Poly(ethylene glycol)-block-poly(ε-caprolactone)-and phospholipid-based stealth nanoparticles with enhanced therapeutic efficacy on murine breast cancer by improved intracellular drug delivery

Abstract

Background: Effective anticancer drug delivery to the tumor site without rapid body clearance is a prerequisite for successful chemotherapy. 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-(methoxy[polyethyleneglycol]-2000) (DSPE-PEG2000) has been widely used in the preparation of stealth liposomes. Although PEG chains can efficiently preserve liposomes from rapid clearance by the reticuloendothelial system (RES), its application has been hindered by poor cellular uptake and unsatisfactory therapeutic effect.

Methods: To address the dilemma, we presented a facile approach to fabricate novel stealth nanoparticles generated by poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL), soybean phosphatidylcholine (SPC), and cholesterol, namely LPPs (L represented lipid and PP represented PEG-b-PCL), for the delivery of anticancer drug paclitaxel (PTX). LPPs were prepared using the thin film hydration method. Two PEG-b-PCL polymers with different molecular weights (MW; PEG2000-b-PCL2000, MW: 4,000 Da and PEG5000-b-PCL5000, MW: 10,000 Da) were used to fabricate stealth nanoparticles. Conventional PEGylated liposome (LDP2000, L represented lipid and DP2000 represented DSPE-PEG2000) composed of SPC, cholesterol, and DSPE-PEG2000 was used as the control. The physical properties, cellular uptake, endocytosis pathway, cytotoxicity, pharmacokinetics, tumor accumulation, and anticancer efficacy of free PTX, PTX-loaded LPPs, and LDP2000 were systemically investigated after injection into 4T1 breast tumor-bearing mice.

Results: LPPs were vesicles around 100 nm in size with negative zeta potential. With enhanced stability, LPPs achieved sustainable release of cancer therapeutics. The cellular uptake level was closely related to the PEG chain length of PEG-b-PCL; a shorter PEG chain resulted in higher cellular uptake. Moreover, the cellular internalization of LPP2000 modified by PEG2000-b-PCL2000 on 4T1 cells was 2.1-fold higher than LDP2000 due to the improved stability of LPP2000. The cytotoxicity of PTX-loaded LPP2000 was also higher than that of LDP2000 and LPP5000 as observed using a WST-8 assay, while blank LPPs showed negligible toxicity. Consistent with the results of the in vitro study, in vivo experiments showed that LPPs allowed significantly improved bioavailability and prolonged T1/2β as compared to free PTX injection. More importantly, LPPs mainly accumulated at the tumor site, probably due to the enhanced permeation and retention effect (EPR effect). As a nanomedicine, LPP2000 (tumor inhibition rate of 75.1%) significantly enhanced the therapeutic effect of PTX in 4T1 breast tumor-bearing mice by inhibiting tumor growth compared to LDP2000 and LPP5000 (tumor inhibition rates of 56.3% and 49.5%, respectively).

Conclusion: Modification of liposomes with PEG2000-b-PCL2000 can simultaneously improve drug accumulation at the target tumor site and tumor cells, showing great promise for utilization as a PEG modification tool in the fabrication of stealth nanoparticles for cancer chemotherapy.

Keywords: murine breast cancer chemotherapy; nanoparticles PEG-b-PCL; paclitaxel; phospholipid.

Figure 1

(A) Representative images of LPPs visualized by TEM. (B) Cumulative release of PTX from PEGylated nanoparticles in water containing 0.1% Tween 80 at 37°C. Note: The error bars represent the SD (n=3). Abbreviations: PTX, paclitaxel; SD, standard deviation; LPP, nanoparticles modified by poly(ethylene glycol)-b-poly(ε-caprolactone); LDP2000, nanoparticles modified by 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-(methoxy[polyethyleneglycol])-2000; TEM, transmission electron microscope.

Figure 2

(A) Uptake of DiI-loaded nanoparticles by 4T1 cells was investigated qualitatively by CLSM. Scale bars represent 10 μm. (B, C) Uptake of DiI-loaded nanoparticles by 4T1 cells was investigated qualitatively by FCM. (D) The endocytosis inhibition assay on 4T1 cells is shown. Notes: For uptake evaluation, 4T1 cells were treated with DiI-loaded PEGylated nanoparticles for 4 hours before measurements. For endocytosis inhibition assay, after pre-incubation with different inhibitors for 30 minutes, cells were treated with DiI-loaded PEGylated nanoparticles for another 4 hours (n=3). * and ** indicate P<0.05 and P<0.01 versus the control group. Abbreviations: CLSM, confocal laser scanning microscopy; FCM, flow cytometry.

Figure 3

In vitro cytotoxicity of PTX formulations and blank formulations against 4T1 cell line. Notes: (A) Represents the cytotoxicity of PTX-loaded formulations and free PTX. (B) Represents the cytotoxicity of blank formulations (x-axis shows corresponding PTX concentrations of blank formulations) and solvents for free PTX (ethanol mixed with Cremophor ELP 35 with a volume ratio of 1:1) on 4T1 cell lines for 48 hours. Data were given as mean ± SD (n=5). *Represents statistically significant difference P<0.05 versus LDP2000, ^#represents statistically significant difference P<0.05 versus free PTX, and $represents statistically significant difference P<0.05 versus LPP5000. ^&Represents statistically significant difference P<0.05. Abbreviations: PTX, paclitaxel; SD, standard deviation.

Figure 4

(A) The PTX concentration–time curves in rat blood plasma after intravenously administrated free PTX and PTX-loaded PEGylated nanoparticles at a PTX dose of 10 mg/mL. (B) Representative images of breast tumor–bearing BALB/c mice 24 hours after injection of free DiR- and DiR-labeled nanoparticles. The arrows indicate the tumors. Abbreviation: PTX, paclitaxel.

Figure 5

Intravenous administration of PTX formulations inhibited the growth of 4T1 murine breast tumors in vivo and prolonged the survival of treated mice. Notes: (A) Suppression of tumor growth in each treatment group. The arrows indicate the injection of PTX formulations. (B) PTX groups versus the saline or LPP2000 group, with tumor growth inhibition of PTX formulations. (C) Weight of subcutaneously transplanted tumors in each treatment group. (D) Survival curves of mice in each group. * and ** represented P<0.05 and P<0.01, respectively. Abbreviation: PTX, paclitaxel.