Biolabeling and Binding Evaluation of Amphiphilic Nanocrystallopolymers

Hindawi Publishing Corporation Journal of Nanomaterials Volume 2016, Article ID 7416532, 7 pages http://dx.doi.org/10.1155/2016/7416532 Research Article Biolabeling and Binding Evaluation of Amphiphilic Nanocrystallopolymers Kwang-Suk Jang Department of Chemical Engineering and Research Center of Chemical Technology, Hankyong National University, Anseong 17579, Republic of Korea Correspondence should be addressed to Kwang-Suk Jang; Received 1 April 2016; Accepted 25 May 2016 Academic Editor: Christian Brosseau Copyright © 2016 Kwang-Suk Jang. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Surfactant-like inorganic-organic hybrid molecules named as nanocrystallopolymers were designed by conjugation of the hydrophilic synthetic poly(amino acid), poly-𝛼,𝛽-(N-(2-hydroxyethyl)l-aspartamide), with hydrophobic inorganic nanoparticles. In aqueous media, amphiphilic nanocrystallopolymers form self-aggregates with unique morphologies. Here, a simple biolabeling method of nanocrystallopolymers was developed. Biotin was selected as a model biomolecule. The specific binding of biotin-labeled nanocrystallopolymers to the targeted surface was evaluated with a surface plasmon resonance sensor. 1. Introduction Inorganic nanoparticles have been extensively studied for biomedical applications such as cellular imaging, cancer diagnosis and therapy, cell and protein separation, and biosensors [1–11]. Enhancing the stabilities of inorganic nanoparticles in aqueous media is one of the most challenging aspects in the field of biomedical engineering. Recently, amphiphilic polymers have been widely used for fabrication of inorganic nanoparticle-loaded capsules [8–11]. A dispersion of inorganic nanoparticles with hydrophobic ligands in an organic solvent can be loaded in polymeric shells forming emulsions. After evaporation of the organic solvent with a low boiling point, nanoparticle-loaded polymeric micelles can be obtained. Hydrophilic polymers chemically bound to hydrophobic nanoparticles forming micelle-like aggregates have been reported [12–14]. Surfactant-like inorganic-organic hybrid molecules named as nanocrystallopolymers were designed by conjugating the hydrophilic synthetic poly(amino acid), poly-𝛼,𝛽-(N-(2-hydroxyethyl)l-aspartamide) (PHEA) with hydrophobic Au nanoparticles (PHEA-g-Au NC). Because dodecanethiolate-protected Au nanocrystals have hydrophobic surfaces, the conjugated Au nanoparticles act as the hydrophobic part of the amphiphilic nanocrystallopolymers. In aqueous media, amphiphilic nanocrystallopolymers form spherical aggregates, core-shell unimolecular micelles, and cylindrical aggregates according to their hydrophilic/hydrophobic compositions. The self-aggregates are composed of a core part of inorganic nanoparticles and a shell part of water-soluble biocompatible polymers; therefore, they are expected to be advantageous for use in biological systems. Au nanoparticles exhibit unique optoelectric properties and can be readily synthesized by several different methods and easily tagged to diverse biomolecules or chemicals. Therefore, Au nanoparticles are among the most widely studied inorganic nanoparticles, together with magnetic nanoparticles and quantum dots, in biological detection, cancer treatment, and so forth. In this study, nanocrystallopolymers were labeled with biotin as a model biomolecule. Self-aggregates of the nanocrystallopolymers have unique morphologies. Biolabeling of nanocrystallopolymers is expected to expand the potentials for biomedical applications such as cellular imaging, cancer diagnosis and therapy, cell and protein separation, and biosensors. Synthesis of poly(amino acid) and biotin-functionalization were verified by 1 H-nuclear magnetic resonance (NMR) spectra, and the morphologies of selfaggregates of the nanocrystallopolymers were confirmed by transmission electron microscopy (TEM). The specific binding of biotin-labeled nanocrystallopolymers to the targeted 2 Journal of Nanomaterials O Ethanolamine (excess amount) O N DMF, 40∘ C HN O O n PSI Ethanolamine (appropriate amount), DMF, 40∘ C HS-(CH2 )10 -COOH DCC, DMAP DMF, 40∘ C O O HN O HN O NH NH NH (CH2 )2 (CH2 )2 (CH2 )2 OH PHEA OH O C O (CH2 )10 SH PHEA-g-C11 SH O N O O O O HN O HN O HN O HN O O O N HN O HN O O NH NH NH NH (CH2 )2 (CH2 )2 (CH2 )2 (CH2 )2 (CH2 )2 OH OH O OH O HS-(CH2 )10 -COOH DCC, DMAP HN DMF, 40∘ C O O NH PSI-PHEA NH2 -EO2 -biotin Ethanolamine DMF, 40∘ C C O C O (CH2 )10 (CH2 )10 SH SH O O O H S PSI-PHEA-g-C11 SH HN PHEA-g-C11 SH-EO2 -biotin O NH N H H O Figure 1: Synthetic routes of PHEA-g-C11 SH and PHEA-g-C11 SH-EO2 -biotin. surface was evaluated with a surface plasmon resonance (SPR) sensor. 2. Experimental Dodecanethiolate-protected Au nanocrystals were synthesized according to the Brust-Schiffrin method as previously reported, and their average core diameter was confirmed to be 1.5 nm average core diameter by TEM [12, 15–17]. PHEA conjugated with the undecanethiols, PHEA-g-C11 SH (P-gC11 SH), was prepared by the simple esterification reaction [12]. The biotin-conjugated backbone, PHEA-g-C11 SH-EO2 biotin (P-g-C11 SH-biot), was prepared by the aminolysis of the partially converted backbone poly(succinimide)- (PSI-) PHEA with amine-terminated biotin having a short ethylene oxide (EO2 ) spacer. All synthetic routes are shown in Figure 1. The synthesis of both backbone polymers was verified by 1 H NMR spectra. Au nanocrystallopolymeric aggregates in aqueous media were prepared as previously reported [12]. The Au nanocrystal-THF solution was added dropwise to the polymerwater solution under vigorous stirring, and the mixture was stirred continuously for 1 day to achieve ligand placeexchange. By removing the THF and unused Au nanocrystals, the Au nanocrystallopolymer forming the Au nanocrystalloaggregates, P-g-Au NC and P-g-Au NC-biot, was obtained in the aqueous solution. The solution was filtered through a 0.45 𝜇m polyvinylidene fluoride (PVDF) filter to remove impurities for further characterizations. The formation of micelles was visualized by TEM. For the TEM analysis of negatively stained nanocrystallopolymers, the solution containing 0.1% (w/v) phosphotungstic acid was placed on a copper grid covered with a formvar carbon membrane. Then, the grid was exposed for removing the solvent. Hydrodynamic diameter was measured by dynamic light scattering (DLS) and calculated with nonnegative least squares algorithms. 1 H-NMR spectra of P-g-Au NC and P-g-Au NC-biot were obtained with D2 O as a solvent. Specific binding of biotin-labeled Au nanocrystallopolymers to avidin was verified by SPR measurement. P-g-Au NC and P-g-Au NC-biot were dissolved in phosphate buffered saline (PBS; 0.01 M, pH 7.4) solution at a concentration of 1 (...truncated)