Journal of Materials Chemistry B
Paper
8
2
2
1
4.24, 84.20, 73.1, 61.3, 52.9, 45.6, 45.5, 38.0, 37.8, 36.6, 31.8, stretched appropriately and the lower fluid cell was filled with
9.8, 29.2, 29.1, 25.7, 22.7, 14.2; IR (KBr): 3426, 3057, 2930, the electrolyte. On applying voltage across the electrodes, ions
861, 1780, 1746, 1708, 1663, 1516, 1429, 1337, 1266, 1190, move between the cells and give a base line current. After
À1
115, 1010, 898, 820, 744 cm ; HRMS (ESI) exact mass calcd calibration with particles of a standard size, the sample
+
+
for C H N O (M + H) 517.2550, found (M + H) 517.2561.
(40 mL, prepared in PBS) was loaded onto the upper fluid cell.
As the aggregates from the sample move through the nanopores,
there would be a temporary decrease in current which can be
2
7
37 2 8
1
f 3
Compound 1d. Yield, 89%; R (EtOAc), 0.44; H NMR (CDCl ,
4
4
3
2
1
0
00 MHz): d 6.04 (d, 1H, J = 8.8 Hz), 4.66 (s, 1H), 4.60 (s, 1H), measured. It takes particle by particle measurements, and gives
.48–4.41 (m, 2H), 4.14–4.10 (m, 1H), 3.98 (d, 1H, J = 6.0 Hz), information on the particle size distribution in the samples.
.94 (d, 1H, J = 5.6 Hz), 3.86–3.79 (m, 1H), 3.75–3.70 (m, 1H),
A morphological study of niosomes was done using HR-SEM
.85 (s, 2H), 2.24 (sext, 2H, J = 4 Hz), 2.10 (sext, 1H, J = 6.4 Hz), and TEM methods. For SEM, the samples were diluted with
.65–1.55 (m, 2H), 1.35–1.20 (m, 8H), 0.91 (d, 3H, J = 6.8 Hz), water or an appropriate solvent, drop-cast onto a piranha-treated
1
3
.88–0.84 (m, 6H), –2OH protons did not appear; C NMR silica substrate, air dried and sputter-coated before analysis. For
, 100 MHz): d 176.1 (2C), 174.1, 171.7, 84.3, 84.2, 73.1, HRTEM, the samples were drop-cast onto copper grids and then
3.0, 60.9, 56.9, 45.6, 45.5, 38.0, 36.9, 31.8, 31.2, 29.8, 29.3, 29.1, stained with 1% uranyl acetate.
(CDCl
3
7
2
2
5.8, 22.7, 19.2, 17.7, 14.2; IR (KBr): 3681, 3299, 3056, 2928,
To get a deeper understanding on the native state of
860, 2360, 2312, 1709, 1518, 1429, 1266, 1193, 1009, 897, 746 these aggregates, CTEM analysis was also performed on one
À1
+
cm ; HRMS (ESI) exact mass calcd for C23
H
37
N
2
O
8
(M + H)
of the samples (NC1c). For this, diluted samples were placed on
the holey polymer coated TEM grid and bolted to form a thin
sample layer. After attaining thermal fixation by flash freezing
+
469.2550, found (M + H) 469.2570.
1
Compound 1e. Yield, 95%; R (EtOAc), 0.5; H NMR (CDCl , into liquid ethane, these grids were transferred to a cryoTEM
f
3
4
4
00 MHz): d 5.95 (d, 1H, J = 8.8 Hz), 4.66 (s, 1H), 4.60 (s, 1H), grid holder previously cooled and maintained in liquid nitrogen.
.55 (dt, 1H, J = 9.2, 4.8 Hz), 4.41–4.35 (m, 1H), 4.19–4.14 Subsequently, these grids were transferred to CTEM for
(
m, 1H), 3.97 (q, 2H, J = 6 Hz), 3.84–3.61 (m, 2H), 2.85 (d, 2H, examination at a voltage of 100 kV.
J = 1.6 Hz), 2.21 (dt, 2H, J = 7.2, 4.0 Hz), 1.65–1.55 (m, 4H), 1.44 Drug entrapment and drug release studies. The suspension
sext, 2H, J = 9.2 Hz), 1.35–1.20 (m, 8H), 0.93 (d, 6H, J = 6.8 Hz), containing the drug-loaded samples NC1a–e, N1a–e and LCPC
(
0
1
3
.87 (t, 3H, J = 6.5 Hz) –OH proton did not appears; C NMR (1 mL) was first centrifuged at 10 000 rpm at 4 1C for 1 h and the
(
CDCl , 100 MHz): d 176.2 (2C), 173.8, 172.7, 84.3, 84.2, 73.1 unentrapped drug in the supernatant was quantified by noting
3
(2C), 61.1, 50.6, 45.6, 45.5, 41.6, 38.1, 36.7, 31.8, 29.3, 29.1, 25.7, its absorbance at 223 nm using a UV spectrophotometer. The
2
2
8
C
5.0, 23.0, 22.7, 21.8, 14.2; IR (KBr): 3659, 3430, 3057, 2930, absorbance was converted to concentration per mL using a
863, 1708, 1519, 1430, 1400, 1335, 1267, 1194, 1111, 1010, standard calibration curve. The percentage of drug encapsulated
À1
98, 818, 742, 611 cm ; HRMS (ESI) exact mass calcd for was calculated using the following equation:
+
+
24
H
39
N
2
O
8
(M + H) 483.2628, found (M + H) 483.2644.
Niosome preparation. Niosomes were prepared using thin-
Encapsulation efficiency = (drug encapsulated/total drug) Â 100
film hydration method. Towards this, the lipid (1a–e) alone or To evaluate the drug release, pellets of NC1c–e and LCPC
in combination with cholesterol and ibuprofen in 1 : 0.5 : 1 ratio (liposome of soyaphosphatidylcholine) formed via centrifuga-
was dissolved in methanol. The solvent was then removed tion at 10 000 rpm at 4 1C for 2 h were placed in previously
by rotary evaporation under reduced pressure to get a thin activated dialysis tubing (HiMedia with MWCO 12 000–14 000),
film which was subsequently hydrated using phosphate buffer and immersed in a phosphate buffer of pH 7.2. The tempera-
(
pH 7.2). The resulting suspension was sonicated and then ture during the experiment was maintained at 37 Æ 0.5 1C to
extruded through 1000 nm filters to get more-or-less uniformly- simulate physiological conditions. 1 mL each of the samples
sized niosomes. Cholesterol is known to increase the rigidity were then withdrawn at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12 and
of niosomes and lower the drug release rate. It is generally 24 hours, and replaced with an equivalent amount of buffer.
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used as a stabilizer in different lipid-based formulations.
These samples were analyzed spectrophotometrically at
Soyaphosphatidylcholine-based formulation in the same lipid– 223 nm, and from the absorbance values, the percentage of
drug–stabilizer ratio was used as a reference in our experiments. drug released at each of these time points was calculated using
Analysis of size and morphology of nano-aggregates. Sizes a calibration curve.
of niosomes were measured mainly using the DLS method
Preparation of the sample for confocal microscopy. The
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(Malvern Zetasizer nano series at 25 1C, in a cell of 1 cm path sample was prepared using a thin film hydration method
length). The wavelength of the laser used was 632.8 nm and the using the lipid 1c, cholesterol and fluorescein in the ratio
scattering angle was 901. One of the ibuprofen formulations 1 : 0.5 : 1. The film was hydrated using Milli-Q water and the
(NC1c) which showed a better performance was analyzed also resulting suspension was sonicated and extruded through a
using qNano (iZON) which works on the Coulter principle. The 1000 nm membrane filter. 1 mL of this sample was centrifuged
equipment has an upper and lower fluid cell fitted with silver– at 12 000 rpm at 4 1C for 1 h to remove unentrapped dye. It was
silver chloride electrodes. Initially, a membrane of the required suspended in Milli-Q water and a drop was placed on a
pore-size (NP1000, NP800 and NP300 from iZON) was fitted, microscope slide and observed under a confocal microscope.
This journal is ©The Royal Society of Chemistry 2016
J. Mater. Chem. B, 2016, 4, 8025--8032 | 8027