End-group effects on the properties of PEG-co-PGA hydrogels

Abstract

A series of resorbable poly(ethylene glycol)-co-poly(glycolic acid) (PEG-co-PGA, 4KG5) macromonomers have been synthesized with the chemistries from three different photopolymerizable end-groups (acrylates, methacrylates and urethane methacrylates). The aim of the study is to examine the effects of the chemistry of the cross-linker group on the properties of photocross-linked hydrogels. 4KG5 hydrogels were prepared by photopolymerization with high vinyl group conversion as confirmed by (1)H nuclear magnetic resonance spectrometry using a 1D diffusion-ordered spectrometry pulse sequence. Our study reveals that the nature of end-groups in a moderately amphiphilic polymer can adjust the distribution and size of the micellar configuration in water, leading to changes in the macroscopic structure of hydrogels. By varying the chemistry of the cross-linker group (diacrylates (DA), dimethacrylates (DM) and urethane dimethacrylates (UDM)), we determined that the hydrophobicity of a single core polymer consisting of poly(glycolic acid) could be fine-tuned, leading to significant variations in the mechanical, swelling and degradation properties of the gels. In addition, the effects of cross-linker chemistry on cytotoxicity and proliferation were examined. Cytotoxicity assays showed that the three types of hydrogels (4KG5 DA, DM and UDM) were biocompatible and the introduction of RGD ligand enhanced cell adhesion. However, differences in gel properties and stability differentially affected the spreading and proliferation of myoblast C2C12 cells.

Figure 1

Reaction scheme for the synthesis of photolymerizable 4KG5DA, 4KG5DM, and 4KG5UDM. Poly (ethylene glycol)-co-poly-(glycolic acid) (4KG5) was synthesized by ring-opening polymerization of glycolide/poly(ethylene glycol) using stannous octoate as a catalyst. The molar ratio of glycolide to PEG is 5:1, which means 2.5 of glycolide per chain end. However, since 2 glycolic acid moieties are generated from one glycolide, 5 glycolic acid units are extended per chain end. 4KG5 copolymer, which has α- and ω-terminated hydroxyl groups, was subsequently end-capped with acrylate, methacrylate, and 2-isocyanatoethyl methacrylate.

Figure 2

Covalent attachment of the GRGDS bioactive peptide to photopolymerizable ACRL-PEG-NHS macromer.

Figure 3

¹H NMR for 4KG5 DA, DM, and UDM with their characteristic vinylic peaks (∼ 5.5-6.5 ppm).

Figure 4

MALDI-TOF of a series of 4k PEG, 4KG5 copolymer, and functionalized 4KG5UDM.

Figure 5

MALDI-TOF MS of ACRL-PEG-NHS and ACRL-PEG-Peptide. MS spectrum of ACRL-PEG-NHS is shifted to the right (higher molecular mass) after covalent attachment of the peptide.

Figure 6

Characterization of the micellar sizes by DLS for the three macromonomers in deionized water at a concentration of 5 mg/mL. The 4KG5 DA, UDM, and DM micelles were found to have diameters of 33.5 ± 1.4 nm, 40.3 ± 1.6 nm, and 56.3 ± 2.3 nm, respectively. The sizes were calculated by volume measurement and expressed as Dav ± S (average diameter ± standard deviation).

Figure 7

¹H NMR of uncross-linked (top) and ¹H DOSY 1D of photocross-linked (bottom) macromonomers (4KG5DA, 4KG5DM, and 4KG5UDM) in D₂O at two mass fractions (10% and 20%). Photopolymerization is induced directly in an NMR tube. 1mL of macromonomer solution containing the photoinitiator (I2959) was transferred into the NMR tube before curing with a long wavelength UV source (365 nm, 300 μW/cm²) to obtain hydrogel. The vinylic peaks (between 5.5-6.5 ppm) disappeared after 10 min irradiation.

Figure 8

Shear modulus (G*) of hydrogels prepared using various 4KG5 end groups (DA, UDM, and DM) and at various mass fractions (10% and 20%).

Figure 9

Volumetric swelling ratio (Qv) versus time of a series of 4KG5 DA, DM, and UDM hydrogels in PBS at two mass fractions (10% and 20%). The Qv value is reported for each type of gels and mass fraction.

Figure 10

Live stain (left) and dead stain (right) of 4KG5 DA, DM, and UDM hydrogels seeded with C2C12 cells after 4 d of incubation.

Figure 11

Nuclear stain of C2C12 cells cultured on 20% 4KG5 DA, DM, and UDM hydrogels after 4 d of incubation.

Figure 12

Proliferation of C2C12 cells cultured on GRGDS-modified 4KG5 DA, DM, and UDM hydrogels after 1, 4, and 7 d of incubation. Cells were stained with Hoechst dye. Data represent the mean of three experiments ± standard deviation of cell number/normalized section (5mm²). A statistical significance of p ≤ 0.005 was obtained for all the comparisons (except for the pair comparison with the symbol *). Statistical significance was calculated using the student's t-test for multiple comparisons.