Tuning the mechanical properties of self-assembled mixed-peptide tubes.

In this study, nano- and microscale fibrillar and tubular structures formed by mixing two aromatic peptides known to self-assemble separately, (diphenylalanine and di-D-2-napthylalanine) have been investigated. The morphology, mechanical strength and thermal stability of the tubular structures formed have been studied. The tubes are shown to consist of both peptides with some degree of nanoscale phase separation. The ability of the mixed peptides to form structures, which display variable mechanical properties dependent on the percentage composition of the peptides is presented. Such materials with tuneable properties will be required for a range of applications in nanotechnology and biotechnology.

Structural study of DNA condensation induced by novel phosphorylcholine-based copolymers for gene delivery and relevance to DNA protection.

Poly[2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine] (DMA-MPC) is currently under investigation as a new vector candidate for gene therapy. The DMA block has been previously demonstrated to condense DNA effectively. The MPC block contains a phosphorylcholine (PC) headgroup, which can be found naturally in the outside of the cell membrane. This PC-based polymer is extremely hydrophilic and acts as a biocompatible steric stabilizer. In this study, we assess in detail the morphologies of DNA complexes obtained using the diblock copolymer series DMA(x)MPC30 (where the mean degree of polymerization of the MPC block was fixed at 30 and the DMA block length was systematically varied) using transmission electron microscopy (TEM) and liquid atomic force microscopy (AFM). Both techniques indicate more compact complex morphologies (more efficient condensation) as the length of the cationic DMA block increases. However, the detailed morphologies of the DMA(x)MPC30-DNA complexes observed by TEM in vacuo and by AFM in aqueous medium are different. This phenomena is believed to be related to the highly hydrophilic nature of the MPC block. TEM studies revealed that the morphology of the complexes changes from loosely condensed structures to highly condensed rods, toroids, and oval-shaped particles as the DMA moiety increases. In contrast, morphological changes from plectonemic loops to flower-like and rectangular block-like structures, with an increase in highly condensed central regions, are observed by in situ AFM studies. The relative population of each structure is clearly dependent on the polymer molecular composition. Enzymatic degradation assays revealed that only the DMA homopolymer provided effective DNA protection against DNase I degradation, while other highly condensed copolymer complexes, as judged from TEM and gel electrophoresis, only partially protected the DNA. However, AFM images indicated that the same highly condensed complexes have less condensed regions, which we believe to be the initiation sites for enzymatic attack. This indicates that the open structures observed by AFM of the DNA complexation by the DMA(x)MPC30 copolymer series are closer to in vivo morphology when compared to TEM.

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica.

Multivalent ions in solution are known to mediate attraction between two like-charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either 'salt bridging' or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re-explore current theories of like-charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.

AFM studies of the crystallization and habit modification of an excipient material, adipic acid.

Atomic force microscopy (AFM) has been used to investigate the (1 0 0) face of crystalline adipic acid, both in air and liquid environments. In air, surface reorganization occurred during scanning of the AFM probe, which has been investigated using single point force-distance analysis under a controlled relative humidity (RH) environment. We suggest such reorganization can be attributed to the influence of a network of water molecules bound to the hydrophilic (1 0 0) surface permitting local AFM tip-enhanced dissolution and reorganization of the solute. In situ imaging was also carried out on the crystals, revealing etch-pit formation during dissolution, and rapid growth at higher levels of supersaturation (sigma), both of which are direct consequences of the hydrophilic nature of the (1 0 0) face. Also presented here are nanoscale observations of the effect of octanoic acid, a structurally-related habit modifier, on crystalline adipic acid. Using AFM, we have been able to show that the presence of octanoic acid at low concentration has little observable affect on the development of the (1 0 0) face; however, as this concentration is increased, there are clear changes in step morphology and growth mode on the (1 0 0) face of the crystal. At a concentration of 1.26 mmol dm(-3) (a concentration corresponding to a molar ratio of approximately 1:175 octanoic acid:adipic acid), growth on the (1 0 0) face is inhibited, with in situ AFM imaging indicating this is a direct consequence of octanoic acid binding to the surface, and pinning the monomolecular growth steps.

Using microscopic techniques to reveal the mechanism of anion exchange in crystalline co-ordination polymers.

Co-ordination polymers are currently attracting extensive interest due to their potential applications as supramolecular hosts, vessels, and frameworks for storage and separations. Many applications rely on the ion exchange capabilities of these compounds, and considerable debate surrounds the mechanism by which ion exchange occurs in co-ordination polymers. Here AFM and SEM were applied, for the first time, to investigate this class of materials. In situ AFM studies revealed the mechanism by which anion exchange and the subsequent structural transformations of the crystalline co-ordination polymers [[Ag(4,4'-bipy)]BF(4)](infinity) and [[Ag(4,4'-bipy)]NO(3)](infinity) occur. The process is initiated by the dissolution of the metastable crystalline polymer, followed by the subsequent crystallization of the new stable phase on the surface of the original crystal. The formation of deep clefts in the metastable polymer crystal during the transformation allows the solution to access the successive crystalline layers. Thus, the entire process can be viewed as a self-perpetuating cascade of dissolution and recrystallization throughout the macroscopic crystal. SEM data consolidate the findings of AFM. These techniques collectively illustrate that the anion exchange, and subsequent structural transformation, proceeds via a solvent-mediated mechanism, rather than a purely solid-state one.

Electrostatic interactions observed when imaging proteins with the atomic force microscope.

The atomic force microscope (AFM) is now an established and valuable tool for the study of biological macromolecules in aqueous environments. In this paper we form a patterned boundary via the microcontact printing of individually isolated proteins, covalently attached to a solid support. We use this boundary to investigate electrostatic interactions that can occur between an AFM tip and a protein surface during imaging in solution. The observed height variations of the protein film are found to be a combination of not only structural considerations and thickness of the protein film, but also the repulsive contribution from electrostatic interactions between the AFM tip and the sample. These variations in measured heights of the protein surface can be described by Derjaguin, Landau, Verway, Overbeek (DLVO) theory. Our experimental results show that height measurements can be manipulated either negatively or positively by adjusting the pH and concentration of the electrolyte buffer that is utilised.

Interactions of 3T3 fibroblasts and endothelial cells with defined pore features.

The colonization of biodegradable polymer scaffolds with cell populations has been established as the foundation for the engineering of a number of tissues, including cartilage, liver, and bone. Within these scaffolds, the cells encounter a porous environment in which they must migrate across the convoluted polymer surface to generate a homogenous cell distribution. Predicting the interactions between cells and pores is important if scaffold characteristics are to be optimized. Therefore, we investigated the behavior of two model cell types over a range of defined pore features. These pore features range from 5 to 90 microm in diameter and have been fabricated by photolithographic techniques. Quantitatively, the behavior of the cells is dependent on three factors: 1) percentage cell coverage of the surface; 2) pore size; and 3) cell type. Fibroblast cells displayed a co-operative pattern of cell spreading in which pores with diameters greater than the cell dimensions were bridged by groups of cells using their neighbors as supports. Endothelial cells were unable to use neighbors as support structures and failed to bridge pores greater than the cell diameter.

Synthesis and characterisation of a degradable poly(lactic acid)-poly(ethylene glycol) copolymer with biotinylated end groups.

Poly(lactic acid)-poly(ethylene glycol)-biotin (PLA-PEG-biotin) is a degradable polymer with protein resistant properties that can undergo rapid surface engineering in aqueous media to create biomimetic surfaces. Surface engineering of this polymer is dependent on biomolecular interactions between the biotin end group and the protein avidin. Given the vigorous conditions of synthesis, it is essential that the manufacture of the polymer does not alter the biotin structure or its molecular recognition. Equally, it is important that the incorporation of biotin does not adversely affect the physicochemical properties of the polymer. (1)H NMR provides evidence of biotin attachment and structural integrity. (1)H NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analysis shows there is no significant effect on bulk properties induced by the biotin end group. Surface plasmon resonance (SPR) and fluorescent spectroscopy studies using the 2-(4'-hydroxyazobenzene) benzoic acid (HABA)/avidin complex show that the biotin moieties binding capabilities are not impaired by the synthesis.

Investigation of microcontact transfer of proteins from a selectively plasma treated elastomer stamp by fluorescence microscopy and force microscopy.

Selective plasma treatment of the recessed regions of the elastomer stamps is shown to alter the resultant protein patterns. Fluorescence microscopy is demonstrated to be an excellent tool to discriminate between regions of microcontact printed fluorescent dye-labelled albumin in polystyrene. Atomic force microscopy and shear force microscopy are used to provide high-resolution images of the patterned protein layers. The formation and characteristics of the patterns formed by these alternative strategies is discussed.

An in situ dissolution study of aspirin crystal planes (100) and (001) by atomic force microscopy.

PURPOSE: To observe in situ and on individual aspirin crystal faces the comparative rates and processes of dissolution of the dominant faces. METHODS: The kinetics of the dissolution rate of two aspirin crystal planes (001) and (100) under 0.05M HCl are studied in situ at room temperature using Atomic Force Microscopy. The dissolution process of each crystal plane was followed by observed changes in topographic features. RESULTS: The results revealed that crystal plane (001) dissolves by receding step edges, and has a dissolution rate of 0.45 nm s(-1). Conversely. plane (100) displays crystal terrace sinking at an average rate of 2.93 nm s(-1). Calculated intrinsic dissolution values (g s(-1) cm(-2)) for planes (001) and (100) are 1.37 x 10(-7) gs(-1) cm(-2) and 8.36 x 10(-7) gs(-1) cm(-2), respectively. CONCLUSIONS: These values indicate that the rate of flux of material from plane (100) is approximately six times greater than that from plane (001), under 0.05M HCl. Interpretation of the data, based upon intrinsic dissolution rates and dissolution rate velocities, correlate with reported variations in the dissolution behavior of commercial aspirin products. These observations illustrate the suitability of the technique for characterizing the dissolution behavior of crystalline drugs.

Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability.

A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

Force-induced melting of a short DNA double helix.

The dynamic behaviour of DNA is of fundamental importance to many cellular processes. One principal characteristic, central to transcription and replication, is the ability of the duplex to "melt". It has recently been shown that dynamic force spectroscopy provides information about the energetics of biomolecular dissociation. We have employed this technique to investigate the unbinding of single dodecanucleotide molecules. To separate the duplex to single-stranded DNA, forces ranging from 17 to 40 pN were required over a range of loading rates. Interpretation of the dependence of melting force on loading rate revealed that the energy barrier to rupture is between 9 and 13 kcal mol-1 in height and situated 0.58 nm from an intermediate structural state. Thermal melting studies show that, prior to dissociation, the oligonucleotide underwent a transition which required between 7 and 11 kcal mol-1 in energy. Through combined dynamic force spectroscopy and thermal melting studies we show the derivation of an energy landscape to dissociate a 12-mer duplex. Until very recently, this type of information was only accessible by computational analysis. Additionally, the force spectroscopy data allow an estimation of the kinetics of duplex formation and melting.

Molecular patterning on carbon based surfaces through photobiotin activation.

We have demonstrated the site-specific adhesion of photobiotin as a method of producing protein micropatterns. These patterns were created by the selective UV irradiation of a thin film of deposited photobiotin. The UV activated areas of photobiotin were then developed using fluorescently labelled avidin. The size of pattern produced is an order of magnitude smaller than those previously reported by this method. The patterns were characterised, using atomic force microscopy (AFM) to determine their microstructure. It was found that the AFM could discriminate between the areas of protein immobilised to the surface through the activated photobiotin, and the bare substrate surface where the inactivated photobiotin had been removed during the washing process. The potential of these patterns as sensing surfaces is demonstrated through the creation of a spatially patterned immunosensing surface. In this case, a biotinylated antibody was bound to the surface and the pattern developed using a second antibody specific to the immobilised biotinylated antibody. This technique could thus provide a simple and efficient method of producing high density immunoassay systems.

Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lactic acid).

The immobilization of adhesion peptide sequences (such as RGD) at the surfaces of poly(alpha-hydroxyacid)s, including poly(lactic acid) (PLA), is complicated by an absence of functional groups to support covalent attachment. We demonstrate a method to overcome this problem, by attaching the peptide to poly(L-lysine) (PLL), which immobilizes the sequence through adsorption at the poly(alpha-hydroxyacid) surface. When coated using a 0.01% w/v solution of PLL-GRGDS, bovine aortic endothelial cells seeded upon the modified PLA showed a marked increase in spreading over unmodified PLA. However, inhibition of the cell-spreading effect occurred when using higher concentrations of PLL-GRGDS, which we attribute to the PLL component. This inhibitory effect can be challenged by increasing the amount of GRGDS attached to each PLL molecule. Potentially, this is a flexible method of surface modification that can engineer many different types of tissue engineering scaffolds with a variety of biomolecules, thus allowing initial cell adhesion to be controlled.

Observation of DNA-polymer condensate formation in real time at a molecular level.

Dynamic real time assembly of toroidal and rod-like DNA condensates has been visualised using atomic force microscopy. Imaging has been conducted in an aqueous environment allowing the visualisation of hydrated, pegylated-polymer DNA condensates undergoing dynamic structural movement and conformational change. A major hurdle in the field of gene delivery is cellular transfection and the subsequent transfer of condensed genetic material to the cell nucleus. An increased understanding of the process of DNA condensation will aid the development and optimisation of gene delivery vectors.

Surface plasmon resonance analysis of dynamic biological interactions with biomaterials.

Surface plasmon resonance (SPR) is an optical technique that is widely gaining recognition as a valuable tool to investigate biological interactions. SPR offers real time in situ analysis of dynamic surface events and, thus, is capable of defining rates of adsorption and desorption for a range of surface interactions. In this review we highlight the diversity of SPR analysis. Examples of a wide range of applications of SPR are presented, concentrating on work relevant to the analysis of biomaterials. Particular emphasis is given to the use of SPR as a complimentary tool, showing the broad range of techniques that are routinely used alongside SPR analysis.

Surface characterization of aspirin crystal planes by dynamic chemical force microscopy.

Tapping mode (TM) atomic force microscopy (AFM) has been applied in a novel fashion to characterize and distinguish the (001) and (100) surfaces of individual aspirin crystals. The surface characterization was achieved by amplitude-phase, distance (a-p,d) measurements employing gold-coated AFM probes functionalized with self-assembled monolayers (SAM). Experiments using model probes coated with -CH3 and -COOH terminated SAMs have been performed on the two aspirin crystal planes (001) and (100). Results indicate that the hydrophobic -CH3 terminated AFM probes had a greater degree of interaction with the crystal plane (001), whereas the -COOH terminated AFM probes had a larger interaction with the crystal plane (100). Interpretation of these data, based upon the chemistries of the probes, correlates with current understanding of the crystal surface chemistry derived from X-ray diffraction data and dissolution rate studies.

Atomic force microscopy studies of intercalation-induced changes in plasmid DNA tertiary structure.

Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide:base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic:toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.

Atomic force microscopy of gastric mucin and chitosan mucoadhesive systems.

Atomic force microscopy has been utilized to probe, at a molecular level, the interaction between purified pig gastric mucin (PGM) and a mucoadhesive cationic polymer, chitosan (sea cure 210+), with a low degree (approx. 11%) of acetylation. Images were produced detailing the structures of both PGM and chitosan in 0.1 M acetate buffer (pH 4.5), followed by the complex of the two structures in the same buffer. PGM in 0.1 M acetate buffer revealed long linear filamentous structures, consistent with earlier electron microscopy and scanning tunnelling micoscopy studies. The chitosan molecules also adopted a linear conformation in the same buffer, although with a smaller average length and diameter. They appeared to adopt a stiff-coil conformation consistent with earlier hydrodynamic measurements. The complexes formed after mixing PGM and chitosan together revealed large aggregates. In 0.1 M ionic strength buffer they were of the order of 0.7 microm in diameter, consistent with previous electron microscopy studies. The effect of ionic strength of the buffer on the structure of the complex was also studied and, together with molecular hydrodynamic data, demonstrates that the interaction is principally electrostatic in nature.