Micellar mixing system for enhancing treatment effect of irreversible electroporation on pancreatic cancer and preparation method thereof

Abstract

The invention discloses a micelle mixing system for enhancing the treatment effect of irreversible electroporation on pancreatic cancer and a preparation method of the micelle mixing system, the injectable micelle mixing system is formed by mixing CGP37157-loaded PEG-PCL micelle CGP37157-coated PEG-PCL and a CaCl2 solution, and a CGP37157 sodium-calcium exchange inhibitor which is insoluble in water can be delivered to tumor cells; during electroporation treatment, calcium ions in mitochondria are inhibited from being discharged along with increase of permeability of tumor cells, calcium overload of the mitochondria is further caused, death of the tumor cells is accelerated, the efficiency of IRE treatment can be effectively improved, and good clinical application prospects are achieved.

Description

Technical field:

[0001] The invention relates to the field of biomedical materials, and in particular to a micelle capable of enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer and a preparation method thereof. Background technology:

[0002] Electroporation is a non-thermal ablation technique that generates nanoscale pores in the plasma membrane of tumor cells by emitting short, high-intensity electrical pulses, thereby inducing cell death. Compared with other traditional thermal ablation methods, electroporation has the advantages of minimal damage to blood vessels or bile ducts and retaining the regenerative potential of treated tissues. According to its working mechanism, electroporation can be divided into reversible electroporation (RE) and irreversible electroporation (IRE). IRE induces irreversible cell membrane perforation by using higher electric fields and pulse numbers, thereby leading to tumor cell death. The action characteristics of electroporation are most suitable for minimally invasive treatment of locally advanced pancreatic cancer (LAPC). The US FDA has approved the use of IRE for the treatment of pancreatic cancer. In 2015, the China National Drug Administration also approved IRE for the clinical treatment of pancreatic cancer. However, the electric field strength is often unevenly distributed during electroporation treatment of pancreatic cancer. When it is below the cell killing threshold, electroporation only disturbs the cell membrane, causing repairable perforations but failing to kill tumor cells. This is also an important reason for tumor recurrence after electroporation treatment. Summary of the invention:

[0003] 1. Technical issues to be resolved

[0004] In view of the defects of current irreversible electroporation treatment, the present invention provides a micelle and a preparation method thereof for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer, which can greatly improve the efficiency of electroporation treatment of tumors and solve the problem that the existing electroporation treatment is prone to tumor cell perforation repair, resulting in easy tumor recurrence and limited treatment effect.

[0005] (II) Technical solution

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer. The micellar mixed system is composed of a mixture of PEG-PCL micelles loaded with CGP37157 and a CaCl2 solution. The PEG-PCL micelles are amphiphilic polyethylene glycol PEG and polycaprolactone PCL block copolymers. The CGP37157 is a sodium-calcium exchange inhibitor with a CAS number of 75450-34-9 and a molecular formula of C15H11Cl2NOS.

[0008] CGP37157 is a benzothiazepine, a Na+-Ca2+ exchange inhibitor, and acts as a selective inhibitor of the mitochondrial sodium-calcium exchanger (IC50=0.36μM in isolated mitochondria). It does not affect channels, exchangers, or ATPases on the cardiac sarcolemma or ATPases on the sarcoplasmic reticulum. CGP37157 is often used to study the role of mitochondrial-derived calcium in cytoplasmic calcium homeostasis. CGP37157 inhibits sodium-dependent calcium efflux through the mitochondrial exchanger NCLX in multiple cell types.

[0009] The structural formula of CGP37157 is

[0010] The present invention also proposes a method for preparing a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer, comprising the following steps:

[0011] S1, Synthesis of carrier PEG-PCL:

[0012] 1 g polyethylene glycol, 1 mL toluene and 1.5 g polycaprolactone were added to the reaction bottle, stirred in an oil bath at 105°C until clear, stannous isooctanoate Sn(oct)2 was added to evacuate, nitrogen was protected, stirred in an oil bath at 105°C for 12 hours, cooled to room temperature, petroleum ether was added, stirred until a white solid precipitated, washed 3 times, filtered, nitrogen blown, and freeze-dried to obtain PEG-PCL;

[0013] The chemical structure of polyethylene glycol is:

[0014]

[0015] The chemical structure of polycaprolactone is:

[0016]

[0017] The synthesis process of carrier PEG-PCL is:

[0018]

[0019] S2, Synthesis of micelles CGP37157@PEG-PCL:

[0020] Take 200 mg of PEG-PCL carrier, add 2 mL of acetone, sonicate to completely dissolve, add 100 ul of CGP37157 (50 mg / mL dissolved in DMSO), vortex until clear, add 2 ml of water, and a clear and transparent micellar solution is obtained. The acetone is evaporated and dialyzed at 4 ° C for 2 hours to obtain the final product CGP37157@PEG-PCL. The concentration of CGP37157 is 2 mg / mL.

[0021] S3, mixing the micelles obtained in step S2 with the CaCl2 solution to make the CaCl2 concentration be 168 mM and the CGP37157 concentration be 1 mg / mL, which is the micelle mixed system.

[0022] In the above step S1, the stirring speed of the oil bath stirring is 200 rpm.

[0023] In the above step S2, the molecular weight cut-off of the dialysis bag used for dialysis is 3500Da.

[0024] In the above step S2, after obtaining the final product CGP37157@PEG-PCL, 50% sucrose solution is added to the micelle solution to make the final concentration of sucrose 5% for easy storage.

[0025] The present invention also provides a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer. preparation Used in drugs for treating pancreatic cancer.

[0026] The pancreatic cancer cell type is one of the following: PANC-1, KPC mouse spontaneous pancreatic ductal adenocarcinoma cell line A548.

[0027] The working principle of the micellar mixed system provided by the present invention is that the cell permeability increases during electroporation IRE treatment, and calcium ions can quickly enter the cell, resulting in the destruction of the intracellular calcium ion balance, especially the mitochondrial calcium ion balance. CGP37157 is a mitochondrial Na+ / Ca2+ exchange inhibitor, which can inhibit the discharge of calcium ions in the mitochondria, further leading to mitochondrial calcium overload, leading to cell death. However, simple CGP37157 is extremely difficult to dissolve in water. By encapsulating CGP37157 in PEG-PCL micelles, a micellar mixed system is prepared, and a CaCl2 solution is mixed. The micellar mixed system of the mixed CaCl2 solution can greatly improve the effect of calcium ions penetrating into tumor cells during electroporation treatment, and can effectively improve the effect of synergistic treatment. When used specifically, after IRE treatment, the mixed system is injected intratumorally, and the injection dose is approximately equal to the size of the tumor volume, which can effectively improve the efficiency of electroporation treatment and prevent tumor cell repair.

[0028] (III) Beneficial effects

[0029] The present invention forms an injectable micelle mixed system by mixing CGP37157-loaded PEG-PCL micelles CGP37157@PEG-PCL and a CaCl2 solution, so that the poorly water-soluble CGP37157 sodium-calcium exchange inhibitor can be delivered into tumor cells. During electroporation treatment, as the permeability of tumor cells increases, the efficiency of calcium ion penetration into tumor cells is greatly improved, and at the same time, the excretion of calcium ions in mitochondria is effectively inhibited, further leading to mitochondrial calcium overload and accelerating tumor cell death. The efficiency of IRE treatment can be effectively improved, and the invention has good clinical application prospects. Description of the drawings:

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below.

[0031] Figure 1 This is a schematic diagram of the chemical equation for the synthesis of the carrier PEG-PCL;

[0032] Figure 2 is the NMR image of PEG-PCL;

[0033] Figure 3 This is a schematic diagram of micelle CGP37157@PEG-PCL;

[0034] Figure 4 is the HPLC chromatogram of micelle CGP37157@PEG-PCL;

[0035] Figure 5 is the particle size distribution diagram of micelle CGP37157@PEG-PCL;

[0036] Figure 6 This is the transmission electron microscopy image of micelle CGP37157@PEG-PCL;

[0037] Figure 7 The release graph of CGP37157 under different pH conditions;

[0038] Figure 8 It is the killing effect on pancreatic cancer cells under reversible electroporation (RE) conditions;

[0039] Fig. 9 The cytotoxic effects of different calcium ion concentrations on pancreatic cancer cells;

[0040] Fig.10 is the cytotoxic effect of different CGP37157 concentrations on pancreatic cancer cells;

[0041] Fig.11 The cell-killing effect of RE combined with calcium ions and CGP37157 pancreatic cancer cells;

[0042] Fig.12 This is a graph showing the killing effect of RE combined with low-concentration micelle mixed system on pancreatic cancer cells;

[0043] Fig.13 This is a diagram showing the tumor size after the IRE combined with micelle mixed system treated the mouse orthotopic pancreatic cancer model;

[0044] Fig.14 This is a graph showing the tumor weight after the IRE combined with micelle mixed system treated the mouse orthotopic pancreatic cancer model;

[0045] Fig.15 This is the survival curve of mice after the IRE combined with micelle mixed system treated the mouse orthotopic pancreatic cancer model;

[0046] Fig.16 This is a diagram showing the tumor size after the IRE combined with micelle mixed system treated the subcutaneous pancreatic cancer model in mice;

[0047] Fig.17 This is a graph showing the tumor weight after the IRE combined with micelle mixed system treated the subcutaneous pancreatic cancer model in mice;

[0048] Fig.18 This is the tumor growth curve of the mouse subcutaneous pancreatic cancer model after the IRE combined with micelle mixed system was treated;

[0049] Fig.19 This is the survival curve of mice after the IRE combined with micelle mixed system treated the subcutaneous pancreatic cancer model in mice; Specific implementation method:

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0051] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0052] (1) Prepare the sample. The sample solution in the NMR tube is generally kept at a certain height of 4 to 5 cm, about 500 to 600 μL;

[0053] (2) Place the sample to be tested into the instrument;

[0054] (3) Locking the field. To ensure that the frequency of the static magnetic field provided by the magnet does not shift;

[0055] (4) Tuning. To ensure that the resonant frequency in the resonant circuit is consistent with the pulse frequency emitted by the spectrometer to the probe, the probe can receive all the transmitted power, thereby obtaining a better signal-to-noise ratio;

[0056] (5) Field shimming. Adjust the magnetic field gradients in different directions in the shimming coil to compensate for the inhomogeneity of the static magnetic field, thereby obtaining test results with satisfactory resolution and sensitivity;

[0057] (6) Collect and process data (see Figure 2 ).

[0058] Example 2 Detection of CGP37157 drug loading in micelles by high performance liquid chromatography

[0059] (1) Prepare DMSO solutions of CGP37157 at standard concentrations of 1 mg / ml, 0.5 mg / ml, 0.2 mg / ml, 0.1 mg / ml, and 0.05 mg / ml, respectively;

[0060] (2) The synthesized CGP37157@PEG-PCL micelle solution was diluted 10 times with DMSO to prepare the sample to be loaded on the machine;

[0061] (3) Setting liquid phase conditions: Phase A: 0.1% trifluoroacetic acid aqueous solution; Phase B: 0.1% trifluoroacetic acid acetonitrile solution;

[0062] Time (min) A(％) B(％) 0 90 10 10 10 90 20 90 10

[0063] (4) Liquid chromatography: record the peak time and absorption peak area of ​​different standard concentrations, and draw a standard curve; (5) Detect the sample to be tested (see Figure 4 ), the concentration was calculated based on the absorption peak area and the standard curve. The experiment showed that the concentration of CGP37157 in the undiluted micelles was 1.96 mg / ml, the drug loading was 2.3%, and the encapsulation efficiency was 84.3%.

[0064] Example 3 Determination of CGP37157@PEG-PCL micelle particle size

[0065] (1) Sample preparation: The synthesized CGP37157@PEG-PCL micelles were diluted 10 times with distilled water to prepare a dispersion of appropriate concentration, and filtered to remove large aggregates and dust particles to ensure that the dispersion was clear and transparent;

[0066] (2) On-line: Turn on the dynamic light flash instrument, check that the laser, detector and other components are working properly, draw an appropriate amount of sample (2 mL) into a clean glass cuvette, and slowly insert it into the sample chamber to avoid bubbles. Wait for 2-5 minutes to allow the sample to reach thermal equilibrium and release all bubbles, then start measuring. Each sample is usually measured 3-5 times;

[0067] (3) Data processing and statistical analysis of micelle size distribution. Experiments show that the micelle size of CGP37157@PEG-PCL is concentrated between 60 and 90 nm, with good uniformity (see Figure 5).

[0068] Example 4 Transmission electron microscopy observation of CGP37157@PEG-PCL micelle morphology

[0069] (1) preparing samples according to the above synthesis method;

[0070] (2) Pipette 10 μl of the sample and drop it onto a copper grid to precipitate for 1 min. Use filter paper to remove the floating liquid.

[0071] (3) 10 μl of uranyl acetate (or phosphotungstic acid staining solution) was added dropwise onto the copper grid to allow precipitation for 1 min, and the floating liquid was removed by filter paper.

[0072] (4) Dry at room temperature for several minutes, and perform electron microscopy imaging at 80-120 kV.

[0073] (5) Observation under a transmission electron microscope and image analysis showed that most CGP37157@PEG-PCL micelles were spherical and had good uniformity (see Figure 6 ).

[0074] Example 5 Release of CGP37157 from CGP37157@PEG-PCL micelles under different pH conditions

[0075] (1) preparing PBS buffer solution systems of pH 7.4 and pH 5.2 (pH adjusted with acetic acid-sodium acetate buffer) respectively;

[0076] (2) CGP37157@PEG-PCL micelles were synthesized according to the above method. 500 μl of micelle solution was taken at pH 7.4 and

[0077] The pH 5.2 PBS buffer solution was diluted 1:1 and placed in two dialysis bottles. The dialysis bottles were placed in 500 ml beakers containing pH 7.4 and pH 5.2 PBS buffer solutions respectively. The beakers were placed in a 37°C water bath for heating.

[0078] (3) Pipette 20ul of sample from the dialysis bottle at five time points: 0h, 2h, 4h, 6h, and 12h, and add the corresponding buffer to the original volume after each sampling;

[0079] (4) 80 μl of DMSO was added to all samples to prepare samples for HPLC, and the concentration of CGP37157 was determined according to the steps in Example 2;

[0080] (5) Based on the drug concentration-time curve, the cumulative release percentage was calculated and the drug release curve was drawn. The experiment showed that CGP37157 was released slightly faster under acidic conditions, but there was no significant difference in the cumulative release amount (see Figure 7 ).

[0081] Example 6 Killing effect on pancreatic cancer cells under reversible electroporation conditions

[0082] (1) Cell culture: KPC A548 cells and Panc-1 cells were revived using DMEM complete medium containing 5% and 10% fetal bovine serum, respectively, inoculated into T25 culture flasks, and cultured in a 37°C, 5% CO2 incubator. The medium was changed after 24 h, and then every 48 h;

[0083] (2) Cell treatment: When the cell confluence in the culture flask was about 90%, the culture medium was removed, the cells were rinsed twice with sterile PBS, and KPC A548 (5 min) and Panc-1 (2 min) were digested with 1 ml of trypsin. The cell suspension was collected and centrifuged, and resuspended with culture medium to adjust the cell density to 5 × 10 5 / mL. Set up a control group (control) and different voltage electroporation groups. RE treatment: Take 700ul of cell suspension and place it in a clean cuvette, which is placed in the base of the electroporator. The voltage is set to 0, 200, 400, 600, and 800V / cm, the number of pulses is 20, the duration of the electric pulse in each pulse cycle is 0.001 seconds, and the interval time is 0.999 seconds. After RE treatment, the cell density is adjusted to 5×10 4 / ml, inoculated in 96-well plates, 200ul (10,000 cells) per well.

[0084] (3) CCK-8 assay: 24 h after inoculating the cells, remove the old culture medium, wash gently with sterile PBS once, add 60 μl of serum-free culture medium containing 10% CCK-8 assay solution to each well, incubate in a 37°C incubator for 1 h, and measure the absorbance at 450 nm with a microplate reader;

[0085] (4) Statistical analysis, experiments show that as the voltage increases, cell activity gradually decreases (see Figure 8 ), indicating that the killing effect of electroporation on cells gradually increased.

[0086] Example 7 Cytotoxic effects of different calcium ion concentrations on pancreatic cancer cells

[0087] (1) Cell culture is the same as above;

[0088] (2) Cell treatment: resuspend the cells and adjust the cell density to 5×10 4 / ml, adjust the calcium ion concentration in the culture medium so that the final calcium ion concentration is distributed between 0 and 100 mM, and inoculate in a 96-well plate, 200ul (10,000 cells) per well;

[0089] (3) CCK-8 detection method is the same as above;

[0090] (4) Statistical analysis showed that tumor cell activity was significantly reduced only at extremely high calcium ion concentrations (see Fig. 9 ).

[0091] Example 8 Cytotoxic effects of different CGP37157 concentrations on pancreatic cancer cells

[0092] (1) Cell culture is the same as above;

[0093] (2) Cell treatment: resuspend the cells and adjust the cell density to 5×10 4 / ml, adjust the concentration of CGP37157 in the culture medium so that the final concentration of CGP37157 is distributed between 0 and 100uM, and inoculate in a 96-well plate, 200ul (10,000 cells) per well;

[0094] (3) CCK-8 detection method is the same as above;

[0095] (4) Statistical analysis showed that tumor cell activity was significantly reduced only at extremely high CGP37157 concentrations (see Fig.10 ).

[0096] Example 9 Cell killing effect of RE combined with calcium ions and CGP37157 pancreatic cancer cells () Cell culture was the same as above;

[0097] (2) Cell treatment: a control group, a reversible electroporation group (RE), and a reversible electroporation combined with Ca 2+ group and reversible electroporation combined with Ca 2+ RE treatment: 700ul of cell suspension was placed in a clean cuvette, which was placed in the base of the electroporator. The voltage was set to 700V / cm, the number of pulses was 20, the duration of the electric pulse in each pulse cycle was 0.001 seconds, and the interval time was 0.999 seconds. After RE treatment, the cell density was adjusted to 5×10 4 / ml, and immediately added different concentrations of Ca 2+ or CGP37157, seeded in 96-well plates, 200 ul (10,000 cells) per well;

[0098] (3) CCK-8 detection method is the same as above;

[0099] (4) Statistical analysis showed that compared with the RE alone group, RE combined with Ca 2+ The cell activity of the group decreased, and with the increase of calcium ion concentration, the cell activity decreased more significantly. On this basis, CGP37157 can significantly enhance the toxic effect of calcium ions on cells ( Fig.11 ); indicating that RE and Ca 2+Example 10 Killing effect of RE combined with low concentration micelle and calcium ion mixed system on pancreatic cancer cells

[0100] (1) Cell culture: KPC A548 cells and Panc-1 cells were revived using DMEM complete medium containing 5% and 10% fetal bovine serum, respectively, inoculated into T25 culture flasks, and cultured in a 37°C, 5% CO2 incubator. The medium was changed after 24 h, and then every 48 h;

[0101] (2) Cell treatment: When the cell confluence in the culture flask was about 90%, the culture medium was removed, the cells were rinsed twice with sterile PBS, and KPC A548 (5 min) and Panc-1 (2 min) were digested with 1 ml of trypsin. The cell suspension was collected and centrifuged, and resuspended with culture medium to adjust the cell density to 5 × 10 5 / mL. Set up a control group (control), a drug-treated group (Ca 2+ and micelle mixed system: CaCGP), reversible electroporation group (RE), reversible electroporation combined with Ca 2+ Group (RE+Ca) and reversible electroporation combined with Ca 2+ and micelle mixed system group (RE+CaCGP). RE treatment: 700ul of cell suspension was placed in a clean cuvette, which was placed in the base of the electroporator. The voltage was set to 700V / cm, the number of pulses was 20, the duration of the electric pulse in each pulse cycle was 0.001 seconds, and the interval time was 0.999 seconds. After RE treatment, the cell density was adjusted to 5×10 4 / ml, and immediately added Ca to the combined group 2+ or CaCGP (Ca 2+ The final concentration was 1 mM, and the final concentration of CGP37157 was 10 uM), and the cells were seeded in a 96-well plate, with 200 ul (10,000 cells) per well.

[0102] (3) CCK-8 assay: 24 h after inoculating the cells, remove the old culture medium, wash gently with sterile PBS once, add 60 μl of serum-free culture medium containing 10% CCK-8 assay solution to each well, incubate in a 37°C incubator for 1 h, and measure the absorbance at 450 nm with a microplate reader;

[0103] (4) Statistical analysis showed that the cell activity of the RE+CaCGP group was lower than that of the other groups (see Fig.12 ), indicating that it has a strong killing effect on tumor cell lines.

[0104] Example 11 Therapeutic effect of IRE combined with micelle mixed system on mouse orthotopic pancreatic cancer model

[0105] (1) Mouse preparation: Healthy 8-week-old female C57 mice were purchased and housed in an SPF animal room. The skin was prepared and ear-tagged one day before inoculation.

[0106] (2) Preparation of inoculated cells: KPC A548 cells were cultured in a 15 cm culture dish as described above. When the cell confluence in the culture flask was about 90%, the culture medium was removed, the cells were rinsed twice with sterile PBS, and KPC A548 was digested with 2 ml of trypsin (5 min). The cell suspension was collected and centrifuged, and resuspended with PBS to adjust the cell density to 2 × 10 7 / mL;

[0107] (3) Inoculation: Use isoflurane inhalation anesthesia, adjust the anesthesia machine to the appropriate ventilation volume, and after complete anesthesia, fix the patient in the lateral position on the operating table, fix the head and limbs with paper tape, disinfect the skin on the left side of the abdomen, expose the surgical area, and use scissors and forceps to cut the skin and peritoneum to a length of about 1 cm to expose the spleen and pancreas. Gently lift the pancreas with forceps, and use a microsyringe to aspirate 15 μl of cell suspension (3×10 5 Cells) were injected into the pancreatic body. During the injection, a small bubble-like bulge was observed at the injection site. After the injection, a cotton swab was used to gently press the injection site for 1 minute to prevent the cell fluid from overflowing. The peritoneum and skin were sutured in sequence with 5-0 absorbable sutures with needles, and the mice were disinfected again. The mice were kept warm until they woke up and then put back into the cage. The mice were observed every 2-3 days.

[0108] (4) Treatment of mice: Eight days after inoculation, mice were enrolled for treatment. A control group (control) and a drug-treated group (Ca 2+ and micelle mixed system: CaCGP), irreversible electroporation group (IRE), irreversible electroporation combined with Ca 2+ Group (IRE+Ca) and irreversible electroporation combined with Ca 2+ IRE treatment: According to the above method, mice were anesthetized, pancreatic tumors were exposed, and the tumor diameter was observed to be about 5 mm. Electrode needles were connected to the electroporator and placed at both ends of the tumor. The voltage was set to 1500 V / cm, the number of pulses was 100, the duration of the electric pulse in each pulse cycle was 0.001 seconds, and the interval time was 0.999 seconds. Tumors were treated with IRE along the long axis and short axis, for a total of 200 pulses. Immediately after IRE treatment, Ca was injected into the tumor. 2+ or Ca 2+ and micelle mixed system 100ul (Ca 2+ The final concentration was 168 mM, and the final concentration of CGP37157 was 1 mg / ml). The peritoneum and skin were then sutured in sequence with absorbable sutures, disinfected again, and kept warm until the mice woke up and were returned to the cage. The mice were observed every 2-3 days and their weights were recorded.

[0109] (5) One week after treatment, the mice were sacrificed, the tumors were removed, and the tumor weight was measured. The experiment showed that the tumors in the IRE+CaCGP group were significantly smaller than those in the other groups (see Figure 13-14 );

[0110] (6) The modeling and treatment methods were the same as above, and the survival curve of mice was recorded. The experimental results showed that the survival time of the IRE+CaCGP group was significantly longer than that of the other groups (see Fig.15 )

[0111] Example 12 Therapeutic effect of IRE combined with micelle mixed system on subcutaneous pancreatic cancer model in mice

[0112] (1) Mice and cells were prepared as described above and inoculated on the right back with 100 μl of cell suspension (2

[0113] ×10 6 cells), and were enrolled for treatment 8 days after inoculation;

[0114] (2) Treatment groups and methods were the same as above. Two weeks after treatment, mice were sacrificed, tumors were removed, and tumor weights were measured. The tumors in the IRE+CaCGP group were significantly smaller than those in the other groups (see Figure 16-17 )

[0115] (3) Modeling and treatment methods were similar to those described above, and the tumor size of mice was measured every 3 days.

[0116] Tumor volume = long diameter × short diameter 2 / 2, draw the tumor growth curve (see Fig.18 ) and mouse survival curve (see Fig.19 ), the experiment showed that the tumor growth in the IRE+CaCGP group was slower than that in other groups, and the survival time of mice was prolonged.

[0117] Through the above experiments, pancreatic cancer cell lines derived from mice were injected into the pancreas or subcutaneously of mice to establish an in situ or subcutaneous model of pancreatic cancer. It was found that the mixed micelle system provided by the present invention can significantly enhance the therapeutic effect of irreversible electroporation on in situ and subcutaneous pancreatic cancer models. The combined treatment can make the tumor shrink more significantly and significantly prolong the survival of mice.

[0118] In summary, the present invention provides a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer and a preparation method thereof, which can greatly improve the efficiency of electroporation treatment of tumors and solve the problem that the existing electroporation treatment is prone to tumor cell perforation repair, resulting in easy tumor recurrence and limited treatment effect.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the protection scope of the present invention. In addition, after reading the technical content of the present invention, those skilled in the art may make various changes, modifications or variations to the present invention, and all of these equivalent forms also fall within the protection scope defined by the present application.

Claims

1. A micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer, characterized in that: The micelle mixed system is composed of a mixture of PEG-PCL micelles loaded with CGP37157 and a CaCl2 solution. The PEG-PCL micelles are amphiphilic polyethylene glycol PEG and polycaprolactone PCL block copolymers. The CGP37157 is a sodium-calcium exchange inhibitor with a CAS number of 75450-34-9 and a molecular formula of C15H11Cl2NOS.

2. The method for preparing a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer according to claim 1, characterized in that: The following steps are involved: S1, Synthesis of carrier PEG-PCL: 1 g polyethylene glycol, 1 mL toluene and 1.5 g polycaprolactone were added to the reaction bottle, stirred in an oil bath at 105°C until clear, stannous isooctanoate Sn(oct)2 was added to evacuate, nitrogen was protected, stirred in an oil bath at 105°C for 12 hours, cooled to room temperature, petroleum ether was added, stirred until a white solid precipitated, washed 3 times, filtered, nitrogen blown, and freeze-dried to obtain PEG-PCL; S2, Synthesis of micelles CGP37157@PEG-PCL: Take 200 mg of PEG-PCL carrier, add 2 mL of acetone, and completely dissolve it by ultrasound. Add 100 ul of CGP37157, which is dissolved in DMSO at a concentration of 50 mg / mL, vortex until clear, add 2 ml of water, and a clear and transparent micellar solution is obtained. The acetone is evaporated and dialyzed at 4°C for 2 hours to obtain the final product CGP37157@PEG-PCL. S3, mixing the CGP37157@PEG-PCL micelles obtained in step S2 with the CaCl2 solution to make the CaCl2 concentration be 168 mM and the CGP37157 concentration be 1 mg / mL, which is the micelle mixing system.

3. The method for preparing a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer according to claim 2, characterized in that: The stirring speed of the oil bath stirring is 200 rpm.

4. The method for preparing a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer according to claim 2, characterized in that: The molecular weight cut-off of the dialysis bag used in the dialysis is 3500Da.

5. The method for preparing a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer according to claim 2, characterized in that: In step S2, after obtaining the final product CGP37157@PEG-PCL, 50% sucrose solution is added to the micelle solution to make the final concentration of sucrose 5% for easy storage.

6. Use of the micellar mixed system according to claim 1 or the micellar mixed system prepared by the method according to any one of claims 2 to 5 in the preparation of a drug for treating pancreatic cancer.

7. The use according to claim 6, characterized in that The pancreatic cancer cell type is one of the following: PANC-1, KPC mouse spontaneous pancreatic ductal adenocarcinoma cell line A548.

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