Nanobody-albumin nanoparticles (NANAPs) for the delivery of a multikinase inhibitor 17864 to EGFR overexpressing tumor cells

Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Journal of Controlled Release (Impact Factor: 7.71). 11/2012; 165(2). DOI: 10.1016/j.jconrel.2012.11.007
Source: PubMed


A novel, EGFR-targeted nanomedicine has been developed in the current study. Glutaraldehyde crosslinked albumin nanoparticles with a size of approximately 100nm were loaded with the multikinase inhibitor 17864-L(x) -a platinum-bound sunitinib analogue- which couples the drug to methionine residues of albumin and is released in a reductive environment. Albumin nanoparticles were surface-coated with bifunctional polyethylenglycol 3500 (PEG) and a nanobody -the single variable domain of an antibody- (Ega1) against the epidermal growth factor receptor (EGFR). EGa1-PEG functionalized nanoparticles showed a 40-fold higher binding to EGFR-positive 14C squamous head and neck cancer cells in comparison to PEGylated nanoparticles. 17864-L(x) loaded EGa1-PEG nanoparticles were internalized by clathrin-mediated endocytosis and ultimately digested in lysosomes. The intracellular routing of EGa1 targeted nanoparticles lead to successful release of the kinase inhibitor in the cell and inhibition of proliferation whereas the non-targeted formulations had no antiproliferative effects on 14C cells. The drug loaded targeted nanoparticles were as effective as the free drug in vitro. These results demonstrate that multikinase inhibitor loaded nanoparticles are interesting nanomedicines for the treatment of EGFR-positive cancers.

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    • "Significant reduction of PIK1 mRNA and protein expression was observed by ASOs-loaded trastuzumab-modified nanoparticles in BT474 breast cancer cells using real-time polymerase chain reaction (PCR) and Western blot analyses. The multikinase inhibitor 17864, a platinum-bound sunitinib analogue, was loaded to albumin nanoparticles via chemical conjugation to the albumin backbone (Altintas et al., 2013). Then, nanoparticles were surface-coated with heterobifunctional PEG and a nanobody (the single variable domain of an antibody) against epidermal growth factor receptor (EGFR). "

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    • "The collected fractions were centrifuged at 15 000 g for 15 min at 4 C and the supernatant was collected and stored at 4 C. Quantification of total protein content was performed using a Micro BCA Kit (Thermo Scientific) and a fraction corresponding to 5 mg protein was loaded on a reducing SDS-PAGE gel (4e12% crosslinked, Invitrogen). Western Blot analysis was performed as previously described [16]. Total and phosphorylated Met were detected by immunoblotting with rabbit anti-phospho-Met (pY1234/pY1235) and anti-Met antibody (Cell Signaling, 1:1000 diluted) in 5% BSA-TBS-T for 2 h on the roller bench at RT. Beta-actin rabbit antibody (Cell Signaling, 1:1000 diluted) was used as a loading control. "
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    ABSTRACT: The hepatocyte growth factor receptor (HGFR, c-Met or Met) is a receptor tyrosine kinase that is involved in embryogenesis, tissue regeneration and wound healing. Abnormal activation of this proto-oncogene product is implicated in the development, progression and metastasis of many cancers. Current therapies directed against Met, such as ligand- or, dimerization-blocking antibodies or kinase inhibitors, reduce tumor growth but hardly eradicate the tumor. In order to improve anti-Met therapy, we have designed a drug delivery system consisting of crosslinked albumin nanoparticles decorated with newly selected anti-Met nanobodies (anti-Met-NANAPs). The anti-Met NANAPs bound specifically to and were specifically taken up by Met-expressing cells and transported to lysosomes for degradation. Treatment of tumor cells with anti-Met NANAPs also resulted in downregulation of the total Met protein. This study shows that anti-Met NANAPs offer a potential system for lysosomal delivery of drugs into Met-positive tumor cells.
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