Biodegradable Polymersomes for the Delivery of Gemcitabine to Panc-1 Cells

Abstract

Traditional anticancer chemotherapy often displays toxic side effects, poor bioavailability, and a low therapeutic index. Targeting and controlled release of a chemotherapeutic agent can increase drug bioavailability, mitigate undesirable side effects, and increase the therapeutic index. Here we report a polymersome-based system to deliver gemcitabine to Panc-1 cells in vitro. The polymersomes were self-assembled from a biocompatible and completely biodegradable polymer, poly(ethylene oxide)-poly(caprolactone), PEO-PCL. We showed that we can encapsulate gemcitabine within stable 200 nm vesicles with a 10% loading efficiency. These vesicles displayed a controlled release of gemcitabine with 60% release after 2 days at physiological pH. Upon treatment of Panc-1 cells in vitro, vesicles were internalized as verified with fluorescently labeled polymersomes. Clonogenic assays to determine cell survival were performed by treating Panc-1 cells with varying concentrations of unencapsulated gemcitabine (FreeGem) and polymersome-encapsulated gemcitabine (PolyGem) for 48 hours. 1 μM PolyGem was equivalent in tumor cell toxicity to 1 μM FreeGem, with a one log cell kill observed. These studies suggest that further investigation on polymersome-based drug formulations is warranted for chemotherapy of pancreatic cancer.

Figure 1

Schematic representation of PolyGem. In aqueous solution, poly(ethylene oxide)-b-poly(caprolactone) (PEO-PCL) self-assemble into spherical polymer vesicles (polymersomes), with the hydrophobic PCL chains orienting end to end to form the bilayer. The figure represents a uniaxial cross section of the polymersome, with gemcitabine (⎔) encapsulated in the aqueous lumen. Vesicles can also be made to include PZn₃  (◯) in the hydrophobic membrane.

Figure 2

Cumulative in situ release of gemcitabine from PolyGem at pH 5.0 and 7.4 and 37°C, as measured via UV/Vis spectroscopy for 7 days. n = 3 for each data point and error bars represent standard deviation.

Figure 3

Cryo-TEM micrographs of PEO-PCL vesicles incubated for 12 hours. (a) pH 7.4. (b)–(d) pH 5.0. Arrows indicate areas of membrane degradation. Scale bar = 100 nm.

Figure 4

PolyGem internalization by Panc-1 cells. (a) Fluorescence intensity of PZn₃-polymersomes internalized by cells in well plates corresponding to 48 hour time point. (b) Concentration of PZn₃ uptake as a function of solution PZn₃ concentration (n = 3). Error bars indicate standard deviation. (c) CLSM z-stack images of Panc-1 cells incubated with PZn₃-polymersomes for 12 hours. Z-slices (Δz = 3 μm) are presented from left to right. Scale bar = 50 μm.

Figure 5

Panc-1 viability after 48 hour treatment with varying concentrations of FreeGem or PolyGem, as measured by a clonogenic assay. Error bars indicate standard deviation (n = 3). ∗P < 0.05.

Figure 6

Cell phenotype as visualized by DIC. (a) Media control, (b) 2.5 μM empty polymersomes, (c) 5 μM FreeGem, and (d) 5 μM PolyGem. Scale bar = 100 μm.