Introduction

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The targeted delivery of anti-cancer agents by nanoparticles simultaneously increases the efficacy and half-life of drugs, reduces toxic side effects of conventional therapeutics. In recent years, magnetic nanoparticles (MNPs) which can be targeted to the tumor tissue under magnetic field, gained importance on cancer therapy for the magnetically controlled delivery of anticancer drugs, as well as hyperthermia. These magnetic particles are generally composed of magnetite (Fe3O4) core and a polymeric shell where the drugs are bound. Unlike classical polymers, dendrimers have a high degree of molecular uniformity, narrow molecular weight distribution, specific size and shape characteristics, and a highly- functionalized terminal surface. Polycationic dendrimers such as Polyamidoamine (PAMAM) are known to be an efficient DNA delivery system since dendrimers bear primary amine groups on theirsurface. PAMAM derndrimers have flexible structure and efficient interaction with DNA. Due to these features PAMAM dendrimers can be used for RNA delivery.

In our ongoing project, focusing on targeted siRNA delivery to breast cancer cells, G4 PAMAM dedrimer coated magnetic nanoparticles have been improved.1 Monodisperse dendrimers are synthesized by step-wise chemical methods to give distinct generations (G0, G1, G2,G3, G4, …) of molecules with narrow molecular weight distribution, uniform size and shape, and multiple (multivalent) surface groups.2 The manufacturing process is a series of repetitive steps starting around a central initiator magnetite core. Each subsequent growth step represents a new “generation” of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately double the molecular weight of the preceding generation.

Small interfering RNA (siRNA) is small pieces of double-stranded (ds) RNA, about 21 nucleotides long, with 3’ overhangs (2 nucleotides) at each end that can be used to “interfere” with the translation of proteins by binding to 3’ UTR region of messenger RNA. Therefore, siRNA can be used for therapeutic application of gene silencing.35 In our study, we used survivin siRNA. Survivin RNAi can partly suppress the expression of survivin in cancer cells, inhibit the cell proliferation and promote cell apoptosis. Survivin RNAi can enhance the cell sensitivity to apoptosis, which implies that survivin RNAi may partly reverse the drug resistance of cancer. Survivin, an inhibitor of apoptosis protein, is highly expressed in most cancers and associated with chemotherapy resistance. It is transcribed from BIRC5 gene. This gene is a member of the inhibitor of apoptosis (IAP) gene family, which encode negative regulatory proteins that prevent apoptotic cell death.6

Materials and Methods

MCF-7 and Doxorubicin resistant MCF-7 cells were cultured in commercially defined RPMI 1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS). Flasks were incubated at 37°C in a 95% (v/v) humidified atmosphere of 5% (v/v) CO2. Cells were grown in filter cap culture flasks. When attached cell concentration on flask surface reached 80% confluency, cells were passaged.

In order to observe the cellular internalization of Survivin siRNA loaded G4 MNPs by MCF-7 and Doxorubicin resistant MCF-7 cell lines, 50.000 cells/well were seeded in 24 well plates and incubated at 37°C overnight. Survivin siRNA loaded G4 nanoparticles were added at different concentrations. After 5-6 hour incubation, cells were visualized under fluorescent microscopy (Life Sciences).

Toxicity of Survivin siRNA loaded G4 MNPs on MCF-7 and Doxorubicin resistant MCF-7 breast cancer cell lines were determined by XTT based cell proliferation assay, which is a colorimetric test based on the reduction tetrazolium salt to colored formazan products by mitochondrial enzymes of living cells. Cells were seeded to 96-well plates at a concentration of 5.000 cells/well. Highest drug dose was 20 nM Survivin siRNA loaded G4 MNPs.

Results and Discussion

Survivin siRNA loading on G4 MNPs was achieved in phosphate buffer (pH 6) by rotating for 5 hours at room temperature. Agarose gel electrophoresis applied to analyze the binding efficiency of siRNA on G4 MNPs (Figure 1). Survivin concentration was fixed at 20 nM and G4 MNP concentrations were increased by 1 to 4 folds. 20 nM concentration of siRNA was completely loaded on nanoparticles from 2 to 4 folds concentrations of G4 MNPs.

FIG_01_PAMAM Dedrimer Coated MNPs for siRNA Delivery and Gene Silencing

Fig. 1: Agarose gel electrophoresis for Survivin siRNA loading optimization of G4 MNPs at pH 6. Survivin siRNA concentration was 20 nM with 1 to 4 folds of G4 MNPs at each well

Survivin siRNA loaded G4 MNPs were incubated on MCF-7 and Doxorubicin resistant MCF-7 cells for 4.5 hours so that the intracellular drug delivery of Survivin siRNA could be visualized (Figure 2).

FIG_02_PAMAM Dedrimer Coated MNPs for siRNA Delivery and Gene Silencing

Fig. 2: Cellular internalization of Survivin siRNA loaded G4 MNPs on MCF-7 and Doxorubicin resistant MCF-7 cells

Survivin siRNA – G4 MNP complex was succesfully penetrated to the cells at first 4.5 hours. Cellular internalization of nanoparticles was higher at DOX resistant MCF-7 cells than normal MCF-7 cells. It was known that nanoparticles facilitate intracellular drug delivery for overcoming drug resistance in cancer cells. Loading of anticancer agents such as siRNA can be a suitable way to bypass resistance mechanism since nanoparticles enter to the cell by endosomal pathway.7

According to the XTT analyses of siRNA unloaded G4 MNPs on MCF-7 and MCF-7/Dox cells, the significant toxicity was observed around and over 350 μg/ml (Figure 03). At lower concentrations of nanoparticles, there was no toxic effect determined. Cell proliferation was not effected by G4 MNPs up to 0.5 μg/ml. The proliferation rate even increased at the concentrations between 1.5 and 40 μg/ml. (Figure 3)

FIG_03_PAMAM Dedrimer Coated MNPs for siRNA Delivery and Gene Silencing

Fig. 3: XTT assay graph of siRNA unloaded G4 MNP on MCF-7 cell line. G4 MNPs had no effect on cell proliferation.

Dose dependent toxicity of Survivin siRNA loaded G4 MNPs were investigated on MCF-7 and Doxorubicin resistant MCF-7 cell lines with XTT assay. Survivin siRNA loaded G4 MNPs were found as highly apoptotic to those breast cancer cells. Inhibitory concentration of 50% (IC50) cell proliferation values were calculated. The IC50 value of Survivin siRNA loaded G4 MNPs were 0.5 nM and 0.9 nM on MCF-7 and MCF-7/Dox cells, respectively (Figure 4 ).

FIG_4a_PAMAM Dedrimer Coated MNPs for siRNA Delivery and Gene Silencing

Fig. 4: A)XTT graph of Survivin siRNA- loaded G4 MNP on MCF-7 cells.

FIG_4b_PAMAM Dedrimer Coated MNPs for siRNA Delivery and Gene Silencing

Fig. 4: B)XTT graph of Survivin siRNA- loaded G4 MNP on MCF-7/Dox cells.

Control siRNA loaded G4 MNPs was not revealed any toxic effect on MCF-7 and MCF-7/Dox cells (Figure 5). Control siRNA, a non-targeting 20-25 nucleotide siRNA designed as a negative control, consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA.

FIG_05_PAMAM Dedrimer Coated MNPs for siRNA Delivery and Gene Silencing

Fig. 5: XTT graph of control siRNA- loaded on MNP G4 (MCF-7 cell line).

Conclusion

In conclusion Survivin siRNA is triggering cell death when delivered by G4 PAMAM dendrimer coated magnetic nanoparticles. Survivin siRNA loaded G4 MNPs have potential to overcome drug resistance in Doxorubicin resistant breast cancer cells.