## 3 Results
### Characterization of Au Nanopeanuts
The resulting TBO-conjugated PEGylated (TBO-PEGylated) nanopeanuts dispersed in H\({}_{2}\)O lead to a light blue colloidal solution, which exhibits the same UV-Vis-NIR absorption spectral contour as the absorption spectral contour of nanopeanuts alone (Figure 2d). Interestingly, an additional peak appears at -660 nm, a slight redshift from the absorption band (630 nm) of pure TBO. We think that this additional peak may arise from the interaction of the surface plasmon of nanopeanuts with TBO molecules [26; 27]. Then we evaluated the cytotoxicity of TBO-PEGylated nanopeanuts (Figure 3). A series of particle concentrations were incubated with A549 cells in 96-well plates for 24 h and subjected to MTT measurements. The nanopeanut alone has shown a certain degree of toxicity at a concentration larger than 10 \(\upmu\)g/mL, which is likely due to the Ag composition in nanoshells. Fourier transform infrared (FTIR) spectroscopy measurements show no CTAB features on the nanopeanut surface. In contrast, neither PEGylated nor TBO-PEGylated nanopeanuts displayed toxic effects at concentrations up to 100 \(\upmu\)g/mL. Before performing two-photon activation experiments, we employed a 633 nm diode laser to verify that TBO-PEGylated nanopeanuts can generate sufficient singlet oxygen to cause photodynamic damage in malignant cells. Since the absorption band of TBO is located at 620-670 nm, a 633-nm diode laser can directly excite TBO (one-photon excitation) to yield singlet oxygen. With the Singlet Oxygen Sensor Green Reagent (Ex/Em: 505/525 nm) (Invitrogen), the direct detection of singlet oxygen was performed using a fluorescence spectrophotometer. Singlet Oxygen Sensor Green is a nonfluorescent molecule, but it can react with singlet oxygen to become a fluorescent one. The fluorescence intensity of Singlet Oxygen Sensor Green is proportional to the length of irradiation time, indicating that a longer irradiation time produces more singlet oxygen (Figure S5). Figure 3: Cell viability studies of A549 cells incubated individually with different concentrations of nanopeanuts at 37 \({}^{\circ}\)C for 24 h.  
Then, we used a dark field and fluorescence imaging system (CytoViva microscope) to monitor cell viability under illumination with a 633-nm diode laser (Figure 4). Laser irradiation at 633 nm had no effect on cell viability. After treating cells with TBO-PEGylated nanopeanuts (10 \(\upmu\)g/mL) for 4 h at 37 \({}^{\circ}\)C, we observed cell uptake under darkfield scattering images. Without laser irradiation, it also showed no damage in cells. After removing uninternalized colloids by PBS wash, A549 cells were subjected to 3 min of laser exposure (200 mW/cm\({}^{2}\)), followed by an additional 16 h incubation. Significant cell destruction was observed in the darkfield image. The disintegration of cytoplasm and the condensation of cell nuclei appeared in fluorescent images as well. These results indicate effective singlet oxygen generation and cancer cell damage upon 633-nm diode laser exposure. The low laser power (200 mW/cm\({}^{2}\)) did not cause a temperature elevation of the colloidal solution, remaining at 25 \({}^{\circ}\)C after 20 min irradiation. There was no hyperthermic effect in the course of irradiation. After characterizing the PDT capability of the TBO-PEGylated nanopeanut, we demonstrated its nonlinear optical property using a 1230-nm femtosecond Cr: forsterite laser. The multiphoton fluorescence spectra of Au nanopeanuts are broad and enhanced by the surface-plasmon resonance effect in the nanopeanuts (Figure 5, red curve). An additional band was observed at ~670 nm when TBO-PEGylated nanopeanuts were excited (Figure 5, black curve). Such TBO fluorescence was also observed when the TBO-PEGylated nanopeanuts were excited by a xenon arc lamp from a conventional fluorescence spectrometer (Figure S6).