DISCUSSION

For the photodynamic therapy light source was used to activate the nanocomposite and the
activated nanocomposite was used as cytotoxic factor for the cultured cancer cell.

The intensity and wavelength of the light source were important parameters for the
photodynamic therapy. The intensity of light normal Led was unknown so it was measured using
radio-spectrometer. Upon the measurement it was known that the wavelength and intensity of
normal led varies largely even if it produce same colour of light and same power is supplied. The
Leds with similar wavelength and intensity were selected and used for the light source.

The normal blue Led were arranged in the matrix. The intensity of the Led was kept constant
around by controlling the current. The constant current of 20 mA was produced by using the
power source that was designed to give around 25 V output. The components used for designing
the power source were LM 317 IC, transformer, resistor and potentiometer. The output voltage
was supplied parallel to each row of the Led so that each row gets the equal potential. The
potentiometers were individually arranged in each row was so adjusted that the current in each
row was 20 mA. Since the power source was meant to be used for long time period heat sinks
were used as a necessary precaution.

The Fe doped TiO2 nano composites was prepared using sol-gel method. The reagents used for
preparation of nano composites were Titanium tetra isopropoxide (TTIP), ethanol, nitric acid,
distilled water and the material used were Beakers, burette, burette clamp, magnetic stirrer and
its fish. Titanium tetra isopropoxide was the main precursor used. The particle size was mainly
controlled by making the pH of water 2. Nitric acid was used to control pH. The pH of the acid
water solution was made by taking appropriate amount of nitric acid. The amount of nitric acid
was used as obtained from the equation pH=-log[H+ ]. The concentration of hydrogen ion was
taken as the concentration of nitric acid as 1 mole of HNO3 produces 1 mole of H+. During
preparation of nanoparticles special attention was given to the temperature of the mixture. The
ethanol was at first heated to 70˚C and then TTIP and water was added dropwise. The volume of
water was taken as the sum of volume of ethanol and TTIP. For more effective chemical reaction
the reactants were mixed dropwise and the mixture was stirred using the magnetic stirrer. The
reaction process was made slow such as 50ml of water was used up using 3 hours of time period.
After TTIP and water reaction ferric nitrate solution was added in the reaction mixture for
doping process. The volume of TTIP and Ferric nitrate was kept contant but their molar ratio
was varied as per the doping ratio. Assumed particle size of the product was obtained after 2%
and 5% doping of Ferric nitrate but the large particle size was obtained when it was 10% doped.
It may be due to increase in doping of iron particles in titanium dioxide. The particles size was
determined by X-ray diffraction method. The graph obtained was compared with the graphs
observed in various literatures and the particle sizes were calculated accordingly by using
Scherrer’s Formula. UV spectroscopy was taken for 3 differently doped samples and one
undoped sample. The absorbance of the doped samples was found to be red shifted with respect
to the increased doping ratio. The main problem faced during UV spectroscopy was preparation
of the nanoparticle solution as it doesn’t get dissolved in both water and ethanol solvent. For
preparing the proper solution it requires Sonodynamic Therapy but it wasn’t available. Thus, the
colloidal suspension was only used for UV spectroscopy due to which the variation on the
obtained graph was observed although it was red shifted.

During cancer cell culture, it was difficult to obtain the required confluency of the cell. The cell
was cultured for 4 times though the required division and confluency wasn’t observed during
each culture. It was detected that the problem was due to the culture media which we then
replaced it with RPMI1640 media. After the replacement, the division and growth of the cancer
cell was adequate and required confluency was obtained.

The nanoparticles were introduced in the 96 well plates. Each well plate contained 100 l of
culture media containing 20000 cells. The problem faced was during the preparation of the
solution of media with required concentration of nanoparticles. As the appropriate solvent wasn’t
found, the colloidal suspension of nanoparticles and media was prepared and introduced into
each well plate. The concentration of the suspension was made 50, 100 and 200 ppm for each 2%
and 5% ferric nitrate doped nanoparticles. The well plates were then incubated for 4 hours and
then light exposure was given to the well plates containing the cell culture, media and
nanoparticles solution. . 3 different well plates were used for 3 different time exposures i.e. 10
minutes, 20 minutes and 1 hour respectively. The problem faced was the heating of transformer
of the power supply of the light source. The power supply was kept outside of the incubator
which might create the condition for the contamination. After exposure, the well plates were
incubated for next 24 hours.

MTT dye was prepared using DMSO and MTT powder but DMSO is also cytotoxic in nature so
special attention was given during its preparation. MTT dye was introduced to each well plate
and was incubated for 4 hours. Then formazol formation was observed and was dissolved using
DMSO. After it was dissolved, absorbance was taken using ELISA reader. The large value of
absorbance represented less number of cell death while the smaller value represented increased
value of cell death. The data obtained from the ELISA reader was used for statistical analysis
and variance was determined using one way ANOVA method.