Citation: Eswaran, L.; Kazimirsky, G.; Byk, G. New Biocompatible Nanohydrogels of Predefined Sizes for Complexing Nucleic Acids. Pharmaceutics 2023, 15, 332. https://doi.org/10.3390/ pharmaceutics15020332 Academic Editors: Bogdan Stefan Vasile and Ionela Andreea Neacsu Received: 28 November 2022 Revised: 15 January 2023 Accepted: 16 January 2023 Published: 19 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article New Biocompatible Nanohydrogels of Predefined Sizes for Complexing Nucleic Acids Lakshmanan Eswaran, Gila Kazimirsky and Gerardo Byk * Laboratory of Nanobiotechnology, Department of Chemistry, Bar-Ilan University, Ramat Gan 52900, Israel * Correspondence: gerardo.byk@biu.ac.il Abstract: The advent of protein expression using m-RNA applied lately for treating the COVID pandemic, and gene editing using CRISPR/Cas9 technology for introducing DNA sequences at a specific site in the genome, are milestones for the urgent need of developing new nucleic acid delivery systems with improved delivery properties especially for in vivo applications. We have designed, synthesized, and characterized novel cross-linked monodispersed nanohydrogels (NHG’s) with well- defined sizes ranging between 50–400 nm. The synthesis exploits the formation of self-assemblies generated upon heating a thermo-responsive mixture of monomers. Self-assemblies are formed and polymerized at high temperatures resulting in NHGs with sizes that are predetermined by the sizes of the intermediate self-assemblies. The obtained NHGs were chemically reduced to lead particles with highly positive zeta potential and low cell toxicity. The NHGs form complexes with DNA, and at optimal charge ratio the size of the complexes is concomitant with the size of the NHG’s. Thus, the DNA is fully embedded inside the NHGs. The new NHGs and their DNA complexes are devoid of cell toxicity which together with their tunned sizes, make them potential tools for gene delivery and foreign protein expression. Keywords: self-assembly; polymerization; cationic nanohydrogels; non-viral gene delivery 1. Introduction The synthesis of monodispersed nanoparticles with well tunned sizes is a complex process of great interest since size and dispersity determine the feasibility of different appli- cations. Their generation has been tackled mostly by synthesizing nanoparticles made of inorganic materials such as silica, titanium oxide, iron, gold, and silver or by more complex mixtures of metals such as quantum dots (for example CdSe/ZnS) whose precursors can drive a controlled formation of monodispersed particles. These materials are known as having significant cell toxicity and the tendency to leak out from the nanoparticles [1]. Therefore, most of the available approaches adapt these materials to different biological applications by developing further coating methods that render them more biocompatible. Coatings are composed of organic materials, mainly polymers that are adsorbed, anchored, or polymerized on the surface of the inorganic nanoparticles resulting in composite parti- cles with lower toxicity and better bioavailability [2]. In summary, the syntheses of these materials are tedious and time-consuming. During the last few years, we have considered the possibility of generating new types of cross-linked nanoparticles composed of polymeric hydrogels. Nanohydrogels (NHGs) have gained significant attention in recent years for drug delivery and tissue engineering, owing to their peculiar properties that combine the characteristics of hydrogel systems (e.g., high water content) with a very small size (nanometric dimension). Their properties enable reaching the smallest capillary vessels, not accessible to macroscale hydrogels, and penetrate tissues either through the paracellular or transcellular pathways. The size and surface properties of NHGs can be manipulated to avoid rapid clearance by phagocytic cells, and so extending their circulation times, allowing both passive and active drug Pharmaceutics 2023, 15, 332. https://doi.org/10.3390/pharmaceutics15020332 https://www.mdpi.com/journal/pharmaceutics