Design and In Vitro Evaluation of Layer by Layer siRNA Nanovectors Targeting Breast Tumor Initiating Cells

<div><p>Efficient therapeutics and early detection has helped to increase breast cancer survival rates over the years. However, the recurrence of breast cancer remains to be a problem and this may be due to the presence of a small population of cells, called tumor initiating cells (TICs). Breast TICs are resistant to drugs, difficult to detect, and exhibit high self-renewal capabilities. In this study, layer by layer (LBL) small interfering RNA (siRNA) nanovectors (SNVs) were designed to target breast TICs. SNVs were fabricated using alternating layers of poly-L-lysine and siRNA molecules on gold (Au) nanoparticle (NP) surfaces. The stability, cell uptake, and release profile for SNVs were examined. In addition, SNVs reduced TIC-related STAT3 expression levels, CD44⁺/CD24⁻/EpCAM⁺ surface marker levels and the number of mammospheres formed compared to the standard transfection agent. The data from this study show, for the first time, that SNVs in LBL assembly effectively delivers STAT3 siRNA and inhibit the growth of breast TICs <i>in vitro</i>.</p></div

Figure 3

<p>(<b>A</b>) Number of cells emitting FITC after uptake of FITC tagged PLL layer (Layer 3-green) from SNVs after 4, 24 and 48 h treatment. (<b>B</b>) Plot of the FITC intensity measured at each time point, 4, 24, and 48 h (n = 9, n is number of regions of interest (ROI) from fluorescent images) Images correspond to fluorescent image of SUM159 cells (nuclei stained with DAPI – blue color) displaying the release of FITC tagged PLL layer (Layer 1-green) from SNVs, scale bar = 10 µm.</p

Figure 4

<p>(<b>A</b>) Protein expression of p-STAT3 pathway in triple negative breast cancer cell lines, SUM159 and MDA-MB-231, after treatment with AuNPs, non-targeting siRNA in siPORT, STAT3 targeting siRNA in siPORT, non-targeting SNVs, and STAT3 targeting siRNA in SNVs. (<b>B</b>) Primary mammosphere forming efficiency from breast cancer cells after 3 days treatment with AuNPs, STAT3 siRNA in siPORT, in PLL and STAT3 targeting siRNA in SNVs. (<b>C</b>) Forming efficiency of secondary mammospheres was plotted after primary mammospheres were collected, disassociated, and re-seeded in fresh mammosphere media for 3 days. (* = statistical significance, n = 6, p<0.05).)</p

Schematic representation on the fabrication method for SNVs using AuNPs.

<p>Plot of (<b>B</b>) zeta potential, (<b>C</b>) hydrodynamic diameter, (<b>D</b>) the amount of siRNA loaded onto SNVs and (<b>E</b>) absorbance for bare AuNPs stabilized by citrate and every subsequent layer added on to the SNVs. (<b>F</b>) AFM height image of SNVs.</p

Figure 2

<p>(<b>A</b>) Absorption spectra of SNVs in FBS and in water. (<b>B</b>) Release profile of the first layer (FITC tagged poly-L-Lysine) released from LBL SNVs in pH 5.5 and 7.2 at 37°C. (<b>C</b>) Release profile of the STAT3 siRNA layer (Cy3-tagged siRNA molecules) released from the SNVs in pH 5.5 and pH 7.2 at 37°C.</p