Alginate–lavender nanofibers with antibacterial and anti-inflammatory activity to effectively promote burn healing

One of the current challenges in wound care is the development of multifunctional dressings that can both protect the wound from external agents and promote the regeneration of the new tissue. Here, we show the combined use of two naturally derived compounds, sodium alginate and lavender essential oil, for the production of bioactive nanofibrous dressings by electrospinning, and their efficacy for the treatment of skin burns induced by midrange ultraviolet radiation (UVB). We demonstrate that the engineered dressings reduce the risk of microbial infection of the burn, since they stop the growth of Staphylococcus aureus. Furthermore, they are able to control and reduce the inflammatory response that is induced in human foreskin fibroblasts by lipopolysaccharides, and in rodents by UVB exposure. In particular, we report a remarkable reduction of pro-inflammatory cytokines when fibroblasts or animals are treated with the alginate-based nanofibers. The down-regulation of cytokines production and the absence of erythema on the skin of the treated animals confirm that the here described dressings are promising as advanced biomedical devices for burn management

Time evolution of the <i>“elongated”</i> (red), <i>“hooked”</i> (yellow), and <i>“curved”</i> conformations of anandamide (first row), oleamide (second row), and PEA (third row), shown for Monomer-A (first column) and Monomer-B (second column) of the <i>wt</i>-FAAH (upper graphs) and <i>mut</i>-FAAH (lower graphs) systems.

<p>Time windows for the substrates location are shown with different background colors: red (MA channel), yellow (AB channel), and cyan (T region). The green background indicates the time windows for the oleamide unbinding in the <i>mut-</i>FAAH. Full details are in the main text, in the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004231#pcbi.1004231.s014" target="_blank">S1 Text</a> and in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004231#pcbi.1004231.s002" target="_blank">S2 Fig</a>.</p

Competition assays performed for the <i>wt</i> (A), W531A (B), and F432A (C) proteins in the presence of both anandamide (black) and PEA (red).

<p>Each set of data was fitted using simple exponential decay functions, whose parameters are reported in S6 Table in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004231#pcbi.1004231.s015" target="_blank">S2 Text</a>.</p

Overview of the FAAH protein (pdb 1MT5) [1] in complex with anandamide, embedded in a 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayer.

<p>The enzyme is a homodimer, which is shown in gray ribbons. The lipids of the membrane are represented in cyan lines with the phosphate atoms highlighted as spheres. A close view of the binding site is shown on the right. The substrate anandamide is shown in yellow sticks, while the catalytic triad (Ser241-Ser217-Lys142) and the oxyanion hole (Ser241-Gly239-Gly240-Ser241) residues are represented in cyan sticks. The so called “membrane access” (MA—red) and the “acyl chain binding” (AB—orange) channels, as well as the “cytosolic port” (CP—cyan) are depicted in molecular surface representation. The interface region between the MA and AB channels is indicated as transition region (T). The Asp403 and Arg486 residues of the MA channel, which favor the substrates entrance within FAAH active site, are also shown as sticks. Key residues—Phe432 (green) and Trp531 (magenta)—are shown in space-filling representation. For clarity, explicit water molecules included in the simulations are omitted. The chemical structure of the FAAH substrates here considered—anandamide, oleamide, and palmitoylethanolamide (PEA)—is also shown.</p

Polar plots of the <i>φ</i> angle (dihedral angle along the Cα-Cβ axis) of Phe432 (<i>φF</i>—green dots) and Trp531 (<i>φW</i>—violet dots) with respect to the <i>d-MA</i> (red background), <i>d-T</i> (cyan background), and <i>d-AB</i> (yellow background) distances for pre-reactive conformations of the <i>wt</i>FAAH/anandamide (first row), <i>wt</i>FAAH/oleamide (second row), and <i>wt</i>FAAH/palmitoylethanolamide (PEA—third row) systems.

<p>The polar (<i>d-MA</i>, <i>d-T</i> and <i>d-AB</i>) and angular (<i>φ—</i>in red on the plot) coordinates are explicitly indicated on the plots. The approximate values of <i>φF</i> of Phe432 for the <i>“open”</i> and <i>“closed”</i> MA channel configurations are highlighted with blue dashed bars. Distances and angles are expressed in Å and degrees, respectively. Definitions of the <i>d-MA</i>, <i>d-T</i> and <i>d-AB</i> distances are reported in the Methods section. Selected snapshots from MD simulations indicating the <i>“open”</i> (left) and <i>“closed”</i> (right) MA channel configurations, as induced by the rotation of the <i>φ</i> angle of Phe432 and the cooperative Trp531, are shown at the bottom of the polar plots. The MA (red) and AB (orange) channels are represented in molecular surfaces. Phe432 (green) and Trp531 (violet) are shown in space-filling representation. The <i>φ</i> angle of the two residues is explicitly reported.</p

K_m obtained from the fitting of the kinetic assays (S12 Fig).

<p>y = V_max*x/(K_m+x) (Origin Pro 8.6)</p><p>K_m obtained from the fitting of the kinetic assays (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004231#pcbi.1004231.s012" target="_blank">S12 Fig</a>).</p

Activity-Based Probe for <i>N</i>‑Acylethanolamine Acid Amidase

<i>N</i>-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-<i>N</i>-[(2<i>S</i>,3<i>R</i>)-2-methyl-4-oxo-oxetan-3-yl]­carbamate) and ARN726 (4-cyclohexylbutyl-<i>N</i>-[(<i>S</i>)-2-oxoazetidin-3-yl]­carbamate). The former is active <i>in vivo</i> by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the <i>in vivo</i> detection of NAAA. The newly synthesized molecule <b>1</b> is an effective <i>in vitro</i> and <i>in vivo</i> click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with <b>1</b> confirmed that ARN726 and ARN077 inhibit NAAA <i>in vitro</i> and <i>in vivo</i>. Compound <b>1</b> is a useful new tool to identify activated NAAA both <i>in vitro</i> and <i>in vivo</i> and to investigate the physiological and pathological roles of this enzyme

La Lanterne : journal politique quotidien

01 octobre 18971897/10/01 (N7467,A21)

Activity-Based Probe for <i>N</i>‑Acylethanolamine Acid Amidase

<i>N</i>-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-<i>N</i>-[(2<i>S</i>,3<i>R</i>)-2-methyl-4-oxo-oxetan-3-yl]­carbamate) and ARN726 (4-cyclohexylbutyl-<i>N</i>-[(<i>S</i>)-2-oxoazetidin-3-yl]­carbamate). The former is active <i>in vivo</i> by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the <i>in vivo</i> detection of NAAA. The newly synthesized molecule <b>1</b> is an effective <i>in vitro</i> and <i>in vivo</i> click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with <b>1</b> confirmed that ARN726 and ARN077 inhibit NAAA <i>in vitro</i> and <i>in vivo</i>. Compound <b>1</b> is a useful new tool to identify activated NAAA both <i>in vitro</i> and <i>in vivo</i> and to investigate the physiological and pathological roles of this enzyme

Synthesis of Highly Fluorescent Copper Clusters Using Living Polymer Chains as Combined Reducing Agents and Ligands

We present the synthesis of colloidally stable ultrasmall (diameter of 1.5 ± 0.6 nm) and fluorescent copper clusters (Cu-clusters) exhibiting outstanding quantum efficiencies (up to 67% in THF and approximately 30% in water). For this purpose, an amphiphilic block copolymer poly(ethylene glycol)-<i>block</i>-poly(propylene sulfide) (MPEG-<i>b</i>-PPS) was synthesized by living anionic ring-opening polymerization. When CuBr is mixed with the living polymer chains in THF, the formation of Cu-clusters is detected by the appearance of the fluorescence. The cluster growth is quenched by the addition of water, followed by THF removal. The structural features of the MPEG-<i>b</i>-PPS copolymer control the cluster formation and the stabilization: the poly(propylene sulfide) segment acts as coordinating and reducing agent for the copper ions in THF, and imparts a hydrophobic character. This hydrophobic block protects the Cu-clusters from water exposure, thus allowing to obtain a stable emission in water. The PEG segment instead provides the hydrophilicity, rendering the Cu-clusters water-soluble. To obtain fluorescent and stable Cu-clusters exhibiting outstanding quantum efficiencies, the removal of the excess of free polymer and copper salt was crucial. The Cu-clusters are also colloidally and optically stable in physiological media and showed bright fluorescence even when taken up by HeLa cells, being noncytotoxic when administered at a Cu dose between 10 nM and 1.6 μM. Given the very small size of the Cu-clusters, localization and fluorescent staining of cell nucleus is achieved, as demonstrated by confocal cell imaging performed at different Cu-cluster doses and at different incubation temperatures