DOI 10.1007/s13233-020-8019-y www.springer.com/13233 pISSN 1598-5032 eISSN 2092-7673 Macromolecular Research Article Macromol. Res. © The Polymer Society of Korea and Springer 2019 Tuning with Phosphorylcholine Grafts Improves the Physicochemical Properties of PLL/pDNA Nanoparticles at Neutral pH Abstract: The improvement of biological properties of polycations is a fundamental step to overcome their limitations as non-viral gene carriers. This work studied the effect of phosphorylcholine (PC) groups on the physicochemical properties of poly(L-lysine) (PLL)/pDNA nanoparticles. Phosphorylcholine-grafted PLL derivatives (PLL-PC) con- taining increasing proportions of PC were obtained by the reductive amination reac- tion with phosphoryl glyceraldehyde and characterized by H NMR, FTIR, and GPC measurements. The PLL-PC derivatives were used to prepare polyplexes with pDNA and their properties were evaluated by fluorescence, gel electrophoresis and dynamic light scattering (DLS) measurements. The PLL-PC derivatives were able to interact with pDNA at low N/P ratios in physiological pH to form stable polyplexes having lower zeta potentials, as evidenced by the gel electrophoresis and zeta potentials measure- ments. A degree of grafting of 10% increased the in vitro transfection efficiency of PLL and a degree of 20 mol% of PC groups provided colloidal stability in physiological saline solution at neutral pH. Overall, the PC-PLL deriv- atives exhibited improved physicochemical properties and have significant potential for further studies as non-viral gene transfer agents. Keywords: PLL, phosphorylcholine, non-viral gene therapy, nanoparticles. 1. Introduction Non-viral gene therapy mediated by polycations has been indi- cated as one of the most promising strategies to deliver genes into cells due to their ability to condense nucleic acids into small nanoparticles, protect them from degradation and promote their transport across cellular membranes 1 . Carriers based on poly- cations have several advantages: e.g ., they can be easily produced, are non-immunogenic and may be tailored to target a variety of diseases. However, as with other non-viral vectors, their appli- cations have been limited by poor in vivo transfection efficien- cies. 2 Poly(L-lysine) (PLL) was one of the first polycations studied for the release of genes and its biodegradability is a particularly important property for in vivo applications. 3,4 Although exten- sively investigated as a gene delivery vector, PLL has limited efficiency owing to its poor circulatory half-life, 5 aggregation and poor endosomal escape. 6 In the past several years, new strate- gies have been adopted seeking to improve the properties of PLL based nanoparticles, 7,8 such as the use of ligands for targeted cell delivery, 9,10 modification with α-amino acids to provide gene carriers responsive to an endosomal pH 11 and inserting acid- cleavable groups into the PLL chain to facilitate the intracellu- lar release. 12 The attaching of small fractions of PEG has provided improvements to the transfection process. 8,13,14 PLL grafted with low percentages of PEG (4-5%) exhibits higher colloidal stabil- ity and also the ability to protect the plasmid from DNase deg- radation. 14 PLL conjugated to palmitic acid (PA) also showed a better performance regarding the protection of DNA against degradation by nucleases and, in some cases, the transfection efficiency of PLL-PA was about 2 to 5 times greater than that mediated by Lipofectamine TM 2000. 15 The polyplex stability is believed to play an important role in transfection efficiency and micelles with controlled degrees of crosslinking have been reported as having ‘improved efficiencies. 16,17 Other strategies to over- come the biocompatibility and toxicity problems of PLL and to increase the transfection efficiency include conjugation with polysaccharides, 18 the preparation of block copolymers 19 and asso- ciation with superparamagnetic iron oxide nanoparticles. 20 Thus, surface modification of the polycations is an important approach to provide low cytotoxicity and to prolong the residence time of the nanocarriers in the bloodstream. 17 Juliana Semensato 1,2 Júlio Cesar Fernandes 2 Mohamed Benderdour 2 Vera Aparecida de Oliveira Tiera 1 Aline Margarete Furuyama Lima 1 Marcio José Tiera* ,1 Departamento de Química e Ciências Ambientais, UNESP-Universidade Estadual Paulista, Brasil Orthopedic Research Laboratory, Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Canada Received March 30, 2019 / Revised June 28, 2019 / Accepted July 1, 2019 Acknowledgments: M. J. Tiera would like to thank the São Paulo Research Foundation (FAPESP- Fundação de Amparo à Pesquisa do Estado de São Paulo) and the National Council for Scientific and Technological Development (CNPq) (Grant 2014/407499). A. M. F. Lima and A. M. M. Junior thank the National Council for the Improvement of Higher Education (CAPES) for its support (Grants PNPD 1267244, finance code 001 and 1743469). The authors report no conflicts of interest in this work. The authors would also like to thank L. F. M. Ferraz (Embrapa Instrumentação-São Carlos) for help with the Scanning Electron Microscopy, M. P. S. Cabrera (Peptides Research Group-UNESP, Grant FAPESP 2012/24259-0) and C. R. Bonini Domingos for access to instrumentation. *Corresponding Author: Marcio José Tiera (marcio.tiera@unesp.br)