Autoren möchte Prof. Maurizio Masi für eine fruchtbare Diskussion und Fräulein Chiara Allegretti für Lektur danken. Autoren Forschung wird von Bando Giovani Ricercatori 2010 (Ministero della Salute GR-2010- 2312573) unterstützt.

Anmerkung: Die verwendeten Chemikalien sind wie empfangen. Linear RGD gekauft wird , aber es kann durch Standard - Fmoc - Festphasen - Peptidsynthese 16,19 hergestellt werden. Lösungsmittel sind von analytischer Qualität. Die Dialyse erfordert die Verwendung von Membran mit einem M w cut-off gleich 3.500 Da. Die synthetisierten Verbindungen werden durch 1 H - NMR - Spektren wurden auf einem 400 MHz - Spektrometer unter Verwendung von Chloroform (CDCl 3) oder Deuteriumoxid (D 2 O) als Lösungsmittel, und die chemischen Verschiebungen sind angegeben als δ - Werte in ppm aufgezeichnet gekennzeichnet. Weiterhin sind Hydrogele unterworfen FT-IR-Analyse KBr Pellet-Technik und ihrer physikalischen Charakterisierung beinhaltet die Gelierung Studien untersucht unter Verwendung der invertierten Teströhrchen bei 37 ° C verwendet wird. 1. Synthese von 4-Azidobutanoyl Chloride 1 <ol><li> Man löst 500 mg 4-azidobutanoic Säure (3,90 mmol) in 10 ml Dichlormethan und 0,5 ml Dimethylformamid gegeben. </li><li> Die Lösung wird bei 0 ° C, Mit einem Eisbad. </li><li> Hinzufügen 505 ul Oxalylchlorid (5,85 mmol) zu 5 ml Dichlormethan gelöst und langsam tropfenweise zu dem Reaktionssystem hinzuzufügen, während gerührt wurde. </li><li> Nach 1 Stunde bei 0 ° C mit einem Eisbad, Rückkehr zur Raumtemperatur. </li><li> Entfernen des Lösungsmittels unter vermindertem Druck am Rotationsverdampfer. </li><li> Charakterisieren des erhaltenen Produkts durch 1 H-NMR - Spektroskopie, um die Probe in CDCl 3 16 aufzulösen. </li></ol> 2. Synthese von RGD-Azid-Derivat 2 <ol><li> Man löst 50 mg RGD (0,145 mmol) in 1 ml 1 M NaOH. </li><li> Man löst 24 mg 1 (0,16 mmol) in 2 ml Tetrahydrofuran gegeben. </li><li> Fügen Sie alle der RGD - Lösung zu Lösung 1 tropfenweise bei 0 ° C mit einem Eisbad. </li><li> Rückkehr auf Raumtemperatur und rühre über Nacht. </li><li> 1 ml 1 M HCl. </li><li> Entfernen des Lösungsmittels unter vermindertem Druck am Rotationsverdampfer. </li><li> Charakterisieren Sie die obtained Produkts durch 1 H-NMR - Spektroskopie, Lösen der Probe in D 2 O 16. </li></ol> 3. PAA Alkin Modifikation 3 <ol><li> Man löst 200 mg von 35% w / w PAA-Lösung (2,8 mmol) in 15 ml destilliertem Wasser. </li><li> Hinzufügen 15,4 mg Propargylamin-hydrochlorid (0,20 mmol). </li><li> Man löst 42,8 mg von 1-Hydroxybenzotriazol-hydrat (HOBt, 0,28 mmol) in 14 ml eines 1: 1 v / v Acetonitril: destilliertes Wasser-Lösung durch Erhitzen auf 50 ° C. </li><li> Fügen alle der HOBt-Lösung PAA-Lösung bei Raumtemperatur. </li><li> Hinzufügen, 53,6 mg Ethyldimethylaminopropylcarbodiimid (EDC, 0,28 mmol) zu dem Reaktionsgemisch. </li><li> Verwenden Sie 1 M HCl, um den pH-Wert auf 5,5 und rührt das Reaktionssystem über Nacht bei Raumtemperatur einzustellen. </li><li> Dialysieren der Lösung. Man löst 11,2 g Natriumchlorid in 2 l destilliertem Wasser und füge dann 0,2 ml 37% w / w HCl. Dialysieren der Lösung mit einer Membran mit einem M w cut-off von 3,5 kDa verwendet wird . </li><li> Perform Dialyse für drei Tage. Ändern, um die Dialyselösung täglich mit 2 l frisch destilliertem Wasser hergestellt, das 0,2 ml 37% w / w HCl. </li><li> Bewahren Sie die endgültige Lösung bei -80 ° C. Lyophilisieren es in einem Lyophilisator nach Herstellerprotokollen. </li><li> Charakterisierung des funktionalisierten Polymers durch 1 H-NMR - Spektroskopie, Lösen der Probe in D 2 O 16. </li></ol> 4. Synthese von PAA-RGD Polymer 4 <ol><li> Man löst 78 mg des modifizierten PAA Alkin 3 (1,083 mmol) in 10 ml destilliertem Wasser. </li><li> Man löst 25 mg des RGD Azid 2 Derivat (0,0722 mmol) in 5 ml Tetrahydrofuran gegeben. </li><li> Fügen alle der RGD Lösung der Polymerlösung. </li><li> Hinzufügen 2,2 mg Kupferiodid (0,0116 mmol) und 2,2 mg Natriumascorbat (0,0111 mmol). </li><li> Rückfluß der resultierenden Mischung über Nacht bei 60 ° C unter Rühren. </li><li> Das Gemisch wird auf 25 ° C. </li><li> Dialyze die Lösung. Man löst 11,2 g Natriumchlorid in 2 l destilliertem Wasser und füge dann 0,2 ml 37% w / w HCl. Dialysieren der Lösung mit einer Membran mit einem M w cut-off von 3,5 kDa verwendet wird . </li><li> Führen Sie die Dialyse für drei Tage. Ändern, um die Dialyselösung täglich mit 2 l frisch destilliertem Wasser hergestellt, das 0,2 ml 37% w / w HCl. </li><li> Bewahren Sie die endgültige Lösung bei -80 ° C. Lyophilisieren es in einem Lyophilisator nach Herstellerprotokollen. </li><li> Charakterisieren des erhaltenen Produkts durch 1 H-NMR - Spektroskopie, Lösen der Probe in D 2 O 16. </li></ol> 5. RGD-funktionalisierten Hydrogel-Synthese <ol><li> Bereiten Sie die PBS. Man löst 645 mg PBS Salz in 50 ml destilliertem Wasser. </li><li> Mischung 40 mg Carbomer und 10 mg funktionalisiertes PAA 4 in 9 ml PBS (Schritt 5.1), bei Raumtemperatur bis zur vollständigen Auflösung (30 min). </li><li> In 400 mg PEG zur Lösung und halten für 45 min gerührt wurde. </li><li> Halten Sie das Rühren und lassen Sie das System für 30 Minuten absetzen. </li><li> Verwenden 1 N NaOH pH auf 7,4 einzustellen. </li><li> Zu 5 ml der erhaltenen Mischung werden 25 mg Agarose-Pulver. </li><li> Bestrahlen, das System mit Mikrowellenstrahlung bei 500 W bis zum Kochen, für eine Zeit, in der Regel zwischen 30 sec und 1 min, und elektromagnetisch erhitzen auf 80 ° C. </li><li> Lassen Sie die Mischung auf Raumtemperatur ausgesetzt, bis die Temperatur auf 50 ° C ab, und mit 5 ml PBS (Schritt 5.1), um eine Lösung mit einer 1: 1 zu erhalten Volumenverhältnis. </li><li> Bereiten Sie 12 Multi-Well-Platte mit Stahlzylinder mit einem Durchmesser von 1,1 cm. </li><li> Nehmen Sie 500 ul Aliquots aus der Lösung und legen Sie sie auf jede Stahlzylinder. </li><li> Lassen Sie für 45 Minuten bis zur vollständigen Gelieren des Systems in Ruhe. </li><li> Entfernen Sie die Zylinder ein Edelstahl-Pinzette, um die Hydrogele erhalten. </li></ol> 6. Laden von Therapeutic Tool (Drug oder Zellen) <ol><li> Wiederholen Sie steps 5,1-5,7. </li><li> Wenn die Mischung (die bereits im Sol-Zustand) 37 ° C erreicht, werden 5 ml der Lösung die gewünschte Arzneimittellösung oder Zellkultur enthält, um ein Endsystem zu einem 1: 1 zu erhalten Volumenverhältnis. </li><li> Wiederholen Sie die Schritte 5,9-5,12 polymere Netzwerke mit Bioverbindungen physikalisch innerhalb des Gels eingeschlossen zu erhalten. </li></ol> 7. Hydrogels Charakterisierung <ol><li> FT-IR-Analyse <ol><li> Nach der Gelbildung, während 24 Stunden in 2,5 ml destilliertem Wasser eine der synthetisierte Hydrogele einweichen. </li><li> Entfernen Sie die wässrigen Medien , wo Hydrogel untergetaucht ist und gefrier trocken mit flüssigem N 2. </li><li> Laminat mit der Hydrogel-Probe gemäß KBr Pellet-Technik. <ol><li> Fügen Sie einen Spachtel voller KBr in einem Achatmörser. Nehmen Sie eine kleine Menge an Hydrogel-Probe (etwa 0,1 bis 2% des KBr Menge oder gerade genug, um die Spitze der Spachtel zu bedecken) und vermischen sich mit dem KBr-Pulver. </li><li> Schleifen Sie die Mischung, bis das Pulver fein und homogen. &lt;/ Li&gt; <li> Verwenden Sie das KBr-Pellet-Kit das IR-Pellet zu bilden. Drücken Sie die Pulver eine manuelle Laborpresse: 3 min bei Druckkapazität in Höhe von 5 Tonnen und dann 3 min bei Druckkapazität von 10 Tonnen. </li><li> Lassen Sie den Druck der endgültige Pellet als homogen und transparent im Aussehen zu erhalten. Legen Sie die Pellets in den IR - Probenhalter und führen Sie das Spektrum 16. </li></ol></li></ol></li><li> Gelieren Studies <ol><li> Füllen 2 ml Mikrozentrifugenröhrchen mit 900 &amp; mgr; l PBS und auf 37 ° C äquilibrieren. </li><li> Füge 100 &amp; mgr; l der hergestellten Polymerlösung, um das Hydrogel bilden, und Inkubieren bei 37 ° C. </li><li> Drehen Sie das Röhrchen und beobachten, ob das Gel bei 1 fließt, 2, 5, 10 und 20 min. Aufzeichnen der Zeit, zu der das Gel fließt nicht als die Gelierzeit. </li></ol></li></ol>

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The authors state no conflict of interest and they have not received any payment in preparation of this manuscript.

The PAA post-polymerization modification with alkyne moieties and the RGD functionalization with the azide group guarantee the formation of a stable bond between the polymer and the peptide. Indeed, triazole serves as a rigid linking unit among the carbon atoms, attached to the 1,4 positions of the 1,2,3-triazole ring and it cannot be cleaved hydrolytically or otherwise. In addition, triazole is extremely difficult to oxidize and reduce, unlike other cyclic structures such as benzenoids and related aromatic heterocycles<...

Hydrogele sind dreidimensionale Netzwerke durch hydrophile vernetzte Polymere gebildet werden, die natürlich oder synthetisch sind, und die durch eine ausgeprägte dreidimensionale Struktur aus. Diese Geräte sind zunehmend attraktiv in den biomedizinischen Bereichen Medikamentenabgabe, Tissue Engineering, Genträger und intelligente Sensoren 1,2. Tatsächlich ihres hohen Wassergehaltes sowie ihre rheologischen und mechanischen Eigenschaften machen sie geeignete Kandidaten Weichgewebe Mikroumgebungen zu imitieren und sie wirksame Werkzeuge für wasserlösliche Zytokin oder Wachstumsfaktor-Lieferung. Einer der vielversprechendsten ist die Verwendung als injizierbare Biomaterial Zellen und bioaktiven Verbindungen trägt. Hydrogele können das Überleben von Zellen und Kontrollstammzell Schicksal durch Halten und präzise Abgabe Stammzellregulationssignale in einer physiologisch relevanten Art und Weise verbessern, wie 3,4 in in vitro und in in vivo Experimenten beobachtet. Der führende Vorteil hierbei ist die Möglichkeit,injizierten Zellen innerhalb der Zone der Impfung (in situ), Minimierung der Menge an Zellen zu erhalten, die das Gebiet und Extravasate in den Kreis Strom verlässt, über den ganzen Körper der Migration und dem angestrebten Ziel 5 zu verlieren. Die Stabilität der dreidimensionalen Netzwerke Hydrogels ist aufgrund seiner Vernetzungsstellen, gebildet durch kovalente Bindungen oder Kohäsionskräfte zwischen den Polymerketten 6. In diesem Rahmen angewendet orthogonal selektive Chemie an Polymerketten ist ein vielseitiges Werkzeug in der Lage 7 Hydrogel Leistungen zu verbessern. Tatsächlich könnte die Modifikation von Polymeren mit geeigneten chemischen Gruppen helfen, geeignete chemische, physikalische und mechanische Eigenschaften bereitzustellen Lebensfähigkeit der Zellen und deren Verwendung in der Gewebebildung zu verbessern. In gleicher Weise zu den Techniken Zellen oder Wachstumsfaktoren innerhalb der Gelmatrix, die Verwendung des RGD-Peptid ermöglicht Verbesserungen in der Zelladhäsion und das Überleben zu laden. RGD ist ein Tripeptid zusammengesetztvon Arginin, Glycin und Asparaginsäure, die bei weitem das wirksamste und oft eingesetzt Tripeptid aufgrund seiner Fähigkeit , mehr als eine Zelladhäsion - Rezeptor und seine biologische Wirkung auf die Zellverankerung, Verhalten und Überleben 8,9 zu adressieren ist. In dieser Arbeit wird die Synthese von RGD-funktionalisierten Hydrogele mit dem Ziel der Netzwerke durch ausreichende biochemische Eigenschaften für eine gastliche Zelle Mikro gekennzeichnet Gestaltung studiert. Die Verwendung von Mikrowellenstrahlung in Hydrogel - Synthese bietet ein einfaches Verfahren , um Nebenreaktionen zu minimieren und höhere Reaktionsgeschwindigkeiten und Ausbeuten in kürzerer Zeit im Vergleich zu den herkömmlichen thermischen Verfahren 10 erhalten. Dieses Verfahren erfordert keine Reinigungsstufen und Ausbeuten sterile Hydrogele aufgrund der Wechselwirkungen der Polymere und der Abwesenheit eines organischen Lösungsmittels in dem Reaktionssystem 11. Daher gewährleistet es einen hohen Prozentsatz von RGD gebunden an das polymere Netzwerk, da kein modkationen werden den Polymer chemischen Gruppen erforderlich Gelbildung beteiligt. Carboxygruppen, von PAA und Carbomer, und Hydroxylgruppen von PEG und Agarose, führen zu dem Hydrogel dreidimensionale Struktur durch eine Polykondensationsreaktion. Die genannten Polymere werden für die Synthese von Hydrogelen in die Rückenmarksverletzung Reparaturbehandlungen 12 verwendet. Diese Geräte, wie in den vorangegangenen berichtet arbeitet 13,14, zeigen eine hohe Biokompatibilität sowie mechanischen und physikalisch - chemische Eigenschaften, die denen von vielen lebenden Geweben und in thixotropen Natur ähneln. Außerdem bleiben sie in situ lokalisiert in der Zone der Injektion. In dieser Arbeit PAA Carboxylgruppen mit einem Alkin - Einheit (1) modifiziert und eine RGD-Azid - Verbindung synthetisiert wird , die Reaktivität des Tripeptids Endgruppe NH 2 mit einer vorbereiteten chemische Verbindung mit der Struktur zu nutzen (CH 2) n - N 3 (&lt;strong&gt; Abbildung 2). Anschließend reagiert das modifizierte PAA mit dem RGD-Azid - Derivat durch CuAAC Klick - Reaktion 15-17 (Abbildung 3). Die Verwendung eines Kupfer (I) -Katalysator führt zu wesentlichen Verbesserungen in sowohl die Reaktionsgeschwindigkeit und die Regioselektivität. Die CuAAC Reaktion wird in der organischen Synthese und in der Polymerwissenschaft weit verbreitet. Es verbindet einen hohen Wirkungsgrad und eine hohe Toleranz gegenüber den funktionellen Gruppen, und es ist unabhängig von der Verwendung von organischen Lösungsmitteln. Eine hohe Selektivität, eine schnelle Reaktionszeit und eine einfache Reinigungsverfahren ermöglichen die Gewinnung von Sternpolymeren, Blockcopolymere oder Ketten gewünschten Einheiten 18 Pfropfen. Dieser Klick-Strategie ermöglicht es, Polymere nach der Polymerisation zu modifizieren, um die physikalisch-chemischen Eigenschaften nach der endgültigen biochemischen Anwendung anpassen. Die CuAAC experimentellen Bedingungen sind leicht reproduzierbar (die Reaktion ist unempfindlich gegen Wasser, während Kupfer Oxidation minimal auftreten kann), und die Art dergebildet Triazol sorgt für Stabilität des Produkts. Die Verwendung von Kupfermetall kann ein kritischer Punkt, aufgrund seiner potentiellen toxischen Wirkung gegenüber Zellen und in der biologischen Mikroumgebung in Betracht gezogen werden, aber der Dialyse wird als Reinigungsverfahren verwendet, um die vollständige Entfernung von katalytischen Rückständen ermöglichen. Schließlich PAA modifizierte RGD in Hydrogel - Synthese (4) und die physikalisch - chemischen Eigenschaften der resultierenden Netzwerken verwendet wird untersucht, um die potentielle Funktionalität dieser Systeme als Zellen oder Drogen Träger zu überprüfen. <img alt="Abbildung 1" src="/files/ftp_upload/54445/54445fig1.jpg" /> Abbildung 1: PAA modifizierte Alkin Synthese ein Schema PAA Funktionalisierung mit Alkin - Gruppe;. &quot;n&quot; gibt die Monomere mit Carboxylgruppe mit Propargylamin reagieren. <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig1large.jpg" target="_blank">Bitte klicken Sie hier um zu sehen</a>eine größere Version dieser Figur. <img alt="Figur 2" src="/files/ftp_upload/54445/54445fig2.jpg" /> Abbildung 2:.. RGD-Azid - Synthese Die Synthese von RGD-Azid - Derivat <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/54445/54445fig3.jpg" /> Abbildung 3: Klicken Sie auf Reaktionsschema der Klick - Reaktion zwischen RGD-Azid - Derivat und Alkin-PAA.. <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 4" src="/files/ftp_upload/54445/54445fig4.jpg" /> Abbildung 4: Hydrogele synthesis. RGD funktionalisierten Hydrogel - Syntheseverfahren. <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="67">
			<td height="67" style="height:67px;width:183px;">Poly(acrylic acid) solution average Mw ~ 100,000, 35 wt % in H2O</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:87px;">523925</td>
			<td style="width:145px;">CAS 9003-01-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol) 2,000</td>
			<td>Sigma Aldrich</td>
			<td>84797</td>
			<td>CAS 25322-68-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbomeer 974P</td>
			<td>Fagron</td>
			<td>1387083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose&#160;</td>
			<td>Invitrogen Corp.</td>
			<td style="width:87px;">16500-500</td>
			<td>UltraPure Agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RGD peptide</td>
			<td style="width:108px;">abcam</td>
			<td>ab142698</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">4-azidobutanoic acid</td>
			<td style="width:108px;">Aurum Pharmatech</td>
			<td style="width:87px;">Z-2421</td>
			<td>&#160;CAS 54447-68-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxalyl chloride</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td>O8801</td>
			<td>CAS 79-37-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">Propargylamine hydrochloride 95%</td>
			<td>Sigma Aldrich</td>
			<td>P50919</td>
			<td>CAS 15430-52-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Copper(I) iodide</td>
			<td>Sigma Aldrich</td>
			<td>3140</td>
			<td>CAS 7681-65-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>Y0000039</td>
			<td>CAS 134-03-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Dialysis Membrane</td>
			<td style="width:108px;">Spectrum Laboratories, Inc.</td>
			<td>132725</td>
			<td style="width:145px;">Spectra/Por 3 Dialysis Membrane&#160; Standard RC Tubing&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; MWCO: 3,5 kD</td>
		</tr>
	</tbody>
</table>

the synthesis of rgd-functionalized hydrogels as a tool for therapeutic applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

Die PAA Alkin - Derivat effizient aus Polyacrylsäure und Propargylamin synthetisiert, wie in Abbildung 1 gezeigt , wobei n die Monomere , deren Carboxyl - Gruppen reagieren mit dem Amin - Etiketten. Die Identität des Produkts wird durch 1 H-NMR - Spektroskopie bestätigt. Abbildung 5 Die 1 H-NMR - Spektrum von PAA zeigt mit Dreifachbindung modifiziert. <img alt="Abbildung 5" src="/files/ftp_upl...

Watch this Scientific Journal Video about The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications at JoVE.com

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

Die Synthese von RGD-funktionalisierten Hydrogele als Werkzeug für therapeutische Anwendungen

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

the-synthesis-rgd-functionalized-hydrogels-as-tool-for-therapeutic

Research

JoVE Journal

Bioengineering

11.1K Aufrufe.  Politecnico di Milano. We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated. 

Serie: The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluoreszenz-Abschrecken eines Liposomenverkapseltes NIR-Fluorophore als Instrument zur<em&gt; In-vivo-</em&gt; Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Forschung

Biotechnik

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Feste Lipidnanopartikel (SLN) für die intrazelluläre Targeting-Anwendungen

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Licht-vermittelte Bildung und Musterung von Hydrogelen für Zellkulturanwendungen

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Tunable Hydrogele aus Pulmonary Extrazellulärmatrix für 3D-Zellkultur

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Synthese von Hydrogelen mit Antifouling-Eigenschaften als Membranen für die Wasserreinigung

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA-Nanoröhren als ein vielseitiges Werkzeug, semiflexiblen Polymere zu studieren

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Ultradünne derzei-elastischen Hydrogele als eine biomimetische Basalmembran für Dual Zellkultur

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Anwendungen für Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Synthese von Nahinfrarot emittierenden Gold-Nanoclustern für biologische Anwendungen

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Gleichzeitige Quantifizierung ausgewählter Kynurenine, analysiert durch Flüssigchromatographie-Massenspektrometrie in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...