Nanovaccine based on tumor antigen covalently coupled gadolinium-doped carbon dots and preparation method and application thereof

Abstract

The application relates to a nanovaccine based on tumor antigen covalent coupling gadolinium doped carbon dots and a preparation method thereof, which is characterized in that: a gadolinium doped carbon dot Gd-CDs with near-infrared fluorescence / magnetic resonance / optical thermal multimodal imaging function is used as a carrier; a tumor antigen TA extracted from tumor cells is covalently coupled to the surface of the Gd-CDs through an amide bond by using an EDC / NHS activation system to obtain a Gd-CDs-TA complex; the loading stability of the antigen is significantly improved; after near-infrared laser irradiation, the Gd-CDs mediate the photothermal effect to induce immunogenic cell death, release tumor-related antigens and damage-related molecular patterns, and together with the covalently loaded exogenous antigens, form a double antigen source to synergistically promote the maturation of dendritic cells and the activation of cytotoxic T cells. In vivo experiments prove that the tumor inhibition rate of the nanovaccine combined with laser irradiation can reach 98%, and the proportion of CD8+ T cells in the tumor tissue can be increased to more than 50%, and the proportion of Tregs can be reduced to less than 3%, so that the nanovaccine can be used for multimodal imaging guided photothermal treatment and antitumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and biomedicine, and relates to a nanovaccine in which tumor antigens are covalently coupled to the surface of gadolinium-doped carbon dots via EDC / NHS-mediated amide bonds, as well as the preparation method of the nanovaccine and its application in tumor multimodal imaging, photothermal therapy and antitumor immunotherapy. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, especially hepatocellular carcinoma (HCC). As a malignant tumor with a continuously rising incidence, its treatment still faces enormous challenges. Not only are traditional surgery, radiotherapy, and chemotherapy limited in their effectiveness for advanced and metastatic lesions, but they are also often accompanied by severe toxic side effects. Although immunotherapy, such as immune checkpoint inhibitors, has brought hope for long-term survival to some patients, its overall response rate remains unsatisfactory. A key reason for this is the insufficient activation and depletion of tumor-specific T cells in the tumor microenvironment.

[0003] Against this backdrop, tumor vaccines have shown great potential as a strategy to stimulate specific and proactive anti-tumor immune responses in the body. An ideal tumor vaccine can efficiently deliver tumor antigens and effectively activate antigen-presenting cells (such as dendritic cells) to initiate robust T-cell immunity. However, traditional vaccine vectors (such as viral vectors and liposomes) face problems such as low antigen loading rates, uncontrollable immunogenicity, potential induction of pre-existing immunity, and poor targeting. Therefore, developing novel, safe, and efficient antigen delivery systems is a key challenge in the field of tumor vaccines.

[0004] In recent years, nanotechnology has provided a new platform for vaccine design. Among them, carbon dots (CDs), as an emerging carbon-based nanomaterial, have attracted much attention in the fields of drug delivery and bioimaging due to their excellent biocompatibility, low toxicity, water solubility, and ease of surface functionalization. Of particular note is that, through proper design, carbon dots themselves can serve as antigen carriers and even as immune adjuvants to enhance immune responses. For example, gadolinium-doped carbon dots (Gd-CDs) can not only serve as contrast agents in magnetic resonance (MR) imaging to visualize the diagnostic and treatment process, but also regulate the immune microenvironment through their specific physicochemical properties (such as photothermal effects).

[0005] The combination of photothermal therapy (PTT) and immunotherapy has proven to be a promising synergistic anti-tumor strategy. PTT, while locally ablating tumors, can induce immunogenic cell death (ICD) in tumor cells. ICD releases a large number of tumor-associated antigens and danger signaling molecules, thereby transforming the tumor in situ into a "vaccine in situ" reservoir, providing an antigen source for and enhancing the strength of subsequent immune responses. However, the antigens released by ICD induced solely by PTT are random and complex, and the resulting immune response may lack sufficient strength and specificity to combat highly heterogeneous tumors.

[0006] Dong et al. prepared Gd-CDs with fluorescence-magnetic resonance dual-modal imaging capabilities using a microwave thermal method with indocyanine green, citric acid, polyethylene glycol, and gadopentetate. These Gd-CDs were placed in a cell membrane suspension extracted from human hepatocellular carcinoma HepG2 cells and extruded using a liposome extruder to construct a Gd-CDs@HCM complex encapsulated in HepG2 cell membranes. Journal of Nanobiotechnology (2026, 24: 217.). This system achieves tumor enrichment through the homologous targeting ability of the cell membrane. After laser irradiation, Gd-CDs generate a photothermal effect that induces immunogenic cell death, and the ablation rate of the primary tumor reaches 84.9%.

[0007] However, the Gd-CDs@HCM system has the following technical shortcomings: 1) The cell membrane and Gd-CDs are bound by physical compression, which is a non-covalent physical coating. The membrane structure may dissociate or detach during in vivo circulation, resulting in loss of targeting function; 2) The types and density of homologous targeting molecules on the cell membrane surface are limited by the cell membrane source, and the targeting efficiency is difficult to precisely control; 3) The system relies on endogenous antigens released by ICDs induced by photothermal activity as the main source of immune activation. It lacks pre-loaded exogenous specific antigens, and the specificity and strength of the immune response are limited by the randomness and complexity of the antigens released by ICDs.

[0008] Liu et al. (Carbon Dots and Tumor Antigen Conjugates as Nanovaccines for Elevated Cancer Immunotherapy[J]. Small The paper (2023, 19(31): e2206683) disclosed nanovaccines GMaal+B16F10-Ag and GMaal+CT26-Ag, constructed by conjugating carbon dots (CDs) with tumor cell-derived antigens B16F10-Ag and CT26-Ag. It further demonstrated that these nanovaccines can be effectively taken up by dendritic cells, promoting DC maturation, cross-presenting antigens to T cells, and specifically targeting B16F10 melanoma or CT26 colon cancer. However, because this technology employs a maleimide-thiol (Mal-SH) chemical conjugation strategy, it requires pre-thiol modification of the antigen or relies on the exposed thiol groups of the antigen itself, limiting the conjugation efficiency and universality. Furthermore, the carbon dots used in this technology lack magnetic resonance imaging capabilities and photothermal effects, making it impossible to achieve synergy between photothermal therapy and immunotherapy. Summary of the Invention

[0009] The purpose of this invention is to provide a nanovaccine based on tumor antigen covalently coupled to Gd-CDs, its preparation method and application. The tumor antigen is covalently coupled to the surface of Gd-CDs through EDC / NHS-mediated amide bonds to improve the loading stability and delivery efficiency of the tumor antigen. While retaining the fluorescence / magnetic resonance / photothermal imaging performance, it generates an in situ vaccine effect by photothermally inducing immunogenic cell death.

[0010] To achieve the above-mentioned objectives, this invention provides a Gd-CDs-TA complex nanovaccine constructed by covalent coupling of gadolinium-doped carbon dots (Gd-CDs) and tumor antigen (TA).

[0011] The Gd-CDs are prepared by microwave heating reaction of indocyanine green, citric acid, polyethylene glycol and gadopentetate, wherein Gd is used as Gd 3+ Gd-CDs are coordinated and doped with diethylenetriaminepentaacetic acid in a carbon dot structure, and possess near-infrared fluorescence emission, magnetic resonance imaging, and photothermal conversion functions. The Gd-CDs and TA are covalently linked by amide bonds, which are formed by the condensation of carboxyl groups on the surface of Gd-CDs with amino groups in TA.

[0012] The Gd-CDs-TA complex constructed in this invention has a particle size of 35-43 nm, a TA protein content of 25-35%, a surface Zeta potential that is at least 2 mV lower than that of Gd-CDs, and retains the fluorescence emission peak of Gd-CDs at 708 nm under 660 nm excitation and the near-infrared absorption in the range of 750-810 nm.

[0013] This invention improves the loading stability of tumor antigens (TA) on the surface of Gd-CDs by replacing the physical coating of the cell membrane with an EDC / NHS-mediated covalent coupling structure, and enhances local tumor enrichment by utilizing surface antigen-related homology recognition and cellular uptake. At the same time, it utilizes the photothermal effect of Gd-CDs to induce immunogenic cell death, forming a dual antigen source and synergistic immune activation mechanism of "stable antigen delivery and homology recognition + photothermal-induced in situ vaccine", thereby realizing imaging-guided tumor photothermal-immunotherapy.

[0014] Furthermore, in the Gd-CDs-TA complex constructed in this invention, the mass ratio of TA to Gd-CDs, based on the protein content of TA, is (1.5-3):1.

[0015] Furthermore, the tumor antigen TA in this invention is extracted from tumor cells, which are selected from mouse liver cancer cells Hepa1-6, human liver cancer cells HepG2, mouse breast cancer cells 4T1, or human lung adenocarcinoma cells A549.

[0016] Furthermore, the present invention also provides a method for preparing the aforementioned nanovaccine based on tumor antigen covalently coupled gadolinium-doped carbon dots, comprising: 1) Gadolinium-doped carbon dots (Gd-CDs) were prepared by dispersing indocyanine green, citric acid, polyethylene glycol, and gadopentetate in water and then reacting them with microwave heating. 2) Add Gd-CDs to cultured tumor cells to a final concentration of 150-250 μg / mL, incubate for 4-6 h, irradiate with 808 nm laser for 3-5 min, and continue incubation in 1640 medium without fetal bovine serum for 36-48 h. Collect the supernatant to obtain tumor antigen (TA). 3) After activating the carboxyl groups on the surface of Gd-CDs with EDC and NHS, they are co-incubated with TA to form amide bonds between the carboxyl groups on the surface of Gd-CDs and the amino groups in TA, thus obtaining the covalently coupled nanovaccine Gd-CDs-TA.

[0017] Specifically, in the preparation method described in this invention, it is preferable to disperse Gd-CDs in PBS, add amide activator EDC and NHS, and activate at room temperature for 2-4 hours.

[0018] More specifically, in the preparation method described in this invention, it is preferable to co-incubate the activated Gd-CDs with TA for 12 to 18 hours.

[0019] More preferably, in the preparation method of the present invention, the mass ratio of EDC to Gd-CDs is (2-3):1, and the mass ratio of NHS to Gd-CDs is (0.8-1.2):1.

[0020] More preferably, in the preparation method of the present invention, the laser power density of the laser irradiation is 0.5 to 1.5 W / cm². 2 .

[0021] More preferably, in the preparation method of the present invention, the culture time of the tumor cells is 12-18 hours.

[0022] Furthermore, the present invention preferably involves centrifuging the collected supernatant to remove non-tumor-derived protein antigens, and then collecting the supernatant again to obtain tumor antigens.

[0023] The Gd-CDs-TA nanovaccine of this invention uses Gd-CDs as a multifunctional carrier. After the carboxyl groups on the surface of Gd-CDs are activated by EDC / NHS, they form amide bonds with the amino groups in TA to achieve covalent loading of TA. Compared with physical coating of cell membranes or simple antigen mixing, the covalent coupling structure of this invention helps to reduce non-specific antigen shedding during preparation, washing, and delivery, and enables antigen delivery and the photothermal effect mediated by Gd-CDs to be achieved on the same nanoplatform.

[0024] Therefore, the nanovaccine based on tumor antigen covalently coupled gadolinium-doped carbon dots described in this invention can be used as a fluorescence / magnetic resonance / photothermal multimodal imaging probe.

[0025] The Gd-CDs-TA complex constructed based on this invention has long-wavelength emission fluorescence imaging, photothermal imaging, and magnetic resonance imaging capabilities, and also has good cell permeability. Therefore, it can be used as a multimodal imaging probe for cell fluorescence / magnetic resonance / photothermal imaging.

[0026] The Gd-CDs-TA complex provided by this invention also has good tumor tissue targeting. It can not only enter the cell to achieve multimodal cell imaging, but also penetrate biological living tissue for in vivo fluorescence, magnetic resonance and photothermal imaging. Therefore, it can be used as a multimodal imaging probe for living animals.

[0027] The Gd-CDs-TA complex described in this invention forms a stable antigen-loaded structure through surface covalent coupling with TA, and exhibits higher tumor cell uptake and local tumor fluorescence signal than that of uncoupled Gd-CDs, which is beneficial for exerting antigen-related homology recognition, local adhesion and immune activation.

[0028] Furthermore, Gd-CDs-TA retains the photothermal properties of Gd-CDs. Upon irradiation with an 808nm laser, its photothermal effect can induce tumor cell death and promote the exposure of calreticulin (CRT) and the release of high-mobility group box 1 (HMGB1), thereby inducing immunogenic cell death (ICD). The tumor-associated antigens and damage-associated molecular patterns released by ICDs can promote the uptake, processing, and presentation of antigens by dendritic cells, and are beneficial for further activation of cytotoxic T cells.

[0029] Therefore, the pre-covalently loaded TA and the antigen released in situ during photothermal therapy constitute a dual antigen source, enabling stable antigen delivery and in situ vaccine effects to synergistically occur on the same platform. Thus, the nanovaccine based on tumor antigen covalently coupled with gadolinium-doped carbon dots described in this invention can also be used to prepare drugs that inhibit tumor cell growth.

[0030] This invention constructs Gd-CDs-TA through covalent coupling of EDC / NHS, which improves the loading stability of TA while retaining multimodal imaging and photothermal performance, and forms a synergistic mechanism of "antigen delivery and homology recognition + photothermal induction of in situ vaccine". Therefore, the complex can be used to prepare imaging-guided tumor photothermal-immunotherapy drugs, especially drugs for photothermal-immunotherapy of hepatocellular carcinoma.

[0031] In vivo and in vitro experiments using Hepa1-6 cells and tumor-bearing mice confirmed that the Gd-CDs-TA complex of this invention has a significant inhibitory effect on liver tumor cells, with tumor inhibition efficiencies of 97.5% and 98.0%, respectively.

[0032] The Gd-CDs-TA complex of this invention possesses trimodal imaging capabilities, including near-infrared fluorescence imaging, magnetic resonance imaging, and photothermal imaging. Its longitudinal relaxation rate is higher than that of gadodiamine, a commonly used contrast agent in clinical practice, providing multidimensional imaging evidence for the timing and efficacy evaluation of photothermal therapy. Attached Figure Description

[0033] Figure 1 This is a Zeta potential diagram of Gd-CDs, Hepa1-6-Ag, Gd-CDs+Hepa1-6-Ag, HepG2-Ag, Gd-CDs+HepG2-Ag, 4T1-Ag, Gd-CDs+4T1-Ag, A549-Ag, and Gd-CDs+A549-Ag.

[0034] Figure 2 This is a TEM image of Gd-CDs-TA.

[0035] Figure 3 This is a graph showing the size changes of Gd-CDs-TA over 7 days in water, PBS, and 10% FBS.

[0036] Figure 4 The images show the UV absorption spectrum (a) and fluorescence emission spectrum (b) of Gd-CDs and Gd-CDs-TA.

[0037] Figure 5 This is an SDS-PAGE gel electrophoresis image (1: Marker; 2: Gd-CDs; 3: Gd-CDs+TA physical mixture; 4: TA; 5: Gd-CDs-TA; 6: Marker).

[0038] Figure 6 T1-weighted images (a) of Gd-CDs-TA and gadodiamine and longitudinal relaxation rate (1 / T1) versus different concentrations of Gd 3+ Linear relationship diagram (b).

[0039] Figure 7 Different concentrations of Gd-CDs-TA were subjected to 808nm (1W / cm) 2 Thermal image (a) and temperature rise curve (b) of laser irradiation (5 min).

[0040] Figure 8 It is physiological saline, Gd-CDs, and Gd-CDs-TA at 808nm (1W / cm) 2 The temperature rise curve of laser irradiation (5 min).

[0041] Figure 9 These are laser confocal microscopy images of Gd-CDs and Gd-CDs-TA in Hepa1-6 cells.

[0042] Figure 10 This is an MR imaging image of Gd-CDs-TA in Hepa1-6 cells.

[0043] Figure 11 These are thermal images (a) and temperature-time curves (b) of different groups of Hepa1-6 cells during photothermal therapy.

[0044] Figure 12 The images show in vivo time-dependent imaging of tumor-bearing mice after Gd-CDs-TA treatment (a) and in vitro fluorescence images of mouse organs and tumor tissues at different time points (b).

[0045] Figure 13 These are in vivo MR imaging images of tumor-bearing mice after intravenous injection of Gd-CDs-TA.

[0046] Figure 14 These are tumor thermography images (a) and temperature-time curves (b) of different groups of tumor-bearing mice during photothermal therapy.

[0047] Figure 15This is a histogram of cell survival rates after co-incubation with Hepa1-6 cells for 24 hours under different conditions.

[0048] Figure 16 The images (a) and histograms (b) of Hepa1-6 cells stained with Calcein AM and PI in different treatment groups are shown.

[0049] Figure 17 This is a statistical graph showing the apoptosis rate of Hepa1-6 cells in different treatment groups.

[0050] Figure 18 These are immunofluorescence images of HMGB1 release and CRT exposure on the cell membrane surface in Hepa1-6 cells after different treatments.

[0051] Figure 19 These are the mature FCM results and statistical analysis charts for DCs in different treatment groups.

[0052] Figure 20 The levels of TNF-α, IL-6, and IFN-γ cytokines produced in the culture medium of different treatment groups were analyzed by ELISA.

[0053] Figure 21 The changes in tumor volume during the treatment period in different treatment groups (a) and photos of Hepa1-6 tumor-bearing mice after 14 days of treatment in each group (b).

[0054] Figure 22 This includes the tumor tissue weight and tumor photographs for each group at the end of the treatment observation period.

[0055] Figure 23 The tumor inhibition rate of each group of Hepa1-6 tumor-bearing mice after treatment is shown.

[0056] Figure 24 This represents the change in mouse body weight during the treatment period in different treatment groups.

[0057] Figure 25 These are pathological sections of the heart, liver, spleen, lungs, and kidneys of mice in different treatment groups 14 days later.

[0058] Figure 26 The results are the blood routine and serum biochemical indicators of C57BL / 6J mice in different treatment groups.

[0059] Figure 27 This is a graph showing the FCM results and statistical analysis of mature DCs (CD11c+CD80+CD86+) in the lymph nodes of mice in different treatment groups.

[0060] Figure 28 This is a graph showing the FCM results and statistical analysis of CD8+ T cells (CD3+CD8+) in tumor tissue.

[0061] Figure 29 This is a graph showing the FCM results and statistical analysis of Tregs (CD4+CD25+Foxp3+) in tumor tissue.

[0062] Figure 30 This is a graph showing the levels of TNF-α, IFN-γ, IL-6, and IL-10 in the peripheral blood of mice that received different treatments. Detailed Implementation

[0063] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It should be noted that the embodiments are merely illustrative and are intended to provide a thorough understanding of the technical solutions of the present invention and to provide guidance for those skilled in the art to implement and apply the present invention. It should be understood that these descriptions do not constitute any limitation on the scope of protection of the present invention.

[0064] Unless otherwise expressly stated, the production processes, experiments, tests or analysis methods involved in the embodiments of the present invention are all considered to be conventional methods known to those skilled in the art, and only need to be implemented in accordance with conventional conditions or relevant product instructions. The steps and names involved are also generally clear and unambiguous in the art.

[0065] The instruments, equipment, raw materials, reagents, or samples used in the embodiments are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels or prepared by known methods, and their source does not have a substantial impact on the implementation results of the present invention.

[0066] Unless otherwise expressly defined, the scientific and technical terms used in this invention have the meanings commonly understood by one of ordinary skill in the art. In case of any conflict, the definitions in this specification shall prevail.

[0067] The terms “comprising,” “including,” “having,” etc., used in this invention should be understood as open-ended, meaning “including but not limited to.” The term “and / or” includes any and all combinations of one or more of the associated listed items. Quantitative terms such as “a,” “one,” etc., do not exclude multiples; “multiple” or “a variety” refers to quantities greater than or equal to two.

[0068] The terms "preferred", "better", and "exemplary" used in this invention are only used to describe specific solutions or effects and are not intended to limit the necessary scope of the solution or the scope of protection.

[0069] This invention relates to the description of numerical parameters (such as dosage, concentration, temperature, time, etc.), and it should be understood that reasonable deviations naturally exist due to measuring instruments, operational errors, statistical fluctuations, etc. The range of such deviations should be within limits acceptable to those skilled in the art based on common sense.

[0070] The nanovaccines based on tumor antigen covalently coupled with gadolinium-doped carbon dots in the following embodiments of the present invention were prepared according to the following methods: 1) Indocyanine green, citric acid, polyethylene glycol-8000 and gadopentetate were added to deionized water in sequence. After ultrasonic treatment for 30-40 min, the mixture was heated in a microwave oven at 700-900W for 3-8 min. The reaction product was filtered through a disposable filter membrane with a pore size of 0.22 μm, dialyzed using a dialysis bag with a molecular weight of 500 Da, and freeze-dried to obtain Gd-CDs powder. 2) Incubate tumor cells in culture dishes for 12–18 h, then add Gd-CDs at a final concentration of 150–250 μg / mL and incubate for 4–6 h, followed by laser incubation (808 nm, 1.0 W / cm²). 2 ) Irradiation for 3 to 5 minutes; 3) The treated tumor cells were incubated with 1640 medium without fetal bovine serum for 36-48 hours. The supernatant was collected and centrifuged to remove non-tumor-derived protein antigens. The tumor antigen TA was obtained by collecting the supernatant. 4) Dissolve Gd-CDs in PBS, add amide activator EDC and NHS, and let stand at room temperature for 2-4 hours; 5) The activated Gd-CDs were co-incubated with TA for 12-18 hours at a mass ratio of TA to Gd-CDs of (1.5-3):1 (based on the protein content of TA). This allowed the carboxyl groups on the surface of Gd-CDs to form amide bonds with the amino groups in TA. After centrifugation, ultrafiltration, and washing, the covalently coupled nanovaccine Gd-CDs-TA was obtained and stored in a refrigerator at 4°C. Example

[0071] Example 1

[0072] Weigh 5 mg of indocyanine green (ICG), 0.2 g of citric acid (CA), 50 mg of polyethylene glycol-8000 (PEG), and 30 mg of gadopentetic acid (Gd-DTPA), and add them sequentially to 15 mL of deionized water. Sonicate the mixture for 40 min to mix thoroughly, and then heat it in a 700 W microwave oven for 3 min to obtain a dark green transparent reaction solution.

[0073] The reaction solution was first filtered through a disposable filter membrane with a pore size of 0.22 μm. The filtrate was then dialyzed using a dialysis bag with a molecular weight of 500 Da. The dialysate was then freeze-dried to obtain green Gd-CDs powder.

[0074] Gd-CDs exhibit uniformly dispersed, near-spherical shapes with an average particle size of 12.24 ± 1.26 nm and a lattice spacing of 0.28 nm. FTIR characterization shows that Gd-CDs have a lattice spacing of 1094 cm⁻¹. -1 1492cm -1 and 1552cm -1 Each of the following has a sulfonic acid group (-SO3) - ), COC bond and Gd 3 + Characteristic peaks of C=O vibration induced by coordination with DTPA. XPS full spectrum shows that C, N, O, S and Gd elements exist in Gd-CDs, with Gd4d being the most abundant. 3 / 2 and Gd 4d 5 / 2 The binding energies are 146.9 eV and 141.7 eV, respectively, indicating that Gd mainly exists as Gd. 3+ The form exists in nanostructures. The photothermal conversion efficiency of Gd-CDs is 46.7%, and the heating performance remains stable after 5 laser on / off cycles.

[0075] Example 2

[0076] Mouse liver cancer cells (Hepa1-6) were seeded in a 90 mm diameter culture dish and incubated overnight. When the cells reached 80-90% confluence, Gd-CDs were added to a concentration of 200 µg / mL and incubated for 4 h.

[0077] Hepa1-6 cells were washed with PBS and then treated with a laser (808 nm, 1.0 W / cm²). 2 Irradiate for 5 min. Then, incubate Hepa1-6 cells with 1640 medium without fetal bovine serum (FBS) for 48 h.

[0078] After incubation, centrifuge at 2000 rpm for 5 min, collect the supernatant and transfer it to an ultrafiltration tube for ultrafiltration concentration. Add PBS to the retentate above the membrane, mix gently, and centrifuge and ultrafilter again to remove non-tumor-derived protein antigens.

[0079] Repeat the above buffer replacement steps, recover the retentate, adjust the volume with an appropriate amount of PBS to obtain Hepa1-6-Ag tumor antigen dispersion, and store at 4°C.

[0080] Weigh 20 mg of Gd-CDs and disperse them in 10 mL of PBS to prepare a Gd-CDs solution with a concentration of 2 mg / mL. Then, add 50 mg of amide activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 20 mg of N-hydroxysuccinimide (NHS) in sequence. After dissolving, let it stand at room temperature for 2 h.

[0081] Take 1 mL of the activated Gd-CDs solution and incubate it overnight with 2 mL of the above Hepa1-6-Ag. After incubation, centrifuge at 2000 r / min for 5 min, collect the supernatant and transfer it to a centrifuge ultrafiltration tube for ultrafiltration concentration. Add PBS to the retentate above the membrane, mix gently, and centrifuge and ultrafilter again to finally prepare the nanovaccine Gd-CDs+Hepa1-6-Ag, which is stored in a refrigerator at 4℃.

[0082] Example 3

[0083] Human hepatocellular carcinoma cells (HepG2) were seeded in culture dishes and incubated overnight, followed by incubation with Gd-CDs (200 µg / mL) for 4 h. HepG2 cells were washed with PBS and then incubated with a laser (808 nm, 1.0 W / cm²). 2 Irradiate for 5 min. Then, incubate HepG2 cells in 1640 medium without FBS for 48 h. Collect the supernatant and centrifuge to remove non-tumor-derived protein antigens to obtain HepG2-Ag tumor antigen.

[0084] Take 1 mL of the Gd-CDs solution activated in Example 2 and incubate it overnight with 2 mL of the above HepG2-Ag. After incubation, centrifuge at 2000 r / min for 5 min, and after ultrafiltration and washing with PBS, prepare the nanovaccine Gd-CDs+HepG2-Ag and store it in a refrigerator at 4°C.

[0085] Example 4

[0086] Mouse breast cancer cells (4T1) were seeded in culture dishes and incubated overnight, followed by incubation with Gd-CDs (200 µg / mL) for 4 h. The 4T1 cells were washed with PBS and then incubated with a laser (808 nm, 1.0 W / cm²). 2 Irradiate for 5 min. Then, incubate 4T1 cells with 1640 medium without FBS for 48 h. Collect the supernatant and centrifuge to remove non-tumor-derived protein antigens to obtain 4T1-Ag tumor antigen.

[0087] Take 1 mL of the Gd-CDs solution activated in Example 2 and incubate it overnight with 2 mL of the above-mentioned 4T1-Ag. After incubation, centrifuge at 2000 r / min for 5 min, and after ultrafiltration and washing with PBS, prepare the nano-vaccine Gd-CDs+4T1-Ag, and store it in a refrigerator at 4°C.

[0088] Example 5

[0089] Human lung adenocarcinoma cells (A549) were seeded in culture dishes and incubated overnight, followed by incubation with Gd-CDs (200 µg / mL) for 4 h. A549 cells were washed with PBS and then incubated with a laser (808 nm, 1.0 W / cm²). 2Irradiate for 5 min. Then, incubate A549 cells in 1640 medium without FBS for 48 h. Collect the supernatant and centrifuge to remove non-tumor-derived protein antigens to obtain A549-Ag tumor antigen.

[0090] Take 1 mL of the Gd-CDs solution activated in Example 2 and incubate it overnight with 2 mL of the above-mentioned A549-Ag. After incubation, centrifuge at 2000 r / min for 5 min, and after ultrafiltration and washing with PBS, prepare the nano-vaccine Gd-CDs+A549-Ag, and store it in a refrigerator at 4°C.

[0091] Example 6

[0092] Gd-CDs, tumor antigens prepared in Examples 2-5 above, and nanovaccines were dispersed in aqueous solution, and the surface zeta potential of different particles was measured by laser Doppler microelectrophoresis.

[0093] Test results are as follows Figure 1 As shown. Figure 1 In Example a, the tumor antigen TA carried a negative charge with a potential of -12.52mV, Gd-CDs carried a positive charge with a potential of +7.63mV, and Gd-CDs-TA after Gd-CDs bound to TA carried a negative charge with a potential of -3.84mV, which preliminarily indicates that the tumor antigen exists in the complex.

[0094] Figure 1 In example b, the tumor antigen HepG2-Ag in Example 3 carries a negative charge with a potential of -13.18mV. Although Gd-CDs + HepG2-Ag after binding to HepG2-Ag still carries a positive charge, its potential is reduced to +5.12mV, which also indicates that the tumor antigen exists in the complex.

[0095] Figure 1 c and Figure 1 In example d, the tumor antigens 4T1-Ag and A549-Ag in Examples 4 and 5 both carry negative charges, with potentials of -15.25mV and -10.31mV, respectively. The nanovaccines Gd-CDs+4T1-Ag and Gd-CDs+A549-Ag after Gd-CDs bind to 4T1-Ag and A549-Ag also carry negative charges, with potentials of -6.59mV and -4.69mV, respectively, showing a similar potential change trend, which again indicates that the tumor antigens exist in the complex.

[0096] Example 7

[0097] Gd-CDs are the core carriers of nanovaccines, exhibiting a uniformly dispersed, near-spherical morphology with an average particle size of approximately 12 nm. Further analysis of the morphology and microstructure of the prepared Gd-CDs-TA complex was conducted using transmission electron microscopy.

[0098] A 10 μL sample of the nano-vaccine was dropped onto a copper grid. After precipitation for 1 min, the floating liquid was removed. Then, 10 μL of uranium acetate was added, and after precipitation for 1 min, the floating liquid was removed again. The sample was dried at room temperature and examined under an electron microscope at 80–120 kV. The results are as follows: Figure 2 The results show that Gd-CDs-TA are spherical, with most particles having a diameter of 35–43 nm and an average diameter of about 38 nm.

[0099] Figure 3 The hydrodynamic diameter of Gd-CDs-TA was determined by dynamic light scattering (DLS), showing that Gd-CDs-TA remained relatively stable in water, PBS and 10% FBS solutions for 7 days.

[0100] Gd-CDs-TA solution and Gd-CDs powder were dispersed in PBS, and the UV-Vis absorption spectrum and fluorescence emission spectrum were measured. Figure 4 In a, Gd-CDs exhibit a characteristic absorption peak in the 750–810 nm range, while Gd-CDs-TA retains this absorption peak; and as Figure 4 As shown in b, under 660 nm excitation, Gd-CDs-TA emits a strong fluorescence signal at 708 nm, and its fluorescence intensity is comparable to that of Gd-CDs, indicating that the coupling of TA does not significantly affect the fluorescence performance of Gd-CDs.

[0101] Example 8

[0102] The protein content in Gd-CDs, TA, and Gd-CDs-TA was tested using SDS-PAGE and BCA protein concentration assay kits, respectively.

[0103] The following groups were set up: Gd-CDs, a physical mixture of untreated Gd-CDs and TA, TA, and a covalently coupled Gd-CDs-TA group. After undergoing the same centrifugation, ultrafiltration, and PBS washing steps, the protein concentration was determined by the BCA method and adjusted to 1 mg / mL.

[0104] Take 20 μL of sample, add 5 μL of 5× protein loading buffer and mix well. Incubate at 95℃ for 5 min. Load 25 μL of sample onto a 10 wt% SDS-PAGE gel. Add 5 μL of protein marker (5-180 kDa) to both lanes. Electrophore at 160V for 40 min. Stain with Coomassie Brilliant Blue. After destaining with destaining solution, use a gel imaging system for imaging.

[0105] Figure 5The results showed that Gd-CDs-TA and TA exhibited similar protein bands, while no corresponding protein bands appeared in the Gd-CDs group; the physical mixture of Gd-CDs and TA without EDC / NHS treatment showed almost no protein bands after the same washing.

[0106] BCA quantification results showed that the TA protein content in Gd-CDs-TA was 30.4%, while the histone content in the physical mixture of untreated Gd-CDs and TA was only 3.4%. These results indicate that TA is mainly stably loaded onto the surface of Gd-CDs through EDC / NHS-mediated covalent bonds, rather than relying on nonspecific physical adsorption.

[0107] Example 9

[0108] The T1 relaxation rate of Gd-CDs-TA was evaluated and compared with that of the commercial product gadodiamine to verify the feasibility of Gd-CDs-TA as a novel MR contrast agent.

[0109] like Figure 6 As shown in figure a, T1-weighted imaging reveals that, with Gd 3+ With increasing concentration, the signal intensity of Gd-CDs-TA gradually increased, exhibiting a concentration-dependent relationship. Quantitative analysis showed that ( Figure 6 b) The longitudinal relaxation rate (r1) of Gd-CDs-TA is 8.95 mM. -1 s -1 The concentration was higher than that of gadodiamine (r1=3.15mM), a commonly used contrast agent in clinical practice. -1 s -1 This confirms the potential of Gd-CDs-TA as a contrast agent for MR imaging.

[0110] Example 10

[0111] The photothermal properties of Gd-CDs-TA were evaluated to verify its feasibility as a photosensitizer.

[0112] 100 μL of ultrapure water and Gd-CDs-TA solutions with concentrations of 50, 100, 200, 400, and 800 μg / mL were respectively placed in 0.2 mL centrifuge tubes. An 808 nm laser was used at 1 W / cm². 2 The solution was irradiated with high power for 5 minutes, and the temperature change of the solution was recorded by an infrared thermal imager. The temperature change trend curves of solutions with different concentrations over time were plotted.

[0113] like Figure 7 As shown in Figures a and b, with the increase of Gd-CDs-TA concentration, the heating rate and the maximum temperature of the solution both increased significantly. At a concentration of 400 μg / mL, the temperature increased by 35 °C within 5 min.

[0114] Subsequently, a 0.4 mg / mL Gd-CDs solution, a Gd-CDs-TA solution, and ultrapure water were placed in a 0.2 mL centrifuge tube, and an 808 nm laser was used at a power of 1 W / cm². 2 After irradiation for 5 minutes, the infrared thermal imager recorded the temperature change of the solution and plotted the temperature change trend curve over time.

[0115] Figure 8 In the study, both Gd-CDs and Gd-CDs-TA exhibited excellent photothermal conversion performance, while the saline group showed no significant temperature increase, confirming that the photothermal effect originates from the inherent properties of Gd-CDs, and that the coupling of TA did not have a significant impact on the photothermal performance.

[0116] Example 11

[0117] In vitro cell imaging experiments were conducted using Hepa1-6 cells. The cell uptake effect of Gd-CDs-TA was observed using a confocal laser scanning microscope (CLSM), and the fluorescence imaging and cell labeling capabilities of Gd-CDs-TA were evaluated.

[0118] Take Hepa1-6 cells in good growth condition and disperse them in complete culture medium at a concentration of 1×10⁻⁶. 4 Cells were suspended at 1 / mL, and 2 mL was transferred to a dedicated culture dish and cultured for 12 h. The culture medium was discarded, the cells were washed with PBS, and then cultured in medium containing 2% FBS for 1 h of starvation. The culture medium was discarded, the cells were washed with PBS, and then complete medium containing 200 µg / mL Gd-CDs and Gd-CDs-TA were added respectively. The cells were cultured under the same conditions for 4 h, fixed with paraformaldehyde for 15 min, and subjected to CLSM imaging. The results are as follows. Figure 9 As shown.

[0119] The bright field in the figure represents the cell distribution under bright field conditions; the 647nm field represents the cell distribution under fluorescent field conditions, where red fluorescence is clearly visible within the cells; the merge image is the image after superimposing the bright field and fluorescent field. As can be seen from the figure, compared with the Gd-CDs group, the Gd-CDs-TA group showed a higher intensity of red fluorescence within the cells, indicating that the uptake of Gd-CDs-TA in Hepa1-6 cells was significantly increased compared to Gd-CDs, and that Gd-CDs-TA possesses fluorescence imaging capabilities.

[0120] Hepa1-6 cells in good growth condition were cultured overnight in 6-well plates. Different concentrations (0, 0.1, 0.2, 0.4, 0.8 mg / mL) of Gd-CDs-TA solution were added to each well and incubated for 4 h. The cells were washed with PBS, digested with trypsin, centrifuged in EP tubes, redispersed in 1% agarose solution, gelled, and then scanned by magnetic resonance imaging (MR) using a magnetic resonance scanner.

[0121] Figure 10 In the study, the MR imaging signal intensity was positively correlated with the concentration of Gd-CDs-TA, indicating that Gd-CDs-TA has MR capability and can be used as an MR contrast agent, and has a high relaxation rate.

[0122] Example 12

[0123] Hepa1-6 cells were planted at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density in six-well plates and incubated overnight.

[0124] No treatment was performed on the Saline group. Gd-CDs powder was added to the cells in the Gd-CDs group, and Gd-CDs-TA was added to the cells in the Gd-CDs-TA group. After incubation for 4 hours, the cells were washed three times with PBS, digested with trypsin, and centrifuged to remove the supernatant.

[0125] Cells were resuspended in 1 mL PBS and transferred to 1.5 mL EP tubes. An 808 nm laser (1 W / cm²) was used for filtration. 2 Cells in the Saline group, Gd-CDs group, and Gd-CDs-TA group were irradiated for 5 minutes, and the temperature changes inside the EP tube were recorded using a photothermal imaging system, with images taken every 1 minute.

[0126] Figure 11 The results showed that, compared with the Saline group, infrared thermal imagers could monitor in real time the temperature of Hepa1-6 cells incubated with Gd-CDs and Gd-CDs-TA increased over time after laser irradiation. Figure 11 b further showed that the temperature increase was most significant in the Gd-CDs-TA group, providing a basis for subsequent photothermal therapy.

[0127] Example 13

[0128] With 1×10 6 A suspension of Hepa1-6 cells was subcutaneously injected into the right anterior axilla of C57BL / 6J mice to establish a tumor-bearing mouse model carrying Hepa1-6 tumors.

[0129] Gd-CDs-TA solution was injected into tumor-bearing mice at a dose of 10 mg / kg. The mice were observed using a small animal in vivo imaging system, and the in vivo fluorescence signals were recorded at 0, 2, 4, 6, 12, and 24 hours.

[0130] from Figure 12 As can be seen from Figure a, a significant fluorescence signal began to appear at the tumor site 4 hours after injection, and reached its peak at 6 hours, indicating that Gd-CDs-TA can be effectively accumulated at the tumor site through the EPR effect.

[0131] Biodistribution in in vivo tissues is also a key factor in assessing the biocompatibility of Gd-CDs-TA. C57BL / 6J mice were sacrificed at different time points after Gd-CDs-TA injection, and major organs (brain, heart, liver, spleen, lung, kidney, and bladder) were collected at each time point. Fluorescence imaging of the isolated tissues was used to study their biodistribution.

[0132] like Figure 12 As shown in b, the fluorescence intensity of the tumor tissue was significantly higher than that of other major organs at 6 h, indicating good tumor targeting.

[0133] Meanwhile, detectable signals were also observed in the liver, kidneys, and lungs. Notably, the fluorescence signal in the lungs significantly decreased within 24 hours, indicating that Gd-CDs-TA is mainly cleared by the liver and kidneys, further demonstrating its good metabolism and strong biocompatibility.

[0134] Subsequently, C57BL / 6J tumor-bearing mice were first scanned using a 3.0T magnetic resonance scanner, and then a dose of 10 mg / Kg of Gd-CDs-TA solution was injected into the tumor-bearing mice via the tail vein. The tumor-bearing mice were scanned again 6 hours later.

[0135] from Figure 13 As can be seen, compared with before injection, a short T1 high signal shadow was visible in the tumor area 6 hours after injection, indicating that the Gd-CDs-TA of the present invention has the characteristic of being able to target liver cancer tissue for MR imaging.

[0136] Simultaneously, physiological saline, Gd-CDs, and Gd-CDs-TA were injected into tumor-bearing mice via tail vein administration. Twelve hours later, the tumors were examined using a laser (808 nm, 1 W / cm²). 2 Photothermal imaging of tumor-bearing mice.

[0137] Using an infrared thermal imager to record temperature changes at the tumor site in real time, the results showed ( Figure 14(a, b) The tumor surface temperature in mice injected with Gd-CDs-TA rapidly increased by 29.4℃ within 5 minutes, which was significantly higher than that in the Saline and Gd-CDs groups, confirming that Gd-CDs-TA still maintains efficient photothermal conversion performance in vivo and can achieve real-time temperature monitoring during photothermal therapy.

[0138] Example 14

[0139] Six experimental groups were set up: Control (G1), Gd-CDs (G2), Gd-CDs-TA (G3), Laser (G4), Gd-CDs+Laser (G5), and Gd-CDs-TA+Laser (G6) to evaluate the in vitro antitumor activity of Gd-CDs-TA on Hepa1-6 cells.

[0140] First, the inhibitory effects of Gd-CDs and Gd-CDs-TA on the proliferation of Hepa1-6 cells were observed using the CCK-8 assay. Then, the targeted inhibitory ability of Gd-CDs-TA on Hepa1-6 cells was further investigated to evaluate its in vitro antitumor activity.

[0141] In this study, Gd-CDs powder was added to cells in the Gd-CDs group and the Gd-CDs+Laser group, while Gd-CDs-TA was added to cells in the Gd-CDs-TA group and the Gd-CDs-TA+Laser group. After co-incubation for 4 hours, the Laser, Gd-CDs+Laser, and Gd-CDs-TA+Laser groups were subjected to 808nm laser (1W / cm²) treatment. 2 The cells were irradiated for 5 minutes, while the Control group received no treatment and were then cultured for 20 hours.

[0142] Figure 15 The figure shows the survival rate of Hepa1-6 cells after 24 hours of incubation in different experimental groups. It shows that compared with other groups, only the Gd-CDs-TA combined with laser irradiation group showed significant cytotoxicity, with the cell survival rate dropping to 2.5%. In contrast, the cell survival rates of the Control group and the Gd-CDs group remained above 80%, indicating that Gd-CDs alone have a weak killing effect on tumor cells.

[0143] Furthermore, the in vitro tumor treatment efficacy of Gd-CDs-TA was evaluated using a live-dead cell staining assay.

[0144] Hepa1-6 cells (5×10) 4Cells (cells / well) were seeded in 12-well plates and cultured overnight, divided into 6 identical groups. 200 μg / mL of Gd-CDs was added to the Gd-CDs and Gd-CDs+Laser groups, while 200 μg / mL of Gd-CDs-TA was added to the Gd-CDs-TA and Gd-CDs-TA+Laser groups. After co-incubation for 4 hours, the Laser, Gd-CDs+Laser, and Gd-CDs-TA+Laser groups were subjected to 808 nm laser (1 W / cm²) treatment. 2 Irradiate for 5 min, then continue culturing for 20 h. Aspirate the culture medium, wash the cells twice with PBS, add Calcein-AM and propidium iodide (PI) solution to stain the cells, incubate at 37°C in the dark for 30 min, aspirate the dye, and fix with paraformaldehyde.

[0145] Cell status was observed and identified under a fluorescence microscope. Cells showing green fluorescence after Calcein-AM staining were considered live cells, while cells showing red fluorescence after PI staining were considered dead cells.

[0146] Figure 16 The results showed that cells co-cultured in the Control group and the Gd-CDs group exhibited green fluorescence with virtually no red fluorescence, indicating that the cells treated by these two methods were mainly viable cells. A small number of red cells appeared in the Gd-CDs-TA group, indicating that the Gd-CDs-TA nanoplatform had less ability to damage cells, further demonstrating its low toxicity.

[0147] Cells in the Laser group treated with near-infrared laser showed significantly enhanced red fluorescence, indicating that near-infrared laser has a damaging effect on Hepa1-6 cells. When near-infrared laser was combined with Gd-CDs and Gd-CDs-TA, the cells exhibited clearly visible and intense red fluorescence, with most cells in an apoptotic state, demonstrating a significant killing effect. In particular, the proportion of dead cells was significantly increased and the number of live cells was significantly reduced in the Gd-CDs-TA+Laser group, forming a stark contrast to the Control group and the Gd-CDs group.

[0148] same, Figure 16 The fluorescence intensity analysis of b directly confirms the highly efficient killing ability of Gd-CDs-TA combined with laser irradiation on Hepa1-6 cells.

[0149] Figure 17Further flow cytometry (FCM) using Annexin V-FITC / PI double staining quantified the apoptosis rates of Hepa1-6 cells induced by different treatments. The results showed that, without laser irradiation, neither Gd-CDs nor Gd-CDs-TA treatments induced significant apoptosis. However, after laser irradiation, the apoptosis rate in the Gd-CDs+Laser group increased to 43.51%, while the apoptosis rate in the Gd-CDs-TA+Laser group reached a high of 74.49%, significantly higher than other groups. This result indicates that Gd-CDs-TA-mediated photothermal therapy can efficiently induce tumor cell apoptosis, and its effect is significantly superior to that of unloaded Gd-CDs. This result suggests that loaded tumor antigens can synergistically enhance the induction of immunogenic cell death, thereby achieving efficient tumor killing.

[0150] The above data indicate that Gd-CDs-TA itself has a weak killing ability against tumor cells, but it can enhance the targeted killing effect of immunotherapy by combining photothermal effects.

[0151] Example 15

[0152] Tumor cells undergoing immunogenic cell death (ICD) release immunogenic damage-associated molecular patterns (DAMPs) to trigger a long-term protective anti-tumor response. In this invention, photothermal therapy-induced ICD is a key component of the treatment-induced anti-tumor immune response; therefore, this study investigates whether Gd-CDs-TA can induce ICD in Hepa1-6 cells under laser irradiation.

[0153] HMGB1 release is one of the hallmark features of ICD (Intracytoplasmic Deposition) in cells. HMGB1 can bind to TLR4 on dendritic cells (DCs), promoting antigen processing and presentation. Therefore, immunofluorescence was used to detect HMGB1 release.

[0154] Hepa1-6 cells were dispersed in complete culture medium at a concentration of 1×10⁶. 4 Cell suspensions of 1 cell / mL were collected, and 2 mL was transferred to a confocal culture dish. Corresponding concentrations of Gd-CDs and Gd-CDs-TA were added to the experimental groups, respectively. After 4 hours of incubation, a dose of 1 W / cm² was used. 2 Cell culture plates were treated with near-infrared laser, and the blank group was replaced with an equal volume of complete culture medium, and then cultured for another 20 hours.

[0155] Cells from each group were collected, fixed with 4% formaldehyde, and then added to 0.1% Triton X-100 in PBS solution. Cell membranes were perforated for 30 min at room temperature, followed by blocking with 10% BSA for 1 h. Primary antibody was then added, and the cells were incubated at room temperature for 2 h, followed by incubation with fluorescently conjugated secondary antibody at room temperature for 1 h. Next, DAPI was added to stain the nuclei for 10 min. Finally, images were taken using a laser confocal microscopy instrument. Green fluorescence indicated the release of HMGB1, and blue fluorescence indicated the localization of the cell nucleus by the DAPI dye.

[0156] The results are as follows Figure 18 As shown, compared to the Control group, there was no significant difference in the green fluorescence intensity of HMGB1 in the cell nuclei between the Gd-CDs group and the Gd-CDs-TA group, indicating that Gd-CDs and Gd-CDs-TA alone had no significant effect on HMGB1 exposure. Compared to the Gd-CDs group, the green fluorescence of HMGB1 in the cell nuclei was significantly weakened in the Laser, Gd-CDs+Laser, and Gd-CDs-TA+Laser groups, proving that near-infrared laser irradiation can promote the release of HMGB1 from the cell nucleus, with the most significant weakening of green fluorescence in the Gd-CDs-TA+Laser group. Therefore, near-infrared laser irradiation of Gd-CDs-TA can lead to ICD.

[0157] During ICD (Intracytoplasmic Deposition), the cellular reticular formation (CRT) can migrate from the endoplasmic reticulum to the cell membrane, and exposed CRT promotes phagocytosis by antigen-presenting cells. Therefore, immunofluorescence is used to detect CRT exposure.

[0158] Hepa1-6 cells were dispersed in complete culture medium at a concentration of 1×10⁶. 4 Cell suspensions of 1 cell / mL were collected, and 2 mL was transferred to a confocal culture dish. Corresponding concentrations of Gd-CDs and Gd-CDs-TA were added to the experimental groups, respectively. After 4 hours of incubation, a dose of 1 W / cm² was used. 2 Cell culture plates were treated with near-infrared laser, and the blank group was replaced with an equal volume of complete culture medium, and then cultured for another 20 hours.

[0159] Cells from each group were collected, fixed with 4% formaldehyde, and blocked with 10% BSA for 1 hour. Primary antibodies were added and incubated at room temperature for 2 hours, followed by incubation with fluorescently conjugated secondary antibodies at room temperature for 1 hour. Next, DAPI was added to stain the nuclei for 10 minutes. Finally, images were taken using a laser confocal microscopy system. Green fluorescence indicates CRT exposure, while blue fluorescence represents the localization of the cell nucleus by the DAPI dye.

[0160] The results are as follows Figure 18As shown, compared to the Control group, there was no significant difference in CRT exposure between the Gd-CDs group and the Gd-CDs-TA group, indicating that Gd-CDs and Gd-CDs-TA alone did not have a significant effect on CRT exposure. Compared to the Gd-CDs group, the Laser, Gd-CDs+Laser, and Gd-CDs-TA+Laser groups showed significantly increased CRT exposure, demonstrating that near-infrared laser irradiation can promote CRT exposure. The Gd-CDs-TA+Laser group showed a significantly increased CRT exposure on the tumor cell surface compared to other groups. Therefore, near-infrared laser irradiation of Gd-CDs-TA can lead to ICD (intracytoplasmic disease).

[0161] Example 16

[0162] Using a 0.4 μm pore size Transwell system, Hepa1-6 cells were seeded in the upper chamber and treated as Control, Gd-CDs, Gd-CDs-TA, Laser, Gd-CDs+Laser, and Gd-CDs-TA+Laser, respectively.

[0163] The material concentration was 200 μg / mL, and after incubation for 4 hours, it was washed three times with PBS; the laser group used 808 nm and 1 W / cm². 2 Irradiate with laser for 5 minutes.

[0164] Immature dendritic cells derived from mouse bone marrow were seeded into the lower chamber and co-cultured for 24 hours. The cells were then stained with CD11c-FITC, CD80-PE, and CD86-APC antibodies, and CD11c was detected using FCM. + CD80 + CD86 + The proportion of mature DCs.

[0165] The results are as follows Figure 19 As shown, the proportion of mature DCs in the Gd-CDs-TA+Laser group reached 30.5%, which was significantly higher than that in other groups.

[0166] TNF-α, IL-6, and IFN-γ in the culture medium were detected by ELISA, and the results are as follows: Figure 20 As shown, compared with the Control group, the levels of the above factors in the Gd-CDs-TA+Laser group increased by 8.6-fold, 7.2-fold, and 2.5-fold, respectively, indicating that this treatment can promote DC maturation and the release of immune-related cytokines.

[0167] Example 17

[0168] To perform in vivo tumor treatment, a tumor with a volume of 100 mm was... 3 The subcutaneous tumor-bearing mice were randomly divided into the following 6 groups:

[0169] G1) Control group; G2) Gd-CDs group; G3) Gd-CDs-TA group; G4) Laser group; G5) Gd-CDs+Laser group; G6) Gd-CDs-TA+Laser group.

[0170] Twelve hours after tail vein injection on days 0 and 3, the tumor sites of the corresponding groups of mice were irradiated with laser (808nm, 1.0W / cm²). 2 The experiment ended on day 14 after 5 minutes. During the treatment period, the long and short diameters of the tumor were measured every 2 days using calipers to calculate the tumor volume and tumor inhibition rate, and the mice were weighed.

[0171] Fourteen days after treatment, mice were anesthetized with tribromoethanol, and blood was collected to detect complete blood count and blood biochemical indicators, and to assess red blood cell (RBC), white blood cell (WBC), platelet (PLT), hemoglobin concentration (HGB), and hematocrit (HCT) levels. Liver and kidney function parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CREA), and blood urea nitrogen (UREA), were monitored.

[0172] Tissue sections were prepared from the heart, liver, spleen, lungs, and kidneys. The tissue sections were observed under a microscope to assess the pathological differences between the organs in each group.

[0173] Figure 21 a represents the trend of tumor volume change in different treatment groups during the treatment cycle. Figure 21 b shows photographs of mice in different treatment groups after treatment. It can be seen that tumor growth was rapid in the Control group and the Gd-CDs group, while tumor growth was slightly inhibited in the Gd-CDs-TA group. The Laser group and the Gd-CDs+Laser group also only partially inhibited tumor growth, indicating that the therapeutic effect of Gd-CDs alone under laser irradiation is limited.

[0174] Notably, tumor growth was most significantly inhibited in the Gd-CDs-TA+Laser group. Simultaneously, the weight of the isolated tumor ( Figure 22 a) and photos ( Figure 22 (b) This conclusion is confirmed in a more intuitive and clear way.

[0175] To quantitatively verify the therapeutic efficiency of Gd-CDs-TA under laser irradiation, Figure 23The calculated tumor growth inhibition rate (TGI) is presented. The inhibition rate of the Laser group alone was not significant (65%), while the Gd-CDs-TA+Laser group had a significant TGI rate (98%), exceeding that of the Gd-CDs-TA group alone (68%). Therefore, it is demonstrated that the Gd-CDs-TA+Laser group achieved the best anti-tumor effect through the synergistic effect of photothermal ablation and in situ vaccine.

[0176] Weight gain curves are also an important indicator for assessing in vivo biocompatibility, such as... Figure 24 As shown, there were no significant differences in body weight among the C57BL / 6J mice in each group during the treatment period.

[0177] After 14 days of treatment, no abnormalities were observed in the images of the major organs of mice in each group after hematoxylin and eosin (H&E) staining. Figure 25 All blood biochemical indicators were within the normal range. Figure 26 These findings indicate that the aforementioned treatment regimens have good biocompatibility in vivo.

[0178] Example 18

[0179] Tumor tissues, spleens, and draining lymph nodes of mice from each group were collected after 14 days of treatment in Example 17, and single-cell suspensions were prepared. FCM analysis was used to determine the proportion of mature dendritic cells (DCs) in the lymph nodes, detecting markers CD11c, CD80, and CD86; the proportion of CD8+ T cells in the tumor tissue was analyzed, detecting markers CD45, CD3, CD4, and CD8; and the proportion of Treg cells in the tumor tissue was analyzed, detecting markers CD45, CD4, CD25, and Foxp3.

[0180] Serum from the eyes of mice in each group was collected, and TNF-α, IFN-γ, IL-6 and IL-10 were measured by ELISA.

[0181] The results are as follows Figure 27 As shown, the percentage of mature DCs in the Gd-CDs-TA and Gd-CDs+Laser groups increased to 34.1% and 42.9%, respectively, which were 2.16 times and 2.72 times higher than the Control group (15.8%). Notably, the increase in mature DCs in the Gd-CDs-TA+Laser group was the most significant, at 68.8%, exceeding the Gd-CDs-TA and Gd-CDs+Laser groups by 2.02 times and 1.60 times, respectively.

[0182] Meanwhile, the proportion of CD8+ T cells in tumor tissue was significantly increased in the Gd-CDs-TA+Laser group (33.4%), while the proportion of Tregs (2.57%) was significantly lower than in other groups. Figure 28 ,29).

[0183] Serum test results as follows Figure 30 As shown, the Gd-CDs-TA+Laser group showed elevated levels of TNF-α, IFN-γ, and IL-6, while the immunosuppressive factor IL-10 decreased, indicating that the nanovaccine combined with photothermal therapy can promote DC maturation and effector T cell infiltration, and alleviate Tregs-mediated immunosuppression.

[0184] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanovaccine based on tumor antigen covalently coupled with gadolinium-doped carbon dots, wherein the nanovaccine is a Gd-CDs-TA complex, composed of gadolinium-doped carbon dots Gd-CDs and tumor antigen TA; The Gd-CDs were prepared by microwave heating reaction of indocyanine green, citric acid, polyethylene glycol and gadopentetate, wherein Gd is in the form of Gd 3+ The Gd-CDs are coordinated and doped with diethylenetriaminepentaacetic acid in a carbon dot structure, and the Gd-CDs have near-infrared fluorescence emission, magnetic resonance imaging and photothermal conversion functions. The Gd-CDs and TA are covalently linked by amide bonds, which are formed by the condensation of carboxyl groups on the surface of Gd-CDs and amino groups in TA. The Gd-CDs-TA complex has a particle size of 35-43 nm, a TA protein content of 25-35%, a surface zeta potential that is at least 2 mV lower than that of Gd-CDs, and retains the fluorescence emission peak of Gd-CDs at 708 nm under 660 nm excitation and the near-infrared absorption in the range of 750-810 nm.

2. The nanovaccine according to claim 1, characterized in that... In the Gd-CDs-TA complex, the mass ratio of TA to Gd-CDs, based on the protein content of TA, is (1.5–3):

1.

3. The nanovaccine according to claim 1, characterized in that... The tumor antigen TA is extracted from tumor cells, which are selected from mouse liver cancer cells Hepa1-6, human liver cancer cells HepG2, mouse breast cancer cells 4T1, or human lung adenocarcinoma cells A549.

4. The method for preparing the nanovaccine based on tumor antigen covalently coupled gadolinium-doped carbon dots as described in claim 1, comprising: 1) Gadolinium-doped carbon dots (Gd-CDs) were prepared by dispersing indocyanine green, citric acid, polyethylene glycol and gadopentetate in water and reacting them with microwave heating. 2) Add Gd-CDs to cultured tumor cells to a final concentration of 150-250 μg / mL, incubate for 4-6 h, irradiate with 808 nm laser for 3-5 min, and continue incubation in 1640 medium without fetal bovine serum for 36-48 h. Collect the supernatant to obtain tumor antigen TA. 3) After activating the carboxyl groups on the surface of Gd-CDs with EDC and NHS, they are co-incubated with TA to form amide bonds between the carboxyl groups on the surface of Gd-CDs and the amino groups in TA, thus obtaining the covalently coupled nanovaccine Gd-CDs-TA.

5. The preparation method according to claim 4, characterized in that: Gd-CDs were dispersed in PBS, and EDC and NHS were added for activation at room temperature for 2-4 hours. The mass ratio of EDC to Gd-CDs was (2-3):1, and the mass ratio of NHS to Gd-CDs was (0.8-1.2):

1.

6. The preparation method according to claim 4, characterized in that: The activated Gd-CDs were co-incubated with TA for 12–18 hours.

7. The preparation method according to claim 4, characterized in that: The power density of the laser is 0.5–1.5 W / cm². 2 .

8. The application of the nanovaccine based on tumor antigen covalently coupled gadolinium-doped carbon dots as described in claim 1 as a fluorescence / magnetic resonance / photothermal multimodal imaging probe.

9. The application of the nanovaccine based on tumor antigen covalently coupled with gadolinium-doped carbon dots as described in claim 1 in the preparation of a drug that inhibits the growth of tumor cells.

10. The application according to claim 9, characterized in that: The drug in question is used for photothermal-immunotherapy combined with hepatocellular carcinoma.

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