Visualizing Light-Triggered Release ofMolecules Inside Living CellsRyan Huschka,|,⊥,¶ Oara Neumann,†,⊥,¶ Aoune Barhoumi,|,⊥ and Naomi J. Halas*,†,‡,§,|,⊥

†Department of Electrical and Computer Engineering, ‡Department of Physics and Astronomy, §Department ofBioengineering, |Department of Chemistry, and ⊥Laboratory for Nanophotonics and the Rice Quantum Institute,Rice University, 6100 Main Street, Houston, Texas 77005

ABSTRACT The light-triggered release of deoxyribonucleic acid (DNA) from gold nanoparticle-based, plasmon resonant vectors, suchas nanoshells, shows great promise for gene delivery in living cells. Here we show that intracellular light-triggered release can beperformed on molecules that associate with the DNA in a DNA host-guest complex bound to nanoshells. DAPI (4′,6-diamidino-2-phenylindole), a bright blue fluorescent molecule that binds reversibly to double-stranded DNA, was chosen to visualize this intracellularlight-induced release process. Illumination of nanoshell-dsDNA-DAPI complexes at their plasmon resonance wavelength dehybridizesthe DNA, releasing the DAPI molecules within living cells, where they diffuse to the nucleus and associate with the cell’s endogenousDNA. The low laser power and irradiation times required for molecular release do not compromise cell viability. This highly controlledco-release of nonbiological molecules accompanying the oligonucleotides could have broad applications in the study of cellular processesand in the development of intracellular targeted therapies.

KEYWORDS Nanoshell, DAPI, controlled release, plasmon, DNA

Strategies for the directed release of controlled quantitiesof molecules inside living cells are in high demand fordrug delivery,1 gene therapy,2,3 and tissue engineering.4,5

The release mechanisms of most delivery vectors depend onprocesses such as diffusion, dissolution, chemical and en-zymatic reactions, or changes in various environmentalfactors such as temperature, pH, solvent, and ionic concentra-tions.2,6-9 For example, transfection reagents such as poly-ethylenimine act as a proton sponge following endocytosis,absorbing protons in the low-pH environment of the endo-some, causing it to swell and eventually rupture, facilitatinggene delivery.10 This type of environmental control of mo-lecular release varies with cellular location and cell type andcan result in unpredictable release. A physical release mech-anism that does not rely on the specific chemical propertiesof the cellular environment would be highly useful and moreeasily generalizable to various cell types.11 Light-inducedrelease is a particularly attractive option; the high spatial andtemporal control that lasers provide would be highly usefulfor initiating and following intracellular processes dynami-cally at the single cell level.12-19

Plasmonic nanoparticles, metal-based nanostructuressupporting collective electronic oscillations, are highly prom-ising potential candidates for facilitating controlled light-triggered release due to their large optical cross sections,their geometrically tunable optical resonances,20-22 andtheir strong photothermal response.23,24 Because of their

large cross sections and extremely low quantum yield,metallic nanoparticles convert optical energy to thermalenergy with high efficiency upon resonant optical illumina-tion.25,26 Resonant optical illumination of the nanoparticletriggers the controlled dehybridization and release of DNAmolecules adsorbed onto the nanoparticle surface.16,18 Goldnanoparticles are also biocompatible, easy to fabricate, andcan be functionalized with a wide variety of host-carriermolecules capable of noncovalent accommodation of guestmolecules.6,18

Au nanoshells, metallodielectric nanoparticles comprisedof a spherical dielectric core covered by a thin Au shell, havebeen successfully employed in a range of biomedical appli-cations27-29 including photothermal cancer therapy.24,30-33

The plasmon resonance wavelength of a nanoshell can bevaried by changing the core size and shell thickness.21,22,34

This tunability is particularly important for biomedical ap-plications because the nanoshell can easily be designed tohave a large absorption efficiency in the near-infrared (NIR)water window (690-900 nm), where blood and tissue aremaximally transparent and light can penetrate tissue todepths of several inches.35

Recently, we demonstrated light-induced dehybridization ofdouble-stranded DNA (dsDNA) attached to Au nanoshells.18

Nanoshells of dimensions [r1, r2] ) [63, 78] nm with theplasmon resonance wavelength at 800 nm were coated withdsDNA, where one strand of the DNA had a thiol moiety onits 5′ end, facilitating covalent attachment to the nanoshellsurface by a Au-thiol bond. The complement DNA sequencewas nonthiolated, and therefore bound only to its comple-mentary DNA sequence and not to the nanoparticle surface.Upon illumination with NIR light at the nanoshell plasmon

* To whom correspondence should be addressed. Phone: (+) 1-(713) 348-5612.Fax: (+) 1-(713) 348-5686. E-mail: halas@rice.edu.¶ These authors contributed equally to this work.Received for review: 06/30/2010Published on Web: 09/21/2010

pubs.acs.org/NanoLett

© 2010 American Chemical Society 4117 DOI: 10.1021/nl102293b | Nano Lett. 2010, 10, 4117–4122

resonance wavelength, the dsDNA was dehybridized, releas-ing the nonthiolated antisense sequence. This process ishighly efficient, resulting in the dehybridization and releaseof nominally 50% of the DNA from the complexes uponillumination with no apparent temperature increase in thesolution ambient. DNA antisense therapy has been exploredextensively as a class of gene therapy and has highlypromising potential to provide safe and effective treatmentsfor a multitude of diseases and genetic disorders.36

Here we show that in addition to light-controlled releaseof DNA the nanoshell-dsDNA complex serves as an effectivehost and light-triggered release vector for other types ofmolecules. Many types of guest molecules can associate withdsDNA, either by intercalating between adjacent base pairsor by binding in either the major or minor groove of the DNAdouble helix.37 The driving forces for association can includeπ-stacking, hydrogen bonding, van der Waals forces, hydro-phobic and polar interactions, and electrostatic attractions;therefore, dsDNA can host a large variety of guest moleculesvia noncovalent bonds.37,38 DAPI (4′,6-diamidino-2-phe-nylindole), a water-soluble blue fluorescent dye that bindsreversibly with dsDNA (Supporting Information Figure S4),is the molecule we chose to deliver to demonstrate andclearly visualize the light-induced intracellular release. DAPIwas chosen because of its bright fluorescent properties,stability, and negligible toxicity.39 DAPI binds preferentiallyto the minor grooves of dsDNA; its association with DNAcauses a large increase in its quantum yield.39-43 Theselectivity of DAPI to dsDNA makes it a frequently used,standard stain for cell nuclei in fluorescence microscopy.42,43

A schematic of the light-triggered molecular release isshown in Figure 1. Initially, nanoshell-dsDNA complexeswere loaded with DAPI by incubation of DAPI with thenanoshell-dsDNA complexes (see Supporting Information).Next, the nanoshell-dsDNA-DAPI complexes were incubatedwith H1299 lung cancer cells (see Supporting Information),where intracellular uptake was verified using both dark-fieldand bright-field microscopy. Upon illumination with an 800nm CW laser, corresponding to the peak resonant wave-length of the nanoshell complexes (Supporting Information-Figure S3), the DAPI molecules were released from thenanoshell complexes. Subsequent to release, the DAPI dif-fused through the cytoplasm and into the cell nucleus, whereit preferentially bound and stained the nuclear DNA. To thebest of our knowledge, this is the first light-controlleddelivery system that can be tailored to release quantifiableamounts of nonbiological molecules within living cells byremote means on demand.

The DAPI fluorescence emission intensity drasticallyincreases as a result of DAPI molecules binding to DNA(Figure 1b). As an isolated molecule, DAPI has a low quan-tum yield (Figure 1b, i),40 however, when DAPI is attachedto single-stranded DNA (ssDNA) (Figure 1b, ii), a weakelectrostatic attraction binds the cationic DAPI molecules tothe negatively charged phosphate backbone of the DNA,

resulting in a slight increase in its fluorescence intensity.40

When the DAPI molecules bind to the minor grooves of thedsDNA (Figure 1b, iii),40,43 the increased rigidity and stabi-lization significantly increases its quantum yield.39 DAPIbinding to the dsDNA also displaces H2O molecules initiallysolvating the DNA oligomers, significantly reducing inter-molecular proton transfer between H2O and DAPI, resultingin an additional increase of DAPI fluorescence intensity.40,41,44

(Additional characterization of DAPI-DNA binding is pro-vided in the Supporting Information, Figure S1).

The specific base-pair composition of the dsDNA playsan important role in determining the number of DAPImolecules that will bind to the dsDNA.45 Previous studieshave shown that DAPI preferentially binds to regions richwith adenine (A) and thymine (T) nucleotide bases becauseDAPI forms hydrogen bonds with A-T bases pairs.43 TheDAPI molecule is 14-15 Å long, corresponding to an overlapof three base pairs.40-42,45 In our experiments, since it isdesirable to bind as many DAPI molecules as possible toimprove the staining of the nucleus after light-inducedrelease, we designed a 26-base pair sequence with multipleA-T-rich regions with segments of three or more consecutiveA-T base pairs to specifically enhance DAPI loading (FigureS2, Supporting Information).

FIGURE 1. Light-induced DAPI release. (a) Schematic diagram of thelight-induced DAPI release and diffusion inside the cell. (b) Fluo-rescence emission of (i) DAPI only, (ii) DAPI with ssDNA, and (iii)DAPI with dsDNA.

© 2010 American Chemical Society 4118 DOI: 10.1021/nl102293b | Nano Lett. 2010, 10, 4117-–4122

To use this light-triggerable complex for molecular releasein live cells, the complex must first be effectively taken upby the cells of interest. To facilitate cell uptake, the nanoshell-dsDNA-DAPI complexes were incubated with H1299 lungcancer cells in serum containing cell culture medium for 1 h.After incubation, the cells were fixed and internalization ofthe nanoshell-dsDNA-DAPI complexes was imaged usingboth dark field (Figure 2a,b) and bright-field (Figure 2c)microscopy. Nanoshells in this size range both absorb andscatter light; their strong scattering cross section enablesthem to be easily visualized by optical microscopy. In Figure2a, H1299 cells with their cell membranes marked by thegreen fluorescence dye Alexa Fluor 488 WGA (wheat germagglutinin) are shown. Internalized nanoshells are easilyseen as diffraction-limited bright spots in this image. As acontrol, cells not incubated with the nanoshell-dsDNA-DAPIcomplexes showed no observable bright spots when imagedin the same manner (Figure 2B).

Because the dark-field images are two-dimensional, theseimages alone do not give clear evidence whether thenanoshell complexes have been endocytosed or are merelyadsorbed onto the outer membrane of the cell. Bright-fieldimaging was used to further investigate cellular uptake.Obtaining images at varying depths of field within anindividual cell allows us to clearly visualize in three dimen-sions the nanoshell distribution within the cell. Figure 2c isa slice from the middle of the cell showing clear diffraction-limited dark spots corresponding to nanoshell complexes,verifying that the nanoparticles are internalized within thecell. A video sequence of two-dimensional projections ob-tained as the depth of field is scanned through the cell

resolves the 3D distribution of nanoshell complexes (pro-vided in the Supporting Information). Internalization ofnanoshells is in agreement with observations by Ochsen-kuhn et al., who used TEM sections of NIH-3T3 fibroblastcells to confirm nanoshell uptake.46

At first thought, it is surprising that the nanoshell-dsDNA-DAPI complex is internalized into cells because the nega-tively charged phosphate backbone on the DNA shouldexperience electrostatic repulsions with the negativelycharged cell membrane.47 However, previous studies byChithrani et al. and Giljohann et al. suggest that Au nano-particles functionalized both with and without DNA adsorbextracellular serum proteins from the cell culture media.47,48

The adsorbed extracellular proteins then interact with thecell membrane and facilitate cellular uptake in an adsorptiveendocytosis pathway. Conversely, recent studies by Ochsen-kuhn et al. show that nanoshell uptake increases in theabsence of extracellular proteins, suggesting the possibilityof a passive, nonendocytotic uptake mechanism.46 While inour studies nanoshell complex uptake is clearly visualizablein H1299 cells, the precise uptake mechanism is not clearlyidentifiable and is likely to depend on a variety of factorsincluding cell type, functionalization of the nanoparticle, andincubation conditions.

To investigate intracellular light-induced molecular re-lease, the H1299 cells incubated with nanoshell-dsDNA-DAPIcomplexes were illuminated with a NIR CW laser (1 W/cm2,800 nm) for 5 min. This irradiation time and laser powerlevel were determined from previous experiments,18 whichdemonstrated after 5 min of laser irradiation that no ad-ditional dehybridization of the DNA occurred. This laserpower and time allow the DAPI to be released while mini-mizing laser exposure to the H1299 cells. After laser irradia-tion, samples were placed in an incubator for one hour toallow time for released DAPI molecules to diffuse to thenucleus. Next, the nuclei of the cells were isolated by lysingthe cell membrane (see Supporting Information) and theDAPI fluorescence intensity was quantified by flow cytom-etry. Nuclei isolation is necessary to ensure that flow cytom-etry only measures fluorescence from DAPI molecules boundto genomic DNA in the nucleus and does not measurefluorescence from DAPI molecules in the cytoplasm.

Evidence of DAPI release is shown by the normalized flowcytometry histograms of DAPI fluorescence intensity versusnumber of nuclei from H1299 cells incubated with nanoshell-dsDNA-DAPI before and after laser treatment (Figure 3).After laser treatment, the fluorescence intensity of the nucleiincreased, demonstrating that DAPI molecules were releasedfrom the nanoshells, diffused through the cytoplasm and intothe cell nuclei, binding with the genomic DNA. Prior to laserirradiation, some DAPI fluorescence is observed within thecells (Figure 3c, left) and measured by flow cytometry (Figure3a,b, before laser). This DAPI fluorescence signal originatesfrom both excess DAPI molecules present in the sample and

FIGURE 2. Nanoshell-dsDNA-DAPI cell uptake. Dark field/epifluo-rescence images of (a) H1299 lung cancer cells incubated withnanoshell-dsDNA-DAPI complexes, (b) nonincubated cells (control).(c) Bright-field image of middle slice of H1299 lung cancer cellsincubated with nanoshell-dsDNA-DAPI complex.

© 2010 American Chemical Society 4119 DOI: 10.1021/nl102293b | Nano Lett. 2010, 10, 4117-–4122

DAPI molecules that were noncontrollably released from thecomplexes during the incubation and prior to laser irradiation.

The bar graph depicts the mean DAPI fluorescence intensity(SEM (standard error of the mean) increase from before laser(59.7( 0.21) to after laser (79.9( 0.33) (Figure 3a) (an ∼33%increase in fluorescence intensity.) An unpaired t test of the twomeans was performed at a 95% confidence level, whichresulted in a two-tailed p value of p < 0.0001, which is statisti-cally significant. This observed increase in DAPI fluorescenceafter laser treatment (∼33%) demonstrates that the nanoshell-dsDNA complex effectively released its guest molecules fromthe dsDNA host carriers inside the cells. Epifluorescence imagesof H1299 cells incubated with nanoshell-dsDNA-DAPI before

(Figure 3c, left) and after (Figure 3c, right) laser treatmentvisually show the increase in DAPI fluorescence intensity. Thecell membrane is marked by the green dye, Alexa-Fluor 488wheat germ agglutinin.

The plasmon resonant illumination of the nanoshells iscrucial for DAPI release into the cells. To test this hypothesis, acontrol experiment consisting of H1299 cells incubated withDAPI only (no nanoshells) was conducted (Figure 3b). The cellswere irradiated with the NIR laser under conditions identicalto the previous experiment. The mean DAPI fluorescenceintensity(SEM did not significantly increase after laser irradia-tion (237 ( 0.86 to 239 ( 0.95, p ) 0.1188), indicating thatDAPI release does not occur without the presence of the

FIGURE 3. Light-induced DAPI release. (a,b) Flow cytometry histograms of DAPI Fluorescence (Ex, 355 nm; Em, 460 nm) versus number ofisolated nuclei from H1299 cells incubated with (a) nanoshell-dsDNA-DAPI and (b) DAPI (control). Negative control (gray), treated cells withoutlaser irradiation (blue) and treated cells with laser irradiation (red). Bar graphs display the mean DAPI fluorescence intensity (SEM beforeand after laser irradiation. (c) Epifluorescence images of H1299 cells incubated with nanoshell-dsDNA-DAPI (left) before and (right) after lasertreatment. The cell membrane is marked by the green dye, Alexa-Fluor 488.

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nanoshell-dsDNA complex. It is important to note that themean fluorescence intensity is higher for the control (Figure 3b)compared to the nanoshell-dsDNA-DAPI sample (Figure 3a) dueto multiple washings of the nanoshell-dsDNA-DAPI sample.

The shape of the flow cytometry histograms for both beforelaser and after laser are consistent with nuclei stained withDAPI.49,50 DAPI is routinely used to study the cell cycle becauseit binds to DNA stoichiometrically. Looking at the before laserhistogram in Figure 3a as an example, the tallest peak (∼40)originates from nuclei with two sets of chromosomes. This peakis the tallest because in a typical cell cycle, a cell spends thelongest portion of time with two sets of chromosomes; there-fore, the probability of a cell having two sets of chromosomesis the highest. The second, smaller peak (∼80), double thefluorescence intensity of the tallest peak, indicates nuclei thathave exactly double the amount of DNA, four sets of chromo-somes, and are ready to enter mitosis and divide. The nucleiwith fluorescence intensities in between these two peaksindicate cells that are currently synthesizing DNA prior tomitosis. The negative control histogram (gray) has a single peakbecause in the absence of DAPI every nuclei essentially fluo-resces identically resulting in a signal that is attributed toautofluorescence (background).

To ensure that this method for light-triggered intracellularmolecular release would be useful for biomedical applications,such as drug delivery, a cytotoxicity assay was performed toinvestigate both the effects of nanoshells and laser irradiationon cell viability. Propidium iodide (PI) was chosen as a markerto distinguish viable from nonviable cells, because it is amembrane-impermeable dye that is excluded from viablehealthy cells.51 When a cell membrane is damaged, PI entersthe cell, stains the dsDNA in the nuclei, and emits red fluores-cence; however, undamaged cells will not fluoresce. Flowcytometry was used to observe changes in PI fluorescenceintensity for a large sample size of 30 000 cells. The negativecontrol (Figure 4a) consisted of cells that were not incubatedwith nanoshell-dsDNA-DAPI and did not undergo laser treat-ment. The fluorescence observed in Figure 4a is attributed toautofluorescence and PI staining caused by apoptotic andnecrotic cells already present in the experiment with damagedmembranes. The nanoshell-dsDNA-DAPI complexes were thenincubated with H1299 cells for 12 h. Following incubation, thecells were divided into two samples: cells not treated with thelaser (Figure 4b) and cells treated with the laser for 10 min(Figure 4c). Figure 4b shows no significant increase in PIfluorescence intensity, demonstrating that nanoshell-dsDNA-DAPI complexes are not cytotoxic under the experimentalconditions of the study.

More interestingly, cells incubated with nanoshell-dsDNA-DAPI complexes and irradiated with the laser for 10 min alsoshow no significant increase in PI fluorescence intensity. Thisdemonstrates that the light-triggered release procedure did notadversely affect the cells. Considering nanoshells are well-known for their use in photothermal therapy, this result maybe surprising; however, the illumination conditions for this

experiment (1W/cm2, 5 min) were significantly below thoseused for photothermal induction of cell death in cell culture(4W/cm2, 4-6 min).24 Figure 4d represents a positive controlsample of cells treated with 0.1% Citrate/0.1% Triton solution,which permeates the cell membrane, allowing PI to enter thecell and stain the nucleus, resulting in a large increase in PIfluorescence intensity.

In conclusion, nanoshells functionalized with dsDNA weresuccessfully used to transport DAPI molecules into living cells.Successful uptake of nanoshells into H1299 cells was achieved.DAPI molecules, initially bound to the dsDNA on the NS surface,are released due to the illumination of the nanoshell-dsDNA-DAPI complex with the appropriate NIR light. DAPI moleculesinitially released in the cell cytoplasm diffuse into the cellnucleus and bind to the genomic DNA of the cell. The stainingof the cell nucleus with the released DAPI was quantified usingflowcytometry.Acytotoxicityassaydemonstratedthatnanoshelluptake is nontoxic and that laser irradiation of nanoshell-ladencells under the conditions where DAPI release occurs does notinduce cell death.

This nanoshell-dsDNA system could be extended to a mul-titude of other guest molecules that associate with the hostdsDNA carrier including small organic fluorophores,38 steroidhormones,52 and therapeutic molecules.37,38,52 For example,the quest to find dsDNA intercalators that inhibit the uncontrol-lable replication of tumor cells comprises an entire field ofcancer research. Currently, there are more than 130 FDAapproved anticancer drugs that specifically target DNA.53 Forin vivo clinical applications, however, before the DNA interca-

FIGURE 4. Flow cytometry cytotoxicity assay. All plots are propidiumiodide (PI) fluorescence intensity versus side-scattered light. (a)Negative control: H1299 cells not incubated with nanoshell-dsDNA-DAPI and no laser treatment. Cells incubated with nanoshell-dsDNA-DAPI for 12 h: (b) without laser treatment and (c) with lasertreatment. (d) Positive control: cells were treated with 0.1% Citrate/0.1% Triton, which permeates the cell membrane, allowing PI tostain the dsDNA in the nucleus.

© 2010 American Chemical Society 4121 DOI: 10.1021/nl102293b | Nano Lett. 2010, 10, 4117-–4122

lator can reach the genomic DNA, it must overcome severalhurdles, such as metabolic pathways and cytoplasmic andnuclear membranes. As a result, the failure of DNA therapiesto offer successful clinical treatments is primarily due a lack ofviable delivery methods rather than effectiveness of the DNAintercalator to treat cancer.37 This nanoshell-dsDNA deliveryvector preserves the guest molecule by minimizing nondesiredinteractions with other molecules and it provides light-triggeredrelease with controllable delivery.

Acknowledgment. The work was supported by the RobertA. Welch Foundation (C-1220). We thank Dr. Lin Ji fromUniversity of Texas MD Anderson Cancer Center for help inproviding and growing H1299 lung cancer cells, KarenRamirez of the University of Texas MD Anderson Cancer,Center Flow Cytometry Facility NCI No. P30CA16672, WalterHittelman of the Department of Experimental TherapeuticsMD Anderson Cancer Center Epifluorescence MicroscopeFacility, Katherine Regan for help with bright-field images,Pernilla Wittung-Stafshede, Benjamin G. Janesko, and SurbhiLal for helpful discussions.

Supporting Information Available. Experimental details,sample preparation, instrumentation used, and experimen-tal results: CD, UV-vis absorbance spectra, fluorescence ofDAPI binding to DNA, base specificity of DAPI binding, andthe effect of buffers on DAPI fluorescence intensity areincluded. This material is available free of charge via theInternet at http://pubs.acs.org.

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