Tunable plasmonic nanoprobes for theranostics of prostate cancer

Abstract

Theranostic applications require coupling of diagnosis and therapy, a high degree of specificity and adaptability to delivery methods compatible with clinical practice. The tunable physical and biological effects of selective targeting and activation of plasmonic nanobubbles (PNB) were studied in a heterogeneous biological microenvironment of prostate cancer and stromal cells. All cells were targeted with conjugates of gold nanoparticles (NPs) through an antibody-receptor-endocytosis-nanocluster mechanism that produced NP clusters. The simultaneous pulsed optical activation of intracellular NP clusters at several wavelengths resulted in higher optical contrast and therapeutic selectivity of PNBs compared with those of gold NPs alone. The developed mechanism was termed "rainbow plasmonic nanobubbles." The cellular effect of rainbow PNBs was tuned in situ in target cells, thus supporting a theranostic algorithm of prostate cancer cell detection and follow-up guided destruction without damage to collateral cells. The specificity and tunability of PNBs is promising for theranostic applications and we discuss a fiber optic platform that will capitalize on these features to bring theranostic tools to the clinic.

Keywords: Plasmonic nanobubble; gold nanoparticle; laser; photothermal; prostate cancer; theranostics..

Figure 1

Principle of optical generation and detection of (A) standard PNB generated with single laser pulse around mono-NP cluster and (B) rainbow PNB generated with several laser pulses (shown with green and purple arrows) and multi-NP cluster (yellow and purple NPs with different plasmon resonances), (C) thermal outputs of single NPs, mono-NP clusters and multi-NP clusters under identical optical excitation with two laser pulses (as in case (B)): only the synergistic effect of two simultaneous plasmon resonances in one NP cluster deliver the thermal energy sufficient for PNB generation. Red arrows in (A) and (B) show PNB detection through optical scattering of an additional probe laser beam.

Figure 2

A,C,E: time-resolved scattering optical images show individual transient PNBs generated around single NP clusters during exposure to the two simultaneous laser pulses (0.5 ns each, 532 nm and 787 nm): A - the mono-NP cluster of gold spheres, C - the mono-NP cluster of gold rods, E - the multi-NP cluster of the same diameter as in A and C but consisting of a mixture of gold spheres and rods; the pixel image amplitude is shown in the gray scale. B, D, F: corresponding time responses obtained simultaneously with the images A,C,E and characterize maximal diameter of the PNB through the duration of the PNB-specific signal. Scale bar is 10μm.

Figure 3

SEM images of the clusters prepared from: (A) gold solid spheres (NSP) 60 nm, (B) gold rods (NR) 25x75 nm, (C) gold NRs and NSPs (multi-NP cluster).

Figure 4

Lifetime and brightness (relative to that of the corresponding NPs) of individual PNBs generated in water with two simultaneous laser pulses (0.5 ns, 532 nm@6 mJ/cm² and 787 nm@14 mJ/cm²): around single NPs, spheres (plasmon peak close to 532 nm) and nanorods (plasmon peak close to 787 nm); around mono-NP clusters consisting of spheres (NSP) or rods (NR) and around multi-NP clusters that included both NSP and NR (rainbow mechanism).

Figure 5

Confocal fluorescent, confocal scattering images of prostate cancer C4-2B (A,B) and stromal HS-5 (D,E) cells: (A,D) - confocal fluorescent images of AlexaFluor488 conjugated to PSMA antibody, (B,E) - confocal scattering images of gold NPs clusters (shown in red on the green fluorescent background that shows cell tracker dye) and (C,F) the corresponding profiles of scattering signal amplitudes by gold NPs clusters in cells.

Figure 6

PNB lifetime-probability of generation diagrams obtained for the cell samples. A - prostate cancer (C4-2B) and stromal (HS-5) cells that were treated with the mixture of gold NP conjugates (NSP60-PSMA and NS110-C225) and were exposed to single and paired laser pulses (532 nm@16 mJ/cm² and 787 nm@19 mJ/cm²): for C4-2B cells: hollow star - single pulse 532 nm, half right star - single pulse 787 nm, solid star - pair of laser pulses at 532 nm and 787 nm (rainbow mechanism); for HS-5: hollow circle - single pulse 532 nm, half right circle - single pulse 787 nm, solid circle - pair of laser pulses at 532 nm and 787 nm (rainbow mechanism). B - prostate cancer cells (C4-2B) that were treated with hollow gold shells 52 nm and were exposed to single and paired laser pulses (532 nm@10 mJ/cm² and 675 nm@8 mJ/cm²): half right square - single pulse 532 nm, hollow square - single 675 nm pulse, solid square - pair of laser pulses at 532 nm and 675 nm.

Figure 7

Microscopy images of living cells: bright field (A,C,E,G), time-resolved optical scattering (F,H) and fluorescent (Alexa Fluor 488 conjugated to PSMA antibody, B,D) images of prostate cancer C4-2B (A,B,E,F) and stromal HS-5 (C,D,G,H) cells; (F) and (H) - time-resolved scattering images of the cells presented in (E) and (G), respectively, that were exposed to the pair of laser pulses at 532 nm and 787 nm: image (F) shows the rainbow PNBs in a C4-2B cell while the image (H) shows no PNB and only the scattering by residual gold NPs in HS-5 cell.

Figure 8

Fluorescent images of the mixture of prostate cancer C4-2B (red) and stromal HS-5 (green) cells: (A) before, (B) after the first scan and (C) 60 s after the second scan, exposure to the pair of laser pulses (532 nm and 787 nm) that selectively generated cell damaging PNBs causing fading of red fluorescence due to leaking of red calcein out through the disrupted membrane. Dashed line shows the area that was exposed to the laser pulses. D - cell population averaged fluorescent signal amplitudes for C4-2B and HS-5 cells before and after treatment with laser pulses. E and F - the time responses of PNBs in C4-2B cell obtained during the first and the second scans.

Figure 9

Fiber optic PNB system for treatment of mulitfocal cancers such as prostate. A: general scheme that shows fiber optical probe connected through the fiber bundle to photodetector, through the multimode fiber to the pump and probe lasers, and through the capillaries to the syringe pump with the NPs and optical buffer fluid. B: scheme of the operation of fiber optic probe in tissue, volume scan is provided by sliding and rotating the probe in the guiding needle. C: the green line shows the attenuation of optical fluence in scattering tissue relative to the PNB thresholds for normal and prostate cancer (PC) cells, the difference between these thresholds determines an effective radius of PNB-based selective ablation of prostate cancer cells.