Enhanced anti-glioma activity of annonaceous acetogenins based on a novel liposomal co-delivery system with ginsenoside Rh2

Figures & data

Scheme 1. Schematic illustration of a novel liposomal co-delivery system with ginsenoside Rh2 for enhancing anti-glioma activity and attenuate systemic toxicity (all details on the picture were created by the author Hui Ao).

Table 1. Optimization of the preparation method of ACGs-Lipo (n = 3, mean ± SD).

Preparation method	Aqueous phase	Average particle size (nm)	PDI
Thin-film dispersion	PBS (pH 7.4)	98.5 ± 2.1	0.33 ± 0.00
Ultrasonic-dripping	PBS (pH 7.4)	159.0 ± 0.1	0.17 ± 0.03
Stirring-dropping	PBS (pH 7.4)	97.7 ± 1.3	0.20 ± 0.02
Stirring-dropping	Deionized water (pH 6.0)	93.1 ± 0.5	0.11 ± 0.03

Figure 1. Preparation of liposomes. The optimal preparation process of ACGs-Lipo (A) and (ACGs + Rh2)-Lipo (B). (C) The appearance of (ACGs + Rh2)-Lipo at different concentrations of phospholipid (from left to right: 8 mg/mL, 10 mg/mL, 12 mg/mL, 15 mg/mL, and 18 mg/mL). All details on A and B were created by the author Hui Ao.

Table 3. Optimization of the concentration of phospholipid in the preparation of (ACGs + Rh2)-Lipo (n = 3, mean ± SD).

Concentration of phospholipid (mg/mL)	Average particle size (nm)	PDI	Zeta potential (mV)
8	289.5 ± 5.3	0.24 ± 0.00	−2.9 ± 0.8
10	112.6 ± 0.0	0.13 ± 0.04	−1.7 ± 0.8
12	115.4 ± 1.3	0.19 ± 0.01	−2.7 ± 0.5
15	79.6 ± 0.7	0.17 ± 0.00	−1.1 ± 0.1
18	199.0 ± 5.1	0.68 ± 0.11	−0.5 ± 0.2

Table 2. Optimization of the preparation method of (ACGs + Rh2)-Lipo (n = 3, mean ± SD).

Preparation method	Average particle size (nm)	PDI	Zeta potential (mV)
Thin-film dispersion combined with probe sonication	112.6 ± 0	0.13 ± 0.04	−1.7 ± 0.8
Thin-film dispersion combined with water bath sonication	128.4 ± 8.3	0.50 ± 0.08	−4.0 ± 0.5
Ultrasonic-dripping	318.0 ± 7.5	0.18 ± 0.02	−2.5 ± 0.6
Stirring-dropping	205.3 ± 3.5	0.23 ± 0.02	−4.2 ± 0.3

Figure 2. Characterization of liposomes. (A) Particle size distributions of ACGs-Lipo and (ACGs + Rh2)-Lipo. The morphology of ACGs-Lipo (B), and (ACGs + Rh2)-Lipo (C). Particle size change curves of ACGs-Lipo (D) and (ACGs + Rh2)-Lipo (E) in physiological media (n = 3, mean ± SD).

Figure 3. The in vitro release profiles of ACGs-Lipo and (ACGs + Rh2)-Lipo (n = 3, mean ± SD).

Figure 4. The in vitro cytotoxicity of free Rh2 (A), Rh2-Lipo (B), free ACGs (C), and ACGs-Lipo and (ACGs + Rh2)-Lipo (D) against U87 MG cells (n = 3, mean ± SD).

Table 4. The IC₅₀ of free drug and liposomes against U87 MG cells (n = 3, mean ± SD).

Group	IC50 (μg/mL)
Free Rh2	26.31 ± 1.76
Rh2-Lipo	0.47 ± 0.05***
Free ACGs	0.0011 ± 0.0008###
ACGs-Lipo	0.00034 ± 0.00006
(ACGs + Rh2)-Lipo	0.00011 ± 0.00003&&

*** p < .001 vs. free Rh2.

^### p < .001 vs. free ACGs.

^&& p < .01 vs. ACGs-Lipo.

Figure 5. In vivo antitumor efficacy in U87 MG tumor‑bearing mice. (A) Schematic diagram of the in vivo antitumor study procedure. (B) The change curve of the mouse tumor volume. (C) The change curve of the mouse body weight. (D) The optical photos of tumors at the end of the experiment. (E) The liver index of the mouse in each group. (F) The spleen index of the mouse in each group. Data represent the mean ± SD (n = 6).

Table 5. The anti-glioma activity and changes in body weight of mice in each group (n = 6, mean ± SD).

Group	Tumor weight (g)	Tumor inhibition rate (%)	Mice body weight at the beginning of the experiment (g)	Mice body weight after dissection of the tumor (g)	Reduced body weight (g)
Normal saline	1.32 ± 0.16		20.3 ± 0.7	19.9 ± 1.3	0.4
TMZ	0.13 ± 0.06***	90.4 ± 4.6	20.4 ± 0.9	15.9 ± 1.9	4.5
Rh2-Lipo	0.79 ± 0.11^***,^###	40.3 ± 8.4###	20.0 ± 0.4	19.9 ± 1.7	0.1
ACGs-Lipo	0.52 ± 0.07^***,##^,@	60.5 ± 5.4###^,@	20.5 ± 1.1	18.7 ± 2.0	1.8
(ACGs + Rh2)-Lipo	0.36 ± 0.07^***,#^,@@^,&	72.9 ± 5.4##^,@@^,&	20.0 ± 0.6	19.7 ± 0.4	0.3

*** p < .001 vs. normal saline.

^### p < .001.

^## p < .01.

^# p < .05 vs. TMZ.

^@@ p < .01.

^@ p < .05 vs. Rh2-Lipo.

^& p < .05 vs. ACGs-Lipo.

Figure 6. In vivo biodistribution in U87 MG tumor‑bearing mice. Comparison of particle size distribution after DiR-labeling of ACGs-Lipo (A) and (ACGs + Rh2)-Lipo (B). (C) The ex vivo distribution of DiR-ACGs-Lipo and DiR-(ACGs + Rh2)-Lipo in U87 MG tumor-bearing mice 24 hours after administration (n = 5). (D) The fluorescence intensity in tumor and liver of ACGs-Lipo and (ACGs + Rh2)-Lipo and their RTTI (n = 5, mean ± SD).

Data availability statement

The data supporting this work are accessible upon reasonable request from the corresponding author.