Contents lists available at ScienceDirect European Polymer Journal journal homepage: www.elsevier.com/locate/europolj IR780-polymer conjugates for stable near-infrared labeling of biodegradable polyester-based nanocarriers Maria Alice de Oliveira a , Marina Guimarães Carvalho Machado a , Sabrina Emanuelle Dias Silva a , Thais Leite Nascimento b , Eliana Martins Lima b , Gwenaelle Pound-Lana a, ⁎ , Vanessa Carla Furtado Mosqueira a a Laboratory of Pharmaceutics and Nanobiotechnology, School of Pharmacy, Universidade Federal de Ouro Preto, Minas Gerais, Brazil b Laboratório de Nanotecnologia Farmacêutica e Sistemas de Liberação de Fármacos, FarmaTec, Faculdade de Farmácia, Universidade Federal de Goiás – UFG, Goiânia, Goiás, Brazil ARTICLEINFO Keywords: Fluorescent label Polylactide Near-infrared probe Polymeric nanocarrier Click chemistry Field fow fractionation ABSTRACT Near-infrared dyes are useful to monitor nanocarriers in vitro and in vivo and can serve as photosensitizers in cancer photodynamic therapy. However, strategies need to be developed to guarantee that the dye photophysical properties and loading within the drug delivery system remain stable for reliable tracking within biological systems. This work reports the facile chemical conjugation of the carbocyanine heptamethine near-infrared dye IR780 to polylactide for stable fuorescent labeling of biodegradable polyester nanocarriers. “Clickable” poly- lactide was synthesized via organocatalyzed ring opening polymerization of D,L-lactide with a cyclooctyne in- itiator. IR780 was derivatized and conjugated to polylactide via a one-pot copper-free azide-alkyne cycloaddi- tion reaction. The synthetic strategy developed was efective to promote conjugation of the near-infrared fuorescent dye to polylactide, as confrmed by high performance liquid chromatography. Nanoparticles con- taining the dye–polymer conjugate were prepared by nanoprecipitation and characterized. Asymmetric fow feld-fow fractionation with light scattering and fuorescence detection revealed that the near-infrared fuor- escence of the nanoparticles remained stable and was not transferred to serum proteins. In contrast, signifcant transfer of the dye to serum proteins was evidenced when the dye was merely encapsulated in similar nano- particles through physical entrapment. Confocal microscopy and fuorescence tomography imaging showed that the polymer-dye conjugate confers fuorescence properties to the NP suitable for further in vitro and in vivo pre- clinical studies. 1. Introduction Fluorescent probes with excitation and emission in the near-infrared region (NIR) can be detected in vivo owing to deep penetration of the light and low auto-fuorescence of biological components at wave- lengths in the 700–900 nm range [1]. In nanomedicine NIR probes are used to label and track nanocarriers via optical in vivo imaging in pre- clinical studies [2]. In addition, some of these probes can restore the energy of the excitation light source via non-radiative processes, whereby they react with their environment to generate reactive oxygen or nitrogen species (ROS or NOS). This property is currently being ex- tensively used in photodynamic therapy [3,4]. However, as pointed out in several recent studies, even lipophilic probes can leak rapidly from their nanocarrier after in vivo administration. Therefore, covalent attachment to the nanocarrier main component is necessary. Among the available NIR probes the lipophilic heptamethine carbocyanine dye IR780 is interesting for both nanocarrier labeling and photodynamic therapy, due to its ability to fuoresce and to promote photo-dependent cytotoxicity [5–8]. Most importantly it is one of the few NIR dyes that are photostable, readily obtained in large amounts (commercially available), and reactive owing to its chlorine substituent, making it an excellent candidate to prepare polymer-dye conjugates [9–11]. In the present study we derivatized IR780 and covalently attached it to preformed reactive polylactide (PLA) via one-pot catalyst-free azide- alkyne cycloaddition. This strategy was designed in order to achieve the stable and versatile labeling of the most commonly used degradable polyester nanoparticles (NP), namely, matrix nanospheres (NS) and oily core nanocapsules (NC) prepared from polyethylene glycol-block- https://doi.org/10.1016/j.eurpolymj.2019.109255 Received 20 June 2019; Received in revised form 12 September 2019; Accepted 15 September 2019 ⁎ Corresponding authors at: Laboratory of Pharmaceutics and Nanobiotechnology, School of Pharmacy, Universidade Federal de Ouro Preto, Campus Universitário Morro do Cruzeiro, Ouro Preto, Minas Gerais 35400-000, Brazil. E-mail addresses: gpoundlana@gmail.com (G. Pound-Lana), mosqueira@ufop.edu.br (V.C.F. Mosqueira). European Polymer Journal 120 (2019) 109255 Available online 16 September 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved. T