Authors would like to thank Prof. Maurizio Masi for fruitful discussion and Miss Chiara Allegretti for language editing. Authors&#39; research is supported by Bando Giovani Ricercatori 2010 (Ministero della Salute GR-2010- 2312573).

Note: The chemicals are used as received. Linear RGD is purchased, but it can be prepared by standard Fmoc solid phase peptide synthesis16,19. Solvents are of analytical grade. The dialysis requires the use of membrane with a Mw cut-off equal to 3,500 Da. The synthesized compounds are characterized by 1H NMR spectra recorded on a 400 MHz spectrometer using chloroform (CDCl3) or deuterium oxide (D2O) as solvents, and chemical shifts are reported as &#948; values in pa...

<ol>
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Drug Deliv. 10 (3), 385-396 (2013).</li><li>Huebsch, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+traction-mediated+manipulation+of+the+cell/matrix+interface+to+control+stem-cell+fate.">Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate.</a> Nat. Mater. 9 (6), 518-526 (2010).</li><li>Mothe, A. J., Tam, R. Y., Zahir, T., Tator, C. H., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+the+injured+spinal+cord+by+transplantation+of+neural+stem+cells+in+a+hyaluronan-based+hydrogel.">Repair of the injured spinal cord by transplantation of neural stem cells in a hyaluronan-based hydrogel.</a> Biomaterials. 34 (15), 3775-3783 (2013).</li><li>Khetan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation-mediated+cellular+traction+directs+stem+cell+fate+in+covalently+crosslinked+three-dimensional+hydrogels.">Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels.</a> Nat. Mater. 12 (5), 458-465 (2013).</li><li>Ashley, G. W., Henise, J., Reid, R., Santi, D. 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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<...

Hydrogels are three-dimensional networks formed by hydrophilic cross-linked polymers, which are natural or synthetic, and characterized by a distinctive three-dimensional structure. These devices are increasingly attractive in the biomedical fields of drug delivery, tissue engineering, gene carriers and smart sensors1,2. Indeed, their high water content, as well as their rheological and mechanical properties make them suitable candidates to mimic soft tissue microenvironments and make them effective tools for water-soluble cytokine or growth factor delivery. One of the most promising use is as an injectable biomaterial carrying cells and bioactive compounds. Hydrogels may improve cell survival and control stem cell fate by holding and precisely delivering stem cell regulatory signals in a physiological relevant fashion, as observed in in vitro and in in vivo experiments3,4. The leading advantage of this is the possibility to maintain injected cells within the zone of inoculation (in situ), minimizing the amount of cells that leaves the area and extravasates into the circulatory torrent, migrating all over the body and losing the target goal5. The stability of the three-dimensional hydrogel networks is due to its cross-linking sites, formed by covalent bonds or cohesive forces among the polymer chains6.
In this framework, orthogonal selective chemistry applied to polymer chains is a versatile tool able to improve hydrogel performances7. Indeed, the modification of polymers with suitable chemical groups could help to provide appropriate chemical, physical and mechanical properties to enhance cell viability and their use in tissue formation. In the same way, among the techniques to load cells or growth factors within the gel matrix, the use of the RGD peptide allows improvements in cell adhesion and survival. RGD is a tripeptide composed of arginine, glycine and aspartic acid, which is by far the most effective and often employed tripeptide due to its ability to address more than one cell adhesion receptor and its biological impact on cell anchoring, behavior and survival8,9. In this work, the synthesis of RGD-functionalized hydrogels is studied with the aim of designing networks characterized by sufficient biochemical properties for a hospitable cell microenvironment.
The use of microwave radiation in hydrogel synthesis offers a simple procedure to minimize side reactions and obtain higher reaction rates and yields in a shorter period of time compared to the conventional thermal processes10. This method does not require purification steps and yields sterile hydrogels due to the interactions of the polymers and the absence of organic solvent in the reaction system11. Therefore, it ensures high percentages of RGD linked to the polymeric network because no modifications are required to the polymer chemical groups involved in gel formation. Carboxyl groups, from PAA and carbomer, and hydroxyl groups, from PEG and agarose, give rise to the hydrogel three-dimensional structure through a polycondensation reaction. The mentioned polymers are used for the synthesis of hydrogels in the spinal cord injury repair treatments12. These devices, as reported in previous works13,14, show high biocompatibility as well as mechanical and physicochemical properties that resemble those of many living tissues and in thixotropic nature. Moreover, they remain localized in situ, at the zone of injection.
In this work, PAA carboxyl groups are modified with an alkyne moiety (Figure 1), and a RGD-azide compound is synthesized exploiting the reactivity of the tripeptide terminal group -NH2 with a prepared chemical compound with structure (CH2)n-N3 (Figure 2). Subsequently, the modified PAA reacts with the RGD-azide derivative through CuAAC click reaction15-17 (Figure 3). The use of a copper(I) catalyst leads to major improvements in both the reaction rate and the regioselectivity. The CuAAC reaction is widely used in organic synthesis and in polymer science. It combines high efficiency and high tolerance to the functional groups, and it is unaffected by the use of organic solvents. A high selectivity, a fast reaction time and a simple purification procedure allow the obtainment of star polymers, block copolymers or chains grafting desired moieties18. This click strategy makes it possible to modify polymers after polymerization to customize the physicochemical properties according to the final biochemical application. The CuAAC experimental conditions are easily reproducible (the reaction is insensitive to water, whereas copper oxidation may occur minimally), and the nature of formed triazole ensures stability of the product. The use of copper metal can be considered a critical point, due to its potential toxic effect against cells and in the biological microenvironment, but dialysis is used as a purification method to allow the complete removal of catalytic residues. Finally, PAA modified RGD is used in hydrogel synthesis (Figure 4) and the physicochemical properties of the resulting networks are investigated, in order to check the potential functionality of these systems as cells or drugs carriers.
<img alt="Figure 1" src="/files/ftp_upload/54445/54445fig1.jpg" /> 
Figure 1: PAA modified alkyne synthesis. A scheme of PAA functionalization with alkyne group; &#34;n&#34; indicates the monomers with carboxyl group reacting with propargylamine. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54445/54445fig2.jpg" /> 
Figure 2: RGD-azide synthesis. The synthesis of RGD-azide derivative. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54445/54445fig3.jpg" /> 
Figure 3: Click reaction. Scheme of click reaction between RGD-azide derivative and alkyne-PAA. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54445/54445fig4.jpg" /> 
Figure 4: Hydrogel synthesis. RGD functionalized hydrogel synthesis procedure. <a href="http://cloudflare.jove.com/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</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.

The PAA alkyne derivative is efficiently synthesized from polyacrylic acid and propargylamine, as showed in Figure 1 where n labels the monomers whose carboxyl groups react with the amine. The identity of the product is confirmed by 1H-NMR spectroscopy. Figure 5 shows the 1H-NMR spectrum of PAA modified with triple bond.
<img alt="Figure 5" src="/files/ftp_upload/54445/54445fig5.jpg" /> 
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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.

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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JoVE Journal

Bioengineering

11.0K Görüntüleme.  Politecnico di Milano. 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 towa...