Design, characterization and optimization of nanostructured vectors for the delivery of DNA and siRNA

Gene therapy represents a highly promising approach for the treatment of severe and currently untreatable diseases. It relies on the introduction of genetic material into target cells for restoring, replacing, or silencing specific genes responsible for pathological conditions [1]. Among the available strategies, RNA interference (RNAi) has emerged as a powerful post-transcriptional gene regulation mechanism mediated by siRNAs [2,3]. Despite remarkable therapeutic potential, the clinical application of siRNAs remains limited due to their instability in biological fluids, susceptibility to nuclease degradation, and poor cellular uptake. Consequently, the development of safe and efficient delivery systems is a key requirement for the successful translation of gene-based therapeutics into clinical practice [1-4]. In this work, we developed and optimized two different nanocarrier systems for gene delivery: Lipid Nanoparticles (LNPs) and β-Cyclodextrin Nanovectors (β-CD NVs). LNPs represent one of the most advanced delivery platforms, offering efficient protection for siRNA molecules with respect to enzymatic degradation and facilitating their intracellular delivery mainly through electrostatic interactions [5-7]. In this study, LNPs were prepared using the Thin Layer Evaporation (TLE) method and characterized for particle size (size), polydispersity index (PdI), and surface charge (Z) by Dynamic Light Scattering (DLS). The objective was to obtain NPs suitable for siRNA complexation and delivery, with size below 200 nm and PdI < 0.3 [4,6]. A Design of Experiments (DoE) approach was applied to optimize critical parameters for LNPs production, identifying optimal conditions at Dioleoylphosphatidylethanolamine (DOPE) = 35%, four sonication cycles, and no extrusion step [5,12]. Based on these optimized parameters, additional cationic lipids were incorporated, and the resulting LNPs were complexed with either negative control or GFP-targeting siRNA at an N/P (+/-) ratio of 10. N/P ratio represents the molar ratio between the nitrogen atoms in the cationic components and the phosphate groups of the siRNA backbone [13]. Complexation efficiency was verified by agarose gel electrophoresis, which identified two lead formulations (designated B and D) for further study. Both formulations exhibited good stability after seven days of storage at 4 ± 1 °C [6,14]. The biological performance of these systems was evaluated in vitro, assessing their stability in culture medium (with and without serum), cytotoxicity toward HeLa cells, internalization in HeLa-GFP and RAW 264.7 cells, and gene silencing efficiency in HeLa-GFP cells. High resolution structural characterization was performed by Synchrotron Small-Angle X-ray Scattering (SAXS). Both LNP formulations displayed sizes below 200 nm with PdI ≤ 0.3, efficient siRNA complexation, high internalization efficiency in HeLa cells (> 85%), and good GFP silencing after 72 h (~50% reduction for formulation B and ~30% for formulation D). These findings highlight the promising potential of the optimized LNPs as effective carriers for siRNA delivery [6,9,15-17]. β-Cyclodextrins are cyclic oligosaccharides known for their biocompatibility, chemical versatility, and ability to self-assemble into nanostructures, making them attractive candidates for non-viral gene delivery [8-11]. In this work, three novel cationic β-CD derivatives (CD1, CD2, and CD3), synthesized by CarboHyde Zrt, were evaluated as NVs for nucleic acid delivery. These compounds were initially characterized by Differential Scanning Calorimetry (DSC) and Infrared Spectroscopy (IR) to investigate their structural features and degree of crystallinity or amorphousness [19]. Dispersibility studies in various solvents were then conducted to determine the critical micellar concentration (CMC) and identify the optimal formulation and concentration range for NVs production. The resulting β-CD NVs were complexed with a GFP-encoding plasmid (P) at an N/P ratio of 10, and their size, PdI, and Z were measured to assess formulation quality. Selected formulations were further modified with Sodium Hyaluronate (HA) or Polysialic Acid (HP) (10% w/w) to evaluate the impact of these polymers on safety and stability. Comprehensive structural characterization of β-CD NVs was performed by Transmission Electron Microscopy (TEM), Cryogenic Electron Microscopy (Cryo-EM), and Synchrotron SAXS, while agarose gel electrophoresis confirmed efficient plasmid complexation. The physical stability of the NVs was monitored at 4 °C and 25 °C (± 1 °C) for up to one week and in culture medium for 24 h [8-11,15]. Cytotoxicity was assessed via MTT assays in HeLa and RAW 264.7 cell lines, while cellular uptake and transfection efficiency were quantified by flow cytometry and fluorescence microscopy. Stable NVs were obtained for CD1 and CD2, exhibiting particle sizes below 200 nm and PdI < 0.3. Both these formulations were stable over time and in serum-supplemented medium. Internalization efficiency exceeded 85% across all tested conditions, and transfection efficiency surpassed 50%, with CD1 showing higher GFP fluorescence intensity compared to CD2-based NVs [20]. The β-CD NVs were also complexed with GFP-siRNA at an N/P ratio of 10, which led to a moderate increase in size and PdI, except for the formulation containing HA. Gene silencing efficiency in HeLa-GFP cells was comparable to that of commercial Lipofectamine for CD1 NVs (around 90%), while CD2 NVs exhibited good transfection performance. These results underscore the potential of CD1 NVs as effective, biocompatible, and versatile non-viral carriers for gene delivery applications.