Star Polymers as Non-Viral Carriers for Apoptosis Induction

Abstract

Apoptosis is a widely controlled, programmed cell death, defects in which are the source of various diseases such as neurodegenerative diseases as well as cancer. The use of apoptosis in the therapy of various human diseases is of increasing interest, and the analysis of the factors involved in its regulation is valuable in designing specific carriers capable of targeting cell death. Highly efficient and precisely controlled delivery of genetic material by low-toxic carriers is one of the most important challenges of apoptosis-based gene therapy. In this work, we investigate the effect of the star polymer with 28 poly(N,N'-dimethylaminoethyl methacrylate) arms (STAR) on human cells, according to its concentration and structure. We show that star polymer cytotoxicity increases within its concentration and time of cells treatment. Except for cytotoxic effect, we observe morphological changes such as a shrinkage, loss of shape and begin to detach. We also prove DNA condensation after star polymer treatment, one of the most characteristic feature of apoptosis. The results indicate that the use of STAR triggers apoptosis in cancer cells compared to various normal cells, what makes these nanoparticles a promising drug in therapeutic strategy, which targets apoptosis. We demonstrate highlighting potential of star polymers as an innovative tool for anti-cancer therapy.

Keywords: apoptosis; cross-membrane transport; delivery systems; star polymers vectors.

Figure 1

Kinetics of star polymer action on HT-1080 cells. (A) The cytotoxicity assay of the tested star polymer. The assay was performed with HT-1080 cells. The results are presented as a % of cells surviving in the presence of the star polymer in the range of concentrations (5–50 µg/mL). (B) HT-1080 cells after 6 h of treatment with star polymer at indicated concentrations were seen under light microscope (upper panel) and under fluorescence microscope (lower panel) following nuclear staining with acridine orange/ethidium bromide (AO/EtBr) double staining. Cells with features of early apoptosis are indicated by white arrows and late apoptosis by yellow arrows. Scale bars represent 100 µm. (C) Flow cytometry analysis of cell death type in HT-1080 cells treated with star polymer after 6 h using annexin V and PI staining.

Figure 2

Flow cytometry analysis of mitochondrial potential (TMRM staining) and ROS production (DC-FDA) in HT-1080 cells after 6 h of treatment with star polymer at indicated concentrations. CCCP (left panel) and H₂O₂ (right panel) as positive controls for mitochondrial depolarization and ROS production, respectively. Percentage of positive cells are presented on each histogram.

Figure 3

Changes in nuclear morphology of HT-1080 cells after treatment with star polymer. (A) DAPI-stained nuclei of HT-1080 cells after treatment with star polymer. In untreated cells, nuclei are large and chromatin is uncondensed. In treated cells shrinkage of nucleus and chromatin condensation are observed. Nuclei with condensed chromatin (early stage of apoptosis) is indicated by white arrows. Fragmented nuclei are indicated by yellow arrows and nuclei with a ring-like shape are indicated by red arrows. Scale bar represents 50 µm. (B) Flow cytometry analysis of DNA content in HT-1080 cells treated with star polymer for 6 h (left panel) and 24 h (right panel). Percentage of positive cells are presented on each histogram.

Figure 4

Quantitative RT-PCR expression analysis of apoptosis-related and inflammatory-related genes in HT-1080 cells treated with star polymer for 6 h at the concentration 30 µg/mL. GAPDH was used as loading control. Values are mean ± SEM (n = 3).

Figure 5

Kinetics of star polymer action on human bladder cancer cell lines and human dermal fibroblasts (PDF). (A) Cytotoxicity dynamics of star polymer for 5637 and T24 cancer cell lines and PDF after 6 and 24 h of treatment. (B) Cells’ morphology after 6 h of star polymer treatment at the concentration 30 µg/mL. Cells were double stained with acridine orange/ethidium bromide (AO/EtBr) in order to distinguish the cell death type. Scale bars represent 100 µm.

Figure 6

Cell death type assessment. (A) Flow cytometry analysis of 5637, T24 and PDF cells treated with star polymer for 6 h using Annexin V and Propidium Iodide staining. (B) DAPI-stained nuclei of 5637, T24 and PDF cells after treatment with star polymer for 6 h at the concentration 30 µg/mL. Condensed nuclei are indicated by white arrows, fragmented by yellow arrows and highly condensed nuclei by red arrows. Scale bars represent 50 µm.