Exploring lipid nanoparticles as delivery systems for gene-based therapies: from formulation to in vivo testing.

Gene therapy has emerged to overcome several obstacles posed by complex diseases in which genetic contribution makes treatment difficult. These therapies are based on the use of genes from exogenous material (DNA and RNA) as drugs to correct or modify the expression of those genes that ultimately cause the disease. One approach of gene therapy is based on the delivery of plasmids, which results to be more complicated than the delivery of mRNAs, owing their larger size and to the fact they must reach the nucleus of the cells. To achieve this aim one promising delivery system are lipid nanoparticles (LNPs). These systems are biocompatible due their composition similar to cell membrane’s and by their charges are able to complex drugs, DNA and RNA becoming systems capable to transport and target specific sites (intra or extra cellular). The aim of this study is to develop LNPs formulations capable of conjugating and delivering plasmids by using a microfluidic system. In collaboration with the group of Professor Graziella Messina, University of Milan, we focused on the delivery of a plasmid, pCito_shNfix, able to ameliorate the disease Muscular Dystrophy. Therefore, the target of developed LNPs are the muscle cells. The formulations were prepared with various components, such as the ionizable lipid DLin-KC2-DMA, to enable interaction between the particles and DNA; cholesterol to modify and control the membrane's fluidity; HSPC and DSPC neutral lipids to stabilize the structure of the LNPs; and PEG (DSPE-PEG and DMG-PEG) to provide the system with stealth properties. Additionally, A2G80 peptide was conjugated to LNPs to achieve specific targeting into muscle cells. The method used to prepare LNPs involved a microfluidic approach with the NanoAssemblr Benchtop® system. After preparation, all formulations were characterized in terms of size, PDI (polydispersity index), and zeta potential using DLS (dynamic light scattering). The plasmid quality was evaluated using agarose gel electrophoresis, and the encapsulation efficiency was quantified using a PicoGreen™ assay. An additional analysis that was conducted to observe the morphology of the nanoparticles was visualization using TEM (transmission electron microscopy). Finally, the formulations were tested in vitro on cell lines to evaluate transfection and effects, and in vivo on mice. Throughout the project, various conditions were tested, including different lipidic compositions, PEG variants, pH and concentrations to determine which conditions yielded the best results in vitro and in vivo in terms of efficacy and tolerability. Additionally, lyophilization trials were conducted to evaluate a preservation method that could enhance the long-term stability of the formulations.