Gadolinium-doped carbon dot complex based on liver cancer cell membrane coating and preparation method and application of gadolinium-doped carbon dot complex

Abstract

The invention relates to a gadolinium-doped carbon dot complex based on liver cancer cell membrane coating and a preparation method of the gadolinium-doped carbon dot complex. The gadolinium-doped carbon dot complex is prepared on the basis of gadolinium-doped carbon dots Gd-CDs which are prepared from indocyanine green, citric acid, polyethylene glycol and gadolinic acid through a one-step microwave thermal method and have fluorescence-magnetic resonance bimodal imaging capacity. The Gd-CDs complex Gd-CDs coated HCM is placed in a cell membrane suspension extracted from human hepatoma carcinoma cells HepG2, and the Gd-CDs complex Gd-CDs coated HCM which is uniform in particle size and has the capacity of photothermal and immunotherapy of hepatocellular carcinoma is obtained through extrusion construction of a liposome extruder. The Gd-CDs (at) HCM complex constructed by the invention not only has good bimodal imaging ability, but also has a specific inhibition effect on liver tumor cells, can be applied to preparation of drugs for inhibiting the growth of the liver tumor cells, and realizes a good treatment effect on liver cancer at a nanometer level.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and biomedicine, and relates to carbon dots with dual-modal imaging functions, a composite constructed by coating hepatocellular carcinoma cell membranes with the carbon dots, and the application of the composite in the treatment and imaging monitoring of hepatocellular carcinoma. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common pathological subtype of primary liver cancer, and its incidence rate has been increasing year by year.

[0003] Currently, the monitoring of hepatocellular carcinoma in clinical practice mainly relies on traditional examination methods such as ultrasound, CT or MRI. These methods have low sensitivity to small lesions, making it difficult to detect early lesions and intervene at an early stage. At the same time, although traditional treatment methods such as surgery, radiofrequency ablation, transcatheter arterial embolization, and anti-angiogenic drugs can benefit patients with early hepatocellular carcinoma, due to the fast growth rate of hepatocellular carcinoma and the lack of typical early symptoms, most patients are often in the advanced stage at the first diagnosis, and their physical condition is poor and they are difficult to receive radical surgery or liver transplantation.

[0004] Therefore, in view of the problems such as the high proportion of advanced hepatocellular carcinoma in clinical practice, easy distant metastasis and recurrence, it is crucial to explore a tumor diagnosis and treatment technology that integrates the diagnosis and treatment of hepatocellular carcinoma, which can detect and diagnose diseases in real time and give timely treatment, thereby improving the therapeutic effect of hepatocellular carcinoma treatment.

[0005] Photothermal therapy (PTT) is a treatment method that activates a photothermal agent through light irradiation, converts light energy into heat energy, induces cytotoxicity mediated by high heat, and causes apoptosis of cells, thereby ablating local tumor tissues. Studies have found that photothermal therapy mediated by nanomaterials can induce the occurrence of immunogenic cell death (ICD) inside tumor cells, improve the immunogenicity of the tumor microenvironment, and largely solve the problem of low response rate of single therapies.

[0006] Although PTT provides a new direction for precise tumor treatment with its spatiotemporally controllable local thermal ablation ability, its clinical application still faces three major challenges: it is difficult to achieve both high photothermal conversion efficiency (PCE) and biodegradability for traditional photothermal agents (such as noble metal nanomaterials); single PTT cannot eliminate residual microtumors and inhibit distant metastasis; the immunogenic clearance of nanoparticles and low tumor targeting efficiency result in limited delivery efficacy.

[0007] Carbon dots (CDs) have been widely used in the research of bioimaging, fluorescence sensing, photocatalysis, etc. due to their good fluorescence properties, excellent biocompatibility, low toxicity, high stability, easy surface functionalization and low cost, and have also become ideal candidates for a new generation of photothermal agents. Furthermore, by introducing paramagnetic metal ions such as gadolinium (Gd 3+ ), the photothermal conversion and magnetic resonance imaging (MRI) functions of CDs can be achieved simultaneously. However, on the one hand, Gd-CDs prepared by traditional synthesis methods are often rapidly cleared by the reticuloendothelial system (RES) due to non-specific adsorption of surface charges and lack active targeting ability. On the other hand, Gd-CDs synthesized using conventional GdCl3∙6H2O as the gadolinium source do not have good photothermal properties.

[0008] Bionic nanotechnology provides an innovative path for the optimization of nanocarriers by simulating the structure and function of natural biological membranes. For example, the tumor cell membrane coating strategy can significantly improve the enrichment efficiency of nanoparticles at the tumor site by utilizing the homologous targeting property (i.e., the tumor homing effect mediated by homologous cell membrane surface adhesion molecules). However, existing bionic systems mostly focus on single therapeutic functions (such as chemotherapy or photothermal therapy), and the research on integrating imaging detection, local ablation and immune activation into one is still relatively scarce.

[0009] Therefore, to break through the current clinical problems such as poor targeting of anti-hepatocellular carcinoma drugs, low level of imaging monitoring, and poor treatment effect, and to improve the application of nanomaterials in biomedicine, it is of great significance to construct a reasonable carbon dot complex and combine dual-modal imaging with photothermal and immunotherapy for realizing the integrated diagnosis and treatment of targeted therapy and monitoring of hepatocellular carcinoma. Summary of the Invention

[0010] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a gadolinium-doped carbon dot complex based on hepatocellular carcinoma cell membrane coating and its preparation method. The carbon dot complex not only has dual-modal imaging function, but also has good photothermal and immunotherapeutic ability for hepatocellular carcinoma.

[0011] The gadolinium-doped carbon dot complex based on hepatocellular carcinoma cell membrane coating described in the present invention is based on gadolinium-doped carbon dots Gd-CDs prepared by a one-step microwave thermal method using indocyanine green, citric acid, polyethylene glycol and gadopentetic acid, and is placed in a cell membrane suspension extracted from human hepatocellular carcinoma cells HepG2, and a Gd-CDs@HCM complex with uniform particle size wrapped by HepG2 cell membrane is constructed by extrusion using a liposome extruder.

[0012] In the gadolinium-doped carbon dot complex coated with hepatocellular carcinoma cell membrane constructed by the present invention, preferably, the mass ratio of HepG2 cell membrane to gadolinium-doped carbon dots Gd-CDs is (0.25-1):1, so that more HepG2 cell membranes can be coated on Gd-CDs.

[0013] The particle size of the Gd-CDs@HCM complex constructed by the present invention is between 20 and 25 nm, and it has good cell permeability.

[0014] The emission wavelength of the Gd-CDs@HCM complex constructed by the present invention is 706 nm, which can achieve near-infrared emission. At the same time, it also has a high relaxation rate, which can reach 8.57 mM -1 s -1 。

[0015] Furthermore, the present invention also provides a preparation method of the gadolinium-doped carbon dot complex coated with hepatocellular carcinoma cell membrane, which specifically includes: Using indocyanine green, citric acid, polyethylene glycol and gadopentetic acid as raw materials, dissolving them in water and carrying out microwave heating reaction to prepare gadolinium-doped carbon dots Gd-CDs; Using cultured human hepatocellular carcinoma cells HepG2, after hypotonic extraction and fragmentation, HepG2 cell membrane suspension (HepG2 cell membrane, HCM) is obtained by low-temperature differential centrifugation; Placing Gd-CDs in the obtained HepG2 cell membrane suspension and repeatedly extruding with a liposome extruder to construct a uniform particle size HepG2 cell membrane-wrapped Gd-CDs complex Gd-CDs@HCM.

[0016] Further, in the above preparation method, the microwave heating reaction is preferably carried out at a microwave power of 700-900 W for 3-8 min.

[0017] Furthermore, the present invention preferably dissolves indocyanine green, citric acid, polyethylene glycol and gadopentetic acid in water, first performs ultrasonic treatment for 30-40 min, and then carries out microwave heating reaction.

[0018] Furthermore, the present invention prepares gadolinium-doped carbon dot Gd-CDs powder by filtering, dialyzing and freeze-drying the reaction product obtained by the microwave heating reaction.

[0019] Specifically, preferably, a disposable filter membrane with a pore size of 0.22 μm is used to filter the reaction product.

[0020] Specifically, preferably, the reaction product is dialyzed using a dialysis bag with a molecular weight cut-off of 500 Da.

[0021] Furthermore, the extraction of HepG2 cell membrane in the present invention, including hypotonic extraction, disruption, and low-temperature differential centrifugation extraction, is performed according to conventional cell membrane extraction methods.

[0022] The hypotonic extraction and disruption process specifically involves adding a membrane protein extraction reagent containing PMSF to HepG2 cells resuspended in PBS, fully suspending the cells, and then repeatedly homogenizing and disrupting them to obtain a disrupted cell suspension.

[0023] The low-temperature differential centrifugation extraction process specifically comprises collecting the final precipitate by differential centrifugation of the broken cell suspension at a low temperature of 4°C, adding physiological saline and vortexing to obtain the HepG2 cell membrane suspension.

[0024] Furthermore, the repeated extrusion using a liposome extruder in the present invention is specifically to use liposome extruders equipped with polycarbonate (PC) membranes of different pore sizes to perform repeated extrusion in order from large to small pore sizes, so that the HepG2 cell membrane is coated on the Gd-CDs.

[0025] More specifically, the polycarbonate membranes with different pore sizes include polycarbonate membranes with pore sizes of 400 nm, 100 nm and 50 nm, respectively.

[0026] Furthermore, the polycarbonate membranes with each pore size were repeatedly extruded 20 to 30 times.

[0027] The Gd-CDs@HCM complex constructed based on the present invention has both long-wavelength emission fluorescence imaging effect and magnetic resonance imaging capability, so it can first be used as a fluorescence-magnetic resonance dual-modality imaging probe.

[0028] Furthermore, the smaller particle size Gd-CDs@HCM complex constructed based on the present invention has good cell permeability. It can not only penetrate living tissues for living fluorescence imaging and magnetic resonance imaging, but also enter cells for dual-modal imaging of cells. Therefore, it can be further used as a fluorescence-magnetic resonance dual-modal imaging probe for cells.

[0029] The Gd-CDs@HCM complex constructed by the present invention can be precisely targeted to the liver tumor area through the HepG2 cell membrane coated thereon, and can effectively activate the anti-tumor immunity in the tumor microenvironment, thereby enhancing the adhesion of the complex to the tumor and having an excellent immunotherapy effect.

[0030] At the same time, the Gd-CDs@HCM complex also has good photothermal properties and excellent photothermal therapeutic effects. After targeting the liver tumor area, laser irradiation produces a photothermal effect to induce tumor necrosis, which then leads to the release of whole-cell tumor antigens. These tumor antigens can activate CD8 +Cytotoxic T cells (CTL) and reduce Foxp3 + Regulatory T cells, further enhance the anti-tumor immunotherapy against primary and metastatic tumors. While inducing apoptosis, it can also promote the release of damage-associated molecule patterns (DAMPs) such as calreticulin (CRT), high mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP), and promote antigen presentation to dendritic cells (DC) to activate CTL, thereby inducing immunogenic cell death (ICD). ICD can transform dying or dead cancer cells into immune "vaccines" and enhance the ability of anti-tumor immunity.

[0031] Therefore, in addition to having good dual-modal imaging ability, the Gd-CDs@HCM complex constructed in the present invention also has a specific inhibitory effect on liver tumor cells, can be used to prepare anti-liver tumor nano-drugs, and achieve good therapeutic effects against liver cancer at the nano-level.

[0032] Proven by in vitro cell experiments, the Gd-CDs@HCM complex constructed in the present invention shows excellent inhibitory ability against liver tumor cells. Description of the Drawings

[0033] Figure 1 Are the TEM image (a) and HRTEM image (b) of Gd-CDs. The insets are the particle size statistical chart and the lattice diagram at the magnification respectively.

[0034] Figure 2 Is the FTIR spectrum of Gd-CDs.

[0035] Figure 3 Are the ultraviolet absorption spectrum (a) and fluorescence emission spectrum (b) of Gd-CDs.

[0036] Figure 4 Is the XPS full spectrum and high-resolution spectra of each element of Gd-CDs.

[0037] Figure 5 Are the Zeta potentials of HCM, Gd-CDs, and Gd-CDs@HCM.

[0038] Figure 6 Is the TEM image of Gd-CDs@HCM.

[0039] Figure 7 SDS-PAGE gel electrophoresis images of HCM, Gd-CDs, and Gd-CDs@HCM.

[0040] Figure 8 Images related to the photothermal performance of Gd-CDs and Gd-CDs@HCM.

[0041] Figure 9 T1-weighted images of Gd-CDs@HCM and gadodiamide (a) and the linear relationship diagram of transverse relaxation rate (1 / T1) versus different concentrations of Gd 3+ (b).

[0042] Figure 10 Hemolysis experiments (a) and histograms of hemolysis rates (b) of Gd-CDs@HCM solutions with different concentrations.

[0043] Figure 11 Histograms of cell viability of Gd-CDs solutions with different concentrations.

[0044] Figure 12 Pathological section images of nude mouse organs after treatment with normal saline (a), Gd-CDs solution (b), and Gd-CDs@HCM solution (c) for different times.

[0045] Figure 13 Weight-time change curves of nude mice treated with normal saline, Gd-CDs solution, and Gd-CDs@HCM solution.

[0046] Figure 14 Blood analysis of nude mice after treatment with normal saline, Gd-CDs solution, and Gd-CDs@HCM solution for different times.

[0047] Figure 15 Laser confocal microscopy images of Gd-CDs and Gd-CDs@HCM in HepG2 cells.

[0048] Figure 16 MRI images of Gd-CDs@HCM in HepG2 cells.

[0049] Figure 17 Time-dependent imaging diagrams (a) in tumor-bearing mice and in vitro fluorescence images (b) of nude mouse organs at different times after treatment with Gd-CDs@HCM, as well as in vivo MRI images (c) of tumor-bearing mice after intravenous injection of Gd-CDs@HCM.

[0050] Figure 18 Histograms of cell viability after co-incubation with THLE-2 and HepG2 cells for 24 h under different conditions.

[0051] Figure 19It is the effect of Gd-CDs@HCM on the migration of HepG2 cells.

[0052] Figure 20 It is the effect of Gd-CDs@HCM on the invasion of HepG2 cells.

[0053] Figure 21 It is the effect of Gd-CDs@HCM on the survival of HepG2 cells.

[0054] Figure 22 It is the effect of Gd-CDs@HCM on the immunogenic cell death of HepG2 cells. Embodiment

[0055] The following further describes in detail the specific embodiments of the present invention in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can well understand and utilize the present invention, rather than limiting the protection scope of the present invention.

[0056] In the embodiments of the present invention, the production processes, experimental methods or detection methods involved, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art, and are also very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the name and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0057] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0058] The gadolinium-doped carbon dot complex coated with liver cancer cell membrane described in the following embodiments of the present invention is specifically prepared by the following method:

[0059] 1) Add indocyanine green, citric acid, polyethylene glycol and gadopentetic acid to deionized water in sequence, then ultrasonicate it for 30 - 40 min, and then put it into a microwave oven with 700 - 900 W to heat and react for 3 - 8 min. After filtering, dialyzing and freeze-drying the reaction product, Gd-CDs powder is obtained.

[0060] In specific embodiments, the filtration is preferably carried out using a disposable filter membrane with a pore size of 0.22 μm.

[0061] In specific embodiments, dialysis is preferably carried out using a dialysis bag with a molecular weight cut-off of 500 Da.

[0062] 2) Take HepG2 cells, resuspend the cells with ice-cold PBS, wash 2 - 3 times, add a membrane protein extraction reagent supplemented with PMSF, fully suspend the cells and place them on ice for 10 - 15 min, transfer them to a homogenizer pre-cooled on ice and homogenize about 30 - 50 times, collect the precipitate from the broken cell suspension by differential centrifugation at 4 °C, add normal saline and vortex to obtain a HepG2 cell membrane suspension.

[0063] 3) According to the mass ratio of HepG2 cell membrane to Gd-CDs being (0.25 - 1):1, mix the Gd-CDs powder with the HepG2 cell membrane suspension, and successively use a liposome extruder equipped with polycarbonate membranes with pore sizes of 400 nm, 100 nm, and 50 nm to extrude repeatedly 20 - 30 times to construct the Gd-CDs@HCM complex.

[0064] In a specific embodiment, the liposome extruder is preferably an Avanti mini liposome extruder.

[0065] Unless otherwise specified, for the dosage of raw material components, temperature, time and other measurement parameters involved in the embodiments of the present invention, there may be slight deviations within the weighing or measurement accuracy range, and acceptable deviations caused by the instrument test accuracy or operation accuracy are allowed. Example

[0066] Example 1

[0067] Weigh 5 mg of indocyanine green (ICG), 0.2 g of citric acid (CA), 50 mg of polyethylene glycol (PEG), and 30 mg of gadopentetic acid (Gd-DTPA), successively add them to 15 mL of deionized water, ultrasonically treat for 40 min to mix evenly, and place them in a microwave oven with a power of 700 W to heat and react for 3 min to obtain a dark green transparent reaction solution.

[0068] First filter the reaction solution with a disposable filter membrane with a pore size of 0.22 μm, then dialyze the filtrate using a dialysis bag with a molecular weight cut-off of 500 Da, and the dialysis product is freeze-dried to obtain green Gd-CDs powder.

[0069] Take human hepatoma cells HepG2 and add them to a MEM cell culture system containing 10 wt% FBS, and culture them under the conditions of 37 °C and 5% CO2.

[0070] Take about 2×10 7 HepG2 cells in the logarithmic growth phase. After washing with PBS, scrape the cells and blow and beat the cells with a pipette to suspend them, centrifuge to remove the supernatant, and collect the cell precipitate.

[0071] Gently resuspend the cell pellet with an appropriate amount of ice-cold PBS, wash the cells twice, add 1 mL of membrane protein extraction reagent supplemented with 10 μL of PMSF just before use, gently pipette up and down to fully suspend the cells, and place on ice for 10 min; transfer the cell suspension to an ice-cold glass homogenizer and homogenize about 40 times.

[0072] Take the homogenized cell suspension, centrifuge at 1000 g for 10 min at 4 °C to remove unbroken cells and nuclei at the bottom; take the supernatant and centrifuge at 8000 g for 10 min to remove mitochondria in the lower layer; carefully collect the supernatant and continue to ultracentrifuge at 20000 g for 60 min at 4 °C, collect the precipitate, add 1 mL of normal saline, and mix well using a vortex mixer to obtain the cell membrane suspension of human hepatoma cells HepG2 (HepG2 cell membrane, HCM).

[0073] Weigh 400 μg of Gd-CDs powder, add it to 1 mL of HepG2 cell membrane suspension, and use an Avanti mini-extruder to first extrude it through a 400 nm PC membrane 20 times repeatedly, and then repeat the above operation through 100 nm and 50 nm PC membranes in sequence to prepare the complex Gd-CDs@HCM encapsulated by hepatoma cell membranes.

[0074] Example 2

[0075] Weigh 10 mg of ICG, 0.3 g of CA, 70 mg of PEG, and 30 mg of Gd-DTPA, add them to 20 mL of deionized water in sequence, ultrasonically treat for 40 min to mix evenly, and place in a microwave oven with a power of 900 W to heat and react for 3 min to obtain a dark green transparent reaction solution.

[0076] First filter the reaction solution through a disposable filter membrane with a pore size of 0.22 μm, then dialyze the filtrate using a dialysis bag with a molecular weight cut-off of 500 Da, and the dialyzed product is freeze-dried to obtain green Gd-CDs powder.

[0077] Take human hepatoma cells HepG2 and add them to the MEM cell culture system containing 10 wt% FBS, and culture under the conditions of 37 °C and 5% CO2.

[0078] Take about 5×10 7 cells in the logarithmic growth phase of HepG2. After washing with PBS, scrape the cells and pipette to suspend the cells, centrifuge to aspirate the supernatant, and collect the cell pellet.

[0079] Gently resuspend the cell pellet with an appropriate amount of ice-cold PBS, wash the cells 3 times, add 1 mL of membrane protein extraction reagent supplemented with 10 μL PMSF before use, gently pipette up and down repeatedly to fully suspend the cells, and place on ice for 15 min; transfer the cell suspension to an ice-cold glass homogenizer and homogenize about 30 times.

[0080] Take the homogenized cell suspension, centrifuge at 1000 g for 10 min at 4 °C to remove unbroken cells and nuclei at the bottom; take the supernatant, and then centrifuge at 8000 g for 10 min to remove mitochondria in the lower layer; carefully collect the supernatant, continue to ultracentrifuge at 20000 g for 60 min at 4 °C, collect the precipitate, add 1 mL of normal saline, and mix well using a vortex mixer to obtain the cell membrane suspension of human hepatoma cell HepG2.

[0081] Weigh 400 μg of Gd-CDs powder, add it to 1 mL of HepG2 cell membrane suspension, and use an Avanti mini-extruder to first extrude it through a 400 nm PC membrane repeatedly 30 times, and then repeat the above operation by passing through 100 nm and 50 nm PC membranes in sequence to prepare the complex Gd-CDs@HCM with Gd-CDs encapsulated by hepatoma cell membranes.

[0082] Example 3

[0083] Weigh 15 mg of ICG, 0.3 g of CA, 70 mg of PEG, and 45 mg of Gd-DTPA, add them to 20 mL of deionized water in sequence, ultrasonically treat for 40 min to mix evenly, and place in a microwave oven with a power of 800 W to heat and react for 3 min to obtain a dark green transparent reaction solution.

[0084] First filter the reaction solution through a disposable filter membrane with a pore size of 0.22 μm, then dialyze the filtrate using a dialysis bag with a molecular weight cut-off of 500 Da, and the dialysis product is freeze-dried to obtain green Gd-CDs powder.

[0085] Take human hepatoma cell HepG2 and add it to the MEM cell culture system containing 10 wt% FBS, and culture it under the conditions of 37 °C and 5% CO2.

[0086] Take about 2×10 7 HepG2 cells in the logarithmic growth phase. After washing with PBS, scrape the cells and pipette to suspend the cells, centrifuge to remove the supernatant, and collect the cell pellet.

[0087] Gently resuspend the cell pellet with an appropriate amount of ice-cold PBS, wash the cells 2 times, add 1 mL of membrane protein extraction reagent supplemented with 10 μL PMSF before use, gently pipette up and down repeatedly to fully suspend the cells, and place on ice for 15 min; transfer the cell suspension to an ice-cold glass homogenizer and homogenize about 50 times.

[0088] Take the broken cell suspension, centrifuge at 1000 g for 10 min at 4 °C to remove the unbroken cells and cell nuclei at the bottom; take the supernatant, and then centrifuge at 8000 g for 10 min to remove the lower-layer mitochondria; carefully collect the supernatant, continue to ultracentrifuge at 20000 g for 60 min at 4 °C, collect the precipitate, add 1 mL of physiological saline, and mix evenly using a vortex mixer to obtain the cell membrane suspension of human hepatocellular carcinoma cell line HepG2.

[0089] Weigh 800 μg of Gd-CDs powder, add it to 1 mL of HepG2 cell membrane suspension, and use an Avanti mini extruder to first extrude it through a 400 nm PC membrane repeatedly for 30 times, and then sequentially extrude it through 100 nm and 50 nm PC membranes and repeat the above operation to prepare the complex Gd-CDs@HCM with Gd-CDs encapsulated by hepatocellular carcinoma cell membranes.

[0090] Example 4

[0091] Take the Gd-CDs powder prepared in the above example, disperse it in absolute ethanol, drop it onto a special copper grid, and after volatilization and drying, use a transmission electron microscope (TEM) to observe the morphology, size and microstructure of Gd-CDs, as Figure 1 shown in a, the morphology of Gd-CDs is spherical-like, with good dispersion and no obvious aggregation phenomenon; it can be seen from the particle size statistical chart in the small window of the figure that its particle diameter is between 9.0 and 16.0 nm, and the average particle size is 12.24 ± 1.26 nm. Further through Figure 1 observation with the high-resolution transmission electron microscope (HRTEM) in b, its lattice spacing is 0.28 nm, corresponding to the graphite (100) crystal plane.

[0092] Using Figure 2 the Fourier transform infrared spectroscopy (FTIR) spectrum can clearly analyze the relationship between the characteristic group peaks in the prepared Gd-CDs and each raw material.

[0093] In the FTIR spectrum of ICG, the broad peak at 3412 cm -1 corresponds to the O-H stretching vibration absorption peak, while the peaks at 2935 cm -1 , 1417 - 1606 cm -1 and 1295 cm -1 belong to the stretching vibrations of C-H, C=C / C=N and C-N respectively; the bands at 1094 cm -1 and 667 cm -1 describe the stretching vibrations of the -SO3 - group and C-S. Compared with ICG, the FTIR spectrum of Gd-CDs at 1094 cm -1and 667 cm -1 showed the same characteristic peaks at, indicating that they might inherit the same -SO3 - functional group.

[0094] In the FTIR spectrum of CA, the broad peak at 3412 cm -1 corresponded to the O-H stretching vibration absorption peak; the peak at 1700 cm -1 corresponded to the C=O stretching vibration, but this peak did not appear significantly in Gd-CDs, indicating that C=O might coordinate with Gd 3+ to form a coordination.

[0095] In the FTIR spectrum of PEG, the peak at 1492 cm -1 belonged to the C-O-C stretching vibration and was clearly retained in the spectrum of Gd-CDs, indicating that PEG, as a stabilizer, was successfully modified on the surface of the nanoparticles.

[0096] In the FTIR spectrum of Gd-DTPA, since there are five -COOH groups in the DTPA molecule, it usually shows C=O stretching vibration (1700 cm -1 ) in the infrared spectrum, but a strong absorption peak appeared at 1552 cm -1 , which might be due to the coordination of Gd 3+ with DTPA, slightly changing the position of this peak; in the spectrum of Gd-CDs, there was also the same peak at this position.

[0097] Therefore, the above Figure 2 FTIR mapping results proved the successful preparation of Gd-CDs nanoparticles.

[0098] Gd-CDs powder was dispersed in PBS, and its ultraviolet-visible absorption spectrum (UV-Vis) and fluorescence emission spectrum were tested, and the results were as Figure 3 shown. Among them, Figure 3 a showed that Gd-CDs had absorption at 782 nm, and the absorption peak belonged to the typical absorption band of the aromatic structure, indicating the existence of a large conjugated structure in the structure. And Figure 3 b showed that the fluorescence spectrum of the Gd-CDs solution at different excitation wavelengths proved that the Gd-CDs solution had the property of excitation dependence. When the excitation wavelength was 660 nm, the emission wavelength was 706 nm, enabling near-infrared emission, which laid the foundation for its research on photothermal therapy in vivo.

[0099] Furthermore, X-ray photoelectron spectroscopy (XPS) was also used to analyze the elemental composition of Gd-CDs.

[0100] As Figure 4As shown in a), the binding energies of C1s, N1s, O1s, and Gd4d exist in the full spectrum of Gd-CDs, indicating that it contains elements C, N, O, and Gd. Among them Figure 4 b is the high-resolution spectrum of C1s. The peaks at 285.58 and 283.90 eV correspond to C-O / C=C and C-C, respectively; from Figure 4 The high-resolution spectrum of N1s in c shows that there are two types of chemical states of N, located at 398.75 and 400.23 eV, corresponding to pyridine N and pyrrole N, respectively; from the high-resolution spectrum of O1s of Gd-CDs ( Figure 4 d), it can be seen that the peaks at 530.10 and 531.58 eV correspond to -C-OH and C=O, respectively; Figure 4 e is the high-resolution spectrum of Gd4d, showing two peaks centered at 163.40 and 158.12 eV, which are similar to the spectral patterns of Gd(2p 5 / 2 ) and Gd(2p 3 / 2 ) in Gd-DTPA.

[0101] Example 5

[0102] Gd-CDs, HCM, and Gd-CDs@HCM were respectively dispersed in aqueous solution, and the surface Zeta potential of different particles was measured by laser Doppler microelectrophoresis.

[0103] The test results are as Figure 5 shown. HCM carries a negative charge with a potential of -4.08 mV, Gd-CDs carry a positive charge with a potential of +7.63 mV, while Gd-CDs@HCM after coating Gd-CDs with HCM still carries a positive charge, but its potential drops to +3.57 mV, preliminarily indicating the presence of cell membranes in the complex.

[0104] Using a biological transmission electron microscope, not only the morphological information of the material can be observed, but also the microstructure of the material can be analyzed.

[0105] The prepared Gd-CDs@HCM complex was analyzed by a biological transmission electron microscope. 10 μL of the sample was pipetted onto a copper grid, and after precipitation for 1 min, the supernatant was aspirated. Then 10 μL of uranyl acetate was added and precipitated for 1 min. After aspirating the supernatant, it was dried at room temperature and imaged by an electron microscope at 80 - 120 kV. The results are as Figure 6 shown. Gd-CDs@HCM is spherical, and most of the particle sizes are distributed in the range of 20 - 25 nm, with an average particle size of about 22 nm.

[0106] The cell membrane usually has poor rigidity and generally appears vesicular under TEM. However, the Gd-CDs@HCM complex appears spherical rather than vesicular under TEM due to the supporting effect of Gd-CDs inside, which indirectly proves that the cell membrane is coated on the surface of Gd-CDs.

[0107] Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to test the protein content in the above-prepared Gd-CDs, HCM, and Gd-CDs@HCM.

[0108] The total protein concentration of each group was quantified with a BCA kit and the protein concentration was diluted to 1 mg / mL. Take 20 μL of the sample, add 5 μL of 5× protein loading buffer, mix well, incubate at 95 °C for 5 min, then load onto a 10 wt% SDS-PAGE gel, add 25 μL to each well, and add 5 μL of protein marker (5 - 180 kDa) to one end lane. Electrophorese at 100 V for 1 h, stain with Coomassie Brilliant Blue, and image with a gel imager after decolorization with decolorizing solution.

[0109] The results are as Figure 7 shown. No lane with protein stripes was run in the Gd-CDs group, while similar protein stripe lanes were run in both the Gd-CDs@HCM and the cell membrane suspension HCM, further proving the successful coating of the cell membrane on the surface of Gd-CDs.

[0110] Example 6

[0111] By evaluating the photothermal properties of Gd-CDs, the feasibility of Gd-CDs as a photosensitizer was verified.

[0112] Take 100 μL of Gd-CDs solutions with concentrations of 0.8, 0.4, 0.2, 0.1, 0.05 mg / mL and ultrapure water respectively, place them in 0.2 mL centrifuge tubes, and irradiate with an 808 nm laser at a power of 1 W / cm 2 for 5 min. An infrared thermal imager records the temperature change of the solution, and a trend curve of the temperature of different concentration solutions over time is made.

[0113] As Figure 8 shown in a, the temperature of ultrapure water under near-infrared laser irradiation increases slowly, and almost no temperature change can be observed. The temperature after 5 min is only 27.1 °C, while the Gd-CDs solution irradiated with the same power shows an obvious temperature increase effect, and the temperature increase effect also increases significantly with the increase of the solution concentration. Among them, the solution with a concentration of 0.4 mg / mL can rapidly heat up to 64.7 °C within 5 min under laser irradiation. Figure 8b is the temperature change curve of Gd-CDs solutions with different concentrations relative to ultrapure water, showing that the temperature increase amplitude of the 0.4 mg / mL solution is the largest, and the temperature increase amplitude slows down after continuing to increase the concentration.

[0114] Irradiate 100 μL of 0.4 mg / mL Gd-CDs solution with an 808 nm laser at a power of 1 W / cm 2 for 5 min, then cool it to the initial temperature, and repeat the operation 5 times. Record the cyclic temperature change with an infrared thermal imager to explore the photothermal stability of Gd-CDs at this concentration, and calculate its photothermal conversion efficiency to be 28.7% according to the following formula: where, h represents the heat conduction coefficient of the container, A represents the surface area of the container, T max is the maximum temperature of the solution, T surr is the initial temperature of the solution, Q s =(5.4×10 -4 )IJs -1 , I is the laser output power, A λ is the absorption value of the nanoplatform at 808 nm.

[0116] Furthermore, according to Figure 8 the temperature-time curve of the 5-cycle irradiation of the 0.4 mg / mL Gd-CDs solution in c, it can be seen that Gd-CDs has good photothermal stability.

[0117] Continue to place 0.4 mg / mL Gd-CDs solution, Gd-CDs@HCM solution and ultrapure water in a 0.2 mL centrifuge tube, and irradiate with an 808 nm laser at a power of 1 W / cm 2 for 5 min. Record the temperature change of the solution with an infrared thermal imager, and make the temperature-time trend curve of different solutions. As Figure 8 shown in d, the temperature increase of Gd-CDs and Gd-CDs@HCM is similar, indicating that the photothermal performance of Gd-CDs is not affected after coating with cell membranes.

[0118] Example 7

[0119] This example evaluated the relaxation rate of Gd-CDs and compared it with the relaxation rate of the commercial product gadodiamide to verify the feasibility of Gd-CDs as a new type of MRI contrast agent.

[0120] As Figure 9As shown in a, the MRI signal intensities of Gd-CDs@HCM and gadodiamide both increase with the increase in Gd 3+ concentration, indicating that the prepared Gd-CDs@HCM can be used as a new candidate material for MRI, and the image signal of Gd-CDs@HCM is higher than that of gadodiamide.

[0121] By Figure 9 plotting the linear relationship between the transverse relaxation rate (1 / T1) and the concentrations of Gd 3+ and gadodiamide in b, the relaxation rate (slope) values of Gd-CDs@HCM and gadodiamide can be calculated to be 8.57 and 2.87 mM -1 s -1 , further indicating that Gd-CDs@HCM has a high relaxation rate and can be used for MRI, providing a basis for bimodal imaging in vivo.

[0122] Example 8

[0123] In this example, UV-Vis detection was used to quantitatively analyze the hemolysis rates of Gd-CDs@HCM solutions with different concentrations to investigate their biocompatibility. The results are as Figure 10 shown. As the concentration of Gd-CDs@HCM increased from 6.25 μg / mL to 400 μg / mL, the hemolysis rates were 1.2%, 1.1%, 1.6%, 1.7%, 2.6%, 2.5%, and 3% in sequence. The measured values were all less than 5%, proving that Gd-CDs@HCM as a carrier has good blood compatibility.

[0124] In this example, the standard CCK-8 method was further used to evaluate the in vitro cytotoxicity of Gd-CDs.

[0125] Take well-grown HepG2 cells (human hepatoma cells) and THLE-2 cells (normal human liver cells). After trypsin digestion, dilute the cells to 1×10 5 / mL with complete medium and inoculate 100 μL per well into a 96-well plate. Culture the cells in a cell incubator until they adhere to the wall.

[0126] Prepare Gd-CDs solutions with different concentrations (0, 12.5, 25, 50, 100, 200 μg / mL) in advance, and co-incubate them with HepG2 and THLE-2 cells for 24 h. After the culture, wash twice with PBS, add the pre-prepared CCK-8 solution to each well, continue to incubate for 1 h, and then use a multifunctional microplate reader to measure the OD value of the solution.

[0127] Figure 11The cell viability of different concentrations of Gd-CDs co-incubated with different cells for 24 h was given. Among them, even when the concentration of Gd-CDs was 200 μg / mL, the viability of both types of cells was still greater than 80%, indicating that Gd-CDs had low cytotoxicity and good biocompatibility.

[0128] At the same time, from the above experimental results, it was also observed that the viability of THLE-2 cells even exceeded 100% under some low-concentration conditions, indicating that low-concentration Gd-CDs also had a certain effect on promoting cell proliferation.

[0129] Furthermore, 6- to 8-week-old BALB / c nude mice were selected as experimental animals to verify the biosafety of Gd-CDs and Gd-CDs@HCM.

[0130] Taking normal saline as the control group and Gd-CDs and Gd-CDs@HCM solutions as the experimental groups, with an injection dose of 10 mg / Kg for each. Then, the BALB / c nude mice were dissected after being fed for 0, 1, 14, and 28 days respectively, and the heart, liver, spleen, lungs, and kidneys were taken to make tissue sections, which were observed under a microscope to evaluate the pathological differences between the experimental groups and the control group; at the same time, the biosafety of Gd-CDs and Gd-CDs@HCM was indirectly verified by monitoring the body weights of three groups of BALB / c nude mice at different times (1, 3, 7, 14, 21, 28 days).

[0131] Figure 12 In (a) is the control group, (b) is the Gd-CDs experimental group, and (c) is the Gd-CDs@HCM experimental group. No obvious changes in the cell state and inflammatory infiltration were found in each tissue in both the control group and the experimental groups, which proved that Gd-CDs and Gd-CDs@HCM had excellent biocompatibility.

[0132] In addition to the tissue microscopic morphology, the body weight growth curve is also an important indicator for testing in vivo biosafety. As Figure 13 shown, the body weight growth trends of each group of BALB / c nude mice were similar within 28 days.

[0133] By performing serum biomarker analysis, the biosafety of Gd-CDs and Gd-CDs@HCM can be further clarified.

[0134] After deeply anesthetizing BALB / c nude mice 1, 3, 14, and 28 days after injecting normal saline, Gd-CDs, and Gd-CDs@HCM solutions respectively, blood was collected from the eyeballs, and routine blood and blood biochemical analyses were performed to evaluate the contents of white blood cells (WBC), hemoglobin concentration (HGB), and platelets (PLT); liver and kidney function parameters were monitored, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and urea nitrogen (UREA) indicators.

[0135] Figure 14 Among them, there were no significant differences in WBC, PLT, HGB, AST, ALT, CREA, and UREA of BALB / c nude mice treated with normal saline, Gd-CDs, and Gd-CDs@HCM in the short term and long term after injection; no death of BALB / c nude mice occurred during the observation period, indicating that Gd-CDs and Gd-CDs@HCM have high in vivo biosafety, laying a foundation for their use in in vivo research.

[0136] Example 9

[0137] An in vitro cell imaging experiment was carried out using HepG2 cells. The cell transfection effect of Gd-CDs@HCM was observed with a confocal laser scanning microscope (CLSM), and the bioimaging and cell labeling abilities of Gd-CDs@HCM were evaluated.

[0138] Take HepG2 cells in good growth state, disperse them into a cell suspension of 1×10 4 cells / mL with complete medium. Pipette 2 mL and transfer it to a special culture dish, culture it in an incubator for 12 h, discard the medium, wash it with PBS, add the medium containing 2% fetal bovine serum (FBS) again, culture it under starvation for 1 h, discard the medium, wash it with PBS, and add complete medium containing Gd-CDs and Gd-CDs@HCM (200 μg / mL) respectively, culture under the same conditions for 4 h, fix the cells with paraformaldehyde for 15 min, and perform CLSM imaging. The results are as Figure 15 shown.

[0139] In the figure, Bright filed shows the cell distribution under bright field; 647 nm shows the cell distribution in the fluorescence field, and it can be clearly seen that red fluorescence appears in the cells; Merge image is the image after superimposing the bright field and the fluorescence field. It can be seen from the figure that compared with Gd-CD, Gd-CDs@HCM can enter the cells more, and are mainly distributed in the cytoplasm, indicating that Gd-CDs@HCM has excellent ability to target HepG2 cells, laying a foundation for subsequent therapeutic research.

[0140] Take HepG2 cells in good growth state, culture them overnight in a 6-well plate, add Gd-CDs@HCM solutions with different concentrations (0, 0.1, 0.2, 0.4, 0.8 mg / mL) to each well and co-incubate for 4 h. Wash the cells with PBS, digest them with trypsin, centrifuge in an EP tube, re-disperse them in 1% agarose solution for gelation, and then perform MRI scanning on the cell solution with a magnetic resonance scanner.

[0141] Figure 16 Among them, after treatment with Gd-CDs@HCM, the cells showed bright images, and the brightness was positively correlated with the concentration of Gd-CDs@HCM, indicating that Gd-CDs@HCM has MRI ability, can be used as an MRI contrast agent, and has a high relaxation rate.

[0142] Inject the Gd-CDs@HCM solution into HepG2 tumor-bearing mice at an injection dose of 10 mg / Kg, observe with a small animal in vivo imager, and record the in vivo fluorescence signals at 0, 2, 4, 6, 12, and 24 h. Figure 17 As can be seen from a, Gd-CDs@HCM reached all parts of the body with the blood 4 h after injection, and the fluorescence intensity gradually increased with time, reaching the strongest at 6 h, gradually decreasing after 12 h, and basically disappearing after 24 h, proving that Gd-CDs@HCM has been basically excreted from the body. Among them, stronger fluorescence could be seen at the tumor site at 6 h, and the fluorescence intensity was the strongest at 12 h, proving that the Gd-CDs@HCM with long-wavelength emission of the present invention can penetrate living tissues for fluorescence imaging and target liver cancer tissues.

[0143] The biodistribution in vivo tissues is also a key factor for evaluating the in vivo biocompatibility of Gd-CDs@HCM. Sacrifice BALB / c nude mice at different times after injecting Gd-CDs@HCM, collect the main organs of the brain, heart, liver, spleen, lung, kidney, and bladder at each time period, and study their biodistribution through fluorescence imaging of ex vivo tissues.

[0144] As Figure 17 shown in b, fluorescence began to appear in the liver 2 h after injection, reached the strongest fluorescence intensity at 6 h, and the fluorescence gradually disappeared after 24 h, indicating that Gd-CDs@HCM can be cleared through rapid liver metabolism, further proving its good metabolism and strong biocompatibility. Fluorescence appeared in the lungs at 6 h after injection, but the accumulation rate was relatively low, indicating that Gd-CDs@HCM can penetrate the biological barrier of BALB / c nude mice and enter the lungs. Fluorescence began to appear in the kidneys after 6 h and decreased significantly or even disappeared after 24 h, indicating that Gd-CDs@HCM can be cleared through kidney metabolism, further proving its good metabolism and strong biocompatibility.

[0145] Furthermore, first, a 3.0T magnetic resonance scanner was used to scan BALB / c tumor-bearing mice, and then a Gd-CDs@HCM solution with a dose of 10 mg / Kg was injected into the tumor-bearing mice through the tail vein. The tumor-bearing mice were scanned again 6 h after the injection. From Figure 17 As can be seen from c, the subcutaneous tumor region of the tumor-bearing mice without Gd-CDs@HCM injection showed a long T1 hypointense signal. After intravenous injection of the Gd-CDs@HCM solution for 6 h, a short T1 hyperintense signal was visible in the subcutaneous tumor region of the tumor-bearing mice, indicating that Gd-CDs@HCM of the present invention can not only penetrate living tissues, but also has the property of targeting liver cancer tissues for MRI imaging.

[0146] Example 10

[0147] Six experimental groups including a control group, a Gd-CDs group, a Gd-CDs@HCM group, a Laser group, a Gd-CDs + Laser group, and a Gd-CDs@HCM+Laser group were set up to evaluate the in vitro anti-tumor activity of the Gd-CDs@HCM complex against HepG2 cells.

[0148] First, the CCK-8 method was used to observe the proliferation inhibitory effects of Gd-CDs and Gd-CDs@HCM on HepG2 and THLE-2 cells, further investigate the targeting inhibitory ability of Gd-CDs@HCM on HepG2 cells, and evaluate its in vitro anti-tumor activity.

[0149] Among them, Gd-CDs powder was added to the cells in the Gd-CDs group and the Gd-CDs + Laser group, and the complex Gd-CDs@HCM was added to the cells in the Gd-CDs@HCM group and the Gd-CDs@HCM+Laser group. After co-incubation for 4 h, the Laser, Gd-CDs + Laser, and Gd-CDs@HCM+Laser groups were irradiated with 808 nm laser (1 W / cm 2 ) for 5 min, and the control group was not treated with anything. Then, the cells were cultured for another 20 h.

[0150] Figure 18 The survival rate results of HepG2 and THLE-2 cells after incubation for 24 h in different experimental groups are given. The results show that the direct killing rates of Gd-CDs on HepG2 and THLE-2 cells are 13.3% and 4.7% respectively, and the direct killing rates of Gd-CDs@HCM on HepG2 and THLE-2 cells are 14.9% and 9.2% respectively, indicating the low toxicity of both to normal hepatocytes and liver cancer cells. Furthermore, near-infrared laser has a certain damaging effect on both THLE-2 and HepG2 cells, causing non-selective photothermal damage. Therefore, the selectivity of photothermal therapy needs to be improved.

[0151] However, when near-infrared laser is used in combination with Gd-CDs or Gd-CDs@HCM, a significant selective killing effect is demonstrated.

[0152] It may be due to the fact that the rapid proliferation of cancer cells leads to weakened DNA repair ability and unstable mitochondrial membrane potential, making them more sensitive to thermal damage, while normal cells have stronger thermal tolerance. Therefore, when near-infrared laser is used in combination with Gd-CDs, it has a stronger killing effect on HepG2 cells. Furthermore, the combination of Gd-CDs@HCM and near-infrared laser further increases the apoptosis rate of HepG2 cells to 98.6%, while the survival rate of THLE-2 cells remains above 70%. This difference indicates that through the coating of the cell membrane, Gd-CDs achieve active targeting of HepG2 cells and precise photothermal and immune synergistic therapy under near-infrared laser irradiation.

[0153] Secondly, the migration of HepG2 cells in vitro was studied by scratch assay.

[0154] HepG2 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 2×10 5 cells / well, ensuring that the seeding density of each group of cells was consistent and evenly distributed, and cultured at 37°C, 5% CO2, and relative humidity of 95%.

[0155] After the cell confluence rate reached 100%, a scratch was made vertically with a 200 μL sterile long pipette tip to form a scratch. The old medium was discarded, and the cells scraped off were rinsed clean with PBS.

[0156] According to different groups, 200 μg / mL of Gd-CDs was added to the Gd-CDs and Gd-CDs+Laser groups, 200 μg / mL of Gd-CDs@HCM was added to the Gd-CDs@HCM and Gd-CDs@HCM+Laser groups, and an equal amount of complete medium was added to the blank and Laser groups. Microscopic photographs were taken as the 0 h control.

[0157] After co-incubation in a 37°C, 5% CO2 incubator for 4 h, the Laser, Gd-CDs+Laser, and Gd-CDs@HCM+Laser groups were irradiated with 808 nm laser (1 W / cm 2 ) for 5 min, and then continued to be cultured in the incubator for 20 h. Finally, the cells were taken out, and the scratch width at the same position was observed and photographed under the microscope.

[0158] As Figure 19As shown, the scratch widths of each group were the same at 0 h. After 24 h, the changes in scratch widths of the Gd-CDs group and the blank group were similar, indicating that Gd-CDs alone could not inhibit cell migration. Compared with the Gd-CDs group, after 24 h, the changes in scratch widths of the other groups had varying degrees of delay compared to the initial time point, and the migration distance of the cells decreased, indicating that they could all inhibit the migration of HepG2 cells. Among them, the Gd-CDs@HCM+Laser group had the most significant effect, demonstrating that the synergistic effect of Gd-CDs@HCM and near-infrared laser might inhibit HepG2 migration through multi-dimensional mechanisms such as physically destroying the cytoskeleton, inhibiting key migration signaling pathways, depleting energy supply, and inducing programmed cell death.

[0159] The above results showed that the bioactive Gd-CDs@HCM complex exhibited good anti-tumor cell migration ability, and for the Gd-CDs@HCM+Laser group, that is, after the combination of photothermal and immunotherapy, its anti-tumor migration effect was greatly improved, suggesting that the combination of photothermal therapy and immunotherapy might be a new method for hepatocellular carcinoma metastasis.

[0160] Next, the metastatic ability of HepG2 cells under various treatment methods was further analyzed through a cell invasion experiment.

[0161] Matrigel was evenly coated on the bottom of the upper chamber of Transwell in advance, and HepG2 cells (2.5×10 4 cells / well) were inoculated in serum-free MEM medium and placed in the upper chamber, and the lower chamber was set with MEM medium containing 10% fetal bovine serum, and cultured at 37 °C, 5% CO2, and relative humidity of 95% for 24 h.

[0162] The experiment was also divided into 6 groups: a control group, a Gd-CDs group, a Gd-CDs@HCM group, a Laser group, a Gd-CDs+Laser group, and a Gd-CDs@HCM+Laser group.

[0163] Gd-CDs with a concentration of 200 μg / mL were added to the Gd-CDs and Gd-CDs+Laser groups respectively, Gd-CDs@HCM with a concentration of 200 μg / mL were added to the Gd-CDs@HCM and Gd-CDs@HCM+Laser groups respectively, and the blank group and the Laser group were replaced with an equal amount of complete medium. After co-incubation for 4 h, the Laser, Gd-CDs+Laser, and Gd-CDs@HCM+Laser groups were irradiated with 808 nm laser (1 W / cm 2 ) for 5 min, and then continued to be cultured for 20 h. Finally, paraformaldehyde was added for fixation and stained with crystal violet solution.

[0164] Count the invaded HepG2 cells under an inverted fluorescence microscope, and quantify the number of invaded cells by calculating the cell number in randomly selected fields of view at a magnification of ×100 ( Figure 20 a). As Figure 20 shown in b, the number of invaded cells in the Gd-CDs group was similar to that in the blank group, indicating that Gd-CDs alone could not inhibit cell invasion. Compared with the Gd-CDs group, the Gd-CDs@HCM, Laser, Gd-CDs+Laser, and Gd-CDs@HCM+Laser groups all significantly inhibited the invasion of HepG2 cells. Among them, the Gd-CDs@HCM+Laser group, which combines photothermal and immunotherapy, produced the most significant inhibitory effect on cell invasion ( p <0.001).

[0165] The above cell invasion experiment also showed that the bioactive Gd-CDs@HCM exhibited good anti-invasive activity. After the combination of photothermal and immunotherapy in Gd-CDs@HCM+Laser, the anti-tumor metastasis effect was greatly improved.

[0166] Furthermore, the in vitro tumor treatment effect of Gd-CDs@HCM was evaluated by a live / dead cell staining assay.

[0167] HepG2 cells (5×10 4 cells / well) were seeded in 12-well plates and cultured overnight. They were also divided into 6 groups. Among them, 200 μg / mL of Gd-CDs was added to the Gd-CDs and Gd-CDs+Laser groups, and 200 μg / mL of Gd-CDs@HCM was added to the Gd-CDs@HCM and Gd-CDs@HCM+Laser groups. After co-incubation for 4 h, the Laser, Gd-CDs+Laser, and Gd-CDs@HCM+Laser groups were irradiated with 808 nm laser (1 W / cm 2 ) for 5 min, and then cultured for another 20 h. The culture medium was aspirated, the cells were washed twice with PBS, and the cells were stained with the fluorescent dyes Calcein-AM and propidium iodide (PI) solution. After incubation at 37 °C in the dark for 30 min, the dyes were aspirated, and paraformaldehyde was added for fixation.

[0168] The cell status was observed and identified under a fluorescence microscope. Among them, the cells stained with Calcein-AM and showing green fluorescence were live cells, and the cells stained with PI and showing red fluorescence were dead cells.

[0169] Figure 21a shows that the cells co-cultured in the control group and the Gd-CDs group showed green fluorescence, with basically no red fluorescence, which means that the cells treated by these two methods were mainly living cells; a small number of red cells appeared in the Gd-CDs@HCM group, indicating that the Gd-CDs@HCM nanoplatform has less ability to damage cells, which further proves its low toxicity. The red fluorescence of cells in the Laser group treated with near-infrared laser was significantly enhanced, indicating that near-infrared laser has a damaging effect on HepG2 cells. When the near-infrared laser was used in combination with Gd-CDs and Gd-CDs@HCM, the cells showed clearly visible strong red fluorescence, and most of the cells were in a state of apoptosis, showing a significant killing effect, especially in the Gd-CDs@HCM+Laser group, where almost all cells were in a state of apoptosis. Similarly, Figure 21 The fluorescence intensity analysis diagram in b also shows that the PI intensity is the strongest and the cell apoptosis is the most when the near-infrared laser and Gd-CDs@HCM are combined for treatment.

[0170] Embodiment 11

[0171] Tumor cells undergoing immunogenic cell death (ICD) release immunogenic damage-associated molecular patterns (DAMPs) to trigger long-term protective anti-tumor responses. In the present invention, ICD induced by photothermal therapy is a key part of the treatment-induced anti-tumor immune response, so whether Gd-CDs@HCM can induce ICD in HepG2 cells under laser irradiation was studied.

[0172] HMGB1 release is one of the hallmark features of ICD in cells. HMGB1 can bind to TLR4 on DCs to promote antigen processing and presentation. Therefore, enzyme-linked immunosorbent assay was used to detect HMGB1 release.

[0173] HepG2 cells were grown at a density of 3 × 10 5 The cells were inoculated into 6-well plates. The corresponding concentrations of Gd-CDs and Gd-CDs@HCM were added to the experimental groups. After 4 h, half of the experimental groups were treated with 1W / cm 2 The blank group was treated with an 808 nm laser, and an equal amount of complete culture medium was added to half of the culture medium, and then cultured for another 20 h.

[0174] The cell culture supernatant was collected, and the samples were processed according to the instructions of the HMGB1 kit. Finally, the HMGB1 content of each group was detected using a multifunctional microplate reader. Figure 22As shown in a, compared with the Gd-CDs group, the HMGB1 content in the Laser, Gd-CDs+Laser, and Gd-CDs@HCM+Laser groups increased, indicating that near-infrared laser irradiation can increase the release of HMGB1. Among them, the increase in HMGB1 content in the Gd-CDs@HCM+Laser group was the most significant ( p <0.01), suggesting that the encapsulation of Gd-CDs@HCM in the cell membrane improved the targeting to liver cancer tissues, thereby promoting the release of HMGB1 from tumor cells to the extracellular space.

[0175] During ICD, ATP is also released extracellularly, and the release of ATP can recruit dendritic cells. Therefore, in this example, an ATP detection kit was further used to analyze the release amount of ATP. The results are as Figure 22 shown in b. Compared with the control group, there was no significant difference in the release amount of ATP between the Gd-CDs group and the Gd-CDs@HCM group, indicating that Gd-CDs alone and Gd-CDs@HCM had no obvious effect on the release of ATP. Compared with the Gd-CDs group, the release amount of ATP increased by 3.5 times after simple near-infrared laser irradiation ( p <0.05); the release amount of ATP in the Gd-CDs+Laser group increased by 3.9 times ( p <0.01); the release amount of ATP in the Gd-CDs@HCM+Laser group increased by 4.3 times ( p <0.01); indicating that tumor cells can promote the release of ATP from tumor cells to the extracellular space after near-infrared laser irradiation.

[0176] When cells undergo ICD, CRT can migrate from the endoplasmic reticulum to the cell membrane, and the exposed CRT promotes the phagocytosis of antigen-presenting cells. Therefore, immunofluorescence was used to detect the exposure of CRT.

[0177] Disperse HepG2 cells into a cell suspension of 1×10 4 cells / mL with complete medium, and use a pipette gun to aspirate 2 mL and transfer it to a confocal culture dish. In the experimental groups, add the corresponding concentrations of Gd-CDs and Gd-CDs@HCM respectively. After acting for 4 h, treat the cell culture plate with near-infrared laser at a dose of 1 W / cm 2 . Replace the blank group with an equal amount of complete medium, and then continue to culture for 20 h.

[0178] Collect the cells of each group, fix them with 4% formaldehyde, and then add 10% BSA to block for 1 h. Then add the primary antibody and incubate at room temperature for 2 h. Next, incubate with the secondary antibody conjugated with a fluorescent substance at room temperature for 1 h. Then add DAPI to stain the nuclei for 10 min. Finally, take pictures with a confocal laser scanning microscope. The green fluorescence shows the exposure of CRT, and the blue fluorescence is the localization of the DAPI dye to the cell nuclei.

[0179] The results are as Figure 22 shown in c. Compared with the control group, there was no significant difference in the exposure of CRT between the Gd-CDs group and the Gd-CDs@HCM group, indicating that neither Gd-CDs alone nor Gd-CDs@HCM had an obvious effect on the exposure of CRT. Compared with the Gd-CDs group, the exposure of CRT was significantly increased in the Laser, Gd-CDs+Laser, and Gd-CDs@HCM+Laser groups, demonstrating that near-infrared laser irradiation could promote the exposure of CRT. Among them, the exposure of CRT on the surface of tumor cells in the Gd-CDs@HCM+Laser group was significantly increased compared with other groups. Therefore, near-infrared laser irradiation of Gd-CDs@HCM could induce ICD.

[0180] The above embodiments of the present invention do not describe all the details in detail, nor do they limit the present invention to the above-described embodiments. All changes, modifications, substitutions, and variations made by those of ordinary skill in the art to these embodiments without departing from the principle and spirit of the present invention shall be included within the protection scope of the present invention.

Claims

1. A gadolinium-doped carbon dot complex based on liver cancer cell membrane coating is based on gadolinium-doped carbon dots Gd-CDs prepared by a one-step microwave thermal method using indocyanine green, citric acid, polyethylene glycol and gadopentetate, placed in a cell membrane suspension extracted from human liver cancer cells HepG2, and extruded by a liposome extruder to construct a HepG2 cell membrane-encapsulated Gd-CDs complex Gd-CDs@HCM with a particle size between 20 and 25 nm. It has fluorescence-magnetic resonance dual-modality imaging function and the ability to photothermally and immunotherapeutically treat hepatocellular carcinoma.

2. The gadolinium-doped carbon dot complex based on liver cancer cell membrane coating according to claim 1 is characterized in that The mass ratio of HepG2 cell membrane to Gd-CDs is (0.25~1):

1.

3. The method for preparing the gadolinium-doped carbon dot complex based on liver cancer cell membrane coating according to claim 1 comprises: Indocyanine green, citric acid, polyethylene glycol and gadopentetate were used as raw materials, dissolved in water and subjected to microwave heating reaction to prepare gadolinium-doped carbon dots Gd-CDs. The cultured human liver cancer cells HepG2 were used for hypotonic extraction and disruption, and then HepG2 cell membrane suspension was obtained by low temperature differential centrifugation. Gd-CDs were placed in the obtained HepG2 cell membrane suspension and repeatedly extruded using a liposome extruder to construct a HepG2 cell membrane-wrapped Gd-CDs complex Gd-CDs@HCM with uniform particle size.

4. The preparation method according to claim 3, characterized in that The reaction was heated at a microwave power of 700 to 900 W for 3 to 8 minutes.

5. The preparation method according to claim 3, characterized in that Indocyanine green, citric acid, polyethylene glycol and gadopentetate are dissolved in water, firstly subjected to ultrasonic treatment for 30 to 40 minutes, and then subjected to microwave heating reaction.

6. The preparation method according to claim 3, characterized in that The microwave heating reaction product is filtered, dialyzed and freeze-dried to obtain gadolinium-doped carbon dots Gd-CDs powder.

7. The preparation method according to claim 3, characterized in that Liposome extruders equipped with polycarbonate membranes with pore sizes of 400 nm, 100 nm and 50 nm were used in sequence for repeated extrusion, and each polycarbonate membrane with a pore size was repeatedly extruded 20 to 30 times.

8. Use of the gadolinium-doped carbon dot complex based on liver cancer cell membrane coating as claimed in claim 1 as a fluorescence-magnetic resonance dual-modality imaging probe.

9. Use of the gadolinium-doped carbon dot complex based on liver cancer cell membrane coating as claimed in claim 1 as a cell fluorescence-magnetic resonance dual-modality imaging probe.

10. Use of the gadolinium-doped carbon dot complex based on liver cancer cell membrane coating according to claim 1 in the preparation of a drug for inhibiting the growth of liver tumor cells.

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