Nuclide silk film as well as preparation method and application thereof

Abstract

The invention discloses a nuclide silk film as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The nuclide silk film provided by the invention is a silk film marked with radionuclide iodine 125. The preparation method comprises the following steps: labeling mulberry silk with iodine 125, and then spinning to form a film; or the mulberry silk is woven into a film and then labeled with iodine 125. The invention discloses an application of a nuclide silk film in preparation of a tumor local radiotherapy material. The nuclide silk film can cover wounds after various cancer resection operations, gamma rays released by radioactive isotope iodine 125 on the nuclide silk film are used for killing residual cancer cells in an operation area or remaining essence and cross sections of visceral organs and cancer cells possibly remaining in adjacent vascular nerve gaps, and therefore the cancer resection effect is achieved. Therefore, the purpose of brachytherapy is achieved, tumor recurrence after cancer operation can be effectively reduced, and external radiation therapy after operation can be partially replaced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a nuclide silk film, a preparation method and application thereof. Background Art

[0002] The principle of tumor radiotherapy is to use the ionizing radiation of high-energy rays to destroy the separated DNA chains in cells during the DNA synthesis period (G phase) and kill cancer cells to treat tumors. Radiotherapy, surgical resection and chemotherapy are the main methods for treating malignant tumors. Tumor radiotherapy can directly damage DNA, and it can also indirectly damage DNA by producing charged groups (such as free radicals) in cells. When the DNA of cancer cells is damaged and cannot be repaired, the cancer cells stop dividing and eventually die. In recent years, with the rapid progress of tumor radiotherapy equipment and the implementation of technologies such as precise positioning, the efficacy of radiotherapy for malignant tumors has been continuously improved, and there is a trend of surpassing and replacing traditional surgery abroad.

[0003] Tumor radiotherapy can be basically divided into two types: external irradiation therapy (hereinafter referred to as radiotherapy) and internal drug radiotherapy (hereinafter referred to as radionuclide therapy):

[0004] External beam radiation therapy: uses various external radiation sources (x-rays, electron beams, protons, heavy ions), which are emitted by a machine outside the body. The machine rotates to a specific angle, allowing the radiation to enter the tumor tissue and kill the tumor to achieve the treatment goal. External beam radiation therapy has the following advantages: (1) A variety of radiation can be used as a radiation source for external radiation; (2) It has a history of nearly 100 years of application, has been put into clinical treatment for a long time, and has mature technology; (3) It has reliable efficacy and is effective in treating some tumors. It has become the preferred treatment method for some malignant tumors, such as nasopharyngeal carcinoma; (4) The discipline is mature. Most major hospitals have tumor radiotherapy departments with corresponding mature systems, complete sets of treatment equipment, corresponding technical processes and technicians.

[0005] At the same time, external radiotherapy has some serious disadvantages: (1) The treatment cycle is long. Due to the cyclical growth of tumors, only 5-10% of the total tumor cells are in the mitotic stage within a certain period of time. Only a certain proportion of tumor cells can be destroyed each time, and the treatment must be repeated. Usually a cycle of treatment takes more than 4 to 6 weeks. (2) The rays must penetrate multiple layers of normal tissue, causing damage to the surface and adjacent tissues. (3) Because organs such as the liver and intestines in the abdomen are more sensitive to radiation, multiple layers of organs overlap, and it is difficult to accurately locate the anatomy, the abdomen with a high incidence of malignant tumors has become a relatively forbidden area for external radiotherapy. (4) Long-term radiotherapy will inevitably lead to radiation damage to the hematopoietic system and multiple systems such as digestion and reproduction. It leads to serious toxic side effects such as pancytopenia, nausea, vomiting, and a series of physiological function impairments.

[0006] Radionuclide therapy refers to the direct use of oral or injected radionuclides, or the use of a variety of carriers (such as metal-coated particles, glass, resin microspheres, etc., or various hormones, antibody targeting proteins) combined with corresponding radionuclides, which are concentrated and retained in the tumor area by perfusion or implantation. The α, β, and γ radiation produced by the radionuclides kill malignant tumor cells at close range, thereby achieving the purpose of treatment. The advantages of radionuclide therapy are: (1) Most treatments are completed in one operation (a few can be more than two times), and the treatment time is short. For example, iridium 90 microspheres can be used to treat liver cancer as a day surgery, and patients do not even need to be hospitalized. (2) Because the radiation source is placed in the tumor, it can release radiation continuously for 24 hours. In theory, it is faster and more efficient than intermittent external radiation therapy. (3) Because the radionuclides used in internal radiation mostly release β and γ rays, the radiation range is limited, and the damage to adjacent tissues is small, it can be used more safely for radionuclide treatment of abdominal tumors. (4) Compared with radiotherapy, radionuclide therapy does not require expensive large equipment (most imported large radiotherapy equipment costs tens of millions or even hundreds of millions of yuan) and high-standard hospital conditions, and is more suitable for the national conditions of my country where most patients are in small and medium-sized cities and rural areas.

[0007] Although radionuclide therapy has obvious advantages over traditional internal radiotherapy, it has not received due attention and has not been widely used. The reason is that most radionuclides may have strong side effects such as bone marrow suppression and cell damage when entering the body. Almost only some iodine-based radionuclides can be used alone in the body, such as iodine-131 to treat thyroid diseases. Other radionuclides such as phosphorus and iridium must be combined with non-degradable materials such as glass and metal to avoid their release.

[0008] In the prior art, the research on using iodine-based nuclides and degradable materials as carriers is relatively in-depth, including the patent with the authorization announcement number CN101940797B of the inventor and the invention name "A kind of shelled nuclide labeled protein microspheres", which discloses microspheres prepared from the following weight ratios of raw materials and excipients: Na 131 I 1-5 parts, Na 125 I 1-5 parts, chitosan 300-1000 parts, sodium alginate 100-500 parts, gelatin 300-800 parts. The actual half-life of the shelled nuclide microspheres in the animal body is longer, closer to the half-life of the nuclide itself, the release of the nuclide is stable, and the degradation time of the microspheres in the body is prolonged. The inventor's patent with the authorization announcement number CN101829340B and the invention name "A pharmaceutical composition for treating solid tumors and its preparation method and use" discloses a pharmaceutical composition for treating solid tumors, which is a preparation prepared from the following raw materials in weight ratio: Na 131 I 1-4 parts, Na 1251 5 parts, can play a local radiation effect for a long time and inhibit the recurrence and metastasis of tumors. Similar iodine 131-carbon microspheres and radioactive resin microspheres have also entered preclinical research at home and abroad. These results of study have laid a very solid technical foundation for the development of radioactive medical polymer materials. But at present, these radioactive polymer materials are all concentrated on the production of microspheres, and radioactive microspheres are mainly concentrated on the treatment of liver cancer by vascular intervention technology. In the treatment field of other multiple malignant tumors, there is no corresponding polymer radioactive material research and application report. Therefore, on the mature technology basis of existing radioactive polymer materials, it is expanded to make other medical appliances, and it is used to treat more types of malignant tumors, which has become a problem urgently needed to be solved for those skilled in the art. Summary of the invention

[0009] One of the purposes of the present invention is to provide a radionuclide silk film that can be covered on the wound surface of various cancer resection operations, and utilize the gamma rays released by the radioactive isotope iodine 125 on the radionuclide silk film to eliminate the remaining cancer cells in the substance and cross-section of the surgical area or the remaining preserved organs, as well as the cancer cells that may remain in the adjacent blood vessel and nerve space, so as to achieve the purpose of close-range radiotherapy and effectively reduce tumor recurrence after cancer surgery.

[0010] The second object of the present invention is to provide a method for preparing the nuclide silk film.

[0011] The third object of the present invention is to provide applications of the nuclide silk film.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0013] The invention discloses a radionuclide silk film, which is a silk film labeled with radionuclide iodine 125.

[0014] In some embodiments of the present invention, the thickness of the nuclide silk is 0.5-1.0 mm.

[0015] In some embodiments of the present invention, the specific activity of the nuclide silk film is 5 to 15 μCi / mg, preferably 7 to 14 μCi / mg.

[0016] The invention discloses a method for preparing a radionuclide silk film, comprising the following steps: labeling mulberry silk with iodine 125 and then weaving it into a film; or weaving mulberry silk into a film and then labeling it with iodine 125.

[0017] In some embodiments of the present invention, weaving mulberry silk into a film and then labeling it with iodine-125 comprises the following steps:

[0018] Take the film spun from mulberry silk and add Na 125I aqueous solution, react to obtain a radionuclide labeled silk film.

[0019] In some embodiments of the present invention, the steps of labeling the silk with iodine-125 and then spinning it into a film include:

[0020] Take mulberry silk, add Na125I aqueous solution, react to obtain radionuclide-labeled mulberry silk, and then weave it into a film.

[0021] In some embodiments of the present invention, Na 125 The activity of the aqueous solution of I is 0.5-1.0 mCi / mL, more preferably 0.8-0.9 mCi / mL, and further preferably 0.865 mCi / mL.

[0022] In some embodiments of the present invention, the reaction temperature is room temperature, and the reaction time is 10 to 60 minutes, preferably 30 minutes.

[0023] In some embodiments of the present invention, the mulberry silk is degummed mulberry silk.

[0024] The invention discloses the use of the above-mentioned nuclide silk film in the preparation of local tumor treatment materials.

[0025] In some embodiments of the present invention, the invention is used in the preparation of materials for local radiotherapy after tumor surgery; preferably, the invention is a wound covering material for tumor resection surgery.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The invention is scientifically designed and ingeniously conceived. Silk, which has been used in clinical practice for a long time and has good safety and tissue compatibility, is used as a carrier, and then the radioactive nuclide iodine 125 with a relatively high radiation dose is labeled to produce an iodine 125 silk film, which is used as a covering for wounds after surgical operations and can achieve the purpose of close-range radiotherapy on the local wounds of surgical resection of malignant tumors.

[0028] The use of the radionuclide silk film of the present invention has the following advantages: (1) Immediately after surgery, local brachytherapy is performed on the cancer resection wound surface, which can timely and efficiently destroy the tumor cells remaining during the operation due to various reasons, and prevent recurrence; it can organically combine surgical resection and internal radiotherapy, and significantly improve the survival rate of cancer patients. (In my country, the recurrence rate after cancer surgery is very high, averaging 40-60%, and individual cancers such as pancreatic cancer and ovarian cancer can reach 90%. The residual cancer cells after cancer surgery are the main cause of recurrence. Therefore, postoperative radiotherapy is an important measure to prevent recurrence.) (2) Partially or completely replace postoperative external radiotherapy. In the prior art, some malignant tumors such as esophageal cancer, thyroid cancer, breast cancer, etc. that are routinely treated with external radiotherapy after surgery can be partially exempted from postoperative radiotherapy by this method, reducing the interference of postoperative radiotherapy on the patient's life and reducing the corresponding medical expenses. For abdominal malignant tumors that cannot be treated with radiotherapy, the radionuclide silk film of the present invention is the only measure to replace postoperative radiotherapy. (3) The silk used in the present invention is a mature natural polymer medical material that has been used for many years and has good tissue compatibility and biodegradability. At the same time, it covers the wound surface and also has auxiliary functions such as wound hemostasis, adsorption and sustained release of anticancer drugs.

[0029] The iodine-125 radionuclide silk film of the present invention is applied in tumor surgery to form an optimal comprehensive treatment model of surgery plus intraoperative close-range radiotherapy; it is expected to significantly reduce residual cancer cells after surgery, increase the patient's postoperative survival rate, and bring good social benefits.

[0030] At the same time, the iodine 125 nuclide silk film of the present invention can be applied to most malignant tumor removal surgeries and has universal applicability. There are nearly 3 million cancer surgeries in the country every year. Even if 10% of the surgeries can use the film, it will generate tens of billions of economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the results of the CCK-8 test for cell viability in Experimental Example 1;

[0032] Figure 2 This is a graph showing the results of the LDH cytotoxicity test for cell mortality in Experimental Example 2;

[0033] Figure 3 This is the Calcein AM / PI live and dead cell double staining result of Experimental Example 3;

[0034] Figure 4 This is the HE staining result of breast cancer cells in Experimental Example 4. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0036] The raw silk described in the embodiments of the present invention is mulberry silk produced in Yibin or Mianyang, Sichuan.

[0037] The degumming step of the mulberry silk in the embodiment of the present invention is a prior art, and the degumming can be carried out by a conventional method.

[0038] Example 1

[0039] This embodiment discloses a method for preparing the nuclide silk film of the present invention, which is specifically as follows:

[0040] S1. Film preparation: raw silk is taken, degummed conventionally, and then spun into film;

[0041] S2. Nuclide labeling: Take the membrane obtained in step S1, cut it into several circular membrane pieces with a diameter of 1.5 cm, add a radioactive nuclide solution, and perform nuclide labeling, which specifically includes the following steps:

[0042] S21. Add the silk woven circular membrane of step S1 to the EP tube respectively;

[0043] S22. Add a certain amount of Na to the EP tube according to the experimental formula design. 125 I solution, for specific data, see Data Table 1;

[0044] S23. According to the experimental formula design, a certain amount of PBS solution (0.3 mol / L, PH = 7.46) was added to the EP tube;

[0045] S24. Add a certain amount of deionized water solution according to the experimental formula design;

[0046] S25. Shake and react at room temperature for 30 minutes;

[0047] S26. Collect the reaction solution, wash the sample twice with pure water, 1 mL each time, and collect all the liquid.

[0048] S27. Measure the activity of the silk membrane and the cleaning solution (reaction solution + first cleaning solution + second cleaning solution) according to conventional methods.

[0049] S28. Analysis of experimental data is shown in the table below.

[0050] Table 1

[0051]

[0052] It can be seen from Table 1 that after the above treatment, the iodine-125 labeling rate of the iodine-125-nuclide silk film is 33±2.11%, and the specific activity after labeling is 7.77-14.08 μCi / mg.

[0053] Anti-cancer experimental series

[0054] Test Example 1

[0055] This test example discloses a CCK-8 cell viability test of the nuclide silk film of the present invention.

[0056] 1. Experimental Grouping

[0057] Control group: only culture medium was added.

[0058] Film group: The culture medium is the same as the control group. In addition, a round iodine 125-silk sheet with a diameter of 1.5 cm, which is the same size as the culture well, is placed one-to-one under the culture well and close to it. (The same below). The diameter of this test example is

[0059] The 1.5 cm round iodine-125-silk sheet was prepared according to the preparation method of the nuclide silk film No. 1 in Table 1 in Example 1.

[0060] 2. Experimental Methods

[0061] (1) Human breast cancer cells (MDA-MB-231 cells) in the logarithmic growth phase were digested with trypsin and then the cells were counted;

[0062] (2) Cells were inoculated in 24-well culture plates, with 3 replicate wells in each group and approximately 2×10 cells per well. 4 When the cells are growing well, they are grouped and treated according to the above experimental plan (an iodine 125-silk film is placed under each well of the treatment group).

[0063] The control group and the film group were treated as follows:

[0064] (3) According to the instructions of the CCK-8 kit, 500 μL of the prepared CCK-8 solution (CCK-8 reagent accounts for 10% of the total volume) was added to each well, and 3 blank replicate wells were set up. The cells were incubated at 37°C in the dark for 1 h.

[0065] (4) After the incubation, the 24-well culture plate was taken out and shaken to mix well. The optical density (OD value) of each well at 450 nm was measured using a microplate reader and the cell survival rate was calculated.

[0066] 3. Experimental results

[0067] The experimental results are as attached Figure 1 shown.

[0068] Depend on Figure 1 It can be seen that the cell viability of the breast cancer experimental group (iodine 25-silk film treatment group) was only 53%; while the cell viability of the control group exceeded 150%, indicating that under the same culture conditions, the untreated cancer cells continued to grow, while the cell viability of the treatment group decreased significantly. Comparing the two, the difference was very significant.

[0069] Test Example 2

[0070] This test example discloses an LDH cytotoxicity test of the nuclide silk film of the present invention.

[0071] 1. Experimental Grouping

[0072] Control group: culture medium only.

[0073] Film group: The culture medium is the same as the control group. In addition, a round iodine 125-silk sheet with a diameter of 1.5 cm, which is the same size as the culture well, is placed one-to-one under the culture well and closely attached. The iodine 125-silk film used in the film group is the film No. 1 in Table 1 of Example 1 (the same as Test Example 1).

[0074] 2. Experimental Methods

[0075] (1) Human breast cancer cells (MDA-MB-231 cells) in the logarithmic growth phase were digested with trypsin and then counted. The cells were inoculated in a 24-well culture plate, with 3 replicate wells in each group and approximately 2×10 cells per well. 4 When the cells are in good growth condition, they are divided into experimental group and control group according to the above experimental scheme.

[0076] (2) One hour before the scheduled detection time, remove the cell culture plate from the cell culture incubator and add the LDH release reagent provided by the kit to the "sample maximum enzyme activity control well" at an amount of 10% of the original culture medium volume. After adding the LDH release reagent, repeatedly pipette several times to mix, and then continue to incubate in the cell culture incubator at 37°C in the dark for 1 hour;

[0077] (3) When the detection time is reached, the supernatant of each well is aspirated and centrifuged at 400 g for 5 min. After centrifugation, 120 μL of supernatant from each well is transferred to a new 96-well plate;

[0078] (4) According to the instructions of the LDH kit, add 60 μL of the prepared LDH working solution to each well and incubate at room temperature in the dark for 30 min;

[0079] (5) After incubation, use a microplate reader to measure the OD value of each well at 490 nm and calculate the cell death rate.

[0080] Cytotoxicity or mortality (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100%

[0081] 3. Experimental results

[0082] The experimental results are as attached Figure 2 shown.

[0083] Depend on Figure 2 It can be seen that the cell death rate in the experimental group was as high as 14%, while that in the control group was less than 5%. The difference between the two groups was significant.

[0084] Test Example 3

[0085] This test example discloses a Calcein AM / PI live-dead cell double staining test of the nuclide silk film of the present invention.

[0086] 1. Experimental Grouping

[0087] Control group: culture medium only.

[0088] Film group: The culture medium is the same as the control group. In addition, a round iodine 125-silk sheet with a diameter of 1.5 cm, which is the same size as the culture well, is placed one-to-one under the culture well and closely attached. The iodine 125-silk film used in the film group is the film No. 1 in Table 1 of Example 1 (the same as Test Example 1).

[0089] 2. Experimental Methods

[0090] 1. Experimental Procedure

[0091] (1) MKN45 or MDA-MB-231 cells in the logarithmic growth phase were digested with trypsin and then the cells were counted;

[0092] (2) Cells were seeded in a 24-well culture plate with a cover glass, with approximately 2 × 10 cells per well. 4 When the cells were growing well, they were divided into groups and treated according to the above experimental scheme (control group and film group).

[0093] (3) Discard the supernatant and prepare the staining working solution according to the instructions of the Calcein AM / PI live-dead double staining kit. Add 250 μL of the working solution to each well and incubate at 4°C in the dark for 30 min.

[0094] (4) After the incubation, observe and take pictures under a fluorescence microscope. (Calcein is green fluorescence, Ex / Em=494nm / 517nm; PI is red fluorescence, Ex / Em=535nm / 617nm).

[0095] The principle of the kit is that the two probes can detect intracellular esterase activity and cell membrane integrity respectively, thereby reflecting cell activity and cytotoxicity. Calcein AM stains live cells and shows green fluorescence, while propidium iodide (PI) stains dead cells and shows red fluorescence.

[0096] 3. Experimental results

[0097] The experimental results are as attached Figure 3 shown.

[0098] By the attached Figure 3 It can be seen that the cells in the control group did not die, and the cells showed uniform green fluorescence; the red fluorescence staining intensity of the cells in the breast cancer film group was significantly enhanced, the cell membrane was severely damaged, and the number of cell deaths was large.

[0099] Test Example 4

[0100] This test example discloses the HE staining and cytopathological observation test of the nuclide silk film of the present invention. The specific operation is as follows: the culture fluid is aspirated from each culture well of the two groups of test example 1, processed by conventional HE staining method, and then observed using an inverted microscope.

[0101] The results are attached Figure 4 The results showed that the cells in the control group were normal in size, with clear boundaries, round or oval nuclei, and clear nuclear membranes and nucleoli; the cells in the film experimental group were basically normal in size, with unclear boundaries, and some nuclear membrane boundaries were unclear, with nuclear condensation and dissolution.

[0102] Summary: The above anti-cancer results confirm the significant inhibition and killing effect of iodine-125 silk film on cancer cells from different experimental methods. These detection methods are internationally recognized standard experiments for anti-cancer effects. Currently, only the experimental effects after 7 days of treatment are shown. The half-life of iodine-125 is 60 days, and the biological effective half-life is 41 days. Therefore, in actual medical work, the subsequent long-term anti-cancer effect will hopefully completely eliminate the local cancer cells that may remain. Preventing tumor recurrence after surgery

[0103] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims

1. A nuclide silk film, characterized in that: It is a silk film labeled with the radioactive nuclide iodine 125.

2. The nuclide silk film according to claim 1, characterized in that: Its specific activity is 5 to 15 μCi / mg; preferably 7 to 14 μCi / mg; Or / and film thickness is 0.5-1.0mm.

3. The method for preparing a nuclide silk film according to claim 1 or 2, characterized in that: The method comprises the following steps: labeling the mulberry silk with iodine 125 and then weaving the silk into a film; or weaving the mulberry silk into a film and then labeling the silk with iodine 125.

4. The method for preparing a nuclide silk film according to claim 3, characterized in that: The steps of weaving silk into a film and then labeling it with iodine 125 include: Take the film spun from mulberry silk and add Na 125 I aqueous solution, react to obtain a radionuclide labeled silk film.

5. The method for preparing a nuclide silk film according to claim 3, characterized in that: The steps of weaving the silk into a film after labeling it with iodine 125 include: Take mulberry silk and add Na 125 I aqueous solution, react to obtain radionuclide labeled mulberry silk, and then weave into a film.

6. The method for preparing a nuclide silk film according to claim 4 or 5, characterized in that: Na 125 The activity of the aqueous solution of I is 0.5-1.0 mCi / mL, more preferably 0.8-0.9 mCi / mL, and further preferably 0.865 mCi / mL.

7. The method for preparing a nuclide silk film according to claim 4 or 5, characterized in that: The reaction temperature is room temperature, and the reaction time is 10 to 60 minutes, preferably 30 minutes.

8. The method for preparing a nuclide silk film according to claim 3, characterized in that: The mulberry silk is degummed mulberry silk.

9. Use of the nuclide silk film according to claim 1 or 2 in preparing local tumor treatment materials.

10. The use according to claim 9, characterized in that: Application in the preparation of materials for local radiotherapy after tumor surgery; preferably wound covering materials for tumor resection surgery.

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