Preparation method of W1.33Ci-MXene layered transition metal carbon / nitride-chitosan nerve conduit

Abstract

The invention belongs to the field of nerve injury repair scaffold materials, and particularly relates to a preparation method of a W1.33Ci-MXene layered transition metal carbon / nitride-chitosan nerve conduit. Comprising the following steps: S1, preparing a chitosan / acetic acid solution by taking chitosan as a solute and acetic acid as a solvent; the preparation method comprises the following steps: preparing a W1.33Ci-MXene / genipin solution by taking W1.33Ci-MXene and genipin as solutes and pure water as a solvent; s2, mixing the two solutions, injecting the mixed solution into a catheter mold, and gelatinizing the mixed solution; and S3, performing low-temperature freezing, and then performing freeze drying immediately. In-vitro experiments prove that the nerve conduit provided by the invention can promote differentiation of a PC12 cell line through temperature change induced by a photothermal effect by using near infrared (NIR) to stimulate the nerve conduit, so that the nerve conduit has the capability of forming synapses.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nerve injury repair scaffold materials, and particularly relates to a W 1.33 The application belongs to the field of nerve injury repair scaffold materials, and particularly relates to a W BACKGROUND

[0002] Vision is one of the most important senses of human beings, which is formed from the visible light reflected by the objects in the external environment, enters the eyeball through the cornea, and is focused on the retina through the cornea, aqueous humor, lens and vitreous body and other refractive systems, is converted into nerve impulses by photoreceptors such as cone cells and rod cells, and is conducted along the optic nerve, can be projected to the visual cortex by the lateral geniculate body or directly conducted to the superior colliculus, and vision is formed. In this process, the optic nerve (ON) formed by the extension and convergence of the axons of retinal ganglion cells (RGCs) plays an important role in conducting visual nerve impulses. After the optic nerve injury, the damage of the axons of RGCs and the death of RGCs are the primary causes of blindness of patients, and therefore, promoting the regeneration and repair of RGCs and their axons after the optic nerve injury is a necessary way to restore the visual function of patients with optic nerve injury.

[0003] For a long time, in order to promote the regeneration and repair of RGCs and their axons, many researchers have focused on inducing in vivo nerve regeneration by electrical stimulation. This method makes the stimulated optic nerve produce nerve impulses by electrical stimulation, and adjusts the nerve proteins related to regeneration, so as to promote the development and maturation of nerves, and has been proved to have great potential in the treatment of neurodegenerative diseases, nerve injury and retinal degeneration. However, due to the direct use of electrical stimulation requires electrode-tissue interface, it has the disadvantages of invasive operation and complications at the interface generated by electrical stimulation, and its in vivo application is still challenging. Therefore, the non-invasive stimulation method as an alternative way has been more and more widely studied, among which the more common ones are magnetic signals, piezoelectric signals or photoelectric signals generated by using specific wavelengths of light. At present, these signals are mostly generated by using inorganic semiconductor substrate materials such as gold, copper, silicon, etc. However, there are still limitations in the use of these materials, such as low photo-thermal absorption efficiency, low conversion efficiency and the same may cause complications of surrounding tissues.

[0004] As a two-dimensional material of transition metal carbide, nitride and carbonitride, MXene has more excellent electrical and mechanical properties than the above-mentioned inorganic semiconductor materials. There has been a related research on the promotion of neuronal electrical activity by MXene under photothermal stimulation, and it is determined that the toxicity generated by MXene under light is almost negligible. MXene has more than 70 predicted morphologies, including solid solution groups and MXene with multi-layer thin layer structure. According to the thickness of the thin layer, the material can absorb light and accumulate in a wide band in the ultraviolet region. Photons are excited after absorption, forming a light-induced electric field charge transport. This phenomenon occurs depending on its light collection ability.

[0005] The light-heat conversion efficiency of MXene structure is very excellent, but it will still be affected by the localized surface plasmon resonance effect (LSPR effect) generated by the material contained therein, that is, free electrons collapse into photons or other non-equilibrium charge carriers due to the LSPR effect, and the excited carriers are relaxed through electron-photon scattering, while the nanoparticles themselves are heated. Tungsten (W) based nanosystems are widely used in medical implants due to three key factors: high atomic number, high absorption in the near-infrared region, and strong X-ray attenuation characteristics. At the same time, due to its hydrophilicity and good electrical conductivity, it is also predicted and proven to promote the self-renewal and differentiation of neural stem cells. Therefore, tungsten is an ideal element for MXene structure.

[0006] Chitosan, as an inherently biocompatible and biodegradable polymer, has various advantages such as from affordable to efficient antibacterial properties. It has been widely used in the biomedical field and can be used as a related carrier for drug delivery, implant scaffolds, nerve conduits or platforms, etc. for the regeneration and repair of damaged nerves. However, the extremely low mechanical properties of these materials hinder their further use. Therefore, the combination of tungsten-based MXene and natural polymers such as chitosan has great potential in generating and transmitting such wireless photothermal signals. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a W 1.33 The preparation method of the Ci-MXene layered transition metal carbon / nitride-chitosan nerve conduit.

[0008] The technical solutions adopted by the present application are as follows:

[0009] A W 1.33 The preparation method of the Ci-MXene layered transition metal carbon / nitride-chitosan nerve conduit comprises the following steps:

[0010] S1: Prepare a chitosan / acetic acid solution with chitosan as the solute and acetic acid as the solvent; and prepare a W 1.33Ci-MXene and genipin are solutes, and pure water is solvent to configure W 1.33 Ci-MXene / Genipin solution;

[0011] S2: Mix the above two solutions, inject into the catheter mold, and make it gel;

[0012] S3: Freeze at low temperature, and then freeze dry immediately.

[0013] In an optional embodiment, in S1,

[0014] The concentration of chitosan is 3%-5% w / v, the concentration of acetic acid is 1.5%-2.5% v / v; the W 1.33 The concentration of Ci-MXene is 20-50 mg / mL, and the concentration of genipin is 1%-3% w / v.

[0015] In an optional embodiment, in S1,

[0016] The concentration of chitosan is 3% w / v, and the concentration of acetic acid is 2% v / v; the W 1.33 The concentration of Ci-MXene is 20 mg / mL, and the concentration of genipin is 1% w / v.

[0017] In an optional embodiment, in S2,

[0018] The volume ratio of the chitosan / acetic acid solution to the W 1.33 The volume ratio of the Ci-MXene / genipin solution to the W 1.33 The ratio of the Ci-MXene / genipin solution to the W

[0019] In an optional embodiment, the bottom of the catheter mold is sealed; the inner diameter of the catheter mold is 3-4 mm; the catheter mold uses a cylindrical copper strip with a diameter of 1-1.5 mm as a central axis, and the two ends are wrapped with adhesive tape as a fixation.

[0020] In an optional embodiment, the inner diameter of the catheter mold is 3.33 mm; the catheter mold uses a cylindrical copper strip with a diameter of 1.1 mm as a central axis.

[0021] In an optional embodiment,

[0022] In S2, the catheter mold injected with the mixed solution is placed in a constant temperature environment at 37 degrees Celsius overnight to make it gel;

[0023] In S3, the catheter mold with the gelled mixed solution is placed in a low temperature environment at -80 degrees Celsius for 5 hours, and then freeze dried for 9 hours.

[0024] In an optional embodiment, the W 1.33 The preparation of Ci-MXene layered transition metal carbon / nitride comprises the following steps:

[0025] S11: (W 2 / 3 Y 1 / 3 )2AlC material was added to a mixed solution of deionized water, hydrochloric acid, and LiF, and stirred in an oil bath at 35°C for 48 hours;

[0026] S12: Etching is performed under conditions of a pH value close to 6, and then centrifugation is performed at 3500 rpm for 3 minutes, the acidic supernatant is decanted, and the precipitate is redispersed in deionized water to remove impurities and HCl. This process is repeated until ultra-thin W is obtained by layering. 1.33 C nanosheets;

[0027] S13: The obtained MXene product was collected by MF-Millipore 0.45 μm vacuum assisted filtration and placed in a vacuum desiccator and dried at 25 °C for 18 h to obtain W 1.33 Ci-MXene layered transition metal carbon / nitride products.

[0028] In an optional embodiment, the (W 2 / 3 Y 1 / 3 ) A method for preparing 2AlC material, comprising the following steps:

[0029] S111: Tungsten, yttrium, aluminum, and graphite were mixed in a mortar and cold pressed. The mixture was placed in a flowing argon atmosphere and heated at a rate of 8°C / min to 1450°C for 2 hours.

[0030] S112: The obtained product was cooled to room temperature and dissolved in 50 mL of HCl for 18 hours to dissolve impurities, and then the impurities and HCl were removed by centrifugation at 3500 rpm for 3 minutes, decanting the acidic supernatant, and redispersing the precipitate in deionized water;

[0031] S113: The obtained MAX powder was dried in a vacuum dryer at 25° C. for 18 hours to obtain a product.

[0032] In an optional embodiment, in S111,

[0033] The mass ratio of the mixed powders of tungsten, yttrium, aluminum and graphite is 1.33:1.33:1:1.

[0034] The beneficial effects of the present invention are as follows:

[0035] (1) The present invention induces W by near-infrared photothermal 1.33The temperature change generated by the Ci-MXene layered transition metal carbon / nitride-chitosan nerve conduit can promote the proliferation and neuron growth of PC12 cells, and changes the morphological characteristics of PC12 cells.

[0036] (2) The present application induces W 1.33 The temperature change generated by the Ci-MXene layered transition metal carbon / nitride-chitosan nerve conduit can promote the proliferation and neuron growth of PC12 cells, and changes the morphological characteristics of PC12 cells.

[0037] (3) The present application provides W 1.33 The C-MXene layered transition metal carbon / nitride-chitosan nerve conduit can promote the growth, proliferation and differentiation of PC12 cells through calcium ion channels, which is verified by in vitro experiments. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings obtained according to these drawings without creative labor are still within the scope of the present application.

[0039] Figure 1 A is W 1.33 Ci-MXene synthesis process diagram.

[0040] Figure 1 B is (W 2 / 3 Y 1 / 3 )2AlC phase scanning electron microscope image.

[0041] Figure 1 C is the scanning electron microscope image after (W 2 / 3 Y 1 / 3 )2AlC phase treated with hydrofluoric acid solution.

[0042] Figure 1 i- Figure 1 iv respectively are Figure 1 C element map shows W, Y, Al, C element signal diagram.

[0043] Figure 1 D is W 1.33 Ci-MXene transmission electron microscope image.

[0044] Figure 1E is a high-resolution transmission electron microscope (HRTEM) image and a selective area electron diffraction (SAED) image of an embodiment of the present application.

[0045] Figure 1 F is a WMC conduit pore size distribution graph of an embodiment of the present application. 1.33 X-ray diffraction (XDR) image of a Ci-MXene.

[0046] Figure 1 G is a WMC conduit pore size distribution graph of an embodiment of the present application. 1.33 X-ray photoelectron spectroscopy (XPS) image of a Ci-MXene.

[0047] Figure 2 A is a schematic diagram of the process of making a nerve conduit of an embodiment of the present application.

[0048] Figure 2 B is a cross-sectional image of a WMC conduit of an embodiment of the present application.

[0049] Figure 2 C is a SEM image of the porous structure of a WMC conduit of an embodiment of the present application.

[0050] Figure 2 D is Figure 2 Magnified image of the red dashed line portion in C.

[0051] Figure 2 E is Figure 2 Magnified image of the red dashed line portion in D.

[0052] Figure 2 F is a pore size distribution graph of a simple chitosan (CH) conduit of an embodiment of the present application.

[0053] Figure 2 G is a pore size distribution graph of a WMC conduit of an embodiment of the present application.

[0054] Figure 2 H is a schematic diagram of the dynamic swelling of a conduit at 37 degrees Celsius and pH 7.4 of an embodiment of the present application.

[0055] Figure 2 I is a schematic diagram of the degradation of a WMC conduit in PBS and PBS+lysozyme of an embodiment of the present application.

[0056] Figure 2 J is a Fourier transform infrared spectroscopy (FTIR) graph of CH and WMC conduits of an embodiment of the present application.

[0057] Figure 2 K is a tensile modulus graph of CH and WMC conduits (n=3, ****p<0.0001) of an embodiment of the present application.

[0058] Figure 3 A is a schematic diagram of the catheter using near-infrared light in an embodiment of the present application.

[0059] Figure 3 B is a temperature rise curve diagram of the CH and WMC catheter after 3 minutes of near-infrared light (808 nm, 0.5 Wcm -2 ) irradiation in an embodiment of the present application.

[0060] Figure 3 C is a temperature rise curve diagram of the CH and WMC catheter after 3 minutes of near-infrared light (808 nm, 1.0 Wcm -2 ) irradiation in an embodiment of the present application.

[0061] Figure 3 D is a temperature rise curve diagram of the CH and WMC catheter after 3 minutes of near-infrared light (808 nm, 2.0 Wcm -2 ) irradiation in an embodiment of the present application.

[0062] Figure 3 E is a temperature change curve diagram of the catheter in a 3 near-infrared light (808 nm, 0.5 Wcm -2 ) switching cycle in an embodiment of the present application.

[0063] Figure 3 F is a temperature change curve diagram of the catheter in a 3 near-infrared light (808 nm, 1.0 Wcm -2 ) switching cycle in an embodiment of the present application.

[0064] Figure 3 G is a temperature change curve diagram of the catheter in a 3 near-infrared light (808 nm, 2.0 Wcm -2 ) switching cycle in an embodiment of the present application.

[0065] Figure 3 H is a digital image of the real-time temperature rise of the near-infrared light irradiation under different power densities in an embodiment of the present application.

[0066] Figure 3 I is a photothermal stability heat map under 0.5 Wcm -2 of near-infrared light irradiation in an embodiment of the present application.

[0067] Figure 4 A is a CLSM image of PC12 cell co-stained with calcein AM / PI after 5 days of culture in an embodiment of the present application.

[0068] Figure 4 B is a schematic diagram of the cell survival rate of PC12 cells after 5 days of culture on the CH and WMC catheter in an embodiment of the present application.

[0069] Figure 4 C is a schematic diagram of the CCK-8 detection results of PC12 cell culture for 5 days in an embodiment of the application (n = 3).

[0070] Figure 4 D is a CLSM image of cytoskeleton (red) / nucleus (blue) co-staining of PC12 cells after culture for 3 days, 5 days and 14 days in an embodiment of the application.

[0071] Figure 4 E is a bar chart of the percentage of PC12 cells with neurite cells in an embodiment of the application (*p < 0.05, **p < 0.01, ***p < 0.001).

[0072] Figure 4 F is a bar chart of the length of PC12 cell neuron axons in an embodiment of the application (*p < 0.05, **p < 0.01, ***p < 0.001).

[0073] Figure 5 A is an immunofluorescence staining image of NF / DAPI, PSD95 / DAPI and GFAP / DAPI in an embodiment of the application.

[0074] Figure 5 B is a representative image of Western Blot protein imprint analysis of NF, PSD95 and GFAP in an embodiment of the application.

[0075] Figure 5 C is a schematic diagram of quantitative statistical analysis results of NF in WB analysis in an embodiment of the application (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

[0076] Figure 5 D is a schematic diagram of quantitative statistical analysis results of PSD95 in WB analysis in an embodiment of the application (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

[0077] Figure 5 E is a schematic diagram of quantitative statistical analysis results of GFAP in WB analysis in an embodiment of the application (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

[0078] Figure 6 A is an immunofluorescence staining image of L-VGCC / DAPI and SYN1 / DAPI in an embodiment of the application.

[0079] Figure 6 B is a representative image of WB analysis of L-VGCC and SYN1 in an embodiment of the application.

[0080] Figure 6 C is a schematic diagram of the quantitative statistical analysis results of L-VGCC in WB analysis in an embodiment of the application (n = 3).

[0081] Figure 6 D is a schematic diagram of the quantitative statistical analysis results of SYN1 in WB analysis in an embodiment of the application (n = 3).

[0082] Figure 6 E is a calcium ion imaging result image in an embodiment of the application.

[0083] Figure 6 F is a schematic diagram of the quantitative analysis results of calcium ion fluorescence intensity in an embodiment of the application (n≥20, *p<0.05, **p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0084] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings.

[0085] Example 1: (W 2 / 3 Y 1 / 3 )2AlC (i-MAX phase) preparation:

[0086] S311: Mix tungsten (W), yttrium (Y), aluminum (Al) and graphite (C) powders in a ratio of 1.33:1.33:1:1, and grind them into uniform powders using a maroon mortar and pestle. Cold-press the obtained mixed powders, and then heat them to 1450℃ at a speed of 8℃ / min -1 in a flowing argon environment, and react for 2 hours;

[0087] S312: Cool the above powders to room temperature, dissolve them in 50mL of hydrochloric acid (HCl) for 18 hours to dissolve other metal and oxide impurities;

[0088] S313: Centrifuge the mixed solution at 3500rpm for 3 minutes, then decant the acidic supernatant, and redisperse the precipitate in deionized water to remove excess HCl;

[0089] S314: Finally, dry it in a vacuum dryer at 25℃ for 18 hours to obtain the final i-MAX phase powder.

[0090] Example 2: W 1.33 Ci-MXene preparation:

[0091] S31: 2 g of the i-MAX phase powder described above was weighed into a mixed solution consisting of deionized water (6 mL), hydrochloric acid (50 M, 12 mL), and LiF (4 g), and was stirred thoroughly in an oil bath at 35°C for 48 hours;

[0092] S32: Etching was performed under conditions close to pH 6, and then the mixed solution was centrifuged at 3500 rpm for 3 minutes, after which the acidic supernatant was decanted, and the precipitate was redispersed in deionized water to remove excess HCl;

[0093] S33: The washing was repeated until an ultra-thin W 1.33 Ci-MXene nanosheet was obtained. The resulting product was collected by vacuum filtration using a MF-Millipore 0.45 pm filter membrane;

[0094] S34: The product MXene was placed in a vacuum dryer at 25°C for 18 hours, and finally a W 1.33 Ci-MXene powder was obtained.

[0095] Example 3: W 1.33 Ci-MXene layered transition metal carbide / nitride-chitosan nerve conduit was prepared:

[0096] S1: Chitosan (3% w / v) was dissolved in an acetic acid solution (2% v / v) at room temperature, and stirred for 2 hours to obtain a uniform chitosan / acetic acid solution;

[0097] S2: The chitosan / acetic acid solution was degassed at a speed of 3000 rpm, and was stored at 24°C for 12 hours;

[0098] S3: 170: μL of the W 1.33 Ci-MXene solution (20 mg / mL) was mixed with the genipin solution (1% w / v) and was ultrasonically stirred for 15 minutes. 830 μL of the W 1.33 Ci-MXene / genipin mixed solution was added to 830 μL of the chitosan / acetic acid solution to obtain a mixture with a final volume of 1 mL;

[0099] S4: The mixture was slowly stirred and immediately added to the conduit mold, preferably a tube with a sealed bottom and an inner diameter of 3.33 mm, and a cylindrical copper strip with a diameter of 1.20 mm fixed at both ends with adhesive tape. The copper strip was placed in the tube, and then the uniform mixture was injected into the gap between the plastic tube and the copper strip to prevent the formation of air bubbles;

[0100] S5: The filled conduit mold is wrapped with aluminum foil and placed vertically in a constant temperature incubator at 37°C overnight to gelate. After gelling, the filled nerve conduit mold is removed and frozen in a refrigerator at -80°C for 5 hours;

[0101] S6: The conduit mold is removed and the formed nerve conduit is gently pulled out of the conduit mold and immediately freeze-dried for 9 hours.

[0102]

[0103] The above disclosure is only the preferred embodiment of the present application, of course, cannot be limited to the scope of the present application, therefore, the equivalent changes made in accordance with the claims of the present application, still falls within the scope of the present application.​

Claims

1. A W 1.33 The preparation method of Ci-MXene layered transition metal carbon / nitride-chitosan nerve conduit is characterized in that: The following steps are involved: S1: Chitosan as solute and acetic acid as solvent were used to prepare chitosan / acetic acid solution; 1.33 Ci-MXene and genipin were used as solutes and pure water as solvent to prepare W 1.33 Ci-MXene / genipin solution; S2: Mix the above two solutions, inject them into the catheter mold, and gel them; S3: Low temperature freezing followed by immediate freeze drying.

2. The method for preparing a nerve conduit according to claim 1, characterized in that: In S1, The concentration of chitosan is 3%-5% w / v, the concentration of acetic acid is 1.5%-2.5% v / v; 1.33 The concentration of Ci-MXene is 20-50 mg / mL, and the concentration of genipin is 1%-3% w / v.

3. The method for preparing a nerve conduit according to claim 2, characterized in that: In S1, The concentration of chitosan is 3% w / v, the concentration of acetic acid is 2% v / v; 1.33 The concentration of Ci-MXene was 20 mg / mL, and the concentration of genipin was 1% w / v.

4. The method for preparing a nerve conduit according to claim 1, wherein: In S2, The chitosan / acetic acid solution and the W 1.33 The volume ratio of the Ci-MXene / genipin solution mixture is 15-20:85-80; the chitosan vinegar / acid solution and the W 1.33 The mixing ratio of Ci-MXene / genipin solution is 17:

83.

5. The method for preparing a nerve conduit according to claim 1, wherein: The bottom of the catheter mold is sealed; the inner diameter of the catheter mold is 3-4 mm; the catheter mold uses a cylindrical copper bar with a diameter of 1-1.5 mm as the central axis, and its two ends are wrapped with tape for fixing.

6. The method for preparing a nerve conduit according to claim 5, characterized in that: The inner diameter of the catheter mold is 3.33 mm; the catheter mold uses a cylindrical copper bar with a diameter of 1.1 mm as the central axis.

7. The method for preparing a nerve conduit according to claim 4, characterized in that: In S2, the catheter mold filled with the mixed solution is placed in a constant temperature environment at 37 degrees Celsius overnight to allow it to gel; In S3, the catheter mold in which the mixed solution has been gelled is placed in a low-temperature environment of -80 degrees Celsius and frozen for 5 hours, and then freeze-dried for 9 hours.

8. The method for preparing a nerve conduit according to claim 1, characterized in that: The W 1.33 The preparation of Ci-MXene layered transition metal carbon / nitride comprises the following steps: S11: (W 2 / 3 Y 1 / 3 )2AlC material was added to a mixed solution of deionized water, hydrochloric acid, and LiF, and stirred in an oil bath at 35°C for 48 hours; S12: Etching is performed under conditions of a pH value close to 6, and then centrifugation is performed at 3500 rpm for 3 minutes, the acidic supernatant is decanted, and the precipitate is redispersed in deionized water to remove impurities and HCl. This process is repeated until ultra-thin W is obtained by layering. 1.33 C nanosheets; S13: The obtained MXene product was collected by MF-Millipore 0.45 μm vacuum assisted filtration and placed in a vacuum desiccator and dried at 25 °C for 18 h to obtain W 1.33 Ci-MXene layered transition metal carbon / nitride products.

9. The method for preparing a nerve conduit according to claim 8, characterized in that: The (W 2 / 3 Y 1 / 3 ) A method for preparing 2AlC material, comprising the following steps: S111: Tungsten, yttrium, aluminum, and graphite were mixed in a mortar and cold pressed. The mixture was placed in a flowing argon atmosphere and heated at a rate of 8°C / min to 1450°C for 2 hours. S112: The obtained product was cooled to room temperature and dissolved in 50 mL of HCl for 18 hours to dissolve impurities, and then the impurities and HCl were removed by centrifugation at 3500 rpm for 3 minutes, decanting the acidic supernatant, and redispersing the precipitate in deionized water; S113: The obtained MAX powder was dried in a vacuum dryer at 25° C. for 18 hours to obtain a product.

10. The method for preparing a nerve conduit according to claim 9, characterized in that: In S111, The mass ratio of the mixed powders of tungsten, yttrium, aluminum and graphite is 1.33:1.33:1:1.

Images