Development of innovative tools and strategies for gene delivery purposes

INTRODUCTION: The main challenge of gene delivery is the design of effective and non-cytotoxic non-viral vectors and other tools capable of targeted delivery of nucleic acids (NAs) to intended sites to alter cellular function and/or structure at the molecular level.1 METHODS: Non-viral gene-delivery systems, i.e., cationic lipids (e.g., triazine-based gemini surfactants, aminoglycoside-grafted-calix[arene])2 and polymers (e.g., chitosan-grafted-polyethylenimine (PEI) and aminoglycoside-grafted poly(amidoamine) (PAMAM))3, were synthesized ad-hoc and complexed with NAs (i.e., pDNA or siRNA) at different transfectant nitrogen-to-nucleic acid phosphate (N/P) ratios to give rise to nano- and micro-particles called lipoplexes and polyplexes. The physico-chemical properties of such assemblies (DH and ζP) were evaluated by means of Dynamic Light Scattering (DLS) and fluorophore titration exclusion assay. In vitro cell transfection studies were performed on different cell types (e.g., HeLa, U87-MG, HEK 293 GFP+, primary chondrocytes) to assess the cytotoxicity and transfection efficiency (TE). Molecular Dynamics (MD) simulations, fluorescence lifetime (FLS) and infrared (IR) spectroscopy were used to shed light on the interaction at the very molecular level between transfectants and NAs and evaluate the stability of the resulting complexes. In order to speed up the optimization process of transfectants, Lab-on-Chip (LoC) platforms were designed.4 Besides, a novel in vitro transfection strategy relying on the combined use of a physical method (vibrational loading to cells to induce a transient membrane poration) and non-viral gene-delivery vectors was envisioned as well. RESULTS: We propose an integrated approach to face the problem of low transfection efficiency (TE) and (relatively) high cytotoxicity of currently available transfectants. On one hand, we developed novel, more effective gene delivery vectors and strategies. On the other hand, by tuning complexation conditions of transfection agents with NAs (e.g., by varying pH and ionic strength of buffers, temperature, order of addition of components during complexation), we were able to shape gene-carrying assemblies of different DH (from 100 nm to few μm), and ζP (over a wide range of +mV), displaying the same chemical composition but differing in physico-chemical features, and thus in their TE. Aminoglycoside-grafted-transfectants-based vectors were found to be effective antimicrobial agents as well. MD simulations, together with FLS and IR evaluations of complexes, allowed to screen and optimize pre- and post-synthesis the TE of gene delivery vectors. LoC platforms were found to be reliable tools for the unbiased, straightforward, and quantitative assessment of transfection efficiency and cytotoxicity at once. Besides, high-frequency-vibrational stimulation (from 100 Hz onward) was shown to dramatically enhance the TE of complexes, leading to ≈50-100-fold increase in transgene expression. DISCUSSION & CONCLUSIONS: Herein, we summarizes some of the main gene delivery strategies that we have that we have focused on in the last decade. Our multidisciplinary approach at the crossroad of physics, materials science, bioengineering and biology has been paving the way towards the rational design and the development of innovative tools and strategies for gene delivery and antibacterial purposes