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. 2013 Jul;13(7):913-20.
doi: 10.1002/mabi.201300030. Epub 2013 Apr 30.

Caveolae-mediated endocytosis of conjugated polymer nanoparticles

Junghan Lee  1 , Megan Twomey, Christian Machado, Giselle Gomez, Mona Doshi, Andre J Gesquiere, Joong Ho Moon
Affiliations

Caveolae-mediated endocytosis of conjugated polymer nanoparticles

Junghan Lee et al. Macromol Biosci. 2013 Jul.

Abstract

Understanding the cellular entry pathways of synthetic biomaterials is highly important to improve overall labeling and delivery efficiency. Herein, cellular entry mechanisms of conjugated polymer nanoparticles (CPNs) are presented. CPNs are intrinsic fluorescent materials used for various biological applications. While CPNs cause no toxicity, decreased CPN uptake is observed from cancer cells pretreated with genistein, which is an inhibitor of caveolae-mediated endocytosis (CvME). CvME is further confirmed by high co-localization with caveolin-1 proteins found in the caveolae and caveosomes. Excellent photophysical properties, non-toxicity, and non-destructive delivery pathways support that CPNs are promising multifunctional carriers minimizing degradation of contents during delivery.

Keywords: conjugated polyelectrolytes; conjugated polymer nanoparticles; conjugated polymers; endocytosis mechanism; small interfering RNA delivery.

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Figures

Figure 1
Figure 1
Atomic force microscopic images of CPNs (inset: image taken at higher magnification) on a freshly-cut mica surface (a), a lateral size analysis histogram (b), and a height histogram (c).
Figure 2
Figure 2
Cellular toxicity of CPNs determined by WST-1 assay (a) and cellular entry kinetics of CPNs after 2 h incubation (b). Trypan blue treatment reduces fluorescence intensity [before (■) and after (○)] by quenching CPNs adsorbed on extracellular membranes.
Figure 3
Figure 3
Energy independent cellular entry of CPNs (a) and endocytosis inhibition of CPNs (20 μM) by pharmacological inhibitors (b). The relative amount of CPNs in HeLa cells in the absence (2) and presence (3–7) of pharmacological inhibitors. (1) HeLa cell control, (2) CPNs only, (3) chlorpromazine (CME inhibitor, 24 μM), (4) genistein (CvME inhibitor, 210μM), (5) methyl-β-cyclodextrin (Caveolae or macropinocytosis inhibitor, 1000 μM), (6) LY294002 (macropinocytosis inhibitor, 120 μM), and (7) cytochalasin D (macropinocytosis inhibitor, 0.04 μM). CPN uptake was significantly inhibited by genistein treatment, implying that CPNs’ entry is related to CvME. CPNs use both energy dependent and independent pathways to enter cells. Error bar represents standard deviation [n=6 for (a) and n=4 for (b)].*p < 0.05.
Figure 4
Figure 4
Fluorescence microscopic images of HeLa cell incubated with CPNs in the presence (a) and absence (b) of genistein. Nucleus stained with a blue dye and CPNs were seen as green dots. CPN uptake was decreased when the cells pretreated with genistein. Scale bar: 10 μm.
Figure 5
Figure 5
Colocalization (appeared as yellow spots in the merged images) of CPNs with (a) LysoTracker Red DND for lysosomes and (b) BODIPY-TR C5-ceramide-BSA complex for golgi staining, respectively, in Hela cells after 24 h CPN incubation.
Figure 6
Figure 6
Fluorescence microscopic images of HeLa cells incubated with CPNs (green) for 24 h followed by immunostaining with caveolin-1 antibodies (red). CPNs overlap with caveolin-1 throughout cytoplasm. Nucleus was stained with a blue dye.
Scheme 1
Scheme 1
Synthetic scheme of N-Boc protected poly(phenyleneethynylene)
See this image and copyright information in PMC

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