Kinetically controlled cellular interactions of polymer-polymer and polymer-liposome nanohybrid systems

Abstract

Although bioactive polymers such as cationic polymers have demonstrated potential as drug carriers and nonviral gene delivery vectors, high toxicity and uncontrolled, instantaneous cellular interactions of those vectors have hindered the successful implementation In Vivo. Fine control over the cellular interactions of a potential drug/gene delivery vector would be thus desirable. Herein, we have designed nanohybrid systems (100-150 nm in diameter) that combine the polycations with protective outer layers consisting of biodegradable polymeric nanoparticles (NPs) or liposomes. A commonly used polycation polyethylenimine (PEI) was employed after conjugation with rhodamine (RITC). The PEI-RITC conjugates were then encapsulated into (i) polymeric NPs made of either poly(lactide-co-glycolide) (PLGA) or poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA); or (ii) PEGylated liposomes, resulting in three nanohybrid systems. Through the nanohybridization, both cellular uptake and cytotoxicity of the nanohybrids were kinetically controlled. The cytotoxicity assay using MCF-7 cells revealed that liposome-based nanohybrids exhibited the least toxicity, followed by PEG-PLGA- and PLGA-based NPs after 24 h incubation. The different kinetics of cellular uptake was also observed, the liposome-based systems being the fastest and PLGA-based systems being the slowest. The results present a potential delivery platform with enhanced control over its biological interaction kinetics and passive targeting capability through size control.

Figure 1

Schematic diagram of preparation of the three nanohybrid systems used in this study.

Figure 2

Scanning Electron Microscopy (SEM) images of (A) PEI-RITC-encapsulated PEG-PLGA NPs and (B) PLGA NPs, scale bar = 100 nm. (C) A Transmission Electron Microscopy (TEM) image of PEI-RITC-encapsulated liposomes, scale bar = 100 nm.

Figure 3

Release profiles of PEI-RITC from the three nanohybrids in PBS buffer with pH 7.4 and acetate buffer with pH 4.0 at 37 °C. Little to no burst release was observed across all nanohybrids, with a sustained release profile up to 23 days. The overall release behavior was faster in pH 4.0 compared to pH 7.4 between the same type of nanohybrid formulation. The inset represents the zoomed-in release profiles for the first 2 days.

Figure 4

Cytotoxicity of PEI-RITC and the three nanohybrids after incubation with MCF-7 cells for a) 1 h, b) 4 h, c) 24 h, and d) 48 h. PEI-RITC exhibits cytotoxicity in a concentration and incubation time dependent manner whereas all nanohybrids show a marked decrease in cytotoxicity kinetics. After 48 h of treatment, all nanohybrids become comparatively toxic to PEI-RITC. * denotes statistical significance (p < 0.05) between PEI-RITC and the three nanohybrids, based on a 1-way ANOVA followed by Tukey’s post hoc test.

Figure 5

CLSM images of MCF-7 cells following treatment with PEI-RITC and the three nanohybrids (PEI-RITC-encapsulated liposomes, PEG-PLGA NPs, and PLGA NPs) all at a concentration of 0.5 µg/ml based on PEI-RITC for 1 h, 4 h, 24 h, and 48 h (red: PEI-RITC, blue: cell nuclei stained by DAPI, green: cellular membrane stained by WGA-AF 488; scale bar = 20 µm). Kinetic control over the internalization of PEI-RITC from the three nanohybrid formulations is demonstrated as cellular uptake was the second fastest for liposomes compared to unencapsulated PEI-RITC, followed by PEG-PLGA NPs, and lastly PLGA NPs. The liposome-based and PEG-PLGA NP-based nanohybrids start to internalize into the cells within 4 h, with complete uptake within 24 h. On the other hand, the PLGA NP-based nanohybrids do not interact with cell membranes until 4 h incubation and start to internalize into the cells from the 24 h incubation point.

Figure 6

Mean fluorescence following treatment of MCF-7 cells with PEI-RITC and the three nanohybrids at a concentration of 0.5 µg/ml based on PEI-RITC up to 48 h. Kinetic control over PEI-RITC internalization is further confirmed as unencapsulated PEI-RITC shows the fastest uptake, as indicated by having the highest fluorescence count compared to the three nanohybrid formulations. Cell binding and uptake of the nanohybrids occur in the order of liposomes, PEG-PLGA NPs, and PLGA NPs, which is consistent with the confocal data shown in Figure 5.