Adhesion of antibody-functionalized polymersomes

Polymersomes are vesicles made from synthetic block copolymers. The adhesiveness of micron-sized polymersomes, functionalized with antibodies that bind to vascular cell adhesion molecules, which could be useful for vascular targeting, was measured. Intercellular adhesion molecule-1 (ICAM-1) is an endothelial cell adhesion molecule whose expression increases during inflammatory disease, and is therefore a natural target for vascular delivery. We functionalized polymersomes with an anti-ICAM-1 antibody, using modular biotin-avidin chemistry. Micropipet aspiration was used to confirm specific adhesion and measure the adhesion strength between an anti-ICAM-1-coated polymersome and an ICAM-1-coated polystyrene microsphere at various surface densities of adhesion molecules. The adhesion is kinetically trapped, and adhesion strength is quantified by the critical tension for detachment. The adhesion strength increases in proportion to the surface density of anti-ICAM-1 molecules, in contrast to results seen previously when measuring adhesion between biotinylated vesicles and avidin-coated beads (Lin et al. Langmuir 2004, 20, 5493). The difference in dependence on the density of functional groups is likely due to the molecular presentation at the vesicle surface; in the current study, the presentation of biotinylated anti-ICAM-1 on a layer of avidin leads to the effective presentation of the anti-ICAM-1 and, thus, a monotonic increase in adhesiveness with antibody density.     

The adhesion kinetics of sticky vesicles in tension: the distinction between spreading and receptor binding

We investigate the kinetics of spreading and adhesion between polymer vesicles decorated with avidin and biotin, held in micropipettes to maintain fixed tension and suppress membrane bending fluctuations. In this study, the density of avidin (actually Neutravidin) and biotin was varied, but was always sufficiently high so that lateral diffusion in the membrane was unimportant to the adhesive mechanism or rate. For a stunning result, we report a concentration-dependent distinction between adhesion and spreading: At low surface densities of avidin and biotin, irreversible vesicle adhesion is strong enough to break the membrane when vesicle separation is attempted, yet there is no spreading or "wetting". By this we mean that there is no development of an adhesion plaque beyond the initial radius of contact and there is no development of a meaningful contact angle. Conversely, at 30% functionalization and greater, membrane adhesion is manifest through a spreading process in which the vesicle held at lower tension partially engulfs the second vesicle, and the adhesion plaque grows, as does the contact angle. Generally, when spreading occurs, it starts abruptly, following a latent contact period whose duration decreases with increasing membrane functionality. A nucleation-type rate law describes the latency period, determined by competition between bending and sticking energy. The significance of this result is that, not only are membrane mechanics important to the development of adhesion in membranes of nanometer-scale thickness, mechanics can dominate and even mask adhesive features such as contact angle. This renders contact angle analyses inappropriate for some systems. The results also suggest that there exist large regions of parameter space where adhesive polymeric vesicles will behave qualitatively differently from their phospholipid counterparts. This motivates different strategies to design polymeric vesicles for applications such as targeted drug delivery and biomimetic scavengers.     

Adhesion plaque formation dynamics between polymer vesicles in the limit of highly concentrated binding sites

This work examines the process of adhesion plaque formation between pairs of copolymer vesicles presenting dense surface concentrations of avidin (NeutrAvidin) and biotin. Micropipet aspiration maintains constant membrane tension, as the low-tension vesicle membrane spreads over a second, more tensed vesicle. Spreading rates near 1 microm/s but as high as 7 microm/s (the adhesion plaque diameter) and contact angle growth rates of 2-14 deg/s are observed. The ultimate contact angles, in the range of 120-140 degrees, are independent of membrane tension and also exceed those previously reported. Adhesion plaque formation occurs in three phases: an initial step in which contact is established, typically lasting from a few seconds to a minute, an abrupt jump into contact in which both vesicles undergo substantial deformation, and a slower continued growth of the contact angle and area. Vesicle pairs are irreversibly bound at the plaque such that attempts to peel them apart cause membrane rupture at critical tensions as high as 4 mN/m, setting a lower bound on the interfacial strength. When the quantity tau(1 - cos theta) (with tau the membrane tension and theta the contact angle) is plotted as a function of time during plaque formation for different values of tau, the curves fail to collapse, indicating the chemical driving force for adhesion greatly exceeds the mechanical resisting tension.     

Designer Extracellular Matrix Based on DNA–Peptide Networks Generated by Polymerase Chain Reaction

Cell proliferation and differentiation in multicellular organisms are partially regulated by signaling from the extracellular matrix. The ability to mimic an extracellular matrix would allow particular cell types to be specifically recognized, which is central to tissue engineering. We present a new functional DNA‐based material with cell‐adhesion properties. It is generated by using covalently branched DNA as primers in PCR. These primers were functionalized by click chemistry with the cyclic peptide c(RGDfK), a peptide that is known to predominantly bind to αvβ3 integrins, which are found on endothelial cells and fibroblasts, for example. As a covalent coating of surfaces, this DNA‐based material shows cell‐repellent properties in its unfunctionalized state and gains adhesiveness towards specific target cells when functionalized with c(RGDfK). These cells remain viable and can be released under mild conditions by DNase I treatment.     

Ultrafast excited-state dynamics of nanoscale near-infrared emissive polymersomes

Formed through cooperative self-assembly of amphiphilic diblock copolymers and electronically conjugated porphyrinic near-infrared (NIR) fluorophores (NIRFs), NIR-emissive polymersomes (50 nm to 50 microm diameter polymer vesicles) define a family of organic-based, soft-matter structures that are ideally suited for deep-tissue optical imaging and sensitive diagnostic applications. Here, we describe magic angle and polarized pump-probe spectroscopic experiments that: (i) probe polymersome structure and NIRF organization and (ii) connect emitter structural properties and NIRF loading with vesicle emissive output at the nanoscale. Within polymersome membrane environments, long polymer chains constrain ethyne-bridged oligo(porphinato)zinc(II) based supermolecular fluorophore (PZn n ) conformeric populations and disperse these PZn n species within the hydrophobic bilayer. Ultrafast excited-state transient absorption and anisotropy dynamical studies of NIR-emissive polymersomes, in which the PZn n fluorophore loading per nanoscale vesicle is varied between 0.1-10 mol %, enable the exploration of concentration-dependent mechanisms for nonradiative excited-state decay. These experiments correlate fluorophore structure with its gross spatial arrangement within specific nanodomains of these nanoparticles and reveal how compartmentalization of fluorophores within reduced effective dispersion volumes impacts bulk photophysical properties. As these factors play key roles in determining the energy transfer dynamics between dispersed fluorophores, this work underscores that strategies that modulate fluorophore and polymer structure to optimize dispersion volume in bilayered nanoscale vesicular environments will further enhance the emissive properties of these sensitive nanoscale probes.     

Palladium(II)-mediated assembly of biotinylated ion channels

Simple synthetic methodology has been used to create biotinylated pyridyl cholate lipids that can undergo multiple self-assembly events when inserted into phospholipid vesicles; Pd(II) links cholates into transmembrane lipids, while avidin laterally clusters these complexes together and concomitantly assembles the vesicles into aggregates. The transmembrane assembly of cholates by Pd(II) "opened" the ion channels, whereas avidin addition produced vesicle aggregates, giving a system that mimicked both transmembrane transport and cellular adhesion. Complexation of these Pd(II)-linked cholates by avidin gave a measurable decrease in ion flow, suggesting some channels became blocked or were prevented from adopting the optimum geometry for ion conduction. This reflects the importance of spatially appropriate preorganisation when generating active supramolecular assemblies.     

Click‐chemistry of polymersomes on nanoporous polymeric surfaces

A facile click chemistry method of immobilizing surface‐functionalized polymer vesicles on casted polymeric PAN substrates is described. Microporous PAN membranes were subjected to hydrochloric acid hydrolysis to obtain surface carboxylates. The carboxylic groups were activated with EDC/NHS‐solution and were then reacted with propargylamine to introduce alkyne groups for CuAAC reactions. The alkyne functionality of the modified membrane surface was verified by reaction with an azide functional click dye both before and after the immobilization of azide‐functionalized ABA vesicles. The efficient postfunctionalization of the membrane with alkyne allowed quantitative coverage of the membrane surface with a polymersome monolayer, as confirmed by immobilization of polymerzomes loaded with a fluorescent dye. Polymersome monolayers immobilized on alkyne functionalized PAN‐membranes were characterized by cryo‐SEM and monolayers were confirmed by atom force microscopy. These methods opens up new avenues for preparing membrane based filtration and sensor technologies. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2032–2039     

Polymer vesicles with a colloidal armor of nanoparticles

The fabrication of polymer vesicles with a colloidal armor made from a variety of nanoparticles is demonstrated. In addition, it is shown that the armored supracolloidal structure can be postmodified through film-formation of soft polymer latex particles on the surface of the polymersome, hereby effectively wrapping the polymersome in a plastic bag, as well as through formation of a hydrogel by disintegrating an assembled polymer latex made from poly(ethyl acrylate-co-methacrylic acid) upon increasing the pH. Furthermore, ordering and packing patterns are briefly addressed with the aid of Monte Carlo simulations, including patterns observed when polymersomes are exposed to a binary mixture of colloids of different size.     

The Level of Hydrophobic Substitution and the Molecular Weight of Amphiphilic Poly-L-lysine-Based Polymers Strongly Affects Their Assembly into Polymeric Bilayer Vesicles

With the aim of producing new materials for drug and gene delivery, the variables associated with the preparation of poly-L-lysine-based vesicles were investigated. Amphiphilic poly-L-lysine graft copolymers with varying levels of grafted methoxypolyethylene glycol (mPEG) and palmitic acid were synthesized using two-step grafting reactions of the macromonomer, mPEG-p-nitrophenyl carbonate (mPEG, MW=5,000), and palmitic acid N-hydroxysuccinimide ester onto poly-L-lysine hydrobromide (MW=4,000 and 19,600). Polymers were characterized by gel permeation chromatography/light scattering. (1)H NMR, and an assay for unreacted varepsilon-amino groups. Polymeric unilamellar vesicles were produced by probe sonication of the amphiphilic poly-L-lysine-based polymers in the presence of cholesterol. Vesicles were characterized by electron microscopy and photon correlation spectroscopy. Vesicle formation was favored by a low molecular weight and a low level of palmitoyl substitution. A vesicle formation index has been derived, F ~ H/L DP, where H is the %molar level of unreacted L-lysine units, L is the %molar level of substituted palmitoyl units, and DP is the square root of the degree of polymerization of the polymer. Additionally, the size of these vesicles may be controlled by controlling the initial molecular weight of the parent poly-L-lysine/resulting amphiphilic polymer. Hence, amphiphilic poly-L-lysine-based polymers of molecular weight=89,000 and 25,000 produced polymeric vesicles of z-average mean diameter 570 nm and 252 nm, respectively. Vesicle encapsulation efficiency for the hydrophilic macromolecule, fluorescein isothiocyanate-dextran (MW=4,400), increased with vesicle size. Copyright 2001 Academic Press.     

Mechanical stability of micropipet-aspirated giant vesicles with fluid phase coexistence

Micropipet aspiration of phase-separated lipid bilayer vesicles can elucidate physicochemical aspects of membrane fluid phase coexistence. Recently, we investigated the composition dependence of line tension at the boundary between liquid-ordered and liquid-disordered phases of giant unilamellar vesicles obtained from ternary lipid mixtures using this approach. Here we examine mechanical equilibria and stability of dumbbell-shaped vesicles deformed by line tension. We present a relationship between the pipet aspiration pressure and the aspiration length in vesicles with two coexisting phases. Using a strikingly simple mechanical model for the free energy of the vesicle, we predict a relation that is in almost quantitative agreement with experiment. The model considers the vesicle free energy to be proportional to line tension and assumes that the vesicle volume, domain area fraction, and total area are conserved during aspiration. We also examine a mechanical instability encountered when releasing a vesicle from the pipet. We find that this releasing instability is observed within the framework of our model that predicts a change of the compressibility of a pipet-aspirated membrane cylinder from positive (i.e., stable) to negative (unstable) values, at the experimental instability. The model furthermore includes an aspiration instability that has also previously been experimentally described. Our method of studying micropipet-induced shape transitions in giant vesicles with fluid domains could be useful for investigating vesicle shape transitions modulated by bending stiffness and line tension.     

Microfluidic fabrication of monodisperse biocompatible and biodegradable polymersomes with controlled permeability

We describe a versatile technique for fabricating monodisperse polymersomes with biocompatible and biodegradable diblock copolymers for efficient encapsulation of actives. We use double emulsion as a template for the assembly of amphiphilic diblock copolymers into vesicle structures. These polymersomes can be used to encapsulate small hydrophilic solutes. When triggered by an osmotic shock, the polymersomes break and release the solutes, providing a simple and effective release mechanism. The technique can also be applied to diblock copolymers with different hydrophilic-to-hydrophobic block ratios, or mixtures of diblock copolymers and hydrophobic homopolymers. The ability to make polymer vesicles with copolymers of different block ratios and to incorporate different homopolymers into the polymersomes will allow the tuning of polymersome properties for specific technological applications.     

Real-time membrane fusion of giant polymer vesicles

Membrane fusion is very important for the formation of many complex organs in metazoans throughout evolution, such as muscles, bones, and placentae. Lipid vesicles (liposomes) are frequently used as model membranes to study the fusion process. This work demonstrates for the first time the real-time membrane fusion of giant polymer vesicles by directly displaying a series of high-resolution and real-time transformation images of individual vesicles. The fusion process includes the sequential steps of membrane contact, forming the center wall, symmetric expansion of fusion pore and complete fusion, undergoing the intermediates of "8" shape with a protruding rim at the contact site, peanut (pear) shape, and oblate sphere. The vesicle swells during fusion, and the fusing vesicle only deforms in the neck domain around the fusion pore in the lateral direction, which verifies the importance of the lateral tension on the fusion pore at the vesicle deformation level. The successful fusion of the synthetic and protein-free polymer vesicles reported here also supports that vesicle proximity combined with membrane perturbation suffices to induce membrane fusion, and that the protein is not necessary for the fusion process.     

The effect of receptor clustering on vesicle-vesicle adhesion

As part of our studies into how the localization of cell adhesion molecules into lipid rafts may affect cell adhesion, we developed Cu(1), a synthetic copper(iminodiacetate)-capped receptor able to phase separate from fluid phospholipid bilayers. The extent to which Cu(1) clustered into adhesive patches on the surface of vesicles could be controlled by changing vesicle composition. Extensive receptor phase separation significantly enhanced vesicle-vesicle adhesion; only vesicles with adhesive patches (blue fluorescence) adhered to their conjugate histidine-coated vesicles (red fluorescence) to form large vesicle aggregates (shown).     

A physical approach to specifically improve the mobility of alkane liquid drops

Seamless control of resistance to liquid drop movement for polar (water) and nonpolar alkane (n-hexadecane, n-dodecane, and n-decane) probe liquids on substrate surfaces was successfully demonstrated using molten linear poly(dimethylsiloxane) (PDMS) brush films with a range of different molecular weights (MWs). The ease of movement of liquid drops critically depended on polymer chain mobility as it relates to both polymer MW and solvent swelling on these chemically- and topographically identical surfaces. Our brush films therefore displayed lower resistances to liquid drop movement with decreasing polymer MW and surface tension of probe liquid as measured by contact angle (CA) hysteresis and tilt angle measurements. Subsequently, while mobility of water drops was inferior and became worse at higher MWs, n-decane drops were found to experience little resistance to movement on these polymer brush films. Calculating CA hysteresis as Δθ(cos) = cos θ(R) - cos θ(A) (θ(A) and θ(R) are the advancing and receding CAs, respectively) rather than the standard Δθ = θ(A) - θ(R) was found to be advantageous for estimation of the actual dynamic dewetting behavior of various probe liquids on an inclined substrate.     

Division and fusion, extension, adhesion, and retraction of vesicles created through Langmuir monolayer collapse: cell-like behavior

The motion of vesicles created through Langmuir monolayer collapse has been investigated. The vesicles grow only in a narrow molecular area range, and they exhibit remarkable, various biological cell-like behaviors such as division (cell division in cell biology, cytokinesis) and self-propulsion (motility). The vesicle division includes some dynamic modes: (i) an expulsion of a single satellite vesicle from an initial vesicle, (ii) a hierarchical and a sequential expulsion of a satellite vesicle, and (iii) a successive expulsion of two satellite vesicles from an initial vesicle. Two neighboring vesicles often show alternate fusion and division between them. Strong shape fluctuations dominate through vesicle division. The vesicles created exhibit distinct motions depending on the molecular area. At a large molecular area where most initial vesicles are created, they show a continuous, random motion on a few tens of micrometers length scale with a strong shape fluctuation and a constant velocity fluctuation profile. At a small molecular area they cease to move and shape fluctuations also become suppressed. At an intermediate molecular area there coexist vesicles with different dynamic modes: some vesicles show random motion similar to that at a large molecular area, but in a less fluctuating manner, while others exhibit a directional motion with an intermittent velocity jump. The directional motion is characterized by three distinct steps, i.e., extension, adhesion, and retraction. The characteristic motion is discussed from the viewpoint of haptotaxis, or the motion driven by adhesion gradients on the monolayer created by the local transfer of charged surfactant molecules between the vesicle and the monolayer, which the vesicle adheres to.     

Gold nanoparticle/polymer nanocomposites: dispersion of nanoparticles as a function of capping agent molecular weight and grafting density

The dispersion of polymer-covered gold nanoparticles in high molecular weight (MW) polymer matrixes is reported. Complete particle dispersion was achieved for PS125-Au in the polystyrene (PS) matrixes studied (up to and including Mn = 80 000 g/mol). PS19-Au, on the other hand, exhibits complete dispersion in a low MW PS matrix (Mn = 2000 g/mol) but only partial dispersion in higher MW matrixes (up to 80 000 g/mol). Similarly, PEO45-Au is fully dispersed in a low MW poly(ethylene oxide) (PEO) matrix (Mn = 1000 g/mol) but only partially in a higher MW PEO matrix (Mn = 15 000 g/mol). Wetting of the polymer-Au brushes by the polymer matrix is associated with dispersibility. Theory predicts that, for dense polymer brushes, wetting is achieved when the MW of the polymer brush equals (and is greater than) that of the polymer matrix. The observed partial dispersion of the PS19-Au and PEO45-Au nanoparticles in matrixes whose MW is greater than the brush MW is attributable to the existence of a high volume fraction of voids within the brush. These voids arise from the unique geometry of the nanoparticle surface arising from the juxtaposed facets of the gold nanoparticle. PS125-Au brushes are wetted by PS matrixes whose degree of polymerization is larger than 125, probably because of their lower grafting density on the gold core or the high fraction of void volumes caused by the facets on the gold cores. Dispersion thus occurs when the matrix MW is greater than that of the brush.     

Biodegradable Polypeptide-based Vesicles with Intrinsic Blue Fluorescence for Antibacterial Visualization

Fluorescence imaging has been an indispensable tool to provide dynamic information about the localization and quantity of organisms.Meanwhile,due to the intrinsic hollow structure and modularized biofunctionalities,polymer vesicles have been widely applied in biomedical field.However,most polymer vesicles are embedded with organic fluorophores for fluorescence imaging,which have certain drawbacks such as leakage and possible cytotoxicity.Here,we present a biodegradable polypeptide-based vesicle with intrinsic blue fluorescence without introducing any fluorophore for real-time visualization of antibacterial process.Through modular design to integrate multiple functional fragments,poly(ε-caprolactone)-block-poly(tryptophan)-block-poly(lysine-stat-phenylalanine)[PCL25-b-PTrP2-b-P(Lys13-stat-Phe4)]was synthesized,where PCL chains form the hydrophobic membrane,P(Lys-stat-Phe) and PTrp provide intrinsic fluorescence and broad-spectrum antibacterial activity.It is noteworthy that the fluorescence emission was shifted from invisible ultraviolet range of amino acids to visible range (emission maximum at 436 nm),which makes it possible to visualize the antibacterial process.In addition,through utilizing the intrinsic fluorescence of vesicles,confocal fluorescent imaging of vesicles with bacteria validated the specific adhesion of vesicle towards bacteria,and the bacterial death through membrane disruption.Overall,we provided a novel approach to developing biodegradable fluorescent polypeptide-based vesicles for real-time visualization of antibacterial process.     

Determination of the line tension of giant vesicles from pore-closing dynamics

Giant vesicles generated from synthetic and natural lipids such as phosphatidylcholines are useful models for understanding mechanical properties of cell membranes. Line tension is the one-dimensional force enabling the closing of transient pores on cell membranes. Transient pores were repeatedly and reproducibly formed on the membrane edge of giant vesicles generated from synthetic and natural phosphatidylcholines employing a nitrogen-pumped coumarin dye laser (440 nm). Line tension was determined at room temperature from closing of these pores that occurred over several seconds when the radius of the vesicle could be considered to be constant. The value of line tension depends on the nature of the lipid for single lipid systems, which, at room temperature, yielded a vesicle bilayer region in the gel, fluid, or mixed gel and fluid phases. The line tension for vesicles generated from phosphatidylcholines with saturated acyl chains of lengths of 12-18 carbon atoms ranges from 1 to 12 pN, exhibiting an increase with chain length. Vesicles generated from the natural Egg-PC, which is a mixture of lipids, are devoid of phase transition and exhibited the largest value of line tension (32 pN). This value is much larger than that estimated from the line tensions of vesicles obtained from lipids with homologous acyl chains. This study, to our knowledge, is the first to employ laser ablation to generate transient pores and determine line tension from the rate of pore closure and demonstrate a relationship between line tension and acyl chain length.     

Probing ligand-protein recognition with sum-frequency generation spectroscopy: the avidin-biocytin case.

Infrared/visible sum-frequency generation (SFG) spectroscopy is used to study the recognition of a protein (avidin) by a derived vitamin (biocytin) adsorbed on a calcium fluoride substrate. The specificity of the process is tested by replacing avidin with bovine serum albumin or presaturated avidin. The SFG spectroscopy shows drastic modifications in the CH and NH spectral ranges only upon exposure of the biocytin film to avidin. The comparison of the SFG data with Fourier transform infrared reflection absorption spectra (FT-IRRAS) in the same spectral ranges illustrates the advantages of nonlinear spectroscopy for studying and detecting recognition between biomolecules.     

Surface tension and aggregation properties of novel cationic gemini surfactants with diethylammonium headgroups and a diamido spacer

A series of novel cationic gemini surfactants with diethylammonium headgroups and a diamido spacer were synthesized, and their surface and bulk properties were investigated by surface tension, electrical conductivity, fluorescence, viscosity, dynamic light scattering (DLS), and transmission electron microscopy (TEM) measurements. An interesting phenomenon, that is, the obvious decline in surface tension upon increasing concentration above the critical micelle concentration (cmc), was found in these gemini surfactant solutions, and two explanations were proposed. This surface tension behavior could be explained by the rapid increase in the counterion activity in the bulk phase or the continued filling of the interface with increasing surfactant concentration above the cmc. More interestingly, not only vesicles but also the surfactant-concentration-induced vesicle to larger aggregate (spongelike aggregate) transition and the salt-induced vesicle and spongelike aggregate to micelle transition were found in the aqueous solutions of these gemini surfactants. The spongelike aggregate that is first reported in the cationic gemini surfactant-water binary system is probably caused by the adhesion and fusion of vesicles at high surfactant concentration.