470377 In Vitro Characterization of a Novel MMP-2 Activatable Peptide for Photoacoustic Lifetime Imaging of Tumor Tissue

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Sadie M. Johnson1, Ekaterina Morgounova2, Michael Wilson3, Shai Ashkenazi2 and Benjamin J. Hackel1, (1)Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, (2)Biomedical Engineering, University of Minnesota - Twin Cities, Minneapolis, MN, (3)Laboratory Medicine and Pathology, University of Minnesota - Twin Cities, Minneapolis, MN

Many molecular imaging agents suffer from uptake and retention in healthy clearance organs. Due to constant signal emission by the contrast agent, this accumulation results in high off-target signal in these organs which significantly inhibits image specificity and sensitivity and hinders imaging of tumors near these organs. Additionally, many of the contrast agents used are handicapped by either poor resolution due to tissue-related scattering (fluorescence imaging), or require expensive equipment and radioactive agents (PET/SPECT). To address these issues, we have created an activatable peptide probe that only generates signal in the presence of the cancer biomarker matrix metalloproteinase 2 (MMP-2). This probe utilizes the novel photoacoustic lifetime imaging (PALI) modality, which decreases background signal, increases imaging depth penetration, lowers costs, and avoids ionizing radiation associated with current molecular imaging methods.

There are two key components to this probe: a dual-state imaging contrast agent, and a biocompatible, activatable motif. The contrast agent for the PALI imaging modality is methylene blue, which was shown to exhibit quenching of the PA signal lifetime upon dimerization but a long lifetime as a monomer. This enables differentiation between the inactive and MMP-2-activated states. The activatable motif used is a peptide design – adapted from the literature (Levi, et al. J Am Chem Soc.2010) – which will induce dimerization of MB to quench the monomer signal until cleavage of the peptide by MMP-2. The peptide contains poly(glutamic acid), an MMP-2-cleavable peptide, and poly(arginine) segments with methylene blue at each terminus. The ionic segments align in a ‘zipper’, which dimerizes the attached methylene blues. The zipper and associated methylene blues dissociate upon cleavage of the MMP-2 recognition peptide.

The initial peptide design, Ac-Keeee[Ahx]PLGLAGrrrrrK (E4R5), displayed a kcat/Km = 1.2 x 105 M-1s-1, comparable to previously published values for similar constructs. Flash photolysis and PALI demonstrate dimerization and effective quenching of the lifetime signal. A systematic series of additional peptides of varying designs of zipper and methylene blue linker were synthesized and tested for their dimerization efficacy and activation rates. Within the E4R5 peptide, the addition of glycine (GE4R5G) or 6-aminohexanoic acid (XE4R5X) between zipper and methylene blue decreased the dimerization efficacy (ratio of dimer/monomer absorptivities, ε613 nm668 nm) from 2.57 ± 0.03 to 2.40 ± 0.01 and 2.09 ± 0.01, respectively. Shorter and longer zipper sequences (E2R3, E3R4, E4R4, E4R5 and E5R6) were shown to dimerize the attached methylene blue at equivalent efficacies. Initial activation rates of the various zipper constructs at 10 μM peptide, 0.2 ng/μL MMP-2, and 37 ºC decrease with increasing zipper length from 269 ± 19 nM/min for E2R3 to 207 ± 12 nM/min for E5R6, demonstrating a potential increase in the strength of the interaction between the two ionic peptides and their increasing length. Notably, the E4R4 and E4R5 peptides have indistinguishable rates of 221 ± 12 nM/min and 226 ± 8 nM/min, respectively, potentially demonstrating the initial design of N poly(arginine) to N+1 poly(glutamic acid) does not affect peptide activity compared to equivalent amino acid numbers. The extended linker peptides (GE4R5G and XE4R5X) also showed decreased rates of 178 ± 13 nM/min and 173 ± 20 nM/min, compared to E4R5 at 226 ± 8 nM/min. Ongoing studies, including additional peptide design comparisons and evaluation of PALI signal before and after activation will be discussed. Additional results to be shared include evaluating the in vitro cellular activation and internalization of select peptides, as well as their in vivo activation via murine tumor models.

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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division