Engineered nanogold hydrogel for realizing precise photo-thermal-immune synergistic treatment and on-demand regulation and control of immune microenvironment as well as preparation method and application of engineered nanogold hydrogel

Abstract

The invention discloses an engineered nanogold hydrogel capable of realizing precise photo-thermal-immune synergistic treatment and on-demand regulation and control of an immune microenvironment, and a preparation method and application thereof, and the preparation method comprises the following steps: carrying out cell membrane coating on nucleic acid modified gold nanoparticles to obtain cell membrane coated nucleic acid modified gold nanoparticles; the preparation method comprises the following steps: dispersing cell membrane coated nucleic acid modified gold nanoparticles in an oxidized sodium alginate aqueous solution, stirring to obtain an SNAs (at) M-OSA hydrogel stock solution, and then adding a CaCl2 solution for calcium crosslinking to obtain the engineered nanogold hydrogel capable of realizing precise photothermal-immune synergistic treatment and on-demand regulation and control of an immune microenvironment. According to the invention, the oxidized sodium alginate is used as a gel for Careaction in a tumor microenvironment, and nucleic acid modified gold nanoparticles packaged by cell membranes are embedded into a gel framework, so that long-acting miRNA capture and accurate tumor targeted therapy are realized. Due to the efficient long-acting release and targeting specific design, the nano-material becomes an excellent candidate scheme for tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanobiomaterials and tumor immunotherapy, and specifically relates to an engineered nanogold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment, as well as a preparation method and application thereof. Background Art

[0002] Cancer immunotherapy shows good prospects by activating the host immune system and selectively attacking tumor cells. However, existing immunotherapies face problems such as the imbalance between effector T cells and regulatory T cells (Tregs) in the tumor immune microenvironment and the uncertainty of efficacy caused by individual differences, which seriously affect the effect of immunotherapy. Especially in tumor tissues, Tregs cells inhibit the recruitment and function of effector T cells through direct or indirect mechanisms, thereby weakening the anti-tumor effect of immunotherapy. In addition, the activation of the PD-1 / PD-L1 pathway also allows tumor cells to escape the attack of the immune system, further limiting the effectiveness of immunotherapy.

[0003] In recent years, photothermal therapy (PTT) has been widely studied for tumor treatment. It uses near-infrared irradiation to locally heat up nanomaterials, directly kill tumor cells, and induce immunogenic cell death (ICD), release tumor-associated antigens, and activate anti-tumor immune responses. However, the nonspecific thermal effect of traditional photothermal agents at the tumor site can easily damage surrounding healthy tissues and immune cells, affecting the treatment effect. In addition, a single PTT is difficult to overcome the problem of immunosuppression in the tumor immune microenvironment. Summary of the invention

[0004] In order to overcome the problem of immunosuppression in the tumor immune microenvironment that is difficult to overcome in the prior art, the purpose of the present invention is to provide an engineered nanogold hydrogel, a preparation method and an application that can achieve precise photothermal-immunotherapy synergistic therapy and on-demand regulation of the immune microenvironment. The engineered nanogold hydrogel has high selectivity for photothermal therapy, inhibits PD-1-mediated immune escape and restores the balance between T cells and Tregs, thereby improving the therapeutic effect of cancer photothermal immunotherapy.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing an engineered nanogold hydrogel for achieving precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment comprises the following steps: Coating the nucleic acid-modified gold nanoparticles with cell membranes to obtain cell membrane-coated nucleic acid-modified gold nanoparticles; The cell membrane-coated nucleic acid-modified gold nanoparticles were dispersed in an oxidized sodium alginate aqueous solution and stirred to obtain a SNAs@M-OSA hydrogel stock solution, and then CaCl was added. 2The solution is calcium cross-linked to obtain an engineered nanogold hydrogel that can achieve precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment.

[0006] 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.

[0007] 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-214-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-214-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-214-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-214-DNA2 is shown in SEQ ID NO. 3; The nucleotide sequence of miR-130a-DNA2 is shown in SEQ ID NO.4.

[0008] A further improvement of the present invention is that the mass ratio of the tumor cell membrane to the nucleic acid-modified gold nanoparticles is 1:1 to 5:1.

[0009] A further improvement of the present invention is that the ultrasonic time is 5 minutes to 30 minutes.

[0010] A further improvement of the present invention is that the oxidized sodium alginate aqueous solution is prepared by the following process: sodium alginate is oxidized at room temperature for 4 hours to 12 hours using sodium periodate to obtain the oxidized sodium alginate aqueous solution, wherein the mass ratio of sodium alginate to sodium periodate is 5:1 to 20:1.

[0011] A further improvement of the present invention is that the mass ratio of the cell membrane-coated nucleic acid-modified gold nanoparticles to the oxidized sodium alginate is 1:2-1:5.

[0012] The present invention further improves the CaCl in the engineered nanogold hydrogel to achieve precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment.2 The concentration is 1.8 × 10 −3 -1.8× 10 −1 mol / L.

[0013] An engineered nanogold hydrogel that achieves precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment.

[0014] Application of an engineered nanogold hydrogel for achieving precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment in the preparation of anti-tumor drugs.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses oxidized sodium alginate as a gelling agent for Ca²⁺ reaction in the tumor microenvironment and embeds cell membrane-encapsulated nucleic acid-modified gold nanoparticles into the gel framework to achieve long-term miRNA capture and precise tumor targeted therapy. Its efficient long-term release and targeted specificity design make it an excellent candidate for tumor immunotherapy. The hydrogel preparation method of the present invention does not produce organic waste liquid and is a green and environmentally friendly technology. In addition, no expensive large-scale equipment is required in the production process, which reduces production costs and is conducive to promotion and application.

[0016] Furthermore, the present invention has constructed two nucleic acid-modified gold nanoparticles (SNA1 and SNA2), which, based on the complementary pairing of miRNA, effectively capture miR-214 and miR-130a, inhibit their expression in tumor cells, and simultaneously mediate in situ photothermal effects. This innovative design significantly improves the high selectivity of photothermal therapy, inhibits PD-1-mediated immune escape, and restores the balance between T cells and Tregs, thereby improving the therapeutic effect of cancer photothermal immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the TEM image of SNA1 provided in Example 1.

[0018] Figure 2 This is the TEM image of SNA2 provided in Example 1.

[0019] Figure 3 This is the TEM image of SNAs@M provided in Example 2.

[0020] Figure 4 This is a scanning electron microscope image of SNAs@M-Gel provided in Example 3.

[0021] Figure 5 This is the gel degradation and release diagram of fluorescently labeled SNAs@M nanoparticles (SNAs@M-PKH67) of SNAs@M-Gel provided in Example 3.

[0022] Figure 6 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-214.

[0023] Figure 7 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-214.

[0024] 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.

[0025] Fig. 9 This is a TEM image of the SNAs (a mixture of SNA1 and SNA2) provided in Example 4 after the addition of miR-214.

[0026] Fig.10 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-214.

[0027] Fig.11 This is the expression of miR-130a in 4T1 cells after different groups of treatment provided in Example 5.

[0028] Fig.12 This is the expression of miR-214 in 4T1 cells after different groups of treatment provided in Example 5.

[0029] Fig.13 This is the survival rate of 4T1 cells after treatment with different formulations provided in Example 5.

[0030] Fig.14 The photothermal images of tumor-bearing mice with and without Light irradiation after intravenous injection of PBS, SNAs@M, and SNAs@M-Gel provided in Example 6.

[0031] Fig.15 This is the final tumor photo on the 21st day after different treatments provided in Example 7.

[0032] Fig.16 This is a statistical diagram of tumor mass on the 21st day after different treatments provided in Example 7.

[0033] Fig.17 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] Certain oncogenic miRNAs (such as miR-130a and miR-214) in tumor cells and Tregs are closely related to PD-L1 expression and Tregs proliferation. By targeting and inhibiting the expression of these oncogenic miRNAs, it is possible to prevent the excessive proliferation of Tregs, restore the activity of effector T cells, and inhibit tumor immune escape. Therefore, the development of an intelligent nanoplatform that can generate photothermal agents in situ in tumor cells and regulate oncogenic miRNAs on demand will help achieve precise photothermal immunotherapy and effectively improve the tumor immune microenvironment to achieve better therapeutic effects.

[0037] The engineered nanogold hydrogel of the present invention, which realizes precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment, is used to realize precise photothermal immunotherapy and on-demand regulation of tumor-related T cells. The injectable hydrogel carrier constructed by oxidized sodium alginate (OSA) is loaded with cell membrane-coated nucleic acid-modified gold nanoparticles (SNAs@M); by regulating the overexpressed miR-130a and miR-214 in tumor cells, the inhibition of PD-L1 expression and the inhibition of Tregs cells are achieved. The nanohydrogel releases SNAs@M in the acidic tumor microenvironment, and SNAs@M enters the tumor cells through tumor cell membrane-mediated internalization, targets and inhibits miR-130a and miR-214, thereby significantly reducing the expression of PD-L1 and restoring the effector T cell (CTL) function inhibited by Tregs.

[0038] At the same time, the capture process of miRNAs will also induce SNAs to aggregate in tumor cells to form photothermal agents, thus achieving specific photothermal therapy in tumor cells. This photothermal effect can not only directly kill primary tumors, but also release a large number of tumor-associated antigens, activate dendritic cells (DCs), further stimulate anti-tumor T cell responses, and reshape the tumor immune microenvironment.

[0039] The engineered nanogold hydrogel of the present invention not only effectively inhibits tumor growth and metastasis through synergistic photothermal therapy and immune regulation, but also reverses the immunosuppressive microenvironment of the tumor, significantly improves the efficacy of photothermal immunotherapy, and provides a promising comprehensive cancer treatment strategy.

[0040] The present invention provides a method for preparing an engineered nanogold hydrogel that realizes precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment, comprising the following steps: (1) Using tetrachloroauric acid as a raw material, gold nanoparticles were prepared by sodium citrate reduction method as the gold nanoparticle 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); and the concentration of the gold nanoparticle solution can be 2 nM-10 nM.

[0041] (2) The gold nanoparticle solution was functionalized with DNA through gold-thiol bonds, and nucleic acid-modified gold nanoparticles with different DNA modifications were prepared by regulating the DNA concentration, freezing temperature, freezing time and other conditions; Specifically, the obtained gold nanoparticle solution was fully mixed with the thiolated DNA and then 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 includes the miR-214-DNA1 fragment (5'-SH-TTTTTTTTTTACTGCCTGTCT-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a nucleotide sequence as shown in SEQ ID NO.1), the miR-130a-DNA1 fragment (5'-ACATTGCACTGTTTTTTTTTT-SH-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a nucleotide sequence as shown in SEQ ID NO.2), the thiolated miR-214-DNA2 fragment (5'-GTGCCTGCTGTAAAAAAAAAA-SH-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a nucleotide sequence as shown in SEQ ID NO.3) and the 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 SNAs (a mixture of SNA1 and SNA2 with a molar ratio of 1:1) were dispersed in PBS solution. o CSave.

[0042] The mass ratio of the gold nanoparticles to the thiolated 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 have good dispersion and stability.

[0043] (3) Cell membrane coating of nucleic acid-modified gold nanoparticles, synthesizing cell membrane-coated nucleic acid-modified gold nanoparticles by regulating the ratio of cell membrane and nanoparticles and coating technology; Specifically, first, the tumor cell membrane (M) was obtained by hypotonic lysis. Then, SNAs (a mixture of SNA1 and SNA2 at a molar ratio of 1:1) were mixed with the cell membrane, and then intermittently and gently sonicated in a bath sonicator for 5 minutes to 30 minutes to successfully coat the cell membrane on the surface of the SNAs to obtain cell membrane-coated nucleic acid-modified gold nanoparticles SNAs@M.

[0044] Among them, the mass ratio of cell membrane to nucleic acid-modified gold nanoparticles is 1:1~5:1.

[0045] (4) The synthesized cell membrane-encapsulated nucleic acid-modified gold nanoparticles (SNAs@M) were dispersed in an oxidized sodium alginate aqueous solution (OSA) to obtain a SNAs@M-OSA hydrogel stock solution, and CaCl was added. 2 solution, and the OSA surface -COO- and Ca 2+ The chelation between them forms an engineered nanogold hydrogel that realizes precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment, namely SNAs@M-Gel hydrogel. 2 The concentration is 1.8 × 10 −3 -1.8× 10 −1 mol / L.

[0046] Specifically, first, sodium periodate (NaIO 4 ) Sodium alginate (SA) was oxidized at room temperature for 4-12 hours to obtain oxidized sodium alginate (OSA) with aldehyde groups. Then, SNAs@M was slowly dripped into the oxidized sodium alginate solution with aldehyde groups to obtain a mixture. The mass ratio of cell membrane-coated nucleic acid-modified gold nanoparticles to oxidized sodium alginate was 1:2-1:5. Then, the mixture was continuously magnetically stirred at room temperature, and the aldehyde groups on the surface of oxidized sodium alginate formed Schiff base bonds with the amino groups on the cell membrane to obtain SNAs@M-OSA hydrogel stock solution. Finally, the SNAs@M-OSA hydrogel stock solution was mixed with CaCl 2The solutions were mixed for 10-30 minutes, and then calcium cross-linked to obtain an engineered nanogold hydrogel, namely SNAs@M-Gel hydrogel, which can achieve precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment.

[0047] Among them, the mass ratio of sodium alginate (SA) to sodium periodate (NaIO4) is 5:1~20:1.

[0048] The present invention uses gold nanoparticles (AuNPs), DNA (miR-214-DNA1, miR-214-DNA2, miR-130a-DNA1, miR-130a-DNA2), cell membrane and oxidized sodium alginate to prepare engineered nanogold hydrogel (SNAs@M-Gel), which significantly inhibits Treg cell proliferation and PD-1 / PDL-1-mediated immune escape by regulating various oncogenic miRNAs on demand, and performs miRNAs-dependent photothermal immunotherapy, achieving efficient personalized treatment by in situ generation of customized tumor-associated antigens.

[0049] In the constructed hydrogel (SNAs@M-Gel), SNAs@M nanoparticles are specifically targeted and accumulated in tumor tissues based on the Schiff base cleavage between the cell membrane and OSA and the homing ability of CCM. Once internalized by tumor cells, the exposed SNAs immediately hybridize with overexpressed miR-130a and miR-214, resulting in significant aggregation of SNAs, which effectively inhibits these oncogenic miRNAs. This not only enables precise photothermal immunotherapy to damage primary tumors and produce tumor-associated antigens to activate T cells, but also inhibits the expression of PD-1 and the proliferation of Treg cells through gene therapy, promoting T cell recognition of tumor cells. This strategy can intelligently respond to overexpressed oncogenic miRNAs in tumor cells, perform photothermal immunotherapy on demand, reshape immune homeostasis, and thus eliminate primary tumors and their metastases, and is expected to become a treatment for various malignant tumors.

[0050] Example 1 The glassware was cleaned with freshly prepared aqua regia and dried. 4 ·3H 2 O 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), i.e., gold nanoparticle solution. Finally, the obtained AuNPs solution was stored at 4℃ for further experiments.

[0051] 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 used directly in subsequent experiments without further purification. The synthesis of DNA-modified gold nanoparticles was prepared by adding DNA to the AuNPs solution by freezing. Specifically, AuNPs (13nm, 10nM, 100µL) were thoroughly mixed with thiolated miR-214-DNA1 fragments (100µM, 6µL) and miR-130a-DNA1 (100µM, 2µL) 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 thio-miR-214-DNA2 (100µM, 6µL) and miR-130a-DNA2 (100µM, 2µL) and placed in a laboratory freezer (set at -20°C) for 2 hours, and then thawed at room temperature to obtain unpurified SNA2. Finally, the mixture of unpurified SNA1 and unpurified SNA2 was centrifuged at 10,000rpm 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 and SNA2 were dispersed in PBS solution and washed three 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.

[0052] Example 2 The homologous 4T1 tumor cell membrane (M) was obtained by hypotonic lysis method. Specifically, the tumor cells were placed in 10mM tris (pH7.4), 10mM MgCl 2and 1mM phenylmethylsulfonyl fluoride, resuspended at 4°C for 1 hour, and then subjected to gentle ultrasonic treatment for 5s (VCX130PB, Sonics, USA). Next, after centrifugation at 100g for 10 minutes to remove debris, the cell membrane 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 cell membrane was dispersed in water and stored at 4°C for subsequent experiments. A mixture of 1mg SNA1 and SNA2 (molar ratio 1:1) prepared in Example 1 (SNAs) was mixed with 2mg cell membranes, and then intermittently and gently ultrasonicated in a bath sonicator (SY25-12, Shengyuan Supersonic, China) for 5 minutes to successfully coat the cell membrane on the surface of the SNAs to obtain SNAs@M. The morphology of SNAs@M was determined by transmission electron microscopy, as shown in Figure 3 As shown, it can be seen that the cell membrane structure is clear and SNAs are efficiently encapsulated by CCM.

[0053] Example 3 SA (1.003 g) was dissolved in 80 mL of deionized water and NaIO 4 (54.4 mg), stirred for 4 h in a dark environment at room temperature. Add 2 mL of ethylene glycol, stir for 1 h, then add 0.2 g of NaCl and extract with ethanol. The above extract was then redissolved and dialyzed with water for 3 days. OSA was obtained after freeze-drying. The SNAs@M suspension prepared in Example 2 (containing 10 mg of membrane protein) was slowly dripped into 1 mL of PBS containing 20 mg of OSA (2%). Then, the mixture was continuously magnetically stirred at room temperature for 6 h, and a Schiff base bond was formed between the amine group of the tumor membrane protein and the aldehyde group of OSA to obtain the SNAs@M-OSA hydrogel stock solution. By mixing the SNAs@M-OSA hydrogel stock solution with CaCl 2 The solutions were mixed at a mass ratio of 100:1 to oxidize the -COO- and Ca 2+ The chelation reaction between the two forms SNAs@M-Gel hydrogel. The CaCl 2 The concentration is physiological concentration (1.8 × 10 −3 M). The morphology of SNAs@M-Gel hydrogel was characterized by SEM. Figure 4 As shown, the typical porous network structure of the hydrogel can be clearly seen.

[0054] In order to study the release performance of SNAs@M, green fluorescent labeled SNAs@M (SNAs@M-PKH67NPs) were prepared. 0.2 mL of SNAs@M-Gel hydrogel was shaken and cultured in PBS (2 mL, pH 6.8, 37°C). At the designed time, 2 mL of release medium was collected and then supplemented with PBS. The in vitro release behavior of SNAs@M nanoparticles and the degradation behavior of gel were measured by fluorescence spectrophotometer. The results are shown in Figure 2. Figure 5 As shown, it can be seen that the degradation rate of the hydrogel reached more than 80% on the 10th day, and the release rate of SNAs@M from the hydrogel exceeded 90%.

[0055] Example 4 Take 20ul of each of the SNA1 and SNA2 aqueous solutions prepared in Example 1 and mix them evenly, add 2μL miR-214 (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-214 can induce the aggregation of gold nanoparticles. Similarly, 20ul of each of the SNA1 and SNA2 aqueous solutions were taken and mixed evenly, and 2μL miR-130a (20uM) was added. After reacting at room temperature for 12 hours, no obvious aggregation of SNAs was observed, 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-214 and miR-130a was evaluated by gel electrophoresis. Pure miR-21 / miR-130a, SNA1, SNA2, SNAs and interfering miRNA were used as the control group. Figure 6 As shown, free miR-21 / miR-130a exhibited a clear emission band, while the pure SNAs group (mixture of SNA1 and SNA2) had no emission band. When miR-214 / miR-130a was mixed with SNA1 or SNA2, the emission band of miR-214 / 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-214 / miR-130a. When miR-214 / miR-130a was mixed with SNA (SNA1+SNA2), the content of miR-214 / 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-214 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 spectrum of the hybridization of miR-214 and miR-130a after being captured by smart gold nanoparticles is shown in Figure 2. Figure 7, it can be seen that only after capturing miR-214, the SNAs aggregates showed significantly enhanced absorption in the near-infrared region. TEM results of smart gold nanoparticles after capturing miR-130a and miR-214 are shown in Figure 8 , Fig. 9 It can be clearly seen that after capturing miR-130a, SNAs slightly aggregated, while after capturing miR-214 hybridization, SNAs showed obvious aggregation. Fig.10 It was shown that the SNAs after capturing miR-214 hybridization exhibited excellent photothermal effect under near-infrared light irradiation.

[0056] Example 5 The SNAs@M hydrogel prepared in Example 3 was co-cultured with 4T1 cells. Fig.11 , Fig.12 As shown in the figure, the control groups SNA1@M, SNA2@M, SNAs@M and the hydrogel SNAs@M-Gel can effectively reduce the expression levels of miR-214 and miR-130a in tumor cells. Fig.13 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.

[0057] Example 6 The SNAs@M hydrogel in Example 3 was injected into tumor-bearing mice at a concentration of 200 ul / mouse. One hour later, the tumor area of ​​the mice was illuminated by NIR (808 nm, 1.25 W cm -2 , 5 minutes) to observe the therapeutic effect of photothermal therapy in vivo. The tumor-bearing mice injected with the same volume of PBS and SNAs@M were used as the control group. The photothermal effect was as follows Fig.14 As shown in Figure 2, it can be proved that SNAs@M hydrogels aggregate at the tumor site in mice and produce significant photothermal effect.

[0058] Example 7 Study on the therapeutic effect of engineered nanogold hydrogel 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 3At 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, Gel solution, (SNAs@M) solution in Example 2, and (SNAs@M-Gel) solution in Example 3, respectively. In order to quantitatively study the anti-tumor effect of SNAs@M-Gel nanocomposite hydrogel in vivo, the changes in tumor volume of all mice under different treatment strategies were monitored, and then the final tumors after these treatments were collected and weighed. Fig.15 , Fig.16 It can be seen that SNAs@M-Gel (gene therapy group) showed a certain anti-tumor efficiency, while the single spherical nucleic acid groups (SNA1@M-Gel and SNA2@M-Gel) could only play their gene trapping function even under near-infrared irradiation, highlighting the importance of oncogenic miRNAs as cross-linkers to induce SNA1 and SNA2 aggregation and thus provide conditions for photothermal therapy. As expected, the SNAs@M-Gel+laser group successfully activated photothermal immunotherapy after near-infrared irradiation, and combined with gene therapy, further improved the treatment efficiency and completely inhibited tumor growth, which has broad application prospects.

[0059] Tumor tissues were collected after treatment and analyzed histologically. Fig.17 The results showed that the tumor disintegration area of ​​the SNAs@M-Gel+laser group was the largest (HE staining) and the expression of Ki67 (proliferation marker) was the lowest, indicating that it had the best therapeutic effect on tumors. These results indicate that SNAs@M-Gel-mediated tumor nucleic acid-dependent photothermal immunotherapy plays an important role in inhibiting primary tumors and their metastasis.

[0060] Example 8 Same as Example 2, except that 1 mg of the mixture of SNA1 and SNA2 (molar ratio 1:1) (SNAs) prepared in Example 1 was mixed with 5 mg of cell membranes and then intermittently and gently sonicated in a bath sonicator (SY25-12, Shengyuan Supersonic, China) for 30 minutes.

[0061] Example 9 Same as Example 2, except that 1 mg of the mixture of SNA1 and SNA2 (molar ratio 1:1) (SNAs) prepared in Example 1 was mixed with 3 mg of cell membranes and then intermittently and gently sonicated in a bath sonicator (SY25-12, Shengyuan Supersonic, China) for 15 minutes.

[0062] Example 10 SA (1.003 g) was dissolved in 80 mL of deionized water and NaIO 4, SA and NaIO 4 The mass ratio of SNAs@M is 20:1 and stirred for 4 h in a dark environment at room temperature. Add 2 mL of ethylene glycol, stir for 1 h, then add 0.2 g of NaCl and extract with ethanol. The above extract is then redissolved and dialyzed with water for 3 days. OSA is obtained after freeze-drying. The SNAs@M suspension (containing 10 mg of membrane protein) prepared in Example 2 is slowly dripped into 1 mL of PBS containing 20 mg of OSA (2%). Then, the mixture is continuously magnetically stirred at room temperature for 6 h, and a Schiff base bond will be formed between the amine group of the tumor membrane protein and the aldehyde group of OSA to obtain the SNAs@M-OSA hydrogel stock solution. By adding CaCl 2 The concentration was adjusted to 1.8 × 10 -2 mol / L, by oxidizing the -COO- and Ca 2+ The chelation effect between them forms SNAs@M-Gel hydrogel.

[0063] Embodiment 11 SA (1.003 g) was dissolved in 80 mL of deionized water and NaIO 4 , SA and NaIO 4 The mass ratio of was 10:1, and stirred for 12 hours in a dark environment at room temperature. Add 2 mL of ethylene glycol, stir for 1 hour, then add 0.2 g of NaCl and extract with ethanol. The above extract was then redissolved and dialyzed with water for 3 days. OSA was obtained after freeze-drying. The SNAs@M suspension (containing 10 mg of membrane protein) prepared in Example 2 was slowly dripped into 1 mL of PBS containing 20 mg of OSA (2%). Then, the mixture was continuously magnetically stirred at room temperature for 6 hours, and a Schiff base bond was formed between the amine group of the tumor membrane protein and the aldehyde group of OSA to obtain the SNAs@M-OSA hydrogel stock solution. By adding CaCl 2 The concentration was adjusted to 1.8 × 10 -1 mol / L, by oxidizing the -COO- and Ca 2+ The chelation effect between them forms SNAs@M-Gel hydrogel.

[0064] Example 12 SA (1.003 g) was dissolved in 80 mL of deionized water and NaIO 4 , SA and NaIO 4The mass ratio of was 5:1, and stirred for 8 hours in a dark environment at room temperature. Add 2 mL of ethylene glycol, stir for 1 hour, then add 0.2 g of NaCl and extract with ethanol. The above extract was then redissolved and dialyzed with water for 3 days. OSA was obtained after freeze-drying. The SNAs@M suspension (containing 10 mg of membrane protein) prepared in Example 2 was slowly dripped into 1 mL of PBS containing 20 mg of OSA (2%). Then, the mixture was continuously magnetically stirred at room temperature for 6 hours, and a Schiff base bond was formed between the amine group of the tumor membrane protein and the aldehyde group of OSA to obtain the SNAs@M-OSA hydrogel stock solution. By adding CaCl 2 The concentration was adjusted to 1.8 × 10 -2 mol / L, by oxidizing the -COO- and Ca 2+ The chelation effect between them forms SNAs@M-Gel hydrogel.

[0065] The engineered gold nanoparticles based on photothermal immunotherapy in the present invention achieve precise tumor treatment and on-demand regulation of T cell / Treg cell balance through the regulation of multiple tumor-related miRNAs, and are suitable for the treatment of primary and metastatic malignant tumors.

[0066] 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.

[0067] 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 an engineered nanogold hydrogel for achieving precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment, 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; The cell membrane-coated nucleic acid-modified gold nanoparticles were dispersed in an oxidized sodium alginate aqueous solution and stirred to obtain a SNAs@M-OSA hydrogel stock solution, and then a CaCl2 solution was added for calcium cross-linking to obtain an engineered nanogold hydrogel that can achieve precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment.

2. The method for preparing an engineered nanogold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment 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 nucleic acid-modified gold nanoparticles are mixed with tumor cell membranes and then sonicated to obtain cell membrane-coated nucleic acid-modified gold nanoparticles.

3. The method for preparing an engineered nanogold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment according to claim 1, 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-214-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-214-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-214-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-214-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 an engineered nanogold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment according to claim 1, characterized in that: The mass ratio of tumor cell membrane to nucleic acid-modified gold nanoparticles was 1:1~5:

1.

5. The method for preparing an engineered nanogold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment according to claim 1, characterized in that: The ultrasound time is 5 minutes to 30 minutes.

6. The method for preparing an engineered nano-gold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment according to claim 1, characterized in that: The oxidized sodium alginate aqueous solution is prepared by the following process: sodium alginate is oxidized by sodium periodate at room temperature for 4 hours to 12 hours to obtain the oxidized sodium alginate aqueous solution, wherein the mass ratio of sodium alginate to sodium periodate is 5:1 to 20:

1.

7. The method for preparing an engineered nano-gold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment according to claim 1, characterized in that: The mass ratio of cell membrane-coated nucleic acid-modified gold nanoparticles to oxidized sodium alginate is 1:2-1:

5.

8. The method for preparing an engineered nano-gold hydrogel for realizing precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment according to claim 1, characterized in that: The concentration of CaCl2 in the engineered nanogold hydrogel for precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment is 1.8 × 10 −3 -1.8× 10 −1 mol / L.

9. An engineered nanogold hydrogel prepared according to the method according to any one of claims 1 to 8 for achieving precise photothermal-immunotherapy and on-demand regulation of the immune microenvironment.

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

Images