Cellular uptake of fluorophore-labeled glyco-DNA–gold nanoparticles
- 340 Downloads
DNA-functionalized gold nanoparticles (AuNP–DNA) were hybridized with complementary di-N-acetyllactosamine-(di-LacNAc, [3Gal(β1-4)GlcNAc(β1-]2)-modified oligonucleotides to form glycol-functionalized particles, AuNP–DNA–di-LacNAc. While AuNP–DNA are known to be taken up by cells via scavenger receptors, glycol-functionalized particles have shown to be taken up via asialoglycoprotein receptors (ASGP-R). In this work, the interaction of these new particles with HepG2 cells was analyzed, which express scavenger receptors class B type I (SR-BI) and ASGP-R. To study the contribution of these receptors as potential mediators for cellular uptake, receptor-blocking experiments were performed with d-lactose, a ligand for ASGP-R, Fucoidan, a putative ligand for SR-BI, and a SR-BI blocking antibody. Labeling with Cy5-modified DNA ligands enabled us to monitor the particle uptake by confocal fluorescence microscopy and flow cytometry, in order to discriminate the two putative pathways by competitive binding studies. While SR-BI-antibody and d-lactose had no inhibiting effects on particle uptake Fucoidan led to a complete inhibition. Thus, a receptor-mediated uptake by the two receptors studied could not be proven and therefore other uptake mechanisms have to be considered.
KeywordsGold nanoparticles DNA Carbohydrates Cellular uptake Hepatocytes
We thank Prof. E. Weinhold and Dr. S. Charrak (Institute of Organic Chemistry, RWTH Aachen University) for HPLC, Dr. W. Bettray (Institute of Organic Chemistry, RWTH Aachen University) for ESI–MS, Dr. Yu Pan (Biomedical Engineering, Biointerface Laboratory RWTH Aachen University) for Nanodrop analytics and the Immunohistochemistry, and Confocal Laser Scanning Microscopy Facility, a core facility of the IZKF within the Faculty of Medicine, RWTH Aachen University. We are grateful to Liangliang Hao for valuable comments on this manuscript. This research is a part of the project “ForSaTum”, co-funded by the European Union (European Regional Development Fund—Investing in your future) and the German federal state North Rhine-Westphalia (NRW). Furthermore, the work was financially supported by the Deutsche Forschungsgemeinschaft Graduate School “Biointerface” (No. 1035) and the Excellence Initiative of the German federal and state Governments (ERS Seed Fund Project).
- Hangeland JJ, Flesher JE, Deamond SF, Lee YC, Ts’O PO, Frost JJ (1997) Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleoside methylphosphonate-neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the mouse. Antisense Nucleic Acid Drug Dev 7:141–149CrossRefGoogle Scholar
- Sauerzapfe B, Křenek K, Schmiedel J, Wakarchuk WW, Pelantová, Křen V, Elling L (2009) Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces. Glycoconj J 26:141–159CrossRefGoogle Scholar
- Stockert RJ (1995) The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol Rev 75:591–609Google Scholar
- Takae S, Akiyama Y, Otsuka H, Nakamura T, Nagasaki Y, Kataoka K (2005) Ligand density effect on biorecognition by PEGylated gold nanoparticles: regulated interaction of RCA120 lectin with lactose installed to the distal end of tethered PEG strands on gold surface. Biomacromolecules 6:818–824CrossRefGoogle Scholar
- Westerlind U, Westman J, Törnquist E, Smith CIE, Oscarson S, Lahmann M, Norberg T (2004) Ligands of the asialoglycoprotein receptor for targeted gene delivery, Part 1: synthesis of and binding studies with biotinylated cluster glycosides containing N-acetylgalactosamine. Glycoconj J 21:227–241CrossRefGoogle Scholar
- Witten KG (2012) Glyko-DNA-funktionalisierte goldnanopartikel: synthese und wechselwirkung mit lektinen und zellen. PhD Thesis RWTH, Aachen UniversityGoogle Scholar