In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

Hybrid lipid membranes incorporating amphiphilic copolymers have gained significant attention due to their potential applications in various fields, including drug delivery and sensing. By combining the properties of copolymers and lipid membranes, such as enhanced chemical tunability and stability, environmental responsiveness, and multidomain nature, novel membrane architectures have been proposed. In this study, we investigated the potentialities of hybrid membranes made of two distinct components: the rigid fully saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the soft copolymer poly(butadiene-b-ethyleneoxide) (PBD-b-PEO). The objective was to explore the interaction of citrate-coated gold nanoparticles (AuNPs) and the hybrid membrane, aiming at constructing AuNPs-hybrid vesicles suprastructures with controlled and adjustable plasmonic properties. A series of experimental techniques were employed to investigate hybrid free-standing and supported membranes. The results revealed that the incorporation of the copolymer into the lipid membrane promotes AuNPs clustering, demonstrating a distinctive aggregative phenomenon of citrate-coated AuNPs on multidomain membranes. Importantly, we show that the size and morphology of AuNPs clusters can be precisely controlled in non-homogeneous membranes, enabling the formation of hybrid suprastructures with controlled patch properties. These results highlight the potential of lipid-copolymer hybrid membranes for designing functional materials with tailored plasmonic properties, with potential applications in nanomedicine and sensing.

Lipid liquid crystalline mesophases, resulting from the self-assembly of polymorphic lipids in water, have been widely explored as biocompatible drug delivery systems. In this respect, non-lamellar structures are particularly attractive: they are characterized by complex 3D architectures, with the coexistence of hydrophobic and hydrophilic regions that can conveniently host drugs of different polarities. The fine tunability of the structural parameters is nontrivial, but of paramount relevance, in order to control the diffusive properties of encapsulated active principles and, ultimately, their pharmacokinetics and release. In this work, we investigate the reaction kinetics of p-nitrophenyl phosphate conversion into p-nitrophenol, catalysed by the enzyme Alkaline Phosphatase, upon alternative confinement of the substrate and of the enzyme into liquid crystalline mesophases of phytantriol/H₂O containing variable amounts of an additive, sucrose stearate, able to swell the mesophase. A structural investigation through Small-Angle X-ray Scattering, revealed the possibility to finely control the structure/size of the mesophases with the amount of the included additive. A UV–vis spectroscopy study highlighted that the enzymatic reaction kinetics could be controlled by tuning the structural parameters of the mesophase, opening new perspectives for the exploitation of non-lamellar mesophases for confinement and controlled release of therapeutics.

Protocells, by breaking down the intricate complexity of eukaryotic cells, are innovative tools for mimicking and understanding cellular phenomena. Protocells based on giant unilamellar vesicles (GUVs) supplemented with active species facilitate in situ biologically relevant enzymatic reactions. However, replicating intercellular communication in synthetic systems remains a major challenge for bottom‐up approaches. Here, this challenge is addressed by creating two different populations of protocells that communicate in a controlled and directional manner. By using double emulsion microfluidics, each protocell type consists of GUVs containing distinct biomolecules and artificial organelles (AOs) with unique structures and functions. A series of chemical reaction networks controlling the activity of the AOs provides the framework for the directional, lactate‐mediated intercellular communication between protocells. The coordinated series of compartmentalized enzymatic reactions demonstrate that the intercellular communication is i) selective for lactate, ii) tunable according to its concentration; and iii) sensitive to the sender/receiver protocell ratio and thus, the distance between them. These protocells capable of collective behavior by intercellular signaling have unique advantages for understanding complex biological processes and serve as a basis for the development of advanced therapeutic strategies by interfacing synthetic protocells with native cells.

Protocells offer a versatile material for dissecting cellular processes and developing simplified biomimetic systems by combining biological components with synthetic ones. However, a gap exists between the integrity and complex functionality of native organelles such as nuclei, and bottom‐up strategies reducing cellular functions within a synthetic environment. Here, this gap is bridged by incorporating native nuclei into polymeric giant unilamellar vesicles (pGUVs) using double‐emulsion microfluidics. It is shown that the nuclei retain their morphology and nuclear envelope integrity, facilitating the import of co‐encapsulated peptide‐based multicompartment micelles (MCMs) via nuclear localization signals (NLS). Importantly, it is demonstrated that the nuclear import machinery remains functional inside the protocells, and by enriching the GUV interior with nuclear import‐promoting factors, the delivery efficiency of NLS‐MCMs significantly increases. The findings reveal that nucleated protocells preserve nuclear function and integrity for extended periods, providing a new platform for studying nuclear processes in a simplified, yet biologically relevant, environment. This approach opens avenues for creating advanced biohybrid materials, offering opportunities to investigate organelle behavior and their interactions with cellular components in greater detail. The findings establish a foundation for high‐throughput applications in synthetic biology and contribute valuable insights into sustaining complex cellular functions in engineered systems.