Name: the synthesis of rgd-functionalized hydrogels as a tool for therapeutic applications
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Description: Watch this Scientific Journal Video about The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications at JoVE.com

Författarna vill tacka professor Maurizio Masi för givande diskussion och Miss Chiara Allegretti för språkgranskning. Författarnas forskning stöds av Bando Giovani Ricercatori 2010 (Minis della Salute GR-2010- 2.312.573).

Obs: Kemikalierna används som mottagna. Linjär RGD köps, men det kan framställas genom standard Fmoc fast fas peptidsyntes 16,19. Lösningsmedel är av analytisk kvalitet. Dialysen kräver användning av membran med en Mw cut-off lika med 3500 Da. De syntetiserade föreningarna kännetecknas av ett H-NMR-spektra registrerades på en 400 MHz-spektrometer med användning av kloroform (CDCI3) eller deuteriumoxid (D2O) som lösningsmedel, och kemiska skift rapporter...

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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<...

Hydrogeler är tredimensionella nätverk som bildats av hydrofila tvärbundna polymerer, som är naturliga eller syntetiska, och kännetecknas av en distinkt tredimensionell struktur. Dessa enheter är alltmer attraktiva i biomedicinska områdena drug delivery, vävnadsteknik, genbärare och smarta sensorer 1,2. Faktum är att deras höga vattenhalt, liksom deras reologiska och mekaniska egenskaper gör dem lämpliga kandidater att efterlikna mjukdelsmikromiljöer och göra dem effektiva verktyg för vattenlösligt cytokin eller tillväxtfaktor leverans. En av de mest lovande användning är som ett injicerbart biomaterial bärande celler och bioaktiva föreningar. Hydrogeler kan förbättra cellöverlevnad och kontroll stamcells öde genom att hålla och exakt leverera stamcellsregleringssignaler i ett fysiologiskt relevant sätt, såsom observerats i in vitro- och i in vivo-experiment 3,4. Den ledande Fördelen med detta är möjlighetenatt upprätthålla injicerade cellerna inom zonen av inokulering (in situ), vilket minimerar den mängd celler som lämnar området och extravasates i cirkulations flöde, migrerar över hela kroppen och förlora målet mål 5. Stabiliteten hos de tre-dimensionella hydrogelpartiklar nätverk beror på dess tvärbindningsställen, bildade genom kovalenta bindningar eller kohesiva krafter mellan polymerkedjorna 6. I detta sammanhang tillämpas ortogonala selektiv kemi till polymerkedjor är ett mångsidigt verktyg kan förbättra hydrogel föreställningar 7. I själva verket modifiering av polymerer med lämpliga kemiska grupper kan bidra till att tillhandahålla lämplig kemiska, fysikaliska och mekaniska egenskaper för att förbättra cellviabilitet och deras användning i vävnadsbildning. På samma sätt, bland de tekniker för att ladda celler eller tillväxtfaktorer inom gelmatrisen, användningen av RGD-peptiden medger förbättringar i celladhesion och överlevnad. RGD är en tripeptid sammansattarginin, glycin och asparaginsyra, som är den i särklass mest effektiva och ofta anställda tripeptid på grund av dess förmåga att adressera mer än en cell adhesion receptor och dess biologiska effekter på cell förankring, beteende och överlevnad 8,9. I detta arbete studeras syntesen av RGD-funktion hydrogeler i syfte att utforma nätverk som kännetecknas av tillräckliga biokemiska egenskaper för en gäst cell mikromiljö. Användningen av mikrovågsstrålning i hydrogel syntes ger ett enkelt förfarande för att minimera bireaktioner och få högre reaktionshastigheter och utbyten på kortare tid jämfört med konventionella termiska processer 10. Denna metod kräver inte reningssteg och ger sterila hydrogeler på grund av de interaktioner mellan polymererna och frånvaron av organiska lösningsmedel i reaktionssystemet 11. Därför ser det höga procentandelar av RGD kopplade till polymera nätverket eftersom ingen modfikationerna är skyldiga att polymer kemiska grupper som är inblandade i gelbildning. Karboxylgrupper, från PAA och karbomer, och hydroxylgrupper, från PEG och agaros, ge upphov till hydrogelen tredimensionella strukturen genom en polykondensationsreaktion. De nämnda polymerer användes för syntes av hydrogeler i ryggmärgsskada reparation behandlingar 12. Dessa enheter, som rapporterats i tidigare verk 13,14, visar hög biokompatibilitet samt mekaniska och fysikalisk-kemiska egenskaper som liknar de hos många levande vävnader och tixotrop natur. Dessutom förblir de lokaliserad in situ, vid zonen för injektionen. I detta arbete är PAA karboxylgrupper modifierad med en alkyn molekyldel (figur 1), och en RGD-azid förening syntetiseras utnyttjar reaktiviteten hos tripeptid terminala gruppen -NH2 med en förberedd kemisk förening med strukturen (CH2) n - N 3 (&lt;strong&gt; Figur 2). Därefter reagerar den modifierade PAA med RGD-azidderivat genom CuAAC klick reaktion 15-17 (Figur 3). Användningen av en koppar (I) katalysator leder till stora förbättringar i både reaktionshastigheten och regioselektivitet. Den CuAAC reaktion används ofta i organisk syntes och polymervetenskap. Den kombinerar hög effektivitet och hög tolerans mot de funktionella grupperna, och det är opåverkad av användningen av organiska lösningsmedel. En hög selektivitet, en snabb reaktionstid och en enkel procedur renings tillåter erhållandet av stjärnpolymerer, segmentsampolymerer eller kedjor ympning önskade delar 18. Denna klick strategi gör det möjligt att modifiera polymerer efter polymerisation att anpassa fysikalisk-kemiska egenskaper enligt den slutliga biokemiska ansökan. De CuAAC experimentella betingelser är lätt reproducerbar (reaktionen är okänslig för vatten, medan koppar oxidation kan förekomma minimalt), och naturen avbildade triazol säkerställer produktens stabilitet. Användningen av kopparmetall kan anses vara en kritisk punkt, på grund av dess potentiella toxiska effekt mot celler och i den biologiska mikromiljö, men dialys används som en reningsmetod för att tillåta fullständigt avlägsnande av katalysatorrester. Slutligen PAA modifierad RGD används i hydrogel-syntes (Figur 4) och de fysikalisk-kemiska egenskaperna hos de erhållna näten undersöks, i syfte att kontrollera den potentiella funktionaliteten hos dessa system som celler eller droger bärare. <img alt="Figur 1" src="/files/ftp_upload/54445/54445fig1.jpg" /> Figur 1: PAA modifierad alkyn syntes Ett system av PAA funktionalisering med alkyn grupp;. &quot;n&quot; anger monomerer med karboxylgrupp reagera med propargylamin. <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig1large.jpg" target="_blank">Klicka här för att se</a>en större version av denna siffra. <img alt="figur 2" src="/files/ftp_upload/54445/54445fig2.jpg" /> Figur 2:.. RGD-azid syntes Syntesen av RGD-azidderivat <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">Klicka här för att se en större version av denna siffra.</a> <img alt="Figur 3" src="/files/ftp_upload/54445/54445fig3.jpg" /> Figur 3: Klicka på reaktionsschema klick reaktion mellan RGD-azidderivat och alkyn-PAA.. <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">Klicka här för att se en större version av denna siffra.</a> <img alt="figur 4" src="/files/ftp_upload/54445/54445fig4.jpg" /> Figur 4: hydrogel Synthesis. RGD funktion hydrogel syntesförfarandet. <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">Klicka här för att se en större version av denna siffra.</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>
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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.

PAA alkyn derivatet effektivt syntetiseras från polyakrylsyra och propargylamin, som visade i figur 1, där n etiketter monomerer vars karboxylgrupper reagerar med amin. Identiteten hos produkten bekräftades genom ett H-NMR-spektroskopi. Figur 5 visar ett H-NMR-spektrum av PAA modifierad med trippelbindning. <img alt="figur 5" src="/files/ftp_upload/54445/54445fig5.jpg" /> <stron...

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.

Syntes av RGD-funktion Hydrogeler som ett verktyg för terapeutiska tillämpningar

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

The overall goal of this methodology is to synthesize hydrogels for tissue engineering applications very easily. This method can help answer key questions in the tissue engineering field, such as the possibility to maintain high cell viability in three dimensional cell culture systems. The main advantage of this technique is that is using quick reaction, we can easily modify hydrogel systems to improve their ability to maintain cell viable.The implications of this technique extend toward the therapy of spinal cord injury because the cells loaded within the scaffold can rebuild the damaged tissue. For this method can provide significant insight into cell therapies as regenerative medicine and their implication in many methodologies. It can also be applied in several other systems like bone, heart, and cartilage.To synthesize the RGD-azide derivative, dissolve 50 milligrams of RGD in one milliliter of one molar sodium hydroxide. Then dissolve 24 milligrams of 4-azidobutanoyl chloride in two milliliters of tetrahydrofuran. Add all of the RGD solution into the 4-azidobutanoyl chloride solution at zero degrees Celsius dropwise.Return to room temperature and stir overnight. Add one milliliter of one molar HCL. Remove the solvent under reduced pressure using a rotary evaporator.Characterize the obtained product by proton nuclear magnetic resonance spectroscopy, dissolving the sample in deuterium oxide or D2O. To perform the PAA alkine modification, dissolve 200 milligrams of 35%in weight PAA solution in 15 milliliters of distilled water. Add 15.4 milligrams of propargylamine hydrochloride.Then dissolve 42.8 milligrams of HOBt in 14 milliliters of a one to one ratio of acetonytryle distilled water solution by heating to 50 degrees Celsius. Add all of the HOBt solution to the PAA solution at room temperature. Next, add 53.6 milligrams of EDC to the reaction mixture.Use one molar HCL to adjust the PH to 5.5 and stir the reaction system overnight at room temperature. Next, dissolve 11.2 grams of sodium chloride in two liters of distilled water. And then add 200 microliters of 37%weight by weight HCL.Dialyze the solution using a membrane with a molecular weight cutoff of 3.5 kilodaltons. Perform dialysis for three days. Change the dialysis solution daily with two liters of freshly prepared distilled water containing 200 microliters of 37%weight by weight HCL.Store the final solution at minus 80 degrees Celsius. Characterize the functionalized polymer by proton nuclear magnetic resonance spectroscopy, dissolving the sample in D2O. To synthesize the PAA-RGD polymer, dissolve 78 milligrams of PAA modified alkine in 10 milliliters of distilled water.Then dissolve 25 milligrams of the RGD-azide derivative in five milliliters of tetrahydrafuran. Add all of the RGD solution to the polymeric solution. Next, add 2.2 milligrams of copper iodide.And 2.2 milligrams of sodium ascorbate. Reflux the resulting mixture overnight at 60 degrees Celsius with stirring. Cool the mixture to 25 degrees Celsius.To dialyze the solution, dissolve 11.2 grams of sodium chloride in two liters of distilled water and add 200 microliters of 37 percent weight by weight HCL. Perform dialysis for three days. Change the dialysis solution daily with two liters of freshly prepared distilled water containing 200 microliters of 37%weight by weight HCL.Then place the final solution at minus 80 degrees Celsius. Characterize and store the resulting PAA-RGD polymer as done for its constituents. Blend 40 milligrams of carbomer and 10 milligrams of the functionalized PAA in nine milliters of phosphate-buffered saline at room temperature until complete dissolution.Add 400 milligrams of PEG to the solution and keep stirring for 45 minutes. Stop the stirring and allow the system to settle for 30 minutes. Then use one molar sodium hydroxide to adjust the PH to 7.4.To five milliliters of the obtained mixture, add 25 milligrams of agarose powder. Irradiate the system with microwave radiation at 500 watts for 30 seconds to one minute until boiling. And electromagnetically heat up the solution to 80 degrees Celsius.Leave the mixture exposed to room temperature until its temperature decreases to 50 degrees Celsius. Then add five milliliters of PBS to obtain a solution at a one to one volumetric ratio. Prepare a 12-multiwell plate containing steel cylinders with a diameter of 1.1 centimeters.Take 500 microliters aliquots from the solution and place them into each steel cylinder. Leave the plate at rest for 45 minutes until complete gelification of the system. Remove the cylinders using stainless steel forceps to obtain the hydrogels.See the text protocol for characterizing the physicochemical properties of the RGD-functionalized hydrogels. Carbomer 974P, PAA-RGD, and PEG, together with agarose, take part in a statistical random polycondensation after microwave irradiation. Hydroxyl groups contained in PEG and agarose react with carboxyl groups present in Carbomer 947P and PAA-RGD to form ACPEG-RGD hydrogels.Considering rheological properties, the storage modulus was found to be approximately one order of magnitude higher than the loss modulus, indicating an elastic rather than viscous material. Moreover, it is clearly visible that hydrogel behavior is dominated at low strain values by the elastic modulus. Scanning electron microscopy shows the morphology of an RGD functionalized hydrogel sample and a hydrogel without functionalization.Both hydrogels revealed that they possess a highly entangled structure of interconnected pores, with some bigger pores containing small pores and some fibular networks on the pore walls. The similar properties indicate that the presence of RGD does not alter the polymer network. After this development, this technique paved the way for research in the field of tissue engineering to explore the stability of these cell culture systems in several other diseases.After watching this video, you should have a good understanding of how to functionalize hydrogels. Improving their ability to maintain viable stem cells.

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Bioengineering

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