Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 May 27;22(1):292.
doi: 10.1186/s12951-024-02561-x.

Effective delivery of miR-150-5p with nucleus pulposus cell-specific nanoparticles attenuates intervertebral disc degeneration

Hua Jiang #  1   2 , Hongyu Qin  3 , Qinghua Yang  3 , Longao Huang  3 , Xiao Liang  3 , Congyang Wang  3 , Abu Moro  3 , Sheng Xu  4 , Qingjun Wei #  5
Affiliations

Effective delivery of miR-150-5p with nucleus pulposus cell-specific nanoparticles attenuates intervertebral disc degeneration

Hua Jiang et al. J Nanobiotechnology. .

Abstract

Background: The use of gene therapy to deliver microRNAs (miRNAs) has gradually translated to preclinical application for the treatment of intervertebral disc degeneration (IDD). However, the effects of miRNAs are hindered by the short half-life time and the poor cellular uptake, owing to the lack of efficient delivery systems. Here, we investigated nucleus pulposus cell (NPC) specific aptamer-decorated polymeric nanoparticles that can load miR-150-5p for IDD treatment.

Methods: The role of miR-150-5p during disc development and degeneration was examined by miR-150-5p knockout (KO) mice. Histological analysis was undertaken in disc specimens. The functional mechanism of miR-150-5p in IDD development was investigated by qRT-PCR assay, Western blot, coimmunoprecipitation and immunofluorescence. NPC specific aptamer-decorated nanoparticles was designed, and its penetration, stability and safety were evaluated. IDD progression was assessed by radiological analysis including X-ray and MRI, after the annulus fibrosus needle puncture surgery with miR-150-5p manipulation by intradiscal injection of nanoparticles. The investigations into the interaction between aptamer and receptor were conducted using mass spectrometry, molecular docking and molecular dynamics simulations.

Results: We investigated NPC-specific aptamer-decorated polymeric nanoparticles that can bind to miR-150-5p for IDD treatment. Furthermore, we detected that nanoparticle-loaded miR-150-5p inhibitors alleviated NPC senescence in vitro, and the effects of the nanoparticles were sustained for more than 3 months in vivo. The microenvironment of NPCs improves the endo/lysosomal escape of miRNAs, greatly inhibiting the secretion of senescence-associated factors and the subsequent degeneration of NPCs. Importantly, nanoparticles delivering miR-150-5p inhibitors attenuated needle puncture-induced IDD in mouse models by targeting FBXW11 and inhibiting TAK1 ubiquitination, resulting in the downregulation of NF-kB signaling pathway activity.

Conclusions: NPC-targeting nanoparticles delivering miR-150-5p show favorable therapeutic efficacy and safety and may constitute a promising treatment for IDD.

Keywords: Intervertebral disc degeneration; Nanoparticles; Ubiquitination; microRNA.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing of interests.

Figures

Fig. 1
Fig. 1
Knockout of miR-150-5p potentially relieves intervertebral disc aging and degeneration. A Selection strategy for miRNAs in nucleus pulposus (NP) tissues based on microarray-based sequencing. B, C Nucleus pulposus cells (NPCs) were obtained from intervertebral discs with and without degeneration (Co6/Co7 vs. Co5/Co6). A heatmap and volcano plot were generated to show the differences in the expression profiles of miRNAs between the intervertebral disc degeneration (IDD) and normal control (NC) groups. There were 22 significantly dysregulated miRNAs in the IDD group (13 miRNAs upregulated and 9 miRNAs downregulated). D Principal component analysis (PCA) of miRNA-normalized expression data from NPCs. PCA of the data revealed strong separation between the two groups and good homogeneity within each group for the miRNA arrays. E Histological examination of NP tissues from NCs and IDD patients. (Scale bar, 100 μm.) F Compared with those in controls (n = 30), miR-150-5p expression levels were increased in NP cells and tissues from IDD patients (n = 67). ***P < 0.001 by the Mann‒Whitney U test. G FISH analysis demonstrated that the levels of miR-150-5p were greater in the NPCs of IDD patients than in those of controls. (Scale bar, 50 μm.) H Targeting strategy for generating miR-150-5p knockout (KO) mice. I The genotypes of the miR-150-5p KO mice were confirmed by Southern blot analysis. J Intervertebral discs were harvested from wild-type (WT) and miR-150-5p KO littermate embryos (E16.5 and E18.5). Hematoxylin and eosin (HE) staining was used to visualize the intervertebral discs of WT and miR-150-5p KO littermate embryos (E16.5 and E18.5). The development of intervertebral discs was not significantly different between WT and miR-150-5p KO littermates. (Scale bar, 100 μm.) K WT and miR-150-5p KO littermates were subjected to needle puncture-induced IDD surgery. Representative images of 6- and 12-week post-IDD surgery intervertebral disc sections. (Scale bar, 100 μm.) L Histological score indicating that miR-150-5p KO could attenuate IDD development (n = 6 per group). P < 0.01 was determined by two-tailed unpaired Student′s t test. M HE and Safranin O staining of intervertebral discs from WT or miR-150-5p KO mice at 6, 15 and 22 months. (Scale bar, 100 μm.) N Histological analysis indicated that miR-150-5p KO could ameliorate age-related IDD in mice. (n = 6 per group) P < 0.01 by two-tailed unpaired Student’s t test
Fig. 2
Fig. 2
Downregulation of miR-150-5p attenuates nucleus pulposus cell senescence. A Cy5-miR-150-5p was taken up by nucleus pulposus cells (NPCs) at 24, 48, and 72 h. (Scale bar, 20 μm.) B EdU assays showed that the level of cellular proliferation was increased in human NPCs transfected with the miR-150-5p inhibitor. (Scale bar, 50 μm.) C Flow cytometry showed that the apoptosis rate was decreased in human NPCs transfected with the miR-150-5p inhibitor. D Senescence-associated β-galactosidase (SA-β-gal) staining demonstrating that cellular senescence was decreased in human NPCs transfected with the miR-150-5p inhibitor. (Scale bar, 20 μm.) E Bar graphs showing the quantification of the relative number of SA-β-gal-positive cells (n = 3). F Western blot analysis showed that the expression levels of P53, P21, P16, MMP3 and MMP9 were affected by the upregulation or downregulation of miR-150-5p. G, H Immunofluorescence analysis showed that the expression levels of P21 and MMP3 were downregulated in human NPCs transfected with the miR-150-5p inhibitor. (Scale bar, 20 μm.) I Intradiscal injection of miR-150-5p mimics, mimic control, miR-150-5p inhibitor, or control in the needle puncture-induced IDD mouse model. The disc tissues were then harvested at the 12th week after IDD surgery. Immunohistochemistry showed the P21 and MMP3 expression levels in the four groups. (Scale bar, 100 μm.)
Fig. 3
Fig. 3
FBXW11 can potentially regulate the targeting of miR-150-5p. A–C Nucleus pulposus cells (NPCs) were harvested from wild-type (WT) mice (n = 3) and miR-150-5p knockout (KO) mice (n = 3). RNA sequencing of NPCs indicated that FBXW11 is a potential regulatory target of miR-150-5p. A heatmap (A) and volcano plot (B) showing the genes differentially expressed between the NPCs of the WT and miR-150-5p KO mice. C The PCA of the data revealed strong separation between the two groups and good homogeneity within each group for mRNA arrays. D, E Bioinformatics data showing that miR-150-5p was computationally predicted to target FBXW11 by different algorithms. F The 3′-UTR of FBXW11 mRNA contains potential binding sites for miR-150-5p. This sequence alignment showed a high level of sequence conservation and complementarity with miR-150-5p. G Relative luciferase activity detection in human NPCs cotransfected with miR-150-5p mimic/inhibitor and FBXW11 3′-UTR-wild/mutant-type reporter plasmids. H, I The qRT‒PCR and Western blot results showed that the FBXW11 mRNA and protein expression levels were decreased and increased, respectively, in human NPCs following transfection with miR-150-5p mimics and inhibitors
Fig. 4
Fig. 4
The effects of miR-150-5p on the FBXW11/TAK1/NF-κB pathway. A, B The NF-κB signaling pathway was predicted to be the pathway most enriched with upregulated miRNAs by KEGG analysis. C The expression levels of proteins in the NF-kB signaling pathway, including p-P65, P65, p-IKKγ, IKKγ, p-IKKα and IKKα, in nucleus pulposus cells (NPCs) from wild-type and miR-150-5p knockout (KO) mice. D TAK1 was identified as a molecular target that interacts with FBXW11 by mass spectrometry (LC‒MS/MS). E Coimmunoprecipitation (co-IP) indicated that FBXW11 may directly interact with TAK1. F–H FBXW11 negatively regulated TAK1 through K48-linked ubiquitination at the K446 residue. I The FBXW11/TAK1/NF-κB pathway may be the fundamental mechanism underlying the progression of IDD induced by miR-150-5p
Fig. 5
Fig. 5
Identification of the binding proteins and selection of nucleus pulposus cell-specific aptamers. A Schematic illustration of aptamer selection in nucleus pulposus cells (NPCs). B–D Essential strategies for optimizing PCR conditions to generate ssDNA aptamers in SELEX. The highest proportion of DNA of interest relative to unwanted products was considered the optimum condition. The results revealed that the best values for final template concentration (B), annealing temperature (C) and number of PCR cycles (D) were 100 ng, 60 °C and 20 cycles, respectively. E Proposed secondary structure of the EY1-4 aptamer. F The subcellular localization of EY3 in NPCs, chondrocytes and fibroblasts. (Scale bar, 20 μm.) G Annotated MS/MS spectrum assigned to the FasL peptide. The data were acquired from the analysis of the samples by high-sensitivity LC‒MS/MS on a Q Exactive mass spectrometer. H Coomassie blue-stained SDS‒PAGE was used to analyze aptamer-assisted target purification. I–L Model of the interaction between EY3 and FasL.
Fig. 6
Fig. 6
The PLGA-PLL-PEG-EY3 nanoparticles facilitate miR-150-5p delivery into nucleus pulposus cells. A Electrophoretic mobility of miRNAs in an agarose gel. B The surface zeta potential and diameter of PLGA-PLL-PEG/miR-150-5p-Cy5 with or without an aptamer (EY3). C Transmission electron microscopy (TEM) image of the PLGA-PLL-PEG/miR-150-5p-Cy5 nanoplatform with or without an aptamer (EY3). (Left: PLGA-PLL-PEG/miR-150-5p-Cy5; Right: PLGA-PLL-PEG-EY3/miR-150-5p-Cy5) (scale bar, 500 nm). D Release profile of PLGA-PLL-PEG/miR-150-5p-Cy5 and PLGA-PLL-PEG-EY3/miR-150-5p-Cy5 within 168 h. Drug release analysis revealed the controlled release of PLGA-PLL-PEG-EY3/miR-150-5p-Cy5, for which approximately 70% of the material was released after 96 h. E In vitro viability of nucleus pulposus cells (NPCs) treated with PLGA-PLL-PEG/miR-150-5p-Cy5 and PLGA-PLL-PEG-EY3/miR-150-5p-Cy5. F Flow cytometry image of the PLGA-PLL-PEG/miR-150-5p-Cy5 and PLGA-PLL-PEG-EY3/miR-150-5p-Cy5 binding peaks. G Cellular uptake of PLGA-PLL-PEG/miR-150-5p-Cy5 with or without an aptamer (EY3) after 8 h of incubation. (Scale bar, 100 μm.) H In vivo fluorescence imaging of intervertebral discs 3 months after intradiscal injection of PLGA-PLL-PEG/miR-150-5p-Cy5 or PLGA-PLL-PEG-EY3/miR-150-5p-Cy5. (Scale bar, 100 μm.) I Hematoxylin and eosin (HE) staining of the lungs, livers and kidneys of mice 3 months after intradiscal injection of PLGA-PLL-PEG/miR-150-5p-Cy5 or PLGA-PLL-PEG-EY3/miR-150-5p-Cy5. (Scale bar, 20 μm.) J Intracellular distribution of PLGA-PLL-PEG-EY3/miR-150-5p-Cy5 in nucleus pulposus cells. (Scale bar, 20 μm.)
Fig. 7
Fig. 7
Intradiscal delivery of miR-150-5p inhibitor by the PLGA-PLL-PEG-EY3 nanoparticles alleviates intervertebral disc degeneration in a mouse model. A Schematic representation of the experimental design. B, C X-ray and MRI images showing an alleviation of intervertebral space collapse in mice treated with PLGA-PLL-PEG-EY3/antagomiR-150-5p (6 weeks, 12 weeks post intradiscal injection). D, E The disc height index and Pfirrmann score were significantly lower in mice treated with PLGA-PLL-PEG-EY3/antagomiR-150-5p (6 weeks and 12 weeks after intradiscal injection). F Safranin-O staining images of intervertebral discs from the different treatment groups. G Histological scores were significantly lower in mice treated with PLGA-PLL-PEG-EY3/antagomiR-150-5p (6 weeks, 12 weeks after intradiscal injection). H, I Immunohistochemistry assays showed increased Col II and decreased MMP13 expression in mice treated with PLGA-PLL-PEG-EY3/antagomiR-150-5p
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Knezevic NN, Candido KD, Vlaeyen JWS, Van Zundert J, Cohen SP. Low back pain. Lancet. 2021;398(10294):78–92. doi: 10.1016/S0140-6736(21)00733-9. - DOI - PubMed
    1. Vlaeyen JWS, Maher CG, Wiech K, Van Zundert J, Meloto CB, Diatchenko L, Battié MC, Goossens M, Koes B, Linton SJ. Low back pain. Nat Rev Dis Primers. 2018;4(1):52. doi: 10.1038/s41572-018-0052-1. - DOI - PubMed
    1. Francisco V, Pino J, González-Gay M, Lago F, Karppinen J, Tervonen O, Mobasheri A, Gualillo O. A new immunometabolic perspective of intervertebral disc degeneration. Nat Rev Rheumatol. 2022;18(1):47–60. doi: 10.1038/s41584-021-00713-z. - DOI - PubMed
    1. Lyu FJ, Cui H, Pan H, Mc Cheung K, Cao X, Iatridis JC, Zheng Z. Painful intervertebral disc degeneration and inflammation: from laboratory evidence to clinical interventions. Bone Res. 2021;9(1):7. doi: 10.1038/s41413-020-00125-x. - DOI - PMC - PubMed
    1. Zhang X, Hu Y, Hao D, Li T, Jia Y, Hu W, Xu Z. New strategies for the treatment of intervertebral disc degeneration: cell, exosome, gene, and tissue engineering. Am J Transl Res. 2022;14(11):8031–8048. - PMC - PubMed
-