CX3CR1-Targeted PLGA Nanoparticles Reduce Microglia Activation and Pain Behavior in Rats with Spinal Nerve Ligation

Abstract

Activation of CX3CR1 in microglia plays an important role in the development of neuropathic pain. Here, we investigated whether neuropathic pain could be attenuated in spinal nerve ligation (SNL)-induced rats by reducing microglial activation through the use of poly(D,L-lactic-co-glycolic acid) (PLGA)-encapsulated CX3CR1 small-interfering RNA (siRNA) nanoparticles. After confirming the efficacy and specificity of CX3CR1 siRNA, as evidenced by its anti-inflammatory effects in lipopolysaccharide-stimulated BV2 cells in vitro, PLGA-encapsulated CX3CR1 siRNA nanoparticles were synthesized by sonication using the conventional double emulsion (W/O/W) method and administered intrathecally into SNL rats. CX3CR1 siRNA-treated rats exhibited significant reductions in the activation of microglia in the spinal dorsal horn and a downregulation of proinflammatory mediators, as well as a significant attenuation of mechanical allodynia. These data indicate that the PLGA-encapsulated CX3CR1 siRNA nanoparticles effectively reduce neuropathic pain in SNL-induced rats by reducing microglial activity and the expression of proinflammatory mediators. Therefore, we believe that PLGA-encapsulated CX3CR1 siRNA nanoparticles represent a valuable new treatment option for neuropathic pain.

Keywords: CX3CR1; Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles; microglia; neuropathic pain; siRNA; spinal nerve ligation.

Figure 1

CX3CR1 siRNA inhibits CX3CR1 expression in BV2 cells and attenuates expression of proinflammatory mediators in lipopolysaccharide stimulated BV2 cells. (A,B) After transfection of BV2 cells with CX3CR1 siRNA or scrambled control siRNA (sc siRNA) for 2 days, the expression of CX3CR1 protein was detected by Western blot using an anti-CX3CR1 antibody, and the CX3CR1/ACTB ratio was quantified. Data are presented as the mean ± SEM (t-test ***p < 0.001 versus sc siRNA). (C) sc siRNA or CX3CR1 siRNA was transfected into BV2 cells for 2 days. mRNA levels of TNF-α, IL-1β, and COX-2 were compared based on the presence or absence of LPS and quantified by qRT-PCR. Data are presented as the mean ± SEM (one-way ANOVA with Tukey’s post hoc test, **p < 0.01 versus sc siRNA + LPS). Cont, control; LPS, lipopolysaccharide.

Figure 2

Characterization of siRNA-encapsulated Poly(D,L-lactic-co-glycolic acid) (PGLA) nanoparticles. (A) siRNA-encapsulated PLGA nanoparticles were prepared by sonicating a mixture of PGLA and CX3CR1 siRNA. (B) Nanoparticles were assessed by scanning electron microscope (SEM), and particle size (C) and zeta potential (D) were examined using a Zetasizer Nano ZS. Scale bar = 300 nm.

Figure 3

Mechanical allodynia and upregulated microglia activation in spinal nerve ligation-induced rats. (A) Neuropathic pain in rats was induced by spinal nerve ligation at the L5 vertebra. (B) Afterwards, the rats were subjected to a pain behavior test using von Frey filaments to evaluate the development of neuropathic pain. Data are presented as the mean ± SEM (one-way analysis of variance (ANOVA) with Dunnett’s post hoc test, ***p < 0.001 versus Ipsi), n = 8 per group (C,D) At 3 days post-SNL surgery, L5 spinal sections were made and immuno-stained with anti-Iba-1 antibody (a microglia-specific marker). Scale bar = 150 μm (top), 100 μm (bottom). Data are presented as the mean ± SEM (t-test ***p < 0.001 versus Contra), n = 8 per group SNL, spinal nerve ligation; Contra, contralateral; Ipsi, ipsilateral; DH, dorsal horn; VH, ventral horn.

Figure 4

Intrathecal injection of PLGA-encapsulated CX3CR1 siRNA nanoparticles alleviates mechanical allodynia in spinal nerve ligation-induced rats. (A) sc siRNA or CX3CR1 siRNA nanoparticles were injected intrathecally at 4 days post-SNL nerve injury. (B) Rats were then subjected to pain behavior testing using von Frey filaments to evaluate the effect of CX3CR1 siRNA on neuropathic pain. Data are presented as the mean ± SEM (one-way ANOVA with Dunnett’s post hoc test, **p < 0.01 versus sc NPs-Ipsi), n = 7 per group. (C) At 6 days post-intrathecal injection, L5 spinal sections were made, and expression of CX3CR1 protein was detected by Western blot using an anti-CX3CR1 antibody, and the CX3CR1/ACTB ratio was quantified. (D) Data are presented as the mean ± SEM (t-test, one-way ANOVA, **p < 0.01 versus sc siRNA NPs), n = 7 per group NPs, nanoparticles; i.t, intrathecal; sc, scrambled; Ipsi, ipsilateral.

Figure 5

PLGA-encapsulated CX3CR1 siRNA nanoparticles attenuate expression of proinflammatory mediators in SNL rats. (A,B) At 6 days post-intrathecal injection, L5 spinal sections were made and incubated with anti-Iba-1 and anti-CX3CR1 antibodies. Data are presented as the mean ± SEM (t-test, **p < 0.01 versus sc siRNA NPs), n = 7 per group. Scale bar = 50 μm (C) On day 6 post-injection, total mRNA was extracted from the ipsilateral spinal dorsal horn at L4–5 (0.7 cm) and utilized for cDNA synthesis. mRNA levels of TNF-α, IL-1β, and cyclooxygenase (COX) 2 were then measured using qRT-PCR. Data are presented as mean ± SEM (one-way ANOVA with Tukey’s post hoc test, **p < 0.01 versus sc siRNA NPs), n = 7 per group. sc, scrambled; NPs, nanoparticles.