No abstract

The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful.Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36–0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

Time-resolved and temperature-dependent photoluminescence (PL) spectroscopy of ternary compound copper indium disulfide (CuInS2, or CIS) core materials, CIS/ZnS coreshells, and quaternary compound ZnCuInS2 (ZnCIS) revealed their optical properties with spectral, temporal, and thermal characteristics, which were closely linked to surface-related recombination, and shallow or deep defect-related donor-acceptor transitions. The PL peaks of semiconductor nanocrystals (SNCs) with sizes near Bohr radius displayed at ∼775 nm for CIS, ∼605 nm for CIS/ZnS, and ∼611 nm for ZnCIS. The spectral blue shift and spectral narrowing with CIS/ZnS and ZnCIS are assigned to the increased spatial confinement and surface regularity with the etching of core materials. Both the shorter lifetime at surface-trapped states or interface states and the longer lifetime at intrinsic defect-related states of CIS, CIS/ZnS, and ZnCIS SNCs were widely distributed across the entire PL spectral region. The surface or interface-trapped electrons were thermally active even at low temperatures, but the electrons at intrinsic defect-related states were relatively stable, which was attributable to the strong Coulomb energy between the charge carriers.

BSA protected gold nanoclusters (Au25) are attracting great deal of attention due to their unique spectroscopic properties and possible use in biophysical applications. Although there are reports on synthetic strategies, spectroscopy and applications, little is known about their polarization behavior. In this study, we synthesized the BSA protected Au25 nanoclusters and studied their steady state and time resolved fluorescence properties including polarization behavior in different solvents: glycerol, propylene glycol and water. We demonstrated that the nanocluster absorption can be separated from the extinction spectrum by subtraction of Rayleigh scattering. The nanocluster absorption spectrum is well approximated by three Gaussian components. By a comparison of the emissions from BSA Au25 clusters and rhodamine B in water, we estimated the quantum yield of nanoclusters to be higher than 0.06. The fluorescence lifetime of the BSA Au25 cluster is long and heterogeneous with an average value of 1.84 μs. In glycerol at −20°C the anisotropy is high, reaching a value of 0.35. However, the excitation anisotropy strongly depends on the excitation wavelengths indicating a significant overlap of the different transition moments. The anisotropy decay in water reveals a correlation time below 0.2 μs. In propylene glycol the measured correlation time is longer and initial anisotropy depends on the excitation wavelength. The BSA Au25 cluster, due to long lifetime and high polarization, can potentially be used in studying large macromolecules such as protein complexes with large molecular weight.

Bovine serum albumin (BSA) protected nanoclusters (Au and Ag) represent a group of nanomaterials that holds great promise in biophysical applications due to their unique fluorescence properties and lack of toxicity. While, these metal nanoclusters have -utility in a variety of disciplines including catalysis, biosensing, photonics, imaging and molecular electronics. However, they suffer from several certain disadvantages such as low fluorescence quantum efficiency (typically near 6%) and broad emission spectrum (540nm to 800nm). We describe an approach to enhance the apparent brightness of BSA Au clusters by linking it with high extinction donor organic dye pacific blue (PB). In this conjugate PB acts as a donor to BSA Au clusters and enhances its brightness by resonance energy transfer (RET). We found that the emission of BSA Au clusters can be enhanced by a magnitude of two-folds by resonance energy transfer (RET) from the high extinction donor PB, and BSA Au clusters can act as an acceptor to nanosecond lifetime organic dyes. By pumping the BSA Au clusters using a high extinction donor, one can increase the effective brightness of less bright fluorophores like BSA Au clusters. Moreover, we prepared another conjugate of BSA Au clusters with the near infra-red (NIR) dye Dylight 750 (Dy750), where BSA Au cluster act as a donor to Dy750. We observed that BSA Au clusters can function as a donor, showing 46% transfer efficiency to the NIR dye Dy750 with long lifetime component in acceptor decay through RET. Such RET-based probes can be used to prevent the problems of broad emission spectrum associated with the BSA Au clusters. Moreover, transferring energy from BSA Au cluster to Dy750 will result in a RET probe with narrow emission spectrum and long lifetime component which can be utilized in imaging applications.

Fluorescence properties of a novel homodimeric BODIPY dye rotor for Fluorescence Lifetime Imaging Microscopy (FLIM) are reported. Steady state and time resolved fluorescence measurements established the viscosity dependant behaviour in vitro. Homodimeric BODIPY embedded in different membrane mimicking lipid vesicles (DPPC, POPC and POPC plus cholesterol) demonstrated to be a viable sensor for fluorescence lifetime based viscosity measurements. Moreover, SKOV3 cells readily endocytosed the dye, which accumulated in membranous structures inside cytoplasm thereby allowing viscosity mapping of internal cell components.

This work reports the surface functionalization of polymeric PLGA nanoparticles by non-covalent insertion of a homo-bifunctional chemical crosslinker, bis(sulfosuccinimidyl) suberate (BS3) for targeted cancer therapy. We dissolved BS3 in aqueous solution of PVA during formulation of nanoparticles by a modified solid/oil/water emulsion solvent evaporation method. The non-covalent insertion of BS3 was confirmed by Fourier transform infrared (FTIR) spectroscopy. Curcumin and annexin A2 were used as a model drug and a cell specific target, respectively. Nanoparticles were characterized for particle size, zeta potential and surface morphology. The qualitative assessment of antibody attachment was performed by transmission electron microscopy (TEM) as well as confocal microscopy. The optimized formulation showed antibody attachment of 86%. However, antibody attachment was abolished upon blocking the functional groups of BS3. The availability of functional antibodies was evaluated by the presence of a light chain fraction after gel electrophoresis. We further evaluated the in vitro release kinetics of curcumin from antibody coated and uncoated nanoparticles. The release of curcumin is enhanced upon antibody attachment and followed an anomalous release pattern. We also observed that the cellular uptake of nanoparticles was significantly higher in annexin A2 positive cells than in negative cells. Therefore, these results demonstrate the potential use of this method for functionalization as well as to deliver chemotherapeutic agents for treating cancer.

Drug delivery to malignant tumors is limited by several factors, including off-target toxicities and suboptimal benefits to cancer patient. Major research efforts have been directed toward developing novel technologies involving nanoparticles (NPs) to overcome these challenges. Major obstacles, however, including, opsonization, transport across cancer cell membranes, multidrug-resistant proteins, and endosomal sequestration of the therapeutic agent continue to limit the efficiency of cancer chemotherapy. Lipoprotein-based drug delivery technology, “nature’s drug delivery system,” while exhibits highly desirable characteristics, it still needs substantial investment from private/government stakeholders to promote its eventual advance to the bedside. Consequently, this review focuses specifically on the synthetic (reconstituted) high-density lipoprotein rHDL NPs, evaluating their potential to overcome specific biological barriers and the challenges of translation toward clinical utilization and commercialization. This highly robust drug transport system provides site-specific, tumor-selective delivery of anti-cancer agents while reducing harmful off-target effects. Utilizing rHDL NPs for anti-cancer therapeutics and tumor imaging revolutionizes the future strategy for the management of a broad range of cancers and other diseases.