Nanoparticles to Knockdown Osteoporosis-Related Gene and Promote Osteogenic Marker Expression for Osteoporosis Treatment

Abstract

Osteoporosis is the most common disease involving bone degeneration. Current clinical treatments are not able to offer a satisfying curative effect, so the development of effective treatments is desired. Gene silencing through siRNA delivery has gained great attention as a potential treatment in bone diseases. SOST gene inhibits the Wnt signaling pathway reducing osteoblast differentiation. Consequently, silencing SOST genes with a specific siRNA could be a potential option to treat osteoporosis. Generally, siRNAs have a very short half-life and poor transfection capacity, so an effective carrier is needed. In particular, mesoporous silica nanoparticles (MSNs) have attracted great attention for intracellular delivery of nucleic acids. We took advantage of their high loading capacity to further load the pores with osteostatin, an osteogenic peptide. In this study, we developed a system based on MSNs coated with poly(ethylenimine), which can effectively deliver SOST siRNA and osteostatin inside cells, with the consequent augmentation of osteogenic markers with a synergistic effect. This established the potential utility of MSNs to co-deliver both biomolecules to promote bone formation, this being a potential alternative to treat osteoporosis.

Keywords: gene silencing; mesoporous nanoparticles; osteogenic stimulation; osteoporosis; therapeutic co-delivery.

Figure 1

Schematic representation of the designed nanocarrier based on mesoporous silica nanoparticles loaded with osteostatin and siRNA to knockdown SOST and promote the expression of early markers of osteogenic differentiation both in vitro and in vivo.

Figure 2

PEI grafting to MSNs surface. TEM micrographs of the nanoparticles (A) before and (B) after coating with 5 kDa PEI polymer. The ζ potential before and after coating with 5, 8, and 10 kDa PEI polymer (bottom right corner inset).

Figure 3

Effective SOST siRNA model molecule binding to MSNs@PEI and cell viability in mouse embryonic fibroblast (MEF) cells. (A) MEF cell viability (measured by Alamar Blue) in contact with different concentrations of MSNs@PEI nanoparticles at 48 h of cell culture. Data are mean ± SEM of three independent experiments performed in triplicate. Pound signs indicate p < 0.01 vs MSN, MSNs@PEI 5kD, and MSNs@PEI 8kD. (B) Agarose gel electrophoresis of MSNs@PEI and complexed siGLO siRNA in different nanoparticle to nucleic acid (N/P) ratios. M: molecular weight marker. The ϕ lane contains only siRNA. After the loading of osteostatin, the N/P ratio and the electrophoretic mobility did not change. The data showed that all siRNA was bound when the N-to-P ratio was over 16 in MSNs@PEI 5 kDa, and over 32 in the case of PEI 8 kDa and PEI 10 kDa.

Figure 4

MSNs@PEI-siGLO uptake by mouse embryonic fibroblast (MEF) cells by flow cytometry and fluorescence microscopy. (A) Cellular uptake of different fluorescein-labeled MSNs, MSNs@PEI, and MSNs@PEI-siGLO was measured by flow cytometry at 2 h of internalization in MEF cells. Representative flow cytometry images are shown on the top. Data are mean ± SEM of three independent experiments performed in triplicate. Asterisks indicate p < 0.03 vs MSN; pound signs indicate p < 0.01 vs MSN and MSNs@PEI. (B) Representative confocal laser scanning microscopy images of MEF cells incubated with Rhodamine-B-labeled MSNs, MSNs@PEI, and MSNs@PEI-siGLO nanoparticles at 2 h of internalization. Blue fluorescence (nuclei), red fluorescence (Rh-MSNs@PEI), and green fluorescence (siGLO).

Figure 5

SiGLO release from MSNs@PEI in MEF cells. Representative fluorescence microscopy images of MEF cells incubated 2 h with MSNs@PEI nanoparticles with siGLO at 0 and 48 h after nanoparticle incubation. Blue fluorescence (nuclei), red fluorescence (MSNs@PEI), and green fluorescence (siGLO). Arrows denote the siGLO released.

Figure 6

Changes in SOST mRNA levels and Runx2 and Alp bone osteogenic markers in MEF cells. (A) SOST, (B) Alp, and (C) Runx2 gene expression (measured by real-time PCR) in MEF cells at different times. Data are mean ± SEM of three independent experiments performed in triplicate. Single asterisks indicate p < 0.01 vs 3 days; triple asterisks indicate p < 0.001 vs 3 and 7 days.

Figure 7

SOST, Runx2, and ALP gene expression in the presence of SiRNA-Sost bound to MSNs@PEI in MEF cells. (A) SOST mRNA expression (measured by real-time PCR) in MEF cells at 14 days of cell culture. (B) Alp and (C) Runx2 mRNA expression (measured by real-time PCR) in MEF cells at 14 days of cell culture. A negative-control siRNA (SiCtl) was used. MSNs@PEI nanoparticles bound to NeControl (MSNs@PEI SiCtl). Data are mean ± SEM of three independent experiments performed in triplicate. Asterisks indicate p < 0.01 vs MSNs@PEI MEF control cells.

Figure 8

Time-dependent osteostatin (OST) release from MSNs@PEI in PBS at pH 7.4, simulating the physiological fluids. Nanoparticles were loaded with OST and afterwards coated with PEI (OST-MSNs@PEI). Points to trace the curves are the means of three independent measurements per time period.

Figure 9

(A) SOST, (B) Alp, and (C) Runx2bone osteogenic markers gene expression in the presence of SiRNA-SOST bound to MSNs@PEI in mouse embryonic fibroblast (MEF) cells in the presence or absence of osteostatin (OST). SOST mRNA expression (measured by real-time PCR) in MEF cells at 14 days of cell culture and Alp and Runx2 mRNA expression (measured by real-time PCR) in MEF cells at 14 days of cell culture. To optimize the experiment, one control was used: a negative control siRNA (SiCtl) (“non-targeting control”, which targets a site that is absent in human, mouse, and rat genomes); MSNs@PEI nanoparticles were bound to SiCtl (MSNs@PEI SiCtl). Data are mean ± SEM of three independent experiments performed in triplicate. Triple asterisks indicate p < 0.001 vs MSNs@PEI MEF; pound signs indicate p < 0.05 vs OST-MSNs@PEI and MSNs@PEI-SiRNA.

Figure 10

In vivo injection of OST-MSNs@PEI-siRNA. SOST, Alp, and Runx2 bone osteogenic markers gene expression in the presence of SiRNA-SOST bound to MSNs@PEI in ovariectomized mice (OVX) in the presence or absence of osteostatin. (A) Femur bone-marrow injection in ovariectomized female mice and cyanine-7 labeled nanoparticles accumulation. (B) SOST mRNA expression (measured by real-time PCR) in femur bone. (C) Alp and (D) Runx2 mRNA expression (measured by real-time PCR) in femur bone. To optimize the experiment, one control was used: a negative control siRNA (SiCtl) (“non-targeting control”, which targets an absent site in human, mouse, and rat genomes) and MSNs@PEI nanoparticles bound to SiCtl (MSNs@PEI SiCtl). Data are mean ± SEM of three independent experiments performed in triplicate. Triple asterisks indicate p < 0.001 vs MSNs@PEI; pound signs indicate p < 0.05 vs OST-MSNs@PEI and MSNs@PEI-SiRNA.