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Original article
01 2021
:15;
103534
doi:
10.1016/j.arabjc.2021.103534

The effect of tacrolimus-containing polyethylene glycol-modified maghemite nanospheres on reducing oxidative stress and accelerating the healing spinal cord injury of rats based on increasing M2 macrophages

, , , , , , , ,
a Department of Emergency, Renmin Hospital of Wuhan University, Wuhan, Hubei, China
b Department of General Practice, Guanggu Zone, Renmin Hospital of Wuhan University, Wuhan, Hubei, China
c Wuhan Britain-China School, Wuhan, Hubei, China
d Department of Emergency, Guanggu Zone, Renmin Hospital of Wuhan University, Wuhan, Hubei, China

⁎Corresponding author. lvjingjun@whu.edu.cn (Jingjun Lyu)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

One of the key factors in repairing spinal cord injury is reducing inflammation and oxidative stress. So, it was tried to increase the growth of neurons more rapidly by enhancing the macrophage M2 and reducing the M1 to M2 ratio by using the possible role of tacrolimus (TAC) loaded on polyethylene glycol-modified maghemite nanospheres (PEG-MNs). In this regard, after designing PEG-MNs-TAC in a three-step process and examining their properties through TEM, DLS and XRD analyses. A toxicity assay was then performed by using MTT test. To investigate the healing role of TAC on damaged tissue, a rat model was used to induce injury in vertebral T6 through physical injury. Then, rats were allocated into five groups: negative control, MNs, PEG-MNs , TAC (and PEG-MNs-TAC. Next, motor scale index, behavioral test, macrophage markers expression, concentration of IL-6, IL-2 and TNF-α, and oxidative stress on rats were analyzed. The results revealed that TAC and PEG-MNs-TAC significantly improved mobility and neuropathic tests. Furthermore, the decrease in TNF-α, IL-6, and IL-2 along with the decrease in ROS levels in the injured spinal cord were considerable in both TAC- and PEG-MNs-TAC-treated groups. Nonetheless, the PEG-MNs-TAC exposed more favorable outcomes compared to TAC. Overall, PEG-MNs-TAC could provide a new treatment for damaged spinal cord care by reducing oxidative stress and decreasing M1 to M2 ratio.

Keywords

Spinal cord injury
Tacrolimus
Maghemite nanosphere
Oxidative stress
Macrophages
1

1 Background

Injured spinal cord is a disabling disease that reduces the quality of life by temporarily or permanently altering spinal cord function (Furlan et al., 2013; Krueger et al., 2013; Singh et al., 2014; Lynch and Cahalan, 2017; Badhiwala et al., 2019). The severity of this abnormality based on complete or incomplete cessation of nerve signals can occur in any part of the spinal cord (Furlan et al., 2013; Badhiwala et al., 2019). In general, primary and secondary spinal cord injury is caused directly by mechanical events, and biochemical or pathological changes, respectively (Witiw and Fehlings, 2015; Eckert and Martin, 2017). Various causes can be very effective in causing the second type of injury, such as edema and extensive bleeding in the spine area, and ischemia-perfusion injury which includes immune responses to inflammation. Inflammation caused by the immune system, which manifests itself by the release of inflammatory cytokines (Fan et al., 2018; David et al., 2019), ultimately leads to an imbalance of macrophages and microglia, which are very suitable in diagnosing the secondary type of damage (Milich et al., 2019). In this line, the accumulation of M1 macrophages maintains and regulates the level of inflammation in the affected area (Gensel et al., 2017; Honjoh et al., 2019). Thus, higher rates of M1 to microglia residing (M2) induce neuronal apoptosis, autophagy, and neurotoxicity, which may increase rehabilitation time or exacerbate neural signaling (Anwar et al., 2016; Tsuda, 2018; Sharifi et al., 2019). M2 in damaged tissue manages tissue regeneration as well as prevents inflammatory responses (Tsuda, 2018; Kroner and Rosas Almanza, 2019).

Tacrolimus (TAC) or Fujimicin is an immunosuppressive drug by inhibiting the production of interleukin-2 (Pamuk et al., 2015), calcineurin and calmodulin-dependent phosphatase (Tung, 2010) to prevent allograft rejection with FDA approval. TAC has been shown to increase nerve regeneration and enhance nerve graft sharing (Saffari et al., 2019). By inhibiting calcineurin via TAC, a decrease in inflammatory cytokines of tumor necrosis factor (TNF)-α and kind of interleukin (IL) such as IL-2, and IL-6 can be observed (Hoshina et al., 2008). In vitro (Saffari et al., 2021) and in vivo (de Mesquita Coutinho et al., 2016) experimental models confirm that TAC not only increases neuritis length and nerve regeneration rate, but also plays a protective role in the nervous system by having a direct effect on cell populations. The use of 6 µg/kg TAC improves the myelin residues in autologous neural transplants (Fansa et al., 1999), which could be clarified by the reduced penetration of M1 macrophage. However, the exact mechanism of the effects of TAC on central and peripheral nerves is not well understood.

Because the use of free drugs is associated with the challenge of long-term instability, the use of nanocarriers based on nanotechnology is very common. However, due to high ambiguities in the biocompatibility and biodegradation of nanocarriers (Khan et al., 2021a), the use of iron oxide nanospheres such as magnetite (Fe3O4) and maghemite (Fe2O3) with low toxicity (Sharifi et al., 2020b), high biocompatibility (Khan et al., 2021b), easy production (Sharifi et al., 2020c), synchronization of therapeutic activities such as imaging, drug delivery and photothermal therapy (Khan et al., 2021b; Wang et al., 2021) along with antibacterial properties (Armijo et al., 2020) have been given special attention. The two compounds magnetite and magnetite are structurally very similar and differ only slightly in the amount of saturated magnetism and toxicity (Sharifi et al., 2020a; Sharifi et al., 2020b). In this regard, Sharifi et al. (2020a) illustrated that nanospheres, in addition to reducing toxicity through targeting, cause greater drug stability in the blood. Furthermore, Kim et al. (2011) reported that iron oxide nanoparticles modified with liposomes improved neurite growth in a dose-dependent proceed in the presence of exogenous nerve growth factor (NGF). In this regard, Marcus et al. (2015) applying NGF-iron oxide nanoparticles increased neurite length, branch number and expression of neuronal differentiation markers in comparison with NGF.

In this study, based on the assumption of the possible effect of TAC on the signaling pathway through macrophage polarization M1 and M2 and induction of neuronal cell growth by maghemite, polyethylene glycol modified-maghemite nanospheres containing TAC (PEG-MNs-TAC) was designed and used in vivo.

2

2 Materials and methods

2.1

2.1 Animal model of spinal cord injury

In order to provide an animal model of spinal cord injury, Wistar rats weighing ∼ 283 g and aged 15 weeks was selected and all medical activities including anesthesia, surgery, injury, recovery and treatment was approved by the Institutional Animal Care and Use Committee of Experimental Animal Center of Wuhan University. During the experimental activities, the rats were kept in a completely isolated, controllable condition with free water and food available and with 12 h of light. Surgical activity began with anesthesia through the combination of xylazine (10 mg/100 g of rats) and ketamine (10 mg/100 g of rats). After shaving and cleaning the area under surgery, surgery was performed by making a skin incision in the area of the T6 vertebral segment and removing the muscle. In addition, bilateral laminectomy was carried out by applying Rongeur under a microscope. In order to cause damage to the spinal cord, the contusion method with the sudden fall of a 12 g metal cylinder from a height of 3 cm on the T6 piece was used. To advance post-operative research, rats received buprenorphine (0.01 mg/100 g of rats) and cefazolin (2 mg/100 g of rat) daily up to 6th day. After induction of spinal cord injury, rats were divided into five groups and 8 rats were placed in each group: Control, MNs (1 mg/kg), PEG-NMs (containing 1 mg/kg), TAC (0.5 mg/kg) and PEG-MNs-TAC (containing 1 mg/kg). Except for the control group, the rats were treated daily with drugs by subcutaneous injection at the injured site.

2.2

2.2 Synthesis of TAC-functionalized PEG-MNs

In order to produce PEG-MNs-TAC, a three-step method was used, including the hydrothermal method for fabrication of magnetite nanospheres, the thermal method for the production of MNs, and finally TAC loading by co-precipitation method. Briefly, the first step involved the production of nanospheres containing poly[acrylic acid] based on the report of Sharifi et al. (2020a) was carried out by thermal method (10 h at 250 °C) using Iron(III) chloride (1.08 g), sodium acrylate (3 g), Sodium acetate (3 g) in 30 ml of ethylene glycol and diethylene glycol (with the same volume ratio) solution. Then, after coating the samples with silicate via ammonia solution (4 ml) and tetraethyl orthosilicate solution (0.4 ml), they were placed in an electric oven at 700 °C to remove the polymer coating. Then, the samples from the electric oven were placed in NaOH solution (0.5 M) to remove the silicate coating. To form maghemite nanospheres in the second stage, the samples provided from the first stage based on the method of Sharifi et al. (2020b) were heated to 250 °C for 4 h. After cooling, the MNs entered the third phase for the loading polyethylene glycol (PEG) and TAC. At this stage, 1 mg of MNs dissolved in 50 ml of sterile water and was added to 1 mg of PEG dissolved in 50 ml of sterile water with the pH = 10. Then, the prepared suspension was placed on a magnetic stirrer for 48 h. The precipitate was washed 5 times with sterile water and dried in a vacuum oven. The PEG-MNs with 2 ml of dimethyl sulfoxide solution and TAC (2 mg) were then mixed and shaken for 24 h. The solution containing the PEG-MNs-TAC was then dried under vacuum for 24 h. Finally, the samples were washed with PBS (three times) to remove excess TAC.

2.3

2.3 Characterization of PEG-MNs-TAC

Morphology and particle size of PEG-MNs-TAC were assayed via High-resolution transmission electron microscope (TEM, JEM-2010) at 100 kV. In addition, the hydrodynamic dimensions of PEG-MNs-TAC were evaluated through dynamic light scattering (DLS). Moreover, the XRD model based on the Cu/kα anode (λ = 1.54178 Å) was used to study the PEG-MNs-TAC structure in the range 2θ between 20 and 80° using a continuous scan manner (XRD-6000; Japan). Brunauer-Emmett-Teller (BET) specific surface area and pore size distribution were calculated through nitrogen adsorption-adsorption isotherm modules at 77 K (Micromeritics ASAP). The pore size distribution for the sample was evaluated using the Halsey equation, from the desorption branch of the isotherms.

2.4

2.4 Loading efficiency and drug releasing

To evaluate the TAC loading efficiency, 100 µg of PEG-MNs was mixed with different concentrations of TAC including 100, 200, 300 and 400 µg at 21 °C for 24 h. Afterwards, the PEG-MNs-TAC was separated from the solution by magnet and the TAC loading efficiency was estimated using the fluorescence spectrum (Hitachi F 2500 spectrometer) based on Eq. (1).

(1)
TAC L o a d i n g e f f i c i e n c y ( % ) = [ ( T o t a l a m o u n t o f T A C i n t h e s o l u t i o n - A m o u n t o f T A C r e m i n i n g i n t h e f i n a l s o l u t i o n ) / A m o u n t o f T A C r e m i n i n g i n t h e f i n a l s o l u t i o n ] × 100

In order to determine the TAC-releasing ability from the nanocarrier, the PEG-MNs-TAC was dissolved in 15 ml of PBS solution at 37 °C in pH 6 and 7. The prepared solution was placed in a dialysis bag (MWCO 3500) and dialyzed against 60 ml of the same buffer via shaking at 120 rpm. Test time points included 1.5, 3, 6, 12, 24, 36, 48, 72 and 96 h, at which time 6 ml of dialysis bag solution was separated for evaluation by adsorption at 490 nm and the same buffer was replaced. Finally, the cumulative TAC release rate was determined according to Eq. (2):

(2)
C u m u l a t i v e d r u g r e l e a s e ( % ) = 6 × ∑ i - 1 n - 1 C i + 60 × C n w e i g h t o f I V M o n I V M - M N s × 100 Where, Ci and Cn refer to the of TAC concentrations at time i and n, respectively.

2.5

2.5 In vitro trial

2.5.1

2.5.1 Cytotoxicity assay

To assess cytotoxicity, primary fibroblasts cells were provided based on reports of Khan and Gasser (2016). 5 × 104 cells/ml were cultured in 96-well microtiter plate with DMEM medium containing 10% bovine fetal serum (FBS), penicillin (100 IU/ml), and streptomycin (100 μg/ml) along with concentrations of 200, 400, 600 and 800 μg/ml PEG-MNs-TAC. Then, the culture medium was incubated in 5% CO2 at 37 °C. After 24 h, the culture medium was detached by PBS and then 1 mg/ml MTT solution was supplemented to each well. The plates were shaken and incubated at 5% CO2 for 4 h at 37 °C. Then, the supernatant was eliminated and 100 μl of dimethyl sulfoxide was added to dissolve the formed formazan. Finally, cell viability was estimated at λ = 540 nm.

2.6

2.6 In vivo trials

2.6.1

2.6.1 Behavioral-motor performance

Performance of motor behaviours was evaluated and recorded by three judges in an open space on the day before surgery and after surgery on days 0, 1, 5, 10, 15, 20, 30 and 40 based on the Basso-Bresnahan-Beattie (BBB) rating scale. During the scoring scale by the judges, the judges were not only unaware of the evaluation of other judges, but were also blind to the treatment groups. Their scoring scale according to the BBB method was between 0 and 21 for the leaned motions, rhythm, weight bearing, joint motions, and single joint operation. In this rating, lack of movement had a zero score and normal movement of the hind limbs had a 21 score. End-limb locomotor evaluation was performed for each rat and the mean score for each rat was recorded at the appointed time.

2.6.2

2.6.2 Evaluation of neuropathic pain

Hot plate, tail-flick latency, and mechanical allodynia methods were used to evaluate nerve pain based on Deuis et al. (2017). Briefly, to evaluate the reaction time of the rats, the time required to jump or lick the paw was determined by the hot plate. In this regard, the rats were placed on a hot plate caused by an electrical flow with a normal temperature of 39 °C, and the time of the first reaction to pain including lifting or licking the hind paw and even jumping, was determined. This experiment was performed 4 times (0, 5, 10, 20, 30 and 40 days) to evaluate modular and brain reflexes during the experimental period. In addition, the study of pain activity in rats was performed using the tail-flick technique (Tjølsen et al. 1989). In this regard, after placing the tail tip in a heat source, the rat's behavioural response including pulling the tail by increasing the radiant heat, was tested. To prevent damage due to heat increase, the heat test time was approximately 10 s. Also, to evaluate the severity of pain in rats before surgery, an electronic von Frey test was performed for each of the rats. Also, after surgery on days 0, 5, 10, 20, 30 and 40 days, the test was repeated and the results were recorded. Results before surgery were considered as a 25 score. To assess hyperalgesia, skin sensitivity to von Frey's hair was recorded as a 50% withdrawal threshold per paw.

2.6.3

2.6.3 Enzyme-linked immunosorbent assay (ELISA)

In order to determine the level of TNFα and IL-6, and IL-2 in spinal cord injury ELISA was used based on the manufacturer's instructions (HyCult Biotechnology, Netherlands). Spinal cord tissue at the lesion epicenter was separated in the groups at 40th day after injury. Spinal cord tissue specimens to be applied for ELISA test were homogenized in the buffer (Tris-HCl:10 mmol/l; NaCl: 150 mmol/l; EDTA: 1 mmol/l; PMSF: 1 mmol/l; Triton X-100: 1%; Aprotinine: 10 mg/ml; Pepstatin: 1 mg/ml). The samples were centrifuged at 12,000 g for 15 min at 4 °C, and the supernatant was harvested for assay.

2.6.4

2.6.4 Flow cytometry assay

After tissues harvested from the injured site in L6 of the spinal cord at the 40th day, the flow cytometry method was carried out to recognize the rate of macrophage polarization M1 and M2 based on a report of Watanabe et al. (2015). The cells of M1 and M2 were identified as CD45+/CD11b+/GR-1−/iNOS + cells and CD45+/CD11b+/GR-1−/CD206 + cells. In order to intracellular staining, the collected cells were resuspended in a stabilization buffer and treated with a permeability buffer based on Stirling and Yong (2008). The samples were again re-suspended in PBS and incubated for 90 min with the antibodies of BV510 rat anti-CD11b (562950, Piscataway, USA), FITC rat anti-CD45 (Abcam plc, ab25670), and PerCP/Cy5.5 rat anti-Ly-6G/Ly-6C, equivalent to Gr-1 (108427, San Diego, USA), rabbit anti-iNOS antibody-primary antibody (ab15323), Alexafluor 647 equivalent to EPR25A secondary antibody (ab199093) for M1 or EPR6828(B) rabbit anti-mannose receptor antibody equivalent to CD206 (ab195192) for M2. Afterwards, flow cytometry (BD Biosciences) was carried out with the removed cellular residue. CD45+/CD11b+/GR-1−/iNOS + cells and CD45+/CD11b+/GR-1−/CD206 + were discovered as M1and M2 types, respectively.

2.6.5

2.6.5 Oxidative stress assessments

ROS measurement in spinal cord injury was used to evaluate oxidative stress. In this regard, to investigate the effect of control, P-control, N-control, PEG-MNs, TAC, and PEG-MNs-TAC on ROS production in spinal cord injury, a commercial fluorescent dihydroethidium (DHE) dye kit (keyGEN BioTECH, Nanjing, China) used as a superoxide probe according to the manufacturer's instructions. 3.87 ml of dimethyl sulfoxide was used to dissolve 6 mg of DHE as a reagent stock solution. Next, the stock produced in the PBS was diluted to produce a 50 μM DHE working solution. The sample spinal cord segments in the 40th day were obtained and then frozen at −80 °C for 20 min. Afterwards, the samples were cut at 15 µm, and incubated with DHE working solution for 30 min at 37 °C in a dark humid room, and ROS were measured at 540 nm excitation and emission of 590 nm using a fluorescence micro-reader.

3

3 Statistical analysis

Obtained values were analyzed using SPSS (SPSS 16; SPSS Chicago, Illinois) via one-way and two-way ANOVA, and Tukey’s test for paired comparison. A possibility level of P < 0.05 was evaluated to be statistically significant. Also, the significance difference was fixed at *P < 0.05, **P < 0.01 and ***P < 0.001.

4

4 Results

4.1

4.1 Structural analysis of PEG-MNs-TAC

The results of the TEM technique illustrate that the PEG-MNs-TAC is spherical in shape with an average size of 85 nm along with a rough surface containing cracks or cavities on it (Fig. 1a). Whereas, the DLS outcomes show that the MNs and PEG-MNs sizes range are between 60 and 115 nm and 65 to 120 nm (average 90 nm) with a polydispersity of 0.245 ± 0.089 and 0.311 ± 0.025, respectively (Fig. 1b). Furthermore, the outcomes of Fig. 1b reveal that loading the TAC increases the size of nanoparticles with a range of 65 to 125 nm and PDI: 0.405 ± 0.075. This data confirmed the loading of PEG and TAC on the MNs by increasing the nanoparticles dimensions between 5 and 10 nm. Also, the output of XRD pattern represents the crystalline phase of the MNs, which had the six main diffraction peaks              without any additional band (Data not shown), which was in agreement with previous report (Sharifi et al., 2020a). After PEG and TAC loading, the peak intensities were relatively reduced, which confirms loading of PEG and TAC on the MNs. Furthermore, the structure of the MNs was investigated using the N2 adsorption–desorption method (Fig. 1c) and the N2 adsorption–desorption isotherm showed an IV behavior with a ring in the range 0.45–0.95, which indicates the porous structure of the MNswith a surface area of 113.21 m2/g, which is an average size compared to other nanospheres. In addition, the pore size distribution curve represents a dominant peak of about 10.5 nm, which confirms the mesoporous structure.

(a) TEM images of poly[ethylene glycol]-maghemite nanospheres-tacrolimus (PEG-MNs-TAC), (b) MNs, PEG-MNs and PEG-MNs-TAC sizes as determined by DLS, c) N2 adsorption–desorption isotherms.
Fig. 1 (a) TEM images of poly[ethylene glycol]-maghemite nanospheres-tacrolimus (PEG-MNs-TAC), (b) MNs, PEG-MNs and PEG-MNs-TAC sizes as determined by DLS, c) N2 adsorption–desorption isotherms.

4.2

4.2 Drug loading efficiency

As can be seen in Fig. 2a, increasing the TAC concentration increases the amount of drug loading with the main postulate that the PEG-MNs concentration is constant (100 μg/ml). However, increasing the drug concentration decreased the percentage of TAC loading efficiency in the PEG-MNs. Ultimately, Fig. 2a exhibits that the highest loading efficiency of TAC on the PEG-MNs is in 200 μg/ml with an efficiency of more than 50%. Also, the results of drug release from the PEG-MNs-TAC illustrate that drug release follows a time-dependent profile (Fig. 2b). On the other hand, the drug release rate in pH 6 and 7 are approximately 78.2% and 55.1%, respectively. In addition, the initial burst release at acidic and neutral pH was 34.5% and 25.2% at 180 min, respectively. On the whole, the outputs of Fig. 2b indicate the release of the TAC based on the pH-sensitivity of the PEG-MNs-TAC, which is very desirable in damaged tissue due to reduced pH.

(a) Drug loading and efficiency, (b) Quantitative analyses of tacrolimus (TAC) release at 37 °C at pH 6 and 7, (c) cytotoxicity assay of TAC, maghemite nanospheres (MNs), Poly[ethylene glycol]-MNs and PEG-MNs-TAC on fibroblasts cells by MTT assay. *P < 0.05, and **P < 0.01 for a difference of treatment groups. a,b,c,d,e Least square means with different letters in superscripts are different at *P < 0.05.
Fig. 2 (a) Drug loading and efficiency, (b) Quantitative analyses of tacrolimus (TAC) release at 37 °C at pH 6 and 7, (c) cytotoxicity assay of TAC, maghemite nanospheres (MNs), Poly[ethylene glycol]-MNs and PEG-MNs-TAC on fibroblasts cells by MTT assay. *P < 0.05, and **P < 0.01 for a difference of treatment groups. a,b,c,d,e Least square means with different letters in superscripts are different at *P < 0.05.

4.3

4.3 Cytotoxicity assay

The output of MNs, PEG-MNs, TAC and PEG-MNs-TAC toxicities evaluation indicates their toxic effects at high concentrations. Among different concentrations of PEG-MNs-TAC, the highest and lowest toxicities were detected at the concentrations of 800 µg/ml and 400 µg/ml, respectively (Fig. 2c). Although no significant effect was seen between 200 and 400 µg/ml, the probability of cytotoxicity at 400 µg/ml is higher than 200 µg/ml compared to the control group. Also, the results of toxicity indicated the toxicity of TAC at all concentrations. While the use of PEG coating reduces the toxicity of MNs and even TAC loaded in nanocarriers. Meanwhile, the toxicity output shows that no significant difference is observed between the MNs and TAC at all concentrations except at 300 µg/mL of TAC with 600 µg/mL of MNs. In this line, the toxicity result indicates no significant difference between the PEG-MNs and PEG-MNs-TAC at all concentrations except 800 µg/mL. Generally, the outcomes demonstrated that the interaction of the PEG-MNs with the TAC could reduce the toxicity of TAC to some extent.

4.4

4.4 Locomotor activity and hot plate assessments

As shown in Fig. 3a, BBB scores did not differ in preoperative rats. It was found that the BBB scores of rats in the PEG-MNs-TAC-treated were group improved significantly compared to the TAC and PEG-MNs-treated group from the third day onwards. These outcomes indicate the effectiveness of PEG-MNs-TAC in improving BBB score (Fig. 3a). The final day results also confirm the higher effectiveness of PEG-MNs-TAC compared to TAC and PEG-MNs. In addition, hot plate test outputs illustrated that rats had the same reaction on the hot plate before surgery (Fig. 3b). After surgery and reduction of the least reaction to the hot plate on the 5th day, from the 15th day onwards, rats treated with PEG-MNs-TAC showed a more significant recovery process compared to other groups. Although hot plate analysis on the final day indicates a positive effect of TAC compared to the PEG-MNs, examination of the duration of treatment shows that there is no significant difference betweenthese groups from the 5th to the 30th day. However, the output indicates a positive effect of TAC and PEG-MNs in the treatment of rats compared to the control. Finally, the increase in the response of rats on the hot plate in the PEG-MNs-TAC was much faster and more promising.

(a) The outputs of locomotor activity, (b) the outcomes of reaction time, (c) the outcomes of latency, and (d) the outputs of mechanical allodynia scoring in Positive control (P-control), Negative control (N-control), Tacrolimus (TAC), Poly[ethylene glycol](PEG)-maghemite nanospheres (MNs) and PEG-MNs-TAC. PEG-MNs-TAC compared to the control group: #P < 0.10, *P < 0.05, **P < 0.01, and ***P < 0.001. a,b,c,dLeast square means with different letters in superscripts are different at *P < 0.05.
Fig. 3 (a) The outputs of locomotor activity, (b) the outcomes of reaction time, (c) the outcomes of latency, and (d) the outputs of mechanical allodynia scoring in Positive control (P-control), Negative control (N-control), Tacrolimus (TAC), Poly[ethylene glycol](PEG)-maghemite nanospheres (MNs) and PEG-MNs-TAC. PEG-MNs-TAC compared to the control group: #P < 0.10, *P < 0.05, **P < 0.01, and ***P < 0.001. a,b,c,dLeast square means with different letters in superscripts are different at *P < 0.05.

4.5

4.5 Tail-flick latency scaling and mechanical allodynia

The results of Fig. 3c revealed that PEG-MNs-TAC had a higher effect on tail-flick than TAC on 10 and 20 days. However, the tail-flip reaction of TAC-treated rats was similar to that of PEG-MNs-TAC on 30th and 40th days. On the other hand, the results show that TAC has no priority over PEG-MNs in the treatment of rats, whereas, PEG-MNs have low effect in the treatment of mice compared to PEG-MNs-TAC. Furthermore, the mechanical allodynia scoring output exhibited that not only PEG-MNs-TAC improved the mechanical allodynia state more effectively in rats than the TAC, but the trend in the chart showed that the PEG-MNs-TAC was rather rapid in healing the spinal cord injury (Fig. 3d). In the following, the trials presented that there were no significant effects between the PEG-MNs and negative control on 1 to 20 days. But, on 30 and 40 days, the PEG-MNs had a significant effect on improving the mechanical allodynia state. On the whole, these results indicate a positive effect of the PEG-MNs-TAC on the treatment of injured rats after induction of spinal cord injury.

4.6

4.6 Inflammatory cytokine assessment

The results of TNF-α, IL-6 and IL-2 pro-inflammatory cytokines on 40th day revealed that TNF-α was significantly lower in PEG-MNs-TAC and TAC-treated rats than in the PEG-MNs (**P < 0.01) and negative control groups (***P < 0.001) (Fig. 4). However, lower levels of TNF-α were seen in the PEG-MNs-TAC-treated group. In line with TNF-α changes, the concentrations of IL-6 and IL-2 in the affected tissue of the PEG-MNs-TAC was lower compared to the TAC (**P < 0.01) and PEG-MNs (*P < 0.05) (Fig. 4). The use of PEG-MNs reduced the concentrations of IL-6 and IL-2 at the level of #P < 0.10 compared to the negative control, while at the levels of **P < 0.01 or *P < 0.05 there was no significant difference with the negative control. Overall, the results of TNF-α, IL-6 and IL-2 pro-inflammatory cytokines showed that the use of PEG-MNs-TAC drastically reduces the inflammation of damaged spinal cord tissue.

The TNF-α, IL-6 and IL-2 pro-inflammatory cytokines after post-spinal cord injury operation. *P < 0.05, **P < 0.01 and ***P < 0.001 for a difference of treated groups.
Fig. 4 The TNF-α, IL-6 and IL-2 pro-inflammatory cytokines after post-spinal cord injury operation. *P < 0.05, **P < 0.01 and ***P < 0.001 for a difference of treated groups.

4.7

4.7 M1 To M2 ratio and oxidative stress

As shown in Fig. 5a, the ratio of M1 to M2 is noticeably lower in positive control. While spinal cord injury drastically increases the M1 to M2 ratio (***P < 0.001) (negative control). However, the use of PEG-MNs (*P < 0.05) and TAC (**P < 0.01) decreases the expression of M1 compared to the negative control. But, the use of PEG-MNs-TAC resulted in a lower expression of M1/M2 compared to the TAC -treated group (**P < 0.01). Moreover, the generation of ROS (Fig. 5b) in the injured spinal cord group treated with PEG-MNs-TAC was clearly reduced compared to the TAC-(* P < 0.05) and PEG-MNs-(**P < 0.01) treated groups. Interestingly, PEG-MNs (*P < 0.05) and TAC (**P < 0.01) separately reduce generation of ROS compared to negative control. Therefore, the combined use of PEG-MNs and TAC in PEG-MNs-TAC form can reduce generation of ROS more effectively than TAC or PEG-MNs alone.

(a) The outcomes of M1 to M2 ratio according to flow cytometry. (b) The evaluation of reactive oxygen specious (ROS) activity after inducing spinal cord injury. *P < 0.05, **P < 0.01 and ***P < 0.001 for a difference of treated groups.
Fig. 5 (a) The outcomes of M1 to M2 ratio according to flow cytometry. (b) The evaluation of reactive oxygen specious (ROS) activity after inducing spinal cord injury. *P < 0.05, **P < 0.01 and ***P < 0.001 for a difference of treated groups.

5

5 Discussion

Although repair of damaged spinal cord is an inherent feature with a very long process, increased levels of apoptotic-inducing cytokines in the affected areas distrupts this process. Due to the limitations of treatment in this area, developing an effective treatment to trigger nerve regeneration is necessary. Since TAC is an anti-transplant drug by reducing the inflammatory activity of the immune system, and it has been successful in neuroregenerative tissues (Sosa et al., 2005; Ibarra and Martiñón, 2009; Saffari et al., 2021), it can be considered as a new way to repair spinal cord injury. The outputs of Gold et al. (1998) showed that using TAC increases the expression of neuronal protein 43, which is associated with the growth (CAP-43) of sensory neurons in the dorsal root ganglia. In the following, the integration of drugs with PEG-MNs can enhance their permeability in CNS and PNS tissues as well as their effectiveness through solubility (Di Shi et al., 2016). The outcomes of this study revealed that although PEG-MNs-TAC has a more significant effect on the treatment of damaged spinal cord than TAC and PEG-MNs alone, both TAC and PEG-MNs-TAC significantly accelerate the repair of damaged tissue based on a significant increase in transmission indices and neuropathic tests such as locomotor activity, hot plate, tail-flick latency, and mechanical allodynia. Moreover, the reduction of IL-6, IL-2 and TNF-α pro-inflammatory cytokines in damaged spinal cord by TAC and PEG-MNs-TAC was considerable. However, PEG-MNs-TAC provided better conditions for reducing inflammatory cytokines. On the other hand, reduction of ROS and modulation of oxidative stress by TAC and PEG-MNs-TAC strengthened the possibility of faster repair of damaged tissue. Interestingly, rats treated with PEG-MNs-TAC showed better response for reducing ROS and oxidative stress activities compared to other groups.

There is ample evidence that macrophages in the affected spinal cord are predominantly polar M1 pro-inflammatory cells (Kroner et al., 2014; Cairns et al., 2018; Kroner and Rosas Almanza, 2019). Reports indicate that the number of M2 macrophages decreases significantly 7 days after spinal cord injury (Kigerl et al., 2009). Shechter et al. (2013) explained that the a part of M2 macrophages reach the spinal cord injury site through the choroid plexus blocking this pathway to improve motor mobility due to the slowing down of tissue regeneration. On the other hand, many iron-containing macrophages migrate to the center of the damaged spinal cord, which is effective in repairing the damaged tissue and transmitting nerve signals (Rathore et al., 2008). In fact, inflammatory agents such as interferon-γ, IL-6, and IL-2 can stimulate pro-inflammatory and neurotoxic M1 macrophages that are involved in exacerbating nerve and axonal damage (Orihuela et al., 2016; Xie et al., 2016; Gensel et al., 2017). Anti-inflammatory mediators like a IL-4 induce anti-inflammatory macrophages (M2) that promote axon development, re-myelination, and nerve tissue repair (Gensel and Zhang, 2015; Gensel et al., 2017). Therefore, the use of markers of the macrophages M1 to M2 ratio and their transcription factors in determining the strength of anti-inflammatory agents in the repair of damaged spinal cord tissue is very useful (Orihuela et al., 2016; Gensel et al., 2017). In this study, similar to the findings of Kannegieter et al. (2017), it was revealed that the groups treated with TAC and PEG-MNs-TAC had a higher ratio of M1/M2 in the damaged spinal cord compared to the control rats. In this line, Bai et al. (2010) have previously shown that the use of low-dose TAC (FK506) in atherosclerosis not only suppresses T cell activation, but also enhances M2 macrophage polarization. While, their report shows that the increase in blood cholesterol causes the deviation of M2 to M1 and is meaningless in protecting atherosclerosis (Bai et al., 2010). In addition to increasing the M2 to M1 ratio, TAC protects axons and repairs spinal cord lesions by restoring ATP level in mitochondria, lowering the cellular oxidation index, and raising glutathione levels (Bavetta et al., 1999; Yousuf et al., 2011; Saffari et al., 2021).

On the other hand, this study, in agreement with the findings of Stover et al. (2001)demonstrated that TAC reduced the generation of TNF-α, IL-2 and IFN-γ. The mechanism identified involves the direct effect of TAC on calcineurin restriction, which inhibits the production of cytokines such as IL-2, IL-6, IL-10, TNF-α, and IFN-γ by inhibiting p38 mitogen-activated protein kinase phosphorylation (Vafadari et al., 2012). In this regard, Liu et al. (2017) by ELISA and q-PCR assays, explained that the amounts of TNF-α and IL-6 significantly increase by regulation of NF-κB pathway in the first days of spinal cord injury compared to positive control. While by healing the damaged site, cytokine levels decrease significantly over longer periods of time. However, similar to the findings of this paper, they observed that treatment of spinal cord injury with TAC could increase the rate of healing by reducing cytokines (Liu et al., 2017). Increased locomotor activity of rats during treatment confirms the speed of healing of the damaged spinal cord site in this study. In line with this report, de Mesquita Coutinho et al. (2016) confirmed that the TAC, based on improving the locomotor activity, increases the rate of recovery. Remarkably, the outcomes of this study indicated that the use of PEG-MNs-TAC can effectively and significantly increase the speed of recovery of injured rats.

6

6 Conclusion

The outcomes of this study illuminated that TAC can be considered as a desirable treatment in spinal cord injuries by accelerating the growth of nerve cells through activating macrophage M2 and reducing the level of oxidative stress in the damaged tissue. However, despite the effective activities of tacrolimus, the use of PEG-MN nanocarriers can significantly increase the effect of TAC in improving spinal cord injury by enhancing the locomotor activity of rats. Compared to the free drug, nanocarriers effectively accelerated the recovery time of rats. Therefore, the use of PEG-MNs-TAC can be considered as a new treatment for spinal cord injury based on reducing oxidative stress, IL-6, IL-2 and TNF-α inflammatory factors, and the expression ratio of M1 to M2.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81372020), Guiding Foundation of Renmin Hospital of Wuhan University (No. RMYD 2018Z15), and the Foundation of Young Talent Physician Training Project in Wuhan City (No. 2014ZX0001).

8

8 Ethics approval and consent to participate

All procedures were carried out in accordance with the Regulations of Experimental Administration issued by the State Committee of Science and Technology of the People’s Republic of China, with the approval of the Ethics Committee in our university.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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