Preparation method and application of nanometer red blood cell preparation

Abstract

A preparation method and application of a nano red blood cell preparation, AS1411 nucleic acid aptamer is modified on the surface of the nano red blood cell, and the active targeting effect of AS1411 and the tumor cell surface nucleolin is used to realize tumor accumulation and release oxygen to relieve radiotherapy anoxia; the nano red blood cell is endocytosed and degraded to release hematin, and is self-assembled into a peroxidase mimic through the pi-pi interaction of AS1411, enters the nucleus by the nuclear targeting ability of AS1411, catalyzes H2O2 generated by radiotherapy into high-activity •OH, enhances the DNA damage in the nucleus, and thus enhances the radiotherapy of the tumor; the preparation method is simple, stable and reliable, the prepared engineering nano red blood cell preparation has the advantages of active targeting, intelligent assembly and nuclear precise catalysis, is an innovation of radiotherapy treatment drugs, and has practical clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for preparing and applying a nano-red blood cell preparation based on an engineered nano-red blood cell in situ self-assembled peroxidase mimicry to mediate intranuclear H2O2 conversion and sensitize tumor radiotherapy. Background Technology

[0002] Since its first application in cancer treatment in 1895, radiotherapy has become an important clinical method for treating tumors, with approximately 70% of cancer patients receiving it. Its anti-tumor effect is mainly achieved through direct DNA damage caused by high-energy X-rays, or indirectly through free radical-mediated DNA damage. Studies have shown that about two-thirds of DNA damage originates from free radicals. During radiotherapy, X-rays can hydrolyze and generate hydroxyl radicals (•OH) and superoxide anions (•O2-); simultaneously, within one hour after radiation, a large amount of •O2 is generated through reduced coenzyme II and the mitochondrial electron transport chain. - However, to maintain redox homeostasis, tumor cells often highly express superoxide dismutase (SOD), which can convert highly toxic •O2 into oxygen. - It is rapidly converted into hydrogen peroxide (H2O2). However, as a relatively stable non-free radical reactive oxygen species, H2O2 is difficult to effectively cause DNA damage, thus severely limiting the efficiency of radiotherapy in causing free radical damage.

[0003] Therefore, converting the large amount of low-toxicity H2O2 generated by radiotherapy into •OH, which has strong oxidizing properties (oxidation potential 2.8 V), has become a key strategy to enhance radiotherapy-induced DNA damage. In recent years, various nanocatalysts, including Fe3O4 nanozymes and iron / copper-based metal-organic frameworks, have been used for intracellular H2O2 conversion. However, exogenous inorganic metal nanomaterials have high toxicity, and their metabolic mechanisms in vivo are unclear, limiting the clinical application of exogenous nanocatalysts. More importantly, the damage target (DNA) of radiotherapy free radicals is mainly in the cell nucleus, while current conversion strategies focus on the cytoplasm or lysosomes; furthermore, due to the high reaction rate of •OH (10⁻⁶ V), the conversion process is difficult. 9 M -1 s -1 Its half-life is extremely short (~10). -9 The converted •OH radicals are difficult to reach the cell nucleus and are consumed before they can exert their effects, which greatly reduces the DNA damage efficiency of •OH. Therefore, there is an urgent need to develop a nucleus-accessible in situ conversion strategy to improve the efficacy of radiotherapy for free radicals.

[0004] Based on this, the present invention utilizes AS1411 to functionalize erythrocyte nanoparticles, constructing a simple erythrocyte nanoparticle formulation that combines oxygen-carrying and in-situ self-assembly of metabolites. This erythrocyte nanoparticle formulation can efficiently accumulate in tumor tissue through AS1411-mediated active targeting. Simultaneously, it is efficiently taken up by tumor cells by binding to nucleolin highly expressed on the surface of tumor cells. The taken-up erythrocyte nanoparticles are rapidly degraded within tumor lysosomes, releasing a large amount of Heme. The released Heme and AS1411 self-assemble into peroxidase mimics within the tumor cells. These peroxidase mimics can be transported to the tumor cell nucleus via the nucleolin-targeting function of AS1411, continuously catalyzing the large amount of low-toxicity H2O2 generated by radiotherapy into highly active •OH, efficiently damaging intracellular DNA. Simultaneously, the oxygen released by the erythrocyte nanoparticles can effectively fix DNA damage and enhance tumor radiotherapy; however, no such publicly reported method has been found to date. Summary of the Invention

[0005] To address the above shortcomings and overcome the deficiencies of existing technologies, the present invention aims to provide a method for preparing and applying a nano-red blood cell preparation. The method involves modifying the surface of nano-red blood cells with AS1411 nucleic acid aptamers. Through the active targeting of AS1411 with nucleolin on the surface of tumor cells, tumor accumulation is achieved, releasing oxygen to alleviate radiotherapy hypoxia. After being endocytosed by cells, the nano-red blood cells degrade and release heme, which self-assembles with AS1411 through π-π interactions into a peroxidase mimic. This mimic, aided by the nuclear targeting ability of AS1411, enters the cell nucleus and catalyzes the H2O2 generated during radiotherapy into highly active •OH, enhancing nuclear DNA damage and thus sensitizing tumor radiotherapy. The specific steps of the preparation method are as follows: S1. Preparation of nano-red blood cells: Commercially available 10% mouse red blood cells were centrifuged at 500 g for 10 min at 4 ℃. The supernatant was discarded, and the red blood cell precipitate was washed twice with PBS to remove residual impurities, resulting in pure red blood cells. The red blood cells were resuspended in PBS and passed sequentially through polycarbonate membranes with pore sizes of 5 mm, 3 mm, 1 mm, 800 nm, 400 nm, and 220 nm to obtain nano-red blood cells. Chemical synthesis of S2 and DSPE-PEG-AS1411: First, prepare a Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2: Take 745 mL of 1.5 M Tris-HCl stock solution with pH 8.8, add 596.4 mg KCl and 32.5 mg MgCl2, then add 35 mL of distilled water, mix thoroughly to dissolve, and use standard hydrochloric acid solution to precisely adjust the pH of the mixture to 7.4; Subsequently, 10 OD AS1411 was added to 399 mL ddH2O to prepare a 100 mM stock solution, wherein the AS1411 sequence is HS-C6-5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3', which was obtained by commercial gene synthesis method and was custom-synthesized by Qingke Biotechnology Co., Ltd.; then 3.6 mL of Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2 was added to dilute to 10 mM AS1411, heated at 95 ℃ for 5 min and then slowly cooled to room temperature at -1 ℃ / min to form a G-quadruplex structure; 4-10 mM AS1411 G-tetrachain was mixed with 8-20 mM DSPE-PEG-MAL and stirred in the dark for 6 h. Then it was washed three times by ultrafiltration at 5000 g for 40 min each time to obtain DSPE-PEG-AS1411. S3. Preparation of AS1411 functionalized nanored blood cells: The 0.5×10 prepared in step S1 10 -1.5×10 10 The nano-red blood cells were co-incubated with 2-6 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min, and then washed three times with 5000 g ultrafiltration for 40 min each time to obtain the finished nano-red blood cell preparation.

[0006] The nano-red blood cell preparations prepared by the above method can be used in the preparation of tumor radiosensitizing drug injections, drugs to relieve tumor hypoxia, and tumor free radical drugs based on intracellular self-assembled peroxidase mimics mediated reactive oxygen species conversion.

[0007] The preparation method of this invention is simple, stable and reliable. The engineered nano-red blood cell preparation has advantages such as active targeting, intelligent assembly and precise intranuclear catalysis, which can achieve efficient damage to nuclear DNA, inhibit DNA damage repair and enhance the efficacy of radiotherapy. It is an innovation in radiotherapy drugs and has practical clinical application value. Attached Figure Description

[0008] Figure 1 This is a diagram illustrating the metabolic process of hemoglobin within tumor cells according to the present invention. Figure 2 Transmission electron microscopy images of the nano-red blood cells and nano-red blood cell formulations of this invention; Figure 3 The Hb concentration determination of erythrocytes and nano-erythrocytes of the present invention, and their oxygen release curves under hypoxic conditions; Figure 4 This is a circular dichroism spectroscopy diagram illustrating the self-assembly and binding mechanism of AS1411 and Heme. Figure 5 This is the ultraviolet absorption spectrum of the Heme / AS1411 self-assembled assembly of the present invention; Figure 6 This is a graph showing the H2O2 catalytic activity of the Heme / AS1411 self-assembled organism of this invention. Figure 7 Intracellular self-assembly diagram of AS1411 and Heme to verify the FRET effect of this invention; Figure 8 This diagram illustrates the targeting capability of the Heme / AS1411 assembly of the present invention to tumor cell nuclei. Figure 9 This is a diagram showing the in vivo distribution behavior of the nano-red blood cell formulation of the present invention after injection. Detailed Implementation

[0009] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances. Example

[0010] This invention discloses a method for preparing and applying a nano-red blood cell preparation, characterized by comprising the following steps: S1. Preparation of nano-red blood cells: Commercially available 10% mouse red blood cells were centrifuged at 500 g for 10 min at 4 ℃. The supernatant was discarded, and the red blood cell precipitate was washed twice with PBS to remove residual impurities, resulting in pure red blood cells. The red blood cells were resuspended in PBS and passed sequentially through polycarbonate membranes with pore sizes of 5 mm, 3 mm, 1 mm, 800 nm, 400 nm, and 220 nm to obtain nano-red blood cells. Chemical synthesis of S2 and DSPE-PEG-AS1411: First, prepare a Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2: Take 745 mL of 1.5 M Tris-HCl stock solution with pH 8.8, add 596.4 mg KCl and 32.5 mg MgCl2, then add 35 mL of distilled water, mix thoroughly to dissolve, and use standard hydrochloric acid solution to precisely adjust the pH of the mixture to 7.4; Subsequently, 10 OD AS1411 was added to 399 mL ddH2O to prepare a 100 mM stock solution. Then, 3.6 mL of Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2 was added to dilute it to 10 mM AS1411. After heating at 95 °C for 5 min, it was slowly cooled to room temperature at -1 °C / min to form a G-quadruplex structure. 4 mM AS1411 G-tetrachain was mixed with 8 mM DSPE-PEG-MAL and stirred in the dark for 6 h. Then it was washed three times by ultrafiltration at 5000 g for 40 min each time to obtain DSPE-PEG-AS1411. S3. Preparation of AS1411 functionalized nanored blood cells: The 0.5×10 prepared in step S1 10 One nano-red blood cell was co-incubated with 2 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min, and then washed three times with 5000 g ultrafiltration for 40 min each time to obtain the finished nano-red blood cell preparation. Example

[0011] This invention discloses a method for preparing and applying a nano-red blood cell preparation, characterized by comprising the following steps: S1. Preparation of nano-red blood cells: Commercially available 10% mouse red blood cells were centrifuged at 500 g for 10 min at 4 ℃. The supernatant was discarded, and the red blood cell precipitate was washed twice with PBS to remove residual impurities, resulting in pure red blood cells. The red blood cells were resuspended in PBS and passed sequentially through polycarbonate membranes with pore sizes of 5 mm, 3 mm, 1 mm, 800 nm, 400 nm, and 220 nm to obtain nano-red blood cells. Chemical synthesis of S2 and DSPE-PEG-AS1411: First, prepare a Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2: Take 745 mL of 1.5 M Tris-HCl stock solution with pH 8.8, add 596.4 mg KCl and 32.5 mg MgCl2, then add 35 mL of distilled water, mix thoroughly to dissolve, and use standard hydrochloric acid solution to precisely adjust the pH of the mixture to 7.4; Subsequently, 10 OD AS1411 was added to 399 mL ddH2O to prepare a 100 mM stock solution. Then, 3.6 mL of Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2 was added to dilute it to 10 mM AS1411. After heating at 95 °C for 5 min, it was slowly cooled to room temperature at -1 °C / min to form a G-quadruplex structure. 8 mM AS1411 G-tetrachain was mixed with 16 mM DSPE-PEG-MAL and stirred in the dark for 6 h. Then it was washed three times by ultrafiltration at 5000 g for 40 min each time to obtain DSPE-PEG-AS1411. S3. Preparation of AS1411 functionalized nanored blood cells: The 1.0×10 prepared in step S1 10 One nano-red blood cell was co-incubated with 4 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min, and then washed three times with 5000 g ultrafiltration for 40 min each time to obtain the finished nano-red blood cell preparation. Example

[0012] This invention discloses a method for preparing and applying a nano-red blood cell preparation, characterized by comprising the following steps: S1. Preparation of nano-red blood cells: Commercially available 10% mouse red blood cells were centrifuged at 500 g for 10 min at 4 ℃. The supernatant was discarded, and the red blood cell precipitate was washed twice with PBS to remove residual impurities, resulting in pure red blood cells. The red blood cells were resuspended in PBS and passed sequentially through polycarbonate membranes with pore sizes of 5 mm, 3 mm, 1 mm, 800 nm, 400 nm, and 220 nm to obtain nano-red blood cells. Chemical synthesis of S2 and DSPE-PEG-AS1411: First, prepare a Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2: Take 745 mL of 1.5 M Tris-HCl stock solution with pH 8.8, add 596.4 mg KCl and 32.5 mg MgCl2, then add 35 mL of distilled water, mix thoroughly to dissolve, and use standard hydrochloric acid solution to precisely adjust the pH of the mixture to 7.4; Subsequently, 10 OD AS1411 was added to 399 mL ddH2O to prepare a 100 mM stock solution. Then, 3.6 mL of Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2 was added to dilute it to 10 mM AS1411. After heating at 95 °C for 5 min, it was slowly cooled to room temperature at -1 °C / min to form a G-quadruplex structure. 10 mM AS1411 G-tetrachain was mixed with 20 mM DSPE-PEG-MAL and stirred in the dark for 6 h. Then it was washed three times by ultrafiltration at 5000 g for 40 min each time to obtain DSPE-PEG-AS1411. S3. Preparation of AS1411 functionalized nanored blood cells: The 1.5×10 prepared in step S1 10 The nano-red blood cells were co-incubated with 6 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min, and then washed three times with 5000 g ultrafiltration for 40 min each time to obtain the finished nano-red blood cell preparation.

[0013] The nano-red blood cell preparations prepared by the above method can be used in the preparation of tumor radiosensitizing drug injections, drugs to relieve tumor hypoxia, and tumor free radical drugs based on intracellular self-assembled peroxidase mimics mediated reactive oxygen species conversion.

[0014] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any person skilled in the art who can make changes or modifications to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention shall fall within the protection scope of the present invention.

[0015] The present invention features a simple construction method. It utilizes the active targeting ability of AS1411 to achieve efficient accumulation of nano-red blood cells in tumor tissue, effectively alleviating tumor hypoxia through the oxygen transport function of hemoglobin. After the nano-red blood cells are internalized and degraded, the released heme and AS1411 self-assemble intracellularly into a highly active peroxidase mimic, which is then transported to the cell nucleus via the nuclear targeting ability of AS1411. This mimic converts radiotherapy-induced low-toxicity H2O2 in situ into highly damaging •OH, thereby achieving efficient damage to nuclear DNA and significantly enhancing the efficacy of radiotherapy. Repeated experiments have verified that this system consistently exhibits a stable and consistent radiosensitizing effect. For example, in Example 3, the characterization data of the nano-red blood cell formulation are as follows: 1. Experiment on the intracellular metabolism of hemoglobin in tumor cells: 4T1 cells were co-incubated with PBS, nano-erythrocytes, and cysteine ​​protease inhibitor (MG-101) for 8 h. Then, the drug-containing medium was removed, the cells were washed twice with PBS, and the cell pellet was collected. 200 mL of 20 mM oxalic acid was added and incubated at 4 °C for 16 h. After incubation, 800 mL of 1 M oxalic acid was added, and the cell lysate was divided into two equal portions. One portion was incubated at room temperature for 30 min, and the other portion was incubated in a metal shaking bath at 95 °C for 30 min. After incubation, the supernatant was collected by centrifugation and fluorescence was measured using an ELISA reader.

[0016] The results showed that, compared with the control group, the intracellular Heme content in the nano-red blood cell group was significantly increased, while the intracellular Heme content in the nano-red blood cell group decreased after treatment with MG-101. This preliminarily indicates that Hb in nano-red blood cells can be metabolized into Heme in the tumor cells.

[0017] 2. Transmission electron microscopy observation of nano-red blood cells and nano-red blood cell preparations: Taking Example 3 as an example, the prepared nano-red blood cells (NE) and nano-red blood cell preparations (NEA) samples were dropped onto a copper grid. After drying, the dropping was repeated three times. The samples were observed and photographed at 80 kV.

[0018] The transmission electron microscopy (TEM) results show that the average particle size of the prepared NE is approximately 100 nm. The particle size of NEA is not significantly different from that of NE.

[0019] 3. Determination of Hb concentration in erythrocytes and nano-erythrocytes, and their oxygen release performance under hypoxic conditions. First, the changes in intracellular Hb content of red blood cells (RBCs) before and after compression were examined using a complete blood count analyzer, and the changes in oxygen concentration in the solution were monitored using a dissolved oxygen meter.

[0020] like Figure 3 As shown in Figure A, the Hb content of NE did not change significantly compared to RBC, preliminarily indicating that the prepared nano-red blood cells still retained the key proteins of natural red blood cells, and NE had excellent oxygen release performance similar to RBC. Figure 3 B).

[0021] 4. Circular dichroism (CD) chromatography was used to investigate the self-assembly and binding of AS1411 with Heme. AS1411 was first annealed and denatured in Tris-HCl buffer, and then stirred with Heme. The self-assembly and binding mode of AS1411 and Heme were detected by circular dichroism spectroscopy.

[0022] like Figure 4 As shown, the negative peak at 238 nm and the positive peak at 262 nm in the CD spectrum of the Heme / AS1411 assembly still exist, indicating that AS1411 still retains the G-tetramer structure. In addition, the CD spectrum signal in the range of 400-500 nm indicates the formation of the Heme / AS1411 assembly, and the signal at this location also indicates that AS1411 and Heme are combined through external π-π interactions.

[0023] 5. UV absorption spectrum of AS1411 / Heme self-assemblies 300 µL of 10 µM AS1411 was stirred with 1.2 mL of 10 µM Heme for 2 h, and the Heme / AS1411 self-assemblies were collected by ultrafiltration. The absorption spectra of AS1411, Heme, and Heme / AS1411 mixtures were scanned in the range of 200–800 nm using a UV-Vis spectrometer.

[0024] like Figure 5As shown, the UV absorption spectrum of the Heme / AS1411 assembly shows characteristic absorption peaks for AS1411 (260 nm) and Heme (220 nm), respectively, further indicating that self-assembly occurred between AS1411 and Heme. Furthermore, the solubility of Heme assembled with AS1411 in PBS is significantly improved, suggesting that Heme may have transformed from an aggregated state to a dispersed monomeric form after assembly, thereby improving its solubility.

[0025] 6. H2O2 catalytic activity experiment of Heme / AS1411 self-assembled assembly The performance of the Heme / AS1411 peroxidase mimic in catalyzing the production of •OH from H2O2 was systematically evaluated using electron spin resonance (ESR) technology. Four experimental systems were designed: the H2O group, the AS1411 group, the Heme group, and the Heme / AS1411 assembly group. The radical scavenger 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) and H2O2 were added sequentially to each reaction system. ESR spectra were acquired and signals analyzed immediately after 5 min of reaction in the dark.

[0026] like Figure 6 As shown, in the presence of H2O2, the Heme / AS1411 assembly exhibited a significantly enhanced characteristic ESR signal, indicating that Heme / AS1411 can efficiently generate •OH; while the Heme assembly alone produced only a very weak ESR signal. These results demonstrate that the Heme / AS1411 assembly possesses excellent peroxidase activity and can efficiently catalyze the decomposition of H2O2 to produce •OH.

[0027] 7. FRET effect verification assay of the intracellular self-assembly performance of AS1411 and Heme This experiment set up three treatment systems: TCPP treatment group, BBQ 650-labeled AS1411 co-treated group with TCPP, and BBQ 650-labeled Heme / AS1411 assembly co-treated group with TCPP. The specific experimental procedure is as follows: 4T1 cells were pre-incubated with BBQ 650-labeled AS1411 or BBQ 650-labeled Heme / AS1411 assembly for 12 h. After thorough washing with PBS, TCPP was added and incubation continued for another 12 h. Subsequently, the cells were fixed and incubated with DAPI staining solution for 15 min. Finally, the fluorescence intensity of intracellular TCPP was observed using a confocal laser scanning microscope (CLSM).

[0028] like Figure 7As shown, 4T1 cells treated with TCPP alone exhibited a significant red fluorescence signal; however, cells pretreated with BBQ650-labeled AS1411 before adding TCPP showed only a very weak red fluorescence; conversely, cells pretreated with BBQ650-labeled Heme / AS1411 assemblies showed a significant recovery of their red fluorescence signal. These results indicate that TCPP can self-assemble with AS1411, leading to TCPP fluorescence quenching; however, when Heme preemptively occupies the AS1411 binding site, it prevents TCPP from self-assembling with AS1411, thus restoring TCPP fluorescence.

[0029] 8. Targeting ability of the Heme / AS1411 assembly to the cell nucleus (experiment) AS1411 and random sequence aptamers were labeled with FAM and annealed for denaturation. They were then reacted with Heme under light-protected conditions and washed with ultrafiltration to obtain the corresponding assemblies. The Heme / AS1411 assemblies or assemblies formed from random sequences were added to cell culture dishes and co-incubated with cells for 12 h. The cells were then fixed, incubated with DAPI staining solution for 15 min, washed three times with PBS, and finally the intracellular TCPP fluorescence intensity was observed using CLSM.

[0030] Depend on Figure 8 It was observed that after incubation with the Heme / AS1411 assembly, the AS1411 fluorescence signal exhibited significant co-localization with the cell nucleus; while in the non-target sequence assembly treatment group, the fluorescence signal was mainly distributed in the cytoplasm. These results indicate that the Heme / AS1411 assembly can achieve nuclear targeting with the aid of AS1411.

[0031] 9. Experimental study on the tumor targeting ability of the nano-red blood cell formulation of the present invention. Equal amounts of NE and NEA were stained with DiD, with a Free DiD dye group serving as a control. Mice in each group were injected intravenously with NE-DiD and NEA-DiD, respectively. Mice in each group were then photographed using a small animal in vivo multimodal imaging system at 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h post-injection. After imaging, the mice were dissected, and images of the heart, liver, spleen, lungs, kidneys, and tumor tissues were taken using the same system.

[0032] from Figure 9As can be seen, no fluorescence signal was detected at the tumor site in the free DiD group after injection; conversely, strong fluorescence signals were detected at the tumor sites of mice treated with DiD-NE and DiD-NEA. Furthermore, the fluorescence intensity in the DiD-NEA group reached its maximum 24 h after injection, providing experimental evidence for the X-ray irradiation time. In vitro imaging results after 48 h showed that, compared with other treatment groups, the tumor sites of mice treated with DiD-NEA exhibited the strongest fluorescence, indicating that it could effectively accumulate in tumor tissue.

[0033] While conducting experiments on Example 3, the same experiments were also conducted on other examples, and the same or similar results were obtained. These will not be described in detail here.

[0034] As can be seen from the above experiments, the present invention has the following advantages compared with the prior art: (1) The AS1411 functionalized nano-red blood cell preparation constructed in this invention has both the active targeting of nucleic acid aptamers and the passive targeting characteristics of nano-red blood cells, and can be efficiently accumulated in tumor tissue; (2) This invention fully utilizes the dual functional characteristics of hemoglobin: on the one hand, the oxygen it carries can effectively alleviate tumor hypoxia and reduce radiotherapy resistance; on the other hand, the heme released by its intracellular metabolism self-assembles with AS1411 to form a peroxidase mimic, which targets the cell nucleus to catalyze the production of highly toxic •OH. Through the synergistic effect of in situ generation of free radicals in the nucleus and the relief of hypoxia and fixation of DNA damage, highly efficient damage to tumor cell DNA is achieved.

[0035] The preparation method of this invention is simple, stable, and reliable. The resulting engineered nano-red blood cell preparation has advantages such as active targeting, intelligent assembly, and precise intranuclear catalysis. After being internalized by tumor cells, the preparation is degraded in lysosomes to release hemoglobin, which then self-assembles with intracellular AS1411 through π-π interactions to form a highly active peroxidase mimic. This peroxidase mimic further enters the cell nucleus with the nuclear targeting ability of AS1411, catalyzing the low-toxicity H2O2 generated by radiotherapy in situ into highly damaging •OH, thereby achieving highly efficient damage to nuclear DNA. In addition, it utilizes the natural oxygen-carrying function of red blood cells to improve tumor oxygenation and inhibit DNA damage repair, thereby significantly enhancing the efficacy of radiotherapy. This is an innovation in radiotherapy drugs and has practical clinical application value.

Claims

1. A method for preparing a nano-red blood cell preparation, characterized in that, Includes the following steps: S1. Preparation of nano-red blood cells: Commercially available 10% mouse red blood cells were centrifuged at 500 g for 10 min at 4 ℃. The supernatant was discarded, and the red blood cell precipitate was washed twice with PBS to remove residual impurities and obtain pure red blood cells. The red blood cells were resuspended in PBS and passed through polycarbonate membranes with pore sizes of 5 mm, 3 mm, 1 mm, 800 nm, 400 nm and 220 nm in sequence to obtain nano-red blood cells. Chemical synthesis of S2 and DSPE-PEG-AS1411: First, prepare a Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2: Take 745 mL of 1.5 M Tris-HCl stock solution with pH 8.8, add 596.4 mg KCl and 32.5 mg MgCl2, then add 35 mL of distilled water, mix thoroughly to dissolve, and use standard hydrochloric acid solution to precisely adjust the pH of the mixture to 7.4; Subsequently, 10 OD AS1411 was added to 399 mL ddH2O to prepare a 100 mM stock solution, wherein the AS1411 sequence is HS-C6-5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3', which was obtained through a commercial gene synthesis method and was custom-synthesized by Qingke Biotechnology Co., Ltd.; then 3.6 mL of Tris-HCl buffer solution containing 200 mM KCl and 4 mM MgCl2 was added to dilute to 10 mM AS1411, heated at 95 ℃ for 5 min and then slowly cooled to room temperature at -1 ℃ / min to form a G-quadruplex structure; 4-10 mM AS1411 G-tetrachain was mixed with 8-20 mM DSPE-PEG-MAL and stirred in the dark for 6 h. Then it was washed three times by ultrafiltration at 5000 g for 40 min each time to obtain DSPE-PEG-AS1411. S3. Preparation of AS1411 functionalized nanored blood cells: The 0.5×10 prepared in step S1 10 -1.5×10 10 The nano-red blood cells were co-incubated with 2-6 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min, and then washed three times with 5000 g ultrafiltration for 40 min each time to obtain the finished nano-red blood cell preparation.

2. The method for preparing the nano-red blood cell preparation according to claim 1, characterized in that, In step S2: 4 mM AS1411 G-tetrachain is mixed with 8 mM DSPE-PEG-MAL and stirred in the dark for 6 h. In step S3: the 0.5×10⁻⁶ prepared in step S1 is... 10 One nano-red blood cell was co-incubated with 2 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min.

3. The method for preparing the nano-red blood cell preparation according to claim 1, characterized in that, In step S2: 8 mM AS1411 G-tetrachain is mixed with 16 mM DSPE-PEG-MAL and stirred in the dark for 6 h. In step S3: the 1.0×10⁻⁶ prepared in step S1 is... 10 One nano-red blood cell was co-incubated with 4 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min.

4. The method for preparing the nano-red blood cell preparation according to claim 1, characterized in that, In step S2: 10 mM AS1411 G-tetrachain is mixed with 20 mM DSPE-PEG-MAL and stirred in the dark for 6 h. In step S3: the 1.5×10⁻⁶ prepared in step S1 is... 10 One nano-red blood cell was co-incubated with 6 mmol DSPE-PEG-AS1411 prepared in step S2 at 37 °C for 30 min.

5. The use of the nano-red blood cell preparation prepared by the method according to any one of claims 1-4 in the preparation of tumor radiosensitizing drug injections.

6. The use of the nano-red blood cell preparation prepared by the method of any one of claims 1-4 in the preparation of drugs to alleviate tumor hypoxia.

7. The use of the nano-red blood cell formulation prepared by the method of any one of claims 1-4 in the preparation of tumor free radical drugs based on intracellular self-assembled peroxidase mimics mediated reactive oxygen species conversion.

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