Phase association and binding energetics of SWCNTs into phospholipid
Langmuir monolayers
Peter N. Yaron1, Philip A. Short2, Brian D. Holt2, Goh Haw-Zan3, Mohammad F. Islam1,4, Mathias Lösche2,3, Kris Noel Dahl1,2
1Chemical Engineering, 2Biomedical Engineering, 3Physics, 4Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA
 Single-walled Carbon nanotubes (SWCNTs) have
been identified as promising candidates for targeted
drug delivery due to their low toxicity and ability to
be functionalized using various bioactive groups
 Currently undetermined what mechanical and
biological mechanism(s) are responsible for uptake
into cells
 Objective: Determine the predominant membrane
insertion and cellular uptake mechanism of
SWCNTs
Langmuir Monolayers
Fluorescence Lifetime Imaging
Microscopy (FLIM)
 Fluorescence emission lifetime is a characteristic
of every fluorophore
 Lifetime also sensitive to the nanoenvironment:
pH, [O2], binding to macromolecules, etc.
 HeLa cells transfected with pAcGFP1-Endo
 Incubated with SWCNTs at 100 µg/ml for various
time points
 Changes in fluorescence lifetimes were observed in
SWCNT-treated cells
Tethered Bilayer Membrane (tBLM)
 Lipid phase behavior can be controlled changing
surface area, A, affecting surface pressure, P
Image courtesy of H. Nanda NCNR NIST
Solvent
Isotherm and Phase Diagram of
DPPC monolayer
Distal
leaflet
Proximal
leaflet
liquid condensed, LC
Aqueous
Reservoir
Tether
Lateral
Spacer
liquid expanded, La
P
Equivalent Circuit
two-dimensional gas, LG
stray
capacitance
FLIM of GFP Labeled Endosomes + SWCNTs
A
25 min
=
EIS Spectra
7
10
16:0 PC (DPPC)
3
10
SWCNT Dimensions
Image Statistics of Fluorescence Lifetimes
1
0.8
0.8
0.8
0.4
0.6
0.4
0.6
0.4
0.2
0.2
0.2
0
0
0
0-500
500-1000 > 1000
t1 [ps]
N 2
It    ai e
mean length : 145 ± 17 nm
0-1000
0-2250 2250-3000 > 3000
N 2
t m   ait i
ti
i 1
i 1
Maximum Insertion Pressure
> 2000
tm [ps]
t2 [ps]
t
1000-2000
N 2
a
i 1
30
Control
5 min.
25 min.
i
 Measuring the change in surface pressure after
exposure to SWCNTs from different starting
pressures one can extrapolate the maximum
insertion energy needed for a SWCNT to penetrate
a phospholipid monolayer
P (mN/m)
0.6
Frequency
1
Frequency
Frequency
radius : 0.7 – 1.3 nm
1
Maximum Insertion Pressure (MIP)
20
SWCNTs MIP
10
A
0
endosomes/cell
Endosome count after SWCNT incubation
200
180
160
140
120
100
80
60
40
20
0
0
Error bars are the
standard deviation from
the average values of
the data sets
0
control
1
0
2
n = 30
53
15 20 25
Pi (mN/m)
30
35
Electrochemical Impedance
Spectroscopy (EIS)
n = 17
n = 35
10
n = 32 n = 33
n = 18
n = 33
5
4
10
15
5
20
6
time after treatment (min)
25
7
8
 EIS was performed on tethered bilayer membranes
before and after incubation with SWCNTs
 changes in tBLM due to inclusion of SWCNTs can
be related to changes in capacitance and resistance
(A-C)
(degrees)
A)
0000 ≤ tm ≤ 1000 ps
1000 ≤ tm ≤ 2000 ps
2000 ≤ tm ≤ 3000 ps
B
5
10
-1
10
0
1
10
10
-100
-80
-60
-40
-20
0
B)
2
3
10
10
f (Hz)
4
5
10
10
C)
3
10
4
5
10
10
f (Hz)
6
10
substrate
interfacial
impedance
100x10
80
60
40
20
0
0.0
2
5 min
|Z|
Control
tBLM
resistance
3
f (Hz)
 Synthesized by HiPCO (high-pressure carbon
monoxide conversion synthesis)
 Size selected using density gradient length sorting
 Highly purified sorting to remove carbonaceous
polymorphs and metallic catalyst particles
 Stabilized and dispersed using a biocompatible triblock co-polymer Pluronic F127
 HeLa cells were transfected
with pAcGFP1-Endo and
incubated with 100 mg/ml of
SWCNTs (A)
 Endocytotic vessels were
determined
by
intensity
maxima
in
the
GFP
fluorescence filter range using
Image J (B)
spreading
resistance
=
SWCNT synthesis
Fixed Cell Imaging
tBLM
capacitance
Im(Y"/mF/cm )
Introduction
-0.4
-0.8
-1.2
0.0
0.4
0.8
2
Re(Y"/mF/cm )
1.2
Bode plots (A & B) of tBLMs with
SWCNTs (red) and without (black), (C)
Cole-Cole plot (C) of the tBLM after
incubation with SWCNTs
Conclusions
 Fixed cell imaging shows an increase in the number
of endocytotic vessels
 FLIM shows altered lifetime of GFP labeled
endosomes suggesting SWCNT uptake via
endocytosis
 Langmuir monolayers yield a maximum insertion
pressure of 28 mN/m which is below MIP needed
for BLM insertion (~30 mN/m)
 EIS shows negligible changes in capacitance and
resistance indicating minimal incorporation of
SWCNTs by purely physical mechanisms
References and Acknowledgements
[1] Holt et al. ACS Nano. 4, (2010): 4872-4878
[2] Bianco, et al. Curr. Opin. Chem. Bio. 9, (2005): 674–679
[3] Kostarelos et al. Nature nano. 108, (2007): 108-113
[4] Gao, et al. Proc. Nat.Acad. Sci. 102, (2005): 9469-9474
[5] S. Pogodin et al. ACS Nano. 4, (2010): 5293–5300
Funding: NSF CAREER, NIH (1P01AG032131)
Biological & Biophysical Basis of Membrane Dynamics and Organization workshop, Nov. 5 & 6, Mellon Institute of Science