Ferritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe²⁺-loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe²⁺ uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe²⁺ through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H′ protein and some of its variants as case studies. We describe the structural features that determine Fe²⁺ translocation with atomistic detail, and we propose a putative mechanism for Fe²⁺ transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin.

Among the plethora of recently proposed molecular sensors, those belonging to the class of fluorescent molecular rotors (FMRs) have attracted much attention owing to their peculiar photophysical properties that enable an unprecedented sensitivity towards environmental microviscosity. The usual FMR synthetic design prescribes chromophores characterized by an intramolecular rotation between two well-defined excited states, a locally excited state and a twisted internal charge transfer (TICT) state, where the sensing capabilities arise from a dual competition of the corresponding radiative/non-radiative decay processes. However, we have recently demonstrated a different modus operandi of a new subclass of solvatochromic FMRs, which exploit a solvent-independent, barrier-free intramolecular rotation of the excited dye. The rotational dynamics is modulated by local viscosity and, in turn, manifested through a variable spectral signal. In order to translate the same rotational mechanism in a versatile sensor of polarity and viscosity, we designed and thoroughly characterized a novel FMR, namely 4-(triphenylamino)-phthalonitrile (TPAP). Remarkably, in addition to a high sensitivity versus solvent polarity and viscosity, TPAP is also able to form stable fluorescent nanoparticles characterized by aggregation-induced emission, via a simple sonochemical treatment. Such peculiar features are tested in different applications aiming at illustrating its capability to report on solvatochromic and vapochromic effects, as well as to provide detailed intracellular information through bioimaging studies.

Lipid lateral diffusion in membrane bilayers is a fundamental process exploited by cells to enable complex protein structural and dynamic reorganizations. For its importance, lipid mobility in both cellular and model bilayers has been extensively investigated in recent years, especially through the application of time-resolved, fluorescence-based, optical microscopy techniques. However, one caveat of fluorescence techniques is the need to use dye-labeled variants of the lipid of interest, thus potentially perturbing the structural and dynamic properties of the native species. Generally, the effect of the dye/tracer molecule is implicitly assumed to be negligible. Nevertheless, in view of the widespread use of optically modified lipids for studying lipid bilayer dynamics, it is highly desirable to well assess this point. Here, fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations have been combined together to uncover subtle structural and dynamic effects in DOPC planar membranes enriched with a standard Rhodamine-labeled lipid. Our findings support a non-neutral role of the dye-labeled lipids in diffusion experiments, quantitatively estimating a decrease in lipid mobility of up to 20% with respect to the unlabeled species. Moreover, results highlight the existing interplay between dye concentration, lipid lateral diffusion and membrane permeability, thus suggesting possible implications for future optical microscopy studies of biophysical processes occurring at the membrane level.

A prototype for a new class of fluorescent molecular rotors (FMRs), namely 4-(diphenylamino)phthalonitrile (DPAP), was synthesized and its sensitivity towards solvent polarity and viscosity probed using photophysical and computational methods. DPAP is characterized by a pronounced fluorosolvatochromism in polarity-dependent absorption, emission and fluorescent lifetime experiments. At the same time, a strong viscosity response is observed, especially in polar and protic solvents. Quantum mechanical calculations assisted in interpreting the unusual solvent sensitivity of DPAP in terms of its high flexibility, giving rise to solvent-independent, barrier-free rotations. As a matter of fact, the modus operandi in DPAP contrasts that of traditional FMRs involving twisted intramolecular charge transfer (TICT) states. The influence of this unusual flexible character on excitation and emission energies was studied using computational methods upon considering twisting of the molecule in solvents of different polarity. Furthermore, a detailed characterization of the excited state profile was attained using time resolved spectroscopy techniques. In particular, a contrasting deactivation pattern of the intramolecular charge transfer (ICT) state was observed in low and high polar media. Moreover, in low and medium polar solvents strong emission is accompanied by triplet excited state formation, while in high polar and protic solvents the ICT state is highly stabilized and decays primarily non-radiatively. Notably, a viscosity increase in the latter solvents hampers rotations leading to a strong emission enhancement. This latter behavior, coupled to a remarkable solvatochromic character, makes DPAP a promising probe for biological and environmental sensing and imaging applications.

The unprecedented ultrasound-induced transformation of fluorescent organic nanoparticles based on a molecular rotor into rhomboidal nanocrystals with enhanced stability and increased emission is presented.