Preparation of AuNR/plasmid DNA complexes

As-synthesized AuNRs22 were first treated with oleate23 or poly(styrene sulfonate) (PSS)17, followed by one of the following cationic dispersants: DOTAP, poly(diallyldimethylammonium chloride) (PDDAC), polyethyleneimine (PEI), poly-L-lysine (PLL), or cationized HDL (catHDL) (Fig. 1a). We have previously reported that catHDL is a highly biocompatible and cell-interactive dispersant for AuNRs23. Oleate-treated AuNRs (oleate-AuNRs)s were only used for catHDL and DOTAP, because the other cationic polyelectrolytes caused aggregation (Supplementary Fig. S1). DOTAP-AuNRs were prepared first by mixing oleate-AuNRs and DOTAP/sodium cholate micelles at a DOTAP/AuNR weight ratio of 10, followed by dialysis against PBS to remove cholate. All cationic AuNRs had intense absorption in the near infrared region, suggesting good colloidal stability in PBS (Fig. 1b). The presence of the cationic dispersant on the AuNR surface was confirmed by Zeta potential analysis (Fig. 1d) and IR spectroscopy (Supplementary Fig. S2).

Figure 1 Characterization of cationic AuNRs. (a) Schematic of preparation of cationic AuNR/pDNA complex. (b) UV-vis-NIR absorption spectra of various cationic AuNRs in PBS ([Au] = 20 µg/mL). All AuNRs have intense absorption in the NIR region, suggesting their high colloidal stability. (c) UV-vis-NIR absorption spectra of various cationic AuNRs after complexation with control plasmid DNA (pCMV-DsRed) in Opti-MEM ([Au] = 20 µg/mL). AuNR/pCMV-DsRed complexes were prepared by simply mixing AuNRs and pCMV-DsRed (w/w ratio = 10) for 20 min at room temperature. Significant broadening of the NIR plasmon peak is observed only for PLL-AuNRs. (d) Zeta potential data (mV, n = 3, average ± SD) of AuNRs before and after complexation with pCMV-DsRed in 10 mM Tris-HCl (pH 7.4). Only DOTAP-AuNRs show a large decrease in the zeta potential upon pCMV-DsRed addition. (e) Agarose gel shift assay. AuNRs were added to 10 µg/mL pCMV-DsRed solution at various w/w ratios. pCMV-DsRed was visualized by Gel Green. Numbers in the image indicate the w/w ratios of AuNRs to pCMV-DsRed. (f) Densitometric analysis of gel bands in (e). Values were calculated from the residual fluorescence intensity of free pCMV-DsRed (n = 3, average ± SD). Full size image

Next, the pDNA-binding capacity of the cationic AuNRs was evaluated by agarose gel electrophoresis. Even in the presence of a control pDNA (pCMV-DsRed), colloidal stability was only slightly decreased for all AuNRs, except the PLL-treated AuNRs (PLL-AuNRs) (Fig. 1c). Furthermore, the amount of free pCMV-DsRed gradually decreased with increasing amount of cationic AuNRs, except in the case of catHDL-treated AuNRs (catHDL-AuNRs) (Fig. 1e,f, Supplementary Fig. S3), clearly demonstrating that all AuNRs other than catHDL-AuNRs showed significant DNA binding capability. At an AuNR/DNA weight ratio of 10, more than 90% of the pCMV-DsRed added appeared to be bound to the cationic AuNRs, in all cases except the catHDL-AuNRs. Interestingly, the zeta potentials amongst the AuNR/pCMV-DsRed complexes at this ratio were dissimilar, despite using an equivalent amount of pCMV-DsRed (Fig. 1d). Only in the case of DOTAP-AuNRs, the zeta potential was significantly negatively shifted upon pCMV-DsRed binding. The reason for this apparent disparity is unclear at present, but these results may reflect possible differences in binding sites for pDNA, such as the outer surface or within the dispersant layer surrounding the AuNRs.

pDNA transfection efficiency

The transfection efficiency of AuNR/pCMV-DsRed complexes was evaluated using HEK293T cells and HeLa cells. Lipofection by Lipofectamine 2000 (LF2000) was used as a positive control. After 24 h of treatment, only DOTAP-AuNRs and PEI-treated AuNRs (PEI-AuNRs) showed significant transfection abilities in both HEK293T cells and HeLa cells, as observed by DsRed expression (Fig. 2a,b). In particular, DOTAP-AuNRs showed a comparable efficiency to LF2000 (Fig. 2b), while showing a lower cytotoxicity than LF2000 (Fig. 2c). To the best of our knowledge, this is the first demonstration that AuNRs are comparable to LF2000 in terms of pDNA transfection efficiency.

Figure 2 Intracellular gene delivery by cationic AuNRs. (a) Fluorescence images of HEK293T cells treated with cationic AuNR/pCMV-DsRed complexes. LF2000 was used as a positive control. Scale bar = 100 µm. (b) Transfection efficiency determined by flow cytometry analysis. Data indicate the mean fluorescence intensity of DsRed (n = 3, average ± SD). HEK293T cells and HeLa cells were treated with AuNR/pCMV-DsRed complexes ([Au] = 20 µg/mL, pCMV-DsRed = 2 µg/mL) for 24 h. DOTAP-AuNRs show a high transfection efficiency, comparable to that of LF2000. (c) Cell Count Kit-8 (CCK-8) assay data for HEK293T cells and HeLa cells treated with AuNR/pCMV-DsRed complexes for 24 h. Data indicate the mean cell viability (n = 3, average ± SD). The cytotoxicity of cells treated with DOTAP-AuNRs is lower than that of those treated with LF2000. (d) Time-dependent change of the localization of AuNRs in HEK293T cells. DOTAP- and PEI-AuNRs were labeled with Rho-PE and Alexa Fluor 546, respectively. AuNR localization (red signal) was analyzed after 2, 4 and 24 h treatment. Late endosomes/lysosomes were stained with LysoTracker Green DND-26 (LysoTracker, green). (e) The colocalization ratios of the red pixels to green pixels after 4 and 24 h were calculated, respectively (n = 10, average ± SD). Asterisks indicate P values < 0.05 determined using Student’s t-test. Scale bar = 10 µm. Full size image

To gain insight into the mechanism of the high transfection ability of DOTAP-AuNRs, DOTAP- and PEI-AuNRs were compared from various viewpoints. Under our transfection condition (20 µg/mL AuNRs), all pCMV-DsRed added was bound to the AuNR surface (Fig. 1e). When compared by inductively coupled plasma (ICP) analysis (Supplementary Fig. S4), cells were observed to take up 20% more DOTAP-AuNRs than PEI-AuNRs, indicating higher pCMV-DsRed delivery by DOTAP-AuNRs. Next, we investigated the intracellular localization of the two AuNRs by confocal microscopy. We observed that the red fluorescence signal, indicating AuNR localization, was localized at the plasma membrane after 2 h of transfection (Fig. 2d, Supplementary Fig. S5), and then moved from the plasma membrane to the late endosomes/lysosomes (green) by 4 h of transfection in both DOTAP- and PEI-AuNR-treated cells (Fig. 2d, Supplementary Fig. S5). The colocalization ratios of the red pixels to green pixels after 4 h were calculated to be 79.0 ± 7.2% for DOTAP-AuNRs and 75.6 ± 8.2% for PEI-AuNRs (Fig. 2e). After 24 h, the DOTAP- and PEI-AuNRs appeared to have exited from the late endosomes/lysosomes and were dispersed in the cytoplasm. The colocalization ratios of the red and green fluorescence signals at 24 h decreased to 33.6 ± 8.2 and 48.1 ± 10.3% for DOTAP- and PEI-AuNRs, respectively (Fig. 2e). These results indicate that the higher transfection efficiency of DOTAP-AuNRs may be, at least in part, due to the higher cell uptake and endosomal escape. While the mechanism of the higher cell uptake of the negatively charged DOTAP complexes compared to that of the positively charged PEI complexes (Fig. 1d) remains to be elucidated, the higher endosomal escape may be explained by the ability of oleate to function as a helper lipid24.

To investigate whether our surface modification method using oleate and DOTAP may be applicable for other nanoparticles, we similarly treated magnetite nanoparticles. When oleate-coated magnetite nanoparticles were mixed with DOTAP at a weight ratio of 20, a stable colloidal dispersion was obtained (Supplementary Fig. S6). The transition of the zeta potential from negative to positive suggested surface modification with DOTAP (Supplementary Fig. S6). Like DOTAP-AuNRs, DOTAP-treated magnetite nanoparticles also showed higher transfection efficiency than PEI-treated magnetite nanoparticles, and was comparable to that of LF2000 (Supplementary Fig. S6). These results clearly demonstrate that our method may be generally applicable for any oleate-coated nanoparticle.

Before the laser illumination experiments, we sought to optimize the conditions for DOTAP-AuNR preparation by changing the DOTAP/AuNR weight ratio, based on transfection efficiency. At mixing ratios lower than 10, the colloidal stability and transfection efficiency of DOTAP-AuNRs were reduced (Supplementary Fig. S7). At a higher mixing ratio of 20, DOTAP-AuNRs also showed lower transfection efficiency, despite higher colloidal stability. Thus, DOTAP-AuNRs prepared at a DOTAP/AuNR weight ratio of 10 were used for the following laser illumination experiments.

Induction of protein expression by intracellular photothermal heating

We next performed photoinduced transgene expression using the DOTAP/AuNRs (Fig. 3a). We constructed a plasmid vector under the control of the human HSP70b’ promoter and inserted the EGFP gene (pHSP70-EGFP). HEK293T cells cotransfected with pHSP70-EGFP and pCAG-tdTomato using LF2000 exhibited only tdTomato expression under normal conditions, while they showed a significant increase in EGFP expression following heat shock at 42 °C for 30 min or longer (Supplementary Fig. S8). Next, pHSP70-EGFP and pCMV-DsRed were cotransfected in HEK293T cells using DOTAP-AuNRs. After 24 h, DsRed-positive cells in the targeted area (100 µm dia.) were illuminated at 780 nm using a NIR laser, and the resultant EGFP expression was evaluated after another 24 h. As shown in Fig. 3b, illuminated cells showed a marked increase in EGFP expression, whereas the surrounding off-target cells showed little to no EGFP expression. The induction of EGFP expression could be detected as soon as 6 h after NIR illumination by time-lapse imaging (Fig. 3c). When compared with the induction efficiency after heat-shock treatment at 42 °C for 30 min (positive control), maximal photoinduction was found to be achieved by illumination for 10 s at 6 W/mm2 (Fig. 3d, left panel). Under this laser condition, the temperature rise of the culture media was negligible and induction of EGFP expression was not detected in cells transfected using LF2000 (Fig. 3d, right panel). On the other hand, no significant changes were observed in the number of DsRed positive cells after illumination (Supplementary Fig. S9). Phototoxicity was low when cells were illuminated at 6 W/mm2, with less than 5% cells dying in 24 h. In contrast, cell death was significantly increased at 9 W/mm2 (Fig. 3e).

Figure 3 Intracellular photothermal heating of HEK293T cells by DOTAP-AuNRs. (a) Schematic of photothermal induction of protein expression by AuNRs. HSP70b’ promoter-driven expression vector (pHSP70b’) is transfected into cells by DOTAP-AuNRs and activated by brief NIR illumination. (b,c) Photoinduced EGFP expression in HEK293T cells. pHSP70-GFP and pCMV-DsRed were cotransfected by DOTAP-AuNRs. After 24 h, DsRed-positive cells were illuminated at 780 nm (6 W/mm2, 10 s). White broken circles indicate the illuminated area. Representative fluorescence images 24 h after illumination are shown in (b) and time lapse images after illumination are shown in (c). Scale bars = 100 µm in (b), 40 µm in (c). (d) Photoinduction efficiency for DOTAP-AuNRs (left) and LF2000 (right). Data were calculated based on the number of EGFP-positive cells/the number of DsRed positive (=photoinducible) cells within illuminated areas (n = 3, average ± SD). Data for the positive control were from cells incubated at 42 °C for 30 min (whole-cell heating). (e) Phototoxicity by intracellular photothermal heating. After illumination, cells were stained with Annexin-V and propidium iodine to detect apoptosis and necrosis, respectively. (f) Intracellular photothermal heating of AuNRs (780 nm, 6 W/mm2). The temperature around intracellular DOTAP-AuNRs was estimated utilizing the temperature dependence of Rho-PE fluorescence intensity (Fig. S9). Within a few seconds of illumination, the temperature reaches approximate 42 °C, which is the threshold temperature (42 °C) of HSP70b’ promoter activation. (g) Laser power intensity-dependence of the maximum temperature around illuminated AuNRs (n = 3, average ± SD). Full size image

To estimate the intracellular temperature around the AuNRs during illumination, the temperature dependency of the fluorescence intensity of 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rho-PE) was utilized (Supplementary Fig. S10)21. Cells were treated with Rho-PE-labeled DOTAP-AuNRs and illuminated. The intracellular temperature quickly increased to reach a plateau of approximately 42 °C (Fig. 3f), which is the threshold temperature for activating the HSP70b’ promoter. When the maximum temperatures achieved at various laser power intensities were plotted, a linear relationship was observed (Fig. 3g). Based on this calibration curve, it was found that at laser power intensities of 4.5 W/mm2 or higher, the intracellular temperature could be increased to higher than 42 °C. These estimated temperature data agree well with the results in Fig. 3d, and demonstrate that intracellular photothermal heating by AuNRs is the main mechanism of HSP promoter-driven EGFP expression induction. These results clearly demonstrate that the HSP70b’ promoter can be safely driven by NIR illumination of the transfected carrier AuNRs in the cells.

As a proof of concept of the time- and site-specific activation of gene expression achievable by use of our system, we next attempted to develop a new method for therapeutic gene delivery to cancer cells. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type-II transmembrane ligand that specifically induces apoptosis in many transformed cell lines by activating the death receptors DR4 and DR5, while having little effect on normal cells25, 26. We constructed an expression vector for an N’terminus-tagged eGFP-TRAIL fusion product, which has been shown to be effective in killing various tumor cell lines27, driven by the HSP70b’ promoter (pHSP70-EGFP-TRAIL) and transfected it into HeLa cells by LF2000. HeLa cells are derived from human cervical cancer and are thus sensitive to apoptotic activity of TRAIL28. We confirmed that heat shock induced significant cell death of HeLa cells transfected with pHSP70-EGFP-TRAIL, but not those with pHSP70-EGFP (Supplementary Fig. S11). Next, pHSP70-EGFP-TRAIL was cotransfected with pCMV-DsRed using DOTAP-AuNR and photoactivated by NIR laser under a time-lapse fluorescent microscope. DsRed-positive cells in the illuminated area (200 µm dia.) progressively showed typical morphological changes of apoptotic cells within 6 h of observation (Supplementary Movie S1). We also occasionally detected similar apoptotic changes of neighboring cells (arrowhead in Fig. 4a). The cell death rate of TRAIL-expressing cells was significantly higher than control cells (Fig. 4b), demonstrating that NIR-induced transgene expression, and not photoheating itself, could induce cell death. Thus, our DOTAP-AuNRs may have potential application in the molecular therapy of cancer