CN111166892A

CN111166892A - Biotin and cell-penetrating peptide co-mediated breast cancer targeted intelligent liposome material

Info

Publication number: CN111166892A
Application number: CN201911271608.7A
Authority: CN
Inventors: 海俐; 吴勇; 郭丽; 卢润鑫; 刘启俊; 周琳; 杨春艳; 王思琪
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2020-05-19

Abstract

The invention discloses a biotin and cell-penetrating peptide co-mediated breast cancer targeted intelligent liposome material. The intelligent liposome material comprises: 1. modifying biotin on a PEG long chain, and connecting the PEG long chain with cholesteric phase through an acid sensitive bond (general formula I, ligand material a); 2. the cell-penetrating peptide R8 was attached to the phospholipid by Michael addition (formula II, ligand material b). After the liposome is prepared by combining the two ligand materials, biotin exposed on the surface of the liposome can specifically recognize SMVT transporters overexpressed on the surface of breast cancer cells; after the liposome reaches the breast tumor part, the PEG long chain connected with the hydrazone bond is broken and separated, and the R8 mediated membrane penetration and cell entry connected with the short chain are exposed, so that the effect of strongly treating breast cancer is realized. The novel intelligent lipid material can be used for different dosage forms including liposome, nanoparticles, micelles and the like, and the prepared paclitaxel-loaded liposome has strong breast cancer penetrability and treatment effect and wide application prospect.

Description

Biotin and cell-penetrating peptide co-mediated breast cancer targeted intelligent liposome material

Technical Field

The invention relates to preparation and characterization of a novel biotin and cell-penetrating peptide co-mediated breast cancer targeted intelligent lipid material, and application of the material as a drug carrier in drug delivery, and belongs to the technical field of medicines.

Background

Breast cancer is a major public health problem in today's society, a major disease that endangers women's physical and mental health, and is called "first red killer". In 2019, 1 month, the national cancer center publishes the latest national cancer statistical data of the first stage, wherein the first malignant tumor in female aspect is breast cancer. Moreover, the incidence of breast cancer in women is first in both developed and developing countries worldwide. So far, various treatment methods such as operation treatment, radiotherapy, chemotherapy, endocrine treatment, small molecule targeted therapy, immunotherapy and the like are widely applied in clinical breast cancer. Chemotherapy is still one of the most effective means in clinical treatment of breast cancer at present, and can significantly improve the survival rate of patients. However, the traditional chemotherapy drugs not only have the disadvantages of poor solubility, insufficient targeting property, serious toxic and side effects on the whole body, and the like, but also are easy to generate drug resistance in the chemotherapy process, so that the clinical application of the traditional chemotherapy drugs is limited.

In order to reduce the distribution of antitumor drugs in normal tissues and organs, reduce the toxic and side effects of antitumor drugs, increase the effective concentration of antitumor drugs in tumor sites, and better exert the curative effect, the research of Nano-delivery system (NDS) targeting tumor is increasingly emphasized. 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 is widely researched in tumor treatment research. Among a plurality of nano drug-carrying systems, the liposome is the most mature nano carrier researched at present, and besides the characteristics, the liposome has the advantages of degradability in vivo, high biocompatibility, low toxicity, convenience in preparation and the like. Therefore, the treatment of breast cancer by using the liposome encapsulated chemotherapeutic drug becomes a hot spot of breast cancer treatment internationally, and is expected to become a main development direction of targeted breast cancer treatment.

Due to the characteristics of tumor vessel abnormality, compact extracellular matrix, tumor cell compact accumulation, high Interstitial Fluid Pressure (IFP) and the like of the solid tumor, the movement of the nanoparticle tumor interstitial space is still hindered. To achieve better cancer treatment, not only active targeting of the drug to the tumor site is required, but also the rate of tissue penetration after reaching the tumor site is critical. Octa-arginine (R8), a typical cationic cell-penetrating peptide, has been shown to have high membrane-penetrating efficiency. The R8 peptide is used to modify liposome, which can not only promote cellular uptake, but also improve the permeability of cell membrane. However, due to its non-specific affinity for different cells, the R8-mediated targeted drug delivery system for breast cancer shows high cell and tissue toxicity in vivo after systemic drug delivery, and is not widely used because of its cationic property, which is likely to cause red blood cell lysis and hemoglobin release. Therefore, if the R8 modified on the liposome can be hidden in the circulation process in vivo, the liposome is exposed after reaching the tumor site, so as to generate the membrane penetrating effect, and thus, the safe and effective accurate target treatment of the tumor can be realized.

In recent years, people develop a plurality of microenvironment responsive liposomes by utilizing the characteristics of tumor microenvironment, and the liposomes can be stably transported in vivo and can generate structural change after reaching tumor parts, thereby achieving ideal anticancer curative effect. Faintly acid (pH is less than 7.2) is a remarkable characteristic of a tumor microenvironment, and liposomes sensitive to different pH conditions can be designed by utilizing the acid environment in tumor tissues, so that accurate targeted therapy is realized. Currently, acid labile chemical bonds such as hydrazone bonds (i.e., stable at neutral pH and cleaved in acidic media) are widely used in this field. Therefore, the invention designs that an acid-sensitive hydrazone bond is used for modifying a polyethylene glycol long chain (PEG 3350) on cholesterin, and the properties of the polyethylene glycol long chain (PEG 3350) that the polyethylene glycol long chain is stable under physiological conditions and is broken under a weak acid environment are utilized to achieve the purposes of concealing R8 in systemic circulation and exposing R8 when reaching a tumor part, thereby realizing safe and effective treatment.

In recent years, fructose, folic acid, biotin, glycyrrhetinic acid, diphosphate and other small molecules are used as targeting groups of targeting vectors of anticancer drugs. Among numerous small molecule ligands, biotin has a simple structure, a single functional group, strong specificity with a receptor, and no immunogenicity, and is considered as a promising important tumor targeting molecule. Biotin is an indispensable vitamin for cell division and proliferation, and is mainly transported into cells by relying on the multi-vitamin transporter SMVT. The study shows that the SMVT is highly expressed on the surface of breast cancer cells such as MCF-7, 4T1, JC and MMT 06056.

Biotin used as an active targeting ligand of liposome for targeted therapy of breast cancer has made a certain research progress, for example, Tian and other researchers prepare biotinylated chitosan nanoparticles carrying bufalin, and the in vivo and in vitro anti-tumor effects show that the biotinylated chitosan nanoparticles can increase the tumor selectivity and reduce the toxicity of bufalin. Therefore, the invention utilizes the capacity of biotin to be specifically recognized by the high-expression SMVT transporter on breast cancer cells to design and modify biotin on the long-chain end of polyethylene glycol to form a ligand material a, thereby improving the targeting capacity of liposome breast cancer. The ligand material a and the ligand material b modified by R8 are modified on the liposome together, so that the liposome has the advantages of in vivo breast cancer targeting property, in vivo safety, breast cancer cell penetrability and the like, and more anticancer drugs can be delivered to tumors, the curative effect of the drugs is improved, and the toxic and side effects are reduced.

Disclosure of Invention

Based on the research and hypothesis, the invention aims to synthesize the biotin-modified intelligent breast cancer targeted lipid material a, and use the biotin-modified intelligent breast cancer targeted lipid material a and the ligand material b modified by the cell-penetrating peptide R8 for preparing the liposome. Meanwhile, the high affinity of the highly expressed multi-vitamin transporter SMVT of the breast cancer cells to biotin and the strong penetrability of the cell-penetrating peptide R8 to tumor cells are utilized, so that the liposome has active targeting property of the breast cancer and strong solid tumor penetrability, the drug amount of tumor parts is further improved, the curative effect is improved, and the toxic and side effects are reduced.

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

wherein, the molecular weight of the PEG is equal to but not limited to 2000, 3350, 5000 and the like; the acid sensitive bond X is not limited to acylhydrazone, phenylboronate, and the like.

The specific preparation method of the compound shown in the general formula (I) is illustrated by taking PEG molecular weight 3350 and X as an acylhydrazone bond as an example:

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

The liposome is characterized by comprising phospholipid, cholesterol, a general formula I (Biotin-PEG-PE-Chol), a formula II (R8-DSPE-PEG 2000) and an active agent.

The liposome mainly comprises a membrane material and an active agent, wherein the membrane material is a phospholipid bilayer and consists of lecithin, cholesterol and a liposome ligand. Wherein, the proportion relation of each component is as follows: the molar ratio of cholesterol to phospholipid is 1:2, and the molar contents of liposome ligands a and b are respectively 8% and 1.2% of the total molar number of cholesterol and phospholipid. The active agent of the present invention is preferably a therapeutic agent or imaging agent, and the dosage of the active agent can be adjusted according to the active agent contained in the carrier, wherein the active agent accounts for 0.1-50% 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. Antiepileptic drugs including, but not limited to barbiturates, epicyclylureas, double-stranded fatty acids, succinimides, benzodiazepines, iminoglycosides, sulfonamides, oxazolidinediones, piperines, corticosteroids, immunoglobulins, and the like. Antidepressant drugs including, but not limited to, norepinephrine reuptake inhibitors, monoamine oxidase inhibitors, 5-hydroxytryptamine reuptake inhibitors.

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 35-40 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.

Paclitaxel in the preferred step (one): the ratio of the lipid materials is 1: 30.

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

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

The material is prepared into specific liposome, and has the following advantages: 1. targeting property: the liposome realizes the targeted therapy of the breast cancer by fully utilizing the passive targeting and active targeting capabilities of the liposome through the intrinsic EPR effect of the liposome and the specific action of the biotin and the SMVT transporter highly expressed on the breast cancer cells. 2. Film penetration property: r8 exhibits higher transmembrane efficiency, increasing the amount of drug in solid tumors; 3. safety: the drug is encapsulated by using a biocompatible carrier material, so that immunoreaction and phagocytosis of a reticuloendothelial system can be reduced, and meanwhile, as the PEG chain of the ligand a exposed on the surface of the liposome is longer, R8 of the ligand b is concealed in the systemic circulation process, so that the nonspecific combination of R8 and negatively charged protein in blood is avoided, the systemic circulation time is prolonged, and the systemic nonspecific toxic and side effects are reduced; 4. for Biopharmaceutical Classification System (BCS) class IV drugs such as paclitaxel, bioavailability can be significantly improved; 5. the lipid material and the encapsulated drug integrally enter tumor cells and then release the drug, so that the action of the drug and the efflux protein can be reduced, and the drug resistance and the like are reduced. The lipid material can be combined with various medicines to perform combined administration on breast cancer. Therefore, the intelligent lipid material shown as the general formula I is designed, the paclitaxel-encapsulated breast cancer targeted intelligent liposome is prepared by combining the ligand material b, the cholesterol part of the liposome material is embedded into the liposome phospholipid bilayer, the ligand a exposed on the surface of the liposome has a longer PEG chain, R8 in the ligand b can be effectively concealed, and meanwhile, the biotin target head of the ligand a can realize breast cancer tumor targeting. After reaching the tumor part, hydrazone bonds of the ligand a are rapidly broken to expose sufficient R8, and the ligand a is mediated to efficiently penetrate through a membrane and enter cells, so that stronger breast cancer targeting and penetrating effects are achieved. The lipid material can be used for different dosage forms such as liposome, nanoparticles, micelle and the like, and has great application prospect.

Drawings

FIG. 1: change in light transmittance after incubation of different ligand-modified paclitaxel-loaded liposomes in 50% serum (n =3, mean ± SD)

FIG. 2: uptake of CFPE-labeled liposomes in 4T1 cells (A: neutral pH = 7.4; B: acidic pH = 6.0) and MCF7 cells (A: neutral pH = 7.4; B: acidic pH = 6.0) (. sup.P <0.05,. sup.P <0.01,. sup.P <0.001, versus CFPE-Lip; n =3, mean. + -. SD)

FIG. 3: confocal pictures taken of CFPE labeled liposomes in 4T1 cells (a: neutral pH = 7.4; B: acidic pH = 6.0) and MCF7 cells (a: neutral pH = 7.4; B: acidic pH = 6.0).

Detailed description of the invention

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

Monobenzyl succinate (2.84 g, 13.62 mmol) was weighed into a two-necked flask, argon was replaced three times, dissolved in 55 mL of anhydrous dichloromethane, stirred at-5 deg.C, isobutyl chloroformate (IBCF, 1.86 g, 13.62 mmol) and N-methylmorpholine (NMM, 1.38 g, 13.62 mmol) were added under argon and after addition activation continued for 30 min. Compound 1 (1.50 g, 11.35 mmol) was dissolved in 2.5 mL of anhydrous dichloromethane and added dropwise to the reactionIn the liquid. After the dropwise addition, the reaction mixture was transferred to room temperature for reaction for 3 hours, and the completion of the reaction was monitored by TLC. The solvent in the reaction system was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate =4: 1) to obtain 2.77 g of a colorless transparent oil with a yield of 75.70%.¹H-NMR (400 MHz, DMSO-d ₆,ppm)δ: 1.39 (s, 9 H), 2.28 (t,J= 6.0 Hz, 2H), 2.43 (t,J= 6.0 Hz, 2H),5.14 (s, 2 H), 7.40-7.31 (m, 5 H), 8.68 (s, 1H), 9.50 (s, 1 H).

EXAMPLE 2 preparation of Compound 3

Compound 2 (2.25 g, 6.98 mmol) was dissolved in 16 mL of degassed methanol, 330 mg of palladium on carbon was added, hydrogen gas was substituted for 5 times, and the mixture was stirred at room temperature. TLC monitored the reaction was complete, the reaction solution was filtered through celite, the celite was washed 2-3 times with methanol, the filtrate was collected and the solvent was removed by concentration under reduced pressure to give 1.60 g of a colorless oil in 98.70% yield. The product was carried on to the next step without purification.¹H-NMR (400 MHz, DMSO-d ₆, ppm)δ: 1.38 (s, 9H), 2.29 (t,J= 6.0 Hz, 2H),2.41 (t,J= 6.0 Hz, 2H), 8.68 (s, 1H), 9.52 (s, 1H), 11.0 (brs, 1H)。

EXAMPLE 3 preparation of Compound 5

Cholesterol 4 (30.00 g, 77.59 mmol) was dissolved in 100 mL of anhydrous pyridine and a solution of p-toluenesulfonyl chloride (TsCl, 23.67 g, 124.14 mmol) in pyridine (50 mL) was slowly added dropwise at 0 ℃. After the completion of the dropwise addition, the reaction mixture was transferred to 55 ℃ for overnight reaction. TCL monitored the completion of the reaction of the starting materials, pyridine was removed by distillation under reduced pressure, the residue was dissolved in ethyl acetate (300 mL), washed with dilute hydrochloric acid (1N, 100 mL. times.2) and saturated aqueous sodium chloride (100 mL. times.2) in that order, dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure to give 37.79 g of a white solid in 90.06% yield. The product is directly used for the next reaction without purification. Mp: 129-132 deg.C (Mp: 130-132 deg.C).

EXAMPLE 4 preparation of Compound 6

Compound 5 (20.00 g, 36.98 mmol) was dissolved in 120 mL dioxane, triethylene glycol (TEG, 27.79 g, 185.05 mmol) was added, refluxing was carried out for 6 hours, and TLC was used to monitor completion of the starting material reaction. The solvent was removed under reduced pressure, the residue was dissolved in 200mL of dichloromethane, washed with a saturated aqueous solution of sodium chloride (100 mL × 2), the organic layer was dried over anhydrous sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/acetone = 8/1) to give 11.68 g of a colorless oil with a yield of 60.87%.¹H-NMR (400 MHz, CDCl3, ppm) δ: 0.67 (s, 3H), 0.86 (d,6H, J=4.4 Hz), 0.91 (d, 3H,J= 4.4 Hz), 1.00 (s, 3H), 0.86-2.38 (remainingcholesterol protons), 3.16-3.21 (m, 1H), 3.62-3.63 (m, 2H), 3.65 (s, 4H),3.68 (d, 4H, J=2.4 Hz), 3.73-3.74 (m, 2H), 5.34 (s, 1H)。

EXAMPLE 5 preparation of Compound 7

The compound fluorenylmethoxycarbonyl-6-aminocaproic acid (5.00 g, 14.15 mmol) was dissolved in 140 mL of anhydrous dichloromethane, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 4.52 g, 23.58 mmol), 4-dimethylaminopyridine (DMAP, 2.88 g, 23.58 mmol) and N, N-diisopropylethylamine (DIPEA, 5.17mL, 31.29 mmol) were added successively under argon protection and activated at-5 ℃ for 30 min. Compound 6 (4.09 g, 7.88 mmol) was dissolved in 16 mL of anhydrous dichloromethane, and after dissolution, the solution was slowly added dropwise to the reaction mixture, after the dropwise addition, the mixture was allowed to warm to room temperature and stirred for 4 hours. TLC to monitor the completion of the reaction, the reaction solvent was removed by concentration under reduced pressure, and the residue was dissolved in 100 mL of dichloromethane and successively treated with 1N HCl (100 mL. times.2) and H₂0、Saturated sodium chloride solution (50 mL × 1) was washed, the organic layer was dried over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate =5: 1) to obtain 5.48 g of a transparent colorless oily product with a yield of 81.38%.¹H NMR (400 MHz, CDCl₃) δ 0.67 (s, 3H, CHOLE CH₃-18), 0.86 (d, 6H,J=6.4 Hz, CHOLE CH₃-26 and -27), 0.91 (d, 3H,J= 6.4 Hz, CHOLE CH₃-21), 0.98(s, 3H, CHOLE CH₃-19),0.92-2.50 (36H, including 28H remain CHOLE protons),3.17-3.20 (m, 3H), 3.62-3.73 (m, 10H), 4.21-4.23 (m, 2H), 4.40 (d,J= 6.8Hz, 2H), 4.88 (s, 1H), 5.33 (s, 1H), 7.31 (t,J= 7.2 Hz, 2H), 7.39 (t,J=7.2 Hz, 2H), 7.59 (d,J= 7.2 Hz, 2H), 7.76 (d,J= 7.2 Hz, 2H)。

EXAMPLE 6 preparation of Compound 8

Compound 7 (3 g, 3.51 mmol) was dissolved in 30 mL of dichloromethane and 1, 8-diazabicyclo [5.4.0 ] was added at room temperature]Undec-7-ene (DBU, 1.60 g, 10.54 mmol), reacted at room temperature for 30 minutes. TLC, the reaction was completed, the reaction solvent was removed by concentration under reduced pressure, the residue was dissolved in 100 mL of dichloromethane, the reaction solvent was removed by concentration under reduced pressure, the residue was dissolved in 30 mL of dichloromethane, washed with water (30 mL × 2) and a saturated sodium chloride solution (30 mL × 1), respectively, the dichloromethane layers were combined and collected, after drying over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane: methanol =40: 1) to obtain 1.25 g of a yellow oily product with a yield of 56.36%.¹H NMR (400 MHz, CDCl₃) δ0.67 (s, 3H, CHOLE CH₃-18), 0.86 (d, 6H,J= 6.4 Hz, CHOLE CH₃-26 and -27),0.91 (d, 3H,J= 6.4 Hz, CHOLE CH₃-21), 0.98 (s, 3H, CHOLE CH₃-19), 0.92-2.50(38H, including 28H remain CHOLE protons), 3.16-3.21 (m, 1H), 3.62-3.73 (m,10H), 4.21-4.23 (m, 2H), 5.34 (s, 1H)。

EXAMPLE 7 preparation of Compound 9

Compound 3 (0.47 g, 2.02 mmol) was placed in a reaction flask, argon replaced 3 times, dissolved in 8 mL of anhydrous dichloromethane, isobutyl chloroformate (IBCF, 295 mg, 2.16 mmol) and N-methylmorpholine (NMM, 218.70 mg, 2.16 mmol) were added under argon, and after addition, activation was carried out at-5 ℃ for 30 min. Compound 8 (0.85 g, 1.35 mmol) was dissolved in 5mL of anhydrous dichloromethane and added dropwise to the reaction mixture. After the dropwise addition, the reaction mixture is transferred to room temperature for reaction for 18 h. TLC monitored the reaction was complete, the reaction solvent was removed by concentration under reduced pressure, the residue was dissolved in 20 mL of dichloromethane, washed with 1N HCl (25 mL × 2), pure water (10 mL × 1), and saturated sodium chloride solution (30 mL × 1), respectively, the dichloromethane layers were collected and combined, the organic layers were dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: acetone =4: 1) to give 566 mg of a transparent colorless oily product with a yield of 49.56%.¹H-NMR (400 MHz, CDCl₃, ppm)δ:0.67(s, 3H), 0.86 (d, 6H,J= 5.6 Hz), 0.91 (d, 3H,J= 6.4 Hz), 0.99 (s, 3H),0.67-2.24 (m, 58H), 1.49 (s, 9H), 2.53-2.89 (m, 6H), 3.18 (s, 1H), 3.34 (t,2H,J= 6.8 Hz), 3.63-3.70 (m, 10H), 4.23-4.26 (m, 2H), 5.33 (s, 1H), 6.42(s, 1H)。

EXAMPLE 8 preparation of Compound 10

Compound 9 (120 mg) was dissolved in 24 mL of dichloromethane, and trifluoroacetic acid (2.4 mL, 10% V/V) was added thereto at room temperature to conduct reaction at room temperature for 30 minutes. The reaction was monitored by TLC for completion, and a large amount of saturated sodium bicarbonate solution was added to the reaction solution to adjust the solution pH =9, extracted with dichloromethane (25 mL × 2), the dichloromethane layer was collected and dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure to give 95 mg of a pale yellow oily product with a yield of 89.79%. The product was carried on to the next step without purification.

EXAMPLE 9 preparation of Compound 12

Compound 11 (41.04 mg, 0.25 mmol) was placed in a reaction flask, argon was substituted for 3 times, the mixture was dissolved with a mixed solvent (2 mL of anhydrous dichloromethane +0.2 mL of N, N-dimethylformamide), the mixture was transferred to-5 ℃ and stirred, dicyclohexylcarbodiimide (DCC, 103.16 mg, 0.50 mmol) and 4-dimethylaminopyridine (DMAP, 6.11 mg, 0.05 mmol) were added under argon protection, and activation was continued at that temperature for 30 min. Dissolving polyethylene glycol (PEG 3350, 837.5 mg, 0.25 mmol) in 2 mL of dichloromethane, dissolving, slowly adding the activated solution dropwise into the polyethylene glycol solution, and moving to room temperature for reaction for about 16 h after the dropwise addition. The reaction was monitored by TLC for completion, and washed with aqueous solution (10 mL × 3), 1N HCl (10 mL × 1), and saturated sodium chloride solution (10 mL × 1), respectively, and the dichloromethane layers were collected and combined, and after the organic layer was dried over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane: methanol =30: 1) to obtain 524 mg of a white solid with a yield of 59.95%.¹H-NMR (400 MHz, CDCl₃, ppm)δ:2.65 (s, 3H), 3.44-3.49 (m, 2H), 3.63-3.72 (m, 296H), 3.81-3.86 (m, 4H), 4.48-4.52 (m, 2H), 8.01 (d,J= 8.0 Hz,2H), 8.14 (d,J= 8.0 Hz, 2H)。

EXAMPLE 10 preparation of Compound 13

Biotin (97.72 mg, 0.40 mmol) was dissolved in 18 mL of a mixed solvent (8 mL of anhydrous dichloromethane +10 mL of anhydrous N, N-dimethylformamide) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 115.02 mg, 0.60 mmol), 4-dimethylaminopyridine (DMAP, 73.30 mg, 0.60 mmol) and N, N-diiso-pyridine (EDCI, and EDCI, respectively) were added under argon protectionPropylethylamine (DIPEA, 116.32 mg, 0.90 mmol), after addition was activated at 0 ℃ for 30 min. Compound 12 (358 mg, 0.10 mmol) was dissolved in 5mL of anhydrous dichloromethane, and after dissolution, the solution was slowly added dropwise to the above activating solution, after completion of the dropwise addition, the solution was allowed to stand at room temperature and stirred for 24 hours. TLC monitored the reaction was complete, concentrated under reduced pressure to remove the reaction solvent, the residue was dissolved in 20 mL of dichloromethane, washed with aqueous solution (30 mL × 2) and saturated sodium chloride solution (25 mL × 2), respectively, the dichloromethane layers were collected and combined, the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (dichloromethane: methanol =10: 1) to give 239 mg of a white solid in 64.21% yield.¹H-NMR (400 MHz, DMSO-d ₆, ppm)δ:1.31-1.34 (m, 2H), 1.42-1.51 (m, 3H), 1.59-1.61 (m, 1H), 2.19 (t,J= 4.8 Hz,2H), 2.57 (d,J= 8.4 Hz, 1H),2.65 (s, 3H), 2.81 (dd,J ₁= 8.4 Hz,J ₂= 2.8Hz, 1H), 3.09 (d,J= 2.8 Hz, 1H), 3.44-3.49 (m, 2H), 3.63-3.71 (m, 296H),3.81-3.89 (m, 4H), 4.14 (s, 1H), 4.31 (s, 1H), 4.50-4.53 (m, 2H), 6.40 (s,1H), 6.50 (s, 1H), 8.01 (d,J= 8.0 Hz, 2H), 8.14 (d,J= 8.0 Hz, 2H)。

EXAMPLE 11 preparation of Compound I

Compound 13 (43 mg, 0.01 mmol) was dissolved in 1.9 mL of a mixed solvent (1 mL of anhydrous dichloromethane +0.9 mL of anhydrous tetrahydrofuran) and replaced with argon three times. Compound 10 (45 mg, 0.06 mmol) was dissolved in 1 mL of anhydrous dichloromethane, replaced with argon three times, added dropwise to the mixed solution of compound 13, and one drop of anhydrous formic acid was added to the reaction system. After the addition, the reaction is carried out for 72 hours at a constant temperature of 20 ℃. The reaction was monitored by TLC, the reaction solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel flash column chromatography (petroleum ether: acetone: methanol =7:2: 1) to give 32 mg of a pale yellow oily product in 62.25% yield. HRMScalcaulated for C₂₁₁H₃₉₃N₅O₈₇S [M + H]⁺4421.5346, found 4409.4862。

EXAMPLE 12 preparation of Compound II (ligand Material b)

DSPE-PEG2000-Mal (1 mmol) and Cys-R8 (1.5 mmol) were dissolved in a mixed solvent of chloroform and methanol (v/v =2: 1), and then a few drops of triethylamine were added to the reaction solution and stirred at room temperature for 24h with exclusion of light. After the DSPE-PEG2000-Mal reaction is completed through TLC monitoring, the solvent in the reaction system is removed through decompression and concentration, the residue is redissolved by chloroform and filtered, and the filtrate is dried by spinning to obtain the compound II. ESI-MS calculated 4295, found 4312 (and literature)BiomaterialsResults are consistent for 2014, 35, 4835-4847).

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: soybean lecithin: ligand =27: 65: 8, the mass ratio of the medicine lipid is lipid: paclitaxel =30:1, hydration was phosphate buffered saline (PBS, 0.01M) pH 7.4. We prepared 4 paclitaxel-loaded liposomes using the above formula: PTX-Lip, PTX-R8-Lip, PTX-PEG-Lip, PTX-R8-PEG-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 30min, 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 and measurement of particle size and potential of liposomes

According to literature reports, the application adopts a freezing centrifugation method to separate the unencapsulated free paclitaxel from the paclitaxel loaded liposome. PTX-Lip, PTX-R8-Lip, PTX-PEG-Lip and PTX-R8-PEG-Lip were prepared as described in example 13, respectively. And (3) centrifuging part of the paclitaxel-loaded liposome solution for 20 minutes at 10000 rpm under the condition of 4 ℃, and obtaining supernate, namely the liposome without free paclitaxel. Taking 30 mu L of centrifuged supernatant and liposome sample before centrifugation respectively, adding 270 mu L of methanol, performing vortex shaking for 10 minutes to completely break the emulsion, centrifuging at 10000 rpm for 10 minutes again, taking the supernatant, injecting the supernatant into a high performance liquid chromatograph for analysis, and calculating the encapsulation efficiency (EE%) of the paclitaxel liposome according to a formula: EE% = post-a centrifugation/pre-a centrifugation × 100%, where post-a centrifugation and pre-a centrifugation refer to peak areas of liposome samples after centrifugation and before centrifugation, respectively. In addition, the particle size and Zeta potential of the 4 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 ligand-modified paclitaxel loaded liposomes (n =3, mean + -SD)

The results show that the 4 paclitaxel-loaded liposomes have good encapsulation efficiency which is more than 85 percent. The particle size is about 110 nm, the dispersion index (PDI) is about 0.1, and the liposome is uniformly distributed; the Zeta potential shows that R8-Lip is electropositive, the rest groups of liposomes are electronegative, and PTX-R8-PEG-Lip shows electronegativity (about 10 mV) under neutral condition and electropositive under weak acid environment, which shows that the liposome material of the invention is stable under physiological environment, can show weak acid response at tumor parts, and the hydrazone bond is broken to expose R8, thereby mediating the liposome to enter cells strongly.

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, 2h, 4h, 6 h, 8 h, 12 h, 24h 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 90% after the liposomes are incubated with fetal calf serum for 48 hours, no obvious aggregation phenomenon exists, and the prepared liposomes have good serum stability and lay the foundation for later in vitro and in vivo experiments. Meanwhile, the light transmittance of R8-Lip is obviously lower than that of other groups of liposomes due to the electropositivity of R8, and the light transmittance of PTX-R8-PEG-Lip is obviously improved compared with that of R8-Lip, so that the lipid material disclosed by the invention has improved in-vivo medication safety when the R8 is used for positively penetrating tumors.

Example 16 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. In order to firstly verify the breast cancer targeting of each group of liposomes from the in vitro cell level and test the uptake condition of the liposomes under different pH values, the murine breast cancer 4T1 and the human breast cancer MCF7 cells are treated in a 3X 10 mode⁵The density of each hole is inoculated in a 12-hole plate, after 24 hours of culture, various liposomes marked by CFPE are respectively added into a cell plate, and the cells are diluted by serum-free culture medium and then added into the hole plate, so that the final concentration of the CFPE in the cell hole plate is 2 mu g/ml. After 2h incubation at 37 ° C, pH ═ 7.4/6.0, drug-containing medium was discarded and washed 2 times with pre-cooled PBS, cells were collected by digestion, centrifuged at 4 ℃ (2000 rpm × 3 min), supernatant was discarded and cells were washed three times with ice PBS, cells were resuspended in PBS, fluorescence intensity of both cells was measured by flow cytometry,the results are shown in FIG. 2.

While quantitatively detecting the liposome entering the cells, the uptake of each group of liposomes in 4T1 and MCF7 cells is directly observed from a qualitative angle by using a confocal laser scanning fluorescence microscope. The specific operation is as follows: 4T1, MCF7 cells at 5X 10⁵The density of each well was inoculated into 6-well plates pre-coated with glass slides at 37 ℃ with 5% CO₂Culturing for 24h under the condition. The CFPE-labeled liposomes were added separately to the cell plates so that the final concentration of CFPE in the cell-well plate was 2 μ g/ml. Incubating at 37 deg.C C, pH ═ 7.4/6.0 for 2h, discarding the drug-containing medium, washing with cold PBS three times (5 min each time), adding 4% paraformaldehyde, fixing at room temperature for 30min, discarding paraformaldehyde, and washing with PBS 3 times (5 min each time). And (3) staining cell nuclei for 5min by using 5 mu g/mL DAPI, discarding the dye, washing by using PBS for 3 times, sealing by using glycerol, inversely placing cover slips on glass slides, and observing and imaging under a laser confocal microscope after sealing. The results are shown in FIGS. 3A and 3B.

The qualitative uptake result of confocal shooting is consistent with the quantitative uptake result of a flow cytometer, and data shows that under the physiological condition of a human body (pH = 7.4), the liposome modified by the intelligent ligand material a designed by the invention is improved by about one time compared with a blank liposome, which indicates that the biotin ligand designed by the invention can target breast cancer and improve the targeting property. Meanwhile, compared with blank liposomes, the uptake of the liposome prepared by jointly using the intelligent ligand materials a and b is improved, and is greatly reduced compared with the uptake of the liposome modified by the single ligand material b, so that the liposome modified by the intelligent ligand material a and b has significant difference; under the weakly acidic condition, because the acid-sensitive hydrazone bond is broken to expose sufficient R8 to mediate the liposome to enter the cell, and the uptake strength of the liposome modified by the single ligand material b is almost the same, the design success of the invention is demonstrated, the electropositivity of R8 can be shielded under the physiological condition, the biotin ligand is used for specifically targeting breast cancer, after the biotin ligand reaches the tumor environment, because of the weak acidity of the environment, the acid-sensitive hydrazone bond is broken to expose sufficient R8 to mediate the cell to penetrate the membrane, so that more liposomes can enter the breast cancer cell, and the stronger drug effect is exerted.

Claims

1. A biotin-modified novel breast cancer targeted intelligent liposome ligand material a has a structure shown in a general formula I or a pharmaceutically acceptable salt or hydrate thereof:

wherein: the molecular weight of the PEG used is equal to, but not limited to, 2000, 3350, 5000; x is an acid sensitive bond and is not limited to acylhydrazone and phenylboronic acid ester bonds.

2. The structure of the biotin-modified novel breast cancer-targeted intelligent liposome material I as claimed in claim 1 is characterized in that: amino caproic acid is used as a bridge chain, one end of the amino caproic acid is connected with a polyethylene glycol-biotin compound with an acid sensitivity function, and the other end of the amino caproic acid is connected with cholesterol.

3. The acid-labile polyethylene glycol-biotin complex of claim 2, which is structurally characterized in that: polyethylene glycol is used as basic skeleton, one end is connected with biotin, and the other end is connected with a fragment containing acid-sensitive bond (such as hydrazone bond).

4. The structure of the novel breast cancer targeted intelligent liposome material I as claimed in claim 1, wherein the synthetic route is characterized in that: polyethylene glycol and biotin compound part with acid-sensitive function, coupling with biotin and p-acetylbenzoic acid respectively through condensation reaction by polyethylene glycol, and then reacting acetyl with hydrazide to form acylhydrazone bond; the cholesterol part is prepared by using cholesterol as an initial raw material, firstly extending by using triethylene glycol, and then coupling with fluorenylmethoxycarbonyl-6-aminocaproic acid through a condensation reaction.

5. The novel breast cancer targeted intelligent liposome material as claimed in claim 1, which is used as a drug carrier in the preparation of breast cancer targeted drugs.

6. The breast cancer targeted liposome prepared from the novel breast cancer targeted intelligent liposome material according to claim 1, which comprises a membrane material and an active agent, wherein the membrane material is a phospholipid bilayer and consists of lecithin, cholesterol and a liposome ligand, and the ratio of the components is as follows: the molar ratio of cholesterol to phospholipid is 1:2, the molar content of the liposome ligand is 0.8-10% of the total molar number of the cholesterol and the phospholipid; the active agent of the invention adopts a therapeutic agent or a developing 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 percent of the total lipid by weight percent; the hydration solution was 0.01M Phosphate Buffered Saline (PBS) at pH 7.4.

7. The breast cancer targeted liposome prepared from the novel breast cancer targeted intelligent liposome material according to claim 1, which is characterized in that the breast cancer targeted liposome with stable particle size and Zeta potential can be prepared by adopting a film method according to the component proportion relation, the particle size of the liposome is about 110 nm, and the entrapment rate is more than 85%.

8. The breast cancer targeted liposome made of the novel breast cancer targeted intelligent liposome material according to claim 1, wherein the phospholipid is lecithin.

9. The breast cancer targeted liposome prepared from the novel breast cancer targeted intelligent liposome material as claimed in claim 1, wherein the active agent is paclitaxel as the therapeutic agent, and CFPE as the developing agent.