Introduction

The optical properties of gold nanoparticles (AuNPs) have been greatly attractive since their useful biological and medical applications such as CT contrast Raman imaging, and radio-sensitization1,2,3,4,5,6. Especially, the highly efficient photothermal effect and relative bio-safety of gold nanoparticles have driven extensive studies for noninvasive cancer therapeutic applications7,8,9. As hyperthermia agents, diverse structures of AuNPs including spheres, rod, branched shapes and cages have been developed10,11,12,13. For higher therapeutic efficiency of the AuNPs for cancers in deep tissue, it has been important to improve the resonant absorption for near-infrared (NIR) which has superior tissue penetration depth14,16. In terms of the size of AuNPs, relatively large sizes (>100 nm) are required to achieve the high absorption for NIR

Figure 2
figure 2

(A) Absorption peak wavelengths of CytC/ssDNA-AuNPs in various pHs. The numbers on the symbols represent the ratios of AuNPs vs. ssDNA vs. cytochrome c of the modification reaction. (B) Responsive pHs vs. reaction ratio of cytochrome c over AuNPs of each CytC/ssDNA-AuNP.

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pH responsiveness of the synthesized AuNPs (CytC/ssDNA-AuNP) has been confirmed by measuring plasmonic absorption peak shift, size of clusters, and taking SEM images at various pH values. As shown in Fig. 3, Tables S1 and S2, the various physical properties of CytC/ssDNA-AuNPs have changed accordingly with the solution pH values and the changes have been also reversible. The plasmonic absorption peak has shifted to a longer wavelength implying that the particles aggregated to larger plasmonic clusters when the pH was reduced to 5.5 which is similar to the characteristic pH value of cancer cells (Fig. 3A). Remarkably, the absorption peak reversibly blue shifted to its original one accordingly with the pH restoring to 7.4 which is normal physiological condition (Fig. 3B). This means that the aggregated nanoparticles in low pH have been disassembled and back to individually existing status. Considering there is no significant pH variation-associated response of ssDNA-AuNPs, which is modified with only single stranded DNA without cytochrome c on the particle surfaces (Fig. S3), the pH responsive behavior of CytC/ssDNA-AuNPs can be regarded as a result of the surface charge variation influenced by combined electrical properties of cytochrome c and ssDNA as expected in above description and Fig. 3C. The pH responsiveness of CytC/ssDNA-AuNP has been consistently confirmed by measuring the particle size in the solution and taking SEM images (Fig. 3D,E). According to the dynamic light scattering experiment, the size of the AuNPs in a solution has been increased from 12 nm to larger than 600 nm in diameter with the reducing pH from 7.4 to 5.5. The increased size of AuNPs is attributed to the electrostatic clustering of the nanoparticles due to the decreased zeta potentials at low pH.

Figure 3
figure 3

(A) Red shift of absorption peak accordingly with reducing pH (7.4, 6.5. 6.0 to 5.5). (B) Blue shift of absorption peak with returning to higher pH (5.5, 6.0, 6.5 to 7.4). (C) Sizes, and (D) Zetapotentials of CytC/ssDNA-AuNPs measured during the pH lowering and elevating process. (E) TEM (upper panel) and SEM (lower panel) images of CytC/ssDNA-AuNPs taken at a cycle of pH 7.4 → 6.5 → 5.5 → 7.4 (scale bar: 300 nm). Error bars refer to standard deviations from three replicates.

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The reversibility of pH induced assembly, and disassembly also has been confirmed by the absorption spectroscopy (Fig. 4A,B) and size measurements (Fig. 4C). When the solution pH was returned to normal physiological pH (~7.4), the absorption spectrum of AuNPs has returned to its original shape implying that the clusters were disassembled and this behavior was well correspondent with the measured particle sizes. This reversible assembly and disassembly behavior of the CytC/ssDNA-AuNPs and correspondingly repeated surface charge variations (Fig. 4D) remained reproducibly over several repeated experiments with pH changes. These results imply that the synthesized and delivered nanoparticles have a potential of showing repeated therapeutic effects without additional introduction of the nanoparticles into a body.

Figure 4
figure 4

(A) Reversible absorption spectrum shift of CytC/ssDNA-AuNPs accordingly with pH variations. Blue lines: 1st cycle of pH 7.4 to 5.5 to 7.4, Green lines: 2nd cycle of pH 5.5 to 7.4, and Red lines: 3rd cycle of pH 5.5 to 7.4. (B) Absorption peaks at each pH cycles extracted from (A). (C) Particle sizes and (D) surface potentials of the particles at each cycles of pH. Error bars refer to standard deviations from three replicates.

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In an environment with high salt concentration such as cytoplasm or biological fluid, the electrostatic interaction between the CytC/ssDNA-AuNPs can be relatively diminished compared with that in deionized water30. In such case, pH responsive behavior of assembly and disassembly of the synthesized particles also may be deactivated due to the charge shielding. To show the feasibility of the CytC/ssDNA-AuNPs in high ionic strength environment, pH responsiveness of the particles has been evaluated in a cell culture media. The particles were suspended in a DMEM media containing serum and penicillin with the same concentration as in deionized water for the experiment. According to the results (Fig. 5A,B), the CytC/ssDNA-AuNPs remain their pH responsive behavior even in a cell media showing plasmonic absorption peak shift accordingly with the pH variations. The absorption peak wavelength of the particles in media was 520 nm at pH 7.8 which meant that the particles existed individually. As the pH of the media was lowered to 5.5, the absorption peak shifted to 550 nm immediately and further red shifted to 600 nm in 24 hours of incubation. When the pH has been raised back to 7.4 by adding NaOH solution to the media, the absorption peak has been shifted to 550 nm implying that the assembled nanoparticles were not completely but partially disassembled. This partial disassembly of nanoparticles may have attributed to the charge screening effect of ions on the ssDNA and cytochrome c of the gold nanoparticles.

Figure 5
figure 5

(A) Red and (B) blue shift of absorption peak accordingly with reducing and elevation pH in DMEM media solution. (C) Solution temperature changes with 660 nm laser irradiation times. Purple: PBS blank solution, yellow: ssDNA-AuNP in pH 7.4 DMEM media, green: ssDNA-AuNP in pH 5.5 DMEM media, blue: CytC/ssDNA-AuNP in pH 7.4 DMEM media, and red: CytC/ssDNA-AuNP in pH 5.5 DMEM media. (D) IR camera images of laser irradiated nanoparticle solutions. Error bars refer to standard deviations from three replicates.

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The plasmonic absorption peak shift to the longer wavelength induced by CytC/ssDNA-AuNPs’ aggregation in acidic pH has a benefit for photothermal therapy of cancer cells. To demonstrate the higher photothermal effect of CytC/ssDNA-AuNPs over non-pH responsive gold nanoparticles, firstly, temperature increase of cell culture media in presence of nanoparticles has been monitored. The media temperatures were measured using a thermal imaging camera for 5 minutes of laser (660 nm, 4 W/cm2) exposure time at two different pHs, 7.4 and 5.5. As shown in temperature profiles and thermal camera images in Fig. 5C,D, the temperature of media containing CytC/ssDNA-AuNPs in a pH condition adjusted to be acidic, 5.5, has been elevated more than 30 °C after 5 minutes of laser illumination on the solution. In contrast, temperature increase was not significant in other media. More specifically, a media containing the same nanoparticles, CytC/ssDNA-AuNPs, in pH 7.4 and medias containing ssDNA-AuNPs in both pH 7.4 and 5.5 did not show significant temperature increase even after 5 minutes of illuminations. In those media, the temperature increase reached only about 9~12 °C. The photothermal conversion efficiency (η) of CytC/ssDNA-AuNPs and ssDNA-AuNPs in both pH 7.4 and 5.5 was evaluated. The η values were calculated by the following Eqs. (1) and (2)31,32;

$${\rm{\eta }}=(h{\rm{S}}({T}_{\max }-{T}_{\max })-{Q}_{dis})/I(1-{10}^{-{\rm{A660}}})$$
(1)
$$h{\rm{S}}={m}_{D}{C}_{D}/\tau {\rm{S}}$$
(2)

where ℎ is heat transfer coefficient, S is the surface area of the container, Tmax is the maximum steady temperature of the nanoparticle solution, Tsur is the environmental temperature, is the heat dissipation from the light absorbed by the solvent and container, I is the laser power, A660 is the absorbance of the particles at 660 nm, mD and CD are the mass and heat capacity of the solvent, and τ is the sample-system time constant. According to the calculation, the conversion efficiencies of CytC/ssDNA-AuNPs are 17.8% and 41.2% at pH 7.4 and 5.5, respectively. The efficiencies of ssDNA-AuNPs are 21.7% and 18.5% at pH 7.4 and 5.5, respectively.

The high photothermal efficiency of CytC/ssDNA-AuNP in acidic condition can be directly reflected to a photothermal therapy of cancer cells of which pH is lower than that of normal physiological value. Here, the nanoparticles were applied to B16F10 skin melanoma cells and ssDNA-AuNPs were used as a control. The in vitro phototherapeutic effect has been evaluated for both of the particles by monitoring the dead cells after illuminating laser light on each of the cells adhered on a culture plate. Before the photothermal therapy, the cellular uptake abilities of the particles were evaluated using dark field microscopy. As seen in Fig. 6A, the pH sensitive CytC/ssDNA-AuNPs were seemed to be accumulated within the cancer cells with remarkably higher efficiency compared with ssDNA-AuNPs. These pH sensitive clustering of CytC/ssDNA-AuNPs and corresponding high accumulation within the cell can contribute to photothermal therapeutic efficiency. The laser has been illuminated on the cells incubated in a presence of 20 nM of nanoparticles and the power has been increased from zero to 14 W/cm2 stepwise. As shown in Fig. 6B, the B16F10 cells incubated with ssDNA-AuNPs have not shown any dead cells which are supposed be blue colored by staining with Trypan blue at any of the laser power illuminated on the cells. In contrast, the cells co-incubated with CytC/ssDNA-AuNPs for 24 hours were killed by laser illumination with power of higher than 10 W/cm2 (Fig. 6C). At 12 W/cm2 of the laser power, almost of the cells positioned within the illuminated area were killed by the photothermal effect. Furthermore, the cells even out of the illumination range were observed to be dead with 14 W/cm2 of laser power which is might be attributed to media overheated with high photothermal effect of CytC/ssDNA-AuNPs. In addition, cytotoxicity tests on normal (MDCK-GFP) and cancer (B16F10 melanoma) cells were performed separately to further confirm that the cancer cell selective death was entirely due to the photothermal effect of pH sensitive CytC/ssDNA-AuNPs. Each cell was treated with blank buffer, ssDNA-AuNP, CytC/ssDNA-AuNP, and CytC/ssDNA-AuNP with laser irradiation (14 W/cm2 for 5 min), respectively. As shown in Fig. 6D,E, normal cells did not response to any of the stimuli and showed 100% cell viability for all cases. The cancer cells, however, were killed only by the combined treatment of CytC/ssDNA-AuNP with laser irradiation. These results clearly demonstrated the superior photothermal efficiency and therapeutic applicability of the CytC/ssDNA-AuNPs as cancer cells specific photothermal therapeutic agents.

Figure 6
figure 6

(A) Dark field microscope images of B16F10 cells (left), co-incubated with ssDNA-AuNP (middle), and CytC/ssDNA-AuNP (right). Photothermal destruction of the cells co-incubated with (B) ssDNA-AuNP and (C) CytC/ssDNA-AuNP for 12 h followed by laser irradiation for 5 min at different power densities (scale bar: 100 µm). Viabilities of (D) MDCK-GFP cells and (E) B16F10 cells co-incubated with each particle with no laser or with laser irradiation.

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