Multifunctional nanoprobe for cancer cell targeting and simultaneous fluorescenc
Multifunctional nanoprobe for cancer cell targeting and simultaneous?uorescence/magnetic resonance imaging
Zhenzhen Wei a,Yafeng Wu b,Yuewu Zhao a,Li Mi a,Jintao Wang c,Jimin Wang c, Jinjin Zhao d,Lixin Wang d,Anran Liu a,Ying Li a,Wei Wei a,Yuanjian Zhang a, Songqin Liu a,*
a Key Laboratory of Environmental Medicine Engineering,Ministry of Education,Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering,Southeast University,Nanjing211189,People's Republic of China
b Laboratory of Bio-inspired Smart Interfacial Science,Technical Institute of Physics and Chemistry,Chinese Academy of Sciences,Beijing100190, People's Republi
c of China
c Taixing People's Hospital,Taixing225400,People's Republic of China
d Department of Microbiology and Immunology,Medical School of Southeast University,Nanjing211189,People's Republic of China
h i g h l i g h t s g r a p h i c a l a b s t r a c t
Multifunctional nanoprobe exhibits good dispersion,low cytotoxicity and excellent biocompatibility.
The nanoprobe targets cancer cells, providing for simultaneous?uores-cence and magnetic resonance imaging.
The nanoprobe is used for real-time imaging in early liver cancer
diagnosis.
a r t i c l e i n f o
Article history:
Received6June2016 Received in revised form
22July2016
Accepted25July2016 Available online28July2016
Keywords:
Multifunctional nanoprobe Fluorescence imaging Magnetic resonance imaging Aptamer
HepG2cells a b s t r a c t
Multifunctional nanoprobes with distinctive magnetic and?uorescent properties are highly useful in accurate and early cancer diagnosis.In this study,nanoparticles of Fe3O4core with?uorescent SiO2shell (MFS)are synthesized by a facile improved St€o ber method.These nanoparticles owning a signi?cant core-shell structure exhibit good dispersion,stable?uorescence,low cytotoxicity and excellent biocompatibility.TLS11a aptamer(Apt1),a speci?c membrane protein for human liver cancer cells which could be internalized into cells,is conjugated to the MFS nanoparticles through the formation of amide bond working as a target-speci?c moiety.The attached TLS11a aptamers on nanoparticles are very stable and can't be hydrolyzed by DNA hydrolytic enzyme in vivo.Both?uorescence and magnetic resonance imaging show signi?cant uptake of aptamer conjugated nanoprobe by HepG2cells compared to4T1, SGC-7901and MCF-7cells.In addition,with the increasing concentration of the nanoprobe,T2-weighted MRI images of the as-treated HepG2cells are signi?cantly negatively enhanced,indicating that a high magnetic?eld gradient is generated by MFS-Apt1which has been speci?cally captured by HepG2cells. The relaxivity of nanoprobe is calculated to be11.5mgà1sà1.The MR imaging of tumor-bearing nude mouse is also con?rmed.The proposed multifunctional nanoprobe with the size of sub-100nm has the potential to provide real-time imaging in early liver cancer cell diagnosis.
?2016Elsevier B.V.All rights reserved.
*Corresponding author.School of Chemistry and Chemical Engineering,Southeast University,Nanjing,211189,People's Republic of China.
E-mail address:liusq@https://www.wendangku.net/doc/7f2832327.html,(S.
Liu).
Contents lists available at ScienceDirect
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https://www.wendangku.net/doc/7f2832327.html,/10.1016/j.aca.2016.07.037
0003-2670/?2016Elsevier B.V.All rights reserved.
Analytica Chimica Acta938(2016)156e164
1.Introduction
In the past few years,numerous attention has been paid to develop early cancer diagnosis using single or combined imaging modalities,such as optical,computed X-ray tomography(CT),ul-trasound,positron emission tomography(PET)and magnetic resonance imaging(MRI).Among them,MRI,a sophisticated promising three-dimensional noninvasive tomographic diagnostic technique,has gained wide acceptance in diagnosis and medical research.It can penetrate deeply into tissue providing excellent soft tissue contrast with submillimeter resolution on clinical scanners and avoid using harmful ionizing radiation[1e4].Super-paramagnetic iron oxide nanoparticles,a kind of powerful multi-functional T2contrast agents,are commonly used as magnetic nonviral vectors for magnetic resonance imaging(MRI)[5e9].They come into being an induced magnetic?eld under an additional ?eld,and disturb the spin e spin relaxation(T2)courses of protons in the vicinity,leading to negative enhanced(dark)MRI image [9,10].However,their applications in MRI were largely restricted due to their poor dispersion or easy aggregation/deposition and hard surface modi?cation[11,12].In order to achieve the ef?cient imaging application,many efforts have been made to improve the stability and biocompatibility of the superparamagnetic iron oxide nanoparticles by coating a hydrophilic surface,such as silica shell [13],polymers[14,15]and biomolecules[16].With the develop-ment of surface chemistry,silica is considered to be an exceptional candidate for encapsulating magnetic NPs owing to its good biocompatibility,outstanding physicochemical stability,and easy modi?cation[11,17,18].
Optical imaging,on the other hand,has high sensitivity at the cellular level but could not provide spatial resolution and3D tissue details.When the cellular-sensitive?uorescent imaging is com-bined with MR imaging,high resolution/sensitive imaging of both tissues and cells could be obtained.So far,many nanocomposites have been developed for simultaneous?uorescence and MR im-aging.For example,Acharya group designed a novel multifunc-tional healthcare nanocomposite material for?uorescence and simultaneously for MRI imaging,including chitosan encapsulated iron oxide as MRI contrasting agent,CdS nanoparticles as?uores-cent probe and podophyllotoxin as anticancer drug[17].Eghbali and his coworkers reported a stable bimodal contrast agent for both MRI and?uorescence imaging,consisting of rhodamine B as?uo-rescent probe and APTES-modi?ed superparamagnetic iron oxide nanoparticles as MRI contrasting agent[19].Zhu and his colleagues designed a multifunctional peptide-?uorescent-magnetic nano-composites,in which?uorescence dye(Cy5.5)was linked on the surface of Fe3O4@PEI for MRI and?uorescence imaging[20].
Aptamer is the arti?cial single-stranded nucleic acid sequence with extremely high speci?city to certain targets that fold into secondary and tertiary structures.It is able to recognize and spe-ci?cally bind to various targets ranging from small molecules, proteins to entire cells[21].Compared with antibodies,aptamers show the advantages of lower toxicity,better thermally stability and are usually prohibited from being discovered by the immune system as foreign agents[22].More importantly,it provides a po-tential approach for early diagnosis of cancers[23].TLS11a aptamer is the?rst aptamer to be identi?ed as speci?c for human liver cancer cell[24,25]and shows great binding af?nity for corre-sponding hepatocellular carcinoma cell line-LH86.More impor-tantly,TLS11a can also function as a membrane protein to protect the nanocomposites to internalize into cells.In the present study, we prepared a multifunctional?uorescent-magnetic nano-composite modi?ed with TLS11a aptamer(Fe3O4@SiO2(FITC)-Apt1) via a facile method with low cytotoxicity and good biocompability, which was potentially for liver cancer targeting and imaging.Iron oxide magnetic nanoparticles were coated with a?uorescent silica shell which would allow their visualization by optical means,and then TLS11a aptamers were introduced on the surface of nano-particles through the formation of amide bond to achieve cancer targeting capability[26e29].HepG2,MCF-7,4T1and SGC-7901cells were chosen as our model cancer cell lines for the in vitro studies.The cellular presence of the nanoprobe was con?rmed by both?uorescence and MR imaging.Cancer cell targeting and MR imaging ability were further tested in vivo.All the data highlighted that the prepared nanoprobe had good potential for targeted diagnostic biomodal imaging.
2.Experimental section
2.1.Materials and reagents
Magnetic nanoparticles(MNPs,10nm,2mg mLà1),modifying with mercaptopropionic acid on the surface,were obtained from Nanjing Nanoeast Biotech Co.LTD(Nanjing,China).1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC),N-hydroxysuccinimide(NHS),tetraethoxysilane(TEOS),(3-aminopropyl)-triethoxysilane(APTES)and?uorescein isothiocya-nate isomer I(FITC)were obtained from Sigma-Aldrich Inc.(USA). 40,60-Diamidino-2-phenylindole solution(DAPI)was purchased from TCI Moving Your Chemistry Forward(Japan).3-(4,5-Dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide(MTT)was received from KeyGen Biotech.Phosphate buffer solution(PBS)was prepared by mixing8g NaCl,0.2g KCl,1.42g Na2HPO4and0.27g KH2PO4in1L of ultrapure water.Dulbecco's modi?ed eagle me-dium(DMEM)and Roswell park memorial institute(RPMI-1640) medium both containing10%foetal bovine serum(FBS)and1% penicillin-streptomycin were gained from KeyGen Biotech.Human hepatoma(HepG2),human mammary cancer(MCF-7),mouse mammary tumor cell(4T1),gastric cancer(SGC-7901)cells were obtained from Department of Microbiology and Immunology, Medical School of Southeast University.All of other chemicals were analytical grade.Ultrapure water(18.2M U cm)was got through Thermo puri?cation system.
Aptamers were purchased from Sangon Biological Engineering Technology&Co.Ltd.(Shanghai,China)and puri?ed using high performance liquid chromatography(HPLC).TLS11a aptamer could speci?cally bind to the membrane surface of hepatocellular carci-noma cells.The TLS11a aptamer modi?ed with e COOH at the end of 50was named to Apt1and used for coupling with nanoparticles, while the intact complementary DNA of TLS11a aptamer was modi?ed with a?uorescent dye of Cy3at the5'-end and named to Apt2.Their sequences were:
TLS11a aptamer(Apt1):50e COOH e ACA GCA TCC CCA TGT GAA CAA TCG CAT TGT GAT TGT TAC GGT TTC CGC CTC ATG GAC GTG CTG-30.
Complementary to Apt1(Apt2):50-Cy3-CAG CAC GTC CAT GAG GCG GAA ACC GTA ACA ATC ACA ATG CGA TTG TTC ACA TGG GGA TGC TGT-30.
2.2.Apparatus
UV e vis spectra were carried on a UV-2450spectrophotometer (SHIMADZU,Japan).The morphology and size of the nanoparticles were observed with a transmission electronmicroscope(TEM, Model S-2400N,Hitachi,Japan)and a?eld emission scanning electron microscope(FESEM,Hitachi S-4800,Japan).Fluorescence spectra were carried out on a FluoroMax-4spectro?uorometer with Xenon discharge lamp excitation(HORIBA,USA).Confocal laser microscopy images were carried out by a confocal laser scanning microscopy(CLSM,FluoView?FV1000,Olympus,Japan).MRI was
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obtained in a clinical3.0T Clinical Siemens Trio Scanner at room temperature(Magnetom Verio3T,Germany).
2.3.Synthesis of multifunctional?uorescent-magnetic nanocomposite
The preparation of multifunctional?uorescent-magnetic nano-composite was performed according to the previously described procedure with some minor modi?cations[11,12,30].Typically, 2mg of FITC was mixed with50m L of APTES in1mL of anhydrous ethanol and gently stirred in dark at room temperature for12h.The resulting mixture of FITC-APTES complex(The reaction and mo-lecular structure was showed in Scheme S1)was stored at4 C for future use.100m L of TEOS was added into10mL of anhydrous ethanol under continuous slight shaking for10min.At the same time,1mL of H2O and500m L of MNPs were added into10mL of anhydrous ethanol under bath sonication treatment.After soni-cation for another10min,the resulted mixture was dropwisely added to above TEOS solution.Then1mL of aqueous ammonia (25wt%)was added to initiate hydrolyzation.The mixed solution was kept stirring for1h,followed by addition of500m L FITC-APTES, 300m L TEOS and stirring for another2h at room temperature. Finally,the Fe3O4@SiO2(FITC)nanoparticles(donated to MFS)were collected by magnetic separation and washed with anhydrous ethanol and ultrapure water for several times to remove excess reactants.Then they were re-dispersed in anhydrous ethanol to a ?nal volume of1mL and kept at4 C for following experiments.
2.4.Coupling of Apt1to MFS
For synthesis of Apt1modi?ed MFS nanoparticles,the amine-functional MFS nanoparticles were prepared in advance.Brie?y, 40m L of APTES was added into1mL of MFS nanoparticles sus-pension under stirring.The hydrolyzation reaction was triggered by adding0.1mL of aqueous ammonia(25wt%).After sonication for 1h,the nanoparticles were obtained by centrifugation and washed several times with anhydrous ethanol and deionized water to remove excess reactants.The resulting amine-functional MFS nanoparticles were dried in vacuum overnight.
For covalent coupling Apt1with MFS,10mg of amine-functionalized MFS nanoparticles were?rstly dispersed in5mL of PBS at room temperature.Then5mg of EDC,5mg of NHS were added into the above suspension.After incubation for15min, 100m L of Apt1(1m M)was added and shaken slightly for another 3h.The resulting nanoparticles were washed with ultrapure water and PBS to remove unbounded Apt1by magnetic?eld and centri-fugation.Finally,the conjugates of MFS-Apt1were dispersed in 2mL of PBS and stored at4 C for future use.
2.5.Cell culture and cytotoxicity assay
The HepG2,MCF-7cells were grown in DMEM,while4T1,SGC-7901cells were in RPMI-1640medium supplemented with10% fetal calf serum and1%penicillin-streptomycin at37 C in a hu-midi?ed atmosphere containing5%CO2.For cytotoxicity assay,cells were cultivated at5?103cells in100m L media per well of a96-well plate for12h.After discarding the media,100m L of0,10,20, 40and80m g/mL of MFS-Apt1suspension in media were added to each well and incubated for24,48,and72h,respectively.Then,the media was discarded,20m L of5mg mLà1MTT solution was added to each well and incubated for4h.After that,150m L dime-thylsulfoxide was added into each well and shaking for15min to solubilize the formazan dye.The absorbance of the plate was recorded on a microplate reader at a wavelength of450nm(Bio-Rad,USA).And the data was expressed as a ratio of treated to untreated cells(control).
To study the cellular uptake of MFS-Apt1nanoprobes,HepG2 cells were incubated with MFS-Apt1suspension in DMEM medium at various concentrations.The cells were harvested by trypsiniza-tion after the indicated time.The cellular uptake of nanoprobes was determined by the incorporated?uorescence intensity through ?ow cytometry.
2.6.Confocal laser microscopy assay
For confocal laser microscopy experiments,the cells were seeded on14mm-diameter cell culture dishes at a density of 1?104cells per well.After cultivated at37 C for24h,12m L 5mg mLà1of MFS-Apt1were added into the dishes,followed by incubation at room temperature for3h,washing with PBS for three times to remove redundant reagents.After that,the cells were?xed with PBS containing4%paraformaldehyde for20min.Nuclei were speci?cally stained with diluted DAPI solution in PBS(volume ratio of1:100for DAPI to PBS).
2.7.MRI experiment in vitro and vivo of nude mouse
HepG2cells were dispersed in12-well plates at a density of 2?105cells in each well for24h.Then200m L of MFS-Apt1with various concentrations were added,and cells without addition of nanoprobes was used as control.After incubation for3h,the cells were washed with PBS for?ve times to remove the redundant non-internalized nanoprobes,harvested with trypsinization,re-suspended in DMEM and?xed with1%agarose gel for MR imaging.
All animal experiments were investigated in compliance with the Guidelines for the Care and Use of Research Animals established by the Stanford University Animal Studies Committee.Male athy-mic nude mice in4e6weeks old were obtained from the Shanghai Laboratory Animal Center,Chinese Academy Sciences(Shanghai Slac Laboratory Animal Co.Ltd.)and kept under sterile conditions. Nude mice were in-situ implanted with HepG2cells.When the tumors of the tumor-bearing nude mice reached0.4cm in diam-eter,the tumor bearing mice were hypodermic injected for MR imaging and biodistribution studies.
3.Results and discussion
3.1.Synthesis and characterization of multifunctional nanoprobes
MFS were synthesized by hydrolysis and condensation of tet-raethylorthosilicate(TEOS)and FITC-APTES in an ammonia ethanol/water solution.This synthetic approach enables the co-encapsulation of Fe3O4NPs and a great deal of FITC dyes into the silica shell simultaneously.The TEM images(Fig.1A and Figure S1) showed that the prepared nanoprobes were spherical and uniform with a distinct core-shell structure.Through changing the amount of TEOS and APTES,three different sizes of82±4,146±10and 192±8nm were prepared with the thickness of the silica shell is about36±2,73±5,and91±4nm,respectively,while the Fe3O4 core was around10nm.The EDS energy spectrum analysis demonstrated the existence of element silicon and iron in nanop-robes(Fig.1B).The as-prepared nanoprobes displayed aurantiacus color under sunlight(a)and chartreuse color under UV illumination (b,l ex?365nm).When a magnet was placed close to the vial,the nanoprobes under UV lamp(c)and sunlight(d)were harvested in just a few minutes as observation inserted in Fig.1B.The super-paramagnetic property of nanoprobes was further con?rmed by magnetic hysteresis loops curves(Fig.1C,Figure S2and Table S1). The zero coercivity and zero remanence revealed that the nanop-robes kept superparamagnetism without magnetic hysteresis at
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room temperature.However,SiO 2shell caused the decrease of saturation magnetization of the nanoprobe [31],and it was evi-denced that the saturation magnetization was declining with the SiO 2shell thickness increasing as shown in Figure S2.
The shape of the UV e vis absorption spectrum of FITC depended on its concentration and a shoulder at around 460nm become dominant at high concentration [32].In our case,an absorbance peak and a shoulder were observed around 483and 460nm in
UV e vis spectrum of FITC (curve a in Fig.1D).When FITC was coupled with APTES,the shoulder peak was almost disappeared,while the absorbance peak shifted 20nm to red,con ?rming the successful conjunction of APTES and FITC (curve b in Fig.1D).After being entrapped into silica shell,a little blue shift of absorbance peak was observed for MFS compared with FITC-APTES (curve c in Fig.1D).No absorbance could be observed from the UV e vis spectra of MNPs.Thus,the absorbance at around 495nm for MFS was come from the entrapped FITC.This blue shift could be explicated by changes in the environment polarity and polarizability of ?uo-rophores embedded in the silica matrix [33,34].In addition,MFS nanoprobes had similar emission spectra to pure FITC,demon-strating that the dyes were co-embedded in the nanoprobes and the silica shell did not change the spectra properties of FITC dyestuff.In a word,the core-shell structure of nanoprobes con-taining both Fe 3O 4NPs and ?uorescent dyes was successfully constructed.
To enhance the diagnostic performance of targeting liver cancer,TLS11a aptamer was covalently coupled to the surface of nanoprobe through amido bond (Scheme 1).There were about 2.04mmol/g amino groups on the surface of MFS with diameter about the sub-100nm,which was determined by ninhydrin chromogenic reaction (Figure S3and S4).The coupling of Apt1to MFS was demonstrated by UV e vis measurements.TLS11a aptamer has a strong absorption at 260nm,while MFS displayed a distinct absorption located at
493nm corresponding to the absorption of immobilized FITC (Figure S5).After Apt1coupling,MFS-Apt1showed an absorption at 495nm corresponding to FITC and a new absorption peak around 260nm,which was ascribed to the absorption of Apt1,con ?rming the successfully binding of Apt1to MFS.The binding of Apt1to MFS was also con ?rmed by alteration of zeta potential for each modi-?cation process.After conjugation with amino groups,the zeta potential changed from à41.4mV for Fe 3O 4NPs to à14.1mV for MFS-NH 2with particle size of 82±4nm (Figure S6and Table S1).When TLS11a aptamer was coated to MFS,the zeta potential decreased to à20.2mV due to negative charge of aptamer.All these results veri ?ed the successful coupling of TLS11a aptamer to MFS.
The ?uorescence stability of the nanoprobes was then investi-gated.The ?uorescence intensity of MFS and MFS-Apt1maintained about 84.5%and 77.1%of their initial value after 1h excitation.The luminescence decay of MFS-Apt1were 2.95,3.01,and 2.40ns for the particle size of 82±4,146±10,192±8nm,respectively,while FITC and APTES-FITC had the luminescence decay of 3.63and 3.18ns,respectively (Figure S7and Table S1).The change of lumi-nescence decay was attributed to the interaction between close pairs of dye molecules.It resulted in the energy transferring during dye molecules,shortened the time that molecule remain in its excited state,and ?nally resulting the reduction of ?uorescence lifetimes [33,34].The high ?uorescence stability and acceptable luminescence decay facilitated the nanoprobes to be used as cancer cell imaging without additional modi ?cation.
3.2.Cytotoxicity,biocompatibility and targeting ability of MFS-Apt1nanoprobes
HepG2cells were employed to assess the cytotoxicity of MFS-Apt1nanoprobes by MTT assays.After incubation with nanop-robes for 72h,cancer cell lines could retain above 87%viability in
Fig.1.(A)TEM images of MFS.(B)EDS energy spectrum analysis of MFS evidencing the presence of Fe and Si.Insert:Photograph of MFS under (a)sunlight and (b)UV light without additional magnetic ?eld,(c)UV light (l ex ?365nm)and (d)sunlight with an additional magnetic ?eld.(C)Magnetic hysteresis loops of MNPs and MFS at room temperature.(D)UV e vis spectrum of (a)FITC,(b)FITC-APTES,(c)MFS,(d)MNPs and (e)emission spectra of MFS recorded at 467nm excitation.
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the presence of MFS-Apt1of 82±4nm even at the concentration up to 80m g mL à1,while the larger size nanoprobe only kept about 69%and 65%viability at the same circumstances (Fig.2).This suggested that sub-100nm nanoprobes exhibited lower cytotox-icity and better biocompatibility than that of large ones [12].
To better understand the biocompatibility of the nanoprobe in vivo ,hemolysis assay was performed.Hemoglobin would release into the solution,which was directly proportional to hemolytic activity of the carrier in the hemolysis assay.Therefore,the UV e vis absorbance at 541nm can be served as an
index to quantify the hemolytic activity [12,35].As exhibited in Figure S8and Figure S9,no signi ?cant visible hemolysis (4.87%)could be observed for nanoprobes with size of 82±4nm at 80m g/mL compared with negative and positive controls.On the other hand,the nanoprobes with sizes of 146±10and 192±8nm showed hemolysis of 15.43%and 17.60%.The signi ?cant hemolysis effect might be attributed to size effect [12].No visible hemolysis effect demonstrated that the nanoprobes with small size of 82±4nm could be applied to research in vivo potentially.For the ultimate use of the nanoprobes as targeted carrier in vivo ,the nanoprobes should not only retain their low toxicity and negligible hemolytic activity,but also need to keep its targeting ability.In order to evaluate the targeting of nanoprobes in vivo ,the nanoprobes were magnetic separated and collected after incubated with human blood for 3h.Then,the nanoprobes were mixed with different concentrations of Apt2and incubated for 30min.The Apt2was completely complementary to Apt1and labelled with a ?uo-rophore (Cy3)at the end of 5’.The nanoparticles were collected by repeated centrifugation and washing to remove redundant Apt2oligonucleotide.When the nanoparticles were excited with 467nm light,the ?uorescence intensity at 561nm was growing with the increased amount of Apt2,while the intensity at ~510nm was decreased gradually (Figure S10).The emission band located at 561nm could be attributed to Cy3dyes labelled with Apt2.Both nanoprobes and Cy3labelled Apt2had a negative zeta potential respectively.Therefore,they could not be combined via an elec-trostatic interaction.In addition,the emission spectrum of nanop-robes was overlapped with the absorption spectrum of Apt2
Scheme 1.Synthetic route of MFS-Apt1.
Fig.2.Viability of HepG2cells after incubation with various concentrations of nanoprobes with the size of (A)82±4,(B)146±10and (C)192±8nm at 24,48and 72h.
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(Figure S10C ).This indicated that energy could transfer from nanoprobe to Cy3of Apt2,where the ?uorescein nanoprobes were regarded as ?uorescent donor while Cy3labelled with Apt2acted as the ?uorescent acceptor [36,37].This led to the decrease of the ?uorescent intensity at ~510nm corresponding to nanoprobes with the increasing amount of Apt2.This phenomenon indirectly demonstrated that Apt1was not hydrolyzed by DNA hydrolytic enzyme in vivo to some extent and was completely complementary to Apt2through complementary base pairing.
3.3.Feasibility assessment of MFS-Apt1nanoprobes in ?uorescent and MRI imaging
For ?uorescent and MRI imaging,the amount of nanoprobes added into each wells and the incubation time were optimized.As shown in Fig.3A,the ?uorescence intensity increased with the increasing amount of nanoprobes and reached a maximum value at 60m g/2?105cells.After that,the ?uorescence intensity gradually decreased with increasing labeling density due to the
self-
Fig.3.(A)Fluorescence intensity of 2?105HepG2cells incubation with different amount of nanoprobes for 2h and (B)2?105HepG2cells incubation with 60m g of nanoprobes for 10,20,40,60,120,180,240,300
min.
Fig.4.Confocal laser scanning microscopy images of HepG2,4T1,SGC-7901,MCF-7cells after 3h incubation with nanoprobes.The internalized nanoparticles were green (MFS-Apt1)and nuclei were blue (DAPI staining).(For interpretation of the references to colour in this ?gure legend,the reader is referred to the web version of this article.)
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quenching of ?uorescein on the surface of cells [38,39].Therefore,the optimum amount of nanoprobes was chosen to be 60m g/2?105cells (Fig.3A).On the other hand,the ?uorescence intensity increased accompanied with increasing incubation time and reached a plateau after 2h owing to the completely labeling of nanoprobes with cells.Consequently,the optimum incubation time was selected to be 2h (Fig.3B).
To evaluate the cell-targeting speci ?city of the prepared nanoprobes,the uptake of three nanoparticles (the nanoprobes,MFS and MNPs)by HepG2cells (Apt1positive)and the uptake of the prepared nanoprobes by HepG2cells and three Apt1negative cells (4T1,SGC-7901and MCF-7cells)were compared through confocal microscopy analysis.After a 2h incubation of HepG2cells with the nanoprobes,numbers of green spots of FITC were observed around the cells and the ?uorescence intensity was strongly position dependent (Fig.4A).After the nanoprobe-incubated cells were co-stained with a nucleus dye,DAPI,the blue ?uorescence of entrapping DAPI and FITC green ?uorescence could be observed,which demonstrated that the ?uorescence activation of the nanoprobe took place on the surface or internal-ized into cells.In contrast,some ?uorescence spots was observed after HepG2cells were treated with MFS nanoparticles,but these green spots could not coincide with the blue nucleus.Therefore,the green ?uorescence spots for MFS was due to the nonspeci ?c attaching of nanoprobe on the substrate,con ?rming that the nonspeci ?c uptake of nanoparticles was negligible (Figure S11).In addition,Apt1negative cells of 4T1,SGC-7901and MCF-7were treated with MFS-Apt1under similar conditions to the HepG2cells.
Only very little green ?uorescence was observed for SGC-7901and MCF-7cells due to low expression level of recognize sites on these cells,further verifying the target-cell-speci ?c delivery (Fig.4B,C and D).
To estimate the potential of nanoprobes as a targeted MR contrast agent,HepG2cells were cultured with various concen-trations of nanoprobes for 3h,and cells without addition of nanoprobes were used as control.Cells were harvested and re-suspended in PBS and ?xed with 1%agarose gel.T 2-weighted MRI images of resulting cells in agarose gel were investigated.As shown in Fig.5A,the T 2-weighted MR signal was signi ?cantly negatively enhanced with the increasing concentration of the nanoprobes,indicating that a high magnetic ?eld gradient was generated by MFS-Apt1which has been speci ?cally captured by HepG2cells.The relaxivity of MFS-Apt1was calculated to be 11.5mg à1s à1(the slope of the ?tted curve in Fig.5A,using HepG2cells without addition of nanoprobes as control).Moreover,when MFS-Apt1was incubated with 4T1,SGC-7901and MCF-7cells under the same conditions as HepG2cells,only very weak T 2-weighted MR signal was observed,respectively (Figure S12).This was in good agreement with the confocal microscopy analysis,suggesting good target-cell-speci ?city of MFS-Apt1.
3.4.The magnetic sensitivity of MFS-Apt1in living mice
The mice was implanted HepG2cells in situ of liver to evaluate the magnetic sensitivity of MFS-Apt1in vivo .When the tumors reached 0.4e 0.6cm in diameter (6weeks of age and 18±2g body
Fig.5.In vitro and in vivo MRI study of MFS-Apt1.(A)Relaxation (1/T 2,R 2)plot of samples as a function of nanoprobes in agarose gel.Insert:T 2-eighted MR images of HepG2cells treated with MFS-Apt1nanoprobes at different concentrations.(B)Time course T 2-weighted MRI of tumor-bearing nude mouse before and after subcutaneous injection of MFS-Apt1.(C)In vivo T 2-weighted MRI of a mouse before (left)and after (right)subcutaneous injection of MFS-Apt1.
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weight),the tumor bearing mice were hypodermic subjected of MFS-Apt1on the lower back of mice for MR imaging.The time course spatial distribution of the nanoprobes could be monitored in real time without sacri?cing the subjects through axial scan.T2-weighted MRI images were received from the organ of liver at various time points,i.e.1,2,4,6,and24h(Fig.5B).The circulation time of nanoprobe in the mouse body getting into tumors region was within1h upon hypodermic injection.Then,HepG2tumors region exhibited negatively enhanced signals in the course of time subcutaneous injection of MFS-Apt1.During this period,a signi?-cant accumulation of negatively enhanced signal in the tumor re-gion was also found in the mouse based on sagittal view in Fig.5C. All these studies demonstrated that MFS-Apt1could be effectively used as a nanoprobe platform for MRI despite of the minor tumor size.The nanoprobe with excellent biocompatibility,low toxicity and appropriate circulation life time,suggests that the multifunc-tional?uorescent-magnetic nanoprobe can be a promising candi-date for further studies both on the early diagnosis and image-guided therapy/surgery of cancer.
4.Conclusions
In summary,a multifunctional?uorescent-magnetic nanoprobe modi?ed with aptamer was successfully constructed by a facile improved St€o ber method.The resulting nanoprobes exhibited good dispersion,stable?uorescence,low cytotoxicity and excellent biocompatibility.Besides,the multifunctional nanoprobes were easily coupled with TLS11a aptamer,which could be used for the speci?c cancer cell targeting and simultaneous?uorescent and MR imaging both in vivo and in vitro.The MR imaging of tumor-bearing nude mouse was also con?rmed.The proposed nanoprobe may be exploited as a potential platform for biomedical imaging,early diagnosis and disease therapy.
Acknowledgments
The project was supported by National Natural Science Foun-dation of China(Grant No.21375014and21475020).
Appendix A.Supplementary data
Supplementary data related to this article can be found at http:// https://www.wendangku.net/doc/7f2832327.html,/10.1016/j.aca.2016.07.037.
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to与for的用法和区别
to与for的用法和区别 一般情况下, to后面常接对象; for后面表示原因与目的为多。 Thank you for helping me. Thanks to all of you. to sb.表示对某人有直接影响比如，食物对某人好或者不好就用to; for表示从意义、价值等间接角度来说，例如对某人而言是重要的，就用for. for和to这两个介词，意义丰富，用法复杂。这里仅就它们主要用法进行比较。 1. 表示各种“目的” 1. What do you study English for? 你为什么要学英语？ 2. She went to france for holiday. 她到法国度假去了。 3. These books are written for pupils. 这些书是为学生些的。 4. hope for the best, prepare for the worst. 作最好的打算，作最坏的准备。 2．对于 1．She has a liking for painting. 她爱好绘画。 2．She had a natural gift for teaching. 她对教学有天赋/ 3．表示赞成同情，用for不用to. 1. Are you for the idea or against it? 你是支持还是反对这个想法？ 2. He expresses sympathy for the common people.. 他表现了对普通老百姓的同情。 3. I felt deeply sorry for my friend who was very ill. 4 for表示因为，由于（常有较活译法） 1 Thank you for coming. 谢谢你来。 2. France is famous for its wines. 法国因酒而出名。 5．当事人对某事的主观看法，对于（某人），对…来说（多和形容词连用）用介词to，不用for.. He said that money was not important to him. 他说钱对他并不重要。 To her it was rather unusual. 对她来说这是相当不寻常的。 They are cruel to animals. 他们对动物很残忍。 6．for和fit, good, bad, useful, suitable 等形容词连用，表示适宜，适合。 Some training will make them fit for the job. 经过一段训练，他们会胜任这项工作的。 Exercises are good for health. 锻炼有益于健康。 Smoking and drinking are bad for health. 抽烟喝酒对健康有害。 You are not suited for the kind of work you are doing. 7. for表示不定式逻辑上的主语，可以用在主语、表语、状语、定语中。 1．It would be best for you to write to him. 2．The simple thing is for him to resign at once. 3．There was nowhere else for me to go. 4．He opened a door and stood aside for her to pass.
of与for的用法以及区别
of与for的用法以及区别 for 表原因、目的 of 表从属关系 介词of的用法 （1）所有关系 this is a picture of a classroom （2）部分关系 a piece of paper a cup of tea a glass of water a bottle of milk what kind of football，American of soccer？ （3）描写关系 a man of thirty 三十岁的人 a man of shanghai 上海人 （4）承受动作 the exploitation of man by man．人对人的剥削。 （5）同位关系 It was a cold spring morning in the city of London in England． （6）关于，对于 What do you think of Chinese food？ 你觉得中国食品怎么样？ 介词 for 的用法小结 1. 表示“当作、作为”。如: I like some bread and milk for breakfast. 我喜欢把面包和牛奶作为早餐。What will we have for supper? 我们晚餐吃什么?
2. 表示理由或原因,意为“因为、由于”。如: Thank you for helping me with my English. 谢谢你帮我学习英语。 Thank you for your last letter. 谢谢你上次的来信。 Thank you for teaching us so well. 感谢你如此尽心地教我们。 3. 表示动作的对象或接受者,意为“给……”、“对…… (而言)”。如: Let me pick it up for you. 让我为你捡起来。 Watching TV too much is bad for your health. 看电视太多有害于你的健康。 4. 表示时间、距离,意为“计、达”。如: I usually do the running for an hour in the morning. 我早晨通常跑步一小时。We will stay there for two days. 我们将在那里逗留两天。 5. 表示去向、目的,意为“向、往、取、买”等。如: let’s go for a walk. 我们出去散步吧。 I came here for my schoolbag.我来这儿取书包。 I paid twenty yuan for the dictionary. 我花了20元买这本词典。 6. 表示所属关系或用途,意为“为、适于……的”。如: It’s time for school. 到上学的时间了。 Here is a letter for you. 这儿有你的一封信。 7. 表示“支持、赞成”。如: Are you for this plan or against it? 你是支持还是反对这个计划? 8. 用于一些固定搭配中。如: Who are you waiting for? 你在等谁? For example, Mr Green is a kind teacher. 比如,格林先生是一位心地善良的老师。
常用介词用法(for to with of)
For的用法 1. 表示“当作、作为”。如: I like some bread and milk for breakfast. 我喜欢把面包和牛奶作为早餐。 What will we have for supper? 我们晚餐吃什么? 2. 表示理由或原因,意为“因为、由于”。如: Thank you for helping me with my English. 谢谢你帮我学习英语。 3. 表示动作的对象或接受者,意为“给……”、“对…… (而言)”。如: Let me pick it up for you. 让我为你捡起来。 Watching TV too much is bad for your health. 看电视太多有害于你的健康。 4. 表示时间、距离,意为“计、达”。如: I usually do the running for an hour in the morning. 我早晨通常跑步一小时。 We will stay there for two days. 我们将在那里逗留两天。 5. 表示去向、目的,意为“向、往、取、买”等。如: Let’s go for a walk. 我们出去散步吧。 I came here for my schoolbag.我来这儿取书包。 I paid twenty yuan for the dictionary. 我花了20元买这本词典。 6. 表示所属关系或用途,意为“为、适于……的”。如: It’s time for school. 到上学的时间了。 Here is a letter for you. 这儿有你的一封信。 7. 表示“支持、赞成”。如: Are you for this plan or against it? 你是支持还是反对这个计划? 8. 用于一些固定搭配中。如: Who are you waiting for? 你在等谁? For example, Mr Green is a kind teacher. 比如,格林先生是一位心地善良的老师。 尽管for 的用法较多，但记住常用的几个就可以了。 to的用法: 一：表示相对，针对 be strange (common, new, familiar, peculiar) to This injection will make you immune to infection. 二：表示对比，比较 1：以-ior结尾的形容词，后接介词to表示比较，如：superior ,inferior,prior,senior,junior 2: 一些本身就含有比较或比拟意思的形容词，如equal，similar，equivalent，analogous A is similar to B in many ways.
(完整版)介词for用法归纳
介词for用法归纳 用法1：(表目的)为了。如： They went out for a walk. 他们出去散步了。 What did you do that for? 你干吗这样做？ That’s what we’re here for. 这正是我们来的目的。 What’s she gone for this time? 她这次去干什么去了？ He was waiting for the bus. 他在等公共汽车。 【用法说明】在通常情况下，英语不用for doing sth 来表示目的。如： 他去那儿看他叔叔。 误：He went there for seeing his uncle. 正：He went there to see his uncle. 但是，若一个动名词已名词化，则可与for 连用表目的。如： He went there for swimming. 他去那儿游泳。(swimming 已名词化) 注意：若不是表目的，而是表原因、用途等，则其后可接动名词。(见下面的有关用法) 用法2：(表利益)为，为了。如： What can I do for you? 你想要我什么？ We study hard for our motherland. 我们为祖国努力学习。 Would you please carry this for me? 请你替我提这个东西好吗？ Do more exercise for the good of your health. 为了健康你要多运动。 【用法说明】(1) 有些后接双宾语的动词(如buy, choose, cook, fetch, find, get, order, prepare, sing, spare 等)，当双宾语易位时，通常用for 来引出间接宾语，表示间接宾语为受益者。如： She made her daughter a dress. / She made a dress for her daughter. 她为她女儿做了件连衣裙。 He cooked us some potatoes. / He cooked some potatoes for us. 他为我们煮了些土豆。 注意，类似下面这样的句子必须用for： He bought a new chair for the office. 他为办公室买了张新办公椅。 (2) 注意不要按汉语字面意思，在一些及物动词后误加介词for： 他们决定在电视上为他们的新产品打广告。 误：They decided to advertise for their new product on TV. 正：They decided to advertise their new product on TV. 注：advertise 可用作及物或不及物动词，但含义不同：advertise sth＝为卖出某物而打广告；advertise for sth＝为寻找某物而打广告。如：advertise for a job=登广告求职。由于受汉语“为”的影响，而此处误加了介词for。类似地，汉语中的“为人民服务”，说成英语是serve the people，而不是serve for the people，“为某人的死报仇”，说成英语是avenge sb’s death，而不是avenge for sb’s death，等等。用法3：(表用途)用于，用来。如： Knives are used for cutting things. 小刀是用来切东西的。 This knife is for cutting bread. 这把小刀是用于切面包的。 It’s a machine for slicing bread. 这是切面包的机器。 The doctor gave her some medicine for her cold. 医生给了她一些感冒药。 用法4：为得到，为拿到，为取得。如： He went home for his book. 他回家拿书。 He went to his friend for advice. 他去向朋友请教。 She often asked her parents for money. 她经常向父母要钱。
of和for的用法
of 1....的,属于 One of the legs of the table is broken. 桌子的一条腿坏了。 Mr.Brown is a friend of mine. 布朗先生是我的朋友。 2.用...做成的;由...制成 The house is of stone. 这房子是石建的。 3.含有...的;装有...的 4....之中的;...的成员 Of all the students in this class,Tom is the best. 在这个班级中,汤姆是最优秀的。 5.(表示同位) He came to New York at the age of ten. 他在十岁时来到纽约。 6.(表示宾格关系) He gave a lecture on the use of solar energy. 他就太阳能的利用作了一场讲演。 7.(表示主格关系) We waited for the arrival of the next bus. 我们等待下一班汽车的到来。
I have the complete works of Shakespeare. 我有莎士比亚全集。 8.来自...的;出自 He was a graduate of the University of Hawaii. 他是夏威夷大学的毕业生。 9.因为 Her son died of hepatitis. 她儿子因患肝炎而死。 10.在...方面 My aunt is hard of hearing. 我姑妈耳朵有点聋。 11.【美】(时间)在...之前 12.(表示具有某种性质) It is a matter of importance. 这是一件重要的事。 For 1.为,为了 They fought for national independence. 他们为民族独立而战。 This letter is for you. 这是你的信。
for和to区别
1.表示各种“目的”，用for （1）What do you study English for 你为什么要学英语？ （2）went to france for holiday. 她到法国度假去了。 （3）These books are written for pupils. 这些书是为学生些的。 （4）hope for the best, prepare for the worst. 作最好的打算，作最坏的准备。 2．“对于”用for （1）She has a liking for painting. 她爱好绘画。 （2）She had a natural gift for teaching. 她对教学有天赋/ 3．表示“赞成、同情”，用for （1）Are you for the idea or against it 你是支持还是反对这个想法？ （2）He expresses sympathy for the common people.. 他表现了对普通老百姓的同情。 （3）I felt deeply sorry for my friend who was very ill. 4. 表示“因为，由于”（常有较活译法），用for （1）Thank you for coming. 谢谢你来。
（2）France is famous for its wines. 法国因酒而出名。 5．当事人对某事的主观看法，“对于（某人），对…来说”，（多和形容词连用），用介词to，不用for. （1）He said that money was not important to him. 他说钱对他并不重要。 （2）To her it was rather unusual. 对她来说这是相当不寻常的。 （3）They are cruel to animals. 他们对动物很残忍。 6．和fit, good, bad, useful, suitable 等形容词连用，表示“适宜，适合”，用for。（1）Some training will make them fit for the job. 经过一段训练，他们会胜任这项工作的。 （2）Exercises are good for health. 锻炼有益于健康。 （3）Smoking and drinking are bad for health. 抽烟喝酒对健康有害。 （4）You are not suited for the kind of work you are doing. 7. 表示不定式逻辑上的主语，可以用在主语、表语、状语、定语中。 （1）It would be best for you to write to him. （2） The simple thing is for him to resign at once.
双宾语 to for的用法
1.两者都可以引出间接宾语，但要根据不同的动词分别选用介词to 或for：(1) 在give, pass, hand, lend, send, tell, bring, show, pay, read, return, write, offer, teach, throw 等之后接介词to。 如： 请把那本字典递给我。 正：Please hand me that dictionary. 正：Please hand that dictionary to me. 她去年教我们的音乐。 正：She taught us music last year. 正：She taught music to us last year. (2) 在buy, make, get, order, cook, sing, fetch, play, find, paint, choose,prepare, spare 等之后用介词for 。如： 他为我们唱了首英语歌。 正：He sang us an English song. 正：He sang an English song for us. 请帮我把钥匙找到。 正：Please find me the keys. 正：Please find the keys for me. 能耽搁你几分钟吗(即你能为我抽出几分钟吗)? 正：Can you spare me a few minutes? 正：Can you spare a few minutes for me? 注：有的动词由于搭配和含义的不同，用介词to 或for 都是可能的。如：do sb a favour＝do a favour for sb 帮某人的忙 do sb harm＝do harm to sb 对某人有害
双宾语tofor的用法
1. 两者都可以引出间接宾语，但要根据不同的动词分别选用介词to 或for： (1) 在give, pass, hand, lend, send, tell, bring, show, pay, read, return, write, offer, teach, throw 等之后接介词to。 如： 请把那本字典递给我。 正：Please hand me that dictionary. 正：Please hand that dictionary to me. 她去年教我们的音乐。 正：She taught us music last year. 正：She taught music to us last year. (2) 在buy, make, get, order, cook, sing, fetch, play, find, paint, choose,prepare, spare 等之后用介词for 。如： 他为我们唱了首英语歌。 正：He sang us an English song. 正：He sang an English song for us. 请帮我把钥匙找到。 正：Please find me the keys. 正：Please find the keys for me. 能耽搁你几分钟吗(即你能为我抽出几分钟吗)? 正：Can you spare me a few minutes? 正：Can you spare a few minutes for me? 注：有的动词由于搭配和含义的不同，用介词to 或for 都是可能的。如： do sb a favou r do a favour for sb 帮某人的忙 do sb harnn= do harm to sb 对某人有害
for和of的用法
for的用法： 1. 表示“当作、作为”。如: I like some bread and milk for breakfast. 我喜欢把面包和牛奶作为早餐。 What will we have for supper? 我们晚餐吃什么? 2. 表示理由或原因,意为“因为、由于”。如: Thank you for helping me with my English. 谢谢你帮我学习英语。 Thank you for your last letter. 谢谢你上次的来信。 Thank you for teaching us so well. 感谢你如此尽心地教我们。 3. 表示动作的对象或接受者,意为“给……”、“对…… (而言)”。如: Let me pick it up for you. 让我为你捡起来。 Watching TV too much is bad for your health. 看电视太多有害于你的健康。 4. 表示时间、距离,意为“计、达”。如:
I usually do the running for an hour in the morning. 我早晨通常跑步一小时。 We will stay there for two days. 我们将在那里逗留两天。 5. 表示去向、目的,意为“向、往、取、买”等。如: Let’s go for a walk. 我们出去散步吧。 I came here for my schoolbag.我来这儿取书包。 I paid twenty yuan for the dictionary. 我花了20元买这本词典。 6. 表示所属关系或用途,意为“为、适于……的”。如: It’s time for school. 到上学的时间了。 Here is a letter for you. 这儿有你的一封信。 7. 表示“支持、赞成”。如: Are you for this plan or against it? 你是支持还是反对这个计划? 8. 用于一些固定搭配中。如:
英语形容词和of for 的用法
加入收藏夹 主题： 介词试题It’s + 形容词 + of sb. to do sth.和It’s + 形容词 + for sb. to do sth.的用法区别。 内容： It's very nice___pictures for me. A.of you to draw B.for you to draw C.for you drawing C.of you drawing 提交人：杨天若时间：1/23/2008 20:5:54 主题：for 与of 的辨别 内容：It's very nice___pictures for me. A.of you to draw B.for you to draw C.for you drawing C.of you drawing 答：选A 解析：该题考查的句型It’s + 形容词+ of sb. to do sth.和It’s +形容词+ for sb. to do sth.的用法区别。 “It’s + 形容词+ to do sth.”中常用of或for引出不定式的行为者，究竟用of sb.还是用for sb.，取决于前面的形容词。 1) 若形容词是描述不定式行为者的性格、品质的，如kind，good，nice，right，wrong，clever，careless，polite，foolish等，用of sb. 例： It’s very kind of you to help me. 你能帮我，真好。 It’s clever of you to work out the maths problem. 你真聪明，解出了这道数学题。 2) 若形容词仅仅是描述事物，不是对不定式行为者的品格进行评价，用for sb.，这类形容词有difficult，easy，hard，important，dangerous，（im）possible等。例： It’s very dangerous for children to cross the busy street. 对孩子们来说，穿过繁忙的街道很危险。 It’s difficult for u s to finish the work. 对我们来说，完成这项工作很困难。 for 与of 的辨别方法： 用介词后面的代词作主语，用介词前边的形容词作表语，造个句子。如果道理上通顺用of，不通则用for. 如： You are nice.(通顺，所以应用of)。 He is hard.(人是困难的，不通，因此应用for.) 由此可知，该题的正确答案应该为A项。 提交人：f7_liyf 时间：1/24/2008 11:18:42
to和for的用法有什么不同(一)
to和for的用法有什么不同（一） 一、引出间接宾语时的区别 两者都可以引出间接宾语，但要根据不同的动词分别选用介词to 或for，具体应注意以下三种情况： 1. 在give, pass, hand, lend, send, tell, bring, show, pay, read, return, write, offer, teach, throw 等之后接介词to。如： 请把那本字典递给我。 正：Please hand me that dictionary. 正：Please hand that dictionary to me. 她去年教我们的音乐。 正：She taught us music last year. 正：She taught music to us last year. 2. 在buy, make, get, order, cook, sing, fetch, play, find, paint, choose, prepare, spare 等之后用介词for 。如： 他为我们唱了首英语歌。 正：He sang us an English song. 正：He sang an English song for us. 请帮我把钥匙找到。 正：Please find me the keys. 正：Please find the keys for me. 能耽搁你几分钟吗(即你能为我抽出几分钟吗)? 正：Can you spare me a few minutes?
正：Can you spare a few minutes for me? 3. 有的动词由于用法和含义不同，用介词to 或for 都是可能的。如： do sb a favor＝do a favor for sb 帮某人的忙 do sb harm＝do harm to sb 对某人有害 在有的情况下，可能既不用for 也不用to，而用其他的介词。如： play sb a trick＝play a trick on sb 作弄某人 请比较： play sb some folk songs＝play some folk songs for sb 给某人演奏民歌 有时同一个动词，由于用法不同，所搭配的介词也可能不同，如leave sbsth 这一结构，若表示一般意义的为某人留下某物，则用介词for 引出间接宾语，即说leave sth for sb；若表示某人死后遗留下某物，则用介词to 引出间接宾语，即说leave sth to sb。如： Would you like to leave him a message? / Would you like to leave a message for him? 你要不要给他留个话？ Her father left her a large fortune. / Her father left a large fortune to her. 她父亲死后给她留下了一大笔财产。 二、表示目标或方向的区别 两者均可表示目标、目的地、方向等，此时也要根据不同动词分别对待。如： 1. 在come, go, walk, move, fly, ride, drive, march, return 等动词之后通常用介词to 表示目标或目的地。如： He has gone to Shanghai. 他到上海去了。 They walked to a river. 他们走到一条河边。
202X中考英语：to和for的区别与用法.doc
202X中考英语：to和for的区别与用法中考栏目我为考生们整理了“202X中考英语：to和for的区别与用法”，希望能帮到大家，想了解更多考试资讯，本网站的及时更新哦。 202X中考英语：to和for的区别与用法 to和for的区别与用法是什么 一般情况下, to后面常接对象; for后面表示原因与目的为多。 Thank you for helping me. Thanks to all of you. to sb. 表示对某人有直接影响比如，食物对某人好或者不好就用to; for 表示从意义、价值等间接角度来说，例如对某人而言是重要的，就用for. for和to这两个介词，意义丰富，用法复杂。这里仅就它们主要用法进行比较。 1. 表示各种“目的” 1. What do you study English for? 你为什么要学英语? 2. She went to france for holiday. 她到法国度假去了。 3. These books are written for pupils. 这些书是为学生些的。 4. hope for the best, prepare for the worst. 作最好的打算，作最坏的准备。
2.对于 1.She has a liking for painting. 她爱好绘画。 2.She had a natural gift for teaching. 她对教学有天赋。 3.表示赞成同情，用for不用to. 1. Are you for the idea or against it? 你是支持还是反对这个想法? 2. He expresses sympathy for the common people.. 他表现了对普通老百姓的同情。 3. I felt deeply sorry for my friend who was very ill. 4 for表示因为，由于(常有较活译法) 1.Thank you for coming. 谢谢你来。 2. France is famous for its wines. 法国因酒而出名。 5.当事人对某事的主观看法，对于(某人)，对?来说(多和形容词连用)用介词to，不用for.. He said that money was not important to him. 他说钱对他并不重要。 To her it was rather unusual. 对她来说这是相当不寻常的。 They are cruel to animals. 他们对动物很残忍。
keep的用法及of 、for sb.句型区别
keep的用法 1. 用作及物动词 ①意为"保存；保留；保持；保守"。如： Could you keep these letters for me, please? 你能替我保存这些信吗？ ②意为"遵守；维护"。如： Everyone must keep the rules. 人人必须遵守规章制度。 The teacher is keeping order in class.老师正在课堂上维持秩序。 ③意为"使……保持某种(状态、位置或动作等)"。这时要在keep的宾语后接补足语，构 成复合宾语。其中宾语补足语通常由形容词、副词、介词短语、现在分词和过去分词等充当。如： 例：We should keep our classroom clean and tidy.(形容词) 我们应保持教室整洁干净。 You'd better keep the child away from the fire.(副词)你最好让孩子离火远一点。 The bad weather keeps us inside the house.(介词短语)坏天气使我们不能出门。 Don't keep me waiting for long.(现在分词)别让我等太久。 The other students in the class keep their eyes closed.(过去分词) 班上其他同学都闭着眼睛。 2. 用作连系动词 构成系表结构：keep＋表语，意为"保持，继续(处于某种状态)"。其中表语可用形容词、副词、介词短语等充当。如： 例：You must look after yourself and keep healthy.(形容词) 你必须照顾好自己，保持身体健康。 Keep off the grass.(副词)请勿践踏草地。 Traffic in Britain keeps to the left.(介词短语)英国的交通是靠左边行驶的。 注意：一般情况下，keep后接形容词较为多见。再如： She knew she must keep calm.她知道她必须保持镇静。 Please keep silent in class.课堂上请保持安静。 3. ①keep doing sth. 意为"继续干某事"，表示不间断地持续干某事，keep后不 能接不定式或表示静止状态的v-ing形式，而必须接延续性的动词。 例：He kept working all day, because he wanted to finish the work on time. 他整天都在不停地工作，因为他想准时完成工作。 Keep passing the ball to each other, and you'll be OK.坚持互相传球，你们就
to of和for的区别
to , of 和for的区别 1.to有到的意思，常常和go，come，get连用引出地点。Go to school , go to the shop , go to the cinema. 常见的短语：the way to 去---的路 On one’s way to 在某人去---的路上 以上的用法中，当地点是副词home，here，there等是to 要去掉。如：get home，the way here To后跟动词原形，是不定式的标志 It is +形容词+(for/of +人+)to do sth.(括号内部分可以省略) It is easy for me to learn English. It is very kind of you to lend me your money. 当形容词表示人的行为特征时用of表示to do的性质时用for Want, hope ,decide, plan , try , fail等词后跟to do I want to join the swimming club. Would like to do I’d like to play basketball with them. It is time to have a break. Next to , close to , from ---to--- 2.for 为，表示目的。 Thank you for Buy sth for sb =buy sb sth It is time for bed. Here is a letter for you.
I will study for our country. 3.of表示所属关系意思是：---的 a map of the world a friend of mine
for和of引导的不定式结构的区别
for和of引导的不定式结构的区别 不定式是一种非谓语动词，不能单独作谓语，因此没有语法上的主语。但由于不定式表示的是动作，在意义上可以有它的主体。我们称之为逻辑主语。 提起不定式逻辑主语，人们首先想到的会是“for+名词（宾格代词）+不定式”的复合结构。如：It is important for us to study English well．然而，有时不定式的逻辑主语须要用“of+名词（代词宾格）”才行。例如：It is kind of you to help me．而不能说：It is kind for you to help me．在选择介词“for”还是“of”时，人们往往总是凭感觉而定。有时受习惯影响，多选介词“for”。于是常出现这样的错误：It was careless for him to lose his way．It is cruel for you to do so．由于众多语法书对这种结构中使用“for”与“of”的区别介绍甚少，一些人对其概念认识尚不完全清楚，笔者认为有必要就这一问题作些探讨与介绍。 一、在句中的语法作用不同 a．不定式for结构在句中可以作主、宾、表、定、状、同位语： 1．It is easy for Tom to do this work．（主语）汤姆做此工作是容易的。 2．I'd like for him to come here．（宾语）我喜欢他来这里。 3．His idea is for us to travel in two different groups．（表语）他的想法是：我们分成两组旅行。 4．Have you heard about the plan for you to go abroad．（定语）你听到让你出国的计划吗？ 5．The word is too difficult for him to pronounce well．（状语）这单词太难，他念不准。 6．In the most schools，it is the custom for the headmaster to declare the newterm start．在大部分学校，校长宣布新学期开始是一个习惯。 b．不定式of结构只能在句中作主语。 1．It was careless of him to leave his umbrella in the train．他把伞丢在火车上真是太粗心了。 2．It is awfully good of you to come to see me off at the station．谢谢你来车站送我。 二、逻辑主语的名词有所不同
of和for的用法
for 表原因、目的 of 表从属关系 介词of的用法 （1）所有关系 this is a picture of a classroom （2）部分关系 a piece of paper a cup of tea a glass of water a bottle of milk what kind of football，american of soccer？ （3）描写关系 a man of thirty 三十岁的人 a man of shanghai 上海人 （4）承受动作 the exploitation of man by man．人对人的剥削。 （5）同位关系 it was a cold spring morning in the city of london in england． （6）关于，对于 what do you think of chinese food？ 你觉得中国食品怎么样？ 介词for 的用法小结 1. 表示“当作、作为”。如: i like some bread and milk for breakfast. 我喜欢把面包和牛奶作为早餐。what will we have for supper? 我们晚餐吃什么? 2. 表示理由或原因,意为“因为、由于”。如: thank you for helping me with my english. 谢谢你帮我学习英语。 thank you for your last letter. 谢谢你上次的来信。 thank you for teaching us so well. 感谢你如此尽心地教我们。 3. 表示动作的对象或接受者,意为“给……”、“对…… (而言)”。如:
介词for和to用法完全归纳
介词for用法完全归纳 ？ 本站特约作者陈根花 ？ 用法1：(表目的)为了。如： They went out for a walk. 他们出去散步了。 What did you do that for 你干吗这样做？ That’s what we’re here for. 这正是我们来的目的。 What’s she gone for this time 她这次去干什么去了？ He was waiting for the bus. 他在等公共汽车。 【用法说明】在通常情况下，英语不用 for doing sth 来表示目的。如： 他去那儿看他叔叔。 误：He went there for seeing his uncle. 正：He went there to see his uncle. 但是，若一个动名词已名词化，则可与 for 连用表目的。如： He went there for swimming. 他去那儿游泳。(swimming 已名词化) 注意：若不是表目的，而是表原因、用途等，则其后可接动名词。(见下面的有关用法) 用法2：(表利益)为，为了。如： What can I do for you 你想要我什么？ We study hard for our motherland. 我们为祖国努力学习。 Would you please carry this for me 请你替我提这个东西好吗？ Do more exercise for the good of your health. 为了健康你要多运动。【用法说明】(1) 有些后接双宾语的动词(如 buy, choose, cook, fetch, find, get, order, prepare, sing, spare 等)，当双宾语易位时，通常用 for 来引出间接宾语，表示间接宾语为受益者。如： She made her daughter a dress. / She made a dress for her daughter. 她为她女儿做了件连衣裙。 He cooked us some potatoes. / He cooked some potatoes for us. 他为我们煮了些土豆。 注意，类似下面这样的句子必须用 for： He bought a new chair for the office. 他为办公室买了张新办公椅。 (2) 注意不要按汉语字面意思，在一些及物动词后误加介词 for： 他们决定在电视上为他们的新产品打广告。 误：They decided toadvertise for their new product on TV. 正：They decided to advertise their new product on TV. 注：advertise 可用作及物或不及物动词，但含义不同：advertise sth＝为卖出某物而打广告；advertise for sth＝为寻找某物而打广告。如：advertise for a job=登广告求职。由于受汉语“为”的影响，而此处误加了介词 for。类似地，汉语中的“为人民服务”，说成英语是 serve the people，而不是 serve for the people，“为某人的死报仇”，说成英语是avenge sb’s death，而不是 avenge for sb’s death，等等。