Protein Oriented Ligation on Nanoparticles Exploiting O₆‐Alkylguanine‐DNA Transferase (SNAP) Genetically Encoded Fusion

Abstract

A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the site-specific covalent conjugation of targeting peptides to nanoparticles. An scFv mutant selective for HER2 tumor antigen is chosen as the targeting ligand. SNAP-scFv is immobilized on magnetofluorescent nanoparticles and its targeting efficiency against HER2-positive cells is assessed by flow cytometry and immunofluorescence.

In vitro interaction of colloidal nanoparticles with mammalian cells: What have we learned thus far?

“…For quantitative studies NPs and their bioconjugates should be as monodisperse as possible with regard to all relevant parameters, such as charge and size, well-defined and well-characterized. Moreover, in the case of bioconjugates, the biological molecule, be it protein or drug, should be attached to the NP with control over orientation [84–85], density, affinity, and number or ratio per NP [85]. Although these goals are extremely hard to achieve, the more they can be fulfilled, the less heterogeneity is present in the NP material and the easier the results (i.e., the correlation between the properties of the NPs and the observed interaction of NPs with cells) can be interpreted [13,18,86].…”

“…By applying existing techniques correctly, the hydrodynamic diameters of NPs can be determined with remarkable accuracy, in particular if relative size changes are determined. Detection can be sensitive enough to resolve size-changes due to the attachment of individual macromolecules to the NPs [84–85 111–112]. Besides size, also shape has been proven to modulate the NP uptake of cells.…”

“…The effect of ligands immobilized on the surface of NPs designated for ligand–receptor-mediated uptake is diminished by the protein corona, which partly overcoats the ligands [134]. However, due to the fact that specific targeting still is possible [84], enough ligands still are biologically active. For highly defined NPs, such as nearly monodisperse NPs overcoated with a shell of an amphiphilic polymer [135], the corona formed by special model proteins can be surprisingly well organized.…”

Orientation‐Controlled Conjugation of Haloalkane Dehalogenase Fused Homing Peptides to Multifunctional Nanoparticles for the Specific Recognition of Cancer Cells

“…As a proof of concept, we have immobilized an scFv antibody fused to a SNAP tag onto MNPs. [20] The same approach was exploited for ligand functionalization of pegylated capsules. [21] In principle, this bimodular orthogonal bioreaction could present two important advantages when the homing ligand is a short peptide (i.e., 5-30 aa): 1) the peptide is separated from the nanoparticle surface by a protein spacer, which prevents undesired interactions and thus optimizes the ligand availability for molecular recognition; 2) the introduction of globular proteins (i.e., the reacting enzyme) enhances the solubility of the nanoconstruct.…”

“…Magnetite nanoparticles with narrow size distribution (10.1 AE 1.3 nm, MNP0) capped by oleate surfactant were obtained by solvothermal decomposition in organic solvents, [26] and they were transferred to the water phase by coating them with an amphiphilic polymer (PMA) in sodium borate buffer (pH 12). [20,27] The resulting PMA-coated nanoparticles (MNP1) were superparamagnetic and exhibited excellent solubility in aqueous media. Amino groups were introduced on MNP1 by using a homobifunctional linker (2,2-(ethylenedioxy)bisethylamine; EDBE) to give MNP2.…”