Leuko-polymersomes

Abstract

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.

Fig. 1

Overall goal of creating a functionalized polymersome to mimic a leukocyte (a leukopolymersome). Biotinylated sialyl-Lewis^x or biotinylated antibody against ICAM-1, or both, can be added to the polymersome shell to mediate adhesion. For clarity, the biotin on the vesicle or the ligands is not shown.

Fig. 2

Left: chemical scheme for biotinylatyion of 4F3 NB functionalized polymer. Upper right: giant biotinylated vesicles with NeutrAvidin, primary (biotinylated) antibody, and secondary antibody associated imaged using HDIC microscopy. Lower right: NeutrAvidin-coated vesicles labeled with Alexa Fluor® 488-biocytin show uniform labeling.

Fig. 3

Flow cytometry of vesicles bearing anti-ICAM-1. Negative controls were vesicles coated with NeutrAvidin, and then labeled with biotinylated goat anti-mouse IgG antibody (left column). Positive controls employed a biotinylated murine anti-human ICAM-1 (right column). A secondary FITC-rat anti-mouse IgG was then added. The vesicles were prepared with embedded porphyrin dimer. The first row indicates the FITC fluorescence plotted against the porphyrin fluorescence. The second row reports the histogram of FITC labeling for the negative (left) and positive (right) control. Both top and bottom row clearly indicate and increase in FITC fluorescence in the presence of the anti-ICAM-1, indicating polymersome labeling with this antibody.

Fig. 4

Displacement versus time for leukopolymersomes adhering to surfaces of different chemistry. Vesicles were prepared with a mixture of sLe^x and anti-ICAM-1 on their surface. At a shear rate of 100 s⁻¹, these vesicles were convected over surfaces with either P-selectin (pink) or P-selectin + ICAM-1 (all other colors). The vesicles flowing over P-selectin flow the fastest, on the order of 20 microns s⁻¹. All other vesicles flow more slowly with intermittent stopping, as mediated by adding ICAM-1 interactions to selectin-mediated interactions.

Fig. 5

Total particles interacting indicate the specificity of the adhesion measurement. Black bars indicate vesicles coated with only an antibody against ICAM-1, green bars indicate vesicles bearing only sLe^x, and red bars indicate vesicles bearing both ligands. These vesicles were convected over surfaces bearing only ICAM-1/Fc (left). Both ICAM-1/Fc and P-selectin/Fc (center) and P-selectin/Fc only (right). On surfaces bearing P-selectin/Fc, only vesicles bearing sLe^x showed strong interaction (right). On surfaces bearing only ICAM-1/Fc, the strongest interaction is displayed by vesicles with anti-ICAM-1 (left). On mixed surfaces, the strongest interaction is supported by vesicles bearing both ligands, consistent with the idea that both ligands are needed for superior adhesion (center).

Fig. 6

For sLe^x-bearing vesicles on P-selectin coated surfaces at 100 s⁻¹, rolling is observed, and the rolling velocity can be correlated with particle size. The different colors indicate different adhesive surface functionalization, either mixed ICAM-1/Fc + P-selectin/Fc (black) or P-selectin/Fc alone (red). While particles rolling on both surfaces average similar velocities (20 μm s⁻¹), particles rolling on the mixed surface roll faster and exhibit a stronger correlation between rolling velocity and particle diameter. This phenomena is a result of a lower density of P-selectin on the adhesive surface with which to form bonds with sLe^x on the vesicle surfaces.