Biodegradable nanoparticles sequentially decorated with Polyethyleneimine and Hyaluronan for the targeted delivery of docetaxel to airway cancer cells

Abstract

Background: Novel polymeric nanoparticles (NPs) specifically designed for delivering chemotherapeutics in the body and aimed at improving treatment activity and selectivity, cover a very relevant area in the field of nanomedicine. Here, we describe how to build a polymer shell of Hyaluronan (HA) and Polyethyleneimine (PEI) on biodegradable NPs of poly(lactic-co-glycolic) acid (PLGA) through electrostatic interactions and to achieve NPs with unique features of sustained delivery of a docetaxel (DTX) drug cargo as well as improved intracellular uptake.

Results: A stable PEI or HA/PEI shell could be obtained by careful selection of layering conditions. NPs with exquisite stability in salt and protein-rich media, with size and surface charge matching biological requirements for intravenous injection and endowed with sustained DTX release could be obtained. Cytotoxicity, uptake and activity of both PLGA/PEI/HA and PLGA/PEI NPs were evaluated in CD44(+) (A549) and CD44(-) (Calu-3) lung cancer cells. In fact, PEI-coated NPs can be formed after degradation/dissociation of the surface HA because of the excess hyaluronidases overexpressed in tumour interstitium. There was no statistically significant cytotoxic effect of PLGA/PEI/HA and PLGA/PEI NPs in both cell lines, thus suggesting that introduction of PEI in NP shell was not hampered by its intrinsic toxicity. Intracellular trafficking of NPs fluorescently labeled with Rhodamine (RHO) (RHO-PLGA/PEI/HA and RHO-PLGA/PEI NPs) demonstrated an increased time-dependent uptake only for RHO-PLGA/PEI/HA NPs in A549 cells as compared to Calu-3 cells. As expected, RHO-PLGA/PEI NP uptake in A549 cells was comparable to that observed in Calu-3 cells. RHO-PLGA/PEI/HA NPs internalized into A549 cells showed a preferential perinuclear localization. Cytotoxicity data in A549 cells suggested that DTX delivered through PLGA/PEI/HA NPs exerted a more potent antiproliferative activity than free DTX. Furthermore, DTX-PLGA/PEI NPs, as hypothetical result of hyaluronidase-mediated degradation in tumor interstitium, were still able to improve the cytotoxic activity of free DTX.

Conclusions: Taken together, results lead us to hypothesize that biodegradable NPs coated with a PEI/HA shell represent a very promising system to treat CD44 overexpressing lung cancer. In principle, this novel nanocarrier can be extended to different single drugs and drug combinations taking advantage of the shell and core properties.

Figure 1

Structure and properties of NPs during layering procedure. A) Schematic representation of the double coating process. B) Size, polydispersity (PI) and zeta potential of NPs during coating and after dispersion of NPs freeze-dried with trehalose in water. Results are the mean of three measurements obtained on three different NP batches ± SD. C) TEM micrographs of PLGA/PEI/HA NPs.

Figure 2

Properties of DTX-PLGA/PEI and DTX-PLGA/PEI/HA NPs freeze-dried with trehalose. A) Size and zeta potential in different media. B) Release profile (37°C) of DTX from NPs dispersed in DMEM FBS+. External dialysis medium was a PBS solution at pH 7.4. Free DTX is reported as control. Results are the mean of three experiments ± SD.

Figure 3

Cytotoxicity of unloaded NPs. A549 and Calu-3 cells were exposed to increasing concentrations of PLGA/PEI/HA and PLGA/PEI NPs for 24 h. After incubation, cell viability and released LDH were evaluated using the MTT (A, B) and LDH (C, D) assays. The cell viability and LDH release from untreated control were set to 100% and 0%, respectively. Results are presented as percentage (mean ± SEM) (n = 3) of the control cells. Differences were considered not statistically significant (P >0.05).

Figure 4

Cellular uptake of fluorescent NPs. Intracellular levels of fluorescent RHO-PLGA/PEI and RHO-PLGA/PEI/HA NPs. A549 and Calu-3 cells were incubated with 0.5 mg/ml of RHO-PLGA/PEI/HA NPs (A) and RHO-PLGA/PEI NPs (B) for 4 h and 24 h. All measurements were normalized to the fluorescence of RHO-labeled NPs in cell medium set as 100%. Results are presented as percentage (mean ± SEM) (n = 3) of the control cells.

Figure 5

Confocal microscopy images of A549 cells after incubation with fluorescent NPs. A549 cells were incubated with RHO-PLGA/PEI/HA NPs (A) and RHO-PLGA/PEI NPs (E) for 24 h. Confocal microscopy images 100X: A549 cell nuclei stained with DAPI (B, F). Merge of the same field for composite images (C, G), scale bar = 10 μm. Pictures were processed using ImageJ Software to reconstruct the x-axis projection using stack images (D, H). Quantification of fluorescence intensity is shown. Bars represent mean values ± SEM of experiments done in triplicate. *P < 0.05.

Figure 6

Subcellular distribution of fluorescent NPs in A549 cells. A549 cells were incubated with RHO-PLGA/PEI/HA NPs for 24 h. Confocal microscopy images 100X: RHO-PLGA/PEI/HA NPs (A), lysosomes of A549 cells stained with LysoTracker Green (B), A549 cell nuclei stained with DAPI (C). Merge of the same field for composite images (D), scale bar = 10 μm. Pictures were processed using ImageJ Software to reconstruct the x-axis projection using stack images (E). Composite image demonstrated colocalization of RHO-PLGA/PEI/HA NPs with lysosomes.

Figure 7

Cytotoxicity of DTX loaded-NPs in A549 cells. A549 cells were exposed to increasing concentrations of free DTX, DTX-PLGA/PEI/HA NPs or DTX-PLGA/PEI NPs for 72 h. After incubation, cell viability and released LDH were evaluated using the MTT (A, B) and LDH (C, D) assays. The cell viability and LDH release from untreated cells were set to 100% and 0%, respectively. Results are presented as percentage (mean ± SEM) (n = 3) of the control cells. *P <0.05, ^# P < 0.001.