Impact of the Structure of Biocompatible Aliphatic Polycarbonates on siRNA Transfection Ability Antoine Fre ̀ re, † Michal Kawalec, ‡ Sarah Tempelaar, ‡ Paul Peixoto, § Elodie Hendrick, ∥ Olivier Peulen, § Brigitte Evrard, † Philippe Dubois, ‡ Laetitia Mespouille, ‡,⊥ Denis Mottet, ∥,⊥ and Ge ́ raldine Piel* ,†,⊥ † Laboratory of Pharmaceutical Technology and Biopharmacy − CIRM, § Metastasis Research Laboratory − GIGA, and ∥ Protein Signalisation and Interaction − GIGA, University of Liege, Avenue de l′Hopital 1 - 4000, Liege, Belgium ‡ Laboratory of Polymeric and Composite Materials, Center of Innovation and Research in Materials and Polymers (CIRMAP), Research Institute for Health Sciences and Technology, University of Mons, Place du Parc 20 - 7000, Mons, Belgium * S Supporting Information ABSTRACT: RNAi therapeutics are promising therapeutic tools that have sparked the interest of many researchers. In an effort to provide a safe alternative to PEI, we have designed a series of new guanidinium- and morpholino-functionalized biocompatible and biodegradable polycarbonate vectors. The impact of different functions (morpholino-, guanidinium-, hydrophobic groups) of the architecture (linear homopolymer to dumbbell-shape) and of the molecular weight of these copolymers on their capacity to form polyplexes and to decrease the expression of two epigenetic regulators of gene expression, HDAC7 and HDAC5, was evaluated. The use of one of these polymers combining morpholine and guanidine functions at the ratio >1 and hydrophobic trimethylene carbonate groups showed a significant decrease of mRNA and protein level in HeLa cells, similar to PEI. These results highlight the potential of polycarbonate vectors for future in vivo application as an anticancer therapy. 1. INTRODUCTION Owing to their specific and effective gene silencing, RNA interference (RNAi) has become a vital tool for gene down regulation in molecular medicine for the treatment of a variety of diseases, such as viral infections, cancer, and neuro- degenerative diseases. 1−3 A RNA interference approach to antiangiogenic therapy specifically targets the mRNA of histone deacetylases 7 (HDAC7). The shutdown of the HDAC7 protein disturbs the angiogenic process, making HDAC7 an attractive target that would directly interfere with the growth of cancerous tumors and metastasis development. 4,5 Another possible target in the histone deacetylases family is HDAC5, controlling the cell-cycle progression and survival of human cancer cells. 6 Although the use of siRNA in gene therapy has reached clinical evaluation, 7 several problems remain with respect to its in vivo use. Complications include an inability to cross the cytoplasmic membrane, instability in the blood, and an inability to specifically target abnormal cells. 1 The use of gene carrier systems able to drive genetic materials toward targeted cells, although challenging, could overcome these issues. Successful gene therapy relies on the development of vectors that can effectively, selectively and safely carry the oligonucleotide sequences to targeted sites. 8−10 Synthetic polymeric vectors are an excellent alternative to viral vectors with their safe handling and their upscalability as obvious advantages. Additionally, the possibility to modify their structure and composition and the ability to decorate them with suitable ligands allows for specifically targeted therapy, siRNA payload maximization and the preparation of nanoparticles that have a specific pharmacokinetic and biodistribution profile. 11,12 Usually polycations are employed as vectors as their positive charges can easily interact with the phosphate groups of the oligonucleotide through electrostatic interactions 13 leading to the condensation of oligonucleotides into structures called polyplexes. 14 Although many polymers have been reported to form stable polyplexes, to date none have matched the efficiency of viral vectors. This gap is related to factors such as cytotoxicity, nonbiodegradability, and (too) strong interaction of polymeric vectors with plasma proteins or oligonucleotides preventing release into the cytoplasm. 15−17 These major limitations remain a great challenge in the design of polycations. To address the cytotoxicity of polycations, biocompatible sequences such as aliphatic polycarbonates 18−20 or poly(ethylene oxides) (PEO) could be introduced, 8,21 while the escape of nanoparticles from endo- somes can be facilitated by the introduction of functional groups onto the polymer that can act as “proton pumps”. 22 Received: November 19, 2014 Revised: January 13, 2015 Published: January 20, 2015 Article pubs.acs.org/Biomac © 2015 American Chemical Society 769 DOI: 10.1021/bm501676p Biomacromolecules 2015, 16, 769−779