Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration.
We have developed collagenase-sensitive hydrogels by incorporating a collagenase-sensitive fluorogenic substrate (CS-FS) within the backbone of a polyethylene glycol (PEG) copolymer to visualize collagenase activity during three-dimensional cell migration. CS-FS was synthesized by conjugating Bodipy dyes to a peptide with collagenase-sensitive sequence, Leu-Gly-Pro-Ala (LGPA), and the products were grafted into the collagenase-sensitive PEG hydrogels. CS-FS both in solution and hydrogels had an increase in the fluorescence intensity after proteolytic degradation by collagenase, but not by non-targeted proteases nor in the absence of an enzyme. Fibroblasts inside the hydrogels conjugated with CS-FS spread and extended lamellipodia in three dimensions over several days, and their pericellular collagenase-mediated proteolysis of the hydrogel was visualized via confocal microscopy. A matrix metalloproteinase inhibitor, served as a negative control, significantly reduced the degradation rate of CS-FS by collagenase and prevented cell migration and cell-mediated collagenase activity inside these hydrogels. In summary, we have fabricated collagenase-sensitive hydrogels incorporated with CS-FS and successfully visualized the collagenase activity during three-dimensional cell migration.
Lee
Soo-Hong
SH
Department of Bioengineering, Rice University, P.O. Box 1892, MS 142, Houston, TX 77251-1892, USA.
Moon
James J
JJ
Miller
Jordan S
JS
West
Jennifer L
JL
D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005341:Fibrinolysin / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D020100:Hydrogels / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism; D018613:Microscopy, Confocal; D008956:Models, Chemical; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry*
D005456:Fluorescent Dyes; D020100:Hydrogels; D011092:Polyethylene Glycols; D019286:Endopeptidase K; D005341:Fibrinolysin; D017364:Collagenases; D020782:Matrix Metalloproteinases