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. 2015 Sep 18:10:5853-63.
doi: 10.2147/IJN.S88371. eCollection 2015.

Neurite outgrowth stimulatory effects of myco synthesized AuNPs from Hericium erinaceus (Bull.: Fr.) Pers. on pheochromocytoma (PC-12) cells

Jegadeesh Raman  1 , Hariprasath Lakshmanan  1 , Priscilla A John  2 , Chan Zhijian  3 , Vengadesh Periasamy  3 , Pamela David  4 , Murali Naidu  4 , Vikineswary Sabaratnam  2
Affiliations

Neurite outgrowth stimulatory effects of myco synthesized AuNPs from Hericium erinaceus (Bull.: Fr.) Pers. on pheochromocytoma (PC-12) cells

Jegadeesh Raman et al. Int J Nanomedicine. .

Abstract

Background: Hericium erinaceus has been reported to have a wide range of medicinal properties such as stimulation of neurite outgrowth, promotion of functional recovery of axonotmetic peroneal nerve injury, antioxidant, antihypertensive, and antidiabetic properties. In recent years, the green synthesis of gold nanoparticles (AuNPs) has attracted intense interest due to the potential use in biomedical applications. The aim of this study was to investigate the effects of AuNPs from aqueous extract of H. erinaceus on neurite outgrowth of rat pheochromocytoma (PC-12) cells.

Methods: The formation of AuNPs was characterized by UV-visible spectrum, energy dispersive X-ray (EDX), field-emission scanning electron microscope (FESEM), transmission electron microscopy (TEM), particle size distribution, and Fourier transform-infrared spectroscopy (FTIR). Furthermore, the neurite extension study of synthesized AuNPs was evaluated by in vitro assay.

Results: The AuNPs exhibited maximum absorbance between 510 and 600 nm in UV-visible spectrum. FESEM and TEM images showed the existence of nanoparticles with sizes of 20-40 nm. FTIR measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the nanoparticles. The purity and the crystalline properties were confirmed by EDX diffraction analysis, which showed strong signals with energy peaks in the range of 2-2.4 keV, indicating the existence of gold atoms. The synthesized AuNPs showed significant neurite extension on PC-12 cells. Nerve growth factor 50 ng/mL was used as a positive control. Treatment with different concentrations (nanograms) of AuNPs resulted in neuronal differentiation and neuronal elongation. AuNPs induced maximum neurite outgrowth of 13% at 600 ng/mL concentration.

Conclusion: In this study, the AuNPs synthesis was achieved by a simple, low-cost, and rapid bioreduction approach. AuNPs were shown to have potential neuronal differentiation and stimulated neurite outgrowth. The water-soluble bioconstituents could be responsible for the neuroactivity. This is the first report for the biosynthesis of AuNPs using the hot aqueous extract of H. erinaceus.

Keywords: AuNPs; Hericium erinaceus; PC-12; nanoparticles; neurite outgrowth.

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Figures

Figure 1
Figure 1
Absorption spectra of AuNPs after bioreduction by HAE of H. erinaceus. Abbreviations: AuNPs, gold nanoparticles; h, hours; au, absorbance units; H. erinaceus, Hericium erinaceus; HAE, hot aqueous extract.
Figure 2
Figure 2
Electron microscopic micrographs of AuNPs mycosynthesized by HAE of H. erinaceus. Notes: (A) TEM image of AuNPs solution formed by the reaction of gold (III) chloride with HAE of H. erinaceus for 36 hours. (B) Individual nanoparticles with clear lattice fringes through high-resolution TEM. (C) FESEM image of AuNPs. Abbreviations: H. erinaceus, Hericium erinaceus; TEM, transmission electron microscopy; AuNPs, gold nanoparticles; FESEM, field emission scanning electron microscopy; HAE, hot aqueous extract.
Figure 3
Figure 3
Size-distribution analysis by dynamic light scattering. Note: The particle size-distribution analysis revealed that particle size was approximately 28 nm. Abbreviation: d, diameter.
Figure 4
Figure 4
EDX image of AuNPs mycosynthesized by HAE of H. erinaceus. Abbreviations: EDX, energy dispersive X-ray; AuNPs, gold nanoparticles; H. erinaceus, Hericium erinaceus; HAE, hot aqueous extract.
Figure 5
Figure 5
FTIR image of AuNPs mycosynthesized by HAE of H. erinaceus. Notes: (A) Control: HAE of H. erinaceus, and (B) AuNPs mycosynthesized by HAE of H. erinaceus. Abbreviations: FTIR, Fourier transform-infrared spectroscopy; AuNPs, gold nanoparticles; H. erinaceus, Hericium erinaceus; HAE, hot aqueous extract.
Figure 6
Figure 6
Determination of monosaccharide and protein composition in HAE of H. erinaceus by TLC and SDS-PAGE methods. Notes: (A) TLC analysis of monosaccharide, (B) protein molecular markers, and (C) polypeptide profile of HAE on SDS-PAGE shows at least 14 different molecular mass proteins. Abbreviations: TLC, thin layer chromatography; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; H. erinaceus, Hericium erinaceus; HAE, hot aqueous extract.
Figure 7
Figure 7
Effects of AuNPs mycosynthesized by HAE of H. erinaceus on the neurite outgrowth on PC-12 cells. Notes: The percentage of neurite-bearing cells of PC-12 cells treated with various concentrations of AuNPs ranged from 200 to 2,000 ng/mL. Cells in complete F-12 K medium without AuNPs served as a negative control. Cells treated with 50 ng/mL of NGF or 50 μg/mL extract of aqueous extract of H. erinaceus served as positive controls. Data were expressed as means ± standard deviation of three experiments. *Significant difference (P<0.05). Abbreviations: AuNPs, gold nanoparticles; NGF, nerve growth factor; NEG, negative control; H. erinaceus, Hericium erinaceus; HAE, hot aqueous extract.
Figure 8
Figure 8
Phase-contrast micrographs of PC-12 neurites at day 3. Notes: (A) Treatment with 50 ng/mL NGF. (B) Treatment with 50 μg/mL of HAE of H. erinaceus. (C) Negative control (complete F-12 K medium). (D–F) Various concentrations of AuNPs ranged from 200 to 600 ng/mL. Scale bar =20 μm. Arrows indicate neurite extensions. Abbreviations: AuNPs, gold nanoparticles; NGF, nerve growth factor; H. erinaceus, Hericium erinaceus; HAE, hot aqueous extract.
Figure 9
Figure 9
Morphology of PC-12 cells in different treatment groups observed under fluorescent microscopy. Notes: (A) Treatment with 50 ng/mL NGF. (B) Treatment with 50 μg/mL of HAE of H. erinaceus. (C) Negative control, (complete F-12 K medium). (D–F) Various concentrations of AuNPs ranged from 200 to 600 ng/mL. Nuclei stained blue and neurofilaments stained green. Scale bar =40 μm. Arrows indicate neurite extensions. Abbreviations: AuNPs, gold nanoparticles; NGF, nerve growth factor; H. erinaceus, Hericium erinaceus; HAE, hot aqueous extract.
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