Evaluation of potential environmental toxicity of polymeric nanomaterials and surfactants

Journal Pre-proof Evaluation of potential environmental toxicity of polymeric nanomaterials and surfactants Indra Hering, Elke Eilebrecht, Michael J. Parnham, Nazende ¨ Gunday-T ¨ ureli, ¨ Akif Emre Tureli, ¨ Marc Weiler, Christoph Schafers, Martina Fenske, Matthias G. Wacker

PII:

S1382-6689(20)30029-6

DOI:

https://doi.org/10.1016/j.etap.2020.103353

Reference:

ENVTOX 103353

To appear in:

Environmental Toxicology and Pharmacology

Received Date:

2 November 2019

Accepted Date:

5 February 2020

Please cite this article as: Hering I, Eilebrecht E, Parnham MJ, Gunday-T ¨ ureli ¨ N, Tureli ¨ AE, ¨ Weiler M, Schafers C, Fenske M, Wacker MG, Evaluation of potential environmental toxicity of polymeric nanomaterials and surfactants, Environmental Toxicology and Pharmacology (2020), doi: https://doi.org/10.1016/j.etap.2020.103353

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Title: Evaluation of potential environmental toxicity of polymeric nanomaterials and surfactants Authors: Indra Hering*1,2, Elke Eilebrecht2, Michael J. Parnham1, Nazende GündayTüreli3, Akif Emre Türeli3 , Marc Weiler3, Christoph Schäfers2, Martina Fenske1**, Matthias G. Wacker4 1

Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie IME, 65926 Frankfurt/Main

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Fraunhofer Institut für Molekularbiologie und Angewandte Oekologie IME, 57392 Schmallenberg

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MJR, PharmJet GmbH, 66802 Überherrn

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National University of Singapore, Department of Pharmacy, Faculty of Science, 6 Science Drive 2,

*Corresponding Author: Indra Hering Fraunhofer Institute for Molecular Biology and Applied Ecology IME

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Auf dem Aberg 1

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Singapore 117546, Singapore

57392 Schmallenberg, Germany

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e-mail adress: [email protected]

**Current affiliation:

German Federal Institute of Hydrology (BfG), Am Mainzer Tor 1, 65068 Koblenz, Germany.

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[email protected]

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Highlights

•Surfactants and polymers used in nanoformulations were tested in fish embryo tests •The most ecotoxic polymer was Eudragit® E100 compared to PLGA and HPMCP

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•Pluronics were environmentally less toxic than Cremophors.

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•Pluronics caused a conspicuous phenotype of shriveled chorion

Abstract:

Nanomaterials have gained huge importance in various fields including nanomedicine. Nanoformulations of drugs and nanocarriers are used to increase pharmaceutical potency. However, it was seen that polymeric nanomaterials can cause negative effects. Thus, it is

essential to identify nanomaterials with the least adverse effects on aquatic organisms. To determine the toxicity of polymeric nanomaterials, we investigated the effects of poly(lactic-coglycolid) acid (PLGA), Eudragit® E 100 and hydroxylpropyl methylcellulose phthalate (HPMCP) on zebrafish embryos using the fish embryo toxicity test (FET). Furthermore, we studied Cremophor® RH40, Cremophor® A25, Pluronic® F127 and Pluronic® F68 applied in the generation of nanoformulations to identify the surfactant with minimal toxic impact. The order of ecotoxicty was HPMCP
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the least toxic impact, thus suggesting adequate environmental compatibility for the generation of nanomedicines.

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Keywords: nanomaterials, polymers, surfactants, ecotoxicity, Danio rerio

1. Introduction

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Nanomaterials have become crucial components in several diverse fields, from electronics to

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agriculture to nanomedicine (Kumari, Yadav et al. 2010). In nanomedicine, these materials are often utilized as vehicles for drug delivery, contrast agents, biosensors as well as to facilitate drug targeting to specific organs and tissues such as the brain, the arterial walls or lungs

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(Duncan 2006, Fako and Furgeson 2009, Mahapatro and Singh 2011, Tao, Hu et al. 2011). Biodegradable polymeric nanomaterials offer several advantages in terms of their

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biocompatibility, sustained drug release and reduced toxicity to humans (Kumari, Yadav et al. 2010, Meyer and Green 2016).

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The dimensions of nanomaterials ranging between 1 and 100 nm (as defined by 2011/696/EU) result in a higher surface area and increased dissolution rate (Malam, Loizidou et al. 2009, Chen, Sonaje et al. 2011, Mahapatro and Singh 2011). The enhanced surface reactivity leads to an increase in bioactivity which in turn, aids drug delivery (Oberdörster, Oberdörster et al. 2005). However, this increased bioactivity may also lead to ecotoxic effects, highlighting the importance to test for toxicity of nanomaterials for medical applications (Oberdörster, Oberdörster et al. 2005, Fischer and Chan 2007, Makadia and Siegel 2011). Therefore, it is

essential to assess the toxicity of polymeric nanomaterials as well as of pharmaceutical excipients, which are widely used in the production of nanosized drug-delivery systems. In this study, we investigated the ecotoxicity of polymeric nanomaterials composed of Eudragit® E100, hydroxypropyl methylcellulose phthalate (HPMCP) or poly(lactic-co-glycolic) acid (PLGA) as well as several surfactants in order to compare their ecotoxic potential and to identify a polymeric nanocarrier with comparatively low ecotoxic potential. PLGA is known for its biocompatibility and biodegradability (Makadia and Siegel 2011). The

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polymer is hydrolyzed to glycolic acid and lactic acid, which are metabolized in the human body, thus diminishing the risk of toxic side effects (Mahapatro and Singh 2011, Makadia and Siegel 2011, Danhier, Ansorena et al. 2012). PLGA nanoparticles have been studied with respect to vaccine delivery, imaging techniques, treatment of various diseases including

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inflammatory diseases and anti-cancer treatments (Cheng, Teply et al. 2007, Dinarvand,

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Sepehri et al. 2011, Yallapu, Jaggi et al. 2013, Arshad, Yang et al. 2015, Prabhu, Patravale et al. 2015, Ghalamfarsa, Hojjat-Farsangi et al. 2016, Colzani, Pandolfi et al. 2018, Malam,

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Loizidou et al. 2009, Danhier, Ansorena et al. 2012, Kalluru, Fenaroli et al. 2013). The degradation of the polymer results in a complete drug release, thus making PLGA suitable for

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drug delivery (Zhu and Braatz 2015). Specifically, PLGA has been used in pH-sensitive microcapsules to study insulin delivery and was used in a drug product approved by the Food and Drug Administration of the United States (US-FDA) for the sustained release of leuprolide,

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a hormone antagonist, and triptorelin which is used for the treatment of breast cancer (Chen,

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Sonaje et al. 2011, Sun, Liang et al. 2015, Frampton 2017). Basic butylated methacrylate copolymer, commercially available as Eudragit® E 100 or Eudragit® E PO, are approved by the FDA and have been utilized for masking taste and odor (Eisele, Haynes et al. 2011). Eudragit® is also applied in the production of nanomedicines including various pharmaceuticals. For example, a nanosuspension of Eudragit® RL 100 and the active compound gefitinib was prepared for anti-cancer treatment, Eudragit® L100 was used to generate diclofenac sodium-loaded nanoparticles and an Eudragit® S 100

nanosuspension of rifaximin was studied in connection with inflammatory bowel disease (Cetin, Atila et al. 2010, Kola Srinivas, Verma et al. 2016, Kumar and Newton 2016). Eudragit® nanoformulations were further investigated concerning drug delivery of curcumin and in antiHIV treatment (Yallapu, Jaggi et al. 2013, Hari, Narayanan et al. 2016). The Eudragit® E 100 nanosuspension we tested in the present study, however, differs in physicochemical properties and chemical structure from other Eudragit® formulations. HPMCP is used in enteric coatings of oral drug products to protect the drug from the acidic

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conditions in the stomach and release of the compound in the intestinal tract (Hussan Singh Deep Roychowdhury Santanu 2012). The polymer has been studied with regard to nanoencapsulation of insulin and the production of curcumin nanoparticles for cancer treatment as well as enzyme treatment and the dissolution of hardly soluble drugs (Sharma,

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Sharma et al. 2011, Jin, Xia et al. 2012, Yallapu, Jaggi et al. 2013, Dinh, Tran et al. 2017

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Zhang, Li et al. 2018).

Surfactants are used in huge quantities in industry and domestic households and it was shown

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that they reach the aquatic environment via sewage treatment plants or direct discharge into surface waters (Ivankovic and Hrenovic 2010, Olkowska, Ruman et al. 2014). For

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nanotechnology-applications such as the production and testing of nanomedicines, a wide variety of surfactants are used either to stabilize the dispersion of nanoparticles or to provide

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the formulation with specific characteristics. These surfactants were used in concentrations of up to 20% (m/V) (Atyabi and Dinarvand 2012). Tween 80, for instance, enables particles to

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cross the blood brain barrier and was therefore used to generate poly-ε-caprolactone-Tween 80 nanoparticles for brain cancer treatment (Kreuter 2001, Das and Lin 2005, Ma, Zheng et al. 2011). Surfactants can thus enter the environment by disposal of nanodrugs, from hospital discharges or during the production process. Therefore, toxic effects of these surfactants on the aquatic organisms are possible. Pluronic® F68 was studied in a breast cancer formulation and more recently in a PLGA-Ciprofloxacin formulation (Mei, Zhang et al. 2009, Gunday Tureli, Tureli et al. 2016). Various other surfactants including Pluronic® F127, Tween 20 and

Cremophor® RH40 were used in the production of curcumin nanoparticles with respect to anticancer treatment (Yallapu, Jaggi et al. 2013, Baskaran, Madheswaran et al. 2014). The embryos of zebrafish, Danio rerio, were used as a model organisms in this study, as they offer a wide range of well-known advantages for ecotoxicity testing (Spence, Gerlach et al. 2008, Fako and Furgeson 2009) and are considered an alternative to adult fish (Strahle, Scholz et al. 2012). The zebrafish has already proven to be a suitable model for testing ecotoxic effects of nanomaterials including metal and fluorescent silica nanoparticles (Griffitt, Luo et al. 2008,

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Fako and Furgeson 2009, Fent, Weisbrod et al. 2010, Muth-Köhne, Sonnack et al. 2013, Ozel, Alkasir et al. 2013, Sonnack, Kampe et al. 2015). Using the fish embryo toxicity test (FET), negative effects of polymeric nanoparticles (poly ethylene imine, poly(2-(dimethylamino) ethylmethacrylate,

poly(-2-hydroxypropyl)

methacrylamide,

polyamidoamine)

on

the

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development of the embryos have been detected (Rizzo, Golombek et al. 2013, Bodewein, Schmelter et al. 2016). Therefore, we hypothesized that other materials used in the production

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of nanomedicines may also be able to cause detrimental effects in aquatic organisms such as

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fish. The aim of this study was thus, to compare the nanomaterials and surfactants used in the

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generation of nanomedicines to identify those substances with the least ecotoxic potential.

2. Materials and Methods

2.1 Fish husbandry and egg collection Wildtype zebrafish Danio rerio (originally obtained from West Aquarium GmbH, Bad Lauterberg, Germany) were kept in UV-sterilized and activated carbon filtrated water at 27 °C ± 1 °C in a 10h/14h dark-light cycle. The fish were fed in the morning with Tetramin flakes (Tetra GmbH, Melle, Germany) and with live nauplia of Artemia salina in the afternoon. After beginning of the light cycle, spawning trays were placed in the fish tanks (50-350 fish per

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aquarium) and left for approximately 60-90 min. After removal of the spawning trays from the tanks, the eggs were collected in a tea strainer and rinsed with fresh aquarium water to remove

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debris. The eggs were transferred to a vessel filled with aquarium water.

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2.2 Test compounds

The polymeric nanosuspensions comprising either Eudragit® E100, hydroxypropyl

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methylcellulose phthalate (HPMCP) and poly(lactic-co-glycolic) acid (PLGA), were produced by MJR Pharmjet GmbH (Ueberherrn, Germany). These suspensions were diluted with ISO

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water which was five times diluted in distilled water (20% ISO water) according to DIN EN ISO 7346-3 (DIN, 1996) to obtain test concentrations of 0.0125, 0.125; 1.25 mg·ml-1 for PLGA and Eudragit® E100, respectively, and 0.019, 0.188,1.88 mg·ml-1 for HPMCP. The concentrations

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are based on the solid concentration of the polymers in the nanosuspensions. The surfactants

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were purchased from BASF (Germany) and diluted in 20% ISO water. Range finding tests were performed to establish concentrations suitable to derive EC50- and LC50-values from concentration-response evaluations (see Table 1 for concentrations). All tested chemicals (nanosuspensions and surfactants) were kindly provided by MJR PharmJet GmbH (Ueberherrn, Germany). The test concentrations of the surfactans are given in % for better comparison to the literature. All test concentrations given are nominal.

2.3 Fish Embryo Toxicity Test (FET) The FET is in accordance with the German Tierschutz-Versuchstierverordnung (TierVersV 1.08.2013) and the European guidelines 2010/63/EU (European Union, 2010) not considered an animal procedure, meaning that zebrafish embryos until the onset of independent feeding (at 120 hours post fertilization (hpf) in zebrafish) are exempt from legal obligations regarding the protection of animals used for scientific purposes. The tests were conducted under standardized conditions as described previously (Muth-

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Köhne, Sonnack et al., 2013, Sonnack, Kampe et al., 2015) in compliance with the test guideline OECD 236 (OECD, 2013) and the DIN EN ISO 15088 (DIN, 2009).

Briefly, embryos without any defects were individually transferred into unsaturated 96-well U-

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bottom-shaped polystyrene microtiter test plates (Greiner Bio-one, Frickenhausen, Germany). Each well contained 200 µl of the test substance or 20% ISO water as control. The pH of the

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test solutions should range between 6.5 and 8.0 and was tested at the start (0 hpf) and end of testing (72 hpf). For the two highest concentrations of HPMCP, the pH needed to be adjusted

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prior testing.

Every 24 h, embryos were assessed for sub-lethal and lethal effects according to the

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description by Braunbeck and Lammer (Braunbeck and Lammer, 2006). Coagulation, no formation of somites, no detachment of tail, teratoma and lack of a heartbeat at the 48 hpf-

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stage were counted as lethal endpoints (Braunbeck and Lammer, 2006). Sub-lethal effects encompassed deformed somites, chorda dorsalis and yolk sac as well as edema, no formation

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of eyes, no spontaneous movement, malformation of tail, head and otholits as well as a general growth retardation (Braunbeck and Lammer, 2006). Additionally, malformation of heart, reduced blood flow and lack in pigmentation were counted as sub-lethal effects (Braunbeck and Lammer, 2006). Each treatment was repeated in parallel on a separate well plate for each test. The tests were repeated three to four times. The exposure time for each test was 72 hours. At the end of testing, the embryos were euthanized on ice.

2.4 Yeast Estrogen Screen (YES) The Yeast Estrogen Screen (YES) was performed according to the ISO guideline 19040-1 (DIN, 2018) in the laboratory of Prof. Dr. Jörg Oehlmann (Goethe-University Frankfurt a.M., Germany). Briefly, the possible estrogenic effect of a test substance can be assessed photometrically with the Sumpter strain of Saccharomyces cerevisiae which was transformed with the human estrogen receptor and the lacZ gene as reporter and expresses β-

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galactosidase when estrogenic substances are present (DIN, 2018). The results are stated as estrogenic activity in 17β-estradiol equivalent concentration (EEQ in ng·L-1). The limit of quantification (LOQ) in this test was 0.42 ng·L-1. This means that substances with a measured

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value below 0.42 ng·L-1 do not display an estrogenic effect. Of the three tested polymers, we only examined HPMCP for any possible estrogenic effects, as it belongs to the group of

2.5 Analysis and statistics

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phthalates which display endocrine activity.

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The data were initially analyzed with Excel (Microsoft, Redmond, USA) to obtain an overview of the effects. Subsequently, ToxRat® (Version 2.10; ToxRat Solutions GmbH, Alsdorf,

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Deutschland) was used to obtain concentration-response curves as well as EC50- and LC50values via Probit analysis including 95% confidence intervals (CI). Testing for normal

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distribution was done with the Shapiro-Wilk-Test (p=0.01) and variance homogeneity was tested by the Levene’s Test (p=0.01). After this, a non-parametric trend analysis by contrast was performed (p=0.05) and the Step-Down-Jonckheere-Terpstra-Test was applied to determine the differences between exposure conditions (p=0.05). If the data was normally distributed, but no variance homogeneity present, the multiple sequentially-rejective Welsh-ttest after Bonferroni-Holm was conducted. If the data was neither normally distributed nor

displayed variance homogeneity, the multiple sequentially-rejective median (2x2-Table) test

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after Bonferroni-Holm was applied. The tests were performed with ToxRatPro 3.1.0.

3. Results To assess possible ecotoxic effects of polymeric nanoformulations for medical treatment, it is essential to test the single compounds of the nanomedicines. The carrier materials PLGA, HPMCP and Eudragit® E100 as well as the surfactants Cremophor® A25, Cremophor® RH40, Pluronic® F68 and Pluronic® F127 were each tested separately using zebrafish embryos in FETs. In this way, sub-lethal and lethal effects can be evaluated to compare the test substances regarding their environmentally toxic potential.

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3.1 Eudragit® E 100

All embryos exposed to Eudragit® E 100 showed effects in all concentrations after 72 h of exposure (Fig.1A). The mortality at 72 hpf ranged from 79% to 95% at 0.0125 mg·ml-1 and

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0.125 mg·ml-1, respectively. At 1.25 mg·ml-1, mortality reached 100% (p<0.001), all embryos were found dead already at 24 hpf at this concentration. The EC50-value at 72 hpf is <0.0125

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mg·ml-1 (Fig.2A). The LC50-value of Eudragit® E 100 was 0.002 mg·ml-1 at 72 hpf (Fig.2B). However, there was no statistically significant difference concerning mortality between the

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treatments and the control at 72 hpf due to wide confidence intervals (p=0.098).

3.2 Hydroxypropyl methylcellulose phthalate

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Embryos exposed to HPMCP displayed a concentration-dependent increase in effects at 72hpf (Fig.1B). The lowest concentration of HPMCP was comparable to the control. At a

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concentration of 0.1875 mg·ml-1, 33% of the embryos showed effects (p=0.008). At the highest concentration, the number of embryos with effects was >90% (p<0.001), with mostly edema (66%) and slow blood flow (31%). Mortality at all three concentrations was below 6% at 72 hpf. The EC50-value of HPMCP was determined to be 0.269 mg·ml-1 at 72 hpf (Table 2). For HPMCP, no LC50-value could be determined (LC50-value> highest test concentration) (Fig.2B). As HPMCP includes a phthalate-residue which could cause estrogenic effects in the zebrafish embryos, we also tested for estrogenicity using the YES-assay. The limit to detect estrogenicity

in this test was 0.425 ng·L-1 EEQ. The maximum measured activity of HPMCP was 0.18 ng·L1

EEQ (Fig.3) and therefore below estrogenic activity.

3.3 Poly lactic co glycolic acid The cumulative effect in the embryos exposed to PLGA at a concentration of 0.0125 mg·ml-1 was below 20%at 72 hpf (Fig.1C). The concentrations of 0.125 mg·ml-1and 1.25 mg·ml-1 caused 87% and 100% of the embryos to display sub-lethal effects, indicating a concentrationdependent increase in effects after 72 hpf of exposure, though only the highest concentration

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1.25 mg·ml-1 was significantly different from the control (p≤0.05). The predominant effect was a lack of hatching in the two highest test concentrations, whereas control embryos and embryos exposed to 0.0125 mg·ml-1 were hatched at 72 hpf (Fig.1C). Moreover, the chorion of the embryos exposed to 0.125 mg·ml-1 and 1.25 mg·ml-1 PLGA displayed a brownish

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discoloration, presumably caused by an accumulation of the substance at the surface of the

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chorion (Fig.4). For PLGA, the EC50-value of 0.063 mg·ml-1 at 72 hpf was four times lower than the EC50-value of HPMCP. No LC50-value could be determined as only sub-lethal

concentrations.

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3.4 Cremophor® A25

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morphological effects were observed (Table 2). Thus, the LC50-value is higher than the tested

Embryos exposed to all concentrations of Cremophor® A25 displayed effects significantly

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different from the control (p=0.024 for 0.0005% and p<0.001 for the other concentrations). Exposure to 0.05% Cremophor® A25 leads to a mean mortality of 99% at 72 hpf (Fig. 5A).

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The majority of embryos was already coagulated after 24 hours (data not shown). At a concentration of 0.005%, 59% of embryos displayed edema, slow blood flow and coagulation at 72 hpf. The LC50- and EC50-values of Cremophor® A25 were 0.006% and 0.003%, respectively, at 72 hpf (Fig.6A and B).

3.5 Cremophor® RH40 All embryos exposed to Cremophor® RH40 at a concentration of 0.5% were dead at 72 hpf At the lower concentration of 0.05%, effects in 52% of the embryos were observed (p<0.001) of which 30% were lethal (Fig.5B). At concentrations lower than 0.05%, Cremophor® RH 40 caused no sub-lethal or lethal effects in more than 20% of the exposed embryos. Effects encompassed slow blood flow (10.2 %) and edema (15.1 %) and other sub-lethal effects such as malformation of tail, head and yolk sac deformation (data not shown). The LC50- and EC50-

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value of Cremophor® RH40 are 0.081% and 0.037%, respectively, at 72 hpf (Table 3).

3.6 Pluronic® F68

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Pluronic® F68 was tested at higher concentrations (>1%) compared to the Cremophors, based on the results of the range finding tests. Over 90% of the embryos exposed to Pluronic® F68

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displayed effects at a concentration of 3% or higher at 72 hpf (p<0.001) (Fig.5C). The effects

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consisted of slower blood flow and edema as well as malformations of head and tail. Additionally, malformations of the head with 22.8% as well as tail deformations with 21.7% (data not shown) of embryos exposed to 4.5% of Pluronic® F68 were observed. Notably,

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directly after the start of the exposure to Pluronic® F68, the chorion lost its spherical shape and appeared creased (Fig. 7). At 24 hpf, this effect was seen in all concentrations of Pluronic®

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F68 tested. This effect did not occur in embryos exposed to the two Cremophors. For Pluronic® F68, an EC50-value of 1.6% could be calculated (Table 3). The LC50-value could not be

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determined in the range of the tested concentrations and is thus higher than 10% Pluronic® F68 (Fig.6C).

3.7 Pluronic® F127 Pluronic® F127 was tested at higher concentrations compared to the surfactants from the Cremophor family, based on the results of the range finding tests. The mean effect values for embryos exposed to 5% and 10% of Pluronic® F127 at 72 hpf were 87% and 98% (p<0.001),

respectively (Fig. 5D). For both, Pluronic® F68 and Pluronic® F127, a significant increase in the effects compared to the controls was determined from the lowest concentration onwards (p<0.001 and p=0.002, respectively). After starting the exposure to Pluronic® F127, the chorion lost its spherical shape and appeared creased (Fig. 7). At 72 hpf, the EC50-value of Pluronic® F127 was 2.4%. For Pluronic® F127 a LC50-value of 5.21% could be determined at

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72 hpf (Table 3).

4. Discussion Several polymeric nanoformulations of drugs are approved by the FDA for treatment of diseases such as cancer (Gliadel®, Guilford Pharm.Inc.) or schizophrenia (Risperdal Consta®, Johnson and Johnson) (Marcato and Durán, 2008). Since these nanomedicines may enter the environment, it is essential not only to test the pharmaceutical compounds of nanomedicines, but also the excipients used during the production process concerning their ecotoxic potential. With regard to the usage and exploration of nanopharmaceuticals, it is important to consider

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potential issues with toxicity also for the environment (Berkner, Schwirn and Voelker, 2016). So far, very few studies have specifically addressed the environmental effects of excipients commonly used for the formulation of nanopharmaceuticals. The present study is, to our knowledge, the first to explore the toxic effects of the polymers Eudragit®, HPMCP and PLGA

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and the surfactants of the Cremophor and Pluronic type in zebrafish embryos.

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4.1 Ecotoxic effects of polymeric nanosuspensions on the zebrafish embryo We tested Eudragit® E 100, HPMCP and PLGA for sub-lethal and lethal effects on zebrafish embryos and larvae until 72 hpf. Whereas Eudragit® E 100 mainly caused mortality at the

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concentrations tested, neither HPMCP nor PLGA showed lethal effects. However, most larvae exposed to  0.125% PLGA did not hatch at 72 hpf. Further, the polymeric particles seemed

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to accumulate at the chorion causing a coloration of the chorion (Fig.3). We suppose that this may have impaired the hatching process. Although the pore channels of the chorion are wide

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enough to let nanoparticles pass (Rawson, Zhang et al., 2000, Bai, Zhang et al., 2009), agglomerates of particles might accumulate on the surface of the chorion and impair oxygen transport through the chorion pores which could lead to delay of hatching as observed in our study (Bai, Zhang et al., 2009, Cheng, Flahaut and Cheng, 2007, Duan, Yu et al., 2013). Hypoxia has been stated before to delay hatching in fish (Cheung, Chiu and Wu, 2014, Mu, Chernick et al., 2017).

Eudragit® E100 was found to be the most toxic nanomaterial tested in our study. The toxicity showed concentration- as well as time-dependency. After hatching at 72 hpf, mortality at the lowest exposure concentration of 0.0125 mg·ml-1 increased from 6% to 79% at 24 and 48 hpf (data not shown), suggesting that the chorion protected the embryos. The chorion acts as a fortification and the permeability of high molecular weight substances is limited (Henn et al., 2011). Nevertheless, the highest concentration of 1.25 mg·ml-1 Eudragit® E 100 was still potent enough to cause 100% mortality already at 24 hpf. However, it was observed that the particles not only precipitated and accumulated at the bottom of the wells, they also accumulated around

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the chorion wall. This may have led to the clogging of the chorion pores. This assumption is supported by Lee et al., 2007 and Bai, Zhang et al., 2009, who showed that silver nanoparticles as well as zinc oxide- nanoparticles block the chorion pore canals and hinder the transport of

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oxygen (Lee, Nallathamby et al. 2007, Bai, Zhang et al. 2009). Hypoxia caused by blocked chorion pore canals could therefore be another contributing factor to the observed mortality at

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high concentrations. Moreover, the Eudragit® E 100 particles are cationic (Chang, Peng and Shukla, 2006, Quinteros, Rigo et al., 2008, Quinteros, Allemandi and Manzo, 2012), whereas

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the chorion is negatively charged (Bodewein, Schmelter et al., 2016). In an earlier study in our laboratory, cationic dendrimers caused 100% mortality in embryos exposed to concentrations

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of ≤10 µM and it was also hypothesized that the cationic dendrimers interacted with the negatively charged chorion (Bonsignorio, Perego et al., 1996). Blocking of the chorion pores

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and subsequent hypoxia was also proposed for the PLGA-nanosuspenion, but no mortality occurred in embryos exposed to the PLGA-nanosuspension. It might be possible that at least

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some molecules of the nanosuspensions had passed the chorion (Bai, Zhang et al., 2009). For PLGA, no further negative effect besides the previously described effect on hatching was observed, which was assumed to be mainly caused by hypoxia (Fig.1C). For Eudragit® E 100, however, a study conducted by Alasino et al. (2005) demonstrated that Eudragit® E 100 caused lytic activity in synthetic liposomes and human red blood cells (Alasino, Ausar et al. 2005). A lytic effect of the Eudragit® E100 molecules, which might have partly passed through the chorion, could have therefore caused the mortality, an effect not seen in embryos exposed

to PLGA. The blocking of the chorion pores and the subsequent hypoxia can be a mechanical effect which occurs independently from the substances and can be the same for PLGA and Eudragit® E 100. Since mortality in embryos exposed to the higher concentrations of Eudragit® E 100 occurred at 24 hpf and only after hatching in the lowest concentration, mortality is independent from the mechanical effect, and likely due to the specific lytic effect of Eudragit® E 100.

However, it needs to be clarified, whether the observed mortality was induced by Eudragit®

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E100 by a single of the described effects or by a combination of effects. Higher concentrations of nanoparticles might cause clogging of the chorion pore canals, leading to hypoxia and thus coagulation. At lower concentrations, the chorion might still act as a fortification without

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inhibiting oxygen exchange. Then the main exposure and effect on the embryos would occur after the hatch event. Furthermore, nanomaterials which enter through the chorion pores might

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differ in their toxicity on the embryo as suggested for PLGA and Eudragit® E100.

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HPMCP belongs to the same group of phthalates, which also include dibutyl phthalate and diethyl phthalate, which display adverse reproductive and developmental effects (European

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Medicines Agency, 2014). However, toxicity studies in rats showed minor toxicity of HPMCP with a no-effect-concentrations of 4.5 g·kg-1 and 6 g·kg-1 and the US-FDA approved oral

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formulations containing HPMCP at a ratio of 302.4 mg per unit dose (U.S. Food and Drug Administration, 2007). In our experiments, the HPMCP nanosuspension displayed the lowest

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ecotoxicity (lowest EC50-value, mortality > 10%) compared to PLGA and Eudragit® E100 nanosuspensions. With regard to wider use of HPMCP in the coating of pharmaceuticals (Hussan Singh Deep Roychowdhury Santanu 2012), it is advisable to extend the present very limited knowledge on its ecotoxic impact. We observed no estrogenic effect in the YES- assay at the HPMCP concentrations tested but this does not rule out any reproductive or developmental toxicity effects in fish or other aquatic

organisms. To investigate possible negative effects of this substance further, studies on the cellular (e.g. apoptosis), enzymatic or gene expression level should be conducted. As far as possible effects of degradation products are concerned, the degradation time of e.g., PLGA can vary from weeks up to months, depending on various parameters such as the molecular weight or the lactide / glycolide ratio, (Gentile, Chiono et al. 2014). The nanosuspensions used for our tests were estimated to be stable for at least a week, and therefore, effects of degradation products of the tested nanomaterials were not taken into

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consideration.

In summary, for PLGA, the EC50-value at 72 hpf of 0.063 mg·ml-1 was four times lower than the EC50-value of HPMCP. Overall, the lowest EC50- and LC50-values were found for Eudragit®

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E100. Therefore, Eudragit® E100 is the most toxic of the three tested nanomaterials (Table

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2).

Thus, Eudragit® E 100 appeared to be the least suitable substance for polymeric

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nanoformulations in terms of their ecotoxicity. The other polymeric nanosuspensions of HPMCP and PLGA seemed to be better candidates for the generation of nanoformulations.

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HPMCP exhibited lower toxicity after 72 hpf and, compared to PLGA, embryos exposed to HPMCP hatched at the highest concentration at 72 hpf. Our findings thus indicate, that HPMCP

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is the least fish embryo toxic polymer of the three types tested.

4.2 Teratogenic effects of various surfactants We also investigated the effects of surfactants used in the production of nanoformulations on the development of zebrafish embryos and larvae. Surfactants are applied to modify the surface properties of nanoformulations for example, to increase their circulating time in the human blood stream or to enable the passage through physiological barriers such as the blood brain barrier (Heinz, Pramanik et al. 2017). The surfactants tested in this study were nonionic.

Nonionic surfactants are used as emulsifiers, to enhance the stability of drug carriers or to increase the effect of pesticides (Ivankovic and Hrenovic 2010). In terms of the aquatic toxicity, the literature reports that the nonionic surfactant class of alkyl ethoxylates caused negative effects on growth and hatching of the fathead minnow (Pimephales promelas) (NOEC from 0.18 mg·ml-1 to 0.32 mg·ml-1), and other nonionic surfactants were observed to alter swimming and avoidance behavior in fish and invertebrates (Lewis, 1991). In a study conducted by Wang et al. (2015), zebrafish embryos were exposed to three different classes of surfactants cationic, anionic and nonionic - and all tested materials led to a developmental retardation

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(Wang, Zhang et al. 2015). The class of nonionic surfactants further affected larval sleeping behavior and locomotion (Wang, Zhang et al. 2015). Thus, there is evidence that nonionic surfactants may have a negative impact on aquatic organisms.

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In our study, we tested four nonionic surfactants known by the trademarks Cremophor® and Pluronic®. Comparing Cremophor® A25 and Cremophor® RH40, we found that the LC50-value

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(72 hpf) of Cremophor® A25 was 13 times lower than the LC50-value of Cremophor® RH40

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(0.081%) and the EC50-value (72 hpf) was 12 times lower reflecting higher toxicity of Cremophor® A25. Mortality of the embryos reached 100% at 0.05% of Cremophor® A25 and at 0.5% of Cremophor® RH40. Cremophor® A25 was more toxic and revealed acute toxicity

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already at a lower concentration (0.05%) than the lowest concentration used in some nanoformulations (Madheswaran, Baskaran et al. 2014). Nanoformulations have been

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investigated as promoters for the skin permeation of finasteride, containing 0.5% - 2.5% of Cremophor® RH40 (Madheswaran, Baskaran et al. 2014). In another study, Cremophor®

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RH40 was used in a concentration of 19% (Erdal, Ozhan et al. 2016) for the generation of microemulsions.

Pluronic® F68 has been used in the preparation of nanoformulations at concentrations of 1% and 10% to enhance the drug release as well as to stabilize nanoparticles during the manufacturing process (Mei, Zhang et al. 2009, Tian, Lin et al. 2011). In our study, the highest tested concentration was 6%. Up to this concentration, mortality above 50% did not occur. However, severe body malformations were detected in the embryos and the EC50-value at 72

hpf was 1.6%. In contrast to Pluronic® F68, the predominant effect of Pluronic® F127 at 72 hpf was mortality due to coagulation and was observed in up to 70% of embryos at the highest concentration (10%) and 53% at 5% Pluronic® F127 (Fig.5D). Pluronic® F 127 is applied in polymeric nanoformulations for wound healing and cancer (Kalita, Devi et al., 2015, Hosseinzadeh et al., 2012). In the study by Atyabi et al. (2012), Pluronic® F 127 has been incorporated in the nanoparticles in concentrations of 10 %, 15 % and 20 % (w/w %) (Atyabi and Dinarvand 2012). The zebrafish

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embryos in our study were exposed to concentrations of Pluronic® F127 ranging from 1.25% to 10%, with the highest concentration matching the concentration used in the formulation of Atyabi et al. (Atyabi and Dinarvand 2012). The LC50-value (72 hpf) for Pluronic® F127 was 5.21%. Compared to Pluronic® F68, the mortality rate of embryos exposed to Pluronic® F127

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was more than twice as high at the two highest concentrations. Pluronic® F68 caused more sub-lethal effects than Pluronic® F127 (see 3% Pluronic® F68 and 2.5% Pluronic® F127),

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whereas Pluronic® F127 caused more lethal effects (Fig.5D). Most interestingly, both

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Pluronic® F127 and Pluronic® F68 caused a deformation of the chorion within minutes of exposure. The chorion appeared “creased”. We propose that this might be due to an osmotic loss of the chorion fluid, which reduces the pressure and thus “wrinkles” the chorion. A similar

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effect was observed in one of our earlier studies where we investigated the toxicity of dendrimeric PAMAM (polyamidoamine) nanoparticles which was also hypothesized to be due

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to osmotic alterations (Bodewein, Schmelter et al. 2016). With the chorion “creased”, the space for the developing embryo inside the egg was clearly reduced and most likely had an influence

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on the observed malformations of head and chorda. Comparing Pluronic® F127 and Pluronic® F68 to Cremophor® A25 and Cremophor® RH40, the results of the present study demonstrated that the Pluronics were less toxic, with Pluronic® F127 showing the highest EC50-value (2.4%). However, Pluronic® F127 caused predominantly mortality, whereas Pluronic® F68 caused mainly sub-lethal effects at the tested concentrations. Pluronic® F127 and Pluronic® F68 impaired the development of the zebrafish embryos, causing severe malformations (3% Pluronic® F68 at 24 hpf) and deformations of the

chorion (at the lowest concentration at 24 hpf). As sub-lethal effects such as edema which were caused by Pluronic® F68 can recede during further development and embryos exposed to Pluronic® F68 displayed lesser mortal effects at the tested concentrations compared to Pluronic® F127, Pluronic® F68 was identified to have the overall lowest toxic effect of the tested surfactants. It should be mentioned that the amounts of surfactants used in the present study are considerably higher than the amounts of surfactants commonly used in the final drug products

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and the even lower concentrations usually found in the environment.

5. Conclusion Our comparison of the nanomaterials and surfactants used in the manufacture of nanomedicines indicated that, with regards to their ecotoxic potential, HPMCP and Pluronic® F68 are the most suitable excipients. These results of the study provide valuable advice for the types of polymers and surfactants and the range of concentrations to be used in future formulations of nanomedicines. For further investigations, it would be important to test for any interdependency of the polymeric

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nanomaterials and the surfactants and to study the effects of mixtures. It was shown that the toxicity of metal nanoparticles can, for instance, be altered by the presence of surfactants (Oleszczuk, Josko et al. 2015). Toxic effects of the tested substances were observed and substances such as nonionic surfactants are used in large quantities in other fields of

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application and are thus likely to enter the aquatic environment. Therefore, more data should be acquired to conduct an environmental risk assessment. Moreover, the effects of polymeric

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nanomaterials, surfactants and their mixtures on other aquatic organisms, such as Daphnia

trophic levels.

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Formatting of funding sources

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magna or algae, need to be investigated to gain a better understanding of the toxicity at all

The authors acknowledge the German Environmental Foundation for financial support to

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project no. AZ32725 and the research funding program Landes-Offensive zur Entwicklung Wissenschaftlich ökonomischer Exzellenz (LOEWE) of the State of Hessen, Research Center

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for Translational Medicine and Pharmacology TMP. Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We want to thank MJR Pharmjet GmbH for providing the test substances and Prof. Dr. Oehlmann and Andrea Misovic (Goethe University, Frankfurt a.M., Germany ) for conducting the YES-assay and for providing the results of the YES-assay. Matthias G. Wacker also acknowledges the National University of Singapore, Office of the Deputy President for Research & Technology (WBS R-148-000-282-133) and the Faculty of Science (R-148-000-

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282-733) for financial support.

References

Alasino, R. V., S. F. Ausar, I. D. Bianco, L. F. Castagna, M. Contigiani and D. M. Beltramo (2005). "Amphipathic and membrane-destabilizing properties of the cationic acrylate polymer Eudragit E100." Macromol Biosci 5(3): 207-213. doi.org/10.1002/mabi.200400168. Arshad, A., B. Yang, A. S. Bienemann, N. U. Barua, M. J. Wyatt, M. Woolley, D. E. Johnson, K. J. Edler and S. S. Gill (2015). "Convection-Enhanced Delivery of Carboplatin PLGA Nanoparticles for the

ro of

Treatment of Glioblastoma." PLoS One 10(7): e0132266. doi: 10.1371/journal.pone.0132266. Atyabi, F. and R. Dinarvand (2012). "Chitosan-Pluronic nanoparticles as oral delivery of anticancer gemcitabine: preparation and in vitro study." International Journal of Nanomedicine 7: 1851-1863. doi: 10.2147/IJN.S26365.

Bai, W., Z. Zhang, W. Tian, X. He, Y. Ma, Y. Zhao and Z. Chai (2009). "Toxicity of zinc oxide nanoparticles

-p

to zebrafish embryo: a physicochemical study of toxicity mechanism." Journal of Nanoparticle Research 12(5): 1645-1654. doi: 10.1007/s11051-009-9740-9.

re

Baskaran, R., T. Madheswaran, P. Sundaramoorthy, H. M. Kim and B. K. Yoo (2014). "Entrapment of curcumin into monoolein-based liquid crystalline nanoparticle dispersion for enhancement of stability

lP

and anticancer activity." Int J Nanomedicine 9: 3119-3130. doi: 10.2147/IJN.S61823. Berkner, S., K. Schwirn, and D. Voelker (2016). "Nanopharmaceuticals: Tiny challenges for the environmental risk assessment of pharmaceuticals". Environmental Toxicology and Chemistry 35: 780-

na

787. doi: 10.1002/etc.3039.

Bodewein, L., F. Schmelter, S. Di Fiore, H. Hollert, R. Fischer and M. Fenske (2016). "Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines."

ur

Toxicol Appl Pharmacol 305: 83-92. doi: 10.1016/j.taap.2016.06.008. Bonsignorio, D., L. Perego, L. Del Giacco and F. Cotelli (1996). "Structure and macromolecular

Jo

composition of the zebrafish egg chorion." Zygote 4: 101-108. doi:10.1017/S0967199400002975. Braunbeck, T. and E. Lammer (2006). "Background Paper on Fish Embryo Toxicity Assays. Prepared for German Federal Environment Agency". UBA (UBA Contract Number 203 85 422). Cetin, M., A. Atila and Y. Kadioglu (2010). "Formulation and in vitro characterization of Eudragit(R) L100 and Eudragit(R) L100-PLGA nanoparticles containing diclofenac sodium." AAPS PharmSciTech 11(3): 1250-1256. doi: 10.1208/s12249-010-9489-6. Chang, R. K., Y. Peng and A. J. Shukla (2006). "Polymethacrylates", in: Rowe, R.C., P.J. Sheskey and S. C. Owen (Eds.), Handbook of Pharmaceutical Excipients, fifth ed. Pharmaceutical Press and American Pharmacists Association , London : Washington D.C., pp. 553–560.

Chen, M. C., K. Sonaje, K. J. Chen and H. W. Sung (2011). "A review of the prospects for polymeric nanoparticle

platforms

in

oral

insulin

delivery."

Biomaterials

32(36):

9826-9838.

doi:10.1016/j.biomaterials.2011.08.087. Cheng J., E. Flahaut and S. H. Cheng (2007). "Effect of carbon nanotubes on developing zebrafish (Danio rerio) embryos." Environmental Toxicology and Chemistry 26(4): 708-716. doi: 10.1897/06-272r.1. Cheng, J., B. A. Teply, I. Sherifi, J. Sung, G. Luther, F. X. Gu, E. Levy-Nissenbaum, A. F. Radovic-Moreno, R. Langer and O. C. Farokhzad (2007). "Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery." Biomaterials 28(5): 869-876. doi:10.1016/j.biomaterials.2006.09.047. Cheung, C. H., J. M. Chiu, R. S. Wu (2014). "Hypoxia turns genotypic female medaka fish into phenotypic males". Ecotoxicology 23 (7): 1260-1269. doi: 10.1007/s10646-014-1269-8.

ro of

Colzani, B., L. Pandolfi, A. Hoti, P. A. Iovene, A. Natalello, S. Avvakumova, M. Colombo and D. Prosperi (2018). "Investigation of antitumor activities of trastuzumab delivered by PLGA nanoparticles." Int J Nanomedicine 13: 957-973. doi: 10.2147/IJN.S152742.

Commission Recommendation of 18 October 2011 on the definition of nanomaterial Text with EEA

-p

relevance (2011/696/EU) OJ L 275, 20.10.2011, p. 38–40 (BG, ES, CS, DA, DE, ET, EL, EN, FR, IT, LV, LT, HU, MT, NL, PL, PT, RO, SK, SL, FI, SV)

re

Danhier, F., E. Ansorena, J. M. Silva, R. Coco, A. Le Breton and V. Preat (2012). "PLGA-based nanoparticles: an overview of biomedical applications." J Control Release 161(2): 505-522. doi:10.1016/j.jconrel.2012.01.043.

lP

Das, D. and S. Lin (2005). "Double-coated poly (butylcynanoacrylate) nanoparticulate delivery systems for brain targeting of dalargin via oral administration." J Pharm Sci 94(6): 1343-1353. doi: 10.1002/jps.20357.

na

DIN EN ISO 7346-3. (1996-06). Water Quality - Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish (Brachydanio Rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) - Part 3: FlowThrough Method.

ur

DIN EN ISO 15088. (2009-06). Water quality - Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio).

Jo

DIN EN ISO 19040-1 (2018-08). Water quality – Determination of the estrogenic potential of water and waste water – Part 1: Yeast Estrogen Screen (Saccharomyces cerevisiae). Dinarvand, R., N. Sepehri, S. Manoochehri, H. Rouhani and F. Atyabi (2011). "Polylactide-co-glycolide nanoparticles for controlled delivery of anticancer agents." Int J Nanomedicine 6: 877-895. doi: 10.2147/IJN.S18905. Dinh, H. T. T., P. H. L. Tran, W. Duan, B. J. Lee and T. T. D. Tran (2017). "Nano-sized solid dispersions based on hydrophobic-hydrophilic conjugates for dissolution enhancement of poorly water-soluble drugs." Int J Pharm 533(1): 93-98. doi: 10.1016/j.ijpharm.2017.09.065.

Duan, J., Yu, Y., Shi, H., Tian, L. Guo, C. Huang, P. Zhou, X. Peng, S. Sun, Z. (2013). "Toxic effects of silica nanoparticles on zebrafish embryos and larvae". PLoS One 8 (9): e74606. doi: 10.1371/journal.pone.0074606. Duncan, R. (2006). "Polymer conjugates as anticancer nanomedicines." Nat Rev Cancer 6(9): 688-701. doi:10.1038/nrc1958. Eisele, J., G. Haynes and T. Rosamilia (2011). "Characterisation and toxicological behaviour of Basic Methacrylate Copolymer for GRAS evaluation." Regul Toxicol Pharmacol 61(1): 32-43. doi:10.1016/j.yrtph.2011.05.012. Erdal, M. S., G. Ozhan, M. C. Mat, Y. Ozsoy and S. Gungor (2016). "Colloidal nanocarriers for the enhanced cutaneous delivery of naftifine: characterization studies and in vitro and in vivo evaluations."

ro of

Int J Nanomedicine 11: 1027-1037. doi: 10.2147/IJN.S96243. European Union, 2010. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes L 276/33. The European Parliament and the Council of the European Union.Off. J. Eur. Union L276.

-p

European Medicines Agency: EMA/CHMP/SWP/362974/2012 Corr. 2 - Guideline on the use of phthalates as excipients in human medicinal products, 2014. Assessed: August, 2019. https://www.ema.europa.eu/en/use-phthalates-excipients-human-medicinal-products

re

Fako, V. E. and D. Y. Furgeson (2009). "Zebrafish as a correlative and predictive model for assessing biomaterial nanotoxicity." Adv Drug Deliv Rev 61(6): 478-486. doi:10.1016/j.addr.2009.03.008.

lP

Fent, K., C. J. Weisbrod, A. Wirth-Heller and U. Pieles (2010). "Assessment of uptake and toxicity of fluorescent silica nanoparticles in zebrafish (Danio rerio) early life stages." Aquat Toxicol 100(2): 218228. doi:10.1016/j.aquatox.2010.02.019.

na

Fischer, H. C. and W. C. Chan (2007). "Nanotoxicity: the growing need for in vivo study." Curr Opin Biotechnol 18(6): 565-571. doi: 10.1016/j.copbio.2007.11.008. Frampton, J. E. (2017). "Triptorelin: A Review of its Use as an Adjuvant Anticancer Therapy in Early

ur

Breast Cancer." Drugs 77(18): 2037-2048. doi: 10.1007/s40265-017-0849-3. Gentile, P., V. Chiono, I. Carmagnola and P. V. Hatton (2014). "An overview of poly(lactic-co-glycolic)

Jo

acid (PLGA)-based biomaterials for bone tissue engineering." Int J Mol Sci 15(3): 3640-3659. doi:10.3390/ijms15033640. Ghalamfarsa, G., M. Hojjat-Farsangi, M. Mohammadnia-Afrouzi, E. Anvari, S. Farhadi, M. Yousefi and F. Jadidi-Niaragh (2016). "Application of nanomedicine for crossing the blood-brain barrier: Theranostic opportunities in multiple sclerosis." J Immunotoxicol 13(5): 603-619. doi: 10.3109/1547691X.2016.1159264.

Griffitt, R. J., J. Luo, J. Gao, J.C. Bonzongo and D. S. Barber (2008). "Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms." Environmental Toxicology and Chemistry 27(9): 1972–1978. doi: 10.1897/08-002.1. Gunday Tureli, N., A. E. Tureli and M. Schneider (2016). "Counter-ion complexes for enhanced drug loading in nanocarriers: Proof-of-concept and beyond." Int J Pharm 511(2): 994-1001. doi: 10.1016/j.ijpharm.2016.08.004. Hari, B. N. V., N. Narayanan, K. Dhevendaran and D. Ramyadevi (2016). "Engineered nanoparticles of Efavirenz using methacrylate co-polymer (Eudragit-E100) and its biological effects in-vivo." Mater Sci Eng C Mater Biol Appl 67: 522-532. doi: 10.1016/j.msec.2016.05.064. Heinz, H., C. Pramanik, O. Heinz, Y. Ding, R. K. Mishra, D. Marchon, R. J. Flatt, I. Estrela-Lopis, J. Llop, S.

ro of

Moya and R. F. Ziolo (2017). "Nanoparticle decoration with surfactants: Molecular interactions, assembly, and applications." Surface Science Reports 72(1): 1-58. doi: 10.1016/j.surfrep.2017.02.001. Henn, K. and T. Braunbeck (2011). "Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)". Comp Biochem Physiol C Toxicol Pharmacol 153: 91-98. doi: 10.1016/j.cbpc.2010.09.003.

re

-p

Hosseinzadeh, H., F. Atyabi, R. Dinarvand and S. N. Ostad (2012). "Chitosan–Pluronic nanoparticles as oral delivery of anticancer gemcitabine: preparation and in vitro study.” Int J Nanomedicine 7:185163. doi: 10.2147/IJN.S26365. Hussan Singh Deep Roychowdhury Santanu, V. P. and V. Bhandari (2012). "A review on recent advances of enteric coating." IOSR Journal of Pharmacy 2(6): 5-11. doi:10.9790/3013-2610511.

lP

Ivankovic, T. and J. Hrenovic (2010). "Surfactants in the environment." Arh Hig Rada Toksikol 61(1): 95110. doi: 10.2478/10004-1254-61-2010-1943.

na

Jin, H. Y., F. Xia and Y. P. Zhao (2012). "Preparation of hydroxypropyl methyl cellulose phthalate nanoparticles with mixed solvent using supercritical antisolvent process and its application in coprecipitation of insulin." Advanced Powder Technology 23(2): 157-163. doi:10.1016/j.apt.2011.01.007.

ur

Kalita, S., B. Devi, R. Kandimalla, K. K. Sharma, A. Sharma, K. Kalita, A. C. Kataki and J. Kotoky (2015). " Chloramphenicol encapsulated in poly-epsilon-caprolactone-pluronic composite: nanoparticles for treatment of MRSA-infected burn wounds". Int J Nanomedicine 10: 2971-2984. doi:

Jo

10.2147/IJN.S75023.

Kalluru, R., F. Fenaroli, D. Westmoreland, L. Ulanova, A. Maleki, N. Roos, M. Paulsen Madsen, G. Koster, W. Egge-Jacobsen, S. Wilson, H. Roberg-Larsen, G. K. Khuller, A. Singh, B. Nystrom and G. Griffiths (2013). "Poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear Mycobacterium bovis BCG infection in macrophages and remain membrane-bound in phago-lysosomes." J Cell Sci 126(Pt 14): 3043-3054. doi: 10.1242/jcs.121814.

Kola Srinivas, N. S., R. Verma, G. Pai Kulyadi and L. Kumar (2016). "A quality by design approach on polymeric nanocarrier delivery of gefitinib: formulation, in vitro, and in vivo characterization." International Journal of Nanomedicine 12: 15-28. doi: 10.2147/ijn.s122729. Kreuter, J. (2001). "Nanoparticulate systems for brain delivery of drugs." Advanced Drug Delivery Reviews 47: 65-81. Kumar, J. and A. M. J. Newton (2016). "Colon Targeted Rifaximin Nanosuspension for the Treatment of Inflammatory Bowel Disease (IBD)." Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry 15(2): 101-117. doi: 10.2174/1871523015666160720103732 Kumari, A., S. K. Yadav and S. C. Yadav (2010). "Biodegradable polymeric nanoparticles based drug delivery systems." Colloids Surf B Biointerfaces 75(1): 1-18. doi: 10.1016/j.colsurfb.2009.09.001.

ro of

Lee, K. J., P. D. Nallathamby, L. M. Browning, C. J. Osgood and X. H. Xu (2007). "In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos." ACS Nano 1(2): 133-143. doi: 10.1021/nn700048y.

Lewis, M. A. (1991). "Chronic and sublethal toxicities of surfactants to aquatic animals: a review and

-p

risk assessment." Wat. Res. 25(1): 101-113.

Ma, Y., Y. Zheng, X. Zeng, L. Jiang, H. Chen, R. Liu, L. Huang and L. Mei (2011). "Novel docetaxel-loaded

re

nanoparticles based on PCL-Tween 80 copolymer for cancer treatment." Int J Nanomedicine 6: 26792688. doi: 10.2147/IJN.S25251.

Madheswaran, T., R. Baskaran, C. S. Yong and B. K. Yoo (2014). "Enhanced topical delivery of finasteride

lP

using glyceryl monooleate-based liquid crystalline nanoparticles stabilized by cremophor surfactants." AAPS PharmSciTech 15(1): 44-51. doi: 10.1208/s12249-013-0034-2. Mahapatro, A. and D. K. Singh (2011). "Biodegradable nanoparticles are excellent vehicle for site

na

directed in-vivo delivery of drugs and vaccines." J Nanobiotechnology 9: 55. doi: 10.1186/1477-31559-55.

ur

Makadia, H. K. and S. J. Siegel (2011). "Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier." Polymers (Basel) 3(3): 1377-1397. doi:10.3390/polym3031377. Malam, Y., M. Loizidou and A. M. Seifalian (2009). "Liposomes and nanoparticles: nanosized vehicles

Jo

for drug delivery in cancer." Trends Pharmacol Sci 30(11): 592-599. doi:10.1016/j.tips.2009.08.004. Marcato, P. D. and N. Durán (2008). "New Aspects of Nanopharmaceutical Delivery Systems." Journal of Nanoscience and Nanotechnology 8(5): 2216-2229. doi: 10.1166/jnn.2008.274. Mei, L., Y. Zhang, Y. Zheng, G. Tian, C. Song, D. Yang, H. Chen, H. Sun, Y. Tian, K. Liu, Z. Li and L. Huang (2009). "A Novel Docetaxel-Loaded Poly (epsilon-Caprolactone)/Pluronic F68 Nanoparticle Overcoming Multidrug Resistance for Breast Cancer Treatment." Nanoscale Res Lett 4(12): 1530-1539. doi: 10.1007/s11671-009-9431-6.

Meyer, R. A. and J. J. Green (2016). "Shaping the future of nanomedicine: anisotropy in polymeric nanoparticle design." Wiley Interdiscip Rev Nanomed Nanobiotechnol 8(2): 191-207. doi: 10.1002/wnan.1348. Mu, J., M. Chernick, W. Dong, R. T. Di Giulio and Hinton, D. E. (2017). " Early life co-exposures to a realworld PAH mixture and hypoxia result in later life and next generation consequences in medaka (Oryzias latipes)." Aquat Toxicol 190: 162-173. doi: 10.1016/j.aquatox.2017.06.026. Muth-Köhne, E., L. Sonnack, K. Schlich, F. Hischen, W. Baumgartner, K. Hund-Rinke, C. Schäfers and M. Fenske (2013). "The toxicity of silver nanoparticles to zebrafish embryos increases through sewage treatment processes." Ecotoxicology 22(8): 1264-1277. doi: 10.1007/s10646-013-1114-5. Oberdörster, G., E. Oberdörster and J. Oberdörster (2005). "Nanotoxicology: An Emerging Discipline

ro of

Evolving from Studies of Ultrafine Particles." Environmental Health Perspectives 113(7): 823-839. doi: 10.1289/ehp.7339.

OECD, 2013. Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Publishing, Paris, France, pp. 1–22.

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Oleszczuk, P., I. Josko and E. Skwarek (2015). "Surfactants decrease the toxicity of ZnO, TiO2 and Ni nanoparticles to Daphnia magna." Ecotoxicology 24(9): 1923-1932. doi: 10.1007/s10646-015-1529-2. Olkowska, E., M. Ruman and Ż. Polkowska (2014). "Occurrence of Surface Active Agents in the

re

Environment." Journal of Analytical Methods in Chemistry 2014: 769708. doi: 10.1155/2014/769708. Ozel, R. E., R. S. Alkasir, K. Ray, K. N. Wallace and S. Andreescu (2013). "Comparative evaluation of

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intestinal nitric oxide in embryonic zebrafish exposed to metal oxide nanoparticles." Small 9(24): 42504261. doi: 10.1002/smll.201301087.

Prabhu, R. H., V. B. Patravale and M. D. Joshi (2015). "Polymeric nanoparticles for targeted treatment

na

in oncology: current insights." Int J Nanomedicine 10: 1001-1018. doi: 10.2147/IJN.S56932.

ur

Quinteros, D. A., V. R Rigo, A. F.Kairuz, M. E. Olivera, R. H. Manzo and D. A. Allemandi (2008). "Interaction between a cationic polymethacrylate (Eudragit E100) and anionic drugs." Eur J Pharm Sci 33(1): 72-79. doi:10.1016/j.ejps.2007.10.002.

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Quinteros, D. A., D. A. Allemandi and R. H. Manzo (2012). "Equilibrium and release properties of aqueous dispersions of non-steroidal anti-inflammatory drugs complexed with polyelectrolyte Eudragit E 100." Sci Pharm 80(2): 487-496. doi:10.3797/scipharm.1107-17. Rawson D. M., T. Zhang, D. Kalicharan and W. L. Jongebloed (2000). " Fieldemissionscanningelectronmicroscopyandtransmissionelectronmicroscopystudiesofthechorion,pl asmamembraneandsyncytiallayersofthegastrula stageembryoofthezebra®shBrachydaniorerio:aconsiderationofthestructuralandfunctionalrelationship swithrespecttocryoprotectantpenetration". Aquaculture Research 31: 325-336. doi: 10.1046/j.13652109.2000.00401.x Rizzo, L. Y., S. K. Golombek, M. E. Mertens, Y. Pan, D. Laaf, J. Broda, J. Jayapaul, D. Mockel, V. Subr, W. E. Hennink, G. Storm, U. Simon, W. Jahnen-Dechent, F. Kiessling and T. Lammers (2013). "In Vivo

Nanotoxicity Testing using the Zebrafish Embryo Assay." J Mater Chem B 1: 3918–3925. doi:10.1039/C3TB20528B. Sharma, M., V. Sharma, A. K. Panda and D. K. Majumdar (2011). "Development of enteric submicron particle formulation of papain for oral delivery." Int J Nanomedicine 6: 2097-2111. doi: 10.2147/IJN.S23985. Sonnack, L., S. Kampe, E. Muth-Kohne, L. Erdinger, N. Henny, H. Hollert, C. Schafers and M. Fenske (2015). "Effects of metal exposure on motor neuron development, neuromasts and the escape response of zebrafish embryos." Neurotoxicol Teratol 50: 33-42. doi: 10.1016/j.ntt.2015.05.006. Spence, R., G. Gerlach, C. Lawrence and C. Smith (2008). "The behaviour and ecology of the zebrafish,

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Danio rerio." Biological Reviews 83(1): 13-34. doi: 10.1111/j.1469-185X.2007.00030.x Strahle, U., S. Scholz, R. Geisler, P. Greiner, H. Hollert, S. Rastegar, A. Schumacher, I. Selderslaghs, C. Sun, S., N. Liang, H. Yamamoto, Y. Kawashima, F. Cui and P. Yan (2015). "pH-sensitive poly(lactide-coglycolide) nanoparticle composite microcapsules for oral delivery of insulin." Int J Nanomedicine 10: 3489-3498. doi: 10.2147/IJN.S81715.

-p

Tao, L., W. Hu, Y. Liu, G. Huang, B. D. Sumer and J. Gao (2011). "Shape-specific polymeric nanomedicine: emerging opportunities and challenges." Exp Biol Med (Maywood) 236(1): 20-29. doi:

re

10.1258/ebm.2010.010243.

Tian, X. H., X. N. Lin, F. Wei, W. Feng, Z. C. Huang, P. Wang, L. Ren and Y. Diao (2011). "Enhanced brain

lP

targeting of temozolomide in polysorbate-80 coated polybutylcyanoacrylate nanoparticles." Int J Nanomedicine 6: 445-452. doi: 10.2147/IJN.S16570.

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Tierschutz-Versuchstierverordnung vom 1. August 2013 (BGBl. I S. 3125, 3126), die zuletzt durch Artikel 394 der Verordnung vom 31. August 2015 (BGBl. I S. 1474) geändert worden ist. U.S. Food and Drug Administration/Center for Drug Evaluation and Research (2007). New Drug Application 22-210 Zenpep (pancrelipase) Delayed-Release Capsules. PHARMACOLOGY REVIEW(S).

ur

Wang, Y., Y. Zhang, X. Li, M. Sun, Z. Wei, Y. Wang, A. Gao, D. Chen, X. Zhao and X. Feng (2015). "Exploring the Effects of Different Types of Surfactants on Zebrafish Embryos and Larvae." Sci Rep 5:

Jo

10107. doi: 10.1038/srep10107.

Yallapu, M. M., M. Jaggi and S. C. Chauhan (2013). "Curcumin Nanomedicine: A Road to Cancer Therapeutics." Curr Pharm Des. 19(11): 1994-2010. doi: 10.2174/138161213805289219. Zhang, Z., H. Li, G. Xu and P. Yao (2018). "Liver-targeted delivery of insulin-loaded nanoparticles via enterohepatic

circulation

of

bile

acids."

Drug

Deliv

25(1):

1224-1233.

doi:

10.1080/10717544.2018.1469685. Zhu, X. and R. D. Braatz (2015). "A mechanistic model for drug release in PLGA biodegradable stent coatings coupled with polymer degradation and erosion." J Biomed Mater Res A 103(7): 2269-2279. doi:10.1002/jbm.a.35357.

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List of Figures/ Tables Figures Fig.1: Lethal and sub-lethal effects of the polymeric nanosuspensions of Eudragit® E100 (A), HPMCP (B) and PLGA (C) at 72 hpf. The white columns depict the cumulative effects caused by the substances, expressed as percentage of embryos affected by any effect. The mortality and the sub-lethal effects edema, slower blood flow and no-hatching are shown individually. Concentrations are given in mg·ml-1. The lethal effects encompass coagulation, no detachment of tail and the absence of a heart

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beat after 48 hpf. * – significant difference to the control (p≤0.05).

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Fig.2: EC50- (A) and LC50-values (B) of the tested nanomaterials. Concentrations are given in mg·ml-1. Regression analysis of the EC50- and LC50-curves was performed by probit-analysis (ToxRat). The error bars display the 95%confidence intervals. * – significant difference to the control (p≤0.05).

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Fig.3: Estrogenic activity of HPMCP. Estrogenic activity was measured in 17β-estradiol equivalent concentration EEQ [ng·L-1] using the YES-assay. The x-axis describes the dissolution-ratio of the HPMCP-nanosuspenson. HPMCP did not display endocrine

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activity.

Fig.4: LM-micrographs of zebrafish embryo exposed to PLGA. Embryos exposed to1.25 mg·ml-1 PLGA-nanosuspensions at 48 hpf and 72 hpf display a discolored chorion compared to the control embryo.

Fig.5: Lethal and sub-lethal effects of the surfactants Cremophor® A25 (A), Cremophor® RH40 (B), Pluronic® F68 (C) and Pluronic® F127 (D) at 72 hpf. The white columns depict the overall effects caused by the substances. The sub-lethal effects of

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edema, slower blood flow as well as lethal effects are shown individually. The lethal effects were coagulation and no heartbeat. *

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– significant difference to the control (p≤0.05).

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Fig.7: LM-micrographs showing teratogenic effects in zebrafish embryo exposed to Pluronic® F68 at a concentration of 4.5 % at 24 hpf (A) and 72 hpf (B) and a concentration of 6 % at 24 hpf (C) and 72 hpf (D). Effects of Pluronic® F127 on a zebrafish embryo exposed to concentration of 5 % are shown for 24 hpf (E) and 72 hpf (F). The chorion appears creased and lost its spherical shape. The embryos display sub-lethal effects ranging from malformations of head and tail to edema (shown by arrows).

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Precipitation of the surfactants is shown at the bottom of the wells.

Tables Table 1: Nominal concentrations of tested excipients in w/v % and ‰, respectively.

Name of Excipient

Tested Concentration [w/v %]

1.5 / 3.0 / 4.5 / 6

Pluronic F68

1.25 / 2.5 / 5.0 / 10

Pluronic F127

0.005 ‰/ 0.05 ‰/ 0.5 ‰ / 5 ‰

Cremophor A25

0.005 ‰/ 0.05 ‰/ 0.5 ‰/ 5 ‰

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Cremophor RH40

Table 2: EC50- and LC50-values in [mg·ml-1 ] of PLGA, HPMCP and Eudragit® E 100 with the corresponding 95 % confidence

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intervals (CI) in brackets for sub-lethal and lethal morphological effects calculated using probit analysis (ToxRat v2.10, ToxRat Germany). The analysis was performed for the two replicates of each test repetition. For HPMCP and PLGA no LC50-value could

Table 2

72 hpf

n.d. (n.d.)

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Eudragit® E100

0.063 (n.d.) 0.269 (n.d.)

72 hpf

0.002 (0.000/0.004) n.d. (n.d.) n.d. (n.d.)

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PLGA HPMCP

LC 50 [mg/ml]

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EC 50 [mg/ml]

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be determined, as under 20% of the exposed embryos displayed lethal effects.

Table 3: EC50- and LC50-values in [%] of embryos exposed to Cremophor® A25, Cremophor® RH40, Pluronic® F68 and Pluronic®

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F127. CI – 95% Confidence Intervals in brackets below. Analysis was performed with ToxRat v2.10, ToxRat Germany.

Table 3

Cremophor® RH 40 Cremophor® A25 Pluronic® F68

EC50 [%]

LC50 [%]

72 hpf

72 hpf

0.037 (n.d.) 0.003 (0.001 / 0.007) 1,599 (1.444/1.740)

0.081 (n.d.) 0.006 (n.d.) n.d. (n.d.)

2,389 (1.611/ 3.127)

5,209 (3.445/ 10.214)

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Pluronic® F127