Hybrid vesicles of lipid and amphiphilic block copolymer combine the biological and functional versatility of lipid delivery systems with the mechanical stability and robustness of polymersomes. While studies of hybrid systems for ease of characterization have focused on giant vesicles, most encapsulation and release applications require nanoscale (large) unilamellar vesicles. We investigate the structure and physical characteristics important for thermal actuation of vesicle‐forming blends of saturated phospholipid, polybutadiene‐b‐poly(ethylene oxide) and thermoresponsive polyisoprene‐b‐poly(N‐isopropylacryl amide) that are additionally loaded with hydrophobic superparamagnetic nanoparticles in the vesicle membrane and resized to large unilamellar vesicles. Lipid/diblock copolymer hybrid vesicles are shown to be the most efficient option to trigger release of encapsulated compounds by application of an external magnetic field causing local hyperthermia. This superiority is shown to depend on the controlled formation of nanoscale lipid domains in the hybrid vesicle membrane and the efficient loading of hydrophobic nanoparticles into the diblock copolymer membrane that retains its stability.

The influence of Escherichia coli rough lipopolysaccharide chemotype on the membrane activity of the mammalian antimicrobial peptides (AMPs) human cathelicidin (LL37) and bovine lactoferricin (LFb) was studied on bilayers using solid state (2)H NMR (ssNMR) and on monolayers using the subphase injection technique, Brewster angle microscopy (BAM) and neutron reflectivity (NR). The two AMPs were selected because of their differing biological activities. Chain-deuterated dipalmitoylphosphatidylcholine (d62-DPPC) was added to the LPS samples, to highlight alterations in the system properties caused by the presence of the different LPS chemotypes and upon AMP challenge. Both LPS chemotypes showed a temperature dependent influence on the packing of the DPPC molecules, with a fluidizing effect exerted below the DPPC phase transition temperature (Tm), and an ordering effect observed above the Tm. The magnitude of these effects was influenced by LPS structure; the shorter Rc LPS promoted more ordered lipid packing compared to the longer Ra LPS. These differential ordering effects in turn influenced the penetrative activity of the two peptides, as the perturbation induced by both AMPs to Ra LPS-containing models was greater than that observed in those containing Rc LPS. The NR data suggests that in addition to penetrating into the monolayers, both LL37 and LFb formed a non-interacting layer below the LPS/DPPC monolayer. The overall activity of LL37, which showed a deeper penetration into the model membranes, was more marked than that of LFb, which appeared to localise at the interfacial region, thus providing evidence for the molecular origins of their different biological activities.

The biophysical analysis of the aggregates formed by different chemotypes of bacterial lipopolysaccharides (LPS) before and after challenge by two different antiendotoxic antimicrobial peptides (LL37 and bovine lactoferricin) was performed in order to determine their effect on the morphology of LPS aggregates. Small-angle neutron scattering (SANS) and cryogenic transmission electron microscopy (cryoTEM) were used to examine the structures formed by both smooth and rough LPS chemotypes and the effect of the peptides, by visualization of the aggregates and analysis of the scattering data by means of both mathematical approximations and defined models. The data showed that the structure of LPS determines the morphology of the aggregates and influences the binding activity of both peptides. The morphologies of the worm-like micellar aggregates formed by the smooth LPS were relatively unaltered by the presence of the peptides due to their pre-existing high degree of positive curvature being little affected by their association with either peptide. On the other hand, the aggregates formed by the rough LPS chemotypes showed marked morphological changes from lamellar structures to ordered micellar networks, induced by the increase in positive curvature engendered upon association with the peptides. The combined use of cryoTEM and SANS proved to be a very useful tool for studying the aggregation properties of LPS in solution at biologically relevant concentrations.

Synchrotron resonance-enhanced infrared atomic force microscopy (RE-AFM-IR) is a near-field photothermal vibrational nanoprobe developed at Diamond Light Source (DLS), capable of measuring mid-infrared absorption spectra with spatial resolution around 100 nm. The present study reports a first application of synchrotron RE-AFM-IR to interrogate biological soft matter at the subcellular level, in this case, on a cellular model of drug-induced phospholipidosis (DIPL). J774A-1 macrophages were exposed to amiodarone (10 μM) or medium for 24 h and chemically fixed. AFM topography maps revealed amiodarone-treated cells with enlarged cytoplasm and very thin regions corresponding to collapsed vesicles. IR maps of the whole cell were analyzed by exploiting the RE-AFM-IR overall signal, i.e., the integrated RE-AFM-IR signal amplitude versus AFM-derived cell thickness, also on lateral resolution around 100 nm. Results show that vibrational band assignment was possible, and all characteristic peaks for lipids, proteins, and DNA/RNA were identified. Both peak ratio and unsupervised chemometric analysis of RE-AFM-IR nanospectra generated from the nuclear and perinuclear regions of untreated and amiodarone-treated cells showed that the perinuclear region (i.e., cytoplasm) of amiodarone-treated cells had significantly elevated band intensities in the regions corresponding to phosphate and carbonyl groups, indicating detection of phospholipid-rich inclusion bodies typical for cells with DIPL. The results of this study are of importance to demonstrate not only the applicability of Synchrotron RE-AFM-IR to soft biological matters with subcellular spatial resolution but also that the spectral information gathered from an individual submicron sample volume enables chemometric identification of treatment and biochemical differences between mammalian cells.

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Polymersomes, vesicles composed of block copolymers, are promising candidates as membrane alternatives and functional containers, e.g., as potential carriers for functional molecules because of their stability and tunable membrane properties. In the scope of possible use for membrane protein delivery to cells by electrofusion, we investigated the cytotoxicity of such polymersomes as well as the effects of nanosecond electric pulses with variable repetition rate on the shape and permeability of polymersomes in buffers with different conductivities. The polymersomes did not show cytotoxic effects to CHO and B16-F1 cells in vitro in concentrations up to 250 µg/mL (for 48 h) or 1.35 mg/mL (for 60 min), which renders them suitable for interacting with living cells. We observed a significant effect of the pulse repetition rate on electrodeformation of the polymersomes. The electrodeformation was most pronounced in low conductivity buffer, which is favorable for performing electrofusion with cells. However, despite more pronounced deformation at higher pulse repetition rate, the electroporation performance of polymersomes was unaffected and remained in similar ranges both at 10 Hz and 10 kHz. This phenomenon is possibly due to the higher stability and rigidity of polymer vesicles, compared to liposomes, and can serve as an advantage (or disadvantage) depending on the aim in employing polymersomes such as stable membrane alternative architectures or drug vehicles.

Lipid membranes play an essential role in biology, acting as host matrices for biomolecules like proteins and facilitating their functions. Their structures, and structural responses to physiologically relevant interactions, i.e. with membrane proteins, provide key information for understanding biophysical mechanisms. Hence, there is a crucial need of methods to understand the effects of membrane host molecules on the lipid bilayer structure. Here, we present a purely experimental method for obtaining the absolute scattering length density (SLD) profile and the area per lipid of liposomal bilayers, by aiding the analysis of small angle X-ray scattering (SAXS) data with the volume of bare headgroups obtained from fast (20-120s) grazing incidence off-specular scattering (GIXOS) data from monolayers of the same model membrane lipid composition. The GIXOS data experimentally demonstrate that the variation of the bare headgroup volume upon lipid packing density change is small enough to allow its usage as a reference value without knowing the lipid packing stage in a bilayer. This approach also bares the advantage that the reference volume is obtained at the same aqueous environment as used for the model membrane bilayers. We demonstrate the validity of this method using several typical membrane compositions, as well as one example of a phospholipid membrane with an incorporated transmembrane peptide. This methodology allows to obtain absolute scale values rather than relative scale by using solely X-ray-based instrumentation, retaining a similar resolution of SAXS experiments. The presented method has high potential to understand structural effects of membrane proteins on the biomembrane structure.