Synthesis, Characterization and In Vitro Study of Biocompatible Cinnamaldehyde Functionalized Magnetite Nanoparticles (CPGF Nps) For Hyperthermia and Drug Delivery Applications in Breast Cancer

<div><p>Cinnamaldehyde, the bioactive component of the spice cinnamon, and its derivatives have been shown to possess anti-cancer activity against various cancer cell lines. However, its hydrophobic nature invites attention for efficient drug delivery systems that would enhance the bioavailability of cinnamaldehyde without affecting its bioactivity. Here, we report the synthesis of stable aqueous suspension of cinnamaldehyde tagged Fe₃O₄ nanoparticles capped with glycine and pluronic polymer (CPGF NPs) for their potential application in drug delivery and hyperthermia in breast cancer. The monodispersed superparamagnetic NPs had an average particulate size of ∼20 nm. TGA data revealed the drug payload of ∼18%. Compared to the free cinnamaldehyde, CPGF NPs reduced the viability of breast cancer cell lines, MCF7 and MDAMB231, at lower doses of cinnamaldehyde suggesting its increased bioavailability and in turn its therapeutic efficacy in the cells. Interestingly, the NPs were non-toxic to the non-cancerous HEK293 and MCF10A cell lines compared to the free cinnamaldehyde. The novelty of CPGF nanoparticulate system was that it could induce cytotoxicity in both ER/PR positive/Her2 negative (MCF7) and ER/PR negative/Her2 negative (MDAMB231) breast cancer cells, the latter being insensitive to most of the chemotherapeutic drugs. The NPs decreased the growth of the breast cancer cells in a dose-dependent manner and altered their migration through reduction in MMP-2 expression. CPGF NPs also decreased the expression of VEGF, an important oncomarker of tumor angiogenesis. They induced apoptosis in breast cancer cells through loss of mitochondrial membrane potential and activation of caspase-3. Interestingly, upon exposure to the radiofrequency waves, the NPs heated up to 41.6°C within 1 min, suggesting their promise as a magnetic hyperthermia agent. All these findings indicate that CPGF NPs prove to be potential nano-chemotherapeutic agents in breast cancer.</p></div

Molecular mechanism underlying anticancer activity of CPGF NPs.

<p>CPGF NPs decreased migration and expression of tumor marker proteins in breast cancer cells. (<b>A</b>) The NPs reduced migration of MDAMB231 and MCF7 cells. The upper panel of each figure shows the wound made at 0 h and the lower panel shows the migration of cells after 18 h. (<b>B</b>) Graphical representation of wound closure in MDAMB231 and MCF7 at 18 h after CPGF NPs treatment. Values are represented as mean±SD of three independent experiments at p<0.01 for MDAMB231 and p<0.0005 for MCF7, indicating statistically significant differences compared to the untreated control cells. (<b>C</b>) Gelatin zymography shows downregulation of MMP-2 expression in MDAMB231 and MCF7 after treatment with CPGF NPs with their corresponding densitometric analysis. The values are represented as mean±SD of three independent experiments at p<0.001, indicating statistically significant differences compared to the untreated control cells. (<b>D</b>) Western blot analysis shows decrease in VEGF expression in MDAMB231 and MCF7 with their corresponding densitometric analysis. Values are represented as mean±SD of three independent experiments with p<0.0001, indicating statistically significant differences compared to the untreated control cells.</p

Cytotoxicity analysis of CPGF NPs and cinnamaldehyde.

<p>The breast cancer (MDAMB231 and MCF7) and non-cancerous (MCF10A and HEK293) cells were treated with different concentrations of (<b>A</b>) CPGF NPs and (<b>B</b>) cinnamaldehyde for 24 h and anlayzed for viability by MTT assay. Lower panel of X-axis in (<b>A</b>) refers to amount of cinnamaldehyde (µM) present in the corresponding concentrations of CPGF NPs (µg/ml) (as calculated from TGA data). All the data are presented as mean±SD of five independent experiments at p<0.0001, indicating statistically significant differences compared to the control untreated group.</p

Physical characterization of CPGF NPs.

<p>(<b>A</b>) XRD patterns of F, GF, PGF and CPGF NPs. (<b>B</b>) TEM image with SAED of CPGF NPs. (<b>C</b>) FTIR patterns of F, GF, PGF and CPGF NPs.</p

Cinnamaldehyde loading and release profiles along with magnetic behavior of CPGF NPs.

<p>(<b>A</b>) Thermogravimetric analysis of F, GF, PGF and CPGF NPs. (<b>B</b>) Release profile of cinnamaldehyde from CPGF NPs at different pH values at different time points. (<b>C</b>) Inset shows uniform suspension of CPGF NPs (i) and (ii) response of the NPs to the externally placed magnet. VSM data shows the magnetic behavior of all the NPs.</p

Response of CPGF NPs to the radiofrequency waves for hyperthermia application.

<p>CPGF NPs exhibit hyperthermia potential. Response of Fe₃O₄ (F), Glycine (G), Pluronic (P), Cinnamaldehyde (C) and CPGF NPs to the radiofrequency waves have been depicted. The NPs showed a significant rise in the temperature to 41.6°C within a time span of 1 min.</p

Schematic illustration of synthesis of CPGF NPs and their release in breast cancer cell.

<p>Schematic illustration of synthesis of CPGF NPs and their release in breast cancer cell.</p

Stability, biocompatibility and uptake of CPGF NPs.

<p>(<b>A</b>) Stability of CPGF NPs in different solutions was monitored by UV-vis spectroscopy. The data is presented as mean±SD of three independent experiments at p<0.0001, indicating statistically significant differences between different solutions. (<b>B</b>) Biocompatibility of CPGF NPs performed in freshly prepared human blood. DW and saline were used as positive and negative controls, respectively. The data is presented as mean±SD of three independent experiments at p<0.001, indicating statistically significant differences compared to positive control group. (<b>C</b>) In vitro uptake of CPGF NPs in MDAMB231 and MCF7, shown by Prussian blue staining, and compared with their respective untreated control cells. The stained cells were photographed with Sony DSC-S75 cyber-shot camera.</p

CPGF NPs induce apoptosis in breast cancer cells.

<p>(<b>A</b>) CPGF NPs decreased the mitochondrial membrane potential of the breast cancer cell lines as observed by JC-1 staining. The data was analyzed by the MARS data analysis software 2.10R3 (BMG Labtech). All data are presented as means±SD of three independent experiments. p<0.0001 indicate statistically significant differences compared to the control untreated group. (<b>B</b>) Western blot data shows increase in caspase-3 expressions in both MDAMB231 and MCF7. α-Tubulin was used as a loading control. The histogram depicts densitometric analysis of western blots of caspase-3. Values are represented as mean±SD of three independent experiments. p<0.001 indicate statistically significant differences compared to the untreated control cells.</p

CPGF NPs decrease the growth kinetics of breast cancer cells.

<p>(<b>A</b>) The NPs decreased the number of cells in MDAMB231 and MCF7, indicated by cell growth assay. Cells were counted from the four quadrants and the average of each has been plotted. The data represents mean±SD of three independent experiments at p<0.0001 indicating statistically significant differences compared to the control untreated group. (<b>B</b>) Reduction in the number of soft agar colonies was observed in MDAMB231 and MCF7 after treatment with CPGF NPs. Colonies were counted from at least 10 different areas and the average of each has been plotted. The data represents mean±SD of three independent experiments at p<0.0001 indicating statistically significant differences compared to the control untreated group.</p