CN104483296B - Breast cancer molecular probe and its manufacturing method - Google Patents

Abstract

The invention discloses a kind of breast cancer molecular probes, are made of signal component and affine component, it is characterised in that its is affine, and component is the component specifically bound to breast carcinoma stem cell target spot；Signal component is made of near-infrared fluorescent signal element and magnetic resonance signal unit.Preparation method is：The specific surfaces marker monoclonal antibody of semiotic function unit and breast carcinoma stem cell is coupled using chemical covalent coupling method, using purifying.The present invention is the molecular probe to breast carcinoma stem cell multiple target point specific bond, it can be solved to breast carcinoma stem cell in physical examination survey and quantitative analysis by the molecular probe, foundation is provided for solid tumor stem cell imaging diagnosis and targeted therapy curative effect evaluation new strategy, it is provided " new model " for early diagnosis of tumor and classification, precise information and appraisal procedure are provided for oncotherapy, and further to solve the problems, such as that Preventive pad determines diagnostic message basis.

Description

Breast cancer molecular probe and its manufacturing method

Technical field

The invention belongs to medical imaging technology fields, and specifically a kind of molecular probe, especially a kind of breast cancer are divided Sub- probe, the imaging for medically breast cancer diagnosis and treatment.

Background technology

Breast cancer is one of the most common malignant tumors in women, and incidence accounts for the 7-10% of the various malignant tumours of whole body, Women is only second to uterine cancer, it has also become threatens the Etiological of WomanHealth.It is that one kind being usually happened at breast galandular epithelium group It knits, seriously affects one of the most common malignant tumour of the physically and mentally healthy even threat to life of women.Breast cancer male is rare, only about The mammary gland patient of 1-2% is male.

The principle of reatment of breast cancer is clinically first choice with operative treatment to morning, mid-term, based on radical excision. Middle and advanced stage is based on complex treatment.The injury and pain that the side effect of Radiotherapy chemotherapy in treatment of cancer is brought to patient's normal body Hardship is obvious to all.

Treatment for cancer, it is early to find that the effect of early treatment is better than treatment of late stage.However, the early stage of breast cancer can nothing Symptom may show part and constitutional symptom with progression of the disease.Such as lump, pain, skin of breast change, mammary gland wheel Corridor change, nipple-areola change, nipple discharge, regional lymph nodes enlargement and DISTANT METASTASES IN performance etc..Therefore, for breast cancer Early stage Accurate Diagnosis, be the early basis for finding and effectively treating and guarantee.There are many diagnostic method of breast cancer, conventional method master Including：X-ray diagnosis, Ultrasonography, cytology and histodiagnosis etc., although these methods are examined in the routine of breast cancer Important function is played in survey, but its detection is all that the dissection in disease whole latter stage changes, it is difficult to realize morning truly Phase diagnoses.

Molecular imaging is to use High-precision image imaging technique, non-invasively in vivo participating in physiology or pathologic process The variation of molecule, gene and cell carry out visual qualitative and quantitative detection.Molecular imaging main application fields are swollen Tumor, elementary tactics is can be with the molecular probe of target specific bond (by signal component and affine component two to internal introduce Part is constituted), after signal component in direct detection probe or the outer effect in the cell of indirect detection molecular probe Product obtains molecular information, to reach tracer and diagnostic purpose.Molecular imaging is led in true, complete physiological environment It crosses image and is directly monitored by cell and molecular pathway, real time imagery is carried out to generation, the evolution of biological activity.Molecular image skill Art can be preferably in the mechanism and feature of Molecular level study disease compared with conventional method, can be with early stage on live body, continuous Therapy mechanism and curative effect are observed in ground, are a kind of real cancer early detection means, to improving diagnosing and treating effect, reduce multiple Hair rate, reducing the death rate all has weight meaning.

Key technology in molecular image is the preparation and application of molecular probe.Molecular probe refers to a certain particular organisms Molecule (such as albumen, DNA and RNA) has labeled compound molecule that is specificity and can carrying out in vivo or in vitro tracer, these Labeled compound molecule can reflect the amount or function of its target biomolecule in body or in vitro.Molecular probe is usually by signal component It is constituted with affine component two parts.Signal component refers to pair that can be generated iconography signal and can be detected by high-precision imaging technique Than agent or marker part (such as radionuclide, fluorescein or paramagnetism molecule), affine component is and imaging target spot specificity In conjunction with part (such as ligand or antibody).

The chemical coupling method such as Li Xubin, by superparamagnetic iron oxide particle (SPIO) and Somat analog Octreotide (OCT) is coupled, and prepares the magnetic resonance point of the specific binding to the long-living hormone receptor of breast cancer cell epidermis (SSTR) Sub- probe SPIO-OCT, the molecular probe cell positive mark rate reach 96.15% (Peking University's journal medicine, Vol.41No.2Apr.2009)。

Zhu Yuan etc. for target spot, is prepared using paramagnetic particles gadolinium as carrier with breast cancer epidermal growth factor acceptor 2 (HER2) Magnetic resonance (MR) molecular probe, provide imaging evidence by MR targeted imagings for breast cancer individualized treatment.Material and side The fluorescence labeling probe FITC-LTVSPWY and Gadolinium-DTPA (Gd- for HER2 that method is prepared early period using seminar DTPA) coupling obtain MR targeted probes (" preparation of breast cancer HER2 targeted molecular probes and external MRI Primary Studies ",《China Medical Imaging magazine》The 5th phases of volume 22 in 2014).

Chinese patent literature CN102399772A disclose to Plasmid DNA carry out fluorescent marker, obtain HER2, TOP2A, AGTRE gene probes.

However, the breast cancer molecular probe announced at present, also common to have following deficiency：(1) obtain molecular image not It can more comprehensively reflect the biological behaviour of breast cancer, diagnosing and treating, breast cancer are carried out based on current molecular probe imaging technique High recurrence rate, chemicotherapy are ineffective.(2) current its imaging of different breast cancer molecular probes respectively has deficiency.Wherein fluorescence at Although as noninvasive, sensibility is high, can carry out being imaged in body real-time multi-target, that there are spatial resolutions is low, anatomical background is unclear Clear weakness.In contrast, magnetic resonance imaging has high spatial resolution and tissue resolving power, noninvasive, radiationless, still Sensibility is relatively poor.(3) most of tumours such as breast cancer are polygenes diseases, average each tumour patient have 8-10 with The related gene unconventionality variation of tumour, the tumour or tumor stem cell targeted therapy majority curative effect of currently employed single target are not It is good.

It with being successfully separated of breast carcinoma stem cell (breast cancer stem cell, BCSC), identifies, and to it The research of physiological action and process, show in breast cancer cell there is a part have spontaneous update, multi-lineage potential with And cell subsets --- the breast carcinoma stem cell of height tumorigenesis ability.Breast carcinoma stem cell accounting in breast cancer tissue is small (flat About 2-9%), absolute content is less in transfer stove or lymphnode metastatic, therefore its detection difficulty is big.But itself and tumors invading Attack transfer, recurrence and treatment resist it is closely related (" progress of breast carcinoma stem cell ", Liu Mingming etc.,《Cancer in China is miscellaneous Will》, the 8th phase of volume 23 in 2013).It was published according on 2 13rd, 2012《Stem cell》An article on periodical, the U.S. adds The researcher in state university San Francisco branch school reports radiotherapy and is killing tumour cell simultaneously for the first time, also can be by other Cancer cell is changed into the breast carcinoma stem cell for resisting treatment.And some researchers think that breast carcinoma stem cell is breast cancer relapse Exclusive source.Although however, the separation and discriminating of breast carcinoma stem cell, and many is had been achieved for the research that it is acted on Scientific achievement, but do not have been reported that the molecular probe to breast carcinoma stem cell targeted imaging also, therefore providing one kind can be to breast The molecular probe of gland cancer stem cell target imaging is breast cancer to solve breast carcinoma stem cell living imaging and quantitative analysis Early diagnosis and curative effect evaluation provide new method, provide new strategy for breast cancer targeted therapy, are of great significance.

Invention content

In view of this, the purpose of the present invention is to provide a kind of novel breast cancer molecular probe, by dry to breast cancer Cell-targeting molecular imaging provides new method for the early diagnosis and curative effect evaluation of breast cancer, is breast carcinoma stem cell and/or breast The oncotherapy of gland cancer targeting provides new strategy.

The technology that the present invention the uses case for：A kind of breast cancer molecular probe, is made of signal component and affine component, It is characterized in that its affine component is the component specifically bound to breast carcinoma stem cell target spot, signal component is by near-infrared fluorescent Signal element and magnetic resonance signal unit composition.Although the signal component of breast cancer molecular probe can be existing molecular imaging In existing signal component, but the present invention is in view of influence problem of the radiation to live body, and for solve single fluorescence imaging and Deficiency of the single magnetic resonance imaging on resolving power and sensibility, the preferred fluorescence of the present invention-magnetic resonance bimodal imaging are that is, described Signal component is made of that the fluorescent material for being suitable for molecular probe is coupled integral signal component with magnetic resonance imaging substance Bimodal molecular probe.

Further, the affine component be breast carcinoma stem cell specific surfaces marker CD44+, ESA+ or Ligand in CD24-.Due on other cell membranes without or rarely CD44+ and ESA+, therefore select this two targets that can make this Probe targeting specific is high.It is preferred that CD44+ and/or ESA+ ligands, especially select specificity preferably CD44 monoclonal antibodies CD44_mAbAnd/or ESA monoclonal antibodies ESA_mAb, using the molecular imaging strategy of Ag-Ab specific binding.

Since common fluorescent penetrates, living tissue ability is weaker, and optimal wavelength of the present invention is in the close red of 700nm-1200nm For outer fluorescent material as signal component, the near-infrared fluorescent in the wave-length coverage is strong to the penetration power of tissue, and raw within the scope of this The autofluorescence of object tissue itself is weaker, to avoid background interference, improves detection sensitivity.Near-infrared fluorescent component can be Organic fluorescent dye is preferably used as fluorescent material with the quantum dot (quantum dots, QDs) with excellent optical, QDs is made of II-VI group or iii-v or I-III-VI races element, diameter about 1nm-10nm, exciting light production can be received The semiconductor nano crystal grain of raw fluorescence, QDs overcome it is previous be usually used in cell and the imaging of biomolecule fluorescent marker have engine dyeing (excitation spectrum is narrow, emission spectrum is very wide and asymmetric for the larger defect of material molecule；Very big overlapping, limits between fluorescence spectra The fluorescence probe quantity that can be applied simultaneously；Photostability is poor, photobleaching and photodissociation easily occurs, photolytic product to biomolecule often Have lethal effect etc.), there is exciting light spectrum width and continuously distributed, can be excited with the light of various different wave lengths；Emit light Compose it is narrow, symmetrically without be overlapped；A variety of advantages such as good light stability, various colors are adjustable.Its medium wavelength 650nm-900nm's is close red Outer QDs is relatively easily-synthesized, and can bear multiple excitation and light emitting, there is lasting photochemical stability, can be in vivo and in vitro Prolonged continuous imaging observation.Simultaneously because the wavelength of fluorescence of near-infrared QDs can carry out essence according to quantum point grain diameter size It really adjusts, therefore is the best probe of visualized distinguishing difference target.

Further, presently preferred embodiments of the present invention is that the QDs of different-grain diameter is marked different targets, in same excitation The fluorescence that different colours can be presented under light, to realize more targeted molecular imagings.

In existing QDs, research finds that QDs containing cadmium (such as CdTe) contains light toxicity, to improve the safety of molecular probe Property, the preferably non-cadmium near-infrared QDs of the present invention, such as InP, CuInS.QDs of the present invention preferably uses Zn-Cu-In-S nuclear quantum dots, most It preferably can also carry out surface inorganic layer (such as ZnS) to the nuclear quantum dot to coat, the nucleocapsid near-infrared QDs of formation can be improved The fluorescence efficiency and photochemical stability of core-shell quanta dots.

Superparamagnetic Iron Oxide (SPIO) and paramagnetism gadolinium can be used for magnetic resonance molecular imaging probe signals component (Gd³⁺).The present inventor has found that SPIO nano particles have very strong light absorption, couple QDs being coupled with it in present study Fluorescence has stronger quenching effect, influences fluorescence imaging effect in QDs bodies.Therefore the present invention preferably paramagnetism gadolinium (Gd³⁺) conduct The MR imaging signal functional units of bimodal molecular probe.

As the preferred embodiment of the present invention, the present invention is by Gd³⁺Work and two functional units of near-infrared QDs, are assembled into advantage The non-cadmium quantum dot nano-particle of near-infrared (paramagnetic QDs, pQDs) that complementary fluorescence efficiency is high, paramagnetism is good is made For the signal component of bimodal probe.The non-cadmium QDs difference of near-infrared of different-grain diameter (corresponding different emission) is chosen simultaneously With Gd³⁺) the different targets of structure bimodal molecular probe label, realize more targeted molecular bimodals imagings.

To realize that more targeted molecular imagings, currently preferred affine component are to be based on specific surfaces marker CD44+ With the ligand of ESA+, by CD44_mAbAnd ESA_mAbTwo kinds of monoclonal antibodies are coupled with the near-infrared pQDs of different wave length respectively, are formed Two kinds of bimodal molecular probes of BCSC targetings, near-infrared pQDS selection the wavelength such as 700nm and 800nm two of different wave length Kind, it is expressed as pQD700, pQD800, accordingly two kinds of bimodal molecular probes of the invention are pQD₇₀₀-CD44_mAbWith pQD₈₀₀-ESA_mAb, or be pQD₈₀₀-CD44_mAbAnd pQD₇₀₀-ESA_mAb。

For bimodal probe, before coupling, magnetic resonance imaging material is needed first to be coupled on fluorescent material surface.Such as It is first passed through before coupling in QDs surface modification Gd3+ chelating agent DOTA, by Gd³⁺It is sequestered in nanometer surfaces QDs.This method by QDs surface modification Gd3+ chelating agent DOTA, provide highdensity Gd³⁺Binding site reduces Gd3⁺Rotation relaxes in advance to improve it Rate improves sensibility.

In a kind of preferred embodiment of the present invention, to hide the capture of reticuloendothelial system, increases probe and follow in vivo The time of ring eliminates the non-specific adsorption in non-lesions position, reduces background signal and false positive, also such as with high molecular material Polyethylene glycol (PEG), acrylic resin (PAA) or polyhydroxy-alcohol etc. are surface modified pQDs.

The present invention also provides the preparation methods of aforementioned breast cancer molecular probe：It will be glimmering using chemical covalent coupling method The specific surfaces marker of light/magnetic resonance dual signal functional unit and breast carcinoma stem cell is coupled, using purifying.

The technique effect and meaning of the present invention is embodied in following aspects：

(1) different from existing disclosed breast cancer molecular probe, the present invention is specifically tied to breast carcinoma stem cell multiple target point The molecular probe of conjunction can be solved to breast carcinoma stem cell in physical examination survey and quantitative analysis by the molecular probe, be entity Tumor stem cell imaging diagnosis and targeted therapy curative effect evaluation new strategy provide foundation, are provided for early diagnosis of tumor and classification " new model " provides precise information and appraisal procedure for oncotherapy, and further to solve the problems, such as that Preventive pad diagnoses surely Information base.

(2) bimodal molecular probe is used, so that fluorescence and MR is imaged and is able to maximize favourable factors and minimize unfavourable ones and have complementary advantages, can be multiplied The quantity and density of signal component, improve the sensibility and resolution ratio of molecular probe, can detect lower loading on single BCSC Even micro BCSC.

(3) position, content and distributions etc. of the BCSC on live body can be obtained in conjunction with bimodal using more targeting technologies Important information, and the important information of BCSC in sentinel lymph node is detected, it is provided accurately for selection, the radiotherapy planning etc. of modus operandi Information provides foundation for personalized treatment.In addition, according to BCSC inactivations and remaining situation, carrys out early stage no-traumatic evaluation treatment and treat Effect, in time adjustment and booster injection, can more effective prevention of breast cancer relapse and transfers to the treatment of the BCSC case.

Description of the drawings

Fig. 1 is that the TEM of quantum dot schemes.

Fig. 2 is the fluorescence emission spectrum of Zn-Cu-In-S and its Zn-Cu-In-S/ZnS core-shell quanta dots.Illustration therein For fluorescence efficiency of the Zn-Cu-In-S/ZnS core-shell quanta dots under different emission.

Fig. 3 is the fluorescence decay figure of Zn-Cu-In-S and Zn-Cu-In-S/ZnS core-shell quanta dots.

ZCIS in Fig. 2,3 is the abbreviation of Zn-Cu-In-S quantum dots.

Fig. 4 is that the internal living body fluorescent of the non-cadmium Zn-Cu-In-S/ZnS core-shell quanta dots of hydrophily is imaged.

Fig. 5 a are that control group (QDs is modified through PAA but do not chelated Gd3+) T1 measures figure；

Fig. 5 b are that the T1 of experimental group (pQDs, i.e. QDs are modified through PAA and chelated Gd3+) is measured；

Fig. 5 c show that wherein I is deionized water signal, and II is not for T1 weighted imagings (TR=100ms, TE=3.1ms) Chelate the QDs signals of Gd3+ probes；III is pQDs signals.

Specific implementation mode

With reference to most preferred embodiment, the invention will be further described by taking bimodal molecular probe as an example, to help reason Solve present disclosure.

1, the preparation of molecular probe

It is prepared by 1.1 paramagnetism quantum dots

1.1.1 the preparation of lipophile quantum dot

The present embodiment uses I-III-VI races element (such as copper, indium, sulphur, zinc etc.), and single point is prepared by high temperature oil phase method Dissipate the Zn-Cu-In-S nuclear quantum dots that property is good and crystallinity is high.To obtain the quantum dot for having more photostability, then to the core amount Son point carries out surface inorganic layer (such as ZnS) and coats, and improves the fluorescence efficiency and photochemical stability of core-shell quanta dots.In design shell The forbidden band of Lattice Matching parameter and Shell Materials that shell and nuclear quantum dot need to be considered when the component and structure of layer is wide The core-shell quanta dots of degree, Rational structure and component have very strong resistant function to extraneous physics, chemical environment, are repaiied in hydrophily Original fluorescence efficiency can be maintained during decorations well.Specific building-up process is as follows：

Zn-Cu-In-S nuclear quantum dot preparation process：Copper acetate (0.1mmol), indium acetate (0.2mmol), vinegar are weighed respectively Sour zinc (0.1mmol) measures 0.5mL oleic acid, is mixed and is dissolved in 10mL octadecylenes.Entire mixed system vacuumizes 30 points Zhong Houtong argon gas is heated to 120 degrees Celsius, until mixed system becomes optical clear, adds 1.0mL dodecyl sulphur Alcohol.Reaction continues to be warming up to 200 degrees Celsius, and adding 0.3mmol sulphur sources, (9.7mg is dissolved in 0.5mL oleyl amines and 1.0mL 18 In alkene), whole system maintains 200 degrees Celsius and reacts 2 hours.

Zn-Cu-In-S/ZnS core-shell quanta dots preparation process：The zinc precursor for first having configured shell, that is, weigh 0.1mmol's Zinc acetate dissolves it in oleyl amine/octadecylene solution of volume ratio (1/4).Take 4.0mL Zn-Cu-In-S obtained above Nuclear quantum dot adds 5mL octadecylenes and 1mL n-dodecyl mercaptans, and logical argon gas is begun to warm up after 30 minutes to be warming up to 220 and take the photograph Family name's degree reinjects the good zinc precursor of configured in advance, and for maintenance system temperature at 220 degrees Celsius, reaction cools to room temperature after 1 hour, Absolute ethyl alcohol is added, centrifugal purification obtains Zn-Cu-In-S/ZnS core-shell quanta dots.

Using high resolution transmission electron microscopy (HRTEM), atomic force microscope (AFM), photoelectron spectroscopy, grain size analysis Instrument, fluorescence and ultraviolet-visible spectrophotometer etc. characterize the pattern of quantum dot, structure, grain size, spectrum etc..

As seen from Figure 1, the size of quantum dot is in 4 rans, size uniformity.

The nuclear quantum dot fluorescence efficiency that do not wrapped up using the core-shell quanta dots ratio after ZnS packages it can be seen from Fig. 2 and 3 Several times are improved, the rate of decay is substantially reduced.

1.1.2 quantum dot is hydrophilically modified

The preparation of amphiphilic macromolecular：Poly- (tertiary butyl acrylate ethylacrylic acid fat methacrylic acid) triblock copolymer Object is with octylame with 1：40 molar ratio mixed dissolution is added coupling agent EDC and reacts 12h in DMF, and obtained mixture dialysis is pure Change, is preserved after freeze-drying for use.

Take suitable lipophile quantum dot powder and amphiphilic three block macromolecule dissolution in chloroform/ethanol mixed solution In, stirring a few hours are to remove chloroform organic solvent.Add suitable PBS buffer solutions, ultrasonic disperse 3 minutes.It is acquired Solution cross 0.22um films to remove largely unbonded macromolecule, solution becomes optical clear after crossing film.Acquired solution is excessively solidifying Glue chromatographic column (G200) further removes unbonded macromolecule, finally obtains purified hydrophily quantum dot.

1.1.3 prepared by paramagnetism quantum dot

By hydrophily quantum dot and DOTA-NH₂Molecule coupling labeled adds GdCl₃Carry out Gd³⁺Chelating obtains paramagnetism amount Sub- point.Specifically preparation process is：According to molar ratio 1:100：4000 by hydrophily quantum dot, DOTA-NH₂And carbodiimide hydrochloride Salt mixes, and reacts at room temperature 2 hours, ultracentrifugation purifies to obtain QDs-DOTA.Again on the basis of the QDs-DOTA obtained, it is added Gd³⁺, room temperature chelatropic reaction 4 hours purifies to obtain paramagnetism quantum dot using ultracentrifugation.

It is different according to nano-quantum point grain size, pQD700, pQD800 are obtained respectively.

To investigate the MR imaging characteristics of paramagnetism quantum dot of the present invention, MR imaging experiments are carried out.As shown in figure 5, Fig. 5 a are Control group (QDs is modified through PAA but do not chelated Gd3+) T1 is measured.Fig. 5 b are that (pQDs, i.e. QDs are modified and chelated through PAA experimental group Gd3+ T1) is measured.The two after comparison, it was found that, the T1 times of pQDs are obviously shortened, and relaxation rate is significantly raised.Fig. 5 c weight for T1 It is imaged (TR=100ms, TE=3.1ms) display, pQDs signal strengths (III) are significantly higher than deionized water (I) and do not chelate Gd3 The QDs (II) of+probe.

It is prepared by the paramagnetism quantum dot of 1.2 targeting breast carcinoma stem cells

By paramagnetism quantum dot and monoclonal antibody molecule CD44_mAb、ESA_mAbCoupling, acquisition can specific recognition breast cancer The paramagnetism quantum dot of stem cell.Specifically preparation process is：According to molar ratio 1:10：4000 by paramagnetism quantum dot, antibody and Carbodiimide hydrochloride mixes, and reacts at room temperature 2 hours.Ultracentrifugation purifies to obtain the paramagnetism quantum of targeting breast carcinoma stem cell Point pQD700-CD44_mAbAnd pQD800-ESA_mAb。

2, live body is tested

Molecular probe in order to verify the present invention targets bimodal imaging effect and safety more for BCSC, carries out as follows Experiment：

The 2.1 internal living body fluorescent imagings based on the non-cadmium Zn-Cu-In-S/ZnS core-shell quanta dots of hydrophily

Hydrophily Zn-Cu-In-S/ZnS core-shell quanta dots cytotoxicity experiment is subjected to first, the results showed that prepared Hydrophily Zn-Cu-In-S/ZnS core-shell quanta dots have good biocompatibility.Then internal fluorescence imaging is carried out Research, the results are shown in Figure 4, the results showed that the non-cadmium Zn-Cu-In-S/ZnS core-shell quanta dots of near-infrared of preparation can live Body fluorescence imaging.

2.2 safety experiment

The molecular probe for configuring various concentration, by cytotoxicity 3- (4,5-dimethylthiazol-2-yl) -2,5- Diphenyltetrazolium bromide (MTT) are tested, and molecular probe of the invention has good biological safety.With Cu content calculations, Cu ion concentrations are incubated altogether at 2 μM, by 96 hours, and cell survival rate is 95% or more.

2.3 imaging sensitivity experiments

To equipped with through bimodal molecular probe label but BCSC numbers it is different (BCSC cell numbers are respectively 1,5,1 × 10¹、50、1×10²、1×10³、1×10⁴、1×10⁵) row fluorescence imaging, the MR T1 imaging respectively of Ep pipes, observe simultaneously more glimmering Light imaging, MR T1 are imaged minimum cell number that can be detected.Experimental data shows the minimum distinguishable of fluorescence imaging mode Imaging cells number is 5；The minimum distinguishable imaging cells number of MR T1 imaging patterns is 50.

2.4 targeting specifics are tested

It is compareed with the breast cancer cell of CD44-/ESA-, BCSC is visited with pQD700-CD44mAb bimodal molecules respectively Needle, pQD700 co-culture be incubated, after washing under fluorescence microscope observe BCSC and CD44-/ESA- breast cancer cell it is glimmering Optical signal, then row MR T1W are imaged respectively, evaluate fluorescence signal and whether magnetic resonance signal is consistent, and judge the targeting of the probe Performance.Above-mentioned experiment is equally carried out to pQD800-ESAmAb bimodals molecular probe.By cell-targeting imaging experiment, this hair Bright bimodal molecular probe has excellent targeting detection result.

Claims (3)

1. a kind of breast cancer molecular probe, is made of signal component and affine component, it is characterised in that its is affine, and component is to breast The component of gland cancer stem cell target spot specific binding；Signal component is by near-infrared fluorescent signal element and magnetic resonance signal unit Composition；

The probe is more targeting bimodal molecular probes, is pQD₁-CD44_mAbAnd pQD₂-ESA_mAb, pQD₁With pQD₂In near-infrared Wave band but wavelength difference；

The affine component is CD44 monoclonal antibodies CD44_mAbAnd/or ESA monoclonal antibodies ESA_mAb；

The fluorescence signal unit is near-infrared quantum dots, and the near-infrared quantum dots are Zn-Cu-In-S nuclear quantum dot outsourcings Include the nuclear shell structure quantum point Zn-Cu-In-S/ZnS core-shell quanta dots of ZnS；The magnetic resonance signal material is paramagnetism gadolinium.

2. the manufacturing method of breast cancer molecular probe described in a kind of claim 1, it is characterised in that：Using chemical covalent coupling side The specific surfaces marker monoclonal antibody of semiotic function unit and breast carcinoma stem cell is coupled by method, using purifying, tool Production procedure is as follows：

（1）The preparation of lipophile quantum dot

Zn-Cu-In-S nuclear quantum dot preparation process：0.1 mmol of copper acetate, 0.2 mmol of indium acetate, zinc acetate are weighed respectively 0.1 mmol measures 0.5 mL oleic acid, is mixed and is dissolved in 10 mL octadecylenes；Entire mixed system vacuumizes 30 minutes Lead to argon gas afterwards, be heated to 120 degrees Celsius, until mixed system becomes optical clear, adds 1.0 mL dodecyl sulphur Alcohol；Reaction continues to be warming up to 200 degrees Celsius, adds 0.3 mmol sulphur sources, the sulphur source is dissolved in 0.5 mL oleyl amines by 9.7mg It is made in 1.0 mL octadecylenes；Whole system maintains 200 degrees Celsius and reacts 2 hours；

Zn-Cu-In-S/ZnS core-shell quanta dots preparation process：The zinc precursor for first having configured shell, that is, weigh the vinegar of 0.1 mmol Sour zinc dissolves it in oleyl amine/octadecylene solution of volume ratio 1/4；4.0 mL Zn-Cu-In-S nuclear quantum dots are taken, then are added Enter 5 mL octadecylenes and 1mL n-dodecyl mercaptans, logical argon gas is begun to warm up after 30 minutes is warming up to 220 degrees Celsius, reinjects The good zinc precursor of configured in advance, for maintenance system temperature at 220 degrees Celsius, reaction cools to room temperature after 1 hour, and anhydrous second is added Alcohol, centrifugal purification obtain Zn-Cu-In-S/ZnS core-shell quanta dots；

（2）Quantum dot is hydrophilically modified

The preparation of amphiphilic macromolecular：It is poly-（Tertiary butyl acrylate ethylacrylic acid fat methacrylic acid）Triblock copolymer with Octylame is with 1：40 molar ratio mixed dissolution is in DMF, addition coupling agent EDC reaction 12h, obtained mixture dialysis purification, It is preserved after freeze-drying for use；

It takes Zn-Cu-In-S/ZnS core-shell quanta dots powder and amphiphilic macromolecular to be dissolved in chloroform/ethanol mixed solution, stirs A few hours are mixed to remove chloroform organic solvent；Add suitable PBS buffer solutions, ultrasonic disperse 3 minutes；Obtained solution 0.22um films are crossed to remove largely unbonded macromolecule, solution becomes optical clear after crossing film；Acquired solution crosses gel chromatography Column G200 further removes unbonded macromolecule, finally obtains purified hydrophily quantum dot；

（3）It is prepared by paramagnetism quantum dot

By hydrophily quantum dot and DOTA-NH₂Molecule coupling labeled adds GdCl₃Carry out Gd³⁺Chelating obtains paramagnetism quantum dot, Specifically preparation process is：According to molar ratio 1:100：4000 by hydrophily quantum dot, DOTA-NH₂It is mixed with carbodiimide hydrochloride It closes, reacts at room temperature 2 hours, ultracentrifugation purifies to obtain QDs-DOTA；Again on the basis of the QDs-DOTA obtained, Gd is added³⁺, Room temperature chelatropic reaction 4 hours purifies to obtain paramagnetism quantum dot using ultracentrifugation；It is different according to nano-quantum point grain size, PQD700, pQD800 are obtained respectively；

（4）It is prepared by the paramagnetism quantum dot for targeting breast carcinoma stem cell

By paramagnetism quantum dot and monoclonal antibody molecule CD44_mAb、ESA_mAbCoupling, acquisition can specific recognition breast cancer it is dry thin The paramagnetism quantum dot of born of the same parents, specific preparation process are：According to molar ratio 1:10：4000 by paramagnetism quantum dot, antibody and carbon two Inferior amine salt hydrochlorate mixes, and reacts at room temperature 2 hours；Ultracentrifugation purifies to obtain the paramagnetism quantum dot of targeting breast carcinoma stem cell pQD700-CD44_mAbAnd pQD800-ESA_mAb。

3. manufacturing method as claimed in claim 2, it is characterised in that：Also use polyethylene glycol, acrylic resin or polyhydroxy-alcohol PQDs is surface modified.