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

Abstract

Purpose: Polymeric micelles are attractive nanocarriers for tumor-targeted delivery of paclitaxel (PTX). High antitumor efficacy and low toxicity require that PTX mainly accumulated in tumors with little drug exposure to normal tissues. However, many PTX-loaded micelle formulations suffer from low stability, fast drug release, and lack of tumor-targeting capability in the circulation. To overcome these challenges, we developed a micellar formulation that consists of sodium cholate (NaC) and monomethoxy poly (ethylene glycol)-block-poly (d,l-lactide) (mPEG-PDLLA).

Methods: PTX-loaded NaC-mPEG-PDLLA micelles (PTX-CMs) and PTX-loaded mPEG-PDLLA micelles (PTX-Ms) were formulated, and their characteristics, particle size, surface morphology, release behavior in vitro, pharmacokinetics and in vivo biodistributions were researched. In vitro and in vivo tumor inhibition effects were systematically investigated. Furthermore, the hemolysis and acute toxicity of PTX-CMs were also evaluated.

Results: The size of PTX-CMs was 53.61±0.75 nm and the ζ-potential was -19.73±0.68 mV. PTX was released much slower from PTX-CMs than PTX-Ms in vitro. Compared with PTX-Ms, the cellular uptake of PTX-CMs was significantly reduced in macrophages and significantly increased in human cancer cells, and therefore, PTX-CMs showed strong growth inhibitory effects on human cancer cells. In vivo, the plasma AUC_0-t of PTX-CMs was 1.8-fold higher than that of PTX-Ms, and 5.2-fold higher than that of Taxol. The biodistribution study indicated that more PTX-CMs were accumulated in tumor than PTX-Ms and Taxol. Furthermore, the significant antitumor efficacy of PTX-CMs was observed in mice bearing BEL-7402 hepatocellular carcinoma and A549 lung carcinoma. Results from drug safety assessment studies including acute toxicity and hemolysis test revealed that the PTX-CMs were safe for in vivo applications.

Conclusion: These results strongly revealed that NaC-mPEG-PDLLA micelles can tumor-target delivery of PTX and enhance drug penetration in tumor, suggesting that NaC-mPEG-PDLLA micelles are promising nanocarrier systems for anticancer drugs delivery.

Keywords: enhanced; polymeric micelles; sodium cholate; tumor-targeting delivery.

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.

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.