Carbon dot material prepared from donkey-hide gelatin and its preparation method and application

Abstract

The present invention discloses a carbon point material prepared from donkey-hide gelatin and a preparation method and application thereof, the method comprising the following steps: S1. grinding the block donkey-hide gelatin into powder by a grinder, the mass ratio of donkey-hide gelatin powder to water is 1:50, a mixed solution is obtained, and the mixed solution is dissolved by an ultrasonic device to obtain a powder solution; S2. the powder solution is transferred to a polytetrafluoroethylene autoclave to obtain a reaction solution; after the reaction solution is cooled to room temperature, a cooling solution is obtained; S3. the cooling solution is filtered and dialyzed for 3-5 days to obtain a purified solution; S4. the purified solution is dried to obtain a freeze-dried powder donkey-hide gelatin carbon point material after freeze-drying. The donkey-hide gelatin carbon point material of the present invention can not only promote erythropoiesis in mice with spinal cord injury, improve anemia, and effectively reduce inflammatory response, thereby promoting functional recovery of spinal cord injury.

Description

Technical Field

[0001] The present invention relates to a carbon dot preparation technology, and in particular to a carbon dot material prepared from donkey-hide gelatin, and a preparation method and application thereof. Background Art

[0002] Inflammation is the main factor of secondary spinal cord injury, leading to local edema, blood-brain barrier (BBB) ​​destruction and neurological deterioration. At present, a variety of treatments have been developed for spinal cord injury inflammation, including hormone therapy, cytokines and interferons, antibodies, miRNA, cell transplantation, etc. These treatments usually target a certain cell type or cytokine, but lack the regulation of the source of inflammation-hematopoietic stem cells. Considering the interruption of central nervous fibers caused by spinal cord injury, a dispersed spinal cord autonomic neural network is generated, causing immune dysfunction in the bone marrow and spleen, including imbalance in the production of myeloid and lymphoid cells in the bone marrow, disorder in the number and function of immune cells in the spleen, and excessive infiltration and cytotoxicity of mature leukocytes and destruction of leukocyte homing after SCI. Therefore, how to affect hematopoietic stem cells in the bone marrow after spinal cord injury, regulate the peripheral immune system, and then reduce the level of inflammation in the acute and subacute phases of local spinal cord injury, and ultimately achieve the purpose of protecting nerves and promoting motor function recovery has become an urgent problem to be solved.

[0003] Hematopoietic stem cells, as the apex of the lineage that produces red blood cells and immune cells, support the regeneration of cells in all blood and immune systems. The latest studies have shown that hematopoietic stem cells play a key role in central nervous system diseases and immune system diseases. The lineage differentiation of hematopoietic stem cells is a strictly regulated process. When a single HSC passes through a discrete, hierarchically organized progenitor cell population, it gradually acquires lineage preferences along multiple directions. This process is affected by infection or inflammatory stimulation, hormones, sympathetic nerves, etc., and is regulated by factors such as IL1β, TNFα, TGFβ, CCL2, CXCL12 and CXCR4. After spinal cord injury, the bone marrow is affected by trauma, stress, neural regulation, and inflammation, and the hematopoietic function is disordered, showing an increase in the number of GMPs, causing a large number of myeloid cells to infiltrate the injury site and impair nerve function.

[0004] Regarding the above issues, the NASCIS II guidelines recommend methylprednisolone for the treatment of acute SCI, which plays a neuroprotective role by inhibiting the infiltration of hematogenous macrophages and reducing the secretion of inflammatory factors. However, it has many side effects and its clinical application is limited. Summary of the invention

[0005] In view of the above-mentioned defects or deficiencies in the prior art, it is desirable to provide a carbon dot material prepared from donkey-hide gelatin and a preparation method and application thereof.

[0006] According to the technical solution provided in the embodiment of the present application, a method for preparing a carbon dot material is provided, wherein the preparation method obtains donkey-hide gelatin carbon dot material, and the donkey-hide gelatin carbon dot material is uniformly dispersed spherical nanoparticles with an average particle size of 2.13 nm.

[0007] A method for preparing donkey-hide gelatin carbon dot material, the method comprising the following steps:

[0008] S1. Grind the block of donkey-hide gelatin into powder by a grinder to obtain donkey-hide gelatin powder, disperse the donkey-hide gelatin powder in deionized water, the mass ratio of donkey-hide gelatin powder to water is 1:50, and obtain a mixed solution, and then ultrasonically treat the mixed solution for 10min-15min by an ultrasonic device to promote the dissolution of the mixed solution to obtain a powder solution;

[0009] S2. The powder solution was transferred to a polytetrafluoroethylene autoclave, and the reaction was carried out at a constant temperature of 100 ℃-200 ℃ for 12h-16h to obtain a reaction solution; the reaction solution was cooled to room temperature to obtain a cooling solution;

[0010] S3. The cooled solution was filtered through a 0.22 μm cellulose filter membrane and dialyzed with a dialysis bag for 3-5 days to obtain a purified solution;

[0011] S4. The purified solution is dried by a vacuum freeze dryer to obtain freeze-dried powder donkey-hide gelatin carbon dot material, and the donkey-hide gelatin carbon dot material is stored at 4°C.

[0012] An application of donkey-hide gelatin carbon dot material, which regulates immune function after spinal cord injury and promotes the reduction of inflammatory cells in the spinal cord injury site.

[0013] In summary, the beneficial effects of the present application are as follows: the donkey-hide gelatin carbon dot material prepared by the method described in the present application can increase the proliferation of hematopoietic stem cells in the spinal cord, and shift their differentiation toward erythroid progenitor cells, while reducing their differentiation toward macrophages / monocytes (inflammatory cells); functional, imaging, and electrophysiological tests all show that carbon dots have the effect of promoting functional recovery in mice with spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0015] Figure 1 Transmission electron microscopy (TEM) and high-resolution TEM images of carbon dots;

[0016] Figure 2 Schematic diagram of the particle size distribution of carbon dots determined by TEM images;

[0017] Figure 3Schematic diagram of using a nanoparticle size potentiometer to measure the Zeta potential of carbon dots in aqueous solution;

[0018] Figure 4 is the X-ray diffraction (XRD) pattern;

[0019] Figure 5 It is a schematic diagram of the total x-ray photoelectron spectroscopy (XPS) spectrum, the high-resolution C1s spectrum, the high-resolution O1s spectrum, and the high-resolution N1s spectrum;

[0020] Figure 6 is a schematic diagram of Fourier transform infrared spectroscopy (FTIR) of carbon dots;

[0021] Figure 7 Schematic diagram of the optical properties of carbon dots;

[0022] Figure 8 Schematic diagram of the UV-visible absorption spectrum of carbon dots;

[0023] Fig. 9 Schematic diagram of fluorescence spectrum of carbon dots;

[0024] Fig.10 Mouse bone marrow cells were treated with different concentrations of carbon dots for 24 h, and cell viability and mortality were detected by live / dead staining flow cytometry.

[0025] Fig.11 Mouse bone marrow cells were treated with different concentrations of carbon dots for 24 h. Each group included n = 4 biologically independent cell samples, 3 × 10 5 Schematic diagram of the number of BFU-E and CFU-GMEE colonies formed by mouse bone marrow cells cultured for 10 days under the action of different concentrations of carbon dots;

[0026] Fig.12 Flow cytometry was used to detect the proportion of Indo 1 violet-positive cells in the mouse bone marrow 3 days after intraperitoneal injection of 50 mg / kg carbon dots; each group included n = 6 biologically independent cell samples;

[0027] Fig.13 3 × 10 5 Schematic diagram of the formation of BFU-E and CFU-GMI colony numbers in mouse bone marrow cells after 10 days of culture;

[0028] Fig.14 Schematic diagram of detecting the proportion of edu-positive cells in the bone marrow cell population. DETAILED DESCRIPTION

[0029] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0030] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0031] Embodiment 1:

[0032] The block of donkey-hide gelatin is ground into powder by a grinder to obtain donkey-hide gelatin powder, the donkey-hide gelatin powder is dispersed in deionized water with a mass ratio of donkey-hide gelatin powder to water of 1:50 to obtain a mixed solution, and then the mixed solution is ultrasonically treated for 10 min-15 min by an ultrasonic device to promote the dissolution of the mixed solution to obtain a powder solution; the powder solution is transferred to a polytetrafluoroethylene high-pressure reactor, and isothermally reacted at a temperature of 100°C-200°C for 12h-16h to obtain a reaction solution; the reaction solution is cooled to room temperature to obtain a cooling solution; the cooling solution is filtered with a 0.22 μm cellulose filter membrane, and dialyzed with a dialysis bag for 3-5 days to obtain a purified solution; the purified solution is dried by a vacuum freeze dryer to obtain a freeze-dried powder donkey-hide gelatin carbon dot material, and the donkey-hide gelatin carbon dot material is stored at 4°C.

[0033] Figure 1 Transmission electron microscopy (TEM) of donkey-hide gelatin carbon dot materials (such as Figure 1 (a)) and high-resolution TEM images (as shown Figure 1 (b) Using high-resolution TEM, we found a lattice fringe spacing of 0.21 nm, and both the particle size and spacing are small.

[0034] From the particle size distribution of carbon dots determined by TEM images, it was observed that the donkey-hide gelatin carbon dots were uniformly dispersed spherical nanoparticles with an average particle size of (2.13±0.635) nm. Figure 2 As shown, this particle size helps to smoothly penetrate various biological barriers.

[0035] The Zeta potential of carbon dots in aqueous solution was measured using a nanoparticle size potentiometer and was -17.53 ± 0.6839 mV. Figure 3 As shown, negatively charged nanoparticles exhibited greater stability in the blood.

[0036] from Figure 4From the X-ray diffraction (XRD) pattern, it can be seen that the elements are mainly composed of carbon, nitrogen and oxygen, and their energy peaks are approximately 284.39 eV, 399.24 eV and 531.04 eV, respectively, proving its basic constituent elements.

[0037] from Figure 5 (a) Total X-ray photoelectron spectroscopy (XPS) spectrum, Figure 5 (b) High-resolution C1s spectrum, Figure 5 (c) High-resolution O1s spectrum, Figure 5 (d) The high-resolution N1s spectrum shows that the X-ray diffraction pattern displays a broad peak at 2θ = 22°, indicating the amorphous nature of graphitic carbon on its (100) plane.

[0038] The surface composition of carbon dots was further analyzed using Fourier transform infrared spectroscopy (FTIR), and a broad absorption peak related to the stretching vibration of OH and NH was found at 3430 cm-1. Continuous peaks were observed at 2940 and 2872 cm-1 for the symmetric and asymmetric stretching vibrations of carbon-hydrogen bonds, respectively. The peaks at 1661, 1406, and 1088 cm-1 correspond to the stretching vibrations of C=C, C-n, and C-o bonds, respectively. Figure 6 As shown in Figure 2, both FTIR and XPS results indicate that there are functional groups containing C, O, and N derived from amino acids in EJCDs, including amino, carboxyl, hydroxyl, and methyl groups.

[0039] Optical properties of carbon dots: Figure 7 The UV-visible absorption spectrum, fluorescence excitation spectrum (blue line), and emission spectrum (purple line) are shown; it can be seen that photoluminescence is a characteristic of carbon dots, making them suitable for cell imaging and tracking. Under light excitation, carbon dots emit light, which is convenient for cell labeling, tracking drug distribution, and evaluating treatment effects. The three-dimensional fluorescence emission spectrum shows that the emission and excitation wavelengths increase due to the normal particle size distribution of carbon dots.

[0040] The three-dimensional fluorescence spectrum of carbon dots and the UV-visible absorption spectrum show that there are two weak absorption bands near 271 nm and 330 nm, such as Figure 8 As shown, it corresponds to the π-π* transition of the conjugated C=C bond in the carbon core, and the n-π* electronic transition of the surface functional groups, such as C=O and C= n. The optimal excitation wavelength is 395 nm, and the maximum emission peak is at 472 nm, as shown in Fig. 9 (b) shown.

[0041] Embodiment 2

[0042] The donkey-hide gelatin carbon dot material prepared in Example 1 was taken out, and after the spinal cord of mice was injured, 50 mg / kg of carbon dots were administered into the abdominal cavity of the mice via a syringe for 14 days, once a day.

[0043] Mouse bone marrow cells were treated with different concentrations of carbon dots for 24 h, and cell viability and mortality were detected by live / dead staining flow cytometry. Fig.10 (a) and Fig.10 (b) shown.

[0044] 3 × 10 5 Mouse bone marrow cells, cultured for 10 days to form BFU-E and CFU-GMEE colonies, such as Fig.13 (a) and Fig.13 As shown in (b), the EdU co-staining ratio was observed, that is, the proliferation ability of bone marrow cells after intervention with donkey-hide gelatin carbon dots was significantly increased compared with other groups. The number of BFU-E and CFU-GMEE colonies are both indicators for evaluating the function of hematopoietic stem cells. The proliferation increased after intervention with donkey-hide gelatin carbon dots, indicating that donkey-hide gelatin carbon dots enter the bone marrow to stimulate hematopoiesis.

[0045] Three days after intraperitoneal injection of 50 mg / kg carbon dots, the proportion of Indo 1 violet-positive cells in the mouse bone marrow was detected by flow cytometry. Fig.12 As shown in (a), each group includes n = 6 biologically independent cell samples; considering that photoluminescence is a characteristic of carbon dots, flow cytometry was used to monitor the uptake of donkey-hide gelatin carbon dots by bone marrow cells in vivo. The results showed that Indo 1violet-A (450 / 50 filter) was the best channel for detecting donkey-hide gelatin carbon dots, and bone marrow cells reached peak fluorescence within 1 h, as shown in Figure 2. Fig.12 As shown in (b). Using its luminescent properties, it was found that donkey-hide gelatin carbon dots can mark living bone marrow cells.

[0046] The hematopoietic stem cells in the bone marrow are affected by carbon dots, proliferate more, and differentiate toward erythroid progenitor cells, while differentiation toward macrophages / monocytes (inflammatory cells) decreases.

[0047] Embodiment 3

[0048] After the mice were injected with donkey-hide gelatin carbon dots in Example 2, inflammatory cells were found to decrease at the site of spinal cord injury 3, 7, and 14 days after spinal cord injury. Functional, imaging, and electrophysiological tests all showed that carbon dots have the effect of promoting functional recovery in mice with spinal cord injury.

[0049] Extract bone marrow cells from mice in the relevant experimental groups

[0050] Mouse bone marrow cells were treated with different concentrations of carbon dots for 24 h, and cell viability and mortality were detected by live / dead staining flow cytometry. Each group included n = 4 biologically independent cell samples; 3 × 10 5The number of BFU-E colonies formed by mouse bone marrow cells cultured for 10 days under the action of different concentrations of carbon dots, such as Fig.11 (a) The number of CFU-GMEE colonies was shown as Fig.11 (b) It can be seen that donkey-hide gelatin carbon dots can induce the proliferation of hematopoietic stem cells.

[0051] Bone marrow cells were incubated with 400 µg / ml carbon dots for 24 h, and the number of Fig.14 As shown in (d), the cells were incubated with 10M EdU for 24 hours, and edu was stained red and the nuclei were stained blue with Hoechst 33342. The proportion of edu-positive cells in the bone marrow cell population was detected, as shown in Fig.14 (a) Fig.14 (b) Fig.14 As shown in (c), it can be seen that donkey-hide gelatin carbon dots can induce the proliferation of hematopoietic stem cells.

[0052] The above description is only an explanation of the preferred embodiments of the present application and the technical principles and other schemes used. At the same time, the scope of the invention involved in the present application is not limited to the technical scheme formed by a specific combination of the above technical features, but also includes other technical schemes formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with the technical features with similar functions disclosed in the present application (but not limited to) to form a technical scheme.

Claims

1. An application of donkey-hide gelatin carbon dot material, characterized by: The preparation method of the donkey-hide gelatin carbon dot material comprises the following steps: S1. Grind the block of donkey-hide gelatin into powder by a grinder to obtain donkey-hide gelatin powder, disperse the donkey-hide gelatin powder in deionized water, the mass ratio of donkey-hide gelatin powder to water is 1:50, and obtain a mixed solution, and then ultrasonically treat the mixed solution for 10min-15min by an ultrasonic device to promote the dissolution of the mixed solution to obtain a powder solution; S2. The powder solution was transferred to a polytetrafluoroethylene autoclave, and the reaction was carried out at a constant temperature of 100 ℃-200 ℃ for 12h-16h to obtain a reaction solution; the reaction solution was cooled to room temperature to obtain a cooling solution; S3. The cooled solution was filtered through a 0.22 μm cellulose filter membrane and dialyzed with a dialysis bag for 3-5 days to obtain a purified solution; S4. The purified solution is dried by a vacuum freeze dryer to obtain freeze-dried powder donkey-hide gelatin carbon dot material, which is stored at 4 ° C; the donkey-hide gelatin carbon dot material obtained by the preparation method is a uniformly dispersed spherical nanoparticle with an average particle size of 2.13nm; The donkey-hide gelatin carbon dot material is used to regulate immune function after spinal cord injury, thereby promoting the reduction of inflammatory cells in the spinal cord injury site; it increases the proliferation of hematopoietic stem cells in the spinal cord, and shifts their differentiation toward red blood cell progenitor cells, while reducing their differentiation toward macrophages / monocytes.

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