siRNA-Loaded Hydroxyapatite Nanoparticles for KRAS Gene Silencing in Anti-Pancreatic Cancer Therapy

Abstract

Pancreatic carcinoma (PC) is greatly induced by the KRAS gene mutation, but effective targeted delivery for gene therapy has not existed. Small interfering ribonucleic acid (siRNA) serves as an advanced therapeutic modality and holds great promise for cancer treatment. However, the development of a non-toxic and high-efficiency carrier system to accurately deliver siRNA into cells for siRNA-targeted gene silencing is still a prodigious challenge. Herein, polyethylenimine (PEI)-modified hydroxyapatite (HAp) nanoparticles (HAp-PEI) were fabricated. The siRNA of the KRAS gene (siKras) was loaded onto the surface of HAp-PEI via electrostatic interaction between siRNA and PEI to design the functionalized HAp-PEI nanoparticle (HAp-PEI/siKras). The HAp-PEI/siKras was internalized into the human PC cells PANC-1 to achieve the maximum transfection efficiency for active tumor targeting. HAp-PEI/siKras effectively knocked down the expression of the KRAS gene and downregulated the expression of the Kras protein in vitro. Furthermore, the treatment with HAp-PEI/siKras resulted in greater anti-PC cells' (PANC-1, BXPC-3, and CFPAC-1) efficacy in vitro. Additionally, the HAp-PEI exhibited no obvious in vitro cytotoxicity in normal pancreatic HPDE6-C7 cells. These findings provided a promising alternative for the therapeutic route of siRNA-targeted gene engineering for anti-pancreatic cancer therapy.

Keywords: KRAS gene; anticancer; gene silence; hydroxyapatite; pancreatic cancer cells; siRNA delivery.

Figure 1

(a) TEM images of HAp, HAp@PEG and HAp@PEG-PEI, (b) SEM image, (c) hydrodynamic size distributions, (d) zeta potential, and (e) TEM elemental color mapping of HAp@PEG-PEI. Elements: N (yellow), O (green), P (red) and Ca (blue).

Figure 2

(a) XRD pattern, (b) FTIR and (c) Raman spectra of HAp-PEI, and (d) TG curves of HAp and HAp-PEI.

Figure 3

In vitro degradation profiles of HAp-PEI in PBS with various pH values of 5.6, 6.5 and 7.4 at 37 °C.

Figure 4

Cell viability of pancreatic cancer cells (PANC-1, BXPC-3, CFPAC-1) and pancreatic ductal epithelial cells (HPDE6-C7) co-incubated with HAp-PEI and HAp at various concentrations (0.05 mg/L–5 mg/L) for 48 h.

Figure 5

Agarose gel electrophoresis map of siRNA in different groups of siRNA loading. The mass of siRNA was 2 μg, and HAp-PEI were 0, 2, 4, 8, 12, 16, 20, and 24 μg, respectively, in 1 to 8 groups. The weight ratios of HAp-PEI and siRNA (HAp-PEI/siRNA) were 0, 1/1, 2/1, 4/1, 6/1, 8/1, 10/1, and 12/1, respectively, in 1 to 8 groups. Group 1–5: siRNA bands are gradually shallow; Red rectangular marked group 6: HAp-PEI/siRNA (w/w) = 8/1, siRNA band has disappeared.

Figure 6

The pancreatic cancer cell PANC-1 transfected siRNA-FAM (green fluorescence) by (a) siRNA-Mate and HAp-PEI for 8 h and (b) HAp-PEI for 6, 12, and 24 h. Cell lysosome was stained with Lysotracker^TM Red (red fluorescence).

Figure 7

Expression of Kras mRNA in PANC-1 and CFPAC-1 cells after treatment by (1) siRNA-Mate/siKras, (2) HAp-PEI/siKras, (3) siKras, (4) HAp-PEI, and (5) HAp/siKras for 48 h. DECP H₂O was used as control. Values are represented as mean ± SD (n = 3). Statistical significance relative to the group of control: ** p < 0.01.

Figure 8

Expression of Kras protein in (a) PANC-1 and (b) CFPAC-1 cells after siKras transfection for 48 h by HAp-PEI, Hap, and siRNA-Mate. DECP H₂O, siKras and HAp-PEI treatment were used as control.

Figure 9

In vitro anti-pancreatic cancer effect. PANC-1, BXPC-3, and CFPAC-1 cell viability after treatment by (1) siRNA-Mate/siKras, (2) HAp-PEI/siKras, (3) DEPC H₂O, (4) siKras, (5) HAp-PEI, (6) HAp/siKras, and (7) HAp-PEI/siNC for 72 h. Untreated cells were used as control. Values are represented as mean ± SD (n = 3). Statistical significance relative to the group of control: * p < 0.05, ** p < 0.01.