Shape dependent cytotoxicity of PLGA-PEG nanoparticles on human cells

Abstract

We investigated the influence of nanoparticles' shape on the physiological responses of cells, when they were fed with spherical and needle-shaped PLGA-PEG nanoparticles (the volume of the nanoparticles had been chosen as the fixed parameter). We found that both types of NPs entered cells via endocytosis and upon internalization they stayed in membrane bounded vesicles. Needle-shaped, but not the spherical-shaped NPs were found to induce significant cytotoxicity in the cell lines tested. Our study evidenced that the cytotoxicity of needle-shaped NPs was induced through the lysosome disruption. Lysosome damage activated the signaling pathways for cell apoptosis, and eventually caused DNA fragmentation and cell death. The present work showed that physiological response of the cells can be very different when the shape of the fed nanoparticles changed from spherical to needle-like. The finding suggests that the toxicity of nanomaterials also depends on their shape.

Figure 1

PLGA-PEG NPs with different shapes. SEM images of (a) spherical-shaped and (b) needle-shaped PLGA-PEG NPs, insert are the magnified TEM images; (c,d) Corresponding confocal images showing the fluorescence coming from incorporated Nile Red dye.

Figure 2

Chemical characterization of PLGA-PEG NPs. (a) 1H-NMR spectrum of PLGA-PEG copolymer showing their chemical composition, (b) Typical FTIR spectrum of PLGA, PEG, and PLGA-PEG NPs (both spherical and needle-shaped).

Figure 3

Shape dependent PLGA-PEG NPs intracellular distribution. Confocal images of cellular uptake of (a) spherical- and (b) needle-shaped NPs at 50 µg/mL by HepG2 cells for 24 hours. (Green: Lysosomes, Red: NPs, Yellow: overlaid. Scale bar: 10 µm).

Figure 4

The effects of PLGA-PEG NPs’ shape on cell morphology. TEM images of the morphologies of HepG2 cells after their being fed with (a) none (control sample), (b) spherical-, and (c) needle-shaped NPs at 250 µg/mL for 24 hours. (Scale bar: 2 µm).

Figure 5

Shape dependent PLGA-PEG NPs’ cytotoxicity. (a) LDH release (normalized to lysed control cell), (b) Caspase 3 activity and (c) MTT assay after HepG2 cells were fed with spherical- and needle-shaped PLGA-PEG NPs.

Figure 6

Needle-shaped PLGA-PEG NPs induced DNA fragmentation by TUNEL assay. Confocal images (merged with transmitted channel) of HepG2 cells (stained with TUNEL Red) after their being fed with (a) & (b) none, (negative and positive control), (c) spherical- and (d) needle-shaped NPs at 250 µg/mL for 24 hours. (Red: TUNEL. All the figures share the same scale bar).

Figure 7

Schematic representation of the proposed cytotoxicity pathways for PLGA-PEG NPs with different morphological features. Upper panel: A spherical-shaped PLGA NP entered the cell via endocytosis, stably resided in the lysosomes, and had no apparent cytotoxicity. Lower panel: A needle-shaped PLGA NP entered the cell via endocytosis, resided in lysosomes and induced lysosomal membrane perturbation, caused caspase-3 activation and DNA damage, and finally apoptosis.