Amplification of reactive oxygen species (ROS) generation through covalent conjugation of bovine serum albumin (BSA) with newly synthesized, ROS-producing carbon dots (CDs) upon visible light irradiation is reported for the first time. Derivatization of surface carboxyl functional groups of Anthrarufin-derived, green-emitting CD with the amine functionality of BSA ushers distinct changes in the photophysics of CD including an unprecedented ∼50 nm shift in its excitation maxima, decrease in fluorescence lifetime, and concomitant increase in ROS generation. Substantial conformational changes of BSA were witnessed upon conjugation with CD, rendering the BSA-CD conjugate resistant to pepsinolysis. A protease-proof nanoassembly was derived from the BSA-CD conjugate through desolvation that simultaneously hosts a prototype antibiotic and generates ROS with excellent efficiency, making it an attractive platform for antibacterial photodynamic therapy (A-PDT) applications. Systemic annihilation of both Gram-positive and -negative bacteria was achieved with the BSA-CD nanoassembly and envisioned as alternatives to traditional photosensitizers.

BackgroundAmphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity.MethodsCitrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet–visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB.ResultsInfrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01).ConclusionGNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

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Contrary to the belief of the chemistry community, intense charge transfer is observed between meta-oriented donor–acceptor moieties in ultrasmall single-benzenic fluorophores. Red emission in any solvent has so far been reported with molecular fluorophores having the lowest molecular weight (MW) of 252.5 Da; however, we have achieved the same with a meta-fluorophore having an MW of only 203.1 Da. Red emission in a nonpolar solvent has so far been reported with a fluorophore having a minimum MW of 464.5 Da, but we have achieved the same with a meta-fluorophore having an MW of only 255.3 Da. Intense Stokes (260 nm) and solvatochromic (160 nm) shifts, high magnitudes of fluorescence quantum yield (>0.4), and excited-state lifetime (>16 ns) have been obtained in these single-benzenic meta-fluorophores, and these values are comparatively much higher in comparison to corresponding o-/p-fluorophores. These extraordinary meta-fluorophores have been employed to measure subcellular nanopolarity in live stem cells toward cost-effective white fluorescent ink and white light-emitting diodes (LEDs) in solid state.

The biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor from tyrosine at the active site of copper amine oxidases is believed to proceed along a pathway that includes a conjugate addition of water to the corresponding o-quinone intermediate, followed by autoxidation of the resulting benzenetriol to the hydroxyquinone cofactor. The water addition reaction has been presumed to occur not only in previous model studies reported for cofactor biogenesis starting with either catechol or o-quinone, but also for generation of the neurotoxin 6-hydroxydopamine during autoxidation of dopamine. We here report the surprising finding that water addition does not occur under solution chemistry conditions. The production of hydroxyquinone from catechol arises instead from reaction of the o-quinone with H 2 O 2 generated during autoxidation of catechol. When starting with the o-quinone itself, production of hydroxyquinone still arises from autoxidation of the catechol, generated either by reduction of the o-quinone by its decomposition products at moderate pH, or by a novel base-mediated redox disproportionation of the o-quinone at high pH. These conclusions are supported by the behavior of independently studied o-quinone intermediates, the observed effects of added catalase, and 18 O-labeling studies utilizing both [ 18 O]H 2 O and [ 18 O]O 2 . The failure to observe water addition to the o-quinone has broad implications for aqueous o-quinone chemistry, and suggests that in TPQ biogenesis, this hydration is being catalyzed at the enzyme active site, possibly by the bound copper.