Figure 1.
PNBs and NPs in target (left panels) vs non-target (right panels) cells.
A: gold NP conjugates are collected at cellular membranes and are clustered during endocytosis resulting in the largest NP clusters in target cells. B: Excitation laser pulse (green) of low fluence induces PNBs only around the largest NP clusters (i.e. only in target cells) because the PNB generation threshold fluence for single NPs and small clusters (non-target cells) is higher than the fluence of the laser pulse. C: Optical scattering of the probe laser radiation (red) by PNBs provides its real-time imaging and monitoring in the individual cell (ID: image detector, RD: response detector).
Table 1.
Cell models and conditions of their treatment with NP and laser pulses.
Figure 2.
Parameters of PNBs generated around gold NP clusters in water for gold nanoshells.
A: PNB generation threshold fluence of the excitation laser pulse as function of NP cluster size (measured through optical scattering amplitude of NP cluster image for individual clusters); B: PNB lifetime and scattering brightness as function of the NP cluster size (measured through optical scattering amplitude of NP cluster image) at specific fluence of the excitation pulse (778 nm, 22 mJ/cm2).
Figure 3.
Images and signals of gold NPs and PNBs in co-culture of target (HN31, labeled with Green Fluorescent Protein for identification) and non-target (NOM9) cells identically treated with 60 nm gold NSP-C225 conjugates (specific to EGFR that is overexpressed in target cells).
A: overlay of bright field, fluorescent and scattering images shows target cells (green) and gold NPs (red) that can be found in both types of cells (the arrows show NP clusters in non-target cells); B: time-resolved scattering image of the same field shows PNB images (bright white spots) only in target cells; C,D: optical scattering time-responses of individual target (C) and non-target (D) cells show the PNB-specific signal only for target cell and the definition of the PNB lifetime of PNBs; time is measured from the moment of the exposure to the excitation laser pulse.
Figure 4.
Cell population-averaged levels of optical scattering signals obtained for individual target (solid bar) and non-target (hollow bar) cells in six cell models represented by target/non-target cells/molecular targets:
Squamous cell carcinoma, HN31/NOM9/EGFR (treated with 50 nm NS-Panitumumab conjugates); Lung cancer, A549/Fibroblast/EGFR (treated with 60 nm NSP-C225 conjugates); Epithelial cancer, HES/HS5/MUC1 (treated with 60 nm NSP-214D4 conjugates); Prostate cancer, C2-4B/HS5/PSMA (treated with 60 nm NSP-anti-PSMA conjugates); Leukemia, J32/JRT3-T3.5/CD3 and human T-cells, T-cell/BM/CD3 (treated with 60 nm NSP-OKT3 conjugates) for: Row A (red): gold NP amplitude of scattering image of gold NPs (a metric for the uptake of NPs by cells; Row B (purple): time-resolved scattering image amplitudes of PNBs; Row C (blue): PNB lifetimes. The ratio of the signals for target/non-target cell is shown for each parameter and cell model and indicates the cellular specificity of NPs (row A) and PNBs (rows B,C).
Figure 5.
Influence of targeting vectors on NP scattering amplitude (red) and PNB lifetime (blue) in individual target (solid bars) and non-target (hollow bars) cells.
A: Target (HN31) and non-target (NOM9) cells identically treated with bare 60 nm gold NSPs and NSP-Panitumumab conjugates (antibody specific to EGFR that is overexpressed in HN31 cells); B: Effects of EGFR-specific antibodies C225 and Panitumumab as targeting vectors in HN31 cell model show 5 different combinations of the two antibodies; C: Effects of single and dual targeting antibodies against PSMA and EGFR (C225) in C4-2B cell model applied in combination with dual simultaneous optical excitation (so called rainbow PNB method) show synergistic enhancement of PNB lifetime in the rainbow mode.