Avidin-conjugated calcium phosphate nanoparticles as modular system for the attachment of biotinylated molecules

Introduction: Calcium phosphate nanoparticles are biocompatible, biodegradable and bioactive due to their similarity to nanocrystalline hydroxyapatite in hard tissue of the human organism.[1] Avidin is a tetrameric protein... [ view full abstract ]

Introduction: Calcium phosphate nanoparticles are biocompatible, biodegradable and bioactive due to their similarity to nanocrystalline hydroxyapatite in hard tissue of the human organism.[1] Avidin is a tetrameric protein that forms a very strong complex with the biomolecule biotin. The avidin-biotin-complex is the strongest known non-covalent bond between a protein and a ligand.[2] In this work, avidin-conjugated calcium phosphate nanoparticles were used for the non-covalent attachment of biotinylated compounds via the avidin-biotin-complex formation. The versatility of the complex formation onto the surface of the nanoparticles was demonstrated with various biotinylated molecules, including fluorescent dyes and antibodies.

Methods: Calcium phosphate nanoparticles were first coated with a thin layer of silica and then covalently functionalized with 3-mercaptopropyltrimethoxysilane. This led to a sulfhydryl-terminated nanoparticle.[3] Avidin was then activated with the cross-linker sulfo-SMCC at amino groups and subsequently conjugated to the nanoparticle by the sulfhydryl groups. This resulted in an avidin-terminated nanoparticles. The freshly prepared avidin-conjugated calcium phosphate nanoparticle dispersion was combined with the biotinylated compounds and incubated overnight. After each reaction step the dispersion was purified by centrifugation and the pellet was dispersed by ultrasonication in ultrapure water. (Figure 1)

Results: Avidin can be covalently attached to calcium phosphate nanoparticle. The hydrodynamic diameter of this nanoparticles was about 200 nm (DLS). The zeta potential was +21 mV. A spherical morphology of the nanoparticles was found in SEM. After the attachment of the biotinylated molecules, an increased diameter was observed by DLS and SEM. The capacity of biotin binding sites for the conjugated avidin was assessed by a fluorescence quenching assay.[4] Each conjugated avidin molecule on average bound 2.6 biotin molecules. A variety of biotinylated molecules can be loaded on the avidin-conjugated calcium phosphate nanoparticle. Furthermore, different biotinylated molecules can be coupled to the same nanoparticle. Fluorescently labelled avidin-conjugated calcium phosphate nanoparticles were detected intracellularly by CLSM (HeLa cells). The co-localization of fluorescent avidin and fluorescent biotin indicated the stability of the complex during the in vitro studies. The dissolved complex alone (i.e. without nanoparticles) is not taken up by HeLa cells.

References: [1]M.Epple,K.Ganesan,R.Heumann,J.Klesing,A.Kovtun,S.Neumann,V.Sokolova, J.Mater.Chem.20 10,20,18–23.
[2]N.M.Green,Adv.Prot.Chem. 1975,29,85–133.
[3]D.Kozlova,S.Chernousova,T.Knuschke,J.Buer, A.M.Westendorf, M.Epple, J.Mater.Chem. 2012,22,396–404.
[4]G.Kada,H.Falk,H.J.Gruber,BBA 1999,1427,33–43.