Unveiling NIR Aza-Boron-Dipyrromethene (BODIPY) Dyes as Raman Probes: Surface-Enhanced Raman Scattering (SERS)-Guided Selective Detection and Imaging of Human Cancer Cells.

The development of new Raman reporters has attracted immense attention in diagnostic research based on surface enhanced Raman scattering (SERS) techniques, which is a well established method for ultrasensitive detection through molecular fingerprinting and imaging. Herein, for the first time, we report the unique and efficient Raman active features of the selected aza-BODIPY dyes 1-6. These distinctive attributes could be extended at the molecular level to allow detection through SERS upon adsorption onto nano-roughened gold surface. Among the newly revealed Raman reporters, the amino substituted derivative 4 showed high signal intensity at very low concentrations (ca. 0.4 µm for 4-Au). Interestingly, an efficient nanoprobe has been constructed by using gold nanoparticles as SERS substrate, and 4 as the Raman reporter (4-Au@PEG), which unexpectedly showed efficient recognition of three human cancer cells (lung: A549, cervical: HeLa, Fibrosarcoma: HT-1080) without any specific surface marker. We observed well reflected and resolved Raman mapping and characteristic signature peaks whereas, such recognition was not observed in normal fibroblast (3T3L1) cells. To confirm these findings, a SERS nanoprobe was conjugated with a specific tumour targeting marker, EGFR (Epidermal Growth Factor Receptor), a well known targeted agent for Human Fibrosarcoma (HT1080). This nanoprobe efficiently targeted the surface marker of HT1080 cells, threreby demonstrating its use as an ultrasensitive Raman probe for detection and targeted imaging, leaving normal cells unaffected.

Carbazole-Linked Near-Infrared Aza-BODIPY Dyes as Triplet Sensitizers and Photoacoustic Contrast Agents for Deep-Tissue Imaging.

Four new N-ethylcarbazole-linked aza-boron-dipyrromethene (aza-BODIPY) dyes (8 a,b and 9 a,b) were synthesized and characterized. The presence of the N-ethylcarbazole moiety shifts their absorption and fluorescence spectra to the near-infrared region, λ≈650-730 nm, of the electromagnetic spectrum. These dyes possess strong molar absorptivity in the range of 3-4×104 m-1 cm-1 with low fluorescence quantum yields. The triplet excited state and singlet oxygen generation of these dyes were enhanced upon iodination at the core position. The core-iodinated dyes 9 a,b showed excellent triplet quantum yields of about 90 and 75 %, with singlet oxygen generation efficiency of about 70 and 60 % relative to that of the parent dyes. Derivatives 8 a,b showed dual absorption profiles, in contrast to dyes 9 a,b, which had the characteristic absorption band of aza-BODIPY dyes. DFT calculations revealed that the electron density was spread over the iodine and dipyrromethene plane of 9 a,b, whereas in 8 a,b the electron density was distributed on the carbazole group and dipyrromethene plane of aza-BODIPY. The uniqueness of these aza-BODIPY systems is that they exhibit efficient triplet-state quantum yields, high singlet oxygen generation yields, and good photostability. Furthermore, the photoacoustic (PA) characteristics of these aza-BODIPY dyes was explored, and efficient PA signals for 8 a were observed relative to blood serum with in vitro deep-tissue imaging, thereby confirming its use as a promising PA contrast agent.

Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies, and the Encapsulation and Release of Dyes through DNA-Mediated Disassembly.

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di- and monoimide chromophores and the investigation of their photophysical, self-assembly and DNA-binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG⋅dC)-poly(dG⋅dC) and poly(dA⋅dT)-poly(dA⋅dT). The interaction of these conjugates with DNA was evaluated by photo- and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5-8×10(4) M(-1) . Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM, which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.

Simultaneous binding of a cyclophane and classical intercalators to DNA: observation of FRET-mediated white light emission.

DNA-assisted Förster resonance energy transfer (FRET) between an anthracene-based cyclophane (CP) and mono- and bis-intercalators such as propidium iodide (PI) and ethidium homodimer-1 (EHD), respectively, has been studied using various photophysical and biophysical techniques. The cyclophane and PI exhibited simultaneous binding to DNA at all concentrations studied and showed DNA-assisted FRET from the excimer of cyclophane with a FRET efficiency of ca. 71%. On the other hand, the bis-intercalator EHD, only at lower concentrations (<3 µM), can act as an acceptor for the energy transfer process with a lower efficiency of ca. 44%. At higher concentrations (>15 µM), EHD, on account of its higher binding affinity, displaces cyclophane from the DNA scaffold. Employing the ternary system comprising of the cyclophane, DNA and PI and fine-tuning the concentrations of the components in a molar ratio of 1 : 0.75 : 0.05 (CP : DNA : PI) we have demonstrated white light emission with CIE coordinates (0.35, 0.37).

Sensitive naked eye detection of hydrogen sulfide and nitric oxide by aza-BODIPY dyes in aqueous medium.

With an objective to develop optical probes for biologically important anions and neutral molecules, we synthesized three novel NIR absorbing aza-BODIPY derivatives, 3a-3c, and have systematically tuned their photophysical properties by changing the peripheral substitution. A profound red-shift was observed in the absorption and fluorescence spectra of the aza-BODIPY dyes with the change in substitution from azido (3a) to amino (3b) to dimethylamino (3c) groups. Theoretical calculations of 3a-3c showed a consistent decrease in bandgap, which supports the observed spectral changes. The study of their interactions with various analytes revealed that the azido-aza-BODIPY 3a selectively interacts with hydrogen sulfide (H2S) when compared to other molecules. Uniquely, the detection of H2S can be visualized through a change in color from bright blue to purple with a detection limit of 0.5 ppm. The sensitivity of the probe was observed to be ~20-fold higher than the allowed exposure limits of H2S as defined by EPA (10 ppm). The aza-BODIPY derivative 3b, on the other hand, exhibited selective interactions with nitrite ions (NO2(-)) and nitric oxide (NO) in aqueous medium through a visible color change from blue to green with a sensitivity of 20 and 0.15 ppb, respectively. In contrast, the dimethylamino-aza-BODIPY derivative, 3c, showed negligible affinity for the anions and neutral molecules tested. By tuning the photophysical properties through the judicious functionalization, the aza-BODIPY dyes thus synthesized can be utilized for the sensitive on-site detection and analysis of H2S, NO2(-), and NO in the aqueous medium.

Efficient reaction based colorimetric probe for sensitive detection, quantification, and on-site analysis of nitrite ions in natural water resources.

We have developed a novel aza-BODIPY probe for the sensitive colorimetric detection of the nitrite ions in the aqueous medium by a simple and direct method. This probe selectively recognizes the nitrite ions through a distinct visual color change from bright blue to intense green with a sensitivity of 20 ppb. Uniquely, this probe can be coated on a glass surface to fabricate a simple solid-state dipstick device that can be used for the visual detection of the nitrite ions in the presence of other competing anions in distilled as well as natural water resources like a sea, lake, and river. Furthermore, this probe can be used for the sensitive detection of the nitrate ions when coupled to a reduction step. Our results demonstrate that this probe not only can be used for the on-site analysis and quantification but also can replace the conventional spot test carried out for the nitrite ions in the laboratory practical experiments.

Biocompatible Aza-BODIPY-Biotin Conjugates for Photodynamic Therapy of Cancer.

With an objective to develop efficient photosensitizers to cancerous tissues, we synthesized two novel biocompatible sensitizers based on aza-BODIPYs incorporated with heavy atoms and biotin moieties. The bioconjugates DPR2a and DPR2b exhibited a favorable absorption range (600-750 nm) with excellent triplet-state quantum yields (up to 79%) and singlet oxygen generation yields (up to 75%). In vitro photobiological investigations employing MDA-MB-231 breast cancer cell lines exhibited rapid cellular uptake, negligible dark toxicity, and high photocytotoxicity. The mechanism of cell death of these systems was predominantly due to the mitochondrial damage, leading to apoptosis mediated via the generation of singlet oxygen-triggered reactive oxygen species. The in vivo studies with the representative conjugate DPR2a employing female NOD/SCID mice models showed inhibition in tumor growth and significantly decreased tumor volume post photodynamic therapy (PDT) treatment. Our results validate that both DPR2a and DPR2b with iodine incorporation exhibit favorable and superior photophysical and photobiological aspects and demonstrate thereby their potential applications in imaging and PDT of cancer.

Aza-BODIPY derivatives: enhanced quantum yields of triplet excited states and the generation of singlet oxygen and their role as facile sustainable photooxygenation catalysts.

A new series of aza-BODIPY derivatives (4 a-4 c, 5 a,c, and 6 b,c) were synthesized and their excited-state properties, such as their triplet excited state and the yield of singlet-oxygen generation, were tuned by substituting with heavy atoms, such as bromine and iodine. The effect of substitution has been studied in detail by varying the position of halogenation. The core-substituted dyes showed high yields of the triplet excited state and high efficiencies of singlet-oxygen generation when compared to the peripheral-substituted systems. The dye 6 c, which was substituted with six iodine atoms on the core and peripheral phenyl ring, showed the highest quantum yields of the triplet excited state (Φ(T) =0.86) and of the efficiency of singlet-oxygen generation (Φ(Δ) =0.80). Interestingly, these dyes were highly efficient as photooxygenation catalysts under artificial light, as well as under normal sunlight conditions. The uniqueness of these aza-BODIPY systems is that they are stable under irradiation conditions, possess strong red-light absorption (620-680 nm), exhibit high yields of singlet-oxygen generation, and act as efficient and sustainable catalysts for photooxygenation reactions.

Squaraine dyes in PDT: from basic design to in vivo demonstration.

The design and development of novel squaraine dyes as sensitisers for photodynamic therapy (PDT) applications has grown tremendously in the last decade from the time when a squaraine dye was proposed to be a potential candidate, to-date when the use of such dyes have been demonstrated in animal models for skin cancer. This perspective article highlights the basic design, tuning of absorption, triplet excited state and two-photon absorption properties and recent developments of the squaraines as PDT sensitisers.

Study of cavity size and nature of bridging units on recognition of nucleotides by cyclophanes.

We synthesized a few novel cyclophanes CP-1 to CP-4 containing anthracene units linked together through different bridging and spacer groups and have investigated their interactions with various nucleosides and nucleotides. Of these systems, CP-1 and CP-3 showed selectivity for 5'-GTP and 5'-ATP as compared to other nucleotides and nucleosides, whereas negligible selectivity was observed with CP-2 and CP-4. Interestingly, CP-1, CP-2 and CP-3 exhibited significant binding interactions with the fluorescent indicator, 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS), resulting in the formation of non-fluorescent complexes. Titration of these complexes with nucleosides and nucleotides resulted in the displacement of HPTS, leading to the revival of its fluorescence intensity. It was observed that 5'-GTP induced the maximum displacement of HPTS from the complex [CP-1·HPTS] with an overall fluorescence enhancement of ca. 150-fold, while 5'-ATP induced ca. 45-fold. Although the displacement of HPTS from the complexes [CP-2·HPTS] and [CP-3·HPTS] was found to be similar to that of [CP-1·HPTS], these complexes showed lesser selectivity and sensitivity. In contrast, negligible displacement of HPTS was observed from the complex [CP-4·HPTS] under similar conditions. These results indicate that CP-1, having a well-defined cavity and good electron acceptor (viologen), is capable of forming selective and stable complexes. Though CP-2 and CP-3 retain the good electron acceptor (viologen), their reduced aromatic surface and larger cavity, respectively, resulted in lesser sensitivity. In contrast, CP-4 having a large cavity and a poor acceptor (1,2-bis(pyridin-4-yl)ethene) showed negligible selectivity, thereby indicating the importance of cavity size, bridging unit and aromatic surface on biomolecular recognition properties of cyclophanes.

Functional cyclophanes: promising hosts for optical biomolecular recognition.

Cyclophanes possess a defined cavity size and are efficient in encapsulating and stabilising guest molecules inside the cavity through various non-covalent interactions. This unique property of the cyclophanes has been widely exploited for the development of selective probes for a variety of guest molecules. The present tutorial review highlights the use of various interesting functionalised cyclophane architectures for the sensitive and selective optical recognition of important biomolecules.

Fluorescence ratiometric selective recognition of Cu(2+) ions by dansyl-naphthalimide dyads.

Chimeric dyads 1a and 1b based on dansyl and naphthalimide units linked through the polymethylene group were synthesized, and their photophysical and interactions with various metal ions were investigated under different conditions. These dyads showed dual emission centered at around 375 and 525 nm, respectively, due to the locally excited state of the naphthalimide chromophore and energy-transfer-mediated emission (FRET) from the dansyl moiety. When titrated with various metal ions, these systems exhibited unusual selectivity for Cu(2+) ions as compared to Na(+), Li(+), K(+), Zn(2+), Pb(2+), Hg(2+), Co(2+), Fe(2+), Cd(2+), Mg(2+), and Ba(2+) ions and signaled the binding event through inhibition of FRET mediated emission at 525 nm with concurrent enhancement in the emission intensity of the naphthalimide chromophore at 375 nm. The uniqueness of these dyads is that they form stable 2:1 stoichiometric complexes involving sulfonamide functionality and act as visual fluorescence ratiometric probes for the selective recognition of Cu(2+) ions.

Picolyl Porphyrin Nanostructures as a Functional Drug Entrant for Photodynamic Therapy in Human Breast Cancers.

The major challenge in photodynamic therapy (PDT) is to discover versatile photosensitizers (PSs) that possess good solubility in biological media, enhanced singlet oxygen generation efficacy, and photodynamic activity. Working in this direction, we synthesized a picolylamine-functionalized porphyrin conjugate, compound 1, and its zinc complex compound 2. Compound 1 forms spherical structures in methanol, whereas compound 2 exhibited vesicular structures. Compared to the existing PSs like foscan and photofrin, compound 2 exhibited a high singlet oxygen generation efficiency and triplet quantum yield. The complex also showed good water solubility, and its PDT activity was demonstrated through in vitro studies using MDA-MB 231 breast cancer cells. The mechanism of biological activity evaluated using various techniques proved that the active compound 2 induced predominantly singlet oxygen-triggered apoptosis-mediated cancerous cell death. Our results demonstrate that zinc insertion in the picolyl porphyrin induces an enhanced triplet excited state, and the singlet oxygen yields quantitatively and imparts excellent in vitro photodynamic activity, thereby demonstrating their pertinence as a nanodrug in future photobiological applications.

Aza-BODIPY nanomicelles as versatile agents for the in vitro and in vivo singlet oxygen-triggered apoptosis of human breast cancer cells.

Herein, we synthesised four aza-BODIPY dyes (1-4) with the singlet oxygen generation quantum yield values of ca. 65-85%. Furthermore, we formulated a nanomedicine by encapsulating these dyes into an amphiphilic micelle, DSPE. The spherical nanomicelles formed were characterized using photophysical and morphological analyses, and their in vitro and in vivo photodynamic efficacies were investigated. One of the conjugates, DSPE-1, showed the lowest IC50 value of 2 µM against a human breast cancer cell line (MDA MB 231). The mechanism of photodynamic activity has been evaluated by employing different biophysical and morphological assays, which confirmed apoptotic cell death (ca. 80-90%) predominantly through the involvement of reactive oxygen species. Interestingly, we observed that 2 mg kg-1DSPE-1 induced enhanced apoptosis and efficient inhibition of the growth of breast tumor xenografts in NOD/SCID mice models. Herein, we demonstrated the application of aza-BODIPY nanomicelles in photodynamic therapy for the first time, and our results revealed that the DSPE-BODIPY nanomicelles enhanced the cellular uptake as well as the photodynamic activity, thereby demonstrating the use of these nanomicelles as efficient sensitizers in biological applications.

Blue-fluorescent and biocompatible carbon dots derived from abundant low-quality coals.

Coal is one of the most abundant natural carbonaceous materials. This paper reports a novel oxidative chemical method for the synthesis of high-value carbon dots (CDs) from cheap and abundant low-quality high­sulfur coals for use in high-end applications. These CDs were synthesized by using wet-chemical ultrasonic stimulation-induced process which is environmentally facile and less drastic compared to other chemical methods of production of CDs. The sizes of the synthesized CDs from different types of coal samples were estimated to be in the range of 1-4 nm, 1-6 nm, 2-5 nm, and 10-30 nm. The quantum yield (QY) of the CDs was determined and it was found to be around 3-14%. For high-end field application, the CDs were further tested for toxicity and were reported to be safe for environmental and biological applications. The cell image analysis under the fluorescence microscope further indicated that the synthesized CDs could be used as a promising bio-compatible material for optical-imaging as well as bio-imaging. The CDs showed promising fluorescent sensing property and can be utilized as a good probe for silver ion detection/sensing. The CDs is also found to be a promising reagent for silver nanoparticles synthesis. The results provide a new avenue for large-scale synthesis of CDs.

In Vitro and In Vivo Demonstration of Human-Ovarian-Cancer Necrosis through a Water-Soluble and Near-Infrared-Absorbing Chlorin.

With the objective of developing efficient sensitizers for therapeutic applications, we synthesized a water-soluble 5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin (TDC) and investigated its in vitro and in vivo biological efficacy, comparing it with the commercially available sensitizers. TDC showed high water solubility (6-fold) when compared with that of Foscan and exhibited excellent triplet-excited-state (84%) and singlet-oxygen (80%) yields. In vitro photobiological investigations in human-ovarian-cancer cell lines SKOV-3 showed high photocytotoxicity, negligible dark toxicity, rapid cellular uptake, and specific localization of TDC in neoplastic cells as assessed by flow-cytometric cell-cycle and propidium iodide staining analysis. The photodynamic effects of TDC include confirmed reactive-oxygen-species-induced mitochondrial damage leading to necrosis in SKOV-3 cell lines. The in vivo photodynamic activity in nude-mouse models demonstrated abrogation of tumor growth without any detectable pathology in the skin, liver, spleen, or kidney, thereby demonstrating TDC application as an efficient and safe photosensitizer.

Selective recognition of tryptophan through inhibition of intramolecular charge-transfer interactions in an aqueous medium.

[reaction: see text] A novel donor-acceptor conjugate 1 was synthesized, and its interactions with various amino acids have been investigated as compared to the model system 2. The conjugate 1 unusually forms an intramolecular charge-transfer complex in the aqueous medium and undergoes selective binding interactions with tryptophan. The uniqueness of this system is that it selectively recognizes tryptophan among all other amino acids and involves synergistic effects of pi-stacking, electrostatic, and donor-acceptor interactions.

Dual-mode semisquaraine-based sensor for selective detection of Hg2+ in a micellar medium.

[reaction: see text] A novel chemosensor based on semisquaraine dye (SSQ) for selective detection of Hg2+ is described. SSQ is obtained in quantitative yields from the reaction between squaric acid and 6-ethoxy-2-quinaldinium iodide. SSQ in combination with surfactant shows a dual chromogenic and fluorogenic response selectively toward Hg2+ as compared to Li+, Na+, K+, Ag+, Ca2+, Mg2+, Zn2+, Pb2+, Cd2+, Cu2+, and Fe3+ due to the soft acid nature and size of the mercuric ion.

Selective interactions of a few acridinium derivatives with single strand DNA: study of photophysical and DNA binding interactions.

Novel acridinium derivatives 1-3, wherein steric factors have been varied systematically through substitution at the ninth position of the acridinium ring, were synthesized and their interactions with single strand and double strand DNA have been investigated through photophysical, biophysical, and microscopic techniques. The acridinium derivative 1 exhibited quantitative fluorescence yields (phi f approximately =1) and high lifetime of 35 ns, while significantly lower fluorescence yields of 0.11 and 0.02 and lifetimes of 3.5 and 1.2 ns were observed for 2 and 3, respectively. The derivatives 1 and 2 having 2-methylphenyl and 2,4-dimethylphenyl substituents at the ninth position of the acridinium ring showed selective interactions with single strand DNA (ssDNA) with association constants of KssDNA = 6.3-6.6 x 10(4) M(-1), while negligible interactions were observed with double strand DNA (dsDNA). In contrast, the derivative 3 with 2,6-dimethylphenyl substitution showed negligible interactions with both ssDNA and dsDNA. Studies with a series of 19-mer oligonucleotides indicate that these derivatives exhibit significant selectivity for the sequences rich in guanosine (ca. 3-fold) as compared to the cytosine-rich sequences. These derivatives with high water solubility and the ability to distinguish between ssDNA and dsDNA through changes in fluorescence emission can be used as fluorescent probes for understanding the role of ssDNA in various biological processes and to study various DNA-ligand interactions.

Encapsulation of electron donor-acceptor dyads in beta-cyclodextrin cavity: unusual planarization and enhancement in rate of electron-transfer reaction.

Interaction of beta-cyclodextrin (beta-CD) with a few novel electron donor acceptor dyads 1a-c and 2a-c, having aryl and flexible methylene spacer groups, has been investigated through photophysical, chiroptical, electrochemical, NMR, and microscopic techniques. Dyads 1a and 1c, with p-tolyl and biphenyl spacer groups, respectively, exhibited significantly decreased fluorescence quantum yields and lifetimes in the presence of beta-CD, while negligible changes were observed for dyad 1b with an o-tolyl spacer. In contrast, spacer-length-dependent significant enhancement in fluorescence quantum yields and lifetimes was observed for dyads 2a-c, with flexible polymethylene (n = 1, 3, 11) spacer groups. Association constants of beta-CD encapsulated complexes have been determined and the contrast behavior observed in these systems is explained through an electron transfer (kET) mechanism based on calculated favorable change in free energy (DeltaGET = -1.27 eV) and the redox species characterized through laser flash photolysis studies. Rates of kET have been estimated and are found to increase ca. 2-fold in the case of dyads 1a and 1c when encapsulated in beta-CD, while significantly decreased kET values were observed for the dyads 2a-c with flexible spacer (ca. 9-fold for 2c). As characterized through cyclic voltammetry, 2D NMR [correlated (COSY) and nuclear Overhauser enhancement (NOESY) spectroscopy], and laser flash photolysis studies, the beta-CD encapsulation of dyads with aliphatic spacer groups leads to the conformational unfolding of a sandwich type of structure, whereas dyads with rigid aryl spacer groups undergo unusual planarization as compared to the uncomplexed dyads, resulting in enhanced electron-transfer reaction between the donor and acceptor moieties.