1. Phase association and binding energetics of SWCNTs into phospholipid Langmuir monolayers Peter N. Yaron 1, Philip A. Short 2, Brian D. Holt 2, Goh Haw-Zan 3, Mohammad F. Islam 1,4, Mathias Lösche 2,3, Kris Noel Dahl 1,2 1 Chemical Engineering, 2 Biomedical Engineering, 3 Physics, 4 Materials 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): 5293–5300 Funding: 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) two-dimensional gas, L G liquid expanded, L  liquid condensed, L C 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, [O 2 ], 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 ps 1000 ≤  m  ≤ 2000 ps 2000 ≤  m  ≤ 3000 ps Control5 min25 min FLIM of GFP Labeled Endosomes + SWCNTs Image Statistics of Fluorescence Lifetimes Control 5 min. 25 min. control 0 5 10 15 20 25 endosomes/cell time after treatment (min) n = 33 n = 35 n = 30 n = 17 n = 18 n = 32n = 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 = Maximum Insertion Pressure SWCNTs MIP = Distal leaflet Tether Lateral Spacer Proximal leaflet Solvent Aqueous Reservoir Tethered Bilayer Membrane (tBLM) Equivalent Circuit 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 capacitance spreading resistance tBLM capacitance tBLM resistance substrate interfacial impedance Image courtesy of H. Nanda NCNR NIST