ARTICLE DNA delivery systems based on copolymers of poly (2-methyl-2-oxazoline) and polyethyleneimine: Effect of polyoxazoline moieties on the endo-lysosomal escape Emi Haladjova 1 | Maroš Smolíc ˇ ek 2,3 | Iva Ugrinova 4 | Denitsa Momekova 5 | Pavletta Shestakova 6 | Zuzana Kroneková 2 | Juraj Kronek 2 | Stanislav Rangelov 1 1 Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria 2 Department for Biomaterials Research, Polymer Institute, Slovak Academy of Sciences, Bratislava, Slovakia 3 Department of Inorganic Chemistry, Faculty of Natural Sciences, Comenius University Mlynská dolina, Bratislava, Slovakia 4 Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria 5 Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria 6 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria Correspondence Emi Haladjova and Stanislav Rangelov, Institute of Polymers, Bulgarian Academy of Sciences, Akad. G. Bonchev St. bl. 103A, Sofia 1113, Bulgaria. Email: ehaladjova@polymer.bas.bg (E. H.) and rangelov@polymer.bas.bg (S. R.) Abstract Poly(2-methyl-2-oxazoline)-polyethylenimine (PMeOx-co-PEI) copolymers dif- fering by degree of polymerization (DP = 50 and 200) and PEI content (from 37 to 99 mol%) were synthesized by living cationic ring-opening polymerization of 2-methyl-2-oxazoline, followed by partial hydrolysis. Upon mixing with DNA in a wide range of N/P ratios, they formed well-defined polyplex particles of small size (typically below 100 nm) and narrow size distribution. The polyplexes demonstrated good colloidal stability and very low in vitro cytotoxicity. The copolymers exhibited buffering capacity of over 50% relative to that of the refer- ence PEI implying effective endo-lysosomal escape of the polyplexes. Increased cellular internalization of both PCR fragments and plasmid DNA, attributable to the strongly positive ζ potential and small size of the polyplexes, was observed. In spite of these favorable prerequisites, the transfection efficiency was low (below 20% relative to the control PEI) and was attributed to retarded swelling of the polyplex particles, endo-lysosomal rupture, and DNA release. KEYWORDS biomedical applications, colloids, drug delivery systems, nucleic acids, polyelectrolytes 1 | INTRODUCTION Gene therapy is a strategy to treat refractory diseases in genetic level. [1–3] Its basic principle consists of delivery of a foreign genetic material into target cells to promote a thera- peutic effect in the patients. The gene delivery, however, is a rather complex and complicated process that usually requires a vector system able to simultaneously carry thera- peutic molecules (DNA or RNA) and protect them from the external environment. [4,5] The main obstacles these systems have to overcome are the successful internalization into the cells, escape from the endo-lysosomal compartment, and effective release of therapeutic molecules. [6–9] Furthermore, the formulations should remain stable under physiological conditions as well as they should be biocompatible and safe for the patients. [6–9] In the past two decades a subject of intense research interest have been nonviral gene delivery vector systems based on polymers. [9–13] Polymer vectors are typically polycations that interact electrostatically with the poly- anionic chain of nucleic acids resulting in formation of nanosized particles known as polyplexes. [9–13] They are typically less active than the viral systems but relatively safer and more cost-effective. Tailorability of the systems, [12,14] flexibility to formulation design, [14,15] ame- nability to various modifications, [9,10] and possibilities for targeted design to overcome specific barriers [9,10] are additional advantages of the polymer gene delivery Received: 13 January 2020 Revised: 22 April 2020 Accepted: 8 May 2020 DOI: 10.1002/app.49400 J Appl Polym Sci. 2020;e49400. wileyonlinelibrary.com/journal/app © 2020 Wiley Periodicals LLC. 1 of 16 https://doi.org/10.1002/app.49400