A bone-resorption surface-targeting nanoparticle to deliver anti-miR214 for osteoporosis therapy

Abstract

With increasing fracture risks due to fragility, osteoporosis is a global health problem threatening postmenopausal women. In these patients, osteoclasts play leading roles in bone loss and fracture. How to inhibit osteoclast activity is the key issue for osteoporosis treatment. In recent years, miRNA-based gene therapy through gene regulation has been considered a potential therapeutic method. However, in light of the side effects, the use of therapeutic miRNAs in osteoporosis treatment is still limited by the lack of tissue/cell-specific delivery systems. Here, we developed polyurethane (PU) nanomicelles modified by the acidic peptide Asp⁸. Our data showed that without overt toxicity or eliciting an immune response, this delivery system encapsulated and selectively deliver miRNAs to OSCAR⁺ osteoclasts at bone-resorption surface in vivo. With the Asp⁸-PU delivery system, anti-miR214 was delivered to osteoclasts, and bone microarchitecture and bone mass were improved in ovariectomized osteoporosis mice. Therefore, Asp⁸-PU could be a useful bone-resorption surface-targeting delivery system for treatment of osteoclast-induced bone diseases and aging-related osteoporosis.

Keywords: bone resorption; microRNA; nanoparticle; osteoporosis; targeting delivery.

Figure 1

Design, synthesis and characterization of Asp⁸-PU. Notes: (A) Schematic diagram of Asp⁸-PU; (B) transmission electron microscopy of Asp⁸-PU (bar 100 nm); (C) dynamic light scattering measurements of Asp⁸-PU size in water and ζ-potential of Asp⁸-PU determined by dynamic light scattering; (D) nucleic acid-binding capacity of Asp⁸-PU measured by electrophoretic mobility assays; (E) serum stability test of naked anti-miR214 and Asp⁸-PU–anti-miR214 complexes. Abbreviation: PU, polyurethane.

Figure 2

Safety evaluation of Asp⁸-PU in vivo. Notes: (A) Hemagglutination assay of mouse blood cells incubated with PU or Asp⁸-PU for 1 hour (bars 50 μm); (B) quantification of serum CK-MB, ALT, and BUN levels by clinical chemistry analysis (data shown as means ± SE, n=5 per group); (C) H&E images of heart, liver, spleen, and kidney collected from mouse tail vein injected with PU or Asp⁸-PU (bars 20 μm); (D) quantification analysis of serum TNFα, IFNγ, IL2, and IL6 by enzyme-linked immunosorbent assay. Abbreviations: PU, polyurethane; BUN, blood urea nitrogen; ALT, alanine aminotransferase; H&E, hematoxylin-eosin; CK-MB, creatine kinase isoenzyme; BUN, blood urea nitrogen.

Figure 2

Safety evaluation of Asp⁸-PU in vivo. Notes: (A) Hemagglutination assay of mouse blood cells incubated with PU or Asp⁸-PU for 1 hour (bars 50 μm); (B) quantification of serum CK-MB, ALT, and BUN levels by clinical chemistry analysis (data shown as means ± SE, n=5 per group); (C) H&E images of heart, liver, spleen, and kidney collected from mouse tail vein injected with PU or Asp⁸-PU (bars 20 μm); (D) quantification analysis of serum TNFα, IFNγ, IL2, and IL6 by enzyme-linked immunosorbent assay. Abbreviations: PU, polyurethane; BUN, blood urea nitrogen; ALT, alanine aminotransferase; H&E, hematoxylin-eosin; CK-MB, creatine kinase isoenzyme; BUN, blood urea nitrogen.

Figure 3

Tissue-selective distribution and cell-selective delivery of miRNA by Asp⁸-PU in vivo. Notes: (A) Tissue-selective distribution of siRNA delivered by Asp⁸-PU in mouse organs. (B) Representative fluorescence micrography of proximal tibiae from mice treated with PU-Cy³-miR214 or Asp⁸-PU-Cy³-miR214. The Cy³-miR214 signal was red, and the bone-formation surfaces were labeled with calcein (green). Bars 100 μm. (C) Immunohistostaining was performed to detect CTSK-positive cells (green). Merged images with DAPI staining showed colocalization of Cy³-miR214 and CTSK-positive cells. Bars 100 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole or dihydrochloride; PU, polyurethane.

Figure 4

Dose–response pattern and persistence of miR214 knockdown in vivo. Notes: *P<0.05. (A) Dose-dependent anti-miR214 knockdown determined by real-time PCR and normalized to baseline after tail-vein injection of PU–anti-miR214 or Asp⁸-PU–anti-miR214 at doses of 2–32 mg/kg; (B) persistence of miR214 knockdown examined by real-time PCR and normalized to baseline after tail-vein injection of PU–anti-miR214 or Asp⁸-PU–anti-miR214 at dose of 16 mg/kg (data presented as means ± SE, n=6 per group); (C) representative bright-field microscopy of sections from distal femora with TRAP staining in mice treated with PU-Cy³-miR214 or Asp⁸-PU-Cy³-miRNA (bars 100 μm); (D) representative bright-field microscopy of sections from distal femora with TRAP staining in mice treated with Asp⁸-PU-Cy³-miR214 (bars 100 μm). Abbreviations: PCR, polymerase chain reaction; PU, polyurethane.

Figure 5

Cell-specific knockdown efficiency of miR214 in vivo. Notes: *P<0.05. (A) Real-time qPCR analysis for miR214-knockdown efficiency in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice administered free anti-miR214 injection of PU–anti-miR214 and Asp⁸-PU–anti-miR214 at 24 and 48 hours after administration; (B) real-time qPCR analysis for mRNA expression of TRAP and CTSK in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice administered PU–anti-miR214 and Asp⁸-PU–anti-miR214 at 48 hours after administration; (C) real-time qPCR analysis for miR214-knockdown efficiency in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice pretreated with PBS or Asp⁸ followed by administration of Asp⁸-PU–anti-miR214 at 24 and 48 hours; (D) real-time qPCR analysis for mRNA expression of TRAP and CTSK in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice pretreated with PBS or Asp⁸ followed by administration of Asp⁸-PU–anti-miR214 at 48 hours. All data presented as means ± SE, n=6 per group. Abbreviations: qPCR, quantitative polymerase chain reaction; PU, polyurethane; FACS, fluorescence-activated cell sorting; OSCAR, osteoclast associated receptor; Ovx, ovariectomized; NS, not significant.

Figure 5

Cell-specific knockdown efficiency of miR214 in vivo. Notes: *P<0.05. (A) Real-time qPCR analysis for miR214-knockdown efficiency in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice administered free anti-miR214 injection of PU–anti-miR214 and Asp⁸-PU–anti-miR214 at 24 and 48 hours after administration; (B) real-time qPCR analysis for mRNA expression of TRAP and CTSK in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice administered PU–anti-miR214 and Asp⁸-PU–anti-miR214 at 48 hours after administration; (C) real-time qPCR analysis for miR214-knockdown efficiency in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice pretreated with PBS or Asp⁸ followed by administration of Asp⁸-PU–anti-miR214 at 24 and 48 hours; (D) real-time qPCR analysis for mRNA expression of TRAP and CTSK in OSCAR⁺ and OSCAR⁻ cells sorted from bone marrow by FACS from Ovx mice pretreated with PBS or Asp⁸ followed by administration of Asp⁸-PU–anti-miR214 at 48 hours. All data presented as means ± SE, n=6 per group. Abbreviations: qPCR, quantitative polymerase chain reaction; PU, polyurethane; FACS, fluorescence-activated cell sorting; OSCAR, osteoclast associated receptor; Ovx, ovariectomized; NS, not significant.

Figure 6

Antiosteoporosis efficacy of Asp⁸-PU–anti-miR214. Notes: *P<0.05. (A) Real-time qPCR analysis of miR214 levels in osteoclasts isolated by FACS in tibiae collected from the groups of mice indicated; (B) representative images showing three-dimensional trabecular architecture by micro-CT reconstruction in distal femora (bars 1 mm); (C) micro-CT measurements of BMD and BV/TV in distal femora; (D) micro-CT measurements of Tb.Th, Tb.Sp, Tb.N, and SMI in distal femora; (E) histomorphometry analysis of Oc.S/BS and Oc.N/BPm in distal femora. All data presented as means ± SE, n=6 per group. **Indicates significant difference at P<0.01. Abbreviations: PU, polyurethane; qPCR, quantitative polymerase chain reaction; FACS, fluorescence-activated cell sorting; CT, computed tomography; BMD, bone-mineral density; BV, bone volume; TV, total volume; Tb.Th, trabecular thickness; Tb.Sp, Tb spacing; Tb.N, Tb number; SMI, structure model index; Oc.S/BS, osteoclast surface/bone surface; Oc.N/BPm, Oc number/bone perimeter; Ovx, ovariectomized; NC, anti-scramble group.