Controlled release of tethered peptides from alginate hydrogels via enzymatic linker degradation

  • Mariana Moreira da Silva Alves Barbosa (Student)

Student thesis: Master's Thesis


Regenerative medicine requires innovative therapeutic designs to accommodate high morphogen concentrations in local depots and provide their sustained delivery for enhanced effectiveness. The aim of this study was to develop a MMP-responsive hydrogel-based delivery vehicle for the active fragment of the osteogenic growth peptide (OGP10-14, YGFGG), to be used in bone regeneration therapies. For this purpose two alternative peptide sequences were designed. These were synthesized using a solid-phase strategy (SPPS), by extending the original YGFGG sequence at its amine terminus with either a MMP-sensitive (PVGLIG) or a MMP-insensitive (scrambled, GIVGPL) domain. The designed sequences were then chemically conjugated to alginate, a hydrogel-forming polymer. The hypothesis behind the study was that in presence of specific MMPs (namely MMP-2) the PVLIG linker could be enzymatically cleaved leading to the release of OGP10-14 from the alginate hydrogel vehicle. In contrast, OGP would remain grafted when using the MMP-insensitive (scrambled) domain as linker. The oligopeptide sequences were successfully synthesized by SPPS, and analyzed by liquid chromatography-mass spectrometry (LC-MS). Both were subsequently characterized in terms of their susceptibility to proteases. The extent of peptide cleavage upon incubation with MMP-2 was determined using the fluorescamine assay, and changes in the peptides molar mass were determined by LC-MS. The PVGLIG sequence was effectively hydrolyzed by MMP-2 at the predicted cleavage site (G↓L). The scrambled sequence was also partially cleaved, albeit unspecifically and at much lower rates. The kinetics of peptide digestion was further analyzed using custom-made FRET sequences, by monitoring the temporal increase in fluorescence resulting from peptide cleavage. Both peptides were partially cleaved by proteases present in serum, but the PVGLIG peptide was much more responsive to MMP-2 than the scrambled peptide. Preliminary studies were carried out to evaluate the bioactivity of OGP10-14 when presented to human mesenchymal stem cells (hMSC) in both peptide sequences. Osteogenic differentiation was more efficiently promoted when OGP was incorporated in the MMP-sensitive oligopeptide. Both peptides were conjugated to alginate via carbodiimide-mediated chemical grafting. The amount of immobilized peptide was quantified by UV-Vis and by the Total Protein Biccinchoninic acid (BCA) assay, which demonstrated that the coupling procedure was successful, with an efficiency around 41% (PVGLIG) to 62% (scrambled). MMP-2 digestion of peptide-alginate conjugates was analyzed by the fluorescamine assay. Higher enzymatic cleavage was observed in peptides-alginate conjugates with the MMP-sensitive PVGLIG linker. Overall, this study provided proof-of-concept on the correct design of alginate-PVGLIG-conjugates, in the sense that, as expected, these were sensitive to MMP-2 mediated cleavage and may be a useful platform for the in situ delivery of OGP.
Date of Award2012
Original languageEnglish
Awarding Institution
  • Universidade Católica Portuguesa
Supervisor Cristina Carvalho Barrias (Supervisor)


  • Mestrado em Engenharia Biomédica

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