PD-L1 / MUC1 double-antibody modified doxorubicin-loaded liposome as well as preparation method and application thereof

Abstract

The invention belongs to the field of cell biology, and particularly relates to a preparation method and application of PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposome. Comprising the following steps: jointly modifying lipidosome with MUC1 and PD-L1 antibodies, and entrapping adriamycin in the lipidosome to prepare the adriamycin-loaded double-antibody modified lipidosome. The physicochemical properties of liposomes such as DOX-aMUC1 / aPDL1-Lip synthesized by the invention meet the requirements of a normal range through detection, and the liposomes have good encapsulation efficiency and drug loading capacity; the strongest cell targeting property and tumor tissue targeting property are realized on tumor cells NCI-H441; compared with other dosage form groups of lipidosome, especially a free DOX group, the anti-tumor activity is the strongest.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, and specifically relates to a preparation method and application of PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposomes. Background Art

[0002] Cancer is still a difficult problem to solve, and malignant tumors are the second leading cause of death. We extracted cancer-related death data from the National Mortality Surveillance System. In 2023, the population who died of cancer accounted for 24.3% of the total national death population. According to statistics from the International Agency for Research on Cancer, there will be 19.3 million new cancer cases in the world in 2023, and nearly 10 million people will die from cancer and its complications. It is estimated that by 2040, new cancer cases will increase by 47%, and nearly 28.4 million people will die from cancer and its complications. At present, the main treatment methods for cancer are still chemotherapy, radiotherapy and surgery, but the poor prognosis, high recurrence rate, low survival rate and poor quality of life of patients are still a major problem in current cancer treatment. In addition, the drug has low selectivity for tumors and is prone to multi-drug resistance, which needs to be solved urgently. Therefore, it is urgent to develop a new treatment method.

[0003] Liposomes are self-assembled vesicles that are non-toxic and have good biocompatibility. They form a closed double-layer structure in an aqueous system. The inside of the double-layer structure is an aqueous phase, and the middle of the membrane is a fat-soluble organic phase, which can encapsulate drugs. Liposomes have stable drug loading, prolonged pharmacokinetics, reduced off-target side effects, and higher drug delivery efficiency for strong blood-brain barriers or plasma membrane barriers. Therefore, the liposome drug delivery system became the first drug delivery system approved for clinical use. Targeted drug delivery system refers to a drug delivery system in which drugs are specifically aggregated and located in specific tissues, organs and cells by suitable carriers through local administration or systemic blood circulation. In addition, since the components that make up liposomes are relatively simple and the structure is relatively stable, the modification of liposomes brings unlimited possibilities.

[0004] Programmed cell death ligand 1 (PD-L1) is a type I transmembrane protein of 40 kDa that is associated with the suppression of the immune system. In the GEPIA2 database, bioinformatics analysis of the expression of PD-L1 in normal tissues and tumor tissues showed that PD-L1 is expressed in most solid tumor tissues, such as pancreatic cancer, lung cancer, cervical cancer, and gastric cancer. Moreover, Chen et al. demonstrated through immunohistochemistry that in patients with gastric cancer, PD-L1 is mainly expressed on the tumor cell membrane, but not in adjacent tissues. Studies have found that patients with tumors that highly express PD-L1 are 4.5 times more likely to die from cancer than those with low PD-L1 expression.

[0005] Mucins 1 (MUC1) is a member of the mucin family and is a type I transmembrane glycoprotein with a highly glycosylated extracellular domain. ] . In the GEPIA2 database, bioinformatics analysis of the expression of MUC1 in normal tissues and tumor tissues revealed that MUC1 is highly expressed in a variety of cancers, including breast cancer, pancreatic cancer, cervical cancer, lung adenocarcinoma, and bile duct cancer, and is associated with a poor prognosis of cancer. Currently, the extracellular segment of MUC1 has been used as a diagnostic indicator for breast cancer and pancreatic cancer in clinical practice, and can also be used as a target for tumor immunotherapy. Therefore, targeting PD-L1 and MUC1 proteins that are highly expressed in most tumor cells can achieve the goal of targeted treatment of most tumors.

[0006] Antibodies (Anti-body, Ab) are specific immune proteins produced by the body through antigen stimulation and can specifically bind to antigens. More and more studies have found that monoclonal antibodies and antibody-drug conjugates play a key role in preventing tumor cell recurrence and resistance and improving the prognosis of patients with recurrence and resistance. Monoclonal antibodies play a very important role in tumor targeted therapy because of their long blood circulation half-life and high specificity for specific antigens on the surface of malignant tumor cells. For example, Li et al. connected anti-CD123 antibodies to camptothecin (CPT) through disulfide bonds to prepare anti-CD123 antibody-drug conjugates. Hatakeyama H et al.'s anti-MT1-MMP antibodies can enhance the endocytosis of drug-loaded liposomes, thereby increasing their cytotoxicity. Therefore, the development of antibody-modified liposomes targeting PD-L1 and MUC1 proteins as drug delivery systems may provide new strategies for targeted therapy of most tumors.

[0007] Doxorubicin (DOX) is a broad-spectrum anticancer drug. Doxorubicin is an anthracycline chemotherapy drug that can insert into DNA fragments, inhibit the synthesis of DNA and RNA, and inhibit the activity of topoisomerase II. It has a killing effect on tumor cells in various growth cycles. Although doxorubicin, as a world-recognized chemotherapy drug, has a good broad-spectrum anti-tumor effect, its killing effect on normal cells is relatively high, and the cardiotoxicity it brings will also affect the normal physiological functions of the body. Doxorubicin, as a water-soluble chemotherapy drug, is consistent with the aqueous phase environment inside the liposome. Therefore, encapsulating it inside the liposome can significantly reduce the side effects caused by doxorubicin. Summary of the invention

[0008] The present invention discloses a doxorubicin-loaded liposome (DOX-aMUC1 / aPDL1-Lip) modified with two antibodies, i.e., an antibody against programmed cell death ligand 1 and an antibody against mucin 1, which are modified on the surface of the liposome. It is found that the liposome can enhance the targeting of the liposome while reducing the cardiotoxicity of doxorubicin. The present invention also provides the in vitro and in vivo targeting and anti-tumor effects of the double-antibody modified doxorubicin-loaded liposome.

[0009] The first purpose of the present invention is to provide a PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposome, comprising co-modifying liposomes with antibodies of MUC1 and PD-L1 and encapsulating doxorubicin therein to prepare a double antibody modified liposome loaded with doxorubicin.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposomes, comprising the following steps: 1) Preparation of antibody-coupled phospholipids

[0011] Dissolve 1-2 mg of phospholipid in phospholipid solution, dissolve 1 mg of antibody or protein in 1 ml of reaction buffer, mix 50 μl of phospholipid solution and 1 ml of reaction buffer evenly, react at room temperature for 2 hours, after the reaction is completed, transfer the reaction system to a 10 kDa ultrafiltration tube, centrifuge and ultrafilter at 9500 rpm, 4°C to remove all solvents, wash twice with pure water to remove unreacted phospholipids, and store the antibody / protein coupling at 4°C;

[0012] 2) Preparation of blank liposomes

[0013] Blank liposomes were prepared by thin film dispersion method, and antibody-free liposomes Lip, single antibody-modified liposomes aMUC1-Lip, aPDL1-Lip, and double antibody-modified liposomes aMUC1 / aPDL1-Lip were prepared. Phosphatidylcholine, cholesterol and antibody-coupled phospholipids were dissolved in a mixture of chloroform and methanol, wherein chloroform / methanol = 2 / 1. Different dosage forms were prepared according to different antibody modifications (aMUC1-Lip, aPDL1-Lip, aMUC1 / aPDL1-Lip). The mixture (lecithin 30mg, cholesterol 7.5mg) is transferred to an eggplant-shaped bottle, chloroform and methanol are removed by evaporation using a rotary evaporator to form a lipid film, when the lipid film is about to form, the rotating speed of the suspension evaporator and the pressure in the eggplant-shaped bottle are quickly adjusted to the maximum to remove the volatilized chloroform in the eggplant-shaped bottle as much as possible after the last drop of liquid in the eggplant-shaped bottle disappears, the maximum pressure and the rotating speed are maintained for 15min, and the lipid film is hydrated with 5ml of 70mg / ml ammonium sulfate solution afterwards, the hydration time is 1h, the temperature is set at 55°C, the rotating speed is set at 40rpm / min, the collected lipid film is transferred to an ultrasonic cell crusher, and the working time is 3 seconds and paused for 3 seconds, the ultrasonic cell crusher temperature is maintained at 20°C, the power is set at 20%, the total working time is 15min, the syringe is passed through 0.45μm and 0.22μm filter membranes three times, the filtered liposomes are dialyzed with ultrapure water with 8000-14000Da dialysis bags, the single dialysis time is 2h, and the dialysis is performed three times in total, and the blank liposomes are collected after the dialysis is completed;

[0014] 3) Preparation of doxorubicin-loaded liposomes

[0015] The ammonium sulfate concentration gradient method was used to prepare DOX-loaded liposomes of various dosage forms (unmodified DOX-Lip loaded with doxorubicin, two types of DOX-aMUC1-Lip and DOX-aPDL1-Lip loaded with doxorubicin modified with single antibodies, and DOX-aMUC1 / aPDL1-Lip loaded with doxorubicin modified with double antibodies). The modified blank liposome solution was mixed with the DOX solution, and the mixed solution was transferred to an eggplant-shaped bottle. The rotary evaporator was set to a temperature of 63°C, a speed of 50rpm / min, and a time of 20min. The solution was then transferred to a 8000-14000Da dialysis bag for dialysis for 2h per dialysis. It was necessary to dialyze three times to remove the unloaded DOX and obtain completely drug-loaded liposomes. The entire experiment needed to be protected from light.

[0016] The third aspect of the present invention provides the use of the above-mentioned PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposomes in the preparation of anti-tumor drugs.

[0017] Application of the above-mentioned PD-L1 / MUC1 dual antibody modified doxorubicin-loaded liposomes in the preparation of drugs for the treatment of non-small cell lung cancer.

[0018] The above-mentioned PD-L1 / MUC1 dual antibody modified doxorubicin-loaded liposomes can also be used in drugs for treating tumors with high surface expression of PD-L1 and MUC1.

[0019] The above-mentioned PD-L1 / MUC1 dual antibody modified drug delivery system is used as a highly targeted system for drug encapsulation and presentation, which can better solve the problem of drug resistance.

[0020] The beneficial effects of the present invention are: (1) the conjugate molecules aPDL1-Lip and aMUC1-Lip are successfully synthesized; (2) the physical and chemical properties of the synthesized DOX-aMUC1 / aPDL1-Lip and other liposomes are tested to be within the normal range requirements, and have good encapsulation efficiency and drug loading; (3) DOX-aMUC1 / aPDL1-Lip has the strongest cell targeting and tumor tissue targeting to tumor cells NCI-H441; (4) DOX-aMUC1 / aPDL1-Lip has the strongest anti-tumor activity compared with liposomes in other dosage forms, especially the free DOX group. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The aPDL1 conjugate and aMUC1 are connected by an amide bond. The synthetic route of the conjugate

[0022] In the figure: (A) aPDL1 coupling route, (B) aMUC1 coupling route;

[0023] Figure 2 Infrared spectrum analysis of conjugate

[0024] In the figures: (A) infrared spectrum of aPDL1-Lip, (B) infrared spectrum of aMUC1-Lip;

[0025] Figure 3 Standard curve of doxorubicin and fluorescein Cy7

[0026] In the figure: (A) Doxorubicin standard curve at 478 nm, (B) Cy7 standard curve at 680 nm;

[0027] Figure 4 Transmission electron microscopy images of doxorubicin-loaded liposomes in various dosage forms;

[0028] Figure 5 Analysis of particle size and Zeta potential stability of doxorubicin-loaded liposomes in various dosage forms within 14 days

[0029] In the figure: (A) particle size results of liposomes of each dosage form group in normal saline, B) particle size results of liposomes of each dosage form group in 3% BSA, (C) Zeta potential results of liposomes of each dosage form group in normal saline, (D) Zeta potential results of liposomes of each dosage form group in 3% BSA;

[0030] Figure 6 In vitro cumulative drug release profiles of doxorubicin-loaded liposomes of various dosage forms;

[0031] Figure 7 Western blot results show the expression of MUC1 and PD-L1 in each cell line;

[0032] Figure 8 Immunofluorescence staining results of PD-L1 and MUC1 protein expression in each cell line

[0033] In the figure: (A) NCI-H441 lung cancer cells highly express both MUC1 and PD-L1, (B) NCI-H460 lung cancer cells are PDL1 positive and MUC1 negative, (C) ZR-75-1 breast cancer cells are MUC1 positive and PDL1 negative, (D) HUH7 liver cancer cells are both negative for MUC1 and PD-L1;

[0034] Fig. 9 Cellular uptake and quantification of different cell lines

[0035] In the figure: (A) NCI-H441 cell uptake fluorescence image, (B) NCI-H441 cell uptake quantitative analysis, (C) NCI-H460 cell uptake fluorescence image, (D) NCI-H460 cell uptake quantitative analysis, (E) ZR-75-1 cell uptake fluorescence image, (F) ZR-75-1 cell uptake quantitative analysis image, (G) HUH7 cell uptake fluorescence image, (H) HUH7 cell uptake quantitative analysis, * P<0.1, ** P<0.01, *** P<0.001, **** P < 0.0001; Fig.10 Cytotoxicity of antibody-modified doxorubicin-loaded liposomes on different cells

[0036] In the figure: (A) NCI-H441 cytotoxicity and IC50 value, (B) NCI-H460 cytotoxicity and IC50 value, (C) ZR-75-1 cytotoxicity and IC50 value, (D) HUH7 cytotoxicity and IC50 value;

[0037] Fig.11 Analysis of tumor targeting of different antibody-modified liposomes in vivo

[0038] In the figure: (A) In vivo imaging of various liposome formulations in tumor-bearing nude mice within 48 hours, (B) Fluorescence imaging of the main organs and tumor tissues of tumor-bearing nude mice, (C) Quantitative analysis of the fluorescence imaging results of tumor tissues of tumor-bearing nude mice, ****P < 0.0001;

[0039] Fig.12 In vivo anti-tumor analysis of doxorubicin-loaded liposomes modified with different antibodies

[0040] In the figure: (A) Schematic diagram of in vivo anti-tumor experiment, (B) Tumor image after treatment with liposomes loaded with doxorubicin of various dosage forms, (C) Quantification of tumor weight in nude mice bearing tumors, (D) Line graph of tumor volume change within 18 days, (E) Line graph of weight change within 18 days, * P<0.1, *** P < 0.001, **** P < 0.0001;

[0041] Fig.13 Pathological analysis of different organs and tumor tissues;

[0042] Fig.14 Diagram of the in vivo antitumor mechanism of various dosage forms of doxorubicin-loaded liposomes. DETAILED DESCRIPTION

[0043] Explanation of symbols

[0044]

[0045]

[0046] Experimental Materials 1 Main Instruments and Equipment

[0047] Electronic balance Sartius GmbH, Germany Small Animal In Vivo Imaging System Perkin Elmer Rotary evaporator Shanghai Yamato Technology Co., Ltd. Ultrapure water instrument Nanjing E-Tech Co., Ltd. UV spectrophotometer Shanghai Mtash Instrument Co., Ltd. Upright fluorescence microscope Nikon Corporation Ultra-low temperature storage box Qingdao Haier Co., Ltd. Autoclave ZEALWAY ELECTRONICS ELISA reader BIO-TEK INSTRUMENTS Clean bench Jiangsu Purification Equipment Co., Ltd. Haier Refrigerator Qingdao Haier Co., Ltd. Constant temperature shaker Changzhou Noki Instrument Co., Ltd. Deep freezer Qingdao Haier Co., Ltd. Inverted fluorescence microscope Olympus Japan Ice Maker SANYO Japan TEM Microscope Japan Jeol Corporation Ultrasonic cell disruptor Ningbo Xinzhi Technology Co., Ltd. Mass spectrometer Thermo Fisher Scientific Blast drying oven Shanghai Zhicheng Instrument Co., Ltd. Laser confocal microscopy German Leica Company

[0048] 2. Reagents and drugs

[0049]

[0050]

[0051] 3. Preparation of main reagents

[0052] (1) 70mg / ml ammonium sulfate solution

[0053] Ammonium sulfate 1.4g

[0054] ddH2O 20ml

[0055] (2) 0.9% saline

[0056] ddH2O 100ml

[0057] Sodium chloride 0.9g

[0058] (3) PBS (PH7.4)

[0059] PBS powder 20g

[0060] ddH2O 2000ml

[0061] High temperature and high pressure standby

[0062] (4)TBST

[0063]

[0064] (5) Cell freezing medium

[0065] DMSO 9ml

[0066] Complete medium 1 ml

[0067] (6) 0.1% crystal violet

[0068] Methanol 1g

[0069] Crystal violet 1000ml

[0070] (7) MTS solution

[0071] MTS powder 50mg

[0072] PBS buffer 10ml

[0073] (8)Phosphotungstic acid solution

[0074] ddH2O 0.1g

[0075] Phosphotungstic acid 10ml

[0076] (9) Protein transfer solution

[0077] 10× transfer buffer 100ml

[0078] ddH2O 700ml

[0079] Methanol 200ml

[0080] (10) 0.1% Triton X-100 solution

[0081] PBS buffer 10 μl

[0082] Triton X-100 10ml

[0083] (11) Electrophoresis buffer

[0084] 10× electrophoresis buffer 100ml

[0085] ddH2O 900ml

[0086] Example 1

[0087] 1 Cell line

[0088] Human lung cancer cell line (NCI-H441) was purchased from Wuhan Pronocell Life Science Co., Ltd., human lung cancer cell line (NCI-H460) and human breast cancer cell line (ZR-75-1) were from the Key Laboratory of Biology, Shandong Second Medical University. Human hepatoma cell line (HUH7) was from Sun Yat-sen University. NCI-H441 and ZR-75-1 cells were cultured in RPMI1640 complete medium containing 10% FBS + 1% PS, and NCI-H460 and HUH7 cells were cultured in DMEM high glucose complete medium containing 10% FBS + 1% PS.

[0089] 2 Experimental animal materials

[0090] 60 BALB / c-nu male mice (4-5 weeks old), weighing 15-17 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., and the experimental animal license number is scxk (Lu) 20220006. They were kept in the Specific Pathogen Free (SPF) grade experimental animal room of the Medical Experimental Animal Center of Shandong Second Medical University. The mouse food and bedding used in the breeding room were all SPF grade, and the drinking water was autoclaved ultrapure water. The temperature of the SPF grade animal room was 25℃±1℃, and the relative humidity was controlled at 40-60%.

[0091] 3 Preparation and evaluation of physicochemical properties of double antibody modified doxorubicin-loaded liposomes

[0092] 3.1 Preparation of antibody-conjugated phospholipids

[0093] First, the modular ligand is synthesized. The covalent coupling of the protein or antibody to the phospholipid can be achieved simply and quickly through the primary amine groups on the antibody or protein, such as lysine. Take 1-2 mg of phospholipid and dissolve it in the phospholipid solution, and take 1 mg of antibody or protein and dissolve it in 1 ml of reaction buffer. Mix 50 μl of phospholipid solution with 1 ml of reaction buffer and react at room temperature for 2 hours. After the reaction is completed, transfer the reaction system to an ultrafiltration tube (10 kDa), centrifuge and ultrafilter at 9500 rpm and 4 ° C to remove all solvents, and wash twice with pure water to remove unreacted phospholipids. The antibody / protein coupling is stored at 4 ° C.

[0094] Synthesis and characterization of antibody conjugates

[0095] The synthesis of aPDL1 conjugate and aMUC1 conjugate was as follows Figure 1 As shown, the primary amine groups (-NH2) on aPDL1 and aMUC1 are covalently coupled to the active lipid bonds (-NHS) on the ligands to form amide bonds (-CO-NH-). Figure 2 As shown, the characteristic peak of phospholipids is around 1600 waves ( Figure 2A), the characteristic peak of aPDL1 is around 3400 waves ( Figure 2 A), while the characteristic peaks of the single curve of aPDL1 phospholipid conjugate appeared at 3500 and 1600 waves, indicating that the aPDL1 phospholipid conjugate was successfully synthesized. The characteristic peak of aMUC1 is at 3400 waves, and the aMUC1 phospholipid conjugate appears at 3400 and 1600 waves ( Figure 2 B), indicating that the synthesis of aMUC1 phospholipid conjugate was successful.

[0096] 3.2 Preparation of blank liposomes

[0097] Blank liposomes were prepared by thin film dispersion method, and non-antibody modified liposomes (Lip), single antibody modified liposomes (aMUC1-Lip, aPDL1-Lip), and double antibody modified liposomes (aMUC1 / aPDL1-Lip) were prepared. Phosphatidylcholine, cholesterol, and antibody-coupled phospholipids were dissolved in a mixture of chloroform and methanol (chloroform / methanol = 2 / 1), and different dosage groups were prepared according to different antibody modifications (aMUC1-Lip, aPDL1-Lip, aMUC1 / aPDL1-Lip). The mixture (30 mg phosphatidylcholine, 7.5 mg cholesterol) was transferred to an eggplant-shaped bottle, and a rotary evaporator was used to evaporate and remove chloroform and methanol to form a lipid film. When the lipid film was about to form, the rotation speed of the suspension evaporator and the pressure in the eggplant-shaped bottle were quickly adjusted to the maximum after the last drop of liquid in the eggplant-shaped bottle disappeared to remove the volatilized chloroform in the eggplant-shaped bottle as much as possible. It is usually necessary to maintain the maximum pressure and rotation speed for 15 minutes. Afterwards, the lipid film was hydrated with 5 ml of ammonium sulfate solution (70 mg / ml), the hydration time was 1 h, the temperature was set at 55 ° C, the speed was set at 40 rpm / min, and the collected lipid film was transferred to an ultrasonic cell crusher with a working time of 3 seconds and a pause of 3 seconds. The temperature of the ultrasonic cell crusher was maintained at 20 ° C, the power was set to 20%, and the total working time was 15 min. The syringe was passed through 0.45 μm and 0.22 μm filter membranes three times. The filtered liposomes were dialyzed with ultrapure water using a dialysis bag (8000-14000 Da), the single dialysis time was 2 h, and the total dialysis was three times. After dialysis, blank liposomes were collected.

[0098] 3.3 Preparation of doxorubicin-loaded liposomes

[0099] The ammonium sulfate concentration gradient method was used to prepare DOX-loaded liposomes of various dosage forms (unmodified doxorubicin-loaded liposomes DOX-Lip, two types of doxorubicin-loaded liposomes DOX-aMUC1-Lip and DOX-aPDL1-Lip modified with single antibodies, and doxorubicin-loaded liposomes DOX-aMUC1 / aPDL1-Lip modified with double antibodies). The modified blank liposome solution was mixed with the DOX solution, and the mixed solution was transferred to an eggplant-shaped bottle. The rotary evaporator was set to a temperature of 63°C, a speed of 50rpm / min, and a time of 20min. The solution was then transferred to a dialysis bag (8000-14000Da) for dialysis. The single dialysis time was 2h, and three dialysis cycles were required. In order to remove the unloaded DOX and obtain completely drug-loaded liposomes, the entire experiment needed to be protected from light.

[0100] 3.4 Preparation of Antibody-modified Cy7-loaded Liposomes

[0101] In order to achieve the targeting effect of each dosage form liposome in the animal body, thiocyanate-7-carboxylic acid (Cy7) was used as a fluorescent probe instead of doxorubicin to be encapsulated in the liposomes of each dosage form. Preparation (unmodified liposome Cy7-Lip, two liposomes modified with single antibody Cy7-aMUC1-Lip, Cy7-aPDL1-Lip, double antibody modified liposome Cy7-aMUC1 / aPDL1-Lip). The modified un-drug-loaded blank liposome solution was mixed with the Cy7 solution, and then the mixed solution was transferred to an eggplant-shaped bottle and the rotary evaporator temperature was set to 63°C, the speed was 50rpm / min, and the time was set to 20min. Then the solution was transferred to a dialysis bag (8000-14000Da) and dialyzed for 2h per dialysis, and a total of three dialysis. In order to remove the unencapsulated Cy7 and obtain the completely encapsulated Cy7 liposome, the entire experiment needs to be protected from light.

[0102] 3.5 Characterization of blank liposomes and liposomes loaded with doxorubicin or Cy7 and analysis of encapsulation efficiency and drug loading

[0103] The standard curves of DOX and Cy7 were drawn by ultraviolet spectrophotometry. DOX and Cy7 concentrated solutions (1 mg / ml) were prepared and diluted according to the concentration gradient of 0, 10, 20, 40, 60, 80, 100 μg / ml. The standard curve of doxorubicin was measured at 478 nm by setting the ultraviolet spectrophotometer, and the standard curve of Cy7 was measured at 680 nm by setting the ultraviolet spectrophotometer. The standard curves of DOX and Cy7 were drawn with the set concentration gradient as the horizontal axis and the measured OD value as the vertical axis. Each group of samples was crushed in a cell crusher, and the cell crusher was ultrasonically crushed with a pulse of 2 seconds on and 3 seconds off, an operating temperature of 20°C, a power of 20%, and a working time of 10 minutes. After crushing, the ultraviolet spectrophotometer was preheated half an hour in advance, and 0.45 μm and 0.22 μm filters were passed through a syringe three times. After that, the liposomes of different dosage forms were diluted 10 times for later use, and 2 ml of liposomes of different dosage forms were transferred to 5 ml centrifuge tubes. 20 μl of 0.1% Triton was added to each group for membrane permeation treatment, and the treatment time was 30 minutes. The liposomes of different dosage forms were measured on the machine using a cuvette. The liposomes of the blank treatment group were normalized before the measurement. The OD values ​​of the liposomes of different dosage forms were brought into the previously drawn DOX and Cy7 standard curves to calculate the drug content of different dosage forms. Each experimental data needs to be repeated three times. The encapsulation efficiency and drug loading of doxorubicin-loaded liposomes and Cy7-loaded liposomes were calculated according to the following formula:

[0104] EE (%) = DOX content in liposomes / initial dosage × 100%

[0105] DL (%) = DOX content in liposomes / total mass of drug-loaded liposomes × 100%

[0106] The liposomes of different dosage forms were characterized and measured after being filtered and diluted 10 times. The Malvern particle size analyzer was preheated 30 minutes in advance, 1.5 ml of liposome solution of each dosage form was taken into the particle size cup and the potentiometric cup, the scattering angle was set to 90°, the working temperature was set to 25°C, the particle size cup and the potentiometric cup were placed according to the machine settings, and the Malvern particle size analyzer was started to measure the particle size and point position of the liposomes of each dosage form. Each set of data must be repeated three times.

[0107] Results (3.2-3.5 Result Analysis): Blank non-antibody modified liposomes (Lip), single antibody modified liposomes (aMUC1-Lip, aPDL1-Lip), and double antibody modified liposomes (aMUC1 / aPDL1-Lip) were successfully prepared. The mass ratio of lecithin, cholesterol, and antibody was 30:7.5:1, the optimal film-forming time was 15min, the optimal film-forming temperature was 40℃, the optimal rotation speed was 100rpm / min, and the optimal pressure was 0.04-0.05MPa. The optimal hydration time was 1h, the optimal hydration temperature was 55℃, the optimal hydration rotation speed was 40rpm / min, and no pressure was applied during hydration.

[0108] Doxorubicin-loaded liposomes without antibody modification (DOX-Lip), doxorubicin-loaded liposomes modified with single antibody (DOX-aMUC1-Lip, DOX-aPDL1-Lip), and doxorubicin-loaded liposomes modified with double antibodies (DOX-aMUC1 / aPDL1-Lip) were successfully prepared, and the optimal drug-to-mass ratio was determined to be 10:1, the optimal incubation time was 1h, the optimal incubation temperature was 60°C, the optimal rotation speed was 100rpm / min, and no pressure was applied for drug encapsulation.

[0109] Fluorescein Cy7-loaded antibody-free liposomes (Cy7-Lip), single antibody-modified Cy7-loaded liposomes (Cy7-aMUC1-Lip, Cy7-aPDL1-Lip), and double antibody-modified Cy7-loaded liposomes (Cy7-aMUC1 / aPDL1-Lip) were successfully prepared. The optimal drug-to-drug ratio was determined to be 10:1, the optimal incubation time was 1h, the optimal incubation temperature was 60°C, the optimal rotation speed was 100rpm / min, and no pressure was applied during drug encapsulation.

[0110] Doxorubicin was detected at a wavelength of 478 nm and a standard curve was drawn. The results are as follows Figure 3 (A) shows that the standard curve formula of doxorubicin is: y = 0.009x + 0.0074, R 2 =0.9971. Cy7 was detected at a wavelength of 680nm and a standard curve was drawn. The results are as follows Figure 3 (B) shows that the formula of the Cy7 standard curve is: y = 0.0105x + 0.0019, R 2 =0.9943. The characterization results of the particle size (Size), Zeta potential, polydispersity index (PDI), EE and DL of each dosage form liposome group are shown in Table 1. According to statistics, the particle size is about 110nm, and the particle size distribution is uniform, the polydispersity is good, the Zeta potential value is about -15mV, the EE average is above 86%, and the DL average is above 7%. The results confirm that the prepared dosage forms of doxorubicin-loaded liposomes have good effects and the physical and chemical properties meet the requirements.

[0111] Table 1. Liposome Size, PDI, Zeta potential, EE and DL of different dosage forms

[0112]

[0113]

[0114] 3.6 Evaluation of liposome morphology in each dosage form group

[0115] Morphological evaluation of liposomes in each dosage form group was performed by observing their size and morphology using TEM electron microscope. First, the samples were ultrasonicated in an ultrasonic cell disruptor using the phosphotungstic acid counterstaining method. The ultrasonic cell disruptor was ultrasonically disrupted with a working pulse of 2 seconds on and 3 seconds off, a working temperature of 20°C, a power of 20%, and a total working time of 10 minutes. After the disruption, the UV spectrophotometer was preheated half an hour in advance, and a 5ml syringe was used to pass through 0.45μm and 0.22μm filter membranes three times. After that, the liposomes in different dosage forms were diluted 10 times. A copper mesh with a polyvinyl alcohol formal lipid film on the surface of the TEM was used. The edge of the copper mesh with the polyvinyl alcohol formal lipid film was clamped with tweezers to make it suspended, and a small amount of liposomes of the test group was sucked and dropped on the copper mesh. After 5 minutes, the remaining liposomes were sucked away with absorbent paper after the liposomes were fully stained. Then the copper mesh was placed on absorbent paper in a glass dish. Finally, the copper mesh of the sample to be tested was dried at low temperature for 2 hours to make it completely dry. Take pictures and record with a TEM microscope.

[0116] Morphological evaluation of doxorubicin-loaded liposomes modified with different antibodies

[0117] The transmission electron microscopy images of each dosage form of doxorubicin-loaded liposomes and blank liposomes are as follows Figure 4 As shown in the results, the doxorubicin-loaded liposomes and blank liposomes of each dosage form are basically three-dimensional spherical and relatively uniform in size. The particle size of DOX-aPDL1-Lip is slightly larger than that of other dosage forms, and the transmission electron microscopy results are basically consistent with the Malvern particle size analyzer results.

[0118] 3.7 Evaluation of the stability of doxorubicin-loaded liposomes

[0119] The liposomes in good condition were placed in NS solution and 3% BSA solution. The mixed solution was stored at 4°C, and its particle size, PDI, Zeta potential, EE and DL were measured at 0, 1, 2, 4, 6, 8, 10, 12 and 14 days, and each set of data was measured three times.

[0120] Different antibody-modified doxorubicin-loaded liposomes have good stability

[0121] Different antibody modified doxorubicin-loaded liposomes have good stability. Figure 5As shown in the figure: After different antibody-modified doxorubicin-loaded liposomes were stored in (NS) solution and 3% BSA solution at 4°C for 14 days, the particle size and Zeta potential of the liposomes in each dosage form were detected by Malvern particle size analyzer and found to be obviously uniform and stable. Figure 5 As shown in A and B, the particle size of doxorubicin-loaded liposomes in each dosage form did not change significantly within 14 days, indicating good stability. Figure 5 As shown in C and D, the Zeta potential of each dosage form of doxorubicin liposomes in the NS solution group was significantly lower than that in the 3% BSA solution group, but each group fluctuated within a small range and was generally stable. The Zeta potential of the 3% BSA group was relatively high, and the higher the absolute value of the Zeta potential, the stronger the stability. Therefore, it was proved that each dosage form of doxorubicin liposomes had higher stability in the 3% BSA solution. Although the absolute value of the potential decreased slightly over time, the overall absolute value of the potential was still high, proving that it had good stability. The results showed that each dosage form of doxorubicin liposomes had good stability within 14 days when stored at low temperature in NS or 3% BSA.

[0122] 3.8 Evaluation of in vitro drug release from doxorubicin-loaded liposomes

[0123] Drug release was evaluated by dialysis. Take 2 ml of each dosage group liposome and free DOX group in a (8000-14000Da) dialysis bag, seal both ends of the dialysis bag and put them into a dark bottle with 20 ml PBS and 3% BSA solution, place the dark bottle on a 37°C shaking table, set the speed to 80 rpm / min, and take samples at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 36 hours, and take 2 ml of the solution in the dark bottle each time. After sampling, immediately add 2 ml of PBS and 3% BSA solution to the dark bottle. Use an ultraviolet spectrophotometer to detect the OD value of the liposomes in each dosage group at 478 nm, calculate the cumulative release percentage, and draw the DOX cumulative release curve with time as the horizontal axis and the calculated cumulative release percentage as the vertical axis.

[0124] Different antibody-modified doxorubicin-loaded liposomes release doxorubicin well in vitro

[0125] Different antibody modified doxorubicin loaded liposomes have good drug release performance in vitro. The in vitro cumulative drug release diagram of different antibody modified doxorubicin loaded liposomes is as follows Figure 6As shown in the figure, compared with free doxorubicin, the antibody-modified doxorubicin-loaded liposomes have a significant sustained release effect. The drug release is divided into two stages. The first stage is a burst release within the first 5 hours, and the second stage reaches a plateau after the 5th hour. Compared with the free doxorubicin group, the antibody-modified doxorubicin-loaded liposome group has a significant sustained release effect. The results showed that at 36 hours, free doxorubicin had basically been released, and the antibody-modified doxorubicin-loaded liposomes were still in a sustained release state. At the same time, the sustained release effect of different antibody-modified doxorubicin-loaded liposome groups was not significantly different.

[0126] 4 Cell culture

[0127] 4.1 Cell recovery

[0128] First, preheat the water bath to 37°C, and preheat fetal bovine serum FBS, DMEM high glucose medium, RPMI1640 medium, penicillin-streptomycin PS, and PBS in the water bath at 37°C in advance. Open the clean bench, wipe the bench clean with 75% alcohol, and place 200μl, 1250μl pipette tips, 2ml, 5ml, 50ml centrifuge tubes, and T25 cell culture bottles in the clean bench for UV sterilization for 30 minutes. Take out NCI-H441, NCI-H460, ZR-75-1, and HUH7 cells from the liquid nitrogen tank, quickly place the cells in a 37°C water bath and shake slowly until the cells melt. Wipe the water off the wall of the cell cryopreservation tube, then disinfect it with 75% alcohol and place it in the clean bench. Prepare DMEM complete medium, place 5ml FBS and 500μl PS in a 50ml centrifuge tube, and fill the total volume to 50ml with DMEM high glucose medium to obtain DMEM complete medium. Prepare RPMI1640 complete medium, 5ml FBS, 500μl PS in a 50ml centrifuge tube, and use RPMI1640 medium to make the total volume 50ml to obtain RPMI1640 complete medium. Transfer the thawed cells in the cryopreservation tube to a 5ml centrifuge tube, add 1ml RPMI1640 complete medium to NCI-H441 and ZR-75-1, and add 1ml DMEM complete medium to NCI-H460 and HUH7. Transfer the total volume of 2ml centrifuge tube to the centrifuge, the speed is 1000rpm / min and the centrifugation time is 5min. After centrifugation, remove the supernatant and take 1ml complete medium to resuspend the cells, and add 5ml complete medium to the T25 culture flask. Transfer the resuspended cells to the culture flask and mix well. Place the culture flask in a cell culture incubator with a carbon dioxide concentration of 5% and 37℃. When the cells proliferate to 80% of the total volume of the culture flask, they can be passaged.

[0129] 4.2 Cell passaging

[0130] First, preheat the water bath to 37°C, and preheat fetal bovine serum FBS, DMEM high glucose medium, RPMI1640 medium, penicillin-streptomycin PS, PBS, and trypsin Try in the water bath at 37°C. Open the clean bench, wipe the bench clean with 75% alcohol, and place 200μl, 1250μl pipette tips, 2ml, 5ml, 50ml centrifuge tubes, and T25 cell culture flasks in the clean bench for UV sterilization for 30 minutes. Prepare DMEM complete medium, place 5ml FBS and 500μl PS in a 50ml centrifuge tube, and fill the total volume to 50ml with DMEM high glucose medium to obtain DMEM complete medium. Prepare RPMI1640 complete medium, place 5ml FBS and 500μl PS in a 50ml centrifuge tube, and fill the total volume to 50ml with RPMI1640 medium to obtain RPMI1640 complete medium. Observe the cells that need to be passaged under a microscope, and passage them when they are 80% attached. Aspirate the previous culture medium in the culture flask, rinse twice with 3ml PBS, absorb 1ml of trypsin, and make the trypsin completely soak the bottom surface. After disinfecting the culture flask, put it in a 37℃ incubator for incubation for 1min-2min. In the middle, you can observe the cell digestion. When the cell morphology changes to a contracted state, immediately add 1ml of culture medium to terminate the digestion, gently blow down the attached cells, and then transfer them to a 5ml centrifuge tube and wait for centrifugation. The centrifuge is 1000rpm / min, and the centrifugation time is set to 5min. After centrifugation, discard the supernatant, resuspend the cell pellet with 1ml complete culture medium, and then passage the resuspended cell solution at a ratio of 1:2 to 1:4. Add 5ml of complete culture medium to the T25 culture flask. Transfer the resuspended cells to the culture flask and mix well. Place the culture flask in a cell culture incubator at 37℃ with 5% carbon dioxide.

[0131] 4.3 Cell cryopreservation

[0132] First, preheat the water bath to 37°C, and preheat fetal bovine serum FBS, DMEM high glucose medium, RPMI1640 medium, penicillin-streptomycin PS, PBS, and trypsin Try in the water bath at 37°C. Open the clean bench, wipe the bench clean with 75% alcohol, and place 200μl, 1250μl pipette tips, 2ml, 5ml, 50ml centrifuge tubes, and cell cryopreservation tubes in the clean bench for UV sterilization for 30 minutes. Prepare DMEM complete medium, place 5ml FBS and 500μl PS in a 50ml centrifuge tube, and fill the total volume to 50ml with DMEM high glucose medium to obtain DMEM complete medium. Prepare RPMI1640 complete medium, place 5ml FBS and 500μl PS in a 50ml centrifuge tube, and fill the total volume to 50ml with RPMI1640 medium to obtain RPMI1640 complete medium. Discard the culture medium of the cells to be subcultured, wash twice with 3ml PBS, absorb 1ml pancreatin to make the pancreatin completely soak the bottom surface, sterilize the culture bottle and put it in a 37℃ incubator for 1min-2min. You can observe the cell digestion in the middle, and stop the digestion immediately when the cell morphology changes to a contracted state. Gently blow down the adherent cells, and then transfer them to a 5ml centrifuge tube for centrifugation. The centrifuge is 1000rpm / min, and the centrifugation time is set to 5min. After centrifugation, discard the supernatant and prepare the cell freezing solution. Add 900μl FBS to 100μl DMSO and mix well, and resuspend the cells with the mixed cell freezing solution. Transfer the cell suspension into a cell freezing tube, use a programmed cell cooling freezing box to store the cells at -80℃, and wait for the cells to cool down before transferring them to a liquid nitrogen tank for storage.

[0133] 4.4 Cell counting

[0134] Resuspend the cells after digestion and centrifugation with complete medium, mix thoroughly and take 100μl of the cell suspension to make up the volume to 1ml with complete medium, which is dilute ten times. Take the diluted cell suspension to the cell counting plate and wait for the liquid to completely infiltrate the counting area before starting to count under an inverted microscope. The counting principle is to count the top but not the bottom, and count the left but not the right. Each count needs to be repeated three times.

[0135] Identification of PD-L1 and MUC1 protein expression in 5 cell lines

[0136] 5.1 Western blot identification of PD-L1 and MUC1 expression

[0137] NCI-H441, NCI-H460, ZR-75-1, and HUH7 cells in the logarithmic growth phase were inoculated into cell culture dishes (5×10 5 / dish), change the medium every two days, and extract the total cell protein when the cells grow to 80% of the culture dish. For the extraction of total cell protein, use RIPA lysis method to extract cell protein. Add 1ml RIPA lysis solution to the culture dish, incubate on ice for 10 minutes, scrape the culture solution and cells together with a cell scraper, transfer to a centrifuge tube, 4℃, 11000rpm, centrifuge time for 15min. After centrifugation, discard the precipitate and take the supernatant. The protein concentration is determined by BCA method, then add 25% volume of 5× loading buffer and mix evenly, and place in a metal bath at 95℃ for 5min. Then store the protein at -20℃. Then perform Western blot. First, configure protein gel. After the glass plate is assembled, configure 10% separation gel. After the separation gel is filled, let it stand for 15 minutes. After the separation gel solidifies, configure the concentrated gel. After the concentrated gel is filled, let it stand for 15 minutes before loading the sample for electrophoresis. After the electrophoresis tank is assembled, load the sample. Load 5μl and 3μl of protein maker in the first hole and the last empty space respectively, and load 10μl of protein sample in the remaining holes. First, adjust the voltage to 20V for 10min to remove inorganic salts and metal ions in the sample. The remaining concentrated gel voltage is 80V for 30min. When the separation gel is to be separated, the voltage is adjusted to 110V for 80min. The electrophoresis can be terminated when the target protein is electrophoresed to the appropriate position. Take out the separation gel for transfer. The transfer conditions are voltage 100V, current 400mA, and time 65min. Then take out the NC membrane and block it with the configured 5% skim milk powder at room temperature for 2h, and then the primary antibody at room temperature for 2h. The membrane was washed three times with TBST solution, each time for 15 min. The secondary antibody was incubated at room temperature for 2 h, and the membrane was washed three times with TBST solution, each time for 15 min. The membrane was then developed and photographed using a chemiluminescence instrument.

[0138] 5.2 Identification of PDL1 and MUC1 expression by cell immunofluorescence

[0139] NCI-H441, NCI-H460, ZR-75-1, and HUH7 cells in the logarithmic growth phase were inoculated into 24-well plates containing 14 mm slides (inoculation density was 5 × 10 4 / well) After 24 hours, the cell morphology is stable and subsequent experiments can be carried out. After washing the cells twice with PBS solution, they can be fixed with 4% PFA, 500μl per well, and fixed for 15min. Wash twice with PBS solution, add 0.5ml 0.2% Triton-X 100 to each well to permeate the membrane for 15min. Wash twice with PBS solution, add 0.5ml 3% BSA solution to each well for blocking and incubate overnight at 4℃. Wash three times with PBS solution and incubate with secondary antibody at room temperature for 1h. Wash three times with PBS, stain the cell nucleus with 500μl DAPI (5μg / ml) solution for 15min, wash three times with PBS solution, add 7μl anti-fluorescence attenuation mounting agent to the slide, turn the side with cells down, and then seal with nail polish, and observe and take pictures under an upright fluorescence microscope.

[0140] Identification of PD-L1 and MUC1-positive cell lines

[0141] In this experiment, the expression of PD-L1 and MUC1 in cell lines NCI-H441, NCI-H460, ZR-75-1 and HUH7 was detected by WB and immunofluorescence. The WB results are as follows Figure 7 As shown, the HUH7 cell line expressed neither MUC1 nor PD-L1 protein, the NCI-H460 cell line expressed PD-L1 protein but not MUC1 protein, the ZR-75-1 cell line expressed MUC1 protein but not PD-L1 protein, and the NCI-H441 cell line expressed both MUC1 protein and PD-L1 protein. The results of immunofluorescence verification are as follows Figure 8 As shown, green fluorescence (FITC) shows the expression of the target protein, and blue fluorescence DAPI shows the cell nucleus. Figure 8 Results A showed that NCI-H441 cells simultaneously expressed PD-L1 and MUC1 proteins; Figure 8 The results in B showed that NCI-H460 cells only expressed PD-L1 protein, but not MUC1 protein. Figure 8 C The results showed that ZR-75-1 cells only expressed MUC1 protein, but not PD-L1 protein. Figure 8 D The results showed that HUH7 cells expressed neither PD-L1 nor MUC1 protein. The immunofluorescence results were consistent with the WB results. The above results identified NCI-H441 and NCI-H460 as PD-L1 positive expression lines, and NCI-H441 and ZR-75-1 as MUC1 positive expression lines. HUH7 was a negative expression line. Therefore, NCI-H441, NCI-H460, and ZR-75-1 were selected as positive cell lines, and HUH7 was selected as a negative control cell line.

[0142] 6 In vitro study on targeting of antibody-modified doxorubicin-loaded liposomes

[0143] In order to explore the targeting efficiency of antibody-modified liposomes for cells in the logarithmic growth phase, tumor cells NCI-H441, NCI-H460, ZR-75-1, and HUH7 cells in the logarithmic growth phase were inoculated into 24-well plates containing 14 mm cell slides (seeding density was 5 × 10 4 After 24 hours, when the cell morphology is stable, discard the culture medium, add liposomes containing 10μg / ml (DOX, DOX-Lip, DOX-aMUC1-Lip, DOX-aPDL1-Lip, DOX-aMUC1 / aPDL1-Lip), incubate for 3 hours, wash the cells twice with PBS and then fix them with 4% PFA, 500μl per well, fix for 15 minutes. Wash twice with PBS, add 0.5ml 0.2% Triton-X 100 to each well to permeate the membrane for 15 minutes. Wash twice with PBS, add 0.5ml 3% BSA to each well for blocking and incubate overnight at 4℃. Wash three times with PBS and incubate with secondary antibody at room temperature for 1 hour. The slides were washed three times with PBS, the cell nuclei were stained with 500 μl DAPI (5 μg / ml) solution for 10 min, and the slides were washed three times with PBS solution. 7 μl anti-fluorescence attenuation mounting medium was added to the slides, and the slides were mounted with nail polish with the side with cells facing down. The slides were observed and photographed under an upright fluorescence microscope.

[0144] Evaluation of cellular uptake targeting

[0145] In order to explore the tumor cell targeting of doxorubicin-loaded liposomes modified with different antibodies, we conducted cell uptake experiments. The cell uptake results are as follows Fig. 9 As shown, doxorubicin can spontaneously display red fluorescence, blue fluorescence DAPI represents the cell nucleus, and the red fluorescence intensity represents the content of doxorubicin taken up by the cell. The superposition of red and blue is purple. Fig. 9 A and B show the cell uptake of the double-positive cell line NCI-H441. The red fluorescence intensity of the DOX-aMUC1 / aPDL1-Lip group was the strongest, followed by the DOX-aMUC1-Lip group and the DOX-aPDL1-Lip group, and the DOX-Lip group and the free doxorubicin group had the lowest red fluorescence intensity (P<0.001). This is because NCI-H441 cells express not only PD-L1 but also MUC1, and the two proteins enhance targeting through antigen-antibody binding at the same time. Fig. 9 The results of C and D showed that in the PD-L1-positive cell line NCI-H460, the red fluorescence intensity of the DOX-aMUC1 / aPDL1-Lip group and the DOX-aPDL1-Lip group was significantly increased compared with that of the DOX-Lip group (P<0.001), but there was no difference between the two groups. Fig. 9 The results of E and F showed that in the MUC1-positive cell line ZR-75-1, the red fluorescence intensity of the DOX-aMUC1 / aPDL1-Lip group and the DOX-aMUC1-Lip group was significantly increased compared with that of the DOX-Lip group (P<0.001), but there was no significant difference between the two groups. Fig. 9 The results of G and H showed that the fluorescence intensity of the DOX-aMUC1 / aPDL1-Lip group was slightly stronger than that of other groups in the double-negative cell line HUH7, and there was no significant difference between the other groups. The results showed that the double antibody-modified doxorubicin-loaded liposome DOX-aMUC1 / aPDL1-Lip had obvious tumor cell targeting.

[0146] In vitro study on the effect of 7-antibody modified doxorubicin-loaded liposomes on tumor cell activity

[0147] The MTS method was used to determine the cytotoxicity of antibody-modified doxorubicin-loaded liposomes to cancer cells. NCI-H441, NCI-H460, ZR-75-1, and HUH7 cells in the logarithmic growth phase were inoculated into 96-well plates (inoculation density was 5000 cells / well). After 24 hours of stable cell morphology and adherence, the culture medium was discarded and replaced with liposomes containing 10μg / ml doxorubicin (DOX, DOX-Lip, DOX-aMUC1-Lip, DOX-aPDL1-Lip, DOX-aMUC1 / aPDL1-Lip), and incubated together for 72h according to different concentrations. Add 100μl of the mixed solution (MTS, PMS, and transparent culture medium) to each well and continue incubation at 37°C for 15min-2h. After the trend is obvious, the OD value is measured by setting the wavelength of the microplate reader to 489nm. Repeat the measurement three times for each well plate. The IC50 calculation formula is as follows:

[0148] IC50 (%) = (1-OD value of drug-treated group / OD value of control group) × 100%

[0149] Evaluation of cytotoxicity of doxorubicin-loaded liposomes modified with different antibodies

[0150] The MTS method was used to detect the cytotoxicity of each dosage form of doxorubicin-loaded liposomes on cell lines NCI-H441, NCI-H460, ZR-75-1 and HUH7. Fig.10As shown, in the double-positive cell line NCI-H441, compared with free doxorubicin and other antibody-modified doxorubicin-loaded liposomes, the IC50 value of the DOX-aMUC1 / aPDL1-Lip group was the lowest at 181.1 ng / ml, indicating that DOX-aMUC1 / aPDL1-Lip had the strongest cytotoxicity to the double-positive cell line NCI-H441. In the PD-L1-positive cell line NCI-H460, compared with free doxorubicin and other antibody-modified doxorubicin-loaded liposomes, the IC50 value of the DOX-aMUC1 / aPDL1-Lip group was the lowest at 80.38 ng / ml, followed by the DOX-aPDL1-Lip group, with an IC50 value of 101.2 ng / ml, indicating that the DOX-aMUC1 / aPDL1-Lip group had the strongest cytotoxicity to the PD-L1-positive cell line NCI-H460. In the MUC1-positive cell line ZR-75-1, compared with free doxorubicin and other antibody-modified doxorubicin-loaded liposome groups, the IC50 value of the DOX-aMUC1 / aPDL1-Lip group was the lowest at 15.36 ng / ml, followed by the DOX-aMUC1-Lip group, with an IC50 value of 18.38 ng / ml, indicating that the DOX-aMUC1 / aPDL1-Lip group had the strongest cytotoxicity to the MUC1-positive cell line ZR-75-1. In the double-negative cell line HUH7, the DOX-aMUC1 / aPDL1-Lip group was basically the same as the other antibody-modified liposome groups. The above results show that the double-antibody-modified doxorubicin-loaded liposome DOX-aMUC1 / aPDL1-Lip can be taken up by tumor cells in large quantities due to its strong tumor cell targeting, thereby improving its anti-tumor cell effect.

[0151] Evaluation of in vivo targeting and antitumor effects of antibody-modified doxorubicin-loaded liposomes

[0152] 8.1 Establishment of NCI-H441 tumor-bearing nude mouse model

[0153] BALB / C-nu mice were purchased and stabilized for one week before the establishment of the tumor-bearing nude mouse model. The NCI-H441 cells that were cultured in advance and in sufficient quantity and in good condition in the logarithmic growth phase were digested and centrifuged at 1000 rpm for 5 minutes, resuspended in NS solution and counted. The resuspended cells were diluted to a concentration of 2.5×10 7 / ml, transfer the cell suspension to the experimental animal center for inoculation, and inoculate 200μl under the axilla of the right forelimb of the nude mouse. After the tumor tissue is visible on day 12, measure the weight and tumor volume of the nude mouse every other day. The tumor volume is measured uniformly using an electronic vernier caliper to count to two decimal places. According to the measured long radius: a and short radius: b, the calculation formula is: tumor volume = ab 2 / 2(mm 3) Each numerical measurement was repeated three times. When the tumor volume of the tumor-bearing nude mice reached the standard of 100 mm 3 At the same time, the tumor-bearing nude mice were divided into groups for small animal living imaging experiments and in vivo anti-tumor experiments.

[0154] 8.2 Verification of in vivo targeting of antibody-modified Cy7-loaded liposomes

[0155] Evaluation of the targeting effect of antibody modification Therefore, the doxorubicin encapsulated in each antibody-modified dosage form group was replaced with Cy7, and the NCI-H441 tumor-bearing nude mice raised in the SPF-level experimental animal center were taken out and randomly divided into 5 groups, with 6 tumor-bearing nude mice in each group, and injected through the tail vein (injection standard: 200μl 10mg / kg Cy7, Cy7-Lip, Cy7-aMUC1-Lip, Cy7-aPDL1-Lip, Cy7-aMUC1 / aPDL1-Lip), and then the small animal in vivo imaging instrument was set to a wavelength of 680nm to detect the in vivo fluorescence distribution, and photos were taken at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, and 48h after drug injection. After 48h, the tumor-bearing nude mice were killed and their hearts, livers, spleens, lungs, kidneys, and tumors were taken out. Photographs were taken to observe the distribution of Cy7 in various organs in different dosage forms.

[0156] Liposomes modified with different antibodies enhance tumor targeting in vivo

[0157] To further investigate the targeting effect of dual antibody modified liposomes in NCI-H441 tumor-bearing nude mice, Cy-7 was loaded into liposomes as a fluorescent probe and monitored using a small animal in vivo imaging system. The results are as follows Fig.11 As shown in A, tumor-bearing nude mice were injected with free Cy7 and Cy7 liposomes modified with different antibodies through the tail vein. One hour after injection, fluorescence (Cy7) was distributed throughout the body of tumor-bearing nude mice in all experimental groups. At 4 hours after injection, the fluorescence intensity gradually weakened, and compared with the free Cy7 group, the fluorescence of the Cy7 liposome group gradually accumulated in the tumor tissue. At 8 hours after injection, the fluorescence intensity of the free Cy7 group and the Cy7-Lip group was significantly reduced. Compared with the free Cy7 group and the Cy7-Lip group, the Cy7-aMUC1-Lip, Cy7-aPDL1-Lip and Cy7-aMUC1 / aPDL1-Lip groups showed more obvious tumor tissue aggregation.

[0158] At 12 hours after injection, Cy7-Lip showed a weak fluorescence signal, which may be due to the transient aggregation of liposomes caused by enhanced permeability and retention (EPR). After 24 hours after injection, the fluorescence signal of the Cy7-Lip group completely disappeared, while Cy7-aMUC1-Lip, Cy7-aPDL1-Lip and Cy7-aMUC1 / aPDL1-Lip groups still had strong fluorescence signals in tumor tissues, and the fluorescence signal of the Cy7-aMUC1 / aPDL1-Lip group was the strongest in tumor tissues. After 48 hours after injection, the antibody-modified liposome group still had strong fluorescence signal accumulation at the tumor site. The above results show that the constructed Cy7-aMUC1-Lip, Cy7-aPDL1-Lip and Cy7-aMUC1 / aPDL1-Lip have good tumor targeting effects and can deliver drugs to tumor tissues. At the same time, the sustained release effect of liposomes enhances the therapeutic effect and achieves the maximum therapeutic effect by accumulating drugs in tumor tissues. 48 hours after injection, tumor-bearing nude mice were killed to observe the fluorescence signals of their main internal organs and tumor tissues. The results are as follows Fig.11 As shown in Figure B, 48 hours after injection, Cy7-Lip had strong signal accumulation in the liver and kidneys. This was due to the accumulation in the liver and kidneys without antibody modification and lack of targeting. However, in the liver, no obvious fluorescence signals were found in the Cy7-aMUC1-Lip, Cy7-aPDL1-Lip and Cy7-aMUC1 / aPDL1-Lip groups, and their fluorescence signals were mainly concentrated in tumor tissues. Moreover, compared with the Cy7-aMUC1-Lip and Cy7-aPDL1-Lip groups, the Cy7-aMUC1 / aPDL1-Lip group showed very strong fluorescence signals in tumor tissues. At the same time, the fluorescence intensity of the tissue in Figure B was quantitatively analyzed, and the results showed that compared with the other groups, Cy7-aMUC1 / aPDL1-Lip had the strongest fluorescence intensity, with significant statistical differences ( Fig.11 C). The results are consistent with the in vivo targeting results, indicating that Cy7-aMUC1 / aPDL1-Lip has the strongest targeting to tumor cells and tissues.

[0159] 8.3 Antitumor evaluation of antibody-modified liposomes loaded with doxorubicin

[0160] Tumor-bearing nude mice kept in an SPF-grade experimental animal room were numbered and randomly divided into 6 groups of 6 nude mice each, ensuring that the average tumor volume and body weight of tumor-bearing nude mice in each group were basically the same. The injection conditions were: (NS, DOX, DOX-Lip, DOX-aMUC1-Lip, DOX-aPDL1-Lip) liposomes were injected into tumor-bearing nude mice at (5 mg / ml) every 4 days, and the injection method was: intraperitoneal injection. The tumor volume and body weight changes of tumor-bearing nude mice were measured every other day and a line graph was drawn. On the 18th day, the tumor-bearing nude mice were killed and the tumors were removed by dissection, weighed, and photographed. The main organs and tumors were stored in 4% paraformaldehyde for fixation for subsequent histological evaluation.

[0161] In vivo antitumor effects of doxorubicin-loaded liposomes modified with different antibodies

[0162] The experimental schedule of the in vivo anti-tumor effect of doxorubicin-loaded liposomes modified with different antibodies is as follows Fig.12 As shown in A, the NCI-H441 cells cultured to the required number were placed on the right forelimb of nude mice for tumor bearing, and the in vivo anti-tumor experiment was performed 20 days after tumor bearing, and the histological evaluation was performed 18 days after the anti-tumor experiment. The results of the in vivo anti-tumor effect showed that the tumor in the normal saline group was the largest; compared with the antibody-modified doxorubicin liposome group, the tumor in the free doxorubicin group was the largest; the tumor sizes of the single antibody-modified doxorubicin liposome group DOX-aMUC1-Lip and DOX-aPDL1-Lip were similar, but smaller than the free doxorubicin group; the tumor in the double antibody-modified doxorubicin liposome DOX-aMUC1 / aPDL1-Lip group was the smallest, indicating that it has the strongest inhibitory effect on tumors ( Fig.12 B). Fig.12 C is the anatomical weighing analysis of tumors in nude mice. Fig.12 The results of B were consistent, with the normal saline group having the largest tumor weight. The tumor weights of the two groups of single antibody modified doxorubicin liposomes DOX-aMUC1-Lip and DOX-aPDL1-Lip groups were basically the same, but smaller than the free doxorubicin group. The tumor weight of the double antibody modified doxorubicin liposomes DOX-aMUC1 / aPDL1-Lip group was the lightest, which was significantly different from other dosage forms of doxorubicin liposomes. Fig.12 D is a line graph of tumor volume measurement in nude mice bearing tumors on day 18. The results show that the tumor volume in the saline group is significantly larger than that in the doxorubicin liposome groups of each dosage form, and the difference in tumor volume change in the double antibody-modified doxorubicin liposome DOX-aMUC1 / aPDL1-Lip group within 18 days is the smallest, and its tumor volume is also the smallest compared to other antibody-modified groups. The results are consistent with the above tumor size and weight results. Fig.12E is a line graph of the weight changes of tumor-bearing nude mice over 18 days. Free doxorubicin has the greatest impact on the weight of tumor-bearing nude mice due to its own cytotoxicity, but after being encapsulated in antibody-modified liposomes, the weight changes of tumor-bearing nude mice are significantly reduced, indicating that after being encapsulated in antibody-modified liposomes, the toxicity of doxorubicin as a chemotherapy drug is significantly reduced while its anti-tumor activity is enhanced.

[0163] 8.4 Histological evaluation of the toxicity of antibody-modified doxorubicin-loaded liposomes on tumor-bearing nude mice

[0164] Cut the main organs and tumor tissues of the fixed tumor-bearing nude mice into as large a block as possible, put them into the embedding box, put the embedding box into a beaker of pure water to rinse overnight, and put the embedding box into the dehydration treatment method after 12 hours (70% ethanol solution 2h converted to 80% ethanol solution 2h converted to 95% ethanol solution 2h converted to anhydrous ethanol solution I 2h converted to anhydrous ethanol solution II 2h converted to xylene solution I 1h converted to xylene II 30min) after dehydration is completed, immerse in wax liquid for 2h, and put it into the embedding machine for embedding. Put the embedded tissue into the slicer in the direction of the maximum cross section, cut into 4μm slices, and bake the slices at 60℃ for 12h until the wax liquid disappears. After the wax liquid disappears, prepare for staining, the steps are (xylene solution I for 2 minutes, transfer to xylene solution II for 2 minutes, transfer to anhydrous ethanol solution I for 2 minutes, transfer to anhydrous ethanol II for 2 minutes, and finally transfer to 75% ethanol solution for 2 minutes), then rinse with pure water for 2 minutes and start staining (hematoxylin staining for 3-5 minutes, quickly transfer to hydrochloric acid aqueous solution for 5 minutes, rinse with pure water, and then let the slices pass through 85% and 95% ethanol solutions in turn for dehydration again, transfer to eosin staining solution for staining for 5 minutes) after staining is completed, perform transparent treatment (put the slices into anhydrous ethanol solution I for 2 seconds, quickly transfer to anhydrous ethanol solution II for 2 seconds, quickly transfer to xylene solution I for 2 seconds, transfer to xylene solution II for 2 seconds), after transparentization is completed, seal the slides with neutral resin, collect the slides after the neutral resin is dry, observe under an upright microscope and take pictures.

[0165] According to the above experimental results, the main organs and tumor tissues of tumor-bearing nude mice were sliced ​​and histopathologically analyzed. The results of HE staining showed that no obvious tissue necrosis was found in the heart, liver, spleen, lung, and kidney. The tumor tissues in the antibody-modified liposome group had obvious tissue necrosis, especially the tumor necrosis degree in the DOX-aMUC1 / aPDL1-Lip group was significantly higher than that in the other groups ( Fig.13 ). The above results indicate that the dual antibody-modified doxorubicin-loaded liposome system we constructed can significantly improve the anti-tumor efficacy while significantly reducing the physical toxicity of doxorubicin.

[0166] In vivo antitumor mechanism of antibody-modified doxorubicin-loaded liposomes

[0167] like Fig.14As shown, the antibody, with its unique advantages such as specificity, is covalently bound to a special phospholipid and a thin film dispersion method and ammonium sulfate concentration gradient method are used to construct antibody-modified specific liposomes containing doxorubicin. It is injected into the tumor-bearing nude mice of NCI-H441 through the tail vein. Through the blood circulation and the specific recognition of aPDL1 and aMUC1 by NCI-H441 cancer cells that highly express PD-L1 and MUC1, the phospholipids fuse with the cell membrane through receptor-mediated endocytosis and release doxorubicin into the cell. Doxorubicin can combine with DNA analysis in the cell nucleus to inhibit nucleic acid synthesis and achieve anti-tumor effects.

Claims

1. A PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposome, characterized in that: The method comprises co-modifying liposomes with antibodies of MUC1 and PD-L1 and encapsulating doxorubicin therein to prepare doxorubicin-loaded double-antibody modified liposomes.

2. A method for preparing PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposomes, characterized in that: The following steps are involved: 1) Preparation of antibody-conjugated phospholipids Dissolve 1-2 mg of phospholipid in phospholipid solution, dissolve 1 mg of antibody or protein in 1 ml of reaction buffer, mix 50 μl of phospholipid solution and 1 ml of reaction buffer evenly, react at room temperature for 2 hours, after the reaction is completed, transfer the reaction system to a 10 kDa ultrafiltration tube, centrifuge and ultrafilter at 9500 rpm, 4°C to remove all solvents, wash twice with pure water to remove unreacted phospholipids, and store the antibody / protein coupling at 4°C; 2) Preparation of blank liposomes Blank liposomes were prepared by thin film dispersion method, and antibody-free liposomes Lip, single antibody-modified liposomes aMUC1-Lip, aPDL1-Lip, and double antibody-modified liposomes aMUC1 / aPDL1-Lip were prepared. Phosphatidylcholine, cholesterol and antibody-coupled phospholipids were dissolved in a mixture of chloroform and methanol, wherein chloroform / methanol = 2 / 1. Different dosage forms were prepared according to different antibody modifications (aMUC1-Lip, aPDL1-Lip, aMUC1 / aPDL1-Lip). The mixture (lecithin 30mg, cholesterol 7.5mg) is transferred to an eggplant-shaped bottle, chloroform and methanol are removed by rotavapor evaporation to form a lipid film, when the lipid film is about to form, the rotating speed of the suspension evaporator and the pressure in the eggplant-shaped bottle are quickly adjusted to the maximum to remove the volatilized chloroform in the eggplant-shaped bottle as much as possible after the last drop of liquid in the eggplant-shaped bottle disappears, and the maximum pressure and rotating speed are maintained for 15min, and the lipid film is hydrated with 5ml of 70mg / ml ammonium sulfate solution afterwards, and the hydration time is 1h, the temperature is set at 55°C, and the rotating speed is set at 40rpm / min. The collected lipid film is transferred to an ultrasonic cell crusher and paused for 3 seconds with a working time of 3 seconds, and the ultrasonic cell crusher temperature is maintained at 20°C, and the power is set at 20%, and the total working time is 15min. The syringe is passed through 0.45μm and 0.22μm filter membranes three times, and the filtered liposomes are dialyzed with ultrapure water with 8000-14000Da dialysis bags, and the single dialysis time is 2h, and the dialysis is performed three times in total. After dialysis, blank liposomes are collected; 3) Preparation of doxorubicin-loaded liposomes The ammonium sulfate concentration gradient method was used to prepare DOX-loaded liposomes of various dosage forms (unmodified DOX-Lip loaded with doxorubicin, two types of DOX-aMUC1-Lip and DOX-aPDL1-Lip loaded with doxorubicin modified with single antibodies, and DOX-aMUC1 / aPDL1-Lip loaded with doxorubicin modified with double antibodies). The modified blank liposome solution was mixed with the DOX solution, and the mixed solution was transferred to an eggplant-shaped bottle. The rotary evaporator was set to a temperature of 63°C, a speed of 50rpm / min, and a time of 20min. The solution was then transferred to a 8000-14000Da dialysis bag for dialysis for 2h per dialysis. It was necessary to dialyze three times to remove the unloaded DOX and obtain completely drug-loaded liposomes. The entire experiment needed to be protected from light.

3. Use of the PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposomes according to claim 1 in the preparation of anti-tumor drugs.

4. Use of the PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposomes according to claim 1 in the preparation of a drug for the treatment of non-small cell lung cancer.

5. Use of the PD-L1 / MUC1 double antibody modified doxorubicin-loaded liposomes according to claim 1 in a drug for treating tumors with high surface expression of PD-L1 and MUC1.

6. The PD-L1 / MUC1 dual antibody modified drug delivery system described in claim 1 is used as a highly targeted system for drug encapsulation and presentation, thereby better solving the problem of drug resistance.

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