Synthesis and functionalization of protease-activated nanoparticles with tissue plasminogen activator peptides as targeting moiety and diagnostic tool for pancreatic cancer

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-pep1Lac interlocking steps strategy of NPs delivery and deposition in pancreatic tumor is promising

Maghemite-containing PLGA–PEG-based polymeric nanoparticles for siRNA delivery: toxicity and silencing evaluation

Gene therapy based on siRNA has emerged as an exciting new therapeutic approach. In this work, incorporation of PEI into PLGA-b-PEG and encapsulation of magnetic NPs as MRI contrast agent, resulted in unique theranostic nanoparticles

Matrix metalloproteinase-9 (MMP-9) as an activator of nanosystems for targeted drug delivery in pancreatic cancer

Specific cancer cell targeting is a pre-requisite for efficient drug delivery as well as for high-resolution imaging and still represents a major technical challenge. Tumor-associated enzyme-assisted targeting is a new concept that takes advantage of the presence of a specific activity in the tumor entity. MMP-9 is a protease found to be upregulated in virtually all malignant tumors. Consequently, we hypothesized that its presence can provide a de-shielding activity for targeted delivery of drugs by nanoparticles (NPs) in pancreatic cancer. Here, we describe synthesis and characterization of an optimized MMP-9-cleavable linker mediating specific removal of a PEG shield from a PLGA-b-PEG-based polymeric nanocarrier (Magh@PNPs-PEG-RegaCP-PEG) leading to specific uptake of the smaller PNPs with their cargo into cells. The specific MMP-9-cleavable linker was designed based on the degradation efficiency of peptides derived from the collagen type II sequence. MMP-9-dependent uptake of the Magh@PNPs-PEG-RegaCP-PEG was demonstrated in pancreatic cancer cells in vitro. Accumulation of the Magh@PNPs-PEG-RegaCP-PEG in pancreatic tissues in the clinically relevant KPC mouse model of pancreatic cancer, as a proof-of-concept, was tumor-specific and MMP-9-dependent, indicating that MMP-9 has a strong potential as a specific mediator of PNP de-shielding for tumor-specific uptake. Pre-treatment of mice with Magh@PNPs-PEG-RegaCP-PEG led to reduction of liver metastasis and drastically decreased average colony size. In conclusion, the increased tumor-specific presence and activity of MMP-9 can be exploited to deliver an MMP-9-activatable NP to pancreatic tumors specifically, effectively, and safely.publisher: Elsevier articletitle: Matrix metalloproteinase-9 (MMP-9) as an activator of nanosystems for targeted drug delivery in pancreatic cancer journaltitle: Journal of Controlled Release articlelink: http://dx.doi.org/10.1016/j.jconrel.2016.08.016 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved.status: publishe

Matrix metalloproteinase-9 (MMP-9) as an activator of nanosystems for targeted drug delivery in pancreatic cancer (vol 239, pg 39, 2016 )

© 2017 Elsevier B.V. The authors regret that a representative tumor photograph used in Fig. 4B (left) was published in parallel in another study (Grünwald et al., Mol Cancer Res. 2016 Nov; 14(11):1147–1158. doi:10.1158/1541-7786.MCR-16-0180, Epub 2016 Aug 3). The corrected Fig. 4B, containing another representative tumor photograph, appears below. The studies do not contain any overlapping experiments or conclusions. All conclusions drawn in either publication are correct and unaffected by the mistake. The authors apologize for the inconvenience caused.[figure presented] Corrected Fig. 4. Evaluation of MMP-9-dependent uptake of Magh@PNPs-PEG-RegaCP-PEG into pancreatic tumors in vivo. (A) Tumor-specific uptake of Magh@PNPs-PEG-RegaCP-PEG. Prussian Blue staining of PNPs in pancreatic tissue; arrows indicate nanomaterial. Tumor −, Healthy littermates (n = 5); Tumor +, KPC mice (n = 5); Representative images; bars, 100 μm. (B) MMP-9-deficient KPC mice were able to form pancreatic tumors. Left, Representative pancreas whole mount; bar, 1 cm; Right, HE staining of pancreatic sections; representative image; bar, 100 μm. (C) MMP-9- and tumor-dependent uptake of Magh@PNPs-PEG-RegaCP-PEG into pancreatic tissue as determined by Prussian Blue staining-based quantification. Tumor −, Healthy littermates; Tumor +, KPC mice; MMP9 +, wild-type mice; MMP9 −, MMP9 knock-out mice (n = 5 per group). Columns, mean; bars, SEM. One-way ANOVA: n.s., not significant; **p < 0.01; ***p < 0.001.status: publishe

Matrix metalloproteinase-9 (MMP-9) as an activator of nanosystems for targeted drug delivery in pancreatic cancer

Specific cancer cell targeting is a pre-requisite for efficient drug delivery as well as for high-resolution imaging and still represents a major technical challenge. Tumor-associated enzyme-assisted targeting is a new concept that takes advantage of the presence of a specific activity in the tumor entity. MMP-9 is a protease found to be upregulated in virtually all malignant tumors. Consequently, we hypothesized that its presence can provide a de-shielding activity for targeted delivery of drugs by nanoparticles (NPs) in pancreatic cancer. Here, we describe synthesis and characterization of an optimized MMP-9-cleavable linker mediating specific removal of a PEG shield from a PLGA-b-PEG-based polymeric nanocarrier (Magh@PNPs-PEG-RegaCP-PEG) leading to specific uptake of the smaller PNPs with their cargo into cells. The specific MMP-9-cleavable linker was designed based on the degradation efficiency of peptides derived from the collagen type II sequence. MMP-9-dependent uptake of the Magh@PNPs-PEG-RegaCP-PEG was demonstrated in pancreatic cancer cells in vitro. Accumulation of the Magh@PNPs-PEG-RegaCP-PEG in pancreatic tissues in the clinically relevant KPC mouse model of pancreatic cancer, as a proof-of-concept, was tumor-specific and MMP-9-dependent, indicating that MMP-9 has a strong potential as a specific mediator of PNP de-shielding for tumor-specific uptake. Pre-treatment of mice with Magh@PNPs-PEG-RegaCP-PEG led to reduction of liver metastasis and drastically decreased average colony size. In conclusion, the increased tumor-specific presence and activity of MMP-9 can be exploited to deliver an MMP-9-activatable NP to pancreatic tumors specifically, effectively, and safely

Acute <i>in Vivo</i> Toxicity Mitigation of PEI-Coated Maghemite Nanoparticles Using Controlled Oxidation and Surface Modifications toward siRNA Delivery

A ceric ammonium nitrate (CAN)-based doping step was used for the fabrication of core maghemite nanoparticles (NPs) that enabled the obtainment of colloid particles with a view to a high-level nanoparticle (NP) surface doping by Ce­(III/IV). Such doping of Ce­(III/IV) cations enables one to <i>exploit their quite rich coordination chemistry</i> for ligand coordinative binding. In fact, they were shown to act as <i>powerful Lewis acid centers</i> for attaching any organic (Lewis base) ligand such as a 25 kDa branched PEI polymer. Resulting _conPEI₂₅-CAN-<i>γ</i>-Fe₂O₃ NPs have been fully characterized before a successful implementation of siRNA loading and cell delivery/gene silencing using a well-known dual luciferase system. This attractive result emphasized their significant potential as an NP platform technology toward additional MRI and/or drug delivery (peptide)-relating end applications. However, due to their high positive charge, PEI polymers can cause severe <i>in vivo</i> toxicity due to their interaction with negatively charged red blood cells (RBC), resulting in RBC aggregation and lysis, leading to thrombosis and, finally, to animal death. In order to mitigate these acute toxic effects, two different types of surface modifications were performed. One modification included the controlled oxidation of 0.1–5% of the PEI amines before or after conjugation to the NPs, using hydrogen peroxide or potassium persulfate. The other type of modification was the addition of a second biocompatible polyanionic polymer to the PEI grafted NPs, based on the concept of a layer-by-layer (LbL) technique. This modification is based on the coordination of another polyanionic polymer on the NPs surface in order to create a combined hybrid PEI and polyanionic polymer nanosystem. In both cases, the surface modification successfully mitigated the NP acute <i>in vivo</i> toxicity, without compromising the silencing efficiency

Intradermal air pouch leukocytosis as an in vivo test for nanoparticles

The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that – after first-line in vitro assays – characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay