Selenium-doped prussian blue nanoparticles, preparation method thereof and application thereof in preparation of radiosensitizers

Abstract

The application provides a preparation method of selenium-doped Prussian blue nanoparticles modified by a targeting polypeptide, and comprises the following steps: (1) synthesizing Prussian blue nanoparticles by a microwave method; (2) synthesizing selenium-containing Prussian blue by a gas-phase atomic deposition method; and (3) preparing selenium-doped Prussian blue nanoparticles modified by a targeting polypeptide: dissolving a tumor-targeting RGD cyclic peptide in ultrapure water, adding a catalyst N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and then stirring, then performing dialysis, adding the selenium-containing Prussian blue into the dialysate, stirring, centrifuging, resuspending and obtaining the selenium-doped Prussian blue nanoparticles modified by the targeting polypeptide. According to the preparation method, the selenium is naturally deposited on the surface of the Prussian blue by the gas-phase atomic deposition method, monodisperse selenium with controllable doping amount is formed, and the selenium-doped Prussian blue modified by the targeting polypeptide is prepared.

Description

Technical Field

[0001] This invention belongs to the technical field of cancer treatment drugs, specifically relating to a selenium-doped Prussian blue nanoparticle modified with a targeted peptide, a method for preparing the selenium-doped Prussian blue nanoparticle modified with the targeted peptide, and its application as a radiosensitizer in cervical cancer radiotherapy. Background Technology

[0002] Radiotherapy is one of the most effective treatments for pelvic malignancies. Radiation plays a crucial role in killing cancer cells, inhibiting their spread and metastasis, and prolonging patient survival. However, during radiotherapy for pelvic malignancies, healthy intestinal tissue is inevitably exposed to the radiation field. Rapidly regenerating intestinal epithelial cells are highly sensitive to radiation, leading to radiation-induced intestinal damage. Approximately 50% of patients develop radiation enteritis (RE). RE is one of the most significant complications of pelvic radiotherapy, clinically manifesting as abdominal pain, diarrhea, bloody stools, tenesmus, etc. In severe cases, intestinal wall ulcers, perforation, and even sinus tract formation may occur. RE affects patient adherence to radiotherapy, reduces tumor control effectiveness, and lowers patients' quality of life. Among pelvic malignancies, cervical cancer patients have the highest rate of radiation enteritis. Therefore, the proportion of cervical cancer patients with concurrent radiation enteritis is increasing daily.

[0003] Radiation enteritis (RE) refers to an inflammatory process in the intestinal mucosa caused by radiation exposure. Previous studies have suggested that the pathogenesis of radiation enteritis may be related to abnormal expression of inflammatory factors, gut microbiota dysbiosis, increased permeability of the intestinal epithelium, and vascular damage. Recent studies have found that the pathogenesis of radiation enteritis is mainly related to apoptosis of intestinal mucosal stem cells and vascular endothelial damage. During intestinal inflammation, the innate immune system recognizes and responds to danger signals such as pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and reactive oxygen species (ROS). These danger signals trigger a pro-inflammatory signaling cascade, disrupting intestinal mucosal tissue homeostasis and promoting the progression of colitis. Due to the complexity of the intestinal mucosal inflammatory microenvironment and the fact that some patients are unresponsive or become unresponsive to single anti-inflammatory therapies, developing effective treatments for radiation enteritis is challenging. Long-term use of small molecule therapies or bio-based immunosuppressive drugs can lead to off-target systemic side effects and serious complications, including autoimmune diseases, opportunistic infections, and organ damage. A "drug-free" treatment strategy, which does not include any small molecule therapies or bio-based immunosuppressive drugs, involves clearing multiple danger signals. This simple design reduces toxicity and facilitates eventual conversion.

[0004] Some epidemiological studies have shown that patients with ulcerative colitis and Crohn's disease have decreased serum selenium levels. Crohn's disease patients exhibit decreased serum selenoprotein P and glutathione peroxidase activity. When selenium levels are reduced, plasma prostaglandin E2 increases in ulcerative colitis patients. In mouse models of colitis, selenium supplementation resulted in symptom relief, and decreased levels of pro-inflammatory cytokines such as IL-1β, tumor necrosis factor-α, and interferon-γ, while anti-inflammatory markers such as arginase 1 increased. Therefore, selenium supplementation can reduce inflammation and promote active immunity.

[0005] In recent years, various nanoparticles with antioxidant functions have been discovered and designed to effectively treat diseases such as inflammation, Alzheimer's disease, and stroke. These nanoparticles possess the ability to mimic the activity of natural enzymes, such as superoxide dismutase and catalase. By loading drugs and modifying their surfaces, these antioxidant nanoparticles can deliver drugs to the lesion site, thereby scavenging free radicals in the lesion area and restoring its redox balance. However, the application of some antioxidant nanoparticles in the field of radiosensitization for tumor radiotherapy is currently limited, and they suffer from short drug half-lives, poor biocompatibility, and low utilization rates, falling far short of achieving effective tumor treatment.

[0006] Based on this, the present invention provides a method for preparing selenium-doped Prussian blue nanoparticles modified with targeted peptides. By structurally designing Prussian blue and depositing selenium on its surface using vapor-phase atomic deposition, the amount of selenium doping can be controlled. Selenium-doped Prussian blue, which targets and kills cancer cells, was prepared through peptide modification. Furthermore, combined with X-ray analysis, it was found to have a radiosensitizing effect, synergistically enhancing the killing effect on cervical cancer tumors (HeLa cells). In summary, this invention aims to innovate a method for quantitative selenium doping in Prussian blue nanomaterials to achieve radiosensitization therapy for tumors. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing selenium-doped Prussian blue nanoparticles modified with targeted peptides, which solves the technical problem that the existing MOF-based selenium-containing nanomaterials have shortcomings such as uncontrollable selenium content and low efficiency.

[0008] The present invention also aims to provide selenium-doped Prussian blue nanoparticles modified with targeted peptides prepared by the above method.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A method for preparing selenium-doped Prussian blue nanoparticles with targeted peptide modification includes the following steps:

[0011] (1) Microwave synthesis of Prussian blue nanoparticles;

[0012] (2) Synthesis of selenium-containing Prussian blue by vapor phase atomic deposition of Prussian blue nanoparticles;

[0013] (3) Preparation of selenium-doped Prussian blue nanoparticles modified with targeted peptides: The tumor-targeting RGD cyclic peptide was dissolved in ultrapure water, and N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added and stirred. The mixture was placed on a magnetic stirrer and reacted for several hours. Selenium-containing Prussian blue nanoparticles were added, stirred, centrifuged, and the precipitate was resuspended in ultrapure water. The precipitate was dispersed in ultrapure water under ice bath conditions and stored to obtain selenium-doped Prussian blue nanoparticles modified with targeted peptides.

[0014] In this invention, the mass-to-volume ratio of the tumor-targeting RGD cyclic peptide to ultrapure water in step (3) is 0.1 to 1:1 mg / mL.

[0015] Furthermore, the mass-to-volume ratio of the tumor-targeting RGD cyclic peptide to ultrapure water is 0.25:1 mg / mL.

[0016] The final concentration of N-hydroxysuccinimide in step (3) is 0.1-1 mg / mL; the final concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.1-0.2 mg / mL.

[0017] Further, the final concentration of N-hydroxysuccinimide is 0.1 mol / mL; the final concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.1 mol / mL.

[0018] In this invention, the mass-to-volume ratio of selenium-containing Prussian blue to RGD-containing polypeptide solution in step (3) is 0.1-5 mg / mL.

[0019] In this invention, the molecular weight cutoff for dialysis in step (3) is 5000 Da.

[0020] In step (3), the product is stored at 4°C.

[0021] The present invention can be improved as follows: In step (1), Prussian blue nanoparticles are synthesized by microwave method. The specific process is as follows: weigh potassium hexacyanoferrate(II) trihydrate and polyvinylpyrrolidone are dissolved in ultrapure water, dilute hydrochloric acid solution is added, and the mixture is stirred until a clear solution is obtained. The reaction is carried out in a microwave reactor, centrifuged and resuspended, and dried to obtain Prussian blue nanoparticles.

[0022] Furthermore, the mass-to-volume ratio of potassium hexacyanoferrate(II) trihydrate to water is 5-7:1 mg / mL; the mass-to-volume ratio of polyvinylpyrrolidone to water is 0.1-0.3 g / mL.

[0023] Preferably, the mass-to-volume ratio of potassium hexacyanoferrate(II) trihydrate to water is 6.585:1 mg / mL; and the mass-to-volume ratio of polyvinylpyrrolidone to water is 0.15 g / mL.

[0024] Further, the concentration of dilute hydrochloric acid is 0.01–0.05 mol / L. More preferably, it is 0.02 mol / L.

[0025] Furthermore, in the microwave reactor, the reaction temperature is 70-90℃ and the reaction time is 10-40 min.

[0026] Preferably, in the microwave reactor, the reaction temperature is 80°C and the reaction time is 20 min.

[0027] Furthermore, the drying process employs vacuum drying at a temperature of 20-60℃ for 3-12 hours.

[0028] Preferably, the vacuum drying temperature is 30°C and the time is 8 hours.

[0029] The present invention can also be improved as follows: In step (2), the specific process of synthesizing selenium-containing Prussian blue by vapor phase atomic deposition is as follows: weigh Prussian blue nanoparticles and selenium source, mix them evenly and put them into a quartz glass tube under sealed vacuum, place them in a Marfie furnace for calcination reaction, and cool down to obtain selenium-containing Prussian blue.

[0030] In this invention, the selenium source is one or more of selenium powder, sodium selenite, selenocysteine, and selenomethionine.

[0031] Preferably, the selenium source is selenium powder.

[0032] Furthermore, the mass ratio of the Prussian blue nanoparticles to selenium powder is 5-20:1.

[0033] Furthermore, the calcination reaction in the Marfie furnace is carried out by a programmed temperature increase of 5℃ / min to 230-280℃, and then maintaining a constant temperature of 260℃ for 2-3 hours.

[0034] Furthermore, the calcination reaction in the Marfé furnace was carried out by a programmed temperature increase of 5°C / min to 260°C, and then maintained at a constant temperature of 260°C for 1 hour.

[0035] In this invention, the centrifugation is carried out at low temperature and high speed, with the centrifuge speed being 10,000 to 12,000 rpm and the temperature being 10-20℃ for more than 10 minutes.

[0036] Furthermore, the centrifugation was carried out at a low temperature and high speed, with the centrifuge speed at 12,000 rpm and the temperature at 10°C for 15 minutes.

[0037] A type of selenium-doped Prussian blue nanoparticle modified with a targeted peptide was prepared by the above method.

[0038] Furthermore, the Prussian blue in the targeted peptide-modified selenium-doped Prussian blue nanoparticles is ferriferrocyanide with the chemical formula Fe4[Fe(CN)6]3.

[0039] The application of the above-mentioned targeted peptide-modified selenium-doped Prussian blue nanoparticles in cervical cancer radiotherapy drugs.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The present invention provides a method for preparing selenium-doped Prussian blue nanoparticles with targeted peptide modification. By modifying the structure of Prussian blue nanoparticles, a structure is constructed, and selenium is naturally deposited onto the surface of Prussian blue by vapor phase atomic deposition to form monodisperse selenium with controllable selenium doping amount. Prussian blue is then modified with targeted peptide to obtain selenium-doped Prussian blue with targeted peptide modification.

[0042] (2) This invention uses catalytically active MOF nanomaterials (such as PB) combined with selenium to fully leverage the advantages of metal-organic framework materials, such as large specific surface area and high porosity, thereby optimizing the selenium loading. Furthermore, Prussian blue nanoparticles themselves possess beneficial antioxidant properties, and under the synergistic effect of X-rays, they can kill cancer cells and protect radiation-sensitive intestinal cells.

[0043] (3) This invention synthesizes Prussian blue via a microwave reactor, solving the problem of low yield in the muffle furnace method (i.e., the traditional hydrothermal method). By comparing Prussian blue nanoparticles synthesized via the traditional hydrothermal method with those synthesized via the microwave reactor, it was found that the yield of nanomaterials synthesized via the microwave reactor is significantly higher than that of the traditional hydrothermal method. See [link to relevant documentation]. Figure 3 Therefore, it can effectively reduce raw material waste and achieve efficient conversion and utilization, providing an innovative solution for the large-scale synthesis of selenium MOF nanomaterials.

[0044] (4) The selenium-doped Prussian blue nanoparticles modified with targeted peptides in this invention are based on the excellent antioxidant activity of Prussian blue nanoparticles. Through the introduction of selenium, under X-ray irradiation, due to the Compton effect of selenium, a radiosensitizing effect is achieved, ultimately manifesting as a killing effect on cervical cancer cells. The experimental demonstration of the application of the synthesized selenium-containing Prussian blue in the treatment of cervical cancer also provides scientific guidance for the subsequent application of nanomedicines in cancer radiotherapy. Attached Figure Description

[0045] Figure 1 Transmission electron microscopy (TEM) images of Prussian blue composite materials with different selenium doping contents synthesized by vapor phase atomic deposition (VPD). In Figures a, b, c, and d, the selenium doping ratios are 0%, 5%, 10%, and 20%, respectively.

[0046] Figure 2 Elemental mapping diagrams of Prussian blue composite materials with different selenium contents synthesized by vapor phase atomic deposition.

[0047] Figure 3 Comparison of yields of nanomaterials synthesized by traditional hydrothermal method and microwave reaction method (same solution volume);

[0048] Figure 4 The selenium content in selenium-containing Prussian blue synthesized by vapor phase atomic deposition was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0049] Figure 5 The electron paramagnetic resonance spectrum of hydroxyl radicals of selenium-containing Prussian blue synthesized by vapor deposition.

[0050] Figure 6 This is a diagram showing the cytotoxicity of Prussian blue with different selenium contents synthesized by vapor phase atomic deposition on colonic epithelial cells (NCM460).

[0051] Figure 7 The IC50 assay was conducted to investigate the cytotoxicity of Prussian blue with different selenium contents synthesized by vapor phase atomic deposition against cervical cancer cells (HeLa) at different radiotherapy doses (2 / 4 Gy). 50 surface;

[0052] Figure 8 Images showing the cytotoxic effects of Prussian blue with different selenium contents on cervical cancer cells (SiHa);

[0053] Figure 9 MTT assay for the cytotoxicity of selenium-doped Prussian blue to HeLa cells with different selenium doping ratios: Figure a shows the effect without X-ray irradiation, and Figure b shows the effect under 4 Gy X-ray irradiation.

[0054] Figure 10 Equivalent image of radiosensitization of HeLa cells by RGD@PB-Se 20 nanoparticles. Detailed Implementation

[0055] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention.

[0056] Example 1

[0057] A method for preparing selenium-doped Prussian blue nanoparticles with targeted peptide modification includes the following steps:

[0058] (1) Microwave synthesis of Prussian blue nanoparticles

[0059] At ambient temperature and pressure (15–35 °C, standard atmospheric pressure), 3 g of polyvinylpyrrolidone and 131.7 mg of potassium hexacyanoferrate(II) trihydrate (potassium ferrocyanide) were dissolved in 20 mL of ultrapure water, and 20 mL of 0.02 M hydrochloric acid was added and stirred for 30 minutes. The mixture was then placed in a microwave reactor and reacted at 80 °C for 20 minutes. After cooling, the mixture was centrifuged at 12,000 rpm for 15 minutes at 10 °C. The supernatant was removed by pipette, and the lower precipitate was retained and washed and resuspended with ultrapure water. This process was repeated three times. The lower precipitate was then dried in a vacuum drying oven at 30 °C for 8 hours to obtain deep blue Prussian blue nanoparticles (PB).

[0060] (2) Synthesis of selenium-containing Prussian blue by vapor phase atomic deposition of Prussian blue nanoparticles

[0061] The Prussian blue nanoparticles prepared in step (1) and selenium powder (PB:selenium powder = 10:1) were mixed evenly and poured into a quartz glass tube with a diameter of 14 mm and a length of 280 mm. A hydrogen flame was prepared using an oxyhydrogen generator and a vacuum pump. The quartz glass tube was sealed at high temperature by the hydrogen flame under vacuum conditions. Finally, the sealed quartz tube was placed in a muffle furnace and heated to 260 °C at a programmed rate of 5 °C / min, and kept at a constant temperature for 1 hour. After natural cooling, the quartz glass tube was removed. After opening the glass tube, the target product, which is selenium-containing Prussian blue (PB-Se10), was collected using an EP tube.

[0062] (3) Preparation of selenium-doped Prussian blue nanoparticles modified with targeted peptides

[0063] 20 mg of tumor-targeting RGD cyclic peptide (DSPE-mPEG-NH2) was dissolved in 9 mL of ultrapure water. N-hydroxysuccinimide (NHS) and 0.2 gm / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to a final concentration of 0.6 mg / mL. The mixture was magnetically stirred for 24 hours. The solution was then transferred to a dialysis bag and dialyzed for 24 hours. The ultrapure water in the dialysis bag was replaced every 8 hours. The molecular weight cutoff for dialysis was 5000, and a clear solution was obtained. 10 mg of selenium-containing Prussian blue prepared in step (2) was dissolved in 2.5 mL of the clear solution. After stirring magnetically for 24 hours, the solution was centrifuged at low temperature and high speed at 12000 rpm for 15 minutes at 10°C. The supernatant was discarded, and the precipitate was resuspended in ultrapure water. This process was repeated 3 times. Ultrapure water was then added, and the precipitate was ultrasonically dispersed in ultrapure water under ice bath conditions using an ultrasonic cleaner. The solution was stored at 4°C to obtain selenium-doped Prussian blue nanoparticles (PB-Se RGD@PB-Se10) modified with targeted peptides.

[0064] Example 2

[0065] A method for preparing selenium-doped Prussian blue nanoparticles with targeted peptide modification includes the following steps:

[0066] (1) Microwave synthesis of Prussian blue nanoparticles

[0067] At ambient temperature and pressure (15–35 °C, standard atmospheric pressure), 3 g of polyvinylpyrrolidone and 131.7 mg of potassium hexacyanoferrate(II) trihydrate (potassium ferrocyanide) were dissolved in 20 mL of ultrapure water, and 20 mL of 0.02 M hydrochloric acid was added and stirred for 30 minutes. The mixture was then placed in a microwave reactor and reacted at 80 °C for 20 minutes. After cooling, the mixture was centrifuged at 12,000 rpm for 15 minutes at 10 °C. The supernatant was removed by pipette, and the lower precipitate was retained and washed and resuspended with ultrapure water. This process was repeated three times. The lower precipitate was then dried in a vacuum drying oven at 30 °C for 8 hours to obtain deep blue Prussian blue nanoparticles (PB).

[0068] (2) Synthesis of selenium-containing Prussian blue by vapor phase atomic deposition of Prussian blue nanoparticles

[0069] The Prussian blue nanoparticles prepared in step (1) and selenium powder (PB:selenium powder = 20:1) were mixed evenly and poured into a quartz glass tube with a diameter of 14 mm and a length of 280 mm. A hydrogen flame was prepared using an oxyhydrogen generator and a vacuum pump. The quartz glass tube was sealed at high temperature by the hydrogen flame under vacuum conditions. Finally, the sealed quartz tube was placed in a muffle furnace and heated to 260 °C at a programmed rate of 5 °C / min, and kept at a constant temperature for 1 hour. After natural cooling, the quartz glass tube was removed. After opening the glass tube, the target product, which is selenium-containing Prussian blue (PB-Se5), was collected using an EP tube.

[0070] (3) Preparation of selenium-doped Prussian blue nanoparticles modified with targeted peptides

[0071] 20 mg of tumor-targeting RGD cyclic peptide (DSPE-mPEG-NH2) was dissolved in 9 mL of ultrapure water. N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to a final concentration of 0.6 mg / mL. The mixture was magnetically stirred for 8 hours. The solution was then transferred to a dialysis bag and dialyzed for 24 hours. The ultrapure water in the dialysis bag was replaced every 8 hours. The molecular weight cutoff for dialysis was 5000, and a clear solution was obtained. 10 mg of selenium-containing Prussian blue prepared in step (2) was dissolved in 2.5 mL of the clarified solution. After stirring magnetically for 24 hours, the solution was centrifuged at low temperature and high speed at 12000 rpm for 15 minutes at 10°C. The supernatant was discarded, and the precipitate was resuspended in ultrapure water. This process was repeated 3 times. Ultrapure water was then added, and the precipitate was ultrasonically dispersed in ultrapure water under ice bath conditions using an ultrasonic cleaner. The solution was stored at 4°C to obtain selenium-doped Prussian blue nanoparticles (PB-Se RGD@PB-Se 5) modified with targeted peptides.

[0072] Example 3

[0073] A method for preparing selenium-doped Prussian blue nanoparticles with targeted peptide modification includes the following steps:

[0074] (1) Microwave synthesis of Prussian blue nanoparticles

[0075] At ambient temperature and pressure (15–35 °C, standard atmospheric pressure), 3 g of polyvinylpyrrolidone and 131.7 mg of potassium hexacyanoferrate(II) trihydrate (potassium ferrocyanide) were dissolved in 20 mL of ultrapure water, and 20 mL of 0.02 M hydrochloric acid was added and stirred for 30 minutes. The mixture was then placed in a microwave reactor and reacted at 80 °C for 20 minutes. After cooling, the mixture was centrifuged at 12,000 rpm for 15 minutes at 10 °C. The supernatant was removed by pipette, and the lower precipitate was retained and washed and resuspended with ultrapure water. This process was repeated three times. The lower precipitate was then dried in a vacuum drying oven at 30 °C for 1 hour to obtain deep blue Prussian blue nanoparticles (PB).

[0076] (2) Synthesis of selenium-containing Prussian blue by vapor phase atomic deposition of Prussian blue nanoparticles

[0077] The Prussian blue nanoparticles prepared in step (1) and selenium powder (PB:selenium powder = 5:1) were mixed evenly and poured into a quartz glass tube with a diameter of 14 mm and a length of 280 mm. A hydrogen flame was prepared using an oxyhydrogen generator and a vacuum pump. The quartz glass tube was sealed at high temperature by the hydrogen flame under vacuum conditions. Finally, the sealed quartz tube was placed in a muffle furnace and heated to 260 °C at a programmed rate of 5 °C / min, and kept at a constant temperature for 1 hour. After natural cooling, the quartz glass tube was removed. After opening the glass tube, the target product, which is selenium-containing Prussian blue (PB-Se 20), was collected using an EP tube.

[0078] (3) Preparation of selenium-doped Prussian blue nanoparticles modified with targeted peptides

[0079] 20 mg of tumor-targeting RGD cyclic peptide (DSPE-mPEG-NH2) was dissolved in 9 mL of ultrapure water. N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to a final concentration of 0.6 mg / mL. The mixture was magnetically stirred for 8 hours. The solution was then transferred to a dialysis bag and dialyzed for 24 hours. The ultrapure water in the dialysis bag was replaced every 8 hours. The molecular weight cutoff for dialysis was 5000, and a clear solution was obtained. 10 mg of selenium-containing Prussian blue prepared in step (2) was dissolved in 2.5 mL of the clear solution. After stirring magnetically for 24 hours, the solution was centrifuged at low temperature and high speed at 12000 rpm for 15 minutes at 10°C. The supernatant was discarded, and the precipitate was resuspended in ultrapure water. This process was repeated 3 times. Ultrapure water was then added, and the precipitate was ultrasonically dispersed in ultrapure water under ice bath conditions using an ultrasonic cleaner. The solution was stored at 4°C to obtain selenium-doped Prussian blue nanoparticles (PB-Se RGD@PB-Se 20) modified with targeted peptides.

[0080] Performance testing

[0081] I. Elemental mapping and transmission electron microscopy characterization of Prussian blue composite materials with different selenium doping contents

[0082] Prussian blue composite materials with different selenium doping contents, namely PB, PB-Se5, PB-Se10, and PB-Se20 nanoparticles (with selenium doping ratios of 0%, 5%, 10%, and 20%, respectively), were synthesized by vapor phase atomic deposition (VPD) and their properties were characterized. Specifically, the properties were characterized using a Hitachi H-7650 transmission electron microscope. Figure 1 The results show that PB, PB-Se 5, PB-Se10 and PB-Se20 nanoparticles have stable morphology and good dispersibility. Figure 2 This is an elemental mapping diagram of Prussian blue composite materials with different selenium contents synthesized by vapor phase atomic deposition.

[0083] II. Detection of selenium content in RGD@PB-Se by inductively coupled plasma mass spectrometry (ICP-MS) (selenium doping ratios of 0%, 5%, 10%, and 20%)

[0084] 1. Digestion: The night before, add an appropriate amount of 10 μL LPB-Se (selenium doping ratios of 0%, 5%, 10%, and 20% respectively) to beakers labeled with different sample names. In a fume hood, add 4-5 mL of aqua regia (nitric acid: concentrated hydrochloric acid = 3:1) and cover with a glass slide (convex side down). Perform cold digestion overnight, with the ventilation system running continuously. The next day, place the cold-digested sample on a hot plate and heat to 180°C. Continue heating until the solution volume in the beaker is reduced to approximately 1-2 mL. Remove the beaker and cool to room temperature. Add 8-9 mL of ultrapure water, transfer / pour into a 15 mL centrifuge tube, and finally bring the volume to 10 mL.

[0085] 2. Detection: The selenium content in PB-Se (with selenium doping ratios of 0%, 5%, 10%, and 20%) was detected using inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the selenium content of the samples with different doping ratios was 3.2%, 10.2%, and 16.6%, respectively. The results are as follows: Figure 4 As shown.

[0086] III. Electron Spin Resonance (ESR) Detection of RGD@PB-Se Hydroxyl Radical Generation Ability (Selenium Doping Ratios of 0%, 5%, 10%, and 20%)

[0087] ① First, we used the Fenton reaction to generate hydroxyl radicals (·OH): A 1.8 mM (final concentration) ferrous sulfate solution was mixed with a 5 mM hydrogen peroxide solution and reacted for 10 minutes. RGD@PB-Se (selenium doping ratios of 0%, 5%, 10%, and 20% were diluted with ultrapure water, and then added to the ferrous sulfate and hydrogen peroxide mixture to achieve a final concentration of 10 μg / mL. After shaking thoroughly, the mixture was allowed to stand for 30 minutes. After 30 minutes, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was added to capture the remaining free radicals. The solution was aspirated using a 0.5 mm capillary tube, and the remaining free radicals in the solution were detected using an electron spin resonance (EPR) instrument (with Fenton reaction, i.e., a mixture of ferrous sulfate and hydrogen peroxide, and DMPO as controls). The ability of RGD@PB-Se (with selenium doping ratios of 0%, 5%, 10%, and 20%) and PB hydroxyl radicals to be generated at the same concentration can be determined based on the EPR intensity.

[0088] ② To simulate the pelvic and abdominal radiotherapy environment, we irradiated a solution in a 0.5 mm capillary tube with X-rays at an intensity of 4 Gy. Then, we used an electron spin resonance (ESR) analyzer to detect the free radicals generated under radiotherapy conditions, observing its ability to produce hydroxyl radicals. The results are as follows: Figure 5 As shown.

[0089] The results showed that, without X-ray irradiation, selenium-doped Prussian blue did indeed produce more hydroxyl radicals than selenium-free blue, and the amount of hydroxyl radicals produced increased slightly with increasing selenium doping ratio. Figure 5 a). Under 4GyX-ray radiotherapy, the generation of hydroxyl radicals increased significantly with increasing selenium doping ratio. Figure 5 a). Therefore, this data demonstrates that under high concentration conditions, selenium doping does indeed increase the generation of hydroxyl radicals, and that under X-ray radiation, increasing the selenium doping ratio significantly enhances hydroxyl radical generation, providing an important experimental basis for the subsequent treatment of radiation-induced enteritis caused by cervical cancer radiotherapy. Figure 5 b is the quantification of hydroxyl radicals.

[0090] IV. Culture and Cytotoxicity Evaluation of Colonic Epithelial Cells NCM460, Cervical Cancer Cells SiHa and HeLa Cells

[0091] Colonic epithelial cell line NCM460, cervical cancer cell lines SiHa and HeLa were purchased from the American Type Culture Collection. NCM460, SiHa, and HeLa cell lines were incubated at 37°C in a 5% CO2 incubator in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 50 U / mL streptomycin. Then, 10% (v / v) fetal bovine serum (FBS, Gibco) and 1% (v / v) penicillin-streptomycin mixture (Beyotime, Code No. C0222) were added to the DMEM medium, and the cells were cultured in a humidified incubator at 37°C with 5% (v / v) CO2.

[0092] NCM460 cells in logarithmic growth phase were seeded at a density of 50,000 cells / mL in 96-well plates and allowed to adhere for 24 hours. After cell adhesion, the cells were treated with RGD@PB-Se (selenium doping ratios of 0%, 5%, 10%, and 20%) at concentration gradients of 0.125, 0.25, 0.5, 1, 2, and 4 μg / mL. After 72 hours of treatment, the cytotoxicity of the drug treatment was analyzed using the MTT assay. The data are shown below. Figure 6 The results showed that under low concentration conditions, increasing the selenium doping ratio resulted in lower cytotoxicity to normal epithelial cells.

[0093] Similar to the above method, SiHa and HeLa cells in logarithmic growth phase were seeded at a density of 20,000 cells / mL in 96-well plates and allowed to adhere for 24 hours. After cell adhesion, the cells were treated with RGD@PB-Se (selenium doping ratios of 0%, 5%, 10%, and 20%) at concentration gradients of 1, 2, 4, 8, 16, and 32 μg / mL. After 72 hours of treatment, the cytotoxicity of the drug treatment was analyzed using the MTT assay. The data are shown below. Figure 7 , Figure 8 , Figure 9 The radiosensitization effect of RGD@PB-Se 20 nanoparticles on HeLa cells is as follows: Figure 10 The results showed that under high concentration conditions, the cytotoxicity of selenium doping to cervical cancer cells SiHa and HeLa gradually increased with the increase of selenium doping ratio.

[0094] This invention first synthesizes Prussian blue precursor materials via a microwave method, and then synthesizes a Prussian blue-based composite system with controllable selenium content using vapor phase atomic deposition. This preparation method overcomes the problems of low yield and long preparation time in traditional methods for preparing Prussian blue-like materials, and solves the shortcomings of uncontrollable selenium content and low efficiency in MOF-based selenium-containing nanomaterials. Furthermore, this method is simple and highly practical. It broadens the development process of MOF-based nanomaterials similar to Prussian blue, facilitating large-scale production. Through selenium doping, the Compton effect of selenium is fully utilized. Under X-ray irradiation, the ability of this nanomedicine to generate hydroxyl radicals is greatly enhanced. In vitro cell experiments have demonstrated that, combined with X-ray irradiation, it exhibits excellent cell-killing effects against cancer cells, and shows a certain protective effect on intestinal cells at low doses.

[0095] The above examples illustrate different implementation processes of the present invention in detail. However, the implementation methods of the present invention are not limited thereto. Those skilled in the art can achieve the purpose of the present invention based on the content disclosed in the present invention. Any improvements and modifications made based on the concept of the present invention fall within the protection scope of the present invention. The specific protection scope is subject to the claims.

Claims

1. A method for preparing selenium-doped Prussian blue nanoparticles modified with targeted peptides, characterized in that, Includes the following steps: (1) Microwave synthesis of Prussian blue nanoparticles; (2) Synthesis of selenium-containing Prussian blue by vapor phase atomic deposition of Prussian blue nanoparticles; (3) Preparation of selenium-doped Prussian blue nanoparticles modified with targeted peptides: The tumor-targeting RGD cyclic peptide was dissolved in ultrapure water, and the catalysts N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added and stirred. Then, dialysis was performed, and selenium-containing Prussian blue was added to the dialysate. The mixture was stirred, centrifuged, and the precipitate was resuspended in ultrapure water. The precipitate was dispersed in ultrapure water under ice bath conditions and stored to obtain selenium-doped Prussian blue nanoparticles modified with targeted peptides. In step (1), Prussian blue nanoparticles are synthesized by microwave method. The specific process is as follows: potassium hexacyanoferrate(II) trihydrate and polyvinylpyrrolidone are weighed and dissolved in ultrapure water. Dilute hydrochloric acid solution is added and stirred until a clear solution is obtained. The reaction is carried out in a microwave reactor, centrifuged and resuspended, and dried to obtain Prussian blue nanoparticles. In step (2), the specific process of synthesizing selenium-containing Prussian blue by vapor phase atomic deposition is as follows: weigh Prussian blue nanoparticles and selenium source, mix them evenly, put them into a sealed vacuum quartz glass tube, place them in a Marfie furnace for calcination reaction, and cool them down to obtain selenium-containing Prussian blue.

2. The method for preparing selenium-doped Prussian blue nanoparticles modified with targeted peptides according to claim 1, characterized in that, The mass-to-volume ratio of the tumor-targeting RGD cyclic peptide to ultrapure water in step (3) is 0.1 to 1:1 mg / mL.

3. The method for preparing selenium-doped Prussian blue nanoparticles modified with targeted peptides according to claim 1 or 2, characterized in that, In step (3), the molecular weight cutoff for dialysis is 5000 Da.

4. The method for preparing selenium-doped Prussian blue nanoparticles with targeted peptide modification according to claim 3, characterized in that, In the microwave reactor, the reaction temperature is 70-90℃ and the reaction time is 10-40 min.

5. The method for preparing selenium-doped Prussian blue nanoparticles modified with targeted peptides according to claim 4, characterized in that, The selenium source is one or more of selenium powder, sodium selenite, selenocysteine, and selenomethionine.

6. The method for preparing selenium-doped Prussian blue nanoparticles modified with targeted peptides according to claim 5, characterized in that, The calcination reaction in the Marfie furnace is carried out by increasing the temperature at a programmed rate of 5℃ / min to 230-280℃ and maintaining a constant temperature of 260℃ for 2-3 hours.

7. A targeted peptide-modified selenium-doped Prussian blue nanoparticle, prepared by any one of the methods of claims 1-6.

8. The use of selenium-doped Prussian blue nanoparticles modified with targeted peptides as described in claim 7 in the preparation of radiotherapy drugs for cervical cancer.

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