Featured Article: Nanoenhanced matrix metalloproteinase-responsive delivery vehicles for disease resolution and imaging

Abstract

The wide array of proteases, including matrix metalloproteinases, produced in response to many pathogenic insults, confers a unique proteolytic signature which is often disease specific and provides a potential therapeutic target for drug delivery. Here we propose the use of collagen-based nanoenhanced matrix metalloproteinase-responsive delivery vehicles that display matrix metalloproteinase-specific degradation in diverse in vitro models of proteolysis. We demonstrate that collagen particles comprised of protease substrates (primarily collagen) can be made of uniform size and loaded efficiently with assorted cargo including fluorescently labeled mesoporous silica, magnetic nanoparticles, proteins and antioxidants. We also demonstrate that pathologic concentrations of proteases produced in situ or in vitro display protease-specific cargo release. Additionally, we show that the collagen-based particles display bright fluorescence when loaded with a fluorophore, and have the potential to be used as vehicles for targeted delivery of drugs or imaging agents to regions of high proteolytic activity.

Keywords: Collagen; antioxidants; burn injury; infection; matrix metalloproteinase; nanoparticles; proteases; senescence.

Figure 1

Components of nanoenhanced MMP-responsive delivery vehicle (NMRDV). The image depicts the basic components of NMRDVs. In this study, the matrix components were type I collagen particles, the cargo was either antioxidants (Trolox, Didox, pomegranate extract, apple peel extract, catalase) or the MMPI Ilomostat, and the enhancing FNPs were either GFP-NP, FeOx-NPs, or TRITC-NP. (A color version of this figure is available in the online journal.)

Figure 2

Physical features and characterization of NMRDV. (a) Scanning Electron Microscopy (SEM) of type I collagen particles dried on a silicon wafer and sputter coated with a layer of gold/palladium alloy. (b) Collagen particles loaded with GFP-labeled BSA were analyzed using imaging flow cytometry and particle size characteristics determined. (c) Left panel, Radius of unloaded and catalase loaded particles across different batches. Right Panel, Aspect ratios of unloaded (0.817 µm) and catalase-loaded particles (0.7958 µm) averaged (n = 4). (d) Immunofluorescent image of FITC-BSA-loaded collagen particles. (e) Cellular distribution of Collagen-FeOx-NPs. IMR-90 primary human fibroblasts Cells were untreated or exposed to FeOx-NPs or Col- FeOx-NPs. Cells were fixed and then probed with FITC-phalloidin DAPI. Immunofluorescence microscopy was performed with the appropriated filter sets to distinguish distinct fluorophores. Actin fibers (green), Nuclei (blue) and Cy5.5 (red). (A color version of this figure is available in the online journal.)

Figure 3

Hyperspectral imaging of collagen and FNP-loaded collagen. Upper panels, DNV display distinct spectral angle mapping (SAM) characteristics that can be used to interrogate absence or presence of FNP. Middle panels, SAM of collagen particles display presence of collagen but not FNP. Lower panels, SAM of Collagen-FNP display both collagen and FNP fingerprint. (A color version of this figure is available in the online journal.)

Figure 4

NMRDV cargo release properties. (a) Collagen particle degradation was measured by bicinchoninic acid assay (BCA) as relative absorbance of total protein present in supernatant after intact particles were separated out following normalization for collagenase input. (b) Collagen particles were loaded with TRITC-labeled C-specs (Hybrid Silica Technologies) and fluorescence was monitored from supernatants after treatment with the indicated concentrations of collagenase. n ≥ 3 ± SEM., *P < 0.05, **P < 0.01 relative to untreated control or as indicated. (c) Inset, Catalase-loaded collagen nanoparticles were treated with 3 µg/mL collagenase and supernatants collected, and immunoblotting for catalase as indicated. Densitometric quantification of n ≥ 3 ± SEM representative experiments

Figure 5

Senescent NMRDV. Top blot – Western blot analysis of secreted MMP-1 in pre-senescent and senescent IMR90 fibroblasts. Middle blots – Western blot analysis of internalized catalase in pre-senescent and senescent fibroblasts incubated with collagen particles loaded with 500 U/mL recombinant catalase or with 500 U/mL recombinant catalase alone. GAPDH is used as a loading control. Bottom blot – Western blot analysis of internalized catalase in senescent fibroblasts incubated with collagen particles loaded with 500 U/mL recombinant catalase in the presence or absence of a broad spectrum MMP inhibitor GM6001 (25 µm). Lower Graph – All data normalized to GAPDH controls and expressed as a percentage of maximal loading that was observed in senescent cells as a result of exposure to catalase-loaded particles. Relative densitometric analysis of immunoblots in inset was performed using NIH ImageJ

Figure 6

Infection NMRDV. (a) qRT-PCR of muMMP-9 and muMMP-13 transcripts from LPS treated and untreated murine alveolar macrophage cell line (MHS). Inset – MMP-13 immunoblot; (b) Catalase–collagen particle degradation in response to supernatants from LPS-treated macrophages. Cargo release is impaired when supernatants are treated with the broad spectrum MMP inhibitor GM6001. Inset, representative immunoblots. Data represent n ≥ 3. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 relative to untreated control