Filippo Gambinossi

Modulation of density and orientation of amphiphilic DNA anchored to phospholipid membranes. I. Supported lipid bilayers.

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipids bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper describes the anchoring of novel cholesterol-modified DNA-18mers in supported lipid bilayers (SLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: we compared the behavior of two anchoring functionalities differing in the number of cholesterol units and in the length of a spacer group. Quartz Crystal Microbalance with impedance monitoring (QCM-Z) measurements showed that both oligonucleotides insert into the bilayer membrane through cholesterol anchoring; however, dramatic differences, in terms of surface organization and thickness, are found as the number of anchoring units increases. In the case of multiple cholesterol units, a peculiar three-regimes concentration dependence was revealed and correlated to the effective size of the adsorbing units. Interestingly, for high oligonucleotide concentration, the adsorption process was rationalized in terms of a compaction model of amphiphilic DNA molecules. QCM-Z temperature cycles of the SLB-anchored double strands provided clear evidence for reversible hybridization at the bilayer interface.

Closed nanoconstructs assembled by step-by-step ss-DNA coupling assisted by phospholipid membranes

We report experimental evidence of the construction of closed pseudo-hexagonal DNA nanostructures grafted onto lipid membranes (supported planar bilayers or liposomes). The anchoring agent is a cholesteryl tetraethylenglycol-18-mer oligonucleotide, which recruits the complementary sequences added in a sequential fashion. A closed rather than an open nanostructure was chosen as a test for possible future applications in nanoelectronics and for the fact that a more rigid and defined structure can offer many advantages in terms of addressability and spatial control. The so-built hybrid soft nanomaterial has never been reported previously and merges the unique features offered by DNA building blocks in nanotechnology, to the characteristics of amphiphilic self-assembly, in terms of responsiveness and further hierarchical aggregation in functional arrays of nano-units. The liposomes decorated with DNA pseudo-hexagons are structurally stable for weeks and can thus be further exploited or specifically addressed. The structural features of the final nanoobjects are independent on the sequence of preparation, i.e. step-wise on the membrane or addition of preformed hexagons, up to a threshold of density on the surface or vesicle number density.

Enzymatic hydrolysis reaction of phospholipids in monolayers

Abstract The hydrolysis reaction of l- , d- and d , l -dipalmitoylphosphatidylcholine (DPPC) catalized by bee venom phospholipase A 2 was studied in spreading monolayer at the water/air interface. DPPC and the hydrolysis products, palmitic acid and l -lysophosphatidylcholine, palmitoyl were characterized at the interface by means of surface pressure, surface potential and ellipsometric measurements. Furthermore, mixed monolayers of reagents and products were investigated to ascertain their miscibility. The results show that the hydrolysis reaction can be followed by the decrease of surface pressure with time on subphases containing β-cyclodextrin, a well-known complexing agent of many amphiphilic compounds. The order of the reaction, the kinetic constant and other kinetic parameters are deduced.

DNA closed nanostructures: a structural and Monte Carlo simulation study

DNA nanoconstructs are obtained in solution by using six unique 42-mer DNA oligonucleotides, whose sequences have been designed to form a pseudohexagonal structure. The required flexibility is provided by the insertion of two non-base-paired thymines in the middle of each sequence that work as flexible hinges and constitute the corners of the nanostructure when formed. We show that hexagonally shaped nanostructures of about 7 nm diameter and their corresponding linear open constructs are formed by self-assembly of the specifically designed linear oligonucleotides. The structural and dynamical characterization of the nanostructure is obtained in situ for the first time by using dynamic light scattering (DLS), a noninvasive method that provides a fast dynamic and structural analysis and allows the characterization of the different synthetic DNA nanoconstructs in solution. A validation of the LS results is obtained through Monte Carlo (MC) simulations and atomic force microscopy (AFM). In particular, a mesoscale molecular model for DNA, developed by Knotts et al., is exploited to perform MC simulations and to obtain information about the conformations as well as the conformational flexibilities of these nanostructures, while AFM provides a very detailed particle analysis that yields an estimation of the particle size and size distribution. The structural features obtained by MC and AFM are in good agreement with DLS, showing that DLS is a fast and reliable tool for characterization of DNA nanostructures in solution.

Spectroscopic investigation of tetracycline interaction with phospholipid Langmuir–Blodgett films

Abstract A widely used veterinary antibiotic, tetracycline (TC), has been incorporated in Langmuir–Blodgett (LB) films of dipalmitoylphosphatidic acid (DPPA) by means of two different procedures: co-transfer and incubation in solution. The resulting structures were characterized by means of contact angle and ellipsometric measurements. The presence of the antibiotic in the phospholipid film was evidenced by means of UV–Vis electronic absorption and infrared vibrational spectroscopy. The two sets of measurements unambiguously indicated the presence of the drug in the LB layer films obtained with both methods, although incubation led to a smaller content of immobilized tetracycline. In both cases, the drug was found to reside in the hydrophilic portion of the layers due to specific interactions of the dimethylamino group of the molecule with the polar head groups of the phospholipid.

Modulation of Density and Orientation of Amphiphilic DNA on Phospholipid Membranes. II. Vesicles

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipid bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper compares the behavior of two cholesteryl modified oligonucleotides, differing in the architecture and hydrophobicity of the lipophilic moiety, in the self-aggregation, hybridization, and insertion in phospholipid vesicle membranes. We have focused our attention on a singly substituted derivative (SC-ON(1)) and a multicholesterol (MC-ON(1)) derivative, where the cholesteryl units are inserted at the desired positions along a noncoupling T-sequence. The self-aggregation properties in solution are also explicitly taken into account and evaluated as competitive with respect to the adsorption at fluid or solid interfaces and to hybridization with the complementary ON(2) sequence. By exploring a wide range of ON derivative concentrations, different peculiar scenarios emerge for different hydrophobicity of the amphiphilic DNA guest molecules on the vesicles, in terms of distribution and conformation of the single strand and consequent coupling properties with the complementary strand in solution.

Antibiotic interaction with phospholipid monolayers

Abstract We studied the interactions of tetracycline (TC) antibiotic molecules with phospholipid monolayers with the two-fold aim of elucidating the mechanism of action and providing a first step for the realization of bio-mimetic sensors for such drugs by means of the Langmuir–Blodgett technique. We examined spreading monolayers of three phospholipids in the presence of tetracycline in the subphase by means of surface pressure–area and surface potential–area isotherms as a function of bulk pH. We selected phospholipids with hydrophobic chains of the same length but polar head groups differing either in dimensions and protonation equilibria, i.e. dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE) and dipalmitoylphosphatidic acid (DPPA). The interaction of tetracycline with the three phospholipids was found to be highly dependent on the electric charge of the antibiotic and on the ionization state of the lipid. Significant interactions are established between the negatively charged form of dipalmitoylphosphatidic acid and the zwitterionic form of tetracycline. The drug was found to migrate at the interface where it is adsorbed underneath or/and among the head groups, depending on the surface pressure of the film, whereas penetration through the hydrophobic layer was excluded for all the three phospholipids.

Self-aggregation and phase separation of a styryl dye in monolayer at the liquid–air interface and in Langmuir–Blodgett films

The styryl dye 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium inner salt, (Di-8-ANEPPS), was incorporated in mixed Langmuir, (L), monolayers with the cationic surfactant, octadecyltrimethylammonium bromide, (OTMA). The stability of the monolayers was found to vary with the surface pressure and the amount of the diluent in the mixtures. The organization of the dye at the interface, deduced from surface pressure–area, π–A, and surface potential–area, ΔV–A, isotherms, is compared with the spectroscopic data in Langmuir and Langmuir–Blodgett, (LB), films. UV–visible absorption spectra of the pure dye and of the mixtures in L and LB monolayers show the formation of aggregates or domains of Di-8-ANEPPS within the matrix at high amphiphile content. On the contrary, successful transfer of the monomeric form of the dye was achieved using small molar fraction of the cationic matrix.