3.1. HPLC Assay
A validated high-performance liquid chromatography (HPLC) method was used to quantify the phyllanthin in the samples from in vitro and in vivo studies [4, 6, 17]. A quaternary gradient HPLC (Waters Delta Prep HPLC system, USA) system equipped with a Photo Diode Array (PDA) Detector (Waters 2998 PDA, USA) and a manual injector (Rheodyne, Cotati, CA, USA) with a 100 µL sample loop was used. HPLC method conditions were shown in Table 1.
Table 1
HPLC parameters for phyllanthin assay
Parameter
|
Required Setting
|
Elution mode
|
Isocratic
|
Mobile phase
|
Water to Methanol in the ratio of 34:66;
(Filtered and Degassed)
|
Sample diluent
|
Mobile phase
|
Column
|
Waters Symmetry C18 250 mm × 4.6 mm, 5 µm
|
Flow rate
|
1.0 mL/min
|
Injection Volume
|
20 µL
|
Column temperature
|
30°C
|
Detection wavelength (λ max)
|
225 nm
|
Run time
|
40 minutes
|
[Table 1: About Here]
3.1.1. Preparation of Standard Solutions
The stock solution of phyllanthin was prepared by transferring 100 mg phyllanthin in a 100 mL volumetric flask containing 80 mL methanol, sonicated for 10 minutes and the volume was made up with methanol. A minimum of twelve standard solutions with concentrations ranging from 0.25 to 50 µg/mL were prepared by diluting the stock solution appropriately with the mobile phase. Phyllanthin spiked plasma samples were prepared by mixing 1 mL blank plasma with appropriate volumes of the standard phyllanthin solutions (100 µL) on the day of analysis. A blank was also prepared to contain 1 mL blank plasma. After preparation, the samples were stored at -20°C till the time of analysis. The phyllanthin concentration in samples was determined by the HPLC-UV method on the same day to avoid any degradation.
3.1.2 Preparation of Samples
The blood samples (0.3–0.4 mL) were withdrawn from the retro-orbital plexus and samples were collected into microcentrifuge tubes containing K2-EDTA (CML Biotech (P) Ltd, India). Then the samples were centrifuged at 4000 rpm for 5 min at 15˚C using a cold centrifuge (Remi Equipment Pvt. Limited, India). To 125 µL of plasma, 25 µL of internal standard (carbamazepine stock solution 10 µg mL− 1 in methanol) was added and then vortexed (vortex mixer, Genei, Mumbai) for 60 seconds. The internal standard was added to ensure no analyte loss while sample preparation and the method produces a consistent response. Then 500µL of methanol was added to precipitate proteins and vortexed for 5 min and centrifuged at 5000 rpm for 10 min. The supernatant was taken and dried in a vacuum oven at 40˚C. Dried samples were then redispersed in 100µL methanol and vortexed. The supernatant was separated and analyzed for drug content by the validated HPLC method.
1.2. Preparation of Conventional and Pegylated Liposomes
[Table 2: About Here]
Table 2
Composition of different formulations (conventional and pegylated liposomes) tested in this study
Formulation
|
Phyllanthin
(mg)
|
DSPC
(mg)
|
Cholesterol
(mg)
|
DSPE
MPEG2000
(mg)
|
Stearic
Acid
(mg)
|
Ratio of
Phyllanthin:
DSPC:
Cholesterol: DSPEMPEG2000:
Stearic Acid
|
PHL1
|
2.5
|
60
|
40
|
0
|
10
|
1:24:12:0:4
|
PHL2
|
5
|
60
|
40
|
0
|
10
|
1:12:8:0:2
|
PHL3
|
10
|
60
|
40
|
0
|
10
|
1:6:4:0:1
|
PHL4
|
5
|
70
|
30
|
0
|
10
|
1:14:6:0:2
|
PHL5
|
10
|
70
|
30
|
0
|
10
|
1:7:3:0:1
|
PHL6
|
5
|
60
|
40
|
5
|
10
|
1:12:8:1:2
|
PHL7
|
5
|
60
|
40
|
10
|
10
|
1:12:8:2:2
|
PHL8
|
5
|
60
|
40
|
15
|
10
|
1:12:8:3:2
|
Conventional and Pegylated liposomes containing phyllanthin were prepared using DSPC, Cholesterol and DSPE-MPEG2000 by film hydration technique. Stearic acid was added to impart charge on the surface of the vesicles and its concentration was kept constant in all the formulations. Different ratios of phyllanthin, DSPC, Cholesterol (CHOL), stearic acid and DSPE-MPEG2000 were mixed according to Table 2. Each composite was dissolved in chloroform/methanol (9:1 v/v) in a round-bottomed flask. The flask was then connected to a rotary evaporator (Buchi Rotavapor, Switzerland) and was rotated at a rate of 40 rpm at 65°C for 25 minutes before starting the vacuum pump.
Then Chloroform/methanol mixture was completely evaporated by applying a very low vacuum which left a thin lipid film on the walls of the flask. The lipid film was then hydrated at 65ºC (approximately 10ºC above the phase transition temperature (Tg) of DSPC) with 10 mL of phosphate-buffered saline (PBS; pH-7.4) and vortexed (Vortex mixer, Genei Mumbai) for one hour. This resulted in large unilamellar vesicles (LUVs) being extruded 10 times through an extruder having 200 nm polycarbonate filter papers (Whatman, Denmark) at 65ºC above Tg of DSPC to obtain small unilamellar vesicles (SUVs). The extruded SUV suspension was then freeze-dried to obtain a fine powder of liposomes.
3.2.1 Lyophilization Cycle
The 5 mL of each extruded liposomal suspension was separately filled in 15 mL glass vials (Borosil, India) and subjected to freeze-drying using 5% (w/v) of mannitol as Cryoprotectant. The Freeze dryer (Lyodel, India) was pre-cooled to -40°C. 0.5 mL mannitol solution was added to all vials with liposomal formulation and the temperature was decreased at a rate of 0.5°C/min to -70°C and held at this temperature for 30 min. Then chamber pressure was decreased to 0.5 Torr and temperature was maintained at -16°C for 10 hrs. During secondary drying, the temperature was increased in a stepwise manner up to -16° C and to a final temperature of + 20°C, holding the temperature for 2 hrs at each step. Finally, the vials were screw-crimped immediately after withdrawing from the freeze dryer [15, 16, 18].
3.2.2. Reconstitution of lyophilized liposomes
The lyophilized liposomes were reconstituted in phosphate-buffered saline (PBS; pH-7.4) by gentle shaking for 30 seconds. After reconstitution, the freeze-dried formulations were characterized for the appearance of the cake, vesicle size, size distribution (Polydispersity index-PDI), Encapsulation Efficiency (EE), and In-vitro drug release.
3.3. Evaluation of Liposomes
Several parameters were measured to evaluate the formulated liposomes such as Encapsulation Efficiency (%EE), Particle Size, Zeta Potential, Poly Dispersity Index (PDI), X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy, Transmission Electron Microscopy (TEM), Differential Scanning Calorimetry and Thermogravimetric analysis (DSC-TGA). In-Vitro drug release, toxicity, and stability studies were also performed. All animal experiments were performed in compliance with the institutional ethics committee regulations and guidelines on animal welfare.
3.3.1 Encapsulation Efficiency (% EE) Determination
The % EE of liposomes was determined by using the ultracentrifugation technique. 2mL of liposomes suspension was centrifuged at 25,000 rpm for two hours at a controlled temperature of 4°C (Remi cooling centrifuge). The supernatant containing free drug was withdrawn and estimated by the HPLC method described in section 3.1. The % EE was estimated using Eq. 1. This procedure was repeated three times per formulation and average of three replicates was reported.
$$\text{Encapsulation Efficiency (\% }\text{EE}\text{) }\text{= }\frac{\text{Amount of drug in supernatant}}{\begin{array}{c}\text{A}\text{m}\text{o}\text{u}\text{n}\text{t}\text{ }\text{o}\text{f}\text{ }\text{d}\text{r}\text{u}\text{g}\text{ }\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\text{l}\text{y}\text{ }\text{t}\text{a}\text{k}\text{e}\text{n}\text{ }\\ \text{f}\text{o}\text{r}\text{ }\text{l}\text{i}\text{p}\text{o}\text{s}\text{o}\text{m}\text{e}\text{ }\text{p}\text{r}\text{e}\text{p}\text{a}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\end{array}}\text{ ×100}\text{ }\text{Eq: 1}$$
3.3.2 Size, Size Distribution, polydispersity index, and Zeta Potential Analysis
The liposomal suspension was diluted 10 times with 0.1 M sodium chloride solution. The size and size distribution of liposomes was determined using DLS (dynamic light scattering) (Nano partica, Horiba Scientific, Japan), by taking the average of 3 measurements, whereas zeta potential was estimated based on electrophoretic mobility under an electric field, as an average of 3 measurements. The polydispersity index (PDI) which is a dimensionless number indicating the width of the size distribution was also measured.
3.3.3 Shape and Surface Morphology by TEM
The particle size, shape, and surface layers of the optimal liposomal formulations were examined by transmission electron microscopy. 20µL of the sample was deposited over carbon-coated copper grids with 200 mesh for 60 s and dried. Excess dispersion was blotted from the grid with filter paper to form a thin-film specimen. Then they were stained with 2%uranyl acetate, air-dried, and examined under a transmission electron microscope (Hitachi H-7500, Japan).
3.3.4 FTIR Analysis
FT-IR spectra were recorded on a Bruker Spectrophotometer, Switzerland. Phyllanthin, a pure drug, and its liposomal formulations were prepared in KBr discs prepared at a hydrostatic pressure of 5 tones cm − 2 for 2 mins. The scanning range was 400–4000 cm− 1 with a resolution of 1 cm− 1. The FT-IR spectra of pure phyllanthin, DSPC, cholesterol, DSPEMPEG2000, stearic acid, mannitol, and their corresponding selected lyophilized liposomal formulation were compared.
3.3.5 DSC-TGA Analysis
DSC-TGA analysis was performed to study the thermal stability and changes in crystallinity over a range of temperatures. Thermographs of pure drug, blank formulations, and selected lyophilized liposomal formulations were recorded using SDT Q600 V20.9 Build 20, TA Instruments, USA. A known mass of sample (4–6 mg) was transferred to an aluminum pan and crimped sealed with a lid. An empty aluminum pan sealed with a lid was used as a reference sample. Samples were scanned from 25°C to 400°C at a rate of 10°C min− 1 under an N2 purge.
3.3.6 XRD Analysis
The powder X-ray diffraction pattern of phyllanthin and selected liposomes in lyophilized powder forms was recorded using an X-RD instrument (PANalytical XPERT-PRO, Netherlands). The selected samples were analyzed in the 2Ө angle range of 5 to 89.99.
3.4. In vitro Dissolution Studies
The in vitro drug release for pure drug and liposomal formulations was performed using the dialysis membrane method. Before use, the dialysis membrane (Himedia, molecular weight cut off 12,000) was activated by washing the membrane under running water for 6 hrs to remove glycerol. Subsequently, sulfur was removed by treatment with 0.3% sodium sulfide for 1 min, followed by acidification (0.2% sulfuric acid) of the membrane. Finally, it was washed with water repeatedly to remove the excess acid. The in-vitro diffusion studies were carried out in two 500 mL beakers one containing 250 mL of 0.1N HCl (pH 1.2) and the other with 250mL of phosphate buffer (pH 7.4). Both the buffer systems contain 0.1% Tween 80 to solubilize the released drug and were equilibrated to 37 ± 0.5 ºC. The dialysis membrane was filled with test products, and both ends of the membrane were sealed. The dialysis membrane containing liposomes was suspended in the release medium (0.1 N HCl for 2 hrs followed by pH 7.4 phosphate buffer for 22 hrs) which was under slow constant stirring. The concentration of phyllanthin in the release medium was determined by withdrawing 2 mL of the sample at regular intervals for 24 hours and immediately replaced with a respective medium by the same quantity. The samples were analyzed using the HPLC method (section 3.1) and % cumulative drug release was calculated. All the drug release studies were conducted in triplicate.
3.5. Stability of Liposomal Formulations
The stability of selected freeze-dried conventional and pegylated liposomal formulations containing phyllanthin in different simulated Gastrointestinal fluids (Simulated Gastric Fluid (SGF), pH 1.2, and Simulated Intestinal Fluid (SIF), pH 6.8) was evaluated. The simulated gastric fluid (SGF) contains 0.2% NaCl, pepsin, 0.7%, and HCl with a pH of 1.2. Simulated intestinal fluid (SIF) consists of 0.685 M monobasic potassium phosphate, 1% NaOH, and 1% pancreatin with pH 6.8. To simulate the effect of bile salts, 3 mM sodium taurocholate was added to the SIF.
The Phyllanthin conventional liposomes (PHL2) and pegylated liposomes (PHL7) were reconstituted with the required volume of PBS (pH 7.4) before use. 1mL of each reconstituted formulation was added to 9 mL of each simulated GI fluid. The samples were incubated in a water bath at 37 ± 1°C and shaken at 100 rpm for 2 hrs and 6 hrs in SGF and SIF, respectively. The enzyme activity in the samples was completely inhibited by the addition of an equal volume of 0.1 M ice-cold sodium hydroxide to SGF and 0.1 M HCl to SIF. The samples were then evaluated for vesicle size, PDI, zeta potential, and % encapsulation efficiency as described in section 3.3 [18].
3.6. Toxicity Studies
Swiss mice (female) weighing 20-25g (Mahaveer enterprises, Hyderabad, India) were randomly divided into three groups (6 mice per group). All formulations were administered orally at a dose of 1000 mg of phyllanthin/kg body weight. Group I served as the control group and was administered drug-free pegylated liposomes, Group II and III were administered phyllanthin-loaded conventional liposomes (PHL2) and phyllanthin-loaded pegylated liposomes (PHL7) respectively. After 14 days of the administration, blood samples from the retro-orbital plexus were collected in K2-EDTA microcentrifuge tubes and centrifuged at 3000 rpm for 5 min to separate plasma. The plasma was then analyzed for the levels of various markers. Blood urea nitrogen (BUN), plasma urea, and plasma creatinine levels were determined for evaluating nephrotoxicity while plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels were determined for evaluating hepatotoxicity. All the biochemical parameters were determined by using an autoanalyzer (RM4000 Biochemical systems International, Italy) according to the manufacturer’s guidelines for the diagnostic kits obtained from Span diagnostics (Span diagnostics LTD., India).
3.7. Pharmacokinetic Evaluation of Phyllanthin Liposomes
Healthy Sprague Dawley rats of either sex weighing about 220–250 g were procured from Mahaveer enterprises, Hyderabad. The animals were acclimatized under standard environment conditions (23 ± 2ºC, 55 ± 5% relative humidity, 12 hrs light/ dark cycle) for one week. The pure phyllanthin and selected liposomal formulations (PHL2 & PHL7) were administered orally at a dose equivalent to 5 mg/kg body weight. The freeze-dried formulations were suitably reconstituted with saline to achieve the desired drug dose in 0.5mL volume, which was administered to animals and the pure drug was suspended in the suspension of Sodium CMC and administered to animals. Group I was administered with pure phyllanthin whereas groups II & III were administered reconstituted phyllanthin conventional liposomes (PHL2) and pegylated liposomes containing phyllanthin (PHL7) respectively. The blood samples (0.3 mL) were withdrawn from the retro-orbital plexus at 0, 0.5, 1, 2, 4, 8, 12, 24, and 36 hours after oral administration. The blood samples were prepared as described in section 3.1.2 and analyzed for drug content by the validated HPLC method. The plasma concentration over time was analyzed by non-compartmental extravascular method, using Kinetica software (Thermo scientific USA). The mean pharmacokinetic parameters (AUC0→∞, AUMC0→∞, Cmax, Tmax, t1/2, Kel, MRT, Cl, and Vz) were calculated from plasma drug concentration (ng·mL− 1) - time (hrs) plots using Kinetica software by applying a log-linear trapezoidal rule. This study is reported in accordance with ARRIVE guidelines. The experiment was performed in accordance with relevant guidelines and regulations.