2.1. Materials
Polysorbate 60 (Tween®60), Sorbitan monostearate (Span®60), Dicetyl phosphate (DCP), Chitosan (Low molecular weight), cholesterol (Chol), heparin, dialysis bag (MWCO = 12000 Da (, and Cn were purchased from Sigma-Aldrich (Germany). Moreover, Absolute ethanol, glycerol, carboxymethylcellulose, and methanol were purchased from Merck (Germany). All these materials were analytically graded. Normal saline was obtained from Shahid Ghazi (Iran). Ketamine and Xylazine were obtained from Alfasan (Netherland).
2.2. Mixture Design (M.D.)
In this study, with the aim of altering the formulations, M.D. with three-level, three-factor and seven experimental runs were adopted. Table 1 shows the independent and dependent variables as well as their levels.
Table 1
The independent and dependent variables of MD with statistical parameters.
Independent Variables (Factors)
|
Levels
|
Units
|
Low
|
High
|
Span 60 concentration
|
0.06
|
0.28
|
G
|
Tween 60 concentration
|
0.06
|
0.28
|
G
|
Chol concentration
|
0.06
|
0.28
|
G
|
Dependent variables (Response)
|
Units
|
|
Goal
|
Mean vesicle size
|
Nm
|
|
Minimize
|
Poly disperse index (PDI)
|
Value
|
|
Minimize
|
Zeta potential
|
mV
|
|
Maximize
|
Entrapment
|
%
|
|
Maximize
|
2.
3. Vesicle preparation
Different niosome formulations, (Table 2), were synthesized in terms of the heating method explained by Mozaffari (2008) (Mozafari et al. 2008) and Rajabzadeh (2017)(Basiri et al. 2017a) with some modifications. Briefly, to hydrate the appropriate amounts of surfactants applying phosphate- buffered saline (PBS, pH 7.4, autoclaved), they were stirred for 60 min at 25°C. Afterward, the Chol dispersion was vigorously stirred for 30 min at 120°C on a hotplate.
Table 2
MD in the process of niosome preparation and physicochemical characteristics of Cn loaded noisome (mean ± SD, n = 3).
Sample
|
Independent Variables
|
Dependent variables
|
Span 60
|
Tween 60
|
Cholesterol
|
Z average (nm)
|
PDI (value)
|
Zeta potential (mV)
|
EE% (Cn)
|
NHM1000
|
0.134
|
0.133
|
0.133
|
111 ± 0.32
|
0.188 ± 0.002
|
-30 ± 0. 8
|
75 ± 0.458
|
NHM1100
|
0.206
|
0.097
|
0.097
|
128 ± 0.32
|
0.251 ± 0.015
|
-26 ± 0.2
|
33 ± 0.1
|
NHM1200
|
0.06
|
0.28
|
0.06
|
70 ± 0.44
|
0.21 ± 0.01
|
-23 ± 0.1
|
88 ± 0.42
|
NHM1300
|
0.097
|
0.201
|
0.097
|
97 ± 0.32
|
0.182 ± 0.002
|
-26 ± 0. 1
|
60 ± 0.22
|
NHM1400
|
0.097
|
0.097
|
0.206
|
153 ± 0.43
|
0.37 ± 0.001
|
-27 ± 0.3
|
90 ± 0.23
|
NHM1500
|
0.28
|
0.06
|
0.06
|
117 ± 0.43
|
0.30 ± 0.003
|
-36 ± 0.2
|
64 ± 0.31
|
NHM1600
|
0.06
|
0.06
|
0.28
|
125 ± 0.46
|
0.140 ± 0.001
|
-30 ± 0.6
|
32 ± 0.12
|
The obtained colloid was added to a preheated (5 min, 60°C) mixture of Cn (100 µM(Sharma et al. 2015b)), glycerol (final concentration of 3%, v/v), DCP (DCP: Surfactant, 0.1 molar ratio) and ethanol (1 cc), which were stirred ( at 60°C, 1000rpm) on a hotplate stirrer (IKA®C. MAG HS7 Safety Control, IKA, Malaysia) for 60 min. The reaction was performed under a nitrogen atmosphere in a handmade glass vessel introduced by Mozaffari. Subsequently, sonication of the sample was performed using a probe sonicator (Sonopuls HD-3100, BANDELIN electronic GmbH & Co. Germany) for 16 min (180 sec “pulse on” and 30 sec “pulse off”). After the preparation of the loaded niosome, they were kept for 30 min at room temperature.
2.4. Preparation of Chitosan Coated CLN (CH-CLN).
The CH-CLNs were prepared according to Marianecci with some modifications(Rinaldi et al. 2020). At first, CH-CLNs were made by the addition of optimum formulation of CLNs into CH solution (1:2, v/v). The mixture was then stirred for 60 min at 25°C. Thereafter, the obtained mixture was adjusted to pH 4.5 (NaOH, 1M), and then sonication of the sample was performed using a probe sonicator for 5 min (180 sec “pulse on” and 30 sec “pulse off).
2.5. Cn Determination
The Cn measurement was performed at 40°C on a High-performance liquid chromatography (Waters, USA). The HPLC system contained a Waters 1525 binary pump, and 2489 Uv-Vis detectors at 420 nm. Also, a reversed-phase Inertsustian Swift C18 column (4.6 × 250 mm, particle size 5 µm) was used for HPLC analysis. The mobile phase was composed of 5 % acetonitrile solution in water buffered to pH 2.7 by 10 % ortho-phosphoric acid (90:10 v/v) at a flow rate of 1 ml/min.
The stock solution was prepared by the dissolution of 5mg Cn in 2.5 ml of methanol (2000 µg/mL). The obtained solution was then diluted by the mobile phase to make a serial concentration of the working standard solution (0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 µg/mL). All the solutions were stored at 4°C in amber glassware and then sonicated for 15 min before usage.
The limit of detection (LOD) and the limit of quantification (LOQ) were 0.186712 and 0.565794, respectively.
2.6 Encapsulation efficiency
CLN and CH-CLN suspensions (5ml) were loaded in a centrifuge Amicon MPS (Millipore, USA) filtration tube and then centrifuged at 6000 rpm (Hettich centrifuge, model EBA 20, Germany) to separate niosomes from the unloaded Cn. In this regard, the following equation was used to calculate the Cn encapsulation efficiency(Moghddam et al. 2016).
EE%= (total Cn-Cn in supernatant)/ (total Cn) ×100
2.7 Size, polydispersity index and zeta potential analysis
The sample sizes, polydispersity index (PDI) and zeta potential of diluted CLN and CH-CLN (1:10 v/v) were measured using dynamic light scattering method (DLS) on Zeta sizer Nano ZS (Malvern Instruments Ltd., United Kingdom) by a helium-neon laser at 630 nm at room temperature(Kassem et al. 2017).
2.9. Fourier Transform Infrared (FT-IR) spectroscopy
The functional groups of the samples’ components were studied by FT-IR (Thermo Nicolet, AVATAR, 370 FT-IR, USA). The samples were scanned with a resolution of 4 cm− 1 between 4000 cm− 1 and 400 cm− 1.
2.11. Transmission electron microscopy (TEM)
TEM image of CLN and CH-CLN have been taken by utilized 2% uranyl acetate solution as a staining agent (Leo 912 Omega TEM ,Germany).
2.13. Cn release studies
The release of Cn was accomplished using the dialysis technique(Le and Kim 2019), (Xu et al. 2016). 5 mL of sample were wrapped in a dialysis bag (D0666, Sigma). In order to immerse the dialysis bag, 50 mL of a gastric fluid (SGF, HCl solution 0.1 N, pH = 1.2) and intestinal fluid (SIF, PBS, pH 7.4) have been simulated for 2 and 6 h respectively in a digital shaker incubator (Hanyang, SI-64A, 50 rpm, 37°C). Accordingly, both matrices contained 0.1% (v/v) Tween80.
To evaluate the kinetic behavior and release mechanism, the result derived from release studies was fitted into different mathematical equations as demonstrated in Table 4.
Table 4
In vitro release of Cn in niosome and chitosan coated niosome R2 and k values.
Function
|
R2
|
K
|
CLN
|
CH-CLN
|
CLN
|
CH-CLN
|
Zero order
|
0.88
|
0.88
|
5.75
|
1.57
|
First order
|
0.92
|
0.92
|
0.125
|
0.6
|
Higuchi
|
0.95
|
0.97
|
16.25
|
4.44
|
Hixson-Crowell
|
0.96
|
0.90
|
0.44
|
0.02
|
2.14 Animal study
2.14.1 Animals
Wistar rats (12 male individuals, 190 g ± 10g in body weight) were obtained from house breeding colonies at the Neuroscience Department of Mashhad University of Medical Sciences for drug control. Afterward, they were kept under standard conditions (12h light and 12h dark cycle, 23 ± 1°C, with easy access to food and water sources). Animal handling and all the other related procedures were approved by Medical Sciences, Ethical Committee Acts (Mashhad, Iran, IR.mums.Rec.1399.287). The minimum number of animals was used to respect animal rights.
2.14.2 Drug administration and accessing its bioavailability
The rats were randomly divided into the following three groups (4 rats in each group): (1) treated with free Cn (group I), (2) treated with CLN (group II), and (3) treated with CH-CLN (group III).
0.5%w/v aqueous solution of carboxymethylcellulose (CMC) was then applied for preparing free Cn solution(Hoppe et al. 2013). Subsequently, the 50 mg/kg of freshly Cn suspension, CLN, and CH- CLN were administered by intraperitoneal injection ( i.p.) to the rats(Tsai et al. 2011). By passing 15 minutes from the injection, the experimental rats were anesthetized by a mixture containing 10% ketamine (100 mg/kg body weight) and 2% xylazine (30 mg/kg body weight) (Tiwari et al. 2014). After anesthetizing, the cardiac puncture was utilized to collect 400 µl blood with a heparinized syringe. Thereafter, the rats were perfused with normal saline to remove their brain and liver. Moreover, to evaluate crossing the BBB, the cerebral cortex, hippocampus, cerebellum, and striatum were collected.
Afterward, the samples were stored in microtubes at -80°C. Cn was extracted from plasma, and different regions of the brain and liver according to the protein precipitation technique proposed by Ravi (2018) with some modifications(Dalvi et al. 2018). Briefly, 400 µL methanol was added to 90 µL Plasma. The mixture was then vortexed for 2 min. Subsequently, the sample was centrifuged at 14000g for 20 min at 4°C. Following that, the supernatant was dried under N2 gas at 40°C. Finally, 100 µL mobile phase was added for reconstitution, which was then analyzed by high-performance liquid chromatography (HPLC, Waters equipped with pump1525 binary pump along with UV2485and FLD2475 detector, USA).
Different regions of brain and liver tissues were homogenized using a probe sonicator for 1min (25son 15 s off) at 4°C in PBS (1:4 W/V, pH: 7.4). Next, the mixtures were centrifuged at 14000 g for 20 min at 4°C. Then, 100 µL of supernatant was moved to a new microtube, and 200 µL of methanol was added to it. After 2 minutes of vortexing, the samples followed the producer same as a plasma sample.
2.15 Statistical data analysis
In this study, all data were presented as mean ± standard deviation. The responses obtained from experiments were analyzed using the software Mini Table 18. Also, the possible mathematic models were analyzed using ANOVA one way. The best-fitting model was selected for each response-based on P values. Also, P values of < 0.05 were considered as statistically significant. All figures were sketched using the Microsoft Excel version 2016.
The experiments were conducted in triplicate with the obtained results presented as a mean ± standard deviation.