αS1-Casein-Loaded Proteo-liposomes as Potential Inhibitors in Amyloid Fibrillogenesis: In Vivo Effects on a C. elegans Model of Alzheimer's Disease.

According to the amyloid hypothesis, in the early phases of Alzheimer's disease (AD), small soluble prefibrillar aggregates of the amyloid ß-peptide (Aß) interact with neuronal membranes, causing neural impairment. Such highly reactive and toxic species form spontaneously and transiently in the amyloid building pathway. A therapeutic strategy consists of the recruitment of these intermediates, thus preventing aberrant interaction with membrane components (lipids and receptors), which in turn may trigger a cascade of cellular disequilibria. Milk αs1-Casein is an intrinsically disordered protein that is able to inhibit Aß amyloid aggregation in vitro, by sequestering transient species. In order to test αs1-Casein as an inhibitor for the treatment of AD, it needs to be delivered in the place of action. Here, we demonstrate the use of large unilamellar vesicles (LUVs) as suitable nanocarriers for αs1-Casein. Proteo-LUVs were prepared and characterized by different biophysical techniques, such as multiangle light scattering, atomic force imaging, and small-angle X-ray scattering; αs1-Casein loading was quantified by a fluorescence assay. We demonstrated on a C. elegans AD model the effectiveness of the proposed delivery strategy in vivo. Proteo-LUVs allow efficient administration of the protein, exerting a positive functional readout at very low doses while avoiding the intrinsic toxicity of αs1-Casein. Proteo-LUVs of αs1-Casein represent an effective proof of concept for the exploitation of partially disordered proteins as a therapeutic strategy in mild AD conditions.

Isolation of Extracellular Vesicles From Microalgae: A Renewable and Scalable Bioprocess.

Extracellular vesicles (EVs) play a crucial role as potent signal transducers among cells, with the potential to operate cross-species and cross-kingdom communication. Nanoalgosomes are a subtype of EVs recently identified and isolated from microalgae. Microalgae represent a natural bioresource with the capacity to produce several secondary metabolites with a broad range of biological activities and commercial applications. The present study highlights the upstream and downstream processes required for the scalable production of nanoalgosomes from cultures of the marine microalgae Tetraselmis chuii. Different technical parameters, protocols, and conditions were assessed to improve EVs isolation by tangential flow filtration (TFF), aiming to enhance sample purity and yield. The optimization of the overall bioprocess was enhanced by quality control checks operated through robust biophysical and biochemical characterizations. Further, we showed the possibility of recycling by TFF microalgae cells post-EVs isolation for multiple EV production cycles. The present results highlight the potential of nanoalgosome production as a scalable, cost-effective bioprocess suitable for diverse scientific and industrial exploitations.

Extracellular Vesicles From Microalgae: Uptake Studies in Human Cells and Caenorhabditis elegans.

Extracellular vesicles (EVs) are lipid membrane nano-sized vesicles secreted by various cell types for intercellular communication, found in all kingdoms of life. Nanoalgosomes are a subtype of EVs derived from microalgae with a sustainable biotechnological potential. To explore the uptake, distribution and persistence of nanoalgosomes in cells and living organisms, we separated them from a culture of the chlorophyte Tetraselmis chuii cells by tangential flow filtration (TFF), labelled them with different lipophilic dyes and characterized their biophysical attributes. Then we studied the cellular uptake of labelled nanoalgosomes in human cells and in C. elegans, demonstrating that they enter the cells through an energy dependent mechanism and are localized in the cytoplasm of specific cells, where they persist for days. Our data confirm that nanoalgosomes are actively uptaken in vitro by human cells and in vivo by C. elegans cells, supporting their exploitation as potential nanocarriers of bioactive compounds for theranostic applications.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Agarose/κ-carrageenan-based hydrogel film enriched with natural plant extracts for the treatment of cutaneous wounds.

Hydrogels for complex and chronic wound dressings must be conformable, absorb and retain wound exudates and maintain hydration. They can incorporate and release bioactive molecules that can accelerate the healing process. Wound dressings have to be in contact with the wound and epidermis, even for long periods, without causing adverse effects. Hydrogel dressing formulations based on biopolymers derived from terrestrial or marine flora can be relatively inexpensive and well tolerated. In the present article hydrogel films composed by agarose (1.0 wt%), κ-carrageenan at three different concentrations (0.5, 1.0 and 1.5 wt%) and glycerol (3.0 wt%) were prepared without recourse to crosslinking agents, and characterized for their mechanical properties, morphology, swelling and erosion behavior. The films resulted highly elastic and able to absorb and retain large amounts of fluids without losing their integrity. One of the films was loaded with the aqueous extract from Cryphaea heteromalla (Hedw.) D. Mohr for its antioxidant properties. Absence of cytotoxicity and ability to reduce the oxidative stress were demonstrated on NIH-3T3 fibroblast cell cultures. These results encourage further biological evaluations to assess their impact on the healing process.

Water Extract of Cryphaea heteromalla (Hedw.) D. Mohr Bryophyte as a Natural Powerful Source of Biologically Active Compounds.

Bryophytes comprise of the mosses, liverworts, and hornworts. Cryphaea heteromalla, (Hedw.) D. Mohr, is a non-vascular lower plant belonging to mosses group. To the date, the most chemically characterized species belong to the liverworts, while only 3.2% and 8.8% of the species belonging to the mosses and hornworts, respectively, have been investigated. In this work, we present Folin-Ciocalteu and oxygen radical absorbance capacity (ORAC) data related to crude extracts of C. heteromalla obtained by three different extraction solvents: pure water (WT), methanol:water (80:20 v/v) (MET), and ethanol:water (80:20 v/v) (ETH). The water extract proved to be the best solvent showing the highest content of biophenols and the highest ORAC value. The C. heteromalla-WT extract was investigated by HPLC-TOF/MS (High Performance Liquid Chromatography-Time of Flight/Mass Spectrometry) allowing for the detection of 14 compounds, five of which were phenolic compounds, derivatives of benzoic, caffeic, and coumaric acids. Moreover, the C. heteromalla WT extract showed a protective effect against reactive oxygen species (ROS) generation induced by tert-butyl hydroperoxide (TBH) on the murine NIH-3T3 fibroblast cell line.

Recombinant mussel protein Pvfp-5ß: A potential tissue bioadhesive.

During their lifecycle, many marine organisms rely on natural adhesives to attach to wet surfaces for movement and self-defense in aqueous tidal environments. Adhesive proteins from mussels are biocompatible and elicit only minimal immune responses in humans. Therefore these proteins have received increased attention for their potential applications in medicine, biomaterials, and biotechnology. The Asian green mussel Perna viridis secretes several byssal plaque proteins, molecules that help anchoring the mussel to surfaces. Among these proteins, protein-5ß (Pvfp-5ß) initiates interactions with the substrate, displacing interfacial water molecules before binding to the surface. Here, we established the first recombinant expression in Escherichia coli of Pvfp-5ß. We characterized recombinant Pvfp-5ß, finding that despite displaying a CD spectrum consistent with features of a random coil, the protein is correctly folded as indicated by MS and NMR analyses. Pvfp-5ß folds as a ß-sheet-rich protein as expected for an epidermal growth factor-like module. We examined the effects of Pvfp-5ß on cell viability and adhesion capacity in NIH-3T3 and HeLa cell lines, revealing that Pvfp-5ß has no cytotoxic effects at the protein concentrations used and provides good cell-adhesion strength on both glass and plastic plates. Our findings suggest that the adhesive properties of recombinant Pvfp-5ß make it an efficient surface-coating material, potentially suitable for biomedical applications including regeneration of damaged tissues.

Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures.

The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (α-La). α-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize α-La-membrane mechanisms of interaction and α-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of α-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an increase in membrane fluidity.

Comparison between iMSD and 2D-pCF analysis for molecular motion studies on in vivo cells: The case of the epidermal growth factor receptor.

Image correlation analysis has evolved to become a valuable method of analysis of the diffusional motion of molecules in every points of a live cell. Here we compare the iMSD and the 2D-pCF approaches that provide complementary information. The iMSD method provides the law of diffusion and it requires spatial averaging over a small region of the cell. The 2D-pCF does not require spatial averaging and it gives information about obstacles for diffusion at pixel resolution. We show the analysis of the same set of data by the two methods to emphasize that both methods could be needed to have a comprehensive understanding of the molecular diffusional flow in a live cell.