CD44-targeting hyaluronic acid-selenium nanoparticles boost functional recovery following spinal cord injury

Abstract

Background: Therapeutic strategies based on scavenging reactive oxygen species (ROS) and suppressing inflammatory cascades are effective in improving functional recovery after spinal cord injury (SCI). However, the lack of targeting nanoparticles (NPs) with powerful antioxidant and anti-inflammatory properties hampers the clinical translation of these strategies. Here, CD44-targeting hyaluronic acid-selenium (HA-Se) NPs were designed and prepared for scavenging ROS and suppressing inflammatory responses in the injured spinal cord, enhancing functional recovery.

Results: The HA-Se NPs were easily prepared through direct reduction of seleninic acid in the presence of HA. The obtained HA-Se NPs exhibited a remarkable capacity to eliminate free radicals and CD44 receptor-facilitated internalization by astrocytes. Moreover, the HA-Se NPs effectively mitigated the secretion of proinflammatory cytokines (such as IL-1β, TNF-α, and IL-6) by microglia cells (BV2) upon lipopolysaccharide-induced inflammation. In vivo experiments confirmed that HA-Se NPs could effectively accumulate within the lesion site through CD44 targeting. As a result, HA-Se NPs demonstrated superior protection of axons and neurons within the injury site, leading to enhanced functional recovery in a rat model of SCI.

Conclusions: These results highlight the potential of CD44-targeting HA-Se NPs for SCI treatment.

Keywords: CD44 targeting; Inflammation; Reactive oxygen species; Selenium nanoparticles; Spinal cord injury.

Scheme 1

Schematic illustration of the preparation of CD44-targeting HA-Se NPs for promoting functional recovery after SCI

Fig. 1

Characterization of hyaluronic acid-selenium nanoparticles (HA-Se NPs). A Dynamic light scattering measurement of HA-Se NPs. B Transmission and C scanning electron microscopy imaging of HA-Se NPs. D The efficacy of HA-Se NPs in scavenging free radicals was evaluated at varying concentrations

Fig. 2

The efficacy of HA-Se NPs in preventing H₂O₂-induced oxidative stress and inflammation was evaluated. A Live/dead staining of astrocytes. Scale bar = 20 μm. The concentration of H₂O₂ was 100 µM. B Quantitative analysis of astrocyte cell death. *P < 0.05. C Levels of reactive oxygen species within astrocytes were determined via DCFH-DA staining. D Quantitative analysis of DCF fluorescence intensity in astrocytes. **P < 0.01. Quantification of E interleukin (IL)-1β, F IL-6, and G tumor necrosis factor (TNF)-α levels in BV2 cell culture medium in the presence or absence of HA-Se NPs. *P < 0.05

Fig. 3

CD44 expression in the injured spinal cord. A Representative western blot images of CD44 expression in the control and SCI groups. B Densitometric analysis of CD44 levels based on data in (A). **P < 0.01 in comparison to the control group. C CD44 immunofluorescence staining of injured spinal cord samples. Mag: Magnification. D Immunofluorescence staining of injured spinal cord samples for GFAP (red) and CD44 (green). E Immunofluorescence staining of injured spinal cord samples for CD44 (green) and Iba-1 (red). F Immunofluorescence staining of injured spinal cord samples for NeuN (green) and CD44 (red). G Immunofluorescence staining of injured spinal cord samples for CD68 (green) and CD44 (red)

Fig. 4

CD44 immunofluorescence staining in astrocytes and internalization of Cy5-HA-Se NPs. A Astrocytes stained for CD44 and Dil. Scale bar = 50 μm. B Cy5-HA-Se NPs internalization by astrocytes after 4 and 8 h of incubation. Scale bar = 50 μm. C Targeting efficiency of HA-Se NPs in vivo. Fluorescence images of the major organs after intravenous injection of rats with Cy5-labeled HA-Se NPs. The organs were collected at 24 h post-injection from non-treated rats and at 6, 12, and 24 h post-injection from SCI rats

Fig. 5

Neuroprotective effects of HA-Se NPs. A Basso Beattie Bresnahan (BBB) scores of SCI rats. B Gross images of the spinal cord in different groups at 12 weeks post-injury. The lesion site is denoted by the red circle. C Images of hematoxylin and eosin (H&E) right, three magnified images of the areas marked by the blue squares. Scale bars = 500 and 50 μm, respectively. Dotted line and arrows indicate the lesion cavity and inflammatory cells, respectively. D Images of an injured spinal cord stained with LFB. Scale bars = 500 and 50 μm, respectively. Dotted line indicates the lesion cavity. E Representative TEM images of different experimental groups after treatment with saline or HA-Se NPs at the indicated concentrations. Scale bar = 2 μm. Arrows indicate myelin sheaths. F Western blot analysis of cleaved caspase-3 in injured spinal cord tissues 24 h after injury. GAPDH was used as the internal control. G Densitometric analysis of cleaved caspase-3 levels based on (D). **P < 0.01 in relation to the saline group

Fig. 6

Immunohistochemistry of scar tissue labeled with NeuN (green) and NF200 (red). The group that received a dosage of 10 mg/kg exhibited greater preservation of both neurons and axons within the affected spinal cord tissue, even at 12 weeks post-SCI. Scale bar = 50 μm