WO2018145588A1 - 细胞囊泡制剂与低剂量放疗联合在制备抗肿瘤药物中的应用 - Google Patents

细胞囊泡制剂与低剂量放疗联合在制备抗肿瘤药物中的应用 Download PDF

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WO2018145588A1
WO2018145588A1 PCT/CN2018/074522 CN2018074522W WO2018145588A1 WO 2018145588 A1 WO2018145588 A1 WO 2018145588A1 CN 2018074522 W CN2018074522 W CN 2018074522W WO 2018145588 A1 WO2018145588 A1 WO 2018145588A1
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radiotherapy
tumor
group
cancer
vesicle
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黄波
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湖北盛齐安生物科技股份有限公司
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A—HUMAN NECESSITIES
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    • A61N5/00—Radiation therapy
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Definitions

  • the present invention relates to a novel application of a cell vesicle preparation.
  • Chinese patent application 20151091237.7 discloses that the chemotherapeutic drug has the effect of sensitizing the above-mentioned tumor cell-derived vesicles, reducing or reversing the drug resistance of the tumor cells, and improving the killing efficacy of the cell vesicles on the tumor cells.
  • Radiotherapy is a treatment that uses a variety of different energy rays to illuminate a tumor to inhibit and kill cancer cells.
  • Radiation therapy has the potential to enhance anti-tumor immunity. The reason is that radiation therapy may cause tumor antigens to be released from tumor cells that are about to die, promote tumor antigen presentation and tumor-specific T cell activation, and contribute to systemic anti-tumor immunity.
  • radiation therapy was initially considered to be immunosuppressive due to its damage to immune cells after irradiation. It should be noted that radiation therapy can also promote immunosuppression, leading to malignant domestication of tumor infiltrating macrophages and tumor-regulated cell-mediated immunosuppression. The dose of radiation therapy is a key factor in reconciling this apparent contradiction.
  • radiotherapy used clinically is greatly limited.
  • a single dose of conventional radiotherapy is 1.8-2.0 Gy, 5 times a week.
  • the total dose of radiotherapy it is divided into high-dose radiotherapy, medium-dose radiotherapy and low-dose radiotherapy.
  • High-dose radiotherapy is used for the precise treatment of tumors. It requires precise positioning and dosimetry of the lesions. The operation is complicated and difficult.
  • Low-dose radiotherapy agents have small side effects but are susceptible to immune tolerance.
  • the chemotherapeutic drug / radiotherapy dose in the conventional dose or even lower than the conventional dosage on the tumor cells, especially tumor regenerative cells, a strong killing effect, improve the tumor
  • the microenvironment repairs the immune system and enhances the body's anti-tumor immune response, thereby achieving tumor control.
  • the cell vesicle preparation of the present invention is a tumor cell derived from apoptosis and is coated with a chemotherapeutic drug, also called a drug-loaded vesicle; the low-dose radiotherapy referred to in the present invention is lower than the treatment. Radiation therapy of the total dose of tumor radiation.
  • the tumor cell vesicle (carrier) for encapsulating the chemotherapeutic drug is derived from apoptosis of the tumor cell, and the chemotherapeutic drug encapsulated in the tumor cell vesicle described in the present invention may be before the application date of the present invention.
  • a chemotherapeutic drug that is clinically applied in the future may also be an active ingredient (which may or may not contain a pharmaceutical excipient) in a chemotherapeutic drug that is clinically applied before and/or after the application date of the present invention, that is, the cell of the present invention is prepared.
  • an active ingredient in a clinical drug composition for treating a tumor can be used, and various commercially available drugs which have been clinically applied to tumor treatment can be directly used. Therefore, the dose of the drug involved in the present invention should be understood as the amount of the active ingredient of the drug.
  • the specific chemotherapeutic drug can be a chemotherapeutic drug for clinical treatment of various tumors, such as a chemotherapeutic drug for treating ovarian cancer, breast cancer, lung cancer, stomach cancer, colon cancer, liver cancer, bladder cancer or rectal cancer, and may be a single chemotherapy drug or A combination of multiple chemotherapy drugs.
  • the preferred chemotherapeutic agent of the present invention is cisplatin, methotrexate or doxorubicin. The most preferred solution is to encapsulate the chemotherapeutic drug cisplatin into tumor cell vesicles to prepare drug-loaded vesicles.
  • the preparation method of the vesicles of the present invention can be referred to the Chinese patent ZL201110241369.8, which is incorporated herein by reference.
  • the low-dose radiotherapy according to the present invention means that when the tumor is subjected to radiotherapy alone at the dose of radiation, the radiotherapy fails to exhibit the desired therapeutic effect, or the dose is not apparent to the tumor. Killing effect.
  • a preferred single therapeutic dose may be 2 Gy, which may be administered to the patient in a single or divided dose, and the total radiation dose administered may be determined based on the weight of the subject and the particular condition being treated.
  • the tumor suitable for treatment using the method of the present invention is preferably ovarian cancer, breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer, bladder cancer or rectal cancer, and more preferably the tumor is lung cancer, colon cancer or liver cancer.
  • 8 x 10 6 cell vesicles may be included in the unit preparation of the cell vesicle preparation, and the drug-loaded vesicles are preferably administered intravenously.
  • the inventors first verified the effects of different radiation doses on H22 liver cancer model mice, and found that compared with the control group, 4Gy radiotherapy mice did not differ in body weight and survival rate, and mouse hair was supple and behavioral. Normal and safe.
  • the present invention also isolates tumor-regenerating cells of tumor-bearing mice, and verifies the effective killing effect of vesicles combined with low-dose radiotherapy agents on tumor regenerative cells, indicating that the combined method of the present invention has the potential to cure tumors.
  • the present invention also studied the therapeutic effects of vesicles loaded with different chemotherapeutic drugs in combination with low-dose radiotherapy, and found that they are also effective in killing tumor cells.
  • FIG. 1 Effect of radiotherapy on body weight and survival of mice
  • 1A effects on mouse body weight
  • 1B effects on survival of mice
  • Figure 2 The killing effect of radiotherapy on tumor cells
  • Figure 3 Effect of vesicle combined with radiotherapy on liver cancer
  • 3A inhibition of mouse tumors
  • 3B effects on survival of mice
  • Figure 4 Effect of vesicle combined with radiotherapy on colon cancer
  • 4A inhibition of mouse tumors
  • 4B effects on survival of mice
  • Figure 5 Comparison of vesicle combined with radiotherapy for lung cancer
  • 5A inhibition of mouse tumors
  • 5B effects on survival of mice
  • 6A effects on mouse body weight
  • 6B effects on kidney function in mice
  • 6C effects on liver function in mice
  • Figure 7A Three-dimensional fibrin glue of tumor regenerative cell clonal populations under different treatment treatments
  • Figure 7B-C Effect of different treatment treatments on the number and volume of tumor regenerative cell clonal populations
  • 7B number of clonal populations
  • 7C volume of clonal populations
  • Figure 8 Effect of vesicle combined with radiotherapy on the retention time of drugs in tumor cells
  • Figure 9 Vesicle combined with radiotherapy for tumor-bearing mice to promote benign domestication of macrophages in vivo;
  • Figure 10 Killing of H22 hepatoma cell lines by tumor vesicles combined with radiotherapy with different drugs
  • 10A cisplatin-bearing tumor vesicles
  • 10B methotrexate-bearing tumor vesicles
  • 10C doxorubicin-containing tumor vesicles.
  • Figure 11 Comparison of the killing effect of the inventive protocol and other common protocols on tumor cells.
  • Mouse liver cancer cell line H22 (BALB/c genetic background), mouse colon cancer cell line CT26 (BALB/c genetic background), mouse lung cancer cell line Lewis (C57BL/6 genetic background), can be preserved from Chinese typical species Center CTCCC purchase.
  • mice C57BL/6 mice, BALB/c mice, were purchased from the Medical Laboratory Animal Center of Wuhan University. The age was 5-6 weeks and the body weight was about 18 grams.
  • the vesicles of the present invention are cell vesicle preparations described in Chinese Patent No. 201110241369.8, unless otherwise specified, and vesicles not coated with chemotherapeutic drugs are called empty vesicles.
  • Low-dose radiation therapy refers to radiation therapy that is lower than the total dose of radiation for treating the tumor, and the radiation therapy has no significant killing effect on the tumor alone; specifically, the total dose of the above experimental animals is 4 Gy of gamma radiation.
  • UV device is a conventional biological safety cabinet; carbon dioxide constant temperature incubator; cisplatin; 3 ⁇ m counting microspheres; flow cytometry.
  • H22 mouse hepatoma cells were cultured in RPMI-1640 cell culture medium to a cell volume of 2 ⁇ 10 7 /ml; H22 mouse liver cancer cells were subjected to ultraviolet irradiation for 60 minutes; and cisplatin at a final concentration of 200 ⁇ g/ml was added. Place in the incubator and continue to culture.
  • the vesicles of the above apoptotic tumor cells were collected by centrifugation, including: first centrifuging at 1300 rpm and 5000 rpm for 10 minutes, taking the supernatant, and then centrifuging at 14,000 g for 1 minute to remove cell debris, and taking the supernatant after centrifugation. After centrifugation at 14,000 g for 1 hour, the precipitate was collected and resuspended in PBS to obtain cisplatin-containing vesicles (Cis-MPs) produced by apoptosis of H22 mouse liver cancer cells.
  • Cis-MPs cisplatin-containing vesicles
  • the collected vesicles were mixed with a known concentration of 3 ⁇ m count microspheres in equal volumes, and counted by a flow meter to obtain a ratio of vesicles to microspheres.
  • Concentration of vesicles concentration of microspheres ⁇ ratio of vesicles to microspheres.
  • H22 mouse liver cancer cells H22 mouse liver cancer cells; experimental BALB/c mice were purchased from Wuhan University Medical Laboratory Animal Center, weighing 18-20 g; optical microscope; counting plate; mouse holder; anti-radiation lead plate; ⁇ -radiation precision treatment instrument (EKAKTA, England); Farmer2570 reference grade dosimeter.
  • the H22 cells were diluted into a cell suspension with PBS, counted under a microscope using a cell counting plate, and diluted to 1 ⁇ 10 6 /ml, and 200 ⁇ l of the cell suspension was subcutaneously injected into the right abdomen of BALB/c mice.
  • the tumor size is as long as 8 ⁇ 8mm, which takes about 10 days.
  • mice 32 subcutaneous tumor-forming BALB/c mice were randomly divided into 4 groups: blank group, control group; radiotherapy group, 3 groups, 4Gy radiotherapy group; 10Gy radiotherapy group; 20Gy radiotherapy group (referring to radiotherapy used during treatment) Total dose).
  • control group does not use radiotherapy treatment
  • the radiotherapy group irradiated the tumor site with ⁇ -rays with a total dose of 4 Gy, 10 Gy, and 20 Gy, respectively. Specifically, it is 1 course every 3 days, lasting 2 courses, that is, a total of 6 days, and the irradiation doses of one course are 2Gy, 5Gy and 10Gy respectively.
  • the specific procedure of radiotherapy is to use a mouse fixator to fix the subcutaneous tumor-forming BALB/c mice, shield the normal tissues of the mice with lead plates, allow the tumor sites to be exposed, and open the aperture of the radiation source to expose the entire tumor.
  • the lowest level. Irradiation was performed at room temperature using a gamma-radiation treatment by a precision therapeutic apparatus at a dose rate of about 500 cGy/min, and the total irradiation dose was adjusted according to time.
  • the 4Gy radiotherapy group needs to be irradiated twice every 3 days for 24s each time; the 10Gy radiotherapy group needs to be irradiated twice every 3 days for 1 minute; the 20Gy radiotherapy group needs to be irradiated twice every 3 days for 2 minutes each time;
  • the gamma-radiation precision therapy device was dose-calibrated with the Farmer 2570 reference meter before each use.
  • mice From the date of radiotherapy, the body weight of each group of mice was weighed daily, and the living state and survival time of the mice were observed and continued until the 30th day.
  • Fig. 1A and Fig. 1B there was no difference in body weight and survival rate between mice treated with a single dose of 2 Gy and a total dose of as low as 4 Gy compared to control mice not treated with radiotherapy. And the mouse hair is supple and has no bow and back.
  • the high-dose radiotherapy group 10Gy radiotherapy group and 20Gy radiotherapy group, the body weight gradually decreased and the survival time was shortened.
  • Mouse liver cancer cell H22 cells Mouse liver cancer cell H22 cells; experimental BALB/c mice were purchased from Wuhan University Medical Laboratory Animal Center, weighing 18-20 g; mouse fixator; anti-radiation lead plate; ⁇ -radiation precision therapeutic instrument (EKAKTA, England) ; flow cytometry (BD); apoptosis detection kit; Farmer2570 reference grade dosimeter. 2, the experimental steps
  • the control group did not receive radiotherapy.
  • the subcutaneous tumor cells were isolated 24 hours after chemotherapy for apoptosis ratio analysis.
  • the specific operation was as follows: the subcutaneous tumor tissue was separated, ground and dispersed, and 0.25% trypsin was added for 2 min, the digestion was terminated with fetal bovine serum, washed with PBS and resuspended into a single cell suspension. Conventional apoptosis detection staining was performed using PI and FITC-annexin V antibodies in the apoptosis kit, and the proportion of apoptotic cells was detected by flow cytometry.
  • mice in the control group were treated as above.
  • mice treated with 4Gy radiotherapy did not promote apoptosis of tumor cells in subcutaneous tumors compared to control mice.
  • Mouse liver cancer cell H22 cells were purchased from Wuhan University Medical Laboratory Animal Center, body weight 18-20 g; mouse holder; anti-radiation lead plate; ⁇ -radiation precision treatment instrument (EKAKTA, England ); Farmer2570 reference grade dosimeter.
  • mice 32 subcutaneous tumor-forming BALB/c mice were randomly divided into 4 groups: blank group, control group; radiotherapy group; vesicle group; vesicle combined with radiotherapy group.
  • control group was not treated with radiotherapy; an equal volume of PBS was injected at the same time point when the vesicle group was injected with vesicles.
  • the total dose of radiotherapy in the vesicle combined with radiotherapy group was 4Gy, the single radiotherapy dose was 2Gy, and the radiotherapy was completed every 3 days for 2 courses.
  • 2 ⁇ 10 6 Cis-MPs were intravenously injected 24 hours after the end of each radiotherapy.
  • the third injection was performed 72 hours after the end of the second radiotherapy, and the fourth injection was performed 72 hours after the third injection of Cis-MPs.
  • the treatment time for the entire vesicle combined with radiotherapy group was 12 days.
  • the radiotherapy group was treated in the same manner as the 4Gy radiotherapy group of Example 3. After 24 hours of the end of each radiotherapy, an equal volume of PBS was administered intravenously and in combination with the vesicle combined with radiotherapy. The second injection was performed 72 hours after the second injection of PBS. The fourth injection was performed 72 hours after the second injection of PBS;
  • the vesicle group was not treated with radiotherapy, and the same amount of Cis-MPs was injected intravenously with the vesicle combined with radiotherapy group;
  • the tumor growth of the mice in the radiotherapy group, the vesicle group, and the vesicle combined with the radiotherapy group was inhibited, and the survival time was prolonged.
  • the therapeutic effect of the vesicle combined with radiotherapy group was better than that of mice treated with vesicles or radiotherapy alone.
  • the tumor volume was atrophied.
  • the survival rate of the mice remained at 87.5%, while the other three groups died. .
  • Mouse colon cancer cell line CT26 (BALB/c genetic background); experimental BALB/c mice were purchased from Wuhan University Medical Laboratory Animal Center, weighing 18-20 g; mouse holder; anti-radiation lead plate; ⁇ -radiation Precision therapy device (EKAKTA, England); Farmer2570 reference grade dosimeter.
  • CT26 was cultured in RPMI-1640 cell culture medium containing 10% serum.
  • CT26 cells were diluted into a cell suspension with PBS, counted and diluted to 1 ⁇ 10 6 cells/ml, and 200 ⁇ l of the cell suspension was subcutaneously injected into the right abdomen of BALB/c mice until the tumor size was as long as 8 ⁇ 8 mm.
  • the tumor growth was inhibited and the survival rate was prolonged in the radiotherapy group, the vesicle group, and the vesicle combined with the radiotherapy group.
  • the therapeutic effect of the vesicle combined with radiotherapy group was better than that of mice treated with vesicles or radiotherapy alone.
  • the tumor growth rate was significantly slowed down.
  • the survival rate of the mice remained at 75%, while the other three groups died.
  • Mouse lung cancer cell line Lewis cells (C57BL genetic background); experimental C57BL mice were purchased from Wuhan University Medical Laboratory Animal Center, weighing 18-20 g; mouse holder; anti-radiation lead plate; ⁇ -radiation precision treatment instrument ( EKAKTA, England); Farmer2570 reference grade dosimeter.
  • Lewis cells were diluted with PBS into cell suspension, counted and diluted to 1 ⁇ 10 6 cells/ml, and 200 ⁇ l of cell suspension was subcutaneously injected into the right abdomen of C57BL mice until the tumor size was as long as 8 ⁇ 8 mm.
  • the tumor growth was inhibited in the radiotherapy group, the vesicle group, and the vesicle combined with the radiotherapy group, and the survival rate was prolonged.
  • the therapeutic effect of the vesicle combined with radiotherapy group was better than that of mice treated with vesicles or low-dose radiotherapy alone.
  • the tumor growth rate was significantly slowed down.
  • the survival rate of the mice remained at 75%, while the other three groups died.
  • mice H22 mouse liver cancer cells; experimental BALB/c mice were purchased from Wuhan University Medical Laboratory Animal Center, weighing 18-20 g; mouse fixator; anti-radiation lead plate; ⁇ -radiation precision therapeutic instrument (EKAKTA, England); Farmer2570 reference grade dosimeter.
  • mice 32 subcutaneous tumor-forming BALB/c mice were randomly divided into 4 groups: blank group, control group; radiotherapy group; vesicle group; vesicle combined with radiotherapy group.
  • mice were weighed daily for 24 days. On the 24th day after the treatment, the mice were intraperitoneally injected with 100 ⁇ l of 1% sodium pentobarbital for general anesthesia, the mice were fixed, the abdominal cavity was opened, and the peripheral blood was collected by the abdominal aorta to be placed in the coagulation tube. The serum was separated by centrifugation at 2000 rpm for 8 min, and the content of creatinine and alanine aminotransferase was detected by an automatic biochemical analyzer. The degree of damage to liver and kidney function was evaluated.
  • the body weights of the mice in the radiotherapy group, the vesicle group, and the vesicle combined with the radiotherapy group did not change significantly during the treatment compared with the control group mice (Fig. 6A).
  • the contents of creatinine and alanine aminotransferase were all within the normal range, and there was no significant difference from the control group.
  • H22 mouse liver cancer cells H22 mouse liver cancer cells; optical microscopy; experimental BALB/c mice purchased from Wuhan University Medical Laboratory Animal Center, weight 18-20 g; radiation-proof lead plate; ⁇ -radiation precision therapeutic instrument (EKAKTA, England); Farmer2570 reference Dose meter.
  • mice 32 subcutaneous tumor-forming mice were randomly divided into 4 groups: blank group, control group; radiotherapy group; vesicle group; vesicle combined with radiotherapy group.
  • the subcutaneous tumors of the tumor-bearing mice of each group were isolated, ground and dispersed, and digested with 0.25% trypsin for 2 min. The digestion was terminated with fetal bovine serum, washed with PBS and resuspended into a single cell suspension. Counting, the tumor cells were cultured with three-dimensional fibrin glue, and as time progressed, only the tumor regenerative cells survived the hardness environment of the three-dimensional fibrin glue.
  • Three-dimensional fibrin glue culture method is a three-dimensional fibrin glue culture method
  • the single cell suspension was resuspended in RPMI-1640 medium containing 10% fetal calf serum, and the cell concentration was adjusted to 10 4 /ml.
  • Fibrinogen was diluted to 2 mg/ml with T7 buffer (50 mM Tris, pH 7.4, 150 mM NaCl).
  • the fibrinogen solution and the cell solution were mixed 1:1.
  • the final concentration of fibrinogen in the mixture was 1 mg/ml and the cell concentration was 5000/ml.
  • 250 ⁇ l of the mixture was mixed into a 24-well plate and mixed well with 5 ⁇ l of thrombin.
  • the culture was transferred to a 37 ° C incubator for 30 min.
  • 1 ml of RPMI-1640 medium containing 10% fetal bovine serum and the corresponding concentration of antibiotics were added to continue the culture.
  • the tumor regenerating cells form a cell population.
  • the tumor regenerative cell clones formed by the tumor cells of the vesicle combined with the radiotherapy group had the smallest volume and the least number (7B) compared with the control group, the radiotherapy group and the vesicle group.
  • Tumor regenerative cells grew the slowest in three-dimensional fibrin glue, which was significantly different from the other three groups.
  • Vesicle combined with low-dose radiotherapy inhibits tumor growth by killing tumor-regenerating cells.
  • doxorubicin Dox, chemotherapeutic drug with red fluorescence
  • ⁇ -radiation precision therapeutic apparatus EKAKTA, England
  • Farmer2570 reference dose Instrument flow cytometry.
  • H22 mouse hepatoma cells were cultured in RPMI-1640 cell culture medium containing 10% serum to achieve a cell volume of 2 ⁇ 10 7 /ml; H22 mouse liver cancer cells were irradiated with ultraviolet light for 60 minutes; and the final 100 ⁇ g/ml Amygdalus was added. Prime (Dox). Incubate in an incubator and incubate.
  • the cell vesicles of the above apoptotic tumor cells were collected by centrifugation, including: centrifugation at 1300 rpm and 5000 rpm for 10 minutes, followed by centrifugation at 14,000 g for 1 minute to remove cell debris, and the supernatant after centrifugation was again 14,000 g. After centrifugation for 1 hour, the precipitate was collected to obtain doxorubicin-containing vesicles (H22-Dox-MPs) produced by apoptosis of H22 mouse liver cancer cells. The PBS was resuspended and counted. The counting method is the same as in the first embodiment.
  • CT26 mouse colon cancer cells and Lewis mice lung cancer cell-derived vesicle-encapsulated doxorubicin treatments were the same as above, and the doxorubicin-containing vesicles (CT26-Dox-MPs) produced by apoptosis of CT26 mouse colon cancer cells were obtained. And doxorubicin-containing vesicles (Lewis-Dox-MPs) produced by Lewis mouse lung cancer cell apoptosis.
  • H22 mouse hepatoma cells, CT26 mouse colon cancer cells, and Lewis mouse lung cancer cells were cultured in RPMI-1640 cell culture medium, counted, and seeded into 24-well plates at 5 ⁇ 10 4 /ml, 1 ml per well. .
  • Various cell lines were divided into 4 groups, namely, control group, radiotherapy group, vesicle group and vesicle combined radiotherapy group.
  • Radiotherapy group 4Gy was used for radiotherapy. The specific operation was to irradiate the cells with ⁇ -radiation precision therapy instrument, and 2Gy was irradiated twice, 24s each time, 24h apart.
  • Vesicle group Doxorubicin-containing vesicles (Dox-MPs) were added to a 24-well plate at a ratio of vesicles to a cell ratio of 5:1.
  • H22 cells, CT26 cells and Lewis cells were treated with radiotherapy at a total dose of 4 Gy, specifically for 2 times, 24 s each time, 24 hours apart. Then, H22-Dox-MPs, CT26-Dox-MPs, and Lewis-Dox-MPs were separately added, and 24-well plates were added at a ratio of 5:1 vesicles.
  • the average fluorescence intensity of the cells in the vesicle combined with the radiotherapy group was the slowest compared to the control group, the radiotherapy group, and the vesicle group.
  • Mouse liver cancer cell H22 cells Mouse liver cancer cell H22 cells; experimental BALB/c mice were purchased from Wuhan University Medical Laboratory Animal Center, weighing 18-20 g; mouse fixator; anti-radiation lead plate; ⁇ -radiation precision therapeutic instrument (EKAKTA, England) Flow cytometry (BD); Farmer 2570 reference grade dosimeter; mouse antibodies anti-CD11b, anti-F4/80, anti-IL-1 ⁇ and anti-IL-10 were purchased from Sigma.
  • mice On the 13th day, each group of mice was dissected, and the lymphoid tissues of the spleen and the tumor infiltrating site were separated, and the function of macrophage was analyzed.
  • the spleen and tumor infiltrated tissue were ground and dispersed into a single cell suspension, the cells were counted, the cell concentration was adjusted to 1 ⁇ 10 6 /100 ⁇ l, and 1 ⁇ l of CD11b and F4/80 antibody were added in the dark, shaken and mixed, and 4°C was avoided. The light was incubated for 30 min. After the incubation, 850 ⁇ l of PBS was added to each tube, shaken and mixed, and centrifuged at 500 g for 5 min.
  • the proportion of IL-1 ⁇ -secreting macrophages in the spleen and tumor infiltrating tissues of the mice treated with the vesicle combined with the radiotherapy group was significantly increased, and inhibition was observed.
  • the proportion of IL-10 secreting macrophages in the tumor immune response was significantly reduced.
  • Vesicle combined with radiotherapy can enhance the benign acclimation of tumor infiltrating macrophages, improve tumor microenvironment and promote immune response.
  • H22 mouse liver cancer cells H22 mouse liver cancer cells; cisplatin; methotrexate; doxorubicin; gamma-radiation precision therapeutic apparatus (EKAKTA, England); Farmer2570 reference grade dosimeter.
  • H22 mouse hepatoma cells were cultured in RPMI-1640 cell culture medium containing 10% serum to achieve a cell volume of 2 ⁇ 10 7 /ml; H22 mouse liver cancer cells were irradiated with ultraviolet light for 60 minutes; Cisplatin at a final concentration of 200 ⁇ g/ml, 1 mg/ml methotrexate (MTX), and 100 ⁇ g/ml doxorubicin (Dox). Place in the incubator and continue to culture.
  • MTX methotrexate
  • Dox doxorubicin
  • the vesicles of the above apoptotic tumor cells were collected by centrifugation, including: centrifugation at 1300 rpm and 5000 rpm for 10 minutes, followed by centrifugation at 14,000 g for 1 minute to remove cell debris, and the supernatant after centrifugation was centrifuged at 14,000 g.
  • Cis-MPs cisplatin vesicles
  • MTX-MPs methotrexate vesicles
  • Dox doxorubicin-containing vesicles
  • H22 mouse hepatoma cells were cultured in RPMI-1640 cell culture medium, counted, and diluted at 5 ⁇ 10 4 /ml. Seed in a 24-well plate, 1 ml per well. They were divided into 4 groups: control group, radiotherapy group, vesicle group and vesicle combined radiotherapy group.
  • the radiotherapy group used 4Gy for radiotherapy to treat cells, and the specific operation was the same as in Example 9;
  • Vesicle group cisplatin vesicles (Cis-MPs), methotrexate vesicles (MTX-MPs), doxorubicin vesicles (Dox-MPs) according to the number of cells in the vesicle ratio of 5:1 The proportion is added to the 24-well plate;
  • the vesicle combined with radiotherapy group radiotherapy was first performed with 4 Gy, and the specific operation was the same as in Example 9. Then, cisplatin-containing vesicles (Cis-MPs), methotrexate vesicles (MTX-MPs), and doxorubicin-containing vesicles (Dox-MPs) were added in a ratio of 5:1 vesicles per cell. 24-well plate.
  • the 4 groups of cells were further placed in an incubator for cultivation.
  • the cell survival rate of the vesicle combined with the radiotherapy group was significantly lower than that of the control group, the radiotherapy group, and the vesicle group. It is indicated that low-dose radiotherapy combined with vesicles containing various types of chemotherapy drugs has toxic effects on tumor cells.
  • H22 mouse liver cancer cells H22 mouse liver cancer cells; cisplatin; ⁇ -radiation precision therapeutic apparatus (EKAKTA, England); Farmer2570 reference grade dosimeter.
  • H22 mouse hepatoma cells were cultured in RPMI-1640 cell culture medium containing 10% serum to achieve a cell volume of 2 ⁇ 10 7 /ml; H22 mouse liver cancer cells were irradiated with ultraviolet light for 60 minutes; divided into 2 groups, one group Cisplatin was added at a final concentration of 200 ⁇ g/ml, and the other group was left untreated. Place in the incubator and continue to culture.
  • the cell vesicles of the above apoptotic tumor cells were collected by centrifugation, including: centrifugation at 1300 rpm and 5000 rpm for 10 minutes, followed by centrifugation at 14,000 g for 1 minute to remove cell debris, and the supernatant after centrifugation was again 14,000 g.
  • Cis-plated vesicles (Cis-MPs) and empty vesicles (MPs) were obtained.
  • H22 mouse hepatoma cells were cultured in RPMI-1640 cell culture medium, counted, and diluted at 5 ⁇ 10 4 /ml. Seed in a 24-well plate, 1 ml per well. They were divided into 8 groups: control group, empty vesicle group, cisplatin group, vesicle group, radiotherapy group, empty vesicle combined with radiotherapy group, cisplatin combined with radiotherapy group, and vesicle combined radiotherapy group.
  • the radiotherapy group used 4Gy for radiotherapy to treat cells, and the specific operation was the same as in Example 9;
  • the empty vesicle group and the vesicle group were added to the 24-well plate according to the ratio of the vesicle to the cell number of 5:1;
  • the cisplatin group was added with cisplatin at a final concentration of 1 ⁇ g/ml per well;
  • Cisplatin combined with radiotherapy group radiotherapy was first performed with a total dose of 4 Gy, and the specific operation was the same as in Example 9. Then a final concentration of 1 ⁇ g/ml cisplatin was added to the 24-well plate;
  • the vesicle combined with radiotherapy group radiotherapy was first performed with a total dose of 4 Gy, and the specific operation was the same as in Example 9. Cis-plated vesicles (Cis-MPs) were then added to the 24-well plates at a ratio of 5:1 vesicles per cell.

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Abstract

细胞囊泡制剂与低剂量放疗联合在制备治疗肿瘤药物中的应用，所述细胞囊泡制剂源自凋亡的肿瘤细胞并包裹了化疗药物，所述低剂量放疗为以治疗所述肿瘤的放射线总剂量较低的放射治疗，可选地，所述化疗药物选自顺铂、甲氨蝶呤和阿霉素。

Description

细胞囊泡制剂与低剂量放疗联合在制备抗肿瘤药物中的应用 技术领域
本发明涉及一种细胞囊泡制剂的新应用。
背景技术
临床上传统的、常规的、应用最多的恶性肿瘤治疗方法是利用化疗/放疗类消灭肿瘤细胞。这两种手段在抗肿瘤治疗中具有一定的优势，但是缺点也十分明显。
无论是全身化疗还是局部灌注给药，多数化疗药物在杀伤肿瘤细胞的同时，对正常人体细胞也有较大损伤，产生毒副反应，从而使化疗不能顺利进行，严重影响了疗效。并且多次使用化疗药物可以使肿瘤细胞特别是其中具有细胞干性的这部分肿瘤细胞(肿瘤再生细胞)产生较强的耐药性，进而使化疗药物失去其对肿瘤细胞的杀伤作用。中国专利ZL20111024-1369.8公开了肿瘤细胞来源的囊泡包裹低剂量化疗药物对肿瘤细胞的杀伤效果比高剂量的化疗药直接杀伤效果更好。
中国专利申请20151091237.7公开了化疗药具有对上述肿瘤细胞来源的囊泡增敏的作用，降低或逆转肿瘤细胞耐药性，提高细胞囊泡对肿瘤细胞的杀伤效力。
放疗是指用各种不同能量的射线照射肿瘤，以抑制和杀灭癌细胞的一种治疗方法。放射治疗具有增强抗肿瘤免疫的潜在能力。原因在于放射治疗可能导致肿瘤抗原从即将死亡的肿瘤细胞释放，促进肿瘤抗原呈递和肿瘤特异性T细胞活化，有助于全身抗肿瘤免疫。然而，放射治疗因其在照射后对免疫细胞的损害在最初被认为是具有免疫抑制作用的。应当注意的是，放射治疗也可以促进免疫抑制，导致肿瘤浸润巨噬细胞的恶性驯化和肿瘤再生细胞介导的免疫抑制。放射治疗的剂量是调和这种明显矛盾的关键因素。所以，临床上使用的放疗剂量受到很大限制。目前，常规放疗的单次剂量为1.8-2.0Gy，1周5次。按照放疗的剂量总剂量分为高剂量放疗、中剂量放疗和低剂量放疗。高剂量放疗用于肿瘤的精准治疗，需要对病灶部位进行精确定位和剂量学测算，操作复杂、难度大。低剂量放疗剂副作用小，但是易产生免疫耐受。
综上，人们期望保留化疗和放疗优势的同时降低毒副作用，使化疗药物/放疗剂量在常规用量甚至低于常规用量的情况下，对肿瘤细胞特别是肿瘤再生细胞产生强效杀伤作用，改善肿瘤微环境，修复免疫系统，提高机体的抗肿瘤免疫应答，从而达到控制肿瘤的效果。
发明内容
本发明的目的是提供细胞囊泡制剂与低剂量放疗联合在制备抗肿瘤药物中的应用。
根据本发明的一方面，本发明所述的细胞囊泡制剂为源自凋亡的肿瘤细胞并包裹了化疗药物，也称载药囊泡；本发明所称的低剂量放疗为以低于治疗所述肿瘤放射线总剂量的放射治疗。
优选地，用于包裹化疗药的肿瘤细胞囊泡(载体)来源于肿瘤细胞凋亡而成，本发明中所述的包裹于肿瘤细胞囊泡中的化疗药，可以为本发明申请日以前和\或以后被临床应用的化疗药，也可以是本发明申请日以前和\或以后被临床应用的化疗药中的有效成分(可以不含有或不完全含有药物辅料)，即制备本发明的细胞囊泡制剂时，可以使用治疗肿瘤的临床用药组成中的有效成分，也可以直接使用已经临床应用于肿瘤治疗的各种商购药。所以，本发明中所涉及的药物剂量，均应理解为该药物的有效成分量。具体化疗药可以是临床上用于治疗各类肿瘤的化疗药，如：治疗卵巢癌、乳腺癌、肺癌、胃癌、结肠癌、肝癌、膀胱癌或直肠癌的化疗药，可以是单一化疗药或多种化疗药联合。本发明优选的化疗药物为顺铂、甲氨蝶呤或阿霉素，最优选的方案为将化疗药物顺铂包裹于肿瘤细胞囊泡中制备成载药囊泡。
本发明所述囊泡的制备方法可以参考中国专利ZL201110241369.8，在此引入作为参考。
本发明所述的低剂量放疗指当以该剂量的放射线对所述的肿瘤进行单独的放射治疗时，所述放射治疗未能体现出应有的治疗效果，或者该剂量对所述肿瘤没有明显杀伤作用。优选的单次治疗剂量可以为2Gy，该放射剂量可以单次或分次给予患者，给予的放射总剂量可以根据治疗对象的体重和具体治疗状况确定。
适于使用本发明方法治疗的肿瘤优选为卵巢癌、乳腺癌、肺癌、胃癌、结 肠癌、肝癌、膀胱癌或直肠癌，更优选所述肿瘤为肺癌、结肠癌或肝癌。
本发明的应用中，所述细胞囊泡制剂的单位制剂中可以包括8×10 6个细胞囊泡，优选以静脉注射给予载药囊泡。
为实现上述目的，发明人首先验证不同辐射剂量对H22肝癌模型小鼠的影响，发现与对照组相比，4Gy放疗处理的小鼠在体重和生存率上无差异，且小鼠毛发柔顺、行为正常，安全性高。
但单独4Gy放疗处理疗效不高，所以决定联合使用化疗药物。
申请人之前的研究发现给予包裹药物的囊泡比给予单纯药物效果好，在此以H22细胞系为试验对象，进行了空囊泡(MPs)、顺铂(Cis)、载顺铂囊泡(Cis-MPs)的药物效果对比试验，以细胞凋亡率判断，发现使用载顺铂囊泡的效果最好；同时也发现，只采取化疗不放疗的效果远低于两者联用。
接着，验证了本发明包裹药物的囊泡与低剂量放疗联用，对H22肝癌模型、CT26结肠癌模型和Lewis肺癌模型的疗效。发现，相较于单独低剂量放疗和单独囊泡，囊泡与低剂量放疗剂联用效果最好，有效控制肿瘤生长、显著提升荷瘤小鼠生存率。最关键的是，该联用方法并没有影响小鼠的体重、肝肾功能和行为。
本发明还分离出荷瘤小鼠的肿瘤再生细胞，验证了囊泡与低剂量放疗剂联用对肿瘤再生细胞的有效杀伤作用，说明本发明的联用方法有根治肿瘤的潜力。
此外，本发明还研究了装载不同化疗药物的囊泡联合低剂量放疗的治疗效果，发现它们同样能有效杀死肿瘤细胞。
有益效果：细胞水平和皮下动物肿瘤模型验证结果表明，本发明所述的囊泡联合低剂量放疗的效果比单独使用囊泡和低剂量放疗好，安全、无毒副作用。
附图说明
图1：放疗对小鼠的体重和生存期的影响；
1A：对小鼠体重的影响；1B：对小鼠生存期的影响；
图2：放疗对肿瘤细胞的杀伤效果；
图3：囊泡联合放疗治疗肝癌的效果；
3A：对小鼠肿瘤的抑制；3B：对小鼠生存期的影响；
图4：囊泡联合放疗治疗结肠癌的效果；
4A：对小鼠肿瘤的抑制；4B：对小鼠生存期的影响；
图5：囊泡联合放疗治疗肺癌效果对比；
5A：对小鼠肿瘤的抑制；5B：对小鼠生存期的影响；
图6：囊泡联合放疗治疗对小鼠的影响；
6A：对小鼠体重的影响；6B：对小鼠肾功能的影响；6C：对小鼠肝功能的影响；
图7A：不同治疗处理下的肿瘤再生细胞克隆群的三维纤维蛋白胶；
Ⅰ对照组Ⅱ放疗组Ⅲ囊泡组Ⅳ囊泡联合放疗组
图7B-C：不同治疗处理对肿瘤再生细胞克隆群的数目和体积影响；
7B：克隆群的数目；7C：克隆群的体积；
图8：囊泡联合放疗治疗对药物在肿瘤细胞内存留时间的影响；
图9：囊泡联合放疗治疗荷瘤小鼠促进其体内巨噬细胞的良性驯化；
9A：脾脏中具有分泌IL-1β功能的巨噬细胞的比例；
9B：脾脏中具有分泌IL-10功能的巨噬细胞的比例；
9C：肿瘤浸润淋巴组织中具有分泌IL-1β功能的巨噬细胞的比例；
9D：肿瘤浸润淋巴组织中具有分泌IL-10功能的巨噬细胞的比例；
图10：载不同药的肿瘤囊泡联合放疗对H22肝癌细胞系的杀伤；
10A：载顺铂肿瘤囊泡；10B：载甲氨蝶呤肿瘤囊泡；10C：载阿霉素肿瘤囊泡。
图11：本发明方案与其他常见方案对肿瘤细胞的杀伤效果比较。
具体实施方式
下述实施例中使用的各种肿瘤细胞、药物及实验动物：
小鼠肝癌细胞系H22(BALB/c遗传背景)、小鼠结肠癌细胞系CT26(BALB/c遗传背景)、小鼠肺癌细胞系Lewis(C57BL/6遗传背景)、均可从中国典型物保藏中心CCTCC购买。
C57BL/6小鼠，BALB/c小鼠，购自武汉大学医学实验动物中心，周龄在5-6周，体重约18克左右。
本发明所述的囊泡如果没有特别说明就是中国专利201110241369.8中所述的细胞囊泡制剂，未包裹化疗药的囊泡称为空囊泡。
低剂量放疗是指低于治疗所述肿瘤的放射线总剂量的放射治疗，该放射治疗单独对所述肿瘤无明显的杀伤作用；具体对于上述实验动物的总剂量为4Gy的γ辐射。
【实施例1】囊泡的制备
1、实验材料和试剂
H22小鼠肝癌细胞；紫外线装置为常规生物安全柜所有；二氧化碳恒温培养箱；顺铂；3μm计数用微球；流式细胞仪。
2、实验步骤
在RPMI-1640细胞培养液中培养H22小鼠肝癌细胞，使细胞量达到2×10 7/ml；将H22小鼠肝癌细胞进行紫外线照射60分钟；加入终浓度为200μg/ml的顺铂。置于培养箱中继续培养。
18-20小时后，显微镜下观察H22小鼠肝癌细胞凋亡情况。通过离心收集上述凋亡肿瘤细胞的囊泡，包括：先以1300rpm和5000rpm转速各离心10分钟，取上清，之后以14000g的离心力离心1分钟，以除去细胞碎片，取离心后的上清再以14000g的离心力离心1小时，收集沉淀，用PBS重悬，得到来自H22小鼠肝癌细胞凋亡所产生的载顺铂囊泡(Cis-MPs)。
3、囊泡的计数
将收集到的囊泡和已知浓度的3μm计数用微球等体积混合，流式仪进行计数，得到囊泡与微球的比例。囊泡的浓度＝微球的浓度×囊泡与微球的比例。
【实施例2】低剂量放疗对小鼠的影响
1、实验材料和试剂
H22小鼠肝癌细胞；实验用BALB/c小鼠购自武汉大学医学实验动物中心，体重18-20克；光学显微镜；计数板；小鼠固定器；防辐射铅板；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)通过实施例1方法培养H22小鼠肝癌细胞；
2)将H22细胞用PBS稀释成细胞悬液，用细胞计数板在显微镜下计数，并稀释为1×10 6个/ml，对BALB/c小鼠右侧腹部皮下注射200μl细胞悬液，待 肿瘤大小长到8×8mm，大约需要10天；
3)将这些皮下成瘤的32只BALB/c小鼠随机分成4组：空白组即对照组；放疗组共3组，4Gy放疗组；10Gy放疗组；20Gy放疗组(指处理期间所用放疗的总剂量)。
4)其中对照组不使用放疗处理；
放疗组分别使用总剂量为4Gy、10Gy、20Gy的γ-射线照射肿瘤部位。具体为每3天1个疗程，持续2个疗程，即共6天，1个疗程照射剂量分别为2Gy、5Gy和10Gy。
放疗疗程具体操作为：使用小鼠固定器固定皮下成瘤的BALB/c小鼠，用铅板屏蔽小鼠正常组织，允许其肿瘤部位暴露，将射线源的孔径打开到暴露整个肿瘤所需的最低水平。使用γ-放射治疗通过精准治疗仪以约500cGy/min的剂量率在室温下进行照射，照射总剂量根据时间进行调整。例如，4Gy放疗组每3天需照射2次，每次照射24s；10Gy放疗组每3天需照射2次，每次照射1min；20Gy放疗组每3天需照射2次，每次照射2min；γ-放射精准治疗仪每次使用前用Farmer2570参考级剂量仪进行剂量校准。
4)至放疗之日起，每天称量各组小鼠的体重，观察小鼠的生活状态和生存期，延续至第30天。
3、实验结果
由图1A和图1B可以看出，相比于未使用放疗处理的对照组小鼠，经单次剂量2Gy，总剂量低至4Gy的放射剂量处理的小鼠在体重和生存率上无差异，且小鼠毛发柔顺、无弓腰耸背现象。而高剂量放疗处理组，即10Gy放疗组、20Gy放疗组小鼠体重逐渐减轻，生存期缩短。
结论：4Gy放疗对小鼠无毒副作用。
故选择安全性较高，无毒副作用的较低剂量放疗，即次低剂量2Gy，总剂量4Gy放疗联合囊泡进行肿瘤治疗。
【实施例3】低剂量放疗对肿瘤的杀伤
1、实验材料和试剂
小鼠肝癌细胞H22细胞；实验用BALB/c小鼠购自武汉大学医学实验动物中心，体重18-20克；小鼠固定器；防辐射铅板；γ-放射精准治疗仪(EKAKTA， England)；流式细胞仪(BD)；凋亡检测试剂盒；Farmer2570参考级剂量仪。2、实验步骤
1)通过实施例2获得BALB/c小鼠皮下动物模型。
2)将这些皮下成瘤的16只小鼠随机分成2组：空白组即对照组；4Gy放疗组。
3)放疗组处理方式同实施例2；
对照组不做放疗处理。
4)化疗后24小时即分离皮下瘤细胞进行细胞凋亡比例分析。
具体操作如下：将皮下瘤组织分离，研磨分散，加0.25％的胰蛋白酶消化2min，用胎牛血清终止消化，PBS洗涤并重悬成单细胞悬液。用凋亡试剂盒内的PI和FITC-annexin V抗体进行常规凋亡检测染色，流式仪上机检测凋亡细胞比例。
对照组荷瘤小鼠同以上处理。
3、实验结果
由图2可以看出，相比于对照组小鼠，经4Gy放疗处理小鼠并未促进皮下瘤内肿瘤细胞的凋亡。
结论：4Gy放疗无杀伤肿瘤效果。
【实施例4】囊泡联合低剂量放疗治疗肝癌
1、实验材料和试剂
小鼠肝癌细胞H22细胞；实验用BALB/c小鼠购自武汉大学医学实验动物中心，体重体重18-20克；小鼠固定器；防辐射铅板；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)包裹顺铂的囊泡制备方法同实施例1。
2)通过实施例2的方法获得BALB/c小鼠皮下动物模型。
3)将32只皮下成瘤的BALB/c小鼠随机分成4组：空白组即对照组；放疗组；囊泡组；囊泡联合放疗组。
4)对照组不做放疗处理；在囊泡组行注射囊泡相同的时间点注射等体积的PBS。
囊泡联合放疗组放疗总剂量为4Gy，单次放疗剂量为2Gy，放疗为每3天完成一个疗程，持续2个疗程。在放疗2个疗程内，每次放疗结束24小时后静脉注射2×10 6个Cis-MPs。第二次放疗结束后72小时行第三次注射，第三次注射Cis-MPs后间隔72小时行第四次注射。整个囊泡联合放疗组的治疗时间为12天。
放疗组处理方式同实施例3的4Gy放疗组，每次放疗结束24小时后静脉注射与囊泡联合放疗组等体积的PBS，第二次注射PBS后间隔72小时行第三次注射，第三次注射PBS后间隔72小时行第四次注射；
囊泡组不做放疗处理，与囊泡联合放疗组同时间点静脉注射等量Cis-MPs；
5)观察4组小鼠的皮下瘤体积大小和生存时间。
3、实验结果
由图3可以看出，相比于对照组小鼠，放疗组、囊泡组和囊泡联合放疗组小鼠的肿瘤生长受到抑制，生存时间延长。囊泡联合放疗组的治疗效果较单独使用囊泡或放疗的小鼠治疗效果更佳，表现在肿瘤体积萎缩，在第60天，小鼠的存活率保持在87.5％，而其他三组均死亡。
结论：囊泡联合低剂量放疗治疗H22肝癌皮下肿瘤效果显著优于单独使用囊泡和单独使用低剂量放疗的方法。
【实施例5】囊泡联合低剂量放疗治疗结肠癌
1、实验材料和试剂
小鼠结肠癌细胞系CT26(BALB/c遗传背景)；实验用BALB/c小鼠购自武汉大学医学实验动物中心，体重18-20克；小鼠固定器；防辐射铅板；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)在含10％血清的RPMI-1640细胞培养液中培养CT26。
2)CT26肿瘤细胞来源的囊泡包裹顺铂的制备方法同实施例1。
3)将CT26细胞用PBS稀释成细胞悬液，计数并稀释为1×10 6个/ml，对BALB/c小鼠右侧腹部皮下注射200μl细胞悬液，待肿瘤大小长到8×8mm。
4)将32只皮下成瘤的小鼠随机分成4组：空白组即对照组；放疗组；囊泡组；囊泡联合放疗组。
5)各组处理方式同实施例4。
6)观察4组小鼠的皮下瘤体积大小和生存时间。
3、实验结果
由图4可以看出，相比于对照组小鼠，放疗组、囊泡组和囊泡联合放疗组小鼠肿瘤生长受到抑制，生存率延长。囊泡联合放疗组的治疗效果较单独使用囊泡或放疗的小鼠治疗效果更佳。表现为肿瘤生长速度明显减慢，在第60天，小鼠的存活率保持在75％，而其他三组均死亡。
结论：囊泡联合低剂量放疗治疗CT26结肠癌皮下肿瘤效果显著优于单独使用囊泡和单独使用低剂量放疗的方法。
【实施例6】囊泡联合低剂量放疗治疗肺癌
1、实验材料和试剂
小鼠肺癌细胞系Lewis细胞(C57BL遗传背景)；实验用C57BL小鼠购自武汉大学医学实验动物中心，体重18-20克；小鼠固定器；防辐射铅板；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)在RPMI-1640细胞培养液中培养Lewis细胞。
2)Lewis细胞来源的囊泡包裹顺铂的制备方法同实施例1。
3)将Lewis细胞用PBS稀释成细胞悬液，计数并稀释为1×10 6个/ml，对C57BL小鼠右侧腹部皮下注射200μl细胞悬液，待肿瘤大小长到8×8mm。
4)将32只皮下成瘤的C57BL小鼠随机分成4组，空白组即对照组；放疗组；囊泡组；囊泡联合放疗组。
5)各组处理方式同实施例4。
5)观察4组小鼠的皮下瘤体积大小和生存时间。
3、实验结果
由图5可以看出，相比于对照组小鼠，放疗组、囊泡组和囊泡联合放疗组小鼠的肿瘤生长受到抑制，生存率有所延长。囊泡联合放疗组的治疗效果较单独使用囊泡或低剂量放疗的小鼠治疗效果更佳。表现为肿瘤生长速度显著减慢，在第60天，小鼠的存活率保持在75％，而其他三组均死亡。
结论：囊泡联合低剂量放疗治疗Lewis肺癌皮下肿瘤效果显著优于单独使 用囊泡和单独使用低剂量放疗的方法。
【实施例7】囊泡联合低剂量放疗治疗对小鼠的影响
1、实验材料和试剂
H22小鼠肝癌细胞；实验用BALB/c小鼠购自武汉大学医学实验动物中心，体重18-20克；小鼠固定器；防辐射铅板；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)包裹顺铂的囊泡制备方法同实施例1。
2)通过实施例2的方法获得BALB/c小鼠皮下动物模型。
3)将32只皮下成瘤的BALB/c小鼠随机分成4组：空白组即对照组；放疗组；囊泡组；囊泡联合放疗组。
4)各组处理方式同实施例4。
5)至治疗之日起，每天将各组小鼠分别称重，持续24天。治疗之后第24天，将小鼠腹腔注射100μl 1％的戊巴比妥钠进行全身麻醉，固定小鼠，打开腹腔，腹主动脉取血法采集外周血置于促凝管内。2000rpm离心8min分离血清，全自动生化分析仪检测肌酐和谷丙转氨酶的含量。评价肝肾功能的损伤程度。
3、实验结果
由图6可以看出，相比于对照组小鼠，放疗组、囊泡组和囊泡联合放疗组小鼠的体重在治疗期间均未发生明显变化(图6A)。肌酐和谷丙转氨酶的含量均处于正常值范围内，与对照组无显著差异。
结论：囊泡联合低剂量放疗治疗无毒副作用。
【实施例8】囊泡联合低剂量放疗对肿瘤再生细胞的杀伤
1、实验材料和试剂
H22小鼠肝癌细胞；光学显微镜；实验用BALB/c小鼠购自武汉大学医学实验动物中心，体重18-20克；防辐射铅板；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)包裹顺铂的囊泡制备方法同实施例1。
2)通过实施例2的方法获得BALB/c小鼠皮下动物模型。
3)将32只皮下成瘤的小鼠随机分成4组：空白组即对照组；放疗组；囊泡组；囊泡联合放疗组。
4)各组处理方式同实施例4。
5)治疗疗程完毕，分离各组荷瘤小鼠的皮下瘤，研磨分散，加0.25％的胰蛋白酶消化2min，用胎牛血清终止消化，PBS洗涤并重悬成单细胞悬液。计数，用三维纤维蛋白胶培养其中的肿瘤细胞，随着时间的延长，只有肿瘤再生细胞能耐受三维纤维蛋白胶的硬度环境而存活下来。
三维纤维蛋白胶培养方法：
将单细胞悬液重悬在含10％胎牛血清的RPMI-1640培养基中，并将细胞浓度调节到10 4个/ml。用T7缓冲液(50mM Tris，pH 7.4，150mM NaCl)将纤维蛋白原稀释到2mg/ml。将纤维蛋白原溶液和细胞溶液按照1：1混合。混合液中纤维蛋白原的终浓度是1mg/ml，细胞浓度为5000个/ml。将250μl混合液种入24-孔板与5μl凝血酶充分混匀。将培养物移至37℃培养箱孵育30min。最后加入1ml含10％胎牛血清的RPMI-1640培养基和相应浓度的抗生素继续培养。待肿瘤再生细胞形成细胞群。
6)观察三维纤维蛋白胶中肿瘤再生细胞所形成的细胞群大小，并进行测量。
3、实验结果
由图7A和图7C可以看出，相比于对照组小鼠、放疗组和囊泡组，囊泡联合放疗组小鼠肿瘤细胞形成的肿瘤再生细胞克隆群体积最小，数目最少(7B)。肿瘤再生细胞在三维纤维蛋白胶中生长的速度最慢，与另外三组相比有显著差异。
结论：囊泡联合低剂量放疗通过杀伤肿瘤再生细胞抑制肿瘤生长。
【实施例9】低剂量放疗对囊泡在肿瘤细胞存留的影响
H22小鼠肝癌细胞；CT26小鼠结肠癌细胞；Lewis小鼠肺癌细胞；阿霉素(Dox，带有红色荧光的化疗药)；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪；流式细胞仪。
2、实验步骤
1)包裹化疗药的囊泡制备方法同实施例1。具体如下：
在含10％血清的RPMI-1640细胞培养液中培养H22小鼠肝癌细胞，使细胞量达到2×10 7/ml；将H22小鼠肝癌细胞进行紫外线照射60分钟；加入终100μg/ml阿霉素(Dox)。置于培养箱中孵育培养。
18-20小时后，显微镜下观察H22小鼠肝癌细胞出现凋亡现象。通过离心收集上述凋亡肿瘤细胞的细胞囊泡，包括：先以1300rpm和5000rpm转速各离心10分钟，之后以14000g的离心力离心1分钟，以除去细胞碎片，取离心后的上清再以14000g的离心力离心1小时，收集沉淀得到来自H22小鼠肝癌细胞凋亡所产生的载阿霉素囊泡(H22-Dox-MPs)。PBS重悬后计数。计数方法同实施例1。
CT26小鼠结肠癌细胞和Lewis小鼠肺癌细胞来源的囊泡包裹阿霉素的处理方法同上，分别得到CT26小鼠结肠癌细胞凋亡所产生的载阿霉素囊泡(CT26-Dox-MPs)和Lewis小鼠肺癌细胞凋亡所产生的载阿霉素囊泡(Lewis-Dox-MPs)。
2)在RPMI-1640细胞培养液中分别培养H22小鼠肝癌细胞、CT26小鼠结肠癌细胞、Lewis小鼠肺癌细胞，计数，按照5×10 4/ml种入24-孔板，每孔1ml。各种细胞系分别分为4组，即对照组、放疗组、囊泡组和囊泡联合放疗组。
3)对照组不做处理；
放疗组：采用4Gy进行放疗处理，具体操作为使用γ-放射精准治疗仪照射细胞，2Gy照射两次，每次24s，间隔24h。
囊泡组：将载阿霉素囊泡(Dox-MPs)按照囊泡比细胞数为5:1的比例加入24-孔板。
囊泡联合放疗组：先用总剂量4Gy进行放疗处理H22细胞、CT26细胞、Lewis细胞，具体为分2次照射，每次24s，间隔24小时。然后分别加入H22-Dox-MPs，CT26-Dox-MPs，Lewis-Dox-MPs，按照囊泡比细胞数为5:1的比例加入24-孔板。
4)各组处理结束后分别在0、4、8、12小时收集细胞。PBS洗涤一次，流式细胞仪上机检测各组细胞的平均荧光强度。
3、实验结果
由图8可以看出，相比于对照组、放疗组和囊泡组，囊泡联合放疗组细胞 平均荧光强度下降速度最慢。
结论：低剂量放疗联合增强了化疗药物在肿瘤细胞内的停留，延长了化疗药物的作用时间。
【实施例10】囊泡联合低剂量放疗增强巨噬细胞的抗肿瘤免疫
1、实验材料和试剂
小鼠肝癌细胞H22细胞；实验用BALB/c小鼠购自武汉大学医学实验动物中心，体重18-20克；小鼠固定器；防辐射铅板；γ-放射精准治疗仪(EKAKTA，England)；流式细胞仪(BD)；Farmer2570参考级剂量仪；小鼠抗体anti-CD11b、anti-F4/80、anti-IL-1β和anti-IL-10购自Sigma公司。
2、实验步骤
1)通过实施例2获得BALB/c小鼠皮下动物模型。
2)将这些皮下成瘤的32只小鼠随机分成4组：空白组即对照组；放疗组；囊泡组；囊泡联合放疗组。
3)各组处理方式同实施例4。
4)第13天解剖各组小鼠，分离脾脏和肿瘤浸润部位组织淋巴组织，进行巨噬细胞功能的分析。
具体方法如下：
将脾脏和肿瘤浸润部位组织研磨分散成单细胞悬液，将细胞计数，调整细胞浓度至1×10 6/100μl，避光条件下加入1μl CD11b和F4/80抗体，震荡混匀，4℃避光孵育30min。孵育结束后每管加入850μl PBS，震荡混匀，500g离心5min。弃上清，每管加入100μl 4％多聚甲醛固定液，常温避光孵育20min孵育结束后直接每管加入100μl 0.1％Trixton-100处理10min，震荡混匀后500g离心5min。弃上清，每管加入1μl小鼠抗体IL-1β和IL-10。常温避光染色30min。孵育结束后每管加入900μl PBS，震荡混匀后500g离心5min，弃上清，每管加入100μl PBS，震荡混匀，避光，流式上机检测。
3、实验结果
由图9可以看出，相比于对照组、放疗组和囊泡组，囊泡联合放疗组处理的小鼠脾脏和肿瘤浸润组织分泌IL-1β的巨噬细胞的比例显著增加，而具抑制肿瘤免疫应答的分泌IL-10的巨噬细胞的比例显著减少。
结论：囊泡联合放疗处理能增进导致肿瘤浸润巨噬细胞的良性驯化，改善肿瘤微环境，促进免疫应答。
【实施例11】载其它化疗药的囊泡联合低剂量放疗对肿瘤细胞的杀伤
1、实验材料和试剂
H22小鼠肝癌细胞；顺铂；甲氨蝶呤；阿霉素；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)包裹化疗药的囊泡制备方法同实施例1。具体如下：
在含10％血清的RPMI-1640细胞培养液中培养H22小鼠肝癌细胞，使细胞量达到2×10 7/ml；将H22小鼠肝癌细胞进行紫外线照射60分钟；分为3组，分别加入终浓度200μg/ml的顺铂、1mg/ml甲氨蝶呤(MTX)、100μg/ml阿霉素(Dox)。置于培养箱中继续培养。
18-20小时后，显微镜下观察H22小鼠肝癌细胞出现凋亡现象。通过离心收集上述凋亡肿瘤细胞的囊泡，包括：先以1300rpm和5000rpm转速各离心10分钟，之后以14000g的离心力离心1分钟，以除去细胞碎片，取离心后的上清再以14000g的离心力离心1小时，收集沉淀得到来自H22小鼠肝癌细胞凋亡所产生的载顺铂囊泡(Cis-MPs)、载甲氨蝶呤囊泡(MTX-MPs)、载阿霉素囊泡(Dox-MPs)。PBS重悬后计数。计数方法同实施例1。
2)在RPMI-1640细胞培养液中培养H22小鼠肝癌细胞，计数，按照5×10 4/ml稀释。种入24-孔板，每孔1ml。共分为4组，分别为对照组、放疗组、囊泡组和囊泡联合放疗组。
3)对照组不做处理；
放疗组采用4Gy进行放疗处理细胞，具体操作同实施例9；
囊泡组：将载顺铂囊泡(Cis-MPs)、载甲氨蝶呤囊泡(MTX-MPs)、载阿霉素囊泡(Dox-MPs)按照囊泡比细胞数为5:1的比例加入24-孔板；
囊泡联合放疗组：先用4Gy进行放疗，具体操作同实施例9。然后将载顺铂囊泡(Cis-MPs)、载甲氨蝶呤囊泡(MTX-MPs)、载阿霉素囊泡(Dox-MPs)按照囊泡比细胞数为5:1的比例加入24-孔板。
4组细胞继续放置培养箱中培养。
4)24小时后通过MTT法检测各孔细胞的凋亡率。
3、实验结果
由图10可以看出，相比于对照组、放疗组和囊泡组，囊泡联合放疗组的细胞存活率显著下降。说明低剂量放疗联合载各类化疗药物的囊泡对肿瘤细胞都具有毒性效果。
结论：低剂量放疗联合载其它肿瘤化疗药的囊泡对肿瘤细胞杀伤比单独放疗疗和单独囊泡治疗具有增强的效果。
【实施例12】单纯顺铂、单纯空囊泡与本发明方案比较
1、实验材料和试剂
H22小鼠肝癌细胞；顺铂；γ-放射精准治疗仪(EKAKTA，England)；Farmer2570参考级剂量仪。
2、实验步骤
1)包裹化疗药的囊泡制备方法同实施例1。具体如下：
在含10％血清的RPMI-1640细胞培养液中培养H22小鼠肝癌细胞，使细胞量达到2×10 7/ml；将H22小鼠肝癌细胞进行紫外线照射60分钟；分为2组，一组加入终浓度200μg/ml的顺铂，另一组不作处理。置于培养箱中继续培养。
18-20小时后，显微镜下观察H22小鼠肝癌细胞出现凋亡现象。通过离心收集上述凋亡肿瘤细胞的细胞囊泡，包括：先以1300rpm和5000rpm转速各离心10分钟，之后以14000g的离心力离心1分钟，以除去细胞碎片，取离心后的上清再以14000g的离心力离心1小时，收集沉淀得到来自H22小鼠肝癌细胞凋亡所产生的载顺铂囊泡(Cis-MPs)、载甲氨蝶呤囊泡(MTX-MPs)、载阿霉素囊泡(Dox-MPs)。PBS重悬后计数。计数方法同实施例1。得到载顺铂囊泡(Cis-MPs)和空囊泡(MPs)。
2)在RPMI-1640细胞培养液中培养H22小鼠肝癌细胞，计数，按照5×10 4/ml稀释。种入24-孔板，每孔1ml。共分为8组，分别为对照组、空囊泡组、顺铂组、囊泡组、放疗组、空囊泡联合放疗组、顺铂联合放疗组、囊泡联合放疗组。
3)对照组不做处理；
放疗组采用4Gy进行放疗处理细胞，具体操作同实施例9；
空囊泡组和囊泡组按照囊泡比细胞数为5:1的比例加入24-孔板；
顺铂组每孔加入终浓度1μg/ml的顺铂；
顺铂联合放疗组：先用总剂量4Gy进行放疗，具体操作同实施例9。然后将终浓度1μg/ml顺铂加入24-孔板；
囊泡联合放疗组：先用总剂量4Gy进行放疗，具体操作同实施例9。然后将载顺铂囊泡(Cis-MPs)按照囊泡比细胞数为5:1的比例加入24-孔板。
4)加入囊泡24小时后通过凋亡试剂盒染色，流式上机检测各孔细胞的凋亡率。
3、实验结果
由图11可以看出，相对于对照组、空囊泡组、顺铂组、囊泡组、放疗组、空囊泡联合放疗组和顺铂联合放疗组，囊泡联合放疗促进细胞凋亡的效率最高，与其他单独处理组具有显著性差异。
结论：单纯药物、空囊泡、空囊泡联合放疗以及化疗药物联合放疗都不如囊泡联合放疗的效果好。

Claims (10)

  1. 细胞囊泡制剂与低剂量放疗联合在制备治疗肿瘤药物中的应用，其中所述细胞囊泡制剂源自凋亡的肿瘤细胞并包裹了化疗药物，所述低剂量放疗为低于治疗所述肿瘤的放射线总剂量的放射治疗。
  2. 根据权利要求1所述的应用，其特征在于，所述化疗药物为选自治疗卵巢癌、乳腺癌、肺癌、胃癌、结肠癌、肝癌、膀胱癌或直肠癌的化疗药物中的一种或多种。
  3. 根据权利要求1所述的应用，其特征在于，所述化疗药物选自顺铂、甲氨蝶呤和阿霉素。
  4. 根据权利要求3所述的应用，其特征在于，所述化疗药物为顺铂。
  5. 根据权利要求4所述的应用，其特征在于，所述低剂量放疗为对所述肿瘤无杀伤作用的放射线剂量。
  6. 根据权利要求5所述的应用，其特征在于，所述放疗为分次间隔进行。
  7. 根据权利要求1所述的应用，其特征在于，所述肿瘤为卵巢癌、乳腺癌、肺癌、胃癌、结肠癌、肝癌、膀胱癌或直肠癌。
  8. 根据权利要求7所述的应用，其特征在于所述肿瘤为肺癌、结肠癌或肝癌。
  9. 根据权利要求8所述的应用，其特征在于，所述肿瘤为肝癌。
  10. 根据权利要求1所述的应用，其特征在于，所述细胞囊泡制剂的单位制剂中包括8×10 6个细胞囊泡，给药方式为静脉注射。
PCT/CN2018/074522 2017-02-09 2018-01-30 细胞囊泡制剂与低剂量放疗联合在制备抗肿瘤药物中的应用 WO2018145588A1 (zh)

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