Rolipram-Loaded Polymeric Micelle Nanoparticle Reduces Secondary Injury after Rat Compression Spinal Cord Injury

Abstract

Among the complex pathophysiological events following spinal cord injury (SCI), one of the most important molecular level consequences is a dramatic reduction in neuronal cyclic adenosine monophosphate (cAMP) levels. Many studies shown that rolipram (Rm), a phosphodiesterase IV inhibitor, can protect against secondary cell death, reduce inflammatory cytokine levels and immune cell infiltration, and increase white matter sparing and functional improvement. Previously, we developed a polymeric micelle nanoparticle, poly(lactide-co-glycolide)-graft-polyethylenimine (PgP), for combinatorial delivery of therapeutic nucleic acids and drugs for SCI repair. In this study, we evaluated PgP as an Rm delivery carrier for SCI repair. Rolipram's water solubility was increased ∼6.8 times in the presence of PgP, indicating drug solubilization in the micelle hydrophobic core. Using hypoxia as an in vitro SCI model, Rm-loaded PgP (Rm-PgP) restored cAMP levels and increased neuronal cell survival of cerebellar granular neurons. The potential efficacy of Rm-PgP was evaluated in a rat compression SCI model. After intraspinal injection, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indotricarbocyanine Iodide-loaded PgP micelles were retained at the injection site for up to 5 days. Finally, we show that a single injection of Rm-PgP nanoparticles restored cAMP in the SCI lesion site and reduced apoptosis and the inflammatory response. These results suggest that PgP may offer an efficient and translational approach to delivering Rm as a neuroprotectant following SCI.

Keywords: cAMP; compression spinal cord injury; polymeric micelle nanoparticle; rolipram; secondary injury.

FIG. 1.

Proposed target-specific poly(lactide-co-glycolide)-graft-polyethylenimine (PgP) micelle nanotherapeutics. Color image is available online at www.liebertpub.com/neu

FIG. 2.

(A) Rolipram (Rm) loading into poly(lactide-co-glycolide)-graft-polyethylenimine (PgP) micelle nanoparticles by the solvent evaporation method. 100 μL containing various concentrations of Rm (0–2 mg/mL in ethanol) was added into PgP (1 mg/mL in water) solution, incubated to allow ethanol evaporation, and filtered to remove free Rm, and the concentration of loaded Rm measured by high-performance liquid chromatography (HPLC). The maximum water solubility of free Rm is indicated by dashed red line (0.2 mg/mL). (B) The long-term stability of Rm-PgP incubated at 37°C for 6 weeks. Each week, solutions containing Rm-PgP were sampled and filtered with 0.2 μm syringe filter to remove released precipitated Rm and then Rm amount was measured by HPLC. The % Rm remained was calculated as: % Rm remained at given time-point = (amount of Rm in PgP at given time-point)/ (amount of Rm in PgP at time 0) × 100.

FIG. 3.

Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) increases cyclic adenosine monophosphate (cAMP) levels of cerebellar granular neurons (CGNs) cultured in hypoxia condition. Rat CGNs were cultured in hypoxia for 24 h and then treated with Rm-PgP (10 μg Rm/well), Rm dissolved in dimethylsulfoxide (Rm-DMSO, 10 μg Rm/well) and PgP only (10 μg PgP/well), and cultured an additional 24 h in hypoxia. Untreated CGNs cultured in hypoxia and CGNs cultured in normoxia was used as negative and positive control, respectively. cAMP levels were measured by enzyme-linked immunosorbent assay. Data presented as mean ± standard error of the mean (n = 9). *p < 0.05, compared with normoxia.

FIG. 4.

Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) increases neurite length of cerebellar granular neurons (CGNs) cultured in hypoxia condition. Rat CGNs were cultured in hypoxia for 24 h and then treated with Rm-PgP (10 μg Rm/well), Rm dissolved in dimethylsulfoxide (Rm-DMSO, 10 μg Rm/well) and PgP only (10 μg PgP/well) and cultured an additional 24 h in hypoxia. Untreated CGNs cultured in hypoxia and CGNs cultured in normoxia was used as negative and positive control, respectively. CGNs were immunostained for beta-III tubulin and imaged. (A) Representative images for β-3 tubulin stained CGNs cultured in (i) normoxia, and (ii) untreated, (iii) PgP-only treated, (iv) Rm-PgP treated, and (v) Rm-DMSO treated CGNs after culture in hypoxia condition. Scale bars represent 100 μm. (B) Neurite length of CGNs cultured in various conditions. Data presented as mean ± standard error of the mean (n = 9). *p < 0.05, compared with normoxia. Color image is available online at www.liebertpub.com/neu

FIG. 5.

Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) is retained at spinal cord injury lesion site after local injection. (A) 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine Iodide (DiR) loading in PgP solution and water. (B) Visualization of DiR-PgP nanoparticles by live animal imaging system at 2, 4, 6, 24, 72, and 120 h post-injection. Left: untreated control animal. Right: DiR-PgP injected animal. (C) Images of spinal cord isolated at 2, 4, 6, 24, 72, and 120 h post-injection. Color image is available online at www.liebertpub.com/neu

FIG. 6.

Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) restores cyclic adenosine monophosphate (cAMP) levels after compression spinal cord injury. Rm-PgP (10 μg Rm) was injected immediately after clip compression injury and the spinal cord was harvested at 1, 2, 3, and 7 days post-injection. cAMP levels were measured by enzyme-linked immunosorbent assay assay. Sham and untreated spinal cord injury animal groups were used for comparison. Data presented as mean ± standard error of the mean (n = 6). *p < 0.05, compared with the sham group, indicated by the dashed red line. Color image is available online at www.liebertpub.com/neu

FIG. 7.

Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) reduces apoptosis after compression spinal cord injury (SCI). Rm-PgP (10 μg Rm) was injected immediately after clip compression injury. At 3 days post-injection, spinal cords were explanted, embedded, sectioned, and stained for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) + cells (green) and nuclei (blue). Sham and untreated SCI animal groups were used for comparison. (A) Representative images of TUNEL stained longitudinally sectioned spinal cord (0.5 cm-long piece from the center of the injury). Scale bar indicates 500 μm. Top: sham. Middle: untreated SCI. Bottom: Rm-PgP treated SCI rats. (B) Representative images of TUNEL+ cells in the spinal cord at the lesion epicenter and 2 and 4 mm rostral and caudal. Scale bar indicates 50 μm, Top: sham. Middle: untreated SCI. Bottom: Rm-PgP treated SCI animals. (C) The % TUNEL+ cells quantified from total 18 different sections of spinal cords from each group (six sections/rat, three rats/group). *p < 0.05, compared with untreated SCI. Color image is available online at www.liebertpub.com/neu

FIG. 8.

Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) increases the ratio of Bcl-2+/Bax+ cells after compression spinal cord injury (SCI). Rm-PgP (10 μg Rm) was injected immediately after clip compression injury. At 3 days post-injection, spinal cords were explanted, embedded, sectioned, and stained for Bax, pro-apototic protein and Bcl-2, anti-apoptotic protein. Sham and untreated SCI animal groups were used for comparison. Representative images of Bcl-2+ cells (A) and Bax+ cells (B) in the spinal cord at the lesion epicenter, 2, and 4 mm rostral and caudal. Scale bar indicates 50 μm, Top: sham. Middle: untreated SCI. Bottom: Rm-PgP treated SCI animals. (C) The ratio of Bcl-2+/Bax+ cells were calculated from a total of nine different sections of spinal cords from each group (three sections/rat, three rats/group). Sections were digitally imaged and the numbers of Bax+ and Bcl-2 + cells were counted. *p < 0.05, compared with untreated SCI. Color image is available online at www.liebertpub.com/neu

FIG. 9.

Rolipram-loaded poly(lactide-co-glycolide)-graft-polyethylenimine (Rm-PgP) reduces inflammatory cell accumulation after compression spinal cord injury (SCI). Rm-PgP (10 μg Rm) was injected immediately after clip compression injury. At 3 days post-injection, spinal cords were explanted, embedded, sectioned, and stained for ED1+ microglia/macrophages (red) and nuclei (blue). Sham and untreated SCI animal groups were used for comparison. (A) Representative images of immunostaining for ED1 in longitudinally sectioned spinal cord (0.5 cm-long piece from the center of the injury). Scale bar indicates 500 μm. Top: sham. Middle: untreated SCI. Bottom: Rm-PgP–treated SCI rats. (B) Representative images of ED1+ cells in the spinal cord at the lesion epicenter, 2 and 4 mm rostral and caudal. Scale bar indicates 50 μm. Top: sham. Middle: untreated SCI. Bottom: Rm-PgP–treated SCI animals. (C) The % ED1+ cells was quantified from a total of 18 different sections of spinal cords from each group (six sections/rat, three rats/group). *p < 0.05, compared with untreated SCI. Color image is available online at www.liebertpub.com/neu