Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrates

Transcription

1 Supplementary Materials for Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrates Nabiha Saklayen *, Marinus Huber, Marinna Madrid, Valeria Nuzzo, Daryl I. Vulis, Weilu Shen, Jeffery Nelson, Arthur A. McClelland, Alexander Heisterkamp *, Eric Mazur * *Corresponding author. nsaklayen@gmail.com (N.S.); heisterkamp@iqo.unihannover.de (A.H.); mazur@seas.harvard.edu (E.M.) This PDF file includes: Figs. S1 to S11 Tables S1 to S6

2 Fig. S1. Laser setup. An Nd:YAG laser with pulses of 11 ns duration, repetition rate of 50 Hz, and 1064 nm wavelength passes through an optical isolator to prevent back reflections, a half-wave plate and a polarizer to control the laser transmission with a computer. A pelican beam splitter measures a small portion of the laser energy during experiments. A lens finally loosely focuses the beam to the desired beam diameter of 1.2 mm. The stage moves at an average speed of 10 mm/s. 1

3 Fig. S2. Fabrication steps for template-stripped thermoplasmonic substrates 2

4 Reflection Transmission Absorption percentage ,000 1,200 wavelength (nm) Fig. S3. Absorption, reflection and transmission spectra of plasmonic substrates illuminated from above. 3

5 Fig. S4. Temperature simulations. (A) Temporal evolution of the maximum water temperature near the nano-hotspot for different laser fluences. The envelope of the excitation pulse is indicated in gray. (B) Top view of the temperature profile of a pyramid. (C) Temporal evolution of the maximum water and gold temperatures for a laser fluence of 55 mj/cm 2. The envelope of the excitation pulse is indicated in gray. (D) The maximum water and gold temperatures for different laser fluences. 4

6 E-field Fig. S5. Damage image. SEM image of a pyramid after laser illumination at a fluence above 200 mj/cm 2. Severe melting is observed on the pyramid apex irradiated at high fluence. The damage indicates that melting only occurs in the thin metal film and there is no visible effect on the polymer layer under the gold film. The pyramid has a base length of 2.4 µm. 5

7 48 mj/ cm 2 52 mj/ cm 2 56 mj/ cm 2 60 mj/ cm 2 Fig. S6. SEM images of pyramids after intracellular delivery. No damage observed on substrate after laser experiment at different fluence scans. Pyramids have base lengths of 2.4 µm. The dark lines and spots on the pyramids are portions of cells that remain after chemical fixing. 6

8 Fig. S7. Distribution of cargo within the cell. This image shows uniform distribution of delivered cargo within each cell. Structured Illumination Imaging of cell in FITC channel (left) and calcein AM channel (right). FITC-Dextran 150 kda is distributed evenly within each cell. Scale bar is 50 µm. 7

9 Fig. S8. Cells proliferate over 48 hours, with cargo inside. (A) FITC-Dextran 150 kda delivery after 24 hours. (B) Calcein AM channel viability after 24 hours. (C) Dextran 150 kda retention after 48 hours of the same region. (D) Viability after 48 hours. Cell density is higher after 48 hours, indicating that the cells not only remain viable but continue to divide after the laser experiment. Scale bar is 50 µm. 8

10 B0 G0R 0 G1 E1 Fig. S9. FITC-dextran 10 kda representative flow cytometry data. (details in Table S2) 9

11 Fig. S10. FITC-dextran flow cytometry for different-sized molecules. (A) Calcein green. (B) FITC-Dextran 10 kda. (C) FITC-Dextran 70 kda. (D) FITC-Dextran 150 kda. (E) FITC-Dextran 500 kda, and (F) FITC-Dextran 2000 kda. 10

12 Fig. S11. FITC-dextran 150 kda flow cytometry for area normalization. The average efficiency for the standard samples (with a flat edge) was 49%. The average efficiency for a sample with no flat edge was 68%. Therefore a normalization factor of 1.4 (68%/49%) was used to normalize the delivery efficiency for flow cytometry data. 11

13 Table S1. Viability of cells with or without Calcein Green dye incubation and with or without laser illumination at 54 mj/cm 2. Data represent mean ± standard error from n = 3 independent experiments. Laser illumination Calcein Green added Viability No No 97.7±0.9 Yes No 97.8±0.5 Yes Yes 99.3±0.6 No Yes 98.7±0.2 Table S2. Reagent list for flow cytometry. Probe Fluorochrome Vendor/ Cat. No. Calcein green FITC ThermoFisher Scientific/ C481 FITC-dextran 10 kda FITC Sigma-Aldrich/ FD10S FITC-dextran 70 kda FITC Sigma-Aldrich/ FD70S FITC-dextran 150 kda FITC Sigma-Aldrich/ FD150S FITC-dextran 500 kda FITC Sigma-Aldrich/ FD500s FITC-dextran 2000 kda FITC Sigma-Aldrich/ FD2000S Live-dead discriminator BD CS&T beads used for QC CellTrace Calcein red-orange, AM (PE channel in flow cytometer) BD TM Cytometer Setup & Tracking Beads (used with FACSDiva software v 6.x) ThermoFisher Scientific/ C34851 BD Biosciences Catalog #; Table S3. Sample information for flow cytometry data sets. Sample ID Description Laser scanned FITC channel Calcein red-orange AM channel B0 Control: blank sample to set initial gates. No No No G0R0 Control: to determine background of green No Yes Yes dye and viability of cells when not laser scanned. G1 Control: negative control for viability. Yes Yes No E1-E3 Experiments: in triplicate. Yes Yes Yes 12

14 Table S4. Flow cytometry settings on BD LSRFortessaSORP TM cell analyzer running BD FACSDiva software version Laser Parameter name Long pass filter Bandpass filter 488 nm SSC n/a 400/ nm FITC-Cargo / nm Calcein AM redorange n/a 582/15 *FSC detector is a photodiode Table S5. Viability of cells with or without Dextran dye incubation and with or without laser illumination. Data represent mean ± standard error from n = 3 independent experiments. Laser illumination Dextran added Viability No No 98.6±0.2 Yes No 98.5±0.3 Yes Yes 97.3±0.3 No Yes 98.5±0.3 Table S6. ICP-MS for Dextran 10 kda Sample ID Final concentration of Au (parts per billion) Control on glass 20 Gold nanoparticle control 171 B0 20 G1 18 G0R0 20 E1 17 E2 17 E