Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nano‐delivery system for therapeutic nucleic acids. The great effort put in the development of ionizable lipids with increased in vivo potency brought LNPs from the laboratory benches to the FDA approval of patisiran in 2018 and to the ongoing clinical trials for mRNA‐based vaccines against SARS‐CoV‐2. Despite these success stories, several challenges remain in RNA delivery, including what is known as “endosomal escape”. Reaching the cytosol is mandatory for unleashing the therapeutic activity of RNA molecules, as their accumulation in other intracellular compartments would simply result in efficacy loss. In LNPs, the ability of ionizable lipids to form destabilizing non‐bilayer structures at acidic pH is recognized as the key for endosomal escape and RNA cytosolic delivery. This is motivating a surge in studies aiming at designing novel ionizable lipids with improved biodegradation and safety profiles. In this work, we describe the journey of RNA‐loaded LNPs across multiple intracellular barriers, from the extracellular space to the cytosol. In silico molecular dynamics modeling, in vitro high‐resolution microscopy analyses and in vivo imaging data are systematically reviewed to distill out the regulating mechanisms underlying the endosomal escape of RNA. Lastly, a comparison with strategies employed by enveloped viruses to deliver their genetic material into cells is also presented. The combination of a multidisciplinary analytical toolkit for endosomal escape quantification and a nature‐inspired design could foster the development of future LNPs with improved cytosolic delivery of nucleic acids.

Microfluidic mixing is recognized as a convenient method to produce liposomes for its scalability and reproducibility. Numerous studies have described the effect of process parameters such as flow rate ratios and total flow rate on size and size distribution of vesicles. In this work, we focused our attention on the effect of flow rate ratios on the encapsulation efficiency of liposomes, as we hypothesized that different amount of residual organic solvent could affect the retention of lipophilic drug molecules within the bilayer. In a further step, we investigated how the liposomes integrity and loading were impacted by different methods of solvent removal: direct dialysis and dilution & dialysis. Liposomes were prepared by rapidly mixing an ethanolic solution of lipids and a model drug with buffer in a herringbone micromixer, employing four different flow rate ratios (FRR, 4:1, 7:3, 3:2, 1:1). Quercetin, resveratrol and ascorbyl palmitate were used as model antioxidant drugs with different lipophilicity. Data showed that liposomes produced using lower flow rate ratios (i.e., with more residual ethanol) had lower encapsulation efficiencies as well as a more prominent loss of lipids from the bilayer following purification with direct dialysis. If the amount of residual ethanol was reduced to 5% (dilution & dialysis method), the lipids and drug leakage was prevented. Such effect was correlated with the drug aggregation propensity in different ethanol/water mixtures measured by molecular dynamics simulations. Overall, these results highlight the need to tailor the purification method basing on the molecular properties of the loaded drug to ensure high encapsulation and limit the waste of material.

Topical application of the anti-inflammatory drug diclofenac (DCF) reduces the severity of systemic unwanted effects compared to its oral administration. A number of transdermal formulations are available on the market and routinely used in clinical and home-care settings. However, the amount of DCF delivered across the skin remains limited and often insufficient, thus making the oral route still necessary for achieving sufficient drug concentration at the inflamed site. In attempting to improve the transdermal penetration, we explored the combined use of DCF nanosuspensions with a microneedle roller. Firstly, DCF nanosuspensions were prepared by a top-down media milling method and characterized by spectroscopic, thermal and electron microscopy analyses. Secondly, the pore-forming action of microneedle rollers on skin specimens (ex vivo) was described by imaging at different scales. Finally, DCF nanosuspensions were applied on newborn pig skin (in vitro) in combination with microneedles roller treatment, assessing the DCF penetration and distribution in the different skin layers. The relative contribution of microneedle length, nanosuspension stabilizer and application sequence could be identified by systemically varying these parameters.