Cellular burial-like signal bionic composite fiber scaffold as well as preparation method and application thereof

Abstract

The invention relates to the field of biological pharmacy, in particular to a cell burial-like signal bionic composite fiber scaffold and a preparation method and application thereof. The invention provides a composite fiber scaffold. The composite fiber scaffold comprises a fiber skeleton wrapped by a neutrophile granulocyte membrane; a brain-derived neurotrophic factor is encapsulated in the fiber skeleton. The present invention provides additional induction of neutrophile granulocyte membranes on macrophage polarization in addition to inflammatory factor adsorption. According to the invention, the structure, inflammation regulation and control and nerve growth promoting ability of the composite fiber scaffold are researched in multiple levels, so that not only is the neutrophile granulocyte membrane coating technology further improved and explored, but also a new idea is provided for spinal cord injury nerve function recovery.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a cell death signal-like bionic composite fiber scaffold and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) is a destructive neurological condition that occurs after acute spinal cord compression or transection. It brings lasting physical and economic burdens to patients due to the sensory and motor dysfunction that is difficult to recover. In spinal cord injury, secondary injury following direct physical injury will trigger a continuous inflammatory cascade, leading to progressive neuronal death and failure of neurorepair. Secondary injury is closely related to the sequential infiltration of inflammatory cells in the injured area. As the first cells to infiltrate the injured area in the inflammatory response, neutrophils, on the one hand, release a variety of enzymes such as myeloperoxidase (MPO), matrix metalloproteinases (MMPs), and neutrophil elastase (NE) through degranulation to aggravate tissue damage, and on the other hand, activate macrophages and microglia by releasing a variety of inflammatory factors to further amplify inflammation and induce neuronal damage. Subsequently, under the stimulation of such proinflammatory signals, macrophages recruited to the injured area are over-polarized to the M1 phenotype, and further secrete inflammatory factors such as IL-1β and TNF-α, leading to an uncontrollable inflammatory storm. Therefore, inhibiting the recruitment of neutrophils in the early stage of spinal cord injury and promoting the change of macrophages from a pro-inflammatory phenotype (M1) to an anti-inflammatory phenotype (M2) are considered to be effective measures to reduce secondary injury and promote tissue repair.

[0003] However, although in the traditional understanding, the crosstalk between local neutrophils and macrophages in the injury is the culprit that causes cytokine storms and aggravates secondary nerve damage, with the continuous deepening of research in recent years, the anti-inflammatory repair effect of neutrophils displayed by macrophage burial has gradually attracted people's attention. Studies have found that apoptotic neutrophils in macrophage burial under inflammatory conditions can activate anti-inflammatory responses by inhibiting proinflammatory cytokines and inducing the production of interleukin (IL)-10 and transforming growth factor (TGF)-β, promoting their own polarization to the M2 phenotype. Extracellular vesicles secreted by neutrophils also have similar effects.

[0004] The recruitment of neutrophils is coordinated by multiple ligand-receptor axes, and strategies targeting a single receptor often fail to effectively inhibit the recruitment of neutrophils and the progression of inflammation. Neutrophil membranes, because of their multiple receptors inherited from source cells, can neutralize multiple chemokines and inflammatory factors in the injured area and inhibit the infiltration of autologous inflammatory cells into the injured area. As cytokine decoys, they have been successfully used in the fields of spinal cord injury, osteoarthritis, and intervertebral disc degeneration. However, in previous studies, only the inflammatory adsorption effect of neutrophil membranes was focused on, and no one has studied the fate of neutrophil membranes after completing this mission. Given the anti-inflammatory effects triggered by macrophage burial of neutrophils and their extracellular vesicles under pathological and physiological conditions, we suspect that neutrophil membranes have similar efficacy and play an additional regulatory role in the changes of their inflammatory phenotype after completing the mission of immune adsorption and being phagocytosed by local macrophages in the injured area.

[0005] After spinal cord injury, local tissue cavitation hinders the connection of neuronal axons, and the inflammatory factor storm triggered by the infiltration of multiple inflammatory cells and the depletion of local neurotrophic factors lead to continuous neuronal death and failure of neural repair. As the two key elements of tissue engineering, biomaterial scaffolds and biological factors can provide a good cell growth platform and a suitable biological microenvironment for injury repair, and have been widely used in various injury scenarios. The directional microsol electrospinning of encapsulated BDNF developed by the research group in the early stage has achieved good therapeutic effects in the field of spinal cord injury repair. Its oriented spun fibers fill the tissue defects caused by injury, and with its unique topological structure, it induces macrophages to polarize to the M2 phenotype and improves neuroinflammation. At the same time, it can simulate the structure of natural spinal cord tissue, guide axon growth, reduce the infiltration of astrocytes, promote neural repair, and reduce the formation of fibrotic scars. BDNF under the protection of the core-shell structure is continuously released, supplements neurotrophy, guides endogenous neural stem cells to differentiate into neurons, and guides neural regeneration. However, natural nerve fibers are highly hydrophilic, and the hydrophobicity of PLLA spinning may limit its biological efficacy, so coating modification of the spinning surface is a feasible measure. Summary of the invention

[0006] In view of this, the present invention provides a biomimetic composite fiber scaffold with a cell burial signal and a preparation method and application thereof. The present invention provides an additional induction effect of neutrophil membrane on macrophage polarization in addition to the adsorption of inflammatory factors. The potential mechanism of neutrophil membrane affecting the functional changes of macrophage membrane was explored by joint analysis of transcriptomics and metabolomics. Based on this, we designed and constructed a biomimetic fiber scaffold with immunomodulatory function and neural differentiation potential to meet the needs of inflammation control and nerve growth at different stages after SCI. The neutrophil membrane was connected to the microsol electrospinning encapsulating BDNF through polylysine. In the acute phase, various chemokines are adsorbed by related receptors on the surface of neutrophil membranes to inhibit the infiltration of neutrophils at the injury site. At the same time, various pro-inflammatory cytokines are naturally shed after adsorption in the environment, and are engulfed by macrophages like "nanocapsules", providing cell burial signals to induce macrophage polarization and metabolic state to change to an anti-inflammatory phenotype, thereby achieving the purpose of inhibiting inflammation in the acute phase and reducing secondary injuries. In the chronic stage, BDNF is released slowly and continuously, promoting the differentiation of neural stem cells into neurons and creating suitable nutritional conditions for nerve repair. The structure, inflammation regulation and nerve growth-promoting ability of the composite fiber scaffold were studied at multiple levels, which not only further improved and explored the neutrophil membrane coating technology, but also provided new ideas for the recovery of nerve function in spinal cord injury.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a composite fiber scaffold, comprising: a fiber skeleton wrapped by a neutrophil membrane;

[0009] The fiber skeleton encapsulates brain-derived neurotrophic factor.

[0010] In some embodiments of the present invention, the composite fiber skeleton is wrapped with polylysine.

[0011] In some embodiments of the present invention, in the composite fiber skeleton, the concentration of polylysine is 0.005%-0.1% (w / v).

[0012] In some embodiments of the present invention, in the composite fiber skeleton, the concentration of polylysine is 0.005% (w / v), 0.01% (w / v), 0.05% (w / v) or 0.1% (w / v).

[0013] In some embodiments of the present invention, the composite fiber skeleton is obtained by electrospinning carboxylated L-polylactic acid.

[0014] The present invention also provides a method for preparing the composite fiber scaffold, comprising the following steps:

[0015] S1: mixing the brain-derived neurotrophic factor with a hyaluronic acid solution, performing ultrasonic emulsification, and then mixing with carboxylated L-polylactic acid and NN dimethylformamide to obtain a carboxylated L-polylactic acid microsol spinning solution;

[0016] S2: using the carboxylated L-polylactic acid microsol spinning solution to obtain the fiber skeleton by an electrospinning method;

[0017] S3: placing the fiber skeleton in the polylysine, mixing, washing, and then mixing with the neutrophil membrane to obtain the composite fiber scaffold.

[0018] In some embodiments of the present invention, in the above preparation method, the brain-derived neurotrophic factor is added in the form of a solution; the volume ratio of the brain-derived neurotrophic factor solution to the hyaluronic acid solution is 1:5.

[0019] In some embodiments of the present invention, in the above preparation method, the concentration of polylysine is 0.005%~0.1% (w / v).

[0020] In some embodiments of the present invention, in the above preparation method, the concentration of polylysine is 0.005% (w / v), 0.01% (w / v), 0.05% (w / v) or 0.1% (w / v).

[0021] In some embodiments of the present invention, in the above preparation method, the protein mass ratio of the fibrous skeleton and the neutrophil membrane is 50:1.

[0022] In some embodiments of the present invention, in the above preparation method, the brain-derived neurotrophic factor is mixed with bovine serum albumin to obtain the brain-derived neurotrophic factor solution.

[0023] In some embodiments of the present invention, in the above preparation method, the concentration of the brain-derived neurotrophic factor solution is 100 μg / mL.

[0024] In some embodiments of the present invention, in the above preparation method, the emulsification uses Span-80 and DCM.

[0025] In some embodiments of the present invention, in the above preparation method, the conditions of the electrospinning include: a pushing speed of 60 μL / min, a voltage of 15 kV, and a receiving distance of 15-20 cm.

[0026] In some embodiments of the present invention, the mixing time in the above preparation method S3 is 30 minutes.

[0027] The present invention also provides the use of the composite fiber skeleton and / or the fiber skeleton obtained by the above preparation method in preparing products for treating spinal cord injury.

[0028] The present invention also provides a product, comprising: the composite fiber skeleton and / or the fiber skeleton obtained by the above preparation method.

[0029] The present invention provides a composite fiber scaffold, comprising: a fiber skeleton wrapped by a neutrophil membrane;

[0030] The fiber skeleton encapsulates brain-derived neurotrophic factor.

[0031] The cell membrane coating technology and microsol electrospinning technology provided by the present invention construct a composite bionic scaffold that regulates inflammatory cell metabolic remodeling and promotes nerve regeneration through cell burial-like signals. Through a series of characterization measurements, cell experiments and in vivo animal experiments, it is proved that the neutrophil membrane coating of the scaffold effectively inhibits the infiltration of inflammatory cells by adsorbing inflammatory factors in the environment. After shedding, it provides "cell burial-like" signals, promotes macrophage metabolic remodeling, alleviates inflammatory responses, reduces neuronal apoptosis, and provides a good platform for nerve repair; the inner layer of directional fibers continuously supplements brain-derived neurotrophic factor (BDNF), contact-guides the growth of sprouting axons, promotes neuronal regeneration, and achieves multi-effect superposition to promote nerve function recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0033] Figure 1 The figure shows the construction process of the composite fiber scaffold and its mechanism of precisely regulating macrophage metabolic remodeling and promoting nerve regeneration after implantation into the injury site; A shows the preparation of neutrophil membrane vesicles NMVs and the schematic diagram of its potential mechanism of promoting macrophage M2 polarization; B shows the construction of NM-MS and its immune regulation and nerve regeneration mechanism;

[0034] Figure 2 Figure 2 shows the characterization of extracted neutrophil membranes (NMVs); wherein: A shows the TEM image and particle size analysis of NMVs; B shows the SDS-PAGE Coomassie Brilliant Blue staining of NMVs membrane proteins; C shows the Western blot bands of key membrane proteins of NMVs;

[0035] Figure 3Figure 4 shows that NMVs regulate the polarization phenotype transformation of macrophages; A shows a schematic diagram of the NMVs-Raw264.7 co-culture system; B shows a fluorescent image of macrophages co-cultured with DiR-labeled NMVs for 3 days; C shows flow cytometry analysis of CD86 or CD206 positive macrophages (using F4 / 80 gate) under different culture systems; D shows PCR analysis of IL-1β mRNA, TNF-α mRNA, IL-10 mRNA and TGF-β mRNA gene expression in macrophages under different culture systems;

[0036] Figure 4 Figure 2 shows the analysis of RNA-Seq results; A shows the volcano map of differentially expressed genes; B shows the results of GO analysis; C shows the results of KEGG analysis; D shows the heat map analysis results of differentially expressed genes in glycolysis and oxidative phosphorylation; E shows the heat map analysis results of inflammation-related genes; FH show the results of GESA analysis;

[0037] Figure 5 Figure 2 shows metabolomics analysis and mechanism verification; A shows KEGG analysis; B shows TCA cycle-related metabolite quantitative analysis; CD shows glutamine metabolism and α-KG content analysis; E shows Glud1 protein expression and quantification; F shows a schematic diagram of NMVs intervention in macrophage polarization pathway; GH shows NF-κB pathway-related protein expression and quantification; IJ shows p-p65 fluorescence images and semi-quantitative analysis in macrophages;

[0038] Figure 6 Figure 3 shows the morphological characterization of fiber scaffolds; wherein: A shows a schematic diagram of NM-MS preparation; B shows SEM images of different fiber scaffolds; C shows a frequency distribution histogram of different fiber scaffold diameters (n=100 fibers / sample); D shows TEM images of different fiber scaffolds; E shows XPS indicating chemical elements on the surface of different fiber scaffolds; F shows the distribution of neutrophil membranes labeled with FITC-BSA and DiR in NM-MS scaffolds; G shows Zeta potential analysis of the surface of different fiber scaffolds;

[0039] Figure 7 The performance characterization of composite fiber scaffolds is shown; wherein: A shows stress-strain curves of different scaffolds; B shows Young's modulus analysis of different scaffolds; C shows water contact angle measurement of different fiber scaffolds; D shows porosity analysis of different scaffolds; E shows WB inspection of key membrane proteins on the surface of NM-MS spinning; FG shows TNF-α and IL-1β adsorption curves of NM-MS spinning; H shows degradation curves of different scaffolds; I shows BDNF release curves of different scaffolds;

[0040] Figure 8Figure 2 shows the biocompatibility study of composite fiber scaffolds; A shows the live / dead cell staining fluorescence images of Raw cells on different scaffolds; B shows the live / dead cell staining fluorescence semi-quantitative analysis; C shows the CCK-8 test of Raw cells on different scaffolds;

[0041] Fig. 9 Figure 2 shows the regulation of macrophage phenotype; A shows the flow cytometry analysis of Raw cells cultured on different fiber scaffolds for 3 days; BC shows the flow cytometry analysis and quantification of CD86+ or CD206+ Raw cells on different fiber scaffolds; DE shows the immunofluorescence staining and semi-quantitative analysis of iNOS and CD206 of Raw cells on different fiber scaffolds; FG shows the expression levels and quantitative analysis of iNOS and CD206 detected by Western blotting; H shows the real-time OCRs of macrophages in the cell mitochondrial stress test; I shows the semi-quantitative analysis of basal respiration, maximum respiration and ATP production;

[0042] Fig.10 Figure 2 shows the detection of neural stem cell differentiation and neuron growth indexes; wherein: A shows a schematic diagram of the co-culture system; B shows the detection of inflammatory factors in the co-culture system by ELISA and C shows the expression pattern of growth factors; D shows the immunofluorescence image of Tuj-1 and GFAP in neural stem cells and E and F show semi-quantitative analysis; G shows the immunofluorescence image of NFH, GAP43 and MAP2 in neuronal cells and HJ shows semi-quantitative analysis; KM shows the use of Western blotting to detect the expression levels of Tuj-1, GFAP, NFH, GAP43 and MAP2 and quantitative analysis;

[0043] Fig.11 Figure 1 shows animal experimental images and postoperative motor function recovery; A shows the experimental images of T9 spinal cord hemisection; B shows the BBB score to evaluate the motor function recovery of the hind limbs; C shows the representative footprints used to evaluate the motor function recovery of the hind limbs, with the forelimb footprints shown in blue and the hind limb footprints shown in red, and D shows the quantitative analysis of the stride length in the footprints; E shows the angle in the inclined plane test; F shows the electrophysiological analysis and G shows the quantification of the amplitude of the motor evoked potential of rats in different groups;

[0044] Fig.12 In vivo immune regulation evaluation; wherein: A shows fluorescence imaging of SCI rats implanted with DiR-labeled NM-MS for in vivo imaging and B shows semi-qualitative analysis of fluorescence intensity; C shows immunofluorescence images of iNOS and ARG1 and D and E show semi-quantitative analysis; F shows immunofluorescence images of Glud1 around the lesion site and G shows semi-quantitative analysis; H shows immunofluorescence images of p-p65 around the lesion site and I shows semi-quantitative analysis;

[0045] Fig.13In vivo neural repair assessment; wherein: A shows H&E staining of longitudinal sections of the spinal cord; B shows quantitative analysis of cavitation area; C shows immunofluorescence images of Tuj-1 and GFAP in the lesion site and D and E show semi-quantitative analysis; F shows immunofluorescence images of GAP43 and G shows semi-quantitative analysis; H shows immunofluorescence images of CD31 and I shows semi-quantitative analysis;

[0046] Fig.14 The graph shows the time curve of neutrophil membrane coating shedding on NM-MS prepared with poly-lysine solutions of different concentrations. DETAILED DESCRIPTION

[0047] The invention discloses a cell burial signal bionic composite fiber scaffold and a preparation method and application thereof.

[0048] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0049] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0050] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0051] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0052] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0053] The present invention discloses for the first time the additional induction effect of neutrophil membrane on macrophage polarization in addition to the adsorption of inflammatory factors. The potential mechanism of neutrophil membrane affecting the functional changes of macrophage membrane was explored by the joint analysis of transcriptomics and metabolomics. Based on this, we designed and constructed a bionic fiber scaffold with immunomodulatory function and neural differentiation potential to meet the needs of inflammation control and nerve growth at different stages after SCI. The neutrophil membrane was connected to the microsol electrospinning encapsulating BDNF through polylysine. In the acute stage, various chemokines were adsorbed by related receptors on the surface of neutrophil membrane to inhibit the infiltration of neutrophils at the injury site. At the same time, various pro-inflammatory cytokines in the adsorption environment naturally fall off and are phagocytosed by macrophages like "nanocapsules", providing burial-like signals to induce macrophage polarization and metabolic state to change to anti-inflammatory phenotypes, thereby achieving the purpose of inhibiting inflammation in the acute stage and reducing secondary damage. In the chronic stage, BDNF is continuously and slowly released to promote the differentiation of neural stem cells into neurons, creating suitable nutritional conditions for neural repair. The structure, inflammation regulation and ability to promote nerve growth of the composite fiber scaffold were studied at multiple levels, which not only further improved and explored the neutrophil membrane coating technology, but also provided new ideas for the recovery of nerve function after spinal cord injury.

[0054] The preparation method of RM-MS involved in the present invention is relatively simple for the extraction of red blood cell membranes because they are anucleated cells. The washed red blood cells are resuspended in 0.25×PBS on ice for 20 minutes, then centrifuged at 800g for 5 minutes, the supernatant is discarded, and the precipitate is resuspended in 0.25×PBS on ice for 20 minutes. Repeat this process until hemoglobin is completely removed. The red blood cell membrane is resuspended, quantified, and prepared into red blood cell membrane vesicles RMVs by the same method, and stored at -80°C for subsequent research.

[0055] The preparation of RM-MS was the same as that of NM-MS, except that the cell membrane source was replaced by RMVs.

[0056] In Examples 1 to 4 of the present invention and Verification Example 1, all the raw materials and reagents used can be purchased from the market.

[0057] The present invention will be further described below in conjunction with embodiments:

[0058] Example 1 Preparation of Neutrophil Membrane-Coated Microsol Electrospinning (NM-MS)

[0059] 1. Extraction of Neutrophil Membranes

[0060] Neutrophils were isolated from the bone marrow of SD rats (4 weeks) by density gradient centrifugation. Specifically, before the rats were sacrificed, 2 mg*kg-1 LPS was injected intraperitoneally to activate neutrophils in vivo. After 8 h, the rats were sacrificed by an overdose of 2% pentobarbital intraperitoneally. The femurs and tibias on both sides were removed, and the bone marrow cavity was flushed with pre-warmed 1640 medium to prepare the bone marrow cell suspension and counted. Neutrophils were isolated using the Rat Bone Marrow Neutrophil Isolation Kit (Solebol, Beijing, China). The purified cells were immediately labeled with CD11b (Abcam) and HIS48 (Abcam) antibodies, washed with PBS, and analyzed by flow cytometry. The purified cells were resuspended in a hypotonic cell solution containing 30 mM Tris-HCl (pH 7.5), 225 mM D-mannitol, 75 mM sucrose, 0.2 mM EGTA and 4% protease phosphatase mixture, and disrupted using an ultrasonic cell disruptor (Xinzhi, Ningbo, Zhejiang). The disrupted solution was centrifuged at 20,000 gRCF, 4°C for 20 min, and the supernatant was centrifuged at 100,000 gRCF, 4°C for 40 min. The precipitate was collected, washed twice with 0.2 mM EDTA aqueous solution, resuspended, and then prepared into neutrophil membrane vesicles NMVs by ultrasound and lipid extrusion. The quantification was performed using a BCA quantitative kit (Solarbio, China), and the prepared membrane solution was stored in a refrigerator at -80°C.

[0061] 2. Preparation of electrospinning solution

[0062] Preparation of carboxylated L-polylactic acid (PLLA) electrospinning solution: 0.5 g PLLA was added to 4 g dichloromethane (DCM) solution, and stirred at high speed for 4 h at 20 °C ± 5 °C to form a uniform solution; 2 g NN dimethylformamide (DMF) solution was added to the above solution and continued to stir for 2 h to prepare a transparent and uniform carboxylated L-polylactic acid electrospinning solution as the control group PLLA.

[0063] Preparation of PLLA microsol electrospinning solution: 0.1g sodium hyaluronate (HA) was dissolved in 9.9g deionized water and stirred at 20℃±5℃ until completely dissolved to obtain a 1wt% sodium hyaluronate aqueous solution; rat BDNF was dissolved in 0.1wt% bovine serum albumin (BSA) solution to obtain a 100μg / mL final concentration BDNF solution. 10μL BDNF solution was mixed with 50μL 1wt% hyaluronic acid solution to obtain a uniform HA-BDNF hydrosol; 0.01g Span-80 and 4g DCM were added, and ultrasonic emulsification was performed at 20℃±5℃ for 20min to obtain a uniform milky white emulsion containing BDNF microsol particles; finally, 0.5g PLLA and 2g DMF were added in turn and stirred at room temperature to obtain a carboxylated L-polylactic acid microsol (MS) spinning solution.

[0064] 3. Preparation of oriented electrospun fibers

[0065] A 10mL syringe (10cm long, 0.9mm inner diameter of the needle) was used to extract a certain amount of carboxylated L-lactic acid electrospinning solution and carboxylated L-lactic acid microsol (MS) spinning solution. The syringe was fixed on the propulsion device, the propulsion speed was set to 60μL / min, and the voltage was set to 15kV. The distance between the needle tip and the electrode receiving end was maintained at 15~20cm, and the two ends were fixed with metal clips of a DC high-voltage power supply. After applying the voltage, parallel oriented spinning fiber scaffolds (PLLA and MS) can be collected between the electrode rods. The prepared electrospun fibers were vacuum dried overnight to remove residual solvent. The oriented fiber membrane was used for subsequent in vitro studies, and the oriented fiber bundles were used for subsequent in vivo studies.

[0066] 4. Preparation of Neutrophil Membrane-Coated Microsol Electrospinning (NM-MS)

[0067] The MS prepared above was soaked in 0.01% (w / v) poly-lysine (Aladdin, China) solution for 30 min, washed with deionized water for 3 times, and then mixed with the above neutrophil membrane solution at a protein mass ratio of 1:50, allowed to stand at 20℃±5℃ for 30 min, washed with deionized water for 3 times to remove excess cell membrane, and the finished product was frozen in a -80℃ refrigerator for use.

[0068] Example 2 Characterization of composite fiber scaffolds

[0069] 1. Scanning Electron Microscope (SEM)

[0070] Four groups of fiber scaffolds, PLLA, MS, RM-MS, and NM-MS, were fixed on the sample stage with conductive glue and gold-sprayed. The voltage was set to 10 kV, and the fiber scaffolds were photographed and recorded using SEM in a vacuum state. ImageJ was used to process the images and randomly calculate the fiber diameters of 100 fiber scaffolds.

[0071] 2. Elemental analysis

[0072] X-ray photoelectron spectroscopy (XPS) was used to compare the chemical element compositions and their ratio changes of the four groups of scaffolds.

[0073] 3. Transmission electron microscopy (TEM)

[0074] The spun fibers from each group were randomly collected on a double-layer copper mesh and dried at 20°C±5°C. The fiber morphology and internal structure were observed using a transmission electron microscope (TEM) at 120 kV.

[0075] 4. Visualized fluorescent microsol spinning

[0076] Fluorescence-visualized microsol electrospun fibers were prepared using bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (FITC) instead of BDNF, and DiR was used to label the neutrophil membrane, and the green fluorescent core and red cell membrane coating were observed by fluorescence microscopy.

[0077] 5. Water contact angle measurement

[0078] The hydrophilicity / hydrophobicity was evaluated by measuring the contact angle of water droplets on the surfaces of different fiber scaffolds using a contact angle meter.

[0079] 6. Mechanical properties tensile test

[0080] A 15.0×1.0×0.1 mm fiber bundle was prepared using a mold and stretched at a speed of 2.5 mm / min using a universal mechanical tester. The stress-strain curve was plotted and the Young's modulus of each group of scaffolds was calculated.

[0081] 7. Porosity determination

[0082] The porosity of each fiber scaffold was measured by the pycnometer method. The pycnometer was filled with ethanol and its mass was recorded as W1. The fiber scaffold with a mass of Ws was immersed in the pycnometer filled with ethanol. The ethanol would penetrate into the fiber pores. Additional ethanol was added to fill the pycnometer and its mass was recorded as W2. The fiber scaffold filled with ethanol was taken out and the remaining mass of the pycnometer and ethanol was recorded as W3. The porosity of the fiber scaffold was then calculated using the following formula:

[0083] p=

[0084] 8. Degradation performance determination

[0085] In order to test the degradation performance of different fiber scaffolds, each group of fiber scaffolds was immersed in a centrifuge tube containing 30 mL of PBS at 20°C ± 5°C, and the PBS was replaced every day. The fiber scaffolds were freeze-dried and weighed at a fixed time point every week, and the ratio of the fiber scaffold mass (Wt) to the initial mass (W0) at each time point was calculated and recorded as the degradation rate, and the degradation curve was drawn.

[0086] 9. Determination of drug release rate

[0087] Regarding the release measurement of BDNF, the MS, RM-SM, and NM-MS fiber scaffolds were immersed in centrifuge tubes containing PBS, placed in a constant temperature shaker at 37°C, and shaken at a speed of 120 times / min. The release solution was collected at 12h, 1d, 2d, 4d, 6d, 8d, 10d, 14d, 18d, 22d, 26d, 30d, 35d, and 40d. After each collection of the release solution, an equal volume of fresh PBS was added and continued to shake at 37°C. The concentration of BDNF in each release solution was determined using a BDNF ELISA kit, and the cumulative release curve of BDNF was drawn.

[0088] Example 3 Cell growth and differentiation on the surface of composite fiber scaffold

[0089] 1. Cell extraction and culture

[0090] Extraction of primary rat NSCs: After anesthetizing 16-day-old gestational rats, soak them in 75% alcohol for 2 minutes, remove the fetus from the rat abdomen, soak them in alcohol for disinfection, and then transfer them to sterile pre-cooled PBS solution. Dissect the fetal brain on ice in a clean bench, and peel off the attached meninges and blood vessels. Cut the brain tissue into pieces. Transfer to a new culture dish containing 2mL sterile PBS, add 2mL 0.25% trypsin and 200μL 50μg / mL DNase, and place it in a 37℃ incubator for digestion for 20min, shaking it gently every five minutes. After terminating the digestion, transfer the tissue fragments to a 50mL centrifuge tube and centrifuge at 1200rpm / min for 5min; discard the supernatant, wash three times with sterile PBS, discard the supernatant again, blow the tissue fragments with 2mL NSCs complete medium, collect the cell suspension in a new centrifuge tube, and add an appropriate amount of DNase to help blow it during the process. After repeating 3-4 times, the tissue was completely digested and the cell suspension was collected by filtering with a 75 μL cell strainer. NSCs cell suspension was plated at 1×10 7 The cells were seeded into 6-well plates and cultured with complete NSCs medium.

[0091] 2. Preparation of cell test materials

[0092] Before the cell experiment, the cell slides carrying each group of fiber scaffolds were placed at the bottom of the well plate for soaking in 75% ethanol and irradiation sterilization overnight. After washing three times with sterile PBS, they were soaked in the corresponding cell culture medium overnight for subsequent cell inoculation.

[0093] 3. Live / dead staining

[0094] 1×10 4 Raw264.7 cells were seeded in 24-well plates with different fiber membranes and cultured for 3 days. The cells were then stained with live / dead cell working staining solution for 30 minutes at room temperature in the dark. The staining solution was removed, and the cell staining was observed and photographed under a fluorescence microscope. The number of live cells was quantified using ImageJ software.

[0095] 4. CCK8 cell proliferation assay

[0096] The proliferation of Raw264.7 cells was evaluated by CCK8 kit. 5 Raw264.7 cells were seeded in 24-well plates with different fiber membranes. The culture medium was removed at 1, 3, and 5 days of culture, and CCK-8 reagent diluted 1:10 with culture medium was added. After incubation in a cell culture incubator for 4 hours, 100 μL of the mixed solution was transferred to a 96-well plate, and the absorbance at 450 nm was measured using an ELISA reader. All experiments were repeated three times.

[0097] 5. Inflammatory immune regulation

[0098] Raw264.7 cell polarization immunofluorescence staining: 3×10 4 Raw264.7 cells were seeded on different groups of scaffolds and cultured with 200 ng / mL LPS for 3 days. The cells were fixed, perforated, and blocked. Raw264.7 cells were labeled with F4 / 80-488, and M1 and M2 were labeled with iNOS and CD206 primary antibodies, respectively. The cells were incubated overnight at 4°C, washed, and incubated with corresponding secondary antibodies and DAPI. The cells were observed and photographed using an inverted fluorescence microscope. ImageJ software was used for semi-quantitative analysis of fluorescence intensity.

[0099] Raw264.7 cell polarization flow cytometry analysis: 5×10 4 Raw264.7 cells were seeded on different groups of scaffolds and cultured with 200 ng / mL LPS for 3 days. The cells were collected and incubated with anti-F4 / 80-APC, anti-CD86-PE and anti-CD206-PE at 4°C in the dark for 30 minutes, and analyzed using a flow cytometer and FlowJo software.

[0100] Metabolic analysis of hippocampal cells: The hippocampal extracellular flux XFe24 analyzer (Agilent, USA) was used to study mitochondrial oxygen consumption rates (OCRs). Cells were plated at 1 × 10 5 Cells were seeded in Seahorse XF-24 plates cultured in each spinning permeate. Cell plates were first incubated in a CO2-free incubator for 1 hour before analysis. For OCR analysis, Oligo (1 μM), FCCP (1.5 μM), and rotenone / antimycin (R / A) (2 μM) were injected into cells sequentially, and basal respiration was assessed as the last OCR before Oligo injection minus non-mitochondrial respiration; maximal respiration was assessed as the maximum OCR after FCCP injection minus the minimum OCR after R / A injection; and ATP production was assessed as the final OCR before Oligo injection minus the minimum OCR after Oligo injection.

[0101] 6. Neuronal Differentiation

[0102] Immunofluorescence staining of neuronal differentiation and growth indicators: NSCs and Raw264.7 cells were co-cultured using Transwell plates. NSCs were inoculated on the fiber membranes of each group in the upper chamber, and BMMs were inoculated on the corresponding fiber membranes in the lower chamber. Both chambers were treated with 200 ng / mL LPS and co-cultured for 10 days. According to the above method, neural stem cells were immunostained for MAP2, Tuj-1, GAP43, NF200 and GFAP. A confocal microscope was used to observe the differentiation status and staining, and ImageJ was used for semi-quantitative analysis of fluorescence intensity.

[0103] WB determination of neuronal differentiation and growth indicators: The above Transwell co-cultured NSCs were subjected to protein extraction, quantification, electrophoresis and membrane transfer. After blocking, the membrane was incubated with primary antibodies against MAP2, Tuj-1, GAP43, NF200 and GFAP, and then incubated with secondary antibodies for band imaging and quantitative analysis.

[0104] ELISA: Based on the above culture system, the supernatant of the cell culture medium was collected after 5 days of intervention. The expression levels of TNF-α, IL-1β, TGF-β, NT-3 and PDGF in the supernatant were detected using ELISA kits.

[0105] Example 4 In vivo evaluation of composite fiber scaffolds

[0106] 1. Establishment of rat spinal cord hemisection model

[0107] Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg). After anesthesia, a 3 cm longitudinal incision was made in the middle of the rat's back with T9 as the center, and the paraspinal muscles were exposed and bluntly separated. The T9 lamina was exposed and the right lamina was bitten off to expose the spinal cord. The right spinal cord was cut with the tip of a scalpel to form a 3 mm defect. The wound was rinsed with normal saline, and a 2 mm × 2 mm × 3 mm fiber scaffold was implanted into the spinal cord defect, and the muscle and skin were tightly sutured. In the sham operation group, only the lamina was removed without cutting the spinal cord; in the blank control group, no material was implanted after cutting the spinal cord; in the negative control group, PLLA, MS, and RM-MS scaffolds were implanted, and in the experimental group, NM-MS scaffolds were implanted. After surgery, each rat was injected intramuscularly with penicillin (2 × 10 5 After the model was successfully established, the rats' urination reflex was impaired and the bladder was manually emptied every 12 hours until spontaneous urination was restored.

[0108] 2. Specimen collection

[0109] At 1 week and 6 weeks after surgery, 15 rats were randomly selected from each group for euthanasia. Spinal cords were collected after cardiac perfusion with PBS and 4% paraformaldehyde, fixed, gradient dehydrated, immersed, and paraffin-embedded, and 5 μm-thick longitudinal sections were cut for histological analysis.

[0110] 3. Evaluation of animal motor function

[0111] The motor function of rats was evaluated using the BBB and IPT scoring systems, footprint analysis, and electrophysiological testing. The BBB score evaluates the movement and coordination of the rat's hind limb joints, ranging from 0 to 21 points. The IPT score measures the maximum tilt angle at which the rat can stay on the inclined board. Eight rats were randomly selected from each group at a fixed time point each week, and the BBB and IPT scores were measured by two blinded observers. At 8 weeks after surgery, three rats were randomly selected from each group, and the forelimbs were painted blue and the hind limbs were painted red. The rats were asked to walk on a narrow runway on white paper, and their footprints were recorded. Eight weeks after surgery, the rats were anesthetized, and the stimulating electrode was inserted into the spinal cord above the injury site, and the receiving electrode was inserted into the right gastrocnemius muscle. The stimulation intensity was 0.5V, the stimulation duration was 0.5s, and the MEPs amplitude was recorded.

[0112] 4. DiR in vivo imaging

[0113] NM-MS were labeled with DiR membrane probe before implantation. Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg) at designated time points (0, 1, 3, 5, 7, and 9 days), and bioluminescent images were obtained using an in vivo imaging system (Spectral Instrumentsimaging, USA).

[0114] 5. Histological analysis

[0115] The 6-week tissue sections were dewaxed with xylene and ethanol, and hematoxylin and eosin (H&E) staining was used to evaluate the repair of spinal cord tissue structure. ImageJ was used to measure the area of ​​spinal cord cavitation in each group. After xylene and ethanol dewaxing, antigen retrieval was performed with 0.3% hydrogen peroxide, followed by 5% BSA blocking treatment, and DHE staining was performed on the 1-week tissue sections. Immunofluorescence staining of iNOS, Arg-1, Glud1, and p-p65 was performed on the 1-week tissue sections to evaluate early immune regulation in vivo. Immunofluorescence staining of GFAP was used to evaluate the activation of astrocytes in vivo at 6 weeks. Immunofluorescence staining of Tuj-1, NeuN, and GAP43 was used to evaluate the in vivo neural regeneration at 6 weeks.

[0116] Verification Example 1 Poly-L-lysine Concentration

[0117] The lysine concentration in step 4 of Example 1 was adjusted to 0.005% to 0.1%, and the neutrophil membrane-coated microsol electrospinning (NM-MS) obtained at different concentrations was used to test the cell membrane coating shedding rate. The experimental results are as follows: Fig.14 As shown in the figure, over time, the cell membrane coating on the surface of each group of NM-MS gradually fell off, and the higher the concentration of the polylysine solution used in the preparation, the slower the shedding rate. Given that the peak of macrophage recruitment under spinal cord injury conditions is 7 days after spinal cord injury, NM-MS prepared at a polylysine concentration of 0.01% was selected for subsequent experiments, because the cell membrane coating on it, after staying for a sufficient time to complete the mission of adsorbing inflammatory factors, was mostly shed at 7 days, making it easier to be engulfed by macrophages recruited to the injury site.

[0118] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite fiber scaffold, characterized in that: include: The membrane-encased fibrous skeleton of neutrophils; The fiber skeleton encapsulates brain-derived neurotrophic factor.

2. The composite fiber skeleton according to claim 1, characterized in that The encapsulation adopts polylysine.

3. The composite fiber skeleton according to claim 2, characterized in that The concentration of the polylysine is 0.005%-0.1% (w / v).

4. The composite fiber scaffold according to any one of claims 1 to 3, characterized in that: The fiber skeleton is obtained by electrostatic spinning of carboxylated L-polylactic acid.

5. The method for preparing the composite fiber scaffold according to claim 4, characterized in that: The steps include: S1: mixing the brain-derived neurotrophic factor with a hyaluronic acid solution, performing ultrasonic emulsification, and then mixing with carboxylated L-polylactic acid and NN dimethylformamide to obtain a carboxylated L-polylactic acid microsol spinning solution; S2: using the carboxylated L-polylactic acid microsol spinning solution to obtain the fiber skeleton by an electrospinning method; S3: placing the fiber skeleton in the polylysine, mixing, washing, and then mixing with the neutrophil membrane to obtain the composite fiber scaffold.

6. The preparation method according to claim 5, characterized in that: The brain-derived neurotrophic factor is added in the form of a solution; the volume ratio of the brain-derived neurotrophic factor solution to the hyaluronic acid solution is 1:

5.

7. The preparation method according to claim 5 or 6, characterized in that: The concentration of the polylysine is 0.005%-0.1% (w / v).

8. The preparation method according to any one of claims 5 to 7, characterized in that: The protein mass ratio of the fiber skeleton to the neutrophil membrane is 50:

1.

9. Use of the composite fiber skeleton according to any one of claims 1 to 4 and / or the fiber skeleton obtained by the preparation method according to any one of claims 5 to 8 in preparing products for treating spinal cord injuries.

10. A product, characterized in that include: The composite fiber skeleton as described in any one of claims 1 to 4 and / or the fiber skeleton obtained by the preparation method as described in any one of claims 5 to 8.

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