Butylcyanoacrylate (BCA) monomer was purchased from Guangzhou Baiyun Medical Adhensive Co., Ltd. (Guangzhou, China; 6606-65-1). Dextran-70 (D0736000), Tween-80 (P1754) and hexadecyltrimethylammonium bromide (CTAB) (H6269) were obtained from Sigma-Aldrich Company (Saint Louis, MO, USA). Prime STAR Max DNA Polymerase (639241), Hind III (1060A) and Sal I double digestion (1080A), high-purity gel extraction kit (9760) and DNA markers (DL2000) were obtained from Takara Biotechnology Co., Ltd. (Shiga, Japan).

The method for synthesizing PBCA NPs is described in [22]. Briefly, 1% (w/v) Dextran-70 and 0.6% (w/v) Tween-80 were dissolved in deionized water, and the pH was adjusted to 2.5 by adding 1 N HCl (North Weiye Measurement Co., Ltd., Henan, China; BWZ7351). A BCA monomer (100 µL) was pipetted slowly into this solution until the final concentration was 1.0% at 1000 rpm and at room temperature for two hours, adjusted the pH to 6.5 with 0.1 N NaOH (FeiMoBio, Beijing, China; FB30092-500) and stirred for one hour. The NPs were separated by ultracentrifugation at 40,000 rpm for two hours at 4 °C and rinsed with distilled water three times. Thereafter, the NPs were dissolved in 0.25% CTAB at 1000 rpm and room temperature for two hours, at 4 °C, 20,000 rpm ultracentrifugation for one hour, then washed with distilled water three times. Finally, the large aggregates and other impurities were removed by 0.22 µm microfiltration bilayer membrane filtration. The colloid was freeze-dried and stored at –20 °C before use. The morphology of NPs was observed by transmission electron microscopy (Kyky Technology Co., Ltd., Beijing, China) and the surface charge (zeta potential) and PNs particle size determined by the Malvern Zetasizer (ZEN3600, Malvern Panalytical, Malvern, UK) laser diffraction light scattering method.

A synthesized eukaryotic expression vector was created as previously described [23]. Briefly, based on mouse genomic DNA, the DNA fragment was PCR amplified (CFX Opus 96, Bio-Rad Laboratories, Hercules, CA, USA). The amplified DNA fragments were purified, the pEGFP vector and BDNF cDNA fragments were digested with Hind III and Sal I, respectively, and T4 DNA ligase was then used to construct the pEGFP-BDNF eukaryotic expression vector. The pEGFP-BDNF DNA was transformed into competent cells derived from Escherichia coli JM109; the white single colonies were collected, placed into liquid luria-bertani (LB) culture medium, and incubated on a shaking table overnight. The recombinant extraction plasmid, pEGFP-BDNF, was then digested by Hind III and Sal I to verify its accuracy and then detected by DNA automatic sequencing analysis (MGI Tech Co. Ltd., Guangdong, China; MGISEQ-2000). Finally, PBCA NPs and pEGFP-BDNF were mixed at a mass ratio of 9:1 at pH 7.0, then stirred at 20,000 rpm for 30 minutes at 25 °C. The NPs were separated by ultracentrifugation at 20,000 rpm for 20 minutes at 4 °C then rinsed with distilled water three times. The colloid was freeze-dried and stored at 4 °C before use.

Fifty-six healthy male C57BL/6J wild-type mice were obtained from the Laboratory Animal Center of Southwest Medical University (Sichuan, China). A collagenase-induced ICH animal model was adopted in this study [24]. The mice (eight weeks, 30 $\pm$ 5 g) were randomly divided into four groups (sham, ICH, ICH+PBCA NPs, and ICH+ PBCA-pEGFP-BDNF NPs; n = 14/group) and anesthetized with 2,2,2-tribromoethanol (Avertin, 100 mg/kg; Sigma-Aldrich; T48402-25G) by intraperitoneal injection. Bacterial collagenase VII (0.03 U in 2.0 µL sterile saline; Sigma-Aldrich) was injected into the right striatum (coordinates 0.2 mm posterior, 2.2 mm lateral to bregma, 3.5 mm below the dura), and the needle was left inserted for 10 minutes after injection. After 12 hours, the ICH+PBCA NPs and ICH+PBCA-pEGFP-BDNF NPs groups were each respectively intraperitoneally injected with PBCA NPs and PBCA-PEGFP-BDNF NPs (10%, 1 mL) once a day for three days. As controls, the sham and ICH groups were intraperitoneally injected with the same amount of sterile saline.

The neurological function of mice was blind assessed by two investigators with no knowledge of the experimental group using the modified Garcia score after ICH modeling and at 24, 48 and 72 hours after treatment [24]. The Garcia score ranged from 3 (severely impaired neurological function) to 18 (normal neurological function). Following anesthesia with Avertin (100 mg/kg), the mice were euthanized via decapitation. The brains were then carefully removed and serially sectioned into one mm thick slices using a vibrating microtome. Consecutive brain slices were photographed, and the ICH volumes were calculated by multiplying the area of the blood clot in each slice by the distance between the slices, using Image J software (1.52s version, National Institutes of Health, Bethesda, MD, USA) for analysis. As described previously, the wet/dry method was used to assess cerebral edema: (wet weight – dry weight)/(wet weight) $\times$ 100% [24].

First, total RNA isolation was performed using TRIzol reagent (Vazyme, Nanjing, China; R401-01). Oligomer (dT) primer and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific, Grand Island, NY, USA; 28025013) were then used for transcription. qPCR was performed with Archimed-X6 real-time PCR (Rocgene, Beijing, China) equipment after preparing the reaction solution according to the SYBR Green PCR kit (Accurate Biology, Hunan, China; AG11701). Each 10 µL reaction contained 5 µL SYBR Green mix, 0.5 µM each primer, 1 µL cDNA template, and 3 µL double distilled water. Cycling conditions were: 95 °C for 30 s (initial denaturation), 40 cycles of 95 °C for 5 s and 60 °C for 30 s. GAPDH was used as the reference gene throughout this procedure. Based on mouse genomic DNA, the following primers (Table 1) were then used.

Specimens were fixed in neutral formalin, embedded in paraffin and then sectioned (5 µm). Sections were dewaxed with Xylene (Aladdin, Shanghai, China; 1330-20-7), endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Aladdin; 7722-84-1) and then washed with DPBS (Thermo Fisher Scientific; 21600069). After blocking with 10% goat serum for one hour, sections were washed, then incubated overnight at 4 °C with either primary antibody rabbit anti-BDNF (Abcam, Cambridge, UK; ab108319, 1:500) or rabbit anti- neuron-specific nuclear protein (NeuN) (Abcam; ab177487, 1:500), washed and then incubated with Alexa Fluor™ 647 donkey anti-rabbit IgG (Abcam; ab150075, 1:500) for one hour. Immunofluorescence images were captured by confocal laser-scanning microscopy (Leica TCS SP5 II, Wetzlar, Germany). Image J software was used to calculate the number of NeuN-positive cells per unit area around the hematoma. Immunohistochemically stained sections were photographed after incubation with 3, 30-diaminobenzidine substrate kit (Beyotime, Shanghai, China; P0202). Image J software was used to calculate the percentage of BDNF positive cells per unit area.

The cerebral tissue of the same hemisphere of ICH were homogenized in a volume of 10$\times$ radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime; P0013B) and then centrifuged at 4 °C and 12,000 g for 15 minutes to obtain supernatants. Finally, the levels of IL-6 (BMS603-2), TNF-$\alpha$ (BMS607-3), and IL-1$\beta$ (BMS6002-2) were measured according to ELISA kits instructions (Invitrogen, Carlsbad, CA, USA).

The cerebral tissue of the same hemisphere of ICH were homogenated and centrifuged at 12,000 g for 30 minutes at 4 °C. A bicinchoninic acid assay protein assay kit (Beyotime; P0010) was used to determine protein concentration. Equal amounts of protein were extracted from each group, separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane. The sealed membrane of milk was incubated with primary antibody mouse anti-p65 (Cell Signaling Technology, Danvers, MA, USA; CST6956T, 1:500) and mouse anti-GAPDH (Beyotime; AF0006, 1:2000) at 4 °C overnight, after washing with PBS at pH 8 to eliminate non-specific binding, membranes were incubated with a biotinylated goat anti-mouse IgG (Sangon Biotech, Shanghai, China; D110087, 1:1000) at 37 °C for one hour. Finally, protein bands were displayed by chemiluminescence. To verify the level of phospho-p65 (p-p65) on the same polyvinylidene fluoride membrane, the membrane was stripped after the initial immunoblotting to remove primary and secondary antibodies, and re-incubated with primary antibody rabbit anti-p-p65 (Cell Signaling Technology; CST3033T, 1:500) and a biotinylated goat anti-rabbit IgG (Sangon Biotech; D110058, 1:1000). Finally, the phosphorylation level of p65 was quantified by p-p65/p65.

Numeric variables are given as mean $\pm$ standard deviation. All statistical analyses were performed with Stata statistical software (version 11.0, Stata, College Station, TX, USA). Two-way analysis of variance and Tukey’s HSD test were used to analyze the neural function data at different time points. The mRNA level of DNF was analyzed by t-test and other data were analyzed by one-way analysis of variance followed by Holm-Sidak multiple comparison tests. A p-value of 0.05 or less was assumed to indicate significant statistical difference.

The plasmid PEGFP-BDNF was cut by Hind III and Sal I double digestion. PBCA NPs and PBCA–pEGFP-BDNF NPs exhibited monodispersed spheres with uniform particle size (Fig. 1A). The mean diameters and zeta potentials of the PBCA NPs and PBCA-pEGFP-BDNF NPs complexes are given in Table 2. Mean diameters and absolute value of zeta potentials of the PBCA NPs increased significantly after complex formation with pEGFP-BDNF (p 0.05). This was attributed to the adsorption of pEGFP-BDNF on the surface of PBCA NPs, which both increased particle size and imparted a negative charge. Finally, the synthesized PBCA-pEGFP-BDNF NPs were digested with Hind III and Sal I to verify their accuracy (Fig. 1B).

Fig. 1.

Preparation and characterization of PBCA-pEGFP-BDNF NPs. (A) Electron micrographs of PBCA NPs and PBCA-pEGFP-BDNF NPs exhibiting monodispersed spheres with uniform particle size. Scale bar: 200 nm. (B) Agarose gel electrophoresis of the product of pEGFP-BDNF complexes after restriction and digestion. PBCA, polybutylcyanoacrylate; pEGFP, enhanced green fluorescent protein plasmid; NPs, nanoparticles.

Inverted fluorescence microscopy assay confirmed that PBCA-pEGFP-BDNF NPs treated mice had green fluorescent protein (GFP) expression in their brain tissues (Fig. 2A). To further illustrate the effect of PBCA-pEGFP-BDNF NPs on BDNF expression in mouse brain tissues, the mRNA of BDNF was assessed using qPCR analysis. The mRNA level of BDNF was significantly increased in brain tissues of mice treated with PBCA-pEGFP-BDNF NPs when compared with PBCA NPs (p 0.05) (Fig. 2B). This indicates that the pEGFP-BDNF plasmid was successfully carried to brain tissues by PBCA NPs and achieved high transfection efficiency.

Fig. 2.

PBCA-pEGFP-BDNF NPs enter the mouse brain and increase BDNF transcription. (A) The expression of enhanced green fluorescent protein (EGFP) was detected by fluorescence microscopy (scale bar: 100 µm). (B) qPCR analysis of BDNF expression. mRNA of BDNF was increased in mouse brain tissue after treatment with PBCA-pEGFP-BDNF NPs (n = 3, *p 0.05). qPCR, real-time quantitative polymerase chain reaction.

Experimental ICH mice exhibited significant neurological deficits when compared to the sham-operated group (p 0.0001). Compared with the PBCA NPs group (13 $\pm$ 0.756), the Garcia score of ICH mice in the PBCA-pEGFP-BDNF NPs group (15.38 $\pm$ 1.408) was significantly increased at 72 hours (p 0.0001) (Fig. 3A). This suggests that PBCA-pEGFP-BDNF NPs treatment improves the impaired neurological function in ICH mice. Additionally, compared with the ICH+vehicle group, the volume of intracerebral hematoma was significantly reduced in mice treated with PBCA-pEGFP-BDNF NPs (p 0.0001) (Fig. 3B) and cerebral edema was improved considerably (p 0.001) (Fig. 3C). To further clarify whether the therapeutic effect of PBCA-pEGFP-BDNF NPs was related to BDNF, BDNF levels in each group of mice were detected by immunohistochemistry. It was found that when compared with the sham group, BDNF expression in ICH mice brain tissue was significantly decreased, but BDNF expression was significantly up-regulated after PBCA-pEGFP-BDNF NPs treatment (p 0.0001) (Fig. 3D). This suggests that PBCA-pEGFP-BDNF NPs treatment may have improved neurological function in experimental ICH mice by upregulating BDNF expression.

Fig. 3.

Effect of PBCA-pEGFP-BDNF NPs treatment on neurologic function in experimental ICH mice. (A) Garcia scores of mice in each group. (B) Representative cerebral hemorrhage brain slices and statistical results (scale bar: 5 mm). (C) Results of quantitative analysis of brain water content. (D) The level of BDNF was detected by immunohistochemistry and analyzed statistically (scale bar: 50 µm). (n = 8, “ns” indicates no statistical difference, **p 0.01, ***p 0.001, ****p 0.0001 vs. ICH+vehicle). ICH, intracerebral hemorrhage.

Neuronal loss in the brain tissue surrounding hematomas was analyzed. Immunofluorescence staining revealed a significant decrease in NeuN-positive cells in ICH mice (192 $\pm$ 19.435) when compared with the sham group (437 $\pm$ 31.774) (p 0.001) (Fig. 4A,B). There was no substantial change in NeuN-positive cells around the hematoma in ICH mice after treatment with PBCA NPs, and a significant increase in NeuN-positive cells around the hematoma in ICH mice after treatment with PBCA-pEGFP-BDNF NPs (371 $\pm$ 69.584) (p 0.01) (Fig. 4A,B). These results suggested that PBCA-pEGFP-BDNF NPs treatment reduced neuronal loss in ICH mice.

Fig. 4.

Effect of treatment with PBCA-pEGFP-BDNF NPs on neuronal loss due to ICH. (A) Immunofluorescence staining was used to assess the number of NeuN-positive cells in the tissues surrounding cerebral hematomas in each group of mice (scale bar: 50 µm). Brain sample with schematic showing the area around the hematoma used for NeuN-positive cell counts (indicated by black squares). (B) Quantitative analysis of NeuN-positive cells. (n = 3, “ns” indicates no statistical difference, **p 0.01, ***p 0.001 vs. ICH+vehicle).

To assess whether the improvement of neurologic function in ICH mice by treatment with PBCA-pEGFP-BDNF NPs is associated with neuroinflammation, the mRNA levels and concentration of IL-6, TNF-$\alpha$, and IL-1$\beta$ were detected by qPCR and ELISA, respectively. When compared with the sham group, the mRNA levels and concentration of IL-6, TNF-$\alpha$, and IL-1$\beta$ were significantly elevated in mice in the ICH+vehicle group (p 0.01) (Fig. 5A,B). Treatment with PBCA NPs had no significant effect on inflammatory factors in ICH mice. However, PBCA-pEGFP-BDNF NPs treatment significantly reduced ICH-induced the mRNA and protein levels of IL-6, TNF-$\alpha$, and IL-1$\beta$ (p 0.05) (Fig. 5A,B). These data suggest that PBCA-pEGFP-BDNF NPs treatment has an inhibitory effect on ICH-induced neuroinflammation.

Fig. 5.

Effect of treatment with PBCA-pEGFP-BDNF NPs on pro-inflammatory cytokines. (A) qPCR and (B) ELISA were performed to detect the mRNA and protein levels of IL-6, TNF-$\alpha$ and IL-1$\beta$ in each group, respectively. (n = 3, “ns” indicates no statistical difference, *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001 vs. ICH+vehicle).

BDNF is a key factor for neuronal survival and normal function and is neuroprotective in ICH [25, 26, 27]. High BDNF levels have been reported to inhibit inflammation levels by modulating p65 NF-$\kappa$B signaling [28]. In this study, it was found that phosphorylated p65 levels were significantly elevated in the ICH + vehicle group, but this trend was reversed by PBCA-pEGFP-BDNF NPs (p 0.01) (Fig. 6A,B). For original western blotting figures in Fig. 6A see Supplementary Material. This suggests that in addition to direct neuroprotection, treatment with PBCA-pEGFP-BDNF NPs may attenuate nerve injury in ICH mice by inhibiting p65 NF-$\kappa$B-mediated inflammatory factor expression.

Fig. 6.

Effect of treatment with PBCA-pEGFP-BDNF NPs on p65 NF-$\kappa$B signaling. (A) Representative Western blot bands and (B) quantitative analyses of p-p65, p65 and GAPDH. The protein expression level was standardized as GAPDH (n = 3, “ns” indicates no statistical difference, **p 0.01, ***p 0.001 vs. ICH+vehicle).