Permeability-selective double-sided anisotropic electrostatic spinning membrane, graft, preparation method and application

Abstract

The invention discloses a selective permeability double-sided anisotropic electrostatic spinning membrane, a graft, a preparation method and application. The double-sided anisotropic electrostatic spinning membrane comprises a hydrophilic inner membrane and a hydrophobic outer membrane which are attached together, the hydrophilic inner membrane is obtained by performing electrostatic spinning on a synthetic biodegradable high polymer and a natural bioactive protein from a biological source as raw materials to form a membrane, and the hydrophobic outer membrane is obtained by performing electrostatic spinning on a synthetic biodegradable high polymer as a raw material to form a membrane; the aperture of the hydrophilic inner membrane is smaller than the diameter of immune cells so as to block the immune cells, the aperture of the hydrophilic inner membrane is larger than the diameter of nutrient substances and active substances secreted by functional cells so as to enable the nutrient substances and the active substances to penetrate in or out, and the aperture of the hydrophilic inner membrane is smaller than that of the hydrophobic outer membrane. The physical structure of filament pores of the inner membrane forms the characteristic of selective permeability, attack of immune cells is hindered, and the hydrophobic outer membrane can prevent adhesion of the immune cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials and biomedical engineering, and particularly to a selectively permeable double-sided anisotropic electrospun membrane, a graft, and a preparation method and application thereof. Background Art

[0002] Regenerative medicine aims to restore the normal functions of damaged cells, tissues, and organs by repairing or replacing them, providing revolutionary treatment strategies for various refractory diseases. In this field, cell transplantation, as an important treatment method, implants functional cells into patients' bodies to secrete drugs of interest or directly participate in tissue repair, providing a promising approach for treating a series of chronic diseases. However, the direct transplantation of exogenous cells faces a major challenge: transplant rejection and / or graft-versus-host disease, and these immune responses may lead to transplant failure, limiting the clinical application of cell transplantation. Therefore, developing effective immune protection strategies to prolong the survival time of transplanted cells and maintain their functions has become an important research direction in the field of regenerative medicine.

[0003] Cell encapsulation technology is another important immune protection strategy. By encapsulating therapeutic cells in a protective device made of biomaterials, the cells can be physically isolated from the host immune system, thus avoiding transplant rejection reactions. Encapsulation technology provides immune protection for cells and promotes oxygen and nutrient exchange through macro-encapsulation, micro-encapsulation, and nano-encapsulation strategies. However, the existing encapsulation technologies still have the following problems: Macro-encapsulation has a huge encapsulation size, which is not conducive to the exchange of nutrients and oxygen between cells and the outside world, nor is it conducive to the transmission of active substances secreted by cells; while micro-capsules and nano-capsules are small in size, which is not conducive to the removal of grafts in subsequent treatments, and the protective effect and the number of transplants are also limited by the size.

[0004] Therefore, it is necessary to provide a system with selective permeability and capable of supporting cell encapsulation. Summary of the Invention

[0005] The purpose of the present invention is to provide a selectively permeable double-sided anisotropic electrospun membrane and a preparation method thereof in view of the technical defects existing in the prior art.

[0006] Another purpose of the present invention is to provide the application of the selectively permeable double-sided anisotropic electrospun membrane.

[0007] The present invention also provides a graft prepared by using the selectively permeable double-sided anisotropic electrospun membrane.

[0008] The present invention also provides a preparation method of the graft.

[0009] The technical solution adopted to achieve the purpose of the present invention is as follows: A selectively permeable double-sided anisotropic electrospinning membrane comprises a hydrophilic inner membrane and a hydrophobic outer membrane bonded together, wherein the hydrophilic inner membrane is formed by electrospinning a synthetic biodegradable polymer and a natural bioactive protein of biological origin as raw materials, and the hydrophobic outer membrane is formed by electrospinning a synthetic biodegradable polymer as raw materials, the pore size of the hydrophilic inner membrane is smaller than the diameter of immune cells to form a barrier to them, the pore size of the hydrophilic inner membrane is larger than the diameter of nutrients and active substances secreted by functional cells to allow them to penetrate in or out, and the pore size of the hydrophilic inner membrane is smaller than the pore size of the hydrophobic outer membrane.

[0010] In the above technical scheme, the synthetic biodegradable polymers used in the hydrophilic inner membrane and the hydrophobic outer membrane are the same, which are polycaprolactone, lactide and caprolactone copolymer or lactide and glycolide copolymer; the biologically derived natural bioactive protein in the hydrophilic inner membrane is one or more of type IV collagen, type I collagen, type II collagen and type III collagen, and the mass ratio of the synthetic biodegradable polymer to the biologically derived natural bioactive protein in the hydrophilic inner membrane is (9-19):1; In the above technical solution, the pore size of the hydrophilic inner membrane is 0.6~0.9 μm, the fiber diameter is 0.2~0.3 μm, and the thickness is 50~200 μm, preferably 150~200 μm; the pore size of the hydrophobic outer membrane is 4.0~5.5 μm, and the fiber diameter is 1.0~1.5 μm.

[0011] Another aspect of the present invention also includes a method for preparing the selective permeability double-sided anisotropic electrospinning membrane, comprising the following steps: Step 1, preparation of hydrophilic inner membrane electrospinning solution: Dissolving a synthetic biodegradable polymer and a natural bioactive protein in a solvent, and stirring them thoroughly to obtain a hydrophilic inner membrane electrospinning solution; Step 2, preparing a hydrophobic outer membrane electrospinning solution: Dissolving a synthetic biodegradable polymer in a solvent and stirring the solvent thoroughly to obtain a hydrophobic outer membrane electrospinning solution; Step 3, preparation of hydrophilic inner membrane: Taking the hydrophilic inner membrane electrospinning solution prepared in step 1, setting the electrospinning solution flow rate, working voltage, the distance between the syringe needle and the receiver, and the receiver rotation speed, and then performing electrospinning to obtain a hydrophilic inner membrane; Step 4, preparation of double-sided anisotropic electrospinning membrane: Before the solvent on the surface of the hydrophilic inner membrane prepared in Step 3 volatilizes, take the electrospinning solution of the hydrophobic outer membrane prepared in Step 2, set the flow rate of the electrospinning solution, the working voltage, the distance between the syringe needle and the aluminum foil receiver, and the rotational speed of the receiver, and perform electrospinning. Due to the similar solubility between the solvents in Step 1 and Step 2, the hydrophobic outer membrane naturally adheres to the hydrophilic inner membrane during electrospinning. After electrospinning, a double-sided anisotropic electrospun membrane is obtained.

[0012] In the above technical solution, the total concentration of the synthetic biodegradable polymer and the bio-derived natural bioactive protein in the electrospinning solution of the hydrophilic inner membrane in Step 1 is 5-10%, preferably 7-10%, and the concentration of the synthetic biodegradable polymer in the electrospinning solution of the hydrophobic outer membrane in Step 2 is 12-16%.

[0013] In the above technical solution, the solvent in Step 1 is HFIP, and the solvent in Step 2 is hexafluoroisopropanol or a mixed solution of chloroform and methanol.

[0014] In the above technical solution, the flow rate of the electrospinning solution of the hydrophilic inner membrane in Step 3 is 0.45-0.55 mL / h, the working voltage is 16-20 kV, the rotational speed of the receiver is 45-50 r / min, the distance between the syringe needle and the receiver is 15-17 cm, and the needle gauge is 27-30 G; the flow rate of the electrospinning solution of the hydrophobic outer membrane in Step 4 is 1.5-2.5 mL / h, the working voltage is 14-16 kV, the rotational speed of the receiver is 45-50 r / min, the distance between the syringe needle and the receiver is 15-17 cm, and the needle gauge is 21-24 G.

[0015] On the other hand, the present invention also includes the application of the selective permeable double-sided anisotropic electrospun membrane as an encapsulation body in the preparation of a functional cell graft.

[0016] On the other hand, the present invention also includes a graft, comprising the selective permeable double-sided anisotropic electrospun membrane and functional cells encapsulated by the selective permeable double-sided anisotropic electrospun membrane.

[0017] In the above technical solution, the functional cells are islet cells, mesenchymal stem cells or transgenic cells.

[0018] In the above technical solution, the graft is prepared by the following steps: take two pieces of the selective permeable double-sided anisotropic electrospun membrane with a predetermined area, make the hydrophilic inner membranes of the two pieces of the selective permeable double-sided anisotropic electrospun membrane face each other, and bond the edges by heat sealing or ultrasonic welding to obtain a double-sided anisotropic electrospun membrane encapsulation body, and encapsulate the functional cells in the double-sided anisotropic electrospun membrane encapsulation body to obtain the graft.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. In terms of preparation process: The raw materials of the selective permeability double-sided anisotropic electrospun membrane prepared by the present invention are a synthetic biodegradable polymer and a natural bioactive protein of biological origin, respectively. The synthetic biodegradable polymer (such as polycaprolactone) is a commonly used biomedical material that has been approved by the US FDA and is usually used as an in vivo implant material and a drug controlled release material; the natural bioactive collagen of biological origin has good biocompatibility and biodegradable safety, is the main component of most extracellular matrices, and has good immunogenicity. The present invention prepares a membrane material by electrospinning, without the presence of toxic substances such as chemical cross-linking agents, and the prepared product has the characteristics of non-toxicity and high biocompatibility. The preparation process is simple, the conditions are mild, the raw material sources are wide, the cost is low, and it is suitable for large-scale production and application; 2. In terms of material properties: The selective permeability double-sided anisotropic electrospun membrane prepared by the present invention uses a synthetic biodegradable polymer as the main raw material and adds a certain proportion of collagen, which significantly improves the mechanical properties of the membrane and changes the characteristics of high toughness and low elasticity of the synthetic biodegradable polymer, making it more potential in cell encapsulation and protection. At the same time, adding collagen improves the pore size of the membrane and forms selective permeability; 3. In terms of product function: The selective permeability double-sided anisotropic electrospun membrane prepared by the present invention exerts different functions through different pore sizes and hydrophilic and hydrophobic properties of the inner and outer membranes. The physical structure of the fine pores of the inner membrane forms the characteristic of selective permeability, which hinders the attack of immune cells while not preventing cells from performing normal physiological functions. The addition of natural bioactive protein improves the hydrophilicity of the inner membrane, which is beneficial to cell adhesion on the inner wall surface and reduces the distance for cells to exchange nutrients and gases with the outside in the double-sided anisotropic electrospun membrane. The hydrophobic outer membrane plays a supporting role. Its synthetic biodegradable polymer is not conducive to cell adhesion, can prevent the adhesion of immune cells, and plays an important role in reducing fibrotic deposition. At the same time, its pore size range is 4.0 - 5.5 μm, and this pore size range has good permeability, which is beneficial for the cells encapsulated inside to exchange nutrients and the like with the outside. Description of the Drawings

[0020] Figure 1 It is an observation diagram of the inner and outer membrane morphologies of the selective permeability double-sided anisotropic electrospun membrane of the present invention.

[0021] Figure 2 It is the test result of the mechanical properties of the PCL membrane, 5% COL membrane, and 10% COL membrane.

[0022] Figure 3 It is the test result of the hydrophilic properties of the PCL membrane, 5% COL membrane, and 10% COL membrane.

[0023] Figure 4 Selective permeability test results of PCL membranes, 5% COL membranes, 10% COL membranes, and 10% COL membranes with different thicknesses for 40 kDa-Dextran and 150 kDa-Dextran.

[0024] Figure 5 Cell adhesion performance test results of PCL membranes, 5% COL membranes, and 10% COL membranes. Detailed implementation manners

[0025] The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] Unless otherwise specified, the normal temperature in the present invention is calculated as 25 ± 2 °C.

[0027] All raw materials used in the embodiments of the present invention are obtained by purchasing commercially.

[0028] In some embodiments of the present invention, the synthetic biodegradable polymers include polycaprolactone (PCL), copolymer of lactide and caprolactone or copolymer of lactide and glycolide. The copolymer of lactide and caprolactone is preferably poly(L-lactide-caprolactone) (PLCL); the naturally occurring bioactive proteins of biological origin include at least one of type IV collagen, type I collagen, type II collagen, and type III collagen. For example, the naturally occurring bioactive protein of biological origin is human recombinant type IV collagen, preferably COL IV. The synthetic biodegradable polymers have good mechanical properties, showing characteristics of high toughness and low elasticity, while some of the naturally occurring bioactive proteins of biological origin have good elasticity and the property of being able to quickly return to the original state after being subjected to external forces, which is beneficial to the protection of cells. The addition of the naturally occurring bioactive protein improves the hydrophilicity of the inner membrane, which is beneficial to cell adhesion and reduces the distance for cells to exchange nutrients and gases with the outside in the double-sided anisotropic electrospun membrane; the physical structure of the hydrophilic inner membrane filaments with small pores forms the characteristic of selective permeability, which hinders the attack of immune cells and at the same time does not prevent cells from performing normal physiological functions. The hydrophobic outer membrane prepared from the synthetic biodegradable polymer is not conducive to cell adhesion and plays an important role in reducing fibrotic deposition. In the following typical embodiments of the present invention, PCL, PLCL, COL IV, and COL I are taken as examples for illustration.

[0029] The abbreviations involved in the following embodiments specifically refer to: PCL: Polycaprolactone; PLCL: Poly(L-lactide-caprolactone); COL I: Type I collagen derived from bovine Achilles tendon COL Ⅳ: Recombinant human type Ⅳ collagen HFIP: Hexafluoroisopropanol PC1: PCL / COL Ⅰ PC4: PCL / COL Ⅳ PLC1: PLCL / COL Ⅰ PLC4: PLCL / COL Ⅳ

[0030] Example 1 A preparation method of a selective permeability double-sided anisotropic electrospun membrane, comprising the following steps: Step 1, preparing a PC4 inner membrane electrospinning solution: Dissolve 0.68 g of PCL and 0.12 g of COL Ⅳ in 10 mL of HFIP, stir well for 8 h to obtain an 8 wt% PC4 inner membrane electrospinning solution, where the mass ratio of PCL to COL Ⅳ is 9:1; Step 2, preparing a PCL outer membrane electrospinning solution: Dissolve 1.5 g of PCL in a 10 mL mixed solution of chloroform and methanol with a volume ratio of 5:1, stir well for 8 h to obtain a 15 wt% PCL outer membrane electrospinning solution; Step 3, preparing a PC4 inner membrane: Take 4 mL of the PC4 inner membrane electrospinning solution prepared in Step 1, set the flow rate of the PC4 inner membrane electrospinning solution to 0.5 mL / h, the working voltage to 17 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min. After electrospinning, obtain a PC4 inner membrane (i.e., a hydrophilic inner membrane with 10% COV IV); Step 4, preparing a selective permeability double-sided anisotropic electrospun membrane: Before the solvent on the surface of the PC4 electrospun inner membrane obtained in Step 3 volatilizes, take 2 mL of the PCL outer membrane electrospinning solution prepared in Step 2, set the flow rate of the PCL outer membrane electrospinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min, then perform electrospinning, and make the outer membrane adhere to the inner membrane by the principle of similar solubility between solvents. After electrospinning, obtain a selective permeability double-sided anisotropic electrospun membrane.

[0031] Example 2 A preparation method of a selective permeability double-sided anisotropic electrospun membrane, comprising the following steps: Step 1, preparing a PC1 inner membrane electrospinning solution: Dissolve 0.68 g of PCL and 0.12 g of COL Ⅰ in 10 mL of HFIP, and stir well for 8 h to obtain an 8 wt% PC1 electrospinning solution with a mass ratio of PCL to COL Ⅰ of 9:1; Step 2, prepare the electrospinning solution for the PCL outer membrane: Dissolve 1.5 g of PCL in a 10 mL mixture of chloroform and methanol with a volume ratio of 5:1, and stir well for 8 h to obtain a 15 wt% PCL outer membrane electrospinning solution; Step 3, prepare the PC1 inner membrane: Take 4 mL of the PC1 inner membrane electrospinning solution prepared in Step 1, set the flow rate of the PC1 inner membrane electrospinning solution to 0.5 mL / h, the working voltage to 17 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min. After electrospinning, obtain the PC1 inner membrane; Step 4, prepare the selectively permeable double-sided anisotropic electrospun membrane: Before the solvent on the surface of the PC1 electrospun inner membrane prepared in Step 3 volatilizes, take 2 mL of the PCL outer membrane electrospinning solution prepared in Step 2, set the flow rate of the PCL outer membrane electrospinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min, and then perform electrospinning. Use the principle of similar solubility between solvents to make the outer membrane adhere to the inner membrane. After electrospinning, obtain the selectively permeable double-sided anisotropic electrospun membrane.

[0032] Example 3 A method for preparing a selectively permeable double-sided anisotropic electrospun membrane, comprising the following steps: Step 1, prepare the electrospinning solution for the PLC4 inner membrane: Dissolve 0.68 g of PLCL and 0.12 g of COL Ⅳ in 10 mL of HFIP, and stir well for 8 h to obtain an 8 wt% PLC4 inner membrane electrospinning solution with a mass ratio of PLCL to COL Ⅳ of 9:1; Step 2, prepare the electrospinning solution for the PLCL outer membrane: Dissolve 1.5 g of PLCL in a 10 mL mixture of chloroform and methanol with a volume ratio of 5:1, and stir well for 8 h to obtain a 15 wt% PLCL outer membrane electrospinning solution; Step 3, prepare the PLC4 inner membrane: Take 4 mL of the PLC4 inner membrane electrospinning solution prepared in Step 1. Set the flow rate of the PLC4 inner membrane electrospinning solution to 0.5 mL / h, the working voltage to 17 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min. After electrospinning, obtain the PLC4 inner membrane; Step 4, prepare a semi-permeable double-sided anisotropic electrospun membrane: Before the solvent volatilizes on the surface of the PLC4 electrospun inner membrane prepared in Step 3, take 2 mL of the PLCL outer membrane electrospinning solution prepared in Step 2. Set the flow rate of the PLCL outer membrane electrospinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min, and then perform electrospinning. Utilize the principle of similar solubility between solvents to make the outer membrane adhere to the inner membrane. After electrospinning, obtain the semi-permeable double-sided anisotropic electrospun membrane.

[0033] Example 4 A method for preparing a semi-permeable double-sided anisotropic electrospun membrane: Step 1, prepare the PLC1 inner membrane electrospinning solution: Dissolve 0.68 g of PLCL and 0.12 g of COL I in 10 mL of HFIP, and stir well for 8 h to obtain an 8 wt% PLC1 inner membrane electrospinning solution, with the mass ratio of PLCL to COL I being 9:1; Step 2, prepare the PLCL outer membrane electrospinning solution: Dissolve 1.5 g of PLCL in a 10 mL mixture of chloroform and methanol with a volume ratio of 5:1, and stir well for 8 h to obtain a 15 wt% PLCL outer membrane electrospinning solution; Step 3, prepare the PLC1 inner membrane: Take 4 mL of the PLC1 solution, set the flow rate of the spinning solution to 0.5 mL / h, the working voltage to 17 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min. After electrospinning, obtain the PLC1 inner membrane; Step 4, prepare the semi-permeable double-sided anisotropic electrospun membrane: Before the solvent volatilizes on the surface of the PLC1 electrospun inner membrane obtained in Step 3, take 2 mL of the PLCL outer membrane electrospinning solution prepared in Step 2. Set the flow rate of the PLCL outer membrane electrospinning solution to 2 mL / h, the working voltage to 15 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the rotation speed of the aluminum foil receiver to 50 r / min, and then perform electrospinning. Utilize the principle of similar solubility between solvents to make the outer membrane adhere to the outer membrane, and obtain the semi-permeable double-sided anisotropic electrospun membrane.

[0034] Example 5 A method for preparing a graft, which is prepared by the following steps: Take two pieces of the selectively permeable double-sided anisotropic electrospun membranes prepared in Example 1 with an area of 1.5 cm × 3 cm, place the inner membranes of the two selectively permeable double-sided anisotropic electrospun membranes opposite to each other, and bond the edges by heat sealing or ultrasonic welding to obtain a double-sided anisotropic electrospun membrane encapsulation body. Resuspend 5.0×10^6 functional cells in 500 μL of 10% gelatin solution, inject the functional cell suspension into the double-sided anisotropic electrospun membrane encapsulation body, soak it in 2% TG enzyme solution, and treat it at 37 °C for 10 min to crosslink the gelatin to obtain a graft. When the functional cells are islet cells, the graft can be used for subcutaneous implantation as a graft for treating diabetes.

[0035] After testing, the properties of the selectively permeable double-sided anisotropic electrospun membranes prepared in Examples 1-4 above are similar and there are no significant differences. Hereinafter, only the selectively permeable double-sided anisotropic electrospun membrane with the components of Example 1 will be taken as an example to illustrate the performance. Using the same method as in Example 1, prepare PCL inner membranes, 5% COL Ⅳ hydrophilic inner membranes and 10% COL Ⅳ hydrophilic inner membranes, and hydrophobic outer membranes PCL with concentrations of 12%, 14% and 16% respectively.

[0036] Test Example 1 1. Surface morphology observation: Observe the microscopic morphology of the selectively permeable double-sided anisotropic electrospun membrane by SEM. Refer to Figure 1 , the fibers of the inner and outer membranes both show a smooth, dense and randomly oriented morphology. As the content of COL Ⅳ increases, the fiber diameter decreases significantly, the inner membrane of the selectively permeable double-sided anisotropic electrospun membrane becomes denser, the pore size decreases significantly, and a denser structure is formed. The fiber diameter of the PCL inner membrane is 0.7037 ± 0.1673 μm, the fiber diameter of the 5% COL Ⅳ hydrophilic inner membrane is 0.2571±0.05282 μm, and the fiber diameter of the 10% COL Ⅳ hydrophilic inner membrane is 0.2574±0.04092 μm. It can be seen that after incorporating COL Ⅳ, the fiber diameter of the hydrophilic inner membrane decreases significantly. The pore size of the PCL inner membrane is 1.829±0.4508 μm, the pore size of the 5% COL Ⅳ hydrophilic inner membrane is 0.7963±0.1933 μm, and the pore size of the 10% COL Ⅳ hydrophilic inner membrane is 0.6386 ± 0.09475 μm. A suitable pore size is beneficial for the hydrophilic inner membrane to form selective permeability, so as to ensure the release of nutrients secreted by the encapsulated functional cells and the blocking of immunoglobulins, and is beneficial to the formation of selective permeability to nutrients and immunoglobulins.

[0037] The concentrations of the hydrophobic outer membrane PCL were 12%, 14%, and 16% respectively, and the pore sizes of the obtained hydrophobic outer membranes ranged from 4 to 5.5 μm. This pore size range has good permeability, which is conducive to the exchange of nutrients and the like between the internally encapsulated cells and the outside world, and reduces the distance from the inside to the outside while playing a supporting role.

[0038] Test Example 2 To test the performance changes of the hydrophilic inner membrane after adding COL Ⅳ, hydrophilic inner membranes of PCL, 5% COL Ⅳ, and 10% COL Ⅳ were prepared, and were respectively denoted as PCL membrane sheet, 5% COL membrane sheet, and 10% COL membrane sheet.

[0039] 1. Mechanical property experiment of the hydrophilic inner membrane Good mechanical properties can maintain the morphology of the double-sided anisotropic electrospun membrane after transplantation. The significance of the differences between each experimental group and the control group is represented by *, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, referring to Figure 2 In (a), the elastic modulus of the PCL membrane sheet was 24.66 ± 0.4008 MPa, the elastic modulus of the 5% COL membrane sheet was 31.50 ± 12.46 Mpa, and the elastic modulus of the 10% COL membrane sheet was 56.97 ± 2.019 Mpa. It can be seen that the membrane sheets with added COL Ⅳ have higher elastic moduli compared to the PCL membrane sheet. Referring to Figure 2 In (b), it can be seen that the elongation at break of the PCL membrane sheet was 92.42 ± 4.670%, the elongation at break of the 5% COL membrane sheet was 135.0 ± 5.337%, and the elongation at break of the 10% COL membrane sheet was 104.9 ± 0.9599%. Compared with the PCL membrane sheet, the elongation at break of the hydrophilic inner membrane increased significantly after incorporating COL Ⅳ. Referring to Figure 2 In (c), it can be seen that the ultimate load of the PCL membrane sheet was 4.750 ± 0.1106 N, the ultimate load of the 5% COL membrane sheet was 8.510 ± 1.212 N, and the ultimate load of the 10% COL membrane sheet was 9.492 ± 0.07350 N. Compared with the PCL membrane sheet, the ultimate load of the hydrophilic inner membrane increased significantly after incorporating COL Ⅳ. Referring to Figure 2As shown in Fig. (d), the tensile strength of the PCL film was 12.222 ± 0.1278 MPa, the tensile strength of the 5% COL film was 15.28 ± 1.503 Mpa, and the tensile strength of the 10% COL film was 27.10 ± 2.910 MPa. In summary, adding an appropriate amount of COL Ⅳ can improve the elastic modulus, elongation at break, tensile strength, and ultimate load of the hydrophilic inner membrane, and the mechanical properties of the obtained hydrophilic inner membrane have been significantly improved.

[0040] 2. Hydrophilicity test of hydrophilic inner membrane Refer to Figure 3 for the water contact angle results of the PCL film, 5% COL film, and 10% COL film. When a water droplet touches the material surface, the water contact angle of the PCL film is 103.0 ± 1.879°, the water contact angle of the 5% COL film is 85.12 ± 4.283°, and the water contact angle of the 10% COL film is 79.04 ± 0.4409°. It can be seen that the greater the COL Ⅳ content in the inner membrane, the smaller the water contact angle and the better the hydrophilicity of the inner membrane. This is because COL Ⅳ contains abundant hydrophilic groups such as amino, hydroxyl, and carboxyl groups, which can effectively improve the hydrophilicity of the inner membrane and can quickly absorb the water droplets dripping on the surface of the hydrophilic inner membrane. Good hydrophilicity can promote cell adhesion and proliferation and can keep cells on the inner wall of the hydrophilic inner membrane, thus ensuring that cells can survive and function normally.

[0041] 3. Selective permeability test of hydrophilic inner membrane Use 40 kDa-Dextran to simulate VEGF and 150 kDa-Dextran to simulate immunoglobulins to test the selective permeability of the PCL film, 5% COL film, 10% COL film, and 10% COL films with different thicknesses (50 μm, 100 μm, 150 μm). Refer to Figure 4 Fig. (a). For 40 kDa-Dextran, the PCL film and the 10% COL film showed good release properties and completed the release within 12 h. The 5% COL film released more slowly than the other two groups, but still released more than 75% of 40 kDa-Dextran within 12 h; for 150 kDa-Dextran, the PCL film was completely released within 12 h, the 10% COL film released less than 75% within 12 h, and the 5% COL film released less than 50% within 12 h. In summary, the 5% COL-containing film has better selective permeability. Refer to Figure 4(b)There was no significant difference in the release of 40 kDa-Dextran by 10% COL membranes with thicknesses of 150 μm and 100 μm (about 50%). The 150-μm-thick membrane had a better controlled release effect on 150 kDa-Dextran (about 20%). The 10% COL membrane with a thickness of 150 μm had better selective permeability.

[0042] 4. Hydrophilic Inner Membrane Cell Adhesion Test Good cell adhesion on the scaffold material is beneficial for subsequent proliferation and differentiation. L929 cells were cultured on the surfaces of PCL, 5% COL membranes, and 10% COL membranes for 3 days, and the adhesion was observed by SEM. Referring to Figure 5 each group of cells could adhere well to the hydrophilic inner membrane, the cell morphology was relatively spread, and obvious pseudopodia were visible. In addition, the cells on the 5% COL membrane and 10% COL membrane were more spread than those on the PCL membrane, indicating that the addition of COL Ⅳ improved the cell affinity of the hydrophilic inner membrane.

[0043] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A selective permeability double-sided anisotropic electrospun membrane, characterized in that, It comprises a hydrophilic inner membrane and a hydrophobic outer membrane bonded together, wherein the hydrophilic inner membrane is formed by electrospinning a synthetic biodegradable polymer and a natural bioactive protein of biological origin as raw materials, and the hydrophobic outer membrane is formed by electrospinning a synthetic biodegradable polymer as raw materials, the pore size of the hydrophilic inner membrane is smaller than the diameter of immune cells to form a barrier to them, the pore size of the hydrophilic inner membrane is larger than the diameter of nutrients and active substances secreted by functional cells to allow them to penetrate in or out, and the pore size of the hydrophilic inner membrane is smaller than the pore size of the hydrophobic outer membrane.

2. The selectively permeable double-sided anisotropic electrospun membrane according to claim 1, characterized in that, The hydrophilic inner membrane and the hydrophobic outer membrane use the same synthetic biodegradable polymer, which is polycaprolactone, a copolymer of lactide and caprolactone, or a copolymer of lactide and glycolide; the biologically derived natural bioactive protein in the hydrophilic inner membrane is one or more of type IV collagen, type I collagen, type II collagen, and type III collagen; the mass ratio of the synthetic biodegradable polymer to the biologically derived natural bioactive protein in the hydrophilic inner membrane is (9-19):

1.

3. The selective-permeability double-sided anisotropic electrospun membrane according to claim 1, wherein The pore size of the hydrophilic inner membrane is 0.6-0.9 μm, the fiber diameter is 0.2-0.3 μm, and the thickness is 50-200 μm; the pore size of the hydrophobic outer membrane is 4.0-5.5 μm, and the fiber diameter is 1.0-1.5 μm.

4. The preparation method of the selectively permeable double-sided anisotropic electrospun membrane according to any one of claims 1-3, characterized in that, The following steps are involved: Step 1, preparation of hydrophilic inner membrane electrospinning solution: Dissolving a synthetic biodegradable polymer and a natural bioactive protein in a solvent, and stirring the solvent thoroughly to obtain a hydrophilic inner membrane electrospinning solution; Step 2, preparing a hydrophobic outer membrane electrospinning solution: Dissolving a synthetic biodegradable polymer in a solvent and stirring the solvent thoroughly to obtain a hydrophobic outer membrane electrospinning solution; Step 3, preparation of hydrophilic inner membrane: Taking the hydrophilic inner membrane electrospinning solution prepared in step 1, setting the electrospinning solution flow rate, working voltage, the distance between the syringe needle and the receiver, and the receiver rotation speed, and then performing electrospinning to obtain a hydrophilic inner membrane; Step 4, preparation of selective permeability double-sided anisotropic electrospinning membrane: Before the solvent on the surface of the hydrophilic inner membrane prepared in step 3 evaporates, take the hydrophobic outer membrane electrospinning solution prepared in step 2, set the flow rate of the electrospinning solution, the working voltage, the distance between the syringe needle and the aluminum foil receiver, and the receiver speed, and perform electrospinning. Since the solvents in step 1 and step 2 are similar and miscible, the hydrophobic outer membrane naturally adheres to the hydrophilic inner membrane during electrospinning. After the electrospinning is completed, a selectively permeable double-sided anisotropic electrospinning membrane is obtained.

5. The preparation method of the selectively permeable double-sided anisotropic electrospun membrane according to claim 4, characterized in that, The total concentration of the synthetic biodegradable polymer and the natural bioactive protein of biological origin in the hydrophilic inner membrane electrospinning solution in step 1 is 5-10%, and the concentration of the synthetic biodegradable polymer in the hydrophobic outer membrane electrospinning solution in step 2 is 12-16%.

6. The preparation method of the selective-permeability double-sided anisotropic electrospun membrane according to claim 4, characterized in that, The solvent in step 1 is HFIP, and the solvent in step 2 is hexafluoroisopropanol or a mixed solution of chloroform and methanol.

7. The preparation method of the selective transmission double-sided anisotropic electrospun membrane according to claim 4, wherein In step 3, the flow rate of the hydrophilic inner membrane electrospinning solution is 0.45 - 0.55 mL / h, the working voltage is 16 - 20 kV, the rotational speed of the receiver is 45 - 50 r / min, the distance between the syringe needle and the receiver is 15 - 17 cm, and the needle gauge is 27 - 30 G; in step 4, the flow rate of the hydrophobic outer membrane electrospinning solution is 1.5 - 2.5 mL / h, the working voltage is 14 - 16 kV, the rotational speed of the receiver is 45 - 50 r / min, the distance between the syringe needle and the receiver is 15 - 17 cm, and the needle gauge is 21 - 24 G.

8. Use of the selective permeability double-sided anisotropic electrospun membrane as described in any one of claims 1 - 3 as a package in the preparation of a functional cell graft.

9. A graft, characterized in that, Comprising the selective permeability double-sided anisotropic electrospun membrane as described in any one of claims 1 - 3 and functional cells encapsulated by the selective permeability double-sided anisotropic electrospun membrane, wherein the functional cells are islet cells, mesenchymal stem cells or transgenic cells.

10. A method for preparing a graft as described in claim 9, characterized in that, Prepared by the following steps: taking a selective permeability double-sided anisotropic electrospun membrane with a predetermined area, making the hydrophilic inner membranes of two selective permeability double-sided anisotropic electrospun membranes face each other, and bonding the edges by heat sealing or ultrasonic welding to obtain a double-sided anisotropic electrospun membrane package, and encapsulating functional cells in the double-sided anisotropic electrospun membrane package to obtain a graft.

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