Nanoscale. 2015 Nov;7(42):17836−17847.
Cell-specific optoporation with near-infrared ultrafast laser and functionalized gold nanoparticles
Éric Bergeron1, Christos Boutopoulos1,2, Rosalie Martel1, Alexandre Torres1, Camille Rodriguez1, Jukka Niskanen3, Jean-Jacques Lebrun4, Françoise M. Winnik3,5, Przemyslaw Sapieha6 and Michel Meunier1.
1 Laser Processing and Plasmonics Laboratory, Department of Engineering Physics, Polytechnique Montréal, C.P. 6079, Succursale Centre-ville, Montreal, QC, H3C 3A7, Canada
2 SUPA, School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews, KY16 9SS, United Kingdom
3 Faculty of Pharmacy and Department of Chemistry, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montreal, QC, H3C 3J7, Canada
4 Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, H3A 1A1, Canada
5 World Premier International (WPI) Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA) and National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan
6 Department of Ophthalmology, Hôpital Maisonneuve-Rosemont Research Centre, Université de Montréal, Montreal, QC, H1T 2M4, Canada
Selective targeting of diseased cells can increase therapeutic efficacy and limit off-target adverse effects. We developed a new tool to selectively perforate living cells with functionalized gold nanoparticles (AuNPs) and near-infrared (NIR) femtosecond (fs) laser. The receptor CD44 strongly expressed by cancer stem cells was used as a model for selective targeting. Citrate-capped AuNPs (100 nm in diameter) functionalized with 0.01 orthopyridyl-disulfide-poly(ethylene glycol) (5 kDa)-N-hydroxysuccinimide (OPSS-PEG-NHS) conjugated to monoclonal antibodies per nm2 and 5 μM HS-PEG (5 kDa) were colloidally stable in cell culture medium containing serum proteins. These AuNPs attached mostly as single particles 115 times more to targeted CD44+ MDA-MB-231 and CD44+ ARPE-19 cells than to non-targeted CD44− 661W cells. Optimally functionalized AuNPs enhanced the fs laser (800 nm, 80–100 mJ/cm2 at 250 Hz or 60–80 mJ/cm2 at 500 Hz) to selectively perforate targeted cells without affecting surrounding non-targeted cells in co-culture. This novel highly versatile treatment paradigm can be adapted to target and perforate other cell populations by adapting to desired biomarkers. Since living biological tissues absorb energy very weakly in the NIR range, the developed non-invasive tool may provide a safe, cost-effective clinically relevant approach to ablate pathologically deregulated cells and limit complications associated with surgical interventions.
Gold nanoparticles (AuNPs) have found numerous applications in nanomedicine, in view of their robustness, ease of functionalization and low toxicity. Our group has demonstrated that near-infrared (NIR) femtosecond (fs, 10-15 s) laser excitation of 100 nm (10-7 m) citrate-capped AuNPs enables high localized transient cell membrane perforation (optoporation), while maintaining high cell viability and enabling transfection of DNA plasmids into cancer cells.[2,3] Such laser irradiation of AuNPs minimizes the heat absorption from AuNPs and biological tissues since they absorb energy very weakly in the NIR range.[4-6] The amplification of the electromagnetic field around the irradiated AuNP can cause the generation of nanoplasma leading to a nanobubble, thus inducing cell perforation without AuNP fragmentation.[2-4]
Since selective targeting of diseased cells can increase therapeutic efficacy and limit off-target adverse effects, the next step for selective cell optoporation is to develop stable AuNPs which labeled targeted cells without affecting surrounding non-targeted cells. Here the receptor CD44 strongly expressed by cancer cells was used as a model for selective targeting and optoporation (Figure 1).
Antibodies (Abs) targeting the CD44 biomarker were conjugated to the biopolymer orthopyridyl-disulfide-poly(ethylene glycol) (5 kDa)-N-hydroxysuccinimide (OPSS-PEG-NHS). The OPSS end’s disulfide group of this OPSS-PEG-Ab complex binds strongly to the AuNP surface. Thus, AuNPs were functionalized with 0.01 OPSS-PEG-Ab/nm2 and 5 µM thiolated PEG chains (HS-PEG). The stability of functionalized AuNPs was evaluated by ultraviolet-visible-NIR spectroscopy and zeta potential measurements. The functionalized AuNPs with HS-PEG (5kDa) were colloidally stable in cell culture medium containing serum proteins, while functionalized AuNPs with HS-PEG (2kDa) were unstable.
Selective targeting with stable functionalized AuNPs was confirmed by flow cytometry, and darkfield, fluorescence and scanning electron microscopy on targeted CD44+ human cells (MDA-MB-231 breast cancer and ARPE-19 retinal pigmented epithelium) and on non-targeted CD44− mouse 661W photoreceptors. The functionalized AuNPs attached mostly as single particles 115 times more to targeted cells than to non-targeted cells.
Selective optoporation of targeted cells was demonstrated with stable functionalized AuNPs enhancing 45 fs laser pulses at 800 nm without affecting surrounding non-targeted cells. Perforated cells in the laser-irradiated area were mainly targeted cells which accumulated the membrane impermeable green fluorescent dye Lucifer Yellow, while untreated cells remained non-perforated.
The proposed novel highly versatile treatment paradigm can be adapted to target and perforate other cell populations by adapting to desired specific biomarkers. Since living biological tissues absorb energy very weakly in the NIR range, the developed non-invasive tool may provide a safe, cost-effective clinically relevant approach to ablate pathologically deregulated cells and limit complications associated with surgical interventions.
This work was supported by Le Fonds de recherche du Québec, Consortium québécois sur la découverte du médicament (CQDM), the Natural Science and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR). EB received funding from Fonds de recherche du Québec – Santé (FRQS) and CB acknowledges funding from the European Union under a Marie Curie Fellowship, FP7-PEOPLE-2013-IOF, project reference 624888. David Rioux, Sergiy Patskovsky, Rémi Lachaine, Alexandra Thibeault-Eybalin, Ariel Wilson, Flavie Lavoie-Cardinal and Yves Drolet are acknowledged for technical assistance and fruitful discussions. The authors also thank Danièle Gagné and Gaël Dulude from the Institute for Research in Immunology and Cancer (IRIC) flow cytometry platform for assistance with flow cytometry.
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