A chlorogenic acid carbon dot magnetic resonance nanoprobe targeting liver fibrosis and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a chlorogenic acid carbon dot magnetic resonance nanoprobe for targeting liver fibrosis and a preparation method and application thereof. The chlorogenic acid carbon dot magnetic resonance nanoprobe is prepared by taking a natural product chlorogenic acid as a carbon source, and is surface-modified by polydopamine, and is further coupled with a cyclic arginine-glycine-aspartic acid peptide (cRGD) targeting peptide and coordinated with manganese ions, so as to construct a magnetic resonance nanoprobe with activated hepatic stellate cell targeting capability. The results of examples show that the nanoprobe has good biocompatibility, stable magnetic resonance imaging (MRI) performance and excellent lesion targeting enrichment capability, can significantly improve the magnetic resonance imaging contrast of liver fibrosis tissue, and realizes noninvasive precise diagnosis and grading of liver fibrosis. Further, the chlorogenic acid carbon dot magnetic resonance nanoprobe provides a new technical scheme for precise image diagnosis of liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chlorogenic acid carbon dot magnetic resonance nanoprobe targeting liver fibrosis, its preparation method, and its application. Background Technology

[0002] Liver fibrosis ( Liver fibrosis Liver fibrosis is a crucial pathological stage in the progression of various chronic liver injuries to cirrhosis and even hepatocellular carcinoma. Its main characteristics include abnormal deposition of extracellular matrix within the liver and persistent activation of hepatic stellate cells. Numerous studies have shown that early-stage liver fibrosis is somewhat reversible; therefore, early diagnosis and accurate grading are of great significance for improving patient prognosis. On the other hand, the activation of hepatic stellate cells (…) during liver fibrosis… Activated hepatic stellate cells The hepatic stellate cells (aHSCs) express integrin αvβ3 in large quantities. Therefore, constructing novel MRI molecular probes that can actively target and activate hepatic stellate cells is of great significance for improving the diagnostic sensitivity of liver fibrosis.

[0003] In recent years, MRI molecular probes based on paramagnetic ions such as Gd and Mn have gradually become a research hotspot. These probes can enhance local T1 signals, thereby improving lesion visualization. However, existing MRI probes generally suffer from insufficient targeting ability, limited biocompatibility, and insufficient ability to recognize early fibrosis.

[0004] Magnetic resonance imaging (MRI) Magnetic Resonance Imaging MRI (radiological imaging) has advantages such as being non-invasive, having high spatial resolution, and being repeatable, making it an important imaging method for the clinical diagnosis of liver diseases. However, traditional MRI mainly relies on changes in tissue structure for judgment, and has low sensitivity to early liver fibrosis, making it difficult to accurately distinguish between different stages of fibrosis.

[0005] Chlorogenic acid is a natural polyphenol compound with good biocompatibility and abundant oxygen-containing functional groups, making it an excellent carbon source for carbon dot preparation; polydopamine has excellent surface modification and metal ion coordination capabilities; the cyclic arginine-glycine-aspartic acid peptide cRGD can recognize αvβ3 receptors on the surface of activated hepatic stellate cells; Mn 2+ It has excellent T1 imaging capabilities.

[0006] Currently, the clinical diagnosis of liver fibrosis mainly relies on methods such as liver biopsy, ultrasound elastography, and magnetic resonance elastography. However, these methods still have certain limitations in early identification of liver fibrosis, lesion-specific visualization, and accurate grading. Conventional magnetic resonance imaging mainly reflects changes in liver morphology and tissue signals, and its sensitivity to early fibrosis-related molecular and cellular changes is insufficient. Existing magnetic resonance contrast agents mostly lack the ability to actively target liver fibrosis lesions, making it difficult to achieve specific recognition of activated hepatic stellate cells, and also making it difficult to accurately judge the degree of disease based on signal differences at different stages of fibrosis.

[0007] Therefore, there is an urgent need to develop a novel nanoprobe that combines the ability to target and activate hepatic stellate cells with T1-weighted magnetic resonance imaging enhancement to improve the detection of liver fibrosis, especially early liver fibrosis, and to provide reliable imaging evidence for non-invasive grading of liver fibrosis. Summary of the Invention

[0008] The purpose of this invention is to provide a chlorogenic acid carbon dot magnetic resonance nanoprobe, its preparation method, and its application in the diagnosis of liver fibrosis using magnetic resonance imaging. The chlorogenic acid carbon dot magnetic resonance nanoprobe provided by this invention not only has good water dispersibility and structural stability, but also good biocompatibility.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a chlorogenic acid carbon dot magnetic resonance nanoprobe, which is prepared by using chlorogenic acid as a carbon source to obtain CA-CDs through hydrothermal reaction, and then loading manganese ions onto the CA-CDs after reacting with dopamine hydrochloride and cyclic arginine-glycine-aspartic peptide cRGD in Tris-HCl buffer in the dark.

[0010] Preferably, the chlorogenic acid carbon dot magnetic resonance nanoprobe targets activated hepatic stellate cells in liver fibrotic tissue.

[0011] This invention also provides a method for preparing the above-mentioned chlorogenic acid carbon dot magnetic resonance nanoprobe, specifically including the following steps: S1. Chlorogenic acid solution was prepared using chlorogenic acid as raw material. After hydrothermal synthesis reaction, the chlorogenic acid solution was filtered and purified by light-protected dialysis to obtain CA-CDs nano-formulation. S2. A mixed solution of cyclic arginine-glycine-aspartic peptide and maleimide polyethylene glycol dopamine and dopamine hydrochloride were dissolved in Tris-HCl solution at pH 8.3. After adding CA-CDs nano-formulation and stirring the reaction, the mixture was ultrafiltered and washed with deionized water to obtain carbon dot nanocomposite. S3. The carbon dot nanocomposite was prepared into a dispersion with a concentration of 0.5 mg / mL and mixed with manganese chloride tetrahydrate solution. After stirring and reacting, centrifugation and washing, chlorogenic acid carbon dot magnetic resonance nanoprobes were obtained.

[0012] Preferably, the process of preparing chlorogenic acid solution in step S1 is as follows: chlorogenic acid is dissolved in deionized water, sonicated for 5 min at room temperature in the dark, and then magnetically stirred for 5 min at 500 rpm; the concentration of the chlorogenic acid solution is 10 mg / mL.

[0013] Preferably, the hydrothermal synthesis reaction in step S1 is carried out at 200°C for 6 hours.

[0014] Preferably, the weight ratio of the cyclic arginine-glycine-aspartic peptide, maleimide polyethylene glycol dopamine, dopamine hydrochloride and CA-CDs nano-formulation in step S2 is 1:10:10:80.

[0015] Preferably, the volume ratio of the dispersion to the manganese chloride tetrahydrate solution in step S3 is 2:1; and the concentration of the manganese chloride tetrahydrate solution is 0.5 mg / mL.

[0016] Preferably, the stirring reaction in step S3 is magnetic stirring at 500 rpm for 12 h; the centrifugation speed is 14000 rpm for 10 min; and the washing is done with deionized water twice.

[0017] The present invention also provides the application of the above-mentioned chlorogenic acid carbon dot magnetic resonance nanoprobe or the chlorogenic acid carbon dot magnetic resonance nanoprobe prepared by the above preparation method in the preparation of contrast agents for magnetic resonance imaging diagnosis of liver fibrosis.

[0018] The beneficial effects of this invention are: The synthesis method of this invention is simple, convenient to operate, and highly reproducible. The resulting nanoprobes have good water dispersibility, structural stability, and biocompatibility.

[0019] In the liver fibrosis microenvironment, the CPRM prepared in this invention can recognize and activate integrin αvβ3 highly expressed on the surface of hepatic stellate cells through the cyclic arginine-glycine-aspartic peptide cRGD targeting peptide, thereby increasing the enrichment of nanoprobes in liver fibrosis tissue. At the same time, the manganese ions coordinated in the probe can shorten the longitudinal relaxation time of local tissues, enhance the T1-weighted magnetic resonance imaging signal, and improve the image contrast between liver fibrosis tissue and normal liver tissue.

[0020] The CPRM prepared by this invention can be used for targeted magnetic resonance imaging of liver fibrosis, and can assist in judging the degree of liver fibrosis based on the changes in magnetic resonance signals at different stages of fibrosis, so as to realize early identification, non-invasive diagnosis and accurate grading of liver fibrosis. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Transmission electron microscopy images of CA-CDs@PDA-RGD-Mn (CPRM); Figure 2 The UV-Vis absorption spectra of CA-CDs and CA-CDs@PDA are shown. Figure 3 The graph shows the zeta potential detection results for CPRM. Figure 4 The diagram shows the docking analysis of the cyclic arginine-glycine-aspartic peptide with integrin αvβ3; where A is the overall view and B is a magnified view of a part. Figure 5 Gel magnetic resonance imaging and pseudocolor images prepared for CPRM; Figure 6 Graph showing the longitudinal relaxation time of CPRM gels at different concentrations; Figure 7 Magnetic resonance imaging of CPRM in a rat model of liver fibrosis; Figure 8 This is a graph showing the concentration and uptake of CPRM. Figure 9 This is a graph showing the hemolysis of blood cells after different treatments. Detailed Implementation

[0023] This invention provides a chlorogenic acid carbon dot magnetic resonance nanoprobe targeting liver fibrosis, organically combining material construction with magnetic resonance targeted imaging functionality. The invention uses chlorogenic acid, a natural product, as a carbon source to prepare carbon dots, constructs a polydopamine functional layer on their surface, and further couples a cyclic arginine-glycine-aspartic peptide (cRGD) targeting peptide and coordinating manganese ions to form a nanoprobe possessing both the ability to activate hepatic stellate cells and the ability to enhance T1-weighted magnetic resonance imaging. The resulting nanoprobe exhibits good water dispersibility, structural stability, and biocompatibility. Specifically, the cyclic arginine-glycine-aspartic peptide (cRGD) targeting peptide can recognize integrin αvβ3, which is highly expressed on the surface of activated hepatic stellate cells, improving the targeted accumulation of the nanoprobe in liver fibrosis tissue; the coordinating manganese ions can shorten the longitudinal relaxation time of local tissues, enhance the T1-weighted magnetic resonance signal of liver fibrosis tissue, and improve the image contrast between diseased tissue and normal liver tissue.

[0024] This invention integrates liver fibrosis lesion targeting and magnetic resonance imaging enhancement into a single nanoprobe. This not only improves the imaging detection capability of early liver fibrosis, but also assists in judging the degree of disease based on changes in magnetic resonance signals at different stages of fibrosis. It provides a new technical means for the early identification, non-invasive diagnosis, accurate grading, and dynamic monitoring of liver fibrosis. It overcomes the problems of existing magnetic resonance contrast agents lacking lesion specificity, insufficient contrast of liver fibrosis tissue, and limited sensitivity in early diagnosis. This invention provides a new nanoprobe platform and diagnostic strategy for precise magnetic resonance imaging of liver fibrosis.

[0025] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0027] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0028] Example 1: Preparation of chlorogenic acid carbon dots (CA-CDs) 200 mg (0.565 mmol) of chlorogenic acid was dissolved in 20 mL of deionized water and sonicated for 5 min at room temperature in the dark, followed by magnetic stirring at 500 rpm for 5 min to obtain a homogeneous chlorogenic acid solution. The chlorogenic acid solution was transferred to a polytetrafluoroethylene-lined reactor and reacted at 200 °C for 6 h. After the reaction was completed, the solution was allowed to cool naturally to room temperature, and then filtered twice through a 0.22 μm filter membrane. It was then placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in deionized water in the dark for 48 h, with the dialysate changed every 8 h for a total of 6 water changes. After dialysis, chlorogenic acid carbon dot nanoparticles (CA-CDs) were obtained.

[0029] Example 2: Preparation of manganese-coated polydopamine-coated chlorogenic acid carbon dots (CPRM) 0.3 mg of cyclic arginine-glycine-aspartic peptide (cRGD) and 3 mg of maleimide polyethylene glycol dopamine (Mal-PEG-Do) were dissolved in 1 mL of pure water and magnetically stirred at 500 rpm for 30 min under ice bath conditions in the dark. A mixed solution of cRGD and Mal-PEG-Do and 3 mg of dopamine hydrochloride (DA) were dissolved in 4 mL of Tris-HCl solution at pH 8.3. This solution was then added to a solution containing 24 mg of CA-CDs. The mixture was magnetically stirred at 500 rpm for 6 h in the dark. After stirring, the mixture was ultrafiltered and washed three times with water. To prevent further polymerization of dopamine, ultrafiltration purification was performed using a centrifugal ultrafiltration tube with a molecular weight cutoff of 5 kDa under light-protected conditions and at 4°C. The centrifugal force was 3800 g, and the single ultrafiltration time was 10 min. After each ultrafiltration concentration, deionized water was added to the original volume before the next ultrafiltration (i.e., perfiltration and washing) was performed. A total of three washings were performed to fully remove unreacted dopamine hydrochloride and free oligomers, resulting in a polydopamine-coated carbon dot nanocomposite, namely CA-CDs@PDA-RGD, abbreviated as CPR.

[0030] Using deionized water as the dispersion medium, the concentration of the carbon dot nanocomposite was diluted or adjusted according to its actual content to prepare a CA-CDs@PDA-RGD dispersion with a concentration of 0.5 mg / mL. 4 mL of manganese chloride tetrahydrate solution (0.5 mg / mL) was added to 8 mL of the CA-CDs@PDA-RGD dispersion (0.5 mg / mL). After stirring at 500 rpm for 12 hours in the dark at room temperature, the mixture was collected by centrifugation at 14000 rpm for 10 min. The mixture was washed twice with deionized water, and unbound excess Mn was removed by centrifugation. 2+ Ultimately, we obtain CA-CDs@PDA-RGD-Mn, abbreviated as CPRM.

[0031] Example 3: Performance Analysis of CA-CDs@PDA-RGD-Mn (CPRM) 3.1 CPRM Shape Detection Take an appropriate amount of CPRM solution, prepare a concentration of 1 mg / mL, and use a pipette to transfer 20 µL onto a copper grid. Dry the sample to prepare a transmission electron microscope (TEM) sample, and observe it using a TEM. Figure 1 As shown, the results indicate that the prepared CPRM exhibits a uniform spherical shape under low magnification.

[0032] 3.2 Ultraviolet-Visible Absorption Spectrum Appropriate amounts of CA-CDs and CA-CDs@PDA-RGD were prepared into sample solutions of the same concentration using deionized water as the dispersion medium. Their UV-Vis absorption spectra were measured under the same test conditions using a UV-Vis spectrophotometer. The results are as follows: Figure 2 As shown, compared with CA-CDs, the UV-Vis absorption spectrum of CA-CDs@PDA-RGD changed significantly and exhibited polydopamine-related characteristic absorption, indicating that a polydopamine coating layer was successfully formed on the surface of CA-CDs.

[0033] 3.3 Zeta potential detection Take appropriate amounts of CA-CDs, CP, CPR and CPRM solutions, prepare them to a concentration of 20 μg / mL, sonicate the probe for 2 min, and then use a Zeta potentiometer to measure the surface potential of CA-CDs, CP, CPR and CPRM respectively.

[0034] The results are as follows Figure 3 As shown, the surface potential of nanoparticles changes regularly at different modification stages. CPRM maintains a good negative potential, indicating that polydopamine coating, RGD modification, and manganese ion coordination have all been successfully completed. At the same time, the obtained nanoprobe has good colloidal stability.

[0035] 3.4 Molecular docking analysis Molecular docking analysis of cRGD and integrin αvβ3 was performed using AutoDock software. Figure 4 As shown in AB, cRGD can stably bind to the αvβ3 receptor binding site and form multiple hydrogen bonds and hydrophobic interactions, indicating that the CPRM constructed in this invention has good theoretical targeting binding ability and can achieve active targeting of activated hepatic stellate cells.

[0036] 3.5 Gel Magnetic Resonance Scan Images and Pseudocolor Different concentrations of CPRM were added to agarose gels, and T1-weighted imaging was performed using a 3.0 T magnetic resonance scanner. Figure 5As shown, with the increase of Mn concentration, the T1-weighted magnetic resonance signal of CPRM gradually increases, and the pseudo-color image shows that the color gradually changes from blue to red.

[0037] 3.6 Functional Testing The longitudinal relaxation time (T1) of CPRM gels at different concentrations was measured, and the longitudinal relaxation rate (r1) was calculated. Figure 6 As shown, 1 / T1 has a good linear relationship with Mn concentration, indicating that CPRM has high longitudinal relaxation efficiency and can be used as a contrast agent for T1-weighted MRI.

[0038] 3.7 Constructing a rat model of liver fibrosis for imaging detection Six-week-old male SD rats were intraperitoneally injected with 20% carbon tetrachloride (carbon tetrachloride: olive oil ratio 1:4, 6 mL / kg / injection, twice a week, for 0 / 2 / 4 / 8 weeks) to establish rat models of liver fibrosis at different stages, including healthy group, ultra-early stage, early stage, and late stage. T1-weighted imaging was performed using a 3.0 T magnetic resonance imaging scanner before injection and at 0.5 h, 1 h, and 1.5 h after injection.

[0039] The results are as follows Figure 7 As shown, the liver MRI signal of all groups of animals was significantly enhanced after injection of CPRM. The signal enhancement in the liver fibrosis group was significantly higher than that in the healthy group, and the enhancement effect was further improved with the increase of fibrosis.

[0040] This indicates that the CPRM prepared in this invention can effectively accumulate in liver fibrosis lesions and significantly improve MRI imaging contrast, enabling non-invasive detection and auxiliary grading of liver fibrosis at different stages.

[0041] 3.8 Cell uptake detection HSC cells activated by TGF-β (aHSCs) were seeded in confocal microplates at a density of 2 × 10-1 per plate. 5 Cells were cultured and adhered to the culture medium for 24 h. Then, they were incubated with PBS, 25 μg / mL Cy5.5-labeled CPRM, and 50 μg / mL Cy5.5-labeled CPRM for 4–6 h, respectively. Subsequently, the CPRM-containing medium was removed, and the cells were washed twice with PBS. The nuclei were then stained with a 1 / 6000 dilution of the Hoechst 33342 fluorescent probe. After 20 min, the cells were washed 2–3 times with PBS to remove excess dye. Finally, the uptake of nanomaterials by the cells was observed using an inverted fluorescence microscope or a confocal microscope.

[0042] The results are as follows Figure 8As shown, aHSC cells treated with Cy5.5-labeled CPRM exhibited a certain red fluorescence signal, and the intracellular fluorescence signal further increased with increasing CPRM concentration from 25 μg / mL to 50 μg / mL, showing a concentration-dependent effect. These results indicate that CPRM can be effectively taken up by activated hepatic stellate cells in a concentration-dependent manner.

[0043] 3.9 Hemolysis Detection CPRM was added to red blood cell suspensions to prepare final concentrations of 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg / mL. Red blood cell suspensions diluted with PBS served as negative controls (-), and red blood cell suspensions diluted with ultrapure water served as positive controls (+). The hemolysis rate was calculated. Each group of solutions was incubated at 37°C for 4 h, then centrifuged at 3000 rpm for 15 min. The hemolysis phenomenon was photographed, and the absorbance of the samples at 542 nm was measured using a microplate reader. Finally, the hemolysis rate was calculated using the formula: Hemolysis rate (%) = [(Sample A - Negative Control A) / (Positive Control A - Negative Control A)] * 100%.

[0044] The results are as follows Figure 9 As shown, the supernatant after co-incubation of different concentrations of CPRM with red blood cells showed no significant changes compared to the negative control group, and the hemolysis rate was less than 5%. This indicates that the prepared CPRM has good blood compatibility.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A chlorogenic acid carbon dot magnetic resonance nanoprobe, characterized in that, The chlorogenic acid carbon dot magnetic resonance nanoprobe is prepared by hydrothermal reaction of chlorogenic acid as carbon source to obtain CA-CDs, and then loading manganese ions onto the carbon dot magnetic resonance nanoprobe after a light-shielded reaction with dopamine hydrochloride and cyclic arginine-glycine-aspartic acid peptide in Tris-HCl buffer.

2. The chlorogenic acid carbon dot magnetic resonance nanoprobe according to claim 1, characterized in that, The chlorogenic acid carbon dot magnetic resonance nanoprobe targets activated hepatic stellate cells in liver fibrosis tissue.

3. The method for preparing a chlorogenic acid carbon dot magnetic resonance nanoprobe as described in claim 1, characterized in that, Specifically, the following steps are included: S1. Chlorogenic acid solution was prepared using chlorogenic acid as raw material. After hydrothermal synthesis reaction, the chlorogenic acid solution was filtered and purified by light-protected dialysis to obtain CA-CDs nano-formulation. S2. A mixed solution of cyclic arginine-glycine-aspartic peptide and maleimide polyethylene glycol dopamine and dopamine hydrochloride were dissolved in Tris-HCl solution at pH 8.

3. After adding CA-CDs nano-formulation and stirring the reaction, the mixture was ultrafiltered and washed with deionized water to obtain carbon dot nanocomposite. S3. The carbon dot nanocomposite was prepared into a dispersion with a concentration of 0.5 mg / mL and mixed with manganese chloride tetrahydrate solution. After stirring and reacting, centrifugation and washing, chlorogenic acid carbon dot magnetic resonance nanoprobes were obtained.

4. The preparation method according to claim 3, characterized in that, The process of preparing chlorogenic acid solution in step S1 is as follows: chlorogenic acid is dissolved in deionized water, sonicated for 5 min at room temperature in the dark, and then magnetically stirred for 5 min at 500 rpm; the concentration of the chlorogenic acid solution is 10 mg / mL.

5. The preparation method according to claim 3, characterized in that, The hydrothermal synthesis reaction described in step S1 is carried out at 200°C for 6 hours.

6. The preparation method according to claim 3, characterized in that, The weight ratio of the cyclic arginine-glycine-aspartic peptide, maleimide polyethylene glycol dopamine, dopamine hydrochloride and CA-CDs nano-formulation in step S2 is 1:10:10:

80.

7. The preparation method according to claim 3, characterized in that, In step S3, the mixing volume ratio of the dispersion to the manganese chloride tetrahydrate solution is 2:1; the concentration of the manganese chloride tetrahydrate solution is 0.5 mg / mL.

8. The preparation method according to claim 3, characterized in that, The stirring reaction in step S3 is magnetic stirring at 500 rpm for 12 h; the centrifugation speed is 14000 rpm for 10 min; the washing is done with deionized water, and the washing is performed twice.

9. The application of the chlorogenic acid carbon dot magnetic resonance nanoprobe according to claim 1 or the chlorogenic acid carbon dot magnetic resonance nanoprobe prepared by any one of claims 3-8 in the preparation of contrast agents for magnetic resonance imaging diagnosis of liver fibrosis.

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