CN117244073B - Application of high-density lipoprotein in preparation of anti-liver cancer drugs - Google Patents

Abstract

The application discloses an application of high-density lipoprotein in preparing anti-liver cancer drugs, which is found that under the in vitro condition, the proliferation and clone formation of liver cancer cells are obviously inhibited by adding the high-density lipoprotein into a culture medium. Subcutaneous injection of hepatoma cell HCCLM9 into nude mice can form tumor; compared with normal saline control group, the intratumoral injection of the high-density lipoprotein can effectively slow down the volume increase of subcutaneous tumor of the mice. The liver cancer cell H22 injected in the spleen of the mouse can transfer to the liver to form in-situ liver cancer of the mouse; while intravenous injection of lenvatinib significantly inhibits H22 liver cancer cell invasion and liver cancer tumor formation, intravenous injection of recombinant high-density lipoprotein coated with lenvatinib and esterified cholesterol ester has better inhibition effect on H22 liver cancer cell invasion and liver cancer tumor formation. The application proves that the high-density lipoprotein can be used as a medicament and a carrier for interfering the primary hepatocellular carcinoma for the first time on an animal level.

Description

Application of high-density lipoprotein in the preparation of anti-liver cancer drugs

Technical Field

The present invention relates to the field of biomedical technology, and in particular to an application of high-density lipoprotein in the preparation of anti-liver cancer drugs.

Background technique

Liver cancer is a highly malignant tumor that grows rapidly and has strong infiltration and metastasis capabilities. 75%-90% of liver cancers are pathologically classified as hepatocellular carcinoma. Liver cancer is a global health threat, with an increasing incidence and high mortality. Although there are many treatment methods, such as surgical resection, radiotherapy, chemotherapy, etc., the treatment of liver cancer still faces many challenges. The lack of highly sensitive and specific biomarkers leads to low early diagnosis rates and lack of curative means in the middle and late stages, which together lead to poor prognosis for liver cancer patients.

Anti-liver cancer drugs mainly include Western medicine and Chinese patent medicine. Western medicine: The main Western medicines for anti-liver cancer are sunitinib, erlotinib, and cetuximab. Chinese patent medicine: When fighting cancer, you can also use Chinese medicine to regulate. Commonly used drugs include Huachansu oral liquid, Shendan Sanjie capsule, and Fufang Banbian capsule. However, these drugs are not targeted at liver cancer and have greater side effects.

Therefore, there is an urgent need to develop an anti-liver cancer drug with good liver targeting.

Summary of the invention

The purpose of the present invention is to provide an application of high-density lipoprotein in the preparation of anti-liver cancer drugs. The present invention finds that high-density lipoprotein has good liver targeting and the ability to inhibit the proliferation of liver cancer cells.

In order to achieve the above object, the present invention adopts the following technical solution:

In a first aspect of the present invention, there is provided a use of high-density lipoprotein in preparing a drug carrier for treating liver cancer.

In the second aspect of the present invention, a use of high-density lipoprotein in the preparation of an anti-liver cancer drug is provided.

In the third aspect of the present invention, a use of a high-density lipoprotein and lenvatinib combination in the preparation of an anti-liver cancer drug is provided.

Furthermore, the anti-liver cancer drug also includes pharmaceutically acceptable excipients.

Furthermore, the auxiliary material includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

Furthermore, the dosage form of the anti-liver cancer drug includes at least one of granules, tablets, pills, capsules, injections or dispersions.

Furthermore, the anti-liver cancer method of the anti-liver cancer drug includes: inhibiting the proliferation of liver cancer cells.

The above high-density lipoprotein can be purchased commercially (for example, it can be purchased from Solebow Technology Co., Ltd., item number H7940); it can also be recombinantly prepared under in vitro conditions, and the preparation method includes mixing lecithin, esterified cholesterol, and triglycerides in a certain proportion, and the preparation principle requires that the esterified cholesterol and triglycerides have the same mass, and the lecithin mass accounts for not less than 65%). As a specific embodiment, lecithin, esterified cholesterol, and triglycerides are mixed in a volume ratio of 4:1:1.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. The application of high-density lipoprotein provided by the present invention in the preparation of anti-liver cancer drugs, the level of high-density lipoprotein cholesterol (HDL-C) in the blood of liver cancer patients shows a significant positive correlation with the prognosis of liver cancer patients. In the field of liver cancer treatment, the use of HDL as a therapeutic drug and drug carrier has unique advantages. HDL has good biocompatibility and is not easily recognized as a foreign body by the body and phagocytosed and cleared; the liver has HDL-specific receptors, which can achieve liver targeting; HDL is a nanoparticle (20 nanometers) with a high permeability and long retention effect (enhanced permeability and retention effect, EPR), which makes it easier to penetrate into liver cancer tissue and stay for a long time (compared with normal liver tissue), thereby enhancing the effect of treatment and reducing damage to normal liver tissue. Therefore, HDL has unique advantages as a liver cancer treatment drug and/or drug carrier, which can achieve precise targeted treatment of liver cancer cells and provide a more effective and low-toxic treatment option for liver cancer patients. The present invention is the first to confirm in vitro that HDL has the ability to inhibit the proliferation of liver cancer cells, and is the first to confirm that high-density lipoprotein can be used to prepare drugs for treating liver cancer.

2. The present invention is the first to demonstrate in mice that the combination of purified high-density lipoprotein and lenvatinib has a synergistic effect in the treatment of liver cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

FIG1 shows the results of cell counting reagent-8 for liver cancer cells in the saline (Saline) and high-density lipoprotein (HDL) treatment groups (**: p≤0.01, *: 0.01<p≤0.05, n.s.: p>0.05).

FIG2 shows the results of the clone formation experiment of liver cancer cells in the saline (Saline) and high-density lipoprotein (HDL) treatment groups (**: p≤0.01, *: 0.01<p≤0.05, n.s.: p>0.05).

Figure 3 is a graph showing the changes in tumor volume of subcutaneous tumors in nude mice treated with saline (Saline) and high-density lipoprotein (HDL) (**: p≤0.01, *: 0.01<p≤0.05, n.s.: p>0.05).

FIG. 4 is a macroscopic photograph of subcutaneous tumors in nude mice treated with saline and high-density lipoprotein (HDL).

Figure 5 shows representative images (left) of Ki67 immunohistochemical staining results of subcutaneous tumors in nude mice treated with saline (Saline) and high-density lipoprotein (HDL) and statistical results of Ki67 staining positivity (right) (**: p≤0.01, *: 0.01<p≤0.05, n.s.: p>0.05).

Figure 6 shows the body weight, liver weight and the ratio of liver weight to body weight of mice with orthotopic liver cancer model at the final sampling time in the saline, Lenvatinib and HDL-coated Lenvatinib treatment groups (**: p≤0.01, *: 0.01<p≤0.05, n.s.: p>0.05).

Figure 7 shows representative gross liver photos (left) and calculation statistics of the number of liver cancer masses (right) at the final sampling of mice with orthotopic liver cancer model in the saline, Lenvatinib and HDL-coated Lenvatinib treatment groups (**: p≤0.01, *: 0.01<p≤0.05, n.s.: p>0.05).

FIG8 is a representative liver H&E staining result of the final sampling of the orthotopic liver cancer model mice in the saline, Lenvatinib and HDL-coated Lenvatinib treatment groups.

Figure 9 shows the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and high-density lipoprotein cholesterol (HDL-C) at the final sampling of mice with orthotopic liver cancer model in the saline, Lenvatinib and HDL-coated Lenvatinib treatment groups (**: p≤0.01, *: 0.01<p≤0.05, n.s.: p>0.05).

Detailed ways

The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

In order to solve the technical problem of the present invention, the overall idea of the present invention is as follows:

In the present invention, we added purified high-density lipoprotein to liver cancer cells cultured in vitro to evaluate the application potential of high-density lipoprotein in reducing the proliferation of liver cancer cells. The results showed that high-density lipoprotein significantly inhibited the proliferation rate of liver cancer cells.

Furthermore, human liver cancer HCCLM9 cells were transplanted into nude mice subcutaneously to form tumors in vivo. Purified high-density lipoprotein was injected intratumorally to evaluate the potential of high-density lipoprotein to inhibit tumor formation. The study found that injection of purified high-density lipoprotein can significantly block the volume growth of subcutaneous tumors, proving that purified high-density lipoprotein inhibits the proliferation of hepatocellular carcinoma and shows the potential for application in inhibiting hepatocellular carcinoma.

Recombinant high-density lipoprotein coated with lenvatinib and esterified cholesterol was constructed by in vitro assembly. H22 liver cancer cells derived from mouse ascites were injected into the spleen of BABL/C mice, and then metastasized to the liver to form in situ liver cancer. Mice were treated by tail vein injection of lenvatinib solution and recombinant high-density lipoprotein loaded with lenvatinib and esterified cholesterol. The results showed that the use of lenvatinib alone significantly reduced the number of liver cancer masses on the surface of the liver, the invasion area of liver cancer cells, and the levels of serum markers of liver damage, aspartate aminotransferase and alanine aminotransferase; and the drug-loaded recombinant high-density lipoprotein had a stronger therapeutic effect.

Based on the above experimental evidence, we found that high-density lipoprotein can be used as a drug and drug carrier for liver cancer treatment. Aiming at the characteristics of high-density lipoprotein, intravenous injection of high-density lipoprotein can achieve the purpose of treating primary hepatocellular carcinoma. Aiming at the hepatocyte targeting characteristics and in vitro recombinant characteristics of high-density lipoprotein, we provide a recombinant high-density lipoprotein loaded with drugs for intravenous injection to achieve the purpose of treating primary hepatocellular carcinoma.

The above results indicate a new use of high-density lipoprotein: it can be used to prepare anti-liver cancer drugs or carriers.

The application of a high-density lipoprotein in the preparation of an anti-liver cancer drug of the present application will be described in detail below in combination with examples and experimental data.

Example 1: High-density lipoprotein can directly inhibit the malignant proliferation of liver cancer cells in vitro

1. Purification of porcine serum high-density lipoprotein

(1) Serum preparation: Contact the slaughterhouse to purchase fresh pig blood. After the blood has coagulated for more than 12 hours, centrifuge at 3000 rpm for 10 minutes and carefully aspirate the upper yellow serum. Filter and sterilize with a 0.22 μm pore size filter membrane to reduce the risk of serum deterioration in subsequent operations.

(2) Removal of VLDL and chyloprotein: Adjust the serum density to 1.006 g/mL with sodium bromide, and centrifuge at 40,000 rpm for 10 hours at 4°C in a Beckman xe-100 centrifuge with a sw70ti rotor (acceleration: 4, deceleration: 6). After centrifugation, obvious stratification can be seen. Carefully remove the upper pale white liquid, which is the VLDL and chyloprotein stratification. Store at 4°C.

(3) LDL purification: adjust the serum density to 1.06 g/mL with sodium bromide, and centrifuge at 40,000 rpm for 10 hours at 4°C in a Beckman xe-100 centrifuge with a sw70ti rotor (acceleration: 4, deceleration: 6). After centrifugation, obvious stratification can be seen. Carefully aspirate the upper pale white liquid, which is the IDL and LDL layers. Store at 4°C.

(4) HDL purification: Sodium bromide was used to adjust the serum density to 1.21 g/mL, and the serum was centrifuged at 40,000 rpm for 10 hours at 4°C in a Beckman XE-100 centrifuge with a SW70TI rotor (acceleration: 4, deceleration: 6). After centrifugation, obvious stratification was observed. Carefully aspirate the pale white liquid on the upper layer, which is the HDL layer.

(5) Dialysis: Boil a regenerated cellulose dialysis bag (3.5 kD) in ddH2O containing 2% NaHCO3 and 1 mM EDTA for 10 minutes, and place the dialysis bag in a 30% ethanol aqueous solution for long-term storage. Before using the dialysis bag, carefully clean the dialysis bag with distilled water, clamp one end with a dialysis bag clamp, carefully add the HDL layer liquid, and then seal the other end with a dialysis bag clamp. The dialysis solution is prepared by dissolving _Na2HPO3 • _12H2O (3.61 _g ), NaCl (8.76 g), and EDTA (0.1 g) in 1 L of distilled water. After dialysis at 4°C for 12 hours, replace the dialysis solution with new one and dialyze three times.

(6) Measurement of LDL-C and HDL-C: Take 200 μL of purified LDL and HDL, add 1 mL of organic solvent (chloroform: methanol = 2:1), vortex and shake, mix thoroughly and let stand for 5 minutes. Centrifuge at 1500g for 20 minutes, take the lower organic phase, about 800 μL, take 400 μL and place in a 55°C metal bath, heat until the liquid is fully evaporated. Add 200 μL of isopropanol to the tube again, rotate overnight at 4°C with a windmill to fully dissolve the lipids, and then start total cholesterol measurement.

2. Cell counting

(1) Collect cells in the logarithmic phase, count them using a hemocytometer, and add 100 μL of culture medium containing 3,000 cells to each well of a 96-well plate. Fill the edge wells with sterile PBS solution. Culture the cells at 5% CO ₂ and 37°C until the cells adhere to the wall and expand in shape. Add LCAT protein solution with a final concentration of 5 μg/mL to the LCAT group, and add an equal volume of physiological saline to the Saline group.

(2) Add 10 μL of cell counting reagent-8 (A311-01, Vazyme), incubate at 37°C for 2 hours under 5% CO ₂ . Measure the absorbance at 450 nm on an ELISA reader. Repeat the above operation at 0, 24, 48, and 72 hours after the cell morphology expands.

(3) Count the absorbance values corresponding to different time points and different groups and calculate their statistical differences.

3. Plate colony formation

(1) Take cells from each group in the logarithmic growth phase, digest them with 0.25% trypsin and beat them into single cells, and suspend the cells in culture medium and count them for later use.

(2) The cell suspension was diluted to 5000 cells/mL. The LCAT group was added with LCAT protein solution with a final concentration of 5 μg/mL, and the Saline group was added with an equal volume of normal saline.

(3) 500 cells were inoculated into a six-well plate and cultured in a cell culture incubator at 37°C, 5% CO ₂ and saturated humidity for 1 week, with fresh culture medium replaced every 2-3 days. LCAT group was added with LCAT protein solution with a final concentration of 5 μg/mL, and Saline group was added with an equal volume of physiological saline.

(4) When visible clones appear in the culture dish, terminate the culture. Discard the supernatant and carefully wash twice with PBS. Add 4% paraformaldehyde and fix at room temperature for 30 minutes.

(5) Remove the fixative solution, add an appropriate amount of 0.5% crystal violet staining solution and stain for 5-10 minutes, slowly wash off the staining solution with running water, and air dry.

(6) After taking pictures with a scanner, the number of clones was counted using ImageJ software, and the clone generation rate and statistical difference were calculated.

According to the results in Figures 1 and 2, compared with the condition of adding physiological saline, adding high-density lipoprotein at a final concentration of 0.2mmol/mL to the culture medium significantly reduced the proliferation rate and clone formation ability of liver cancer cells, proving that high-density lipoprotein can directly inhibit the malignant proliferation ability of liver cancer cells under in vitro conditions.

Example 2: High-density lipoprotein can directly inhibit the proliferation of liver cancer cells in vivo

1. Subcutaneous tumor formation and intratumor injection experiments in nude mice

(1) Basic feeding conditions: 4-week-old male BALB/c-nude mice weighing about 14 grams were purchased from Beijing Huafukang Biotechnology Co., Ltd., and the mouse production license number is SYXK(鄂)-2019-0050. The bedding, drinking water, complete pellet feed and other items that come into contact with animals are sterilized by high pressure. The experimental and feeding conditions are strictly in accordance with the SPF grade specifications. All animal experiments were approved by the Animal Care and Use Committee of the School of Life Sciences, Wuhan University.

(2) Subcutaneous tumor modeling: 4-week-old mice were injected subcutaneously after one week of adaptive feeding. Each mouse was injected into the skin under the armpits on both sides. The single injection volume was 200 μL, containing 7.5×10 ⁶ HCCLM9 cells, in which the cell suspension and matrix gel (0827045, ABW) were mixed at a ratio of 1:1.

(3) Recording tumor volume: Tumors formed in subcutaneous tumor modeling are mostly ellipsoidal. Five days after injection, when the matrix gel is basically absorbed, the tumor volume is measured every two days.

(4) Intratumoral injection: After the tumor size reaches about ²⁰⁰ mm3, the purified LCAT protein is injected once every two days. A total of four injections are given, with an injection volume of 50 μL. The control group is injected with normal saline, and the high-density lipoprotein group is injected with 50 μL of normal saline containing 2 mg/mL HDL-C.

(5) Final sampling: Use forceps to clamp the mouse tumor skin, cut the skin, remove the tumor, and place it in a pre-cooled PBS solution on ice. After all tumor samples are collected, place the tumors in a unified manner and take photos. Cut part of the tumor, place it in a cryopreservation tube, and quickly freeze it in liquid nitrogen. Then take it out and store it at -80℃ for a long time.

2. Ki67 immunohistochemistry

(1) The sections can be dewaxed and rehydrated according to the H&E staining procedure.

(2) EDTA high-temperature antigen repair: Place the rehydrated sections into a repair box containing EDTA repair solution adjusted to pH 9.0; place the repair box in a pressure cooker and add water, heat to boiling, and maintain for 15 minutes; remove the repair box and leave it at room temperature for more than 20 minutes, waiting for the sample to return to room temperature.

(3) Cleaning: Take out the repaired slices and place them in a container filled with deionized water of sufficient height. Shake gently, pour out the water and then repeat the cleaning with new deionized water, each cleaning lasting 5 minutes.

(4) Endogenous peroxidase blocking: After antigen repair, shake the slices gently to remove most of the water, carefully absorb the water droplets with absorbent paper, then circle the tissue position with a histochemical pen, and place the slices flat in a moist incubation box. Add 3% hydrogen peroxide ( _H2O2 ) to the tissue position, then place the incubation box in an incubator and incubate at 37°C for 20 _minutes . Take out the slices and soak them in PBS buffer for three washes, each wash for 5 minutes.

(5) Blocking: Add a blocking solution containing 10% BSA to the tissue, place the slices in a wet incubation box, and block for 1 hour at room temperature.

(6) Primary antibody incubation: discard the blocking solution and put the incubation box back into the wet box. Add Ki67 and HMGCR antibodies diluted in PBS solution containing 1% BSA to the tissue of the slice (the dilution ratio of both is 1:200) and incubate overnight at 4°C. On the second day, place the slices in an incubator and incubate at 37°C for 30-60 minutes; remove the primary antibody, wash the slices with PBS, and wash three times at room temperature with gentle shaking for 10 minutes each; then put the slices back into the wet box.

(7) Secondary antibody incubation: Add biotin-labeled secondary antibody (dilution ratio of 1:200) to the sliced tissue and incubate at 37°C for 30 minutes. Then discard the secondary antibody, soak the slices in PBS, wash three times on a shaker at room temperature for 5 minutes each time, and finally put the slices back into the humidified incubation box.

(8) Add HRP-labeled streptavidin to the tissue in the slice, and then incubate in a 37°C incubator for 30 minutes. Discard the remaining liquid, soak the slice in PBS, and gently shake and wash three times at room temperature, each time for 5 minutes. Finally, put the slice back into the wet incubation box.

(9) Color development: First, color a slide in advance and determine the color development time. Specifically, add DAB working solution to the slice tissue and incubate at room temperature. Use a timer to count the time and observe the time when the positive signal appears on the slice under a microscope. Finally, after the positive signal basically appears, determine the color development time. All subsequent slices will be colored according to this time. After all slices are colored, soak the slices in PBS and wash them three times on a shaker at room temperature, each time for 5 minutes.

(10) Hematoxylin counterstaining: Place the slices on a rack, immerse them in hematoxylin dye, take them out after 1-3 seconds, and quickly place the slices in running water and rinse carefully for 10 minutes.

(11) Dehydration: Soak the slides in the following solutions in sequence: 70% ethanol (soak for 3 minutes), 95% ethanol (soak for 3 minutes), 100% ethanol (soak for 5 minutes), 100% ethanol (soak for 5 minutes), and 100% ethanol (soak for 5 minutes).

(12) Clearing: After dehydration, immerse the slides in xylene, repeat 3 times, each time for 2 minutes.

(13) Sealing: Before xylene dries, add an appropriate amount of neutral gum to the tissue and cover it with a glass slide to press it tightly, ensuring that the tissue is completely covered without bubbles. Then leave it at room temperature for several hours to air dry, and finally take photos with a microscope or scan it with a slide scanner.

The results of the in vivo experiments are shown in Figures 3 and 4. Under in vitro conditions, high-density lipoprotein has the ability to inhibit the proliferation of liver cancer cells. However, it is unknown whether the same biological effect exists in vivo. The results of Figures 3 and 4 prove that high-density lipoprotein can also directly inhibit the proliferation of liver cancer cells in vivo, and ultimately show that the rate of increase of tumor volume in subcutaneous tumors in nude mice is slowed down (Figure 3), and the tumor volume is significantly smaller when the final sample is taken (Figure 4).

Example 3: Combination use of high-density lipoprotein and lenvatinib

High-density lipoprotein has good biocompatibility and liver targeting, and the in vitro recombinant technology of high-density lipoprotein is mature. Based on the above information, we use high-density lipoprotein as a drug carrier to see if we can improve the therapeutic effect of existing liver cancer treatment drugs. Using the in vitro recombinant method, we obtained recombinant high-density lipoprotein coated with lenvatinib. Physiological saline, lenvatinib and drug-loaded recombinant high-density lipoprotein were injected intravenously to intervene in liver cancer. The specific experimental methods are as follows:

1. Preparation of recombinant high-density lipoprotein coated with lenvatinib and esterified cholesterol

(1) Take 4 mg of lecithin, 1 mg of esterified cholesterol, 1 mg of triglyceride, and 1 mg of lenvatinib and dissolve them in 0.5 mL of anhydrous ethanol. Use a skin test syringe to draw up the solution and then quickly inject it into 12.1 mL of PBS. Stir for 15 min under _N2 .

(2) Dissolve 5.4 mg sodium cholate in 0.5 mL PBS; dissolve 10 mg rhApoA-I and 5 mg rhApoE in 0.8 mL PBS and add to the above liquid under stirring.

(3) Leave at room temperature for 30 min; move to 4°C and incubate for 12 h to allow the components to polymerize and form rhHDL.

(4) The liquid was added to a protein dialysis bag with a pore size of 10 kD, and the dialysate (PBS) was fully dialyzed at 4°C to remove sodium cholate.

2. Construction of mouse orthotopic liver cancer model

(1) Basic feeding conditions: 6-week-old male C57BL/6 mice weighing about 20-22 grams were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and the mouse production license number is SCXK (Su)-2018-0008. The bedding, drinking water, complete pellet feed and other items that come into contact with animals are sterilized by high pressure. The experimental and feeding conditions are strictly in accordance with the SPF grade specifications. All animal experiments were approved by the Animal Care and Use Committee of the School of Life Sciences, Wuhan University.

(2) Anesthesia and skin preparation: 5% chloral hydrate was injected into the peritoneal cavity of BABL/c mice at a dose of 200 μL/20 g body weight for anesthesia. The mouse was pinched at the fingertips to confirm that it had lost its pain sensation. The hair corresponding to the spleen position at 0.5 cm below the sternum on the left side of the mouse abdomen was carefully cut with scissors, taking care not to cause trauma. 75% alcohol was sprayed on the mouse and the remaining hair was carefully wiped off.

(3) Carefully mix the H22 cells, count and adjust the cell density to 1.4×108 cells/mL, add an equal volume of Matrigel and mix by pipetting, then place on ice for later use.

(4) First, carefully wipe the surgical area with an iodine cotton ball for disinfection, carefully lift the skin with tweezers, carefully cut a 1 cm window in the epidermis with scissors, and then carefully cut the peritoneum to obtain a window of about 1 cm in length. Use tweezers to carefully explore and pick out the spleen.

(5) Place a 10 μL microinjection needle in an ice box to keep it cold. Aspirate 5 μL of 7 × 107 cells/mL H22 cells with the microinjection needle. Pull out the spleen with forceps in the left hand and inject slowly and carefully from the end of the spleen. Release the forceps with the left hand. At the moment when the right hand withdraws the needle, hold the pipette with the left hand and quickly drip 5 μL of Compat brand medical glue at the needle insertion site. Use the forceps in the right hand to fix the spleen to prevent the glue from adhering to other tissues.

(6) After the medical glue has solidified, gently release the forceps and carefully return the spleen from the wound to the abdominal cavity. Use the sterilized medical suture needle and suture thread to suture the wound, one stitch every 0.3 cm. After suturing, wipe the wound with a 75% alcohol cotton ball to prevent infection. Carefully place the mouse that has completed the operation in a cage, and try to make it lie on its side close to the objects placed to prevent its body temperature from dropping too quickly. Usually, after 2-3 hours of recovery, the mouse will wake up and start to move around in the cage.

(7) After the surgery, the mice were observed every other day. If the mice were still alive and performing well (no significant weight loss and no obvious movement disorder) three days after the surgery, they were considered to have successfully established the model and could continue to be raised for future use. After five days of recovery from the surgery, H22 cells could be transferred to the liver of the mice through the blood circulation and form dispersed white masses, thereby establishing a mouse in situ liver cancer model.

(8) The drug treatment started on the third day after surgery, and was administered once every two days. According to the specific surface area calculation, the dosage of lenvatinib for mice was calculated to be approximately 2.6 mg/kg body weight, and the administration method was intravenous injection. The control group was treated with tail vein injection of normal saline.

(9) Sacrifice and sampling: Samples were collected 10 days after tumor cell injection. The mice were fasted for 16 hours before sampling. Blood was drawn from the eyeballs and the mice were killed by cervical dislocation. The livers were removed and photographed, and the liver cancer and adjacent tissues of the mice were separated for subsequent analysis, experimental testing and pathological testing.

3. Mouse sampling and pathological staining analysis

(1) Final sampling

① After weighing the mouse, collect 0.6-1mL of blood from the eyeball. Fix the mouse in supine position and moisten the hair on the chest and abdomen of the mouse with distilled water.

② Use forceps to clamp the skin in the middle of the mouse's abdomen, cut the skin along the middle of the abdomen towards the head to under the xiphoid process, and cut the skin towards the tail end, exposing the subcutaneous fascia, muscles, etc. layer by layer, open the abdominal cavity, and fully expose the organs.

③ Quickly find and remove the mouse's liver, place the removed liver specimen on sterile gauze, wipe off any residual blood on the surface of the liver, place the liver in a sterile culture dish, quickly take a photo, and weigh it.

④Collect fresh samples: Cut the cancerous and adjacent parts of the liver, place them in cryopreservation tubes, and quickly freeze them in liquid nitrogen. Then take them out and store them at -80℃ for a long time.

⑤ Paraffin specimen: Part of the liver was cut and fixed in 10% neutral formalin.

⑥ Serum collection: Blood was collected from the eyeball of the mouse and placed in a non-anticoagulant tube at room temperature for 1 hour, centrifuged at 3000 rpm for 10 minutes, and the supernatant was the serum. The collected supernatant was stored at -80°C. Serum-related indicators were tested by Wuhan Wellcome Animal Testing Co., Ltd.

(2) Liver tissue processing and pathological staining experiments

① Liver dehydrated, transparent, wax-soaked

Cut part of the liver lobe tissue fixed in 10% neutral formalin into the marked embedding frame and rinse it in a small flow of running water for more than 30 minutes. Set the following programs on the machine according to the following process: ① Dehydration: 75% alcohol (45 minutes) → 75% alcohol (45 minutes) → 85% alcohol (45 minutes) → 85% alcohol (45 minutes) → 95% alcohol (45 minutes) → 95% alcohol (45 minutes) → anhydrous alcohol (1 hour) → anhydrous alcohol (1 hour); ② Transparency: xylene (1 hour) → xylene (1 hour); ③ Wax immersion (65°C): paraffin (1 hour) → paraffin (1 hour). After the tissue is rinsed, put the embedding frame containing the tissue into the machine basket and start the above program. After the above program is completed, take out the tissue embedding frame and send it to the pathology room to embed the tissue, and clean the machine for standby use.

② Liver tissue sections were sliced using a microtome (slice thickness 5 μm).

③Hematoxylin-eosin (HE) staining of liver tissue

Place the liver tissue paraffin sections in a 65°C oven (30 minutes) → xylene (5 minutes × 3 times) → 100% alcohol (1 minute) → 90% alcohol (1 minute) → 70% alcohol (1 minute) → wash with distilled water → hematoxylin (5 minutes) → wash with tap water to remove floating color on the sections → 1% hydrochloric acid alcohol (1 to 3 seconds) → wash with tap water several times → Scott blueing solution (0.35g sodium bicarbonate, 2g magnesium sulfate, 100mL distilled water) (1 minute) → wash with tap water several times → eosin (1 minute) → wash with distilled water to remove floating color on the sections → 70% alcohol once → 90% alcohol once → 100% alcohol (30 seconds × 3 times) → xylene (2 minutes × 3 times) → seal the sections before the xylene dries up and take pictures.

4. Experimental results

The body weight, liver weight and the ratio of liver weight to body weight of mice with orthotopic liver cancer model at the final sampling of the saline, lenvatinib and high-density lipoprotein-coated lenvatinib treatment groups are shown in Figure 6. It can be seen that lenvatinib significantly reduced the liver weight and the ratio of liver weight to body weight of mice without significantly changing the body weight of mice; and the degree of reduction of liver cancer burden in mice by drug-loaded recombinant high-density lipoprotein was higher than that of the group treated with lenvatinib alone.

Representative liver macrophotographs (left) and the calculation and statistics of the number of liver cancer masses at the final sampling of mice with orthotopic liver cancer model in the saline, lenvatinib and high-density lipoprotein-coated lenvatinib treatment groups are shown in Figure 7. It can be seen that the macrophotographs of the mouse liver and the statistical results of the number of liver cancer masses also suggest that the inhibitory effect of drug-loaded recombinant high-density lipoprotein on liver cancer is better than that of lenvatinib treatment alone.

Representative liver H&E staining results of the terminal samples of mice with orthotopic liver cancer model in the saline, Lenvatinib and HDL-coated Lenvatinib treatment groups are shown in Figure 8. It can be seen that the H&E staining results of liver pathological sections show that drug-loaded recombinant high-density lipoprotein has a stronger inhibitory effect on inhibiting the intrahepatic invasion of liver cancer cells than Lenvatinib.

The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and high-density lipoprotein cholesterol (HDL-C) levels of mice with orthotopic liver cancer model at the final sampling of the saline, lenvatinib and high-density lipoprotein-coated lenvatinib treatment groups are shown in Figure 9. It can be seen that both drug-loaded recombinant high-density lipoprotein and lenvatinib treatment significantly reduced the levels of aspartate aminotransferase and alanine aminotransferase in the mouse serum.

The above results show that high-density lipoprotein, as a drug carrier, has the ability to significantly improve the efficacy of lenvatinib in the treatment of liver cancer. Recombinant high-density lipoprotein has great application potential as a drug carrier in the treatment of liver cancer.

Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (4)

1. Use of a combination of high-density lipoprotein and lenvatinib in the preparation of anti-liver cancer drugs. 2. The use according to claim 1, characterized in that the anti-liver cancer drug also includes a pharmaceutically acceptable excipient. 3. The use according to claim 2, characterized in that the auxiliary material comprises at least one of a disintegrant, a binder, a diluent, a lubricant, a sweetener or a colorant. 4. The use according to claim 1, characterized in that the dosage form of the anti-liver cancer drug includes at least one of granules, tablets, pills, capsules or injections.