Mathematical modeling and optimization technique of anticancer antibiotic adsorption onto carbon 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.

Ultrafast spectroscopy study of DNA photophysics after proflavine intercalation.

Proflavine (PF), an acridine DNA intercalating agent, has been widespread applied as an anti-microbial and topical antiseptic agent due to its ability to suppress DNA replication. On the other hand, various studies show that PF intercalation to DNA can increase photogenotoxicity and has potential chances to induce carcinomas of skin appendages. However, the effects of PF intercalation on the photophysical and photochemical properties of DNA have not been sufficiently explored. In this study, the excited state dynamics of the PF intercalated d(GC)9 • d(GC)9 and d(AT)9 • d(AT)9 DNA duplex are investigated in an aqueous buffer solution. Under 267 nm excitation, we observed ultrafast charge transfer (CT) between PF and d(GC)9 • d(GC)9 duplex, generating a CT state with an order of magnitude longer lifetime compared to that of the intrinsic excited state reported for the d(GC)9 • d(GC)9 duplex. In contrast, no excited state interaction was detected between PF and d(AT)9 • d(AT)9. Nevertheless, a localized triplet state with a lifetime over 5 µs was identified in the PF-d(AT)9 • d(AT)9 duplex.

Nanoscale Three-Dimensional Imaging of Drug Distributions in Single Cells via Laser Desorption Post-Ionization Mass Spectrometry.

Exploring the three-dimensional (3D) drug distribution within a single cell at nanoscale resolution with mass spectrometry imaging (MSI) techniques is crucial in cellular biology, yet it remains a great challenge due to limited lateral resolution, detection sensitivities, and reconstruction problems. Herein, a microlensed fiber laser desorption post-ionization time-of-flight mass spectrometer (MLF-LDPI-TOFMS) was developed for the 3D imaging of two anticancer drugs within single cells at a 500 × 500 × 500 nm3 voxel resolution. Nanoscale desorption was obtained with a microlensed fiber (MLF), and a 157 nm post-ionization laser was introduced to enhance the ionization yield. Furthermore, a new type of alignment method for 3D reconstruction was developed on the basis of our embedded uniform circular polystyrene microspheres (PMs). Our findings demonstrate that this 3D imaging technique has the potential to provide information about the 3D distributions of specific molecules at the nanoscale level.

Synthesis of Novel Biologically Active Proflavine Ureas Designed on the Basis of Predicted Entropy Changes.

A novel series of proflavine ureas, derivatives 11a-11i, were synthesized on the basis of molecular modeling design studies. The structure of the novel ureas was obtained from the pharmacological model, the parameters of which were determined from studies of the structure-activity relationship of previously prepared proflavine ureas bearing n-alkyl chains. The lipophilicity (LogP) and the changes in the standard entropy (ΔS°) of the urea models, the input parameters of the pharmacological model, were determined using quantum mechanics and cheminformatics. The anticancer activity of the synthesized derivatives was evaluated against NCI-60 human cancer cell lines. The urea derivatives azepyl 11b, phenyl 11c and phenylethyl 11f displayed the highest levels of anticancer activity, although the results were only a slight improvement over the hexyl urea, derivative 11j, which was reported in a previous publication. Several of the novel urea derivatives displayed GI50 values against the HCT-116 cancer cell line, which suggest the cytostatic effect of the compounds azepyl 11b-0.44 µM, phenyl 11c-0.23 µM, phenylethyl 11f-0.35 µM and hexyl 11j-0.36 µM. In contrast, the novel urea derivatives 11b, 11c and 11f exhibited levels of cytotoxicity three orders of magnitude lower than that of hexyl urea 11j or amsacrine.

Dynamical Recrossing in the Intercalation Process of the Anticancer Agent Proflavine into DNA.

Intercalation into DNA is the interaction mode of some anthracycline antibiotics. Recently, the molecular mechanism of this process was explored using the static free energy landscape. Here we explore the dynamical effects in the intercalation of proflavine into DNA by calculating the transmission coefficient κ-providing a measure of the departure from transition state theory for the reaction rate constant-by examination of the recrossing events at the transition state. For that purpose, we first found the accurate transition state of this complex system-as judged by a committor analysis-using a set of all-atom simulations of total length 6.3 ms. In a subsequent calculation of the transmission coefficient κ in another extensive set of simulations the small value κ = 0.1 was found, indicating a significant departure from TST. Comparison of this result with Grote-Hynes and Kramers theories shows that neither theory is able to capture this complex system's recrossing events; the source of this striking failure is discussed, as are related aspects of the mechanism. This study suggests that, for biomolecular processes similar to this, dynamical effects essential for the process are complex in nature and require novel approaches for their elucidation.

Structure and dynamics of proflavine association around DNA.

Proflavine is a small molecule that intercalates into DNA and, thereby, acts as an anticancer agent. Intercalation of proflavine is shown to be a two-step process in which the first step is believed to be the formation of a pre-intercalative outside bound state. Experimental studies so far have been unable to capture the nature of the outside bound state. However, the sub-millisecond timescale observed in fluorescence kinetic experiments is often attributed to the binding of proflavine outside of DNA. Here, we have performed molecular dynamics simulations with multiple proflavine molecules to study the structure and dynamics of the formation of the outside bound state of DNA at different ion concentrations. We observed that the timescale of the outside bound state formation is, at least, five orders of magnitude faster (in nanoseconds) than the experimentally reported timescale (sub-milliseconds) attributed to binding outside DNA. Moreover, we also observed the stacked arrangement of proflavine all around DNA, which is different from the experimentally predicted stacking arrangement perpendicular to the helical axis of DNA in the close vicinity of the phosphate groups. This study, therefore, provides insight into the molecular structure and dynamics of the pre-intercalative outside bound state and will help in understanding the overall intercalation mechanism.

Physical and chemical stability of proflavine contrast agent solutions for early detection of oral cancer.

BACKGROUND AND PURPOSE: Proflavine hemisulfate solution is a fluorescence contrast agent to visualize cell nuclei using high-resolution optical imaging devices such as the high-resolution microendoscope. These devices provide real-time imaging to distinguish between normal versus neoplastic tissue. These images could be helpful for early screening of oral cancer and its precursors and to determine accurate margins of malignant tissue for ablative surgery. Extemporaneous preparation of proflavine solution for these diagnostic procedures requires preparation in batches and long-term storage to improve compounding efficiency in the pharmacy. However, there is a paucity of long-term stability data for proflavine contrast solutions. METHODS: The physical and chemical stability of 0.01% (10 mg/100 ml) proflavine hemisulfate solutions prepared in sterile water was determined following storage at refrigeration (4-8℃) and room temperature (23℃). Concentrations of proflavine were measured at predetermined time points up to 12 months using a validated stability-indicating high-performance liquid chromatography method. RESULTS: Proflavine solutions stored under refrigeration were physically and chemically stable for at least 12 months with concentrations ranging from 95% to 105% compared to initial concentration. However, in solutions stored at room temperature increased turbidity and particulates were observed in some of the tested vials at 9 months and 12 months with peak particle count reaching 17-fold increase compared to baseline. Solutions stored at room temperature were chemically stable up to six months (94-105%). CONCLUSION: Proflavine solutions at concentration of 0.01% were chemically and physically stable for at least 12 months under refrigeration. The solution was chemically stable for six months when stored at room temperature. We recommend long-term storage of proflavine solutions under refrigeration prior to diagnostic procedure.

Oil and drug control the release rate from lyotropic liquid crystals.

The control of the diffusion coefficient by the dimensionality d of the structure appears as a most promising lever to efficiently tune the release rate from lyotropic liquid crystalline (LLC) phases and dispersed particles towards sustained, controlled and targeted release. By using phosphatidylcholine (PC)- and monolinoleine (MLO)-based mesophases with various apolar structural modifiers and water-soluble drugs, we present a comprehensive study of the dimensional structural control of hydrophilic drug release, including 3-d bicontinuous cubic, 2-d lamellar, 1-d hexagonal and 0-d micellar cubic phases in excess water. We investigate how the surfactant, the oil properties and the drug hydrophilicity mitigate or even cancel the effect of structure variation on the drug release rate. Unexpectedly, the observed behavior cannot be fully explained by the thermodynamic partition of the drug into the lipid matrix, which points out to previously overlooked kinetic effects. We therefore interpret our results by discussing the mechanism of structural control of the diffusion rate in terms of drug permeation through the lipid membrane, which includes exchange kinetics. A wide range of implications follow regarding formulation and future developments, both for dispersed LLC delivery systems and topical applications in bulk phase.

Spectroscopic and thermodynamic insights into the interaction between proflavine and human telomeric G-quadruplex DNA.

The G-quadruplex (GQ-DNA), an alternative structure motif of DNA, has emerged as a novel and exciting target for anticancer drug discovery. GQ-DNA formed in the presence of monovalent cations (Na(+)/K(+)) by human telomeric DNA is a point of interest due to their direct relevance for cellular aging and abnormal cell growths. Small molecules that selectively target and stabilize G-quadruplex structures are considered to be potential therapeutic anticancer agents. Herein, we probe G-quadruplex and proflavine (a well-known DNA intercalator, hence acting as an anticarcinogen) association through steady state and time-resolved fluorescence spectroscopy to explore the effect of stabilization of GQ-DNA by this well-known DNA intercalator. The structural modifications of G-quadruplex upon binding are highlighted through circular dichroism (CD) spectra. Moreover, a detailed insight into the thermodynamics of this interaction has been provided though isothermal titration calorimetry (ITC) studies. The thermodynamic parameters obtained from ITC help to gain knowledge about the nature as well as the driving forces of binding. This present study shows that proflavine (PF) can act as a stabilizer of telomeric GQ-DNA through an entropically as well as enthalpically feasible process with high binding affinity and thereby can be considered as a potential telomerase inhibitor.

Intercalation and de-intercalation pathway of proflavine through the minor and major grooves of DNA: roles of water and entropy.

DNA intercalation is a clinically relevant biophysical process due to its potential to inhibit the growth and survival of tumor cells and microbes through the arrest of the transcription and replication processes. Extensive kinetic and thermodynamic studies have followed since the discovery of the intercalative binding mode. However, the molecular mechanism and the origin of the thermodynamic and kinetic profile of the process are still not clear. Here we have constructed the free energy landscape of intercalation, de-intercalation and dissociation from both the major and minor grooves of DNA using extensive all-atom metadynamics simulations, capturing both the free energy barriers and stability in close agreement with fluorescence kinetic experiments. In the intercalated state, an alternate orientation of proflavine is found with an almost equal stability compared to the crystal orientation, however, separated by a 5.0 kcal mol(-1) barrier that decreases as the drug approaches the groove edges. This study provides a comprehensive picture in comparison with experiments, which indicates that the intercalation and de-intercalation of proflavine happen through the major groove side, although the effective intercalation barrier increases because the path of intercalation goes through the stable (abortive) minor groove bound state, making the process a millisecond long one in excellent agreement with the experiments. The molecular origin of the higher barrier for the intercalation from the minor groove side is attributed to the desolvation energy of DNA and the loss of entropy, while the barrier from the major groove, in the absence of desolvation energy, is primarily entropic.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

Molecular mechanism of direct proflavine-DNA intercalation: evidence for drug-induced minimum base-stacking penalty pathway.

DNA intercalation, a biophysical process of enormous clinical significance, has surprisingly eluded molecular understanding for several decades. With appropriate configurational restraint (to prevent dissociation) in all-atom metadynamics simulations, we capture the free energy surface of direct intercalation from minor groove-bound state for the first time using an anticancer agent proflavine. Mechanism along the minimum free energy path reveals that intercalation happens through a minimum base stacking penalty pathway where nonstacking parameters (Twist→Slide/Shift) change first, followed by base stacking parameters (Buckle/Roll→Rise). This mechanism defies the natural fluctuation hypothesis and provides molecular evidence for the drug-induced cavity formation hypothesis. The thermodynamic origin of the barrier is found to be a combination of entropy and desolvation energy.

Rapid CE-UV binding tests of environmentally hazardous compounds with polymer-modified magnetic nanoparticles.

Hazardous compounds and bacteria in water have an adverse impact on human health and environmental ecology. Polydopamine (or polypyrrole)-coated magnetic nanoparticles and polymethacrylic acid-co-ethylene glycol dimethacrylate submicron particles were investigated for their fast binding kinetics with bisphenol A, proflavine, naphthalene acetic acid, and Escherichia coli. A new method was developed for the rapid determination of % binding by sequential injection of particles first and compounds (or E. coli) next into a fused-silica capillary for overlap binding during electrophoretic migration. Only nanolitre volumes of compounds and particles were sufficient to complete a rapid binding test. After heterogeneous binding, separation of the compounds from the particles was afforded by capillary electrophoresis. % binding was influenced by applied voltage but not current flow. In-capillary coating of particles affected the % binding of compounds.

[Energetics of complex formation of the DNA hairpin structure d(GCGAAGC) with aromatic ligands].

The energy contributions of various physical interactions to the total Gibbs energy of complex formation of the biologically important DNA hairpin d(GCGAAGC) with aromatic antitumor antibiotics daunomycin and novantron and the mutagens ethidium and proflavine have been calculated. It has been shown that the relatively small value of the total energy of binding of the ligands to the hairpin is the sum of components great in absolute value and different in sign. The contributions of van der Waals interactions and both intra- and intermolecular hydrogen bonds and bonds with aqueous environment have been studied. According to the calculations, the hydrophobic and van der Waals components are energetically favorable in complex formation of the ligands with the DNA pairpin d(GCGAAGC), whereas the electrostatic (with consideration of hydrogen bonds) and entropic components are unfavorable.

DNA binding of a proflavine derivative bearing a platinum hanging residue.

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I=0.1M and 25°C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides.

Magnetic field effect corroborated with docking study to explore photoinduced electron transfer in drug-protein interaction.

Conventional spectroscopic tools such as absorption, fluorescence, and circular dichroism spectroscopy used in the study of photoinduced drug-protein interactions can yield useful information about ground-state and excited-state phenomena. However, photoinduced electron transfer (PET) may be a possible phenomenon in the drug-protein interaction, which may go unnoticed if only conventional spectroscopic observations are taken into account. Laser flash photolysis coupled with an external magnetic field can be utilized to confirm the occurrence of PET and authenticate the spin states of the radicals/radical ions formed. In the study of interaction of the model protein human serum albumin (HSA) with acridine derivatives, acridine yellow (AY) and proflavin (PF(+)), conventional spectroscopic tools along with docking study have been used to decipher the binding mechanism, and laser flash photolysis technique with an associated magnetic field (MF) has been used to explore PET. The results of fluorescence study indicate that fluorescence resonance energy transfer takes place from the protein to the acridine-based drugs. Docking study unveils the crucial role of Ser 232 residue of HSA in explaining the differential behavior of the two drugs towards the model protein. Laser flash photolysis experiments help to identify the radicals/radical ions formed in the due course of PET (PF(•), AY(•-), TrpH(•+), Trp(•)), and the application of an external MF has been used to characterize their initial spin-state. Owing to its distance dependence, MF effect gives an idea about the proximity of the radicals/radical ions during interaction in the system and also helps to elucidate the reaction mechanisms. A prominent MF effect is observed in homogeneous buffer medium owing to the pseudoconfinement of the radicals/radical ions provided by the complex structure of the protein.

Synthesis, characterization and cytotoxicity studies of palladium(II)-proflavine complexes.

An investigation of the reaction of Pd(II) complexes with proflavine (3,6-diaminoacridine) resulted in the isolation of the compounds [Pd(terpy)(proflavine)](NO(3))(HSO(4))*3H(2)O, 1, (terpy = 2,2':6',2″-terpyridine), [Pd(en)(proflavineH))](NO(3))(SO(4)), 2, (en = ethylenediamine), and [Pd(proflavineH)Cl(2)](SO(4))(0.5)*H(2)O, 3. They have been isolated and characterized by NMR, IR, and electro-spray ionization mass spectrometry techniques and by elemental analyses. The proflavine was bonded to the Pd(II) through the endocyclic nitrogen in 1, but through the proflavine NH(2) in 2. Compound 3 appeared to be polymeric in the solid state with a 1:1 mole ratio of Pd(II):proflavine. Upon solution of 3 in DMSO, two unique species were formed. In one species the Pd(II) was bonded to two proflavines through the endocyclic nitrogen (1:2 mole ratio) and in the other species, a Pd(II) was bonded to each NH(2) group of a single proflavine (2:1 mole ratio). Molecular modeling of the equilibrium geometry by Spartan 8 produced structures which were consistent with the experimental data on the solutions of the three compounds. In vitro cytotoxicity testing against two breast cancer cell lines and one ovarian cancer cell line showed that compounds 1 and 3 had significant activity.

Photo-inducible cytotoxic and clastogenic activities of 3,6-di-substituted acridines obtained by acylation of proflavine.

The cytotoxicity and photo-enhanced cytotoxicity of a series of 18 3,6-di-substituted acridines were evaluated on both tumour CHO cells and human normal keratinocytes, and compared to their corresponding clastogenicity as assessed by the micronucleus assay. Compounds 2f tert-butyl N-[(6-tert-butoxycarbonylamino)acridin-3-yl]carbamate and 2d N-[6-(pivalamino)acridin-3-yl]pivalamide displayed a specific cytotoxicity on CHO cells. These results suggested that the two derivatives could be considered as interesting candidates for anticancer chemotherapy and hypothesized that the presence of 1,1-dimethylethyl substituents was responsible for a strong nonclastogenic cytotoxicity. Compounds 2b and 2c, on the contrary, displayed a strong clastogenicity. They indicated that the presence of nonbranched aliphatic chains on positions 3 and 6 of the acridine rings tended to induce a significant clastogenic effect. Finally, they established that most of the acridine compounds could be photo-activated by UVA-visible rays and focussed on the significant role of light irradiation on their biological properties.

Cytotoxic activity of proflavine diureas: synthesis, antitumor, evaluation and DNA binding properties of 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas.

The synthesis of novel 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas was reported. Their biological activity to inhibit cell proliferation was assessed by a MTT assay on two cell lines, HeLa and HCT-116, at micromolar concentration. 1',1''-(Acridin-3,6-diyl)-3',3''-dihexyldiurea hydrochloride was active on a HCT-116 cell line with an IC(50) value of 3.1 microM. The interaction of these compounds with calf thymus DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence and CD spectroscopy. From spectrofluorimetric titrations, binding constants for the DNA-drug complexes were determined (K=0.9-4.2x10(5) M(-1)). Antiproliferative activity of synthesized derivatives might be related to their intercalation into DNA.

Quantifying RNA-peptide interaction by single-quantum dot-based nanosensor: an approach for drug screening.

The sequence-specific RRE RNA-Rev binding is essential for HIV-1 replication and provides a useful in vitro system for real-time evaluating the inhibitory effect of drugs on the RRE-Rev interaction. The rapid and sensitive detection of RRE-Rev interaction in complex biological systems represents a fundamental challenge. Here we report the development of a single-quantum-dot (QD)-based nanosensor for sensitively quantifying Rev peptide-RRE interaction and characterizing the potential inhibitors by virtue of single-molecule detection and QD-based fluorescence resonance energy transfer (FRET). We demonstrate that the stoichiometry of Rev peptide binding to RRE can be accurately determined by using this single-QD-based nanosensor. Importantly, this single-QD-based nanosensor can sensitively quantify the inhibitory efficacy of proflavin on the Rev peptide-RRE binding, even in the presence of substantial levels of interference fluorescence from high-concentration proflavin, which usually prevents the discrimination of FRET signals in ensemble measurements. The application of this nanosensor in the screening of libraries of small-molecule drugs will facilitate the development of new drugs against various diseases, cancers, and HIV.