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.
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 rate 7.5 times higher than 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.
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.
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 β 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 integrinindependent 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.
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