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Objective To investigate the methods of screening specific aptamers for (EpCAM)Gpositive prostate cancer (PCa)cells by cellGSELEX technique.Methods A random DNA library was designed to screen EpCAMGspecific DNA aptamers from human prostate cancer cells expressing EpCAM molecule by cellGSELEX technique.After 12 rounds of in vitro screening,DNA products were cloned and sequenced.Flow cytometry and cellular immunofluorescence were used to detect the specific binding ability of aptamers to target cells.Results Two aptamers of Ep1 and Ep2 were selected.Both of them could specifically bind to EpCAMGpositive cancer cells LNCap,PCG3 ,DU1 45 , and HEK293T cells transfected with target molecule.The binding rates of Ep1 were 61.0%,74.3%,5 9.1% and 60.3%.The binding rates of Ep2 were 65.1%,77.8%,54.2% and 58.3%.Neither of them could bind to HEK293T cells transfected with empty vector with the binding rate of 5.4% in Ep1 and 3.3% in Ep2,respectively.Flow cytometry analysis and confocal images indicated that the EpCAM aptamers could specifically recognize human PCa cells expressing EpCAM,but could not bind to EpCAMGnegative cells.Conclusion EpCAM aptamers derived from cellGSELEX technology can recognize and bind to EpCAMGpositive PCa cells specifically,which may provide new ideas for the specific diagnosis and targeted therapy of prostate cancer,and lay an experimental basis for the other specific diagnosis and treatment schemes of malignant tumors.
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Objective TostudythevalueofquantitativeparametersofDCE-MRIandthreedimensionalarterialspinlabeling(3D-ASL) inpreoperativegliomagrading.Methods 70patientsdiagnosedpathologicallywithinitialgliomawereassessedretrospectively,including 32caseswithlow-gradeglioma(LGG)and38caseswithhigh-gradeglioma(HGG).Allpatientsunderwentconventional,enhanced, DCEand3D-ASL MRIat3.0Tbeforesurgery.TheparametricvaluesofDCEsuchasvolumetransferconstant(Ktrans),extravascular extracellularspacevolumefraction(Ve),therateconstant(Kep),fractionalplasmavolume(Vp),cerebralbloodflow (CBF)andcerebral bloodvolume(CBV)wereobtainedbycorrespondingpost-processingsoftware.ThecerebralbloodflowofASL (ASL-CBF)wasalso obtained.Ttestoftwoindependentsampleswasusedtoanalyzewhetherthemaximumandaveragevaluesofeachparameterwere statisticallydifferentbetweenLGGand HGG.Thediagnosticaccuracyofdifferenttechniqueforgliomagradingwasdeterminedby ROCcurveanalysis.Results ThemaximumvaluesofDCE-Ktrans,Ve,rCBVandmaximumvalueofASL-rCBFwerestatisticallydifferent betweentheHGGandLGG (P＜0.05).AlltheparametricaveragevalueswerestatisticallydifferentbetweentheHGGandLGG (P＜0.05).ThemaximumandaveragevaluesofKtranshadarelativelyhighestdiagnosticefficiencyinallparameters,withtheAUCwere0.986 and0.971,theoptimumthresholdwere0.264and0.068,thesensitivitywere93.3%and94.1%,andthespecificitywere100%and 100%,respectively.ThemaximumvaluesofVe,rCBV,ASL-rCBFandtheaveragevaluesofallparametershadarelativelyhigher diagnosticefficiency.Conclusion ThemaximumvaluesofKtrans,VeandrCBFofDCE,themaximumvalueofASL-rCBFandtheaverage valueofeachparameterwereusefultodistinguishbetweenLGGand HGG.ThemaximumandaveragevaluesofKtransarethebest parametersforidentifyingHGGandLGG.
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Super hydrophobic interface modified with silver nanoparticles was fabricated for the detection of pesticide residues.By using a chemical reduction method,silver nanoparticles were deposited on the substrate surfaces with different microscopic pore structures.Two kinds of composite substrates,including regular stainless steel mesh and cellulose polyester film,were used.The pre-treatment of the substrate with fluoridated reagents was used to form a super hydrophobic interface,which made the target molecules on the surface concentrate effectively.The surface with the cellulose polyester substrate was used to detect Rhodamine 6G (R 6G) effectively with surface enhanced Raman scattering (SERS) technique.The results showed that the detection hmit was 10-16 mol/L.In addition,the surfaces based on the stainless steel mesh and cellulose polyester substrate were used to detect trichlorfon pesticide with detection limits of 1 × 10-15 mol/L and 1 × 10-16 mol/L,respectively.
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Objective To prepare dual?modality single?photon emission computed tomography (SPECT)?MRI molecular nanoprobes targeting HAb18G/CD147 expressed on breast cancer cell membranes and investigate the physicochemical and biological properties in vitro. Methods Superparamagnetic iron oxide nanoparticles (SPIOs) were prepared by one?pot reaction method as described. The single?chain antibody fragments HAb18F(ab')2 were conjugated to SPIOs via chemical method and then labeled with 125I using Iodogen method. The final 125I?SPIO?HAbF18(ab')2 nanoprobes were purified. SPIOs or 125I?HAb18F(ab')2 were used as control. We carried preliminary evaluation on their physicochemical properties and biological characteristics in vitro: transmission electron microscope (TEM) and dynamic light scattering (DLS) were used to measure these nanoparticle sizes and the hydrodynamic diameters. The MRI T2 transverse relaxation efficiency of these nanoprobes at different Fe2+concentrations were measured with 1.5 T clinical MR scanner. The 125I?SPIO?HAb18F(ab')2 and 125I?HAb18F(ab')2 radiochemical purity were measured by thin layer chromatography and the radio chemical yield was calculated. We also conducted stability tests in vitro and octanol/water partition coefficient experiments. Two breast tumor cell lines, MDA?MB?231 (HAb18G?overexpressing cells,experimental group) and MDA?MB?468 (control), were used for assessment of cells viability at different Fe2 + concentrations (1, 5, 10, 20, 40 μg/ml) by methyl thiazolyl tetrazolium assay. Specific binding experiments in vitro included two parts:magnetic resonance imaging and radionuclide tests, the above?mentioned breast cancer cell lines were incubated with 125I?SPIO?HAb18F(ab')2 nanoprobes respectively and took MDA?MB?231 cells which were not treated as blank group. First comparing the MR signal intensity differences among experimental group, the control group and blank group, then calculated the rate of MRI signal changes;Two breast tumor cell lines, MDA?MB?231 and MDA?MB?468 were incubated with 125I?SPIO?HAb18F(ab')2 nanoprobes too, then measured radioactivity counting byγcounter at different time and calculated the cell binding rates, and did statistical analysis by using one?way ANOVA. Results The SPIOs were fairly homogeneous with an average core size of (10.32±1.30) nm;the SPIO and 125I?SPIO?HAb18F(ab')2 hydrodynamic diameter of 44.80 and 52.64 nm, and MRI scanning showed that the transverse relaxation efficiency of SPIO and 125I?SPIO?HAb18F(ab')2 were 38.79 and 106.73 mM-1 · s-1, respectively. The radio chemical yield of 125I?SPIO?HAbF18(ab')2 and 125I?HAb18F(ab')2 were 41.90% and 85.50%, respectively. The radio chemical yield of the two groups were >95%, suggesting well stability in vitro. The lipo?hydro partition coefficient values were -0.99 ± 0.03 and-1.49 ± 0.08, respectively, which demonstrated that they were both water?soluble substances. Different Fe2+concentrations (1,5,10,20,40μg/ml) of 125I?SPIO?HAb18F(ab')2 on breast cancer cell lines MDA?MB?231 and MDA?MB?468 showed no significant inhibition of cell proliferation (F values were 0.78, 0.66; P values were 0.58, 0.66). The cell?specific binding experiment showed: MRI signal intensity values on experimental group, the control group and the blank group were (1 670 ± 5), (1 930 ± 8), (2 349 ± 14), respectively, significant differences existed among these groups (F=4 408.48,P=0.000), the rate of signal intensity change of experimental group and the control group were 28.87%,17.78%. SPECT:MDA?MB?231 could uptake 125I?SPIO?HAb18F(ab')2, the cell binding rates were (6.52 ± 0.60)% and (10.52 ± 2.04)% in 20 min and 4 h, respectively.Conclusions Our results suggested that the dual?modality SPECT?MRI nanoprobes 125I?SPIO?HAb18F(ab')2 were prepared successfully with good physicochemical properties and biological characteristics in vitro. These dual?modality molecular imaging nano?probes may have potential to improvearly detection and diagnosis of HAb18G/CD147?expressing cancers and to facilitate the development of HAb18G/CD147?directed interventions.
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Objective To develop an MR optical dual-modality probe targeting angiogenesis of gastric cancer and to study its physical characteristics , in vitro cytotoxicity and magnetic effects of different pulse sequences on 3 T clinical MR scanner.Methods We conjugated GX1-Cy5.5, a novel gastric cancer neo-vasculature targeted peptide labeled with Cy 5.5, to the surface functionalized magnetic nanoparticles according to different molecular weights (1∶100, 1∶500),resulting in dual-modality probe DPs100 and DPs500 (named DPs).The hydrodynamic size and zeta potential of DPs and DPs 500 were analyzed by nano-ZS.The human umbilical vein endothelial cells (HUVECs) and BGC-823 cells were treated with DPs for 24 h, and methyl thiazol tetrazolium ( MTT) method was used to detect the survival rate of cells.DPs with different concentrations were scanned on different MR sequences , and then the relative signal intensity was observed.The absorbance of HUVECs and BGC823 cells treated with DPs of different concentration (0.00, 1.25, 2.50, 15.00, 50.00, 100.00 and 150.00 μg/ml) were compared with single factor analysis of variance.Relative signal intensity of different MR sequences was compared using a paired Wilcoxon signed-rank test.Results The dual-modality probe targeting angiogenesis of gastric cancer was successfully constructed.The hydrodynamic size of iron oxide nanoparticles , DPs100 and DPs500 was (35.23 ±0.07), (39.49 ±0.16) and (40.43 ±1.70) nm and the Zeta potential was (0.31 ±0.20), ( -4.15 ±0.79) and ( -10.51 ± 2.37) mV.The coupled rates of DPs 100 and DPs500 with polypeptide were 92%and 94% respectively.The absorbance of HUVECs and BGC823 cells treated with DPs of different concentrations were 0.76 ±0.04, 0.80 ±0.03, 0.79 ±0.05, 0.75 ±0.06, 0.74 ±0.05, 0.77 ±0.01,0.71 ±0.04 and 0.38 ±0.04, 0.43 ±0.04, 0.41 ±0.03, 0.43 ±0.07, 0.44 ±0.04, 0.41 ±0.07 and 0.40 ±0.04, there was no statistical significance ( F=0.94, 0.51;P>0.05).The signal intensity increased first and then decreased following the increasing concentrations of DPs on T 1WI,especially on FSPGR T1WI (Z =-3.294,P 10μg/ml( Z=-7.110,P>0.05).With iron concentration≤10 μg/ml,the signal intensity on SSFSE T 2*WI was significantly decreased compared to FSE T2 WI ( Z =-2.023, P <0.05 ) .Conclusions DPs may be potential dual-modal probes for characterization of tumor angiogenesis by MR and optical imaging noninvasively , without causing significant effects on the cell activity in vitro , and SSFSE T2*WI may be the most sensitive sequence for DPs evaluation on MR.