Remote activation of biomolecules in deeptissues using near-infrared-to-UVupconversion nanotransducersMuthu Kumara Gnanasammandhan Jayakumara, Niagara Muhammad Idrisa, and Yong Zhanga,b,1

aDepartment of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 117576; and bGraduate School for IntegrativeSciences and Engineering, National University of Singapore, Singapore 117456

Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved March 29, 2012 (received for review September 6, 2011)

Controlled activation or release of biomolecules is very crucial invarious biological applications. Controlling the activity of biomole-cules have been attempted by various means and controlling theactivity by light has gained popularity in the past decade. The ma-jor hurdle in this process is that photoactivable compounds mostlyrespond to UV radiation and not to visible or near-infrared (NIR)light. The use of UV irradiation is limited by its toxicity and verylow tissue penetration power. In this study, we report the exploita-tion of the potential of NIR-to-UV upconversion nanoparticles(UCNs), which act as nanotransducers to absorb NIR light havinghigh tissue penetration power and negligible phototoxicity andemit UV light locally, for photoactivation of caged compounds and,in particular, used for photo-controlled gene expression. Both ac-tivation and knockdown of GFP was performed in both solutionand cells, and patterned activation of GFP was achieved success-fully by using upconverted UV light produced by NIR-to-UV UCNs.In-depth photoactivation through tissue phantoms and in vivo ac-tivation of caged nucleic acids were also accomplished. The successof this methodology has defined a unique level in the field ofphoto-controlled activation and delivery of molecules.

siRNA ∣ mesoporous silica ∣ uncaging

Photocaging involves the caging of molecules of interest by alight-sensitive molecule which can then be destroyed by light

irradiation to make it functional. Various molecules, like pro-teins, peptides, nucleic acids, amino acids, and drugs, have beenphotocaged and delivered to the cells/animals, and photolysisis done in the area of interest, enabling activation of thesemolecules with very high spatial and temporal resolution (1–7).Unfortunately, most of these current photoactivable systems areonly suitable for in vitro applications because UV light necessaryfor the uncaging process is very harmful, besides having a poortissue penetration depth, which made it unacceptable for in vivoand clinical use. Near-infrared (NIR) light has the deepest tissuepenetration compared to visible and UV light (8, 9). It is also safeand is expected to cause minimal photodamage to the biologicalspecimen involved. However, to bridge this gap, a transducerwhich is capable of converting NIR light to UV light is required.Converting high-energy UV light to visible light and NIR lightis done by various materials; however, the opposite process wherelow-energy NIR light is converted to visible or UV light is re-stricted to few rare earth materials which show a unique propertycalled upconversion. These upconversion nanoparticles areusually made of host lattices of ceramic materials, such as LaF3,YF3, Y2O3, LaPO4, NaYF4 embedded with trivalent lanthanideions like Yb3þ, Er3þ, and Tm3þ and show a unique phenomenonof absorbing NIR light and emitting UV, visible, and NIR light(10). There are a few reports exploiting NIR-to-NIR and NIR-to-visible upconversion nanoparticles (UCNs) (11–14); however,the use of NIR-to-UV UCNs is minimally explored (15–17).Here, we have successfully demonstrated the use of NIR-to-UVupconversion nanoparticles as a transducer for activating cagednucleic acids in solution and in cells, and deep-tissue activation of

the same was tested using different approaches like tissue phan-toms and animal models, which altogether overcomes the currentlimitations of photo-controllable gene expression in particularand photo-controllable biomolecules in general. Nanocrystalsmade of sodium yttrium fluoride (NaYF4) and codoped withytterbium (Yb3þ) and thulium (Tm3þ), which can emit in theUV-visible region were used for this study. The nanocrystals arethen coated with thin layers of mesoporous silica as reportedpreviously by our group (11), and caged DNA/siRNA was loadedinto these mesopores, which enables delivery of higher payloadand protection of the nucleic acids from the harsh environment.This method also increases the loading efficiency of nucleic acidswhen compared to chemical crosslinking, which is hampered byunstable attachment and low attachment efficiency. UpconvertedUV light emitted from the NIR irradiated UCNs will act as aswitch to uncage the caged molecules, thereby rendering it fullyfunctional for exerting its effect in the host cells. Apart fromacting as a transducer to activate photocaged nucleic acids, themesoporous silica coating on the surface of the UCNs help in thedelivery of nucleic acids into the cells, protecting them fromharmful enzymatic environment which might cause the degrada-tion of nucleic acids. Lanthanide-based upconversion nanoparti-cles are also comparatively less toxic (18) and studies have provedthem to be safe for long-term usage in vivo (19), thus providing asafe approach for use in biological applications. All the afore-mentioned properties make NIR-to-UV UCNs ideal candidatesfor in-depth control of biomolecules in vitro and in vivo.

ResultsThe nanoparticle core of NaYF4 UCNs and their various coatingsis shown in Fig. 1. The nanoparticle core (Fig. 1B) is initiallycoated with an amorphous layer of silica (Fig. 1C) and then amesoporous layer of silica (Fig. 1 D and E) is coated onto thenanoparticles as shown by the transmission electron micrographswith an overall size of approximately 80 nm. The emission spec-trum of these nanoparticles can be easily tuned by varying theconcentrations of the lanthanides used (20, 21). Fig. 1 F–H showthe emission spectrum of NaYF4 nanocrystals doped with differ-ent concentrations of Yb, Er, and Tm and the emission variesfrom UV, visible, and NIR light, and they have been used for var-ious applications, depending on their emission. MonodisperseNaYF4∶Yb, Tm NIR-to-UV UCNs were synthesized by a proto-col as reported previously (11) and the fluorescence emissionspectrum of them is shown in Fig. 1H, and the Inset shows the

Author contributions: M.K.G.J. and Y.Z. designed research; M.K.G.J. and N.M.I. performedresearch; M.K.G.J. and Y.Z. analyzed data; and M.K.G.J. and Y.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: biezy@nus.edu.sg.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1114551109/-/DCSupplemental.

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total fluorescence of NIR-to-UV UCNs dispersed in water whenexcited by a 980-nm continuous wave (CW) NIR laser.

UCNs were used to prove that caged siRNAs and caged plas-mid DNA can be uncaged and activated using NIR-to-UVupcon-verted light. Various strategies have been undertaken for cagingnucleic acids with light-sensitive compounds (22) and caging with4,5-dimethoxy-2-nitroacetophenone (DMNPE) was found to beeffective in caging plasmid DNA and siRNA (7, 23). The prob-able site of attachment of DMNPE to the phosphate backboneof DNA was proposed by Haselton and coworkers (24) Detailedschematic on caging of these nucleic acids with the chemicalDMNPE and their uncaging with upconverted UV light emittedfrom the UCNs is as illustrated in Fig. 2A. Fig. 2 also illustratesthe transducing ability of UCNs to produce upconverted UVlight in deep tissues where direct UV irradiation cannot pene-trate. The emission peak at 350 nm coincides well with the wa-velength needed to uncage DMNPE from the nucleic acids whichmakes them ideal transducers for activation. After the cagingof nucleic acids, they are loaded into the mesopores of the UCNsby physical adsorption (Fig. 2B). The loading was checked by UVabsorbance spectrophotometry and was found to be more than70% for both DNA and siRNA. DNA loading was also confirmedby gel retardation assay as shown in Fig. S1. Typical release pro-file of DNA is given in Fig. S2 and the protection of DNA by themesopores of the UCNs against harsh biological conditions waschecked by the DNAse protection assay as shown in Fig. S3.

Admittedly though, UV light is known for its harmful effectsand may thus draw a parallel conjecture and possible concern onthe use of upconverted UV light emitted from excited NIR-to-UVUCNs in living organisms. This issue was therefore addressedhere by assessing the viability of cells that were exposed to up-converted UV light. B16-F0 cells incubated overnight with0.5 mg∕mL of NIR-to-UV UCNs and then irradiated with differ-ent light doses using a 980 nm CW NIR laser were examined forsigns of cytotoxicity 48 h post treatment using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium, inner salt] assay. Cells without the nanopar-ticles were also irradiated with the NIR laser and used as a controlto check the phototoxicity. As shown in Fig. 3A, no apparent re-duction in cell viability was detected in any of the cell sampleswith or without the nanoparticles. Lactate dehydrogenase (LDH)release and live/dead cell staining done on cells exposed toNIR-to-UV UCNs and exposed to NIR laser also showed similar

results (Fig. S4). The above studies show that both NIR-to-UVUCN transfection and NIR laser exposure are safe for the cellsand the upconverted UV light does not lower the cell viability,which might be attributed to the highly localized UV radiationproduced by the UCNs compared to conventional whole-cellirradiation. Furthermore, the phototoxicity of NIR light was alsoevaluated using single cell gel electrophoresis assay as shown inFig. 3B by assessing the extent of DNA damage after cells wereexposed to the NIR laser, UCNs, and UCNsþNIR laser and wasfound to be minimal. Hence, this result ascertained the safe useof NIR irradiation and the upconverted UV light produced byNIR-to-UV UCNs for photoactivation in living organisms.

Upon establishing the safe use of upconverted UV light, theefficacy of UCNs in producing sufficient such UV light to uncagenucleic acids in solution was next examined by absorbance spec-

Fig. 1. Characterization of UCNs. Schematic showing UCNcore and its silica coatings (A); transmission electron micro-graph (TEM) images of monodisperse NaYF4 (Yb20%,Er2%) UCNs (Scale bar, 100 nm) (B); with silica coating (Scalebar, 100 nm) (C); and mesoporous silica coating (Scale bar D,50 nm; Scale bar E, 25 nm) (D and E). Fluorescence emissionspectra of UCNs, NIR-to-VIS, NaYF4∶Yb 20%, Er 2% (F); NIR-to-NIR, NaYF4∶Yb 0.2%, Tm 0.02% (G); and NIR-to-UV,NaYF4∶Yb 25%, Tm 0.3% UCNs (H), under excitation byan NIR laser at 980 nm, and Inset shows the total fluores-cence of NIR-to-UV UCNs in a cuvette, excited by a980 nm NIR laser.

Fig. 2. Schematic illustration. Plasmid DNA and siRNA are caged withDMNPE and then uncaged by upconverted UV light from NIR-to-UV UCNs.Inset shows the penetration depth of UVand NIR light in the skin (A). Loadingof caged plasmid DNA/siRNA into the mesopores of UCNs (B).

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trophotometry (23). In Fig. 3C, DMNPE-caged siRNA revealed acharacteristic plateau at 355 nm, which confirms the attachmentof DMNPE caging groups to the siRNAmolecules. Upon irradia-tion with direct UV light for 12 min though, a significant drop inthe 355 nm peak was observed, resembling the spectra of native(uncaged) siRNA molecules. DMNPE-caged siRNA molecules,which were premixed with NIR-to-UV UCNs and then irradiatedwith a 980 nm NIR laser, also showed a similar shift in the trendof the spectra that is reminiscent of the uncaging phenomenon,thereby confirming the ability of NIR-to-UV UCNs to uncagesiRNA molecules. The ability of UCNs to activate photocagedsiRNA with and without loading into the mesopores was checkedby absorbance spectrophotometry (Fig. S5).

With the aim of assessing UCNs’ ability to photoactivate cagednucleic acids in an in vitro platform, experiments were performedto check if both gene expression and gene silencing can be regu-

lated by this setup in cells. First, caged plasmid DNA encodingGFP (test) and noncaged plasmid encoding enhanced greenfluorescent protein (pEGFP) (control) were separately loadedinto the mesoporous silica shell of NIR-to-UV UCNs and usedthem as a vehicle to deliver these plasmids into B16-F0 cells.UCNs endocytosed into the cells were then irradiated with a980 nm CWNIR laser for various doses. Cellular uptake of UCNswas checked by its blue emission as shown in Fig. S6. Absorptionof NIR light by the UCNs produced upconverted UV emissionwhich facilitates uncaging of the delivered caged plasmid DNA,thereby rendering them functional. Indeed, at 32 h post-NIRirradiation, successful expression of the encoded GFP was quan-titated using a scanning fluorescence microplate reader, as shownin Fig. 4B. Similar experiments were performed with NIR-to-UVUCNs loaded with DMNPE-caged GFP siRNA and noncagedGFP siRNA, which was used to transfect B16F0 cells pretrans-fected with EGFP plasmid. The cells were then exposed to980 nm CW NIR laser and the gene knockdown was checkedafter 32 h, as stated above, and shown in Fig. 4A.

To further show that this technique can activate genes withvery high spatial and temporal resolution, cells were transfectedwith NIR-to-UV UCNs loaded with photocaged plasmids, asmentioned previously. Instead of irradiating the whole well witha NIR laser, the NIR laser was shined through a stencil withthe pattern NUS for 12 min (Fig. 4C). The expression of GFPwas checked after 32 h using a confocal fluorescence microscope,and the pattern was visualized as shown in Fig. 4D. Live cellDAPI staining was also done to show the presence of cells andits confluence. Similar control experiments were performed,using caged GFP plasmids without NIR irradiation and uncagedGFP plasmids with NIR irradiation (Fig. S7 A and B).

The main advantage of this method to activate photocagednucleic acids in deep tissues was demonstrated in several ways.Photoactivation was tested using a tissue phantom model withsimilar optical properties to that of muscle tissues, by placingthe tissue phantom between the cells loaded with NIR-to-UVUCNs/caged EGFP and the laser, as shown in Fig. 5A. The cagedplasmids are activated only if the NIR laser penetrates throughthe tissue phantom and reaches the cell monolayer. For this pur-pose, agarose gel-based solid tissue phantoms were casted usingIntralipids as the scatterer and Indian ink as the absorber (25, 26).The absorbance data of the tissue phantom and the mouse tissuesamples are shown in Fig. 5B. The GFP expression results fromthe experiments with and without the tissue phantom were com-pared. The results showed that the UCNs inside the cells pro-duced upconverted UV light upon excitation by the NIR lightpenetrating into the tissue phantoms of different thickness, suchas 2 mm and 4 mm, and the caged plasmids were activated, asshown in Fig. 5C. This study showed that UCNs had the abilityto activate photocaged nucleic acids at depths and further experi-ments were designed to show the same in vivo. NIR-to-UVUCNsloaded with photocaged GFP plasmids were injected beneath theskin in the flank of Balb/C mice and given an interval of 32 h, foruptake and expression. Control mice received a saline injection.The mice were then placed on a petri dish, and the injected sitewas imaged using a confocal fluorescence microscope. Two-di-mensional and three-dimensional Z-stack images were obtainedto show the GFP expression and its persistence across the opticalsections Fig. S8 A and B). The mice were then euthanized, andthe skin tissue with the injected UCNs was harvested and fixed.Cryosectioning of the tissues was done and immunostained withanti-GFP antibody labeled with TRITC. Counterstaining wasdone with DAPI and imaged using a confocal fluorescence micro-scope as shown in Fig. S8C. In addition, cells transfected withNIR-to-UV UCNs with photocaged GFP plasmids were loadedinto a polydimethylsiloxane (PDMS) device and transplantedinto the flank of Balb/C mice under the skin and sutured. NIRirradiation was done to activate the cells in the device through

Fig. 3. Phototoxicity of NIR light and absorbance spectrophotometry ofDMNPE-caged siRNA. Viability of B16F0 cells without (black) and with (gray)NIR-to-UV UCNs exposed to various doses of 980 nm CW NIR laser (A). Meanpercentage of tail DNA in cells treated with NIR, UCNs, and UCNþ NIR laser,determined by single-cell gel electrophoresis assay (B). Absorbance spectro-photometry of native siRNA (black) DMNPE-caged siRNA before (green) andafter UV irradiation (violet) and DMNPE-caged siRNA mixed with NIR-to-UVUCNs and activated with NIR irradiation (red) (C).

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the skin (Fig. 5A). The device was then explanted after 48 h andex vivo imaging was performed, as shown in Fig. 5D. GFP expres-sion was checked using a confocal fluorescence microscope andquantitation was done using a microplate reader and the resultsare given in Fig. 5E.

DiscussionIn this study, a unique dimension to remote control of biomole-cules by light using NIR-to-UV UCNs has been added. We pro-vide proof-of-concept of our unique approach to activate photo-caged siRNAs and photocaged plasmid DNA using indirect UVlight emitted from the NIR-to-UV UCNs. Indeed, these NIR-to-UV UCNs are foreseen to overcome the drawback of currentsystems in using low penetrating UV light for activating photo-caged nucleic acids. The use of UCNs is therefore anticipated toincrease not only the depth of penetration but also its associatedtherapeutic efficiency rather significantly. Initial reports on ex-ploiting upconverted UV light for photoactivation shows preli-minary studies on the activation of the caged compound (3′5′-dicarboxymethoxy benzoin) attached to the surface of the UCNsand release of carboxylic acids in solution. Here, we used meso-porous silica-coated NIR-to-UV UCNs encapsulating DNA/siRNA molecules in the porous silica shell with an improvedbiocompatibility and increased payload capacity, thus offeringa more efficient loading and delivery of the DNA/siRNA cargomolecules. It is a unique report on such UCN-based delivery andphotoactivation of biomolecules (nucleic acids) in solution, cells,and animal models. The DNA and siRNA photocaged with aphotolabile DMNPE group was found to have a caging efficiencyof 2–3%, similar to that reported previously (24). It was also de-monstrated that these caged nucleic acids were efficiently loadedinto the mesopores of the porous silica shell by physical adsorp-tion, which is in agreement with recent reports elucidating themechanism of DNA adsorption onto mesoporous silica thatshows more efficient adsorption of DNA in mesoporous silica,as compared to solid silica (27). Additionally, incorporating themwithin the mesoporous silica shell gives the benefit of beingprotected from the surrounding harsh biological conditions, asassessed by DNAse protection assay. Sustained release of theloaded caged nucleic acids from the mesoporous silica-coatedUCNs for prolonged durations was also successfully demon-strated here. When used on cells, these UCNs were able to absorbNIR light with good tissue penetration and extremely low photo-

toxicity. No statistically significant difference in the percentageviability of cells exposed to different concentrations of NIR-to-UV UCNs was observed when compared to those that werenot exposed to the nanoparticles. Interestingly, though, in thosecells not treated with the nanoparticles, there was a mild butstatistically insignificant increase in their percentage cell viabilitywith increase in the duration of NIR irradiation, presumably dueto the photobiostimulation effects that NIR has on cells that isknown to increase their proliferation (28, 29). Besides the inci-dent NIR light, the upconverted UV produced by the irradiatednanoparticles was also found to be relatively safe for the cells,within the nanoparticle dosage and duration of NIR laser irradia-tion range used in the present work, as proved by the results fromthe MTS assay, LDH assay, live/dead cell staining and cometassay. Although the comet assay showed some extent of DNAdamage in cells treated with the UCN-based system, the damageis very minimal when compared to using direct UV light even atvery low doses (30). Moreover, the comet assay includes DNAdamage that is repairable by the cell machinery if given time,hence the actual extent to which these cells are affected is verylow as ascertained by other cytotoxicity tests. Besides the upcon-verted UV light, the nanoparticles’ visible emission can also beused for optional imaging capabilities with superior quality dueto UCNs’ inherent unique optical properties. The blue emissionat 450 nm was used to track the nanoparticle uptake by cells dur-ing their delivery and activation process with almost nil back-ground autofluorescence and extremely high photostability.

Experiments were performed to confirm the ability of NIR-to-UV UCNs in activating photocaged nucleic acids (DNA andsiRNA) in both solution cells. In fact, uncaging of DMNPE-caged siRNA in solution had demonstrated an efficiency thatwas comparable to those activated by conventional direct UVlight (23, 24). Nonetheless, a direct comparison on factors, suchas specific dose, duration of irradiation, and percentage restora-tion of activity between the samples photoactivated using directUV light and that using NIR light, cannot be made here becausethe intensity of upconverted UV is comparatively much lowerthan that from direct UV irradiation, mainly attributed to thesignificantly low quantum yield of the upconversion process(0.005–0.3%) (31). Hence, even when the power of both the NIRlight and direct UV light are kept identical, the intensity of UVlight eventually received by the cells is very different.

Fig. 4. Photoactivation and patterned activation ofcaged nucleic acids in cells. Total GFP fluorescenceintensity of B16-F0 cells after cell lysis. Cells are trans-fected with NIR-to-UV UCNs/caged GFP siRNA com-plex (gray) and with NIR-to-UV UCNs/noncagedGFP siRNA complex (black) and irradiated with differ-ent light doses using a 980 nm CW NIR laser whichcorresponds to 0, 4, 8, 12 min (I-IV) of irradiationto uncage GFP siRNA. GFP fluorescence is normalizedto control cells expressing GFP without any siRNAtreatment (A). Cells are transfected with NIR-to-UVUCNs/caged pEGFP complex (gray) and with NIR-to-UV UCNs/noncaged pEGFP complex (black) and irra-diated with different light doses using a 980 nm CWNIR laser, which corresponds to 0, 4, 8, 12 min (I-IV) ofirradiation to uncage pEGFP. GFP fluorescence is nor-malized to the fluorescence from normal cells with-out GFP transfection (B). *P < 0.05 versus 0 min NIRlaser exposure. Schematic of setup showing the sten-cil and the position of the laser for patterning of cellstransfected with caged pGFP. Inset shows the pattern(NUS) on the stencil (C). Composite image showingthe GFP fluorescence from three different wells(one letter in each well of a 96-well plate) (Scalebar, 200 μm) and live-cell DAPI staining of the sameto show the cell confluence (D).

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Additional experiments conducted in this study also revealedUCNs’ ability to efficiently photoactivate nucleic acids both whenthey were still in the nanoparticles and when they have been re-leased out of the particles’ porous shell. Indeed, this result sug-gests that UCNs can activate nucleic acids even after their releaseinto the cellular environment. Gene expression and knockdownof GFP were accomplished in cells by using caged EGFP plasmidsand caged GFP siRNA and the amount of protein expressed wascontrolled. Control experiments to check the influence of NIRirradiation on the variation of gene expression was also donein parallel by delivering noncaged DNA and siRNA. The NIR

laser power and the concentration of nanoparticles were keptconstant, but the light dosage was varied to bring about variousdegrees of uncaging. Significant control was achieved in both ex-pression and knockdown as shown by the reporter gene assayafter cell lysis and was again comparable to caging systems acti-vated by direct UV light (4, 7). There was a statistically significantincrease in GFP fluorescence for caged pEGFP delivery from672 J∕cm2 of NIR laser exposure when compared to nonirra-diated control, and there was a statistically significant decreasein GFP fluorescence for caged GFP siRNA delivery from1;344 J∕cm2 of NIR laser exposure when compared to nonirra-

Fig. 5. Deep-tissue photoactivation. Experimentalsetup showing the uncaging of photocaged genesin cells through a tissue phantom and in a microfab-ricated device in an animal model (A). UV-visible-NIRabsorbance of hind limb muscle tissue from furlessblack mouse of 3 mm thickness (⦁) and agarose geltissue phantoms of 2 mm (×) and 4 mm thickness(▪) (B). GFP expression in cells transfected withNIR-to-UV UCNs loaded with photocaged pEGFPand activated by an NIR laser through tissue phan-toms of different thickness. Bars show fluorescenceintensity of GFP in cells with (black) and without(gray) NIR irradiation. *P < 0.05 versus cells irra-diated with NIR laser without any tissue phantomin between (C). Ex vivo imaging shows GFP expres-sion in cells with and without NIR irradiation, respec-tively (Scale bar, 50 μm) (D), quantitativemeasurement of GFP fluorescence (E). *P < 0.05 ver-sus cells without NIR irradiation.

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diated control. In a similar experiment, patterned activation ofGFP was done to show the versatility of UCNs not only in acti-vating the photocaged nucleic acids but also to show the activa-tion with very high spatial and temporal resolution as shown bythe word NUS patterned by GFP expressing cells.

Finally, our main hypothesis, which highlights the necessity ofusing UCNs for photo-controllable gene expression is that NIR-to-UV UCNs can be used for activating photocaged nucleic acidsin deeper tissues compared to conventional systems. The first ser-ies of photoactivation studies were tested initially using agarose-based tissue phantoms with similar optical properties as muscletissue. The degree of activation reduces with increase in thicknessof the tissue but significant activation (more than 50%) was seeneven with a tissue phantom thickness of 0.4 cm. However, therewas a statistically significant difference in GFP fluorescence in-tensity of the sample with 0.4 cm tissue phantom when comparedto the sample without the tissue phantom. The system can be im-proved by further increasing the quantum yield of UCNs, andthere is a huge scope for improvement enabling activation in verydeep tissues. This technique was then used to activate photocagednucleic acids in animal models. Photocaged GFP-loaded UCNswere injected under the skin of mice and activated using the NIRlaser. GFP fluorescence was checked by using a confocal fluores-cence microscope and immunohistochemistry. Injection of nano-particles under the skin and activation of the nucleic acids arenot routine procedures. To prove the versatility of UCNs in actualapplications, an experiment was devised to mimic the clinical set-ting as closely as possible. Cell therapy has gained popularity in thepast decade, and it usually involves cells encapsulated in a scaffold/device and transplanted into the body, after which the activation isdone to produce the proteins of interest; this application requiresdeep-tissue activation. So cells transfected with UCNs containingphotocaged GFP plasmid were loaded into a PDMS device andtransplanted into mice. The cells in the device were then activatedusing NIR light through the skin and PDMS layer, and efficientactivation was observed even at that depth. All these results provethat this technique has enormous potential in the fields such asgene therapy for controlled and specific gene delivery/knockdown,

developmental biology for site-specific gene knockdown and forpatterning of biomolecules using safe NIR light.

Materials and MethodsPhotoactivation of Caged siRNA in Cells. Caged GFP siRNA was first loadedonto the mesoporous coating of NIR-to-UV UCNs. Mesoporous NIR-to-UVUCNs (0.5 mg∕mL) were added dropwise to 20 μg of siRNA and agitatedgently at room temperature for 4 h. The solution was then centrifuged at6;300 × g for 10 min and the pellet containing the UCNs loaded with cagedplasmids was resuspended in deionized water and used for further experi-ments. The loading was checked by absorbance spectrophotometry and agar-ose gel electrophoresis and was found to be approximately 7.23 μg siRNA/mgUCN. B16-F0 cells were grown as described previously and transfected withpEGFP using Lipofectamine 2000, according to manufacturer’s instructions.The transfected cells were trypsinized and 0.5 mg∕mL mesoporous NIR-to-UV UCNs loaded with the caged siRNA were then added into the cell suspen-sion, and the mixture was plated onto culture plates at 62;500 cells∕cm2 for24 h. A similar set of cells were also plated with the addition of NIR-to-UVUCNs loaded with noncaged siRNA as a control. After which, the cells werewashed twice with culture medium and then irradiated with a 980 nm CWNIR laser for 0, 4, 8, and 12 min to make a specific dose of 0, 672, 1,344, and2;016 J∕cm2. The control wells received no NIR irradiation. Confocal fluores-cence imaging of the cells was done after 32 h to check for GFP fluorescence.GFP expression was quantitated after cell lysis using a fluorescence micro-plate reader (FLUOstar Optima; BMG Labtech GmbH). Further informationon methods for caging nucleic acids and activating them in solution, micemodel, and through tissue phantoms can be found in SI Materials andMethods.

Statistical Analysis. To compare the mean values of experimental group tothat of control ones, one-way ANOVA, at an alpha level of 0.05 (a P valueof less than 0.05 is considered as statistically significant), was performed usingOriginPro 8.5.

ACKNOWLEDGMENTS.We thank Sounderya Nagarajan for providing the tissuephantoms, Qingqing Dou for providing the upconversion nanoparticles,Shashi Ranjan for PDMS device fabrication, Kerwin Kwek Zeming for stencilpreparation, Akshaya Bansal for help with cytotoxicity studies, and SelvaRajan for support in preparing the schematic illustrations. Financial supportfrom Singapore Ministry of Health National Medical Research Council(NMRC) Grant R-397-000-105-275 andMinistry of Education AcRF Tier 1 GrantR-397-000-075-112 are also gratefully acknowledged.

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8488 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1114551109 Jayakumar et al.

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