3.1 HPLC Assay
High-performance liquid chromatography (HPLC) was used to quantify the hypohyllanthin in the in vitro and in vivo samples [28]. The HPLC system consists of a quaternary pump (Waters Delta Prep HPLC system, USA), a manual injector (Rheodyne, Cotati, CA, USA) equipped with a 100µL sample loop and a variable wavelength programmable photo diode array (PDA) detector (Waters 2998 PDA, USA). The system was equipped with the EMPOWER 2 software (Waters, Milford, MA, USA) for data acquisition and processing. Analyses was performed on C-18 column {(250mm×4.6mm; particle size 5µm) (Waters symmetry)} with methanol: water (66:34 v/v) as mobile phase flowing at 1.0 ml min− 1. The injection volume of 20 µL and absorption wavelength (λmax) of 225 nm was used.
3.1.1 Preparation of standard solutions
Hypophyllanthin stock solution was prepared by transferring 100 mg hypophyllanthin to a 100 mL volumetric flask containing 70 mL methanol. Then solution was sonicated for 10 minutes and finally made volume to 100 mL with methanol. A minimum of twelve standard solutions with concentrations ranging from 0.5 to 100 µg mL− 1 were prepared by diluting the stock solution appropriately with the mobile phase. The sample concentration was estimated by external calibration.
3.1.2 Preparation of samples
Blood samples (0.3–0.4 mL) were withdrawn from the retro-orbital plexus, and samples were collected into microcentrifuge tubes containing K2-EDTA (CML Biotech (P) Ltd, India).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 during sample preparation, and the method produced a consistent response. Then, 500 µL of methanol was added to precipitate the proteins, which were 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 100 µL methanol and vortexed. The supernatant was separated and analyzed for drug content by the validated HPLC method. Samples were analyzed on the day of preparation and were stored at -20°C until the analysis.
3.2. Preparation of conventional and PEGylated liposomes
Table 1
Composition of different formulations (conventional and pegylated liposomes) tested in this study
Formulation
|
Hypophyllanthin
(mg)
|
DSPC
(mg)
|
Cholesterol
(mg)
|
DSPE
MPEG2000
(mg)
|
Stearic
Acid
(mg)
|
Ratio of
Hypophyllanthin:
DSPC:
Cholesterol: DSPEMPEG2000:
Stearic Acid
|
HPL1
|
2.5
|
60
|
40
|
0
|
10
|
1:24:12:0:4
|
HPL2
|
5
|
60
|
40
|
0
|
10
|
1:12:8:0:2
|
HPL3
|
10
|
60
|
40
|
0
|
10
|
1:6:4:0:1
|
HPL4
|
5
|
70
|
30
|
0
|
10
|
1:14:6:0:2
|
HPL5
|
10
|
70
|
30
|
0
|
10
|
1:7:3:0:1
|
HPL6
|
5
|
60
|
40
|
5
|
10
|
1:12:8:1:2
|
HPL7
|
5
|
60
|
40
|
10
|
10
|
1:12:8:2:2
|
HPL8
|
5
|
60
|
40
|
15
|
10
|
1:12:8:3:2
|
Film hydration technique was used to synthesize the conventional and pegylated liposomes by using DSPC, cholesterol and DSPE-MPEG2000. Stearic acid concentration was kept constant in all the formulations and was added to impart the charge on surface of the vesicles. As mentioned in the Table 1 the different ratios of hypophyllanthin, DSPC, cholesterol, stearic acid and DSPE-MPEG2000 were mixed and chloroform/methanol (9:1 v/v) was used to dissolve each composite in a round-bottomed flask. The rotary evaporator (Buchi Rotavapor, Switzerland) was then connected to the flask and before starting the vacuum pump the flask with rotary was rotated at a rate of 40 rpm at 65°C for 25 minutes. By applying a very low vacuum the chloroform/methanol mixture was completely evaporated which lead to form a thin lipid film on the walls of the flask. Next step is to hydrate the lipid film and was performed by using with 10 mL of phosphate-buffered saline (PBS; pH 7.4) and vortexed (Vortex mixer, Genei Mumbai) for one hour at 65°C (approximately 10°C above the phase transition temperature (Tg) of DSPC). Large unilamellar vesicles were resulted and extruded 10 times through an extruder having 200 nm polycarbonate filter papers (Whatman, Denmark) at 65ºC above the Tg of DSPC to obtain small unilamellar vesicles (SUVs). Finally, the freeze drying was performed on the extruded SUV suspension to obtain the fine powder of liposomes.
3.2.1 Lyophilization cycle
Extruded liposomal suspension (5.0ml) was separated and placed in a 15 mL glass vials (Borosil, India) and freeze drying was performed by using 5% (w/v) mannitol as a cryoprotectant. Initially, the freeze dryer (Lyodel, India) was precooled to -40°C and later 0.5 ml mannitol solution was added to vials with liposomal formulation and then the temperature was decreased to -70°C at a rate of 0.5°C/min and the set temperature was held for 30 min. Then, for 10 hrs the chamber temperature and pressure were maintained at -16°C and 0.5 Torr, respectively. During secondary drying, the final temperature was set to + 20°C with a stepwise increment of -16°C, the holding temperature is for 2 hrs at each step. At last, after withdrawing the vials from the freeze dryer they were screw-crimped immediately[29–31].
3.2.2. Reconstitution of lyophilized liposomes
The lyophilized liposomes were reconstituted by gently shaking for 30 seconds in a phosphate-buffered saline (PBS; pH 7.4). After reconstitution, the appearance of the cake, vesicle size, size distribution (polydispersity index-PDI), encapsulation efficiency (EE), and in vitro drug release characterization was performed on freeze-dried formulations.
3.3. Evaluation of liposomes
The formulated liposomes were evaluated based on several parameters such as encapsulation efficiency (%EE), particle size, zeta potential, polydispersity index (PDI), X-ray diffraction (XRD), Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), differential scanning calorimetry and thermogravimetric analysis (DSC-TGA). Other studies like stability, toxicity and In vitro drug release were also studied. All animal experiments were performed in compliance with the institutional ethics committee regulations and guidelines on animal welfare.
3.2.1 Encapsulation efficiency (% EE) determination
The ultracentrifugation technique was used to determine the % EE of the liposomes. The liposome suspension of 2.0 ml was centrifuged at 25, 000 rpm at a controlled temperature of 4°C (Remi cooling centrifuge) for 2.0 hrs. The HPLC method as described in section 3.1 was used to determine free drug content that is withdrawn from supernatant. The % EE was estimated using Eq. 1. For each formulation, the procedure was repeated three times and the average of three replicates was reported.
$$\text{Encapsulation Efficiency (% EE) = }\frac{\text{Amount of drug in supernatant}}{\begin{array}{c}\text{A}\text{m}\text{o}\text{u}\text{n}\text{t}\text{ }\text{o}\text{f}\text{ }\text{d}\text{r}\text{u}\text{g}\text{ }\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\text{l}\text{y}\text{ }\text{t}\text{a}\text{k}\text{e}\text{n}\text{ }\\ \text{f}\text{o}\text{r}\text{ }\text{l}\text{i}\text{p}\text{o}\text{s}\text{o}\text{m}\text{e}\text{ }\text{p}\text{r}\text{e}\text{p}\text{a}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\end{array}}\text{ ×100 Eq: 1}$$
3.2.2 Size, size distribution, polydispersity index, and zeta potential analysis
The liposomal suspension was diluted 10 times with 0.1 M sodium chloride solution. DLS (dynamic light scattering) (Nano partica, Horiba Scientific, Japan) was used to determine the size and size distribution of liposomes by taking the average of 3 measurements. Also, zeta potential and 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
Transmission electron microscopy (Hitachi H-7500, Japan) was used to examine the liposomal formulation size, shape, and surface layers. 20 µL of the sample was deposited over carbon-coated copper grids with 200 mesh for 60 s and dried. Excess liquid was blotted from the grid with filter paper to form a thin film and finally stained with 2% uranyl acetate, air-dried, and examined under a transmission electron microscope.
3.3.4 FTIR analysis
FT-IR spectra of pure hypophyllanthin and liposomal formulations were recorded on a Bruker Spectrophotometer (Switzerland). The samples of interest were prepared as KBr discs by applying 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 hypophyllanthin, 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. 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. Samples were scanned from 25°C to 400°C at a rate of 10°C min− 1 under an N2 purge. Thermographs of pure drug, blank formulations, and selected lyophilized liposomal formulations were recorded using SDT Q600 V20.9 Build 20, TA Instruments, USA.
3.3.6 XRD analysis
The powder X-ray diffraction pattern of hypophyllanthin and selected liposomes in lyophilized powder forms was analyzed in the 2θ angle range of 5 to 89.99 using an XRD instrument (PANalytical XPERT-PRO, Netherlands).
3.4. In vitro dissolution studies
Dialysis membrane method has been employed to evaluate in vitro drug release for pure drug and liposomal formulations. Dialysis membrane (Himedia, molecular weight cut off 12,000) was washed under running water for 6 hrs to remove glycerol. Subsequently, sulfur from the membrane was removed by treating 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 by using 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 release media contains 0.1% Tween 80 to solubilize the released drug. The beakers were set at slow constant stirring and equilibrated to 37 ± 0.5 ºC. The activated dialysis membrane was filled with test products, and both ends of the membrane were sealed and suspended in the release medium (0.1 N HCl for 2 hrs followed by pH 7.4 phosphate buffer for 22 hrs). 2 mL of release medium was withdrawn from the beakers at regular intervals to estimate the concentration of hypophyllanthin by HPLC method (section 3.1), and the % cumulative drug release was calculated. All drug release studies were conducted in triplicate.
3.5. Stability of liposomal formulations
Selected freeze dried conventional and pegylated liposomal formulations containing hypophyllanthin (HPL2 & HPL7) were evaluated for their stability in different simulated GIT fluids (SGF, pH 1.2 and SIF, pH 6.8). 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 at pH 6.8. To simulate the effect of bile salts, 3 mM sodium taurocholate was added to the SIF.
The hypophyllanthin conventional liposomes (HPL2) and pegylated liposomes (HPL7) were reconstituted in PBS (pH 7.4) before use. One milliliter 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 in SGF and and 6 hrs in SIF. Then equal volume of 0.1 M ice-cold sodium hydroxide was added to SGF and 0.1 M HCl to SIF to inhibit the enzyme activity. The samples were then analyzed for vesicle size, PDI, zeta potential, and % encapsulation efficiency as described in section 3.3[31].
3.6. Toxicity Studies
Swiss mice (female) weighing 20–25 g (Mahaveer Enterprises, Hyderabad, India) were randomly divided into three groups of six. All formulations were administered orally at a dose of 1000 mg of hypophyllanthin per kilogram body weight. Group I served as the control group and was administered drug-free pegylated liposomes. Groups II and III were administered conventional liposome formulation (HPL2) and pegylated liposome formulation (HPL7), respectively. After 14 days of administration, blood samples from the retro-orbital plexus were collected in K2-EDTA microcentrifuge tubes. Blood samples were centrifuged at 3000 rpm for 5 min to separate plasma. The plasma was then analyzed for the levels of various markers. Nephrotoxicity was evaluated using the levels of Blood urea nitrogen (BUN), plasma urea, and plasma creatinine levels, while plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels to evaluate hepatotoxicity. Afore-mentioned biochemical parameters were determined by using an autoanalyzer (RM4000 Biochemical systems International, Italy) using diagnostic kits obtained from Span Diagnostics (Span Diagnostics Ltd., India).
3.7. Pharmacokinetic evaluation of hypophyllanthin liposomes
The pharmacokinetics studies were carried out in the Phytopharmacology laboratory, at the University college of Pharmaceutical Sciences, Andhra University which was approved by the institutional animal ethics committee and CPCSEA (Regd. No.516/PO/c/01/ CPCSEA) for experimentation on animals. The pure hypophyllanthin, hypophyllanthin Conventional liposomes (HPL2) and hypophyllanthin Pegylated liposomes (HPL7) were selected for pharmacokinetics study in sprague dawley rats. The selected formulations were administered orally at a dose equivalent to 5 mg per kg body weight of hypophyllanthin respectively, by oral gavage. The freeze-dried formulations were suitably reconstituted with saline to achieve the desired drug dose in 0.5 mL volume, which was administered to animals. The free hypophyllanthin was suspended in sodium CMC and administered to rats. The healthy Sprague dawley rats were acclimatized to standard environmental conditions (23 ± 2°C, 55 ± 5% relative humidity, 12 h light/dark cycle) for one week before administering the drug. The rats were divided into six groups of 4 each. Group I was administered pure hypophyllanthin, whereas groups II & III were administered reconstituted hypophyllanthin conventional liposomes (HPL2) and pegylated liposomes (HPL7), respectively at a dose of 5 mg/kg body weight. 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 the noncompartmental 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 (hr) plots using Kinetica software by applying a log-linear trapezoidal rule.