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Electrochemical DNA sensors for DNA damage detection based on electroactive polymer poly(proflavine) (PPFL) that was synthesized at screen-printed carbon electrodes (SPCEs) from phosphate buffer (PB) and two natural deep eutectic solvents (NADESs) consisting of citric or malonic acids, D-glucose, and a certain amount of water (NADES1 and NADES2) were developed. Poly(proflavine) coatings obtained from the presented media (PPFLPB, PPFLNADES1, and PPFLNADES2) were electrochemically polymerized via the multiple cycling of the potential or potentiostatic accumulation and used for the discrimination of thermal and oxidative DNA damage. The electrochemical characteristics of the poly(proflavine) coatings and their morphology were assessed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). The working conditions for calf thymus DNA implementation and DNA damage detection were estimated for all types of poly(proflavine) coatings. The voltammetric approach made it possible to distinguish native and chemically oxidized DNA while the impedimetric approach allowed for the successful recognition of native, thermally denatured, and chemically oxidized DNA through changes in the charge transfer resistance. The influence of different concentrations of conventional antioxidants and pharmaceutical preparations on oxidative DNA damage was characterized. [ABSTRACT FROM AUTHOR]

Abstract This study employs a combination of mathematical derivation and optimization technique to investigate the adsorption of drug molecules on nanocarriers. Specifically, the chemotherapy drugs, fluorouracil, proflavine, and methylene blue, are non-covalently bonded with either a flat graphene sheet or a spherical $${\rm C}_{60}$$ C 60 fullerene. Mathematical expressions for the interaction energy between an atom and graphene, as well as between an atom and $${\rm C}_{60}$$ C 60 fullerene, are derived. Subsequently, a discrete summation is evaluated for all atoms on the drug molecule utilizing the U-NSGA-III algorithm. The stable configurations’ three-dimensional architectures are presented, accompanied by numerical values for crucial parameters. The results indicate that the nanocarrier’s structure effectively accommodates the atoms on the drug’s carbon planes. The three drug types’ molecules disperse across the graphene surface, whereas only fluorouracil spreads on the $${\rm C}_{60}$$ C 60 surface; proflavine and methylene blue stack vertically to form a layer. Furthermore, all atomic positions of equilibrium configurations for all systems are obtained. This hybrid method, integrating analytical expressions and an optimization process, significantly reduces computational time, representing an initial step in studying the binding of drug molecules on nanocarriers.

This study employs a combination of mathematical derivation and optimization technique to investigate the adsorption of drug molecules on nanocarriers. Specifically, the chemotherapy drugs, fluorouracil, proflavine, and methylene blue, are non-covalently bonded with either a flat graphene sheet or a spherical C 60 fullerene. Mathematical expressions for the interaction energy between an atom and graphene, as well as between an atom and C 60 fullerene, are derived. Subsequently, a discrete summation is evaluated for all atoms on the drug molecule utilizing the U-NSGA-III algorithm. The stable configurations' three-dimensional architectures are presented, accompanied by numerical values for crucial parameters. The results indicate that the nanocarrier's structure effectively accommodates the atoms on the drug's carbon planes. The three drug types' molecules disperse across the graphene surface, whereas only fluorouracil spreads on the C 60 surface; proflavine and methylene blue stack vertically to form a layer. Furthermore, all atomic positions of equilibrium configurations for all systems are obtained. This hybrid method, integrating analytical expressions and an optimization process, significantly reduces computational time, representing an initial step in studying the binding of drug molecules on nanocarriers. [ABSTRACT FROM AUTHOR]

Electrochemical DNA sensors for DNA damage detection based on electroactive polymer poly(proflavine) (PPFL) that was synthesized at screen-printed carbon electrodes (SPCEs) from phosphate buffer (PB) and two natural deep eutectic solvents (NADESs) consisting of citric or malonic acids, D-glucose, and a certain amount of water (NADES1 and NADES2) were developed. Poly(proflavine) coatings obtained from the presented media (PPFLPB, PPFLNADES1, and PPFLNADES2) were electrochemically polymerized via the multiple cycling of the potential or potentiostatic accumulation and used for the discrimination of thermal and oxidative DNA damage. The electrochemical characteristics of the poly(proflavine) coatings and their morphology were assessed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). The working conditions for calf thymus DNA implementation and DNA damage detection were estimated for all types of poly(proflavine) coatings. The voltammetric approach made it possible to distinguish native and chemically oxidized DNA while the impedimetric approach allowed for the successful recognition of native, thermally denatured, and chemically oxidized DNA through changes in the charge transfer resistance. The influence of different concentrations of conventional antioxidants and pharmaceutical preparations on oxidative DNA damage was characterized.

Proflavine (PF)—2,6-diaminoacridine—is a mutagen and, as one of the dyes of the acridine series, it is able to form complexes with DNA, which leads both to the appearance of optical activity in the long-wave absorption band of the dye and to a change in optical activity in the UV region of the spectrum, where the absorption of nitrogenous bases of DNA is observed. An experimental study of optical activity in the form of circular dichroism (CD) spectra shows that in the visible region of the spectrum, optical activity is caused by the exciton chromophore-chromophore interaction of dye molecules that have formed a complex with an asymmetric helical DNA molecule. The appearance of an additional CD in the UV region is related to the exciton interaction of short-wavelength optical transitions of dye molecules with similar frequencies of the UV transitions of nucleic bases. The decomposition of the CD spectra into components makes it possible to isolate the contribution from the interaction of long-wavelength dye transitions with the short-wavelength (UV) transitions of the neighboring chromophores. In particular, a contribution was found from the interaction of the magnetic transition moment of chromophores in higher vibrational states with the dipole transition moments of the neighboring chromophores. This previously undescribed effect is related to the violation of the symmetry plane of the aromatic system of the dye in higher vibrational states; it is absent in the region of the zero vibrational band. The constructed decomposition procedure makes it possible to separate from the total CD spectra the contributions of the optical interactions previously described theoretically, which potentially allows us to estimate the geometric parameters of the complex. [ABSTRACT FROM AUTHOR]

New four 3,6-bis[(alkylamino)ureido]acridines were synthesized and thoroughly characterized using NMR spectra. In vitro DNA binding studies of acridines carried out employing absorption, fluorescence and circular dichroism measurements revealed their binding into DNA via intercalation. The UV–Vis absorbance data were analysed to obtain the binding constant (Kb). Kb values were found in the range of 4.46 − 6.73 × 104 M−1. The DNA viscosity measurements were also performed to confirm the binding mode of the investigated acridines. [ABSTRACT FROM AUTHOR]

Proflavine is a well-known antiseptic and bacteriostatic drug, however, it has the potential to be hazardous and mutagenic. Proflavine enters cells and intercalates between DNA base pairs, resulting in mutation and replication inhibition. Previously several investigators demonstrated that photo-activated proflavine generated double-stranded DNA breakage and protein structural alterations. The present study investigated the role of hydroxyl radical (·OH) due to activation of proflavine in the breakdown of protein and enzyme by photo-activated proflavine. The results show that the formation of hydroxyl radicals increased as the photo-illumination period increased, as did the concentrations of proflavine and Cu (II). As demonstrated by SDS-PAGE, the excess of free radicals due to proflavine resulted in oxidative modifications and degradation of BSA protein and trypsin enzyme. Additionally, with an increase in Cu (II) concentration, photo-illuminated proflavine induced a considerable loss of enzyme activity and also accelerated the degradation of the enzyme. Bathocuproine, a particular Cu (I)-sequestering agent, prevented protein degradation and enzyme inactivation. Hydroxyl radical scavengers inhibited the protein-damaging process, indicating that hydroxyl radicals play a substantial role in protein damage. The tryptophan moiety was quenched by proflavine, demonstrating that it binds to proteins and enzymes, changing their structure and activity. As a result, this study helps to better understand proflavine's deleterious influence on protein and enzyme degradation by oxygen-free radicals.

Proflavine is an acridine derivative which was discovered as one of the earliest antibacterial agents, and it has been proven to have potential application to fields such as chemotherapy, photobiology and solar-energy conversion. In particular, it is well known that proflavine can bind to DNA with different modes, and this may open addition photochemical-reaction channels in DNA. Herein, the excited-state dynamics of proflavine after intercalation into DNA duplex is studied using femtosecond time-resolved spectroscopy, and compared with that in solution. It is demonstrated that both fluorescence and the triplet excited-state generation of proflavine were quenched after intercalation into DNA, due to ultrafast non-radiative channels. A static-quenching mechanism was identified for the proflavine-DNA complex, in line with the spectroscopy data, and the excited-state deactivation mechanism was proposed. [ABSTRACT FROM AUTHOR]

Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is mainly limited by the spectral overlap of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405 and 488 nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that the addition of 2,3,4,5,6-pentafluoroaniline to Atto 465 NHS ester, creating Atto 465-pentafluoroaniline (Atto 465-p), generates a bright nuclear stain in the violet-blue region of the visible spectrum. This allows the 405 nm excitation and emission, classically used for nuclear counterstains, to be used for the detection of another target protein. This increases the flexibility of the mIF panel and, with appropriate staining and microscopy, enables the quantitative analysis of at least six targets in one tissue section. (J Histochem Cytochem XX: XXX–XXX, XXXX) [ABSTRACT FROM AUTHOR]

Background: Minor groove binding is a rate-limiting step in proflavine-DNA intercalation reaction. This step is believed also to be responsible for the sequence-dependent kinetics of proflavine binding to DNA. At the same time, most studies are focused on the final stage of the reaction – the intercalation complex, and there is a lack of data concerning the structure and stability of proflavine-DNA minor groove-bound complexes. Objectives: The objective of this study was to investigate the stability of proflavine minor groove-bound complexes with DNA oligonucleotides of different sequence by molecular dynamics simulation and to analyze the DNA conformational changes caused by the proflavine binding. Materials and methods: The molecular dynamics simulations of proflavine minor groove-bound complexes with poly(dA)·poly(dT) and poly(dCG)·poly(dCG) oligonucleotides of 30 bp length were done in program package AMBER12 with explicit water (SPC/E) and ions (NaCl 0.15 M) using force fields FF14SB for DNA and GAFF for ligand. The starting configurations of complexes were obtained by docking method in AutoDock 3.05. After multi-stage equilibration protocol, each system was simulated at T=300 K and p=1 bar for a 50 ns production phase. Then trajectories were post-processed in AMBERTools17 and VMD-1.9.3 packages. Results: Our simulations confirm that proflavine-DNA minor groove-bound complexes are stable in the 50 ns time range but there are some structural rearrangements in them with respect to the initial structures. The narrowing of the DNA minor groove is observed in the proflavine binding site. In proflavine-poly(dCG)·poly(dCG) complex it is more pronounced and is accompanied by the BI/BII transitions in DNA and the reorientation of ligand. In proflavine-poly(dA)·poly(dT) complex the specific intermolecular hydrogen bonds are formed, which are optimized by the changes in opening and propeller twisting of involved AT-base pairs. Complexes are stabilized by the van der Waals and hydrophobic interactions, which are more favorable in the proflavine-poly(dA)·poly(dT) complex. Conclusions: Our results show that the binding of proflavine to a minor groove of DNA induces the conformational changes in the DNA that are important for the resulting complex stability.

The present work highlights the broad range of oxygen and nitrogen heterocycles and their applications in medicinal field. A facial approach has been developed for the synthesis of coumarinyl xanthenes and acridine derivatives via one pot multicomponent reaction using green chemical technique. All the isolated compounds were characterized by spectral analysis such as IR NMR and mass. Compounds were screened for their antibacterial and anti-inflammatory activity. While, biological activity results of synthesized coumarin derivatives are quite significant and contribute to the medicinal chemistry. [ABSTRACT FROM AUTHOR]

Proflavine, an acridine dye, is a known DNA intercalating agent. In the present study, we show that proflavine alone on photoillumination can generate reactive oxygen species (ROS). These proflavine-derived ROS cause damage to proteins, and this effect is enhanced when the divalent metal ion Cu (II) is included in the reaction. Bathocuproine, a specific Cu (I) sequestering agent, when present in the reaction mixture containing Cu (II), was found to inhibit the protein degradation, showing that Cu (I) is an essential intermediate in the reaction. The effect of several scavengers of ROS such as superoxide dismutase, sodium azide, potassium iodide, and thiourea were examined on the protein damaging reaction. Potassium iodide was found to be the most effective in inhibiting protein damage followed by sodium azide and thiourea. Our results indicate the involvement of superoxide, singlet oxygen, triplet oxygen, and hydroxyl radicals in proflavine-induced damage to proteins.

Proflavine derivatives are extremely interesting chemotherapeutic agents, which have shown promising pharmaceutical potential due to their wide range of biological activities. This review summarizes the current state of research into the anticancer, antimicrobial, antimalarial and antileishmanial properties of these attractive compounds. Our attention has focused on new classes of proflavine conjugates, which display significant levels of anticancer activity. Highly promising cytotoxic properties have been identified in proflavine conjugates with imidazolidinones, ureas and thioureas. In particular, proflavine‐dialkyldithioureas displayed substantial cytotoxic effect against the human leukemia HL‐60 cells with IC50 values from 7.2 to 34.0 μm. As well, palladium complexes with proflavine ligand have important biologic activity. The LC50 values of these complexes were significantly lower than that of cisplatin against the SK‐BR‐3 cell line. Proflavine derivatives are extremely interesting chemotherapeutic agents, which have shown promising pharmaceutical potential due to their wide range of biological activities. This review summarizes the current state of research into the anticancer, antimicrobial, antimalarial and antileishmanial properties of these attractive compounds. [ABSTRACT FROM AUTHOR]

Highlights • Energetics of the binding of quinacrine and proflavine with poly(I).poly(C) was studied. • The binding was exothermic in both the cases. • Enthalpy-entropy compensation and negative heat capacity value was observed in both the cases. • The binding was driven largely by non-polylectrolytic forces for both quinacrine and proflavine. Abstract Thermodynamics of the interaction of quinacrine with poly(I).poly(C) was studied in comparison to proflavine using microcalorimetry. Isothermal titration calorimetry results suggested that the binding of quinacrine to poly(I).poly(C) was enthalpy dominated with a smaller but favourable entropic contribution at 298.15 K. For proflavine, the binding was predominantly entropy dominated at 298.15 K. The equilibrium constant for quinacrine-poly(I).poly(C) complexation was calculated to be (5.11 ± 0.88)·105 M−1 while for proflavine the equilibrium constant was (3.45 ± 0.85)·105 M−1 at 298.15 K. With increasing temperature the equilibrium constant decreased, the standard molar enthalpic contribution increased in magnitude, the positive standard molar entropic contribution decreased while standard molar Gibbs energy remained almost invariant for both the systems. An enthalpy-entropy compensation phenomenon was observed for the complexation of both quinacrine and proflavine with poly(I).poly(C). Negative standard molar heat capacity values were also observed for the complexation of both quinacrine and proflavine with poly(I).poly(C) which suggested the involvement of hydrophobic forces in the binding reaction. Salt dependent calorimetric data and the dissection of the standard molar Gibbs energy suggested the involvement of a dominant non-electrostatic component in the binding reaction for both quinacrine and proflavine. [ABSTRACT FROM AUTHOR]

It is well known that proflavine binds to DNA. Here we investigate the binding mode of proflavine to native DNA using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy as well as by fluorescence techniques. The observed changes of proflavine upon complexation with DNA can be summarized as a red shift and hypochromism in the absorption spectrum. The negative LDr in the proflavine absorption region has a magnitude comparable to or larger than that of the DNA absorption region, confirming the intercalative binding mode of proflavine to DNA. Saturation of the LD spectrum in the proflavine absorption region at R = 0.25 and a decrease in the fluorescence intensity provide further evidence of intercalation. Furthermore, the coupling of electric transition of intercalated proflavine is observed. Although proflavine has been reported to position itself along the DNA stem at high [proflavine]/[DNA base] ratios, the spectral characteristics, which include a clear isosbestic point in the absorption spectrum and proportionality in the LD magnitude in the proflavine absorption region, do not show any possibility of exterior binding. The observed change in proflavine upon the formation of a complex with DNA can be summarized as a red‐shift and hypochromism in the absorption spectrum, The negative LDr in the proflavine absorption region with its magnitude comparable or larger than that in the DNA absorption region confirm the intercalative binding mode of proflavine to DNA. [ABSTRACT FROM AUTHOR]

Methylene blue and proflavine are fluorescent dyes used to stain nucleic acid from the molecular level to the tissue level. Already clinically used for sentinel node mapping, detection of neuroendocrine tumors, methemoglobinemia, septic shock, ifosfamide-induced encephalopathy, and photodynamic inactivation of RNA viruses, the antimicrobial, anti-inflammatory, and antioxidant effect of methylene blue has been demonstrated in different in vitro and in vivo studies. Proflavine was used as a disinfectant and bacteriostatic agent against many gram-positive bacteria, as well as a urinary antiseptic involved in highlighting cell nuclei. At the tissue level, the anti-inflammatory effects of methylene blue protect against pulmonary, renal, cardiac, pancreatic, ischemic-reperfusion lesions, and fevers. First used for their antiseptic and antiviral activity, respectively, methylene blue and proflavine turned out to be excellent dyes for diagnostic and treatment purposes. In vitro and in vivo studies demonstrated that both dyes are efficient as perfusion and tissue tracers and permitted to evaluate the minimal efficient concentration in different species, as well as their pharmacokinetics and toxicity. This review aims to identify the optimal concentrations of methylene blue and proflavine that can be used for in vivo experiments to highlight the vascularization of the skin in the case of a perforasome (both as a tissue tracer and in vascular mapping), as well as their effects on tissues. This review is intended to be a comparative and critical presentation of the possible applications of methylene blue (MB) and proflavine (PRO) in the surgical field, and the relevant biomedical findings from specialized literature to date are discussed as well.

Proflavine, an acridine dye, is a known DNA intercalating agent. In the present study, we show that proflavine alone on photoillumination can generate reactive oxygen species (ROS). These proflavine-derived ROS cause damage to proteins, and this effect is enhanced when the divalent metal ion Cu (II) is included in the reaction. Bathocuproine, a specific Cu (I) sequestering agent, when present in the reaction mixture containing Cu (II), was found to inhibit the protein degradation, showing that Cu (I) is an essential intermediate in the reaction. The effect of several scavengers of ROS such as superoxide dismutase, sodium azide, potassium iodide, and thiourea were examined on the protein damaging reaction. Potassium iodide was found to be the most effective in inhibiting protein damage followed by sodium azide and thiourea. Our results indicate the involvement of superoxide, singlet oxygen, triplet oxygen, and hydroxyl radicals in proflavine-induced damage to proteins.

Proflavine could recognise HIV RRE (Rev response element) RNA, thus inhibiting its interaction with Rev peptide. Owing to the non-significant difference in fluorescence polarization (FP) signal between the proflavine and proflavine-RRE complex, it is not practicable for the identification of antagonists of HIV Rev peptide using proflavine as a FP probe. In the present study, graphene oxide (GO) was introduced to amplify the FP signal to enhance the sensitivity for the identification of Rev antagonists, thereby enabling the development of a primary GO-enhanced FP biosensor for the discovery of Rev antagonists. The effect of the amount of GO on the FP changes was investigated via displacement assay using a known Rev antagonist, neomycin B. The mechanism underlying the changes in FP caused by the addition of neomycin B was explored in the presence of GO. Finally, the performance of this biosensor was further evaluated using other 4 test drugs. The introduction of GO to this interaction system increased the practicability for the discovery of Rev antagonists using proflavine as a probe, and this label-free method does not suffer from limitations caused by fluorescent covalent labeling, thus providing a novel strategy for the characterization of ligand-RNA binding interactions. [ABSTRACT FROM AUTHOR]

Proflavine, an acridine dye, is a known DNA intercalating agent. In the present study, we show that proflavine alone on photoillumination can generate reactive oxygen species (ROS). These proflavine-derived ROS cause damage to proteins, and this effect is enhanced when the divalent metal ion Cu (II) is included in the reaction. Bathocuproine, a specific Cu (I) sequestering agent, when present in the reaction mixture containing Cu (II), was found to inhibit the protein degradation, showing that Cu (I) is an essential intermediate in the reaction. The effect of several scavengers of ROS such as superoxide dismutase, sodium azide, potassium iodide, and thiourea were examined on the protein damaging reaction. Potassium iodide was found to be the most effective in inhibiting protein damage followed by sodium azide and thiourea. Our results indicate the involvement of superoxide, singlet oxygen, triplet oxygen, and hydroxyl radicals in proflavine-induced damage to proteins. [ABSTRACT FROM AUTHOR]

A novel pretreatment-free method involving laser desorption postionization (LDPI) coupled with time-of-flight mass spectrometry (MS) was developed for the monitoring of proflavine level in rat whole blood. It comprises a protocol for dosing via intravenous administration and collection of whole blood, followed by direct LDPI-MS analysis without any sample pretreatment. An intense ion signal at m/ z 209 was observed from whole blood without any interference signals, except some background signals below m/ z 100. The calibration curve was established with use of 9-phenylacridine as the internal standard for proflavine determination from the plotting of the peak ratios of proflavine to the internal standard, with a correlation coefficient ( R ) greater than 0.99. The limit of detection was estimated to be 0.48 pmol/mm and the quantification range was 0.5-16.5 μg/mL for proflavine. In addition, only a minimal matrix effect was observed, as expected from considerations of the desorption and ionization mechanism. Interday and intraday accuracy and precision were calculated to be within 13% and 82-114%, respectively. Estimated concentrations of proflavine residue in whole blood were also successfully obtained at selected time points after dosing. The proposed method is simple, low cost, and sensitive, and should be seen as a complementary method for monitoring drug levels in blood. [ABSTRACT FROM AUTHOR]

Proflavine, a nitrogen-containing dye is used for DNA staining and as an efficient marker, however, it is harmful to body cells, and its efficient removal is essential. In this work, graphene oxide (GO) has been used as an adsorbent for the removal of the dye through the process of adsorption. Though GO is now being used as an adsorbent for the removal of industrial dyes, very few experiments have been done on biologically useful dyes. GO is synthesized from graphite powder by the modified Hummer's method and is characterized by X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), Raman and ultra-violet spectroscopies, etc. These characterization techniques indicate the presence of oxygen containing functional groups, e.g., carbonyl, carboxyl and hydroxyl groups on the graphene sheets. These sheets are held well apart by these groups. Parameters like concentration of the adsorbent, pH and temperature have been varied to find the optimum conditions for the most efficient removal of the dye molecules from the solution. Then, for the optimum external conditions, the time dependence of the adsorption was analyzed using pseudo-first and pseudo-second orders kinetics. The adsorption isotherms were also obtaining throws light on the mechanism of the adsorption process which we investigated further by using FTIR characterization. [ABSTRACT FROM AUTHOR]

Interaction of proflavine with hemoglobin (Hgb) was studied employing spectroscopy, calorimetry, and atomic force microscopy. The equilibrium constant was found to be of the order 10 4 M − 1 . The quenching of Hgb fluorescence by proflavine was due to the complex formation. Calculation of the molecular distance (r) between the donor (β-Trp37 of Hgb) and acceptor (proflavine) suggested that energy can be efficiently transferred from the β-Trp37 residue at the α1β2 interface of the protein to the dye. Proflavine induced significant secondary structural changes in Hgb. Synchronous fluorescence studies showed that proflavine altered the microenvironment around the tryptophan residues to a greater extent than the tyrosine residues. Circular dichroism spectral studies showed that proflavine caused significant reduction in the α-helical content of Hgb. The esterase activity assay further complemented the circular dichroism data. The Soret band intensity of Hgb decreased upon complexation. Differential scanning calorimetry and circular dichroism melting results revealed that proflavine induced destabilization of Hgb. The binding was driven by both positive entropy and negative enthalpy. Atomic force microscopy studies revealed that the essential morphological features of hemoglobin were retained in the presence of proflavine. Overall, insights on the photophysical aspects and energetics of the binding of proflavine with Hgb are presented. [ABSTRACT FROM AUTHOR]