Plant-derived exosomes extracted from Lycium barbarum L. loaded with isoliquiritigenin to promote spinal cord injury repair based on 3D printed bionic scaffold

Abstract

Plant-derived exosomes (PEs) possess an array of therapeutic properties, including antitumor, antiviral, and anti-inflammatory capabilities. They are also implicated in defensive responses to pathogenic attacks. Spinal cord injuries (SCIs) regeneration represents a global medical challenge, with appropriate research concentration on three pivotal domains: neural regeneration promotion, inflammation inhibition, and innovation and application of regenerative scaffolds. Unfortunately, the utilization of PE in SCI therapy remains unexplored. Herein, we isolated PE from the traditional Chinese medicinal herb, Lycium barbarum L. and discovered their inflammatory inhibition and neuronal differentiation promotion capabilities. Compared with exosomes derived from ectomesenchymal stem cells (EMSCs), PE demonstrated a substantial enhancement in neural differentiation. We encapsulated isoliquiritigenin (ISL)-loaded plant-derived exosomes (ISL@PE) from L. barbarum L. within a 3D-printed bionic scaffold. The intricate construct modulated the inflammatory response following SCI, facilitating the restoration of damaged axons and culminating in ameliorated neurological function. This pioneering investigation proposes a novel potential route for insoluble drug delivery via plant exosomes, as well as SCI repair. The institutional animal care and use committee number is UJS-IACUC-2020121602.

Keywords: 3D bioprinting; M2 polarization; nerve regeneration; plant‐derived exosomes; spinal cord injury.

FIGURE 1

Characterization of plant‐derived exosomes (PE) and isoliquiritigenin (ISL)‐loaded plant‐derived exosomes (ISL@PE) in hydrogel. (a) Transmission electron micrographs of PE and ISL@PE. (b) The size distribution of PE and ISL@PE. (c) The in vitro cumulative release rate of ISL@PE and free ISL at pH 1.2, 6.8, and 7.4. (d) 3D‐printed spinal cord hydrogel scaffold. (A) Model design of mimic spinal cord segment. (B, C) 3D‐printed hydrogel (scale bar equals to 3 mm). (e) Young's modulus of GelMA. (f) The scanning electron micrographs of the 3D‐printed functional hydrogel (scale bar equals to 20 μm). The arrows indicate the ISL@PE. (g) The in vitro release of ISL@PE in hydrogel under various pH conditions.

FIGURE 2

Neuro differentiation effect of isoliquiritigenin (ISL)‐loaded plant‐derived exosomes (ISL@PE). (a) Fluorescent staining of neural stem cells expressing GFAP, Nestin, Sox2, and Tuj1. (b) Fluorescent staining of neural stem cells expressing Sox2, Tuj1, and MAP2 in control, PE, ME, free ISL, and ISL@PE formulations. Scale bar equals to 50 μm.

FIGURE 3

Quantitative analysis of neuro differentiation effect of ISL@PE. (a) Quantitative analysis of relative positive cell expressed in immune fluorescent staining. (b) Neurite length of differentiated neural stem cells in different groups. *p > 0.05; **p < 0.05; ***p < 0.01.

FIGURE 4

Effect of different groups on polarization of lipopolysaccharide (LPS)‐treated microglial N9 cells. Scale bar equals to 50 μm. Inducible nitric oxide synthase (iNOS) production is an important effect of antimicrobial M1 macrophage activity, whereas anti‐inflammatory (M2) macrophages high expressed arginase‐1 (Arg‐1) and CD206. The LPS‐induced group termed normal group did not express CD206 and Arg‐1, suggesting that the N9 cells could hardly transition from M1 to M2 without intervention. After PE, free ISL, and ISL@PE treatment, N9 cells expressed low levels of the M2 proteins CD206 and Arg‐1, indicating that free ISL and ISL@PE had a beneficial effect on the transition of N9 cells from M1 to M2. No significant changes could be found in ME group.

FIGURE 5

Effect of isoliquiritigenin (ISL)‐loaded plant‐derived exosomes (ISL@PE) on immune reaction in lipopolysaccharide (LPS)‐treated microglial N9 cells. (a) Immunofluorescent staining of oxidative stress in N9 cells. (b) Flow cytometry detection of oxidative stress in N9 cells. (c, d) The statistical results of ROS‐immunofluorescent staining (ROS‐IF) and ROS‐flow cytometry detection (ROS‐FCS). (e) Western blot of iNOS, CD206, and Arg‐1 expression in N9 cells on Day 3 of different treatment groups. (f) Western blot of AKT and pAKT expression in N9 cells on Day 3 of different treatment groups. Scale bars are equal to 50 μm, ***p < 0.01.

FIGURE 6

The positive effect of 3D‐printed hydrogel with isoliquiritigenin (ISL)‐loaded plant‐derived exosomes (ISL@PE) in treating spinal cord injury. (a) Basso–Beattle–Bresnahan (BBB). (b) Inclined plane test. (c) Open field test. (d) Nissl staining. (e) HE staining. Scale bar equals to 50 μm.

FIGURE 7

Fluorescent staining of GFAP, Tuj1, NF200, Nestin, MAP2, and MBP proteins in the injured spinal cord of different treatment groups. Scale bars are equal to 50 μm. A positive expression of Tuj1, NF200, Nestin, MAP2, and MBP could be found in 3D‐printed hydrogel group with isoliquiritigenin (ISL)‐loaded plant‐derived exosomes (ISL@PE) compared to the model group.

FIGURE 8

Immune staining of GFAP, GAP43, NF200, and MBP proteins of the injured spinal cord in different treatment groups. (a) Immunohistochemical staining of relative protein expressed in different group. (b) Quantitative analysis of immunohistochemical staining. There is a higher expression of GAP43, NF200, and MBP in the spinal cord 3D printed hydrogel containing isoliquiritigenin (ISL)‐loaded plant‐derived exosomes (ISL@PE) compared to that containing ISL. Scale bar equals to 50 μm. ***p < 0.01.

FIGURE 9

Relative expression of GFAP, GAP43, NF200, MBP, and GAPDH proteins in the injured spinal cord in different treatment groups. Western Blot protein expressions (a) and quantification data (b) of GFAP, Tuj1, NF200, Nestin, MAP2, and MBP in each group. (c) Quantification data of IL‐10, IL‐1β, IL‐6, and TNF‐α of injured spinal cord in different treatment groups. ***p < 0.01.