阅读说明：本技术 一种19f-穴蕃纳米乳的磁共振成像显影剂的制备方法 (Preparation method of 19F-Boehringer nanoemulsion magnetic resonance imaging developer ) 是由 周欣 张怀彬 陈世桢 娄昕 于 2019-10-28 设计创作，主要内容包括：本发明公开了一种<Sup>19</Sup>F-穴蕃微乳的磁共振成像显影剂的制备方法,步骤是：(1)纳米乳的制备：由磷脂、普朗尼克F-68、<Sup>19</Sup>F-穴蕃、<Sup>19</Sup>F-卟啉和1,1,1-三(全氟叔丁氧基甲基)乙烷经超声乳化法得到纳米乳；(2)<Sup>19</Sup>F-穴蕃的制备：穴蕃和F-聚乙二醇反应得化合物<Sup>19</Sup>F-穴蕃。(3)<Sup>19</Sup>F-卟啉的制备：卟啉与F-聚乙二醇反应得到化合物<Sup>19</Sup>F-卟啉。(4)靶向分子的制备:胆固醇-聚乙二醇2000-马来酰亚胺与精氨酸-甘氨酸-天冬氨酸反应得靶向分子。(5)靶向纳米乳的制备：将胆固醇-聚乙二醇2000-精氨酸-甘氨酸-天冬氨酸加入到纳米乳液中,振荡得到靶向纳米乳。方法简单高效,显影剂具有高灵敏度、生物相容性好、能靶向肺癌肿瘤进行MR成像及光学成像,应用于肺癌早期诊断和治疗。(The invention discloses a 19 The preparation method of the magnetic resonance imaging developer of the F-Botana microemulsion comprises the following steps: (1) preparing the nano-emulsion: consists of phospholipid, pluronic F-68, 19 F-the tomato in the cave, 19 The F-porphyrin and the 1,1, 1-tri (perfluoro-tert-butoxymethyl) ethane are processed by an ultrasonic emulsification method to obtain the nano-emulsion; (2) 19 F-Boehu tomatoThe preparation of (1): the reaction of the Boehmeria cinerea and the F-polyethylene glycol to obtain the compound 19 F-Bo Fan. (3) 19 Preparation of F-porphyrin: porphyrin reacts with F-polyethylene glycol to obtain a compound 19 F-porphyrin. (4) The preparation of the targeting molecule comprises the reaction of cholesterol-polyethylene glycol 2000-maleimide and arginine-glycine-aspartic acid to obtain the targeting molecule. (5) Preparing targeted nano-emulsion: adding cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid into the nano emulsion, and oscillating to obtain the targeted nano emulsion. The method is simple and efficient, the developing agent has high sensitivity and good biocompatibility, can target lung cancer tumors to carry out MR imaging and optical imaging, and is applied to early diagnosis and treatment of lung cancer.)

1. a kind of¹⁹the preparation method of the magnetic resonance imaging developer of the F-Boehringer nanoemulsion comprises the following steps:

(1) Preparing the nano-emulsion: and (3) mixing the components in a mass ratio of 40: 5: 3.1: 7.6 phospholipids/Pluronic F-68¹⁹F-Bo tomato¹⁹Dissolving F-porphyrin in an organic solvent, removing the organic solvent by using a rotary evaporator at room temperature to enable the raw material to form a lipid membrane on the wall of a flask, then drying the lipid membrane in vacuum at 40-50 ℃ for 20-30min to remove the organic solvent completely, adding ultrapure water into the flask to enable the membrane to be dissolved, adding 78mg of 1,1, 1-trichloroethane, carrying out ultrasonic treatment for 10-20min by using an ultrasonic crusher, and then carrying out extrusion for 3-5 times by using a 0.22 mu m microporous filter membrane to obtain the nanoemulsion;

The organic solvent is chloroform/methanol which is 3/1;

(2)¹⁹F-preparation of the Boehmeria cinerea: under the conditions of no water and nitrogen protection, 50mg of the Boehringer-Guerin, 51 mu mol of the 1-hydroxybenzotriazole and 24.7mg of 183 mu mol of the N, N-dimethylformamide are added into a reaction bottle, 2mL of the N, N-dimethylformamide solution is added, 35mg of the carbodiimide and 183 mu mol of the N, N-dimethylformamide solution are added under ice bath conditionsAnd (3) mL of solution, stirring and reacting for 28-32min, adding 1mL of solution of F-polyethylene glycol and 112 mu mol of N, N-dimethylformamide, heating the reaction solution to 48-52 ℃, reacting for 11-13h, and after the reaction is finished, concentrating the reaction solution under reduced pressure of 0.05-0.1Mpa, and purifying by column chromatography: dichloromethane/methanol 10:1 to obtain the compound¹⁹F-Bo Fan;

(3)¹⁹Preparation of F-porphyrin: under the protection of anhydrous and nitrogen, adding 182mg of porphyrin compound, 0.3mmol of 1-hydroxybenzotriazole and 243mg of 1.8mmol of N, N-dimethylformamide into a reaction bottle, adding 345mg of carbodiimide and 30mL of 1.8mmol of N, N-dimethylformamide under the condition of ice bath, stirring for reacting for 28-32min, adding 3.9g of F-polyethylene glycol and 20mL of 1.8mmol of N, N-dimethylformamide, heating the reaction solution to 44-46 ℃, reacting for 22-26 h, concentrating the reaction solution under reduced pressure of 0.05-0.1MPa after the reaction is finished, and purifying by column chromatography: dichloromethane/methanol 10:1 to obtain the compound¹⁹(ii) an F-porphyrin;

(4) adding cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid into 1mL of phosphate buffer solution according to a molar ratio of 10:1-1:1, placing the mixture on a shaking table at room temperature, shaking at a speed of 300rpm for 20-24 hours, dialyzing by using a dialysis bag in ultrapure water to remove unreacted arginine-glycine-aspartic acid molecules, and freeze-drying by using a freeze dryer to obtain targeted molecules cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid;

(5) Preparing targeted nano-emulsion: adding cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid into the nano emulsion prepared in the step (1), and then placing the nano emulsion on a shaking table to oscillate for one hour at the speed of 300rpm to obtain the targeted nano emulsion.

2. A method as claimed in claim 1¹⁹The preparation method of the magnetic resonance imaging developer of the F-Boehringer nanoemulsion is characterized by comprising the following steps of: the phospholipid is soybean lecithin Lipoid S75.

3. a method as claimed in claim 1¹⁹Magnetic resonance of F-Botana nano-emulsionA method of making an imaging developer, comprising: the tomato is¹⁹F. Polyethylene glycol modified water soluble tomato.

4. A method as claimed in claim 1¹⁹The preparation method of the magnetic resonance imaging developer of the F-Boehringer nanoemulsion is characterized by comprising the following steps of: the molar ratio of the addition amount of the cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid to the phospholipid is 1: 20.

5. A method as claimed in claim 1¹⁹the preparation method of the magnetic resonance imaging developer of the F-Boehringer nanoemulsion is characterized by comprising the following steps of: the mass ratio of the phospholipid to the F-68 in the step (1) is 8: 1.

6. A method as claimed in claim 1¹⁹The preparation method of the magnetic resonance imaging developer of the F-Boehringer nanoemulsion is characterized by comprising the following steps of: the mass ratio of the phospholipid to the 1,1, 1-trichloroethane is 1:1.9-1: 4.

Technical Field

The invention belongs to the technical field of nuclear magnetic resonance imaging, and particularly relates to a nuclear magnetic resonance imaging system¹⁹The preparation method of the magnetic resonance imaging developer of the F-Botanus nanoemulsion is applied to targeted magnetic resonance imaging, optical imaging and photodynamic therapy of lung cancer tumors.

Background

In clinical disease diagnosis, Nuclear Magnetic Resonance (NMR) has advantages such as no radiation, multiple functions, high temporal spatial resolution, and the like, and is widely used. But is conventional¹H MRI has strong interference due to background signals in vivo and low sensitivity. Heteronuclear MRI (e.g., magnetic resonance imaging) with the continuing development of magnetic resonance imaging technology³¹P，¹³C，¹⁹F，¹²⁹Xe) are widely focused and studied.

The nano-emulsion as a novel nano-carrier has the advantages of good biocompatibility, stability, easy modification and the like, and has great application potential in diagnosis and treatment of tumors. Perfluorocarbon (PFCs) nanoemulsions due to their high content¹⁹the F content can provide strong¹⁹F signal, and is widely applied to¹⁹F MRI tracer. 1,1, 1-tris (perfluoro-tert-butoxymethyl) ethane is a nontoxic PFCs with 27 symmetries in the molecule¹⁹F atom, which can provide a single, strong¹⁹f signal, and¹⁹The F signal has no interference of background signal in organism, and can be used for tumor cells and solid tumors¹⁹And F, MRI detection. Xenon is an inert, non-toxic gas that can be used as an ideal medium without background signals in the human body.¹²⁹The natural abundance of Xe is 26.4%, and hyperpolarization is generated by a home-made polarization device¹²⁹Xe gas, can improve the sensitivityis 10000 times higher. Will then be hyperpolarised¹²⁹Xe and CEST technology can be combined to further improve the sensitivity by 100 times, and the sensitivity is ultrahigh. The Boehringer molecule is a commonly used molecule¹²⁹Xe MRI contrast agents, widely used as¹²⁹Xe MRI probes.

By carrying out on the Boehmeria molecules¹⁹f and polyethylene glycol modification, so that the problem of poor water solubility of the compound is solved, and the compound can be well applied to organisms. Will be provided with¹⁹The nano emulsion prepared from F-Boehmeria and 1,1, 1-tri (perfluoro-tert-butoxymethyl) ethane has good stability, good biocompatibility, no toxicity and can be used as a nano emulsion¹²⁹Xe/¹⁹F dual channel multifunctional developer for use in vivo¹²⁹Xe MRI and¹⁹F MRI imaging.

Disclosure of Invention

the invention aims to overcome the defects of the prior art and provides a method for preparing a novel anti-counterfeiting bottle¹⁹A preparation method of a magnetic resonance imaging developer of the F-Boehringer nanoemulsion; provided with¹⁹F-Botana nano-emulsion is combined with¹²⁹Xe MRI and¹⁹F MRI two magnetic resonance imaging methods, which have the function of targeted diagnosis of tumors by introducing arginine-glycine-aspartic acid (RGD) ligand to be specifically combined with receptors on cancer cells. By passing¹²⁹Xe MRI and¹⁹The F MRI technology realizes the target diagnosis of the lung cancer tumor. The developer has ultrahigh sensitivity and better biocompatibility, and is suitable for in-vivo magnetic resonance imaging.

in order to achieve the purpose, the invention adopts the following technical scheme:

A kind of¹⁹The preparation method of the magnetic resonance imaging developer of the F-Boehringer nanoemulsion comprises the following steps:

A. Preparing the nano-emulsion: firstly, the mass ratio is 40: 5: 3.1: 7.6 phospholipids/Pluronic F-68¹⁹F-Bo tomato¹⁹Dissolving F-porphyrin in organic solvent, removing organic solvent with rotary evaporator at room temperature (20-25 deg.C) to form lipid membrane on the wall of flask, vacuum drying at 40-50 deg.C for 20-30min to remove organic solvent, dissolving membrane in ultrapure water, adding into flask, and adding into the flaskAdding 78mg of 1,1, 1-tri (perfluoro-tert-butoxymethyl) ethane, performing ultrasonic treatment for 10-20min by using an ultrasonic crusher (ultrasonic crusher SCQ-900F), and then extruding for 3-5 times by using a 0.22 mu m microporous filter membrane (Jinteng polyethersulfone filter membrane) to finally obtain the nanoemulsion;

The organic solvent is chloroform/methanol which is 3/1;

B、¹⁹F-preparation of the Boehmeria cinerea: adding a solution of Ipomoea batatas (50mg,51 mu mol) and 1-Hydroxybenzotriazole (HOBT) (24.7mg,183 mu mol) in N, N-Dimethylformamide (DMF) (2mL) into a reaction bottle under the protection of anhydrous and nitrogen gas, adding a solution of carbodiimide (EDC) (35mg,183 mu mol) in N, N-Dimethylformamide (DMF) (2mL) under ice-bath conditions, stirring for reaction for 28-32min, adding a solution of F-polyethylene glycol (HFBB) (0.3g,112 mu mol) in N, N-Dimethylformamide (DMF) (1mL), heating the reaction solution to 48-52 ℃, reacting for 11-13h, and concentrating the reaction solution under reduced pressure (0.05-0.1MPa) after the reaction is finished to obtain the compound by purification (dichloromethane/methanol is 10:1)¹⁹F-Bo Fan.

C、¹⁹Preparation of F-porphyrin: adding a porphyrin compound (182mg,0.3mmol) and a 1-Hydroxybenzotriazole (HOBT) (243mg,1.8mmol) solution of N, N-Dimethylformamide (DMF) (30mL) into a reaction bottle under the protection of anhydrous and nitrogen gas, adding a carbodiimide (EDC) (345mg,1.8mmol) solution of N, N-Dimethylformamide (DMF) (30mL) under ice bath conditions, stirring for reaction for 28-32min, adding a F-polyethylene glycol (HFBB) (3.9g,1.8mmol) solution of N, N-Dimethylformamide (DMF) (20mL), heating the reaction solution to 44-46 ℃, reacting for 22-26 h, and concentrating the reaction solution under reduced pressure (0.05-0.1MPa) after the reaction is finished to obtain the compound through purification (dichloromethane/methanol is 10:1)¹⁹F-porphyrin.

D. Preparation of Cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid (RGD) Cholesterol-polyethylene glycol 2000-maleimide and arginine-glycine-aspartic acid (RGD) were added in a molar ratio of 10:1-1:1 to 1mL of Phosphate Buffered Saline (PBS), and the mixture was placed on a shaker at room temperature and shaken at 300rpm for 20-24 hours. Then dialyzing in ultra-pure water by using a dialysis bag (common) to remove unreacted arginine-glycine-aspartic acid (RGD) molecules, and freeze-drying by using a freeze dryer (common) to obtain the targeted molecule cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid (RGD).

E. Preparing targeted nano-emulsion: adding cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid (RGD) into the nano emulsion prepared in the step (1), and then placing the nano emulsion on a shaking table to oscillate at the speed of 300rpm for about 1 hour to obtain the targeted nano emulsion.

Preferably, the phospholipid is soybean lecithin (Lipoid S75).

Preferably, the pore size of the microfiltration membrane is 0.22 μm.

Preferably, the mole ratio of the cholesterol-polyethylene glycol 2000-arginine-glycine-aspartic acid (RGD) to the phospholipid is 1: 20.

Preferably, the mass ratio of the phospholipid to the F-68 in the step (1) is 8: 1.

Preferably, the mass ratio of the phospholipid to the 1,1, 1-tris (perfluoro-tert-butoxymethyl) ethane is 1: 1.9.

In addition, the invention also claims the arginine-glycine-aspartic acid (RGD) -nanoemulsion MRI developer prepared by the method.

The magnetic resonance imaging developer obtained by the technical measures is applied to preparing early MRI medicines (diagnosis and treatment) for treating or preventing lung cancer tumors.

Compared with the prior art, the invention has the following obvious advantages and beneficial effects:

(1) The polyethylene glycol modified ipomoea batatas molecule prepared by the invention solves the problem of poor water solubility, so that¹²⁹The Xe MRI contrast agent can be well applied in organisms and has good biocompatibility;

(2) Prepared by the invention¹⁹F-Boeheng nano-emulsion is prepared from¹⁹F is introduced into the Boehmeria molecules to act as both¹²⁹Xe MRI and¹⁹F MRI contrast agent. The introduction of the porphyrin compound enables the nano-emulsion to be used as an optical imaging developer¹²⁹Xe/¹⁹F a dual channel, bimodal MRI contrast agent;

(3) The nanoemulsion has ultrahigh sensitivity and can be used for nano-emulsionLung cancer cells were MRI imaged at molar concentrations. Also by¹²⁹Xe MRI and¹⁹F MRI and optical imaging prove that the nanoemulsion has better specific recognition on lung cancer tumors and better photodynamic therapy effect on tumor cells. The imaging agent can be applied to early MRI diagnosis and treatment of lung cancer tumors.

Drawings

FIG. 1 is a drawing¹⁹preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹F-Botana nano-emulsion dynamic light scattering diagram.

The prepared nano-emulsion has uniform particle size distribution.

FIG. 2 is a drawing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹transmission electron micrograph of F-Botanh nanoemulsion.

The transmission electron microscope image shows that the nano-emulsion particle size is about 100nm and is consistent with the dynamic light scattering particle size.

FIG. 3 is a drawing of a¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹F-Botana nano-emulsion ultraviolet absorption spectrum.

The RGD modified nano-emulsion has a characteristic ultraviolet absorption peak at 275nm, which indicates that RGD molecules are successfully modified on the surface of the nano-emulsion.

FIG. 4 is a drawing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹F-the fluorescence emission spectrogram of the Boehringer's nanoemulsion,

The nanoemulsion has fluorescence emissions at 655nm and 722 nm.

FIG. 5 is a drawing showing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹The F-Boehna nanoemulsion¹⁹f NMR spectrum.

The nanoemulsion of¹⁹The F signal is at-72 ppm.

FIG. 6 is a drawing of a¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹The F-Boehna nanoemulsion¹²⁹Xe Hyper CEST spectrum.

The nanoemulsion of¹²⁹Xe Hyper CEST signalaround 72ppm and has an ultra-high sensitivity.

FIG. 7 is a drawing showing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹Fluorescence confocal images of the F-Botana nanoemulsion after incubation with A549 cells and MCF7 cells, respectively.

The result shows that the targeted nanoemulsion has good specific targeting property on lung cancer cells A549.

FIG. 8 is a drawing showing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹After the F-Bosch nanoemulsion is respectively incubated with A549 cells and MCF7 cells¹²⁹Xe MRI and¹⁹F, MRI spectrogram.

The magnetic resonance imaging result also shows that the targeted nanoemulsion has good targeting property on lung cancer cells A549.

FIG. 9 is a drawing showing¹⁹preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹The photodynamic treatment effect of the F-Botana nanoemulsion on A549 cells is shown.

the nanoemulsion can obviously kill tumor cells after entering the tumor cells and being irradiated by laser, and has good phototherapy effect.

FIG. 10 is a drawing showing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹Living body of F-Botan nano-emulsion for A549 tumor mouse¹⁹F MRI and fluoroscopic imaging.

The nanoemulsion can be well targeted to tumors after being injected into a mouse body through tail vein¹⁹F MRI and fluorescence imaging.

FIG. 11 is a drawing showing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹F-dynamic light scattering pattern of the Botana nanoemulsion.

the average hydrated particle size of the nanoemulsion is 120 nm.

FIG. 12 is a drawing¹⁹Preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹F-dynamic light scattering pattern of the Botana nanoemulsion.

The average hydrated particle size of the nanoemulsion is 134 nm.

FIG. 13 is a drawing showing¹⁹preparation method of magnetic resonance imaging developer of F-Boehringer nanoemulsion¹⁹F-dynamic light scattering pattern of the Botana nanoemulsion.

The average hydrated particle size of the nanoemulsion is 128 nm.

Detailed Description

The invention is further illustrated by the following specific examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.