Gold Nanoshell-Functionalized Polymer Nanoswimmer for

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Gold Nanoshell-Functionalized Polymer Nanoswimmer for Photomechanical Poration of Single Cell Membrane Wei Wang, Zhiguang Wu, Xiankun Lin, Tieyan Si, and Qiang He J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13882 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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Journal of the American Chemical Society

Gold Nanoshell-Functionalized Polymer Nanoswimmer for Photomechanical Poration of Single Cell Membrane Wei Wang, Zhiguang Wu*, Xiankun Lin, Tieyan Si, Qiang He* Key Laboratory of Micro-systems and Micro-structures Manufacturing (Ministry of Education), Harbin Institute of Technology, Yikuangjie 2, Harbin 150080 (China) KEYWORDS: nanoswimmer • nanomotor • self-propulsion • layer-by-layer • cell-membrane-poration

ABSTRACT: We report an ultrasound-driven gold nanoshell-functionalized polymer multilayer tubular nanoswimmer that can photomechanically perforate the membrane of a cancer cell by assistance of near infrared (NIR) light. The nanoswimmers were constructed by template-assisted layer-by-layer technique and subsequent functionalization of Au nanoshells inside the big opening. The nanoswimmers exhibit efficient and controllable movement toward target cells through the manipulation of acoustic field. Next, the nanoswimmers with end-on attachment onto the HeLa cells achieve the poration of cell membrane within 0.1 s under the irradiation of NIR light. The experimental and theoretical results suggest that the instantaneous photothermal effect provides enough photomechanical force to open the cell membrane. Such NIR light-assisted nanoswimmers-enabled cell membrane poration possesses various advantages including active targeting, short time and precision in single cell that conventional chemical and physical cell poration techniques could not achieve, and thus provides considerable promises of a variety of biomedical applications such as gene delivery and artificial insemination.

1. INTRODUCTION Poration of a cell membrane plays an important role in intracellular delivery of diverse large molecules for various medical and biological strategies. During the past decades, various chemical and physical strategies to open various types of cell membranes have been widely developed, and have been applied in many fields of biotechnologies such as subcellular surgery, targeted cancer therapy, and genome editing.1, 2 Particularly, physically-triggered cell poration mainly relies on disturbance of lipid molecules under the instantaneous physical fields to generate holes on cell membranes and a change in membrane permeability. To perform intracellular delivery toward a specific type of cells, a significant number of molecules such as antibodies, nucleic acids, and aptamers are utilized to functionalize on the materials. However, these strategies are limited in precise poration of cell membranes and passive diffusion. For example, artificial insemination requires the active poration of membrane in single cell level. Recent advances in self-propelled synthetic nanomachines such as nanoswimmers, molecular machines and molecular cars have opened up new horizons for potential biomedical domains,3-10 including drug targeted delivery, cell operation, and cell manipulation.11-15 Learning from the chemically-powered autonomous biomotors such as kinesin, various types of artificial nanoswimmers were developed to overcome the low Reynolds number fluids and

perform efficient propulsion through converting chemical or physical energies into mechanical movement.16-25 Many nanoswimmers have demonstrated the active transportation of various drugs and genes toward the targeted cells and subsequently are internalized by cells.26,27 However, the delivery into the interior of cells is not realized by mechanically opening the cell membrane as expected. It is because the maximum applied stress of these nanoswimmers is almost one to two orders of magnitude smaller than the critical ruptured stress of cell membranes.28, 29 Therefore, the active poration of cell membranes by the nanoswimmers is still challenging. Here we present an ultrasound-driven gold nanoshellfunctionalized polymer multilayer tubular nanoswimmer for photomechanically opening single cell membrane by assistance of the near-infrared (NIR) light. The polymer nanoswimmer with a cone shape was fabricated by the layer-by-layer (LbL) deposition into a nanoporous template and subsequently the big opening was functionalized with Au nanoshells (AuNSs) by a seed growth method.30-32 Compared to previous techniques such as template-based electrodeposition of microtubes and rolled up nanotechnology,33, 34 the polymer multilayer tubular nanoswimmers fabricated by the porous template-assisted LbL technology can not only control the length, wall thickness, outside and inner diameters of the resulting nanoswimmers, but also conveniently vary the wall A

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components and properties without extra surface chemistry by assembling the corresponding components.35 The efficient movement and controlled cell targeting of the polymer multilayer nanoswimmers, with controllable velocity and orientation, was facilitated with the external acoustic field. Next, the photothermal effect of AuNSs at the big opening lead to fast perforation within 0.1 s at the attaching point. The surrounding fluorescence molecules could percolate into the cells along the nanopores opened by the nanoswimmers. Such nanoswimmers capable of opening a cell membrane is prospective to be utilized for intracellular drug delivery, artificial insemination and subcellular surgery process.

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mg mL-1 in 0.5 mol L-1 NaCl solution), respectively. Each step of immersion was maintained for 20 min. The template was washed with pure water for 3 times between each immersion step. After the deposition of 10 bilayers of PSS and PAH into the pores of the template (denoted as (PSS/PAH)10), the polishing step was executed to remove the useless materials on the template by polishing with wet swabs. The template was placed at the air/liquid interface of gold clusters solution for 20 min. After washing 3 times, repeat this cycle for 4 times. 1.5 mL HAuCl4 (20 mg mL-1) was added into 200 mL K2CO3 (1.8 mmol L-1). The solution was stored for more than 48 h. The template with gold clusters was immersed into the solution (15 mL). 100 μL formaldehyde was added in the solution. The solution and template were stored for 12 h in 4 °C. AuNSs were formed in this process and mainly distributed on the big opening, because the template was half immersed in gold clusters solution. The resulting AuNS-functionalized (PSS/PAH)10 tubular nanoswimmer was released by dissolving the template in dichloromethane. Washing step was executed by centrifugation at 6000 g for 30 s, then, treated with dichloromethane, ethyl alcohol and water sequentially. The AuNS-functionalized (PSS/PAH)6, (PSS/PAH)14, (PSS/PAH)18, (PAA/PDDA)10 and (ALG/CHI)10 tubular nanoswimmers were prepared according to the abovementioned procedures. The immersion solutions were PAA (2 mg mL-1 in 0.5 mol L-1 NaCl solution), PDDA (2 mg mL-1 in 0.5 mol L-1 NaCl solution), ALG (1 mg mL-1 in 0.1 mol L-1 NaCl solution) and CHI (1 mg mL-1 in 0.1 mol L-1 NaCl solution, 0.02 mol L-1 acetic acid). The AuNS-functionalized (PSS/PAH)10 swimmers with diameter of 1 μm were fabricated by the same method of nanoswimmer and the only difference was the usage of PC membrane (catalog no. 110610). Fabrication of the gold nanorod swimmers. The Au nanorods with diameter of 200 nm and 800 nm were fabricated using a template-assisted electrodeposition protocol. A 200 nm AAO and a PC membrane (catalog no. 110609) were served as template. A gold film was sputtered on one side of the templates to serve as working electrode. Then, the templates were plated by a three electrodes cell. A Pt wire was served as counter electrode. Au was deposited from gold plating solution with 3 C at -1 V (vs Ag/AgCl). The sputtered layer was removed by a swab and polished with aluminate powder. The AAO templates were dissolved in 1 mol L-1 NaOH for 15 min under shaking and the 200 nm Au nanorods were separated by centrifugation at 6000 g for 30 s, then, treated with water repeatedly until a neutral pH was obtained. The PC templates with nanorods were served same as PC templates with nanoswimmers in above section. Acoustic equipment. The acoustic field setup to drive the nanoswimers was built as previous report.37 A piezoelectric ceramic (Shenzhen Huajingda electronics CO., Ltd., catalog no. H4P163000) was attached by cyanoacrylate glue on a glass plate. Kapton tape with a rectangle hole which was used as acoustic chamber with a cover glass was placed beside the piezoelectric ceramic. The piezoelectric ceramic

2. EXPERIMENTAL SECTION Materials. Polycarbonate (PC) filtering membrane (catalog no. 7060-2513, with a diameter of 200 nm at the small opening), anodic aluminum oxide (AAO) (catalog no. 68097023, with equal diameters of 200 nm), PC filtering membrane (catalog no. 110609, with equal diameters of 800 nm) and PC filtering membrane (catalog no. 110610, with equal diameters of 1 μm) were purchased from Whatman Inc.. Poly (allylamine hydrochloride) (PAH, Mw = 70000), poly (styrene sulfonate) (PSS, Mw = 70000), Chitosan (CHI, Lot # STBF8219V), Alginic acid sodium (ALG, from brown algae, Lot # SLBR3536V), poly (diallyl dimethyl ammonium chloride) (PDDA, Typical Mw = 100000 200000) and poly (acrylic acid) (PAA, Lot # 120M5074V) were purchased from Sigma-Aldrich, Co.. Propidium iodide (PI) was purchased from Shanghai Yuanye Biological Technology Co., Ltd.. Gold chloride trihydrate (HAuCl4·3H2O, ≥ 99.9% trace metals basis) was purchased from Aladdin Co., Ltd.. Tetrakis (hydroxymethyl) phosphonium Chloride (THPC, ca. 80% in Water) was purchased from TCI (Shanghai) Development Co., Ltd.. All chemical composites, including sodium chloride (NaCl), sodium hydroxide (NaOH), potassium carbonate (K2CO3), ethyl alcohol, were analytical reagent without further purification. The water in all experiments was obtained by a Milli-Q purification system with 18.2 MΩ cm. Roswell Park Memorial Institute (RPMI) 1640 medium and phosphate buffer saline (PBS) were purchased from HyClone. Gold plating solution was purchased from Technic Inc. (Orotemp 24 RTU RACK). Fabrication of the nanoswimmers. The gold clusters were synthesized according to the previous report.30 In order to prepare the gold clusters, 1.5 mL NaOH aqueous solution (0.2 mol L-1) was added into 45.5 mL water with stirring. 1 mL THPC solution (1.2 mL of 80% THPC added to 100 mL water) was added. After 5 min, 1 mL HAuCl4 (20 mg mL-1) was added and the solution turned to pale brown. The resulting gold clusters solution was refrigerated for at least 2 weeks prior to the usage. The AuNS-functionalized polymer multilayer tubular nanoswimmer was fabricated by template-assisted layer-by-layer technique and gold seed growth method.36 PC filtering membrane with a diameter of 200 nm at the small opening and pore length of 10 μm was served as template and alternatively immersed into PSS (2 mg mL-1 in 0.5 mol L-1 NaCl solution) and PAH (2 B


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Journal of the American Chemical Society plane. By adjusting the objective stage, the cells or nanoswimmers could be shifted to the record position of the laser spot in the field of view. By shifting and rotating objective table, the ellipse spot was vertical to the tip or middle of a nanoswimmer in order to test the influence of the irradiation regions on the poration of cell membranes. The poration of the two nanoswimmers on the cell membrane in Figure S17 and S18 was executed successively by shifting objective stage at XY plane. Unless otherwise stated, each experiment of NIR irradiation was maintained 0.1 s. Simulation and computation. The temperature distribution of the nanoswimmer is computed by the following equation.39

was connected to a function generator system, including a signal generator (Tekrtonix Inc., AFG1062) and a signal amplifier (Toellner Co., Ltd., TOE 7607). Characterization. Scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDX) analysis of the nanoswimmers were acquired by Merlin Compact (Carl Zeiss AG) at an operating voltage of 20 kV. For the characterization of the nanoswimmers, a drop of the solution was dropped on a silicon wafer. For the characterization of the HeLa cells after poration, the coverslip with HeLa cells and nanoswimmers were washed three times with PBS, and then 4 ml of 2.5 vol % glutaraldehyde PBS solution (PH 7.2) was added and kept 4 °C overnight. After that, the coverslips were washed twice to remove glutaraldehyde and undergone the dehydrate process by the immersion into ethanol solution of different concentrations of 30, 50, 70, 90 and 100% for 15 minutes by step. The cell sample then was dried in a vacuum oven overnight. Then the coverslip with HeLa cells and nanoswimmers can be detected by SEM after the vacuum gold coating. Transmission electron microscopy (TEM) images of the nanoswimmers were acquired by JEM1400 (Japan Electron Optics Laboratory Co., Ltd.). Ultraviolet-visible-nearinfrared (UV-vis-NIR) spectrum was performed by U-4100 spectrophotometer (HITACHI Co., Ltd.). Videos were captured by inverted microscope (OLYMPUS Co., CKX71, OLYMPUS SLMPLAN 40x, 20x and 10x) with a Complementary Metal Oxide Semiconductor (CMOS) camera (China Daheng Group Inc., DH-HV315UC-ML). The fluorescence images were captured by inverted microscope (OLYMPUS Co., IX71, OLYMPUS SLMPLAN 20x) with a Charge-Coupled Device (CCD) camera (QImaging Co., QICAM B series 33527). Velocity analysis of nanoswimmer tracking and colormap image was performed by Image J. Confocal laser scanning microscopy (CLSM) images of the cell poration were acquired by Leica TCS SP5 Ⅱ (Leica Microsystems GmbH). Inverting false color bright field images and fluorescence images were overlaid to stack 3D rebuilding CLSM images by Image J. Cell culture. All cell culture was performed according to the standard vendor procedure. The HeLa cells were cultured on a coverslip in RPMI 1640 medium with 1% penicillin, streptomycin and 10% fetal bovine serum at 37 °C, 5% CO2. Cell poration. The experiment setup of NIR laser was built up as previous report.38 In this home-made setup, the laser light path overlapped with optical path of the microscope (Figure S1). Before the cell poration, the NIR spot would be signed by a slide with NIR-absorbed gold-sputtering layer to focus and record position in field of view. After focusing, the laser spot is elliptical like with 2.5 μm in short axis and 20 μm in long axis. The shape of the laser spot was produced by the C-type laser diode (Shanghai Laser & Optics Century Co., Ltd. IRM808TA), as shown in Figure S2. To investigate cell membrane poration of the swimmers, after removing cell medium with PBS, 5 μL of PI (1 mg/mL in PBS) was added onto 1.13 cm2 cell coverslip before NIR irradiation. The laser spot could remain invariant in field of view and it could remain focus when shifting objective stage at XY

𝜌(𝑟)𝑐(𝑟)

∂𝑇(𝑟) ∂𝑡

= ▽𝑘(𝑟)▽𝑇(𝑟) + 𝑄(𝑟,𝑡)

(1)

T(r) is temperature change as a function of coordinate. ρ(r) is the mass density of the nanoswimmer. c(r) is specific heat. k(r) is thermal conductivity. Q(r,t) = Q0 + dQ, while Q0 is the energy source power of laser and dQ is heat transfer through convection, dQ = hcAdT. hc is convective heat transfer coefficient of the process, dT is the temperature difference between the nanoswimmer surface and the medium. The computation is performed by Mamthematica and MATLAB, as shown in Figure S21. Then, the instantaneous photothermal force is calculated to be ~10-8 N to ~10-9 N according to F = -C∂T(r,t) which is proportional to the temperature gradient (Figure S22).40 3. RESULTS AND DISCUSSION As schematically illustrated in Figure 1A, the fabrication process of the nanoswimmers consists of two main steps: assembly of polymer multilayers as the framework of the nanoswimmers, and formation of AuNSs inside the big opening of the nanoswimmers. The framework of the nanoswimmer was fabricated through a template-assisted LbL technique as described in our previous report.36 Briefly, a conically porous PC membrane with a diameter of 200 nm at the small opening was used as a template to alternatingly deposit 10 bilayers of poly (styrene sulfonate) (PSS) and poly (allylamine hydrochloride) (PAH) (in details, please see experimental section). Functionalization of AuNSs includes the deposition of gold nanoclusters at the big opening, and subsequent seed growth of gold nanoclusters into AuNSs through addition of gold growth solution and reducing agents. Scanning electron microscopy (SEM) image in Figure 1B illustrates mass production of the AuNSfunctionalized (PSS/PAH)10 nanoswimmers based on the template deposition method, and millions of the nanoswimmers can be obtained from one template. The length of the nanoswimmers is ~10 μm, and the diameters of the two opening are ~200 nm and ~800 nm, respectively. The transmission electron microscopy (TEM) images in Figure 1C exhibit the conical architecture. Enlarged TEM images in Figure 1C reveal that the Au mainly distributed in the big opening of the nanoswimmers. Energy dispersive Xray (EDX) elemental mapping analysis in Figure 1D further confirms the distribution of Au within the nanoswimmers as expected. The UV-vis-NIR spectrum of the nanoswimmer C



Figure 1. Fabrication and characterization of the AuNSfunctionalized (PSS/PAH)10 tubular nanoswimmers. (A) Schematic illustration of the preparation of the nanoswimmers. (B) SEM image of the nanoswimmers. Scale bar, 2 μm. (C) TEM image showing a single nanoswimmer (left). Scale bar, 1 μm. (Right top) enlarged TEM image of big opening. Scale bar, 200 nm. (Right bottom) enlarged TEM image near the small opening, Scale bar, 100 nm. (D) SEM image and corresponding EDX elemental mapping analysis of the single nanoswimmer for distribution of gold (Au) and carbon (C). Scale bar, 1 μm.

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Figure 2. Movement characterization of the AuNSfunctionalized (PSS/PAH)10 nanoswimmers. (A) Scheme of acoustically-powered controllable movement of the nanoswimmers. The blue and red wave indicate the frequency to drive the forward (blue) and backward (red) movement of the nanoswimmers. (B) Velocity response of the nanoswimmers upon acoustic field with different frequencies. Signal amplitude, 10 Vp-p. Signal-amplifier, 10 dB. (C and D) Time-lapse images illustrating the controllable forward/ backward movement of a single nanoswimmer (C) and multiple nanoswimmers (D) in pure water in response to the acoustic field with Ff/Fb. Blue line and red line indicate the forward movement and backward movement, respectively. Scale bar, 10 μm. (E) Evaluation of the orientation of the nanoswimmers under acoustic field. (F) Velocity modulation of acoustically-powered nanoswimmers in response to an expected 30/10/30 μm s-1 (8/6/8 Vp-p) potential steps with a frequency of 450 kHz. Red and blue dash lines show the trajectories at applied signal amplitude of 8 and 6 Vp-p, respectively. Scale bar, 10 μm. (G) Velocity response of signal amplitude. Frequency, 450 kHz. Signal-amplifier, 10 dB.

in Figure S3 shows the characteristic absorption of AuNSs in the NIR region. The movement of the AuNS-functionalized (PSS/PAH)10 nanoswimmers upon external acoustic field involves a similar mechanism as various metallic micro- and nanosized particles, which utilizes stable standing wave region in the acoustic chamber from external acoustic field with the resonant frequency to drive the nanoswimmers suspend to levitation plane and move.37 Not only can the nanoswimmers perform efficient movement upon the acoustic field with a resonant frequency of around 0.5 MHz, the directional movement can also be controlled through the manipulation of the acoustic frequency in the resonant region (Figure 2A).41 Figure 2B displays the influence of the frequency on the velocity of the nanoswimmers. The nanoswimmers display a high average velocity of over 25 μm s-1 under the acoustic field with different frequencies (450, 486, 490, 512 and 525 kHz) under the intensity of 10 Vp-p and 10 dB. Such influence on the acoustically-powered movement can be exploited for modulation of the nanoswimmers. For example, the time-lapse images of Figure 2C, illustrate the forward/backward movement by the modulation of the frequency of the acoustic field. We next examined the forward frequency (Ff) - defined as the frequency of acoustic field to drive the forward movement of the nanoswimmers, and backward frequency (Fb) defined as the frequency to drive the backward movement of the nanoswimmers. The nanoswimmers rapidly changed from a forward movement at a forward frequency (Ff) to a backward movement at the corresponding frequency (Fb). Additionally, controllable forward/ backward movement of multiple nanoswimmers were achieved by utilizing Ff (486 kHz) and Fb (490 kHz) (Figure 2D). Similar to the acoustically-powered movement of the micro/nanorods which perform the directional motion, orbit or spin in the suspending plane with light end leading orientation,42 the quantification of nanoswimmer orientation under acoustic field indicates that the majority

of nanoswimmers (90.56% and 92.31%) display movement with small opening leading (Figure 2E). The critical frequency for the forward (486 kHz)/backward (490 kHz) motion of the nanoswimmers was identical even if it was manipulated for 10 times repeatedly (Figure S4). The asymmetric opening of the swimmers and metal components with a higher density endow the efficient movement in the direction of the front small-opening and roughly maintains a linear trajectory. As a control, the timelapse images in Figure S5 show that both of the gold nanorod swimmers could be propelled by ultrasound field and directionally reversed due to higher density and asymmetric concave end as previously reported.37,42 Also, AuNS-functionalized (PSS/PAH)6, (PSS/PAH)10, (PSS/PAH)14, (PSS/PAH)18, (PAA/PDDA)10 and (ALG/CHI)10 tubular nanoswimmers with a diameter of 200 nm at the small opening show a controllable orientation behavior, indicating that both the wall thickness and composition of the as-prepared nanoswimmers did not obviously influence their orientation (Figure S6). As the dependence of the velocity upon the ultrasound D


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strength, the average speed of the nanoswimmers increases from 5 μm s-1 at 2 Vp-p to 80 μm s-1 at 10 Vp-p (Figure 2G). Such dependence can be utilized to control the velocity of the nanoswimmers. The time-lapse image in Figure 2F illustrates the on-demand acceleration or deceleration of nanoswimmers by modulating the strength of acoustic field. Such precise and rapid speed control holds promise for a variety of practical applications of the nanoswimmers. To investigate the interaction between the nanoswimmers and the cells, the nanoswimmers were dispersed onto the cell coverslip containing HeLa cells and then the acoustic field was launched to trigger the movement of the nanoswimmers. The nanoswimmers can approach to the HeLa cells by the manipulation of the acoustic field. For example, the time-lapse images of Figure 3A exhibit that one nanoswimmer with the big opening orientation approached toward one HeLa cell under the acoustic field, while the nanoswimmer was bounced off once touched to the HeLa cell. It should be noted that the side of the nanoswimmers possesses minimal association with cells due to the highly hydrated, non-adhesive properties of the polymer multilayer.43 In contrast, the time-lapse images in Figure 3B, illustrate that the nanoswimmers with the small opening orientation could move and accomplish an end-on attachment onto the membrane of HeLa cells. Also, we found that two nanoswimmers could separately target to two cells in the same systems (Figure S7) and two nanoswimmers subsequently attached to one cell (Figure S8). Once the nanoswimmers attached on the HeLa cells, the detachment was not observed over 30 minutes, suggesting that the cell media hardly causes adverse effect to the acousticallypowered movement. Additionally, the nanoswimmers can be acoustically manipulated to approach to the cell (Figure 3C and Video S3). The corresponding enlarged images demonstrate the position change of the big opening with a small vane, confirming the periodic rotation (Figure S9). It should be noted that the spinning movement of the nanoswimmer is attributed to the role of AuNSs with higher density under acoustic field, which is in agreement with the previous publications.37, 44 Here, PI was added in the medium as a fluorescence indicator of cell poration since it can display a red fluorescence once binding with the DNA in the cell. Surprisingly, the red fluorescence in the PI channel was hardly detected from the cell even if the nanoswimmer drilled more than 100 s, indicating that the poration could not be achieved only by the energy of acoustically-powered motion. This is different with the previous publication for the magnetically driven biotube-based microswimmer (~60 μm in length and 2.5 μm in width, much larger than our nanoswimmer) for cell poration.45 To investigate this difference, we first evaluated the driving force of the acoustically powered nanoswimmers. When the nanoswimmer moves freely in a low Reynolds number fluid under acoustic pressure, its acoustic driving force (Fdriving) reaches a balance with the Stokes drag force in fluid. The Stokes drag force (FStokes) is calculated by following:

Figure 3. Interaction between HeLa cells and the AuNSfunctionalized (PSS/PAH)10 nanoswimmers upon different acoustically-powered movement behaviors. (A) Time-lapse images illustrating the non-attachment of big openingorientated nanoswimmer toward to HeLa cells. Scale bar, 10 μm. (B) Time-lapse images illustrating the end-on attachment of small opening-orientated nanoswimmer onto HeLa cells. Scale bar, 10 μm. (C) Time-lapse images displaying the motion of the nanoswimmer toward to HeLa cells with small opening orientation and shifting to spin drilling on the surface of the cell under acoustic field. Scale bar, 10 μm. 𝐹𝑆𝑡𝑜𝑘𝑒s =

2𝜋𝜂𝐿𝑉 𝐿

ln( 𝑅) - 0.72

(1)

Where η is the viscosity coefficient. R is the big opening radius of the nanoswimmers, R = 400 nm. L is the length of the nanoswimmer, L = 10 μm. V is the velocity of the nanoswimmers. The estimated Fdriving of the nanoswimmers is Fdriving = FStokes ≈ 2.01 × 10-12 N.46 The order of magnitude of Fdriving is comparable with other similar tubular swimmers.47, 48 If the applied force by the nanoswimmer is beyond the critical force of the cell membrane, the cell poration should occur. Given that the critical force is also dependent on cell types and variability, we next employed, to the best of our knowledge, the reported minimum critical stress, σ = 0.6 × 104 N m-2.28, 29 The contact area (S) between the cell membrane and the nanoswimmer is approximated by the cross section of the wall of the small opening (the diameter D is 200 nm and the thickness l is 24.5 nm, as shown in Figure 1C), S = π(D/2)2 – π(D/2 - l)2= 1.35 × 10-14 m2. The minimum critical force on the cell membrane is thus estimated as Fcritical = σS = 0.81 × 10-10 N, which is about 40 times higher than the acoustic driving force Fdriving. To address this issue, NIR laser was conducted to assist the nanoswimmers to open the cell membrane as illustrated in Figure 4A and Figure S10. The acoustically-powered movement endows the nanoswimmer attachment on the HeLa cell membrane with the orientation of small opening. The AuNSs on the big opening converted NIR laser energy into heat and help to penetrate the cell membrane. As shown in Figure 4B and Figure 4C, the nanoswimmer was acoustically driven toward the HeLa cell, and obviously the small opening of the nanoswimmer attached to the surface E



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The timescale of the poration from the nanoswimmer is comparable to physically-triggered cell poration and much faster than that of Au nanorods, liquid metal nanomachine and SiO2 helix swimmer internalization.49-52 The colormap images in Figure 4E-G exhibit that the PI fluorescence appeared in the nuclei close to the small opening of the nanoswimmer, and the intensity gradually increased after the NIR-irradiation, indicating that poration occurred at the contact area. In contrast, the adjacent HeLa cell without the nanoswimmer did not display a red fluorescence upon exposure of NIR light (Figure 4B-4G). Additionally, we did not observe a red fluorescence under irradiation of the NIR laser on the small opening (Figure S12A) or the center part (Figure S12B) of the nanoswimmer, suggesting that the NIR irradiation on the AuNS-functionalized big opening of the nanoswimmer is essential for the cell poration. In absence of nanoswimmers, the irradiation of NIR light on the cell did not result in the appearance of a red fluorescence in the nuclei (Figure S12C), suggesting that the NIR irradiation could not open the cell membrane lonely. Figure S12D shows that the nanoswimmer could open the cell membrane in the absence of acoustic field, but requested longer irradiation time or higher power. By comparison, the Au nanorod swimmers with diameters of 200 nm and 800 nm could not open the cell membranes, indicating the insufficient stress on the cell poration under the same conditions (Figure S13A and S13B). Also, the swimmer with a diameter of 1 μm could not perforate the cell membrane since the contact area is 5 times larger than that of the nanoswimmer with a diameter of 200 nm at the small opening (Figure S13C). Moreover, we found that there is a critical laser power of 1 mJ/μm2 for the occurrence of the photomechanical poration of the AuNSfunctionalized (PSS/PAH)10 nanoswimmer (Figure S14). Next, we investigated the effect of the wall thickness and polymer composition of AuNS-functionalized polymer multilayer nanoswimmers with a diameter of 200 nm at the small opening on the cell poration. Figure S15 shows that AuNS-functionalized (PSS/PAH)6, (PSS/PAH)10, (PSS/PAH)14, (PSS/PAH)18 tubular nanoswimmers also required a similar critical laser power for the occurrence of cell poration. Likewise, AuNS-functionalized (PAA/PDDA)10 and (ALG/CHI)10 tubular nanoswimmers exhibited a similar poration rate in the same conditions (Figure S16). Taken together, the wall thickness and polymer composition of AuNS-functionalized polymer multilayer nanoswimmers could not obviously influence their poration ability on the cell membranes. Furthermore, the photomechanical poration of two AuNS-functionalized (PSS/PAH)10 nanoswimmers on the two different cells in the same system (Figure S17), or two nanoswimmers on different positions of one cell (Figure S18) did not show obvious difference. To further characterize the poration of the nanoswimmer, the confocal laser scanning microscope (CLSM) was conducted and the corresponding images indicate the spatial insertion of the nanoswimmer into the HeLa cells (Figure 4H-I). The SEM image in Figure 4J shows that the corresponding nanoswimmer was partially lied on

Figure 4. NIR light-assisted cell poration. (A) Schematic cell poration of the AuNS-functionalized (PSS/PAH)10 nanoswimmers upon the exposure of NIR light. (B-D) Timelapse images showing the movement of the nanoswimmers toward HeLa cell under the acoustic field and the perforation with NIR irradiation. Scale bars, 10 μm. Blue dash line shows the trajectory of acoustic driving and red circle indicates the region of laser spot. (E-G) Time-lapse colormap images showing the dynamic intracellular distribution of fluorescence intensity posterior to the NIR irradiation to the nanoswimmers. Scale bars, 10 μm. (H) CLSM image of the nanoswimmers after cell poration. Yellow dash line indicates the frontier of the cell. Scale bar, 10 μm. (I) Corresponding 3D rebuilding CLSM image of (H). Scale bar, 10 μm. (J) SEM image of the nanoswimmer after cell poration. Scale bar, 5 μm.

of the cell membrane. The NIR laser with a power of 1.5 mJ/μm2 was focused on the big opening of the nanoswimmer, and subsequently the nanoswimmer penetrated the cell membrane within 0.1 s, as shown in Figure 4D and Video S5. It can be seen that the nanoswimmer did not go straight deeper but jumped by an angle. As schematically illustrated in Figure S11, acoustic field might induce the nanoswimmers deviating away from the perpendicular line of the cell membrane even after attaching onto the cell membrane. The small opening of the nanoswimmer was nailed down on cell membrane, and the photomechanical force was generated on the big opening which was functionalized with AuNSs. Therefore, a component of the photomechanical force vertically to the long axis from a deviation could lead to the sudden jump by an angle. The nanoswimmer remained on the cell membrane after the NIR irradiation and sonication. F


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Journal of the American Chemical Society indicate the photomechanical force vertical toward the cell membrane achieved the cell poration. Compared with the conventional technique such as electro-poration, these polymer multilayer tubular nanoswimmers with autonomous movement and multifunctional capacity can actively target a single cell and open the cell membrane, and also could deliver various cargoes into the cell. Such cellmembrane-opening nanoswimmer with motion control may provide a novel concept to develop nanomachines for rapidly drug delivery, subcellular surgery, and therapy in future.

the HeLa cell, indicating that the poration of the HeLa cell results in the insertion of the nanoswimmer. To better understand the mechanism of cell poration by the nanoswimmers, we also estimated the photomechanical force by the photothermal effect upon exposure of NIR. We firstly irradiated the AuNS-functionalized (PSS/PAH)10 nanoswimmers in pure water under different powers of NIR laser (Figure S19) and then correlated the maximal splashing diameter on the energy of NIR irradiation (Figure S20). We computed the temperature distribution on the cross section of the nanoswimmer by solving the heat diffusion equation according to previously reported method (Figure S21).38 In this case, the instantaneous photothermal force (Fp) is estimated to be ~10-9 N to ~10-8 N according to F = -C∂T(r,t), and proportional to the temperature gradient. Compared with the Fcritical (0.81 × 10-10 N) of a cell membrane, the instantaneous photothermal Fp (~10-9 N to ~10-8 N) is theoretically sufficient to open the cell membrane as schematically illustrated in Figure S22. This direct photomechanical poration mechanism of the nanoswimmer is different from that of gold nanoparticles, Janus nanomotor, molecular machine and biotube-based microswimmer.10, 45, 53, 54 The cell membrane poration of gold nanoparticle relies on thermal effect which causes local high-pressure from instantaneous nanobubbles around the gold nanoparticle or enhanced fluidity of the cell membrane.53 As for NIR light-powered Janus nanomotor, insufficient propulsion force of the Janus nanomotor could not mechanically open the cell membrane lonely. Thermal effect around the binding point assists the cell-membraneopening.54 So the Janus nanomotor is thermomechanical mechanism. The opening of cell membranes through a molecular machine is attributed to their megahertzrotation-caused mechanical force tangential toward the cell membrane.10 The mechanical force of biotube-based microswimmers is tangential toward cell membrane.45 In contrast, the cell membrane poration mechanism of the nanoswimmer exclusively relies on mechanical injection. Also, our simulation in Figure S21 shows that the elevated temperature caused by the photothermal effect is dependent on the distance from the nanoswimmer. The distance between the HeLa cell and the big opening of the nanoswimmer should not bring sufficient heat to influence the cell membrane, and thus the cell poration by the nanoswimmer is so-called photomechanical.

ASSOCIATED CONTENT Supporting Information. This Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Descriptions of additional data (word) The movement of the nanoswimmer in pure water under acoustic field. (AVI) Controlled forward/backward movement of the nanoswimmer under acoustic field. (AVI) Nanoswimmer rotating on the surface of cell steadily for 106 s. (AVI) Nanoswimmer moving towards to HeLa cell. (AVI) NIR irradiation onto the nanoswimmer attached into the HeLa cell. (AVI) Dynamic intracellular fluorescence as the function of time after the NIR irradiation of the nanoswimmer with 10x acceleration. (AVI) Nanoswimmer rotating on the surface of the cell for 2 s with 4x deceleration. (AVI)

AUTHOR INFORMATION Corresponding Author *[email protected]; *[email protected]

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT This work is financially supported by the National Science Foundation of China (21603047 and 21573053), China Postdoctoral Science Foundation funded project (2016M590286), and China Postdoctoral Innovation Talents Support Program (BX201700065).

4. CONCLUSION We have demonstrated an AuNS-functionalized polymer nanoswimmer-based strategy for the photomechanical perforation of single cell membrane. The controllable movement of nanoswimmer toward the target cell could be achieved through the manipulation of external acoustic field. The orientation of the small opening endows the stable attachment of the nanoswimmers on the cells, and the following NIR irradiation onto the big opening accomplishes the photomechanical poration of the cell membrane. Unlike the cell poration of gold nanoparticles, Janus nanomotor, molecular machine and biotube-based microswimmer, both experimental and theoretical results

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Gold Nanoshell-Functionalized Polymer Nanoswimmer for

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