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Cite this: J.Mater. Chem. B, 2016,

4, 4455

Self-assembled gold nanostar–NaYF4:Yb/Erclusters for multimodal imaging, photothermaland photodynamic therapy†

Liangcan He,a Joseph Dragavon,b Suehyun Cho,c Chenchen Mao,c Adem Yildirim,a

Ke Ma,a Rajarshi Chattaraj,d Andrew P. Goodwin,ae Wounjhang Parkce andJennifer N. Cha*ae

A grand challenge for medicine is to develop tools to selectively image and treat diseased cells. Rare

earth doped upconverting nanoparticles (UCNPs) have been extensively studied for imaging applications

because of their ability to absorb near infrared radiation (NIR) and emit visible light, but these particles

cannot induce therapy alone. Recently, we developed methods to couple the UCNPs to visible and

NIR-absorbing gold nanostructures through nucleic acid interactions. Here, we show that gold–UCNP

clusters with optimized plasmon resonance and particle compositions provide both in vitro imaging

contrast and combination cell killing through simultaneous photothermal (PTT) and photodynamic (PDT)

therapy. PDT was induced by embedding singlet oxygen photosensitizers in silica shells on the UCNPs.

Upon photoexcitation with 980 nm light, the NIR absorbing gold–UCNP clusters both increased

the local temperature and generated singlet oxygen, increasing cell killing relative to either modality

alone. The multifunctional polyethylene glycol (PEG) coated gold–NaYF4:Yb/Er clusters exhibited high

biocompatibility without irradiation but synergistic cell killing of MCF-7 cancer cells under light excita-

tion. Finally, we also demonstrate that an optimal gold plasmon resonance is critical for minimizing

absorbance overlap with the photosensitizers.

Introduction

Because upconverting materials tend to exhibit low backgroundauto-fluorescence, high photostability, low toxicity, and tunableemission, upconverting nanoparticles (UCNPs) have garneredsignificant interest for nanomedicine applications.1–7 In addition toimaging, the emissive wavelengths of UCNPs can excite photo-sensitizers with matching absorption profiles that generatesinglet oxygen capable of inducing cell toxicity via photodynamictherapy (PDT).8–13 In addition, NIR-responsive noble metal nano-structures have been utilized to promote hyperthermic responses inthe body.14–21 Therefore, formulations of gold–UCNP nanoparticle

clusters can impart both imaging and simultaneous PDT andphotothermal (PTT) therapy in the local vicinity of diseasedcells.22 However, because the non-resonant energy transferprocesses between metal surfaces and UCNPs cause significantdecreases in UCNP fluorescence, we recently developed strate-gies to control the number of Au conjugated to each UNCP tooptimize interparticle energy transfer.23–26 In the prior study,gold nanostars (AuNSs) and nanoparticles (AuNPs) werecoupled to silica coated UCNPs by DNA interactions.23 AuNSswere used because these structures exhibit three-dimensionalanisotropy that can respond independently of light polariza-tion, and the plasmon resonances are also easily tuned to reachthe NIR region.15,19,23,27–31 Furthermore, by tuning the molarratio of AuNP or AuNS to the UCNP, rapid 10–15 1C increases intemperature were observed under 980 nm excitation withminimal photoluminescence quenching. In doing so, we obtainedhighly emissive imaging agents still capable of localized heating bya single excitation wavelength, as compared to other systems suchas graphene oxide–UCNPs which require additional 808 nm laserirradiation for PTT.18,22,32,33

In this report, the photosensitizer zinc phthalocyanine (ZnPc)was loaded into the silica shells on the UCNPs in order to obtainhighly emissive agents that are capable of both PTT and PDT in

a Department of Chemical and Biological Engineering, University of Colorado,

Boulder, CO, 80303 USA. E-mail: Jennifer.Cha@colorado.edub BioFrontiers Advanced Light Microscopy Core, BioFrontiers Institute,

University of Colorado, Boulder, CO 80303, USAc Department of Electrical, Computer and Energy Engineering,

University of Colorado, Boulder, CO, 80303 USAd Department of Mechanical Engineering, University of Colorado, Boulder, CO,

80303 USAe Materials Science and Engineering Program, University of Colorado, Boulder, CO,

80303 USA

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tb00914j

Received 12th April 2016,Accepted 7th June 2016

DOI: 10.1039/c6tb00914j

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in vitro cell killing assays. ZnPc was utilized because the absorptionspectra matched well with the emission wavelengths of the UCNPsto generate singlet oxygen for PDT. Next, polyethylene glycol(PEG) chains were conjugated to the nanoparticles to impartbiocompatibility and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) cell assays were run using theUCNPs alone or the Au–UCNP clusters under NIR illumination.From these studies, we observed significantly higher cell deathrates (22.85% higher compared with PTT alone) with combinedPTT and PDT. It was also found however that the synergistic effectof PTT and PDT was only seen when the absorption overlapbetween the photosensitizers and the gold was minimized.

Materials and methodsReagents and materials

PEG-SH (2000), L-ascorbic acid (98%), silver nitrate (99%), gold(III)chloride trihydrate (99.9+%), Triton X-100, yttrium(III) chloride(99.99%), ytterbium(III) chloride (99.99%), erbium(III) chloride(99.9%), oleic acid (90%), ammonium fluoride (99.99%), tetra-ethylorthosilicate (99%), 3-aminopropyltriethoxysilane (99%),bis-p-sulfonatophenyl phenylphosphine dihydrate dipotassiumsalt (97%), cyclohexane (99.5%), Igepal CO-520, ammoniumhydroxide solution (30–33%), propidium iodide (94%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and 40-6-diamidino-2-phenylindole (DAPI) were all bought fromSigma Aldrich. 5 nm and 10 nm gold colloid were bought fromTED Pella. Sodium borohydride, sodium hydroxide (98.8%),dimethyl sulfoxide (99.7%), tris(2-carboxyethyl) phosphinehydrochloride and succinimidyl 4-(N-maleimidomethyl) cyclo-hexane-1-carboxylate (SMCC) were bought from Thermo Scientific.1-Octadecene (90%) was bought from Acros. Zinc phthalocyanine(95%) and fluorescein diacetate (97%) were bought form Alfa Aesar.DNA1 (50-SH-C6-TTA TAA CTA TTC CTA AAA AAA AAA A-30) andDNA2 (50-SH-C6-TTT TTT TTT TTA GGA ATA GTT ATA A-30) werebought from Integrated DNA Technologies, Inc. All the chemicalswere used as received without further purification. Deionized waterwas used throughout the experiments.

Synthesis of 25 nm NaYF4:Yb,Er upconverting nanoparticles(UCNPs)

Monodisperse NaYF4:18%Yb,2%Er UCNPs were synthesizedusing a previously reported method. In a typical procedure,0.1562 g YCl3, 0.0503 g YbCl3, and 0.0055 g ErCl3 were mixedwith 6 mL oleic acid and 15 mL octadecene. The solution wasslowly heated to 160 1C with vigorous stirring under argon for50 min to form a homogeneous transparent solution, and thenallowed to cool to room temperature. Then, a 10 mL methanolsolution of NaOH (0.1 g) and NH4F (0.148 g) was added drop-by-drop and the solution was stirred for another 40 min. Thesolution was then slowly heated and degassed at 110 1C for20 min, then refilled with argon and degassed three timestotally. After that, the solution was heated to 300 1C within 10 minand reacted for 70 min under argon. After the solution was cooledback to room temperature, the products were precipitated from the

solution with ethanol and then washed with ethanol and water(1 : 1) three times.

Synthesis of silica coated UCNPs

In a typical procedure, 1.5 mL oleic acid stabilized UCNPs(about 6.2 mg) in toluene was dried in 70 1C. After that, 2 mLcyclohexane was added, the solution was sonicated for 10 min.Then, 10 mL water which contains 0.1 g CTAB was added intothe above solution and stirred vigorously for overnight toevaporate the cyclohexane, resulting in a clear CTAB–UCNPswater solution. For coating mesoporous silica onto UCNPs, 2.5 mLof the above CTAB–UCNPs solution was added to the mixture of25 mL water, 3 mL ethanol and 40 mL 1 M NaOH solution. And then500 mL 10% tetraethylorthosilicate (TEOS) ethanol solutionwas added into the above solution dropwise and the reactionwas maintained for 14 hours. The as-obtained products werecentrifuged and washed with ethanol three times.

Synthesis of DNA modified silica coated UCNPs

Briefly, 1.8 mg silica coated UCNPs were first reacted with3-aminopropyltriethoxysilane (APTES) in ethanol (VAPTES : Vethanol =5 : 95) for 5 h. Then the amine modified silica coated UCNPs(in 100 mL H2O) was reacted with 1.8 mg succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) in500 mL DMSO solution for 5 h. The mixture was centrifugedand washed three times, then it was resuspended in 500 mLDMSO solution. 187 mL 200 mM of DNA2 (DNA2 : UCNP = 200 : 1)was mixed with 30 mL tris(2-carboxyethyl) phosphine (TCEP)(50 mM in 200 mM NaHCO3), then added to the SMCC modifiedUCNPs. The reaction was allowed to proceed overnight. Finally,the above products were purified and dispersed in water forfurther use. The procedure used for preparation of DNA modified520 nm AuNPs, 700 nm AuNSs and 980 nm AuNSs is the samewith our previous methods.23

Preparation of ZnPc loaded Au–UCNP PEGylated clusters

Firstly, 45 mL DMSO solution which contained 2 mg ZnPc wasprepared. Then, 15 mL ZnPc solution was mixed with each kindof Au–UCNP clusters and was sonicated for 5 min then shakefor 24 hours. After which, the products were spun by centrifu-gation at 10 000 rpm for 12 min. The supernatant was carefullydiscarded and the pellet was washed by ethanol and PBS for fivetimes. Then 4 mg HS-PEG2000 was added into each tube and thesolution was then reacted for 12 hours. The PEG modifiedclusters were then centrifuged and washed with water for threetimes. The final samples were kept in 4 1C for further use.

Cell culture

The MCF-7 cells were grown to confluence at 37 1C and with 5% CO2

in Eagle’s Minimum Essential Medium (EMEM) medium containing10% fetal bovine serum and 1% penicillin/streptomycin.

Cytotoxicity assay

The viability of MCF-7 cells were determined by using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. MCF-7 cells were seeded into a 96-well cell culture plate

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at 104 per well and then incubated for 24 h at 37 1C under 5%CO2. Then, Au–UCP clusters dispersed in PBS were added toeach well to give final particle concentrations of 0, 10, 40, 100and 250 mg mL�1. The cells were then incubated for 4 h at 37 1Cunder 5% CO2. After incubation, particle containing media wasremoved, wells were washed using PBS to remove the non-uptakenparticles and 90 mL of fresh medium was added. Then 20 mL offilter-sterilized MTT reagent (5 mg mL�1 in PBS) was added to eachwell, and the plates were incubated at 37 1C for further 4 h. Afterincubation, medium was removed and the precipitated formazancrystals were dissolved by adding acidic isopropyl alcohol (40 mMHCl). Absorption values of the dissolved formazan crystals in eachwell were measured at 570 and 750 nm using a microplate reader.All the samples were prepared in triplicate.

Photothermal test

MCF-7 cells were incubated with 250 mg mL�1 of Au–UCNP clustersfor 4 hours, and excess Au–UCNP clusters were removed by PBSwashing. Then, 980 nm laser (laser spot size: 1 mm in diameter;laser power: 500 mW; laser confluence: 15.9 W cm�2) was used toirradiate the cells for 15 min and then replaced with fresh culturemedium and cultivated for 12 h at 37 1C in order to determine theeffects of the 980 nm laser irradiation and laser induced 1O2 on cellviability. Cell viability was determined by MTT assay (as describedabove). All the samples were prepared in triplicate.

Confocal fluorescence imaging

250 mg mL�1 of Au–UCNP clusters were incubated with MCF-7cells (104 per well) for 4 hours at 37 1C under 5% CO2. Thenthe wells were washed by fresh PBS three times and fixed with4% paraformaldehyde for 15 min. After that the nuclei werestained with 2 mg mL�1 40-6-diamidino-2-phenylindole (DAPI)for 15 min and then the wells were washed with PBS threetimes. Luminescence signals were collected in the wavelengthregions of 500–550 nm and 570–620 nm.

Fluorescein diacetate (green)/propidium iodide (red) (FDA/PI)double staining

The hyperthermic and 1O2 induced potential effect of Au–UCNPclusters on MCF-7 cells was roughly checked by monitoringthe changes of lysosomal membranes. The Au–UCNP clusters(250 mg mL�1) incubated MCF-7 cells were irradiated by thesame laser for 15 min and then replaced with fresh culturemedium and cultivated for 12 h at 37 1C. After that, the cellculture medium was removed and the cells were washed byPBS. Then, 100 mL staining solution was added to each wellsand incubated for 10 min in the dark. At last, the stainingsolution was removed and the cells were washed by PBS threetimes. The samples were then checked by fluorescent micro-scopy. FDA/PI staining solution: 50 mL 2 mg mL�1 PI PBSsolution and 8 mL 5 mg mL�1 FDA acetone solution were addedinto 5 mL PBS solution, the mixture should be freshly preparedand kept in dark.

Results and discussion

NaYF4:Yb/Er upconversion nanoparticles (UCNPs) were firstsynthesized using a thermal decomposition method to producemonodisperse nanocrystals B25 nm in diameter (Fig. 1).23,34–37

Since the as-prepared NaYF4:Yb/Er UCNPs are capped with oleicacid, the particles were first transferred into water usingcetyltrimethylammonium bromide (CTAB) (Fig. S1 and S2, ESI†).Next, to increase stability in biological media and permit photo-sensitizer loading onto the UCNPs, mesoporous silica shellsB10 nm thickness were condensed around the UCNPs, followedby removal of CTAB through treatment with ammonium nitrite(NH4NO2) at 60 1C for 2 h followed by washing with ethanol.38–41

In order to conjugate the gold nanoparticles (AuNPs) andgold nanostars (AuNSs) to the UCNPs, the silica-coated UCNPswere further reacted with aminopropyltriethoxysilane (APTES) inethanol for 5 hours followed by N-hydroxysuccinimide (NHS)-maleimide (SMCC) in DMSO.23 The SMCC-modified UCNP werethen reacted overnight with thiol-terminated DNA oligonucleo-tides to produce DNA-modified UCNPs. After removal of excessDNA by centrifugation, UV-Vis absorption measurements of thesupernatant showed that B71 DNA strands were attached perUCNP. Concurrently, three kinds of DNA-conjugated Au nano-structures with plasmon resonances at 520 nm, 700 nm, and980 nm were prepared using our previously published procedures(Fig. 2).23,42,43 The DNA-stabilized Au nanostructures were thenhybridized to the DNA-modified UCNPs using 1 : 10 Au : UCNPmolar ratios (Scheme 1). These loadings had previously been shownto minimize photoluminescence quenching from the UCNPs.23 Asshown in Fig. 2, TEM analysis showed that the AuNPs and AuNSswere well conjugated to the UCNPs, with very few unbound AuNPsand AuNSs. With the AuNSs, most of the interactions with theUCNPs appeared to occur at the tips of the AuNS branches, whichcan be attributed to the high anisotropy of the AuNSs. Next, inorder to improve biocompatibility with cells, the Au–UCNPclusters were reacted with 5 mg thiolated-PEG2000 overnight inwater solution, after which the excess PEG was removed bycentrifugation.44–46 While the presence of DNA on the Au andUCNP could potentially prevent or decrease the amounts of PEGconjugated to the nanoparticles, as will be discussed in moredetail below, in vitro cell assays showed that coating the nano-particles with PEG did improve cell viability.

In order to evaluate the Au–UCNP clusters as imaging andtherapy agents, we first measured the relative cell cytotoxicity ofthe UCNP and Au–UCNP clusters against breast cancer MCF-7

Fig. 1 TEM images of as-synthesized UCNPs and silica coated UCNPs.

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cells in the absence of any photoirradiation. For this, a typicalMTT cell viability assay was performed by first incubating theUCNP and Au–UCNP clusters with the cells at concentrationsup to 250 mg mL�1 for 4 h. Next, filtered MTT solutions in PBSwere added to the wells and incubated for another 4 h, afterwhich the media was carefully removed and mixed with 100 mLacidic isopropyl alcohol (IPA, 40 mM HCl). The mixtures weregently agitated at room temperature overnight to completelydissolve the dye crystals, followed by absorbance measurementon a plate reader. As shown in Fig. 3, by coating the UCNP andAu–UCNP nanoparticles with PEG, the overall cell viabilitygreatly improved as compared to particles with no PEG attached.Coating nanoparticles with PEG both reduces the nonspecificbinding of blood proteins and macrophages to nanoparticleswhile potentially increasing the particle circulation life-time forin vivo applications.47,48 At the highest nanoparticle concentrationstested (250 mg mL�1), both the PEGylated UCNPs and Au–UCNPs

retained cell viabilities greater than 88% after 4 h incubation.In comparison, particles without PEG produced lower cellviabilities at only 56% after 4 h (Fig. S3, ESI†).

Next, we investigated the PEGylated Au–UCNP clusters asimaging agents. As mentioned earlier, clusters of 1 : 10 Au :UCNP demonstrated strong visible emission under 980 nm excita-tion.23 For the imaging studies in this work, the PEGylated UCNPand Au–UCNP clusters were cultured with MCF-7 cells for 4 hat 37 1C under 5% CO2 using nanoparticle concentrations of250 mg mL�1.49 Excess nanoparticles were removed and thecells were prepared for imaging studies by fixing the cells with4% paraformaldehyde for 15 min.49 The UCNP and Au–UCNPtreated cells were then imaged by confocal microscopy using405 nm and 980 nm excitation. As shown in Fig. 4 and Fig. S6(ESI†), the cell nuclei were easily identified by 40-6-diamidino-2-phenylindole (DAPI) staining. The quantified photolumines-cence intensity of the images in Fig. 4 is shown in Table S1 andFig. S7 (ESI†). Under 980 nm light, both the UCNPs alone andthe Au–UCNP clusters showed strong visible emission in thegreen, with weaker fluorescence in the red; this emissionprofile was consistent with photoluminescence studies runpreviously.23,35,50 Since no autofluorescence was observed fromthe cells upon NIR excitation,51 the Au–UCNP clusters showclear promise as agents for both imaging and PDT.

Next we coupled UCNPs to NIR-absorbing Au to study theeffect of simultaneous PDT and PTT, since it was previouslydemonstrated that combined therapy treatments could not onlyincrease efficacy but could also lower toxic side effects incomparison to two light source cancer therapies.18,22,32,33 Thespectral overlap between the UCNP emission with the absor-bance profile of zinc phthalocyanine (ZnPc) allows a series ofenergy transfers to convert NIR absorption into singlet oxygengeneration (1O2), which can induce cell cytotoxicity by 1O2-induced apoptosis (Fig. 5 and Scheme 1).9,12,13 First, ZnPcwas incubated with the mesoporous silica coated UCNPs inDMSO for 24 h, followed by repeated centrifuge washing toproduce a blue-purple UCNP solution, demonstrating success-ful ZnPc incorporation within the silica shells. The dye9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) wasused to measure the amount of singlet oxygen production upon380 nm excitation (Fig. S4 and S5, ESI†). Upon singlet oxygen

Fig. 2 TEM images of (a1) 520 nm AuNPs, (b1) 700 nm Au NSs, (c1) 980 nmAu NSs and the corresponding conjugated Au–UCNP clusters images of (a2)AuNP–UCNP, (b2) 700 AuNS–UCNP, (c2) 980 NS–UCNP (insets are theenlarged TEM images). The molar ratios of AuNP : UCNP and AuNS : UCNPused in the images were 5 : 1 and 1 : 10 respectively.

Scheme 1 Schematic illustration for the assembly of the DNA conjugatedsilica coated UCNPs and DNA conjugated gold nanostructures that arealso loaded with photosensitizers for simultaneous imaging, PDT and PTT.After 980 nm laser excitation, the UCNPs can upconvert NIR light to visiblelight. Spectral overlap between the emission light of the UCNPs with theabsorption spectra of the photosensitizer ZnPc generates cytotoxic 1O2 forPDT. At the same time, the NIR absorbing gold nanostructures can convertlight to heat which can be utilized for PTT.

Fig. 3 In vitro cell viability results of MCF-7 cells incubated with Au–UCNPclusters at different concentrations (0, 10, 40, 100 and 250 mg mL�1) for 4 h.

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generation, the ABDA fluorescence becomes quenched, andthis decay can be measured and plotted as a function ofirradiation time to NIR laser (Fig. 5).12

The applicability of the Au–UCNP clusters for both PDT andPTT was next assessed in in vitro cell assays. For this, PEGylatedUCNPs were incubated with MCF-7 cell cultures for 4 h at 37 1Cfollowed by replacement of cell media with fresh PBS. TheUCNP treated cells were then photoirradiated for 15 min using a980 nm laser (15.9 W cm�2), followed by MTT assays to measurecell viability. As shown in Fig. 6, in the absence of photoirradiation

more than 90% of the cells remained viable in the presence ofthe UCNPs, which was consistent with the previous toxicitystudies (Fig. 3). Conversely, more than 86% of the cells remainedviable under photoirradiation without UCNPs, indicating thatthe NIR laser light itself did little to harm the cells. However,photoirradiation of MCF-7 cells with ZnPc-loaded UCNPs causedcell viability to decrease significantly to 78.05% and 44.08% for40 mg mL�1 and 250 mg mL�1 concentrations, respectively(Fig. 6). These results therefore show the utilization of theUCNPs as agents for PDT with NIR excitation.

The effect of PTT alone was studied via the incubation ofMCF-7 cells with the AuNP–UCNP and AuNS–UCNP clusters(without ZnPc) at concentrations of 250 mg mL�1, followed byirradiation for 15 min with 980 nm laser light. As shown inFig. 6, while only about 10% cell death was observed from thecells treated with the AuNP–UCNP clusters (Au plasmon atB520 nm), for the 700 AuNS–UCNP (Au plasmon at B700 nm)and 980 AuNS–UCNP (Au plasmon at B980 nm) treated cells, cellviability sharply dropped to 42.44% and 51.02%, respectively. Thismatches the results from our previous work, which demonstratedrapid temperature gains from the AuNS–UCNP materials butalmost no increase from the AuNP–UCNP system.23

Finally, the effect of the combined synergistic PDT and PTTeffects on cell viability was illustrated by treating the MCF-7cells with ZnPc loaded AuNP–UCNP and AuNS–UCNP clusters,followed by the same irradiation conditions. MTT assaysshowed that combined PDT and PTT led to significant gainsin cell killing, as the cell viabilities dropped substantially to70.17%, 19.59%, and 30.68% for the AuNP–UCNP, 700 AuNS–UCNP, and 980 AuNS–UCNP, respectively (Fig. 6). Thus combi-nation therapy led to approximately 20% more cell death thanPTT alone. Because of the partial spectral overlap between theZnPc and the AuNPs (520 nm plasmon), less of the emitted lightfrom the UCNPs was available for the ZnPc photosensitizer,

Fig. 4 Confocal laser scanning microscopy imaging of MCF-7 cells irra-diated by 980 nm laser: (a) control samples (no UCNP or Au–UCNPadded), (b) incubated with UCNPs, (c) incubated with AuNP–UCNP clusters,(d) incubated with 700 AuNS–UCNP clusters and (e) incubated with 980AuNS–UCNP clusters. All of the nanoparticles were added at 250 mg mL�1

for 4 h. Green channel images were obtained within the wavelength of500–550 nm, red channel images were collected within the wavelength of570–620 nm. The cell nucleus was stained by DAPI (blue fluorescence). Thebright field images and corresponding merged images were also shownabove. All the images have the same scale bar: 20 mm.

Fig. 5 (a) Fluorescence emission spectra (dashed line) of the UCNP under980 nm laser irradiation and the ZnPc absorption spectra (solid line). (b) 1O2

produced by the ZnPc loaded UCNPs under 980 nm laser irradiation(15.9 W cm�2) as determined by the fluorescence decay of ABDA (excita-tion wavelength: 380 nm). ABDA fluorescence decay (measured its peakintensity at 407 nm) is plotted vs. laser irradiation time (ABDA: 2 mM, UCNP–ZnPc: 533 mg mL�1).

Fig. 6 Quantitative analysis of cell viabilities with the ZnPc loaded UCNP–Au nanoclusters from 980 nm laser irradiation for 15 min (unit of legendunderneath is mg mL�1). As demonstrated, a strong synergistic effect wasobserved with the dual use of PTT and PDT as compared to either alone.Furthermore, this was clearly dependent on the type of Au nanostructureutilized.

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which accounts for the B70% viability for these cells ascompared to ZnPc-loaded UCNPs alone (44.08%). However,with the NIR responsive AuNS–UCNP clusters, significantincreases in cell death were induced, presumably both to thelimited overlap in optical signatures between the AuNS andZnPc as well as the combined utilization of PTT and PDT. Inaddition, larger cell death was induced with the 700 AuNS–UCNP as compared to the 980 AuNS–UCNP which we canattribute to the larger increases in temperature caused by the700 AuNSs than the 980 AuNSs upon NIR absorption.23 Inaddition to MTT assays, live-dead cell assays were also run byusing fluorescein diacetate and propidium iodide.18 As shownin Fig. 7, the cell staining assays matched the results obtainedfrom the MTT studies. Lastly, to assess the ability of using theAu–UCNP clusters as potential imaging agents in vivo, a piece of3% agarose was placed on top of the wells of the MCF-7 cultures

incubated with the 700 AuNS–UCNPs followed by confocalmicroscopy. As shown in Fig. 8, the UCNPs showed strongemission through the agarose gel demonstrating the possibilityof utilizing the AuNS–UCNP clusters as theranostics, tissueengineering and cellular imaging.

Conclusions

In summary, a multifunctional nanotheranostic has beendesigned for PL imaging, PTT and PDT. We first show thatattaching PEG chains to the UCNP and Au–UCNP clustersimparted high biocompatibility. Furthermore, the AuNP–UCNPand AuNS–UCNP still demonstrated strong emission in in vitro cellassays through careful loading of the AuNP and UCNP components.We showed that NIR laser light was absorbed by the Au–UCNPclusters and transferred into local photothermal energy to inducecell cytotoxicity. By loading the photosensitizer ZnPc into the silicacoatings on the UCNPs, singlet oxygen could also be generated tocause cell death. Finally, we demonstrate in this work that the ZnPcloaded AuNS–UCNP clusters could synergistically induce bothPTT and PDT to effectively cause a 20% increase in cell death ascompared to using either modality alone. These results thereforeshow the potential of using multifunctional nanotheranostics ashighly efficient agents for tumor eradication.

Acknowledgements

The research was primarily supported by the National Instituteof Health (1R21EB020911-01) U.S. Dept. of Energy (DOE), Officeof Science, Basic Energy Sciences (BES) under Award # DE-SC0006398, Army Research Office under Award # W911NF-14-1-0211 and the National Science Foundation Award # DMR1420736.

Fig. 7 Images of fluorescein diacetate (green)/propidium iodide (red)(FDA/PI) double stained cells after 980 nm laser irradiation for 15 min (a)images of blank MCF-7 cells (b) images of UCNPs incubated MCF-7 cells(c) images of 520 AuNP–UCNP clusters incubated MCF-7 cells (d) imagesof 700 AuNS–UCNP clusters incubated MCF-7 cells (e) images of 980AuNS–UCNP clusters incubated MCF-7 cells. Left is green channel, right isred channel. All the images have the same scale bar: 50 mm.

Fig. 8 Confocal images of: (a) MCF-7 cells incubated with 700 AuNS–UCNP clusters; (b) the cells shown in (a) through a 1 mm thick 3% agarosegel (a1 and b1: green channel, a2 and b2: red channel).

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