Search

Three new fluorescent molecular rotors were synthesized with the aim of using them as sensors to dose thymidine phosphorylase, one of the target enzymes of 5-fluorouracil, a potent chemotherapic drug largely used in the treatment of many solid tumors, that acts by hindering the metabolism of pyrimidines. 5-Fluorouracil has a very narrowtherapeutic window, in fact, its optimal dosage is strictly related to the level of its target enzymes that vary significantly among patients, and it would be of the utmost importance to have an easy and fast method to detect and quantify them. The three molecular rotors developed as TP sensors differ in the length of the alkylic spacer joining the ligand unit, a thymine moiety, and the fluorescent molecular rotor, a [4-(1-dimethylamino)phenyl]-pyridinium bromide. Their ability to trigger an optical signal upon the interaction with thymidine phosphorylase was investigated by fluorescent measurements.

Identification and quantification of bacteria affecting human life, directly causing diseases and indirectly contaminating natural ecosystems, are mostly carried out by expensive and time-consuming methods. There is the need for rapid and economic ways for detecting bacteria in the environment and in clinics. We propose the use of engineered liposomes for detecting bacteria in drinking water. Our approach exploits cationic liposomes functionalized with a surface potential-sensitive fluorophore, 4-heptadecylumbelliferone (C17-HC). The interaction between liposomes and bacteria involves a change in the surface potential experienced by C17-HC and switches on an optical signal. We investigated, by DLS, zeta-potential and fluorescence experiments, a large number of cationic liposomes formulated with a natural phospholipid 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC) or 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), C17-HC, and one of three synthetic cationic components, differing from each other for the number of unsaturations on the polar ammonium head, two of which ad hoc synthesized. Then we evaluated the ability of liposomes to produce a fluorescent signal upon interaction with three bacterial strains, Staphylococcus aureus , Escherichia coli and Enterococcus faecalis ; moreover, we analyzed the fluorescent response of each liposome formulation in the presence of the three bacterial strains at the same time, in order to simulate a real scenario. We found that interaction with bacteria triggers an optical signal in six of the evaluated formulations, resulting responsive down to 10 2 CFU/mL of bacteria suspended in pipeline water coming from the water main of Rome (Italy).

5-Fluorouracil (5-FU) is a chemotherapic drug widely employed to treat a wide range of solid tumors. Unfortunately, it has a narrow therapeutic window and the level of its target enzymes in biological fluids of patients can vary considerably. On these premises, a new fluorescent lipid based sensor for the detection of thymidine phosphorylase, one of the target enzymes of 5-FU, was developed, to optimize patient treatment. Both cationic and anionic fluorescent liposomes containing both an amphiphile tail-tagged with a pyrene residue and a 5‐FU derivative were investigated. The effect of the presence of a bulky quencher (the bromine atom) covalently linked to the end of the alkyl chain of the anionic component on the emission signal was also evaluated. The interaction of liposomes with the target enzyme induces the occurrence of a fluorescent signal, at an extent that depends on the formulation, due to the variation of the excimer/monomer ratio. In particular, a promising specific result was obtained upon the interaction of the target enzyme with liposomes formulated with DOPC, the cationic fluorescent surfactant, the 5-FU derivative and 11-bromoundecaonic acid at 5/1/1/3 molar ratio. Langmuir compression isotherms allowed clarifying the influence of lipid organization on the response of the sensor.

The aggregation properties of a new cationic fluorescent amphiphile tagged on the hydrophobic tail with a pyrene moiety and bearing two hydroxyethyl functionalities on the polar headgroup were investigated by fluorescence experiments as pure components or in mixed liposomes containing an unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine, at different molar ratios. The obtained results put in evidence that the conformation and the miscibility of the lipids in the aggregates strongly influence the excimer/monomer ratio. Mixed monolayers at the same composition were investigated by Langmuir compression isotherms to deepen the understanding of lipid organization and miscibility, both in the polar and in the hydrophobic regions. The presence of two hydroxyethyl functionalities on the polar headgroup of the newly synthesized amphiphile exerts a shielding effect of the charge of the amphiphile increasing the compressibility of lipid components in contrast with the disturbing effect of the unsaturated acyl chains of the phospholipid.

In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N(3) position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3'-azido-2'-fluoro-dideoxyuridin. Theoretical and experimental results showed that all the derivatives exert the same inhibition activity of 5-FU either as monomer and when embedded in lipid bilayer.

Pyrene lipids are useful tools to investigate membrane organization and intracellular lipid trafficking. The molecular interactions controlling the organization of lipid monolayers composed of a cationic amphiphile tagged with a pyrene residue and a saturated or unsaturated phospholipid, namely, 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine, were investigated by Langmuir trough isotherms to understand how the molecular structure of the components and their relative amount affect the physicochemical properties of lipid monolayers. The obtained results show that the cationic headgroups and unsaturation of hydrophobic chains strongly affect the organization of the lipid monolayer as a function of the amount of components. On the other hand, the presence of the pyrene moiety does not seem to have a marked influence on the interaction within lipid assembly.

Phase properties and lateral distribution of components in lipid membranes are frequently investigated by fluorescence of pyrene-labeled lipids. In particular conditions, at specific component molar fractions, lipid bilayers can present regions organized in regular arrangements, hexagonal lattices, where the acyl chain form regular patterns around the pyrene tagged chain of the labeled component. Liposomes composed of a pyrenyl twin type amphiphile (1) bearing a pyrrolidinium headgroup and either 1,2-dimyristoyl-sn-glycero-phosphocholine or 1,2-dioleoyl-sn-glycero-phosphocholine were investigated by fluorescence spectroscopy and Langmuir compression isotherm techniques in a restricted range of molar fraction, X1, (0.005-0.15) to evaluate the influence of composition, temperature and curvature radius on lipid organization and to assess the eventual organization in super lattice arrangements. Amphiphile 1 presents different characteristics with respect to the Pyr-PC usually used in similar investigations, both in terms of charge and conformational freedom, and its presence has a significant effect on membrane organization. Our results put in evidence that both temperature and the presence of unsaturation strongly influence the presence of hexagonal lattice in lipid bilayers.

The investigation of certain features of liposomes such as their lipid bilayer permeability or their propensity to fuse, quite often relies on the entrapment in the internal aqueous compartment of hydrophilic molecules whose optical properties change when they are released into the bulk. However, though defined as probes, these molecules bear functional groups that can interact with the liposome components thus having the potential of affecting the properties of the aggregate. To the best of our knowledge, this issue is scarcely hashed in the literature, despite its importance for an appropriate use of hydrophilic probes in liposome characterization. The influence of calcein, rhodamine B and phenol red, three hydrophilic dyes generally employed in liposome characterization, on the features of some cationic liposomes was investigated by dynamic laser light scattering, fluorescence and absorption measurements. The presence of the hydrophilic dye in the internal aqueous compartment of liposomes strongly affects liposome stability, dimension and permeability depending on the molecular structure of probe and lipid components.

5-Fluorouracil (5-FU) is widely employed in the treatment of some of the most frequently occurring malignant tumours such as breast, colon, and skin cancer. Unfortunately, 5-FU exhibits high toxicity and low tumor affinity with the risk of limited effectiveness and severe side effects for the patients. Numerous derivatives have been synthesized to improve the physicochemical, biopharmaceutical and pharmacokinetic properties of 5-FU, diminishing or circumventing some of its disadvantages. Three 4-substituted 5-fluoropyrimidines characterized by the presence of a polyoxyethylene chain of different lengths and a hydrophobic alkyl chain were synthesized and characterized. The new 5-FU derivatives were included in cationic liposomes formulated with a natural phospholipid and a cationic gemini surfactant to be delivered to cancer cells. The cytotoxicity of the formulations was evaluated by MTT assay on the HCT116 cancer cell line in vitro. The investigated liposome formulations showed a higher cytotoxicity compared to the free drug, thus showing great potential in cancer therapy.