Photothermal nanotherapeutics and nanodiagnostics for selective killing of bacteria targeted with gold nanoparticles

Abstract

We describe a new method for selective laser killing of bacteria targeted with light-absorbing gold nanoparticles conjugated with specific antibodies. The multifunctional photothermal (PT) microscope/spectrometer provides a real-time assessment of this new therapeutic intervention. In this integrated system, strong laser-induced overheating effects accompanied by the bubble-formation phenomena around clustered gold nanoparticles are the main cause of bacterial damage. PT imaging and time-resolved monitoring of the integrated PT responses assessed these effects. Specifically, we used this technology for selective killing of the Gram-positive bacterium Staphylococcus aureus by targeting the bacterial surface using 10-, 20-, and 40-nm gold particles conjugated with anti-protein A antibodies. Labeled bacteria were irradiated with focused laser pulses (420-570 nm, 12 ns, 0.1-5 J/cm(2), 100 pulses), and laser-induced bacterial damage observed at different laser fluences and nanoparticle sizes was verified by optical transmission, electron microscopy, and conventional viability testing.

FIGURE 1

The principle of selective laser killing of S. aureus targeted with gold nanoparticles.

FIGURE 2

Schematics of integrated PT setup for diagnosis and therapy.

FIGURE 3

Images of S. aureus with attached gold nanoparticles: (a) phase contrast image (100×, NA 1.3); (b) PT image of bacteria alone (100×, NA 1.25, laser: 520 nm, 100 μJ, with a fluence 20 J/cm²); (c) PT images of bacteria with 40-nm gold particles at laser energy 2 μJ (0.4 J/cm²), time delay between pump and probe laser pulse 30 ns; and (d) PT images of bacteria with 40-nm gold particles at laser energy 10 μJ (2 J/cm²), time delay 120 ns. Dashed lines represent the bacterial boundary in c and d. Arrows in d indicate PT images of single nanoparticles, whereas the arrowhead shows a bubble around one nanocluster.

FIGURE 4

Integral PT responses from single S. aureus bacterium at a laser energy of 100 μJ (a) and S. aureus with 40-nm gold nanoparticles at laser energies/fluence of 0.5 μJ/0.1 J/cm² (b), 3.5 μJ/0.7 J/cm² (c), and 10 μJ/2 J/cm² (d). Amplitude (vertical axis)/timescale (horizontal axis) for images a–d: 50 mV/400 ns/div; 100 mV/100 ns/div; 200 mV/1 μs/div; and 500 mV/1 μs/div, respectively.

FIGURE 5

TEM images of S. aureus conjugated with gold nanoparticles before (a) and after (b–e) multilaser exposure of 100 pulses, wavelength of 532 nm, and pulse duration of 12 ns at a different conditions (see text): laser fluence of 0.5 J/cm² and no clusters (b); laser fluence of 0.5 J/cm² with clustered nanoparticles (c); and laser fluence of 3 J/cm² at one (d) and several (e) nanocluster numbers. A dashed line represents the bacterial boundary in e. Arrows in b and c indicate penetration of nanoparticles into the wall, and in d, arrows indicate local cell-wall damage.

FIGURE 6

Phase-contrast images of S. aureus with 40-nm nanoparticles before (a) and after (b) laser exposure (532 nm; 12 ns; 3 J/cm², 100 pulses).

FIGURE 7

Nonlinear PT response from S. aureus as a function of nanoparticles size. Laser parameters: wavelength, 525 nm; pulse width, 8 ns; and pulse energy, 1 μJ (0.2 J/cm²).

FIGURE 8

Selectivity of binding S. aureus with bioconjugated gold nanoparticles. The phase contrast (a) and fluorescent images of S. aureus labeled with chicken anti-goat IgG as secondary antibodies with Alexa Fluor 594 without (b) and with (c) the primary chicken-anti-mouse IgG antibody labeled with Alexa Fluor 488. Bacterial viability was assessed after laser exposure (532 nm, 12 ns, 3 J/cm²) using 40-nm gold nanoparticles by plating the sample on tryptic soy agar (d). The results of plate counting of samples without laser treatment were assumed as 100%. Statistical values were reported as means ± SD of two independent experiments in triplicates. Differences between means were calculated by Student's t-test for paired values. (*p = 0.0006.)

FIGURE 9

Efficiency of laser killing of S. aureus targeted with 40-nm gold nanoparticles as a function of laser fluence (0–5 J/cm²). Bacterial viability was assessed by plate counting. Laser parameters are: wavelength, 532 nm; pulse width, 12 ns; and number of pulses, 100. The open squares represent samples irradiated without gold nanoparticles; the solid squares represent samples irradiated with attached gold nanoparticles. The plate-counts of samples without laser treatment were assumed 100% viable. Statistical values are reported as means ± SD of three independent experiments in duplicates.