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. 2012 Jun 15;429(1-2):31-7.
doi: 10.1016/j.ijpharm.2012.03.012. Epub 2012 Mar 13.

Improving matrix metalloproteinase-2 specific response of a hydrogel system using electrophoresis

Yu Zhang  1 , Richard A Gemeinhart
Affiliations

Improving matrix metalloproteinase-2 specific response of a hydrogel system using electrophoresis

Yu Zhang et al. Int J Pharm. .

Abstract

Matrix metalloproteinases (MMPs) overexpression plays a critical role in cancer invasion and metastasis. We utilized this key feature of tumor microenvironment to develop a disease-stimuli triggered drug delivery system. Poly(acrylic acid) hydrogels were synthesized by UV polymerization and pendant MMP-2 sensitive peptides (Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys) conjugated throughout using EDC/sulfo-NHS chemistry. There were significantly more peptides released in the presence of MMP-2 compared with the control groups. The released peptide fragments were analyzed by HPLC and MALDI-MS and confirmed to be the expected fragments. In order to avoid nonspecific release of nonconjugated (i.e. unreacted) peptides, a novel method of electrophoretic washing was developed disrupting the strong electrostatic interactions between the peptides and the pendant groups of the hydrogel. After electrophoresis, the nonspecific peptide release in the absence of MMP-2 was minimized. This newly developed purification system significantly improved the control of release to be in response of the magnitude of the stimuli, i.e. MMP. Specifically, peptides were released proportionally to the concentration of MMP-2 present. Now that many of the design parameters have been examined, anticancer drugs will be conjugated to the MMP sensitive peptide linkers with the goal of implantation in a tumor void releasing anticancer reagent in response to elevated level of MMPs.

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Figures

Figure 1
Figure 1
Schematic illustration of the formation, optimization, and characterization of the MMP-sensitive drug delivery system.
Figure 2
Figure 2
Response surface plot of MMP-2 sensitive peptide conjugation amount as a function of pH and acrylic acid to peptide feed ratio derived from three independent samples examined at each condition. The optimal conditions are predicted by the red regions ranging to the least optimal conditions in the regions of blue following the color spectrum.
Figure 3
Figure 3
Peptide extraction capacities under various conditions. Peptide was extracted using aqueous solvents at varying pH (A), different ionic strength (B), and different polarity (C) where greater extraction (Peptide-μmol) indicates more thorough removal of unreacted peptides. Data represent the mean plus or minus (±) the standard deviation of three independent samples where ††† indicates statistical difference between the groups at p < 0.001.
Figure 4
Figure 4
Removal of nonconjugated peptides with electrophoresis. (A) Schematic illustration of the electrophoresis apparatus and regions measured. The agarose gel was cut consecutively from the loading well to the edge of the gel tray after electrophoresis. (B) The fluorescence intensity of hydrogel (red, — -), peptide (nonconjugated) in hydrogel (blue; — —), and conjugated peptide hydrogel (green; —) before and after electrophoresis at varying times where ††† indicates statistical significant (p < 0.001) compared with the pre-electrophoresis condition. (C–E) Peptide distribution into the agarose gels downstream of the hydrogel (red, — —), peptide (nonconjugated) in hydrogel (blue; — —), and conjugated peptide hydrogel (green; —) after electrophoresis for 1 (C), 2 (D), and 3 (E) hours. Data represent the mean plus or minus (±) the standard error of the mean of three independent samples.
Figure 5
Figure 5
Effect of MMP-2 concentrations on peptide release from hydrogel. Peptide release over time from hydrogels incubated in 0 nM (red, solid bars), 9 nM (blue, diagonally crossed bars), and 27 nM (green, dotted bars) MMP-2. Data represent the mean plus or minus (±) the standard deviation of three independent samples where †, ††, and ††† indicate statistical difference between the groups at p < 0.05, 0.01, and 0.001, respectively.
Figure 6
Figure 6
Identification of peptide release fragments with HPLC and MS. (A) HPLC chromatograms of the cleaved peptide releasate following addition of MMP-2 (red) and nonspecific peptide released without addition of MMP-2 (blue). (B) MS spectrograms of the fractions corresponding to the 14 min HPLC peak of MMP-2 free sample (blue) and the 16 min peak of MMP-2 supplemented group (red). The expected fragments are marked with the one-letter peptide abbreviations and mca for methoxycoumarin.
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