Finanziamento: US Army Medical Research e Materiel Command (USAMRMC) e gestito dalla Telemedicina e Advanced Technology Research Center. Shunxi Ji ha contribuito alla modifica delle siringhe come descritto qui.

1. System Set-up Il sistema è costituito da un&#39;azienda e concentrando serbatoio (HCT) dotato di un miscelatore stadio, un taglio elevato omogeneizzatore in-linea, una pompa a rulli per spostare fluido tra l&#39;HCT e l&#39;omogeneizzatore, e uno scambiatore di calore (Figura 1). <ol><li> Posizionare un sterilizzato, bocca larga 4 L recipiente di raccolta di vetro munito di 2 porte base e 3 porte laterali sotto il mixer a singolo stadio. Abbassare la testa del mixer alla bocca della nave e garantire un raccordo a tenuta di gas con guarnizioni in gomma o nastro adesivo (al fine di evitare che l&#39;aria ambiente di contaminare lo spazio di testa). </li><li> Montare una delle porte di base (Figura 1, porta # 1) della HCT con sterile 3/8 &quot;(ID) tubo trasparente, di circa 10&quot; di lunghezza, dotata di rubinetto a 3 vie sulla punta per la raccolta della emulsione concentrata . </li><li> Montare la seconda porta base (Figura 1, porta # 2) con sterile 3/8 &quot;(ID) tubi, ufficio intornoy 36 &quot;di lunghezza. Alimentare questo tubo attraverso una pompa a rulli. Montare l&#39;ingresso di alto omogeneizzatore taglio con un raccordo a T comprese le due porte e collegare come segue: collegare il tubo dal porto # 2, attraverso la pompa rullo e collegarlo alla porta laterale del T-pezzo. Attaccare l&#39;altra porta ad una bombola di ossigeno utilizzando un misuratore di flusso di gas di ossigeno a basso flusso. </li><li> Collegare la porta di uscita del omogeneizzatore ad alta taglio alla porta di ingresso di uno scambiatore termico in linea mantenuta a 4 ° C. Collegare la porta di uscita dello scambiatore di calore per l&#39;apertura di ritorno del HCT (Figura 1, porta # 3), creando un sistema a circuito chiuso. </li><li> Attaccare un serbatoio di ossigeno (tramite un flussometro) alla HCT (Figura 1, porta # 4). Collegare un monitor composizione del gas che è aperto all&#39;atmosfera alla porta all&#39;inizio della HCT (Figura 1, port # 5). </li><li> Se sterilità è desiderato, sterilizzare i componenti in vetro e metallo prima di ogni utilizzo in autoclave. Sterilizzare i componenti di tubi e co plasticannectors di ossido di etilene prima di ogni utilizzo. Ciò è particolarmente importante se il prodotto viene testato in vivo. </li></ol> 2. LOM Fabbricazione <ol><li> Porre 20 g di 1,2-GMP distearoil-sn-glicero-3-fosfocolina (DSPC) e 10 g di colesterolo nella base del HCT. Aggiungere 1 L di plasma-Lyte A alla HCT e mescolare a mano per 1 min, integrando il più possibile lipidi in fase acquosa. </li><li> Abbassare il mixer monostadio nella fase acquosa, assicurando che l&#39;intera testa del mixer è coperto dalla fase acquosa. Assicurarsi che la parte superiore del HCT è a tenuta di gas (vedi punto 1.1) e che non ci sono le porte laterali aperte. Accendere la sorgente di gas collegato alla porta # 4 e attendere che la frazione di ossigeno della spazio di testa HCT raggiunge il&gt; 95%. Al 10 L / min (LPM), questo dovrebbe prendere ~ 10 min. </li><li> Uso del mixer monostadio, mescolare l&#39;emulsione precursore per 5 minuti a 5000 rpm. La miscela risultante dovrebbe apparire bianco pallido e non contengono lipi visibiled grumi. Una volta mescolato, miscela lipidica acqua non utilizzata può essere conservato a 4 ° C fino a 30 giorni prima monouso. </li><li> Prime dell&#39;intero sistema a ciclo chiuso con l&#39;emulsione precursore attivando la pompa a rulli a 1.3 LPM. Una volta che il sistema è innescato, tenere la pompa a 1,3 LPM. </li><li> Per iniziare fabbricazione dei LOMS, accendere il taglio elevato omogeneizzatore in-linea a 7.500 giri. Subito dopo, attivare il flusso di ossigeno alla porzione di ingresso del omogeneizzatore a 0.5 LPM. Tenere il mixer di palco singolo (in HCT) su a 3.500 giri. LOMS si formano all&#39;interfaccia di pale del rotore e gli schermi emulsor ai omogeneizzatore in-linea (Figura 2). In pochi minuti, il liquido dovrebbe diventare visibilmente più viscoso. Un approccio più rigoroso è quello di determinare la viscosità in funzione del tempo, che può essere fatto rimuovendo aliquote da Port 1 durante la fabbricazione e l&#39;analisi con un viscosimetro. Nota: Se bolle d&#39;aria visibili presenti nella tubazione in uscita dal mixer, ossigenofruiti omogeneizzatore in-linea è troppo alta. Titolare basso flusso di gas fino a quando il fluido è opaco e non contiene bolle di gas visibile. </li><li> Funzionare il sistema per 15 minuti, quindi spegnere l&#39;alto omogeneizzatore taglio e l&#39;ingresso di ossigeno ad esso. Continuare a eseguire il miscelatore singola fase nel HCT fino a quando l&#39;emulsione viene rimosso; questo mitiga separazione di fase e mantiene il prodotto relativamente uniforme all&#39;interno della HCT. Nota: Il volume dell&#39;emulsione riempito di gas dovrebbe aumentare di circa 2-3x durante la fase di concentrazione seriale. In caso contrario, verificare che l&#39;ossigeno fluisce in alto omogeneizzatore taglio e che le concentrazioni di lipidi in emulsione precursore siano corrette. Efficienza della produzione diminuisce concentrazione di lipidi diminuisce. </li></ol> 3. Collection, Concentrazione, valutazione e conservazione di LOMS <ol><li> Collegare un sterile, modificato 140 ml siringa luer-lock per il rubinetto collegato alla porta di base # 1 sulla nave collezione. Redigere 100 ml di liquido. Cap Strettamente siringa e ripetere fino a quando tutto il liquido è stato rimosso. <ol><li> Modifica siringhe ritirando 100 ml di aria nella siringa e poi segare lo stantuffo eccesso e materiale siringa sopra il marchio 140 ml. Riempire e siringhe vuote con pinze dentate di redigere lo stantuffo. Questa modifica permette facile centrifugazione. </li></ol></li><li> Siringhe centrifuga con la fine capped orientato verso il basso in un frigorifero (4 ° C) Benna centrifugare a 225 xg per 10 min. </li><li> Tre strati di materiale appariranno dopo la centrifugazione. Espellere lo strato inferiore di un eccesso di fase acquosa nuvoloso e scartare. Il secondo strato è bianco luminoso e contiene LOMS concentrati. Trasferire schiuma concentrata ad una siringa impermeabile ai gas utilizzando un rubinetto a tre vie per prevenire la contaminazione gas ambiente. Eliminare lo strato finale, che contiene il gas ossigeno libero da LOMS rottura. </li><li> Qualità della schiuma può essere valutata raggiungendo ≥ 90% del gas di schiuma concentrato. Calculate concentrazione di gas come segue: Volume% gas = [(peso piuma / volume Foam) - 1] x 100 <ol><li> Come controllo secondo qualità, microbolle dimensioni di oscuramento della luce per determinare se la dimensione delle particelle è nel range previsto. Va notato che un cambiamento nel tempo omogeneizzazione o formulazione potrebbe alterare dimensione delle bolle. </li></ol></li><li> Tappare ermeticamente la siringa di vetro con un raccordo luer-lock. LOMS concentrati possono essere diluiti con Plasma-Lyte A al momento dell&#39;uso. Le siringhe possono essere conservati a 22, 4, o -20 ° C; temperature più fredde possono fornire maggiore stabilità shelf-life 7. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

I passi più importanti per la creazione, LOMS altamente ossigenati concentrate le seguenti: 1) garantire che lo spazio di testa all&#39;interno del HCT rimane completamente ossigenato; 2) garantire che la purezza degli eccipienti lipidi è ottimale (comprese le condizioni di conservazione e utilizzo dei prodotti GMP); 3) garantire che i lipidi polvere mescolano completamente con la fase acquosa prima di innesco del sistema; e 4) prestando attenzione all&#39;aumento della frazione di gas all&#39;interno del HCT per gara...

Microbolle composto da proteine, polimero, e conchiglie lipidi sono stati sviluppati come vettori per il drug delivery, terapia genica, e mezzi di contrasto ecografici 1-5. Poiché questi usi terapeutici richiedono intravascolare persistenza microbolle, tali microbolle sono comunemente pieni di inerti, alti gas peso molecolare come perfluorocarburi 6, che hanno bassa solubilità nel sangue e stabilizzano il 3,4 bolla. Recentemente, a base di lipidi microbolle di ossigeno (LOMS) sono stati progettati per fornire dosi terapeutiche di ossigeno, che può preservare la consegna di ossigeno d&#39;organo e prevenire instabilità emodinamica durante i periodi di ostruzione delle vie aeree o ipossiemia 7. Emulsioni progettati per la consegna di gas endovenosa richiedono diverse caratteristiche di design rispetto a quelli utilizzati per gli agenti di contrasto ecografici o somministrazione di farmaci mirati. In primo luogo, perché il corpo consuma grandi quantità di gas ossigeno (~ 200 ml / min), LOMS devono essere prodotti einiettato su larga scala. Ciò richiede che il processo di fabbricazione sia efficiente. In secondo luogo, il processo di fabbricazione deve essere a circuito chiuso per evitare la contaminazione azoto attraverso l&#39;esposizione di LOMS (che deve essere riempito con 100% di ossigeno) all&#39;aria ambiente. Terzo, poiché lo scopo di LOMS è consegna gas endovenosa, la frazione di gas LOMS dovrebbe essere massimizzata, riconoscendo i limiti imposti dalla emulsione viscosità 7. Infine, come con qualsiasi iniettabili per via endovenosa, un controllo preciso sulla distribuzione granulometrica è essenziale per evitare l&#39;ostruzione microvascolare 8. Ci sono diversi metodi stabiliti per la produzione di microbolle. Sonicazione utilizza alta intensità, ultrasuoni a bassa frequenza applicato all&#39;interfaccia aria-liquido di una emulsione che comprende un tensioattivo, come ad esempio un fosfolipide anfipatica, in presenza di un gas spazio di testa per produrre microbolle 7,9. Questo processo è controllabile variando ultrafrequenza del suono, potenza e durata dell&#39;impulso, e la distribuzione granulometrica risultante possono essere adattate per produrre microbolle di una specifica distribuzione dimensione, anche se sonicazione è raramente usato nella fabbricazione di microbolle clinicamente utilizzati. Fusione è l&#39;agitazione meccanica intenso di un tensioattivo e gas in un sistema chiuso, che è anche difficile da scalare fino a grandi volumi 2. Microfluidica basata su Droplet permette un controllo preciso della distribuzione delle dimensioni di microbolle 10-13. Anche se tradizionalmente difficili da scalare, multi-canale, microfluidica ad alta velocità sono stati descritti che aumentano l&#39;efficienza di produzione di microbolle 13. Le microbolle fabbricati utilizzando uno di questi metodi possono richiedere processi formato di riduzione post-produzione, come frazionamento centrifuga 14,15 e microbolle galleggiamento 16,17. Un altro metodo stabilito per la fabbricazione di microbolle altamente stabili è homogeniz shearzione 6, che può risultare in una stabilizzazione reticolo esagonale fosfolipide sulla superficie di microbolle 18. Basandosi su questo concetto, si descrive l&#39;incorporazione di un taglio elevato omogeneizzatore in-linea per creare auto-assemblaggio LOMS 19. In questo processo, l&#39;omogeneizzatore utilizza lame a rapida rotazione in prossimità doppio sottili schermi emulsor maglia, creando alto taglio meccanico ed idraulico per la creazione di microbolle. Concentrazione Serial dell&#39;emulsione lipidica attraverso questo sistema produce una frazione di gas sempre più concentrata, che può essere ulteriormente concentrato mediante centrifugazione.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:239px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:405px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)</td>
			<td style="width:141px;">Avanti Polar Lipids</td>
			<td style="width:119px;">770365</td>
			<td>Alternate product: non-GMP from NOF America (Coatsome MC-8080)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Cholesterol</td>
			<td style="width:141px;">Sigma Aldrich</td>
			<td style="width:119px;">C75209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plasma-Lyte A</td>
			<td style="width:141px;">VWR</td>
			<td style="width:119px;">80089-818</td>
			<td>Alternatively can use NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Glass collection vessel</td>
			<td style="width:141px;">Specialty Glass, Inc.</td>
			<td style="width:119px;">Custom</td>
			<td>Contact: Pam Zurbrick - 281-595-2210</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gas composition (oxygen) monitor</td>
			<td style="width:141px;">Precision Medical</td>
			<td style="width:119px;">PM5900L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Sarns 8000 roller pump</td>
			<td style="width:141px;">Calicut Medical</td>
			<td style="width:119px;">16407</td>
			<td>Part of a modular perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">BIOtherm Heat Exchanger</td>
			<td style="width:141px;">Medtronic</td>
			<td style="width:119px;">ECMOtherm-II</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Verso laboratory in-line mixer</td>
			<td style="width:141px;">Silverson Machines, Inc</td>
			<td style="width:119px;">TH-IL-102-VERSO</td>
			<td>Use multistage workheads and front-end extension with T piece</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">T-piece for Silverson Verso inlet port</td>
			<td style="width:141px;">Process Innovations</td>
			<td style="width:119px;">Custom</td>
			<td>Contact: Brian Leavitt - 508-423-2266</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">L5M-A laboratory mixer</td>
			<td style="width:141px;">Silverson Machines, Inc</td>
			<td style="width:119px;">NC0136483</td>
			<td>Use mesh emulsor screen (fine)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Rochester-Ochsner toothed forceps</td>
			<td style="width:141px;">Fisher Scientific</td>
			<td style="width:119px;">13-812-18</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">140 mL syringe</td>
			<td style="width:141px;">Kendall Healthcare Monoject&#160;</td>
			<td style="width:119px;">8881114030</td>
			<td>Ensure there is a luer lock.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">IX71 Inverted light microscope</td>
			<td style="width:141px;">Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Retiga-2000R microscope camera</td>
			<td style="width:141px;">QImaging</td>
			<td style="width:119px;">RET-2000R-F-M-12</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;">Accusizer 780A Autodilution</td>
			<td style="width:141px;">PSS-NICOMP Particle Sizing Systems</td>
			<td style="width:119px;">Out of production</td>
			<td></td>
		</tr>
	</tbody>
</table>

manufacture of concentrated, lipid-based oxygen microbubble emulsions by high shear homogenization and serial concentration

Microbolle di gas sono stati sviluppati come contrasto ecografico e agenti di consegna della droga. Le microbolle possono essere prodotti dalla lavorazione di tensioattivi mediante sonicazione, agitazione meccanica, dispositivi microfluidici, o omogeneizzazione. Recentemente, a base di lipidi microbolle di ossigeno (LOMS) sono stati progettati per fornire ossigeno per via endovenosa durante le emergenze mediche, invertendo pericolo di vita ipossiemia, e prevenire lesioni a organi, arresto cardiaco e morte. Vi presentiamo i metodi per la produzione in scala-up di microbolle altamente ossigenati utilizzando un circuito chiuso ad alta-shear omogeneizzatore. Il processo può produrre 2 L di LOMS concentrati (90% in volume) in 90 min. Bolle risultanti hanno un diametro medio di circa 2 micron, e un profilo reologico coerente con quella del sangue quando diluito al 60% in volume. Questa tecnica produce LOMS a capacità elevata e con la purezza di ossigeno, suggerendo che questa tecnica può essere utile per laboratori di ricerca traslazionale.

Alta omogeneizzazione taglio permette efficiente (cioè entro un pomeriggio) la produzione di LOMS sufficienti per uno studio sugli animali e non richiede competenze tecniche. Una volta abile, fino a 2 L di LOMS concentrati può essere fabbricato in 90 min. Dimensioni microbolle e la morfologia è stata valutata mediante microscopia ottica e oscuramento della luce. Quando è stato visualizzato un campione di 10 microlitri di LOMS, sono stati notati LOMS sferici, nonché una scarsit?...

Watch this Scientific Journal Video about Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration at JoVE.com

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Descriviamo metodi per la produzione di grandi volumi di base di lipidi microbolle ossigeno (LOMS) progettati per la consegna di ossigeno endovenosa utilizzando high-shear omogeneizzazione e concentrazione seriale.

Fabbricazione di concentrato, ossigeno a base di lipidi microbolle emulsioni da High Shear omogeneizzazione e concentrazione seriale

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

manufacture-concentrated-lipid-based-oxygen-microbubble-emulsions

Research

JoVE Journal

Bioengineering

14.9K Views.  Harvard Medical School.  Descriviamo metodi per la produzione di grandi volumi di base di lipidi microbolle ossigeno (LOMS) progettati per la consegna di ossigeno endovenosa utilizzando high-shear omogeneizzazione e concentrazione seriale. 

Video: Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

Introduzione alla tecnica di distruzione mirata ad ultrasuoni microbolle

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

Ricerca

Bioingegneria

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Generazione di Shear Adesione mappa usando SynVivo reti microvascolari sintetico

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Ad alta risoluzione Spatiotemporal Analisi del recettore Dynamics per singola molecola di microscopia a fluorescenza

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Sintesi di nanoparticelle d&#39;oro liposomi Integrated Photo-reattiva e la misurazione del loro microbolle cavitazione su impulso laser di eccitazione

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Fabbricazione e consegna della droga Applicazioni di seta nanoparticelle

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Quantifying Intermembrane Distances with Serial Image Dilations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for electrical conduction between cardiomyocytes. The current method for quantifying this space by manual segmentation is slow and has low spatial resolution.We developed an algorithm that uses serial image dilations of a binary outline to count the number of pixels between two opposing 2 dimensional edges.This algorithm requires fewer man hours and has a higher spatial resolution than the manual method while preserving the reproducibility of the manual process.In fact, experienced and novice investigators were able to recapitulate the results of a previous study with this new algorithm.The algorithm is limited by the human input needed to manually outline the perinexus and computational power mainly encumbered by a pre-existing pathfinding algorithm.However, the algorithm&#39;s high-throughput capabilities, high spatial resolution and reproducibility make it a versatile and robust measurement tool for use across a variety of applications requiring the measurement of the distance between any 2-dimensional (2D) edges.

The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering.

Quantificare intermembrana distanze con le dilatazioni immagine seriale

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Lo Scavenging catalitica di pianta reattive dell'ossigeno specie In Vivo di nanoparticelle di ossido di cerio anionici

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Rilevamento dell'endotossina in Nano-formulazioni utilizzando dosaggi di Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

Fabbricazione di guide d'onda in modalità zero per microscopia ad alta concentrazione a singola molecola

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...