Realization of differential release of minocycline hydrochloride from electrosprayed polymeric or biomacromolecular microparticles for the repair of traumatic brain injury

Abstract

Traumatic brain injury (TBI) occurs when an external force impacts the brain and can result in various serious consequences. Currently, there are no clinical therapies to satisfy the different pathophysiological stages of TBI, realizing a combination of anti-inflammatories in the acute stage and polarization of M2 phenotype microglia. Herein, poly(lactic-co-glycolic acid) (PLGA) and silk fibroin (SF) were selected as shell layers, respectively, to fabricate minocycline hydrochloride (MH)-loaded core-shell microparticles through coaxial electrospray. MH@SF and MH@PLGA microparticles exhibited a differential release profile to promote nerve repair and regeneration after TBI. MH@SF achieved a fast release in 7 days to suppress neuroinflammatory response, while the sustained release of MH@PLGA up to 25 days allowed for modulating polarization of M2 phenotype microglia. The obtained microparticles promoted cell viability and neurite growth of primary neurons and SH-SY5Y cells by establishing neurite transection (NT) and oxygen-glucose deprivation (OGD) models to simulate primary and secondary injury after TBI. In vivo experiments further proved that MH@SF and MH@PLGA microparticles alleviate the neuroinflammation microenvironment and enhance motor, learning, and memory functions of mice after TBI. In summary, this work proposed a promising strategy to regulate the neuroimmune microenvironment through matching different pathophysiological stages of TBI, demonstrating potential for clinical translation to treat TBI.

Keywords: Differential release; Electrosprayed microparticle; Immune microenvironment; Neuroprotection; Traumatic brain injury.

Graphical abstract

Fig. 1

Schematic illustration, characterization, and release behaviors. (A) Schematic illustration of core-shell microparticles (MH@PLGA and MH@SF) prepared by coaxial electrospaying. (B) SEM images of MH@PLGA microparticles and MH@SF microparticles. The enlarged images are in the upper right, the scale bar = 1 μm. (C–E) Fluorescence micrographs showing the structure of microparticles, BSA/FITC was loaded in the core layer (green), the enlarged images are in the upper right, the scale bar = 5 μm. (F) FTIR spectra of MH, PLGA, SF, MH@PLGA microparticles, and MH@SF microparticles. (G) Release profiles of MH from MH@PLGA microparticles. (H) Release profiles of MH from MH@SF microparticles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Effects of microparticles on the viability and phenotype of BV2 cells. (A) Cell viability of BV2 cells (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the control group). (B) NO release from the supernatant of BV2 cell culture (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the LPS group). (C–F) ELISA analysis of IL-1β, TNF-α, IL-4 and IL-10 from the supernatant of BV2 cell culture (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared with the control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the LPS group). (G) Western blot analysis of iNOS (M1) and Arg-1 (M2) protein levels in BV2 cells across different groups. (H) Immunofluorescence staining for iNOS (red) and Arg-1 (green) was utilized to characterize BV2 cell polarization across various groups. The nuclei were stained blue. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The impact of BV2 cell polarization on the viability, neurite length, and number of primary neurons after NT and OGD treatments. (A) After NT treatment, the viability of primary neurons (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the control group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the LPS group). (B) After NT treatment, the immunofluorescence images of primary neurons. DAPI (blue), SYP (green), MAP2 (red), and merged illustration. (C) After OGD treatment, the viability of primary neurons (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the control group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the LPS group). (D) After OGD treatment, the immunofluorescence images of primary neurons. DAPI (blue), SYP (green), MAP2 (red), and merged illustration. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

In vivo recovery assessment of each group. (A) Timeline of in vivo experiments. (B) Experimental procedure for establishing the TBI model. (C) The mNSS test evaluates the motor abilities and mNSS scores of mice at different points in time (mean ± SD, n = 8). (D) Escape latency in mice from day 7 to day 11 after treatment (mean ± SD, n = 8). (E) The timeline of the MWM test. (F) Escape latency of mice on day12 after treatment (mean ± SD, n = 8, ∗P < 0.05, ∗∗P < 0.01 compared to the MH@PLGA group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the MH@SF group). (G) Time in novel arm of mice at day 12 after treatment (mean ± SD, n = 8, ∗P < 0.05, ∗∗P < 0.01 compared to the MH@PLGA group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the MH@SF group). (H) Representative movement tracks on day 12. (I–K) The expression of inflammatory factors (IL-6, IL-1β, and IL-4) in the serum of mice after 14 days following the injury. (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the MH@PLGA group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the MH@SF group).

Fig. 5

MH-loaded microparticles inhibited inflammation following TBI. (A) Immunofluorescence staining of Arg-1 (green) and DAPI (blue), along with a merged illustration, in the damaged brain tissues at 14 days post-injury. (B) Immunofluorescence staining of iNOS (red), DAPI (blue), and a merged illustration in the damaged brain tissues at 14 days post-injury. (C) Fluorescence micrographs of mature neurons in the DG region, with NeuN (green), DAPI (blue), and a merged illustration at 14 days post-injury. (D–F) Quantification of fluorescence intensity of Arg-1, iNOS and NeuN (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the MH@PLGA group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the MH@SF group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

MH-loaded microparticles reduced tissue loss and promoted neuronal regeneration following TBI. (A) Representative photographs of injured brains. (B) HE staining of tissue sections. (C) Nissl staining of tissue sections. (D) Quantitative analysis of lesion volume in each group after 14 days of treatment (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the MH@PLGA group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the MH@SF group). (E) Quantitative analysis of the number of Nissl bodies in each group after 14 days of treatment (mean ± SD, n = 3, ∗P < 0.05, ∗∗P < 0.01 compared to the MH@PLGA group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the MH@SF group).