Double-target modified liposome as well as preparation method and application thereof

Abstract

The invention discloses a double-target modified liposome as well as a preparation method and application thereof. The double-target modified liposome is formed by modifying a CL4 aptamer and a VTP polypeptide on the surface of a liposome. The invention proves that the CL4 and VTP modified liposome has an obvious active targeting drug delivery effect on colorectal cancer, the double-target modification targeting effect is superior to that of a single target, in-vivo and in-vitro experiments show that the uptake rate of the double-target liposome is improved by 10%-20% compared with that of the single target liposome, and a synergistic interaction effect is achieved. According to the invention, the dual-target modified liposome preparation simultaneously loaded with docetaxel and kaempferol is synthesized for the first time, and a research report that a nano-carrier simultaneously loads the docetaxel and the kaempferol is not found yet. In-vivo and in-vitro experiments prove that the CL4 and VTP modified liposome co-loaded with docetaxel and kaempferol has a remarkable effect of inhibiting the proliferation of colorectal cancer compared with a pure drug combination / unmodified double-loading / single-loading liposome, and has a good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a dual-target modified liposome and a preparation method and application thereof. Background Art

[0002] In addition to surgery, colorectal cancer patients currently receive systemic treatment mainly through chemotherapy, radiotherapy, etc. However, due to the low specificity of chemotherapy drugs to cancer cells, the low response rate of chemotherapy and serious side effects, the conventional treatment effect of colorectal cancer, especially for patients with advanced metastasis, is not good.

[0003] Small molecule chemotherapy drugs are easily recognized by the reticuloendothelial system in the human body and phagocytosed by macrophages, showing a faster in vivo clearance rate and a shorter blood circulation time. After intravenous administration, the donor concentration in the blood circulation system will decrease rapidly, resulting in ineffective efficacy at the tumor and low bioavailability. With the development of nanomedicine, liposome delivery system has become one of the most promising nanocarriers for cancer treatment. Liposomes are a new type of drug delivery carrier. They are spherical structures composed of phospholipid bilayers and membrane materials such as cholesterol. They have the advantages of improving the therapeutic index of drugs, reducing the therapeutic dose of drugs, and reducing the toxicity of drugs. For drugs with low polarity, they can also enhance stability. Due to the particle size of liposomes, they are mainly phagocytosed by the reticuloendothelial system after entering the body, making it difficult for liposomes to extravasate into tissues with tight endothelial connections, which can significantly reduce the side effects of liposome drugs. Compared with free (i.e., unencapsulated) drugs, the improvement of liposomes on drug therapy is more about reducing toxicity rather than improving efficacy. Therefore, it is necessary to further transform liposomes to improve the interaction between liposomes and diseased cells. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the non-specificity of existing liposomes and colorectal cancer cells and provide a dual-target modified liposome.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned dual-target modified liposome.

[0006] Another object of the present invention is to provide applications of the dual-target modified liposomes.

[0007] Another object of the present invention is to provide a drug for treating colorectal cancer.

[0008] The purpose of the present invention is achieved by the following technical scheme: a dual-target modified liposome, wherein the liposome surface is modified with a CL4 aptamer and a VTP polypeptide. The present invention firstly finds that the efficiency of liposomes modified with a CL4 aptamer and a VTP polypeptide co-modified in targeting colorectal cancer cells is higher than that of liposomes modified with a single target, thereby facilitating the delivery of anti-tumor drugs loaded by the liposomes to colorectal cancer cells.

[0009] The sequence of the CL4 aptamer is as follows:

[0010] 5'-GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3'; preferably as follows:

[0011] 5'-GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-Chol-3', wherein Chol is the abbreviation for cholesterol.

[0012] The amino acid sequence of the VTP is as follows: CGKRK; is Cys-Gly-Lys-Arg-Lys.

[0013] The method for preparing the dual-target modified liposomes is to introduce the CL4 aptamer and the VTP polypeptide during the thin film hydration method for preparing the liposomes; preferably, the method comprises the following steps:

[0014] (1) mixing lecithin, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-Mal, dissolving them with a mixed solution obtained by dichloromethane and ethanol in a volume ratio of 3-5:1, decompressing and drying to obtain a film, further drying, adding PBS for hydration, ultrasonication, adding VTP, incubating, and then dialyzing to obtain VTP-modified liposomes; wherein the lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-Mal and 90-110:15-25:3.5-4.5:8-9:8-9 in a molar ratio;

[0015] (2) Dissolving the cholesterol-modified CL4 aptamer in the VTP-modified liposomes obtained in step (1), incubating, and dialyzing to obtain dual-target modified liposomes.

[0016] The lecithin described in step (1) is preferably egg yolk lecithin.

[0017] The dichloromethane and ethanol in the mixed solution described in step (1) are preferably mixed in a volume ratio of 4:1.

[0018] The mixed solution described in step (1) is used as a reaction medium for dissolving the reaction components and does not participate in the reaction. The amount used can be conventional; preferably, the ratio of the mixed solution to the total mass of the reaction components is 5 mL: 20-30 mg; more preferably, the ratio of the mixed solution to the total mass of the reaction components is 5 mL: 23-25 ​​mg.

[0019] The conditions for the reduced pressure spin drying in step (1) are preferably: spin drying at a rotation speed of 80 to 100 rpm for 10 to 20 min; more preferably: spin drying at a rotation speed of 90 rpm for 15 min.

[0020] The drying described in step (1) is preferably vacuum drying; the drying conditions are preferably vacuum drying at 35-40° C. for 1-3 h; most preferably vacuum drying at 37° C. for 2 h.

[0021] The purpose of the PBS in step (1) is to dissolve the film.

[0022] The amount of PBS used in step (1) is preferably the same volume as the mixed solution.

[0023] The hydration conditions described in step (1) are preferably: hydration at 35-40°C, 80-100 rpm for at least 15 min; more preferably: hydration at 35-40°C, 80-100 rpm for 20-40 min; most preferably: hydration at 37°C, 90 rpm for 30 min.

[0024] The ultrasonic conditions in step (1) are preferably 200-250 W for 1-3 s, stop for 1-s, and then ultrasonicate for 2-4 min; more preferably 220 W for 1 s, stop for 1 s, and then ultrasonicate for 3 min.

[0025] The incubation conditions in step (1) are preferably: incubating at 2-8°C overnight; more preferably: incubating at 4°C overnight.

[0026] The dialyzed solution in step (1) is preferably PBS containing 0.4-0.6% by volume of Tween-80; more preferably PBS containing 0.5% by volume of Tween-80.

[0027] The dialysis conditions in step (1) are preferably: dialysis at 2-8°C for 6-10 hours; more preferably: dialysis at 4°C for 8 hours.

[0028] The molar ratio described in step (1) is preferably 100:20:4:8.56:8.56.

[0029] The amount of the CL4 aptamer in step (2) is preferably in a molar ratio of 1:9000 to 11000 with respect to the phosphatidylcholine in step (1); more preferably in a molar ratio of 1:10000 with respect to the phosphatidylcholine in step (1).

[0030] The incubation conditions described in step (2) are preferably as follows: incubation at 35-40°C for 40-80 min, incubation at 2-8°C for 40-80 min as one cycle, and incubation in cycles 2 to 4 times; more preferably as follows: incubation at 37°C for 60 min, incubation at 4°C for 60 min as one cycle, and incubation in cycles 3 times.

[0031] The dialyzed solution in step (2) is preferably PBS containing 0.4-0.6% by volume of Tween-80; more preferably PBS containing 0.5% by volume of Tween-80.

[0032] The dialysis conditions in step (2) are preferably: dialysis at 2-8°C for 6-10 hours; more preferably: dialysis at 4°C for 8 hours.

[0033] Application of the above dual-target modified liposomes in the treatment of colorectal cancer.

[0034] A drug for treating colorectal cancer is prepared by loading an anti-tumor drug onto the above-mentioned dual-target modified liposome.

[0035] The anti-tumor drug is preferably docetaxel (DTX) and kaempferol (KA); more preferably, it is a compound of docetaxel and kaempferol in a molar ratio of 1:1.

[0036] The method for preparing the drug for treating colorectal cancer is to add an anti-tumor drug when preparing the dual-target modified liposomes by thin film hydration method; preferably, it comprises the following steps:

[0037] 1) mixing lecithin, cholesterol, an anti-tumor drug, DSPE-PEG2000 and DSPE-PEG2000-Mal, dissolving them with a mixed solution obtained by dichloromethane and ethanol in a volume ratio of 3-5:1, decompressing and drying to obtain a film, further drying, adding PBS for hydration, ultrasonication, adding VTP, incubating, and then dialyzing to obtain VTP-modified liposomes loaded with the anti-tumor drug; wherein the lecithin, cholesterol, anti-tumor drug, DSPE-PEG2000, DSPE-PEG2000-Mal and VTP are in a molar ratio of 90-110:15-25:7-9:3.5-4.5:8-9:8-9;

[0038] 2) Dissolving the CL4 aptamer modified with cholesterol in the VTP-modified liposome loaded with the anti-tumor drug obtained in step 1), incubating, and dialyzing to obtain a drug for treating colorectal cancer.

[0039] The lecithin described in step 1) is preferably egg yolk lecithin.

[0040] The anti-tumor drugs described in step 1) are preferably docetaxel (DTX) and kaempferol (KA); more preferably, a mixture of docetaxel and kaempferol in a molar ratio of 1:1.

[0041] In the mixed solution described in step 1), dichloromethane and ethanol are preferably mixed in a volume ratio of 4:1.

[0042] The mixed solution described in step 1) is used as a reaction medium for dissolving the reaction components and does not participate in the reaction. The amount used can be conventional; preferably, the ratio of the mixed solution to the total mass of the reaction components is 5 mL: 20-30 mg; more preferably, the ratio of the mixed solution to the total mass of the reaction components is 5 mL: 23-25 ​​mg.

[0043] The conditions for the reduced pressure spin drying in step 1) are preferably: spin drying at a rotation speed of 80 to 100 rpm for 10 to 20 min; more preferably: spin drying at a rotation speed of 90 rpm for 15 min.

[0044] The drying in step 1) is preferably vacuum drying; the drying conditions are preferably vacuum drying at 35-40° C. for 1-3 h; most preferably vacuum drying at 37° C. for 2 h.

[0045] The purpose of the PBS in step 1) is to dissolve the film.

[0046] The amount of PBS used in step 1) is preferably the same volume as the mixed solution.

[0047] The hydration conditions in step 1) are preferably: hydration at 35-40°C, 80-100 rpm for at least 15 min; more preferably: hydration at 35-40°C, 80-100 rpm for 20-40 min; most preferably: hydration at 37°C, 90 rpm for 30 min.

[0048] The ultrasonic conditions in step 1) are preferably 200-250W for 1-2s, stop for 1-2s, and then ultrasonic for 2-4min; more preferably 220W for 1s, stop for 1s, and then ultrasonic for 3min.

[0049] The incubation conditions in step 1) are preferably: incubating at 2-8°C overnight; more preferably: incubating at 4°C overnight.

[0050] The dialyzed solution in step 1) is preferably PBS containing 0.4-0.6% by volume of Tween-80; more preferably PBS containing 0.5% by volume of Tween-80.

[0051] The dialysis conditions in step 1) are preferably: dialysis at 2-8°C for 6-10 hours; more preferably: dialysis at 4°C for 8 hours.

[0052] The molar ratio in step 1) is preferably 100:20:8:4:8.56:8.56.

[0053] The amount of the CL4 aptamer in step 2) is preferably in a molar ratio of 1:9000 to 11000 with respect to the phosphatidylcholine in step 1); more preferably in a molar ratio of 1:10000 with respect to the phosphatidylcholine in step (1).

[0054] The incubation conditions described in step 2) are preferably as follows: incubation at 35-40°C for 40-80 min, incubation at 2-8°C for 40-80 min as one cycle, and incubation in cycles 2 to 4 times; more preferably as follows: incubation at 37°C for 60 min, incubation at 4°C for 60 min as one cycle, and incubation in cycles 3 times.

[0055] The dialyzed solution in step 2) is preferably PBS containing 0.4-0.6% by volume of Tween-80; more preferably PBS containing 0.5% by volume of Tween-80.

[0056] The dialysis conditions in step 2) are preferably: dialysis at 2-8°C for 6-10 hours; more preferably: dialysis at 4°C for 8 hours.

[0057] Compared with the prior art, the present invention has the following advantages and effects:

[0058] (1) The present invention proves that CL4 and VTP modified liposomes have a significant active targeted drug delivery effect on colorectal cancer, and the dual-target modification targeting effect is better than that of a single target. In vitro and in vivo experiments show that the dual-target liposome uptake rate is 10%-20% higher than that of a single target liposome, which has a synergistic effect. In the present invention, the free thiol group on the cysteine ​​residue at the end of the VTP polypeptide and the polyethylene glycol 2000-maleimide in the liposome membrane material form a stable thioether bond through a Michael-type addition reaction to construct the VTP-modified liposome surface; the 3' end of CL4 is modified with a cholesterol group, and CL4 is labeled to the liposome by utilizing the hydrophobic interaction between the cholesterol molecule and the phospholipid bilayer.

[0059] (2) The dual drug-loaded liposomes prepared by the present invention have a simple preparation method and mild experimental conditions.

[0060] (3) The present invention synthesizes for the first time a liposome preparation simultaneously loaded with docetaxel and kaempferol. There has been no research report on nanocarriers simultaneously loaded with these two drugs.

[0061] The present invention demonstrates through in vitro and in vivo experiments that, compared with simple combined drug administration / unmodified dual-loaded / single-drug-loaded liposomes, CL4 and VTP-modified liposomes co-loaded with docetaxel and kaempferol have a significant inhibitory effect on colorectal cancer proliferation and have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 1 is a transmission electron micrograph of the liposome of the present invention.

[0063] Figure 2 Graph showing the long-term stability analysis results of each group of liposomes in the examples of the present invention, wherein A to D are the particle size and polydispersity coefficient data on the 1st day, the 8th day, the 15th day, and the 22nd day, respectively.

[0064] Figure 3 The results of in vitro targeting analysis of liposomes modified with CL4 and VTP in the examples of the present invention are shown; wherein A is the fluorescence distribution of cells after co-incubation with fluorescent liposomes, and B is a quantitative histogram of the fluorescence intensity of cells; *** indicates P<0.001, and **** indicates P<0.0001.

[0065] Figure 4 The in vivo targeting analysis diagram of the non-target liposomes and single / double-target modified liposomes in the examples of the present invention; wherein A is the real-time distribution of fluorescent liposomes in mice, and B is the quantitative histogram of fluorescence intensity at each time point; * indicates P<0.05.

[0066] Figure 5 The graph is a research result of the inhibition of colorectal cancer cell proliferation by drug monomers, blank liposomes, drug-loaded liposomes and single / double-target modified drug-loaded liposomes in the examples of the present invention; wherein A is the survival rate of MC38 cells under the action of drug monomers / liposomes, and B is the survival rate of MC38 cells under the action of blank liposomes.

[0067] Figure 6 The figures are the results of the in vivo anti-tumor effect study of drug monomers, blank liposomes, drug-loaded liposomes and single / double-target modified drug-loaded liposomes in mice in the examples of the present invention; wherein A is the bioluminescence image of colorectal liver metastases in mice at different drug administration times, and B is the tumor growth curve drawn by the fluorescence amount of the bioluminescence image. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0069] The reagents and consumables used are as follows:

[0070] High-purity egg yolk lecithin PC was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., product number LR-51;

[0071] Cholesterol (Chol), purchased from Macklin, catalog number C804519;

[0072] Distearoylphosphatidyl acetamide-polyethylene glycol 2000 (DSPE-PEG2000) was purchased from Shanghai Fanshuo Biotechnology Co., Ltd., catalog number PZ1-E1-2K;

[0073] Docetaxel (DTX) was purchased from Shanghai Bioengineering Co., Ltd., catalog number A606561;

[0074] Kaempferol (KA), purchased from aladdin, product number K107144;

[0075] Coumarin 6 (C6), purchased from aladdin, product number C100929;

[0076] The cell membrane red fluorescent probe DIR was purchased from Shanghai Weihuan Biotechnology Co., Ltd., catalog number B8806;

[0077] CL4 was commissioned to Shanghai Bioengineering Co., Ltd. for synthesis and modified with a cholesterol at its 3′ end with the sequence of GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-Chol;

[0078] The VTP peptide was commissioned to be synthesized by Shanghai Bioengineering Co., Ltd., and the peptide sequence is Cys-Gly-Lys-Arg-Lys;

[0079] Distearoylphosphatidyl acetamide-polyethylene glycol 2000-maleimide (DSPE-PEG2000-Mal) was purchased from Xi'an Ruixi Biotechnology Co., Ltd., catalog number R-0039-2k;

[0080] The mNeonGreen-Luc dual-labeled lentiviral vector was commissioned to be synthesized by Heyuan Biotechnology (Shanghai) Co., Ltd.;

[0081] Polybrene was provided by Heyuan Biotechnology (Shanghai) Co., Ltd.;

[0082] Puromycin was purchased from Jiangsu Biotech Biotechnology Research Institute, catalog number ST551;

[0083] D-luciferin sodium salt was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 40901ES02.

[0084] Example 1: Preparation of liposomes

[0085] (1) Preparation of blank liposomes

[0086] The thin film hydration method was used to accurately weigh 15 mg PC, 1.53 mg Chol, and 2.22 mg DSPE-PEG2000, that is, the molar ratio of PC:Chol:DSPE-PEG2000 was 100:20:4, and the mixture was dissolved in 4 mL of dichloromethane and 1 mL of ethanol. The mixture was placed in a 250 mL round-bottom flask and dried under reduced pressure to form a uniform thin film. The operating conditions were 90 r / min for 15 min, and then the mixture was dried in a vacuum at 37°C for 2 h. 5 mL PBS (pH 7.4, 0.01 M) was added to the round-bottom flask, and the mixture was hydrated in a rotary evaporator at 37°C and 90 r / min for 30 min. The cell disruptor probe was ultrasonicated for 3 min. The ultrasonic conditions were 220 W, 1 s, and 1 s. Blank liposomes Blank-lp were prepared.

[0087] (2) Preparation of drug-loaded liposomes

[0088] Using the thin film hydration method, accurately weigh 15 mg PC, 1.53 mg Chol, 2.22 mg DSPE-PEG2000, 0.64 mg DTX and 0.23 mg KA, that is, the molar ratio of PC: Chol: DSPE-PEG2000: DTX: KA is 100:20:4:4:4, dissolve them in 4 mL of dichloromethane and 1 mL of ethanol, place them in a 250 mL round-bottom flask and spin dry them under reduced pressure to form a uniform thin film (90 r / min, 15 min), and dry them in vacuum at 37 °C for 2 h. 5 mL of PBS (pH 7.4, 0.01 M) was added to a round-bottom flask, hydrated in a rotary evaporator at 37°C and 90 r / min for 30 min, and ultrasonicated with a cell disruptor probe for 3 min. The ultrasonic conditions were 220 W, ultrasonicated for 1 s, and stopped for 1 s to obtain the drug-loaded liposome PEG-DTX-KA-lp, referred to as PEG-DK-lp.

[0089] (3) Preparation of CL4-modified liposomes

[0090] 0.026 mg CL4 (i.e., the molar ratio of PC:CL4 is 10000:1) was dissolved in the drug-loaded liposomes prepared in step (2), placed in a vial, incubated at 37°C for 60 min and 4°C for 60 min as one cycle, and the cycle was repeated 3 times. Then, the liposomes were transferred to a dialysis bag (8000-14000 Da), and dialyzed in 1 L of PBS containing 0.5% v / v Tween-80 at 4°C for 8 h to remove the unencapsulated target, thereby obtaining CL4-modified liposomes (CL4-DTX-KA-lp), referred to as CL4-DK-lp.

[0091] (4) Preparation of VTP-modified liposomes

[0092] Using the thin film hydration method, accurately weigh 15 mg PC, 1.53 mg Chol, 2.22 mg DSPE-PEG2000, 4.9 mg DSPE-PEG2000-Mal, 0.64 mg DTX, and 0.23 mg KA, dissolve them in 4 mL dichloromethane and 1 mL ethanol, place them in a 250 mL round-bottom flask, and spin dry them under reduced pressure to form a uniform thin film (90 r / min, 15 min), and then dry them in a vacuum at 37 °C for 2 h. 5 mL of PBS (pH 7.4, 0.01 M) was added to a round-bottom flask, and the mixture was hydrated in a rotary evaporator for 30 min (37° C., 90 r / min). The cell disruptor probe was ultrasonicated for 3 min under the ultrasonic conditions of 220 W, ultrasonication for 1 s, and stop for 1 s. 1 mg of VTP was added, and the mixture was incubated at 4° C. overnight. The mixture was then transferred to a dialysis bag (8000-14000 Da) and dialyzed in 1 L of PBS containing 0.5% v / v Tween-80 at 4° C. for 8 h to remove unencapsulated drugs and targets, thereby obtaining drug-loaded liposomes VTP-DTX-KA-lp, referred to as VTP-DK-lp; wherein the molar ratio of PC:Chol:DSPE-PEG2000:DSPE-PEG2000-Mal:DTX:KA:VTP was 100:20:4:8.56:4:4:8.56.

[0093] (4) Preparation of CL4 and VTP modified liposomes

[0094] 0.026 mg CL4 (i.e., the molar ratio of PC:CL4 is 10000:1) was dissolved in the drug-loaded liposome VTP-DTX-KA-LP prepared in step (4), placed in a vial, incubated at 37°C for 60 min and 4°C for 60 min as one cycle, and the cycle was repeated 3 times. Then, the liposome was transferred to a dialysis bag (8000-14000 Da), and dialyzed in 1 L of PBS containing 0.5% v / v Tween-80 at 4°C for 8 h to remove the unencapsulated target, thereby obtaining a dual-target modified liposome (CL4-VTP-DTX-KA-lp, referred to as CL4-VTP-DK-LP).

[0095] (5) Preparation of fluorescent liposomes

[0096] The fluorescent liposomes used in the cell experiment used the fluorescent probe coumarin 6 (C6) instead of the drug, the dosage of C6 was 0.1 mg, and the other steps were the same as above to obtain C6-PEG-lp, C6-CL4-lp, C6-VTP-lp, and C6-CL4-VTP-lp, respectively. The fluorescent liposomes used in the animal experiment used the fluorescent probe DIR cell membrane red fluorescent probe (DIR) instead of the drug, the dosage of DIR was 0.5 mg, and the other steps were the same as above, prepared in the dark, and obtained DIR-PEG-lp, DIR-CL4-lp, DIR-VTP-lp, and DIR-CL4-VTP-lp, and stored.

[0097] Example 2 Morphological Investigation

[0098] The morphology of CL4-VTP-DK-LP liposomes was observed by transmission electron microscopy. The sample was added to the carbon film copper mesh, and after the material evaporated, 1% w / v phosphotungstic acid solution (pH 7.0) was added for dyeing, and excess water was absorbed with filter paper. After the sample was dried, the sample was loaded and vacuum tested with an acceleration voltage of 200 kV.

[0099] Figure 1 The liposomes were shown to be spherical in shape, and the particle size observed by TEM was basically consistent with that measured by Malvern nanoparticle size potential analyzer, which was about 120 nm.

[0100] Example 3 Liposome Particle Size and Potential Characterization

[0101] The prepared liposomes were placed in a quartz cuvette for the particle size analyzer, and the particle size, PDI, Zeta potential and distribution were measured using a Malvern nanoparticle size potential analyzer. Three measurements were performed for each sample. avg denoted by , which represents the average hydrodynamic diameter relative to the intensity range measured using dynamic light scattering.

[0102] The results are shown in Table 1: the particle size of the drug-loaded targeted liposome CL4-VTP-DK-LP is about 120 nm, and the potential is -8.31±0.67.

[0103] Example 4 Calculation of the encapsulation efficiency of targeted liposomes co-loaded with docetaxel and kaempferol

[0104] The encapsulation efficiency is a key quality attribute of liposomes. It refers to the percentage of drug content encapsulated in the lipid bilayer to the total drug dosage. It can reflect the degree of drug encapsulation in liposomes and guide the improvement of the preparation process.

[0105] Determine the encapsulation of liposomes by dialysis: accurately draw 1mL of drug-loaded liposomes, place them in a dialysis bag, suspend the dialysis bag in 5mL of PBS (0.01M, pH 7.4) containing 0.5% Tween-80, shake and dialyze at room temperature for 8h, take out 100μL of the inner solution in the dialysis bag, add 250μL of methanol, ultrasonically break the emulsion for 3min, pass through a microporous filter membrane (0.22μm), and then draw 10μL for injection. Use high-performance liquid chromatography to detect the absorption peak area of ​​the sample at 232nm and 360nm respectively, and bring them into the standard curve of docetaxel and kaempferol to calculate the encapsulation rate, and determine the concentration C of docetaxel and kaempferol encapsulated in the liposomes. The concentration of docetaxel and kaempferol in the preparation of liposomes is C 0 The calculation formula is: EE (%) = C / C 0 ×100%.

[0106] The results are shown in Table 1: the encapsulation efficiency (EE%) of CL4-VTP-DK-LP for docetaxel and kaempferol was 87.83±2.01% and 84.42±3.41%, respectively, both of which meet the requirements of the Chinese Pharmacopoeia that the liposome drug encapsulation efficiency should be higher than 80%.

[0107] Table 1

[0108]

[0109] Example 5 Long-term stability analysis of targeted liposomes co-loaded with docetaxel and kaempferol

[0110] The drug-loaded liposomes retained in PBS were stored in a refrigerator at 4°C for three weeks, and the particle size and potential were measured every 7 days. Each measurement was repeated three times, and the stability of the co-drug-loaded modified liposomes was analyzed.

[0111] The results are as follows Figure 2 As shown: Both the liposomes without added target and the liposomes with single / double target modification stored at 4°C for three weeks showed no change in particle size.

[0112] Example 6 In vitro targeting study of targeted liposomes co-loaded with docetaxel and kaempferol

[0113] Mouse colon cancer MC38 cells (purchased from Wuhan Sewell Biotechnology Co., Ltd.) were cultured at 5×10 4 / well were inoculated in a 12-well plate and cultured for 24 hours. The culture medium was discarded and the original culture medium in the 12-well plate was replaced with 1 mL of complete culture medium containing fluorescent liposomes of coumarin 6, the concentration of coumarin 6 in the culture medium was 20 μg / mL. After incubation for 4 hours, the old culture medium was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and then 200 μL of DAPI with a concentration of 5 μg / mL was added and incubated at room temperature in the dark for 10 minutes, washed with PBS, and the areas with uniform cell growth were selected under a fluorescence microscope and photographed at the same exposure time to evaluate the cellular uptake of liposomes in each group.

[0114] MC38 cells in logarithmic growth phase were cultured at 3×10 3 The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured for 12 hours. The original medium in the 96-well plate was replaced with RPMI 1640 complete medium containing fluorescent liposomes of coumarin 6, and the concentration of coumarin 6 in the medium was 20 μg / mL. The cells were incubated for 4 hours. The cells were washed with PBS, 100 μL of DMSO was added, and the fluorescence intensity was measured by a fluorescence microplate reader. Three replicate wells were set for each group.

[0115] The results are as follows Figure 3 As shown: Compared with non-target modified or single-target modified fluorescent liposomes, dual-target modified fluorescent liposomes more significantly targeted colorectal cancer cells, among which the fluorescence intensity of C6-CL4-lp, C6-VTP-lp, and C6-CL4-VTP-lp in MC38 cells was 1.31 times, 1.33 times, and 1.42 times that of C6-PEG-lp. The results showed that compared with the single-target group, dual-target liposomes increased the drug uptake by colorectal cancer cells.

[0116] Example 7 In vivo targeting study of targeted liposomes co-loaded with docetaxel and kaempferol

[0117] Construction of nude mouse metastatic tumor model: 6-week-old male nude mice BALB / C-nu were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. to establish the experimental liver metastasis model. 6 A single cell suspension of MC38 cells / mL was injected into the spleen of nude mice at a volume of 100 μL / mouse. Three weeks after the model was established, 200 μL of fluorescent liposomes labeled with the fluorescent probe DIR were injected into the tail vein of the mice. The in vivo fluorescence was detected at different times using a small animal in vivo imager to analyze the in vivo distribution of the liposomes.

[0118] The results are as follows Figure 4As shown: At each time period after injection, the fluorescence intensity of the tumor site of each group was compared. At 48 hours after injection, the fluorescence intensity of the tumor site of DIR-CL4-lp, DIR-VTP-lp, and DIR-CL4-VTP-lp was 1.06 times, 1.13 times, and 1.21 times that of DIR-PEG-lp. Compared with non-target modified or single-target modified fluorescent liposomes, dual-target modified fluorescent liposomes more significantly targeted the colorectal cancer liver metastasis site, verifying the in vivo specific targeting of the liposomes.

[0119] Example 8 In vitro study on the anti-proliferative effect of targeted liposomes co-loaded with docetaxel and kaempferol on colorectal cancer

[0120] In order to determine the anti-colorectal cancer effect of docetaxel and kaempferol, the toxicity of liposomes on MC38 cells was detected by CCK8 method. 3 / well inoculated in 96-well plates, wait for cells to adhere to the wall, discard the culture medium, add a series of concentrations of RPMI 1640 complete culture medium containing drugs or drug-loaded liposomes, the DTX content of each group is 0.39, 0.78, 1.56, 3.13, 6.25μmol / L, the KA content is 0.78, 1.56, 3.13, 6.25, 12.5μmol / L, 100μL per well, 5 replicates per group. Set up blank wells (only culture medium) and control wells (only PBS), culture continuously at 37℃ for 48h, add 10μL CCK8 reagent to each well, continue incubation for 2h, avoid light and shake, measure the OD value of each well at 450nm with an enzyme reader, draw a cell inhibition rate curve, and calculate the IC of each group 50 value.

[0121] The results are as follows Figure 5 As shown: The IC50 of free DTX, DTX+KA and liposome PEG-DK-lp, CL4-DK-lp, VTP-DK-lp, CL4-VTP-DK-lp were calculated to be 4.993μmol / L, 4.452μmol / L, 3.852μmol / L, 2.696μmol / L, 1.344μmol / L, and 0.956μmol / L, respectively. Compared with the drug monomer combination and the liposomes without modified targets, the targeted liposomes co-loaded with docetaxel and kaempferol can more effectively inhibit the proliferation of colorectal cancer cells in vitro.

[0122] Example 9 Construction of a C57 Mouse Model of Colorectal Cancer Liver Metastasis

[0123] According to the lentiviral operation manual provided by Heyuan Biotechnology (Shanghai) Co., Ltd., the mNeonGreen-Luc double-labeled lentiviral vector was used; MC38 cells were cultured at 1×10 6The number of cells / well was plated in a six-well plate. When the cells were fused to 30%-40%, the original culture medium was discarded, and the virus transfection solution was added according to the optimal MOI value. At the same time, 5 μg / mL of polybrene was added to the cell culture medium. After 72 hours of transfection, 2 μg / mL of puromycin was added to screen out the successfully transfected cell clones to obtain MC38 cells transfected with luciferase.

[0124] Six-week-old male C57 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and injected into the spleen with 200 μL of MC38 cell suspension transfected with luciferase at a concentration of 5×10 6 / mL, a colorectal cancer liver metastasis model was established, and drug administration began 15 days after model construction. Docetaxel in each group was 10 mg / kg, and kaempferol was 0.27 mg / kg. The drug monomer was dissolved in PBS containing 0.2% v / v Tween-80, and 200 were injected into the tail vein every 3 days. Bioluminescence was continuously detected with a small animal living imager every two days after the start of drug administration. Mice were intraperitoneally injected with 200 μL of 15 mg / mL D-luciferin sodium salt solution, and bioluminescence was detected with a small animal living imager after 10 minutes, and the fluorescence intensity value was recorded.

[0125] The results are as follows Figure 6 As shown: On the eighth day of administration, the tumors in the control group and the blank liposome group grew rapidly, while those in the drug monomer group and each liposome group shrank. The tumor sizes of the DTX+KA, PEG-DK-lp, CL4-DK-lp, VTP-DK-lp, and CL4-VTP-DK-lp groups were 25.20%, 22.72%, 21.18%, 19.37%, and 15.66% of that of the control group, respectively. The dual-target modified liposomes co-loaded with docetaxel and kaempferol showed the best effect in inhibiting colorectal cancer liver metastases.

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A dual-target modified liposome, characterized in that: The liposome surface is modified with CL4 aptamer and VTP peptide; The sequence of the CL4 aptamer is as follows: 5'-GCCUUAGUAACGUGCUUUGAUGUCGAUUCGACAGGAGGC-3'; The amino acid sequence of the VTP is as follows: CGKRK.

2. The method for preparing the dual-target modified liposome according to claim 1, characterized in that The method comprises the following steps: introducing CL4 aptamer and VTP polypeptide into the process of preparing liposome by thin film hydration method.

3. The method for preparing the dual-target modified liposome according to claim 1, characterized in that The steps include: (1) mixing lecithin, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-Mal, dissolving them with a mixed solution obtained by dichloromethane and ethanol in a volume ratio of 3 to 5:1, decompressing and drying to obtain a film, further drying and adding PBS for hydration; then ultrasonicating, adding VTP, incubating, dialyzing, and obtaining VTP-modified liposomes; wherein the lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-Mal and 90 to 110:15 to 25:3.5 to 4.5:8 to 9:8 to 9 in a molar ratio; (2) Dissolving the cholesterol-modified CL4 aptamer in the VTP-modified liposome obtained in step (1), incubating, and dialyzing to obtain CL4 and VTP dual-target modified liposomes.

4. The method for preparing dual-target modified liposomes according to claim 3, characterized in that: The lecithin described in step (1) is egg yolk lecithin; The mixed solution in step (1) comprises dichloromethane and ethanol in a volume ratio of 4:1; The amount of the mixed solution in step (1) is calculated based on the ratio of the total mass of the mixed solution to the total mass of the reaction components: 5 mL: 20-30 mg; The dialyzed solution in step (1) is PBS containing 0.4-0.6% by volume of Tween-80; The CL4 aptamer in step (2) is used in a molar ratio of 1:9000 to 11000 with respect to the lecithin in step (1); The dialyzed solution in step (2) is PBS containing 0.4-0.6% Tween-80 by volume.

5. The method for preparing dual-target modified liposomes according to claim 3, characterized in that: The conditions for the reduced pressure spin drying in step (1) are: spin drying at a speed of 80 to 100 rpm for 10 to 20 min; The drying in step (1) is vacuum drying; The hydration conditions in step (1) are: hydration at 35-40° C. and 80-100 rpm for at least 15 min; The ultrasonic conditions in step (1) are as follows: ultrasonication at a power of 200 to 250 W for 1 to 2 seconds, stopping for 1 to 2 seconds, and ultrasonication for 2 to 4 minutes; The incubation conditions described in step (1) are: incubating at 2-8°C overnight; The dialysis conditions in step (1) are: dialysis at 2-8° C. for 6-10 hours; The molar ratio in step (1) is 100:20:4:8.56:8.56; The incubation conditions in step (2) are as follows: incubation at 35-40° C. for 40-80 min and incubation at 2-8° C. for 40-80 min as one cycle, and the cycle incubation is repeated 2-4 times; The dialysis conditions in step (2) are: dialysis at 2-8°C for 6-10 hours.

6. Use of the dual-target modified liposomes according to claim 1 for treating colorectal cancer.

7. A drug for treating colorectal cancer, characterized in that: The dual-target modified liposomes described in claim 1 are loaded with anti-tumor drugs.

8. The drug for treating colorectal cancer according to claim 7, characterized in that: The anti-tumor drugs are docetaxel and kaempferol.

9. The method for preparing the drug for treating colorectal cancer according to claim 7 or 8, characterized in that The steps include: 1) mixing lecithin, cholesterol, an anti-tumor drug, DSPE-PEG2000 and DSPE-PEG2000-Mal, dissolving them with a mixed solution obtained by dichloromethane and ethanol in a volume ratio of 3-5:1, decompressing and drying to obtain a film, further drying and adding PBS for hydration; then ultrasonicating, adding VTP, incubating, dialyzing, and obtaining VTP-modified liposomes loaded with the anti-tumor drug; wherein the lecithin, cholesterol, anti-tumor drug, DSPE-PEG2000, DSPE-PEG2000-Mal and VTP are in a molar ratio of 90-110:15-25:7-9:3.5-4.5:8-9:8-9; 2) Dissolving the CL4 aptamer modified with cholesterol in the VTP-modified liposome loaded with the anti-tumor drug obtained in step 1), incubating, and dialyzing to obtain a drug for treating colorectal cancer.

10. The method for preparing a drug for treating colorectal cancer according to claim 9, characterized in that: The lecithin described in step 1) is egg yolk lecithin; The mixed solution in step 1) is prepared by mixing dichloromethane and ethanol in a volume ratio of 4:1; The conditions for the reduced pressure spin drying in step 1) are to spin dry at a speed of 80 to 100 rpm for 10 to 20 minutes; The drying in step 1) is vacuum drying; The hydration conditions in step 1) are: hydration at 35-40° C. and 80-100 rpm for at least 15 min; The ultrasonic conditions in step 1) are as follows: ultrasonication for 1 to 2 seconds at a power of 200 to 250 W, stopping for 1 to 2 seconds, and ultrasonication for 2 to 4 minutes; The incubation conditions described in step 1) are: incubating at 2-8°C overnight; The dialyzed solution in step 1) is PBS containing 0.4-0.6% Tween-80 by volume; The dialysis conditions in step 1) are: dialysis at 2-8° C. for 6-10 hours; The molar ratio in step 1) is 100:20:8:4:8.56:8.56; The CL4 aptamer in step 2) is used in a molar ratio of 1:9000 to 11000 with respect to the lecithin in step 1); The incubation conditions in step 2) are as follows: incubation at 35-40° C. for 40-80 min and incubation at 2-8° C. for 40-80 min as one cycle, and the cycle incubation is repeated 2-4 times; The dialyzed solution in step 2) is PBS containing 0.4-0.6% Tween-80 by volume; The dialysis conditions in step 2) are: dialysis at 2-8°C for 6-10 hours.

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