Nanoparticle-Encapsulated Tamoxifen Inducing Cytotoxic Effect on Mcf-7 Breast Cancer Cell Lines A.M. Akim1, E.E. Tung1, P.P. Chong1, M.Y. Hamzah2, and K.Z.M. Dahlan2 1
Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400, Selangor, Malaysia 2 General Research and Technology Development, Malaysian Nuclear Agency, Bangi 43000 Selangor, Malaysia
Abstract –– Nanoparticle in drug delivery offers major improvements to those limitations by providing longer circulation times, greater specificity, and enhances anticancer effects. Thus, the objective of this study is to synthesize a polymeric nanoparticle encapsulating TMX, and investigate the in vitro characteristics on breast cancer cell line, MCF-7. Materials and Methods. The nanoparticles were synthesized through gamma irradiation polymerization between Nisopropylacrylamide (NIPAAM), N-vinyl-2-pyrrolidone (VP), poly(ethyleneglycol) diacrylate (PEG-DA). TMX was directly loaded into the nanoparticles by random physical entrapment. Photon Cross Correlation Spectroscopy Nanophox (PCCSNanophox) and Transmission Electron Microscopcy (TEM) were used to observe size and morphology of the nanoparticles. To study the in vitro effects, MCF-7 cells were treated with TMX-nanoparticles and its cytotoxicity effects were investigated through MTT assay. Results and Discussion. From the results, it was confirmed that nanoparticles at 0.055M subjected to 8kGy of gamma irradiation posses a consistent size distribution of 49.89 nm SD ±1.82. This was further confirmed through pictures taken with TEM in which the nanoparticles had a clearly distinguishable core-shell structure, with a spherical or elliptical shape with smooth surface. Through the cytotoxicity assay, it showed that TMXnanoparticles had a greater inhibitory effect compared to TMX alone at 48 hrs and 72 hrs. There was also no evidence of toxicity by void polymeric nanoparticles. Keywords — Polymeric nanoparticle, gamma irradiation, Tamoxifen, TMX-nanoparticles, MCF-7, breast cancer.
I.
INTRODUCTION
Many new materials and devices were created in a wide range of applications. One of the emerging strategies that have been receiving a lot of attention is the use of nanoparticle systems in the treatment of cancer. Through prior studies, it was suggested that these nanoparticles in drug delivery offers major improvements to those limitations by providing longer circulation times, greater specificity, and enhances anticancer effects[1][2]. Hence, nanoparticles conjugated anticancer drugs can be a promising and potential
strategy in the treatment of breast cancer. Tamoxifen (TMX) is one of the most common anti-cancer drug in breast cancer treatment. On breast cancer tissue, it acts as an anti-estrogen. However, upon other tissue such as the uterus, it acts as an estrogen [3]. Other side effects include liver cancer, blood clotting, retinopathy and corneal opacities which is dependent to be dose dependent. This suggests the use of a lower dose of TMX and a better delivery system to enhance its efficiency in breast cancer treatment. II. MATERIALS AND METHODS A.
Reagent
NIPAAM, VP, PEG-DA, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenul tetrazolium bromide] (MTT) and tamoxifen were purchased from Sigma, USA. B.
Cell Line
MCF-7 breast cancer cell line was purchased from American Type Culture Collection (ATCC), Rockville, Maryland, USA. The cell lines were maintained in DMEM 4.5g/l Glucose culture medium supplemented with 10% FBS and 1% of antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin). The cells were grown in tissue culture flasks in the incubator at humidified atmosphere containing 5% CO2 and 95% air, at 37°C. C.
Synthesis of Polymeric Nanoparticles
Polymeric nanoparticles were prepared from NIPAAM, VP and PEG-DA at 90:5:5 molar ratios ranging from 0.01M-0.08M. The samples were subjected to gamma irradiation at doses ranging from 2kGy to 50kGy. The size and stability of the nanoparticles were detected through Photon Cross Correlation Spectroscopy Nanophox (PCCSNanophox).
V. Van Toi et al. (Eds.): 4th International Conference on Biomedical Engineering in Vietnam, IFMBE Proceedings 40, pp. 226–228, 2013. www.springerlink.com
Nanoparticle-Encapsulated Tamoxifen Inducing Cytotoxic Effect on Mcf-7 Breast Cancer Cell Lines
D.
Transmission Eletron Microscopy (TEM)
TEM pictures of the nanoparticles sample were taken under JEM 2100 TEM instrument operating at magnification of 100kV. A drop of aqueous solution of the sample was placed on a membrane grid surface. Excess fluid was removed using a filter paper. The grid surface was air dried at room temperature before loaded into the microscope.
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nanoparticles across the dose range. On the other hand, both TMX and TMX-nanoparticles effectively inhibited the growth of MCF-7 cells when compared to untreated cells and cells exposed to void nanoparticles. At 48 hours and 72 hours, the effect of TMX-nanoparticles was more pronounced than TMX alone, giving a lower IC50 20.5 20.0 19.5 19.0 18.5
NIPAAM-VP-PEGDA 0.05M at 8kGy
18.0 17.5 17.0 16.5 16.0 15.5 15.0 14.5 14.0 13.5 13.0
Loading of Drug into Nanoparticles
12.5 12.0 11.5 Correlation / %
E.
9.5 9.0 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0.0001
0.0005
0.0010
0.005
0.010
0.05 0.10 Lag time / ms
0.5
1.0
5
10
Fig. 1 Size particles and stability graph of void nanoparticles from Photon
Cell Viability Assays
Cross Correlation Spectroscopy-Nanophox.
Growth inhibition of cells was measured through MTT assay, which relies on the conversion of MTT, 3-[4,5dimethylthiazol-2-yl]-2,5-diphenul tetrazolium bromide], to a purple coloured formazan product by the activity of living cells. 1 x105 cells/ml of MCF-7 cells were plated in 96 well plates. The cells were treated with void nanoparticles, TMX and TMX-nanoparticles at concentrations ranging from 0 to 60 µM for 24, 48 and 72 hours. MTT assay was performed by adding 50ul of MTT (2mg/ml) into each well. After 4 hours incubation, the treatment were removed and 100ul of dimentyl sulphoxide (DMSO) were added to solubilize the purple formazen formed. The readings were measured by microplate reader. From the results, the relative growth inhibition was determined. III.
10.0
8.5
TMX were loaded in the polymeric nanoparticles by using a post-polymerization method where TMX is directly loaded into the nanoparticles by random physical entrapment through constant vortexing or mild sonication. F.
11.0 10.5
Fig. 2 Picture of void nanoparticle taken from Tranmission Electron Microscope.
RESULTS AND DISCUSSIONS
The particle size and stability of nanoparticles measured with PCCS-Nanophox as shown in Fig. 1. At a concentration of 0.055M of NIPAAM-VP-PEGDA at 8kGy of gamma irradiation, the nanoparticles that were formed have a very consistent size with average of 49.89nm SD± 1.82. This is further confirmed through TEM of the nanoparticles is illustrated in Fig. 2. From the picture, it was demonstrated that the nanoparticles synthesized have a spherical morphology, with clear distinguishable core-shell structure. It has a smooth surface with an approximate size of 47nm core and 86nm outer shell. It is important that a drug delivery platform must be biodegradable, biocompatible and non toxic towards cells. Therefore, the toxicity of void nanoparticles was also studied. From the results showed in Fig. 3, 4 and 5 there was no evidence of toxicity associated with the void
Fig. 3 MTT result of nanoparticle after 24 hours.
Fig. 4 MTT result of nanoparticle after 48 hours.
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Fig. 5 MTT result of nanoparticle after 72 hours. IV. CONCLUSIONS The nanoparticles are non-toxic ideal as a drug delivery platform and provide an opportunity for other diseases and drugs.
ACKNOWLEDGMENT The author would like to acknowledge Universiti Putra Malaysia and Agensi Nuklear Malaysia for the facilities that they provide for this research.
REFERENCES 1. Byrne, J. D., Betancourt, T., and Peppas, L. B. (2008). Active targeting schemes for nanoparticle systems in cancer therapeutics. Advanced Drug Delivery Review 60, 1615-1626. 2. Wang, A. and Li, S. (2008). Hydroxycamptothecin-loaded nanoparticles enhance target drug delivery and anticancer effect. BMC Biotechnology 8 (46). 3. Cohen, I. 2004. Endometrial pathologies associated with postmenopausal tamoxifen treatment, Gynecologic Oncology 94,256–266. Author: Institute: Street: City: Country: Email:
Abdah Md Akim Universiti Putra Malaysia Faculty of Medicine and Health Sciences Serdang Malaysia
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