CN105079826A - Preparation method and application of RGD@BBN double-targeted MR (magnetic resonance)/optical dual-mode molecular probe - Google Patents

Preparation method and application of RGD@BBN double-targeted MR (magnetic resonance)/optical dual-mode molecular probe Download PDF

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CN105079826A
CN105079826A CN201510585362.6A CN201510585362A CN105079826A CN 105079826 A CN105079826 A CN 105079826A CN 201510585362 A CN201510585362 A CN 201510585362A CN 105079826 A CN105079826 A CN 105079826A
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molecular probe
rgdbbn
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CN105079826B (en
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杨蕊梦
江新青
唐文洁
韦敏燕
杨惠康
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Guangzhou First Peoples Hospital
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Abstract

The invention discloses a preparation method and application of an RGD@BBN double-targeted MR (magnetic resonance)/optical dual-mode molecular probe, wherein the RGD@BBN double-targeted MR/optical dual-mode molecular probe is prepared by the following steps: coating water-soluble superparamagnetic iron oxide ( SPIO) particles in hydrophilic core of lipidosome and coating oil-soluble QDs particles in hydrophobic lipid bilayer of the lipidosome by taking lipidosome as a carrier, and linking a double-targeted RGD@BBN ligand by virtue of a post-interpolation method. The RGD@BBN contained in the dual-mode molecular probe disclosed by the invention is interpolated and immobilized on the surface of the lipid bilayer by virtue of self-assembly, and the preparation method is mild in condition and is efficient and practical. According to the invention, a relatively independent space is coated by two different image contrast agents without changing the properties of the image contrast agents; and the prepared double-target dual-mode molecular probe, which simultaneously has RGD and BBN targeting functions, has targeting functions on RGD or (and) BBN receptor-positive tumors.

Description

The Synthesis and applications of the two targeting MR/ optics bimodal molecular probe of a kind of RGDBBN
Technical field
The invention belongs to molecular image field, be specifically related to the preparation method and application of the two targeting MR/ optics bimodal molecular probe of a kind of RGDBBN.
Background technology
The different morphology and function formation method of multi-mode molecule imaging technical combinations, MR/ optical imagery, SPECT-CT/MR imaging, optics/CT imaging, optics/PET imaging, PET/CT imaging, CT/SPECT imaging and MRI/PET imaging are common multi-modality imaging technical combinations means, and what wherein range of application was the widest is MR/ optical imagery.
In molecular probe builds, the conventional tumor cells image probe for single target spot, its cell surface target spot (receptor) must not expressed or low expression in normal structure at tumor tissue specificity high expressed.But, in the growth course of tumor, tumor cell surface present heterogeneity and inhomogeneity by cognition, even if same tumor patient, the acceptor type of its expression of tumor tissue or expression also can be inconsistent.In addition, two or more receptors also can be expressed in a kind of surface of tumor cell simultaneously.Therefore, the application of two target spot or Mutiple Targets molecular probe more can strengthen the targeting of molecular probe affinity groups, thus improves the accuracy rate of early diagnosis of tumor.
Magainin (Bombesin, BBN) is a kind of containing 14 amino acid whose polypeptide.GRPR is one of hypotype of BBN receptor, and it presents high expressed in kinds of tumors tissue (as small cell lung cancer, colon cancer, carcinoma of prostate, cancer of pancreas and breast carcinoma etc.), and in the low expression of normal structure or do not express.This characteristic becomes a kind of tumour-specific target spot, for diagnosing tumor and targeted therapy.
α vβ 3be the important forming member of integrin family, play an important role in the control etc. of tumor growth, angiogenesis, local infiltration, metastatic potential.Integral protein α vβ 3receptor high expressed in invasive tumor (as late period glioma, breast carcinoma, carcinoma of prostate, malignant melanoma, ovarian cancer) and tumor neogenetic blood vessels, and do not express in already present blood vessel and normal structure or express very low, this has become a target spot had a great attraction of Tumor receptor imaging.
In recent years, a series of nucleic ( 99mtc, 111in, 90y, 64cu, 177lu, 18f, 68ga etc.) BBN or RGD of labelling or both analog be widely used in clinical before and the tumor imaging of clinical stage and Therapy study.The radionuclide imaging that multiple BBN and RGD is correlated with is also in breast carcinoma imaging research.There is report to be coupled on RGD-BBN heterodimer molecule by bifunctional linking reagent NOTA, use 18f, 64cu, 68ga difference labelling RGD-BBN heterodimer is studied for the microPET of breast carcinoma, and result proves that the molecular probe of radioisotope labeling RGD-BBN can delicately to integrin alpha vβ 3carry out video picture with the tumor of any one receptor high expressed of GRPR, and have higher tumor uptake compared with monomer whose, this conclusion is that breast carcinoma " two target spot " imaging of RGD-BBN provides solid theoretical basis.But up to now, the research about the numerator mediated tumor amboceptor targeted imaging of RGD-BBN heterodimer is few, and is all at nuclear medicine or optical field.
Summary of the invention
For solving the shortcoming and defect part of prior art, primary and foremost purpose of the present invention is to provide the two targeting MR/ optics bimodal molecular probe of a kind of RGDBBN.
Another object of the present invention is to provide the above-mentioned RGDBBN preparation method of two targeting MR/ optics bimodal molecular probe.
Another object of the present invention is to provide the above-mentioned RGDBBN application of two targeting MR/ optics bimodal molecular probe.
For achieving the above object, the present invention adopts following technical scheme:
The two targeting MR/ optics bimodal molecular probe of a kind of RGDBBN, this bimodal molecular probe is wrap to carry oil-soluble quantum dot and Superparamagnetic Iron Oxide nanoparticle and in the MR/ optics bimodal liposome nanoparticle of the two targeting part of finishing RGDBBN simultaneously, it is using liposome as carrier, carry Superparamagnetic Iron Oxide nanoparticle (SPIO) at liposome hydrophilic core bag, carry oil-soluble quantum dot and at the two targeting part of finishing RGDBBN at the hydrophobic phospholipid bilayer bag of liposome.
Above-mentioned bimodal molecular probe possesses the fluorescence property of superparamagnetism and infrared band simultaneously, and possesses RGD and BBN target function.Dynamic light scattering, transmissioning electric mirror determining result show elaioplast nanometer particle be spherical, size is more homogeneous, good stability.
The preparation method of the two targeting MR/ optics bimodal molecular probe of described RGDBBN, comprises the steps:
(1) prepare Superparamagnetic Iron Oxide nanoparticle (SPIO), and be mixed with Superparamagnetic Iron Oxide nanoparticle solution;
(2) the oil-soluble quantum dot of 200 ~ 700 mass parts soybean phospholipids, 100 ~ 200 mass parts cholesterol and 1 ~ 2 mass parts is dissolved in dehydrated alcohol and chloroform mixed liquor, then reduction vaporization is to eliminate organic solvent, makes lipid form uniform thin film on reaction vessel inwall; Add the Superparamagnetic Iron Oxide nanoparticle solution that step (1) is obtained, the lipid be attached on reaction vessel inwall fully comes off dissolving by vortex a period of time, rapid Ultrasonic Pulverization, obtained magnetic/fluorescent dual module state nano-probe (lipo (QDs)-SPIO); Superparamagnetic Iron Oxide nanoparticle wherein in Superparamagnetic Iron Oxide nanoparticle solution is 5 ~ 8 mass parts;
(3) by sulfhydrylation arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) and DSPE-PEG 2000-MAL (DSPE-PEG-maleimide) fully mixes, room temperature reaction 24 ~ 48h, dialyses 2 ~ 3 days, to remove unnecessary sulfhydrylation RGD, obtains DSPE-PEG 2000-RGD;
By sulfhydrylation Magainin (Bombesin, BBN) and DSPE-PEG 2000-MAL fully mixes, room temperature reaction 24 ~ 48h, dialyses 2 ~ 3 days, to remove unnecessary sulfhydrylation BBN, obtains DSPE-PEG 2000-BBN;
Insertion after adopting, by 1 ~ 4 mass parts DSPE-PEG 2000-RGD, 1 ~ 4 mass parts DSPE-PEG 2000-BBN, 2 ~ 4 mass parts DSPE-PEG 2000magnetic/fluorescent dual module state nano-probe that (DSPE-PEG 2000) obtains with 70 ~ 90 mass parts steps (2) fully mixes, room temperature reaction 24 ~ 48h; Use again MiniExtruder liposome extruder successively from large aperture to small-bore (successively from aperture 0.8 μm, 0.4 μm, 0.2 μm) extrude back and forth, obtain the two targeting MR/ optics bimodal molecular probe of described RGDBBN.
The concrete steps of step (1) are: by the FeCl of 3100 ~ 6140 mass parts 36H 2the FeSO of O and 2100 mass parts 47H 2o, is dissolved in 40 parts by volume deionized waters; Solution is transferred in reaction vessel, puts into small magnet; Pass into nitrogen, heating in water bath, and add HCl solution adjustment pH to 2 ~ 4, after mix homogeneously, add 20 ~ 40 parts by volume strong aqua ammonia, after reaction a period of time, be warming up to 70 ~ 100 DEG C, continue reaction 1h, obtain pitchy turbid solution; Be 7 ~ 8 with deionized water wash to pH, obtain Superparamagnetic Iron Oxide nanoparticle (SPIO); To be dissolved in certain deionized water and ultrasonic, to be obtained Superparamagnetic Iron Oxide nanoparticle solution.
In the concrete steps of step (1):
Described water bath heating temperature is preferably 20 ~ 40 DEG C;
Described HCl solution concentration is preferably 0.1 ~ 0.5mmol/L;
Described strong aqua ammonia mass concentration is preferably 20 ~ 30%, heats up after adding strong aqua ammonia reaction 30min again;
The preferred 1h of described ultrasonic time.
In step (2):
In described dehydrated alcohol and chloroform mixed liquor, dehydrated alcohol and chloroform volume ratio are preferably 1 ~ 3:1; Mixeding liquid volume does not need to be particularly limited to;
Described reduction vaporization temperature is preferably 35 ~ 65 DEG C;
Described vortex time is preferably 15 ~ 30min;
Described ultrasound condition is preferably 20KHz, and ultrasonic time is preferably 30 ~ 40min.
In step (3):
Described sulfhydrylation arginine-glycine-aspartic acid and DSPE-PEG 2000the mass ratio of-MAL is (1.0 ~ 1.9): 4.0;
Described sulfhydrylation Magainin and DSPE-PEG 2000the mass ratio of-MAL is (1.4 ~ 2.9): 4.0;
Described dialysis refers to carries out separation and purification with the bag filter of molecular cut off 2kDa, and after 12h, every 4h changes dialysis medium, dialyses continuously 2 ~ 3 days, to remove unnecessary sulfhydrylation RGD and sulfhydrylation BBN;
Described DSPE-PEG 2000-RGD, DSPE-PEG 2000-BBN, DSPE-PEG 20003:2:3:80 is preferably with the mass parts ratio of magnetic/fluorescent dual module state nano-probe (lipo (QDs)-SPIO).
The extrusion passes in the described each aperture of MiniExtruder liposome extruder is preferably more than 20 times.
The two targeting MR/ optics bimodal molecular probe of described RGDBBN is applied with image nanoparticle assembling form, can be used as image molecular probe and applies in field of medicaments.
Compared with prior art, the present invention has the following advantages and beneficial effect:
(1) bimodal molecular probe of the present invention, using liposome as carrier, core loads hydrophilic SPIO, phospholipid bilayer loads oil-soluble QDs: liposome vectors is phospholipid, cholesterol is filmogen, employing film dispersion method obtains, and has excellent biocompatibility and biodegradability; Contained QDs granule is before film forming, adds simultaneously, be directly connected on thin film after film forming in phospholipid, these filmogens of cholesterol, and its tissue penetration depths is comparatively large, fluorescence intensity is high; Contained SPIO granule is add after film forming, and adopt Probe Ultrasonic Searching to be wrapped in liposome hydrophilic core, it has significant T2/T2* relaxation rate.
(2) the contained RGDBBN of bimodal molecular probe of the present invention adopts self-assembling method insert and be fixed on phospholipid bilayer surface, and preparation condition is gentle, highly effective.Two kinds of different image contrast agent have been wrapped up relatively independent space, and have not been changed their character by the encapsulation processes that the present invention adopts, and preparation method is simple, highly effective.Two target spot bimodal molecular probes that the present invention prepares, there is RGD and BBN target function simultaneously, to RGD or (with) tumor of BBN receptor positive all has target function, the novel molecular probe be expected to be used as early diagnosis of tumor, detecting curative effect; And this probe has superparamagnetism and fluorescence property, can be used as the New video molecular probe of function admirable, function uniqueness.
Accompanying drawing explanation
Fig. 1 is the two targeting MR/ optics bimodal molecular probe structural models figure of RGDBBN of the present invention.
Fig. 2 is that the two targeting MR/ optics bimodal molecular probe of RGDBBN of embodiment 1 is to the envelop rate of variable concentrations SPIO.
Fig. 3 is the two targeting MR/ optics bimodal molecular probe grain size distribution in aqueous of RGDBBN of embodiment 2.
Fig. 4 is the two targeting MR/ optics bimodal molecular probe shape appearance figure in aqueous of RGDBBN of embodiment 2.
Fig. 5 is the T of the two targeting MR/ optics bimodal molecular probe of RGDBBN of embodiment 3 2mapping schemes.
Fig. 6 is the T47D breast cancer cell prussian blue staining figure of embodiment 4.
Fig. 7 is the MDA-MB-435 breast cancer cell prussian blue staining figure of embodiment 5.
Fig. 8 is DSPE-PEG 2000the structural formula of-RGD.
Fig. 9 is DSPE-PEG 2000the structural formula of-BBN.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.1 mass parts in the present invention: 1 parts by volume=1mg:1mL.
Embodiment 1
A preparation method for the two targeting MR/ optics bimodal molecular probe of RGDBBN, comprises the steps:
(1) Superparamagnetic Iron Oxide nanoparticle (SPIO) solution is prepared:
Take 3100mgFeCl6H 2o and 2100mgFeSO 47H 2o, is dissolved in 40mL deionized water, is fully uniformly mixed dissolving; Above-mentioned solution is transferred in three mouthfuls of round-bottomed flasks, puts into magnetic stir bar, be placed on constant-temperature heating magnetic stirring apparatus; Pass into nitrogen, 25 DEG C of heating in water bath, and the HCl solution adding 0.15mmol/L regulates about pH to 2, after mix homogeneously, add the strong aqua ammonia of 20mL20%, after 30min, be warming up to 70 DEG C, continue reaction 1h, obtain pitchy turbid solution, washing 6 times to pH with deionized water is about 7; After be dissolved in 800mL deionized water, and be placed in ultrasonic washing unit effect 1h, obtain the Superparamagnetic Iron Oxide nanoparticle SPIO solution that concentration is 0.5mg/mL.
(2) film dispersion method is adopted to prepare magnetic/fluorescent dual module state nano-probe (lipo (QDs)-SPIO):
Get the soybean phospholipid of 700mg and the cholesterol of 200mg, with oil-soluble CdSe/ZnS quantum dot (the model Q1605 of 1.0mg, purchased from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd.), add the mixed liquor 60mL be made up of 40mL dehydrated alcohol and 20mL chloroform and raw material is dissolved completely; Above-mentioned reactant is proceeded in round-bottomed flask, flask to be placed on Rotary Evaporators in 35 DEG C of reduction vaporizations, to eliminate organic solvent, make lipid form uniform thin film on flask inwall; Add the SPIO solution 10mL that step (1) is obtained, after vortex 20min, fully come off the lipid be attached on inwall dissolving, rapidly in Probe Ultrasonic Searching ripple pulverizer, power is adjusted to 20KHz, effect 30min, obtained magnetic/fluorescent dual module state nano-probe;
(3) " two target spot " " bimodal " molecular probe (RGDBBN-lipo (QDs)-SPIO) is prepared:
A () is by 1.4mg sulfhydrylation RGD (the biochemical company limited of Shanghai gill) and 4.0mg DSPE-PEG-maleimide (DSPE-PEG 2000-MAL) (U.S. Avanti) fully mix, and adds magnetic stir bar, room temperature reaction 24h, the bag filter retaining 2kDa with molecular weight carries out separation and purification, and every 4h changes dialysis medium, continuously dialysis 2 days, to remove unnecessary sulfhydrylation RGD, obtain DSPE-PEG 2000-RGD (structure as shown in Figure 8);
B () is by 2.1mg sulfhydrylation BBN (the biochemical company limited of Shanghai gill) and 4.0mgDSPE-PEG 2000-MAL (U.S. Avanti) fully mixes, and adds magnetic stir bar, room temperature reaction 24h, the bag filter retaining 2kDa with molecular weight carries out separation and purification, and every 4h changes dialysis medium, continuously dialysis 2 days, to remove unnecessary sulfhydrylation BBN, obtain DSPE-PEG 2000-BBN (structural formula as shown in Figure 9);
C () adopts rear insertion, by 1.0mgDSPE-PEG 2000-RGD, 4.0mgDSPE-PEG 2000-BBN, 2.0mgDSPE-PEG 2000magnetic/fluorescent dual module state the nano-probe (lipo (QDs)-SPIO) obtained with 70mg step (2) fully mixes, and adds magnetic stir bar, room temperature reaction 24h; Again with MiniExtruder liposome extruder successively from large aperture to small-bore 0.8 μm, 0.4 μm, 0.2 μm extrude back and forth, the extrusion passes in each aperture is more than 20 times, obtains the two targeting MR/ optics bimodal molecular probe of described RGDBBN.
The two targeting MR/ optics bimodal molecular probe structural models figure of obtained RGDBBN as shown in Figure 1, using liposome as carrier, carry water solublity Superparamagnetic Iron Oxide nanoparticle (SPIO) at hydrophilic core bag, carry oil-soluble quantum dot and at the two targeting part of finishing RGDBBN at hydrophobic phospholipid bilayer bag.
Embodiment 2
A preparation method for the two targeting MR/ optics bimodal molecular probe of RGDBBN, comprises the steps:
(1) Superparamagnetic Iron Oxide nanoparticle (SPIO) solution is prepared:
Take the FeCl6H of 4090mg 2the FeSO of O and 2100mg 47H 2o, is dissolved in 40mL deionized water, is fully uniformly mixed dissolving; Above-mentioned solution is transferred in three mouthfuls of round-bottomed flasks, puts into magnetic stir bar, be placed on constant-temperature heating magnetic stirring apparatus; Pass into nitrogen, 35 DEG C of heating in water bath, and the HCl solution adding 0.35mmol/L regulates about pH to 3, after mix homogeneously, adds the strong aqua ammonia of 30mL25%, after 30min, is warming up to 85 DEG C, continue reaction 1h, obtain pitchy turbid solution; Washing 7 times to pH with deionized water is about 8; After be dissolved in 800mL deionized water, and be placed in ultrasonic washing unit effect 1h, obtain the Superparamagnetic Iron Oxide nanoparticle SPIO solution that concentration is 1.5mg/mL;
(2) film dispersion method is adopted to prepare magnetic/fluorescence two
Get the soybean phospholipid of 400mg and the cholesterol of 150mg, with 1.5mg oil-soluble CdSe/ZnS quantum dot (model Q1605, purchased from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd.), add the mixed liquor be made up of 40mL dehydrated alcohol and 20mL chloroform, raw material is dissolved completely; Above-mentioned reactant is proceeded in round-bottomed flask, flask to be placed on Rotary Evaporators in 50 DEG C of reduction vaporizations, to eliminate organic solvent, make lipid form uniform thin film on flask inwall; Add the SPIO solution 5mL that step (1) is obtained, after vortex 20min, fully come off the lipid be attached on inwall dissolving, rapidly in Probe Ultrasonic Searching ripple pulverizer, power is adjusted to 20KHz, effect 35min, obtained magnetic/fluorescent dual module state nano-probe;
(3) " two target spot " " bimodal " molecular probe (RGDBBN-lipo (QDs)-SPIO) is prepared:
A () is by 1.9mg sulfhydrylation RGD and 4.0mg DSPE-PEG-maleimide (DSPE-PEG 2000-MAL) fully mix, add magnetic stir bar, room temperature reaction 36h, carry out separation and purification with the bag filter of molecular cut off 2kDa, every 4h changes dialysis medium, and dialysis 3 days, to remove unnecessary sulfhydrylation RGD, obtains DSPE-PEG continuously 2000-RGD (structure as shown in Figure 8);
B () is by 2.9mg sulfhydrylation BBN and 4.0mgDSPE-PEG 2000-MAL fully mixes, and adds magnetic stir bar, room temperature reaction 36h, carries out separation and purification with the bag filter of molecular cut off 2kDa, and every 4h changes dialysis medium, and dialysis 3 days, to remove unnecessary sulfhydrylation BBN, obtains DSPE-PEG continuously 2000-BBN (structural formula as shown in Figure 9);
C () adopts rear insertion, by 3.0mgDSPE-PEG 2000-RGD, 2.0mgDSPE-PEG 2000-BBN, 3.0mgDSPE-PEG 2000magnetic/fluorescent dual module state the nano-probe (lipo (QDs)-SPIO) obtained with 80.0mg step (2) fully mixes, and adds magnetic stir bar, room temperature reaction 36h; Again with MiniExtruder liposome extruder successively from large aperture to small-bore 0.8 μm, 0.4 μm, 0.2 μm extrude back and forth, the extrusion passes in each aperture is more than 20 times, obtains the two targeting MR/ optics bimodal molecular probe of described RGDBBN.
Embodiment 3
A preparation method for the two targeting MR/ optics bimodal molecular probe of RGDBBN, comprises the steps:
(1) Superparamagnetic Iron Oxide nanoparticle (SPIO) solution is prepared:
Take the FeCl6H of 6140mg 2the FeSO of O and 2100mg 47H 2o, is dissolved in 40mL deionized water, is fully uniformly mixed dissolving; Above-mentioned solution is transferred in three mouthfuls of round-bottomed flasks, puts into magnetic stir bar, be placed on constant-temperature heating magnetic stirring apparatus; Pass into nitrogen, 40 DEG C of heating in water bath, and the HCl solution adding 0.5mmol/L regulates about pH to 4, after mix homogeneously, adds the strong aqua ammonia of 40mL30%, after 30min, is warming up to 90 DEG C, continue reaction 1h, obtain pitchy turbid solution; Under the adsorption of external magnetic field, washing 10 times to pH with deionized water is about 8; After be dissolved in 800mL deionized water, and be placed in ultrasonic washing unit effect 1h, obtain the Superparamagnetic Iron Oxide nanoparticle SPIO solution that concentration is 2mg/mL;
(2) film dispersion method is adopted to prepare magnetic/fluorescent dual module state nano-probe (lipo (QDs)-SPIO):
Get the soybean phospholipid of 400mg and the cholesterol of 200mg, with 2.0mg oil-soluble CdSe/ZnS quantum dot (model Q1605, purchased from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd.), add the mixed liquor of 60mL dehydrated alcohol and 20mL chloroform, raw material is dissolved completely; Above-mentioned reactant is proceeded in round-bottomed flask, flask to be placed on Rotary Evaporators in 60 DEG C of reduction vaporizations, to eliminate organic solvent, make lipid form uniform thin film on flask inwall; Add the SPIO solution 4mL of step (1), after vortex 30min, fully come off the lipid be attached on inwall dissolving, and rapidly in Probe Ultrasonic Searching ripple pulverizer, power is adjusted to 20KHz, effect 35min, obtained magnetic/fluorescent dual module state nano-probe;
(3) " two target spot " " bimodal " molecular probe (RGDBBN-lipo (QDs)-SPIO) is prepared:
A () is by 1.2mg sulfhydrylation RGD and 4.0mg DSPE-PEG-maleimide (DSPE-PEG 2000-MAL) fully mix, add magnetic stir bar, room temperature reaction 48h, carry out separation and purification with the bag filter of molecular cut off 2kDa, after 12h, every 4h changes dialysis medium, and dialysis 3 days, to remove unnecessary sulfhydrylation RGD, obtains DSPE-PEG continuously 2000-RGD (structure as shown in Figure 8);
B () is by 1.8mg sulfhydrylation BBN and 4.0mgDSPE-PEG 2000-MAL fully mixes, and adds magnetic stir bar, room temperature reaction 48h, carries out separation and purification with the bag filter of molecular cut off 2kDa, and every 4h changes dialysis medium, and dialysis 3 days, to remove unnecessary sulfhydrylation BBN, obtains DSPE-PEG continuously 2000-BBN (structural formula as shown in Figure 9);
C () adopts rear insertion, by 4.0mgDSPE-PEG 2000-RGD, 1.0mgDSPE-PEG 2000-BBN, 4.0mgDSPE-PEG 2000mix with the magnetic/fluorescent dual module state nano-probe (lipo (QDs)-SPIO) of 90.0mg step (2), add magnetic stir bar, room temperature reaction 48h; Again with MiniExtruder liposome extruder successively from large aperture to small-bore 0.8 μm, 0.4 μm, 0.2 μm extrude back and forth, the extrusion passes in each aperture is more than 20 times, obtains the two targeting MR/ optics bimodal molecular probe of described RGDBBN.
Embodiment 4
The breast carcinoma targeting application of the two targeting MR/ optics bimodal molecular probe of RGDBBN.
To the T47D breast cancer cell of logarithmic (log) phase be in 5 × 10 5/ hole is inoculated in 24 well culture plates, 37 DEG C, 5%CO 2condition, hatches after 24 hours, washs three times with PBS liquid, adds RGDBBN-lipo (QDs)-SPIO molecular probe.The Fe concentration that every hole adds is 40 μ g/mL, 1mL, hatching 24h hour.Adopt the method detection molecules probe of prussian blue staining in the distribution situation of cell.Culture plate is taken out, three times are fully washed by PBS solution, it is that 4% paraformaldehyde solution fixes 20 minutes that every hole adds 1mL mass concentration, after wash three times with PBS solution, the mass concentration adding equivalent is leave standstill 5 minutes after 2% potassium ferrocyanide solution and 2%HCl mixed liquor, and under 37 DEG C of conditions, lucifuge hatches 30 minutes, deionized water washes away dyeing liquor, core fast red is redyed, and remove supernatant, PBS washes three times, percentage by volume is adopted to be respectively 50%, each 1 time of 75%, 85%, 95% and 100% Gradient elution using ethanol, transparent 2 times of dimethylbenzene, each 10 minutes; Neutral gum mounting, observes under being placed in inverted microscope and takes pictures.
Embodiment 5
The breast carcinoma targeting application of the two targeting MR/ optics bimodal molecular probe of RGDBBN.
To the MDA-MB-435 breast cancer cell of logarithmic (log) phase be in 5 × 10 5/ hole is inoculated in 24 well culture plates, 37 DEG C, 5%CO 2condition, hatches after 24 hours, washs three times with PBS liquid, adds RGDBBN-lipo (QDs)-SPIO molecular probe.The Fe concentration that every hole adds is 40 μ g/mL, 1mL, hatching 24h hour.Adopt the method detection molecules probe of prussian blue staining in the distribution situation of cell.Culture plate is taken out, three times are fully washed by PBS solution, it is that 4% paraformaldehyde solution fixes 20 minutes that every hole adds 1mL mass concentration, after wash three times with PBS solution, the mass concentration adding equivalent is 2% potassium ferrocyanide solution and mass concentration is leave standstill 5 minutes after 2%HCl mixed liquor, under 37 DEG C of conditions, lucifuge hatches 30 minutes, deionized water washes away dyeing liquor, core fast red is redyed, remove supernatant, PBS washes three times, percentage by volume is adopted to be respectively 50%, 75%, 85%, each 1 time of 95% and 100% Gradient elution using ethanol, transparent 2 times of dimethylbenzene, each 10 minutes, neutral gum mounting, observes under being placed in inverted microscope and takes pictures.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (10)

1. the two targeting MR/ optics bimodal molecular probe of RGDBBN, it is characterized in that, this bimodal molecular probe is wrap to carry oil-soluble quantum dot and Superparamagnetic Iron Oxide nanoparticle and in the MR/ optics bimodal liposome nanoparticle of the two targeting part of finishing RGDBBN simultaneously, it is using liposome as carrier, carry Superparamagnetic Iron Oxide nanoparticle at liposome hydrophilic core bag, carry oil-soluble quantum dot and at the two targeting part of finishing RGDBBN at the hydrophobic phospholipid bilayer bag of liposome.
2. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 1, is characterized in that, comprise the steps:
(1) prepare Superparamagnetic Iron Oxide nanoparticle, and be mixed with Superparamagnetic Iron Oxide nanoparticle solution;
(2) be dissolved in dehydrated alcohol and chloroform mixed liquor by 200 ~ 700 mass parts soybean phospholipids, 100 ~ 200 mass parts cholesterol and 1 ~ 2 mass parts oil-soluble quantum dot, reduction vaporization, makes lipid form uniform thin film on reaction vessel inwall; Add the Superparamagnetic Iron Oxide nanoparticle solution that step (1) is obtained, the lipid be attached on reaction vessel inwall fully comes off dissolvings by vortex a period of time, and Ultrasonic Pulverization, obtains magnetic/fluorescent dual module state nano-probe; Superparamagnetic Iron Oxide nanoparticle wherein in Superparamagnetic Iron Oxide nanoparticle solution is 5 ~ 8 mass parts;
(3) by sulfhydrylation arginine-glycine-aspartic acid and DSPE-PEG 2000-MAL mixes, and room temperature reaction 24 ~ 48h, dialyses 2 ~ 3 days, obtain DSPE-PEG 2000-RGD;
By sulfhydrylation Magainin and DSPE-PEG 2000-MAL mixes, and room temperature reaction 24 ~ 48h, dialyses 2 ~ 3 days, obtain DSPE-PEG 2000-BBN;
By 1 ~ 4 mass parts DSPE-PEG 2000-RGD, 1 ~ 4 mass parts DSPE-PEG 2000-BBN, 2 ~ 4 mass parts DSPE-PEG 2000magnetic/fluorescent dual module state the nano-probe obtained with 70 ~ 90 mass parts steps (2) mixes, room temperature reaction 24 ~ 48h; Again by MiniExtruder liposome extruder successively from aperture 0.8 μm, 0.4 μm, 0.2 μm extrude back and forth, obtain the two targeting MR/ optics bimodal molecular probe of described RGDBBN.
3. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 2, is characterized in that, by the FeCl of 3100 ~ 6140 mass parts 36H 2the FeSO of O and 2100 mass parts 47H 2o, is dissolved in 40 parts by volume deionized waters; Solution is transferred in reaction vessel, puts into Magnet; Pass into nitrogen, heating in water bath, and add HCl solution adjustment pH to 2 ~ 4, after mix homogeneously, add 20 ~ 40 parts by volume strong aqua ammonia, after reaction a period of time, be warming up to 70 ~ 100 DEG C, continue reaction 1h, obtain pitchy turbid solution; Be 7 ~ 8 with deionized water wash to pH, obtain Superparamagnetic Iron Oxide nanoparticle; To be dissolved in certain deionized water and ultrasonic, to be obtained Superparamagnetic Iron Oxide nanoparticle solution.
4. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 3, is characterized in that, in the concrete steps of step (1):
Described water bath heating temperature is 20 ~ 40 DEG C;
Described HCl solution concentration is 0.1 ~ 0.5mmol/L;
Described strong aqua ammonia mass concentration is 20 ~ 30%, heats up after adding strong aqua ammonia reaction 30min again;
Described ultrasonic time is 1h.
5. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 2, is characterized in that, in step (2):
In described dehydrated alcohol and chloroform mixed liquor, dehydrated alcohol and chloroform volume ratio are 1 ~ 3:1;
Described reduction vaporization temperature is 35 ~ 65 DEG C;
Described vortex time is 15 ~ 30min;
Described ultrasound condition is 20KHz, and ultrasonic time is 30 ~ 40min.
6. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 2, is characterized in that, the sulfhydrylation arginine-glycine-aspartic acid described in step (3) and DSPE-PEG 2000the mass ratio of-MAL is (1.0 ~ 1.9): 4.0; Described sulfhydrylation Magainin and DSPE-PEG 2000the mass ratio of-MAL is (1.4 ~ 2.9): 4.0.
7. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 2, it is characterized in that, dialysis described in step (3) refers to carries out separation and purification with the bag filter of molecular cut off 2kDa, after 12h, every 4h changes dialysis medium, dialyses continuously 2 ~ 3 days.
8. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 2, is characterized in that, DSPE-PEG described in step (3) 2000-RGD, DSPE-PEG 2000-BBN, DSPE-PEG 2000be 3:2:3:80 with the mass parts ratio of magnetic/fluorescent dual module state nano-probe.
9. the preparation method of the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 2, it is characterized in that, the extrusion passes in each aperture of MiniExtruder liposome extruder described in step (3) is more than 20 times.
10. the two targeting MR/ optics bimodal molecular probe of RGDBBN according to claim 1 is applied in field of medicaments as image molecular probe.
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