Iron oxide- Indocyanine green based magneto-optical nanocomposites for potential multi-modal applications in cancer therapeutics

Lamichhane, Nisha (2022) Iron oxide- Indocyanine green based magneto-optical nanocomposites for potential multi-modal applications in cancer therapeutics. Doctoral thesis, University of Central Lancashire.

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Magneto-optical nanocomposites possess properties of both magnetic and optical components as novel nanomedicines and demonstrate an immense potential in cancer therapeutics under the application of external stimuli such as alternating magnetic field (AMF) and near-infrared (NIR) laser irradiation. In this thesis, two different hydrophilic and one hydrophobic iron oxide nanoparticles (IONPs) types synthesised by modified co-precipitation methods, acted as a magnetic component. Synthesised IONPs cores (IO1, IO2 and IO3) were further coated with mesoporous silica shell using hexadecyl trimethyl ammonium bromide (CTAB) as a surface directing agent. Upon removal of CTAB from the mesopores by acidic ethanolic washing, the resultant magnetic silica (MS) nanocomposites (MS1, MS2 and MS3) were utilised for loading indocyanine green (ICG), acting as an optical component. The resultant ICG loaded magnetic silica nanocomposites (MSICG) nanocomposites (MS1ICG, MS2ICG and MS3ICG) were novel magneto-optical nanocomposites. Synthesised IONPs, MS and MSICG nanocomposites were extensively characterised and tested for their performance in magnetic hyperthermia therapy (MHT), photothermal therapy (PTT) and photodynamic therapy (PDT) in-vitro using commercial MCF7 breast cancer cell lines.
All the synthesised IONPs were spherical in morphology with superparamagnetic properties. Hydrophilic IONPs (IO1 and IO2) exhibited higher saturation magnetisation of 63.6 emu/g and 59.4 emu/g compared to hydrophobic IONPs (IO3) of 49.3 emu/g. Zeta potential measurements indicated that the surface of the IONPs were positively charged. The distinct XRD patterns corresponds to iron oxides. All IONPs showed a distinct Fe-O bond vibration at 550 cm-1 from FTIR analysis due to magnetite phase. In addition, hydrophobic IONPs showed peaks at 2925 and 2852 cm-1, corresponding to -CH2 stretching vibrations due to the presence of oleic acid. Following silica coating, the MS nanocomposites were nearly spherical with increased average size. The MS nanocomposites with hydrophobic IONPs had average size of 38 nm from TEM analysis and showed a thin layer of silica coating around magnetic core. Upon silica coating, the surface charge of the MS nanocomposites reversed from positive (around +19 mV for IONPs) to negative (around -23 mV) charge confirming the formation of core-shell nanocomposites. The presence of a silica shell around magnetic core was further confirmed with characteristic bond vibrations at 1080 and 795 cm-1 equivalent to Si-O-Si stretching by FTIR. The mesoporous silica shell dramatically enhanced the surface area of magnetic core due to the internal porosity in the nanocomposites. MS1 nanocomposites had the highest value of BET surface area 965 m2g-1 with mesopores diameter of around 3 nm. The saturation magnetisation values reduced significantly in MS nanocomposites when hydrophilic IONPs were used as cores. Whereas MS3 nanocomposites containing hydrophobic magnetic core showed relatively high saturation magnetisation value of 44.51 emu/g. Furthermore, ICG loading efficiency was dependent on surface area and showed higher loading in MS1ICG nanocomposites with encapsulation efficiency of 68.6% compared to MS2ICG (23.4%) and MS3ICG (32.2%)., The presence of encapsulated ICG in MSICG nanocomposites was confirmed by FTIR (a small peak at 1409 cm-1 due to N-H bending) and TGA weight loss of about 1.55% equivalent to calculated loading value from UV-vis spectrophotometer.
IO2 nanoparticles showed better heating efficiency by reaching the maximum set temperature of 42 ℃ within 88 seconds compared to IO1 at 196 seconds with SPA values of IO1- 35.8 W/g and IO2- 94.1 W/g under an AMF. Similarly, the MS2 nanocomposites showed better heating efficiency by reaching 42 ℃ within 129 seconds with SPA values of 58.1 W/g compared to MS1 (6.8 W/g) and MS3 (25.2 W/g). The results indicated the materials ability to magnetic hyperthermia therapy (MHT) as potential cancer therapeutics. Similarly, the MSICG showed heating efficiency under NIR laser irradiation (wavelength- 808 nm, power density- 1.2 W/cm2). An increase in temperature of up to 22 ℃ in 6 minutes for MS3ICG when compared with MS1ICG (19 ℃) and MS2ICG (13 ℃). The results indicated the presence of ICG as a photosensitiser increased the materials ability for PDT/PTT. Therefore, application of both AMF and laser as external stimuli can be considered as multimodal routes in cancer therapeutics.
Furthermore, MS2ICG and MS3ICG nanocomposites were systematically studied for their therapeutic efficiency in-vitro using a commercial breast cancer cell line, MCF7. Cultured MCF7 cells treated with MS2ICG and MS3ICG nanocomposites in the presence of AMF and laser irradiation showed higher cancer cell killing efficiency with potential for dual cancer therapeutics. Further evaluation of MCF7 cells treated with laser irradiation alone showed effective dose and time dependent cancer cells killing efficiency. Further investigation of endocytosis using different endocytic inhibitors suggested the nanocomposites internalisation was an active energy dependent pathway followed by multiple other pathways, mainly clathrin-mediated endocytosis.
Furthermore, the assessment of oxidative stress in MCF7 cells upon treatment with nanocomposites in the absence and presence of external stimuli (laser irradiation) using cellular integrity markers and oxidative stress markers showed the presence of different reactive oxygen species (ROS) and reactive nitrogen species (RNS), responsible for cellular damage. The elevated value of lactase dehydrogenase (LDH) and lipid peroxidation (LPO) suggested the cellular damage caused due to the ROS generation. The DCFDA (2’,7’-dichlorodihydrofluorescein diacetate) assay provided further evidence of ROS generation via higher fluorescence in cells treated with MSICG nanocomposites. Finally, both MSICG nanocomposites tested for apoptotic gene expression by RTPCR showed the elevated pro-apoptotic genes such as p53, Bax and a decrease in anti-apoptotic gene Bcl-2.
In conclusion, the synthesised magneto-optical nanocomposites (MSICG) exhibited efficient MHT/PDT/PTT effects due to the presence of both magnetic and optical components and opened an avenue for further investigation in-vivo with potential for cancer therapeutics.

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