Long-Lasting Exendin-4-Coated Gold Nanoparticles: Synthesis and In Vivo Evaluation of Hypoglycemic Activity

Abstract

Background: Gold nanoparticles (NPs) have drawn great attention in the area of biomedical research with their relatively safe and versatile properties. This study aimed to synthesize long-lasting exendin-4-coated gold NPs (EX-ABD-AFF-GoldNPs) and evaluate their anti-diabetic effects in vivo.

Methods: In the present study, EX-ABD-AFF-GoldNPs were synthesized using a simple one-step aqueous reduction method. The physical characterization of the prepared particles verified the successful formation of the EX-ABD-AFF-GoldNPs through dynamic light scattering (DLS), transmission electron microscopy (TEM), ultraviolet-visible (UV-VIS) light spectroscopy, and Fourier transform infrared spectroscopy (FTIR). The anti-hyperglycemic and anti-obesity effects were assessed in high-fat diet (HFD)-fed obese diabetic mice. Additionally, pharmacokinetics (PK) and biodistribution studies were performed to verify the long-lasting properties.

Results: The EX-ABD-AFF-GoldNPs were conglomerates of smaller globular-shaped particles, and the average size was 110(±14) nm, based on the TEM images. Safety assessments using Min6, HepG2, and B16F10 cell lines demonstrated low cytotoxicity, with over 80% cell viability up to the highest tested concentration of 150 μg/mL (as EX-ABD-AFF). Notably, the animal studies showed that the EX-ABD-AFF-GoldNPs exhibited significant hypoglycemic activity, comparable to the EX-ABD-AFF, in the HFD-fed mice. A 4-week treatment with EX-ABD-AFF-GoldNPs produced similar reductions in blood glucose and body weight to the EX-ABD-AFF, without any apparent toxicity. Furthermore, the PK and biodistribution study results confirmed the long-lasting properties (plasma half-life: 43.6 h) of the particles.

Conclusions: Overall, this study demonstrated that the preparation of therapeutic protein-loaded gold NPs is feasible and, despite their much larger size compared with the protein, EX-ABD-AFF-GoldNPs can be successfully absorbed through the subcutaneous route and show nearly equivalent hypoglycemic activity to the EX-ABD-AFF protein. Finally, this study showed that long-lasting properties could be acquired by only coating EX-ABD-AFF onto gold NPs.

Keywords: diabetes; exendin-4; gold; long-lasting; nanoparticle.

Figure 1

Physical characterization of EX-ABD-AFF-GoldNPs. (A) Image of the EX-ABD-AFF-GoldNP suspension. (B) Hydrodynamic size distribution and (C) size stability by DLS. (D) BIO-TEM image of EX-ABD-AFF-GoldNPs, (E) High-resolution transmission electron microscopic (HR-TEM) image of the particles and elemental distribution of carbon (C), nitrogen (N), and gold (Au) in the TEM particle images. (F) UV-VIS spectrum of EX-ABD-AFF-GoldNPs (EX-ABD-AFF-GoldNPs: EX-ABD-AFF-coated gold nanoparticles).

Figure 2

Fourier transform infrared (FT-IR) spectroscopic profiles of EX-ABD-AFF-GoldNPs.

Figure 3

Cytotoxicity profile of EX-ABD-AFF-GoldNPs for (A) Min6, (B) HepG2, and (C) B16F10 cells.

Figure 4

Glucose tolerance test (GTT) results in high-fat-diet-fed C57BL/6 obese mice. (A) Blood glucose level vs. time profile after subcutaneous injection of EX-ABD-AFF-GoldNPs and glucose. (B) Comparison of the area under the curves (AUCs). The statistically significant differences among the groups were compared by one-way ANOVA (Tukey’s multiple comparison test as the post hoc test). * p < 0.05, ** p < 0.01.

Figure 5

Hypoglycemic efficacy study results in high-fat-diet-fed C57BL/6 obese mice. (A) Blood glucose levels along the time after a single administration of EX-ABD-AFF-GoldNP. The mice’s blood glucose level profiles (B) and body weight changes (C) during the 4-week long-term efficacy study (twice-weekly administration of EX-ABD-AFF-GoldNPs). (D) Average fat weights of the mice after termination of the long-term study. The statistically significant differences among the groups were compared by one-way ANOVA (Tukey’s multiple comparison test as the post hoc test). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

PK and biodistribution profiles of EX-ABD-AFF-GoldNPs. (A) Plasma-concentration-versus-time profile of RITC-labeled EX-ABD-AFF-GoldNPs after subcutaneous injection in C57BL/6 mice (n = 3). (B) Representative mouse organ images after subcutaneous administration of RITC-labeled-EX-ABD-AFF-GoldNPs. (C) Fluorescent intensity profiles of RITC-labeled-EX-ABD-AFF-GoldNPs in major organs at days 1, 4, and 7 post-administration (RITC: rhodamine B isothiocyanate).