Cancer Research Tumor Site Specific Silencing of NF-κB p65 by … · blockage of the initial siRNA...

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Therapeutics, Targets, and Chemical Biology Tumor SiteSpecific Silencing of NF-κB p65 by Targeted Hollow Gold NanosphereMediated Photothermal Transfection Wei Lu, Guodong Zhang, Rui Zhang, Leo G. Flores II, Qian Huang, Juri G. Gelovani, and Chun Li Abstract NF-κB transcription factor is a critical regulator of the expression of genes involved in tumor formation and progression. Successful RNA interference (RNAi) therapeutics targeting NF-κB is challenged by small interfering RNA (siRNA) delivery systems, which can render targeted in vivo delivery, efficient endolysoso- mal escape, and dynamic control over activation of RNAi. Here, we report near-IR (NIR) lightinducible NF-κB downregulation through folate receptortargeted hollow gold nanospheres carrying siRNA recogniz- ing NF-κB p65 subunit. Using micropositron emission tomography/computed tomography imaging, the targeted nanoconstructs exhibited significantly higher tumor uptake in nude mice bearing HeLa cervical cancer xenografts than nontargeted nanoparticles following i.v. administration. Mediated by hollow gold nanospheres, controllable cytoplasmic delivery of siRNA was obtained on NIR light irradiation through photothermal effect. Efficient downregulation of NF-κB p65 was achieved only in tumors irradiated with NIR light but not in nonirradiated tumors grown in the same mice. Liver, spleen, kidney, and lung were not affected by the treatments, in spite of significant uptake of the siRNA nanoparticles in these organs. We term this mode of action photothermal transfection.Combined treatments with p65 siRNA photo- thermal transfection and irinotecan caused substantially enhanced tumor apoptosis and significant tumor growth delay compared with other treatment regimens. Therefore, photothermal transfection of NF-κB p65 siRNA could effectively sensitize the tumor to chemotherapeutic agents. Because NIR light can penetrate the skin and be delivered with high spatiotemporal control, therapeutic RNAi may benefit from this novel transfection strategy while avoiding unwanted side effect. Cancer Res; 70(8); 317788. ©2010 AACR. Introduction Small interfering RNA (siRNA) molecules are the function- al mediators of RNA interference (RNAi), which can induce posttranscriptional gene silencing (1). RNAi-mediated gene silencing is now an attractive tool for interfering with the ex- pression of undesired genes, such as oncogenes. Owing to the relatively high molecular weight and negative charge of siR- NAs, their endolysosomal escape and systemic delivery are major challenges confronting efficient RNAi. Several cationic polymers and lipids have been used to form nanoparticulate polyelectrolyte complexes with negatively charged siRNA on the basis of enhanced endocytosis, and escape of siRNA from endolysosomal vesicles is achieved by membrane destabiliza- tion through proton sponge effect (24). These techniques, however, lack temporal and spatial control of RNAi. Once siRNA is taken up by cells, gene silencing becomes an irre- versible process. Light-induced RNAi has enormous advantages because RNAi can be confined only in the illuminated area, thereby reducing nonspecific effects and providing tools for kinetic studies, off-target effect assessment, and phenotypic assay (58). To date, regulation of siRNA by light is achieved pho- tochemically by either using photosensitizing compounds to induce disruption of the endocytic vesicles (5, 6) or removal of photolabile moiety covalently attached to siRNA for the blockage of the initial siRNA interaction with the RNA- induced silencing complex (7, 8). However, these techniques are difficult to apply to in vivo gene silencing because the UV/visible light (350450 nm) used to activate siRNA cannot penetrate the skin (6). Near-IR (NIR) light (700850 nm), on the other hand, can penetrate deep into the tissue because tissue absorption of light in the NIR region is minimal (9). Nanostructures of no- ble metals such as gold and silver exhibit unique optical properties due to strong surface plasmon resonance (SPR) absorption at visible and NIR wavelengths, which can present photothermal effects to trigger a variety of biological activi- ties (1016). In particular, core/shell-structured hollow gold nanospheres (HAuNS, 40 nm) consist only of a thin gold Authors' Affiliation: Department of Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Houston, Texas Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Chun Li, Department of Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Unit 59, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-5182; Fax: 713-794-5456; E-mail: [email protected]. doi: 10.1158/0008-5472.CAN-09-3379 ©2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 3177 on May 29, 2020. © 2010 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-3379

Transcript of Cancer Research Tumor Site Specific Silencing of NF-κB p65 by … · blockage of the initial siRNA...

Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-3379

Therapeutics, Targets, and Chemical Biology

Cancer

Research

Tumor Site–Specific Silencing of NF-κB p65 by TargetedHollow Gold Nanosphere–MediatedPhotothermal Transfection

Wei Lu, Guodong Zhang, Rui Zhang, Leo G. Flores II, Qian Huang, Juri G. Gelovani, and Chun Li

Abstract

Authors' AThe Univer

Note: SupResearch

CorresponImaging, Th1515 HolcoFax: 713-7

doi: 10.115

©2010 Am

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NF-κB transcription factor is a critical regulator of the expression of genes involved in tumor formationand progression. Successful RNA interference (RNAi) therapeutics targeting NF-κB is challenged by smallinterfering RNA (siRNA) delivery systems, which can render targeted in vivo delivery, efficient endolysoso-mal escape, and dynamic control over activation of RNAi. Here, we report near-IR (NIR) light–inducibleNF-κB downregulation through folate receptor–targeted hollow gold nanospheres carrying siRNA recogniz-ing NF-κB p65 subunit. Using micro–positron emission tomography/computed tomography imaging, thetargeted nanoconstructs exhibited significantly higher tumor uptake in nude mice bearing HeLa cervicalcancer xenografts than nontargeted nanoparticles following i.v. administration. Mediated by hollow goldnanospheres, controllable cytoplasmic delivery of siRNA was obtained on NIR light irradiation throughphotothermal effect. Efficient downregulation of NF-κB p65 was achieved only in tumors irradiated withNIR light but not in nonirradiated tumors grown in the same mice. Liver, spleen, kidney, and lung werenot affected by the treatments, in spite of significant uptake of the siRNA nanoparticles in these organs.We term this mode of action “photothermal transfection.” Combined treatments with p65 siRNA photo-thermal transfection and irinotecan caused substantially enhanced tumor apoptosis and significant tumorgrowth delay compared with other treatment regimens. Therefore, photothermal transfection of NF-κB p65siRNA could effectively sensitize the tumor to chemotherapeutic agents. Because NIR light can penetratethe skin and be delivered with high spatiotemporal control, therapeutic RNAi may benefit from this noveltransfection strategy while avoiding unwanted side effect. Cancer Res; 70(8); 3177–88. ©2010 AACR.

Introduction

Small interfering RNA (siRNA) molecules are the function-al mediators of RNA interference (RNAi), which can induceposttranscriptional gene silencing (1). RNAi-mediated genesilencing is now an attractive tool for interfering with the ex-pression of undesired genes, such as oncogenes. Owing to therelatively high molecular weight and negative charge of siR-NAs, their endolysosomal escape and systemic delivery aremajor challenges confronting efficient RNAi. Several cationicpolymers and lipids have been used to form nanoparticulatepolyelectrolyte complexes with negatively charged siRNA onthe basis of enhanced endocytosis, and escape of siRNA fromendolysosomal vesicles is achieved by membrane destabiliza-tion through proton sponge effect (2–4). These techniques,

ffiliation: Department of Experimental Diagnostic Imaging,sity of Texas M.D. Anderson Cancer Center, Houston, Texas

plementary data for this article are available at CancerOnline (http://cancerres.aacrjournals.org/).

ding Author: Chun Li, Department of Experimental Diagnostice University of Texas M.D. Anderson Cancer Center, Unit 59,mbe Boulevard, Houston, TX 77030. Phone: 713-792-5182;94-5456; E-mail: [email protected].

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however, lack temporal and spatial control of RNAi. OncesiRNA is taken up by cells, gene silencing becomes an irre-versible process.Light-induced RNAi has enormous advantages because

RNAi can be confined only in the illuminated area, therebyreducing nonspecific effects and providing tools for kineticstudies, off-target effect assessment, and phenotypic assay(5–8). To date, regulation of siRNA by light is achieved pho-tochemically by either using photosensitizing compounds toinduce disruption of the endocytic vesicles (5, 6) or removalof photolabile moiety covalently attached to siRNA for theblockage of the initial siRNA interaction with the RNA-induced silencing complex (7, 8). However, these techniquesare difficult to apply to in vivo gene silencing because theUV/visible light (350–450 nm) used to activate siRNA cannotpenetrate the skin (6).Near-IR (NIR) light (700–850 nm), on the other hand, can

penetrate deep into the tissue because tissue absorption oflight in the NIR region is minimal (9). Nanostructures of no-ble metals such as gold and silver exhibit unique opticalproperties due to strong surface plasmon resonance (SPR)absorption at visible and NIR wavelengths, which can presentphotothermal effects to trigger a variety of biological activi-ties (10–16). In particular, core/shell-structured hollow goldnanospheres (HAuNS, ∼40 nm) consist only of a thin gold

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wall with a hollow interior and displayed strong SPR tunabil-ity in the NIR region (17–19). Spatiotemporal silencing of areporter gene (green fluorescence protein) was recently re-ported in vitro through the NIR laser–induced release ofsiRNA from the HAuNS (20).NF-κB transcription factor is a critical regulator of the ex-

pression of genes involved in the immune and inflammatoryresponse and also regulates the tumor development (21, 22).NF-κB has been connected to many aspects of tumor forma-tion and progression, including inhibition of apoptosis, by in-creasing the expression of antiapoptotic and survival factors(22). Inhibition of NF-κB has been associated with increasedsensitivity to chemotherapeutic agents, including irinotecan(23). In this study, we introduced siRNA duplex againstNF-κB p65 subunit to the surface of HAuNS (HAuNS-siRNA)to construct folate receptor–targeted nanoparticles. Usingthis targeted siRNA delivery system, we investigated theNIR light–triggered efficient p65 downregulation. We termthis mode of action “photothermal transfection.” Further-more, applying this photothermal transfection technique,we explored tumor site–specific RNAi and enhanced chemo-sensitivity to irinotecan in nude mouse bearing HeLa cervicalcancer xenografts.

Materials and Methods

Synthesis of HAuNS-siRNA and folate-targeted HAuNS-siRNA (F-PEG-HAuNS-siRNA). Double-stranded siRNAs withtwo thymidine residues (dTdT) at the 3′ end of the sequenceextending between amino acid residues 347 and 353 were de-signed for the NF-κB p65 subunit (sense, 5′-GCCCUAUCC-CUUUACGUCA-3′; ref. 23). Nontargeting control siRNAsequence (siRNAluc) was designed against firefly luciferasemRNA with two UU overhangs (sense, 5′-AUGAACGU-GAAUUGCUCAA-3′). The custom-synthesized siRNA wasfunctionalized with sulfhydryl group at the 5′ end of thesense chain so that it would couple with HAuNS (Dharma-con). In the fluorescence tracking analysis, the above thio-lated siRNA duplex was labeled with Dy547 (excitation, 549nm; emission, 563 nm) at the 3′ end of the sense chain. In allexperiments of this work, “siRNA” refers to siRNA againstp65, except when otherwise specified.HAuNS were synthesized according to our previously pub-

lished methods (18, 19). For the conjugating reaction, 2 nmolof thiolated siRNA duplex (siRNA-SH) were added into theHAuNS solution (6 × 1012 nanoparticles in 1.0 mL water;siRNA-SH:HAuNS molar ratio, ∼200:1). The solution wasshaken for 24 h at room temperature and then centrifugedat 7,000 rpm for 15 min. The HAuNS-siRNA nanoparticleswere washed three times with water and collected for furthercharacterization (see Supplementary Data). Folic acid was in-troduced to the gold surface of HAuNS-siRNA through athioctic acid–terminated polyethylene glycol linker (F-PEG-TA; Supplementary Fig. S1) to afford F-PEG-HAuNS-siRNA,which was prepared by mixing HAuNS (6 × 1012 nanoparti-cles) with both siRNA-SH (2 nmol) and F-PEG-TA (0.4 nmol)in 1 mL water (HAuNS:siRNA:F-PEG-TA molar ratio,∼1:200:40). For the preparation of nontargeting PEG-

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HAuNS-siRNA, the same amount of PEG-SH (Sigma-Aldrich)was added instead of F-PEG-TA.p65 interference with F-PEG-HAuNS-siRNA activated by

NIR light irradiation in vitro. HeLa cells (American TypeCulture Collection) were seeded in a 24-well plate (∼3 ×104 per well). The cells were incubated with F-PEG-HAuNS-siRNA, PEG-HAuNS-siRNA, or F-PEG-HAuNS-siRNAluc

(4 × 1011 nanoparticles in 1 mL folate-free RPMI 1640)at 37°C for 2 h. The cells were then irradiated with NIR laser(32 or 50 mW/cm2) for different time periods. Transfectionwith Lipofectamine 2000 (Invitrogen) was used as a positivecontrol. Each treatment was performed in pentaplicate. Thesamples were collected 48 h later for Western blot analysisand immunohistochemistry (see Supplementary Data). To ex-amine the effect of time intervals after NIR irradiation onRNAi, the HAuNS-treated and NIR laser–treated cells werecultured for additional 24, 48, 72, or 96 h.Combined treatment with p65 RNAi and irinotecan

in vitro. HeLa cells in a 96-well plate were either left un-treated or treated with F-PEG-HAuNS-siRNA (3 × 1011 na-noparticles/mL, 0.2 mL RPMI 1640 folate-free medium) for2 h in the presence or absence of the NIR laser (50 mW/cm2,60 s) delivered at the end of incubation period. Cells withoutboth laser and nanoparticles were used as a control. After24 h, the cells were incubated with irinotecan (Sigma-Aldrich)for another 24 h at various concentrations in triplicate. Thecell survival was determined using MTT (Sigma-Aldrich) as-say (24). The percentage of surviving cells relative to the non-treated cells was plotted as a function of concentration. Inaddition, the apoptotic response to irinotecan was analyzedusing both flow cytometry and immunohistochemistry (seeSupplementary Data).Biodistribution, micro–positron emission tomography/

computed tomography imaging, and intratumor fluores-cent tracking. All experiments involving animals were donein accordance with the guidelines of the Institutional AnimalCare and Use Committee. See Supplementary Data for de-tailed labeling and imaging method.NIR light–activated p65 RNAi in vivo. Nude mice

(Charles River) were s.c. inoculated with 2.5 × 106 HeLa cellsin both sides of rear legs 10 d before the experiment. Tumor-bearing mice were randomly allocated into four groups (n = 6).Mice in group A were i.v. injected with F-PEG-HAuNS-siRNA (0.25 μmol/kg siRNA), group B with PEG-HAuNS-siRNA(0.25 μmol/kg siRNA), group C with F-PEG-HAuNS-siRNAluc

(0.25 μmol/kg siRNAluc), and group D with saline. After 6 h,the tumor in the left leg of each mouse was irradiated withNIR laser (50 mW/cm2) for 60 s. Following another 48 h, themice were sacrificed. Major organs, such as liver, spleen, kid-ney, lung, and tumors, from three mice of each group were dis-sected for frozen sectioning. The two adjacent slices receivedp65 and H&E staining, respectively. For p65 immunohisto-chemistry, the slices were stained with rabbit anti-p65 poly-clonal antibody (1:100; Santa Cruz Biotechnology) followedby IRDye800CW goat anti-rabbit IgG (1:800; Li-Cor). Cell nucleiwere counterstained with 4′,6-diamidino-2-phenylindole(DAPI). The whole tissues were scanned under an Odyssey im-aging system (Li-Cor) and visualized under fluorescence

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Silencing of NF-κB p65 by Photothermal Transfection

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microscope (Zeiss Axio Observer.Z1, Carl Zeiss MicroImagingGmbH) at highmagnification. The tumors from the other threemice of each group were removed for p65 Western blot analy-sis. The fluorescence was scanned under Odyssey imaging sys-tem with intensity analysis.Chemosensitivity to irinotecan in vivo. Nude mice were

s.c. inoculated with 2.5 × 106 HeLa cells in right rear legs. Forhistologic study, tumor-bearing mice were randomly allocat-ed into four groups (n = 5) on day 10 when tumors reached0.4 to 0.6 cm in average diameter and received the followingtreatment. Mice in groups A and B were i.v. injected withF-PEG-HAuNS-siRNA (0.25 μmol/kg siRNA), group C withF-PEG-HAuNS-siRNAluc (0.25 μmol/kg siRNAluc), and groupD with saline. After 6 h, mice in groups A and C were irradi-ated with NIR laser (50 mW/cm2) for 60 s. On days 11 and 13,all mice received 0.25 mL (4 mg/mL) of i.p. irinotecan. Micewere sacrificed on day 17, and the tumors were removed.Two adjacent slices were analyzed by H&E and terminaldeoxynucleotidyl transferase–mediated dUTP nick end label-ing (TUNEL; R&D Systems) staining, respectively. For tumorgrowth delay study, the tumors were measured every 2 or 3 dstarting on day 10 (n = 8–10). The largest and shortest di-mensions of each tumor were measured using a caliper.The tumor size was calculated as the average diameter ofthe two dimensions. Mice with tumors >1.2 cm were sacri-ficed. An additional group of tumor-bearing mice withoutany treatment served as control.

Results

NIR light–triggered decoupling of siRNA from HAuNS.Scheme for the bioconjugation of HAuNS-siRNA was illus-trated in Fig. 1A. After irradiation with a pulsed NIR laserlight (800 nm) at different output powers, the absorptionspectra of the HAuNS-siRNA solutions dramatically changed.The solution containing HAuNS-siRNA showed an intenseabsorption peak ∼800 nm before the laser irradiation. How-ever, the solution of laser-irradiated nanoconstructs dis-played significantly reduced SPR absorbance as well asconsiderable blueshift in a dose- and time-dependent man-ner (Fig. 1B). At a dose of 76 mW/cm2 for 60 seconds, theSPR band in the NIR region completely disappeared, whereasan absorption peak at ∼520 nm resembling a SPR band ofspherical solid gold nanoparticles appeared (Fig. 1B, left).The spectral change indicates that HAuNS lost its structuralintegrity on NIR irradiation, possibly collapsing into solidgold nanoparticles. The structural transformation of HAuNSwas confirmed by transmission electron microscopy (TEM)(Supplementary Fig. S2). The phenomenon of shape changeas a result of NIR laser irradiation was also observed in othergold-based nanostructures (12–14, 25, 26).Structural change on NIR light irradiation was accompa-

nied by dissociation of siRNA from HAuNS. TEM images ofuranyl acetate–stained HAuNS-siRNA clearly showed that thesiRNA molecules were attached to the surface of the HAuNS,forming a “corona” (Fig. 1C, middle left). On laser irradiationat 50 or 76 mW/cm2 for 60 seconds, most of the coronasiRNA layer was lost (Fig. 1C, two right graphs), suggesting

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detachment of siRNA from HAuNS. The siRNA release fromHAuNS-siRNA was further confirmed by analyzing surfaceatomic composition using X-ray photoelectron spectroscopy(XPS). Untreated HAuNS-siRNA exhibited substantial surfaceN and P atoms (5.5% and 2.0%), owing to the presence of nu-cleotides and phosphates of siRNA molecules. However, thesurface N and P contents drastically decreased to 0.2% and0.05%, respectively, after NIR laser irradiation (Fig. 1D; Sup-plementary Table S1). Moreover, PAGE analysis revealed therelease of siRNA from F-PEG-HAuNS-siRNA only when thenanoparticles were irradiated with NIR laser (SupplementaryFig. S3). The released siRNA exhibited mobility similar to thatof free siRNA, suggesting that the siRNA was intact afterirradiation. A possible explanation for NIR laser–triggeredsiRNA release is the Au-S bond breakage as a result of thelocal temperature elevation on absorption of NIR light byHAuNS (Fig. 1A; refs. 18–20). A similar mechanism was be-lieved to operate in NIR laser–triggered release of plasmidDNA from gold nanorod–plasmid DNA conjugates (13, 14).NIR light–activated endolysosomal escape of HAuNS-

siRNA through disruption of endolysosomal membrane.We next constructed folate-targeted F-PEG-HAuNS-siRNAfor their intracellular trafficking following NIR laser irradia-tion (Fig. 2A). To track the intracellular distribution of siRNAmolecules, the 3′ end of the sense chain of siRNA was labeledwith Dy547. F-PEG-HAuNS-siRNA was selectively taken up byHeLa cells that express high levels of folate receptors. Littlefluorescent signal was observed in HeLa cells treated withnontargeted PEG-HAuNS-siRNA or free siRNA or in receptor-negative A549 cells treated with F-PEG-HAuNS-siRNA(Supplementary Fig. S4). Before NIR light irradiation, thefluorescent signal from siRNA in HeLa cells treated withF-PEG-HAuNS-siRNA colocalized with endolysosomal com-partments stained with LysoTracker Green (Fig. 2B, left),indicating receptor-mediated endocytosis of F-PEG-HAuNS-siRNA (27). However, 15 minutes after laser irradiation(50 mW/cm2, 60 seconds) of F-PEG-HAuNS-siRNA–treatedHeLa cells, the red fluorescence from siRNA displayed amore diffused pattern (Fig. 2B, middle), suggesting endoly-sosomal escape of siRNA. Without laser treatment, only afew red fluorescent spots were found located outside theendolysosomes over the same period (Fig. 2B, right). Thisobservation is in accordance with the previous report thatfolate-conjugated gold nanoparticles were located primarilyin endocytic vesicles, with only a few particles entering thecytosol over a period of 6 hours (27).TEM images show that F-PEG-HAuNS-siRNA nanoparti-

cles were located in the endocytic vesicles (endosomes andlysosomes) with intact compartment membrane (Fig. 2C,top). Some particles remained attached to the rim of endocyticvesicles (Fig. 2C, top, arrows), suggesting that a high-affinitymembrane binding site still existed for the nanoparticles inthis compartment (27). However, in laser-treated group, theboundary of the endocytic vesicular membrane could not beclearly visualized (Fig. 2C, bottom, left, arrows). Imagesacquired at higher magnification (Fig. 2C, bottom, right threeimages) showed disruption of the endocytic vesicularmembrane, resulting in the escape of nanoparticles from the

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endolysosomes. The disruption of the endolysosomal com-partment membrane was also evidenced by the decrease inthe endolysosomal distribution of LysoTracker Green fluores-cence (Fig. 2B, middle).We further examined whether siRNA molecules were de-

tached from HAuNS within HeLa cells on NIR laser irradia-

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tion. For this purpose, the scattering signal from HAuNSnanoparticles was visualized under a dark field (18). Z-stackimages showed that Dy547-labeled siRNA colocalized withHAuNS in cells treated with F-PEG-HAuNS-siRNA (Fig. 2D,top, arrows). However, the fluorescent signal from siRNAand scattering signal from HAuNS were completely separated

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Figure 1. Photothermal-inducedsiRNA release. A, scheme forbioconjugation of HAuNS-siRNAand photothermal-inducedsiRNA release. B, absorptionspectra of the HAuNS-siRNAsolution before and after NIR laserirradiation (800 nm) at differentenergy power levels for 60 s (left)and at 50 mW/cm2 for differenttime periods (right). C, TEM imagesof uranyl acetate–stained HAuNSand HAuNS-siRNA before andafter NIR laser irradiation. Bottom,TEM images at a highermagnification. Arrow, integratedsiRNA layer present on the surfaceof HAuNS. Scale bar, 50 nm. D,XPS spectra of HAuNS-siRNAshowing the detachment of siRNAfrom HAuNS after NIR laserirradiation at 50 mW/cm2 for 60 s.

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Figure 2. Intracellular trafficking ofF-PEG-HAuNS-siRNA following NIR laserirradiation. A, scheme for the synthesisof F-PEG-HAuNS-siRNA and theirproposed intracellular itineraryfollowing NIR light irradiation. B,photothermal-induced endolysosomalescape of Dy547-labeled siRNA.Green, LysoTracker Green–labeledendolysosomes; red, Dy547-labeledsiRNA. Scale bar, 10 μm. C, TEM imagesof intracellular distribution ofF-PEG-HAuNS-siRNA with or withoutNIR light treatment. Arrows in the top panelindicate that the nanoparticles remainattached to the rim of endocytic vesicles.Arrows in the bottom panel representindiscernible membrane boundary of theendocytic vesicles. Higher-magnificationimages (bottom, right three images)show that some parts of the membrane ofendocytic vesicles disappeared, resultingin endolysosomal escape of thenanoparticles as depicted by the schemesbelow. D, Z-stack images showing thedissociation of siRNA from the HAuNSafter laser irradiation. Red, Dy547-labeledsiRNA; green, scattering signal ofHAuNS; blue, cell nuclei counterstainedwith DAPI. Arrows, siRNA colocalized withHAuNS. Scale bar, 10 μm.

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after NIR laser irradiation (Fig. 2D, bottom), confirming thedecoupling of siRNA from HAuNS by NIR light.HAuNS-induced efficient photothermal transfection and

RNAi in vitro. We investigated the effect of irradiation doseon cell viability to obtain an optimal laser dose for photo-thermal transfection study (Supplementary Fig. S5). The cellviability decreased with increasing laser output power andwith increasing exposure time. At an output power of50 mW/cm2, cellular damage was minimal when the irradia-tion time was 60 seconds. At this dose, photothermal-induced membrane disruption was limited to endolysosomes(Fig. 2C). Most F-PEG-HAuNS-siRNAluc–treated cells (∼87%)on this irradiation were impermeable to ethidium homodimer-1,suggesting intact plasma membrane integrity (SupplementaryFig. S5). At output powers >50 mW/cm2, significantly morecells were killed, possibly as a result of photothermal ablationmediated by HAuNS inside the cells (18, 19). Thus, 50mW/cm2

for 60 seconds was selected as a safe dose for further genesilencing experiments.Following photothermal transfection, the cellular expres-

sion of p65 was significantly inhibited (Fig. 3A, left). Thep65 silencing effect was time dependent, as the fluorescentintensity decreased by about 30%, 92%, 95%, and 84% at 24,48, 72, and 96 hours, respectively (Fig. 3A, right). Immunoflu-orescence staining further confirmed efficient silencing ofp65 on NIR light irradiation (Fig. 3B). Silencing of p65 expres-sion was observed only in HeLa cells treated with F-PEG-HAuNS-siRNA plus NIR laser irradiation but not in cells trea-ted with F-PEG-HAuNS-siRNA alone or in cells treated withnontargeted PEG-HAuNS-siRNA plus NIR laser. Moreover,siRNA-mediated p65 gene silencing significantly enhancedchemosensitivity of HeLa cells to irinotecan (Fig. 3C andD). The fraction of Annexin V–labeled apoptotic cells in-creased from 3.9% to 7.8% using various treatment schemesto 26.6% using a combined irinotecan and NIR light–triggered photothermal transfection protocol, representinga 3.4- to 6.8-fold increase in apoptotic response (Fig. 3D).These results indicate that receptor-mediated endocytosisof HAuNS is necessary but not sufficient for efficient genesilencing and that effective downregulation of p65 expressioncould be activated by NIR light through HAuNS-mediatedphotothermal transfection.

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NIR light–controllable tumor site–specific RNAi in vivo.The subnanometer size (∼40 nm in diameter), excellent col-loidal stability, and versatile surface chemistry open up pos-sibility for targeted delivery of HAuNS-siRNA in vivo forselective RNAi therapy (19, 28). Toward this end, we conju-gated 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-[acetic acid-N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-lipoicacid mono amide] (DOTA-LA) to F-PEG-HAuNS-siRNA,which allowed labeling with 64Cu (t1/2 = 12.7 hours) for pos-itron emission tomography (PET) imaging (SupplementaryFig. S6). Instant TLC analysis showed that the radiochemicalpurity of 64Cu-labeled F-PEG-HAuNS-siRNA [F-PEG-HAuNS-siRNA(DOTA-64Cu)] was >99%. After incubation in full mouseserum for 24 hours, the radiochemical purity of F-PEG-HAuNS-siRNA(DOTA-64Cu) remained >95% (SupplementaryFig. S7), indicating that the radiolabel on HAuNS was stable.Micro-PET/computed tomography (CT) imaging revealedthat the tumor uptake of F-PEG-HAuNS-siRNA(DOTA-64Cu)was significantly higher than that of nontargeted PEG-HAuNS-siRNA(DOTA-64Cu) at 6 hours following i.v. injectioninto nude mice bearing s.c. HeLa cervical cancer xenografts(Fig. 4A; Supplementary Videos S1 and S2). This was con-firmed by biodistribution study, which showed 4.7-fold highertumor uptake of F-PEG-HAuNS-siRNA(DOTA-64Cu) com-pared with PEG-HAuNS-siRNA(DOTA-64Cu) (5.26 ± 1.25%ID/g versus 1.11 ± 0.50% ID/g). Liver and spleen were the ma-jor organs taking up significant amount of both targeted andnontargeted nanoparticles (Fig. 4B).To track intratumor distribution of siRNA, we labeled

HeLa cells with cell lipid intercalating dye PKH67 (Sigma)in vitro before their inoculation (29). Mice bearing PKH67-labeled HeLa xenografts were i.v. injected with Dy547-labledHAuNS-siRNA conjugates. Z-stack images of tumor sectionsshowed significantly higher tumor uptake of Dye547-labeledF-PEG-HAuNS-siRNA than that of Dye547-labeled PEG-HAuNS-siRNA at 6 hours after injection (Fig. 4C). Further-more, whereas PEG-HAuNS-siRNA was distributed mainlyin the tumor interstitial fluid space, most F-PEG-HAuNS-siRNA colocalizedwith PKH67-labeled cellular lipids, indicatingthat targeted nanoparticles were internalized through folatereceptor–mediated endocytosis (Fig. 4C, arrowheads). Asshown in our in vitro study, cellular internalization of

Figure 3. Photothermal transfection of F-PEG-HAuNS-siRNA and enhanced chemosensitivity to irinotecan in HeLa cells. A, Western blot of p65 expressionin HeLa cells. Left, p65 expression at 48 h following different transfection procedures. M, molecular weight marker. Lane 1, cells without any treatment;lane 2, Lipofectamine 2000 plus siRNA; lane 3, free siRNA; lanes 4 and 5, PEG-HAuNS-siRNA and F-PEG-HAuNS-siRNAluc both plus NIR laserirradiation (50 mW/cm2 for 60 s); lanes 6 to 8, F-PEG-HAuNS-siRNA plus NIR laser irradiation (50 mW/cm2) for 60, 30, and 0 s; lane 9, F-PEG-HAuNS-siRNAplus NIR laser irradiation (32 mW/cm2 for 60 s). Right, p65 expression at different time periods after photothermal transfection (50 mW/cm2, 60 s)with F-PEG-HAuNS-siRNA (lanes 1, 3, 5, and 7) or with F-PEG-HAuNS-siRNAluc (lanes 2, 4, 6, and 8). Green, p65; red, β-actin. B, immunohistochemicalanalysis of p65 expression with different transfection procedures. Laser dose: 50 mW/cm2, 60 s. Control, cells without any treatment. Red, p65; green,cell nuclei counterstained with DAPI. Scale bar, 20 μm. C, cells preincubated with or without F-PEG-HAuNS-siRNA in the presence or absence of NIR laser(50 mW/cm2, 60 s) were treated with different concentrations of irinotecan for 24 h. Cell viability was plotted as a percentage of the nontreated cells.Points, mean (n = 3); bars, SD. D, apoptotic analysis of cells untreated or treated with F-PEG-HAuNS-siRNA or F-PEG-HAuNS-siRNAluc, NIR laser (50 mW/cm2, 60 s), in the presence or absence of irinotecan treatment (6 μmol/L). Cells were stained with PhiPhiLux G1D2 (caspase-3 substrate, green),Annexin V–allophycocyanin (Annexin V-APC; pseudo red), and propidium iodide (PI; pseudo blue). Top graph of each group, flow cytometry analysiswith Annexin V-APC/PI; bottom graph, immunofluorescent images merged with differential interference contrast images. Control, cells without anytreatment. Scale bar, 25 μm.

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HAuNS-siRNA is the prerequisite for effective photothermaltransfection.Following i.v. injection of F-PEG-HAuNS-siRNA, significant

downregulation of NF-κB p65 subunit was achieved only inthe HeLa xenografts irradiated with NIR laser but not in thecontralateral tumors grown in the same mice not exposed toNIR laser (Fig. 5A, top two rows, samples 1 and 2). This wasconfirmed by Western blot analysis of exercised tumors(Fig. 5B, samples 1 and 2). The fluorescent intensity ratioof p65 to β-actin from tumors irradiated with NIR laser wasreduced to 23% of that from the contralateral tumors not ex-posed to NIR laser (Fig. 5C, samples 1 and 2). There was nochange in the level of p65 expression in all other controlgroups, including tumors of mice injected with nontargetedPEG-HAuNS-siRNA and targeted F-PEG-HAuNS-siRNAluc con-jugatedwith a control siRNAluc, regardless whether the tumorswere irradiated with NIR light (Fig. 5). Liver, spleen, kidney,

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and lung did not show significant downregulation of p65,in spite of significant uptake of the siRNA nanoparticles inthese organs (Supplementary Fig. S8). Histologic examinationof tumors showed that no significant ablation of tumor cellsoccurred at the laser dose applied (50 mW/cm2, 60 seconds;Fig. 5A, bottom two rows).Finally, combined treatment with irinotecan and F-PEG-

HAuNS-siRNA directed at NF-κB p65 in the presence ofNIR light induced substantially enhanced apoptotic responseto HeLa cells in vivo compared with tumors of mice treatedwith irinotecan alone, irinotecan plus F-PEG-HAuNS-siRNAbut without NIR laser exposure, or irinotecan plus a controlsiRNAluc nanoconstruct (F-PEG-HAuNS-siRNAluc) in thepresence of NIR light (Fig. 6A). Irinotecan treatment withthe combination of p65 siRNA photothermal transfectionconsequently led to a significant tumor growth delay com-pared with other treatment groups (Fig. 6B).

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Figure 4. Tumor targeting ofF-PEG-HAuNS-siRNA directed atfolate receptor. A, micro-PET/CTimaging of nude mice bearingHeLa cervical cancer xenograftsin right rear leg 6 h after i.v.injection of F-PEG-HAuNS-siRNA(DOTA-64Cu) or PEG-HAuNS-siRNA(DOTA-64Cu). Arrowheads,tumors. B, biodistribution of F-PEG-HAuNS-siRNA(DOTA-64Cu) andPEG-HAuNS-siRNA(DOTA-64Cu)6 h following injection. Data wereplotted as percentage ofinjected dose per gram of tissue(%ID/g). Columns, mean (n = 5);bars, SD. *, P < 0.01. C, Z-stackimages of tumor sections frommicebearing PKH67-labeled HeLaxenografts 6 h after i.v. injection ofDy547-labeled F-PEG-HAuNS-siRNA or PEG-HAuNS-siRNA. Blue,cell nuclei stained with DAPI; green,PKH67-labeled HeLa cell lipids;red, Dy547-labeled siRNA.Arrowheads, colocalization ofsiRNA with cellular lipids, indicatingthe intracellular distribution ofsiRNA. Scale bar, 20 μm.

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Silencing of NF-κB p65 by Photothermal Transfection

Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-3379

Discussion

Our current work showed NIR light–induced tumor site–specific downregulation of NF-κB p65 expression in nude

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mice bearing HeLa cervical cancer xenografts based onphotothermal transfection technology. Success of this site-specific RNAi relies on targeted delivery of HAuNS-siRNAnanoconstructs into tumor cells as well as HAuNS-mediated

Figure 5. NIR light–controllable site-specific p65 RNAi in HeLa xenografts. Tumor-bearing mice were randomly allocated into four groups and receiveddifferent treatments as described in Materials and Methods. The tumor samples were numerated from 1 to 8. L, tumor in left rear leg; R, tumor inright rear leg. Tumor samples 1 and 2 were from the same mouse in group A, samples 3 and 4 from the same mouse in group B, samples 5 and 6 fromthe same mouse in group C, and samples 7 and 8 from the same mouse in group D. A, representative micrographs from each sample showing p65expression and histology. Top two rows, immunofluorescent staining of p65. Green, NF-κB p65 subunit; blue, cell nuclei. Bottom two rows, H&E staining.Scale bars, 1 mm (first row), 20 μm (second row), 1 mm (third row), and 50 μm (fourth row). B, Western blot of p65 expression in HeLa xenograftsfrom samples 1 to 8. Green, p65; red, β-actin. C, quantitative analysis of fluorescent intensities as the ratio of p65 to β-actin (n = 3).

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photothermal effect. HAuNS have the desired combinationof optimal plasmon absorption tunable in the NIR region(∼800 nm), small size (40–50 nm), and spherical shape ca-pable of efficient delivery of siRNA to cancer cells (19, 30).Smaller pegylated gold nanoparticles display less uptake by

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the cells of the reticuloendothelial system and longer circu-lation time in the blood than gold nanoparticles of largersizes (28). Tagged with folic acid, HAuNS as siRNA carriershad a better chance of achieving active in vivo targetingthrough folate receptor–mediated endocytosis (Fig. 4). The

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Figure 6. Effect of p65 siRNA photothermaltransfection combined with irinotecan onnude mice bearing HeLa cancer xenografts.Mice received various treatments asdescribed in Materials and Methods. A, red,representative micrographic images oftumors stained with H&E and TUNEL. Scalebars, 1 mm (H&E), 1 mm (TUNEL; wholetissue), and 50 μm (TUNEL; enlargedimages). B, tumor size versus time curve.Points, mean (n = 8–10); bars, SD. Control,tumor-bearing mice did not receive anytreatment.

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Silencing of NF-κB p65 by Photothermal Transfection

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entrapment of the nanoparticles in the endolysosomal com-partments is critical for a high level of spatial and temporalcontrol over transfection. Selective endolysosomal escape ofthe nanoparticles is activated only when a population ofcells is irradiated with a NIR laser. Simultaneous shapetransformation of HAuNS and breakage of the Au-S bondon NIR irradiation lead to the release of free siRNA intocytosol and induction of RNAi-mediated gene silencing. Aproposed mechanism is shown schematically in Fig. 2A.The breakage of Au-S bond could be explained by thermalenergy transfer mechanism due to the localized absorptionof laser energy by HAuNS. Alternatively, the instantaneoustemperature increase of free electrons, called hot electrons,in HAuNS may result in Au-S bond breakage (14).As shown in Fig. 5, downregulation of p65 with F-PEG-

HAuNS-siRNA was achieved at a moderate laser dose thatwas not sufficient to cause photothermal ablation of cancercells. Moreover, the other major organs, including liver andspleen, did not show significant downregulation of p65, inspite of significant uptake of the siRNA nanoparticles inthese organs (Supplementary Fig. S8), implying that the re-lease of siRNA from F-PEG-HAuNS-siRNA in nontarget tis-sues not exposed to NIR light was insignificant. BecauseHAuNS are made of pure gold expected to be nontoxic(24), our photothermal transfection strategy could be appliedsafely and effectively in vivo to induce RNAi with high spatio-temporal control. As shown by enhanced chemosensitivityand apoptotic response to irinotecan both in vitro and in vivo(Figs. 3 and 6), the photothermal transfection technique maybe used in rational design of nanoscale delivery system forsuccessful chemotherapy. It also suggests a new methodolo-gy for targeting other key genes in cancer development withsiRNA-based therapeutics. Because NIR light can penetratedeep into tissues and can be delivered at predetermined site,and because many target siRNAs have overlapping/redun-dant features in biological systems, RNAi regulated by NIR

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laser irradiation should offer a significant advantage in avoid-ing unwanted side effect.A potential limitation of the photothermal transfection

technique is the possible release of F-PEG or PEG into thecytosol on NIR laser irradiation. Although PEG is generallyconsidered as a biocompatible material, its side effect afterintracellular release should be carefully examined in futurestudies. In an ideal carrier system, there should be no releaseof both the PEG and the siRNA from the surface of HAuNSwithout NIR laser exposure and no PEG release while siRNAmolecules are released. Future studies toward the design ofNIR light–inducible siRNA delivery system should be directedat improving the selectivity of siRNA release by introducingsiRNA to the surface of gold via a more specific photolabilebond that is responsive only to NIR light.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Geng Ku (Washington University) for his help in the use ofNIR laser system, Markeda Wade for editing the manuscript, and KennethDunner for assisting in TEM studies.

Grant Support

NIH grant R01 CA119387 (C. Li) and John S. Dunn Foundation. The TEM,animal, and small animal imaging facilities are supported by the NIH throughM.D. Anderson’s Cancer Center Support Grant CA016672. XPS analysis wasperformed by Evans Analytical Group. 64Cu was provided by Washington Uni-versity Medical School, which was partially funded through National CancerInstitute grant R24 CA86307.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 09/11/2009; revised 12/18/2009; accepted 01/28/2010; publishedOnlineFirst 04/06/2010.

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