D-α-tocopherol polyethylene glycol succinate-based derivative nanoparticles as a novel carrier for paclitaxel delivery

Figures & data

Figure 1 Synthetic route of 4-arm-PEG-TPGS.

Abbreviations: PEG, polyethylene glycol; TPGS, D-α-tocopherol polyethylene glycol succinate; SA, succinic anhydride; DMAP, 4-dimethylamino pyridine; DCC, dicyclohexylcarbodiimide; RT, room temperature.

Figure 2 Characterization of 4-arm-PEG-TPGS.

Notes: (A) ¹H-NMR spectra and (B) FTIR spectra.

Abbreviations: PEG, polyethylene glycol; TPGS, D-α-tocopherol polyethylene glycol succinate; NMR, nuclear magnetic resonance; FTIR, Fourier transform infrared spectroscopy; SA, succinic anhydride.

Figure 3 GPC results of 4-arm-PEG and TPGS-based derivative 4-arm-PEG-TPGS.

Abbreviations: GPC, gel permeation chromatography; PEG, polyethylene glycol; TPGS, D-α-tocopherol polyethylene glycol succinate; MW, molecular weight.

Table 1 Characterization of PTX-loaded 4-arm-PEG-TPGS nanoparticles

Polymers	Drug feeding concentration (wt%) Particle	Particle sizea (nm)	PDIa	ζ potential(mV)	EEb (%)	PTX concentration (mg/mL)
TPGS	10	134.8±3.7	0.22±0.07 is	−3.44 ±0.04	18.2±0.3	0.090
TPGS	15	158.2±9.3	0.25±0.04	−3.65±0.21	12.7±0.6	0.095
TPGS	20	205.0±5.7	0.23±0.05	−3.78±0.39	9.9±0.5	0.10
4-arm-PEG5K -TPGS	10	260.0±1.1	0.24±0.06	−10.02±1.1	91.7±6.5	0.45
4-arm-PEG5K -TPGS	15	283.6±1.1	0.16±0.03	−9.81±0.22	79.8±2.4	0.60
4-arm-PEG5K -TPGS	20	307.3±3.7	0.21±0.03	−9.72±1.20	71.1±3.4	0.70
4-arm-PEG10K -TPGS	10	295.2±0.9	0.18±0.02	−13.02±1.5	32.6±3.2	0.16
4-arm-PEG10K -TPGS	15	305.4±3.7	0.22±0.01	−12.02±1.1	32.5±1.4	0.15
4-arm-PEG10K -TPGS	20	316.9±4.4	0.19±0.02	−11.99±1.1	29.4±3.6	0.30
4-arm-PEG20K -TPGS	10	313.7±4.7	0.15±0.02	−14.03±2.2	43.1±2.5	0.22
4-arm-PEG20K-TPGS	15	317.1±4.8	0.14±0.01	−13.67±1.7	35.3±1.6	0.27
4-arm-PEG20K-TPGS	20	329.7±5.5	0.20±0.02	−13.50±1.6	31.6±2.3	0.32

Notes:

a Measured by DLS.

b Measured by HPLC.

Abbreviations: PTX, paclitaxel; PEG, polyethylene glycol; TPGS, D-α-tocopherol polyethylene glycol succinate; PDI, polydispersity index; EE, encapsulation efficacy; DLS, dynamic light scattering; HPLC, high-performance liquid chromatography.

Figure 4 Characterization of 4-arm-PEG-TPGS nanoparticles.

Notes: (A and B) DLS result and TEM image of 4-arm-PEG_5K-TPGS nanoparticles, (C and D) DLS result and TEM image of 4-arm-PEG_10K-TPGS nanoparticles, (E and F) DLS result and TEM image of 4-arm-PEG_20K-TPGS nanoparticles, (G) plot of the intensity ratio I₃₃₉/I₃₃₅ as a function of log C for TPGS, 4-arm-PEG_5K-TPGS, 4-arm-PEG_10K-TPGS, and 4-arm-PEG_20K-TPGS nanoparticles, (H and I) the stability of TPGS micelles and PTX-NP dispersed in PBS and FBS, (J) hemolysis assay of blank 4-arm-PEG-TPGS nanoparticles of various concentrations incubated with RBCs for 4 hours at 37°C in an incubator shaker.

Abbreviations: PEG, polyethylene glycol; TPGS, D-α-tocopherol polyethylene glycol succinate; DLS, dynamic light scattering; TEM, transmission electron microscope; PTX, paclitaxel; NP, nanoparticles; PBS, phosphate buffered saline; FBS, fetal bovine serum; RBCs, red blood cells; PEI, polyethylenimine.

Figure 5 In vitro release of PTX from PTX-NP_5K in PBS (pH 7.4) and FBS.

Abbreviations: PTX, paclitaxel; NP, nanoparticles; PBS, phosphate buffered saline; FBS, fetal bovine serum.

Figure 6 Cellular uptake of coumarin-6-NP_5K by A2780 cells.

Notes: (A) CLSM images after 2 hours incubation and (B) MFI value analyzed by flow cytometry.

Abbreviations: NP, nanoparticles; CLSM, confocal laser scanning microscopy; MFI, mean fluorescence intensity; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 7 In vitro cytotoxicity of Taxol^®, PTX-NP_5K, and free PTX against (A) A2780, (B) A549, and (C) MCF-7 cells after treatment for 24, 48, and 72 hours.

Abbreviations: PTX, paclitaxel; NP, nanoparticles.

Table 2 IC₅₀ values (μg/mL) of Taxol^®, PTX-NP_5K, and free PTX after 24, 48, and 72 hours incubation with A2780, A549, and MCF-7 cells at 37°C

Incubation time	Taxol^®			PTX-NP5K			Free PTX
	A2780	A549	MCF-7	A2780	A549	MCF-7	A2780	A549	MCF-7
24 hours	2.40±0.34	7.83±0.46	1.35±0.05	3.97 ±0.12	20.69±1.94	17.0±1.52	>100	>100	>100
48 hours	0.50±0.08	4.86±0.60	0.13±0.12	1.12±0.01	5.05±0.24	0.31±0.06	>100	>100	>100
72 hours	0.05±0.01	0.11±0.02	0.03±0.01	0.19±0.07	0.46±0.08	0.20±0.06	>25	>25	>25

Abbreviations: PTX, paclitaxel; NP, nanoparticles; IC 50, half maximal inhibitory concentration.

Figure 8 Cell apoptosis analysis of A2780 cells with Taxol^®, PTX-NP_5K, and free PTX after 24 hours treatment.

Notes: (A) Nucleus apoptosis assay and (B) annexin V-FITC/PI double staining by flow cytometry.

Abbreviations: PTX, paclitaxel; NP, nanoparticles; V-FITC, V-fluorescein isothiocyanate; PI, propidium iodide.

Figure 9 Cell cycle distribution in A2780 cells treated with various formulations.

Notes: (A) Control, (B) Taxol^®, (C) PTX-NP_5K, and (D) free PTX for 24 hours.

Abbreviations: PTX, paclitaxel; NP, nanoparticles; PI, propidium iodide.

Figure 10 In vivo antitumor efficacy in tumor-bearing Kunming mice treated with Taxol^® (10 mg/kg), PTX-NP_5K (10 mg/kg), and PTX-NP_5K (30 mg/kg) (n=5).

Notes: (A) Relative tumor growth ratio (*P,0.05), (B) tumor weight, (C) images of tumor tissues, (D) relative body weight and (E) HE staining assay of the tumor sections.

Abbreviations: PTX, paclitaxel; NP, nanoparticles; HE, hematoxylin and eosin.

Figure S1 Hemolysis assay of Cremophor EL-based vehicle (Cremophor ELand dehydrated alcohol, 1:1, v/v) of various concentrations incubated with RBCs for 4 hours at 37°Cin an incubator shaker.

Abbreviation: RBCs, red blood cells.

Figure S2 In vitro cytotoxicity of Taxol®, PTX-NP_5K, and free PTX against S180 cells after treatment for 24, 48, and 72 hours.

Abbreviations: PTX, paclitaxel; NP, nanoparticles.

Figure S3 Cell apoptosis analysis of S180 cells with Taxol®, PTX-NP_5K, and free PTX after 24 hours treatment.

Abbreviations: PTX, paclitaxel; NP, nanoparticles; FITC, fluorescein isothiocyanate; PI, propidium iodide.