July 2009
681
cohol from Carlo Erba (Milano, Italy). All other solvents and chemicals priate concentration of particles and sonicated for 10 s. All measurements
were of analytical grade. were done in triplicate and data were expressed as mean valueꢂstandard de-
General Melting points (mp) were determined using an electrothermal viation (S.D.).
or a Köfler apparatus and are uncorrected. Infrared (IR) spectra were
Drug Content and Yields of Production The amount of ECN en-
recorded in nujol with a Perkin-Elmer 781 IR spectrophotometer and are ex- trapped in the lipid matrix was determined as follows: the freeze-dried
pressed in v (cmꢁ1). Nuclear magnetic resonance (1H-NMR) spectra were nanoparticles (50 mg) were dissolved in methanol (10 ml) at 80 °C and then
determined in CDCl3 on a Varian XL-200 (200 MHz) spectrometer. Chemi- slowly cooled to room temperature to precipitate the lipid. After centrifuga-
cal shifts (d scale) are reported in parts per million (ppm) downfield from tion (3000 rpm, 5 min), an aliquot of supernatant was diluted 100 times with
tetramethylsilane (TMS) used as an internal standard. Electron ionization methanol and analyzed by HPLC. The drug content and the encapsulation
mass spectra (EI-MS) (70 eV) were recorded on a Hewlett-Packard 5989 efficiency (EE) were expressed as percentage, and calculated according to
Mass Engine Spectrometer. Elemental analyses were performed on a Perkin- the following equations:
Elmer 2400 instrument; all values are given as percentages, and the results
were within ꢂ0.4% of the theoretical values.
drug loading content (%)ꢄ(weight of drug in nanoparticles/weight of
prepared nanoparticles)ꢅ100
Chemical Procedure for the Synthesis of Fatty Esters (I—VI)
A
mixture of the appropriate fatty acid (1—6) (10 mmol) and p-toluene sul-
fonic acid (1 mmol) was dissolved in isopropyl alcohol (50 ml) and stirred
for 72 h at 60 °C. Then, the solution was evaporated to dryness and extracted
with ethyl acetate by using 10% (w/v) sodium hydroxide as the aqueous
phase. The organic layers were dried over anhydrous sodium sulphate and
evaporated under reduced pressure to give the desired isopropyl esters (I—
VI) as oil or amorphous solid, which were used without further purification.
Spectral and analytical data of all the synthesized title compounds are given
below.
EE (%)ꢄ(weight of drug entrapped in nanoparticles
/weight of drug used for nanoparticle preparation)ꢅ100
The percentage of production yield was calculated as the weight percent-
age of the final product after lyophilization, with respect to the initial total
amount of solid materials used for the preparation. The results were ex-
pressed as the mean of three replicates for each batch.
HPLC Analysis HPLC analyses were carried out using a previously re-
ported modified method.19) The equipment consisted of a Varian Prostar 210
HPLC system that included an autosampler Varian 410 and a diode array de-
tector (DAD) Varian 330 (Varian Deutschland GmbH, Dramstad, Germany)
set to 200 nm. The chromatographic separation was performed using a
spherisorb octyl 5 mm RP-C8 column (250ꢅ4.6 mm, Supelco, Milano,
Italy). A mixture of methanol and 0.05 M NH4H2PO4 (85/15, v/v) was used
as the mobile phase, at a flow of 1.0 ml/min. Injection volume was 20.0 ml.
Differential Scanning Calorimetry (DSC) Thermal measurements
were performed on freeze-dried particles using a DSC Q100 calorimeter (TA
Instrument, New Castle, U.S.A.). The instrument was calibrated using an in-
dium standard and the sample was analysed against a hermetic empty refer-
ence pan. The measurements were obtained at a scanning rate of 10 °C/min
and performed under an Ar purge (50 ml/min). DSC analysis was carried out
on ECN, on PCR, on a physical mixture of ECN–PCR–isopropyl esters (I—
VI), and on drug loaded nanoparticles.
Ex-Vivo Permeation Experiments After preparation, the nanoparticles
were formulated into hydrogels by addition of HPMC K100M (2%, w/w),
selected as gelling agent, to the nanoparticles dispersions. The mixture was
stirred at room temperature at approximately 1000 rpm for 15 min. The ob-
tained hydrogels contained a final ECN concentration of 1% w/w.
Porcine skin is a good model frequently used for human skin showing a
similar penetration for topically applied compounds.20,21) Pig ears were col-
lected immediately after animal death from a local slaughterhouse. Hairs on
both the surfaces of ears were removed using a hair clipper. The epidermis
was separated by heating the skin in distilled water at 60 °C for 2 min and
SC was gently peeled off. Skin samples were frozen at ꢁ20 °C in an alu-
minum foil until using.
For permeation studies, the frozen skin was hydrated for 30 min at 32 °C
in receptor medium. The SC was cut and mounted on the bottom of a cylin-
drical plastic support connected to a drive shaft of the modified dissolution
apparatus (Erweka DT 70, Erweka GmbH, Heusenstamm, Germany) as pre-
viously reported.19) A methanol/water solution (70/30, v/v) (200 ml) was
used as the receptor medium. Manually, 1 ml samples were taken hourly for
8 h and after 24 h, and replaced with the same volume of fresh medium. The
amount of drug permeated through the SC was determined by HPLC analy-
sis.
The permeation of ECN was investigated for 24 h and plots of the accu-
mulated amount of ECN (mg/cm2) against time (h) were constructed. The
steady state flux, representing the absorption rate per unit area, was esti-
mated from the slope of the linear region of the plot.
Isopropyl Decanoate (IPD, I): Yield 91.4%; colorless oil. 1H-NMR
(CDCl3) d: 5.09—4.91 (1H, m), 2.25 (2H, t), 1.61 (2H, t), 1.23 (6H, d),
1.42—1.16 (18H, m), 0.88 (3H, t). IR (Nujol) cmꢁ1: 1735. MS m/z: 214
(Mꢃ). Anal. Calcd for C13H26O2: C, 72.84; H, 12.23. Found: C, 72.71; H,
12.45.
Isopropyl Laurate (IPL, II): Yield 88.0%; yellow oil. 1H-NMR (CDCl3) d:
5.09—4.91 (1H, m), 2.25 (2H, t), 1.61 (2H, t), 1.40—1.18 (22H, m), 0.88
(3H, t). IR (Nujol) cmꢁ1: 1735. MS m/z: 242 (Mꢃ). Anal. Calcd for
C15H30O2: C, 74.32; H, 12.473. Found: C, 74.03; H, 12.41.
Isopropyl Myristate (IPM, III): Yield 93.5%; yellow oil. 1H-NMR
(CDCl3) d: 5.09—4.91 (1H, m), 2.25 (2H, t), 1.61 (2H, t), 1.40—1.18 (26H,
m), 0.88 (3H, t). IR (Nujol) cmꢁ1: 1735. MS m/z: 270 (Mꢃ). Anal. Calcd for
C17H34O2: C, 75.50; H, 12.67. Found: C, 75.59; H, 12.84.
Isopropyl Palmitate (IPP, IV): Yield 89.3%; yellow oil. 1H-NMR (CDCl3)
d: 5.13—4.94 (1H, m), 2.25 (2H, t), 1.61 (2H, t), 1.44—1.18 (30H, m), 0.88
(3H, t). IR (Nujol) cmꢁ1: 1735. MS m/z: 298 (Mꢃ). Anal. Calcd for
C19H38O2: C, 76.45; H, 12.83. Found: C, 76.54; H, 13.09.
Isopropyl Stearate (IPS, V): Yield 94.4%; yellow oil. 1H-NMR (CDCl3) d:
5.09—4.93 (1H, m), 2.29—2.02 (2H, br s), 1.68—1.55 (2H, t), 1.45—1.16
(34H, m), 0.94—0.82 (3H, br s). IR (Nujol) cmꢁ1: 1735. MS m/z: 326 (Mꢃ).
Anal. Calcd for C21H42O2: C, 77.24; H, 12.96. Found: C, 76.11; H, 12.72.
Isopropyl Arachidate (IPA, VI): Yield 92.3%; white crystal. mp 53—
55 °C. 1H-NMR (CDCl3) d: 5.07—4.93 (1H, m), 2.29—2.02 (2H, br s), 2.25
(2H, t), 1.45—1.16 (38H, m), 0.94—0.82 (3H, br s). IR (Nujol) cmꢁ1: 1735.
MS m/z: 354 (Mꢃ). Anal. Calcd for C23H46O2: C, 77.90; H, 13.07. Found: C,
77.81; H, 13.22.
Preparation of Nanoparticles ECN-loaded nanoparticles were pre-
pared by high shear homogenization method, as previously described,19) by
using a mixture of various isopropyl fatty esters (I—VI) and PCR as the
lipid phase. Their compositions are reported in Table 1.
Briefly, the oil phase consisting of PCR (3% w/w), fatty ester (2% w/w)
and drug (1% w/w) was heated at 80 °C. After dispersion in a surfactant so-
lution (Tween 80, 2.5% w/w) having the same temperature, the mixture was
treated by high shear homogenization (Silverson L4R mixer, Crami, Italy),
emulsifying at 6200 rpm for 5 min. The resulting nanoemulsions were rap-
idly cooled to room temperature to obtain nanoparticle dispersions. Each
preparation was carried out in triplicate.
Freeze-Drying of Nanoparticles A weighed amount of nanoparticle
aqueous dispersion was rapidly frozen below ꢁ80 °C in a deep-freezer
(Dairei Co., Ltd., Tokyo, Japan) and lyophilized using a 5Pascal LIO 5P ap-
paratus (Cinquepascal srl, Milano, Italy). The freeze-drying process was car-
ried out at ꢁ54.5 °C under vacuum (0.909 mbar) for 8 h and then the lipid
powders were collected for yields of production, encapsulation efficiency
and DSC measurements.
The permeability coefficient (Kp) was calculated from the following equa-
tion22)
:
KpꢄJ/C
Particle Size Determination The mean particle size and polydispersity where J is the flux (mg/cm2/h) and C the concentration of ECN in the donor
index (PI) of nanoparticle dispersions were measured by photon correlation compartment (mg/cm3).
spectroscopy (Coulter N5, Beckman Coulter, Miami, FL, U.S.A.). The
analyses were performed using the following conditions: fluid refractive
Results were reported as meanꢂS.D. of five determinations.
Statistical Analysis All data were subjected to one-way analysis of vari-
index: 1.333; temperature: 20 °C; viscosity: 0.890 centipoises; fixed scatter- ance (ANOVA) (Origin®, version 7.0 SR0, OriginLab Corporation, MA,
ing angle of 90°; sample time: 3.0 ms; sample run time: 300 s. Before the U.S.A.). In all cases, individual differences between formulations were eval-
analysis, each sample was diluted with double-distilled water to the appro- uated using a nonparametric post hoc test (Tukey’s test). The differences