Investigation of Genetıcal Material Decorated Nanoparticles Usability as a New Generation Biochemical Strategy in Cancer Therapy

Abstract

Breast cancer (BC) is the second most frequently diagnosed cancer type worldwide. Triple negative breast cancer (TNBC) type is a considerably aggressive one compared to other breast cancer subtypes with high metastasis, relapse and mortality rate in patients. Due to its significant genetic heterogeneity and deficiency of molecular targets such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), no effective targeted therapy strategies are available for TNBC. In recent years, emerging evidence has revealed that short non-coding microRNAs (miRNA) are broadly involved in various cancer pathophysiological processes including cell cycle, proliferation, differentiation, migration/invasion and aging, via regulating the expression of the target genes that are active in those pathways. miRNAs gain an important role as therapeutics by enabling the degradation of the target mRNA or the inhibition of translation. Therefore, miRNA-based therapeutics may be a novel therapeutic approach, especially for TNBC patients. However, safe and efficient delivery systems are required for miRNAs to be successfully used in clinical trials.

In the presented study, poly-L-lysine (PLL) modified sericin (Ser) coated superparamagnetic iron oxide nanoparticles (SPIONs) (PLL/Ser-SPIONs) were synthesized and characterized as miRNA delivery vehicle for TNBC therapy. PLL/Ser-SPIONs were obtained in a size range of 25-30 nm with a cationic zeta potential value that is +13 mV. The nanoparticles were interacted with different concentrations of miRNA (miR) control and the range of the binding efficiencies was found to be as 97-99%. Transmission electron microscopy (TEM) and confocal microscopy analyses exhibited that miR-control or control small interfering RNA (siRNA) loaded PLL/Ser-SPIONs were successfully uptaken by TNBC (MDA-MB-231) cells. miR-329 was chosen as a target miRNA after analyzing several miRNA databases and discovering that its overexpression is related to high survival rate in patients with TNBC. It was demonstrated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) that miR-329 expression level is significantly reduced in many TNBC cell lines. In vitro delivery of miR-329 either using PLL/Ser-SPIONs or a commercial transfection reagent inhibited proliferation, invasion/migration and enhanced apoptosis rate in TNBC cells. The underlying molecular mechanism of miR-329 based therapy was investigated via a number of target prediction algorithms and they showed that miR-329 has binding sites at three prime untranslated region (3’-UTR) of eukaryotic elongation factor-2 kinase (eEF2K) and AXL genes. In addition, by Western Blot assay, upregulated levels of both eEF2K and AXL were observed in TNBC cells, and it was also found that these genes are associated with shorter overall survival in TNBC patients. More important, after miR-329 delivery in TNBC cells, both eEF2K and AXL expressions were remarkably inhibited. The TNBC cells were also treated with specific siRNAs for eEF2K and AXL, and silencing these genes recapitulated the effects of ectopic expression of miR-329 by reducing the cell growth and invasion/migration. Furthermore, in vivo delivery of miR-329 loaded PLL/Ser-SPIONs led to a drastic inhibition in tumor growth in TNBC orthotopic xenograft models (MDA-MB-231 and MDA-MB-436) in mice. A significant knockdown of eEF2K and AXL levels was also observed in tumor samples by Western Blot analysis. In addition, PLL/Ser-SPIONs-miR 329 treatment in mice did not cause any detectable side effects. These obtained results suggest that miR-329 acts as a tumor suppressor (TS) and its reduced expression results in an increase in eEF2K and AXL expressions that lead to progression of TNBC. Moreover, this work also suggests PLL/Ser-SPION as a potential theranostic (therapy+diagnostic) agent with its multifunctional property. Taken all together, this study shows that PLL/Ser-SPIONs-miR 329 system may be a promising and novel miR-therapy strategy against TNBC providing a safe and high antitumor efficacy.