Asymmetric cationic liposomes designed for heat-activated association with cells.

Improved anticancer drugs and drug carriers are needed in combination therapies, such as hyperthermia-assisted chemotherapy. Liposomal drug carriers with advanced functions are attractive candidates for targeted accumulation and drug release in response to heat stimulus. We report on the design of liposomes with a heat-activated surface function. Our design is based on asymmetric lipid membranes with a defined gel to liquid-crystalline phase-transition temperature around 41°C. Asymmetry between the inner and the outer membrane leaflets was generated through selective PEGylation of cationic lipids in the outer membrane leaflet. In a physiological buffer, the PEGylated asymmetric liposomes had a neutral zeta potential and did not bind to planar anionic model membranes. In contrast, following upon heat-activation, binding of liposomes to the model membranes occurred. Release of a hydrophilic dye encapsulated in the asymmetric liposomes occurred at 40°C. Enhanced uptake of the asymmetric liposomes by hypopharyngeal carcinoma cells (FaDu cells) was observed when hyperthermia was applied compared to experiments performed at 37°C. These results show the potential of asymmetric liposomes for localized delivery of drugs into cells in response to (external) temperature stimulus.

Graphene Oxide and Lipid Membranes: Size-Dependent Interactions.

We have investigated the interaction of graphene oxide (GO) sheets with supported lipid membranes with focus on how the interaction depends on GO sheet size (three samples in the range of 90-5000 nm) and how it differs between small and large liposomes. The layer-by-layer assembly of these materials into multilamellar structures, as discovered in our previous research, is now further explored. The interaction processes were monitored by two complementary, real time, surface-sensitive analytical techniques: quartz crystal microbalance with dissipation monitoring (QCM-D, electroacoustic sensing) and indirect nanoplasmonic sensing (INPS, optical sensing). The results show that the sizes of each of the two components, graphene oxide and liposomes, are important parameters affecting the resulting multilayer structures. Spontaneous liposome rupture onto graphene oxide is obtained for large lateral dimensions of the graphene oxide sheets.

Effects of Al(3+) on Phosphocholine and Phosphoglycerol Containing Solid Supported Lipid Bilayers.

Aluminum has attracted great attention recently as it has been suggested by several studies to be associated with increased risks for Alzheimer's and Parkinson's disease. The toxicity of the trivalent ion is assumed to derive from structural changes induced in lipid bilayers upon binding, though the mechanism of this process is still not well understood. In the present study we elucidate the effect of Al(3+) on supported lipid bilayers (SLBs) using fluorescence microscopy, the quartz crystal microbalance with dissipation (QCM-D) technique, dual-polarization interferometry (DPI), and molecular dynamics (MD) simulations. Results from these techniques show that binding of Al(3+) to SLBs containing negatively charged and neutral phospholipids induces irreversible changes such as domain formation. The measured variations in SLB thickness, birefringence, and density indicate a phase transition from a disordered to a densely packed ordered phase.

Synthesis and Evaluation of Astatinated N-[2-(Maleimido)ethyl]-3-(trimethylstannyl)benzamide Immunoconjugates.

Effective treatment of metastasis is a great challenge in the treatment of different types of cancers. Targeted alpha therapy utilizes the short tissue range (50-100 µm) of α particles, making the method suitable for treatment of disseminated occult cancers in the form of microtumors or even single cancer cells. A promising radioactive nuclide for this type of therapy is astatine-211. Astatine-211 attached to tumor-specific antibodies as carrier molecules is a system currently under investigation for use in targeted alpha therapy. In the common radiolabeling procedure, astatine is coupled to the antibody arbitrarily on lysine residues. By instead coupling astatine to disulfide bridges in the antibody structure, the immunoreactivity of the antibody conjugates could possibly be increased. Here, the disulfide-based conjugation was performed using a new coupling reagent, maleimidoethyl 3-(trimethylstannyl)benzamide (MSB), and evaluated for chemical stability in vitro. The immunoconjugates were subsequently astatinated, resulting in both high radiochemical yield and high specific activity. The MSB-conjugate was shown to be stable with a long shelf life prior to the astatination. In a comparison of the in vivo distribution of the new immunoconjugate with other tin-based immunoconjugates in tumor-bearing mice, the MSB conjugation method was found to be a viable option for successful astatine labeling of different monoclonal antibodies.

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction.

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.

Non-Invasive Acoustical sensing of Drug-Induced Effects on the Contractile Machinery of Human Cardiomyocyte Clusters.

There is an urgent need for improved models for cardiotoxicity testing. Here we propose acoustic sensing applied to beating human cardiomyocyte clusters for non-invasive, surrogate measuring of the QT interval and other characteristics of the contractile machinery. In experiments with the acoustic method quartz crystal microbalance with dissipation monitoring (QCM-D), the shape of the recorded signals was very similar to the extracellular field potential detected in electrochemical experiments, and the expected changes of the QT interval in response to addition of conventional drugs (E-4031 or nifedipine) were observed. Additionally, changes in the dissipation signal upon addition of cytochalasin D were in good agreement with the known, corresponding shortening of the contraction-relaxation time. These findings suggest that QCM-D has great potential as a tool for cardiotoxicological screening, where effects of compounds on the cardiomyocyte contractile machinery can be detected independently of whether the extracellular field potential is altered or not.

Heat-activated liposome targeting to streptavidin-coated surfaces.

There is a great need of improved anticancer drugs and corresponding drug carriers. In particular, liposomal drug carriers with heat-activated release and targeting functions are being developed for combined hyperthermia and chemotherapy treatments of tumors. The aim of this study is to demonstrate the heat-activation of liposome targeting to biotinylated surfaces, in model experiments where streptavidin is used as a pretargeting protein. The design of the heat-activated liposomes is based on liposomes assembled in an asymmetric structure and with a defined phase transition temperature. Asymmetry between the inside and the outside of the liposome membrane was generated through the enzymatic action of phospholipase D, where lipid head groups in the outer membrane leaflet, i.e. exposed to the enzyme, were hydrolyzed. The enzymatically treated and purified liposomes did not bind to streptavidin-modified surfaces. When activation heat was applied, starting from 22°C, binding of the liposomes occurred once the temperature approached 33±0.5°C. Moreover, it was observed that the asymmetric structure remained stable for at least 2 weeks. These results show the potential of asymmetric liposomes for the targeted binding to cell membranes in response to (external) temperature stimulus. By using pretargeting proteins, this approach can be further developed for personalized medicine, where tumor-specific antibodies can be selected for the conjugation of pretargeting agents.

Sensing conformational changes in DNA upon ligand binding using QCM-D. Polyamine condensation and Rad51 extension of DNA layers.

Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59 bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film deformation allows comparative studies of two or more analytes using the same DNA layer as demonstrated here by spermine and spermidine.

Construction and modeling of concatemeric DNA multilayers on a planar surface as monitored by QCM-D and SPR.

The sequential hybridization of a 534 base pair DNA concatemer layer was monitored by QCM-D and SPR, and the QCM-D data were analyzed by Voigt viscoelastic models. The results show that Voigt-based modeling gives a good description of the experimental data but only if shear viscosity and elasticity are allowed to depend on the shear frequency. The derived layer thickness, shear viscosity and elasticity of the growing film give a representation of the DNA film in agreement with known bulk properties of DNA, and reveal a maximum in film viscosity when the molecules in the layer contain 75 base pairs. The experimental data during construction of a 3084 bp DNA concatemer layer were compared to predictions of the QCM-D response of a 1 µm thick film of rod-like polymers. A predicted nonmonotonous variation of dissipation with frequency (added mass) is in qualitative agreement with the experiments, but with a quantitative disagreement which likely reflects that the flexibility of such long DNA molecules is not included in the model.

Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size.

DPPC liposomes ranging from 90 nm to 160 nm in diameter were prepared and used for studies of the formation of supported lipid membranes on silica (SiO2) at temperatures below and above the gel to liquid-crystalline phase transition temperature (Tm = 41 °C), and by applying temperature gradients through Tm. The main method was the quartz crystal microbalance with dissipation (QCM-D) technique. It was found that liposomes smaller than 100 nm spontaneously rupture on the silica surface when deposited at a temperature above Tm and at a critical surface coverage, following a well-established pathway. In contrast, DPPC liposomes larger than 160 nm do not rupture on the surface when adsorbed at 22 °C or at 50 °C. However, when liposomes of this size are first adsorbed at 22 °C and at a high enough surface coverage, after which they are subject to a constant temperature gradient up to 50 °C, they rupture and fuse to a bilayer, a process that is initiated around Tm. The results are discussed and interpreted considering a combination of effects derived from liposome-surface and liposome-liposome interactions, different softness/stiffness and shape of liposomes below and above Tm, the dynamics and thermal activation of the bilayers occurring around Tm and (for liposomes containing 33% of NaCl) osmotic pressure. These findings are valuable both for preparation of supported lipid bilayer cell membrane mimics and for designing temperature-responsive material coatings.

Real-time monitoring of surface-confined platelet activation on TiO2.

For the development of advanced hemocompatible biomaterial functions, there is an unmet demand for in vitro evaluation techniques addressing platelet-surface interactions. We show that the quartz crystal microbalance with dissipation (QCM-D) monitoring technique, here combined with light microscopy, provides a surface sensitive technique that allows for real-time monitoring of the activation and aggregation of the surface-confined platelets on TiO2. The QCM-D signal monitored during adhesion and activation of platelets on TiO2 coated surfaces was found to be different in platelet-poor and platelet-rich environment although light microscopy images taken for each of the two cases looked essentially the same. Interestingly, aggregation of activated platelets was only observed in a protein-rich environment. Our results show that a layer of plasma proteins between the TiO2 surface and the platelets strongly influences the coupling between the platelets and the underlying substrate, explaining both the observed QCM-D signals and the ability of the platelets to aggregate.

Study on multilayer structures prepared from heparin and semi-synthetic cellulose sulfates as polyanions and their influence on cellular response.

Multilayer coatings of polycationic chitosan paired with polyanionic semi-synthetic cellulose sulfates or heparin were prepared by the layer-by-layer method. Two different cellulose sulfates (CS) with high (CS2.6) and intermediate (CS1.6) sulfation degree were prepared by sulfation of cellulose. Multilayers were fabricated at pH 4 and the resulting films were characterized by several methods. The multilayer 'optical' mass, measured by surface plasmon resonance, showed little differences in the total mass adsorbed irrespective of which polyanion was used. In contrast, 'acoustic' mass, calculated from quartz crystal micro balance with dissipation monitoring, showed the lowest mass and dissipation values for CS2.6 (highest sulfation degree) multilayers indicating formation of stiffer layers compared to heparin and CS1.6 layers which led to higher mass and dissipation values. Water contact angle and zeta potential measurements indicated formation of more distinct layers with using heparin as polyanion, while use of CS1.6 and CS2.6 resulted into more fuzzy intermingled multilayers. CS1.6 multilayers significantly supported adhesion and growth of C2C12 cells where as only few cells attached and started to spread initially on CS2.6 layers but favoured long term cell growth. Contrastingly cells adhered and grew poorly on to the layers of heparin. This present study shows that cellulose sulfates are attractive candidates for multilayer formation as potential substratum for controlled cell adhesion. Since a peculiar interaction of cellulose sulfates with growth factors was found during previous studies, immobilization of cellulose sulfate in multilayer systems might be of great interest for tissue engineering applications.

Phase transition-controlled flip-flop in asymmetric lipid membranes.

Lipid membrane asymmetry is of fundamental importance for biological systems and also provides an attractive means for molecular control over biomaterial surface properties (including drug carriers). In particular, temperature-dependent changes of surface properties can be achieved by taking advantage of distinct phase transitions in lipid membrane coatings where lipids exchange (flip-flop) between leaflets. In this study, temperature is used to control flip-flop of lipids in asymmetric lipid membranes on planar solid supports, where the two leaflets of the lipid membrane are in different phase states. More specifically, the lower leaflet is prepared from a supported lipid membrane composed of a high Tm lipid mixture of phosphocholine (PC), phosphatidylserine (PS), and a bioactive lipid on TiO2, followed by selective removal of the top leaflet by detergent. Next, at a lower temperature, where the remaining leaflet is in the gel state, a top leaflet of a different lipid composition and in the fluid phase is formed. Phase transition-induced changes in membrane surface properties following upon temperature-activation of the prepared asymmetric membrane are demonstrated by the detection of biotinylated lipids, which were initially located (thus "hidden") in the lower-gel phase leaflet, at the surface of the top leaflet. These processes were monitored in real-time by the quartz crystal microbalance with dissipation (QCM-D) and the dual polarization interferometry (DPI) techniques, allowing modeling of the mass and the anisotropic property of the lipid structures in different phase states.

Effect of molecular composition of heparin and cellulose sulfate on multilayer formation and cell response.

Here, the layer-by-layer method was applied to assemble films from chitosan paired with either heparin or a semisynthetic cellulose sulfate (CS) that possessed a higher sulfation degree than heparin. Ion pairing was exploited during multilayer formation at pH 4, while hydrogen bonding is likely to occur at pH 9. Effects of polyanions and pH value during layer formation on multilayers properties were studied by surface plasmon resonance ("dry layer mass"), quartz crystal microbalance with dissipation monitoring ("wet layer mass"), water contact angle, and zeta potential measurements. Bioactivity of multilayers was studied regarding fibronectin adsorption and adhesion/proliferation of C2C12 myoblast cells. Layer growth and dry mass were higher for both polyanions at pH 4 when ion pairing occurred, while it decreased significantly with heparin at pH 9. By contrast, CS as polyanion resulted also in high layer growth and mass at pH 9, indicating a much stronger effect of hydrogen bonding between chitosan and CS. Water contact angle and zeta potential measurements indicated a more separated structure of multilayers from chitosan and heparin at pH 4, while CS led to a more fuzzy intermingled structure at both pH values. Cell behavior was highly dependent on pH during multilayer formation with heparin as polyanion and was closely related to fibronectin adsorption. By contrast, CS and chitosan did not show such dependency on pH value, where adhesion and growth of cells was high. Results of this study show that CS is an attractive candidate for multilayer formation that does not depend so strongly on pH during multilayer formation. In addition, such multilayer system also represents a good substrate for cell interactions despite the rather soft structure. As previous studies have shown specific interaction of CS with growth factors, multilayers from chitosan and CS may be of great interest for different biomedical applications.

Cell membrane damage and protein interaction induced by copper containing nanoparticles--importance of the metal release process.

Cu-containing nanoparticles are used in various applications in order to e.g. achieve antimicrobial activities and to increase the conductivity of fluids and polymers. Several studies have reported on toxic effects of such particles but the mechanisms are not completely clear. The aim of this study was to investigate the interactions between cell membranes and well-characterized nanoparticles of CuO, Cu metal, a binary Cu-Zn alloy and micron-sized Cu metal particles. This was conducted via in vitro investigations of the effects of the nanoparticles on (i) cell membrane damage on lung epithelial cells (A549), (ii) membrane rupture of red blood cells (hemolysis), complemented by (iii) nanoparticle interaction studies with a model lipid membrane using quartz crystal microbalance with dissipation monitoring (QCM-D). The results revealed that nanoparticles of the Cu metal and the Cu-Zn alloy were both highly membrane damaging and caused a rapid (within 1h) increase in membrane damage at a particle mass dose of 20 µg/mL, whereas the CuO nanoparticles and the micron-sized Cu metal particles showed no such effect. At similar nanoparticle surface area doses, the nano and micron-sized Cu particles showed more similar effects. The commonly used LDH (lactate dehydrogenase) assay for analysis of membrane damage was found impossible to use due to nanoparticle-assay interactions. None of the particles induced any hemolytic effects on red blood cells when investigated up to high particle concentrations (1mg/mL). However, both Cu and Cu-Zn nanoparticles caused hemoglobin aggregation/precipitation, a process that would conceal a possible hemolytic effect. Studies on interactions between the nanoparticles and a model membrane using QCM-D indicated a small difference between the investigated particles. Results of this study suggest that the observed membrane damage is caused by the metal release process at the cell membrane surface and highlight differences in reactivity between metallic nanoparticles of Cu and Cu-Zn and nanoparticles of CuO.

Tuning cell adhesion and growth on biomimetic polyelectrolyte multilayers by variation of pH during layer-by-layer assembly.

Polyelectrolyte multilayers of chitosan and heparin are assembled on glass where heparin is applied at pH = 4, 9 and 4 during the formation of the first layers followed by pH = 9 at the last steps (denoted pH 4 + 9). Measurements of wetting properties, layer mass, and topography show that multilayers formed at pH = 4 are thicker, contain more water and have a smoother surface compared to those prepared at pH = 9 while the pH = 4 + 9 multilayers expressed intermediate properties. pH = 9 multilayers are more cell adhesive and support growth of C2C12 cells better than pH = 4 ones. However, pH 4 + 9 conditions improve the bioactivity to a similar level of pH = 9 layers. Multilayers prepared using pH 4 + 9 conditions form thick enough layers that may allow efficient loading of bioactive molecules.

Probing the biofunctionality of biotinylated hyaluronan and chondroitin sulfate by hyaluronidase degradation and aggrecan interaction.

Molecular interactions involving glycosaminoglycans (GAGs) are important for biological processes in the extracellular matrix (ECM) and at cell surfaces, and also in biotechnological applications. Enzymes in the ECM constantly modulate the molecular structure and the amount of GAGs in our tissues. Specifically, the changeable sulfation patterns of many GAGs are expected to be important in interactions with proteins. Biotinylation is a convenient method for immobilizing molecules to surfaces. When studying interactions at the molecular, cell and tissue level, the native properties of the immobilized molecule, i.e. its biofunctionality, need to be retained upon immobilization. Here, the GAGs hyaluronan (HA) and chondroitin sulfate (CS), and synthetically sulfated derivatives of the two, were immobilized using biotin-streptavidin binding. The degree of biotinylation and the placement of biotin groups (end-on/side-on) were varied. The introduction of biotin groups could have unwanted effects on the studied molecule, but this aspect that is not always straightforward to evaluate. Hyaluronidase, an enzyme that degrades HA and CS in the ECM, was investigated as a probe to evaluate the biofunctionality of the immobilized GAGs, using both quartz crystal microbalance and high-performance liquid chromatography. Our results showed that end-on biotinylated HA was efficiently degraded by hyaluronidase, whereas already a low degree of side-on biotinylation destroyed the degrading ability of the enzyme. Synthetically introduced sulfate groups also had this effect. Hence hyaluronidase degradation is a cheap and easy way to investigate how molecular function is influenced by the introduced functional groups. Binding experiments with the proteoglycan aggrecan emphasized the influence of protein size and surface orientation of the GAGs for in-depth studies of GAG behavior.

Acoustical sensing of cardiomyocyte cluster beating.

Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cell quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to µm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66-168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.

Characterization of nanoparticle-lipid membrane interactions using QCM-D.

In vitro characterization of nanoparticles is becoming increasingly important due to the rapid development of novel nanoparticle formulations for applications in the field of nanomedicine and related areas. Commonly, nanoparticles are simply characterized with respect to their size and zeta potential, and additional in vitro characterization of nanoparticles is needed to develop useful nanoparticle structure-activity relationships. In this context it is highly interesting to characterize the interactions between nanoparticles and model interfaces, such as lipid membranes. Here, we describe a methodology to study such interactions using the quartz crystal microbalance with dissipation monitoring technique (QCM-D). In order to mimic some aspects of the native cell membrane, a supported lipid membrane is formed on the QCM-D sensor surface. Subsequently the membrane is exposed to nanoparticles, and the nanoparticle-lipid membrane interactions are monitored in real time. The outcome of such analysis provides information on the adsorption process (importantly kinetics and adsorbed amounts) as well as on the integrity of both the nanoparticles and the lipid membrane upon interaction. QCM-D analyses are suitable for screening of nanoparticle-lipid membrane interactions due to the fair throughput of the technique, which can be complemented, when needed, by additional analyses by other surface-sensitive analytical techniques.