Engineered nanogold for realizing precise photothermal-immune synergistic treatment and immune escape inhibition of tumors as well as preparation method and application of engineered nanogold

Abstract

The invention discloses engineered nanogold for realizing precise photothermal-immune synergistic treatment and immune escape inhibition of tumors as well as a preparation method and application of the engineered nanogold. Nucleic acid modified gold nanoparticles are subjected to cell membrane coating, and cell membrane coated nucleic acid modified gold nanoparticles are obtained; and grafting an immunologic adjuvant on the cell membrane surface of the cell membrane coated with the nucleic acid modified gold nanoparticles. The engineered gold nanoparticles prepared by the invention have tumor cell specific targeting ability, and the engineered gold nanoparticles are guided to gather in tumor tissues and be specifically endocytosed; the nanoparticles can only be in tumor cells, and miRNAs capture mediated photo-thermal immunotherapy can be realized. The engineering gold nanoparticles not only destroy primary tumors, but also activate an immunogenic death (ICD) process by photothermal immunotherapy, and generate tumor-associated antigens locally, so that DCs are mature, T cells are activated, and enhanced immunotherapy is performed under the assistance of a pre-released immunologic adjuvant.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and tumor immunotherapy, and specifically relates to an engineered nanogold for realizing precise photothermal-immunotherapy of tumors and inhibition of immune escape, as well as a preparation method and application thereof. Background Art

[0002] Existing cancer treatments, such as surgery, chemotherapy, and radiotherapy, although effective to a certain extent, often face problems such as high recurrence rates, significant side effects, and uneven efficacy. As an emerging cancer treatment strategy, immunotherapy has shown broad application prospects by activating the host's immune system to identify and eliminate tumor cells. However, due to the strong immune escape ability of tumor cells, existing immunotherapies still have difficulty in achieving lasting and effective anti-tumor effects in many clinical situations.

[0003] In particular, tumor cells inhibit T cell activity through the programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway, further escaping the surveillance of the immune system, which greatly limits the efficacy of immunotherapy. Although immune blockade through anti-PD-1 or anti-PD-L1 antibodies has achieved certain results, the stability and effectiveness of antibodies are difficult to maintain during treatment. Oncogenic nucleic acids (such as miR-21 and miR-130a) in tumor cells play an important role in tumor growth and immune escape, so inhibiting the expression of these miRNAs has become a potential therapeutic strategy.

[0004] In addition, photothermal therapy (PTT) has received widespread attention in recent years as a method of directly killing tumor cells by generating local high temperatures using nanomaterials under near-infrared light irradiation. Studies have shown that PTT can not only kill tumor cells by physical means, but also induce the release of tumor-associated antigens and activate systemic anti-tumor immune responses. However, traditional photothermal preparations cannot achieve tumor-specific photothermal conversion and are prone to damage normal cells and immune cells, thereby affecting the treatment effect and producing side effects. Therefore, the development of a material that can specifically generate photothermal agents in tumor cells to achieve precise photothermal immunotherapy has become a hot topic in current research. Summary of the invention

[0005] The present invention aims to address the limitations of existing immunotherapy and photothermal therapy in tumor treatment, and provides an engineered nanogold, preparation method and application for achieving precise photothermal-immunotherapy and immune escape inhibition for tumors.

[0006] 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 of tumors and immune escape inhibition comprises the following steps: Coating the nucleic acid-modified gold nanoparticles with cell membranes to obtain cell membrane-coated nucleic acid-modified gold nanoparticles; By grafting immune adjuvants onto the cell membrane surface of cell membrane-coated nucleic acid-modified gold nanoparticles, we obtain engineered nanogold that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0007] A further improvement of the present invention is that the nucleic acid-modified gold nanoparticles are cell membrane-coated to obtain cell membrane-coated nucleic acid-modified gold nanoparticles, comprising the following steps: Tumor cell membranes were obtained using hypotonic lysis method; The nucleic acid-modified gold nanoparticles are mixed with tumor cell membranes and then sonicated to obtain cell membrane-coated nucleic acid-modified gold nanoparticles.

[0008] A further improvement of the present invention is that the nucleic acid-modified gold nanoparticles are a mixture of SNA1 and SNA2 in a molar ratio of 1:1; The gold nanoparticle solution, miR-21-DNA1, and miR-130a-DNA1 were mixed and frozen for 2 h and then thawed at room temperature to obtain SNA1; The gold nanoparticle solution, miR-21-DNA2, and miR-130a-DNA2 were mixed and 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-130a-DNA1 is shown in SEQ ID NO.2; The nucleotide sequence of miR-21-DNA2 is shown in SEQ ID NO. 3; The nucleotide sequence of miR-130a-DNA2 is shown in SEQ ID NO.4.

[0009] 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.

[0010] A further improvement of the present invention is that the ultrasonication time is 5 minutes to 30 minutes.

[0011] A further improvement of the present invention is that an immune adjuvant is grafted onto the cell membrane surface of the cell membrane-coated nucleic acid-modified gold nanoparticles to obtain engineered nanogold that realizes precise photothermal-immunotherapy of tumors and immune escape inhibition, comprising the following steps: Phospholipid-polyethylene glycol-R837 was added to the cell membrane-coated nucleic acid-modified gold nanoparticles and incubated at room temperature for 1 to 12 hours to obtain engineered nanogold that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0012] A further improvement of the present invention is that the molecular weight of PEG is 2000-5000.

[0013] A further improvement of the present invention is that the mass ratio of the cell membrane-coated nucleic acid-modified gold nanoparticles to the phospholipid-polyethylene glycol-R837 is 1:1-5:1.

[0014] An engineered gold nanoparticle that achieves precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0015] The application of engineered gold nanoparticles in the preparation of anti-tumor drugs for achieving precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0016] Compared with the prior art, the present invention has the following beneficial effects: First, the present invention grafts immune adjuvants on the cell membrane surface of the cell membrane-coated nucleic acid-modified gold nanoparticles, and the engineered nanogold particles prepared have tumor cell-specific targeting ability, guide the engineered gold nanoparticles to aggregate in 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. Furthermore, the engineered nanogold particles not only destroy the primary tumor, but also activate the immunogenic death (ICD) process by photothermal immunotherapy, produce tumor-associated antigens locally, thereby maturing DCs and activating T cells, and enhancing immunotherapy with the assistance of the pre-released immune adjuvant. At the same time, the inhibition of immune escape also greatly improves the therapeutic effect of tumors, and has excellent targeting. The present invention does not produce organic waste liquid during the preparation process, and is a green and environmentally friendly preparation method. The present invention does not require large, expensive, and high-precision instruments and equipment, and has low production costs.

[0017] 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 nucleic acid-modified 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

[0018] Figure 1 This is the TEM image of SNA1 provided in Example 1.

[0019] Figure 2 This is the TEM image of SNA2 provided in Example 1.

[0020] Figure 3 This is the TEM image of SNAs@CCM provided in Example 2.

[0021] Figure 4 This is the fluorescence spectrum of SNAs@CCM-R837 provided in Example 3.

[0022] Figure 5 This is the gel electrophoresis test result of the SNAs (mixture of SNA1 and SNA2) provided in Example 4 before and after adding miR-130a and miR-21.

[0023] Figure 6 This is the UV–Vis spectra of the SNAs (a mixture of SNA1 and SNA2) provided in Example 4 before and after the addition of miR-130a and miR-21.

[0024] Figure 7 This is the TEM aggregation state of the SNAs (a mixture of SNA1 and SNA2) provided in Example 4 after adding miR-21.

[0025] Figure 8 This is a TEM image of the SNAs (a mixture of SNA1 and SNA2) provided in Example 4 after the addition of miR-130a.

[0026] Fig. 9 The temperature rise curves and photothermal photographs of the SNAs (a mixture of SNA1 and SNA2) provided in Example 4 before and after the addition of miR-130a and miR-21.

[0027] Fig.10 The expression of miR-21 in 4T1 cells after treatment with PBS, SNA1, SNA2, SNAs, SNAs@CCM, and SNAs@CCM-R837 is provided in Example 5.

[0028] Fig.11 The expression of miR-130a in 4T1 cells after treatment with PBS, SNA1, SNA2, SNAs, SNAs@CCM, and SNAs@CCM-R837 as provided in Example 5.

[0029] Fig.12 This is the survival rate of 4T1 cells after treatment with different formulations provided in Example 5.

[0030] Fig.13 These are the photothermal images of tumor-bearing mice with and without Light irradiation after intravenous injection of PBS, AuNPs, SNAs, SNAs@CCM, and SNAs@CCM-R837 provided in Example 6.

[0031] Fig.14 This is the final tumor photo on the 21st day after different treatments provided in Example 7.

[0032] Fig.15 This is a statistical diagram of tumor mass on the 21st day after different treatments provided in Example 7.

[0033] Fig.16 The immunohistochemistry diagram of mice after different treatments provided in Example 7. DETAILED DESCRIPTION

[0034] 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.

[0035] The unit M in the present invention is mol / L, and μM is μmol / L.

[0036] The engineered nanogold in the present invention for realizing precise photothermal-immunotherapy and immune escape inhibition for tumors can realize tumor-specific photothermal therapy (PTT), efficient immune activation and immune escape inhibition, and can be applied to the precise treatment of primary and metastatic tumors.

[0037] The engineered nanogold uses miR-21 and miR-130a, two miRNAs related to tumor migration and PD-1-mediated immune escape, to design two complementary paired nucleic acid-modified gold nanoparticles (SNAs), named SNA1 and SNA2 respectively. These two SNAs can specifically capture miR-21 and miR-130a in tumor cells, inhibit their expression, and thus inhibit tumor migration and immune escape.

[0038] At the same time, SNA1 and SNA2 aggregate under the action of excessive miRNAs in tumor cells, producing a tumor-specific photothermal effect, thereby effectively achieving photothermal therapy. In addition, the outer layer of engineered nanogold encapsulates the homologous cancer cell membrane and combines with the immune adjuvant imiquimod (R837), which can further enhance the activity of immune cells, promote anti-tumor immune response, and avoid immune escape.

[0039] The present invention also provides a method for preparing engineered nanogold, which combines gold nanoparticles with antisense oligonucleotides through simple self-assembly technology and wraps homologous cancer cell membranes to finally form SNAs@CCM-R837 nanoparticles. The nanoparticles can respond to the overexpression of specific miRNAs in the tumor microenvironment, accurately locate and kill tumor cells, and activate the immune system, thereby achieving accurate and efficient treatment of malignant tumors.

[0040] The present invention provides an engineered nano-gold for realizing precise photothermal-immunotherapy and immune escape inhibition for tumors and a preparation method thereof, 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) Gold nanoparticles were functionalized with DNA via gold-thiol bonds, and nucleic acid-modified gold nanoparticles with different DNA modifications were prepared by regulating the concentration of DNA, freezing temperature, freezing time and other conditions; Specifically, the obtained gold nanoparticles were fully mixed with thiolated DNA (100 µM), frozen in a laboratory freezer (-20°C), and then thawed at room temperature to obtain unpurified nucleic acid-modified gold nanoparticles. The thiolated DNA includes miR-21-DNA1 fragment (5'-SH-TTTTTTTTTTTCAACATCAGT-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a nucleotide sequence as shown in SEQ ID NO.1), miR-130a-DNA1 (5'-ACATTGCACTGTTTTTTTTTT-SH-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a nucleotide sequence as shown in SEQ ID NO.2), miR-21-DNA2 fragment (5'-CTGATAAGCTAAAAAAAAAAA-SH-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a nucleotide sequence as shown in SEQ ID NO.3) and miR-130a-DNA2 fragment (5'-SH-TTTTTTTTTTATGCCCTTTTA-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a nucleotide sequence as shown in SEQ ID NO.4). Then, the unmodified DNA was removed by centrifugation and the washed nucleic acid-modified gold nanoparticles SNA1 and SNA2 were dispersed in PBS solution. o CSave.

[0041] 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 after cleaning have a nanometer-level particle size of 11-20nm, and have good dispersion and stability.

[0042] (3) Coating the washed nucleic acid-modified gold nanoparticles with cell membranes, and synthesizing cell membrane-coated nucleic acid-modified gold nanoparticles by adjusting the ratio of cell membranes and nanoparticles and coating technology; Specifically, first, tumor cell membrane (CCM) was obtained by hypotonic lysis method. Then, nucleic acid-modified gold nanoparticles 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 the surface of SNAs with CCM to obtain cell membrane-coated nucleic acid-modified gold nanoparticles SNAs@CCM.

[0043] The mass ratio of tumor cell membrane to nucleic acid-modified gold nanoparticles is 1:1~5:1.

[0044] (4) The immune adjuvant R837 was grafted onto the cell membrane surface of the cell membrane-coated nucleic acid-modified gold nanoparticles to obtain engineered nanogold that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0045] Specifically, phospholipid-polyethylene glycol-R837 (DSPE-PEG-R837) was added to the synthesized SNAs@CCM and incubated at room temperature for 1-12 hours to allow R837 to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation, thereby obtaining engineered nanogold SNA2@CCM-R837 that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0046] Among them, the molecular weight of PEG is 2000~5000; the mass ratio of SNAs@CCM to DSPE-PEG-R837 is 1:1~5:1.

[0047] The present invention uses gold nanoparticles (AuNPs), DNA (miR-21-DNA1, miR-21-DNA2, miR-130a-DNA1, miR-130a-DNA2), cell membrane (CCM) and DSPE-PEG-R837 to prepare engineered gold nanoparticles (SNAs@CCM-R837), which significantly inhibit PD-1 / PDL-1-mediated immune escape by regulating various oncogenic microRNAs on demand, and perform miRNAs-dependent photothermal immunotherapy by in situ generating customized tumor-associated antigens, so as to achieve precise and efficient treatment that meets the individual needs of specific patients.

[0048] During the treatment process, the acidic environment of the tumor tissue cleaves the Schiff base to generate free R837 and SNAs@CCM. SNAs@CCM further enter the tumor cells through CCM-mediated internalization and specifically hybridize with overexpressed miR-130a and miR-21, effectively inhibiting the migration of tumor cells and the expression of PDL-1, avoiding the immune escape of tumor cells. At the same time, the capture of miRNAs also caused a significant aggregation of SNAs, which immediately produced photothermal agents in tumor cells and performed highly selective photothermal therapy under near-infrared irradiation. These chain processes not only destroyed the primary tumor, but also produced a large number of tumor-associated antigens, which matured the surrounding dendritic cells (DCs) and activated anti-tumor T cells along with the released R837, thereby enhancing immunotherapy and inhibiting immune escape.

[0049] 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 citrate-covered AuNPs (13nm). Finally, the obtained AuNPs solution was stored at 4℃ for further experiments.

[0050] The thiolated DNA was incubated in buffer (0.1M ECDP, 1x TE Buffer) for 30 min, which would protect all disulfide bond functions of the thiolated DNA, and the resulting sample could be used directly 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 (100µM, 6µL) and miR-130a-DNA1 (100µM, 2µL) (3:1) 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 (13 nm, 10 nM, 100 µL) were mixed with thio-miR-21-DNA2 (100 µM, 6 µL) and miR-130a-DNA2 (100 µM, 2 µL) (3:1) and placed in a laboratory freezer (set at -20 °C) for 2 h, 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. The lower DNA-AuNPs (SNA1 and SNA2) were washed with 10 × 10−3 The final samples of nucleic acid-modified gold nanoparticles SNA1 and SNA2 were obtained by washing with 4 M phosphate buffer for 3 times. The nucleic acid-modified gold nanoparticles SNA1 and SNA2 were dispersed in PBS solution. 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.

[0051] 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, the cell membrane structure can be clearly seen and SNAs are efficiently encapsulated by CCM.

[0052] Example 3 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 (pH 7.4), 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 SNA2 to obtain SNAs@CCM.

[0053] An equal amount of DSPE-PEG2000-R837 was added to the synthesized SNAs@CCM and incubated for 15 minutes to allow R837 to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation, thereby obtaining engineered nanogold SNAs@CCM-R837 that can achieve precise photothermal-immunotherapy and immune escape inhibition for tumors. The fluorescence spectrum of SNAs@CCM-R837 is shown in Figure 2. Figure 4 As shown, it can be proved that R837 is successfully modified on the cell membrane surface.

[0054] Example 4 Take 20ul of each of the SNA1 and SNA2 aqueous solutions prepared in Example 1 and mix them evenly, add 2μL miR-21 (20uM), and react at room temperature for 12 hours. Aggregation of SNAs was observed, and the color changed from wine red to purple, indicating that miR-21 can induce the aggregation of gold nanoparticles. Similarly, take 20ul of each of the SNA1 and SNA2 aqueous solutions and mix them evenly, add 2μL miR-130a (20uM), and react at room temperature for 12 hours. It was observed that no obvious aggregation of SNAs occurred, indicating that the traction force of miR-130a on SNA1 and SNA2 was weak and did not induce the aggregation of gold nanoparticles. First, the ability of SNAs to capture miR-21 and miR-130a was evaluated by gel electrophoresis. Pure miR-21 / miR-130a, SNA1, SNA2, SNAs and interfering miRNA were used as the control groups. Figure 5As shown, free miR-21 / miR-130a showed a clear emission band, and the pure SNAs group (mixture of SNA1 and SNA2) had no emission band. When miR-21 / miR-130a was mixed with SNA1 or SNA2, the emission band of miR-21 / miR-130a was 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 / miR-130a. When miR-21 / miR-130a was mixed with SNA (SNA1+SNA2), the content of miR-21 / miR-130a was further reduced due to the synergistic capture effect. However, by comparison, it was found that the capture efficiency of SNAs (SNA1+SNA2) for miR-21 was slightly stronger than that of miR-130a. In addition, scrambled miRNA could not pair with the oligonucleotides on SNAs, and the emission band intensity remained unchanged. Next, the UV absorption spectra of the hybridization of miR-21 and miR-130a after being captured by nucleic acid-modified gold nanoparticles are shown in Figure 2. Figure 6 , it can be seen that only after capturing miR-21, the SNAs aggregates showed significantly enhanced absorption in the near-infrared region. The TEM results of nucleic acid-modified gold nanoparticles capturing miR-21 and miR-130a are shown in Figure 2. Figure 7 , Figure 8 It can be clearly seen that after capturing miR-130a, SNAs slightly aggregated, while after capturing miR-21 and hybridizing, SNAs showed obvious aggregation. Fig. 9 It was shown that the SNAs after capturing miR-21 hybridization exhibited excellent photothermal effect under near-infrared light irradiation.

[0055] Example 5 The nucleic acid-modified gold nanoparticles (SNA1 and SNA2) synthesized in Example 1 were co-cultured with 4T1 cells. Fig.10 , Fig.11 As shown in Figure 2, nucleic acid-modified gold nanoparticles SNA1 and SNA2 can effectively reduce the expression levels of miR-21 and miR-130a in tumor cells. Fig.12 As shown, 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.

[0056] Example 6 The SNAs@CCM-R837 in Example 3 was injected into tumor-bearing mice at a concentration of 10 mg / kg. One hour later, the tumor area of ​​the mice was illuminated by NIR (808 nm, 1.25 W cm -2The tumor-bearing mice injected with the same volume of PBS, Au NPs, SNAs and SNAs@CCM were used as the control group, and the photothermal effect was as follows: Fig.13 As shown. It can be proved that nucleic acid-modified gold nanoparticles will aggregate in the tumor site in mice and produce a significant photothermal effect.

[0057] Example 7 Study on the therapeutic effect of engineered nanogold in mice with 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) solution in Example 1, (SNAs@CCM) solution in Example 2, and (SNAs@CCM-R837) solution in Example 3, respectively. In order to quantitatively study the anti-tumor effect of SNAs@CCM-R837 in vivo, the changes in tumor volume of all mice receiving different treatment strategies were monitored, and then all final tumors after treatment were collected and weighed. Fig.14 , Fig.15 It can be seen that SNAs (gene therapy group) have a certain anti-tumor effect, while the membrane-coated SNAs@CCM group has a stronger anti-tumor effect due to its homologous targeting of cancer cell membranes. After near-infrared irradiation, the tumor inhibition effect of the dual-effect group (SNAs@CCM+Light) combining photothermal immunotherapy and gene therapy was further enhanced. The tumor growth in the SNAs@CCM-R837+Light group was completely inhibited, indicating that the additional immune activation of R837 further improved the treatment efficiency and is expected to be used in tumor treatment.

[0058] 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-R837 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-R837 can improve the tumorigenic microenvironment through miRNAs regulation, inhibit immune escape, and exert excellent anti-tumor function.

[0059] Example 8 (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 solution, miR-21-DNA1, and miR-130a-DNA1 were mixed and frozen at -20°C for 2 hours and then thawed at room temperature to obtain SNA1; The gold nanoparticle solution, miR-21-DNA2, and miR-130a-DNA2 were mixed and frozen at −20 °C for 2 h and then thawed at room temperature to obtain SNA2; A mixture of SNA1 and SNA2 with a molar ratio of 1:1 was nucleic acid-modified gold nanoparticles; The mass ratio of the gold nanoparticles to DNA is 1:200; the freezing time is 20 minutes; the nucleic acid-modified gold nanoparticles prepared by the invention after cleaning have a particle size of nanometer level, a particle diameter of 11-20nm, and good dispersion and stability.

[0060] (3) Coating the washed nucleic acid-modified gold nanoparticles with cell membranes, and synthesizing cell membrane-coated nucleic acid-modified gold nanoparticles by adjusting the ratio of cell membranes and nanoparticles and coating technology; Specifically, first, tumor cell membrane (CCM) was obtained by hypotonic lysis method. Then, nucleic acid-modified gold nanoparticles SNAs 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 cell membrane-coated nucleic acid-modified gold nanoparticles SNAs@CCM.

[0061] The mass ratio of tumor cell membrane to nucleic acid-modified gold nanoparticles is 1:1.

[0062] (4) Phospholipid-polyethylene glycol-R837 (DSPE-PEG-R837) was added to SNAs@CCM and incubated at room temperature for 12 h to allow R837 to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation, thereby obtaining engineered nanogold SNA2@CCM-R837 that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0063] Among them, the molecular weight of PEG is 5000; the mass ratio of SNAs@CCM to DSPE-PEG-R837 is 3:1.

[0064] Example 9 (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 solution, miR-21-DNA1, and miR-130a-DNA1 were mixed and frozen at -20°C for 2 hours and then thawed at room temperature to obtain SNA1; The gold nanoparticle solution, miR-21-DNA2, and miR-130a-DNA2 were mixed and frozen at −20 °C for 2 h and then thawed at room temperature to obtain SNA2; A mixture of SNA1 and SNA2 with a molar ratio of 1:1 was nucleic acid-modified gold nanoparticles; 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 after cleaning have a particle size of nanometer level, a particle diameter of 11-20nm, and good dispersion and stability.

[0065] (3) Coating the washed nucleic acid-modified gold nanoparticles with cell membranes, and synthesizing cell membrane-coated nucleic acid-modified gold nanoparticles by adjusting the ratio of cell membranes and nanoparticles and coating technology; Specifically, first, tumor cell membrane (CCM) was obtained by hypotonic lysis method. Then, nucleic acid-modified gold nanoparticles SNAs 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 cell membrane-coated nucleic acid-modified gold nanoparticles SNAs@CCM.

[0066] The mass ratio of tumor cell membrane to nucleic acid-modified gold nanoparticles is 5:1.

[0067] (4) Phospholipid-polyethylene glycol-R837 (DSPE-PEG-R837) was added to SNAs@CCM and incubated at room temperature for 6 hours to allow R837 to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation, thereby obtaining engineered nanogold SNA2@CCM-R837 that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0068] Among them, the molecular weight of PEG is 4000; the mass ratio of SNAs@CCM to DSPE-PEG-R837 is 5:1.

[0069] 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 solution, miR-21-DNA1, and miR-130a-DNA1 were mixed and frozen at -20°C for 2 hours and then thawed at room temperature to obtain SNA1; The gold nanoparticle solution, miR-21-DNA2, and miR-130a-DNA2 were mixed and frozen at −20 °C for 2 h and then thawed at room temperature to obtain SNA2; A mixture of SNA1 and SNA2 with a molar ratio of 1:1 was nucleic acid-modified gold nanoparticles; The mass ratio of the gold nanoparticles to DNA is 1:500; the freezing time is 1 hour; the nucleic acid-modified gold nanoparticles prepared by the invention after cleaning have a particle size of nanometer level, a particle diameter of 11-20nm, and good dispersion and stability.

[0070] (3) Coating the washed nucleic acid-modified gold nanoparticles with cell membranes, and synthesizing cell membrane-coated nucleic acid-modified gold nanoparticles by adjusting the ratio of cell membranes and nanoparticles and coating technology; Specifically, first, tumor cell membrane (CCM) was obtained by hypotonic lysis method. Then, nucleic acid-modified gold nanoparticles SNAs 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 cell membrane-coated nucleic acid-modified gold nanoparticles SNAs@CCM.

[0071] The mass ratio of tumor cell membrane to nucleic acid-modified gold nanoparticles is 3:1.

[0072] (4) Phospholipid-polyethylene glycol-R837 (DSPE-PEG-R837) was added to SNAs@CCM and incubated at room temperature for 1 hour to allow R837 to be modified on the surface of the 4T1 cell membrane through phospholipid intercalation, thereby obtaining engineered nanogold SNA2@CCM-R837 that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

[0073] Among them, the molecular weight of PEG is 2000; the mass ratio of SNAs@CCM to DSPE-PEG-R837 is 1:1.

[0074] 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.

[0075] 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 of tumors and immune escape inhibition, characterized in that: The following steps are involved: Coating the nucleic acid-modified gold nanoparticles with cell membranes to obtain cell membrane-coated nucleic acid-modified gold nanoparticles; By grafting immune adjuvants onto the cell membrane surface of cell membrane-coated nucleic acid-modified gold nanoparticles, we obtain engineered nanogold that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

2. The method for preparing engineered nanogold for realizing precise photothermal-immunotherapy and immune escape inhibition of tumors according to claim 1, characterized in that: The nucleic acid-modified gold nanoparticles are cell membrane-coated to obtain cell membrane-coated nucleic acid-modified gold nanoparticles, comprising the following steps: Tumor cell membranes were obtained using hypotonic lysis method; The gold nanoparticles modified with nucleic acid are mixed with tumor cell membranes and then sonicated to obtain cell membrane-coated nucleic acid-modified gold nanoparticles.

3. The method for preparing engineered nanogold for realizing precise photothermal-immunotherapy and immune escape inhibition of tumors according to claim 2, characterized in that: The nucleic acid-modified gold nanoparticles were a mixture of SNA1 and SNA2 at a molar ratio of 1:1; The gold nanoparticle solution, miR-21-DNA1, and miR-130a-DNA1 were mixed and frozen for 2 h and then thawed at room temperature to obtain SNA1; The gold nanoparticle solution, miR-21-DNA2, and miR-130a-DNA2 were mixed and 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-130a-DNA1 is shown in SEQ ID NO.2; The nucleotide sequence of miR-21-DNA2 is shown in SEQ ID NO. 3; The nucleotide sequence of miR-130a-DNA2 is shown in SEQ ID NO.

4.

4. The method for preparing engineered nanogold for realizing precise photothermal-immunotherapy and immune escape inhibition of tumors according to claim 2, 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 realizing precise photothermal-immunotherapy and immune escape inhibition of tumors according to claim 2, characterized in that: The ultrasound time is 5 minutes to 30 minutes.

6. The method for preparing engineered nanogold for realizing precise photothermal-immunotherapy and immune escape inhibition of tumors according to claim 1, characterized in that: The immune adjuvant is grafted onto the cell membrane surface of the cell membrane-coated nucleic acid-modified gold nanoparticles to obtain engineered nanogold that realizes precise photothermal-immunotherapy and immune escape inhibition for tumors, including the following steps: Phospholipid-polyethylene glycol-R837 was added to the cell membrane-coated nucleic acid-modified gold nanoparticles and incubated at room temperature for 1 to 12 hours to obtain engineered nanogold that can achieve precise photothermal-immunotherapy of tumors and inhibition of immune escape.

7. The method for preparing engineered nanogold for realizing precise photothermal-immunotherapy and immune escape inhibition of tumors according to claim 6, characterized in that: The molecular weight of PEG is 2000~5000.

8. The method for preparing engineered nanogold for realizing precise photothermal-immunotherapy and immune escape inhibition of tumors according to claim 6, characterized in that: The mass ratio of cell membrane-coated nucleic acid-modified gold nanoparticles to phospholipid-polyethylene glycol-R837 is 1:1~5:

1.

9. An engineered nanogold prepared according to the method according to any one of claims 1 to 8 for achieving precise photothermal-immunotherapy of tumors and inhibition of immune escape.

10. Use of engineered nanogold prepared according to the method according to any one of claims 1 to 8 for achieving precise photothermal-immunotherapy of tumors and inhibition of immune escape in the preparation of anti-tumor drugs.

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