Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb Research paper Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity Osama Saher a,e, ⁎ , Cristina S.J. Rocha a,1 , Eman M. Zaghloul a , Oscar P.B. Wiklander a , Susanna Zamolo b , Marc Heitz b , Kariem Ezzat a , Dhanu Gupta a , Jean-Louis Reymond b , Rula Zain a,d , Florian Hollfelder c , Tamis Darbre b , Karin E. Lundin a , Samir EL Andaloussi a , C.I. Edvard Smith a, ⁎ a Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Huddinge, Sweden b Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland c Department of Biochemistry, University of Cambridge 80 Tennis Court Road, Cambridge CB2 1GA, UK d Department of Clinical Genetics, Centre for Rare Diseases, Karolinska University Hospital, SE-171 76 Stockholm, Sweden e Department Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt ARTICLE INFO Keywords: Splice-switching oligonucleotides Gene therapy Delivery vectors Transfection Peptide dendrimers Synergism ABSTRACT Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best den- drimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum- free conditions. Interestingly, addition of sucrose to the formulation enabled – for the first time – peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on trans- fection efficacy. 1. Introduction By 2005, at least one hundred different dendrimer structures and more than 1000 surface modifications were reported [1]. The rapidly growing field of dendrimers started when polypropylenimine (PPI) was successfully synthesized by Fritz Vögtle and his team in the late 1970s [2]. Several structures and modifications followed afterwards [3]. However, it was not until the early 1990s when dendrimers were in- troduced to the field of gene delivery [4]. Unlike many widely used transfection reagents, dendrimers are synthesized with a well-defined molecular structure and are thus monodisperse, so that control over their size and structure [5] makes structure-function relationships possible [6,7] Secondly, the buffering capacity of dendrimers has been postulated to enhance drug/gene delivery: The ‘proton sponge hypothesis’ in- troduced by Hansler and Szoka [4] and confirmed by Sonawane et al. [8] highlighted the functional role of the tertiary amines of dendrimers under acidic conditions in the endosome. Tertiary amines get https://doi.org/10.1016/j.ejpb.2018.09.002 Received 9 May 2018; Received in revised form 15 August 2018; Accepted 3 September 2018 ⁎ Corresponding authors at: Department of Laboratory Medicine (LABMED), H5, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Huddinge, Sweden. 1 Present address: New Modalities, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden. E-mail addresses: osama.ahmed@ki.se (O. Saher), edvard.smith@ki.se (C.I.E. Smith). European Journal of Pharmaceutics and Biopharmaceutics 132 (2018) 29–40 Available online 04 September 2018 0939-6411/ © 2018 Elsevier B.V. All rights reserved. T