Subscriber access provided by UNIVERSITY OF KENTUCKY
Article
Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation Sergio Dominguez-Medina, Lydia Kisley, Lawrence J. Tauzin, Anneli Hoggard, Bo Shuang, A.Swarnapali D. S. Indrasekara, Sishan Chen, Lin-Yung Wang, Paul J Derry, Anton Liopo, Eugene R Zubarev, Christy F. Landes, and Stephan Link ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06439 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 13, 2016
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.
ACS Nano 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 33
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
ACS Nano
1
Adsorption and Unfolding of a Single Protein
2
Triggers Nanoparticle Aggregation
3
Sergio Dominguez-Medina,1†# Lydia Kisley,1†∆ Lawrence J. Tauzin,1 Anneli Hoggard,1 Bo
4
Shuang,1 A. Swarnapali D. S. Indrasekara,1 Sishan Chen,1 Lin-Yung Wang,1 Paul J. Derry,1
5
Anton Liopo,1 Eugene R. Zubarev,1,2 Christy F. Landes,1,3* Stephan Link1,3*
6
1 Department of Chemistry, Rice University, Houston, TX 77251
7
2 Department of Materials Science and NanoEngineering, Rice University, Houston, TX 77251
8
3 Department of Electrical and Computer Engineering, Rice University, Houston, TX 77251
9
† These authors contributed equally to this work
10
*Corresponding author's email:
[email protected],
[email protected] Page 1 of 33
ACS Paragon Plus Environment
ACS Nano
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
11
ABSTRACT
12
The response of living systems to nanoparticles is thought to depend on the protein corona,
13
which forms shortly after exposure to physiological fluids and which is linked to a wide array of
14
pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and
15
nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however,
16
often missing mainly due to the inadequacies in current ensemble experimental approaches.
17
Through the application of a variety of single molecule and single particle spectroscopic
18
techniques in combination with ensemble level characterization tools, we have been able to
19
identify different interaction pathways between gold nanorods and bovine serum albumin
20
depending on the protein concentration. Overall, we found that local changes in protein
21
concentration influence everything from cancer cell uptake to nanoparticle stability and even
22
protein secondary structure. We envision that our findings and methods will lead to strategies to
23
control the associated pathophysiology of nanoparticle exposure in vivo.
24
25
KEYWORDS: Protein corona, nanorods, super-localization microscopy, correlation
26
spectroscopy, surface plasmon
Page 2 of 33
ACS Paragon Plus Environment
Page 2 of 33
Page 3 of 33
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
ACS Nano
27
The interaction of nanoparticles with living organisms has received growing attention in the
28
past decade1–3 due to the development of novel therapeutic and diagnostic tools,4–6 and the
29
growing concerns regarding the safety of nanomaterials in vivo.7–11 It is understood that
30
nanoparticles adsorb proteins in biological fluids forming a “protein corona”, which influences
31
the nanoparticle physicochemical properties and subsequent interactions with the physiological
32
environment.12–27 Both the possibility that in vivo administration of therapeutic nanoparticles can
33
disturb the structural integrity of proteins and the current knowledge of the direct link between
34
denatured proteins and severe neurodegenerative diseases suggest that nano-therapeutic
35
platforms potentially pose a health risk.2,28–30 Therefore understanding the interaction of proteins
36
with nanoparticles on a molecular level is paramount for the efficient and safe application of
37
engineered nanoparticles.
38
The quantification and identification of the protein corona composition for different types of
39
nanoparticles with varying surface chemistries have been undertaken.12,14,30–34 Ex-situ ensemble
40
techniques such as gel electrophoresis coupled with mass spectrometry have been widely used to
41
characterize of chemical identity and abundance of constituent corona proteins after separation
42
from the nanoparticles.12,29 Less disruptive, complementary optical spectroscopy approaches can
43
be used to study the protein-nanoparticle complex in-situ and were able to determine the
44
thermodynamics of protein binding as well as to provide insight into the interactions between
45
protein coated nanoparticles and cells.15,35–38 For instance, the micromolar affinity of serum
46
albumin to colloidal nanoparticles resulting in a protein monolayer15 at physiological
47
concentrations and enhanced colloidal stability in plasma39 has been revealed by a combination
48
of UV-VIS and fluorescence correlation spectroscopic techniques. Payne and co-workers have
49
used super-resolution optical microscopy imaging under in vitro conditions, and demonstrated Page 3 of 33
ACS Paragon Plus Environment
ACS Nano
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
50
that what cellular receptors actually screen are the adsorbed proteins on the nanoparticles and not
51
the initially synthesized nanoparticles themselves.40,41 Spectroscopic methods are furthermore
52
ideally suited to characterize changes in protein structure when bound to a nanoparticle surface.
53
The structural integrity of the corona proteins - which domains of the protein bind and which
54
ones potentially change their structure28,42–48 - has been studied by using far and near UV circular
55
dichroism (CD),28,42,43,49,50 IR,43,50 and Raman44 spectroscopy.
56
While the protein-nanoparticle community has begun to transition from the mere identification
57
of adsorbed proteins to the understanding of the physiological responses that can arise due to the
58
presence of the protein corona, it is still not fully understood how protein adsorption affinities,
59
relative concentrations, and surface chemistries are ultimately linked to the corona composition
60
and possible structural changes of the proteins. A mechanistic bridge between these aspects is
61
lacking due to the low spatio-temporal resolution and ex-situ requirements of most current
62
techniques.18 Ensemble analytical techniques that assume quasi equilibrium conditions with
63
exchange between free and bound proteins have furthermore led to controversial results.12,19,30–
64
33,51
65
Here we have used a combined approach of ensemble and single protein/single nanoparticle
66
methods to answer the following questions about the nanoparticle protein corona: Is
67
thermodynamic equilibrium a valid concept for the protein corona as local changes in protein
68
concentrations occur? How does protein adsorption influence protein chemistry? How is
69
nanoparticle stability altered?
70
protein/nanoparticle interactions influence physiological outcomes? Mechanistic understanding
71
of the physicochemical properties of bovine serum albumin (BSA) adsorbed to cationic-ligand
72
functionalized gold nanorods (AuNRs) is used as the experimental system. While an ensemble
Finally, how might equilibrium and non-equilibrium
Page 4 of 33
ACS Paragon Plus Environment
Page 4 of 33
Page 5 of 33
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
ACS Nano
73
protein adsorption isotherm implicates the adsorption of several proteins to the nanoparticle
74
surface, single-protein/single-nanoparticle interactions visualized through super-localization
75
imaging reveal that in fact only one protein is irreversibly adsorbed at a time. The conflicting
76
findings are resolved through surface plasmon coupled circular dichroism (CD) spectroscopy,
77
and demonstrated that the local changes in protein concentration affect the colloidal stability of
78
nanoparticles, protein secondary structure and eventually cellular uptake of nanoparticles.
79 80
RESULTS AND DISCUSSION
81
When nanoparticles are pre-incubated in bovine serum albumin (BSA) at lower than
82
physiological concentrations, properties such as uptake by MCF-7 cancer cells, BSA adsorption,
83
and
84
mercaptoundecyltrimethylammonium bromide coated AuNRs (MUTAB-AuNRs) incubated in
85
10 % fetal bovine serum (FBS) are uptaken by MCF-7 cancer cells (Figure 1a, b).52 However, by
86
pre-incubating the MUTAB-AuNRs in 1 % w/v BSA the uptake is increased three-fold (Figure
87
1b). This increase is not due to MUTAB-AuNRs adhered to the outside of the cells as the cells
88
were washed several times with PBS following a previously published protocol.52 As the most
89
abundant serum protein, BSA is present in higher concentrations in FBS than 1 % w/v. Pre-
90
incubation of MUTAB-AuNRs with 1 % w/v BSA, however, seems to influence their interaction
91
with cancer cells and cause increased uptake. An important question to ask is therefore if and
92
how a BSA-based protein corona is concentration dependent. This question will be addressed
93
next with a combination of in situ ensemble and single molecule and single particle
94
spectroscopic techniques.
nanoparticle
aggregation
are
strongly
influenced
Page 5 of 33
ACS Paragon Plus Environment
(Figure
1).
Cationic
ACS Nano
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
Figure 1 | Pre-incubation of MUTAB-AuNRs with low concentrations of BSA strongly influences AuNR cell uptake, BSA protein adsorption, and NR aggregation. a, Cartoon representation of (left, blue) pre-incubation of MUTAB-AuNRs with low concentration of BSA compared to (right, orange) MUTAB-AuNRs exposed to high concentration of proteins in serum without preincubation. b, Number of MUTAB-AuNRs uptaken per MCF-7 cell for MUTAB-AuNRs preincubated with 1% BSA before suspending in 10 % FBS and Eagle’s minimum essential media (EMEM), compared to MUTAB-AuNRs only suspended in 10% FBS and EMEM. Cells suspended in 10% FBS and EMEM without MUTAB-AuNRs are shown as a control. c, Increases in MUTAB-AuNR fluctuation correlation decay time caused by BSA adsorption. Luminescence correlation decays for MUTAB-AuNRs alone (gray), incubated with low concentrations of BSA (blue) vs. in higher serum-level BSA concentrations (orange). Autocorrelation curves with respective decay fits shown as solid lines. Under low BSA concentration the fit did not follow a predictable model; dashed line is instead shown as a guide for the eye. d, UV/vis spectra under similar conditions as c suggest MUTAB-NR aggregation occurs when incubated at low concentrations of BSA.
95
Luminescence correlation spectroscopy provides strong support that the BSA protein corona
96
that forms on MUTAB-AuNRs is concentration dependent (Figure 1c). At high protein
97
concentrations (20 µM), the increase in measured hydrodynamic radius matches the dimensions
98
of native BSA53 and hence implies the formation of a protein monolayer, consistent with earlier
Page 6 of 33
ACS Paragon Plus Environment
Page 6 of 33
Page 7 of 33
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
ACS Nano
99
reports of BSA monolayer formation on nanoparticles (Table S1).15,54,55 In contrast, at lower
100
BSA concentrations (2 nM) the luminescence correlation decay shifts to longer average decay
101
times, indicating that surprisingly the nanoparticle-protein complex has become even larger than
102
what is observed under the high BSA concentration conditions. Most importantly, the data could
103
not be fit to an equilibrium adsorption model, suggesting non-equilibrium behavior, consistent
104
with nanoparticle aggregation as verified further below.
105
Further support for the strong deviation between low and high BSA concentrations is found in
106
the UV-VIS spectra (Figure 1d). At high BSA concentration the extinction spectrum of the
107
MUTAB-AuNRs is slightly red-shifted, due to a change in dielectric environment and consistent
108
with equilibrium formation of a stable BSA monolayer. However, at low BSA concentrations the
109
decreased intensity and resonance shift of the plasmon resonance indicate that the MUTAB-
110
AuNRs aggregate in the presence of low BSA concentrations.
111
The combination of our results on cancer cell uptake, protein adsorption, and nanoparticle
112
colloidal stability strongly suggest that local protein concentrations must be considered when
113
assessing corona chemistry and its influence on nanoparticles’ ultimate physiological fate, as
114
suggested previously.11,20 Of particular interest in Figure 1 is the case of low protein
115
concentrations. Low protein-to-nanoparticle ratios can, for example, occur under controlled pre-
116
incubation conditions after injection into a living organism and upon accumulation in cellular
117
compartments. Thus, we examine more closely the structure of protein and nanoparticles when
118
they interact at low concentrations and suggest how these processes lead to the ultimate fate of
119
the protein-nanoparticle colloid.
Page 7 of 33
ACS Paragon Plus Environment
ACS Nano
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
120
BSA adsorbs at low concentrations to cationic ligand-coated gold nanomaterials of varying
121
geometries (Figure 2). A 2 nM concentration of Alexa647-labeled BSA (3 dyes per protein) is
122
flowed over individual nanomaterials of varying surface chemistries (cationic MUTAB, cationic
123
amine poly(ethylene glycol) [NH2-PEG], and anionic citrate) and different geometries
124
(nanospheres [~50 nm diameter], nanowires [width: ~70 nm; length: 1 - 10 µm], and nanorods
125
[width: 20 nm, length: 58 nm]). BSA binding to these different nanostructures is visualized via
126
an increase in fluorescence intensity due to adsorption of the fluorescently labeled protein. Due
Figure 2 | Single molecule imaging of BSA interactions with nanomaterials of varying geometries (sphere, wire, rod) and surface chemistries (cationic MUTAB, cationic NH2-PEGSH, anionic citrate). Images show nanomaterial highlighted with dashed red outline (left) before and (right) after 2 nM Alexa-BSA is introduced. Images are binned, ranging from 10-100 frames. These results show that BSA binding occurs independent of the nanoparticle geometry. Furthermore, a different surface chemistry also facilitates binding as long as the surface charge remains positive. No significant adsorption is seen for negative charged citrate capped nanoparticles at this BSA concentration.
Page 8 of 33
ACS Paragon Plus Environment
Page 8 of 33
Page 9 of 33
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
ACS Nano
127
to total internal refection excitation, only individual molecules at the surface are registered while
128
freely diffusing proteins only contribute to a slightly larger average background signal.56 We find
129
that BSA binds to the cationic-ligand functionalized nanomaterials at this low concentration. On
130
the other hand, negligible binding of BSA to the citrate capped nanospheres is observed under
131
the same conditions. Much higher µM concentrations are needed for this ligand.35 To understand
132
the mechanistic details of protein adsorption at low concentrations to cationic nanomaterials, we
133
will concentrate on the interactions between BSA and MUTAB-NRs as example nanoparticle
134
supports and serum proteins, respectively. Further analysis of other nanoparticle chemistries and
135
serum proteins are presented in the Supplementary Information (SI).
136
Single molecule analysis of the fluorescence imaging data reveals that only one BSA protein
137
binds irreversibly to each MUTAB-AuNR (Figure 3). BSA adsorption onto single MUTAB-
138
AuNRs is identified by co-localization57 of dye fluorescence and intrinsic AuNR luminescence
139
(Figure 3a, Figure S1, S2, S3, Video S1). Scanning electron microscopy (SEM) image analysis
140
confirms that 99% of the analyzed BSA adsorption events occur at single MUTAB-AuNRs
141
(Figure S4) with little non-specific BSA interaction with the substrate because of surface
142
passivation (Figure S5).
143
Although in its native form smaller than a AuNR, we find no evidence for multiple BSA
144
adsorption by analyzing fluorescence time transients (Figure 3b,c). Protein adsorption is
145
observed as an increase in fluorescence intensity, followed by a step-wise decrease as each of the
146
dye molecules on the protein photobleach (Figure 3b). Photobleaching step and intensity
147
distribution analyses are commonly used in single molecule experiments to detect single vs.
148
oligomer states of proteins,58,59 including proteins with multiple dyes.60,61 The number of
Page 9 of 33
ACS Paragon Plus Environment
ACS Nano
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
149
photobleaching steps identified by an 46
150
established step-finding algorithm
151
thus a direct measure of the BSA
152
molecules per AuNR (Figure 3b, line).
153
Analysis for 86 AuNRs yields an
154
average of 3.2 ± 1.2 photobleaching
155
steps (Figure 3c), matching the labeling
156
density
157
(Molecular Probes, A34785). Further
158
controls
involve
159
presence
of
160
photobleaching
of
161
(Figure
reversible
162
desorption/re-adsorption (Figure S6), or
163
dye fluorescence quenching close to the
164
AuNRs (Video S2). In particular,
165
Figure 3d confirms that despite the
166
labeling with 3 dyes the BSA protein
167
molecule
168
multichromophoric system capable of
169
simultaneous off/on blinking of all dyes
170
due to energy transfer.
171
Page 10 of 33
indicated
3d),
does
by
the
is
supplier
eliminating
the
simultaneous
not
multiple
behave
dyes protein
as
a
Figure 3 | Single BSA proteins adsorb to single MUTABAuNRs. a, Co-localization of single MUTAB-AuNRs, confirmed by SEM (inset; scale bar, 50 nm), with fluorescently labeled BSA. Fluorescence images are 74 frames binned with the same intensity; scale bar 500 nm. AuNRs are visible through their weak intrinsic photoluminescence using long exposure times before introducing BSA b, Intensity transient (black) of BSA adsorbed to a MUTAB-AuNR with state and step identification (teal dashed line) of photobleaching steps. Simultaneous photobleaching was discounted by probability analysis (Figure S6). c, Distribution of the number of photobleaching steps for 86 transients. d, Simultaneous photobleaching steps are unlikely. For a labeling density of 3 fluorophores per BSA molecule, the average photobleaching step size as a percent of the maximum intensity should be ~33%, which is indeed confirmed by the observed average step size of 30 ± 10%. A value of > 66% indicates simultaneous photobleaching events. Out of the 213 photobleaching steps analyzed, < 4% of steps had values greater than 60%.
The adsorption of single BSA Page 10 of 33
ACS Paragon Plus Environment
Page 11 of 33
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
ACS Nano
172
proteins to single MUTAB-AuNRs is
173
irreversible on a time scale of 8 hours as
174
demonstrated by a constant percentage of
175
AuNR-BSA
176
continuously rinsing with buffer solution
177
without proteins (Figure 4). It is not
178
clear, however, if this observation of only
179
one
180
immobilized MUTAB-AuNR extends to
181
the solution case. The exposed AuNR
182
surface area is about twice as large in
183
solution, but the protein-to-nanoparticle
184
ratio is only ~26, as ~75 pM nanorods are
185
exposed to 2 nM BSA. It is not trivial to
186
quantify this ratio for MUTAB-AuNRs
187
immobilized on a substrate, but we estimate this number to be orders of magnitude larger given
188
that 2 nM BSA (~108 BSA proteins per µl) is flowed over single nanorods spaced out at least 1
189
µm from each other at a flow rate of 5 µl/min for 15 min. Regardless of the experimental
190
geometry (i.e. free MUTAB-AuNRs in solution vs. immobilized on a surface), there is space for
191
many dozens of proteins to bind to each AuNR (width = 20 nm, length = 58 nm; resulting
192
surface area of ~ 3000 nm2), assuming a native BSA conformation that can be approximated by
193
an equilateral triangular prism with 2 triangular facets of ~ 28 nm2 and 3 smaller rectangular
194
sides of ~ 12 nm2.53 The only hypothesis that explains how the strong, irreversible adsorption of
protein
complexes
adsorbing
while
to
each
Figure 4 | Irreversible adsorption of BSA to MUTABAuNRs. a, Representative images of BSA/MUTABAuNRs identified based on the stronger BSA signal (circles) before (No BSA), after (t = 0 min) adding a 2 nM BSA solution, and after 500 min of rinsing with buffer. False identification (No BSA) is likely due to 1 MΩ) and
314
placed overnight in a water bath at 35° C. Excess MUTAB was removed by centrifugation at
315
7,500 rpm for 10 minutes and replaced with Millipore H2O for storage. We estimate that the final
316
concentration of free MUTAB in solution after this centrifugation step was less than 0.01 mg/ml.
317
The MUTAB-AuNR solutions were positively charged (ζ = 35 ± 5 mV) at pH 7.2 according to
318
zeta-potential measurements (Malvern Zen 3600). Further characterization of the MUTAB-
319
AuNRs in the buffer conditions of the experiments (20 mM HEPES, 20 mM NaCl in Molecular
320
Biology Grade H2O) by UV/vis spectroscopy (Figure S17-S18) and by transmission electron
321
microscopy (Figure S19) are included in the SI.
322
Commercially available citrate-coated gold nanospheres (AuNPs) with a nominal diameter of
323
50 nm were purchased from BBI solutions (Cardiff, UK). Sizing performed by the manufacturer
324
using transmission electron microscopy (TEM) showed that their actual size is 48 ± 4 nm (Batch
325
# 16659, ~ 75 pM concentration based on the mass of gold per milliliter used for the synthesis).
326
For the control experiments shown in Figure S7-9, citrate-AuNPs were functionalized with
327
MUTAB using the same procedure as described above for the AuNRs. The zeta-potential of the
328
AuNPs went from negatively charged (ζ = -35 ± 5 mV) in the presence of citrate, to positively
329
charged once functionalized with MUTAB (ζ = 40 ± 5 mV), confirming the ligand exchange
330
reaction occurred. Page 18 of 33
ACS Paragon Plus Environment
Page 19 of 33
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
ACS Nano
331
Gold nanowires (Au nanowires, diameter ~ 70 nm, length 1 ~ 10 µm) were synthesized using a
332
modified three-step seeding synthesis originally developed for AuNRs.71 Seed particles were
333
prepared by a rapid reduction of HAuCl4 (gold precursor) using NaBH4 (reducing agent). These
334
particles were then grown in a solution containing HAuCl4, ascorbic acid, and CTAB. The
335
presence of ascorbic acid reduces the gold salt from its Au(III) to a stable Au(I) state. Further
336
addition of seeds to this solution induces an autocatalytic reaction of Au(I) that enlarges the seed
337
particles. While this method would typically yield nanowires with an aspect ratio smaller than
338
10, aspect ratios larger than 100 can be achieved when the reaction takes place in an acidic
339
environment (pH ~2) and less seeds are added to the growth solution. CTAB on the as-
340
synthesized Au nanowires was replaced with MUTAB. The CTAB-Au nanowires sedimented at
341
the bottom of the vial after 1-2 hours at room temperature without mixing or shaking. Carefully,
342
CTAB was removed out of solution without removing the nanowires. An equal volume of
343
MUTAB (1 mg/ml in Millipore H2O) was added to fill the volume left by removed CTAB. The
344
solution was mixed using a micro-pipette and placed overnight in a water bath at 35° C. The
345
MUTAB-Au nanowires were then re-suspended in solution by gently shaking the vial, and then
346
left untouched for 1-2 hours at room temperature to allow for sedimentation. Finally, excess
347
MUTAB was removed out of solution (carefully without removing the nanowires), and an equal
348
volume of Millipore H2O was added to fill the volume left by excess MUTAB.
349
Cell Culture. BSA (7.5% in DBPS), penicillin-streptomycin (10,000 U-10 mg/mL), HCl
350
(Trace Metals Grade) and HNO3 (Trace Metals Grade) were purchased from Sigma-Aldrich.
351
MCF-7 cells were acquired from ATCC (HTB-22.) EMEM (with EBSS and L-Glutamine) was
352
purchased from Lonza. FBS (heat-inactivated) was purchased from Seradigm. Lactate
Page 19 of 33
ACS Paragon Plus Environment
ACS Nano
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
353
dehydrogenase (LDH) activity assay kit was purchased from Thermo Scientific. All chemicals
354
were used without further purification.
Page 20 of 33
355
Microscope glass coverslips. Glass coverslips (22 x 22 mm2, VWR) were cleaned by
356
sonication for 10 minutes in acetone, 10 minutes in water/detergent mixture, and 10 minutes in
357
deionized water. The coverslips were then immersed in a bath of base (6:1:1 water, 30% H2O2,
358
NH4OH) for 90 seconds at 80 °C followed by rinsing with copious amounts of deionized water.
359
The glass was then cleaned for 2 minutes in O2 plasma (Harrick Plasma). Cleaned coverslips
360
were placed under vacuum for long term storage. Details of the passivation of the surface for
361
correlation spectroscopy experiments and single protein binding experiments can be found in the
362
caption of Figure S5.
363
Luminescence correlation spectroscopy. Luminescence of MUTAB-AuNRs was recorded
364
with a home-built confocal microscope described elsewhere.72 The instrument is based on an
365
inverted epifluorescence microscope (Observer.D1, Zeiss) equipped with a 638 nm diode laser
366
(CUBE, Coherent). The laser light was collimated and delivered to the back aperture of an oil
367
immersion objective (Apochromat 64X, NA = 1.4, Zeiss). The objective focused the light onto a
368
small liquid cell placed on top of a glass coverslip containing 45 µl of sample solution. The
369
emitted light was collected by the same objective and passed through a dichroic mirror (ZT
370
532/638 rpc, Chroma Technolgoy), a long pass filter (XLP-647, CVI Melles-Griot), and a 50-
371
µm-diameter pinhole (Thorlabs) placed at the focal plane of the microscope. The luminescence
372
signal was detected by a single element diode detector (PDM 50ct, Micro-Photon-Devices) and
373
processed by a home-built photon counting module (LabView). All measurements were recorded
374
at a controlled laboratory temperature of 20 ± 1 °C, and using an excitation power of 1.5 x 103 Page 20 of 33
ACS Paragon Plus Environment
Page 21 of 33
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
ACS Nano
375
mW/cm2 at the sample. This excitation power ensured minimal heating and negligible auto-
376
fluorescence background of unlabeled BSA (Figure S20). For protein concentration dependent
377
measurements as summarized in Table S1, ~75 pM MUTAB-AuNR solutions with varying
378
concentrations of BSA (Catalog #A7906, Sigma-Aldrich; BSA concentrations: 0.1 nM, 0.5 nM,
379
1 nM, 1.5 nM, 2 nM, 3 nM, 4 nM, 5 nM; 10 nM; BSA:AuNR ratios: 1.33, 6.66, 13.33, 20, 26.66,
380
40, 53.33, 66.66, 133.33) were prepared by mixing equal volumes of both solutions in buffer.
381
The dimensions of the observation volume were determined using a calibration sample with a
382
known diffusion coefficient at 20 °C (Figure S21).
383
Data acquisition and analysis for correlation spectroscopy experiments. The luminescence
384
intensity was recorded in data sets of 40 s at a temporal resolution of 10 µs. Each data set was
385
autocorrelated using an automated routine in Matlab. At least seven data sets were averaged per
386
measurement and the experiment was repeated independently three times at each protein
387
concentration. The autocorrelation functions G(τ) were analyzed in Matlab using a one species,
388
three dimensional diffusion model with an additional exponential term that accounts for nanorod
389
rotation:73
390
1 G (τ ) = N
τ 1 + τD
−1
r 2 τ 1 + 0 z 0 τ D
−
1 2
τ − τR ⋅ 1 + Ae
391
where denotes the average number of particles in the three-dimensional Gaussian-shaped
392
observation volume, with radial and axial dimensions r0 and z0, respectively. The translational
393
diffusion time τD is related to the translational diffusion coefficient of the particles, Dtr =
394
r02/(4τD). An additional exponential term of amplitude A and rotational diffusion time τR was
395
used to account for nanorod rotational diffusion. The translational diffusion coefficient was used Page 21 of 33
ACS Paragon Plus Environment
ACS Nano
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 33
396
to estimate the equivalent hydrodynamic radius of the nanoparticles using the Stokes-Einstein
397
relationship Rh = kT/6πηDtr. Changes in viscosity due to the presence of BSA were calculated
398
using the intrinsic viscosity of this protein5 (4.2 cm3/g) assuming a linear relationship in the range
399
of concentrations used. Results of correlation spectroscopy experiments before and after BSA
400
binding at different concentrations are shown in Table S1.
401
Super-localization single molecule microscopy. Samples containing single immobilized
402
MUTAB-AuNRs (Figures 2, 3) and MUTAB-AuNPs (Figure 2) were prepared by dropcasting
403
~40 µl of ~12.5 pM of nanoparticle solutions (1:6 dilution of the concentration used for
404
luminescence correlation spectroscopy experiments) on microscope coverslips passivated with
405
unlabeled BSA (see caption of Figure S5 for details on the passivation procedure). The droplet
406
was then dried for 10 minutes at room temperature, and the coverslips were rinsed with 1 ml of
407
Millipore H2O and gently dried with nitrogen gas. A home-built total internal reflection wide
408
field microscope with 637 nm excitation (Coherent OBIS-FP 637 LX) as described elsewhere74
409
was used with a custom flow chamber (1 mm height, elliptical opening of 12×5 mm; 43018C,
410
Grace BioLabs) placed over the sample containing immobilized MUTAB-nanoparticles. A
411
solution of 2 nM Alexa647-labeled BSA (A34785, Molecular Probes, 3 dyes/protein) was flowed
412
over the sample at 5 µl/min using a Genie Plus flow system (Kent Scientific). This flow rate does
413
not impart strong forces on protein-binding site interactions and results in effectively negligible
414
net directed diffusion due to flow.75 The sample was allowed to equilibrate for 15 min with
415
protein flow before measuring, ensuring that an excess of proteins is exposed to each
416
nanoparticle (2 nmol BSA/l x 5 ul/min x 15 min x NA = 9 x 1010 BSA molecules compared to
417
~10 particles/5 µm2 in Figure S3 x 60 mm2 area of sample = 12 x 105 particles/sample). Data was
418
collected at an incident excitation intensity of 5 mW/cm2, an integration time of 100 ms, frame Page 22 of 33
ACS Paragon Plus Environment
Page 23 of 33
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
ACS Nano
419
rate of 7.5 Hz, and electron multiplying gain of 300. Analysis was possible at the super-
420
localization level, as the fluorescent BSA can be selectively observed only when adsorbed to the
421
interface, and was unobservable by motion blur when freely diffusing (D ∼ 60 µm2/s). Increases
422
in signal to noise ratio of each frame, identification of adsorbed BSA, and super-localization
423
analysis of the location by radial symmetry fitting was performed using MATLAB 2011b as
424
described previously.57,68 Samples containing single immobilized MUTAB-Au nanowires
425
(Figure 2, 5c, S14) were prepared using the same drying procedure, but because the
426
concentration was unknown, the solution was repeatedly diluted until single isolated nanowires
427
were observed on the microscope coverslip under the optical microscope.
428
Circular dichroism (CD) spectroscopy. Ensemble CD measurements were taken on a J-815
429
circular dichroism spectrometer (JASCO) with a 1 mm pathlength quartz cuvette at 20 ± 0.1 °C
430
using a Peltier temperature controller. Equal volumes of ~1 nM nanoparticle solutions (MUTAB-
431
AuNRs or AuNPs) and 0.1 mg/ml BSA solutions were mixed ~30 minutes before each
432
measurement. Wavelength ranges of 190-260 nm and 500-750 nm were collected every 1 nm
433
under standard sensitivity, bandwidth of 1.00 nm, and rate of 50 nm/min. Four scans were
434
collected for each trial and averaged. The spectra were baseline-corrected and presented as mean
435
residue ellipticity. The solvent for CD measurements was 1 mM phosphate buffer (pH 7.2) due
436
to its low absorbance in the far UV. This solvent did not affect the stability of MUTAB-AuNRs,
437
MUTAB-AuNPs or BSA.
438
SEM imaging. SEM images were taken using a FEI Quanta 400 ESEM FEG, operated at 10
439
kV under low vacuum conditions. Analysis of aggregate size was performed in ImageJ.
440
MUTAB-AuNR uptake by MCF-7 cells. Page 23 of 33
ACS Paragon Plus Environment
ACS Nano
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
441
(a)
Pre-incubation of MUTAB-AuNRs under different media conditions before
442
incubation with MCF-7 cells. For the experiments shown in Figure 1a, 1b, MUTAB-
443
AuNRs were incubated in two different media conditions: 1) 1 mL of MUTAB-AuNRs
444
were mixed with 133 μl of 7.5% (w/v) BSA (final concentration of BSA ~1 % w/v) and
445
incubated overnight. This formed a BSA-corona onto the MUTAB-AuNRs before exposure
446
to serum. Next, the BSA-MUTAB-AuNRs were diluted (1:10) in EMEM with 10% FBS
447
(with 1% PCN) and incubated overnight. 2) 1 mL of MUTAB-AuNRs were directly diluted
448
(1:10) in EMEM with 10% FBS (with 1% PCN) and incubated overnight. Both MUTAB-
449
AuNR solutions were found to be stable in EMEM over the timescale of the experiments,
450
as observed via UV/Vis spectroscopy.
451
(b)
Page 24 of 33
Nanoparticle incubation with MCF-7 Cells. MCF-7 cells were seeded into two 6-well
452
plates with each well containing 2 ml of 100,000 cells/ml solution and cultured for three
453
days in EMEM with 10% FBS and 1% PCN (same as incubation conditions for MUTAB-
454
AuNRs). On the third day, the media was removed and each well was carefully washed
455
three times with PBS to remove adhered proteins. Two wells were filled with 2 ml media
456
containing either 1% BSA, 10% FBS in EMEM with 1% PCN, or 10% FBS in EMEM with
457
1% PCN. The remainder of the wells contained 2 ml of the corresponding BSA-MUTAB-
458
AuNR solutions, each in triplicate. The plates were incubated at 37° C in 4.5% CO2 for 6
459
hours. After incubation for 6 hours, 50 μl of media from each well was removed in
460
triplicate using a pipette and kept for the LDH assay. The wells were washed with PBS
461
three times to remove adhered proteins and nanorods. The cells were then detached via
462
incubation in 0.05% trypsin with EDTA and counted using a standard hemacytometer
Page 24 of 33
ACS Paragon Plus Environment
Page 25 of 33
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
ACS Nano
463
(Figure S22). After counting, the cells were transferred to a solution of 0.1% Triton X-100
464
and placed in a -20 °C freezer overnight.
465
(c)
LDH cytotoxicity assay of MCF-7 cells incubated with BSA-MUTAB-AuNRs. A LDH
466
assay was performed by adding 50 μl of formazan dye (Thermo Scientific) to each well
467
containing media from the nanoparticle-cell incubation experiments (Figure S23). The
468
plates were placed in an incubator at 37 °C with 4.5% CO2 for 30 minutes and read on a
469
BioTek Uniread 800 plate reader. The purpose of this assay was to measure the cytotoxicity
470
of the MUTAB-AuNRs under the different incubation conditions with BSA and FBS,
471
compared to MCF-7 cells alone.
472
(d)
ICP-MS measurement of BSA-MUTAB-AuNRs uptaken by MCF-7 cells. The lysed
473
cells were thawed and centrifuged at 8000 g for 10 minutes. The supernatant was removed
474
and the pellets were digested using aqua regia (3:1 solution of HNO3 and HCl). Following
475
digestion, the pellets were diluted to 10 ml using 5% HCl in milli-Q water. The samples
476
were measured for Au concentration using a Perkin-Elmer Nexion 300 ICP-MS. The
477
concentration of Au was determined for each cell using the cell count collected after
478
detaching the cells (Figure S24).
479
Calculation of the number of AuNRs uptaken by each cell. The average number of AuNRs
480
uptaken per cell was calculated by first determining the number of AuNRs in the different
481
solutions with cells from the concentration of Au determined via ICP-MS. The number of
482
nanorods was then divided by the average number of cells. The addition of BSA to the MUTAB-
483
AuNRs increases the uptake by MCF-7 cells by 320% compared to the conventional incubation
484
conditions in EMEM with 10% FBS. Page 25 of 33
ACS Paragon Plus Environment
ACS Nano
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
485
CONFLICT OF INTEREST
486
The authors declare no competing financial interests.
Page 26 of 33
487 488
ACKNOWLEDGEMENTS
489
This work was funded by the National Science Foundation (Grants CBET-1134417 and CHE-
490
1151647 to C. Landes; Grant CBET-1438634 to S. Link and C. Landes; Grant DMR-1105878 to
491
E. Zubarev), the Welch Foundation (Grants C-1787 to C. Landes and C-1664 to S. Link), and the
492
National Institute of Health (Grant GM94246-01A1 to C. Landes). L. Kisley and A. Hoggard
493
acknowledge the National Science Foundation Graduate Research Fellowship (Grant 0940902).
494
S. Dominguez-Medina acknowledges partial support from a Lodieska Stockbridge Vaughn
495
Fellowship from Rice University. We would like to thank Professors Peter Wolynes, Naomi
496
Halas, and Carsten Sönnichsen for their valuable feedback.
497 498
SUPPORTING INFORMATION AVAILABLE
499
Details regarding experimental & computational methods and data analysis. This material is
500
available free of charge via the Internet at http://pubs.acs.org.
501 502
AUTHOR PRESENT ADDRESSES
503
# - CEA-Grenoble; 17 Rue des Martyrs; F-38054 Grenoble Cedex 9, France
504
∆ - University of Illinois at Urbana-Champaign; School of Chemical Sciences; 600 S. Mathews
505
Ave.; MC-712, Box C-3; Urbana, IL 61801
506
Page 26 of 33
ACS Paragon Plus Environment
Page 27 of 33
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
ACS Nano
507 508
(1)
Xia, X.-R.; Monteiro-Riviere, N. A.; Riviere, J. E. An Index for Characterization of Nanomaterials in Biological Systems. Nat. Nanotechnol. 2010, 5, 671–675.
509 510 511
(2)
Nel, A. E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding Biophysicochemical Interactions at the Nano-Bio Interface. Nat. Mater. 2009, 8, 543–557.
512 513
(3)
Moyano, D. F.; Rotello, V. M. Nano Meets Biology: Structure and Function at the Nanoparticle Interface. Langmuir 2011, 27, 10376–10385.
514 515
(4)
Pissuwan, D.; Valenzuela, S. M.; Cortie, M. B. Therapeutic Possibilities of Plasmonically Heated Gold Nanoparticles. Trends Biotechnol. 2006, 24, 62–67.
516 517 518
(5)
Zharov, V. P.; Mercer, K. E.; Galitovskaya, E. N.; Smeltzer, M. S. Photothermal Nanotherapeutics and Nanodiagnostics for Selective Killing of Bacteria Targeted with Gold Nanoparticles. Biophys. J. 2006, 90, 619–627.
519 520 521
(6)
Gobin, A. M.; Watkins, E. M.; Quevedo, E.; Colvin, V. L.; West, J. L. Near-InfraredResonant Gold/gold Sulfide Nanoparticles as a Photothermal Cancer Therapeutic Agent. Small 2010, 6, 745–752.
522 523
(7)
Nel, A.; Xia, T.; Mädler, L.; Li, N. Toxic Potential of Materials at the Nanolevel. Science 2006, 311, 622–627.
524 525 526
(8)
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.
527 528 529 530
(9)
Dutta, D.; Sundaram, S. K.; Teeguarden, J. G.; Riley, B. J.; Fifield, L. S.; Jacobs, J. M.; Addleman, S. R.; Kaysen, G. A.; Moudgil, B. M.; Weber, T. J. Adsorbed Proteins Influence the Biological Activity and Molecular Targeting of Nanomaterials. Toxicol. Sci. 2007, 100, 303–315.
531 532
(10)
Colvin, V. L. The Potential Environmental Impact of Engineered Nanomaterials. Nat. Biotechnol. 2003, 21, 1166–1170.
533 534 535
(11)
Kim, J. A.; Salvati, A.; Åberg, C.; Dawson, K. A. Suppression of Nanoparticle Cytotoxicity Approaching in Vivo Serum Concentrations: Limitations of in Vitro Testing for Nanosafety. Nanoscale 2014, 6, 14180–14184.
536 537
(12)
Casals, E.; Pfaller, T.; Duschl, A.; Oostingh, G. J.; Puntes, V. Time Evolution of the Nanoparticle Protein Corona. ACS Nano 2010, 4, 3623–3632.
538 539
(13)
Dobrovolskaia, M. A.; Patri, A. K.; Zheng, J.; Clogston, J. D.; Ayub, N.; Aggarwal, P.; Neun, B. W.; Hall, J. B.; McNeil, S. E. Interaction of Colloidal Gold Nanoparticles with Page 27 of 33
ACS Paragon Plus Environment
ACS Nano
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
540 541
Human Blood: Effects on Particle Size and Analysis of Plasma Protein Binding Profiles. Nanomedicine 2009, 5, 106–117.
542 543 544
(14)
Lacerda, S. H. D. P.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. Interaction of Gold Nanoparticles with Common Human Blood Proteins. ACS Nano 2010, 4, 365–379.
545 546 547
(15)
Röcker, C.; Pötzl, M.; Zhang, F.; Parak, W. J.; Nienhaus, G. U. A Quantitative Fluorescence Study of Protein Monolayer Formation on Colloidal Nanoparticles. Nat. Nanotechnol. 2009, 4, 577–580.
548 549 550 551 552
(16)
Tenzer, S.; Docter, D.; Rosfa, S.; Wlodarski, A.; Kuharev, J.; Rekik, A.; Knauer, S. K.; Bantz, C.; Nawroth, T.; Bier, C.; Sirirattanapan, J.; Mann, W.; Treuel, L.; Zellner, R.; Maskos, M.; Schild, H.; Stauber, R. H. Nanoparticle Size Is a Critical Physicochemical Determinant of the Human Blood Plasma Corona: A Comprehensive Quantitative Proteomic Analysis. ACS Nano 2011, 5, 7155–7167.
553 554 555
(17)
Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Probing BSA Binding to Citrate-Coated Gold Nanoparticles and Surfaces. Langmuir 2005, 21, 9303– 9307.
556 557 558
(18)
Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S. Protein-Nanoparticle Interactions: Opportunities and Challenges. Chem. Rev. 2011, 111, 5610–5637.
559 560 561
(19)
Walkey, C. D.; Chan, W. C. W. Understanding and Controlling the Interaction of Nanomaterials with Proteins in a Physiological Environment. Chem. Soc. Rev. 2012, 41, 2780–2799.
562 563 564
(20)
Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F. B.; Dawson, K. A. Physical-Chemical Aspects of Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. J. Am. Chem. Soc. 2011, 133, 2525–2534.
565 566 567
(21)
Ritz, S.; Schöttler, S.; Kotman, N.; Baier, G.; Musyanovych, A.; Kuharev, J.; Landfester, K.; Schild, H.; Jahn, O.; Tenzer, S.; Mailänder, V. Protein Corona of Nanoparticles: Distinct Proteins Regulate the Cellular Uptake. Biomacromolecules 2015, 16, 1311–1321.
568 569 570 571
(22)
Sisco, P. N.; Wilson, C. G.; Chernak, D.; Clark, J. C.; Grzincic, E. M.; Ako-Asare, K.; Goldsmith, E. C.; Murphy, C. J. Adsorption of Cellular Proteins to PolyelectrolyteFunctionalized Gold Nanorods: A Mechanism for Nanoparticle Regulation of Cell Phenotype? PLoS One 2014, 9, e86670.
572 573 574
(23)
Pino, P. del; Pelaz, B.; Zhang, Q.; Maffre, P.; Nienhaus, G. U.; Parak, W. J. Protein Corona Formation around Nanoparticles – from the Past to the Future. Mater. Horiz. 2014, 1, 301–313. Page 28 of 33
ACS Paragon Plus Environment
Page 28 of 33
Page 29 of 33
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
ACS Nano
575 576
(24)
Treuel, L.; Docter, D.; Maskos, M.; Stauber, R. H. Protein Corona - from Molecular Adsorption to Physiological Complexity. Beilstein J. Nanotechnol. 2015, 6, 857–873.
577 578 579
(25)
Winuprasith, T.; Chantarak, S.; Suphantharika, M.; He, L.; McClements, D. J. Alterations in Nanoparticle Protein Corona by Biological Surfactants: Impact of Bile Salts on ΒLactoglobulin-Coated Gold Nanoparticles. J. Colloid Interface Sci. 2014, 426, 333–340.
580 581 582 583
(26)
Landgraf, L.; Christner, C.; Storck, W.; Schick, I.; Krumbein, I.; Dähring, H.; Haedicke, K.; Heinz-Herrmann, K.; Teichgräber, U.; Reichenbach, J. R.; Tremel, W.; Tenzer, S.; Hilger, I. A Plasma Protein Corona Enhances the Biocompatibility of Au@Fe3O4 Janus Particles. Biomaterials 2015, 68, 77–88.
584 585 586
(27)
Darabi Sahneh, F.; Scoglio, C.; Riviere, J. Dynamics of Nanoparticle-Protein Corona Complex Formation: Analytical Results from Population Balance Equations. PLoS One 2013, 8, e64690.
587 588 589
(28)
Deng, Z. J.; Liang, M.; Monteiro, M.; Toth, I.; Minchin, R. F. Nanoparticle-Induced Unfolding of Fibrinogen Promotes Mac-1 Receptor Activation and Inflammation. Nat. Nanotechnol. 2011, 6, 39–44.
590 591
(29)
Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Biomolecular Coronas Provide the Biological Identity of Nanosized Materials. Nat. Nanotechnol. 2012, 7, 779–786.
592 593 594 595
(30)
Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nanotechnol. 2013, 8, 772–781.
596 597 598 599
(31)
Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the Nanoparticle-Protein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 2050–2055.
600 601 602
(32)
Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14265–14270.
603 604 605
(33)
Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. W. Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake. J. Am. Chem. Soc. 2012, 134, 2139–2147.
606 607 608
(34)
Mahmoudi, M.; Lohse, S. E.; Murphy, C. J.; Fathizadeh, A.; Montazeri, A.; Suslick, K. S. Variation of Protein Corona Composition of Gold Nanoparticles Following Plasmonic Heating. Nano Lett. 2014, 14, 6–12.
Page 29 of 33
ACS Paragon Plus Environment
ACS Nano
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
609 610 611
(35)
Dominguez-Medina, S.; McDonough, S.; Swanglap, P.; Landes, C. F.; Link, S. In Situ Measurement of Bovine Serum Albumin Interaction with Gold Nanospheres. Langmuir 2012, 28, 9131–9139.
612 613
(36)
Nienhaus, G. U.; Maffre, P.; Nienhaus, K. Studying the Protein Corona on Nanoparticles by FCS. Methods Enzymol. 2013, 519, 115–137.
614 615 616
(37)
Ament, I.; Prasad, J.; Henkel, A.; Schmachtel, S.; Sönnichsen, C. Single Unlabeled Protein Detection on Individual Plasmonic Nanoparticles. Nano Lett. 2012, 12, 1092– 1095.
617 618 619
(38)
Zijlstra, P.; Paulo, P. M. R.; Orrit, M. Optical Detection of Single Non-Absorbing Molecules Using the Surface Plasmon Resonance of a Gold Nanorod. Nat. Nanotechnol. 2012, 7, 379–382.
620 621 622
(39)
Dominguez-Medina, S.; Blankenburg, J.; Olson, J.; Landes, C. F.; Link, S. Adsorption of a Protein Monolayer via Hydrophobic Interactions Prevents Nanoparticle Aggregation under Harsh Environmental Conditions. ACS Sustain. Chem. Eng. 2013, 1, 833–842.
623 624
(40)
Doorley, G. W.; Payne, C. K. Cellular Binding of Nanoparticles in the Presence of Serum Proteins. Chem. Commun. 2011, 47, 466–468.
625 626
(41)
Doorley, G. W.; Payne, C. K. Nanoparticles Act as Protein Carriers during Cellular Internalization. Chem. Commun. 2012, 48, 2961–2963.
627 628 629
(42)
Deng, Z. J.; Liang, M.; Toth, I.; Monteiro, M. J.; Minchin, R. F. Molecular Interaction of Poly(acrylic Acid) Gold Nanoparticles with Human Fibrinogen. ACS Nano 2012, 6, 8962– 8969.
630 631 632 633
(43)
Chakraborty, S.; Joshi, P.; Shanker, V.; Ansari, Z. A.; Singh, S. P.; Chakrabarti, P. Contrasting Effect of Gold Nanoparticles and Nanorods with Different Surface Modifications on the Structure and Activity of Bovine Serum Albumin. Langmuir 2011, 27, 7722–7731.
634 635 636
(44)
Zhang, D.; Neumann, O.; Wang, H.; Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N. J. Gold Nanoparticles Can Induce the Formation of Protein-Based Aggregates at Physiological pH. Nano Lett. 2009, 9, 666–671.
637 638
(45)
Pan, H.; Qin, M.; Meng, W.; Cao, Y.; Wang, W. How Do Proteins Unfold upon Adsorption on Nanoparticle Surfaces? Langmuir 2012, 28, 12779–12787.
639 640 641 642
(46)
Medintz, I. L.; Konnert, J. H.; Clapp, A. R.; Stanish, I.; Twigg, M. E.; Mattoussi, H.; Mauro, J. M.; Deschamps, J. R. A Fluorescence Resonance Energy Transfer-Derived Structure of a Quantum Dot-Protein Bioconjugate Nanoassembly. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9612–9617. Page 30 of 33
ACS Paragon Plus Environment
Page 30 of 33
Page 31 of 33
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
ACS Nano
643 644
(47)
Roach, P.; Farrar, D.; Perry, C. C. Interpretation of Protein Adsorption: Surface-Induced Conformational Changes. J. Am. Chem. Soc. 2005, 127, 8168–8173.
645 646 647
(48)
Wangoo, N.; Suri, C. R.; Shekhawat, G. Interaction of Gold Nanoparticles with Protein: A Spectroscopic Study to Monitor Protein Conformational Changes. Appl. Phys. Lett. 2008, 92, 133104.
648 649
(49)
Fleischer, C. C.; Payne, C. K. Nanoparticle-Cell Interactions: Molecular Structure of the Protein Corona and Cellular Outcomes. Acc. Chem. Res. 2014, 47, 2651–2659.
650 651 652
(50)
Chakraborti, S.; Joshi, P.; Chakravarty, D.; Shanker, V.; Ansari, Z. A.; Singh, S. P.; Chakrabarti, P. Interaction of Polyethyleneimine-Functionalized ZnO Nanoparticles with Bovine Serum Albumin. Langmuir 2012, 28, 11142–11152.
653 654 655
(51)
Cedervall, T.; Lynch, I.; Foy, M.; Berggård, T.; Donnelly, S. C.; Cagney, G.; Linse, S.; Dawson, K. A. Detailed Identification of Plasma Proteins Adsorbed on Copolymer Nanoparticles. Angew. Chem., Int. Ed. Engl. 2007, 46, 5754–5756.
656 657 658 659
(52)
Vigderman, L.; Manna, P.; Zubarev, E. R. Quantitative Replacement of Cetyl Trimethylammonium Bromide by Cationic Thiol Ligands on the Surface of Gold Nanorods and Their Extremely Large Uptake by Cancer Cells. Angew. Chem., Int. Ed. Engl. 2012, 124, 660–665.
660
(53)
Peters, T. All About Albumin; Elsevier, 1995.
661 662 663
(54)
Treuel, L.; Brandholt, S.; Maffre, P.; Wiegele, S.; Shang, L.; Nienhaus, G. U. Impact of Protein Modification on the Protein Corona on Nanoparticles and Nanoparticle-Cell Interactions. ACS Nano 2014, 8, 503–513.
664 665 666 667
(55)
Pelaz, B.; del Pino, P.; Maffre, P.; Hartmann, R.; Gallego, M.; Rivera-Fernández, S.; de la Fuente, J. M.; Nienhaus, G. U.; Parak, W. J. Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake. ACS Nano 2015, 9, 6996–7008.
668 669 670
(56)
Chen, J.; Bremauntz, A.; Kisley, L.; Shuang, B.; Landes, C. F. Super-Resolution mbPAINT for Optical Localization of Single-Stranded DNA. ACS Appl. Mater. Interfaces 2013, 5, 9338–9343.
671 672 673
(57)
Shuang, B.; Chen, J.; Kisley, L.; Landes, C. F. Troika of Single Particle Tracking Programing: SNR Enhancement, Particle Identification, and Mapping. Phys. Chem. Chem. Phys. 2014, 16, 624–634.
674 675 676
(58)
Das, S. K.; Darshi, M.; Cheley, S.; Wallace, M. I.; Bayley, H. Membrane Protein Stoichiometry Determined from the Step-Wise Photobleaching of Dye-Labelled Subunits. Chembiochem 2007, 8, 994–999. Page 31 of 33
ACS Paragon Plus Environment
ACS Nano
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
677 678 679
(59)
Ding, H.; Wong, P. T.; Lee, E. L.; Gafni, A.; Steel, D. G. Determination of the Oligomer Size of Amyloidogenic Protein Beta-amyloid(1-40) by Single-Molecule Spectroscopy. Biophys. J. 2009, 97, 912–921.
680 681 682
(60)
Kastantin, M.; Langdon, B. B.; Chang, E. L.; Schwartz, D. K. Single-Molecule Resolution of Interfacial Fibrinogen Behavior: Effects of Oligomer Populations and Surface Chemistry. J. Am. Chem. Soc. 2011, 133, 4975–4983.
683 684 685
(61)
Casanova, D.; Giaume, D.; Moreau, M.; Martin, J.-L.; Gacoin, T.; Boilot, J.-P.; Alexandrou, A. Counting the Number of Proteins Coupled to Single Nanoparticles. J. Am. Chem. Soc. 2007, 129, 12592–12593.
686 687 688
(62)
Whitmore, L.; Wallace, B. A. DICHROWEB, an Online Server for Protein Secondary Structure Analyses from Circular Dichroism Spectroscopic Data. Nucleic Acids Res. 2004, 32, W668–W673.
689 690 691
(63)
Andrade, M. A.; Chacón, P.; Merelo, J. J.; Morán, F. Evaluation of Secondary Structure of Proteins from UV Circular Dichroism Spectra Using an Unsupervised Learning Neural Network. Protein Eng. Des. Sel. 1993, 6, 383–390.
692 693
(64)
George, J.; Thomas, K. G. Surface Plasmon Coupled Circular Dichroism of Au Nanoparticles on Peptide Nanotubes. J. Am. Chem. Soc. 2010, 132, 2502–2503.
694 695 696
(65)
Lu, F.; Tian, Y.; Liu, M.; Su, D.; Zhang, H.; Govorov, A. O.; Gang, O. Discrete Nanocubes as Plasmonic Reporters of Molecular Chirality. Nano Lett. 2013, 13, 3145– 3151.
697 698 699 700
(66)
Wang, R.-Y.; Wang, P.; Liu, Y.; Zhao, W.; Zhai, D.; Hong, X.; Ji, Y.; Wu, X.; Wang, F.; Zhang, D.; Zhang, W.; Liu, R.; Zhang, X. Experimental Observation of Giant Chiroptical Amplification of Small Chiral Molecules by Gold Nanosphere Clusters. J. Phys. Chem. C 2014, 118, 9690–9695.
701 702 703 704
(67)
Govorov, A. O.; Fan, Z.; Hernandez, P.; Slocik, J. M.; Naik, R. R. Theory of Circular Dichroism of Nanomaterials Comprising Chiral Molecules and Nanocrystals: Plasmon Enhancement, Dipole Interactions, and Dielectric Effects. Nano Lett. 2010, 10, 1374– 1382.
705 706
(68)
Parthasarathy, R. Rapid, Accurate Particle Tracking by Calculation of Radial Symmetry Centers. Nat. Methods 2012, 9, 724–726.
707 708 709 710
(69)
Su, L.; Lu, G.; Kenens, B.; Rocha, S.; Fron, E.; Yuan, H.; Chen, C.; Van Dorpe, P.; Roeffaers, M. B. J.; Mizuno, H.; Hofkens, J.; Huchison, J. A.; Uji-i, H. Visualization of Molecular Fluorescence Point Spread Functions via Remote Excitation Switching Fluorescence Microscopy. Nat. Commun. 2015, 6, 6287.
Page 32 of 33
ACS Paragon Plus Environment
Page 32 of 33
Page 33 of 33
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
ACS Nano
711 712
(70)
Cukalevski, R.; Ferreira, S.; Dunning, C. IgG and Fibrinogen Driven Nanoparticle Aggregation. Nano Res. 2015, 8, 2733.
713 714
(71)
Khanal, B. P.; Zubarev, E. R. Purification of High Aspect Ratio Gold Nanorods: Complete Removal of Platelets. J. Am. Chem. Soc. 2008, 130, 12634–12635.
715 716 717 718
(72)
Tcherniak, A.; Dominguez-Medina, S.; Chang, W.-S.; Swanglap, P.; Slaughter, L. S.; Landes, C. F.; Link, S. One-Photon Plasmon Luminescence and Its Application to Correlation Spectroscopy as a Probe for Rotational and Translational Dynamics of Gold Nanorods. J. Phys. Chem. C 2011, 115, 15938–15949.
719 720 721
(73)
Tirado, M. M.; Martínez, C. L.; de la Torre, J. G. Comparison of Theories for the Translational and Rotational Diffusion Coefficients of Rod-like Macromolecules. Application to Short DNA Fragments. J. Chem. Phys. 1984, 81, 2047.
722 723 724
(74)
Kisley, L.; Chang, W.-S.; Cooper, D.; Mansur, A. P.; Landes, C. F. Extending Single Molecule Fluorescence Observation Time by Amplitude-Modulated Excitation. Methods Appl. Fluoresc. 2013, 1, 037001–37001.
725 726 727 728
(75)
Kisley, L.; Chen, J.; Mansur, A. P.; Shuang, B.; Kourentzi, K.; Poongavanam, M.-V.; Chen, W.-H.; Dhamane, S.; Willson, R. C.; Landes, C. F. Unified Superresolution Experiments and Stochastic Theory Provide Mechanistic Insight into Protein IonExchange Adsorptive Separations. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 2075–2080.
729 730
TABLE OF CONTENTS GRAPHIC
731
Page 33 of 33
ACS Paragon Plus Environment
