Efficiency of newly formulated camptothecin with β-cyclodextrin-EDTA-Fe3O4 nanoparticle-conjugated nanocarriers as an anti-colon cancer (HT29) drug

Abstract

Camptothecin (CPT) is an anti-cancer drug that effectively treats various cancers, including colon cancer. However, poor solubility and other drawbacks have restricted its chemotherapeutic potential. To overcome these restrictions, CPT was encapsulated in CEF (cyclodextrin-EDTA-FE₃O₄), a composite nanoparticle of magnetic iron oxide (Fe₃O₄), and β-cyclodextrin was cross-linked with ethylenediaminetetraacetic acid (EDTA). This formulation improved CPT's solubility and bioavailability for cancer cells. The use of magnetically responsive anti-cancer formulation is highly advantageous in cancer chemotherapy. The chemical characterisation of CPT-CEF was studied here. The ability of this nano-compound to induce apoptosis in HT29 colon cancer cells and A549 lung cancer cells was evaluated. The dose-dependent cytotoxicity of CPT-CEF was shown using MTT. Propidium iodide and Annexin V staining, mitochondrial membrane depolarisation (JC-1 dye), and caspase-3 activity were assayed to detect apoptosis in CPT-CEF-treated cancer cells. Cell cycle analysis also showed G1 phase arrest, which indicated possible synergistic effects of the nano-carrier. These study results show that CPT-CEF causes a dose-dependent cell viability reduction in HT29 and A549 cells and induces apoptosis in colon cancer cells via caspase-3 activation. These data strongly suggest that CPT could be used as a major nanocarrier for CPT to effectively treat colon cancer.

Figure 1

FT-IR spectra for (A) Fe₃O₄, (B) β-CD-EDTA-Fe₃O₄, and (C) β-CD-EDTA-Fe₃O₄/CPT nanocarriers.

Figure 2

TEM of (a) Fe₃O_4; (b) β-CD-EDTA; (c) β-CD-EDTA-Fe₃O₄; (d) β-CD-EDTA-Fe₃O₄/CPT Nanocarriers.

Figure 3

In-vitro drug release analysis of β-CD-EDTA-Fe₃O₄/CPT at pH 2.4 (a) and at pH 7.0 (b).

Figure 4

Magnetic properties of CPT-CEF determined through magnetometer.

Figure 5

(a) The effect of CPT-CEF on cell viability of HT29 colon cancer cells and (b) The effect of CPT-CEF on cell viability of A549 lung cancer cells. Cells were treated with various concentrations of CPT-CEF, CPT, CEF and Fe₃O₄ for 48 h, and cell viability was assessed via MTT assay. CPT-CEF reduced cell viability in HT29 cells in a dose-dependent manner. Results (mean ± SD) were calculated as percent of corresponding control values. *P < 0.05 is significant. Statistical analysis was performed via ANOVA. Each point represents three independent experiments.

Figure 6

Microscopy of HT29 (a,b,c,d,e,f) and A549 (g,h,i,j,k,l) cells treated with CPT-CEF to show changes in cell morphology. Cells were exposed to the IC₅₀ concentration of CPT-CEF and viewed at 24 and 48 h. Panel a and g (Untreated cells at 24 h), b and h (IC₅₀ treated cells for 24 h), c and i (Untreated cells at 48 h), and d and j (IC₅₀ treated cells for 48 h). Under phase contrast microscopy, cell morphology changes in treated cells are clearly visible in comparison to untreated cells; the cell membrane is evidently damaged. Panels’ e and f show fluorescent micrographs of AO/PI double-stained human colorectal cancer cells (HT29). Cells were exposed to the IC₅₀ concentration of CPT-CEF (panel f and i) or vehicle (panel e and k) for 48 h. Cell apoptosis was assayed by AO/PI staining to detect chromosomal condensation (CC), late stage apoptosis (LS), necrosis (N), and membrane blebbing (MB), as shown in the micrograph. Microscope magnification × 100 and scale bar of 10 µm (HT29) and scale bar of 100 µm (A549) were applied for the images.

Figure 7

Annexin V/PI assay showing the apoptosis-inducing effect of CPT-CEF. HT29 cells were treated with various concentrations of CPT-CEF: ¼ IC₅₀ (panel b), ½IC₅₀ (panel c), IC₅₀ (panel d), or untreated (panel a) for 48 h, and viability was assessed via Annexin V/PI assay and flow cytometry.

Figure 8

Annexin V/PI assay showing the apoptosis-inducing effect of CPT-CEF. A549 cells were treated with various concentrations of CPT-CEF: ¼ IC₅₀ (panel b), IC₅₀ (panel c), or untreated (panel a) for 48 h, and viability was assessed via Annexin V/PI assay and flow cytometry.

Figure 9

Mitochondrial depolarisation initiated by CPT-CEF. As analysed with JC-1 dye and flow cytometry, CPT-CEF treatments of HT29 cells with concentrations of ¼ IC₅₀ (panel b), ½ IC₅₀ (panel c), IC₅₀ (panel d), or untreated (panel a) for 48 h are shown.

Figure 10

Mitochondrial depolarisation initiated by CPT-CEF. As analysed with JC-1 dye and flow cytometry, CPT-CEF treatments of A549 cells with concentrations of ½ IC₅₀ (panel b), IC₅₀ (panel c), or untreated (panel a) for 48 h are shown.

Figure 11

Caspase-3 induction in various CPT-CEF-treated HT29 cells. Graph shows the percentage of induction of caspase-3 activity in CPT-CEF-treated HT29 cells at a concentration of ¼ IC₅₀, ½ IC₅₀, IC₅₀ and untreated for 48 h. Results are presented as mean ± S.D. (n = 3). *P < 0.05, compared with the control group (Untreated).

Figure 12

The cell cycle arrest caused by CPT-CEF in a concentration-dependent manner in HT29 cells. (a) Untreated, (b) 1/2 IC₅₀ treated cells and (c) IC₅₀ treated HT29 cells. (d) The graphical representation of the trend in cell cycle arrest of HT29 cells in response to CPT-CEF treatment.

Figure 13

The cell cycle arrest caused by CPT-CEF in a concentration-dependent manner in A549 cells. (a) Untreated, (b) 1/2 IC₅₀ treated cells and (c) IC₅₀ treated A549 cells. (d) The graphical representation of the trend in cell cycle arrest of A549 cells in response to CPT-CEF treatment.

Figure 14

Microscopy of HT29 cells treated with CPT-CEF with magnets. Cells were exposed to the IC₅₀ concentration of CPT-CEF and viewed at 48 h. Panel a (untreated HT29 cells at 48 h), (b) (IC₅₀ treated HT29 cells for 48 h with magnets), (c) (IC₅₀ treated HT29 cells for 48 h without magnets), (d) (Treated HT29-T75 flask with magnets placed under one side of the flask and another side of the flask without magnets). Under phase contrast microscopy, cells treated under magnetic environment shows significant changes in cell to cell attachment in comparison to treated cells without the influence of magnets. Microscope magnification × 100 and scale bar of 100 µm were applied for all the images.