Reactive Oxygen Species-Responsive Chitosan-Bilirubin Nanoparticles Loaded with Statin for Treatment of Cerebral Ischemia

Abstract

Cerebral ischemia impairs blood circulation, leading to elevated reactive oxygen species (ROS) production. A ROS-responsive delivery of drugs can enhance the therapeutic efficacy and minimize the side effects. There is insufficient evidence on the impact of ROS-responsive nanoparticles on ischemic stroke. We developed ROS-responsive chitosan-bilirubin (ChiBil) nanoparticles to target acute ischemic lesions and investigated the effect of atorvastatin-loaded ROS-responsive ChiBil. We randomly assigned rats with transient middle cerebral artery occlusion (MCAO) to 4 groups: saline, Statin, ChiBil, and ChiBil-Statin. These groups were treated daily via the tail vein for 7 d. Behavioral assessment, magnetic resonance (MR) imaging, evaluation of neuroinflammation, blood-brain barrier (BBB) integrity, apoptosis, and neurogenesis after stroke were conducted. In vitro, results showed nanoparticle uptake and reduced intracellular ROS, lipid peroxidation, and inflammatory cytokines (IL-6 and TNF-α). In vivo, results showed improved motor deficits and decreased infarct volumes on MR images in the ChiBil-Statin group compared with the Control group on day 7 (P < 0.05). Furthermore, the expression of inflammatory cytokines such as IL-1β and IL-6 was reduced in the ChiBil-Statin group compared with the Control group (P < 0.05). Improvements in BBB integrity, apoptosis, and neurogenesis were observed in the ChiBil-Statin group. The findings demonstrated that intravenous ROS-responsive multifunctional ChiBil-Statin could effectively deliver drugs to the ischemic brain, exerting marked synergistic pleiotropic neuroprotective effects. Therefore, ChiBil-Statin holds promise as a targeted therapy for ischemic vascular diseases characterized by increased ROS production, leading to new avenues for future research and potential clinical applications.

Fig. 1.

Nanoparticle synthesis and experimental scheme. (A) Micellar structure of ChiBil, loading of atorvastatin calcium, and its properties and effects in treating cerebral ischemia. (B) Experimental scheme. Rats with transient MCAO were randomly assigned to 4 groups: Control, Statin, ChiBil, and ChiBil-Statin. Each group received daily administration of the respective formulations intravenously for 7 d. Treatment effects were assessed using micro-MRI, rotarod test, NDS, PCR, Western blot (WB), Evans blue (EB), and immunofluorescence (IF) to evaluate functional recovery, BBB integrity, neuro-inflammation, apoptosis, and neurogenesis after stroke.

Fig. 2.

Nanoparticle characterization. (A) Size and (B) zeta potential of ChiBil-Statin nanoparticles. (C) TEM image of ChiBil nanoparticles loaded with SPIONs showing the micellar structure and size of ChiBil. (D) Three independent batches show comparable interbatch reproducibility. (E) Encapsulation efficiency of atorvastatin calcium in ChiBil evaluated by loading 1, 3, and 6 mg of statin in the same amount of micelles of 10 mg. (F) Nanoparticle stability shown in different conditions using PBS and 10% FBS for 24 h. (G) UV–visible spectra of atorvastatin calcium release with and without ROS stimuli (100 mM AAPH) in normal PBS at pH 7 and 37 °C. (H) Images showing the oxidation of ChiBil-Statin before and after incubation with a ROS generator.

Fig. 3.

In vitro experiments for the effects of ChiBil-Statin. (A) MTS assay of the cell viability of BV-2 cells exposed to ChiBil and ChiBil-Statin for 24 h. (B) Cellular uptake study of BV-2 cells treated with ChiBil nanoparticles loaded with IR780 for 4 h under normal and OGD conditions. Scale bar, 100 μm. (C) ROS-quenching effects of ChiBil-Statin measured under OGD conditions after incubation with DCF-DA. (D) Lipid peroxidation levels were assessed by MDA detection using lysed cell samples. (E) TNF-α and (F) IL-6 levels were detected in the supernatant of BV-2 cells. Data are presented as mean ± SEM. Data were analyzed by one-way ANOVA. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.

In vivo micro-MRI analysis of infarct volume. (A) MR images of ischemic rats were imaged from day 1 to day 7 on every alternate day. (B) Quantified infarct volume in ischemic regions on different days shows a significantly lower infarct volume in the ChiBil-Statin group than in the Control group (P = 0.013, Tukey’s post hoc analysis of repeated-measures ANOVA). Group average values of the infarct volume at 7 d after stroke (Control: 320.81 ± 50.10 mm³; Statin: 137.65 ± 41.13 mm³; ChiBil: 197.15 ± 47.49 mm³; ChiBil-Statin: 137.65 ± 41.13 mm³). (C) The ratio of infarct volume to baseline across different groups on different days shows a significant difference in the ChiBil-Statin group starting from day 3. Group average values of the ratio of infarct volumes to baseline at 7 d after stroke (Control: 0.769 ± 0.101; Statin: 0.570 ± 0.060; ChiBil: 0.523 ± 0.106; ChiBil-Statin: 0.348 ± 0.061). Data are presented as mean ± SEM. Data at each time point were analyzed using ANOVA with Tukey’s multiple comparisons. n = 8 in each group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

Behavioral assessment of cerebral ischemia-induced rats according to the treatment. (A) Rotarod test results show a significant interaction between treatment and time based on their coordination, balance, and endurance (P = 0.024; repeated-measures ANOVA). The ChiBil-Statin group demonstrated significantly longer times of fall than the Control group (P = 0.042, post hoc analysis using Tukey’s test). (B) Neurologic deficit scores for the different groups on days 1 and 7. Repeated-measures ANOVA for the NDS shows lesser neurological deficit for the ChiBil-Statin (P = 0.002) and ChiBil (P = 0.006) groups than for the Control group (Tukey’s post hoc analysis). Data are presented as mean ± SEM, with n = 17 in each group. The number of animals that died during the 7-d treatment was 12, 8, 5, and 3 in the Control, Statin, ChiBil, and ChiBil-Statin groups, respectively. *P < 0.05; **P < 0.01.

Fig. 6.

RT-PCR for inflammatory, endothelial, and neurogenesis markers. (A) IL-1β, (B) IL-6, (C) TNF-α, (D) MCP-1, (E) ICAM-1, (F) SOX2, (G) Iba-1, (H) eNOS, and (I) Nestin mRNA expression. The ChiBil-Statin treatment led to decreased gene expression of IL-1β, IL-6, and ICAM-1, along with increased gene expression of Nestin compared to that in the Control group. The ChiBil treatment led to decreased gene expression of IL-6 compared to the Control group. The statin treatment led to increased gene expression of Nestin compared to that in the Control group. Nonparametric data are analyzed using the Kruskal–Wallis test with Dunn’s post hoc analysis and are presented as median with interquartile range, with minimum to maximum whiskers in box plots. Parametric data are analyzed using one-way ANOVA with Tukey’s post hoc analysis and presented as mean ± SEM. n = 6 in each group. *P < 0.05; **P < 0.01.

Fig. 7.

Western blotting and IVIS® imaging for BBB integrity. (A) Images of Western blot. (B) IVIS® fluorescence images of ischemic rat brain on day 7. Western blot quantification of (C) Cav-1, (D) claudin-5, (E) occludin, and (F) JAM-A. The ChiBil-Statin treatment increased tight junction and regulatory protein expression of Cav-1, claudin-5, occludin, and JAM-A compared to the Control group. The ChiBil treatment also led to higher protein expression of claudin-5 and occludin than those in the Control group. The statin treatment led to increased protein expression of JAM-A compared to that in the Control group. (G) Quantified data of fluorescence accumulation reflecting the extent of BBB disruption in the ischemic parts. The ChiBil-Statin, ChiBil, and Statin treatments show decreased BBB disruption compared to the Control group, with the lowest disruption observed in the ChiBil-Statin group. Parametric data are analyzed using one-way ANOVA with Tukey’s post hoc analysis and presented as mean ± SEM. n = 6 in each group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8.

Immunofluorescence staining on ischemic penumbra in the subventricular zone. Brain tissues stained for (A) CC3 (red)/NeuN (green), (B) BrdU (red)/DCX (green), and (C) BrdU (red)/NeuN (green). Scale bar, 20 μm. (D) A CC3/NeuN-positive population in the ischemic region shows a quantitative percentage of apoptotic neurons on day 7. The number of CC3/NeuN-positive cells is significantly lower in the ChiBil-Statin group than in the Control, Statin, and ChiBil-Statin groups. (E) A quantitative percentage of survival of newly generated cells was shown by BrdU/DCX-positive cells in the ischemic regions on day 14. BrdU/DCX-positive cells are significantly higher in the ChiBil-Statin group than in the Control, Statin, and ChiBil groups. (F) BrdU/NeuN-positive cells in the ischemic regions showed a quantified percentage of neurogenesis on day 28. BrdU/NeuN staining shows markedly improved survival of newly generated neurons in the ChiBil-Statin group compared to that in the Control, Statin, and ChiBil groups. Parametric data are analyzed using one-way ANOVA with Tukey’s post hoc analysis and presented as mean ± SEM. n = 6 in each group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.