title: Synthesis and functionalization of protease-activated nanoparticles with tissue plasminogen activator peptides as targeting moiety and diagnostic tool for pancreatic cancer.
abstract: BACKGROUND
  Functionalized nanoparticles (NPs) are one promising tool for detecting specific molecular targets and combine molecular biology and nanotechnology aiming at modern imaging. We aimed at ligand-directed delivery with a suitable target-biomarker to detect early pancreatic ductal adenocarcinoma (PDAC). Promising targets are galectins (Gal), due to their strong expression in and on PDAC-cells and occurrence at early stages in cancer precursor lesions, but not in adjacent normal tissues.
  
  
  RESULTS
  Molecular probes (10-29 AA long peptides) derived from human tissue plasminogen activator (t-PA) were selected as binding partners to galectins. Affinity constants between the synthesized t-PA peptides and Gal were determined by microscale thermophoresis. The 29 AA-long t-PA-peptide-1 with a lactose-functionalized serine revealed the strongest binding properties to Gal-1 which was 25-fold higher in comparison with the native t-PA protein and showed additional strong binding to Gal-3 and Gal-4, both also over-expressed in PDAC. t-PA-peptide-1 was selected as vector moiety and linked covalently onto the surface of biodegradable iron oxide nanoparticles (NPs). In particular, CAN-doped maghemite NPs (CAN-Mag), promising as contrast agent for magnetic resonance imaging (MRI), were selected as magnetic core and coated with different biocompatible polymers, such as chitosan (CAN-Mag-Chitosan NPs) or polylactic co glycolic acid (PLGA) obtaining polymeric nanoparticles (CAN-Mag@PNPs), already approved for drug delivery applications. The binding efficacy of t-PA-vectorized NPs determined by exposure to different pancreatic cell lines was up to 90%, as assessed by flow cytometry. The in vivo targeting and imaging efficacy of the vectorized NPs were evaluated by applying murine pancreatic tumor models and assessed by 1.5 T magnetic resonance imaging (MRI). The t-PA-vectorized NPs as well as the protease-activated NPs with outer shell decoration (CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1Lac) showed clearly detectable drop of subcutaneous and orthotopic tumor staining-intensity indicating a considerable uptake of the injected NPs. Post mortem NP deposition in tumors and organs was confirmed by Fe staining of histopathology tissue sections.
  
  
  CONCLUSIONS
  The targeted NPs indicate a fast and enhanced deposition of NPs in the murine tumor models. The CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1Lacinterlocking steps strategy of NPs delivery and deposition in pancreatic tumor is promising.
authors.affiliation: Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany.|Department of Radiation Oncology, Technische Universität München, Munich, Germany.
authors.forename: Sophie
authors.initials: S
authors.lastname: Dobiasch
authors.affiliation: Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany.
authors.forename: Szilard
authors.initials: S
authors.lastname: Szanyi
authors.affiliation: Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany.
authors.forename: Aleko
authors.initials: A
authors.lastname: Kjaev
authors.affiliation: Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany.|Department of General-, Visceral-, Transplantations-, Vascular- and Thorax-Surgery LMU Munich, München, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Werner
authors.affiliation: Department of Radiology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Strauss
authors.affiliation: Department of Radiology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Weis
authors.affiliation: Department of Radiology, University of Heidelberg, Heidelberg, Germany.|Diagnostik München, Diagnostic Imaging and Prevention Center, Munich, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Grenacher
authors.affiliation: Nanomaterials Research Center, Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel.
authors.forename: Katya
authors.initials: K
authors.lastname: Kapilov-Buchman
authors.affiliation: Nanomaterials Research Center, Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel.
authors.forename: Liron-Limor
authors.initials: LL
authors.lastname: Israel
authors.affiliation: Nanomaterials Research Center, Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Lellouche
authors.affiliation: Department of Industrial Chemistry Toso Montanari, University of Bologna, Bologna, Italy.
authors.forename: Erica
authors.initials: E
authors.lastname: Locatelli
authors.affiliation: Department of Industrial Chemistry Toso Montanari, University of Bologna, Bologna, Italy.
authors.forename: Mauro Comes
authors.initials: MC
authors.lastname: Franchini
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Vandooren
authors.affiliation: Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.
authors.forename: Ghislain
authors.initials: G
authors.lastname: Opdenakker
authors.affiliation: Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany. klaus.felix@med.uni-heidelberg.de.
authors.forename: Klaus
authors.identifier: http://orcid.org/0000-0001-9825-2684
authors.initials: K
authors.lastname: Felix
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D048271:Chitosan / Q000737:chemistry; D003287:Contrast Media / Q000737:chemistry; D005260:Female; D005290:Ferric Compounds / Q000737:chemistry; D037161:Galectins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D053758:Nanoparticles / Q000737:chemistry* / Q000633:toxicity; D010190:Pancreatic Neoplasms / Q000175:diagnosis* / Q000000981:diagnostic imaging / Q000473:pathology; D010316:Particle Size; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011100:Polyglycolic Acid / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000737:chemistry*; D014183:Transplantation, Heterologous
chemical_list: D003287:Contrast Media; D005290:Ferric Compounds; D037161:Galectins; D010455:Peptides; C000499:ferric oxide; D011100:Polyglycolic Acid; D011092:Polyethylene Glycols; D048271:Chitosan; D010959:Tissue Plasminogen Activator; D020780:Matrix Metalloproteinase 9
keywords: Galectins; Nanotheranostics; Pancreatic cancer; Tissue plasminogen activator