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. 2016 Aug 31;11:4247-59.
doi: 10.2147/IJN.S113407. eCollection 2016.

Arginine-glycine-aspartic Acid-Polyethylene Glycol-Polyamidoamine Dendrimer Conjugate Improves Liver-Cell Aggregation and Function in 3-D Spheroid Culture

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Arginine-glycine-aspartic Acid-Polyethylene Glycol-Polyamidoamine Dendrimer Conjugate Improves Liver-Cell Aggregation and Function in 3-D Spheroid Culture

Zhanfei Chen et al. Int J Nanomedicine. .
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Abstract

The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system.

Keywords: ammonia metabolism; arginine–glycine–aspartic acid (RGD); dendrimer; liver cell; spheroid culture.

Figures

Figure 1
Figure 1
Synthesis and identification of RGD-PEG-PAMAM conjugates. Notes: +, RGD-PEG structure linking on the amino terminal of PAMAM. (A) Synthetic scheme for the synthesis of RGD-PEG-PAMAM conjugates. (B) NMR spectra of RGD-PEG-PAMAM, PEG-PAMAM, and PAMAM. The emergence of peptide peaks indicates coupling of peptides to PAMAM. Abbreviations: RGD, arginine–glycine–aspartic acid; PEG, polyethylene glycol; PAMAM, polyamidoamine; NMR, nuclear magnetic resonance; Fmoc, fluorenylmethyloxycarbonyl; Boc, butyloxycarbonyl; FITC, fluorescein isothiocyanate.
Figure 2
Figure 2
Effect of RGD-PEG-PAMAM on cell-spheroid formation. Notes: (A) Cytotoxicity assay on HepG2, Huh7, and 293A cells at various concentrations of RGD-PEG-PAMAM by CCK-8 assay. Data represent mean ± standard deviation (n=5) of cell viability (%). (B) Confocal microscopy showing the binding of RGD-PEG-PAMAM with Huh7 cells at 24 hours: representative fluorescent image of RGD-PEG-PAMAM (a); representative phase contrast image superimposed with DAPI staining of nuclei (blue) and fluorescein-labeled RGD-PEG-PAMAM (green) (b); incorporation of RGD-PEG-PAMAM conjugates in Huh7 cell-spheroid culture (RGD-PEG-PAMAM [c and e]; representative phase-contrast image superimposed with DAPI staining of nuclei [blue] and fluorescein-labeled RGD-PEG-PAMAM [green] [d and f]). Abbreviations: RGD, arginine–glycine–aspartic acid; PEG, polyethylene glycol; PAMAM, polyamidoamine; DAPI, 4′,6-diamidino-2-phenylindole.
Figure 3
Figure 3
RGD-PEG-PAMAM-induced accumulation of intracellular ROS levels. Notes: *P<0.05. (A) Fluorescence micrographs of intracellular ROS generation in Huh7 cells following 2 hours’ exposure to negative control, positive control (0.5 mg/mL loxapine), PAMAM (2 μM), PEG-PAMAM (100 μg/mL), and RGD-PEG-PAMAM (100 μg/mL). (B) Intracellular ROS levels were measured using DCFH-DA. The Huh7 and 293A cells were treated with PAMAM (2 μM) and different concentrations of PEG-PAMAM and RGD-PEG-PAMAM for 1, 2, 4, and 6 hours. Data expressed as percentage of positive control cells. Data presented as mean ± standard deviation (n=3). Abbreviations: RGD, arginine–glycine–aspartic acid; PEG, polyethylene glycol; PAMAM, polyamidoamine; ROS, reactive oxygen species; DCFH-DA, 2′,7′-dichlorodi-hydrofluorescin diacetate.
Figure 4
Figure 4
RGD-PEG-PAMAM promotes the proliferation of HepG2, Huh7, and 293A cells. Notes: *Statistically significant difference compared to HepG2, Huh7, and 293A cells treated with nothing (blank) at culture days 2, 3, 5, and 7 (P<0.05). (A) Cell-proliferation curves. (B) Incorporation of RGD-PEG-PAMAM conjugates in Huh7 spheroid-cell culture for 1, 2, and 3 days. The phase-contrast images show the Huh7 spheroid cells of the blank (treated with nothing, left pictures, ×200), PEG-PAMAM treatment (middle pictures, ×200), and RGD-PEG-PAMAM treatment (right pictures, ×400, from light to fluorescence). (C) Representative immunoblots from three independent studies for p-FAK, FAK, p-AKT, AKT, p-ERK, ERK, and β-actin in HepG2 and Huh7 cells treated with 100 μg/mL RGD-PEG-PAMAM for 0, 3, and 16 hours. Enhanced p-AKT and p-ERK expression was eliminated in both cell types when they were pretreated with 50 μM LY294002 for 1 hour. Abbreviations: RGD, arginine–glycine–aspartic acid; PEG, polyethylene glycol; PAMAM, polyamidoamine; OD, optical density.
Figure 5
Figure 5
Effects of RGD-PEG-PAMAM on the synthesis of albumin and urea in Huh7 cells. Notes: (A) Albumin level in the supernatant of different cells in cultivation for 1, 3, and 5 days. Group data represent mean ± standard deviation (n=3). *P<0.05 (n=3), **P<0.01 (n=3) compared to Huh7 cells treated with nothing (control). (B) Comparison of urea production. Data represent mean ± standard deviation (n=3) of urea production. *P<0.05 (n=3) compared to Huh7 cells treated with nothing or PEG-PAMAM. (C) Representative immunoblots from three independent studies for Arg1, OTC, and β-actin in Huh7 cells treated with 100 μg/mL RGD-PEG-PAMAM for 1, 3, and 5 days. The densitometry data were normalized to β-actin. *P<0.05 (n=3), **P<0.01 (n=3) compared to treatment for 1 or 5 days. (D) Representative immunoblots from three independent studies for Arg1, OTC, and β-actin in Huh7 cells treated with 100 μg/mL RGD-PEG-PAMAM, PEG-PAMAM, or nothing for 3 days. The densitometry data were normalized to β-actin. *P<0.05 (n=3), **P<0.01 (n=3) compared to treatment with PEG-PAMAM or nothing. Abbreviations: RGD, arginine–glycine–aspartic acid; PEG, polyethylene glycol; PAMAM, polyamidoamine.

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