Preparation of hippurate-zinc layered hydroxide nanohybrid and its synergistic effect with tamoxifen on HepG2 cell lines

Figures & data

Figure 1 Powder x-ray diffraction patterns of (A) ZnO, (B) HAN, and (C) HA.

Abbreviations: ZnO, zinc oxide; HAN, hippuric acid nanohybrid; HA, hippuric acid.

Figure 2 Molecular structure of HA and three-dimensional molecular size of (A) HA and (B) spatial orientation of hippuric acid in ZLH inorganic interlayers.

Abbreviations: HA, hippuric acid; ZLH, zinc layered hydroxide.

Table 1 Fourier transform infrared assignments for HA and HAN25,27,30,31

Assignments	HA	HAN
ν (O–H) in the layer; H2O	–	3428
ν (N–H)	3342	–
	3072
ν (C–H)	2938	2930
ν (C=O) in COOH	1760
	1744	–
νas (COO^−)	–	1578–1551
νs (COO^−)	–	1412
δb (N–H)	1621
	1608
	1613
C–H deformation	1490	1492
δb (CH2)	1336	1297
	1318
	1304
ν (C–C)	1258	922
	943	833
	851
δb (CH), in plane bend	1180	1187
		1160
δr (CH2)	1079	1078
	1030	1030
	1000	1004
δb (COO^−)	–	717

Abbreviations: HA, hippuric acid; HAN, hippuric acid nanohybrid.

Figure 3 Fourier transform infrared spectra of (A) HA and (B) HAN.

Abbreviations: HA, hippuric acid; HAN, hippuric acid nanohybrid.

Figure 4 Thermogravimetric and differential thermogravimetric analyses of (A) HA and (B) HAN.

Abbreviations: HA, hippuric acid; HAN, hippuric acid nanohybrid.

Table 2 Physicochemical properties of ZnO and HAN

Sample	C%	H%	N%	Zn (%w/w)	Anion (%w/w)	BET surface area m^2/g	BJH pore volume cm^3/g	BET average pore diameter Å	BJH average pore diameter Å
ZnO	–	–	–	(80.3)	–	6	0.01	64	111
HAN	23.3	3.5	3.1	38.3b	38.7a	12	0.04	107	130

Notes: Values in parentheses are theoretical value.

a Estimated from CHNS analysis;

b estimated from ICP analysis.

Abbreviations: HA, hippuric acid; HAN, hippuric acid nanohybrid; ZnO, zinc oxide; BJH, Barret-Joyner-Halenda; BET, Brunauer, Emmett, and Teller.

Figure 5 Solid-state ultraviolet-visible spectra of (A) pure HA and its nanohybrid, HAN and (B) a Kubelka-Munk plot of HA, HAN, and ZnO.

Abbreviations: HA, hippuric acid; HAN, hippuric acid nanohybrid; ZnO, zinc oxide.

Figure 6 (A) Adsorption-desorption isotherms and (B) BJH pore size distribution for HAN and ZnO.

Abbreviations: BJH, Barret-Joyner-Halenda; HAN, hippuric acid nanohybrid; ZnO, zinc oxide.

Figure 7 Field emission scanning electron microscopic images of (A) ZnO with 50,000× magnification, (B) HAN with 25,000× magnification and (C) HAN at higher magnification 50,000×.

Abbreviations: ZnO, zinc oxide; HAN, hippuric acid nanohybrid.

Figure 8 Release profiles of HA from the HAN into (A) phosphate-buffered solution at pH 4.8 and (B) pH 7.4 and in (C) aqueous solution containing 0.08 M Na₂CO₃. Inset shows release profiles of physical mixture of HA with ZLH into phosphate-buffered solution at (D) pH 4.8 and (E) pH 7.4 and (F) aqueous solution of Na₂CO₃.

Abbreviations: HA, hippuric acid; HAN, hippuric acid nanohybrid; ZLH, zinc- layered hydroxide.

Table 3 Correlation coefficient (r2), rate constant (K), and half-time (t1/2) obtained by fitting the data of the release of HA from HAN into 0.08 M Na2CO3 and in phosphate-buffered solution at pH 7.4 and pH 4.8

Aqueous solution	Saturation release (%)	r^2			Rate constant (k)	t1/2 (hours)
		First-order	Pseudo-second order	Bhaskar equation
Na2CO3	95.7	0.9865	0.9907	0.9802	0.001 g mg^−1 · h^−1,a	9.35a
pH 7.4	56.4	0.9937	0.9252	0.9817	0.012 h^−1,b	57.8b
pH 4.8	99.8	0.7361	0.1298	0.8133	_	0.09c

a Notes: Estimated using pseudo-second order kinetics;

b estimated using first-order kinetics;

c estimated using the Bhaskar equation.

Figure 9 Fitting of the data for HA release from HAN into various solutions to the first-order, pseudo-second order kinetics and the Bhaskar equation for 0.08 M Na₂CO₃ (A and B), pH 7.4 (C, D, and G) and pH 4.8 (E, F, and H).

Abbreviations: HA, hippuric acid; HAN, hippuric acid nanohybrid.

Figure 10 MTT assays of HepG2 cells after 72 hours of treatment with combination of tamoxifen with HA (B), HAN (C), and ZLH (D). Inset shows MTT for free tamoxifen (A).

Abbreviations: Tam, tamoxifen; HA, hippuric acid; HAN, hippuric acid nanohybrid; ZLH, zinc-layered hydroxide.

Table 4 Untreated HepG2 cells during incubation with tamoxifen alone, and combination of tamoxifen with HA, HAN, and ZLH

Concentration of Tam (μg/mL)	Untreated cells (%)	Concentration of tam: HA or HAN or ZLH (μg/mL)	Untreated cells (%)
			HA	HAN	ZLH
20.0	25.1	20:20	20.8	11.9	16.4
10.0	37.1	10:10	35.7	17.7	33.2
5.0	38.5	5:5	35.5	19.0	34.4
2.5	48.6	2.5:2.5	44.8	23.9	44.7
1.25	55.0	1.25:1.25	54.0	27.7	55.0
0.625	74.1	0.625:0.625	72.1	33.3	73.3
0.3125	80.8	0.3125:0.3125	77.2	52.4	83.2
0.00	100.0	0:0	100.0	100.0	100.0

Abbreviations: Tam, tamoxifen; HA, hippuric acid; HAN, hippuric acid nanohybrid; ZLH, zinc-layered hydroxide.

Table 5 Antiproliferative assay of HepG2 cells after 24, 48 and 72 hours of treatment with free tamoxifen and combination of tamoxifen with HA and with HAN

Concentrations	×10^5 cell count in 24 hours	×10^5 cell count in 48 hours	×10^5 cell count in 72 hours
Untreated	5.6	12.3	21.2
5 μg Tam	4.9	5.2	7.5
5 μg Tam + 5 μg HA	4.9	5.1	7.2
5 μg Tam + 5 μg HAN	3.7	3.4	2.9

Abbreviations: Tam, tamoxifen; HA, hippuric acid; HAN, hippuric acid nanohybrid.

Figure 11 Antiproliferative assay of HepG2 cells after, 24, 48 and 72 hours of treatment with free tamoxifen and combination of tamoxifen with HA and with HAN.

Abbreviations: Tam, tamoxifen; HA, hippuric acid; HAN, hippuric acid nanohybrid.