Chiral photoimmunoregulation gold nanoclusters, preparation method and application thereof

Abstract

The present application relates to a kind of chiral photoimmunoregulation gold nanoclusters and its preparation method and application, it is related to nanoprobe technical field.The structure general formula of the chiral photoimmunoregulation gold nanoclusters is Au 25 (SR) 18 , SR is thiol ligand, the thiol ligand is one or several in L-cysteine, D-cysteine, in the chiral photoimmunoregulation gold nanocluster, thiol ligand and gold atom form coordination structure by Au-S bond.The chiral photoimmunoregulation gold nanocluster of the present application has immunoregulation capacity, can use its optical characteristics as the photothermal treatment preparation or photodynamic therapy agent of tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of nanoprobe materials technology, and more specifically, to a chiral photoimmunomodulated gold nanocluster, its preparation method, and its application. Background Technology

[0002] Malignant tumors are the second leading cause of death worldwide. Data released by the World Health Organization (WHO) in 2023 shows that approximately 9.7 million deaths globally were related to malignant tumors, accounting for nearly one-sixth of all deaths that year. Current clinical treatments for malignant tumors include surgical resection, chemotherapy, and radiotherapy. However, these traditional treatments still face many challenges: chemotherapy drugs generally suffer from insufficient targeting, damaging normal tissue cells while killing tumor cells; radiotherapy easily leads to fibrosis in the irradiated area; and surgery often fails to completely remove all lesions. The primary goal of cancer treatment should be to improve treatment efficacy while minimizing adverse reactions. In recent years, the rapid development of nanomedicine has brought new research directions to the detection and treatment of malignant tumors. However, traditional nanomaterials, such as gold nanoparticles, still have limitations such as uneven particle size distribution and poor biocompatibility. Therefore, developing novel nanoprobes that can simultaneously achieve diagnostic and therapeutic functions is of significant research importance.

[0003] Atomic-scale precision gold nanoclusters (Au NCs) are ultra-small nanomaterials composed of a metallic core and an organic ligand shell. The core typically contains tens to hundreds of gold atoms, while the outer organic ligands not only maintain structural stability but also effectively prevent aggregation between core particles. Differences in the number of gold atoms in different gold nanoclusters lead to significant variations in their optical properties and other physicochemical properties. When the particle size of gold nanoclusters approaches their Fermi level de Broglie wavelength, they exhibit a pronounced quantum confinement effect, displaying specific absorption spectra and luminescence characteristics. These unique physicochemical properties make gold nanoclusters a highly promising candidate material for tumor diagnosis and treatment. Therefore, it is necessary to provide a gold nanocluster that can be fabricated into nanoprobes, possesses immunomodulatory capabilities, and whose optical properties can be utilized as a photothermal or photodynamic therapy agent for tumor treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a chiral photoimmunomodulatory gold nanocluster with immunomodulatory capabilities, which can be used as a photothermal therapy agent or photodynamic therapy agent for treating tumors by utilizing its optical properties.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution: A chiral photoimmunoregulatory gold nanocluster, wherein the general structural formula of the chiral photoimmunoregulatory gold nanocluster is Au. 25 (SR) 18 SR is a thiol ligand, which is one or more of L-cysteine ​​and D-cysteine. In the chiral photoimmunoregulated gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

[0006] The electronic effects of ligands and steric hindrance energy jointly regulate the nucleation kinetics and size distribution of Au NCs.

[0007] The type of ligand determines the cluster size and optical properties. L-cysteine ​​(L-CYS) and D-cysteine ​​(D-CYS) contain monothiol groups, amino and carboxylic acid groups, which enhance water solubility. 3-Mercaptopropionic acid (3MPA) contains a monothiol group, and the carboxylic acid group provides negative charge repulsion, reducing aggregation. This invention stabilizes the structure of gold nanoclusters through specific thiol ligands, preventing the aggregation and oxidation of gold nanoclusters. The Au of this invention... 25 Gold nanoclusters exhibit high stability, with strong surface rigidity of the Au core inhibiting cluster aggregation and oxidation, allowing for stable storage in physiological environments.

[0008] The chiral photoimmunomodulatory gold nanoclusters of this invention possess immunomodulatory capabilities and can be utilized as photothermal or photodynamic therapy agents for tumor treatment based on their optical properties. This is because gold nanoclusters prepared using L-cysteine ​​or D-cysteine ​​as thiol ligands can induce the formation of chiral signals at the gold-sulfur interface. These chiral signals play a crucial role in immunomodulation, activating the immune system and synergistically complementing the near-infrared imaging-guided photothermal and photodynamic therapy of the gold nanoclusters themselves, thereby killing tumor cells while inhibiting tumor recurrence and distant metastasis.

[0009] Preferably, the thiol ligand is L-cysteine.

[0010] Compared to D-cysteine, L-cysteine, as a thiol ligand, exhibits stronger immunomodulatory capabilities in gold nanoclusters.

[0011] This invention also protects a method for preparing the chiral photoimmunomodulated gold nanoclusters, comprising the following steps: adding a thiol ligand to an aqueous tetrachloroauric acid solution while stirring; adding NaOH while stirring to adjust the pH; adding a strong reducing agent NaBH4 while stirring; and stirring to react, thereby obtaining Au. 25 (SR) 18 Chiral photoimmunoregulatory gold nanoclusters; the thiol ligand is one or more of L-cysteine ​​and D-cysteine.

[0012] This invention uses tetrachloroauric acid (HAuCl4) as a gold precursor to provide Au. 3+Ions. Under the action of a reducing agent, Au 3+ Reduced to Au atoms, they self-assemble to form atomically precise gold nanoclusters. Thiol ligands (-SH) and gold atoms (Au0) form a stable coordination structure through Au-S bonds, inhibiting the excessive growth of clusters into nanoparticles.

[0013] Sodium hydroxide can adjust the pH of the reaction system, control the reduction effect of sodium borohydride, and optimize the coordination ability of ligands.

[0014] In the preparation method of chiral photoimmunoregulatory gold nanoclusters, the reaction conditions determine the stability, size uniformity and yield of Au NCs by regulating the protonation state and coordination ability of the ligands, the reduction rate and the balance of nucleation growth.

[0015] Preferably, in the preparation method, the molar ratio of S element in the thiol ligand to Au element in tetrachloroauric acid is (1~4):1.

[0016] More preferably, the molar ratio of sulfur (S) in the thiol ligand to Au in tetrachloroauric acid is (1.5~2.5):1. Controlling the molar ratio of S in the thiol ligand to Au in tetrachloroauric acid can yield higher yields. Appropriate ligands help prevent excessive ligand coverage of the nuclear active site, which could inhibit nucleation kinetics and lead to a decrease in yield.

[0017] Ligand concentration directly affects the size of gold nanoclusters. An appropriate ligand concentration can prevent excessive growth of gold nanoclusters into large particles due to excessive ligand concentration, and can also prevent insufficient nucleation due to insufficient ligand concentration.

[0018] Preferably, in the preparation method, the molar ratio of Au element in tetrachloroauric acid to reducing agent NaBH4 is (0.5~50):1.

[0019] More preferably, the molar ratio of Au to reducing agent in tetrachloroauric acid is (10~30):1.

[0020] Preferably, in the preparation method, the molar ratio of Au to NaOH in tetrachloroauric acid is 1:(5~12).

[0021] Sodium borohydride exhibits a vigorous reducing effect. Sodium hydroxide, acting as a regulator, can inhibit this reducing action. It possesses the ability to modulate the reduction reaction, controlling the uniform formation of gold nanoclusters. The amount of sodium hydroxide is generally adjusted based on the amount of sodium borohydride used.

[0022] Preferably, the reaction pH is 8-12. When pH=10, it can promote the deprotonation of thiol ligands and enhance the Au-S bond strength.

[0023] Preferably, the concentration of Au is 0.0009~0.00461 mol / L before stirring the reaction.

[0024] Preferably, the temperature of the stirring reaction is 10~30 ℃, and the stirring reaction time is 2~12 h.

[0025] This invention also protects a composite nanoprobe comprising gold nanoclusters, mesoporous silica, and a targeting peptide, wherein the gold nanoclusters are loaded in the pores of the mesoporous silica, and the targeting peptide is attached to the surface of the mesoporous silica, and the gold nanoclusters are the chiral photoimmunomodulated gold nanoclusters described above.

[0026] Because gold nanoclusters are too small and metabolize too quickly, in order to improve the long-term circulation capacity of the clusters, mesoporous silica is used to load gold nanoclusters, and nasopharyngeal carcinoma-targeting peptides are attached to the silica surface to form a composite nanoprobe that can be effectively taken up by nasopharyngeal carcinoma cells.

[0027] Simultaneously, the mesoporous silica in the composite nanoprobe will decompose under the action of photothermal photodynamics and enzymes, releasing the Au of this invention. 25 (SR) 18 Chiral photoimmunomodulatory gold nanoclusters regulate and activate the immune system.

[0028] Preferably, the preparation method of mesoporous silica includes the following steps: dissolving concentrated ammonia in deionized water, adding hexadecyltrimethylammonium bromide (CTAB), adding polyethylene glycol, stirring to dissolve at room temperature, and then adding tetraethyl orthosilicate. After stirring at room temperature, centrifuging is performed, and the sample is collected. The sample is then placed in a vacuum drying oven for vacuum drying. After drying is complete, the sample is placed in a resistance furnace for calcination to evaporate the mesoporous silica template powder.

[0029] This invention also protects a method for preparing a composite nanoprobe, comprising the following steps: ultrasonically dispersing silica powder in a gold nanocluster solution, stirring, centrifuging at high speed, and collecting sample A; fully dispersing sample A in deionized water, adding 3-aminopropyltriethoxysilane, stirring, washing, and centrifuging to obtain an aminated sample B; dispersing the aminated sample B in deionized water, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and carboxylated nasopharyngeal carcinoma peptide NTP, stirring in the dark to obtain the composite nanoprobe.

[0030] This invention also protects the application of the above-mentioned composite nanoprobes in near-infrared optical imaging, preparation of photothermal therapy agents, and preparation of photodynamic therapy agents.

[0031] The chiral photoimmunomodulatory gold nanoclusters of this invention possess a unique atomically precise structure and strong chiral optical activity, enabling them to function simultaneously as photothermal / photodynamic therapeutic agents and immunoadjuvants, achieving multimodal synergistic treatment of nasopharyngeal carcinoma. These chiral photoimmunomodulatory gold nanoclusters exhibit strong immunomodulatory capabilities, balancing photodynamic effects with immune activation. By loading targeted peptides onto mesoporous silica, these gold nanoclusters not only enhance tumor-specific enrichment but also sustainably release chiral gold clusters under photothermal action, synergistically triggering immunogenic cell death and dendritic cell activation, thereby inhibiting metastasis. This integrated "diagnosis-therapy-immunotherapy" design overcomes the limitations of traditional gold-25 clusters, which suffer from single-function limitations and insufficient chiral signaling, providing an innovative solution for nasopharyngeal carcinoma treatment.

[0032] This invention presents a composite nanoprobe based on gold nanoclusters, offering multiple advantages in tumor therapy: First, the well-defined atomic composition of gold nanoclusters allows for precise modulation of their fluorescence emission and photothermal / photodynamic response properties, making them suitable for multimodal image-guided precision treatment. Second, due to their particle size of less than 5 nanometers, gold nanoclusters can be excreted through the kidneys, reducing long-term in vivo toxicity. Furthermore, their surface ligands can be further modified to target molecules, improving their accumulation efficiency at tumor sites. In addition, the atomically precise gold nanoclusters demonstrate significant capabilities in the integration of diagnosis and therapy. In diagnosis, their near-infrared fluorescence imaging (NIR) possesses deep tissue penetration, enabling real-time observation of tumor boundaries and metastatic lesions. In therapy, they can ablate tumor tissue through the synergistic effects of photothermal and photodynamic effects, and can also load chemotherapeutic drugs, achieving synergistic treatment through multiple mechanisms.

[0033] Compared with the prior art, the present invention has the following beneficial technical effects: This invention provides a chiral photoimmunomodulated gold nanocluster, the general structural formula of which is Au. 25 (SR) 18 SR is a thiol ligand, which is one or more of L-cysteine ​​and D-cysteine. In the chiral photoimmunoregulated gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

[0034] The chiral photoimmunomodulatory gold nanoclusters of the present invention have immunomodulatory capabilities and can be used as photothermal therapy agents or photodynamic therapy agents for the treatment of tumors by utilizing their optical properties. Attached Figure Description

[0035] Figure 1 The UV-Vis absorption spectra of the gold nanoclusters in Examples 1-6 and Comparative Example 1 are shown.

[0036] Figure 2Transmission electron microscopy (TEM) images and UV-Vis absorption spectra of gold nanoclusters from Examples 1, 6, and Comparative Example 1.

[0037] Figure 3 These are circular dichroism (CD) spectra of the gold nanoclusters in Examples 1 and 6.

[0038] Figure 4 To detect the expression levels of CD80, CD86, and CD40, PBS solutions of gold nanoclusters, monophosphate, and ovalbumin from Examples 1, 6, and Comparative Example 1 were co-incubated with mouse bone marrow-derived dendritic (DC) cells.

[0039] Figure 5 Transmission electron microscopy (TEM) images of mesoporous silica dried before loading chiral gold nanoclusters, and of the prepared chiral gold nanoclusters loaded into the pores of mesoporous silica, with nasopharyngeal carcinoma-targeting peptides attached to the surface of the mesoporous silica, forming a gold nanocluster optical probe.

[0040] Figure 6 This is a near-infrared II optical imaging image of the composite nanoprobe from Example 7.

[0041] Figure 7 To excite the composite nanoprobe of Example 7 using an 808nm laser, an indium gallium arsenide near-infrared camera was used to capture images of the composite nanoprobe of Example 7, which showed a significant near-infrared emission compared to the pure dielectric solution.

[0042] Figure 8 The photothermal conversion curve and photothermal cycling curve of the composite nanoprobe in Example 7 are shown under 808nm laser excitation in a medium solution.

[0043] Figure 9 The image shows the intensity of reactive oxygen species generated under 808 nm laser irradiation after mixing 1,3-diphenylisobenzofuran (DPBF) reactive oxygen indicator with the composite nanoprobe solution of Example 7 or dimethyl sulfoxide medium.

[0044] Figure 10 To use 20 μg ml -1 and 40 μg ml -1 In Example 7, the composite nanoprobe was co-incubated with poorly differentiated nasopharyngeal squamous cell carcinoma CNE2 cells and highly metastatic nasopharyngeal carcinoma cell line 5-8f cells for 2 hours, and then reactive oxygen species were tested using DCFH dye.

[0045] Figure 11 To use 20 μg ml -1 and 40 μg ml -1In Example 7, the composite nanoprobe was co-incubated with poorly differentiated nasopharyngeal squamous cell carcinoma CNE2 cells and highly metastatic nasopharyngeal carcinoma cell line 5-8f cells for 2 hours, and tumor cell liveness imaging was performed using Calcein-AM and propidium iodide PI dual dyes. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise stated, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0047] Carboxylated nasopharyngeal carcinoma peptide NTP, commercially available, manufactured by Shanghai Chutai Biotechnology Co., Ltd.

[0048] Example 1 A chiral photoimmunoregulatory gold nanocluster with the general structural formula Au 25 (CYS) 18 The thiol ligand is L-cysteine. In the chiral photoimmunoregulated gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

[0049] The preparation method of the above-mentioned chiral photoimmunomodulated gold nanoclusters includes the following steps: 0.395 mL of tetrachloroauric acid (HAuCl4) solution (0.1166 mol / L) was mixed with 1.382 mL of L-cysteine ​​solution (the molar ratio of sulfur in the L-cysteine ​​thiol ligand to Au in tetrachloroauric acid was 1.5:1), and the mixture was homogenized at 500 rpm for 30 min on a magnetic stirrer. Then, 0.921 mL of NaOH solution (0.3 mol / L, the molar ratio of Au in tetrachloroauric acid to NaOH was 1:6, pH=10) was added, and the mixture was stirred for another 30 min to ensure thorough mixing. The reaction solution was then sonicated for 30 min to promote ligand exchange. 46 μL of a reducing agent NaBH4 solution (0.05 mol / L, with a molar ratio of Au in tetrachloroauric acid to NaBH4 of 20:1) was slowly added dropwise, and the volume was adjusted to 10 mL with deionized water to achieve an Au concentration of 0.00461 mol / L. The reaction was then carried out with stirring at room temperature (25°C) for 6 h to obtain gold nanoclusters, abbreviated as Au. 25 (CYS) 18 -1.

[0050] Example 2 A chiral photoimmunoregulatory gold nanocluster with the general structural formula Au 25 (CYS) 18The thiol ligand is L-cysteine. In the chiral photoimmunoregulated gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

[0051] The difference between the preparation method of the chiral photoimmunoregulatory gold nanoclusters described above and Example 1 is that deionized water was used to bring the volume to 20 mL so that the concentration of Au element was 0.0023 mol / L.

[0052] The obtained gold nanoclusters are abbreviated as Au. 25 (CYS) 18 -2.

[0053] Example 3 A chiral photoimmunoregulatory gold nanocluster with the general structural formula Au 25 (CYS) 18 The thiol ligand is L-cysteine. In the chiral photoimmunoregulated gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

[0054] The difference between the preparation method of the chiral photoimmunoregulatory gold nanoclusters described above and Example 1 is that: the volume was adjusted to 50 mL with deionized water so that the concentration of Au element was 0.0009 mol / L.

[0055] The obtained gold nanoclusters are abbreviated as Au. 25 (CYS) 18 -3.

[0056] Example 4 A chiral photoimmunoregulatory gold nanocluster with the general structural formula Au 25 (CYS) 18 The preparation method differs from that in Example 1 in that the molar ratio of Au to NaOH in tetrachloroauric acid is 1:5, and the pH is 9.

[0057] The obtained gold nanoclusters are abbreviated as Au. 25 (CYS) 18 -4.

[0058] Example 5 A chiral photoimmunoregulatory gold nanocluster with the general structural formula Au 25 (CYS) 18 The preparation method differs from that in Example 1 in that the molar ratio of Au to NaOH in tetrachloroauric acid is 1:12, and the pH is 11.5.

[0059] The obtained gold nanoclusters are abbreviated as Au. 25 (CYS) 18 -5.

[0060] Example 6 A chiral photoimmunoregulatory gold nanocluster with the general structural formula Au 25 (CYS) 18 The thiol ligand is D-cysteine. In the chiral photoimmunoregulated gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

[0061] The preparation method of the above-mentioned chiral photoimmunomodulated gold nanoclusters includes the following steps: 0.825 mL of 0.02426 mol / L tetrachloroauric acid aqueous solution, 0.6 mL of 0.05 mol / L D-cysteine ​​ligand solution, 0.6 mL of 0.3 mol / L NaOH solution, and 20 μL of 50 mmol / L NaBH4 solution were added sequentially to a 10 mL glass bottle under slow stirring at approximately 2-minute intervals. The solution was then diluted to a total volume of 10 mL to achieve an Au concentration of 0.002 mol / L. The solution was then gently stirred under sealed conditions at room temperature for 6 hours to obtain gold nanoclusters. The molar ratio of sulfur (S) in the D-cysteine ​​thiol ligand to Au in tetrachloroauric acid is 1.5:1. The molar ratio of Au to the reducing agent NaBH4 in tetrachloroauric acid is 20:1. The molar ratio of Au to NaOH in tetrachloroauric acid is 1:6.

[0062] Example 7 A composite nanoprobe includes gold nanoclusters, mesoporous silica, and a targeting peptide. The gold nanoclusters are loaded in the pores of the mesoporous silica, and the targeting peptide is attached to the surface of the mesoporous silica. The gold nanoclusters are the chiral photoimmunomodulation gold nanoclusters described in Example 1.

[0063] The preparation method of the composite nanoprobe includes the following steps: S1. Preparation of mesoporous silica: Dissolve 0.2 ml of concentrated ammonia (25%) in 10 ml of deionized water, add 0.02 g of cetyltrimethylammonium bromide (CTAB), add 0.2 ml of 1% polyethylene glycol, stir to dissolve at room temperature, and then add 0.1 ml of tetraethyl orthosilicate. Stir at room temperature for 1 h, centrifuge, and collect the sample. Place in a vacuum drying oven for vacuum drying. After drying, place in a resistance furnace at 520℃ for 5 h to evaporate the mesoporous silica powder template agent.

[0064] S2. Take 0.02 g of the mesoporous silica powder template agent from step S1, and ultrasonically disperse it in 10 ml of the gold nanocluster solution prepared in Example 1. The ultrasonic dispersion time is 30 min, and the mixture is stirred at room temperature for 12 h. After centrifugation at 11000 rpm for 15 min, the sample is collected. The sample is thoroughly dispersed in 10 ml of deionized water, and 0.5 ml of 3-aminopropyltriethoxysilane (APTES) is added. After rapid stirring for 12 h, excess APTES is removed by washing and centrifugation in the same manner to obtain an aminated sample, which is then dispersed in 10 ml of deionized water. Take 1 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2 mg of N-hydroxysuccinimide (NHS), and 50 μl of carboxylated nasopharyngeal carcinoma peptide (NTP) and add them to the prepared suspension. Stir rapidly in the dark for 2 h. Excess other reagents are removed by washing and centrifugation in the same manner to obtain the final sample dispersed in 10 ml of deionized water.

[0065] Comparative Example 1 A chiral photoimmunoregulatory gold nanocluster with the general structural formula Au 25 (3MPa) 18 The thiol ligand is 3-mercaptopropionic acid. In the chiral photoimmunoregulatory gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

[0066] The difference between the preparation method of the chiral photoimmunomodulated gold nanoclusters described above and Example 1 is that the ligand is 3-mercaptopropionic acid (3MPA).

[0067] Result detection The materials of each embodiment were subjected to the following structural characterization and performance tests.

[0068] (1) Absorption spectrum The UV-Vis absorption spectra of the gold nanoclusters in Examples 1-3 are as follows: Figure 1 As shown in (a) in the figure, from Figure 1 As can be seen from (a) in Examples 1 to 3, the absorption peak of Example 1 is more obvious and the absorption value is higher, indicating that the concentration of gold nanoclusters prepared by the method of Example 1 is higher, which can make the subsequent detection signal stronger.

[0069] The UV-Vis absorption spectra of gold nanoclusters in Examples 1, 6, and 1 are shown below. Figure 1 As shown in (b), experiments have demonstrated that the gold nanoclusters prepared using cysteine ​​(CYS) and 3-mercaptopropionic acid (3MPA) are Au nanoclusters. 25 The clusters exhibit distinct characteristic absorption peaks near 445 and 675 nm. A common feature of these ligands is that a thiol group is linked to a secondary carbon atom.

[0070] from Figure 1 (b) It can be seen that the UV-Vis absorption spectra of Examples 1 and 6 are basically identical, therefore, Figure 1 In (b), the UV-Vis absorption spectra that overlap from Examples 1 and 6 are referred to as Au. 25 (CYS) 18 This is because L-cysteine ​​and D-cysteine ​​are enantiomers with identical chemical compositions, differing only in spatial configuration. In the synthesis of atomically precise gold nanoclusters, the thiol groups of L-cysteine ​​and D-cysteine ​​have the same coordination ability with Au, both stabilizing the gold core through Au-S bonds. The difference in chirality does not alter the electronic properties of the ligands, thus having minimal impact on the electronic transitions of Au NCs. Under the same synthetic conditions, both L-cysteine ​​and D-cysteine ​​lead to the formation of Au NCs of the same size, resulting in highly consistent absorption spectra.

[0071] from Figure 1 (b) As can be seen from Example 1 and Comparative Example 1, the absorption peak of the gold nanoclusters prepared with L-cysteine ​​as a ligand red-shifts to 680 nm, belonging to Au 25 Localized surface plasmon resonance (LSPR) was observed. The single thiol group exhibited weak coordination ability, but the amino group stabilized the dispersion through hydrogen bonding. In contrast, the gold clusters prepared using 3-mercaptopropionic acid as a ligand showed broad absorption peaks with low intensity, indicating product size dispersion and low yield, attributed to insufficient electrostatic repulsion of the carboxylic acid group and the single coordination site.

[0072] The UV-Vis absorption spectra of gold nanoclusters in Examples 1, 4, and 5 are as follows: Figure 1 As shown in (c), the absorption peak (Au) of Example 1 can be observed. 25 (CYS) 18 -1) Both are more obvious and have higher absorption values, indicating that the gold nanoclusters prepared by the method in Example 1 at pH 10 have a better ratio, which can make the subsequent detection signal stronger.

[0073] Figure 2 The images show transmission electron microscope (TEM) images and UV-Vis absorption spectra of the L-type gold nanoclusters prepared in Example 1, the D-type gold nanoclusters prepared in Example 4, and the gold nanoclusters in Comparative Example 1. Experimentally, gold nanoclusters with sizes ranging from 1.5 to 1.7 nm were successfully prepared, and their atomically precise structure was confirmed by optical absorption spectroscopy. 25 (SR) 18 The TEM images show that the atomically precise gold nanoclusters prepared in Example 1 have uniform shapes and good dispersion. This may be because L-cysteine ​​is a monothiol ligand, and the amino and carboxylic acid groups can stabilize the dispersion of Au NCs through a hydrogen bond network.

[0074] Figure 3 These are the circular dichroism (CD) spectra of the gold nanoclusters from Examples 1 and 6. Figure 3 This indicates that regulating chiral cysteine ​​can induce Au. 25 The cluster exhibits a chiral signal, which is mainly induced by gold-sulfur bonds at the interface between the gold cluster core and the surface ligands.

[0075] (2) Immune regulation L-type gold nanoclusters prepared in Example 1, D-type gold nanoclusters prepared in Example 4, and gold nanoclusters from Comparative Example 1, along with monophosphate and ovalbumin in PBS solution, were co-incubated with mouse bone marrow-derived dendritic (DC) cells. The expression levels of CD80, CD86, and CD40 were detected. The results are as follows: Figure 4 As shown. From Figure 4 As can be seen, the expression levels of CD80, CD86, and CD40 in dendritic cells stimulated by the L-type gold nanoclusters prepared in Example 1 and the D-type gold nanoclusters prepared in Example 6 were significantly higher than those in the gold nanoclusters of Comparative Example 1 and the control group, indicating that the gold nanoclusters of the present invention have excellent immunomodulatory capabilities. Furthermore, the expression levels of CD80, CD86, and CD40 in dendritic cells stimulated by the L-type gold nanoclusters prepared in Example 1 were also significantly higher than those in the D-type gold nanoclusters prepared in Example 6.

[0076] Figure 5 Transmission electron microscopy (TEM) images show the dried mesoporous silica before loading chiral gold nanoclusters, the loaded chiral gold nanoclusters within the pores of the mesoporous silica, and the nasopharyngeal carcinoma-targeting peptides attached to the surface of the mesoporous silica to form a gold nanocluster optical probe. It can be seen that the mesoporous silica has distinct strip-shaped pores, with the gold nanoclusters loaded within them. After loading the gold nanoclusters, the silica pores are blocked, and the TEM images clearly show a large quantity of gold nanoclusters loaded.

[0077] (3) Near-infrared II optical imaging Figure 6 This is a near-infrared II optical imaging image of the composite nanoprobe from Example 7. It can be seen that the composite nanoprobe of Example 7 has obvious emission peaks at 840 nm and 1060 nm, with an optimal emission wavelength of 465 nm. This excitation light induces electrons in Au NCs to transition from the ground state to an excited state, subsequently relaxing to a lower energy level through nonradiative relaxation, and finally releasing energy as near-infrared photons (840 nm or 1060 nm). The emission peak is located at 1060 nm (near-infrared II, NIR-II), with a full width at half maximum (FWHM) of approximately 100 nm, exhibiting a typical quantum confinement effect. Light in the near-infrared II window (1000-1700 nm) has deeper tissue penetration and lower autofluorescence interference, making it suitable for in vivo deep imaging.

[0078] The composite nanoprobe from Example 7 was placed in a solution and excited by irradiation with an 808 nm laser. Images were then captured using an indium gallium arsenide near-infrared camera. The results are as follows: Figure 7 As shown, Figure 7 (a) in the text is a pure medium solution. Figure 7 (b) shows a solution containing the composite nanoprobe from Example 7. It can be seen that... Figure 7 (b) in the example exhibits obvious near-infrared luminescence, indicating that the composite nanoprobe of Example 7 has the ability to target nasopharyngeal carcinoma with near-infrared optical imaging.

[0079] (4) Photothermal conversion efficiency test The composite nanoprobe from Example 7 was placed in a 0.5 ml glass vial and tested using an 808 nm near-infrared laser (0.5 W cm⁻¹). -2 Irradiation was performed, and temperature changes were recorded in real time using an infrared thermal imager. The trends of irradiation time and solution temperature, photothermal conversion curves, and photothermal cycle curves are shown below. Figure 8 As shown, from Figure 8 It can be seen that the temperature rises by more than 50°C after 3 minutes of irradiation, and reaches approximately 57°C after 210 seconds. This demonstrates that the probe has excellent photothermal conversion capability and photothermal stability.

[0080] Temperatures of 42-48°C can induce reversible damage to cancer cells, while temperatures above 50°C lead to irreversible necrosis through direct protein denaturation and cell membrane rupture, significantly improving treatment efficacy. Photothermal heating to above 50°C can directly induce tumor cell necrosis, while the high penetrability of near-infrared light can precisely target deep lesions, reducing thermal damage to normal tissues.

[0081] (5) Detection of reactive oxygen species and photodynamic properties 1,3-Diphenylisobenzofuran (DPBF) is an aromatic compound containing electron-rich double bonds. The conjugated double bonds in its molecule are highly sensitive to singlet oxygen, and it can combine with singlet oxygen via a cycloaddition reaction to form an internal peroxide. DPBF exhibits characteristic absorption peaks in the UV-Vis spectrum. When it reacts with singlet oxygen, its conjugated structure is disrupted, leading to a decrease in absorbance at the characteristic absorption peak. The degree of absorbance reduction is proportional to the amount of singlet oxygen generated; therefore, the concentration of singlet oxygen can be detected by monitoring changes in absorbance. Furthermore, DPBF exhibits high selectivity for singlet oxygen, showing almost no reaction with other reactive oxygen species, making it suitable for real-time monitoring of singlet oxygen.

[0082] Dimethyl sulfoxide (DMSO) dissolves DPBF. DMSO is a strongly polar aprotic solvent that effectively dissolves DPBF, preventing probe aggregation or precipitation due to insufficient solvent polarity. Furthermore, DMSO itself does not significantly react with singlet oxygen, thus not interfering with the specific binding of DPBF to singlet oxygen. The absorption peaks of DMSO in the UV-Vis region do not overlap with the characteristic peaks of DPBF, ensuring the accuracy of the detection signal.

[0083] 1,3-Diphenylisobenzofuran (DPBF) was used as a singlet oxygen probe. DPBF was dissolved in dimethyl sulfoxide (DMSO). The composite nanoprobe solution from Example 7 was then mixed with DPBF. The sample was irradiated with an 808 nm laser for different times, and the changes in the absorption spectrum of the sample after irradiation for different times were detected by UV-Vis spectroscopy.

[0084] Test results are as follows Figure 9 As shown. From Figure 9 As can be seen, reactive oxygen species can be generated under 808 laser irradiation, thereby quenching the absorption peak of DPBF at 410 nm and causing its peak intensity to drop rapidly. This indicates that the composite nanoprobe of Example 7 has excellent singlet oxygen generation ability and excellent photodynamic performance.

[0085] (6) Reactive oxygen species test for nasopharyngeal carcinoma cells Figure 10 To use 20 μg ml -1 and 40 μg ml -1 In Example 7, the composite nanoprobe was co-incubated with poorly differentiated nasopharyngeal squamous cell carcinoma CNE2 cells and highly metastatic nasopharyngeal carcinoma cell line 5-8f cells for 2 hours, and then reactive oxygen species were tested using DCFH dye.

[0086] After co-incubating nasopharyngeal carcinoma cells with the composite probe from Example 7 for 2 hours, reactive oxygen species (ROS) were measured using DCFH dye. The ROS levels were determined after irradiation of the nasopharyngeal carcinoma cells with an 808 laser (0.5 W cm⁻¹). -2 In the control group, no reactive oxygen species (ROS) signal was detected on the cell surface. However, nasopharyngeal carcinoma cells co-incubated with the sample produced significant ROS signals after 5 minutes of 808 nm laser irradiation, indicating that the sample possesses photodynamic therapy capabilities for nasopharyngeal carcinoma. The experiment used two different types of nasopharyngeal carcinoma cells. It was found that for poorly differentiated nasopharyngeal squamous cell carcinoma CNE2 cells, a probe concentration of only 20 μg / mL was sufficient to generate adequate ROS, while for the highly metastatic nasopharyngeal carcinoma cell line 5-8f, a probe concentration of 40 μg / mL was required to generate sufficient ROS.

[0087] (7) Nasopharyngeal carcinoma cell live-death imaging test Figure 11 To use 20 μg ml-1 and 40 μg ml -1 In Example 7, the composite nanoprobe was co-incubated with poorly differentiated nasopharyngeal squamous cell carcinoma CNE2 cells and highly metastatic nasopharyngeal carcinoma cell line 5-8f cells for 2 hours, and tumor cell liveness imaging was performed using Calcein-AM and propidium iodide PI dual dyes.

[0088] After co-incubating nasopharyngeal carcinoma cells with the 20 μg / mL nanocomposite probe from Example 7 for 2 hours, cell viability imaging was performed using Calcein-AM and propidium iodide (PI) dual dyes. CNE2 nasopharyngeal carcinoma cells irradiated with an 808 laser showed green color after irradiation, indicating viable cells. However, nasopharyngeal carcinoma cells co-incubated with the sample showed red color after 5 minutes of 808 laser irradiation, indicating almost complete cell death, demonstrating the sample's ability to inactivate nasopharyngeal carcinoma cells. The highly metastatic nasopharyngeal carcinoma cell line 5-8f showed a low cell death rate after treatment with the 40 μg / mL probe, but almost complete cell death after treatment with the 40 μg / mL probe, demonstrating the probe's excellent therapeutic effect on highly metastatic cells at a certain dose. Both types of nasopharyngeal carcinoma cells remained viable under both laser irradiation and 40 μg / mL probe treatment, indicating the probe's excellent cell biocompatibility.

[0089] The experiments described above demonstrate that the photothermal-photodynamic synergistic effect of the Au NCs composite nanoprobes in this invention can be precisely activated by near-infrared light, achieving highly efficient ablation of tumor cells. The photothermal effect expands the tumor vascular space, promoting the accumulation of nanoprobes. The photodynamic effect induces oxidative damage through reactive oxygen species, and the two synergistically enhance the therapeutic effect. Simultaneously, the fluorescence imaging function of Au NCs allows for real-time navigation of the treatment area, promoting integrated diagnostic and therapeutic applications.

[0090] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of a chiral photoimmunomodulatory gold nanocluster in the preparation of a drug for treating nasopharyngeal carcinoma, characterized in that, The general structural formula of the chiral photoimmunomodulated gold nanoclusters is Au. 25 (SR) 18 SR is a thiol ligand, and the thiol ligand is L-cysteine. In the chiral photoimmunoregulated gold nanoclusters, the thiol ligand and gold atoms form a coordination structure through Au-S bonds.

2. The application according to claim 1, characterized in that, The preparation method of the chiral photoimmunomodulated gold nanoclusters includes the following steps: adding a thiol ligand to a tetrachloroauric acid aqueous solution with stirring, adding NaOH with stirring to adjust the pH, adding a strong reducing agent NaBH4 with stirring, and stirring to react, thereby obtaining Au. 25 (SR) 18 Chiral photoimmunoregulatory gold nanoclusters; the thiol ligand is L-cysteine.

3. The application according to claim 2, characterized in that, The molar ratio of sulfur in the thiol ligand to Au in tetrachloroauric acid is (1~4):

1.

4. The application according to claim 2, characterized in that, The molar ratio of Au to the reducing agent NaBH4 in tetrachloroauric acid is (0.5~50):

1.

5. The application according to claim 2, characterized in that, The molar ratio of Au to NaOH in tetrachloroauric acid is 1:(5~12).

6. The application according to claim 2, characterized in that, Before the reaction was stirred, the concentration of Au was 0.0009~0.00461 mol / L.

7. A composite nanoprobe, characterized in that, The invention comprises gold nanoclusters, mesoporous silica, and a targeting peptide, wherein the gold nanoclusters are loaded in the pores of the mesoporous silica, and the targeting peptide is attached to the surface of the mesoporous silica. The gold nanoclusters are chiral photoimmunomodulatory gold nanoclusters used in claims 1 to 6.

8. The composite nanoprobe according to claim 7, characterized in that, The preparation method of the composite nanoprobe includes the following steps: ultrasonically dispersing silica powder in a gold nanocluster solution, stirring, centrifuging at high speed, and collecting sample A; fully dispersing sample A in deionized water, adding 3-aminopropyltriethoxysilane, stirring, washing, and centrifuging to obtain aminated sample B; dispersing aminated sample B in deionized water, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and carboxylated nasopharyngeal carcinoma peptide, stirring in the dark to obtain the composite nanoprobe.

9. The application of the composite nanoprobe according to claim 7 in the preparation of near-infrared optical imaging reagents, photothermal therapy agents, and photodynamic therapy agents.

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