Bio fabrication of Fe₂O₃ NPs and their analgesia efficiency for post-operative epidural anaesthetic applications- study of chronic inflammatory pain model in mice

ABSTRACT

The current study reported the synthesis of ferric oxide nanoparticles (Fe₂O₃ NPs) using the leaf extract of Amaranthus spinosus Linn and studied their analgesia efficiency in chronic inflammatory pain model in mice. X-RD, TEM results showed that the spherical Fe₂O₃ NPs having rhombohedral crystalline structure with an average particle size of 10 nm. Additionally, inflammatory pain models were designed through FCA injection on the CD1 mice hind paw with various Fe₂O₃ NPs doses and analgesic behaviour was monitored. The prepared Fe₂O₃ NPs showed a dose-dependent analgesic activity with considerable decrease in inflammatory cells, ROS production and pro-inflammatory markers. Additionally, the expression of enzymes responsible for ROS production were decreased. The outcomes of present work revealed the local administration of Fe₂O₃ NPs caused the substantial analgesia through reduction of pro-inflammatory signalling and inflammatory cellular infiltration along with micro-environmental free radical scavenging in mouse inflammatory pain model.

KEYWORDS:

• Fe₂O₃ NPs
• analgesia
• pain
• mouse

Introduction

Pain is an important healthcare issue globally. The two most popular classes of drugs recommended for moderate to mild pain are steroidal and non-steroidal anti-inflammatory drugs (NSAID’s) because of their anti-inflammatory activity. However, drugs having effective control on pain along with minimum side reactions are desired. In addition, functional nanomaterials have gained extreme industrial and scientific attention because of their broad ranged potential uses in biomedicine, material engineering, optoelectronics as well as in various other areas [1]. In a drug delivery system, the utilization of nano-scaled materials as carriers has various advantages such as targeted engineering, surface alteration and selectivity of lesion/tissue by appropriate control of size. Although, the usage of nano-scaled materials as delivery vehicle has been more prevalent, only a few organic nanoparticles (NPs) have been employed to control the pain [2–4]. For instance, diclofenac sodium and ethyl cellulose/iron have been combined for the treatment of arthritis [2]. Similarly, nasal morphine NPs (Biovector) have showed a better analgesic activity [3], and the nano carriers were utilized in the topical pain conditions [4].

The crystalline ferric oxide nanoparticles (Fe₂O₃ NPs) are the most effective nanomaterials for photo-electrochemical and photo-catalytic applications because of their small band gap (2.0–2.2 eV) and their ability to take upto 40% of the solar spectral energy. These α-Fe₂O₃ NPs exhibits excellent magnetic properties and higher stability in the aqueous solutions [5,6]. In addition, Hematite, an n-type semiconductor, has gained significant attention due to its potential uses in medicine, optical devices, gas sensors, pigments, catalysis, and their use as anode material [7–13]. Moreover, α- Fe₂O₃ NPs were prepared using various established procedures which include electrochemical anodization [14], magnetic sputtering [15], ultrasonic spray pyrolysis [16], sol – gel process [17], vapour – solid growth method [18] and hydrothermal production [19]. On the other hand, Fe₂O₃ NPs exhibit a variety of properties which gave rise to their important applications in science and technology such as antimicrobial [20], vaccine delivery [21].

In recent times, green nanotechnologies have gained huge attention in the field of research mainly because of their simplified manufacturing procedures for preparation of nanoproducts. These products are safe to all the human beings which are environment friendly with sustainable viability [22]. The synthesis of NPs utilizing chemicals leads to the presence of certain hazardous chemical species which get adsorbed onto the NP’s surface that may exhibit poor biological activity when used in various applications. To prevent these disadvantages, scientists have developed new green approaches for NP’s synthesis. The process of green synthesis is simple and different compared to the chemical process, employing the use of either plant extracts or microorganisms. Biofabrication also provides improvements compared to the chemical process as it is not expensive for large-scale preparation. Generally, the biofabrication process involves three major steps: (1) selection of solvent medium (2) selection of environmental benign reductant and (3) selection of non-hazardous substances as stabilizing agents [23]. On the other hand, various nanoparticles such as ZnO NPs, Fe NPs, MgO NPs, Au NPs, CuO NPs, NiO NPs, Cu-Ni NPs, Co₃O₄-NPs, Ag NPs, CeO₂ NPs were synthesized using biological approach such as using plant extracts, algae, and biomolecules which later studied for their biological applications [24–46].

Many research investigations have foregrounded the analgesic effects related with applications of NPs. A renowned study involves the use of core-shell superparamagnetic iron oxide nanoparticles with a photonic ZnO shell, which applied to human-adipose-tissue-derived stem cells, demonstrating a significant reduction in neuropathic pain in animal model [47]. In addition, the use of cationic NPs for effective delivery of Piroxicam has shown the enhanced localization pursuing intra-articular injections [48]. Moreover, the encapsulation of anaesthetics within nanoliposomes has been considered as an important strategy for improving the administration of local anaesthesia, thereby contributing to more effective pain management [49]. However, there were no reports on use of Fe₂O₃ NPs for their analgesic studies without drug loading.

The current study reported the synthesis of Fe₂O₃ NPs using leaf extract of Amaranthus spinosus Linn. The biomolecules present in the leaf extract act as stabilizing and reducing agents. The current study also revealed that an analgesic effect can be produced by a low dosage of ultra-small Fe₂O₃ NPs, possibly through significant reduction of reactive oxygen species (ROS) and inhibition effect on the activity of macrophage upon applying on lesion area in mice having severe inflammatory pain.

Experiments

Materials

Ferric nitrate nonahydrate [Fe(NO₃)₃], Potassium bromide (KBr), Hydrogen peroxide (H₂O₂), O- Dianisidine, dimethyl sulphoxide and other chemicals, solvents were procured from Sigma Aldrich, Shanghai.

Preparation of Fe₂O₃ samples

Amaranthus spinosus Linn fresh leaves were collected and thoroughly rinsed multiple times using double distilled water and dried under sunlight. About 4 g of dried leaves were added to 100 mL of double distilled water. This mixture was undergone to a water bath maintained at 60°C for a duration of 30 min and left at room temperature for cooling. Post-cooling, the extracts were filtered using cellulose nitrate filter paper and preserved at a temperature of 4°C for future experiments.

Fe₂O₃ NPs were fabricated utilizing Amaranthus spinosus Linn leaf extract as reducing and stabilizing agent. Briefly, 1 mM Fe(NO₃)₃ was dissipated in 100 mL distilled water and stirred continuously for about 6 h using magnetic stirrer. Later 10 mL Amaranthus spinosus Linn leaf extract was mixed with ferric nitrate solution and continuously stirred at a temperature of 50°C for 24 h. Then the resultant solution was centrifuged for 20 min at 15,000 rpm and washed five times for removing the residue of nitrate ions. Subsequently, the precipitate was kept for drying under vacuum at 70°C. Then the dried sample was calcinated at a temperature of 200°C for about 4 h to obtain α- Fe₂O₃ NPs.

Characterization

X-ray diffraction (X-RD) analysis of powdered sample was performed with the help of X-ray diffractometer with Cu Kα₁ radiation at 1.5406 A° and the samples were examined at a range of 10° to 80° (2θ) with 1 min⁻¹ scanning rate, step size of 0.02°, operation voltage of 40 kV, and an electric current of about 40 mA. Raman spectra was studied utilizing Renishaws InVia Raman microscope. A dried powder of NPs was used for raman analysis. Transmission electron microscope (TEM) analysis was recorded using JEOL TEM instrument, Japan. An aqueous diluted sample of NPs dispersion was dropped on a Cu grid with ultrathin Cu on a holey C-film and allowed to dry in vacuum for TEM analysis. Fourier transform infrared spectrophotometer (FT-IR) spectrum was recorded at a wavenumber ranging from 400 to 4000 cm⁻¹ with the help of Bruker spectrophotometer (IFS 66/S) utilizing KBr pellet method. Optical features were determined utilizing Ultraviolet-Visible (UV-Vis) spectrophotometer (PerkinElmer LAMBDA 35). An aqueous diluted sample of NPs dispersion was used for optical absorbance measurements.

Animal inflammatory pain model

In current study, CD1 mice (male) weighing about 20–30 g were utilized for animal experiments. All the animal studies were carried out according to the ethical guidelines of Affiliated Hospital of Hubei University of Arts and Science. A Freund’s complete adjuvant (FCA) was used to induce the inflammation- associated pain condition. FCA (also known as irritative chemical) of 0.01 mL quantity was injected using a 27 G needle into the plantar foot pad (left side) for inducing pain by local inflammation.

Behavioural analysis

Animals were daily adapted to an environment in which they were tested for minimum of two days prior to baseline testing. To test the mechanical pain sensitivity, animals were placed in cases on an overhead mesh floor (metal) and then permitted to habituation for about 30 min prior to initiation of the examination. To test mechanical allodynia, von Frey hairs were used. Pressing the mice paw using one such series of von Frey hairs by increasing stiffness logarithmically (0.02 to 2.56 g) which is present at right angle to the plantar surface was performed with the application of each hair on the paw of mice for 1 s. Dixon’s up-down method was used to determine the half of the threshold withdrawal.

Further, a heat sensitivity test was performed where the animals were placed in a plastic box which was kept on glass plate and later subjected to the radiant beam of heat over a clear surface. In addition, the base line latency was adjusted between 10–12 s, with a maximum cut-off of 20 s for preventing the injury. These latencies divided by an interval of 3–5 min were made into an average of over three trials.

Immunohistochemical analysis

Sections of paw skin were collected during last stage of experiment. Then animals were lethally anaesthetized for 5 min using 4% isoflurane and then infusion with 4% paraformaldehyde which followed by removal of paw skin and postfixed. Immunohistochemistry (IHC) analysis was carried out on the frozen sections with 12 μm thickness inserted in Cryoma- trix™compound. Rat anti-CD68 (1:200 dilution) and Rabbit anti-myeloperoxidase enzyme (anti-MPO) (1:500 dilution) were utilized as primary antibodies. All the experimental methods were performed by following the protocol using Bond-Max Automated IHC stainer. With the help of Bond Polymer Refine Detection Kit, the tissues were blocked with H₂O₂ for 5 min. Later, incubation of primary antibodies for a period of 15 min at ambient temperature was performed. The secondary antibody, Anti-rabbit/mouse poly-HRP enzyme (horseradish peroxidase), was incubated at ambient temperature for a period of 8 min followed by developing with 3,3′-diaminobenzidine chromogens. Hematoxylin was used for counterstaining. The staining intensity of CD86 and MPO in the mice paw skin was calculated by using an imaging analysis system assisted by computer (ImageJ, NIH).

Measurement of MPO and ROS in skin tissues

The MPO and ROS levels were calculated for investigating a possible method. For detection of free radical production, an assay of lucigenin-based chemiluminescence was performed. Skin was collected and isolated the weighed homogenized skin with trypsin EDTA followed by centrifugation. Later, the supernatant layer was removed, and 1 mL Tyrode’s solution was added to pellet. Pellet was thoroughly mixed and then immediately obtained to measure oxygen using back grounded chemiluminescence in lucigenin consisting of 2 mL buffer and was assessed for 5 min. Then chemiluminescence was measured at ambient temperature for 2.5 min using a Luminometer. After calculating the oxygen production, the chemiluminescence was represented as relative ratio in comparison with control. For identifying neutrophil activity in skin, measurement of MPO was carried out. In brief, the weighed tissue was lysed using 0.005% triton X-100 and frozen at −70°C temperature until experimental usage. For assay, the substrate cocktail was prepared using 20 μL of 26.4 mM H₂O₂, 5 mL 0.1 M citrate buffer, 50 μL of O-Dianisidine in 82.4 mM dimethyl sulphoxide and 32 μL of 20%Triton X-100. 0.7 mL citrate buffer of 0.1 M concentration (pH 5.5) was added to 0.5 mL cell lysate followed by addition of 4.8 mL of previously obtained assay mixture. Then the mixture was placed for 1 h at ambient temperature. MPO intensity was known by measuring optical density at 450 nm.

Western blot technique

After MPO and ROS measurements, more amount of skin tissue was obtained and then homogenized using sample buffer sodium dodecyl sulphate (SDS) which comprises phosphatase inhibitors and proteinase. Protein samples weighing 25 μg are divided on polyacrylamide gel electrophoresis and then moved into nitro-cellulose western blots. Blocking of blots was carried out using 5% of milk followed by incubation at 4°C overnight with 1° antibodies against phagocyte superoxide (p47phox) and inducible nitric oxide synthase (iNOS). These 1° antibodies were utilized as anti-p47phox rabbit polyclonal antibody and mouse anti-iNOS monoclonal-168 antibody. Endogenous beta-actin was utilized as a loading control together with an anti-human beta-actin mouse monoclonal antibody. After incubating 1° antibody, blots were subjected to washing and then identified with a corresponding 2° antibody like anti-rabbit/mouse IgG, which was linked to HRP for 1 h at ambient temperature. Detection of chemiluminescence was carried out using Immobilon Western Chemiluminescent HRP Substrate and therefore, directly calculated by BioSpectrum Imaging System. Based upon apparent molecular sizes, specific bands were evaluated.

Quantification and statistics

For studying animal behaviour, paw withdrawal thresholds data which was determined with the help of up-down technique, qualified normal test are considered appropriate for parametric statistical analysis. The obtained data was evaluated with the help of Student’s t-test (with two groups) or ANOVA. Also, Newman – Keuls method was utilized for the post hoc test. Therefore, the obtained data was represented as an average ± standard error of mean (SEM), whereas p < 0.05 was taken into consideration as statistically relevant in every case. For measurement of western blot technique, the specific band density of β-actin, p47phox and iNOS were quantified using image-analysis software (Image J). The rectangular size had been fixed for every single band and subtracted from the background around the band. Each protein expression level was standardized to the loading control (β-actin). The data from western blot technique was analysed with the help of ANOVA and later by Newman – Keuls method for the post hoc test. In addition, immunohistochemistry staining intensity in tissues of paw skin was quantified from the microscope captured images to measure the positive biomarker cell level. Five paw skin tissue slices were selected randomly from every mouse. The level of intensity for every skin tissue slice was made into average on the fixed three stained areas. The background was removed to measure the biomarker negative cells intensity. Then, the average positive biomarker cells intensity by separating background was determined for every section. Therefore, similar intensities of illumination were used to get all the images for quantifying the test. All the obtained images were evaluated with the help of computer-aided image analysis system. # represents p ≤ 0.05 versus Fe₂O₃ 1.25 mg group, * represents p ≤ 0.05 versus PBS sham group, + represents p ≤ 0.05 versus naïve group.

Results and discussion

Characterization of Fe₂O₃ NPs

The crystal phase and crystallinity of the fabricated Fe₂O₃ NPs are studied using XRD analysis as depicted in Figure 1. Well-defined XRD reflections were observed at 2θ values of 72.26°, 63.99°, 62.41°, 57.59°, 54.09°, 49.48°, 40.85°, 35.612°, 33.15° and 24.13° which can be indexed to Bragg’s reflections of (300), (214), (018), (116), (024), (113), (110), (104) and (012) respectively. The obtained XRD results have confirmed the rhombohedral structural phase of Fe₂O₃ NPs. These XRD findings were matched well with JCPDS No: 33–0664. On the other hand, there were no additional diffraction patterns correlated to any impurities have been observed in the XRD pattern excluding rhombohedral structure of Fe₂O₃ NPs. The XRD findings indicated that the fabricated materials were crystalline in nature with pure phase. The crystallite size found to be 9 nm and was evaluated utilizing following Debye-Scherrer equation.

Figure 1. XRD pattern of prepared α- Fe₂O₃ NPs.

$\mathit{D}=\frac{0.89\mathit{K}}{\mathit{\beta }\mathit{cos\theta }}$

where β, θ, k and D represents full width at half maximum (FWHM) peak in radians, Braggs angle, wavelength of X-rays and mean crystallite particle size respectively.

The surface morphological characteristics of the fabricated Fe₂O₃ powder samples were studied utilizing TEM analysis as depicted in Figure 2(a,b), which displayed that the synthesized NPs were spherical in shape with uniform particle size distribution and average particle size of 10 nm analysed using image J software (also confirmed by DLS histogram shown in Figure 2(d)). Elemental composition of the fabricated Fe₂O₃ NPs was studied using EDX spectroscopy as depicted in Figure 2(c). The EDX spectra displayed that the synthesized NPs comprised oxygen and iron. No additional peaks correlated to any impurity, or any additional element was determined in the spectrum, which showed the fabrication of pure Fe₂O₃ NPs.

Figure 2. HR-TEM images (a, b) and EDS spectrum (c) and DLS histogram (d) of prepared Fe₂O₃ NPs.

Raman technique was used to know the structure of Fe₂O₃ NPs which was depicted in Figure 3. The absorption peaks observed at 610, 495, 409, 292, 243 and 224 cm⁻¹ were assigned to the characteristic rhombohedral phase of Fe₂O₃ NPs. These results were consistent with the findings of X-RD measurements. The peak positions in raman spectra of Fe₂O₃ NPs were in accordance with the previously reported literature [50].

Figure 3. Raman spectrum of prepared α- Fe₂O₃ NPs.

Figure 4(a) showed the UV-Vis absorption spectra of Fe₂O₃ NPs. The spectra obtained displayed an absorbance band peak found in the UV region at 272, 430 and 551 nm. These findings were assigned to photo excitation of electrons from the valence band to conduction band. Absorption band gap (E_g) of fabricated Fe₂O₃ NPs could be determined using Tauc equation: αhv = C (hv - E_g)^1/2 where E_g represents band gap energy used in direct transitions, hv is the photon energy and α represents optical absorption coefficient. A graph of (αhv)² vs photon energy was depicted in Figure 4(b). The optical band gap energy for direct transitions of the Fe₂O₃ NPs was observed as E_g = 2.25 eV. These results were in accordance with the values reported in the literature [51].

Figure 4. UV–visible optical absorption spectrum (a) and band gap plot (b) for prepared Fe₂O₃ NPs.

Figure 5 displayed the FTIR spectrum of the fabricated samples. In the FTIR spectra, the absorption peaks observed at 3400 and 1637 cm⁻¹ attribute to stretching vibrations of – OH and C=O bonds respectively. The absorption bands noticed at 1138 and 1394 cm⁻¹ were assigned to stretch vibration of C-O bond. Additionally, the absorption bands found at 525 and 458 cm⁻¹ were attributed to α- Fe₂O₃ [52].

Figure 5. FTIR spectrum of prepared Fe₂O₃ NPs.

The fundamental process behind the formation of Fe₂O₃ NPs, encompassing particle nucleation and growth through plant extracts, remains largely elusive. Nonetheless, research suggests that phytochemicals, both primary and secondary metabolites present in plant extracts, are instrumental in the biosynthesis process. It is hypothesized that Amaranthus spinosus Linn leaf extract might not directly reduce Fe^{3 +} to Fe⁰. Instead, the organic constituents within the extract likely interact with iron ions, leading to the formation of iron oxide nanoparticles. This is particularly plausible considering that first-row transition metals are inherently susceptible to oxidation [53].

Analgesia produced by Fe₂O₃ NPs in mice chronic pain model

Figure 6 depicted the analgesic behaviour produced by Fe₂O₃ NPs through the effect of anti-allodynia (Figure 6(a), Fe₂O₃_5: 2.56 ± 0.01, Fe₂O₃_1.25: 0.82 ± 0.13, PBS: 0.29 ± 0.03 at 90 min) and anti-hyperthermia (Figure 6(b), Fe₂O₃_5: 5.37 ± 0.17, Fe₂O₃_1.25: 3.87 ± 0.18, PBS: 3.87 ± 0.13 at 90 min) in mice having severe inflammatory pain on injecting different drugs of 10 μL (Fe₂O₃_1.25/Fe₂O₃ _5, PBS) into lesion paw. These results indicated the dose-dependent analgesic effect of Fe₂O₃ NPs.

Figure 6. Mechanical (a) And thermal response (b) Studies for pain sensitivity recorded following von Frey microfilaments and radiant heat.

Reduced macrophage activity and inflammatory cell marker expression in the mice inflammatory skin produced by Fe₂O₃ NPs

On administration of Fe₂O₃ NPs, a reduction in inflammatory cell markers was observed. The immuno-histological staining signal for MPO expression (neutrophil marker) (PBS: 3.16 ± 0.32, naïve: 1.00 ± 0.06, Fe₂O₃ _5: 1.89 ± 0.20, Fe₂O₃ _1.25: 2.84 ± 0.33) (Figure 7) and CD68 (macrophage marker) (PBS: 3.07 ± 0.22, naïve:1.00 ± 0.10, Fe₂O₃ _5: 1.68 ± 0.09, Fe₂O₃ _1.25: 2.34 ± 0.20) (Figure 8) were inhibited upon injecting NPs into the lesion paw. Moreover, the MPO activity of whole skin was inhibited on administration of Fe₂O₃ into the lesion paw (PBS: 1.15 ± 0.06, naïve: 1.00 ± 0.05, Fe₂O₃ _5: 0.80 ± 0.06, Fe₂O₃ _1.25: 0.94 ± 0.06) (Figures 9, 10). However, it was reported in literature that Fe₂O₃ NPs have been shown to adversely affect macrophage viability, exhibiting a dose-dependent trend [54]. Given this precedent, it stands to reason that Fe₂O₃ NPs might also impede macrophage marker expression and functionality.

Figure 7. Immunostaining images showing the macrophage marker.

Figure 8. Immunostaining mean pixel intensity of CD68 expression.

Figure 9. Immunostaining images showing the MPO activity.

Figure 10. Immunostaining mean pixel intensity (a) and total skin MPO activity (b).

Fe₂O₃ supressed the production of total ROS

The complete amount of skin ROS generation was checked to further investigate the probable role of NPs in treatment of pain. It is found that a slight reduction in the ROS level on injecting NPs into the lesion paw (PBS: 0.97 ± 0.03, naïve: 1.00 ± 0.02, Fe₂O₃ _5: 0.82 ± 0.02, Fe₂O₃ _1.25: 0.84 ± 0.02) (Figure 11). Numerous research initiatives have highlighted the potential of Fe₂O₃ NPs to elevate ROS production. Naqvi and colleagues investigated macrophage (J774) cell culture with varying Fe₂O₃ NPs concentrations (25–500 μg/mL) resulting dose-responsive cell toxicity, yet there wasn’t a proportional increase in ROS production [55]. In the context of our study, it is plausible that minimal doses of Fe₂O₃ NPs could generate analgesia while mildly diminishing ROS generation. This observed impact might correlate with the inhibition of macrophage activity. On the other hand, a recent report also noted that Fe₂O₃ NPs safeguarded cultured cells across diverse stress scenarios, encompassing H₂O₂ exposure, emulating a catalase-like function [56]. In animal experiments, these Fe₂O₃ NPs contributed to the equilibrium of ROS, lessened intracellular oxidative stress, and curtailed cellular harm. Although these outcomes agree with our observations, they don’t elucidate the potentially unique mechanism in pain transmission and ensuing therapeutic interventions.

Figure 11. Chemiluminescence approach showing the suppression of ROS production after paw injection with Fe₂O₃ NPs.

Administration of Fe₂O₃ into the lesion paw altered the enzymes related to ROS generation

Blotting method was utilized to verify the effect of NPs on certain enzymes related to ROS-generation (p47phox and iNOS) (Figure 12). The expression of certain enzymes related to ROS generation was inhibited (p47phox, PBS: 0.99 ± 0.05, naïve: 1.00 ± 0.03, Fe₂O₃_5: 0.85 ± 0.02, Fe₂O₃_1.25: 0.87 ± 0.04, Figure 12(b)) (iNOS, PBS: 1.63 ± 0.14, naïve: 1.00 ± 0.11, Fe₂O₃ _5:1.19 ± 0.06, Fe₂O₃ _1.25: 1.21 ± 0.07, Figure 12(a)). The decline in ROS might result from the diminished generation or the stimulation of the ROS clearing mechanism. However, a potential mechanism could be that Fe₂O₃ NPs induced the analgesia in mice by inhibiting enzymes related to ROS production. Due to the complex enzyme system for ROS generation, the total enzyme system was not verified and hence only one probable mechanism was checked.

Figure 12. Western blot results representing the expression of (a) iNOS, (b) p47phox in paw skin.

Conclusions

In conclusion, the current study reported the synthesis of Fe₂O₃ NPs using leaf extract of Amaranthus spinosus Linn. The biomolecules present in the leaf extract act as stabilizing and reducing agents. X-RD patterns exhibited that the prepared Fe₂O₃ NPs were rhombohedral crystal structure with an average particle size of 10 nm. The Fe₂O₃ NPs showed a dose-dependent analgesic activity with considerable decrease in inflammatory cells, ROS production and pro-inflammatory markers in lesion tissue of the paw. The current study also revealed that an analgesic effect can be produced by a low dosage of Fe₂O₃ NPs. The outcomes of present work revealed the local administration of Fe₂O₃ NPs caused the substantial analgesia through reduction of pro-inflammatory signalling and inflammatory cellular infiltration along with micro-environmental free radical scavenging in mouse inflammatory pain model. Hence the prepared Fe₂O₃ NPs may exhibit the possible post-operative epidural anaesthetic applications in future.

Acknowledgments

Authors are thankful to Affiliated Hospital of Hubei University of Arts and Science for their support to do this research.

Disclosure statement

No potential conflict of interest was reported by the author(s).