title: Fluorescence techniques used to measure interactions between hydroxyapatite nanoparticles and epidermal growth factor receptors.
abstract: The potential applications of nanomaterials in therapeutics are immense and to fully explore this potential, it is important to understand the interaction of nanoparticles with cellular components. To examine the interaction between nanoparticles and cell membrane receptors, this report describes the use of advanced fluorescence techniques to measure interactions between hydroxyapatite (HA) nanoparticles and epidermal growth factor receptors (EGFRs), as a model system. FITC-labelled HA nanoparticles and monomeric red fluorescent protein (mRFP)-conjugated EGFRs expressed in Chinese hamster ovary cells (CHO-K1) were generated and their interaction measured using acceptor photobleaching-fluorescence resonance energy transfer (AP-FRET) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET). Results confirmed that hydroxyapatite nanoparticles not only interacted with EGFR but also attenuated downstream EGFR signalling, possibly by hindering normal dimerization of EGFR. Furthermore, the extent of signal attenuation suggested correlation with specific surface area of the nanoparticles, whereby greater specific surface area resulted in greater downstream signal attenuation. This novel demonstration establishes fluorescence techniques as a viable method to study nanoparticle interactions with proteins such as cell surface receptors. The approach described herein can be extended to study interactions between any fluorescently labelled nanoparticle-biomolecule pair.
authors.affiliation: School of Materials Science and Engineering, Nanyang Technological University, Singapore.
authors.forename: Mustafa H
authors.initials: MH
authors.lastname: Kathawala
authors.forename: Stella P K
authors.initials: SP
authors.lastname: Khoo
authors.forename: Thankiah
authors.initials: T
authors.lastname: Sudhaharan
authors.forename: Xinxin
authors.initials: X
authors.lastname: Zhao
authors.forename: Joachim
authors.initials: J
authors.lastname: Say Chye Loo
authors.forename: Sohail
authors.initials: S
authors.lastname: Ahmed
authors.forename: Kee
authors.initials: K
authors.lastname: Woei Ng
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D045744:Cell Line, Tumor; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D017886:Durapatite / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry* / Q000633:toxicity; D011485:Protein Binding
chemical_list: D017886:Durapatite; D066246:ErbB Receptors
keywords: Cell-nanoparticle interaction; Epidermal growth factor receptor; Fluorescence lifetime imaging microscopy; Fluorescence resonance energy transfer (FRET); Hydroxyapatite