Reconstruction of the cervical spinal cord based on motor function restoration and mitigation of oxidative stress and inflammation through eNOS/Nrf2 signaling pathway using ibuprofen-loaded nanomicelles

2.1 Materials

Ibuprofen and PEG-block- PLA methyl ether, PEG average Mn 350, PLA average Mn 1000 diblock copolymer were obtained from Sigma-Aldrich Co. dissolved in 1% ethanol and diluted with phosphate buffered saline (PBS). All remaining materials were purchased from either Sigma-Aldrich Co. (St. Louis, MO, USA) or Merck Co. (Darmstadt, Germany).

2.2 Methods

2.3 Characterization of nanomicelle

The diameter of prepared nanomicelles were determined by transmission electron microscopy (TEM) analysis (JEM 2100F, Japan). The mean hydrodynamic radius and zeta potential of samples were also evaluated by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 instrument (Malvern, UK).

2.4 Entrapment efficiency (EE) and drug loading (DL)

The loading amount of ibuprofen in PEG-PLA diblock copolymer nanomicelles was explored by LC-20AT HPLC system at 264 nm.

The percentage DL of ibuprofen-PEG/PLA diblock copolymer nanomicelles was analyzed through separating the unentrapped cargo from nanomicelles by centrifugation (10,000 rpm, 15 min). Ibuprofen contents in the supernatant was then assessed through high-performance liquid chromatography (HPLC, LC-20AT, Shimadzu, Japan) at 264 nm equipped with Diamonsil 5 μm C18 column at 30 °C column temperature. The mobile phase was a mixture of acetonitrile and distilled water with a 60:40 vol ratio with a flow rate of 1.0 mL/min. The DL% and EE% were determined by the following equations (Li et al., 2018). $$\mathrm{E}\mathrm{E}(\%)=\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{b}\mathrm{u}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{m}\mathrm{i}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{e}\mathrm{s}/\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{b}\mathrm{u}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{n}\times 100\%$$ $$\mathrm{D}\mathrm{L}(\%)=\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{b}\mathrm{u}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{m}\mathrm{i}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{e}\mathrm{s}/\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{m}\mathrm{i}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{g}\times 100\%$$

2.5 Serum stability of prepared nanomicelles

The ibuprofen-PEG/PLA diblock copolymer nanomicelles were stored at pH 7.4 PBS and 10% FBS for 120 h at 4 °C. The stability of the nanomicelles was then monitored by the alterations in particle size in the samples over time monitored by DLS.

2.6 Drug release kinetic

The in vitro release assay of ibuprofen from PEG-PLA diblock copolymer nanomicelles was assessed using dialysis method. In briefly, free ibuprofen (100 μg) or -ibuprofen-PEG/PLA diblock copolymer nanomicelles (with 100 μg ibuprofen) were poured into the dialysis bags (MWCO = 2500 Da, Rancho Dominguez, CA) and placed in 20 mL of PBS (pH 7.4, 10% FBS) at 37 °C with stirring at 100 rpm for 120 h and at different time intervals, the samples were examined by HPLC method.

2.7 Animal handling

All experiments were carried our based on the animal ethics committees’ guidelines approved at our University. Female Sprague–Dawley rats (220–250 g) were anesthetized using ketamine and xylazine and cervical laminectomy was done through the modified approach of SCI (Kim, 2016). Briefly, the muscles over the vertebral column C4–6 and the C5 spine segment were carefully removed, raised the cervical cord, severance was induced with surgical sharp blades #11.

The experimental groups (n = 8) were treated with PEG-PLA diblock copolymer, PEG-PLA diblock copolymer nanomicelle, ibuprofen, and ibuprofen-PEG/PLA diblock copolymer nanomicelles directly poured on the cervical cord and on the blades before severance for fast drug absorption. The group with SCI which received PBS was considered as the control group.

2.8 Electrophysiology assay

Five randomly rats per group were selected for electrophysiology assay 1 h after surgery. To examine electrophysiological function of the spinal motor, rats were fixed on a stereotaxic device and motor-evoked potentials (MEPs) were read employing a bipolar disk electrode in the sciatic nerve after stimulation (8 mA) of the hindlimb area based on the previous report (Kim, 2016).

2.9 Basso, Beattie and Bresnahan (BBB) locomotor rating scale

The motor function of the hind limbs was assessed by the BBB assay based on the standard BBB grading assessment. Scoring standards were as follows: 0–7, joint activity; 8–13, gait and coordination function; 14–21, claw movement.

2.10 Reactive oxygen species (ROS) assay

The level of ROS in spinal cord tissue (0.2 cm of the spinal cord) was explored using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) by spectrophotometer (λex/λem: 495/523 nm). The cells were centrifuged at 1500 rpm for 10 min at 4 °C, followed by addition of H₂DCF-DA probe with a final concentration 10 μM, and incubation (30 min) in a dark. Finally, the DCF fluorescence was assessed.

2.11 Reactive nitrogen species (RNS) measurement

4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM Diacetate) was used to assess the level of RNS generation in investigated spinal cords by spectrophotometer (λex/λem: 493/517 nm) as above described. The cells were incubated with the DAF-FM Diacetate (10 μM) for 30 min in a dark. Finally, the fluorescence intensity was measured.

2.12 Elisa assay

Animals were killed after SCI by CO₂ inhalation and 0.2 cm of the spinal cord were sectioned, at the injured site. The level of cytokine and oxidative stress markers were then assessed by homogenation of the samples in PBS (pH 7.4), centrifuging (18,000 rpm, 30 min), and using the supernatant. The levels of TNF-α, IL-1β, IL-6, superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were then assessed with commercially available enzyme-linked immunosorbent assay (ELISA) (Biosource International, Camarillo, CA) kits and samples were run in duplicate.

2.13 Quantitative polymerase chain reaction (qPCR) analysis

The extraction of total RNA and synthesis of cDNA were carried out using commercially available kit (Thermo Fisher Scientific, Inc., USA) according to the manufacturer's instructions, in a real-time PCR system (RR820A; Takara Bio, Inc., Otsu, Japan). Primers (Table 1) were designed based on genes presented in GenBank.

The relative gene expression was assessed using the 2^ΔΔCt assay.

2.14 Statistical analysis

Statistical analysis was performed using SPSS 25.0 software. The comparison between the two groups was performed by One-Way ANOVA. Paired t-test was used for comparison and Pearson correlation analysis was used for the correlation between the data, and t test was used for clinical efficacy. P < 0.05 was considered as statistically significant difference.

3.1 Nanomicelle characterization

Diblock copolymer could self-assemble to form nanomicelles by filming-rehydration strategy. Under TEM, the morphology of ibuprofen-PEG/PLA diblock copolymer nanomicelles was illustrated in Fig. 1(A), showing mostly spheroidal shape with small size (Tong et al., 2011). The average diameter of ibuprofen-PEG/PLA diblock copolymer nanomicelles presented in Fig. 1A were analyzed to be in size range of 10–30 nm. Average hydrodynamic radius of blank PEG-PLA diblock copolymer nanomicelles was 32.4 ± 2.18 nm and ibuprofen-PEG/PLA diblock copolymer nanomicelles presented in Fig. 1B were found to be in size range of 36.2 ± 2.01 nm. The hydrodynamic radius of ibuprofen-PEG/PLA diblock copolymer nanomicelles was primarily consistent with that of free micelles, which indicated that the structure of PEG-PLA diblock copolymer nanomicelles was not disintegrated by encapsulating ibuprofen. Also, the hydrophobic forces among the nanomicelles moiety hold ibuprofen together to induce the formation of spherical nanomicelles with small geometry (Lu and Park, 2013). Moreover, the hydrodynamic radius of this nanomicelles was large enough to prevent being filtered out or metabolized by kidneys or liver, yet small enough to escape the macrophage engulfment (Wang et al., 2013). Therefore, the ibuprofen-PEG/PLA diblock copolymer nanomicelles with the particle size of 36.2 ± 2.01 nm could show slow metabolism and great stability and enough circulation time (Raza et al., 2016). Besides, zeta potential values of blank PEG-PLA diblock copolymer nanomicelles and ibuprofen-PEG/PLA diblock copolymer nanomicelles were −21.73 ± 4.21 mV and −34.48 ± 3.38 M, respectively (Fig. 1C), indicating the potential loading of cargo and promising colloidal stability of fabricated nanomicells. Indeed, interparticle electrostatic interactions result in the colloidal stability of nanomicelles (Hu et al., 2014; Szilagyi et al., 2014).

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Fig. 1

Characterization of PEG/PLA diblock copolymer nanomicelles and ibuprofen-PEG/PLA diblock copolymer nanomicelles. (A) TEM image. (B) Size distribution. (C) Zeta potential.

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3.2 EE%, DL%, and in vitro stability

The DL% and EE% for ibuprofen-PEG/PLA diblock copolymer nanomicelles were around 9.3% and 72.09%, respectively. Ibuprofen-PEG/PLA diblock copolymer nanomicelles remained stable when stored at 4 °C for 120 h. Indeed, no nanomicelle hydrodynamic radius distribution changes were detected during 120 h. The high drug loading capacity and potential stability of ibuprofen-PEG/PLA diblock copolymer nanomicelles can be assosiated with the hydrophobic interaction of the drugs and copolymers in solubilizing ibuprofen (Abouzeid et al., 2014). In consistent with previous reports, ibuprofen-PEG/PLA diblock copolymer nanomicelles incubation in the presence of 10% FBS 120 h demonstrated no difference in hydrodynamic radius or size distribution (Roby et al., 2006). Nanomicelles with potential colloidal stability over time can be promisingly stable in the blood over time (Dong et al., 2017).

3.3 In vitro drug release assay

As shown in Fig. 2, free ibuprofen was released rapidly, about 96% of the ibubrofen has been released during the first 10 h. In contrast, ibuprofen-PEG/PLA diblock copolymer nanomicelles revealed a slight burst release within the first 20 h, followed by a continuous release phase over 12 h. Only about 83% of ibuprofen was slowly released from ibuprofen-PEG/PLA diblock copolymer nanomicelles even after 120 h (Fig. 2). These data indicated that ibuprofen-PEG/PLA diblock copolymer nanomicelles provide relatively low leakage at physiological pH which can lead to a sustained high drug accumulation at the target site. Since the ibuprofen release is relatively slow, it suggests that the core of the ibuprofen-PEG/PLA diblock copolymer nanomicelles is stiff and glassy (Torchilin, 2005), which indicates less mobility to the encapsulated ibuprofen in comparison with the mobile cores (Gill et al., 2011 Oct 1). Therefore, the ibuprofen loaded into the inner core of PEG-PLA diblock copolymer nanomicelles was released in a sustained approach.

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Fig. 2

In vitro study of ibuprofen-PEG/PLA diblock copolymer nanomicelles. Release profile of ibuprofen and ibuprofen-PEG/PLA diblock copolymer nanomicelles.

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3.4 Electrophysiological assay

To analysis electrophysiological function of the spinal motor pathway, MEPs were read in the sciatic nerve upon stimulation (8 mA) of the hindlimb site of the sensorimotor cortex (Kim, 2016). MEP signal of sham -treated rats exhibited a positive/negative deflection pattern (Fig. 3A), however did not display a similar pattern in all treated groups after surgery. However, as shown in Fig. 3B, it can be determined that the recorded amplitudes were relatively recovered in the case of ibuprofen and ibuprofen-PEG/PLA diblock copolymer nanomicelles. Fig. 3B shows the average MEP amplitudes recorded following SCI. In control group, MEP amplitude decreased to 17.41 ± 3.75 mV at 1 h after surgery, whereas MEP amplitudes in ibuprofen- and ibuprofen-PEG/PLA -treated animals were remarkably increased in comparison with the control group, and this enhancement was more pronounced for ibuprofen-PEG/PLA -treated animals (104.47 ± 18.76 mV; P < 0.05).

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Fig. 3

Motor-evoked potential (MEP) analysis. (A) MEP graphs of different groups after surgery. (B) The average amplitude of different groups after surgery. Data represent the mean ± S.D. of five rats in each group. ^@@@P < 0.001, relative to sham-treated group, *P < 0.05, **P < 0.01, ***P < 0.001, relative to control group.

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3.5 Hind limb motor function assay

As shown in Fig. 4, on time 5 min, none of the rats in the SCI and treated groups both with free ibuprofen of ibuprofen-PEG/PLA diblock copolymer nanomicelles scored higher than 3 points, suggesting that the spinal cord was completely injured and hind limb motor deactivation was significant. On time 10 min the motor function of the hind limbs in the treated group was improved, with higher scores for ibuprofen-PEG/PLA diblock copolymer nanomicelles-treated group than those in the free ibuprofen treated-group.

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Fig. 4

BBB scores after SCI at different time intervals.

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3.6 Assessment of ROS and RNS levels

The molecular mechanisms behind the protective effects of the ibuprofen in the free or nano-formulated states was then assessed using fluorimetric assay exploring the generation of free radicals: ROS and RNS. The level of ROS (Fig. 5A) and RNS (Fig. 5B) generated by SCI was significantly higher than sham-treated group (P < 0.001). However, the obtained data indicated that the both free ibuprogen and ibuprofen-PEG/PLA diblock copolymer nanomicelles compounds can reduce the generation of ROS and RNS. Indeed, the marked reduction in the generation of ROS and RNS was more pronounced in the case of samples incubated with ibuprofen-PEG/PLA diblock copolymer nanomicelles than free ibuprofen.

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Fig. 5

SCI-induced changes in the spinal cord levels of ROS (A) and RNS (b) as determined by fluorimetric assay. Data represent the mean ± S.D. of five rats in each group. ^@@@P < 0.001, relative to sham-treated group, *P < 0.05, **P < 0.01, relative to control group.

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3.7 Elisa assay

Compared with that in the control group, the levels of inflammatory cytokines including TNF-α (Fig. 6A), IL-1β (Fig. 6B) and IL-6 (Fig. 6C) were significantly decreased, and the levels of SOD (Fig. 6D), CAT (Fig. 6E) and GSH (Fig. 6F) were significantly increased in the both free ibuprofen- and ibuprofen-PEG/PLA diblock copolymer nanomicelles-treated groups.

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Fig. 6

SCI-stimulated changes in the spinal cord levels of TNF-α (A), IL-1β (B), IL-6 (C), SOD (D), CAT (E), and GSH (F) as determined by ELISA. Data represent the mean ± S.D. of five rats in each group. ^@@@P < 0.001, relative to sham-treated group, *P < 0.05, **P < 0.01, ***P < 0.001, relative to control group.

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However, ibuprofen-PEG/PLA diblock copolymer nanomicelles significantly reduced the levels of inflammatory cytokines, and increased the levels of non-enzymatic and enzymatic antioxidant systems compared with that in the free ibuprofen group (Fig. 6).

3.8 qPCR assay

Compared with that in the sham group, the expression of eNOS (Fig. 7A) and Nrf2 (Fig. 7B) mRNA in spinal cord sample of the SCI group (control) was remarkably increased (P < 0.001). However, both free ibuprofen and ibuprofen-PEG/PLA diblock copolymer nanomicelles significantly reduced eNOS and Nrf2 mRNA expression levels in comparison with that in the SCI group. Also, the decrement rate of ibuprofen-PEG/PLA diblock copolymer nanomicelles on the expression level of both mRNA was more pronounced than free drug.

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Fig. 7

SCI-stimulated changes in the spinal cord expression of eNOS mRNA (A) and Nrf2 mRNA (B) as determined by qPCR. Data represent the mean ± S.D. of five rats in each group. ^@@@P < 0.001, relative to sham-treated group, *P < 0.05, **P < 0.01, relative to control group.

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