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