Name: the synthesis of rgd-functionalized hydrogels as a tool for therapeutic applications
Uploaded: 2016-10-07T14:00:00.000+00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications at JoVE.com

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 parts per million. Furthermore, hydrogels are subjected to FT-IR analysis using KBr pellet technique and their physical characterization involves gelation studies assessed using the inverted test tube at 37 &#176;C.
1. Synthesis of 4-Azidobutanoyl Chloride 1
<ol>
	<li>Dissolve 500 mg of 4-azidobutanoic acid (3.90 mmol) in 10 ml of dichloromethane and 0.5 ml of dimethylformamide.</li>
	<li>Cool the solution at 0 &#176;C, using an ice bath.</li>
	<li>Add 505 &#181;l of oxalyl chloride (5.85 mmol) to 5 ml of dichloromethane and slowly add dropwise to the reaction system, while stirring.</li>
	<li>After 1 hr at 0 &#176;C using an ice bath, return to room temperature.</li>
	<li>Remove the solvent under reduced pressure using a rotary evaporator.</li>
	<li>Characterize the obtained product by 1H-NMR spectroscopy, dissolving the sample in CDCl316.</li>
</ol>
2. Synthesis of RGD-azide Derivative 2
<ol>
	<li>Dissolve 50 mg of RGD (0.145 mmol) in 1 ml of 1 M NaOH.</li>
	<li>Dissolve 24 mg of 1 (0.16 mmol) in 2 ml of tetrahydrofuran.</li>
	<li>Add all of the RGD solution to solution 1 dropwise at 0 &#176;C using an ice bath.</li>
	<li>Return to room temperature and stir overnight.</li>
	<li>Add 1 ml of 1 M HCl.</li>
	<li>Remove the solvent under reduced pressure using a rotary evaporator.</li>
	<li>Characterize the obtained product by 1H-NMR spectroscopy, dissolving the sample in D2O16.</li>
</ol>
3. PAA Alkyne Modification 3
<ol>
	<li>Dissolve 200 mg of 35% w/w PAA solution (2.8 mmol) in 15 ml of distilled water.</li>
	<li>Add 15.4 mg of propargylamine hydrochloride (0.20 mmol).</li>
	<li>Dissolve 42.8 mg of 1-hydroxybenzotriazole hydrate (HOBt, 0.28 mmol) in 14 ml of a 1:1 v/v acetonitrile:distilled water solution by heating to 50 &#176;C.</li>
	<li>Add all of the HOBt solution to PAA solution at room temperature.</li>
	<li>Add 53.6 mg of ethyldimethylaminopropylcarbodiimide (EDC, 0.28 mmol) to the reaction mixture.</li>
	<li>Use 1 M HCl to adjust the pH to 5.5 and stir the reaction system overnight at room temperature.</li>
	<li>Dialyze the solution. Dissolve 11.2 g of sodium chloride in 2 L of distilled water and then add 0.2 ml of 37% w/w HCl. Dialyze the solution using a membrane with a Mw cut-off of 3.5 kDa.</li>
	<li>Perform dialysis for three days. Change the dialysis solution daily with 2 L of freshly prepared distilled water containing 0.2 ml of 37% w/w HCl.</li>
	<li>Store the final solution at -80 &#176;C. Lyophilize it in a lyophilizer according to manufacturer&#39;s protocols.</li>
	<li>Characterize the functionalized polymer by 1H-NMR spectroscopy, dissolving the sample in D2O16.</li>
</ol>
4. Synthesis of PAA-RGD Polymer 4
<ol>
	<li>Dissolve 78 mg of PAA modified alkyne 3 (1.083 mmol) in 10 ml of distilled water.</li>
	<li>Dissolve 25 mg of the RGD azide 2 derivative (0.0722 mmol) in 5 ml of tetrahydrofuran.</li>
	<li>Add all of the RGD solution to the polymeric solution.</li>
	<li>Add 2.2 mg of copper iodide (0.0116 mmol) and 2.2 mg of sodium ascorbate (0.0111 mmol).</li>
	<li>Reflux the resulting mixture overnight at 60 &#176;C, with stirring.</li>
	<li>Cool the mixture to 25 &#176;C.</li>
	<li>Dialyze the solution. Dissolve 11.2 g of sodium chloride in 2 L of distilled water and then add 0.2 ml of 37% w/w HCl. Dialyze the solution using a membrane with a Mw cut-off of 3.5 kDa.</li>
	<li>Perform dialysis for three days. Change the dialysis solution daily with 2 L of freshly prepared distilled water containing 0.2 ml of 37% w/w HCl.</li>
	<li>Store the final solution at -80 &#176;C. Lyophilize it in a lyophilizer according to manufacturer&#39;s protocols.</li>
	<li>Characterize the obtained product by 1H-NMR spectroscopy, dissolving the sample in D2O16.</li>
</ol>
5. RGD-functionalized Hydrogel Synthesis
<ol>
	<li>Prepare the PBS. Dissolve 645 mg of PBS salt in 50 ml of distilled water.</li>
	<li>Blend 40 mg of carbomer and 10 mg of functionalized PAA 4 in 9 ml of PBS (step 5.1), at room temperature, until complete dissolution (30 min).</li>
	<li>Add 400 mg of PEG to the solution and keep stirring for 45 min.</li>
	<li>Stop the stirring and allow the system to settle for 30 min.</li>
	<li>Use 1 N NaOH to adjust pH to 7.4.</li>
	<li>To 5 ml of the obtained mixture, add 25 mg of agarose powder.</li>
	<li>Irradiate the system with microwave radiation at 500 W until boiling, for a time usually between 30 sec and 1 min, and electromagnetically heat up to 80 &#176;C.</li>
	<li>Leave the mixture exposed to room temperature until its temperature decreases to 50 &#176;C and add 5 ml of PBS (step 5.1), in order to obtain a solution at a 1:1 volumetric ratio.</li>
	<li>Prepare 12 multiwell plate containing steel cylinders with a diameter of 1.1 cm.</li>
	<li>Take 500 &#181;l aliquots from the solution and place them to each steel cylinders.</li>
	<li>Leave at rest for 45 min until complete gelification of the system.</li>
	<li>Remove the cylinders using a stainless steel forceps to obtain the hydrogels.</li>
</ol>
6. Loading of Therapeutic Tool (Drug or Cells)
<ol>
	<li>Repeat steps 5.1-5.7.</li>
	<li>When the mixture (already at sol state) reaches 37 &#176;C, add 5 ml of the solution containing the desired drug solution or cell culture, in order to obtain a final system at a 1:1 volumetric ratio.</li>
	<li>Repeat steps 5.9-5.12 to obtain polymeric networks with biocompounds physically entrapped within the gel.</li>
</ol>
7. Hydrogel Characterization
<ol>
	<li>FT-IR Analysis
	<ol>
		<li>After gel formation, soak one of the synthetized hydrogels in 2.5 ml of distilled water for 24 hr.</li>
		<li>Remove the aqueous media where hydrogel is submerged and freeze-dry with liquid N2.</li>
		<li>Laminate the hydrogel sample according to KBr pellet technique.
		<ol>
			<li>Add a spatula full of KBr into an agate mortar. Take a small amount of hydrogel sample (about 0.1-2% of the KBr amount, or just enough to cover the tip of spatula) and mix with the KBr powder.</li>
			<li>Grind the mixture until the powder is fine and homogenous.</li>
			<li>Use the KBr pellet kit to form the IR pellet. Press the powder using a manual laboratory press: for 3 min at pressure capacity equal to 5 tons and then for 3 min at pressure capacity of 10 tons.</li>
			<li>Release the pressure to obtain the final pellet as homogeneous and transparent in appearance. Insert the pellet into the IR sample holder and run the spectrum16.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Gelation Studies
	<ol>
		<li>Fill 2 ml microcentrifuge tube with 900 &#181;l of PBS and equilibrate to 37 &#176;C.</li>
		<li>Add 100 &#181;l of the prepared polymer solution to form the hydrogel and incubate at 37 &#176;C.</li>
		<li>Invert the tube and observe if the gel flows at 1, 2, 5, 10 and 20 min. Record the time at which the gel does not flow as the gelation time.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Slaughter, B. V., Khurshid, S. S., Fisher, O. Z., Khademhosseini, A., Peppas, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+in+Regenerative+Medicine.">Hydrogels in Regenerative Medicine.</a> Adv. Mater. 21 (32-33), 3307-3329 (2009).</li><li>Rossi, F., Perale, G., Papa, S., Forloni, G., Veglianese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+options+for+drug+delivery+to+the+spinal+cord.">Current options for drug delivery to the spinal cord.</a> Expert Opin. 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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+drug+delivery+system+with+predictable+and+tunable+drug+release+and+degradation+rates.">Hydrogel drug delivery system with predictable and tunable drug release and degradation rates.</a> Proc. Natl. Acad. Sci. U S A. 110 (6), 2318-2323 (2013).</li><li>Rossi, F., van Griensven, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Functionalization+as+a+Powerful+Tool+to+Improve+Scaffold+Performances.">Polymer Functionalization as a Powerful Tool to Improve Scaffold Performances.</a> Tissue Eng. Part A. 20 (15-16), 2043-2051 (2014).</li><li>Gould, S. T., Darling, N. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+peptide+functionalized+thiol-ene+hydrogels+as+culture+substrates+for+understanding+valvular+interstitial+cell+activation+and+de+novo+tissue+deposition.">Small peptide functionalized thiol-ene hydrogels as culture substrates for understanding valvular interstitial cell activation and de novo tissue deposition.</a> Acta Biomater. 8 (9), 3201-3209 (2012).</li><li>Azagarsamy, M. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength-Controlled+Photocleavage+for+the+Orthogonal+and+Sequential+Release+of+Multiple+Proteins.">Wavelength-Controlled Photocleavage for the Orthogonal and Sequential Release of Multiple Proteins.</a> Angew. Chem. Int. Edit. 52 (51), 13803-13807 (2013).</li><li>Larra&#241;eta, E., 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=Microwave-Assisted+Preparation+of+Hydrogel-Forming+Microneedle+Arrays+for+Transdermal+Drug+Delivery+Applications.">Microwave-Assisted Preparation of Hydrogel-Forming Microneedle Arrays for Transdermal Drug Delivery Applications.</a> Macromol. Mater. Eng. 300 (6), 586-595 (2015).</li><li>Cook, J. P., Goodall, G. W., Khutoryanskaya, O. V., Khutoryanskiy, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-Assisted+Hydrogel+Synthesis:+A+New+Method+for+Crosslinking+Polymers+in+Aqueous+Solutions.">Microwave-Assisted Hydrogel Synthesis: A New Method for Crosslinking Polymers in Aqueous Solutions.</a> Macromol. Rapid Comm. 33 (4), 332-336 (2012).</li><li>Perale, G., 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=Multiple+drug+delivery+hydrogel+system+for+spinal+cord+injury+repair+strategies.">Multiple drug delivery hydrogel system for spinal cord injury repair strategies.</a> J. Control. Release. 159 (2), 271-280 (2012).</li><li>Rossi, F., Perale, G., Storti, G., Masi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Library+of+Tunable+Agarose+Carbomer-Based+Hydrogels+for+Tissue+Engineering+Applications:+The+Role+of+Cross-Linkers.">A Library of Tunable Agarose Carbomer-Based Hydrogels for Tissue Engineering Applications: The Role of Cross-Linkers.</a> J. Appl. Polym. Sci. 123 (4), 2211-2221 (2012).</li><li>Frith, J. E., 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=An+injectable+hydrogel+incorporating+mesenchymal+precursor+cells+and+pentosan+polysulphate+for+intervertebral+disc+regeneration.">An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration.</a> Biomaterials. 34 (37), 9430-9440 (2013).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry:+Diverse+chemical+function+from+a+few+good+reactions.">Click chemistry: Diverse chemical function from a few good reactions.</a> Angew. Chem. Int. Edit. 40 (11), (2001).</li><li>Sacchetti, A., Mauri, E., Sani, M., Masi, M., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-assisted+synthesis+and+click+chemistry+as+simple+and+efficient+strategy+for+RGD+functionalized+hydrogels.">Microwave-assisted synthesis and click chemistry as simple and efficient strategy for RGD functionalized hydrogels.</a> Tetrahedron Lett. 55 (50), 6817-6820 (2014).</li><li>Ossipov, D. A., Hilborn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(vinyl+alcohol)-based+hydrogels+formed+by+&amp;#34;click+chemistry&amp;#34.">Poly(vinyl alcohol)-based hydrogels formed by &amp;#34;click chemistry&amp;#34.</a> Macromolecules. 39 (5), 1709-1718 (2006).</li><li>Truong, V., Blakey, I., Whittaker, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+and+Amphiphilic+Polyethylene+Glycol-Based+Hydrogels+with+Tunable+Degradability+Prepared+by+&amp;#34;Click&amp;#34;+Chemistry.">Hydrophilic and Amphiphilic Polyethylene Glycol-Based Hydrogels with Tunable Degradability Prepared by &amp;#34;Click&amp;#34; Chemistry.</a> Biomacromolecules. 13 (12), 4012-4021 (2012).</li><li>Hou, R. Z., 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=New+synthetic+route+for+RGD+tripeptide.">New synthetic route for RGD tripeptide.</a> Prep. Biochem. Biotechnol. 36 (3), 243-252 (2006).</li><li>Rossi, F., Chatzistavrou, X., Perale, G., Boccaccini, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Degradation+of+Agar-Carbomer+Based+Hydrogels+for+Tissue+Engineering+Applications.">Synthesis and Degradation of Agar-Carbomer Based Hydrogels for Tissue Engineering Applications.</a> J. Appl. Polym. Sci. 123 (1), 398-408 (2012).</li><li>Mauri, E., Rossi, F., Sacchetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+drug+delivery+using+chemoselective+functionalization+of+hydrogels.">Tunable drug delivery using chemoselective functionalization of hydrogels.</a> Mater. Sci. Eng. C. 61, 851-857 (2016).</li><li>Joaquin, A., Peppas, N. A., Zoldan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+Polymer+Library+for+Developing+Induced+Pluripotent+Stem+Cell+Derived+Cardiac+Patches.">Hydrogel Polymer Library for Developing Induced Pluripotent Stem Cell Derived Cardiac Patches.</a> Tissue Eng. Part A. 20, S55-S55 (2014).</li><li>Rossi, F., 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=Tunable+hydrogel-Nanoparticles+release+system+for+sustained+combination+therapies+in+the+spinal+cord.">Tunable hydrogel-Nanoparticles release system for sustained combination therapies in the spinal cord.</a> Colloids Surf. B Biointerfaces. 108, 169-177 (2013).</li><li>Kolb, H. C., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+impact+of+click+chemistry+on+drug+discovery.">The growing impact of click chemistry on drug discovery.</a> Drug Discov. Today. 8 (24), 1128-1137 (2003).</li><li>Ossipov, D. A., Yang, X., Varghese, O., Kootala, S., Hilborn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+approach+to+functional+hyaluronic+acid+hydrogels+using+orthogonal+chemical+reactions.">Modular approach to functional hyaluronic acid hydrogels using orthogonal chemical reactions.</a> Chem. Commun. 46 (44), 8368-8370 (2010).</li><li>Anderson, S. B., Lin, C. C., Kuntzler, D. V., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+performance+of+human+mesenchymal+stem+cells+encapsulated+in+cell-degradable+polymer-peptide+hydrogels.">The performance of human mesenchymal stem cells encapsulated in cell-degradable polymer-peptide hydrogels.</a> Biomaterials. 32 (14), 3564-3574 (2011).</li><li>Caron, I., 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=A+new+three+dimensional+biomimetic+hydrogel+to+deliver+factors+secreted+by+human+mesenchymal+stem+cells+in+spinal+cord+injury.">A new three dimensional biomimetic hydrogel to deliver factors secreted by human mesenchymal stem cells in spinal cord injury.</a> Biomaterials. 75, 135-147 (2016).</li><li>Lee, J. W., Kim, H., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+spacer+arm+length+between+adhesion+ligand+and+alginate+hydrogel+on+stem+cell+differentiation.">Effect of spacer arm length between adhesion ligand and alginate hydrogel on stem cell differentiation.</a> Carbohyd. Polym. 139, 82-89 (2016).</li><li>Liu, Y., Fan, Z., Wang, Y., Yu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Release+of+Low+Molecular+Protein+Insulin-like+Growth+Factor-1+through+Self-Assembling+Peptide+Hydrogel+with+Biotin+Sandwich+Approach.">Controlled Release of Low Molecular Protein Insulin-like Growth Factor-1 through Self-Assembling Peptide Hydrogel with Biotin Sandwich Approach.</a> J.Biomed. Eng. 32 (2), 387-392 (2015).</li></ol>

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 heterocycles24. In this way, the chemical lability of PAA-RGD product is almost absent under the experimental conditions of hydrogel synthesis. The peptide is grafted to the 3D polymeric network due to the nature of its chemical bond and it is not physically entrapped (therefore, it could leave the system).
The alkyne PAA is prepared using propargylamine in an aqueous solution, in the presence of EDC and HOBt, to obtain the amine derivative. Experimental results indicate that the percentage of triple bond linking to the PAA is proportional to the amount of amine used. Referring to the NMR analysis, the polymer functionalization degree is between 90% and 100% when the molar ratio between the reagents PAA and propargylamine is 1:1 and is about 50% when the ratio is 1:0.5. Due to the fact that the formation of the gel network involves an esterification reaction, it is necessary to maintain some PAA carboxyl groups capable of reacting with hydroxyl groups of PEG and agarose. The modification of half PAA carboxyl groups is adequate for this, but hydrogels with improved physicochemical properties are obtained when the polymer functionalization degree is equal to 10% (molecular ratio PAA:propargylamine 1:0.1)25. The purification of the alkyne PAA is performed by dialysis, using a dialysis membrane with molecular weight cut-off (MWCO) of 3.5 kDa, allowing the removal of by-products and unreacted species. The product is finally isolated using lyophilization. 
RGD is characterized by the amino terminal group -NH2, which can be used as a coupling point to link the spacer-N3. The linkage between these compounds is obtained via an amide bond that is very stable and not too difficult to form16. In order to proceed in this way, 4-azidobutanoyl chloride is prepared as the linker of the spacer-N3 system. 
The alkyne moieties react with the RGD-azide derivative, through a click reaction, using copper iodide as a catalyst and sodium ascorbate as an antioxidant agent due to the possible oxidation of copper (Cu+ &#8594; Cu2+) in experimental atmospheric conditions. After screening different solvents and temperatures, the best results are performed by using a tetrahydrofuran/water mixture at 60 &#176;C16. According to the biological application, the post-synthesis retention of copper poses potential toxicity risks, especially for applications where the cells must remain viable. As a solution, dialysis is used to remove the copper catalyst and other by-products, obtaining a purified PAA-RGD compound suitable for hydrogel synthesis without any possible side effects. Click chemistry ensures a quantitative yield of the polymeric product grafting RGD. 
Due to the same reactivity of each PAA carboxyl group, the polymer functionalization is statistically evaluated. The exact number of modified carboxyl groups per PAA chain cannot be established and the reported functionalization degree is referred as the total number of carboxyl groups that have reacted with propargylamine. This estimation affects the subsequent assessment of the RGD functionalization and its effective amount loaded into each hydrogel. In addition, GPC analysis is not performed because in this work, the polymer is used and the protocol does not begin from monomers. The percentage of RGD functionalization is commonly verified with 1H-NMR and it is not possible to obtain reliable data from HPLC analysis. However, considering that each step of this work is replicated many times and the same experimental values and results are recorded, the illustrated results are reliable.
Another limitation is the use of copper as the catalyst as previously reported. The possible interaction of the copper with the PAA carboxyl groups during the click reaction and its subsequent presence as an entrapped molecule in the hydrogel network would reduce the biocompatibility of the system and would generate toxic effects in cell cultures. The three-day dialysis minimizes this potential limitation of the technique.
The functionalized PAA is used as a subunit in hydrogel formation, with carbomer, PEG and agarose. Carbomer and agarose are chosen because of their well-known biocompatibility in therapeutic approaches and tissue engineering. Carbomer also has anti-inflammatory properties while agarose is a suitable polymer for tissue repair strategies and provides a good environment for cell adhesion, spreading, proliferation, and protein drugs. Carbomer is a cross-linked poly-acrylic acid containing carboxyl groups (65%) that make it an ionizable molecule. The molecular weight is 106 Da. Its use is fundamental for gel synthesis due to its high number of reactive carboxyl groups. However, at the same time, it is very difficult to estimate a quantitative yield of any possible chemical functionalization because of its complex structure. For this reason, PAA is used due to its lower molecular weight (105 Da) and its linear structure. The combination of carbomer and PAA allows the design of gel networks with a RGD moiety that, otherwise, would not be viable. PEG is also introduced into the gel network due to its high biocompatibility, its ability to increase stem cell adhesion, and its high elasticity (being a small polymer) that can create elastic junctions between the carbomer and the agarose to form local interconnections through their own hydroxyl groups26. It also has a physical effect by increasing system viscosity and thus enhancing tight macromer cross-linking.
The use of the four polymers leads to a 3D network that is highly biocompatible, injectable and capable of maintaining its biochemical functionality with improved cell adhesion due to the presence of RGD. The latter is decisive in generating a suitable cell microenvironment within the hydrogel that otherwise would not give a satisfactory result27.
The hydrogel network can be consider as a "carbon skeleton" of macromolecules forming a stable structure comprised mostly of C-C bonds, where the cross-linking points are C-O bonds formed by esterification between carboxyl and hydroxyl groups. The microwave irradiation is a key point to ensure the agarose solubility in the aqueous reagent system and to avoid any further purification in the synthesis of sterile hydrogels via the sol/gel transition, because any initiators or monomers can be used and by-products are not formed. The microwave-assisted reaction also guarantees a high efficiency, and the functionalization degree of polymer chains with bioactive compounds or drugs generally does not affect the morphology, the structure, and the physical properties of the networks. These hydrogels are able to form in situ due to their ability to easily conform to any shape and be injected using minimally invasive surgical procedures. This is a significant advantage compared to the use of rigid and preformed scaffolds.
The synthesized networks can be used as an enhanced cell survival biomaterial, thanks to its high biocompatibility and the ability to create a hospitable cell microenvironment27. The improvement of this work is the ability to design a three-dimensional structure where the RGD is chemically linked through a stable and strong bond that is not cleavable in physiological conditions, thus avoiding RGD diffusion through the hydrogel pores to maintain its therapeutic effect at a high level for a long and sustained period of time. Alternative synthesis of chemical linkers, such as the ester or the hydrazone bonds, do not allow the same effect towards cells because they are cleavable, or pH/temperature sensitive under the cell microenvironment21,28,29. In addition, the tripeptide release is mostly driven by a pure diffusion mechanism that is very quick due to its small steric hindrance. The alternatives to modifications of the physical and mechanical parameters of the hydrogel network, for example reducing pores size, cannot be enough to maintain an adequate concentration of RGD within the matrix.
A possible additional functionalization is the use of fibronectin, which can optimize the hydrogel compatibility with the extracellular matrix in order to make viable living cells. One of the most important aspects is maintaining cell viability within hydrogel networks for days. In this way, cells can leave the matrix able to proliferate and differentiate in vivo. The first experimental results are published27. This highlights the comparison between RGD-functionalized hydrogels and non-functionalized hydrogels showing promising results. In particular, the RGD in the gel network improves stem cell survival and guarantees the immunomodulation of the pro-inflammatory and pro-regenerative effect at the lesion site in vivo. The presence of RGD linked to the hydrogel is a crucial point to achieve suitable therapeutic results. The RGD maintains its availability for cell interactions and it does not escape rapidly through hydrogel pores, maintaining its bioactive impact.

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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Poly(acrylic acid) solution average M&lt;sub&gt;w&lt;/sub&gt; ~100,000, 35 wt% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>523925</td><td>CAS 9003-01-4</td></tr><tr><td>Poly(ethylene glycol) 2,000</td><td>Sigma Aldrich</td><td>84797</td><td>CAS 25322-68-3</td></tr><tr><td>Carbomeer 974P</td><td>Fagron</td><td>1387083</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Invitrogen Corp.</td><td>16500-500</td><td>UltraPure Agarose</td></tr><tr><td>RGD peptide</td><td>abcam</td><td>ab142698</td><td></td></tr><tr><td>4-azidobutanoic acid</td><td>Aurum Pharmatech</td><td>Z-2421</td><td>&amp;nbsp;CAS 54447-68-6</td></tr><tr><td>Oxalyl chloride</td><td>Sigma Aldrich</td><td>O8801</td><td>CAS 79-37-8</td></tr><tr><td>Propargylamine hydrochloride 95%</td><td>Sigma Aldrich</td><td>P50919</td><td>CAS 15430-52-1</td></tr><tr><td>Copper(I) iodide</td><td>Sigma Aldrich</td><td>3140</td><td>CAS 7681-65-4</td></tr><tr><td>Sodium ascorbate</td><td>Sigma Aldrich</td><td>Y0000039</td><td>CAS 134-03-2</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td></td></tr><tr><td>Dialysis Membrane</td><td>Spectrum Laboratories, Inc.</td><td>132725</td><td>Spectra/Por 3 Dialysis Membrane&amp;nbsp; Standard RC Tubing&lt;br /&gt;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" /> 
Figure 5: 1H-NMR spectrum of the PAA modified alkyne. The signal related to the alkyne moiety is highlighted. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The signals of the polymer chain can be observed in the range 2.75-1.50 ppm; whereas a peak at 2.80 ppm, representative of alkyne&#39;s H, and a peak at 4.20 ppm, related to the 2 H of the -CH2, characterize the propargyl moiety. This confirms that PAA has been properly modified. The evaluation of the degree of alkyne functionalization has been carried out by integrating the area under the PAA peaks (set to 3.00, according to the number of hydrogens per monomer) and propargyl moiety, as illustrated in Figure 5. The degree of functionalization f is calculated as:
<img alt="Equation" src="/files/ftp_upload/54445/54445eq1.jpg" />
<img alt="Equation" src="/files/ftp_upload/54445/54445eq2.jpg" /> represents the integral area of the propargyl residue, the sum of the alkyne&#39;s H area (labelled as <img alt="Equation" src="/files/ftp_upload/54445/54445eq3.jpg" />) and the -CH2 area (indicated as <img alt="Equation" src="/files/ftp_upload/54445/54445eq4.jpg" />), whereas <img alt="Equation" src="/files/ftp_upload/54445/54445eq5.jpg" /> refers to the integral area of the polymer signals. The degree of functionalization is calculated to be 10% and it is consider satisfactory according to the hydrogel synthesis, where PAA has to react through its residual carboxyl groups to form the 3D network. A quantitative yield is obtained for the modified polymer16.
In a similar manner, Figure 6 shows the 1H-NMR spectrum of the product after the CuAAC click reaction between the alkyne modified PAA and RGD-azide. The peak of the formed triazole at 8.15 ppm confirms that the reaction occurs in a quantitative yield and RGD is strongly linked to the PAA chains. Figure 6 illustrates all of the characteristic signals of the PAA chain and the RGD.
<img alt="Figure 6" src="/files/ftp_upload/54445/54445fig6.jpg" /> 
Figure 6: 1H-NMR spectrum of the RGD linked to PAA. The signal of triazole is indicated (labelled as &#34;A&#34;). RGD polymer functionalization via CuAAC click reaction is performed. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
RGD-functionalized hydrogels are prepared through chemical cross-linking of the four polymers (PAA, carbomer, agarose and PEG) by microwave-assisted free radical polymerization. Heating to 80 &#176;C leads to a higher macromer mobility, and thus enhances the short-range interconnections among the carboxyl and hydroxyl groups of the polymers. The esterification reaction takes place between these functional groups and produces local networks called &#34;microgels&#34;.
As the polycondensation proceeds, the system viscosity increases continuously, while the probability of interaction between macromer reactive sites decreases. Nevertheless, the closer functional groups still interact efficiently due to a slower mobility. The resulting physicochemical condition is characterized by a &#34;welding&#34; between microgel surfaces that produces the final 3D macrostructure of the hydrogel. The esterification, hydrogen bonding and carboxylation bring the polymer chains statistically closer, thus creating a stable heterogeneous structure. The resulting system exhibits sol/gel behavior and it transitions to a gel state within 5 min. This time interval is reported as gelation time.
The chemical nature of the RGD-functionalized hydrogels is studied using FT-IR analysis. Figure 7 shows the comparison among FT-IR spectra of the RGD-azide compound (green line), the hydrogel synthesized without RGD functionalization (black line), and the hydrogel with peptide modification (blue line). The hydrogel spectra are both characterized by a broad signal in the 3,600-3,200 cm-1 range, representative of the stretching vibration of residual O-H bonds and by a peak around 2,940 cm-1 of the C-H stretch. The validation that esterification occurs among the carboxyl and hydroxyl polymer groups is given by the peaks around 1,600 cm-1 and 1,400 cm-1, corresponding, respectively, to the symmetric and asymmetric stretching of CO2 moiety. These peaks are more visible in the spectrum of the non-functionalized hydrogel, whereas in the RGD-hydrogel spectrum they are partially covered by the signals indicated as amide bands I and II.
<img alt="Figure 7" src="/files/ftp_upload/54445/54445fig7.jpg" /> 
Figure 7: Comparison of FT-IR spectra. FT-IR spectra of RGD (green line), hydrogel without RGD functionalization (black line) and RGD functionalized hydrogel (blue line). The signal related to the amide RGD is indicated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The stretching of C=O, labelled as amide band I (&#34;Amide I&#34; in Figure 7), presents a peak at 1,650 cm-1 in the tripeptide spectrum and it is shifted to about 1,670 cm-1 in the RGD-hydrogel sample. The bending of N-H, related to amide band II (&#34;Amide II&#34; in Figure 7), can be recorded with the signal around 1,550 cm-1 in the RGD spectrum and it is also recognizable in the hydrogel sample, at around 1,600 cm-1. Because there are no amide components in the standard hydrogel formulation, the presence of peaks of an amidic nature suggests that the PAA is really functionalized with the RGD and it is able to form a hydrogel with peptide sites within the polymeric network.
The hydrogel FT-IR spectrum also shows the peaks related to the stretching vibration of C-O-C of glycosidic bond (900-1,000 cm-1 range) between the monosaccharide units of the agarose and the ester groups.
To obtain insight into the 3D structure and physical and mechanical properties of these hydrogels, SEM analysis, gelation, swelling kinetics and rheological studies are performed, as discussed in previous works13,20. SEM results (Figure 8) show that hydrogels are characterized by a complex microscopic structure with some bigger pores containing small pores and some fibrillar networks on the pore walls. In addition, most of the pores are interconnected. The entangled structure is similar to the 3D network of hydrogels prepared in the same manner but without RGD functionalization. This demonstrates that the RGD does not alter the polymer network. Using the inverted test tube test, the hydrogel sample solidifies within 5 min, as observed in the hydrogel sample without RGD functionalization21. This short gelation time underlines its suitability for biomedical applications.
<img alt="Figure 8" src="/files/ftp_upload/54445/54445fig8.jpg" /> 
Figure 8: SEM analysis. SEM images show the morphology of a RGD-functionalized hydrogel sample (A) and a hydrogel without functionalization (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The swelling equilibrium ratio indicates the ability to absorb and retain a large amount of water and it is one of the leading features of hydrogel systems20,22. The analyzed samples exhibit fast swelling kinetics and they reach swelling equilibrium within the first hour. Their swelling equilibrium value Q is reported in our previous work16 and it is similar to the value obtained by analysis of hydrogels without RGD, confirming that the tripeptide is integrated with the polymeric network and does not create a high hindrance to the gelation process.
With the rheological studies, the gel storage modulus (G&#180;) is found to be approximately one order of magnitude higher than the loss modulus (G&#180;&#180;), indicating an elastic rather than viscous material23 and both are essentially independent from frequency. Similar values of G&#180; and G&#180;&#180; are recorded with the gel sample without a peptide modification16. This demonstrates that the presence of RGD within the polymeric network does not affect the rheological properties of the material, maintaining the peculiar features competing to injectable system for biomedical application.

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.2K Views.  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. 

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

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Chemistry

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochemistry

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

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.

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

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

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.

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

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video