Silvia Franzè

Purpose: Lipopolyplexes (LPP), i.e. hybrid ternary complexes of cationic polymers, nucleic acids and liposomes, represent a second-generation non-viral vector aiming to overcome the limitations of the first-generation polyplexes and lipoplexes like in vivo toxicity and ineffective transfection efficiency. Although their potential has already been proven in vitro and in vivo, lipopolyplexes are still poorly explored as gene delivery systems. Here, we provid evidence of the effect of lipopolyplexes composition on their physicochemical features, cytotoxicity, and biological activity (i.e. cell uptake, endosomal escape, and transfection efficiency). Methods: Lipopolyplexes were prepared by either bulk mixing or a two-step microfluidic process consisting of i) the formation of polyplexes by complexing a plasmid DNA encoding the green fluorescence protein with a panel of cationic polymers (either chitosan, poly-L-lysine (PLL) or polyethyleneimine (PEI)) followed by ii) the formation of the ternary complex by mixing polyplexes with neutral liposomes. The optimal polymer/DNA/lipid Nitrogen/Phosphate ratios and microfluidic operating parameters (volume ratio and total flow rate (TFR) were preliminarily defined to obtain lipopolyplexes with desired properties. Results: The optimized conditions led to obtain lipopolyplexes with a mean diameter of ~180 nm, a PDI < 0.2 and a slightly positive or neutral z-potential. FRET, SAXS and Cryo-EM analyses demonstrated the formation of a ternary complex in which the type of polymer dictated particles’ structure. Lipopolyplexes displayed negligible toxicity in vitro, while promoting higher protein expression compared to the corresponding polyplexes and control 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) lipoplexes. Moreover, despite the three lipopolyplexes displaying similar uptake kinetics, those made of PEI showed the highest endosomolytic activity and promoted the most effective DNA transfection. Conclusion: Overall, this study demonstrates that lipopolyplexes are a valid platform for pDNA delivery, with PEI lipopolyplexes being the best performing formulation, and that the type of cationic polymer plays a major role in the nanoparticles intercellular trafficking.

New and innovative nanomedicines have been developed and marketed over the past half‐century, revolutionizing the prognosis of many human diseases. Although a univocal regulatory definition is not yet available worldwide, the term “nanomedicines” generally identifies medicinal products that use nanotechnology in their design or production. Due to the intrinsic high structural complexity of these products, the scientific and regulatory communities are reflecting on how to revise the regulatory framework to provide a more appropriate benefit/risk balance to authorize them on the market, considering the impact of their peculiar physicochemical features in the evaluation of efficacy and safety patterns. Herein, a critical perspective is provided on the current open issues regarding regulatory qualification and physicochemical characterization of nanosystems considering the current European regulatory framework on nanomedicine products. Practicable paths for improving their quality assurance and predicting their fate in vivo are also argued. Strengthening the multilevel alliance among academic institutions, industrial stakeholders, and regulatory authorities seems strategic to support innovation by standard approaches (e.g., qualification, characterization, risk assessment), and to expand current knowledge, also benefiting from the new opportunities offered by artificial intelligence and digitization in predictive modelling of the impact of nanomedicine characteristics on their fate in vivo.

Deformable liposomes represent valuable drug carriersfor cutaneousadministration. Nevertheless, the fluid lipid membrane can favor thedrug leakage during storage. Proliposomes may represent a suitablestrategy to solve this issue. As an alternative, a novel carrier,which encloses hydrophobic drugs in the inner core of vesicles, namely,a drug-in-micelles-in-liposome system (DiMiL), has been proposed.In this work, we investigated the possible advantages of combiningthese two approaches to obtain a formulation able to enhance the skinpenetration of cannabidiol (CBD). Proliposomes were prepared by spray-dryingor slurry method testing lactose, sucrose, and trehalose as carriersat different sugar/lipid weight ratios. The ratio between soy-phosphatidylcholine(main lipid) and Tween 80 was instead fixed at 85:15 w/w. DiMiL systemswere extemporaneously obtained by the hydration of proliposomes witha Kolliphor HS 15 micellar dispersion (containing CBD, when appropriate).Based on the technological properties, sucrose and trehalose at 2:1sugar/lipid ratio resulted in the best carriers for spray-dried and"slurried" proliposomes, respectively. Cryo-EM imagesclearly showed the presence of micelles in the aqueous core of lipidvesicles and the presence of sugars did not alter the structural organizationof DiMiL systems, as demonstrated by SAXS analyses. All formulationswere highly deformable and able to control CBD release regardlessof the presence of sugar. The permeation through human epidermis ofCBD carried by DiMiL systems was significantly improved comparedto that obtained loading the drug in conventional deformable liposomeswith the same lipid composition or in an oil solution. Furthermore,the presence of trehalose led to a further slight increase of theflux. Altogether, these results demonstrated that proliposomes maybe a valuable intermediate for the preparation of deformable liposome-basedcutaneous dosage forms, improving the stability without compromisingthe overall performances.

Psoriasis is a lifelong disease which requires treatment adherence for successful management. Considering the complexity of this pathology, the combination of active pharmaceutical ingredients with a synergistic mechanism of action can improve the safety and efficacy of the treatment with respect to the conventional monotherapy. Moreover, a fixed dose of therapeutic agents in a topical formulation offers the possibility to simplify administration, reduce the doses of each active ingredient, and improve patient’s compliance. Among the first-line treatments in mild to moderate psoriasis, the formulation of calcipotriol (Cal) and betamethasone dipropionate (BD) in a single vehicle is challenging due to their chemical incompatibility in an aqueous environment and the formation of degradation products. Based on these considerations, this review aims to provide an overview on the biopharmaceutical properties of Cal/BD fixed-dose combination products available on the market (namely ointment, oleogel, foam, and O/W cream), highlighting also the novel approaches under evaluation. The main differences among topical formulations are discussed considering the different features of the anatomic districts involved in psoriasis and the patient’s adherence. Moreover, since in vitro experiments are fundamental to evaluate the skin permeation profile during the development of an efficacious medicinal product, special emphasis is given to models proposed to mimic psoriatic lesions.

The administration of drug fixed combinations by nanocarriers is a new attractive approach since it can allow improvements in both the skin penetration of cargo compounds and their synergistic effects. The cutaneous administration of lidocaine (LD) and cannabidiol (CBD) combination can be useful for the local treatment of neuropathic pain. In fact, these drugs might exert a complementary effect on pain acting on sodium and calcium channels. In this study, the feasibility to deliver this combination in the deeper layers of the skin using deformable liposomes was studied. Based on a study of the drug affinity for lipid components performed by DSC, CBD was loaded in the lipid bilayer for limiting the leakage, while LD was loaded in the inner core by a pH gradient method (G-liposomes) or after previous encapsulation in micelle (DiMiL). The effect of the presence of Tween 80 in the liposome membrane was also evaluated. DiMiL increased both the skin permeation and the retention in the dermis of CBD and LD with respect to G-liposomes (R_24dermis: 11.52 ± 2.4 against 4.51 ± 0.8 µg/cm² for CBD; 19.6 ± 2.9 against 3.2 ± 0.1 µg/cm² for LD). Moreover, both DiMiL and G-liposomes were more efficient than control formulations carrying free drugs in improving drug skin permeation. Interestingly, in the presence of a drug exerting a fluidizing effect such as CBD, the removal of Tween 80 from the composition led to an improved control of drug release and a higher extent of drug retention in the dermis layer.

Pyridobenzothiazolone derivatives are a promising class of broad-spectrum antivirals. However, the mode of action of these compounds remains poorly understood. The HeE1-17Y derivative has already been shown to be a potent compound against a variety of flaviviruses of global relevance. In this work, the mode of action of HeE1-17Y has been studied for West Nile virus taking advantage of reporter replication particles (RRPs). Viral infectivity was drastically reduced by incubating the compound with the virus before infection, thus suggesting a direct interaction with the viral particles. Indeed, RRPs incubated with the inhibitor appeared to be severely compromised in electron microscopy analysis. HeE1-17Y is active against other enveloped viruses, including SARS-CoV-2, but not against two non-enveloped viruses, suggesting a virucidal mechanism that involves the alteration of the viral membrane.

Moving towards a real mass vaccination in the context of COVID-19, healthcare professionals are required to face some criticisms due to limited data on the stability of a mRNA-based vaccine (Pfizer-BioNTech COVID-19 Vaccine in the US or Comirnaty in EU) as a dose in a 1 mL-syringe. The stability of the lipid nanoparticles and the encapsulated mRNA was evaluated in a “real-life” scenario. Specifically, we investigated the effects of different storing materials (e.g., syringes vs. glass vials), as well as of temperature and mechanical stress on nucleic acid integrity, number, and particle size distribution of lipid nanoparticles. After 5 h in the syringe, lipid nanoparticles maintained the regular round shape, and the hydrodynamic diameter ranged between 80 and 100 nm with a relatively narrow polydispersity (<0.2). Samples were stable independently of syringe materials and storage conditions. Only strong mechanical stress (e.g., shaking) caused massive aggregation of lipid nanoparticles and mRNA degradation. These proof-of-concept experiments support the hypothesis that vaccine doses can be safely prepared in a dedicated area using an aseptic technique and transferred without affecting their stability.

The possible application of a hot-melt ram extrusion printing to the preparation of diclofenac orodispersible films (ODF) made of maltodextrin was studied focusing the attention on the effects of t...