PEGylation Does Not Impair Insulin Efficacy in Three-Dimensional Cartilage Culture: An Investigation toward Biomimetic Polymers

Abstract

A major goal in tissue engineering is the controlled application of growth factors. As a novel application system, we are currently developing biomimetic polymers that are processed into three-dimensional scaffolds. Bioactive proteins will be covalently bound to the polymers via a poly(ethylene glycol) (PEG) linker. Of paramount importance is the maintenance of the biological activity of the protein after PEGylation and covalent binding to the polymer. Therefore, within this study, insulin used as a model protein was PEGylated with an active succinimidyl ester of poly(ethylene glycol) (SS-NH-PEG) (MW ~2000) and biological effects of the protein-PEG conjugate were monitored in comparison with unmodified insulin. No significant differences in chondrocyte proliferation were observed in a conventional proliferation assay after treatment with insulin or PEGylated insulin. In a complex three-dimensional cartilage-engineering model the effects of insulin and PEGylated insulin were investigated over a wide concentration range (0.025-25 microg/mL). Insulin and PEGylated insulin at equivalent concentrations resulted in cartilaginous tissue constructs exhibiting identical wet weight, cell number, biochemical composition of the extracellular matrix, and histological appearance, both compounds significantly improving tissue quality as compared with control constructs. In conclusion, the presented study demonstrates that PEGylation of insulin using SS-NH-PEG did not change the activity of the protein in a complex biological environment and is regarded as a step toward the development of biomimetic polymers.

Covalently immobilized biosignal molecule materials for tissue engineering

“…Some studies using the spacer chains were performed and the increase in the activity is reported. 26,[29][30][31][32]48 However, possible implications of steric hindrance in spacing growth factor from the substrate and the possible impact on growth factor binding to its cell surface receptor should be taken into consideration. In fact, the POE conjugation reduced the biological activity of insulin.…”

Micronization of Insulin by High Pressure Homogenization

“…Bovine chondrocytes were harvested from knee joints by collecting fresh articular cartilage from the surfaces of the patellar grove under aseptic conditions. The cartilage was cut into small pieces and enzymatically digested by collagenase type II in cell culture medium (21). The cell digest was filtered through a 150 mm filter, centrifuged at 1,200 rpm for 5 min and washed three times with PBS.…”

Evidence for Metabolic Cleavage of a PEGylated Protein in Vivo Using Multiple Analytical Methodologies

“…A NMR spectrum of PEG is relatively straightforward to interpret as the majority of protons along the PEG backbone are in the same coupling environment and hence produce a single peak at a chemical shift of ∼3.65 ppm. NMR has also been utilized to characterize PEGylated proteins − and could prove to be a useful tool in providing a quantitative assay for measuring the metabolism of PEG since it is possible to integrate all PEG moieties.…”