Adsorption of lipid-functionalized poly(ethylene glycol) to gold surfaces as a cushion for polymer-supported lipid bilayers

Inclusion of a polymer cushion between a lipid bilayer membrane and a solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, the properties of a heterofunctional, telechelic PEG lipopolymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N- [3-(2-(pyridyldithio)propionate]) (DSPE-PEG-PDP) adsorbed from ethanol and water solutions onto gold surfaces were studied using a variety of surface-sensitive techniques. X-ray photoelectron spectroscopy showed that the PEG molecules are tethered to the gold surface via thiolate bonds. When adsorbed from water, ethanol, or their mixtures, reflection-absorption infrared spectroscopy showed that amorphous PEG layers with disordered DSPE alkyl chains were formed, independent of adsorption time or solution concentration. On the basis of advancing and receding water and hexadecane contact angles on the lipopolymer films, the DSPE lipid groups appear to segregate from the PEG layer and become exposed at the surface of the polymer films. Swelling observed in surface plasmon resonance experiments and the large contact angle hysteresis observed indicate that highly swellable, mobile films capable of molecular rearrangements are formed. The self-assembling and amorphous properties of these PEG layers make them ideal candidates as polymer cushions for polymer-supported lipid bilayers. The DSPE surface concentration can be controlled, to a limited degree, by varying the adsorption time of DSPE-PEG-PDP from ethanol. A more effective strategy is to coadsorb DSPE-PEG-PDP with a non-lipid-functionalized PEG-PDP from an ethanol/water mixture, which allows the PEG thickness and density to remain constant while decreasing the density of DSPE groups.     

Surface-supported bilayers with transmembrane proteins: role of the polymer cushion revisited

Protein lateral mobility in surface-supported bilayers is often much lower than the mobility of the lipids. In the present study we explore whether the incorporation of a PEG cushion between the bilayer and the substrate increases the lateral mobility of transmembrane proteins in bilayers produced via directed assembly, a method based on Langmuir-Blodgett deposition techniques. In our experiments, the PEG cushions were incorporated by adding PEG lipids to the protein/lipid monolayer at the air/water interface, at the first step of bilayer assembly. The protein and lipid mobilities in 160 different bilayers, with various PEG molecular weights and PEG lipid concentrations, were measured and compared. We found that the measured diffusion coefficients do not depend on the PEG molecular weight or the PEG lipid concentration and are very similar to the values measured in the absence of PEG. Therefore, contrary to our expectations, we found that a PEG cushion does not necessarily increase protein mobility, suggesting that the low protein mobility is not a consequence of protein-substrate interactions. Furthermore, we showed that the low protein mobility is not due to protein aggregation. The major determinant of protein mobility in surface-supported bilayer systems appears to be the method of bilayer assembly. While proteins were always mobile if the bilayers were prepared using the directed assembly method, in the presence and absence of a PEG cushion, other bilayer assembly protocols resulted in complete lack of protein mobility.     

Diffusive dynamics of vesicles tethered to a fluid supported bilayer by single-particle tracking

We recently introduced a method to tether intact phospholipid vesicles onto a fluid supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). Once tethered, the vesicles can diffuse in two dimensions parallel to the supported membrane surface. The average diffusion coefficient, D, is typically 0.2 microm(2)/s; this is 3-5 times smaller than for individual lipid or DNA-lipid conjugate diffusion in supported bilayers. In this article, we investigate the origin of this difference in the diffusive dynamics of tethered vesicles by single-particle tracking under collision-free conditions. D is insensitive to tethered vesicle size from 30 to 200 nm, as well as a 3-fold change in the viscosity of the bulk medium. The addition of macromolecules such as poly(ethylene glycol) reversibly stops the motion of tethered vesicles without causing the exchange of lipids between the tethered vesicle and supported bilayer. This is explained as a depletion effect at the interface between tethered vesicles and the supported bilayer. Ca ions lead to transient vesicle-vesicle interactions when tethered vesicles contain negatively charged lipids, and vesicle diffusion is greatly reduced upon Ca ion addition when negatively charged lipids are present both in the supported bilayer and tethered vesicles. Both effects are interesting in their own right, and they also suggest that tethered vesicle-supported bilayer interactions are possible; this may be the origin of the reduction in D for tethered vesicles. In addition, the effects of surface defects that reversibly trap diffusing vesicles are modeled by Monte Carlo simulations. This shows that a significant reduction in D can be observed while maintaining normal diffusion behavior on the time scale of our experiments.     

Supported lipid bilayers on biocompatible polysaccharide multilayers

Formation of supported lipid bilayers on soft polymer cushions is a useful approach to decouple the membrane from the substrate for applications involving membrane proteins. We prepared biocompatible polymer cushions by the layer-by-layer assembly of two polysaccharide polyelectrolytes, chitosan (CHI) and hyaluronic acid, on glass and silicon substrates. (CHI/HA)(5) films were characterized by atomic force microscopy, giving an average thickness of 57 nm and roughness of 25 nm in aqueous solution at pH 6.5. Formation of zwitterionic lipid bilayers by the vesicle fusion method was attempted using DOPC vesicles at pH 4 and 6.5 on (CHI/HA)(5) films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; a combination of fluorescence and AFM indicated that this was attributable to formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. By contrast, more uniform bilayers with mobile lipids were produced at pH 4. Fluorescence recovery after photobleaching gave diffusion coefficients that were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. These results demonstrate that polysaccharides provide a useful alternative to other polymer cushions, particularly for applications where biocompatibility is important.     

Impact of membrane fluidity on steric stabilization by lipopolymers

In this work, the impact of lipid lateral mobility on the steric interaction between membranes containing poly(ethylene glycol) (PEG) functionalized lipids was investigated using the surface force apparatus. The force-distance profiles show the presence of electrostatic and steric repulsion that arise from the presence of negatively charged PEG functionalized lipids. Fluid-phase bilayers have high lateral diffusion relative to gel-phase bilayers; however, a quantitative comparison of the interaction forces between membranes in these two different phase states demonstrates a reduced rate of diffusion in the fluid phase for the PEG-lipids under constrained geometries. Thus, the amount of polymer in the contact zone can be modulated and is reduced with fluid membranes; however, complete exclusion was not achieved. As a result, the steric repulsion afforded by PEG chains or binding affinity of ligated PEG chains can only be modestly tailored by the phase state of the liposome.     

Fluid and air-stable lipopolymer membranes for biosensor applications

The behavior of poly(ethylene glycol) (PEG) conjugated lipids was investigated in planar supported egg phosphatidylcholine bilayers as a function of lipopolymer density, chain length of the PEG moiety, and type of alkyl chains on the PEG lipid. Fluorescence recovery after photobleaching measurements verified that dye-labeled lipids in the membrane as well as the lipopolymer itself maintained a substantial degree of fluidity under most conditions that were investigated. PEG densities exceeding the onset of the mushroom-to-brush phase transition were found to confer air stability to the supported membrane. On the other hand, substantial damage or complete delamination of the lipid bilayer was observed at lower polymer densities. The presence of PEG in the membrane did not substantially hinder the binding of streptavidin to biotinylated lipids present in the bilayer. Furthermore, above the onset of the transition into the brush phase, the protein binding properties of these membranes were found to be very resilient upon removal of the system from water, rigorous drying, and rehydration. These results indicate that supported phospholipid bilayers containing lipopolymers show promise as rugged sensor platforms for ligand-receptor binding.     

Stability of DNA-tethered lipid membranes with mobile tethers

We recently introduced two approaches for tethering planar lipid bilayers as membrane patches to either a supported lipid bilayer or DNA-functionalized surface using DNA hybridization (Chung, M.; Lowe, R. D.; Chan, Y-H. M.; Ganesan, P. V.; Boxer, S. G. J. Struct. Biol.2009, 168, 190-9). When mobile DNA tethers are used, the tethered bilayer patches become unstable, while they are stable if the tethers are fixed on the surface. Because the mobile tethers between a patch and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions, we have investigated the sources of instability, focusing on membrane composition. The most stable patches were made with a mixture of saturated lipids and cholesterol, suggesting an important role for membrane stiffness. Other factors such as the effect of tether length, lateral mobility, and patch membrane edge were also investigated. On the basis of these results, a model for the mechanism of patch destruction is developed.     

Creating fluid and air-stable solid supported lipid bilayers

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.     

Double cushions preserve transmembrane protein mobility in supported bilayer systems

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)-conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 x 10(-8) cm2/s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 x 10(-8) cm2/s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

A facile approach for assembling lipid bilayer membranes on template-stripped gold

Lipid vesicles are designed with functional chemical groups to promote vesicle fusion on template-stripped gold (TS Au) surfaces that does not spontaneously occur on unfunctionalized Au surfaces. Three types of vesicles were exposed to TS Au surfaces: (1) vesicles composed of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids; (2) vesicles composed of lipid mixtures of 2.5 mol % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio)propionate] (DSPE-PEG-PDP) and 97.5 mol % of POPC; and (3) vesicles composed of 2.5 mol % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG) and 97.5 mol % POPC. Atomic force microscopy (AFM) topography and force spectroscopy measurements acquired in a fluid environment confirmed tethered lipid bilayer membrane (tLBM) formation only for vesicles composed of 2.5 mol % DSPE-PEG-PDP/97.5 mol % POPC, thus indicating that the sulfur-containing PDP group is necessary to achieve tLBM formation on TS Au via Au-thiolate bonds. Analysis of force-distance curves for 2.5 mol % DSPE-PEG-PDP/97.5 mol % POPC tLBMs on TS Au yielded a breakthrough distance of 4.8 ± 0.4 nm, which is about 1.7 nm thicker than that of POPC lipid bilayer membrane formed on mica. Thus, the PEG group serves as a spacer layer between the tLBM and the TS Au surface. Fluorescence microscopy results indicate that these tLBMs also have greater mechanical stability than solid-supported lipid bilayer membranes made from the same vesicles on mica. The described process for assembling stable tLBMs on Au surfaces is compatible with microdispensing used in array fabrication.     

Multinuclear NMR studies of single lipid bilayers supported in cylindrical aluminum oxide nanopores

Lipid bilayers were deposited inside the 0.2 microm pores of anodic aluminum oxide (AAO) filters by extrusion of multilamellar liposomes and their properties studied by 2H, 31P, and 1H solid-state NMR. Only the first bilayer adhered strongly to the inner surface of the pores. Additional layers were washed out easily by a flow of water as demonstrated by 1H magic angle spinning NMR experiments with addition of Pr3+ ions to shift accessible lipid headgroup resonances. A 13 mm diameter Anopore filter of 60 microm thickness oriented approximately 2.5 x 10(-7) mol of lipid as a single bilayer, corresponding to a total membrane area of about 500 cm2. The 2H NMR spectra of chain deuterated POPC are consistent with adsorption of wavy, tubular bilayers to the inner pore surface. By NMR diffusion experiments, we determined the average length of those lipid tubules to be approximately 0.4 microm. There is evidence for a thick water layer between lipid tubules and the pore surface. The ends of tubules are well sealed against the pore such that Pr3+ ions cannot penetrate into the water underneath the bilayers. We successfully trapped poly(ethylene glycol) (PEG) with a molecular weight of 8000 in this water layer. From the quantity of trapped PEG, we calculated an average water layer thickness of 3 nm. Lipid order parameters and motional properties are unperturbed by the solid support, in agreement with existence of a water layer. Such unperturbed, solid supported membranes are ideal for incorporation of membrane-spanning proteins with large intra- and extracellular domains. The experiments suggest the promise of such porous filters as membrane support in biosensors.     

Assembly of lipid bilayers on silica and modified silica colloids by reconstitution of dried lipid films

A method is presented for the assembly of lipid bilayers on silica colloids via reconstitution of dried lipid films solvent-cast from chloroform within packed beds of colloids ranging from 100 nm to 10 μm in diameter. Rapid solvent evaporation from the packed bed void volume results in uniform distribution of dried lipid throughout the colloidal bed. Fluorescence measurements indicate that significant, if not quantitative, retention of DOPC or DPPC films cast between sub-bilayer and multilayer quantities occurs when the colloids are redispersed in aqueous solution. Phospholipid bilayers assembled in this manner are shown to effectively passivate the surface of 250 nm colloids to nonspecific adsorption of bovine serum albumin. The method is shown to be capable of preparing supported bilayers on colloid surfaces that do not generally support vesicle fusion such as poly(ethylene glycol) (PEG) modified silica colloids. Bilayers of lipids that have not been reported to self-assemble by vesicle fusion, including gel-phase lipids and single-chain diacetylene amphiphiles, can also be formed by this method. The utility of the solid-core support is demonstrated by the facile assembly of supported lipid bilayers within fused silica capillaries to generate materials that are potentially suitable for the analysis of membrane interactions in a microchannel format.     

Structure and thermodynamics of lipid bilayers on polyethylene glycol cushions: fact and fiction of PEG cushioned membranes

In developing well hydrated polymer cushioned membranes, structural studies are often neglected. In this work, neutron and X-ray reflectivity studies reveal that hybrid bilayer/polyethylene glycol (PEG) systems created from mixtures of phospholipids and PEG conjugated lipopolymers do not yield a hydrated cushion beneath the bilayer unless the terminal ends of the lipopolymers are functionalized with reactive end groups and can covalently bind (tether) to the underlying support surface. While reactive PEG tethered systems yielded bilayers with near complete surface coverage, a bimodal distribution of heights with sub-micrometer lateral dimensions was observed consisting of cushioned membrane domains and uncushioned regions in close proximity to the support. The membrane fraction cushioned by the hydrated polymer could be controlled by adjusting the molar ratio of lipopolymer in the bilayer. A general phase diagram based on the free energy of the various configurations is derived that qualitatively predicts the observed behavior and the resulting structure of such systems a priori. As further evidenced by ellipsometry, atomic force and fluorescence microscopy, the tethered system provides a simple means for fabricating small cushioned domains within a membrane.     

Protein adsorption on supported phospholipid bilayers

Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO(2) surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3-4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed.     

E-cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion

Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 +/- 0.3 microm(2)/s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.     

Creation of lipid partitions by deposition of amphipathic viral peptides

Phospholipid vesicles exhibit a natural characteristic to fuse and reform into a continuous single bilayer membrane on hydrophilic solid substrates such as glass, mica, and silica. The resulting solid-supported bilayer mimics physiological tendencies such as lipid flip-flop and lateral mobility. The lateral mobility of fluorescently labeled lipids fused into solid-supported bilayers is found to change upon deposition on the membrane surface of an amphipathic alpha-helical peptide (AH) derived from the hepatitis C virus (HCV) NS5A protein. The binding of the AH peptide to a phospholipid bilayer, with the helical axis parallel to the bilayer, leads to immobilization of the bilayer. We used AFM to better understand the mechanistic details of this specific interaction, and determined that the diminished fluidity of the bilayer is due to membrane thinning. Utilizing this specific interaction between AH peptides and lipid molecules, we demonstrate a novel process for the creation of lipid partition by employing AH peptides as agents to immobilize lipid molecules, thus creating a patterned solid support with partition-defined areas of freely mobile lipid bilayers. This architecture could have a wide range of applications in novel sensing, biotechnology, high-throughput screening, and biomimetic strategies.     

Self-assembly of solid-supported membranes using a triggered fusion of phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane

A general procedure for the formation ofsolid-supported artificial membranes containing transmembrane proteins is reported. The main objective was to directly use the pool of proteins of the native biomembrane (here the inner membrane from mitochondria of human carcinogenic hepatic cells) and to avoid purification steps with detergent. Proteoliposomes of phospholipid-enriched inner membranes from mitochondria were tethered and fused onto a tailored surface via a streptavidin link. The failure of some preliminary experiments on membrane formation was attributed to strong nonspecific interactions between the solid surface and the protuberant hydrophilic parts of the transmembrane complexes. The correct loading of uniform membranes was performed after optimization of a tailored surface, covered with a grafted short-chain poly(ethylene glycol), so that nonspecific interactions are reduced. Step-by-step assembly of the structure and triggered fusion of the immobilized proteoliposomes were monitored by surface plasmon resonance and fluorescence photobleaching recovery, respectively. The long-range lateral diffusion coefficient (at 22 degrees C) for a fluorescent lipid varies from 2.5 x 10(-8) cm2 s(-1) for a tethered lipid bilayer without protein to 10(-9) cm2 s(-1) for a tethered membrane containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The decrease in the diffusion coefficient in the tethered membrane with increase in protein area fraction was too pronounced to be fully explained by the theoretical models of obstructed lateral diffusion. Covalent tethering links with the solid are certainly involved in the decrease of the overall lateral mobility of the components in the supported membrane at the highest protein-to-lipid ratios.     

The Effect of Ligand Mobility on the Cellular Interaction of Multivalent Nanoparticles

Multivalent nanoparticle binding to cells can be of picomolar avidity making such interactions almost as intense as those seen with antibodies. However, reducing nanoparticle design exclusively to avidity optimization by the choice of ligand and its surface density does not sufficiently account for controlling and understanding cell–particle interactions. Cell uptake, for example, is of paramount significance for a plethora of biomedical applications and does not exclusively depend on the intensity of multivalency. In this study, it is shown that the mobility of ligands tethered to particle surfaces has a substantial impact on particle fate upon binding. Nanoparticles carrying angiotensin‐II tethered to highly mobile 5 kDa long poly(ethylene glycol) (PEG) chains separated by ligand‐free 2 kDa short PEG chains show a superior accumulation in angiotensin‐II receptor type 1 positive cells. In contrast, when ligand mobility is constrained by densely packing the nanoparticle surface with 5 kDa PEG chains only, cell uptake decreases by 50%. Remarkably, irrespective of ligand mobility and density both particle types have similar EC50 values in the 1–3 × 10^?9 m range. These findings demonstrate that ligand mobility on the nanoparticle corona is an indispensable attribute to be considered in particle design to achieve optimal cell uptake via multivalent interactions.     

Protein resistance of titanium oxide surfaces modified by biologically inspired mPEG-DOPA

In the present study, we have utilized X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry (ELM), and optical waveguide lightmode spectroscopy (OWLS) to examine the surface adsorption and protein resistance behavior of bio-inspired polymers consisting of poly(ethylene glycol) (PEG) conjugated to peptide mimics of mussel adhesive proteins. Peptides containing up to three residues of 3,4-dihydroxyphenylalanine (DOPA), a key component of mussel adhesive proteins, were conjugated to monomethoxy-terminated PEG polymers. These mPEG-DOPA polymers were found to be highly adhesive to TiO2 surfaces, with quantitative XPS analysis providing useful insight into the binding mechanism. Additionally, the antifouling properties of immobilized PEG were reflected in the excellent resistance of mPEG-DOPA-modified TiO2 surfaces to protein adsorption. Measurements of mPEG-DOPA and human serum adsorption were related in terms of ethylene glycol (EG) surface density and serum mass adsorbed and demonstrated a threshold of approximately 15-20 EG/nm2, above which substantially little protein adsorbs. With respect to surface density of adsorbed PEG and the associated nonfouling behavior of the adlayers, strong parallels exist between the nonfouling properties of the surface-bound mPEG-DOPA polymers and PEG polymers immobilized to surfaces using other approaches. Peptide anchors containing three DOPA residues resulted in PEG surface densities higher than those achieved using several existing PEG immobilization strategies, suggesting that peptide mimics of mussel adhesive proteins may be useful for achieving high densities of protein-resistant polymers on surfaces.