Novel design and combination strategy of minocycline and OECs-loaded CeO₂ nanoparticles with SF for the treatment of spinal cord injury: In vitro and in vivo evaluations

2.1 Chemicals used

The chemicals (NH₄)₂Ce(NO₃)₆, ethylenediamine, hydrazine monohydrate, ethanol, Na₂CO₃, LiBr, and PVP were purchased from Jiangsu Zhongshan Chemical Co., Ltd. Silk worm cocoons were obtained from Jiangsu Academy of Agricultural Science, PR China. Biological mediums (BSA, DMEM, FBS, and EDTA) were obtained from Tansoole Co., Ltd., Shanghai, China. The purchased chemicals were used as analytical grade (AR) reagents.

2.2 Synthesis of CeO₂ NPs by hydrothermal method

For the synthesis of CeO₂ NPs, 0.25 g of (NH₄)₂Ce(NO₃)₆ was added into 30 mL of distilled water and stirred for 20 min to get a completely dissolved solution. Thereafter, 0.15 mL of ethylenediamine was dissolved in 25 mL of deionised water and was added into the above solution in a drop-wise manner under constant stirring. Then, 1 mL of hydrazine monohydrate was added, continuously stirring for 15 min at room temperature. Finally, the solution was transferred into an autoclave and heated at 100°C for 24 h. After the completion of reaction, the autoclave was completely cooled to room temperature. The final product mixture was centrifuged at 5,000 rpm for 20 min to get white precipitate which was washed with water (3 × 10) and ethanol (3 × 10) thrice, filtered, and dried at 60°C for 2 h. Finally, the white powder was calcined at 500°C for 2 h to get CeO₂ NPs.

2.3 Synthesis of SF hydrogel

SF solution was prepared from a natural B. mori silkworm cocoons through a previously reported literature [39,40]. First, the cocoons were cut into tiny pieces and boiled in Na₂CO₃ (0.02 M) for 30 min. The degumming process was repeated thrice using deionized water in order to remove sericin and wax from the silk. Then, the silk fibres were dried at room temperature and dissolved in LiBr (9.3 M) solution at 60°C for 2 h. The solution was dialysed in distilled water for 3 days using dialysis membrane at ambient condition to remove LiBr salt. Thereafter, the SF solution was centrifuged at 8,000 rpm for 30 min. Then, the final SF solution was stored at 4°C for further studies.

2.4 Preparation of CeO₂ NPs embedded SF hydrogel

In this preparation, 100 mg of synthesized CeO₂ NPs (white powder) was dispersed in 10 mL of ethanol. Meanwhile, 5 wt% of PVP was prepared in ethanol and was added to the CeO₂ NPs’ solution at constant stirring. Thereafter, the prepared SF solution was poured into the above mixture under ultrasonication for 3 h to get CeO_2-loaded SF hydrogel. Then, the SF hydrogel nanocomposite was freeze dried at −40°C and preserved at 4°C for further studies.

2.5 Characterisation

Functional groups and other important moieties present in the synthesized SF hydrogel and CeO₂ incorporated hydrogel were studied by Infrared Spectrophotometer (Shimadzu –8400, Japan) using KBr pellet method. SF hydrogel and CeO₂/SF were investigated by Ultra-High-Resolution Schottky Scanning Electron Microscope SU7000. The size and morphology of CeO₂ NPs were analysed by TEM, JEOL JEM-2100 with an accelerating voltage of 200 kV. Panalytical X-ray diffractometer with a Cu-kα X-ray source operated at 40 kV with a current of 30 mA (λ = 0.15406 nm) was used to investigate the crystalline behaviour of NPs.

2.6 The OECs culture method

The Nash method was used to attain OEC from the sources of adult rats’ nerve fibres and olfactory bulbs as per previous reports [41]. Chiefly, the adult rats were successfully anesthetized using an appropriate dose of chloral hydrate. Thereafter, the olfactory nerves were separated and bulbs were placed into the magnesium- and calcium-free HBSS solution obtained from Sigma-Aldrich, USA. The isolated blood vessels- and meninges-removed tissues were pulverized and incubated using DMEM medium supplemented with trypsin (0.1%) at biological conditions (37°C; 5% of CO₂) for 30 min. After that, the suspension was centrifuged with FBS solution after inactivation of trypsinization. Then, the suspension was placed into a culture flask with DMEM medium added with FBS (10%), standard antibiotics, and l-glutamine (2 mM). After 48 h of incubation, OECs were attached onto the precoated poly l-lysine plates.

2.7 The spinal cord in vivo animal model

The adult female rat models (230–260 g) were purchased from Slac Laboratory Animal Co. Ltd., PR china for the animal testing of SCI treatments. All animal experiments and protocols were in agreement with the policies of the Institutional Animal Care and Use Committee, Nantong University, Jiangsu, China. The adult rat animal models were anesthetized using chloral hydrate (450 mg per 1 kg) by intraperitoneal injection. The spinal cord of rats was exposed under laminectomy at vertebral level T11 and spinal injury was created, which was followed by animal guidelines of college, China. After producing the SCI, the operated muscles and skin of animal models were bolted distinctly and kept in chamber for one night. Then, commercial gentamicin was successfully administered for 2 days to avoid infections of wound and bladder and standard acetaminophen was supplemented into drinking water for one week, and expressions of urinary bladder was obtained daily for evacuation of reflexing bladder [42].

2.8 Administration of MCN

The MCN with dissolving sterile PBS solution was administered intraperitoneally to the injury created animal models. The SCI created animal models proximately received 80 mg/kg of MCN after SCI injury and then administered after one day as established by previous reports. The animal injected with bare PBS solution with absence of MCN was considered as control sample. At the same time, the rat without injury with non-therapeutic approaches was sacrificed and was considered as sham.

2.9 Cell transplantation

The cell transplantation was done 7 days after initial SCI surgery with treatment of MCN. In brief, the adult rats were re-exposed through laminectomy after being anesthetized. The cultured OECs (6 µL/450,000) cells were transplanted to the defected SCI site by using syringe, and the injected place was marked. The OEC culture was injected at the places of lesion (0.8 mm), rostral (1 mm), and caudal (1 mm) epicentres. The animal injected with DMEM biological medium was considered as control sample.

2.10 The measurement of BBB score

The BBB scores were evaluated to measure the recovery function of MCN and transplanted cells followed by BBB scale technique, which measured the hind limb function in the ranges between 0 and 21. The augmented score indicated the improvement in the recovery functions including joint movements, limb movements, weight behaviours, and other recovery functions. The scores were obtained and calculated following SCI locomotion recovery progression in different time intervals (1, 7, 14, 21, 28, and 35 days).

2.11 Histological analysis

The treated animal models were sacrificed feasibly with an overdose of anaesthetic cocktail (ketamine and xylazine) at the end of the experiment day. After that, the treated spinal cords of rats were carefully detached and stored in sucrose solution (30%) dissolved in PBS medium (pH 7.4) for 12 h. The spinal cords with injury site of 3 cm in size were separated sensibly. Then, the samples were cut into small square shape sections (10 mm) using the cryostat device and stained by haematoxylin and eosin (H and E) dye. The presence of connective and nervous tissues on the treated spinal cords were observed at 10× and 40× magnifications and visualized using the light microscopic technique.

2.12 Statistical analysis

The statistical significance value of the presented results was examined using the one-way analysis of variance technique. All the values are mentioned as the mean value ± SD (standard deviations).

Figure 1

Schematic representation of the present investigation icluding synthetic and structural schemes.

3.1 Characterisation of CeO₂ NPs

Herein we described a fabrication of drug-loaded novel therapeutic agent containing SF/CeO₂ for SCI recovery (Figure 1). CeO₂ NPs were synthesized by well-established hydrothermal method and the formation of NPs were confirmed by the combination of x-ray diffraction studies (XRD) and Transmission electron microscopic studies (TEM. Figure 2a shows the XRD pattern for CeO₂ NPs and the sharp peaks present in between the angles 20° and 80° clearly indicates the formation of NPs. Moreover, the spectrum shows intense sharp peaks appearing at 28.4, 33.2, 47.5, 56.4, 59.6, 70.3, and 76.7 having the miller indices (111), (200), (220), (311), (222), (154), and (331), respectively. These peaks vividly indicate the crystalline nature of CeO₂ NPs. Further, the size and structural morphology of CeO₂ NPs were examined under TEM and the TEM images showed that the NPs are spherical structure having ∼20–25 nm (Figure 2b). Along with spherical particles, some thread like morphologies were also observed in the TEM micrograph.

Figure 2

(a) XRD pattern for CeO₂ NPs and (b) TEM images of CeO₂ NPs.

3.2 Characterisation of CeO₂-loaded SF hydrogel

The functional groups present in the SF were confirmed by FTIR spectroscopy. An overlapped FTIR spectrum for SF hydrogel and CeO₂ NPs-loaded SF hydrogel is given in Figure 3a. The spectrum (a) displays a band at 1,632 cm⁻¹ for N–H stretching which confirmed the presence of (amide groups) amino acids such as glycine, alanine, and so on. These characteristic bands were considered as the absorption peak of the peptide backbone. The important peaks at 1,632, 1,519, and 1,231 were assigned to amide I, amide II, and amide III bands for β-sheet conformation. The characteristic band appeared at 1,519 cm⁻¹ due to N–H bending vibration and additional peaks were observed at 1,231 and 1,336 cm⁻¹ due to C–N stretching vibrations. Moreover, these characteristic absorbance peaks are clearly the evidence for the hydrogen bonding in N–H groups. We observed that a small peak at 3,289 cm⁻¹ is ascribed to O–H hydroxyl groups in SF hydrogel material. In addition, two peaks appeared at wavenumber 2,983 and 2,895 cm⁻¹ which are attributed to C–H symmetric and C–H asymmetric vibrations, respectively. As seen in spectrum (b), in addition to all the peaks obtained for SF hydrogel, a small sharp peak appeared at around 600 cm⁻¹ for Ce–O metal to oxide linkage revealing the incorporation of CeO₂ NPs in SF hydrogel.

Figure 3

(a) Overlapped spectra for SF hydrogel and CeO₂-loaded SF hydrogel; (b) XRD patterns for: (a) SF hydrogel, (b) CeO₂ NPs, and (c) CeO₂-loaded SF hydrogel.

Further, CeO₂-loaded SF hydrogel was studied by X-ray diffraction studies. The amorphous structure of freeze-dried SF hydrogel exhibiting a broad peak at around 20° is shown in Figure 3b (pattern a), and the obtained spectrum is closely analogous to the XRD pattern for β-sheet SF structure. The lyophilized SF hydrogel displays only typical broad peak at around 20°, obviously no other peaks were observed in the spectrum which specify the possibility of more α-helical structure. As seen in Figure 3b (pattern b), the XRD pattern for CeO₂-loaded SF hydrogel showing broad peak at 20° reveals the amorphous nature of SF material, and along these wide peaks at small angle region, seven more additional peaks appeared owing to the existence of CeO₂ NPs in hydrogel.

Besides, the structural morphology of SF hydrogel and CeO₂ NPs-loaded SF hydrogel was investigated through scanning electron microscopic (SEM) technique. Figure 4a shows that the SEM image of SF hydrogel are porous in nature and have exposed interconnected heterogeneous porous architecture. Moreover, the freeze-dried process also enhances the porosity of the hydrogel. The surface morphology of CeO₂ NPs-loaded SF hydrogel was examined under SEM and Figure 4b shows that the microstructure of hydrogel composite have porous architecture and CeO₂ NPs were embedded in the surface of hydrogel matrix. Figure 4c displays vividly that the CeO₂ NPs were properly blended to the SF solution, and the NPs’ spherical as well as elongated thread like morphologies are well matched to the TEM images of CeO₂ NPs. These results strongly support the existence of NPs in the hydrogel matrices. Additionally, we carried out TEM analysis (Figure 5) of CeO₂/SF hydrogel which confirmed the incorporation of NPs in hydrogel. TEM images obtained for CeO₂/SF hydrogel displayed a dark portion indicating CeO₂ NPs entrenched in NPs in the porous architecture of hydrogel matrix. Moreover, Figure 5c indicates a zoom version of image b and displays an intense image of CeO₂ NPs.

Figure 4

(a) SEM image of SF hydrogel; (b and c) SEM of CeO₂ NPs-loaded SF hydrogel.

Figure 5

(a and b) different magnified TEM images of CeO₂ loaded SF hydrogel; (c) an expanded version of subfigure (b).

After the cell cultures in appropriate time, we can observe the successful forms of OECs through the optical and fluorescence microscopic images as shown in Figure 6. The fluorescence microscopic observation of OECs exhibits the findings of NGFRp75 (Figure 6). The production of repressive glial scars and spinal cord neurons have been significantly damaged by the secondary injury after SCI. The current single strategic therapies of SCI have targeted its own complications that could limit the functions of post-injury recovery, which provides inadequate developments in the recovery functions of SCI. Hence, we have designed and developed combined therapies for SCI recovery using OECs’ transplantation and distribution of MCN to overwhelm the treatment of SCI that enhances therapeutic potential. The present investigation results established that combination potential of OECs’ transplantation and MCN have great effects than treating separately with cells’ transplantation and MCN. The cell compatibility of the prepared nanomaterials was evaluated on cultured OECs at different concentrations using CCK-8 assay. The results of cell viability exhibited that the prepared nanomaterials displayed non-toxicity on the OECs at appropriate concentrations, which demonstrated greater biological compatibility with normal cells as exhibited in Figure 6a. Specifically, the prepared CeO₂ NPs and CeO₂/SF nanovesicles displayed cell viability percentages of 89.45% and 94.65%, respectively, at higher concentration levels, which revealed that molecular structures and concentrations of prepared nanoformulated materials have not disturbed the normal cell lines. These observed results of in vitro drug release and cell viability demonstrated that therapeutic dose of nanomaterials could provide effective treatment for SCI recovery, signifying the combination therapy of MCN and OECs. The analysis result of in vitro drug release was evaluated using simulated body fluid (PBS; pH 7.4 supplemented with 2% of DMSO) with treatment of MCN-loaded CeO₂ NPs and CeO₂/SF composite vesicles. The drug molecules’ (MCN) release from CeO₂ NPs were significantly faster when compared to the CeO₂/SF nanovesicles, which confirm that the developed CeO₂/SF nanoformulation provides controlled release platform. The drug release rate of MCN is 75% and 100% from CeO₂/SF nanocomposite and CeO₂ NPs, respectively, at 5 days, which revealed that CeO₂/SF composite have effective and sustained release platform as shown in Figure 6b. The anti-inflammatory efficiency of MCN-loaded CeO₂ NPs and CeO₂/SF composite was investigated by quantification-based measurements using ELISA method. Both nanomaterials distributed with MCN have significantly reduced expressions of IL-1β, TNF-α, and IL-10 when compared with control (LPS) group as exhibited in Figure 7. The prepared MCN-loaded CeO₂/SF composite displayed that IL-1β and TNF-α expressions significantly assuaged, due to the effective anti-inflammatory action of MCN and CeO₂ nanoparticulate system.

Figure 6

Optical and Immuno-fluorescence microscopic images of cultured OECs: (a) in vitro cell compatibility evaluations of prepared materials on OECs and (b) in vitro drug release ability of the nanomaterials with MCN.

Figure 7

Evaluations of pro-inflammatory expressions on the TNF-α (a), IL-10 (b), and IL-1β (c) in treated groups of SCI, MCN, MCN@CSF, and OEC + MCN@CSF.

The improvement of SCI recovery function was evaluated by the investigation of locomotor behaviour of hind limbs such as BBB score. The results of locomotor function demonstrated that animal models of SCI have gradual enhancements in the hind limb movements at increasing time intervals at about 35 days. Though the hind limb movement and motor functions were progressively developed, which was treated separately with bare OECs and MCN on different days (14, 21, 28, and 35 days) when compared with SCI group, the combination results of OECs and MCN exhibited augmented BBB scores at increasing days post-treatment, which confirms the amplified functional recovery as displayed in Figure 8. The results established that the combination of MCN and OECs’ transplantation have significant progress in BBB scores and tissue sparing than SCI models treated with bare MCN and OECs alone. High recovery rate after SCI was observed and recorded after 2–5 weeks with the treatment of MCN + OECs group, which revealed that the transplantation of OECs has significantly influenced the functional recovery improvement. Meanwhile, the grafting of OECs provides favourable biological setting for the SCI recovery mechanisms with decreasing actions of pro-inflammatory factors and reducing formation of glial scars. At the same time, the transplantation of OECs may regulate the expressions of glial fibrillary acidic protein (GFAP), which avert the glial scar formation and inhibitory molecules secretion. Previous reports recorded that OECs’ graftings have greatly influenced the GFAP expressions after SCI. The treatment of MCN to SCI therapy has providing modulation of apoptosis, microglia, and caspases, which proved the auspicious therapeutic potential for regeneration of SCI. In agreement with the previous reports, we have investigated and confirmed that MCN combined with OECs have importantly enhanced the functional recovery and regeneration effects after SCI.

Figure 8

Evaluation of BBB behaviour motor function: (a) on hind limb of rat models and bladder weight (b) treated with different samples including MCN, OEC, and combined OEC + MCN materials loaded on CeO₂/SF nanomaterials.

The histopathological observation was performed to demonstrate the visualization of SCI recovery after administration of MCN and MCN + OECs-loaded CeO₂/SF nanomaterial as displayed in Figure 9. The H and E staining observation of combination therapeutic approach exhibited significant histological enhancement when compared to the nanomaterial with MCN (alone), OECs (alone), and SCI control group. The SCI control group without any bio-pharmaceutical treatment exhibited more pseudocysts with very less progressed development. In treatment group of MCN + OECs-loaded CeO₂/SF nanomaterial, the recovery of spinal injury was significantly improved, and successfully re-established SCI section as exhibited in Figure 9. These results of histopathological observation have clearly demonstrated that controlled release of CeO₂/SF hydrogel sample and recovery action of OECs greatly influenced the SCI treatment. The recovery efficiency of the prepared material was established by the reduction in pseudocyst volume in the treatment group when compared to the SCI control group.

Figure 9

The analysis of histopathological study of the SCI (untreated sample), MCN, OEC, and MCN + OEC loaded on CSF nanomaterial; H and E and MTS staining images were observed after 8 weeks of post-injury under microscopic technique.