Encapsulation of Inositol Hexakisphosphate with Chitosan via Gelation to Facilitate Cellular Delivery and Programmed Cell Death in Human Breast Cancer Cells

Abstract

Inositol hexakisphosphate (InsP₆) is the most abundant inositol polyphosphate both in plant and animal cells. Exogenous InsP₆ is known to inhibit cell proliferation and induce apoptosis in cancerous cells. However, cellular entry of exogenous InsP₆ is hindered due to the presence of highly negative charge on this molecule. Therefore, to enhance the cellular delivery of InsP₆ in cancerous cells, InsP₆ was encapsulated by chitosan (CS), a natural polysaccharide, via the ionic gelation method. Our hypothesis is that encapsulated InsP₆ will enter the cell more efficiently to trigger its apoptotic effects. The incorporation of InsP₆ into CS was optimized by varying the ratios of the two and confirmed by InsP₆ analysis via polyacrylamide gel electrophoresis (PAGE) and atomic absorption spectrophotometry (AAS). The complex was further characterized by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) for physicochemical changes. The data indicated morphological changes and changes in the spectral properties of the complex upon encapsulation. The encapsulated InsP₆ enters human breast cancer MCF-7 cells more efficiently than free InsP₆ and triggers apoptosis via a mechanism involving the production of reactive oxygen species (ROS). This work has potential for developing cancer therapeutic applications utilizing natural compounds that are likely to overcome the severe toxic effects associated with synthetic chemotherapeutic drugs.

Keywords: apoptosis; breast cancer; chitosan; cytotoxicity; encapsulation; inositol polyphosphates; reactive oxygen species.

Figure 1

Schematic depiction of the concept of cellular entry of exogenously administered InsP₆ after encapsulation with chitosan by ionic gelation to shield off the negative charge. Note that the encapsulated InsP₆ enters the cell through cell membrane, whereas negatively charged free InsP₆ is unable to enter the cell membrane.

Figure 2

Schematic illustration of the preparation of the CS:InsP₆ nanomaterial complex by ionic gelation. Chitosan dissolved in acetic acid (5.0 mg/mL) and InsP₆ dissolved in deionized water (5.0 mg/mL) were mixed in varying proportions and stirred for 30 min followed by pH adjustment. The CS:InsP₆ complex was purified by centrifugation and washing with ethanol and lyophilized to dry powder.

Figure 3

Detection of InsP₆ contents in the CS:InsP₆ complex by PAGE. Optimization of InsP₆ incorporation in chitosan was carried out by varying the ratios of CS:InsP₆ (B). Standard InsP₆ with known concentrations were also run in parallel to establish the linearity of detection (A). Band densities were analyzed by image J software. (B) shows the amounts of InsP₆ detected in the samples with various ratios of CS:InsP₆ applied on the gel. The maximum amount of InsP₆ (0.49 μg) was detected in the sample with a CS:InsP₆ ratio of 2.5:1.0. This amount (0.49 μg), when calculated using the dilution factor of the samples loaded on the gel, provides a total incorporation of 49 ug InsP₆ per mg CS. Data shown are a representative of at least three independent experiments with similar results. The error bars are not shown as the data shown are from a single experiment repeated at least three times.

Figure 4

SEM images of CS (A) and the CS:InsP₆ complex (B). Arrows point to the empty spaces in CS (A) that were perhaps filled by InsP₆ (B), showing morphological changes following encapsulation. The CS:InsP₆ complex at a ratio of 2.5:1.0 was used for SEM analysis in (B). Electron micrographs shown are representative images seen in replicate experiments with similar results.

Figure 5

FTIR spectra of (a) InsP₆, (b) CS, and (c) encapsulated complex with a CS:InsP₆ ratio of 2.5:1.0. Note that the spectral properties of the characteristic bands at specific wavenumbers in InsP₆ (a) and CS (b) are changed upon encapsulation (c).

Figure 6

Cellular uptake of encapsulated InsP₆. Band intensity was analyzed by image J software. (A) shows quantitative detection of InsP₆ in the CS:IsP₆ complex with a CS:InsP₆ ratio of 2.5:1.0. A volume of 5, 10, and 20 µL of the complex loaded on the gel gave 0.15, 0.36, and 0.64 µg InsP₆, respectively, showing a concentration-dependent linear increase in the detection of InsP₆ in the complex. (B) shows a significant increase in InsP₆ uptake by MCF-7 cells using the encapsulated complex with a CS:InsP₆ ratio of 2.5:1.0 as compared to corresponding free InsP₆ and CS. Data shown are representative of experiments performed independently at least three times with similar results. Statistical analysis is not shown as the data are from a representative experiment.

Figure 7

Dose- and time-dependent induction of cell viability in MCF-7 cells by encapsulated InsP₆ treatment. Cell viability was determined at 24 h (A), 48 h (B), and 72 h (C) by MTT assay using the given doses of free InsP₆ (1.0–4.0 µM) and equivalent amounts of the CS: InsP₆ complex that would give similar doses of free InsP₆. Data are shown as means ± SD from three independent experiments. All experimental values were statistically compared with their respective controls to determine any significant differences. Only treatment with 4 µM encapsulated InsP₆ gave a significant difference as compared with 4 µM free InsP₆. * p value ≤ 0.001 or ** p ≤ 0.0001 show significantly different values as compared to the respective controls.

Figure 8

Effect of encapsulated InsP₆ on apoptosis. MCF-7 cells were incubated with 4 µM of encapsulated InsP₆ for 72 h to induce apoptosis. Etoposide (100 µM) was used as a positive control. (A) MCF-7 cells were stained with acridine orange/ethidium bromide and visualized under UV light using a fluorescent microscope. (B) The percentage of apoptosis was determined by counting 200–300 live (green) and/or dead (red) cells. Values shown are mean ±SD from three experiments, each performed in triplicate. ** p value ≤ 0.001 as compared to the control.

Figure 9

Effect of encapsulated InsP₆ on ROS generation. MCF-7 cells were treated with 4 µM free InsP₆ equivalent of the encapsulated CS:InsP₆ complex for 72 h in a 96-well microplate. Etoposide (100 µM) was used as a positive control. Cells were then stained with 10 µM DCFH-DA and fluorescence intensity was recorded using a fluorescence microplate reader. Values shown are mean ± SD from three independent experiments, each performed in triplicate. * p value of ≤0.0001 was considered significantly different compared to the control.

Figure 10

Determination of specificity of encapsulated InsP₆-induced apoptosis by flow cytometry (A). Apoptosis was measured by using a commercially available Vybrant apoptosis assay kit #4. Live cells - are shown as green in lower left quadrant and apoptotic cells are shown as blue in lower and upper right quadrant. Necrotic cells give a red color which are expected to show up in upper left quadrant. The data shown are representative of an experiment repeated at least three times with similar results. (B) shows statistical analysis results of the flow cytometry data showing mean ± standard deviation (SD) from three independent experiments. The % apoptosis values were obtained by combining early and late apoptosis values from the lower and upper right quadrants, respectively. One-way ANOVA with multiple comparisons was used to determine values that were statistically significant. **** p < 0.0001 was considered statistically significant values compared with their respective controls.