Supporting Information For Nanostraw Electroporation System for Highly Efficient Intracellular Delivery and Transfection. Nicholas A.

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1 Supporting Information For Nanostraw Electroporation System for Highly Efficient Intracellular Delivery and Transfection Xi Xie 1, Alexander M. Xu 1, Sergio Leal-Ortiz 2, Yuhong Cao 1, Craig C. Garner 2, Nicholas A. Melosh 1, * 1 Department of Materials Science and Engineering, Stanford University, Stanford, California 94305, USA 2 Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, Corresponding author: Nicholas A. Melosh 476 Lomita Mall McCullough Bldg. Stanford, CA Phone: (650) Fax: (650) nmelosh@stanford.edu California 94304, USA

2 Supporting Information Figures Figure S1. Schematic of nanostraw-electroporation. (a) Cellular engulfment of the nanostraws provides an intimate contact, offering highly effective electroporation and delivery. (b) Porous membranes provide non-uniform cell-to-substrate contact, limiting delivery efficiency for a given cell viability. Figure S2. Schematic of nanostraw fabrication. (a) Nanoporous track-etched polycarbonate membranes were (b) coated with 20-nm-thick alumina on all the surfaces, and then (c) the alumina on the top surface was removed with a directional reactive ion etch (RIE). Finally (d) the polymer was selectively etched by oxygen RIE to expose the resultant nanostraw.

3 Figure S3. Spatial selectivity of delivery. Delivery was highly selective on cells positioned over the microfluidic channel were delivered with PI dye. (a) PI dye was delivered into cells (red). (b) Live cells were labeled with calcein AM (green). (c) Phase image shows the channel boundary. Figure S4. Cell viability after electroporation. (a) Cell viability in 3 days after electroporation, compared with (b) viability of cells cultured on nanostraw membranes without electroporation. Live cells were labeled with calcein AM (green) and dead cells labeled with ethidium homodimer-1 (red).

4 Figure S5. Supplemental PI delivery and prfp transfection figures. (a) HEK transfection data. HEK cells were successfully tranfected with prfp (red) with efficiency of ~ 67%. (b) Control experiment data. Nanostraws (diameter 250 nm) were unable to deliver PI dye into cell without electroporation. (c) Porous membrane electroporation system is capable of delivering PI dye (red) into cells. (d) Low voltage (6 V / 20 µs / 2000 pulses) was sufficient to achieve efficient transfection (71%) into CHO cells. (e) Testing of membrane resealing after electroporation. Injection of RFP plasmid 10 s after electroporation yielded positive transfection signals (red), while (f) injection of plasmid 10 min after electroporation showed no transfection. In (a)-(f), cell nuclei were stained with hoechst (blue).

5 Figure S6. Plasmids were electrophoretically driven through the nanostraw. Figures showed prfp transfection results. (a) RFP plasmid was added 10 s after electroporation, (b) plasmid was present in the microfluidic channel during electroporation, and (c) plasmid was present during electroporation but electrode polarity was reversed. Figure S7. (a)-(b) Gene expression level (measured by RFP fluorescent intensity) can be controlled by adjusting (a) the plasmid concentration or (b) pulse number. In (a), fluorescent intensity of the three samples were normalized to the sample of "plasmid 0.32 µg/µl". In (b), fluorescent intensity of the three samples were normalized to the sample of "800 pulses".cell viabilities over 3 days after transfection at varied (c) plasmid concentrations and (d) pulse number were evaluated.

6 Figure S8. Correlation of GFP and RFP expression in single cells. Each dot represents a single cell fluorescence measurement, where the x-axis indicates the normalized GFP intensity and y-axis indicates the normalized RFP intensity. (a) During co-transfection where both plasmid factors were delivered at the same time, RFP and GFP fluorescent intensities were correlated (a linear function), such that suggesting both plasmids were delivered in equal quantities into the same recipient cells. (b) In sequential transfection where each plasmid was delivered 24 hours apart, fluorescent intensities of RFP and GFP in the same cell were uncorrelated, showing cell access over this time period consisted of two independent events.