Sodium cholate-enhanced polymeric micelle system for tumor-targeting delivery of paclitaxel

Figures & data

Table 1 Physicochemical characterization of PTX-loaded micelles (n=3)

	Size (nm)	PDI	ZP (mV)	EE (%)	DL (%)
PTX-Ms	62.6±0.9	0.121±0.013	−3.1±0.1	85.6±2.1	4.1±0.1
PTX-CMs	53.6±0.8	0.024±0.011	−19.7±0.7	91.3±0.7	3.8±0.1

Note: Data are presented as mean ± SD.

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; PDI, polydispersity index; ZP, ζ-potential; DL, drug loading; EE, encapsulation efficiency.

Figure 1 Particle-size distribution and morphology of PTX-Ms and PTX-CMs.

Notes: Size distribution of PTX-Ms and PTX-CMs measured by DLS (A). TEM images of PTX-Ms (B) and PTX-CMs (C). Scale bar 200 nm. Plot of intensity ratios (I₁:I₃) as the function of micelle concentrations (D).

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; DLS, dynamic light scattering; TEM, transmission electron microscopy.

Figure 2 In vitro evaluation of micelles.

Notes: (A) In vitro PTX-release profiles of Taxol and PTX-loaded micelles in PBS solution (pH 7.4) with 0.2% Tween 80 at 37°C. Fluorescence-adsorption peaks of cells treated with FITC-Ms and FITC-CMs: (B) bone-marrow macrophages; (C) A549 cells. In vitro cytotoxicity of different formulations of PTX against human cancer cells: (D) BEL7402; (E) A549.

Abbreviations: PTX, paclitaxel; FITC-Ms, fluorescein isothiocyanate-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.

Table 2 IC₅₀ of various PTX-loaded formulations against BEL7402 cells and A549 cells after 48 hours’ incubation

	IC50 (ng/mL)
	BEL7402	A549
Taxol	10.07±0.44	16.65±0.71
PTX-Ms	23.47±0.38	28.72±0.33
PTX-CMs	14.81±0.27	22.86±0.23

Note: Data are presented as mean ± SD.

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.

Table 3 Pharmacokinetic parameters of Taxol, PTX-Ms, and PTX-CMs after intravenous administration in Sprague Dawley rats at PTX dose of 8 mg/kg

	t½	Cmax	AUC0−t	AUC0−∞	MRT	Vz	Cl
	hours	μg/mL	mg/L⋅h	mg/mL⋅h	hours	L/kg	L/kg/h
Taxol	2.36	10.21	8.92	9.02	3.25	4.16	0.89
PTX-Ms	8.97	16.06	26.27	30.51	9.72	3.39	0.26
PTX-CMs	17.19	18.48	46.46	67.79	19.96	2.92	0.12

Note: Data are presented as mean ± SD, n=4.

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; t_½, half-life; C_max, maximum plasma concentration; AUC, area under the plasma concentration–time curve; MRT, mean residence time; V_z, apparent volume of distribution; Cl, clearance (total body).

Figure 3 Time courses of PTX levels in rat plasma after intravenous administration of PTX-Ms, PTX-CMs, and Taxol at PTX dose of 8 mg/kg (n=4).

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.

Figure 4 In vivo biodistribution of micelles in BEL7402 xenograft mice.

Notes: In vivo imaging of mice (A, B) and ex vivo imaging of tumors and major organs (C, D) at 2, 6, and 24 hours after mice had been treated with DiR-CMs (A, C) and DiR-Ms (B, D). (E) Tissue distribution of PTX at 2 hours after mice had been treated with Taxol, PTX-Ms, and PTX-CMs at PTX dose of 10 mg/kg.

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; DiR, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide.

Figure 5 Antitumor activity of PTX-CMs in BEL7402 xenograft mice.

Notes: Tumor-growth curves of early-stage (A) and late-stage (C) tumor model. Body weight changes in early-stage (B) and late-stage (D) tumor model. Data are presented as mean ± SD (n=8). (E) Survival rates of mice in different treatment groups within 28 days. Data are presented as mean ± SD (n=8). (F) Representative images of mice treated with PTX-CMs at PTX dose of 30 mg/kg in late-stage tumor model at 0, 4, 8, 18, 25, and 32 days. Arrows indicate time when mice received intravenous injections.

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.

Figure 6 In vitro hemolysis of PTX-CMs.

Notes: Taxol (A); PTX-Ms (B); PTX-CMs (C). (D) Hemolysis percentage of Taxol, PTX-Ms, and PTX-CMs. Each data point is represented as mean ± SD (n=3). −, negative control; +, positive control.

Abbreviations: PTX-Ms, paclitaxel-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.

Figure 7 Systemic toxicity of PTX-CMs.

Notes: (A) Body weight changes after male ICR mice had received signal injections of PBS and PTX-CMs at PTX dose of 30 mg/kg. (B–G) Indicators of hepatic and renal functions in toxicity assay measured at 14 days posttreatment. Data are represented as mean ± SD (n=10). (H) H&E staining of major organs after single injection of PBS and PTX-CMs at PTX dose of 30 mg/kg. Magnification ×400.

Abbreviations: PTX-CMs, paclitaxel-loaded sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; TP, total protein; Alb, albumin; Glob, globulin; T-Bil, total bilirubin; BUN, blood urea nitrogen; A/G, Alb/Glob; UA, uric acid; ALT, alanine transaminase.

Scheme 1 Preparation of PTX-CMs and the fate of PTX-CMs in vivo.

Abbreviations: PTX, paclitaxel; mPEG, monomethoxy(polyethylene glycol); PLA, polylactic acid; CMs, sodium cholate and mPEG-block-poly(d,l-lactide) micelles; DiR, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide; EPR, enhanced permeability and retention.

Figure S1 Cumulative PTX-release profile.

Notes: Four commercially available PTX formulations and PTX-CMs in PBS solution (pH 7.4) with 0.2% Tween 80 at 37°C. Data are shown as mean ± SD (n=3).

Abbreviations: PTX, paclitaxel; CMs, sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.

Figure S2 Fluorescence microscopy images of bone-marrow macrophage cells and A549 cells at 4 hours after incubation with FITC-Ms and FITC-CMs.

Abbreviations: FITC-Ms, fluorescein isothiocyanate-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy (polyethylene glycol)-block-poly(d,l-lactide) micelles.

Figure S3 Quantitative flow-cytometry analysis of bone-marrow macrophage cells and A549 cells at 4 hours after incubation with FITC-Ms and FITC-CMs.

Abbreviations: FITC-Ms, fluorescein isothiocyanate-loaded monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles; CMs, sodium cholate and monomethoxy (polyethylene glycol)-block-poly(d,l-lactide) micelles.

Figure S4 Antitumor effect of PTX-CMs in A549 late-stage tumor model.

Notes: (A) Tumor growth curve, and (B) body weight changes. Arrows indicate time points when mice received intravenous injections. Data are presented as mean ± SD (n=7).

Abbreviation: PTX-CMs, paclitaxel-loaded sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.

Table S1 Hematological test of whole blood collected from healthy ICR mice treated with single injections of PBS and PTX-CMs at PTX dose of 300 mg/kg (n=10)

Parameters	Reference values	PBS	PTX-CMs
WBC, 10^9/L	4–12	4.4±1.11	5.52±1.43
Ne, 10^9/L	0.7–4	0.56±0.36	1.19±0.56
Ly, 10^9/L	3–8.5	3.83±0.88	4.3±1.05
Mo, 10^9/L	0–1.3	0±0	0.01±0.03
Eo, 10^9/L	0–0.5	0.02±0.06	0.02±0.06
Ba, 10^9/L	0–0.1	0±0	0±0
Ne, %	12–44	12.41±5.41	20.78±7.16
Ly, %	54–85	87.01±5.85	78.36±7.22
Mo, %	0–15	0.1±0.15	0.38±0.36
Eo, %	0–5	0.48±0.8	0.47±0.54
BA, %	0–1	0±0	0.01±0.03
RBC, 10^12/L	7.7–12.5	8.82±0.65	9.04±0.64
Hb, g/L	120–200	168.1±7.28	180.5±13.44
Hct	25–56	45.66±2.28	47.78±2.8
MCV, fL	40–48	51.79±1.72	52.89±1.99
MCH, pg	20–32	19.08±1.15	19.96±0.83
MCHC, g/L	300–370	368.5±12.4	377.6±11.56
RDW, %	11.2–16	13.29±0.29	14.03±0.54
Plt, 10^9/L	100–900	523±37.51	671.7±159.55
MPV, fL	5–10	6.76±0.27	7.06±0.71
PDW, fL	9–17	12.21±0.53	12.34±0.81

Abbreviations: WBC, white blood cell; Ne, neutrophil; Ly, lymphocyte; Mo, monocyte; Eo, eosinophil; Ba, basophil; RBC, red blood cell; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean content of Hb; MCHC, mean concentration of Hb; RDW, RBC distribution width; Plt, platelet; MPV, mean Plt volume; PDW, Plt distribution width; PTX-CMs, paclitaxel-loaded sodium cholate and monomethoxy(polyethylene glycol)-block-poly(d,l-lactide) micelles.