Steady-state Fluorescence Anisotropy Research Articles

Impact of Ionic Liquids on the Physicochemical Behavior of Vesicles.

The effects of two ionic liquids (ILs), 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) and 1-butyl-1-methyl pyrrolidinium tetrafluoroborate ([bmp]BF4), on a mixture of phospholipids (PLs) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) (6:3:1, M/M/M, 70% PL) in combination with 30 mol % cholesterol (CHOL) were investigated in the form of a solvent-spread monolayer and bilayer (vesicle). Surface pressure (π)-area (A) isotherm studies, using a Langmuir surface balance, revealed the formation of an expanded monolayer, while the cationic moiety of the IL molecules could electrostatically and hydrophobically bind to the PLs on the palisade layer. Turbidity, dynamic light scattering (size, ζ-potential, and polydispersity index), electron microscopy, small-angle X-ray/neutron scattering, fluorescence spectroscopy, and differential scanning calorimetric studies were carried out to evaluate the effects of IL on the structural organization of bilayer in the vesicles. The ILs could induce vesicle aggregation by acting as a "glue" at lower concentrations (<1.5 mM), while at higher concentrations, the ILs disrupt the bilayer structure. Besides, ILs could result in the thinning of the bilayer, evidenced from the scattering studies. Steady-state fluorescence anisotropy and lifetime studies suggest asymmetric insertion of ILs into the lipid bilayer. MTT assay using human blood lymphocytes indicates the safe application of vesicles in the presence of ILs, with a minimal toxicity of up to 2.5 mM IL in the dispersion. These results are proposed to have applications in the field of drug delivery systems with benign environmental impact.

Revealing the Improved Binding Interaction of Plant Alkaloid Harmaline with Human Hemoglobin in Molecular Crowding Condition.

Harmaline and harmine are two structurally similar β-carboline alkaloids with several therapeutic activities, such as anti-inflammatory, antioxidant, neuroprotective, nephroprotective, antidiabetic, and antitumor activities. It has been previously reported that the interaction between harmaline and hemoglobin (Hb) is weak in buffer media compared to harmine. Crowding agents induce a molecular crowding environment in the ex vivo condition, which is almost similar to the intracellular environment. In this present study, we have investigated the nature of the interactions of harmaline and harmine with Hb by increasing the percentage of the crowding agent in buffer solution. The results of the UV-vis and fluorescence spectroscopy analysis have showed that with an increasing proportion of crowding agents, the interaction between harmaline and Hb is steadily improving in comparison to harmine. It has been found that the binding constant of Hb-harmaline reaches 6.82 × 105 M-1 in the 40% polyethylene glycol 200-mediated crowding condition, indicating high affinity compared to very low interaction in buffer media. Steady-state fluorescence anisotropy along with fluorescence lifetime measurements further revealed that the rotational movement of harmaline is maximally restricted by Hb in high crowding environments. Stoichiometry results represent that Hb and harmaline interacts in a 1:1 ratio in different percentages of the crowding agent. The circular dichroism spectroscopic results predict stronger interaction of harmaline with Hb (secondary structure alterations) in a higher crowding environment. From the melting study, it was found that the reactions between Hb and harmaline in crowding environments are endothermic (ΔH > 0) and disordering (ΔS > 0) in nature, indicating that hydrogen bonding and van der Waals interactions are the main interacting forces between Hb and harmaline. Harmaline molecules are more reactive in molecular crowding conditions than in normal buffer condition. This study represents that the interaction between harmaline and Hb is stronger compared to the structurally similar harmine in a molecular crowding environment, which may enlighten the drug discovery process in cell-mimicking conditions.

The effect of trans-resveratrol on the physicochemical properties of lipid membranes with different cholesterol content

Resveratrol is one of the most popular phytoalexins, which naturally occurs in grapes and red wine. This compound not only has beneficial effects on the human body, especially on the cardiovascular system, but also has antiviral, antibacterial and antifungal properties. In addition, resveratrol may have therapeutic effects against various types of cancer. The mechanism of action of resveratrol is not fully understood, but it is suspected that one of the most important steps is its interaction with the cell membrane and changing its molecular organization. Therefore, in the present study, we investigated the effects of resveratrol at different concentrations (0–75 μM) on model membranes composed of POPC, SM and cholesterol, in systems with different cholesterol contents and a constant POPC/SM molar ratio (1:1). Our tests included systems containing 5, 15 and 33.3 mol% cholesterol. Tests were carried out for monolayers using the Langmuir monolayer technique supported by Brewster angle microscopy and penetration experiments. Bilayer (liposome) experiments included calcein release, steady-state DPH fluorescence anisotropy and partition coefficients. The results showed that resveratrol interacts with model cell membranes (lipid monolayers and lipid bilayers), and its incorporation into membranes is accompanied by changes in their physicochemical parameters, such as lipid packing, fluidity and permeability. Furthermore, we showed that the cholesterol content of the membrane significantly affects the degree of incorporation of resveratrol into the model membrane, which may indicate that the molecular mechanism of action of this compound is closely related to its interactions with lipid rafts, domains responsible for regulating various cellular functions.

Water-induced fluorescence turn-on imidazole derivative and its interaction with bovine serum albumin

A new trityl imidazole derivative L was synthesized in one-step by reacting 1H-imidazole with triphenylmethyl chloride. The receptor L was applied for the detection of trace amounts of water in DMSO. The weakly emissive L in DMSO showed a significant fluorescence enhancement upon the addition of water due to the suppression of the photo-induced electron transfer process and aggregation-induced emission. The sensitivity of L to detect water in DMSO was estimated as 0.002 wt%. Further, the protein-ligand binding interaction between L and BSA was explored by steady-state fluorescence, fluorescence anisotropy and fluorescence lifetime under physiological conditions at pH 7. The formation of the BSA-L complex quenched the fluorescence emission of BSA. The molecular docking analysis was performed to establish the binding mode and stability of the BSA-L complex. The multiple non-covalent interactions occurred between the residues of BSA and the receptor L stabilized the BSA-L complex.

Apollo-IRE1: A genetically encoded sensor for live cell and multiplexed imaging of ER stress.

Several metabolic diseases, such as Alzheimer's, diabetes, and arthritis, are caused in part by the cellular accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER). In response to protein misfolding, a transmembrane stress-sensor called IRE1α activates the Unfolded Protein Response (UPR) to maintain protein folding homeostasis. To measure ER stress response in living beta-cells with spatial and temporal resolution, we developed a genetically encoded sensor based on fluorescent protein-tagged IRE1α. This sensor homo-oligomerizes to dimeric and tetrameric conformations under early and chronic ER stress, respectively, resulting in homoFRET and an associated decrease in steady-state fluorescence anisotropy (i.e., decreased IRE1 anisotropy/polarisation of fluorescence due oligomerization (Apollo-IRE1)). Apollo-IRE1 responds to chemical and physiological (high glucose) stress in a rat beta-cell line (INS-1E) and mouse islets. Preliminary data suggest that UPR-associated XBP1 mRNA splicing is highly correlated with sensor response. Furthermore, individual cells with the highest sensor response (correlating to late-stage UPR) show elevated levels of pro-apoptotic protein TXNIP. The single colour design of Apollo sensors also makes them ideal for multiplexing, offering an exciting opportunity to explore multiparametric analysis of cellular metabolic characteristics. To demonstrate the multiplexing capability of Apollo-IRE1, we will explore simultaneously imaging of a sensor for NADPH (Apollo-NADP+) and by immunofluorescence with other ER stress markers. These data validate and demonstrate the utility of Apollo-IRE1 in sensing early and chronic ER stress through live cell imaging and multiplexed analysis.

Gallic Acid-Triethylene Glycol Aptadendrimers Synthesis, Biophysical Characterization and Cellular Evaluation.

Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid-triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.

Thin-Plate Superstructures of the Immunogenic 33-mer Gliadin Peptide.

Gluten related-disorders have a prevalence of 1-5 % worldwide triggered by the ingestion of gluten proteins in wheat, rye, barley, and some oats. In wheat gluten, the most studied protein is gliadin, whose immunodominant 33-mer amino acid fragment remains after digestive proteolysis and accumulates in the gut mucosa. Here, we report the formation of 33-mer thin-plate superstructures using intrinsic tyrosine (Tyr) steady-state fluorescence anisotropy and cryo-TEM in combination with water tension measurements. Furthermore, we showed that fluorescence decay measurements of 33-mer intrinsic fluorophore Tyr provided information on the early stages of the formation of the thin-plate structures. Finally, conformational analysis of Tyr residues using minimalist models by molecular dynamic simulations (MD) demonstrated that changes in Tyr rotamer states depend on the oligomerization stage. Our findings further advance the understanding of the formation of the 33-mer gliadin peptide superstructures and their relation to health and disease.

Bactericidal Activity of Carvacrol against Streptococcus pyogenes Involves Alteration of Membrane Fluidity and Integrity through Interaction with Membrane Phospholipids

Background: Carvacrol, a mono-terpenoid phenol found in herbs, such as oregano and thyme, has excellent antibacterial properties against Streptococcus pyogenes. However, its mechanism of bactericidal activity on S. pyogenes has not been elucidated. Objectives: This study investigated the bactericidal mechanism of carvacrol using three strains of S. pyogenes. Methods: Flow cytometry (FCM) experiments were conducted to determine carvacrol’s membrane permeabilization and cytoplasmic membrane depolarization activities. Protoplasts of S. pyogenes were used to investigate carvacrol’s effects on the membrane, followed by gel electrophoresis. The carvacrol-treated protoplasts were examined by transmission electron microscopy (TEM) to observe ultrastructural morphological changes. The fluidity of the cell membrane was measured by steady-state fluorescence anisotropy. Thin-layer chromatographic (TLC) profiling was conducted to study the affinity of carvacrol for membrane phospholipids. Results: Increased membrane permeability and decreased membrane potential from FCM and electron microscopy observations revealed that carvacrol killed the bacteria primarily by disrupting membrane integrity, leading to whole-cell lysis. Ultra-structural morphological changes in the membrane induced by carvacrol over a short period were confirmed using the S. pyogenes protoplast and membrane isolate models in vitro. In addition, changes in the other biophysical properties of the bacterial membrane, including concentration- and time-dependent increased fluidity, were observed. TLC experiments showed that carvacrol preferentially interacts with membrane phosphatidylglycerol (P.G.), phosphatidylethanolamine (P.E.), and cardiolipins (CL). Conclusions: Carvacrol exhibited rapid bactericidal action against S. pyogenes by disrupting the bacterial membrane and increasing permeability, possibly due to affinity with specific membrane phospholipids, such as P.E., P.G., and CL. Therefore, the bactericidal concentration of carvacrol (250 µg/mL) could be used to develop safe and efficacious natural health products for managing streptococcal pharyngitis or therapeutic applications.

New Two-Photon Absorbing Squaraine Derivative with Efficient Near-Infrared Fluorescence, Superluminescence, and High Photostability

The nature of linear photophysical and nonlinear optical properties of a new squaraine derivative 2,4-bis[4-(azetidyl)-2-hydroxyphenyl]squaraine (1) with efficient near-infrared (NIR) emission was comprehensively analyzed based on spectroscopic, photochemical, and two-photon absorption (2PA) measurements, along with quantum chemical analysis. The steady-state absorption, fluorescence, and excitation anisotropy spectra of 1 and its fluorescence emission lifetimes revealed the multiple aspects of the electronic structure of 1, including the relative orientations of the main transition dipoles, effective rotational volumes in solvents of different polarities, and a maximum molar extinction of 1.35 × 10-5 M-1·cm-1, which is unusually small for similar symmetric squaraines. The degenerate 2PA spectrum of 1 was obtained over a broad spectral range under femtosecond excitation, using standard open-aperture Z-scan and two-photon induced fluorescence methods, revealing maximum 2PA cross sections of ∼400 GM. Squaraine 1 exhibited efficient superluminescence emission in the polar solvent (dichloromethane) at room temperature under femtosecond pumping conditions. Quantum chemical analysis of the electronic structure of 1 was performed using the DFT/TD-DFT level of theory and found to be in good agreement with experimental data. The new squaraine derivative 1 displayed high fluorescence quantum yield, efficient NIR superluminescence, large 2PA cross sections, and high photostability with a photodecomposition quantum yield ∼4 × 10-6, suggesting its potential for applications in two-photon fluorescent bioimaging and lasing.

Photophysical Behaviour of Novel Quaternary Pyrenoyl Salts and Their Sensitivity Towards Bile Salt Micellization.

The present work describes the synthesis and characterization of pyrene derivatives, N-(1-Pyrenoylmethyl) pyridinium bromide (PM-PB) and N-(1-Pyrenoylmethyl)-N,N,N-triethylammonium bromide (PM-TAB). The photophysical behavior of these molecules has been studied in various protic and aprotic solvents. Using steady state fluorescence intensity, fluorescence anisotropy and dynamic fluorescence lifetime studies, the sensitivity of these molecules towards the micellization process of bile salts has been monitored. These derivatives have been effectively used in estimating critical micellar concentration (CMC) of bile salt, sodium deoxycholate (NaDC).

Hyaluronan interactions with cationic surfactants – Insights from fluorescence resonance energy transfer and anisotropy techniques

Interactions of hyaluronan with micelles formed by cationic surfactants were studied by the time-resolved measurement of fluorescence resonance energy transfer (FRET) using perylene and fluorescein as probes. Two surfactants were studied – Cetyltrimethylammonium bromide (CTAB) and Septonex. In pure micellar solutions, the same values of FRET efficiency were found for both surfactants, but values for the binding of the probe pair were lower for Septonex micelles than in the case of CTAB. This was attributed to steric effects of the carbethoxy group in the Septonex polar head. Upon the addition of hyaluronan, decreased FRET efficiency and increased binding were detected in comparison with pure surfactants. To resolve the structure of the formed aggregates, steady state and time-resolved fluorescence anisotropy was employed as an additional technique. All results indicated that cationic micelles bind to hyaluronan forming a pearl necklace structure with micelles of smaller size compared to pure surfactant. Besides theoretical interest, the studied polyelectrolyte-surfactant system may be interesting for the formulation of drug delivery systems.

Modulation of Cholesterol Pathways in Human Macrophages Infected by Clinical Isolates of Leishmania infantum.

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite’s ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF.

Transformation of an Aqueous Micellar Phase to a Bilayer of Gemini Surfactants on Gold Nanoparticles: A Steady-State and Time-Resolved Fluorescence and Fluorescence Anisotropy Study by Tuning the Precise Locations of Probes

The present work reports the transformation of aqueous micelles of gemini surfactants (12-4-12,2Br– or 12-8-12,2Br–) to a bilayer of surfactants on the surface of gold nanoparticles (AuNPs) capped with citrate during their in situ synthesis. The synthesized AuNPs are characterized using surface plasmon resonance (SPR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), scanning transmission electron microscopy (STEM), and high-resolution transmission electron microscopy (HR-TEM) techniques. The transformation of micelles to a surfactant bilayer with time is understood with the help of fluorescence measurements such as steady-state and time-resolved fluorescence and fluorescence anisotropy, probed by tuning the precise locations of two fluorophores, Coumarin-480 (C-480) and rhodamine 6G (Rh6G), present in the micelles. As the formation of AuNPs initiates, the dyes get relocated from micelles to a site near the NP surface, which results in fluorescence quenching and a decrease in lifetime due to the nanomaterial surface energy transfer (NSET) from the donor dye to the acceptor AuNPs. The results indicated that Rh6G lies close to the head groups of the surfactant in micelles. Fast segmental/tumbling motions of Rh6G in micelles/bilayers are primarily responsible for the decay of anisotropy to zero. Lateral diffusion is responsible for slow rotational relaxation. With the growth of rod/needle-shaped NPs, the average lifetime and rotational relaxation time increase with an increase in fluorescence intensity due to the transfer of dye molecules from the NP surface to the interior of the bilayer. A significant change in the weightage of the slow component of C-480 compared to that of Rh6G with the formation of the bilayer supports that the former dye is located deep inside the bilayer with an equal contribution to depolarization from both the rotational motions at equilibrium. The excitation wavelength-dependent rotational relaxation time of Rh6G in the bilayer supports the prolate ellipsoidal shape of AuNPs surrounded by the bilayer. This study can help understand the mechanism of drug loading in forming metal NP-based hybrid drug delivery systems in an aqueous micellar solution of gemini surfactants.

On the synergy/antagonism of selected terpenes in the effect on lipid membranes studied in model systems

In this work the influence of the mixtures of four terpenes on the lipid monolayers and bilayers imitating bacteria membrane was analyzed. The aim of these investigations was to verify possible synergistic/antagonistic effects occurring between terpenes and affecting their influence on membranes. The studies were performed for major and minor components of the hop essential oil, namely: β-myrcene, α-humulene, E-β-Farnesene and limonene. The experiments on monolayers were based on the studies on penetration of terpenes and their mixtures into model systems, on the surface pressure-area measurements, and Brewster angle microscopy studies, while for bilayers they involved the hydrodynamic diameter, zeta potential and steady-state fluorescence anisotropy measurements of liposomes. Based on the collected results it was concluded that the synergy occurring between selected terpenes increases the effect of their mixtures on monolayers and bilayers as compared to particular components. Among the studied mixed terpene systems the equimolar myrcene/humulene/farnesene/limonene system was found to penetrate the most strongly into the monolayer and change its morphology as well as to make the bilayer more fluid. Additionally, from the comparison of these results with those published for the hop oil it was summarized that neither the studied mixtures nor the individual studied terpenes change the properties of the model membranes as effectively as the total extract. Thus, the action of the hop oil on membrane is not determined by its major components but it is rather the result of the interactions and synergy between major and minor components.

Pressure and temperature dependence of fluorescence anisotropy of green fluorescent protein.

We have studied the effect of high hydrostatic pressure and temperature on the steady state fluorescence anisotropy of Green Fluorescent Protein (GFP). We find that the fluorescence anisotropy of GFP at a constant temperature decreases with increasing pressure. At atmospheric pressure, anisotropy decreases with increasing temperature but exhibits a maximum with temperature for pressure larger than 20 MPa. The temperature corresponding to the maximum of anisotropy increases with increasing pressure. By taking into account of the rotational correlation time changes of GFP with the pressure–temperature dependent viscosity of the solvent, we argue that viscosity increase with pressure is not a major contributing factor to the decrease in anisotropy with pressure. The decrease of anisotropy with pressure may result from changes in H-bonding environment around the chromophore.

A modern, simple, and economical accessory for continuous L-format measurement of steady-state fluorescence anisotropy

In this study, the pros and cons of the most relevant L-format devices reported in the literature for measuring steady-state fluorescence polarization/anisotropy are identified. Combining all this information, and with the use of modern elements for the acquisition, treatment, and recording of signals, a modern, simple, and economical L-format accessory is implemented to rapidly and continuously record steady-state fluorescence anisotropy. This device can be adapted to the majority of the commercial spectrofluorometers (or fluorometers). During the measurement, the emission polarizer is in permanent rotation by means of a Gimbal brushless DC motor, and as a result the recorded fluorescence signal is sinusoidal. The maximums and minimums of this signal, which are obtained with the help of LabVIEW tools, allow recording the fluorescence anisotropy. The LabVIEW applications developed for this investigation are freely available, so it is not necessary to have LabVIEW software.

Interaction of Dopamine with Zwitterionic DMPC and Anionic DMPS Multilamellar Vesicle Membranes.

Dopamine (DA), a naturally occurring neurotransmitter, plays a crucial role in the function of the mammalian nervous system. DA-lipid-membrane interaction is inevitable during the neurotransmission process. In this report, we have studied the interaction of DA with anionic 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS), neutral (zwitterionic) 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and synaptic membrane-mimicking mixed DMPC/DMPS (3:1 molar ratio) model multilamellar vesicle (MLV) membranes. Differential scanning calorimetry (DSC) studies suggest a strong specific interaction of DA with the anionic DMPS membrane, a weak interaction with the zwitterionic DMPC membrane, and a moderate interaction with the mixed DMPC/DMPS (3:1) membrane. The intrinsic fluorescence of DA was used as a new approach to gain a molecular-level understanding of DA-lipid-membrane interaction. Toward this end, a detailed photophysical study of DA, including its steady-state fluorescence anisotropy and fluorescence lifetime, was undertaken for the first time. The partition coefficient, location, and distribution of DA in the DMPS and DMPC model membranes were studied by employing intrinsic fluorescence. The effect of DA on the phase transition of the model membranes was also examined using the intrinsic fluorescence of DA. Zeta potential studies suggest a strong electrostatic interaction of DA with the anionic DMPS membrane and a nonspecific, relatively weak interaction of DA with the zwitterionic DMPC membrane. In addition, we observed cholesterol-induced DA expulsion from both DMPS and DMPC membranes. We believe that this work will provide a more in-depth understanding of DA-membrane interaction at a molecular level.

Effect of lipopolymer (DSPE-PEG750) on phospholipid monolayers and bilayers differing in the structure of the polar head group

Phospholipid liposomes are the most popular kind of vesicles used in drug delivery due to their high biocompatibility with biomembranes. However, this type of liposomes show some limitations related with their short half-life in bloodstream. This problem can be resolved by the coating of their surface with the application of PEG molecules which form steric and entropic barrier, avoiding recognition of encapsulated liposomes by the reticuloendothelial system (RES). Such modification of liposomes significantly affects the physicochemical characteristics of PEGylated liposomes, which is related to the intermolecular interactions between the lipopolymer molecules and the phospholipid that builds the liposome membrane. In our work we investigated the influence of the PEG-ylated lipid (DSPE-PEG750) on physicochemical properties of phospholipid monolayers and bilayers with different structure of the polar group (DPPC, DPPE, DPPS). The phospholipid monolayers were examined with the application of Langmuir monolayer technique and Brewster angle microscopy (BAM) as well as grazing incidence x-ray diffraction (GIXD). The studies performed on phospholipid bilayer systems were related to dynamic light scattering and zeta potential measurements and the experiments with the calcein release and steady-state fluorescence anisotropy of DPH. The obtained results showed that the type of polar group of phospholipid as well as the addition of PEG-ylated lipid significantly changes the molecular organization of phospholipid monolayers. Moreover, these parameters also influence the properties of phospholipid bilayers such as size, surface charge, stability and permeability.

Nature of Linear Spectral Properties and Fast Electronic Relaxations in Green Fluorescent Pyrrolo[3,4-c]Pyridine Derivative.

The electronic nature of 4-hydroxy-1H-pyrrolo[3,4-c]pyridine-1,3,6(2H,5H)-trione (HPPT) was comprehensively investigated in liquid media at room temperature using steady-state and time-resolved femtosecond transient absorption spectroscopic techniques. The analysis of the linear photophysical and photochemical parameters of HPPT, including steady-state absorption, fluorescence and excitation anisotropy spectra, along with the lifetimes of fluorescence emission and photodecomposition quantum yields, revealed the nature of its large Stokes shift, specific changes in the permanent dipole moments under electronic excitation, weak dipole transitions with partially anisotropic character, and high photostability. Transient absorption spectra of HPPT were obtained with femtosecond resolution and no characteristic solvate relaxation processes in protic (methanol) solvent were revealed. Efficient light amplification (gain) was observed in the fluorescence spectral range of HPPT, but no super-luminescence and lasing phenomena were detected. The electronic structure of HPPT was also analyzed with quantum-chemical calculations using a DFT/B3LYP method and good agreement with experimental data was shown. The development and investigation of new pyrrolo[3,4-c]pyridine derivatives are important due to their promising fluorescent properties and potential for use in physiological applications.

The effect of the polyethylene glycol chain length of a lipopolymer (DSPE-PEGn) on the properties of DPPC monolayers and bilayers

Due to the growing importance of controlled drug delivery systems (DDS), the main task of nanotechnology is to develop stable, effective and non-toxic nanocarriers in which the drug can be encapsulated and delivered to a specific diseased site in the patient's body. Currently, one of the most popular ways to improve the pharmacokinetic and physicochemical properties of liposomes is introducing into their structure poly(ethylene glycol) chains conjugated with 1,2-disteroil-sn-glycero-3-phosphoethanolamine (DSPE) molecules. Because the research so far does not give an unequivocal answer which length of PEG chains is more beneficial for liposomes properties, the aim of this work was to investigate the influence of this parameter (DSPE-PEG350, DSPE-PEG750 and DSPE-PEG2000) on model DPPC membrane. The studies were performed on monolayer and bilayer systems and were related to the surface pressure measurements, Brewster angle microscopy experiments, Grazing Incidence X-ray Diffraction studies, dynamic light scattering and zeta potential measurements and the experiments with the calcein release and steady-state fluorescence anisotropy of DPH. The obtained results proved that the molecular organization of the DPPC membrane strongly depends on the length of poly(ethylene glycol) chains conjugated with DSPE. Moreover, the addition of different lengths of polymer chains changes the properties of formulated liposomes, especially their stability, permeability, size and surface charge.