View Article Online View Journal
Nanoscale Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: B. Ding, Y. Tian, Y. Pan, Y. Shan, M. Cai, H. Xu, Y. Sun and H. Wang, Nanoscale, 2015, DOI: 10.1039/C5NR01020A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/nanoscale
Page 1 of 4
Nanoscale
Nanoscale
View Article Online
DOI: 10.1039/C5NR01020A
RSC Publishing
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2015, Accepted 00th January 2015
Recording the dynamic endocytosis of single gold nanoparticles by AFM-based Force Tracing Bohua Dinga,# , Yongmei Tianb,c,#, Yangang Panb,c, Yuping Shanb, Mingjun Caib, Haijiao Xub, Yingchun Suna,* and Hongda Wangb,c,*
DOI: 10.1039/x0xx00000x www.rsc.org/
We utilized Force Tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NPs invagination are accurately determined at the single-particle and microsecond level unprecedentedly. Nanoparticles (NPs) are attracting considerable attentions because of their potential applications in biology and medicine. Gold nanoparticles (Au NPs), which possess interesting physicochemical properties, have a long history dating back to Roman times and to the pioneering work of Faraday on the synthesis of stable aqueous dispersions of gold nanoparticles (gold hydrosols) 1. The strongly binding of Au NPs to amine 2 and thiol 3 groups allows the surface modification of Au NPs with amino acids 4 and proteins 5, leading to important biomedical applications from diagnostics 6, drug delivery 7, cell imaging 8, immunostaining 9 and biosensing 10 to electron microscopy markers 11. One fundamental question that requires to be tackled prior to wide biomedical application of Au NPs is the endocytic mechanism of nanoparticles in the cells and their subsequent intracellular transport. Understanding the internalization process of Au NPs in living cells is tremendously important not only for revealing the cellular mechanisms underlying the endocytosis of NPs, but also for new developments in sensing, imaging and therapy. In the past decades, the endocytosis of Au NPs has been extensively studied 12-15. Chan and co-workers studied the internalization of pristine and protein-coated Au NPs with sizes ranging from a few to hundreds of nanometers 16. Shang et al. systematically analyzed the cellular uptake of fluorescent NPs using spinning disk confocal microscopy in combination with quantitative image analysis 17. However, in these bulk experiments, the effect of the microenvironment faced by single Au NPs upon contact with a heterogeneous cell surface is lost in the average outcome of an ensemble assay. Thus there is an extreme need for single molecule technique capable of studying the dynamics of single Au NPs. Up to date, there is few report on the force and instantaneous kinetics of single Au NP entry into living cells 18, 19, owing to the limitation of temporospatial resolution.
This journal is © The Royal Society of Chemistry 2015
Fig. 1 Schematic illustration of the Force Tracing technique. (A) The scheme of the Au NPs-functionalized AFM tip. The Au NP was covalently coupled to AFM tip via a heterobifunctional PEG linker. (B) Image of silicon substrate with tethered Au NPs (dashed circles). (C) The setup of Force Tracing. (D) Typical Force Tracing curve. The tracing curve begins from the left, and the arrow indicates the endocytic force signal. The abscissa (X axis) represents the time of Force Tracing, and the ordinate (Y axis) represents the deflection of the AFM tip corresponding to the force.
Force Tracing technique, based on atomic force microscopy (AFM), utilizes the high sensitivity of a fine AFM cantilever to detect forces down to piconewton at the microsecond level, which provides a unique opportunity to explore the dynamic interactions between Au NPs and cell membranes at single particle level. In previous work, this newly developed Force Tracing technique has been successfully employed to study the endocytic dynamics of single virion through the apical membrane of host cells (Pan et al., in press). Here we further applied this ultrafast and highly sensitive technique to track the dynamic process of cellular endocytosis of single Au NP with the average diameters of 5 nm, 10 nm, and 20 nm under physiological conditions, which could reveal new aspects of the dynamic mechanism of NPs entry into living cells.
Nanoscale, 2015, 00, 1-3 | 1
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
COMMUNICATION
Nanoscale
In order to investigate the endocytosis of single Au NPs with different sizes precisely, we utilized an ultrafast and sensitive Force Tracing technique to directly record this complicated process. The prerequisite for using Force Tracing technique to measure Au NPs endocytosis force is the linkage of Au NPs with the AFM tip. To ensure the flexibility of Au NPs movement, they were covalently conjugated onto an AFM tip via a long heterobifunctional PEG linker (aldehyde-PEG-NHS) (Fig. 1A). The PEG linker was immobilized on an aminated AFM tip through the NHS ester terminus, and the amino groups capped Au NPs reacted with the benzaldehyde moiety of the immobilized linker. During the experiment, the total length of the linker and APTES (about 30 nm) was suitable for measuring the force of Au NPs internalization into the cell through the cell membrane (about 20 nm) 20. To verify Au NPs attachment on the AFM probe, the silicon surface was functionalized with Au NPs according to the same procedure. As shown in the AFM topographical image (Fig. 1B), the silicon surface is covered with a dense layer of nanoparticles (dashed circles). Usually only 1-3 Au NPs were located at the AFM tip apex owing to steric resistance 21-23, ensuring a single Au NP to interact with the cell membrane. Once the endocytosis initiates, the interaction force between Au NPs tethered with the AFM tip and the cell membrane will pull the Au NPs downwards, inducing the extension of the PEG linker and the deflection of the AFM cantilever, which is detected and recorded as endocytosis force signal. As depicted in Fig. 1C, the whole process of single Au NPs endocytosis by living cells can be tracked in situ via Force Tracing technique. African green monkey kidney (Vero) cells were used to study the dynamic process of Au NPs endocytosis. To perform Force Tracing measurements, the Au NPs modified AFM tip was positioned on the top of Vero cell with the help of a CCD camera. We first measured force-distance curves on Vero cell to locate the contact point between the AFM tip and cell surface, and then approached the AFM tip to this contact point through the fine adjustment of the feedback system. The AFM tip tethered with Au NPs would stay above the cell surface as the feedback system was switched off. Upon Au NPs endocytosis, the AFM cantilever bent downwards, and the deflection was recorded using a PCI card (shown in Fig. 1C). The sampling rate of the data acquisition card is 2 MS/s per channel, which is suitable for tracking the rapid process of Au NPs entry into a living cell. Figure 1D shows a typical Force Tracing curve with a sudden step indicating endocytosis force signal, which is clearly distinguished from the cellular activity (Fig.S1). The Force Tracing curve begins from the left. At the beginning, Au NP interacted with cells gently and the curve was approximately flat. As the Au NP on the tip was captured by the living cell, the AFM cantilever would bend downward and a sudden fall in the curve (indicated by the arrow) appeared. When the bending force of AFM cantilever counterbalanced the endocytosis force, the Au NP couldn’t move further into the cell and the curve became flat again. From Force Tracing curves, we can easily measure the force and duration of Au NPs endocytosis. Taking advantage of this highly sensitive Force Tracing technique, we measured the endocytosis force and duration of single Au NPs with the diameters of 5 nm, 10 nm, and 20 nm in living Vero cells, as shown in Fig. 2A, 2B, and 2C, respectively. The sudden steps in the Force Tracing curves (arrows) represent the deflections of the AFM cantilever during internalization of the tiptethered Au NPs into the cell, corresponding to endocytosis forces of the Au NPs with different sizes. It can be seen that the force signals of 5 nm Au NPs are not obvious; however, with the increase of Au NP sizes, the force signals become more and more evident (Fig. 2B and 2C). To make a quantitative comparison of the endocytosis
2 | Nanoscale, 2015, 00, 1-3
Page 2 of 4
Nanoscale
Fig. 2 Comparison of the endocytosis of single Au NPs with different sizes by Vero cells. (A-C) Typical Force Tracing curves showing the endocytosis of 5 nm, 10 nm, and 20 nm Au NPs. The arrows indicate the endocytic force signals. (D-F) The distributions of endocytic forces for 5 nm, 10 nm, and 20 nm Au NPs. (G-I) The distributions of endocytic duration for 5 nm, 10 nm, and 20 nm Au NPs, respectively. (J, K) Statistical endocytic force and duration distribution of 5 nm (red), 10 nm (green), and 20 nm (blue) Au NPs, respectively. The boxes mean the main distribution (50%), with the vertical lines through boxes representing the overall distribution and the horizontal lines in every box representing the average values.
forces among different sizes of Au NPs, the histograms of force distributions were plotted (Fig.2D-F), which show that the forces of Au NPs endocytosis into the living cells vary from 20 pN to 140 pN, with the average values of 45 ± 14 pN, 67 ± 16 pN, 126 ± 25 pN for 5 nm, 10 nm, and 20 nm Au NPs, respectively. The corresponding distributions of duration are shown in Fig.2G, 2H, and 2I, respectively, which range between 20 ms and 160 ms, and the average duration for 5 nm, 10 nm, and 20 nm Au NPs are 45 ±18 ms, 55 ± 18 ms, 81 ± 20 ms, respectively. As shown in Fig.2J and 2K, the statistical comparison of endocytosis force and duration for Au NPs with three different sizes discloses the correlation between the endocytosis process and the sizes of Au NPs. Both the overall distributions and the average values of endocytosis force and duration display an upward tendency from 5 nm, 10 nm to 20 nm Au NPs. The size-dependent endocytosis feature of single Au NP could be attributed to the increased interaction area with the increase of Au NP sizes. To confirm the specificity of the force tracing events, blocking experiments were performed. Cytochalasin B, a cell-permeable mycotoxin, was widely used to inhibit cellular endocytosis of Au NPs by disrupting the cytoskeletons. After the Vero cells were pretreated with 5 μg/mL cytochalasin B for 30 minutes at 37 °C, only a few force signals appeared in about eight hundred randomly chosen Force Tracing curves (Fig.3A, Fig.S2), demonstrating that the force signals were specifically related to the cellular endocytosis of Au NPs. We further testified the endocytosis pathway of single
This journal is © The Royal Society of Chemistry 2015
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
COMMUNICATION
View Article Online
DOI: 10.1039/C5NR01020A
Page 3 of 4
Nanoscale
COMMUNICATION
Fig. 3 Blocking Au NPs endocytosis by cytochalasin B and methyl-β-cyclodextrin (MβCD). (A) The typical Force Tracing curves of Au NPs endocytosis by Vero cells (Au), cytochalasin B-pretreated Vero cells (CB) and MβCD-pretreated Vero cells (MβCD). Control experiments: the typical force curves obtained by the PEGtethered AFM tip (PEG) and the unmodified AFM tip (clean tip). (B) The corresponding probability of tracing curves with force signals. Values are represented by mean ± standard deviation (n=800).
Au NP by cholesterol depletion with methyl-β-cyclodextrin. After pretreatment with 5 mM methyl-β-cyclodextrin for 20 minutes at 37 °C to remove the cholesterol in Vero cell plasma membrane, the endocytosis force signals were largely abolished (Fig.3A, Fig.S3), indicating the involvement of cholesterol-dependent endocytosis pathway. As shown in Fig.3B, the probability of tracing curves with endocytosis force signals are 1% and 2% for cytochalasin Bpretreated and methyl-β-cyclodextrin-pretreated Vero cells, respectively, both of which are distinctly lower than that of untreated Vero cells (15%). As control experiments, the PEG functionalized AFM tip and the bare AFM tip (without modification) were used to collect Force Tracing curves on Vero cells, and no force signal was observed in thousands of force curves with the probability less than 1%. All above results suggest that the force signals in the Force Tracing curves definitely result from the endocytosis of single Au NP by Vero cell, which is a process largely dependent on plasma membrane cholesterol. For a better understanding of the process of Au NPs endocytosis, the Au NPs displacement D is calculated following the equations below. The Au NPs displacement D depends on the two variances: the bending distance of the cantilever, d; and the stretching length of the PEG linker, x.
D d x
(1)
The force-dependent stretching behavior of the PEG linker can be most approximately described by the extended worm-like chain (WLC) model as follows:
FL p 1 k BT 4
x F 1 L0 K 0
2
1 x F 4 L0 K0
(2)
Where kB is the Boltzmann constant, T is the absolute temperature, Lp is the persistence length, K0 is the enthalpic correction, x is the extension, and L0 is the contour length. From previous report 24, the persistence length Lp is 3.8 ± 0.02 Å, and the enthalpic correction K0 is 1561 ± 33 pN. Given that the PEG unit length is 4.2 Å and the terminus is 5.25 Å, the estimated contour length L0 for PEG of 76-77 mers is about 326 Å. According to Hooke’s law, the bending distance d of the cantilever can be calculated using the equation:
F kd
This journal is © The Royal Society of Chemistry 2015
(3)
Fig. 4 Quantifying Au NP displacements by Force Tracing curves. (A) The total displacements versus endocytic forces, in which the corresponding displacements and endocytic forces of different sized Au NPs are shown. (B) The diagram of Au NPs internalization into living cells, which involves the recruitment of membrane cholesterol (red).
Where F is the force measured from the Force Tracing curve, and k is the spring constant of the cantilever. Based on equations (1), (2), and (3), the correlation between Au NPs displacement D and the endocytosis force was obtained. Figure 4A shows the endocytosis force as a function of the Au NPs displacement. From the measured values of endocytosis force, the total displacement is determined to be 22 nm, 24 nm, 27 nm for 5 nm, 10 nm, 20 nm Au NPs. So the average velocity of Au NPs movement during endocytosis process can be calculated as 0.489 µm/s, 0.436 µm/s and 0.333 µm/s for 5 nm, 10 nm, 20 nm Au NPs, respectively (the total displacement D divided by the endocytosis duration). On the basis of Force Tracing curves, a scheme was drawn to illustrate the process of Au NPs entry into living cells. As shown in Figure 4B, the Au NPs-tethered AFM tip first comes to contact with the cell surface, where the Au NPs interact with the cell membrane in the cholesterol-rich domain, inducing the subsequent membrane invagination. The vesicle-wrapped Au NPs are finally internalized into the cell. With the increase of Au NP diameters, the interaction area between Au NPs and the cell membrane as well as the local membrane curvature at the contact region become larger, which result in the increasing endocytosis force and duration from 5 nm, 10 nm to 20 nm Au NPs. In summary, the process of single Au NPs endocytosis was quantitatively analyzed utilizing a novel Force Tracing technique. The dynamic biophysical parameters, such as the endocytosis force, duration and velocity of Au NPs with different sizes, are directly measured at the single-particle and microsecond level unprecedentedly. The results reveal that both the endocytosis force and duration increase with the increasing sizes of Au NPs. The function relationship between the endocytosis displacement and the endocytosis force can be plotted as an exponential curve, from which the average velocity of Au NPs movement during endocytosis process as well as the displacements of different sized Au NPs were readily calculated. The displacements and average velocities of Au NPs also display size-dependent endocytosis feature, except that the displacements increase but the average velocities decrease as the size of Au NPs ranges from 5 nm, 10 nm to 20 nm. All the values of the displacements are larger than Au NPs sizes, suggesting the complete invagination of Au NPs into endocytic vesicles and following movement in a short distance. The endocytosis durations are below 100 ms, manifesting very fast Au NPs internalization, which is unachievable by other approaches. The endocytosis signals of Au NPs were totally blocked by cholesterol depletion with methyl-β-cyclodextrin,
Nanoscale, 2015, 00, 1-3 | 3
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
Nanoscale
View Article Online
DOI: 10.1039/C5NR01020A
View Article Online
Nanoscale
Page 4 of 4
DOI: 10.1039/C5NR01020A
which confirms the dependence of Au NP endocytosis on membrane cholesterol, indicating a clathrin- or caveolaemediated endocytosis pathway for Au NPs. Compared with traditional single-particle track method based on confocal microscopy 25 and electron microcopy 26, Force Tracing technique has the advantage of high temporospatial resolution (microsecond and subnanometer levels). Another advantage of Force Tracing is the capability of measuring the endocytic force and obtaining kinetic parameters, including displacement, velocity, binding energy density of endocytosis. The endocytosis of single particle can be accurately described by a quantitative way via Force Tracing. Although other single molecule techniques (e.g. magnetic tweezer, optical tweezers and glass microneedles) are comparable to Force Tracing in kinetic measurements 27, they can not be used to track faster events (less than 1 ms). With the advantages of high temporospatial resolution and single-particle track, Force Tracing technique demonstrates to be an excellent approach to investigate the dynamic mechanism of single NPs endocytosis in living cells.
Nanoscale 10 L. Olofsson, T. Rindzevicius, I. Pfeiffer, M. Kall and F. Hook, Langmuir, 2003, 19, 10414-10419. 11 W. Baschong and N. G. Wrigley, J. Electron Microsc.y Tech., 1990, 14, 313-323. 12 A. Chaudhuri, G. Battaglia and R. Golestanian, Phys. Biol., 2011, 8, 046002. 13 R. Vacha, F. J. Martinez-Veracoechea and D. Frenkel, Nano Lett., 2011, 11, 5391-5395. 14 Y. Shan, X. Hao, X. Shang, M. Cai, J. Jiang, Z. Tang and H. Wang, Chem. Commun., 2011, 47, 3377-3379. 15 X. Hao, J. Wu, Y. Shan, M. Cai, X. Shang, J. Jiang and H. Wang, J. Phys..Condens. Matter, 2012, 24, 164207. 16 B. D. Chithrani and W. C. W. Chan, Nano Lett., 2007, 7, 1542-1550. 17 L. Shang, K. Nienhaus, X. Jiang, L. Yang, K. Landfester, V. Mailaender, T. Simmet and G. U. Nienhaus, Beilstein J. Nanotech., 2014, 5, 2388-2397 18 Y. Shan, S. Ma, L. Nie, X. Shang, X. Hao, Z. Tang and H. Wang, Chem. Commun., 2011, 47, 8091-8093. 19 F. Tian, T. Yue, Y. Li and X. Zhang, Sci. China Chem., 2014, 57, 1662-1671. 20 W. Zhao, Y. Tian, M. Cai, F. Wang, J. Wu, J. Gao, S. Liu, J. Jiang, S.
Acknowledgements
Jiang and H. Wang, Plos One, 2014, 9, e91595. 21 C. K. Riener, F. Kienberger, C. D. Hahn, G. M. Buchinger, I. O. C.
This work was supported by Ministry of Science and Technology of China (Grant no. 2011CB933600 to H.W.), National Natural Science Foundation of China (Grant no. 21373200 to H. W., no. 21303181 to Y. S.).
22 J. Jiang, X. Hao, M. Cai, Y. Shan, X. Shang, Z. Tang and H. Wang,
Notes and references
23 H. Wang, X. Hao, Y. Shan, J. Jiang, M. Cai and X. Shang,
Egwim, T. Haselgrübler, A. Ebner, C. Romanin, C. Klampfl, B. Lackner, H. Prinz, D. Blaas, P. Hinterdorfer and H. J. Gruber, Anal. Chim. Acta, 2003, 497, 101-114. Nano Lett., 2009, 9, 4489-4493.
a
School of physics, Northeast Normal University, Changchun, Jilin
130024, P.R. China. b
State Key Laboratory of Electroanalytical Chemistry, Changchun
Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P.R. China. c
University of Chinese Academy of Sciences, Beijing, 100049, China
E-mail: [email protected] (Y. Sun); [email protected] (H. Wang). # These authors contributed equally to this paper.
Ultramicroscopy, 2010, 110, 305-312. 24 F. Kienberger, V. P. Pastushenko, G. Kada, H. J. Gruber, C. Riener, H. Schindler and P. Hinterdorfer, Single Mol., 2000, 1, 123-128. 25 X. Hao, X. Shang, J. Wu, Y. Shan, M. Cai, J. Jiang, Z. Huang, Z. Tang, H. Wang, Small, 2011, 7, 1212-1218. 26 S. Watanabe, B. R. Rost, M. Camacho-Perez, M. W. Davis, B. SoehlKielczynski, C. Rosenmund, E. M. Jorgensen, Nature, 2013, 504, 242-247 .
† Electronic Supplementary Information (ESI) available: [details of the
27 H. Clausen-Schaumann, M. Seitz, R. Krautbauer, H. E. Gaub, Force
experimental procedures and the results of the control experiments.]. See
spectroscopy with single bio-molecules. Curr. Opin. Chem. Biol.,
DOI: 10.1039/c000000x/
2000, 4, 524-530.
1
R. Shukla, V. Bansal, M. Chaudhary, A. Basu, R. R. Bhonde and M.
2
P. R. Selvakannan, S. Mandal, S. Phadtare, R. Pasricha and M. Sastry,
3
A. C. Templeton, M. P. Wuelfing and R. W. Murray, Accounts Chem.
4
H. Joshi, P. S. Shirude, V. Bansal, K. N. Ganesh and M. Sastry, J.
5
A. Gole, C. Dash, C. Soman, S. R. Sainkar, M. Rao and M. Sastry,
6
J. J. Storhoff and C. A. Mirkin, Chem. Rev., 1999, 99, 1849-1862.
7
C. M. Niemeyer, Angew. Chem. Int. Edit., 2001, 40, 4128-4158.
8
X. Shi, S. Wang, S. Meshinchi, M. E. Van Antwerp, X. Bi, I. Lee and
9
J. Roth, Histochem. Cell Biol., 1996, 106, 1-8.
Sastry, Langmuir, 2005, 21, 10644-10654. Langmuir, 2003, 19, 3545-3549. Res., 2000, 33, 27-36. Phys. Chem. B, 2004, 108, 11535-11540. Bioconjugate Chem., 2001, 12, 684-690.
J. R. Baker, Jr., Small, 2007, 3, 1245-1252.
4 | Nanoscale, 2015, 00, 1-3
This journal is © The Royal Society of Chemistry 2015
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
COMMUNICATION
Nanoscale Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: B. Ding, Y. Tian, Y. Pan, Y. Shan, M. Cai, H. Xu, Y. Sun and H. Wang, Nanoscale, 2015, DOI: 10.1039/C5NR01020A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/nanoscale
Page 1 of 4
Nanoscale
Nanoscale
View Article Online
DOI: 10.1039/C5NR01020A
RSC Publishing
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2015, Accepted 00th January 2015
Recording the dynamic endocytosis of single gold nanoparticles by AFM-based Force Tracing Bohua Dinga,# , Yongmei Tianb,c,#, Yangang Panb,c, Yuping Shanb, Mingjun Caib, Haijiao Xub, Yingchun Suna,* and Hongda Wangb,c,*
DOI: 10.1039/x0xx00000x www.rsc.org/
We utilized Force Tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NPs invagination are accurately determined at the single-particle and microsecond level unprecedentedly. Nanoparticles (NPs) are attracting considerable attentions because of their potential applications in biology and medicine. Gold nanoparticles (Au NPs), which possess interesting physicochemical properties, have a long history dating back to Roman times and to the pioneering work of Faraday on the synthesis of stable aqueous dispersions of gold nanoparticles (gold hydrosols) 1. The strongly binding of Au NPs to amine 2 and thiol 3 groups allows the surface modification of Au NPs with amino acids 4 and proteins 5, leading to important biomedical applications from diagnostics 6, drug delivery 7, cell imaging 8, immunostaining 9 and biosensing 10 to electron microscopy markers 11. One fundamental question that requires to be tackled prior to wide biomedical application of Au NPs is the endocytic mechanism of nanoparticles in the cells and their subsequent intracellular transport. Understanding the internalization process of Au NPs in living cells is tremendously important not only for revealing the cellular mechanisms underlying the endocytosis of NPs, but also for new developments in sensing, imaging and therapy. In the past decades, the endocytosis of Au NPs has been extensively studied 12-15. Chan and co-workers studied the internalization of pristine and protein-coated Au NPs with sizes ranging from a few to hundreds of nanometers 16. Shang et al. systematically analyzed the cellular uptake of fluorescent NPs using spinning disk confocal microscopy in combination with quantitative image analysis 17. However, in these bulk experiments, the effect of the microenvironment faced by single Au NPs upon contact with a heterogeneous cell surface is lost in the average outcome of an ensemble assay. Thus there is an extreme need for single molecule technique capable of studying the dynamics of single Au NPs. Up to date, there is few report on the force and instantaneous kinetics of single Au NP entry into living cells 18, 19, owing to the limitation of temporospatial resolution.
This journal is © The Royal Society of Chemistry 2015
Fig. 1 Schematic illustration of the Force Tracing technique. (A) The scheme of the Au NPs-functionalized AFM tip. The Au NP was covalently coupled to AFM tip via a heterobifunctional PEG linker. (B) Image of silicon substrate with tethered Au NPs (dashed circles). (C) The setup of Force Tracing. (D) Typical Force Tracing curve. The tracing curve begins from the left, and the arrow indicates the endocytic force signal. The abscissa (X axis) represents the time of Force Tracing, and the ordinate (Y axis) represents the deflection of the AFM tip corresponding to the force.
Force Tracing technique, based on atomic force microscopy (AFM), utilizes the high sensitivity of a fine AFM cantilever to detect forces down to piconewton at the microsecond level, which provides a unique opportunity to explore the dynamic interactions between Au NPs and cell membranes at single particle level. In previous work, this newly developed Force Tracing technique has been successfully employed to study the endocytic dynamics of single virion through the apical membrane of host cells (Pan et al., in press). Here we further applied this ultrafast and highly sensitive technique to track the dynamic process of cellular endocytosis of single Au NP with the average diameters of 5 nm, 10 nm, and 20 nm under physiological conditions, which could reveal new aspects of the dynamic mechanism of NPs entry into living cells.
Nanoscale, 2015, 00, 1-3 | 1
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
COMMUNICATION
Nanoscale
In order to investigate the endocytosis of single Au NPs with different sizes precisely, we utilized an ultrafast and sensitive Force Tracing technique to directly record this complicated process. The prerequisite for using Force Tracing technique to measure Au NPs endocytosis force is the linkage of Au NPs with the AFM tip. To ensure the flexibility of Au NPs movement, they were covalently conjugated onto an AFM tip via a long heterobifunctional PEG linker (aldehyde-PEG-NHS) (Fig. 1A). The PEG linker was immobilized on an aminated AFM tip through the NHS ester terminus, and the amino groups capped Au NPs reacted with the benzaldehyde moiety of the immobilized linker. During the experiment, the total length of the linker and APTES (about 30 nm) was suitable for measuring the force of Au NPs internalization into the cell through the cell membrane (about 20 nm) 20. To verify Au NPs attachment on the AFM probe, the silicon surface was functionalized with Au NPs according to the same procedure. As shown in the AFM topographical image (Fig. 1B), the silicon surface is covered with a dense layer of nanoparticles (dashed circles). Usually only 1-3 Au NPs were located at the AFM tip apex owing to steric resistance 21-23, ensuring a single Au NP to interact with the cell membrane. Once the endocytosis initiates, the interaction force between Au NPs tethered with the AFM tip and the cell membrane will pull the Au NPs downwards, inducing the extension of the PEG linker and the deflection of the AFM cantilever, which is detected and recorded as endocytosis force signal. As depicted in Fig. 1C, the whole process of single Au NPs endocytosis by living cells can be tracked in situ via Force Tracing technique. African green monkey kidney (Vero) cells were used to study the dynamic process of Au NPs endocytosis. To perform Force Tracing measurements, the Au NPs modified AFM tip was positioned on the top of Vero cell with the help of a CCD camera. We first measured force-distance curves on Vero cell to locate the contact point between the AFM tip and cell surface, and then approached the AFM tip to this contact point through the fine adjustment of the feedback system. The AFM tip tethered with Au NPs would stay above the cell surface as the feedback system was switched off. Upon Au NPs endocytosis, the AFM cantilever bent downwards, and the deflection was recorded using a PCI card (shown in Fig. 1C). The sampling rate of the data acquisition card is 2 MS/s per channel, which is suitable for tracking the rapid process of Au NPs entry into a living cell. Figure 1D shows a typical Force Tracing curve with a sudden step indicating endocytosis force signal, which is clearly distinguished from the cellular activity (Fig.S1). The Force Tracing curve begins from the left. At the beginning, Au NP interacted with cells gently and the curve was approximately flat. As the Au NP on the tip was captured by the living cell, the AFM cantilever would bend downward and a sudden fall in the curve (indicated by the arrow) appeared. When the bending force of AFM cantilever counterbalanced the endocytosis force, the Au NP couldn’t move further into the cell and the curve became flat again. From Force Tracing curves, we can easily measure the force and duration of Au NPs endocytosis. Taking advantage of this highly sensitive Force Tracing technique, we measured the endocytosis force and duration of single Au NPs with the diameters of 5 nm, 10 nm, and 20 nm in living Vero cells, as shown in Fig. 2A, 2B, and 2C, respectively. The sudden steps in the Force Tracing curves (arrows) represent the deflections of the AFM cantilever during internalization of the tiptethered Au NPs into the cell, corresponding to endocytosis forces of the Au NPs with different sizes. It can be seen that the force signals of 5 nm Au NPs are not obvious; however, with the increase of Au NP sizes, the force signals become more and more evident (Fig. 2B and 2C). To make a quantitative comparison of the endocytosis
2 | Nanoscale, 2015, 00, 1-3
Page 2 of 4
Nanoscale
Fig. 2 Comparison of the endocytosis of single Au NPs with different sizes by Vero cells. (A-C) Typical Force Tracing curves showing the endocytosis of 5 nm, 10 nm, and 20 nm Au NPs. The arrows indicate the endocytic force signals. (D-F) The distributions of endocytic forces for 5 nm, 10 nm, and 20 nm Au NPs. (G-I) The distributions of endocytic duration for 5 nm, 10 nm, and 20 nm Au NPs, respectively. (J, K) Statistical endocytic force and duration distribution of 5 nm (red), 10 nm (green), and 20 nm (blue) Au NPs, respectively. The boxes mean the main distribution (50%), with the vertical lines through boxes representing the overall distribution and the horizontal lines in every box representing the average values.
forces among different sizes of Au NPs, the histograms of force distributions were plotted (Fig.2D-F), which show that the forces of Au NPs endocytosis into the living cells vary from 20 pN to 140 pN, with the average values of 45 ± 14 pN, 67 ± 16 pN, 126 ± 25 pN for 5 nm, 10 nm, and 20 nm Au NPs, respectively. The corresponding distributions of duration are shown in Fig.2G, 2H, and 2I, respectively, which range between 20 ms and 160 ms, and the average duration for 5 nm, 10 nm, and 20 nm Au NPs are 45 ±18 ms, 55 ± 18 ms, 81 ± 20 ms, respectively. As shown in Fig.2J and 2K, the statistical comparison of endocytosis force and duration for Au NPs with three different sizes discloses the correlation between the endocytosis process and the sizes of Au NPs. Both the overall distributions and the average values of endocytosis force and duration display an upward tendency from 5 nm, 10 nm to 20 nm Au NPs. The size-dependent endocytosis feature of single Au NP could be attributed to the increased interaction area with the increase of Au NP sizes. To confirm the specificity of the force tracing events, blocking experiments were performed. Cytochalasin B, a cell-permeable mycotoxin, was widely used to inhibit cellular endocytosis of Au NPs by disrupting the cytoskeletons. After the Vero cells were pretreated with 5 μg/mL cytochalasin B for 30 minutes at 37 °C, only a few force signals appeared in about eight hundred randomly chosen Force Tracing curves (Fig.3A, Fig.S2), demonstrating that the force signals were specifically related to the cellular endocytosis of Au NPs. We further testified the endocytosis pathway of single
This journal is © The Royal Society of Chemistry 2015
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
COMMUNICATION
View Article Online
DOI: 10.1039/C5NR01020A
Page 3 of 4
Nanoscale
COMMUNICATION
Fig. 3 Blocking Au NPs endocytosis by cytochalasin B and methyl-β-cyclodextrin (MβCD). (A) The typical Force Tracing curves of Au NPs endocytosis by Vero cells (Au), cytochalasin B-pretreated Vero cells (CB) and MβCD-pretreated Vero cells (MβCD). Control experiments: the typical force curves obtained by the PEGtethered AFM tip (PEG) and the unmodified AFM tip (clean tip). (B) The corresponding probability of tracing curves with force signals. Values are represented by mean ± standard deviation (n=800).
Au NP by cholesterol depletion with methyl-β-cyclodextrin. After pretreatment with 5 mM methyl-β-cyclodextrin for 20 minutes at 37 °C to remove the cholesterol in Vero cell plasma membrane, the endocytosis force signals were largely abolished (Fig.3A, Fig.S3), indicating the involvement of cholesterol-dependent endocytosis pathway. As shown in Fig.3B, the probability of tracing curves with endocytosis force signals are 1% and 2% for cytochalasin Bpretreated and methyl-β-cyclodextrin-pretreated Vero cells, respectively, both of which are distinctly lower than that of untreated Vero cells (15%). As control experiments, the PEG functionalized AFM tip and the bare AFM tip (without modification) were used to collect Force Tracing curves on Vero cells, and no force signal was observed in thousands of force curves with the probability less than 1%. All above results suggest that the force signals in the Force Tracing curves definitely result from the endocytosis of single Au NP by Vero cell, which is a process largely dependent on plasma membrane cholesterol. For a better understanding of the process of Au NPs endocytosis, the Au NPs displacement D is calculated following the equations below. The Au NPs displacement D depends on the two variances: the bending distance of the cantilever, d; and the stretching length of the PEG linker, x.
D d x
(1)
The force-dependent stretching behavior of the PEG linker can be most approximately described by the extended worm-like chain (WLC) model as follows:
FL p 1 k BT 4
x F 1 L0 K 0
2
1 x F 4 L0 K0
(2)
Where kB is the Boltzmann constant, T is the absolute temperature, Lp is the persistence length, K0 is the enthalpic correction, x is the extension, and L0 is the contour length. From previous report 24, the persistence length Lp is 3.8 ± 0.02 Å, and the enthalpic correction K0 is 1561 ± 33 pN. Given that the PEG unit length is 4.2 Å and the terminus is 5.25 Å, the estimated contour length L0 for PEG of 76-77 mers is about 326 Å. According to Hooke’s law, the bending distance d of the cantilever can be calculated using the equation:
F kd
This journal is © The Royal Society of Chemistry 2015
(3)
Fig. 4 Quantifying Au NP displacements by Force Tracing curves. (A) The total displacements versus endocytic forces, in which the corresponding displacements and endocytic forces of different sized Au NPs are shown. (B) The diagram of Au NPs internalization into living cells, which involves the recruitment of membrane cholesterol (red).
Where F is the force measured from the Force Tracing curve, and k is the spring constant of the cantilever. Based on equations (1), (2), and (3), the correlation between Au NPs displacement D and the endocytosis force was obtained. Figure 4A shows the endocytosis force as a function of the Au NPs displacement. From the measured values of endocytosis force, the total displacement is determined to be 22 nm, 24 nm, 27 nm for 5 nm, 10 nm, 20 nm Au NPs. So the average velocity of Au NPs movement during endocytosis process can be calculated as 0.489 µm/s, 0.436 µm/s and 0.333 µm/s for 5 nm, 10 nm, 20 nm Au NPs, respectively (the total displacement D divided by the endocytosis duration). On the basis of Force Tracing curves, a scheme was drawn to illustrate the process of Au NPs entry into living cells. As shown in Figure 4B, the Au NPs-tethered AFM tip first comes to contact with the cell surface, where the Au NPs interact with the cell membrane in the cholesterol-rich domain, inducing the subsequent membrane invagination. The vesicle-wrapped Au NPs are finally internalized into the cell. With the increase of Au NP diameters, the interaction area between Au NPs and the cell membrane as well as the local membrane curvature at the contact region become larger, which result in the increasing endocytosis force and duration from 5 nm, 10 nm to 20 nm Au NPs. In summary, the process of single Au NPs endocytosis was quantitatively analyzed utilizing a novel Force Tracing technique. The dynamic biophysical parameters, such as the endocytosis force, duration and velocity of Au NPs with different sizes, are directly measured at the single-particle and microsecond level unprecedentedly. The results reveal that both the endocytosis force and duration increase with the increasing sizes of Au NPs. The function relationship between the endocytosis displacement and the endocytosis force can be plotted as an exponential curve, from which the average velocity of Au NPs movement during endocytosis process as well as the displacements of different sized Au NPs were readily calculated. The displacements and average velocities of Au NPs also display size-dependent endocytosis feature, except that the displacements increase but the average velocities decrease as the size of Au NPs ranges from 5 nm, 10 nm to 20 nm. All the values of the displacements are larger than Au NPs sizes, suggesting the complete invagination of Au NPs into endocytic vesicles and following movement in a short distance. The endocytosis durations are below 100 ms, manifesting very fast Au NPs internalization, which is unachievable by other approaches. The endocytosis signals of Au NPs were totally blocked by cholesterol depletion with methyl-β-cyclodextrin,
Nanoscale, 2015, 00, 1-3 | 3
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
Nanoscale
View Article Online
DOI: 10.1039/C5NR01020A
View Article Online
Nanoscale
Page 4 of 4
DOI: 10.1039/C5NR01020A
which confirms the dependence of Au NP endocytosis on membrane cholesterol, indicating a clathrin- or caveolaemediated endocytosis pathway for Au NPs. Compared with traditional single-particle track method based on confocal microscopy 25 and electron microcopy 26, Force Tracing technique has the advantage of high temporospatial resolution (microsecond and subnanometer levels). Another advantage of Force Tracing is the capability of measuring the endocytic force and obtaining kinetic parameters, including displacement, velocity, binding energy density of endocytosis. The endocytosis of single particle can be accurately described by a quantitative way via Force Tracing. Although other single molecule techniques (e.g. magnetic tweezer, optical tweezers and glass microneedles) are comparable to Force Tracing in kinetic measurements 27, they can not be used to track faster events (less than 1 ms). With the advantages of high temporospatial resolution and single-particle track, Force Tracing technique demonstrates to be an excellent approach to investigate the dynamic mechanism of single NPs endocytosis in living cells.
Nanoscale 10 L. Olofsson, T. Rindzevicius, I. Pfeiffer, M. Kall and F. Hook, Langmuir, 2003, 19, 10414-10419. 11 W. Baschong and N. G. Wrigley, J. Electron Microsc.y Tech., 1990, 14, 313-323. 12 A. Chaudhuri, G. Battaglia and R. Golestanian, Phys. Biol., 2011, 8, 046002. 13 R. Vacha, F. J. Martinez-Veracoechea and D. Frenkel, Nano Lett., 2011, 11, 5391-5395. 14 Y. Shan, X. Hao, X. Shang, M. Cai, J. Jiang, Z. Tang and H. Wang, Chem. Commun., 2011, 47, 3377-3379. 15 X. Hao, J. Wu, Y. Shan, M. Cai, X. Shang, J. Jiang and H. Wang, J. Phys..Condens. Matter, 2012, 24, 164207. 16 B. D. Chithrani and W. C. W. Chan, Nano Lett., 2007, 7, 1542-1550. 17 L. Shang, K. Nienhaus, X. Jiang, L. Yang, K. Landfester, V. Mailaender, T. Simmet and G. U. Nienhaus, Beilstein J. Nanotech., 2014, 5, 2388-2397 18 Y. Shan, S. Ma, L. Nie, X. Shang, X. Hao, Z. Tang and H. Wang, Chem. Commun., 2011, 47, 8091-8093. 19 F. Tian, T. Yue, Y. Li and X. Zhang, Sci. China Chem., 2014, 57, 1662-1671. 20 W. Zhao, Y. Tian, M. Cai, F. Wang, J. Wu, J. Gao, S. Liu, J. Jiang, S.
Acknowledgements
Jiang and H. Wang, Plos One, 2014, 9, e91595. 21 C. K. Riener, F. Kienberger, C. D. Hahn, G. M. Buchinger, I. O. C.
This work was supported by Ministry of Science and Technology of China (Grant no. 2011CB933600 to H.W.), National Natural Science Foundation of China (Grant no. 21373200 to H. W., no. 21303181 to Y. S.).
22 J. Jiang, X. Hao, M. Cai, Y. Shan, X. Shang, Z. Tang and H. Wang,
Notes and references
23 H. Wang, X. Hao, Y. Shan, J. Jiang, M. Cai and X. Shang,
Egwim, T. Haselgrübler, A. Ebner, C. Romanin, C. Klampfl, B. Lackner, H. Prinz, D. Blaas, P. Hinterdorfer and H. J. Gruber, Anal. Chim. Acta, 2003, 497, 101-114. Nano Lett., 2009, 9, 4489-4493.
a
School of physics, Northeast Normal University, Changchun, Jilin
130024, P.R. China. b
State Key Laboratory of Electroanalytical Chemistry, Changchun
Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P.R. China. c
University of Chinese Academy of Sciences, Beijing, 100049, China
E-mail: [email protected] (Y. Sun); [email protected] (H. Wang). # These authors contributed equally to this paper.
Ultramicroscopy, 2010, 110, 305-312. 24 F. Kienberger, V. P. Pastushenko, G. Kada, H. J. Gruber, C. Riener, H. Schindler and P. Hinterdorfer, Single Mol., 2000, 1, 123-128. 25 X. Hao, X. Shang, J. Wu, Y. Shan, M. Cai, J. Jiang, Z. Huang, Z. Tang, H. Wang, Small, 2011, 7, 1212-1218. 26 S. Watanabe, B. R. Rost, M. Camacho-Perez, M. W. Davis, B. SoehlKielczynski, C. Rosenmund, E. M. Jorgensen, Nature, 2013, 504, 242-247 .
† Electronic Supplementary Information (ESI) available: [details of the
27 H. Clausen-Schaumann, M. Seitz, R. Krautbauer, H. E. Gaub, Force
experimental procedures and the results of the control experiments.]. See
spectroscopy with single bio-molecules. Curr. Opin. Chem. Biol.,
DOI: 10.1039/c000000x/
2000, 4, 524-530.
1
R. Shukla, V. Bansal, M. Chaudhary, A. Basu, R. R. Bhonde and M.
2
P. R. Selvakannan, S. Mandal, S. Phadtare, R. Pasricha and M. Sastry,
3
A. C. Templeton, M. P. Wuelfing and R. W. Murray, Accounts Chem.
4
H. Joshi, P. S. Shirude, V. Bansal, K. N. Ganesh and M. Sastry, J.
5
A. Gole, C. Dash, C. Soman, S. R. Sainkar, M. Rao and M. Sastry,
6
J. J. Storhoff and C. A. Mirkin, Chem. Rev., 1999, 99, 1849-1862.
7
C. M. Niemeyer, Angew. Chem. Int. Edit., 2001, 40, 4128-4158.
8
X. Shi, S. Wang, S. Meshinchi, M. E. Van Antwerp, X. Bi, I. Lee and
9
J. Roth, Histochem. Cell Biol., 1996, 106, 1-8.
Sastry, Langmuir, 2005, 21, 10644-10654. Langmuir, 2003, 19, 3545-3549. Res., 2000, 33, 27-36. Phys. Chem. B, 2004, 108, 11535-11540. Bioconjugate Chem., 2001, 12, 684-690.
J. R. Baker, Jr., Small, 2007, 3, 1245-1252.
4 | Nanoscale, 2015, 00, 1-3
This journal is © The Royal Society of Chemistry 2015
Nanoscale Accepted Manuscript
Published on 02 April 2015. Downloaded by University of California - Santa Cruz on 02/04/2015 17:51:50.
COMMUNICATION
