The characterization of materials at the atomic-, nano-, and microscales is of crucial importance in understanding and then tailoring their macroscale properties and function for end-use applications and for effective modern cradle-to-reuse materials cycling. Synchrotron light, as well as the complementary neutron beams, offer exquisite microscopy probes to look into the heart of materials. This chapter presents some examples of pharma-oriented nanoparticle characterization highlighting the possibilities of synchrotron light and neutron beams. Small-angle X-ray scattering (SAXS) is a well-established technique to probe nanoscale structures. SAXS can also deliver valuable information on the structure of self-assembled nanovectors, such as liposomes, which are recognized as efficient platforms for drug delivery. Future developments for neutron characterization will be driven in parallel with instrumental developments at existing sources and future facilities such as the European Spallation Source (ESS) being built in Sweden.  

In this review, the authors discuss the challenges of studying supported lipid bilayers (SLBs) deposited by vesicle fusion in terms of (1) evaluating SLB formation and quality using quartz crystal microbalance with dissipation and (2) analyzing the composition and asymmetry of SLBs composed by lipid mixtures using complementary surface sensitive techniques. An overview of the literature is presented and the inconsistencies on this topic are discussed with the objective to expand beyond simple lipid compositions and set the basis for forming and analyzing SLBs of complex natural lipid extracts formed via the vesicle fusion method. The authors conclude by providing some guidelines to successfully form SLBs of complex lipid mixtures including natural extracts.

The interaction mechanism of a novel amphiphilic antimicrobial peptide dendrimer, BALY, with model lipid bilayers was explored through a combination of neutron reflection and molecular dynamics simulations. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phos-phocholine (DPPC) lipid bilayers were examined at room temperature to extract information on the interaction of BALY with fluid and gel phases, respectively. Furthermore, a 1:4 mixture of POPC and DPPC was used as a model of a phase-separated membrane. Upon interaction with fluid membranes, BALY inserted in the distal leaflet and caused thinning and disordering of the headgroups. Membrane thinning and expansion of the lipid cross-sectional area were observed for gel phase membranes, also with limited insertion to the distal leaflet. However, dendrimer insertion through the entire lipid tail region was observed upon crossing the lipid phase transition temperature of DPPC and in phase separated membranes. The results show clear differences in the interaction mechanism of the dendrimer depending on the lipid membrane fluidity, and suggest a role for lipid phase separation in promoting its antimicrobial activity.

Two monoclonal antibodies from the IgG subclasses one and two were compared in their adsorption behavior with hydrophobic surfaces upon dilution to 10 mg/mL with 0.9% NaCl. These conditions simulate handling of the compounds at hospital pharmacies and surfaces encountered after preparation, such as infusion bags and i.v. lines. Total internal reflection fluorescence and quartz crystal microbalance with dissipation monitoring were used to follow and quantify this. Furthermore, the influence of the nonionic surfactant polysorbate 80 (PS80) on the adsorption process of these two antibodies was investigated. Despite belonging to two different IgG subclasses, both antibodies displayed comparable adsorption behavior. Both antibodies readily adsorbed in the absence of PS80, whereas adsorption was reduced in the presence of 30 mg/L surfactant. The sequence of exposure of the surfactant and protein to the surface was found to have a major influence on the extent of protein adsorption. Although only a fraction of adsorbed protein could be removed by rinsing with 30 mg/L surfactant solution, adsorption was entirely prevented when surfaces were pre-exposed to PS80

Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.

Nanodisc films are a promising approach to study the equilibrium conformation of membrane bound proteins in native-like environment. Here we compare nanodisc formation for NADPH-dependent cytochrome P450 oxidoreductase (POR) using two different scaffold proteins, MSP1D1 and MSP1E3D1. Despite the increased stability of POR loaded MSP1E3D1 based nanodiscs in comparison to MSP1D1 based nanodiscs, neutron reflection at the silicon–solution interface showed that POR loaded MSP1E3D1 based nanodisc films had poor surface coverage. This was the case, even when incubation was carried out under conditions that typically gave high coverage for empty nanodiscs. The low surface coverage affects the embedded POR coverage in the nanodisc film and limits the structural information that can be extracted from membrane bound proteins within them. Thus, nanodisc reconstitution on the smaller scaffold proteins is necessary for structural studies of membrane bound proteins in nanodisc films.

Antimicrobial drug resistance is a major human health threat. Among the many attempts to tackle this problem, the synthesis of antimicrobial compounds that mimic natural antimicrobial peptides appears as a promising approach. Peptide-based dendrimers can be designed to have higher potency than natural antimicrobial peptides and at the same time they can evade the bacterial defense system. Novel dendrimers with similar chemical structure but varying potency in terms of minimum inhibitory concentration were designed. The dependency between dendrimer structure and antibacterial activity as well as their capacity to attack model cell membranes was studied. The data suggests that supramolecular structure in terms of charge distribution and amphiphilicity, rather than net charge, is the main driver for disruption of cellular membranes and this correlates well with dendrimer hemolytic activity.

A bio-inspired coating consisting of pectin (polygalacturonic acid) and cationic cellulose nanofibers were successfully produced by the layer-by-layer method. The build-up and the morphology of the resulting coatings were studied with spectroscopic ellipsometry and atomic force microscopy, respectively. The coating was able to survive the exposure of a simulated gastric fluid, but was partially degraded upon exposure to pectinase enzyme, which simulate the action of the microbial symbionts present in the human colon. Prior to exposure, the oxygen permeability coefficient of the coating (0.033 ml (STP) mm m−2 day−1 atm−1 at 23 °C and 20% RH) was in the same order of magnitude as for ethylene vinyl alcohol films (0.001–0.01 ml (STP) mm m−2 day−1 atm−1). However, after exposure to the mimicked gastrointestinal (GI) tract conditions, the contribution of coating to the overall barrier properties was not measurable.

We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main phase-transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid bilayers formed below or above Tm by neutron reflection and investigated the effect of subsequent cooling to below the Tm. Our results clearly show that a continuous supported bilayer can be formed with high surface coverage below the lipid Tm. We also demonstrate that the high dissipation responses observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported.

Hydroxyapatite is a very interesting material given that it is the main component in tooth enamel and because of its uses in bone implant applications. Therefore, not only the characterization of its surface is of high relevance but also designing reliable methods to study the interfacial properties of films adsorbed onto it. In this paper we apply the colloidal probe atomic force microscopy method to investigate the surface properties of commercially available hydroxyapatite surfaces (both microscopic particles and macroscopic discs) in terms of interfacial and frictional forces. In this way, we find that hydroxyapatite surfaces at physiological relevant conditions are slightly negatively charged. The surfaces were then exposed to human whole saliva, and the surface properties were re-evaluated. A thick film was formed that was very resistant to mechanical stress. The frictional measurements demonstrated that the film was indeed highly lubricating, supporting the argument that this system may prove to be a relevant model for evaluating dental and implant systems.

The interactions of the bile salt sodium taurocholate (TC) in 50 mM Trizma–HCl buffer and 150 mM NaCl (pH 9) at 37 °C with membranes composed of sphingomyelin (SM) were studied by dynamic light scattering, cryogenic transmission electron microscopy (cryo-TEM) and turbidity measurements. Small unilamellar SM vesicles were prepared by extrusion. Below the CMC of TC, taurocholate addition leads to vesicle growth due to incorporation of the taurocholate molecules into the vesicle bilayer. At around half the CMC of the bile salt, the SM vesicles are transformed into SM/TC mixed worm-like micelles, which are visualized by cryo-TEM for the first time. Further increase in the taurocholate concentration leads to the rupture of these structures into small spherical micelles. Interestingly, large non-spherical micelles were also identified for pure taurocholate solutions. Similar threadlike structures have been reported earlier for the bile salt sodium taurodeoxycholate [Rich, A., Blow, D., 1958. Nature 182, 1777; Blow, D.M., Rich, A., 1960. J. Am. Chem. Soc. 82, 3566–3571; Galantini, L., Giglio, E., La Mesa, C., Viorel-Pavel, N., Punzo, F., 2002. Langmuir 18, 2812] and for mixtures of taurocholate and phosphatidylcholate [Ulmius, J., Lindblom, G., Wennerström, H., Johansson, L.B.-Å., Fontel, K., Söderman, O., Ardvisson, G., 1982. Biochemistry 21, 1553; Hjelm, R.P., Thiyagarajan, P., Alkan-Onyuksel, H., 1992. J. Phys. Chem. 96, 8653] as determined by various scattering methods.

Films formed from saliva on surfaces are important for maintenance of oral health and integrity by serving as barriers against chemical and/or biological agents. OBJECTIVES: The aim of the present study was to investigate the structure of films formed from human whole saliva onto alumina surfaces. METHODS: In situ ellipsometry was used to investigate adsorbed amounts and thickness of the adsorbed layers. Neutron reflectivity and atomic force microscopy (AFM) were used to study the density profile within the adsorbed layer and morphology, respectively, of the adsorbed salivary film onto alumina (Al2O3). RESULTS: The results show that saliva adsorbed rapidly on alumina. First a thin and dense layer covered the surfaces. With time, a thicker and diffuser layer was developed. The film morphology described a uniformly covering dense layer and a second outer layer containing polydisperse adsorbed macromolecules or aggregates. CONCLUSION: The film was found to be composed of two layers: an inner and dense thin region, that forms a uniform layer, and an outer, more diffuse and thicker region that protrudes towards the bulk of the solution. This study was supported by research grants from the Knowledge Foundation, Malmö University, The Swedish Dental Society, The Swedish Patent Revenue Fund for Research in Preventive Dentistry, The Swedish Research Council and Uppsala University. We are grateful also to the Institute Laue Langevin, Grenoble for an allocation of neutron beam time.

Films formed from saliva on surfaces are important for maintenance of oral health and integrity by protection against chemical and/or biological agents. The aim of the present study was to investigate adsorbed amounts, thickness and the structure of films formed from human whole saliva on alumina surfaces by means of in situ ellipsometry, neutron reflectivity and atomic force microscopy. Alumina (Al2O3, synthetic sapphire) is a relevant and interesting substrate for saliva adsorption studies as it has an isoelectric point close to that of tooth enamel. The results showed that saliva adsorbs rapidly on alumina. The film could be modelled in two layers: an inner and dense thin region which forms a uniform layer, and an outer, more diffuse and thicker region that protrudes towards the bulk of the solution. The film morphology described a uniformly covering dense layer and a second outer layer containing polydisperse adsorbed macromolecules or aggregates.

This paper describes a combined investigation of the salivary and MUC5B films structure and topography in conditions similar to those found in the oral cavity in terms of ionic strength, pH, and protein concentration. AFM and ellipsometry were successfully used to give a detailed picture of the film structure and topography both on hydrophilic and on hydrophobic substrata. Regardless of the substrata, the salivary film can be described as having a two sublayer structure in which an inner dense layer is decorated by large aggregates. However, the shape and height of these larger aggregates largely depend on the type of substrata used. Additionally, we show that the adsorption of MUC5B is controlled by the type of substrata and the MUC5B film topography is similar to that of the larger aggregates present in the salivary films, especially on hydrophobic substrates. Therefore, we conclude that MUC5B is a major component in the salivary film when formed on hydrophobic substrates. Furthermore, we studied how resistant the salivary and MUC5B films are against elutability by buffer rinsing and addition of SDS solution. We conclude that the adsorbed proteins contain fractions with varying binding strengths to the two types of surfaces. Specifically, we have shown that the large MUC5B biomacromolecules on the hydrophobic substrates are especially resistant to both elution with buffer solution and SDS. Therefore, these large mucins can be responsible for the increased resistance of HWS films on hydrophobic substrates and can protect the intraoral surfaces against surface-active components present in oral health care products.

Abstract In situ ellipsometry was used to study layer-by-layer film formation on hydrophilic and hydrophobized silica surfaces by alternating sequential adsorption of human mucin MUC5B and cationic proteins lysozyme, lactoferrin, lactoperoxidase or histatin 5, respectively. The stability of the multilayers was investigated by addition of sodium dodecyl sulfate solution (SDS). Atomic force microscopy was employed to investigate morphological structures on the surfaces during the layer-by-layer film build-up. It was clearly shown that, on both hydrophilic and hydrophobized silica, only MUC5B and lactoperoxidase showed the ability for multilayer formation, resulting in an approximately linear increase in adsorbed amount and film thickness with each deposition cycle. The net increase in amounts per cycle was larger on the hydrophilic silica. Further, MUC5B needs to be adsorbed first on the hydrophilic substrates to obtain this fast build-up behavior. Generally, addition of SDS solution showed that a large fraction of the adsorbed film could be desorbed. However, films on the hydrophobized silica were more resistant to surfactant elution. In conclusion, MUC5B–cationic protein multilayers can be formed on hydrophilic and hydrophobized silica, depending on the choice of the cationic protein as well as in which order the build-up is started on hydrophilic silica. Additionally, SDS disrupts the layer-by-layer film formed by MUC5B and lactoperoxidase.

Bovine submaxillary mucin (BSM) and chitosan were used to build layer-by-layer structures on solid substrates. The build-up was monitored using in situ ellipsometry to obtain time resolved values of the thickness and adsorbed amount. Additionally surface morphology during build-up was studied by atomic force microscopy (AFM). It was found that the adsorbed amount of the film increases approximately linearly with each deposition cycle on hydrophobized silica whereas construction on silica was found not to be possible at the experimental conditions used. We conclude that sufficient amount of the first mucin layer is crucial for the subsequent multilayer formation. The complex build-up kinetics on hydrophobized silica is characterized by adsorption and redissolution processes and the overall growth is the sum of both processes. AFM imaging on hydrophobized silica also confirmed the presence of redissolution processes and chitosan addition led to a reduction both in the number of surface aggregates and in the roughness of the surface. The present work also shows that by adjusting the relative concentrations of the polyelectrolytes it is possible to change the growth rate considerably. The final structures after deposition of 8 bilayers were found to have a high content of water and film stability test revealed that a substantial amount dissolves when increasing electrolyte concentration or pH of the ambient solution. Human mucin from saliva (MUC5B) was also used to create multilayers with chitosan on hydrophobized silica and it was revealed that no redissolution appears to be present in this system.

The contribution of nonspecific interactions to the overall interactions of thiol-ssDNA and dsDNA macromolecules with gold nanoparticles was investigated. A systematic investigation utilizing dynamic light scattering and cryogenic transmission electron microscopy has been performed to directly measure and visualize the changes in particle size and appearance during functionalization of gold nanoparticles with thiol-ssDNA and nonthiolated dsDNA. The results show that both thiol-ssDNA and dsDNA do stabilize gold nanoparticle dispersions, but possible nonspecific interactions between the hydrophobic DNA bases and the gold surface promote interparticle interactions and cause aggregation within rather a short period of time. We also discuss the adsorption mechanisms of dsDNA and thiol-ssDNA to gold particles.