Photodynamic activity of Temoporfin nanoparticles induces a shift to the M1-like phenotype in M2-polarized macrophages.

The monocyte/macrophage cell lineage reveals an enormous plasticity, which is required for tissue homeostasis, but is also undermined in various disease states, leading to a functional involvement of macrophages in major human diseases such as atherosclerosis and cancer. We recently generated in vivo evidence that crystalline, nonfluorescent nanoparticles of the hydrophobic porphyrin-related photosensitizer Aluminum phthalocyanine are selectively dissolved and thus may be used for specific fluorescent labelling of rejected, but not of accepted xenotransplants. This led us to hypothesize that nanoparticles made of planar photosensitizers such as porphyrins and chlorins were preferentially taken up and dissolved by macrophages, which was verified by in vitro studies. Here, using an in vitro system for macrophage differentiation/polarization of the human monocyte THP-1 cell line, we demonstrate differential uptake/dissolution of Temoporfin-derived nanoparticles in polarized macrophages, which resulted in differential photosensitivity. More importantly, low dose photodynamic sensitization using Temoporfin nanoparticles can be used to trigger M1 re-polarization of THP-1 cells previously polarized to the M2 state. Thus, sublethal photodynamic treatment using Temoporfin nanoparticles might be applied to induce a phenotypic shift of tumor-associated macrophages for the correction of an immunosuppressive microenvironment in the treatment of cancer, which may synergize with immune checkpoint inhibition.

Chlorin Nanoparticles for Tissue Diagnostics and Photodynamic Therapy.

BACKGROUND: Organic crystalline nanoparticles (NPs) are not fluorescent due to the crystalline structure of the flat molecules organized in layers. In earlier experiments with Aluminum Phthalocyanine (AlPc)-derived NPs, the preferential uptake and dissolution by macrophages was demonstrated [3]. Therefore, inflamed tissue or cancer tissue with accumulated macrophages may exhibit specific fluorescence in contrast to healthy tissue which does not fluoresce. The present study addresses the photobiological effects of NP generated from Temoporfin (mTHPC), a clinically utilized photosensitizer belonging to the chlorin family. METHODS: In-vitro investigations addressing uptake, dissolution and phototoxicity of mTHPC NP vs. the liposomal mTHPC formulation Foslip were performed using J774A.1 macrophages and L929 fibroblasts. For total NP uptake analysis, the cells were lysed, the nanoparticles dissolved and the fluorescence quantified. The intracellular molecular dissolution was measured by flow cytometry. Fluorescence microscopy served for controlling intracellular localization of the dissolved fluorescing molecules. Reaction mechanisms after PDT (mitochondrial activity, apoptosis) were analyzed using fluorescent markers in cell-based assays and flow cytometry. RESULTS: Organic crystalline NP of different size were produced from mTHPC raw material. NP were internalized more efficiently in J774A.1 macrophages when compared to L929 fibroblasts, whereas uptake and fluorescence of Foslip was similar between the cell lines. NP dissolution correlated with internalization levels for larger particles in the range of 200-500 nm. Smaller particles (45 nm in diameter) were taken up at high levels in macrophages, but were not dissolved efficiently, resulting in comparatively low intracellular fluorescence. Whereas Foslip was predominantly localized in membranes, NP-mediated fluorescence also co-localized with acidic vesicles, suggesting endocytosis/phagocytosis as a major uptake mechanism. In macrophages, phototoxicity of NPs was stronger than in fibroblasts, even exceeding Foslip when administered in identical amounts. In both cell lines, phototoxicity correlated with mitochondrial depolarization and enhanced activation of caspase 3. CONCLUSIONS: Due to their preferential uptake/dissolution in macrophages, mTHPC NP may have potential for the diagnosis and photodynamic treatment of macrophage-associated disorders such as inflammation and cancer.

Broadband Optical Properties of Milk.

Dairy products play an important role in our daily nutrition. As a turbid scattering medium with different kinds of particles and droplets, each alteration of these components changes the scattering properties of milk. The goal of this work is the determination of the amount of main scattering components, the fat droplets and the casein micelles, by understanding the light propagation in homogenized milk and in raw milk. To provide the absolute impact of these milk components, the geometrical and optical properties such as the size distribution and the refractive index (RI) of the components have to be examined. We determined the reduced scattering coefficient [Formula: see text] and the absorption coefficient [Formula: see text] from integrating sphere measurements. By use of a collimated transmission setup, the scattering coefficient [Formula: see text] was measured. Size measurements were performed to validate the influence of the fat droplet size on the results of the scattering properties; also, the RI of both components was determined by the said coefficients. These results were used to determine the absolute impact of the milk components on the scattering behavior. By fitting Mie theory calculations on scattering spectra [Formula: see text] and [Formula: see text] from different raw milk samples, it was possible to get reliable values for the concentrations of fat and casein and for the size of the fat droplets. By destroying the casein micelles, it was possible to separate the influence of the different scattering components on scattering behavior.

Evaluation of photodynamic treatment using aluminum phthalocyanine tetrasulfonate chloride as a photosensitizer: new approach.

Photodynamic therapy (PDT) has been the subject of several clinical studies. Evidence to date suggests that direct cell death may involve apoptosis. T(24) cells (bladder cancer cells, ATCC-Nr. HTB-4) were subjected to PDT with aluminum phthalocyanine tetrasulfonate chloride (AlS(4)Pc-Cl) and red laser light at 670 nm. Morphological changes after PDT were visualized under confocal microscopy. Raman microspectroscopy is considered as one of the newly established methods used for the detection of cytochrome c as an apoptotic marker. Results showed that PDT treated T(24) cells seem to undergo apoptosis after irradiation with 3 J cm(-2). Cytochrome c could not be detected from cells incubated with AlS(4)Pc-Cl using Raman spectroscopy whereas AlS(4)Pc-Cl seems to interfere with the Raman spectrum of cytochrome c.