phase association and binding energetics of swcnts into phospholipid langmuir monolayers peter n....
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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
Introduction
[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): 5293–5300Funding: NSF CAREER, NIH (1P01AG032131)
References and Acknowledgements
Electrochemical Impedance Spectroscopy (EIS)
Langmuir Monolayers
Biological & Biophysical Basis of Membrane Dynamics and Organization workshop, Nov. 5 & 6, Mellon Institute of Science
SWCNT synthesis 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 tri-block co-polymer Pluronic F127
mean length : 145 ± 17 nm
radius : 0.7 – 1.3 nm
SWCNT Dimensions
16:0 PC (DPPC)
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)
Lipid phase behavior can be controlled changing surface area, A, affecting surface pressure,
Fixed Cell Imaging
HeLa cells were transfected with pAcGFP1-Endo and incubated with 100 g/ml of SWCNTs (A)
Endocytotic vessels were determined by intensity maxima in the GFP fluorescence filter range using Image J (B)
A
two-dimensional gas, LG
liquid expanded, L
liquid condensed, LC
Isotherm and Phase Diagram of DPPC monolayer
Maximum Insertion Pressure (MIP) 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
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
0000 ≤ m ≤ 1000 ps1000 ≤ m≤ 2000 ps2000 ≤ m≤ 3000 ps
Control 5 min 25 min
FLIM of GFP Labeled Endosomes + SWCNTs
Image Statistics of Fluorescence Lifetimes
Control5 min.25 min.
control
0 5 10
15 20 25
en
doso
mes/
cell
time after treatment (min)
n = 33
n = 35
n = 30
n = 17
n = 18
n = 32
n = 33
Endosome count after SWCNT incubation
Error bars are the standard deviation from the average values of the data sets
A
B
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
Conclusions
=
2
1
tIN
i
t
iiea
2
1
2
1
N
ii
N
iiim aa
Maximum Insertion Pressure
SWCNTs MIP
30
20
10
0Δ
( / )mNm
35302520151050i( / )mNm
=
Distal leaflet
Tether
LateralSpacer
Proximal leaflet
Solvent
AqueousReservoi
r
Tethered Bilayer Membrane (tBLM)
Equivalent Circuit
-100-80-60-40-20
0
(d
eg
rees)
103 104 105 106
f (Hz)
103
105
107
|Z|
10-1 100 101 102 103 104 105
f (Hz)
EIS Spectra
A)
C)
B)
Bode plots (A & B) of tBLMs with SWCNTs (red) and without (black), (C) Cole-Cole plot (C) of the tBLM after incubation with SWCNTs
stray capacitan
ce
spreading
resistance
tBLM capacitanc
e
tBLM resistanc
e
substrate interfaci
al impedan
ce
-1.2
-0.8
-0.4
0.0
Im(Y"/
ω)(
/F cm2 )
1.20.80.40.0( "/Re Y ω)( /F cm2)
100 10x 3
806040200
( )f Hz
Image courtesy of H. Nanda NCNR NIST