Sodium alginate and naloxone loaded macrophage-derived nanovesicles for the treatment of spinal cord injury

Abstract

Spinal cord injury (SCI) causes Ca²⁺ overload, which can lead to inflammation and neuronal apoptosis. In this study, we prepared a nanovesicle derived from macrophage membrane (MVs), which encapsulated sodium alginate (SA) and naloxone (NAL) to inhibit inflammation and protect neurons by reducing the free Ca²⁺concentration at the SCI site. Based on the transmission electron microscopy (TEM) image, the encapsulated sample (NAL-SA-MVs) had a particle size of approximately 134 ± 11 nm and exhibited a sustained release effect. The encapsulation rate of NAL and SA was 82.07% ± 3.27% and 72.13% ± 2.61% in NAL-SA-MVs, respectively. Targeting tests showed that the NAL-SA-MVs could accumulate in large quantities and enhance the concentration of SA and NAL at the lesion sites. In vivo and in vitro studies indicated that the NAL-SA-MVs could decrease the concentration of free Ca²⁺, which should further alleviate the inflammatory response and neuronal apoptosis. Anti-inflammation results demonstrated that the NAL-SA-MVs could reduce the pro-inflammation factors (iNOS, TNF-α, IL-1β, IL-6) and increase the expression of anti-inflammation factors (IL-10) at the cell and animal level. Concurrently, fluorescence, flow cytometry and western blot characterization showed that the apoptotic condition of the neurons was significantly inhibited. In addition, the motor function of C57 mice were significantly improved after NAL-SA-MVs treatment. In conclusion, it is suggested that the NAL-SA-MVs has tremendous potential in the treatment of SCI.

Keywords: Inflammation; Macrophage membrane; Naloxone; Neuroprotection; Sodium alginate; Spinal cord injury.

Graphical abstract

Scheme 1

Sodium alginate and naloxone loaded macrophage-derived nanovesicles preparation and spinal cord injury therapy.

Fig. 1

The characterization of NAL-SA-MVs. (A) TEM imaging of NAL-SA-MVs. (B) The DLS size distribution diagrams of MVs, SA-MVs, NAL-SA-MVs. (C) The ζ potential distribution of MVs, SA-MVs, NAL-SA-MVs. (D) 7 d stability test of NAL-SA-MVs. (E) The vitro release assay of NAL and SA. (F) The pharmacokinetics of free NAL and NAL-SA-MVs in vivo. (G) The cytotoxicity of blank MVs in VSC4.1 and BV₂. (H) Western blotting analysis of protein markers of RAW264.7, RAW264.7 cell membranes, SA-MVs and NAL-SA-MVs. All data represented the mean ± SD (n = 3).

Fig. 2

The uptake of NAL-SA-MVs in vitro and the targeting of NAL-SA-MVs in vivo. (A, B) Confocal fluorescence imaging of BV₂ cells treated with FITC-ReVs and FITC-MVs. FITC-ReVs/FITC-MVs (green), nucleus (blue) and cell membrane (red). (C, D) Confocal fluorescence imaging of VSC4.1 cells treated with FITC-ReVs and FITC-MVs. FITC-ReVs/FITC-MVs (green), nucleus (blue) and cell membrane (red). (E) The spinal cord fluorescence imaging of SCI mice after FITC-ReVs and FITC-MVs injected by tail vein, respectively, at different time points. (F) Fluorescence quantitative analysis of spinal cord in different groups over time. (G) The fluorescence imaging of the major organs (heart, liver, spleen, lung and kidney) from different groups mice at 12 h after tail vein injection. (H) Fluorescence quantitative analysis of organs in different groups. All data represented the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

SA binds to Ca²⁺ and reduces the concentration of free Ca²⁺ in the damaged microenvironment. (A, B) Intracellular free Ca²⁺ was determined by Fura-2AM fluorescent probe and its statistical analysis. (C, D) Determination and statistical analysis of free calcium ion concentration in injured spinal cord tissue sections by Fura-2AM fluorescent probe. (E, F) Two-photon detection of free calcium ion concentration in hyaline spinal cord and its statistical analysis. All data represented the mean ± SD (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

NAL-SA-MVs inhibited the secretion of inflammatory mediators and achieved anti-inflammatory effect in vitro. (A) Fluorescence microscopy images for immunofluorescence staining of BV₂ cells were untreated, treated with PBS, NAL, SA-MVs and NAL-SA-MVs. Cell nuclei were stained with DAPI (blue), cell cytoskeleton was stained with β- tubulin (red), pro-inflammatory protein were stained with iNOS (green). (B) Fluorescence quantitative analysis in different groups. (C) Western blot detection of the inflammatory factors (iNOS, IL-6, IL-10) released by BV₂ cells in different groups. (D–F) Quantitative protein expression statistical analysis of (iNOS, IL-6 and IL-10 in figure C. All data represented the mean ± SD (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

NAL-SA-MVs inhibited the inflammatory response at the site of injuried spinal cord. (A) Western blot detection of the inflammatory factors (iNOS, IL-6, TNF-α, IL-1β, IL-10) from spinal cord tissue of different groups. (B-F) Quantitative protein expression statistical analysis of iNOS, IL-6, TNF-α, IL-1β, IL-10 in Fig. 5A. (G, H) Fluorescence microscopy images for immunofluorescence staining of spinal cord tissue sections. Cell nuclei were stained with DAPI (blue), pro-inflammatory protein were stained with IL - 1β (green). All data represented the mean ± SD (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

NAL-SA-MVs can inhibit the expression of apoptotic factors and has anti-apoptotic effect in vitro. (A, B) Representative flow cytometry analysis of the proportion of apoptotic cells in each treatment group and the corresponding quantitative analysis. (C, D) Fluorescence microscopy images of VSC4.1 cells were untreated and treated with PBS, NAL, SA-MVs, NAL-SA-MVs. Cell nuclei were stained with DAPI (blue), cell cytoskeleton was stained with β - tubulin (red), pro-apoptotic protein were stained with Caspase-3 (green). (E) Western blot detection of the apoptotic factors (Caspase-3, Bax, Bcl-2) released by VSC4.1 cells in different groups. (F–I) Quantitative protein expression statistical analysis of Caspase-3, Bax, Bcl-2 and Bax/Bcl-2 in figure E. All data represented the mean ± SD (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7

NAL-SA-MVs inhibited apoptosis after spinal cord injury in mice. (A) Western blotting detection of the cytokines (Caspase-3, Bcl-2, Bax) from spinal cord tissue of different groups. (B–E) Quantitative protein expression statistical analysis of Caspase-3, Bax, Bcl-2 and Bax/Bcl-2 in Fig. 7A. (F, G) Fluorescence microscopy images of spinal cord tissue sections of sham, SCI, NAL, SA-MVs and NAL-SA-MVs. Cell nuclei were stained with DAPI (blue), neurons were stained with neun (green), and apoptin were stained with Caspase-3 (red). (H, I) Fluorescence microscopy images and statistical analysis of TUNEL-DAPI assay. All data represented the mean ± SD (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8

NAL-SA-MVs could promote the survival of neurons and the recovery of motor function in mice after spinal cord injury, and the evaluation of the preparation safety. (A) The wound surface of a mouse in the process of SCI model. (B) Statistical analysis of BMS scores in different groups before and after spinal cord injury in mice. (C) Statistical analysis of body weight of mice before and after spinal cord injury. (D) Footprints of SHAM, SCI, NAL, SA-MVs and NAL-SA-MVs group. The forepaws and hindpaws of each group were stained, respectively, with black and red dye. (E, F) The statistical analysis of step length (SL) and stride width (SW). (G) Neun-labeled neurons at 5 × and 10 × of the different groups. (H) Number of surviving neurons at 7d after spinal cord injury in each group by Nissl staining. (I) HE staining of heart, liver, spleen, lung and kidney tissue sections of each group for safety evaluation. All data represented the mean ± SD (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.