Singlet oxygen lifetime changes in dying glioblastoma cells.

Time-resolved phosphorescence detection was employed to determine the lifetime of singlet oxygen in live cells. Using hypericin as a photosensitizer, singlet oxygen was generated in U87MG glioblastoma cells. The phosphorescence of singlet oxygen was detected in aqueous cell suspensions following pulsed laser excitation. Our goal was to eliminate or reduce the problems associated with lifetime measurements in water-based cell suspensions. The apparatus enabled simultaneous singlet oxygen phosphorescence and transient absorption measurements, reducing uncertainty in lifetime estimation. The changes in singlet oxygen lifetime were observed during early and late apoptosis induced by photodynamic action. Our findings show that the effective lifetime of singlet oxygen in the intracellular space of the studied glioblastoma cells is 0.4 µs and increases to 1.5 µs as apoptosis progresses. Another group of hypericin, presumably located in the membrane blebs and the plasma membrane of apoptotic cells, generates singlet oxygen with a lifetime of 1.9 µs.

Nanocomposite based on Gd2O3 nanoparticles and drug 5-fluorouracil as potential theranostic nano-cargo system.

We have prepared silica matrix with hexagonal symmetry of pores (SBA-15) and loaded it with anticancer drug 5-Fluorouracil (5-FU) to promote it as a drug delivery system. Gd2O3 nanoparticles were incorporated into the matrix to enhance nanosystems applicability as contrast agent for MRI, thus enabled this nanocomposite to be used as multifunctional nano-based therapeutic agent. Drug release profile was obtained by UV-VIS spectroscopy, and it indicates the prolongated release of 5-FU during the first hours and the total release after 5 h. The cytotoxicity tests using MTT-assay, fluorescent microscopy, bright-field microscopy, and flow cytometry were carried out using human glioma U87 MG cells and SK BR 3 cells. The nanocomposite with anticancer drug (Gd2O3/SBA-15/5FU) showed toxic behaviour towards studied cells, unlike nanocomposite without drug (Gd2O3/SBA-15) that was non-toxic. Our drug delivery system was designed to minimalize negative effect of Gd3+ ions at magnetic resonance imaging and drug 5-FU on healthy cells due to their encapsulation into biocompatible silica matrix, so the Gd3+ ions are more stable (in comparison to chelates), lower therapeutic dose of 5-FU is needed and its prolongated release from silica pores was confirmed. Very good T1 contrast in MR images was observed even at low concentrations, thus this nanosystem can be potentially used as contrast imaging agent.

Imaging of heterogeneity in 3D spheroids of U87MG glioblastoma cells and its implications for photodynamic therapy.

BACKGROUND: In recent years, pharmacology and toxicology have emphasised the intention to move from in vivo models to simplified 3D objects represented by spheroidal models of cancer. Mitochondria are one of the subcellular organelles responsible for cell metabolism and are often a lucrative target for cancer treatment including photodynamic therapy (PDT). METHODS: Hanging droplet-grown glioblastoma cells were forced to form spheroids with heterogeneous environments that were characterised by fluorescence microscopy and flow cytometry using fluorescent probes sensitive to oxidative stress and apoptosis. PDT was induced with hypericin at 590 nm. RESULTS: It was found that the metabolic activity of the cells in the periphery and core of the spheroid was different. Higher oxidative stress and induction of caspase-3 were observed in the peripheral layers after PDT. These parts were more destabilised and showed higher expression of LC3B, an autophagic marker. However, the response of the whole system to the treatment was controlled by the cells in the core of the spheroids, which were hardly affected by the treatment. It has been shown that the depth of penetration of hypericin into this system is an important limiting step for PDT and the induction of autophagy and apoptosis. CONCLUSIONS: In this work, we have described the fluorescence imaging of vital mitochondria, caspase-3 production and immunostaining of autophagic LC3B in cells from glioblastoma spheroids before and after PDT. Overall, we can conclude that this model represents an in vitro and in vivo applicable alternative for the study of PDT in solid microtumours.

Effective transport of aggregated hypericin encapsulated in SBA-15 nanoporous silica particles for photodynamic therapy of cancer cells.

Photodynamic therapy (PDT) represents an interesting modality for the elimination of damaged biomaterials and cells. This treatment takes advantage of the photosensitizing properties of molecules that are active only when irradiated with light. In the present work, a dual property of hypericin, a hydrophobic molecule with high performance in photodiagnostics and photodynamic therapy, was exploited. The non-fluorescent and photodynamically inactive form of hypericin aggregates was loaded into the nanopores of SBA-15 silica particles. The synthesized particles were characterized by infrared spectroscopy, thermogravimetry, differential thermal analysis, small-angle X-ray scattering and transmission electron microscopy. Hypericin aggregates were confirmed by absorption spectra typical of aggregated hypericin and by its short fluorescence lifetime. Release of hypericin from the particles was observed toward serum proteins, mimicking physiological conditions. Temperature- and time-dependent uptake of hypericin by cancer cells showed gradual release of hypericin from the particles and active cellular transport by endocytosis. A closer examination of SBA-15-hypericin uptake by fluorescence lifetime imaging showed that aggregated hypericin molecules, characterized by a short fluorescence lifetime (∼4 ns), were still present in the SBA-15 particles upon uptake by cells. However, monomerization of hypericin in cancer cells was observed by extending the hypericin fluorescence lifetime by ∼8 ns, preferentially in lipid compartments and the plasma membrane. This suggests a promising prognosis for delayed biological activity of the entire cargo, which was confirmed by effective PDT in vitro. In summary, this work presents an approach for safe, inactive delivery of hypericin that is activated at the target site in cells and tissues.

Redistribution of hydrophobic hypericin from nanoporous particles of SBA-15 silica in vitro, in cells and in vivo.

Nanoporous silica is nowadays used in various fields of nano- and micro-materials research. The advantage of nanoporous material is that it can be filled with various hydrophilic and hydrophobic molecules, which are then delivered to the target cells and tissues. In the present study, we have studied the interaction of nanoporous silica with hydrophobic and photodynamically active molecule - hypericin. Hypericin was adsorbed on/in SBA-15 silica, which led to the disappearance of its fluorescence due to hypericin aggregate formation. However, it was observed here that hypericin can be easily redistributed from these particles towards proteins and lipids in serum and cells in vitro and in vivo. Moreover, the charged surface character of SBA-15 pores forced the creation of protein/lipid corona on particles. Such complex enabled monomerization of hypericin on the surface of particles presented by fluorescence in the corona and singlet oxygen production suitable for photodynamic therapy (PDT). The PDT efficacy achieved by introducing the new construct into the PDT protocol was comparable to the efficacy of hypericin PDT. In conclusion, this study demonstrates a promising approach for the delivery of hydrophobic photosensitizers to cancer cells by nanoporous silica using fluorescence techniques.

Biocompatible zeolite-dye composites with anti-amyloidogenic properties.

One of the most attractive approaches in biomedicine and pharmacy is the application of multifunctional materials. The mesoporous structure of clinoptilolite (CZ) absorbs various types of substances and can be used as a model for studying the carriers for targeted drug delivery with controlled release. CZ-dye composites are fabricated by incorporation into clinoptilolite pores commonly used dyes, aluminum phthalocyanine, zinc porphine, and hypericin. We examined and compared the effect of pure dyes and CZ-dye composites on insulin amyloidogenesis. The formation of insulin amyloid fibrils and the disassembly of preformed fibrils is significantly affected by any of the three compounds, however, the strongest effect is observed for aluminum phthalocyanine indicating a structurally-dependent anti-amyloidogenic activity of the dyes. The incorporation of dyes into CZ particles resulted in enhanced anti-amyloidogenic activity in comparison to pure CZ particles. The cell metabolic activity, biocompatibility and fluorescence biodistribution of the dyes entrapped in the composites were tested in vitro (U87 MG cells) and in vivo in the quail chorioallantoic membrane model. Considering the photoactive properties of the dyes used, we assume their applicability in photodiagnostics and photodynamic therapy. It can also be expected that their anti-amyloidogenic potential can be enhanced by photodynamic effect.

Effect of Photobiomodulation on Protein Kinase Cδ, Cytochrome C, and Mitochondria in U87 MG Cells.

Photobiomodulation (PBM) therapy is a relatively new modality for the combined treatment of cancer. Pre-treatment of certain types of cancer cells with PBM potentiates the treatment efficacy of photodynamic therapy (PDT). The mechanism of action of this synergetic effect is not yet fully understood. In the present study, we focused on protein kinase Cδ (PKCδ) as a proapoptotic agent that is highly expressed in U87MG cells. The distribution of PKCδ in the cytoplasm was changed and its concentration was increased by PBM using radiation at 808 nm (15 mW/cm2, 120 s). This process was accompanied by the organelle specific phosphorylation of PKCδ amino acids (serine/tyrosine). Enhanced phosphorylation of serine 645 in the catalytic domain of PKCδ was found in the cytoplasm, whereas the phosphorylation of tyrosine 311 was mainly localized in the mitochondria. Despite a local increase in the level of oxidative stress, only a small amount of cytochrome c was released from the mitochondria to cytosol. Although a partial inhibition of mitochondrial metabolic activity was induced in PBM-exposed cells, apoptosis was not observed. We hypothesized that PBM-induced photodamage of organelles was neutralized by autophagy maintained in these cells. However, photodynamic therapy may effectively exploit this behaviour to generate apoptosis in cancer treatment, which may increase the treatment efficacy and open up prospects for further applications.

The Remodulation of Actin Bundles during the Stimulation of Mitochondria in Adult Human Fibroblasts in Response to Light.

ß-actin belongs to cytoskeletal structures that change dynamically in cells according to various stimuli. Human skin can be considered as an organ that is very frequently exposed to various stress factors, of which light plays an important role. The present study focuses on adult human fibroblasts exposed to two types of light stress. Orange light with a wavelength of 590 nm was used here to stimulate the photosensitizer localized in the cells as a residual dose of photodynamic therapy (PDT). On the other hand, near-infrared light with a wavelength of 808 nm was considered for photobiomodulation (PBM), which is often used in healing processes. Confocal fluorescence microscopy was used to observe changes in intercellular communication, mitochondrial structures, and cytoskeletal dynamics defined by the remodulation of ß-actin of fibroblasts. The number of ß-actin bundles forming spherical structures was detected after light exposure. These structures as ß-actin oligomers were confirmed with super-resolution microscopy. While PDT led to the disintegration of actin oligomers, PBM increased their number. The interaction of ß-actin with mitochondria was observed. The combination of PDT and PBM treatments is important to minimize the side effects of cancer treatment with PDT on healthy cells, as shown by the cell metabolism assay in this work. In this work, ß-actin is presented as an important parameter that changes and is involved in the response of cells to PDT and PBM.

Influence of Hydrophobic Side-Chain Length in Amphiphilic Gradient Copoly(2-oxazoline)s on the Therapeutics Loading, Stability, Cellular Uptake and Pharmacokinetics of Nano-Formulation with Curcumin.

Due to the simple one-step preparation method and a promising application in biomedical research, amphiphilic gradient copoly(2-oxazoline)s are gaining more and more interest compared to their analogous block copolymers. In this work, the curcumin solubilization ability was tested for a series of amphiphilic gradient copoly(2-oxazoline)s with different lengths of hydrophobic side-chains, consisting of 2-ethyl-2-oxazoline as a hydrophilic monomer and 2-(4-alkyloxyphenyl)-2-oxazoline as a hydrophobic monomer. It is shown that the length of the hydrophobic side-chain in the copolymers plays a crucial role in the loading of curcumin onto the self-assembled nanoparticles. The kinetic stability of self-assembled nanoparticles studied using FRET shows a link between their integrity and cellular uptake in human glioblastoma cells. The present study demonstrates how minor changes in the molecular structure of gradient copoly(2-oxazoline)s can lead to significant differences in the loading, stability, cytotoxicity, cellular uptake, and pharmacokinetics of nano-formulations containing curcumin. The obtained results on the behavior of the complex of gradient copoly(2-oxazoline)s and curcumin may contribute to the development of effective next-generation polymeric nanostructures for biomedical applications.

Spheroidal Model of SKBR3 and U87MG Cancer Cells for Live Imaging of Caspase-3 during Apoptosis Induced by Singlet Oxygen in Photodynamic Therapy.

Aspects related to the response of cells to photodynamic therapy (PDT) have been well studied in cell cultures, which often grow in monolayers. In this work, we propose a spheroidal model of U87MG and SKBR3 cells designed to mimic superficial tumor tissue, small spheroids (<500 µm) suitable for confocal fluorescence microscopy, and larger spheroids (>500 µm) that can be xenografted onto quail chorioallantoic membrane (CAM) to study the effects of PDT in real time. Hypericin was used as a model molecule for a hydrophobic photosensitizer that can produce singlet oxygen (1O2). 1O2 production by hypericin was detected in SKBR3 and U87MG spheroid models using a label-free technique. Vital fluorescence microscopy and flow cytometry revealed the heterogeneity of caspase-3 distribution in the cells of the spheroids. The levels of caspase-3 and apoptosis increased in the cells of spheroids 24 h after PDT. Lactate dehydrogenase activity was evaluated in the spheroids as the most reliable assay to detect differences in phototoxicity. Finally, we demonstrated the applicability of U87MG spheroids on CAM in photodiagnostics. Overall, the variability and applicability of the prepared spheroid models were demonstrated in the PDT study.

Photobiomodulation and photodynamic therapy-induced switching of autophagy and apoptosis in human dermal fibroblasts.

Nowadays, photobiomodulation (PBM) in combination with chemotherapy or other therapeutic approaches is an attractive adjuvant modality for cancer treatment. Targeted destruction of cancer cells is one of the main advantages of photodynamic therapy (PDT). We have shown in previous studies that the combination of PBM at 808 nm and hypericin-mediated PDT increases PDT efficacy in human glioblastoma cells U87 MG. The study presented here shows significant differences between U87 MG and non-cancerous human dermal fibroblasts (HDF) cells treated by PBM and PDT. This study focuses on mitochondria because PBM mainly affects these organelles. We demonstrated that an interplay between mitochondrial and autophagic proteins plays a crucial role in the response of HDF cells to PBM and PDT. Fluorescence microscopy, flow cytometry, and Western blot analysis were used to examine the autophagic profile of HDF cells after these treatments. An increase in ubiquitin, SQSTM1, LC3BII, and cytochrome c was accompanied by a decrease in M6PR, ATG16L1, and Opa1 in HDF cells exposed to PBM and PDT. Overall, we observed that the switching of autophagy and apoptosis is dose-dependent and also occurs independently of PBM in HDF cells after hypericin-mediated PDT. However, PBM might preferentially induce autophagy in noncancer cells, which might escape apoptosis under certain conditions.

The Japanese quail chorioallantoic membrane as a model to study an amphiphilic gradient copoly(2-oxazoline)s- based drug delivery system for photodynamic diagnosis and therapy research.

Amphiphilic gradient copoly(2-oxazoline)s are widely researched in the field of drug delivery. They could be used as a transport system for hydrophobic drugs such as hypericin (HYP). We prepared six gradient copolymers (EtOx)-grad-(ROPhOx) by living cationic ring-opening polymerization of a hydrophilic comonomer 2-ethyl-2-oxazoline (EtOx) and a hydrophobic comonomer 2-(4-alkyloxyphenyl)-2-oxazoline (ROPhOx), with different composition ratio (88:12 and 85:15) and three different alkyl chain lengths of alkyl (R) substituents. As an experimental model, Japanese quail chorioallantoic membrane (CAM) was used. The effect of nanoparticles loaded with HYP was evaluated by the changes of fluorescence intensity during photodynamic diagnosis (PDD) monitored under 405 nm LED light before administration, and 0,1,3 and 24 h after topical administration. The effectiveness of photodynamic therapy (PDT) (405 nm, 285 mW/cm2) applied 1h after the administration of HYP-loaded nanoparticles was evaluated using vascular damage score and histological sections. Molecular analysis was done by measuring angiogenesis-related gene expression by qPCR. The application of nanoparticles unloaded or loaded with HYP proved to be biocompatible, non-toxic, and undamaging to the CAM tissue, while they successfully altered the HYP fluorescence. We observed a possible anti-angiogenic potential of prepared nanoparticles, which could present an advantage for PDT used for tumour treatment.

Dual-Functional Antioxidant and Antiamyloid Cerium Oxide Nanoparticles Fabricated by Controlled Synthesis in Water-Alcohol Solutions.

Oxidative stress is known to be associated with a number of degenerative diseases. A better knowledge of the interplay between oxidative stress and amyloidogenesis is crucial for the understanding of both, aging and age-related neurodegenerative diseases. Cerium dioxide nanoparticles (CeO2 NPs, nanoceria) due to their remarkable properties are perspective nanomaterials in the study of the processes accompanying oxidative-stress-related diseases, including amyloid-related pathologies. In the present work, we analyze the effects of CeO2 NPs of different sizes and Ce4+/Ce3+ ratios on the fibrillogenesis of insulin, SOD-like enzymatic activity, oxidative stress, biocompatibility, and cell metabolic activity. CeO2 NPs (marked as Ce1-Ce5) with controlled physical-chemical parameters, such as different sizes and various Ce4+/Ce3+ ratios, are synthesized by precipitation in water-alcohol solutions. All synthesized NPs are monodispersed and exhibit good stability in aqueous suspensions. ThT and ANS fluorescence assays and AFM are applied to monitor the insulin amyloid aggregation and antiamyloid aggregation activity of CeO2 NPs. The analyzed Ce1-Ce5 nanoparticles strongly inhibit the formation of insulin amyloid aggregates in vitro. The bioactivity is analyzed using SOD and MTT assays, Western blot, fluorescence microscopy, and flow cytometry. The antioxidative effects and bioactivity of nanoparticles are size- or valence-dependent. CeO2 NPs show great potential benefits for studying the interplay between oxidative stress and amyloid-related diseases, and can be used for verification of the role of oxidative stress in amyloid-related diseases.

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

Autophagy and Apoptosis Induced in U87 MG Glioblastoma Cells by Hypericin-Mediated Photodynamic Therapy Can Be Photobiomodulated with 808 nm Light.

Glioblastoma is one of the most aggressive types of tumors. Although few treatment options are currently available, new modalities are needed to improve prognosis. In this context, photodynamic therapy (PDT) is a promising adjuvant treatment modality. In the present work, hypericin-mediated PDT (hypericin-PDT, 2 J/cm2) of U87 MG cells is combined with (2 min, 15 mW/cm2 at 808 nm) photobiomodulation (PBM). We observed that PBM stimulates autophagy, which, in combination with PDT, increases the treatment efficacy and leads to apoptosis. Confocal fluorescence microscopy, cytotoxicity assays and Western blot were used to monitor apoptotic and autophagic processes in these cells. Destabilization of lysosomes, mitochondria and the Golgi apparatus led to an increase in lactate dehydrogenase activity, oxidative stress levels, LC3-II, and caspase-3, as well as a decrease of the PKCα and STAT3 protein levels in response to hypericin-PDT subcellular concentration in U87 MG cells. Our results indicate that therapeutic hypericin concentrations can be reduced when PDT is combined with PBM. This will likely allow to reduce the damage induced in surrounding healthy tissues when PBM-hypericin-PDT is used for in vivo tumor treatments.

Alkyl Chain Length in Poly(2-oxazoline)-Based Amphiphilic Gradient Copolymers Regulates the Delivery of Hydrophobic Molecules: A Case of the Biodistribution and the Photodynamic Activity of the Photosensitizer Hypericin.

Self-assembled nanostructures of amphiphilic gradient copoly(2-oxazoline)s have recently attracted attention as promising delivery systems for the effective delivery of hydrophobic anticancer drugs. In this study, we have investigated the effects of increasing hydrophobic side chain length on the self-assembly of gradient copolymers composed of 2-ethyl-2-oxazoline as the hydrophilic comonomer and various 2-(4-alkyloxyphenyl)-2-oxazolines as hydrophobic comonomers. We show that the size of the formed polymeric nanoparticles depends on the structure of the copolymers. Moreover, the stability and properties of the polymeric assembly can be affected by the loading of hypericin, a promising compound for photodiagnostics and photodynamic therapy (PDT). We have found the limitation that allows rapid or late release of hypericin from polymeric nanoparticles. The nanoparticles entering the cells by endocytosis decreased the hypericin-induced PDT, and the contribution of the passive process (diffusion) increased the probability of a stronger photoeffect. A study of fluorescence pharmacokinetics and biodistribution revealed differences in the release of hypericin from nanoparticles toward the quail chorioallantoic membrane, a preclinical model for in vivo studies, depending on the composition of polymeric nanoparticles. Photodamage induced by PDT in vivo well correlated with the in vitro results. All formulations studied succeeded in targeting hypericin at cancer cells. In conclusion, we demonstrated the promising potential of poly(2-oxazoline)-based gradient copolymers for effective drug delivery and sequential drug release needed for successful photodiagnostics and PDT in cancer therapy.

In vitro cellular activity of maghemite/cerium oxide magnetic nanoparticles with antioxidant properties.

Magnetic γ-Fe2O3/CeO2 nanoparticles were obtained by precipitation of Ce(NO3)3 with ammonia in the presence of γ-Fe2O3 seeds. The formation of CeO2 nanoparticles on the seeds was confirmed by transmission electron microscopy linked with selected area electron diffraction, energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy, and dynamic light scattering. The γ-Fe2O3/CeO2 particle surface was functionalized with PEG-neridronate to improve the colloidal stability in PBS and biocompatibility. Chemical and in vitro biological assays proved that the nanoparticles, due to the presence of cerium oxide, effectively scavenged radicals, thus decreasing oxidative stress in the model cell line. PEG functionalization of the nanoparticles diminished their in vitro aggregation and facilitated lysosomal cargo degradation in cancer cells during autophagy, which resulted in concentration-dependent cytotoxicity of the nanoparticles. Finally, the iron oxide core allowed easy magnetic separation of the particles from liquid media and may enable monitoring of nanoparticle biodistribution in organisms using magnetic resonance imaging.

Magnetic Characterization and Moderate Cytotoxicity of Magnetic Mesoporous Silica Nanocomposite for Drug Delivery of Naproxen.

In this study, we describe the magnetic and structural properties and cytotoxicity of drug delivery composite (DDC) consisting of hexagonally ordered mesoporous silica, iron oxide magnetic nanoparticles (Fe2O3), and the drug naproxen (Napro). The nonsteroidal anti-inflammatory drug (NSAID) naproxen was adsorbed into the pores of MCM-41 silica after the ultra-small superparamagnetic iron oxide nanoparticles (USPIONs) encapsulation. Our results confirm the suppression of the Brownian relaxation process caused by a "gripping effect" since the rotation of the whole particle encapsulated in the porous system of mesoporous silica was disabled. This behavior was observed for the first time, to the best of our knowledge. Therefore, the dominant relaxation mechanism in powder and liquid form is the Néel process when the rotation of the nanoparticle's magnetic moment is responsible for the relaxation. The in vitro cytotoxicity tests were performed using human glioma U87 MG cells, and the moderate manifestation of cell death, although at high concentrations of studied systems, was observed with fluorescent labeling by AnnexinV/FITC. All our results indicate that the as-prepared MCM-41/Napro/Fe2O3 composite has a potential application as a drug nanocarrier for magnetic-targeted drug delivery.

Influence of Oxidative Stress on Time-Resolved Oxygen Detection by [Ru(Phen)3]2+ In Vivo and In Vitro.

Detection of tissue and cell oxygenation is of high importance in fundamental biological and in many medical applications, particularly for monitoring dysfunction in the early stages of cancer. Measurements of the luminescence lifetimes of molecular probes offer a very promising and non-invasive approach to estimate tissue and cell oxygenation in vivo and in vitro. We optimized the evaluation of oxygen detection in vivo by [Ru(Phen)3]2+ in the chicken embryo chorioallantoic membrane model. Its luminescence lifetimes measured in the CAM were analyzed through hierarchical clustering. The detection of the tissue oxygenation at the oxidative stress conditions is still challenging. We applied simultaneous time-resolved recording of the mitochondrial probe MitoTrackerTM OrangeCMTMRos fluorescence and [Ru(Phen)3]2+ phosphorescence imaging in the intact cell without affecting the sensitivities of these molecular probes. [Ru(Phen)3]2+ was demonstrated to be suitable for in vitro detection of oxygen under various stress factors that mimic oxidative stress: other molecular sensors, H2O2, and curcumin-mediated photodynamic therapy in glioma cancer cells. Low phototoxicities of the molecular probes were finally observed. Our study offers a high potential for the application and generalization of tissue oxygenation as an innovative approach based on the similarities between interdependent biological influences. It is particularly suitable for therapeutic approaches targeting metabolic alterations as well as oxygen, glucose, or lipid deprivation.