Cell-binding specificity of gold nanoparticle–antibody conjugates
To evaluate the conjugation stability of gold and antibodies and the binding specificity of different conjugates to L-428 cells, the two conjugates, gold-BerH2 (anti-CD30 receptor) and gold-ACT1(anti-CD25 receptor), and the goat anti-mouse Alexa 488 were adopted in this experiment. Both CD30 and CD25 are cell membrane proteins of the tumor necrosis factor receptors, but only CD30 has a high overexpression on the surface of L-428. Through the specific coupling of goat anti-mouse immunoglobulin G antibody to monoclonal antibody, stable coupling was achieved between aM-A488 and BerH2/ACT1. Whether or not aM-A488 attaches to L-428 depends on the expression of protein on L-428. Flow cytometry measurements are shown in . About 50,000 events were acquired per sample. The results indicate that only for the cells in the gold-BerH2 group, the detection of the fluorescence signal was positive. The fluorescence signal on the cells in the gold-ACT1 group was the same as that for the control cell group (negative). L-428 was positive for CD30, but negative for CD25. As a result, the gold-BerH2 conjugates bound specifically to CD30 receptors on the surface of L428 cells, which were also stained with aM-A488. A conceptual diagram of this binding process is shown in the inset box in . The conjugation stability was confirmed on one hand, but the different binding abilities of different conjugate-bound cells would bring a different performance in the cell-killing experiment.
Flow cytometry detection of the binding specificity of different gold nanoparticle–antibody conjugates.
Cytotoxicity of gold nanoparticles
shows cell viability of L428 cells after some hours of exposure to a certain concentration of gold, or gold-BerH2 conjugates. The ratio of pure gold to cells was 104:1, and the ratios of gold conjugates to cells were 104:1 and 108:1, respectively. The concentration of conjugates was increased to determine gold’s potential cellular toxicity. After incubation, the number of viable cells were stained with calcein-AM (2 μg/mL) and then measured using flow cytometry. For all samples, 20,000 events were acquired.
Viability of L-428 cells exposed to either unbound golds or gold-BerH2 conjugates for 6, 12, and 24 hours, as evaluated by flow cytometry.
For the groups of unbounded gold and gold-BerH2 conjugates incubated with L-428 cells at a ratio of 104, the cell viability was greater than 90% after 24 hours of incubation, which had no significant difference compared with the control cell group. At the ratio of 108:1, a bigger decrease in cell viability was observed after incubation for 24 hours and L-428 cell viability dropped to 84%. Therefore, for a short incubation period (less than 12 hours), there was no significant increase in the number of dead cells incubated with gold at a relatively low concentration. To avoid the cytotoxicity induced by gold with high doses and a long incubation period, the highest ratio of gold to cells used for the photothermal killing experiment was not more than 104:1, and the incubation period was less than 6 hours.
The effect of laser-irradiated power in cell viability in the presence of gold–antibody conjugates
To monitor the necrotic effect of laser irradiation on living cells in the presence of gold, we performed cell damage experiments on L-428 cells, which were incubated with gold-BerH2 conjugates at a 104:1 ratio for 20 minutes, and then irradiated with a laser at 50 mW, 5 pulses. After irradiation, cell viability was assessed using calcein-AM (2 μg/mL) and PI (1 μg/mL) staining, and tested using flow cytometry and optical fluorescence microscopy. The events for FACS were 20,000 events. The living cells were stained positively with calcein-AM solution. The cells positively stained with PI presumably represented the later stages of cell death, when membrane integrity was lost. The percentage of death was calculated as the number of PI positive cells divided by the total number of cells.
shows the photothermal treatment results, where severe destruction of L-428 cells was observed when the cells were exposed to laser irradiation (50 mW, 5 pulses) with gold-BerH2 conjugates. The flow cytometry results of L-428 cells incubated with gold-BerH2 conjugates treated with or without laser irradiation are shown on the left side. The corresponding fluorescent images are shown on the right side. Without laser irradiation, about 93% of the cell population was alive, which exhibited bright green fluorescence after calcein-AM staining. More than 95% of the cell population was dead after laser irradiation at 50 mW, 5 pulses, and the dead targets exhibited red fluorescence after PI staining.
Photothermal treatments of L-428 cells with gold-BerH2 conjugates. (A) With 532 nm laser irradiation with 50 mW, 5 pulses; (B) without laser irradiation.
Furthermore, the effect of laser influence on laser-induced cell damage has been considered in this section. To test whether the increased cell death rate is linked to laser irradiated power, we treated cell samples with different laser power settings. L-428 cells incubated with gold-BerH2 conjugates and the control cell group were seeded into the sample micro-cuvette respectively. The cell concentration was 4 × 106/mL, and the gold-BerH2 conjugate to cell ratio was 104:1. The cell samples were irradiated with 5 pulses and different laser power settings, and then tested using the flow cytometry by calcein-AM/PI double staining. show a representative flow cytometry dot plot illustrating the change in cell viability induced by different power laser irradiation. Right lower quadrants expressed the calcein-AM positive cells as a percentage of the total cell population. Left upper quadrants expressed the PI positive cells as a percentage of the total cell population. As shown in , for the control cell group, there was no remarkable change in cell viability when increasing the laser-irradiated power from 0 mW to 50 mW. But for the cells in the gold-BerH2 conjugates group as shown in , cell damage efficiency was affected significantly by the laser-irradiated power. The cell death rate was about 40% when using 15 mW laser irradiation; most of the cells died when the laser power was increased to 50 mW. In the presence of gold–antibody conjugates, the cell death rate mostly depends on the laser irradiated power.
Figure 5 Flow cytometry dot plot and statistical analysis diagram of L-428 cells after being irradiated at different laser power settings. (A) L-428 cells; (B) L-428 cells incubated with gold-BerH2 conjugates. (C) The results were analyzed by flow cytometry data. (more ...)
The statistical analysis of the repeated experiments is shown in . For the control cell group incubated without gold, the cell viability curve did not change much with the variation of laser-irradiated power; however, the curve of the cells with gold-BerH2 conjugates showed a rapid decrease in cell viability as the laser power increased. Excessive irradiated laser power caused devastating damage or complete necrosis of L-428 cells incubated with gold–antibody conjugates. Due to the specific aggregation of the CD30 receptor and BerH2 antibodies, gold-BerH2 conjugates were bound tightly to the surface of L-428 cells. Efficient conversion of light strongly absorbed by the gold to heat energy induced a significant increase in cell death.
Cell viability induced by different binding modes of gold to cells
The effect of the photothermal treatment of L-428 cells was evaluated in the above experiments by combining laser irradiation and gold nanoparticles. In this section, we compared the efficiency of different binding modes of gold to cells and cell viability. There were four experimental groups for comparing the killing efficiency with or without gold nanoparticles: the control cell group, cells with gold, cells with gold-BerH2 conjugates, and cells with gold-ACT1 conjugates. From the cell-binding specificity tests, it was known that BerH2 antibodies could bind specifically on the surface of L-428 cells, but ACT1 antibodies could not. Thus, the above four experimental groups represented four combination conditions between gold and cells: pure cells, cells with unbound gold, cells targeted by few nonspecific gold–antibody conjugates, and cells targeted by many specific gold–antibody conjugates. The cell samples were treated by laser irradiation with 40 mW, 5 pulses, which would be compared with the nonirradiated part. Then, 10,000 events were tested using flow cytometry by calcein-AM/PI double-staining.
The experimental analysis results are shown in , and were divided into two parts: irradiated and non-irradiated. In the non-irradiated part, there was no difference of cell viability between the four cell samples without laser irradiation. The presence of gold did not influence cell viability. In the irradiated part, there was no significant difference from the control group in the cell viability. For the cells in the unbound gold group and in the gold-ACT1 conjugates group, a slight increase in the death rate can be found. Due to the small volume of the sample micro-cuvettes, gold or gold-ACT1 conjugates in the sample volume might distribute in the surrounding cells, which could induce some heat energy shift to the cells. Compared with the other three groups, due to the specific coupling of CD30 antigen and BerH2 antibody, gold-BerH2 conjugates have a tight binding on the surface of L428 cells. Under laser irradiation with 40 mW, 5 pulses, a large damage rate of about 96% was observed in the L-428 cells.
The influence of gold nanoparticles on the viability of L428 cells under four different conjugation conditions: without gold (Control), with unbound golds (Golds), with gold-ACT1 conjugates (Gold-ACT1), and with gold-BerH2 conjugates (Gold-BerH2).