Gli autori riconoscono la borsa di studio Arabia Saudita-finanziato (I821) per aver reso possibile la ricerca. Gli autori sono grati al dott Xianwei Liu per il supporto di esperti in analisi TEM presso la Cranfield University.

1. Preparazione di emulsioni olio-in-acqua <ol><li> Pesare gliceril palmitostearato (120 mg), isopropilmiristato (60 mg), D-α-tocoferolo polietilene glicole succinato (30 mg) e lecitina di soia (30 mg), e aggiungerli al 12,5 ml di solventi organici (6,25 ml di acetone e 6,25 ml di etanolo). sciogliere rapidamente la miscela alla temperatura di 70 ° C (5 ° C sopra il punto di fusione del lipide solido) in bagno d&#39;acqua. </li><li> Aggiungere sia 125, 250 o 375 mg di Poloxamer188 in 12,5 ml di DDH 2 O per ottenere 1-3% (rispettivamente) Poloxamer 188 soluzione, che è soggetto a riscaldamento alla stessa temperatura come sopra. </li><li> Aggiungere la soluzione fase acquosa dalla fase 1.2 alla soluzione fase olio dal punto 1.1 a gocce per formare emulsioni sotto agitazione magnetica a 400 rpm. Mescolare l&#39;emulsione a 400 rpm per altri 4 ore per consentire l&#39;evaporazione dei solventi organici. </li></ol> 2. solidificazione e omogeneizzazione <ol><li> lasciare tha emulsione in una stanza fredda (4 ° C) per 2 ore a solidificare / cristallizzare. </li><li> Per ottenere NLC, sottoporre l&#39;emulsione di elevata dispersione pura (HSD), con un omogeneizzatore a 10.000-15.000 rpm per 10-40 min. </li></ol> 3. Ottimizzazione del NLC Preparazione <ol><li> Prelevare campioni dal punto 2.2 con una concentrazione di tensioattivo di 1, 2 e 3% sottoposti HSD a velocità di 10.000, 15.000 e 20.000 rpm, rispettivamente, e gli intervalli di tempo di 10, 20, 30 e 40 minuti, rispettivamente, </li><li> Esaminare i campioni per dimensione delle particelle (PS), poli indice di dispersione (PDI), morfologia e ultrastruttura 20. Nota: I parametri che producono particelle con il più piccolo formato (155 nm) e il valore di PDI (0,2) sono determinati come ottimale. </li></ol> 4. Preparazione di Dac-caricato NLC (NLC-Dac) <ol><li> Preparare la soluzione fase olio come descritto al punto 1.1 con l&#39;aggiunta di Dac (70 mg) prima sciogliendo la miscela a 70 ° C a bagnomaria. </li><li> Prepare una soluzione fase acquosa come descritto al punto 1.2 con 1% di tensioattivo, e aggiungere questa soluzione a quello preparato nella fase 4.1 a gocce per formare una emulsione sotto agitazione magnetica a 400 rpm. Mescolare l&#39;emulsione per ulteriori 4 ore per evaporare i solventi organici. </li><li> Lasciare l&#39;emulsione in una stanza fredda (4 ° C) per 2 ore a solidificare / cristallizzare come descritto al punto 2, e, infine, sottoporre l&#39;emulsione di elevata dispersione pura (HSD) utilizzando i parametri ottimali determinati nella fase 3. </li></ol>

<ol>
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Part III. physicochemical characterization.</a> AAPS PharmSciTech. 11 (3), 1243-1249 (2010).</li><li>Lei, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+drug+encapsulation+in+a+novel+nano-vesicular+carrier+for+the+treatment+of+cutaneous+melanoma:+Characterization+and+in+vitro/in+vivo+evaluation.">Dual drug encapsulation in a novel nano-vesicular carrier for the treatment of cutaneous melanoma: Characterization and in vitro/in vivo evaluation.</a> RSC Advances. 5 (26), (2015).</li></ol>

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particelle nanostrutturati a base di lipidi sono stati utilizzati per fornire un supporto altamente lipofilo per la consegna dei farmaci idrofobici. Un NLC è la seconda generazione del vettore lipidico nanostrutturato solido, che sono solidi a temperatura ambientale e del corpo. L&#39;incorporazione di un lipide solido in un lipide liquido in un risultato NLC in una cristallizzazione meno perfetta, aumentando così l&#39;efficienza droga carico e riducendo anche l&#39;espulsione di farmaci incapsulati durante la conser...

Dacarbazina (DAC) è un agente alchilante che esercita l&#39;attività anti-tumorale attraverso acidi nucleici metilazione del DNA o danni diretti, portando ad arresto del ciclo cellulare e la morte cellulare 1. Come primo agente chemioterapico linea, Dac è stato usato da solo o in combinazione con altri farmaci chemioterapici per il trattamento di vari cancri 2-6. Esso è l&#39;agente più attivo finora usato nel trattamento di melanoma cutaneo e metastatico, che è la forma più aggressiva di 3,7,8 cancro della pelle. Il tasso di risposta, tuttavia, è solo il 20% al massimo, e gli effetti terapeutici sono spesso accompagnate da gravi effetti collaterali sistemici. Nella sua forma naturale, DAC è idrofilo ed è instabile a causa della sua fotosensibilità 9. L&#39;unica formula disponibile per uso clinico attualmente è una polvere sterile per essere utilizzato in sospensione per via endovenosa 7,8 infusione. Il basso tasso di risposta ed elevata tossicità sistemica rmangiato del farmaco è in gran parte attribuibile alla sua scarsa solubilità in acqua, quindi a basso disponibilità al sito di destinazione, e alta la distribuzione in siti non bersaglio, che limita la dose massima del farmaco 10. La rapida degradazione e metabolismo dopo il ricovero endovenosa insieme allo sviluppo di resistenza ai farmaci limitano l&#39;applicazione clinica ed effetto terapeutico del farmaco 11. Pertanto, non vi è un urgente bisogno di sviluppare formulazioni alternative DAC per il trattamento di melanoma maligno. Sistemi colloidali contenenti liposomi, micelle o particelle nanostrutturate sono stati intensamente studiati per il loro uso nella somministrazione di farmaci come recensito da Marilene et al. 12 particelle nanostrutturati come portatori potenziali di farmaci sono state attirando sempre maggiore attenzione negli ultimi dieci anni a causa della loro capacità di aumentare carico di droga efficienza, controllo di rilascio del farmaco, migliorare la farmacocinetica di droga e la biodistribuzione, e quindi restrarre droga tossicità sistemica 13. Solo pochi nanoformulazioni, tuttavia, sono state studiate finora per la consegna Dac, che mostra la protezione del farmaco dal foto degenerazione, maggiore solubilità della droga, e migliorato effetto terapeutico 10,14,15. Tuttavia queste formulazioni sofferto di scarsa efficienza di incapsulamento, mentre alcuni anche utilizzando nanoparticelle polimeriche di sintesi che non sono convenienti. Vettori nanostrutturati lipidici (NLC), costituiti da una miscela di lipidi solidi e liquidi, sono stati sviluppati per drug delivery 16,17. I farmaci devono essere incapsulate sono spesso solubili in entrambi i lipidi liquidi e fasi lipidi solidi 18, con un conseguente elevato carico e rilascio controllato 19. Questo studio mira a sviluppare una nuova formulazione Dac sulla base di NLC-incapsulamento utilizzando gliceril palmitostearato e isopropilmiristato come lipidi. La preparazione coinvolti olio in emulsione acquosa, evaporazione, solidificazione, e homogenization. I preparativi sono stati caratterizzati per la dimensione NLC, forma, ultrastruttura e dispersity, l&#39;efficienza di incapsulamento di droga e di carico di droga 20.

<table border="0" cellpadding="0" cellspacing="0" width="968">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dacarbazine (DAC)</td>
			<td style="width:467px;">Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">D2390-100MG</td>
			<td style="width:167px;">drug used for uploading</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glyceryl palmitostearate&#160;</td>
			<td>Gattefoss&#233; (Saint_Priest_c&#233;dex, France)</td>
			<td style="width:119px;">85251-77-0</td>
			<td>solid lipid&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">d-&#945;- Tocopherol polyethylene glycol succinate (TPGS)</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">57668</td>
			<td>lipid phase surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poloxamer 188</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">15759-1KG</td>
			<td>liqiud phase surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetone&#160;</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">650501-1L</td>
			<td>organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol&#160;</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">459836-1L</td>
			<td>organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soybean lecithin (SL)</td>
			<td>Cuisine Innovation (Dijon, France)</td>
			<td style="width:119px;">SLL1402</td>
			<td>lipid phase surfactant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Double-distilled water was collected in our laboratory from</td>
			<td>Millipore-Q Gradient A10 ultra-pure water system (Millipore, France)</td>
			<td style="width:119px;">SAS - 67120&#160;</td>
			<td>aqueous phase&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T 25 digital ULTRA-TURRAX</td>
			<td style="width:467px;">IKA</td>
			<td style="width:119px;">3725000</td>
			<td>as high shear disperser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hotplate Magnetic Stirrer</td>
			<td>Scientific Support, Inc</td>
			<td style="width:119px;">1454&#160;</td>
			<td>emulsion homogenization</td>
		</tr>
	</tbody>
</table>

encapsulation of cancer therapeutic agent dacarbazine using nanostructured lipid carrier

L&#39;unica formula di dacarbazina (DAC) in uso clinico è l&#39;infusione endovenosa, presenta un profilo terapeutico povero a causa della bassa dispersità del farmaco in soluzione acquosa. Per ovviare a questo, un vettore nanostrutturato lipidica (NLC) costituito gliceril palmitostearato e isopropil miristato è stato sviluppato per incapsulare Dac. NLCS con dimensione controllata sono stati raggiunti con elevata dispersione di taglio (HSD) dopo la solidificazione di emulsione di olio in acqua. I parametri di sintesi, tra cui la concentrazione di tensioattivi, la velocità e il tempo di HSD sono stati ottimizzati per ottenere il NLC più piccolo con la dimensione, indice di polidispersione e potenziale zeta di 155 ± 10 nm, 0,2 ± 0,01, e -43,4 ± 2 mV, rispettivamente. I parametri ottimali sono stati anche impiegati per la preparazione NLC DAC-caricato. Il CNT risultante caricato con Dac possedeva dimensioni, l&#39;indice di polidispersione e potenziale zeta di 190 ± 10 nm, 0,2 ± 0,01, e -43,5 ± 1,2 mV, rispettivamente. L&#39;ef incapsulamento della drogaFICIENCY e caricamento del farmaco ha raggiunto il 98% e il 14% rispettivamente. Questo è il primo rapporto sulla incapsulamento del Dac utilizzando NLC, il che implica che NLC potrebbe essere un nuovo candidato potenziale come vettore di droga per migliorare il profilo terapeutico di Dac.

I preparativi della NLC e NLC-Dac utilizzando gliceril palmitostearato e isopropilmiristato con diversi parametri sono stati caratterizzati per PS, PDI, morfologia e ultrastruttura 20. Il PS e PDI del NLCS erano concentrazione di tensioattivi, la velocità HSD e la durata dipendente. Come giudicato da PS e PDI del NLCS, i migliori risultati sono stati ottenuti con 1% di tensioattivo e una velocità di dispersione pura di 15.000 rpm per 30 min (Figura 1A, <stro...

Watch this Scientific Journal Video about Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier at JoVE.com

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

Il metodo più comunemente utilizzato per nanostrutturato carrier lipidico sintesi (NLC) coinvolge olio in emulsione acquosa, omogeneizzazione e solidificazione. Questo è stato modificato qui applicando alta dispersione di taglio dopo la solidificazione di raggiungere un NLC con la dimensione auspicabile, una migliore incapsulamento di droga e l&#39;efficienza farmaco carico come un potenziale vettore per la consegna dacarbazina.

Incapsulamento del Cancro agente terapeutico dacarbazina Utilizzando Nanostrutturati Lipid Carrier

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug ...

encapsulation-cancer-therapeutic-agent-dacarbazine-using

Research

JoVE Journal

Chemistry

11.0K Views.  Cranfield University.  Il metodo più comunemente utilizzato per nanostrutturato carrier lipidico sintesi (NLC) coinvolge olio in emulsione acquosa, omogeneizzazione e solidificazione. Questo è stato modificato qui applicando alta dispersione di taglio dopo la solidificazione di raggiungere un NLC con la dimensione auspicabile, una migliore incapsulamento di droga e l'efficienza farmaco carico come un potenziale vettore per la consegna dacarbazina. 

Video: Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Metabolomica non mirati da fonti biologiche Uso UltraPerformance cromatografia liquida-spettrometria di massa ad alta risoluzione (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Ricerca

Chimica

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Isolamento e Caratterizzazione chimica del lipide A da batteri Gram-negativi

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

La proteomica quantitativa, utilizzando riduttiva dimethylation per Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Un colorimetrico metodo che misura particolare Granzyme B proteolitici attività: idrolisi di Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

Preparazione facile di Internamente auto-assemblato Lipid particelle stabilizzati da nanotubi di carbonio

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

Spettroscopia NMR come uno strumento robusto per la rapida valutazione del profilo lipidico di olio di pesce integratori

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

Dalle molecole ai materiali: nuovi cristalli liquidi ionici attraverso legame alogeno di ingegneria

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Peptidi e proteine quantificazione utilizzando Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

Analisi di minerali prodotto da hFOB 1.19 e Saos-2 cellule mediante microscopia elettronica a trasmissione con microanalisi a raggi x dispersiva di energia

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Organic Structure-directing Agent-free Synthesis for *BEA-type Zeolite Membrane

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in petroleum and petrochemical fields because of their high thermal, chemical, and mechanical strength. A *BEA-type zeolite is an interesting membrane material because of its large pore size and wide Si/Al range. This manuscript presents a protocol for *BEA membrane preparation by a secondary growth method that does not use an organic structure-directing agent (OSDA). The preparation protocol consists of four steps: pretreatment of support, seed preparation, dip-coating, and membrane crystallization. First, the *BEA seed crystal is prepared by conventional hydrothermal synthesis using OSDA. The synthesized seed crystal is loaded on the outer surface of a 3 cm long tubular &#945;-Al2O3 support by a dip-coating method. The loaded seed layer is prepared with the secondary growth method using a hydrothermal treatment at 393 K for 7 days without using OSDA. A *BEA membrane having very few defects is successfully obtained. The seed preparation and dip-coating steps strongly affect the membrane quality.

A *BEA seed crystal was loaded on a porous &#945;-Al2O3 support by the dip-coating method, and hydrothermally grown without using an organic structure-directing agent. A *BEA-type zeolite membrane having very few defects was successfully prepared by the secondary growth method.

Sintesi senza agenti che disorienta la struttura organica per la membrana zeolite di tipo

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in ...