Arginine carbon dots and preparation method and application thereof

Abstract

The application relates to arginine carbon dots and a preparation method and application thereof. Citric acid and L-arginine are added into ultrapure water, ultrasonic treatment is carried out after sufficient stirring, then full reaction is carried out at 180 DEG C-220 DEG C, centrifugal separation is carried out after cooling, the supernatant is filtered, the product is dialyzed, the purified product is freeze-dried to obtain arginine carbon dots (Arg-CDs). By constructing an ION-CCI rat model, the Arg-CDs are applied to the rat in vivo, and the results of hemolysis experiments and HE staining prove that the Arg-CDs have no obvious toxic side effects and good biocompatibility. In the rat ION-CCI model experiment, whether on the molecular level or the behavior evaluation, it is indicated that the Arg-CDs can effectively relieve trigeminal neuralgia. The Arg-CDs can effectively inhibit the microglia-mediated neuroinflammation in the Sp5C of rats. It is proposed that the arginine carbon dots (Arg-CDs) can be used as a carbon-based nanomaterial with good biological safety, and the treatment of trigeminal neuralgia can be realized by inhibiting the activation of central microglia.

Description

Technical Field

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

[0002] As fluorescent nanomaterials, carbon dots not only retain the functional active groups of the raw materials but also combine their inherent biocompatibility, ease of preparation, excellent fluorescence properties, and considerable biofunctionality. For example, MCDs synthesized using metformin not only retain the raw material's anti-inflammatory effects but can also guide bone regeneration in inflammatory microenvironments. Furthermore, ginger charcoal nanoparticles isolated from ginger charcoal are highly safe and have analgesic properties.

[0003] L-arginine, a non-essential amino acid, can inhibit active oxidative stress and regulate microglial inflammatory responses, playing an important role in analgesia. Therefore, developing carbon dots synthesized from L-arginine could retain the functional groups of L-arginine and fully utilize the advantages of carbon dots, potentially playing 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 its preparation method and application, to obtain a carbon-based nanomaterial with good biosafety, and to 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, comprising adding citric acid and L-arginine to ultrapure water, stirring thoroughly and then sonicating; then reacting thoroughly at 180°C-220°C, cooling and centrifuging, filtering the supernatant and dialyzing the product, and lyophilizing the purified product 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 prepares functional fluorescent carbon dots (Arg-CDs) by mixing L-arginine and citric acid. Using an ION-CCI rat model, Arg-CDs were applied to rats. Hemolysis experiments and HE staining results demonstrated that Arg-CDs had no significant toxic side effects and exhibited good biocompatibility. Both molecular and behavioral assessments in the ION-CCI rat model suggest that Arg-CDs can effectively alleviate trigeminal neuralgia. Arg-CDs can effectively inhibit microglia-mediated neuroinflammation in rat Sp5C cells. Therefore, the present invention proposes that arginine carbon dots (Arg-CDs) can be used as a biosafe carbon-based nanomaterial to treat trigeminal neuralgia by inhibiting central microglial activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : (A) TEM image, (B) size distribution histogram and (C) HR-TEM image of Arg-CDs;

[0013] Figure 2 : FI-TR spectrum of Arg-CD;

[0014] Figure 3 : Ultraviolet absorption spectrum (UV-vis) and fluorescence spectrum (PL) of Arg-CD;

[0015] Figure 4 :(A)Full spectrum XPS analysis of Arg-CDs, high resolution (B) C1s, (C) N1s, and (D) O1s peaks of Arg-CDs;

[0016] Figure 5 : Zeta potential diagram of Arg-CD;

[0017] Figure 6 : XRD pattern of Arg-CDs;

[0018] Figure 7 : (A) Calcein-AM / PI staining (100×, scale bar 200 μm) and (B) CCK-8 assay results after co-culture of BV2 cells with different concentrations of Arg-CDs (0, 1, 10, 100 μg / ml) for 24 h. ns indicates no statistical significance ( P >0.05);

[0019] Figure 8 : Internalization of 100 µg / ml Arg-CDs into BV2 cells after co-culture for 24 h (400×, scale bar 50 µm);

[0020] Figure 9: Arg-CDs affect the 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;

[0021] Figure 10 : Effects of Arg-CDs on the expression of IBA1 in LPS-treated BV2 (400×, scale bar 50µm);

[0022] Figure 11 :(A) Changes in 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;

[0023] Figure 12 : Painful face-rubbing behavior in patients with chronic constriction injury of the infraorbital nerve, ns represents P>0.05, ** represents P<0.01, **** represents P<0.0001;

[0024] Figure 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 significantly decreased ( 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;

[0025] Figure 14 : Immunohistochemical detection of c-FOS levels in Sp5C (200×, scale bar 100µm);

[0026] Figure 15 Immunofluorescence detection of IBA1 levels in brain Sp5C (100×, scale bar 200µm). The white area represents rat Sp5C; the white arrows indicate activated microglia.

[0027] Figure 16 : Immunohistochemical staining of rat Sp5C and IL-1β (400×, scale bar 50µm). DETAILED DESCRIPTION

[0028] 1. Synthesis of Arginine Carbon Dots (Arg-CDs)

[0029] 0.4 g of citric acid and 0.8 g of L-arginine were added to 30 ml of ultrapure water, stirred thoroughly, and sonicated for 15 minutes. The mixed solution was transferred to a 50 ml autoclave and reacted in an oven at 200°C for 10 hours. After the reaction, the solution was cooled to room temperature, and a yellow, transparent solution was obtained. After high-speed centrifugation, the supernatant was collected and filtered through a 0.22 µm microporous filter membrane. The product was then transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed for 12 hours. Finally, the purified solution was lyophilized using a vacuum freeze dryer to obtain a yellow-brown powder of Arg-CDs. Testing showed that the quantum yield (QY) of the Arg-CDs nanofluorescent material was 13.6%.

[0030] The synthesized Arg-CD was characterized by TEM. Figure 1 As shown, the obtained Arg-CDs exhibit spherical morphology and are uniformly dispersed with no apparent agglomeration. The Arg-CDs display a narrow size distribution from 2 nm to 5 nm, with an average diameter of 3.09 ± 0.58 nm. Furthermore, a highly crystalline carbon structure was observed in HRTEM images. The intercrystalline fringe spacing was approximately 0.16 nm. The Arg-CDs exhibit typical characteristics of carbon dots, including small size, spherical morphology, and crystalline structure.

[0031] 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 are attributed to the stretching vibrations of the OH and NH bonds. -1 The sharp peaks at 1576 cm 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.

[0032] like Figure 3As shown, UV-visible and PL measurements of Arg-CDs were performed to investigate their optical properties. Two typical absorption bands were observed near 220 nm and 330 nm, attributed to the π-π* transitions of the aromatic C=C bond and the n-π* transitions of the C=O bond. Arg-CDs exhibited blue fluorescence under UV illumination (365 nm). The maximum fluorescence excitation and emission wavelengths were 325 nm and 406 nm, respectively. Furthermore, Arg-CDs exhibited excitation wavelength-dependent fluorescence, with the emission peak shifting significantly with increasing excitation wavelength. Sakaguchi reagent turned the Arg-CDs solution red, and the resulting product exhibited a strong absorption peak at 525 nm, confirming that Arg-CDs are rich in the unique guanidino groups on the L-arginine side chain. Arg-CDs solutions appear pale yellow under sunlight but emit blue fluorescence upon UV illumination, facilitating their research and application in bioimaging.

[0033] The elemental composition and chemical bonds of Arg-CDs were determined by XPS analysis. Figure 4 As shown, Arg-CDs exhibit three main characteristic peaks near 284.08 eV (C1S), 399.08 eV (N1s), and 531.08 eV (O1s), confirming successful doping of nitrogen into the C1s. The high-resolution C1s spectrum reveals four fitting peaks for Arg-CDs: 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 reveals three fitting peaks for Arg-CDs: CNC, NH, and C=N, corresponding to 399.4 eV, 400.5 eV, and 401.8 eV, respectively. The O1s spectrum reveals two fitting peaks for Arg-CDs: 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%).

[0034] 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 of Arg-CDs shows that there is a broad peak at 21.87°, which corresponds to the (002) crystal plane of graphitic carbon, indicating that the center of Arg-CDs has a highly carbonized graphite structure.

[0035] 2. In vitro study of Arg-CDs inhibiting BV2 cell inflammation

[0036] 2.1 Cytocompatibility

[0037] The effects of different concentrations of Arg-CDs (0, 1, 10, 100 μg / mL) on the survival rate of BV2 cells were determined by CCK-8 assay. Figure 7 As shown in the figure, as the concentration of Arg-CDs in the culture medium increased, cell viability was basically unaffected. Even after exposure to 100µg / mL Arg-CDs for 24 hours, the survival rate of BV2 did not decrease significantly and remained above 85%. There was no statistical difference among the groups ( P >0.05). The above results indicate that Arg-CDs has good low toxicity in the concentration range of 0-100µg / ml.

[0038] 2.2 Cellular uptake

[0039] 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.

[0040] Arg-CDs inhibit BV2 cell inflammation

[0041] (1) Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR)

[0042] In order to observe the inhibitory effect of Arg-CDs on microglial inflammation, BV2 cells were treated with LPS (1µg / ml) for 6 hours to establish a neuroinflammatory cell model. Figure 9 As shown in the results, compared with the LPS group, pretreatment with 100 μg / ml Arg-CDs 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 ameliorate the inflammatory response of BV2 cells induced by LPS.

[0043] (2) Immunofluorescence (IF)

[0044] 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. Figure 10 As shown in the results, compared with the CON group, LPS treatment significantly increased the expression of IBA1 in microglia, while Arg-CDs inhibited the LPS-induced high expression of IBA1 in BV2 cells.

[0045] In summary, CCK-8 and Calcein-AM / PI staining assays demonstrated that Arg-CDs possessed good cytocompatibility. Under Arg-CD co-culture conditions, CLSM clearly visualized Arg-CDs within BV2 cells, demonstrating that BV2 cells efficiently uptake Arg-CDs. Arg-CDs modulated LPS-induced BV2 cell activation and downregulated the expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), thereby inhibiting microglia-mediated inflammation.

[0046] 3. Study on the antinociceptive effect of Arg-CDs on the trigeminal neuralgia model in rats

[0047] Adult male Sprague-Dawley (SD) rats weighing 200–300 g 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 a standard rodent chow diet with free access to food and water.

[0048] To evaluate the effects of Arg-CDs on trigeminal neuralgia, a commonly used chronic constriction injury (ION-CCI) model of the infraorbital nerve was established in male Sprague-Dawley rats. The ION-CCI model was established as follows: After anesthesia, rats were placed in the lateral decubitus position. The surgical area was disinfected, and a 10-mm incision was made approximately 8 mm from the nasal margin to the left vibrissae pad. A sterile glass needle was used to separate a portion of the infraorbital nerve, exposing it to the visual field. The exposed infraorbital nerve was then ligated with 4-0 absorbable surgical sutures, approximately 2 mm apart, with appropriate force. Following ligation, the nerve diameter was significantly reduced without disrupting the nerve's external circulation. The incision was sutured with 4-0 sutures, and the surgical area was disinfected again. Rats in the sham group underwent the same procedure, except that the exposed infraorbital nerve was not ligated. All procedures performed in this study were performed in accordance with protocols 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.

[0049] 3.1 Effects of Arg-CDs on the Behavior of ION-CCI Rats

[0050] (1) Mechanical pain threshold

[0051] Mechanical stimulation was performed using Von Frey hairs (North Coast Medical Inc). Briefly, rats were placed in a homemade wire cage for 10 minutes to acclimate to the experimental environment until they resumed their normal sniffing / non-movement posture. After the acclimatization period, the examiner applied von Frey hairs to the ipsilateral ION area (whisker pad) of the rat to the injury, starting with 0.008g, until a clear behavioral response was elicited. When the rat showed a positive response, its intensity, i.e., the pain threshold, was recorded. Positive responses included:

[0052] ① Rapid head-retraction reaction;

[0053] ② Escape or attack behavior, manifested as running away, curling up, hiding the head, biting or scratching stimuli;

[0054] ③ Asymmetric facial grooming: rats performed at least three uninterrupted face-washing movements in the stimulated facial area.

[0055] 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.

[0056] 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 levels, with no statistically significant difference ( P >0.05). The pain threshold on the sixth day after surgery was significantly decreased (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.

[0057] The ION-CCI group experienced a short plateau period after surgery, with no significant change in pain threshold. The pain threshold then gradually decreased and reached a stable level 14 days after surgery. There was no statistically significant 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.

[0058] 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 injected intraperitoneally with Arg-CDs 30 minutes later, and the changes in the facial mechanical pain threshold of the rats were observed and compared with those in the saline group. Figure 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 showed no significant change (2.63±1.85g, P >0.05).

[0059] (2) Face-rubbing behavior

[0060] Prepare a transparent plastic box and install a monitoring recorder at a height above the side of the box to ensure that the monitoring field of view covers the entire rat activity area. Place SD rats individually in the box. After acclimating to the environment for 10 minutes, begin recording the time and frequency of face rubbing by the rats' paws for 20 minutes. After recording is completed, return the rats to their original breeding boxes.

[0061] On the 14th day after surgery, 30 minutes after intraperitoneal injection of Arg-CDs, the frequency and duration of face rubbing in the 10 mg / kg CDs group were significantly shortened (2.50 ± 1.51 times, 49.00 ± 6.57 s) compared with the ION-CCI+Vehicle group (5.83 ± 1.47 times, 49.00 ± 6.57 s). P <0.01; 27.50±4.76s, P <0.0001), while there was no significant difference in the 1 mg / kg CDs group (4.00±1.55 times, P >0.05;47.17±7.60s, P >0.05).

[0062] 3.2 Changes in c-FOS protein expression in SpC5 of ION-CCI rats

[0063] (1) Immunoblotting

[0064] like Figure 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-quantified by immunoblotting. Compared with the ION-CCI+Vehicle group, the expression of c-FOS in the 1 mg / kg CDs group did not change significantly ( 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.

[0065] (2) Immunohistochemistry (IHC)

[0066] The nuclei of neurons immunoreactive for c-FOS protein appeared brown and round or oval under a light microscope. Immunohistochemical staining of c-FOS protein in Sp5C of rats in each group revealed that, compared with the high expression of c-FOS in the ION-CCI+Vehicle group, c-FOS protein expression was significantly reduced after intervention with 10 mg / kg Arg-CDs, while protein expression remained unchanged after 1 mg / kg CDs.

[0067] Effects of Arg-CDs on neuroinflammation in ION-CCI rats

[0068] (1) Immunofluorescence (IF)

[0069] Immunofluorescence was used to visualize the positive expression of IBA1 in the trigeminal nerve conduction center. Figure 15 Compared with the ION-CCI+Vehicle group, the number of Sp5C-positive cells in the 10 mg / kg CDs group was significantly reduced.

[0070] (2) Immunohistochemistry (IHC)

[0071] like Figure 16 By immunohistochemical staining of IL-1β protein in Sp5C of rats in each group, it was 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.

[0072] In summary, hemolysis assays and HE staining results demonstrate that Arg-CDs have no significant toxic side effects and exhibit good biocompatibility. In the ION-CCI rat model, both molecular and behavioral assessments suggest that Arg-CDs effectively alleviate trigeminal neuralgia. Arg-CDs also effectively inhibit microglia-mediated neuroinflammation in rat Sp5C.

Claims

1. Application of arginine carbon dots in the preparation of a drug for treating trigeminal neuralgia, characterized in that: The preparation method of the arginine carbon dots is as follows: citric acid and L-arginine are added to ultrapure water, fully stirred and then ultrasonicated; then fully reacted at 180°C-220°C, cooled and then centrifuged, the supernatant is filtered and the product is dialyzed, and the purified product is freeze-dried to obtain arginine carbon dots (Arg-CDs).

2. The use according to claim 1, characterized in that: The mass ratio of citric acid to L-arginine is 1:

2.

3. The use according to claim 1 or 2, characterized in that: The reaction temperature was 200°C.

4. The use according to claim 3, characterized in that: The supernatant was filtered through a 0.22 µm microporous membrane.

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

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