Synthesis and Characterization of the Immuno Au Nanocages
For this study, Au nanocages of 65 ± 7 nm in edge length and 7.5 ± 1 nm in wall thickness were synthesized through a galvanic replacement reaction using silver nanocubes (~54 nm in edge length) as the sacrificial template and chloroauric acid, HAuCl4
, as the precursor to Au.10–14
shows scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the Au nanocages. These Au nanocages were shown to contain 37% residual Ag in the form of an alloy with Au by energy dispersive X-ray analysis. If necessary, the residual Ag can be selectively removed through a dealloying process with the use of Fe(NO3
Using the DDA method, the absorption, scattering, and extinction spectra were calculated for a Au nanocage with an edge length of 65 nm, a wall thickness of 7.5 nm, and a pore diameter of 20 nm at the corners. The results are shown in . The Cabs
were found to be peaked at 6.16 × 10−14
and 2.02 × 10−14
, respectively, with a Cabs
ratio of ~3.0. The measured extinction spectrum of the nanocages shown in the inset is consistent with the calculation. The discrepancy in peak width can be ascribed to the variations in edge length, wall thickness, the degree of corner truncation, and the porosity for the Au nanocages contained in a bulk sample. The Au nanocages were then conjugated to anti-HER2 antibodies using a two-step procedure.21,22
After conjugation, the extinction peak of these immuno Au nanocages slightly red-shifted from 800 to 805 nm, which was expected due to the small change of refractive index on the surface of the nanocages. It is worth pointing out that this small shift can be taken into account during the preparation of the pristine Au nanocages to ensure that there will be an exact overlap between the resonance peak of the immuno Au nanocages and the central wavelength of the laser. Recently, Au nanocages with similar optical properties were demonstrated by us to be effective agents for the in vitro
photothermal destruction of cancer cells.19
Thus, it was reasoned that these immuno Au nanocages would work well for the quantitative studies described here.
Figure 1 (A) SEM and TEM (inset) images of Au nanocages synthesized using the galvanic replacement reaction in which Ag nanocubes serve as a sacrificial template. The scale bar in the inset represents 100 nm. (B) The absorption (Cabs), scattering (Csca), and extinction (more ...)
Quantification of the Targeting Process In Vitro
Previously, the targeting selectivity of our bioconjugation protocol was demonstrated by immunofluorescence imaging and SEM.21,22
However, no information with regards to the number of nanocages attached to the cell surface was obtained. This information is important as it can tell us the amount of nanocages necessary to induce a photothermal therapeutic effect and allow for better comparisons between our nanocages and other nanostructures. Thus, in the first part of this study, we sought to quantify the average number of Au nanocages attached per cell. In addition to Au nanocages, Au nanospheres of 40 nm in diameter were used as a reference to validate the experimental procedure due to their well-defined size, shape, and composition. As a first approximation, SEM images were taken of the SK-BR-3 cells treated with immuno Au nanocages or nanospheres. The number of nanostructures observed in a randomly selected 2 µm × 1.5 µm portion of the cell was counted from multiple samples. Upon the basis of the number of nanostructures measured in that section and the dimensions of the cell as revealed by SEM imaging, the surface coverage was estimated to be approximately 200–2000 Au nanostructures per cell. This broad range could be attributed to the inhomogeneous distribution of receptors on the cell surface.23
Panels A and B of show SEM images of individual cells that had been targeted with the immuno Au nanospheres and nanocages, respectively. Interestingly, TEM imaging of a microtomed sample revealed that the Au nanocages were not solely immobilized on the surface of the cell. As shows, some of them were internalized into the cell, although none appeared to enter the nucleus. The HER2 antibodies have been reported to trigger endocytosis, but the exact mechanism for nanocage uptake has yet to be elucidated.24,25
Figure 2 SEM images of SK-BR-3 cells targeted with immuno Au nanospheres (A) and nanocages (B). SEM images at higher magnification (insets) reveal that the bright spots in the SEM images are indeed nanospheres and nanocages, respectively. The scale bar in the (more ...)
While electron microscopy allows us to see where the immuno Au nanostructures are located (on the surface vs inside) and roughly at what coverage, this technique is of rather limited power when dealing with large numbers of cells. To more accurately quantify the targeting efficiency of the immuno Au nanostructures, we decided to use flow cytometry coupled with inductively coupled plasma mass spectroscopy (ICP-MS). With this approach, the amount of Au contained in a specific number of cells can be determined. Then, from the geometric parameters of the nanostructures, the number of nanostructures per cell can be calculated. Specifically, the amount of Au can be easily determined by analyzing the sample with ICP-MS, while the number of cells in the sample can be easily quantified by spiking it with a known amount of Sphero Ultra Rainbow beads, followed by flow cytometry counting. shows a typical flow cytometry graph where forward scatter (x-axis) and right angle scatter (y-axis) can be used to differentiate the size difference between the beads and the cells. Then the number of cells can be quantified using FCS Express software to gate the populations of both cells and beads. For the standard in vitro targeting procedure described in the Experimental Methods, we found that there were approximately 460 ± 130 Au nanospheres per cell and roughly 400 ± 90 Au nanocages per cell; both numbers fall within the initial estimates from SEM images. In future in vivo studies, this information should allow for the administration of immuno Au nanocages in proper dosages to induce a photothermal effect for tumors of known sizes.
Quantification of the Photothermal Treatment Process In Vitro
The photothermal treatment with the immuno Au nanocages was implemented in vitro with SK-BR-3 cells. The central wavelength of the radiation was 805 nm with a bandwidth of 54 nm, so there was an optimal overlap with the absorption peak of the immuno Au nanocages. Yet, good spectral overlap between the light source and the light absorbing nanostructure will not alone ensure a good therapeutic effect. Factors such as time of cellular response to laser irradiation, laser power density, and time of laser exposure could also influence the efficiency of the photothermal effect. Thus, we systematically varied these parameters and quantified the amount of cellular death under each condition using flow cytometry coupled with propidium iodide (PI) staining.
PI is a popular nuclear or chromosomal counterstain in multicolor fluorescence techniques and is commonly used in flow cytometry to differentiate cell cycles. When PI binds to DNA by intercalating between the bases, it fluoresces about 20–30-fold stronger than unbound PI.26
Since PI is not permeable to live cells, it works well as a marker to quantify the number of dying or dead cells in a sample. In addition, it only requires a short incubation time (<15 min) and can generate well-separated populations of live and dead/dying cells. In a typical flow cytometry measurement, one can use PI fluorescent and forward scattering signals to quantify the cellular death caused by the photothermal effect.
shows the experimental setup, where cells in the center of a well (6.38 mm in diameter; 96-well plate) were irradiated with the Ti:sapphire laser with a spot size of 2 mm. The SK-BR-3 cells are adherent, hence their position is fixed on the surface of the well. It should be pointed out that the laser only irradiated 9.8% of the cells in each well (note cells are not drawn to scale). We also tried to use smaller wells (e.g
., 3.7 mm in diameter; 384-well plate), but they were found to increase the experimental error, so the 96-well plate was used. In one study, the treated cells were harvested at various times after irradiation to investigate when cells start to respond to the photothermal treatment. In , the cells were irradiated at a laser power density of 4.77 W/cm2
for 5 min. After that, the cells were returned to a 37 °C incubator for a specific duration of time before the percentage of cellular damage was quantified. Cells targeted with the immuno Au nanocages (●) exhibited more cellular damage at early harvest times. The cellular damage decreased at harvest times greater than 3 h after laser exposure, which could possibly be attributed to (i
) the cells untouched by the laser proliferated during their course in the incubator, resulting in a dilution of the destructed cells, and/or (ii
) some of the damaged cells recovered from the photothermal effect. Cells irradiated under the same conditions but without the immuno Au nanocages (
) exhibited no significant response during the given time. A small, unavoidable percentage of cellular death was observed, probably due to the pipetting and handling of cells during sample preparation. The results of this study indicate that the cells respond immediately to the photothermal treatment and should be harvested within 3 h after irradiation to better reflect the treatment. Harvest times shorter than 1 h after laser irradiation are not feasible due to the time required to prepare the sample for flow cytometry.
Figure 3 (A) Illustration depicting the experimental setup used for photothermal therapy of SK-BR-3 cells. The cellular growth vessel had a diameter of 6.38 mm. The cells were irradiated with an 805 nm Ti:sapphire laser with a spot size of 2 mm. Thus, only 9.8% (more ...)
Panels C and D of show the flow cytometry graphs of the cells with and without targeting by the immuno Au nanocages, respectively, at a harvest time of 3 h after laser irradiation. Forward scatter is plotted on the x-axis, and PI emission signal is plotted on the y-axis. The flow cytometry graphs were sectioned into four quadrants. Quadrant I, exhibiting a large forward scattering and low PI signal, represents the population of live cells. Since the live cells have intact cell membranes, PI could not stain their DNAs. Quadrant II, displaying large forward scattering and high PI signal, corresponds to the population of dying cells. The membranes of these cells had been compromised so PI could penetrate and then hybridize with their DNAs. The population of dead cells, or stained DNAs, exhibits a high PI and small forward scattering signal as shown in quadrant III. These are free DNAs, no longer enclosed within a membrane. Quadrant IV reveals a population with small forward scattering and low PI signal that could be attributed to instrumental background signals and debris from ruptured cells. The population in quadrant IV does not representatively correspond to live or damaged cells, so the percentage of cellular damage was normalized to the population in quadrants I, II, and III. A higher population of damage was observed when the cells were targeted with the immuno Au nanocages. These results exemplify that the immuno Au nanocages are effective photothermal agents capable of absorbing light and converting it into heat. The flow cytometry data also reveal that cells targeted by the immuno Au nanocages display irreversible damage upon photothermal treatment, as shown by the larger population in quadrant III, where the cellular membrane is broken to such an extent that the cell can no longer function nor recover from the damage. Although a small portion of damaged cells are present in quadrant II, the majority is observed in quadrant III, suggesting that the decrease of cellular damage over time was caused by the proliferation of cells outside the spot size and not by the recovery of the compromised cells as speculated earlier.
We also investigated the effect of the laser power density. shows a plot of cellular damage against the laser power density for cells irradiated for 5 min and harvested 3 h after irradiation. In the control, the cells without Au nanocages (
) maintained viability and the values are consistent with the control experiments shown in . Cells treated with the immuno Au nanocages (●) exhibited little or no damage at power densities less than 1.6 W/cm2
. At some threshold between 1.6 and 2.4 W/cm2
, the damage becomes significantly greater than the control. At power densities greater than 1.6 W/cm2
, the damage for cells treated with the immuno Au nanocages increases linearly, similar to what was found in our previous publication.19
As the power density increased to 6.4 W/cm2
, the cellular damage increased to 55%. Since the laser irradiated only 9.8% of the well, the cellular damage extended beyond the spot size of the laser, as is expected because the amount of heat generated increases with increasing laser power. As will be discussed later, the heat generated within the spot size can transfer to surrounding regions. Depending on the stage and type of cancer, cancerous cells can invade local regions of tumor sites and removal of nearby regions may be necessary, thus this linear relationship provides a means of calibrating the treatment to kill cancerous cells that have broken away from the primary tumor. Alternatively, by keeping the power density low, collateral damage to nearby healthy cells can be minimized.
Figure 4 Plots of cellular damage versus laser power density for SK-BR-3 cells incubated with immuno Au nanocages (●) and for the control in which cells were not incubated with immuno Au nanocages (). Cells were irradiated for 5 min and harvested (more ...)
The cells were also exposed to the laser for different periods of time. shows a plot of cellular damage against the duration of laser exposure for cells irradiated at 4.77 W/cm2
and harvested 3 h after irradiation. In the control, cells without immuno Au nanocage treatment (
) maintained viability, which is consistent with the results from the control experiments shown in and . The percentage of damage for cells treated with immuno Au nanocages (●) continuously increased for the first 5 min. After 5 min, the cellular death reached a steady percentage at about 35%. An exposure time of 1 min resulted in cellular damage (18%) larger than the percentage of cells irradiated by the laser (9.8%), suggesting that Au nanocages in the spot size of the laser irradiation responded quickly to the exposure. As the exposure time increased to 5 min, the cellular death increased, signifying that the death outside the spot size of the laser depends on the amount of time necessary for heat generated from the immuno Au nanocages to transfer to cells outside the irradiated regions. The cellular death becomes steady after 5 min, which could be attributed to other temperature gradients in the surroundings, thus establishing equilibrium. This relationship is important for calibrating the laser parameters for practical applications of in vivo
photothermal treatment. For example, a highpowered laser can be expensive, so rather than increasing the power of the laser, the time of laser exposure could be easily prolonged; however, after some extended period of time, the cellular death will become steady. This parameter provides another approach to treat the local invasion of tumor.
Figure 5 Plots of cellular damage versus laser exposure time for SK-BR-3 cells incubated with immuno Au nanocages (●) and for the control in which cells were not incubated with immuno Au nanocages (). In both cases, the cells were irradiated with (more ...)