Polymeric micelles are emerging as a highly integrated nanoplatform for cancer targeting, drug delivery and tumor imaging applications. In this study, we describe a multifunctional micelle (MFM) system that is encoded with a lung cancer-targeting peptide (LCP), and encapsulated with superparamagnetic iron oxide (SPIO) and doxorubicin (Doxo) for MR imaging and therapeutic delivery, respectively. The LCP-encoded MFM showed significantly increased αvβ6-dependent cell targeting in H2009 lung cancer cells over a scrambled peptide (SP)-encoded MFM control as well as in an αvβ6-negative H460 cell control. 3H-Labeled MFM nanoparticles were used to quantify the time- and dose-dependent cell uptake of MFM nanoparticles with different peptide encoding (LCP vs SP) and surface densities (20% and 40%) in H2009 cells. LCP functionalization of the micelle surface increased uptake of the MFM by more than 3-fold compared to the SP control. These results were confirmed by confocal laser scanning microscopy, which further demonstrated the successful Doxo release from MFM and accumulation in the nucleus. SPIO clustering inside the micelle core resulted in high T2 relaxivity (>400 Fe mM−1 s−1) of the resulting MFM nanoparticles. T2-weighted MRI images showed clear contrast differences between H2009 cells incubated with LCP-encoded MFM over the SP-encoded MFM control. An ATP activity assay showed increased cytotoxicity of LCP-encoded MFM over SP-encoded MFM in H2009 cells (IC50 values were 28.3 ± 6.4 nM and 73.6 ± 6.3 nM, respectively; p < 0.005). The integrated diagnostic and therapeutic design of MFM nanomedicine potentially allows for image-guided, target-specific treatment of lung cancer.

Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the ␣1(V)436 -450 collagen region, which is hydrolyzed at the Gly2Val bond selectively by MMP-2 and MMP-9, or the Gly2Leu cleavage site within the consensus interstitial collagen sequence ␣1(I-III)769 -783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.Collagen catabolism (collagenolysis) is normally a well regulated physiological process critical to tissue and organ development, morphogenesis, and wound healing (1). Pathological conditions resulting from aberrant collagenolysis include primary and metastatic tumor growth, arthritis, arteriosclerosis, and periodontitis (1-4). A number of proteases have been described as exhibiting collagenolytic behavior, requiring that they catalyze the hydrolysis of the collagen triple helix (1). The most extensively studied are the matrix metalloproteinases (MMPs).2 MMPs that catalyze the hydrolysis of one or more of the interstitial collagens (types I-III) within their triple-helical domain include the secreted proteases MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 and the membrane-bound proteases MT1-MMP and MT2-MMP (5-8).Considerable work has been performed to define the MMP domains and regions that participate in collagenolysis. In the cases of MMP-1, MMP-8, MMP-13, MT1-MMP, and MT2-MMP, efficient collagenolytic activity in the isolated enzyme requires both the catalytic (CAT) and hemopexin (HPX)-like domains ( Fig. 1) (9 -14). The linker region between these domains also participates in collagenolysis, either by direct binding of substrate (15) or by allowing for the proper orientation of the CAT and HPX domains (16). The "gelatinase" members of the MMP family (MMP-2 and MMP-9)...

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P1-P1' subsites of the triple-helical peptide. Inhibition studies revealed Ki values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).

Phage display is commonly used to isolate peptides that bind to a desired cell type. While chemical synthesis of selected peptides often results in ligands with low affinity, a multivalent tetrameric presentation of the peptides dramatically improves affinity. One of the primary uses of these peptides is conjugation to nanoparticle-based therapeutics for specific delivery to target cell types. We set out to optimize the path from phage display peptide selection to peptide presentation on a nanoparticle surface for targeted delivery. Here, we examine the effects of peptide valency, density, and affinity on nanoparticle delivery and therapeutic efficacy, using the αvβ6-specific H2009.1 peptide as a model phage-selected peptide and liposomal doxorubicin as a model therapeutic nanoparticle. Liposomes displaying the higher affinity multivalent H2009.1 tetrameric peptide demonstrate 5-10 fold higher drug delivery than liposomes displaying the lower affinity monomeric H2009.1 peptide, even when the same number of peptide subunits are displayed on the liposome. Importantly, a 6-fold greater toxicity is observed towards αvβ6-expressing cells for liposomes displaying tetrameric verses monomeric H2009.1 peptides. Additionally, liposomal targeting and toxicity increase with increasing concentrations of H2009.1 tetrameric peptide on the liposome surface. Thus, both the multivalent peptide and the multivalent liposome scaffold work together to increase targeting to αvβ6-expressing cells. This multi-layered approach to developing high affinity targeted nanoparticles may improve the utility of moderate affinity peptides. As tetramerization is known to increase affinity for a variety of phage-selected peptides, it is anticipated that the tetrameric scaffold may act as a general method for taking peptides from phage display to nanoparticle display.