Tunable plasmonic nanobubbles for cell theranostics

Abstract

Combining diagnostic and therapeutic processes into one (theranostics) and improving their selectivity to the cellular level may offer significant benefits in various research and disease systems and currently is not supported with efficient methods and agents. We have developed a novel method based on the gold nanoparticle-generated transient photothermal vapor nanobubbles, that we refer to as plasmonic nanobubbles (PNB). After delivery and clusterization of the gold nanoparticles (NP) to the target cells the intracellular PNBs were optically generated and controlled through the laser fluence. The PNB action was tuned in individual living cells from non-invasive high-sensitive imaging at lower fluence to disruption of the cellular membrane at higher fluence. We have achieved non-invasive 50-fold amplification of the optical scattering amplitude with the PNBs (relative to that of NPs), selective mechanical and fast damage to specific cells with bigger PNBs, and optical guidance of the damage through the damage-specific signals of the bubbles. Thus the PNBs acted as tunable theranostic agents at the cellular level and in one process that have supported diagnosis, therapy and guidance of the therapy.

Figure 1

PNB cell theranostic with multi-stage tunable PNB: (a) cell is targeted with NP-antibody conjugates and intracellular NP clusters are formed through the receptor-mediated endocytosis, (b) the 1st (diagnostic) PNB provides the data on a cell and allows one to determine the parameters of the next laser pulse, (c) the 2nd PNB delivers mechanical impact (cell damage though membrane disruption is shown) and this action is guided through the increased optical scattering (red arrows) of the 2nd PNB; the PNB is tuned by varying the fluence of the pump pulse (green arrows).

Figure 2

Experimental setup: single gold NPs in water or individual cells in the sample chamber were mounted on the stage of an inverted optical microscope; PNB generation was provided by focused single pulses (532 nm, 0.5 ns); a pulsed probe laser (690 nm, 0.5 ns) provided time-resolved optical scattering imaging of the PNB and a continuous probe laser (633 nm, 1 mW) provided monitoring of the optical scattering of PNBs though their time responses. An additional pulsed laser (532 nm, 10 ns, 1 mJ cm⁻²) was used for excitation of fluorescence in the cells.

Figure 3

Targeting the cell with gold NPs (a) and optical generation and detection of the intracellular PNBs: the 1st one non-invasively amplifies optical scattering (b), while increasing the fluence of the pump laser pulse induces the 2nd PNB that mechanically damages the cell (c); I—stages of the PNB theranostic action, II—optical pulsed scattering images of one cell with the membrane border shown with a white line, III—optical time response of the PNB shows its lifetime, IV—bright field and V—fluorescent (ethidium bromide-specific) images of the cell show it before (a) and after the generation of the 1st (b) and the 2nd (c) PNBs.

Figure 4

Influence of the fluence of a single pump laser pulse (532 nm, 0.5 ns) on the PNB parameters and on the damage as measured in individual A549 cells: (a) PNB generation probability (PRB): (◆)—cells incubated with NP-C225 conjugates, (■)—intact cells; cell damage probability (PD): (◇)—cells incubated with NP-C225 conjugates, (□)—intact cells; (b) PNB lifetime: (◆)—cells incubated with NP-C225 conjugates, (■)—intact cells; (c) amplification of optical scattering amplitude by the PNB (relatively to gold NPs) in the NP-treated cells as function of the PNB lifetime (i.e. maximal size of the PNB).

Figure 5

Future clinical application scheme for PNB theranostics for treating superficial and subcutaneous tumors and metastases. (a) Injection of the buffer suspension of gold NP conjugates that selectively cauterize around cancer cells (dark green); (b) diagnosis stage: optically transparent buffers flush unbound NPs and create an optically transparent medium for irradiation of the targeted cancer cells with a short pump pulse (green) that selectively generates the PNB around the NP clusters; small nanobubbles act as optical scattering probes for the detection of cancer cells with the probe laser (red); (c) therapy: selective mechanical destruction of the cancer cells is provided by the 2nd, bigger PNB generated with the second pump laser pulse, and is optically guided though the destruction-specific scattering of the probe laser radiation by the 2nd PNB; (d) as a result of the 2-pulse process the cancer cells are selectively eliminated without damage to collateral normal cells.