CN110917139A

CN110917139A - Preparation and application of multi-branch biotin modified breast cancer targeted liposome

Info

Publication number: CN110917139A
Application number: CN201911214065.5A
Authority: CN
Inventors: 吴勇; 海俐; 郭丽; 乐其明; 蒲妍池; 彭瑶
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2020-03-27

Abstract

The invention discloses a novel lipid material for realizing the delivery of a breast cancer targeted drug. The novel lipid material passes through the branch framework, one end of the novel lipid material is connected with cholesterol extended by polyethylene glycol, the other end of the novel lipid material is connected with different quantities of biotin with a breast cancer targeting function, and the affinity between the novel lipid material and a receptor can be utilized, so that stronger tumor targeting is realized, and more effective breast cancer treatment effect is exerted. The novel lipid material can be used for different dosage forms including liposome, nanoparticles, micelle and the like, and the prepared paclitaxel-loaded liposome has obvious breast cancer targeting property and wide application prospect.

Description

Preparation and application of multi-branch biotin modified breast cancer targeted liposome

Technical Field

The invention relates to a novel lipid material and application thereof in a drug delivery system, has the function of breast cancer targeted drug delivery, comprises the preparation and characterization of the material, and application thereof as a drug carrier in drug delivery, and belongs to the technical field of medicines.

Background

Breast cancer is one of the common malignant tumors in women, seriously affecting women's survival and quality of life, and is called the first killer of women's health. According to the statistics of national cancer data issued by the national cancer center in 2018, the first malignant tumor in female aspects is breast cancer. Moreover, the incidence of breast cancer in women is first in both developed and developing countries worldwide. Statistically, the incidence rate of breast cancer is rising from the end of the 70 th 20 th century, and the onset age is younger, which is now the second most common cancer in the world, second only to lung cancer.

The treatment of breast cancer includes operation treatment, endocrine treatment, chemotherapy, radiotherapy and the like, and at present, radical operation and postoperative chemotherapy are mainly used. The commonly used drugs for breast cancer adjuvant chemotherapy in the present clinical stage mainly include anthracyclines, taxoids, capecitabine, gemcitabine, cyclophosphamide, platinum and vinorelbine. Although chemotherapy plays a very important role in the comprehensive treatment of breast cancer, can control the systemic dissemination of early breast cancer, prevent and relieve the recurrence and metastasis of breast cancer after operation, and effectively prolong the life of patients, many problems still exist. The lack of tumor specificity is one of the major problems with chemotherapeutic drugs. The non-selective distribution of chemotherapeutic drugs in normal and cancer cells not only induces excessive systemic toxicity, but also reduces drug accumulation in tumor cells, thereby reducing drug efficacy, and also resulting in tumor resistance to chemotherapeutic drugs. In addition, the unrestricted interaction of the drug with plasma and tissue proteins may lead to rapid inactivation of the chemotherapeutic drug, thereby further reducing the efficacy of the chemotherapeutic drug. In recent years, with the deepening of molecular biological methods such as signal path, apoptosis and the like in tumor research, breast cancer molecular targets and targeted therapy gradually become trends and hot spots for anti-breast cancer research. Therefore, the design of a novel targeted drug delivery system plays a significant role in developing a drug for treating breast cancer.

The nano material has the characteristics of easy surface modification, adjustable particle size and surface charge, high porosity, large specific surface area and the like, and has remarkable advantages in various aspects in tumor treatment research. The functionalization of the surface ligand of the nano-carrier directly influences the targeting capability of the nano-drug delivery system and is always considered as an important factor for determining the delivery efficiency of the nano-carrier. However, for the functionalization of the surface ligand of the nano-carrier, the attention of researchers mainly focuses on the type of the ligand, for example, the targeting abilities of different ligands are compared or a plurality of ligands are introduced simultaneously to improve the targeting property of a drug-carrying system, and the like, while the factors such as the density, the structure and the distribution of the ligand on the surface of the nano-carrier are rarely studied, and the density of the ligand is combined with the structure for comparative analysis.

In recent years, many studies have been made on tumor-targeted drug delivery systems using various types of targeting ligands such as cyclic rgd (crgd), Folic Acid (FA), biotin (biotin), human epidermal growth factor receptor-2 (Her 2), galactose, and glycyrrhizic acid (glycyrrhizin). Among them, biotin is not synthesized in any mammalian cell, and is a 244 Da small-molecule water-soluble vitamin, so biotin has great advantages compared with other ligands: the structure is simple, the functional group is single, the steric hindrance is small, and a large amount of modification is easy to be carried out on the surface of the nano carrier, so that the density of sites on the surface of the carrier for recognition of a transporter can be improved. On the other hand, the sodium ion-dependent vitamin complex transporter (SMVT) has been confirmed to be the main transporter of biotin. Biotin receptors have been reported to be highly expressed in several malignant cell lines, such as breast cancer (MCF-7, 4T1, JC and MMT 06056), ovarian cancer (OV 2008, ID 8), colon cancer (Colo-26), mast cell tumor (P815), lung cancer (M109), kidney cancer (RENCA, RD 0995) and leukemia cell line (L1210 FR); but low in normal cells. Therefore, if biotin is used as a targeting molecule to modify the nano-carrier, the influence of the targeting molecule density and the ligand structure on the breast cancer targeting capability of the nano-carrier is discussed, and a new thought and method is expected to be provided for the targeted therapy research of breast cancer chemotherapeutic drugs.

Disclosure of Invention

In our previous studies, it has been found that the targeting ability of the double-branched biotin-modified lipid material (Bio-Bio-Chol) to breast cancer is better than that of the single biotin-modified lipid material (Bio-Chol) and better than that of the liposome modified by 2 times of the ligand Bio-Chol in a physical mixing manner, therefore, the biotin density, structure and distribution on the surface of the liposome are further discussed, and the targeting ability of biotin to breast cancer is expected to be exerted to the maximum extent.

Based on the research and hypothesis, the invention aims to provide a series of novel biotin-modified lipid materials, apply the biotin-modified lipid materials to the preparation of breast cancer targeted liposomes, realize stronger tumor targeting by utilizing stronger specific combination of biotin and breast cancer cells, increase the concentration of the drug in tumor tissues, play a more effective role in treating breast cancer, reduce the distribution of the drug in peripheral organs and reduce the toxic and side effects of the drug. Therefore, the lipid materials shown in the general formulas (I) and (II) are further designed, the cholesterol part of the lipid material is embedded into the liposome phospholipid bilayer, and the biotin part with breast cancer targeting is exposed on the surface of the liposome, so that the liposome has the breast cancer targeting function. The lipid material can be used for different dosage forms such as liposome, nanoparticles, micelle and the like, has the functions of breast cancer targeting and toxicity reduction, and has great application prospect when being applied to a drug delivery system.

The invention provides a compound with a structure shown in a general formula (I) or a pharmaceutically acceptable salt or hydrate thereof:

(I)

wherein, the molecular weight of the PEG is equal to but not limited to 150, 200, 400, 600, 800, 1000, 1500, 2000, 4000, etc.

The specific preparation method of the compound shown in the general formula (I) is as follows:

the invention also provides a compound with the structure shown in the general formula (II) or pharmaceutically acceptable salt or hydrate thereof:

wherein:

the molecular weight of the PEG used is equal to, but not limited to, 150, 200, 400, 600, 800, 1000, 1500, 2000, 4000, etc.

The specific preparation method of the compound represented by the general formula (II) is as follows:

the novel lipid material can be used as a ligand for preparing breast cancer targeted liposomes.

The liposome is characterized by comprising phospholipid, cholesterol, Tri-Biotin-Chol, Tetra-Biotin-Chol and an active agent.

The liposome mainly comprises a membrane material and an active agent, wherein the membrane material is a phospholipid bilayer and comprises lecithin, cholesterol and a liposome ligand, and the ratio of the components is as follows: the molar ratio of the cholesterol to the phospholipid is 1-2: 1-10, and the molar content of the liposome ligand is 1-25% of the total molar number of the cholesterol and the phospholipid. The active agent of the present invention is preferably a therapeutic agent or a contrast agent, and the dosage of the active agent can be adjusted according to the active agent contained in the steroid, wherein the active agent accounts for 0.1% to 50% of the total lipid by weight percent, as known in the art.

The phospholipids in the liposomes include all types of phospholipids, including but not limited to soybean phospholipids, lecithin, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol; lecithin is preferred.

The active agent in the liposomes can be an antineoplastic agent, including but not limited to alkylating agents, antimetabolites, antitumor antibiotics, anthracyclines, plant alkaloids, paclitaxel derivatives, topoisomerase inhibitors, monoclonal antibodies, photosensitizers, kinase inhibitors, and platinum-containing compounds.

The preparation method of the breast cancer targeted liposome comprises the following steps:

weighing phospholipid, cholesterol and paclitaxel, dissolving in solvent, adding liposome ligand (without blank liposome) at a certain proportion, and rotary evaporating in constant temperature water bath at 20-40 deg.C to remove organic solvent.

And (II) placing the eggplant-shaped bottle in a vacuum drier for vacuum drying overnight to remove residual solvent.

And (III) adding hydration liquid such as phosphate buffer solution or ammonium sulfate solution into the eggplant-shaped bottle, hydrating for about 0.5-2 hours by using a constant-temperature air bath shaker at 20 ℃, performing ultrasonic treatment by using an ice-water bath probe, and controlling the particle size of the liposome to be about 110 nm by using a method such as extrusion film-coating or ultrasonic treatment.

Preferably, the ratio of paclitaxel to lipid material in step (one) is 1: 30.

Preferably, the solvent in step (two) is chloroform, and the molar ratio of the lipid is 1:2 (cholesterol: soybean phospholipid).

The preferred hydration solution in step (III) is 0.01M Phosphate Buffered Saline (PBS) at pH 7.4.

The invention realizes the aim through the following technical scheme:

detailed description of the invention

The following examples are intended to illustrate the invention without further limiting it. The present invention will be further illustrated in detail with reference to examples, but the present invention is not limited to these examples and the preparation method used. Also, equivalent substitutions, combinations, improvements or modifications of the invention may be made by those skilled in the art based on the description of the invention, but these are included in the scope of the invention.

The novel lipid material is prepared by the following steps:

example 1

Preparation of Compound 2

Diethanolamine 1 (1 g, 9.51 mmol) was dissolved in 30 mL acetonitrile, di-tert-butyl dicarbonate (2.49 g, 11.41 mmol) was added with stirring at room temperature, and the reaction was continued for 4 hours at room temperature with TLC monitoring. After completion of the reaction, concentration was performed under reduced pressure, and the residue was purified by silica gel flash column chromatography (petroleum ether: ethyl acetate = 5: 1) to obtain 1.82 g of compound 2 as a colorless oil in a yield of 93.50%.¹H NMR (400 MHz, CDCl₃, ppm) δ: 1.47 (s, 9H), 2.83 (s, 2H), 3.44 (d,4H,J= 11.6 Hz), 3.80 (s, 4H)。

Example 2

Preparation of Compound 3

Biotin (2.38 g, 9.76 mmol) was dissolved in a mixed solvent of dichloromethane and N, N-dimethylformamide (v/v =45 mL/15 mL), EDCI (2.80 g, 14.61 mmol), DMAP (1.78 g, 14.61 mmol), DIPEA (4.84 mL, 29.28 mmol) were added, and 10mL of a dichloromethane solution of compound 2 (500 mg, 2.44 mmol) was added dropwise with stirring at room temperature, and after completion of the dropwise addition, the reaction solution was yellow, followed by reaction at room temperature for 10 hours and monitoring by TLC. After the reaction was complete, the reaction solution was washed with 1N hydrochloric acid solution (50 mL. times.1) and saturated NaCl solution (50 mL. times.8) in this order, and anhydrous Na₂SO₄Drying, concentration of the solvent under reduced pressure and purification of the residue by silica gel flash column chromatography (dichloromethane: methanol =30: 1) gave 1.24 g of a pale yellow solid in 77.33% yield. mp: 108-.¹H NMR (400 MHz, CDCl₃, ppm)δ: 1.26-1.29 (m, 4H), 1.47(s, 9H), 1.65-1.76 (m, 8H), 2.38 (s, 4H), 2.75 (d, 2H,J= 7.2 Hz), 2.90-2.95(m, 2H), 3.12-3.18 (m, 2H), 3.43-3.54 (m, 4H), 4.19 (s, 4H), 4.35 (s, 2H),4.52-4.54 (m, 2H)。

Example 3

Preparation of Compound 4

3.22 g of Compound 3 are dissolved in 20 mL of dichloromethane, and 10mL of trifluoroacetic acid are added. The reaction was carried out at room temperature for 4 hours, the reaction solution changed from yellow to brick red, and the completion of the reaction was monitored by TLC. Concentration under reduced pressure was carried out to remove methylene chloride and trifluoroacetic acid, thereby obtaining 3.47 g of a greenish black oil, and 30 mL of ethyl glacial ether was added to precipitate a pale yellow solid, which was centrifuged and dried, thereby obtaining 2.94 g of a pale yellow trifluoroacetate compound 4.

Example 4

Preparation of Compound 5

Succinic anhydride (1.12 g, 11.2 mmol) was dissolved in 25 mL of dichloromethane, and triethylamine (3.12 mL, 22.4 mmol) and 45 mL of a solution of compound 4 (3.76 g, 5.59 mmol) in dichloromethane were added. The reaction was carried out at room temperature for 6 hours, TLC monitored for completion, washed 1 time with 1N HCl solution (50 mL), 2 times with saturated sodium chloride solution (50 mL), and the organic phase was concentrated under reduced pressure to give a yellow oil which was purified by silica gel flash column chromatography (dichloromethane: methanol = 20: 1) to give 2.14 g of compound 5 as a colorless oil in 58.10% yield.¹H NMR (400MHz, CDCl₃, ppm)δ: 1.26-1.37 (m, 4H), 1.42-1.64 (m, 8H), 2.25-2.35 (m, 4H), 2.41-2.44 (m, 2H), 2.50 (s, 4H), 2.56-2.59(m, 4H), 2.80-2.85 (m, 2H), 3.10 (s, 2H), 3.12-3.18 (m, 2H), 3.50 (t, 2H,J=5.6 Hz), 3.61 (t, 2H,J= 5.6 Hz), 4.06-4.09 (m, 2H), 4.12-4.19 (m, 4H),4.29-4.33 (m, 2H), 12.09 (s, 1H)。

Example 5

Preparation of Compound 7

Cholesterol 6 (32.00 g, 82.76 mmol) was dissolved in 100mL of anhydrous pyridine, and 50 mL of pyridine-dissolved p-toluenesulfonyl chloride (TsCl, 23.67 g, 124.14 mmol) solution was added dropwise to the reaction solution at 0 ℃ and then reacted at 50 ℃ for 5 hours, monitored by TLC. After the reaction is completed, the reaction solution is concentrated under reduced pressure, 300 mL of ethyl acetate (ethyl acetate) is taken to dissolve the residue again, and then 100mL of 1 mol/L HCl solution is taken to wash for 2 times and 100 timesmL of saturated NaCl solution 2 times without Na₂SO₄Drying, and concentrating the solvent under reduced pressure to obtain 42.35 g of white solid with the yield of 94.62%, wherein the product can be directly used for the next reaction without column chromatography purification.

Example 6

Preparation of Compound 8

130 mL of dioxane was taken to dissolve Compound 7 (22.48 g, 41.56 mmol) and polyethylene glycol (27.86 mL, 207.82 mmol) was added to the reaction solution which was then moved to reflux for additional 6 hours and monitored by TLC. Concentrating under reduced pressure when the reaction is complete, dissolving the residue again with 200 mL of dichloromethane, washing with 100mL of saturated NaCl solution for 2 times, and adding anhydrous Na₂SO₄After drying and concentration of the solvent under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15: 1) to obtain 12.06 g of compound 8 as a colorless oil in 55.92% yield.¹H NMR (600 MHz, CDCl₃, ppm)δ:0.66 (s, 3H), 0.85 (d, 6H,J= 6.4 Hz), 0.91 (d, 3H,J= 6.4 Hz), 0.99 (s,3H), 0.67-2.38 (remaining cholesterol protons), 3.16-3.21 (m, 1H), 3.59-3.75(m, 12H), 5.32 (m, 1H)。

Example 7

Preparation of Compound 9

N-Boc-N' -Fmoc-L-lysine (5 g, 10.67 mmol) was dissolved in 25 mL of dichloromethane, and dicyclohexylcarbodiimide (DCC, 2.93 g, 14.23 mmol) and DMAP (174 mg, 1.42 mmol) were added in this order at-5 ℃ to activate for 30 minutes. After the activation was completed, 15mL of a dichloromethane solution of Compound 8 (3.69 g, 7.11 mmol) was added dropwise to the reaction solution, and after completion of the addition, the reaction was allowed to warm to room temperature and stirred for 8 hours, followed by TLC. After completion of the reaction, the by-product dicyclohexylurea was removed by filtration, and the solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20: 1) to obtain 6.41 g of compound 9 as a pale yellow oil with a yield of 93%.¹H NMR (400MHz, CDCl₃, ppm) δ: 0.67 (s, 3H), 0.86 (d,6H,J= 6.8 Hz), 0.91 (d, 3H,J= 6.4 Hz), 0.98 (s, 3H), 1.43 (s, 9H), 0.87-2.37 (remaining cholesterol&lys protons), 3.10-3.19 (m, 3H), 3.62-3.72 (m,10H), 4.23 (t, 1H,J= 6.8 Hz), 4.31-4.35 (m, 2H), 4.39 (t, 3H,J= 6.8 Hz),5.32 (d, 1H,J= 4.8 Hz), 7.32 (t, 2H,J= 7.2 Hz), 7.40 (t, 2H,J= 7.2 Hz),7.61 (d, 2H,J= 7.2 Hz), 7.76 (d, 2H,J= 7.6 Hz)。

Example 8

Preparation of Compound 10

Compound 9 (6.41 g, 6.61 mmol) was dissolved in 20 mL of methylene chloride, 2.96 mL of 1, 8-diazabicycloundecen-7-ene (DBU) was added, and the mixture was allowed to react at room temperature for 20 minutes. TLC to monitor completion of the reaction, the reaction solution was washed with water (50 mL. times.2) and saturated NaCl solution (50 mL. times.2) in this order, and anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave a pale yellow oil, and the residue was purified by silica gel column chromatography (petroleum ether: acetone =1: 1) to give 4.71 g of compound 10 as a colorless oil in a yield of 95.32%.¹H NMR (400MHz, CDCl₃, ppm) δ: 0.67 (s, 3H), 0.87 (d, 6H,J= 6.4 Hz),0.91 (d, 3H,J= 6.4 Hz), 0.99 (s, 3H), 1.43 (s, 9H), 0.85-2.35 (remainingcholesterol&lys protons), 3.12-3.18 (m, 3H), 3.63-3.76 (m, 10H), 4.35-4.39(m, 2H), 5.34 (d, 1H,J= 4.8 Hz), 8.69 (s, 2H)。

Example 9

Preparation of Compound 11

Compound 5 (1.16 g,1.76 mmol) was dissolved in a mixed solvent of dichloromethane and N, N-dimethylformamide (v/v =40 mL/10 mL), and 2- (7-benzotriazole oxide) -N, N' -tetramethyluronium hexafluorophosphate (HATU, 802 mg, 2.11 mmol) and N, N-diisopropylethylamine (873 μ L, 5.28 mmol) were added and activated at-5 ℃ for 30 minutes. After activation was complete, 12 mL of Compound 10 (876 mg, 1.17 mmol) in dichloromethane were added dropwise and the reaction was continued at room temperature for 6 hours, monitored by TLC. After the reaction was completed, the reaction solution was washed with 1N hydrochloric acid solution (50 mL. times.1) and saturated NaCl solution (50 mL. times.8) in this order, and the organic layer was washed with anhydrous Na₂SO₄And (5) drying. Filtration and concentration of the solvent under reduced pressure gave a pale yellow oil which was purified by silica gel column chromatography (dichloromethane: methanol = 20: 1) to give 1.32 g of a pale yellow oilCompound 11 was yellow gum in 81.26% yield.¹HNMR (600 MHz, CDCl₃, ppm)δ: 0.68 (s, 3H), 0.87 (d, 6H,J= 6.0 Hz), 0.92 (d,3H,J= 6.6 Hz), 0.99 (s, 3H), 1.44 (s, 9H), 0.86-2.23 (remaining cholesterol&lys&biotin protons), 2.36 (d, 4H,J= 10.2 Hz), 2.66-2.92 (m, 8H), 3.07(t, 2H,J= 6 Hz), 3.15-3.20 (m, 3H), 3.39-3.75 (m, 14H), 4.09-4.57 (m, 11H),5.34 (s, 1H)。

Example 10

Preparation of Compound 12

Compound 11 (600 mg) was dissolved in 6 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 30 minutes. And monitoring by TLC. After the reaction is completed, the mixture is concentrated under reduced pressure, 50 mL of dichloromethane is added for redissolution, and saturated NaHCO is used for sequential use₃The solution (20 mL), saturated NaCl solution (20 mL. times.2) was washed, and the organic layer was washed with anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure gave 525 mg of compound 12 as a pale yellow gum in 94.25% yield.¹H NMR (600MHz, CDCl₃, ppm) δ: 0.67 (s, 3H), 0.86 (d,6H,J= 10.8 Hz), 0.91 (d, 3H,J= 10.2 Hz), 0.99 (s, 3H), 1.44 (s, 9H),0.86-2.29 (remaining cholesterol&lys&biotin protons), 2.32-2.37 (m, 4H),2.66-2.92 (m, 10H), 3.12-3.18 (m, 3H), 3.42-3.72 (m, 14H), 4.06-4.54 (m,11H), 5.35 (s, 1H)。

Example 11

Preparation of ligand tri-Biotin-Chol (I)

Biotin (150 mg, 0.61 mmol) was dissolved in a mixed solvent of dichloromethane and N, N-dimethylformamide (v/v =24 mL/8 mL), and after adding 2- (7-benzotriazole oxide) -N, N' -tetramethyluronium hexafluorophosphate (HATU, 278 mg, 0.73 mmol) and N, N-diisopropylethylamine (202 μ L, 1.22 mmol), the mixture was activated at-5 ℃ for 30 minutes. After activation, 13 mL of Compound 12 (525 mg, 0.41 mmol) in dichloromethane was added dropwise and the reaction was continued at room temperature for 10 h, monitored by TLC. When the reaction was complete, the reaction solution was washed with 1N HCl solution (20 mL. times.2) and saturated NaCl solution (20 mL. times.8) in that order, and the organic layer was washed with anhydrous Na₂SO₄Drying, filtering, and reducing pressureConcentration gave a light yellow oil which was purified by silica gel column chromatography (dichloromethane: methanol =12: 1) to give 521 mg of a light yellow solid with a yield of 84.47%. mp: 121 and 123 ℃.¹HNMR (600MHz, CDCl₃, ppm) δ: 0.68 (s, 3H), 0.87 (d, 3H,J= 6.6 Hz), 0.91 (d,3H,J= 6.0 Hz), 0.99 (s, 3H), 0.86-2.24 (remaining cholesterol&lys&biotin protons), 2.36 (d, 4H,J= 12 Hz), 2.72-2.91 (m, 10H), 3.16-3.20 (m,6H), 3.52-3.92 (m, 14H), 4.05-4.54 (m, 13H), 5.34 (s, 1H). HR-MS calculatedfor C₇₇H₁₂₅N₉O₁₅S₃Na [M+Na]⁺1534.8426, found 1534.8431。

Example 12

Preparation of ligand tetra-Biotin-Chol (II)

Compound 5 (408 mg, 0.62 mmol) was dissolved in 10mL of dichloromethane, and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 354 mg, 0.93 mmol) and N, N-diisopropylethylamine (308. mu.L, 1.86 mmol) were added and activated at-5 ℃ for 30 minutes. After activation, 10mL of Compound 12 (397 mg,0.31 mmol) in dichloromethane were added dropwise and the reaction was continued at room temperature for 10 h, monitored by TLC. After the reaction was completed, the reaction solution was washed with 1N hydrochloric acid solution (20 mL. times.2) and saturated NaCl solution (20 mL. times.1) in this order, and the organic layer was washed with anhydrous Na₂SO₄Drying, filtration, concentration of the solvent under reduced pressure and purification of the residue concentration by silica gel column chromatography (dichloromethane: methanol =10: 1) gave 415 mg of a pale yellow solid with a yield of 69.81%. mp: 154-.¹H NMR (600MHz, CDCl₃, ppm) δ: 0.68 (s, 3H),0.87 (d, 3H,J= 6.6 Hz), 0.91 (d, 3H,J= 6.6 Hz), 0.99 (s, 3H), 0.86-2.27(remaining cholesterol&lys&biotin protons), 2.33-2.37 (m, 10H), 2.61-2.90(m, 14H), 3.14-3.19 (m, 7H), 3.40-3.90 (m, 18H), 4.04-4.55 (m, 19H), 5.34 (s,1H). HR-MS calculated for C₉₅H₁₅₂N₁₂O₂₁S₄Na [M+Na]⁺1949.0037, found 1949.0052。

The specific preparation method of the breast cancer targeted liposome comprises the following steps:

example 13

Preparation of liposomes

The film-hydration ultrasonic method is used as a classical liposome preparation method, has the advantages of most extensive application and simple operation, and the prepared liposome has a typical structure. Therefore, the thin film-hydration ultrasonic method is selected to prepare the paclitaxel loaded liposome.

According to the previous search of the paclitaxel liposome-loaded prescription in the subject group, an optimized prescription is selected: the lipid material molar ratio is cholesterol to soybean phospholipid: ligand =33:64:3, the drug lipid mass ratio is lipid to paclitaxel =30:1, and the hydration solution is phosphate buffered saline (PBS, 0.01M) with pH 7.4. We prepared 5 paclitaxel-loaded liposomes using the above formula: PTX-tetra-Bio-Lip, PTX-tri-Bio-Lip, PTX-Bio-Bio-Lip.

The specific operation is as follows: accurately weighing the lipid material and paclitaxel in the prescribed amount in a 50 mL eggplant-shaped flask, dissolving with a proper amount of chloroform-methanol mixed solution (V/V = 2/1), performing rotary evaporation in a constant-temperature water bath at 37 ℃ to remove the solvent to obtain a uniform and complete lipid film, and performing vacuum drying overnight to remove the residual solvent. Adding PBS buffer solution with pH of 7.4, hydrating in constant temperature air bath shaker at 20 deg.C and 180 rpm for 30 min, and performing ultrasonic treatment (80W, 5S, 5S) with probe in ice water bath for 3 min to obtain slightly opalescent liposome solution.

Example 14

Encapsulation efficiency of liposome and measurement of particle size and potential

According to literature reports, the application adopts a freezing centrifugation method to separate the unencapsulated free paclitaxel from the paclitaxel loaded liposome. PTX-tetra-Bio-Lip, PTX-tri-Bio-Lip, PTX-Bio-Lip, respectively, were prepared as described in example 13. And (3) centrifuging part of the paclitaxel-loaded liposome solution for 20 minutes at 10000rpm under the condition of 4 ℃, and obtaining supernate, namely the liposome without free paclitaxel. Respectively taking 40 μ L of centrifuged supernatant and liposome sample before centrifugation, adding 960 μ L of methanol, vortex shaking for 10 min to completely break emulsion, centrifuging at 10000rpm for 10 min, collecting supernatant, injecting into high performance liquid chromatograph for analysis, and calculating UV loading according to formulaEncapsulation efficiency of paclitaxel liposome (EE%): EE% = a_{After centrifugation}/A_{Before centrifugation}X 100%, wherein A_{After centrifugation}And A_{Before centrifugation}Refer to the peak areas of the liposome samples after centrifugation and before centrifugation, respectively. Furthermore, the particle size and Zeta potential of the 5 kinds of paclitaxel-loaded liposomes were measured. The prepared liposomes were diluted with ultrapure water to an appropriate concentration, and the particle size and potential of the liposomes were measured by a laser particle size and Zeta potential analyzer, and the particle size, potential and encapsulation efficiency of each group of liposomes were as shown in table 1.

Table 1: particle size, potential and encapsulation efficiency of different types of paclitaxel-loaded liposomes

As shown in Table 1, the particle size of the 5 groups of paclitaxel-loaded liposomes is 102-114 nm, PDI is less than 0.2, and the liposomes are uniformly distributed; each group of paclitaxel-loaded liposomes is negatively charged, and the Zeta potential is between-2 to-4 mV, so that the paclitaxel-loaded liposomes are beneficial to avoiding the adsorption of plasma proteins, and can be prevented from being removed by a reticuloendothelial system in vivo.

Example 15

Evaluation of serum stability

The light transmittance of the paclitaxel loaded liposome modified by different ligands in 50% fetal calf serum is measured by a turbidity method, and the specific operation is as follows: and (3) uniformly mixing each group of paclitaxel-loaded liposome with the fetal calf serum with the same volume, slowly shaking in a constant-temperature shaking table at 37 ℃ (45 rpm), sampling at 0 h, 1 h, 2 h, 4 h, 8h, 12 h, 24 h and 48 h respectively, measuring the absorbance value of the sample at 750 nm by an enzyme labeling instrument, and converting into light transmittance.

The results (figure 1) show that the light transmittance of all the liposomes is still more than 91% after the liposomes are incubated with fetal calf serum for 48 hours, no obvious aggregation phenomenon exists, and the results show that the interaction between PTX-Lip, PTX-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip and serum protein is small, so that the serum stability is good, and a foundation is laid for later in vitro and in vivo experiments.

Example 16

Evaluation of hemolytic Properties

18-22 g Kunming mice are taken, blood is taken from the orbit and placed in a centrifuge tube coated with heparin sodium, the centrifuge tube is centrifuged at 4 ℃ (10000 rpm is multiplied by 10 min), supernatant is discarded, lower layer red blood cells are washed three times by normal saline, the supernatant is colorless, and finally the red blood cells are resuspended into 2% (w/v) solution by the normal saline. Five paclitaxel-loaded liposomes were prepared as described in example 13 and gradually diluted with physiological saline to lipid concentrations of 800, 600, 400, 300, 200, 150, 100, 75, 50, 25, 10 μmol/L. Respectively taking 0.2 mL of the liposome with different concentrations, mixing the liposome with an equal volume of 2% erythrocyte suspension, incubating the mixture in a constant temperature shaking table at 37 ℃ for 1 hour, centrifuging the mixture at 10000rpm for 10 minutes, taking supernatant, and detecting the absorbance A at the wavelength of 540 nm by using a microplate reader. The result of 1% of Triton (polyethylene glycol octyl phenyl ether, Triton X-100) and red blood cells were used as a positive control, i.e., the hemolysis rate was 100%; the result of incubation of PBS with erythrocytes was used as a negative control, i.e., the hemolysis rate was 0%. The hemolysis rate of each group of liposomes was calculated as: hemolysis rate (percent hemolysis)% = (A)_{Sample (I)}–A_{Negative of}）/（A_{Positive for}–A_{Negative of}）×100%。

As shown in FIG. 2, the hemolysis rate of 5 groups of paclitaxel loaded liposomes was below 10% with increasing lipid concentration, and the liposome had good biological safety without significant increase. With increasing density of targeting molecules in the liposomal ligands, there was no significant difference in hemolysis of PTX-Lip, PTX-Bio-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip.

Example 17

Evaluation of drug Release in vitro

The in vitro drug release of different ligand modified liposomes was investigated by dialysis. 0.4 mL of each paclitaxel liposome-loaded group or paclitaxel (solvent is ethanol: polyoxyethylene castor oil =1:1, v/v) with equal concentration as a free drug is respectively placed in a dialysis bag of 8000-12000 Da, the dialysis bag is sealed and placed in 40 mL of dialysis medium (1% Tween 80 PBS solution, v/v), the dialysis bag is slowly shaken in a constant temperature shaking table (37 ℃ and 45 rpm), 0 h, 0.5 h, 1 h, 2 h, 4 h, 8h, 12 h, 24 h and 48 h are respectively sampled for 0.1 mL and added with release medium with equal volume, and after 48 h, the liquid in the dialysis bag is uniformly mixed with the release medium and sampled to be used as a sample with complete release of paclitaxel. The removed sample was analyzed according to the HPLC chromatographic conditions described above. And the cumulative release of each sample was calculated at each time point and the release behavior was plotted (n = 3), and the results are shown in fig. 3.

As shown in fig. 3, the release of free paclitaxel was relatively rapid, and more than 85% of the drug was released into the culture medium after 12 hours of incubation; the drug-loaded liposomes of each group release slowly, the release characteristics are not obviously different, the cumulative release amount of PTX is lower than 72 percent after the drug-loaded liposomes are incubated with PBS for 48 hours, and no obvious burst release phenomenon exists. The results show that the prepared 5 groups of liposomes PTX-Lip, PTX-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip can obviously improve the release behavior of the drugs and have the slow release effect; with the increase of the density of the targeting molecules in the liposome ligand, the drug release behaviors of the paclitaxel loaded liposome of each group have no obvious difference.

Preliminary targeting evaluation

Example 18

Cell uptake assay

The liposome labeled with CFPE was prepared by replacing paclitaxel with the fluorescent agent CFPE according to the method for preparing paclitaxel-loaded liposome of example 13.

4T1 cells and MCF-7 cells highly expressing biotin receptors were plated in 12-well plates at 1X 10⁵The cells were seeded at a concentration of one cell per well and cultured in a cell incubator at 37 ℃ and 5% carbon dioxide for 24 h to achieve 80% confluency of the cells. The medium was discarded, and CFPE-labeled Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip were diluted with the medium, respectively, to give a final CFPE concentration of 2. mu.g/mL after addition to the well plate. Then continuously incubating for 4 hours in a cell culture box, discarding the culture medium containing the liposome, washing with precooled PBS for 2 times, digesting and collecting the cells, centrifuging for 3 min at 4 ℃ and 2000 rpm, discarding the supernatant after centrifugation, washing with precooled PBS for 3 times, and resuspendingCells were in 0.3 mL PBS. The fluorescence intensity in 4T1 cells and MCF-7 cells was measured by flow cytometry.

On the basis of quantitative determination by flow cytometry, we also qualitatively investigated the uptake of Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip in 4T1 cells and MCF-7 cells more intuitively by laser scanning confocal microscopy. The specific operation is as follows: 4T1 cells and MCF-7 cells were plated at 5X 10 in 6-well plates, respectively⁵The cells/well were seeded at 37 ℃ with 5% CO₂Culturing for 24 hours at the concentration, discarding the medium, diluting CFPE-labeled Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip with the medium, respectively, and adding to a well plate to obtain a final concentration of CFPE of 2 μ g/mL. Incubation was continued for 4 hours in a cell incubator, the liposome-containing medium was discarded and washed 2 times with ice-PBS 5 min each, fixed with 4% paraformaldehyde at room temperature for 30 min, discarded, and washed 3 times with PBS 5 min each. Adding 0.1 microgram/mL DAPI dye to stain the nucleus for 5 minutes, discarding the dye, washing with PBS for 3 times, sealing with glycerol, and shooting under a laser confocal microscope.

As shown in FIGS. 4A and B, 4T1 cells and MCF-7 cells both showed the strongest uptake capacity for the trimodal biotin-modified liposome tri-Bio-Lip. In 4T1 cells, the fluorescence intensity of tri-Bio-Lip was 5.21 times that of Lip, 2.60 times that of Bio-Lip, 1.67 times that of Bio-Bio-Lip, and 1.17 times that of tetra-Bio-Lip, respectively. Uptake of tri-Bio-Lip by MCF-7 cells was 2.90-fold, 2.27-fold, 1.70-fold, and 1.33-fold for Lip, Bio-Bio-Lip, and tetra-Bio-Lip, respectively. The qualitative uptake experiment result of laser confocal shows that the tripartite biotin-modified liposome tri-Bio-Lip shows the highest uptake level in 4T1 cells and MCF-7 cells with high SMVT receptor expression, and is consistent with the quantitative result of flow cytometry.

Example 19

Cytotoxicity test

Respectively diluting PTX-Lip, PTX-Bio-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip with culture medium to prepare taxol with the concentration of 20 microgram/mL, 5 microgram/mL and 1 microgram/mL, 0.5 mug/mL, 0.1 mug/mL and 0.01 mug/mL of liposome solution for standby. Dissolving appropriate amount of free paclitaxel in DMSO, and diluting with culture medium according to the above concentration. 4T1 cells and MCF-7 cells were plated at 5X 10 in 96-well plates, respectively³The individual cells/well were seeded at 37 ℃ with 5% CO₂Culturing for 24 hours under the conditions of (1), replacing the culture medium with the above-mentioned free paclitaxel solution and PTX-Lip, PTX-Bio-Bio-Lip, PTX-tri-Bio-Lip, PTX-tetra-Bio-Lip of different concentrations, discarding the culture medium after CO-culturing for 48 hours, washing 2 times with PBS, adding 20 μ L of MTT solution and 5 mg/mL of serum-free culture medium into a 96-well plate in sequence, and culturing at 37 ℃ with 5% CO₂Cultured for 4 hours under the conditions of (1). Discarding the culture medium, adding 150 muL DMSO into the cell hole, placing in an enzyme-linked immunosorbent assay (ELISA) instrument for oscillation, and measuring the absorbance value A at 490 nm after uniform mixing_Sample. Absorbance value A in DMSO_BlankAs a blank, absorbance values A of non-drug-treated wells were measured_ControlAs a control, by the formula: survival rate (cell viability)% = (a)_Sample– A_Blank)/(A_Control–A_Blank) X 100%, the cell viability of the dosing wells was calculated.

The results showed that the inhibitory activity of PTX-Lip, PTX-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip on 4T1 cells and MCF-7 cells was gradually increased with the increase of the concentration of paclitaxel administration. Compared with other types of paclitaxel-loaded liposomes, the trimodal biotin-modified liposome PTX-tri-Bio-Lip shows stronger cytotoxicity; in particular, PTX-tri-Bio-Lip showed stronger inhibitory activity against 4T1 cells than free paclitaxel. Free paclitaxel also shows better inhibition capability under different administration concentrations, because each group of paclitaxel loaded liposome has a process of releasing the drug, and the time for exerting the drug effect is delayed compared with the free drug, so the free paclitaxel shows better inhibition activity in a short time, and the sustained release effect of the liposome is also indirectly shown.

Example 20

Apoptosis assay

Free paclitaxel, PTX-Lip, PTX-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip ability to promote apoptosis in 4T1 were tested by annexin V-FITC/PI double staining. 4T1 cells were plated at 5X 10 in 6-well plates⁵The cells/well were seeded at 37 ℃ with 5% CO₂Culturing for 24 hours, replacing the culture medium with free paclitaxel solution with PTX concentration of 0.01 mug/mL or PTX-Lip, PTX-Bio-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip, respectively, and continuing to incubate for 24 hours in the cell culture box. After the cells are digested respectively, the cells are washed 3 times by using a cold PBS solution, 500 muL binding buffer is used for resuspending the cells, 5 muL annexin V-FITC and 5 muL PI are sequentially added into a pore plate, then the cells are dyed for 15 min in a dark place at room temperature, and the capability of the free paclitaxel solution and the PTX-Lip, PTX-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip solution for promoting the apoptosis of 4T1 cells is investigated by a flow cytometer.

The results showed that the percentages of apoptosis and necrosis induced by PTX, PTX-Lip, PTX-Bio-Bio-Lip, PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip were 40.01. + -. 1.98%, 23.75. + -. 2.85%, 25.55. + -. 1.59%, 35.26. + -. 1.68%, 43.29. + -. 0.84% and 36.84. + -. 1.69%, respectively. Compared with free paclitaxel, PTX-Lip, PTX-Bio-Bio-Lip and PTX-tetra-Bio-Lip, the trimodal biotin modified liposome PTX-tri-Bio-Lip has stronger effect of inducing apoptosis of 4T1 and is consistent with the in vitro cytotoxicity experimental result.

Example 21

Evaluation of in vivo targeting

DiD-labeled liposomes were prepared according to the procedure for paclitaxel liposome preparation in example 13, substituting paclitaxel for the fluorescent agent DiD. After the 4T1Balb/C bearing mice are established for 14 days, DiD-loaded liposome Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip with the dose of 200 mug DiD/kg are administered to the bearing mice by tail vein injection. And hair at the tumor site of the mouse was removed to facilitate observation of the fluorescence intensity at the tumor site. At 1 h, 4 h, 8h, 12 h and 24 h post-dose, each group of mice was anesthetized with 4% chloral hydrate and placed in a small animal in vivo imager for imaging. After imaging is finished, the anesthetized mouse is subjected to heart perfusion by using normal saline at a corresponding time point, each organ and tumor tissue are separated, the anesthetized mouse is respectively placed in the normal saline for three times to be cleaned so as to remove residual blood on the surface, then, the water on the surfaces of each organ and tumor tissue is sucked dry by using filter paper, and the anesthetized mouse is placed in a small animal living body imager for imaging.

In vivo imaging results show that 4 hours after administration, each group of biotin-modified liposomes are aggregated at the tumor site, but the fluorescence intensity is weak; fluorescence reached maximum intensity 8h after dosing. In vitro imaging results of isolated organs show that after the DiD-loaded liposomes of each group enter blood, the DiD-loaded liposomes are mainly distributed at liver and spleen parts along with in vivo circulation, especially in livers with vigorous metabolism. In vitro imaging results of the isolated tumor tissues show that 8h after administration, each group of DiD-loaded liposomes has the strongest fluorescence in the isolated tumor tissues, especially Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip; the results of semiquantitative determination of fluorescence intensity of ex vivo tumor tissues at 8h after administration showed that the fluorescence intensity of tri-Bio-Lip was 3.59 times, 2.97 times, 1.40 times and 1.29 times that of Lip, Bio-Bio-Lip and tetra-Bio-Lip, respectively.

The results of the above studies show that the in vivo breast cancer targeting ability of 5 groups of DiD-loaded liposomes is as follows: the tri-Bio-Lip > tetra-Bio-Lip > Bio-Lip > Bio-Lip > Lip, which is consistent with the in vitro targeting results. The tri-Bio-Lip > Lip further proves that the targeting molecule density is improved, and the breast cancer targeting property of the liposome can be effectively enhanced; meanwhile, tetra-Bio-Lip < tri-Bio-Lip, combined with the analysis of the cell uptake experiment result, shows that factors such as the branched structure and the space distance of biotin residues in the ligand may have important influence on the breast cancer targeting capability of the liposome besides the targeting molecule density.

Drawings

FIG. 1: change in light transmittance of different types of paclitaxel-loaded liposomes incubated in 50% serum

FIG. 2: hemolytic rates of different types of paclitaxel-loaded liposomes

FIG. 3: in vitro drug release behavior of different types of paclitaxel-loaded liposomes and free paclitaxel

FIG. 4: (A) and (B) represents uptake of CFPE-labeled liposomes of different types by 4T1 cells and MCF-7 cells, respectively, as measured by flow cytometry, and p represents<0.05、p<0.01 and p<0.001, Lip group as control group, n. s. indicating no significant difference, (mean ± SD,n= 3)。

Claims

1. a multi-branch biotin modified breast cancer targeted lipid material is a structure shown in general formulas (I) and (II) or a pharmaceutically acceptable salt or hydrate thereof:

wherein, the molecular weight of the PEG is equal to but not limited to 150, 200, 400, 600, 800, 1000, 1500, 2000, 4000 and the like;

2. The class of multi-branched biotin-modified breast cancer-targeting lipid materials of claim 1, wherein lipid material (I) is characterized by: cholesterol is taken as a liposome carrier, polyethylene glycol (PEG) is taken as a bridge chain, lysine is taken as a branched skeleton, one end of the cholesterol is connected with the cholesterol extended by the polyethylene glycol, and the other two ends of the cholesterol are respectively connected with single biotin and double-branched biotin which is coupled by diethanolamine and extended by succinic acid, so that the biotin becomes triple-branched biotin; the lipid material (II) is characterized in that: cholesterol is used as a liposome carrier, polyethylene glycol is used as a bridge chain, lysine is used as a branch skeleton, one end of the cholesterol is connected with cholesterol extended by the polyethylene glycol, and the other two ends of the cholesterol are connected with double-branch biotin which is coupled by diethanol amine and extended by succinic acid, so that the double-branch biotin becomes the four-branch biotin.

3. The breast cancer targeted lipid material according to claim 1, and application of the breast cancer targeted lipid material as a drug carrier in a breast cancer targeted drug delivery system.

4. The structure of lipid material (I) of breast cancer according to claim 1, whose synthetic route is characterized in that: coupling two biotins by using diethanol amine as a branching reagent of a branched monomer, and extending the diethanol amine by using succinic acid to reduce the steric hindrance of a condensation reaction to obtain a key intermediate (compound 5); on the other hand, cholesterol is used as a starting material, a side chain is extended by polyethylene glycol and then coupled with branched skeleton fluorenylmethyloxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) double-protected lysine Fmoc-L-Lys (Boc) -OH, then the Fmoc is removed under alkaline conditions, and then the coupled compound is connected with the compound 5, and finally the Boc protecting group is removed under trifluoroacetic acid conditions, and then the coupled compound is connected with biotin to obtain the lipid material (I).

5. The structure of lipid material (II) of breast cancer according to claim 1, whose synthetic route is characterized in that: coupling two biotins by using diethanol amine as a branching reagent of a branched monomer, and extending the diethanol amine by using succinic acid to reduce the steric hindrance of a condensation reaction to obtain a key intermediate (compound 5); on the other hand, cholesterol is used as a starting material, a side chain is extended by polyethylene glycol and then coupled with branched skeleton fluorenylmethyloxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) double-protected lysine Fmoc-L-Lys (Boc) -OH, then the Fmoc is removed under alkaline conditions and then connected with the compound 5, and finally the Boc protecting group is removed under trifluoroacetic acid conditions and then connected with the compound 5 again to obtain the lipid material (II).

6. The novel breast cancer-targeting lipid material as claimed in claim 1, wherein said active agent is paclitaxel as the therapeutic agent and CFPE or DiD as the imaging agent.