title: Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation.
abstract: Activatable cell-penetrating peptides (ACPPs) are a new class of promising molecular imaging probes for the visualization of enzymes in vivo. The cell-penetrating function of a polycationic peptide is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a cleavable linker present between the polycationic cell-penetrating peptide and polyanionic peptide affords dissociation of both domains and enables the activated cell-penetrating peptide to enter cells. Here, we aimed to develop an ACPP sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for nuclear imaging purposes.
  
  
  METHODS
  MMP-2/9 ACPPs and nonactivatable cell-penetrating peptides (non-ACPP) were prepared by 9-fluorenylmethyloxycarbonyl solid-phase peptide synthesis and labeled with (177)Lu or (177)Lu/(125)I for dual-isotope studies. The in vivo biodistribution of these probes was assessed in MMP-2/9-positive tumor-bearing mice (n = 6) and healthy mice (n = 4) using γ-counting. Furthermore, a radiolabeled cell-penetrating peptide serving as a positive control was evaluated in tumor-bearing mice (n = 6).
  
  
  RESULTS
  Biodistribution studies showed a 5-fold-higher retention of ACPP in tumor than in muscle (P < 0.01) and a 6-fold-higher tumor retention relative to non-ACPP (P < 0.01), supporting earlier studies on fluorescently labeled ACPPs proposing activation by tumor-associated MMP-2/9. Surprisingly, however, the uptake of ACPP was significantly higher than that of non-ACPP in almost all tissues (P < 0.01). To unravel the activation process of ACPP in vivo, we developed dual-isotope ACPP analogs (dACPPs) that allowed us to discriminate between uncleaved dACPP and activated dACPP. In vivo biodistribution of dACPP indicated that the tissue-associated counts originated from activated dACPP. Interestingly, dACPP administration to healthy mice, compared with MMP-2/9-positive tumor-bearing mice, resulted in a similar dACPP biodistribution. Furthermore, a radiolabeled cell-penetrating peptide showed tumor-to-tissue ratios equal to those found for ACPP (P > 0.05).
  
  
  CONCLUSIONS
  This study demonstrates that the tumor targeting of radiolabeled MMP-2/9 ACPPs is most likely caused by the activation in the vascular compartment rather than tumor-specific activation, as suggested earlier. The results in the present paper indicate that different and more tissue-specific enzyme-ACPP combinations are needed to unleash the full potential of the elegant ACPP concept in living animals.
authors.affiliation: Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors.forename: Sander M J
authors.initials: SM
authors.lastname: van Duijnhoven
authors.forename: Marc S
authors.initials: MS
authors.lastname: Robillard
authors.forename: Klaas
authors.initials: K
authors.lastname: Nicolay
authors.forename: Holger
authors.initials: H
authors.lastname: Grüll
mesh_terms: D000818:Animals; D001711:Biotransformation; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008187:Lutetium; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000000981:diagnostic imaging* / Q000201:enzymology*; D011868:Radioisotopes / Q000737:chemistry; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D014018:Tissue Distribution
chemical_list: D057846:Cell-Penetrating Peptides; D007457:Iodine Radioisotopes; D011868:Radioisotopes; D019275:Radiopharmaceuticals; D008187:Lutetium; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9