Synthesis and Characterization of Lipid Immuno-Nanocapsules for Directed Drug Delivery: Selective Antitumor Activity against HER2 Positive Breast-Cancer Cells

Lipid nanocapsules (LNC) are usually developed as nanocarriers for lipophilic drug delivery. The surface characteristics of these colloidal particles are determinant for a controlled and directed delivery to target tissues with specific markers. We report the development of immuno-nanocapsules, in which some antibody molecules with different immuno-specificity are conjugated to the nanocapsule surface, offering the standardization of a simple method to obtain vectorized nanosystems with specific recognition properties. Nanocapsules were prepared by a solvent-displacement technique, producing an oily core coated by a functional shell of different biocompatible molecules and surface carboxylic groups. Three different antibodies (one a specific HER2 oncoprotein antibody) were conjugated with these nanoparticles by the carbodiimide method, which allows the covalent immobilization of protein molecules through carboxylic surface groups. The immuno-nanocapsules were completely characterized physico-chemically via electrokinetic and colloidal stability experiments, confirming the correct immobilization of these antibody molecules on the colloidal nanoparticles. Also, additional immunological analyses verified that these IgG-LNC complexes showed the expected specific immuno-response. Finally, different healthy and tumoral breast-cell lines were cultured in vitro with Nile-Red-loaded and docetaxel-loaded HER2 immuno-nanocapsules. The results indicate that our immuno-nanocapsules can increase their uptake in HER2 overexpressing tumoral cell lines

IC₅₀ values derived from curves analysis showed in Fig. 3.

<p>IC₅₀ values derived from curves analysis showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023887#pone-0023887-g003" target="_blank">Fig. 3</a>.</p

PKR regulation by 5-FU treatment in HCT116 p53^+/+ and HCT116 p53^−/− cells.

<p>(<b>A</b>) HCT116 p53^+/+, HCT116 p53^−/− cells were mock-treated, treated with 500 IU/ml of human IFNα during 16 hours, or treated with 10 µM of 5-FU during 4, 8, 16 and 24 hours. Total proteins were extracted for immunoblot analysis using anti whole p53, PKR, anti phospho PKR, anti whole PKR, anti phospho eIF2α, anti whole eIF2α, anti cleaved caspase-3, and anti β-actin antibodies. (<b>B</b>) HCT116 p53^+/+, HCT116 p53^−/− cells were mock treated or treated with 10 µM of 5-FU or 500 IU/ml of IFNα during 16 hours. Expression of endogenous mRNA of PKR was analyzed by real-time RT-PCR (means±SEM, n = 3) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023887#s2" target="_blank">material and methods</a>. (<b>C</b>) HCT116 p53^+/+, HCT116 p53^−/− cells expressing shRNA against PKR or control shRNA mock-treated, or treated with 10 µM of 5-FU during 48 hours. After treatment cells were trypsinized and analyzed by flow cytometry to AnnexinV positive determination. Total proteins of both cell lines were extracted for immunoblot analysis using anti whole PKR and β-actin antibodies. The asterisks “**” designates p<0.01 in HCT116 p53^+/+ cells expressing shRNA against PKR versus cells expressing the control shRNA. The asterisk “*” designates p<0.05 in HCT116 p53^−/− cells expressing shRNA against PKR versus cells expressing the control shRNA.</p

Apoptosis induced in MEFs and in human colon and breast cancer cell lines during the 5-FU/IFNα-combined treatment.

<p>(<b>A</b>) 5×10⁵ human colon and breast cancer cells, (<b>B</b>) 5×10⁵ PKR^+/+ or PKR^−/− MEFs and (<b>C</b>) 5×10⁵ HCT-116 colon cancer cells expressing shRNA against PKR or control shRNA, were mock-treated, treated with 500 IU/ml of IFNα, treated with 5 µM of 5-FU or treated with combination of IFNα plus 5-FU during 48 hours. Thereafter treated cells were trypsinized and analyzed by flow cytometry to Annexin V positive determination. The asterisks “**” designates p<0.01 and the asterisk “*” designates p<0.05 in cells with PKR knockdown versus cells expressing PKR.</p

PKR implication in the cell cycle arrest and in the apoptosis induced by 5-FU.

<p>(<b>A</b>) PKR^+/+ and PKR^−/− MEFs were mock treated or treated with 5 µM of 5-FU during 48 hours. Cell cycle was analyzed by flow cytometry after staining with PI. (<b>B</b>) PKR^+/+ MEFs, PKR^−/− MEFs and eIF2α^A/A MEFs were mock-treated, treated with 10 µM of 5-FU or with 100 µg/ml of Irinotecan during 48 hours. Thereafter treated cells were trypsinized and analyzed by flow cytometry to AnnexinV positive determination. The asterisks ‘‘**” designates p<0.01 and the asterisk ‘‘*” designates p<0.05 in mutated cells versus wild type cells. (<b>C</b>) MCF-7 breast cell line expressing shRNA against PKR (shRNA-PKR) or expressing a control shRNA (shRNAc) and (<b>D</b>) colon and breast cancer cell lines SW-480, T84, MCF-7 and T47D, were mock-treated, or treated with 10 µM of 5-FU during 48 hours. Subsequently, cells were trypsinized and analyzed by flow cytometry to AnnexinV positive determination. Values shown represent the mean of the triplicate determinations calculated from a single experiment. Experiments were repeated at least three times. The asterisks ‘‘**” designates p<0.01 in T84 versus SW-480 cells and T47D versus MCF-7 cells.</p

Localization of phospho-PKR and total-PKR in HCT116 p53^+/+ and HCT116 p53^−/− cells.

<p>(<b>A</b>) HCT116 p53^+/+ cells and (<b>B</b>) HCT116 p53^−/− cells were mock treated or treated with 5 µM of 5-FU. At the indicated times cells were fixed and processed for immunofluorescence analysis by confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023887#s2" target="_blank">Material and Methods</a> using antibodies against phospho-PKR (green) and anti- whole PKR (green) and DAPI for staining nuclei (blue).</p

Cytotoxicity effect of 5-FU- and 5-FU/IFNα-combined therapy.

<p>(<b>A</b>) PKR^−/− and PKR^+/+ MEFs, (<b>B</b>) Human colon cancer cell lines T84 and SW-480, and (<b>C</b>) Human breast cancer cell lines, T47D and MCF-7 were treated with increasing amounts of 5-FU alone (triangles) or in combination with 50 IU/ml of IFNα (square) during 6 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023887#s2" target="_blank">Material and Methods</a>. The curve of cell survival was represented as percentage referred to mock-treated cells. The concentration of 5-FU was different depending on cell lines sensitivity. Values shown represent the mean of the triplicate determinations calculated from a single experiment. Cell survival was statistically different when comparing cell lines expressing PKR with the cell lines deficient in PKR expression (p≤0.05 in A and B; p≤0.025 in C). Experiments were repeated at least three times.</p