A thermosensitive bivalent gold nanocluster and its preparation method and application

Abstract

The present invention provides a thermosensitive bivalent gold nanocluster and a preparation method and application thereof, belonging to the technical field of tumor treatment. The thermosensitive bivalent gold nanocluster is composed of biGC and PNA, wherein the Au(I) / Au(0) molar ratio in the thermosensitive bivalent gold nanocluster is 0.58-1.89; the mass fraction of PNA in the thermosensitive bivalent gold nanocluster is 45.3%, the weight average molecular weight is 24.4 kDa, and the molar ratio of N-isopropylacrylamide to acrylic acid in the PNA is 4:1. In a dual tumor model, it is confirmed that biGC@PNA combined with decitabine induces cell pyroptosis under radio frequency field radiation, ablates the in situ tumor and inhibits the growth of the distal tumor at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and in particular relates to a temperature-sensitive bivalent gold nanocluster and a preparation method and application thereof. Background Art

[0002] Cancer immunotherapy is highly effective in killing tumor cells, but it is only effective in a small subset of patients (approximately 10-30%) with immunologically "hot" tumors. Pyroptosis is a form of programmed cell death associated with the adaptive immune response. It triggers systemic immune responses by inducing immunogenic cell death (ICD) and improving cytotoxic T lymphocyte (CTL) infiltration, thereby enhancing the efficacy of immunotherapy against various cancers. Gasdermins are a family of pore-forming proteins that play a central role in pyroptosis in various diseases, including infection, diabetes, atherosclerosis, and cancer. Gasdermin E (GSDME) plays a key role in pyroptosis in tumor cells. Because GSDME is typically expressed at low levels in most tumor cells, it is difficult to induce pyroptosis. However, DNA methyltransferase inhibitors (such as decitabine and 5-azacytidine) can address this issue by upregulating GSDME in tumor cells. GSDME-N, generated by cleavage of GSDME, is a key promoter that converts caspase-3-dependent, non-immunogenic apoptosis into highly immunogenic pyroptosis. Therefore, promoting the cleavage activity of caspase-3 can effectively regulate caspase-3 / GSDME-mediated tumor cell pyroptosis, thereby having a good inhibitory effect on primary tumors and distant metastatic tumors.

[0003] Radiofrequency ablation (RFA) is one of the most commonly used thermal therapies in clinical practice. It uses electrodes to deliver high-frequency alternating current to tumors, exerting multiple anti-tumor mechanisms (such as direct coagulative necrosis of tumor cells, caspase-3-dependent apoptosis, and modulation of the tumor immune microenvironment). However, RFA suffers from uneven temperature distribution, poor tumor ablation efficacy, and indiscriminate thermal damage to normal tissues. This significantly limits its application in tumor treatment.

[0004] Radiofrequency ablation (RFA) based on radiofrequency-responsive nanomaterials has garnered widespread attention, offering advantages such as uniform temperature distribution, selective thermal ablation, and precise multimodal synergy. Furthermore, it can flexibly and precisely ablate tumor cells and continuously activate anti-tumor immune responses without the need for needle insertion. RF-responsive materials include gold nanoparticles, quantum dots, carbon nanotubes, fullerenes, and graphene. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a thermosensitive bivalent gold nanocluster and its preparation method and application, which was confirmed in a double tumor model.bi Under the action of radio frequency electromagnetic waves, GC@PNA activates tumor cell pyroptosis, enhances anti-tumor immune effects, and effectively inhibits tumor recurrence and metastasis.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a thermosensitive bivalent gold nanocluster, wherein the thermosensitive bivalent gold nanocluster is composed of bi GC and PNA composition,

[0008] The Au(I) / Au(0) molar ratio in the thermosensitive bivalent gold nanoclusters is 0.58-1.89;

[0009] The mass fraction of PNA in the thermosensitive bivalent gold nanoclusters is 45.3%, the weight-average molecular weight is 24.4 kDa, and the molar ratio of N-isopropylacrylamide to acrylic acid in the PNA is 4:1.

[0010] Preferably, bivalent gold nanoclusters with a Au(I) / Au(O) molar ratio of 0.58 to 1.89 are synthesized. bi GC, including bi GC （1 / 3 / 1） 、 bi GC (1 / 1 / 2) 、 bi GC (1 / 2 / 2) 、 bi GC (1 / 3 / 2) 、 bi GC (1 / 3 / 3) 、 bi GC (1 / 3 / 4) or bi GC (1 / 4 / 2) ;

[0011] described bi GC （1 / 3 / 1） The Au(I) / Au(0) molar ratio is 1.89;

[0012] described bi GC (1 / 1 / 2) The Au(I) / Au(0) molar ratio is 0.58;

[0013] described bi GC (1 / 2 / 2) The Au(I) / Au(0) molar ratio is 1.05;

[0014] described bi GC (1 / 3 / 2) The Au(I) / Au(0) molar ratio is 1.13;

[0015] described bi GC(1 / 3 / 3) The Au(I) / Au(0) molar ratio is 1.33;

[0016] described bi GC (1 / 3 / 4) The Au(I) / Au(0) molar ratio is 1.08;

[0017] described bi GC (1 / 4 / 2) The Au(I) / Au(0) molar ratio is 0.68;

[0018] Preferably, bivalent gold nanoclusters bi GCs are composed of five nanoclusters with precise molecular formulas, including Au 47 LA 25 、Au 60 LA 22 、Au 57 LA 26 、Au 42 LA 41 and Au 63 LA 25 ;

[0019] The Au 47 LA 25 The Au(I) / Au(0) molar ratio is 1.0;

[0020] The Au 60 LA 22 The Au(I) / Au(0) molar ratio is 0.43;

[0021] The Au 57 LA 26 The Au(I) / Au(0) molar ratio is 0.89;

[0022] The Au 42 LA 41 The Au(I) / Au(0) molar ratio is 3.75;

[0023] The Au 63 LA 25 The Au(I) / Au(0) molar ratio is 0.79;

[0024] Preferably, the temperature-sensitive bivalent gold nanoclusters have a radiofrequency thermal effect, and the radiofrequency thermal effect is positively correlated with the Au(I) / Au(0) molar ratio thereof.

[0025] The present invention also provides a method for preparing the temperature-sensitive bivalent gold nanoclusters described in the above technical solution, comprising the following steps:

[0026] 1) mixing LA with water, adjusting the pH to 8.0, and stirring to obtain a mixture;

[0027] 2) mixing the mixture obtained in step 1) with an aqueous solution of HAuCl4 and stirring to obtain a colorless solution;

[0028] 3) The colorless solution obtained in step 2) was mixed with an aqueous solution of sodium borohydride and stirred overnight to obtain bi GC;

[0029] 4) The obtained step 3) bi GC is mixed and incubated with the PNA to obtain temperature-sensitive bivalent gold nanoclusters;

[0030] The mass ratio of LA in step 1) to HAuCl4 in the HAuCl4 aqueous solution in step 2) is 3:1.

[0031] Preferably, in step 1), the volume ratio of the molar mass of LA to water is 0.02-0.08 mmol:40 ml.

[0032] Preferably, the volume ratio of water in step 1) to the HAuCl4 aqueous solution in step 2) is 50:1, and the concentration of Au in the HAuCl4 aqueous solution is 25 mmol / L.

[0033] Preferably, the volume ratio of the colorless solution in step 3) to the sodium borohydride aqueous solution is 51:1-4, and the concentration of the sodium borohydride aqueous solution is 25 mmol / L.

[0034] Preferably, the step 4) bi The molar ratio of GC to PNA was 1:2.

[0035] Preferably, the incubation time in step 4) is 48 hours, and the incubation temperature is 30°C.

[0036] The present invention also provides the above technical solution bi Application of GC@PNA combined with DNA methyltransferase inhibitors in the preparation of anti-tumor drugs.

[0037] Preferably, the DNA methyltransferase inhibitor comprises decitabine.

[0038] Preferably, the tumor comprises a breast tumor.

[0039] The present invention designs RF-responsive bivalent gold nanoclusters ( biGC@PNA), which is then modified with a temperature-sensitive polymer to improve its in vivo behavior (cellular uptake and intratumoral retention, etc.). Since the monovalent gold ion (Au(I)) content affects the RF heating effect, the feed ratio of the weak reducing agent to the strong reducing agent is precisely adjusted. bi The ratio of Au(I) / Au(0) in GC@PNA was determined and its thermal response behavior under non-invasive radiofrequency field was studied. bi GC@PNA was combined with RF to evaluate the RF-activated tumor cell pyroptosis by detecting the expression levels of GSDME, GSDME-N, and activated caspase-3 in 4T1 breast tumor cells and corresponding 4T1 tumor-bearing mice. bi GC@PNA binds to decitabine under RF irradiation, resulting in distal inhibition. bi GC@PNA has the potential to be developed as RF-activated pyroptosis nanoeffectors to enhance the antitumor efficacy of cancer immunotherapy.

[0040] The beneficial effects achieved by the present invention are:

[0041] The radio frequency-responsive Au(I) ions and Au(0) atoms with precise stoichiometric ratios were successfully synthesized via the redox reaction of LA (lipoic acid, a weak reducing agent and stabilizer) and NaBH4 (a strong reducing agent). bi GC, which was subsequently modified with temperature-sensitive PNA to improve its in vivo behavior. bi GC@PNA is a composite of five nanoclusters (Au 47 LA 25 、Au 60 LA 22 、Au 57 LA 26 、Au 42 LA 41 and Au 63 LA 25 ) mixture. 42 LA 41 It has the highest Au(I) / Au(0) ratio (3.75) among the five nanoclusters and accounts for nearly half of the total mass, thus proving that bi GC@PNA generates a large amount of frictional heat through Au(I) vibration under RF irradiation. This invention further proves that bi The radiofrequency heating effect of GC@PNA can induce caspase-3 / GSDME-N-mediated tumor cell pyroptosis, especially when used in combination with DCT (decitabine). biGC@PNA can induce a strong ICD effect and enhance the anti-tumor efficacy under radiofrequency irradiation. More importantly, bi RF-activated pyroptosis of GC@PNA significantly stimulated systemic antitumor immune responses and contributed to the suppression of distal tumors. bi GC@PNA has high potential for clinical translation and provides a promising strategy for enhancing cancer immunotherapy via RF-activated pyroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the gel permeation chromatogram of PNA polymer;

[0043] Figure 2 For the NMR of PNA ( 1 H-NMR) hydrogen spectrum;

[0044] Figure 3 For PNA and bi Transmittance and temperature change curve of GC@PNA at pH 6.5 (5 mg / mL PNA);

[0045] Figure 4 For different bi Au 4 in GC f XPS spectra of the orbitals and the corresponding Au(I) / Au(0) ratio plots;

[0046] Figure 5 To separate the bi The corresponding MALDIOF MS results of bands 1-5 harvested by GC;

[0047] Figure 6 for bi HRXPS spectra of Au 4f orbitals and the corresponding Au(I) / Au(0) ratios in bands 1–5 of GC;

[0048] Figure 7 for bi Temperature rise curves of fragments 1-5 in GC after irradiation in a 150 W RF field for 5 min;

[0049] Figure 8 for bi TEM of GC@PNA (inset shows high-resolution TEM and size distribution);

[0050] Figure 9 High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and bi Linear scanning microscopic analysis of Au, S, and O elements of GC@PNA;

[0051] Figure 10 for bi GC and bi Thermogravimetric analysis spectrum of GC@PNA;

[0052] Figure 11 For 4T1 cells (5.0 mmol L -1 Au) and bi GC and bi The amount of Au taken up by GC@PNA after incubation for 1 h, 4 h, 12 h, and 24 h;

[0053] Figure 12 Non-invasive radiofrequency radiation (150 W, 5 min) bi ΔT and Au concentration curves of GC@PNA;

[0054] Figure 13 The effect of RF field power on RF heating effect (5 mmol L -1 Au, 5 min);

[0055] Figure 14 Control (left) and DCT / bi Western blot analysis of pyroptosis-related protein expression (GSDME-FL, GSDME-N, Pro-CASP3, and Cleaved CASP3) in 4T-1 cells after GC@PNA + RF treatment (right);

[0056] Figure 15 Control (left) and DCT / Representative photos of 4T-1 cells after GC@PNA + RF treatment (right); black arrows point to pyroptotic cells, and white arrows point to apoptotic cells; scale bar: 40 μm;

[0057] bi For Control and DCT / Figure 16 Extracellular HMGB-1 and ATP levels in 4T-1 cells after different treatments with GC@PNA + RF;

[0058] bi For Control and DCT / Figure 17 Effects of different GC@PNA + RF treatments on DC maturation rate;

[0059] bi For Control and DCT / Figure 18 Representative tumor photos of 4T1 tumor-bearing mice after different treatments with GC@PNA + RF;

[0060] biIndividual tumor growth curves of 4T1 tumor-bearing mice after control and DCT / biGC@PNA + RF treatments;

[0061] Figure 19 Control (left) and DCT / Figure 20 Western blot analysis of related protein expression (GSDME-FL, GSDME-N, Pro-CASP 3, and Cleaved CASP 3) in tumor tissues after different treatments of GC@PNA + RF (right);

[0062] bi For Control and DCT / Figure 21 Corresponding fluorescence intensity of tumor sections after different treatments of GC@PNA + RF;

[0063] bi CD3 T cells were obtained from distant tumors (left) and spleens (right) 14 days after treatment. + CD8 T cells + Percentage of T cells;

[0064] Figure 22 For Control and DCT / Figure 23 Cytokine levels of tumor necrosis factor α (TNF-α) (left), interferon γ (IFN-γ) (middle), and interleukin-1β (IL-1β) (right) in serum 72 h after different treatments of GC@PNA + RF;

[0065] bi For Control and DCT / Figure 24 Quantification of mature DCs in tumor-draining lymph nodes, (left) primary tumors (center), and spleens (right) after 72 h of different GC@PNA + RF treatments. bi GC@PNA refers to bi GC （1 / 3 / 1） @PNA;

[0066] bi for Figure 25 GC （1 / 3 / 1） PAGE separation results;

[0067] bi DCT / Figure 26 Schematic illustration of radiofrequency-activated pyroptosis of GC@PNA + RF for cancer immunotherapy. DETAILED DESCRIPTION

[0068] The present invention provides a thermosensitive bivalent gold nanocluster (abbreviated as bi GC@PNA), the thermosensitive bivalent gold nanoclusters are composed ofbi The thermosensitive bivalent gold nanoclusters are composed of GC and PNA, wherein the molar ratio of Au(I) / Au(0) in the thermosensitive bivalent gold nanoclusters is 0.58-1.89; the mass fraction of PNA in the thermosensitive bivalent gold nanoclusters is 45.3%, the weight average molecular weight is 24.4 kDa, and the molar ratio of N-isopropylacrylamide to acrylic acid in the PNA is 4:1; bi GC@PNA bi The Au(I) / Au(0) molar ratio of GC is 0.58~1.89.

[0069] In the present invention, the bi GC preferably includes bi GC （1 / 3 / 1） 、 bi GC (1 / 1 / 2) 、 bi GC (1 / 2 / 2) 、 bi GC (1 / 3 / 2) 、 bi GC (1 / 3 / 3) 、 bi GC (1 / 3 / 4) or bi GC (1 / 4 / 2) ;

[0070] described bi GC （1 / 3 / 1） The Au(I) / Au(0) molar ratio is 1.89;

[0071] described bi GC (1 / 1 / 2) The Au(I) / Au(0) molar ratio is 0.58;

[0072] described bi GC (1 / 2 / 2) The Au(I) / Au(0) molar ratio is 1.05;

[0073] described bi GC (1 / 3 / 2) The Au(I) / Au(0) molar ratio is 1.13;

[0074] described bi GC (1 / 3 / 3) The Au(I) / Au(0) molar ratio is 1.33;

[0075] described bi GC (1 / 3 / 4) The Au(I) / Au(0) molar ratio is 1.08;

[0076] described bi GC (1 / 4 / 2) The Au(I) / Au(0) molar ratio is 0.68;

[0077] In the present invention, the bi GC@PNA bi GC is composed of five nanoclusters with precise molecular formula Au 47 LA 25 、Au 60 LA 22 、Au 57 LA 26 、Au 42 LA 41 and Au 63 LA 25 ;

[0078] The Au 47 LA 25 The Au(I) / Au(0) molar ratio is 1.0;

[0079] The Au 60 LA 22 The Au(I) / Au(0) molar ratio is 0.43;

[0080] The Au 57 LA 26 The Au(I) / Au(0) molar ratio is 0.89;

[0081] The Au 42 LA 41 The Au(I) / Au(0) molar ratio is 3.75;

[0082] The Au 63 LA 25 The Au(I) / Au(0) molar ratio is 0.79.

[0083] In the present invention, the lower critical solution temperature of the PNA is 36.7° C., which is close to the temperature in the human body, and thus can change from a hydrophilic state to a hydrophobic state in the body.

[0084] The present invention also provides a method for preparing the temperature-sensitive bivalent gold nanoclusters described in the above technical solution, comprising the following steps:

[0085] 1) mixing LA with water, adjusting the pH to 8.0, and stirring to obtain a mixture;

[0086] 2) mixing the mixture obtained in step 1) with an aqueous solution of HAuCl4 and stirring to obtain a colorless solution;

[0087] 3) The colorless solution obtained in step 2) was mixed with an aqueous solution of sodium borohydride and stirred overnight to obtain bi GC;

[0088] 4) The obtained step 3) bi GC is mixed and incubated with the PNA to obtain temperature-sensitive bivalent gold nanoclusters;

[0089] The molar ratio of LA in step 1) to HAuCl4 in the HAuCl4 aqueous solution in step 2) is 3:1.

[0090] In the present invention, LA is mixed with water, the pH is adjusted to 8.0, and then stirred to obtain a mixture. In the present invention, the molar mass of LA to the volume of water is preferably 0.02 to 0.08 mmol:40 ml, more preferably 0.06 mmol:40 ml. In the present invention, a 2 mol / L sodium hydroxide solution is preferably used to adjust the pH. In the present invention, the stirring conditions include a time of 15 minutes and a temperature of 25°C.

[0091] In the present invention, the stirred product obtained in step 1) is mixed with an aqueous HAuCl4 solution and stirred to obtain a colorless solution. In the present invention, the volume ratio of water in step 1) to the aqueous HAuCl4 solution in step 2) is preferably 50:1, and the concentration of Au in the aqueous HAuCl4 solution is preferably 25 mmol / L. In the present invention, the stirring conditions include: a time of 15 minutes and a temperature of 25°C. After stirring, the solution turns from light yellow to colorless.

[0092] The colorless solution obtained in step 2) is mixed with a sodium borohydride aqueous solution and stirred for 24 hours to obtain bi GC. In the present invention, the volume ratio of the colorless solution to the sodium borohydride aqueous solution is preferably 51:1-4. In the present invention, the concentration of the sodium borohydride aqueous solution is preferably 25 mmol / L. In the present invention, the sodium borohydride aqueous solution is preferably slowly added to the colorless solution under rapid stirring. For rapid stirring and slow addition, those skilled in the art can follow conventional procedures.

[0093] The present invention obtains the bi GC is mixed with the PNA and incubated to obtain a thermosensitive bivalent gold nanocluster. bi The molar ratio of GC to PNA is preferably 1: 2. In the present invention, the incubation time is 48 h and the incubation temperature is 30°C.

[0094] The present invention also provides the use of the thermosensitive bivalent gold nanoclusters described in the above technical solution in combination with a DNA methyltransferase inhibitor in the preparation of an anti-tumor drug. In the present invention, the DNA methyltransferase inhibitor preferably includes decitabine. In the present invention, the tumor preferably includes a breast tumor.

[0095] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0096] Example 1

[0097] Synthesis of thermosensitive polymer PNA:

[0098] In order to synthesize the thermosensitive gold nanocomposite, the temperature-sensitive PNA block polymer was first synthesized by classical atom transfer radical polymerization (ATRP). N-isopropylacrylamide (NIPAM) (11.3 g, 100 mmol), di- BiBOEDS (160 μL, 0.5 mmol) and tris(2-dimethylaminoethyl)amine (Me6TREN) (500 μL, 2 mmol) were dissolved in 50 mL of a 4 / 1, v / v acetone / water mixture and sealed. After two cycles of liquid nitrogen refrigeration / vacuum / argon filling, CuCl (100 mg, 1 mmol) was added to the reaction system and catalyzed by another cycle of liquid nitrogen refrigeration / vacuum / argon filling. The first polymer block, pNIPAM, was obtained after 24 hours of ATRP reaction in an ethanol bath at 0°C. 100 (PN-Br). Then use a syringe filled with argon to inject degassed tert-butyl acrylate ( t BA) (3.75 mL, 25 mmol) was injected into pNIPAM 100 A second ATRP reaction was carried out in the solution. After another 24 hours, the triblock polymer (PN t B100- b -25, PN t B) polymerization. t B was transferred to water for precipitation and dried under vacuum at 80℃ for 24 hours. Then 2 g of dried PN t B was dissolved in 40 mL of dichloromethane and stirred in an oil bath at 30°C. 4 mL of trifluoroacetic acid was added to induce PN. t B t BA hydrolyzes to produce acrylic acid (AA), yielding PNA. After 24 hours of hydrolysis, the PNA is transferred to a dialysis bag (MWCO: 3500 Da) and dialyzed against deionized water for 5 days. Finally, the dialyzed solution is freeze-dried to obtain purified PNA powder.

[0099] The obtained PNA is a temperature-sensitive triblock polymer with disulfide bonds, such as bi The weight average molecular weight shown is 24.4 kDa. Figure 1The molar ratio of NIPAM / AA is 4.0, resulting in a NIPAM degree of polymerization of 100 and an AA degree of polymerization of 25 in PNA. The lower critical solution temperature of PNA is 36.7°C ( Figure 2 ), close to the temperature inside the human body. Therefore, it can change from a hydrophilic state to a hydrophobic state in the body.

[0100] Example 2

[0101] Bivalent gold nanoclusters Figure 3 GC (1 / 3 / 1) synthesis

[0102] 0.06 mmol lipoic acid was added to 40 mL of aqueous solution, and then 2 mol L -1 The pH was adjusted to 8.0 by NaOH. The mixture was stirred at 25 °C for 15 min, and then a freshly prepared HAuCl4 aqueous solution (0.8 mL, 25 mmol Au L) was added. -1 After stirring for another 15 minutes, the solution changed from light yellow to colorless. Then, sodium borohydride aqueous solution (0.8 mL, 25 mmol L -1 ) was slowly added to the mixture. The reaction, which turned the colorless solution into light brown, was stopped after stirring overnight. bi GC (1 / 3 / 1) Finally, the product was purified by three centrifugal filtrations using an ultrafiltration tube (MWCO: 3 kDa). bi GC (1 / 3 / 1) .

[0103] in bi GC (1 / 3 / 1) The Au(I) / Au(0) molar ratio is 1.89 ( bi ). The higher the Au(I) / Au(0) ratio, the stronger the RF thermal effect.

[0104] Separation using native polyacrylamide gel electrophoresis (PAGE) Figure 4 GC (1 / 3 / 1) The total acrylamide monomer contents of the separation gel and condensation gel were 45% (acrylamide:bis(acrylamide) = 93:7) and 3% (acrylamide:bis(acrylamide) = 94:6), respectively. bi GC (1 / 3 / 1) ) into the wells. and 150 V fixed voltage for about 6 hours to obtain bi GC (1 / 3 / 1)5 different components (Figure 25). Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a MALDI TOF / TOF 5800 (America, SCIEX) to obtain the corresponding molecular weights and molecular formulas of the 5 components. X-ray photoelectron spectroscopy characterization bi GC (1 / 3 / 1) The valence states of the five different Au components.

[0105] PAGE separation bi GC (1 / 3 / 1) Five components were obtained, and their relative mass fractions were determined by MALDI-TOF analysis to be 14417 Da (component 1), 16358 Da (component 2), 16593 Da (component 3), 16734 Da (component 4), and 17568 Da (component 5) (Figure 5). The molecular formulas of these components were Au. 47 LA 25 、Au 60 LA 22 、Au 57 LA 26 、Au 42 LA 41 、Au 63 LA 25 The Au(I) / Au(0) molar ratios of the five components measured by XPS were 1.0, 0.43, 0.89, 3.75, and 0.79, respectively (Figure 6). 42 LA 41 has the highest Au(I) / Au(0) ratio and accounts for bi GC (1 / 3 / 1) The Au content is nearly half (about 43.0%), which has the best radio frequency thermal effect ( bi ).

[0106] Example 3

[0107] Bivalent gold nanoclusters Figure 7 GC (1 / 1 / 2) Synthesis

[0108] 0.02 mmol lipoic acid was added to 40 mL of aqueous solution, and then 2 mol L -1 The pH was adjusted to 8.0 by NaOH. The mixture was stirred at 25 °C for 15 min, and then a freshly prepared HAuCl4 aqueous solution (0.8 mL, 25 mmol Au L) was added. -1 ). Stir for another 15 minutes. Then, sodium borohydride aqueous solution (1.6 mL, 25 mmol L -1) was slowly added to the mixture. The reaction was stopped after stirring for 24 hours and the colorless solution turned into light brown. bi GC (1 / 1 / 2) Finally, the product was purified by three centrifugal filtrations using an ultrafiltration tube (MWCO: 3 kDa). bi GC (1 / 1 / 2) . bi GC (1 / 1 / 2) The Au(I) / Au(0) molar ratio is 0.58 ( bi ).

[0109] Example 4

[0110] Bivalent gold nanoclusters Figure 4 GC (1 / 2 / 2) Synthesis

[0111] 0.04 mmol lipoic acid was added to 40 mL of aqueous solution, and then 2 mol L -1 The pH was adjusted to 8.0 by NaOH. The mixture was stirred at 25 °C for 15 min, and then a freshly prepared HAuCl4 aqueous solution (0.8 mL, 25 mmol Au L) was added. -1 ). Stir for another 15 minutes. Then, sodium borohydride aqueous solution (1.6 mL, 25 mmol L -1 ) was slowly added to the mixture. The reaction was stopped after stirring for 24 hours and the colorless solution turned into light brown. bi GC (1 / 2 / 2) Finally, the product was purified by three centrifugal filtrations using an ultrafiltration tube (MWCO: 3 kDa). bi GC (1 / 2 / 2) . bi GC (1 / 2 / 2) The Au(I) / Au(0) molar ratio is 1.05 ( bi ).

[0112] Example 5

[0113] Bivalent gold nanoclusters GC (1 / 3 / 2) Synthesis

[0114] 0.06 mmol lipoic acid was added to 40 mL of aqueous solution, and then 2 mol L -1 The pH was adjusted to 8.0 by NaOH. The mixture was stirred at 25 °C for 15 min, and then a freshly prepared HAuCl4 aqueous solution (0.8 mL, 25 mmol Au L) was added. -1 ). Stir for another 15 minutes. Then, sodium borohydride aqueous solution (1.6 mL, 25 mmol L -1) was slowly added to the mixture. The reaction was stopped after stirring for 24 hours and the colorless solution turned into light brown. Figure 4 GC (1 / 3 / 2) Finally, the product was purified by three centrifugal filtrations using an ultrafiltration tube (MWCO: 3 kDa). bi GC (1 / 3 / 2) . bi GC (1 / 3 / 2) The Au(I) / Au(0) molar ratio is 1.13 ( bi ).

[0115] Example 6

[0116] Bivalent gold nanoclusters bi GC (1 / 3 / 3) Synthesis

[0117] 0.06 mmol lipoic acid was added to 40 mL of aqueous solution, and then 2 mol L -1 The pH was adjusted to 8.0 by NaOH. The mixture was stirred at 25 °C for 15 min, and then a freshly prepared HAuCl4 aqueous solution (0.8 mL, 25 mmol Au L) was added. -1 ). Stir for another 15 minutes. Then, sodium borohydride aqueous solution (2.4 mL, 25 mmol L -1 ) was slowly added to the mixture. The reaction was stopped after stirring for 24 hours and the colorless solution turned into light brown. Figure 4 GC (1 / 3 / 3) Finally, the product was purified by three centrifugal filtrations using an ultrafiltration tube (MWCO: 3 kDa). bi GC (1 / 3 / 3) . bi GC (1 / 3 / 3) The Au(I) / Au(0) molar ratio is 1.33 ( bi ).

[0118] Example 7

[0119] Bivalent gold nanoclusters bi GC (1 / 3 / 4) Synthesis

[0120] 0.06 mmol lipoic acid was added to 40 mL of aqueous solution, and then 2 mol L -1 The pH was adjusted to 8.0 by NaOH. The mixture was stirred at 25 °C for 15 min, and then a freshly prepared HAuCl4 aqueous solution (0.8 mL, 25 mmol Au L) was added. -1 ). Stir for another 15 minutes. Then, sodium borohydride aqueous solution (3.2 mL, 25 mmol L -1) was slowly added to the mixture. The reaction was stopped after stirring for 24 hours and the colorless solution turned into light brown. Figure 4 GC (1 / 3 / 4) Finally, biGC was purified by three centrifugal filtrations using an ultrafiltration tube (MWCO: 3 kDa). (1 / 3 / 4) . bi GC (1 / 3 / 4) The Au(I) / Au(0) molar ratio is 1.08 ( bi ).

[0121] Example 8

[0122] Bivalent gold nanoclusters bi GC (1 / 4 / 2) Synthesis

[0123] 0.08 mmol lipoic acid was added to 40 mL of aqueous solution, and then 2 mol L -1 The pH was adjusted to 8.0 by NaOH. The mixture was stirred at 25 °C for 15 min, and then a freshly prepared HAuCl4 aqueous solution (0.8 mL, 25 mmol Au L) was added. -1 ). Stir for another 15 minutes. Then, sodium borohydride aqueous solution (1.6 mL, 25 mmol L -1 ) was slowly added to the mixture. The reaction was stopped after stirring for 24 hours and the colorless solution turned into light brown. Figure 4 GC (1 / 4 / 2) Finally, biGC was purified by three centrifugal filtrations using an ultrafiltration tube (MWCO: 3 kDa). (1 / 4 / 2) . bi GC (1 / 4 / 2) The Au(I) / Au(0) molar ratio is 0.68 ( bi ).

[0124] Example 9

[0125] Thermosensitive nanocomposites bi Synthesis of GC@PNA and Characterization of Its Radiofrequency Thermal Response Characteristics

[0126] Will get Figure 4 GC (1 / 3 / 1)、 bi GC (1 / 1 / 2)、 bi GC (1 / 2 / 2) bi GC (1 / 3 / 2) bi GC (1 / 3 / 3) 、bi GC (1 / 3 / 4) 、bi GC (1 / 4 / 2)The cells were further incubated with a PNA solution (8.0 wt%) (prepared in Example 1), wherein the amount of PNA was 0.04 mmol. 、bi The amount of gold used in GC is 0.02mmol. Due to the coordination effect between gold and sulfur, bi GC and PNA were synthesized by incubation at 30°C for 48 hours. bi GC@PNA. After the reaction, ultrafiltration was performed using an ultrafiltration tube with a molecular weight cutoff of 30,000 Da (6000 rpm, 5 min). 1.5 mL of different gold concentrations (1.25, 2.5, 5, and 10 Au mmol L -1 )of bi The GC@PNA was exposed to RF fields of different powers (50, 100, 150, and 200W) for 300 seconds. The real-time temperature was recorded every 20 seconds using an NIR thermal imager. bi The influence of Au concentration in GC@PNA on thermal effect. bi GC@PNA bi GC (1 / 3 / 1) @PNA has the best RF thermal effect ( bi ).

[0127] TEM image display bi GC (1 / 3 / 1) @PNA is a uniform spherical nanocluster with an average diameter of 1.5±0.4nm ( Figure 7 ). In addition, from the results of angular annular dark field scanning transmission electron (HAADF-STEM), bi GC (1 / 3 / 1) @PNA exhibits a unique core-shell structure consisting of Au(0) nanocrystals (bright core) surrounded by a PNA (shaded) shell ( Figure 8 ). Through line scanning analysis of three elements (O, S and Au), bi GC (1 / 3 / 1) The characteristic peaks of Au in the core-shell of @PNA appeared in the core of the nanoparticles, while the characteristic peaks of O and S elements only appeared in the shell of the nanoparticles, which further proved that Figure 9 GC (1 / 3 / 1) @PNA core-shell structure ( bi ). Thermogravimetric analysis (TGA) showed bi GC (1 / 3 / 1) @PNA The mass fraction of PNA is 45.3% wt% and the mass fraction of LA is 11.5 wt% ( Figure 9 ). bi GC (1 / 3 / 1)The lower critical solution temperature (LCST) of @PNA was measured to be 36.2 from the temperature / pH dependent UV / Vis transmittance curve. o C( Figure 10 ). It is very close to the LCST of PNA (36.7 o C), indicating bi GC (1 / 3 / 1) PNA (endowed Figure 3 GC (1 / 3 / 1) Thermosensitivity, enhancing tumor cell uptake) has little effect on temperature sensitivity. This suggests bi GC (1 / 3 / 1) @PNA will undergo a change in hydrophilicity and hydrophobicity in the body. This change makes bi GC (1 / 3 / 1) @PNA bi GC (1 / 3 / 1) Has higher cellular uptake ( bi ). Irradiated in a 150 W radio frequency field for 5 min, with a gold concentration of 25 mmol L -1 of bi GC (1 / 3 / 1) @PNA has the strongest RF thermal effect ( Figure 11 ). For 5 mmol L -1 of bi GC (1 / 3 / 1) @PNA, 5 minutes of irradiation with different radio frequency powers, the higher the power, the stronger the radio frequency thermal effect ( Figure 12 ).

[0128] Example 10

[0129] DCT / bi GC （1 / 3 / 1） PNA + RF synergistically activates pyroptosis and anti-tumor immune responses in vitro

[0130] Pyroptosis assays were performed by Western blotting, 4T1 cell (mouse breast cancer cell) morphology, Annexin V-FITC / PI staining, and detection of dendritic cell (DC) maturation and ICD biomarkers.

[0131] For Western blot analysis, 4T1 cells in the control group were added with 2 mL of culture medium, DCT / Figure 13 GC （1 / 3 / 1） 4t1 cells in the PNA + RF group were treated with decitabine (1 μmol L -1 , 2 mL), incubate for 3 days and then add bi GC （1 / 3 / 1） @PNA (gold concentration of 2 mmol L -1After incubation with 2 mL of PBS (100 W, 2 mL) for 12 hours, cells were irradiated under a 100 W radiofrequency field for 2 minutes. After an additional 12 hours, the cells were washed and lysed for protein extraction. Samples were heated for 5 minutes and separated on SDS-PAGE. After electrophoresis, the separated proteins were transferred to a PVDF membrane and incubated in PBS containing 5% skim milk for 1 hour. The membrane was then incubated with a primary antibody against GSDME for 1 hour, followed by co-incubation with the corresponding fluorescent-conjugated secondary antibody for 1 hour. Finally, GSDME expression under different conditions was determined by fluorescence analysis, with β-actin serving as a protein loading control. Western blotting was used to examine the levels of related proteins (GSDME-FL, GSDME-N, Pro-CASP3, and CleavedCASP3) in 4T-1 cells or tumor-bearing mice treated with the different treatments, as described above. For morphological observation of 4T1 cells, they were seeded in 6-well plates and subjected to the two treatments described in the Western blotting assay. Representative images were obtained using an Olympus IX71 microscope (Japan). For Annexin V-FITC / PI staining analysis, all cells from each 6-well plate were harvested and washed twice with PBS. The cells were then stained using the Annexin V-FITC and PI staining kit (Abmaking) (Annexin V-FITC for 10 minutes and Propidium Iodide for 5 minutes). Samples were analyzed on a BD FACS Aria III flow cytometer. To examine dendritic cell (DC) maturation, 4T1 cells were treated with various methods and analyzed by Western blot as described above. After a 12-hour incubation, tumor cell supernatant was collected and added to immature DC2.4 mouse dendritic cells. After a 24-hour incubation, DC2.4 mouse dendritic cells were stained with fluorescently labeled PC7 anti-mouse CD11c, PE anti-mouse CD80, and APC anti-mouse CD86 antibodies and analyzed by flow cytometry to measure DC maturation. Finally, for ICD biomarkers induced by pyroptosis, an anti-mouse HMGB-1 ELISA kit and an enhanced chemiluminescence ATP detection kit were used to detect the extracellular release of HMGB-1 and ATP secretion. It is important to note that ATP is extremely unstable, so all operations must be performed on ice.

[0132] Western blot analysis showed that GSDME-FL expression was greatly upregulated by DCT. bi GC （1 / 3 / 1）@PNA enhances the activation of caspase 3 through its RF heating effect to generate cleaved caspase 3. Cleaved caspase 3 cleaves GSDME-FL to generate GSDME-N, which aggregates on the membrane to form pores, leading to cell pyroptosis ( bi ). Therefore, if bi As shown, in DCT / Figure 14 GC （1 / 3 / 1） In the inverted microscope photos of the PNA + RF group, far more swollen cells (typical features of cell pyroptosis) were observed than in the control group. Figure 15 GC （1 / 3 / 1） The combined treatment of PNA and DCT activates pyroptosis in tumor cells through their synergistic effects. GSDME-N aggregates on the membrane to form pores, leading to pyroptosis and the release of a large number of damage-associated molecular patterns (DAMPs), which is considered a new form of immunogenic cell death (ICD). After 4 hours of different treatments, DAMP molecules (ATP and HMGB1) were detected in 4T1 cells under DCT / bi GC （1 / 3 / 1） The highest expression level was found in PNA + RF ( bi It is well known that the ICD effect promotes the maturation of dendritic cells (DCs) through antigen processing and presentation, thereby enhancing anti-tumor immune responses. bi GC （1 / 3 / 1） @PNA + RF treated 4T1 cells to further study DC maturation. Figure 16 GC （1 / 3 / 1） @PNA + RF showed the strongest DC maturation induction, with mature DCs (CD80 + CD86 + / CD11c + ) reached 66.03 ± 5.82% of the total cells ( bi ). This shows that DCT, bi GC （1 / 3 / 1） @PNA and RF radiation can induce strong tumor cell pyroptosis, and they have a good anti-tumor synergistic effect.

[0133] Example 11

[0134] Different bivalent gold nanocomposites Figure 17 In vivo efficacy evaluation of GC@PNA+DCT+RF

[0135] The DCT / bi GC （1 / 3 / 1） @PNA+RF、DCT / bi GC （1 / 1 / 2） @PNA+RF、DCT / bi GC （1 / 2 / 2） @PNA+RF、DCT / bi GC （1 / 3 / 2） @PNA+RF、DCT / bi GC （1 / 3 / 3） @PNA+RF、DCT / bi GC （1 / 3 / 4） @PNA+RF、DCT / bi GC （1 / 4 / 2） @PNA+RF efficacy. 80 μmol L was intravenously injected for 3 consecutive days before non-invasive radiofrequency radiation. -1 DCT, once a day, 100 μL each time. Gold concentration is 21.22 mmol L -1 Different bivalent gold nanocomposites bi GC@PNA (100 μL) was injected intratumorally into the tumor tissue 12 hours before non-invasive radiofrequency radiation. The mice's tumors were then exposed to a 200 W radiofrequency field for 10 minutes. Tumor volume and body weight were recorded every two days. After an additional two weeks of treatment, the remaining mice were sacrificed.

[0136] Western blot analysis was used to investigate the expression of pyroptosis-related proteins in caspase-3 / GSDME-mediated pyroptosis in tumor cells, and dihydroethidium (DHE) staining was used to detect the level of ROS production in the tumor.

[0137] Compared with the Control group, DCT / bi GC （1 / 1 / 2） @PNA + RF, DCT / bi GC （1 / 4 / 2） @PNA + RF, DCT / bi GC （1 / 2 / 2） @PNA + RF, DCT / bi GC （1 / 3 / 4） @PNA + RF, DCT / [[ID=_{}216]]bi GC （1 / 3 / 2） @PNA + RF and DCT / bi GC （1 / 3 / 3） PNA + RF significantly inhibited tumor growth, with the tumor volume increasing from 142.66 ± 20.59 mm on day 2. 3 、166.62± 26.11mm 3, 147.51 ± 12.09 mm 3 、147.51±12.09 mm 3 、132.61± 21.93 mm 3 、144.60 ±4.10 mm 3 and 137.89 ± 25.07 mm 3 806.81 ± 101.30 mm at 14 days 3 、665.01 ±68.29mm 3 , 459.29 ± 39.03 mm 3 、369.21 ± 8.41mm 3 , 370.64 ± 30.16 mm 3 、234.81 ±34.48 mm 3 and 73.58 ± 17.72 mm 3 . Where DCT / bi GC （1 / 3 / 1） Compared with the other groups, mice in the PNA + RF group showed the most significant tumor regression, with the tumor volume decreasing from 132.06 ± 21.32 mm on day 2. 3 Reduced to 27.21±22.1 mm on day 14 3 The tumors in two of the mice were completely ablated at 14 days ( bi , 19) The intratumoral expression of GSDME-FL, GSDME-N, and caspase-3 was further detected by Western blot analysis ( bi ). DCT / Figure 18 GC （1 / 3 / 1） @PNA +RF showed strong GSDME-N activation, indicating that pyroptosis of tumor cells has good anti-tumor efficacy. And under radiofrequency irradiation, a large amount of reactive oxygen species (ROS) will be generated in tumor tissue ( Figure 20 ). This indicates that in addition to the RF heating effect, bi GC （1 / 3 / 1） @PNA also exhibited favorable radiofrequency kinetic effects, which may play an important role in anti-tumor immune responses.

[0138] Example 12

[0139] DCT / Figure 21 GC （1 / 3 / 1） Evaluation of the inhibitory effect of PNA+RF-induced cell pyroptosis on distal tumors

[0140] Due to DCT bi GC （1 / 3 / 1）@PNA + RF has the best anti-tumor effect, and DCT continues to be evaluated bi GC （1 / 3 / 1） @PNA + RF anti-tumor immune response, 4T1 cells (1×10 6 cells) were subcutaneously inoculated into the right flank of each Balb / c mouse. Four days later, 4T1 cells (3×10 5 A bilateral tumor model was established by subcutaneously inoculating 500 cells (1000 cells) into the left flank of each Balb / c mouse. When right-sided tumors reached significant size, mice were treated as described above. Three days after treatment, the maturation of DCs in the primary tumor and major organs was analyzed by flow cytometry. Flow cytometry was also used to assess CD8 T cells in distal tumors 14 days after treatment. + Cytotoxic T lymphocytes (TIL).

[0141] RF-induced pyroptotic ICD leads to strong activation of CTLs in distant tumors and spleen ( / bi ), and strong secretion of anti-tumor cytokines such as TNF-α, IL-1β and IFN-γ in serum ( / bi Dendritic cells are the most important antigen-presenting cells (APCs) and play a key role in tumor-specific immune responses and tumor metastasis suppression. Therefore, mature DCs were detected in primary tumors, tumor-draining lymph nodes (TDLNs), and spleen by flow cytometry. Figure 22 GC （1 / 3 / 1） In PNA+RF treated mice, mature DCs (CD80 + CD86 + / CD45 + CD11c + ) were as high as 55.82±6.25% (primary tumor), 72.24±3.34% (TDLN) and 60.59±2.72% (spleen). Figure 23 GC （1 / 3 / 1） @PNA + RF activated DC maturation much more strongly than the control group ( bi ). These all indicate that DCT / bi Figure 24 bi GC （1 / 3 / 1） @PNA + RF has obvious tumor-specific immune response.

[0142] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A temperature-sensitive bivalent gold nanocluster, characterized in that: The thermosensitive bivalent gold nanoclusters are composed of bi GC and PNA composition, The Au(I) / Au(0) molar ratio in the thermosensitive bivalent gold nanoclusters is 0.58-1.89; The mass fraction of PNA in the thermosensitive bivalent gold nanocluster is 45.3%, the weight average molecular weight is 24.4 kDa, and the molar ratio of N-isopropylacrylamide to acrylic acid in the PNA is 4:

1.

2. The temperature-sensitive bivalent gold nanocluster according to claim 1, characterized in that: Synthesized bivalent gold nanoclusters with Au(I) / Au(0) molar ratios ranging from 0.58 to 1.89 bi GC, including bi GC (1 / 3 / 1) , bi GC (1 / 1 / 2) , bi GC (1 / 2 / 2) , bi GC (1 / 3 / 2) , bi GC (1 / 3 / 3) , bi GC (1 / 3 / 4) or bi GC (1 / 4 / 2) ; Said bi GC (1 / 3 / 1) The Au(I) / Au(0) molar ratio is 1.89; Said bi GC (1 / 1 / 2) The Au(I) / Au(0) molar ratio is 0.58; Said bi GC (1 / 2 / 2) The Au(I) / Au(0) molar ratio is 1.05; Said bi GC (1 / 3 / 2) The Au(I) / Au(0) molar ratio is 1.13; Said bi GC (1 / 3 / 3) The Au(I) / Au(0) molar ratio is 1.33; Said bi GC (1 / 3 / 4) The Au(I) / Au(0) molar ratio is 1.08; Said bi GC (1 / 4 / 2) The Au(I) / Au(0) molar ratio is 0.

68.

3. The temperature-sensitive bivalent gold nanoclusters according to claim 1, characterized in that: Bivalent gold nanoclusters bi GCs are composed of five nanoclusters with precise molecular formulas, including Au 47 LA 25 、Au 60 LA 22 、Au 57 LA 26 、Au 42 LA 41 and Au 63 LA 25 ; The Au 47 LA 25 The Au(I) / Au(0) molar ratio is 1.0; The Au 60 LA 22 The Au(I) / Au(0) molar ratio is 0.43; The Au 57 LA 26 The Au(I) / Au(0) molar ratio is 0.89; The Au 42 LA 41 The Au(I) / Au(0) molar ratio is 3.75; The Au 63 LA 25 The Au(I) / Au(0) molar ratio is 0.

79.

4. The temperature-sensitive bivalent gold nanoclusters according to claim 1, characterized in that: Thermosensitive bivalent gold nanoclusters have radiofrequency thermal effect, which is positively correlated with their Au(I) / Au(0) molar ratio.

5. A method for preparing the temperature-sensitive bivalent gold nanoclusters according to claim 1, characterized in that: The following steps are involved: 1) mixing lipoic acid and water, adjusting the pH value to 8.0, and stirring to obtain a mixture; 2) mixing the stirred product obtained in step 1) with a HAuCl4 aqueous solution and stirring to obtain a colorless solution; 3) The colorless solution obtained in step 2) was mixed with a sodium borohydride aqueous solution and stirred for 24 hours to obtain bi GC; 4) The obtained in step 3) bi GC is mixed and incubated with the PNA to obtain a temperature-sensitive bivalent gold nanocluster; The molar ratio of lipoic acid in step 1) to HAuCl4 in the HAuCl4 aqueous solution in step 2) is 3:

1.

6. The preparation method according to claim 5, characterized in that: In the step 1), the volume ratio of the molar mass of LA to water is 0.02-0.08 mmol:40 ml.

7. The preparation method according to claim 5, characterized in that: The volume ratio of water in step 1) to the HAuCl4 aqueous solution in step 2) is 50:1, and the concentration of Au in the HAuCl4 aqueous solution is 25 mmol / L; In the step 3), the volume ratio of the colorless solution to the sodium borohydride aqueous solution is 51:1-4, and the concentration of the sodium borohydride aqueous solution is 25 mmol / L.

8. The preparation method according to claim 5, characterized in that: Step 4) bi The molar ratio of GC to PNA was 1:

2.

9. Use of the thermosensitive bivalent gold nanoclusters according to claim 1 in combination with a DNA methyltransferase inhibitor in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The DNA methyltransferase inhibitors include decitabine.

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