Paclitaxel loading in PLGA nanospheres affected the in vitro drug cell accumulation and antiproliferative activity

Abstract

Background: PTX is one of the most widely used drug in oncology due to its high efficacy against solid tumors and several hematological cancers. PTX is administered in a formulation containing 1:1 Cremophor EL (polyethoxylated castor oil) and ethanol, often responsible for toxic effects. Its encapsulation in colloidal delivery systems would gain an improved targeting to cancer cells, reducing the dose and frequency of administration.

Methods: In this paper PTX was loaded in PLGA NS. The activity of PTX-NS was assessed in vitro against thyroid, breast and bladder cancer cell lines in cultures. Cell growth was evaluated by MTS assay, intracellular NS uptake was performed using coumarin-6 labelled NS and the amount of intracellular PTX was measured by HPLC.

Results: NS loaded with 3% PTX (w/w) had a mean size < 250 nm and a polydispersity index of 0.4 after freeze-drying with 0.5% HP-Cyd as cryoprotector. PTX encapsulation efficiency was 30% and NS showed a prolonged drug release in vitro. An increase of the cytotoxic effect of PTX-NS was observed with respect to free PTX in all cell lines tested.

Conclusion: These findings suggest that the greater biological effect of PTX-NS could be due to higher uptake of the drug inside the cells as shown by intracellular NS uptake and cell accumulation studies.

Figure 1

Scanning electron microscopy of 3% PTX-NS. Scanning electron micrographs of PTX-NS without cryoprotector (1A), freeze-dried without cryoprotector (1B), with the addition of HP-Cyd as a cryoprotector (1C) and freeze-dried with HP-Cyd (1D). Magnifications: 1 μm (A and D), 0.5 μm (B), 5μm (C).

Figure 2

In vitro release of PTX-NS. PTX 3% loaded NS in pH 7.4 phosphate buffer solution, at 37°C for 360 h. Data are expressed as the mean ± SD of five separate experiments.

Figure 3

Intracellular distribution of NS in ARO cells. ARO cells were incubated for 2 h with PLGA NS loaded with the fluorescent coumarin-6 probe and analyzed by fluorescent microscopy. Nuclei were stained with PI and are visible in red (1A). The uptake of coumarin-6-loaded NS is visible in green (1B). Figure 1C displays an overlaying images obtained combining the FITC and the PI filters. A representation of two experiments is shown. Magnification: 63 ×

Figure 4

Cytotoxicity of free PTX and PTX-NS in ARO cells. ARO cells were treated with increasing concentrations (0.2 – 4.27 μg/ml) of either free or NS-loaded PTX for different times (4A = 24 h, 4B = 48 h, 4C = 2 h). Cell viability was assessed by the MTS assay. Data are expressed as % of viable cells (mean ± SD of three separate experiments, each performed in triplicate) exposed to PTX in respect to untreated controls. ^• free PTX vs. PTX-NS, *treated vs. not treated ^•*p < 0.05 ^••**p < 0.01

Figure 5

Accumulation of PTX in ARO cells. ARO cells were incubated for 48 h with 2.13 or 4.27 μg/ml of either free PTX or PTX-NS. The amount of active PTX present in the cells was analyzed by HPLC. Data are expressed as mean ± SD of three replicates.

Figure 6

Cytotoxicity of free PTX and PTX-NS in MDA MB231 cells. MDA MB231 cells were treated with increasing concentrations (0.1 – 2.0 μg/ml) of either free PTX or PTX-NS for 24 (6A) or 48 (6B) h and cell viability assessed by the MTS assay. Data are expressed as % of viable cells (mean ± SD of three separate experiments, each performed in triplicate) exposed to PTX in respect to untreated controls. ^• free PTX vs. PTX-NS, *treated vs. not treated *p < 0.05 ^••**p < 0.01

Figure 7

Cytotoxicity of free PTX and PTX-NS in RT 112 cells. RT 112 cells were treated with increasing concentrations (0.1 – 2.0μg/ml) of either free PTX or PTX-NS for 24 (7A) or 48 (7B) h and cell viability was assessed by the MTS assay. Data are expressed as % of viable cells (mean ± SD of three separate experiments, each performed in triplicate) exposed to PTX in respect to untreated controls. ^• free PTX vs. PTX-NS, *treated vs. not treated ^•*p < 0.05 ^••**p < 0.01