Engineered gold nanoparticles for realizing all-directional precise photo-thermal therapy of tumor tissues as well as preparation method and application of engineered gold nanoparticles

Abstract

The invention discloses an engineered gold nanoparticle for realizing all-dimensional precise photothermal therapy of tumor tissues, and a preparation method and application thereof. The preparation method comprises the following steps: modifying a thiolated copper ion trapping agent, a thiolated piperidine compound and sulfydryl-undecanoic acid on a gold nanoparticle; the engineered gold nanoparticles for realizing all-directional precise photo-thermal therapy of tumor tissues are obtained. According to the engineered gold nanoparticles prepared through the method, by means of the characteristic that the pH of tumor tissue is continuously reduced from the shallow layer to the deep layer, through the protonation process of a tertiary amine group in a piperidine compound, charge overturning in different levels of tumors is achieved, and therefore the engineered gold nanoparticles are infiltrated into the whole tumor tissue in an all-dimensional mode. After the engineered gold nanoparticles are endocytosed by tumor cells, excessive copper ions in the cells can trigger precise photothermal-chemical kinetics therapy of the engineered gold nanoparticles and induce apoptosis of the tumor cells, and generated apoptosis fragments further cause immunogenic death, so that anti-tumor immunotherapy is realized.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical materials, nanomaterial technology and synergistic tumor treatment, and specifically to an engineered gold nanoparticle for achieving all-round precise photothermal treatment of tumor tissues, as well as a preparation method and application thereof. Background Art

[0002] In recent years, cancer is one of the diseases with the highest mortality rate, which seriously threatens human life and health. Although traditional cancer treatments, such as chemotherapy, radiotherapy and surgery, have made significant progress, they still cannot meet current clinical needs due to their inherent defects. As an emerging cancer treatment method, photothermal therapy can use photothermal conversion agents to convert light energy into heat energy, thereby effectively destroying tumor cells, showing a bright prospect for tumor treatment. However, while conventional photothermal therapy eliminates tumor cells, it will inevitably damage surrounding normal cells, causing systemic side effects. Therefore, it is urgent to construct a selective photothermal therapy system so that it can only exert photothermal effects in tumor cells and avoid additional damage to normal cells.

[0003] Although a variety of targeting ligands have been modified onto the surface of photothermal agents to improve their tumor targeting ability, these modified photothermal agents are still quickly cleared in the blood circulation and have difficulty reaching the tumor site. In order to overcome this challenge, some new treatment strategies, especially pH-responsive charge reversal technology, have attracted widespread attention. This strategy enables photothermal agents to be negatively charged in a neutral physiological environment, avoiding adsorption by negatively charged proteins in the blood, thereby achieving long-term circulation. When reaching the tumor site, the acidic tumor microenvironment causes the charge on the surface of the photothermal agent to change from negative to positive, and it is efficiently taken up by tumor cells with negatively charged cell membranes, thereby being retained at the tumor site.

[0004] However, these photothermal agents with charge reversal properties are mainly retained in the superficial layer of tumor tissue, mainly because they are quickly internalized by superficial tumor cells after binding, making it difficult for the photothermal agents to penetrate deeper into the tumor. Although some strategies have been developed to improve the tumor penetration performance of charge reversal agents through further functionalization, the comprehensive infiltration of these agents in tumors is still difficult to achieve. Summary of the invention

[0005] In view of the problems of the prior art, the purpose of the present invention is to provide an engineered gold nanoparticle, a preparation method and an application for realizing all-round and precise photothermal therapy of tumor tissue. The engineered gold nanoparticles (Au-MBP NPs) have charge flipping ability and can fully infiltrate into the entire tumor tissue to realize tumor-specific photothermal-chemodynamic-immunotherapy.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing engineered gold nanoparticles for achieving all-round precision photothermal therapy of tumor tissues comprises the following steps:

[0008] Thiolated copper ion capture agents, thiolated piperidine compounds and mercapto-undecanoic acid were modified onto gold nanoparticles to obtain engineered gold nanoparticles that can achieve all-round precise photothermal therapy of tumor tissues.

[0009] Furthermore, the specific process is: adding the thiolated copper ion capture agent, the thiolated piperidine compound and the mercapto-undecanoic acid to the gold nanoparticle solution, mixing and reacting for 2 to 4 hours, to obtain engineered gold nanoparticles that can achieve all-round precise photothermal therapy of tumor tissue.

[0010] Furthermore, the molar ratio of the gold nanoparticles to the mercapto-undecanoic acid, the thiolated copper ion capture agent, and the thiolated piperidine compound is 1:0.007-0.03:0.005:0.007-0.009; and the particle size of the gold nanoparticles is 20-50 nm.

[0011] Further, the thiol-containing copper ion capture agent is prepared by the following process:

[0012] Mercapto-undecanoic acid, a copper ion capture agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are mixed and subjected to amide reaction to obtain a thiolated copper ion capture agent.

[0013] Further, the copper ion capture agent is N-aminoethyl-N'-benzoylthiourea;

[0014] The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:copper ion capture agent is 1 to 2:1:1:1.2 to 1.5.

[0015] Further, the thiolated piperidine compound is prepared by the following process:

[0016] Mercapto-undecanoic acid, a piperidine compound, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are mixed and subjected to an amide reaction to obtain a mercapto-piperidine compound.

[0017] Further, the piperidine compound is 1-(2-aminoethyl)-piperidine;

[0018] The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:piperidine compound is 1 to 2:1:1:1.2 to 1.5.

[0019] Furthermore, the gold nanoparticle solution is prepared by the following process: using sodium citrate reduction method to reduce tetrachloroauric acid into gold to obtain gold nanoparticles.

[0020] An engineered gold nanoparticle that enables all-round precision photothermal therapy of tumor tissue.

[0021] Application of engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue in the preparation of anti-tumor drugs.

[0022] Furthermore, the anti-tumor drug is an anti-breast cancer drug.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The Au-MBP NPs prepared in the present invention can utilize the protonation process of the tertiary amine group to achieve charge reversal in different levels of tumors. They can not only stay in the shallow tumor tissue, but also penetrate into the deep tumor, thereby being able to fully infiltrate the entire tumor tissue.

[0025] (2) The Au-MBP NPs prepared by the present invention can utilize the excess copper ions and H2O2 in tumor cells to achieve selective photothermal-chemodynamic-immunotherapy targeting only tumors. The contents of copper ions and H2O2 in normal cells are very low and will not trigger these three treatment methods, nor will they damage normal cells. They have good biosafety and will not cause systemic toxicity.

[0026] (3) The Au-MBP NPs constructed by the present invention can exert the effects of photothermal therapy, chemodynamic therapy and immunotherapy. The synergy of the three treatment methods will greatly exceed the effect of a single treatment method and significantly promote the killing of tumors.

[0027] (4) The Au-MBP NPs constructed in the present invention can not only achieve ablation of primary tumors, but also reduce the copper ion concentration in tumors, and further inhibit tumor metastasis by blocking copper metabolism.

[0028] (5) The present invention uses a one-step method to simultaneously modify the thiolated copper ion capture agent, the thiolated piperidine compound and the mercaptoundecanoic acid onto the surface of the gold nanoparticles through the Au-S bond. The method is simple to operate, highly repeatable, and the obtained engineered gold nanoparticles have high storage stability. In addition, this method does not require large-scale, advanced equipment, has low production costs, does not generate waste liquid, and is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the TEM image of Au-MBP NPs provided in Example 1.

[0030] Figure 2 This is a diagram of the particle size of Au-MBP NPs provided in Example 1.

[0031] Figure 3 This is the Fourier infrared spectrum of Au-MBP NPs provided in Example 1.

[0032] Figure 4 This is a graph showing the potential changes of Au-MBP NPs at different pH values ​​provided in Example 6.

[0033] Figure 5 This is the UV–Vis spectrum of Au-MBP NPs provided in Example 7 that converts cupric ions into cuprous ions.

[0034] Figure 6 This is the UV–Vis spectrum of ROS produced by the reaction of Au-MBP NPs with H2O2 provided in Example 7.

[0035] Figure 7 This is a TEM image of copper ion-induced Au-MBP NPs aggregation provided in Example 7.

[0036] Figure 8 This is the time-temperature curve of the photothermal conversion of Au-MBP NPs provided in Example 7 before and after the addition of copper ions.

[0037] Fig. 9 is a flow cytometry analysis of the uptake of Au-MBP NPs by 4T1 cells at different pH values ​​provided in Example 8, wherein A is the uptake of Au-MBP NPs by 4T1 cells. Cy5 Flow cytometry of Au-M7B5P9, B is the quantitative statistics of Figure A, and C is the uptake of 4T1 cells Cy5 Au-M 20 Flow cytometry of B5P9, D is the quantitative statistics of Figure C, and E is the uptake of 4T1 cells Cy5 Au-M 20 Figure 5P7 is the flow cytometry statistics. Figure 5P7 is the flow cytometry statistics. Figure 5P7 is the flow cytometry statistics.

[0038] Fig.10 This is a concentration analysis diagram of the effect of Au-MBP NPs on copper ion concentration in 4T1 cells provided in Example 9.

[0039] Fig.11 It is a graph showing the survival rates of 4T1 cells and MCF-10A cells in different treatment groups provided in Example 10, wherein A is a graph showing the survival rate of 4T1 cells, and B is a graph showing the survival rate of MCF-10A cells.

[0040] Fig.12This is a graph showing changes in tumor volume of mice in different treatment groups provided in Example 11.

[0041] Fig.13 These are photos of mouse tumors in different treatment groups provided in Example 11.

[0042] Fig.14 The different treatment groups provided in Example 12 promoted CD8 + Immunohistochemistry of T cell recruitment.

[0043] Fig.15 These are photos of lung tissues showing inhibition of tumor metastasis in different treatment groups provided in Example 13.

[0044] Fig.16 This is a statistical diagram of lung nodules in different treatment groups that inhibit tumor metastasis provided in Example 13. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with examples and accompanying drawings, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications and replacements made to the inventive method, steps or conditions all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0046] The present invention provides a method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue, comprising the following steps:

[0047] (1) Mercapto-undecanoic acid (MUA) is added to a copper ion capture agent (BTU, N-aminoethyl-N'-benzoylthiourea), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC and NHS are used as catalysts), and DMSO is used as a solvent to form a first reaction system. The thiol group is modified onto the copper ion capture agent by an amide reaction between an amino group and a carboxyl group, thereby obtaining a thiolated copper ion capture agent SH-BTU.

[0048] Mercapto-undecanoic acid is added to a piperidine compound (PD, 1-(2-aminoethyl)-piperidine, a charge flipping group), and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC and NHS are used as catalysts), and DMSO is used as a solvent to form a second reaction system. The thiol group is modified onto the piperidine compound by utilizing the amide reaction of the amino group and the carboxyl group to obtain the thiolated piperidine compound SH-PD.

[0049] Furthermore, the concentration of mercapto-undecanoic acid in the first reaction system and the second reaction system in step (1) is 0.025 to 0.25 mol / L.

[0050] Furthermore, in the first reaction system and the second reaction system described in step (1), the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) is 0.05-0.5 mol / L, and the concentration of N-hydroxysuccinimide (NHS) is 0.025-0.25 mol / L.

[0051] Furthermore, the copper ion capture agent in step (1) is N-aminoethyl-N'-benzoylthiourea, and the concentration is 0.03-0.3 mol / L.

[0052] Furthermore, the piperidine compound described in step (1) is 1-(2-aminoethyl)-piperidine, and the concentration is 0.03 to 0.3 mol / L.

[0053] Furthermore, in the first reaction system in step (1), the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC): N-hydroxysuccinimide (NHS): mercapto-undecanoic acid: copper ion capture agent is 1 to 2: 1: 1: 1.2 to 1.5.

[0054] Furthermore, in the second reaction system in step (1), the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC): N-hydroxysuccinimide (NHS): mercapto-undecanoic acid: piperidine compound is 1 to 2: 1: 1: 1.2 to 1.5.

[0055] Furthermore, in step (1), the reaction time of the amide reaction between the first reaction system and the second reaction system is 12 to 24 hours, and the reaction is carried out at room temperature.

[0056] (2) Using the sodium citrate reduction method, tetrachloroauric acid is reduced to gold. The specific process is: tetrachloroauric acid solution is mixed with sodium citrate solution, and then a reduction reaction is carried out. By controlling the concentration of sodium citrate and tetrachloroauric acid and the reaction time, gold nanoparticle solutions with different particle sizes are obtained.

[0057] Furthermore, the concentration of the tetrachloroauric acid solution in step (2) is 0.25 to 2.5 mmol / L.

[0058] Furthermore, the concentration of the sodium citrate solution in step (2) is 1.5 to 15 mmol / L.

[0059] Furthermore, the molar ratio of tetrachloroauric acid to sodium citrate in step (2) is 1:6-10.

[0060] Furthermore, the temperature of the reduction reaction in step (2) is 100-140°C.

[0061] Furthermore, the reduction reaction time in step (2) is 10 to 40 minutes.

[0062] Furthermore, the concentration of gold nanoparticles in the gold nanoparticle solution in step (2) is 2 to 20 nmol / L.

[0063] Furthermore, the particle size of the gold nanoparticles described in step (2) is 10 to 40 nm.

[0064] (3) SH-BTU, SH-PD and MUA were modified onto the gold nanoparticle solution using gold-sulfur bonds. By controlling the ratio of gold nanoparticles to SH-BTU, SH-PD and MUA and the reaction time, a series of Au-MBP NPs with charge reversal properties and copper ion capture capabilities were prepared, namely, engineered gold nanoparticles that can achieve all-round precise photothermal therapy of tumor tissues.

[0065] Furthermore, the concentration of gold nanoparticles in the gold nanoparticle solution in step (3) is 10 to 100 nmol / L.

[0066] Furthermore, the molar ratio of the gold nanoparticles, mercapto-undecanoic acid, thiolated copper ion capture agent, and thiolated charge flip group compound described in step (3) is 1:0.007-0.03:0.005:0.007-0.009, preferably 1:0.007:0.005:0.009; 1:0.01:0.005:0.009; 1:0.02:0.005:0.009; 1:0.03:0.005:0.009 or 1:0.02:0.005:0.007.

[0067] Furthermore, the reaction time in step (3) is 2 to 4 hours, and the reaction is carried out at room temperature.

[0068] Furthermore, the particle size of the engineered gold nanoparticles described in step (3) is 20 to 50 nm.

[0069] The engineered gold nanoparticles prepared by the above method can be used for the preparation of anti-tumor drugs. Specifically, they can be used to fully infiltrate tumor tissues by utilizing charge reversal, and achieve synergistic treatment of selective photothermal therapy, chemodynamic therapy and immunotherapy in primary and metastatic tumors.

[0070] The present invention uses gold nanoparticles as the core, and uses Au-S bonds to modify the surface of the gold nanoparticles with thiolated copper ion capture agents, thiolated piperidine compounds and mercapto-undecanoic acid. By changing the ratio of the thiolated copper ion capture agents, thiolated piperidine compounds and mercapto-undecanoic acid, a series of engineered gold nanoparticles (Au-MBP NPs) are obtained. The increasingly strong acidity of the tumor tissue from the shallow to the deep layer will trigger the protonation process of the tertiary amine groups on the surface of these Au-MBP NPs, realizing step-by-step charge reversal, thereby being retained in tumor tissues at different depths to achieve all-round tumor infiltration. After being internalized, the copper ion capture agents on these Au-MBP NPs will use the thiourea group to chelate excess copper ions, resulting in the aggregation of gold nanoparticles, and exerting the effect of selective photothermal therapy. At the same time, copper ions are reduced to cuprous ions, which react with H2O2 in a Fenton-like reaction to produce ·OH, realizing the chemokinetic therapy process. Furthermore, photothermal therapy and chemodynamic therapy induced tumor apoptosis, which in turn triggered immunogenic death and promoted CD8 + T cells are recruited to obtain anti-tumor immunotherapy. In addition, after copper ions are chelated, the available copper ion content in the tumor is reduced, affecting the subsequent epithelial-mesenchymal transition, thereby inhibiting tumor metastasis.

[0071] The following are specific embodiments.

[0072] Example 1: Preparation of engineered gold nanoparticles Au-MBP NPs

[0073] (1) Preparation of thiolated copper ion capture agent and thiolated piperidine compound:

[0074] Weigh 28.7 mg of EDC (0.15 mmol), 8.6 mg of NHS (0.075 mmol) and 16.4 mg of mercapto undecanoic acid (0.075 mmol) and add them to 3 mL of DMSO, stir at room temperature for 30 min to activate the carboxyl group. Then, add 21.3 mg of BTU (N-aminoethyl-N'-benzoylthiourea, 0.09 mmol), continue stirring for 24 h, and after the reaction is completed, purify the thiolated copper ion capture agent (SH-BTU) by extraction. Add the reaction solution to deionized water, add dichloromethane, mix well to separate the solution, discard the water layer, and add MgSO4 to absorb the remaining moisture. Finally, vacuum dry to obtain the purified thiolated copper ion capture agent (SH-BTU).

[0075] The thiolation process of the piperidine compound is consistent with the above method, and it is only necessary to replace BTU with 11.6 mg of 1-(2-aminoethyl)-piperidine (0.09 mmol). After the reaction is completed, the thiolated piperidine compound (SH-PD) is purified by extraction. The reaction solution is added to deionized water, and then dichloromethane is added, and the solution is fully mixed to separate into layers, the water layer is discarded, and MgSO4 is added to absorb the residual water. Finally, the purified thiolated piperidine compound (SH-PD) is obtained by vacuum drying.

[0076] (2) Preparation of gold nanoparticles:

[0077] All glassware was cleaned with fresh aqua regia and dried before use. First, 10 mL of HAuCl4·3H2O (2.5 mmol / L) was added to a beaker containing 80 mL of ultrapure water, stirred on a magnetic stirrer, and heated to 120-140°C. After the solution boiled, 10 mL of trisodium citrate (0.015 mol / L) solution was added, and continued to heat for 20-30 min. It can be observed that the solution turned into wine red. After the reaction stopped, it was cooled at room temperature to obtain a gold nanoparticle solution, which was stored at 4°C.

[0078] (3) Preparation of Au-MBP NPs:

[0079] The gold nanoparticle solution was centrifuged at 12000rpm for 15min to concentrate the gold nanoparticle solution five times. Subsequently, 2mL of the concentrated gold nanoparticle solution (1.25mmol / L) was taken, and 200μL of MUA (0.25mmol / L), 50μL of SH-BTU (0.25mmol / L) and 70μL of SH-PD (0.25mmol / L) solution were added to make the ratio of the amount of gold nanoparticles: mercapto undecanoic acid: SH-BTU: SH-PD to be 1:0.020:0.005:0.007, and then stirred at room temperature for 4h. After the reaction stopped, the solution was centrifuged at 12000rpm for 10min, the supernatant was removed, and ultrapure water was added to resuspend, and finally the engineered gold nanoparticles for all-round precision photothermal therapy of tumor tissue were obtained.

[0080] In order to distinguish different gold nanoparticles, they are labeled as Au-M x B y P z (x, y and z represent the ratios of MUA, SH-BTU and SH-PD, respectively), so the above gold nanoparticles are Au-M 20 B5P7.

[0081] Example 2

[0082] The same as Example 1, except that the molar ratio of gold nanoparticles: MUA: SH-BTU: SH-PD is 1:0.007:0.005:0.009, recorded as Au-M7B5P9.

[0083] Example 3

[0084] Same as Example 1, except that the ratio of the amount of gold nanoparticles: MUA: SH-BTU: SH-PD is 1: 0.01: 0.005: 0.009, denoted as Au-M 10 B5P9.

[0085] Example 4

[0086] Same as Example 1, except that the ratio of the amount of gold nanoparticles: MUA: SH-BTU: SH-PD is 1:0.02:0.005:0.009, denoted as Au-M 20 B5P9.

[0087] Example 5

[0088] Same as Example 1, except that the ratio of the amount of gold nanoparticles: MUA: SH-BTU: SH-PD is 1: 0.03: 0.005: 0.009, denoted as Au-M 30 B5P9.

[0089] In Examples 1-5, the ratios of gold nanoparticles, MUA, SH-BTU and SH-PD were changed to produce five types of engineered gold nanoparticles.

[0090] The five nanoparticles prepared in Examples 1 to 5 can complete charge reversal at different pH values, thereby infiltrating into tumor tissues at different levels and achieving all-round infiltration of the tumor.

[0091] In order to study the structure of Au-MBP NPs, transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to investigate the structure of Au-MBP NPs. 20 B5P7 was characterized. Figure 1 As shown, Au-M 20 B5P7 has a uniform spherical morphology and is in a monodisperse state with a diameter of about 20 nm. The DLS results also show that ( Figure 2 ), Au-M 20 The hydrated particle size of B5P7 is about 20 nm, which is consistent with the TEM results. 20 The structure of B5P7 was characterized. Figure 3 As shown, from Au-M 20In the infrared spectrum of B5P7, the C=O characteristic absorption from the carboxyl group (MUA), the characteristic absorption from the tertiary amine group (SH-PD), and the CC characteristic absorption from the benzene ring (SH-BTU) were found, which proved that MUA, SH-PD and SH-BTU were successfully modified on the surface of gold nanoparticles, which also meant that Au-M 20 Successful preparation of B5P7.

[0092] Example 6: Charge reversal performance of Au-MBP NPs at different pH

[0093] The five gold nanoparticles prepared in Example 1 to Example 5 were centrifuged at 12000 rpm for 10 min, the supernatant was removed, and then PBS solutions with different pH values ​​(pH 7.4, pH 6.8, pH 6.0, pH 5.5, pH 5.0) were added and resuspended. After incubation for 4 h, DLS was used to detect the surface potential of the five gold nanoparticles at different pH values.

[0094] In order to achieve charge reversal of these five gold nanoparticles at different pH values, we first adjusted the amount of MUA and SH-PD to change the initial potential of Au-MBP NPs at pH 7.4, and then gradually increased the potential through the protonation process of SH-PD at increasingly smaller pH values, ultimately achieving charge reversal from negative to positive at different pH values. Figure 4 As shown in Figure 2, when the proportion of MUA gradually increased (x increased from 7 to 30), the carboxyl groups on the surface of Au-MBP NPs also increased accordingly, resulting in the four nanoparticles (Au-M7B5P9, Au-M 10 B5P9, Au-M 20 B5P9 and Au-M 30 In addition, keeping the amount of MUA (x = 20) unchanged and reducing the amount of SH-PD (z from 9 to 7), the potential of Au-M 20 The potential of B5P7 was further reduced to -27.57mV. Therefore, by adjusting the ratio of x and z, the potential of the five gold nanoparticles at pH7.4 was first changed. Subsequently, the pH value was gradually reduced, and the tertiary amine groups on the surface of the five gold nanoparticles were protonated, resulting in a gradual increase in the potential, and finally the potential changed from negative to positive. Due to the different initial potentials, the five gold nanoparticles achieved a gradual charge reversal at different pH values ​​(Au-M7B5P9: pH6.8; Au-M 10 B5P9: pH 6.5; Au-M 20 B5P9: pH 6.0; Au-M 30 B5P9: pH 5.8; Au-M 20 B5P7: pH 5.5).

[0095] Example 7: Functional verification of Au-MBP NPs in vitro

[0096] First, the ability of these five Au-MBP NPs to reduce copper ions to cuprous ions was verified by adding copper ions to Au-M7B5P9, Au-M 10 B5P9, Au-M 20 B5P9, Au-M 30 B5P9 and Au-M 20 The samples were incubated in B5P7 solution, and then a specific cuprous ion detection reagent, bathocuproin (BCS), was added. The UV-vis spectra before and after the addition of copper ions were detected using a UV spectrophotometer. Next, the ability of Au-MBP NPs to generate ROS was verified. Similarly, copper ions and H2O2 were added to these five Au-MBP NPs solutions, and then TMB reagent was added to detect ROS, and the UV-vis spectra of the solutions were detected. Subsequently, the ability of copper particles to trigger the aggregation of Au-MBP NPs was verified. Representative Au-M 20 B5P7 added copper ions and then used TEM to detect Au-M 20 The morphology of B5P7. Finally, the photothermal conversion ability of Au-MBP NPs was tested. After copper ions were added to the five gold nanoparticle solutions, the solutions were irradiated with 808nm laser, and the temperature changes of the solutions within 10min were recorded with a thermal imager.

[0097] After Au-MBP NPs capture copper ions, the BTU groups on the surface can reduce copper ions to cuprous ions, while BCS can only form complexes with cuprous ions.

[0098] like Figure 5 As shown in Figure 2, when copper ions were added to the Au-MBP NPs solution, the BCS-Cu + The characteristic absorption peak of the complex indicates that Cu + The newly generated Cu + It can react with H2O2 to produce ·OH. TMB can be used to detect the generation of ·OH.

[0099] from Figure 6 It can be seen that the characteristic absorption peaks of oxidized TMB were observed at 370 nm and 655 nm, which proves that the Au-MBP NPs reduced Cu + It can react with H2O2 to produce ·OH in a Fenton-like reaction. In addition, copper ions can also trigger the aggregation of Au-MBPNPs, and TEM is used to evaluate this aggregation state. 20 B5P7+Cu 2+ TEM image (i.e. Figure 7) It can be observed that the aggregated structure of Au-MBP NPs is clearly presented. Under 808nm laser irradiation, the aggregated Au-MBP NPs can show excellent photothermal effect (see Figure 8 ). Compared with the Au-MBP NPs solution without copper ions, copper ions can trigger the temperature of the Au-MBP NPs solution to rise rapidly, and the temperature eventually rises by more than 20 degrees, which means that Au-MBP NPs has a higher photothermal conversion efficiency.

[0100] Example 8: Evaluation of the uptake behavior of Au-MBP NPs by 4T1 cells at different pH

[0101] In order to simplify the verification process, three gold nanoparticles with a wider charge reversal range (Au-M7B5P9, Au-M 20 B5P9 and Au-M 20 B5P7) to verify the uptake behavior of 4T1. To determine the final location of Au-MBP NPs in 4T1 cells, these three gold nanoparticles ( Cy5 Au-M7B5P9, Cy5 Au-M 20 B5P9 and Cy5 Au-M 20 B5P7). Then, Cy5 Au-M7B5P9 was incubated with 4T1 cells at pH 7.4 and pH 6.8. Cy5 Au-M 20 B5P9 was incubated with 4T1 cells at pH 7.4, pH 6.8, and pH 6.0. Cy5 Au-M 20 B5P7 was incubated with 4T1 cells at pH 7.4, pH 6.8, pH 6.0, and pH 5.5, and the fluorescence signal in 4T1 cells was detected by flow cytometry.

[0102] like Fig. 9 As shown in A and B, at pH 7.4, Cy5 The Cy5 fluorescence of Au-M7B5P9 was negligible, indicating that the uptake efficiency of Au-M7B5P9 by tumor cells was low under neutral physiological environment. However, in 4T1 cells at pH 6.8, Cy5 The fluorescence intensity of Au-M7B5P9 increased significantly. This result means that Au-M7B5P9 can be taken up by tumor cells at pH 6.8, which is also related to the charge reversal of Au-M7B5P9 at pH 6.8. Similarly, under the conditions of pH 7.4 and 6.8, the intracellular Cy5 Au-M 20The fluorescence of B5P9 is very weak, proving that it is not taken up by tumor cells ( Fig. 9 C and D). In contrast, when the pH changes to 6.0, Cy5 Au-M 20 The fluorescence of B5P9 was significantly enhanced, which was attributed to the Au-M 20 At pH 6.0, the charge of B5P9 changes from negative to positive, and a large amount of negatively charged tumor cells take up. 20 B5P7, no fluorescence of Cy5 was observed at pH 7.4, pH 6.8, and pH 6.0 ( Fig. 9 E and F), and obvious red fluorescence appeared at pH 5.5, indicating that Au-M 20 The charge reversal of B5P7 at pH 5.5 can significantly improve the uptake efficiency of 4T1 cells. In short, these results prove that the series of Au-MBP NPs prepared by the present invention can respond to different acidic microenvironments, charge reversal at different pH, and be efficiently taken up by tumor cells at different depths. It is also expected to achieve comprehensive infiltration in the entire tumor tissue and realize comprehensive and precise treatment of tumor tissue.

[0103] Example 9: Verification of Au-MBP NPs capturing copper ions in 4T1 cells

[0104] First use Au-M7B5P9, Au-M 20 B5P9 and Au-M 20 B5P7 was incubated with 4T1 cells, and then the solution was irradiated with 808nm laser, and the cell structure was destroyed with cell lysis solution. Subsequently, the lysed cell solution was centrifuged, the supernatant was carefully collected, and the precipitate was discarded. The copper ion concentration in the supernatant was detected with the help of an atomic absorption spectrometer using the flame method.

[0105] Compared with the PBS group, Au-M7B5P9, Au-M 20 B5P9 and Au-M 20 When B5P7 was incubated with 4T1 cells alone, the intracellular copper ion content decreased significantly. This is because these Au-MBP NPs can use the BTU group to complex copper ions and reduce the free copper ions in the cells (see Fig.10 ). In addition, adding these three Au-MBP NPs separately and then irradiating with 808nm laser will increase the ambient temperature, accelerate the complexation reaction between BTU and copper ions, and further reduce the copper ion level in cells. This result also shows that Au-MBP NPs can effectively complex copper ions and reduce the available copper ion content in tumor cells.

[0106] Example 10: Evaluation of the growth activity of Au-MBP NPs on 4T1 cells and MCF-10A cells

[0107] First use Au-M7B5P9, Au-M 20 B5P9 and Au-M 20 B5P7 was incubated with 4T1 cells and MCF-10A cells, followed by 808 nm laser irradiation. Finally, the MTT method was used to detect the effect of Au-MBP NPs on the survival activity of these two cells.

[0108] Compared with the PBS group, Au-M7B5P9, Au-M 20 B5P9 and Au-M 20 B5P7 significantly inhibited the growth of 4T1 cells, which was mainly due to the ability of Au-MBP NPs to generate ROS and inhibit the activity of tumor cells (see Fig.11 Further 808 nm laser irradiation caused Au-MBP NPs to produce a photothermal effect, which together with the generation of ROS reduced the proliferation activity of 4T1 cells. In addition, whether Au-MBP NPs were added alone or further 808 nm laser irradiation was applied, the growth activity of MCF-10A cells was not affected (see Fig.11 These results suggest that Au-MBP NPs treatment will only kill tumor cells without damaging normal cell growth.

[0109] Example 11: Evaluation of the tumor inhibition effect of Au-MBP NPs in vivo

[0110] First, a 4T1 breast cancer tumor model was established. Healthy BALB / c nude mice (female) aged 5 to 7 weeks were selected and fed adaptively in an SPF animal room for one week. Subsequently, 100 μL of 4T1 cell suspension (10 8 One week later, 50 μL of 4T1 cell suspension (10 7 cells / mL), causing tumor metastasis. When the tumor volume reaches 50-100mm 3 The tumor volume of mice was calculated as V = L × W. 2 / 2, L and W are the long side and short side of the tumor, respectively. Subsequently, tumor-bearing mice were injected with PBS, Au-M7B5P9+Au-M 20 B5P9+Au-M 20 B5P7, Au-M7B5P9+808nm laser irradiation, Au-M 20 B5P9+808nm laser irradiation, Au-M 20B5P7+808nm laser irradiation, Au-M7B5P9+Au-M 20 B5P9+Au-M 20 B5P7 + 808nm laser irradiation. 24h after injection, the specific treatment group was irradiated with 808nm laser. The tumor volume of mice was measured every two days for a total of 21 days. At the end of the treatment, representative tumors of each group were selected for photography.

[0111] In the PBS group, the tumor grew rapidly and eventually exceeded 1000mm 3 (See Fig.12 ). However, Au-M7B5P9+Au-M 20 B5P9+Au-M 20 When these three drugs are injected into mice separately and then irradiated with 808nm laser, they can be locally retained and aggregated in the tumor, exerting the synergistic effect of CDT and PTT, further limiting the tumor growth of mice. Obviously, the combined injection of the three preparations and laser irradiation achieved the most significant tumor inhibition effect, thanks to the all-round infiltration of the three nanoparticles throughout the tumor, showing excellent CDT and PTT synergistic therapeutic effects, thereby greatly inhibiting tumor growth. It can also be seen from the representative tumor photos of each group (see Fig.13 ), the mice in the group treated with the three drugs combined with light had the smallest tumors, indicating the most obvious tumor inhibition effect.

[0112] Example 12: Investigation of the immune activation effect of Au-MBP NPs in vivo

[0113] The treatment process of Au-MBP NPs was referred to Example 11. After the treatment, the mice were euthanized, and the tumors of the mice in each group were collected and sliced. Subsequently, CD8 staining was performed on the tumor slices by immunohistochemical staining.

[0114] Compared with the PBS group, Au-M7B5P9+Au-M 20 B5P9+Au-M 20 Co-injection of B5P7 significantly increased CD8 + The infiltration of T cells is mainly due to the fact that the CDT treatment induced by Au-MBP NPs can activate the ICD process and recruit more CD8 + T cells (see Fig.14 ). When the three were injected separately and then irradiated with light, local CDT and PTT further increased CD8 +More importantly, Au-M7B5P9+Au-M 20 B5P9+Au-M 20 The combination of B5P7 and light treatment resulted in a synergistic therapeutic effect of CDT and PTT throughout the tumor, leading to CD8 + The increase in T cells was the greatest. This result indicated that Au-MBP NPs could induce strong anti-tumor immune activation through the synergistic effects of CDT and PTT.

[0115] Example 13: Evaluation of the performance of Au-MBP NPs in inhibiting tumor metastasis in vivo

[0116] The treatment process of Au-MBP NPs was referred to Example 11. At the end of the treatment, the lung tissues of each treatment group were collected and fixed by immersion in Bouin's solution. Then, they were washed with 70% ethanol, and the lung tissues were displayed in bright yellow to highlight the metastatic tumors in the lungs. The lung tissues were photographed. Subsequently, the metastatic nodules on the lung tissues were counted under a microscope.

[0117] like Fig.15 As shown in Figure 2, in the PBS group, the lungs of mice were covered with a large number of tumor nodules, indicating severe lung metastasis. 20 B5P9+Au-M 20 The combined treatment of Au-MBP NPs with B5P7 significantly reduced the formation of metastatic lesions, which was attributed to the fact that these Au-MBP NPs could reduce the level of copper ions, inhibit the EMT pathway, and cooperate with the ICD process triggered by CDT to jointly inhibit tumor metastasis. When the three Au-MBP NPs were injected separately and irradiated with light, these nanoparticles could be locally retained in the tumor, not only capturing copper ions, but also inducing a stronger ICD immune response using CDT and PTT, further enhancing the inhibition of lung metastasis. More importantly, after Au-M7B5P9+Au-M 20 B5P9+Au-M 20 The combined injection of B5P7 and light irradiation resulted in the largest reduction in tumor nodules in the mouse lungs. This was attributed to the fact that these Au-MBP NPs could fully infiltrate the tumor tissue, resulting in a decrease in the copper ion content of the entire tumor. The immune response caused by CDT and PTT was also the strongest, resulting in the strongest anti-metastatic activity. Subsequently, the tumor nodules were counted using a microscope (see Fig.16 ), compared with about 56 tumor nodules in the PBS group, the number of metastatic nodules dropped dramatically to about 16 after combined treatment with three Au-MBP NPs and light irradiation, with a decrease of 71.43%, further demonstrating that Au-MBP NPs have excellent anti-metastatic properties.

[0118] Embodiment 14

[0119] Same as Example 1, except that the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:copper ion capture agent is 1:1:1:1.5.

[0120] Embodiment 15

[0121] Same as Example 1, except that the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:copper ion capture agent is 2:1:1:1.4.

[0122] Example 16

[0123] Same as Example 1, except that the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:copper ion capture agent is 1.5:1:1:1.2.

[0124] Embodiment 17

[0125] Same as Example 1, except that the ratio of the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:piperidine-containing compound is 1.5:1:1:1.2.

[0126] Embodiment 18

[0127] Same as Example 1, except that the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:piperidine compound is 1:1:1:1.5.

[0128] Embodiment 19

[0129] Same as Example 1, except that the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:piperidine compound is 2:1:1:1.3.

[0130] The above description is only for the best embodiment of the present invention, but it should not be understood as limiting the claims. The present invention is not limited to the above embodiments, and its specific structure is allowed to be changed. However, all changes made within the protection scope of the independent claims of the present invention are within the protection scope of the present invention.

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

Claims

1. A method for preparing engineered gold nanoparticles for achieving all-round and precise photothermal therapy of tumor tissue, characterized in that: The following steps are involved: Thiolated copper ion capture agents, thiolated piperidine compounds and mercapto-undecanoic acid were modified onto gold nanoparticles to obtain engineered gold nanoparticles that can achieve all-round precise photothermal therapy of tumor tissues.

2. The method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue according to claim 1, characterized in that: The specific process is: add thiolated copper ion capture agent, thiolated piperidine compound and mercapto-undecanoic acid to the gold nanoparticle solution, mix and react for 2 to 4 hours to obtain engineered gold nanoparticles that can achieve all-round precise photothermal therapy of tumor tissue.

3. The method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue according to claim 1 or 2, characterized in that: The molar ratio of the gold nanoparticles to mercapto-undecanoic acid, mercapto-copper ion capture agent and mercapto-piperidine compound is 1:0.007-0.03:0.005:0.007-0.009; the particle size of the gold nanoparticles is 20-50nm.

4. The method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue according to claim 1 or 2, characterized in that: The thiolated copper ion scavenger is prepared by the following process: Mercapto-undecanoic acid, a copper ion capture agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are mixed and subjected to amide reaction to obtain a thiolated copper ion capture agent.

5. The method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue according to claim 4, characterized in that: The copper ion capture agent is N-aminoethyl-N'-benzoylthiourea; The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:copper ion capture agent is 1 to 2:1:1:1.2 to 1.

5.

6. The method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue according to claim 1 or 2, characterized in that: The thiolated piperidine compounds are prepared by the following process: Mercapto-undecanoic acid, a piperidine compound, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are mixed and subjected to an amide reaction to obtain a mercapto-piperidine compound.

7. The method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue according to claim 6, characterized in that: The piperidine compound is 1-(2-aminoethyl)-piperidine; The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide:mercapto-undecanoic acid:piperidine compound is 1 to 2:1:1:1.2 to 1.

5.

8. The method for preparing engineered gold nanoparticles for achieving all-round precise photothermal therapy of tumor tissue according to claim 1 or 2, characterized in that: The gold nanoparticle solution is prepared by the following process: using sodium citrate reduction method to reduce tetrachloroauric acid into gold to obtain gold nanoparticles.

9. An engineered gold nanoparticle prepared according to the method according to any one of claims 1-8 for achieving all-round precise photothermal therapy of tumor tissue.

10. Use of engineered gold nanoparticles prepared according to the method according to any one of claims 1 to 8 for achieving all-round precise photothermal therapy of tumor tissue in the preparation of anti-tumor drugs.

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