Optical and Thermophoretic Control of Janus Nanopen Injection into

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Optical and Thermophoretic Control of Janus Nanopen Injection into Living Cells Christoph M. Maier, Maria Ana Huergo, Sara Milosevic, Carla Pernpeintner, Miao Li, Dhruv PRATAP Singh, Debora Walker, Peer Fischer, Jochen Feldmann, and Theobald Lohmueller Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03885 • Publication Date (Web): 23 Nov 2018 Downloaded from http://pubs.acs.org on November 24, 2018

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Optical and Thermophoretic Control of Janus Nanopen Injection into Living Cells Christoph M. Maier1,2, Maria Ana Huergo3, Sara Milosevic1, Carla Pernpeintner1,2, Miao Li1, Dhruv P. Singh4, Debora Walker4, Peer Fischer4, Jochen Feldmann1,2, Theobald Lohmüller,1,2* 1Chair

for Photonics and Optoelectronics, Department of Physics, Ludwig-MaximiliansUniversität München, 80799 Munich, Germany

2Nanosystems

Initiative Munich and Center for Nanoscience (CeNS), Schellingstraße 4, 80799 Munich, Germany

3Instituto

de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Universidad Nacional de La Plata—CONICET, Sucursal 4 Casilla de Correo 16, 1900 La Plata, Argentina 4Max

Planck Institute for Intelligent Systems, Heisenbergstraße 3, 70569 Stuttgart, Germany [email protected]

KEYWORDS Janus nanoparticles, plasmonics, optical tweezer, cell injection, thermophoresis, biomolecule delivery.

ACS Paragon Plus Environment

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ABSTRACT

Devising strategies for the controlled injection of functional nanoparticles and reagents into living cells paves the way for novel applications in nano-surgery, sensing and drug delivery. Here, we demonstrate the light controlled guiding and injection of plasmonic Janus nanopens into living cells. The pens are made of a gold nanoparticle attached to a dielectric alumina shaft. Balancing optical and thermophoretic forces in an optical tweezer allows that single Janus nanopens are trapped and positioned on the surface of living cells. While the optical injection process involves strong heating of the plasmonic side, the temperature of the alumina stays significantly lower, thus allowing the functionalization with fluorescently labelled, single stranded DNA, and hence the spatially controlled injection of genetic material with an untethered nanocarrier.


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The plasma membrane is a semipermeable barrier that separates and protects living cells from the outside environment. Gases and small, uncharged molecules can pass the membrane by diffusion, while the passage of ions, large molecules or particles is tightly regulated by specific proteins and transport pathways. Devising strategies for the targeted and controlled delivery of nanoparticles, which are loaded with specific drugs or reagents into living cells thus constitutes a key challenge for applications in biomedicine1, 2 including sensing3 and nano-theranostics4, 5. Cell transfection and drug delivery are typically achieved by biochemical methods, using liposomes6 and specialized polymers7 that aid the membrane passage, or by physical techniques such as electro-8, 9 and sonoporation10, 11. In recent years, optical approaches have become more relevant, as they combine the benefits of being sterile and specific, meaning a single cell or a controlled number of cells can be selected from cell culture or even tissue with a focused laser beam12-16. Optical transfection, for example, was achieved by using high-energy laser pulses to punch transient holes into the plasma membrane of living cells. Drugs17, nanoparticles18 or genetic material12, 19, 20 were then able to enter the cell through these holes for as long as they remained open. Optothermal methods based on plasmonic heating of gold nanoparticles were proposed as a less harmful strategy to control membrane permeability, as they generally require lower laser intensities21-24. Non-radiative decay of localized surface plasmons results in strong heating of gold nanoparticles when they are irradiated with light at the plasmon resonance frequency25. Particle temperatures of several hundred degrees centigrade can be reached within picoseconds when a single gold particle is irradiated with a focused laser beam26. It has been shown that localized particle heating of gold particles that are attached to a lipid membrane can be applied to improve membrane permeability for molecules or ions21 and even to create membrane pores27. The drawback of optothermal methods (and a concern for most optical approaches, in general) is that


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the transport of material into the cell is only driven by diffusion12. Controlling the exact time, location and direction for the passage of substances across the membrane is therefore challenging. In order to overcome this restraint, we have shown previously that laser heating of gold nanoparticles can be extended towards the active optical injection of single particles into synthetic giant unilamellar vesicles28 and living cells29. This was achieved by a combination of plasmonic heating and optical force. Gold nanoparticles were first optically printed on the surface of a living cell. Nanoparticle printing requires a considerably high laser power to achieve strong enough scattering forces that overcome any electrostatic repulsion between the particles and the cell surface. During the printing step, the particles can already reach very high temperatures and momentum, which might have an impact on cell viability. Achieving a feasible particle deposition rate moreover requires to either increase the particle concentration or the laser power30, 31. Both, nanoparticle printing and injection into the cell was achieved with a focused laser beam at a wavelength resonant to the particle plasmon. High nanoparticle temperatures (>400°C) were required for the injection process in order to create vapor bubbles that rupture the cell membrane and allow the particles to enter, which is potentially harmful for cells. However, viability measurements have shown that a majority (>75 %) of the cells survived the injection process, likely because particle heating is only applied to a small membrane area29. The biggest limitation of plasmon enhanced optical injection stems from the fact that most biomolecules and chemical linkers are destroyed at very high temperatures. Therefore, gold particles could not be functionalized prior to injection, which severely limits the applicability of the technique for biological and biomedical studies. The ideal probe for optical nanoparticle injection and cell transfection would thus combine the benefits of the light controlled positioning of a single plasmonic nanoprobe on a living cell with the possibility of subsequent optical injection.


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For the latter, the particle itself, or at least a part of the particle, should stay cold enough to allow for the attachment of biomolecules. Here, we report a new approach for the controlled optical injection of biomolecules directly into living cells by using plasmonic Janus nanoparticles (JNPs) or ‘nanopens’. The nanopens consist of a gold nanoparticle (~80 nm) attached to a dielectric shaft of Al2O3 (~500 nm in length) (Figure 1a). The novelty of the design is that the nanopens have a ‘hot’ and a ‘cold’ end when they are irradiated with light. On the ‘hot’ side, plasmonic heating of the gold particle induces a thermal gradient along the long axis of the nanopens. When a pen is trapped with an optical tweezer, this temperature gradient leads to the emergence of a thermophoretic force. Balancing optical and thermal forces by adjusting the laser power allows to control the vertical displacement of a single JNPs in a laser trap with high accuracy. We demonstrate that this concept of a light-driven ‘particle-elevator’, which is for the first time reported for Janus particles on the nanoscale, can be used to position individual JNPs on the surface of living cells and represents an important advantage over the previous printing and injection approaches with simple gold nanoparticles. Furthermore, we demonstrate that single stranded DNA (ssDNA) that is attached to the Al2O3 shaft can be injected into the cell without the problem of thermal degradation, since the dielectric part of the nanopen is colder in comparison to the gold nanoparticle under laser irradiation. JNPs were prepared by first depositing an array of gold nanoparticles on a silicon substrate using micellar nanolithography, followed by shadow growth physical vapor deposition as previously described32 to increase the size of the Au nanoparticles. A thin layer (~5 nm) of TiO2 was then deposited on the Au to enhance the adhesion to the alumina, which is grown to yield a nanopen with a length of ~500 nm. Before the experiment, the nanopens were functionalized with a fluorescently labelled oligo-ssDNA 5’6-FAM-dTMP(18)3’ (6-FAM: 6-Carboxyfluorescein, dTMP:


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2-deoxythymidine-5-monophosphate) (supporting information, Figure S1). The thymidine strand binds to alumina primarily through the thymine and phosphate polar groups33. After the functionalization, the substrate was immersed in water and ultra-sonicated to remove the nanopens from the wafer and to disperse them in solution. Single JNPs were identified under the microscope due to the strongly scattered light from the gold nanoparticle at one end of the nanopens (darkfield imaging) or by the fluorescence from the alumina shaft (Figure 1b). The complete injection process of the JNPs into living cells was carried out in two steps. First, individual JNPs were trapped by a focused near-infrared (NIR) laser and carefully positioned on the surface of a living cell (Figure 1c). Second, the nanopens were optically injected, one-by-one. Injection was performed at a laser wavelength close to the particle plasmon resonance (Figure 1d). Notably, trapping and injection of single JNPs is achieved by choosing the right laser wavelength. Two types of optical forces act on a nanopen in a laser beam: A gradient force directed towards the center of the laser focus and a scattering force in the direction of light propagation due to scattered and absorbed photons34, 35. Optical trapping of a nanosized object is only possible if the gradient force is stronger than the scattering force. For the nanopens the overall optical force is a sum of the individual forces acting on the gold particle and the dielectric shaft. For alumina with a relatively high index of refraction, the gradient force is dominant, regardless of the laser wavelength36. Gold nanoparticles, however, display strong absorption and scattering of light at their plasmon resonance, which, in water, is at around ~560 nm for the spherical gold particle at the JNP tip (Figure 2a). Scattering forces acting on a single JNP can thus become substantial when the particle is irradiated with a green laser (λ = 532 nm). Strong scattering forces in the pN range push a nanoparticle in the direction of the propagating light, which forms the basis for optical printing30 or laser injection28, 29 (supporting information, Figure S2). Optical trapping, on the other


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hand, is possible with a focused laser beam at a wavelength that is far red-shifted from the plasmon resonance. In this case, both scattering and absorption of light are weak and gradient forces are overall dominant34 (Figure 2b). Optical trapping of the JNPs was done with a NIR laser (λ = 1064 nm). At this wavelength, gradient forces are strong for both the dielectric and metallic part. Compared to a single spherical gold nanoparticle (diameter: 80 nm), the nanopens thus show a better lateral confinement in an optical trap, when both particles are manipulated at the same laser power (supporting information, Figure S3). Although light absorption by the gold particle is low at 1064 nm, a small plasmonic heating effect is still observed. At a laser power of only 14 mW under the microscope objective, temperature calculations show a rise of the gold particle temperature by 3 K while the temperature of the alumina shaft does not change (Figure 2c). The resulting temperature gradient of 5 ∙ 106 K/m along the long axis of the JNPs in the optical trap leads to the emergence of a thermophoretic force37-39. Self-propulsion of laser-heated Janus particles due to thermophoresis40-42 has been studied intensely as a strategy to control the movement of artificial microswimmers43, 44. For example, it has been demonstrated that a nanodiamond swimmer’s translation and rotational motion could be controlled with light45. The Janus nanoparticle in a laser trap is subject to both optical and a thermophoretic force37. In the optical tweezer, the scattering force orients the Janus particle with the metal side pointing in the direction of the propagating light, while the gradient force pulls the particle towards the laser focus. When the laser is coupled in from the top, like in our experiment, the nanopens thus align along the beam axis with the gold particle pointing downwards (Figure 3a). Increasing the laser power not only leads to stronger optical forces, but also to an increase of the thermophoretic force


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because of plasmonic heating. This results in an upward movement of the nanopen in the laser trap with respect to the laser focus. Lowering the laser power results in a nanopen movement in the opposite direction. Adjusting the laser power thus allows to balance optical and thermophoretic forces with respect to each other, and therefore the control of the position and orientation of an individual nanopen along the beam axis. We analyzed the movement of optically trapped JNPs for different laser intensities. The nanopen displacement was determined by analyzing the diffraction pattern of an optically trapped nanopen according to Speidel et al.46 (supporting information, Figure S4). Changing the laser power between 14 mW and 56 mW allowed the reversible upward and downward movement of a single optically trapped JNP along the beam axis over a range of 4 μm with high positional control (Figure 3b). This light-controlled ‘elevator’ movement enables the accurate positioning of individual nanopens on the surface of a cell. By increasing the laser power, a nanopen is moving upwards in the laser beam, while a cell that is attached to the substrate stays in focus. By moving the microscope stage, a single cell can now be observed in the microscope and precisely positioned below the JNP without the requirement of any re-focusing. By reducing the power of the laser trap, the nanopen moves downwards again and eventually sticks to the cell surface (Figure 4a). After the JNPs deposition, the functionalized nanopens were optically injected into cells. Nanopen injection was performed with a focused laser beam at a wavelength close to the plasmon resonance of the gold particle at the JNP tip (λ=532 nm). A minimum laser power of 6.8 mW was required to achieve strong enough plasmonic heating for the formation of vapor bubbles. The bubble formation itself can be seen in the dark-field microscope by a white flash at the injection point, indicating both bubble generation and collapse (Figure 4b). When the bubbles expand, they


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rupture a small, transient micro-hole into the plasma membrane47. Optical forces (now predominantly scattering forces) that are simultaneously acting on the JNP in the laser focus then inject the nanopen into the cell through this newly formed hole (Figure 4c). The injection is directly visible since the nanopen is immediately moving out of focus. By lowering the focal plane, the injected JNP can be located again inside the cell and below the injection area. The nanopens are not freely diffusing inside the cell, since the average pore size of the cytoskeleton meshwork is < 50 nm48. The motion of larger particles, such as the nanopens is therefore restricted. Scanning electron microscopy (SEM) micrographs of the cells that were chemically fixated directly after the injection process also display that small holes are formed in the cell membrane at the position where the individual pens were injected (Figure 4d). Notably, the nano-bubble formation is not only responsible for rupturing the membrane but also prevents heat induced damage to the cell. As reported before29, 49, the estimated temperature of the vapor around the nanoparticle is moderate compared to the surface temperature reached by the gold particle. The nano-bubble surrounding the JNP thus acts like a protective shield and isolates the cell against unwanted heat transfer. How far exactly the nanopens can be injected into the cell depends on the applied laser power and the properties of the cell itself. A high laser power results in a stronger scattering force that is pushing the particle forward. At the same time, strong plasmonic heating results in a larger opening in the plasma membrane and thus in more cell damage (supporting information, Figure S5). Most importantly, the density of cell organelles and the cytoskeleton itself provide a resistance for the JNP injection. However, these conditions can vary for different cell lines and even for different injection sites on a single cell. Importantly, the injection process does not destroy or strip-off the fluorescently labeled oligossDNA attached to the alumina shaft. Injected nanopens inside the cell could still be identified by


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the fluorescence of the 5’6-FAM-dTMP(18)3’ (Figure 4e, and supporting information, Figure S6). In addition to the functionalization strategy via thymidine, we also tested to possibility to label JNPs with a fluorescently labeled ssDNA sequence of adenosine nucleotides, namely 5’E.N.dAMP(25)3’ (E.N.: Eterneon 394/507, dAMP: 2’-deoxyadenosine-5’-monophosphate). This sequence was chosen, since adenine is the complementary DNA base to thymine and poly-adenine strands had been reported to act as anchor sequence for DNA binding to gold particles and quantum dots50. The 5’E.N.-dAMP(25)3’ functionalized nanopens could also be identified by their fluorescence after injection, similar to the JNPs labelled with 5’6-FAM-dTMP(18)3’, although the cells in this experiment displayed stronger auto-fluorescence due to the excitation at 394 nm (supporting information, Figure S7). The stability of the ssDNA label can be explained by the ten-fold lower thermal conductivity of amorphous Al2O3 compared to gold. The alumina shaft of the nanopen is not subject to direct laser heating. When illuminated with a continuous wave laser, the temperature distribution from the gold nanoparticle surface expands as a function of 1/r to the surrounding 26. Even for a nanoparticle temperature of 500°C, which is already well above the spinodal decomposition temperature of water (~320°C)51, the local temperature at the end of the nanopen would only reach 57°C (Figure 4f). The chemisorption of the thymidine nucleotides on the alumina surfaces is stable for temperatures up to 100°C, as reported before33. Taking this temperature distribution along the JNP under consideration, one can thus assume that only the ssDNA molecules close to the gold particle are likely destroyed. Molecules that are more than a few tens of nanometers away from the gold particle surface, however, will survive the treatment. This ensures that heating does not impact the delivery of biomolecules and genetic material to the cell.


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In support of this analysis, we have also performed independent control experiments to provide experimental proof for the inhomogeneous heating of plasmonic JNPs with laser light. We therefore studied the heat-induced polymerization reaction of polydimethylsiloxane (PDMS) around single irradiated nanopens according to a previously reported approach from our group52. In brief, we found that thermal curing of PDMS was only localized around the gold particle, while the dielectric part of the Janus was sticking out of the PDMS shell (further details on this control experiment and the obtained results are provided as supporting information, Figure S8). This finding verifies the initial idea that the alumina shaft stays indeed much colder than the plasmonic side under laser irradiation. In conclusion, we have devised an all-optical strategy to inject plasmonic Janus nanopens into living cells. The combination of a metal and a dielectric material allows the functionalization with fluorescent molecules selectively on the dielectric part of the pens. The interplay between optical and thermophoretic forces allows to trap and position the Janus nanopens on the surface of individual cells. Although the nanopen injection process involves strong plasmonic heating, we could show that the temperature of the alumina shaft remains significantly lower. Fluorescence imaging revealed that the nanopen functionalization stayed intact after the injection process. Overall, this strategy for optical injection of Janus nanoparticles into living cells paves the way for future applications in cell transfection, sensing, drug delivery or personalized medicine.


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Figure 1. Positioning and injection of functionalized plasmonic Janus nanoparticles (JNPs) into cells. a) SEM micrograph of a JNP consisting of a spherical gold particle (~80nm) connected to a dielectric alumina shaft (length: ~500nm). The Al2O3 part of the JNPs is functionalized with a fluorescently labeled oligo-ssDNA 5’6-FAM-dTMP(18)3’. b) Individual 6-FAM labelled JNPs are identified either by dark-field or fluorescence microscopy. The patterning was obtained by sequential optical deposition of one, two and three (1st row) as well as one and two (2nd row) JNPs at each individual spot. The number of JNPs per spot are resolved in both, the DFM and in the fluorescence image. c) Illustration of the positioning process on a cell membrane using an offresonant NIR focused laser beam (λ=1064nm). d) Schematic of the JNP injection process with a green laser that is close to the particle plasmon resonance (λ=532nm).


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Figure 2. JNPs in an optical trap. a) Single nanoparticle scattering spectrum of a JNP in water. b) Total optical force acting on a JNP trapped with a NIR laser at a laser power of 14mW. The individual optical forces acting on the gold nanoparticle and the alumina shaft are summed in the plot. c) Simulation of the temperature distribution around a JNP trapped with a 1064nm laser in water for P = 14mW.


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Figure 3. Laser power dependent positioning of a JNP in z-direction. a) Illustration of a JNP in an optical trap. The total force acting on a nanopen is the combination of optical and thermophoretic forces that are working in different directions. The z-position is controlled by tuning the laser power. For higher laser powers, thermophoretic forces are more dominant and make the JNP move upwards. A downward movement with respect to the beam axis is observed if the laser power is reduced. Importantly, this process is fully reversible. b) The measured zposition over several cycles exhibits a linear relationship between power and z-position.


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Figure 4. Laser injection of JNPs into CHO cells. After a single JNP is positioned on the cell membrane (a), it is injected into the cell with a focused laser beam at a wavelength resonant to the particle plasmon of the nanopen. Strong plasmonic heating leads to nano-bubble formation at the cell surface (b). During the injection process, the JNP is pushed inside the cell and to a lower focal plane (c). The nanopen is no longer observed at the injection area on the cell surface. The light green spots in the DFM images (dashed arrow in (a)) correspond to secondary reflection from the laser coupling and do not interfere with the sample. (d) Dark-field and scanning electron microscopy images of a cell with JNPs injected around the cell nucleus. (#1-#3) SEM close-ups of the cell membrane at the respective injection sites. Close-up #4 shows a non-injected JNP attached to the cell membrane. This nanopen is not visible in the DFM image since it is not in the same focal plane as the injected particles. (e) Top: DFM image of a cell after injection of a JNP functionalized with 5’6-FAM-dTMP(18)3’. Bottom: epi-fluorescence image of the squared area showing a strong fluorescence of the nanopen inside the cell. (f) Calculated temperature distribution of a JNP for a gold nanoparticle temperature of 500°C. The cold end of the nanopen shaft only heats up to 57°C due to the low thermal conductivity of Al2O3.


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ASSOCIATED CONTENT The Supporting Information is available free of charge. Functionalization of the Janus nanoparticles; Optical forces acting on JNPs during injection; Spatial precision of JNP manipulation; Determination of the JNP displacement along the beam axis; Injection of JNP with different laser powers; JNPs functionalized with 5’6-FAM-dTMP(18)3’; JNPs functionalized with 5’E.N.-dAMP(25)3’; Inhomogeneous heating of JNPs studied by PDMS polymerization; Description of the optical dark field and fluorescence microscopy setup as well as scanning electron microscopy; Details on the numerical methods; Cell culture and fixation protocol (PDF) AUTHOR INFORMATION Corresponding Author *Dr. Theobald Lohmüller ([email protected]) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial support by the ERC through the Advanced Investigator Grant HYMEM, by the DFG through the SFB 1032, project A08 and by the Bayerisches Hochschulzentrum für Lateinamerika through a BAYLAT funding is gratefully acknowledged. We also acknowledge Joachim Rädler for providing cell culture facilities, Gerlinde Schwake for providing technical cell culture support, and Conny Miksch in the growth of the nanostructures.


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Optical and Thermophoretic Control of Janus Nanopen Injection into

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