Acute Aquatic Toxicity to Zebrafish and Bioaccumulation in Marine Mussels of Antimony Tin Oxide Nanoparticles.

Antimony tin oxide (Sb2O5/SnO2) is effective in the absorption of infrared radiation for applications, such as skylights. As a nanoparticle (NP), it can be incorporated into films or sheets providing infrared radiation attenuation while allowing for a transparent final product. The acute toxicity exerted by commercial Sb2O5/SnO2 (ATO) NPs was studied in adults and embryos of zebrafish (Danio rerio). Our results suggest that these NPs do not induce an acute toxicity in zebrafish, either adults or embryos. However, some sub-lethal parameters were altered: heart rate and spontaneous movements. Finally, the possible bioaccumulation of these NPs in the aquacultured marine mussel Mytilus sp. was studied. A quantitative analysis was performed using single particle inductively coupled plasma mass spectrometry (sp-ICP-MS). The results indicated that, despite being scarce (2.31 × 106 ± 9.05 × 105 NPs/g), there is some accumulation of the ATO NPs in the mussel. In conclusion, commercial ATO NPs seem to be quite innocuous to aquatic organisms; however, the fact that some of the developmental parameters in zebrafish embryos are altered should be considered for further investigation. More in-depth analysis of these NPs transformations in the digestive tract of humans is needed to assess whether their accumulation in mussels presents an actual risk to humans.

Protection against Paraquat-Induced Oxidative Stress by Curcuma longa Extract-Loaded Polymeric Nanoparticles in Zebrafish Embryos.

The link between oxidative stress and environmental factors plays an important role in chronic degenerative diseases; therefore, exogenous antioxidants could be an effective alternative to combat disease progression and/or most significant symptoms. Curcuma longa L. (CL), commonly known as turmeric, is mostly composed of curcumin, a multivalent molecule described as having antioxidant, anti-inflammatory and neuroprotective properties. Poor chemical stability and low oral bioavailability and, consequently, poor absorption, rapid metabolism, and limited tissue distribution are major restrictions to its applicability. The advent of nanotechnology, by combining nanosacale with multi-functionality and bioavailability improvement, offers an opportunity to overcome these limitations. Therefore, in this work, poly-Ɛ-caprolactone (PCL) nanoparticles were developed to incorporate the methanolic extract of CL, and their bioactivity was assessed in comparison to free or encapsulated curcumin. Their toxicity was evaluated using zebrafish embryos by applying the Fish Embryo Acute Toxicity test, following recommended OECD guidelines. The protective effect against paraquat-induced oxidative damage of CL extract, free or encapsulated in PCL nanoparticles, was evaluated. This herbicide is known to cause oxidative damage and greatly affect neuromotor functions. The overall results indicate that CL-loaded PCL nanoparticles have an interesting protective capacity against paraquat-induced damage, particularly in neuromuscular development that goes well beyond that of CL extract itself and other known antioxidants.

pH-sensitive nanoliposomes for passive and CXCR-4-mediated marine yessotoxin delivery for cancer therapy.

Background: Yessotoxin (YTX), a marine-derived drug, was encapsulated in PEGylated pH-sensitive nanoliposomes, covalently functionalized (strategy I) with SDF-1α and by nonspecific adsorption (strategy II), to actively target chemokine receptor CXCR-4. Methods: Cytotoxicity to normal human epithelial cells (HK-2) and prostate (PC-3) and breast (MCF-7) adenocarcinoma models, with different expression levels of CXCR-4, were tested. Results: Strategy II exerted the highest cytotoxicity toward cancer cells while protecting normal epithelia. Acid pH-induced fusion of nanoliposomes seemed to serve as a primary route of entry into MCF-7 cells but PC-3 data support an endocytic pathway for their internalization. Conclusion: This work describes an innovative hallmark in the current marine drug clinical pipeline, as the developed nanoliposomes are promising candidates in the design of groundbreaking marine flora-derived anticancer nanoagents.

Novel concept of exosome-like liposomes for the treatment of Alzheimer's disease.

Exosomes are cell-derived vesicles that act as carriers for proteins and nucleic acids, with therapeutic potential and high biocompatibility. We propose a new concept of exosome-like liposomes for controlled delivery. The goal of this work was to develop a new type of liposomes with a unique mixture of phospholipids, similar to naturally occurring exosomes but overcoming their limitations of heterogeneity and low productivity, for therapeutic delivery of bioactive compounds. Curcumin was chosen as model compound, as it is a phytochemical molecule known to have antioxidant and anti-inflammatory properties, which can protect the brain against oxidative stress and reduce ß-amyloid accumulation, major hallmarks of Alzheimer's disease (AD). These new liposomes can efficiently encapsulate hydrophobic curcumin, yielding particles with a size smaller than 200 nm, and a polydispersity index lower than 0.20, which make them ideal for crossing the blood-brain barrier. These particles have a long shelf life, being stable up to 6 months. The curcumin encapsulation efficiency was higher than 85% (up to approximately 94%). Curcumin-loaded liposomes were not cytotoxic (up to 20 µM curcumin, and 200 µM of exo-liposomes), and significantly reduced oxidative stress induced in SH-SY5Y neuronal cells, indicating their potential for neuroprotection. They also do not show any toxicity and are internalized in zebrafish embryos, concentrating in lipid enriched areas, as the brain and the yolk sac. Such innovative carriers are a new effective approach to deliver drugs into the brain, as these are stable, protect the cargo and are uptaken by neuronal cells. Upon internalization, liposomes release the therapeutic biomolecules, resulting in successful neuroprotection, being a positive alternative strategy for AD therapy.

Extraction of Ibuprofen from Natural Waters Using a Covalent Organic Framework.

Ibuprofen is one of the most widely used pharmaceuticals, and due to its inefficient removal by conventional wastewater treatment, it can be found in natural surface waters at high concentrations. Recently, we demonstrated that the TpBD-(CF3)2 covalent organic framework (COF) can adsorb ibuprofen from ultrapure water with high efficiency. Here, we investigate the performance of the COF for the extraction of ibuprofen from natural water samples from a lake, river, and estuary. In general, the complexity of the natural water matrix induced a reduction in the adsorption efficiency of ibuprofen as compared to ultrapure water. The best performance, with over 70% adsorption efficiency, was found in lake water, the sample which featured the lowest pH. According to the theoretical calculations, ibuprofen more favorably interacts with the COF pores in the protonated form, which could partially account for the enhanced adsorption efficiency found in lake water. In addition, we explored the effect of the presence of competing pharmaceuticals, namely, acetaminophen and phenobarbital, on the ibuprofen adsorption as binary mixtures. Acetaminophen and phenobarbital were adsorbed by TpBD-(CF3)2 with low efficiency and their presence led to an increase in ibuprofen adsorption in the binary mixtures. Overall, this study demonstrates that TpBD-(CF3)2 is an efficient adsorbent for the extraction of ibuprofen from natural waters as well.

Are TiO2 nanoparticles safe for photocatalysis in aqueous media?

Although environmental and toxicity concerns are inherently linked, catalysis using photoactive nanoparticles and their hazardous potential are usually addressed independently. A toxicological assessment under the application framework is particularly important, given the pristine nanoparticles tend to change characteristics during several processes used to incorporate them into products. Herein, an efficient TiO2-functionalized macroporous structure was developed using widely adopted immobilization procedures. The relationships between photocatalysis, catalyst release and associated potential environmental hazards were assessed using zebrafish embryonic development as a proxy. Immobilized nanoparticles demonstrated the safest approach to the environment, as the process eliminates remnant additives while preventing the release of nanoparticles. However, as acute sublethal effects were recorded in zebrafish embryos at different stages of development, a completely safe release of TiO2 nanoparticles into the aquatic environment cannot be advocated.

Internalization of Methotrexate Conjugates by Folate Receptor-α.

The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types. The entry of methotrexate into the cell is mediated by two main transport systems: the reduced folate carrier and membrane-associated folate receptors. These transporters differ considerably in their mechanism of (anti)folate uptake, substrate specificity, and tissue specificity. Although the mechanism of action of the reduced folate carrier is fairly well-established, that of the folate receptor has remained unknown. The development of specific folate receptor-targeted antifolates would be accelerated if additional mechanistic data were to become available. In this work, we used two fluorescently labeled conjugates of methotrexate, differently linked at the terminal groups, to clarify the uptake mechanism by folate receptor-α. The results demonstrate the importance of methotrexate amino groups in the interaction with folate receptor-α.

Adsorption of marine phycotoxin okadaic acid on a covalent organic framework.

Phycotoxins, compounds produced by some marine microalgal species, can reach high concentrations in the sea when a massive proliferation occurs, the so-called harmful algal bloom. These compounds are especially dangerous to human health when concentrated in the digestive glands of seafood. In order to generate an early warning system to alert for approaching toxic outbreaks, it is very important to improve monitoring methods of phycotoxins in aquatic ecosystems. Solid-phase adsorption toxin tracking devices reported thus far based on polymeric resins have not been able to provide an efficient harmful algal bloom prediction system due to their low adsorption capabilities. In this work, a water-stable covalent organic framework (COF) was evaluated as adsorbent for the hydrophobic toxin okadaic acid, one of the most relevant marine toxins and the parental compound of the most common group of toxins responsible for the diarrhetic shellfish poisoning. Adsorption kinetics of okadaic acid onto the COF in seawater showed that equilibrium concentration was reached in only 60min, with a maximum experimental adsorption of 61mgg-1. Desorption of okadaic acid from the COF was successful with both 70% ethanol and acetonitrile as solvent, and the COF material could be reused with minor losses in adsorption capacity for three cycles. The results demonstrate that COF materials are promising candidates for solid-phase adsorption in water monitoring devices.

Peptide Anchor for Folate-Targeted Liposomal Delivery.

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.

Folic acid-tagged protein nanoemulsions loaded with CORM-2 enhance the survival of mice bearing subcutaneous A20 lymphoma tumors.

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, is reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly. FROM THE CLINICAL EDITOR: In this research, the authors developed folic acid tagged nanoemulsions containing a carbon monoxide releasing protein molecule for targeted cancer cell treatment. In-vitro and in-vivo experiments showed efficacy against B-cell lymphoma cells. The same nanocarrier platform could be applied to other tumor cells expressing folate receptors on the cell surface.

Phosphorylated silk fibroin matrix for methotrexate release.

Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated ß-sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin.

Phosphorylation of silk fibroins improves the cytocompatibility of silk fibroin derived materials: a platform for the production of tuneable material.

Silk fibroin demonstrates great biocompatibility and is suitable for many biomedical applications, including tissue engineering and regenerative medicine. Current research focuses on manipulating the physico-chemical properties of fibroin, and examining the effect of this manipulation on firobin's biocompatibility. Regenerated silk fibroin was modified by in vitro enzymatic phosphorylation and cast into films. Films were produced by blending, at several ratios, the phosphorylated and un-phosphorylated fibroin solutions. Fourier transform infra-red spectroscopy was used to determine the specific P-OH vibration peak, confirming the phosphorylation of the regenerated silk fibroin solution. Differential scanning calorimetry showed that phosphorylation altered the intra- and inter-molecular interactions. Further experiments demonstrated that phosphorylation can be used to tailor the hydrophylicity/hydrophobicity ratio as well as the crystalinity of silk fibroin films. Release profiling of a model drug was highly dependent on silk modification level. Cytotoxicity assays showed that exposure to lixiviates of phosphorylated films only slightly affected cellular metabolism and proliferation, although direct contact resulted in a strong direct correlation between phosphorylation level and cell proliferation. This new method for tuning silk biomaterials to obtain specific structural and biochemical features can be adapted for a wide range of applications. Phosphorylation of silk fibroins may be applied to improve the cytocompatibility of any silk-based device that is considered to be in contact with live animals or human tissues.

Estrogenic chemical effects are independent from the degree of sex role reversal in pipefish.

Endocrine disrupting chemicals (EDCs) have been reported to disturb several ecological relevant endpoints. Surprisingly, EDC-induced effects on fish sexual behaviour have been poorly studied despite the fact that even subtle alterations might contribute to a disruption of sexual interactions, thus negatively impacting reproduction. As the few assessments on sexual behaviour have been conducted in species with orthodox sex roles, it might be argued that sex-role reversed species might provide a potentially complementary system to further explore the effects of EDCs on reproduction. In the present study, two pipefish species with distinct degrees of sex-role reversal were selected to further elucidate the impact of chronic EE2 exposure on sexual behaviour and reproduction-related endpoints. The obtained results indicate that, independently of the degree of sex role reversal, courtship behaviour seems to resist oestrogenic chemical exposure. However, exposure to environmentally relevant EE2 levels did induce a complete absence of pregnancies at 18 ng/L. Even though pregnancies were observed at intermediate concentrations, the percentage of non-transferred or misplaced oocytes increased and a dose-dependent decrease of oocyte volume was observed. Imbalances in the oogenesis process, induction of vitellogenin in males and the absence of pregnancies highlight that environmental relevant concentrations of EE2 have the potential to negatively affect pipefish populations, most of them inhabiting coastal areas where oestrogenic contamination is more prevalent.