Delivery of pOXR1 through an injectable liposomal nanoparticle enhances spinal cord injury regeneration by alleviating oxidative stress

Abstract

Oxidation resistance 1 (OXR1) is regarded as a critical regulator of cellular homeostasis in response to oxidative stress. However, the role of OXR1 in the neuronal response to spinal cord injury (SCI) remains undefined. On the other hand, gene therapy for SCI has shown limited success to date due in part to the poor utility of conventional gene vectors. In this study, we evaluated the function of OXR1 in SCI and developed an available carrier for delivering the OXR1 plasmid (pOXR1). We found that OXR1 expression is remarkably increased after SCI and that this regulation is protective after SCI. Meanwhile, we assembled cationic nanoparticles with vitamin E succinate-grafted ε-polylysine (VES-g-PLL) (Nps). The pOXR1 was precompressed with Nps and then encapsulated into cationic liposomes. The particle size of pOXR1 was compressed to 58 nm, which suggests that pOXR1 can be encapsulated inside liposomes with high encapsulation efficiency and stability to enhance the transfection efficiency. The agarose gel results indicated that Nps-pOXR1-Lip eliminated the degradation of DNA by DNase I and maintained its activity, and the cytotoxicity results indicated that pOXR1 was successfully transported into cells and exhibited lower cytotoxicity. Finally, Nps-pOXR1-Lip promoted functional recovery by alleviating neuronal apoptosis, attenuating oxidative stress and inhibiting inflammation. Therefore, our study provides considerable evidence that OXR1 is a beneficial factor in resistance to SCI and that Nps-Lip-pOXR1 exerts therapeutic effects in acute traumatic SCI.

Keywords: DNA condensed agent; Gene therapy; OXR1; Oxidative stress; Spinal cord injury.

Graphical abstract

Fig. 1

Expression of OXR1 is increased in neurons in vivo and in vitro following injury. (a-b) Representative western blotting and quantification of OXR1 protein in spinal cord at specified times after SCI. (c-d) Immunofluorescence staining and quantitative analysis of OXR1 expression in neurons in the indicated groups after SCI. Scale bar = 50 μm. Scale bar (Enlarged) = 10 μm. (e-f) Immunoblots and quantification of TBHP-subjected PC12 cells at different time points. GAPDH was the loading control. Data are presented as the means ± SD, n = 3. Significant differences among the different groups are indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Sham was used as a control.

Fig. 2

Characterization of Nps-pOXR1-Lip. (a) Schematic diagram of the production process of Nps-pOXR1-Lip. (b) Transmission electron microscopy images of Nps-pOXR1-Lip. Scale bars = 50 nm. (c) Hydrodynamic diameter, PDI and (d) Zeta of Nps, Nps-pOXR1, Nps-pOXR1-Lip.n = 3. (e) Gel retardation assay of Nps-pOXR1-Lip prepared at varying weight ratios. (f) Hydrodynamic diameter of Nps-pOXR1-Lip at various weight ratios. n = 3. (g) Gel retardation assay of Nps-pOXR1-Lip incubated with DNase I. (h) At specified points, the hydrodynamic diameter and (i) PDI of Nps-pOXR1-Lip incubated with pH 6.5 PBS containing 10% human serum albumin at 37 °C was detected by DLS. n = 3.

Fig. 3

In vitro cellular uptake and transfection assay. (a) Cell viability and (b) transfection efficiency of two types of complexes with different concentrations of Nps (VES-g-PLL micelle, concentration of VES-g-PLL in complexes) against PC12 cells after 24 h of incubation (n = 60. (c-d) Confocal images and quantitative analysis of PC12 cells after incubation with various compounds containing pDNA for 24 h. Scale bar = 50 μm (n = 3). (e-f) Immunoblots and quantification of OXR1 protein in PC12 cells after the same treatment as above. PEI/pOXR1 (0.5 μg/mL) was used as a positive control. GAPDH was the loading control. Data represent mean ± SD. n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

Nps-pOXR1-Lip enhances OXR1 expression in the spinal cord. (a-d) Immunoblots and immunofluorescence staining of OXR1 in spinal cord tissues of rats after treatment with various formulations at 3 d. (e) Double immunostaining of OXR1 and GFAP or Iba1 at 3 d after SCI. (f-h) Immunofluorescence intensity of OXR1 and NEUN, GFAP and Iba1 across the white dotted lines in each group. Plot profile analysis in ImageJ was used to determine the colocalization of different indicators. Scale bar = 50 μm. Scale bar (Enlarged) = 10 μm. Data represent mean ± SD. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

Nps-pOXR1-Lip promotes functional recovery after SCI. (a) Drug injection model and behavioral experimental design. (b) Images of representative spinal cords of all treatment groups 28 days postinjury. (c) Footprint analysis of each group at 28 dpi (blue, forelimbs; red, hind limbs). (d) Representative images from LFB staining at 28 dpi. Scale bar = 1000 μm. Scale bar (Enlarged) = 100 μm. (e, g) Statistical analysis of the BBB in the different groups at −1, 1, 3, 7, 14, 21, and 28 dpi. n = 6. (f, h) Statistical analysis of the angle of inclined test in the different groups. n = 6. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6

Nps-pOXR1-Lip reduces apoptosis after SCI. (a-b) Immunofluorescence staining and quantitative analysis of TUNEL in spinal cord sections from various groups at 7 dpi. Scale bar = 50 μm. (c-d) Confocal images of transverse spinal cord sections stained with OXR1 and C-caspase-3 at 7 dpi. Scale bar = 50 μm. (e-h) Immunoblots and quantification of C-caspase-3, Bcl-2, and Bax expression in rats after SCI in various groups. GAPDH was used as a loading control. Data represent mean ± SD. n = 3. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7

Nps-pOXR1-Lip alleviates oxidative stress in vivo. (a-b) DHE staining for reactive ROS 3 days after SCI. Immunofluorescence and quantitative analysis of frozen spinal cord sections stained with DHE. Scale bar = 50 μm. (c-g) Representative western blotting and quantification of Nrf-2, HO-1, SOD1, and CAT expression in rats after SCI in various groups at 3 dpi. GAPDH was the loading control. n = 3. Data represent mean ± SD. n = 3. *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8

Nps-pOXR1-Lip reduces fibrotic scar tissue by impacting the inflammatory reaction. (a-b) Immunoblot analysis showing protein expression levels of CD68 in various groups after 7 dpi (n = 3). (c-f) Immunofluorescence staining of monocytic phagocytes (CD68) and astrogliosis (GFAP) in spinal cord horizontal sections. Scale bar = 1000 μm. CD68-positive cells were detected in the rostral, epicentral and caudal sites of the spinal cord in the (a1-a3) SCI, (b1-b3) pOXR1, (c1-c3) Nps-pOXR1, and (d1-d3) Nps-pOXR1-Lip groups. Scale bar = 100 μm. (d-f) Fluorescence intensity quantification of CD68 in different sites. n = 3. (g) Masson staining of horizontal sections of spinal cord in various groups at 28 dpi. Scale bar = 100 μm. (h-k) Immunoblots and quantification of laminin, Neurocan, and NG2 expression in the spinal cord of various groups at 28 dpi. GAPDH was used as a loading control (n = 3). Data represent mean ± SD. n = 3. *p < 0.05, **p < 0.01, ***P < 0.001.

Scheme 1

The Nps-pOXR1-Lip nanoparticle is designed for treat SCI.