Formulation and Evaluation on Synergetic Anti-Hepatoma Effect of a Chemically Stable and Release-Controlled Nanoself-Assembly with Natural Monomers

Figures & data

Table 1 IC₅₀ and CI Values of HepG2 Treated with Drugs in Different Mass Ratios for 24 h

Treatment	IC50 (μg/mL)		CI
	GA	TAN
Free GA	35.37±5.71	—	—
Free TAN	—	27.17±5.65	—
Free GA:TAN (3:1)	19.49±5.18	6.49±1.72	0.790±0.21
Free GA:TAN (2.5:1.5)	13.11±1.13	7.87±0.67	0.660±0.06
Free GA:TAN (2:2)	6.38±0.4	6.38±0.4	0.415±0.03
Free GA:TAN (1.5:2.5)	7.82±1.75	13.04±2.92	0.701±0.16
Free GA:TAN (1:3)	5.08±0.31	15.25±0.93	0.704±0.04

Table 2 The Size, PDI, EE, DLC of the Nano-Self assemblies Before and After Freeze Drying (n=3)

Sample		Size	PDI	Zeta (mV)	EE%	LC%
Before freeze-drying	PD@TAN-Nanos	176.9±5.63	0.254±0.034	−19.7±0.52	57.32±0.46	7.61±0.13
	PD@GA-Nanos	163.4±7.32	0.261±0.014	−16.1±0.52	55.41±0.21	6.87±0.22
	PD@TG-Nanos	185.4±6.46	0.215±0.017	−21.9±1.01	GA:78.63±0.46	GA:6.48±0.36
					TAN:78.97±0.31	TAN:6.58±0.17
	DCA@TG-Nanos	203.1±8.21	0.253±0.031	−28.7±0.48	GA:86.86±1.28	GA:7.12±0.10
					TAN:73.84±2.46	TAN:6.05±0.20
After freeze-drying	PD@TAN-Nanos	181.5±7.91	0.243±0.022	–	54.14±0.53	7.21±0.13
	PD@GA-Nanos	169.7±11.22	0.252±0.011	–	52.58±0.17	6.34±0.19
	PD@TG-Nanos	193.7±7.97	0.227±0.019	–	GA:75.62±0.33	GA:6.13±0.47
					TAN:74.83±0.51	TAN:6.01±0.34
	DCA@TG-Nanos	216.3±12.59	0.261±0.016	–	GA:85.30±2.68	GA:7.05±0.22
					TAN:72.01±1.46	TAN:5.89±0.12

Figure 1 The particle size and morphology of four nanoself-assemblies. (A) PD@TAN-Nanos, (B) PD@GA-Nanos, (C) DCA@TG-Nanos, (D) PD@TG-Nanos.

Table 3 Prediction of Binding Energy

Compound1	Compound2	Entropy
GA	TAN	−4.01
GA	DCA	−8.15
TAN	DCA	−5.5

Figure 2 The DSC thermogram of nanoself-assemblies.

Figure 3 The results of physical stability test in vitro. (A) Dilution stability; (B) Storage stability at room temperature; (C) Storage stability at 4°C; (D) Plasma stability.

Figure 4 The results of chemical stability test in vitro. (A) The thermal stability of TAN in the different formulations, (B) The thermal stability of GA in the different formulations, (C) The photostability of TAN in the different formulations, (D) The photostability of GA in the different formulations, (E) The pH stability of TAN in the different formulations, (F) The pH stability of GA in the different formulations.

Figure 5 TAN release curves from the nanoself-assemblies (A); GA release curves from the nanoself-assemblies (B); In vitro cytotoxicity of the nanoself-assemblies on human normal liver cell line HL-7702 and human hepatoma cell line HepG2 for 24 h (C and E) and 48 h (D and F); The error bars was calculated from the data of three repetitions.

Figure 6 Tissue distributions of TAN (A) and GA (B) in the primary organs and tumor tissues after I.V. administration with the nanoself-assemblies at 15 mg/kg (n=10).

Table 4 Main Pharmacokinetic Parameters of TAN Injection and Its Preparations

Parameters		AUC0-∞ (mg/L·h)	MRT0-∞ (h)	Vz (L/kg)	CLz (L/h/kg)	T1/2 (h)
TAN solution		1.80±0.27	2.29±0.17	18.06±0.33	11.09±0.43	1.13±1.09
GA solution		1.62±0.16	2.18±0.20	17.85±0.14	12.34±0.08	1.00±0.24
DCA@TG-Nanos	TAN	4.58±0.89*	3.85±0.48*	17.08±0.39	4.37±0.74**	2.71±1.06*
	GA	6.36±1.15**	4.56±0.44*	18.19±0.21	3.14±0.51**	3.42±0.09*
PD@TAN-Nanos		7.32±0.17**Δ	4.72±0.08*Δ	9.74±0.15*	2.30±0.41**Δ	2.94±0.75*
PD@GA-Nanos		9.05±1.13**Δ	5.49±1.21*Δ	10.91±0.16*	2.21±0.26**Δ	4.01±0.12*Δ
PD@TG-Nanos	TAN	8.91±1.13**Δ	5.29±1.04*Δ	11.93±0.17*	2.56±0.24**Δ	3.03±0.56*Δ
	GA	10.52±0.14**Δ	5.69±0.91*Δ	11.49±1.02*	1.90±0.18**Δ	4.19±0.08*Δ

Notes: *Represents comparison with the drug solution, ∆ represents comparison with the DCA@TG-Nanos. *Or ∆ imply P<0.05, and **Or ∆∆ imply P<0.01.

Figure 7 (A) Mean plasma concentration vs time curves of various formulations after I.V. administration at 15 mg/kg. Data are expressed as the mean±s.d. (n=5). (B) Tumor growth inhibition curves (n=18), *P<0.05, **P<0.01, ***P<0.001. (C) Body weights of mice bearing H22 tumors after different treatments for 14 days (n=18), *P<0.05. (D) The curve of mice survival for 14 days.

Figure 8 H&E analyses of the main organs and tumor tissues after treatment with various formulations. The scale bar represents 250 μm in the H&E images.