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. 2024 Dec 22;16(12):1623.
doi: 10.3390/pharmaceutics16121623.

Cationic Hydroxyethyl Cellulose Nanocomplexes and RANK siRNA/Zoledronate Co-Delivery Systems for Osteoclast Inhibition

Sohyun Lee  1 , Seoyeon Park  1 , Tae-Il Kim  1   2
Affiliations

Cationic Hydroxyethyl Cellulose Nanocomplexes and RANK siRNA/Zoledronate Co-Delivery Systems for Osteoclast Inhibition

Sohyun Lee et al. Pharmaceutics. .

Abstract

Background/objectives: In this study, HECP2k polymer, polyethylenimine2k (PEI2k)-modified hydroxyethyl cellulose (HEC) was utilized to form the nanocomplexes with receptor activator of nuclear factor k-B (RANK) siRNA and zoledronate (Zol) for osteoclast inhibition. HECP2k/(RANK siRNA + Zol) nanocomplexes prepared by simple mixing were anticipated to overcome the low transfection efficiency of siRNA and the low bioavailability of Zol.

Methods: The characterization of both HECP2k/(pDNA + Zol) nanocomplexes and HECP2k/(RANK siRNA + Zol) nanocomplexes was performed.

Results: The nanocomplexes were successfully formed even in the presence of Zol, showing about 200 nm sizes and about 20 mV of positive zeta potential values suitable for efficient cellular uptake. They also possessed high endosome buffering ability by PEI and Zol, suggesting the potential for efficient endosomal escape. It was found that the low cytotoxic nanocomplexes (>90% cell viability) displayed greater transfection efficiency than PEI25k and even HECP2k polyplexes. Finally, it was found by tartrate-resistant acid phosphatase (TRAP) assay and qPCR analysis that HECP2k/(RANK siRNA + Zol) nanocomplexes could inhibit the TRAP to about 50% value and another characteristic osteoclastic gene expression, increasing FAS gene expression to about 16 times higher than control and more efficiently (about 3 times and 5 times higher, respectively) than HECP2k/siRNA polyplexes and Zol only.

Conclusions: HECP2k/(RANK siRNA + Zol) nanocomplexes formed by simple mixing showed great potential for inhibiting osteoclast differentiation and osteoclast activity, inducing the apoptosis via combinatorial effects of RANK siRNA and Zol.

Keywords: RANK siRNA; drug/gene co-delivery systems; hydroxyethyl cellulose; nanocomplex; osteoclast inhibition; zoledronate.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(A) Agarose gel electrophoresis result of HECP2k/(pDNA + Zol) nanocomplexes. (B) PicoGreen assay result of HECP2k/(pDNA + Zol) nanocomplexes (Zol:HECP2k:pDNA = 0~5:50:1, w/w/w).
Figure 2
Figure 2
(A) Average sizes and (B) Zeta potential values of HECP2k/(pDNA + Zol) nanocomplexes with various weight ratios of Zol (Zol:HECP2k:pDNA = 0~20:50:1, w/w/w).
Figure 3
Figure 3
TEM images of HECP2k polyplexes (A,B) and HECP2k/(pDNA + Zol) nanocomplexes (C,D) (Zol:HECP2k:pDNA = 2:50:1, w/w/w).
Figure 4
Figure 4
MTT assay results of HECP2k polymer and Zol on (A) HeLa and (B) RAW264.7 cells. MTT assay results of HECP2k/(pDNA + Zol) nanocomplexes on (C) HeLa and (D) RAW264.7 cells. WR means the weight ratio of Zol (Zol/pDNA) in the nanocomplexes. PEI25k and HECP2k polyplexes were used as controls.
Figure 5
Figure 5
Transfection efficiency of HECP2k/(pDNA + Zol) nanocomplexes with various Zol weight ratios (WR) on HeLa (A,B) and RAW264.7 cells (C,D). Transfection experiments were performed in serum-free conditions (A,C) and serum conditions (B,D).
Figure 6
Figure 6
Osteoclast inhibition analysis by TRAP staining assay. (A) RAW264.7 cells, (B) osteoclasts differentiated from RAW264.7 cells by RANKL treatment, (C) Zol only-treated osteoclasts, and (D) HECP2k/(RANK siRNA + Zol) nanocomplexes-treated osteoclasts. (E) TRAP quantification result by measuring the absorbance of TRAP at 405 nm (WR means Zol/siRNA).
Figure 7
Figure 7
Relative FAS mRNA levels of HECP2k/(siRNA + Zol) nanocomplexes-treated osteoclasts by qPCR. Zol only and HECP2k/siRNA polyplexes were used as controls. All values were normalized to the value of non-treated osteoclasts.
Figure 8
Figure 8
Relative mRNA levels of (A) RANK, (B) NFATc1, and (C) cathepsin-K for HECP2k/(siRNA + Zol) nanocomplexes-treated osteoclasts by qPCR. Zol only and HECP2k/siRNA polyplexes were used as controls. All values were normalized to the value of non-treated RAW264.7 cells.
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