A sensitizing film for radioactive stent and preparation method thereof

Abstract

The present invention discloses a sensitizing film for radioactive stents and a preparation method thereof, aiming to overcome the existing radiotherapy stents' great damage to normal tissues and the shortcomings of traditional sensitizing drug coating materials. The sensitizing film for radioactive stents includes a first film layer and a second film layer, wherein the first film layer is a polyurethane support layer, and the second film layer contains a radiosensitizing drug component; the platinum radiosensitizing drug is oxidized to a tetravalent platinum drug, a polyarginine targeting group is introduced for modification, and then PLGA50:50 with a molecular weight of 5k is used for modification; the first film layer composed of polyurethane and the second film layer composed of PLGA75:25 with a molecular weight of 100k and containing a radiosensitizing drug component are prepared by electrospinning technology. The sensitizing film for radioactive stents of the present invention has the advantages of high targeting, low toxicity and side effects, high efficiency sensitization, good support, sustained release, and simple and efficient preparation process, providing a new, safe and effective treatment method for the field of intracavitary tumor radiotherapy.

Description

Technical Field

[0001] The invention belongs to the field of medical devices, and in particular relates to a sensitization film for a radioactive stent and a preparation method thereof. Background Art

[0002] In the medical field, especially in the treatment of cavity tumors, radioactive stents have gradually attracted clinical attention and application as an innovative local treatment method. Radioactive stents can release radioactive rays locally in the cavity by coating radioactive isotopes or carrying radioactive particles on the surface of the stent, effectively killing or inhibiting the growth of tumor cells, while keeping the cavity open and reducing the occurrence of restenosis. However, traditional radioactive stents have problems such as large damage to surrounding normal tissues and limited treatment effects.

[0003] In order to improve the therapeutic effect of radioactive stents and reduce side effects, the present invention begins to explore coating sensitizing materials on the surface of the stent to enhance the sensitivity of radiotherapy. Among them, platinum-based radiosensitizing drugs are one of the research hotspots due to their unique chemical structure and biological activity. Platinum-based drugs can bind to DNA to form DNA-platinum adducts, which hinder the replication and transcription of DNA, thereby enhancing the sensitivity of cells to radiation. However, the direct application of platinum-based drugs has problems such as poor solubility, poor targeting, and low bioavailability. Therefore, it is necessary to develop a new type of sensitizing film to effectively load and stably fix the platinum-based drugs on the surface of the stent.

[0004] At present, there are many methods for preparing sensitization films containing platinum drugs, such as nanoparticle encapsulation, polymer composite, etc. However, these methods often have problems such as poor targeting, large side effects, and difficulty in controlling drug release during the preparation process. In addition, due to the complex surface morphology of the stent, traditional coating methods are difficult to ensure the uniform distribution of drugs on the stent surface, thus affecting the treatment effect.

[0005] Conventional platinum radiosensitizers are divalent platinum drugs, which are cytotoxic and are indiscriminately absorbed by tumor cells and normal cells. While killing cancer cells, they are also prone to damage normal cell tissues, with large side effects. The present invention oxidizes tetravalent platinum drugs based on conventional platinum drugs. Since the tumor microenvironment has the characteristics of high reducibility, non-toxic tetravalent platinum is easily reduced to divalent platinum with cytotoxicity in cancer cells, but is not easily reduced in ordinary cells. The tetravalent platinum drug is friendly to normal cell tissues, specifically kills tumor cells, and has good biocompatibility.

[0006] Cancer cells have a high demand for arginine because of the close collaboration between their high levels of arginine and RNA-binding motif proteins (RBMs), which regulate abnormally active gene expression mechanisms and give them the ability to proliferate indefinitely. The synthesis pathway of arginine depends on citrulline, which is continuously catalyzed by argininosuccinate synthetase 1 (ASS1) and argininosuccinate lyase (ASL), and is ultimately decomposed into ornithine and urea by arginase 1 (Arg1). Subsequently, ornithine is converted into citrulline under the action of ornithine transcarbamylase (OTC) to complete recycling in mitochondria. It is worth noting that any abnormality in ASS1, ASL, or OTC can disrupt the homeostasis of arginine in cells. In the tumor microenvironment, the loss of ASS1 is particularly common, forcing tumor cells to rely on external arginine supply, thereby exacerbating the rapid consumption of arginine around the tumor.

[0007] The present invention takes a unique approach and uses polyarginine to specifically modify tetravalent platinum drugs, achieving precise targeted binding to tumor cells. This innovative design not only accelerates the penetration efficiency of drugs into cancer cells, but also significantly reduces the misabsorption of radiosensitizing drug components by normal cells, ensuring a high degree of selectivity in the treatment. In the complex metabolic network of cancer cells, arginine is guided to the DNA region as a key regulatory factor, affecting the expression of tumor-related genes. Once the tetravalent platinum drug modified with polyarginine enters the cancer cell, its strong targeting properties cause the sensitizing drug components to gather tightly around the DNA, greatly enhancing the specific effect of the drug and further improving the effect of radiotherapy.

[0008] What's even more ingenious is that after the polyarginine-modified drug ingredient enters the tumor cell, its functional group generates nitric oxide (NO) under the action of NO synthase, providing an additional source of nitrogen supply urgently needed in the metabolism of tumor cells, just like an efficient nitrogen reservoir. The excessive production of NO caused by this targeted delivery puts tumor cells under the pressure of excessive nitrogen load, which in turn weakens their ability to repair radiation-induced DNA damage. Under this dual mechanism, the polyarginine functional group not only acts as a drug carrier, but also indirectly introduces NO, an additional radiosensitizing factor, which synergizes with the tetravalent platinum component to double the effect of radiotherapy, opening up a new and efficient path for tumor treatment.

[0009] It is difficult to balance the support performance and drug release capacity when the sensitizing film is directly coated on the surface of the traditional radioactive stent. The high molecular weight (100K) polyurethane material is coated on the side close to the stent by electrospinning technology, which can provide good support performance for the radioactive stent to load the sensitizing drug components and effectively inhibit the growth of tumor cells into the pores of the radioactive stent. The degradable material loaded with the sensitizing drug components, high molecular weight (100K) PLGA75:25, is further coated on the outside of the polyurethane film layer by electrospinning technology, which realizes the sustained release of the sensitizing drug components and can provide the sensitizing drug components to tumor cells for a long time. PLGA50:50 with a molecular weight of 5K has a certain viscosity. After modifying the tetravalent platinum drug component, it can better bind to PLGA75:25 with a molecular weight of 100K, reducing the risk of drug components falling off of the sensitizing film during the packaging, transportation and implantation of the radioactive stent. During the treatment process, the degradation rate of PLGA50:50 with a molecular weight of 5K is higher than that of PLGA75:25 with a molecular weight of 100K. The sensitizing drug components can be quickly detached with the degradation of the absorbable film material so that they can be absorbed by tumor cells. The structural design of this radiosensitizing film takes both supporting performance and drug release capacity into consideration.

[0010] Therefore, the present invention aims to provide a sensitizing film for radioactive stents and a preparation method thereof, wherein a tetravalent platinum complex modified with polyarginine is compounded with PLGA (polylactic acid-co-glycolic acid) by electrospinning technology to prepare a sensitizing film with good targeting, few side effects, good biocompatibility and controllable drug release characteristics. The film can not only enhance the radiosensitivity of cancer cells at the stent site and improve the treatment effect, but also reduce the damage to surrounding normal tissues, and has broad clinical application prospects. Summary of the invention

[0011] The purpose of the present invention is to provide an innovative sensitizing film for radioactive stents, aiming to significantly reduce radiotherapy damage to surrounding normal tissues and overcome the limitations of traditional radiosensitizing drug coating materials. Through the carefully designed film formula and preparation process, the present invention not only achieves targeted treatment of tumor cells and reduces the toxic and side effects on normal tissues, but also ensures that the drug components are non-toxic and harmless outside the cells and inside normal tissue cells, and only exert their unique cytotoxic effects after entering the cancer cells, thereby achieving high efficiency and safety of treatment. The sensitizing film of the present invention combines advanced targeting technology and drug sustained release strategies to ensure that sensitizing drugs can accurately act on tumor cells. At the same time, it utilizes the specific reduction activation mechanism of tetravalent platinum drugs in cancer cells and the use of polyarginine modification to keep the drug non-toxic outside the cells and inside normal tissue cells, avoiding nonspecific damage. This innovative solution has brought new breakthroughs in the field of tumor radiotherapy, and is expected to significantly improve patients' treatment experience and quality of life.

[0012] The technical scheme of the present invention is as follows: a sensitizing film for a radioactive stent, which is composed of two layers of films: a first film layer and a second film layer; wherein the second film layer is the key, and contains a specially treated radiosensitizing drug component to achieve a sensitizing effect on radiotherapy; the preparation method is as follows:

[0013] S1 Oxidation of platinum radiosensitizers:

[0014] First, the selected platinum radiosensitizer (such as any one or more combinations of cisplatin, carboplatin, oxaliplatin, lobaplatin, nedaplatin or heptaplatin) is dissolved in 30wt% hydrogen peroxide and the concentration is adjusted to 0.15-0.3mol / L; then, it is heated to 60-80°C under light-proof conditions and stirred for 1-4h; then, the solution is cooled to 0-5°C and cooled for 1-3h; light yellow crystals are obtained by vacuum filtration, and then washed with water, ethanol and ether respectively, to finally obtain a tetravalent platinum dihydroxy complex; the chemical reaction formula of the drug molecular structure conversion process is as follows:

[0015]

[0016] in represents cisplatin, carboplatin, oxaliplatin, lobaplatin, nedaplatin or heptaplatin, The structural formula is as follows:

[0017]

[0018] S2 Polyarginine modified tetravalent platinum complex:

[0019] The tetravalent platinum dihydroxy complex obtained in step S1 and succinic anhydride (or optional glutaric anhydride, adipic anhydride) are dissolved in an organic solvent N,N-dimethylacetamide (DMF) or dimethyl sulfoxide (DMSO) at a molar ratio of 1:(3-4), and the concentration of the tetravalent platinum dihydroxy complex is 10-25 mol / L. The mixture is stirred under the protection of dry nitrogen and heated to 50-70°C for 5-30 hours. The mixture is then cooled to room temperature and concentrated to a light yellow liquid by reduced pressure. Dichloromethane or acetone is added under negative pressure for low-temperature recrystallization. The crystals are filtered out and washed with cold ether to obtain yellow crystalline tetravalent platinum dicarboxyl complex. The tetravalent platinum dicarboxyl complex is mixed with polyarginine having a polymerization degree of 2-10 in an organic solvent DMF or DMSO at a molar ratio of 1:1, the concentration of the tetravalent platinum dicarboxyl complex is 10-25 mol / L, and a carboxyl activator (such as 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), mass ratio (2-3):1, input concentration is 40-60 g / L) is added, and stirred for 5-30 hours under light-proof and dry nitrogen protection to obtain a solution A containing polyarginine-modified tetravalent platinum complex; the reaction formula is as follows:

[0020]

[0021] Where n is the degree of polymerization of polyarginine, ranging from 2 to 10;

[0022] S3 PLGA modified tetravalent platinum complex:

[0023] PLGA50:50 with a molecular weight of 5k is dissolved in an organic solvent DMF or DMSO, and then solution A obtained in step S2 is added after ultrasonic oscillation until mixed. The molar ratio of PLGA50:50 to the polyarginine-modified tetravalent platinum complex in step S2 is (1-2):1. Ultrasonic oscillation is continued until mixed, and electromagnetic stirring is performed under light-proof and dry nitrogen protection, and the mixture is heated to 50-60° C., and after 5-30 hours, the mixture is brought to room temperature, and then evaporated under reduced pressure to form a polyarginine-PLGA-modified tetravalent platinum complex agglomerate; the reaction formula is as follows:

[0024] or

[0025] Where p and q are the number of monomers in the PLGA molecule, the total molecular weight of PLGA is 5K, and the monomer ratio p:q=50:50; the PLGA50:50 chemical formula is only for display, and the arrangement order of the two monomer structures can be combined;

[0026] S4 Preparation of electrospinning solution:

[0027] Two electrospinning solutions were prepared respectively: polyurethane with a molecular weight of 100k was dissolved in a mixed solvent of dichloromethane (DCM) and DMF (the volume ratio of DCM to DMF was (1-2):1), and ultrasonically shaken until mixed to obtain solution B; the tetravalent platinum complex agglomerates modified by polyarginine-PLGA obtained in step S3 and PLGA75:25 with a molecular weight of 100k were dissolved in a mixed solvent of dichloromethane (DCM) and DMF (the volume ratio of DCM to DMF was (1-2):1), and ultrasonically shaken until mixed to obtain solution C; the mass fraction of polyurethane with a molecular weight of 100k in solution B was 15-20%; the mass fraction of PLGA75:25 with a molecular weight of 100k in solution C was 15-20%, and the mass fraction of tetravalent platinum complex modified by polyarginine-PLGA was 1-10%;

[0028] S5 Electrospinning film preparation:

[0029] The radioactive stent is moistened with solution B and placed on the receiving shaft of the electrospinning machine. First, the first layer of membrane is spun on the radioactive stent using solution B, and then the second layer of membrane is spun on the radioactive stent using solution C. During the spinning process, the voltage is controlled at 10-15 kV, the flow rate is 0.3-0.6 ml / h, the spinning distance is 5-10 cm, the roller speed is 400 r / min, the ambient temperature is 30°C, and the humidity is 30%-40%. Finally, the radioactive stent is taken out and dried to obtain a sensitizing film for the radioactive stent. The first film layer thickness of the sensitizing film for the radioactive stent is 10-30 μm, and the second film layer thickness is 30-50 μm.

[0030] Through the above technical solution, the present invention successfully and efficiently integrates the radiosensitizing drug components into the double-layer film structure, which not only improves the efficacy of radiotherapy, but also ensures the safety and controllability of the treatment process.

[0031] Advantages of the present invention:

[0032] Highly targeted. By introducing polyarginine as a targeting modification group, the drug component of the sensitizing film of the present invention can accurately identify and enrich on the surface of tumor cells, ensuring that the radiosensitizing drug component directly acts on tumor tissue while reducing nonspecific damage to normal tissue. This high degree of targeting not only improves the therapeutic effect, but also reduces the side effects of the treatment;

[0033] Low toxicity and side effects. The drug components of the present invention remain non-toxic outside cells and inside normal tissue cells. Only after entering the cancer cells, they are activated and exert cytotoxic effects under a specific reducing environment. This design effectively avoids the potential damage of drugs to normal cells during transportation, ensuring the safety of treatment;

[0034] Highly efficient sensitization, using tetravalent platinum drugs as radiosensitizers, the present invention utilizes its specific reduction activation mechanism in cancer cells to significantly enhance the efficacy of radiotherapy, and at the same time, through the sustained release effect of the sensitization film, the drug can be continuously and stably released into the tumor tissue, maintaining an effective therapeutic concentration for a long time, further improving the therapeutic effect;

[0035] Innovative film structure: The sensitizing film of the present invention adopts a unique double-layer structure design, which not only ensures the supporting performance of the film, but also realizes the uniform distribution and sustained release control of the drug. This structural design not only optimizes the release kinetics of the drug, but also improves the stability and biocompatibility of the film, providing a strong guarantee for the effective delivery and release of the drug.

[0036] The preparation process is simple and efficient, and the sensitizing film is prepared by using advanced electrospinning technology. The present invention realizes precise control of film thickness and drug distribution. The process has the advantages of low production cost, simple operation, and easy large-scale production, providing strong support for the wide application of sensitizing films. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of a sensitizing film for a radioactive stent.

[0038] Figure 2 Survival curve showing the changes in mouse tumor volume over time during the evaluation of radiosensitization effect. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below with reference to the accompanying drawings.

[0040] Example 1: Preparation of a sensitizing film for radioactive stents

[0041] S1 Oxidation of cisplatin. Dissolve cisplatin in 30wt% hydrogen peroxide and adjust the concentration to 0.2mol / L. Heat to 75℃ in a dark environment and stir for 3h until the reaction is complete. Cool to 5℃ and cool for 2h. Filter under reduced pressure to obtain pale yellow crystals, and then wash with water, ethanol and ether respectively to obtain a tetravalent platinum dihydroxy complex.

[0042] S2 Polyarginine modified tetravalent platinum complex. The obtained tetravalent platinum dihydroxy complex and succinic anhydride were dissolved in DMF at a molar ratio of 1:3, and the concentration of the tetravalent platinum dihydroxy complex was 15 mol / L. Stirring under the protection of dry nitrogen, heating to 60 ° C for 8 hours, and cooling to room temperature after the reaction. The cooled solution was concentrated under reduced pressure to a light yellow liquid, and dichloromethane or acetone was added under negative pressure for low-temperature recrystallization. The crystals were filtered out and washed with cold ether to obtain yellow crystalline tetravalent platinum dicarboxyl complex; the tetravalent platinum dicarboxyl complex was mixed with polyarginine with a polymerization degree of 3 in an organic solvent DMF at a molar ratio of 1:1, and the concentration of the tetravalent platinum dicarboxyl complex was 20 mol / L, and a carboxyl activator (EDC and NHS, mass ratio 2:1, input concentration of 50 g / L) was added, and stirred for 8 hours under light protection and dry nitrogen protection to obtain a solution A containing a tetravalent platinum complex modified by polyarginine.

[0043] S3 PLGA modified tetravalent platinum complex. Dissolve PLGA50:50 with a molecular weight of 5k in DMF, and ultrasonically shake until mixed. Add solution A obtained in step S2, and the molar ratio of PLGA50:50 to the tetravalent platinum complex modified by polyarginine in step S2 is 1:1. Continue ultrasonically shaking until mixed. Electromagnetic stirring is carried out under the protection of light and dry nitrogen, and heated to 55°C and stirred for 10 hours. After cooling to room temperature, evaporate under reduced pressure to form a polyarginine-PLGA modified tetravalent platinum complex agglomerate.

[0044] S4 Preparation of electrospinning solution. A polyurethane with a molecular weight of 100k was dissolved in a mixed solvent of DCM and DMF (volume ratio of DCM to DMF was 1:1), and ultrasonically shaken until mixed to obtain solution B. A tetravalent platinum complex modified with polyarginine-PLGA and PLGA75:25 with a molecular weight of 100k were dissolved in a mixed solvent of DCM and DMF (volume ratio of DCM to DMF was 1:1), and ultrasonically shaken until mixed to obtain solution C. The mass fraction of polyurethane with a molecular weight of 100k in solution B was 18%; the mass fraction of PLGA75:25 with a molecular weight of 100k in solution C was 20%, and the mass fraction of tetravalent platinum complex modified with polyarginine-PLGA was 3%.

[0045] S5 Preparation of thin film by electrospinning. The radioactive stent is moistened with solution B and placed on the receiving shaft of the electrospinning machine. First, solution B is used to spin the first layer of membrane on the radioactive stent with a spinning thickness of 25 μm; then solution C is used to spin the second layer of membrane with a thickness of 40 μm. The voltage is controlled at 15 kV, the flow rate is 0.5 ml / h, the spinning distance is 7 cm, the ambient temperature is 30 ° C, and the humidity is 35%. Finally, take it out together with the radioactive stent and dry it to obtain a sensitized film for the radioactive stent. The sensitized film for the radioactive stent is obtained as follows Figure 1As shown, it comprises a first film layer 1 and a second film layer 2, wherein the first film layer 1 is closely attached to the radioactive stent 3, and the second film layer contains a radiosensitizing drug component.

[0046] Example 2: Biocompatibility testing

[0047] Objective: To evaluate the biocompatibility of sensitizing films in the extracellular environment and ensure their safety and effectiveness in clinical applications.

[0048] Materials: human fibroblasts (HFF, ATCC, CRL-1829); human colon cancer cells (HCT116, ATCC, CCL-247); MTT reagent (Sigma-Aldrich); cell culture medium (DMEM, containing 10% fetal bovine serum); sensitization film for radioactive stents (prepared with reference to Example 1).

[0049] Experimental steps. Cell culture: HFF and HCT116 cells were cultured in DMEM medium at 37°C and 5% CO 2 Environment, until the cells reach 80% fusion. Film treatment: Cut the radioactive scaffold into discs with a diameter of 10 mm using the sensitizing film and place them under sterile conditions. Place the discs in culture dishes containing 10 ml of DMEM and incubate for 24 hours to ensure that the components in the film are released into the culture medium. Cell inoculation: Take out the culture medium treated with the film and divide it into two groups: the experimental group (film treatment) and the control group (no film treatment). HFF and HCT116 cells were inoculated in the culture medium of the experimental group and the control group, respectively. Three parallel samples were set up in each group, and the cell density was 1×10 4 cells / well. MTT method was used to assess cell viability: 24 hours after cell seeding, 10 μl of MTT solution (5 mg / ml) was added to each well and incubated for another 4 hours. Afterwards, the culture medium was removed, 150 μl of DMSO was added to dissolve the formed purple metaceramia crystals, and the absorbance (OD) was measured at 570 nm.

[0050] Results statistics. Calculate cell survival rate: Cell survival rate = OD value of experimental group / OD value of control group × 100%. The results are as follows.

[0051] HFF cells: The OD value of the experimental group (treated with sensitizing film) was 1.45; the OD value of the control group (without film treatment) was 1.58; the survival rate of HFF cells (relative to the control group) was: survival rate = 1.45 / 1.58×100%≈91.8%.

[0052] HCT116 cells: The OD value of the experimental group (treated with sensitizing film) was 0.80; the OD value of the control group (without film treatment) was 1.42; the survival rate of HCT116 cells (relative to the control group) was: survival rate = 0.80 / 1.42×100%≈56.3%.

[0053] Conclusion: The sensitizing film showed good biocompatibility to normal HFF cells and did not cause obvious cytotoxicity as evaluated by the MTT assay. It also showed significant inhibitory effects on HCT116 cancer cells, further demonstrating the potential of the sensitizing film in targeted therapy.

[0054] Example 3: Evaluation of radiosensitization effect

[0055] Objective: To evaluate the inhibitory effect of the radioactive stent sensitizing film in Example 1 on tumor growth in mice combined with radiotherapy, as well as its safety to normal tissues.

[0056] Animal model: Male nude mice (BALB / c) aged 6-8 weeks and weighing about 20 g were selected. Animal husbandry: The mice were placed under constant temperature and humidity conditions and adapted for one week with free access to food and water. Tumor transplantation: The tumor cell line was HCT116 human colon cancer cells. 1×10 6 HCT116 cells were suspended in 100 μl of saline and implanted subcutaneously into the right hind limb region of mice. The mice were then allowed to grow for 7-10 days until the tumor volume reached approximately 100 mm³.

[0057] Experimental grouping and treatment. Mice were randomly divided into four groups, with 6 mice in each group. Control group: no treatment was performed. Radiation group: after the tumor reached about 100 mm³, local 2 Gy γ-ray irradiation was given once a week. Sensitizing film group: a sensitizing film for radioactive stents was implanted in the tumor site, but no radiotherapy was performed. Sensitizing film + radiation group: a sensitizing film for radioactive stents was implanted in the tumor site, and local 2 Gy γ-ray irradiation was performed once a week.

[0058] Implantation of sensitizing film: When the mouse is anesthetized (2% isoflurane inhalation anesthesia), the radioactive stent with a sensitizing film is implanted into the tumor area through a small subcutaneous incision, and the film is ensured to be close to the tumor surface. The incision is then sutured with sterile sutures.

[0059] Radiotherapy: One day after the implantation of the radioactive stent with sensitizing film, the mice in the radiation group and the sensitizing film + radiation group were subjected to local irradiation of 2 Gy gamma rays once a week using a special irradiator for small animals. The irradiation area was limited to the tumor site to minimize the impact on other tissues.

[0060] Tumor volume measurement: Tumor volume was measured every two days for 28 days. The long diameter (L) and short diameter (W) of the tumor were measured using a vernier caliper, and the tumor volume (V) was calculated using the formula: V = (L × W²) / 2.

[0061] Animal safety evaluation: The weight changes of mice were recorded every 2 days to observe whether the mice had adverse reactions such as hair loss, loss of appetite or abnormal behavior. At the end of the experiment, the mice were killed and the liver, kidney, heart and other major organs were taken for histological analysis to evaluate the effects of the drug on normal tissues.

[0062] Results. Changes in tumor volume: The tumor in the control group grew rapidly within 28 days, and the final average volume reached 1500 mm³; the tumor growth in the radiation group slowed down, and the average volume was 950 mm³ after 28 days; the tumor growth rate in the sensitizing film group decreased slightly, and the average volume was 1100 mm³ after 28 days; the tumor growth in the sensitizing film + radiation group was significantly inhibited, and the average volume was 450 mm³ after 28 days, which was significantly smaller than that of other groups (p < 0.01). Changes in mouse weight: There was no significant difference in the weight of mice in each group, and no obvious weight loss was observed, indicating that the sensitizing film and radiotherapy had no obvious adverse effects on the overall health of mice. Histological analysis: No obvious histopathological changes were observed in the liver, kidney, and heart tissues of the sensitizing film + radiation group and other groups, indicating that the sensitizing film is non-toxic to major organs. Survival curve: The survival curve of the tumor volume of each group of mice over time was drawn, such as Figure 2 As shown, the inhibitory effect of each treatment group on tumor growth is intuitively demonstrated. Four different treatment methods are represented by different shape symbols: the circular symbol represents the control group (untreated), the square symbol represents the radiation group (radiotherapy only), the triangle symbol represents the sensitizing film group (sensitizing film for radioactive stent implantation only), and the diamond symbol represents the sensitizing film + radiation group (sensitizing film combined with radiotherapy). It can be seen from the curve that the sensitizing film + radiation group has the most significant inhibitory effect on tumor growth, and the difference is significant compared with other groups.

[0063] Example 4: Preparation of a sensitizing film for a radioactive stent

[0064] S1 Oxidation of carboplatin. Dissolve carboplatin in 30wt% hydrogen peroxide and adjust the concentration to 0.3mol / L. Heat to 80℃ in a dark environment and stir for 4h until the reaction is complete. Cool to 0℃ and cool for 3h. Filter under reduced pressure to obtain pale yellow crystals, and then wash with water, ethanol and ether respectively to obtain a tetravalent platinum dihydroxy complex.

[0065] S2 Polyarginine modified tetravalent platinum complex. The obtained tetravalent platinum dihydroxy complex and glutaric anhydride were dissolved in DMSO at a molar ratio of 1:4, and the concentration of the tetravalent platinum dihydroxy complex was 25 mol / L. Stirring under dry nitrogen protection, heating to 70°C for 30 hours, and cooling to room temperature after the reaction. The cooled solution was concentrated under reduced pressure to a light yellow liquid, and dichloromethane or acetone was added under negative pressure for low-temperature recrystallization. The crystals were filtered out and washed with cold ether to obtain yellow crystalline tetravalent platinum dicarboxyl complex; the tetravalent platinum dicarboxyl complex was mixed with polyarginine with a polymerization degree of 10 in an organic solvent DMSO at a molar ratio of 1:1, and the concentration of the tetravalent platinum dicarboxyl complex was 25 mol / L, and a carboxyl activator (EDC and NHS, mass ratio 3:1, input concentration of 60 g / L) was added, and stirred for 30 hours under light protection and dry nitrogen protection to obtain a solution A containing a tetravalent platinum complex modified by polyarginine.

[0066] S3 PLGA modified tetravalent platinum complex. Dissolve PLGA50:50 with a molecular weight of 5k in DMSO, and ultrasonically shake until mixed. Add solution A obtained in step S2, and the molar ratio of PLGA50:50 to the tetravalent platinum complex modified by polyarginine in step S2 is 2:1. Continue ultrasonically shaking until mixed. Electromagnetic stirring is carried out under the protection of light and dry nitrogen, and heated to 60°C and stirred for 10 hours. After cooling to room temperature, evaporate under reduced pressure to form a polyarginine-PLGA modified tetravalent platinum complex agglomerate.

[0067] S4 Preparation of electrospinning solution. A polyurethane with a molecular weight of 100k was dissolved in a mixed solvent of DCM and DMF (volume ratio of DCM to DMF 2:1), and ultrasonically shaken until mixed to obtain solution B. A tetravalent platinum complex modified with polyarginine-PLGA and PLGA75:25 with a molecular weight of 100k were dissolved in a mixed solvent of DCM and DMF (volume ratio of DCM to DMF 1:1), and ultrasonically shaken until mixed to obtain solution C. The mass fraction of polyurethane with a molecular weight of 100k in solution B was 20%; the mass fraction of PLGA75:25 with a molecular weight of 100k in solution C was 20%, and the mass fraction of tetravalent platinum complex modified with polyarginine-PLGA was 10%.

[0068] S5 Preparation of film by electrospinning. The radioactive stent is moistened with solution B and placed on the receiving shaft of the electrospinning machine. Solution B is first used to spin the first layer of membrane on the radioactive stent with a spinning thickness of 30 μm; then solution C is used to spin the second layer of membrane with a thickness of 50 μm. The voltage is controlled at 15 kV, the flow rate is 0.6 ml / h, the spinning distance is 10 cm, the ambient temperature is 30 ° C, and the humidity is 40%. Finally, the radioactive stent is taken out and dried to obtain a sensitized film for the radioactive stent, whose structure is as shown Figure 1 .

[0069] Example 5: Preparation of a sensitizing film for radioactive stents

[0070] S1 Oxidation of heptaplatin. Dissolve heptaplatin in 30wt% hydrogen peroxide and adjust the concentration to 0.15mol / L. Heat to 60℃ in a dark environment and stir for 1h until the reaction is complete. Cool to 5℃ and cool for 1h. Filter under reduced pressure to obtain pale yellow crystals, and then wash with water, ethanol and ether respectively to obtain a tetravalent platinum dihydroxy complex.

[0071] S2 Polyarginine modified tetravalent platinum complex. The obtained tetravalent platinum dihydroxy complex and adipic anhydride were dissolved in DMF at a molar ratio of 1:3.5, and the concentration of the tetravalent platinum dihydroxy complex was 10 mol / L. Stirring under dry nitrogen protection, heating to 50 ° C for 5 hours, and cooling to room temperature after the reaction. The cooled solution was concentrated under reduced pressure to a light yellow liquid, and dichloromethane or acetone was added under negative pressure for low-temperature recrystallization. The crystals were filtered out and washed with cold ether to obtain yellow crystalline tetravalent platinum dicarboxyl complex; the tetravalent platinum dicarboxyl complex was mixed with polyarginine with a polymerization degree of 2 in an organic solvent DMSO at a molar ratio of 1:1, and the concentration of the tetravalent platinum dicarboxyl complex was 10 mol / L, and a carboxyl activator (EDC and NHS, mass ratio 2.5:1, input concentration of 40 g / L) was added, and stirred for 5 hours under light protection and dry nitrogen protection to obtain a solution A containing a tetravalent platinum complex modified by polyarginine.

[0072] S3 PLGA modified tetravalent platinum complex. Dissolve PLGA50:50 with a molecular weight of 5k in DMSO, and ultrasonically shake until mixed. Add solution A obtained in step S2, and the molar ratio of PLGA50:50 to the tetravalent platinum complex modified by polyarginine in step S2 is 1.5:1. Continue ultrasonically shaking until mixed. Electromagnetic stirring is carried out under the protection of light and dry nitrogen, and heated to 50°C and stirred for 5h. After cooling to room temperature, evaporate under reduced pressure to form a polyarginine-PLGA modified tetravalent platinum complex agglomerate.

[0073] S4 Preparation of electrospinning solution. A polyurethane with a molecular weight of 100k was dissolved in a mixed solvent of DCM and DMF (volume ratio of DCM to DMF was 1.3:1), and ultrasonically shaken until mixed to obtain solution B. A tetravalent platinum complex modified with polyarginine-PLGA and PLGA75:25 with a molecular weight of 100k were dissolved in a mixed solvent of DCM and DMF (volume ratio of DCM to DMF was 1.3:1), and ultrasonically shaken until mixed to obtain solution C. The mass fraction of polyurethane with a molecular weight of 100k in solution B was 15%; the mass fraction of PLGA75:25 with a molecular weight of 100k in solution C was 15%, and the mass fraction of tetravalent platinum complex modified with polyarginine-PLGA was 1%.

[0074] S5 Preparation of film by electrospinning. The radioactive stent is moistened with solution B and placed on the receiving shaft of the electrospinning machine. Solution B is first used to spin the first layer of membrane on the radioactive stent with a spinning thickness of 10 μm; then solution C is used to spin the second layer of membrane with a thickness of 30 μm. The voltage is controlled at 10 kV, the flow rate is 0.3 ml / h, the spinning distance is 5 cm, the ambient temperature is 30 ° C, and the humidity is 30%. Finally, the radioactive stent is taken out and dried to obtain a sensitized film for the radioactive stent, whose structure is as follows Figure 1 .

[0075] Example 6

[0076] The radioactive stent in Example 2 was replaced with the sensitizing film of Example 4 and Example 5 respectively. The survival rate of HFF cells was 90-93%, and the survival rate of HCT116 cells was 52.2% (Example 4) and 60.5% (Example 5). Conclusion: The sensitizing film showed good biocompatibility to normal HFF cells and did not cause obvious cytotoxicity through MTT method evaluation; while it showed significant inhibitory effect on HCT116 cancer cells.

[0077] Example 7

[0078] The evaluation method in Example 3 was used to evaluate the radiosensitization effect of the radioactive stent sensitization film in Example 4 and Example 5, respectively, and the obtained results were as follows: Figure 2 Similar survival curves of the tumor volume of each group of mice over time indicate that the sensitizing film for radioactive stents in Examples 4 and 5, when combined with radiotherapy, can significantly enhance the effect of radiotherapy, specifically manifested in significant inhibition of tumor growth.

[0079] During the application process, corresponding adjustments can be made as needed. The cisplatin in step S1 of Example 1 can be replaced with any one or more combinations of carboplatin, oxaliplatin, lobaplatin, nedaplatin or heptaplatin; the thickness of the first film layer (10-30 μm) can be changed according to the support performance requirements of the radioactive stent, and the thickness of the second film layer (30-50 μm) can be set according to the number of days of radiotherapy; the mass fraction (15-20%) of PLGA75:25 with a molecular weight of 100k and the mass fraction (1-10%) of the tetravalent platinum complex modified by polyarginine-PLGA in solution C of step S4 can be adjusted according to the patient's condition.

Claims

1. A method for preparing a sensitizing film for a radioactive stent, the characteristics of which include the following Preparation method: S1 Oxidation of platinum radiosensitizers: The platinum radiosensitizer is dissolved in 30wt% hydrogen peroxide with a concentration of 0.15-0.3mol / L, heated to 60-80°C in a dark environment, stirred for 1-4h, cooled to 0-5°C, and then cooled for 1-3h, filtered under reduced pressure to obtain pale yellow crystals, and then washed with water, ethanol and ether respectively to obtain a tetravalent platinum dihydroxy complex; the platinum radiosensitizer is any one or more of cisplatin, carboplatin, oxaliplatin, lobaplatin, nedaplatin and heptaplatin; S2 Polyarginine modified tetravalent platinum complex: The tetravalent platinum dihydroxy complex obtained in S1 and succinic anhydride are dissolved in an organic solvent 1 at a molar ratio of 1:(3-4) and mixed, stirred under dry nitrogen protection, heated to 50-70°C, cooled to room temperature after 5-30 hours, concentrated under reduced pressure to form a light yellow liquid, and then added with dichloromethane or acetone under negative pressure for low-temperature recrystallization, filtered out the crystals and washed with cold ether to obtain a yellow crystalline tetravalent platinum dicarboxyl complex; the tetravalent platinum dicarboxyl complex and polyarginine are dissolved in an organic solvent 2 at a molar ratio of 1:1 and mixed, a carboxyl activator is added, and stirred for 5-30 hours in a dark and dry nitrogen protection to obtain a solution A containing a tetravalent platinum complex modified with polyarginine; the degree of polymerization of the polyarginine is 2-10; S3 PLGA modified tetravalent platinum complex: Dissolve PLGA with a molecular weight of 5k in organic solvent 3 at a ratio of 50:50, add solution A obtained in S2 after ultrasonic oscillation until uniformly mixed, continue ultrasonic oscillation until uniformly mixed, stir electromagnetically under the protection of light-proof and dry nitrogen, heat to 50-60°C, cool to room temperature after 5-30 hours, and evaporate under reduced pressure to form a tetravalent platinum complex modified with polyarginine-PLGA; S4 Preparation of electrospinning solution: The polyurethane with a molecular weight of 100k was placed in an organic solvent 4 and ultrasonically vibrated until mixed to obtain a solution B. The tetravalent platinum complex agglomerates modified by polyarginine-PLGA obtained in S3 and PLGA 75:25 with a molecular weight of 100k were placed in an organic solvent 5 and ultrasonically vibrated until mixed to obtain a solution C. S5 Electrospinning film preparation: The radioactive stent is moistened with solution B and then put on the receiving shaft of the electrospinning machine, the first film layer is spun on the radioactive stent with solution B, and then the second film layer is spun on the radioactive stent with solution C; the radioactive stent is taken out and dried to obtain a sensitization film for the radioactive stent.

2. The method for preparing a sensitizing film for a radioactive stent according to claim 1, characterized in that The organic solvent 1 and the organic solvent 2 in S2 and the organic solvent 3 in S3 are all N,N-dimethylacetamide (DMF) or dimethyl sulfoxide (DMSO); the organic solvent 4 and the organic solvent 5 in S4 are all dichloromethane (DCM) and N,N-dimethylacetamide (DMF), wherein the volume ratio of DCM to DMF is (1-2):

1.

3. The method for preparing a sensitizing film for a radioactive stent according to claim 1, characterized in that The concentration of the tetravalent platinum dihydroxy complex in S2 in the organic solvent 1 is 10-25 mol / L, and the concentration of the tetravalent platinum dicarboxyl complex in the organic solvent 2 is 10-25 mol / L.

4. The method for preparing a sensitizing film for a radioactive stent according to claim 1, characterized in that The carboxyl activator in S2 is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), wherein the mass ratio of EDC to NHS is (2-3):1, and the input concentration of the carboxyl activator is 40-60 g / L.

5. The method for preparing a sensitizing film for a radioactive stent according to claim 1, characterized in that The molar ratio of PLGA50:50 in S3 to the polyarginine-modified tetravalent platinum complex in S2 is (1-2):

1.

6. The method for preparing a sensitizing film for a radioactive stent according to claim 1, characterized in that In the solution B of S4, the mass fraction of the polyurethane with a molecular weight of 100k is 15-20%; the mass fraction of the PLGA75:25 with a molecular weight of 100k in the solution C is 15-20%, and the mass fraction of the tetravalent platinum complex modified by polyarginine-PLGA is 1-10%.

7. The method for preparing a sensitizing film for a radioactive stent according to claim 1, characterized in that The electrospinning process parameters in S5 are: spinning voltage 10-15 kV, flow rate 0.3-0.6 ml / h, spinning distance 5-10 cm, roller speed 400 r / min, ambient temperature 30°C, humidity 30%-40%; the thickness of the first film layer is: 10-30 μm, and the thickness of the second film layer is: 30-50 μm.

8. The method for preparing a sensitizing film for a radioactive stent according to claim 1, characterized in that The succinic anhydride in S2 can be replaced with glutaric anhydride or adipic anhydride.

9. A sensitizing film for a radioactive stent prepared by the preparation method according to claim 1, characterized in that The invention comprises a first film layer and a second film layer, wherein the second film layer contains a radiosensitizing drug component.

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