PH and Oxygen dual-sensitive magnetic resonance imaging contrast agent and preparation method thereof

Abstract

The present disclosure provides a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent and a preparation method thereof, which is a dual-modal nanoparticle contrast agent with .sup.19F signal and CEST dual signal (.sup.19F-CEST), a CEST contrast agent that is dual-sensitive to pH and oxygen. The preparation method comprises: mixing a variety of phospholipid surfactants and cholesterol well to obtain a blend of phospholipid surfactants, which is dissolved in chloroform or a mixed solvent of chloroform and methanol, evaporated to dryness with a rotary evaporator and dried overnight in a vacuum oven at 40 C. after adding rhodamine, finally, it is dispersed in water containing glycerin by mechanical dispersion or ultrasonic vibration to obtain a lipid modifier; mixing perfluorocarbon, the lipid modifier obtained in step (1), glycerin, and water and ultrasonically mixing well with a probe, and extruding with an extruder to prepare a perfluorocarbon nanoemulsion.

Claims

1. A method for preparing a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent, wherein the method comprises the following steps: (1) preparation of lipid modifier: mixing a variety of phospholipid surfactants and cholesterol well to obtain a blend of phospholipid surfactants, which is dissolved in chloroform or a mixed solvent of chloroform and methanol, evaporated to dryness with a rotary evaporator and dried overnight in a vacuum oven at 40 C. after adding rhodamine, finally, dispersing in water containing glycerin by mechanical dispersion or ultrasonic vibration to obtain a lipid modifier; the phospholipid surfactant blend is composed of phosphatidylcholine liposome, phosphatidylglycerol liposome and cholesterol; (2) preparation of perfluorocarbon nanoemulsion: mixing perfluorocarbon, the lipid modifier obtained in step (1), glycerin, and water and ultrasonically mixing well with a probe, and extruding with an extruder to prepare a perfluorocarbon nanoemulsion. (3)

2. The method for preparing a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent according to claim 1, wherein in the perfluorocarbon nanoemulsion obtained in step (2), perfluorocarbon and glycerin account for 10-40% of the total mass, water accounts for 55-85% of the total mass, and phospholipid surfactants account for 1-5% of the total mass.

3. The method for preparing a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent according to claim 2, wherein in the perfluorocarbon nanoemulsion obtained in step (2), the mass ratio of perfluorocarbon to glycerin 10-20:1.

4. The method for preparing a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent according to claim 1, wherein the perfluorocarbon is selected from one or more of brominated perfluorooctane, perfluoro-15-crown ether-5, FC-3280 and FC-77.

5. The method for preparing a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent according to claim 1, wherein in the lipid modifier obtained in step (1), the mass ratio of the blend of phospholipid surfactant, the mixed solvent of chloroform and methanol, and rhodamine is (80-95):(35-45):(0.5-2).

6. The method for preparing a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent according to claim 1, wherein in step (1), the ultrasonic oscillation time is 5 seconds to 10 seconds, and the power P.sub.1 is 400 W>P1>300 W; in step (2), the ultrasonic treatment time is 60 seconds-70 seconds, and the power P2 is: P2>P1 and 450 W>P2400 W.

7. The method for preparing a pH and oxygen dual-sensitive magnetic resonance imaging contrast agent according to claim 1, wherein the molar ratio of phosphatidylcholine liposome, phosphatidylglycerol liposome and cholesterol is (60-80):(10-15):(10-25).

8. A pH and oxygen dual-sensitive magnetic resonance imaging contrast agent prepared by the method according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1. Hydrated particle size diagram of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles.

[0033] FIG. 2. ZETA potential diagram of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles.

[0034] FIG. 3. FIG. 3-1 is the .sup.1H magnetic resonance spectrum of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles; FIG. 3-2 is an enlarged view of A shown in FIG. 3-1.

[0035] FIG. 4. .sup.1H-MR CEST imaging Z-spectrum of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles.

[0036] FIG. 5. Transmission electron microscopy (TEM) image of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles.

[0037] FIG. 6. T1 weighting (6-1) and .sup.19F signal image (6-2) of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles. The volume dilution ratios of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles. are: 1, 1:5, 1:10, 1:20, 1:100, respectively.

[0038] FIG. 7. CEST signal ST%map color map of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles. The pre-saturation pulse power in FIG. (7-1) is 1.2 T and the saturation time is 3 s, the pre-saturation pulse power in FIG. (7-2) is 2.4 T and the saturation time is 3 s. The volume dilution ratio of the pH and oxygen dual-sensitive 19F-MRI/CEST multimodal imaging nanoparticles. are: 1, 1:5, 1:10, 1:20, 1:100, respectively)

[0039] FIG. 8. Histograms of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles with different dilution ratios between different pre-saturation pulses and different saturation time. The pre-saturation pulse power in FIG. (8-1) is 1.2 T, and the pre-saturation pulse power in FIG. (8-2) is 2.4 T.

[0040] FIG. 9. pH sensitive standard curve of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles.

[0041] FIG. 10. O.sub.2 sensitive standard curve of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles.

DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only a part of the embodiments of the present invention, but not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts are all in the protection scope of the present invention.

Embodiment 1

[0043] A pH and oxygen double-sensitive magnetic resonance imaging contrast agent is prepared by the method as follows:

[0044] (1) preparation of lipid modifier: mixing a variety of phospholipid surfactants and cholesterol well to obtain a blend of phospholipid surfactants, which is dissolved in a mixed solvent of chloroform and methanol, evaporated to dryness with a rotary evaporator and dried overnight in a vacuum oven at 40 C. after adding rhodamine, finally, it is dispersed in water containing glycerin by ultrasonic vibration to obtain a lipid modifier.

[0045] The blend of phospholipid surfactant is composed of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG), and cholesterol, and the molar ratio between them is DPPC:DPPG:cholesterol=75:15:20.

[0046] The mass ratio of the blend of phospholipid surfactant, the mixed solvent of chloroform and methanol, and rhodamine is 85:35:0.7.

[0047] The ultrasonic vibration processing time is 5 seconds, and power is 380 W.

[0048] (2) preparation of perfluorocarbon nanoemulsion: mixing perfluorocarbon, the lipid modifier obtained in step (1), glycerin, and water and ultrasonically mixing well with a probe, and extruding with an extruder (Avanti mini extruder) to prepare a perfluorocarbon nanoemulsion.

[0049] Perfluorocarbon accounts for 31.25% of the total mass, glycerin accounts for 2% of the total mass, water accounts for 64.75% of the total mass, and phospholipid surfactants account for 2% of the total mass.

[0050] Perfluorocarbon (PFC) is selected from perfluoro-15-crown ether-5.

[0051] The ultrasonic processing time is 65 seconds, and power is 400 W.

[0052] Dialysis is used to remove components that are not effectively coated in the emulsion in step (2) to obtain a CEST contrast agent probe.

Embodiment 2

[0053] A pH and oxygen double-sensitive magnetic resonance imaging contrast agent is prepared by the method as follows:

[0054] (1) preparation of lipid modifier: mixing a variety of phospholipid surfactants and cholesterol well to obtain a blend of phospholipid surfactants, which is dissolved in a mixed solvent of chloroform and methanol, evaporated to dryness with a rotary evaporator and dried overnight in a vacuum oven at 40 C. after adding rhodamine, finally, it is dispersed in water containing glycerin by ultrasonic vibration to obtain a lipid modifier.

[0055] The blend of phospholipid surfactant is composed of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG), and cholesterol, and the molar ratio between them is DPPC:DPPG:cholesterol=75:15:20.

[0056] The mass ratio of the blend of phospholipid surfactant, the mixed solvent of chloroform and methanol, and rhodamine is 90:35:1.5.

[0057] The ultrasonic vibration processing time is 8 seconds, and power is 360 W.

[0058] (2) preparation of perfluorocarbon nanoemulsion: mixing perfluorocarbon, the lipid modifier obtained in step (1), glycerin, and water and ultrasonically mixing well with a probe, and extruding with an extruder (Avanti mini extruder) to prepare a perfluorocarbon nanoemulsion.

[0059] Perfluorocarbon accounts for 31.25% of the total mass, glycerin accounts for 2% of the total mass, water accounts for 64.75% of the total mass, and phospholipid surfactants account for 2% of the total mass.

[0060] Perfluorocarbon (PFC) is selected from perfluoro-15-crown ether-5.

[0061] The ultrasonic processing time is 70 seconds, and power is 430 W.

[0062] Dialysis is used to remove components that are not effectively coated in the emulsion in step (2) to obtain a CEST contrast agent probe.

Embodiment 3

[0063] A pH and oxygen double-sensitive magnetic resonance imaging contrast agent is prepared by the method as follows:

[0064] (1) preparation of lipid modifier: mixing a variety of phospholipid surfactants and cholesterol well to obtain a blend of phospholipid surfactants, which is dissolved in a mixed solvent of chloroform and methanol, evaporated to dryness with a rotary evaporator and dried overnight in a vacuum oven at 40 C. after adding rhodamine, finally, it is dispersed in water containing glycerin by ultrasonic vibration to obtain a lipid modifier.

[0065] The blend of phospholipid surfactant is composed of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG), and cholesterol, and the molar ratio between them is DPPC:DPPG:cholesterol=75:15:20.

[0066] The mass ratio of the blend of phospholipid surfactant, the mixed solvent of chloroform and methanol, and rhodamine is 85:35:2.

[0067] The ultrasonic vibration processing time is 5 seconds, and power is 350 W.

[0068] (2) preparation of perfluorocarbon nanoemulsion: mixing perfluorocarbon, the lipid modifier obtained in step (1), glycerin, and water and ultrasonically mixing well with a probe, and extruding with an extruder (Avanti mini extruder) to prepare a perfluorocarbon nanoemulsion.

[0069] Perfluorocarbon accounts for 36.45% of the total mass, glycerin accounts for 2% of the total mass, water accounts for 62.55% of the total mass, and phospholipid surfactants account for 2% of the total mass.

[0070] Perfluorocarbon (PFC) is selected from perfluoro-15-crown ether-5.

[0071] The ultrasonic processing time is 60 seconds, and power is 400 W.

[0072] Dialysis is used to remove components that are not effectively coated in the emulsion in step (2) to obtain a CEST contrast agent probe.

[0073] The characterization and effect verification experiments of the CEST contrast agent probe of the embodiment 1 of the present invention are further described below.

(1) MR CEST Imaging of the pH and Oxygen Dual-Sensitive .SUP.19.F-MRI/CEST Multimodal Imaging Nanoparticles

[0074] The synthesized pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoemulsion is the original solution, and is diluted in a 0.25 ml EP tube with a volume ratio of 1, 1/5, 1/10, 1/20, 1/100, respectively, and is fixed it with distilled water in a 40 ml centrifuge tube for .sup.19FMR, T1RARE, T1mapping, T2mapping, CEST EPI sequence scanning, and the CEST signal efficiency of the probe is analyzed and calculated through data processing by MATLAB and Graphpad Prism7. Scanning parameters: Repetition Time: 10000 ms, Echo Time: 20 ms, Slice thickness: 2 mm, FOV: 35*35 mm, Bandwidth: 300000, Averages: 1, Repetitions: 95, Segments: 1, Number Offset Experiment: 95, Min CEST Offset: 4000, Max CEST Offset: 4000, RF Amplitude T: 3, Length: 5000 ms, Duration time: 3 s, 5 s, 8 s, Saturation power: 0.2, 0.5, 0.8, 1.0, 1.2, 2.4, 3.5, 4.7 T.

(2) Characterization of the pH and Oxygen Dual-Sensitive .SUP.19.F-MRI/CEST Multimodal Imaging Nanoparticles

[0075] Characterization of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles use transmission electron microscopy (TEM) to observe the size, morphology and structural characteristics of nanoparticles; use nanoparticle size potential analyzer to measure the hydrated particle size, Zeta potential and polydispersity index of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles at room temperature; the results meet the requirements of nanometer particle size, and the solution has good stability.

[0076] The hydration particle size of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles under dynamic light scattering (DLS) is 123.4 nm, which provides a powerful guarantee for effectively improving the EPR of the tumor area (FIG. 1).

[0077] The content of phosphatidylcholine liposomes modifying the cell model of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles in the solute is extremely small, and it is difficult to detect the proton spectrum peak on the 1H magnetic resonance spectrum (FIG. 3), as shown in FIG. 3-1, almost no proton spectrum peak of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles is detected. After magnifying A shown in FIG. 3-1 at 3-6 ppm (FIG. 3-2), a very small proton spectrum peak can be detected, but under CEST imaging technology, the OH group CEST signal peak derived from phosphatidylcholine liposomes can be easily found (FIG. 4).

[0078] The 19F and CEST dual signal sources of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles can effectively improve the quality of spatial positioning. (When the dilution ratio is 1:5, the .sup.19F signal is significantly weakened on .sup.19F-MRI, but on .sup.1H-MR CEST imaging, when the pre-saturation pulse power is 2.4 T and the saturation time is 3 s, up to 63% of the CEST signal can be detected; when the dilution ratio is 1:100, the .sup.19F signal is basically undetectable on .sup.19F-MRI, but up to 36% of the signal can still be detected on CEST imaging, so it can provide a beacon effect for 19F signal positioning. FIG. (6-2), FIG. (7-2)).

[0079] CEST contrast agent is affected by the dissociation coefficient of acidity of free water. When the pre-saturation pulse is 0.8 T and 3 T, the pH-sensitive standard curve of the pH and oxygen dual-sensitive .sup.19F-MRI/CEST multimodal imaging nanoparticles is obtained by the ratiometer method, which can be further used to detect the acidity of the area of interest.

[0080] Because of the oxygen-carrying properties of perfluorocarbon (PFC) nanoemulsions, we have discovered for the first time the effect and law of oxygen on the signal of perfluorocarbon (PFC) nanoemulsion, which can lay a research foundation for the oxygen carrying and release degree of biological perfluorocarbon (PFC) nanoemulsion.

[0081] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be construed as limitations to the present invention, and those of ordinary skill in the art may make changes, modifications, substitutions, and variations to the above-described embodiments within the scope of the present invention without departing from the principle and purpose of the present invention. The scope of the present invention is defined by the appended claims and equivalents thereof.

[0082] The above are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited to this. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.