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Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrate Nabiha Saklayen, Marinus Huber, Marinna Madrid, Valeria Nuzzo, Daryl Inna Vulis, Weilu Shen, Jeffery Nelson, Arthur A. McClelland, Alexander Heisterkamp, and Eric Mazur ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b08162 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 14, 2017

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Figure 1. Nanosecond-laser excitation of large-area plasmonic substrates for intracellular delivery. A nanosecond laser is scanned across the sample for high-throughput delivery to a million cells cultures on the substrate. The laser spot illuminates thousands of cells simultaneously. Laser illumination cases thermoplasmonic nano-heating at the apex of each pyramid. The heating forms bubbles that create temporary pores in cells at close contact. The pores enable fluorescent cargo in the surrounding solution to diffuse directly into the cytosol. over the course of minutes. 67x15mm (300 x 300 DPI)

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Figure 2. Large-area template-stripped thermoplasmonic substrates. (a) Repeated template-stripping on photolithography templates enables low-cost fabrication of large areas. (b) One substrate consisting of 10 million pyramids next to a U.S. quarter dollar. (c) Top-down view of pyramids under Scanning Electron Microscopy (SEM). (d) SEM tilted view shows uniform base lengths (2.4 µm), heights (1.4 µm), and spacings (1.4 µm). (e) Finite Element Method simulations showing the thermoplasmonic pyramidal apexs reaching maximum temperature of 342 °C at a laser fluence of 45 mJ/cm². Due to the low repetition rate 123x55mm (300 x 300 DPI)


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Figure 3. Cell morphology on substrate. (a) SEM of chemically-fixed HeLa CCL-2 cells on a thermoplasmonic substrate. (b) Confocal laser-scanning microscopy slice of cell with Calcein red-orange AM fluorescence at z = 8.85 µm, as measured from the bottom of the cell. The cell is 40 µm in length. (c) Grid pattern from pyramids. (d) 27 z-slices stacked (with spacings of 0.57 µm) to show the cross section of the same cell taken along the dotted line in (c). but below the point where we d 95x44mm (300 x 300 DPI)


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Figure 4. Characterizing the efficiency and viability of delivery to cells, and confirming no visible substrate damage. (a) Overlay of a bright-field and fluorescent image. Areas I and III have pyramids. Areas II and IV are flat gold. A laser is scanned below the dotted line. Area III is the only region with green molecules delivered to cells. (b) Delivery efficiency and viability as a function of laser fluence. Data represent mean ± standard error from n = 3 independent experiments. (c) Calcein green delivery. (d) Calcein red-orange AM viability. (e) Overlay of images (c) and (d). (f) SEM image after chemical fixing. (g) Overlay of images (e) and (f). (h) Zoom in. Inset of an individual pyramid (2.4 µm base length) confirms no visible damage on pyramid. Filopodia of a cell at the bottom right corner of the pyramid. we used automated cell countin 210x181mm (300 x 300 DPI)


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Figure 5. Quantifying delivery efficiency and viability of different-sized molecules using flow cytometry. (a,b) Representative data from FITC-Dextran 150 kDa experiment. The experimental sample (a) is laser-scanned while the control (b) is not. A density plot of side scatter-area vs. Calcein red-orange AM fluorescence determines viable cells. The viable cells are plotted in a side-scatter vs. FITC-Dextran 150 kDa density plot to measure the delivery efficiency. The background signal is less than 1% in the control in (a). The delivery efficiency is 74% in the experiment (b). (c) Viability and efficiency for FITC-cargo ranging in size from 0.623 kDa to 2000 kDa. Data represent mean ± standard error from n = 3 independent experiments. granularity, respectively 248x372mm (300 x 300 DPI)


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Intracellular

Delivery

Using

Nanosecond-Laser

Excitation of Large-Area Plasmonic Substrates Nabiha Saklayen, †,1,* Marinus Huber,†,2,3 Marinna Madrid,2 Valeria Nuzzo,4 Daryl I. Vulis,2 Weilu Shen,2 Jeffery Nelson,5 Arthur A. McClelland,6 Alexander Heisterkamp, 7, * Eric Mazur1, 2, * 1

2

Department of Physics, Harvard University, Cambridge, MA 02138, USA.

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. 3

Department of Physics, Ludwig Maximilian University of Munich, 80539 Munich, Germany. 4

5

6

7

*

ECE Paris Ecole d’Ingénieurs, 75015 Paris, France.

Division of Science, Harvard University, Cambridge, MA 02138, USA.

Center for Nanoscale Systems, Harvard University, Cambridge, MA 02138, USA.

Institute for Quantum Optics, Leibniz University Hannover, 30167 Hanover, Germany.

Corresponding

authors.

Email:

[email protected]

(N.S.);

[email protected]

hannover.de (A.H.); [email protected] (E.M.)

ABSTRACT. Efficiently delivering functional cargo to millions of cells on the timescale of minutes will revolutionize gene therapy, drug discovery, and high-throughput screening. Recent


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studies of intracellular delivery with thermoplasmonic structured surfaces show promising results, but in most cases require time- or cost-intensive fabrication, or lead to unreproducible surfaces. We designed and fabricated large-area (14 x 14 mm), photolithography-based, template-stripped plasmonic substrates that are nanosecond laser-activated to form transient pores in cells for cargo entry. We optimized fabrication to produce plasmonic structures that are ultrasmooth and precisely patterned over large areas. We used flow cytometry to characterize the delivery efficiency of cargos ranging in size from 0.6 to 2000 kDa to cells (up to 95% for the smallest molecule) and viability of cells (up to 98%). This technique offers a throughput of 50,000 cells/min, which can be scaled up as necessary. This technique is also cost-effective as each large-area photolithography substrate can be used to deliver cargo to millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. The approach we present offers additional desirable features: spatial selectivity, reproducibility, minimal residual fragments, and cost-effective fabrication. This research supports the development of safer genetic and viral disease therapies as well as research tools for fundamental biological research that rely on effectively delivering molecules to millions of living cells.

keywords: plasmonic intracellular delivery, template-stripping, flow cytometry, pulsed lasers, thermoplasmonic substrates The direct intracellular delivery of functional proteins, enzymes, or genetic material is a powerful way to manipulate cell behavior for biomedical research and disease intervention.1,2 For example, functional proteins are delivered to ablate genes in hematopoietic stem cells with high precision for research and therapeutic applications.3,4 Enzymes are delivered for their ability to bind targets with high affinity and specificity.5,6 siRNA delivery facilitates gene silencing for


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biomedical applications.7,8 In the case of blood disorders such as human immunodeficiency virus (HIV) or leukemia, delivering functional molecules into a patient’s stem cells for transplantation therapy could cure them of the disease, circumventing the side effects of chemotherapy, transplantation rejection by the immune system, and the search for a matching donor.9–11 The ability to effectively deliver large and diverse cargos such as amino acids, peptides, proteins, protein cages, antibodies, polysaccharides, nucleic acids, viruses, and DNAs/RNAs directly into cells will transform biomedical research. However, no current intracellular delivery method, either biological, chemical, or physical, can offer a collection of advantageous features at once: (1) high efficiency, viability, and throughput, (2) diverse cargo delivery capability, (3) spatial selectivity (delivering to specific cells on a surface), scalability and reproducibility, (4) minimal post-delivery cytotoxicity, and (5) cost-effectiveness. Viral transduction is the most popular biological method due to decades of extensive research, but has limitations. For example, viral methods offer limited cargo-carrying capacity, only deliver genetic cargo, require customization of the virus for each cargo and cell type, and include immunotoxicity risks.12–14 Among non-viral methods, electroporation techniques offer high throughput and efficiency, but also lead to high cell death.15 Nucleofection offers improved viability, but require expensive customized reagents.16 Lipofection, a chemical method, offers high throughput but is cell-type specific and risks endosomal trapping of the cargo.17 Physical methods such as microinjection, nanowire-mediated delivery, and microfluidic squeezing are promising, but offer limited throughput and/or reproducibility.13,15,18–23 Thermoplasmonic nanostructures have the potential to become excellent tools for biomedical applications.24–26 Laser-activated thermoplasmonic gold nanoparticles form heat-mediated bubbles to make transient pores in cells for intracellular delivery.27–35 Despite being effective


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intracellular delivery agents, the long-term cytotoxicity of gold nanoparticles still remains under debate.36,37 Recent studies of intracellular delivery with thermoplasmonic structured surfaces are encouraging as they do not utilize gold nanoparticles in solution, but in some cases require timeor cost-intensive fabrication; in some instances, each substrate is fabricated using lithography methods, making it difficult to scale up to applications that require many large-area samples.20,38 Some substrate-based studies with gold nanoparticle layers are more easily fabricated, but can be non-reproducible in nature and leave metal fragments behind after the experiment.39 Template stripping is a high-throughput and reproducible approach to producing plasmonic structures in the form of metallic films that are ultrasmooth and precisely patterned over large areas.40–43 This fabrication method is highly advantageous as lithography needs to be performed once to fabricate templates in silicon that can be repeatedly used to generate template-stripped metallic substrates in a few steps. In our previous work, tipless pyramid substrates exhibiting a high near-field enhancement were irradiated with femtosecond laser pulses to show proof-ofprinciple intracellular delivery.44 However, only small areas could be fabricated due to templatestripping from an e-beam lithography template, limiting experiments to the study of tens of cells. The tipless pyramids required angular gold deposition using a thermal evaporator with rotating stage, which affected precision in reproducibility. Using a femtosecond laser made the optical setup complex. These challenges made it difficult to develop large-scale biomedical applications of the technique. We demonstrate that thermoplasmonic substrates made by template-stripping from large-area photolithography templates can be combined with nanosecond laser excitation to achieve intracellular delivery in large numbers of cells, which enables flow cytometry measurements. This technique is cost-effective as large-area photolithography templates can be used repeatedly


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to produce substrates to study millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. We show that template-stripped substrates, consisting of pyramidal nano-heaters, permit intracellular delivery with efficiencies of up to 95% for the smallest molecules, viability up to 98%, and scalable throughput of 50,000 cells/min, as quantified by flow cytometry. Additional desirable features of this technique include cargo delivery capacity up to 2000 kDa, spatial selectivity, scalability, reproducibility, minimized cytotoxicity from gold fragment residue, and no requirement of biological or chemical customization. Intracellular delivery using large-area template-stripped thermoplasmonic substrates will support the ability to effectively study cellular function with improved speed and control, which in the long run could lead to the development of gene therapies to cure chronic diseases. Results and Discussion Nanosecond laser illumination of large-area plasmonic substrates We use pulsed-nanosecond laser illumination of large-area template-stripped thermoplasmonic substrates to deliver cell-impermeable cargo directly into the cytosol of living cells (Fig. 1). The thermoplasmonic substrate consists of microscale pyramids with a continuous gold thin film on top, fabricated using photolithography and template-stripping. We culture cells on the substrate, surround them with a solution containing the delivery cargo, and scan a nanosecond pulsed laser across the substrate to generate intense heating at the apex of each pyramid, forming hotspots. The hotspots lead to bubble formation, creating transient pores in cells adhering to the substrate. These pores enable cargo from the surrounding solution to enter the cytosol via diffusion. A large beam spot illuminates the substrate to generate pores in thousands of cells simultaneously


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(setup shown in Fig. S1). Millions of cells are permeabilized when the laser is scanned across the substrate over the course of minutes.

In our previous research, we used tipless pyramids that were template-stripped from e-beam lithography templates and femtosecond laser-excited to show proof-of-principle intracellular delivery.44 The tipless pyramids were designed to maximize the high nearfield enhancement with femtosecond pulses as the high nearfield enhancement is considered a possible driving mechanism for cell poration.45 We switched to a compact, turn-key nanosecond laser to bring down the cost of the laser setup by a factor of 10 and reduce the setup complexity. Since the nanosecond laser causes heating to be dominant effect, we changed our design to regular pyramids (with tip in place) to ease the fabrication process. The nanosecond laser used has a wavelength of 1064 nm (which is advantageous for reduced scattering effects), repetition rate of 50 Hz, beam spot diameter of 1.2 mm, and a flat top beam profile. We use a large beam spot to directly scan the surface of the substrate instead of focusing through an objective to enable rapid scanning of large areas without worrying about losing focus. Due to the low repetition rate, each pulse can be seen as individual effect. In order to obtain larger pyramid arrays, our challenge was to optimize fabrication for large areas while maintaining uniform precision for approximately 10 million pyramids on a single substrate, across dozens of substrates. We fabricated silicon master templates (14 x 14 mm) using photolithography and anisotropic etching. Each template consists of 10 million inverted pyramids of base length 2.4 µm and edge-to-edge separation 1.2 µm (Details of the fabrication process in Supplementary Materials and Fig. S2). We deposit a 50-nm thick gold film onto dozens of master templates, and add UV-glue and a coverslip on top. After the glue cures, we


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peel off (“template-strip”) the gold, glue, and coverslip composite using a razor blade (Fig. 2A). This results in thermoplasmonic substrates comparable in size to a U.S. quarter dollar (Fig. 2B). By repeating gold deposition and template-stripping, we fabricate dozens of thermoplasmonic substrates with high precision in a single batch. Scanning electron microscopy (SEM) confirms that the template-stripped substrates are highly uniform in base lengths, spacings, and heights across the length of an individual substrate, and consistent from batch to batch (Fig. 2C and 2D). The absorption, reflection, and transmission spectra measured on the substrates are shown in Fig. S3. The master templates can be re-used hundreds of times for template-stripping. This intracellular delivery technique is easily-scalable as photolithography can be extended to make larger samples, and the scanning speed and/or beam spot size can be increased with additional optical components. Plasmonic pyramids are effective nano-heaters due to the combined effect of a thin metal film and sharp apex, making them ideal for thermoplasmonic applications.22,41–43 Sharp metallic nanostructures are ideal for absorbing laser energy and concentrating the energy to hotspots that form bubbles in an aqueous environment.24,46–48 We performed numerical simulations to confirm that the incoming laser energy is concentrated at the pyramidal apex, in a region of hundreds of nanometers, leading to localized heating (Fig. 2E). At fluences above 200 mJ/cm2 we observe damage at the apex of the pyramid, confirming experimentally that the heating is concentrated at the apex of the pyramids (Fig. S4). Using numerical simulations we determined the threshold laser fluence required for the temperature at the pyramidal apex to reach the temperature range for bubble formation. The critical temperature of water at 1 atm is 367 – 377 °C, and thermodynamical theory and experimental studies 46,49 show that explosive boiling occurs where temperatures reach 80 – 90%


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of this critical value, which corresponds to temperatures between 293 – 340 °C. Simulation results show that for fluences above 40 mJ/cm2, the aqueous environment within tens of nanometers surrounding the pyramidal apex reaches temperatures above 300 °C, which is within the temperature range for bubble formation. All laser experiments with cells are performed above the threshold laser fluence necessary for bubble formation (40 mJ/cm2), but below the point where we damage the gold film at the pyramidal apex (200 mJ/cm2).

We imaged the morphology of HeLa cells on the thermoplasmonic substrate using SEM and confocal microscopy. Figure 3A is an SEM image of a chemically-fixed cell on the pyramids. Because chemical fixation kills the cell and alters the cell’s lipid membranes, we additionally used confocal microscopy to image the morphology of a living cell that was fluorescently tagged with a cell-permeant dye for living cells (Fig. 3B-C). A confocal slice at a height of 8.85 µm from the bottom of the cell reveals regular cell morphology (Fig. 3B). A scan closer to the bottom of the cell (at 3.42 µm) makes pyramids appear in a dark grid-like pattern as the pyramids do not fluoresce in this channel (Fig. 3C). A z-stack cross section of the cell along the dotted white line shows the membrane adhering to the pyramids (Fig. 3D). Both imaging methods indicate that each cell adheres to approximately 40 – 50 pyramids, which enables the excitation of specific regions of the cell by targeting local hotspots. Optimizing laser fluence for intracellular delivery without damage Experiments demonstrate that laser-scanning a thermoplasmonic substrate covered in HeLa cells leads to intracellular delivery of cargo that is dissolved in the surrounding solution in a spatially selective manner. In Figure 4A we overlay a bright-field image with a fluorescent image with excitation and emission wavelengths of 495/515 nm, respectively. Areas I and III contain gold-coated pyramids, whereas areas II and IV are flat gold. Areas III and IV were laser-


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scanned, while areas I and II were not. Only area III — the area containing gold-coated pyramids that is irradiated by the laser — demonstrates the successful delivery of cell-impermeable Calcein green molecules. This observation confirms that gold-coated pyramids in combination with laser-irradiation leads to the delivery of molecules into cells. Figure 4A demonstrates that the technique offers spatially-selective delivery, and works only when cells are cultured on pyramids and laser-scanned. Note that the pyramidal surface on the left reflects less light than the flat gold on the right, making the surface appear darker. We determined the efficiency of Calcein green delivery to HeLa cells (Fig. 4C) and used a second dye, Calcein red-orange AM, to check the post-experimental viability of the cells 4 hours after the experiment (Fig. 4D) in a small region of the substrate. The overlay of the efficiency and viability in Figure 4E demonstrates that cells with Calcein green delivered to the cytoplasm survive the experiment. To determine how many cells had Calcein green delivered to the cytoplasm (efficiency), and how many of those cells were alive in the Calcein red-orange AM channel (viability) we used automated cell counting on fluorescent images. We determined the optimum laser fluence for maximum delivery efficiency and viability by repeating each experiment described above on 3 separate substrates, in 3 separate dishes at different laser fluences. Figure 4B shows the fluence dependence of the efficiency and viability. At the lowest fluence (48 mJ/cm2) in Figure 4B, the viability is 98% and the efficiency is 70%. This means that most of the cells are surviving the laser treatment, but only 70% of them have cell-impermeable cargo (Calcein Green) inside them. As the laser fluence is increased to 54 mJ/cm2, 95% of the cells have Calcein Green in them and 98% of them are still viable. We define this point as our optimum fluence as viability and efficiency are maximized. Simulation


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results (Fig. S4) show that the local temperature reaches 370 °C at this fluence, which is in the temperature range for bubble formation. Increasing the laser fluence beyond this point leads to the onset of cell death, as can be seen starting at 56 mJ/cm2, where the viability drops to 80%. The efficiency is also around 80%, and continues to follow the viability curve closely at higher fluences. This trend occurs because Calcein Green can only remain within a cell if the cell is alive and has an intact cell membrane. Since a fraction of the cells begin to die starting at 56 mJ/cm2, the efficiency decreases accordingly with viability. Past the optimum fluence, cells either survived the laser irradiation, had cell-impermeable cargo within, or they were not viable anymore. To determine if the pyramids experience any visible damage or melting during laser exposure at 54 mJ/cm2, we chemically fixed the cells after laser scanning and took an SEM image of the substrate. As Figure 4F illustrates, the pyramids in the laser-treated area experiences no visible damage despite undergoing intense thermoplasmonic heating at the apex (inset Fig. 4H). We observe no damage to the pyramids in the fluence range 48–90 mJ/cm2 (Fig. S6). All subsequent experiments are performed at the optimum fluence of 54 mJ/cm2 because it offers the highest efficiency and viability, and no visible damage. In the Supplementary Materials we show that this technique delivers cargo with uniform distribution within each cell, which is advantageous for applications that need to avoid endosomal trapping (Fig. S7). We observe cells proliferating after thermoplasmonic treatment, indicating high cell viability at 48 hours after laser treatment (Fig. S8). Dextran 150 kDa is retained in the cytoplasm during proliferation over 48 hours (Fig. S8). The viability with and without cargo in solution combined with and without laser scanning was determined to be between 97.7 and 99.3 % for all cases using cell counting (Table S1).


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Even though cells may appear viable after undergoing intracellular delivery with this technique, there is a potential risk of DNA mutations if gold nano-fragments remain in the cell.36 We performed inductively coupled plasma mass spectroscopy (ICP-MS) on laser-scanned cells, along with unirradiated controls, to measure the gold content in the cells after laser-scanning (Table S6). Cells that undergo thermoplasmonic intracellular delivery have a signal that is representative of background noise, as do cells that were not in contact with gold. ICP-MS measurements on cells that are incubated with gold nanoparticles (and then washed several times to remove floating nanoparticles) exhibit greater gold content than background noise. ICP-MS verifies that no gold residue remains in the cells post-experiment as compared to controls, therefore minimizing risks of gold fragment-induced mutagenesis. Quantifying delivery efficiency and viability with flow cytometry We performed flow cytometry experiments to quantify the delivery efficiency to cells their viability for different cargo sizes with triplicate experiments performed on different substrates on the same day. We delivered a range of cargos (Calcein green, Dextran 10 kDa, Dextran 70 kDa, Dextran 150 kDa, Dextran 500 kDa, Dextran 2000 kDa) to 1 million HeLa cells by laser scanning the entire thermoplasmonic substrate in 3 minutes. We then used trypsin to detach the cells from the substrate to prepare them for flow cytometry measurements. Flow cytometry measured the forward and side scattered light and the fluorescence of cells passing through a beam of light. The forward and side scattering gave information about cell size and internal granularity, respectively.50 Figure 5 compares viability and delivery efficiency of cells that were not laser-scanned (Fig. 5A) and cells that were laser-scanned (Fig. 5B). For the substrate that was not laser-scanned, the viability is 98.3% and the background signal for delivery is less than 1% (Fig. 5A). For the laser-


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scanned substrate, the viability is 98.2% and the delivery efficiency is 74% (Fig. 5B). The delivery scatter plots show a range of fluorescence signal, indicating that different amounts of FITC-cargo entered the cell. We performed triplicate independent experiments with flow cytometry for cargo sizes ranging from 0.6 kDa to 2000 kDa (Fig. 5C, Fig. S9 and Fig. S10). The largest cargo, FITC-Dextran 2000 kDa, is delivered with an efficiency of 16%, and a viability of 97% (Fig. 5C). The largest increase in efficiency is between FITC-Dextran 500 kDa (24%) and FITC-Dextran 150 kDa (68%). Calcein green (0.623 kDa) is delivered at 95% efficiency and 98% viability. We attribute the increased efficiency with decreasing cargo size to faster diffusion of smaller molecules. The cargos we delivered match functional proteins in molecular weight (13 kDa to 150 kDa).51 Flow cytometry were highly reproducible, which is important for reliable biological studies that require robust statistical data, indicating a batch-to-batch consistency in substrate fabrication. The viability with and without cargo in solution combined with and without laser scanning was determined to be between 97.3 and 98.6 % for all cases using flow cytometry (Table S5). Consistent fabrication enables different substrates to undertake intracellular delivery to cells at the reproducible efficiencies. The viability of cells after different cargo sizes were delivered to them in Fig. 5c is consistent, even though experiments were performed on different days, indicating that results are reproducible over extended periods of time. Conclusions We demonstrate that nanosecond-laser excitation of template-stripped thermoplasmonic substrates offers an alternative intracellular delivery technique for cargos of different sizes. We use laser irradiation to form hotspots at the apex of gold-covered pyramids on a substrate. These


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hotspots make the surroundings, within a nanoscale region (tens of nanometers), reach high temperatures required for bubble formation. These bubbles permeabilize the membranes of cells adhering to the substrates and the temporary pores allow for cell-impermeable cargo to diffuse into the cytoplasm. The ability to deliver cargos ranging in size from 0.623 kDa to 2000 kDa to cells makes this technique applicable to different research and clinical applications. The cargos delivered (especially the 70 kDa and 150 kDa) are the same size as biologically relevant cargos, including proteins and antibodies, making this technique valuable for studying cellular function, accelerating drug discovery, enhancing gene therapy, and improving high-throughput screening technologies. We characterized the following features of this intracellular delivery technique: (1) efficiency, viability, and throughput, (2) diverse cargo delivery, (3) spatial selectivity, scalability and reproducibility, (4) gold fragment residue, and (5) cost-effective fabrication and laser setup. We quantify delivery efficiency (up to 95% for 0.623 kDa cargo) and viability (98%) using two methods, fluorescent microscopy and flow cytometry. We fabricated highly uniform templatestripped thermoplasmonic substrates over large areas using cost-effective template-stripping of templates made with photolithograpy. The large size of the substrates enabled us to perform flow cytometry measurements on a large number of cells, making our technique useful for further biological studies, which is not possible with many substrate-based intracellular delivery techniques due to complex and/or irreproducible fabrication.13 Even though we achieved promising delivery throughput rates of 50,000 cells/min, this rate can increased by several factors by building scanning mirror setups and/ or increasing the beam spot size. Additional desirable features of the technique include reproducibility across batches, scalability, and spatial selectivity, which can be important for the direct intracellular delivery of


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functional proteins, enzymes, or genetic material, and not always offered by gold nanoparticles and other substrate-based intracellular delivery methods.38,39 We examined the reproducibility across samples by performing flow cytometry in triplicate on the same day and on different days and delivered cargos of wide-ranging sizes (0.6 – 2000 kDa). ICP-MS measurements confirmed that no gold residue remains in the cells post-experiment, therefore minimizing risks of gold fragment-induced mutagenesis in therapeutic applications, which advantageous as compared to nanoparticle-meditated intracellular delivery.36,52 In the future we plan to transpose this technique to other cell types. Intracellular delivery using nanosecond-laser activation of template-stripped thermoplasmonic substrates holds potential to advance personalized medicine for chronic diseases in a costeffective manner; for example, genome-editing cargo could be delivered directly to a patient’s own cells for treatment, bypassing an extensive donor search or experiencing life-threatening toxicity. Methods Fabrication of master templates Silicon wafers were sonicated in acetone for 5 min., and then methanol for 5 min. before being rinsed in IPA. O2 plasma cleaning was performed with the conditions 100 W at 20 mT for 1 min. 15 nm of Cr, used as a hard lithographic mask, was deposited via thermal evaporation (Fig. S2A). The wafer was baked at 200 °C for 3 min. to evaporate all solvents before processing. SPR 700-1 photoresist was spin-coated onto the wafer at 3000 RPM for 45 s (ramp = 1000 RPM/s). The wafer was then softbaked at 115 °C for 60 s (Fig. S2B). The entire area of the silicon wafer was exposed in the autostepper to form a grid-based pattern. A post-exposure bake was performed at 115 °C for 60 s. The wafer was developed in CD-26 developer for 1 minute, and


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then rinsed with DI water for 20 s. Development was repeated until no residue was released into the developer. The plasma stripper was then used to descum the wafer for 15 s (Fig. S2C). A chromium etch was performed for 12 s (1.5 nm/s etched at room temperature) to remove the Cr in the exposed squares. The sample was washed with DI water and dried with a N2 gun. The photoresist was removed in acetone. An O2 plasma clean was performed with the conditions 100 W, at 20 mTorr for 3 min to completely remove residual photoresist (Fig. S2D). KOH etch: A 4.9% HF etch of 15 s was used to remove oxide formed on the silicon. A KOH etch consisting of 2 parts water and 1 part 45% KOH, was performed at 80 °C on a hotplate with a thermometer for 3 minutes to form the inverted pyramid substrate (Fig. S2E). Chromium etching was performed at room temperature for 20 s to remove the hard mask (Fig. S2F). Fabrication of template-stripped substrates 50 nm of gold is deposited via electron-beam evaporator (Fig. S2G). A No. 1.5 coverslip is glued to a master template with UV-curable glue (Norland Adhesive 61) and cured under the UV lamp overnight (Fig. S2H). The thermoplasmonic substrate is peeled off from the template using a razor blade (Fig. S2I) to produce the final template-stripped substrate (Fig. S2J). Temperature simulations The light-substrate interaction was studied in an aqueous environment using a Finite Element Method (Comsol, Multiphyiscs 4.4). The three-dimensional electromagnetic interaction was calculated using the scattered field formulation under the assumptions that the spot size is large compared to the periodic pyramid structure (plane wave approximation) and that the optical properties of the polymer, gold film and adjacent water do not change during the interaction with the linearly polarized 11 ns (FWHM) Gaussian pulse. The geometrical parameters (pyramid base length. 2.4 µm; spacing: 1.2 µm; thickness of the gold layer: 50 nm) were chosen to resemble the


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actual fabricated sample. Periodic boundary conditions were applied in the x and y directions. The simulation domain was truncated in the z-direction using perfectly matched layers (PML). The calculated laser energy absorbed was used as a transient heat source for a one-temperature model. The spatial and temporal evolution of the gold and water temperature was calculated for different laser fluences. More details about the setup of the simulation can be found in Demesy et al.: “Tridimensional multiphysics model for the study of photo-induced thermal effects in arbitrary nano-structures.” The physical properties of gold and water were taken from “Ekici et al.; Thermal Analysis of Gold Nanorods Heated with Femtosecond Laser Pulses.” Properties of the UV glue were provided by the manufacturer. Seeding cells on substrates HeLa CCL-2 cells are cultured in DMEM containing 10% (FBS) and 1% penicillin streptomycin, and are incubated at 37 ºC and 5% CO2. Cells are passaged every other day and used for experiments at 80% confluence, between passage numbers 15 and 30. Cells are washed with 8 mL PBS and incubated for 3 minutes with trypsin (5 mL), before being neutralized with cell media (13 mL). Pipetting is used to wash the bottom of flask 5 times with the cell mixture before transferring to a 15 mL tube and centrifuging for 5 min. at 125 g. The supernatant is removed gently with vacuum pipette and cells are re-suspended in 8 mL of fresh media and pipetted 30 times (up and down counted as 1 time). We use Countessa cell counter to measure cell density and viability. Healthy cells have 90–99% viability. Eight template-stripped substrates are taped lightly to the bottom of a 100-mm petri dish with double-sided Kapton tape. 5 million cells are suspended in 15 mL of fresh cell medium and added to the petri dish for overnight incubation. Laser scanning experiments


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Substrates with cells on the surface are transferred to a 35-mm petri dish and 2 mL of prewarmed PBS (37 °C) solution containing the molecules to be introduced into the cells (Calcein green at 500 µM or FITC-Dextrans at 25 mg/mL) is added. The laser setup is shown in Fig. S1. A Nd:YAG laser emits 11-ns pulses at a wavelength of 1064 nm with a repetition rate of 50 Hz. The laser beam passes through an optical isolator, a half-wave plate (HWP) and a polarizer (P) controlling the beam energy passing through the setup. A pelican beam splitter sends 8% of the beam to an energy detector (ED) to monitor the energy during an experiment. A lens (L) loosely focuses the beam (1.2 mm in diameter) on the sample, which sits on an x-y translational stage. The petri dish contains the dye to be delivered in PBS solution. For the fluence experiments (Fig. 4), the laser was scanned in a line across the sample (scan speed = 10 mm/s; line length ∆x = 100 mm), with each line at a different fluence. We used the maximum scanning speed on our stage (10 mm/s) to make the experimental time as short as possible. We then optimized the fluence of the laser for this scanning speed. We used a seeding density to ensure 80% cell confluency at the time of experiments. Distances longer than the width of the sample were scanned to avoid cells being affected by the acceleration and deceleration of the stage at the end of each run, which changes the number of pulses hitting the sample and affects the viability and efficiency of the technique. For flow cytometry, experiments were done using the exact same procedure as fluence scanning experiments and the entire sample was scanned (scan speed = 10 mm/s, ∆x = 100 mm, ∆y = 0.5 mm, pyramid region scanning time is 3 minutes, total time scanning time is 10 mins). A negative control of areas with pyramids and no laser irradiation, and areas with no pyramids and no irradiation were performed on the same sample for each experiment. Before laser scanning, the cells were incubated overnight (16 – 20


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hours). After laser scanning, the cells were incubated for 4 hours before microscopy and 2 hours before flow cytometry. Fluorescent microscopy and cell counting Fluorescence microscopy was done on an upright microscope to image Calcein green and Calcein red-orange AM. Automated cell counting of fluorescent images was done using Fiji Image processing software using fluorescent images of the samples. The final cell count was checked to make sure that none of the cells were missed. Additional cells were added in the cell counter window. The following definitions were used to calculated percentages: Viability = NCAMRed /Naverage and Efficiency = NGreen /Naverage, where NCAMRed is the number of live cells (in red) in the field of view, Naverage is the average number of live cells in a laser-irradiated area, and NGreen is the number cells that appear green due to delivery of green molecules. Triplicate experiments were performed for all data sets. Cell fixing Cells were fixed immediately after fluorescence microscopy, which was about 4.5 hours after laser scanning. Samples were soaked in fixing solution (1 part 25% glutaraldehyde, 1 part 1M Hepes, 8 parts Millipore water) for at least 10 min. Samples were soaked in buffer solution (2 parts 1M Hepes, 8 parts Millipore water) and wrapped with parafilm before overnight storage in fridge at 5 °C. The next day, the samples were rinsed by soaking in Millipore water for at least 5 min. Samples were soaked in 1% osmium for at least 20 minutes, and then rinsed with water for 5 min. The samples were then dehydrated with ethanol by soaking in 50% ethanol for 5 min., 70% ethanol for 5 min., 90% ethanol for 5 min., 100% ethanol 3 times, 7 min. each (total: 20-30 minutes), and 100% ethanol with molecular sieves (grade: 3 Angstrom) for 7 min. Samples were


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finally soaked in HMDS 3 times, at least 7 min. each (total 20–30 min.) before air drying at room temperature. Flow cytometry Flow cytometry measurements were performed to measure viability and delivery efficiency of different cargos to thousands of cells. Each FITC-cargo to be studied (Table S2) had 6 samples associated with it, including 3 control samples and 3 experimental samples using the conditions shown in Table S3. For flow cytometry, we used the maximum FITC-Dextran concentration recommended by the manufacturer of 25 mg/mL to ensure the detection of the smallest levels of fluorescence in a cell in the flow cytometry scatter plots. The samples were washed with 10 mL of PBS and then 1 mL of PBS. 2 mL of trypsin was added in a 35-mm dish and incubated for 7 min. before neutralizing with 5 mL of pre-warmed cell medium. Cells were transferred to a 15mL tube and centrifuged at 125 g for 5 min. The supernatant was removed and cells were resuspended in 1 mL PBS and pipetted up and down 30 times before being transferred to a 5-mL round bottom test tube with a cell strainer snap cap. Flow-cytometry measurements were done using BD LSRFortessaSORPTM cell analyzer running BD FACSDiva software version 6.1.3, using the optical configuration in Table S4. Measurements were done following the daily instrument QC, which utilizes BD CS&T beads (BD Biosciences catalog #; 642412) in the following order: B0, G0R0, G1, E1-E3. Appropriate gates were set for each sample using flow cytometry analysis software version FCS Express 5 Research Edition (DeNovo SoftwareTM; Glendale, CA.). 10,000 events were recorded for each sample. Figure S8 shows representative data from Dextran 10 kDa delivery experiments. The ungated side scattering area (SSC-A) vs. forward scattering area (FSC-A) density plot represents the light-scattering distribution of cells. SSC-A corresponds to


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intracellular composition and FSC-A to size. The gate was set broadly for a monoculture population and serveed to eliminate debris. A side scattering height (SSC-H) vs. side scattering width (SSC-W) density plot, gated on Gate 1, was used to exclude possible doublets (cells stuck together). A SSC-A vs. Calcein AM red-orange (561-nm laser with a 582 ± 15-nm band pass filter) density plot, hierarchically gated on Gate 2, was used to gate on only live cells (Calcein AM red-orange positive). The resulting gate, Gate 3, provided the viability statistics. A SSC-A vs. FITC-Dextran 10 kDa (488-nm laser with a 530 ± 30-nm band pass filter) density plot, hierarchically gated on Gate 3, was used to quantify the number of cells in the live population that had FITC-cargo inside them.

The control sample was used to set the gate for the

experimental samples, where generally < 2% background is desired (less than 1% in our case, shown in sample G0R0, Fig. S9).

We used a SSC-A vs. FSC-A ungated density plot

representing the light scattering distribution of cells that were laser-scanned, and followed the same gating procedure as mentioned above (samples G1 and E1 in Fig. S9). We measured 59% delivery efficiency of the FITC-Dextran 10 kDa cargo. Viability and efficiency flow cytometry was performed from triplicate experiments for cargo sizes ranging from 0.6 kDa to 2000 kDa (Fig. 5C). The efficiency was normalized by the pyramid area. The data represent mean ± standard error from three independent experiments. Because the pyramids cover 60% of the substrate, additional experiments were performed to normalize the efficiency by the pyramid-covered region of the substrate (Fig. S11). The thermoplasmonic substrates are 18 x 18 mm in size with a perimeter of flat gold (Fig. 2B) that acts as a control region during experiments (Fig. 4A). We fabricated additional substrates that were smaller, 14 x 14 mm, and did not have the perimeter of flat gold. Flow cytometry experiments were performed for FITC-Dextran 150 kDa cargo using both substrates, with and


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without the flat gold perimeter. The average delivery efficiency of a substrate with the flat gold perimeter was 49%, and without the flat gold perimeter was 68%. The efficiency data for all experiments in Figure 5C was therefore normalized by a factor of 1.4 to exclude cells from the flat gold perimeter of the thermoplasmonic substrates. ICP-MS The samples were first weighed out on an analytical balance into clean 15-ml BD Falcon tubes. They were digested using ultrapure hydrochloric acid overnight. The next day, samples were diluted to a volume (usually 5 or 10 ml) with DI water. A fresh calibration curve was run with the sample. A method blank was run with the batch of samples to calculate the detection limit. Calibration verification standards were run to calculate percent recoveries. Author Contributions NS, MH, MM, VN, DIV, WS, JN, and AM designed and carried out the experiments, and analyzed the results. AH and EM supervised the research and the development of the manuscript. NS wrote the first draft of the manuscript; all authors subsequently took part in the revision process and approved the final copy of the manuscript. †These authors contributed equally to work. The additional data presented in this paper is available in Supplementary Materials. Funding Sources The research described in this article was supported by the National Science Foundation under contracts PHY-1219334 and PHY-1205465. NS was funded by the Howard Hughes Medical Institute’s International Fellowship. MM was funded by the Graduate Prize Fellowship at Harvard University. AH received funding from the German Research Foundation through the Cluster of Excellence REBIRTH (DFG EXC62/3).


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Competing interests. The authors declare that they have no competing interests. Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: _____________. Laser setup (Figure S1); Fabrication steps for template-stripped thermoplasmonic substrates (Figure S2); Absorption, reflectance, and transmission of plasmonic substrates (Figure S3); Temperature simulations (Figure S4); Damage image (Figure S5); SEM images of pyramids after intracellular delivery (Figure S6); Distribution of cargo within the cell (Figure S7); Cells proliferate over 48 hours, with cargo inside (Figure S8); FITC-dextran 10 kDa representative flow cytometry data (Figure S9); FITC-dextran flow cytometry for different-sized molecules (Figure S10); FITC-dextran 150 kDa flow cytometry for area normalization (Figure S11); Viability (Calcein green) (Table S1); Reagent list for flow cytometry (Table S2); Sample information for flow cytometry data sets (Table S3); Flow cytometry settings (Table S4); Viability (Dextran) (Table S5); ICP-MS for Dextran 10 kDa (Table S6)

ACKNOWLEDGMENT Substrate fabrication and cell culture was performed at the Center for Nanoscale Systems at Harvard University. Flow cytometry was performed at Harvard Bauer Core. Confocal microscopy was performed at Harvard Center for Biological Imaging. ICP-MS was performed at Harvard T. Chan School of Public Health. Yang Li, Reza Sanatinia, and Hemi Gandhi provided feedback on the manuscript throughout its development. The authors thank Luo Gu, Rahul Palchaudhari, Alain Viel, Adrian Pegoraro, Eric Diebold, Yang Li, Daniel Flicker, Evelyn Hu,


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Hai Bi, Ranhua Xiong for discussions. Eric Diebold, Lauren Milling, Sebastien Courvoisier, Jun Chen and Jean-Pierre Wolf contributed to the preliminary research. References and Notes (1)

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