Selectively permeable double-sided heterogeneous electrospun membrane, grafts, and methods of making and using

Abstract

The application discloses a selective permeation double-sided heterogeneous electrospinning membrane, a graft, and a preparation method and application thereof. The double-sided heterogeneous electrospinning membrane comprises a hydrophilic inner membrane and a hydrophobic outer membrane which are attached together. The hydrophilic inner membrane is formed by electrospinning of a synthetic biodegradable polymer and a natural bioactive protein of biological origin. The hydrophobic outer membrane is formed by electrospinning of a synthetic biodegradable polymer. The pore size of the hydrophilic inner membrane is smaller than the diameter of immune cells so as to form a barrier to the immune cells. The pore size of the hydrophilic inner membrane is larger than the diameter of nutrients and active substances secreted by functional cells so as to enable the nutrients and the active substances to pass in or out. The pore size of the hydrophilic inner membrane is smaller than the pore size of the hydrophobic outer membrane. The physical structure of the small pores of the inner membrane filament forms a selective permeation feature, and the hydrophobic outer membrane can prevent the adhesion of immune cells.

Description

Technical Field

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

[0002] Regenerative medicine aims to restore normal function by repairing or replacing damaged cells, tissues, and organs, providing a revolutionary treatment strategy for a variety of intractable diseases. In this field, cell transplantation, as an important therapeutic approach, provides a promising approach for treating a range of chronic diseases by implanting functional cells into the patient's body to secrete drugs of interest or directly participate in tissue repair. However, the direct transplantation of exogenous cells faces a major challenge: transplant rejection and / or graft-versus-host disease. These immune responses may lead to transplant failure and limit the clinical application of cell transplantation. Therefore, the development of effective immune protection strategies to prolong the survival of transplanted cells and maintain their function 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, thereby avoiding transplant rejection. Encapsulation technology provides immune protection to cells and promotes oxygen and nutrient exchange through macroencapsulation, microencapsulation, and nanoencapsulation strategies. However, existing encapsulation technologies still have the following problems: the large size of macroencapsulation is not conducive to the exchange of nutrients and oxygen between cells and the outside world, nor is it conducive to the delivery of active substances secreted by cells; while the small size of microcapsules and nanocapsules is not conducive to the removal of transplants during subsequent treatment, and the protective effect and number of transplants are also limited by size.

[0004] Therefore, there is a need to provide a system that is selectively permeable and can support cell encapsulation. Summary of the Invention

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

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

[0007] The present invention also provides a transplant prepared by utilizing the selectively permeable double-sided anisotropic electrospinning membrane.

[0008] The present invention also provides a method for preparing the transplant.

[0009] The technical solution adopted to achieve the purpose of the present invention is:

[0010] A selectively permeable double-sided anisotropic electrospun membrane comprises a hydrophilic inner membrane and a hydrophobic outer membrane bonded together. The hydrophilic inner membrane is formed by electrospinning synthetic biodegradable polymers and natural bioactive proteins of biological origin, while the hydrophobic outer membrane is formed by electrospinning synthetic biodegradable polymers. The pore size of the hydrophilic inner membrane is smaller than that of immune cells to block them, while the pore size of the hydrophilic inner membrane is larger than that of nutrients and active substances secreted by functional cells to allow them to penetrate in or out. The pore size of the hydrophilic inner membrane is smaller than that of the hydrophobic outer membrane.

[0011] In the above technical solution, the hydrophilic inner membrane and the hydrophobic outer membrane use the same synthetic biodegradable polymer, which is polycaprolactone, a lactide-caprolactone copolymer, or a lactide-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; the mass ratio of the synthetic biodegradable polymer to the biologically derived natural bioactive protein in the hydrophilic inner membrane is (9-19):1;

[0012] 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.

[0013] Another aspect of the present invention also includes a method for preparing the selectively permeable double-sided anisotropic electrospinning membrane, comprising the following steps:

[0014] Step 1: Preparation of hydrophilic inner membrane electrospinning solution:

[0015] 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;

[0016] Step 2, preparing a hydrophobic outer membrane electrospinning solution:

[0017] Dissolving a synthetic biodegradable polymer in a solvent and stirring thoroughly to obtain a hydrophobic outer membrane electrospinning solution;

[0018] Step 3: Preparation of hydrophilic inner membrane:

[0019] Taking the hydrophilic inner membrane electrospinning solution prepared in step 1, setting the electrospinning solution flow rate, working voltage, distance between the syringe needle and the receiver, and receiver speed, and then performing electrospinning to obtain a hydrophilic inner membrane;

[0020] Step 4: Preparation of double-sided anisotropic electrospinning membrane:

[0021] Before the solvent on the surface of the hydrophilic inner membrane prepared in step 3 evaporates, the hydrophobic outer membrane electrospinning solution prepared in step 2 is taken, and 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 are set to perform electrospinning. Since the solvents in steps 1 and 2 are similar and miscible, the hydrophobic outer membrane naturally adheres to the hydrophilic inner membrane during electrospinning. After the electrospinning is completed, a double-sided anisotropic electrospinning membrane is obtained.

[0022] In the above technical solution, 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%, preferably 7-10%, and the concentration of the synthetic biodegradable polymer in the hydrophobic outer membrane electrospinning solution in step 2 is 12-16%.

[0023] 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.

[0024] In the above technical solution, the flow rate of the hydrophilic inner membrane electrospinning solution in step 3 is 0.45~0.55mL / h, the working voltage is 16~20 kV, the receiver speed is 45~50r / min, the distance between the syringe needle and the receiver is 15~17 cm, and the needle specification is 27~30G; the flow rate of the hydrophobic outer membrane electrospinning solution in step 4 is 1.5~2.5 mL / h, the working voltage is 14~16 kV, the receiver speed is 45~50 r / min, the distance between the syringe needle and the receiver is 15~17cm, and the needle specification is 21~24G.

[0025] Another aspect of the present invention also includes the use of the selectively permeable double-sided anisotropic electrospinning membrane as an encapsulation body in the preparation of functional cell transplants.

[0026] Another aspect of the present invention also includes a transplant comprising the selectively permeable double-sided anisotropic electrospinning membrane and functional cells encapsulated by the selectively permeable double-sided anisotropic electrospinning membrane.

[0027] In the above technical solution, the functional cells are pancreatic islet cells, mesenchymal stem cells or transgenic cells.

[0028] In the above technical solution, the transplant is prepared by the following steps: taking two sheets of selectively permeable double-sided anisotropic electrospun membranes of a predetermined area, aligning the hydrophilic inner membranes of the two sheets of selectively permeable double-sided anisotropic electrospun membranes relative to each other, 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 transplant.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. Preparation process: The raw materials of the selectively permeable double-sided anisotropic electrospun membrane prepared by the present invention are synthetic biodegradable polymers and natural bioactive proteins of biological origin. Synthetic biodegradable polymers (such as polycaprolactone) are commonly used biomedical materials and have been approved by the US FDA. They can usually be used as in vivo implant materials and drug controlled release materials; 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 membrane materials by electrospinning, without the presence of toxic substances such as chemical cross-linking agents, and the prepared products are non-toxic and highly biocompatible. The preparation process is simple, the conditions are mild, the raw materials are widely available, the cost is low, and it is suitable for large-scale production applications;

[0031] 2. Material properties: The selectively permeable double-sided anisotropic electrospun membrane prepared by the present invention uses synthetic biodegradable polymers as the main raw material, and the addition of a certain proportion of collagen significantly improves the mechanical properties of the membrane, changing the high toughness and low elasticity characteristics of synthetic biodegradable polymers, making it more promising in cell encapsulation and protection. The addition of collagen also improves the pore size of the membrane, forming selective permeability;

[0032] 3. Product function: The selectively permeable double-sided anisotropic electrospun membrane prepared by the present invention exerts different functions through the different pore sizes and hydrophilicity of the inner and outer membranes. The physical structure of the inner membrane filament pores forms the characteristics of selective permeability, which hinders the attack of immune cells while not hindering the cells from performing normal physiological functions. The addition of natural bioactive proteins increases the hydrophilicity of the inner membrane, which is conducive to cell adhesion to the inner wall surface and reduces the distance for cells to exchange nutrients and gases with the outside world in the double-sided anisotropic electrospun membrane. The hydrophobic outer membrane plays a supporting role, and its synthetic biodegradable polymer is not conducive to cell adhesion, which can prevent immune cell adhesion and plays an important role in reducing fibrosis deposition. At the same time, its pore size range is 4.0~5.5 μm. This pore size range has good permeability, which is conducive to the exchange of nutrients and other substances between the internally encapsulated cells and the outside world. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1This is an observation diagram of the inner and outer membrane morphology of the selectively permeable double-sided anisotropic electrospun membrane of the present invention.

[0034] Figure 2 The mechanical properties test results of PCL diaphragm, 5% COL diaphragm and 10% COL diaphragm.

[0035] Figure 3 The hydrophilicity test results of PCL membrane, 5% COL membrane and 10% COL membrane are shown.

[0036] Figure 4 The following are the test results of the selective permeability of PCL membrane, 5% COL membrane, 10% COL membrane and 10% COL membranes of different thicknesses to 40kDa-Dextran and 150 kDa-Dextran.

[0037] Figure 5 The cell adhesion test results of PCL membrane, 5% COL membrane, and 10% COL membrane. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] Unless otherwise specified, the normal temperature in the present invention is 25±2°C.

[0040] All raw materials used in the examples of the present invention are commercially available.

[0041] In some embodiments of the present invention, the synthetic biodegradable polymer includes polycaprolactone (PCL), a lactide-caprolactone copolymer, or a lactide-glycolide copolymer, preferably poly-L-lactide-caprolactone (PLCL). The biologically derived natural bioactive protein includes at least one of type IV collagen, type I collagen, type II collagen, and type III collagen. For example, the biologically derived natural bioactive protein is human recombinant type IV collagen, preferably COL IV. Synthetic biodegradable polymers exhibit excellent mechanical properties, exhibiting high toughness and low elasticity, while some biologically derived natural bioactive proteins possess excellent elasticity and the ability to quickly recover after external forces, which is beneficial for cell protection. The addition of natural bioactive proteins increases the hydrophilicity of the inner membrane, facilitating cell adhesion and reducing the distance between cells and the outside world for nutrient and gas exchange in the double-sided anisotropic electrospun membrane. The physical structure of the hydrophilic inner membrane filaments creates a selective permeability characteristic, hindering immune cell attack while not hindering normal cellular function. The hydrophobic outer membrane made of synthetic biodegradable polymers is not conducive to cell adhesion and plays an important role in reducing fibrosis deposition. In the following typical embodiments of the present invention, PCL, PLCL, COL IV and COL I are used as examples for illustration.

[0042] The abbreviations involved in the following embodiments specifically refer to:

[0043] PCL: polycaprolactone;

[0044] PLCL: poly-L-lactide-caprolactone;

[0045] COL Ⅰ: Type Ⅰ collagen from bovine Achilles tendon;

[0046] COL Ⅳ: human recombinant type Ⅳ collagen;

[0047] HFIP: hexafluoroisopropanol;

[0048] PC1: PCL / COL Ⅰ;

[0049] PC4: PCL / COL IV;

[0050] PLC1:PLCL / COL Ⅰ;

[0051] PLC4:PLCL / COL Ⅳ.

[0052] Example 1

[0053] A method for preparing a selectively permeable double-sided anisotropic electrospinning membrane comprises the following steps:

[0054] Step 1, prepare PC4 inner membrane electrospinning solution:

[0055] 0.68 g PCL and 0.12 g COL IV were dissolved in 10 mL HFIP and stirred for 8 h to obtain an 8 wt% PC4 inner membrane electrospinning solution, in which the mass ratio of PCL to COL IV was 9:1;

[0056] Step 2: Preparation of PCL outer membrane electrospinning solution:

[0057] 1.5 g of PCL was dissolved in 10 mL of a mixture of chloroform and methanol (volume ratio: 5:1) and stirred for 8 h to obtain a 15 wt% PCL outer membrane electrospinning solution.

[0058] Step 3, preparation of PC4 inner membrane:

[0059] Take 4 mL of the PC4 inner membrane electrospinning solution prepared in step 1 and set the flow rate of the PC4 inner membrane electrospinning solution to 0.5 mL / h, the operating 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 the electrospinning is completed, the PC4 inner membrane (i.e., the hydrophilic inner membrane with 10% COV IV) is obtained;

[0060] Step 4: Preparation of a selectively permeable double-sided anisotropic electrospinning membrane:

[0061] Before the solvent on the surface of the PC4 electrospinning inner membrane obtained in step 3 evaporates, 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 operating 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. The outer membrane is adhered to the inner membrane by utilizing the principle of like dissolves like between solvents. After the electrospinning is completed, a selectively permeable double-sided anisotropic electrospinning membrane is obtained.

[0062] Example 2

[0063] A method for preparing a selectively permeable double-sided anisotropic electrospinning membrane comprises the following steps:

[0064] Step 1, prepare PC1 inner membrane electrospinning solution:

[0065] 0.68 g PCL and 0.12 g COL Ⅰ were dissolved in 10 mL HFIP and stirred for 8 h to obtain an 8 wt% PC1 electrospinning solution with a PCL to COL Ⅰ mass ratio of 9:1.

[0066] Step 2: Preparation of PCL outer membrane electrospinning solution:

[0067] 1.5 g of PCL was dissolved in 10 mL of a mixture of chloroform and methanol (volume ratio: 5:1) and stirred for 8 h to obtain a 15 wt% PCL outer membrane electrospinning solution.

[0068] Step 3, preparation of PC1 inner membrane:

[0069] 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 operating 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 the electrospinning is completed, the PC1 inner membrane is obtained;

[0070] Step 4: Preparation of a selectively permeable double-sided anisotropic electrospinning membrane:

[0071] Before the solvent on the surface of the PCL electrospinning inner membrane prepared in step 3 evaporates, 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 operating 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. The principle of like dissolves like between solvents is used to make the outer membrane adhere to the inner membrane. After the electrospinning is completed, a selectively permeable double-sided anisotropic electrospinning membrane is obtained.

[0072] Example 3

[0073] A method for preparing a selectively permeable double-sided anisotropic electrospinning membrane comprises the following steps:

[0074] Step 1, preparation of PLC4 inner membrane electrospinning solution:

[0075] 0.68 g PLCL and 0.12 g COL IV were dissolved in 10 mL HFIP and stirred 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.

[0076] Step 2, preparation of PLCL outer membrane electrospinning solution:

[0077] 1.5 g of PLCL was dissolved in 10 mL of a mixture of chloroform and methanol (volume ratio: 5:1) and stirred for 8 h to obtain a 15 wt% PLCL outer membrane electrospinning solution.

[0078] Step 3, preparation of PLC4 inner membrane:

[0079] 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 operating 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 the electrospinning is completed, the PLC4 inner membrane is obtained;

[0080] Step 4: Preparation of a selectively permeable double-sided anisotropic electrospinning membrane:

[0081] Before the solvent on the surface of the PLC4 electrospinning inner membrane prepared in step 3 evaporates, 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 operating 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. The principle of like dissolves like between solvents is used to make the outer membrane adhere to the inner membrane. After the electrospinning is completed, a selectively permeable double-sided anisotropic electrospinning membrane is obtained.

[0082] Example 4

[0083] A method for preparing a selectively permeable double-sided anisotropic electrospinning membrane:

[0084] Step 1, preparation of PLC1 inner membrane electrospinning solution:

[0085] 0.68 g PLCL and 0.12 g COL I were dissolved in 10 mL HFIP and stirred for 8 h to obtain an 8 wt% PLC1 inner membrane electrospinning solution with a mass ratio of PLCL to COL I of 9:1;

[0086] Step 2, preparation of PLCL outer membrane electrospinning solution:

[0087] 1.5 g of PLCL was dissolved in 10 mL of a mixture of chloroform and methanol (volume ratio: 5:1) and stirred for 8 h to obtain a 15 wt% PLCL outer membrane electrospinning solution.

[0088] Step 3, preparation of PLC1 inner membrane:

[0089] Take 4 mL of PLC1 solution, set the spinning solution flow rate to 0.5 mL / h, the operating voltage to 17 kV, the distance between the syringe needle and the aluminum foil receiver to 17 cm, and the aluminum foil receiver rotation speed to 50 r / min. After the electrospinning is completed, the PLC1 inner membrane is obtained;

[0090] Step 4: Preparation of a selectively permeable double-sided anisotropic electrospinning membrane:

[0091] Before the solvent on the surface of the PLC1 electrospinning inner membrane obtained in step 3 evaporates, 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 operating 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. The principle of like dissolves like between solvents is used to make the outer membrane adhere to the outer membrane, and a selectively permeable double-sided anisotropic electrospinning membrane is obtained.

[0092] Example 5

[0093] A method for preparing a transplant, comprising the following steps:

[0094] Two sheets of the selectively permeable double-sided anisotropic electrospun membrane prepared in Example 1 with an area of ​​1.5 cm × 3 cm were taken, the inner membranes of the two sheets of selectively permeable double-sided anisotropic electrospun membrane were opposite each other, and the edges were bonded by heat sealing or ultrasonic welding to obtain a double-sided anisotropic electrospun membrane package. 5.0 × 10^6 functional cells were resuspended in 500 μL of 10% gelatin solution, and the functional cell suspension was injected into the double-sided anisotropic electrospun membrane package. The package was immersed in a 2% TG enzyme solution and treated at 37°C for 10 min to cross-link the gelatin to obtain a graft. When the functional cells are pancreatic islet cells, the graft can be used as a subcutaneous implant for the treatment of diabetes.

[0095] Testing revealed that the performance of the selectively permeable, bifacially anisotropic electrospun membranes prepared in Examples 1-4 was similar, with no significant differences. The following performance description uses the selectively permeable, bifacially anisotropic electrospun membrane prepared in Example 1 as an example. Using the same method as in Example 1, PCL inner membranes, 5% COL IV hydrophilic inner membranes, and 10% COL IV hydrophilic inner membranes were prepared, along with hydrophobic outer membranes at concentrations of 12%, 14%, and 16%, respectively.

[0096] Test Example 1

[0097] 1. Surface morphology observation:

[0098] The microstructure of the selective permeability double-sided anisotropic electrospun membrane was observed by SEM. Figure 1The fibers of both the inner and outer membranes exhibited a smooth, dense, and randomly oriented morphology. As the COL IV content increased, the fiber diameter decreased significantly. The inner membrane of the selectively permeable double-sided anisotropic electrospun membrane became denser, and the pore size decreased significantly, forming a more compact structure. The fiber diameter of the PCL inner membrane was 0.7037 ± 0.1673 μm, while the fiber diameter of the 5% COL IV hydrophilic inner membrane was 0.2571 ± 0.05282 μm, and the fiber diameter of the 10% COL IV hydrophilic inner membrane was 0.2574 ± 0.04092 μm. This indicates that the fiber diameter of the hydrophilic inner membrane decreased significantly after the incorporation of COL IV. The pore size of the PCL inner membrane was 1.829 ± 0.4508 μm, the pore size of the 5% COL IV hydrophilic inner membrane was 0.7963 ± 0.1933 μm, and the pore size of the 10% COL IV hydrophilic inner membrane was 0.6386 ± 0.09475 μm. Appropriate pore size facilitates the formation of selective permeability in the hydrophilic inner membrane, thereby ensuring the release of nutrients secreted by the encapsulated functional cells and blocking immunoglobulins, thus promoting selective permeability to nutrients and immunoglobulins.

[0099] The concentrations of PCL in the hydrophobic outer membrane were 12%, 14% and 16%, respectively. The pore size of the resulting hydrophobic outer membrane ranged from 4 to 5.5 μm. This pore size range has good permeability, which is conducive to the exchange of nutrients and other substances between the encapsulated cells inside and the outside world, playing a supporting role while reducing the distance from the inside to the outside world.

[0100] Test Example 2

[0101] To test the changes in the performance of the hydrophilic inner membrane after adding COL IV, PCL, hydrophilic inner membranes with 5% COL IV and 10% COL IV were prepared and recorded as PCL membrane, 5% COL membrane and 10% COL membrane, respectively.

[0102] 1. Experiment on mechanical properties of hydrophilic inner membrane

[0103] Good mechanical properties can maintain the morphology of the double-sided anisotropic electrospun membrane after transplantation. The significant differences between the experimental groups and the control group are represented by *, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, refer to Figure 2 In (a), the elastic modulus of the PCL diaphragm is 24.66±0.4008 MPa, the elastic modulus of the 5% COL diaphragm is 31.50±12.46 MPa, and the elastic modulus of the 10% COL diaphragm is 56.97±2.019 MPa. It can be seen that the diaphragm with COL Ⅳ has a higher elastic modulus than the PCL diaphragm. Figure 2In (b), it can be seen that the elongation at break of the PCL membrane is 92.42±4.670%, the elongation at break of the 5% COL membrane is 135.0±5.337%, and the elongation at break of the 10% COL membrane is 104.9±0.9599%. Compared with the PCL membrane, the elongation at break of the hydrophilic inner membrane is significantly improved after the addition of COL IV. Figure 2 In (c), it can be seen that the ultimate load of the PCL membrane is 4.750±0.1106 N, the ultimate load of the 5% COL membrane is 8.510±1.212 N, and the ultimate load of the 10% COL membrane is 9.492±0.07350 N. Compared with the PCL membrane, the ultimate load of the hydrophilic inner membrane is significantly improved after the incorporation of COL IV. Figure 2 In (d), the tensile strength of the PCL membrane is 12.222 ± 0.1278 MPa, the tensile strength of the 5% COL membrane is 15.28 ± 1.503 MPa, and the tensile strength of the 10% COL membrane is 27.10 ± 2.910 MPa. In summary, the incorporation of an appropriate amount of COL IV can increase the elastic modulus, elongation at break, tensile strength, and ultimate load of the hydrophilic inner membrane, significantly improving the mechanical properties of the resulting hydrophilic inner membrane.

[0104] 2. Hydrophilicity test of hydrophilic inner membrane

[0105] Reference Figure 3 , which show the water contact angle results for PCL, 5% COL, and 10% COL membranes. When a water droplet contacts the material surface, the water contact angle for the PCL membrane is 103.0±1.879°, the 5% COL membrane is 85.12±4.283°, and the 10% COL membrane is 79.04±0.4409°. This indicates that the greater the COL IV content in the inner membrane, the smaller the water contact angle and the greater the hydrophilicity of the inner membrane. This is because COL IV contains abundant hydrophilic groups, such as amino, hydroxyl, and carboxyl groups, which effectively increase the hydrophilicity of the inner membrane and enable it to quickly absorb water droplets that fall on the hydrophilic inner membrane surface. This good hydrophilicity promotes cell adhesion and proliferation, keeping cells on the inner wall of the hydrophilic inner membrane, thereby ensuring normal cell survival and function.

[0106] 3. Hydrophilic membrane selective permeability test

[0107] 40 kDa-Dextran was used to simulate VEGF and 150 kDa-Dextran was used to simulate immunoglobulin to test the selective permeability of PCL membrane, 5% COL membrane, 10% COL membrane and 10% COL membranes of different thicknesses (50 μm, 100 μm, 150 μm). Figure 4 In (a), for 40 kDa-Dextran, PCL membrane and 10% COL membrane showed good release, and the release was completed within 12 hours. The 5% COL membrane released slower than the other two groups, but still released more than 75% of 40 kDa-Dextran within 12 hours. For 150 kDa-Dextran, PCL membrane released completely within 12 hours, while the 10% COL membrane released less than 75% within 12 hours, and the 5% COL membrane released less than 50% within 12 hours. In summary, the membrane containing 5% COL has better selective permeability. Figure 4 (b) There was no significant difference in the release of 40 kDa-Dextran between 10% COL membranes with a thickness 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 150 μm thick 10% COL membrane had better selective permeability.

[0108] 4. Hydrophilic endothelial cell adhesion test

[0109] Good cell adhesion on the scaffold material is conducive to the subsequent proliferation and differentiation. L929 cells were cultured on the surface of PCL, 5% COL membrane, and 10% COL membrane for 3 days and the adhesion was observed by SEM. Figure 5 Cells in all groups adhered well to the hydrophilic inner membrane, with a relatively spread morphology and visible pseudopodia. Furthermore, cells on 5% and 10% COL membranes were more spread than those on PCL membranes, indicating that the addition of COL IV increased the cell affinity for the hydrophilic inner membrane.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A double-sided anisotropic electrospinning membrane with selective permeability, characterized in that: The double-sided anisotropic electrospun membrane comprises a hydrophilic inner membrane and a hydrophobic outer membrane bonded together, wherein the hydrophilic inner membrane is formed by electrospinning synthetic biodegradable polymers and natural bioactive proteins of biological origin, and the hydrophobic outer membrane is formed by electrospinning synthetic biodegradable polymers, wherein: The hydrophilic inner membrane and the hydrophobic outer membrane use the same synthetic biodegradable polymer, which is polycaprolactone, a lactide-caprolactone copolymer, or a lactide-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; the mass ratio of the synthetic biodegradable polymer to the biologically derived natural bioactive protein in the hydrophilic inner membrane is (9-19):1; The pore size of the hydrophilic inner membrane is smaller than the diameter of the immune cells to form a barrier to them. 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 150~200 μm. 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. The pore size of the hydrophilic inner membrane is smaller than the pore size of the hydrophobic outer membrane. The pore size of the hydrophobic outer membrane is 4.0~5.5 μm, and the fiber diameter is 1.0~1.5 μm.

2. The method for preparing a double-sided anisotropic electrospinning membrane according to claim 1, wherein: 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 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 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, distance between the syringe needle and the receiver, and receiver 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 evaporates, the hydrophobic outer membrane electrospinning solution prepared in step 2 is taken, and 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 are set to perform electrospinning. Since the solvents in steps 1 and 2 are similar and miscible, the hydrophobic outer membrane naturally adheres to the hydrophilic inner membrane during electrospinning. After the electrospinning is completed, a double-sided anisotropic electrospinning membrane is obtained.

3. The method for preparing a double-sided anisotropic electrospinning membrane according to claim 2, wherein: The total concentration of the synthetic biodegradable polymer and the biologically derived natural bioactive protein in the hydrophilic inner membrane electrospinning solution in step 1 is 7-10%, and the concentration of the synthetic biodegradable polymer in the hydrophobic outer membrane electrospinning solution in step 2 is 12-16%.

4. The method for preparing a double-sided anisotropic electrospinning membrane according to claim 2, wherein: The solvent in step 1 is HFIP, and the solvent in step 2 is hexafluoroisopropanol or a mixed solution of chloroform and methanol.

5. The method for preparing a double-sided anisotropic electrospinning membrane according to claim 2, wherein: The flow rate of the hydrophilic inner membrane electrospinning solution in step 3 is 0.45~0.55 mL / h, the operating voltage is 16~20 kV, the receiver speed is 45~50 r / min, the distance between the syringe needle and the receiver is 15~17 cm, and the needle specification is 27~30 G; the flow rate of the hydrophobic outer membrane electrospinning solution in step 4 is 1.5~2.5 mL / h, the operating voltage is 14~16 kV, the receiver speed is 45~50 r / min, the distance between the syringe needle and the receiver is 15~17 cm, and the needle specification is 21~24 G.

6. A transplant, characterized in that The invention comprises the double-sided anisotropic electrospinning membrane according to claim 1 and functional cells encapsulated by the double-sided anisotropic electrospinning membrane.

7. The implant according to claim 6, wherein The functional cells are pancreatic islet cells, mesenchymal stem cells or transgenic cells.

8. A method for preparing a transplant according to claim 6, characterized in that: The preparation is carried out by the following steps: taking two pieces of double-sided anisotropic electrospun membranes of a predetermined area, placing the hydrophilic inner membranes of the two double-sided anisotropic electrospun membranes opposite each other, bonding the edges by heat sealing or ultrasonic welding to obtain a double-sided anisotropic electrospun membrane package, and encapsulating functional cells in the electrospun membrane package to obtain a transplant.

Images