Citation
Ahmed, Hamidu
(2019)
Molecular effects of free and doxorubicin-loaded aragonite calcium carbonate nanoparticles on MCF-7 cell lines.
Doctoral thesis, Universiti Putra Malaysia.
Abstract
Cancer is one of the prime causes of death and breast cancer is the most erratic
malignant disease which causes serious burden to women worldwide. Cockle shell- derived aragonite
calcium carbonate nanoparticles (Ar-CC-NPs) encapsulated with doxorubicin (DOX) has transpire
as an efficacious therapy against breast tumour, but still the use of targeted therapy in
cancer treatment appears to be ineffective and often associated with some set back,
hence necessitates the need for improved targeted therapy. This study determines the
therapeutic potential of doxorubicin- loaded aragonite CaCO3 nanoparticles using proteomic
approach. For the proteomic study, Liquid Chromatography/Mass Spectrometry analysis
(LCMS/MS) was employed to investigate the MCF-7 related protein in human breast cancer cells
after treatment with the DOX and DOX-Ar-CC-NPs. Aragonite CaCO3 nanoparticles are synthesized from
cockle shells and characterized for physiochemical properties using Transmission electron
microscope (TEM), Field emission scanning electron microscope (FESEM), Zeta potential,
Fourier transmission infrared (FTIR) and X- ray diffraction (XRD) techniques. The aragonite
nanoparticles are synthesized from the cheaply available natural sea water cockle shells, which are
cleaned with banana peels, homogenized and then stirred vigorously in dodecyl dimethyl betane
(BS-12) solution using a rotary pulvering blending machine in order to reduce the a stringent
temperature and unsafe chemicals associated with nanoparticles production and are then
characterized for particle geometry using electron microscopy. An IC50 which is inhibitory
concentration of 50% of the tested cells that signifies drug concentration, for the indication
of cell viability was also determined and then, the synthesized aragonite calcium
carbonate nanoparticles (Ar-CC-NPs) are then loaded with doxorubicin (DOX), an
antineoplastic agent, which formed Doxorubicin-aragonite- nanoparticles (DOX-Ar-CC-NPs). The
cytotoxic effect of DOX-Ar-CC-NPs was determined using superoxide dismutase commercial
ELISA kit for cell membrane integrity, and flow cytometry, fluorescent imaging and
electron microscopy for programmed cells death evaluation. Enzymes-linked immunosorbent assay was also
used to assay oxidative stress biomarkers and apoptotic enzymes from MCF-7 cell treated using
DOX-Ar-CC-NPs and the proteomic profile of cancer cell treated with DOX-Ar-CC-NPs are examined
and evaluated the release profile, cytotoxicity and uptake, in pursuance of understand the
molecular sound effects of free and DOX-Ar- CC-NPs on MCF-7 cell line and to further
improve the anticancer effects of doxorubicin through early prediction and resolution of its
drug resistance problems. The results of the study shows that Ar-CC-NPs with average diameter of
35.5 nm, 19.3% loading content and 97% encapsulation efficiency has a surface potential and
intensity of -19 ± 3.9 mV and 100%, respectively. In addition, DOX-Ar-CC-NPs have an
IC50 at 24, 48 and 72 hours of 1.829µg/mL, 0.902µg/mL and 1.0377µg/mL while that of DOX alone
were 0.475µg/mL, 0.2483µg/mL and 0.0723µg/mL, respectively. However, even at higher
concentration, no apparent toxicity was recorded with Ar-CC-NPs, which reveals its anticancer
effect with MCF-7 cells with a viability of 92%. The DOX-Ar-CC-NPs had significant
inhibitory effect on cell feasibility compared to DOX alone (p<0.05), similar trend was
noticed in cellular apoptosis, oxidative stress markers and cellular uptake
evaluation. However, treatment with DOX-Ar-CC-NPs significantly decreased the
elevated level of superoxide dismutase 2 (SOD2) compared to untreated MCF-7 cells.
Thus, the present findings revealed the capability of DOX-Ar-CC-NPs to induce apoptosis in MCF-7
cells, which indicates the high potency of Ar-CC-NPs in drug delivery. For the proteomic study, a
total of 408 MCF-7 related proteins for DOX-Ar-CC-NPs and 128 proteins for DOX alone were
identified from MCF-7 cells. The proteomic profiling analysis outcomes reveals new
developments for the advancement of proteomics technologies which could yield a good result
on discovery of potential significant breast cancer biomarkers for MCF-7 cells pertinent studies
with archival samples. Shotgun LC-MS/MS studies could also serve to determine new biomarkers.
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