Application of plasma activating solution in preparation of medicine for enhancing hypoxic tumor radiotherapy sensitivity

Abstract

The invention discloses an application of a plasma activation solution in preparation of a drug for enhancing hypoxia tumor radiotherapy sensitivity. Atmospheric pressure plasma generated at low power activates a culture solution DMEM, and acts on tumor cells in a hypoxic state, so that the hypoxic state of the tumor cells can be improved, the activity and clone ability of the hypoxic tumor cells can be inhibited, and the growth of tumors can be inhibited by cooperating with radiotherapy. The plasma activating solution can inhibit the activity of hypoxic colorectal cancer cells and regulate the cell cycle, so that the hypoxic colorectal cancer cell cycle is collected in the G2 / M period, the radiotherapy effect is enhanced, and the plasma activating solution has good safety when being applied in vivo.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to the application of a plasma-activated solution in the preparation of a drug for enhancing the radiosensitivity of hypoxic tumors. Background Art

[0002] Worldwide, colorectal cancer (CRC) has become the third most common malignancy (10%) and the second leading cause of cancer death (9.4%). Early-stage colorectal cancer can be effectively treated by surgical intervention or endoscopy. However, patients with colorectal cancer are usually diagnosed at an advanced stage. To improve the prognosis of patients with advanced CRC, combined therapy (chemoradiotherapy + surgery) has become the standard treatment modality.

[0003] Radiotherapy kills tumor cells through direct and indirect effects. Radiotherapy can directly damage various biomolecules, such as proteins, lipids, and most importantly, DNA. Two-thirds of DNA damage is mainly caused by reactive oxygen species (ROS) generated directly by radiation. In cancer treatment, radiotherapy has always been in a very important position, and radiotherapy resistance remains the main cause of treatment failure, while hypoxia is one of the most studied causes of radiotherapy resistance. In fact, hypoxia is associated with poor prognosis after tumor radiotherapy. Therefore, it is crucial to explore ways to improve the radiosensitivity of hypoxic tumors. Currently, to improve hypoxia-induced radiotherapy resistance, some scholars have conducted the following explorations: 1. Increasing oxygen supply; 2. Using hypoxia-activated prodrugs as radiosensitizers; 3. Using hypoxia-mediated signal reprogramming as a radiosensitization strategy; 4. Adopting a high-LET mode as an alternative to oxygen-dependent low-LET ionizing radiation. However, all of the above methods have their limitations.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the Invention

[0005] In view of the above deficiencies of the prior art, the present invention provides the application of a plasma-activated solution in the preparation of a drug for enhancing the radiosensitivity of hypoxic tumors, so as to solve the problem that the existing methods cannot effectively improve hypoxia-induced radiotherapy resistance.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0007] In a first aspect of the present invention, there is provided the application of a plasma-activated solution in the preparation of a drug for enhancing the radiosensitivity of hypoxic tumors, wherein the plasma-activated solution is a liquid treated with plasma.

[0008] Preferably, the preparation method of the plasma-activated solution is as follows:

[0009] Introduce working gas into the atmospheric pressure low-temperature plasma jet generation system. After the gas flow stabilizes, start the high-voltage power supply to excite the plasma jet generation tube, generating a plasma source, and making the plasma source act on the liquid to obtain the plasma-activated liquid.

[0010] Preferably, the working gas includes helium and oxygen. The working flow rate of helium is 2 L / min, and the air flow rate of oxygen is 10 mL / min.

[0011] Preferably, the working voltage is 4.5 - 4.7 kV, the frequency is 6.46 kHz, the discharge gap is 2 - 10 mm, and the discharge power is 0.467 - 0.550 W.

[0012] Preferably, the liquid is selected from one of the complete medium DMEM, the complete medium RPMI1640, and the medium MEM.

[0013] Preferably, the contact time between the plasma source and the liquid is 1 - 20 min, and the distance between the plasma discharge device port and the liquid surface is 0.5 - 2 cm.

[0014] Preferably, the tumor includes colorectal cancer, and the colorectal cancer cells have the characteristics of hypoxic tumor cells.

[0015] In the second aspect of the present invention, there is provided a drug for enhancing the radiosensitivity of hypoxic tumors, and the drug includes the above-mentioned plasma-activated liquid.

[0016] Beneficial effects:

[0017] The present invention discloses the application of the plasma-activated liquid in the preparation of a drug for enhancing the radiosensitivity of hypoxic tumors. The active particles generated by low-temperature plasma discharge can be effectively transferred into the liquid environment to obtain the plasma-activated liquid in the present invention. Then, the plasma-activated liquid is injected into the solid tumor through a colonoscope, which can improve the local hypoxic state of advanced tumors and have a certain inhibitory effect on the activity, migration, and proliferation of tumors, while causing no obvious damage to normal tissues. In addition, this plasma-activated liquid can regulate the cell cycle, cause the cell cycle of hypoxic tumor cells to be recruited in the G2 / M phase, enhance the radiotherapy effect, and its application in vivo has good safety. Although the direct plasma discharge beam can selectively kill superficial tumors such as melanoma and basal cell carcinoma, for tumors in deep organs such as the colon, rectum, and stomach, since the plasma beam cannot enter the body for discharge, the plasma beam cannot kill the tumors in deep organs such as the colon, rectum, and stomach, while using the plasma-activated liquid can effectively enhance the sensitivity of tumor treatment.

[0018] Specifically, the plasma activation solution provided by the present invention shows the following effects in improving the radiosensitivity of hypoxic tumors: By culturing in a hypoxic incubator for 12 hours, multiple hypoxic cell models of colorectal cancer (HCT8, HT29, HCT116, LoVo) are established. The activated DMEM (plasma activation solution) and non-activated DMEM are respectively applied to these cells, and then radiotherapy treatment is carried out. The results show that in the cells treated with the plasma activation solution, the level of HIF-1α decreases significantly, indicating that the hypoxic condition is improved, and the activity and colony formation ability of these cells are significantly lower than those of the cells treated with ordinary DMEM. In addition, for the hypoxic cells treated with the plasma activation solution, the activity of these cells decreases significantly after radiotherapy. These results indicate that the activated DMEM solution can significantly increase the radiosensitivity of hypoxic tumor cells.

[0019] Finally, the plasma activation solution provided by the present invention has no obvious damage to cells and animals and has good safety. At the same time, its production process is simple, the preparation cost is low, it is suitable for large-scale production, and it can be used as a drug to enhance radiosensitivity and applied by intratumoral injection or peritumoral injection. Description of the Drawings

[0020] Figure 1 It is for measuring the physicochemical properties of the plasma activation solution in Example 2 of the present invention and contains rich reactive oxygen and reactive nitrogen substances.

[0021] Figure 2 It is a protein detection chart for different groups to improve the hypoxic state of tumor cells in Example 3 of the present invention.

[0022] Figure 3 It is a numerical analysis chart for different groups to synergistically inhibit the activity of hypoxic tumor cells by radiotherapy in Example 4 of the present invention.

[0023] Figure 4 It is a data analysis chart for different groups to synergistically inhibit the proliferation of hypoxic tumor cells by radiotherapy in Example 5 of the present invention. Detailed Embodiments

[0024] The present invention provides the application of the plasma activation solution in the preparation of a drug for enhancing the radiosensitivity of hypoxic tumors. To make the purpose, technical solution and effects of the present invention clearer and more definite, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Cold atmospheric plasma (CAP) is an ionized gaseous substance composed of atoms that have lost some electrons and positive and negative electrons generated by atomic ionization. It contains a large amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS). With the increasing research and attention in the biomedical field, it has been widely applied to many branches of medicine, especially in the field of oncology. Its anti-cancer properties in in vitro treatment have been widely demonstrated in dozens of cancer types, such as skin cancer, breast cancer, colorectal cancer, brain cancer, lung cancer, cervical cancer, head and neck cancer, etc. Preclinical evidence shows that CAP has selective cytotoxicity to malignant cells compared with normal cells. Among all the components of CAP, ROS and RNS are considered to play an important role in inducing selective death of cancer cells. In addition, treating tumor cells with activated complete medium PAM can arrest their growth cycle at the G2 / M phase, and tumor cells at this stage are the most sensitive to radiation. Therefore, the combination of the two can improve the sensitivity of tumor cells to radiotherapy.

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments, and are only used to illustrate the present invention and in no way limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0027] Example 1 Preparation of Plasma-Activated Saline

[0028] The complete medium used in the present invention is prepared from Dulbecco's Modified Eagle Medium (DMEM, Viva cells) containing 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin-streptomycin (P / s, Gibco USA), and 1% non-essential amino acids x100 (NEAA x100, Gibco).

[0029] The plasma generation system (plasma jet, i.e., plasma jet device, see ① Y. Xu, Y. Li, X. Yang, D. Lu, Y. Zheng, J. Tan, W. Li, Q., Y. Liu, J. Gao, S. Wang. Low-temperature plasma-activated medium enhances the chemosensitivity of colorectal cancer cells by improving hypoxia. American journal of cancer research, 2023, 13(5), 1985-1998; ② Y. Li, Yang Lv, M. Tang, E. H. Choi, J. Wang, G. Lv, Y. Zhu, S. Wang, Y. Liu, Low-temperature plasma-jet-activated medium inhibited tumorigenesis of lung adenocarcinoma in a 3D in vitro culture model. Plasma Processes Polym. 2021; 18:2100049. doi.org / 10.1002 / ppap.202100049.) adopted in the embodiments of the present invention mainly consists of a plasma reaction chamber, a DC high-voltage power supply, and a gas source. The working gases used in this embodiment are high-purity helium (purity: 99.999%) and high-purity oxygen (purity: 99.999%).

[0030] The main preparation process of the plasma-activated liquid is as follows: Using the above plasma generation system, first unscrew the helium and oxygen valves, adjust the gas flows to 2 L / min and 10 L / min respectively, then place a 1.0 - 1.5 ml complete medium on the reaction table under the plasma jet, make the bottom end of the plasma jet device 1 cm away from the liquid surface, turn on the power switch, and turn off the power after reacting for 20 min to obtain the plasma-activated complete medium (Plasma Activated DMEM, PAM).

[0031] Example 2 Determination of the relative concentration of active particles in the plasma-activated liquid (PAM) prepared in Example 1

[0032] To determine the concentration levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the plasma-activated liquid (PAM) prepared in Example 1, a commercial kit was used: ROS, RNS superoxide anion (all purchased from Beijing Biobasic Biotechnology Co., Ltd., product numbers BB-46151, BB-46159, BB-46157 respectively). According to the instructions provided in the kit, 100 μL of the activated plasma-activated liquid was added to a 96-well plate, and the untreated culture medium was used as a control group to measure the concentration levels of the three particles respectively. The readings of the control group and the experimental group were measured by a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 516 nm). Taking the control group as a reference, it was calculated that the concentrations of the experimental group were 1.2 times (ROS concentration) and 1.1 times (superoxide anion concentration) that of the control group (as Figure 1 shown), and there was no significant difference in the total RNS level between the two groups. It was proved that the main reactive particles that might exist in the plasma-activated liquid were ROS superoxide anion and RNS.

[0033] Example 3 The plasma-activated liquid prepared in Example 1 improves the hypoxic state of tumor cells

[0034] In tumor treatment, tumor hypoxia often leads to tumor radioresistance. Research shows that when the partial pressure of O2 (pO2) is lower than 10 mmHg, tumor cells can obtain radio-biological hypoxia, thereby developing relative resistance to radiotherapy. For example, at 1 mmHg, the resistance of cancer cells to radiation is three times that of normal oxygenated cells. Enhancing the oxygen content of cells can effectively improve the hypoxic state to increase the radiotherapy sensitivity. Under hypoxic conditions, the protein of hypoxia-inducible factor (HIF-1α) in tumor cells will increase compared with before to promote angiogenesis and improve the hypoxic state.

[0035] Specific experimental procedures:

[0036] Detection of HIF-1α protein: Colorectal cancer cells were first cultured in complete medium until 80% confluence, and then divided into a CON group (treated with DMEM) and a PAM group (treated with PAM). After culturing at room temperature and normoxia for 12 hours in each group, they were then placed in an anaerobic incubator (O2 1%) for 12 hours. PAM was quickly added to the PAM group and the COM group, and then they were placed in the anaerobic incubator for culture. Subsequently, an appropriate amount of RIPA buffer (Sangon Biotech, China), protease inhibitor (Zhonghui Hercai, China), and PMSF (Zhongui Hercai, China) were used to harvest and lyse the cells. Then, a BCA protein assay kit (Sangon Biotechnology, China) was used to measure the protein concentration. Approximately 30 μg of protein samples were separated on a 12% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, MA, USA). After blocking the membrane with 5% bovine serum albumin (BSA) at room temperature for 1 hour, it was incubated with a primary antibody against HIF-1α (Cell Signaling Technology, 361691:100) at room temperature for 1 hour. Finally, a luminescent solution (Beyotime Institute of Biotechnology, China) was used for protein detection, and a high-sensitivity imaging system (Amersham ImageQuant 800, Cytiva, USA) was used for imaging. The results are as Figure 2 shown.

[0037] The above Western blot results indicated that the expression level of HIF-1α in the PAM group was significantly decreased compared with that in the CON group. Therefore, it can be seen that PAM can improve the hypoxic state of hypoxic tumor cells by inhibiting the expression of HIF-1α.

[0038] Example 4 Inhibitory effect of the plasma activation solution prepared in Example 1 on the activity of hypoxic tumor cells in combination with radiotherapy

[0039] During the occurrence and development of tumors, the enhancement of tumor cell activity often leads to the rapid proliferation of tumors, invasion of other tissues or organs, and the formation of new tumor sites, thus promoting the progression and deterioration of cancer. In this example, a cell activity experiment was used to detect the effect of PAM on the activity of hypoxic tumor cells.

[0040] Cell viability assay: The 2D Cell Counting Kit-8 (CCK-8) was used to evaluate cell viability. Cells cultured under standard conditions were harvested, suspended and seeded into 96-well flat-bottom plates, and divided into 4 groups according to the same method above (CON group: DMEM group; PAM group: PAM treatment group; 2Gy group: 2Gy radiotherapy group; 2Gy+PAM group: 2Gy radiotherapy + PAM treatment group), with a density of 3x10^3 cells per well. After culturing at normal temperature and normoxia for 12 hours, each group was then placed in an anaerobic incubator (O2 1%) for 12 hours. PAM was quickly added to the PAM group and the COM group, and DMEM was added to the CON group and the 2Gy group. At the same time, after incubating in the anaerobic incubator for 12 hours, both the COM and IR groups were quickly treated with 2Gy radiotherapy and then placed in the anaerobic incubator for culture. During the COM and IR treatments, the PAM and CON groups were also placed in the same environment. Then, they were incubated in a normoxic incubator for 48 hours. The proliferation rate was evaluated by adding CCK-8 solution to each well and incubating the cells for 3 hours. Absorbance measurements were performed at 450 nm using a microplate reader (Multiskan GO; Thermo Fisher Scientific). For verification, this experiment was independently performed at least three times. And data analysis was performed by Graphed Pism, as Figure 3 shown.

[0041] Through the Graphed Pism software, data analysis was performed on the absorbance measurement values of each group. It can be seen that in various colorectal cancer cell lines, the activity of hypoxic tumor cells in the PAM group was significantly decreased compared with the CON group, and the 2Gy combined with PAM group significantly inhibited cell activity compared with the simple radiotherapy group.

[0042] Example 5 Inhibitory effect of the plasma activation solution prepared in Example 1 on the proliferation of hypoxic tumor cells in combination with radiotherapy

[0043] Inhibiting tumor colony formation can significantly slow down the growth rate of tumors, because colony formation is a key process for tumor cell proliferation and expansion. In addition, reducing colony formation may also reduce the risk of tumor cell metastasis, thereby reducing the likelihood of metastasis. Inhibition of colony formation usually enhances the effectiveness of existing treatment methods, because it helps to reduce drug resistance after treatment and may ultimately improve the long-term prognosis of patients and increase the survival rate.

[0044] Specific experimental procedure: Tumor cells were divided into 4 groups according to the above method (ctrl group: blank treatment group; PAM group: PAM treatment group; PAM + 2Gy group: 2Gy radiotherapy + PAM treatment group), and then cultured for 2 days. After a series of procedures, such as washing with PBS (Gibco, USA), fixing with 4% paraformaldehyde (Beyo time, China), and staining with crystal violet, cell images were captured using a hypersensitive imaging system (Amersham ImageQuant 800, Cytiva, USA). Subsequently, the collected data were analyzed using ImageJ software, as Figure 4 shown.

[0045] The number of clones formed by hypoxic tumor cells after PAM treatment was significantly lower than that of the CON group, and the number of clones formed by the PAM + 2Gy group was significantly reduced compared with the PAM group and the CON group. Moreover, the number of clones formed was quantified by Image J, and quantitative data analysis was performed. It was found that the 2Gy + PAM group significantly reduced the clone formation ability of hypoxic tumor cells compared with the other groups.

[0046] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, it can be improved or transformed according to the above description. All such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. Application of plasma activated liquid in the preparation of drugs for enhancing the radiotherapy sensitivity of hypoxic tumors, wherein the plasma activated liquid is a liquid treated with plasma.

2. The use according to claim 1, characterized in that: The preparation method of the plasma activation solution is as follows: A working gas is introduced into the atmospheric pressure low-temperature plasma jet generating system, and after the gas flow is stabilized, a high-voltage power supply is started to excite the plasma jet generating tube to generate a plasma source, so that the plasma source reacts with the liquid to obtain the plasma activation liquid.

3. The use according to claim 2, characterized in that: The working gas includes helium and oxygen, the working flow rate of the helium is 2L / min, and the air flow rate of the oxygen is 10mL / min.

4. The use according to claim 2, characterized in that: The operating voltage is 4.5-4.7kV, the frequency is 6.46kHz, the discharge gap is 2-10mm, and the discharge power is 0.467-0.550W.

5. The use according to claim 2, characterized in that: The liquid is selected from one of complete culture medium DMEM, complete culture medium RPMI1640, and culture medium MEM.

6. The use according to claim 2, characterized in that: The contact time between the plasma source and the liquid is 1-20 minutes, and the distance between the plasma discharge device port and the liquid surface is 0.5-2 cm.

7. The use according to claim 1, characterized in that: The tumor includes colorectal cancer, and the colorectal cancer cells have characteristics of hypoxic tumor cells.

8. A drug for enhancing the radiotherapy sensitivity of hypoxic tumors, characterized in that: The medicine comprises the plasma activated liquid of claim 1.

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