In Vitro Photoselective Gene Transfection of Hepatocellular Carcinoma Cells with Hypericin Lipopolyplexes.

The lipopolyplex, a multicomponent nonviral gene carrier, generally demonstrates superior colloidal stability, reduced cytotoxicity, and high transfection efficiency. In this study, a new concept, photochemical reaction-induced transfection, using photosensitizer (PS)-loaded lipopolyplexes was applied, which led to enhanced transfection and cytotoxic effects by photoexcitation of the photosensitizer. Hypericin, a hydrophobic photosensitizer, was encapsulated in the lipid bilayer of liposomes. The preformed nanosized hypericin liposomes enclosed the linear polyethylenimine (lPEI)/pDNA polyplexes, resulting in the formation of hypericin lipopolyplexes (Hy-LPP). The diameters of Hy-LPP containing 50 nM hypericin and 0.25 µg of pDNA were 185.6 ± 7.74 nm and 230.2 ± 4.60 nm, respectively, measured by dynamic light scattering (DLS) and atomic force microscopy (AFM). Gel electrophoresis confirmed the encapsulation of hypericin and pDNA in lipopolyplexes. Furthermore, in vitro irradiation of intracellular Hy-LPP at radiant exposures of 200, 600, and 1000 mJ/cm2 was evaluated. It demonstrated 60- to 75-fold higher in vitro luciferase expression than that in nonirradiated cells. The lactate dehydrogenase (LDH) assay supported that reduced transfection was a consequence of photocytotoxicity. The developed photosensitizer-loaded lipopolyplexes improved the transfection efficiency of an exogenous gene or induced photocytotoxicity; however, the frontier lies in the applied photochemical dose. The light-triggered photoexcitation of intracellular hypericin resulted in the generation of reactive oxygen species (ROS), leading to photoselective transfection in HepG2 cells. It was concluded that the two codelivered therapeutics resulted in enhanced transfection and a photodynamic effect by tuning the applied photochemical dose.

Mechanism of non-phenolic substrate oxidation by the fungal laccase Type 1 copper site from Trametes versicolor: the case of benzo[a]pyrene and anthracene.

Laccases (EC 1.10.3.2) are multicopper oxidases with the capability to oxidize diverse phenolic and non-phenolic substrates. While the molecular mechanism of their activity towards phenolic substrates is well-established, their reactivity towards non-phenolic substrates, such as polycyclic aromatic hydrocarbons (PAHs), remains unclear. To elucidate the oxidation mechanism of PAHs, particularly the activation mechanism of the sp2 aromatic C-H bond, we conducted a density functional theory investigation on the oxidation of two PAHs (anthracene and benzo[a]pyrene) using an extensive model of the T1 copper catalytic site of the fungal laccase from Trametes versicolor.

Construction and synergistic anti-tumor study of a tumor microenvironment-based multifunctional nano-drug delivery system.

To solve the problems existing in the clinical application of hypericin (Hyp) and tirapazamine (TPZ), a nano-drug delivery system with synergistic anti-tumor functions was constructed using mesoporous silica nanoparticles (MSN) and sodium alginate (SA). The system exhibited excellent stability, physiological compatibility and targeted drug release performance in tumor tissues. In the in vitro and in vivo experiments, Hyp released from MSN killed tumor cells through photodynamic therapy (PDT). The degree of hypoxia in the tumor tissue site was exacerbated, enabling TPZ to fully exert its anti-tumor activity. Our studies suggested that the synergistic effects between the components of the nano-drug delivery system significantly improve the anti-tumor properties of Hyp and TPZ.

A spermine-responsive supramolecular chemotherapy system constructed from a water-soluble pillar[5]arene and a diphenylanthracene-containing amphiphile for precise chemotherapy.

Stimuli-responsive supramolecular chemotherapy, particularly in response to cancer biomarkers, has emerged as a promising strategy to overcome the limitations associated with traditional chemotherapy. Spermine (SPM) is known to be overexpressed in certain cancers. In this study, we introduced a novel supramolecular chemotherapy system triggered by SPM. The system featured pyridine salts of a diphenylanthracene derivative (PyEn) and a complementary water-soluble pillar[5]arene (WP5C5) with long alkyl chains. The diphenylanthracene unit of PyEn is effectively encapsulated within the long alkyl chains of WP5C5, resulting in a substantial reduction in the cytotoxicity of PyEn towards normal cells. The therapeutic effect of PyEn is selectively triggered intracellularly through SPM, leading to the endosomal release of PyEn and concurrent in situ cytotoxicity. This supramolecular chemotherapy system exhibits notable tumor inhibition against SPM-overexpressed cancers with reduced side effects on normal tissues. The supramolecular strategy for intracellular activation provides a novel tool with potential applications in chemotherapeutic interventions, offering enhanced selectivity and reduced cytotoxicity to normal cells.

Fluorescence lifetime nanothermometer based on the equilibrium formation of anthracene AIE-excimers in living cells.

The effective measurement of temperature in living systems at the nano and microscopic scales continues to be a challenge to this day. Here, we study the use of 2-(anthracen-2-yl)-1,3-diisopropylguanidine, 1, as a nanothermometer based on fluorescence lifetime measurements and its bioimaging applications. In aqueous solution, 1 is shown in aggregated form and the equilibrium between the two main aggregate types (T-shaped and π-π) is highly sensitive to the temperature. The heating of the medium shifts the equilibrium toward the formation of highly emissive T-shaped aggregates. This species shows a high fluorescence emission and a long lifetime in comparison with the π-π aggregates and the freé monomer. A linear relationship between the fluorescence lifetime and the temperature both in aqueous solution and in a synthetic intracellular buffer was found. Fluorescence lifetime imaging microscopy (FLIM) also showed a linear relationship between lifetime and temperature with an excellent sensitivity in MCF7 breast cancer cells, which opens the door for its potential use as FLIM nanothermometer in the biomedical field.

Carbohydrate-Functionalized Anthracene Carboximides as Multivalent Ligands and Bio-Imaging Agents.

Anthracene carboximides (ACIs) conjugated with gluco-, galacto- and mannopyranosides are synthesized, by glycosylation of N-hydroxyethylanthracene carboximide acceptor with glycosyl donors. Glycoconjugation of anthracene carboximide increases the aq. solubility by more than 3-fold. The glycoconjugates display red-shifted absorption and emission, as compared to anthracene. Large Stokes shift (λabs/λem=445/525 nm) and high fluorescence quantum yields (Φ) of 0.86 and 0.5 occur in THF and water, respectively. The ACI-glycosides undergo facile photodimerization in aqueous solutions, leading to the formation of the head-to-tail dimer, as a mixture of syn and anti-isomers. Solution phase and solid-state characterizations by dynamic light scattering (DLS), microscopic imaging by atomic force (AFM) and transmission electron (TEM) microscopies reveal self-assembled vesicle structures of ACI glycosides. These self-assembled structures act as multivalent glycoclusters for ligand-specific lectin binding, as evidenced by the binding of Man-ACI to Con A, by fluorescence and turbidity assays. The conjugates do not show cellular cytotoxicity (IC50) till concentrations of 50 µM with HeLa and HepG2 cell lines and are cell-permeable, showing strong fluorescence inside the cells. These properties enable the glycoconjugates to be used in cell imaging. The non-selective cellular uptake of the glycoconjugates suggests a passive diffusion through the membrane.

Enhanced photocatalytic degradation of polycyclic aromatic hydrocarbons (PAHs) Using NiO nanoparticles.

The oncogenic and genetic properties of anthracene, a member of the polycyclic aromatic hydrocarbons (PAHs) family, pose a significant health threat to humans. This study aims to investigate the photocatalytic decomposition of anthracene under various conditions, such as different concentrations of PAHs, varying amounts of NiO (nickel oxide) nanoparticles, and different pH levels under ultraviolet light and sunlight. The synthesized NiO nanoparticles showed surface plasma resonance at 230 and 360 nm, while XRD and SEM analysis confirmed the nanoparticles were cubic crystalline in structure with sizes ranging between 37 and 126 nm. NiO nanoparticles exhibited 79% degradation of pyrene at 2 µg/mL of anthracene within 60 min of treatment. NiO at 10 µg/mL concentration showed significant adsorption of 57%, while the adsorption method worked efficiently (72%) at 5 pH. Photocatalytic degradation was confirmed by isotherm and kinetic studies through monolayer adsorption and pseudo-first-order kinetics. Further, the absorption process was confirmed by performing GC-MS analysis of the NiO nanoparticles. On the other hand, NiO nanoparticles showed antimicrobial activity against Gram negative and Gram-positive bacteria. Therefore, the present work is one of its kind proving the dual application of NiO nanoparticles, which makes them suitable candidates for bioremediation by treating PAHs and killing pathogenic bacteria.

In Vivo Imaging of Tumor Hypoxia by Maintaining Green Fluorescence of 9-Aminoanthracene Under Hypoxic Conditions.

In this study, 9-aminoanthracene (9AA) was used as a new fluorescence reagent for the in vivo imaging of tumor hypoxia by taking advantage of the maintenance of its green fluorescence under hypoxic conditions. As 9AA is insoluble in water, polyethylene glycol (PEG)-400 was used to dissolve 9AA in saline. Each organ was successfully stained with 9AA, as observed by green fluorescence using in vivo imaging, following intragastric administration of a 9AA PEG-saline solution in mice. Therefore, the intragastric administration of 9AA can be used for in vivo imaging of normal mice. Tumor hypoxia staining using the 9AA fluorescence method was evaluated by in vivo imaging of mice subcutaneously transplanted with Ehrlich ascites carcinoma cells and compared with conventional pimonidazole (PIMO) staining under hypoxic conditions. The tumor sections were stained with green fluorescence derived from 9AA and the same sections corresponded to hypoxic areas upon immunohistochemical staining with PIMO.

Regeneration of Phytochemicals by Structure-Driven Organization of Microbial Biosynthetic Steps.

Medicinal phytochemicals, such as artemisinin and taxol, have impacted the world, and hypericin might do so if its availability issue could be addressed. Hypericin is the hallmark component of Saint John's wort (Hypericum perforatum L.), an approved depression alleviator documented in the US, European, and British pharmacopoeias with its additional effectiveness against diverse cancers and viruses. However, the academia-to-industry transition of hypericin remain hampered by its low in planta abundance, unfeasible bulk chemical synthesis, and unclear biosynthetic mechanism. Here, we present a strategy consisting of the hypericin-structure-centered modification and reorganization of microbial biosynthetic steps in the repurposed cells that have been tamed to enable the designed consecutive reactions to afford hypericin (43.1 mg L-1 ), without acquiring its biosynthetic knowledge in native plants. The study provides a synthetic biology route to hypericin and establishes a platform for biosustainable access to medicinal phytochemicals.

Hypericin-loaded oil-in-water nanoemulsion synthesized by ultrasonication process enhances photodynamic therapy efficiency.

Hypericin (Hy) is a hydrophobic photosensitizer used in photodynamic therapy for cancer therapeutic. In this study, Hy-loaded oil-in-water (O/W) nanoemulsions (NEs) were produced by the ultrasonication method combing different biocompatible oils and surfactants to enhance Hy aqueous solubility and bioavailability. Experimental parameters were optimized by the characterization of droplet size, zeta potential, and physicochemical properties. In vitro studies based on the release profile, cytotoxicity, cell morphology, and Hy intracellular accumulation were assayed. Hy at 100 mg L-1 was incorporated into the low viscosity (~0.005 Pa s) NEs with spherical droplets averaging 20-40 nm in size and polydispersity index <0.02. Hy release from the NE was significantly higher (4-fold) than its suspension (p < 0.001). The NEs demonstrated good physical stability during storage at 5 °C for at least six months. The Hy-loaded NEs exhibited an IC50 value 6-fold lower than Hy suspension during PDT against breast cancer cell lines (MCF-7). Cell microscopy imaging confirmed the increased cytotoxic effects of Hy-loaded NEs, showing damaged and apoptotic cells. Confocal laser scanning microscopy evidenced greater Hy delivery through NE into MCF-7 cells followed by improved intracellular ROS generation. Our results suggest that the Hy-loaded NEs can improve hypericin efficacy and assist Hy-PDT's preclinical development as a cancer treatment.

The first copper(I) complex of anthrahydrazone with potential ROS scavenging activity showed significant in vitro anticancer activity by inducing apoptosis and autophagy.

Based on the anticancer pharmacophore of anthrahydrazone and quinoline, a new quinolylanthrahydrazone ligand, 9-AQH (anthracene-9-quinolylhydrazone), was synthesized to further afford four metal complexes, [CoII(9-AQH)(NO3)2(H2O)] (1), [NiII(9-AQH)2(H2O)2]·2NO3 (2), [CuI(9-AQH)2]·NO3 (3), [ZnII(9-AQH)2(NO3)]·NO3 (4), determined by X-ray single crystal diffraction analysis. The reaction of Cu(NO3)2 with 9-AQH formed the stable and repeatable copper(I) complex 3. In vitro screening demonstrated only 3 showed significant and broad-spectrum anticancer activity, indicating that Cu(I) played a key role in exerting the anticancer activity. In solution, Cu(I) was not naturally oxidized to Cu(II) suggested by 1H-NMR (Nuclear Magnetic Resonance) and EPR (Electron Paramagnetic Resonance) analysis. The presence of 3 could also catalyze the H2O2 system to give hydroxyl free radicals, suggested by further EPR and electrophoresis assay. At the cellular level, although no obvious Cu(II) signals were detected and the total ROS (Reactive Oxygen Species) scavenging in the tumor cells treated with 3, the potential redox property between Cu(I)/Cu(II), as a key role, should not be denied for the significant anticancer activity of 3, considering the much complicated circumstance and other reductive substances in cells. The anticancer mechanism of 3 on the most sensitive MGC-803 cells pointed to significant cell apoptosis through mitochondrial pathway, rather than cell cycle arrest. While the autophagy observed in tumor cells treated by 3 suggested its complicated anticancer mechanism, and whether there was an intrinsic correlation still needed to be further investigated.

Plasmid DNA-Based Bioluminescence-Activated System for Photodynamic Therapy in Cancer Treatment.

The low depth of tissue penetration by therapeutic light sources severely restricts photodynamic therapy (PDT) in treating deep-seated tumors. Using a luciferase/d-luciferin bioluminescence system to artificially create internal light sources in cells instead of external light sources is an effective means of solving the above problems. However, high-efficiency bioluminescence requires a higher concentration of luciferase in the cell, which poses a considerable challenge to the existing system of enzyme loading, delivery, activity and retention of drugs, and dramatically increases the cost of treatment. We loaded the substrate D-luciferin, and the photosensitizer hypericin into a polyethyleneimine (PEI)-modified nano-calcium phosphate (CaP) to solve this problem. Subsequently, the plasmid DNA containing the luciferase gene was loaded onto it using the high-density positive charge characteristic of PEI from the nanodrug (denoted DHDC). After the DHDC enters the tumor cell, it collapses and releases the plasmid DNA, which uses the intracellular protein synthesis system to continuously and massively express luciferase. Using endogenous ATP, Mg2+ , and O2 in cells, luciferase oxidizes d-luciferin and produces luminescence. The luminescence triggers hypericin excitation to generate ROS and kill cancer cells. This study provides a new strategy for the application of bioluminescence in PDT treatment.

Aggregation-induced emission fluorophores based on strong electron-acceptor 2,2'-(anthracene-9,10-diylidene) dimalononitrile for biological imaging in the NIR-II window.

In this communication, three novel donor-acceptor-donor type second near-infrared AIE fluorophores based on strong electron-acceptor 2,2'-(anthracene-9,10-diylidene) dimalononitrile have been successfully developed for bioimaging, in which they exhibit good photostability and can be used in mouse ear vessel imaging with high resolution (FWHM = 93.4 µm/SBR = 1.5) after capsuling in nanoparticles with good biocompatibility.

Harvest time affects antioxidant capacity, total polyphenol and flavonoid content of Polish St John's wort's (Hypericum perforatum L.) flowers.

The polyphenol content and antioxidant capacity of hyperforin and hypericin-standardized H. perforatum L. extracts may vary due to the harvest time. In this work, ethanol and ethanol-water extracts of air-dried and lyophilized flowers of H. perforatum L., collected throughout a vegetation season in central Poland, were studied. Air-dried flowers extracts had higher polyphenol (371 mg GAE/g) and flavonoid (160 mg CAE/g) content, DPPH radical scavenging (1672 mg DPPH/g), ORAC (5214 µmol TE/g) and FRAP (2.54 mmol Fe2+/g) than lyophilized flowers extracts (238 mg GAE/g, 107 mg CAE/g, 1287 mg DPPH/g, 3313 µmol TE/g and 0.31 mmol Fe2+/g, respectively). Principal component analysis showed that the collection date influenced the flavonoid and polyphenol contents and FRAP of ethanol extracts, and DPPH and ORAC values of ethanol-water extracts. The ethanol extracts with the highest polyphenol and flavonoid content protected human erythrocytes against bisphenol A-induced damage. Both high field and benchtop NMR spectra of selected extracts, revealed differences in composition caused by extraction solvent and raw material collection date. Moreover, we have shown that benchtop NMR can be used to detect the compositional variation of extracts if the assignment of signals is done previously.

Real-Time Ratiometric Imaging of Micelles Assembly State in a Microfluidic Cancer-on-a-Chip.

The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability.

Non-Polymeric Nanogels as Versatile Nanocarriers: Intracellular Transport of the Photosensitizers Rose Bengal and Hypericin for Photodynamic Therapy.

The use of nanocarriers for intracellular transport of actives has been extensively studied in recent years and represents a central area of nanomedicine. The main novelty of this paper lies on the use of nanogels formed by a low-molecular-weight gelator (1). Here, non-polymeric, molecular nanogels are successfully used for intracellular transport of two photodynamic therapy (PDT) agents, Rose Bengal (RB) and hypericin (HYP). The two photosensitizers (PSs) exhibit different drawbacks for their use in clinical applications. HYP is poorly water-soluble, while the cellular uptake of RB is hindered due to its dianionic character at physiological pH values. Additionally, both PSs tend to aggregate precluding an effective PDT. Despite the different nature of these PSs, nanogels from gelator 1 provide, in both cases, an efficient intracellular transport into human colon adenocarcinoma cells (HT-29) and a notably improved PDT efficiency, as assessed by confocal laser scanning microscopy and flow cytometry. Furthermore, no significant dark toxicity of the nanogels is observed, supporting the biocompatibility of the delivery system. The developed nanogels are highly reproducible due to their non-polymeric nature, and their synthesis is easily scaled up. The results presented here thus confirm the potential of molecular nanogels as valuable nanocarriers, capable of entrapping both hydrophobic and hydrophilic actives, for PDT of cancer.

Diels-Alder Additions as Mechanistic Probes-Interception of Silyl-Isoindenes: Organometallic Derivatives of Polyphenylated Cycloheptatrienes and Related Seven-Membered Rings.

The intermediacy of short-lived isoindenes, generated in the course of metallotropic or silatropic shifts over the indene skeleton, can be shown by Diels-Alder trapping with tetracyanoethylene, leading to the complete elucidation of the dynamic behaviour of a series of polyindenylsilanes. Cyclopentadienones, bearing ferrocenyl and multiple phenyl or naphthyl substituents undergo [4 + 2] cycloadditions with diaryl acetylenes or triphenylcyclopropene to form the corresponding polyarylbenzenes or cycloheptatrienes. The heptaphenyltropylium cation, [C7Ph7+], was shown to adopt a nonplanar shallow boat conformation. In contrast, the attempted Diels-Alder reaction of tetracyclone and phenethynylfluorene yielded electroluminescent tetracenes. Finally, benzyne addition to 9-(2-indenyl)anthracene, and subsequent incorporation of a range of organometallic fragments, led to development of an organometallic molecular brake.

Remote-controlled regio- and diastereodifferentiating photodimerization of a dynamic helical peptide-bound 2-substituted anthracene.

Photodimerization of a novel 2-substituted anthracene linked to a right-handed 310-helical nonapeptide induced by long-range chiral information transfer from the remote chiral l-Val residue through a chiral domino effect proceeded in a highly regio- and diastereo-differentiating manner to produce the chiral head-to-head anti-photodimer in 90% relative yield with up to 97% diastereomeric excess.

Heck reaction synthesis of anthracene and naphthalene derivatives as traps and clean chemical sources of singlet molecular oxygen in biological systems.

Studies have previously shown that anthracene and naphthalene derivatives serve as compounds for trapping and chemically generating singlet molecular oxygen [O2(1Δg)], respectively. Simple and efficient synthetic routes to anthracene and naphthalene derivatives are needed, for improved capture and release of O2(1Δg) in cellular environments. Because of this need, we have synthesized a dihydroxypropyl amide naphthlene endoperoxide as a O2(1Δg) donor, as well as five anthracene derivatives as O2(1Δg) acceptor. The anthracene derivatives bear dihydroxypropyl amide, ester, and sulfonate ion end groups connected to 9,10-positions by way of unsaturated (vinyl) and saturated (ethyl) bridging groups. Heck reactions were found to yield these six compounds in easy-to-carry out 3-step reactions in yields of 50-76%. Preliminary results point to the potential of the anthracene compounds to serve as O2(1Δg) acceptors and would be amenable for future use in biological systems to expand the understanding of O2(1Δg) in biochemistry.

Substrate-Dependent Modulation of SIRT2 by a Fluorescent Probe, 1-Aminoanthracene.

Sirtuin isoform 2 (SIRT2) is an enzyme that catalyzes the removal of acyl groups from lysine residues. SIRT2's catalytic domain has a hydrophobic tunnel where its substrate acyl groups bind. Here, we report that the fluorescent probe 1-aminoanthracene (AMA) binds within SIRT2's hydrophobic tunnel in a substrate-dependent manner. AMA's interaction with SIRT2 was characterized by its enhanced fluorescence upon protein binding (>10-fold). AMA interacted weakly with SIRT2 alone in solution (Kd = 37 µM). However, when SIRT2 was equilibrated with a decanoylated peptide substrate, AMA's affinity for SIRT2 was enhanced ∼10-fold (Kd = 4 µM). The peptide's decanoyl chain and AMA co-occupied SIRT2's hydrophobic tunnel when bound to the protein. In contrast, binding of AMA to SIRT2 was competitive with a myristoylated substrate whose longer acyl chain occluded the entire tunnel. AMA competitively inhibited SIRT2 demyristoylase activity with an IC50 of 21 µM, which was significantly more potent than its inhibition of other deacylase activities. Finally, binding and structural analysis suggests that the AMA binding site in SIRT2's hydrophobic tunnel was structurally stabilized when SIRT2 interacted with a decanoylated or 4-oxononanoylated substrate, but AMA's binding site was less stable when SIRT2 was bound to an acetylated substrate. Our use of AMA to explore changes in SIRT2's hydrophobic tunnel that are induced by interactions with specific acylated substrates has implications for developing ligands that modulate SIRT2's substrate specificity.