Development of a selective cell capture and release assay: impact of clustered RGD ligands

Abstract

There is a growing interest in isolating tumor cells from biological samples. Considering that circulating tumor cells can be rare in blood, it appears challenging to capture these cells onto a surface with high selectivity and sensitivity. In the present paper we describe the design of functionalized surfaces aiming to selectively capture tumor cells by using the RGD peptide ligand either with a tetrameric or a monomeric presentation. β-cyclodextrin-coated self-assembled monolayers were used as platforms for the binding of RGD ligands endowed with a redox ferrocene cluster as the dissociation of the inclusion complex on the surface is accounted for the release of the captured cells upon electrochemical oxidation of ferrocene. For this purpose, we determined suitable RGD densities for both monovalent and tetravalent ligand presentations. The present results indicate that the clustered RGD architecture efficiently improves the selective cell capture at a very low RGD-surface density (∼ 10 RGD/μm 2 ) in comparison with the monovalent presentation (∼ 1000 RGD/ μm 2 ). IntroductionMultivalent interactions are ubiquitously observed in biological systems for a wide range of functions including integrinmediated cell adhesion to extracellular matrix (ECM). 1 Among the integrin receptors, the α V β 3 integrin subclass has received special attention as it is involved in tumour progression such as aggressive metastatic cancer.2 Felding-Habermann et al. have shown that this receptor contributes to circulating tumour cell (CTC) arrest. 3 As cells expressing α V β 3 integrin interact with the ECM through the recognition of the ubiquitous triad sequence RGD (Arg-Gly-Asp), a series of peptides containing the RGD sequence were developed for diagnosis and tumour therapy. 4 The cyclopentapeptide derivatives were found to specifically bind the α V β 3 integrin. 5 Additionally, the multimeric presentation of RGD motifs appears to be a prerequisite for efficient integrin targeting. 6 Our group and others have shown that multimeric compounds exhibit attractive biological properties. 7 Recently, we have reported the benefit of tetrameric RGD derivatives for the near-infrared optical guided surgery of solid tumours 8 and for tumour-targeted drug delivery. Although multimeric RGD compounds show better affinity than their corresponding monomers, 7 few reports have tested how important clustered architectures are for cell binding to RGD-functionalized surfaces. Pioneering studies have demonstrated that nanoscale patterning of RGD ligand presentation significantly influences cell adhesion and spreading. 10 Those surfaces were prepared from highmolecular weight RGD-containing polymers or nanoparticles. Journal NameScheme 1 Synthesis of compounds 1 and 3. Reaction conditions: i) TFA/H2O (7:3), rt, 20 min then TFA/H2O (95:5), rt, 120 min, 44%; ii) NaIO4, H2O, rt, 20 min, 46%; iii) TFA/H2O/CH3CN (7:2:1), rt, 20 min, 42%; iv) CuSO4, THPTA, sodium ascorbate, phosphate buffer pH 7.4/DMF (6/4), 45°C, 150 min, 1: 63%, 3 :71 %. SPPS: solid-ph...

Impact of Multimeric Ferrocene‐containing Cyclodecapeptide Scaffold on Host‐Guest Interactions at a β‐Cyclodextrin Covered Surface

“…We noticed a weak release of guest compound upon rinsing step followed by stable signals. The hydrated masses could be compared to the molecular surface density which had been previously measured [9] by spectroscopic ellipsometry for similar Fc-containing scaffolds, we reported values of 22 pmol/cm 2 (83.4 � 7.0 ng/cm 2 before rinsing) and 18.6 pmol/cm 2 (70.3 � 5.9 ng/cm 2 after rinsing). By comparing the dry masses with the hydrated ones extracted from QCM-D measurements, hydration ratios of 49 % and 52 % were respectively obtained.…”

“…It consists in a "one step" procedure directly exposing the gold surface to a mixed solution of β-CD-terminated thiol (CDthiol) and oligo(ethylene glycol)-terminated thiol (hydroxyl-thiol) used as a diluent. [9,20] Wettability studies were carried out by measuring contact angles of water on SAM surfaces previously exposed to various molar ratios of CD-thiol in order to analyze the variation of the surface hydrophilicity. The stepwise increase from 0 to 5 % of the CD-thiol molar ratio in the thiol solution from which the β-CD SAMs were formed is accompanied by a continual decrease of the contact angles (from 36 � 4 deg.…”

“…Previously, we designed functionalized surfaces that permit a selective tumor cell capture and release. [9,10] As the β-CD has a weak binding affinity for ferrocene (K A close to 10 3 M À 1 ), [11] we have exploited a nano-sized cyclopeptide scaffold composed of ten amino acid residues that present in a spatially controlled manner two independent functional domains: [12] a ligand domain for cancer cell recognition and a clustered Fc domain allowing host-guest interactions through a multivalent presentation. [9,10] Multivalent interactions are known to be much stronger than the corresponding monovalent ones, [13] in particular at interfaces.…”

“…[9,10] As the β-CD has a weak binding affinity for ferrocene (K A close to 10 3 M À 1 ), [11] we have exploited a nano-sized cyclopeptide scaffold composed of ten amino acid residues that present in a spatially controlled manner two independent functional domains: [12] a ligand domain for cancer cell recognition and a clustered Fc domain allowing host-guest interactions through a multivalent presentation. [9,10] Multivalent interactions are known to be much stronger than the corresponding monovalent ones, [13] in particular at interfaces. [14] This was highlighted by the group of Huskens that studied the multivalency effect of Fc-containing dendrimers on β-CDmodified self-assembled monolayers (SAMs) surfaces.…”

Biotin-Avidin-Mediated Capture of Microspheres on Polymer Fibers

“…Several physical and biochemical strategies have been described for the capture of moving cells or microspheres. While physical methods such as filtration [4] or acoustic waves can be used to select cells based on their size [5], biochemical methods such as cell-binding to peptide-functionalized surfaces [6], or immunosorting techniques using magnetic beads [7] allow for the capture of cells based on their biological properties. However, biochemical strategies might affect the cell behavior upon capture, since they are based on the biology of captured cells.…”