Preparation of novel DC-loaded phyto-elastosomes
The thin film hydration (TFH) technique has been commonly used for the development of bilayered nanovesicles [17,18,21,25] and thus was employed for this study. A schematic representation of the production the vesicles is displayed in Fig.1. In brief, different amounts of Tween® 80, CHOL and DCP were accurately weighed and kept in a round-bottomed flask (RBF) and dissolved with 10 mL chloroform-ethanol (1:1) mixture. The organic solvents were removed using a rotary evaporator under reduced pressure at a rotary speed of 100 rpm and temperature of 75°C resulting in the formation of a thin lipid film at the base of the RBF. The thin film was then hydrated with PBS containing DC at 100 rpm and 75°C leading to the formation of novel DC-loaded phyto-elastosomes. The dispersions were sonicated for 10 minutes in ice-cold Branson 8510 sonicator (Shelton, USA) and centrifuged for 90 minutes using Eppendorf 3154-C centrifuge (Hamburg, Germany) to separate the unbound harpagoside. The phyto-elastosomal dispersions were stored in vials at 4 ± 2°C. Noteworthy, DC-loaded vesicles were prepared using 30 mg of the extract viz. initial drug concentration was 3mg/mL.
The Critical Quality Attributes (CQAs) monitored were particle size (PS) and polydispersity index (PDI) using a Nano-ZS® Zetasizer (Worcestershire, United Kingdom) set in the Photon Correlation Spectroscopy mode and zeta potential (ZP), setting the zetasizer to Laser Doppler Anemometry mode. The entrapment efficiency (%EE) was determined using a validated reversed-phase HPLC method [26] and the deformability index (DI) using the extrusion method.
Screening studies
The elasticity of vesicular membranes is a unique and crucial parameter for deformable vesicles [21, 27]. The elasticity is based on the type and amount of edge activator(s) used in formulations [17–19]. Ethanol, used in high concentrations of > 10 % v/v is considered to be an effective elasticity enhancer for a number of vesicular systems such as ethoniosomes [28], invasomes [18], ethosomes [19] and elastic niosomes [21]. However, at high ethanol concentrations and beyond certain levels, the bilayers of the vesicles become leaky, which may significantly decrease %EE [29]. The ideal concentration of ethanol to be used throughout this study, was investigated using screening studies by varying the amounts of ethanol used in hydration medium by 5%. The formulation composition and manufacture parameters of the batches prepared during screening studies of ethanol is summarized in Table 1. The elasticity of the phyto-elastosomes was determined using the extrusion method [21, 30]. Phyto-elastosomal dispersions were extruded through a 50 nm polycarbonate membrane filter (Millipore, USA) at constant pressure of 1.5 bar for 10 minutes. The DI was then calculated using Eq. 1 [30, 31].
Where,
j = the weight of bilayered vesicle dispersions extruded
rv = the size of vesicle after extrusion
rp = the pore size of the filter membrane.
Table 1
Formulation parameters during screening of ethanol
Batch
|
Tween® 80 (µmole)
|
CHOL (µmole)
|
DC (mg/mL)
|
pH
|
Ethanol (% v/v)
|
1
|
80
|
20
|
0
|
7.0
|
0
|
2
|
80
|
20
|
0
|
7.0
|
20
|
3
|
80
|
20
|
3
|
7.0
|
0
|
4
|
80
|
20
|
3
|
7.0
|
5
|
5
|
80
|
20
|
3
|
7.0
|
10
|
6
|
80
|
20
|
3
|
7.0
|
15
|
7
|
80
|
20
|
3
|
7.0
|
20
|
8
|
80
|
20
|
3
|
7.0
|
25
|
9
|
80
|
20
|
3
|
7.0
|
30
|
10
|
80
|
20
|
3
|
7.0
|
35
|
11
|
80
|
20
|
3
|
7.0
|
40
|
The influence of pH on the entrapment of harpagoside was determined by screening the pH of hydration medium. Batches of both phyto-elastosomes and phyto-niosomes prepared by the THF method described above (preparation section) were hydrated using hydration medium of different pH. A summary of formulation parameters used during screening of the pH is shown in Table 2.
Table 2. Formulation parameters during screening of pH of hydration medium
Batch
|
Tween® 80 (µmole)
|
CHOL (µmole)
|
DC (mg/mL)
|
pH
|
Ethanol (% v/v)
|
12
|
80
|
20
|
3
|
7.0
|
0
|
13
|
80
|
20
|
3
|
7.0
|
20
|
14
|
80
|
20
|
3
|
6.5
|
0
|
15
|
80
|
20
|
3
|
6.5
|
20
|
16
|
80
|
20
|
3
|
6.0
|
0
|
17
|
80
|
20
|
3
|
6.0
|
20
|
18
|
80
|
20
|
3
|
5.5
|
0
|
19
|
80
|
20
|
3
|
5.5
|
20
|
20
|
80
|
20
|
3
|
5.0
|
0
|
21
|
80
|
20
|
3
|
5.0
|
20
|
The effect of Tween® 80, CHOL and DCP both the stability and %EE of the vesicles was investigated by developing phyto-elastosomes using different amounts of Tween® 80, CHOL effect and additives. The hydration medium used was a 20 % v/v ethanolic PBS (pH 7.0) solution with a DC concentration of 3 mg/mL. The amounts of excipients used during screening of formulation compositions is summarized in Table 3.
Table 3
Formulation parameters during screening of Tween® 80, CHOL and DCP
Batch
|
Tween® 80 µmole
|
CHOL µmole
|
DCP µmole
|
22
|
90
|
10
|
0
|
23
|
60
|
40
|
0
|
24
|
25
|
75
|
0
|
25
|
10
|
90
|
0
|
26
|
90
|
10
|
1
|
27
|
90
|
10
|
5
|
28
|
90
|
10
|
10
|
29
|
180
|
20
|
0
|
30
|
120
|
80
|
0
|
31
|
50
|
150
|
0
|
32
|
20
|
180
|
0
|
33
|
180
|
20
|
2
|
34
|
180
|
20
|
10
|
35
|
180
|
20
|
20
|
Topographical studies
The shape and surface morphology of phyto-elastosomes was studied using TEM. A drop of aqueous phyto-elastosomal suspension was placed onto a copper grid with a carbon film for approximately 30 seconds after which excess liquid was removed using Whatman® 110mm diameter filter paper (Maidstone, England). The grid was placed onto a carbon film and allowed to dry at a room temperature of 22°C overnight. The sample was then visualized using a Zeiss Libra® 120 TEM (Munich, Germany).
The Energy Dispersive X-Ray Scanning Electron Microscope (EDX-SEM) was used to determine the surface elemental compositions of raw DC and that of the novel DC-loaded phyto-elastosomes. Approximately 1 mg of sample was dusted onto a graphite plate and irradiated at an accelerated voltage of 20 kV using SEM. Liquid nitrogen was used to vaporize the sample. Elemental analysis was undertaken using a Vega® SEM (Vega LMU, Tuscan, Czechoslovakia Republic). The surface elements present in the sample were determined by measuring the number of x-rays emitted by the sample versus the energy of the rays.