Effects of Membrane Rheology on Leuko-polymersome Adhesion to Inflammatory Ligands.

A strategy for treating inflammatory disease is to create micro-particles with the adhesive properties of leukocytes. The underlying rheology of deformable adhesive microspheres would be an important factor in the adhesive performance of such particles. In this work the effect of particle deformability on the selectin-mediated rolling of polymer vesicles (polymersomes) is evaluated. The rheology of the polymersome membrane was modulated by cross-linking unsaturated side-chains within the hydrophobic core of the membrane. Increased membrane rigidity resulted in decreased rates of particle recruitment rather than decreased average rolling velocities. Reflective interference contrast microscopy of rolling vesicles confirmed that neither flaccid nor rigid vesicles sustained close contacts with the substrate during rolling adhesion. A variable-shear rate parallel-plate flow chamber was employed to evaluate individual vesicles rolling on substrates under different flow conditions. Analysis of the trajectories of single flaccid vesicles revealed several distinct populations of rolling vesicles; however, some of these populations disappear when the vesicle membranes are made rigid. This work shows that membrane mechanics affects the capture, but not the rolling dynamics, of adherent leuko-polymersomes.

A Generalized System for Photo-Responsive Membrane Rupture in Polymersomes.

Polymersomes are vesicles whose membranes are comprised of self-assembled block co-polymers. We recently showed that co-encapsulating conjugated multi-porphyrin dyes in a polymersome membrane with ferritin protein in the aqueous lumen confers photo-lability to the polymersome. In the present study, we illustrate that the photo-lability can be extended to vesicles containing dextran, an inert and inexpensive polysaccharide, as the luminal solute. Here we explore how structural features of the polymersome/porphyrin/dextran composite affect its photo-response. Increasing dextran molecular weight, decreasing block copolymer molecular weight, and altering fluorophore-membrane interactions results in increasing the photo-responsiveness of the polymersomes. Amphiphilic interactions of the luminal encapsulant with the membrane coupled with localized heat production in the hydrophobic bilayer likely cause differential thermal expansion in the membrane and the subsequent membrane rupture. This study suggests a general approach to impart photo-responsiveness to any biomimetic vesicle system without chemical modification, as well as a simple, bio-inert method for constructing photo-sensitive carriers for controlled release of encapsulants.

Tunable leuko-polymersomes that adhere specifically to inflammatory markers.

The polymersome, a fully synthetic cell mimetic, is a tunable platform for drug delivery vehicles to detect and treat disease (theranostics). Here, we design a leuko-polymersome, a polymersome with the adhesive properties of leukocytes, which can effectively bind to inflammatory sites under flow. We hypothesize that optimal leukocyte adhesion can be recreated with ligands that mimic receptors of the two major leukocyte molecular adhesion pathways, the selectins and the integrins. Polymersomes functionalized with sialyl Lewis X and an antibody against ICAM-1 adhere avidly and selectively to surfaces coated with inflammatory adhesion molecules P-selectin and ICAM-1 under flow. We find that maximal adhesion occurs at intermediate densities of both sialyl Lewis X and anti-ICAM-1, owing to synergistic binding effects between the two ligands. Leuko-polymersomes bearing these two receptor mimetics adhere under physiological shear rates to inflamed endothelium in an in vitro flow chamber at a rate 7.5 times higher than those to uninflamed endothelium. This work clearly demonstrates that polymersomes bearing only a single ligand bind less avidly and with lower selectivity, thus suggesting proper mimicry of leukocyte adhesion requires contributions from both pathways. This work establishes a basis for the design of polymersomes for targeted drug delivery in inflammation.

Photoinitiated destruction of composite porphyrin-protein polymersomes.

Bilayer vesicles assembled from amphiphilic diblock copolymers (polymersomes) adopt asymmetric structures when loaded with moderate concentrations (>or=1.5 mg/mL) of horse spleen ferritin (HSF) or its iron-free variant (HSAF). Incorporation of both ferritin and a zinc porphyrin dimer (PZn(2)) generates photoresponsive vesicles: irradiation with focused light of near-UV to near-IR wavelengths induces polymersome deformation and destruction on the minute time scale. To investigate this phenomenon, polymersomes were loaded with dye-labeled ferritin and PZn(2). Confocal microscopy identified BODIPY-FL-labeled ferritin at the membrane, whereas Cy3-labeled ferritin was found both at the membrane and throughout the aqueous core. Fluorescence recovery after photobleaching (FRAP) experiments confirmed that Cy3- and BODIPY-FL-labeled ferritin and PZn(2) exhibited slow diffusion at the membrane, consistent with membrane association. Furthermore, micropipette aspiration experiments revealed increased elastic moduli and altered bending rigidity in vesicles incorporating HSAF. Finally, a small molecule (biocytin) was encapsulated within the ferritin-PZn(2) vesicles and released upon exposure to light. These data indicate synergy between ferritin, whose membrane association lowers the barrier to deformation, and PZn(2), which embeds in the membrane, harvests light energy and produces local heating that may lead to membrane budding. This appears to be a general protein-polymer membrane phenomenon, as replacement of ferritin with bovine serum albumin or equine skeletal myoglobin resulted in vesicles with similar asymmetric morphology and photosensitivity.

Isolation of αL I domain mutants mediating firm cell adhesion using a novel flow-based sorting method.

The inserted (I) domain of αLß2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure-function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context.

Neutrophil adhesion and chemotaxis depend on substrate mechanics.

Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma, and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micro-machined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K(D) of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against beta(2)-integrins leads to a significant reduction but not an elimination of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

Two-photon microscopy of oxygen: polymersomes as probe carrier vehicles.

Oxygen concentration distributions in biological systems can be imaged by the phosphorescence quenching method in combination with two-photon laser scanning microscopy. In this paper, we identified the excitation regime in which the signal of a two-photon-enhanced phosphorescent probe (Finikova, O. S.; Lebedev, A. Y.; Aprelev, A.; Troxler, T.; Gao, F.; Garnacho, C.; Muro, S.; Hochstrasser, R. M.; Vinogradov, S. A. ChemPhysChem 2008, 9, 1673-1679) is dependent quadratically on the excitation power (quadratic regime), and performed simulations that relate the photophysical properties of the probe to the imaging resolution. Further, we characterized polymersomes as a method of probe encapsulation and delivery. Photophysical and oxygen sensing properties of the probe were found unchanged when the probe is encapsulated in polymersomes. Polymersomes were found capable of sustaining high probe concentrations, thereby serving to improve the signal-to-noise ratios for oxygen detection compared to the previously employed probe delivery methods. Imaging of polymersomes loaded with the probe was used as a test-bed for a new method.

Leuko-polymersomes.

Polymersomes are vesicles whose membranes are comprised of self-assembled amphiphilic block co-polymers. Synthetic control of block co-polymer chemistry provides an advantageous diversity of polymersome functions, ranging from tunable materials strength, superior encaspulation of hydrophobic and hydrophilic drugs and optical dyes, and facile functionalization. We have exploited polymersome tunability to make leuko-polymersomes: polymersomes with the adhesive properties of leukocytes. By functionalizing the terminal groups on the outer shell of the vesicle with biotin, we have used modular avidin-biotin chemistry to attach adhesion ligands that mimic the two critical adhesion pathways that leukocytes utilize to achieve adhesion in the fast fluid flow of blood vessels--selectins and integrins. We demonstrate that adhesion is specific and is supported at hydrodynamic flow rates at which leukocytes adhere. We envision the use of such particles for monitoring or treating inflammation, cancer and cardiovascular disease.