Bionic selenium-doped fiber membrane as well as preparation method and application thereof

Abstract

The invention discloses a bionic selenium-doped fiber membrane as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The preparation method comprises the following steps: (1) mixing and stirring a polylactic acid-glycolic acid copolymer and sodium selenite in a solvent to prepare a spinning solution, then carrying out electrostatic spinning, and finally drying to prepare selenium-containing electrostatic spinning fibers; and (2) mixing the selenium-containing electrostatic spinning fiber obtained in the step (1) with a macrophage membrane, and carrying out ultrasonic oscillation to obtain the selenium-containing electrostatic spinning fiber. The bionic selenium-doped fiber membrane is prepared by adopting an electrostatic spinning technology, the preparation process is simple, the cost is low, large-scale production is easy, and the prepared bionic selenium-doped fiber membrane has good biological activity and selenium element release ability, has excellent immunoregulation ability, can effectively remove inflammatory factors in vitro, regulate and control polarization of macrophages, and can be used for preparing the biomimetic selenium-doped fiber membrane. The polypeptide has bone immune regulation ability, can effectively treat infectious bone defects, and has wide application prospect and practical value.

Description

Technical Field

[0001] The invention relates to the technical field of medical materials, and in particular to a bionic selenium-doped fiber membrane and a preparation method and application thereof. Background Art

[0002] Infected bone defects are a common orthopedic disease, usually caused by trauma, infection or tumor resection. Traditional bone repair materials such as autologous bone transplantation, allogeneic bone transplantation and metal implants have problems such as limited donors, immune rejection and high risk of infection. In recent years, electrospun fibers, as a material with good biocompatibility and degradability, have been widely used in tissue engineering and drug delivery. However, the immunomodulatory ability and bone regeneration effect of existing electrospun fiber materials in inflammatory environments still need to be improved.

[0003] In the prior art, electrospun fiber materials mainly promote tissue repair by loading drugs or growth factors, but in the inflammatory environment of infected bone defects, excessive inflammatory response will inhibit the bone regeneration process. In addition, the existing electrospun fiber materials lack the ability to clear inflammatory factors and cannot effectively regulate the bone immune microenvironment. Therefore, it is of great significance to develop an electrospun fiber material that can regulate the bone immune microenvironment and promote bone regeneration under inflammatory conditions. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a bionic selenium-doped fiber membrane and its preparation method and application, so as to solve the problems that the existing electrospun fiber materials have insufficient immunoregulatory ability in inflammatory environments and cannot effectively eliminate inflammatory factors; the ability to regulate macrophage polarization is limited and cannot effectively inhibit excessive inflammatory responses; the bone regeneration effect in inflammatory environments is not ideal, and the like.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: The first aspect of the present invention provides a method for preparing a bionic selenium-doped fiber membrane, comprising the following steps: (1) firstly, polylactic acid-glycolic acid copolymer and sodium selenite are mixed and stirred in a solvent to prepare a spinning solution, then electrospinning is performed, and finally drying is performed to obtain selenium-containing electrospun fibers; (2) The selenium-containing electrospun fibers obtained in step (1) are mixed with macrophage membranes and subjected to ultrasonic vibration to obtain a product.

[0006] The beneficial effects of the present invention are as follows: the present invention adopts electrospinning technology to prepare selenium-containing fiber membranes, which has simple process, low cost, and is easy to scale up production. By adjusting the spinning parameters (such as voltage, spinning speed, solvent ratio, etc.), the pore size, thickness and mechanical properties of the fiber membrane can be precisely controlled to meet the needs of different application scenarios.

[0007] Furthermore, in step (1), the concentration of the polylactic acid-glycolic acid copolymer in the spinning solution is 10 w / v %-30 w / v %; and the mass ratio of the polylactic acid-glycolic acid copolymer to sodium selenite is (90-95):(5-10).

[0008] Preferably, in step (1), the concentration of the polylactic acid-glycolic acid copolymer in the spinning solution is 20 w / v %; and the mass ratio of the polylactic acid-glycolic acid copolymer to sodium selenite is 92.25:7.75.

[0009] Furthermore, the solvent in step (1) is a mixed solvent consisting of dichloromethane and dimethylformamide; the volume ratio of dichloromethane to dimethylformamide is (1-5):1.

[0010] Furthermore, the conditions for electrospinning in step (1) are as follows: the distance between the nozzle and the receiving roller is 15-20 cm, the spinning speed is 1-5 mL / h, the spinning voltage is 20-25 kV, the spinning environment temperature is 20-30°C; the drying temperature is 50-70°C, and the time is 20-30 h.

[0011] The beneficial effect of adopting the above further technical solution is: the present invention prepares selenium-containing electrospun fibers by chemical synthesis method, which ensures that the fibers have good biological activity and the ability to release selenium.

[0012] Furthermore, the macrophage membrane in step (2) is prepared by the following method: firstly suspending the macrophages in phosphate buffer, then subjecting them to freeze-thaw cycle treatment, and finally centrifuging to obtain the macrophage membrane.

[0013] Furthermore, the freeze-thaw cycle treatment conditions are: first freezing at -90~-70°C, then thawing at 35-40°C, repeating freezing and thawing 2-5 times; the centrifugal speed is 8000-12000 rpm, and the time is 5-15 min.

[0014] The beneficial effect of adopting the above further technical scheme is: the present invention prepares macrophage membranes by repeated freeze-thaw method, which ensures the purity and biocompatibility of the prepared cell membranes, and provides excellent cell compatibility for the subsequently prepared bionic selenium-doped fiber membranes, so that they can support the normal growth of cells.

[0015] Furthermore, in step (2), the mass ratio of selenium-containing electrospun fibers to macrophage membrane is (5-20):1.

[0016] Furthermore, in step (2), the frequency of ultrasonic oscillation is 30-50 kHz, and the time is 10-60 min.

[0017] The beneficial effect of adopting the above further technical solution is: the present invention adopts ultrasonic vibration through physical adsorption and ultrasound-assisted self-assembly technology to achieve efficient coating of macrophage membrane on the fiber surface.

[0018] The second aspect of the present invention provides a bionic selenium-doped fiber membrane, which is prepared by the above-mentioned preparation method.

[0019] The beneficial effects of the present invention are as follows: the bionic selenium-doped fiber membrane prepared by the present invention has a multifunctional integrated design, which integrates the functions of immune regulation, inflammatory factor removal, macrophage polarization regulation and tissue repair promotion, and realizes a multifunctional synergistic effect, so that it can not only regulate the immune microenvironment, but also has potential bone regeneration ability. At the same time, the porous structure and bionic characteristics of the fiber membrane are conducive to cell adhesion, proliferation and differentiation.

[0020] The third aspect of the present invention provides the use of the above-mentioned bionic selenium-doped fiber membrane in the preparation of medical devices or drugs for treating infectious bone defects.

[0021] The beneficial effects of the present invention are as follows: the bionic selenium-doped fiber membrane prepared by the present invention can effectively remove inflammatory factors, regulate macrophage polarization, and has bone immunity regulation ability, and has broad application prospects and practical value in the treatment of infectious bone defects. After the fiber membrane is implanted, the macrophage membrane on its surface can adsorb and remove local inflammatory factors (such as TNF-α, IL-1β, etc.) through membrane surface receptors (such as tumor necrosis factor receptors, interleukin receptors, etc.), thereby restoring the bone immune microenvironment. At the same time, the selenium element slowly released by the fiber membrane regulates the polarization of macrophages from M1 type to M2 type, inhibiting the inflammatory response.

[0022] The present invention has the following beneficial effects: The present invention adopts electrospinning technology to prepare bionic selenium-doped fiber membrane, and the preparation process is simple, the cost is low, and it is easy to mass produce. The prepared bionic selenium-doped fiber membrane has good biological activity and selenium release ability, has excellent immunoregulatory ability, can effectively clear inflammatory factors in vitro, regulate macrophage polarization, has bone immune regulation ability, can effectively treat infectious bone defects, and has broad application prospects and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a transmission electron microscopy image of the bionic selenium-doped fiber membrane obtained in Example 1; Figure 2 This is a scanning electron microscope image of the PLGA fiber membrane prepared in Comparative Example 1; Figure 3 This is a scanning electron microscope image of the selenium-containing fiber membrane prepared in Comparative Example 2; Figure 4This is the result of the ELISA test for clearing inflammatory factors in Experimental Example 1, where a is the IL-1β inflammatory factor and b is the TNF-α inflammatory factor; Figure 5 This is a graph showing the qPCR detection results of the macrophage polarization regulation experiment in Experimental Example 2, wherein a is the macrophage inflammatory differentiation marker IL-1β, b is the macrophage inflammatory differentiation marker IL-6, c is the macrophage pro-repair differentiation marker Arg-1, and d is the macrophage pro-repair differentiation marker CD206. DETAILED DESCRIPTION

[0024] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0025] Embodiment 1: A method for preparing a bionic selenium-doped fiber membrane comprises the following steps: (1) Preparation of selenium-containing spinning solution: First, PLGA (polylactic acid-co-glycolic acid) was weighed using a precision analytical balance, and dichloromethane (DCM) and dimethylformamide (DMF) were used as solvents in a volume ratio of 2.5:1. PLGA was dissolved in the mixed solvent and placed on an electromagnetic stirrer for continuous stirring for 12 hours to prepare a 20 w / v % PLGA spinning solution. Then, a certain amount of sodium selenite (Na 2 SeO 3 ) powder (the mass ratio of sodium selenite to PLGA was 7.75:92.25) was added into the PLGA spinning solution and stirred continuously for 3 h on an electromagnetic stirrer to obtain a selenium-containing spinning solution.

[0026] (2) Electrospinning The selenium-containing spinning solution obtained in step (1) was connected to the spinning nozzle, and the parameters of the electrospinning equipment were set as follows: the distance between the nozzle and the receiving roller was 18 cm, the spinning speed was 3 mL / h, the voltage was 22 kV, the ambient temperature in the electrospinning equipment was 25°C, the roller was rotated at a low speed and covered with aluminum foil to collect the fibers. After spinning, the residual solvent was evaporated in a vacuum drying oven at a drying temperature of 60°C for 24 h to obtain a selenium-containing electrospun fiber membrane.

[0027] (3) Preparation of macrophage membrane Macrophage membranes were extracted from macrophages using a repeated freeze-thaw method. The specific steps were as follows: macrophages were suspended in PBS buffer, subjected to three freeze-thaw cycles (freezing at -80°C and thawing at 37°C), and then centrifuged at 10,000 rpm for 10 min to obtain macrophage membranes.

[0028] (4) Preparation of bionic selenium-doped fiber membrane The macrophage membrane obtained in step (3) and the selenium-containing electrospun fiber obtained in step (2) were mixed in a mass ratio of 1:10, placed in an ultrasonic oscillator, and oscillated at a frequency of 40 kHz for 30 min to ensure that the macrophage membrane was evenly wrapped on the fiber surface, thereby obtaining a bionic selenium-doped fiber membrane (MSe@Fiber).

[0029] The transmission electron microscopy image of the bionic selenium-doped fiber membrane prepared in this embodiment is as follows: Figure 1 As shown, it can be clearly seen that the fiber filaments are surrounded by a clear cell membrane.

[0030] Embodiment 2: A method for preparing a bionic selenium-doped fiber membrane comprises the following steps: (1) Preparation of selenium-containing spinning solution: First, PLGA (polylactic acid-co-glycolic acid) was weighed using a precision analytical balance, and dichloromethane (DCM) and dimethylformamide (DMF) were used as solvents in a volume ratio of 2.5:1. PLGA was dissolved in the mixed solvent and placed on an electromagnetic stirrer for continuous stirring for 12 hours to prepare a 15 w / v % PLGA spinning solution. Then, a certain amount of sodium selenite (Na 2 SeO 3 ) powder (the mass ratio of sodium selenite to PLGA was 7.75:92.25) was added into the PLGA spinning solution and stirred continuously for 3 h on an electromagnetic stirrer to obtain a selenium-containing spinning solution.

[0031] (2) Electrospinning The selenium-containing spinning solution obtained in step (1) was connected to the spinning nozzle, and the parameters of the electrospinning equipment were set as follows: the distance between the nozzle and the receiving roller was 20 cm, the spinning speed was 3 mL / h, the voltage was 22 kV, the ambient temperature in the electrospinning equipment was 25°C, the drum was rotated at a low speed and covered with aluminum foil to collect the fibers. After spinning, the residual solvent was evaporated in a vacuum drying oven at a temperature of 60°C for 24 h to obtain a selenium-containing electrospun fiber membrane.

[0032] (3) Preparation of macrophage membrane Macrophage membranes were extracted from macrophages using a repeated freeze-thaw method. The specific steps were as follows: macrophages were suspended in PBS buffer, subjected to three freeze-thaw cycles (freezing at -80°C and thawing at 37°C), and then centrifuged at 10,000 rpm for 10 min to obtain macrophage membranes.

[0033] (4) Preparation of bionic selenium-doped fiber membrane The macrophage membrane obtained in step (3) and the selenium-containing electrospun fiber obtained in step (2) were mixed at a mass ratio of 1:20, placed in an ultrasonic oscillator, and oscillated at a frequency of 50 kHz for 30 min to ensure that the macrophage membrane was evenly wrapped on the fiber surface, thereby obtaining a bionic selenium-doped fiber membrane.

[0034] Embodiment 3: A method for preparing a bionic selenium-doped fiber membrane comprises the following steps: (1) Preparation of selenium-containing spinning solution: First, PLGA (polylactic acid-co-glycolic acid) was weighed using a precision analytical balance, and dichloromethane (DCM) and dimethylformamide (DMF) were used as solvents in a volume ratio of 2.5:1. PLGA was dissolved in the mixed solvent and placed on an electromagnetic stirrer for continuous stirring for 12 hours to prepare a 25 w / v % PLGA spinning solution. Then, a certain amount of sodium selenite (Na 2 SeO 3 ) powder (the mass ratio of sodium selenite to PLGA was 7.75:92.25) was added into the PLGA spinning solution and stirred continuously for 3 h on an electromagnetic stirrer to obtain a selenium-containing spinning solution.

[0035] (2) Electrospinning The selenium-containing spinning solution obtained in step (1) was connected to the spinning nozzle, and the parameters of the electrospinning equipment were set as follows: the distance between the nozzle and the receiving roller was 15 cm, the spinning speed was 3 mL / h, the voltage was 22 kV, the ambient temperature in the electrospinning equipment was 25°C, the roller was rotated at a low speed and covered with aluminum foil to collect the fibers. After spinning, the residual solvent was evaporated in a vacuum drying oven at a temperature of 60°C for 24 h to obtain a selenium-containing electrospun fiber membrane.

[0036] (3) Preparation of macrophage membrane Macrophage membranes were extracted from macrophages using a repeated freeze-thaw method. The specific steps were as follows: macrophages were suspended in PBS buffer, subjected to three freeze-thaw cycles (freezing at -80°C and thawing at 37°C), and then centrifuged at 10,000 rpm for 10 min to obtain macrophage membranes.

[0037] (4) Preparation of bionic selenium-doped fiber membrane The macrophage membrane obtained in step (3) and the selenium-containing electrospun fiber obtained in step (2) were mixed in a mass ratio of 1:5, placed in an ultrasonic oscillator, and oscillated at a frequency of 30 kHz for 30 min to ensure that the macrophage membrane was evenly wrapped on the fiber surface, thereby obtaining a bionic selenium-doped fiber membrane.

[0038] Comparative Example 1: A method for preparing a PLGA fiber membrane comprises the following steps: (1) Preparation of selenium-containing spinning solution: First, PLGA (polylactic acid-co-glycolic acid) was weighed using a precision analytical balance, and dichloromethane (DCM) and dimethylformamide (DMF) were used as solvents in a volume ratio of 2.5:1. PLGA was dissolved in the mixed solvent and placed on an electromagnetic stirrer for continuous stirring for 12 hours to prepare a 20 w / v % PLGA spinning solution.

[0039] (2) Electrospinning The PLGA spinning solution obtained in step (1) was connected to the spinning nozzle. The parameters of the electrospinning equipment were set as follows: the distance between the nozzle and the receiving roller was 18 cm, the spinning speed was 3 mL / h, the voltage was 22 kV, the ambient temperature in the electrospinning equipment was 25°C, the roller was rotated at a low speed and covered with aluminum foil to collect the fibers. After spinning, the residual solvent was evaporated in a vacuum drying oven at a temperature of 60°C for 24 h to obtain a PLGA fiber membrane (PLGA). The SEM image is shown in FIG. Figure 2 shown.

[0040] Comparative Example 2: A method for preparing a selenium-containing fiber membrane comprises the following steps: (1) Preparation of selenium-containing spinning solution: First, PLGA (polylactic acid-co-glycolic acid) was weighed using a precision analytical balance, and dichloromethane (DCM) and dimethylformamide (DMF) were used as solvents in a volume ratio of 2.5:1. PLGA was dissolved in the mixed solvent and placed on an electromagnetic stirrer for continuous stirring for 12 hours to prepare a 20 w / v % PLGA spinning solution. Then, a certain amount of sodium selenite (Na 2 SeO 3 ) powder (the mass ratio of sodium selenite to PLGA was 7.75:92.25) was added into the PLGA spinning solution and stirred continuously for 3 h on an electromagnetic stirrer to obtain a selenium-containing spinning solution.

[0041] (2) Electrospinning The selenium-containing spinning solution obtained in step (1) was connected to the spinning nozzle. The parameters of the electrospinning equipment were set as follows: the distance between the nozzle and the receiving roller was 18 cm, the spinning speed was 3 mL / h, the voltage was 22 kV, the ambient temperature in the electrospinning equipment was 25°C, the roller was rotated at a low speed and covered with aluminum foil to collect the fibers. After spinning, the residual solvent was evaporated in a vacuum drying oven at a temperature of 60°C for 24 h to obtain a selenium-containing fiber membrane (Se@Fiber). The SEM image is shown in FIG. Figure 3 shown.

[0042] Test Example 1: In vitro inflammatory factor clearance experiment The biomimetic selenium-doped fiber membrane (MSe@Fiber) prepared in Example 1, the PLGA fiber membrane (PLGA) prepared in Comparative Example 1, and the selenium-containing fiber membrane (Se@Fiber) prepared in Comparative Example 2 were co-cultured with high-glucose culture medium (IL-1β concentration: 100 pg / mL, TNF-α concentration: 220 pg / mL) containing inflammatory factors (IL-1β and TNF-α), and a blank control group (control) without adding fiber membrane was set up. The concentration changes of inflammatory factors in the culture medium were detected by ELISA.

[0043] The results are as follows Figure 4 As shown, the results showed that the average IL-1β concentrations of the control group, PLGA group, Se@Fiber group and MSe@Fiber group were 95.2 pg / mL, 92.9 pg / mL, 92.3 pg / mL and 63.3 pg / mL, respectively; the average TNF-α concentrations were 211.7 pg / mL, 209.3 pg / mL, 207.4 pg / mL and 128.1 pg / mL, respectively. Data analysis showed that the MSe@Fiber group had a 33.5% scavenging effect on IL-1β, which was significantly lower than that of Comparative Examples 1 and 2; at the same time, the MSe@Fiber group had a 39.5% scavenging effect on TNF-α, which was also significantly lower than that of Comparative Examples 1 and 2, while the fiber membranes prepared from Comparative Examples 1 and 2 had no such effect. The above results prove that the bionic selenium-doped fiber membrane (MSe@Fiber) prepared by the present invention exhibits significant inflammatory factor clearance ability in in vitro experiments, and its surface macrophage membrane wrapping has significant advantages in regulating the inflammatory microenvironment.

[0044] Experimental Example 2: Macrophage polarization regulation experiment The biomimetic selenium-doped fiber membrane (MSe@Fiber) prepared in Example 1, the PLGA fiber membrane (PLGA) prepared in Comparative Example 1, and the selenium-containing fiber membrane (Se@Fiber) prepared in Comparative Example 2 were co-cultured with macrophages under inflammatory stimulation, with a macrophage cell density of 4×10 4 / cm 2Inflammatory stimulation method: Use medium containing 1 μg / mL lipopolysaccharide for 12 hours. The medium preparation contains 89% high glucose medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. The above percentages are all volume fractions. A blank control group (control) without fiber membrane was set up. After 48 hours of culture, the expression levels of macrophage inflammatory differentiation markers (IL-1β and IL-6) and macrophage repair differentiation markers (Arg-1 and CD206) were detected by qPCR.

[0045] The experimental results are as follows Figure 5 As shown. The results show that compared with the control example and the blank control group, the bionic selenium-doped fiber membrane (MSe@Fiber) prepared in Example 1 of the present invention can significantly reduce the expression of IL-1β and IL-6, and the reduction is significantly higher than that of the Se@Fiber group in the control example 2; at the same time, the expression of Arg-1 and CD206 in the MSe@Fiber group is significantly increased, and the increase is better than that of the Se@Fiber group, and there is no significant difference between the PLGA group in the control example 1 and the blank control group. The results show that the bionic selenium-doped fiber membrane (MSe@Fiber) prepared by the present invention can significantly inhibit the inflammatory differentiation of macrophages and promote their differentiation into pro-repair, further proving its superiority in regulating macrophage polarization.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a bionic selenium-doped fiber membrane, characterized in that: The following steps are involved: (1) firstly, polylactic acid-glycolic acid copolymer and sodium selenite are mixed and stirred in a solvent to prepare a spinning solution, then electrospinning is performed, and finally drying is performed to obtain selenium-containing electrospun fibers; (2) The selenium-containing electrospun fibers obtained in step (1) are mixed with macrophage membranes and subjected to ultrasonic vibration to obtain a product.

2. The method for preparing the bionic selenium-doped fiber membrane according to claim 1, characterized in that: In the step (1), the concentration of the polylactic acid-glycolic acid copolymer in the spinning solution is 10 w / v %-30 w / v %; and the mass ratio of the polylactic acid-glycolic acid copolymer to sodium selenite is (90-95):(5-10).

3. The method for preparing the bionic selenium-doped fiber membrane according to claim 1, characterized in that: The solvent in step (1) is a mixed solvent consisting of dichloromethane and dimethylformamide; the volume ratio of dichloromethane to dimethylformamide is (1-5):

1.

4. The method for preparing the bionic selenium-doped fiber membrane according to claim 1, characterized in that: The conditions for electrospinning in step (1) are as follows: the distance between the nozzle and the receiving roller is 15-20 cm, the spinning speed is 1-5 mL / h, the spinning voltage is 20-25 kV, the spinning environment temperature is 20-30°C; the drying temperature is 50-70°C, and the time is 20-30 h.

5. The method for preparing the bionic selenium-doped fiber membrane according to claim 1, characterized in that: In step (2), the macrophage membrane is prepared by the following method: firstly suspending the macrophages in a phosphate buffer, then subjecting the macrophages to a freeze-thaw cycle, and finally centrifuging to obtain the macrophage membrane.

6. The method for preparing the bionic selenium-doped fiber membrane according to claim 5, characterized in that: The freeze-thaw cycle treatment conditions are: first freezing at -90~-70°C, then thawing at 35-40°C, repeating freezing and thawing 2-5 times; the centrifugal speed is 8000-12000 rpm, and the time is 5-15 min.

7. The method for preparing the bionic selenium-doped fiber membrane according to claim 1, characterized in that: In the step (2), the mass ratio of selenium-containing electrospun fibers to macrophage membrane is (5-20):

1.

8. The method for preparing the bionic selenium-doped fiber membrane according to claim 1, characterized in that: The frequency of the ultrasonic oscillation in step (2) is 30-50 kHz, and the duration is 10-60 min.

9. A bionic selenium-doped fiber membrane, characterized in that: The method is prepared by the method according to any one of claims 1 to 8.

10. Use of the bionic selenium-doped fiber membrane according to claim 9 in the preparation of medical devices or drugs for treating infectious bone defects.

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