Mucoadhesive polymers: Design of S-protected thiolated cyclodextrin-based hydrogels.

AIM: This study aims to design chemically crosslinked thiolated cyclodextrin-based hydrogels and to evaluate their mucoadhesive properties via mucosal residence time studies on porcine small intestinal mucosa and on porcine buccal mucosa. METHODS: Free thiol groups of heptakis(6-deoxy-6-thio)-ß-cyclodextrin (ß-CD-SH) were S-protected with 2-mercaptoethanesulfonic acid (MESNA) followed by crosslinking with citric acid. Cytotoxicity was assessed by hemolysis as well as resazurin assay. Hydrogels were characterized by their rheological and mucoadhesive properties. Ritonavir was employed as model drug for in vitro release studies from these hydrogels. RESULTS: The structure of S-protected ß-CD-SH was confirmed by IR and 1H NMR spectroscopy. Degree of thiolation was 390 ± 7 µmol/g. Hydrogels based on native ß-CD showed hemolysis of 12.5 ± 2.5 % and 13.6 ± 2.7 % within 1 and 3 h, whereas hemolysis of just 3.5 ± 2.8 % and 3.9 ± 3.0 % was observed for the S-protected thiolated CD hydrogels, respectively. Both native and S-protected thiolated hydrogels showed minor cytotoxicity on Caco-2 cells. Rheological investigations of S-protected thiolated ß-CD-based hydrogel (16.2 % m/v) showed an up to 13-fold increase in viscosity in contrast to the corresponding native ß-CD-based hydrogel. Mucosal residence time studies showed that thiolated ß-CD-based hydrogel is removed to a 16.6- and 2.4-fold lower extent from porcine small intestinal mucosa and porcine buccal mucosa in comparision to the native ß-CD-based hydrogel, respectively. Furthermore, a sustained release of ritonavir from S-protected thiolated ß-CD-based hydrogels was observed. CONCLUSION: Because of their comparatively high mucoadhesive and release-controlling properties, S-protected thiolated ß-CD-based hydrogels might be promising systems for mucosal drug delivery.

Self-Emulsifying Drug Delivery Systems (SEDDS) Containing Reverse Micelles: Advanced Oral Formulations for Therapeutic Peptides.

Alternative methods to hydrophobic ion pairing for the formation of lipophilic complexes of peptide drugs to incorporate them in lipid-based nanocarriers such as self-emulsifying drug delivery systems (SEDDS) for oral administration are highly on demand. Such an alternative might be reverse micelles. Within this study, SEDDS containing dry reverse micelles (dRMsPMB ) formed with an anionic (sodium docusate; AOT), cationic (dimethyl-dioctadecyl-ammonium bromide; DODAB), amphoteric (soy lecithin; SL), or non-ionic (polysorbate 85; P85) surfactant loaded with the model peptide drug polymyxin B (PMB) are developed. They are characterized regarding size, payload, release kinetics, cellular uptake, and peptide activity. SEDDS exhibit sizes from 22.2 ± 1.7 (AOT-SEDDS-dRMsPMB ) to 61.7 ± 3.2 nm (P85-SEDDS-dRMsPMB ) with payloads up to 2% that are approximately sevenfold higher than those obtained via hydrophobic ion pairing. Within 6 h P85-SEDDS-dRMsPMB and AOT-SEDDS-dRMsPMB show no release of PMB in aqueous medium, whereas DODAB-SEDDS-dRMsPMB and SL-SEDDS-dRMsPMB show a sustained release. DODAB-SEDDS-dRMsPMB improves uptake by Caco-2 cells most efficiently reaching even ≈100% within 4 h followed by AOT-SEDDS-dRMsPMB with ≈20% and P85-/SL-SEDDS-dRMsPMB with ≈5%. The peptide drug maintains its antimicrobial activity in all SEDDS-dRMsPMB . According to these results, SEDDS containing dRMs might be a game changing strategy for oral peptide drug delivery.

Oral drug delivery: Influence of mucus on cellular interactions and uptake of lipid-based nanocarriers in Caco-2 cells.

This study aimed to investigate the impact of the mucus gel barrier on intestinal mucosal uptake of lipid-based nanocarriers (NCs). Zwitterionic- (ZW), polyglycerol- (PG) and polyethylene glycol- (PEG) surfactant-based o/w nanoemulsions were developed. NCs were assessed regarding their size and zeta potential, stability in biorelevant media and mucus, mucus permeation behavior, cellular interactions and uptake by Caco-2 cells with and without mucus and by a Caco-2/HT29-MTX co-culture. All NCs were in the size range of 178 - 204 nm and exhibited a zeta potential between -4.2 and +1.2 mV. ZW- and PG-NCs demonstrated mucus permeating properties comparable to PEG-NCs. In contrast, ZW- and PG-NCs showed high cellular uptake, whereas limited cellular uptake was observed in case of PEG-NCs. Furthermore, mucus on Caco-2 cells as well as the mucus secreting co-culture had a significant impact on the cellular uptake of all tested NCs. According to these results, ZW- and PG-NCs are advantageous to overcome the mucus and epithelial barrier of the intestinal mucosa. STATEMENT OF SIGNIFICANCE: Within this study the impact of mucus on cellular uptake of lipid-based nanocarriers (NCs) with different surface decorations was investigated. The potential of NCs with zwitterionic-, polyglycerol- and polyethylene glycol-surfactants on their surface to overcome the mucus and epithelial barrier was evaluated. Zwitterionic- and polyglycerol-NCs showed mucus permeating properties similar to PEG-NCs. In contrast, zwitterionic- and polyglycerol-NCs substantially outperformed PEG-NCs in their cellular uptake properties. According to these findings, zwitterionic- and polyglycerol-NCs have the potential to overcome both the mucus and epithelial barrier of the mucosa.

PEG vs. zwitterions: How these surface decorations determine cellular uptake of lipid-based nanocarriers.

AIM: To evaluate the impact of polyethylene glycol (PEG) and zwitterionic surface decoration of lipid-based nanocarriers (NC) on cellular uptake. METHODS: Anionic, neutral and cationic zwitterionic lipid-based NCs based on lecithin were compared with conventional PEGylated lipid-based NCs regarding stability in biorelevant fluids, interaction with endosome mimicking membranes, cytocompatibility, cellular uptake and permeation across intestinal mucosa. RESULTS: PEGylated and zwitterionic lipid-based NCs exhibited a droplet size between 100 and 125 nm with a narrow size distribution. For the PEGylated and zwitterionic lipid-based NCs only minor alterations in size and PDI in fasted state intestinal fluid and mucus containing buffer were observed, demonstrating similar bioinert properties. Erythrocytes interaction studies revealed enhanced endosomal escape properties for zwitterionic lipid-based NCs compared to PEGylated lipid-based NCs. For the zwitterionic lipid-based NCs negligible cytotoxicity on Caco-2 and HEK cells, even in the highest tested concentration of 1 % (v/v) was recorded. The PEGylated lipid-based NCs showed a cell survival of ≥75 % for concentrations ≤0.05 % on Caco-2 and HEK cells, which was considered as non-toxic. For the zwitterionic lipid-based NCs up to 60-fold higher cellular uptake on Caco-2 cells was determined compared to PEGylated lipid-based NCs. For the cationic zwitterionic lipid-based NCs the highest cellular uptake with 58.5 % and 40.0 % in Caco-2 and HEK cells, respectively, was determined. The results were confirmed visually by life cell imaging. Ex-vivo permeation experiments using rat intestinal mucosa demonstrated up to 8.6-fold enhanced permeation of the lipophilic marker coumarin-6 in zwitterionic lipid-based NCs compared to the control. Up to 6.9-fold enhanced permeation of coumarin-6 in neutral zwitterionic lipid-based NCs compared to the PEGylated counterpart was recorded. CONCLUSION: The replacement of PEG surfactants with zwitterionic surfactants is a promising approach to overcome the drawbacks of conventional PEGylated lipid-based NCs regarding intracellular drug delivery.

Beyond the behavioural phenotype: Uncovering mechanistic foundations in aquatic eco-neurotoxicology.

During the last decade, there has been an increase in awareness of how anthropogenic pollution can alter behavioural traits of diverse aquatic organisms. Apart from understanding profound ecological implications, alterations in neuro-behavioural indices have emerged as sensitive and physiologically integrative endpoints in chemical risk assessment. Accordingly, behavioural ecotoxicology and broader eco-neurotoxicology are becoming increasingly popular fields of research that span a plethora of fundamental laboratory experimentations as well as applied field-based studies. Despite mounting interest in aquatic behavioural ecotoxicology studies, there is, however, a considerable paucity in deciphering the mechanistic foundations underlying behavioural alterations upon exposure to pollutants. The behavioural phenotype is indeed the highest-level integrative neurobiological phenomenon, but at its core lie myriads of intertwined biochemical, cellular, and physiological processes. Therefore, the mechanisms that underlie changes in behavioural phenotypes can stem among others from dysregulation of neurotransmitter pathways, electrical signalling, and cell death of discrete cell populations in the central and peripheral nervous systems. They can, however, also be a result of toxicity to sensory organs and even metabolic dysfunctions. In this critical review, we outline why behavioural phenotyping should be the starting point that leads to actual discovery of fundamental mechanisms underlying actions of neurotoxic and neuromodulating contaminants. We highlight potential applications of the currently existing and emerging neurobiology and neurophysiology analytical strategies that should be embraced and more broadly adopted in behavioural ecotoxicology. Such strategies can provide new mechanistic discoveries instead of only observing the end sum phenotypic effects.

Better Information From Survey Data: Filtering Out State Dependence Using Eye-Tracking Data.

Ideally, survey respondents read and understand survey instructions, questions, and response scales, and provide answers that carefully reflect their beliefs, attitudes, or knowledge. However, respondents may also arrive at their responses using cues or heuristics that facilitate the production of a response, but diminish the targeted information content. We use eye-tracking data as covariates in a Bayesian switching-mixture model to identify different response behaviors at the item-respondent level. The model distinguishes response behaviors that are predominantly influenced either positively or negatively by the previous response, and responses that reflect respondents' preexisting knowledge and experiences of interest. We find that controlling for multiple types of adaptive response behaviors allows for a more informative analysis of survey data and respondents.

Analysis of tail coiling activity of zebrafish (Danio rerio) embryos allows for the differentiation of neurotoxicants with different modes of action.

In (eco)toxicology, there is a critical need for efficient methods to evaluate the neurotoxic potential of environmental chemicals. Recent studies proposed analysis of early coiling activity in zebrafish embryos as a powerful tool for the identification of neurotoxic compounds. In order to demonstrate that the analysis of early tail movements of zebrafish embryos allows for the discrimination of neurotoxicants acting via different mechanisms, the present study investigated the effects of four different neurotoxicants on the embryogenesis (fish embryo toxicity test) and early tail coiling movements of zebrafish embryos. Cadmium predominantly increased the frequency of tail coiling at the late pharyngula stage. Dichlorvos delayed embryonic development and caused convulsive tail movements resulting in prolonged duration of tail coils. Embryos exposed to teratogenic concentrations of fluoxetine and citalopram displayed absence of spontaneous tail movements at 24 h post-fertilization. In contrast, a non-teratogenic test concentration of citalopram decreased coiling frequency at multiple time points. Results demonstrated that the analysis of tail coiling movements of zebrafish embryos has the potential to discriminate neurotoxic compounds with different primary modes of action. In addition, chemical-induced effects on coiling activity were shown to potentially overlap with effects on embryogenesis. Further studies are needed to clarify the interplay of unspecific developmental toxicity of neurotoxic chemicals and effects resulting from specific neurotoxic mechanisms.

In search of a comprehensible set of endpoints for the routine monitoring of neurotoxicity in vertebrates: sensory perception and nerve transmission in zebrafish (Danio rerio) embryos.

In order to develop a test battery based on a variety of neurological systems in fish, three sensory systems (vision, olfaction, and lateral line) as well as nerve transmission (acetylcholine esterase) were analyzed in zebrafish (Danio rerio) embryos with respect to their suitability as a model for the screening of neurotoxic trace substances in aquatic ecosystems. As a selection of known or putative neurotoxic compounds, amidotrizoic acid, caffeine, cypermethrin, dichlorvos, 2,4-dinitrotoluene, 2,4-dichlorophenol, 4-nonylphenol, perfluorooctanoic acid, and perfluorooctane sulfonic acid were tested in the fish embryo test (OECD test guideline 236) to determine EC10 values, which were then used as maximum test concentration in subsequent neurotoxicity tests. Whereas inhibition of acetylcholinesterase was investigated biochemically both in vivo and in vitro (ex vivo), the sensory organs were studied in vivo by means of fluorescence microscopy and histopathology in 72- or 96-h-old zebrafish embryos, which are not regarded as protected developmental stages in Europe and thus - at least de jure - represent alternative test methods. Various steps of optimization allowed this neurotoxicity battery to identify neurotoxic potentials for five out of the nine compounds: Cypermethrin and dichlorvos could be shown to specifically modulate acetylcholinesterase activity; dichlorvos, 2,4-dichlorophenol, 4-nonylphenol, and perfluorooctane sulfonic acid led to a degeneration of neuromasts, whereas both vision and olfaction proved quite resistant to concentrations ≤ EC10 of all of the model neurotoxicants tested. Comparison of neurotoxic effects on acetylcholinesterase activity following in vivo and in vitro (ex vivo) exposure to cypermethrin provided hints to a specific enzyme-modulating activity of pyrethroid compounds. Enhancement of the neuromast assay by applying a simultaneous double-staining procedure and implementing a 4-scale scoring system (Stengel et al. 2017) led to reduced variability of results and better statistical resolution and allowed to differentiate location-dependent effects in single neuromasts. Since acetylcholinesterase inhibition and neuromast degeneration can be analyzed in 72- and 96-h-old zebrafish embryos exposed to neurotoxicants according to the standard protocol of the fish embryo toxicity test (OECD TG 236), the fish embryo toxicity test can be enhanced to serve as a sensitive neurotoxicity screening test in non-protected stages of vertebrates.

An optimized method to assess ototoxic effects in the lateral line of zebrafish (Danio rerio) embryos.

In order to clarify the suitability of the lateral line of zebrafish (Danio rerio) embryos as a model for the screening of ototoxic (neurotoxic) effects, existing neuromast assays were adapted, improved and validated with a series of chemicals known or unknown for their ototoxic potential (caffeine copper sulfate, dichlorvos, 2.4-dinitrotoluene, neomycin, 4-nonylphenol, perfluorooctanesulfonic acid). Present methods were improved by (1) the introduction of a 4-step scoring system, (2) the selection of neuromasts from both the anterior and posterior lateral line systems, (3) a combined DASPEI/DAPI staining applied after both a continuous and pulse exposure scenario, and (4) an additional screening for nuclear fragmentation. Acute toxicities of the model substances were determined by means of the fish embryo test as specified in OECD TG 236, and EC10 concentrations were used as the highest test concentration in the neuromast assay. The enhanced neuromast assay identified known ototoxic substances such as neomycin and copper sulfate as ototoxic at sensitivities similar to those of established methods, with pulse exposure leading to stronger effects than continuous exposure. Except for caffeine, all substances tested (dichlorvos, 2.4-dinitrotoluene, 4-nonylphenol, perfluorooctanesulfonic acid) produced significant toxic effects in neuromasts at EC10 concentrations. Depending on the test substances and their location along the lateral line, specific neuromasts differed in sensitivity. Generally, neuromasts proved more sensitive in the pulse exposure scenario. Whereas for neomycin and copper sulfate neuromasts located along the anterior lateral line were more sensitive, posterior lateral line neuromasts proved more sensitive for the other test substances. Nuclear fragmentation could not only be associated with all test substances, but, albeit at lower frequencies, also with negative controls, and could, therefore, not be assigned specifically to chemical damage. The study thus documented that for a comprehensive evaluation of lateral line damage both neuromasts from the anterior and the posterior lateral line have to be considered. Given the apparently rapid regeneration of hair cells, pulse exposure seems more appropriate for the identification of lateral line neurotoxicity than continuous exposure.

Acetylcholinesterase in zebrafish embryos as a tool to identify neurotoxic effects in sediments.

In order to clarify the suitability of zebrafish (Danio rerio) embryos for the detection of neurotoxic compounds, the acetylcholinesterase assay was adapted and validated with a series of priority pollutants listed as relevant for the European water policy (Aroclor 1254, 2,3-benzofuran, bisphenol A, chlorpyrifos, paraoxon-methyl, quinoline, and methyl mercury chloride) as well as acetonic extracts from three sediments of known contamination. The acute toxicities of the model substances and the sediment extracts were determined by means of the fish embryo test as specified in OECD TG 236, and concentrations as low as the effective concentration at 10% inhibition (EC10) were used as the highest test concentration in the acetylcholinesterase test in order to avoid nonspecific systemic effects mimicking neurotoxicity. Among the model compounds, only the known acetylcholinesterase inhibitors paraoxon-methyl and chlorpyrifos produced a strong inhibition to about 20 and 33%, respectively, of the negative controls. For the sediment extracts, a reduction of acetylcholinesterase activity to about 60% could only be shown for the Vering Canal sediment extracts; this could be correlated to high contents of acetylcholinesterase-inhibiting polycyclic aromatic hydrocarbons (PAHs) as identified by chemical analyses. Co-incubation of the Vering Canal sediment extracts with chlorpyrifos at EC10 concentrations each did not significantly increase the inhibitory effect of chlorpyrifos, indicating that the mode of action of acetylcholinesterase inhibition by the sediment-borne PAHs is different to that of the typical acetylcholinesterase blocker chlorpyrifos. Overall, the study documents that zebrafish embryos represent a suitable model not only to reveal acetylcholinesterase inhibition, but also to investigate various modes of neurotoxic action.

The fish embryo test (FET): origin, applications, and future.

Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish (Danio rerio) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If-for ethical reasons-not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has-at least for the substances tested so far-compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight >3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.