Well-Defined Star-Shaped Polyglutamates with Improved Pharmacokinetic Profiles As Excellent Candidates for Biomedical Applications Aroa Duro-Castano, † Richard M. England, † David Razola, ‡ Eduardo Romero, ‡ Marta Oteo-Vives, ‡ Miguel Angel Morcillo, ‡ and María J. Vicent* ,† † Polymer Therapeutics Laboratory, Centro de Investigació n Príncipe Felipe (CIPF), Av. Eduardo Primo Yú fera 3, Valencia 46012, Spain ‡ Biomedical Applications of Radioisotopes and Pharmacokinetics Unit, CIEMAT, Av. Complutense 40, Madrid 28040, Spain * S Supporting Information ABSTRACT: There is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Additionally, limitations with existing systems, such as a lack of biodegradability and biocompatibility, inevitably lead to side effects and poor patient compliance. New polymer therapeutics based on amino acids are excellent candidates for drug delivery, as they do not suffer from these limitations. This article reports on a simple yet powerful methodology for the synthesis of 3-arm star-shaped polyglutamic acid with well- defined structures, precise molecular weights (MW), and low polydispersity (Đ = <1.3). These were synthesized by ring-opening polymerization (ROP) of N-carboxyanhydrides (NCA) in a divergent method from novel multifunctional initiators. Herein, their exhaustive physicochemical characterization is presented. Furthermore, preliminary in vitro evaluation in selected cell models, and exhaustive in vivo biodistribution and pharmacokinetics, highlighted the advantages of these branched systems when compared with their linear counterparts in terms of cell uptake enhancement and prolonged plasma half-life. KEYWORDS: ring-opening polymerization, polypeptides, star-shaped polymers, polyglutamates, NCA polymerization, polymer therapeutics, drug delivery 1. INTRODUCTION There has been a considerable effort devoted to the development of new and more versatile polymeric architectures with specific and predictable properties to be used as targeted drug delivery systems. 1 Such desirable features in these materials include adjustable molecular weights (higher MW to enhance passive targeting by the enhanced permeability and retention (EPR) effect), 2-4 predictable structure and con- formation in solution, lower heterogeneity, and greater possibility for multivalency. Nevertheless, the design and synthesis of new polymeric constructs of relevant MW, together with their physicochemical characterization, conformational studies, and especially their potential for biological applications, still remain to be fully exploited in this area. Toward this aim, polypeptide-based architectures can be considered suitable aspirants. The ring-opening polymerization (ROP) of α-amino-N- carboxyanhydrides (NCAs) 5-7 can be considered an excellent method to produce a wide variety of polypeptide architectures, including homopolymers, copolymers, block copolymers, and branched systems that do not exist naturally, on a multigram scale with relative ease. Since the first NCA synthesis by Leuch and co-workers, 8-10 many methodologies have been described with the aim of improving and overcoming the inherent limitations of this polymerization technique, as reviewed in excellent literature. 5-7 Among them, the use of high vacuum techniques (HVT), 11 amine hydrochloride salts, 12 heavy metal catalysts, 13 hexamethyldisilazanes (HMDS), 14 as well as by means of optimization of reaction conditions (pressure, temperature, etc.) 15 can be considered. Nevertheless, all methods have their own limitations. Previously in our group, we have reported the use of primary amine tetrafluoroborate (BF 4 ) salts as an improvement over the use of hydrochloride salts. This potent alternative is based on the non-nucleophilicity of the BF 4 salts, allowing the synthesis of well-defined polyglutamates (among other polypeptides) on a multigram scale and with low polydispersity (Đ < 1.3), adjustable MW, Received: May 8, 2015 Revised: August 8, 2015 Accepted: September 10, 2015 Article pubs.acs.org/molecularpharmaceutics © XXXX American Chemical Society A DOI: 10.1021/acs.molpharmaceut.5b00358 Mol. Pharmaceutics XXXX, XXX, XXX-XXX