Remote-Controlled Release of DNA in Living Cells via Simultaneous

Sep 25, 2014 - Using photons as external triggers to realize remote-controlled release of oligonucleotide is superior to other intracellular or extern...
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Remote-Controlled Release of DNA in Living Cells via Simultaneous Light and Host−Guest Mediations Jing Zheng,† Yuhong Nie,†,‡ Sheng Yang,† Yue Xiao,† Jishan Li,† Yinhui Li,† and Ronghua Yang*,† †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China ‡ Key Laboratory of Biotechnology in Tobacco Industry, School of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou, 450002, People’s Republic of China S Supporting Information *

ABSTRACT: Using photons as external triggers to realize remote-controlled release of oligonucleotide is superior to other intracellular or external stimulus. UV light is a valid photon-controlled manner due to high efficiency. However, further applications of these approaches in living cells are hampered by the large dose of UV-light irradiation. To address this issue, a simultaneous light and host/guest mediation was proposed in this paper. Gold nanoparticles (AuNPs) encoding with mercapto-β-cyclodextrin (βCD) served as a carried agent. Azobenzene (Azo), which was labeled on a releasing oligonucleotide, acted as a photochemically controlled switch. Ferrocene (Fc), an excellent guest for inclusion complexation by βCD, serves as “enhancers” and shifts the equilibrium of the inclusion−exclusion process between trans-Azo and βCD under UV-light irradiation, thus making the dose of UV-light irradiation reduced obviously. For further application, transfected green fluorescent protein (GFP)-expressing human lung cancer A549 cells were used to determine cellular uptake and gene silencing mediated by our constructed system in vivo. The results demonstrate that by employing Fc host−guest interaction, about 62.4% gene silencing was achieved within 30 min, which is significantly higher than that without Fc competition. Our strategy provides the potential for orthogonal DNA delivery and therapeutic activation that would be capable of achieving higher levels of site-specific activity and reduced amounts of side effects. inclusion of Azo in βCDs has been exploited to various smalldrug and DNA release systems.28−32 However, further applications of these approaches in living cells are seriously hampered by the large dose of UV-light irradiation, which would be sufficient to induce cell apoptotic events or other side effects.33,34 Accordingly, it would be desirable to find an intrinsic mechanism to boost the disassembly of the inclusion complex, and thus reduce the UV-light irradiation dose. To address this issue, herein we proposed a new strategy for regulating Azo release from the cavity of βCD via simultaneous light and host−guest mediations. Ferrocene (Fc), being also an excellent guest for inclusion complexation by βCD, has a higher binding affinity (7.6 × 104)35 than the trans forms Azo moieties for βCD (4 × 103).16 The addition of Fc could serve as supramolecular “enhancers” and shift the equilibrium of the complexation−decomplexation process between trans-Azo and βCD; therefore, this property has potential of accelerating target release, with immediately apparent advantages in UVlight irradiation dosage control and potential utility in biomolecule delivery and controlled release, as shown in Figure 1. As a proof of concept, first, we used Azo-labeled hairpin DNA as a model target and mercapto-β-cyclodextrin (SH−βCD) to encode gold nanoparticles (AuNPs) as delivery vectors. Under visible light, βCD−AuNPs and Azo-labeled

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ene delivery and therapy have made quantum advances in recent years.1−3 During the past decades, various safe and effective nonviral routes have been successfully developed to trap and deliver target therapeutic oligonucleotides into cells.4−7 For unpacking the oligonucleotides from tailored surfaces in a highly controlled manner, much attention has been focused on developing new oligonucleotides trigger release mechanisms, which include either alteration of intracellular environment, such as pH8,9 and the level of enzyme,10 or external means with electrical,11,12 thermal,13 or optical stimulus.14−17 Using photons, such as UV light, near-infrared light (NIR), and two-photon excitation(TPE),18,19 as external triggers is superior to other intracellular or external stimulus, because light does not contaminate the reaction system and can easily be manipulated to provide both spatial and temporal control for oligonucleotide release. Due to high photoefficiency, UV-light irradiation was widely used. On the basis of this, several UV-light photoresponsive molecules that convert UVlight energy to mechanical motion through the change of geometry of the molecules attract more attention in practical application; these include spiropyran,20 diarylethene,21 stilbene,22 and azobenzene (Azo),23 of which, Azo is a well-known class of light-responsive compounds that can be reversibly isomerized from its planar trans form to the nonplanar cis form upon UV-light and visible irradiations.24 Using the noncovalent inclusion interaction of Azo and β-cyclodextrins (βCDs),16,25,26 light-controlled assembly/disassembly of the βCD/Azo complex could be easily achieved.27 This unique light-responsive © XXXX American Chemical Society

Received: June 22, 2014 Accepted: September 25, 2014

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inert and biologically compatible, but also provide a nanoplatform for facile delivery of biomolecules into living cells, with remote-controlled release by UV light and competitive unsheathing.

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EXPERIMENTAL SECTION Preparation of βCD−AuNPs. AuNPs, which were typically about 13 nm in diameter, were synthesized through a previous report;36 the transmission electron microscopy (TEM) image is shown in Supporting Information Figure S1. The concentration of AuNPs was estimated by UV−vis spectroscopy based on an extinction coefficient of 2.7 × 108 M−1·cm−1 at λ = 519 nm for 13 nm particles. The concentration of AuNPs is estimated to be as much as 4.5 nM. For the surface modification process, 5 mg of SH−βCD was added to 2 mL of AuNPs colloidal solutions. The mixture was then incubated for 10 h at room temperature. Then the resulting mixture was subjected to centrifugation at the speed of 12 000g for 10 min and the supernatant was removed, while the oil-like AuNPs precipitate was dissolved by ultrapure water to keep the final volume identical. These modified AuNPs solutions were stored at 4 °C in a freezer. Formation of βCD−AuNPs/DNA and Release of DNA. The Azo-labeled P1 and the solution of βCD−AuNPs were mixed for 24 h at 25 °C in order to make the Azo-labeled P1 enter into the cavity of βCD as much as possible. Then the resulting mixture was subjected to centrifugation at the speed of 10 000g for 15 min and the supernatant was removed with the purpose of removing excess P1, while the oil-like βCD− AuNPs/P1 precipitate was dissolved by ultrapure water to keep the final volume identical. Agarose Gel Electrophoresis. Each DNA sample (2 μM, 10 μL) was mixed with 4 μL of glycerol and analyzed by agarose gel at 90 V for about 45 min in 1× TBE buffer (89 mM tris(hydroxymethyl)aminomethane, 2 mM ethylenediamine tetraacetic acid, and 89 mM boric acid, pH 8.0). The bands were visualized by UV illumination (312 nm) and photographed by a digital camera. Cytotoxicity Test. Cell toxicity was tested by measuring the cell viability using propidium iodide (PI) dye to stain the dead cells, followed by flow cytometry monitoring. Cells without any treatment were considered to be 100% viable. Cellular Incubation for DNA Delivery. Cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin (all reagents from Invitrogen). Cells were plated into chambered coverslides

Figure 1. Design scheme of controlled-release DNA in living cells based on cooperatively mediated host−guest interaction through light and competitive unsheathing. (A) Disassembly of βCD/Azo complex irradiated by UV light and Fc competitive unsheathing. (B) The corresponding target DNA released from βCD/Azo upon light and Fc addition.

target DNA formed a composite system (βCD−AuNPs/DNA) through the host−guest interaction of βCD and Azo. The release of target DNA was triggered by UV light and can function well under large irradiation dose. However, when we investigated the cytotoxicity effect of UV-light irradiation, the result demonstrated that it was essential to reduce the dose of UV-light irradiation further in vivo application. Thus, Fc was employed and coincubated in living cells; we found that, upon cooperative regulation of a competitive guest molecule, a small UV-light irradiation dose can result in high effective target DNA release and low cytotoxicity. As further application, human lung cancer A549 cells which were transfected with epidermal growth factor (EGFR)-GFP plasmids were employed to report the single-stranded DNA antisense oligonucleotide (AON−GFP) delivery and controlled-release performance of our proposed strategy. The Azo-labeled AON−GFP was conjugated with βCD−AuNPs. Upon UV-light irradiation and Fc competitive unsheathing, the liberated AON exhibited silencing activity in A549-GFP cells, which is significantly higher than that without Fc competition. The results suggest that our constructed βCD−AuNPs are not only chemically

Figure 2. (A) TEM images of βCD−AuNPs. (B) Absorption spectra of AuNPs before (a) and after (b) SH−βCD conjugation. Inset: 2% agarose gel electrophoresis image of AuNPs (lane a) and βCD−AuNPs (lane b). B

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DNA, P1 labeled with Azo and fluorescein isothiocyanate (FITC) at the 5′- and 3′- ends, respectively, was designed (sequences are shown in Table S1, Supporting Information). To ensure all Azo molecules can transform into the trans configuration that gets into the cavity of βCD, P1 was first irradiated with visible light for 24 h. In PBS buffer (pH 7.4, containing 5 mM Mg2+) and at room temperature, P1 emits strong FITC fluorescence (curve a, Figure 3A). We mixed

or 24-well plates 24 h prior to incubation with βCD−AuNPs/ DNA or βCD−AuNPs/AON-GFP conjugates and Fc at a concentration of ∼8 × 104 cells per well. Confocal Laser Scanning Microscopy Imaging. Fluorescence imaging was conducted with a confocal laser scanning microscopy setup consisting of a heated specimen holder and an Olympus IX-70 inverted microscope with an Olympus Fluo View 500 confocal scanning system. For the cell uptake experiment, the cells were incubated with 500 μL of 5 nM βCD−AuNPs/P1 or βCD−AuNPs/AON at 4 °C for 3 h. After incubation, cells were washed with 500 μL of PBS buffer for three times and then irradiated by UV light for different times. Flow Cytometry Experiments. After UV-light irradiation for different times, cells were washed twice with 1 mL of PBS containing 5 mM MgCl2, treated with 500 μL of trypsin for 5 min at 37 °C in 5% CO2, dispersed in 500 μL of PBS, and then subjected to flow cytometry analysis using a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, U.S.A.).

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RESULTS AND DISCUSSION β-Cyclodextrins (βCDs) are cyclic oligomers of seven glucose molecules. The outside of the βCDs toroid is hydrophilic due to hydroxyl groups, imparting the molecules with good water solubility, whereas the interior is relatively hydrophobic because of the glycosidic oxygen bridges. As such, βCDs are regarded as a representative of supramolecular host compounds used to functionalize the nanoparticles as a result of their high water solubility, low toxicity, and specific recognition ability toward many model substrates.37−39 In order to encode CD on AuNPs surface, in our experiment, βCD was functionalized with thiol to obtain SH−βCD. TEM images show that the attained βCD−AuNPs possess good monodispersity (Figure 2A), with average diameter of around 20 nm. Figure 2B shows that the UV−vis spectrum of AuNPs exhibits a maximum peak of the surface plasmon resonance absorption at 522 nm, while the peak shifts to 526 nm after modification with SH−βCD. Meanwhile, the average dynamic light scattering (DLS) size of the βCD-modified AuNPs increases slightly from 12 ± 2 to 18 ± 2 nm, concomitant with the ζ-potential change from −27.6 ± 1.2 to −21.7 ± 1.5 mV, indicating that the citric acid salts on the surface of AuNPs were succeeded replaced by βCD. The formation of βCD−AuNP was further confirmed by agarose gel. As shown in the inset of Figure 2B, the AuNPs were aggregated in the inlet of the gel, which was mainly due to the high ion strength of the 1× TBE (lane a). In this case, the electric field is shielded, and the nanoparticles come close to each other until the attractive forces, such as induced dipole interaction, i.e., van der Waals force or hydrogen bonds, eventually cause the particles to agglomerate.40 After βCD modification, because βCD can provide steric stabilization to render AuNPs more stable in regard to possible aggregation at high salt concentrations,41 the attained βCD−AuNPs can subsequently migrate in the gel, and an obvious wine-colored band could be observed. These results can confirm the successful modification of AuNPs with a synthetic molecular host. According to the previous report,42 the surface coverage of βCD molecules on AuNP was evaluated to be 56.3%. To demonstrate the feasibility of our proposed strategy, first, we investigated that the photoisomerization of Azo was totally reversible by monitoring the peak intensity at 326 nm upon alternating UV and visible light irradiation for multiple cycles (Figure S2, Supporting Information).Then, a hairpin structure

Figure 3. (A) Fluorescence emission spectra of (a) free P1; (b) “a” + βCD−AuNPs; (c) free P2 + βCD−AuNPs; (d) “b” under the irradiation with UV light for 90 min; (e) “b” + 100 nM Fc under the irradiation with UV light for 30 min in the PBS buffer at 20 °C. λex = 480 nm. (B) In vitro fluorescence recovery of P1 from βCD−AuNP/ P1 before (green column) and after (wine-red column) 100 nM Fc addition as a function of UV-light irradiation duration time. (C) The effect of various concentrations of Fc on the fluorescence intensity of βCD−AuNPs/P1 under different UV-light irradiation times. (D) The releasing percentage of P1 as a function of UV-light irradiation time for βCD−AuNPs/P1 + 100 nM Fc (a) and βCD−AuNPs/P1 (b). [βCD−AuNPs] = 1 nM. The error bars signify the standard error obtained from three repetitive measurements.

βCD−AuNP (1 nM) and P1 together and then incubated the mixture for 30 min at room temperature, following by centrifugation at the speed of 10 000g for 15 min and washing by PBS buffer for three times. Under this condition, very weak fluorescence can be observed (curve b, Figure 3A), indicating that the fluorophore FITC was closed to the surface of AuNPs by forming a βCD/Azo inclusion complex. We calculated that the loading efficiency of P1 is 106 nM (details seen in Supporting Information). Meanwhile, a control experiment demonstrated that the coincubation of βCD−AuNPs and P2 (the sequence is the same as P1 and without being Azo-labeled; detailed sequence is shown in Table S1, Supporting Information) could not cause obvious fluorescence decrease of P2 (curve c, Figure 3A); this is mainly due to that the βCD−AuNPs could not serve as an effective quencher without βCD/Azo complex formation. These results indicate that the βCD/Azo inclusion complex plays an important role as a high-performance carrier agent for C

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Azo-labeled DNA. When UV light is applied to the mixture of βCD−AuNP and P1 for 90 min (the hand-held UV lamp is 6 W, and all the power of UV-light irradiation in our experiment is kept the same), significant fluorescence recovery of P1 can be observed (curve d, Figure 3A), suggesting that the photons initiate Azo isomerization to disassemble βCD−AuNPs/P1. Simultaneously, the result also indicated that a large UV irradiation dose is necessary for our design. As noted above, the use of more than 1.0 h of UV-light irradiation is essential for maximum release of P1 from βCD− AuNPs/P1. Considering the cytotoxic effect of UV light, we next investigated the cell death upon different UV-light irradiation durations using PI dye to stain dead cells, followed by flow cytometry monitoring. As shown in Supporting Information Figure S3, less than 10% of dead HeLa cells were observed within about 26 min. As the irradiation time increased (120 min), cell viability dramatically decreased and up to 80% of HeLa cells were killed. Thus, it is necessary to shorten the UV-light irradiation time and reduce the irradiation dose for further in vivo application. According to our hypothesis, the addition of the competing guest molecule, Fc, could serve as the enhancer of the disassembly of βCD− AuNPs/P1 complex. Supporting Information Figure S4 shows that the fluorescence of βCD−AuNPs/P1 was hardly affected upon addition of increased concentrations of Fc, suggesting that, though Fc has higher affinity than the trans-Azo for βCD, it is not strong enough to disassemble the complex of βCD− AuNPs/P1 within the time. Upon UV-light irradiation, it is noteworthy that a significant enhancement of fluorescence is observed for the mixture of βCD−AuNPs/P1 and Fc within 30 min (curve e, Figure 3A), which is comparable with UV-light irradiation time with 90 min without Fc coincubation (curve d, Figure 3A). Figure 3B shows the in vitro fluorescence recovery of P1 from βCD−AuNP/P1 as a function of UV-light irradiation duration time. In the absence of Fc, F/F0 (F0 and F are the fluorescence intensity of P1 released from βCD− AuNP/P1 before and after UV-light irradiation, respectively) reaches equilibrium within 100 min, while the equilibrium time of 60 min can be achieved upon 100 nM Fc addition. Figure 3C further investigated the effect of various concentrations of Fc on the fluorescence increment of βCD−AuNPs/P1 under different UV-light irradiation times. The results show that the fluorescence increment is becoming more and more manifest as functions of UV-light irradiation duration time and Fc concentration. As seen from Figure 3C, we can conclude that 100 nM is the optimized concentration in our subsequent experiment. To demonstrate the efficiency of our constructed βCD−AuNPs/P1 + Fc system for controlled-release P1 delivery, the releasing percentage of P1 (defined as (Ft − F0)/(F − F0) × 100%, where F and F0 are the fluorescence intensity of P1 before and after being complexed with βCD− AuNP/P1, respectively; Ft is the fluorescence intensity after UV-light irradiation for different times) is much higher (curve a, Figure 3D) than that of 0 nM Fc addition (curve b, Figure 3D). Thus, a large a UV-light irradiation dose could be reduced for the same release rate, demonstrating the efficiency of Fc unsheathing and the potential to improve the biocompatibility of this controlled-release delivery system. To further confirm that Fc can competitively unsheathe trans-Azo from the surface of βCD−AuNPs, we investigated the reversibility of P1 release irradiated alternately by UV and visible light. As shown in Figure 4A, upon irradiation with UV light for 90 min, βCD−AuNPs/P1 is disassembled to release

Figure 4. (A) Fluorescence intensity change at 520 nm of the βCD− AuNPs/P1 (blue curve) and βCD−AuNPs/P1 + Fc (red curve) after several repeated steps of UV and visible light irradiations. (B) 2% agarose gel electrophoresis image of P1 under different conditions: lane 1, βCD−AuNPs/P1; lane 2, βCD−AuNPs/P1 under UV-light irradiation for 30 min; lane 3, βCD−AuNPs/P1 + 100 nM Fc under UV-light irradiation for 30 min; lane 4, P1. The concentration of P1 was 2.0 μM.

P1 from the surface of AuNPs; thus, obvious fluorescence increment could be observed. Then, alternate visible and UVlight irradiation can realize fluorescent quenching and recovery of P1 (blue curve). With addition of 100 nM Fc to the βCD− AuNPs/P1 solution, the initial UV irradiation can ensure fast release and, thus, fluorescent recovery of P1. However, the fluorescent quench of P1 cannot be achieved under the subsequent visible light irradiation (red curve), indicating that the formation of Fc/βCD complex hinders the inclusion interaction of Azo and βCD due to higher binding affinity of Fc over trans-Azo for βCD. This conclusion was further strengthened through sol−gel electrophoresis, as shown in Figure 4B, considering that, once P1 trapped into the cavity of βCD−AuNPs, the fluorescence of FITC could be quenched by AuNPs and no bright band can be observed (lane 1). When the UV-light irradiation duration time was 30 min, only a small portion of P1 would detach from the surface of AuNPs; therefore, a very weak band can be seen (lane 2). With the combination of Fc addition, due to the cooperative action between competitive unsheathing and trans-to-cis isomer of Azo, a bright band in the gel (lane 3) could be observed and the brightness is comparable with P1 itself (lane 4), which directly unravels, the high proportion of βCD−AuNP/P1 complex could disassemble, and P1 was released. Next, we investigated whether this strategy could function well in cells for effective DNA delivery. To ensure all βCD− AuNPs/P1 were taken up by HeLa cells, we incubated the complex with HeLa for 3.0 h, following by washing with PBS for three times. According to the previous report, we learned the complex βCD−AuNPs/P1 used in our experiment is also located in the cytoplasm.43 Then, the UV light was applied, in this event, Azo were converted to the cis form, and thus P1 escaped from the cavity of βCD; increased fluorescence signal was thus observed for cells treated with prolonging irradiation time (Figure 5A). It is worth noting that, when HeLa cells were irradiated by UV light for 30 min, they did not generate sufficient fluorescence signal to illuminate the P1 release. Given the extracellular unsheathing of trans-Azo with Fc, cells were first incubated with Fc, and the uptake concentration is about 500 nM (this value was optimized by the above-mentioned concentration in vitro, and details of the efficiency of Fc uptake by cells is seen in Supporting Information), then washed three D

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Figure 5. Investigation of βCD−AuNPs/P1 in living cells. Representative confocal laser scanning microscopy images of HeLa cells treated with (A) βCD−AuNPs/P1, (B) βCD−AuNPs/P1 + 500 nM Fc under the UV-light irradiation for different times. The images are the overlay of FITC fluorescence and the bright-field image. Scale bars = 40 μm. (C) Flow cytometry results from HeLa cells treated with βCD−AuNPs/P1 and βCD−AuNPs/P1 + 500 nM Fc under the UV-light irradiation for different times. [βCD−AuNPs] = 5 nM. The error bars signify the standard error obtained from three repetitive measurements.

Finally, RNA interference (RNAi), which is one of the most exciting discoveries in functional genomics during the past decade, has become an important method for analyzing gene functions in eukaryotes and holds great promise for the development of therapeutic gene silencing.44−46 Having successfully established remote-controlled Azo−DNA delivery and release by simultaneous light and host−guest mediations in vitro and in vivo, we extended our work to transport and controlled-release of antisense single-stranded DNA antisense oligonucleotide (AON) via βCD−AuNP.47 We employed a phosphorothioate-modified AON (sequences are shown in Table S1, Supporting Information) known to silence the gene encoding destabilized GFP which marks the EGFR expressing by A549 cells (A549-GFP).48 Destabilized fluorescent proteins were chosen because they have short half-lives that allow for the monitoring of downregulation shortly after administration of the molecular therapeutics.49 Similar to the P1 case, we constructed the βCD−AuNP/AON complex and the A549GFP cells were coincubated with βCD−AuNP/AON and Fc for 3 h. For comparison, we also employed the constructed βCD−AuNP/AON for A549-GFP cells without Fc coincubation. Upon UV-light irradiation for different times, the cells were washed and supplemented with fresh medium and allowed to incubate for an additional 24 h for RNAi to take effect.49 Confocal imaging revealed the GFP expression A549 cells displayed bright green fluorescence (Figure 6A, panel a) and significant reduction in GFP expression by βCD−AuNP/AON + Fc (weak fluorescence in Figure 6A, panel c) relative to the Fc-untreated control cells (Figure 6A, panel b) upon UV-light

times with PBS (the cell cytotoxic effect of 500 nM Fc was investigated and no cell death was demonstrated, Figure S5, Supporting Information). The confocal laser scanning microscopy results revealed that HeLa cells displayed no fluorescence increment upon Fc addition without UV-light irradiation (Figure S6, Supporting Information). To investigate the cooperative regulation of the competitive guest molecule and UV light, the Fc and βCD−AuNPs/P1 pretreated HeLa cells were then exposed to UV light with exposure durations ranging from 0 to 30 min. As can be seen from Figure 5B, longer exposure duration results in stronger fluorescence. The increasing fluorescence intensity over time indicates a continuous P1 release which resulted from the bent cis isomer of Azo and Fc competitive effect. We also used flow cytometry to collect fluorescence data for cells treated with UV light and Fc cooperative addition; the result is demonstrated in Figure 5C. Comparing with confocal laser scanning microscopy, which allows imaging only a small number of cells, flow cytometry can analyze thousands of cells per second, generating a quantifiable statistical average for a large population of cells, while eliminating cell-to-cell variation and experimental artifacts. The flow cytometry results were in excellent agreement with the confocal imaging; about 2-fold enhancement was observed for HeLa cells coincubated with Fc and βCD−AuNPs/P1, then irradiated by UV light for 30 min. Taken together, while synergistic reaction between the trans form to cis form upon UV-light irradiation and competitive unsheathing of the higher binding affinity guest molecule can work well in our buffer system, these results also demonstrate that approach is useful for intracellular DNA delivery in living cells.

Figure 6. (A) Confocal microscopy assay for (a) untreated control A549-GFP cells, (b) cells incubated with βCD−AuNP/AON then irradiated with UV light for 30 min, (c) cells incubated with βCD− AuNP/AON + 500 nM Fc and then irradiated with UV light for 30 min. Scale bars = 40 μm. (B) Silencing efficiency (%) of βCD−AuNP/ AON under UV-light irradiation for different times with (a) and without Fc (b) addition. [βCD−AuNPs] = 5 nM. The error bars signify the standard error obtained from three repetitive measurements. E

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irradiation for 30 min. The weaker fluorescence intensity was due to the silencing of expression of GFP by RNAi. Further, flow cytometry data showed that the potency of AON or percentage of silencing (defined as (I0 − It)/I0, I0 is the flow cytometry result of A549-GFP cells, It is the flow cytometry results of A549-GFP cells incubated with βCD−AuNP/AON + Fc or βCD−AuNP/AON and then irradiated with UV light for different times) followed βCD−AuNP/AON + Fc > βCD− AuNP/AON under the same UV-light irradiation durations, as shown in Figure 6B. Meanwhile, we investigated the Fc itself and βCD−AuNP could not induce of GFP gene silencing under UV-light irradiation (data not shown). These results demonstrated that the βCD−AuNP/AON + Fc complex was successfully used as a highly effective and biocompatible light and host−guest mediations controlled-delivery vector that could controllably release AON to silence the target reporter GFP gene and downregulate GFP expression in vivo.

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CONCLUSIONS The result concluded that the constructed βCD−AuNPs provides a nanoplatform for facile delivery of DNA into living cells, with controlled-release of DNA by UV light and competitive unsheathing. This light-mediated release strategy paves a simple and unique way for delivering therapeutic materials into cells in a spatiotemporally controlled fashion and could be used to enhance the transfection of genetic materials as well as protein and drug delivery. What is more important, considering the cytotoxicity of the UV light, we also demonstrated the use of synthetic host−guest chemistry to provide triggered activation of a therapeutic system by competitive complexation. With this approach, a highly effective and biocompatible remote-controlled biomolecule delivery system can be constructed. Besides orthogonal DNA delivery and therapeutic activation, this strategy also provides the potential for spatiotemporal dynamics of specific biomolecules detection, and the related-work is being carried out in our group.

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ASSOCIATED CONTENT

S Supporting Information *

More experimental details and spectroscopic data as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-731-88822523. Fax: 86-731-88822523. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (21135001, 21305036, 21221003, 21405038, and J1103312) and the National Key Basic Research Program (2011CB91100-0). J. Zheng received financial support from Growth Program for Young Teachers in Hunan University.

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