Docetaxel and curcumin-containing poly(ethylene glycol)-block-poly(ε-caprolactone) polymer micelles

PEG–PLC diblock copolymers were purchased from Sigma-Aldrich with a PEG average of Mn ∼5000 and a PLC average of Mn ∼5000. Pure docetaxel anhydrous was purchased from Shanghai Bioman Pharma Co. Ltd, Shanghai, China. Dulbecco's phosphate-buffered saline (DPBS) was purchased from Sigma. [3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchase from Promega (USA). Curcumin and tissue culture reagents were purchased from Sigma. The hela cell line was obtained from the Department of Animal Cell Culture, Institute of Biotechnology (VAST). All other organic solvents were analytical grade from Fisher Scientific.

2.2.1. Preparation of drug-loaded PEG–PCL NPs.

2.2.2. Morphology of the NPs.

2.2.3. Particle size and zeta potential.

2.2.4. Drug encapsulation efficiency.

2.2.5. In vitro cell uptake assays.

2.2.6. Cytotoxicity assay.

The in vitro cytotoxicity study of drug-free and drug-loaded NPs and the free drug was carried out on Hela cells using the MTS assay. Hela cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 100 IU ml⁻¹ of penicillin G sodium and 100 μg ml⁻¹ of streptomycin sulfate at 37 °C in a humidified environment of 5% CO₂. The cells were seeded in a 96-well plate for 24 h to allow attachment, then the medium was replaced by 100 μl with various concentrations of Doc-free, Doc-loaded, Cur-loaded and Doc–Cur-loaded NPs (ranging from 0.025, 0.25, 2.5, 12.5 and 25 μg ml⁻¹ of Doc or Cur concentration) and free Doc and Cur.

Free Doc was prepared in DMSO and the DMSO concentration in the medium was lower than 0.1% (no effect on cell proliferation). After incubation for 24 or 48 h, the cells were incubated with 20 μl per well of MTS and phenazine methosulfate (PMS) solution (MTS/PMS ratio: 20/1) for 1–4 h at 37 °C in a humidified, 5% CO₂ atmosphere. Absorbance was measured at 490 nm using a BioTek Synergy^® HT microplate reader (USA). Cell viability was calculated by the following equation

$$\begin{eqnarray*}&&{\rm{Cell \ viability }}\left( \% \right) = \left( {{\rm{Abs}}_{{\rm{sample}}} /{\rm{Abs}}_{{\rm{control}}} } \right){\rm{ }} \times {\rm{ 1}}00\% , \end{eqnarray*}$$

where Abs_sample is the absorbance of the cells treated with the drug-loaded NP suspension and Abs_control is the absorbance of the cells treated with the medium only (positive control). All experiments were repeated three times.

The characteristics of the drug-loaded NPs are shown in table 1. Drugs were successfully entrapped in PEG–PCL NPs. In this work we investigated the effect of nanoprecipitation and sonication methods on drug entrapment efficiency. When the Doc/polymer ratio was equal to 1/5, the encapsulation efficiency of Doc was 16 ± 1.2%, and the encapsulation efficiency of Cur was 50 ± 3.5% when the Cur/polymer ratio was equal to 1/10 (by the nanoprecipitation method). But Doc entrapped by nanoprecipitation combined with sonication had an increase of encapsulation efficiency of about 19 ± 1.7% in just 30 min.

Particle size influences the kinetic parameters of drugs, for example drug biodistribution and clearance. The accumulation of drugs with a particle size ranging from 20 to 100 nm in tumor tissues is significantly increased due to the enhanced permeability and retention (EPR) effect and the escape of NPs from the reticulo-endothelial system (RES) [3,17]. Therefore, we investigated the effect on particle size of forming NPs using a combination of nanoprecipitation with sonication for about 30 min. NPs were fabricated with narrower size, lower polydispersity index (PI) and steady encapsulation efficiency. To homogenize the size of NPs, formulated NPs were extruded through a EmulsiFlex™-C5 High Pressure Homogenizer (INVESTIN, Ottawa, Canada) with 100 nm nanoporous membranes (Whatman). Thus we obtained drug-loaded NPs with a narrower mean diameter and size distribution than is shown in table 1.

There was no significant effect on the encapsulation efficiency of Doc and Cur as they were incorporated into PEG–PCL NPs. Furthermore, the size and size distribution of formulated NPs were not influenced by the presence of the drug for drug-unloaded NPs and these characteristics were similar to other drug-loaded NPs (data not shown).

Zeta potential can greatly influence particle stability in suspension and avoid the aggregation of particles. The zeta potential of drug-loaded NPs detected by dynamic light scattering was −6.97 mV.

For surface morphology, the NPs were evaluated by FE-SEM and TEM analysis. As shown in figure 1, the NPs had a spherical shape and a smooth surface with uniform size distribution.

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The feasibility of the drug-loaded PEG–PCL NPs was evaluated by cellular uptake of fluorescent Doc–Cur-loaded PEG–PCL NPs on the Hela cell line. Curcumin has been widely used due to its biocompatibility and high fluorescence activity. Fluorescent images of Doc–Cur-loaded PEG–PCL NPs (figure 2(a)) and cellular uptake images on Hela cells (figures 2(b) and (c)) were taken using the Nikon Eclipse 80i microscope (Japan) with a 40 × objective lens, and Cur in PEG–PCL NPs was used as a probe for marking NPs in the cellular uptake experiment to visualize and measure cellular uptake of polymeric NPs.

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Cellular uptake images of Doc–Cur-loaded NPs on Hela cells after 2 h (figure 2(b)) and 4 h (figure 2(c)) incubation with Doc–Cur-loaded PEG–PCL NPs at 2 μg ml⁻¹ (caculated by Doc) and 4 μg ml⁻¹ (calculated by Cur) are shown in figure 2. We find that the fluorescence of the Doc–Cur-loaded NPs (green) is located in cells, which means that the NPs have been internalized by the cell. The fluorescence intensity of Hela cells increased after 2 h and decreased after 4 h of incubation. So, cellular uptake was dependent on the incubation time. These results are consistent with published data [18].

The cytotoxicity of Doc-loaded NPs with and without Cur and the free Doc and Cur was evaluated on the Hela cell line. Table 2 shows the IC₅₀ values of Hela cells after 24 and 48 h at various Doc concentrations of 0.025, 0.25, 2.5, 12.5 and 25 μg ml⁻¹ (Cur concentration in NPs was 0.05, 0.5, 5.0, 25 and 50 μg ml⁻¹).

The IC₅₀ value for Hela cells decreased from 3.5 μg ml⁻¹ for free Doc to 2.01 μg ml⁻¹ for Doc–NPs and 1.52 μg ml⁻¹ Doc–Cur–NPs after 24 h of incubation. After 48 h of incubation, the significant difference of IC₅₀ values between free Doc, Doc–NPs and Doc–Cur–NPs was 1.16, 0.58 and 0.35 μg ml⁻¹, respectively. No cytotoxic activity was observed for the drug-free NPs. These results suggest that the incorporation of Doc and Cur into NPs enhanced its anti-tumoral activity compared to Doc-NPs and free Doc.

Thus, the higher cytotoxicity of the drug-loaded NPs can be attributed to different mechanisms between free drug molecules and drug molecules entrapped into NPs. The NPs were adsorbed on the cell surface, therefore the drug concentration near the cell membrane significantly increases, resulting in a concentration gradient and promoting penetration of the drug into the cell [19]. However, for free drug molecules, part of these molecules were transported into the cytoplasm by passive diffusion and transported out by P-glycoprotein pumps. Moreover, drug-loaded NPs penetrate into cells through endocytosis and they can escape from the influence of P-glycoprotein pumps, thus leading to a higher cellular uptake compared to free drug molecules [20].

Thus, the combination of Doc and Cur into PEG–PCL NPs is able to increase the synergistic effect on cancer cells. Moreover, fluorescent signals in cancer cells can be observed by the presence of Cur in NPs for further studies in vitro and in vivo.