Codelivery of TGF-β1 and anti-miR-141 by PLGA microspheres inhibits progression of intervertebral disc degeneration

Abstract

Background: Cervical and lumbar pain is usually caused by degeneration of the nucleus pulposus (NP). As a powerful therapeutic strategy, tissue engineering can effectively restore the normal biological properties of the spinal unit. Previous studies suggested that poly(lactic-co-glycolic acid) (PLGA) microspheres are effective carriers of cells and biomolecules in NP tissue engineering. This study aims to explore the therapeutic effect of PLGA microspheres coloaded with transforming growth factor-β1 (TGF-β1) and anti-miR-141 on intervertebral disc degeneration (IDD).

Methods: PLGA microspheres were characterized by scanning electron microscopy, a laser particle size analyzer, and laser confocal microscopy. The in vitro release rate of biomolecules from the microspheres was analyzed by reversed-phase high-performance liquid chromatography and agarose gel electrophoresis. The rat NP cells (NPCs) treated with the solutions released from microspheres for different lengths of time were assigned to a control group (Ctrl), an empty PLGA microsphere group (Mock microsphere, MS), a TGF-β1-loaded PLGA microsphere group (TMS), an anti-miR-141-loaded PLGA microsphere group (AMS), and an anti-miR-141 + TGF-β1-loaded PLGA microsphere group (ATMS). The proliferation and apoptosis of NPCs were observed by alamar blue and flow cytometry. The gene and protein expression of cartilage markers COL2A1 and ACAN were observed by RT-qPCR and Western blot. The rat model of IDD was established by tail puncture. Rats were divided into a control group (Ctrl), a mock operation group (Mock), a TGF-β1 microsphere group (TMS), an anti-miR-141 microsphere group (AMS), and an anti-miR-141 + TGF-β1 microsphere group (ATMS). The degree of rat tail IDD was assessed in each group through magnetic resonance imaging (MRI), safranin O-fast green staining, immunohistochemistry, and Western blotting.

Results: PLGA microspheres were stably coloaded and could sustainably release TGF-β1 and anti-miR-141. The results of in vitro cell experiments showed that the release solution of PLGA microspheres significantly enhanced the proliferation of NPCs without inducing their apoptosis and significantly upregulated cartilage markers in NPCs. The effect of microspheres was greater in the ATMS group than that in the TMS group and AMS group. In vivo experiments showed that IDD could be effectively inhibited and reversed by adding microspheres coloaded with TGF-β1 and/or anti-miR-141, and the effect was greatest in the ATMS group.

Conclusion: PLGA microspheres coloaded with TGF-β1 and anti-miR-141 can reverse IDD by inhibiting the degeneration of NPCs.

Keywords: Intervertebral disc degeneration; Microsphere; Nucleus pulposus; TGF-β1; miR-141.

Fig. 1

Characterization of PLGA microspheres and the loading and release of biomolecules. A, B SEM images of PLGA microsphere. C Localization of TGF-β1 and anti-miR-141 in microsphere. D Release detection of TGF-β1 and anti-miR-141 in vitro

Fig. 2

Effect of PLGA microsphere release solution on NPCs. A Alamarblue was used to detect the proliferation level of nucleus pulposus cells under the release of PLGA microspheres at different time points. B Flow cytometry was used to detect the apoptotic level of nucleus pulposus cells under the release of PLGA microspheres in different groups. C–G RT-qPCR, western blot and immunofluorescence were used to detect the expression levels of phenotypic genes and proteins of nucleus pulposus cells in different groups (n ≥ 3, *p < 0.05, **p < 0.01)

Fig. 3

In vivo repair effect of PLGA microspheres on IDD. A Group description of animal experiment. B, C Observation of rat tail intervertebral disc degeneration in each group by MRI and classification analysis (n ≥ 3, *p < 0.05, **p < 0.01)

Fig. 4

In vivo repair effect of PLGA microspheres on IDD. A, B Observation of rat tail intervertebral disc in each group by safranine and fast green staining and classification analysis. C, D The COL2A1 expression level in NP tissues in the five groups were determined by immunohistochemistry and quantitative analysis. E, F Western blot was used to detect the difference in the expression of ACAN and COL2A1 protein in nucleus pulposus tissues with different degrees of degeneration (n ≥ 3, *p < 0.05, **p < 0.01)