A micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer and its preparation method

Abstract

The present invention discloses a micellar mixed system for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer and a preparation method thereof. By mixing CGP37157-loaded PEG-PCL micelles (CGP37157@PEG-PCL) with a CaCl2 solution to form an injectable micellar mixed system, the poorly water-soluble CGP37157 sodium-calcium exchange inhibitor can be delivered into tumor cells. During electroporation therapy, as the permeability of tumor cells increases, the excretion of calcium ions in mitochondria is inhibited, further leading to mitochondrial calcium overload and accelerating tumor cell death. This can effectively improve the efficiency of IRE treatment and has good clinical application prospects.

Description

Technical field:

[0001] The present 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 uses short, high-intensity electrical pulses to create nanoscale pores in the plasma membrane of tumor cells, thereby inducing cell death. Compared with other traditional thermal ablation methods, electroporation offers advantages such as minimal damage to blood vessels or bile ducts and preservation of the regenerative potential of the treated tissue. Based on its mechanism of action, electroporation can be categorized as reversible electroporation (RE) and irreversible electroporation (IRE). IRE uses a high electric field and pulse frequency to induce irreversible cell membrane perforation, leading to tumor cell death. The characteristics of electroporation make it ideal for minimally invasive treatment of locally advanced pancreatic cancer (LAPC). The US FDA has approved IRE for the treatment of pancreatic cancer, and the China National Medical Products Administration also approved IRE for clinical treatment of pancreatic cancer in 2015. However, the electric field strength during electroporation treatment of pancreatic cancer is often unevenly distributed. 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 problems solved

[0004] In response to the defects of current irreversible electroporation treatment, the present invention provides a micelle that enhances 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 existing electroporation treatment is prone to tumor cell perforation repair, resulting in easy tumor recurrence and limited treatment effect.

[0005] (2) 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-type Na+-Ca2+ exchange inhibitor that acts as a selective inhibitor of the mitochondrial sodium-calcium exchanger (IC50 = 0.36 μM in isolated mitochondria). It does not affect channels, exchangers, or ATPases on the cardiac sarcolemma or on the sarcoplasmic reticulum. CGP37157 is commonly 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] Add 1 g of polyethylene glycol, 1 mL of toluene, and 1.5 g of polycaprolactone to a reaction flask, stir in an oil bath at 105°C until clear, add stannous isooctanoate (Sn(oct)2), evacuate, protect with nitrogen, stir in an oil bath at 105°C for 12 hours, cool to room temperature, add petroleum ether, stir until a white solid precipitates, wash three times, filter, blow with nitrogen, and lyophilize 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 the carrier PEG-PCL is as follows:

[0018]

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

[0020] Take 200 mg of PEG-PCL carrier, add 2 mL of acetone, and sonicate to completely dissolve. Then add 100 μl of CGP37157 (50 mg / mL dissolved in DMSO), vortex until clear, add 2 mL of water, and a clear and transparent micellar solution will appear. The acetone is evaporated and the solution is 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, thereby obtaining 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 was added to the micelle solution to make the final sucrose concentration 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 micelle 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 excretion 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 to make a micelle mixed system, and mixing it with a CaCl2 solution, the micelle mixed system mixed with the CaCl2 solution can greatly improve the effect of calcium ion penetration into tumor cells during electroporation treatment, which can effectively improve the effect of synergistic treatment. In specific use, after IRE treatment, the mixed system is injected intratumorally, and the injection dose is approximately equal to the tumor volume, which can effectively improve the efficiency of electroporation treatment and prevent tumor cell repair.

[0028] (3) 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. This system can deliver the poorly water-soluble CGP37157 sodium-calcium exchange inhibitor into tumor cells. During electroporation therapy, as the permeability of tumor cells increases, the efficiency of calcium ion penetration into tumor cells is greatly improved. 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. This system can effectively improve the efficiency of IRE treatment and 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 following briefly introduces the drawings required for use in the embodiments.

[0031] Figure 1 Schematic diagram of the chemical equation for the synthesis of carrier PEG-PCL;

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

[0033] Figure 3 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 Transmission electron microscopy image of micelle CGP37157@PEG-PCL;

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

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

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

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

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

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

[0043] Figure 13 This is a diagram showing the tumor size after the IRE combined with micelle mixed system was used to treat the orthotopic pancreatic cancer model in mice;

[0044] Figure 14 This is a graph showing the tumor weight after the IRE combined with micelle mixed system was used to treat the orthotopic pancreatic cancer model in mice;

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

[0046] Figure 16 This is a diagram showing the tumor size in a mouse subcutaneous pancreatic cancer model after treatment with the IRE combined with micelle mixed system;

[0047] Figure 17 This is a graph showing the tumor weight in a mouse subcutaneous pancreatic cancer model after treatment with the IRE combined with micelle mixed system;

[0048] Figure 18 This is the tumor growth curve of the mouse subcutaneous pancreatic cancer model after the IRE combined with micelle mixed system was used to treat the model.

[0049] Figure 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 with reference to 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, approximately 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 transmitted 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 shim 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-fold with DMSO to prepare the sample to be loaded onto the instrument;

[0061] (3) Set the liquid phase conditions: Phase A: 0.1% trifluoroacetic acid in water; Phase B: 0.1% trifluoroacetic acid in acetonitrile;

[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-fold with distilled water to prepare a dispersion of appropriate concentration. The dispersion was filtered to remove large aggregates and dust particles to ensure that the dispersion was clear and transparent.

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

[0067] (3) Data processing and statistical analysis of micelle size distribution showed that the CGP37157@PEG-PCL micelle size was 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) Prepare samples according to the above synthesis method;

[0070] (2) Pipette 10 μl of the sample and drop it onto a copper grid for precipitation for 1 min. Absorb the floating liquid with filter paper.

[0071] (3) 10 μl of uranyl acetate (or phosphotungstic acid staining solution) was added dropwise to the copper grid to allow it to settle for 1 minute, and the supernatant was removed by absorbing it with filter paper.

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

[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 at different pH conditions

[0075] (1) Prepare PBS buffer solution systems at 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, and 500 μl of micelle solution was taken at pH 7.4,

[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 to heat;

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

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

[0080] (5) Based on the drug concentration versus 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 supplemented with 5% and 10% fetal bovine serum, respectively, and seeded into T25 culture flasks. The cells were 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 in 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. Place the cuvette in the base of the electroporator. Set the voltage to 0, 200, 400, 600, and 800V / cm respectively. The number of pulses is 20. The duration of the electric pulse in each pulse cycle is 0.001 seconds, and the rest time is 0.999 seconds. After RE treatment, the cell density is adjusted to 5×10 4 / ml, seeded in 96-well plates, 200ul (10,000 cells) per well.

[0084] (3) CCK-8 assay: 24 h after seeding the cells, remove the old culture medium and gently wash once with sterile PBS. Add 60 μl of serum-free culture medium containing 10% CCK-8 assay solution to each well and incubate in a 37°C incubator for 1 h. Measure the absorbance at 450 nm using 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 was 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 into 96-well plates, with 200 μl (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 Figure 9 ).

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

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

[0093] (2) Cell treatment: resuspend the cells and adjust the cell density to 5×10 4 / ml, adjust the CGP37157 concentration in the culture medium so that the final concentration of CGP37157 is distributed between 0 and 100uM, and inoculate into 96-well plates, with 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 Figure 10 ).

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

[0097] (2) Cell treatment: control group, reversible electroporation group (RE), reversible electroporation combined with Ca 2+ group and reversible electroporation combined with Ca 2+ and CGP37157 groups. RE treatment: 700 μl of cell suspension was placed in a clean cuvette, which was placed in the base of the electroporator. The voltage was set to 700 V / cm, the number of pulses was 20, the duration of each pulse cycle was 0.001 seconds, and the rest 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 to the combined group 2+ or CGP37157, seeded in 96-well plates, 200 μl (10,000 cells) per well;

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

[0099] (4) Statistical analysis: Compared with the RE group alone, 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 ( Figure 11 ); indicating that RE and Ca 2+Example 10 Killing effect of RE combined with low concentration micelles 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 supplemented with 5% and 10% fetal bovine serum, respectively, and seeded into T25 culture flasks. The cells were 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 in culture medium to adjust the cell density to 5 × 10 5 / mL. Set up control group (control), drug treatment 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 rest 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 seeding the cells, remove the old culture medium and gently wash once with sterile PBS. Add 60 μl of serum-free culture medium containing 10% CCK-8 assay solution to each well and incubate in a 37°C incubator for 1 h. Measure the absorbance at 450 nm using a microplate reader.

[0103] (4) Statistical analysis showed that the cell activity of the RE+CaCGP combination group was lower than that of the other groups (see Figure 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 in situ 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 the 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 approximately 90%, the culture medium was aspirated, the cells were rinsed twice with sterile PBS, and KPC A548 were digested with 2 ml of trypsin (5 min). The cell suspension was collected and centrifuged, and resuspended in 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 The cells were injected into the pancreatic body. During the injection, a small bubble-like bulge was observed at the injection site. After the injection, the injection site was gently pressed with a cotton swab for 1 minute to prevent the cell fluid from overflowing. The peritoneum and skin were sutured in sequence with 5-0 absorbable sutures with a needle, and the mice were disinfected again. The mice were kept warm until they woke up and then returned to the cage. The mice were observed every 2-3 days.

[0108] (4) Treatment of mice: 8 days after inoculation, mice were enrolled in treatment groups, with control group (control), 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+ There were 5 groups: mice with micelle mixed system (IRE+CaCGP). IRE treatment: According to the above method, mice were anesthetized and pancreatic tumors were exposed. The tumor diameter was observed to be about 5 mm. Electrodes 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 rest 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 mice were then sutured with absorbable sutures to the peritoneum and skin, disinfected again, and kept warm until they regained consciousness. The mice were then returned to their cages and their status and weights were recorded every 2-3 days.

[0109] (5) One week after treatment, the mice were sacrificed, the tumors were removed, and the tumor weight was measured. The results 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 Figure 15 )

[0111] Example 12 Therapeutic Effect of IRE Combined with Micellar Mixture System on Subcutaneous Pancreatic Cancer Model in Mice

[0112] (1) Mice and cells were prepared as described above and inoculated on the right side of the 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) The modeling and treatment methods were the same as above, and the tumor size of the mice was measured every 3 days.

[0116] Tumor volume = long diameter × short diameter 2 / 2, draw the tumor growth curve (see Figure 18 ) and mouse survival curve (see Figure 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 in situ or subcutaneous pancreatic cancer models. 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 and its preparation method for enhancing the therapeutic effect of irreversible electroporation on pancreatic cancer, which can significantly improve the efficiency of electroporation treatment of tumors and solve the problem that existing electroporation treatment is prone to tumor cell perforation repair, resulting in easy tumor recurrence and limited therapeutic effect.

[0119] Finally, it should be noted that the above embodiments are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention. In addition, after reading the technical contents of the present invention, those skilled in the art may make various changes, modifications, or variations to the present invention, and all such equivalent forms also fall within the scope of protection 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: Add 1 g of polyethylene glycol, 1 mL of toluene, and 1.5 g of polycaprolactone to a reaction flask, stir in an oil bath at 105°C until clear, add stannous isooctanoate (Sn(oct)2), evacuate the solution, protect with nitrogen, and stir in an oil bath at 105°C for 12 hours. Cool to room temperature, add petroleum ether, and stir until a white solid precipitates. Wash three times, filter, blow with nitrogen, and lyophilize to obtain PEG-PCL. S2, Synthesis of micelles CGP37157@PEG-PCL: Take 200 mg of PEG-PCL carrier, add 2 mL of acetone, and sonicate to completely dissolve. Then add 100 μl of CGP37157, which is dissolved in DMSO at a concentration of 50 mg / mL. Vortex until clear. Add 2 mL of water to form a clear and translucent micellar solution. Rotary evaporate the acetone and dialyze 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 a CaCl2 solution to make the CaCl2 concentration 168 mM and the CGP37157 concentration 1 mg / mL, thereby obtaining the micelle mixed 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 sucrose concentration 5% for easy storage.

6. Use of the micelle mixed system according to claim 1 or the micelle 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.

Images