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Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

ABSTRACT: Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three-dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real-time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications.

SUBMITTER: Leight JL

PROVIDER: S-EPMC4077845 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

Leight Jennifer L JL Alge Daniel L DL Maier Andrew J AJ Anseth Kristi S KS

Biomaterials 20130702 30

Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated ...[more]

PMID: 23830581

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Project description:Physiological barriers inside of tumor tissue often result in poor interstitial penetration and heterogeneous intratumoral distribution of nanoparticle-based drug delivery systems (DDS). Novel, matrix metalloproteinase (MMP)-sensitive peptide-crosslinked nanogels (pNGs) as multistage DDS are reported with a beneficial size reduction property to promote the process of deep tissue penetration. Methods: The presented pNGs are based on a dendritic polyglycerol (dPG) scaffold crosslinked by a modified MMP-sensitive fluorogenic peptide. The crosslinker integrates degradability in response to proteases present in the tumor microenvironment. Surfactant-free, inverse nanoprecipitation is employed to prepare the nanogels using strain-promoted click chemistry. The size and crosslinking density of the pNGs are controlled by the functionalization degree of dPG with cyclooctyne groups and by the peptide crosslinker fraction. The intrinsic reporter moiety of the crosslinker was used to study the influence of pNG compositions on the degradation profile. The therapeutic drug Doxorubicin was conjugated through a pH-sensitive linkage to dPG to form a multistage DDS. The penetration behavior of the pNGs was studied using agarose matrix and multicellular tumor spheroids (MCTS). Results: Nanogel sizes were controlled in the range of 150-650 nm with narrow size distributions and varying degrees of crosslinking. The pNGs showed stability in PBS and cell media but were readily degraded in the presence of MMP-7. The crosslinking density influenced the degradation kinetic mediated by MMP-7 or cells. Stable conjugation of DOX at physiological pH and controlled drug release at acidic pH were observed. The digestions of nanogels lead to a size reduction to polymer-drug fragments which efficiently penetrated into agarose gels. Moreover, the degradable multistage pNGs demonstrated deeper penetration into MCTS as compared to their non-degradable counterparts. Thus, degradable pNGs were able to deliver their cargo and efficiently reduce the cell viability in MCTS. Conclusion: The triggered size reduction of the pNGs by enzymatic degradation can facilitate the infiltration of the nanocarrier into dense tissue, and thereby promote the delivery of its cargo.

Dataset Information

Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

Publications

Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

Similar Datasets

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