Elina Vuorimaa

Study of PEGylated lipid layers as a model for PEGylated liposome surfaces: Molecular dynamics simulation and Langmuir monolayer studies

We have combined Langmuir monolayer film experiments and all-atom molecular dynamics (MD) simulation of a bilayer to study the surface structure of a PEGylated liposome and its interaction with the ionic environment present under physiological conditions. Lipids that form both gel and liquid-crystalline membranes have been used in our study. By varying the salt concentration in the Langmuir film experiment and including salt at the physiological level in the simulation, we have studied the effect of salt ions present in the blood plasma on the structure of the poly(ethylene glycol) (PEG) layer. We have also studied the interaction between the PEG layer and the lipid bilayer in both the liquid-crystalline and gel states. The MD simulation shows two clear results: (a) The Na(+) ions form close interactions with the PEG oxygens, with the PEG chains forming loops around them and (b) PEG penetrates the lipid core of the membrane for the case of a liquid-crystalline membrane but is excluded from the tighter structure of the gel membrane. The Langmuir monolayer results indicate that the salt concentration affects the PEGylated lipid system, and these results can be interpreted in a fashion that is in agreement with the results of our MD simulation. We conclude that the currently accepted picture of the PEG surface layer acting as a generic neutral hydrophilic polymer entirely outside the membrane, with its effect explained through steric interactions, is not sufficient. The phenomena we have observed may affect both the interaction between the liposome and bloodstream proteins and the liquid-crystalline-gel transition and is thus relevant to nanotechnological drug delivery device design.

Self-assembled films of hydrophobin proteins HFBI and HFBII studied in situ at the air/water interface.

Hydrophobins are a group of surface-active fungal proteins known to adsorb to the air/water interface and self-assemble into highly crystalline films. We characterized the self-assembled protein films of two hydrophobins, HFBI and HFBII from Trichoderma reesei, directly at the air/water interface using Brewster angle microscopy, grazing-incidence X-ray diffraction, and reflectivity. Already in zero surface pressure, HFBI and HFBII self-assembled into micrometer-sized rafts containing hexagonally ordered two-dimensional crystallites with lattice constants of 55 A and 56 A, respectively. Increasing the pressure did not change the ordering of the proteins in the crystallites. According to the reflectivity measurements, the thicknesses of the hydrophobin films were 28 A (HFBI) and 24 A (HFBII) at 20 mN/m. The stable films could also be transferred to a silicon substrate. Modeling of the diffraction data indicated that both hydrophobin films contained six molecules in the unit cell, but the ordering of the molecules was somewhat different for HFBI and HFBII, suggesting specific protein-protein interactions.

Time-resolved fluorescence spectroscopy reveals functional differences of cationic polymer-DNA complexes.

Cationic polymers bind DNA and form compacted nanoparticulates (i.e., polyplexes). Polyplexes augment DNA delivery into the cells as a nonviral method of gene therapy. DNA packing and release are the key factors in polyplex-mediated gene delivery, but they are poorly understood due to the lack of physical methods of investigation. We used time-resolved fluorescence spectroscopy to study poly(ethylenimine) (PEI) and poly(L-lysine) (PLL) polyplexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that DNA exists in several different states in PEI polyplexes and only in one tightly bound state in PLL polyplexes. The observed difference in the nature of the polyplexes may explain why PEI releases DNA more easily than PLL even though both polycations condense DNA effectively. The present method utilizing time-resolved fluorescence spectroscopy gives information on the specific interactions between DNA and the cationic polymers in the polyplexes. This kind of information is very important in the development of biologically effective nonviral systems for DNA delivery.

Role of Polyplex Intermediate Species on Gene Transfer Efficiency: Polyethylenimine−DNA Complexes and Time-Resolved Fluorescence Spectroscopy

Polyethylenimine (PEI) is a cationic DNA condensing polymer that facilitates gene transfer into the mammalian cells. The highest gene transfer with branched PEI is obtained at high nitrogen/phosphate (N/P) ratios with free PEI present. The small molecular weight PEI alone is not able to mediate DNA transfection. Here, we used recently developed time-resolved fluorescence spectroscopic method to study the mechanism of PEI-DNA complex formation and to investigate how free PEI, mean molecular weight, and branching of PEI affect the complexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that for both linear and branched high-molecular-weight PEI the complexation takes place in two steps, but the small-molecular-weight branched PEI complexed DNA at a single step. According to the binding constants obtained from time-resolved spectroscopic measurements, the affinity of N/P complexation per nitrogen atom is highest for LPEI and weakest for BPEI, whereas SPEI-DNA complexation showed intermediate values. Thus, the binding constant alone does not give adequate measure for transfection efficiency. On the other hand, the presence of intermediate states during the polyplex formation seems to be favorable for the gene transfection. Free PEI had no impact on the physical state of PEI-DNA complexes, even though it was essential for gene transfection in the cell culture. In conclusion, the molecular size and topology of PEI have direct influence on the DNA complexation but the free PEI does not. Free PEI must facilitate transfection at the cellular level and not via indirect effects on the PEI-DNA complexes.

Self-assembled structures of hydrophobins HFBI and HFBII

Hydrophobins are small proteins that function in the growth and development of fungi. The structures of class II hydrophobins HFBI and HFBII from Trichoderma reesei were studied using grazing incidence X-ray diffraction. HFBI was weakly ordered but HFBII formed a highly crystalline coating on water surface. Change from monoclinic to hexagonal structure was observed as the sample dried. The three-dimensional structures differed from the oblique two-dimensional structures observed in Langmuir-Blodgett monolayers of both HFBI and HFBII by atomic force microscopy.

A regression technique to analyze the second-order nonlinear optical response of thin films.

We present a new technique, based on regression analysis, to determine the second-order nonlinear optical susceptibility tensor of thin films. The technique does not require the absolute levels or phases of measured signals to be mutually calibrated. In addition it yields indicators that address the quality of theoretical models describing the sample. We use the technique to determine the susceptibility tensor of samples of a nonracemic chiral material which have very low symmetry (both chiral and anisotropic) and have many independent tensor components. The results show the importance of using detailed theoretical models that account for the linear optical properties of the sample.

Curing kinetics and glass-transition temperature of hexamethylene diisocyanate-based polyurethane

The curing process of hexamethylene diisocyanate-based polyurethane has been monitored by applying FTIR and DSC methods. A general relationship between glass-transition temperature (Tg) and conversion of curing process has been obtained. This suggests that the reaction path and the relative reaction rates are independent of the curing temperature. The reaction kinetics of the system is analyzed using the Tg data converted to the conversion of the curing process. A set of experimental data and one theoretical model of Tg versus chemical conversion are presented to prove the assumption where a direct one-to-one relationship between the Tg (as measured) and the chemical conversion is obtained. Apparent activation energies (Ea) obtained by applying three different methods suggest good agreement.

Self-assembled films of hydrophobin protein HFBIII from Trichoderma reesei

Hydrophobins are a group of small amphiphilic proteins which are known to self-assemble on interfaces. They contain eight conserved cysteine residues, which make four disulfide bridges. A new hydrophobin protein, HFBIII, from the fungus Trichoderma reesei contains one extra cysteine residue, giving the protein a naturally reactive site. The self-assembly of hydrophobin protein HFBIII was studied using grazing-incidence X-ray diffraction and reflectivity. HFBIII self-assembles into a hexagonally ordered monolayer at an air/water interface and also forms crystalline coatings on a silicon substrate. The lattice constants for the hexagonal coatings are a = b = 56.5 A, γ = 120°. The self-assembled structure in the HFBIII film is very similar to those formed by two other T. reesei hydrophobins, HFBI and HFBII.

Nonlinear Optical and Structural Properties of Langmuir-Blodgett Films of Thiohelicenebisquinones

We provide a detailed investigation of the second-order nonlinear optical and structural properties of Langmuir-Blodgett (LB) films of nonracemic thiohelicenebisquinone (THBQ). We prepare both X- and Y-type films of different thicknesses and characterize them using optical second-harmonic generation and atomic-force microscopy (AFM). We find that the overall nonlinear properties of the samples are essentially independent of the film thickness and the deposition type and arise from susceptibility tensor components associated with chirality. Both X- and Y-type films can be described by D2 symmetry, which is a higher symmetry than the previously assumed C2 of LB films of THBQ and a similar helicenebisquinone (HBQ). However, the two types of films are shown to differ significantly with respect to the orientation of the in-plane axis. For Y type, the axis follows the direction of vertical sample deposition, but for X type, the direction of the axis varies randomly and significantly between different samples. The Y-type samples are therefore more ordered than the X-type samples. This was confirmed by AFM measurements in which the Y type exhibits uniform ordering into columnar structures. Similar structures in X type, on the other hand, are shorter and more randomly oriented, like those earlier observed for racemic samples of HBQ [Verbiest, T., et al. Science 1998, 282, 913]. The common nonlinear properties and different high-level ordering observed here for two different types of nonracemic samples reinforces that the nonlinearity of THBQ (and probably HBQ, as well) originates from the low-level columnar aggregation of the molecules with the higher-level structures playing a lesser role. In addition, within the columns, the molecules likely assume fairly random azimuthal orientations so that the columns themselves exhibit approximate Dinfinity symmetry.

Advantages of polarized two-beam second-harmonic generation in precise characterization of thin films

Polarized second-harmonic generation using two fundamental beams, instead of one, offers significant advantages for characterizing nonlinear optical thin films. The technique is more precise and allows the internal consistency of the results to be verified. The superiority of the two-beam arrangement over the traditional single-beam arrangement is demonstrated by determining the susceptibility tensors of Langmuir-Blodgett films. We show that, for a well-understood reference sample, the results obtained using two fundamental beams agree qualitatively with those obtained with a single fundamental beam, but are more precise. In a more complicated situation, however, the single-beam technique appears to work well but yields results that are, in fact, incorrect. The two-beam technique, instead, yields clearly inconsistent results, thereby highlighting systematic errors in the experimental arrangement or in the theoretical model used to interpret the results.