nanomaterials Communication Functionalizable Glyconanoparticles for a Versatile Redox Platform Marie Carrière 1,2, Paulo Henrique M. Buzzetti 1 , Karine Gorgy 1, Muhammad Mumtaz 2, Christophe Travelet 2 , Redouane Borsali 2,* and Serge Cosnier 1,* 1 UMR 5250, Département de Chimie Moléculaire, CNRS, Université Grenoble Alpes, CEDEX 09, 38058 Grenoble, France; [email protected] (M.C.); [email protected] (P.H.M.B.); [email protected] (K.G.) 2 CERMAV, UPR 5301, CNRS, Université Grenoble Alpes, CEDEX 09, 38058 Grenoble, France; [email protected] (M.M.); [email protected] (C.T.) * Correspondence: [email protected] (R.B.); [email protected] (S.C.) Abstract: A series of new glyconanoparticles (GNPs) was obtained by self-assembly by direct nano- precipitation of a mixture of two carbohydrate amphiphilic copolymers consisting of polystyrene- block-β-cyclodextrin and polystyrene-block-maltoheptaose with different mass ratios, respectively 0–100, 10–90, 50–50 and 0–100%. Characterizations for all these GNPs were achieved using dynamic light scattering, scanning and transmission electron microscopy techniques, highlighting their spher- ical morphology and their nanometric size (diameter range 20–40 nm). In addition, by using the inclusion properties of cyclodextrin, these glyconanoparticles were successfully post-functionalized using a water-soluble redox compound, such as anthraquinone sulfonate (AQS) and characterized by cyclic voltammetry. The resulting glyconanoparticles exhibit the classical electroactivity of free AQS in solution. The amount of AQS immobilized by host–guest interactions is proportional to the percentage of polystyrene-block-β-cyclodextrin entering into the composition of GNPs. The modulation of the surface density of the β-cyclodextrin at the shell of the GNPs may constitute Citation: Carrière, M.; Buzzetti, an attractive way for the elaboration of different electroactive GNPs and even GNPs modified by P.H.M.; Gorgy, K.; Mumtaz, M.; biotinylated proteins. Travelet, C.; Borsali, R.; Cosnier, S. Functionalizable Glyconanoparticles Keywords: glyconanoparticles; block copolymer; β-cyclodextrin; maltoheptaose; host–guest interaction; for a Versatile Redox Platform. anthraquinone sulfonate Nanomaterials 2021, 11, 1162. https:// doi.org/10.3390/nano11051162 Academic Editor: Josefina Pons 1. Introduction Received: 9 April 2021 Amphiphilic block copolymers (BCPs) are ideal building blocks for materials science. Accepted: 27 April 2021 Their self-assembly has attracted great attention for many years because they can provide Published: 29 April 2021 in solution different three-dimensional morphologies such as spherical micelles, vesicles, cylindrical micelles, lamellae and others structures that have attractive applications in Publisher’s Note: MDPI stays neutral biomaterials, catalysis, photoelectronic materials, biomedicine and more recently, in bio- with regard to jurisdictional claims in electrochemistry applications [1–4]. To obtain nanoparticles with diblock copolymers, published maps and institutional affil- which are composed of two chemically hydrophobic and hydrophilic different blocks, the iations. copolymers were dissolved in a first step in a mixture of solvents, allowing the solubiliza- tion of the different blocks. The next step consists of adding the resulting solution to a large volume of water, followed by organic solvent removal by evaporation [4] or by dialysis [3], leading to self-assembled NPs. Depending on the targeted application, the introduction of a Copyright: © 2021 by the authors. specific group can generate nanoparticles with specific properties. For example, the linkage Licensee MDPI, Basel, Switzerland. of a polystyrene chain with a cyclodextrin, leading to a polystyrene-block-β-cyclodextrin This article is an open access article copolymer as already described, allowed the formation of micelles by nanoprecipitation distributed under the terms and in water with a polystyrene core covered by a shell formed by cyclodextrins. In addition, conditions of the Creative Commons if a hydrophobic compound is added in the organic phase containing the copolymer, it Attribution (CC BY) license (https:// can be incorporated in the core of the nanoparticles as well as inserted in the cyclodextrins creativecommons.org/licenses/by/ (βCD) cavities by host–guest interaction, as previously reported for bis-pyrene-2,20-Azino- 4.0/). Nanomaterials 2021, 11, 1162. https://doi.org/10.3390/nano11051162 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, 1162 2 of 13 bis(3-ethylbenzothiazoline-6-sulfonic acid) [3], 9,10-phenanthrenequinone (PQ) [4], or tetrazines (TZ) [5]. Another approach consists of the post-functionalization of these glyconanoparticles in an aqueous medium with molecules that are soluble or partially soluble in water. In this way, for bioelectrochemical applications, the nanoparticles can constitute a versatile plat- form that may be easily functionalized by compounds capable of orienting or electrically wiring enzymes. In addition, this concept can be extended to biological macromolecules sensitive to organic solvents such as enzymes, antibodies or oligonucleotides. Their func- tionalization by molecules offering binding affinities with βCD will lead to the formation of nanoparticles modified by a shell of biomolecules. Although metallic nanoparticles coated with cyclodextrin have been modified by enzymes such as adamantane modified L-phenylalanine dehydrogenase or bovine pancreatic trypsin [6,7], no modification of polystyrene-block-β-cyclodextrin-based nanoparticles by proteins has been described. With this objective, an interesting parameter may be the surface density of the inclusion site at the surface of the nanoparticle shell. Replacing the βCD with a linear saccharide should allow a reduction in the number of associations with proteins which are large size molecules and therefore limit alterations in their flexibility and their activity. Moreover, compared to the toroidal form of β-cyclodextrin, a linear saccharide could form a more regular layer and thus facilitate the formation of a more compact hydrophilic shell for the nanoparticles. In this context, and in order to modulate the amount of βCD at the surface of gly- conanoparticles (GNPs), we report a series of new GNPs obtained by the self-assembly of two amphiphilic glycopolymers in different proportions, namely polystyrene-block- maltoheptaose copolymer (PS-b-MH) [8] and polystyrene-block-β-cyclodextrin copolymer (PS-b-βCD). PS-b-MH has a linear conformation that does not display specific association such as host–guest interactions. By varying the ratios, 100% PS-b-βCD, 50% PS-b-βCD— 50% PS-b-MH, 10% PS-b-βCD—90% PS-b-MH, it is therefore possible to generate a series of GNPs with different surface densities of βCD groups. The modulation of the surface density of βCD at GNPs and its impact on host–guest properties was investigated by incubation with anthraquinone sulfonate (AQS), a water-soluble model compound. This redox compound forms a stable host–guest complex with βCD with an association constant of 600 L mol−1 and can be easily detected by cyclic voltammetry [9]. 2. Materials and Methods 2.1. Materials p-toluenesulfonyl chloride (regentPlus, ≥99%), NaN3 (regentPlus, ≥99.5%), trimethy- lamine (TEA, 99%), 1,4-dioxane (anhydrous, 99.8%), and calcium hydride (CaH2, 95%), mag- nesium sulfate (anhydrous, reagentPlus, ≥99.5%), ethylene oxide solution (2.5–3.3 mol L−1 in THF), di-n-butylmagnesium solution (1.0 mol L−1 in heptane) and sec-butyllithium solution (1.4 mol L−1 in cyclohexane) were purchased from Sigma Aldrich (Munich, Ger- many)and used as received. Toluene (Biosolve, Dieuze, France) was first distilled over CaH2 and then over polystyryllithium. Styrene (Sigma Aldrich, ReagentPlus, ≥99%) was first distilled over CaH2 and then over di-n-dibutylmagnesium. CH2Cl2 (CP) stabilized by amylene and pyridine (extra dry) was obtained from Biosolve and distilled over CaH2 at 35 ◦C prior to use. Maltoheptaose (MH) (Hayashibara Company, Okayama, Japan) and 6-mono-o-(p-toluenesulfonyl)-β-cyclodextrin (Cyclolab, Budapest, Hungary) were func- tionalized with an alkynyl group according to the literature methods [10,11]. The Cu/CuO nanopowder (CuNP, 20–50 nm, 99.9% metal basis) was purchased from Alfa Aesar (Ward Hill, MA, USA). Tetrahydrofuran (THF), absolute methanol and absolute ethanol were bought from Biosolve. N,N-dimethylformamide (DMF, Fisher Scientific, Waltham, MA, USA) were used as received. Milli-Q water was obtained by water purification to a resistiv- ity of 18.2 MW cm using a Millipore Ultrapure system (MilliporeSigma, Burlington, MA, USA). The deuterated solvents were purchased from Eurisotop (Saint-Aubin, France). The Nanomaterials 2021, 11, 1162 3 of 13 cuprisorb resin was from Seachem (Madison, GA, USA). Sodium anthraquinone-2-sulfonate (AQS, ≥98%) and potassium chloride (KCl, ≥99%) were purchased from Sigma Aldrich. 2.2. Block Copolymers Synthesis (BCPs) Protocols PS-b-MH and PS-b-βCD block copolymers synthesis is preceded by hydroxyl-terminated polystyrene (PS-OH), tosyl terminated polystyrene (PS-OTs) and azido-functionalized polystyrene (PS-N3) preparation. The synthesis descriptions are described, and the charac- terization of the different copolymers can be found in the Supplementary Information. 2.2.1. Synthesis of Hydroxyl-Terminated Polystyrene (PS-OH) Hydroxyl-terminated polystyrene