Neutrophil membrane-derived nanoparticles protect traumatic brain injury via inhibiting calcium overload and scavenging ROS

Abstract

The secondary injury is more serious after traumatic brain injury (TBI) compared with primary injury. Release of excessive reactive oxygen species (ROS) and Ca²⁺ influx at the damaged site trigger the secondary injury. Herein, a neutrophil-like cell membrane-functionalized nanoparticle was developed to prevent ROS-associated secondary injury. NCM@MP was composed of three parts: (1) Differentiated neutrophil-like cell membrane (NCM) was synthesized, with inflammation-responsive ability to achieve effective targeting and to increase the retention time of Mn₃O₄ and nimodipine (MP) in deep injury brain tissue via C-X-C chemokine receptor type 4, integrin beta 1 and macrophage antigen-1. (2) Nimodipine was used to inhibit Ca²⁺ influx, eliminating the ROS at source. (3) Mn₃O₄ further eradicated the existing ROS. In addition, NCM@MP also exhibited desirable properties for T₁ enhanced imaging and low toxicity which may serve as promising multifunctional nanoplatforms for precise therapies. In our study, NCM@MP obviously alleviated oxidative stress response, reduced neuroinflammation, protected blood-brain barrier integrity, relieved brain edema, promoted the regeneration of neurons, and improved the cognition of TBI mice. This study provides a promising TBI management to relieve the secondary spread of damage.

Keywords: Mn3O4; Neutrophil membrane; Nimodipine; Reactive oxygen species; Traumatic brain injury.

Fig. 1

a TEM images of Mn₃O₄. b The lattice of Mn₃O₄ shown in TEM. c HAADF-STEM-EDX mapping of Mn₃O₄. d Dynamic light scattering (DLS) measurements of Mn₃O₄. e X-ray Diffraction (XRD) pattern of Mn₃O₄. f Mn 2p_1/2 and Mn 2p_3/2 XPS spectra of Mn₃O_4. g Representative western blot images of Integrin beta 1, MAC-1 and CXCR4, respectively. h TEM image of NCM@MP. i Dynamic light scattering (DLS) of NCM@MP. j–k The insets were the corresponding T1WI and T2WI of NCM@MP solution, respectively. l–m r1 and r2 relaxivity of NCM@MP at the different concentrations of Mn. Data were mean ± SD (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

a ·O²⁻, b DPPH, and c·OH scavenging rate by MP and NCM@MP, respectively. d Fluorescence values of ROS level in BV2 microglia with different treatments. Indicators for oxidative stress, including e SOD, f MDA activity under different and neuroinflammation indicators g IL-1β, h TNF-α levels in LPS-triggered BV2 cells under different treatment conditions showed significant anti-oxidative stress response and anti-inflammatory of NCM@MP. i Calcium overloading degree tested by Fluo-4/AM Labeling which showed NCM@MP alleviated intracellular calcium levels. j–l Cell apoptosis representative images of H₂O₂-triggered SH-SY5Y cells treated by NCM@Mn₃O₄ and NCM@MP, which revealed neuroprotective function of NCM@MP in vitro. Data were mean ± SD (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

a–b Targeting ability of NCM@MP in vitro evaluated by ICP-MS analysis. c–d In vivo T1WI and relaxation time of TBI lesion pre- and post-injection of NCM@MP at different time points. e The BBB-crossing ability of NCM@MP evaluated by T1 relaxation time. f T1 relaxation time were used to evaluate the ability of NCM@MP to target TBI. Data were mean ± SD (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

a–b Injury area percentage was decreased by NCM@MP treatment on the 1st day after TBI using TTC staining. c–d Representative Ktrans map derived from DCE-MR imaging and quantitative analysis of the brain at 1-day post-treatment. e–f Representative T2WI and quantitative analysis of brain edema at 1-day post-treatment. g–h Representative T2WI and quantitative analysis of lesion volumes at 4 weeks post-injection. i Hematoxylin–eosin staining at 28-day post-treatment. These results showed NCM@MP improved BBB permeability and edema in TBI acute stage, and increased lesion defect recovery in chronic stage. j–k Swimming trajectories of mice and the time to find the platform in the training phase in four groups in the testing phase of water maze test. l–m Latency to first entry and platform entries on the test day. Results illustrated that the NCM@MP promoting the recovery of neuronal cognition and the spatial learning and memory abilities of TBI mice. Data were mean ± SD (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

a–b ELISA analysis of inflammatory factors IL-1β and TNF-α levels of the mice on day 1 post TBI with NCM@Mn₃O₄ or NCM@MP treatment confirmed the abilities on regulating inflammatory factors. c–d The brain SOD and MDA levels of the mice on day 1 after different treatment confirmed the anti-oxidative abilities of the NCM@MP on TBI mice. e Representative immunohistochemical images and quantification of GFAP in astrocytes and lBA1 in microglia from the injured tissues in TBI models at day 3 post-injection, which showed NCM@MP reduced glial cell activation in vivo. The scale bar denotes 50 μm. f Representative immunohistochemical images and quantification of SOX and NeuN in regenerated tissues at day 28 post-injection, which showed NCM@MP promoted neuronal regeneration. The scale bar denotes 20 μm for SOX and 50 μm for NeuN. g Representative western blot images and quantification of signaling pathway. Data were mean ± SD (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

a–c The cell viability of bEnd.3, BV2 and SH-SY5Y treated with a variety of concentrations of NCM@MP and there were no significant vitality changes. d–e Serum biochemical indexes and HE staining of the main organs of the mice at 14 days post-treatment. These results showed great biocompatibility of NCM@MP. Data were mean ± SD (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001