Engineered nanogold for realizing precise photo-thermal-immune synergistic treatment and on-demand regulation of tumor-related macrophages as well as preparation method and application of engineered nanogold

Abstract

The invention discloses engineered nanogold for realizing precise photo-thermal-immune synergistic treatment and on-demand regulation of tumor-related macrophages as well as a preparation method and application of the engineered nanogold. Nucleic acid modified gold nanoparticles are subjected to cell membrane coating, and bionic nanoparticles are obtained; mannose is adopted to modify bionic nanoparticles on the outer side of a cell membrane, and the engineered nanogold capable of achieving precise photo-thermal-immune collaborative treatment and on-demand regulation and control of tumor-related macrophages is obtained. The nucleic acid modified gold nanoparticles are subjected to cell membrane coating, the prepared bionic nanoparticles have tumor cell and immune cell dual-targeting groups, and the bionic nanoparticles are guided to gather around tumor tissues and are specifically endocytosed; and secondly, the nanoparticles can only be in tumor cells to realize miRNAs capture-mediated photo-thermal immunotherapy, and have excellent targeting property.

Description

Technical Field

[0001] The present invention relates to the field of engineered nanomaterial technology and biomedical materials, and specifically to an engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages, as well as a preparation method and application thereof. Background Art

[0002] Current cancer treatments, such as surgery, chemotherapy, and radiotherapy, have problems with insufficient efficacy and high recurrence rates. Although immunotherapy has made some progress, the immunosuppressive effect in the tumor microenvironment severely limits its efficacy. In particular, tumor-associated M2 macrophages promote tumor progression by expressing miR-21. Therefore, the development of photothermal immunotherapy that can utilize miR-21 to regulate tumor cells and macrophages has become a hot topic in current research.

[0003] In the tumor-specific microenvironment, excessive oncogenic miRNAs in tumor tissues, such as miR-21, are one of the most commonly upregulated oncogenic miRNAs, responsible for regulating various downstream effectors associated with tumorigenesis, including invasion, proliferation, migration, and invasion. In addition, many studies have reported that miR-21 can also change the expression levels of proteins related to the macrophage polarization pathway and deeply participate in the regulation of macrophage polarization, leading to macrophage polarization toward M2 type. Therefore, the use of gene capture strategy to downregulate the expression of miR-21 in tumor cells and M2 macrophages is an innovative treatment method to precisely damage tumors and reverse the immunosuppressive microenvironment. However, the easy degradation of nucleic acid fragments and the nonspecific absorption of nanocarriers by the kidneys and liver make intracellular specific delivery the biggest challenge for the clinical application of new drugs based on miRNA interference technology.

[0004] To improve the therapeutic effect, strategies have been developed to combine miRNAs regulation with other therapies, such as chemotherapy or photothermal therapy. However, due to the different mechanisms of action of these therapies, there are temporal and spatial differences in the process of action, making it difficult to achieve a synergistic anti-tumor effect. In addition, traditional photothermal agents have an indiscriminate photothermal effect on tumor cells and healthy cells around tumor tissues, which can produce significant side effects during photothermal therapy.

[0005] Therefore, it is necessary to develop a new type of material that can avoid damage to immune cells by selectively producing photothermal agents only in tumor cells, and at the same time regulate the immunosuppressive microenvironment by controlling the phenotype of macrophages, so as to simultaneously achieve effective gene therapy and photothermal-mediated immunotherapy, thereby achieving precise, efficient and synergistic anti-tumor treatment without affecting normal tissues. Summary of the invention

[0006] In view of the technical problem of obvious side effects in photothermal therapy in the prior art, the purpose of the present invention is to provide an engineered nanogold, preparation method and application for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages, comprising the following steps: The nucleic acid-modified gold nanoparticles are coated with cell membranes to obtain biomimetic nanoparticles; By using mannose to modify bionic nanoparticles on the outside of the cell membrane, we obtained engineered gold nanoparticles that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0008] A further improvement of the present invention is that the nucleic acid-modified gold nanoparticles are modified with DNA on the surface of the gold nanoparticles by freezing; The nucleic acid-modified gold nanoparticles are coated with cell membranes to obtain biomimetic nanoparticles, comprising the following steps: Tumor cell membranes were obtained using hypotonic lysis method; Nucleic acid-modified gold nanoparticles were mixed with tumor cell membranes and then sonicated to obtain SNAs@CCM; SNA2 was mixed with tumor cell membranes and then sonicated to obtain SNA2@CCM; an equal amount of DSPE-PEG2000-Man was added to SNA2@CCM and incubated to obtain SNA2@CCM-Man; SNAs@CCM and SNA2@CCM-Man were mixed to obtain engineered gold nanoparticles that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages. The nucleic acid-modified gold nanoparticles are a mixture of SNA1 and SNA2 at a molar ratio of 1:1; The nucleic acid-modified gold nanoparticles are prepared by the following process: gold nanoparticles, miR-21-DNA1 and miR-21-DNA2 are mixed and then frozen, and then thawed at room temperature to obtain nucleic acid-modified gold nanoparticles; SNA2 was prepared by the following process: the gold nanoparticle solution was mixed with miR-21-DNA2 and then frozen for 2 h and then thawed at room temperature to obtain SNA2; The nucleotide sequence of miR-21-DNA1 is shown in SEQ ID NO.1; The nucleotide sequence of miR-21-DNA2 is shown in SEQ ID NO.2.

[0009] A further improvement of the present invention is that the mass ratio of nucleic acid-modified gold nanoparticles to tumor cell membranes is 1:2; The mass ratio of SNAs@CCM and SNA2@CCM-Mane is 1:1.

[0010] A further improvement of the present invention is that the mass ratio of the tumor cell membrane to the gold nanoparticles modified with nucleic acid is 1:1 to 5:1.

[0011] A further improvement of the present invention is that the ultrasonic time is 5 minutes to 30 minutes.

[0012] A further improvement of the present invention is that mannose is used to modify bionic nanoparticles on the outside of the cell membrane to obtain engineered nanogold that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages, comprising the following steps: adding phospholipid-polyethylene glycol-mannose to the bionic nanoparticles, incubating at room temperature for 1 hour to 12 hours, and obtaining engineered nanogold that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0013] A further improvement of the present invention is that the molecular weight of PEG is 2000-5000.

[0014] A further improvement of the present invention is that the mass ratio of the bionic nanoparticles to the phospholipid-polyethylene glycol-mannose is 1:1-5:1.

[0015] An engineered gold nanoparticle that enables precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0016] An application of engineered gold nanoparticles in the preparation of anti-tumor drugs that achieves precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0017] Compared with the prior art, the present invention has the following beneficial effects: First, the present invention utilizes cell membrane coating of nucleic acid-modified gold nanoparticles to prepare biomimetic nanoparticles with dual targeting groups for tumor cells and immune cells, guiding the biomimetic nanoparticles to gather around tumor tissues and be specifically internalized; secondly, the nanoparticles can only achieve miRNAs capture-mediated photothermal immunotherapy in tumor cells, thereby avoiding damage to normal cells and immune cells. This tumor-specific photothermal therapy greatly reduces the toxic side effects on normal cells around tumor cells, and the targeted regulation of immune cells also greatly improves the therapeutic effect of tumors, with excellent targeting. The present invention does not produce organic waste liquid during the experiment, and is a green and environmentally friendly preparation method. The present invention does not require the use of large, expensive, high-precision instruments and equipment, and has low production costs.

[0018] Furthermore, the present invention modifies DNA on the surface of gold nanoparticles by freezing method. This method is simple, efficient and highly repeatable. The obtained smart gold nanoparticles are stable under physiological conditions and storage conditions, and are easy to realize industrial production and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the TEM image of SNA1 provided in Example 1.

[0020] Figure 2 This is the TEM image of SNA2 provided in Example 1.

[0021] Figure 3 This is the TEM image of SNAs@CCM provided in Example 2.

[0022] Figure 4 This is the fluorescence spectrum of SNA2@CCM-Man (FITC-labeled Man) provided in Example 3.

[0023] Figure 5 This is the gel electrophoresis test result of the SNAs (a mixture of SNA1 and SNA2) provided in Example 5 before and after the addition of miR-21.

[0024] Figure 6 This is the UV–Vis spectrum of the SNAs (a mixture of SNA1 and SNA2) provided in Example 5 before and after the addition of miR-21.

[0025] Figure 7 The SNAs (SNA1 and SNA2 / DOX Aggregation status of the mixture before and after adding miR-21.

[0026] Figure 8 The temperature rise curve and photothermal photograph of the SNAs (a mixture of SNA1 and SNA2) provided in Example 5 before and after the addition of miR-21.

[0027] Fig. 9 The particle sizes of the SNAs (a mixture of SNA1 and SNA2) provided in Example 6 before and after incubation in PBS for 48 hours.

[0028] Fig.10 This is the expression of miR-21 in 4T1 cells after SNA1+SNA2 treatment as provided in Example 7.

[0029] Fig.11 This is the expression of miR-21 in M2 macrophages after SNA1+SNA2 treatment as provided in Example 7.

[0030] Fig.12This is the survival rate of 4T1 cells after treatment with different formulations provided in Example 7.

[0031] Fig.13 These are the photothermal images of tumor-bearing mice with and without Light irradiation after intravenous injection of PBS, SNAs@CCM, SNA2@CCM-Man and SNAs@CCM / SNA2@CCM-Man as provided in Example 7.

[0032] Fig.14 This is the final tumor photo on the 21st day after different treatments provided in Example 8.

[0033] Fig.15 It is the survival rate of mice after different treatments provided in Example 8.

[0034] Fig.16 The immunohistochemistry diagram of mice after different treatments provided in Example 8. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood.

[0036] The unit M in the present invention is mol / L, and μM is μmol / L.

[0037] The engineered gold nanoparticles constructed in the present invention can not only perform miRNAs-dependent photothermal immunotherapy in tumor cells, accurately damage primary tumors and activate anti-tumor immune responses against metastatic tumors, but also inhibit the expression of miRNAs in tumor-associated M2-type macrophages and promote their polarization into tumor-suppressive M1-type macrophages, thereby manipulating the immunosuppressive microenvironment on demand and avoiding immune escape.

[0038] The present invention utilizes gold nanoparticles to deliver antisense nucleotides to reduce the concentration of miRNAs in tumor cells and macrophages, responds to and utilizes the tumorigenic microenvironment to achieve precise tumor treatment, and is a simple and easy-to-operate method for preparing engineered nanogold.

[0039] The present invention provides a method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages, comprising the following steps: (1) Using tetrachloroauric acid as the raw material, gold nanoparticles were prepared by sodium citrate reduction method as the core of the nanocarrier; Specifically, the glassware used was cleaned with aqua regia before the experiment. The chloroauric acid aqueous solution (100 mL, 0.01 wt%) was boiled under stirring, and then sodium citrate solution (3.5 mL, 1.0 wt%) was added and continued to boil for 10 minutes, and the color of the solution turned into wine red. The solution was cooled to room temperature to obtain a gold nanoparticle solution (Au NPs, 2 nM); the concentration of gold nanoparticles in the gold nanoparticle solution can be 2 nM-10 nM; (2) The gold nanoparticle core is functionalized with DNA through a gold-thiol bond, and nucleic acid-modified gold nanoparticles with different DNA modifications are prepared by regulating the DNA concentration, freezing temperature, freezing time and other conditions; Specifically, the obtained gold nanoparticles were fully mixed with thiolated DNA (100µM) and placed in a laboratory freezer (-20°C) for freezing, and then thawed at room temperature to obtain unpurified nucleic acid-modified gold nanoparticles. Different DNAs included miR-21-DNA1 (5'-SH-TTTTTTTTTTTCAACATCAGT-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with the nucleotide sequence shown in SEQ ID NO.1), and miR-21-DNA2 fragment (5'-CTGATAAGCTAAAAAAAAAAA-SH-3, synthesized by Sangon Biotech (Shanghai) Co., Ltd., with the nucleotide sequence shown in SEQ ID NO.2). Unmodified DNA was then removed by centrifugation and washing, and the washed nucleic acid-modified gold nanoparticles SNA1 and SNA2 were dispersed in a PBS solution, 4 o CSave.

[0040] SNA2 was prepared by the following process: the gold nanoparticle solution was thoroughly mixed with miR-21-DNA2 and then placed in a laboratory freezer (-20°C) for 2 h, and then thawed at room temperature to obtain SNA2; The mass ratio of the gold nanoparticles to DNA is 1:200-1:800; the freezing time is 20 minutes-2 hours; the nucleic acid modified gold nanoparticles prepared by the invention have a nanometer-level particle size of 11-20nm, and good dispersion and stability.

[0041] (3) Cell membrane coating of nucleic acid-modified gold nanoparticles, and synthesis of biomimetic nanoparticles by regulating the ratio of cell membrane and nanoparticles, coating technology, etc.; Specifically, first, tumor cell membrane (CCM) was obtained by hypotonic lysis method. Then, SNAs (a mixture of SNA1 and SNA2 with a molar ratio of 1:1) were mixed with CCM, and then intermittently and gently sonicated in a bath sonicator for 5 minutes to 30 minutes to successfully coat CCM on the surface of SNAs to obtain SNAs@CCM.

[0042] The same method can be used to obtain CCM-coated SNA2, namely SNA2@CCM.

[0043] Among them, the mass ratio of cell membrane to the gold nanoparticle solution modified with nucleic acid is 1:1~5:1.

[0044] (4) The macrophage-targeting molecule mannose (Man) was modified on the outside of the cell membrane to obtain miRNAs-capturing biomimetic nanoparticles that can target M2 macrophages.

[0045] Specifically, phospholipid-polyethylene glycol-mannose (DSPE-PEG-Man) was added to the synthesized SNA2@CCM and incubated at room temperature for 1 hour to 12 hours, so that mannose (Man) was modified on the surface of the 4T1 cell membrane through phospholipid intercalation to obtain SNA2@CCM-Man.

[0046] Among them, the molecular weight of PEG is 2000~5000; the mass ratio of SNA2@CCM solution to DSPE-PEG-Man is 1:1~5:1.

[0047] SNAs@CCM and SNA2@CCM-Man were mixed in a mass ratio of 1:1 to obtain engineered gold nanoparticles, SNAs@CCM / SNA2@CCM-Man, which can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0048] The present invention uses gold nanoparticles (AuNPs), DNA (DNA1 and DNA2), cell membrane (CCM) and DSPE-PEG-Man to prepare two types of engineered nanogold (SNAs@CCM and SNA2@CCM-Man), which can be used to achieve a responsive chain reaction of the tumor microenvironment. The system can target and regulate oncogenic miRNAs, reduce excessive miRNAs in tumors, and thus achieve manipulation of the genetic environment. At the same time, it relies on oncogenic miRNAs to trigger photothermal effects to significantly increase the temperature of the tumor site, and regulate the immune microenvironment by inducing macrophage polarization, ultimately achieving efficient and accurate tumor clearance.

[0049] Among them, AuNPs, as a controllable photothermal agent, optimizes its photothermal performance, prolongs its residence time in tumor tissue and enhances its effect by transforming from a monodisperse state to an aggregated state. SNA1 and SNA2 achieve the purpose of gene therapy by base-pairing with oncogenic miRNAs to inhibit their gene expression. When SNA1 and SNA2 bind to miR-21, which is highly expressed in tumor cells, AuNPs aggregate in tumor cells. This reaction is only triggered in tumor cells, thus achieving long-term tumor-specific photothermal therapy. In addition, SNA2 used alone only binds to miR-21 overexpressed in M2 macrophages, prompting them to polarize toward M1 macrophages, while not triggering the aggregation of AuNPs in macrophages, thereby avoiding damage to immune cells by near-infrared light irradiation.

[0050] The following are specific embodiments.

[0051] Example 1 The glassware used was cleaned with freshly prepared aqua regia and dried. First, the HAuCl4·3H2O aqueous solution (100mL, 0.01wt%) was boiled at 100℃ under stirring, and then sodium citrate solution (3.5mL, 1.0wt%) was added and boiled for 10 minutes. The color of the solution gradually changed from yellow to purple within 5 minutes, and finally to wine red. The solution was cooled to room temperature to obtain a citrate-covered AuNPs solution (13nm). Finally, the obtained AuNPs solution was stored at 4℃ for further experiments.

[0052] The thiolated DNA was incubated in a buffer (0.1M ECDP, 1x TE Buffer) for 30 minutes, which will protect all disulfide bond functions of the thiolated DNA, and the obtained sample can be directly used in subsequent experiments without further purification. DNA-modified gold nanoparticles were prepared by freezing by adding DNA to the AuNPs solution. Specifically, AuNPs (13nm, 10nM, 100µL) were thoroughly mixed with thiolated miR-21-DNA1 (100µM, 8µL) and placed in a laboratory freezer (set at -20℃) for 2 hours, and then thawed at room temperature to obtain unpurified SNA1. In the same way, AuNPs (13nm, 10nM, 100µL) were mixed with thiolated miR-21-DNA2 (100µM, 6µL) and placed in a laboratory freezer (set at -20℃) for 2 hours, and then thawed at room temperature to obtain unpurified SNA2. Finally, the mixture was centrifuged at 10,000 rpm for 20 min, and the supernatant with unbound DNA was removed. −3The obtained SNA1 and SNA2 were dispersed in PBS solution and washed 3 times with 4 M phosphate buffer to obtain the final samples SNA1 and SNA2. o C. Transmission electron microscopy of SNA1 and SNA2 is shown in Figure 1 and Figure 2 As shown, the particle size is about 16 nm and the dispersibility is good.

[0053] Example 2 The homologous 4T1 tumor cell membrane (CCM) was obtained by hypotonic lysis. Specifically, the tumor cells were placed in a hypotonic lysis buffer consisting of 10mM tris (pH7.4), 10mM MgCl2 and 1mM phenylmethylsulfonyl fluoride, resuspended at 4°C for 1 hour, and then subjected to gentle sonication for 5s (VCX130PB, Sonics, USA). Then, after centrifugation at 100g for 10 minutes to remove debris, the CCM was concentrated by centrifugation at 10000g for 10 minutes, and then washed in water for more than three times until the intracellular components were removed. Finally, the obtained CCM was dispersed in water and stored at 4°C for subsequent experiments. The 1mg SNA1 and SNA2 (molar ratio 1:1) mixture (SNAs) prepared in Example 1 was mixed with 2mg CCM, and then intermittently and gently sonicated in a bath sonicator (SY25-12, Shengyuan Supersonic, China) for 5 minutes to successfully coat the CCM on the surface of the SNAs to obtain SNAs@CCM. The morphology of SNAs@CCM was determined by transmission electron microscopy. Figure 3 As shown, it can be seen that the cell membrane structure is clear and SNAs are efficiently encapsulated by CCM.

[0054] Example 3 1 mg of SNA2 prepared in Example 1 was mixed with 2 mg of CCM, and then intermittently and gently sonicated in a bath sonicator (SY25-12, Shengyuan Supersonic, China) for 5 minutes to successfully coat the surface of SNA2 with CCM to obtain SNA2@CCM.

[0055] An equal amount of DSPE-PEG2000-Man was added to the synthesized SNA2@CCM and incubated for 15 minutes to allow mannose to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation to obtain SNA2@CCM-Man. DSPE-PEG-Man was labeled with FITC, and the fluorescence spectrum of SNA2@CCM-Man (FITC) is shown in Figure 4 As shown, it can be proved that Man is successfully modified on the cell membrane surface.

[0056] Example 4 The SNAs@CCM prepared in Example 2 and the SNA2@CCM-Man prepared in Example 3 were mixed in a mass ratio of 1:1 to obtain engineered nanogold, SNAs@CCM / SNA2@CCM-Man, which can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0057] Example 5 Take 20ul of each of the SNA1 and SNA2 aqueous solutions of Example 1 and mix them evenly. Add 2μL of miR-21 (20uM). After reacting at room temperature for 12 hours, SNAs were observed to aggregate and the color changed from wine red to purple, indicating that miR-21 can induce the aggregation of gold nanoparticles. First, the ability of SNAs to capture miR-21 was evaluated by gel electrophoresis. Pure miR-21, SNA1, SNA2, SNAs and interfering miRNA were used as the control group. Figure 5 As shown, free miR-21 exhibits a clear emission band, and the pure SNAs group (a mixture of SNA1 and SNA2) has no emission band. When miR-21 is mixed with SNA1 or SNA2, the emission band of miR-21 is significantly weakened due to the specific base pairing between the oligonucleotides on the surface of SNA1 or SNA2 and the first and second halves of free miR-21. When miR-21 is mixed with SNA (SNA1+SNA2), the content of miR-21 is further reduced due to the synergistic capture effect. In addition, scrambled miRNA cannot pair with the oligonucleotides on SNAs, and the emission band intensity remains unchanged. Next, the UV absorption spectra and TEM results of the smart gold nanoparticles before and after hybridization are shown respectively. Figure 6 and Figure 7 It can be clearly seen that after hybridization, the gold nanoparticles showed obvious aggregation and had significantly enhanced absorption in the near-infrared region. Figure 8 It was shown that the hybridized gold nanoparticles exhibited excellent photothermal effect under near-infrared light irradiation.

[0058] Example 6 The glassware used was cleaned with freshly prepared aqua regia and dried. First, the HAuCl4·3H2O aqueous solution (100mL, 0.01wt%) was boiled at 100℃ under stirring, and then sodium citrate solution (3.5mL, 1.0wt%) was added and boiled for 10 minutes. The color of the solution gradually changed from yellow to purple within 5 minutes and finally to wine red. The solution was cooled to room temperature to obtain citrate-covered AuNPs (13nm). Finally, the obtained AuNPs solution was stored at 4℃ for further experiments.

[0059] The thiolated DNA was incubated in a buffer (0.1M ECDP, 1x TE Buffer) for 30 minutes, which will protect all disulfide bond functions of the thiolated DNA, and the resulting sample can be directly used in subsequent experiments without further purification. The synthesis of antisense oligonucleotide-modified gold nanoparticles was prepared by adding antisense oligonucleotides to the AuNPs solution by freezing. Specifically, AuNPs (13nm, 10nM, 100µL) were thoroughly mixed with thiolated miR-21-DNA1 or miR-21-DNA2 (100µM, 8µL) and placed in a laboratory freezer (set at -20℃) for 2 hours, and then thawed at room temperature to obtain unpurified SNA1 or SNA2. Finally, the unpurified SNA1 or SNA2 was centrifuged at 10000rpm for 20 minutes, and the supernatant without DNA was removed. The lower DNA-AuNPs (SNA1 and SNA2) were washed with 10×10 −3 The obtained SNA1 or SNA2 was dispersed in PBS solution for 4 o C Save. Fig. 9 As shown, after incubating SNAs (a mixed solution of SNA1 and SNA2 with a molar ratio of 1:1, i.e., smart gold nanoparticles) in PBS for 48 h, the particle size did not change significantly, indicating that the prepared smart gold nanoparticles have good stability and are suitable for in vivo treatment.

[0060] Example 7 The smart gold nanoparticles in Example 2 and Example 3 were co-cultured with 4T1 cells and M2 macrophages. Fig.10 , Fig.11 As shown, the smart gold nanoparticles SNA1 and SNA2 can effectively reduce the expression level of miR-21 in tumor cells and macrophages. The smart gold nanoparticles SNAs@CCM / SNA2@CCM-Man in Example 4 were co-cultured with 4T1 cells. Fig.12 As shown in the figure, gene therapy can effectively kill tumor cells, but under the synergistic effect of photothermal therapy and cell membrane homologous targeted enhanced delivery, the cell survival rate is the lowest, that is, the killing effect on cancer cells is the best. Next, the smart gold nanoparticles SNAs@CCM / SNA2@CCM-Man in Example 4 were injected into tumor-bearing mice at a concentration of 10 mg / kg. One hour later, the tumor area of ​​the mouse was illuminated by NIR (808 nm, 1.25 W cm -2 , 5 minutes) to observe the therapeutic effect of photothermal therapy in vivo. Tumor-bearing mice injected with the same volume of PBS, SNAs@CCM and SNA2@CCM-Man were used as the control group, and the photothermal effect was as follows Fig.13As shown. It can be proved that smart gold nanoparticles will aggregate at the tumor site in mice and produce a significant photothermal effect.

[0061] Example 8 Study on the therapeutic effect of engineered nanogold in mice with cell membrane or mannose-modified cell membrane as targeting group, antisense oligonucleotide as miRNAs capture agent and gold nanoparticles as core: Balb / C mice were inoculated subcutaneously with 4T1 cells. When the tumor reached approximately 100 mm 3 At the same time, nude mice were randomly divided into 5 groups (n=5) and received tail vein injection of 200 μL of PBS buffer solution, (SNAs@CCM) solution in Example 2, (SNA2@CCM-Man) solution in Example 3, and SNAs@CCM / SNA2@CCM-Man in Example 4 (i.e., engineered nanogold that achieves precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages). In order to quantitatively study the in vivo anti-tumor efficacy of the SNAs@CCM / SNA2@CCM-Man system, the changes in tumor volume of all mice receiving different treatment strategies were monitored, and then the final tumors after these treatments were collected. The results are shown in Fig.14 As shown in the tumor tissue photos, SNAs@CCM (gene therapy group) showed a certain anti-tumor efficiency, while the dual-effect group (SNAs@CCM and near-infrared irradiation) combining photothermal immunotherapy and gene therapy showed a stronger tumor inhibition effect. SNA2@CCM-Man nanoparticles mainly promote macrophage polarization to activate immunity, so the multi-site manipulation system SNAs@CCM / SNA2@CCM-Man can achieve simultaneous immune activation through photothermal ablation of primary tumors and reverse the immunosuppressive microenvironment by regulating macrophage phenotype, thereby completely inhibiting tumor growth. The survival rate of mice is shown in Figure 2. Fig.15 As shown, it is proved that the engineered nanogold can effectively kill tumors and improve the survival rate of mice.

[0062] On the last day, mice in the four groups were killed, and tumor tissues were collected and immunohistochemically stained with hematoxylin-eosin (H&E), a cell activity marker, and Ki67, a cell marker in the proliferation cycle, to detect the cell activity of tumor tissues. Fig.16 As shown in the figure, the SNAs@CCM / SNA2@CCM-Man group had the largest tumor disintegration area (HE staining) and the lowest expression of Ki67 (proliferation marker) under near-infrared irradiation, indicating that it has the best therapeutic effect on tumors. These results show that SNAs@CCM / SNA2@CCM-Man can improve the tumorigenic microenvironment through miRNAs regulation and exert excellent anti-tumor function.

[0063] Example 9 (1) Clean all glassware with aqua regia. Boil the chloroauric acid aqueous solution (100 mL, 0.01 wt%) under stirring, then add sodium citrate solution (3.5 mL, 1.0 wt%) and continue boiling for 10 minutes until the solution turns wine red. Cool the solution to room temperature to obtain a gold nanoparticle solution (Au NPs, 2 nM); (2) After thoroughly mixing the gold nanoparticles and thiolated DNA (100 µM), place them in a laboratory freezer (-20°C) for freezing, and then thaw at room temperature to obtain unpurified nucleic acid-modified gold nanoparticles. The thiolated DNA contains miR-21-DNA1 (5'-SH-TTTTTTTTTTTCAACATCAGT-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd.) and miR-21-DNA2 fragments (5'-CTGATAAGCTAAAAAAAAAAA-SH-3, synthesized by Sangon Biotech (Shanghai) Co., Ltd.). Unmodified DNA is then removed by centrifugation and the washed nucleic acid-modified gold nanoparticles SNA1 and SNA2 are dispersed in a PBS solution for 4 hr. o CSave.

[0064] The gold nanoparticle solution was fully mixed with miR-21-DNA2 and placed in a laboratory freezer (-20°C) for 2 h, and then thawed at room temperature to obtain SNA2; The mass ratio of the gold nanoparticles to DNA is 1:200; the freezing time is 1 hour; the nucleic acid-modified gold nanoparticles prepared by the invention have a nanometer-level particle size of 11-20nm, and good dispersion and stability.

[0065] (3) Tumor cell membrane (CCM) was obtained by hypotonic lysis method. Then, SNAs (a mixture of SNA1 and SNA2 with a molar ratio of 1:1) were mixed with CCM and then intermittently and gently sonicated in a bath sonicator for 5 min to successfully coat CCM on the surface of SNAs to obtain SNAs@CCM.

[0066] The same method can be used to obtain CCM-coated SNA2, namely SNA2@CCM.

[0067] Among them, the mass ratio of cell membrane to the gold nanoparticle solution modified with nucleic acid is 1:1.

[0068] (4) Phospholipid-polyethylene glycol-mannose (DSPE-PEG-Man) was added to SNA2@CCM and incubated at room temperature for 12 h to allow mannose (Man) to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation to obtain SNA2@CCM-Man.

[0069] Among them, the molecular weight of PEG is 3000; the mass ratio of SNA2@CCM solution to DSPE-PEG-Man is 1:3.

[0070] SNAs@CCM and SNA2@CCM-Man were mixed in a mass ratio of 1:1 to obtain engineered gold nanoparticles, SNAs@CCM / SNA2@CCM-Man, which can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0071] Example 10 (1) Using tetrachloroauric acid as the raw material, gold nanoparticles were prepared by sodium citrate reduction method as the core of the nanocarrier; Specifically, all glassware used was cleaned with aqua regia before the experiment. A chloroauric acid aqueous solution (100 mL, 0.01 wt%) was boiled under stirring, and then a sodium citrate solution (3.5 mL, 1.0 wt%) was added and continued to boil for 10 minutes until the color of the solution turned into wine red. The solution was cooled to room temperature to obtain a gold nanoparticle solution (Au NPs, 2 nM); (2) The gold nanoparticle core is functionalized with DNA through a gold-thiol bond, and nucleic acid-modified gold nanoparticles with different DNA modifications are prepared by regulating the DNA concentration, freezing temperature, freezing time and other conditions; Specifically, the obtained gold nanoparticles were fully mixed with thiolated DNA (100 µM) and placed in a laboratory freezer (-20°C) for freezing, and then thawed at room temperature to obtain unpurified nucleic acid-modified gold nanoparticles. The thiolated DNA contained miR-21-DNA1 (5'-SH-TTTTTTTTTTTCAACATCAGT-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd.) and miR-21-DNA2 fragments (5'-CTGATAAGCTAAAAAAAAAAA-SH-3, synthesized by Sangon Biotech (Shanghai) Co., Ltd.). Unmodified DNA was then removed by centrifugation and washing, and the washed nucleic acid-modified gold nanoparticles SNA1 and SNA2 were dispersed in a PBS solution for 4 hr. o CSave.

[0072] SNA2 was prepared by the following process: the gold nanoparticle solution was thoroughly mixed with miR-21-DNA2 and then placed in a laboratory freezer (-20°C) for 2 h, and then thawed at room temperature to obtain SNA2; The mass ratio of the gold nanoparticles to DNA is 1:800; the freezing time is 2 hours; the nucleic acid-modified gold nanoparticles prepared by the invention have a nanometer-level particle size of 11-20nm, and have good dispersion and stability.

[0073] (3) Cell membrane coating of nucleic acid-modified gold nanoparticles, and synthesis of biomimetic nanoparticles by regulating the ratio of cell membrane and nanoparticles, coating technology, etc.; Specifically, first, tumor cell membranes (CCM) were obtained by hypotonic lysis. Then, SNAs (a mixture of SNA1 and SNA2 with a molar ratio of 1:1) were mixed with CCM, and then intermittently and gently sonicated in a bath sonicator for 30 min to successfully coat CCM on the surface of SNAs to obtain SNAs@CCM.

[0074] The same method can be used to obtain CCM-coated SNA2, namely SNA2@CCM.

[0075] Among them, the mass ratio of cell membrane to the gold nanoparticle solution modified with nucleic acid is 5:1.

[0076] (4) Phospholipid-polyethylene glycol-mannose (DSPE-PEG-Man) was added to SNA2@CCM and incubated at room temperature for 7 hours to allow mannose (Man) to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation to obtain SNA2@CCM-Man.

[0077] Among them, the molecular weight of PEG is 5000; the mass ratio of SNA2@CCM solution to DSPE-PEG-Man is 5:1.

[0078] SNAs@CCM and SNA2@CCM-Man were mixed in a mass ratio of 1:1 to obtain engineered gold nanoparticles, SNAs@CCM / SNA2@CCM-Man, which can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0079] Embodiment 11 (1) Using tetrachloroauric acid as the raw material, gold nanoparticles were prepared by sodium citrate reduction method as the core of the nanocarrier; Specifically, all glassware used was cleaned with aqua regia before the experiment. A chloroauric acid aqueous solution (100 mL, 0.01 wt%) was boiled under stirring, and then a sodium citrate solution (3.5 mL, 1.0 wt%) was added and continued to boil for 10 minutes until the color of the solution turned into wine red. The solution was cooled to room temperature to obtain a gold nanoparticle solution (Au NPs, 2 nM); (2) The gold nanoparticle core is functionalized with DNA through a gold-thiol bond, and nucleic acid-modified gold nanoparticles with different DNA modifications are prepared by regulating the DNA concentration, freezing temperature, freezing time and other conditions; Specifically, the obtained gold nanoparticles were fully mixed with thiolated DNA (100 µM) and placed in a laboratory freezer (-20°C) for freezing, and then thawed at room temperature to obtain unpurified nucleic acid-modified gold nanoparticles. The thiolated DNA contained miR-21-DNA1 (5'-SH-TTTTTTTTTTTCAACATCAGT-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd.) and miR-21-DNA2 fragments (5'-CTGATAAGCTAAAAAAAAAAA-SH-3, synthesized by Sangon Biotech (Shanghai) Co., Ltd.). Unmodified DNA was then removed by centrifugation and washing, and the washed nucleic acid-modified gold nanoparticles SNA1 and SNA2 were dispersed in a PBS solution for 4 hr. o CSave.

[0080] SNA2 was prepared by the following process: the gold nanoparticle solution was thoroughly mixed with miR-21-DNA2 and then placed in a laboratory freezer (-20°C) for 2 h, and then thawed at room temperature to obtain SNA2; The mass ratio of the gold nanoparticles to DNA is 1:500; the freezing time is 20 minutes; the nucleic acid-modified gold nanoparticles prepared by the invention have a nanometer-level particle size of 11-20nm, and have good dispersion and stability.

[0081] (3) Cell membrane coating of nucleic acid-modified gold nanoparticles, and synthesis of biomimetic nanoparticles by regulating the ratio of cell membrane and nanoparticles, coating technology, etc.; Specifically, first, tumor cell membranes (CCM) were obtained by hypotonic lysis. Then, SNAs (a mixture of SNA1 and SNA2 with a molar ratio of 1:1) were mixed with CCM, and then intermittently and gently sonicated in a bath sonicator for 15 min to successfully coat CCM on the surface of SNAs to obtain SNAs@CCM.

[0082] The same method can be used to obtain CCM-coated SNA2, namely SNA2@CCM.

[0083] Among them, the mass ratio of cell membrane to the gold nanoparticle solution modified with nucleic acid is 1:3.

[0084] (4) Phospholipid-polyethylene glycol-mannose (DSPE-PEG-Man) was added to SNA2@CCM and incubated at room temperature for 1 hour to allow mannose (Man) to be modified on the surface of 4T1 cell membrane through phospholipid intercalation to obtain SNA2@CCM-Man.

[0085] Among them, the molecular weight of PEG is 2000; the mass ratio of SNA2@CCM solution to DSPE-PEG-Man is 1:1.

[0086] SNAs@CCM and SNA2@CCM-Man were mixed in a mass ratio of 1:1 to obtain engineered gold nanoparticles, SNAs@CCM / SNA2@CCM-Man, which can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

[0087] 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.

[0088] 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 precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages, characterized in that: The following steps are involved: The nucleic acid-modified gold nanoparticles are coated with cell membranes to obtain biomimetic nanoparticles; By using mannose to modify bionic nanoparticles on the outside of the cell membrane, we obtained engineered gold nanoparticles that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

2. The method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages according to claim 1, characterized in that: Nucleic acid-modified gold nanoparticles use a freezing method to modify DNA on the surface of gold nanoparticles; The nucleic acid-modified gold nanoparticles are coated with cell membranes to obtain biomimetic nanoparticles, comprising the following steps: Tumor cell membranes were obtained using hypotonic lysis method; Nucleic acid-modified gold nanoparticles were mixed with tumor cell membranes and then sonicated to obtain SNAs@CCM; SNA2 was mixed with tumor cell membranes and then sonicated to obtain SNA2@CCM; an equal amount of DSPE-PEG2000-Man was added to SNA2@CCM and incubated to obtain SNA2@CCM-Man; SNAs@CCM and SNA2@CCM-Man were mixed to obtain engineered gold nanoparticles that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages. The nucleic acid-modified gold nanoparticles are a mixture of SNA1 and SNA2 at a molar ratio of 1:1; The nucleic acid-modified gold nanoparticles are prepared by the following process: gold nanoparticles, miR-21-DNA1 and miR-21-DNA2 are mixed and then frozen, and then thawed at room temperature to obtain nucleic acid-modified gold nanoparticles; SNA2 was prepared by the following process: the gold nanoparticle solution was mixed with miR-21-DNA2 and then frozen for 2 h and then thawed at room temperature to obtain SNA2; The nucleotide sequence of miR-21-DNA1 is shown in SEQ ID NO.1; The nucleotide sequence of miR-21-DNA2 is shown in SEQ ID NO.

2.

3. The method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages according to claim 2, characterized in that: The mass ratio of nucleic acid-modified gold nanoparticles to tumor cell membranes was 1:2; The mass ratio of SNAs@CCM and SNA2@CCM-Man is 1:

1.

4. The method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages according to claim 1, characterized in that: The mass ratio of tumor cell membrane to nucleic acid-modified gold nanoparticles is 1:1~5:

1.

5. The method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages according to claim 1, characterized in that: The ultrasound time is 5 minutes to 30 minutes.

6. The method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages according to claim 1, characterized in that: Mannose is used to modify bionic nanoparticles on the outside of the cell membrane to obtain engineered nanogold that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages, comprising the following steps: adding phospholipid-polyethylene glycol-mannose to the bionic nanoparticles, incubating at room temperature for 1 hour to 12 hours, to obtain engineered nanogold that can achieve precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

7. The method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages according to claim 1, characterized in that: The molecular weight of PEG is 2000~5000.

8. The method for preparing engineered nanogold for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages according to claim 1, characterized in that: The mass ratio of bionic nanoparticles to phospholipid-polyethylene glycol-mannose is 1:1~5:

1.

9. An engineered gold nanoparticle prepared according to the method of any one of claims 1 to 8 for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages.

10. Use of engineered nanogold prepared according to the method according to any one of claims 1 to 8 for achieving precise photothermal-immunotherapy and on-demand regulation of tumor-associated macrophages in the preparation of anti-tumor drugs.

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