1. Formulation, characterization and in vitro drug release/permeability study
1.1 Preparation of SNEDDS
The preparation of SNEDDS was performed according to the HLB-RSM approach developed by bahloul et al. [18] to encapsulate poor-water soluble drugs and the same excipients (olive oil, Tween 80, Span 85, PEG 300) were used. In order to set the quantitative composition of the formulation, one point from the optimum area of the corresponding HLB-RSM experimental design was chosen based on the demonstrated high efficiency and self-emulsification capacity of this area. TQ (2% w/w) was dissolved in the excipients mixture (24% olive oil, 44% Tween 80, 27% Span 85, 5%PEG 300), by constant stirring at 50°C until obtaining transparent formulation. The obtained SNEDDS+TQ will be visually inspected for TQ precipitation and phase separation before checking its self-emulsification capacity in comparison with Blank SNEDDS.
1.2 Characterization of SNEDDS formulation
1.2.1 Particle Size and Zeta potential of SNEDDS
Following measurements using the DLS, Malvern NanoS instrument (Malvern Instruments Ltd., Malvern, Worcestershire, UK), the particle size and polydispersity index (PDI) were obtained . We employed the Malvern Zetasizer Nano Z (Malvern Instruments Ltd., Malvern, Worcestershire, UK) equipment to measure the Zeta potential. The two formulations, SNEDDS without TQ (Blank SNEDDS) and SNEDDS with TQ (SNEDDS+TQ), were properly diluted to form clear self-emulsions and the measurements were done in triplicates at 25°C[19,20].
1.2.2 Thermodynamic stability of SNEDDS
The physical stability of the Blank SNEDDS and SNEDDS+TQ formulations was tested on the span of 48 hours in a heating-cooling system. Screw-cup glass tubes, containing 5g of the liquid formulations underwent 6 heating and cooling cycles at different temperatures ranging from 4° C to 45° C. As a result, the stability of the formulations was confirmed. These formulations were further studied using a centrifigution test (3500 rpm for 30 min) [21].
The morphology of Blank SNEDDS and SNEDDS+TQ was observed by transmission electronic microscopy (JEM-100S Electron Microscope Tokyo, Japan) as previously described [22]. Briefly, after dilution with 0.1% (1v/1v) distilled water, 5 µL was deposited on the copper grids of the electron microscope during 2 min. The excess of liquid was removed with a paper. After setting two grids for each sample, every one of these grids underwent negative staining using 2% uranyl acetate.
1.3 In vitro TQ dissolution/diffusion tests and TQ dosage
1.3.1 Drug dissolution
A release test of TQ from SNEDDS formula and TQ free drug suspension was accomplished using USP type II dissolution apparatus (Erweka DT-720, Germany) with paddle speed of 100 rpm. Samples (2 mL) were withdrawn at regular time intervals (0, 10, 20, 30, 45, 60 and 120 minutes) and diluted in the mobile phase (v/v ratio), filtration was performed using a 0.45 µm filter . The assays were carried-out in triplicates.
1.3.2 Drug diffusion
The permeability test of TQ from SNEDDS formula and TQ free drug suspension was carried-out using an absorption simulator (Sartorius SM 16750 GmbH, Germany) as previously described [23]. Briefly, the device is divided into donor and receptor compartments separated by a diffusion cell containing a nitrocellulose mixedester membrane (0.45 µm pore size, 47 mm diameter). The membrane was previously impregnated for 1h into caprylic acid and lauric alcohol (1v/1v ratio). The donor compartment was loaded with SNEDDS+TQ formulation. At various times (10, 20, 30, 45, 60 and 90 min), 1 mL of medium was removed from the receptor compartment. The drug absorption was calculated and expressed in percentage along with the apparent permeability coefficient according to the method of Lassoued et al. [24]. The test was performed in triplicates.
1.3.3 Dosage of TQ
The dosage of TQ was performed by HPLC (YL- instrument 9300 module, Anyang, Korea) using the method of Hosseinzadeh et al.[23], with some modifications. Briefly, the C18 column was used anda 1.5 mL/min flow rate was set. Water/methanol (30/70 v/v) was used as the mobile phase. The injection volume was 20 µL and the ultra-violet detection was at 254 nm. All analyzes were performed in triplicates at room temperature (25°C).
2. In vivo study
2.1 Animals
Male Wistar rats were used for the experiment weighing between 250g and 300g. Standard conditions (12/12h light/dar cycle, 22°C constant temperature) where they were housed were maintained. They were provided with adequate food and water access. All the procedures were performed in accordance with the European Union Regulations (Directive 2010/63/EU) for animal experiments and with the approval of the ethic committee on the research in life sciences and health of the Higher Institute of Biotechnology of Monastir (university of Monastir, Tunisia) (CER-SVS/ISBM-017/2020).
2.2 Surgical Procedure
Anesthesia using isoflurane inhalation was performed on the rats (1.5% isoflurane at a rate of 0.8 L/min). They were then placed in a heating pad to maintain their temperature at 37 degrees Celsius and underwent the same surgical procedures that were previously mentioned [15,16]. In brief, the hepatic triad was occluded with a microvascular clip for 60 min in order to induce a partial warm liver ischemia (70% of the liver mass). Perfusion was immediately restored by removal of the clip. After 24h of reperfusion, rats were sacrificed, and blood from the vena cava as well as ischemic tissue from the left and median liver lobes were gently collected. The tissue samples for histological study were stored in the 10 % formalin and those for biochemical and molecular determinations were stored at -80°C.
2.3 Experimental Groups
These rats were divided, randomly, into 5 groups, with n=6 in every group.Sham group: Rats were given an appropriate volume of saline per os for 10 days, they underwent hepatic triad dissection but no to I/R.
- I/R group: As Sham group, rats were given an appropriate volume of saline per os for 10 days. At the 10th day and 1h after their gavage, rats underwent a partial warm liver ischemia for 60 min, followed by 24h of reperfusion.
- Blank SNEDDS group: Same protocol as for the I/R group, but rats received per os the preparation of SNEDDS without TQ for 10 days.
- SNEDDS+TQ group: Same protocol as the Blank SNEDDS group, but the SNEDDS contained TQ (10 mg/Kg of body weight).
- Ethanol+TQ group: Same protocol as for the Blank SNEDDS group, but rats received per os TQ (10 mg/Kg of body weight) dissolved in ethanolic solution (pure ethanol and distilled water, 1/1 v/v ratio).
2.4 Biochemical assays
2.4 1 Determination of transaminase activities
The activities of aspartate aminotransferase (AST) and alanine aminotransferase(ALT) were evaluated as cytolysis markers [16].They were measured in plasma samples using commercial kits as per the manufacturer's instructions, at 340 nm with an ultraviolet spectrophotometer (Biomaghreb REF 40047, Tunisia).
2.4.2 Determination of oxidative stress parameters
● Superoxyde dismutase activity
In order to evaluate the total superoxide dismutase (SOD) activity according to Marklund & Marklund method, the liver samples were homogenized in ice-cold phosphate buffered saline (100 mM KH2PO4, 100 mM K2HPO4 and pH =7.4)
● Malondialdehyde concentration
the lipoperoxidation's end product is malanodialdehyde (MDA) . Hepatic samples were homogenized in ice-cold tris buffered saline (100 mM Tris, pH =7). Thiobarbituric acid reactive substances (TBARS) in hepatic tissue were measured, as previously stated, at 530 nm to determine the MDA concentration [24]. These results were expressed as nmol of MDA/mg of proteins.
2.4.3 Determination of inflammation parameters
● C-Reactive Protein assay
Following the manufacturer's instructions, a CRP assay kit was used to spectrophotometrically determine the concentration of C-reactive protein (CRP) in plasma at 600 nm (REF 969620, Beckman Coulter Inc., USA).
● Myeloperoxidase activity
Myeloperoxidase (MPO) is an enzyme that has both pro-inflammatory and pro-oxidant activities [25]. The activity of MPO was measured spectrophotometrically in tissue homogenates using the Krawisz et al. method at 630 nm and was expressed as U of MPO /mg of proteins [26].
2.5 Histopathological Study
Liver specimens collected from rats were immediately fixed in 10% PBS buffered formalin for 24-48 h, embedded in paraffin, sectioned at 4 µm thick and stained with standard Hematoxylin-Eosin (H&E). Stained tissue sections were randomly observed by an experienced pathologist who had no information about the treatment of groups using a light microscope. Specimens were evaluated for vascular congestion, neutrophil infiltration and necrosis. The liver histological changes were graded, according to Suzuki’s score, on a scale going from 0 to 4 as follows: (0) none, (1) minimal, (2) mild, (3) moderate and (4) severe[27].
2.6 Immunofluorescence Study
The inducible isoform of Nitric Oxide Synthase (iNOS) is upregulated in response to stress conditions and represents a good indicator on oxidative stress and inflammation during I/R injury [28]. Sections of paraffin-embedded hepatic tissue were used. They were deparaffinized in absolute xylene after being incubated at 60°C in the oven. Graded ethanol concentrations were used during the rehydration process. Sections were boiled in a citrate buffer after being washed with distilled water (0.01 M, pH 6). Following that, slides were washed three times with PBST (0.05% tween-20 in PBS, pH 7.4). To enable cell membrane permeation, sections were treated with Triton X 1% (1V Triton X 1%:9V PBS). Sections were treated with a blocking solution (10% goat serum with 1% bovine serum albumin in PBS) to stop the non-specific binding of antibodies. They were then treated with the primary antibody (1:250) mouse monoclonal iNOS overnight at 4 °C. (610329 BD Transduction Laboratories, United kingdom)
2.7 Quantitative Reverse Transcription PCR (RT-PCR) analysis
As per the manufacturer's instructions, the total RNA extraction required the homogenisation of 500 mg of each liver in 0.1 mL of trizol reagent (Invitrogen thermo Fisher scientific). Afterwards, the freshly obtained RNA isolates were quantified using a spectrophotometer of the NanoDrop brand (NanoDrop 2000, Thermo Fisher Scientific). Its reverse transcription was performed using an adequate reverse transcription kit (Moloney Murine Leukemia Virus Reverse Transcriptase, Invitrogen, Thermo Fisher Scientific). The use of a combination of adequate gene primers (Bio Basic, Inc., Canada) and SYBR Green PCR Master Mix (Thermo Fisher Scientific, Molecular Biology) allowed for the quantitative RT-PCR that was performed on the cDNA. These reactions were run on the Applied Biosystems RT-PCR systems. The implementation of a delta-delta CT method normalized gene expression to β-actin.
Table 1. Gene primers used for RT-PCR
Gene |
Forward Sequence |
Reverse Sequence |
β-actin |
5’-TACTCCTGCTTCCTGATCCACAT-3’ |
5’-TATGCCAACACAGTGCTGTCTGG-3’ |
TLR-4 |
5’-CCAGAGCCGTTGGTGTATCT-3’ |
5’-CAGAGCATTGTCCTCCCACT-3’ |
TNF-α |
5’-CTCTTCTCATTCCCGCTCGT-3’ |
5’- GGGAGCCCATTTGGGAACTT-3’ |
NFκB-p65 |
5’-ATCTTGAGCTCGGCAGTGTT-3’ |
5’-TCTGCTTCCAGGTGACAGTG-3’ |
IL-6 |
5’-CCAGTTGCCTTCTTGGGACT-3’ |
5’-TCTGACAGTGCATCATCGCT-3’ |
Bcl-2 |
5’- GAC TGAGTACCTGAACCGGCATC-3’ |
5’- CTGAGCAGCGTCT TCAGAGACA-3’ |
Bax |
5’-AGTCCTCACTGCCTCACTCACC-3’ |
5’-TTTC CCCGTTCCCCATTC-3’ |
Statistical analysis
Data were expressed as means ± standard error (SE). Statistical evaluation of data was performed using ordinary one-way analysis of variances (ANOVA) followed by Tukey post hoc test (GraphPad Prism V7 statistical software for windows). Difference was considered statistically significant when P < 0.05.