The potential effect of leptin co-administration on photodynamic damage using quail chorioallantoic membrane model.

BACKGROUND: The chorioallantoic membrane (CAM) of the Japanese quail is an excellent model for studying photodynamic therapy (PDT) due to its rich vascularization. PDT is used not only in oncological treatment but also in infectious diseases, or psoriasis, where it yields significant advantages. This treatment also has its limitations, such as burning, itching, erythema, redness, swelling, and delayed wound healing. The aim of this study was to analyse the potentially protective properties of the tissue hormone leptin during PDT. METHODS: Japanese quail embryos incubated ex ovo were used in this experiment. On the 9th day of embryonic development, leptin (5 µg) and photosensitiser hypericin (79 µM) were topically applied, followed by irradiation. The effect of leptin co-administration was evaluated from CAM images and histological structure analysis, histological samples, and qPCR, where the expression of genes involved in angiogenesis, apoptosis, and oxidative stress was monitored. RESULTS: We observed vascular damage in all experimental groups, the highest damage was found after the application of hypericin without leptin coadministration. Histological analysis confirmed the protective effect of leptin. qPCR analysis presented differences in FREK gene expression, but also in genes involved in oxidative stress like SOD, NRF-1, NRF-2, and GPX7. The application of leptin significantly reduced the expression of apoptosis regulatory proteins CASP3, cytochrome C, and APAF1. CONCLUSIONS: Our results in the CAM model suggest a possible protective effect of leptin to prevent PDT damage and aid in the subsequent regeneration of target tissues after antimicrobial PDT.

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.

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.

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.

Chorioallantoic Membrane Models of Various Avian Species: Differences and Applications.

The chorioallantoic membrane model (CAM) of an avian embryo is used as an experimental model in various fields of research, including angiogenesis research and drug testing, xenografting and cancer research, and other scientific and commercial disciplines in microbiology, biochemistry, cosmetics, etc. It is a low-cost, low-maintenance, and well-available in vivo animal model that is non-sentient and can be used as an alternative for other mammal experimental models. It respects the principles of the "3R" rule (Replacement, Reduction, and Refinement)-conditions set out for scientific community providing an essential framework for conducting a more human animal research, which is also in line with constantly raising public awareness of welfare and the ethics related to the use of animal experimental models. In this review, we describe the chorioallantoic membrane of an avian embryo, focusing on its properties and development, its advantages and disadvantages as an experimental model, and the possibilities of its application in various fields of biological research. Since the most common chicken CAM model is already well known and described in many publications, we are particularly focusing on the advantages and application of less known avian species that are used for the CAM model-quail, turkey, and duck.