Fluorescence Resonance Energy Transfer in Quantum Dot-Protein Kinase Assemblies

In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and purified the catalytic kinase domain of PDK1 with an N-terminal histidine tag [His6-PDK1(ΔPH)]. We have conjugated this construct to CdSe-ZnS core-shell QDs coated with dihydrolipoic acid (DHLA) and tested the response of the resulting assembly to a molecular dyad incorporating an ATP ligand and a BODIPY chromophore. The supramolecular association of the BODIPY-ATP dyad with the His6-PDK1(ΔPH)-QD assembly encourages the transfer of energy from the QDs to the BODIPY dyes upon excitation. The addition of ATP results in the displacement of BODIPY-ATP from the binding domain of the His6-PDK1(ΔPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking the His6-PDK1(ΔPH), indicates that the BODIPY-ATP dyad adsorbs nonspecifically on the surface of the nanoparticles, promoting the transfer of energy from the CdSe core to the adsorbed BODIPY dyes. Thus, the implementation of FRET-based assays to probe the binding domain of PDK1 with luminescent QDs requires the identification of energy acceptors unable to interact nonspecifically with the surface of the nanoparticles

Emission color tuning and white-light generation based on photochromic control of energy transfer reactions in polymer micelles

We encapsulate a fluorescent donor molecule and a photochromic acceptor unit (photoswitch) in polymer micelles and show that the color of the emitted fluorescence is continuously changed from blue to yellow upon light-induced isomerization of the acceptor. Interestingly, white-light generation is achieved in between. With the photoswitch in the colorless form, intense blue emission from the donor is observed, while UV-induced isomerization to the colored form induces an energy transfer reaction that quenches the donor emission and sensitizes the yellow emission from the colored photoswitch. The process is reversed by exposure to visible light, triggering isomerization to the colorless form

Carrier capability of halloysite nanotubes for the intracellular delivery of antisense PNA targeting mRNA of neuroglobin gene

Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently. Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis.This research received funds from the European Union NextGenerationEU [IR0000010 “ELIXIRxNextGenIT”, PNRR MUR-M4C2—Investimento 3.1, CUP UNINA: B53C22001800006] and [CN00000041 “National Center for Gene Therapy and Drugs based on RNA Technology”, National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4, CUP UNINA: E63C22000940007], EUROSTART (MUR FONDI PNR D.M. 737/2021, CUP UNIPA: B79J21038330001) and PRIN 2022 PNRR “P2022YJZ5F -PE5"

Exploring the cellular uptake of hectorite clay mineral and its drug carrier capabilities

In the last years, the use of clay minerals for pharmaceutical purposes has increased due to their interesting properties. Hectorite (Ht) is a clay belonging to the smectite group which has attracted attention for applications in biology, tissue engineering and as drug carrier and delivery system. However, the mechanisms involved in Ht cellular uptake and transport into cells, are still unclear. Herein, we used a labeled Ht (Ht/1Cl) to study both the cellular uptake, by confocal laser scanning microscopy, and internalization pathways involved in the cellular uptake, by various endocytosis-inhibiting studies and fluorescence microscopy. These studies highlighted that Ht can penetrate the cellular membrane, localizing mainly in the cytoplasm. The main intracellular transport mechanisms are the ATP-dependent ones and those where filaments and microtubules are involved. Finally, as proof of concept for the potential of Ht as carrier system, we envisaged the covalent grafting of the anticancer molecule methotrexate (MTX), chosen as model, to obtain the Ht-MTX nanomaterial. The covalent linkage was confirmed by several techniques and the morphology of the obtained nanomaterial was imaged by SEM and TEM investigations. The kinetic release of the drug from the Ht-MTX nanomaterial in physiological conditions was studied as well. Furthermore, cytotoxic studies on different cell lines, namely, HL-60, HL-60R, MCF-7, 5637, UMUC3 and RT112 showed that Ht could be a promising material for anticancer therapy.The work was financially supported by the University of Palermo. This work was carried out in the frame of the PON ‘‘AIM: Attrazione e Mobilità Internazionale” No. 1808223-1 project. Confocal measurements were performed at ATeN Center – University of Palermo

An all-photonic full color RGB system based on molecular photoswitches

On-command changes in the emission color of functional materials is a sought-after property in many contexts. Of particular interest are systems using light as the external trigger to induce the color changes. Here we report on a tri-component cocktail consisting of a fluorescent donor molecule and two photochromic acceptor molecules encapsulated in polymer micelles and we show that the color of the emitted fluorescence can be continuously changed from blue-to-green and from blue-to-red upon selective light-induced isomerization of the photochromic acceptors to the fluorescent forms. Interestingly, isomerization of both acceptors to different degrees allows for the generation of all emission colors within the red-green-blue (RGB) color system. The function relies on orthogonally controlled FRET reactions between the blue emitting donor and the green and red emitting acceptors, respectively

Computational insights on the isomerization of photochromic oxazines

We investigated the isomerization of the simplest member of a family of photochromic oxazines with the aid of density functional theory, using three different functionals. Specifically, we simulated the thermal interconversion of the two enantiomers, associated with this compound, and established that the opening of the oxazine ring dictates the rate of the overall degenerate process. The M062X functional provides the best match to experimental data, whereas B3LYP calculations fail to model accurately the ground-state potential-energy surface of this system. In addition, we also modeled the absorption spectra of this compound and its photogenerated isomer with time-dependent calculations. The resulting data support the original assignment of the experimental spectra and confirm that the oxazine ring opens upon excitation. The MPW1PW91 functional provides the best match to experimental data, whereas M062X calculations fail to model accurately the spectroscopic parameters of this particular system. Furthermore, the MPW1PW91 calculations demonstrate that the photoinduced opening of the oxazine ring occurs along the potential-energy surface of the first triplet excited state. Indeed, the photoinduced isomerization appears to involve: (1) the initial excitation of one isomer to the second singlet excited state, (2) its thermal relaxation to the first triplet excited state, (3) its ring opening to produce the other isomer, and (4) the thermal relaxation of the product to the ground state. Thus, our calculations provide valuable information on the elementary steps governing the isomerization of this particular photochromic compound in the ground state and upon excitation. These useful mechanistic insights can guide the design of novel members of this family of photoresponsive compounds with specific properties

Photoactivatable synthetic fluorophores

Photoactivatable fluorophores switch from a nonemissive state to an emissive one under irradiation at an activation wavelength and then emit light in the form of fluorescence upon illumination at an excitation wavelength. Such a concatenation of activation and excitation events translates into the possibility of switching fluorescence on within a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence offers the opportunity to monitor dynamic processes in real time as well as to reconstruct images with resolution at the nanometer level. As a result, these photoresponsive molecular switches are becoming invaluable analytical tools to probe the structures and dynamics of a diversity of materials relying on the noninvasive character of fluorescence imaging

Photoactivatable Fluorophores

Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength and then emit after irradiation at an exciting wavelength. The interplay of such activation and excitation events can be exploited to switch fluorescence on in a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence translates into the opportunity to implement imaging and spectroscopic schemes that are not possible with conventional fluorophores. Specifically, photoactivatable fluorophores permit the monitoring of dynamic processes in real time as well as the reconstruction of images with subdiffraction resolution. These promising applications can have a significant impact on the characterization of the structures and functions of biomolecular systems. As a result, strategies to implement mechanisms for fluorescence photoactivation with synthetic fluorophores are particularly valuable. In fact, a number of versatile operating principles have already been identified to activate the fluorescence of numerous members of the main families of synthetic dyes. These methods are based on either the irreversible cleavage of covalent bonds or the reversible opening and closing of rings. This paper overviews the fundamental mechanisms that govern the behavior of these photoresponsive systems, illustrates structural designs for fluorescence photoactivation, and provides representative examples of photoactivatable fluorophores in actions

Photoactivatable Synthetic Dyes for Fluorescence Imaging at the Nanoscale

The transition from conventional to photoactivatable fluorophores can bring the resolution of fluorescence images from the micrometer to the nanometer level. Indeed, fluorescence photoactivation can overcome the limitations that diffraction imposes on the resolution of optical microscopes. Specifically, distinct fluorophores positioned within the same subdiffraction volume can be resolved only if their emissions are activated independently at different intervals of time. Under these conditions, the sequential localization of multiple probes permits the reconstruction of images with a spatial resolution that is otherwise impossible to achieve with conventional fluorophores. The irreversible photolysis of protecting groups or the reversible transformations of photochromic compounds can be employed to control the emission of appropriate fluorescent chromophores and allow the implementation of these ingenious operating principles for superresolution imaging. Such molecular constructs enable the spatiotemporal control that is required to avoid diffraction and can become invaluable analytical tools for the optical visualization of biological specimens and nanostructured materials with unprecedented resolution