Both the waste of edible food and the consumption of non-edible food within the best before date are ongoing concerns in food industry. Until now, no methods are applied to access food quality of packed food without opening of packages. We demonstrate the formulation of a sensor foil comprising of a non-toxic porphyrin on an inorganic matrix in polyethylene. The sensor foil is capable of detecting amines in the gas phase over food products, which could act as spoilage indicators during the shelf life of packaged food. The foil was optimized to prevent reactions with other analytes in the gas phase of food by the alteration of the hydrophobic polymer. We performed experiments, using model packing units, to monitor the behavior of the foil and correlated the change in the fluorescence spectra to the total viable count of bacteria on the fish. The readout of the foils was performed with fluorescence spectroscopy to yield highly accurate results in contrast to less accurate the colorimetric determination.
Along food supply chains, several critical steps can lead to inconsumable food. Especially food of animal origin undergoes rapid aging, when stored inadequately. Quality assessment of packaged food products faces serious problems ranging from the loss of integrity of the package to damage of the food and it is applied only to a low number of samples per batch. As a result, food products are either wasted or not analyzed, which results in a significant decrease in food safety. As a part of an intelligent packaging system, we designed a sensor foil that can detect amines, produced during the food aging process. Change of the fluorescence of the sensor foil can be assessed with spectroscopy or color change from green to red can be detected optically with a camera, e.g. by smartphone. The foil can be incorporated inside the single packaging units and noninvasively measured routinely by the store or consumer. The readout of the foils was performed with steady-state tabletop spectrometers, which were then compared to the results for readouts with different inexpensive handheld devices that could be used during real-life applications, e.g., at any step in a food supply chain. Ideally, the single food product is linked to a single foil at the primary producer, measuring the first spectrum and connecting the data to the specific product, e.g. via distributed ledger. For a transparent process chain, QR-codes could be utilized to allow access to the freshness data along the shelf life of a single package.
Food waste during all stages of the supply chain and at the consumer is an emerging problem. Non-destructive methods to determine the freshness of packaged food products could play an important role in the reduction of this problem. In this study, we developed a chemical sensor, i.e. a sensor foil, capable of detecting amines as food spoilage indicators in reaction vessels and model packaging units by fluorescence spectroscopy. To obtain the foil, a phosphorylated porphyrin was adsorbed to silica and then extruded in polyethylene. The reactivity of the foil was tested with single amines in reaction vessels to demonstrate the behavior under ideal conditions. The sensor foil was applied in model packages containing salmon or cheese to show that fluorescence spectroscopy can be used to detect the emission of spoilage indicators. Lastly, model experiments with cod filets were carried out to obtain data to test the capability of determining the freshness by machine learning algorithms.
Optical coherence tomography (OCT) with axial and lateral resolution of 1 µm, termed microscopic OCT (mOCT), is suited for the investigation of dynamic processes on cellular level. An improvement in contrast to visualize cellular structures can be achieved by evaluating inherent signal fluctuation. This so-called dynamic contrast was recently demonstrated for the widely used scanning frequency domain OCT (FD-OCT). Here we show comparative measurements of dynamic microscopic OCT (dmOCT) and multiphoton autofluorescence imaging of ex-vivo trachea. Compared to multiphoton microscopy, the dmOCT provides greater penetration depth, can visualize structures that are not detectable by autofluorescence imaging and has no risk of photodamage.
A central challenge in the treatment of different diseases is the delivery of therapeutic agents to a specific cellular site. Liposomes that can release their cargo upon an externally controlled trigger are attractive candidates for localized drug release. Light as external trigger can be controlled temporal and spatial with high precision. In this study, we investigate the potential of light sensitive liposomes with four different photosensitizers for light-induced release. To demonstrate permeabilization of the liposomes, we encapsulated calcein in high concentration inside liposomes, that calcein fluorescence is quenched. If calcein is released from the liposome, quenching is diminished and the fluorescence increases. We demonstrated that liposomes with the sensitizers Benzoporphyrine derivative monoacid (BPD), chlorine e6 (Ce6), Al(III) Phthalocyanine chloride disulfonic acid (AlPcS2) and a di-hydroxyphenyl porphyrine (5,10-DiOH) release cargo effectively after irradiation. Liposomes with 5,10-DiOH showed a quicker release compared to the other sensitizers. Further we observed through fractionated irradiation, that most of the release took place during light irradiation, while the permeability of the liposome decreased shortly after light exposure.
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.
Light-induced inhibition of intracellular molecules holds great promise for a selective treatment of cancer and other diseases. Challenges for the targeting of intracellular proteins are the synthesis of effective photoimmuno-conjugates and their functional delivery inside living cells. In earlier studies we have shown, that photodynamic inactivation of the nuclear Ki-67 protein leads to an effective elimination of proliferating tumor cells. Here we show a selective treatment for EGFR and Ki-67 positive cancer cells after light-controlled delivery of indocyanine green (ICG) photo-immunoconjugates. The Ki-67 antibody TuBB-9, which recognizes an active state of the protein, was labeled with different ratios of ICG and encapsulated into immuno-liposomes that selectively deliver the conjugates to EGFR overexpressing cells. To overcome endosomal entrapment of the delivered agents, ovarian carcinoma cells were treated with the photosensitizer benzoporphyrin monoacid derivative (BPD) and irradiated first for endosomal escape of the TuBB-9-ICG constructs. 24 h after irradiation TuBB-9-ICG antibodies showed a relocalization from spots in the cytoplasm to the cell nucleus. A second irradiation of the delivered TuBB-9-ICG led to a significant elimination of cells after Ki-67 inactivation.
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.
Nanomedicine is beginning to impact the treatment of several diseases and current research
efforts include development of integrated nano-constructs (theranostics) which serve as probes
for imaging and therapy in addition to delivering macromolecules intracellularly. In cancer, there
is a vital unmet need for effective alternative treatments with high specificity and low systemic
toxicity. This can be achieved by targeting key molecular markers associated with cancer cells
with reduced effective drug doses. Here, we show an innovative proof-of-principle approach for
efficient killing of proliferating ovarian cancer cells by inactivating a protein associated with cell
proliferation namely, the nuclear Ki-67 protein (pKi-67), using nanotechnology-based
photodynamic therapy (PDT). Antibodies against pKi-67 are widely used as prognostic tools for
tumor diagnosis. In this work, anti pKi-67 antibodies were first conjugated to fluorescein
isothiocyanate (FITC) and then encapsulated inside liposomes. After incubation of OVCAR-5
ovarian cancer cells with these liposomes, confocal microscopy confirmed the localization of the
antibodies to the nucleoli of the cells. Irradiation with a 488 nm laser led to a significant loss of
cell viability. The specificity of this approach for pKi-67 positive cells was demonstrated in
confluent human lung fibroblasts (MRC-5) where only a small population of cells stain positive
for pKi-67 and only minimal cell death was observed. Taken together, our findings suggest that
pKi-67 targeted with nano-platform is an attractive therapeutic target in cancer therapy.
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.
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