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.

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.

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.

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.

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.

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.

Bis(3,5-diiodo-2,4,6-trihydroxyphenyl)squaraine photodynamic therapy disrupts redox homeostasis and induce mitochondria-mediated apoptosis in human breast cancer cells.

Photodynamic therapy (PDT) is a clinically established and highly evolving treatment modality for cancer. PDT utilizes a light responsive drug called photosensitizer that selectively destroys tumor cells upon light irradiation. Squaraines are a class of dyes possessing all favorable characteristics of a photosensitizer and have been considered to be a potent candidate for next generation PDT. In this study we chose an iodo derivative of squaraine called diiodo-squaraine (bis(3, 5-diiodo-2,4,6-trihydroxyphenyl)squaraine) which has been reported for its tumor specificity but least studied for its cellular and molecular functions. Our studies revealed that the iodo derivative of squaraine possess maximum photodynamic activity in human breast cancer cells MDA- MB- 231 and had very little cytotoxicity in normal breast cells MCF-10A. We analyzed its pro and anti-apoptotic events initiated by oxidative stress exploring a proteomic approach and delineated other critical molecular pathways and key proteins involved in regulating the complex network of cellular response upon PDT. Our study showed that, diiodo- squaraines predominantly accumulate in mitochondria and induce mitochondria-mediated apoptosis. Our study also reveals the novel mechanistic role of diiodo-squaraines to induce oxidative stress there by activating both protective and death inducing pathways post PDT.

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.

In Vitro and in Vivo Demonstration of Photodynamic Activity and Cytoplasm Imaging through TPE Nanoparticles.

We synthesized novel tetraphenylethene (TPE) conjugates, which undergo unique self-assembly to form spherical nanoparticles that exhibited aggregation induced emission (AIE) in the near-infrared region. These nanoparticles showed significant singlet oxygen generation efficiency, negligible dark toxicity, rapid cellular uptake, efficient localization in cytoplasm, and high in vitro photocytotoxicity as well as in vivo photodynamic activity against a human prostate tumor animal model. This study demonstrates, for the first time, the power of the self-assembled AIE active tetraphenylethene conjugates in aqueous media as a nanoplatform for future therapeutic applications.

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.

Amino Acid-Porphyrin Conjugates: Synthesis and Study of their Photophysical and Metal Ion Recognition Properties.

Synthesis, photophysical and metal ion recognition properties of a series of amino acid-linked free-base and Zn-porphyrin derivatives (5-9) are reported. These porphyrin derivatives showed favorable photophysical properties including high molar extinction coefficients (>1 × 10(5) m(-1) cm(-1) for the Soret band), quantum yields of triplet excited states (63-94%) and singlet oxygen generation efficiencies (59-91%). Particularly, the Zn-porphyrin derivatives, 6 and 9 showed higher molar extinction coefficients, decreased fluorescence quantum yields, and higher triplet and singlet oxygen quantum yields compared to the corresponding free-base porphyrin derivatives. Further, the study of their interactions with various metal ions indicated that the proline-conjugated Zn-porphyrins (6 and 9) showed high selectivity toward Cu(2+) ions and signaled the recognition through changes in fluorescence intensity. Our results provide insights on the role of nature of amino acid and metallation in the design of the porphyrin systems for application as probes and sensitizers.

Enhancement in intramolecular interactions and in vitro biological activity of a tripodal tetradentate system upon complexation.

Novel biomimetic mononuclear complexes, [Fe()Cl2](+) () and [Cu()(H2O)](2+) () based on naphthalimide appended tripodal tetradentate ligand ( = 2,2',2''-(3,3',3''-(2,2',2''-nitrilotris(methylene)tris(1H-benzo[d]imidazole-2,1-diyl))tris(propane-3,1-diyl))tris(1H-benzo-[de]isoquinoline-1,3(2H)-dione)) have been synthesized and characterized by various analytical and spectral techniques. In addition, the structures of the ligand () and complex were established unambiguously through X-ray crystal structure analysis. Uniquely, the coordination with a metal ion modified the ligand scaffold to interact efficiently with ct-DNA (groove binding) as well as protein (hydrophobic and/or electrostatic interactions). We have determined the affinity of these complexes for DNA/protein and the values are found to be in the range, KDNA = 0.34-1.01 × 10(4) M(-1) and KBSA = 4.1-5.0 × 10(5) M(-1). Furthermore, the fluorescence quenching of BSA with complexes and occurs through a static mechanism and affects the conformation of BSA around the tryptophan residues. The in vitro biological studies of these systems employing HeLa cell lines indicated that both these complexes exhibited enhanced cytotoxicity (IC50 = 32 ± 0.19 and 10 ± 0.21 µM for complexes and , respectively), when compared to the ligand () (IC50 = 150 µM). Interestingly, both the complexes ( and ) were found to be non-toxic to normal H9C2 cell lines. The mechanism of in vitro biological activity of these complexes has been evaluated through a variety of techniques: acridine orange/ethidium bromide, DAPI staining studies, annexin V-FITC/PI and poly(ADP-ribose)-polymerase (PARP) cleavage, which confirmed the apoptotic mediated cell death. Our results demonstrate the importance of complexation of the naphthalimide ligand () as well as the potential of these biomimetic metal complexes as cytotoxic and anticancer agents.

Effective Amyloid Defibrillation by Polyhydroxyl-Substituted Squaraine Dyes.

With an objective to develop ß-amyloid destabilizing agents, we have investigated the interactions of a few water-soluble near-infrared (NIR)-absorbing squaraine dyes 1-3 with lysozyme and its amyloid aggregates through photophysical and biophysical techniques. These dyes exhibited strong interactions with lysozyme and ß-amyloids in addition to serum albumins as evidenced by the absorption and emission changes. The interactions were found to be spontaneous with association constant values in the range of approximately 10(4)-10(5) m(-1), as confirmed through half-reciprocal analysis and isothermal calorimetric measurements. Uniquely, such effective interactions of the dyes have led to the complete disassembly of the ß-amyloid fibrillar structures to form spherical particles approximately 350 nm in size, as confirmed through photophysical, thioflavin assay, circular dichroism (CD), atomic force microscopy (AFM), TEM, and selected-area electron diffraction (SAED) techniques. These results demonstrate that the squaraine dyes 1-3 under investigation act as effective protein-labelling and destabilizing agents of the protein amyloidogenesis.

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.

A reversible dual mode chemodosimeter for the detection of cyanide ions in natural sources.

Herein, we report the synthesis of two indolium probes 1 and 2 based on anthracene and pyrene derivatives and their interactions with various anions. Of these probes, the pyrene conjugate 2 acts as a dual colorimetric and fluorescent chemodosimeter for the selective and sensitive detection of cyanide ions. The detection limit of probe 2 for CN(-) ions was found to be 10 ppb (30 nM). The nature of interaction has been thoroughly studied through various techniques such as (1)H NMR and IR spectroscopy, HRMS, and isothermal calorimetric (ITC) studies. These studies confirm that probe 2 forms a 1,2-adduct in the presence of CN(-) ions. Kinetic studies using probe 2 showed the completion of the reaction within 15 s with a rate constant of k' = 0.522±0.063 s(-1). This probe can be coated on a solid surface (dipstick) and a polymer matrix for the on-site analysis and quantification of endogenous cyanide ions in natural sources such as Indian almonds.

Antimicrobial photodynamic efficiency of novel cationic porphyrins towards periodontal Gram-positive and Gram-negative pathogenic bacteria.

The Gram-negative Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are major causative agents of aggressive periodontal disease. Due to increase in the number of antibiotic-resistant bacteria, antimicrobial Photodynamic therapy (aPDT) seems to be a plausible alternative. In this work, photosensitization was performed on Gram-positive and Gram-negative bacteria in pure culture using new-age cationic porphyrins, namely mesoimidazolium-substituted porphyrin derivative (ImP) and pyridinium-substituted porphyrin derivative (PyP). The photophysical properties of both the sensitizers including absorption, fluorescence emission, quantum yields of the triplet excited states and singlet oxygen generation efficiencies were evaluated in the context of aPDT application. The studied porphyrins exhibited high ability to accumulate into bacterial cells with complete penetration into early stage biofilms. As compared with ImP, PyP was found to be more effective for photoinactivation of bacterial strains associated with periodontitis, without any signs of dark toxicity, owing to its high photocytotoxicity.

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.

Optimization of triplet excited state and singlet oxygen quantum yields of picolylamine-porphyrin conjugates through zinc insertion.

We synthesized a new class of picolylamine-porphyrin conjugates 1-3 and have investigated the effect of heavy atom insertion on their intersystem crossing efficiency through spin-orbit perturbations. By incorporating zinc ions in the core as well as periphery positions of the porphyrin ring, we have successfully optimized their triplet excited state quantum yields and their efficiency to generate singlet oxygen. Uniquely, the picolylamine-porphyrin conjugate 3 having five zinc ions exhibited a triplet excited state quantum yield of ca. 0.97 and a sensitized singlet oxygen generation yield of ca. 0.92. In contrast, the free base porphyrin derivative 1 exhibited ca. 0.64 and 0.5 of the triplet excited state and singlet oxygen quantum yields, respectively. Our results demonstrate that the insertion of zinc metal ions in the picolylamine-porphyrin conjugates not only quantitatively enhances the triplet excited state and singlet oxygen yields but also imparts hydrophilicity, thereby their potential use as sensitizers in photodynamic therapy and green photooxygenation reactions.

White photoluminescence and electroluminescence from a ternary system in solution and a polymer matrix.

Efficient white photoluminescence was obtained from a ternary system in solution and a polymer matrix when excited at 380 nm whereas at 560 nm, it showed intense red emission. The LED device fabricated based on this system exhibited white electroluminescence with a low turn on voltage of 3 V.