Preparation method of host-guest interaction mediated gold nanocluster assembly fluorescent probe

Abstract

The invention discloses a gold nanocluster assembly based on host-guest interaction mediation and preparation and application of the gold nanocluster assembly. The gold nanocluster assembly is prepared by the following steps: firstly, preparing a near-infrared two-region luminescence gold nanocluster protected by a cyclodextrin ligand, and then preparing a near-infrared two-region luminescence gold nanocluster by utilizing the strong host-guest complexation between cyclodextrin and adamantane. A novel gold nanocluster assembly is prepared by introducing a diadamantyl ligand as a connector and adamantane modified hyaluronic acid as a regulator. The prepared gold nanocluster assembly has good water-phase dispersibility, near-infrared two-region luminescence performance and tumor-targeted near-infrared two-region fluorescence imaging performance, can be used as a fluorescence probe for in-vivo imaging, and can be used for in-vivo imaging by utilizing the characteristics of good kidney clearance, low biotoxicity and the like of the gold nanocluster. And photosensitizers, drug molecules and the like can be carried for drug delivery, so that the combination of tumor diagnosis and treatment can be realized, and a wide application prospect is shown.

Description

Technical Field

[0001] The invention belongs to the field of nano material synthesis, and specifically relates to a gold nano cluster assembly mediated by host-guest interaction and the preparation and application thereof. Background Art

[0002] In recent years, with the continuous in-depth research on nanomaterials, the application of nanotechnology in the biomedical field has made significant progress. Gold nanoclusters (Au NCs) are widely used in biomedical fields such as imaging, sensing and disease treatment due to their excellent luminescence properties, low toxicity, high catalytic activity, high electron density, and high excretion rate. However, Au NCs are rapidly excreted through the urinary system, which makes them show good biosafety in the body, but also leads to a short retention time in the body, which in turn hinders their application in tumor enrichment effects and tumor precision imaging.

[0003] At the same time, conventional inorganic nanoparticles used for bioimaging are potentially non-degradable and biotoxic. In order to balance the large size required for passive tumor targeting with efficient in vivo clearance, so as to accurately locate the disease and cancer sites, an effective strategy is to construct gold nanocluster assemblies (Au NCs assemblies, AAs) with enhanced permeability retention (EPR) effect by controlling the size of nanoparticles. However, due to the complex tumor microenvironment, the tumor imaging effect is not proportional to the particle size, and the controllable synthesis of AAs still faces challenges. Summary of the invention

[0004] In order to solve the above problems, the present invention utilizes the host-guest interaction between cyclodextrin and adamantane to develop a method for controllably preparing gold nanocluster assemblies of different sizes. The obtained gold nanocluster assemblies have good aqueous phase dispersibility, near-infrared zone II luminescence performance and tumor-targeted near-infrared zone II fluorescence imaging performance, and are expected to be used in the diagnosis and treatment of tumors.

[0005] To achieve the above object, the present invention adopts the following technical solution: A host-guest interaction-mediated gold nanocluster assembly, which is first synthesized by using thiol-β-cyclodextrin (SH-β-CD) to synthesize surface ligand-protected gold nanoclusters (CD-Au NCs), and then utilizing the host-guest interaction between cyclodextrin and adamantane derivatives, as well as the competitive relationship between different adamantane derivatives, with a bis-adamantane ligand (Ad-linker-Ad) as a connector and adamantane-modified hyaluronic acid (HA-Ad5) as a regulator to synthesize a type of gold nanocluster assembly (AAs).

[0006] The preparation method of the gold nanocluster assembly comprises the following steps: 1. Preparation of CD-Au NCs 1) Take 15 mL of 10 mM 6-mercapto-6-deoxy-β-cyclodextrin solution, add it to 77.9 mL of ultrapure water, and stir evenly; 2) Add 4 mL of 25 mM HAuCl4 solution, the solution is now clear yellow, and continue stirring for 5 min; 3) Add 0.6 mL of 1 M NaOH solution to adjust the pH. After the yellow color of the solution fades, continue stirring for 5 min. 4) Add 2.5 mL of 0.01 M NaOH solution to dissolve the freshly prepared 80 mM NaBH4 solution and continue stirring for 12 h until the solution turns dark brown. 5) The obtained mixed solution was placed in a dialysis bag with a molecular weight cutoff (MWCO) of 8000-14000 Da for 10 h, during which the dialysate was replaced every 2 h; 6) Collect the liquid in the dialysis bag and use an ultrafiltration centrifuge tube with a molecular weight of 50 kDa to ultrafilter and centrifuge at 4°C and 4500 rpm for 15 min to remove the aggregated macromolecules. The filtrate obtained in the ultrafiltration tube is the purified CD-Au NCs solution.

[0007] 2. Preparation of AAs Take 9 mL of the prepared CD-Au NCs solution, add 6 mL of 3 mM 1,3-bis(1-adamantyl)imidazolium tetrafluoroborate aqueous solution and 3 mL of 3-12 mg / mL adamantane-modified hyaluronic acid aqueous solution, oscillate for 12 h, centrifuge at 12000 rpm for 10 min at 4 °C, discard the supernatant, wash the precipitate once with ultrapure water, and re-ultrasonicate and disperse it in ultrapure water to obtain.

[0008] Furthermore, in the adamantane-modified hyaluronic acid used, the modification degree of adamantane is 5% to 33%.

[0009] During the process, AAs with different particle sizes can be obtained by adjusting the ratio of Ad-linker-Ad to HA-Ad, and AAs assemblies with different yields can be obtained by using HA-Ad with different Ad modification degrees.

[0010] The gold nanocluster assembly can be used as a fluorescent probe for in vivo imaging, or can be loaded with photosensitizers and drug molecules to form drug delivery, and is therefore expected to be used in the diagnosis and treatment of tumors.

[0011] The beneficial effects of the present invention are: 1) The synthesis steps of the present invention are complete, highly controllable, and have good reproducibility; 2) The structure of CD-Au NCs in the gold nanocluster assembly prepared by the present invention has not changed significantly compared with the structure of conventional gold nanoclusters, and it has good near-infrared second-zone luminescence performance in the wavelength range of 900-1400nm.

[0012] 3) In the present invention, the dispersibility and biocompatibility of the gold nanocluster assembly can be improved by introducing hyaluronic acid (HA).

[0013] 4) The gold nanocluster assemblies prepared by the present invention can accumulate at the tumor site through active targeting mediated by the CD44 receptor overexpressed on the surface of tumor cells and the EPR effect, achieving precise imaging of the tumor site, which will provide a powerful tool for disease diagnosis and real-time monitoring.

[0014] 5) The gold nanocluster assemblies prepared by the present invention can be loaded with photosensitizers and drug molecules for drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the infrared spectra of CD-Au NCs synthesized in Example 1 and the raw material SH-β-CD.

[0016] Figure 2 TEM image of CD-Au NCs synthesized in Example 1.

[0017] Figure 3 This is the DLS image of CD-Au NCs synthesized in Example 1.

[0018] Figure 4 This is the Zeta potential diagram of CD-Au NCs synthesized in Example 1.

[0019] Figure 5 This is the absorption spectrum of CD-Au NCs synthesized in Example 1.

[0020] Figure 6 This is the fluorescence spectrum of CD-Au NCs synthesized in Example 1.

[0021] Figure 7 The AAs-Ad synthesized in Example 2 33 and TEM images of AAs-Ad5.

[0022] Figure 8 The AAs-Ad synthesized in Example 2 33 and DLS graphs of AAs-Ad5.

[0023] Fig. 9 The AAs-Ad synthesized in Example 2 33, AAs-Ad5 and the absorption spectra comparison of CD-Au NCs synthesized in Example 1.

[0024] Fig.10 TEM images and magnified images of the assemblies AAs-1, AAs-2, and AAs-3 synthesized in Example 3 (the illustration in the lower left corner is the mapping distribution diagram of AAs-1).

[0025] Fig.11 This is the X-ray photoelectron spectrum (Au4f) of the assemblies AAs-1 and AAs-2 synthesized in Example 3.

[0026] Fig.12 This is a graph showing the cell survival rates of L02 and 4T1 cells after incubation with different concentrations of CD-Au NCs, AAs-1 and AAs-2 for 24 h in Example 4.

[0027] Fig.13 This is a graph showing the liver function indicators of mice injected with PBS, CD-Au NC, AAs-1 and AAs-2 in Example 4, respectively, 24 hours later.

[0028] Fig.14 This is a graph showing the renal function indicators of mice injected with PBS, CD-Au NCs, AAs-1 and AAs-2 respectively 24 hours after injection in Example 4.

[0029] Fig.15 These are NIR-II fluorescence images (808 nm laser excitation) of the in vivo organs (heart, liver, spleen, lung, and kidney) of mice injected with CD-Au NCs, AAs-1, and AAs-2 through the tail vein in Example 5 within 24 hours and in vitro 24 hours after the injection.

[0030] Fig.16 This is the NIR-II fluorescence imaging image of CD-Au NCs, AAs-1 and AAs-2 cell uptake in Example 6 (808 nm laser excitation).

[0031] Fig.17 These are NIR-II fluorescence imaging images of cell uptake in Example 7 after incubation with different concentrations of AAs-2 for different times (808 nm laser excitation).

[0032] Fig.18 This is an electron micrograph of cells incubated with 1 mM AAs-2 for 6 h in Example 7 (AAs-2 is within the dotted line).

[0033] Fig.19 This is the NIR-II fluorescence imaging image (808 nm laser excitation) of the specific targeted cellular uptake of CD44 overexpressed on the surface of AAs-2 and 4T1 cells in Example 8.

[0034] Fig. 20 Graph showing the fluorescence spectra of RB, AAs, and AAs-2@R in Example 9.

[0035] Fig.21 This is the absorption spectrum of RB, AAs, and AAs-2@RB in Example 9.

[0036] Fig. 22 This is a laser confocal image of 4T1 and L02 cells incubated with AAs-2@RB in Example 9 (scale bar: 20 μm). DETAILED DESCRIPTION

[0037] A host-guest interaction-mediated gold nanocluster assembly, the preparation steps of which are as follows: 1) Take 15 mL of 10 mM 6-mercapto-6-deoxy-β-cyclodextrin solution, add it to 77.9 mL of ultrapure water, and stir evenly; 2) Add 4 mL of 25 mM HAuCl4 solution, the solution is now clear yellow, and continue stirring for 5 min; 3) Add 0.6 mL of 1 M NaOH solution to adjust the pH until the yellow color of the solution fades, and continue stirring for 5 min; 4) Add 2.5 mL of 0.01 M NaOH solution to dissolve the freshly prepared 80 mM NaBH4 solution and continue stirring for 12 h until the solution turns dark brown. 5) The obtained mixed solution was placed in a dialysis bag with a MWCO of 8000-14000 Da and dialyzed for 10 h, during which the dialysate was replaced every 2 h; 6) Collect the liquid in the dialysis bag and use an ultrafiltration centrifuge tube with a molecular weight of 50 kDa to ultrafilter and centrifuge at 4°C and 4500 rpm for 15 min to remove the aggregated macromolecules. The filtrate obtained in the ultrafiltration tube is the purified CD-Au NCs solution.

[0038] 7) Take 9 mL of the prepared CD-Au NCs solution, add 6 mL of 3 mM 1,3-bis(1-adamantyl)imidazolium tetrafluoroborate aqueous solution and 3 mL of 3-12 mg / mL adamantane-modified hyaluronic acid (the degree of modification of adamantane is 5%-33%) aqueous solution, oscillate for 12 h, centrifuge at 12000 rpm for 10 min at 4 °C, discard the supernatant, wash the precipitate once with ultrapure water, and re-ultrasonicate in ultrapure water to obtain gold nanocluster assemblies.

[0039] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0040] Example 1 The synthesis of SH-β-CD as a ligand-protected CD-Au NCs includes the following steps: Take 15 mL of 10 mM 6-mercapto-6-deoxy-β-cyclodextrin solution in 77.9 mL of ultrapure water and stir evenly; then add 4 mL of 25 mM HAuCl4 solution, which is now a clear yellow solution, and stir for 5 minutes; then add 0.6 mL of 1 M NaOH solution to adjust the pH until the yellow color of the solution fades, and continue stirring for 5 minutes; then add 2.5 mL of 80 mM NaBH4 solution prepared with 0.01 M NaOH solution, continue stirring at room temperature for 12 hours, and the solution finally turns dark brown; place the resulting solution in a dialysis bag with a MWCO of 8000-14000 Da and dialyze for 10 hours, during which the dialysate is replaced every 2 hours; after the dialysis is completed, collect the liquid in the dialysis bag and use an ultrafiltration centrifuge tube with a molecular weight of 50 kDa to ultrafiltrate and centrifuge at 4 ℃ for 15 minutes. min to remove the aggregated macromolecules, and the filtrate obtained in the ultrafiltration tube is the purified CD-Au NCs.

[0041] 1. The prepared CD-Au NCs samples and the used SH-β-CD were subjected to infrared spectroscopy analysis. The results are shown in Figure 1 . Figure 1 It was confirmed that there were SH-β-CD ligands on the surface of CD-Au NCs samples.

[0042] 2. The prepared CD-Au NCs solution was subjected to TEM, DLS and Zeta data testing. The results are shown in Figure 2-4 As can be seen from the figure, the size of the synthesized CD-Au NCs nanomaterials is relatively uniform, and the particle size of CD-Au NCs is consistent with the TEM results, which is about 1.5nm.

[0043] 3. The prepared CD-Au NCs solution was subjected to UV-visible absorption spectroscopy. The results are shown in Figure 5 . Figure 5 This indicates that CD-Au NCs have a wide spectrum absorption performance in the range of 200-1000 nm, and an absorption peak at 340 nm. At the same time, there is no plasma resonance absorption peak of large-sized gold nanoparticles at about 520 nm in the absorption spectrum, indicating that CD-AuNCs have the characteristics of ultra-small size.

[0044] 4. Use 808 nm laser to excite CD-Au NCs sample and test its fluorescence performance in the second near-infrared region. The results are shown in Figure 6 The results show that CD-Au NCs have a wide emission range in the near-infrared region II, with fluorescence emission in the range of 900-1400 nm, and the maximum emission peak position is 1079 nm, which lays a foundation for its application in near-infrared region II biological in vivo fluorescence imaging.

[0045] Example 2 Preparation of AAs-Ad with a size of 50.7 nm 33 and AAs-Ad5 with a size of 78.8 nm, comprising the following steps: Take 9 mL of the CD-Au NCs solution prepared in Example 1, add 6 mL of 3 mM 1,3-bis(1-adamantyl) imidazole tetrafluoroborate aqueous solution, 3 mL of 3 mg / mL HA-Ad 33 (33% hyaluronic acid modified with adamantane) aqueous solution or HA-Ad5 (5% hyaluronic acid modified with adamantane) aqueous solution, shake and react for 12 h. Then, the reaction solution was centrifuged at 12000 rpm for 10 min at 4 °C, the supernatant was discarded, the precipitate was washed once with ultrapure water, and then ultrasonically dispersed in ultrapure water to obtain the gold nanocluster assembly AAs-Ad 33 and AAs-Ad5.

[0046] 1. Assemble the synthesized gold nanoclusters AAs-Ad 33 TEM and DLS tests were performed with AAs-Ad5. The results are shown in Figure 7 , 8 As can be seen from the figure, the gold nanocluster assembly was successfully synthesized, and AAs-Ad 33 The DLS test results of α-Ad5 and AAs-Ad5 were 50.7 nm and 78.8 nm, respectively, which were consistent with the TEM results.

[0047] 2. The gold nanocluster assembly AAs-Ad prepared in Example 2 33 The UV absorption spectra of AAs-Ad5 and CD-Au NCs were compared. Fig. 9 .Depend on Fig. 9 It can be seen that these three materials have a wide absorption range of 200-1000 nm and have absorption peaks at 340 nm, indicating that the assembly of these two materials did not destroy the structure of CD-AuNCs and did not increase the original size of gold nanoclusters.

[0048] 3. The assembly was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The quantitative results revealed that the yield of AAs-Ad5 was as high as 80.5%, while 33 Only 25.4%, probably due to HA-Ad 33 The modification degree of Ad in the HA-Ad5 is high, which leads to strong hydrophobicity. Therefore, HA-Ad5 with an Ad substitution degree of 5% was selected as one of the components in subsequent experiments.

[0049] Example 3 AAs-1 with a size of 37.8 nm, AAs-2 with a size of 78.8 nm, and AAs-3 with a size of 955 nm were prepared, which included the following steps: Synthesis of AAs-1: Take 9 mL of the CD-Au NCs solution synthesized in Example 1, add 6 mL of 3 mM 1,3-bis(1-adamantyl)imidazolium tetrafluoroborate aqueous solution and 3 mL of 12 mg / mL HA-Ad5 aqueous solution, and shake for 12 h. Then, centrifuge the reaction solution at 12000 rpm for 10 min at 4 °C, discard the supernatant, wash the precipitate with ultrapure water once, and re-ultrasonic disperse it in ultrapure water to obtain the AAs-1 assembly.

[0050] Synthesis of AAs-2: Take 9 mL of the CD-Au NCs solution synthesized in Example 1, add 6 mL of 3 mM 1,3-bis(1-adamantyl)imidazolium tetrafluoroborate aqueous solution and 3 mL of 3 mg / mL HA-Ad5 aqueous solution, and shake for 12 h. Then, centrifuge the reaction solution at 12000 rpm for 10 min at 4 °C, discard the supernatant, wash the precipitate with ultrapure water once, and re-ultrasonic disperse it in ultrapure water to obtain the AAs-2 assembly.

[0051] Synthesis of AAs-3: Take 9 mL of the CD-Au NCs solution synthesized in Example 1, add 6 mL of 3 mM 1,3-bis(1-adamantyl)imidazolium tetrafluoroborate aqueous solution and 3 mL of ultrapure water, and shake for 12 h. Then centrifuge the reaction solution at 12000 rpm for 10 min at 4 °C, discard the supernatant, wash the precipitate with ultrapure water once, and re-ultrasonic disperse it in ultrapure water to obtain the AAs-3 assembly.

[0052] AAs-1, AAs-2 and AAs-3 prepared in Example 3 were subjected to TEM test. The results are as follows: Fig.10As shown. The figure shows that three sizes of gold nanocluster assemblies were successfully prepared, and only AAs-3 assembled by Ad-linker-Ad was aggregated together in a network with poor dispersion; after adding a small amount of HA-Ad5, due to the large molecular structure of HA, the steric hindrance was large, resulting in the destruction of the network structure of HA-Ad5 when competing with Ad-linker-Ad, forming a gold nanocluster assembly AAs-2 with a smaller size of 78.8 nm; continuing to increase the amount of HA-Ad5, a smaller AAs-1 gold nanocluster assembly with a size of 37.8 nm can be prepared. In addition, the element mapping analysis results of the AAs-1 sample showed that the Au and S of the gold nanocluster assembly were evenly distributed, indicating the successful construction of the assembly.

[0053] X-ray photoelectron spectroscopy (XPS) analysis was performed on AAs-1 and AAs-2 prepared in Example 3 to determine the content of Au in different valence states in the two. The results are shown in Fig.11 As can be seen from the figure, the Au(0) contents of the two are similar, both 65%, which is not much different from the Au(0) content of CD-Au NCs, indicating that the structure of CD-Au NCs will not be changed after assembly.

[0054] Example 4 Biosafety Study on Gold Nanoclusters and Their Assemblies First, the cytotoxicity of CD-Au NCs, AAs-1 and AAs-2 was studied by CCK-8 experiment to evaluate the cell compatibility of the materials. The specific operation was to incubate different concentrations of CD-Au NCs, AAs-1 and AAs-2 with L02 and 4T1 for 24 hours, and then detect the absorbance at 450 nm after incubation with CCK-8 kit to detect cell survival rate (n=3). The results are shown in Fig.12 As can be seen from the figure, in the concentration range of 0-1000 μM, the survival rate of normal cells of each material is greater than 90%, and the survival rate of cancer cells is also greater than 80%, indicating that the material has little effect on cell survival rate and has good cell safety.

[0055] In addition, the in vivo safety of CD-Au NCs, AAs-1 and AAs-2 in mice was studied. The specific operation was to extract the serum of mice 24 hours after the tail vein injection of PBS, CD-Au NCs, AAs-1 and AAs-2 to detect liver function and kidney function indicators (n=3) to evaluate the biosafety of the materials. The results are shown in Fig.13 , 14 As can be seen from the figure, the results of various indicators of different materials are all within the normal reference value range of serum biochemical indicators of mice, indicating that the materials will not cause damage to the kidneys and liver and have good biosafety.

[0056] Example 5 Distribution characteristics of gold nanoclusters and their assemblies in living mice.

[0057] The prepared CD-Au NCs, AAs-1 and AAs-2 assemblies were injected into the tail vein, and the biodistribution of the materials in normal mice was monitored within 24 h using a near-infrared zone II in vivo imager. The mice were killed 24 h after injection and their hearts, livers, spleens, lungs and kidneys were isolated for monitoring. The results are shown in Fig.15 . As can be seen from the figure, 10 minutes after injection, all three materials were fluorescent in the bladder, and size-dependent fluorescence was shown in the liver. At the same time, CD-Au NCs were brighter in the bladder at 1 hour, and most organs in the body had no fluorescence as time went on. AAs-1 and AAs-2 had almost no fluorescence in the bladder at 1 hour, but showed obvious fluorescence signals at 6 hours, and the fluorescence lasted until 24 hours after injection. This shows that after entering the body, the gold nanocluster assemblies will first disaggregate into small-sized CD-Au NCs in the body and be excreted through the bladder, proving that the preparation of gold nanocluster assemblies can effectively prolong the residence time of gold nanoclusters in the body. Example 6 Cellular uptake of gold nanocluster assemblies of different sizes The prepared CD-Au NCs, AAs-1 and AAs-2 were incubated with 4T1 cells at a concentration of 0.25 mM for 3 h, and the relative cellular uptake was determined by the fluorescence intensity of the near-infrared zone II in vivo imager. Fig.16 The results in the figure show that when the concentration is 0.25 mM, the plate fluorescence of the larger AAs-2 is the brightest, indicating the highest material uptake, which proves that the AAs assembly has an advantage over CD-Au NCs in terms of cell uptake.

[0058] Example 7 Time and concentration dependence of AAs-2 assembly uptake in tumor cells After incubating 4T1 cells with AAs-2 assemblies of different concentrations (0, 0.25, and 1 mM, respectively) for 1 h, 3 h, and 6 h, the incubated cells were collected and subjected to near-infrared second-zone fluorescence imaging to explore the time and concentration dependence of cellular uptake. The results are shown in Fig.17 The results in the figure show that when the incubation time is 1 h, there is almost no fluorescence at each concentration, while the fluorescence of the plate with a concentration of 1 mM is the brightest when the incubation time is 6 h, indicating that with the increase of incubation time and sample concentration, the cell uptake also increases, and it has a positive correlation with time and concentration dependence. Fig.18 Cell electron microscopy results also confirmed that the cells contained ingested AAs-2 assemblies.

[0059] Example 8 Targeting of HA-modified AAs-2 assemblies to tumor cells overexpressing CD44 receptors A group of 4T1 cells overexpressing CD44 receptors were pre-incubated with HA (HA(+)) and then incubated with AAs-2. Another group of cells were not pre-treated with HA (HA(-)) and only incubated with HA-modified AAs-2 assemblies. After incubation for 6 h, the cells were collected and subjected to near-infrared second-zone fluorescence imaging. The results are shown in Fig.19 As can be seen from the figure, the fluorescence of the well plate pretreated with HA is weak, indicating that the cell uptake is significantly reduced. This shows that HA first binds to the cell membrane surface receptor, which to a certain extent prevents the subsequent HA-modified AAs-2 assembly from interacting with it, resulting in a decrease in uptake; while directly incubating with HA-modified AAs-2 assembly has better targeting for cancer cells overexpressing CD44 receptors.

[0060] Example 9 Targeting ability of AAs-2 to tumor cells overexpressing CD44 receptor Synthesis of AAs-2@RB: Weigh 90 mg of the dye rhodamine B (RB), add 9 mL of the CD-AuNCs solution synthesized in Example 1 and mix evenly, then add 6 mL of 3 mM 1,3-bis(1-adamantyl) imidazole tetrafluoroborate aqueous solution and 3 mL of 3 mg / mL HA-Ad5 aqueous solution, and shake and react at room temperature in the dark for 12 h. Then transfer the reacted solution to a dialysis bag with a molecular weight of 8000-14000 Da, stir and dialyze in the dark to remove excess RB solution and other raw materials (change the dialysate every 6 h), dialyze for 3-4 days until the dialysate is colorless and transparent, then collect the liquid in the dialysis bag, and the resulting solution is AAs-2@RB.

[0061] 1. Spectral characterization of synthesized AAs-2@RB. Fig. 20 , 21 . The figure proves that RB is successfully modified in AAs-2@RB. And as can be seen from the figure, the emission spectra of RB, AAs-2@RB and AAs-2 synthesized without RB modification were scanned from 530 to 700 nm at an excitation wavelength of 488 nm. The results show that both AAs-2@RB and RB have emission peaks at 577 nm. At the same time, the UV-visible absorption spectrum shows that both AAs-2@RB and RB have absorption peaks at 553 nm, indicating the successful modification of RB.

[0062] 2. AAs-2@RB was co-incubated with 4T1 (high CD44 expression) and L02 (normal cells, low CD44 expression) cells for 8 h, and fluorescence confocal imaging was performed. The results are shown in Fig. 22(The red fluorescence represents AAs-2@RB, and the stained cell nucleus is shown in blue). The results in the figure show that the red fluorescence intensity of 4T1 cells is the strongest when co-incubated for 6 hours, indicating that the amount of gold nanocluster assemblies taken up is the largest at this time. At the same time, the red fluorescence of 4T1 cells is significantly brighter than that of L02 cells at the same time point, indicating that the gold nanocluster assemblies have good tumor cell targeting ability.

[0063] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing a gold nanocluster assembly mediated by host-guest interaction, characterized in that: First, surface ligand-protected gold nanoclusters CD-Au NCs were synthesized using thiol-β-cyclodextrin. Then, the host-guest interaction between cyclodextrin and adamantane derivatives, as well as the competitive relationship between different adamantane derivatives, were utilized to synthesize a class of gold nanocluster assemblies using diadamantane ligands as connectors and adamantane-modified hyaluronic acid as a regulator.

2. The method for preparing the gold nanocluster assembly according to claim 1, characterized in that: The preparation steps of the surface ligand-protected gold nanoclusters CD-Au NCs are as follows: 1) Take 15 mL of 10 mM 6-mercapto-6-deoxy-β-cyclodextrin solution, add it to 77.9 mL of ultrapure water, and stir evenly; 2) Add 4 mL of 25 mM HAuCl4 solution, the solution is now clear yellow, and continue stirring for 5 min; 3) Add 0.6 mL of 1 M NaOH solution to adjust the pH. After the yellow color of the solution fades, continue stirring for 5 min. 4) Add 2.5 mL of 0.01 M NaOH solution to dissolve the freshly prepared 80 mM NaBH4 solution and continue stirring for 12 h until the solution turns dark brown; 5) The obtained mixed solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed for 10 hours, during which the dialysate was replaced every 2 hours; 6) Collect the liquid in the dialysis bag and use an ultrafiltration centrifuge tube with a molecular weight cutoff of 50 kDa to ultrafilter and centrifuge at 4 ℃ and 4500 rpm for 15 min to remove the aggregated macromolecules. The filtrate obtained in the ultrafiltration tube is the purified CD-Au NCs solution.

3. The method for preparing the gold nanocluster assembly according to claim 1, characterized in that: The preparation of the gold nanocluster assembly is as follows: 9 mL of CD-Au NCs solution is added with 6 mL of 3 mM 1,3-bis(1-adamantyl)imidazolium tetrafluoroborate aqueous solution and 3 mL of 3-12 mg / mL adamantane-modified hyaluronic acid aqueous solution, and after oscillating for 12 h, the mixture is centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant is discarded, the precipitate is washed once with ultrapure water, and then ultrasonically dispersed in ultrapure water to obtain the assembly.

4. The method for preparing gold nanocluster assembly according to claim 3, characterized in that: In the adamantane-modified hyaluronic acid used, the degree of substitution of adamantane is 5% to 33%.

5. A gold nanocluster assembly prepared by the method as claimed in claim 1.

6. Use of the gold nanocluster assembly as claimed in claim 5 as a fluorescent probe for in vivo imaging.

7. Use of the gold nanocluster assembly as claimed in claim 5 in preparing an in vivo drug delivery system.

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