Abstract
The use of fluorescent imaging probes that monitor the activity of proteases that experience an increase in expression and activity in tumors is well established. These probes can be conjugated to nanoparticles of iron oxide, creating a multimodal probe serving as both a magnetic resonance imaging (MRI) agent and an indicator of local protease activity. Previous works describe probes for cathepsin D (CatD) and metalloproteinase-2 (MMP2) protease activity grafted to cross-linked iron oxide nanoparticles (CLIO). Herein, we have synthesized a triply labeled fluorescent iron oxide nanoparticle molecular imaging (MI) probe, including an AF750 substrate concentration reporter along with probes for cathepsin B (CatB) sand MMP2 protease activity. The reporter provides a baseline signal from which to compare the activity of the two proteases. The activity of the MI probe was verified through incubation with the proteases and tested in vitro using the human HT29 tumor cell line and in vivo using female nude mice injected with HT29 cells. We found the MI probe had the appropriate specificity to the activity of their respective proteases, and the reporter dye did not activate when incubated in the presence of only MMP2 and CatB. Probe fluorescent activity was confirmed in vitro, and reporter signal activation was also noted. The fluorescent activity was also visible in vivo, with injected HT29 cells exhibiting fluorescence, distinguishing them from the rest of the animal. The reporter signal was also observable in vivo, which allowed the signal intensities of the protease probes to be corrected; this is a unique feature of this MI probe design.
Highlights
Genomic and proteomic approaches have identified a host of molecular markers associated with disease [1,2,3,4]
The activation of the fluorescence signal of the Triple Fluorochrome Probe (TFP) imaging probe was tested under optimal conditions for cathepsin B (CatB) cleavage by using 20 mM of sodium acetate buffer
The MR capability of the TFP probe can be utilized in future applications to provide additional anatomical context to in vivo visualization of probe activation
Summary
Genomic and proteomic approaches have identified a host of molecular markers associated with disease [1,2,3,4]. The activation of the fluorescence signal of the TFP imaging probe was tested under optimal conditions. The activation of the fluorescence signal of the TFP imaging probe was tested under optimal conditions for CatB cleavage by using 20 mM of sodium acetate buffer (pH 5). MinMtPh2eatcutimvaotrioinndsiegpneanldweintht tohfedperloivbeercyoncocenncternattriaotniosnig. nSailm, MilaMrlPy, iflnuoFriegsucreenc6eBa, ctthiveatCioantBisfdluisoprelasyceendce inactthiveatuiomnosriginadleapnedndAeFn7t5o0fsdueblsivtreartye concennttrraattiioonn.siSginmalilarelys, einenFinguthree 6tuBm, tohreaCreaatBcoflmuopraersecdentoceno afcltuivoaretisocnensciegnaacltiavnitdyAseFe7n50insutbhsetrsautrerocounncdeintgrattiisosnues.igSnimalilaarre tsoeetnheinMthMePt2umacotrivaarteiaoncosmigpnarl,edthteo CnaotB flaucotirveastcieonncseiganctailviistycosrereenctiendtbhye bsueirnrogudnivdiidnegdtibsysuthe.e Spirmobilea’rs tdoetlhiveeMryMsigPn2aalc(tAivFa7t5io0n). signal, the CatB activation signal is corrected by being divided by the probe’s delivery signal (AF750)
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