Anti-Metastatic Effect of Gold Nanoparticle-Conjugated Maclura tricuspidata Extract on Human Hepatocellular Carcinoma Cells

Figures & data

Table 1 Antioxidant Capability of MTR, MTS, MTL and MTF Extracts

Plant Parts	Total Phenolic (µg GAE/g)	Flavonoid (µg QE/g)	Reducing Power (µg AAE/g)	DPPH (%)	ABTS (%)
Root	328.0 ± 16.3	138.6 ± 10.1	12.4 ± 0.9	87.0 ± 4.3	89.3 ± 6.2
Stem	233.5 ± 15.8	117.8 ± 7.0	12.9 ± 1.9	84.4 ± 3.4	84.1 ± 5.6
Leaf	131.5 ± 13.9	110.3 ± 11.3	9.7 ± 1.4	77.2 ± 6.6	84.7 ± 6.9
Fruit	77.5 ± 14.0	117.0 ± 16.3	6.9 ± 1.5	47.4 ± 4.9	87.0 ± 4.3

Abbreviations: MTR, Maclura tricuspidata root; MTS, Maclura tricuspidata stem; MTL, Maclura tricuspidata leaves; MTF, Maclura tricuspidata fruits; GAE, gallic acid equivalent; QE, quercetin equivalent; AAE, ascorbic acid equivalent; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid).

Figure 1 The optimization of MTR-, MTS-, MTL-, and MTF-GNPs.

Notes: (A) UV-Vis spectra of MTR-, MTS-, MTL-, and MTF-GNPs. (B) The hydrodynamic sizes of MTR-, MTS-, MTL-, and MTF-GNPs were determined by DLS. (C) Zeta potential values of MTR-, MTS-, MTL-, and MTF-GNPs were measured by DLS.

Abbreviations: MTR-, MTS-, MTL-, and MTF-GNPs, Maclura tricuspidata root-, stem-, leaf-, and fruit-extract-gold nanoparticles, respectively; UV-Vis, ultraviolet-visible; DLS, dynamic light scattering.

Figure 2 Characterization of MTR-, MTS-, MTL-, and MTF-GNPs.

Notes: HR-TEM images at (A) low and (B) high magnification, (C) SAED patterns, (D) HAADF imaging, and (E) EDS analysis of MTR-, MTS-, MTL-, and MTF-GNPs.

Abbreviations: HR-TEM, high-resolution transmission electron microscopy; SAED, selected area electron diffraction; HAADF, High-angle annular dark field; EDS, energy dispersive spectroscopy; MTR-, MTS-, MTL-, and MTF-GNPs, Maclura tricuspidata root-, stem-, leaf-, and fruit-extract-gold nanoparticles, respectively.

Figure 3 Physicochemical characterization of MTR-, MTS-, MTL-, and MTF-GNPs.

Notes: (A) XRD pattern and (B) FTIR spectra of MTR-, MTS-, MTL-, and MTF-GNPs.

Abbreviations: XRD, X-ray diffraction; FT-IR, Fourier transform-infrared spectroscopy; MTR-, MTS-, MTL-, and MTF-GNPs, Maclura tricuspidata root-, stem-, leaf-, and fruit-extract-gold nanoparticles, respectively.

Figure 4 Photocatalytic properties of MTR-, MTS-, MTL-, and MTF-GNPs.

Notes: UV-Vis spectra of (A) methylene blue and (B) rhodamine B on addition of MTR-, MTS-, MTL-, and MTF-GNPs. Control is non-treated group.

Abbreviations: UV-Vis, ultraviolet-visible; MTR-, MTS-, MTL-, and MTF-GNPs, Maclura tricuspidata root-, stem-, leaf-, and fruit-extract-gold nanoparticles, respectively.

Table 2 Cell Viability of MTR, MTS, MTL and MTF-GNPs

(mg/mL)	HepG2 (%)			SK-Hep-1 (%)
	40	80	100	40	80	100
24 h
MTR-GNP	101.2 ± 0.3	87.9 ± 6.3	69.8 ± 3.1**	94.0 ± 2.2	91.2 ± 2.5	68.9 ± 2.0*
MTS-GNP	98.4 ± 4.5	90.2 ± 1.1	71.9 ± 6.2*	93.2 ± 1.1	83.1 ± 2.3	67.5 ± 2.2**
MTL-GNP	99.1 ± 0.7	100.5 ± 4.0	82.5 ± 2.6**	100.4 ± 1.0	97.2 ± 1.5	86.6 ± 1.5**
MTF-GNP	103.1 ± 0.9	97.5 ± 5.7	79.9 ± 4.7**	100.2 ± 2.2	93.4 ± 2.1	93.2 ± 2.0
48h
MTR-GNP	77.6 ± 1.5**	75.0 ± 5.4**	48.1 ± 4.7**	73.5 ± 2.5**	63.1 ± 2.0**	49.6 ± 3.7**
MTS-GNP	81.9 ± 2.2*	74.9 ± 1.9**	61.4 ± 6.2**	72.9 ± 3.1**	64.6 ± 0.7**	54.2 ± 2.3**
MTL-GNP	83.0 ± 1.5*	85.8 ± 3.3*	67.1 ± 2.5**	77.1 ± 2.2**	68.1 ± 2.3**	62.4 ± 1.9**
MTF-GNP	88.0 ± 0.8*	77.1 ± 2.6**	71.3 ± 2.2**	77.5 ± 2.6**	68.9 ± 2.5**	62.5 ± 2.0**
72h
MTR-GNP	61.5 ± 4.0**	55.6 ± 1.5**	27.6 ± 3.1**	63.9 ± 3.0**	54.1 ± 1.1**	34.0 ± 3.8**
MTS-GNP	53.6 ± 2.0**	40.0 ± 0.5**	31.9 ± 2.8**	66.7 ± 2.3**	38.9 ± 2.6**	34.2 ± 4.0**
MTL-GNP	61.0 ± 1.3**	44.7 ± 1.7**	42.9 ± 3.1**	64.5 ± 3.7**	60.2 ± 1.6**	42.5 ± 2.6**
MTF-GNP	65.7 ± 0.4**	59.9 ± 1.6**	52.4 ± 4.6**	62.8 ± 1.5**	62.9 ± 2.2**	53.9 ± 1.1**

Notes: Cell viability of MTR, MTS, MTL and MTF-GNPs were detected by CCK-8 assay. *p < 0.05 and **p < 0.01 compared to the control group.

Abbreviations: MTR-, MTS-, MTL- and MTF-GNPs, Maclura tricuspidata root-, stem-, leaves- and fruits-extract-gold nanoparticles.

Figure 5 Anti-migration and -invasion effects of MTR-, MTS-, MTL-, and MTF-GNPs.

Notes: (A) Cell migration was detected in HepG2 and SK-Hep-1 cells using the Radius™ 24-Well Cell Migration Assay kit. (B) Cell invasion was measured with the CytoSelect™ Cell Migration and Invasion assay kit. Control is non-treated group.

Abbreviations: MTR-, MTS-, MTL-, and MTF-GNPs, Maclura tricuspidata root-, stem-, leaf-, and fruit-extract-gold nanoparticles, respectively; TGF-β, transforming growth factor-beta.

Table 3 PLD Enzymatic Activity, MMP-9, MMP-9 Promoter Activity of MTR, MTS, MTL and MTF-GNPs

Cells	PLD Activity (% of Control)		MMP-9 Relative Luciferase Activity (Fold)		PLD-1 Relative Luciferase Activity (Fold)
	HepG2	SK-Hep-1	HepG2	SK-Hep-1	HepG2	SK-Hep-1
Control	100	100	1	1	1	1
TGF-β	271.7 ± 10.8	211.4 ± 12.1	3.5 ± 0.2	2.7 ± 0.1	5.5 ± 0.1	5.2 ± 0.5
MTR + TGF-β	208.3 ± 14.0*	163.9 ± 11.7**	2.7 ± 0.3	2.1 ± 0.1*	4.1 ± 0.3*	4.1 ± 0.4*
MTR-GNP + TGF-β	137.1 ± 6.68**	112.4 ± 2.9**	1.7 ± 0.1**	1.4 ± 0.1**	2.6 ± 0.4*	2.8 ± 0.2**
MTS + TGF-β	199.0 ± 14.6*	151.7 ± 9.7**	2.5 ± 0.2*	1.9 ± 0.2*	3.9 ± 0.4*	3.9 ± 0.2*
MTS-GNP + TGF-β	133.2 ± 8.0**	105.9 ±3.3**	1.6 ± 0.1**	1.4 ± 0.1**	2.9 ± 0.2*	2.4 ± 0.1**
MTL + TGF-β	223.5 ± 13.1*	171.0 ± 10.3**	2.9 ± 0.2	2.2 ± 0.2*	5.0 ± 0.2	4.3 ± 0.1
MTL-GNP + TGF-β	187.8 ± 7.4**	150.1 ± 5.8**	2.2 ± 0.1**	1.7 ± 0.2**	4.2 ± 0.2*	3.4 ± 0.2*
MTF + TGF-β	211.3 ± 11.7*	172.2 ± 7.6**	2.7 ± 0.3	2.4 ± 0.3	4.7 ± 0.2	4.2 ± 0.4
MTF-GNP + TGF-β	188.5 ± 5.8**	150.4 ± 4.6**	2.2 ± 0.1*	1.9 ± 0.2**	4.2 ± 0.1*	3.6 ± 0.5*

Notes: PLD enzymatic activity was determined by Amplex Red phospholipase D assay kit in HepG2 and SK-Hep-1 cells. *p < 0.05 and **p < 0.01 compared to the TGF-β group.

Abbreviations: TGF-β, transforming growth factor β; MMP-9, matrix metalloproteinases −9; PLD, phospholipase D; MTR-, MTS-, MTL- and MTF-GNPs, Maclura tricuspidata root-, stem-, leaves- and fruits-extract-gold nanoparticles.

Figure 6 Downregulation of MMP-9, PLD, and other EMT-related proteins by MTR-, MTS-, MTL-, and MTF-GNPs.

Notes: (A) MMP-9 enzymatic activity was detected by gelatin zymography. (B) Effect of MTR-, MTS-, MTL-, and MTF-GNPs on the expression of MMP-9 and PLD1 proteins. (C) Effect of MTR-, MTS-, MTL-, and MTF-GNPs on the protein expression of E-cadherin, N-cadherin, and vimentin. Control is non-treated group.

Abbreviations: MMP-9, matrix metalloproteinase-9; PLD, phospholipase D; TGF-β, transforming growth factor-beta; MTR-, MTS-, MTL-, and MTF-GNPs, Maclura tricuspidata root-, stem-, leaf-, and fruit-extract-gold nanoparticles, respectively.