Eder Lilia Romero

Archaeal lipids are a source of new biomaterials for pharmaceutical and nanomedical applications; however, their classical extraction method relies on chloroform and methanol, toxic solvents that conflict with green chemistry principles. In this paper, we explore the performance of an eco-friendly method for the extraction of total lipids from the haloarchaea Halorubrum tebenquichense. Using the bio-solvents ethyl acetate and ethanol in a two-step procedure, a fraction of total lipids (135 ± 41 mg phospholipids and 1.1 ± 0.4 mg bacterioruberin (BR)/100 g cell paste) was obtained containing the same composition as that resulting from extraction with the classical solvents, as confirmed by electrospray ionization mass spectrometry, although with lower phospholipid (PL) content, thus with a higher proportion of bacterioruberin (BR/PL ratio 9.0 vs. 6.8 µg/mg). The extracted lipids were subsequently utilized for the preparation of archaeosomes, which were characterized by uniform size distribution (406 ± 137 nm, 0.63 ± 0.13 polydispersity index), colloidal stability, and negative ζ potential (−38.2 ± 5.4 mV). The photoprotective potential of these archaeosomes was determined for the first time in human keratinocyte (HaCaT) cells exposed to UVB irradiation (270 mJ/cm²). Treatment with archaeosomes significantly (p < 0.05) enhanced cell viability (from ~43 to ~80%), reduced intracellular ROS generation and proinflammatory cytokine release (TNF-α), and mitigated UVB-induced apoptosis compared to untreated controls, indicating effective cytoprotection. This study demonstrates that ethyl acetate–ethanol-based extraction offers an alternative for archaeal lipid recovery and highlights the potential of archaeosomes as natural photoprotective agents for skincare applications.

Archaea lipids are a source of new biomaterials for pharmaceutical and nanomedical applications; however, their classic extraction method relies on chloroform and methanol, toxic solvents that conflict with green chemistry principles. In this paper we explore the performance of an eco-friendly method for the extraction of total lipids from the haloarchaea Halorubrum tebenquichense. Using the bio-solvents ethyl acetate and ethanol in a two-step procedure, a fraction of total lipids (135 ± 41 mg phospholipids and 1.1 ± 0.4 mg bacterioruberin (BR) / 100 g cell paste) was obtained containing the same composition as that resulting from extraction with the classical solvents as confirmed by Electrospray Ionization Mass Spectrometry, although with lower phospholipid content, thus with a higher proportion of bacterioruberin. The extracted lipids were subsequently utilized for preparation of archaeosomes, which were characterized by uniform size distribution (406 ± 137 nm, 0.63 ± 0.13 polydispersity index), colloidal stability, and negative ζ potential (-38.2 ± 5.4 mV). The photoprotective potential of these archaeosomes was for the first time determined in human keratinocyte (HaCaT) cells exposed to UVB irradiation (270 mJ/cm²). Treatment with archaeosomes significantly (p< 0.05) enhanced cell viability (from ~43 to ~80 %), reduced intracellular ROS generation and proinflammatory cytokine release (TNF-α) and mitigated UVB-induced apoptosis compared to untreated controls, indicating effective cytoprotection. This study demonstrates that ethyl acetate–ethanol-based extraction offers an alternative for archaeal lipid recovery and highlights the potential of archaeosomes as natural photoprotective agents for skincare applications.

The membranes of halophilic archaea are a source of novel biomaterials, mainly of isoprenoid nature, with therapeutic properties practically unraveled. Here, we explored the antitumoral activity of neutral archaeolipids (NAs, such as bacterioruberin, astaxanthin, and dihydrosqualene) present in the total archaeolipids (TAs) (a fraction from the first step of lipid extraction by the modified Blight and Dyer technique) extracted from halophilic archaea Halorubrum tebenquichense, and formulated as TA-nanoarchaeosomes (TA: polar archaeolipids (PAs): Tween 80, 5:5:4 w:w:w, TA-nanoARC). The structure of 300.3 ± 84.2 nm TA-nanoARC of 0.59 ± 0.12 polydispersity index and −20 ± 3.7 mV ζ potential as determined by SAXS modelling, revealed that NA reduced the hydrophobic core and enlarged its hydrophilic section in comparison to TA-lacking bilayers (nanoARC), while preserving the width (~50 Å) and unilamellarity. Stable to storage and nebulization, TA-nanoARC was cytotoxic on A549 cells after 48 h, with an IC50 expressed as [bacterioruberin] of 0.15 μg/mL (~0.20 µM), comparable to or lower than the IC50 of docetaxel or cisplatin. Such cytotoxicity was exerted at a concentration harmless to macrophages (mTHP-1 cells). Besides, the conditioned medium from TA-nanoARC nebulized on A549 cells reduced the expression of the CD204/SRA-1, an M2 phenotype marker, and induced pro-inflammatory activity, comparable to or to a greater extent than that induced by lipopolysaccharide, including IL-6 and TNF-α, in mTHP-1 as a model of tumor-associated macrophages. The endocytosis of TA-nanoARC by A549 cells induced Lysotracker red fluorescence to fade and blur. This suggested the internalization of the highly viscous and ordered TA-nanoARC rich in NAs and subsequent lysosomal dysfunction (and not its antioxidant activity), as responsible for the selective damage on A549 cells. These are the first results showing that nebulized TA-nanoARC, lethal to A549 cells and modulating mTHP-1 cell phenotype, may act as antitumorals in the absence of cytotoxic drugs.

Iron oxide-based nanoparticles, such as magnetic nanoparticles (MNPs), have gained significant attention in the area of drug delivery due to their unique magnetic properties, allowing for precise targeting and controlled release of therapeutic agents. Several successful research studies were reported with combinations of magnetic nanoparticles and polysaccharides such as sodium alginate, chitosan, cellulose, etc. The presented research work is based on synthesising MNPs via the co-precipitation method and their successful encapsulation within alginate beads, serving as a promising drug delivery system for aceclofenac, a model drug. The physical and chemical characteristics of both the prepared magnetite nanoparticles and the aceclofenac-loaded MNPs alginate beads were thoroughly examined using scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), Fourier-transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), and vibrating sample magnetometry (VSM). Furthermore, a drug release study was conducted to evaluate the release kinetics of aceclofenac from the prepared MNP alginate beads. The magnetic characteristics of magnetite and MNP beads shed light on the potential application of novel drug delivery systems for magnetically targeted therapeutic interventions. The present research offers valuable insights into the development of magnetic nanoparticle-based drug carriers, paving the way for enhanced drug delivery strategies in the field of pharmaceutical sciences.

The properties of two hybrid nanoarchaeosomes (hybrid nanoARCs) made of archaeolipids extracted from the halophilic archaea Halorubrum tebenquichense and combining the properties of archaeolipid bilayers with metallic nanoparticles are explored here. BS-nanoARC, consisting of a nanoARC loaded with yerba mate (Ilex paraguariensis) extract (YME)-biogenic silver nanoparticles (BSs), and [BS + BS-nanoARC], consistent of a BS-nanoARC core covered by an outer shell of BSs, were structurally characterized and their therapeutic activities screened. By employing 109 ± 5 µg gallic acid equivalents (GAEs) and 73.4 µg chlorogenic acid/ YME mg as a silver reductive agent, spherical, heterogeneously sized (~80 nm diameter), −27 mV ζ potential, 90% Ag⁰ and λ_max 420 nm BSs were obtained. We further prepared ~100–200 nm diameter, −57 mV ζ potential BS-nanoARC and ~300 nm diameter, −37 mV ζ potential [BS + BS-nanoARCs]. Freshly prepared and nebulized BS-nanoARCs reduced the release of TNF-α, IL-6 and IL-8 by LPS-irritated THP-1-macrophages and were highly anti-planktonic against S. aureus (MIC₉₀: 13 ± 0.8 µg Ag/mL). While the nanoARCs and BS-nanoARCs were innocuous, freshly prepared [BS + BS-nanoARCs] magnified the cytotoxicity of BSs (IC₅₀ 12 µg Ag/mL vs. IC₅₀ ~36 µg Ag/mL) on A549 cells. Such cytotoxicity remained after 30 days in the dark at 4 °C, while that of BSs was lost. Freshly prepared BSs also lost activity upon nebulization, whereas freshly prepared [BS + BS-nanoARCs] did not. However, the cytotoxicity of the [BS + BS-nanoARCs] was also lost when nebulized after 30 days of storage. Despite the harmful effects of storage and mechanical stress on the structure of the more active [BS + BS-nanoARCs], hybrid nanoARCs are promising examples of nanomedicines combining the properties of archaeolipids with antimicrobial silver nanoparticles and anti-inflammatory polyphenols that could complement oncologic therapies, reducing the usage of classical antitumoral agents, corticosteroids, and, importantly, of antibiotics, as well as their waste.

Sodium alendronate (ALN) is a very hydrosoluble and poorly permeable molecule used as an antiresorptive agent and with vascular anticalcifying capacity. Loaded into targeted nanovesicles, its anti-inflammatory activity may be amplified towards extra-osseous and noncalcified target cells, such as severely irritated vascular endothelium. Here cytotoxicity, mitochondrial membrane potential, ATP content, and membrane fluidity of human endothelial venous cells (HUVECs) were determined after endocytosis of ALN-loaded nanoarchaeosomes (nanoARC-Chol(ALN), made of polar lipids from Halorubrum tebenquichense: cholesterol 7:3 w/w, 166 ± 5 nm, 0.16 ± 0.02 PDI, −40.8 ± 5.4 mV potential, 84.7 ± 21 µg/mg ALN/total lipids, TL). The effect of nanoARC-Chol(ALN) was further assessed on severely inflamed HUVECs. To that aim, HUVECs were grown on a porous barrier on top of a basal compartment seeded either with macrophages or human foam cells. One lighter and one more pronounced inflammatory context was modelled by adding lipopolysaccharide (LPS) to the apical or the apical and basal compartments. The endocytosis of nanoARC-Chol(ALN), was observed to partly reduce the endothelial-mesenchymal transition of HUVECs. Besides, while 10 mg/mL dexamethasone, 7.6 mM free ALN and ALN-loaded liposomes failed, 50 μg/mL TL + 2.5 μg/mL ALN (i.e., nanoARC-Chol(ALN)) reduced the IL-6 and IL-8 levels by, respectively, 75% and 65% in the mild and by, respectively, 60% and 40% in the pronounced inflammation model. This is the first report showing that the endocytosis of nanoARC-Chol(ALN) by HUVECs magnifies the anti-inflammatory activity of ALN even under conditions of intense irritation, not only surpassing that of free ALN but also that of dexamethasone.