Boosting Membrane Interactions and Antimicrobial Effects of Photocatalytic Titanium Dioxide Nanoparticles by Peptide Coating.

Photocatalytic nanoparticles offer antimicrobial effects under illumination due to the formation of reactive oxygen species (ROS), capable of degrading bacterial membranes. ROS may, however, also degrade human cell membranes and trigger toxicity. Since antimicrobial peptides (AMPs) may display excellent selectivity between human cells and bacteria, these may offer opportunities to effectively "target" nanoparticles to bacterial membranes for increased selectivity. Investigating this, photocatalytic TiO2 nanoparticles (NPs) are coated with the AMP LL-37, and ROS generation is found by C11 -BODIPY to be essentially unaffected after AMP coating. Furthermore, peptide-coated TiO2 NPs retain their positive ζ-potential also after 1-2 h of UV illumination, showing peptide degradation to be sufficiently limited to allow peptide-mediated targeting. In line with this, quartz crystal microbalance measurements show peptide coating to promote membrane binding of TiO2 NPs, particularly so for bacteria-like anionic and cholesterol-void membranes. As a result, membrane degradation during illumination is strongly promoted for such membranes, but not so for mammalian-like membranes. The mechanisms of these effects are elucidated by neutron reflectometry. Analogously, LL-37 coating promoted membrane rupture by TiO2 NPs for Gram-negative and Gram-positive bacteria, but not for human monocytes. These findings demonstrate that AMP coating may selectively boost the antimicrobial effects of photocatalytic NPs.

Insights into the mechanisms of interaction between inhalable lipid-polymer hybrid nanoparticles and pulmonary surfactant.

Pulmonary delivery of small interfering RNA (siRNA) using nanoparticle-based delivery systems is promising for local treatment of respiratory diseases. We designed dry powder inhaler formulations of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) with aerosolization properties optimized for inhalation therapy. Interactions between LPNs and pulmonary surfactant (PS) determine the fate of inhaled LPNs, but interaction mechanisms are unknown. Here we used surface-sensitive techniques to study how physicochemical properties and pathological microenvironments influence interactions between siRNA-loaded LPNs and supported PS layers. PS was deposited on SiO2 surfaces as single bilayer or multilayers and characterized using quartz crystal microbalance with dissipation monitoring and Fourier-transform infrared spectroscopy with attenuated total reflection. Immobilization of PS as multilayers, resembling the structural PS organization in the alveolar subphase, effectively reduced the relative importance of interactions between PS and the underlying surface. However, the binding affinity between PS and LPNs was identical in the two models. The physicochemical LPN properties influenced the translocation pathways and retention time of LPNs. Membrane fluidity and electrostatic interactions were decisive for the interaction strength between LPNs and PS. Experimental conditions reflecting pathological microenvironments promoted LPN deposition. Hence, these results shed new light on design criteria for LPN transport through the air-blood barrier.

Surface-Functionalized Polystyrene Nanoparticles Alter the Transmembrane Potential via Ion-Selective Pores Maintaining Global Bilayer Integrity.

Although nanoplastics have well-known toxic effects toward the environment and living organisms, their molecular toxicity mechanisms, including the nature of nanoparticle-cell membrane interactions, are still under investigation. Here, we employ dynamic light scattering, quartz crystal microbalance with dissipation monitoring, and electrophysiology to investigate the interaction between polystyrene nanoparticles (PS NPs) and phospholipid membranes. Our results show that PS NPs adsorb onto lipid bilayers creating soft inhomogeneous films that include disordered defects. PS NPs form an integral part of the generated channels so that the surface functionalization and charge of the NP determine the pore conductive properties. The large difference in size between the NP diameter and the lipid bilayer thickness (∼60 vs ∼5 nm) suggests a particular and complex lipid-NP assembly that is able to maintain overall membrane integrity. In view of this, we suggest that NP-induced toxicity in cells could operate in more subtle ways than membrane disintegration, such as inducing lipid reorganization and transmembrane ionic fluxes that disrupt the membrane potential.

Pulmonary surfactant and drug delivery: Vehiculization of a tryptophan-tagged antimicrobial peptide over the air-liquid interfacial highway.

This work evaluates interaction of pulmonary surfactant (PS) and antimicrobial peptides (AMPs) in order to investigate (i) if PS can be used to transport AMPs, and (ii) to what extent PS interferes with AMP function and vice versa. This, in turn, is motivated by a need to find new strategies to treat bacterial infections in the airways. Low respiratory tract infections (LRTIs) are a leading cause of illness and death worldwide that, together with the problem of multidrug-resistant (MDR) bacteria, bring to light the necessity of developing effective therapies that ensure high bioavailability of the drug at the site of infection and display a potent antimicrobial effect. Here, we propose the combination of AMPs with PS to improve their delivery, exemplified for the hydrophobically end-tagged AMP, GRR10W4 (GRRPRPRPRPWWWW-NH2), with previously demonstrated potent antimicrobial activity against a broad spectrum of bacteria under various conditions. Experiments using model systems emulating the respiratory interface and an operating alveolus, based on surface balances and bubble surfactometry, served to demonstrate that a fluorescently labelled version of GRR10W4 (GRR10W4-F), was able to interact and insert into PS membranes without affecting its biophysical function. Therefore, vehiculization of the peptide along air-liquid interfaces was enabled, even for interfaces previously occupied by surfactants layers. Furthermore, breathing-like compression-expansion dynamics promoted the interfacial release of GRR10W4-F after its delivery, which could further allow the peptide to perform its antimicrobial function. PS/GRR10W4-F formulations displayed greater antimicrobial effects and reduced toxicity on cultured airway epithelial cells compared to that of the peptide alone. Taken together, these results open the door to the development of novel delivery strategies for AMPs in order to increase the bioavailability of these molecules at the infection site via inhaled therapies.

Mesoporous silica as a matrix for photocatalytic titanium dioxide nanoparticles: lipid membrane interactions.

In the present study, we investigate the combined interaction of mesoporous silica (SiO2) and photocatalytic titanium dioxide (TiO2) nanoparticles with lipid membranes, using neutron reflectometry (NR), cryo-transmission electron microscopy (cryo-TEM), fluorescence oxidation assays, dynamic light scattering (DLS), and ζ-potential measurements. Based on DLS, TiO2 nanoparticles were found to display strongly improved colloidal stability at physiological pH of skin (pH 5.4) after incorporation into either smooth or spiky ("virus-like") mesoporous silica nanoparticles at low pH, the latter demonstrated by cryo-TEM. At the same time, such matrix-bound TiO2 nanoparticles retain their ability to destabilize anionic bacteria-mimicking lipid membranes under UV-illumination. Quenching experiments indicated both hydroxyl and superoxide radicals to contribute to this, while NR showed that free TiO2 nanoparticles and TiO2 loaded into mesoporous silica nanoparticles induced comparable effects on supported lipid membranes, including membrane thinning, lipid removal, and formation of a partially disordered outer membrane leaflet. By comparing effects for smooth and virus-like mesoporous nanoparticles as matrices for TiO2 nanoparticles, the interplay between photocatalytic and direct membrane binding effects were elucidated. Taken together, the study outlines how photocatalytic nanoparticles can be readily incorporated into mesoporous silica nanoparticles for increased colloidal stability and yet retain most of their capacity for photocatalytic destabilization of lipid membranes, and with maintained mechanisms for oxidative membrane destabilization. As such, the study provides new mechanistic information to the widely employed, but poorly understood, practice of loading photocatalytic nanomaterials onto/into matrix materials for increased performance.

Photocatalytic nanoparticles - From membrane interactions to antimicrobial and antiviral effects.

As a result of increasing resistance among pathogens against antibiotics and anti-viral therapeutics, nanomaterials are attracting current interest as antimicrobial agents. Such materials offer triggered functionalities to combat challenging infections, based on either direct membrane action, effects of released ions, thermal shock induced by either light or magnetic fields, or oxidative photocatalysis. In the present overview, we focus on photocatalytic antimicrobial effects, in which light exposure triggers generation of reactive oxygen species. These, in turn, cause oxidative damage to key components in bacteria and viruses, including lipid membranes, lipopolysaccharides, proteins, and DNA/RNA. While an increasing body of studies demonstrate that potent antimicrobial effects can be achieved by photocatalytic nanomaterials, understanding of the mechanistic foundation underlying such effects is still in its infancy. Addressing this, we here provide an overview of the current understanding of the interaction of photocatalytic nanomaterials with pathogen membranes and membrane components, and how this translates into antibacterial and antiviral effects.

Membrane Interactions of Virus-like Mesoporous Silica Nanoparticles.

In the present study, we investigated lipid membrane interactions of silica nanoparticles as carriers for the antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES). In doing so, smooth mesoporous nanoparticles were compared to virus-like mesoporous nanoparticles, characterized by a "spiky" external surface, as well as to nonporous silica nanoparticles. For this, we employed a combination of neutron reflectometry, ellipsometry, dynamic light scattering, and ζ-potential measurements for studies of bacteria-mimicking bilayers formed by palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol. The results show that nanoparticle topography strongly influences membrane binding and destabilization. We found that virus-like particles are able to destabilize such lipid membranes, whereas the corresponding smooth silica nanoparticles are not. This effect of particle spikes becomes further accentuated after loading of such particles with LL-37. Thus, peptide-loaded virus-like nanoparticles displayed more pronounced membrane disruption than either peptide-loaded smooth nanoparticles or free LL-37. The structural basis of this was clarified by neutron reflectometry, demonstrating that the virus-like nanoparticles induce trans-membrane defects and promote incorporation of LL-37 throughout both bilayer leaflets. The relevance of such effects of particle spikes for bacterial membrane rupture was further demonstrated by confocal microscopy and live/dead assays on Escherichia coli bacteria. Taken together, these findings demonstrate that topography influences the interaction of nanoparticles with bacteria-mimicking lipid bilayers, both in the absence and presence of antimicrobial peptides, as well as with bacteria. The results also identify virus-like mesoporous nanoparticles as being of interest in the design of nanoparticles as delivery systems for antimicrobial peptides.

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer.

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

Oxidation of Polyunsaturated Lipid Membranes by Photocatalytic Titanium Dioxide Nanoparticles: Role of pH and Salinity.

In the present study, UV-induced membrane destabilization by TiO2 (anatase) nanoparticles was investigated by neutron reflectometry (NR), small-angle X-ray scattering (SAXS), quartz crystal microbalance with dissipation (QCM-D), dynamic light scattering (DLS), and ζ-potential measurements for phospholipid bilayers formed by zwitterionic palmitoyloleoylphosphatidylcholine (POPC) containing biologically relevant polyunsaturations. TiO2 nanoparticles displayed pH-dependent binding to such bilayers. Nanoparticle binding alone, however, has virtually no destabilizing effects on the lipid bilayers. In contrast, UV illumination in the presence of TiO2 nanoparticles activates membrane destabilization as a result of lipid oxidation caused by the generation of reactive oxygen species (ROS), primarily •OH radicals. Despite the short diffusion length characterizing these, the direct bilayer attachment of TiO2 nanoparticles was demonstrated to not be a sufficient criterion for an efficient UV-induced oxidation of bilayer lipids, the latter also depending on ROS generation in bulk solution. From SAXS and NR, minor structural changes were seen when TiO2 was added in the absence of UV exposure, or on UV exposure in the absence of TiO2 nanoparticles. In contrast, UV exposure in the presence of TiO2 nanoparticles caused large-scale structural transformations, especially at high ionic strength, including gradual bilayer thinning, lateral phase separation, increases in hydration, lipid removal, and potential solubilization into aggregates. Taken together, the results demonstrate that nanoparticle-membrane interactions ROS generation at different solution conditions act in concert to induce lipid membrane destabilization on UV exposure and that both of these need to be considered for understanding the performance of UV-triggered TiO2 nanoparticles in nanomedicine.

Membrane interactions of antimicrobial peptide-loaded microgels.

In the present study, lipid membrane interactions of anionic poly(ethyl acrylate-co-methacrylic acid) (MAA) microgels as carriers for the cationic antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) were investigated. In doing so, neutron reflectometry (NR), Fourier-transform infrared spectroscopy with attenuated total reflection (FTIR-ATR), zeta potential, ellipsometry, and circular dichroism spectroscopy (CD) experiments were employed to investigate the relative importance of membrane interactions of peptide-loaded microgel particles and of released peptide. For the free peptide, NR results showed membrane binding occurring preferentially in the tail region in a concentration-dependent manner. At low peptide concentration (0.3 µM) only peptide insertion in the outer leaflet was seen, however, pronounced membrane defects and peptide present in both leaflets was observed at higher peptide concentration (5.0 µM). LL-37 loaded into MAA microgels qualitatively mirrored these effects regarding both peptide localization within the membrane and concentration-dependent defect formation. In addition, very limited membrane binding of microgel particles was observed, in agreement with FTIR-ATR and liposome leakage results. FTIR-ATR showed LL-37 to undergo α-helix formation on membrane insertion, also supported by CD results, the kinetics of which was substantially reduced for microgel-loaded LL-37 due to sustained peptide release. Together, these findings demonstrate that membrane interactions for microgel-loaded LL-37 are dominated by released peptide, but also that slow release of microgel-loaded LL-37 translates into kinetic effects on peptide-membrane interactions, relating to both peptide localization within the bilayer, and to bilayer structure.

Effects of oxidation on the physicochemical properties of polyunsaturated lipid membranes.

The exposure of biological membranes to reactive oxygen species (ROS) plays an important role in many pathological conditions such as inflammation, infection, or sepsis. ROS also modulate signaling processes and produce markers for damaged tissue. Lipid peroxidation, mainly affecting polyunsaturated phospholipids, results in a complex mixture of oxidized products, which may dramatically alter membrane properties. Here, we have employed a set of biophysical and surface-chemical techniques, including neutron and X-ray scattering, to study the structural, compositional, and stability changes due to oxidative stress on phospholipid bilayers composed of lipids with different degrees of polyunsaturation. In doing so, we obtained real-time information about bilayer degradation under in situ UV exposure using neutron reflectometry. We present a set of interrelated physicochemical effects, including gradual increases in area per molecule, head group and acyl chain hydration, as well as bilayer thinning, lateral phase separation, and defect formation leading to content loss upon membrane oxidation. Such effects were observed to depend on the presence of polyunsaturated phospholipids in the lipid membrane, suggesting that these may also play a role in the complex oxidation processes occurring in cells.