Deciphering the Broad Antimicrobial Activity of Melaleuca alternifolia Tea Tree Oil by Combining Experimental and Computational Investigations.

Tea Tree Oil (TTO) is an essential oil obtained from the distillation of Melaleuca alternifolia leaves and branches. Due to its beneficial properties, TTO is widely used as an active ingredient in antimicrobial preparations for topical use or in cosmetic products and contains about 100 different compounds, with terpinen-4-ol, γ-terpinene and 1,8-cineole (or eucalyptol) being the molecules most responsible for its biological activities. In this work, the antimicrobial activity of whole TTO and these three major components was evaluated in vitro against fungi, bacteria and viruses. Molecular dynamics simulations were carried out on a bacterial membrane model and a Coxsackievirus B4 viral capsid, to propose an atomistic explanation of their mechanism of action. The obtained results indicate that the strong antimicrobial activity of TTO is attributable to the induction of an altered membrane functionality, mediated by the incorporation of its components within the lipid bilayer, and to a possible ability of the compounds to bind and alter the structural properties of the viral capsid.

Membrane cholesterol depletion in cortical neurons highlights altered NMDA receptor functionality in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-ß-cyclodextrin (MßCD). Interestingly, MßCD chronic treatment has provoked a significant reduction of NMDA-evoked currents in both cellular populations which was dose- and time-dependent but significantly higher in ALS neurons compared to Control. The different MßCD effect on NMDA-evoked currents was not due to a different membrane receptor subunit composition but seemed to cause in both neuronal populations a NMDA receptor membrane redistribution. MßCD treatment effect was receptor-specific since no alterations in the two neuronal populations were detected on AMPA receptors. These results lead us to speculate for an altered proteomic composition of lipid rafts in cortical mutated neurons and suggest the need for further studies on the lipid rafts composition and on their interaction with membrane receptors in ALS cortices.

Selective targeting and degradation of doxorubicin-loaded folate-functionalized DNA nanocages.

Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.

Simulative and experimental investigation on the cleavage site that generates the soluble human LOX-1.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor that mediates the recognition, the binding and internalization of ox-LDL. A truncated soluble form of LOX-1 (sLOX-1) has been identified that, at elevated levels, has been associated to acute coronary syndrome. Human sLOX-1 is the extracellular part of membrane LOX-1 which is cleaved in the NECK domain with a mechanism that has not yet been identified. Purification of human sLOX-1 has been carried out to experimentally identify the cleavage site region within the NECK domain. Molecular modelling and classical molecular dynamics simulation techniques have been used to characterize the structural and dynamical properties of the LOX-1 NECK domain in the presence and absence of the CTLD recognition region, taking into account the obtained proteolysis results. The simulative data indicate that the NECK domain is stabilized by the coiled-coil heptad repeat motif along the simulations, shows a definite flexibility pattern and is characterized by specific electrostatic potentials. The detection of a mobile inter-helix space suggests an explanation for the in vivo susceptibility of the NECK domain to the proteolytic cleavage, validating the assumption that the NECK domain sequence is composed of a coiled-coil motif destabilized in specific regions of functional significance.

Folate-Functionalization Enhances Cytotoxicity of Multivalent DNA Nanocages on Triple-Negative Breast Cancer Cells.

DNA is an excellent programmable polymer for the generation of self-assembled multivalent nanostructures useful for biomedical applications. Herein, we developed (i) folate-functionalized nanocages (Fol-NC), very efficiently internalized by tumor cells overexpressing the α isoform of the folate receptor; (ii) AS1411-linked nanocages (Apt-NC), internalized through nucleolin, a protein overexpressed in the cell surface of many types of cancers; and (iii) nanostructures that harbor both folate and AS1411 aptamer functionalization (Fol-Apt-NC). We analyzed the specific miRNA silencing activity of all types of nanostructures harboring miRNA sequestering sequences complementary to miR-21 and the cytotoxic effect when loaded with doxorubicin in a drug-resistant triple-negative breast cancer cell line. We demonstrate that the presence of folate as a targeting ligand increases the efficiency in miR-21 silencing compared to nanocages functionalized with AS1411. Double-functionalized nanocages (Fol-Apt-NC), loaded with doxorubicin, resulted in an increase of over 51% of the cytotoxic effect on MDA-MB-231 cells compared to free doxorubicin, demonstrating, besides selectivity, the ability of nanocages to overcome Dox chemoresistance. The higher efficiency of the folate-functionalized nanocages is due to the way of entrance, which induces more than four times higher intracellular stability and indicates that the folate-mediated route of cell entry is more efficient than the nucleolin-mediated one when both folate and AS1411 modifications are present.

AS1411 Aptamer Linked to DNA Nanostructures Diverts Its Traffic Inside Cancer Cells and Improves Its Therapeutic Efficacy.

The nucleolin-binding G-quadruplex AS1411 aptamer has been widely used for cancer therapy and diagnosis and linked to nanoparticles for its selective targeting activity. We applied a computational and experimental integrated approach to study the effect of engineering AS1411 aptamer on an octahedral truncated DNA nanocage to obtain a nanostructure able to combine selective cancer-targeting and anti-tumor activity. The nanocages functionalized with one aptamer molecule (Apt-NC) displayed high stability in serum, were rapidly and selectively internalized in cancer cells through an AS1411-dependent mechanism, and showed over 200-fold increase in anti-cancer activity when compared with the free aptamer. Comparison of Apt-NCs and free AS1411 intracellular distribution showed that they traffic differently inside cells: Apt-NCs distributed through the endo-lysosomal pathway and were never found in the nuclei, while the free AS1411 was mostly found in the perinuclear region and in nucleoli. Molecular dynamics simulations indicated that the aptamer, when linked to the nanocage, sampled a limited conformational space, more confined than in the free state, which is characterized by a large number of metastable conformations. A different intracellular trafficking of Apt-NCs compared with free aptamer and the confined aptamer conformations induced by the nanocage were likely correlated with the high cytotoxic enhancement, suggesting a structure-function relationship for the AS1411 aptamer activity.

Combined and selective miR-21 silencing and doxorubicin delivery in cancer cells using tailored DNA nanostructures.

MicroRNAs play an important role in tumorigenesis and, among them, miR-21 is found to be aberrantly up-regulated in various tumors. The tumor-associated antigen, folate receptor alpha is a GPI-membrane protein overexpressed in many malignant tumors of epithelial origin, including ovarian and cervical cancers. Covalently bound octahedral DNA nanocages were functionalized with folate molecules and utilized as scaffolds to engineer four sequestering units with a miR-21 complementary sequence for obtaining biocompatible Fol-miR21-NC non-toxic nanostructures, to be able to selectively recognize folate receptor alpha-overexpressing cancer cells and sequester the oncogenic miR-21. qPCR assays showed that Fol-miR21-NCs reduce the miR-21 expression up to 80% in cancer cells in the first 2 days of treatment. Functional assays demonstrated that miR-21 sequestering leads to up-regulation of miR-21 tumor suppressor targets (i.e., PTEN and Pdcd4), reduction in cancer cell migration, reduction in proliferation, and increase in cell death. Fol-miR21-NCs can be efficiently loaded with the chemotherapeutic agent doxorubicin. Co-delivery of anti-miR-21 and doxorubicin showed additive cytotoxic effects on tumor cells, paving the way for their use as selective nucleic acid drugs.

Entry, fate and degradation of DNA nanocages in mammalian cells: a matter of receptors.

DNA has been used to build nanostructures with potential biomedical applications. However, their use is limited by the lack of information on the mechanism of entry, intracellular fate and degradation rate of nanostructures inside cells. We generated octahedral DNA nanocages functionalized with folic acid and investigated the cellular uptake mediated by two distinctive internalization pathways, using two cellular systems expressing the oxidized low-density lipoprotein receptor-1 (LOX-1) and the α isoform of the folate receptor (αFR), respectively. Here, we report that DNA nanocages are very efficiently and selectively internalized by both receptors with an efficiency at least 30 times higher than that observed in cells not expressing the receptors. When internalized by LOX-1, nanocages traffic to lysosomes within 4 hours and are rapidly degraded. When the uptake is mediated by αFR, DNA nanocages are highly stable (>48 hours) and accumulate inside cells in a time-dependent way. These data demonstrate that the selection of the cellular receptor is crucial for targeting specific sub-cellular compartments and for modulating the DNA nanocage intracellular half-life, indicating that vitamin-mediated uptake may constitute a protected pathway for intracellular drug delivery.

Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells.

DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells.

Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells.

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MßCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding.

Molecular mechanism of statin-mediated LOX-1 inhibition.

Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins.