Polymer encapsulated upconversion nanoparticle/iron oxide nanocomposites for multimodal imaging and magnetic targeted drug delivery
Huan Xu a ,Liang Cheng a ,Chao Wang a ,Xinxing Ma b ,Yonggang Li b ,*,Zhuang Liu a ,*
a
Jiangsu Key Laboratory for Carbon-Bad Functional Materials &Devices,Institute of Functional Nano &Soft Materials Laboratory (FUNSOM),Soochow University,Suzhou,Jiangsu 215123,China b
Department of Radiology,The First Af filiated Hospital of Soochow University,Suzhou,Jiangsu 215006,China
a r t i c l e i n f o
Article history:
Received 4August 2011Accepted 17August 2011Available online xxx Keywords:
Upconversion nanoparticles Iron oxide
flash 培训
Composite nanostructures Multimodal imaging Drug delivery
perfumedMagnetic targeting
a b s t r a c t
Multimodal imaging and imaging-guided therapies have become a new trend in the current develop-ment of cancer theranostics.In this work,we encapsulate hydrophobic upconversion nanoparticles (UCNPs)together with iron oxide nanoparticles (IONPs)by using an amphiphilic block copolymer,poly (styrene-block-allyl alcohol)(PS 16-b -PAA 10),via a microemulsion method,obtaining an UC-IO@Polymer multi-functional nanocomposite system.Fluorescent dye and anti-cancer drug molecules can be further loaded inside the UC-IO@Polymer nanocomposite for additional functionalities.Utilizing the Squaraine (SQ)dye loaded nanocomposite (UC-IO@Polymer-SQ),triple-modal upconversion luminescence (UCL)/down-conversion fluorescence (FL)/magnetic resonance (MR)imaging is demonstrated in vitro and in vivo,and also applied for in vivo cancer cell tracking in mice.On the other hand,a chemotherapy drug,doxorubicin,is also loaded into the nanocomposite,forming an UC-IO@Polymer-DOX complex,which enables novel imaging-guided and magnetic targeted drug delivery.Our work provides a method to fabricate a nanocomposite system with highly integrated functionalities for multimodal biomedical imaging and cancer therapy.
拉登死亡Ó2011Elvier Ltd.All rights rerved.
1.Introduction
Molecular imaging has appeared to be an important tool in basic biomedical rearch as well as dia diagnosis and prognosis in the clinic [1e 3].Current imaging modalities such as optical,X-ray,nuclear,magnetic resonance (MR),and ultrasound imaging each has its own bene fits and disadvantages [4e 7].For example,optical imaging techniques afford nsitive and multiplexed imaging,but are intrinsically limited by the poor tissue penetration ability of light and only able to image small animals [8].MR imaging,in contrast,although allows three-dimensional whole-body imaging with high spatial resolution,its nsitivity in molecular imaging is not satisfactory as it requires high concentrations of contrast agents [9].Therefore,the integration of two or more imaging modalities should offer the synergistic advantages of each and has attracted great interest in the past few years [10e 14].
Upconversion nanoparticles (UCNPs)usually containing Lanthanide ions (Ln 3þ)are able to be excited by multiple low-energy photons and then emit a single high-energy photon at
a shorter wavelength,a process named upconversion luminescence (UCL)[15].Recently,near-infrare
d (NIR)light excited UCNPs have shown potential applications in various fields including biomedical imaging owing to their unique upconversion optical behaviors that offer improved light penetration depth,high chemical and photo stability,as well as the abnce of auto-fluorescence during imaging [16,17].Great progress has been made on UCNPs in biomedicine field,including bio-imaging and cancer therapy [15,18,19].We and others have found that UCL imaging offers excellent signal-to-noi (S/N)ratios and thus ultra-high imaging nsitivity,owing to the auto-fluorescence free nature of UCL imaging [20].By injecting UCNPs with different emission spectra tuned by either Ln 3þdoping or luminescence resonance energy transfer (LRET),multicolor in vivo UCL imaging has been realized [21,22].A number of groups have also developed dual or even triple-modal imaging probes bad on UCNPs for combined UCL/magnetic resonance/nuclear imaging [2,12,23,24].UCNPs may also find applications for drug delivery and cancer therapies [25e 28].Highly ef ficient in vivo photodynamic treatment of cancer using photonsitizer loaded UCNPs upon irradiation by near-infrared (NIR)light has recently been achieved by our group [29].In our latest work,we developed a multi-functional nanostructure with integrated UCL emission,paramagnetic property,and strong NIR
*Corresponding authors.
E-mail address: (Y.Li),zliu@suda.edu (Z.
去黄
Liu).Contents lists available at SciVer ScienceDirect
Biomaterials
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0142-9612/$e e front matter Ó2011Elvier Ltd.All rights rerved.doi:10.1016/j.biomaterials.2011.08.053
Biomaterials xxx (2011)1e 10
absorption,for dual-targeted photothermal therapy and in vivo UCL/MR multimodal imaging [28].
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Herein,we demonstrate a facial synthetic method to prepare a novel nanocomposite for multi-model imaging and imaging combined drug delivery (Fig.1a).UCNPs and IONPs can be encap-sulated by amphiphilic polymers via a microemulsion method into composite nanoparticles with sizes of 150e 200nm (UC-IO@Pol-ymer).By introducing a fluorescent dye during sample preparation,the obtained UC-IO@Polymer-SQ could be ud for triple-modal UCL/down-conversion FL/MR imaging in vitro and in vivo.On the other hand,a chemotherapy drug,doxorubicin,can also be loaded into the nanocomposite together with UCNPs and IONPs,forming an UC-IO@Polymer-DOX complex,which enables magnetic tar-geted drug delivery toward cancer cells.
2.Experimental ction 2.1.Materials
Y 2O 3,Yb 2O 3and Er 2O 3were purchad from Shanghai Chemical Industrial Co.The rare-earth tri
fluoroacetates were prepared by dissolving the respective rare-earth oxides in tri fluoroacetic acid (CF 3COOH,Shanghai Chemical Industrial Co.).Oleic acid (OA,90%),oleyl amine (OM)and 1-octadecene (ODE >90%),benzyl ether (99%),1,2-dexadecanediol (97%),iron acetylacetonate (Fe (acac)3),polyvinyl alcohol (PVA,M.W.9000w 10,000)and poly (styrene-block-allyl alcohol)(PS 16-b -PAA 10,M.W.2200)were all purchad from Sigma e Aldrich.Doxorubicin (DOX)was brought from Beijing HuaFeng United Technology CO.Ltd.The water-insoluble Squaraine (SQ)dye ud in the experiment was obtained from Prof.Xiaohong Zhang ’s group in Technical Institute of Physics and Chemistry,CAS (Beijing,China)[30].All chemicals involved in this work were analytical grade and ud without further puri fication.
2.2.Synthesis of inorganic nanoparticles
Synthesis of UCNPs.The UCNP synthesis was carried out following a literature protocol with slight modi fications [31].Typically,1mmol of Re (CF 3COO)
3(Y:Yb:Er ¼78%:20%:2%);24ml solvent (16ml OA/8ml OM)were brought to a 100ml three-necked flask simultaneously and degasd at 120 C for 30min under vacuum.In the prence of nitrogen,the mixture was rapidly heated to 275 C and kept at this temperature for 30min under magnetic stirring.A
fter cooling down to room temperature,the products were precipitated by adding ethanol,parated by centrifugation,washed by cyclohexane,and then washed three times with ethanol.The nanoparticle products could be easily re-disperd in chloroform.
Synthesis of IONPs.IONPs were synthesized using a literature method with slight modi fications [32].Fe (acac)3(1mmol),1,2-dodecandiol (5mmol,90%),oleic acid (OA,1ml),oleyl amine (OM,1ml),and benzyl ether (BE,10ml)were mixed in a 100ml three-necked flask and magnetically stirred under the protection by nitrogen.The mixture was heated to 200 C for 2h and then,under a flow of nitrogen,rapidly heated to re flux (w 300 C)for 1h.After cooling down to room temperature;the black products were precipitated by adding 20ml ethanol into the reaction mixture and then collected by centrifugation.The obtained IONPs were washed by ethanol for veral times and re-disperd in hexane for further u.
2.3.Synthesis of UC-IO@polymer nanocomposites
PS 16-b -PAA 10(50mg)was first dissolved in 500m l chloroform,followed by addition of UCNPs and IONPs at different quantities.100mg of PVA (MW 9000w 10,000)as a polymer stabilizer was dissolved in 5ml of distilled (D.I.)water at 70 C and then cooled down to room temperature.The organ
indicate是什么意思ic pha was then added to the PVA solution and emulsi fied for 5min by puld sonication (100W).The emulsion was then stirred at room temperature overnight to evaporate the organic solvent.The resulting nanocomposites were puri fied by centrifugation,washed with D.I.water for 3times to remove excess polymers and unincorporated nanoparticles [33].
For the preparation of UC-IO@Polymer-SQ and UC-IO@Polymer-DOX nano-composites,the same procedure was ud except 100m g SQ or 1mg DOX (depro-tonated,dissolved in 100m l DMSO),respectively,was added to the organic pha before the formation of microemulsion.2.4.Determination of DOX loading and relea
To measure the amount of DOX loaded inside the UC-IO@Polymer-DOX complex,different concentrations of DOX were added into the organic pha mentioned above,and then the same procedure was done to obtain the UC-IO@Polymer-DOX with different loading amount of DOX [25]
.
Fig.1.UC-IO@Polymer nanocomposite synthesis and characterization.(a)A schematic illustration for UC-IO@Polymer nanocomposite synthesis and applications.(b)A repre-ntative SEM image of UC-I
O@Polymer.The average particle size was around 200nm (c)A TEM image of UC-IO@Polymer.(d e g)EDX mapping of the UC-IO@Polymer nano-composite.(d)A high-angle annular dark-field scanning TEM (HAADF-STEM)image of a single UC-IO@Polymer nanoparticle.The area within the red rectangle was lected for EDS mapping.(e e g)HAADF-STEM-EDS mapping images of UC-IO@Polymer showing the yttrium L edge (e,green),iron K edge (f,red)and the merged image (g).(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)
H.Xu et al./Biomaterials xxx (2011)1e 10
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The UC-IO@Polymer-DOX nanocomposites were incubated in phosphate buff-ered saline(PBS)solution at PH5and7.4for different periods of time.DOX relead from nanocomposites was collected by dialysis.The amount of relead DOX was measured byfluorescence spectrum.
2.5.Characterizationgreen king
The scanning electron microscopy(SEM)images were obtained by using a FEI Quanta200F scannin
g electron microscope.Transmission electron microscopy(TEM) and high-resolution TEM(HR-TEM)images were taken using a Philips CM300trans-mission electron microscope operating at an acceleration voltage of200kV.The pha and crystallography of the product was characterized by using a Shimadzu XRD-6000X-ray diffractometer equipped with Cu ka radiation(l¼0.15406nm).A scanning rate of 0.05 sÀ1was applied to record the pattern in the2q range of10e80 .Luminescence spectra were obtained on a FluoroMax4luminescence spectrometer(HORIBA Jobin Yvon).UCL emission spectra were obtained on the same luminescence spectrometer with an external980nm lar diode(1.5W,continuous wave with1mfiber,Beijing Hi-Tech Optoelectronics CO.,Ltd)as the excitation source.UV e Vis spectra were acquired by using a PerkinElmer Lambda750UV/Vis spectrophotometer.
UC-IO@Polymer-SQ nanocomposites in the concentration ranging from0.004to 0.08g/L of iron ion were scanned under a3-T clinical MRI scanner(Bruker Biospin Corporation,Billerica,MA,USA)at room temperature.After acquiring the T2-weighted MR images,the magnitudes of image intensities T2were measured within manually drawn regions of interest for each sample.Relaxation rates R2(R2¼1/T2) were calculated from T2values at different iron concentrations.
2.6.Cellular experiments
Cell culture.Human cervical HeLa cell line and murine breast cancer4T1cell lines were obtained from American Type Culture Collection(ATCC).All cell culture related reagents were purchad from Invitrogen.Cells were grown in normal RPMI e1640culture medium with10%fetal bovine rum(FBS)and1%penicillin/ streptomycin.
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Cell cytotoxicity.The in vitro cytotoxicity was measured using a standard methyl thiazolyl tetrazolium(MTT,Sigma e Aldrich)assay.HeLa cells and4T1cells were eded into96-well cell-culture plates at5Â104/well and then incubated for24h at 37 C under5%CO2.After incubating4T1and HeLa cells with various concentrations of UC-IO@Polymer-SQ,UC-IO@Polymer,UC-IO@Polymer-DOX and free DOX for24
h,
Fig.2.Optical and magnetic properties of UC-IO@Polymer-SQ nanocomposite.(a)A SEM image of UC-IO@Polymer-SQ nanocomposite showing the average particle size of around 200nm.(b)Photos of UC-IO@Polymer-SQ in aqueous solutions under the980-nm lar and UV light with(upper)and without(down)the magnet.(c)UV/Vis and FL spectrum of UC-IO@Polymer-SQ nanocomposite.(d)UCL spectra of UC-IO@Polymer and UC-IO@Polymer-SQ.(e)Magnetization loops of IONPs and UC-IO@Polymer-SQ.(f)T2-weighted MR images of UC-IO@Polymer-SQ solutions at different iron concentrations(upper).(g)T2relaxation rates(R2)of UC-IO@Polymer-SQ solutions at different iron concentrations(down).
H.Xu et al./Biomaterials xxx(2011)1e103
the standard MTT assay was carried out to determine the cell viabilities relative to the control untreated samples.
Confocal imaging of labeled cells.Confocal UCL imaging of cells was performed using a modi fied Leica lar scanning confocal microscope,with a NIR lar at 980nm as an additional excitation source.4T1cells were incubated with UC-IO@Polymer-SQ for 4h before confocal fluorescence and U
CL imaging.HeLa cells were incubated with UC-IO@Polymer-DOX for 30min,1h,2h and 6h before confocal fluorescence and UCL imaging.All cells were washed twice with cell culture medium before confocal imaging.Imaging of SQ dye was carried out at 633nm lar excitation,with its emission collected from 620nm to 720nm.DOX imaging was carried out at 488nm lar excitation,while UCNPs was excited by the external lar at 980nm.The emissions were collected in the ranges of 500e 600nm and 500e 700nm for DOX and UCNPs,respectively.
Magnetic drug targeting.For the magnetic drug targeting,UC-IO@Polymer-DOX (20m M DOX)was incubated with HeLa cells for 48h at 37 C,with a magnet placed under the center of the culture dish.Cells were then washed twice with PBS,stained with calcein AM and propidium iodide,and then imaged by the confocal micro-scope;UCL and FL images of magnetic targeting cells were also obtained by the confocal microscope.
MR imaging of cells.4T1cells were incubated with UC-IO@Polymer-SQ over-night.The cells were washed with PBS,centrifuged and then covered with agaro solution before MR imaging.The treated cells were scanned under a 3-T clinical MRI scanner at room temperature to obtain the MR images.2.7.In vivo UCL/FL/MR imaging
Balb/c mice and Athymic nude mice (w 20g)were purchad from Suzhou Belda Bio-Pharmaceutical Co.and ud under protocols approved by Soochow University
Laboratory Animal Center.For in vivo UCL/FL imaging,the UC-IO@Polymer-SQ in 0.9%NaCl saline solution was intravenously injected into the nude mice.4h after injection,in vivo and ex vivo imaging were carried out by a Maestro EX in vivo fluorescence imaging system (CRi,Inc.).MR imaging were accomplished through a 3-T clinical MRI scanner equipped with a special coil ud for small animal imaging.
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For cell labeling,w 2million 4T1cells in each cell culture plate were incubated with UC-IO@Polymer-SQ for 24h at 37 C.After extensively washing three times with rum-free RMPI-1640medium,the cells were detached from the plate by trypsin-EDTA and re-suspended in rum-free RMPI-1640.For in vivo imaging,4T1cells (w 1million cells for each sample)after incubation with UC-IO@Polymer-SQ were injected into mice,which were subquently imaged by the UCL and FL imaging systems.MR imaging was conducted on a 3-T clinical MRI scanner equipped with a special coil designed for small animal imaging.
3.Results and discussion
3.1.Fabrication of UC-IO@Polymer nanocomposites
Yb/Er doped UCNPs (Y:Yb:Er ¼78%:20%:2%)and ultra-small IONPs nanocrystals were synthesized following a literature proce-dure with slight modi fication,both exhibit the cubic pha and the size distribution in the range of 6w 8nm,as shown by the trans-mission electron microscopy (TEM)images (Supporting Informa-tion,Fig.S1&2).The as-prepared UCNPs and IONPs were
then
Fig.3.In vitro cell experiments of UC-IO@Polymer-SQ nanocomposite.(a)Relative viabilities of UC-IO@Polymer-SQ treated 4T1cells normalized to the untreated control.The experiment was conducted 24h after cell incubation.(b)Lar scanning confocal microscopy (LCSM)images of 4T1cells incubated with UC-IO@Polymer-SQ.(c)In vitro T 2-weighted MR images of UC-IO@Polymer-SQ treated 4T1cells (right)and untreated cells (left).(d)The MR signals plot of 4T1cells incubated with and without UC-IO@Polymer-SQ.Error bars were bad on triplicated samples or measurements.
H.Xu et al./Biomaterials xxx (2011)1e 10eleven是什么意思
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encapsulated with an amphiphilic block copolymer PS16-b-PAA10 into composite nanoparticles via a microemulsion method(Fig.1a). To understand how the UCNP/IONP ratio affected the morphologies and sizes of nanocomposites,we synthesized UC-IO@Polymer with different ratios of nanoparticles.From the scanning electron microscopy(SEM)images,when the ratio of UCNPs and IONPs came to1:1,the size and shape of the nanocomposites appeared to be quite uniform(Supporti
ng Information,Fig.S3,a e e).This ratio was thus chon as the optimized condition in our following experi-ments.From the SEM,TEM and dynamic light scattering(DLS)data, the size of UC-IO@Polymer nanocomposites was around200nm and showed excellent water solubility(Fig.1b,Supporting Information, Fig.S4).Energy dispersive X-ray spectroscopy(EDX)mapping of Y and Fe further evidenced the prence of multiple IONPs and UCNPs in one polymer encapsulated nanoparticle(Fig.1c e g).Bad on the inductively coupled plasma-atomic emission spectrometry(ICP-AES)measurement of metal ion concentrations,it was determined that1mg of UC-IO@Polymer contained0.056mg UCNPs(5.6%w/w) and0.08mg IONPs(8%w/w).
3.2.Optical and magnetic properties of UC-IO@Polymer-SQ
The UC-IO@Polymer nanocomposites can be loaded with SQ dye to obtain the UC-IO@Polymer-SQ nanocomposite capable of triple-modal imaging(Fig.1a).SEM and DLS tests showed that the size of UC-IO@Polymer-SQ nanocomposites was around200nm,sug-gesting that the loaded SQ did not significantly affect the size of nanocomposites(Fig.2a,Supporting Information,Fig.S4).Photos of UC-IO@Polymer-SQ in aqueous solutions under the980-nm lar and UV light with and without the magnet clearly
demonstrated
Fig.4.In vivo cell tracking experiments.(a)FL/UCL images of mice subcutaneously injected with UC-IO@Polymer-SQ labeled4T1cells taken0day,5days and10days after injection.(b)T2-weighted MR images of mice inoculated with UC-IO@Polymer-SQ labeled4T1cells and control unlabeled cells after0day and5days.(c)The MR signals plot of tumors on mice inoculated with UC-IO@Polymer-SQ treated4T1cells and untreated cells after0day and5days.Error bars were bad on triplicated measurements.
H.Xu et al./Biomaterials xxx(2011)1e105
