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Somayeh Karimi,1,* Mohamad Hassan Fouani,2,* Ahmad Moshaii,1 Maryam Nikkhah,2 Saman Hosseinkhani,3 Reza Sheikhnejad4 1Department of Physics, Tarbiat Modares University, Tehran, Iran; 2Department of Nanobiotechnology, Tarbiat Modares University, Tehran, Iran; 3Department of Biochemistry, Tarbiat Modares University, Tehran, Iran; 4Department of Molecular Biology, Tofigh Daru Engineering-Research Co., Tehran, Iran*These authors contributed equally to this workCorrespondence: Ahmad Moshaii; Maryam Nikkhah Tel +98 21 82 88 34 59; +98 21 82 88 47 34Email moshaii@modares.ac.ir; m_nikkhah@modares.ac.irIntroduction: Cancer treatment using functionalized vehicles in order to block involved genes has attracted a remarkable interest. In this study, we investigated the cellular uptake and cytotoxic effects of three sizes of anti Bcl-2 DNAi-conjugated gold nanoparticles by MCF-7 cells.Methods: Three different sizes of gold nanoparticles were synthesized by citrate reduction method and after characterization, the nanoparticles were functionalized by Bcl-2 targeted DNAi. Cell internalization of the nanoparticles was analyzed by atomic absorption spectroscopy and light microscopy. The cytotoxic effects of the nanoparticles were investigated by MTT assay, flow cytometry and RT-PCR of the target gene.Results: While poor cell internalization of bare gold nanoparticles was observed, the results demonstrated that cellular uptake of DNAi-conjugated gold nanoparticles is completely size-dependent, and the largest nanoparticle (∼ 42 nm) revealed the highest internalization rate compared to other sizes (∼ 14 and ∼ 26 nm). Experimental findings showed that the DNAi-conjugated gold nanoparticles induced apoptotic pathway by silencing of the targeted Bcl-2 gene. In addition, supplementary theoretical studies demonstrated that the 42 nm DNAi-conjugated gold nanoparticles have great photothermal conversion efficiency for treatment under external illumination and these nanoparticles can be induced further cytotoxic effect by approximately 10°C temperature elevations.Conclusion: Remarkable photothermal properties of DNAi-conjugated 42 nm Au-NPs in parallel with their high cell internalization and cytotoxic effects introduce them as potential dual functional anticancer nanosystems.Keywords: breast cancer, apoptosis, DNAi, Bcl-2, gold nanoparticles

Background: Superoxide dismutases (SODs) are categorized as antioxidant enzymes that are involved in many processes such as stress signalling responses and cell protection against free radical species. The primary function of SOD is the removal of produced radical species like superoxide ions in different physiological processes. There are various isozymes of SODs which are classified according to the metal cofactor in their active sites into four general types of Fe-SOD, Mn-SOD, Cu/Zn-SOD and Ni-SOD. Among metal nanoparticles, gold nanoparticles (AuNPs) are useful for biological purposes as sensing probe for determining critical analysis based on surface plasmon resonance and colorimetric method. In this study, the human Cu-Zn SOD expressed, purified, and its interaction with AuNPs based on a new colorimetric method was investigated. Objectives: In this approach, a colorimetric detection method for SOD activity was developed based on the carboxylic stabilized AuNPs. Material and Methods: The Ni-NTA Sepharose affinity column was performed for the purification process of enzyme. Following SOD purification, the enzyme activity in presence of AuNPs due to the possible etching in the presence of free radicals which are produced by riboflavin, methionine, Na2CO3 and potassium phosphate buffer, have been performed. In addition, Fluorescence spectroscopy analysis toward SOD and gold nanoparticle were performed. Results: Superoxide radicals generated from the enzymatic reaction would preferentially etch AuNPs and resulted in remarkable changes of localized surface plasmon resonance of AuNPs, which is reduced in the presence of SOD. Under the optimized experimental conditions assay (pH~7.8 and 25 ˚C), better selectivity and sensitivity toward SOD activity was shown. Conclusions: In this context, an indirect new colorimetric method for determining of SOD activity based on gold nanoparticles (AuNPs) was evaluated. According to the presented result, it may be concluded that by scavenging of free superoxide radicals in the presence of SOD, the amount of AuNP absorbance can be replenished.