CN105412947A - CT/MR bimodal nanoprobe and preparation method thereof - Google Patents

CT/MR bimodal nanoprobe and preparation method thereof Download PDF

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CN105412947A
CN105412947A CN201510957314.5A CN201510957314A CN105412947A CN 105412947 A CN105412947 A CN 105412947A CN 201510957314 A CN201510957314 A CN 201510957314A CN 105412947 A CN105412947 A CN 105412947A
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王小林
王瑞芝
史向阳
罗宇
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Zhongshan Hospital Fudan University
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Abstract

The invention discloses a CT/MR bimodal imaging contrast agent for hyaluronic acid targeting tumor model early diagnosis and a preparation method of the contrast agent. The preparation method comprises the following steps: by taking a generation 5 dendrimer (G5) as a template, respectively modifying DOTA.NHS, HA and FI to the surface of the template, thus obtaining a semi-finished product G5.NH2-DOTA-FI-HA, then by using the semi-finished product as a template to wrap gold nanoparticles, and finally chelating to link Mn and form an end-product, i.e., multifunctional HA targeted dendrimer nano-composites (Au<0>)n-G5.NH2-DOTA(Mn)-FI-HA. The G5 with the surface combined with tagged molecules, targeted molecules and ligand molecules is obtained by a simple and mild method, and is taken as a platform to combine imaging elements gold and manganese, the obtained bimodal contrast agent can tag a condition that a nanoprobe is swallowed by cancer cells at a cellular level, and has a remarkable targeting effect on a CD44 high-expression cancer cell strain, and early diagnosis on cancer can be realized; meanwhile, as the method is simple in preparation and raw materials are low in cost and easy to get, mass production can be realized.

Description

A kind of CT/MR bimodal nano-probe and preparation method thereof
Technical field
The invention belongs to bimodal molecular image probe field, relate to a kind of CT/MR bimodal nano-probe, particularly relate to a kind of dendrimer trapping gold nano-particle of hyaluronic acid targeting in conjunction with manganio chelate CT/MR bimodal nano-probe and preparation method thereof.
Background technology
Cancer (cancer), also known as malignant tumor, is to one of most threatening killer of human health.And early stage Diagnosis and Treat becomes the key of curing cancer.In the early diagnosis of tumor, traditional image technology can only understand gross tumor volume size and Anatomical orientation, and molecular image learns a skill and can obtain more how valuable information by let us, such as, protein molecular before and after cancerating detects, tumor growth kinetics is assessed, tumor cell marker etc., and living body molecule imaging carries out pathogenetic research under can be implemented in the situation of harmless organism microenvironment, and the molecular motion track helping display complicated.Be applied to clinical molecular image at present to learn a skill and mainly comprise CT imaging, NMR (Nuclear Magnetic Resonance)-imaging (MRI), SPECT imaging and ultra sonic imaging etc.
As the important component part of molecular imaging, select suitable contrast agent can improve susceptiveness, the specificity of image-forming diagnose widely.And as desirable and the nano material system of the early stage targeting diagnosis of clinical cancer can be applied to, while guarantee bio-safety, more want to take into account to carry targeted molecular and imaging agents molecule simultaneously, and possess several key properties such as preparation is easy, raw material is cheap and easy to get.The contrast agent of current clinical practice, the Omnipaque (Omnipaque) of such as CT imaging, six kinds of gadolinio micromolecule contrast agent of MRI imaging all also exist obvious defect, as too short in: blood circulation time, without specificity, especially also there is nephrotoxicity under gadolinio contrast agent finite concentration.Therefore, expect clinically to develop the targeting molecule image probe having two kinds or more of imaging pattern concurrently, and then make up the weak point of single molecular image imaging.
Along with the deep development of nanotechnology, increasing new material is developed to meeting clinical demand.Such as prepare ferroferric oxide nano granules by coprecipitation, and then on its surface layer electrostatic self-assembled polyglutamic acid and polylysine, finally the 5th PAMAM dendrimer (G5) being enclosed with gold nano grain is modified under EDC effect iron oxide surface, achieve T 2weighting MR imaging and CT imaging bimodal radiography (J.Mater.Chem., 2012,22,15110-15120); The 5th PAMAM dendrimer is utilized to be platform, chelating reagent DOTA-NHS is connected to dendrimer surface to be used for chelating gadolinium ion and to be used for MR imaging, utilize the cavity structure that dendrimer is special, trapping gold nano-particle is used for CT imaging, achieves T simultaneously 1the MR/CT bimodal imaging function (Biomaterials, 34, (2013), 1570-1580) that weighting MR strengthens and CT strengthens.Because manganese is in sequestering properties in Ink vessel transfusing transportation, its toxicity can be left in the basket, and cell can safety discharge born of the same parents in manganese (Journalofmagneticresonanceimaging, 41 (2015) 797-805), so paramagnet---manganese can be used to alternative gadolinium and carry out T 1enhanced Imaging, also more meets the custom of clinical diagnosis simultaneously.CD44 is the major cell surface label of malignant progression, transfer, does not express in Non-Invasive or normal cell strain, is also the outstanding feature of tumor stem cell.The ligands specific hyaluronic acid (HA) of its efficiency natural, has good biocompatibility, biodegradability and simple structure and is easy to modify.Targeted molecular HA, ligand molecular DOTA, just based on former works basis, are attached on dendrimer by the present invention, and trapping gold nano-particle (AuNPs) is then by T 1the Mn of reinforced effects is successfully chelated on DOTA, thus prepares the molecular image probe that can realize the imaging of CT/MR bimodal.
Summary of the invention
For achieving the above object, the invention provides a kind of preparation method of manganio chelate CT/MR bimodal probe of trapping gold nano-particle of hyaluronic acid targeting, described preparation method is first respectively by chelating reagent 2, 2 ', 2 "-(10-(2-(2, 5-dioxo pyrrolidin)-1-oxygen base)-2-oxygen ethyl)-1, 4, 7, 10-tetraazacyclododecanand-1, 4, 7-tri-) triacetic acid (DOTA-NHS), targeted molecular hyaluronic acid (HA) and Fluorescein isothiocyanate (FI) modify the 5th PAMAM dendrimer (G5) surface, then sodium borohydride reduction is utilized to react trapping gold nano-particle, Coordinative Chemistry method is finally utilized to be chelated on DOTA molecule by manganese ion.The concrete steps of described method comprise:
Step one, chelating reagent DOTA-NHS modified G5 surface through surperficial covalent modification;
Step 2, targeted molecular HA is connected to G5 surface;
Step 3, by tracer molecule Fluorescein isothiocyanate (FI) labelling on G5;
Step 4, be template trapping gold nano-particle with G5;
Step 5, utilize DOTA coordination Chelated Manganese ion.
The flow chart of above-mentioned steps as shown in Figure 1.
Further, step one adopts DOTA-NHS and G5.NH 2reaction forms G5.NH 2-DOTA, wherein the 5th PAMAM dendrimer G5.NH 2molecular weight be 26010; Preferably, G5.NH 2be 1:33 ~ 37 with the mol ratio of DOTA-NHS, most preferably be 1:35; Preferably, the solvent of described reaction is DMSO; Further preferably, adopt the mode slowly dripped by described G5.NH 2dMSO solution add in the DMSO solution of described DOTA-NHS, and continuous vigorous stirring 20 ~ 30 hours, makes its complete reaction, forms G5.NH 2-DOTA solution.
Further, the HA in step 2 is connected with G5 after first adopting EDC/NHS to react activation again, and the molecular weight of wherein said HA is 5805; In this priming reaction, the mol ratio of HA, EDC and NHS is preferably 1:4 ~ 6:4 ~ 6, is more preferably 1:5:5; The time of priming reaction is preferably 2 ~ 4 hours, and the best is 3 hours.
Preferably, the concrete operations of described priming reaction are: add in the aqueous solution of HA by the DMSO solution of EDC and the DMSO solution of NHS; Wherein, the concentration of HA aqueous solution is preferably 3.5 ~ 4.5 μm of ol/ml, and the best is 4 μm of ol/ml; The concentration of the DMSO solution of EDC is preferably 95 ~ 105 μm of ol/ml, and the best is 100 μm of ol/ml; The concentration of the DMSO solution of NHS is preferably 95 ~ 105 μm of ol/ml, and the best is 100 μm of ol/m.
Preferably, the mode that the HA solution employing in step 2 after activation drips adds the G5.NH that step one is formed 2in-DOTA solution, and stirring makes it react, and obtains G5.NH 2-DOTA-HA solution, the time of this reaction is preferably 60 ~ 80 hours, and optimum is 72 hours; Preferably, wherein the mol ratio of HA and G5 is 18 ~ 22:1, and the best is 20:1.
Preferably, step 2 also comprises the G5.NH that will obtain 2-DOTA-HA solution is dialysed in ultra-pure water, and to remove by-product wherein and impurity, the molecular cut off of bag filter is preferably 7000 ~ 9000, most preferably be 8000, the time of dialysis is preferably 40-50 hour, and the best is 48 hours, wherein change ultra-pure water 4 ~ 8 times, the best is 6 times.
Further, step 3 adopts the G5.NH DMSO solution of FI being added dropwise to step 2 acquisition 2in-DOTA-HA solution, and make its stirring reaction under the condition of lucifuge, form G5.NH 2-DOTA-FI-HA solution, the response time is preferably 20 ~ 28 hours, and the best is 24 hours.Preferably, the mol ratio of FI and G5 is 4 ~ 6:1, and the best is 5:1; The FI concentration be dissolved in DMSO is preferably 4.5 ~ 5.5 μm of ol/mL, and the best is 5 μm of ol/mL.
Further, utilize sodium borohydride reduction to react trapping gold nano-particle in step 4, concrete operations are: by gold chloride (HAuCl 4) aqueous solution adds the G5.NH of step 3 gained 2in-DOTA-FI-HA solution, fully add NaBH again after mixing 4solution, stirred at ambient temperature obtains (Au after reacting a period of time 0) n-G5.NH 2-DOTA-FI-HA solution (wherein n is the molar ratio value of Au and G5); The time of its stirring reaction is preferably 1.5 ~ 3 hours, and the best is 2 hours.
Preferably, the mol ratio of Au and the G5 added in step 4 is 75 ~ 125:1, and the best (obtains product (Au for 100:1 0) 100-G5.NH 2-DOTA-FI-HA); Preferably, the described HAuCl added 4the concentration of solution is 25 ~ 35mg/ml, and optimum is 30mg/ml.
Further, the concrete operations of step 5 are: (the Au aqueous solution of manganese sulfate being added dropwise to step 4 gained 0) n-G5.NH 2in-DOTA-FI-HA solution, and lucifuge vigorous stirring is reacted 20 ~ 28 hours under room temperature, and the best is 24 hours, obtains (Au 0) n-G5.NH 2the solution of-DOTA (Mn)-FI-HA, successively dialyses this solution dialyzer, finally carries out lyophilizing purification, obtain end product (Au in PBS and distilled water 0) n-G5.NH 2-DOTA (Mn)-FI-HA.
Preferably, the MnSO added in step 5 4be 66 ~ 74:1 with the mol ratio of G5, the best is 70:1; Preferably, MnSO 4be 2:1 with the mol ratio of DOTA.
Preferably, in step 5, the time of PBS dialysis is 20 ~ 28 hours, and optimum is 24 hours.
Preferably, in step 5, the distill water dialysis time is 45 ~ 50 hours, and optimum is 48 hours.
The present invention take G5 as platform, targeted molecular HA, CT imaging element Au and MR imaging element (DOTA-Mn chelate) three are organically combined, obtains the dendrimer trapping gold nano-particle of HA targeting in conjunction with manganio chelate CT/MR bimodal nano-probe.Utilize dendrimer surface to there is a large amount of amino thus multifunction can be realized and modify this characteristic, first adopt chemical bonding to be connected in G5 surface amino groups by chelating reagent DOTA, further targeted molecular hyaluronic acid (HA), fluorescence molecule (FI) are modified to G5 surface.HA can realize CD44 high expression tumour cell strain model specific target tropism, FI molecule is study at cellular level the effect that hyaluronic targeting ability serves molecular probe, and the existence of DOTA can play the effect of chelated metal ions manganese, thus realize T 1the MR Enhanced Imaging of weighting.
The synthesis technique that the present invention adopts is simple, and reaction condition gentleness is controlled, is easy to operation.It is well biocompatibility that the CT/MR bimodal molecular image probe prepared has, and good targeting CT/MR bimodal developing function can be realized at cellular level and animal level, final contrast agent can excrete with metabolism, can not accumulate for a long time in vivo.The CT/MR bimodal molecular image probe of HA targeting prepared by the method has good application prospect at molecular image diagnostic field.
Be described further below with reference to the technique effect of accompanying drawing to design of the present invention, concrete structure and generation, to understand object of the present invention, characteristic sum effect fully.
Accompanying drawing explanation
Fig. 1 is the flow chart of steps of preparation method of the present invention;
Fig. 2 is G5.NH obtained in embodiment 1 2the hydrogen nuclear magnetic resonance spectrogram of-DOTA in deuterated water;
Fig. 3 is G5.NH obtained in embodiment 1 2the hydrogen nuclear magnetic resonance spectrogram of-DOTA-HA in deuterated water;
Fig. 4 is G5.NH obtained in embodiment 1 2the hydrogen nuclear magnetic resonance spectrogram of-DOTA-FI-HA in deuterated water;
Fig. 5 is obtained (Au in embodiment 1 0) 100-G5.NH 2the transmission electron microscope photo of-DOTA (Mn)-FI-HA nano-probe;
Fig. 6 is obtained (Au in embodiment 1 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe particle diameter cartogram;
Fig. 7 is obtained (Au in embodiment 1 0) 100-G5.NH 2the T of-DOTA (Mn)-FI-HA nano-probe 1the Linear Fit Chart of relaxation time inverse and manganese concentration;
Fig. 8 is obtained (Au in embodiment 1 0) 100-G5.NH 2the brightness flop figure that the CT image of-DOTA (Mn)-FI-HA nano-probe changes with gold concentration;
Fig. 9 is obtained (Au in embodiment 1 0) 100-G5.NH 2the CT value of-DOTA (Mn)-FI-HA nano-probe and the Linear Fit Chart of gold concentration;
Figure 10 is obtained (Au in embodiment 1 0) 100-G5.NH 2the MTT measurement result of-DOTA (Mn)-FI-HA nano-probe on HCCLM3 cell;
Figure 11 uses variable concentrations (Au 0) 100the fractographic HCCLM3 cell photo of-G5.NH2-DOTA (Mn)-FI-HA nano-probe process Dual culture after 24 hours, wherein a uses PBS process (blank), b ~ f uses nano-probe process (b, the 10 μ g/mL of different manganese concentration respectively; C, 20 μ g/mL; D, 50 μ g/mL; E, 75 μ g/mL; F, 100 μ g/mL);
Figure 12 is that targeting group and non-targeted group use variable concentrations (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA processes the flow cytometry figure of Dual culture after 4 hours, and wherein a uses PBS process (blank), and b ~ c uses the (Au of different manganese concentration respectively 0) 100-G5.NH 2the process of-DOTA (Mn)-FI-HA nano-probe (b, 25 μ g/mL; C, 100 μ g/mL), d ~ e uses the (Au of different manganese concentration respectively 0) 100-G5.NH 2the process of-DOTA (Mn)-FI-HA nano-probe+CD44 receptor blocking (d, 25 μ g/mL; E, 100 μ g/mL);
Figure 13 is that targeting group and non-targeted group use variable concentrations (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA processes the average fluorescent strength comparison diagram of Dual culture after 4 hours;
Figure 14 is (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe and (Au 0) 100-G5.NH 2the CT imaging effect comparison diagram of-DOTA (Mn)-FI nano-probe;
Figure 15 is (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe and (Au 0) 100-G5.NH 2the CT image numerical comparison diagram of-DOTA (Mn)-FI nano-probe;
Figure 16 is (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe and (Au 0) 100-G5.NH 2the MR imaging effect comparison diagram of-DOTA (Mn)-FI nano-probe;
Figure 17 is (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe and (Au 0) 100-G5.NH 2the MR image numerical comparison diagram of-DOTA (Mn)-FI nano-probe;
Figure 18 is injection (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA and (Au 0) 100-G5.NH 2after during-DOTA (Mn)-FI nano-probe 24 in mice with tumor body the distribution situation figure of each internal organs.
Detailed description of the invention
The preparation of embodiment 1CT/MR bimodal nano-probe
(1) DOTA-NHS (35 μm of ol) of 26.25mg is dissolved in the DMSO of 10mL, until completely dissolved, dropwise adds 10mLG5.NH 2solution (26mg, the G5.NH of 1 μm of ol 2be dissolved in 10mLDMSO), continuous vigorous stirring 1 day, forms G5.NH 2-DOTA solution.
(2) 120.49mgHA (20 μm of ol) is dissolved in 5mL70 DEG C of ultra-pure water, add 1mLEDC (19.11mg wherein, 100 μm of ol) DMSO solution and 1mLNHS (11.91mg, 100 μm of ol) DMSO solution, treat that it dissolves completely, and stir-activating 3h.
(3) the HA dropwise of activation is added the product G5.NH of above-mentioned steps (1) 2in-DOTA solution, vigorous stirring reacts 3 days; Dialyse in ultra-pure water 2 days (period changes water 6 times, 2L/ time) with the bag filter that molecular cut off is 8000, removing by-product and impurity, obtain G5.NH 2-DOTA-HA solution.
(4) FI (5 μm of ol) of 1.91mg is dissolved in 1mLDMSO, after dissolving completely, dropwise adds the product G5.NH of above-mentioned steps (3) 2in-DOTA-HA solution, under lucifuge condition, continuing vigorous stirs 1 day, forms G5.NH 2-DOTA-FI-HA solution.
(5) according to the molar equivalent of 100:1 (Au:G5), the chlorauric acid solution (30mg/mL) of 2.78mL is added the G5.NH of above-mentioned steps (4) gained 2in-DOTA-FI-HA solution, stirred at ambient temperature 30min, adds NaBH subsequently 4solution (18.92mg, 0.5mmol, 1mLH 2o), stirred at ambient temperature reaction 2h, obtains (Au 0) 100-G5.NH 2-DOTA-FI-HA solution.
(6) according to the molar equivalent of 70:1 (Mn:G5), the manganese sulfate of 13.52mg (is dissolved in 1mLH 2o) (the Au of above-mentioned steps (5) gained is dropwise added 0) 100-G5.NH 2in-DOTA-FI-HA solution, under room temperature, lucifuge vigorous stirring reaction 24h, obtains (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA solution, dialyse this solution dialyzer in PBS 24h, and then by distill water dialysis 48h, lyophilizing purification, obtains end-product (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA.
The present invention use UV, visible light NMR (Nuclear Magnetic Resonance) spectrum ( 1hNMR), the method such as ICP-AES (ICP-AES), Zeta electric potential, hydration particle diameter characterizes the physicochemical properties of the molecular probe prepared, and by the x-ray damping capacity of CT analyzing molecules probe, the T of nuclear magnetic resonance imaging instrument analyzing molecules probe 1imaging performance and r 1relaxation rate, then evaluates blood compatibility and the cytotoxicity of nano-probe by mtt assay and phase contrast microscope observational method, in recycling flow cytometry, body, CT/MR imaging experiment detects the molecular probe of HA modification to the targeted imaging effect of tumor cell.Concrete test result is as follows:
Embodiment 2 nuclear magnetic resonance spectroscopy ( 1hNMR) test
By analyzing the obtained G5.NH of embodiment 1 step (1) 2hydrogen spectrum spectrum peak (as Fig. 2) of-DOTA in deuterated water is known, G5.NH 2at the spectrum peak that 1.75 ~ 3.25ppm occurs ,-DOTA proves that DOTA is successfully connected on G5, and calculated known by integrated peak areas, each G5 is connected to 23.6 DOTA.By identical method, the G5.NH that step (3) is obtained 2the G5.NH that-DOTA-HA, step (4) are obtained 2-DOTA-FI-HA has finally connect 13.6 HA (as Fig. 3) and 3.7 FI (as Fig. 4) respectively.
Embodiment 3Zeta electromotive force and the test of hydration particle diameter
(the Au that the present invention prepares 0) n-G5.NH 2only there is a small amount of positive charge on-DOTA (Mn)-FI-HA molecular image probe surface, avoids higher positive charge and can restrict the application of this material at biomedical sector.Therefore, the present invention is without the need to carrying out complete acetylation process to the experimental group material after modification and matched group material.Surface potential and the hydration particle size determination result of embodiment 1 are as shown in table 1: synthesize (the Au obtained 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe surface potential and hydration particle diameter are respectively+6.1mV and 245.3nm, and its polydispersity index (PDI) is 0.564.(Au is drawn from experimental result 0) 100-G5.NH 2-DOTA (Mn)-FI-HA has possessed excellent colloidal stability and can be applicable to biomedical sector.
Table 1 (Au 0) 100-G5.NH 2the surface potential of-DOTA (Mn)-FI-HA nano-probe aqueous solution and hydrodynamic diameter
Embodiment 4 transmission electron microscope (TEM) is tested
(the Au that embodiment 1 prepares 0) 100-G5.NH 2size and the form of-DOTA (Mn)-FI-HA nano-probe are observed (Fig. 5) by transmission electron microscope simultaneously.The pattern that test result shows these nano-probes is all spherical or torispherical, and particle size distribution is comparatively homogeneous.The diameter of 300 nano-probes is measured, we obtain (Au by random selecting 0) 100-G5.NH 2the diameter of-DOTA (Mn)-FI-HA nano-probe is 2.1nm (Fig. 6).This result is obviously little than hydrodynamic diameter, and we infer that the reason of this phenomenon is because dynamic light scattering measures is the total diameter of multiple nano-probes of tufted distribution in aqueous solution, and transmission electron microscope is the diameter of the single nanoparticle measured.
Embodiment 5 stability test
(the Au that the present invention prepares 0) n-G5.NH 2-DOTA (Mn)-FI-HA nano-probe will be applied to biomedical aspect, must possess good stability.In order to assess (the Au prepared 0) 100-G5.NH 2the stability of-DOTA (Mn)-FI-HA nano-probe, it is dissolved in ultra-pure water, PBS, cell culture media solution and (does not add the cell culture media solution of nano-probe as blank) by respectively, left at room temperature through one month is put, and can observe in each solution and not find precipitation.This illustrates, this nano-probe has good colloidal stability in above-mentioned solution.
Embodiment 6r 1relaxation rate is measured
R 1relaxation rate reflection (Au 0) n-G5.NH 2-DOTA (Mn)-FI-HA molecular image probe, as the efficiency of MRI contrast agent, is the longitudinal relaxation time of unit molar concentration manganese, by the T under variable concentrations 1the Fitting Calculation reciprocal in relaxation time obtains.Fig. 7 is (Au prepared by embodiment 1 0) 100-G5.NH 2the T of-DOTA (Mn)-FI-HA nano-probe 1(to the Linear Fit Chart of relaxation time inverse and manganese concentration manganese concentration wherein utilize ICP to measure obtained (Au 0) 100-G5.NH 2the Fe content of-DOTA (Mn)-FI-HA nano-probe lyophilized powder is configured to respective concentration with PBS buffer again), can find out that the relaxation time of this material reciprocal has good linear relationship along with the increase (in 0.2 ~ 1.0mM concentration range) of manganese concentration.And by can be calculated (Au prepared by embodiment 1 0) 100-G5.NH 2the r of-DOTA (Mn)-FI-HA molecular image probe 1relaxation rate is 5.42mM -1s -1.Therefore, (the Au prepared by embodiment 1 0) 100-G5.NH 2-DOTA (Mn)-FI-HA can be used as the excellent T in the diagnosis of MRI molecular image 1signal strengthens contrast agent.
Embodiment 7T 1weighting MR imaging measurement
(the Au that the present invention prepares embodiment 1 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe has carried out T 1weighting MR imaging performance is tested, and the display of its result is along with the raising of manganese concentration (in 0.2 ~ 1.0mM concentration range), and MRI signal strengthens gradually, and in good gradient relation.Result illustrates that the material prepared has good MRI signal and strengthens contrast agent application potential quality.
Embodiment 8X line attenuation coefficient detects
(the Au that the present invention prepares 0) n-G5.NH 2wrap up Au nano-particle in-DOTA (Mn)-FI-HA nano-probe, thus possessed good x-ray damping capacity, may be used for CT Enhanced Imaging by this.Obviously being brightened along with the raising of gold concentration by the brightness of Fig. 8, CT image, (gold concentration wherein utilizes ICP to measure obtained (Au 0) 100-G5.NH 2the gold content of-DOTA (Mn)-FI-HA nano-probe lyophilized powder is configured to respective concentration with PBS buffer again).Fig. 9 matched curve shows, and the CT value (HU) of different gold concentration point-blank.Result illustrates, this nano-probe has the potential quality that excellent CT strengthens contrast agent application.
Embodiment 9MTT cell survival rate is tested
(the Au that embodiment 1 prepares is detected by the survival rate of MTT colorimetric method for determining HCCLM3 cell 0) 100-G5.NH 2the cytotoxicity (as Figure 10) of-DOTA (Mn)-FI-HA.HCCLM3 cell and (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA (manganese concentration is 10,25,50,75,100 μ g/mL) Dual culture 24 hours at 37 DEG C.Then, after MTT process, measure light absorption value at 570nm place, with the absorption value of PBS buffer group for benchmark, the absorption value after the material processed of variable concentrations calculates the survival rate of HCCLM3 cell by comparison.Experimental result display (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA does not have significant difference to the survival rate of HCCLM3 cell within the scope of manganese concentration 10 to 75 μ g/mL, cell survival rate is all more than 85%, when concentration is increased to 100 μ g/mL, the survival rate of cell declines slightly, but still remains on 80%.This illustrates (the Au that embodiment 1 prepares 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe has good cell compatibility.
Embodiment 10 morphological observation
Subsequently, to (the Au that embodiment 1 prepares 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe has carried out morphological observation after Dual culture.We are by (the Au of PBS buffer (contrast) and different manganese concentration 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe (10,20,50,75,100 μ g/mL) and HCCLM3 cell are at 37 DEG C after Dual culture 24h, inverted phase contrast microscope is utilized to observe, find except a part of cell become separated from each other except, the cell of positive processed group is healthy, form and PBS matched group basic simlarity (Figure 11).
Embodiment 11 Flow cytometry
Utilize (the Au that Flow cytometry HCCLM3 cell is prepared embodiment 1 0) 100-G5.NH 2flow cytometry figure (adopting free HA to make CD44 receptor blocking and the process of PBS buffer in contrast) (Figure 12) and average fluorescent strength (adopting free HA to make CD44 receptor blocking in contrast) (Figure 13) after-DOTA (Mn)-FI-HA processes cell under variable concentrations detect the Targeting Effect of HA.HCCLM3 cell respectively with (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA (manganese concentration is 25,100 μ g/mL) Dual culture 4 hours at 37 DEG C.Then by the average fluorescent strength of Flow cytometry cell.Figure 12 display is after nano-probe 4 hours Dual culture, and cell all can be engulfed a certain amount of nano-probe and send fluorescence.Observed by average fluorescent strength in Figure 13, along with the raising of manganese concentration, (Au 0) 100-G5.NH 2after-DOTA (Mn)-FI-HA processes, the average fluorescent strength of cell significantly increases, and the average fluorescent strength of matched group CD44 receptor blocking group cell is also in the relation increased progressively simultaneously.It should be noted that, process HCCLM3 cell under identical manganese concentration after, non-blacked group comparatively blocking-up group shows obviously higher fluorescence intensity, this phenomenon is relevant with the endocytosis that hyaluronic acid selectively targeted after birth surface molecular label mediates, and makes the more (Au of HCCLM3 cytophagy 0) 100-G5.NH 2-DOTA (Mn)-FI-HA, thus there is the phenomenon of high fluorescent value.The result shows that modifying HA imparts (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA is to the special target ability of HCCLM3 cell.
Embodiment 12 in-vivo tumour CT imaging
By (Au prepared by embodiment 1 0) 100-G5.NH 2-DOTA (Mn)-FI-HA nano-probe and the (Au utilizing identical method (omitting step 2 and 3) to prepare 0) 100-G5.NH 2-DOTA (Mn)-FI nano-probe contrasts, and is evaluated the CT imaging effect (as Figure 14) of tumor locus by tail vein injection.Observe the CT image of tumor locus in tumor bearing nude mice after injection 0 ~ 2 hour.As shown in figure 14 (in figure, T represents the HA targeted probes group of embodiment 1, and N indicates the non-targeted probe matched group without HA), inject latter 30 minutes, (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA molecular probe can produce enrichment at nude mice liver in situ tumor locus and peak, and between 1h ~ 2h, tumor locus density starts to decline, and tumor's profiles is still high-visible; Simultaneously compared with non-targeted group, tumor display is more clear.Figure 15 supports the discovery of CT image more from the angle quantized, and tumor region peaked and disappears gradually between 1 ~ 2 hour 30 minutes time, and targeting group also exists significant difference at each time point and non-targeted group of CT value.Result illustrates (Au prepared by embodiment 1 0) 100-G5.NH 2the targeting CT imaging that-DOTA (Mn)-FI-HA molecular probe can realize tumor is more conducive to the discovery of diseased region, can be applied to in-vivo tumour CT image-forming diagnose.
The MR imaging of embodiment 13 in-vivo tumour and tissue distribution
By (Au prepared by tail vein injection embodiment 1 0) 100-G5.NH 2-DOTA (the Mn)-FI-HA and (Au utilizing identical method (omitting step 2 and 3) to prepare 0) 100-G5.NH 2the MR imaging effect (as Figure 16) of tumor locus is evaluated in the contrast of-DOTA (Mn)-FI nano-probe.Figure 17 has quantized the result contrasted further, (in figure, T represents the HA targeted probes group of embodiment 1 compared with matched group, N indicates the non-targeted probe matched group without HA), with CT characteristics of image is consistent above, tumor region after injection 30 minutes MR signal enhancements is the most obvious, between 1 ~ 2h, signal intensity starts to lower, and all obviously more non-targeted material is brighter for each time point, and the molecular image probe showing HA modification has obvious MRI diagnosing tumor effect.The tumor MR signal value change of putting corresponding inject time, also consistent with MR image result.These results suggest that (Au prepared by embodiment 1 0) 100-G5.NH 2-DOTA (Mn)-FI-HA molecular probe has good cancer target ability, can be successfully applied to targeting MR tumor imaging diagnosis in body, be easier to pick out tumor tissues.
Embodiment 14 tissue distribution detects
At injection (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA and (Au 0) 100-G5.NH 2-DOTA (Mn)-FI nano-probe is after 24 hours, put to death tumor bearing nude mice, dissect and take out the heart, liver, spleen, lung, kidney and tumor and weigh, then digest 24 hours with chloroazotic acid, get the content of Digestive system for ICP-AES analyzing metal elements gold, thus determine at injection (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA and (Au 0) 100-G5.NH 2after during-DOTA (Mn)-FI nano-probe 24 in mice with tumor body the distribution situation (as Figure 18) of each internal organs.As can be seen from the figure at tail vein injection (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA and (Au 0) 100-G5.NH 2after-DOTA (Mn)-FI, in liver, spleen, lung, gold element content significantly improves, although and at other organs after one's own heart, kidney also slightly increase amplitude little.It is pointed out that injection (Au simultaneously 0) 100-G5.NH 2the content of the tumor bearing nude mice tumor locus gold of-DOTA (Mn)-FI-HA molecular probe is apparently higher than injection (Au 0) 100-G5.NH 2the tumor bearing nude mice of-DOTA (Mn)-FI material.These results not only demonstrate (Au 0) 100-G5.NH 2-DOTA (Mn)-FI-HA has good targeting to tumor locus, and illustrate the molecular image probe prepared of the present invention can in Mice Body normal metabolite clearance.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that the ordinary skill of this area just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technical staff in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.

Claims (10)

1. the preparation method of the manganio chelate CT/MR bimodal probe of the trapping gold nano-particle of a hyaluronic acid targeting, it is characterized in that chelating reagent DOTA-NHS, targeted molecular HA and FI are modified the 5th PAMAM dendrimer surface by described method first respectively, then utilize sodium borohydride reduction to react trapping gold nano-particle, finally utilize Coordinative Chemistry method to be chelated on DOTA molecule by manganese ion.
2. preparation method as claimed in claim 1, is characterized in that said method comprising the steps of:
Step one, chelating reagent DOTA-NHS modified G5 surface through surperficial covalent modification;
Step 2, targeted molecular HA is connected to G5 surface;
Step 3, by tracer molecule FI labelling on G5;
Step 4, be template trapping gold nano-particle with G5;
Step 5, utilize DOTA coordination Chelated Manganese ion.
3. preparation method as claimed in claim 2, is characterized in that step one adopts DOTA-NHS and G5.NH 2reaction forms G5.NH 2-DOTA, wherein the molecular weight of the 5th PAMAM dendrimer G5 is 26010.
4. preparation method as claimed in claim 3, be connected with G5 after the HA that it is characterized in that in step 2 first adopts EDC/NHS to react activation, the molecular weight of wherein said HA is 5805 again.
5. preparation method as claimed in claim 4, the G5.NH that the HA solution that it is characterized in that in step 2 after activation adopts the mode dripped to add step one to be formed 2in-DOTA solution, and stirring makes it react, and obtains G5.NH 2-DOTA-HA solution.
6. preparation method as claimed in claim 5, is characterized in that step 3 adopts the G5.NH DMSO solution of FI being added dropwise to step 2 acquisition 2in-DOTA-HA solution, and make its stirring reaction under the condition of lucifuge, form G5.NH 2-DOTA-FI-HA solution.
7. preparation method as claimed in claim 6, is characterized in that step 4 concrete operations are: G5.NH chlorauric acid solution being added step 3 gained 2in-DOTA-FI-HA solution, fully add NaBH again after mixing 4solution, stirred at ambient temperature obtains (Au after reacting a period of time 0) n-G5.NH 2-DOTA-FI-HA solution.
8. preparation method as claimed in claim 7, is characterized in that the concrete operations of step 5 are: (the Au aqueous solution of manganese sulfate being added dropwise to step 4 gained 0) n-G5.NH 2in-DOTA-FI-HA solution, and under room temperature, lucifuge vigorous stirring is reacted 20 ~ 28 hours, obtains (Au 0) n-G5.NH 2-DOTA (Mn)-FI-HA solution.
9. preparation method as claimed in claim 8, is characterized in that step 5 also comprises (the Au will obtained after reaction 0) n-G5.NH 2-DOTA (Mn)-FI-HA solution dialyzer is successively dialysed in PBS and distilled water, finally carries out lyophilizing purification, obtains end product (Au 0) n-G5.NH 2-DOTA (Mn)-FI-HA.
10. the manganio chelate CT/MR bimodal probe of the trapping gold nano-particle of the hyaluronic acid targeting prepared by preparation method as claimed in any one of claims 1 to 9 wherein.
CN201510957314.5A 2015-12-18 2015-12-18 CT/MR bimodal nanoprobe and preparation method thereof Pending CN105412947A (en)

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