Highly efficient local anesthetic drug-loaded water-soluble carbon dots, preparation method and application thereof

Abstract

This invention relates to the field of fluorescent carbon nanomaterials, specifically to highly efficient water-soluble carbon dots for loading local anesthetic drugs, their preparation method, and applications. Using water-soluble amino acids and carboxylic acid compounds that readily aggregate and carbonize to form six-membered rings as precursors, the precursors are sonicated to dissolve them in an organic solvent to form a homogeneous solution. This solution is then transferred to a reaction vessel and subjected to a solvothermal reaction at 180-240°C for 4-10 hours. The reaction vessel is then allowed to cool naturally to room temperature, directly obtaining a carbon dot solution. The obtained carbon dot solution is filtered, eluted, and dried to obtain a solid carbon dot powder. The functionalized carbon dots prepared by this invention possess both good water solubility and high efficiency in loading local anesthetic drugs, showing broad application prospects in the field of sustained-release analgesia of local anesthetic drug formulations.

Description

Technical Field

[0001] This invention relates to the field of carbon nanomaterials, specifically to water-soluble carbon dots for efficiently loading local anesthetic drugs, their preparation methods, and applications. Background Technology

[0002] Local anesthetics are among the most commonly used anesthetics in clinical practice, such as procaine, lidocaine, and ropivacaine. However, a single dose of local anesthetic can only maintain its effect for 4-6 hours, making it difficult to treat long-term, severe postoperative pain. To ensure sustained analgesia after surgery, frequent injections are often required, leading to fluctuations in blood drug concentration, reduced efficacy, and poor patient compliance. While continuous infusion using an infusion pump can stabilize blood drug concentration, it requires constant monitoring and a long-term catheter connection to the patient, increasing the risk of incision site infection and potential inaccurate drug delivery if the catheter becomes loose. Therefore, it is necessary to develop carriers capable of loading local anesthetics to achieve slow, long-term release and thus long-acting analgesia. Currently, various sustained-release formulation materials have been studied both domestically and internationally, mainly including biodegradable materials such as microspheres, hydrogels, nanoparticles, and liposomes. However, these materials still have some drawbacks, such as low drug loading capacity, drug leakage, and potential toxic side effects of the carrier itself. Therefore, developing a suitable carrier that can effectively load local anesthetics without causing toxic side effects is a key factor in achieving sustained, slow drug release during treatment.

[0003] Carbon dots (CDs) are zero-dimensional nanomaterials primarily composed of carbon, typically smaller than 10 nm in size, and composed of sp... 2 / sp 3 Composed of a carbon skeleton and abundant functional groups, carbon dots (CDs) possess excellent photoluminescence properties, providing a theoretical basis for bioimaging and a foundation for visualizing their localized drug delivery. More importantly, CDs exhibit good biocompatibility, simple synthesis, and the ability to be functionalized, leading to their widespread research and application in the biomedical field, and they hold promise as next-generation sustained-release carriers for local anesthetics. Although CDs have made initial progress in loading small-molecule aromatic anticancer chemotherapeutic drugs, the excellent water solubility and small molecular weight of local anesthetics, which facilitate escape, have prevented reports on long-acting sustained-release analgesia using carbon dot-loaded local anesthetics. Summary of the Invention

[0004] The purpose of this invention is to provide a highly efficient water-soluble carbon dot for loading local anesthetic drugs.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned carbon dot material.

[0006] Another object of the present invention is to provide applications of the aforementioned carbon dots.

[0007] The water-soluble carbon dots for highly effective loading of local anesthetic drugs according to the present invention are prepared by a method comprising the following steps:

[0008] Using water-soluble amino acid precursors and carboxylic acid precursors that easily aggregate and carbonize to form six-membered rings as reactants, they are dissolved in an organic solvent and a homogeneous solution is formed under ultrasonication. The carbon dot solution is obtained by solvothermal reaction at 180-240℃ for 4-10 hours.

[0009] The obtained carbon dot solution was eluted and dried to obtain carbon dot solid powder.

[0010] The water-soluble carbon dots of the present invention, which are highly effective loading local anesthetic drugs, wherein the water-soluble amino acids are cysteine, methionine and / or glutamic acid;

[0011] The precursor of the carboxylic acid compound that is easily aggregated and carbonized to form a six-membered ring is citric acid, malic acid or maleic acid.

[0012] The organic solvent is N,N-dimethylformamide, or a solvent soluble in formamide or a mixture of N,N-dimethylformamide and formamide.

[0013] The water-soluble carbon dots for loading local anesthetic drugs according to the present invention comprise a core structure consisting of a multi-ring array of six-membered rings, each of which is an aromatic or unsaturated five-membered ring, and its edges contain hydrophilic functional groups such as -OH, -COOH and -NH2.

[0014] The water-soluble carbon dots of the highly efficient local anesthetic drug according to the present invention are separated and purified by silica gel column chromatography using a mixture of methanol and dichloromethane as the eluent.

[0015] The method for preparing water-soluble carbon dots loaded with highly effective local anesthetic drugs according to the present invention includes the following steps:

[0016] Using water-soluble amino acids and carboxylic acid compounds that easily aggregate and carbonize to form six-membered rings as precursors, the precursors are sonicated to dissolve them in an organic solvent to form a homogeneous solution (mass ratio of 5:1 to 1:5). The solution is then transferred to a reaction vessel and hydrothermally reacted at 180-240℃ for 4-10 hours. The reaction vessel is then allowed to cool naturally to room temperature to directly obtain a carbon dot solution. The obtained carbon dot solution is filtered, eluted, and dried to obtain carbon dot solid powder.

[0017] According to the present invention, a method for preparing water-soluble carbon dots loaded with highly efficient local anesthetic drugs is used, wherein a mixture of methanol and dichloromethane is used as the eluent, and silica gel column chromatography is employed for separation and purification.

[0018] The carbon dots obtained by this invention through a simple solvothermal method have both large and complete sp...2 The carbon dot solid powder prepared by combining the center of the structural domain with the edges modified with abundant hydrophilic functional groups exhibits good solubility in water and good biocompatibility, making it suitable for further biological applications.

[0019] Another objective of this invention is to use the prepared water-soluble carbon dots for efficiently loading local anesthetic drugs to achieve slow release of the local anesthetic drugs.

[0020] The carbon dots prepared in this invention can be loaded with local anesthetic drugs for anesthesia and analgesia via π-π stacking, including ester-based and amide-based local anesthetic drugs such as procaine, lidocaine, and ropivacaine. This achieves efficient loading and slow release of local anesthetic drugs for use in preoperative anesthesia and long-acting postoperative analgesia.

[0021] Beneficial effects:

[0022] According to the technical solution of this invention, water-soluble amino acids and carboxylic acid compounds that easily aggregate and carbonize to form six-membered rings are selected as precursors. The reaction sites of the precursors and the size of the central core and the number of hydrophilic functional groups at the edges of the carbon dots are controlled. Solvothermal reaction conditions are controlled, such as a precursor mass ratio between 5:1 and 1:5, a reaction time of 4-10 hours, and a reaction temperature of 180-240℃, to synthesize large and complete sp... 2 The carbon dot structure consists of a central domain and edges modified with abundant hydrophilic functional groups. If the reaction time is too short (less than 4 hours) or the reaction temperature is too low (less than 180°C), the designed structure cannot be formed, meaning the carbon dots formed are too small to efficiently load large amounts of local anesthetic drugs. If the reaction time is too long (more than 10 hours) or the reaction temperature is too high (more than 240°C), over-carbonization is likely to occur, and carbon dots cannot be obtained.

[0023] According to the preparation method of the present invention, the purification process using a mixture of methanol and dichloromethane (volume ratio of methanol to dichloromethane 1:2) as the eluent and silica gel column chromatography is crucial. Because there are too many impurities after the reaction, water-soluble carbon dots capable of efficiently loading local anesthetic drugs cannot be obtained without silica gel column purification.

[0024] The water-soluble carbon dots prepared in this invention, which are highly efficient at loading local anesthetic drugs, possess excellent optical properties and good water solubility. They can be loaded with local anesthetic drugs used for anesthesia and analgesia, including ester and amide local anesthetic drugs such as procaine, lidocaine, and ropivacaine, through π-π-stack interactions. The functionalized carbon dots prepared in this invention simultaneously exhibit high efficiency in loading local anesthetic drugs and good water solubility, showing broad application prospects in the field of long-acting analgesia with local anesthetics. As carriers for sustained-release local anesthetic drugs, they possess excellent luminescent properties and biocompatibility, thus achieving the dual function of drug sustained-release carrier and bioimaging probe. They hold promise as a novel, highly biocompatible, and low-cost sustained-release carrier for local anesthetic drugs in clinical applications for long-acting postoperative analgesia. Attached Figure Description

[0025] Figure 1 The fluorescence spectra of the water-soluble cysteine ​​carbon dots prepared in Example 1 under excitation at different wavelengths are shown.

[0026] Figure 2 The image shows the ultraviolet absorption spectrum of the water-soluble cysteine ​​carbon dots prepared in Example 1.

[0027] Figure 3 Transmission electron microscope (TEM) image of the water-soluble cysteine ​​carbon dots prepared in Example 1;

[0028] Figure 4 The X-ray diffraction pattern of the water-soluble cysteine ​​carbon dots prepared in Example 1;

[0029] Figure 5 The Raman spectrum of the water-soluble cysteine ​​carbon dots prepared in Example 1;

[0030] Figure 6 The X-ray photoelectron spectrum of the water-soluble cysteine ​​carbon dots prepared in Example 1 is shown below.

[0031] Figure 7 The infrared spectrum of the water-soluble cysteine ​​carbon dots prepared in Example 1;

[0032] Figure 8 Transmission electron microscope (TEM) images of the amount of water-soluble cysteine ​​carbon dots prepared in Comparative Example 1;

[0033] Figure 9 The fluorescence test results of the solution obtained in Comparative Example 1 are shown;

[0034] Figure 10 The image shows the ultraviolet absorption spectrum of the local anesthetic procaine loaded onto the water-soluble cysteine ​​carbon dots prepared in Example 1.

[0035] Figure 11The fluorescence spectrum of the local anesthetic procaine loaded onto the water-soluble cysteine ​​carbon dots prepared in Example 1 is shown.

[0036] Figure 12 The high-performance liquid chromatogram is shown after the water-soluble cysteine ​​carbon dots prepared in Example 1 are loaded with the local anesthetic procaine.

[0037] Figure 13 The figure shows the results of an in vitro simulated drug release experiment after loading the local anesthetic procaine onto the water-soluble cysteine ​​carbon dots prepared in Example 1.

[0038] Figure 14 The graph shows the cell viability of SH-SY5Y cells after 3 days of incubation with the local anesthetic procaine loaded onto the water-soluble cysteine ​​carbon dots prepared in Example 1.

[0039] Figure 15 The figure shows the flow cytometry results of apoptosis in SH-SY5Y cells after incubation for 3 days with the local anesthetic procaine loaded onto the water-soluble cysteine ​​carbon dots prepared in Example 1.

[0040] Figure 16 The local muscle tissue enrichment of the water-soluble cysteine ​​carbon dots prepared in Example 1 in Kunming mice;

[0041] Figure 17 The sensory nerve blockade in vivo after the water-soluble cysteine ​​carbon dots prepared in Example 1 were loaded with the local anesthetic procaine.

[0042] Figure 18 The study describes the motor nerve blockade in vivo after the water-soluble cysteine ​​carbon dots prepared in Example 1 were loaded with the local anesthetic procaine.

[0043] Figure 19 The graph shows the blood biochemical parameters of Kunming mice after treatment with the water-soluble cysteine ​​carbon dots prepared in Example 1.

[0044] Figure 20 The image shows the H&E staining pathological analysis of the main tissues of Kunming mice after treatment with water-soluble cysteine ​​carbon dots prepared in Example 1. Detailed Implementation

[0045] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0046] A method for preparing water-soluble carbon dots loaded with highly effective local anesthetic drugs according to a specific embodiment of the present invention includes the following steps:

[0047] (1) A water-soluble amino acid precursor and a carboxylic acid precursor that is easy to aggregate and carbonize to form a six-membered ring are sonicated to dissolve them in N,N-dimethylformamide to form a homogeneous solution. The solution is then transferred to a reaction vessel and reacted solvothermically at 160-240℃ for 4-10 hours. The reaction vessel is then allowed to cool naturally to room temperature to obtain a carbon dot solution. The obtained carbon dot solution is filtered, eluted and dried to obtain carbon dot solid powder.

[0048] (2) Collect the solution after the above reaction, filter it through a 0.22 μm filter membrane, and place the filtrate in a dialysis bag (3500-7000 Da) for dialysis in deionized water for two days, changing the deionized water every 4-6 hours. After dialysis, collect the solution in the dialysis bag, filter it again through a 0.22 μm filter membrane, and then evaporate the solvent. Using a mixed solvent of methanol and dichloromethane as the eluent, purify the solution by silica gel column chromatography to obtain a carbon dot solution. After drying, obtain carbon dot solid powder.

[0049] The method uses a mixture of methanol and dichloromethane (volume ratio of methanol to dichloromethane 1:2) as the eluent and employs silica gel column chromatography for purification. This process removes impurities from the product after the reaction, thereby obtaining water-soluble carbon dots for high-efficiency loading of local anesthetic drugs.

[0050] Example 1: Preparation of water-soluble cysteine ​​carbon dots

[0051] Using cysteine ​​and citric acid as precursors, 0.2 g of cysteine ​​and 0.25 g of citric acid were weighed into beakers, and 30 mL of N,N-dimethylformamide solvent was added. A homogeneous solution was formed under ultrasonic treatment, and then transferred to a polytetrafluoroethylene-lined autoclave. The reaction was carried out at 180 °C for 8 hours using a solvothermal method, followed by natural cooling of the autoclave to room temperature, resulting in a brownish-red carbon dot solution. After the reaction, the solution was collected, filtered through a 0.22 μm membrane, and the filtrate was placed in a dialysis bag (3500-7000 Da) and dialyzed against deionized water for two days, changing the deionized water every 4-6 hours. After dialysis, the solution in the dialysis bag was collected and filtered again through a 0.22 μm filter membrane. The solvent was then evaporated to dryness. A mixture of methanol and dichloromethane (volume ratio of methanol to dichloromethane 1:2) was used as the eluent for purification by silica gel column chromatography. The purified carbon dot solution was separated and dried to obtain carbon dot solid powder.

[0052] As an alternative technical solution in this embodiment, the water-soluble amino acid precursor is methionine or glutamic acid.

[0053] As an alternative technical solution in this embodiment, the carboxylic acid compound precursor that is easily aggregated and carbonized to form a six-membered ring can be malic acid or maleic acid.

[0054] As an alternative technical solution in this embodiment, the organic solvent can be formamide or a mixture of N,N-dimethylformamide / formamide.

[0055] As an alternative technical solution in this embodiment, the mass ratio of the water-soluble amino acid precursor to the carboxylic acid compound precursor that is easily aggregated and carbonized to form a six-membered ring is any ratio of 5:1 to 1:5.

[0056] As an alternative technical solution in this embodiment, the reaction temperature can be 160°C, 200°C or 240°C.

[0057] As an alternative technical solution in this embodiment, the solvothermal reaction can be carried out for 4 hours, 6 hours, or 10 hours.

[0058] A dilute methanol solution containing red fluorescent carbon dots emits bright orange-red fluorescence under a handheld ultraviolet lamp (365 nm), exhibiting excitation-independent intrinsic state fluorescence characteristics. Figure 1 The emission peak is located at 600 nm. The characteristic exciton absorption peak of the red carbon dot is located at 560 nm. Figure 2 ).

[0059] Transmission electron microscopy revealed that the intrinsic state red fluorescent carbon dots had a uniform size distribution and an average particle size of 4.0 nm. Figure 3 The X-ray diffraction pattern of the carbon dots shows a broad diffraction peak around 24°, corresponding to the (002) plane of graphite. Figure 4 ). I in carbon dot Raman spectra D / I G The ratio is 1.43 ( Figure 5 This indicates that the carbon dots are highly graphitized, consistent with the high crystallinity characterized by high-resolution transmission electron microscopy.

[0060] X-ray photoelectron spectroscopy results indicate that the carbon dots are mainly composed of three elements: C, N, and O, with C and N atoms accounting for 65.33% and 16.42% respectively. Figure 6 Infrared spectroscopy of carbon dots in solid form confirms the presence of functional groups such as amino and carboxyl groups in carbon dots. Figure 7 ).

[0061] Comparative Example 1

[0062] The specific steps are the same as in Example 1, using equal masses of cysteine ​​and citric acid as precursors, the difference being that the reaction temperature is 130℃. The resulting carbon dots have a uniform size distribution and good dispersion, but the average particle size is 2.5 nm. Figure 8 The size of the carbon dots is significantly smaller than that of the water-soluble cysteine ​​carbon dots prepared in Example 1. Small carbon dots are difficult to efficiently load procaine through π-π stacking.

[0063] The reaction steps were the same as in Example 1, except that the reaction time was 12 hours and the temperature was set at 260°C. After the reaction was complete, the reaction solution was filtered through a 0.22 μm filter membrane. A large amount of black, water-insoluble precipitate was found on the filter membrane, indicating that excessively long reaction times and high temperatures would cause over-carbonization, preventing the formation of carbon dots. The resulting solution was characterized by fluorescence testing. Figure 9 As shown, when the conditions are set to a solvothermal reaction at 260℃ for 14 hours, compared with the standard reaction conditions (reaction at 180℃ for 8 hours), the fluorescence intensity of water-soluble cysteine ​​carbon dots in the aqueous solution of the product of the same concentration is greatly reduced, proving that the carbon dot content generated by the high temperature and excessively long reaction time is extremely low, and a large number of precursors are over-carbonized into black insoluble precipitates.

[0064] Example 2: Preparation of a water-soluble cysteine ​​carbon dot sustained-release formulation loaded with procaine

[0065] Procaine (PrC) aqueous solutions of different mass concentrations were mixed with the water-soluble cysteine ​​carbon dots (Cys-CDs) aqueous solution prepared in Example 1 at mass ratios of 1:2, 1:1, 2:1, 5:1, and 10:1. The mixture was then stirred at room temperature for 12 hours to prepare a procaine-loaded water-soluble cysteine ​​carbon dot sustained-release formulation via liquid-phase mixing. The resulting product was dialyzed against distilled water for 24 hours (dialysis bag 500-1000 Da) to remove unloaded small molecule procaine. The drug-loaded water-soluble cysteine ​​carbon dot sustained-release formulation sample solution was collected by filtration through a 0.22 μm filter membrane. After freeze-drying, the obtained powder was stored in a dry place at room temperature.

[0066] The ability of water-soluble cysteine ​​carbon dots to bind to procaine is determined by ultraviolet absorption and fluorescence spectroscopy. For example... Figure 10 As shown, procaine binds to the carbon dots of water-soluble cysteine ​​via a non-covalent interaction through π-π stacks, exhibiting a redshift of approximately 5 nm in its absorption peak at 290 nm, while in the fluorescence spectrum ( Figure 11 The decrease in fluorescence intensity also confirms the existence of this non-covalent interaction, causing energy transfer along the interface between the water-soluble cysteine ​​carbon dots and procaine, resulting in a decrease in fluorescence intensity. This is confirmed by high-performance liquid chromatography (HPLC). Figure 12 Quantitative calculations of drug loading and loading efficiency demonstrated that the drug loading efficiency of procaine loaded with water-soluble cysteine ​​carbon dots can reach 37.5%. In in vitro simulated drug release experiments... Figure 13The cumulative drug release of the water-soluble cysteine ​​carbon dot sustained-release formulation loaded with procaine was only 64.8% over three days (pH 7.4). When the solution environment was changed to a slightly acidic environment with pH 4.8, the cumulative drug release over three days reached 84.2%. In contrast, procaine alone achieved a cumulative release of 83.8% within 12 hours, demonstrating that loading the drug onto water-soluble cysteine ​​carbon dots can significantly prolong the drug release time and achieve a sustained-release effect.

[0067] The toxicity of a water-soluble cysteine ​​carbon dot sustained-release formulation loaded with procaine was assessed cytologically. Cell viability results were obtained after 3 days of incubation with SH-SY5Y cells. Figure 14 Studies showed that water-soluble cysteine ​​carbon dots alone did not produce any cytotoxicity, and the sustained-release formulation of water-soluble cysteine ​​carbon dots loaded with procaine also did not significantly affect cell proliferation due to the slow release of procaine. In apoptosis experiments (…), Figure 15 Similar results were also observed: neither the standalone water-soluble cysteine ​​carbon dots nor the procaine-loaded water-soluble cysteine ​​carbon dots sustained-release formulation caused significant cell apoptosis, indicating the good biocompatibility of the carbon dots and demonstrating the good drug release effect of the sustained-release formulation, which is crucial for subsequent in vivo treatment.

[0068] Example 3: Application of long-acting in vivo nerve block therapy based on water-soluble cysteine ​​carbon dots

[0069] The water-soluble cysteine ​​carbon dots prepared above were used as a carrier for the local anesthetic procaine in vivo for long-acting nerve block therapy. The experimental mice weighed 28±2g, and the procaine dose in the treatment group was 40mg / kg. Sensory and motor nerve block in the hind limbs were monitored hourly after administration. NIR fluorescence in vivo imaging experiments were used to assess the blockade. Figure 16 Analysis showed that intramuscular injection of water-soluble cysteine ​​carbon dots near the sciatic nerve in the right hind limb of mice resulted in significantly higher fluorescence intensity at the injection site compared to other areas, with a local enrichment time of approximately 8 hours, meeting the local localization requirements for use as a sustained-release drug carrier in vivo. After loading with the local anesthetic procaine, the procaine on these carbon dots prolonged the sensory nerve blockade time in mice through slow release. Figure 17 ) and motor nerve block time ( Figure 18 It is more than four times the duration of nerve blockade by a single drug.

[0070] After treatment, blood biochemical indicators and tissue sections of major organs were analyzed in mice from each group. Enzyme activity indicators reflecting heart, liver, and kidney function were measured to evaluate the effects of each drug on the major organs of mice. Results ( Figure 19The results showed that, compared with the PBS group and the carbon dot group, the carbon dot sustained-release formulation group showed no significant changes, with values ​​remaining within the normal range, indicating no adverse effects on the heart, liver, and kidneys of mice. However, procaine alone induced higher levels of creatinine (CREA), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), indicating some damage to the liver, kidneys, and heart of mice. This is because injecting large amounts of a single local anesthetic can have considerable side effects on the cardiovascular system and metabolic organs (such as the liver and kidneys), leading to elevated ALT and LDH levels. H&E staining analysis revealed that, compared with the blank PBS group, the carbon dot sustained-release formulation group showed normal results in heart, liver, spleen, lung, kidney, and muscle sections, with tightly and neatly arranged cells, and no significant difference between the two groups. Figure 20 No significant underlying pathological changes, inflammation, or myotoxic reactions were observed. This demonstrates that the sustained release of the drug at this carbon dot not only prolongs the nerve blockade effect in vivo but also reduces the systemic toxicity of the drug.

[0071] The above embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and processes, but the protection scope of the present invention is not limited to the above embodiments.

Claims

1. The application of water-soluble carbon dots, which are highly efficient carriers for local anesthetic drugs, as carriers for local anesthetic drugs, is characterized by: The water-soluble carbon dots are prepared by a method comprising the following steps: Using water-soluble amino acids and carboxylic acid compounds that readily aggregate and carbonize to form six-membered rings as precursors, these precursors are dissolved in an organic solvent and subjected to ultrasonic treatment to form a homogeneous solution. The solution is then subjected to a solvothermal reaction at 180-240 °C for 4-10 hours to obtain a carbon dot solution. The water-soluble amino acid is cysteine, methionine, or glutamic acid; the carboxylic acid compound readily aggregates and carbonizes to form a six-membered ring is citric acid, malic acid, or maleic acid; the organic solvent is N,N-dimethylformamide, formamide, or a mixture of N,N-dimethylformamide and formamide; and the mass ratio of the water-soluble amino acid to the carboxylic acid compound readily aggregates and carbonizes to form a six-membered ring is 5:1 to 1:

5. The obtained carbon dot solution was eluted and dried to obtain carbon dot solid powder.

2. The application according to claim 1, characterized in that, The mixture of methanol and dichloromethane was used as the eluent, and the particles were separated and purified by silica gel column chromatography.

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