Combined administration of PTX and S-HM-3 in TPGS/Solutol micelle system for oncotarget therapy

Figures & data

Figure 1 The schematic diagram of PHTSm for oncotarget therapy.

Abbreviations: PTX, pacli taxel; TPGS, tocopheryl polyethylene glycol succinate.

Table 1 The method of dosage regimen (0.2 mL/mouse, caudal vein injection)

Groups	Drugs	Days and dosage
G1	Control group	Days 1, 3, 5, 7
G2	TSm	Days 1, 3, 5, 7
G3	S-HM-3	3.5 mg/kg S-HM-3 twice daily
G4	PTX	Days 1, 3, 5, 7/10 mg/kg PTX
G5	PTX + S-HM-3	Days 1, 3, 5, 7, PTX/10 mg/kg and 3.5 mg/kg S-HM-3
G6	PTSm	Days 1, 3, 5, 7/PTX 10 mg/kg
G7	PHSm	Days 1, 3, 5, 7/PTX 10 mg/kg and 3.5 mg/kg S-HM-3

Abbreviation: PTX, paclitaxel.

Table 2 The amount of PTX feeding for the influence on the particle size, particle size distribution, zeta potential, and encapsulation efficiency of PTSm (n=3)

PTX concentration (mg/mL)	Size (nm)	PDI	ζ (mV)	EE (%)	DL (%)
0.5	21.2±1.1	0.20±0.02	−14.3±0.1	95.24±1.49	1.07±1.37
1.0	20.1±1.1	0.19±0.01	−13.5±0.2	96.58±2.11	2.41±2.11
2.0	22.9±1.0	0.17±0.02	−13.1±0.1	43.03±1.37	2.38±1.49

Abbreviations: DL, drug loading; EE, encapsulation efficiency; PDI, polydispersity index; PTX, paclitaxel.

Table 3 Influence of Solutol and TPGS’s ratio on preparation of PHTSm (n=3)

Solutol:TPGS	Size (nm)	PDI	ζ (mV)	EE of PTX (%)	DL of PTX (%)
19:1	21.5±2.1	0.17±0.02	−10.3±0.1	77.49±1.20	1.93±1.20
17:3	24.4±1.1	0.11±0.01	−19.0±0.1	96.58±2.11	2.41±2.11
16:4	26.1±1.0	0.17±0.01	−19.5±0.1	63.03±1.81	1.57±1.81
15:5	22.2±1.6	0.27±0.21	−21.2±0.1	4.03±1.24	0.85±1.24
14:6	27.3±1.6	0.30±0.21	−24.2±0.1	30.03±1.31	0.75±1.31
13:7	23.5±1.1	0.37±0.11	−26.0±0.1	19.02±1.01	0.47±1.01

Abbreviations: DL, drug loading; EE, encapsulation efficiency; PDI, polydispersity index; PTX, paclitaxel; TPGS, tocopheryl polyethylene glycol succinate.

Figure 2 Characterization of micelles. TEM photo of PTSm (A) and PHTSm (B), and the AFM photo of PHTSm (C). The fluorescence intensity ratio of I1/I3 from pyrene emission spectra vs the log of the concentration (D). The in vitro PTX release profile (E). *P<0.05, **P<0.01.

Abbreviations: AFM, atomic force microscopy; NS, nonsignificant; PTX, paclitaxel; TEM, transmission electron microscopy.

Table 4 IC₅₀ (µg/mL) of different samples (n=3)

Cell line	TSm	S-HM-3	PTX	PTX + S-HM-3	PTSm	PHTSm
A549	>100	>100	1.26	1.40a	1.18a	1.18a
MDA-MB-231	>100	>100	1.37	1.50a	0.84a	0.912a

Note:

a P>0.05 compared with PTX group.

Abbreviation: PTX, paclitaxel.

Figure 3 The cytotoxicity of micelles on cancer cells.

Notes: The results of MTT tests on A549 (A) and MDA-MB-231 cells (B). Confocal microscopic images of MBA-MB-231 cells after incubation with control, C₆-TSm, and C₆-HTSm groups for 1 and 3 hours (C). The FACS analysis of MBA-MB-231 cells treated with C₆-labeled TSm and HTSm micelles for 1 and 3 hours (D). *P<0.05.

Abbreviations: C₆, coumarin-6; FACS, fluorescence-activated cell sorting; PTX, paclitaxel.

Figure 4 Micelles with S-HM-3 inhibits cell migration.

Notes: The image of different samples on the migration of HUVECs for 24 hours (Scale ×100) (A). Effect on the migration amount of HUVECs (B). Effect on the inhibitor ratio of HUVECs migration (C) (n=3, **P<0.01; ***P<0.001; NS means P>0.05).

Abbreviations: HUVEC, human umbilical vein endothelial cells; NS, nonsignificant.

Figure 5 In vivo tumor targeting assays.

Notes: (A) In vivo fluorescence images of different ratios of Solutol and TPGS. (B) Dir-TSm (17:3) and Dir-HTSm (17:3) in MDA-MB-231 tumor-bearing female nude mice.

Abbreviation: TPGS, tocopheryl polyethylene glycol succinate.

Figure 6 In vivo distribution and pharmacokinetic of free S-HM-3, free PTX and S-HM-3, PTX in PHTSm micelles.

Note: S-HM-3 distributions in different organs after injection (A) S-HM-3 (3.5 mg/kg) and (B) PHTSm (containing S-HM-3 3.5 mg/kg); in vivo S-HM-3 plasma concentration–time curves of (C) free S-HM-3 group and (D) PHTSm group; in vivo PTX plasma concentration–time curve of free PTX (E) and PHTSm (F) after caudal vein administration in SD rat.

Abbreviation: PTX, paclitaxel.

Table 5 S-HM-3 pharmacokinetic parameters of free S-HM-3 group and PHTSm group

Parameters	S-HM-3 group	PHTSm group
t1/2β (hours)	0.43±0.10	10.87±1.20
Vd (L/kg)	2.66±0.23	621.53±106.08
CL (L/h/kg)	4.35±0.64	39.50±3.11
AUC0–72 (h·µg/L)	0.47±0.05	61.03±4.61
AUC0–∞ (h·µg/L)	0.50±0.07	61.31±4.62

Abbreviations: AUC, area under the curve; CL, plasma clearance rate.

Table 6 PTX pharmacokinetic parameters of free PTX group and PHTSm group

Parameters	PTX group	PHTSm group
t1/2β (hours)	1.79±0.13	9.93±1.44
Vd (L/kg)	3.34±0.50	1,174.96±195.32
V1 (L/kg)	0.62±0.08	682.20±22.58
CL (L/h/kg)	1.29±0.17	81.87±3.52
AUC0–72 (h·µg/L)	5.30±0.68	85.25±3.73
AUC0–∞ (h·µg/L)	5.47±0.71	85.61±3.72

Abbreviations: AUC, area under the curve; PTX, paclitaxel.

Table 7 Tumor weight and tumor inhibition rate of different groups

Sample number	Tumor weight (g)	Tumor volume (mm^3)	T/C (%)	IR (%)
Saline	0.74±0.27	851.36±187.31	–	–
TSm	0.57±0.11	600.61±129.17	62.38±10.95	22.92
S-HM-3	0.34±0.01**	402.27±130.15	31.70±7.92	53.57
PTX	0.30±0.12**	335.62±98.01	27.24±12.36	59.99
PTX + S-HM-3	0.28±0.04**	337.98±118.57	19.95±8.51	62.52
PTSm	0.19±0.03**	238.84±61.70	13.26±4.86	74.23
PHTSm	0.12±0.03**	169.46±42.31	9.53±1.28	83.05

Note:

** P<0.001.

Abbreviations: IR, inhibition rate; PTX, paclitaxel.

Figure 7 The results of antitumor assay in vivo.

Notes: Tumor weight (A) and inhibition rate of different groups (B) at 14 days. Tumor volume of different groups (C) and change of tumor weight (D) within 14 days. *P<0.05 means significant difference, **P<0.01 or ***P<0.001 means extremely significant difference.

Abbreviations: NS, nonsignificant; PTX, paclitaxel.

Figure 8 H&E and immunohistochemical micrographs of tumor sections.

Notes: H&E and immunohistochemical micrographs of tumor sections at 14 days (magnification ×400) (A). CD31 (B) and P53 (C) immunohistochemical micrographs of tumor sections at 14 days (n=5). *P<0.05, ***P<0.001.

Abbreviations: MVD, microvessel density; NS, nonsignificant; PTX, paclitaxel.

Figure S1 Xenograft tumors harvested at 14 days (G1 of Saline, G2 of PTX + S-HM-3, G3 of S-HM-3, G4 of PTX, G5 of TSm, G6 of PTSm, and G7 of PHTSm).

Abbreviation: PTX, paclitaxel.