(676f) Novel Fluorescent Nano Structures for Bio-Imaging of MCF-7 Cells

Novel
Fluorescent Nano Structures for Bioimaging of MCF-7 Cells

Aishee
Dey*, Lopamudra Giri^# and Sudarsan Neogi*

*
Indian Institute of Technology, Kharagpur

#
Indian Institute of Technology, Hyderabad

Although
nanomaterials have a wide area of application including targeted drug delivery,
diagnostic imaging and controlled release, the  design of nanomaterials having
multimodal applications remains challenging.  The recent trend in the field of
nanomedicine revolves around development of in vivo dignostics and therapeutics
that can be used as theranostics. In order to achieve this, we performed the
optimization of the fabrication process at multiple levels.The major objective
of the current work is to tune unique physicochemical properties in nanostructures
through proper selection of fabrication methods, that can be used as
therapeutic as well as diagnostic tools. Here we choose to develop a fluorescent
imaging sensor based on metal oxide nanoparticles that can be used to study the
nanoparticle uptake in cells and penetration of nanoparticles through tissue through
laser scanning confocal microscopy [1]. Specifically, we choose to use semiconductor
quantum dots or metal oxide nanoparticles as they can be designed to have
optical, magnetic, and structural properties that are different from the same
particles in their bulk form [2].
Here we choose to use ZnO nanoparticles as they are known to provide a targeted
delivery which is beneficial for cancer patients as they can differentiate
between cancer cell and normal cells, and hence avoid their adverse effects on
normal cells.Secondly, we chose to use ZnO nanoparticles because of their
excellent bio-compatibility [3]. Another feature of the proposed work is that
the targeted approach requires minimal drug loading in contrary to large drug
loading required in case of non-targeted approach [4].
The major novelty of the work lies in achieving the multimodalities in a single
nanostructure, where we manipulated the optical properties of ZnO so that it
can be used as therapeutic reagent as well as imaging sensor to study the cell
uptake and penetration of particles.

Here
we propose a surfactant assisted method to synthsize the fluorescent ZnO
nanoparticles. ZnO particles were formed using chemical synthesis method by
mixing zinc nitrate hexahydrate and ammonium carbonate solutions along with a
surfactant Tween 80 [5]. 
Since the objective of the work was to maximize the drug efficacy  along with
maintenance of the fluorescent level of the nanostructures, a series of
experiment were performed for nanoparticle fabrication using various
concentrations of Tween-80.  The result clearly indicates that with increasing amount
of Tween 80 present in the mixture, the morphology of the nanoparticles changed
along with the fluorescence of the particles. The fluorescent nature of the
particles increased with increase in Tween 80 which was evident from the
confocal microscope imaging, the best being observed at Tween 80 concentration
of 14% (v/v) (ZnOT14) . In the excitation wavelength of 488 nm and an emission
range of 508-550 nm, the nanoparticles showed green fluorescence as shown in
figure 1. The nanoparticles were further characterized by SEM, TEM, XRD and
FTIR techniques. Images from the
SEM and TEM show that with change in Tween 80 concentration, the size of the
nanoparticles change as demonstrated in figure 2. XRD results confirm the formation of ZnO due to
presence of prominent peaks at 31.9°, 34.6°, 36.61°, 47.88°, 68.34°, 69.1°
which are in good agreement with JCPDS 89-0511. As per FTIR results, broad peak
at 3500 cm^-1 indicating the presence of hydroxyl groups
intensified on addition of Tween 80, as the surfactant introduces additional hydroxyl groups on the surface of the
nanoparticles. The
ZnO nanoparticle with Tween 80 concentration of 14% (v/v) was found to be
highly haemocompatible (0.031% haemolysis), indicating its non-toxicity. Secondly,
the nanoparticles were used for imaging of MCF-7 cells which are a breast
cancer cell line. Breast cancer is one of the most common cancer found in
women. Breast cancer can lead to metastasis which is the secondary malignant
growth from the primary cancer site. With progress in metastases of breast
cancer, some organs like lungs, liver and bone get damaged [6]. Thus imaging of the cells by nanoparticles is important as
it can lead to proper detection of the cancer tissues. Also the MCF-7 is an
immortal cell line which do not proliferate indefinitely and can be grown in
vitro. The cells were cultured in a media containing 44.5 ml Dulbecco’s
modified eagle’s medium , 5 ml of 10% fetal bovine serum and 0.5 ml of 1%
penstrap antibody in a 50 ml media. The cells were cultured for 4 days. Further
the cells were treated with the synthesized ZnO nanoparticles and imaged using
confocal microscope.

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Figure 1:
Variation of fluorescence of ZnO nanoparticles with Tween 80 concentration of
(a) 0 % (v/v), (b) 6 % (v/v), (c) 12 %(v/v) and (d) 14 %(v/v).

Figure 2:
FESEM images of ZnO nanoparticles with Tween 80 concentration of (a) 0 % (v/v),
(b) 6 % (v/v), (c) 12 %(v/v) and (d) 14 %(v/v).

Figure 3:
(a) Histogram showing percentage haemolysis of positive control, negative
control and ZnOT14;          (b) Original images of haemolysis experiment
showing ZnOT14, positive control and negative contol.

References

[1]       T. Xia et al., “Comparison
of the Mechanism of Toxicity of Zinc Oxide and Cerium oxide Nanoparticles Based
on Dissolution and Oxidative Stress Properties, ” ACS Nano, vol. 2, no.
10, pp. 2121-2134, 2008.

[2]      D.
Groneberg, G. Michael, T. Welte and U. Pison, “Nanoparticle-based diagnosis and
therapy .,”Curr Drug Targets, vol. 7, no. 6, pp. 643-648, 2006.

[3]
     J. W. Rasmussen, E. Martinez, P. Louka,
and D. G. Wingett, "Zinc Oxide Nanoparticles for Selective Destruction of
Tumor Cells and Potential for Drug Delivery Applications ", Expert Opin
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[4]      S.
Nie, Y. Xing, G. J. Kim, and J. W. Simons, “Nanotechnology Applications in
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[5]      Y.
Khan, S. K. Durrani, M. Mehmood, J. Ahmad, M. R. Khan, and S. Firdous, “Low
temperature synthesis of fluorescent ZnO nanoparticles,” Appl. Surf. Sci.,
vol. 257, no. 5, pp. 1756–1761, 2010.

[6]      E.
Goldman et al., “Nanoparticles target early-stage breast cancer
metastasis in vivo,” Nanotechnology, vol. 28, no. 43, pp. 43LT01,
2017.