Volume 112, Issue 6, Pages 1258-1269 (March 2017) Energy Transfer Mechanisms during Molecular Delivery to Cells by Laser-Activated Carbon Nanoparticles  Aritra Sengupta, Michael D. Gray, Sean C. Kelly, Stefany Y. Holguin, Naresh N. Thadhani, Mark R. Prausnitz  Biophysical Journal  Volume 112, Issue 6, Pages 1258-1269 (March 2017) DOI: 10.1016/j.bpj.2017.02.007 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 A schematic diagram showing TNET. The nanosecond laser heats nanoparticles, which produce acoustic, fluid mechanical, and thermal fields that lead to intracellular delivery of molecules. Biophysical Journal 2017 112, 1258-1269DOI: (10.1016/j.bpj.2017.02.007) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Representative fluorescence micrographs showing intracellular uptake of calcein in DU145 cells. (a) Exposure to both CB and laser generated significant intracellular uptake of calcein (iv), whereas exposure to either CB (ii) or laser (iii) alone did not. Addition of calcein 2 min after laser exposure also led to insignificant uptake (v). Laser fluence was 44 mJ/cm2, and CB concentration was 25 mg/L. (b) Intracellular uptake increased with increasing concentration of CB up to 50 mg/L, but then decreased at 75 mg/L (due to increased loss of cell viability (14)). Laser fluence was 44 mJ/cm2. (c) Intracellular uptake increased with increasing laser fluence up to 100 mJ/cm2, but then did not increase further at 200 mJ/cm2 (and did not lose additional cell viability (14)). CB concentration was 25 mg/L and all laser exposures were 7 min long. Scale bars, 100 μm. Biophysical Journal 2017 112, 1258-1269DOI: (10.1016/j.bpj.2017.02.007) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Bulk temperature rise in a cuvette containing CB nanoparticle suspensions at three different concentrations (0, 12.5, and 25 mg/L) and two different laser fluences (25 and 44 mJ/cm2) as measured using an infrared camera. The rate of temperature rise increased when higher CB concentrations were used and when larger laser fluence was applied. (Solid lines) Predicted temperature rise modeled using Eq. 3. Biophysical Journal 2017 112, 1258-1269DOI: (10.1016/j.bpj.2017.02.007) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 (a) Representative time domain acoustic output of a 50 mg/L CB nanoparticle suspension measured using a hydrophone. (Inset) Expanded view of the same data. Data show mean values (n = 121). (b) Peak pressure increased linearly with increasing CB nanoparticle concentration. (c) Increasing laser fluence increased the pressure nonlinearly. Unless otherwise noted, measurement distance from source was 5 mm, CB concentration was 50 mg/L, and fluence was 303 mJ/cm2. Data show mean ± SD (n = 100–115). Biophysical Journal 2017 112, 1258-1269DOI: (10.1016/j.bpj.2017.02.007) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Effect of acetic acid on pressure emission during laser irradiation. When water was replaced with acetic acid (which does not participate in the carbon-steam reaction), there were still significant pressure emissions. CB concentration was 0.4 g/L and laser fluence was 347 mJ/cm2. Data show mean ± SD (n = 34–127). Biophysical Journal 2017 112, 1258-1269DOI: (10.1016/j.bpj.2017.02.007) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Effect of segregating cells and CB nanoparticles during laser exposure. When cells and nanoparticles were mixed, there were significant bioeffects that increased with increasing laser fluence. When cells and nanoparticles were separated by a 10-μm-thick acoustically transparent film, background levels of bioeffects were seen. Bioeffects were measured by adding propidium iodide during and after laser irradiation to label permeabilized cells, whether viable or nonviable. CB nanoparticle concentration was 50 mg/L. Scale bars, 100 μm. Biophysical Journal 2017 112, 1258-1269DOI: (10.1016/j.bpj.2017.02.007) Copyright © 2017 Biophysical Society Terms and Conditions