Rapid developments in laser technology have opened up many possibilities in biomedical applications, such as cell manipulation or elimination, which may jointly be referred to as cell surgery. Still, the precision of direct interaction of laser light with tissue structures on micro- and nanoscales below 500nm is limited by the diffraction of light. Using nanoabsorbers, this limit can be overcome. When irradiated with laser pulses ranging from femto- to nanoseconds, strong nanoabsorbers, such as noble-metal particles, destroy cellular structures through thermal and physical effects.1 Chromophores convert the incident light into chemical processes, which in turn can inactivate proteins with high precision by cross-linking.
Gold nanoparticles mediate physical effects that are mainly related to thermal-energy deposition within the particles. This causes either thermal denaturation or mechanical effects on the cell membrane because of nanocavitation.2 We used pulsed irradiation of spherical gold nanoparticles with wavelengths around their absorption peak at 520nm for cell-membrane permeabilization, cell elimination, and protein inactivation. Using membrane-bound particles, we managed to deliver small molecules or antibodies inside living cells under irradiation.3 To attach the gold particles to the cell membranes, we coated them with antibodies against rejection by membrane proteins: see Figure 1(a). With higher irradiation energies, the same membrane-bound gold particles successfully eliminated tumor cells in vitro.4 We proved the high cell-elimination efficiency of spherical gold nanoparticles in nanosecond-pulsed irradiation experiments using different cell cultures. Figure 1(b) shows selective elimination of mouse lymphoma cells, which were grown in co-culture with fibroblasts.
Figure 1. (a) Microscopy image of L428 lymphoma cells with membrane-bound gold nanoparticles (silver enhancement). Scale bar: 25μm. Gold particles are coated with anti-CD30 antibodies. (b) Fluorescence-microscopy image of a mixed co-culture with mouse fibroblasts (3T3, short for ‘three-day transfer, inoculum 3×105cells’) and CD8-positive lymphoma cells (LB27.4). Scale bar: 50μm. 3T3 cells were stained green with carboxyfluorescein succinimidyl ester (generally known as CFSE) before plating (green). The co-culture was incubated with anti-CD8 gold particles and irradiated. Dead LB27.4 cells are stained red with Hoechst 33258 dye.
On cellular scales, nanoparticle-mediated effects proved very selective. With pulsed excitation, heat confinement should also allow selective effects on molecular scales. We demonstrated fragmentation of proteins attached to gold nanoparticles. However, the effects seemed insufficiently localized for intracellular surgery. In recent experiments with scavengers diluted into conjugates of proteins with gold nanoparticles, we were able to demonstrate nonlocal effects.
Figure 2. (a) Confocal microscopy confirms subnuclear localization of anti-Ki-67-fluorescein isothiocyanate (FITC) antibody conjugates (top panel). Scale bars: 10μm. (b) Subsequent light irradiation causes ovarian-cancer-cell death and loss of spherical structure in 3D cultures (bottom panel), following prior cell incubation with liposomal tubulin beta polypeptide (TuBB)-9-FITC constructs. Scale bar: 200μm.
Inactivating intracellular proteins is also possible using chromophores bound to antibodies, which generate singlet oxygen and free radicals under irradiation of continuous or pulsed light. In biomedical research, this mechanism is known as chromophore-assisted laser inactivation. It is used to analyze cellular functions of proteins. In a joint project with the Research Center Borstel (Germany), we demonstrated the physiological role of the widely used proliferation marker Ki-67.5 (‘Ki’ refers to Kiel, Germany, its city of origin.) This protein is strongly expressed in all proliferating cells and its expression is correlated with poor prognosis and overall survival of cancer patients. Under light irradiation, the antibody-dye conjugate (photo-immunoconjugate) becomes an inhibitor for the target protein, which is involved in ribosomal ribonucleic-acid (rRNA) synthesis. This molecular-level inactivation is epitope specific, since these photo-immunoconjugates—which target different parts of the Ki-67 protein—did not affect RNA synthesis.
Clinically, irradiation of phototoxic dye molecules is applied in photodynamic therapy (PDT), for instance to fight cancers, age-related macula degeneration, and skin and infectious diseases. Although PDT is used for various applications, it lacks sensitivity. Therefore, new targeted approaches are being investigated. Most of these are aimed at membrane proteins, which are at the beginning of signal-transduction pathways. Cells often protect themselves by turning on alternative pathways. This could be prevented by targeting essential proteins directly. The nuclear protein Ki-67 could be a universal target, since it is essential for all proliferating cells. However, delivery of targeting moieties into cell nuclei has thus far been very challenging.
Fortunately, a solution was found by the well-known PDT research group of Tayyaba Hasan. We used the liposomal encapsulation of photo-immunoconjugates they developed to transfer tubulin beta polypeptide (TuBB)-9-fluorescein isothiocyanate (FITC) antibodies into the cytoplasm of ovarian cancer cells.6 From the cytoplasm, the antibodies relocalize to the cell nucleus (see Figure 2). Light irradiation led to a significant loss of viability only in proliferating, Ki-67-expressing cells, while nonproliferating cells remained unaffected. Irradiation of a 3D culture model of ovarian-cancer cells led to disruption of the 3D spheres.
In summary, we demonstrated for the first time a potential role of Ki-67 as a molecular target for cancer therapy, in addition to its important role in tumor diagnostics. Our work also demonstrates PDT with a truly molecular target. This may lead to more effective cancer therapy, which represents one area of further research. With nanoparticle and dye conjugates, a wide range of laser-induced cell manipulations can be achieved for different applications, where gold-particle-derived effects are selective on the cellular level and chromophore-mediated effects are used for intracellular manipulations.
Gereon Hüttmann, Ramtin Rahmanzadeh, Florian Rudnitzki
Institute of Biomedical Optics
University of Lübeck
Institute of Molecular Medicine
University of Bonn
Wellman Center for Photomedicine
Massachusetts General Hospital and Harvard Medical School
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