Ferric Ion Driven Assembly of Catalase-like Supramolecular Photosensitizing Nanozymes for Combating Hypoxic Tumors.

A facile approach to assemble catalase-like photosensitizing nanozymes with a self-oxygen-supplying ability was developed. The process involved Fe3+ -driven self-assembly of fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids. By adding a zinc(II) phthalocyanine-based photosensitizer (ZnPc) and the hypoxia-inducible factor 1 (HIF-1) inhibitor acriflavine (ACF) during the Fe3+ -promoted self-assembly of Fmoc-protected cysteine (Fmoc-Cys), the nanovesicles Fmoc-Cys/Fe@Pc and Fmoc-Cys/Fe@Pc/ACF were prepared, which could be disassembled intracellularly. The released Fe3+ could catalyze the transformation of H2 O2 enriched in cancer cells to oxygen efficiently, thereby ameliorating the hypoxic condition and promoting the photosensitizing activity of the released ZnPc. With an additional therapeutic component, Fmoc-Cys/Fe@Pc/ACF exhibited higher in vitro and in vivo photodynamic activities than Fmoc-Cys/Fe@Pc, demonstrating the synergistic effect of ZnPc and ACF.

Supramolecular Assembly of Acriflavine on Graphene Oxide for the Sensing of Adenosine Phosphates.

An acriflavine-graphene oxide (GAF) supramolecular assembly has been prepared from water-soluble graphene oxide (GO) and a fluorescent dye, acriflavine (AF). Upon binding this non-covalently to the GO, the fluorescence of acriflavine has been "turned off" effectively, competitive binding potential of the sensor substrates such as ATP, ADP, AMP and the pyrophosphate weakens the supramolecular assembly of GAF, which allows the release of acriflavine quantitatively, which also "turns-on" the fluorescence of the dye under UV irradiation. Interestingly, GAF displayed the highest sensitivity towards ATP within the family of adenosine phosphates. We have developed a naked eye detection method for the adenosine phosphates biomolecules. For the first time, acriflavine has been utilized for the sensing of adenosine phosphates in combination with GO, which can be useful for the detection of other biomolecules.

Overcoming Radioresistance in Tumor Therapy by Alleviating Hypoxia and Using the HIF-1 Inhibitor.

Radiotherapy has been extensively used to treat cancer patients because it can effectively damage most solid tumors without penetration limits. A hypoxic microenvironment in solid tumors leads to severe radioresistance and expression of hypoxic inducible factor-1 (HIF-1), which results in poor efficacy of radiotherapy alone. Herein, we report the excellent efficacy of radiotherapy achieved using a new type of yolk-shell Cu2-xSe@PtSe (CSP) nanosensitizer functionalized with the HIF-1α inhibitor acriflavine (ACF). We prepare the CSP nanosensitizer through the interfacial redox reactions between chloroplatinic acid and Cu2-xSe nanoparticles (CS) and then functionalize the nanosensitizer with ACF through their electrostatic interactions. We show that the synthesized CSP nanosensitizer can arrest the cell cycle (i.e., at the gap 2/mitosis (G2/M) phases) of tumor cells to enhance their sensitivity to X-rays and decompose endogenous H2O2 into O2 to reduce hypoxia and increase the production of reactive oxygen species, which leads to severe damage to DNA double strands and apoptosis of tumor cells. We also show that the ACF on the surface of CSP nanoparticles can effectively reduce the expression of HIF-1α. All these effects lead to a low vascular endothelial growth factor, low density of microvessels in tumor, decreased cell proliferation, and increased cell apoptosis, which synergistically and drastically enhance the efficacy of radiotherapy. This work provides insights and guidance for developing novel nanosensitizers to enhance the efficacy of radiotherapy.

Bifunctional liposomes reduce the chemotherapy resistance of doxorubicin induced by reactive oxygen species.

Doxorubicin (DOX) liposome is a widely used nano-medicine for colorectal cancer treatment. However, doxorubicin therapy increases the level of reactive oxygen species (ROS) in tumor cells, such as hydrogen peroxide (H2O2), which can stabilize hypoxia-inducible-factor-1α (HIF-1α). In a tumor hypoxic microenvironment, HIF-1 can up-regulate tumor-resistance related proteins, including P-glycoprotein (P-gp), glucose transporter 1 (GLUT-1), and matrix metalloproteinase 9 (MMP-9), leading to tumor tolerance to chemotherapy. The functional inhibition of HIF-1 can overcome this resistance and enhance the efficacy of tumor therapy. Here, we encapsulated one of the most effective HIF-1 inhibitors, acriflavine (ACF), and DOX in liposomes (DOX-ACF@Lipo) to construct bifunctional liposomes. ACF and DOX, released from DOX-ACF@Lipo, could effectively suppress the function of HIF-1 and the process of DNA replication, respectively. Consequently, the bifunctional liposome has great potential to be applied in clinics to overcome chemotherapy resistance induced by hypoxia.

The evolution of Bordetella pertussis has selected for mutations of acr that lead to sensitivity to hydrophobic molecules and fatty acids.

Whooping cough, or pertussis, is resurgent in numerous countries worldwide. This has renewed interest in Bordetella pertussis biology and vaccinology. The in vitro growth of B. pertussis has been a source of difficulty, both for the study of the organism and the production of pertussis vaccines. It is inhibited by fatty acids and other hydrophobic molecules. The AcrAB efflux system is present in many different bacteria and in combination with an outer membrane factor exports acriflavine and other small hydrophobic molecules from the cell. Here, we identify that the speciation of B. pertussis has selected for an Acr system that is naturally mutated and displays reduced activity compared to B. bronchiseptica, in which the system appears intact. Replacement of the B. pertussis locus with that of B. bronchiseptica conferred higher levels of resistance to growth inhibition by acriflavine and fatty acids. In addition, we identified that the transcription of the locus is repressed by a LysR-type transcriptional regulator. Palmitate de-represses the expression of the acr locus, dependent on the LysR regulator, strongly suggesting that it is a transcriptional repressor that is regulated by palmitate. It is intriguing that the speciation of B. pertussis has selected for a reduction in activity of the Acr efflux system that typically is regarded as protective to bacteria.

Sensitive spectrofluorometric method for the determination of ascorbic acid in pharmaceutical nutritional supplements using acriflavine as a fluorescence reagent.

An easily performed, specific, sensitive, rapid, reliable and inexpensive procedure for the spectrofluorometric quantitation of ascorbic acid was proposed using acriflavine as a fluorescence quenching reagent. The procedure was based on the determined quenching effect of ascorbic acid on the natural fluorescence signal of acriflavine and the reaction between ascorbic acid and acriflavine in Britton-Robinson buffer solution (pH 6) to produce an ion-associated complex. The reduction in acriflavine fluorescence intensity was detected at 505 nm, while excitation occurred at 265 nm. The relationship between quenching fluorescence intensity (∆F) and concentration of ascorbic acid was linear (R2  = 0.9967) within the range 2-10 µg/ml and with a detection limit of 0.08 µg/ml. No significant interference was detected from other materials often found in pharmaceutical nutritional tablets. The obtained results were compared with those from high-performance liquid chromatography and appeared in good agreement, with no important differences in precision or accuracy. The proposed spectrofluorimetric method was used to determine the amount of ascorbic acid in a number of commercial pharmaceutical nutritional supplement tablets with a 95% confidence performance.

Generalized ratiometric fluorescence nanosensors based on carbon dots and an advanced chemometric model.

Probe encapsulated by biologically localized embedding (PEBBLE) has emerged as a new type of sensing technique for complex systems. Generalized ratiometric PEBBLE nanosensors prepared by encapsulating an intensity-based probe and an inert reference dye inside the pores of stable matrix possess advantages of easy synthesis, immunity to interference, lower toxicity, and robustness to variations in probe loading. However, the selection of appropriate reference dyes used in generalized ratiometric PEBBLE nanosensors is a rather difficult task since they should satisfy some stringent requirements. In this contribution, the feasibility of using carbon dots (C-dots) as generic inert references in synthesizing PEBBLE nanosensors was first investigated in detail. And a dual-wavelength monitoring strategy and the quantitative fluorescence model for generalized ratiometric probes (QFMGRP) were adopted to solve the problems brought by the use of carbon dots as inert references. C-dots doped PEBBLE nanosensors (C-PEBBLE nanosensors) for the quantification of NO2- and free Ca2+ were synthesized by encapsulating C-dots and intensity based fluorescence probes (i.e., acriflavine for NO2-, and Rhod-2 for Ca2+, respectively) inside the pores of stable matrix. Experimental results showed that the combination of C-PEBBLEs, the QFMGRP model and the dual-wavelength monitoring strategy achieved accurate quantification of NO2- and the free Ca2+ in real-world samples. Their quantitative results were in good consistence with those determined by HPLC and atomic absorption spectrophotometer, respectively. The strategies proposed in this contribution have generic applicability in the synthesis of PEBBLE nanosensors and their quantitative applications.

Spectroscopic investigation and computational studies on the interaction of Acriflavine with various estrogens.

The fluorescence quenching of Acriflavine (AFN) by certain estrogens was examined in aqueous media by employing steady state and time-resolved fluorescence measurements. The absorption spectra of AFN change with significant bathochromic shift in presence of quencher molecules. The quenching behavior was examined by correlating the bimolecular quenching rate constant (kq) with the free energy change (ΔG). The decrease in quenching rate constant depends on the increase in oxidation potential of quencher molecules. The fluorescence quenching experiments were carried out in different solvents of varying polarities and reveals the possibility of charge transfer quenching mechanism. Lifetime measurements indicate static quenching. The quenching behavior is addressed from bond dissociation enthalpy (BDE) calculations. The antioxidant activity of estrogen compounds were evaluated by deoxyribose oxidation assay.

A sensitive fluorescence quenching method for the detection of tartrazine with acriflavine in soft drinks.

In this work, a simple, rapid, sensitive, selective spectrofluorimetric method was applied to detect tartrazine. The fluorescence of acriflavine could be efficiently quenched by tartrazine. The method manifested real time response as well as presented satisfied linear relationship to tartrazine. The linear response range of tartrazine (R2 = 0.9995) was from 0.056 to 5 µmol L-1 . The detection limit (3σ/k) was 0.017 µmol L-1 , indicating that this method could be applied to detect traces of tartrazine. The accuracy and precision of the method was further assured by recovery studies via a standard addition method, with percentage recoveries in the range of 96.0% to 103.0%. Moreover, a quenching mechanism was investigated systematically by the linear plots at varying temperatures based on the Stern-Volmer equation, fluorescence lifetime and UV-visible absorption spectra, all of which proved to be static quenching. This sensitive, selective assay possessed a great application prospect for the food industry owing to its simplicity and rapidity for the detection of tartrazine.

Aptamer-based electrochemical biosensor by using Au-Pt nanoparticles, carbon nanotubes and acriflavine platform.

Herein, an ultrasensitive electrochemical aptasensor for quantitative detection of bisphenol A (BPA) was fabricated based on a novel signal amplification strategy. This aptasensor was developed by electrodeposition of gold-platinum nanoparticles (Au-PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs-COOH). In this protocol, acriflavine (ACF) was covalently immobilized at the surface of glassy carbon electrode modified with Au-PtNPs/CNTs-COOH nanocomposite. Attachment of BPA-aptamer at the surface of modified electrode was performed through the formation of phosphoramidate bonds between the amino group of ACF and phosphate group of the aptamer at 5'end. By interaction of BPA with the aptamer, the conformational of aptamer was changed which lead to retarding the interfacial electron transfer of ACF as a probe. Sensitive quantitative detection of BPA was carried out by monitoring the decrease of differential pulse voltammetric (DPV) responses of ACF peak current with increasing the BPA concentration. The resultant aptasensor exhibited good specificity, stability and reproducibility, indicating that the present strategy was promising for broad potential application.

Self-assembled nanocomposite film with tunable enhanced fluorescence for the detection of DNA.

In this study, a simple and environmentally friendly, silver nanocomposite film was prepared via the in situ reduction of silver ions in self-assembled chitosan (CS)/sodium alginate film matrixes. Negatively charged DNA containing the fluorescent intercalator acriflavine (Acf) was assembled on the surface of the silver nanocomposite film, to facilitate the detection of DNA. A tunable fluorescence enhancement was achieved for the Acf in the silver nanocomposite film simply by changing the thickness of the interlayer between the DNA and the silver nanocomposite film. Using the interlayer prepared by an assembly of poly(acrylic acid) and CS, a significant enhancement in the fluorescence of Acf was obtained. Owing to the ability of Acf to intercalate into DNA, this hybrid system with an enhanced Acf fluorescence could be used to monitor the template-independent DNA elongation process in a facile, high-efficiency, label-free fashion.

Development of a sensor to study the DNA conformation using molecular logic gates.

This communication reports our investigations on the Fluorescence Resonance Energy Transfer (FRET) between two laser dyes Acriflavine and Rhodamine B in absence and presence of DNA at different pH. It has been observed that energy transfer efficiency is largely affected by the presence of DNA as well as the pH of the system. It is well known that with increase in pH, DNA conformation changes from double stranded to single stranded (denaturation) and finally form random coil. Based on our experimental results two different types of molecular logic gates namely, XOR and OR logic have been demonstrated which can be used to have an idea about DNA conformation in solution.

In vitro membrane protein synthesis inside cell-sized vesicles reveals the dependence of membrane protein integration on vesicle volume.

Giant unilamellar vesicles (GUVs) are vesicles>1 µm in diameter that provide an environment in which the effect of a confined reaction volume on intravesicular reactions can be investigated. By synthesizing EmrE, a multidrug transporter from Escherichia coli, as a model membrane protein using a reconstituted in vitro transcription-translation system inside GUVs, we investigated the effect of a confined volume on the synthesis and membrane integration of EmrE. Flow cytometry was used to analyze multiple properties of the vesicles and to quantify EmrE synthesis inside GUVs composed of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. We found that EmrE was synthesized and integrated into the GUV membrane in its active form. We also found that the ratio of membrane-integrated EmrE to total synthesized EmrE increased with decreasing vesicle volume; this finding is explained by the effect of an increased surface-area-to-volume ratio in smaller vesicles. In vitro membrane synthesis inside GUVs is a useful approach to study quantitatively the properties of membrane proteins and their interaction with the membrane under cell-mimicking environments.

Investigation on the inclusion and toxicity of acriflavine with cyclodextrins: a spectroscopic approach.

Acriflavine hydrochloride (AFN) is a prospective drug worn in the eradication of HIV1 infection. The toxicity and adverse side effects renders the potent drug to limits its usage. However, to overcome the dilemma we have aimed to select carriers with great complexation efficiencies in different cyclodextrins (CDs) of varying cavity size. The interaction of AFN with α, ß and γ-CDs were investigated using absorption and steady state as well as lifetime measurements. From the obtained data it was found that AFN fits in the cavity of α and ß-CDs but unable to form inclusion complex with γ-CD. The effect of quencher molecules during the inclusion phenomena of AFN with CDs was explored via steady state measurements. The nature of binding forces responsible for the inclusion of AFN with CDs was discussed by using thermodynamic parameters. Using Benesi-Hildebrand equation the stoichiometry of AFN with CDs was predominantly found to be 1:1. To get deeper in situ, the in vitro toxicity of AFN and its complexation product were probed by Artemia salina sp. The toxicity of AFN was reduced when complexed with α and ß-CDs.

Development of a DNA sensor using a molecular logic gate.

This communication reports the increase in fluorescence resonance energy transfer (FRET) efficiency between two laser dyes in the presence of deoxyribonucleic acid (DNA). Two types of molecular logic gates have been designed where DNA acts as input signal and fluorescence intensity of different bands are taken as output signal. Use of these logic gates as a DNA sensor has been demonstrated.

Interaction of acriflavine with pyrimidines: a spectroscopic approach.

The interaction of acriflavine with uracils was investigated by using spectroscopic tools viz., UV-Vis absorption, steady state and time resolved fluorescence measurements. The spectroscopic data were analyzed using Stern-Volmer equation to determine the quenching process. The bimolecular quenching rate constant (k(q)), binding constant (K) and number of binding sites (n) were calculated at different temperature from the relevant fluorescence data. The experimental results obtained from life-time measurement indicate that the quenching mechanism was static via the formation of ground state complex. The free energy change (ΔGet) for electron transfer process was calculated by Rehm-Weller equation. The existence of binding forces and the interactions of acriflavine with uracils were examined.

Drug uptake pathways of multidrug transporter AcrB studied by molecular simulations and site-directed mutagenesis experiments.

Multidrug resistance has been a critical issue in current chemotherapy. In Escherichia coli , a major efflux pump responsible for the multidrug resistance contains a transporter AcrB. Crystallographic studies and mutational assays of AcrB provided much of structural and overall functional insights, which led to the functionally rotating mechanism. However, the drug uptake pathways are somewhat controversial because at least two possible pathways, the vestibule and the cleft paths, were suggested. Here, combining molecular simulations and site-directed mutagenesis experiments, we addressed the uptake mechanism finding that the drug uptake pathways can be significantly different depending on the properties of drugs. First, in the computational free energy analysis of drug movements along AcrB tunnels, we found a ligand-dependent drug uptake mechanism. With the same molecular sizes, drugs that are both strongly hydrophobic and lipophilic were preferentially taken in via the vestibule path, while other drugs favored the cleft path. Second, direct simulations realized totally about 3500 events of drug uptake by AcrB for a broad range of drug property. These simulations confirmed the ligand-dependent drug uptake and further suggested that a smaller drug favors the vestibule path, while a larger one is taken in via the cleft path. Moreover, the direct simulations identified an alternative uptake path which is not visible in the crystal structure. Third, site-directed mutagenesis of AcrB in E. coli verified that mutations of residues located along the newly identified path significantly reduced the efflux efficiency, supporting its relevance in in vivo function.

Acriflavine enhances radiosensitivity of colon cancer cells through endoplasmic reticulum stress-mediated apoptosis.

Radiotherapy (RT) is one of the most effective tools in the clinical treatment of cancer. Because the tumor suppressor p53 plays a central role in radiation-mediated responses, including cell cycle-arrest and apoptosis, a number of studies have suggested that p53 could be a useful therapeutic target of anti-cancer agents. Accordingly, we sought to discover a new agent capable of increasing p53 activity. HCT116 colon cancer cells, containing wild-type p53, were stably transfected with a p53 responsive-luciferase (p53-Luc) reporter gene. A cell-based high-throughput screen of 7920 synthetic small molecules was performed in duplicate. Of the screened compounds, acriflavine (ACF) significantly increased p53-Luc activity in a concentration-dependent manner without causing toxicity. Pretreatment with ACF enhanced the induction of p53 protein expression and phosphorylation on serine 15 by γ-irradiation. Clonogenic assays showed that ACF pretreatment also potentiated radiation-induced cell death. The combination of irradiation and ACF treatment induced mitochondrial release of cytochrome c and significant activation of caspase-3 with PARP cleavage in colon cancer cells, demonstrating typical apoptotic cell death. Combined treatment with ACF and radiation increased the expression of Bax and Bad, while decreasing expression of Bcl-2. In addition, the ACF/radiation treatment combination induced endoplasmic reticulum (ER) stress responses mediated by IRE1α (inositol-requiring transmembrane kinase and endonuclease 1α), eIF-2α (eukaryotic initiation factor 2α), caspase-2/12, and CHOP (C/EBP homologous protein). The knockdown of IRE1α by siRNA inhibited the apoptotic cell death induced by ACF/radiation treatment. In vivo studies showed that combined treatment with ACF and radiation significantly inhibited the growth of tumors in colorectal cancer xenografted mice. These results indicate that ACF acts through p53-dependent mitochondrial pathways and ER stress signals, and could be a promising radiosensitizer.

Photocatalytic degradation of acridine dyes using anatase and rutile TiO2.

The adsorption and photodegradation of acridine orange (AO) and acriflavine (AF) dyes on two mesoporous titania crystalline phases, anatase and rutile, were experimentally studied. Anatase and rutile were characterized by nitrogen adsorption, electron scanning and transmission microscopy, and X-ray diffraction. The adsorption capacity of rutile was higher than that of anatase, while the reverse is observed for photodegradation of both dyes. The adsorption of AF on both adsorbents was higher than that of AO, which was related with the smaller size of AF molecules compared with those of AO, therefore the access of AF to the adsorption sites is favored.

c-di-GMP can form remarkably stable G-quadruplexes at physiological conditions in the presence of some planar intercalators.

The ubiquitous bacterial biofilm regulator, c-di-GMP can form G-quadruplexes at physiological conditions in the presence of some aromatic compounds, such as acriflavine and proflavine. The fluorescence of these compounds is quenched upon c-di-GMP binding and some of the formed c-di-GMP G-quadruplexes are stable even at 75 °C.