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

Summary

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.

Abstract

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.

Introduction

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 sensors^1,2. 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 experiments^3,4. 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 survival^8,9. 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 works^13,14, 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 reaction^15-17 (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.

Figure 1: PAA modified alkyne synthesis. A scheme of PAA functionalization with alkyne group; "n" indicates the monomers with carboxyl group reacting with propargylamine. Please click here to view a larger version of this figure.

Figure 2: RGD-azide synthesis. The synthesis of RGD-azide derivative. Please click here to view a larger version of this figure.

Figure 3: Click reaction. Scheme of click reaction between RGD-azide derivative and alkyne-PAA. Please click here to view a larger version of this figure.

Figure 4: Hydrogel synthesis. RGD functionalized hydrogel synthesis procedure. Please click here to view a larger version of this figure.

Protocol

Note: The chemicals are used as received. Linear RGD is purchased, but it can be prepared by standard Fmoc solid phase peptide synthesis^16,19. Solvents are of analytical grade. The dialysis requires the use of membrane with a M_w 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 gelation studies assessed using the inverted test tube at 37 °C.

1. Synthesis of 4-Azidobutanoyl Chloride 1

1. Dissolve 500 mg of 4-azidobutanoic acid (3.90 mmol) in 10 ml of dichloromethane and 0.5 ml of dimethylformamide.
2. Cool the solution at 0 °C, using an ice bath.
3. Add 505 µl of oxalyl chloride (5.85 mmol) to 5 ml of dichloromethane and slowly add dropwise to the reaction system, while stirring.
4. After 1 hr at 0 °C using an ice bath, return to room temperature.
5. Remove the solvent under reduced pressure using a rotary evaporator.
6. Characterize the obtained product by ¹H-NMR spectroscopy, dissolving the sample in CDCl₃¹⁶.

2. Synthesis of RGD-azide Derivative 2

1. Dissolve 50 mg of RGD (0.145 mmol) in 1 ml of 1 M NaOH.
2. Dissolve 24 mg of 1 (0.16 mmol) in 2 ml of tetrahydrofuran.
3. Add all of the RGD solution to solution 1 dropwise at 0 °C using an ice bath.
4. Return to room temperature and stir overnight.
5. Add 1 ml of 1 M HCl.
6. Remove the solvent under reduced pressure using a rotary evaporator.
7. Characterize the obtained product by ¹H-NMR spectroscopy, dissolving the sample in D₂O¹⁶.

3. PAA Alkyne Modification 3

1. Dissolve 200 mg of 35% w/w PAA solution (2.8 mmol) in 15 ml of distilled water.
2. Add 15.4 mg of propargylamine hydrochloride (0.20 mmol).
3. 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 °C.
4. Add all of the HOBt solution to PAA solution at room temperature.
5. Add 53.6 mg of ethyldimethylaminopropylcarbodiimide (EDC, 0.28 mmol) to the reaction mixture.
6. Use 1 M HCl to adjust the pH to 5.5 and stir the reaction system overnight at room temperature.
7. 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 M_w cut-off of 3.5 kDa.
8. 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.
9. Store the final solution at -80 °C. Lyophilize it in a lyophilizer according to manufacturer's protocols.
10. Characterize the functionalized polymer by ¹H-NMR spectroscopy, dissolving the sample in D₂O¹⁶.

4. Synthesis of PAA-RGD Polymer 4

1. Dissolve 78 mg of PAA modified alkyne 3 (1.083 mmol) in 10 ml of distilled water.
2. Dissolve 25 mg of the RGD azide 2 derivative (0.0722 mmol) in 5 ml of tetrahydrofuran.
3. Add all of the RGD solution to the polymeric solution.
4. Add 2.2 mg of copper iodide (0.0116 mmol) and 2.2 mg of sodium ascorbate (0.0111 mmol).
5. Reflux the resulting mixture overnight at 60 °C, with stirring.
6. Cool the mixture to 25 °C.
7. 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 M_w cut-off of 3.5 kDa.
8. 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.
9. Store the final solution at -80 °C. Lyophilize it in a lyophilizer according to manufacturer's protocols.
10. Characterize the obtained product by ¹H-NMR spectroscopy, dissolving the sample in D₂O¹⁶.

5. RGD-functionalized Hydrogel Synthesis

1. Prepare the PBS. Dissolve 645 mg of PBS salt in 50 ml of distilled water.
2. 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).
3. Add 400 mg of PEG to the solution and keep stirring for 45 min.
4. Stop the stirring and allow the system to settle for 30 min.
5. Use 1 N NaOH to adjust pH to 7.4.
6. To 5 ml of the obtained mixture, add 25 mg of agarose powder.
7. 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 °C.
8. Leave the mixture exposed to room temperature until its temperature decreases to 50 °C and add 5 ml of PBS (step 5.1), in order to obtain a solution at a 1:1 volumetric ratio.
9. Prepare 12 multiwell plate containing steel cylinders with a diameter of 1.1 cm.
10. Take 500 µl aliquots from the solution and place them to each steel cylinders.
11. Leave at rest for 45 min until complete gelification of the system.
12. Remove the cylinders using a stainless steel forceps to obtain the hydrogels.

6. Loading of Therapeutic Tool (Drug or Cells)

1. Repeat steps 5.1-5.7.
2. When the mixture (already at sol state) reaches 37 °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.
3. Repeat steps 5.9-5.12 to obtain polymeric networks with biocompounds physically entrapped within the gel.

7. Hydrogel Characterization

1. FT-IR Analysis
   1. After gel formation, soak one of the synthetized hydrogels in 2.5 ml of distilled water for 24 hr.
   2. Remove the aqueous media where hydrogel is submerged and freeze-dry with liquid N₂.
   3. Laminate the hydrogel sample according to KBr pellet technique.
      1. 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.
      2. Grind the mixture until the powder is fine and homogenous.
      3. 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.
      4. 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.
2. Gelation Studies
   1. Fill 2 ml microcentrifuge tube with 900 µl of PBS and equilibrate to 37 °C.
   2. Add 100 µl of the prepared polymer solution to form the hydrogel and incubate at 37 °C.
   3. 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.

Results

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.

Discussion

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<...

Materials

List of materials used in this article

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