CN111888376B

CN111888376B - Cisplatin-loaded fibrin glue composite system combined treatment system

Info

Publication number: CN111888376B
Application number: CN202010983127.5A
Authority: CN
Inventors: 杨茜; 高玲; 易成
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-06-24
Filing date: 2020-09-17
Publication date: 2022-06-24
Anticipated expiration: 2040-09-17
Also published as: KR20210041547A; CN112043834A; CN112043834B; CN111888376A

Abstract

The invention relates to a cisplatin-loaded fibrin glue composite system combined treatment system which comprises a medicine bag and a radiotherapy device, wherein the medicine bag comprises a cisplatin/fibrin glue composite system prepared by taking cisplatin solution, thrombin solution and fibrinogen solution as raw materials, the concentration of the thrombin solution is 80-120 units/mL, and the concentration of the fibrinogen solution is 5-15 mg/mL. The combined treatment system has a synergistic effect on inhibiting the recurrence of locally advanced breast cancer, can effectively promote the apoptosis of breast cancer cells, realizes postoperative synchronous radiotherapy and chemotherapy, effectively inhibits the recurrence of locally advanced breast cancer, and reduces toxic and side effects. The combined treatment system provided by the invention has wide application prospect in preparing equipment for treating tumors (particularly local advanced tumors).

Description

Cisplatin-loaded fibrin glue composite system combined treatment system

Technical Field

The invention belongs to the field of material drug carriers, and particularly relates to a cisplatin-loaded fibrin glue composite system combined treatment system and application of the combined treatment system in preparation of equipment for treating tumors.

Background

The breast cancer is one of the most common malignant tumors of women, and has become a main cause of death of female malignant tumor patients in the global scope, about 140 million people are diagnosed with the breast cancer every year in the global scope, and the incidence rate of the breast cancer is increased year by year, thus seriously threatening the health of women. The locally advanced breast cancer represents a group of tumors with relative heterogeneity, mainly refers to breast cancer with primary focus larger than 5cm, extensive infiltration in breast or skin and chest wall adhesion fixation or/and regional lymph node metastasis, clinical stage III, and poorer prognosis. In recent years, clinical treatment of breast cancer has been greatly developed, and the main treatment strategies include various means such as surgery, chemotherapy, radiotherapy, endocrine therapy, targeted therapy and the like, but the treatment means for locally advanced breast cancer still has various limitations. Due to the characteristics of large tumor volume, deep infiltration, regional lymph node metastasis and the like, although patients with locally advanced breast cancer have the opportunity of surgical treatment, even if the patients are subjected to improved radical surgery combined with axillary lymph node cleaning, the patients are difficult to ensure that the tumors can be completely resected, and high risks of relapse and metastasis exist in parts such as the chest wall, the upper and lower areas of the clavicle and the like. Chemotherapy has the problems of large toxic and side effects of the whole body of the drug, long treatment period, tumor drug resistance and the like, brings limited clinical benefits, and also seriously affects the life quality of patients. Conventional fractionated radiotherapy, while helpful in reducing the postoperative recurrence rate of locally advanced breast cancer, is difficult to work with distant micrometastases. Furthermore, endocrine and targeted therapies benefit only a fraction of locally advanced breast cancer patients, and their efficacy is usually sustained only for a period of time, with eventual recurrence and metastasis of the tumor. In the clinical treatment decision of the locally advanced breast cancer, a specialist can adopt a comprehensive treatment mode according to the conditions of tumor volume, infiltration depth, regional lymph node metastasis and the like, but even if the comprehensive treatment mode is adopted, more than 90 percent of the deaths of locally advanced breast cancer patients are caused by tumor recurrence and metastasis. Therefore, how to effectively inhibit the recurrence and metastasis of locally advanced breast cancer after operation and improve the life quality of patients is one of the key scientific problems which plague clinicians and the field of life medicine at present.

In the actual clinical practice of surgical treatment of locally advanced breast cancer, modified radical surgery (total mastectomy plus axillary lymph node dissection) is often used by specialists, but even then it is difficult to ensure that the tumor can be completely resected and there is still a great risk of recurrence and metastasis. Because young women are in many cases, the requirements of patients on the appearance and the quality of life after operation are obviously improved, and if the classic radical operation (the enlarged excision of the soft tissue of 5cm around the tumor) is adopted, the difficulty that the body and the soul of the patients are aggravated is faced. The medicine can eliminate tumor and reduce normal tissue damage to the maximum extent, improve the life quality of patients and is the direction for treating and developing locally advanced breast cancer. The conventional segmentation mode is mostly adopted for clinical radiotherapy, and although the local control rate of tumors can be improved, the radiotherapy time is long and the compliance of patients is low. With the development of radiotherapy technology, stereotactic Radiotherapy (RT) can realize more accurate and higher-dose irradiation of a tumor target region by using a stereotactic technology, and has been widely applied to the treatment of early lung cancer, early breast cancer, liver cancer and partial oligometastasis; and have been tried for the treatment of some locally advanced tumors such as lung cancer and pancreatic cancer, etc., with good efficacy and without increasing the toxicity associated with radiotherapy. Therefore, the stereotactic radiotherapy used for the treatment after the locally advanced breast cancer surgery has the following advantages: effectively killing tumor cells and reducing the local recurrence rate of tumors; the systemic toxic and side effects are rarely generated; shorten the radiotherapy time and improve the compliance of patients.

Meanwhile, the concept of tumor treatment and tumor heterogeneity is well recognized. The treatment of tumors is gradually changed from single treatment to multiple combination treatments, thereby maximizing the treatment efficacy. Currently, clinical postoperative combined chemotherapy and radiotherapy is a common scheme for treating locally advanced breast cancer. Cisplatin (CDDP), a cell-nonspecific drug, binds to DNA, causes cross-linking, destroys the function of DNA, and inhibits mitosis of cells, and is commonly used for the treatment of head and neck tumors, ovarian cancer, breast cancer, and the like. In recent years, more and more researches show that CDDP plays a role in sensitizing and enhancing the tumor radiotherapy. However, CDDP is not targeted in vivo distribution and may cause adverse effects, most commonly nephrotoxicity, neurotoxicity, ototoxicity and myelosuppression, and this systemic mode of treatment still presents a potential safety hazard. Clinical studies have found that patients treated with CDDP and radiotherapy simultaneously produce more severe toxic side effects than patients treated alone.

Therefore, the research of the treatment system which can effectively reduce the local recurrence rate of the tumor, improve the treatment effect of various local advanced tumors including local advanced breast cancer, improve the prognosis of patients with local advanced tumors and reduce the adverse reaction of the medicine to the patients has very important significance.

Disclosure of Invention

The invention aims to provide a cisplatin-loaded fibrin glue composite system combined treatment system and application of the combined treatment system in preparing equipment for treating tumors.

The invention provides a combined treatment system which comprises a medicine bag and a radiotherapy device, wherein the medicine bag comprises a cisplatin/fibrin glue composite system prepared by taking a cisplatin solution, a thrombin solution and a fibrinogen solution as raw materials, the concentration of the thrombin solution is 80-120 units/mL, and the concentration of the fibrinogen solution is 5-15 mg/mL.

Further, the radiotherapy device is a stereotactic radiotherapy device.

Further, the concentration of the thrombin solution is 100 units/mL, and the concentration of the fibrinogen solution is 10 mg/mL;

and/or the volume ratio of the thrombin solution to the fibrinogen solution is 1: (1 to 9), preferably 1: 9.

further, the thrombin solution is prepared by dissolving thrombin in an aqueous calcium chloride solution, and the fibrinogen solution is prepared by dissolving fibrinogen in physiological saline; wherein the concentration of the calcium chloride aqueous solution is preferably 30-50 mmol/L, and more preferably 40 mmol/L; the mass fraction of the physiological saline is preferably 0.5-1.5%, and more preferably 0.9%.

Further, the concentration of the cisplatin solution is 1-3 mg/mL, preferably 2 mg/mL;

and/or the volume ratio of the cisplatin solution to the thrombin solution is 1: (0.5-2), preferably 1: 1.

further, the cisplatin solution is prepared by dissolving cisplatin in physiological saline, and the mass fraction of the physiological saline is preferably 0.5-1.5%, and more preferably 0.9%.

The invention also provides the application of the combined treatment system in preparing equipment for treating tumors.

Further, the tumor is a locally advanced tumor.

Further, the locally advanced tumor is locally advanced breast cancer, locally advanced osteosarcoma, locally advanced thyroid undifferentiated carcinoma, locally advanced lung cancer, locally advanced melanoma, locally advanced skin squamous carcinoma, locally advanced lung cancer, locally advanced esophageal cancer, locally advanced liver cancer or locally advanced pancreatic cancer;

preferably, the locally advanced tumor is locally advanced breast cancer.

Further, the device is capable of inhibiting recurrence of a tumor.

In the present invention, 0.9% physiological saline refers to 0.9% physiological saline by mass.

The combined treatment system provided by the invention has a synergistic effect on inhibiting the recurrence of locally advanced breast cancer, can effectively promote apoptosis of breast cancer cells, realizes synchronous postoperative radiotherapy and chemotherapy, effectively inhibits the recurrence of locally advanced breast cancer, and reduces toxic and side effects. The combined treatment system provided by the invention has wide application prospect in preparing equipment for treating tumors (particularly local advanced tumors).

Experiments also show that for the CDDP/Fibrin gel complex system forming the drug package in the combined treatment system, the stable CDDP/Fibrin gel complex system can be further formed with the CDDP solution only if the Fibrin glue obtained by mixing the fibrinogen solution and the thrombin solution with specific concentrations and a specific volume ratio (9:1) has good gelling condition.

CDDP is a commonly used and effective chemotherapeutic drug in clinic, and is suitable for treating tumors such as breast cancer, osteosarcoma, thyroid undifferentiated carcinoma, lung cancer and the like. Meanwhile, stereotactic Radiotherapy (RT) is an effective, noninvasive and less toxic and side effect treatment means in clinic, is suitable for superficial tumors (breast cancer, melanoma, skin squamous carcinoma and the like) and deep tumors (lung cancer, esophageal cancer, liver cancer, pancreatic cancer and the like), and has wide application prospect. Therefore, the combined treatment system consisting of the CDDP/Fibrin gel complex system and the radiotherapy device is suitable for treating various local advanced tumors and has larger clinical transformation potential.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a photograph of the Fibrin gel (A) obtained in example 1 and the CDDP/Fibrin gel complex system (B) obtained in example 2.

FIG. 2 is SEM images of the Fibrin gel (A) prepared in example 1 and the CDDP/Fibrin gel complex system (B) prepared in example 2.

FIG. 3 is a rheological diagram of the Fibrin gel (A) obtained in example 1 and the CDDP/Fibrin gel complex system (B) obtained in example 2.

FIG. 4 shows the cytotoxicity of CDDP/Fibrin gel complex against breast cancer cells.

FIG. 5 shows the effect of CDDP/Fibrin gel combination in combination with radiation therapy on breast cancer cells.

FIG. 6 is a graph of in vivo images of mice at various time points after topical injection of the Fibrin gel drug carrier.

FIG. 7 is a photograph showing (A) the procedure for establishing a model for postoperative recurrence of locally advanced breast cancer, (B) the preoperative tumor volume, (C) the tumor weight after surgical resection, and (D) the tumor after surgical resection; the grouping situation is as follows: (1) control, (2) fibre gel, (3) RT, (4) CDDP/fibre gel, (5) CDDP (i.v.) + RT, (6) CDDP/fibre gel + RT.

FIG. 8 is a schematic view of the treatment process (A), (B) a graph showing the change in tumor volume for each group, (C) a graph showing the body weight of mice for each group, (D) photographs of tumors for each group after the treatment is completed, and (E) the mean tumor weight of tumors for each group after the treatment is completed.

FIG. 9 is a picture of Ki-67 and TUNEL staining of tumor tissue.

FIG. 10 shows H & E stained pathological sections of organ tissues after treatment of each group of mice.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1: preparation of Fibrin glue (Fibrin gel)

1. Preparing thrombin solution

Thrombin was purchased from Dalian biotechnology, Inc., product number MB1368, molecular weight 37kD, CAS No. 9002-.

Adding thrombin into 40mmol/L calcium chloride water solution to dissolve, preparing thrombin solution with concentration of 100 units/mL, and storing at-20 deg.C.

2. Preparing fibrinogen solution

Fibrinogen was purchased from Dalian Melam Biotechnology Inc. under product number MB5809, molecular weight 340kDa, CAS No. 9001-32-5.

Dissolving fibrinogen in 0.9% physiological saline to obtain 10mg/mL fibrinogen solution, and storing at-20 deg.C.

3. Preparation of fibrin glue

Mixing the fibrinogen solution and the thrombin solution according to the volume ratio of 9:1 to form fibrin glue.

Example 2: preparation of cisplatin/Fibrin glue (CDDP/Fibrin gel) Complex System

1. Preparing thrombin solution

A thrombin solution having a concentration of 100 units/mL was prepared in the same manner as in step 1 of example 1, and stored at-20 ℃.

2. Preparing fibrinogen solution

A fibrinogen solution having a concentration of 10mg/mL was prepared in the same manner as in step 2 of example 1, and stored at-20 ℃.

3. Preparing CDDP solution

Cisplatin (CDDP) was purchased from Dalian Meiren Biotechnology Ltd.

Adding CDDP into 0.9% physiological saline to dissolve, preparing CDDP solution with concentration of 2mg/mL, and storing at-20 deg.C.

4. Preparation of CDDP/fibre gel composite system

And mixing the fibrinogen solution, the thrombin solution and the CDDP solution according to the volume ratio of 9:1:1 to obtain the CDDP/Fibrin gel composite system.

Example 3: preparation of a combination therapy System

The combination therapy system of the present invention consists of two parts: (1) a stereotactic radiotherapy device, and (2) a drug pack. Wherein, the drug package comprises the CDDP/Fibrin gel complex system prepared in the example 2.

The beneficial effects of the preparation of the invention are demonstrated by the following experimental examples.

Experimental example 1: gelling of fibrin glue prepared in different proportions

1. Experimental methods

100 units/ml of thrombin solution and 10mg/ml of fibrinogen solution were prepared in the same manner as in

steps

1 and 2 of example 1, and then the fibrinogen solution and the thrombin solution were mixed at different volume ratios shown in Table 1 to prepare different fibrin glues, and the gelling of each fibrin glue was observed.

2. Results of the experiment

TABLE 1 gelling behavior of fibrin glues obtained in different proportions

The results are shown in Table 1. It can be seen that the fibrin glue obtained by mixing a fibrinogen solution and a thrombin solution at specific concentrations and at a specific volume ratio (9:1) has a good gelling behaviour.

Experimental example 2: gel state observation of Fibrin gel and CDDP/Fibrin gel composite system

The results of observing the state of the Fibrin gel obtained in example 1 and the CDDP/Fibrin gel complex system obtained in example 2 are shown in FIG. 1.

It can be seen that the Fibrin gel prepared in example 1 is in a non-flowing transparent gel state (FIG. 1A), and the CDDP/Fibrin gel complex prepared in example 2 is in a non-flowing transparent gel state and has good system stability (FIG. 1B). Therefore, the Fibrin gel and the CDDP/Fibrin gel composite system prepared by the invention are hydrogel.

Experimental example 3: morphology observation of Fibrin gel and CDDP/Fibrin gel composite system

1. Experimental methods

The morphology of the Fibrin gel prepared in example 1 and the hydrogel of the CDDP/Fibrin gel complex system prepared in example 2 was observed by a scanning electron microscope.

2. Results of the experiment

FIG. 2A is a photograph of the morphology of Fibrin gel, and it can be seen that Fibrin glue prepared by the present invention has the characteristics of high permeability, porosity and interconnection. FIG. 2B is a photograph showing the appearance of the CDDP/fibre gel complex system, which shows that the complex system also has high permeability and porosity.

Experimental example 4: rheological behavior of Fibrin gel and CDDP/Fibrin gel composite systems

1. Experimental methods

The rheological behavior of the Fibrin gel prepared in example 1 and the CDDP/Fibrin gel composite hydrogel prepared in example 2 was investigated using a rotational rheometer.

2. Results of the experiment

As shown in FIG. 3, the elastic modulus G 'and the viscous modulus G' both tend to be stable, and G 'is higher than G', indicating the completion of the gelation process of the Fibrin gel and CDDP/Fibrin gel complex system.

Experimental example 5: cytotoxicity of CDDP/Fibrin gel complex system on breast cancer cells

1. Experimental method

The in-vitro inhibition effect of the CDDP/Fibrin gel composite system hydrogel on breast cancer cells is detected by an MTT method. Breast cancer cells 4T1 were cultured in 1640 medium (containing 10% fetal bovine serum, penicillin and streptomycin each 50U/mL). 4T1 cells at 3X 10³cells/well density were inoculated into 96-well plates and cultured overnight; after the cells are subjected to combined action for 48 hours by using 100 mu L of CDDP/Fibrin gel complex system with different concentrations, 20 mu L of MTT (5mg/mL) is added into each hole, and the incubation is continued for 4 hours in a carbon dioxide incubator; mu.L of dimethyl sulfoxide was added to each well to dissolve the resulting mixture to produce a purple substance, and the absorbance was measured at a wavelength of 570nm using a microplate reader, where n is 5. When the data is processed, the survival rate of the cells cultured in the blank culture medium is set as 100%, the ratio of the absorbance value of each of the rest experimental groups to the control group is the relative survival rate of each group, and the data is processed and analyzed according to the average value +/-standard deviation.

2. Results of the experiment

The results are shown in FIG. 4. As can be seen, the effect of inhibiting the activity of 4T1 cells of breast cancer cells is gradually enhanced along with the increase of the concentration of CDDP in the CDDP/Fibrin gel complex system, and the IC is₅₀The value was 0.513. mu.g/mL.

Experimental example 6: sensitization of CDDP/Fibrin gel complex system to radiotherapy

1. Experimental methods

The sensitization of the CDDP/Fibrin gel complex system prepared in example 2 to radiotherapy was investigated by colony formation experiments. 4T1 cells with different concentrations are inoculated into a 24-well plate for overnight culture; after irradiation with different radiotherapy doses (0Gy, 2Gy, 4Gy and 6Gy), the cells were continuously exposed in a transwell chamber with 100. mu.L of CDDP/Fibrin gel complex; the percentage of cloned 4T1 cells was finally counted to indicate the viability of the cells and the viability of the cell population, and the sensitization of the CDDP/Fibrin gel complex system to radiotherapy was tested. Controls were irradiated with different radiotherapy doses (0Gy, 2Gy, 4Gy and 6Gy) alone.

2. Results of the experiment

The results are shown in FIG. 5. It can be seen that, compared with the single use of the CDDP/Fibrin gel complex system (corresponding to the data of fig. 5 where the radiotherapy dose is 0) or the single use of radiotherapy, the inhibition effect of the CDDP/Fibrin gel complex system in combination with radiotherapy on the clonogenic capacity of breast cancer cells is significantly enhanced, which indicates that the combined use of the CDDP/Fibrin gel complex system of the present invention and radiotherapy can significantly improve the inhibition effect on the clonogenic capacity of breast cancer cells.

Experimental example 7: in vivo release of Fibrin gel drug loaded systems

1. Experimental methods

The small molecule fluorescent probe Cy5.5 SE is used as a model drug to be entrapped in the Fibrin gel prepared in the example 1, and the in vivo drug slow release condition of the Fibrin gel is observed by taking a picture by using a small animal in vivo imaging technology at different time points (t is 6h, 24h, 48h, 120h and 168h) after the subcutaneous injection of the mouse. The same concentration of Cy5.5 SE in water was used as a control (free drug group).

2. Results of the experiment

The results are shown in FIG. 6, the free drug group and the Fibrin gel hydrogel group carrying Cy5.5 SE have fluorescence enriched in subcutaneous parts and high fluorescence intensity; over time, the free drug group fluorescence greatly decreased, while the Cy5.5 SE-loaded Fibrin gel hydrogel group fluorescence showed a slow decrease trend, and on the 7 th day after injection, the Cy5.5 SE-loaded Fibrin gel hydrogel group still observed a high-intensity fluorescence signal in the subcutaneous region. The results indicate that the Fibrin gel drug-loaded hydrogel has a sustained release effect in vivo after local administration.

Experimental example 8: evaluation of treatment effect of CDDP/Fibrin gel complex system combined with stereotactic radiotherapy in mouse breast cancer postoperative recurrence model

1. Experimental method

The experiment was carried out according to the scheme shown in FIG. 8A. The method comprises the following specific steps:

(1) establishment of a mouse breast cancer postoperative recurrence model (fig. 7A): BALB/c female mice were used as experimental animals, and the right dorsal part of each mouse was inoculated with 1X 10⁶4T1 cells, and establishing a breast cancer subcutaneous tumor model when the tumor grows to 300mm³Surgical resection was performed in the right and left cases, and 10% of tumors remained to simulate tumor recurrence.

(2) Experimental grouping:

normal saline group (Control group), 200 μ L, tumor bed injection;

fibrin gel group, 200 μ L, tumor bed implantation;

RT group, radiotherapy dose 6 Gy;

CDDP/fibre gel group, 200. mu.l, CDDP (2mg/kg), tumor bed implantation;

CDDP (i.v.) + RT group, CDDP (2mg/kg) administered intravenously, RT (radiotherapy dose 6 Gy);

group CDDP/Fibrin gel + RT, 200. mu.l CDDP (2mg/kg), RT (radiotherapy dose 6Gy), tumor bed implantation.

The administration mode of the CDDP/Fibrin gel composite system is tumor bed implantation, and stereotactic radiotherapy is performed within 24h to realize synchronous postoperative chemoradiotherapy.

(3) After successful modeling of locally advanced breast cancer, the drug and/or radiation therapy is administered separately as described above. General (activity, behavior, hair, skin, secretions, etc.) and mortality was observed for each group of mice throughout the treatment period; tumor volume was measured every 2d (V ═ ab)²2; a and b represent the long and short diameters of mouse tumors, respectively) and the body weight of the mouse. After the experiment, the major organs (heart, liver, spleen, lung and kidney) were taken to carry out H&E staining to assess the safety of the combination treatment; tissue sections and immunohistochemical staining assays (Ki67 and TUNEL) were performed to evaluate each group of mice for overall tumor recurrence.

2. Results of the experiment

(1) Establishment of mouse breast cancer postoperative recurrence model

As shown in fig. 7B-7D, there was no significant difference between the preoperative tumor volume and the surgically excised tumor weight average among the six groups of tumor-bearing mice. The experimental result not only proves the successful establishment of the local advanced breast cancer postoperative recurrence model, but also explains the uniformity of the postoperative recurrence model.

(2) CDDP/Fibrin gel complex system combined with radiotherapy for preventing tumor recurrence effect

FIG. 8D is a photograph of tumors from each group after the treatment, and it can be seen that the postoperative recurrence rate of tumors from the Control group, RT group and CDDP/Fibrin gel group was 100%, while the postoperative recurrence rate of tumors from the CDDP/Fibrin gel + RT group was only 20%. The experimental result shows that the CDDP/Fibrin gel composite system is combined with radiotherapy, and the synergistic effect is achieved on the prevention of postoperative recurrence of breast cancer.

In addition, fig. 8C is a graph showing the average body weight change of each group of mice, and it can be seen that there is no significant difference in the body weight change of each group. FIG. 8B is a graph of tumor volume change in mice from each group, showing that tumors in both Control and Fibrin gel groups recurred and grew rapidly, but the Fibrin gel group was slightly slower than the Control group; compared with the Control group, the tumor growth of the CDDP/Fibrin gel + RT group is obviously slowed down, and the statistical difference is significant (p is 0.001); tumor growth was significantly slowed in the CDDP/Fibrin gel + RT group compared to CDDP (i.v.) + RT; compared with the Control group, the tumor volume of the mice in the RT group is reduced by 50.5 percent, the tumor volume of the mice in the CDDP/Fibrin gel group is reduced by 80 percent, and the tumor volume of the mice in the CDDP/Fibrin gel + RT group is reduced by 98.9 percent. FIG. 8E is the mean tumor weights of the groups after the end of treatment, and it can be seen that the tumor weights of the remaining groups after the end of treatment were lower than those of the Control group; compared with the Control group, the tumor weight of the CDDP/Fibrin gel + RT group is obviously reduced, and the statistical difference is significant (p is less than 0.001); a significant reduction in tumor weight in the CDDP/Fibrin gel + RT group compared to CDDP (i.v.) + RT; compared with the Control group, the tumor weight of the mice in the RT group is reduced by 80.8 percent, the tumor weight of the mice in the CDDP/Fibrin gel group is reduced by 87 percent, and the tumor weight of the mice in the CDDP/Fibrin gel + RT group is reduced by 96.5 percent.

(3) Ki67 and TUNEL

The proliferation of tumor cells was evaluated by Ki-67 immunohistochemical staining, and the results are shown in FIG. 9, in which the CDDP/Fibrin gel + RT group Ki-67LI was significantly reduced (p <0.001) compared to the control group. The results show that the CDDP/Fibrin gel complex system can effectively inhibit the proliferation of tumor cells in combination with radiotherapy. Apoptosis in tumor tissue was detected by TUNEL staining and figure 9 shows a typical image of apoptosis in 6 sets of 4T1 tumor sections. More apoptotic positive nuclei were observed in tumor sections from the CDDP/Fibrin gel + RT group compared to the control group (p ═ 0.008). Experimental results show that the CDDP/Fibrin gel complex system can obviously induce apoptosis of 4T1 tumor cells by combined radiotherapy, and the anti-tumor effect is obviously improved.

(4) H & E stained section

Pathological observation was performed on H & E stained sections of the major organs (heart, liver, spleen, lung and kidney) of tumor-bearing mice after the completion of the treatment, and the results are shown in fig. 10. Through comparative analysis, the pathological results of main organs of each group of mice are not obviously different from those of Control group mice, which shows that the local administration of the CDDP/Fibrin gel composite system prepared by the invention is a safe treatment mode and has no obvious toxic or side reaction when being used for treating a postoperative recurrence model of breast cancer.

The experiment result shows that the CDDP/Fibrin gel composite system provided by the invention has an excellent effect of inhibiting postoperative growth of breast cancer. In addition, the CDDP/Fibrin gel composite system provided by the invention is combined with radiotherapy, so that a synergistic effect is achieved in the inhibition of the recurrence of locally advanced breast cancer, and the treatment effect on locally advanced breast cancer can be remarkably improved.

In summary, the present invention provides a combination therapy system comprising a drug pack and a radiation therapy device. The combined treatment system has a synergistic effect on inhibiting the recurrence of locally advanced breast cancer, can effectively promote apoptosis of breast cancer cells, realizes synchronous postoperative radiotherapy and chemotherapy, effectively inhibits the recurrence of locally advanced breast cancer, and reduces toxic and side effects. The combined treatment system provided by the invention has wide application prospect in preparing equipment for treating tumors (particularly local advanced tumors).

Claims

1. Use of a combination therapy system for the manufacture of a device for the treatment of tumours; the tumor is a locally advanced tumor, and the locally advanced tumor is locally advanced breast cancer;

the combined treatment system consists of a medicine bag and a radiotherapy device, wherein the medicine bag is a cisplatin/fibrin glue composite system prepared by taking a cisplatin solution, a thrombin solution and a fibrinogen solution as raw materials, the concentration of the thrombin solution is 100 units/mL, the concentration of the fibrinogen solution is 10mg/mL, and the volume ratio of the thrombin solution to the fibrinogen solution is 1: 9; the concentration of the cisplatin solution is 2mg/mL, and the volume ratio of the cisplatin solution to the thrombin solution is 1: 1.

2. use according to claim 1, characterized in that: the radiotherapy device is a stereotactic radiotherapy device.

3. Use according to claim 1, characterized in that: the thrombin solution is prepared by dissolving thrombin in calcium chloride aqueous solution, and the fibrinogen solution is prepared by dissolving fibrinogen in physiological saline.

4. Use according to claim 3, characterized in that: the concentration of the calcium chloride aqueous solution is 30-50 mmol/L; the mass fraction of the normal saline is 0.5-1.5%.

5. Use according to claim 4, characterized in that: the concentration of the calcium chloride aqueous solution is 40 mmol/L; the mass fraction of the normal saline is 0.9%.

6. Use according to claim 1, characterized in that: the cisplatin solution is prepared by dissolving cisplatin in physiological saline.

7. Use according to claim 6, characterized in that: the mass fraction of the normal saline is 0.5-1.5%.

8. Use according to claim 7, characterized in that: the mass fraction of the normal saline is 0.9%.

9. Use according to any one of claims 1 to 8, characterized in that: the device is capable of inhibiting recurrence of a tumor.