Construction of cholesterol self-targeting carbon dots and application of cholesterol self-targeting carbon dots in atherosclerosis and tumors

Abstract

The invention discloses construction of a cholesterol self-targeting carbon dot and application of the cholesterol self-targeting carbon dot in atherosclerosis and tumors. The carbon dot is synthesized from beta-cyclodextrin, L-arginine and ammonium fluoride through a hydrothermal method. The preparation method comprises the following steps: dissolving beta-cyclodextrin, L-arginine and ammonium fluoride in a sodium hydroxide solution, carrying out ultrasonic dissolution, transferring the solution into a polytetrafluoroethylene liner, putting the liner into a stainless steel high-pressure kettle, and carrying out heating reaction in a muffle furnace; and after the reaction is finished and a reaction system is cooled to room temperature, taking out a solution, putting the solution into a dialysis bag for dialysis purification, and after dialysis is finished, freeze-drying the solution to obtain the carbon dots. The carbon dots prepared by the invention can target cholesterol, have near-red fluorescence emission, and can be used for imaging and treatment of high cholesterol diseases (atherosclerosis and tumors).

Description

Technical Field

[0001] This invention belongs to the field of materials science and technology, and relates to the construction of cholesterol self-targeting carbon dots and their application in atherosclerosis and tumors. Background Technology

[0002] Cholesterol is an essential component of the body, but high cholesterol or abnormal cholesterol accumulation can lead to numerous diseases, such as atherosclerosis and tumors. In atherosclerosis, elevated low-density lipoprotein cholesterol deposits under the arterial intima, and after oxidative modification, triggers a persistent inflammatory response, foam cell formation, and plaque progression, thereby causing vascular stenosis and increasing the risk of thrombosis. In tumorigenesis, abnormal cholesterol metabolism can indirectly promote the occurrence and development of various cancers by regulating cell proliferation, survival signaling pathways, and immune responses in the tumor microenvironment; for example, some tumor cells utilize cholesterol to enhance their invasive and metastatic abilities. Therefore, cholesterol accumulation is a common pathological basis for both of these diseases.

[0003] The progression of diseases such as atherosclerosis and tumors is often closely related to high cholesterol. Therefore, precise targeting of cholesterol not only helps to intervene in the pathological process at its source but also provides a key entry point for achieving high-resolution imaging of lesions. Against this backdrop, a therapeutic platform that combines targeting capabilities with imaging capabilities is particularly important. In recent years, carbon dots, as an emerging carbon-based nanomaterial, have offered new possibilities for precise imaging-guided therapy for diseases such as atherosclerosis and tumors due to their good biocompatibility, excellent optical properties, and flexible structural control.

[0004] The functional realization of carbon dots depends on the selection of carbon source, doping strategy, and synthesis method. For diseases related to high cholesterol, constructing carbon dots with cholesterol-targeting capabilities is particularly crucial. This study selected cyclic compounds as carbon sources, whose hydrophobic cavities can specifically encapsulate the steroidal nucleus structure of cholesterol, achieving targeted recognition. Introducing amino acids as nitrogen sources can regulate physiological factors in vivo, endowing carbon dots with additional therapeutic functions. Furthermore, the incorporation of inorganic halide salts modulates the fluorescence properties of the carbon dots, giving them a broad-spectrum fluorescence emission.

[0005] This study successfully constructed a multifunctional carbon dot nanoplatform integrating cholesterol targeting, multi-disease fluorescence imaging, and regulation of physiological factors. This system not only supports real-time visual monitoring but also enables targeted therapy and multiple synergistic interventions, providing an innovative solution for the precise diagnosis and efficient treatment of cholesterol-related diseases. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by constructing a carbon dot system with autonomous cholesterol targeting function. This material can specifically recognize and bind to cholesterol, enabling multiple fluorescence imaging of atherosclerotic plaques and tumors, and regulating physiological factors, thus providing an innovative solution for the precise diagnosis and efficient treatment of cholesterol-related diseases.

[0007] To achieve the above objectives, the present invention employs the following technical solutions:

[0008] The construction of a cholesterol self-targeting carbon dot includes the following steps:

[0009] Cyclic compounds, amino acids, and halide salts are dissolved separately in an alkaline solution according to a specific feeding ratio. After ultrasonic dissolution, two or more of the three solutions are transferred to a polytetrafluoroethylene (PTFE) liner. The liner is then placed in a stainless steel autoclave, and the reaction is carried out in a muffle furnace with one or more heating cycles. After the reaction is completed, the reaction system is cooled to room temperature, and the solution is removed and placed in a dialysis bag for dialysis purification. After dialysis, the solution is freeze-dried to obtain the β-CDAF of this invention.

[0010] Furthermore, the cyclic compound is one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or cyclodextrin derivatives.

[0011] Furthermore, the amino acid is one of L-arginine, tryptophan, threonine, valine, and leucine.

[0012] Furthermore, the halide salt is one of ammonium fluoride, sodium fluoride, and sodium chloride.

[0013] Furthermore, the alkaline solution is a solution of sodium hydroxide, potassium hydroxide, triethylamine, etc., with a concentration of 0.5-3 mol / L (molar volume ratio).

[0014] Furthermore, the optimal mass ratio of reactants is cyclic compound: amino acid: halide salt = 3:3:2.

[0015] Furthermore, the optimal reaction temperature for the heating reaction is 150-200℃, and the optimal reaction time is 2-10 hours.

[0016] Furthermore, the dialysis bag used in the dialysis has a molecular weight cutoff of 100-500 Da, and the dialysis time is 12-48 hours.

[0017] An application of cholesterol self-targeting carbon dots is used in the diagnosis and treatment of atherosclerosis and tumors.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] First, the prepared carbon dots are modified with halogens, allowing control over their structure and luminescence range. Second, the prepared carbon dots possess unique affinity for alkanes, particularly cholesterol, enabling specific inclusion of cholesterol compounds via host-guest chemistry, enhancing their water solubility and facilitating their clearance from both in vivo and in vitro. Therefore, this allows for in vivo lesion localization and real-time detection and treatment of hypercholesterolemia (such as atherosclerosis and tumors). Attached Figure Description

[0020] To more clearly illustrate the solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments recorded in the present invention. For those skilled in the art, other drawings obtained from these drawings without creative effort still fall within the scope of the present invention.

[0021] Figure 1 This is a schematic diagram of the preparation process in Example 1;

[0022] Figure 2 This is the visible fluorescence emission spectrum of the carbon dots prepared in Example 1;

[0023] Figure 3 This is the near-infrared fluorescence emission spectrum of the carbon dots prepared in Example 1;

[0024] Figure 4 The image shows the XPS spectrum of the carbon dots prepared in Example 1. The full spectrum analysis of the carbon dots shows a total of four peaks: 284.80 eV (C 1s), 400.10 eV (N 1s), 531.40 eV (O 1s), and 684.00 eV (F 1s).

[0025] Figure 5 The images show a transmission electron microscope (TEM) image (left) and a particle size distribution map (right) of the carbon dots prepared in Example 1. As can be seen from the images, the synthesized carbon dots are basically elliptical in shape and have good dispersibility. The particle size distribution histogram obtained through statistical analysis shows that the average particle size of the carbon dots is 1.96 nm.

[0026] Figure 6 The image shows the fluorescence of the carbon dots prepared in Example 1 under a small animal in vivo imaging system. As shown in the figure, the carbon dots exhibit obvious fluorescence compared to water under the in vivo imaging system.

[0027] Figure 7 The images are digital photographs of the carbon dots prepared in Example 1 after binding to cholesterol-rich blood vessels without washing, and the images of the samples after washing with PBS, taken under a 365nm UV lamp and under different channels of an inverted fluorescence microscope.

[0028] Figure 8 After establishing an atherosclerosis model in mice, the carbon dots prepared in Example 1 were injected into the mice via the tail vein. Mice were anesthetized and photographed using a small animal in vivo imaging system at different time points. The images show that the synthesized carbon dots can target and image the carotid artery lesions.

[0029] Figure 9 After establishing a tumor model in mice, the carbon dots prepared in Example 1 were injected into the mice via the tail vein. Mice were anesthetized and photographed using a small animal in vivo imaging system at different time points. The images show that the synthesized carbon dots can target the tumor site for imaging. Detailed Implementation

[0030] The present invention will be further described in detail below through specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention, and are not intended to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention; in addition, some non-essential improvements and adjustments can be made to the present invention based on the above-described invention.

[0031] The instruments and equipment used in the following embodiments of the present invention are as follows:

[0032] Electronic balance ME104E; Fluorescence spectrophotometer RF-5301 (Shimadzu, Japan); Transmission electron microscope JEM-1200EX TEM (JEOL, Japan); X-ray photoelectron spectrometer EscaLab 250Xi (Thermo, USA); Small animal in vivo imaging system VISQUE In vivo Smart-LF (Shanghai, China); Three-in-one ultraviolet analyzer ZF-7; Inverted fluorescence microscope Nikon Ti-S.

[0033] The carbon dots described in the following examples are β-CDAF.

[0034] The specific embodiments of the present invention are further described in detail below:

[0035] Example 1:

[0036] Weigh out 0.6000 g of β-cyclodextrin, 0.6000 g of L-arginine, and 0.4000 g of ammonium fluoride. Dissolve β-cyclodextrin, L-arginine, and ammonium fluoride separately in 0.67 mol / L sodium hydroxide solution, sonicate to dissolve, mix the three solutions, and transfer them to a polytetrafluoroethylene (PTFE) liner. Place the liner in a stainless steel autoclave and heat at 200°C for 6 hours in a muffle furnace. After the reaction is complete, allow the reaction system to cool to room temperature, remove the solution, and place it in a dialysis bag with a molecular weight cutoff of 100-500 Da for dialysis and purification. After dialysis for 24 hours, freeze-dry the solution to obtain the β-CDAF of this invention.

[0037] Examples 2-6:

[0038] The prepared carbon dot powder is resoluble in aqueous solution. Spectroscopic characterization of the aqueous solution revealed that it exhibits dual emission fluorescence in the visible and near-infrared regions. Figure 2 and Figure 3 As shown, its fluorescence emission peak is highly dependent on the excitation light, and the position of the fluorescence peak changes with the excitation light. The structure of the carbon dots was characterized using X-ray photoelectron spectroscopy, as shown... Figure 4 As shown, the prepared carbon dots contain C, N, O, and F elements. The structure of the carbon dots was characterized using transmission electron microscopy, as follows: Figure 5 As shown, the prepared carbon dots exhibit good monodispersity in aqueous solution, are spherical, and have a particle size of approximately 1.96 nm. Their in vivo imaging capability was verified using a small animal in vivo imaging system, such as... Figure 6 As shown, the prepared carbon dots can emit obvious near-infrared signals, which indicates that the prepared carbon dots can be used for in vivo imaging of animals.

[0039] Example 7:

[0040] Mouse arteries were immersed in a cholesterol-ethanol solution. After the ethanol evaporated and cholesterol deposited on the vessel wall, the arteries were incubated in β-CDAF solution for 24 hours. The vessels were then removed and placed on a glass slide, and digital photographs were taken under a 365 nm UV lamp. Fluorescence was detected using an inverted fluorescence microscope. Finally, the arteries were placed in PBS and shaken on a shaker for 1 hour to wash away cholesterol. The images were then taken again under a 365 nm UV lamp, and fluorescence was detected.

[0041] Example 8:

[0042] Example 1 describes the application of a cholesterol self-targeting carbon dot prepared in practical bioimaging. Fluorescence imaging was used to detect the targeting of the carbon dot in vivo. A mouse atherosclerosis model was established, and the carbon dot was injected into the mice via the tail vein. The results showed that the carbon dot could effectively target and image lesions in the carotid artery.

[0043] Example 9:

[0044] Example 1 describes the application of a cholesterol self-targeting carbon dot prepared in practical bioimaging. Fluorescence imaging was used to detect the targeting of the carbon dot in vivo. A mouse 4T1 tumor model was established, and the carbon dot was injected into the mice via the tail vein. The results showed that the carbon dot could effectively target and image the tumor site.

Claims

1. A cyclic compound, amino acid, and halide salt are dissolved separately in an alkaline solution according to a certain feeding ratio. After ultrasonic dissolution, two or more of the three solutions are transferred to a polytetrafluoroethylene (PTFE) liner. The liner is then placed in a stainless steel autoclave, and the reaction is carried out in a muffle furnace with one or more heating cycles. After the reaction is completed, the reaction system is cooled to room temperature, and the solution is removed and placed in a dialysis bag for dialysis purification. After dialysis, the solution is freeze-dried to obtain the β-CDAF of this invention.

2. The method for preparing β-CDAF according to claim 1, characterized in that, The cyclic compound is one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or cyclodextrin derivatives.

3. The method for preparing β-CDAF according to claim 1, characterized in that, The amino acid is one of L-arginine, tryptophan, threonine, valine, and leucine.

4. The method for preparing β-CDAF according to claim 1, characterized in that, The halide salt is one of ammonium fluoride, sodium fluoride, and sodium chloride.

5. The method for preparing β-CDAF according to claim 1, characterized in that, The optimal mass ratio of the reactants for the reaction is 3:3:

2.

6. The method for preparing β-CDAF as described in claim 1, characterized in that: The alkaline solution was a sodium hydroxide solution with a concentration of 0.67 mol / L. The reaction temperature was 200℃ and the reaction time was 6 hours.

7. The method for preparing β-CDAF as described in claim 1, characterized in that: The dialysis bag used in the dialysis has a molecular weight cutoff of 100-500 Da, and the dialysis time is 24 hours.

8. The fluorescent carbon dots as described in claim 1, characterized in that: The fluorescent carbon dots emit wavelengths between 525 and 600 nm in the visible light region and between 730 and 900 nm in the near-infrared region.

9. The fluorescent carbon dots as described in claim 1, characterized in that: It can target cholesterol crystals for imaging.

10. The application of β-CDAF according to any one of claims 1–9 in the diagnosis and treatment of atherosclerosis and tumors, characterized in that: In vivo imaging can be achieved in the near-infrared emission wave range of 600nm-1000nm.

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