Chandrashekhar V. Kulkarni

Engineering bicontinuous cubic structures at the nanoscale—the role of chain splay†

Many amphiphile–water mixtures will self-assemble into three-dimensional soft condensed structures known as inverse bicontinuous cubic phases. These structures are found in nature and have applications in nanotechnology. Here we show that by systematically varying amphiphile chain splay, we are able to control the relative stability of the inverse bicontinuous phases in a homologous series of monoglycerides in a predictable manner. In particular, we demonstrate that decreasing chain splay leads to the appearance of the primitive bicontinuous cubic phase while increasing chain splay reduces the channel size of the remaining two bicontinuous phases and tends to destabilize them with respect to the more curved inverse micellar and inverse hexagonal phases. These observations are consistent with a model in which the energetic stability of these phases is principally governed by the competing demands for homogeneous interfacial curvature and uniform chain packing and points to straightforward rules for engineering these self-assembling nanostructures.

Wettability studies of topologically distinct titanium surfaces

Biomedical implants made of titanium-based materials are expected to have certain essential features including high bone-to-implant contact and optimum osteointegration, which are often influenced by the surface topography and physicochemical properties of titanium surfaces. The surface structure in the nanoscale regime is presumed to alter/facilitate the protein binding, cell adhesion and proliferation, thereby reducing post-operative complications with increased lifespan of biomedical implants. The novelty of our TiO2 nanostructures lies mainly in the high level control over their morphology and roughness by mere compositional change and optimisation of the experimental parameters. The present work focuses on the wetting behaviour of various nanostructured titanium surfaces towards water. Kinetics of contact area of water droplet on macroscopically flat, nanoporous and nanotubular titanium surface topologies was monitored under similar evaporation conditions. The contact area of the water droplet on hydrophobic titanium planar surface (foil) was found to decrease during evaporation, whereas the contact area of the droplet on hydrophobic nanorough titanium surfaces practically remained unaffected until the complete evaporation. This demonstrates that the surface morphology and roughness at the nanoscale level substantially affect the titanium dioxide surface-water droplet interaction, opposing to previous observations for microscale structured surfaces. The difference in surface topographic nanofeatures of nanostructured titanium surfaces could be correlated not only with the time-dependency of the contact area, but also with time-dependency of the contact angle and electrochemical properties of these surfaces.

Water-in-oil nanostructured emulsions: towards the structural hierarchy of liquid crystalline materials

We present novel water-in-oil (W/O) emulsions with a nanostructured oil phase that resemble soft materials such as creams, pastes and spreadable materials used in cosmetics, pharma and food applications. These emulsions have a broad structural hierarchy that involves 50 to 90 volume percent of micron sized water droplets (2–50 µm) confined by a continuous hydrophobic film that itself is made of a lyotropic nanostructure. This nanostructure can be modulated into inverse bicontinuous cubic, micellar cubic, inverse hexagonal or microemulsion phases. The novelty of these W/O nanostructured emulsions lies in (i) their preparation, which does not require any emulsion stabilizer, (ii) their hierarchical structure and intrinsic properties, which can be fine-tuned by varying temperature, water content and amount of oil and (iii) their ability to be loaded with hydrophobic, amphiphilic and hydrophilic functional molecules. Here, we present a systematic study along with the principles behind these dense nanostructured emulsions prepared, for the first time, from a monoglyceride system. High interfacial area, continuous architectural motif, enhanced water storage capacity and extensive tunability of these nanostructured emulsions open up new avenues in various scientific and technological applications.

Nanostructural Studies on Monoelaidin–Water Systems at Low Temperatures

In recent years, lipid based nanostructures have increasingly been used as model membranes to study various complex biological processes. For better understanding of such phenomena, it is essential to gain as much information as possible for model lipid structures under physiological conditions. In this paper, we focus on one of such lipids--monoelaidin (ME)--for its polymorphic nanostructures under varying conditions of temperature and water content. In the recent contribution (Soft Matter, 2010, 6, 3191), we have reported the phase diagram of ME above 30 °C and compared with the phase behavior of other lipids including monoolein (MO), monovaccenin (MV), and monolinolein (ML). Remarkable phase behavior of ME, stabilizing three bicontinuous cubic phases, motivates its study at low temperatures. Current studies concentrate on the low-temperature (<30 °C) behavior of ME and subsequent reconstruction of its phase diagram over the entire temperature-water composition space (temperature, 0-76 °C; and water content, 0-70%). The polymorphs found for the monoelaidin-water system include three bicontinuous cubic phases, i.e., Ia3d, Pn3m, and Im3m, and lamellar phases which exhibit two crystalline (L(c1) and L(c0)), two gel (L(β) and L(β*)), and a fluid lamellar (L(α)) states. The fluid isotropic phase (L(2)) was observed only for lower hydrations (<20%), whereas hexagonal phase (H(2)) was not found under studied conditions. Nanostructural parameters of these phases as a function of temperature and water content are presented together with some molecular level calculations. This study might be crucial for perception of the lyotropic phase behavior as well as for designing nanostructural assemblies for potential applications.

Lipid Nanobilayers to Host Biological Nanopores for DNA Translocations

We characterize a recently introduced novel nanobilayer technique [Gornall, J. L., Mahendran, K. R., Pambos, O. J., Steinbock, L. J., Otto, O., Chimerel, C., Winterhalter, M., and Keyser, U. F. Simple reconstitution of protein pores in nano lipid bilayers. Nano Lett. 2011, 11 (8), 3334-3340] and its practical aspects for incorporating the biological nanopore α-hemolysin from Staphylococcus aureus and subsequent studies on the translocation of biomolecules under various conditions. This technique provides advantages over classical bilayer methods, especially the quick formation and extended stability of a bilayer. We have also developed a methodology to prepare a uniform quality of giant unilamellar vesicles (GUVs) in a reproducible way for producing nanobilayers. The process and the characteristics of the reconstitution of α-hemolysin in nanobilayers were examined by exploiting various important parameters, including pH, applied voltage, salt concentration, and number of nanopores. Protonation of α-hemolysin residues in the low pH region affects the translocation durations, which, in turn, changes the statistics of event types as a result of electrostatics and potentially the structural changes in DNA. When the pH and applied voltage were varied, it was possible to investigate and partly control the capture rates and type of translocation events through α-hemolysin nanopores. This study could be helpful to use the nanobilayer technique for further explorations, particularly owing to its advantages and technical ease compared to existing bilayer methods.

Lipid-hydrogel films for sustained drug release

We report a hybrid system, fabricated from nanostructured lipid particles and polysaccharide based hydrogel, for sustained release applications. Lipid particles were prepared by kinetically stabilizing self-assembled lipid nanostructures whereas the hydrogel was obtained by dissolving kappa-carrageenan (KC) in water. The drug was incorporated in native as well as lipid particles loaded hydrogels, which upon dehydration formed thin films. The kinetics of drug release from these films was monitored by UV-vis spectroscopy while the films were characterized by Fourier transform infra-red (FTIR) spectroscopy and small angle X-ray scattering techniques. Pre-encapsulation of a drug into lipid particles is demonstrably advantageous in certain ways; for instance, direct interactions between KC and drug molecules are prohibited due to the mediation of hydrophobic forces generated by lipid tails. Rapid diffusion of small drug molecules from porous hydrogel network is interrupted by their encapsulation into rather large sized lipid particles. The drug release from the lipid-hydrogel matrix was sustained by an order of magnitude timescale with respect to the release from native hydrogel films. These studies form a strong platform for the development of combined carrier systems for controlled therapeutic applications.

Immobilization of nanostructured lipid particles in polysaccharide films.

Lipid-based equilibrium self-assemblies and their hierarchically ordered forms have been known since the last few decades. Related progress in colloids and interface science led the development of oil-in-water type internally self-assembled lipid particles, known as Isasomes, which have aroused great interest in biotechnological applications. These submicrometer-sized lipid particles are internally nanostructured in a form of various liquid-crystalline or microemulsion phases, which facilitate their loading with hydrophilic, hydrophobic, and amphiphilic molecules. Their internal nanostructure can also be finely tuned. Recently, it has been shown that Isasomes can be entrapped in thermoreversible polysaccharide hydrogels. Herein, we report on the immobilization of Isasomes in solid polysaccharide films prepared by drying particle-loaded κ-carrageenan and methyl cellulose-based hydrogels. These rather simple but elegant media facilitate the storage of these functional particles and their subsequent release by simple resolubilization in water and/or thermal transitions. Systematic rehydration studies of such Isasome-loaded films have shown that the Isasomes can be remobilized and/or recovered after resolubilization of loaded films, even after several months.

Evidence that membrane curvature distorts the tertiary structure of bacteriorhodopsin

The membrane protein bacteriorhodopsin (bR) can be reconstituted into the membrane of the lipid 1-monoolein (MO). This lipid forms a lyotropic liquid crystalline phase whose membrane has hyperbolic interfacial curvature. Using optical absorption spectroscopy and small angle X-ray scattering we have observed retinal unbinding from bR that is correlated with the degree of membrane interfacial curvature. The evidence suggests that bR is susceptible to membrane induced saddle splay for modest perturbations from equilibrium, but for more extreme distortions becomes stiff and resists membrane induced curvature.

Pressure effects on a protein–lipid model membrane

We attempt to mimic cellular biomembrane structures using a mixture of two important biochemical components – a membrane protein and a lipid, in the presence of water. Protein loaded lipid structures were subjected to a wide range of pressures to examine their morphological reorganizations using synchrotron X-ray radiation under stepwise pressure variation and rapid pressure jumps. Here we report the first evidence of a highly swollen gyroid cubic phase under high pressure (gyroid type structures have been seen in ER and Golgi membranes) and the close resemblance of lipid nanostructural behavior with literature studies on compression–decompression of piezophilic biomembranes. These studies are promising for understanding complex biomembrane restructuring and hence functioning, such as how cells cope with extreme conditions of high pressures, and protect their delicate internal organelles.

In Cubo Crystallization of Membrane Proteins

One of the best options to understand the structures and hence functions of membrane proteins is macromolecular crystallography which demands for their three-dimensional (3D) crystals. The current chapter is focused on the lipid bicontinuous cubic phase method which is known to produce such crystals and is also called in cubo method. The lipid cubic phases are 3D self-assemblies consisting of two apposing aqueous regions separated by a lipid bilayer draped onto mathematical minimal surfaces. The dimensions of these phases lie in 6–25 nm range which act as an in vitro matrix for stabilization of membrane proteins from which they are crystallized for determining high resolution structures. The pros and cons of the in cubo method are presented with regard to other crystallization methods and also other lipid mesophases. Recent technological and methodological advances in addition to mechanistic principles are also discussed. The method is further explored for its advantages and potential cautions while going for its optimization and generalization. A brief survey of databases is also presented which may help for ready referral while using the in cubo method.