Diagnosis-treatment integrated nano-probe for .SUP.19.F-MR/fluorescence multi-mode molecular imaging and drug-loading and preparation method and application of nano-probe

Abstract

The present invention provides a .sup.19F-MR/fluorescence multi-mode molecular imaging and drug loading diagnosis-treatment integrated nanoprobe, and a preparation method and an application. The nano-probe is a nanoparticle formed by coating a mixture of a surfactant containing a molecular targeting treatment drug and a fluorescent dye with a Perfluorocarbon (PFC) carrier; and by uniformly dispersing a mixed solution into water and glycerol, processing ultrasonically, removing a component which is not effectively coated, and purifying, the drug-loading nanoparticle capable of being used for 19 F-MR imaging may be prepared. The nano-probe may implement in-vivo 19F-MR molecular imaging; a carried molecular targeting treatment drug can implement targeted binding and targeted treatment; and by virtue of a characteristic that PFC in a nucleus may carry and release oxygen massively, an anaerobious microenvironment in the tumor is improved, a chemosensitization effect is achieved, and thus the diagnosis-treatment integration of the tumor is implemented finally.

Claims

1. A diagnosis-treatment integrated nano-probe for .sup.19F-MR/fluorescence multi-mode molecular imaging and drug-loading, wherein the nanoprobe consists of a core, a core shell that surrounds the core, glycerol, and water, the core is a mixture of a surfactant containing a molecular targeting small-molecule treatment drug and fluorescent dye, and the core shell is Perfluorocarbon (PFC) carrier, wherein the molecular targeting small-molecule treatment drug is a third-generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (EGFR-TKI) AZD9291, and can target a T790M tumor.

2. The diagnosis-treatment integrated nano-probe for .sup.19F-MR/fluorescence multi-mode molecular imaging and drug-loading according to claim 1, wherein the fluorescent dye is 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(ammonium salt) (16:0 LissRhod PE) having a wavelength of 580 nm.

3. The diagnosis-treatment integrated nano-probe for .sup.19F-MR/fluorescence multi-mode molecular imaging and drug-loading according to claim 1, wherein the PFC is Perfluoro-15-Crown-5-Ether (PFCE).

4. A preparation method of the diagnosis-treatment integrated nano-probe for .sup.19F-MR/fluorescence multi-mode molecular imaging and drug-loading according to claim 1, comprising the following steps: obtaining a mixture of a surfactant containing a molecular targeting small-molecule treatment drug and a fluorescent dye, and using Perfluorocarbon (PFC) as a carrier to coat the mixture to form a nanoparticle; and uniformly mixing the nanoparticle with glycerol and water to obtain the 19F-MR/fluorescence multi-mode molecular imaging and drug loading diagnosis-treatment integrated nanoprobe.

5. The preparation method according to claim 4, comprising the following steps I-III: I, uniformly mixing a molecular targeting small-molecule treatment drug with a surfactant and a fluorescent dye to get a pre-mixed mixture, wherein, the surfactant coating the molecular targeting small-molecule treatment drug physically because of a surface tension; and dissolving the pre-mixed mixture into a volatile organic solvent, stirring for 10 min at a room temperature, evaporating the volatile organic solvent via a rotary evaporator to dryness, then drying for 12 h in a vacuum drying oven at 37° C., and at last, dispersing into water via ultrasonic processing to obtain a mixture for later use; II, uniformly dispersing PFC into the mixture obtained in the step I, adding glycerol and the water dropwise, and mixing for 5 min in a high-pressure homogenizer to prepare into an emulsion containing the 19 F-MR/fluorescence multi-mode molecular imaging and drug loading diagnosis-treatment integrated nanoprobe; and III, removing a component, which is uncoated effectively, with a dialysis manner in an environment at a pH of 7.4 and the room temperature from the emulsion obtained in the step II to obtain the 19 F-MR/fluorescence multi-mode molecular imaging and drug loading diagnosis-treatment integrated nanoprobe.

6. The preparation method according to claim 5, wherein the surfactant is lecithin 95% and cholesterol, and taking 5.5 mg of the molecular targeting small-molecule treatment drug in the step I as a standard to weight dosages of the surfactant, the fluorescent dye and the volatile organic solvent, and relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step I, a dosage of the surfactant lecithin 95% is 45-50 mg; relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step I, a dosage of the surfactant cholesterol is 5-6 mg; relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step I, a dosage of the fluorescent dye is 0.1-1 mg; relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step I, a dosage of the volatile organic solvent is 100-300 μL; the volatile organic solvent in the step I is chloroform or a mixed solvent of the chloroform and methanol; the stirring in the step I is carried out away from light; and the ultrasonic processing in the step I has a frequency of 20-40 kHz, a power of 40-90 W and ultrasonic time of 5-10 min.

7. The preparation method according to claim 6, wherein a molar ratio of the PFC to the molecular targeting small-molecule treatment drug in the step II is 50-1000:1; relative to 5.5 mg of molecular targeting small-molecule treatment drug in the step I, a dosage of the glycerol is 0.1-0.5 g; and relative to 5.5 mg of molecular targeting small-molecule treatment drug in the step I, a dosage of the water is 2-5 mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a size distribution test of a probe.

(2) FIG. 2 is an analysis on an apparent zeta potential of a probe.

(3) FIG. 3 is a Transmission Electron Microscope (TEM) photo of a probe.

(4) FIG. 4 is a 19 F-MRI phantom image of a probe.

(5) FIG. 5 is a multinuclear MRI image after a probe is delivered intravenously to an H1975 tumor-bearing mouse with lung adenocarcinoma.

(6) FIG. 6 shows a measurement of a cell vitality after different nanoprobes are used to incubate an H1975 cell for 24 h.

(7) FIG. 7 shows a change of a weight within 24 d when different nanoprobes are used to treat a transplantation tumor of an H1975 tumor-bearing mouse with lung adenocarcinoma.

(8) FIG. 8 shows a change of a relative tumor volume within 24 d when different nanoprobes are used to treat a transplantation tumor of an H1975 tumor-bearing mouse with lung adenocarcinoma.

(9) FIG. 9 shows a tumor histological measurement when different nanoprobes are used to treat a transplantation tumor of an H1975 tumor-bearing mouse with lung adenocarcinoma for evaluation of an anti-tumor efficacy.

DESCRIPTION OF THE EMBODIMENTS

(10) The present invention is further described below in combination with specific embodiments and FIG. 1 to FIG. 9.

Embodiment 1

(11) Preparation Method of 19 F-MR/Fluorescence Multi-Mode Molecular Imaging and Drug Loading Diagnosis-Treatment Integrated Nanoprobe

(12) I. Uniformly mix a molecular targeting treatment drug (small-molecule) with a surfactant and a fluorescent dye, the surfactant coating the molecular targeting treatment drug (small-molecule) physically because of a surface tension; and thereafter, dissolve into a volatile organic solvent, stir for 10 min at a room temperature, evaporate the organic solvent via a rotary evaporator to dryness, then dry for 12 h in a vacuum drying oven at 37° C., and at last, disperse into water via ultrasonic processing to obtain a mixture for later use.

(13) II, Uniformly disperse PFC into the mixture obtained in the step I, add glycerol and the water dropwise, and mix for 5 min in a high-pressure homogenizer to prepare into an emulsion containing the 19 F-MR/fluorescence multi-mode molecular imaging and drug loading diagnosis-treatment integrated nanoprobe.

(14) III, Remove a component, which is uncoated effectively, with a dialysis manner in an environment at a pH of 7.4 and the room temperature from the emulsion obtained in the step II to obtain the 19 F-MR/fluorescence multi-mode molecular imaging and drug loading diagnosis-treatment integrated nanoprobe.

(15) Relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step I, a dosage of the surfactant lecithin 95% (PC) was 45-50 mg.

(16) Relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step I, a dosage of the cholesterol was 5-6 mg.

(17) Relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step I, a dosage of the fluorescent dye was 0-1 mg.

(18) Relative to 5.5 mg of the molecular targeting small-molecule treatment drug in the step 1, a dosage of the volatile organic solvent was 100-300 μL.

(19) The volatile organic solvent in the step I was chloroform or a mixed solvent of the chloroform and methanol.

(20) The stirring in the step I was carried out away from light.

(21) The ultrasonic processing in the step I had a frequency of 20-40 kHz, a power of 40-90 W and ultrasonic time of 5-10 min.

(22) Further, a molar ratio of the PFC to the molecular targeting small-molecule treatment drug in the step II was (70-200): 1.

(23) Relative to 5.5 mg of molecular targeting small-molecule treatment drug in the step 1, a dosage of the glycerol was 0-0.5 g.

(24) Relative to 5.5 mg of molecular targeting small-molecule treatment drug in the step I, a dosage of the water was 2-5 mL.

(25) Or the water was ultrapure water.

(26) By observing the nanoparticle in Embodiment 1 with a TEM, it is found that the prepared drug-loading nanoparticle is of a spherical structure with a uniform and stable size and has an average particle size of about 115 nm (FIG. 3). With dynamic light scattering for measurement, the size distribution is within 110-130 nm (FIG. 1), which complies with the result observed by the TEM; and the apparent zeta potential is about +62 mV (FIG. 2).

Embodiment 2

(27) Application of 19 F-MR/Fluorescence Multi-Mode Molecular Imaging and Drug Loading Diagnosis-Treatment Integrated Nanoprobe (Prepared in Embodiment 1) in Taking as Imaging Contrast Agent

(28) 1. During 19F-MRI phantom test of the probe, the nanoprobe solution prepared in Embodiment 1 was mixed with a 1.7% Agrose sol to prepare into a phantom sample respectively having a final concentration of 7.67 mmol/L, 15.35 mmol/L, 30.69 mmol/L, 61.38 mmol/L and 122.76 mmol/L; and a 19 F imaging capability was tested, with a result shown in FIG. 4. As can be seen from the result, the 19 F signal enhancement capability of the probe is positively correlated with the concentration of the sample; and along with an increase in the concentration of the sample, a signal is enhanced linearly.

(29) 2. MRI after the probe is delivered intravenously

(30) First of all, a naked mouse was anesthetized by isoflurane; and upon successful anesthetization, three naked mice were taken successively, delivered with 300 μL of the probe intravenously according to a numbering sequence, and fixed in a 1H/19 F double-tuned body coil, where a body of each naked mouse was parallel to a scanning bed, a head entered first and a head end was consistent with a direction of a main magnetic field, so that a subcutaneous transplantation tumor was located within a same horizontal line of the center of the 1H119 F body coil. During scanning, a life monitoring system was used to monitor a respiratory rate, and an oxygen supply and a body temperature of 37±0.5° C. for the experimental animal were maintained. The 1H19 F double-tuned body coil was used; a T1W RARE sequence was used for anatomical localization imaging, with an imaging parameter TR=820 ms, TE=12 ms, NA=4, RARE factor=8, matrix=256*256, FOV=38.4*38.4 mm2, and slice thickness=1.5 mm. A 19F RARE sequence was co-localized with 1H, with an imaging parameter TR=2000 ms, TE=10 ms, NA=128, RARE factor=32, matrix=64*64, FOV=38.4*38.4 mm2, and slice thickness=3 mm. The total scanning time was 12 min.

(31) After the probe was delivered to the body of the healthy naked mouse intravenously, an MR multinuclear fused image was shown in FIG. 5, with a highlight in the tumor being a multinuclear imaging signal after the probe of the present invention was delivered intravenously. As can be seen from the figure, the probe of the present invention has an excellent 19 F-MRI capability.

Embodiment 3

(32) Evaluation on Efficacy of Probe (Prepared in Embodiment 1) in Targeted Treatment

(33) 1. Evaluation on Efficacy of Probe (Prepared in Embodiment 1) at Cell Level

(34) A cell was prepared into a single cell suspension by using a culture solution containing 10% of fetal calf serum, and as per 200 μL cells for each pore, the single cell suspension was inoculated to a 96-pore plate, with 200 μL for each pore. Upon overnight incubation, the nanoprobe having different concentrations was added. After 24 h, an MTT solution and DMSO were added, and a mixed solution was placed onto a shaker and vibrated for 15 min at a low speed. A light absorption value of each pore was measured at OD570 nm by using an eliasa.

(35) As can be seen from FIG. 6, with the incubation of PFCE, all cells have a viability of greater than 90%, which indicates that the PFCE does not produce a significant cytotoxicity to an H1975 cell. The PFCE-AZD9291 has a higher level of cytotoxicity than the PFCE and saline at 24 h. The PFCE-AZD9291 shows obvious concentration reliance for an in-vitro anti-tumor activity of the H1975 cell.

(36) 2. Evaluation on Efficacy of Probe (Prepared in Embodiment 1) in Targeted Treatment of Transplantation Tumor of H1975 Tumor-Bearing Mouse with Lung Adenocarcinoma

(37) An H1975 cell at 10 6 was inoculated to a right leg of a Balb/c female naked mouse that was 5-6 weeks old, thus establishing a transplantation tumor. Upon the inoculation of 20 days, when the transplantation tumor was grown to 6-8 mm, a follow-up experiment was carried out. All mice were divided into three groups (n=5), and the mouse in each group was respectively given with the saline, the PFCE and the PFCE-AZD9291. The drug was administered at 50 μL/time every three days. A digital caliper was used to measure a size of the tumor every two days, and a volume was calculated via a formula (L×W2)/2, where the L was a longest diameter of the tumor, and the W was a shortest diameter. Meanwhile, an electronic scale was used to weigh a weight of the mouse. After monitoring of 24 d, the mouse was killed, and the tumor was taken out for histological and immunohistochenmical experiments. A Hematoxylin-Eosin staining (HE staining) tissue slice was used to identify a histopathological change. In order to evaluate proliferation and apoptosis of a tumor cell, proliferating cell nuclear antigen (Ki67) staining and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining were carried out.

(38) With analysis on a tumor growth curve, it may be seen that after the tumor of the mouse is treated by the PFCE-AZD9291 nanoparticle, the volume increase is lower than the other two groups, which indicates that the designed nanoparticle loaded with a chemotherapeutic drug has a good anti-tumor effect (FIG. 7). Additionally, there is no statistically significant difference in weight among all groups during treatment (FIG. 8). Furthermore, the HE staining of the tumor slice shows that the number of necrocytosis in the PFCE-AZD9291 group is greater than that in the other two groups, the TUNEL staining shows that the PFCE-AZD9291 group has a great number of apoptotic cells (green fluorescence), and the proliferating cell nuclear antigen (Ki67) staining shows that the number of proliferated cells in the PFCE-AZD9291 group is obviously smaller than that in the saline group and the PFCE group, all of which indicate that the drug-loading nanoparticle of the present invention has the excellent targeting anti-tumor effect (FIG. 9).