Nanovacuums: Nanoparticle Uptake and Differential Cellular

Subscriber access provided by Washington University | Libraries

Communication

Nano-vacuums: Nanoparticle Uptake and Differential Cellular Migration on a Carpet of Nanoparticles Jie An Yang, Hoa Tri Phan, Shruti Vaidya, and Catherine Jones Murphy Nano Lett., Just Accepted Manuscript • DOI: 10.1021/nl400972r • Publication Date (Web): 11 Apr 2013 Downloaded from http://pubs.acs.org on April 14, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Nano Letters is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Nano-vacuums: Nanoparticle Uptake and Differential Cellular Migration on a Carpet of Nanoparticles Jie An Yang, Hoa Tri Phan, Shruti Vaidya and Catherine J. Murphy* Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA

ABSTRACT

The behavior of prostate carcinoma (PC3) cells and human dermal fibroblast (HDF) cells when incubated with sedimented Au NPs in vitro is studied. Darkfield microscopy demonstrates that both PC3 and HDF cells can ‘vacuum’ Au NPs from the surface. Mean square displacement and mean cumulative square distance of cells shows that PC3 migration decreases in the presence of Au NPs while for HDF, migration is dependent on the surface charge and shape of Au NPs.

KEYWORDS Gold, nanoparticles, migration, uptake, nanocrystals, cellular

ACS Paragon Plus Environment

1

Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 22

Gold nanoparticles (Au NPs) are excellent model NP systems for biological studies because of their low cytotoxicity and large absorption and scattering coefficients.1 These properties have enabled biomedical applications of Au NPs in areas such as chemical sensing, imaging, drug delivery and targeting.

2–7

While the promise of nano/bio-technology is great, the understanding

of nanoparticle mechanisms of interaction with biological entities has only been in recent years probed and questioned. For example, gold nanospheres (Au NSs) are preferentially taken up by HeLa cells as compared to gold nanorods of similar sizes.8 Au NSs of 40-50 nm diameter showed the largest uptake by human breast cancer cells as compared to other sizes.9 A net negative surface charge on the Au NPs was also shown to be preferred for Au NP uptake into human keratinocyte cells.10 Albanese and Chan showed that the degree of Au NP aggregation, a common but unavoidable occurrence when Au NPs are added to cell media, can influence cellular uptake.11 Recently, a study showed that the inherent density of Au NPs created a concentration gradient within the cell chamber, and cells at the bottom of the chamber were exposed to larger amounts of Au NPs as compared to the top.12 Most work on Au NPs has focused on its cytotoxicity.4,13 However, the changes in cellular behavior in the presence of Au NPs is potentially more interesting. For example, the study of cell migration is an area of active research as it has implications in wound healing, remodeling and cancer metastasis.14,15 Cells undergoing repeated polarized extension-contraction cycles, which when coupled with adhesion and de-adhesion to the substrate, causes cell migration.16 In particular, for single cell migration, a 5 step process has been identified: 1) leading edge formation, 2) adhesion and traction force generation, 3) focalized proteolysis, 4) actomyosin contraction and 5) rear-end retraction.17 This model is active in both normal and neoplastic single cell migration, the rate of which is influenced by the cell type and cellular environment.


2

Page 3 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

As long ago as 1977, Au NPs with diameters of 200 – 400 nm were used to visualize cell migration through the formation of tracks under darkfield illumination.18 This assay was used to track 3T3 fibroblast cells and human keratinocytes after fixation.19,20 However, because the Au NPs were plated on the substrate before the cells, cell adhesion to the substrate could be compromised. More recently, it had been shown that the uptake of Au NPs by human dermal fibroblast (HDF) cells can influence the formation of intracellular actin fibers, thus can indirectly affect cell adhesion and spreading.21 Taken together, previous work suggests that with improvements in Au NP size, shape and surface control, the time is ripe to study cellular function such as cell migration with well-defined nanostructures. Here, we study the effect of sedimented Au NPs on cell migration. In a typical setup, prostate carcinoma (PC3) or HDF cells were first plated to about 60-70% confluency. The cells were plated such that cell-cell interactions were minimized and cells were allowed to freely diffuse. The Au NPs then added to the cell media were allowed to sediment slowly based on their intrinsic density onto the glass substrate, and the cellular behavior was observed over a period of at least 8 h (Scheme 1). The media was left intact during the experiments. We show that cells in vitro, using their leading edge, can ‘clean’ a surface deposited with gold nanospheres (Au NSs) and long gold nanorods (Au NRs). The measured mean square displacement (MSD) and mean cumulative square distance (MCSD) values of the cell show that the ‘vacuuming’ of Au NPs by cells is deeply intertwined with cellular migration and may relate to the known differences in migration mechanisms of PC3 and HDF cells.


3

Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 22

Scheme 1. a) Schematics of experimental setup. Cells were first plated at low densities. Au NPs added were allowed to sediment (1-2 h) and cell migration observed. b) Cartoon of the uptake of Au NPs on a substrate by a generic cell. The cell probes the surface for Au NPs. As the cell migrates, Au NPs are taken up via the leading edge and subsequently endocytosized at the cell body.

Gold Nanoparticles and their Surface Modifications In order to probe the sedimentation effect of nanoparticles on cells, large and hence heavier nanoparticles were specifically chosen. Two types of Au NPs were used: Au NSs and Au NRs. (Note: The abbreviation Au NPs will be used to describe both types of nanoparticles.) Au NSs with a diameter of 90.8 ± 0.3 nm and Au NRs with aspect ratio of ~14 (294 ± 112 nm by 21.1 ± 3.6 nm) were synthesized as previously published.22,23 The rate of sedimentation, or settling velocity, for the spherical 90 nm Au NS can be estimated by equating the frictional and buoyancy force to the gravitational force (Stokes’ law):24 2 (ρp -ρ)

vs = 9

η

gR2

(Eq. 1)


4

Page 5 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

where vs is the Au NSs' settling velocity (m/s), ρp is the Au NS density (19,320 kg/m3), ρ is the water density (1,000 kg/m3), η is the viscosity of water (1.002 mPa·s), g is the gravitational acceleration (9.8 m/s2) and R is the radius of the Au NS (45 x 10-9 m). Au NSs of 90 nm have a settling velocity of 81 nm/s. For Au NRs, due to their anisotropic shape, the translational frictional force of each Au NR depends on its orientation in solution.24 However, because the Au NRs are randomly dispersed in solution, it can be assumed that all orientations are present at any given point of time and the average frictional coefficient can be used. This can be obtained from the friction ratio (cylinders:spheres): µ

ft = 6πηR

(Eq. 2) e

where µ is the translational friction coefficient for Au NRs and Re is the equivalent radii of a sphere with equal volume.25 For the case of Au NRs with aspect ratio 14, Re is 28.97 nm and ft is 1.696, giving µ to be 9.28 x 10-10 Pa·s (Supporting Information). The buoyancy force is the volume of solvent that is displaced by the Au NR and the gravitational force is the weight of the Au NR. By equating the upward frictional and buoyancy force to the downward force due to gravity, vs for Au NRs can be estimated: vs =

πr2 hg(ρp -ρ) µ

(Eq. 3)

where r is the radius and h is the length of the Au NR (assumed cylindrical). The value vs for Au NR is 20 nm/s. This translates to ~3.5 h for 90 nm Au NPs and ~14.1 h for 300 nm x 20 nm Au NRs to fall a distance of 1 mm, half the height of the cell chamber, demonstrating that both Au NSs and Au NRs have a high tendency to sediment over time. However, in our experiments, Au


5

Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 22

NP aggregation in cell media increases the rate of sedimentation such that the cells’ ‘vacuuming effect’ can be observed after ~1 – 2 h. The surface chemistry of Au NPs was modified using layer-by-layer polyelectrolyte deposition.26 Au NSs had initial surface ligands comprising of citrate and hydroquinone, imparting an overall negative charge. By incubating positively-charged poly(allylamine hydrochloride) (PAH, M.W. ~15,000 g/mol) followed by negatively-charged poly(acrylic acid) (PAA, M.W. ~15,000 g/mol), the zeta potential of the Au NSs can be varied. For Au NRs, since they are synthesized with the positively charged surfactant, cetyltrimethylammonium bromide (CTAB), a positively-charged polyelectrolyte, poly(diallyldimethylammonium chloride) (PDADMAC, M.W. 500 nm) pathway.30 Therefore, in our studies, we blocked these pathways with chlorpromazine (clathrin inhibitor) and filipin (caveolin inhibitor) and the cellular uptake of Au NPs was still observed. Darkfield images showed that cell migration still resulted in Au NP being ‘vacuumed’ from the surface onto the cell bodies, thus suggesting that clathrin and caveolin are not required for ‘vacuuming’ (Supporting Information, Movie S4). TEM images showed much fewer Au NPs being endocytosized, demonstrating that ‘vacuuming’ and endocytosis are separate events (Supporting Information). These results demonstrate that Au NPs interaction with cells in vitro can be generally divided into three stages: 1) interaction at the cell edge, 2) transfer to the cell body and 3) endocytosis. Interaction of cells and Au NPs mostly occurred via the filopodia and lamellipodia at the leading edge. As the Au NPs were not tightly bound to the substrate, the constant probing by the cells can dislodge the Au NPs, resulting in them being picked up by cells and transported towards the cell body.


12

Page 13 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Gold Nanoparticles’ Influence on Cell Migration. The presence of Au NPs on substrates can potentially assist or hinder cellular adhesion to the substrate, and hence influence cell migration. The usual method to measure cell migration is by calculating the mean square displacement (MSD) of the cells31,32 2

MSDt= 〈[rt+t0 -rt0 ] 〉 = 4Dt

(Eq. 4)

where r(t0) is the initial position of the cell and r(t+t0) is the new position after time t, 〈⋯ 〉 denotes a combined average over all trajectories for all cells and D is the diffusion coefficient. If the cells are sufficiently far apart and do not interfere with each other, random motion is assumed. MSD were measured with respect to the center of each cell’s nucleus. From Eq. 4, a linear relationship is expected when MSD is plotted again time. Results show that for both PC3 and HDF cells incubated with and without Au NPs, a linear dependence is only observed over 100 min intervals (Fig. 3a, b). At longer time intervals, breaks and jumps were observed. Compared to cells incubated in the absence of Au NPs, PC3 cell migration in the presence of Au NPs are slower while for HDF cells, cell migration is faster with all Au NPs except PAA Au NSs. Cellular diffusion coefficients calculated from the entire MSD plots showed that Au NPs did have an influence on cell migration (Table 1). PC3 cellular diffusion coefficients decrease from 12.8 ± 0.2 µm2/min in the absence of Au NPs to a range of 3.2 ± 0.1 µm2/min – 10.6 ± 0.4 µm2/min when incubated with mPEG Au NSs and PSS Au NRs respectively. HDF cellular diffusion coefficients increase from 3.7 ± 0.1 µm2/min to 4.4 ± 0.1 µm2/min and 10.8 ± 0.3 µm2/min when incubated with mPEG Au NRs and PAH Au NSs respectively. When incubated with PAA Au NSs, the cellular diffusion coefficient decreases to 3.2 ± 0.1µm2/min.


13

Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 22

To better represent cell migration over long durations, the mean cumulative square distance (MCSD) is used, 2

MCSDt= 〈∑ni=0 ([rti +τ-rti ] )〉

(Eq. 5)

where r(ti+τ) is the position of cell at time τ from its previous position, r(ti), where τ = 5 min. The summation is taken over all available time points and 〈⋯ 〉 denotes a combined average over all trajectories. The MCSD measures the total distance moved by the cells over the time frame observed, while the slope of the MCSD plot against time gives information about its speed. Results show that PC3 cell migration is reduced in the presence of Au NPs (Fig. 3c), similar to its respective MSD plot, regardless of the surface charge and shape of Au NPs. The MCSD plots show a linear relationship with time, signifying that the cells are migrating at a constant speed. The distributions of the samples are compared at 8 h after incubation and plotted in a box plot (Fig. 3e). On average, PC3 cells have reduced cell migration when incubated with Au NPs. The MCSD box plots at 8 h after incubation show that PC3 cells have a mean MCSD of 2200 µm2. Compared to PC3 incubated with Au NPs, the mean MCSD obtained were 960 µm2 for PAH NPs, 1400 µm2 for PAA NPs, 850 µm2 for mPEG NPs, 1100 µm2 for PDADMAC NRs, 1100 µm2 for PSS NRs and 850 µm2 for mPEG NRs. The average velocity of each cell samples are deduced from the total distance moved over the 8 h incubation period (Table 1). These values reflect that PC3 cells move slower in the presence of Au NPs. Interestingly, HDF cells show a different MCSD pattern when incubated with Au NPs (Fig. 3d, f). HDF cells were observed to migrate faster in the presence of positively charged Au NPs (NSs and NRs) as well as PSS Au NRs than without Au NPs, and slower in the presence of mPEG Au NSs, mPEG Au NRs and PAA Au NSs. The mean MCSD after 8 h incubation for HDF cells was 430 µm2, which increases to 970 µm2 with PAH NPs, 660 µm2 with PDADMAC NRs and 620 µm2 with PSS


14

Page 15 of 22

NRs. The mean MCSD decreases to 320 µm2, 340 µm2 and 290 µm2 when incubated with PAA NSs, mPEG NSs and mPEG NRs respectively. The cell velocity reflects the same trend: PAH Au NSs causes the largest increase of velocity from (0.28 ± 0.01) µm/min to (0.40 ± 0.03) µm/min, followed by PDADMAC Au NRs and PSS Au NRs at (0.37 ± 0.02) µm/min and (0.35 ± 0.02) µm/min respectively.

a)

b)

PC3

8000

6000

6000

5000

5000

MSD (µ m2)

MSD (µ µ m2)

HDF

7000

7000

4000 3000 2000

4000 3000 2000 1000

1000 0

0 0

100

200

300

400

500

0

100

Time (min)

c)

200

300

400

500

400

500

Time (min)

d)

PC3

HDF

1000

2000 800

MCSD (µ µ m2)

1600

MCSD (µ µ m2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

1200 800

600

400

200

400 0

0 0

100

200

300

400

500

0

100

Time (min)

e)

200

300

Time (min)

f)


15

Nano Letters

PC3

10000

HDF

1800 1600

8000

1400

MCSD (µm2)

MCSD (µ m2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

6000

4000

1200 1000 800 600 400

2000

200 0

PC3

PAA NSs PDADMAC LRs PEG LRs PAH NSs PEG NSs PSS LRs

0

HDF

PAA NSs PDADMAC LRs PEG LRs PAH NSs PEG NSs PSS LRs

Figure 3. Influence of Au NPs on the migration of PC3 and HDF cells. a, b) Plot of the meansqaure displacement (MSD) of PC3 and HDF cells with and without Au NPs with respect to time. Au NPs and Au NRs concentrations are fixed at 0.02 nM. Legends are as follows: black solid square, PC3 or HDF alone; red hollow square, PAH NPs; green hollow triangle, PAA NPs; blue hollow triangle, mPEG NPs; red solid square, PDADMAC Au NRs; green solid triangle, PSS Au NRs; blue solid triangle, mPEG Au NRs. c, d) Plot of the mean cumulative sqaure displacement (MCSD) of PC3 and HDF cells with and without Au NPs with respect to time. e) Box plot showing the distribution of PC3’s MCSD at 8 h after incubation. Box represent the 25th to 75th percentile of sample with the line denoting the median. Mean of sample is shown as a solid square. Whiskers represent the lower and upper inner fence limit (1.5 interquartile range). Outliers are denotes as diamonds. Sample size: PC3, 22; PAH, 38; PAA, 41; PEG NPs, 29; PDADMAC NRs, 40; PSS NRs, 24; PEG NRs, 45. f) Box plot showing the distribution of HDF’s MCSD at 8 h after incubation. Sample size: HDF, 21; PAH NPs, 14; PAA NPs, 15; PEG NPs, 19; PDADMAC NRs, 20; PSS NRs, 24; PEG NRs, 19.

The effect of Au NS concentration on the migration of PC3 cells was further investigated. While PC3 cells move slower when incubated with Au NSs, decreasing the Au NS concentration from 0.02 nM to 0.005 nM did not significantly alter PC3 cell migration (Fig. 4). While the mean MCSD decrease from 950 µm2 to 630 µm2 for PAH NPs and 1400 µm2 to 1000 µm2 for PAA NPs when concentration decreases from 0.02 nM to 0.005 nM respectively, no difference was observed for the MCSD box plot between different Au NS concentrations.


16

Page 17 of 22

a)

b) 10000 2000 8000 1500

MCSD (µm2)

MCSD (µ µ m2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

1000

500

6000

4000

2000

0

0 0

100

200

300

400

500

PC3

Time (min)

PAH NSs

PAH NSs

PAA NSs

PAA NSs

0.005 nM

0.02 nM

0.005 nM

0.02 nM

Figure 4. Influence of Au NPs concentration on the migration of PC3 cells. a) Plot of the mean cumulative sqaure displacement (MCSD) of PC3 cells with and without Au NPs with respect to time. Au NPs and Au NRs concentrations are at 0.005 nm and 0.02 nM. Legends are as follows: black solid square, PC3 alone; blue hollow sqaure, PAH NPs (0.005 nM); blue solid square, PAH NPs (0.02 nM); red hollow sqaure, PAA NPs (0.005 nM); red solid square, PAA NPs (0.02 nM). b) Box plot showing the distribution of PC3’s MCSD at 8 h after incubation. Box represent the 25th to 75th percentile of sample with the line denoting the median. Mean of sample is shown as a solid square. Whiskers represent the lower and upper inner fence limit (1.5 interquartile range). Outliers are denotes as diamonds. Sample size: PC3, 22; PAH NPs (0.005 nM), 40; PAH NPs (0.02 nM), 38; PAA NPs (0.005 nM), 28; PAA NPs (0.02 nM), 41.

The results here suggest that the surface charge and shape of Au NPs can potentially influence how cells ‘see’ the glass substrate and ultimately influence their migration. First, the behavior of the Au NPs in cell media (with 10% fetal bovine serum) reflects that a protein corona is most likely forming around the Au NPs. Previous studies have shown that regardless of the surface coatings, Au NPs maintain a net negative zeta potential when incubated in cell media.29 While the initial net charge of the nanoparticles then might be identical, which therefore might make all the nanoparticles have similar electrostatic interactions with the poly-lysine-coated surface the experiments were performed on, the same cannot be said for the protein corona. Because of the difference in initial surface charge, the type, orientation and concentration of proteins that form the corona may differ between nanoparticle sets, and become the driving force that alters cell


17

Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 22

migration. It becomes interesting to consider, then, how tighter binding of the nanoparticles to the surface would influence cellular behavior. Secondly, the inherent cell migration mechanism may influence how the cells interact with the Au NP coated surface. Most carcinoma cells, like PC3 cells, are phenotypically heterogenous and maintain a roundish/elliptoid shape (10-30 µm).33 They have a migration velocity of about 0.1-20 µm/min and their movement is usually characterized by an amoeboid migration mechanism, which is dependent on propulsive, cytoplasmic streaming.34 Such movement is characterized as a crawling type migration achieved by short-lived and relatively weak interactions with the substrate, which pulls the cell forward. The presence of the loosely bound Au NPs implies that when the pseudopods extend, a firm grip on the substrate cannot be established and thus PC3 cells’ migration is reduced. On the other hand, fibroblast cells (large mesenchymal cells 50-200 µm wide) have elaborate cytoskeletal networks.35 They adhere strongly to substrates (via integrin receptors), project broad lamellipodia at the leading edge and use microtubule networks to regulate cell migration, resulting in slow movement with net speed of around 0.25-1 µm/min. HDF cells are thus more susceptible to variations in Au NP surface charges, size and shape. Experiments are currently in progress to understand these differences. In conclusion, we show here that Au NPs, when allowed to sediment and incubate with cells in vitro, can be taken up via the cells’ intrinsic migration mechanism. For PC3 cells, a general decrease in cell migration was observed, while for HDF cells, cell migration is dependent on the surface charge and shape of Au NPs. These results signify that the interaction of Au NPs with cells in vitro is non-trivial and depends critically on cell type. Provocatively, it may be possible that Au NPs have the potential to be used to reduce the metastasis potential of cancer cells as well as to aid in wound healing in mesenchymal cells.


18

Page 19 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

ASSOCIATED CONTENT Supporting Information. Details of the experimental setup, synthesis of gold nanoparticles, cell incubation experiments, derivation of sedimentation velocity, live dead staining, TEM images of PC3 and HDF cells and cell tracking using ImageJ are included in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Email: [email protected] (C.J. Murphy). Phone: +1 217 333 7680. Fax: +1 217 244 3186. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by the National Science Foundation (CHE-1011980). The authors thank the Frederick Seitz Materials Research Laboratory (UIUC) for help with TEM micrographs of gold nanoparticles in cells and the Cell Media Facility (UIUC) for help with cell cultures. J.A.Y. thanks UIUC for the J.C. Bailar Fellowship. H.T.P. acknowledges the Vietnam-UIUC student exchange program for undergraduate summer research at UIUC. S.V. is grateful to the I-STEM High School Summer Research Program for support. ABBREVIATIONS


19

Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 22

Au NP, gold nanoparticle; Au NS, gold nanospheres; Au NR, long gold nanorod; PC3, prostate carcinoma; HDF, human dermal fibroblast.

REFERENCES (1) (2)

(3) (4)

(5) (6)

(7) (8)

(9) (10)

(11) (12) (13)

(14)

(15)

Dreaden, E. C.; Alkilany, A. M.; Huang, X.; Murphy, C. J.; El-Sayed, M. A. The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev. 2012, 41, 2740–2779. Murphy, C. J.; Gole, A. M.; Hunyadi, S. E.; Stone, J. W.; Sisco, P. N.; Alkilany, A.; Kinard, B. E.; Hankins, P. Chemical Sensing and Imaging with Metallic Nanorods. Chem. Comm. 2008, 544–557. Bedford, E. E.; Spadavecchia, J.; Pradier, C.-M.; Gu, F. X. Surface Plasmon Resonance Biosensors Incorporating Gold Nanoparticles. Macromol. Biosci. 2012, 12, 724–739. Alkilany, A. M.; Thompson, L. B.; Boulos, S. P.; Sisco, P. N.; Murphy, C. J. Gold Nanorods: Their Potential for Photothermal Therapeutics, Tempered by the Complexity of the Biological Interactions. Adv. Drug Deliv. Rev. 2012, 64, 190–199. Jans, H.; Huo, Q. Gold Nanoparticle-enabled Biological and Chemical Detection and Analysis. Chem. Soc. Rev. 2012, 41, 2849–2866. Dreaden, E. C.; El-Sayed, M. A. Detecting and Destroying Cancer Cells in More than One Way with Noble Metals and Different Confinement Properties on the Nanoscale. Acc. Chem. Res. 2012, 45, 1854–1865. Dykman, L.; Khlebtsov, N. Gold Nanoparticles in Biomedical Applications: Recent Advances and Perspectives. Chem. Soc. Rev. 2012, 41, 2256–2282. Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Determining the Size and Shape Dependence of Gold Nanoparticle Uptake into Mammalian Cells. Nano Lett. 2006, 6, 662–668. Jiang, W.; Kim, B. Y. S.; Rutka, J. T.; Chan, W. C. W. Nanoparticle-mediated Cellular Response is Size-dependent. Nature Nanotech. 2008, 3, 145–150. Grabinski, C.; Schaeublin, N.; Wijaya, A.; D’Couto, H.; Baxamusa, S. H.; HamadSchifferli, K.; Hussain, S. M. Effect of Gold Nanorod Surface Chemistry on Cellular Response. ACS Nano 2011, 5, 2870–2879. Albanese, A.; Chan, W. C. W. Effect of Gold Nanoparticle Aggregation on Cell Uptake and Toxicity. ACS Nano 2011, 5, 5478–5489. Cho, E. C.; Zhang, Q.; Xia, Y. The Effect of Sedimentation and Diffusion on Cellular Uptake of Gold Nanoparticles. Nature Nanotech. 2011, 6, 385–391. Alkilany, A. M.; Lohse, S. E.; Murphy, C. J. The Gold Standard: Gold Nanoparticle Libraries to Understand the Nano-Bio Interface. Acc. Chem. Res. 2012, DOI: 10.1021/ar300015b. Veiseh, O.; Kievit, F. M.; Ellenbogen, R. G.; Zhang, M. Cancer Cell Invasion: Treatment and Monitoring Opportunities in Nanomedicine. Adv. Drug Deliv. Rev. 2011, 63, 582– 596. Friedl, P.; Gilmour, D. Collective Cell Migration in Morphogenesis, Regeneration and Cancer. Nature Rev. Mol. Cell Biol. 2009, 10, 445–457.


20

Page 21 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

(16) Friedl, P.; Hegerfeldt, Y.; Tusch, M. Collective Cell Migration in Morphogenesis and Cancer. Inter. J. Dev. Biol. 2004, 48, 441–449. (17) Friedl, P.; Alexander, S. Cancer Invasion and the Microenvironment: Plasticity and Reciprocity. Cell 2011, 147, 992–1009. (18) Albrecht-Buehler, G. The Phagokinetic Tracks of 3T3 Cells. Cell 1977, 11, 395–404. (19) Albrecht, G. Daughter 3T3 Cells. Are They Mirror Images of Each Other? J. Cell Biol. 1977, 72, 595–603. (20) Woodley, D. T.; Bachmann, P. M.; O’Keefe, E. J. Laminin Inhibits Human Keratinocyte Migration. J. Cell. Physiol. 1988, 136, 140–146. (21) Pernodet, N.; Fang, X.; Sun, Y.; Bakhtina, A.; Ramakrishnan, A.; Sokolov, J.; Ulman, A.; Rafailovich, M. Adverse Effects of Citrate/Gold Nanoparticles on Human Dermal Fibroblasts. Small 2006, 2, 766–773. (22) Perrault, S. D.; Chan, W. C. W. Synthesis and Surface Modification of Highly Monodispersed, Spherical Gold Nanoparticles of 50-200 nm. J. Am. Chem. Soc. 2009, 131, 17042–17043. (23) Jana, N. R.; Gearheart, L.; Murphy, C. J. Wet Chemical Synthesis of High Aspect Ratio Gold Nanorods. J. Phys. Chem. B 2001, 105, 4065–4067. (24) Sharma, V.; Park, K.; Srinivasarao, M. Shape Separation of Gold Nanorods Using Centrifugation. Proc. Natl. Acad. Sci.USA 2009, 106, 4981–4985. (25) Bloomfield, V., A. Separations and Hydrodynamics: Survey of Biomolecular Hydrodynamics. In On-line Biophysics Textbook; Schuster, T. M.; pp 1–16. (26) Gole, A.; Murphy, C. J. Polyelectrolyte Coated Gold Nanorods: Synthesis, Characterization and Immobilization. Chem. Mater. 2005, 17, 1325–1330. (27) Sisco, P. N.; Wilson, C. G.; Mironova, E.; Baxter, S. C.; Murphy, C. J.; Goldsmith, E. C. The Effect of Gold Nanorods on Cell-Mediated Collagen Remodeling. Nano Lett. 2008, 8, 3409–3412. (28) Niidome, T.; Yamagata, M.; Okamoto, Y.; Akiyama, Y.; Takahashi, H.; Kawano, T.; Katayama, Y.; Niidome, Y. PEG-modified Gold Nanorods with a Stealth Character for in vivo Applications. J. Control. Release 2006, 114, 343–347. (29) Alkilany, A. M.; Nagaria, P. K.; Hexel, C. R.; Shaw, T. J.; Murphy, C. J.; Wyatt, M. D. Cellular Uptake and Cytotoxicity of Gold Nanorods: Molecular Origin of Cytotoxicity and Surface Effects. Small 2009, 5, 701–708. (30) Thurn, K. T.; Brown, E. M. B.; Wu, A.; Vogt, S.; Lai, B.; Maser, J.; Paunesku, T.; Woloschak, G. E. Nanoparticles for Applications in Cellular Imaging. Nanoscale Res. Lett. 2007, 2, 430–441. (31) Qian, H.; Sheetz, M. P.; Elson, E. L. Single Particle Tracking. Analysis of Diffusion and Flow in Two-Dimensional Systems. Biophys. J. 1991, 60, 910–921. (32) Dieterich, P.; Klages, R.; Preuss, R.; Schwab, A. Anomalous Dynamics of Cell Migration. Proc. Natl. Acad. Sci.USA 2008, 105, 459–463. (33) Condeelis, J.; Jones, J.; Segall, J. E. Chemotaxis of Metastatic Tumor Cells: Clues to Mechanisms from the Dictyostelium Paradigm. Cancer Metastasis Rev. 1992, 11, 55–68. (34) Friedl, P.; Wolf, K. Tumor-Cell Invasion and Migration: Diversity and Escape Mechanisms. Nature Rev. Cancer 2003, 3, 362–374. (35) Vorotnikov, A. V. Chemotaxis: Movement, Direction, Control. Biochemistry (Moscow) 2012, 76, 1528–1555.


21

Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 22

TOC:

Time


Nanovacuums: Nanoparticle Uptake and Differential Cellular

Recommend Documents