CN113398276B - Preparation and application of brain glioma targeted berberine and folic acid modified lipid material - Google Patents
Preparation and application of brain glioma targeted berberine and folic acid modified lipid material Download PDFInfo
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Abstract
The invention discloses preparation and application of a brain glioma targeted berberine and folic acid modified lipid material, which can be used for preparing a brain glioma targeted Tween80 coated berberine and folic acid co-modified liposome. The surface of the liposome is coated with Tween80, and the liposome can effectively pass through a blood brain barrier through receptor-mediated endocytosis after being combined with a low-density lipoprotein receptor, so that the medicament is delivered to the brain; in addition, the liposome is modified by folic acid and berberine to improve the brain glioma targeting and mitochondrial targeting capabilities of the liposome, the long-chain polyethylene glycol is introduced into the stable liposome to realize long circulation in vivo, and intelligently trigger the hydrolytic cleavage of an acid-sensitive hydrazone bond under the pH of an acidic tumor cell organelle (endosome and lysosome), so that the problem of multidrug resistance (MDR) of tumors is solved.
Description
Technical Field
The invention relates to preparation of a brain glioma targeted berberine and folic acid modified lipid material and application thereof in a drug delivery system, which has the functions of prolonging in vivo circulation and targeted drug delivery of brain glioma, can realize mitochondrial targeted elimination of multidrug resistance (MDR), and is an intelligent liposome capable of breaking bonds in tumor cells. The invention comprises the preparation of the material and the application of the material as a drug carrier in drug delivery, belonging to the fields of medical technology and chemical synthesis.
Background
The incidence of brain tumor in China is gradually increased, and the brain tumor has the characteristics of rapid development, poor prognosis and easy recurrence, seriously harms human life, particularly brain glioma. Brain glioma (glioma) accounts for approximately 51% of all intracranial tumors, and is one of the most common primary malignancies of the Central Nervous System (CNS). The 2-year overall survival rate is only 3% -5%, the median survival time is about 14 months, and the high malignancy, recurrence rate and disability rate of the traditional Chinese medicine are great threats to human health. The treatment measures usually applied to brain glioma mainly include surgical treatment, radiotherapy and chemotherapy. Because brain glioma is high in malignancy degree and grows infiltratively, and is difficult to distinguish from the boundary of normal brain tissue, the operation is not easy to completely cut off, the technical requirement of the brain operation is high, the wound is serious, and the brain operation is easy to infect, the radiotherapy of a patient after the operation has a certain curative effect, but the central nerve can not be eradicated and is easy to hurt at the same time; chemotherapy has proven to be a crucial link in many tumor treatments, and the success or failure of chemotherapy has a great influence on the quality of life and prognosis of patients. However, chemotherapy has no tissue and organ specificity, so the drugs are distributed throughout the body, and due to the special anatomical structure of the brain, the brain has low drug concentration, poor curative effect and great toxic and side effects. The key to the therapeutic effect of brain tumor chemotherapy is that the chemotherapeutic drug can penetrate the blood-brain barrier (BBB) and selectively transport to the tumor site. The blood brain barrier, which serves as a regulatory interface between the blood and the Central Nervous System (CNS), plays a crucial role in maintaining the environment within the CNS constant. The Blood Brain Barrier (BBB) composed of brain capillary endothelial cells, astrocytes and basement membranes among the brain capillary endothelial cells and astrocytes protects the central nervous system, and simultaneously limits a plurality of substances from entering the brain from blood, almost all macromolecules and 98% micromolecule drugs can not enter the central nervous system through the blood brain barrier, so that the drugs effective to the central nervous system are difficult to enter focus parts and reach effective treatment concentration, and therefore the expected treatment effect can not be achieved.
The nano-carrier is widely applied to chemotherapy administration, and shows great clinical potential in the aspects of targeted drug delivery of brain glioma and treatment of glioma. Based on nano-carriers including polymer NPs, micelles, liposomes, protein nanocages, inorganic NPs and the like, the mature endogenous blood brain barrier transport route is utilized to transport materials into the brain, the method is widely reported to be used for treating brain glioma, and a 'Trojan horse' method based on nano-carriers is a promising non-invasive strategy and is used for penetrating the blood brain barrier to improve the treatment effect. It includes carrier-mediated endocytosis transport (CMT), receptor-mediated endocytosis transport (RMT), adsorption-mediated cell transport (AMT) and cell-mediated. Low-density lipoprotein (LDL) receptor family (LDLRs) are highly expressed in the blood-brain barrier and have high affinity for apolipoprotein B (ApoB) and lipoprotein E (ApoE) fragments, and polypeptides such as angiopep-2 (ANG, TFFYGGSRGKRNNFKTEEY). Tween80 (Tween 80, T-80), a nonionic water-soluble surfactant. Numerous studies have shown that tween 80-covered nanoparticles are able to carry drugs across the blood-brain barrier after administration, thereby delivering the drugs to the brain. The main mechanism is believed to be that apolipoproteins (such as apolipoprotein e (apoe) or apolipoprotein b (apob)) in plasma are adsorbed on Tween80, and then nanocarriers are transported to the brain by low-density lipoprotein (LDL) receptor-mediated endocytosis.
The Folate Receptor (FR) is a 38kDa glycosylphosphatidylinositol glycoprotein, one of the most studied targets in cancer therapy, and is significantly, and in some cases by two orders of magnitude, upregulated in the number of folate receptors in many cancer cells compared to normal tissues. In addition, normal cells can transport less folate across their membranes, but will not transport any type of folate conjugate. Whereas malignant cells can transport folate conjugates through folate receptors. Folic Acid (FA), a low-molecular vitamin, is an important precursor material in the synthesis of purines and pyrimidines, has high affinity with folate receptors, and, in addition, studies have shown that FR is overexpressed in ovarian cancer, lung cancer, brain cancer, head and neck cancer, renal cell carcinoma and breast cancer. The folic acid ligand is not only cheap, nontoxic and non-immunogenic, but also widely applied because of easy combination with a carrier and high affinity maintenance, can be combined with a folic acid receptor over-expressed by a blood brain barrier, and promotes the penetration of a medicament to the blood brain barrier through the receptor-mediated endocytosis. Furthermore, overexpression of FR on gliomas also makes it a good candidate for tumor targeting.
Conventional drug therapy may induce multidrug resistance (MDR) during brain glioma, resulting in systemic distribution of chemotherapeutic drugs with less drug remaining in tumor tissue, which in turn damages normal tissues, causing serious side effects. Mitochondria play a central regulation role in the apoptosis and anti-apoptosis process of tumor, and are ideal anti-tumor targets. Mitochondrially-directed intelligent liposome delivery is considered to be a promising strategy to eliminate multidrug resistance (MDR). Berberine (BBR) is an isoquinoline alkaloid and has a variety of significant pharmacological effects, including antibacterial, antiviral, antidiabetic, antiinflammatory and anticancer. Notably, the BBR can selectively accumulate in the mitochondria of cancer cells, which is associated with the lipophilic structure and delocalized positive charge of the BBR. Furthermore, the action of BBR on mitochondria can also induce apoptosis in cancer cells, which is thought to be associated with BBR's ability to eliminate mitochondrial membrane potential, up-regulate ROS levels, and induce changes in mitochondrial membrane permeability. However, positively charged nanoparticles generally exhibit poor plasma stability, can be rapidly cleared from blood circulation, and can cause severe cytotoxicity.
Disclosure of Invention
The purpose of the research is to provide treatment of liposome user brain glioma co-modified by berberine coated with tween80 and folic acid.
The liposome is coated with Tween80, and can effectively pass through a blood brain barrier through receptor-mediated endocytosis after being combined with a low-density lipoprotein receptor, so that the drug is delivered to the brain, and the brain chemotherapy drug concentration is increased. On the basis, the liposome is modified by folic acid to improve the brain glioma targeting of the liposome, and by utilizing the strong specific combination of the folic acid and brain glioma cells, the brain tumor targeting is stronger, the concentration of the drug in tumor tissues is further increased, a more efficient anti-brain glioma effect is exerted, the distribution of the drug in peripheral organs is reduced, and the toxic and side effects of the drug are reduced. The introduction of the long polyethylene glycol chain stabilizes the liposome, realizes the in vivo long circulation of the liposome, intelligently triggers the hydrolytic breakdown of an acid-sensitive hydrazone bond under the pH value of an acid tumor cell organelle (endosome and lysosome), exposes berberine, utilizes positive charges to combine with tumor mitochondria with high negative potential, realizes the targeting of the mitochondria and eliminates the problem of multidrug resistance (MDR) of the tumor. The material can be used for preparing a multifunctional intelligent liposome with extremely high potential and multiple targeting capability, can provide a new idea and method for targeted therapy of brain glioma, and has wide application prospect.
The invention provides a structure shown in a general formula (I) or pharmaceutically acceptable salt or hydrate thereof, and a lipid material (I) is characterized in that: an acid sensitive bond is taken as a connecting group, one end of the acid sensitive bond is connected with polyethylene glycol (PEG) and folic acid with brain glioma targeting, and the other end of the acid sensitive bond is connected with cholesterol-polyethylene glycol conjugate:
wherein, the molecular weight of the PEG is equal to but not limited to 200, 400, 600, 800, 1000, 1500, 2000, 3350, 4000 and the like; the polyglycols used are equal to, but not limited to, triethylene glycol, tetraethylene glycol, hexaethylene glycol, octaethylene glycol, etc., i.e. n =3, 4, 6, 8; the acid sensitive bond used is equivalent to, but not limited to, a hydrazone bond, an imine bond, an oxime bond, a hemiacetal (ketone), etc.
The specific preparation method of the compound shown in the general formula (I) takes FA-PEG3350-Hz-Chol as an example:
the invention provides a structure shown in a general formula (II) or a pharmaceutically acceptable salt or hydrate thereof, and a lipid material (II) is characterized in that: the preparation method comprises the following steps of (1) taking polyglycols as a framework, connecting cholesterol at one end and berberine with a mitochondrion targeting function at the other end:
wherein, the polyethylene glycol is equal to but not only triethylene glycol, tetraethylene glycol, hexaethylene glycol, octaethylene glycol and the like, i.e. a \ b =3, 4, 6, 8.
The specific preparation method of the compound shown in the general formula (II) takes BBR-Chol as an example:
the novel lipid material can be used as a ligand for preparing a brain glioma targeted liposome.
The liposome is characterized by comprising phospholipid, cholesterol, Tween80, FA-PEG3350-Hz-Chol, BBR-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 soybean phospholipid, 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: 1-5, and the molar content of the liposome ligand is 1-20% 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 preparation method of the brain glioma targeted liposome comprises the following steps:
dissolving phospholipid, cholesterol and paclitaxel in chloroform/methanol mixed solvent (v/v = 2:1), and rotary evaporating at 37 deg.C under vacuum to remove organic solvent to form complete lipid membrane layer.
And (II) vacuum storage overnight, and residual solvent removal.
And (III) adding Tween80, hydrating in a buffer solution at 20 ℃ for 0.5 h, carrying out intermittent ultrasound at 80W by using a probe type ultrasonic instrument, and carrying out ultrasound for 80 s in an ice water bath to obtain a microemulsion liposome solution.
Detailed Description
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
Compound 1 (19.88 g, 51.40 mmol) was placed in a reaction flask, argon gas was substituted for 3 times, and dissolved in 65 mL of a mixed solvent (52 mL of anhydrous dichloromethane +13 mL of anhydrous pyridine), succinic anhydride (3.30 g, 33.60 mmol), DMAP (632.0 mg, 5.170 mmol) were added in this order with stirring at room temperature, after completion of the reaction, the reaction was stirred at room temperature for 16 hours, TLC (thin layer chromatography) monitored for completion of the reaction of the raw materials, the reaction was stopped, 15mL of water was added and stirred for 2 hours, the reaction solution was supplemented with 200mL of dichloromethane, and washed with 1N HCl (150 mL × 2) and saturated sodium chloride solution (100 mL × 1) in this order, 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: dichloromethane =3: 1) to obtain 12.30g of a white solid powder with a yield of 49.15%.1H NMR (400 MHz, Chloroform-d) δ 5.41 – 5.34 (m, 1H), 4.63 (dtd, J = 11.6, 8.4, 4.1 Hz, 1H), 2.72 – 2.65 (m, 2H), 2.60 (dd, J = 10.2, 4.2 Hz, 2H), 2.32 (d, J = 8.1 Hz, 2H), 0.80-2.20 (remaining cholesterol protons)，0.68 (s, 3H)。
Example 2
Preparation of Compound 3
Compound 2 (21.00 g, 2.055 mmol) was placed in a reaction flask and replaced with argon 3 times to be dissolved in 20mL of anhydrous dichloromethane, dicyclohexylcarbodiimide (DCC, 0.635 g, 3.083 mmol) and 4-dimethylaminopyridine (DMAP, 0.076g, 0.620 mmol) were added successively under the protection of argon, and activation was carried out at 0 ℃ for 30 min. Tetraethyleneglycol (1.996 g, 10.275 mmol) was dissolved in 3mL of anhydrous dichloromethane,and slowly dripping the activated reaction solution under the protection of argon, and moving to room temperature and stirring for 2 hours after the addition is finished. TLC, the reaction was completed, insoluble matter was removed by filtration, the filtrate was collected and concentrated under reduced pressure to remove the reaction solvent, the residue was dissolved in 10mL of dichloromethane, the reaction solution was washed with 1N HCl (20 mL × 2), water (10 mL × 1) and saturated sodium chloride solution (10 mL × 1) in this order, the organic layer was dried over anhydrous sodium sulfate, and then the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane: methanol =100: 1) to obtain 762.1 mg of a transparent colorless oily product with a yield of 55.93%.1H NMR (400 MHz, Chloroform-d) δ 5.36 (m, 1H), 4.61 (qd, J = 10.0, 8.1, 4.1 Hz, 1H), 4.30 – 4.23 (m, 2H), 3.75 – 3.64 (m, 12H), 3.61 (dd, J = 5.2, 3.7 Hz, 2H), 2.70 – 2.55 (m, 4H), 0.70-2.40 (remaining cholesterol protons), 0.67 (s, 3H)。
Example 3
Preparation of Compound 4
The compound monobenzyl succinate (315.63 mg, 1.52 mmol) was dissolved in 8 mL of anhydrous dichloromethane, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 368.4 mg, 1.92 mmol), 4-dimethylaminopyridine (DMAP, 234.79 mg, 1.92 mmol) and N, N-diisopropylethylamine (DIPEA, 300.4 mg, 2.32 mmol) were added successively under argon protection and activated at 0 ℃ for 30 min. Compound 3 (670.0 mg, 1.01 mmol) was dissolved in 8 mL of anhydrous dichloromethane, and the solution was transferred to the above reaction solution after dissolution, and the solution was stirred at room temperature for 6 hours. The reaction was stopped by TLC monitoring the reaction was complete, the reaction solution was washed with 1N HCl (20 mL × 2), saturated aqueous sodium bicarbonate (20 mL × 1), and saturated aqueous sodium chloride (10 mL × 1) in this order, the organic layer was collected, 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: ethyl acetate =4: 1) to give 750 mg of a transparent golden yellow oily product in 87.04% yield.1H NMR (400 MHz, Chloroform-d) δ 7.40 – 7.30 (m, 5H), 5.36 (m, 1H), 5.13 (s, 2H), 4.69 – 4.55 (m, 1H), 4.28 – 4.20 (m, 4H), 4.12 (q, J = 7.1 Hz, 2H), 3.72 – 3.61 (m, 12H), 2.68 (s, 4H), 2.66 – 2.58 (m, 4H), 0.70-2.40 (remaining cholesterol protons), 0.67 (s, 3H)。
Example 4
Preparation of Compound 5
Compound 4 (700.0 mg, 0.82 mmol) was dissolved in 2.5 mL of a mixed solvent (0.5 mL of tetrahydrofuran +2 mL of methanol), 100 mg of palladium on carbon was added, the mixture was replaced with hydrogen gas 3 times, and the mixture was stirred at room temperature for 3 hours. TLC to monitor the reaction was complete, the reaction solution was filtered through celite, celite was washed 2-3 times with a small amount of the above mixed solvent, the filtrate was collected and concentrated under reduced pressure to remove the solvent to give 620.5 mg of a colorless oil in 99.12% yield. The product was carried on to the next step without purification.1H NMR (400 MHz, Chloroform-d) δ 5.36 (dt, J = 3.3, 1.6 Hz, 1H), 4.62 (dtt, J = 11.4, 8.0, 4.1 Hz, 1H), 4.31 – 4.21 (m, 4H), 3.73 – 3.62 (m, 12H), 2.72 – 2.56 (m, 8H), 0.70-2.40 (remaining cholesterol protons), 0.67 (s, 3H)。
Example 5
Preparation of Compound 6
Compound 5 (4.936 g, 6.47 mmol) was weighed into a reaction flask, replaced with argon three times, dissolved in 50 mL of dry dichloromethane, stirred at-5 deg.C, isobutyl chloroformate (IBCF, 1.325 g, 9.70 mmol) and N-methylmorpholine (NMM, 1.309 g, 12.94 mmol) were added sequentially under argon, and after addition activation continued for 30 min. The compound tert-butyl carbazate (1.282 g, 9.70 mmol) was dissolved in 5.0 mL of anhydrous dichloromethane and added to the above reaction solution, which was allowed to react at room temperature for 2 h, 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 (dichloromethane: methanol =4: 1) to obtain 3.271 g of a white foamy solid in a yield of 57.34%.1H NMR (400 MHz, Chloroform-d) δ 5.40 – 5.32 (m, 1H), 4.61 (dtd, J = 11.4, 8.5, 7.9, 4.0 Hz, 1H), 4.25 (t, J = 4.8 Hz, 4H), 3.75 – 3.60 (m, 12H), 2.73 (t, J = 6.6 Hz, 2H), 2.62 (dq, J = 11.9, 6.2 Hz, 4H), 2.52 (t, J = 6.7 Hz, 2H), 0.70-2.40 (remaining cholesterol protons), 1.46 (s, 9H) 0.67 (s, 3H)。
Example 6
Preparation of Compound 7
Compound 6 (100 mg, 0.11 mmol) was dissolved in 10mL of dichloromethane at room temperatureTrifluoroacetic acid (2.0 mL, 20% V/V) was added and the reaction was carried out at room temperature for 15 minutes. The reaction was monitored by TLC for completion, and an appropriate amount of saturated sodium bicarbonate solution was added to the reaction solution to adjust the solution pH =10, extracted with dichloromethane (10 mL × 2), the dichloromethane layer was collected and dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure at 30 ℃ to give 81 mg of a light brown transparent oily product with a yield of 91.76%. The product was carried on to the next step without purification.1H NMR (400 MHz, Chloroform-d) δ 7.45 (s, 1H), 5.35 (m, 1H), 4.60 (dtd, J = 11.6, 8.4, 4.2 Hz, 1H), 4.24 (td, J = 4.9, 2.0 Hz, 4H), 3.67 (d, J = 14.2 Hz, 12H), 2.71 (t, J = 6.7 Hz, 2H), 2.62 (dq, J = 11.6, 6.1 Hz, 4H), 2.44 (t, J = 6.7 Hz, 2H), 0.70-2.40 (remaining cholesterol protons), 0.67 (s, 3H)。
Example 7
Preparation of Compound 9
Compound 8 (500.0 mg, 3.05 mmol) was placed in a reaction flask, replaced with argon 3 times, dissolved in a mixed solvent (12 mL dry dichloromethane +2 mL N, N-dimethylformamide), then moved to-5 ℃ and stirred, dicyclohexylcarbodiimide (DCC, 1254.9 mg, 6.09 mmol), 4-dimethylaminopyridine (DMAP, 74.5 mg, 0.61 mmol) were added under argon protection, and activation continued at this temperature for 30 min. Dissolving polyethylene glycol (PEG 3350, 10.216 g, 3.05 mmol) in 45 mL of anhydrous dichloromethane, dissolving, slowly dropwise adding the activated solution into the polyethylene glycol solution, and moving to room temperature for reaction for 16 h after dropwise adding. TLC monitored the reaction completion, and the insoluble matter was removed by filtration, and the collected filtrate was washed with aqueous solution (100 mL. times.3), 1N HCl (100 mL. times.1), and saturated sodium chloride solution (50 mL. times.1), respectively, and the dichloromethane layer was collected, and after the organic layer was dried over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure, and the dichloromethane/ether was recrystallized to obtain 8.920 g of a white solid with a yield of 83.65%.1H NMR (400 MHz, Chloroform-d) δ 8.12 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 14.2 Hz, 12H), 4.51 – 4.45 (m, 2H), 3.81 (m, 5H), 3.62 (s, 350H), 3.44 (dd, J = 5.9, 4.0 Hz, 2H), 2.63 (s, 3H)。
Example 8
Preparation of Compound 10
Folic acid (900.0 mg, 2.04 mmol) was placed in a reaction flask, argon gas was substituted for 3 times in the dark, 30 mL of DMSO was dissolved, then the solution was transferred to-5 ℃ and stirred, dicyclohexylcarbodiimide (DCC, 700.5 mg, 3.40 mmol) and 4-dimethylaminopyridine (DMAP, 41.5 mg, 0.34 mmol) were added under the protection of argon gas, and activation in the dark was continued for 4 h at this temperature. Compound 9 (2.378 g, 0.68 mmol) was dissolved in 10mL of anhydrous dichloromethane, and after completion of the dissolution, the solution was added dropwise to the activated solution, and the solution was allowed to react at room temperature for 36 hours. After the reaction, the insoluble matter was removed by filtration and the filtrate was collected, 200mL of methylene chloride was added to the filtrate, and the mixture was washed with an aqueous solution (100 mL. times.3) and a saturated sodium chloride solution (50 mL. times.1), respectively, and the methylene chloride layer was collected, and after the organic layer was dried over anhydrous sodium sulfate, the solvent was removed by concentration under reduced pressure, and methylene chloride/diethyl ether was recrystallized to obtain 2.122 g of a yellow solid powder with a yield of 79.62%.1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 8.03 (s, 4H), 7.36 (d, J = 8.7 Hz, 2H), 6.56 (d, J = 8.7 Hz, 2H), 4.71 (d, J = 7.8 Hz, 1H), 4.47 (s, 2H), 4.40 – 4.32 (m, 2H), 3.45 (s, 280H), 2.58 (s, 3H), 2.20 (dd, J = 12.8, 8.6 Hz, 2H), 2.02 (m, 2H)。
Example 9
Preparation of compound FA-PEG3350-Hz-Chol
Compound 7 (50.0 mg, 0.064 mmol) was dissolved in 1 mL of anhydrous dichloromethane and replaced three times with argon. Compound 10 (126.0 mg, 0.032 mmol) was dissolved in 0.5 mL of anhydrous dichloromethane, replaced with argon three times, and then added dropwise to the mixed solution of compound 7, and one drop of anhydrous formic acid was added to the reaction system. After the addition, the reaction was carried out at room temperature for 48 hours under dark conditions. TLC monitoring reaction completion, decompression concentrating to remove reaction solvent, and recrystallizing with dichloromethane/diethyl ether to obtain yellow solid powder102.5 mg, yield 68.47%.1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.98 – 7.88 (m, 2H), 7.41 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 8.7 Hz, 2H), 5.61 (d, J = 8.0 Hz, 1H), 5.32 (s, 1H), 4.75 (dd, J = 8.2, 3.7 Hz, 1H), 4.58 – 4.38 (m, 8H), 4.10 (q, J = 4.6 Hz, 8H), 3.49 (s, 252H), 2.53 (s, 3H), 2.35 (t, J = 6.9 Hz, 2H), 2.25 (q, J = 10.4, 7.3 Hz, 6H), 2.06 (d, J = 5.5 Hz, 2H), 0.70-2.0 (remaining cholesterol protons), 0.64 (s, 3H)。
Example 10
Preparation of Compound 11
Cholesterol 1 (10.00 g, 25.86 mmol) was dissolved in a mixed solvent (10 mL of anhydrous pyridine +10 mL of anhydrous chloroform), the solution was brought to 0 ℃ and stirred, and a pyridine solution (10 mL) of p-toluenesulfonyl chloride (TsCl, 7.89 g, 41.38 mmol) was slowly added dropwise at 0 ℃. After the dropwise addition, the reaction solution was reacted at 0 ℃ for 6 hours. TCL monitored completion of the reaction of the starting materials, the solvent was distilled off under reduced pressure, the residue was dissolved in chloroform (100 mL), and washed with dilute hydrochloric acid (1N, 100 mL. times.2) and water (100 mL. times.1) in this order with a saturated aqueous solution of sodium chloride (50 mL. times.1), dried over anhydrous sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and chloroform/methanol was recrystallized to give a white solid powder in a yield of 87.43%, 12.23 g. Directly carrying out the next reaction. Mp: 129 ℃ and 132 ℃ (Mp: 130 ℃ and 132 ℃).
Example 11
Preparation of Compound 12
Compound 11 (5.000 g, 9.24 mmol) was dissolved in 30 mL dioxane, triethylene glycol (TEG, 6.948 g, 46.26 mmol) was added, reflux was performed for 6 hours, and the completion of the starting reaction was monitored by TLC. The solvent was removed under reduced pressure, the residue was dissolved in 50 mL of dichloromethane, washed with a saturated aqueous sodium bicarbonate solution (50 mL × 2), a saturated aqueous sodium chloride solution (50 mL × 1), 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/ethyl acetate = 5/1) to give 3.066 g of a colorless oil with a yield of 63.91%.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 (remaining cholesterol 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 12
Preparation of Compound 13
The compound 4- (tert-butoxycarbonyl) benzoic acid (641.8 mg, 2.89 mmol) was added with 10ml of anhydrous dichloromethane under argon protection with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 831.4 mg, 4.337 mmol), 4-dimethylaminopyridine (DMAP, 529.9 mg, 4.337 mmol) and N, N-diisopropylethylamine (DIPEA, 747.4 mg, 5.78 mmol), respectively, and after completion of the addition, activated at 0 ℃ for 30 min. Compound 12 (1.0 g, 1.93 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 dropwise addition, the solution was allowed to stand at room temperature and stirred for 5 hours. TLC monitored the reaction was complete, the reaction solvent was removed by concentration under reduced pressure, the residue was dissolved in 20mL of dichloromethane, washed with 1mol/L aqueous hydrochloric acid (30 mL × 2) and saturated sodium chloride solution (20 mL), the dichloromethane layer was collected and 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 = 10/1) to give 1168.4 mg of pale yellow oil in 83.82% yield.
Example 13
Preparation of Compound 14
Compound 13 (2.0 g, 2.77 mmol) was dissolved in 1 mL of dichloromethane and a solution of triethylsilane (0.804 g, 6.92 mmol) dissolved in 1 mL of dichloromethane and 10mL of trifluoroacetic acid were added dropwise in succession in an ice-water bath and reacted at room temperature for 2 hours. TLC monitored the reaction completion, concentrated under reduced pressure to remove the reaction solvent, the residue was dissolved in 20mL of dichloromethane and washed successively with water (20 mL. times.1) and saturated aqueous sodium chloride (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was freed of the solvent under reduced pressure to give 1.74g of a clear oil which was used directly in the next reaction in 94.18% yield.
Example 14
Preparation of Compound 15
Compound 14 (1.0 g, 1.50 mmol) was placed in a reaction flask, replaced with argon 3 times, dissolved in 15mL of anhydrous dichloromethane and moved to-5 ℃ with stirring, dicyclohexylcarbodiimide (DCC, 618.0 mg, 3.00 mmol), 4-dimethylaminopyridine (DMAP, 36.7 mg, 0.30 mmol) were added under argon protection, and activation continued at this temperature for 30 min. Tetraethylene glycol (728.4 mg, 3.75 mmol) was dissolved in 5mL of anhydrous dichloromethane, and after dissolution, the activated solution was slowly added dropwise to the tetraethylene glycol solution, and after completion of the addition, the solution was allowed to react at room temperature for 4 hours. TLC monitored the reaction was complete, insoluble materials were removed by filtration, the filtrate was washed with 1N HCl (20 mL × 2) and saturated sodium chloride solution (15 mL × 1), the dichloromethane layer was collected, 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 = 80/1) to give 546.4 mg of a clear oil with a yield of 43.20%.1H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 4H), 5.25 (d, J = 4.8 Hz, 1H), 4.55 (t, J = 5.5 Hz, 1H), 4.41 (dd, J = 5.9, 3.5 Hz, 4H), 3.79 – 3.71 (m, 4H), 3.58 (dd, J = 6.2, 3.6 Hz, 4H), 3.54 – 3.42 (m, 14H), 3.37 (t, J = 5.2 Hz, 2H), 3.05 (ddt, J = 11.3, 9.0, 4.4 Hz, 1H), 0.62 (s, 3H)。
Example 15
Preparation of Compound 16
Compound 15 (380.0 mg, 0.451 mmol) and carbon tetrabromide (448.7 mg, 1.353 mmol) were placed in a reaction flask, dissolved in 5mL of dichloromethane and then moved to 0 ℃ for stirring, triphenylphosphine (354.9 mg, 1.353 mmol) was dissolved in 6 mL of dichloromethane and added dropwise to the above solution, the reaction solution was moved to room temperature for 4 hours, TLC monitored for completion of the reaction, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2/1) to obtain 345.9 mg of a transparent oil with a yield of 83.19%.1H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 4H), 7.58 (m, 2H), 5.20 (s, 1H), 4.37 (dt, J = 7.2, 3.9 Hz, 4H), 3.73 (dd, J = 5.9, 3.3 Hz, 4H), 3.66 (t, J = 5.8 Hz, 2H), 3.55 (d, J = 5.1 Hz, 4H), 3.51 (d, J = 5.5 Hz, 10H), 3.43 (s, 4H), 3.01 (s, 1H), 0.70-2.40 (remaining cholesterol protons), 0.59 (s, 3H)。
Example 16
Preparation of Compound 17
Compound 16 (300.0 mg, 0.33 mmol) and berberrubine (35.0 mg, 0.11 mmol) were placed in a reaction flask, dissolved in 3mL DMF, argon replaced 3 times, the reaction solution was moved to 80 ℃ to react for 18 h, the reaction was stopped, the solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol = 30/1) to give 55.0 mg of yellow solid powder with a yield of 44.38%.1H NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.89 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H), 8.04 – 7.90 (m, 5H), 7.73 (s, 1H), 7.00 (s, 1H), 6.14 (s, 2H), 5.28 – 5.13 (m, 1H), 4.90 (t, J = 6.4 Hz, 2H), 4.38 (dt, J = 9.6, 4.6 Hz, 4H), 4.30 (t, J = 4.6 Hz, 2H), 4.03 (s, 3H), 3.72 (dt, J = 26.3, 4.5 Hz, 6H), 3.56 (ddd, J = 21.7, 8.5, 5.5 Hz, 11H), 3.45 (s, 4H), 3.17 (t, J = 6.4 Hz, 3H), 3.07 – 2.94 (m, 1H), 0.70-2.40 (remaining cholesterol protons), 0.59 (s, 3H)。
The specific preparation method of the breast cancer targeted liposome comprises the following steps:
example 17
The film hydration method is the most classical liposome preparation method and has the characteristics of simple operation and wide application. The task is to prepare a Tween 80-coated BBR and FA co-modified paclitaxel-loaded liposome by adopting a thin film hydration method. We have previously screened liposome formulations.
According to the preparation exploration of paclitaxel liposome, we finally choose the optimized prescription: lipid molar ratio 65:35 (cholesterol: soybean phospholipids), hydration solution was Phosphate Buffered Saline (PBS) of ph7.4 (0.02M), paclitaxel: the ratio of the lipid materials is 1: 30. PTX-FA-Lip (folic acid modified long-circulating acid sensitive liposome), PTX-BBR-Lip (berberine modified liposome), PTX-FA + BBR-Lip (folic acid and berberine co-modified liposome), PTX-Tween80-FA + BBR-Lip (Tween 80 coated BBR and FA co-modified liposome) and a control group PTX-Lip are prepared by the above formula respectively.
All lipid materials were dissolved in chloroform/methanol mixed solvent (v/v = 2:1), and then the organic solvent was removed by rotary evaporation at 37 ℃ under vacuum to form an intact lipid membrane layer. Vacuum preserving overnight, hydrating in PBS (pH 7.4) at 37 deg.C for 0.5 h, performing intermittent ultrasound with probe type ultrasonic instrument at 80W for 80 s in ice water bath to obtain microemulsion liposome solution, centrifuging at 4 deg.C for 5 min at 10000rpmin, and removing unloaded lipid material to obtain liposome. PTX liposomes were prepared by adding PTX to the above lipid organic solution (lipid: paclitaxel = 22:1, mass ratio) before the solvent was evaporated. The Encapsulation Efficiency (EE) of PTX was determined by high performance liquid chromatography. Similarly, CFPE-labeled liposomes were prepared by adding an appropriate amount of CFPE before the solvent was evaporated.
Table 1: particle size, potential and entrapment rate of PTX-loaded liposomes modified by different ligands
Preliminary targeting study
Example 18
To verify the ability of Tween 80-coated BBR and FA CO-modified liposomes to cross the blood brain barrier and target tumor cells at the cellular level, after reviving bEnd.3 cells and C6 cells, 10% fetal calf serum, 1% double-antibody DMEM medium, 37 ℃, 5% CO2The culture was carried out for two weeks with liquid change every other day. Taking cells in logarithmic growth phase according to the ratio of 3X 105Inoculating in 12-well plate at 37 deg.C and 5% CO2Culturing for 24 h, respectively adding five kinds of liposomes containing the lipid fluorescent substance CFPE under the condition of keeping out of the sun after the culture is finished, setting 3 multiple holes to ensure that the concentration of the CFPE in the culture solution is 2 mug/ml, and continuously incubating for 4 h. After the incubation was completed, the medium was discarded, washed three times with cold PBS, trypsinized, centrifuged, washed once more with PBS, 300 ul of lpbs was added to resuspend the cells, placed on a flow cytometer (BD FACS Celesta, BD, USA), the FITC channel was selected, and the average fluorescence intensity of the cells was measured. Further, the uptake of different ligand-modified groups of liposomes in the bEnd.3 cells and the C6 cells was studied. The result shows that the berberine and folic acid co-modified liposome coated with the Tween80 can effectively penetrate the BBB and transport the drug to the brain gliomaA cell.
Example 19
To perform more intuitive studies on mitochondrial targeting, we observed the mitochondrial localization of BBR and each group of drug-loaded liposomes in C6 cells using a laser confocal scanning microscope (CLSM). Taking C6 cells in logarithmic growth phase at 5X 105The density of cells/well was plated on a 6-well plate containing coverslips at 37 ℃ with 5% CO2Culturing for 24 h, respectively adding five kinds of CFPE labeled liposomes under the condition of keeping out of the sun after the culture is finished, ensuring the CFPE concentration to be 2 mug/mL, and continuously incubating for 4 h. After incubation, the cells were washed 3 times with 2 mL of cold PBS in the dark, 1 mL of 150 nM mitochondrial localization reagent Mito-tracker Red was stained for 20 min, the dye-containing culture solution was discarded after staining was completed, the cells were washed 3 times with cold PBS, 4% paraformaldehyde was fixed for 30 min at room temperature, washed 3 times with cold PBS, then the nuclei were stained with DAPI (5 μ g/mL) for 5 min, washed 3 times with cold PBS after staining was completed, mounted, and finally the sample was imaged using a laser scanning confocal microscope (LSM800, carlsa AG, germany). The result shows that the berberine has remarkable mitochondrial targeting capability.
Example 20
The tumor targeting ability of Tween80-FA + BBR-Lip was further examined in a C6-bearing glioma mouse model. After a C6-loaded in-situ brain glioma mouse model is established for two weeks, DiD-loaded liposomes DiD-Lip, DiD-FA-Lip, DiD-BBR-Lip, DiD-FA + BBR-Lip and DiD-Tween-FA + BBR-Lip are administered to the model mouse through tail vein injection according to the dose of 500 micrograms DiD/kg. Groups of mice were anesthetized at 1 h, 4 h, 8 h, 12 and 24 h post-dose and observed in a small animal in vivo imager. The result shows that the fluorescence signal intensity of DiD-Tween-FA + BBR-Lip in the brain of the C6 brain glioma-bearing mouse is obviously stronger than that of other groups of liposomes, and the DiD-Tween-FA + BBR-Lip has the strongest brain glioma targeting capability.
Claims (6)
1. A brain glioma targeting liposome modified by berberine coated by Tween80 and folic acid, the lipid material used for preparing the liposome is a structure shown in general formulas (I) and (II) or pharmaceutically acceptable salt thereof, the lipid material (I) is characterized in that: an acid sensitive bond is taken as a connecting group, one end of the acid sensitive bond is connected with polyethylene glycol (PEG) and folic acid with brain glioma targeting, and the other end of the acid sensitive bond is connected with cholesterol-polyethylene glycol conjugate:
wherein the molecular weight of the PEG is 200, 400, 600, 800, 1000, 1500, 2000, 3350, 4000; the polyethylene glycol is one of triethylene glycol, tetraethylene glycol, hexaethylene glycol and octaethylene glycol, namely n =3, 4, 6 and 8; the acid sensitive bond is one of hydrazone bond, imine bond, oxime bond, hemiacetal and hemiketal;
the lipid material (II) is characterized in that: the preparation method comprises the following steps of (1) taking polyglycols as a framework, connecting cholesterol at one end and berberine with a mitochondrion targeting function at the other end:
wherein, the polyethylene glycol is one of triethylene glycol, tetraethylene glycol, hexaethylene glycol and octaethylene glycol, i.e. a/b =3, 4, 6 and 8.
2. The use of the brain glioma-targeting liposome of claim 1 as a pharmaceutical carrier in the preparation of a brain glioma-targeting drug.
3. The brain glioma-targeting liposome of claim 1, wherein the brain targeting property of Tween80, the glioma targeting property of folic acid and the mitochondrion targeting property of berberine are combined to form a plurality of targeted intelligent liposomes which can penetrate the blood brain barrier and have brain glioma and mitochondrion.
4. The brain glioma-targeting liposome of claim 1, comprising a membrane material and an active agent, wherein the membrane material is a phospholipid bilayer and is composed of a phospholipid, cholesterol and the lipid material of claim 1, wherein the ratio of the components is as follows: the molar ratio of the cholesterol to the phospholipid is 1-2: 1-5, and the molar content of the lipid material in claim 1 is 1-20% of the total molar number of the cholesterol and the phospholipid; the active agent adopts a therapeutic agent or an imaging agent, wherein the active agent accounts for 0.1 to 50 percent of the total lipid by weight percentage.
5. The brain glioma-targeting liposome of claim 4, wherein the brain glioma-targeting liposome prepared by the method of thin film according to the component proportion relationship of claim 4 has stable particle size and Zeta potential, and the liposome has a particle size of about 100nm and an entrapment rate of more than 80%.
6. The brain glioma-targeting liposome of claim 4, wherein the active agent is paclitaxel, temozolomide, fluorouracil, teniposide.
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