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Head and neck squamous cell carcinoma (HNSCC) still represents the world's sixth most common tumor entity, with increasing incidence. The reachability of light makes HNSCC suitable for light-based therapies such as Photochemical Internalization (PCI). The drug Bleomycin is cytotoxic and used as an anti-tumor medication. Since Bleomycin is endocytosed as a relatively large molecule, part of it is degraded in lysosomes before reaching its intracellular target. The goal of our study was to improve the intracellular availability of Bleomycin with PCI. We investigate the intracellular delivery of Bleomycin after PCI with the photosensitizer Fimaporfin. A systematic variation of Bleomycin and Fimaporfin concentrations and light irradiation led to the pronounced cell death of HNSCC cells. After optimization, the same level of tumor cell death of 75% was reached with a 20-fold lower Bleomycin concentration. This would allow treatment of HNSCC with high local tumor cell death and reduce the side effects of Bleomycin, e.g., lung fibrosis, at the same time. This demonstrates the increased efficacy of the anti-tumor medication Bleomycin in combination with PCI. [ABSTRACT FROM AUTHOR]

Effectiveness of radiation treatment for cancer is limited in hypoxic tumors. Previous data shows that UVC‐emitting nanoparticles enhance cytotoxicity of X‐ray irradiation in hypoxic tumor cells. This study examines the impact on cell killing, particle size, uptake into cells, incubation time, and UV emission intensity of LuPO4:Pr3+,Nd3+. A549 cells are treated with LuPO4:Pr3+,Nd3+ and X‐rays. The surviving fraction is evaluated using the colony formation assay after treatment of cells with different particle sizes (D50 = 0.16 and 5.05 µm) and after different incubation times before X‐ray irradiation. Nanoparticle uptake into cells is verified by transmission electron microscopy and quantified by inductively coupled plasma mass spectrometry. The microparticles exhibit a five times higher emission intensity compared to nanoparticles. Both particle sizes show an increased cytotoxic effect after X‐ray excitation with prolonged incubation times. Surprisingly, the smaller nanoparticles show a significantly higher biological effect compared to the larger particles, despite their significantly lower UVC emission. Nanoparticles accumulate more quickly and closer to the nucleus than the microparticles, resulting in higher localized UVC emission and greater lethality. The results suggest that the number of intracellular particles and their proximity to the cell DNA is more important than the emission intensity of the particles. [ABSTRACT FROM AUTHOR]

The delivery of macromolecules into living cells is challenging since in most cases molecules are endocytosed and remain in the endo‐lysosomal pathway where they are degraded before reaching their target. Here, a method is presented to selectively improve cell membrane permeability by nanosecond laser irradiation of gold nanorods (GNRs) with visible or near‐infrared irradiation in order to deliver proteins across the plasma membrane, avoiding the endo lysosomal pathway. GNRs were labeled with the anti‐EGFR (epidermal growth factor receptor) antibody Erbitux to target human ovarian carcinoma cells OVCAR‐3. Irradiation with nanosecond laser pulses at wavelengths of 532 nm or 730 nm is used for transient permeabilization of the cell membranes. As a result of the irradiation, the uptake of an anti‐Ki‐67 antibody was observed in about 50 % of the cells. The results of fluorescence lifetime imaging show that the GNR detached from the membrane after irradiation. [ABSTRACT FROM AUTHOR]

Nanosecond pulsed laser irradiation can trigger a release of nucleic acids from gold nanoparticles, but the involved nanoeffects are not fully understood yet. Here we investigate the release of coumarin labeled siRNA from 15 to 30 nm gold particles after nanosecond pulsed laser irradiation. Temperatures in the particle and near the surface were calculated for the different radiant exposures. Upon irradiation with laser pulses of 4 nanosecond duration release started for both particle sizes at a calculated temperature increase of approximately 500 K. Maximum coumarin release was observed for 15 nm particles after irradiation with radiant exposure of 80 mJ cm−2 and with 32 mJ cm−2 for 30 nm particles. This corresponds to a temperature increase of 815 and 900 K, respectively. Our results show that the molecular release by nanosecond pulsed irradiation is based on a different mechanism compared to continuous or femtosecond irradiation. Local temperatures are considerably higher and it is expected that bubble formation plays a crucial role in release and damage to cellular structures. [ABSTRACT FROM AUTHOR]

The intracellular delivery of therapeutic agents is a central challenge in the treatment of different diseases. Light sensitive liposomes can release their cargo with high temporal and spatial precision. The light sensitivity of the liposomes is based on incorporated photosensitizers. Since light penetration into human tissue is higher at wavelengths greater than 650 nm, we synthesized the chlorin and bacteriochlorin derivative from the new photosensitizer 5,10-Di-(4-hydroxyphenyl)-15,20-diphenyl-porphyrin. Both, chlorin and bacteriochlorin showed a higher absorption peak at 650 nm than the porphyrin. Interestingly, the bacteriochlorin showed a stable additional absorption peak at 740 nm. To investigate the potential of the derivates for light-induced release from liposomes we incorporated them into liposomes. Liposomes with the porphyrin, the chlorin and bacteriochlorin released their cargo effectively after light irradiation. Liposomes with the bacteriochlorin showed release also after irradiation at 740 nm. Additionally, we investigated the potential of the bacteriochlorin as a sensitizer for PDT. The bacteriochlorin showed good cell killing results when irradiated at 740 nm compared to the photosensitizer BPD irradiated at 690 nm. We believe that the synthesized porphyrin 5,10-DiOH and its chlorin and bacteriochlorin derivatives are interesting candidates for photodynamic applications and need further investigation. [ABSTRACT FROM AUTHOR]

The fluorescent dye indocyanine green (ICG) is clinically approved and has been applied for ophthalmic and intraoperative angiography, measurement of cardiac output and liver function, or as contrast agent in cancer surgery. Though ICG is known for its photochemical effects, it has played a minor role so far in photodynamic therapy or techniques for targeted protein-inactivation. Here, we investigated ICG as an antibody-conjugate for the selective inactivation of the protein Ki-67 in the nucleus of cells. Conjugates of the Ki-67 antibody TuBB-9 with different amounts of ICG were synthesized and delivered into HeLa and OVCAR-5 cells through conjugation to the nuclear localization sequence. Endosomal escape of the macromolecular antibodies into the cytoplasm was optically triggered by photochemical internalization with the photosensitizer BPD. The second light irradiation at 690 nm inactivated Ki-67 and subsequently caused cell death. Here, we show that ICG as an antibody-conjugate can be an effective photosensitizing agent. Best effects were achieved with 1.8 ICG molecules per antibody. Conjugated to antibodies, the ICG absorption peaks vary proportionally with concentration. The absorption of ICG above 650 nm within the optical window of tissue opens the possibility of selective Ki-67 inactivation deep inside of tissues. [ABSTRACT FROM AUTHOR]

Using nanotechnology for optical manipulation of molecular processes in cells with high spatial and temporal precision promises new therapeutic options. Especially tumor therapy may profit as it requires a combination of both selectivity and an effective cell killing mechanism. Here we show a dual targeting approach for selective and efficient light-controlled killing of cells which are positive for epidermal growth factor receptor (EGFR) and Ki-67. Liposomes with the covalently linked EGFR antibody Erbitux enabled selective uptake of FITC-labeled Ki-67 antibody TuBB-9 in EGFR-positive cells pre-loaded with the photoactive dye BPD. After irradiation at 690 nm, BPD disrupted the endosomal membranes and delivered the antibodies to the nucleoli of the cells. The second irradiation at 490 nm activated the FITC-labeled TuBB-9, which caused inactivation of the Ki-67 protein and subsequent cell death via apoptosis. Efficient cell killing was possible at nanomolar concentrations of TuBB-9 due to the effective transport by immune liposomes and the high efficacy of the Ki-67 light-inactivation. Delivery of the liposomal constructs and cell destruction correlated well with the EGFR expression pattern of different cell lines (HeLa, OVCAR-5, MCF-7, and human fibroblasts), demonstrating an excellent selectivity.

UV‐C emitting nanoscale scintillators can be used to sensitize cancer cells selectively against X‐rays during radiation therapy, due to the lethal DNA lesions caused by UV‐C photons. Unfortunately, nanoscale particles (NPs) show decreased UV‐C emission intensity. In this paper, the influence of different Nd3+ concentrations on the UV‐C emission of micro‐ and nanoscale LuPO4:Pr3+ is investigated upon X‐ray irradiation and vacuum UV excitation (160 nm). Co‐doped LuPO4 results in increased UV‐C emission independent of excitation source due to energy transfer from Nd3+ to Pr3+. The highest UV‐C emission intensity is observed for LuPO4:Pr3+,Nd3+(1%,2.5%) upon X‐ray irradiation. Finally, LuPO4 NPs co‐doped with different dopant concentrations are synthesized, and the biological efficacy of the combined approach (X‐rays and UV‐C) is assessed using the colony formation assay. Cell culture experiments confirm increased cell death compared to X‐rays alone due to the formation of UV‐specific DNA damages, supporting the feasibility of this approach. [ABSTRACT FROM AUTHOR]

Light can manipulate molecular biological processes with high spatial and temporal precision and optical manipulation has become increasingly popular during the last years. In combination with absorbing dyes or gold nanoparticles light is a valuable tool for cell and protein inactivation with high precision. Here we show distinct differences in the underlying mechanisms whether gold nanoparticles or fluorescent dyes are used for the inactivation of the Ki‐67 protein. The proliferation‐associated protein Ki‐67 was addressed by the antibody MIB‐1. In vitro studies showed a fragmentation of the Ki‐67 protein after laser irradiation of 15 nm gold nanoparticle antibody conjugates with nanosecond pulsed laser, while continuous wave (cw) irradiation of fluorescein isothiocyanate (FITC)‐ and Alexa 488‐labeled antibodies led to specific crosslinking of Ki‐67. The irradiation energy for the gold nanoparticles was above cavitation bubble formation threshold. We observed a fragmentation of the target protein and also of the gold particles. The understanding of the underlying inactivation mechanisms is important for the application and further development of these two techniques, which can harness nanotechnology to introduce molecular selectivity to biological systems. [ABSTRACT FROM AUTHOR]

While optical coherence tomography (OCT) provides a resolution down to 1 µm, it has difficulties in visualizing cellular structures due to a lack of scattering contrast. By evaluating signal fluctuations, a significant contrast enhancement was demonstrated using time-domain full-field OCT (FF-OCT), which makes cellular and subcellular structures visible. The putative cause of the dynamic OCT signal is the site-dependent active motion of cellular structures in a sub-micrometer range, which provides histology-like contrast. Here we demonstrate dynamic contrast with a scanning frequency-domain OCT (FD-OCT), which we believe has crucial advantages. Given the inherent sectional imaging geometry, scanning FD-OCT provides depth-resolved images across tissue layers, a perspective known from histopathology, much faster and more efficiently than FF-OCT. Both shorter acquisition times and tomographic depth-sectioning reduce the sensitivity of dynamic contrast for bulk tissue motion artifacts and simplify their correction in post-processing. Dynamic contrast makes microscopic FD-OCT a promising tool for the histological analysis of unstained tissues.

The delivery of therapeutic drugs to a specific cellular site is a challenge in the treatment of different diseases. Liposomes have been widely studied as vehicles for drug delivery, and recent research begins to show the potential of the light-controlled opening of liposomes. Liposomes with photoactive molecules can release their cargo upon light irradiation for localized drug release. Light as an external trigger can be controlled temporally and spatially with high precision. In this study, we investigate the potential of light-sensitive liposomes with four photosensitizers and two lipid formulations for light-induced release. To investigate the permeabilization of the liposomes, calcein was encapsulated in high concentration inside the liposomes so that the calcein fluorescence is quenched. If calcein is released from the liposome, quenching is avoided, and the fluorescence increases. We demonstrated that liposomes with the sensitizers benzoporphyrine derivative monoacid (BPD), chlorine e6 (Ce6), Al(III) phthalocyanine chloride disulfonic acid (AlPcS 2 ), and 5,10-di-(4-hydroxyphenyl)-15,20-diphenyl-21,23 H -porphyrin (5,10-DiOH) release cargo effectively after irradiation. Liposomes with 5,10-DiOH showed a quicker release compared to the other sensitizers upon irradiation at 420 nm. Further, we observed through fractionated irradiation, that most of the release took place during light application, while the permeability of the liposome decreased shortly after light exposure. This effect was stronger with liposomes containing less cholesterol.

Purpose: Pulsed-laser irradiation of light-absorbing gold nanoparticles (AuNPs) attached to cells transiently increases cell membrane permeability for targeted molecule delivery. Here, we targeted EGFR on the ovarian carcinoma cell line OVCAR-3 with AuNPs. In order to optimize membrane permeability and to demonstrate molecule delivery into adherent OVCAR-3 cells, we systematically investigated different experimental conditions., Materials and Methods: AuNPs (30 nm) were functionalized by conjugation of the antibody cetuximab against EGFR. Selective binding of the particles was demonstrated by silver staining, multiphoton imaging, and fluorescence-lifetime imaging. After laser irradiation, membrane permeability of OVCAR-3 cells was studied under different conditions of AuNP concentration, cell-incubation medium, and cell-AuNP incubation time. Membrane permeability and cell viability were evaluated by flow cytometry, measuring propidium iodide and fluorescein isothiocyanate-dextran uptake., Results: Adherently growing OVCAR-3 cells can be effectively targeted with EGFR-AuNP. Laser irradiation led to successful permeabilization, and 150 kDa dextran was successfully delivered into cells with about 70% efficiency., Conclusion: Antibody-targeted and laser-irradiated AuNPs can be used to deliver molecules into adherent cells. Efficacy depends not only on laser parameters but also on AuNP:cell ratio, cell-incubation medium, and cell-AuNP incubation time., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The selective inhibition of intracellular and nuclear molecules such as Ki-67 holds great promise for the treatment of cancer and other diseases. However, the choice of the target protein and the intracellular delivery of the functional agent remain crucial challenges. Main hurdles are (a) an effective delivery into cells, (b) endosomal escape of the delivered agents, and (c) an effective, externally triggered destruction of cells. Here we show a light-controlled two-step approach for selective cellular delivery and cell elimination of proliferating cells. Three different cell-penetrating nano constructs, including liposomes, conjugates with the nuclear localization sequence (NLS), and conjugates with the cell penetrating peptide Pep-1, delivered the light activatable antibody conjugate TuBB-9-FITC, which targets the proliferation associated protein Ki-67. HeLa cells were treated with the photosensitizer benzoporphyrin monoacid derivative (BPD) and the antibody constructs. In the first optically controlled step, activation of BPD at 690 nm triggered a controlled endosomal escape of the TuBB-9-FITC constructs. In more than 75% of Ki-67 positive, irradiated cells TuBB-9-FITC antibodies relocated within 24 h from cytoplasmic organelles to the cell nucleus and bound to Ki-67. After a second light irradiation at 490 nm, which activated FITC, cell viability decreased to approximately 13%. Our study shows an effective targeting strategy, which uses light-controlled endosomal escape and the light inactivation of Ki-67 for cell elimination. The fact that liposomal or peptide-assisted delivery give similar results leads to the additional conclusion that an effective mechanism for endosomal escape leaves greater variability for the choice of the delivery agent.

When irradiated with nanosecond laser pulses, gold nanoparticles allow for manipulation or destruction of cells and proteins with high spatial and temporal precision. Gold nanorods are especially attractive, because they have an up-to-20-fold stronger absorption than a sphere of equal volume, which is shifted to the optical window of tissue. Thus, an increased efficiency of cell killing is expected with laser pulses tuned to the near infrared absorption peak of the nanorods. In contrast to the higher-absorption, experiments showed a reduced efficacy of cell killing. In order to explain this discrepancy, transient absorption of irradiated nanorods was measured and the observed change of particle absorption was theoretically analyzed. During pulsed irradiation a strong transient and permanent bleaching of the near-infrared absorption band occurred. Both effects limit the ability of nanorods to destroy cells by nanocavitation. The existence of nanocavitation and transient bleaching was corroborated by optoacoustic measurements.

Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67-directed antibody and subsequent light-triggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti-pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67-positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation., (Copyright © 2010 AACR.)

Light-absorbing nanoparticles that are heated by short laser pulses can transiently increase membrane permeability. We evaluate the membrane permeability by flow cytometry assaying of propidium iodide and fluorescein isothiocyanate dextran (FITC-D) using different laser sources. The dependence of the transfection efficiency on laser parameters such as pulse duration, irradiant exposure, and irradiation mode is investigated. For nano- and also picosecond irradiation, we show a parameter range where a reliable membrane permeabilization is achieved for 10-kDa FITC-D. Fluorescent labeled antibodies are able to penetrate living cells that are permeabilized using these parameters. More than 50% of the cells are stained positive for a 150-kDa IgG antibody. These results suggest that the laser-induced permeabilization approach constitutes a promising tool for targeted delivery of larger exogenous molecules into living cells.

Due to their unique optical properties, optical probes, including metal nanoparticles (NPs) and fluorescent dyes, are increasingly used as labeling tools in biological imaging. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) at 750-nm excitation, we recorded intensity and FLIM images from gold NPs (30 nm) and the fluorescent dye Alexa 488 (A488) conjugated with monoclonal ACT-1 antibodies as well as Hoechst 33258 (H258) after incubation with the lymphoma cell line (Karpas-299). From the FLIM images, we can easily discriminate the imaging difference between cells and optical probes according to their distinct fluorescence lifetimes (cellular autofluorescence: 1 to 2 ns; gold NPs: <0.02 ns; A488: 3.5 ns; H258: 2.5 ns). The NP-ACT-1 and A488-ACT-1 conjugates were bound homogeneously on the surface of cells, whereas H258 stained the cell nucleus. We demonstrate that the emission intensity of gold NPs is about ten times stronger than that of the autofluorescence of Karpas-299 cells at the same excitation power. Compared with fluorescent dyes, stronger emission is also observed from gold NPs. Together with their high photostability, these observations suggest that gold NPs are a viable alternative to fluorescent dyes for cellular imaging and cancer diagnosis.

Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkin's disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.