Hybrid Polymer-Grafted Multiwalled Carbon Nanotubes for in vitro Gene Delivery **

Antonio Nunes1, Nadja Amsharov2, Chang Guo1, Jeroen Van den Bossche1, Padmanabhan Santhosh 2,3, Theodoros Karachalios4, Stephanos Nitodas4, Marko Burghard2, Kostas Kostarelos1,*, Khuloud T. Al-Jamal1, * 1 Nanomedicine

Laboratory, Centre for Drug Delivery Research, The School of Pharmacy, University of London, WC1N 1AX, UK 2 Nanoscale Science Department, Max-Planck-Institute for Solid State Research, 70569 Stuttgart, Germany 3 Department of Nanoengineering, University of California San Diego, La Jolla, California 92093-0448, USA 4 Nanothinx S.A, Rio-Patras, 26504, Greece

__________________________________________________ * Correspondence should be addressed to: Dr Khuloud T Al-Jamal (E-mail: khuloud.al-jamal@pharmacy,ac.uk) or Professor Kostas Kostarelos (E-mail: [email protected]) Nanomedicine Laboratory Centre for Drug Delivery Research The School of Pharmacy University of London, 29-39 Brunswick Square London WC1N 1AX United Kingdom

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Supplementary Information D

Figure S1: NMR analysis: A) NMR spectrum of 0.5 mg PEI in the presence of 0.25 mg MWNT-COOH; B) NMR spectrum of the PEI-g-MWNT; (C) PEI protons peaks at top: non-conjugated, and at bottom: PEI-g-MWNT. The calibration curve of PEI integrals was done at different PEI concentrations using tBuOH as an internal standard. The resulting standard curve was used to calculate the amount of polymer conjugated. Analysis of the samples indicated a PEI to PEI-g-MWNT ratio 5.9 % (%w/w). (D) HRTEM shows the structure CNT and coating by the polymer.

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Figure S2: Raman spectra showing the conjugation of the polymer onto CNT backbone. The typical resonant peaks for MWNT that possess overall structural integrity are represented. D-band (disorder) intensity at ~1340 cm-1 increased after the conjugation to PEI or PAA, considered an indication that chemical grafting of the polymer took place. G-band (graphite) intensity at ~1600 cm-1 is a more complex spectral feature related to tangential mode vibrations.

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Figure S3: Gene transfer efficiency in A549 cells using various PEI- and PAA-based vectors and their physical mixtures. Gene expression was assessed 24 h post-transfection and expressed as average RLU/well ± SD (n=3). Vectors used were (A) Individual PEI or PAA polymers; (B) Physical mixtures of PEI/PAA polymers prepared by gradually increasing PAA fraction from 0 (0% PAA) to 1 (100% PAA) at constant total polymer : pDNA mass ratio of 1:1, in the absence or presence of MWNT-COOH (polymer : MWNT-COOH mass ratio of 1:1); (C) Physical mixtures of PEI-g-MWNT/PAA-g-MWNT mixtures prepared by increasing the PAA-g-MWNT fraction from 0 (0% PAA-g-MWNT) to 1 (100% PAA-g-MWNT) at constant total polymer-g-MWNT:pDNA mass ratio of 15:1 and 20:1.

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