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.
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•Hypericin aggregates are formed by adsorption in nanoporous SBA-15 silica particles.•The serum protein/lipid corona formed on SBA-15 allows monomerization of hypericin.•The corona ...facilitates the redistribution of hypericin from SBA-15 into cells.•Hypericin with protein/lipid SBA-15 induces singlet oxygen and photodynamic therapy.
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.
Against the background of divergent political developments across Europe, farm animal welfare (FAW) science has evolved during the last three decades as an inter-disciplinary research area. Recent ...achievements include pan-European research projects and the implementation of animal welfare assessment systems on-farm. The aim of this study was mapping activities for FAW science and investigating geographical differences in FAW research in Europe (EU28 + candidate countries and the European Economic Area) with regard to available resources (e.g. human resources, infrastructure, funding) and research output (e.g. collaborations and publications). Further, we enquired if economic attributes such as the Coefficient of National Innovation Capacity (NIC) were associated with the reported available resources and research output factors (publications and collaborations) of FAW research. Based on questionnaires sent out to a wide researcher network in regions of an enlarged Europe, we found differences with regard to ‘input factors’ such as human resources, animal and laboratory facilities and national and international research funding and ‘output factors’ such as inter/national collaboration, participation in EU-funded projects related to FAW and number of publications. Respondents were allocated to 4 Western and 4 Eastern geographical clusters of countries (‘hubs’). There were a larger number of researchers, students and technical staff per laboratory in Western compared to Eastern hubs. A pronounced difference was found for funding, as 35% of respondents in the Eastern hubs stated that they lack funded FAW projects compared to 4% in the West. In general, respondents from the Western hubs stated significantly more often that they run projects in the field of FAW research (
p
= 0.034). Furthermore they were significantly more involved in EU-funded schemes, such as FP7 (EU’s Research and Innovation Funding Programme for 2007–2013) with 24% (
p
= 0.013) and in ERA-NET Cofund projects (European Research Area—Coordination of Research Programmes) with 5.7% (
p
= 0.042). The average sum of impact factors from 5 self-named citations was 3.0 ± 2.8 (mean, SD) in the Eastern hubs and 7.5 ± 4.4 in the Western Hubs. When investigating associations of the economic status of EU countries with resource factors and achievements in FAW research, the ‘Coefficient of National Innovation Capacity (NIC)’ was moderately correlated with the input factors for FAW research such as the average number of PhDs currently employed in the institutions (
r
s
= 0.66;
p
< 0.001) and the total number of employed researchers (
r
s
= 0.56;
p
< 0.01). Stronger associations were found between the scientific output and the economic ranking, here represented by the cumulative impact factor of their published papers (
r
s
= 0.74;
p
< 0.001), and between the number of EC-project reports published in CORDIS 2015 with NIC (
r
s
= 0.67;
p
< 0.001). We conclude that due to economic disadvantages as represented by the lower NIC or rare participation in EU-funding schemes, the Eastern Hubs could not reach the same level of output factors as the Western Hubs, which negatively impacts on the number of young researchers (PhDs) and impact factors, thus resulting in lower visibility and influence.
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.
•The copoly(2-oxazoline)s can be used as a transport system for hypericin.•They are biocompatile, non toxic and do not cause damage to the tissue.•They have a potential anti-angiogenic effect during ...photodynamic therapy.•Quail chorioallantoic membrane model well reflects the effects of the therapy.
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.
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.
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.
There has been increasing interest in fluorescence-based imaging techniques in clinical practice, with the aim to detect and visualize the tumour configuration and the border with healthy tissue. ...Strong photodynamic activity of hypericin (Hyp) can be improved by various molecular transport systems (e.g. LDL). Our aim was to examine pharmacokinetics of Hyp in the presence of LDL particles on ex ovo chorioallantoic membrane (CAM) of Japanese quail with implanted TE1 tumour spheroids (human squamocellular carcinoma). Spheroids were implanted on CAM surface on embryonal day 7 and after 24 hours formulations of free Hyp and Hyp:LDL 100:1 and 200:1 were topically applied. All experimental formulations in the fluorescent image very well visualized the tumour spheroid position, with gradual increase of fluorescence intensity in 6-h observation period. LDL transportation system exhibited clear superiority in fluorescence pharmacokinetics than free Hyp formulation by increasing tumour-normal difference. Our experimental results confirm that Hyp and Hyp:LDL complex is potent fluorophore for photodynamic diagnosis of squamocellular carcinoma.
Photosensitizing properties of hypericin are well known, and the chicken chorioallantoic membrane has previously been used to test photodynamic effects of hypericin and other substances. In our study ...the photodynamic effect of hypericin in the ex ovo quail chorioallantoic membrane model was evaluated. Steady-state and time-resolved fluorescence spectroscopy of hypericin solution in PEG-400 and its mixture in PBS was performed to assess and characterize the process of aggregation and disaggregation of hypericin during the drug formulation preparation. A therapeutical formulation (2 µg/g of embryo weight) was topically applied on CAM into the silicone ring. Hypericin was excited by diode laser with wavelength 405 nm, fluence rate 140 mW/cm2, and fluence 16.8 J/cm2. Hypericin in 100% PEG-400 exhibits typical fluorescence spectra with a maximum of about 600 nm, while hypericin 10% PEG-400 formulation exhibits almost no fluorescence. Time resolved spectra analysis showed fluorescence decay of hypericin in 100% PEG-400 solution with a mean lifetime of 5.1 ns and in 10% PEG 4.1 ns. Damage of quail chorioallantoic membrane vasculature after photodynamic therapy ranged from hemorrhage and vanishing of capillary vessels to thrombosis, lysis, and hemorrhage of larger vessels.The presented findings suggest that quail embryos can be used as a suitable model to test the effect of hypericin and other photodynamic compounds.
Light during incubation can influence embryonic and postembryonic development of chickens, but the underlying mechanisms are poorly understood. Previous studies have demonstrated that red and blue ...lights during incubation had opposite effects on the development of embryonic melatonin biosynthesis; red light results in the highest and blue light in the lowest amplitude of the daily rhythm. Therefore, in this study, we investigated if exposure to monochromatic red (632 nm) and blue (463 nm) light during incubation can differently influence growth, selected biochemical (glucose, cholesterol, triacylglycerols) and endocrine (corticosterone and thyroid hormones) traits and behavioural parameters during postembryonic development in broiler chickens. For analysis, we used 10 and 11 hatchlings incubated in red and blue light, respectively and 10 birds per each group (six males and four females) in 3-weeks-old broilers. During the rapid growth phase (days 18, 20 and 21 of age), higher body weight was recorded in broilers incubated under red compared to blue light, whereas endocrine and metabolic traits did not differ between the treatments. The improved growth rate was related to behavioural traits, mainly because chickens incubated in red light exhibited more passive (resting, standing, preening, dust bathing) and less active behaviours (walking, foraging, fighting, wing-flapping) than the blue-light incubated birds. The time spent for eating and drinking and the results of the tonic immobility test did not differ between both groups. Our results suggest that red and blue monochromatic light during incubation can differently program the postembryonic development of broilers, with possible consequences for their growth and welfare.
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