Fapi and peg co-modified ultrasmall gold nanoparticles, and preparation method and application thereof

Abstract

The application discloses FAPI and PEG co-modified ultra-small gold nanoparticles and a preparation method and application thereof. The preparation method specifically comprises the following steps: taking chloroauric acid as a gold source, taking a thiolated FAPI molecule as a first targeting ligand, and first preparing FAPI-modified ultra-small gold nanoparticles under the reduction condition of sodium borohydride; then introducing thiol polyethylene glycol with different molecular weights and different proportions as a second stabilizing ligand to obtain a series of FAPI and PEG co-modified ultra-small gold nanoparticles. The ultra-small gold nanoparticles provided by the application have the characteristics of near-infrared luminescence, small particle size, good dispersibility, good biocompatibility and low tumor imaging background. The FAPI ligand can recognize fibroblast activation protein positive tumor-related fibroblasts, and the PEG ligand can improve the colloidal stability and in-vivo circulation behavior of the nanoparticles, so that the differentiation of the fibroblast activation level in the microenvironment of thyroid tumors can be realized.

Description

Technical Field

[0001] This invention belongs to the fields of functional nanomaterials and tumor molecular imaging, specifically involving ultrasmall gold nanoparticles co-modified with FAPI and PEG, their preparation methods, and applications. Background Technology

[0002] Thyroid cancer is one of the most common malignant tumors of the endocrine system. Most differentiated thyroid cancers can achieve a good prognosis after surgery, radioactive iodine therapy, and endocrine suppression therapy. However, undifferentiated and dedifferentiated thyroid cancers are highly invasive, progress rapidly, and have a high risk of recurrence and metastasis. They also have limited response to traditional treatments. Clinically, there is still a lack of highly sensitive, low-background imaging and diagnostic tools that can reflect the differences in the tumor microenvironment.

[0003] Tumor tissue is not solely composed of tumor cells; tumor-associated fibroblasts are an important stromal cell type in the solid tumor microenvironment. Fibroblast activation protein (FAP) is highly expressed in activated fibroblasts of various solid tumors, but expressed at low levels in most normal tissues. Therefore, FAP has become an important molecular target for tumor microenvironment-targeted imaging and therapy. FAPI-like molecules can bind to FAP-positive fibroblasts, possessing the potential to recognize the tumor stroma and reflect the activation state of the tumor microenvironment.

[0004] While existing small-molecule FAPI imaging agents possess rapid target recognition capabilities, their short in vivo residence time limits the imaging window, and they struggle to simultaneously achieve sustained tumor tissue retention, low-background imaging, and multifunctional surface modulation. Nanomaterials can regulate in vivo distribution behavior through size, surface charge, ligand density, and hydrophilicity; however, traditional nanoparticles often suffer from problems such as large particle size, high uptake by the reticuloendothelial system, significant risk of long-term retention, or insufficient exposure of target ligands. Mol.Pharmaceutics 2025,22,1498 1506).

[0005] Ultrasmall gold nanoparticles possess advantages such as high chemical stability, easily modifiable surface, tunable near-infrared luminescence, strong resistance to photobleaching, and potential for good renal clearance. By introducing targeting and stabilizing ligands onto their surface, tumor imaging performance can be improved while maintaining their small size advantage. Among them, FAPI ligands can provide targeted recognition of FAP-positive fibroblasts, while PEG ligands can improve aqueous stability, reduce nonspecific adsorption, and improve in vivo circulation behavior.

[0006] However, the ratio of FAPI and PEG ligands on the surface of ultrasmall gold nanoparticles and the PEG chain length jointly affect the nanoparticles' luminescence properties, colloidal stability, surface potential, in vivo circulation, tumor penetration, and FAPI targeted recognition. If the FAPI ratio is too high, the material may experience non-specific protein adsorption and accelerated in vivo clearance; if the PEG ratio is too high or the chain length is too long, although the cycling stability is enhanced, the targeting ligand may be partially blocked.

[0007] Therefore, developing an ultrasmall gold nanoparticle synergistically modified with FAPI and PEG, and systematically comparing the effects of different PEG chain lengths and ligand ratios on thyroid tumor imaging, is of great significance for achieving targeted imaging of the thyroid tumor microenvironment. Summary of the Invention

[0008] To overcome the limitations of existing technologies, such as the short imaging window of FAPI small molecules and the difficulty in simultaneously achieving tumor targeting and in vivo safety with conventional nanomaterials, this invention provides an ultrasmall gold nanoparticle co-modified with FAPI and PEG, along with its preparation method and applications. This material utilizes FAPI ligands to recognize FAPI-positive fibroblasts in the thyroid tumor microenvironment, and utilizes PEG ligands to improve aqueous dispersibility, reduce non-specific adsorption, and prolong in vivo circulation, thereby enhancing the targeted imaging effect of thyroid tumors.

[0009] The present invention is achieved through the following technical solution.

[0010] This invention provides a method for preparing ultrasmall gold nanoparticles co-modified with FAPI and PEG, comprising the following steps: (1) Mix the aqueous solution of chloroauric acid with the FAPI derivative, an inhibitor of fibroblast activation proteins, until homogeneous; (2) A reducing agent was added to carry out a reduction reaction to obtain FAPI-modified ultrasmall gold nanoparticles; (3) Add mercapto polyethylene glycol to carry out surface ligand regulation reaction; (4) After the reaction is completed, the ultrasmall gold nanoparticles are obtained by dialysis and / or ultrafiltration purification to obtain the fibroblast activation protein inhibitor FAPI and polyethylene glycol PEG co-modified.

[0011] Preferably, in step (1), the molar ratio of chloroauric acid to FAPI derivative is 1:2-1:6.

[0012] Preferably, in step (1), the FAPI derivative is an FAPI-type molecule with a gold surface anchoring group, which includes a thiol group, a disulfide bond, a thioctic acid group, or other groups that can form a stabilizing effect with the gold surface.

[0013] Preferably, in step (2), the reducing agent is one or more of sodium borohydride, ascorbic acid, citrate and triethylamine borane.

[0014] Preferably, in step (3), the molecular weight of mercapto polyethylene glycol is 500-5000 Da; the proportion of mercapto polyethylene glycol in the total molar amount of FAPI derivative and mercapto polyethylene glycol is 10%-95%.

[0015] More preferably, the mercapto polyethylene glycol is HS-PEG1K or HS-PEG2K.

[0016] Preferably, in step (3), the proportion of mercapto polyethylene glycol in the total molar amount of FAPI derivative and mercapto polyethylene glycol is 10%-95%.

[0017] More preferably, the proportion of mercapto polyethylene glycol in the total molar amount of FAPI derivative and mercapto polyethylene glycol is 30%, 60% or 90%.

[0018] The present invention also provides an ultrasmall gold nanoparticle co-modified with FAPI and PEG prepared by the above preparation method.

[0019] Preferably, the ultrasmall gold nanoparticles co-modified with FAPI and PEG are monodisperse nanoparticles with a gold core particle size of 1-3 nm and a hydrated particle size of 3-15 nm. They maintain good dispersion in aqueous solution, have a negative or near-neutral surface potential, and exhibit near-infrared luminescence characteristics, making them suitable for in vivo near-infrared imaging.

[0020] This invention also provides the application of the above-mentioned FAPI and PEG co-modified ultrasmall gold nanoparticles in the preparation of thyroid tumor targeted imaging agents.

[0021] Preferably, FAPI and PEG co-modified ultrasmall gold nanoparticles can be used to prepare imaging formulations for identifying or assessing the expression levels of fibroblast activation proteins in the thyroid tumor microenvironment.

[0022] Preferably, the thyroid tumor-targeting imaging formulation can be used to differentiate between thyroid tumor models with low and high fibroblast expression.

[0023] Preferably, the thyroid tumor includes undifferentiated thyroid carcinoma, dedifferentiated thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, or a xenograft model thereof.

[0024] Preferably, the imaging includes near-infrared fluorescence imaging, near-infrared II imaging, in vitro tissue imaging, detection of gold content in tumor tissue, or silver-enhanced staining for localization.

[0025] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention modifies ultra-small gold nanoparticles with FAPI and PEG dual ligands, while taking into account both FAP targeted recognition and stable circulation in vivo.

[0026] (2) This invention obtains a series of materials from F1-AuNPs to F6-AuNPs by regulating the PEG chain length and PEG ratio, and the materials can be screened according to the FAP expression level and imaging window in the tumor microenvironment.

[0027] (3) The ultra-small gold nanoparticles modified with FAPI and PEG provided by the present invention have the advantages of near-infrared luminescence, low imaging background, good visualization effect of tumor tissue and clear tissue localization.

[0028] (4) In the present invention, the targeting differences of ultra-small gold nanoparticles co-modified by FAPI and PEG were verified in the m1TCp11 low expression thyroid tumor model and the BPC high expression thyroid tumor model, which can reflect the differences in the activation level of fibroblasts in the thyroid tumor microenvironment.

[0029] (5) No obvious tissue damage was observed in HE staining of major organs by the FAPI and PEG co-modified ultrasmall gold nanoparticles provided by the present invention, suggesting that they have good preliminary biosafety. Attached Figure Description

[0030] Figure 1 A schematic diagram of the process for preparing ultrasmall gold nanoparticles co-modified with FAPI and PEG and using them for in vivo near-infrared imaging in a tumor model mouse.

[0031] Figure 2 The ultraviolet absorption spectrum and near-infrared emission spectrum of F1-AuNPs prepared in Example 2 are shown.

[0032] Figure 3 The ultraviolet absorption spectrum and near-infrared emission spectrum of F2-AuNPs prepared in Example 2 are shown.

[0033] Figure 4 The ultraviolet absorption spectrum and near-infrared emission spectrum of F3-AuNPs prepared in Example 2 are shown.

[0034] Figure 5 The ultraviolet absorption spectrum and near-infrared emission spectrum of F4-AuNPs prepared in Example 2 are shown.

[0035] Figure 6 The ultraviolet absorption spectrum and near-infrared emission spectrum of F5-AuNPs prepared in Example 2 are shown.

[0036] Figure 7 The ultraviolet absorption spectrum and near-infrared emission spectrum of F6-AuNPs prepared in Example 2 are shown.

[0037] Figure 8The diagram shows the core size and hydrated particle size of F1-AuNPs to F6-AuNPs prepared in Example 2.

[0038] Figure 9 The zeta potential statistics of F1-AuNPs to F6-AuNPs prepared in Example 2 are shown.

[0039] Figure 10 This is an in vivo near-infrared image of F1-AuNPs to F6-AuNPs in a thyroid tumor model with low expression of m1TCp11, as shown in Example 4.

[0040] Figure 11 The curves showing the change in tumor / background signal ratio over time in the m1TCp11-low-expression thyroid tumor model of F1-AuNPs to F6-AuNPs in Example 4 are shown.

[0041] Figure 12 This is a statistical graph showing the gold content of F1-AuNPs to F6-AuNPs in the m1TCp11-low-expression thyroid tumor model in Example 4.

[0042] Figure 13 This is a silver-enhanced staining image of F1-AuNPs to F6-AuNPs in thyroid tumor tissue with low m1TCp11 expression, as shown in Example 4.

[0043] Figure 14 The images show in vivo near-infrared images of the low-expression model and the high-expression model in Example 5.

[0044] Figure 15 The curves show the tumor / background signal ratio changes over time after F6-AuNPs were injected into the low-expression and high-expression models in Example 5.

[0045] Figure 16 This is a comparison of the gold content in tumor tissues of the low-expression model and the high-expression model 24 hours after injection of F6-AuNPs in Example 5.

[0046] Figure 17 This is a comparison of silver-enhanced staining of tumor tissues from the low-expression model and the high-expression model 24 hours after injection of F6-AuNPs in Example 5.

[0047] Figure 18 HE staining of mouse heart tissue after F1-AuNPs were injected into F6-AuNPs in Example 6.

[0048] Figure 19 HE staining of mouse liver tissue after F1-AuNPs were injected into F6-AuNPs in Example 6.

[0049] Figure 20HE staining of mouse spleen tissue after injection of F1-AuNPs into F6-AuNPs in Example 6.

[0050] Figure 21 HE staining of mouse lung tissue after injection of F1-AuNPs into F6-AuNPs in Example 6.

[0051] Figure 22 HE staining of mouse kidney tissue after F1-AuNPs were injected into F6-AuNPs in Example 6. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the FAPI ligands in the following embodiments are FAPI derivatives with gold surface anchoring groups, and the PEG ligands are mercapto polyethylene glycol. All animal experimental procedures were performed in accordance with relevant regulations on laboratory animal ethics.

[0053] Figure 1 A schematic diagram illustrating the preparation of FAPI and PEG co-modified ultrasmall gold nanoparticles and their application in in vivo near-infrared imaging of a tumor model mouse is shown. The following description, in conjunction with examples, further clarifies the process.

[0054] Example 1: Preparation of FAPI-modified ultrasmall gold nanoparticles In this embodiment, FAPI-modified ultrasmall gold nanoparticles are first prepared as precursor materials for subsequent PEG ligand regulation.

[0055] (1) Add 20 mM of chloroauric acid aqueous solution to the reaction flask, dilute with deionized water to 1 mM concentration, and stir evenly at room temperature.

[0056] (2) Add mercapto FAPI solution to the above chloroauric acid solution so that the molar ratio of chloroauric acid to mercapto FAPI is 1:6, and continue stirring for 10 min to allow mercapto FAPI to fully coordinate with gold ions.

[0057] (3) At room temperature (25°C), 50 mM of freshly prepared sodium borohydride solution was rapidly added to make the molar ratio of chloroauric acid: mercaptoFAPI: sodium borohydride 1:6:10. After addition, stirring was continued for 6 h. During the reaction, the color of the solution gradually changed, and crude product of FAPI-modified ultrasmall gold nanoparticles was obtained.

[0058] (4) After the reaction is completed, the reaction solution is placed in a dialysis bag and dialyzed for 24 hours to remove free FAPI ligands, inorganic salts and small molecule byproducts; then it is washed 10 times by centrifugation using an ultrafiltration tube and concentrated to obtain FAPI-modified ultrasmall gold nanoparticles, which are stored at 4°C for later use.

[0059] Example 2: Preparation of six types of FAPI and PEG co-modified ultrasmall gold nanoparticles Based on the FAPI-modified gold nanoparticles obtained in Example 1, thiol-modified polyethylene glycol with different chain lengths and proportions was further introduced to obtain a series of ultra-small gold nanoparticles co-modified by FAPI and PEG.

[0060] (1) The FAPI-modified gold nanoparticles obtained in Example 1 were divided into six groups, which were named F1, F2, F3, F4, F5 and F6 respectively.

[0061] (2) HS-PEG1K was added to groups F1, F2 and F3 respectively to carry out surface ligand regulation reaction, so that the proportion of HS-PEG1K in the total molar amount of mercapto FAPI and HS-PEG1K was 30%, 60% and 90% respectively. After stirring for 24 h, F1-AuNPs, F2-AuNPs and F3-AuNPs were obtained respectively.

[0062] (3) HS-PEG2K was added to groups F4, F5 and F6 respectively to carry out surface ligand regulation reaction, so that the proportion of HS-PEG2K in the total molar amount of mercapto FAPI and HS-PEG2K was 30%, 60% and 90% respectively. After stirring for 24 h, F4-AuNPs, F5-AuNPs and F6-AuNPs were obtained respectively.

[0063] (4) After the reaction was completed, the six reaction solutions were purified by dialysis and ultrafiltration to remove unbound ligands and concentrate them to obtain F1-AuNPs, F2-AuNPs, F3-AuNPs, F4-AuNPs, F5-AuNPs and F6-AuNPs that were stably dispersed in the aqueous phase, which are six kinds of FAPI and PEG co-modified ultrasmall gold nanoparticles.

[0064] Example 3: Characterization of the optical and colloidal properties of ultrasmall gold nanoparticles co-modified with six types of FAPI and PEG To verify the basic physicochemical properties of the six FAPI and PEG co-modified ultra-small gold nanoparticles, the F1-AuNPs to F6-AuNPs prepared in Example 2 were subjected to ultraviolet absorption spectroscopy, near-infrared emission spectroscopy, hydration particle size and zeta potential tests.

[0065] Specifically, the ultrasmall gold nanoparticles co-modified with six types of FAPI and PEG were diluted to 100 nM, and their absorption spectra were tested using a UV-Vis absorption spectrometer, their emission spectra were tested using near-infrared fluorescence imaging or spectroscopy, their hydrated particle size was tested using a dynamic light scattering instrument, and their zeta potential was tested using an electrophoretic light scattering method.

[0066] Figures 2-7The ultraviolet absorption and near-infrared emission spectra of F1-AuNPs to F6-AuNPs are shown.

[0067] Figures 2-7 The results showed that all six FAPI and PEG co-modified ultrasmall gold nanoparticles exhibited continuous absorption in the visible light region and significant emission signals in the near-infrared region. This indicates that the FAPI and PEG dual-ligand modification did not impair the optical luminescence properties of the gold nanoparticles. After different PEG chain lengths and different PEG ratios, the FAPI and PEG co-modified ultrasmall gold nanoparticles could still maintain the near-infrared optical properties of gold nanoparticles.

[0068] Figure 8 The diagram shows the statistics of core size and hydrated particle size for F1-AuNPs to F6-AuNPs. In fact, the upper curve represents the hydrated particle size data, and the lower curve represents the core size data.

[0069] Figure 8 The results showed that the core size distribution of F1-AuNPs to F6-AuNPs was in the range of about 1-2 nm, and the hydrated particle size (HD) distribution was in the range of about 5-10 nm, all of which are within the nanoscale range suitable for in vivo circulation and tumor penetration. Among them, the overall hydrated particle size of the PEG2K group was increased compared with that of the PEG1K group, indicating that the PEG chain length can effectively regulate the hydrated layer thickness of the nanoparticles.

[0070] Figure 9 The zeta potential statistics of F1-AuNPs to F6-AuNPs are shown.

[0071] Figure 9 The results showed that F1-AuNPs to F6-AuNPs all exhibited negative or near-neutral negative potentials, indicating that they have good aqueous dispersion stability and low risk of strong positive charge toxicity. As the PEG ratio increased, the absolute value of the surface potential decreased overall, indicating that the PEG layer can shield some surface charges and reduce non-specific interactions.

[0072] Example 4: Imaging Validation in a Thyroid Tumor Model with Low M1TCp11 Expression To verify the effects of different PEG chain lengths and ligand ratios on thyroid tumor imaging, this embodiment established an m1TCp11 thyroid tumor model as a low-expression model of fibroblasts, and performed in vivo near-infrared imaging, tumor signal statistics, tumor gold content detection, and silver-enhanced staining of tumor tissue.

[0073] (1) Take 6-week-old mice and subcutaneously inoculate them with m1TCp11 cells on the back or flank until the tumor volume reaches about 80-150 mm. 3 Experiments were conducted at that time.

[0074] (2) F1-AuNPs to F6-AuNPs were prepared into 2μM injection solution using sterile DPBS and injected into tumor-bearing mice via the tail vein.

[0075] (3) In vivo near-infrared imaging was performed at 1h, 3h, 6h, 12h, 24h and 48h after drug administration to record the signals of the tumor area and the normal skin background area, and the tumor / background signal ratio was calculated.

[0076] (4) Mice were sacrificed at the time point with higher imaging signal, tumor tissue was collected, and the gold content in the tumor tissue was determined by inductively coupled plasma mass spectrometry or equivalent gold element detection method. The distribution of FAPI and PEG co-modified ultrasmall gold nanoparticles in the tumor tissue was observed by tissue silver enhancement staining.

[0077] Figure 10 The in vivo near-infrared imaging of F1-AuNPs to F6-AuNPs in a thyroid tumor model with low expression of m1TCp11 is shown.

[0078] Figure 11 The curves showing the tumor / background signal ratio over time in a thyroid tumor model with low m1TCp11 expression of F1-AuNPs to F6-AuNPs are presented.

[0079] like Figure 10 and Figure 11 As shown, in the m1TCp11 low-expression model, all six FAPI and PEG-modified ultrasmall gold nanoparticles could generate certain near-infrared signals in the tumor region, but the tumor signals differed depending on the PEG chain length and ratio. The PEG2K modified group showed better overall imaging performance, with F6-AuNPs exhibiting higher tumor / background signals around 24 hours, indicating that longer PEG chains and higher PEG ratios are beneficial for the cyclic stability of the material and passive tumor enrichment in the low-expression model.

[0080] Figure 12 The graph shows the statistical content of gold in tumor tissues of F1-AuNPs to F6-AuNPs in a thyroid tumor model with low expression of m1TCp11.

[0081] like Figure 12 As shown, the gold content detection results of tumor tissue 24 hours after drug administration were consistent with the trend of in vivo imaging. F6-AuNPs had the highest tumor content in the m1TCp11 low expression model, approximately 5.3 ID% / g. This result indicates that in thyroid tumor models with low FAP expression or weak fibroblast targeting, the PEG chain length, PEG ratio, and in vivo circulation stability of the material contribute more significantly to tumor enrichment.

[0082] Figure 13The image shows a silver-enhanced staining pattern of F1-AuNPs to F6-AuNPs in thyroid tumor tissue with low m1TCp11 expression.

[0083] like Figure 13 As shown, silver-enhanced staining further confirmed that all six FAPI and PEG-modified ultrasmall gold nanoparticles could enter tumor tissue, and F6-AuNPs showed a more obvious distribution of silver-stained particles within the tumor tissue.

[0084] Example 5: Targeting Validation in a BPC-Overexpressing Thyroid Tumor Model To verify the targeting and recognition ability of FAPI and PEG co-modified ultrasmall gold nanoparticles on the FAP-high expression thyroid tumor microenvironment, this embodiment established a BPC thyroid tumor model as a fibroblast high-expression model and compared it with a m1TCp11 low-expression model. Based on the screening results in Example 4, F6-AuNPs with better imaging performance were selected as representative materials for comparison between high- and low-expression models.

[0085] (1) Establish thyroid tumor models with low m1TCp11 expression and high BPC expression respectively, and administer drugs when the tumor volumes of the two groups are similar.

[0086] (2) F6-AuNPs were prepared into a 2μM injection solution and injected into the two groups of tumor-bearing mice via the tail vein.

[0087] (3) In vivo near-infrared imaging was performed at 1h, 3h, 6h, 9h, 12h, 24h, 48h and 72h after drug administration to compare tumor signal changes in the high-expression model and the low-expression model.

[0088] (4) Tumor tissue was collected 24 hours after administration, the gold content of the tumor tissue was detected, and the distribution differences of F6-AuNPs in tumor tissues with different expression levels were compared by silver-enhanced staining.

[0089] Figure 14 In vivo near-infrared images of the low-expression model and the high-expression model are shown.

[0090] Figure 15 The tumor / background signal ratio curves after F6-AuNP injection in low-expression and high-expression models are shown as changes over time.

[0091] like Figures 14-15As shown, compared with the m1TCp11 low-expression model, the BPC high-expression model exhibited stronger near-infrared tumor signal and a higher tumor / background signal ratio at multiple time points, reaching a high level around 24 hours. These results indicate that F6-AuNPs not only rely on PEG-regulated in vivo circulation and tumor penetration, but also recognize FAP-positive tumor-associated fibroblasts through FAPI ligands, thereby achieving stronger targeted enrichment in thyroid tumor models with high fibroblast activation levels.

[0092] Figure 16 The image shows a comparison of the gold content in tumor tissues of low-expression and high-expression models 24 hours after injection of F6-AuNPs.

[0093] like Figure 16 As shown, 24 hours after drug administration, the gold content of F6-AuNPs in tumor tissue of the BPC high-expression model was approximately 1.72 times that of the low-expression model, indicating that FAPI-mediated active targeting can significantly improve the enrichment of materials in FAP-high-expression thyroid tumors.

[0094] Figure 17 The image shows a comparison of silver-enhanced staining of tumor tissues from low-expression and high-expression models 24 hours after injection of F6-AuNPs.

[0095] like Figure 17 As shown, the silver-enhanced staining results further revealed that more FAPI and PEG-modified ultrasmall gold nanoparticles were deposited and distributed in the tumor tissue of the high-expression model, indicating that the material of the present invention can reflect the targeting differences of the tumor microenvironment with high FAPI expression at the tissue level.

[0096] Example 6: Preliminary biosafety evaluation of F1-AuNPs to F6-AuNPs To evaluate the preliminary in vivo safety of the FAPI and PEG co-modified ultrasmall gold nanoparticles provided by this invention, this embodiment performed HE staining analysis on the major organs of mice after injection of F1-AuNPs to F6-AuNPs.

[0097] (1) F1-AuNPs to F6-AuNPs were prepared into 2μM injection solutions and injected into mice via the tail vein; the same volume of PBS was injected as a control and the group was called the PBS group.

[0098] (2) Three days after administration, mice were sacrificed and their main organs, such as heart, liver, spleen, lungs and kidneys, were taken, fixed, embedded, sectioned and stained with HE.

[0099] (3) Observe the structure of major organs and tissues under a microscope to assess whether there is obvious inflammatory infiltration, tissue necrosis, hemorrhage, cell structure destruction or other acute tissue damage.

[0100] Figures 18-22 HE staining images of mouse heart, liver, spleen, lung, and kidney tissues after injection of F1-AuNPs to F6-AuNPs are shown.

[0101] like Figures 18-22 As shown, after treatment with F1-AuNPs to F6-AuNPs, no obvious tissue structural abnormalities were observed in the major organs of mice (heart, liver, spleen, lungs, and kidneys) compared to the PBS group, indicating that the ultrasmall gold nanoparticles co-modified with FAPI and PEG provided in this invention have good preliminary biocompatibility and in vivo application safety.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, and simplifications made within the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for preparing ultrasmall gold nanoparticles co-modified with FAPI and PEG, characterized in that, Includes the following steps: (1) Mix the aqueous solution of chloroauric acid with the FAPI derivative, an inhibitor of fibroblast activation proteins, until homogeneous; (2) A reducing agent was added to carry out a reduction reaction to obtain FAPI-modified ultrasmall gold nanoparticles; (3) Add mercapto polyethylene glycol to carry out surface ligand regulation reaction; (4) After the reaction is completed, the ultrasmall gold nanoparticles are obtained by dialysis and / or ultrafiltration purification to obtain the fibroblast activation protein inhibitor FAPI and polyethylene glycol PEG co-modified.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of chloroauric acid to FAPI derivative is 1:2-1:

6.

3. The preparation method according to claim 1, characterized in that, In step (1), the FAPI derivative is an FAPI-type molecule with gold surface anchoring groups, including mercapto, disulfide bond, thioctic acid group or other groups that can form a stabilizing effect with the gold surface.

4. The preparation method according to claim 1, characterized in that, In step (2), the reducing agent is one or more of sodium borohydride, ascorbic acid, citrate and triethylamine borane.

5. The preparation method according to claim 1, characterized in that, In step (3), the molecular weight of the mercapto polyethylene glycol is 500-5000 Da; the proportion of the mercapto polyethylene glycol in the total molar amount of FAPI derivative and mercapto polyethylene glycol is 10%-95%.

6. FAPI and PEG co-modified ultrasmall gold nanoparticles prepared by the preparation method according to any one of claims 1-5.

7. The ultrasmall gold nanoparticles co-modified with FAPI and PEG according to claim 6, characterized in that, The FAPI and PEG co-modified ultrasmall gold nanoparticles are monodisperse nanoparticles with a gold core particle size of 1-3 nm, a hydrated particle size of 3-15 nm, a negative or near-neutral surface potential, and near-infrared luminescence characteristics.

8. The application of the FAPI and PEG co-modified ultrasmall gold nanoparticles as described in claim 7 in the preparation of thyroid tumor targeted imaging formulations.

9. The application according to claim 8, characterized in that, The thyroid tumors include undifferentiated thyroid carcinoma, dedifferentiated thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, or their xenograft models.

10. The application according to claim 8, characterized in that, The imaging includes near-infrared fluorescence imaging, near-infrared II imaging, in vitro tissue imaging, detection of gold content in tumor tissue, or silver-enhanced staining for localization.

Images