title: A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.
abstract: Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.
authors.forename: Stelios
authors.initials: S
authors.lastname: Florinas
authors.forename: Marc
authors.initials: M
authors.lastname: Liu
authors.forename: Ryan
authors.initials: R
authors.lastname: Fleming
authors.forename: Lilian
authors.initials: L
authors.lastname: Van Vlerken-Ysla
authors.forename: Joanne
authors.initials: J
authors.lastname: Ayriss
authors.forename: Ryan
authors.initials: R
authors.lastname: Gilbreth
authors.forename: Nazzareno
authors.initials: N
authors.lastname: Dimasi
authors.forename: Changshou
authors.initials: C
authors.lastname: Gao
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.affiliation: SynChem, Inc., Elk Grove Village, Illinois 60007, United States.
authors.forename: Ze-Qi
authors.initials: ZQ
authors.lastname: Xu
authors.forename: Shaoyi
authors.initials: S
authors.lastname: Chen
authors.forename: Anjaneyulu
authors.initials: A
authors.lastname: Dirisala
authors.affiliation: Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|The Innovation Center of Nanomedicine, 66-20 Horikawa-cho, Saiwai-ku, Kawasaki 212-0013, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kataoka
authors.forename: Horacio
authors.initials: H
authors.lastname: Cabral
authors.forename: R James
authors.initials: RJ
authors.lastname: Christie
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008823:Micelles; D053758:Nanoparticles / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism*; D036104:Receptor, EphA2 / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D008823:Micelles; D011108:Polymers; D036104:Receptor, EphA2