Questa ricerca è stata sostenuta dal National Science Foundation cinese (21474057 e 21604076).

Attenzione: si prega di consultare tutte le schede di dati di sicurezza (MSDS) prima dell'uso. Si prega di utilizzare pratiche adeguate di sicurezza durante gli esperimenti di chimica, compreso l'uso di una cappa aspirante e dispositivi di protezione individuale (occhiali protettivi, guanti protettivi, camice da laboratorio, ecc.). Il protocollo richiede cella standard gestione tecniche (sterilizzazione, recupero delle cellule, cellule passaggio, congelamento delle cellule, macchiatura delle cellule, ecc.).
 1. preparazione di idrogeli <ol><li>sintesi della benzaldeide terminato funzionalizzati di polietilene glicole (spina DF) <ol><li>pre-essiccazione di PEG polimero <ol><li>pesare 4,00 g PEG (4.000 MW, 1.00 mmol), trasferirla in un pallone a fondo tondo (250 mL) e aggiungere toluene (100 mL). 
			Nota: Altre pioli con diversi pesi molecolari può lavorare per la formazione di idrogel ma porterà a diversa rigidezza.</li>
			<li>Riscaldare la soluzione da una pistola di calore (o un piatto caldo intorno ai 40 ° C) leggermente per aiutare a sciogliere tutti i polimeri. Dopo tutti i polimeri si dissolvono, è possibile utilizzare un evaporatore per rimuovere tutti i solventi. Ripetere questo processo secco e sciogliere due volte per rendere il PEG secche polimeri. 
			Attenzione: Questa operazione deve essere eseguita in una cappa con estrema cura. Il toluene è infiammabile.</li>
		</ol></li> <li>Reazione di terminazione di benzaldeide di PEG <ol><li>aggiungere un agitatore magnetico e tetraidrofurano (THF, 100 mL) al pallone e sciogliere PEG con un agitatore. Aggiungere 4-carboxybenzaldehyde (0,90 g, 6,0 mmol) e 4-dimetilaminopiridina (0,07 g, 0,6 mmol) in sequenza per le soluzioni di sciogliere tutti i solidi completamente.</li>
			<li>Aggiungi N, N &#39;-dicyclohexylcarbodiimide (DCC, 1,25 g, 6,0 mmol) alla soluzione e aggiungere un tubo di essiccazione che viene riempito con anidro CaCl 2 al pallone. Mantenere la reazione sotto agitazione a temperatura ambiente (~ 20 ° C) per circa 12 h.</li>
		</ol></li> <li>Processo di post-reazione <ol><li>filtrare i solidi bianchi generati nella soluzione di vuoto dopo la reazione è completa. Versare la soluzione nel freddo etere dietilico (500 mL) sotto agitazione per precipitare i solidi bianchi. Raccogliere tutti i solidi bianchi dal filtro e la sostanza secca in una cappa aspirante.</li>
			<li>Dissolversi ancora i solidi bianchi in THF, filtrare qualsiasi solidi bianchi insolubili, versare la soluzione nel fresco freddo etere etilico per precipitare i solidi bianchi e asciugarlo. Ripetere questo processo due o tre volte e quindi posizionare i solidi bianchi in un forno di essiccazione sotto vuoto (20 ° C, 0,1 mbar) per asciugarle completamente. Raccogliere la polvere bianca come il prodotto finale: benzaldeide terminato DF PEG.</li>
		</ol></li></ol></li> <li>Preparazione di idrogeli <ol><li>pesare quantità diverse di DF PEG (0,11 g, 0,028 mmol; 0,22 g, 0.055 mmol; 0,44 g, 0.110 mmol) in un tubo (10 mL), aggiungere acqua deionizzata (5,0 mL) e utilizzare un vortice o agitatore magnetico per aiutare sciogliere il polimero.</li>
		<li>Sciogliere glicole chitosano (0,495 g, 6,18 x 10 -3 mmol) in acqua deionizzata (15,0 mL) in un tubo (50 mL) e utilizzare un vortice per pochi minuti per omogeneizzare la soluzione di chitosano (3% in peso).</li>
		<li>Aggiungere la soluzione di chitosano di glicole (0,2 mL, 3% in peso) e soluzioni di PEG DF (0,2 mL) in sequenza in un tubo (2,0 mL). Utilizzare un vortice per mescolare le soluzioni in modo omogeneo a forma idrogel in alcuni minuti. Seguire i rapporti nella tabella 1 per rendere idrogeli di resistenze meccaniche differenti. 
		Nota: Utilizzare il metodo invertente tubo per determinare se l'idrogel è già formato.</li>
	</ol></li> <li>Analisi di reologia <ol><li>effettuare analisi di reologia su un reometro rotazionale con una piastra di acciaio parallela (diametro: 20 mm). Per la prova di gelificazione, pubblicizza chitosano glicolata (0,2 mL, 3% in peso) su una piastra inferiore. Aggiungere soluzioni acquose DF PEG (0,2 mL, 2% in peso) uniformemente goccia a goccia sulla superficie della soluzione del chitosano, quindi mescolare rapidamente con una pipetta. Più basso giù la piastra superiore e cominciano a test.</li>
		<li>Per idrogel &#39; prova di rigidezza s, tagliare un idrogel in un cerchio (diametro: 20 mm) e misurare il modulo elastico (G &#39;) valori contro analisi di frequenza al 1% di deformazione. Tipico G &#39; valori a 6,3 rad s &#8722; 1 sono elencati nella tabella 1. 
		Nota: Effettuare le analisi di reologia di idrogel 0,5 h dopo la formazione di idrogel per garantire la stabilizzazione dinamica legame dell'idrogel.</li>
		<li>Per l'idrogel &#39; s self-sanabile proprietà testare, tagliare un idrogel a pezzi e raccogliere i pezzi insieme a Self-Heal a un pezzo integrale. Quindi tagliare il pezzo di idrogel per fare un cerchio (diametro: 20 mm) e metterlo sul reometro per eseguire l'analisi di reologia.</li>
	</ol></li></ol> 2. colture cellulari 3D in idrogel <ol><li>preparazione delle soluzioni di gelificazione di idrogel <ol><li>pesare DF PEG (0,44 g, 0,11 mmol) in una provetta sterile (4,0 mL), aggiungere in terreni di coltura delle cellule (RPMI-1640, 2.0 mL) e utilizzare un vortice o agitatore per aiutare a sciogliere il polimero per ottenere la soluzione di polimero (20% in peso). Caricare la soluzione in una siringa (10,0 mL) e poi sterilizzarlo facendolo passare attraverso un filtro batteri ritentiva micron (0,22 µm).</li>
		<li>Pesare il chitosano di glicole (0,165 g, 2.06 x 10 -3 mmol) in una provetta sterile (15,0 mL), aggiungere in terreni di coltura delle cellule (RPMI-1640, 4,0 mL) e vortex per aiutare a sciogliere il polimero per ottenere la soluzione di glicole chitosano (4,0% in peso). Caricare la soluzione in una siringa (10,0 mL) e filtrare con un filtro di batteri a rimanenza da 0,22 µm.</li>
	</ol></li> <li>Colture cellulari in idrogel Attenzione: eseguire tutto cella procedure correlate in una cappa di coltura del tessuto. Conoscenza di base della tecnica sterile è previsto.
	<ol><li>Preparazione della sospensione di cellule <ol><li>cultura L929 cellule in RPMI-1640 supplementato con 10% FBS, 5% penicillina (10 mL) in un Petri dish (diametro 10 cm) e incubare a 37 ° C, 5% CO 2. Modificare il mezzo ogni giorno prima dell'uso.</li>
			<li>Raccogliere le celle L929 con PBS contenente tripsina (0,025% w/v) ed EDTA (0,01% w/v), poi centrifugare (5 min a 70 x g) e risospendere le cellule nel medium RPMI-1640 (1,0 mL). Eseguire la conta cellulare utilizzando standard di funzionamento del Consiglio di sangue-conteggio. Risospendere le cellule per regolare la concentrazione cellulare di ~ 3,75 × 10 6 cellule/mL.</li>
		</ol></li> <li>Incapsulamento di cellule in idrogel <ol><li>mescolare le sospensioni di cellula L929 (0,4 mL, 3.75 x 10 6 cellule mL -1) con chitosano glicolata (0,4 mL) in un tubo (4,0 mL) Vortex. Dispensare la soluzione di chitosano L929/glicole (0,8 mL) in al centro di un piatto di Petri confocale (diametro 2,0 cm). Pipettare le soluzioni DF PEG (0,2 mL) nello stesso piatto e pipettare delicatamente per mescolare la soluzione e inducono la formazione di idrogel. 
			Nota: Valutare la formazione di idrogel inclinando il piatto petri.</li>
			<li>Per la coltura cellulare diretto, aggiungere ulteriori quantità di terreni di coltura RPMI-1640 (1,0 mL) in cima l'idrogel. Mettere l'idrogel di cella incorporato (1,0 mL, 1.5 wt % glicole chitosano, 4,0 wt % DF PEG, 1,5 × 10 6 cellule mL -1) in un incubatore (37 ° C, 5% CO 2) e cambiare il mezzo ogni giorno. Preparare per l'imaging delle cellule il giorno 1, 3, 5 e 7 dopo incapsulamento cella.</li>
		</ol></li> <li>Della cultura post-cella 3D in idrogel dopo l'iniezione, preparare cella caricata idrogel (1,0 mL, vedi punto 2.2.2) in una siringa (10,0 mL, ago 48 G). Dopo le forme di idrogel, iniettare lentamente l'idrogel in una capsula Petri per imaging confocale. Aggiungere un importo supplementare di terreni di coltura (1,0 mL) in cima l'idrogel e cambiare ogni giorno. Mettere la capsula di Petri in un incubatore (37 ° C, 5% CO 2) e preparare per l'imaging in seguito. 
		Attenzione: Si prega di controllare e seguire il protocollo di funzionamento di sicurezza di una siringa.</li>
	</ol></li> <li>Analisi di attuabilità delle cellule <ol><li>confocale osservazione <ol>
			<li>Sciacquare l'idrogel con PBS (1,0 mL) per due volte. Macchia nel caso degli idrogeli con diacetato di fluoresceina (FDA, 0,5 mL, 0,05 mg/mL) e soluzioni di propidio ioduro (PI, 0,5 mL, 0,08 mg/mL) per 15 min. Dopo la colorazione, rimuovere tutti i solventi.</li>
			<li>Osservare nel caso degli idrogeli utilizzando un microscopio confocale in lunghezze d'onda di eccitazione di 488 nm e 543 nm di visualizzare dal vivo e morto cellule, rispettivamente. Prendere z-stack attraverso ogni profondità di 2 µm di idrogeli per convalidare una distribuzione uniforme delle cellule in tutto. 
			Nota: FDA macchie cellule vive mentre le cellule morte di macchie PI.</li>
		</ol></li> <li>Degradare l'idrogel (1,0 mL) con acido acetico (HAc, 3% v, 1,0 mL) per 5 min e pipettare in una provetta (4,0 mL). Raccogliere le cellule di centrifuga (70 x g, 5 min) e risospendere le cellule in terreno di coltura cellulare di RPMI-1640 (1,0 mL). Eseguire la conta cellulare utilizzando un sangue conteggio pensione.</li>
	</ol>
	</li>
</ol>

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L'idrogel presentato in questo protocollo (Figura 1) ha due componenti principali: il chitosano di glicole di polimero naturale e un sintetico benzaldeide terminato gelator polimero DF PEG, che sono entrambi materiali biocompatibili. Sintesi di DF PEG sono presentata utilizzando una reazione di modificazione di uno stadio. PEG del peso molecolare 4.000 fu scelto nel presente protocollo per preoccupazioni di solubilità, efficienza di modificazione, come pure la rigidità di idrogel. Una seri...

Idrogeli sono materiali polimerici reticolati con grandi quantità di acqua e morbido proprietà meccaniche, e sono stati utilizzati in molte applicazioni bio-medicali1,2. Idrogeli possono offrire un ambiente morbido e bagnato, che è molto simile all'ambiente fisiologico per cellule in vivo. Di conseguenza, idrogeli sono diventati uno dei più popolari impalcature per cella 3D cultura3,4. Rispetto alla coltura delle cellule 2D di Petri, coltura cellulare 3D ha avanzato velocemente per offrire che una matrice extracellulare (ECM) ha imitato il microambiente per celle da contattare e montare per proliferazione e differenziazione di scopi5. Inoltre, idrogeli contenenti polimeri naturali potrebbero offrire ambienti bio-compatibili e promozione per le cellule di proliferare e differenziare3. Idrogeli derivati da polimeri sintetici sono preferibili per i loro componenti semplici e chiare, che escludono le influenze complesse come proteine di origine animale o virus. Tra tutti i candidati di idrogel per colture cellulari 3D, idrogeli che facilmente sono preparati e hanno una proprietà coerente sono sempre preferiti. La possibilità di regolare le proprietà di idrogel per soddisfare i requisiti di ricerca differenti è importante come ben6.
Qui vi presentiamo una facile preparazione di un idrogel a base di chitosano glicole usando immina dinamica chimica, che diventa una piattaforma versatile idrogel per cella 3D cultura7. In questo metodo, noto bio-compatibili glicole chitosano vengono utilizzati per stabilire cornici delle reti di idrogel. Suoi gruppi amminici sono ha reagiti con un glicole di polietilene benzaldeide terminato come il gelator di polimeri per formare legami immina dinamico come crosslinkages di idrogeli8. Immina dinamico obbligazioni possono formare e si decompongono reversibilmente e responsabilmente ai dintorni, dotando l'idrogel con reticolati regolabile meccanicamente reti9,10,11. A causa del suo contenuto di acqua alta, materiali bio-compatibili e resistenze meccaniche regolabili, l'idrogel è applicato con successo come impalcatura per celle L929 in cella 3D cultura12,13. Il protocollo qui i dettagli delle procedure, tra cui la sintesi del polimero gelator, idrogel preparazione, l'incorporamento delle cellule e coltura delle cellule 3D.
L'idrogel Mostra anche diverse altre caratteristiche grazie alla sua dinamica immina crosslinkages, tra cui il multi-sensible a reagire ai vari bio-stimoli (acido/pH, piradossale derivato di vitamina B6, papaina proteine, ecc.), che indica che potrebbe essere l'idrogel indotto a decomporsi sotto condizioni fisiologiche8. L'idrogel è anche self-sanabile e iniettabili, che significa l'idrogel potrebbe essere amministrato per mezzo di un metodo di iniezione invasiva minimale e ottenere un vantaggio in droga e cella consegne14,15. Con l'aggiunta di additivi funzionali o gelators polimero specifico predefiniti, l'idrogel è compatibile per ottenere proprietà specifiche come magnetico, temperatura, pH sensibile, ecc.16,17, che potrebbe compiere un vasta gamma di esigenze di ricerca. Tali proprietà rivelano la capacità potenziale di idrogel per essere un iniettabile più vettori per farmaci e cellule sia in vitro che in vivo ricerca bio-medica e applicazioni.

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Glycol chitosan</td>
			<td style="width:360px;">Wako Pure Chemical Industries</td>
			<td style="width:131px;">39280-86-9</td>
			<td style="width:385px;">90% degree of deacetylation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Carboxybenzaldehyde</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>619-66-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N, N&#39;-dicyclohexylcarbodiimide</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>538-75-0</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride anhydrous</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>10043-52-4</td>
			<td>96%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-dimethylamiopryidine</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>1122-58</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneglycol</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>5254-43-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrahydrofuran</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>109-99-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toluene</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>108-88-3</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl ether</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>60-29-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>64-19-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anhydrous CaCl2</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>10043-52-4</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>596-09-8</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodide&#160;</td>
			<td>Sigma</td>
			<td>25535-16-4</td>
			<td>94%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI-1640 culture media</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td></td>
			<td>0.25%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Solarbio</td>
			<td></td>
			<td>0.01 M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin streptomycin solution</td>
			<td>Hyclone</td>
			<td></td>
			<td>10,000 U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rheometer</td>
			<td>TA Instrument</td>
			<td></td>
			<td>AR-G2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>710-3channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L929 Cells</td>
			<td>ATCC</td>
			<td></td>
			<td>NCTC clone 929; L cell, L929, derivative of Strain L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Evaporator</td>
			<td>EYELA</td>
			<td></td>
			<td>N-1100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 guage needle</td>
			<td>ShanghaiZhiyu Medical Material Co., LTD</td>
			<td></td>
			<td>48-guage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM3000 B</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope software</td>
			<td>Imaris</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat gun</td>
			<td>Confu</td>
			<td></td>
			<td>KF-5843&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

Il protocollo presenta un metodo facile, efficiente e versatile per preparare idrogel a base di chitosano usando immina dinamica chimica. L'idrogel è preparata mescolando soluzioni di glicole chitosano con un gelator di polimeri sintetizzati benzaldeide terminato e idrogeli sono ottenuti in modo efficiente in diversi minuti a temperatura ambiente. Dai vari rapporti tra glicole chitosano, gelator di polimero e contenuto di acqua, idrogel versatile con tempi di gelificazione differenti e rigidità sono ottenuti. Quando danneggiato, l'idrogel può ritrovare apparenze e modulo elastico, dovuto la reversibilità delle obbligazioni immina dinamico come crosslinkages. Questa proprietà auto-sanabile consente l'idrogel di essere iniettabili, poiché esso può essere auto-guarito da pezzi spremuti da un idrogel di massa integrale dopo il processo di iniezione. L'idrogel è anche multi-sensible a reagire a molti stimoli di bio-attivi a causa di Stati di equilibrazione differenti delle obbligazioni immina dinamico. Questo idrogel è stata confermata come bio-compatibili e cellule di fibroblasti di topo L929 erano incorporate seguenti procedure standard e la proliferazione delle cellule è stata valutata facilmente tramite un processo di coltivazione di cella 3D. L'idrogelo in grado di offrire una piattaforma regolabile per diverse ricerche dove è profittato di un mimo fisiologico di un ambiente 3D per le cellule. Insieme alle sue proprietà multi-reattivo, self-sanabile e iniettabili, nel caso degli idrogeli potenzialmente può essere applicato come elementi portanti multipli per farmaci e cellule in future applicazioni bio-medicali.

Una presentazione schematica di questo protocollo su idrogel preparazione e il suo uso come coltura cellulare 3D è offerto nella Figura 1. Informazioni di idrogel Sommario e rapporti preparati con diversi punti di forza meccaniche sono riassunta nella tabella 1. L'idrogel di self-sanabile e proprietà di reologia presenta rigidità di idrogel di modulo elastico contro frequenza test nella Figura 2. Le immagini confocal cellulare e numeri di cel...

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Qui descriviamo una facile preparazione di idrogeli iniettabili a base di chitosano usando immina dinamica chimica. Metodi per regolare la resistenza meccanica di idrogel e la sua applicazione nella coltura cellulare 3D sono presentati.

Preparazione di idrogeli iniettabili a base di chitosano e la sua applicazione nella coltura cellulare 3D

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Research

JoVE Journal

Chemistry

18.9K Views.  Tsinghua University. Qui descriviamo una facile preparazione di idrogeli iniettabili a base di chitosano usando immina dinamica chimica. Metodi per regolare la resistenza meccanica di idrogel e la sua applicazione nella coltura cellulare 3D sono presentati.

Video: Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

Sintesi, Consegna Cellulare e<em&gt; In vivo</em&gt; Applicazione di sensori di pH basati Dendrimero

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

Ricerca

Chimica

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

Isolamento e preparazione delle pareti cellulari batteriche per l'analisi compositia mediante cromatografia liquida ad alte prestazioni

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

Preparazione e utilizzo di carbeni carbonile-decorate in Attivazione di fosforo bianco

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Il sequenziamento di Plant parete Heteroxylans Utilizzando enzimatica, chimica (metilazione) e (Spettrometria di Massa, Risonanza Magnetica Nucleare) tecniche fisiche

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

Indagini su GA (III) Complesso di EOB-DTPA e la sua<sup&gt; 68</sup&gt; Ga marcata radioattivamente analogico

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Preparazione e caratterizzazione di nanostrutture ibride C60/Graphene

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

Modifica di bio-ispirati Polydopamine superficie di nanodiamanti e sua riduzione delle nanoparticelle d'argento

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Photogeneration of N-Heterocyclic Carbenes: Application in Photoinduced Ring-Opening Metathesis Polymerization

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes and determine the corresponding photochemical mechanism. Then, we describe a protocol to perform ring-opening metathesis polymerization (ROMP) in solution and in miniemulsion using this NHC-photogenerating system. To photogenerate IMes, a system comprising 2-isopropylthioxanthone (ITX) as the sensitizer and 1,3-dimesitylimidazolium tetraphenylborate (IMesH+BPh4-) as the protected form of NHC is employed. IMesH+BPh4- can be obtained in a single step by anion exchange between 1,3-dimesitylimidazolium chloride and sodium tetraphenylborate. A real-time steady-state photolysis setup is described, which hints that the photochemical reaction proceeds in two consecutive steps: 1) ITX triplet is photo-reduced by the borate anion and 2) subsequent proton transfer takes place from the imidazolium cation to produce the expected NHC IMes. Two separate characterization protocols are implemented. Firstly, CS2 is added to the reaction media to evidence the photogeneration of NHC through formation of the IMes-CS2 adduct. Secondly, the amount of NHC released in situ is quantified using acid-base titration. The use of this NHC photo-generating system for the ROMP of norbornene is also discussed. In solution, a photopolymerization experiment is conducted by mixing ITX, IMesH+BPh4-, [RuCl2(p-cymene)]2 and norbornene in CH2Cl2, then irradiating the solution in a UV reactor. In a dispersed medium, a monomer miniemulsion is first formed then irradiated inside an annular reactor to produce a stable poly(norbornene) latex.

We describe a protocol to photogenerate N-heterocyclic carbenes (NHCs) by UV irradiation of a 2-isopropylthioxanthone/imidazolium tetraphenylborate salt system. Methods to characterize the photoreleased NHC and elucidate the photochemical mechanism are proposed. The protocols for ring-opening metathesis photopolymerization in solution and miniemulsion illustrate the potential of this 2-component NHC photogenerating system.

Photogeneration di carbeni N-eterociclico: applicazione a polimerizzazione per metatesi anello-apertura fotoindotti

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

Preparazione e applicazione di un nuovo biosensore batterico per il rilevamento presuntivo di residui di arma da fuoco

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

Preparazione di cellule liquide microwell supportate al grafene per la microscopia elettronica di trasmissione in situ

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...