In Vivo Assessment of Mitochondrial Oxygen Consumption.

The Protoporphyrin IX-Triplet State Lifetime Technique (PpIX-TSLT) has been proposed by us as a potential clinical noninvasive tool for monitoring mitochondrial function. We have been working on the development of mitochondrial respirometry for monitoring mitochondrial oxygen tension (mitoPO2) and mitochondrial oxygen consumption (mitoVO2) in skin. In this work, we describe the principles of the method in small experimental animals.

Broaden sources and reduce expenditure: Tumor-specific transformable oxidative stress nanoamplifier enabling economized photodynamic therapy for reinforced oxidation therapy.

Cancer cells immersed in inherent oxidative stress are more vulnerable to exogenous oxidative damages than normal cells. Reactive oxygen species (ROS)-mediated oxidation therapy preferentially aggravating tumor oxidative stress to disrupt redox homeostasis, has emerged as an effective and specific anticancer treatment. Herein, following an ingenious strategy of "broaden sources and reduce expenditure", we designed a versatile tumor-specific oxidative stress nanoamplifier enabling economized photodynamic therapy (PDT), to achieve synergistic oxidative stress explosion for superior oxidation therapy. Methods: Cinnamaldehyde (CA) as a therapeutic ROS generator was first conjugated to hyaluronic acid (HA) through acid-labile hydrazone bond to synthesize tailored amphiphilic HA@CA conjugates, which could surprisingly self-assemble into uniform nanofibers in aqueous media. Photosensitizer protoporphyrin (PpIX) was efficiently encapsulated into HA@CA nanofibers and transformed HA@CA nanofibers to final spherical HA@CAP. Results: With beneficial pH-responsiveness and morphology transformation, improved bioavailability and selective tumor accumulation, HA@CAP combining ROS-based dual chemo/photodynamic treatment modalities could induce cytotoxic ROS generation in a two-pronged approach to amplify tumor oxidative stress, termed "broaden sources". Moreover, utilizing CA-induced H2O2 production and cascaded Fenton reaction in mitochondria to consume intracellular overloaded Fe(II), HA@CAP could skillfully block endogenic heme biosynthesis pathway on site to restrain undesired elimination of PpIX for economized PDT, termed "reduce expenditure". Both in vitro and in vivo results demonstrated the superior antitumor performance of HA@CAP. Conclusion: This study offered an inspiring strategy of "broaden sources and reduce expenditure" to specifically boost tumor oxidative stress for reinforced oxidation therapy.

Optical Characterization of Neurosurgical Operating Microscopes: Quantitative Fluorescence and Assessment of PpIX Photobleaching.

Protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (5-ALA) is increasingly used as a fluorescent marker for fluorescence-guided resection of malignant gliomas. Understanding how the properties of the excitation light source and PpIX fluorescence interact with the surgical microscope is critical for effective use of the fluorescence-guided tumor resection technique. In this study, we performed a detailed assessment of the intensity of the emitted blue light and white light and the light beam profile of clinical grade operating microscopes used for PpIX visualization. These measurements revealed both recognized fluorescence photobleaching limitations and unrecognized limitations that may alter quantitative observations of PpIX fluorescence obtained with the operating microscope with potential impact on research and clinical uses. We also evaluated the optical properties of a photostable fluorescent standard with an excitation-emission profile similar to PpIX. In addition, we measured the time-dependent dynamics of 5-ALA-induced PpIX fluorescence in an animal glioma model. Finally, we developed a ratiometric method for quantification of the PpIX fluorescence that uses the photostable fluorescent standard to normalize PpIX fluorescence intensity. This method increases accuracy and allows reproducible and direct comparability of the measurements from multiple samples.

Measurements of the optical coefficients of the protoporphyrin IX endogenously producing yeast-based model in the visible and NIR.

Models mimicking the endogenous production of protoporphyrin IX (PpIX), as well as its fluorescence, are of high interest for applied and fundamental studies in the fields of cancer detection by fluorescence imaging, photodynamic therapy (PDT), and photobiomodulation (PBM). Here, we present and describe optical properties of the yeast-based models able to produce PpIX endogenously after the administration of 5-aminolevulinic acid (ALA) and/or 2,2'-bipyridyl. As their optical properties have an important impact on the spatial distribution of the fluence rate in these liquid models, their absorption and reduced scattering coefficients were determined to be between 400 and 808 nm for two yeast solutions previously described by our group. These coefficients were derived from measurements of the total reflectance and light penetration depth using a dedicated Monte Carlo simulation. We observed that absorption and scattering coefficients were smaller than those of soft tissues at all wavelengths. This work will enable the production of a low-cost optical phantom loaded with appropriate amounts of light-absorbing and -scattering particles to mimic tumors containing PpIX, offering a useful tool to optimize the spectral and radiometric design of certain cancer photodetection setups.

Tumor reactive ringlet oxygen approach for Monte Carlo modeling of photodynamic therapy dosimetry.

Photodynamic therapy (PDT) is an emergent technique used for the treatment of several diseases. It requires the interaction of three components: a photosensitizer, a light source and tissue oxygen. Knowledge of the biophysical aspects of PDT is important for improving dosimetry protocols and treatment planning. In this paper we propose a model to simulate the spatial and temporal distribution of ground state oxygen ((3)O2), cumulative singlet excited state oxygen ((1)O2)rx and photosensitizer, in this case protoporphyrin IX (PpIX) in an ALA mediated PDT treatment. The results are analyzed in order to improve the treatment dosimetry. We compute the light fluence in the tissue using Monte Carlo simulations running in a GPU system. The concentration of (3)O2, ((1)O2)rx and the photosensitizer are calculated using this light fluence and a set of differential equations describing the photochemical reactions involved in PDT. In the model the initial photosensitizer concentration depends on tissue depth and type, moreover we consider blood vessel damage and its effect in the ground state oxygen concentration in the tissue. We introduce the tumor reactive single oxygen (TRSO) as a new dosimetry metric. It represents the amount of singlet oxygen per tumor volume that reacts, during the treatment, with the molecules in the tumor. This quantity integrates the effect of the light irradiance, the optical properties of the tumor and the normal tissue, the oxygen consumption and supply, and the photosensitizer biodistribution on the skin.

Optical properties of tumor tissues grown on the chorioallantoic membrane of chicken eggs: tumor model to assay of tumor response to photodynamic therapy.

Herein, the optical adequacy of a tumor model prepared with tumor cells grown on the chorioallantoic membrane (CAM) of a chicken egg is evaluated as an alternative to the mouse tumor model to assess the optimal irradiation conditions in photodynamic therapy (PDT). The optical properties of CAM and mouse tumor tissues were measured with a double integrating sphere and the inverse Monte Carlo technique in the 350- to 1000-nm wavelength range. The hemoglobin and water absorption bands observed in the CAM tumor tissue (10 eggs and 10 tumors) are equal to that of the mouse tumor tissue (8 animals and 8 tumors). The optical intersubject variability of the CAM tumor tissues meets or exceeds that of the mouse tumor tissues, and the reduced scattering coefficient spectra of CAM tumor tissues can be equated with those of mouse tumor tissues. These results confirm that the CAM tumor model is a viable alternative to the mouse tumor model, especially for deriving optimal irradiation conditions in PDT.

Assessment of the action spectrum for photooxidation in full fat bovine milk.

The action spectrum for photooxidation in full fat bovine milk was measured. Samples of milk with air or argon in headspace were exposed to narrow wavelength bands of light in the range 400-700 nm. Photooxidation in terms of off-flavors was measured by a sensory panel, volatile compounds by headspace solid phase micro extraction (SPME-GC-MS), and photobleaching of photosensitizers in milk (riboflavin, protoporphyrin IX and a chlorophyllic compound) by front face fluorescence spectroscopy. The action spectrum deviated significantly from the absorption spectrum of milk. Significant oxidation was induced by wavelengths around 400 nm and 500-650 nm in milk with air in headspace. Argon in headspace gave significant oxidation also at 700 nm. It is suggested that protoporphyrin IX and chlorophyll are responsible for oxidation induced by wavelengths >500 nm, and that also riboflavin is contributing from 400 to 500 nm.

Oxygenation measurement by multi-wavelength oxygen-dependent phosphorescence and delayed fluorescence: catchment depth and application in intact heart.

Oxygen delivery and metabolism represent key factors for organ function in health and disease. We describe the optical key characteristics of a technique to comprehensively measure oxygen tension (PO(2)) in myocardium, using oxygen-dependent quenching of phosphorescence and delayed fluorescence of porphyrins, by means of Monte Carlo simulations and ex vivo experiments. Oxyphor G2 (microvascular PO(2)) was excited at 442 nm and 632 nm and protoporphyrin IX (mitochondrial PO(2)) at 510 nm. This resulted in catchment depths of 161 (86) µm, 350 (307) µm and 262 (255) µm respectively, as estimated by Monte Carlo simulations and ex vivo experiments (brackets). The feasibility to detect changes in oxygenation within separate anatomical compartments is demonstrated in rat heart in vivo. Schematic of ex vivo measurements.

Modelling fluorescence in clinical photodynamic therapy.

Understanding the interactions of non-ionizing radiation with living organisms has been the focus of much research over recent decades. The complex nature of these interactions warrants development of theoretical and experimental studies to gain an insight into predicting and monitoring the success of photodynamic therapy (PDT) protocols. There is a major impetus towards evidence-based recommendations for patient diagnosis, treatment and management. Knowledge of the biophysical aspects of PDT is important for improving dosimetry protocols. Fluorescence in clinical PDT may be used to detect and diagnose pre-malignant and malignant conditions, while photobleaching can monitor changes in fluorescence during treatment. Combining empirical fluorescence photobleaching clinical data with computational modelling enables clinical PDT dosimetry protocols to be investigated with a view to optimising treatment regimes. We will discuss how Monte Carlo radiation transfer (MCRT) modelling has been intercalated in the field of fluorescence detection and PDT. In this paper we highlight important aspects of basic research in PDT by reporting on the current utilisation of fluorescence in clinical PDT from both a clinical and theoretical perspective. Understanding and knowledge of light propagation in biological tissue from these perspectives should have a positive impact on treatment planning.

Factors implicated in the assessment of aminolevulinic acid-induced protoporphyrin IX fluorescence.

BACKGROUND: Photodynamic therapy and photodiagnosis of cancer requires preferential accumulation of fluorescent photosensitizers in tumors. Clinical evidence documents feasibility of ALA-based photodiagnosis for tumor detection. However, false positive results and large variations in fluorescence intensities are also reported. Furthermore, selective accumulation of fluorescent species of photosensitizers in tumor cell lines, as compared to normal ones, when cultured in vitro, is not always observed. To understand this discrepancy we analyzed the impact of various factors on the intensity of detected PpIX fluorescence. METHODS: Impacts of cell type, mitochondrial potential, cell-cell interactions and relocalization of PpIX among different cell types in co-cultures of different cell lines were analyzed by confocal microscopy and flow cytometry. Fluorescence spectroscopy was used to estimate absolute amounts of ALA-induced PpIX in individual cell lines. Immunofluorescence staining was applied to evaluate the ability of cell lines to produce collagen. RESULTS: Higher ALA-induced PpIX fluorescence in cancer cell lines as compared to normal ones was not detected by all the methods used. Mitochondrial activity was heterogeneous throughout the cell monolayers and could not be clearly correlated with PpIX fluorescence. Positive collagen staining was detected in all cell lines tested. CONCLUSIONS: Contrary to in vivo situation, ALA-induced PpIX production by cell lines in vitro may not result in higher PpIX fluorescence signals in tumor cells than in normal ones. We suggest that a combination of several properties of tumor tissue, instead of tumor cells only, is responsible for increased ALA-induced PpIX fluorescence in solid tumors. GENERAL SIGNIFICANCE: Understanding the reasons of increased ALA-induced PpIX fluorescence in tumors is necessary for reliable ALA-based photodiagnosis, which is used in various oncological fields.

Photodynamic effects on basal cell carcinoma with topical Photosensitizer.

Photodynamic therapy (PDT) is a potential cancer therapy used in several clinical fields. Its application in dermatology following a fixed protocol usually generates good results. However, some cases of basal cell carcinoma show tumour persistence. The poor response observed in this type of pathology, whose lesions penetrate in the deeper layers of the skin, could be attributed to an insufficient accumulation of the PS (Photosensitizer) in deeper tissues. The development of accurate models could propose the adequate treatment dosimetry for those problematic cases in order to maximize the efficiency of the PDT treatment outcome. In this work we present a PDT model that tries to predict the photodynamic effect on the skin affected by a basal cell carcinoma with a topically administered photosensitizer. The results obtained allow us to know the evolution of the cytotoxic agent in order to estimate the necrotic area adjusting parameters such as the optical power, the photosensitizer concentration, the incubation and exposition time or the diffusivity and permeability of the damaged tissue.

In situ assessment of porphyrin photosensitizers in Propionibacterium acnes.

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of delta-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 x 10(5) molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.