Pharmaceutical composition for treating liver cancer through arterial embolism as well as preparation method and application of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition for treating liver cancer through arterial embolism, the pharmaceutical composition is composed of iodine oil and tirapazamine-loaded nano-vesicles, and the tirapazamine-loaded nano-vesicles comprise nano-vesicles and tirapazamine coated in the inner cores of the nano-vesicles. The invention also provides a preparation method and application of the pharmaceutical composition for treating liver cancer by arterial embolism, the preparation method comprises the following steps: performing liver left artery microcatheter hyperselection on a test rabbit VX2 liver cancer model through in-vivo test, then confirming tumor responsible blood vessels by radiography, and then applying the iodophol and tirapazamine-loaded nano-vesicle compound for embolism until thorough embolism is realized, thereby obtaining the pharmaceutical composition for treating liver cancer by arterial embolism. According to the present invention, the application of the iodophol and the tirapazamine-loaded nano-vesicle compound in the treatment of liver cancer by TAE is safe and effective, the combination with TAE provides the strong synergistic anti-tumor effect, and the TPZ can be promoted to the deep tumor under the action of the UTMD so as to induce the apoptosis of the hypoxia tumor cells;

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition for arterial embolization treatment of liver cancer, and a preparation method and application thereof. Background Art

[0002] Primary hepatocellular carcinoma has a low early diagnosis rate, high malignancy, high recurrence rate and poor prognosis. Currently, non-surgical options represented by transarterial chemoembolization (TACE) have become the main method recommended by guidelines for the treatment of advanced liver cancer, but the long-term efficacy is poor. Although the development of imaging technologies such as digital subtraction angiography and CBCT has improved the technical performance and effectiveness of TACE, due to the complexity of liver cancer blood supply, the embolization of tumor blood vessels may still be incomplete, which allows some tumors to survive in a relatively hypoxic microenvironment. Studies have shown that hypoxia itself can increase the malignancy of tumors and cause tumor infiltration and metastasis, while affecting the action process of anti-tumor drugs. Although TACE combined with targeted and immunotherapy has further improved the efficacy, the residual hypoxic tumor cells after TACE surgery for liver cancer may be one of the important reasons for tumor recurrence and affecting long-term efficacy. Therefore, embolization combined with hypoxia targeted therapy may be an effective strategy to improve prognosis.

[0003] The purpose of TACE is to selectively deliver the anti-tumor drug-lipiodol emulsion to the tumor artery. The infusion of the emulsion can directly target the delivery of high concentrations of anti-cancer drugs to the tumor and block the terminal arterioles with lipiodol. However, the clinical outcomes of TACE are still unsatisfactory, usually only causing partial remission in 15% to 55% of patients and extending the median survival from 16 months to 20 months. The poor outcomes may be partly due to the low efficiency of tumor drug delivery. In addition, chemotherapy drugs cannot effectively target tumor stem cells, which is the root cause of tumor recurrence after conventional TACE treatment. Their cytotoxic effects may be inhibited in the hypoxic environment created by embolization.

[0004] Tirapazamine (TPZ), a hypoxia-activated prodrug, is a strong bioreductant that can selectively produce cytotoxicity to hypoxic cells and specifically damage hypoxic tumor cells. It causes single-strand or double-strand breaks in the DNA of hypoxic cells, leading to cell death. This can just break through the bottleneck of liver cancer treatment in the hypoxic environment after TACE. However, the main existing administration methods of TPZ are intravenous injection or arterial perfusion before TAE embolization, both of which have some limitations and cannot make TPZ specifically act on the hypoxic microenvironment of liver cancer. For example, intravenous injection will produce many adverse reactions (such as ototoxicity, muscle spasm, bone marrow toxicity, olfactory nerve necrosis, retinal detachment, etc.); and arterial administration before embolization may limit the subsequent distribution of TPZ in the tumor due to embolism, and its clinical efficacy is entirely attributed to the first-pass effect after arterial perfusion of TPZ, which is limited in clinical application. How to increase the efficacy of TPZ while reducing adverse reactions, the choice of drug carrier is particularly important.

[0005] Liposomes have the characteristics of biocompatibility, biodegradability, low toxicity and immunogenicity, and simple preparation. At the same time, the enhanced permeability and retention effect in solid tumors facilitates the passive enrichment of liposomes in tumor tissues. As a carrier of chemotherapeutic drugs, it is of great significance to improve bioavailability, reduce side effects and reduce drug resistance.

[0006] It can be seen that establishing a new TACE drug delivery system is conducive to the accumulation of drugs at the tumor site, which is crucial for enhancing tumor control and reducing side effects. Summary of the invention

[0007] In view of the problems and shortcomings in the prior art, the object of the present invention is to provide a pharmaceutical composition for treating liver cancer by arterial embolization, and a preparation method and application thereof.

[0008] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a pharmaceutical composition for treating liver cancer by arterial embolization, wherein the pharmaceutical composition is composed of iodized oil and tirapazamine-loaded nanovesicles; the tirapazamine-loaded nanovesicles include nanovesicles and tirapazamine coated in the inner core of the nanovesicles.

[0010] Preferably, the volume ratio of the iodized oil to the tirapazamine-loaded nanovesicles is 2:1.

[0011] Preferably, the encapsulation efficiency of the TPZ-NBs is 35.81%.

[0012] The second aspect of the present invention provides a method for preparing the pharmaceutical composition according to the first aspect, comprising the following steps:

[0013] (1) mixing phospholipids and cholesterol, and dissolving them in an organic solvent to obtain a mixed solution;

[0014] (2) evaporating the mixed solution obtained in step (1) to fully volatilize the organic solvent, adding a citrate buffer solution with a pH of 4.0 to fully hydrate the solution to obtain blank nanovesicles;

[0015] (3) dissolving tirapazamine in ultrapure water and mixing thoroughly to prepare a tirapazamine solution;

[0016] (4) adding the tirapazamine solution obtained in step (3) to the blank nanovesicles obtained in step (2), incubating in a water bath to obtain tirapazamine-loaded nanovesicles, and placing the tirapazamine-loaded nanovesicles in an ultrasonic instrument for ultrasonic treatment under ice bath conditions to obtain tirapazamine-loaded nanovesicle spherical particles of uniform size;

[0017] (5) dispersing the tirapazamine-loaded nanovesicles in iodized oil to form a tirapazamine-loaded nanovesicle complex in the iodized oil.

[0018] Preferably, in step (1), the mass ratio of the phospholipid to cholesterol is 4:1; and the organic solvent is chloroform.

[0019] Preferably, the solubility of the tirapazamine solution in step (3) is 0.5-1 mg / ml.

[0020] Preferably, in step (4), the water bath incubation temperature is 40° C., the ultrasonic treatment power is 200 W, the ultrasonic frequency is 2 s for 2 seconds, the interval is 3 s, and it is repeated 240 times.

[0021] Preferably, in step (5), a Voltexer is used to mix the iodized oil and the tirapazamine-loaded nanovesicles in a ratio of 2:1 for at least 5 times, each time for 5 seconds, to obtain a complex of the iodized oil and the tirapazamine-loaded nanovesicles.

[0022] The third aspect of the present invention provides the use of the pharmaceutical composition described in the first aspect in a drug for arterial embolization to treat liver cancer.

[0023] Preferably, the pharmaceutical composition is used for TAE treatment, and iodized oil and tirapazamine-loaded nanovesicle complex embolization are administered, followed by transabdominal UTMD.

[0024] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0025] As a traditional embolic agent, iodized oil can stay in tumor blood vessels for a long time due to its lipophilicity, effectively blocking the blood supply of the tumor, and causing hypoxia and necrosis of the tumor tissue. This physical embolic effect is widely used in the treatment of solid tumors such as liver cancer. On the other hand, the core mechanism of action of nanovesicles loaded with tirapazamine is that it is selectively activated in hypoxic tumor cells to generate free radical forms, causing DNA damage and inducing cell death. This mechanism makes it particularly suitable for the treatment of hypoxic tumor cells; the drug-loaded form of nanovesicles enhances the targeting and penetration of the drug, making it more concentrated on the tumor site. The embolic effect of iodized oil causes the tumor to be in a hypoxic state, thereby increasing the sensitivity of the tumor to tirapazamine, forming a synergistic effect. This combination therapy has significant advantages. The synergistic effect of the dual mechanism not only effectively prevents the growth of the tumor, but also reduces the systemic toxicity of the drug, and the therapeutic effect is better.

[0026] The present invention successfully constructs a drug delivery system of a nanovesicle complex loaded with tirapazamine, which can provide a good drug encapsulation rate and drug sustained release efficiency. The nanovesicle complex loaded with tirapazamine can exert a strong anti-tumor effect in an in vitro hypoxic microenvironment; in an in vivo experiment, the left hepatic artery microcatheter superselection is performed on the experimental rabbit VX2 liver cancer model, and then the tumor-responsible blood vessel is confirmed by angiography, and then iodized oil and the nanovesicle complex loaded with tirapazamine are given for embolization until the embolism is complete. After completion, UTMD is performed through the abdomen, which verifies that the application of iodized oil and the nanovesicle complex loaded with tirapazamine in TAE treatment of liver cancer is safe and effective, and has a stronger synergistic anti-tumor effect in combination with TAE. Under the action of UTMD, it can promote TPZ to the deep part of the tumor and induce apoptosis of hypoxic tumor cells. The present invention overcomes the shortcomings of the prior art and has a high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The morphology of blank NBs and TPZ-NBs under a light microscope at 200 times magnification, the left picture shows NBs, and the right picture shows TPZ-NBs;

[0028] Figure 2 is the cumulative release rate of TPZ at different time points;

[0029] Figure 3 The proliferation of two liver cancer cell lines in different states, different TPZ-NBs concentrations, and different time points (A: 97H, normal conditions; B: 97H, hypoxic conditions; C: HepG2, normal conditions; D: HepG2, hypoxic conditions);

[0030] Figure 4Figure A shows the scratch test results of 97H liver cancer cell line under different TPZ-NBs concentrations (microscope magnification: ×40); Figure B shows the scratch area of ​​HepG2 liver cancer cells at 0, 6h, 12h, 24h, and 48h under different TPZ-NBs concentrations;

[0031] Figure 5 A in the middle shows the results of the scratch test on the HepG2 liver cancer cell line under different TPZ-NBs concentrations. (Microscope magnification: ×40); B shows the scratch area of ​​HepG2 liver cancer cells at 0, 6h, 12h, 24h, and 48h under different TPZ-NBs concentrations;

[0032] Figure 6 A in the middle shows the cell status of 97H transmembrane under different TPZ-NBs concentrations (microscope magnification: ×200); B shows the transmembrane area of ​​97H under different TPZ-NBs concentrations;

[0033] Figure 7 A in the middle shows the status of HepG2 cells penetrating the membrane under different TPZ-NBs concentrations (microscope magnification: ×200); B shows the area of ​​HepG2 cells penetrating the membrane under different TPZ-NBs concentrations;

[0034] Figure 8 Schematic diagram of the experimental rabbit VX2 liver cancer model;

[0035] Fig. 9 This is the CT comparison before and after TAE of VX2 liver cancer experimental rabbits and the TAE process;

[0036] Fig.10 The bar graphs of liver and kidney function of VX2 liver cancer experimental rabbits in each group before and after TAE operation;

[0037] Fig.11 This is the gross appearance of liver tumors in each group of VX2 liver cancer experimental rabbits two weeks after embolization;

[0038] Fig.12 HE staining results of tumor tissue sections of VX2 liver cancer experimental rabbits in each group after surgery;

[0039] Fig.13 The immunohistochemical staining and quantitative analysis results of HIF-1, VEGF, PCNA and CD31 in the tumor tissues of the VX2 liver cancer experimental rabbits in each group two weeks after embolization;

[0040] Fig.14 These are the results of TUNEL immunofluorescence staining and quantitative analysis of tumor tissues of VX2 liver cancer experimental rabbits in each group two weeks after embolization. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below through embodiments in combination with the accompanying drawings. 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.

[0042] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0043] Example 1

[0044] In this example, tirapazamine-loaded nanovesicles (TPZ-NBs) were prepared by dialysis, and their morphology was observed under a microscope.

[0045] 1. Test methods

[0046] 1.1 Preparation of TPZ-loaded nanovesicles (TPZ-NBs)

[0047] (1) Preparation of blank vesicles. Phospholipids and cholesterol were weighed in a mass ratio of 4:1 and mixed, and then 500 mg of the mixture was dissolved in 20 ml of chloroform and rotary evaporated in a rotary evaporator (40°C water bath) to fully evaporate the organic solvent, and 15 ml of pH 4.0 citrate buffer was added to fully hydrate to obtain blank nanovesicles.

[0048] (2) Preparation of tirapazamine-loaded nanovesicles (TPZ-NBs) by dialysis. Dissolve 7.5 mg TPZ in 15 ml ultrapure water, mix thoroughly to make a 0.5 mg / ml TPZ solution, add it to 15 ml blank NBs, incubate in a 40°C water bath, and obtain TPZ-NBs of uneven size. Place TPZ-NBs in an ultrasonic instrument under ice bath conditions, and set the parameters as follows: 200 W, ultrasonic 2 s, interval 3 s, repeat 240 times. Use high shear dispersion method to prepare nano-scale ultrasonic vesicles (NBs). Spherical particles of uniform size can be observed under a microscope, which are TPZ-NBs.

[0049] 1.2 Identification of physicochemical properties of TPZ-NBs

[0050] (1) Sample preparation. The sample net is a copper net covered with a layer of carbon film. Use filtered deionized water to clean and dry glassware and instruments, and use an oxygen plasma cleaner to treat the copper net. Then drop 10ul of NBs sample on the net. Cover the sample net with a culture dish and leave it at room temperature until the sample dries naturally. Carefully rinse the sample net with deionized water, then rinse with ethanol, and finally use filter paper to absorb the ethanol.

[0051] (2) Measurement. Turn on the transmission electron microscope, load and inject samples, adjust the sample height, adjust the instrument, image the standard sample, and select an appropriate magnification so that a large number of nanoparticles appear in the field of view. At the same time, ensure that the resolution at this magnification is much smaller than the size of the nanoparticles. Observe at least 200 nanoparticles in different areas of the sample, repeat the above steps, and image the sample to be tested.

[0052] 2. Results Analysis

[0053] like Figure 1 As shown, the nanovesicles observed under a microscope mainly exhibit a perfect spherical outline; a high degree of size uniformity; their surface is clear at high magnification edges, with no rough or granular structure. Although most of the nanovesicles are dispersed in the form of single particles, a small amount of tiny aggregates are also observed. Under a transmitted light microscope, blank NBs appear transparent and colorless, while TPZ-NBs appear light yellow and can be clearly distinguished from the surrounding medium.

[0054] Example 2

[0055] In this example, ultracentrifugation was used to obtain the encapsulation efficiency of TPZ-NBs, and dynamic dialysis was used to perform the drug release test.

[0056] 1. Test methods

[0057] 1.1 Encapsulation efficiency determination

[0058] The encapsulation efficiency of TPZ-NBs was obtained by ultracentrifugation. 1 ml of TPZ-NBs was placed in an ultracentrifuge and centrifuged at 18000 rpm for 30 min. The supernatant was fixed to 10 ml with ultrapure water and the absorbance was measured. Encapsulation efficiency = (W total amount - W free amount) / W total amount × 100%. W total amount is the total amount of TPZ-NBs, and W free amount is the total amount of free NBs.

[0059] 1.2 Dynamic dialysis method for drug release test

[0060] Take 1ml TPZ-NBs and transfer it to the pre-treated dialysis bag, close the buckle, and place it in a beaker containing 100ml release mediator, then place the beaker in a constant temperature water bath oscillator and shake it at a constant speed (100rpm) at 37℃. After the release begins, take 1ml of drug release medium at different time points of 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, and 48h (add an equal amount of isothermal blank medium at the same time), and calculate the cumulative release rate of the drug.

[0061] 2. Results Analysis

[0062] After 5 mg of tirapazamine (TPZ) was dissolved in 10 ml of ultrapure water, a 0.5 mg / ml TPZ stock solution was prepared. Ultrapure water was used as a control. Under the condition of absorbance at 260 nm, the encapsulation rate was measured according to the formula encapsulation rate = (W total amount - W free) / W total amount × 100%. The encapsulation rate of TPZ-NBs was measured to be 35.81%. Figure 2 As shown, the cumulative release rate of TPZ-NBs decreased significantly in the first 6 hours, slowly decreased from 6 to 24 hours, remained relatively stable at 57.2% after 24 hours, and was 58% at 48 hours.

[0063] Example 3

[0064] In this example, hypoxia models of liver cancer cell lines 97H and HepG2 were established, and the optimal concentration of TPZ-NBs on hypoxic cells was determined by cytotoxicity analysis. Liver cancer cell proliferation assay, scratch assay and Transwell cell invasion assay were used to perform in vitro cell verification of the effects of TPZ-NBs on the proliferation, migration and invasion of liver cancer cells.

[0065] 1. Test methods

[0066] 1.1 Establishment of hypoxia model of liver cancer cell lines 97H and HepG2

[0067] 97H and HepG2 liver cancer cell lines were inoculated into 24-well plates (3×10 5 / well) and incubated in an incubator overnight. The next day, the culture medium was replaced with fresh complete medium, and then 97H and HepG2 liver cancer cells were treated with 20 ng / ml CoCl2 as a hypoxia inducer to construct a cell model under a hypoxia-simulated environment.

[0068] 1.2 Experiment on the effect of TPZ-NBs on the proliferation of liver cancer cells under hypoxic microenvironment.

[0069] 97H and HepG2 liver cancer cell lines were inoculated in 96-well plates (1×104 / well), and after the cells adhered to the wall, TPZ-NBs with different final concentrations of 0μg / ml, 10μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, 80μg / ml, and 100μg / ml were added, and co-cultured with liver cancer cells in a hypoxic cell model. The culture medium contained a final concentration of 20ng / ml CoCl2 and 10% fetal bovine serum DMEM culture medium. The absorbance at 450nm was detected by CCK-8 method at different time points of 6h, 12h, 24h, and 48h, and the cell survival rate was calculated as follows: Cell survival rate % = (experimental group-blank group) / (control group-blank group) × 100%. The effect of TPZ-NBs on the proliferation of liver cancer cells under a hypoxic microenvironment was observed under a 200x microscope.

[0070] 1.3 Scratch test

[0071] TPZ-NBs at concentrations of 0μg / ml, 10μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, 80μg / ml, and 100μg / ml were used to treat 97H and HepG2 liver cancer hypoxic cells. A 200μl sterile pipette tip was used to draw a straight line in the center of the cell growth area at the bottom of the culture dish. The cells were washed three times with PBS to remove the drawn cells, and new culture medium and each group of extracts were added. Photos were taken with a microscope at 0, 6h, 12h, 24h, and 48h.

[0072] 1.4 Transwell cell invasion assay

[0073] TPZ-NBs at concentrations of 0μg / ml, 10μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, 80μg / ml, and 100μg / ml were used to treat 97H and HepG2 liver cancer hypoxic cells, and added to the upper chamber of the Transwell with matrigel, and culture medium containing 20% ​​fetal bovine serum was added to the lower chamber for 48 hours. Non-migrated cells were removed, fixed, stained with crystal violet, and 10 fields of view were randomly selected for counting under a microscope.

[0074] 1.5 Statistical analysis

[0075] SPSS26.0 software was used for statistical analysis. When comparing two groups of data that followed a normal distribution, the t-test was used for analysis. For paired comparisons within and between groups, the LSD or Bonferroni correction method was used according to the situation; the comparison of rates was performed using the Mann-Whitney U test. In the significance analysis, P < 0.05 was used as the standard for statistical significance, *, P < 0.05, **, P < 0.01, ***, P < 0.001.

[0076] 2. Results Analysis

[0077] like Figure 3 The experiment showed that under normal conditions, when the concentration of TPZ-NBs was 5ug / ml and the liver cancer cells were cultured for 24 hours, TPZ-NBs had the greatest inhibitory effect on the proliferation of liver cancer cells; under hypoxic conditions, when the concentration of TPZ-NBs was 2ug / ml and the liver cancer cells were cultured for 24 hours, TPZ-NBs had the greatest inhibitory effect on the proliferation of liver cancer cells. Figure 4 , Figure 5 The scratch test results showed that after 48 hours of co-culture of hypoxic cells with TPZ-NBs, the scratch area gradually increased with the increase of TPZ-NBs concentration, indicating that the migration ability of liver cancer cells was significantly reduced. Figure 6 , Figure 7The results of the Transwell assay showed that after 48 hours of co-culture of hypoxic cells and TPZ-NBs, the number and area of ​​transmembrane cells decreased significantly with the increase in TPZ-NBs concentration, indicating that the invasive ability of liver cancer cells was significantly inhibited.

[0078] Example 4

[0079] In this example, a VX2 liver cancer model of experimental rabbits was established and the rabbits were divided into groups for treatment.

[0080] 1. Animal Source

[0081] 45 experimental New Zealand rabbits were obtained from the School of Medical Sciences of Zhengzhou University, of either sex, with a weight range of 3.0±0.2kg. Five experimental rabbits were used as tumor-bearing rabbits to obtain fresh VX2 tumor tissues, and the remaining 40 experimental rabbits were used for the rabbit VX2 tumor model.

[0082] 2. Test methods

[0083] 2.1 Establishment of the experimental rabbit VX2 liver cancer model

[0084] Obtaining tumor mass: VX2 tumor mass was implanted on the outer side of the rabbit thigh; after 15 days, when an immovable mass could be felt on the outer side of the rabbit thigh, it indicated that the tumor was successfully implanted. After anesthesia, the rabbit was killed and the tumor tissue was freed. After removing the central necrotic tissue, the translucent fish-like tumor was trimmed into about 1mm using tissue scissors. 3 Then New Zealand white rabbits weighing 3.0±0.2kg were selected for the establishment of VX2 rabbit liver cancer model.

[0085] Anesthesia: The rabbits were anesthetized by intramuscular injection of 20 mg of Su Mian Xin II and 2.5 ml of 4% sodium pentobarbital solution. The anesthesia effect was determined by detecting the disappearance of corneal reflex, muscle tension reflex and toe web reflex. When the anesthesia effect was poor, sodium pentobarbital was added in an appropriate amount to ensure that the rabbits were in a fully anesthetized state, so as to prepare for the subsequent model establishment operation.

[0086] Modeling: After the test rabbit is successfully anesthetized, its abdomen is shaved and disinfected, and a longitudinal incision is made along the midline of the abdomen at the lower end of the sternum, and then the subcutaneous tissue, muscle and peritoneum are separated in sequence. After entering the abdominal cavity, the liver tissue is exposed, and both hands use hemostatic forceps to gently pull the left lobe of the liver out of the body and fix it to increase the operating field of view. Lift the tip of the disposable scalpel, and enter the liver tissue 0.5-1cm after forming a 30-degree angle with the liver tissue. Keep the position of the tip unchanged, bury the tumor tissue along the bottom of the tip, then withdraw the tip and use a gelatin sponge of appropriate size to close the puncture. After observing no obvious bleeding, gently return to the abdominal cavity, restore the normal anatomical position of the liver, and then suture the muscle layer and skin layer by layer and disinfect the wound.

[0087] Postoperative management: To prevent infection, all experimental rabbits were given penicillin or ceftazidime (0.1ml / Kg) gluteus maximus injection for 3 consecutive days. The rabbits were fed under constant temperature and humidity conditions, and observed for symptoms such as dyspnea and diarrhea. Two weeks after modeling, the experimental rabbits were anesthetized and enhanced CT was performed to determine the size and location of the VX2 tumor in the liver. Modeling was successful if the tumor was larger than 1cm.

[0088] Schematic diagram of the experimental rabbit VX2 liver cancer model Figure 8 shown.

[0089] 2.2 Imaging examination

[0090] For the successfully established VX2 model, CT plain scan and enhanced scan were performed two weeks after modeling. A Siemens dual-source spiral CT machine was used for scanning, with the tube voltage set to 120kV, the tube current set to 70mA, and the slice thickness set to 1mm. A 22G indwelling needle was inserted through the auricular vein, and iohexol was injected through the auricular vein at a flow rate of 1mL / s through a high-pressure syringe. The injection dose was 1ml / kg calculated based on body weight. The enhancement delay time of the arterial phase, portal phase, and equilibrium phase was set to 10 seconds, 20 seconds, and 30 seconds, respectively. Before performing a CT scan, the rabbit was anesthetized in the same way as the modeling. After successful anesthesia, the experimental rabbit was fixed on the rabbit table, and finally CT images of the liver at different stages were obtained to evaluate the growth of the transplanted tumor.

[0091] 2.3 Treatment options

[0092] The rabbit liver cancer model was randomly divided into 4 groups, with 10 rabbits in each group, namely the Control group, TAE group, TPZ-NBs+TAE group, and TPZ-NBs+UTMD+TAE group. The experimental rabbits with successfully established VX2 tumor models were randomly assigned to four different experimental groups. The Control group was the blank control group, which only underwent angiography and injected 5 ml of normal saline into the hepatic artery. The TAE group was the iodized oil embolization group, in which the micro-guidewire and catheter were superselected to the left hepatic artery and then angiography was performed. After the tumor vessels were confirmed, iodized oil was given for embolization. Only iodized oil was used for embolization throughout the whole process. The TPZ-NBs+TAE group was the tirapazamine nanocapsule combined with TAE group. The left hepatic artery was superselected by microcatheter, and then the tumor-responsible vessels were confirmed by angiography, and then iodized oil TPZ-NBs complex was given for embolization until it was completely embolized. The TPZ-NBs+UTMD+TAE group was the microcatheter superselected to the left hepatic artery and then angiography was performed, and iodized oil TPZ-NBs complex was given for embolization, and UTMD was performed transabdominally after completion.

[0093] Preparation of TPZ-NBs complex in iodized oil: TPZ-NBs complex containing 1 mg / mL tirapazamine was dispersed in iodized oil to form TPZ-NBs complex in iodized oil. TPZ-NBs complex and iodized oil were mixed at least 5 times with a Voltexer machine at a ratio of 1:2 for 5 seconds each time to obtain an emulsion.

[0094] TAE operation process: anesthesia is the same as before. After anesthesia, the right inguinal area is shaved, disinfected, and covered with sterile towels. A 2 cm incision is made along the direction of the artery. Since the artery of the experimental rabbit is relatively thin, the subcutaneous tissue and fascia are bluntly separated to free the right femoral artery. A suture is inserted into each end of the femoral artery and the distal end of the femoral artery is gently lifted. The artery is punctured with a 22G venous puncture needle. After the needle core is withdrawn and arterial blood gushes out of the indwelling cannula, a 2.7F micro-guidewire and catheter are introduced into the descending aorta. The two cooperate to perform angiography of the descending abdominal aorta, hepatic artery, and left hepatic artery. After confirming the tumor staining, the micro-catheter is super-selectively advanced. After the micro-catheter is super-selected to the target artery, embolization treatment of the tumor is performed according to the group. After the operation is completed, the micro-catheter and micro-guidewire are withdrawn, the sheath is pulled out, the two ends of the femoral artery are ligated respectively, and the muscle layer and skin are sutured by interrupted suture method, and disinfected again. Penicillin 800,000 U / d is used for 3 consecutive days before and after the operation.

[0095] UTMD Procedure: Rabbit liver cancer models that required UTMD technical intervention were anesthetized and fixed, and abdominal hair was carefully removed immediately before ultrasound examination. Ultrasound examination was performed using an Aplio500 ultrasound system equipped with a 375BT convex probe transducer with a center frequency of 3.5 MHz. Basic B-mode ultrasound (dynamic range of 65, mechanical index of 1.5, gain of 90, and depth of 4 cm) was used to detect VX2 tumors. After positioning and morphological examination of the tumor, the optimal plane was determined and the skin was marked. The marked skin area was scanned up and down continuously with the probe and UTMD was performed (technical parameters: mechanical index of 0.15, sound intensity of 1.5 W / cm2, and irradiation time of 15 min).

[0096] 3. Results Analysis

[0097] like Fig. 9 As shown in the figure, all rabbits with transplanted tumors were successfully modeled after enhanced CT verification. After 2.7-F microcatheter hepatic artery angiography, abnormal staining groups in the left lobe of the liver were observed. The control group was given sham surgery with saline arterial perfusion, and the TAE group, TPZ-NBs+TAE group, and TPZ-NBs+UTMD+TAE group were given corresponding embolic agents for embolization. After the angiography confirmed that the tumor vessels were not developed, the microcatheter and guidewire were withdrawn, and the femoral artery was ligated. No related surgical complications were observed, and the surgical success rate was 100%.

[0098] Example 5

[0099] In this example, the liver and kidney functions of each group of model rabbits were detected, and the changes in the levels of ALT, AST, and BUN in the serum before and after the operation were detected to evaluate its safety.

[0100] 1. Analysis of liver and kidney toxicity

[0101] Before TAE surgery, and on days 1, 3, 7, and 14 after surgery, blood samples were collected from four groups of experimental rabbits. One milliliter of blood was drawn through the ear vein and centrifuged at 3000 rpm for 15 minutes to separate the supernatant. Liver function tests included aspartate aminotransferase (AST) and alanine aminotransferase (ALT) evaluation. Blood urea nitrogen (BUN) was used to examine renal function.

[0102] 2. Results Analysis

[0103] There was basically no difference in the liver and kidney function tests of the four groups of experimental rabbits before surgery. Fig.10 As shown in the figure, the levels of ALT, AST, and BUN increased to varying degrees on the first day after TAE surgery, and gradually returned to baseline levels 2 weeks after surgery. There was no significant difference in the AST and ALT values ​​measured at each time point between the treatment groups, but they were significantly higher than those in the blank control group. The reason is attributed to the damage to the liver caused by chemotherapy drugs and arterial embolism, AST and ALT in the tissues were released into the blood, and BUN remained stable.

[0104] There was no statistical difference in the changes of serum ALT, AST and BUN among the three groups undergoing TAE embolization. However, the TPZ-NBs+UTMD combined with TAE group had the highest alanine aminotransferase level on the first day after surgery, which may indicate that the TPZ-NBs complex in the TPZ-NBs+UTMD combined with TAE group carried relatively more nanoparticles into the tissue compared with the group not exposed to ultrasound.

[0105] Table 1 Levels of ALT, AST and BUN in the serum of rabbits in each group before and after embolization

[0106]

[0107] Example 6

[0108] In this example, pathological evaluation of the tumors in four groups of test rabbits was performed by pathological analysis and hematoxylin-eosin staining (HE staining).

[0109] 1. Test methods

[0110] 1.1 Pathological analysis

[0111] The treated animals were killed by anesthesia with an overdose of xylazine hydrochloride injection, and the complete liver tissue was obtained after dissection. The left lobe of the liver was separated, washed and photographed for observation. Each tumor was then fixed in 10% formalin buffer. After dehydration, the specimens were embedded in paraffin, cut into 3 μm thick slices and placed in a slide spreader. The slides were loaded with slides, and finally baked and stored at room temperature.

[0112] 1.2 Hematoxylin-eosin staining (HE staining)

[0113] The steps of hematoxylin-eosin staining (HE) are as follows: ① Dewaxing of paraffin sections: treat in xylene I solution and xylene II solution for 20 minutes each. Then, treat the sections in anhydrous ethanol I and II solution for 5 minutes each, and finally treat in 75% alcohol solution for 5 minutes, and then wash with distilled water. ② Hematoxylin staining: First, stain the sections in hematoxylin for 3-5 minutes, then differentiate with hydrochloric acid aqueous solution, then turn blue with ammonia solution, and finally wash. ③ Eosin staining: Put the washed tissue sections into 85% and 95% alcohol for dehydration, and then stain in eosin stain for 5 minutes. ④ Dehydration and sealing: Put the sections into anhydrous ethanol solutions I, II, and III for 5 minutes each, then transfer to xylene I and II for 5 minutes each, and finally seal with neutral gum for later observation under a microscope.

[0114] 2. Results Analysis

[0115] The gross appearance of the tumors in the four groups of experimental rabbits two weeks after embolization was as follows Fig.11 As shown in the figure, the tumor volume of the Control group increased significantly and protruded from the liver capsule. After incision, a large amount of fish-like tumor tissue with a small amount of necrotic tissue was observed in the periphery. After incision, a small amount of fish-like residual tumor and most of the necrotic tissue were observed in the TAE group. In the TPZ-NBs+TAE group and the TPZ-NBs+UTMD+TAE group, iodized oil TPZ-NBs complex was used during surgery, and flake necrosis of liver tissue in the tumor area was observed. After incision, necrotic tumor tissue and normal liver tissue were observed.

[0116] like Fig.12The HE staining results showed that in the sections of the Control group, tumor tissue could be observed to have extensively grown into the liver parenchyma, and some tumor nest structures could be observed; in the sections of the TAE group, TPZ-NBs+TAE group, and TPZ-NBs+UTMD+TAE group, necrotic tumor tissue in the corresponding vascular supply area after embolization and the surrounding fibrotic capsule structure could be observed; in the TPZ-NBs+TAE group and TPZ-NBs+UTMD+TAE group, iodized oil and TPZ-NBs complex and necrotic tumor tissue could be observed, and it was observed that the nanoparticles treated with UTMD had penetrated deeper into the tumor and were more widely distributed.

[0117] Example 7

[0118] In this example, immunohistochemical staining was used to detect the expression of VEGF, HIF-1, and CD31 in the tumors of four groups of tumor rabbits.

[0119] 1. Test methods

[0120] The immunohistochemistry (IHC) staining steps are as follows.

[0121] (1) Dewaxing and hydrating sections: the steps are the same as HE staining.

[0122] (2) Antigen retrieval: Place the sections in a citric acid antigen retrieval buffer repair box and perform antigen retrieval in a microwave oven, heat on medium for 8 minutes until boiling, stop heating for 8 minutes, then switch to medium-low heat and maintain for 7 minutes. After the slides cool, place them in PBS (PH7.4) and wash them three times, 5 minutes each time.

[0123] (3) Blocking endogenous peroxidase: The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. The slides were then washed in PBS (pH 7.4) for 5 min, and washed 3 times in total.

[0124] (4) Serum blocking: Add 3% BSA to the sections and block at room temperature for 30 min.

[0125] (5) Add primary antibody: Gently shake off the blocking solution, add the prepared primary antibody dropwise, and incubate overnight in a humidified chamber at 4°C.

[0126] (6) Adding secondary antibody: Wash the sections three times with PBS, 5 min each time. After the sections are slightly dried, add the secondary antibody and incubate at room temperature for 50 min.

[0127] (7) DAB staining: Wash the sections three times with PBS. After the sections are slightly dried, add DAB staining solution to the circle and observe the color development under a microscope. Stop staining when a positive brown-yellow color appears.

[0128] (8) Re-staining of cell nuclei: Use hematoxylin staining solution to counterstain for 3 min, then wash with tap water, differentiate with hydrochloric acid, wash with tap water, turn blue with ammonia solution, and rinse with running water.

[0129] (9) Dehydration and sealing: Finally, place the slices in anhydrous ethanol solutions I, II, and III in turn for 5 minutes each, then transfer to xylene I and II for 5 minutes each, finally dry slightly and seal with neutral gum.

[0130] 2. Results Analysis

[0131] Depend on Fig.12 It can be seen that the expression of HIF-1 and VEGF in the Control group was significantly lower than that in the TAE group, TPZ-NBs+TAE group and TPZ-NBs+UTMD+TAE group, while there was no statistical difference among the TAE group, TPZ-NBs+TAE group and TPZ-NBs+UTMD+TAE group; the expression of PCNA in the Control group was significantly higher than that in the TAE group, TPZ-NBs+TAC group and TPZ-NBs+UTMD+TAE group, among which the expression of PCNA in the TAE group was significantly higher than that in the TPZ-NBs+UTMD+TAE group.

[0132] MVD tumor microvessels are very important in the evaluation of tumor activity, and CD31 is one of the important expression molecules representing MVD. In this study, MVD was counted by comparing CD31. Fig.13 It can be seen that the expression of CD31 in the TPZ-NBs+UTMD+TAE group was significantly lower than that in the other three groups, indicating that compared with the other three groups, the MVD count value in the TPZ-NBs+UTMD+TAE group was the smallest.

[0133] Example 8

[0134] In this example, tumor apoptosis was detected by TUNEL immunofluorescence staining.

[0135] 1. Test methods

[0136] The steps of TUNEL immunofluorescence staining are as follows:

[0137] (1) Dewaxing of sections: refer to immunohistochemistry staining (IHC)

[0138] (2) Repair: Add proteinase K to the sections and incubate at 37°C for 25 min. Then wash the slides in PBS (pH 7.4) three times, 5 min each time.

[0139] (3) Membrane permeabilization: After the sections are slightly dried, add the membrane permeabilization working solution and incubate at room temperature for 20 min. Place the slides in PBS (pH 7.4) and then wash them on a decolorizing shaker for 5 min each time, for a total of 3 washes.

[0140] (4) Staining: Mix appropriate amounts of reagent 1 (TdT) and reagent 2 (dUTP) in the Tunel kit in a ratio of 1:9, add the mixture dropwise to the slices, and then incubate in a 37°C incubator for 2 hours.

[0141] (5) DAPI counterstaining of cell nuclei: Wash the sections with PBS (pH 7.4) three times, 5 min each time. Add DAPI staining solution onto the tissue sections and incubate at room temperature in the dark for 10 min.

[0142] (6) Mounting: Place the slides in PBS and wash three times, 5 min each time. After the sections are slightly dried, use an anti-fluorescence quenching mounting medium.

[0143] 2. Results Analysis

[0144] Depend on Fig.14 The results of TUNEL immunofluorescence staining showed that the number of apoptotic cells in the TPZ-NBs+UTMD+TAE group was significantly higher than that in the other three groups, and there was no significant difference between the TAE group and the TPZ-NBs+TAE group, but it was significantly higher than that in the control group.

[0145] In summary, the present invention successfully constructed a TPZ-NBs drug delivery system, which can provide a good drug encapsulation rate and drug sustained release efficiency, and can exert a strong anti-tumor effect in vitro; it verifies that the application of TPZ-NBs in TAE to treat liver cancer is safe and effective, and has a stronger synergistic anti-tumor effect in combination with TAE. Under the action of UTMD, it can promote TPZ to the deep part of the tumor and induce apoptosis of hypoxic tumor cells. Therefore, the present invention overcomes the shortcomings of the prior art and has a high application value.

Claims

1. A pharmaceutical composition for treating liver cancer by arterial embolization, characterized in that: The pharmaceutical composition consists of iodized oil and tirapazamine-loaded nanovesicles; the tirapazamine-loaded nanovesicles comprise nanovesicles and tirapazamine coated in the inner core of the nanovesicles.

2. The pharmaceutical composition according to claim 1, characterized in that The volume ratio of the iodized oil to the tirapazamine-loaded nanovesicles is 2:

1.

3. The pharmaceutical composition according to claim 1, characterized in that The encapsulation efficiency of the tirapazamine-loaded nanovesicles was 35.81%.

4. The method for preparing the pharmaceutical composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) mixing phospholipids and cholesterol, and dissolving them in an organic solvent to obtain a mixed solution; (2) evaporating the mixed solution obtained in step (1) to fully volatilize the organic solvent, adding a citrate buffer solution with a pH of 4.0 to fully hydrate the solution to obtain blank nanovesicles; (3) dissolving tirapazamine in ultrapure water and mixing thoroughly to prepare a tirapazamine solution; (4) adding the tirapazamine solution obtained in step (3) to the blank nanovesicles obtained in step (2), incubating in a water bath to obtain tirapazamine-loaded nanovesicles, and placing the tirapazamine-loaded nanovesicles in an ultrasonic instrument for ultrasonic treatment under ice bath conditions to obtain tirapazamine-loaded nanovesicle spherical particles of uniform size; (5) dispersing the tirapazamine-loaded nanovesicles in iodized oil to form a tirapazamine-loaded nanovesicle complex in the iodized oil.

5. The preparation method according to claim 4, characterized in that: In step (1), the mass ratio of phospholipid to cholesterol is 4:

1.

6. The preparation method according to claim 4, characterized in that: The concentration of the tirapazamine solution in step (3) is 0.5-1 mg / ml.

7. The preparation method according to claim 4, characterized in that: In the step (4), the water bath incubation temperature is 40° C., the ultrasonic treatment power is 200 W, the ultrasonic frequency is 2 s, the interval is 3 s, and it is repeated 240 times.

8. The preparation method according to claim 4, characterized in that: In the step (5), a Voltexer is used to mix the iodized oil and the tirapazamine-loaded nanovesicles at a ratio of 2:1 for at least 5 times, each time for 5 seconds, to obtain a complex of the iodized oil and the tirapazamine-loaded nanovesicles.

9. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a drug for arterial embolization to treat liver cancer.

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