Youngjae Byun, Jin Bong Hwang, Sung Hwan Bang

In this research, poly 3-caprolactone (PCL) was used to encapsulate
a-tocopherol within nanospheres. PCL is semi-crystalline
biodegradable and biocompatible polyester with low glass transition
temperature and melting point (Pitt, 1990). It has been investigated
for drug delivery for several years and it is non-toxic and
non-mutagenic (Forrest, Zhao, Won, Malick, & Kwon, 2006; Mora-
Huertas, Fessi, & Elaissari, 2010). Moreover, it is considerably lower
cost than other biodegradable polyesters such as polyglycolide,
polylactide, and their copolymers.
The objective of this work was to entrap a-tocopherol within
PCL nanoparticles by O/W emulsion solvent evaporation with
ultrasonification method and to optimize the encapsulation
formulation. To achieve this goal, this study was designed to assess
the influence of formulation variables on the characteristics of
nanoparticles such as encapsulation efficiency, a-tocopherol
loading, particle size, zeta potential, and morphology. The formulation
variables were as follows: (1) solvent in the oil phase;
(2) concentration of PCL; (3) ultrasonification time.
2. Materials and methods
2.1. Materials
a-tocopherol (Mw 430.7 g/mol) was purchased from EMD
Bioscience (CA, USA). Poly 3-caprolactone (PCL, Mw 65,000 g/mol)
was purchased from SigmaeAldrich (MO, USA). Polyvinyl alcohol
(PVA, Mw 22,000 g/mol) was purchased from Acros organics (NJ,
USA). Methylene chloride (DCM), methanol, and acetonitrile (ACN),
all HPLC grade were purchased from J.T.Baker (USA). Phosphate
Buffered Saline (PBS, 10 liquid concentrate) was purchased from
EMD Bioscience (CA, USA).
2.2. Formulation of nanoparticles containing a-tocopherol
In this study, nanoparticles were prepared using an oil-in-water
emulsion solvent evaporation with ultrasonification technique
(Fig. 1) by modification of previous works (Kim, Hwang, Park, &
Park, 2005 and Konan et al., 2002). Three factors were statistically
examined, solvent in the oil phase, PCL concentration, and
ultrasonification time (Table 1).
In this procedure, specific amount of PCL (300 or 500 mg) was
dissolved in 10 mL of solvent (methylene chloride or methylene
chloride:acetonitrile ¼ 50:50) containing 10 mg of a-tocopherol.
A PVA (2g/100 mL) solution was prepared in PBS solution. The
PCL solution was added to 40 mL of the PVA solution. The total
mixture in 250 mL centrifuge bottle was then placed in an ice bath
and emulsified using a Branson Digital Sonifier (model 250, Connecticut,
USA) with 55 W of energy output for a specific time
(1, 2, and 3 min) to obtain an oil-in-water emulsion. Another 40 mL of
the PVA solution was then added to the emulsion. The final solution
was stirred for 12 h at 300 rpm on a magnetic stir plate to allow the
evaporation of methylene chloride and acetonitrile and to allow
the formation of the nanoparticles. The suspension was then
centrifuged at 4880 g for 20 min. The pellet was resuspended in
distilled water and centrifuged three more times at 1220 g for 20 min
each. These washing steps were performed to remove unencapsulated
PVA and a-tocopherol. The nanoparticles were collected and
frozen at 80 C for at least 2 h and subsequently freeze dried for 2
days. The freeze dried nanoparticles were stored at 4 C.
2.3. a-Tocopherol encapsulation efficiency (%) and loading (%)
Encapsulation efficiency was determined by an extraction
method. Dried nanoparticles (10 mg) were dissolved in 5 mL of
methylene chloride and 5 mL of distilled water. The mixture was
Y. Byun et al. / LWT - Food Science and Technology 44 (2011) 24e28 25
Fig. 1. Flow chart depicting the encapsulation process.
vigorously vortexed for 1 min and sonicated 5 min in order to
extract the a-tocopherol into the organic solution. Then, methylene
chloride was evaporated and replaced with methanol. The 2 mL of
organic solution was filtered and the a-tocopherol content of the
solution was analyzed by HPLC (Waters 1525 Binary HPLC pump,
USA).
The experiment was performed in triplicate and the encapsulation
efficiency was calculated using the ratio of the mass of
Table 1
Batch compositions used for constituting a-tocopheroleloaded nanoparticle.
Code PCL concentration
(g/100 mL)
3M1 3 DCM a
Solvent in the oil
phase
3M2 3 DCM 2
3M3 3 DCM 3
5M1 5 DCM 1
5M2 5 DCM 2
5M3 5 DCM 3
3MA1 3 DCM:ACN b
1
3MA2 3 DCM:ACN 2
3MA3 3 DCM:ACN 3
5MA1 5 DCM:ACN 1
5MA2 5 DCM:ACN 2
5MA3 5 DCM:ACN 3
a DCM: Methylene chloride, ACN: Acetonitrile.
b (DCM:ACN): DCM:ACN ¼ 50:50.
Ultrasonification
time (min)
1