good application prospects in the field of blood-contact biomedical materials.

[0051] Example 1

[0052] Poly(L-lactide-co-caprolactone) and polyurethane were dissolved in a mixed solution of chloroform and N,N-dimethylformamide (DMF) at a mass ratio of 1:1. The volume ratio of chloroform:DMF was 1:1 to prepare a solution with a concentration of 130 mg / mL. The solution was stirred to obtain a stable electrospinning emulsion. CMCS of different proportions was added thereto to make the mass ratios of PU / PLCL / CMCS 4:4:0, 4:4:1, 4:4:2, and 4:4:4, respectively. The stirring was continued to obtain polymer solutions with different compositions. The specific components are as follows:

[0053] Table 1 shows the composition of the polymer solution

[0054]

[0055] The resulting solution was electrospun using the following conditions: a 20G stainless steel needle (ID = 0.6 mm, OD = 0.9 mm), an injection rate of 1.2 mL / h, an electrospinning voltage of 15 kV, a receiving distance of 20 cm, and an electrospinning time of 8 h. The resulting electrospun fiber membrane had a thickness of 70 μm.

[0056] Dry the electrospun membrane in a vacuum drying oven for at least 96 hours to remove any residual solvent. Cut the membrane into equal pieces with scissors and place them on the middle shelf of the desiccator. Add 10% glutaraldehyde solution (10 mL, 50% by weight) below the membrane. Steam crosslink at room temperature for 3 hours.

[0057] After the cross-linking is completed, the material is taken out and immersed in a glycine solution for 30 minutes to react with the residual glutaraldehyde, and then dried in a vacuum drying oven for 12 hours to obtain a biomedical membrane material.

[0058] The electrospun nanofiber scaffolds with PU, PLCL, and CMCS component ratios of 4:4:0 and 4:4:4 were characterized by FTIR. Figure 2 It can be seen that pure CMCS has a peak at 1585 cm -1 The asymmetric and symmetric stretching vibration peaks of -COO- were observed at 1309 cm -1 The stretching vibration of CN stretching group was observed at 1700 cm -1 and 1728cm -1 There are stretching vibration peaks of C=O and C=N at 3330cm -1 There is a broad peak of NH symmetrical vibration at 1181cm -1 The peak at 1728 cm represents the CO stretching vibration in PLCL. It was observed that the addition of CMCS did not form a new peak, but due to the formation of hydrogen bonds, the electron cloud density was averaged and the group stretching vibration frequency was reduced.-1 After cross-linking, the peak at 3330 cm -1 、1728cm -1 The peak intensity at is significantly weakened, indicating that the amino groups in the blend film react with glutaraldehyde to form a network-type polymer.

[0059] In order to verify the biocompatibility of nanofibers, the CCK-8 method was used to evaluate the cell proliferation of nanofiber scaffolds. The absorbance was measured at a wavelength of 450 nm using a microplate reader. Figure 3 It can be seen that the absorbance of the PU / PLCL / CMCS composite scaffold increases with the increase of culture time, and with the increase of CMCS content, the absorbance also shows an upward trend. The addition of CMCS has greatly promoted the proliferation of cells. The SEM image shows the performance of cells after 48 hours of culture on the composite nanofiber scaffold. Figure 4 As shown, cells spread on the surface of the nanofiber scaffold, showing a flat morphology and attaching to the fibers by extending pseudopodia. This result shows that the composite nanofiber scaffold can provide a good environment for cell adhesion and proliferation.

[0060] Example 2

[0061] Poly(L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan were dissolved in a mixed solution of chloroform and N,N-dimethylformamide (DMF) in a mass ratio of 1:9:1, wherein the volume ratio of chloroform:DMF was 4:1, and a solution with a concentration of 100 mg / mL was prepared and stirred to obtain a stable polymer solution.

[0062] The resulting solution was electrospun under the following conditions: an 18G stainless steel needle (ID = 0.6 mm, OD = 0.9 mm), an injection rate of 0.5 mL / h, an electrospinning voltage of 10 kV, a receiving distance of 15 cm, an electrospinning time of 8 h, an electrospun fiber membrane thickness of 10 μm, an ambient temperature of 30°C, and an ambient humidity of 25%.

[0063] The electrospun membrane was dried in a vacuum drying oven for 100 hours to remove residual solvent. The membrane was then cut into equal pieces with scissors and placed on the middle shelf of the desiccator. A 10% glutaraldehyde solution (10 mL, 50% by weight) was added below the membrane. Steam crosslinking was performed at room temperature for 3 hours.

[0064] After the cross-linking is completed, the material is taken out and immersed in a glycine solution for 30 minutes to react with the residual glutaraldehyde, and then dried in a vacuum drying oven for 12 hours to obtain a biomedical membrane material.

[0065] Example 3

[0066] Poly(L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan were dissolved in a mixed solution of chloroform and N,N-dimethylformamide (DMF) in a mass ratio of 1:4:0.5, wherein the volume ratio of chloroform:DMF was 8:1, and a solution with a concentration of 200 mg / mL was prepared and stirred to obtain a stable polymer solution.

[0067] The obtained solution was electrospun under the following electrospinning conditions: a 19G stainless steel needle (ID = 0.6 mm, OD = 0.9 mm), an injection rate of 1.5 mL / h, an electrospinning voltage of 15 kV, a receiving distance of 20 cm, an electrospinning time of 12 h, an electrospun fiber membrane thickness of 70 μm, an ambient temperature of 35° C., and an ambient humidity of 30%.

[0068] The electrospun membrane was dried in a vacuum drying oven for 110 hours to remove residual solvent. The membrane was then cut into equal pieces with scissors and placed on the middle shelf of the desiccator. A 10% glutaraldehyde solution (10 mL, 50% by weight) was added below the membrane. Steam crosslinking was performed at room temperature for 3 hours.

[0069] After the cross-linking is completed, the material is taken out and immersed in a glycine solution for 60 minutes to react with the residual glutaraldehyde, and then dried in a vacuum drying oven for 48 hours to obtain a biomedical membrane material.

[0070] It can be understood that the various technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within the scope of protection of the present application.

Claims

1. A method for preparing an electrospun fiber membrane, characterized in that: The steps include: dissolving poly(L-lactide-co-caprolactone), polyurethane, and carboxymethyl chitosan in an organic solvent at a mass ratio of 1:(1-9):(0.25-1) to obtain a polymer solution; electrospinning the polymer solution to obtain an electrospun membrane; The electrospun membrane is dried and then subjected to steam cross-linking treatment; The electrospun membrane after steam cross-linking treatment is sterilized to obtain the electrospun fiber membrane.

2. The method for preparing an electrospun fiber membrane according to claim 1, wherein: In the step of dissolving poly(L-lactide-co-caprolactone), polyurethane and carboxymethyl chitosan in an organic solvent in a mass ratio of 1:(1-9):(0.25-1) to obtain a polymer solution, the number average molecular weight of the poly(L-lactide-co-caprolactone) is 10-200 kDa, the molar ratio of lactide to caprolactone is 25:75-70:30, the density of the polyurethane is 1.1-1.3 g / mL, and the degree of carboxylation of the carboxymethyl chitosan is 87%-90%.

3. The method for preparing an electrospun fiber membrane according to claim 1 or 2, wherein: The organic solvent includes a mixed solvent of chloroform and N,N-dimethylformamide, and the volume ratio of the chloroform to the N,N-dimethylformamide is (4-8):

1.

4. The method for preparing an electrospun fiber membrane according to claim 1, wherein: The concentration of the polymer solution is 100-200 mg / mL.

5. The method for preparing an electrospun fiber membrane according to claim 1, wherein: In the step of electrospinning the polymer solution to obtain an electrospun membrane, the electrospinning conditions are as follows: the stainless steel needle is 18-20G, the syringe capacity is 5-20mL, the injection rate is 0.5-1.5mL / h, the electrospinning voltage is 10-15kV, the ambient temperature is 30-35°C, the ambient humidity is 25%-30%, the receiving distance is 15-20cm, the electrospinning time is 8-12h, and the thickness of the electrospun fiber membrane is 10-70μm.

6. The method for preparing an electrospun fiber membrane according to claim 1, wherein: The step of steam cross-linking the electrospun membrane after drying specifically includes the following steps: The electrospun membrane was vacuum dried for more than 96 hours to remove the organic solvent remaining on the surface; The dried electrospun membrane is cross-linked with glutaraldehyde vapor at room temperature for 1 to 3 hours.

7. The method for preparing an electrospun fiber membrane according to claim 1, wherein: The step of sterilizing the electrospun membrane after steam cross-linking treatment to obtain the electrospun fiber membrane specifically includes the following steps: The electrospun membrane after steam cross-linking treatment is maintained in a disinfectant for 30 to 60 minutes, and then vacuum dried for 12 to 48 hours to obtain the electrospun fiber membrane, and the disinfectant includes a glycine solution.

8. An electrospun fiber membrane, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 7.

9. The electrospun fiber membrane according to claim 8, characterized in that The tensile strength of the electrostatically spun fiber membrane can reach above 5 MPa, the thickness is 10 to 70 μm, and the electrostatically spun fiber membrane is composed of fibers of 1 to 2 μm.

10. Use of the electrospun fiber membrane according to claim 8 in biomedical materials in contact with blood.

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