Orientation-controlled conjugation of haloalkane dehalogenase fused homing peptides to multifunctional nanoparticles for the specific recognition of cancer cells.

Multifunctional nanoparticles (MNPs) that combine unique superparamagnetic properties and fluorescence emission are promising bimodal tracers for the noninvasive diagnosis of malignant cells both in vitro and in vivo. The selective recognition of specific cancer cells impacts diagnostic sensitivity, and it can be accomplished by the functionalization of MNPs with molecules that have a high affinity for specific membrane receptors. However, the mode in which individual homing ligands are immobilized at the interface between the inorganic core and the biological environment, may strongly affect the actual targeting efficiency of the nanoconjugate. A generally underestimated concern is the molecular organization at the nanoscale, which is a relevant consideration for protein ligands and is even crucial when short peptides are used. To optimize the recognition by a specific biological receptor, the immobilized peptide needs to be stably ligated to the nanoparticle but sufficiently mobile to interact with the receptor. Indeed, peptides tend to bind to the surface of an MNP through hydrophobic residues, which is promoted by entropic stabilization and electrostatic interactions and exploits polar and dissociated groups in the peptide sequence . This often results in a loss or reduction of delivery efficiency and, more importantly, of target selectivity. For this reason, the development of effective and reliable strategies to afford ordered ligand orientation on the nanoparticle surface has attracted a lot of interest in nanomedicine. Several approaches have been explored to control ligand positioning, including conjugation mediated by affinity tags inserted into the protein primary sequence, oriented immobilization on MNPs driven by recombinant protein linkers, and site-specific chemo-selective ligation. Recently, we have proposed a novel approach that relies on engineered proteins consisting of a receptor-targeting domain genetically fused with a nanoparticle-capture domain, in which the capture module should be an enzyme capable of irreversibly reacting with a suicide inhibitor covalently anchored on MNP. As a proof of concept, we have immobilized an scFv antibody fused to a SNAP tag onto MNPs. The same approach was exploited for ligand functionalization of pegylated capsules. 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. Haloalkane dehalogenase (HALO) from Rhodococcus rhodochrous forms an ester bond between aspartate 106 in the enzyme and the substrate, concomitantly removing halides from aliphatic hydrocarbons. Substitution of His272 with a phenylalanine prevents the substrate release, which usually occurs in native HALO, and thus an stable bond can be formed between HALO and an alkyl conjugate (Figure 1). Hence, we reasoned that a chloroalkane linker could be a good candidate to mediate the covalent, oriented immobilization on MNP of homing peptides genetically fused with HALO. We designed a bimodular genetic fusion (HALO–U11) comprising a small peptide of 11 amino acids (U11) that has a high affinity for urokinase plasminogen activator receptor (uPAR), which is overexpressed in several metastasizing tumors, as a targeting module, and HALO, as an MNP capture module (Figure 1). We demonstrate the utility of this method for peptide nanoconjugation and cellular imaging by evaluating the capability of MNP covalently bound with HALO–U11 to specifically recognize uPAR-positive cancer cells. UPAR is up-regulated in a broad range of cancer cell types and tumor-associated stromal cells, and mediates various biological processes at the cell surface, including plasminogen activation, extracellular matrix invasion, cell adhesion, and metastasis. U11 is believed to be the primary [*] Dr. M. Colombo, P. Verderio, E. Rozek, F. Andreata, E. Galbiati, Prof. P. Tortora, Dr. D. Prosperi NanoBioLab, Dipartimento di Biotecnologie e Bioscienze Universit di Milano Bicocca Piazza della Scienza 2, 20126 Milano (Italy) E-mail: [email protected] Homepage: http://www.nanobiolab.btbs.unimib.it