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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 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.
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....
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 δ values in parts per million. Furthermore, hydrogels are subjected to FT-IR analysis using KBr pellet technique and their physical characterization involves gelati....
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.
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<.......
The authors state no conflict of interest and they have not received any payment in preparation of this manuscript.
Authors would like to thank Prof. Maurizio Masi for fruitful discussion and Miss Chiara Allegretti for language editing. Authors' research is supported by Bando Giovani Ricercatori 2010 (Ministero della Salute GR-2010- 2312573).
....Name | Company | Catalog Number | Comments |
Poly(acrylic acid) solution average Mw ~ 100,000, 35 wt % in H2O | Sigma Aldrich | 523925 | CAS 9003-01-4 |
Poly(ethylene glycol) 2,000 | Sigma Aldrich | 84797 | CAS 25322-68-3 |
Carbomeer 974P | Fagron | 1387083 | |
Agarose | Invitrogen Corp. | 16500-500 | UltraPure Agarose |
RGD peptide | abcam | ab142698 | |
4-azidobutanoic acid | Aurum Pharmatech | Z-2421 | CAS 54447-68-6 |
Oxalyl chloride | Sigma Aldrich | O8801 | CAS 79-37-8 |
Propargylamine hydrochloride 95% | Sigma Aldrich | P50919 | CAS 15430-52-1 |
Copper(I) iodide | Sigma Aldrich | 3140 | CAS 7681-65-4 |
Sodium ascorbate | Sigma Aldrich | Y0000039 | CAS 134-03-2 |
Phosphate buffered saline | Sigma Aldrich | P4417 | |
Dialysis Membrane | Spectrum Laboratories, Inc. | 132725 | Spectra/Por 3 Dialysis Membrane Standard RC Tubing MWCO: 3,5 kD |
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