Tumor Targeting of Functionalized Quantum Dot- Liposome Hybrids by Intravenous Administration
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1 Tumor Targeting of Functionalized Quantum Dot- Liposome Hybrids by Intravenous Administration Wafa T. Al-Jamal 1, Khuloud T. Al-Jamal 1, Bowen Tian 1, Andrew Cakebread 2, John M. Halket 2 and Kostas Kostarelos Correspondence to: Professor Kostas Kostarelos Nanomedicine Laboratory, Centre for Drug Delivery Research The School of Pharmacy, University of London Brunswick Square, London WC1N 1AX (UK) tel: fax: kostas.kostarelos@pharmacy.ac.uk 1 Nanomedicine Laboratory, Centre for Drug Delivery Research, The School of Pharmacy, University of London, Brunswick Square, London WC1N 1AX, UK. 2 Department of Forensic Science & Drug Monitoring., Kings College London, Franklin-Wilkins Building, 150 Stamford St, London SE1 9NH, UK. 1
2 Supplementary Information Conocal laser scanning microscopy (CLSM) To assess the encapsulation of f-qd into liposomes, DOPC lipid films were fluorescently labelled with mol% DiI (molecular probe, USA). Liposome labelling was performed by mixing Dil ethanolic solution with DOPC phospholipid mixture prior to lipid film formation. Encapsulation of f-qd within liposomes was studied with Zeiss LSM 510 Meta. The lasers used were 30mW Argon laser (488 nm for green channel) and 1mW 543nm HeNe laser (for red channel). The emission was collected using a band pass filter between nm for green QD and 560nm long pass filter for Dil-labelled liposomes. Supplementary Figure 1A shows the co-localization of the green signal (f- QD) with the red DiI-labelled multilamellar vesicles (MLVs), indicating encapsulation within the MLVs. In a control experiment, pre-formed DiI-labelled MLVs were mixed with f-qd leading to no co-localization or surface association between the MLVs and the f-qd signals (Supplementary Figure 1B). A B 10µm Supplementary Figure 1: CLSM images for 1*10 14 p/ml f-qd (A) encapsulated or (B) mixed with 8mM DiI-labelled DOPC liposomes (scale bar 10μm). 2
3 Agarose Gel Electrophoresis To study the surface interaction between the f-qd and liposome vesicles, aliquots of 40 µl freshly prepared f-qd and QD were mixed with zwitterionic or cationic small unilamellar vesicles (SUV) (8 μl) of 30% (w/v) glycerol in 0.5X TBE buffer and loaded to 1% agarose gel (0.25X TBE) in 0.5X TBE buffer. The gel was run for 60 min at 80V using a Sub-Cell GT agarose gel electrophoresis system (BioRad, USA) and then photographed under UV light using GeneGenius system, PerkinElmer Life and Analytical Sciences (USA). Negatively charged f-qd showed fluorescent band migration towards the positive electrode (Supplementary Figure 2, lane I). A similar result was observed with f-qd mixed with zwitterionic DOPC SUV (Supplementary Figure 2, lane II) which indicates free f-qd. No retardation in f-qd migration was observed which excludes QD interaction with the liposome surface. On the other hand, electrostatic interaction took place once negatively charged QD were mixed with cationic DOTAP SUV. This observation was confirmed by retarded migration of f-qd in the agarose gel (Supplementary Figure 2, lane III). I II III (-) (+) Supplementary Figure 2: Migration profile in 1% agarose and 0.5X TBE buffer of 1*10 14 p/ml: f-qd alone (lane I); f-qd mixed with 8mM DOPC SUV (lane II); and f-qd mixed with 8mM DOTAP SUV (lane III). 3
4 Atomic Force Microscopy (AFM): In order to study the f-qd-l hybrid structure, twenty µl of a liposome or f- QD-L hybrid suspension (2mM) was deposited on the surface of freshly cleaved mica (Agar Scientific, Essex, UK), and samples were allowed to adsorb for 30 seconds. Unbound liposomes were removed by washing with filtered dh 2 O. Samples were then dried under a nitrogen stream. Imaging was carried out in Tapping Mode using a Multimode AFM, E-type scanner, Nanoscope IV controller, Nanoscope 5.31r1 control software (all from Veeco, Cambridge, UK) and a silicon tapping tip (NSG01, NTI-Europe, Apeldoorn, The Netherlands) of 10 nm curvature radius, mounted on a tapping-mode silicon cantilever with a typical resonant frequency of 150 khz and a force constant of 5.5 N/m, to image 5 µm 5 µm square areas of the mica surface with a resolution of pixels and a scan rate of 1 Hz. All AFM images were performed in air. Supplementry Figure 3 represents the AFM images of f-qd-l hybrid vesicles (DSPC:Chol:DSPE-PEG 2000 ; 1.8:1:0.2). The amplitude image showed a clear difference in the vesicle morphology (Supplementry Figure 3B) once compared to the same formulation in the abscence of f-qd (Supplementry Figure 3A). Higher resolution and 3D-images revelaed f-qd encapsulation inside the liposomes with some degree of interaction with the lipid bilayer, resulting a rough, uneven surface (Supplementry Figure 3D). 4
5 A C B D Supplementary Figure 3: AFM of f-qd-l hybrid vesicles. Amplitude images of 2mM DSPC: Chol: DSPE-PEG 2000 (1.8:1:0.2) (A) empty liposomes and (B) f-qd-l hybrid vesicles. Higher resolution amplitude (top) and 3D-image (bottom) of (C) empty liposomes and (D) f-qd-l hybrid vesicles. 5
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