Development of chitosan based ß-carotene mucoadhesive formulation for skin cancer treatment.

Mucopermeating nanoformulations can enhance mucosal penetration of poorly soluble drugs at their target site. In this work, thiolated chitosan (TCS)-lithocholic acid (LA) nanomicelles loaded with ß-carotene, a safe phytochemical with anticancer properties, were designed to improve the pharmaceutical and pharmacological drug profile. The TCS-LA nanomicelles were characterized by FTIR to confirm the presence of the thiol group that favors skin adhesion, and to corroborate the conjugation of hydrophobic LA with hydrophilic CS to form an amphiphilic polymer derivative. Their crystalline nature and thermal behavior were investigated by XRD and DSC analyses, respectively. According to DLS and TEM, their average size was <300 nm, and their surface charge was +27.0 mV. ß-carotene entrapment and loading efficiencies were 64 % and 58 %, respectively. In vitro mucoadhesion and ex vivo mucopenetration analyses further corroborated the potential of the nanoformulation to deliver the drug in a sustained manner under conditions mimicking cancer micro-environment. Anticancer studies in mice demonstrated that the loaded nanomicelles delayed skin cancer growth, as revealed by both morphological and biochemical parameters. Based on the results obtained herein, it can be concluded that drug-loaded TCS-LA is a novel, stable, effective and safe mucoadhesive formulation of ß-carotene for the potential treatment of skin cancer.

New perspectives for triplet-triplet annihilation based photon upconversion using all-organic energy donor & acceptor chromophores.

It is recognized that metal organic complexes that serve as sensitizers can present various degrees of challenges viz. synthesis and stability for photonic applications such as triplet-triplet annihilation based photon upconversion (TTA-PUC). Presently, researchers, including our group, are turning their attention toward purely organic triplet sensitizers, which can be handled more easily for photon management science. In this review, we surveyed recently developed all-organic chromophoric systems that were devised and used for TTA-PUC research. Knowing that TTA-PUC research has mainly been focused on the design and synthesis of the triplet sensitizers, we detailed the underlying photophysics and thermodynamics that served as the starting point for the synthesis of the purely organic chromophores in question. Accordingly, this review details triplet sensitizers that operate on (i) spin-orbit coupling or heavy atom effect, (ii) Baird-type aromaticity and antiaromaticity, (iii) open-shell characteristics or doublet excited state and (iv) thermally activated delayed fluorescence.

A Naphtho-p-quinodimethane Exhibiting Baird's (Anti)Aromaticity, Broken Symmetry, and Attractive Photoluminescence.

We report a novel reductive desulfurization reaction involving π-acidic naphthalene diimides (NDI) 1 using thionating agents such as Lawesson's reagent. Along with the expected thionated NDI derivatives 2-6, new heterocyclic naphtho-p-quinodimethane compounds 7 depicting broken/reduced symmetry were successfully isolated and fully characterized. Empirical studies and theoretical modeling suggest that 7 was formed via a six-membered ring oxathiaphosphenine intermediate rather than the usual four-membered ring oxathiaphosphetane of 2-6. Aside from the reduced symmetry in 7 as confirmed by single-crystal XRD analysis, we established that the ground state UV-vis absorption of 7 is red-shifted in comparison to the parent NDI 1. This result was expected in the case of thionated polycyclic diimides. However, unusual low energy transitions originate from Baird 4nπ aromaticity of compounds 7 in lieu of the intrinsic Hückel (4n + 2)π aromaticity as encountered in NDI 1. Moreover, complementary theoretical modeling results also corroborate this change in aromaticity of 7. Consequently, photophysical investigations show that, compared to parent NDI 1, 7 can easily access and emit from its T1 state with a phosphorescence 3(7a)* lifetime of τP = 395 µs at 77 K indicative of the formation of the corresponding "aromatic triplet" species according to the Baird's rule of aromaticity.

Lamellar Peptide-Cadmium-Doped Zinc Oxide Nanohybrids That Emit White Light.

A variety of hybrid nanostructures have been developed that emit white light. Two different white-light-emitting systems are reported. These are cadmium-doped zinc oxide nanosheets and complex lamellar nanostructures that consist of alternating inorganic cadmium-doped zinc oxide domains with the self-assembled aromatic-capped peptide BPI-FF-OH (BPI: benzo[ghi]perylene monoimide, F: d-phenylalanine). An electrochemical method is employed to synthesize cadmium-doped zinc oxide nanosheets and lamellar organic/cadmium-doped zinc oxide nanoflakes on a gallium-doped ZnO/p-Si (111) substrate. External structural features and internal structural ordering of wurtzite cadmium-doped zinc oxide and lamellar organic/cadmium-doped zinc oxide nanohybrids are characterized by small-angle X-ray scattering, XRD, field-emission SEM, energy-dispersive X-ray spectroscopy, secondary-ion mass spectrometry, ellipsometry, and photoluminescence spectroscopy. Cadmium-doped zinc oxide nanosheets and lamellar organic/cadmium-doped zinc oxide hybrids emit white light with a broad emission covering the visible spectrum from λ=415 to 700 nm. Characteristic white-light emissions of both materials were well characterized by photoluminescence studies. The white-light luminescence is attributed to cadmium doping in the zinc oxide crystal and the presence of the dipeptide-functionalized BPI fluorophore in the lamellar nanohybrid.

Electrodeposited Lamellar Photoconductor Nanohybrids Driven by Peptide Self-Assembly.

Aromatic organic molecules serve as optoelectronic materials owing to their intrinsic optical and electronic properties. Herein, self-assembled lamellar nanostructures as photoconductor hybrids, which are obtained from naphthalene-2-methoxycarbonyl (Nmoc)-capped peptide amphiphiles, are described. Hybrid nanostructures are constructed in a controlled manner by an electrochemical deposition technique in combination with the inorganic Zn(OH)2 phase. Inorganic Zn(OH)2 layers turn into semiconductor ZnO layers upon annealing at 150 °C and lamellar nanostructures are formed in a periodic manner. Synergistic effects of hydrogen bonding and π-π stacking interactions of aromatic peptide amphiphiles are the driving force for the formation of self-assembled lamellar nanostructures. Morphological, structural, and optical studies of such lamellar hybrid nanostructures are reported. Photoconduction of these hybrid nanostructures is also examined in detail.

Peptide-Nanofiber-Supported Palladium Nanoparticles as an Efficient Catalyst for the Removal of N-Terminus Protecting Groups.

Sonication-induced tryptophan- and tyrosine-based peptide bolaamphiphile nanofibers have been used to synthesize and stabilize Pd nanoparticles under physiological conditions. The peptide bolaamphiphile self-assembly process has been thoroughly studied by using several spectroscopic and microscopic techniques. The stiffness of the soft hydrogel matrix was measured by an oscillatory rheological experiment. FTIR and circular dichroism (CD) experiments revealed a hydrogen-bonded ß-sheet conformation of peptide bolaamphiphile molecules in a gel-phase medium. The π-π stacking interactions also played a crucial role in the self-assembly process, which was confirmed by fluorescence spectroscopy. Electron (SEM and TEM) and atomic force microscopy (AFM) studies showed that the peptide bolaamphiphile molecules self-assemble into nanofibrillar structures. Pd nanoparticles were synthesized in the hydrogel matrix in which redox-active tryptophan and tyrosine residues reduce the metal ions to metal nanoparticles. The size of the Pd nanoparticles are in the range of 3-9 nm, and are stabilized by peptide nanofibers. The peptide-nanofiber-supported Pd nanoparticles have shown effective catalytic activity for the removal of N-terminus protecting groups of amino acids and peptides.

Weak glycolipid binding of a microdomain-tracer peptide correlates with aggregation and slow diffusion on cell membranes.

Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous diffusion behavior in the probe population.

Biocompatible glutathione capped gold clusters as one- and two-photon excitation fluorescence contrast agents for live cells imaging.

The one- and two-photon excitation emission properties of water soluble glutathione monolayer protected gold clusters were investigated. Strong two-photon emission was observed from the gold clusters. The two-photon absorption cross section of these gold clusters in water was deduced from the z-scan measurement to be 189 740 GM, which is much higher compared to organic fluorescent dyes and quantum dots. These gold clusters also showed high photo-stability. The MTT assay showed that these gold clusters have low toxicity even at high concentrations. We have successfully demonstrated their applications for both one and two-photon excitation live cell imaging. The exceptional properties of these gold clusters make them a promising alternative for one- and two-photon bio-imaging and other nonlinear optical applications.

Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Abeta, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption.

Alternate raft pathways cooperate to mediate slow diffusion and efficient uptake of a sphingolipid tracer to degradative and recycling compartments.

Several cholesterol-dependent cellular uptake pathways involving microdomain-resident sphingolipids have been characterized, but little is known about what controls the further intracellular trafficking routes of those domains. Here, we present evidence that the uptake and intracellular trafficking of a recently described sphingolipid-binding probe, the sphingolipid binding domain (SBD) peptide, is mediated by two parallel cooperating mechanisms requiring flotillin, dynamin and cdc42, which act in concert to direct a distinct surface behavior and trafficking itinerary. Diffusion measurements of SBD at the cell surface by fluorescence correlation spectroscopy suggest that cdc42- and flotillin-associated uptake sites both correspond to domains of intermediate mobility, but that they can cooperate to form low-mobility, efficiently internalized domains. Interestingly, we find that the choice of uptake mechanism affects subsequent trafficking of SBD, as does cholesterol content. Interference with one or other uptake pathway acts as a toggle switch for the trafficking of SBD to recycling endosomes or endolysosomes, whereas both of these pathways are bypassed if cholesterol is reduced. The data are in accordance with a scenario in which SBD mirrors the trafficking response of raft-borne lipids towards a degradative or recycling target. In summary, we suggest that both the surface behavior of a cargo and its subsequent trafficking are determined by a combination of endocytic accessory proteins and the cholesterol content of different membrane compartments.

A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains.

We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent.