Bionic nano radiotherapy sensitizer as well as preparation method and application thereof

Abstract

The invention discloses a bionic nano radiotherapy sensitizer as well as a preparation method and application thereof, and belongs to the field of tumor treatment. The bionic nano radiotherapy sensitizer comprises a radiotherapy sensitizing material and a nano-material, a small interfering RNA; the tumor cell coating wraps the radiotherapy sensitization material and the small interfering RNA; the near-infrared fluorescent dye is modified on the outer surface of the tumor cell coating. The sensitizer disclosed by the invention can be used for specifically recognizing homotype tumors, delivering the radiotherapy sensitizer after accurate targeting of the tumors, effectively enhancing intracellular radioactive ray energy deposition, improving the hypoxia state of tumor cells, sensitizing radiotherapy, silencing expression of target genes of the tumor cells and inhibiting negative immunoregulation, so that the anti-tumor immune response of effector T cells is enhanced, and the radiotherapy effect is improved. In addition, on the basis of the near-infrared fluorescent material, the in-vivo delivery process of the bionic nano radiotherapy sensitizer can be traced.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and in particular to a bionic nano radiotherapy sensitizer and a preparation method and application thereof. Background Art

[0002] Clinically, X-ray radiotherapy is one of the important means of treating breast cancer. It can be used as an independent treatment method or combined with other treatment methods such as surgery and chemotherapy to provide patients with effective and comprehensive treatment options. However, during the treatment process, the tumor non-specificity of X-ray radiotherapy can cause damage to normal tissue cells in the irradiated area. At the same time, the high proliferation rate of tumor cells and abnormal tumor vascular structure can cause tumor hypoxia and increase radiotherapy resistance. Therefore, there is an urgent need to develop new X-ray radiotherapy programs that are highly specific to tumors and more effective.

[0003] Targeted nanomaterials for sensitizing radiotherapy is a very promising strategy. Nanoparticles with high Z sequences have a high absorption capacity for radiation, which can enhance the energy deposition of ionizing radiation, enhance DNA damage, and improve the effect of radiotherapy. Hainfeld et al. found that compared with the single irradiation group, gold nanoparticles (Z = 79) combined with X-ray irradiation can significantly delay tumor growth and prolong survival in EMT6 tumor-bearing mice. Chattopadhyay et al. reported that Au-T targeting human epidermal growth factor receptor-2 (HER-2) combined with X-ray therapy can significantly inhibit tumor growth in tumor-bearing mice compared with X-ray therapy alone. However, the hypoxic characteristics of tumor cells and the fact that radiotherapy can further aggravate tumor hypoxia by damaging blood vessels can both cause radiotherapy resistance and reduce the effect of radiotherapy. Therefore, it is necessary to develop strategies to improve hypoxia and synergize radiotherapy sensitization.

[0004] X-ray radiotherapy changes the tumor microenvironment, and literature studies have shown that radiotherapy can enhance the immune response. However, it can also be observed that patients with poor immune function have poor control of tumors after radiotherapy and poor prognosis. This shows that the ability of radiotherapy-induced anti-tumor immune response to occur depends on the immune function status of the tumor microenvironment. Radiotherapy-induced immune activation is not enough to counteract the immunosuppressive microenvironment. For example, tumor cells can express inhibitory receptors, PD-L1, after radiotherapy, which bind to PD-1 on the surface of T cells, inhibit T cell activity, lead to immune escape, and ultimately cause tumor progression, metastasis or recurrence. This may be the reason why anti-tumor immune responses caused by single radiotherapy are rare in clinical practice. Therefore, suppressing the negative immune environment while radiotherapy may be a promising development direction.

[0005] In summary, the development of a bionic nanoradiotherapy sensitizer that combines radiosensitization with immune activation and can also be effectively traced is of great significance for the treatment of breast cancer. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a bionic nano-radiotherapy sensitizer and a preparation method and application thereof.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] The first aspect of the present invention relates to a bionic nano-radiotherapy sensitizer, comprising:

[0009] Radiotherapy sensitizing materials;

[0010] Small interfering RNA;

[0011] Tumor cell coatings containing radiosensitizing materials and small interfering RNA; and

[0012] Near-infrared fluorescent dye modified on the outer surface of tumor cell coating.

[0013] Optionally, the radiotherapy sensitizing material includes: any one of gold, gadolinium, hafnium, tantalum, tungsten, bismuth, nanosilver, platinum, and MnO2.

[0014] Optionally, the tumor cell coating is derived from any one of mouse tumor cells B16, 4T1, EMT6, LEWIS, Hepa1-6, OVHM, and human tumor cells HELA, MDA-MB-231, U87, PLC.

[0015] Optionally, the small interfering RNA includes an immune checkpoint blocker or a small interfering RNA of any key molecule capable of causing T cell exhaustion.

[0016] Optionally, the immune checkpoint blocker includes: PD-L1 siRNA or CTLA-4 siRNA.

[0017] Optionally, the small interfering RNA of any key molecule capable of causing T cell exhaustion is indoleamine 2,3-dioxygenase.

[0018] Optionally, the emission wavelength of the near-infrared fluorescent dye is higher than 900 nm.

[0019] Optionally, the near-infrared fluorescent dye comprises a positively charged organic small molecule having hydrophobic properties or amphiphilicity.

[0020] The second aspect of the present invention relates to a method for preparing a bionic nano-radiotherapy sensitizer, comprising the following steps:

[0021] The metal elements are combined with MnO2 through reduction to form radiotherapy sensitizing materials;

[0022] Bridging the radiotherapy sensitization material with the small interfering RNA through electrostatic adsorption;

[0023] The obtained product is ultrasonically mixed with tumor cell membrane coating and near-infrared fluorescent dye to obtain the bionic nano-radiotherapy sensitizer.

[0024] The third aspect of the present invention relates to the use of the above-mentioned bionic nano-radiotherapy sensitizer in the preparation of anti-tumor drugs.

[0025] Beneficial effects of the present invention:

[0026] The radiotherapy sensitizer of the present invention can specifically identify homotype tumors, accurately target tumors and deliver the radiotherapy sensitizer, effectively enhance the intracellular radiation energy deposition, improve the hypoxic state of tumor cells, enhance radiotherapy, and at the same time silence the expression of tumor cell target genes, inhibit negative immune regulation, thereby enhancing the anti-tumor immune response of effector T cells and synergistically improving the treatment effect. In addition, the in vivo delivery process of bionic nano-radiotherapy sensitizers can be traced based on near-infrared fluorescent materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings.

[0028] Figure 1 This is a schematic diagram of the structure of the bionic nano-radiotherapy sensitizer;

[0029] Figure 2 The transmission electron microscope image and protein spectrum of the biomimetic nano-radiotherapy sensitizer prepared in Example 1;

[0030] Figure 3 The particle size distribution of the biomimetic nano-radiotherapy sensitizer prepared in Example 1;

[0031] Figure 4 The optical property diagram of the bionic nano-radiotherapy sensitizer prepared in Example 1 and the cell membrane image labeled with near-infrared dye;

[0032] Figure 5 This is a diagram showing the targeting efficiency of the bionic nano-radiotherapy sensitizer prepared in Example 1 to the same type of cells;

[0033] Figure 6 This is a diagram showing the tumor tracing effect of the bionic nano-radiotherapy sensitizer prepared in Example 1 in tumor-bearing mice. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] In this example, Au / MnO2 and PD-L1 siRNA are used as the core, and the outer layer is coated with 4T1 mouse breast tumor cell membrane carrying near-infrared fluorescent dye as a bionic nanoradiotherapy sensitizer (named R&F@Au / MnO2-CM), and in vivo tracing and the effect on the anti-tumor effect of nanomedicines are studied in tumor-bearing mice.

[0037] 1.4 Extraction of T1 mouse breast tumor cell membrane

[0038] (1) Cell culture: 4T1 cells were cultured in RPMI-1640 complete medium, 5% CO2, and 37°C incubator. When the number of 4T1 cells was sufficient, the cells were collected.

[0039] (2) Digestion of cells: Wash the cells with 1× PBS, add 0.25% trypsin to digest for 6 min, and then add an appropriate amount of complete medium containing serum to terminate the digestion. Blow the bottom of the dish to collect all the cells. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add PBS to resuspend the cell pellet and count.

[0040] (3) Centrifuge again (1000 rpm, 5 min), discard the supernatant, and collect the cell pellet. Add 10×10 -3 M MgCl2, 10×10 -3 M Tris and 1× cold Tris buffer (pH=7.4) without ethylenediaminetetraacetic acid (EDTA) protease inhibitors, and incubate at 4°C for 1 h.

[0041] (4) The suspension was sonicated in an ultrasonic machine at 0°C for 10 min.

[0042] (5) After ultrasonic treatment, centrifuge at 600 rpm for 10 min to remove the precipitate and retain the supernatant.

[0043] (6) The obtained supernatant was further centrifuged at 11,480 rpm for 10 min to remove the precipitate and retain the supernatant.

[0044] (7) The supernatant obtained again was centrifuged at 110,000 rpm for 30 min to collect the precipitate, which was the desired 4T1 cell membrane. The above centrifugation steps must be performed at 4°C.

[0045] (8) The collected cell membranes were resuspended in PBS and squeezed through a 400 nm polycarbonate membrane five times to ensure homogeneity.

[0046] (9) Add protease inhibitors at a ratio of 100:1 and store at -80°C until use.

[0047] 2. Synthesis of multifunctional biomimetic nanoradiotherapy sensitizer

[0048] (1) Under stirring conditions at about 600 rpm, 4.11 mL of HAuCl4 was added to 96 mL of deionized water, heated to 160°C, and the mixed aqueous solution was boiled after waiting for about 10 minutes.

[0049] (2) Quickly add 10 mL of trisodium citrate solution (38.8 mM) and incubate for an additional 15 min. The solution gradually changes from light yellow to colorless, black, and finally to cherry red.

[0050] (3) The solution was cooled to room temperature and then centrifuged at 11,000 rpm for 20 min. A cherry red precipitate was observed. The supernatant was discarded, and the solution was resuspended in deionized water and washed by centrifugation twice before being collected for later use.

[0051] (4) Weigh 24 mg of trisodium citrate and add it to the prepared AuNPs solution to make the volume 80 mL.

[0052] (5) Weigh 15.8 mg of potassium permanganate, add it to 20 mL of deionized water, and mix well.

[0053] (6) While rapidly stirring the AuNPs solution (80 mL), potassium permanganate solution was slowly added dropwise and incubated at room temperature for 1 h.

[0054] (7) The rotation speed of the potassium permanganate-containing mixed solution was reduced to 300 rpm, and the solution was heated to 80°C to promote the growth of monodisperse Au / MnO2 nanosheets. After incubation for 30 min, stirring was stopped and the solution was allowed to cool to room temperature. At this time, the solution changed from purple-red when potassium permanganate was first added to brown.

[0055] (8) The Au / MnO2 nanosheets were collected by centrifugation at 10,000 rpm for 20 min and washed with water to remove unreacted KMnO4.

[0056] (9) Au / MnO2 and PEI were stirred at room temperature for 30 min at a mass concentration ratio of 1:1.

[0057] (10) The mixture was transferred to an ultrafiltration tube, centrifuged at 4500 rpm for 5 min, the resulting sediment was collected, resuspended in ultrapure water, and transferred to an ultrafiltration tube again. After repeated washing twice, the collected sediment was resuspended in ultrapure water to obtain PEI-coated Au / MnO2 (Au / MnO2-PEI).

[0058] (11) Positively charged Au / MnO2-PEI (Au concentration of 1 mg / ml) and negatively charged PD-L1 siRNA were physically mixed at a mass ratio of 1:6 at 4 °C for 30 min to obtain the product R@Au / MnO2.

[0059] (12) 4T1 cell membrane and Au@MnO2-siRNA were mixed by low-temperature ultrasound for 20 min to form R@Au / MnO2.

[0060] (13) NIR-935 with a concentration of 2 mg / mL was added at a ratio of 1:10, and low-temperature ultrasound was performed for 10 min. The unadsorbed excess NIR-935 was removed by centrifugation at 10,000 rpm for 15 min. The resulting precipitate was the final material we desired, which was recorded as R&F@Au / MnO2-CM.

[0061] 3. In vivo tracking of multifunctional bionic nanoradiotherapy sensitizers

[0062] (1) After tail vein injection of 200 μL R&F@Au / MnO2-CM 1 mg / kg (calculated as Au concentration) into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.), near-infrared in vivo imaging was used to collect near-infrared fluorescence signals from the tumor sites of tumor-bearing mice and to track the migration of R&F@Au / MnO2-CM in real time.

[0063] Example 2

[0064] In this example, Au / MnO2 and IDO siRNA are used as the core, and the outer layer is coated with LEWIS mouse lung cancer cell membrane carrying near-infrared fluorescent dye as a bionic nanoradiotherapy sensitizer, and in vivo tracing and the effect on the anti-tumor effect of nanomedicine are studied in tumor-bearing mice.

[0065] 1. Extraction of Lewis mouse lung cancer cell membrane

[0066] (1) Cell culture: Lewis cells were cultured in DMEM-H complete medium, 5% CO2, and 37°C incubator. When the number of Lewis cells was sufficient, the cells were collected.

[0067] (2) Digestion of cells: Wash the cells with 1× PBS, add 0.25% trypsin to digest for 6 min, and then add an appropriate amount of complete medium containing serum to terminate the digestion. Blow the bottom of the dish to collect all the cells. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add PBS to resuspend the cell pellet and count.

[0068] (3) Centrifuge again (1000 rpm, 5 min), discard the supernatant, and collect the cell pellet. Add 10×10 -3 M MgCl2, 10×10 -3 M Tris and 1× cold Tris buffer (pH=7.4) without ethylenediaminetetraacetic acid (EDTA) protease inhibitors, and incubate at 4°C for 1 h.

[0069] (4) The suspension was sonicated in an ultrasonic machine at 0°C for 10 min.

[0070] (5) After ultrasonic treatment, centrifuge at 600 rpm for 10 min to remove the precipitate and retain the supernatant.

[0071] (6) The obtained supernatant was further centrifuged at 11,480 rpm for 10 min to remove the precipitate and retain the supernatant.

[0072] (7) The supernatant obtained is centrifuged again at 110,000 rpm for 30 min to collect the precipitate, which is the desired cell membrane. The above centrifugation steps must be performed at 4°C.

[0073] (8) The collected cell membranes were resuspended in PBS and squeezed through a 400 nm polycarbonate membrane five times to ensure homogeneity.

[0074] (9) Add protease inhibitors at a ratio of 100:1 and store at -80°C until use.

[0075] 2. Synthesis of multifunctional biomimetic nanoradiotherapy sensitizer

[0076] (1) Under stirring conditions at about 600 rpm, 4.11 mL of HAuCl4 was added to 96 mL of deionized water, heated to 160°C, and the mixed aqueous solution was boiled after waiting for about 10 minutes.

[0077] (2) Quickly add 10 mL of trisodium citrate solution (38.8 mM) and incubate for an additional 15 min. The solution gradually changes from light yellow to colorless, black, and finally to cherry red.

[0078] (3) The solution was cooled to room temperature and then centrifuged at 11,000 rpm for 20 min. A cherry red precipitate was observed. The supernatant was discarded, and the solution was resuspended in deionized water and washed by centrifugation twice before being collected for later use.

[0079] (4) Weigh 24 mg of trisodium citrate and add it to the prepared AuNPs solution to make the volume 80 mL.

[0080] (5) Weigh 15.8 mg of potassium permanganate, add it to 20 mL of deionized water, and mix well.

[0081] (6) While rapidly stirring the AuNPs solution (80 mL), potassium permanganate solution was slowly added dropwise and incubated at room temperature for 1 h.

[0082] (7) The rotation speed of the potassium permanganate-containing mixed solution was reduced to 300 rpm, and the solution was heated to 80°C to promote the growth of monodisperse Au / MnO2 nanosheets. After incubation for 30 min, stirring was stopped and the solution was allowed to cool to room temperature. At this time, the solution changed from purple-red when potassium permanganate was first added to brown.

[0083] (8) The Au / MnO2 nanosheets were collected by centrifugation at 10,000 rpm for 20 min and washed with water to remove unreacted KMnO4.

[0084] (9) Au / MnO2 and PEI were stirred at room temperature for 30 min at a mass concentration ratio of 1:1.

[0085] (10) The mixture was transferred to an ultrafiltration tube, centrifuged at 4500 rpm for 5 min, the resulting sediment was collected, resuspended in ultrapure water, and transferred to an ultrafiltration tube again. After repeated washing twice, the collected sediment was resuspended in ultrapure water to obtain PEI-coated Au / MnO2 (Au / MnO2-PEI).

[0086] (11) Positively charged Au / MnO2-PEI (Au concentration of 1 mg / ml) and negatively charged IDO siRNA were physically mixed at a mass ratio of 1:6 at 4 °C for 30 min to obtain a complex with small interfering RNA.

[0087] (12) After the Lewis cell membrane and the above-mentioned complex were mixed by cryo-ultrasound for 20 min, NIR-935 at a concentration of 2 mg / mL was added at a ratio of 1:10, and cryo-ultrasound was performed for 10 min. The mixture was centrifuged at 10,000 rpm for 15 min to remove the excess NIR-935 that was not adsorbed. The resulting precipitate was the desired final material.

[0088] 3. In vivo tracking of multifunctional bionic nanoradiotherapy sensitizers

[0089] (1) After 200 μL of 1 mg / kg (calculated as Au concentration) nano-radiotherapy sensitizer was injected into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.) through the tail vein, the tumor sites of the tumor-bearing mice were observed under near-infrared in vivo imaging, and the near-infrared fluorescence signals were collected to track the migration of the bionic nano-radiotherapy sensitizer in the tumor-bearing mice in real time.

[0090] Example 3

[0091] In this example, Au / MnO2 and CTLA-4 siRNA are used as the core, and the outer layer is coated with B16 mouse melanoma cell membrane carrying near-infrared fluorescent dye as a bionic nanoradiotherapy sensitizer, and in vivo tracing and the effect on the anti-tumor effect of nanomedicine are studied in tumor-bearing mice.

[0092] 1. Extraction of B16 Mouse Melanoma Cell Membranes

[0093] (1) Cell culture: B16 cells were cultured in DMEM complete medium, 5% CO2, and 37°C incubator. When the number of B16 cells was sufficient, the cells were collected.

[0094] (2) Digestion of cells: Wash the cells with 1× PBS, add 0.25% trypsin to digest for 6 min, and then add an appropriate amount of complete medium containing serum to terminate the digestion. Blow the bottom of the dish to collect all the cells. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add PBS to resuspend the cell pellet and count.

[0095] (3) Centrifuge again (1000 rpm, 5 min), discard the supernatant, and collect the cell pellet. Add 10×10 -3 M MgCl2, 10×10 -3 M Tris and 1× cold Tris buffer (pH=7.4) without ethylenediaminetetraacetic acid (EDTA) protease inhibitors, and incubate at 4°C for 1 h.

[0096] (4) The suspension was sonicated in an ultrasonic machine at 0°C for 10 min.

[0097] (5) After ultrasonic treatment, centrifuge at 600 rpm for 10 min to remove the precipitate and retain the supernatant.

[0098] (6) The obtained supernatant was further centrifuged at 11,480 rpm for 10 min to remove the precipitate and retain the supernatant.

[0099] (7) The supernatant obtained is centrifuged again at 110,000 rpm for 30 min to collect the precipitate, which is the desired cell membrane. The above centrifugation steps must be performed at 4°C.

[0100] (8) The collected cell membranes were resuspended in PBS and squeezed through a 400 nm polycarbonate membrane five times to ensure homogeneity.

[0101] (9) Add protease inhibitors at a ratio of 100:1 and store at -80°C until use.

[0102] 2. Synthesis of multifunctional biomimetic nanoradiotherapy sensitizer

[0103] (1) Under stirring conditions at about 600 rpm, 4.11 mL of HAuCl4 was added to 96 mL of deionized water, heated to 160°C, and the mixed aqueous solution was boiled after waiting for about 10 minutes.

[0104] (2) Quickly add 10 mL of trisodium citrate solution (38.8 mM) and incubate for an additional 15 min. The solution gradually changes from light yellow to colorless, black, and finally to cherry red.

[0105] (3) The solution was cooled to room temperature and then centrifuged at 11,000 rpm for 20 min. A cherry red precipitate was observed. The supernatant was discarded, and the solution was resuspended in deionized water and washed by centrifugation twice before being collected for later use.

[0106] (4) Weigh 24 mg of trisodium citrate and add it to the prepared AuNPs solution to make the volume 80 mL.

[0107] (5) Weigh 15.8 mg of potassium permanganate, add it to 20 mL of deionized water, and mix well.

[0108] (6) While rapidly stirring the AuNPs solution (80 mL), potassium permanganate solution was slowly added dropwise and incubated at room temperature for 1 h.

[0109] (7) The rotation speed of the potassium permanganate-containing mixed solution was reduced to 300 rpm, and the solution was heated to 80°C to promote the growth of monodisperse Au / MnO2 nanosheets. After incubation for 30 min, stirring was stopped and the solution was allowed to cool to room temperature. At this time, the solution changed from purple-red when potassium permanganate was first added to brown.

[0110] (8) The Au / MnO2 nanosheets were collected by centrifugation at 10,000 rpm for 20 min and washed with water to remove unreacted KMnO4.

[0111] (9) Au / MnO2 and PEI were stirred at room temperature for 30 min at a mass concentration ratio of 1:1.

[0112] (10) The mixture was transferred to an ultrafiltration tube, centrifuged at 4500 rpm for 5 min, the resulting sediment was collected, resuspended in ultrapure water, and transferred to an ultrafiltration tube again. After repeated washing twice, the collected sediment was resuspended in ultrapure water to obtain PEI-coated Au / MnO2 (Au / MnO2-PEI).

[0113] (11) Positively charged Au / MnO2-PEI (Au concentration of 1 mg / ml) and negatively charged CTLA-4 siRNA were physically mixed at a mass ratio of 1:6 at 4°C for 30 min to obtain a mixture with small interfering RNA.

[0114] (12) After the B16 cell membrane and the above mixture were mixed by low-temperature ultrasound for 20 min, NIR-935 with a concentration of 2 mg / mL was added at a ratio of 1:10, and low-temperature ultrasound was performed for 10 min. The mixture was centrifuged at 10,000 rpm for 15 min to remove the excess NIR-935 that was not adsorbed. The resulting precipitate was the final material we needed.

[0115] 3. In vivo tracking of multifunctional bionic nanoradiotherapy sensitizers

[0116] (1) After 200 μL of 1 mg / kg (calculated as Au concentration) nano-radiotherapy sensitizer was injected into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.) through the tail vein, the tumor sites of the tumor-bearing mice were observed under near-infrared in vivo imaging, and the near-infrared fluorescence signals were collected to track the migration of the bionic nano-radiotherapy sensitizer in the tumor-bearing mice in real time.

[0117] Example 4

[0118] In this example, Au / MnO2 and PD-L1 siRNA are used as the core, and the outer layer is coated with EMT6 mouse breast tumor cell membrane carrying near-infrared fluorescent dye as a bionic nanoradiotherapy sensitizer, and in vivo tracking and the influence of nanomedicine on the anti-tumor effect are studied in tumor-bearing mice.

[0119] 1. Extraction of EMT6 Mouse Breast Tumor Cell Membranes

[0120] (1) Cell culture: EMT6 cells were cultured in a DMEM complete medium, 5% CO2, and 37°C incubator. When the number of EMT6 cells was sufficient, the cells were collected.

[0121] (2) Digestion of cells: Wash the cells with 1× PBS, add 0.25% trypsin to digest for 6 min, and then add an appropriate amount of complete medium containing serum to terminate the digestion. Blow the bottom of the dish to collect all the cells. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add PBS to resuspend the cell pellet and count.

[0122] (3) Centrifuge again (1000 rpm, 5 min), discard the supernatant, and collect the cell pellet. Add 10×10 -3 M MgCl2, 10×10 -3 M Tris and 1× cold Tris buffer (pH=7.4) without ethylenediaminetetraacetic acid (EDTA) protease inhibitors, and incubate at 4°C for 1 h.

[0123] (4) The suspension was sonicated in an ultrasonic machine at 0°C for 10 min.

[0124] (5) After ultrasonic treatment, centrifuge at 600 rpm for 10 min to remove the precipitate and retain the supernatant.

[0125] (6) The obtained supernatant was further centrifuged at 11,480 rpm for 10 min to remove the precipitate and retain the supernatant.

[0126] (7) The supernatant obtained is centrifuged again at 110,000 rpm for 30 min to collect the precipitate, which is the desired cell membrane. The above centrifugation steps must be performed at 4°C.

[0127] (8) The collected cell membranes were resuspended in PBS and squeezed through a 400 nm polycarbonate membrane five times to ensure homogeneity.

[0128] (9) Add protease inhibitors at a ratio of 100:1 and store at -80°C until use.

[0129] 2. Synthesis of multifunctional biomimetic nanoradiotherapy sensitizer

[0130] (13) Under stirring conditions at about 600 rpm, 4.11 mL of HAuCl4 was added to 96 mL of deionized water and heated to 160 °C. After waiting for about 10 min, the mixed aqueous solution boiled.

[0131] (14) 10 mL of trisodium citrate solution (38.8 mM) was quickly added and incubated for an additional 15 min. The solution gradually changed from light yellow to colorless, black, and finally to cherry red.

[0132] (15) The solution was cooled to room temperature and then centrifuged at 11,000 rpm for 20 min. A cherry red precipitate was observed. The supernatant was discarded, and the solution was resuspended in deionized water and washed by centrifugation twice before being collected for later use.

[0133] (16) Weigh 24 mg of trisodium citrate and add it to the prepared AuNPs solution to make the volume 80 mL.

[0134] (17) Weigh 15.8 mg of potassium permanganate, add it to 20 mL of deionized water, and mix well.

[0135] (18) Under the condition of rapid stirring of AuNPs solution (80 mL), potassium permanganate solution was slowly added dropwise and incubated at room temperature for 1 h.

[0136] (19) The rotation speed of the potassium permanganate-containing mixed solution was reduced to 300 rpm, and the solution was heated to 80 °C to promote the growth of monodisperse Au / MnO2 nanosheets. After incubation for 30 min, stirring was stopped and the solution was allowed to cool to room temperature. At this time, the solution changed from purple-red when potassium permanganate was first added to brown.

[0137] (20) The Au / MnO2 nanosheets were collected by centrifugation at 10000 rpm for 20 min and washed with water to remove the unreacted KMnO4.

[0138] (21) Au / MnO2 and PEI were stirred at room temperature for 30 min at a mass concentration ratio of 1:1.

[0139] (22) The mixture was transferred to an ultrafiltration tube and centrifuged at 4500 rpm for 5 min to collect the resulting sediment, which was resuspended in ultrapure water and transferred to an ultrafiltration tube again. After repeated washing twice, the collected sediment was resuspended in ultrapure water to obtain PEI-coated Au / MnO2 (Au / MnO2-PEI).

[0140] (23) Positively charged Au / MnO2-PEI (Au concentration of 1 mg / ml) and negatively charged PD-L1 siRNA were physically mixed at a mass ratio of 1:6 at 4 °C for 30 min to obtain a mixture with small interfering RNA.

[0141] (24) After the EMT6 cell membrane and the above mixture were mixed by low-temperature ultrasound for 20 min, NIR-935 with a concentration of 2 mg / mL was added at a ratio of 1:10, and low-temperature ultrasound was performed for 10 min. The mixture was centrifuged at 10,000 rpm for 15 min to remove the excess NIR-935 that was not adsorbed. The resulting precipitate was the final material we needed.

[0142] 3. In vivo tracking of multifunctional bionic nanoradiotherapy sensitizers

[0143] (2) After tail vein injection of 200 μL of 1 mg / kg (calculated as Au concentration) of nano-radiotherapy sensitizer into tumor-bearing mice (Suzhou Jingweiyu Biotechnology Co., Ltd.), near-infrared in vivo imaging was used to collect near-infrared fluorescence signals from the tumor sites of the tumor-bearing mice and to track the migration of the bionic nano-radiotherapy sensitizer in the tumor-bearing mice in real time.

[0144] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0145] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A bionic nano-radiotherapy sensitizer, characterized in that: include: Radiotherapy sensitizing materials; Small interfering RNA; Tumor cell coatings encapsulating radiosensitizing materials and small interfering RNA; as well as Near-infrared fluorescent dye modified on the outer surface of tumor cell coating.

2. The bionic nano-radiotherapy sensitizer according to claim 1, characterized in that: The radiotherapy sensitizing material includes: any one of gold, gadolinium, hafnium, tantalum, tungsten, bismuth, nanosilver, platinum, and MnO2.

3. The bionic nano-radiotherapy sensitizer according to claim 1, characterized in that: The tumor cell coating is derived from any one of mouse tumor cells B16, 4T1, EMT6, LEWIS, Hepa1-6, OVHM, and human tumor cells HELA, MDA-MB-231, U87, and PLC.

4. The bionic nano-radiotherapy sensitizer according to claim 1, characterized in that: The small interfering RNA includes an immune checkpoint blocker or a small interfering RNA of any key molecule capable of causing T cell exhaustion.

5. The bionic nano-radiotherapy sensitizer according to claim 4, characterized in that: The immune checkpoint blockers include: PD-L1 siRNA or CTLA-4 siRNA.

6. The bionic nano-radiotherapy sensitizer according to claim 4, characterized in that: The small interfering RNA of any key molecule capable of causing T cell exhaustion is indoleamine 2,3-dioxygenase.

7. The bionic nano-radiotherapy sensitizer according to claim 1, characterized in that: The emission wavelength of the near-infrared fluorescent dye is higher than 900 nm.

8. The bionic nano-radiotherapy sensitizer according to claim 1, characterized in that: The near-infrared fluorescent dye includes a hydrophobic or amphiphilic positively charged organic small molecule.

9. A method for preparing a bionic nano-radiotherapy sensitizer, characterized in that: The following steps are involved: The metal elements are combined with MnO2 through reduction to form radiotherapy sensitizing materials; Bridging the radiotherapy sensitization material with the small interfering RNA through electrostatic adsorption; The obtained product is ultrasonically mixed with tumor cell membrane coating and near-infrared fluorescent dye to obtain the bionic nano-radiotherapy sensitizer.

10. Use of the bionic nano-radiotherapy sensitizer according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

Images