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Biomaterials 32 (2011) 2979e2988

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Biomaterials

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Computed tomography imaging of cancer cells using acetylateddendrimer-entrapped gold nanoparticles

Han Wang a,1, Linfeng Zheng a,1, Chen Peng b,1, Rui Guo c, Mingwu Shen c,Xiangyang Shi b,c,d,**, Guixiang Zhang a,*

aDepartment of Radiology, Shanghai Jiaotong University Affiliated First People’s Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road,Shanghai 200080, PR Chinab State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, PR ChinacCollege of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, PR ChinadCQM-Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal

a r t i c l e i n f o

Article history:Received 23 November 2010Accepted 4 January 2011Available online 28 January 2011

Keywords:DendrimersGold nanoparticlesMolecular imagingComputed tomographyCancer cells

* Corresponding author. Tel.: þ86 21 63240090 416** Corresponding author. College of Chemistry,Biotechnology, Donghua University, 2999 North RenmChina. Tel.: þ86 21 67792656; fax: þ86 21 67792306

E-mail addresses: xshi@dhu.edu.cn (X. Shi), guixian1 Authors contributed equally to this work.

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.01.001

a b s t r a c t

We report a new use of acetylated dendrimer-entrapped gold nanoparticles (Au DENPs) for in vitro and invivo computed tomography (CT) imaging of cancer cells. In this study, Au DENPs prepared using amine-terminated generation 5 poly(amidoamine) dendrimers were subjected to an acetylation reaction toneutralize the positive surface potential. The acetylated Au DENPs were used for both in vitro and in vivoCT imaging of a human lung adencarcinoma cell line (SPC-A1 cells). Micro-CT images show that SPC-A1cells can be detected under X-ray after incubation with the acetylated Au DENPs in vitro and thexenograft tumor model can be imaged after both intratumoral and intraperitoneal administration of theparticles. Transmission electron microscopy data further confirm that the acetylated Au DENPs are ableto be uptaken dominantly in the lysosomes of the cells. Combined morphological observation of cellsafter hematoxylin and eosin staining, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay of cell viability, and flow cytometric analysis of cell cycle show that the acetylated AuDENPs do not appreciably affect the cell morphology, viability, and cell cycle, indicating their goodbiocompatibility at the given concentration range. Findings from this study suggest that the developedacetylated Au DENPs have a great potential to be used for CT imaging of cancer cells.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Molecular imaging (MI) represents an advanced technology inmedical imaging area, which was formally introduced in 1999 andrapidly developed in the latest decade. It has the ability to detectand quantitatively measure the function of biological and cellularprocesses in vitro and in vivo. MI probes are one of the mostimportant components in MI technologies. With the developmentsof various functional MI probes, the biochemical and cell biologicalevents that are associated with disease progression and treatmentresponse can be detected [1e12]. The modalities of MI includeoptical imaging, nuclear-based imaging, ultrasound imaging,

6; fax: þ86 21 63240825.Chemical Engineering andin Road, Shanghai 201620, PR804.gzhang@sina.com (G. Zhang).

All rights reserved.

magnetic resonance imaging, and computed tomography (CT)imaging, etc [1e5,8,10,12]. As one of the important MI technologies,CT affords better spatial and density resolution than other imagingmodalities. These advantages become particularly apparent whenCT is used to diagnose diseases in the thoracic region, such as lungcancer. To achieve sensitive CT imaging capability, development ofsuitable CT MI probes is necessary.

Gold nanoparticles (AuNPs) have been used as probes in opticalmolecular imaging for cancer detection and treatment due to theirsurface plasmon absorption and light-scattering properties[10,13e18]. More recently, AuNPs have seen increasing potential asmolecular probes for X-ray CT imaging, as they offer severaladvantages over conventional iodine-based agents [14,19e23].First, because of its higher atomic number and electron density,gold has a higher X-ray absorption coefficient than iodine,endowing it in principle with a greater ability to enhance CTcontrast [24]. Second, AuNPs are reported to be non-cytotoxic[13,25,26]. Third, their surfaces are relatively easy to be modifiedwith functional groups such as targeting molecules or specific

H. Wang et al. / Biomaterials 32 (2011) 2979e29882980

biomarkers, endowing the resulting particles with characteristicsfavorable for a range of MI applications [13,27e30]. In addition,proper treatment of AuNPs can increase their circulation time in thecardiovascular system via effectively decreasing the rapid uptakeand clearance by the reticuloendothelial system (RES) [19,27e29].This is particularly advantageous when treating tumors with leakyvasculature and poor lymphatic drainage through the enhancedpermeation and retention (EPR) effect [31]. With the prolongedblood circulation time, the EPR effect to enhance the transportationof AuNPs to the tumor site via a “passive” mechanism can beexploited. Similarly, with the bound targeting ligands on theparticles, the rate of endocytotic uptake of the particles can besignificantly increased through a receptor-mediated process via an“active” mechanism provided that the particles have prolongedcirculation times [27,28].

Dendrimers are a class of highly branched, monodispersed,synthetic macromolecules with well-defined composition andarchitecture [32]. With the unique physicochemical properties,dendrimers have been used as templates or stabilizers to generatedendrimer-entrapped AuNPs (Au DENPs) [14,33e36] or dendrimer-stabilized AuNPs (Au DSNPs) for biomedical applications [37e41].The art of dendrimer chemistry allows one to synthesize Au DENPsor Au DSNPs with terminal amines transformed to acetyl functionalgroups, significantly avoiding nonspecific cell membrane bindingand toxicity [34,35,39,42,43]. Simultaneously, the developed AuDENPs or Au DSNPs can be afforded with targeting capability bydirectly using amine-terminated Au DENPs as a starting platformfor conjugation with targeting ligands [35,44] or by using den-drimers pre-modified with targeting moieties [39].

In our previous reports [14,33], we show that AuDENPs preparedusing generation 5 amine-terminated poly(amidoamine) (PAMAM)dendrimers (G5.NH2) canbeacetylated for invivoCT imagingofmiceafter intravenous injection. X-ray absorption coefficient measure-ments show that the attenuation of Au DENPs or acetylated AuDENPs aremuch higher than that of the iodine-based contrast agentat the same molar concentration of the active element (Au versusiodine) [14,33]. Themouse’s inferior vena cava and pulmonary veinscan be clearly distinguished after intravenous injection of acetylatedAu DENPs. The good blood pool imaging capability of acetylated AuDENPs leads us tohypothesize that theseparticles could also beusedto for CT imaging of cancer cells in vitro and in vivo.

In the present study, we utilized G5.NH2 dendrimers astemplates to synthesize Au DENPs. The terminal amine groupswereacetylated to generate acetylated Au DENPs with a neutral surface.

Fig. 1. UVeVis spectrum (a) and TEM image (b) of

Then the acetylated Au DENPs were used as an imaging agent for CTimaging of a model cancer cell, human lung adencarcinoma cell eSPC-A1 cell in vitro and the xenograft tumor model in vivo. Thenanoparticle distribution within the cells was observed by trans-mission electron microscopy (TEM). Furthermore, the biocompat-ibility of the acetylated Au DENPs were evaluated by systematicmorphological observation of the cells after stained with hema-toxylin and eosin (HE), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of cell viability, and flowcytometric analysis of the cell cycle. To our knowledge, this is thefirst report related to the CT imaging of cancer cells using acetylatedAu DENPs.

2. Experimental section

2.1. Materials

Ethylenediamine core G5.NH2 PAMAM dendrimers with a polydispersity indexof less than 1.08 were purchased from Dendritech (Midland, MI). All other chemicalswere obtained from Aldrich. SPC-A1 cell line (a kind of human lung adenocarcinomacell) was purchased from Shanghai Cell Bank, Chinese Academy of Sciences, China.Penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Sigma(St. Louis, MO). Trypsin-EDTA, Dulbecco’s PBS, RPMI 1640 medium, and bovineserum albumin were obtained from GIBCO-BRL (Gaithersburg, MD). Water used inall experiments was purified using a Milli-Q Plus 185 water purification system(Millipore, Bedford, MA) with resistivity higher than 18 MU cm. Regeneratedcellulose membranes with molecular weight cutoff (MWCO) of 10,000 wereacquired from Fisher.

2.2. Synthesis and characterization of acetylated Au DENPs

The acetylated Au DENPs were synthesized according to the method publishedpreviously [14,33]. In brief, Au DENPs were prepared with sodium borohydridereduction chemistry with a molar ratio of Au salt to G5.NH2 dendrimer of 50:1. Acertain amount of HAuCl4 solution [2.5mL inwater/methanol (v/v¼ 2:1)] was addedto an aqueous solution of G5.NH2 (20 mg, 10 mL) under vigorous stirring. After30min, an icy cold NaBH4 solution [5mL inwater/methanol (v/v¼ 2:1)] with a threetimes molar excess to the Au salt was added to the Au salt/dendrimer mixture understirring, and the reaction mixture turned a deep red color within a few seconds. Thestirring process was continued for 2 h to complete the reaction. The reactionmixturewas then extensively dialyzed against water (six times, 4 L) for 3 days to remove theexcess reactants; this was followed by lyophilization to obtain the Au DENPs. Thefinal Au DENP product is denoted as [(Au0)50eG5.NH2].

The [(Au0)50eG5.NH2] DENPs were further acetylated to neutralize the den-drimer terminal amine groups. Briefly, triethylamine (54.7 mL) was added to anaqueous solution of [(Au0)50eG5.NH2] DENPs (25 mL, 21.12 mg) under magneticstirring. After 30 min, acetic anhydride (31.0 mL, 324 mM, 400% molar excess of thetotal primary amines of Au DENPs) was added to the DENP/triethylamine mixturesolution with stirring, and the mixture was allowed to react for 24 h. The aqueoussolution of the reaction mixture was extensively dialyzed against phosphate-buffered saline (PBS) buffer (three times, 4 L) and water (three times, 4 L) for 3 days

the acetylated Au DENPs [(Au0)50eG5.NHAc].

Fig. 2. Transection micro-CT images (a) and CT values (b) of the SPC-A1 cell suspensions incubated with acetylated Au DENPs at different concentrations for 4 h (n ¼ 3).

H. Wang et al. / Biomaterials 32 (2011) 2979e2988 2981

to remove excess reactants and byproducts, followed by lyophilization to obtain thedry powder of the [(Au0)50eG5.NHAc] DENP product. Before use, the acetylated AuDENPs were dissolved in PBS, (pH 7.2) and stored at 4 �C.

The synthesized acetylated Au DENPs were characterized using UVeVis spec-trometry and TEM. UVeVis spectra were collected with a Lambda 25 UVeVisspectrometer (PerkinElmer). Samples were dissolved in water before the experi-ments. TEM was performed with a JEOL 2010F analytical electron microscope (JEOL,Japan) operating at 200 kV. An aqueous solution of Au DENPs (1 mg/mL) wasdropped onto a carbon-coated copper grid and air-dried before measurements.

2.3. Cell culture

SPC-A1 cells were cultured in RPMI 1640 medium supplemented with 10% FBSand 1% penicillinestreptomycin at 37 �C and 5% CO2 in a humidified incubator.

2.4. In vitro micro-CT imaging

SPC-A1 cells were incubatedwith acetylated AuDENPswith a concentration of 0,200, 500, 1000, 2000, and 3000 nM, respectively for 4 h at 37 �C. After washed with

H. Wang et al. / Biomaterials 32 (2011) 2979e29882982

PBS buffer for three times, cells were trypsinized, centrifuged, and resuspendedwith100 mL PBS buffer in a 0.5mL Eppendorf tube containing approximately 1.5�106 cellsfor each tube. The cell suspension in each tubewas placed in a self-designed scanningholder, and then scanned using a micro-CT imaging system (eXplore Locus, GEHealthcare, London, Ontario, Canada) with the following operating parameters: tubevoltage, 80 kV; tube current, 450 mA; exposure time, 400 ms; slice thickness, 45 mm;slice space, 0 mm; scan field of view, 45mm� 80mm; effective pixel size, 0.046mm.Imageswere reconstructed on amicro-CT imagingworkstation (GEHCmicroView,GEHealthcare, London, Ontario, Canada) using the following parameters: voxel,45 mm� 45 mm� 45 mm;displayfield of view,10e25mm. CT valueswere acquired onthe same workstation using the software supplied by the manufacturer. Eachexperiment was carried out in triplicate.

2.5. Cellular uptake of acetylated Au DENPs

The cellular uptake of acetylated Au DENPswas confirmed by inductively coupledplasma atomic emission spectroscopy (ICP-AES), TEM, and silver staining. For ICP-AESmeasurement, about 3�105 cells were seeded onto a 6-well cell culture plate for 24 h.The cells were then incubated with acetylated Au DENPs (2000 nM) for 4 h. The cellswere washed with PBS buffer for three times, then typsinized and harvested bycentrifugation. Digestion of the cells was performed in aqua regia, then the amount ofAu uptake in the cells was quantified by ICP-AES (Leeman Prodigy, USA).

The cellular uptake of the particles was then characterized using silver staining.SPC-A1 cells seeded onto polylysine-coated cover slips were incubated with acety-lated Au DENPs with a concentration of 2000 nM for 4 h at 37 �C. Cells were stainedwith a silver enhancement solution kit according to the manufacturer’s instruction.After rinsing three times with PBS, the cell nuclei were stained with 1% Nuclear fastred, and then washed, air-dried, dehydrated, cleaned, and mounted for lightmicroscopy observation.

For further TEM imaging of the distribution of the acetylated Au DENPs withinthe cells, SPC-A1 cells were plated in 6-well cell culture plates with a density of3 � 105 cells per well in RPMI 1640 mediumwith 10% FBS in a humidified incubator(37 �C, 5% CO2) for 24 h to grow tow80% confluence. Then, the acetylated Au DENPswere added to each well with a final concentration of 2000 nM and incubated for12 h at 37 �C. The culturemediumwas discarded and the cells werewashedwith PBSbuffer, trypsinized, centrifuged, washed for three times with PBS buffer again, andfinally fixed with 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 12 h at4 �C and post-fixed with 1% OsO4 in 0.2 M phosphate buffer (pH 7.2) for 2 h at 4 �C.After additional washing in buffer, the cells were dehydrated in a series of ethanolsolutions of 30%, 50%, 70%, 95%, and 100%. The cell samples were then embeddedwith Epon 812 (Shell Chemical, UK), followed by polymerization. Then, theembedded cells were sectioned using a ReicharteUltramicrotome. The sections witha thickness of 75 nmweremounted onto 200-mesh copper grids and counterstainedwith uranyl acetate and lead citrate for 5 min before TEM measurements. The gridswere visualized using an H600 transmission electron microscope (Hitachi, Japan)with an operating voltage of 60 kV.

2.6. Cytotoxicity of acetylated Au DENPs

Themorphology of cells after treatment with acetylated Au DENPs was observedafter HE staining. About 3 � 105 SPC-A1 cells were plated in each well of 6-well cellculture plates, where each well was covered with a poly-L-lysine (PLL)-coated coverslip. The cells were cultured for 24 h to grow to w80% confluence. Then acetylatedAu DENPs (2000 nM) were incubated with the cells for 4 h, followed by washingwith PBS buffer for three times and fixing with 4% paraformaldehyde in 0.1 M

Fig. 3. TEM images of SPC-A1 cell: (a) negative control cells without treatment, (b) cells ina higher-magnification image of the highlight area in (b) to clearly show the acetylated Au

phosphate buffer (pH 7.2) for 2 h. The cell samples on the cover slips were washedtwice for 5 min with PBS buffer, dipped into a Coplin jar containing hematoxylin for30 s, rinsed with water for 1 min, and stained with 1% eosin Y solution for 10e30 s.Finally, the cover slips were dehydrated and mounted onto glass slide. Themorphology of cells was observed using an optical microscope (200�magnification,Nikon, Tokyo, Japan).

The viability of cells treated with the acetylated Au DENPs was evaluated viaMTT assay. The MTT assay was carried out according to the method publishedpreviously [45,46]. SPC-A1 cells were seeded in 96-well culture plates at a density of1 � 104 cells per well in triplicate and allowed to grow for 24 h. Then the mediumwas replaced by RPMI 1640 medium containing different concentrations of acety-lated Au DENPs (200, 500, 1000, 2000, and 3000 nM) and cells were incubated for4 h at 37 �C in CO2 incubator. After treatment, the medium was aspirated and cellswere washed with PBS buffer for three times. And then 200 mL fresh RPMI 1640mediumwas added to each well. Thereafter, 20 mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL in PBS buffer) was added to each welland incubated for 4 h at 37 �C in CO2 incubator. The mediumwas carefully removed,and the cells werewashedwith PBS buffer. Subsequently, DMSO (200 mL) was added.The absorbance values at a wavelength of 490 nm in each well were measured usinga microplate reader (Bio-tek).

The toxicity of the acetylated Au DENPs was further examined by flow cyto-metric detection of cell cycles. SPC-A1 cells were seeded in 6-well cell culture platesat a density of 3 � 105 cells per well in quadruplication and allowed to grow toconfluence for 24 h. Then, after replacing themediumwith freshmedium containingdifferent concentrations of acetylated Au DENPs (1000 and 2000 nM), the cells wereincubated for 4 h at 37 �C in CO2 incubator. After the treatment, cells were harvestedby trypsinization and centrifugation, washed with PBS buffer, and fixed in citratebuffer for 2 h. The cells were then centrifuged to remove citrate buffer and resus-pended with PBS buffer with a cell concentration of 1 � 106 cells/mL. The cellssuspensionwere incubated with trypsogen for 3min and then incubatedwith RNasefor 3 min. Subsequently, the cells were stained with propidium iodide (PI) for 15minand then the PI-stained cells were measured by flowcytometry (FACSCalibur, BectonDickinson, USA) in red (FL2) channel at 488 nm. The cell cycle profiles, including G0-G1, G2-M, and S phases, and sub-G1 fraction were analyzed by using Cellquestsoftware (FACSCalibur, Becton Dickinson, USA).

2.7. In vivo micro-CT imaging of SPC-A1 tumor

Animal experiments and animal care were carried out according to protocolsapproved by the institutional committee for animal care, and also in accordancewiththepolicyof theNationalMinistry ofHealth.Male 4- to6-week-old BALB/C nudemice(n ¼ 5, Shanghai SLAC laboratory Animal Co., Ltd., Shanghai, China) were subcuta-neously injected in the right side of their back with 1 � 106 cells/mouse. When thetumor nodules had reached a volume of 1.0 � 0.15 cm3 after approximately 3 weekspost-injection (Fig. S1(A)), the tumor was confirmed by gross specimen and HEstaining, which showed the SPC-A1 cell features (Fig. S1(B)e(C)). The mice wereplaced in a scanning holder, and then scanned using a micro-CT imaging system(eXplore Locus, GEHealthcare, London, Ontario, Canada)with the parameters similarto those for in vitro experiments. CT scanning was performed both before and afterintratumoral or intraperitoneal injection of Au DENPs (50 mL, [Au] ¼ 0.2 M) at timepoints of 1, 2, 4, and 6 h post-injection. Images were reconstructed on a micro-CTimaging workstation (GEHC microView, GE Healthcare, London, Ontario, Canada)using the following parameters: voxel, 45 mm� 45 mm� 45 mm; display field of view,10e25 mm. CT values were acquired on the same workstation using the softwaresupplied by the manufacturer.

cubated with acetylated Au DENPs at the concentration of 2000 nM for 12 h, and (c)DENPs (arrowhead) in the cytoplasm and the cell nucleus (arrow).

Fig. 5. MTT assay of the viability of SPC-A1 cells treated with acetylated Au DENPs atdifferent concentrations for 4 h (n ¼ 3).

Fig. 4. Optical microscope images of SPC-A1 cells with HE staining: (a) negative control cells without treatment with acetylated Au DENPs (200�), (b) the cells incubated withacetylated Au DENPs at the concentration of 2000 nM for 12 h (200�).

H. Wang et al. / Biomaterials 32 (2011) 2979e2988 2983

2.8. In vivo tumor uptake of acetylated Au DENPs

The tumor was extracted and sectioned. The tumor sections were stained witha silver enhancement solution kit according to themanufacturer’s instructions. Afterrinsing three times with PBS, the sections were stained with 1% Nuclear fast red, andthen washed, air-dried, dehydrated, cleaned, and mounted for light microscopyobservation.

2.9. Statistical analysis

All data were expressed as means� S.D. Comparisons between two groups wereanalyzed by Student’s t-test, and those between multiple groups were evaluated byone-way analysis of variance (ANOVA) followed by LSD’s tests using SPSS 15.0software. The p values <0.05 were considered significant.

3. Results and discussion

3.1. Synthesis and characterization of acetylated Au DENPs

Under the same experimental protocol described in ourprevious reports [14,33], we were able to obtain acetylated AuDENPs with Au salt/G5 dendrimer molar ratio of 50:1. UVeVisspectroscopy and TEM were utilized to characterize the synthe-sized acetylated Au DENPs (Fig. 1). It is clear that the acetylated AuDENPs display a typical surface plasmon band at 510 nm (Fig. 1(a)),indicating the successful formation of AuNPs. TEM imaging data(Fig. 1(b)) shows that the acetylated Au DENPs have a relativelyuniform size distribution with a mean diameter of 2.6 nm, inagreement with our previous results [33]. The acetylated Au DENPsin a powder form can be dissolved in water, PBS buffer, and cellculture medium with good colloidal stability for at least 6 monthsat room temperature. In general, the acetylated Au DENPs werestored at �20 �C in a dried form.

3.2. Micro-CT imaging of SPC-A1 cells in vitro

Theoretically, Au has a higher X-ray attenuation coefficient thaniodine due to its higher atomic number and electron density [24].According to the LamberteBeer law [20], the relationship among aninput X-ray flux I0, a tissue matrix of thickness T with linear attenu-ation coefficient mm, and the transmitted flux Im is described by theformula Im ¼ I0$e�mmT . When both tissue and contrast agent arepresent, theflux Ic thatpasses througha scannedsectionof thickness tis I0$e�mmT�t$e�mct , or Im$e�ðmc�mmÞt, where mc is the linear attenuationcoefficient of the contrast agent. The difference in the signal betweenthe surroundingmatrix and the feature defined by the contrast agentcan then be calculated as C ¼ ðIm � IcÞ=Im ¼ 1 � e�ðmc�mmÞt .Thus the difference in signal intensity induced by a contrast agent isintroduceddependingonlyon the thickness of the contrast agent and

the difference in the linear attenuation coefficients of the contrastagent and the matrix. For this reason, the attenuation coefficient ofa given contrast agent is one of the most important factors thatdetermine its CT imaging efficiency.

Our previous work confirms that Au DENPs or acetylated AuDENPs prepared using G5.NH2 dendrimers as templates havebetter X-ray attenuation property than iodine-based clinicallyused contrast agent. To prove our hypothesis that the acetylatedAu DENPs are able to be used as an effective agent to imagecancer cells via CT. SPC-A1 cells were selected as a model. TheSPC-A1 cell suspensions incubated with acetylated Au DENPs atdifferent concentrations (0, 200, 500, 1000, 2000, and 3000 nM,respectively) were imaged using a micro-CT imaging system(Fig. 2). Fig. 2(a) shows the transection CT images of SPC-A1 cellswith or without incubation of the acetylated Au DENPs. It isclear that with the concentration of acetylated Au DENPs incu-bated, SPC-A1 cells gradually display bright images. The CTimages of the cells incubated with acetylated Au DENPs at highconcentrations (2000 and 3000 nM) were brighter than thoseof the cells incubated with acetylated Au DENPs at lowconcentrations (200, 500, and 1000 nM) and those of the

H. Wang et al. / Biomaterials 32 (2011) 2979e29882984

cells without treatment with acetylated Au DENPs (negativecontrol cells).

A quantitative analysis of the CT values of the cells (Fig. 2(b))further showed that the CT value of SPC-A1 cells incubated withacetylated Au DENPs at the concentration of 2000 nM was signifi-cantly higher than those of the cells incubated with acetylated AuDENPs at lower concentrations (200, 500, and 1000 nM) and that ofnegative control cells, respectively (p < 0.05). Further increasingthe concentration of acetylated Au DENPs to 3000 nM, therewas nosignificant difference in the CT values in comparison to the case ofthe particle concentration at 2000 nM. The brighter CT images ofcells at a higher concentration of acetylated Au DENPs (2000 nM)should be associated with the more cellular uptake of the particles.

Fig. 6. Flow cytometry analysis of SPC-A1 cells treated with acetylated Au DENPs at theconcentration of (a) 1000 nM and (b) 2000 nM, respectively for 4 h at 37 �C (n ¼ 4). Thedata of the non-treated negative control cells is shown in (c).

3.3. In vitro cellular uptake of acetylated Au DENPs

To quantify the cellular uptake of the acetylated Au DENPs, ICP-AES analysis was performed. We showed that the Au elementuptake in the cells incubated with 2000 nM acetylated Au DENPs ismuch higher than that of the negative control cells (0.293 pg percell versus �0.03 pg per cell). This indicates that the high X-rayattenuation of the cells is due to the higher quantity of the acety-lated Au DENPs uptaken by cells.

The uptake of acetylated Au DENPs in the SPC-A1 cells was alsoconfirmed by silver staining (Fig. S2). Accumulated black spots,which associated with the uptake of the acetylated Au DENPs, wereclearly observed in the cytoplasm of the SPC-A1 cells treated withacetylated Au DENPs (2000 nM) for 4 h. In contrast, the SPC-A1 cellswithout treatment did not show black spots.

For further identification of the distribution of the acetylated AuDENPs in the subcellular compartments, TEM imaging of cells wasperformed (Fig. 3). It is clear that after incubatedwith acetylated AuDENPs (2000 nM), numerous high electron-staining particles canbe found in the cytoplasm of the cells (Fig. 3(b)). The higher-magnification images further demonstrate that the uptake ofacetylated Au DENPs is dominantly located in the lysosomes (Fig. 3(c)). In sharp contrast, there are no electron-staining particles in thecytoplasm of the control SPC-A1 cells without treatment (Fig. 3(a)).The TEM results confirmed that the acetylated Au DENPs wereinternalized by the cells instead of sticking to the surface of thecells. The internalization of the acetylated Au DENPs likely occursthrough two distinct mechanisms: both phagocytosis and diffusionvia cell walls may take place, in agreement with literature [47,48].

Table 1Cell cycle analysis of SPC-A1 cells after incubation with the acetylated Au DENPs(mean � S.D., n ¼ 4).

Group Cell cycle (%)G0-G1 G2-M S G2/G1

Control 59.860 � 7.271 11.970 � 2.388 28.170 � 4.896 1.948 � 0.0051000 nM

Au DENPs58.048 � 9.348 12.388 � 2.568 29.568 � 6.784 1.950 � 0.000

2000 nMAu DENPs

58.988 � 6.038 12.235 � 2.271 28.778 � 3.894 1.950 � 0.008

3.4. Cytotoxicity of acetylated Au DENPs

To evaluate the morphology changes of SPC-A1 cells afterincubation with the acetylated Au DENPs, the treated cells werestained with HE (Fig. 4). The results showed that even if theconcentration of acetylated Au DENPs was up to 2000 nM, the cellsdid not display any appreciable morphological changes whencompared with the untreated control cells. These results indicatethat acetylated Au DENPs are non-cytotoxic, and do not affect onthe morphology of the SPC-A1 cells in a certain concentrationwindow.

To further examine the cytotoxicity of the acetylated Au DENPson SPC-A1 cells, the concentration-dependent effect of the acety-lated Au DENPs on the cell viability was analyzed by MTT assay(Fig. 5). It is clear that cells incubated with different concentrationsof acetylated Au DENPs (200, 500, 1000, 2000, and 3000 nM,respectively) have approximately similar cell viability comparedwith the untreated negative control cells (p > 0.05, n ¼ 3). Theresults clearly suggest the good biocompatibility of the acetylatedAu DENPs even at a concentration up to 3000 nM.

Cell cycle is an important parameter of cell biology. Cell cycledamage is one of the important features for the cell toxicity [49].Cell phase distribution is generally analyzed by the determinationof DNA contents, and the fraction of DNA content in sub-G1 phase isan indicator of cell apoptosis [50,51]. To investigate the influence ofacetylated Au DENPs on the cell apoptosis, the treated cells wereanalyzed by flow cytometry (Fig. 6). The sub-G1 fraction of SPC-A1cells incubated with acetylated Au DENPs at the concentration of1000 nM and 2000 nM were 3.87 � 2.39% and 6.08 � 3.23%,respectively in the quadruplication experiment. We showed thatthere was no statistically significant difference with that of theuntreated negative control cells (4.50 � 1.84%, p > 0.05). As shownin Table 1, acetylated Au DENPs had no effect on the cell cycle ofSPC-A1 cells. These results further confirm that acetylated AuDENPs are non-cytotoxic at the given concentration range.

3.5. Micro-CT imaging of SPC-A1 cells in vivo

The excellent in vitro performance of the acetylated Au DENPs aspotential CT imaging agent for SPC-A1 cells along with the greatbiocompatibility of the particles drives us to pursue their applica-bility for in vivo CT imaging of xenotransplanted tumor model inBALB/C nude mice. Fig. 7 shows the tumor CT images before andafter intratumoral injection of acetylated Au DENPs. It’s clear that

Fig. 7. Transection micro-CT images (a) and CT values (b) of the xenograft SPC-A1 tumor in nude mice before and after intratumorally injected with acetylated Au DENPs([Au] ¼ 0.2 M) for 1, 2, 4, and 6 h.

H. Wang et al. / Biomaterials 32 (2011) 2979e2988 2985

the tumor site shows an obvious enhancement with a significantlyhigher CT value after administration of acetylated Au DENPs whencompared with that before injection. Moreover, we found that withthe time, acetylated Au DENPs could diffuse into the entire tumorarea. After analysis of the CT values of the tumor area at differenttime points, we found that the CT value of tumor area decreasedgradually, but even at the time point of 6 h after intratumorinjection of the acetylated Au DENPs, the CT value of tumor areawas still significantly higher than that of the tumor area beforeinjection (194.97 � 20.88 versus 43.26 � 6.18). These results clearlyindicate that acetylated Au DENPs can not only enhance the lungtumor in vivo, but also diffuse in the tumor area. Taken togetherwith the results of in vitro CT cell imaging and the TEM verificationof the cellular uptake, we think that some of the acetylated AuDENPs may diffuse into the SPC-A1 cells to enhance the CT contrastof the tumor.

For testing whether the acetylated Au DENPs can be used toimage the tumor by another clinical route of drug administration,the particles were administrated by intraperitoneal injection intothe tumor mice. The mice were scanned by micro-CT after 1, 2, 4,

and 6 h post-injection. The results were really promising andinspiring. We found that the tumor region and the margin of thetumor region gradually turned to be clearer and sharper with thetime post-injection (Fig. 8(a)). The CT value of the tumor area alsogradually increased with the time post-injection (Fig. 8(b)). Theseresults indicate that after intraperitoneal injection, part of theacetylated Au DENPs could be absorbed into the capillary networkwhich is on the peritoneum, and be transported to the tumor sitevia EPR effect [52e55], then be ingested by tumor cells. Further ICP-AES analysis showed that the amount of Au element in the tumorsite 6 h post intraperitoneal injection was up to 6.91 mg per g. Thisresult confirmed that the acetylated Au DENPs were able to betransported to the tumor site for enhanced CT imaging. We alsoperformed ICP-AES analysis of the biodistribution of the acetylatedAu DENPs in different organs such as liver, spleen, kidney, andintestines at 6 h post intraperitoneal injection. Our preliminarydata revealed that the liver, spleen, kidney, and intestines of themouse contained 39.43, 260.25, 38.29, and 20.02 mg Au/g, respec-tively. Detail pharmacokinetic studies of the acetylated Au DENPswill be carried out in our future efforts.

Fig. 8. Transection micro-CT images (a) and CT values (b) of the xenograft SPC-A1 tumor in nude mice before and after intraperitoneally injected with acetylated Au DENPs([Au] ¼ 0.2 M) for 1, 2, 4, and 6 h.

H. Wang et al. / Biomaterials 32 (2011) 2979e29882986

3.6. In vivo tumor uptake of acetylated Au DENPs

To further confirm that the acetylated Au DENPs were able to bedelivered to the tumor site for CT imaging. The tumor sections weresilver stained andobservedusing lightmicroscope (Fig. S3).Noblackspots associatedwith the acetylated AuDENPswere observed in thenegative control (Fig. S3(A)). In contrast, in the sections of the tumortreated via both intratumoral and intraperitoneal injection of theacetylated Au DENPs at 6 h post-injection, there were numerousblack spots clearly localized in the cytoplasm of the cells, indicatingthe presence of the acetylated Au DENPs (Fig. S3(B)e(C)). The lessstaining in the tumor section administrated via intraperitonealinjection of the particles when comparedwith that via intratumoralinjection suggests that the amount of the accumulated particles inthe tumor site via intraperitoneal injection route is less than that viaintratumoral injection at the same timepoint, in agreementwith theCT imaging data. Our results clearly suggest that the particles can beslowly delivered to the tumor site via intraperitoneal injection,allowing for effective CT imaging of tumors, which is very importantfor early stage diagnosis of unknown tumors.

4. Conclusion

In summary, a new use of acetylated Au DENPs for CT imaging ofcancer cells in vitro and in vivo is reported in this study. Micro-CTimaging studies show that SPC-A1 cells incubated with acetylatedAu DENPs are able to be detected through the attenuation of theX-ray and the xenograft tumor model can be imaged using CT afterboth intratumoral and intraperitoneal administration of the acety-lated Au DENPs. TEM imaging studies show that the acetylated AuDENPs can be uptaken predominantly by the lyososomes of thecells. Combined HE staining of cell morphology, MTT assay of cellviability, and flow cytometry analysis of cell cycle show that theutilized acetylated Au DENPs are non-cytotoxic at the studiedconcentration range. Our results clearly indicate that acetylated AuDENPs could be used as a promising platform for CT imaging ofcancer cells both in vitro and in vivo. Given the unique features ofdendrimer-based nanostructures and the ability to generate tar-geting ligand-modified Au DENPs and Au DSNPs, we anticipate thatthe dendrimer-modified AuNPs should be able to find promisingapplications in targetedCT imagingof tumor cells in vitro and in vivo.

H. Wang et al. / Biomaterials 32 (2011) 2979e2988 2987

Acknowledgments

X. S. gratefully acknowledges the Fundação para a Ciência ea Tecnologia (FCT) and Santander bank for the Chair in Nanotech-nology. This work was financially supported by grants from theNational Natural Science Foundation of China (30901730 and20974019), the National Basic Research Program of China (973Program, 2007CB936000), the Fund of Ministry of Education ofChina (20090073110072), the Fund of the Science and TechnologyCommissionof ShanghaiMunicipality (No.1052nm05800), theFundof Youth Research Programme of Health Administration Bureau ofShanghai Municipality (2008Y108), the Fund of Doctor InnovationProgramme of Medical College, Shanghai Jiaotong University(BXJ0934 and BXJ201043), the Shanghai Pujiang Program(09PJ1400600), and the Shanghai Natural Science Foundation(10ZR1400800).

Appendix

Figures with essential color discrimination. Figs. 2, 7 and 8 inthis article are difficult to interpret in black andwhite. The full colorimage can be found in the online version, at doi:10.1016/j.biomaterials.2011.01.001.

Appendix. Supplementary material

Supplementary data related to this article can be found online atdoi:10.1016/j.biomaterials.2011.01.001.

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