Arginine carbon dots as well as preparation method and application thereof

Abstract

The invention relates to arginine carbon dots as well as a preparation method and application thereof. The preparation method comprises the following steps: adding citric acid and L-arginine into ultrapure water, fully stirring, performing ultrasonic treatment, fully reacting at 180-220 DEG C, cooling, centrifuging, filtering supernate, dialyzing a product, and freeze-drying the purified product to obtain the arginine carbon dots (Arg-CDs). By constructing an ION-CCI rat model, the Arg-CDs are applied to the body of a rat, and hemolysis experiments and HE staining results both prove that the Arg-CDs have no obvious toxic or side effect and good biocompatibility. In a rat ION-CCI model experiment, whether molecular level or behavioral evaluation indicates that the Arg-CDs can effectively relieve the trigeminal neuralgia. The Arg-CDs can be used for effectively inhibiting neuroinflammation mediated by small glial cells in Sp5C of rats. The arginine carbon dots (Arg-CDs) provided by the invention can be used as a carbon-based nano material with good biological safety, and the treatment of the trigeminal neuralgia is realized by inhibiting the activation of central microglial cells.

Description

Technical Field

[0001] The invention relates to functional fluorescent carbon dots, and in particular to arginine carbon dots and a preparation method and application thereof. Background Art

[0002] As a fluorescent nanomaterial, carbon dots not only retain the functional active groups of the raw materials, but also combine the characteristics of carbon dots themselves, such as good biocompatibility, easy preparation, excellent fluorescence properties and considerable biological functionality. For example, MCDs synthesized using metformin as raw material not only retain the anti-inflammatory effect of the raw material, but also can guide bone tissue regeneration under the inflammatory microenvironment; in addition, the ginger charcoal nano-components separated from ginger charcoal are highly safe and have analgesic effects.

[0003] As a non-essential amino acid, L-arginine can inhibit active oxidative stress, regulate microglial inflammatory response, and play an important role in analgesia. Therefore, developing a carbon dot based on L-arginine synthesis can retain the functional groups of L-arginine and give full play to the advantages of carbon dots, which is expected to play a key role in the treatment of trigeminal neuralgia. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an arginine carbon dot and a preparation method and application thereof, so as to obtain a carbon-based nanomaterial with good biosafety and achieve the treatment of trigeminal neuralgia.

[0005] To solve the above technical problems, according to the first aspect of the present invention, a method for preparing arginine carbon dots is provided, wherein citric acid and L-arginine are added to ultrapure water, and the mixture is sufficiently stirred and then ultrasonicated; the mixture is then fully reacted at 180°C-220°C, cooled and centrifuged, the supernatant is filtered and the product is dialyzed, and the purified product is lyophilized to obtain arginine carbon dots (Arg-CDs).

[0006] As a preferred embodiment, the mass ratio of citric acid to L-arginine is 1:2.

[0007] As a preferred embodiment, the reaction temperature is 200°C.

[0008] As a preferred embodiment, the supernatant is filtered through a 0.22 µm microporous filter membrane.

[0009] As a preferred embodiment, during dialysis, the product is transferred to a dialysis bag with MWCO=1000Da and dialyzed for 12 hours.

[0010] According to another aspect of the present invention, provided is the use of the above-mentioned arginine carbon dots in a drug for treating trigeminal neuralgia.

[0011] The present invention mixes L-arginine and citric acid to prepare functional fluorescent carbon dots Arg-CDs. By constructing an ION-CCI rat model, Arg-CDs are applied to rats. The hemolysis test and HE staining results both prove that Arg-CDs have no obvious toxic side effects and good biocompatibility. In the ION-CCI rat model experiment, both molecular and behavioral evaluations indicate that Arg-CDs can effectively relieve trigeminal neuralgia. Arg-CDs can effectively inhibit neuroinflammation mediated by microglia in rat Sp5C. Therefore, the present invention proposes that arginine carbon dots (Arg-CDs) can be used as a carbon-based nanomaterial with good biosafety to achieve the treatment of trigeminal neuralgia by inhibiting the activation of central microglia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : (A) TEM image, (B) size distribution histogram and (C) HR-TEM image of Arg-CDs. Figure 2 : FI-TR spectrum of Arg-CD; Figure 3 :Ultraviolet absorption spectrum (UV-vis) and fluorescence spectrum (PL) of Arg-CD; Figure 4 :(A)Full spectrum XPS analysis of Arg-CDs, high resolution (B) C1s, (C) N1s, (D) O1s peaks of Arg-CDs; Figure 5 : Zeta potential diagram of Arg-CD; Figure 6 : XRD pattern of Arg-CDs; Figure 7 :(A) Calcein-AM / PI staining (100×, scale bar 200µm) and (B) CCK-8 assay results after co-culture of BV2 with different concentrations of Arg-CDs (0, 1, 10, 100µg / ml) for 24 hours. ns indicates no statistical significance ( P >0.05); Figure 8 : Internalization of 100µg / ml Arg-CDs in cells co-cultured with BV2 for 24h (400×, scale bar 50µm); Fig. 9 :Effects of Arg-CDs on gene expression of proinflammatory cytokines (A) TNF-α (B) IL-6 (C) IL-1β in LPS-induced microglia. ** indicates P <0.01, **** indicates P <0.0001; Fig.10: Effect of Arg-CDs on the expression of IBA1 in LPS-treated BV2 (400×, scale bar 50µm); Fig.11 :(A) Changes of pain threshold before and after surgery in the sham group and ION-CCI group;(B) Effects of Arg-CDs on mechanical pain threshold in the ION-CCI trigeminal neuralgia model in rats. ns represents P >0.05,** represents P <0.01, **** represents P <0.0001; Fig.12 : Painful face-rubbing behavior in chronic constriction injury of the infraorbital nerve, ns represents P>0.05, ** represents P<0.01, **** represents P<0.0001; Fig.13 :(A) Western blot detection of c-Fos expression in Sp5C;(B) The expression of c-Fos in the ION-CCI+10mg / kg group was decreased, with statistically significant difference ( P <0.05), and there was no significant change in protein expression in ION-CCI+1mg / kg CDs ( P >0.05). ns represents P >0.05, * represents P <0.05; Fig.14 : Immunohistochemical detection of c-FOS levels in Sp5C (200×, scale bar 100µm); Fig.15 :Immunofluorescence detection of IBA1 levels in brain Sp5C (100×, scale bar 200µm). The white area is rat Sp5C; the white arrows point to activated microglia; Fig.16 : Rat Sp5C IL-1β immunohistochemical staining (400×, scale bar 50µm). DETAILED DESCRIPTION

[0013] 1. Synthesis of Arginine Carbon Dots (Arg-CDs) 0.4g citric acid and 0.8g L-arginine were added to 30ml ultrapure water, stirred thoroughly and ultrasonicated for 15min. The mixed solution was transferred to a 50ml high-pressure reactor and reacted in an oven at 200℃ for 10h. After the reaction, the solution was transferred to room temperature for cooling, and the solution was yellow and transparent. After high-speed centrifugation, the supernatant was taken and filtered using a 0.22µm microporous filter membrane. The product was then transferred to a dialysis bag (MWCO=1000Da) and dialyzed for 12h. Finally, the purified solution was freeze-dried using a vacuum freeze dryer to obtain Arg-CDs yellow-brown powder. After testing, the quantum yield (QY) of the nanofluorescent material Arg-CDs was 13.6%.

[0014] The synthesized Arg-CD was characterized by TEM. Figure 1 As shown in the figure, the obtained Arg-CDs have spherical morphology and are uniformly dispersed without obvious agglomeration. Arg-CDs show a narrow size distribution from 2nm to 5nm, with an average diameter of 3.09±0.58nm. In addition, a highly crystalline carbon structure was observed in the HRTEM image. The distance between the crystal fringes is about 0.16nm. Arg-CDs exhibit typical characteristics of carbon dots, including small size, spherical morphology, and crystalline structure.

[0015] The surface functional groups and structures of Arg-CDs were studied by FT-IR. Figure 2 As shown, at 3100-3500 cm -1 The broad absorption bands in the range of 1672 and 1399 cm-1 are attributed to the stretching vibrations of the OH and NH bonds. -1 The sharp peaks at 1576 cm-1 correspond to the two characteristic absorption bands of -CONH- and CN, respectively, which indicates that nitrogen atoms have been successfully incorporated into the carbon dots. -1 The characteristic peak at 400-1450 cm corresponds to the bending vibration of C=C / N. -1 The low-intensity peak at 1112 cm -1 The peak at can be attributed to the stretching vibration of CO.

[0016] like Figure 3As shown, the UV-vis and PL of Arg-CDs were measured to study their optical properties. Two typical absorption bands were observed near 220 nm and 330 nm, which were attributed to the π-π* transitions of the aromatic C=C bond and the n-π* transitions of the C=O bond. Arg-CDs showed blue fluorescence under UV light (365 nm). The maximum fluorescence excitation and emission wavelengths were 325 nm and 406 nm, respectively. In addition, Arg-CDs also showed fluorescence characteristics dependent on the excitation wavelength, and the emission peak shifted significantly with the increase of the excitation wavelength. Sakaguchi reagent turned the Arg-CDs solution red, and the color product had a strong absorption peak at 525 nm, confirming that Arg-CDs were rich in unique guanidine groups on the side chain of L-arginine. The Arg-CDs solution was light yellow under sunlight and could emit blue fluorescence after irradiation with UV light, which was conducive to its research and application in biological imaging.

[0017] The elemental composition and chemical bonds of Arg-CDs were determined by XPS analysis. Figure 4 As shown, Arg-CDs has three main characteristic peaks near 284.08 eV (C1S), 399.08 eV (N1s) and 531.08 eV (O1s), confirming that the N element has been successfully doped into C1s. The high-resolution C1s spectrum shows that Arg-CDs has four fitting peaks of CC / C=C, CN, CO and C=O, corresponding to 284.2 eV, 285.1 eV, 287.6 eV and 288.6 eV, respectively. The N1s spectrum shows that Arg-CDs has three fitting peaks of CNC, NH and C=N, corresponding to 399.4 eV, 400.5 eV and 401.8 eV, respectively. The O1s spectrum shows that Arg-CDs has two fitting peaks of C=O and CO, corresponding to 531.3 eV and 532.8 eV, respectively. Arg-CDs have higher carbon (C) content (78.13%) and oxygen (O) content (17.40%), and lower nitrogen (N) content (4.47%).

[0018] like Figure 5 As shown in Figure 2, the Zeta potential of Arg-CDs in water is -18.5 mv. Figure 6 As shown, the XRD pattern shows that Arg-CDs has a broad peak at 21.87°, corresponding to the (002) crystal plane of graphitic carbon, indicating that the center of Arg-CDs has a highly carbonized graphite structure.

[0019] 2. In vitro study on the inhibition of BV2 cell inflammation by Arg-CDs 2.1 Cytocompatibility The CCK-8 method was used to determine the effects of different concentrations of Arg-CDs (0, 1, 10, 100 µg / mL) on the survival rate of BV2 cells. Figure 7 As shown in the figure, as the concentration of Arg-CDs in the culture medium increased, the cell viability was basically not affected. Even after exposure to 100µg / mL Arg-CDs for 24 hours, the survival rate of BV2 did not decrease significantly and was still maintained above 85%. There was no statistical difference among the groups ( P >0.05). The above results showed that Arg-CDs had good low toxicity in the concentration range of 0-100µg / ml.

[0020] 2.2 Cellular uptake In order to observe the internalization of Arg-CDs by BV2 cells, CLSM was used to observe the fluorescence emitted by Arg-CDs in BV2 cells. Figure 8 As shown, the blue fluorescence of Arg-CDs in BV2 indicated that Arg-CDs could be taken up into cells.

[0021] 2.3 Arg-CDs inhibit BV2 cell inflammation (1) Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) In order to observe the inhibitory effect of Arg-CDs on microglial inflammation, BV2 cells were treated with LPS (1 µg / ml) for 6 h to establish a neuroinflammatory cell model. Fig. 9 As shown in the figure, compared with the LPS group, 100 µg / ml Arg-CDs pretreatment significantly inhibited the gene expression of proinflammatory cytokines TNF-α, IL-6, and IL-1β in BV2 cells induced by LPS. These observations indicate that Arg-CDs can effectively improve the inflammatory response of BV2 cells induced by LPS.

[0022] (2) Immunofluorescence (IF) To further demonstrate the anti-inflammatory effect of Arg-CDs in vitro, IBA1 (a marker of microglial activation) was detected by in vitro immunofluorescence staining to evaluate the effect of Arg-CDs on LPS-induced BV2 cell activation. Fig.10 As shown, compared with the CON group, LPS treatment significantly increased the expression of IBA1 in microglia, while Arg-CDs inhibited the high expression of IBA1 in BV2 cells induced by LPS.

[0023] In summary, CCK-8 and Calcein-AM / PI staining experiments showed that Arg-CDs had good cell compatibility. Under Arg-CDs co-culture conditions, CLSM could clearly observe that Arg-CDs were located in BV2 cells, indicating that BV2 cells could effectively take up Arg-CDs. Arg-CDs could regulate the activation state of BV2 cells induced by LPS and downregulate the gene expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β) to inhibit microglia-mediated inflammation.

[0024] 3. Study on the antinociceptive effect of Arg-CDs on the trigeminal neuralgia model in rats Adult male Sprague-Dawley (SD) rats weighing between 200 and 300 g were used in the experiment and were provided by the Experimental Animal Center of Shanxi Medical University. All rats were housed in a constant temperature room with a 12-h light-dark cycle and fed with standard rodent chow, with free access to food and water.

[0025] To evaluate the effects of Arg-CDs on trigeminal neuralgia, a commonly used chronic constriction injury of the infraorbital nerve (ION-CCI) model was established in male SD rats. The ION-CCI model was established as follows: after anesthesia, the rats were placed in the lateral position, the skin of the surgical area was disinfected, and the surgical incision was located about 8 mm from the lower edge of the nose to the left vibrissa pad. The incision length was about 10 mm. A sterilized glass needle was used to separate part of the infraorbital nerve and expose it to the visual field. Subsequently, the two ends of the exposed infraorbital nerve were ligated with 4-0 absorbable surgical sutures, about 2 mm apart, and the ligation was performed with appropriate force. After ligation, the diameter of the nerve was significantly reduced, and the extraneural circulation was not interrupted. The surgical incision was sutured with 4-0 sutures, and the surgical area was disinfected again. Rats in the sham group (Sham group) underwent the same operation, but the exposed infraorbital nerve did not need to be ligated with sutures. All procedures performed in the study were performed in accordance with the protocol approved by the Animal Care and Use Committee of Shanxi Medical University. Every effort was made to minimize intraoperative suffering and the number of rats used.

[0026] 3.1 Effects of Arg-CDs on the behavior of ION-CCI rats (1) Mechanical pain threshold Mechanical stimulation was performed using Von Frey hairs (North coast medical Inc). Briefly, a rat was placed in a homemade wire cage for 10 min to acclimate to the experimental environment until they resumed their normal sniffing / non-movement posture. After the acclimatization process, the examiner could apply von Frey hairs to the ipsilateral ION area (whisker pad) of the rat to the injury, starting with 0.008 g, until a clear behavioral response was elicited. When the rat had a positive response, its intensity, i.e., the pain threshold, was recorded. Positive responses included: ① Rapid head-retraction reaction; ② Escape or attack behavior, manifested as running away, curling up, hiding the head, biting or scratching stimuli; ③ Asymmetric face grooming: rats performed at least three uninterrupted face-washing movements in the stimulated face area.

[0027] If the rats did not show any of the above reactions at a stimulation intensity of 15 g, 15 g was taken as the pain threshold.

[0028] The preoperative pain threshold of the sham group was 12.17±3.19g, and the pain threshold at each time point after surgery remained relatively stable. There was no statistically significant difference between the pain threshold at each time point after surgery and that before surgery ( P >0.05). The preoperative pain threshold of the ION-CCI group was 13.33±2.58g. The pain threshold on the second and fourth days after surgery was 14.17±2.04g and 11.50±3.83g, respectively, which remained basically unchanged compared with preoperative pain threshold, with no statistically significant difference ( P >0.05). On the sixth day after surgery, the pain threshold dropped significantly (7.83±4.02g), which was significantly different from that before surgery ( P <0.05). Over time, the pain threshold dropped sharply, and 14 days after surgery, the threshold dropped to 0.79±0.65g, and the pain threshold tended to be stable.

[0029] The ION-CCI group had a short plateau period after surgery, with no significant change in pain threshold, and then the pain threshold gradually decreased and reached a stable level 14 days after surgery. There was no statistical difference in the preoperative threshold between the Sham group and the ION-CCI group ( P >0.05). On the sixth day after surgery, the pain threshold of the ION-CCI group was 7.83±4.02g, and that of the Sham group was 12.67±3.83g, with a significant difference between the two groups ( P <0.05), and the statistical difference persisted until the end of the experiment.

[0030] To evaluate the analgesic effect of Arg-CDs in the ION-CCI rat pain model. On the 14th day after surgery, ION-CCI rats were intraperitoneally injected with Arg-CDs for 30 minutes, and the changes in the mechanical pain threshold of the rats' faces were observed and compared with those in the saline group. Fig.11 As shown, compared with the saline group (1.49±0.65g), the pain threshold of the ION-CCI rats in the 10mg / kg CDs group increased 30min after intraperitoneal injection of Arg-CDs (7.00±1.91g, P <0.01), and the pain threshold of the 1mg / kg CDs group had no significant change (2.63±1.85g, P >0.05).

[0031] (2) Scrubbing behavior Prepare a transparent plastic box, install a monitoring recorder at a certain height above the side of the transparent plastic box to ensure that the monitoring field of view can cover the entire rat activity area, put the SD rats alone in the plastic box, and after adapting to the environment for 10 minutes, start recording the time and number of times the rats rub their faces with their paws, and the recording time is 20 minutes. After the recording is completed, put the rats back to the original breeding box.

[0032] On the 14th day after surgery, 30 minutes after intraperitoneal injection of Arg-CDs, the frequency and duration of face wiping in the 10 mg / kg CDs group were significantly shorter (2.50±1.51 times, 49.00±6.57s) compared with the ION-CCI+Vehicle group (5.83±1.47 times, 49.00±6.57s). P <0.01; 27.50±4.76s, P <0.0001, while there was no significant difference in the 1mg / kg CDs group (4.00±1.55 times, P >0.05;47.17±7.60s, P >0.05).

[0033] 3.2 Changes in c-FOS protein expression in SpC5 of ION-CCI rats (1) Immunoblotting like Fig.13 , 30 minutes after intraperitoneal injection of Arg-CDs in ION-CCI rats, the expression of c-Fos in Sp5C of each group was semi-quantitated by immunoblotting. Compared with the ION-CCI+Vehicle group, there was no significant change in the expression of c-FOS in the 1 mg / kg CDs group ( P >0.05, while the c-FOS expression in the 10 mg / kg CDs group was significantly decreased ( P<0.05). This suggests that intraperitoneal injection of 10 mg / kg Arg-CDs 30 minutes later can significantly inhibit the expression of c-FOS protein in Sp5C of ION-CCI rats.

[0034] (2) Immunohistochemistry (IHC) The c-FOS protein immunoreactive positive neuron nuclei were brown, round or oval under the light microscope. The c-FOS protein of Sp5C in each group of rats was stained by immunohistochemistry, and it was observed that compared with the high expression of c-FOS in the ION-CCI+Vehicle group, the expression of c-FOS protein was significantly reduced after intervention with 10mg / kg Arg-CDs, while there was no significant change in protein expression in the 1mg / kg CDs group.

[0035] Effects of Arg-CDs on neuroinflammation in ION-CCI rats (1) Immunofluorescence (IF) Immunofluorescence was used to visualize the positive expression of IBA1 in the trigeminal nerve conduction center. Fig.15 , compared with the ION-CCI+Vehicle group, the number of Sp5C-positive cells in the 10 mg / kg CDs group was significantly reduced.

[0036] (2) Immunohistochemistry (IHC) like Fig.16 By immunohistochemical staining of IL-1β protein in Sp5C of rats in each group, it can be observed that compared with the high expression of IL-1β in the ION-CCI+Vehicle group, the expression of IL-1β protein was significantly reduced after intervention with 10 mg / kg Arg-CDs.

[0037] In summary, the hemolysis test and HE staining results all proved that Arg-CDs had no obvious toxic side effects and good biocompatibility. In the ION-CCI rat model experiment, both molecular and behavioral assessments suggested that Arg-CDs could effectively relieve trigeminal neuralgia. Arg-CDs could effectively inhibit microglia-mediated neuroinflammation in rat Sp5C.

Claims

1. A method for preparing arginine carbon dots, characterized in that: Citric acid and L-arginine were added into ultrapure water, stirred thoroughly and then sonicated; then fully reacted at 180°C-220°C, centrifuged after cooling, the supernatant was filtered and the product was dialyzed, and the purified product was freeze-dried to obtain arginine carbon dots (Arg-CDs).

2. The method for preparing arginine carbon dots according to claim 1, characterized in that: The mass ratio of citric acid to L-arginine is 1:

2.

3. The method for preparing arginine carbon dots according to claim 1 or 2, characterized in that: The reaction temperature was 200°C.

4. The method for preparing arginine carbon dots according to claim 3, characterized in that: The supernatant was filtered through a 0.22 µm microporous membrane.

5. The method for preparing arginine carbon dots according to claim 1, characterized in that: During dialysis, the product was transferred to a dialysis bag with MWCO = 1000Da and dialyzed for 12h.

6. The arginine carbon dots prepared by the method of any one of claims 1 to 5.

7. Use of the arginine carbon dots according to claim 6 in the preparation of a drug for treating trigeminal neuralgia.

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