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.

Photoactivatable Fluorophores for Bioimaging Applications.

Photoactivatable fluorophores provide the opportunity to switch fluorescence on exclusively in a selected area within a sample of interest at a precise interval of time. Such a level of spatiotemporal fluorescence control enables the implementation of imaging schemes to monitor dynamic events in real time and visualize structural features with nanometer resolution. These transformative imaging methods are contributing fundamental insights on diverse cellular processes with profound implications in biology and medicine. Current photoactivatable fluorophores, however, become emissive only after the activation event, preventing the acquisition of fluorescence images and, hence, the visualization of the sample prior to activation. We developed a family of photoactivatable fluorophores capable of interconverting between emissive states with spectrally resolved fluorescence, instead of switching from a nonemissive state to an emissive one. We demonstrated that our compounds allow the real-time monitoring of molecules diffusing across the cellular blastoderm of developing embryos as well as of polymer beads translocating along the intestinal tract of live nematodes. Additionally, they also permit the tracking of single molecules in the lysosomal compartments of live cells and the visualization of these organelles with nanometer resolution. Indeed, our photoactivatable fluorophores may evolve into invaluable analytical tools for the investigation of the fundamental factors regulating the functions and structures of cells at the molecular level.

Spectroscopic single-molecule localization microscopy: applications and prospective.

Single-molecule localization microscopy (SMLM) breaks the optical diffraction limit by numerically localizing sparse fluorescence emitters to achieve super-resolution imaging. Spectroscopic SMLM or sSMLM further allows simultaneous spectroscopy and super-resolution imaging of fluorescence molecules. Hence, sSMLM can extract spectral features with single-molecule sensitivity, higher precision, and higher multiplexity than traditional multicolor microscopy modalities. These new capabilities enabled advanced multiplexed and functional cellular imaging applications. While sSMLM suffers from reduced spatial precision compared to conventional SMLM due to splitting photons to form spatial and spectral images, several methods have been reported to mitigate these weaknesses through innovative optical design and image processing techniques. This review summarizes the recent progress in sSMLM, its applications, and our perspective on future work.

Photoactivatable BODIPYs for Live-Cell PALM.

Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications.

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.

Nanocarrier based on halloysite and fluorescent probe for intracellular delivery of peptide nucleic acids.

The development of systems able to deliver genetic material into a target site is a challenge for modern medicine. Single-stranded peptide nucleic acids have attracted attention as promising therapeutic molecules for diagnostic and gene therapy. However, their poor cell membrane permeability represents a drawback for biomedical applications. Halloysite nanotubes (HNTs) are emerging materials in drug delivery applications both for their ability to penetrate cell membranes and for enhancing the solubility of drugs in biological media. Herein, we report the first example of the use of a nanocarrier based on halloysite labelled with fluorescent switchable halochromic oxazine molecules, to deliver a single-stranded peptide nucleic acids tetramer (PNAts) into living cells. The PNAts is covalently attached to halloysite (HNTs-PNA), whereas the fluorescent probe supramolecularly interacts with HNTs. The ability of the nanomaterial to bind complementary single-stranded DNA was assessed by resonance light scattering measurements. Finally, studies of cellular uptake were carried out by confocal laser scanning microscopy on normal and tumoral cell lines. This work highlights the usefulness of the covalent approach to generate HNTs-PNA nanomaterials for the potential targeting of future specific nucleic acids in living cells, which could open the doorway to novel possibilities for theranostic and gene therapy applications.

Fluorescence Switching for Temperature Sensing in Water.

A water-soluble thermochromic molecular switch with spectrally resolved fluorescence in its two interconvertible states can be assembled in three synthetic steps by integrating a fluorescent coumarin chromophore, a hydrophilic oligo(ethylene glycol) chain, and a switchable oxazole heterocycle in the same covalent skeleton. Measurements of its two emissions in separate detection channels of a fluorescence microscope permit the noninvasive and ratiometric sensing of temperature at the micrometer level with millisecond response in aqueous solutions and within hydrogel matrices. The ratiometric optical output of this fluorescent molecular switch overcomes the limitations of single-wavelength fluorescent probes and enables noninvasive temperature mapping at length scales that are not accessible to conventional thermometers based on physical contact.

BODIPYs with Photoactivatable Fluorescence.

The borondipyrromethene (BODIPY) chromophore is a versatile platform for the construction of photoresponsive dyes with unique properties. Specifically, its covalent connection to a photocleavable group can be exploited to engineer compounds with photoswitchable fluorescence. The resulting photoactivatable fluorophores can increase their emission intensity or shift their emission wavelengths in response to switching. Such changes permit the spatiotemporal control of fluorescence with optical stimulations and the implementation of imaging strategies that would be impossible to replicate with conventional fluorophores. Indeed, BODIPYs with photoactivatable fluorescence enable the selective highlighting of intracellular targets, the nanoscaled visualization of sub-cellular components, the real-time monitoring of dynamic events and the photochemical writing of optical barcodes.

Far-red photoactivatable BODIPYs for the super-resolution imaging of live cells.

The identification of viable designs to construct switchable fluorescent probes and operate them in the interior of live cells is essential to allow the acquisition of SMLM images and permit the visualization of cellular components with sub-diffraction resolution. Our laboratories developed a mechanism to switch the fluorescence of BODIPY chromophores with the photoinduced cleavage of oxazine heterocycles under mild 405-nm illumination. With appropriate structural modifications, these switchable molecules can be engineered to immobilize covalently on large biomolecules within lysosomal compartments of live COS-7 cells and produce bright far-red fluorescence with optimal contrast upon activation. Such a combination of properties permits the acquisition of PALM images of the labeled organelles with localization precision of ca. 15nm. This article reports the experimental protocols for the synthesis of and live-cell labeling with these compounds as well as for the reconstruction of super-resolution images of the resulting biological preparations.

Photoactivatable fluorophores for single-molecule localization microscopy of live cells.

Photochemical reactions can be designed to convert either irreversibly or reversibly a nonemissive reactant into an emissive product. The irreversible disconnection of a photocleavable group from an emissive chromophore or the reversible interconversion of a photochromic component is generally exploited to implement these operating principles for fluorescence switching. In both instances, the interplay of activating radiation, to convert the nonemissive state into the emissive species, and exciting radiation, to produce fluorescence from the latter, can be exploited to switch fluorescence on in a given area of interest at a precise interval of time. Such a level of spatiotemporal control provides the opportunity to reconstruct sub-diffraction images with resolution at the nanometer level. Indeed, closely-spaced emitters can be switched on under photochemical control at distinct intervals of time and localized independently at the single-molecule level. In combination with appropriate intracellular targeting strategies, some of these photoactivatable fluorophores can be switched and localized inside live cells to permit the visualization of sub-cellular structures with a spatial resolution that would be impossible to achieve with conventional fluorophores. As a result, photoactivatable fluorophores can become invaluable probes for the implementation of super-resolution imaging schemes aimed at the elucidation of the fundamental factors controlling cellular functions at the molecular level.

Compact, "Clickable" Quantum Dots Photoligated with Multifunctional Zwitterionic Polymers for Immunofluorescence and In Vivo Imaging.

We detail the preparation of highly fluorescent quantum dots (QDs), surface-engineered with multifunctional polymer ligands that are compact and readily compatible with strain-promoted click conjugation, and the use of these nanocrystals in immunofluorescence and in vivo imaging. The ligand design combines the benefits of mixed coordination (i.e., thiol and imidazole) with zwitterion motifs, yielding sterically-stabilized QDs that present a controllable number of azide groups, for easy conjugation to biomolecules via the selective click chemistry. The polymer coating was characterized using NMR spectroscopy to extract estimates of the diffusion coefficient, hydrodynamic size, and ligand density. The azide-functionalized QDs were conjugated to anti-tropomyosin receptor kinase B antibody (α-TrkB) or to the brain-derived neurotrophic factor (BDNF). These conjugates were highly effective for labeling the tropomyosin receptor kinase B (TrkB) in pyramidal neurons within cortical tissue and for monitoring the BDNF induced activation of TrkB signaling in live neuronal cells. Finally, the polymer-coated QDs were applied for in vivo imaging of Drosophila melanogaster embryos, where the QDs remained highly fluorescent and colloidally stable, with no measurable cytotoxicity. These materials would be of great use in various imaging applications, where a small size, ease of conjugation, and great colloidal stability for in vivo studies are needed.

Live-Cell Imaging at the Nanoscale with Bioconjugatable and Photoactivatable Fluorophores.

Optical diffraction fundamentally limits the spatial resolution of conventional fluorescence images to length scales that are, at least, 2 orders of magnitude longer than the dimensions of individual molecules. As a result, the development of innovative probes and imaging schemes to overcome diffraction is very much needed to enable the investigation of the fundamental factors regulating cellular functions at the molecular level. In this context, the chemical synthesis of molecular constructs with photoactivatable fluorescence and the ability to label subcellular components of live cells can have transformative implications. Indeed, the fluorescence of the resulting assemblies can be activated with spatiotemporal control, even in the intracellular environment, to permit the sequential localization of individual emissive labels with precision at the nanometer level and the gradual reconstruction of images with subdiffraction resolution. The implementation of these operating principles for subdiffraction imaging, however, is only possible if demanding photochemical and photophysical requirements to enable photoactivation and localization as well as stringent structural requisites to allow the covalent labeling of intracellular targets in live cells are satisfied. Because of these complications, only a few synthetic photoactivatable fluorophores with appropriate performance for live-cell imaging at the nanoscale have been developed so far. Significant synthetic efforts in conjunction with spectroscopic analyses are still very much needed to advance this promising research area further and turn photoactivatable fluorophores into the imaging probes of choice for the investigation of live cells.

High-Throughput Single-Molecule Spectroscopy Resolves the Conformational Isomers of BODIPY Chromophores.

A borondipyrromethene (BODIPY) chromophore is connected to a benzoxazole, benzothiazole, or nitrobenzothiazole heterocycle through an olefinic bridge with trans configuration. Rotation about the two [C-C] bonds flanking the olefinic bridge occurs with fast kinetics in solution, leading to the equilibration of four conformational isomers for each compound. Ensemble spectroscopic measurements in solutions fail to distinguish the coexisting isomers. They reveal instead averaged absorption and emission bands with dependence of the latter on the excitation wavelength. Using high-throughput single-molecule spectroscopy, two main populations of single molecules with distinct spectral centroids are observed for each compound on glass substrates. Computational analyses suggest the two populations of molecules to be conformational isomers with antiperiplanar and periplanar arrangements of the BODIPY chromophores about its [C-C] bond to the olefinic bridge. Thus, statistical analysis of multiple single-molecule emission spectra can discriminate stereoisomers that would otherwise be impossible to distinguish by ensemble measurements alone.

Photopotentiation of the GABAA receptor with caged diazepam.

As the inhibitory γ-aminobutyric acid-ergic (GABAergic) transmission has a pivotal role in the central nervous system (CNS) and defective forms of its synapses are associated with serious neurological disorders, numerous versions of caged GABA and, more recently, photoswitchable ligands have been developed to investigate such transmission. While the complementary nature of these probes is evident, the mechanisms by which the GABA receptors can be photocontrolled have not been fully exploited. In fact, the ultimate need for specificity is critical for the proper synaptic exploration. No caged allosteric modulators of the GABAA receptor have been reported so far; to introduce such an investigational approach, we exploited the structural motifs of the benzodiazepinic scaffold to develop a photocaged version of diazepam (CD) that was tested on basolateral amygdala (BLa) pyramidal cells in mouse brain slices. CD is devoid of any intrinsic activity toward the GABAA receptor before irradiation. Importantly, CD is a photoreleasable GABAA receptor-positive allosteric modulator that offers a different probing mechanism compared to caged GABA and photoswitchable ligands. CD potentiates the inhibitory signaling by prolonging the decay time of postsynaptic GABAergic currents upon photoactivation. Additionally, no effect on presynaptic GABA release was recorded. We developed a photochemical technology to individually study the GABAA receptor, which specifically expands the toolbox available to study GABAergic synapses.

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.

Ratiometric temperature sensing with fluorescent thermochromic switches.

The connection of fluorescent chromophores to switchable heterocycles translates into molecular probes with ratiometric response to temperature. The opening and closing of their heterocyclic component equilibrates two emissive species with resolved fluorescence. Their relative emission intensities change monotonically with temperature to enable the visualization of thermal distributions at the microscale.

Far-Red Photoactivatable BODIPYs for the Super-Resolution Imaging of Live Cells.

The photoinduced disconnection of an oxazine heterocycle from a borondipyrromethene (BODIPY) chromophore activates bright far-red fluorescence. The high brightness of the product and the lack of autofluorescence in this spectral region allow its detection at the single-molecule level within the organelles of live cells. Indeed, these photoactivatable fluorophores localize in lysosomal compartments and remain covalently immobilized within these organelles. The suppression of diffusion allows the reiterative reconstruction of subdiffraction images and the visualization of the labeled organelles with excellent localization precision. Thus, the combination of photochemical, photophysical and structural properties designed into our fluorophores enable the visualization of live cells with a spatial resolution that is inaccessible to conventional fluorescence imaging.

Fluorescence activation with switchable oxazines.

The identification of operating principles to activate fluorescence under the influence of external stimulations is essential to enable the implementation of imaging strategies requiring the spatiotemporal control of emission. In this context, our laboratories designed mechanisms to switch fluorescence with either light or pH based on the unique photochemical and photophysical properties of either photoresponsive or halochromic oxazines respectively. These heterocycles can be connected covalently to fluorescent chromophores and opened with either light or pH to impose a significant bathochromic shift on the main absorption of the emissive appendage. Such a spectral change allows the selective excitation of the resulting species to activate bright fluorescence with infinite contrast and spatiotemporal control. Indeed, these mechanisms for fluorescence activation enable the acquisition of images with subdiffraction resolution, the selective signaling of cancer cells and the monitoring of translocating species in real time. Thus, our structural designs for fluorescence switching under external control can evolve into invaluable probes for the implementation of bioimaging strategies that would be impossible to perform with conventional fluorophores.

A Photoactivatable Far-Red/Near-Infrared BODIPY To Monitor Cellular Dynamics in Vivo.

A mechanism to photoactivate far-red/near-infrared fluorescence with infinite contrast and under mild visible illumination was designed around the photophysical properties of borondipyrromethene (BODIPY) dyes and the photochemical behavior of oxazine heterocycles. Specifically, the photoinduced and irreversible cleavage of an oxazine ring with a laser line at 405 nm extends the electronic conjugation of a BODIPY chromophore over a 3 H-indole auxochrome with a 2-(4-methoxyphenyl)ethenyl substituent in position 5. This structural transformation shifts bathochromically the main absorption band of the BODIPY component to allow the selective excitation of the photochemical product with a laser line of 633 nm and produce fluorescence between 600 and 850 nm. This combination of activation, excitation, and emission wavelengths permits the visualization of the cellular blastoderm of developing Drosophila melanogaster embryos with optimal contrast and essentially no autofluorescence from the biological specimen. Furthermore, the sequential acquisition of images, after the photoactivation event, enables the tracking of individual cells within the embryos in real time. Thus, our structural design and operating principles for the photoactivation of far-red/near-infrared fluorescence can evolve into invaluable probes to monitor cellular dynamics in vivo.

Photochemical Barcodes.

A photochemical strategy to encode fluorescence signals in vivo with spatial control was designed around the unique properties of a photoactivatable borondipyrromethene (BODIPY). The photoinduced disconnection of two oxazines, flanking a single BODIPY, in two consecutive steps produces a mixture of three emissive molecules with resolved fluorescence inside polymer beads. The relative amounts and emission intensities of the three fluorophores can be regulated precisely in each bead by adjusting the dose of activating photons to mark individual particles with distinct codes of fluorescence signals. The visible wavelengths and mild illumination sufficient to induce these transformations permit the photochemical barcoding of beads also in living nematodes. Different regions of the same animal can be labeled with distinct barcodes to allow the monitoring of their dynamics for long times with no toxic effects. Thus, our photochemical strategy for the generation of fluorescence barcodes can produce multiple and distinguishable labels in the same biological sample to enable the spatiotemporal tracking of, otherwise indistinguishable, targets.