Colloids and Surfaces B: Biointerfaces 76 (2010) 16–19

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Factors effect on the loading efficiency of Vitamin C loaded

chitosan-coated nanoliposomes
Nan Liu, Hyun-Jin Park ∗
Graduate School of Biotechnology, Korea University, 1, 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-701, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: Chitosan-coated nano-size liposomes as a new carrier with bioactivity were made from phosphatidyl-
Received 4 March 2009 choline (pc) and cholesterol (chol) by direct injection. Liposomes prepared using ethanol as a solvent with
Received in revised form pc:chol ratios of 40:60 and 60:40 displayed mall mean diameters (97.4 nm and 95.8 nm, respectively).
30 September 2009
Different factors affecting the loading efficiency and payload of Vitamin C for these nano-size liposomes
Accepted 30 September 2009
were investigated by high-pressure liquid chromatography. Liposomes prepared with a pc:chol ratio of
Available online 21 October 2009
60:40 were promising Vitamin C carriers with a maximum loading efficiency about 96.5% and payload
about 46.82%. When liposomes were prepared with 100 mg initial mass of Vitamin C, maximum loading
Keywords:
Vitamin C
efficiency was obtained. Furthermore, with an increasing initial mass of Vitamin C, the payload increased.
Nanoliposomes Based on the experimental results, it appears that the chitosan concentration does not affect the load-
Phosphatidylcholine ing efficiency and payload of liposomes. Liposomes prepared under the above optimum conditions were
Cholesterol stable during 15 weeks storage such that over 85% Vitamin C was protected against oxidation.
Loading efficiency © 2009 Elsevier B.V. All rights reserved.
Stability

1. Introduction However, liposomes that merely consist of amphiphilic phos-

Liposomes are microscopic vesicles consisting of membrane-
like phospholipid bilayers surrounding an aqueous medium.
Liposomes have been widely used in the pharmaceutical, food, and
cosmetics industries [1–4] and have been successfully employed
for the encapsulation of a range of synthetic drugs and biologi-
cals [5–9]. As carriers, liposomes are required to have the ability to
protect the active compound against chemical degradation by the
surrounding dispersion medium [10] and control the release rate of
the incorporated compound. Liposomes were prepared by a mix-
ture of phospholipid and cholesterol (chol). In chol–phospholipid
mixtures, chol is reported to decrease the temperature, enthalpy,
and sharpness of the gel to liquid crystalline phase transition of the
phospholipid; fluidize or disorder the gel phase; rigidify or, order
the fluid lamellar phase; reduce membrane permeability above the
main transition temperature; and decrease the average molecular
surface area of the phospholipid [11–18]. Amphiphilic phospho-
lipid is composed of hydrophilic head domain and hydrophobic tail
domain. It can form the liposome in the aqueous media having a
hydrophilic surface and hydrophobic interior. It would be an ideal
core material, which itself is bioactive, to prepare the carriers for
nutrients.
∗ Corresponding author. Tel.: +82 2 3290 4149; fax: +82 2 953 5892.
E-mail address: hjpark@korea.ac.kr (H.-J. Park).
phatidylcholine (pc), are poor in maintaining their shape mainly
via hydrophobic interaction and are, therefore, not useful as a drug
delivery system [19]. A great deal of research has been focused on
enhancing the stability of liposomes. Complexation or coating of
liposomes with artificial polymers [20–24] and biopolymers such as
polysaccharides [25–27] are representative of some of the methods
attempted.
Chitosan, a linear and abundant polysaccharide, was presently
utilized as the wall material of the delivery system. Due to their
biodegradable, biocompatible, mucoadhesive and non-toxic nature
[28–31], chitosan has been widely used in numerous drug delivery
systems. Compared to other delivery systems, chitosan nanoparti-
cles have a special feature; they can adhere to the mucosal surface
and transiently open the tight junction between epithelial cells.
Some reports have indicated that chitosan can increase membrane
permeability, both in vitro [31–33] and in vivo [34].
Vitamin C (VC) (l-ascorbate) is an essential nutrient for a large
number of higher primate species, a small number of other mam-
malian species, a few species of birds, and some species of fish. VC
presence is required for a range of essential metabolic reactions
in all animals and plants. Its deficiency causes scurvy in humans.
It is also widely used as a food additive. The pharmacophore of
VC is the ascorbate ion. In living organisms, ascorbate is an anti-
oxidant, since it protects the body against oxidative stress, and is a
cofactor in several vital enzymatic reactions. However, VC is unsta-
ble so it needs to be protected against environmentally-mediated
oxidation.

0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2009.09.041
 N. Liu, H.-J. Park / Colloids and Surfaces B: Biointerfaces 76 (2010) 16–19 17

The objectives of this study were to investigate the effect of dif-

ferent characteristics on the loading efficiency and VC payload of
chitosan-coated liposomes. Also, the stability of VC-loaded lipo-
somes during storage was ascertained.

2. Materials and methods

2.1. Materials

Chitosan was provided by KITTO Life Company (Korea). The

degree of deacetylation of the 4 kDa product exceeded 90%.
Phosphatidylcholine (pc), chol and VC were purchased from
Sigma–Aldrich. All other chemicals were of analytical grade.

2.2. Preparation of chitosan-coated nano-size liposomes

Liposomes were prepared from different ratios of pc and chol

(20:80, 40:60, 60:40 and 80:20) at different reaction conditions by
a sonication method. 100 mg of lipid with different pc:chol ratios
was dissolved in 15 ml ethanol, which was added drop-wise to a VC
solution with stirring. The organic solvent was evaporated and sol-
vent traces were removed by maintaining the pH of the phosphate
buffer solution (PBS) at 7.4 and storing under vacuum overnight.
The liposome dispersions were downsized by sonication using an
Ultrasonic Homogenizer UH-600 probe-type sonifier operating at
20 W for 2 min. The sonication was repeated using pulse function
(pulse on for 10.0 s; pulse off for 2.0 s). The experimental conditions
are summarized in detail in Fig. 1. Then, a 0.1% chitosan solution was
added with constant stirring condition to obtain chitosan-coated
nano-size liposomes. The liposome suspension was centrifuged
(15000 rpm, 15 min) to remove free VC.

2.3. Liposome characterization

The physicochemical characteristics of the liposomes were

studied. The shape and surface structure of the chitosan-
coated liposomes were observed by cryo-transmission electron
microscopy (cryo-TEM). One drop of the liposome suspension was
applied to a pre-treated support film with a pipette. Each speci-
men grid was blotted with filter paper to remove excess fluid and
then was rapidly plunged into liquid ethane that had been cooled
to liquid nitrogen temperature to prevent the formation of ice crys-
tals. The grid was transferred to a cryo workstation and then into
a cryo holder, which was inserted into the electron microscope for Fig. 1. Cryo-TEM micrographs of (a) non-coated liposomes and (b) chitosan-coated
examination. The average particle size and size distribution were liposomes. The bar represents 200 nm.
determined by quasielastic laser light scattering with a Malvern
Zetasizer® (Malvern Instruments). 1 ml of the liposome suspension
was diluted to 3ml with distilled water, put into a polystyrene latex The loading efficiency was calculated as the ratio between the VC
cell and measured at a detector angle of 90◦ , wavelength of 633 nm, amount in particles and the initial added drug C:
refractive index of 1.33 and real refractive index of 1.59 at 25◦ .
loading efficiency (%)
C1 [amount of VC in liposomes (mg)]
2.4. VC loading efficiency = × 100 (2)
C [initial added VC (mg)
VC was determined as described previously [37]. The
mobile phase was 0.2% metaphosphoric acid/methanol/acetonitrile 2.5. Stability of VC-loaded liposome suspensions during storage
(90:8:2, v/v/v) and the retention time was about 3.5 min. The drug
content (C1 ) was assayed by HPLC at room temperature at 250 nm, As the carrier, the ability of liposomes to protect the stabil-
using a 250 mm × 3 mm Nucleosil® 100-5 C18 packed column. From ity of VC during storage is also very important. During storage,
the data, the drug payload was calculated as the ratio of amount non-coated and chitosan-coated liposomes were stored at room
of entrapped drug in nanoparticles (mg) C1 /amount of liposomes temperature or 4 ◦ C to investigate the stability of VC in the
(mg): liposomes suspension by HPLC. Stability of VC in the liposomes sus-
pensions was calculated as the ratio between the C2 (mg) amount
C1 [amount of VC (mg) in liposomes] of VC in liposomes at different storage times and C1 (mg) amount
payload (%) = × 100 (1)
amount of liposomes of VC in liposomes before storage:
18 N. Liu, H.-J. Park / Colloids and Surfaces B: Biointerfaces 76 (2010) 16–19

Fig. 2. Influence of different pc:chol ratios and different chitosan concentrations on Fig. 3. Different pc:chol ratios affect loading efficiency and payload of liposomes:
the size of liposomes: ()0 1% chitosan solution; (䊉) 0.2% chitosan solution; () 0.5% () loading efficiency; (䊉) payload (data shown are the mean ± S.D., n = 3).
chitosan solution; () 1.0% chitosan solution (data shown are the mean ± S.D., n = 3).

3.3. Influence of pc:chol ratio on liposome loading efficiency and

Stability of VC
C2 (mg) [amount of VC in liposomes at different storage time]
= × 100%
C1 (mg) [amount of VC in liposomes before storages]
3. Results and discussion
3.1. Characteristics of VC-loaded liposomes
Pc, which is an amphiphilic molecule, forms micelles with the
head domain outside and tail domain inside in the aqueous media
[4]. After sonication, the size of the micelle was reduced to nanome-
ter dimensions and the regular spherical shapes of the liposomes
were revealed by cryo-TEM. Nano-sized liposomes with a mean
diameter of about 80 nm with double layers were routinely evi-
dent (Fig. 1a). Since chitosan is a hydrophilic polymer, it is coated
on the lipid layers of the liposomes (Fig. 1b). It was apparent
that chitosan coating thickened the lipid layers and increased the
number of layers to produce a multi-layer assembly. The result
was marginal but real increase in the size of the liposomes. We
speculate that during the process of chitosan coating, sonication
perturbed the lipid bilayer, allowing for the coating of chitosan,
after which the lipid layers reformed to generate the multi-layer
liposomes.
VC payload
Loading efficiency and payload of 100 mg VC-loaded liposomes
formed with different pc:chol ratios of pc and chol was investigated
by HPLC. A large amount of VC could be loaded into chitosan-coated
liposomes; the nano-sized liposomes formed with a pc:chol ratio
of 60:40 produced the highest loading efficiency (96.55%) and VC
payload (48.28%) (Fig. 3). The results indicated that smaller diam-
eter liposomes were capable of higher VC loading, perhaps due to
the increased presence of carriers.
3.4. Initial mass of VC affects loading efficiency and payload of
liposomes
Liposomes were prepared with different pc:chol and different
initial masses of VC, and the influence of the initial mass of VC
on the loading efficiency and payload was investigated by HPLC.
The initial mass of VC (50 mg, 75 mg, 100 mg and 125 mg) influ-
enced these parameters, producing a maximum loading efficency
exceeding 88% (Fig. 4). The multi-layered liposomes may have
proven to be an ideal carrier for hydrophilic components. When
100 mg of VC was loaded into the liposomes the highest loading effi-
ciency was obtained. As expected, the payload was increased with
increasing VC.

3.2. Factors affecting liposome size

Liposomes were prepared under different reaction conditions

that included different pc:chol ratios and different concentra-
tions of chitosan solutions, and their effects of these factors on
liposome size were investigated. At pc:chol ratios of 40:60 and
60:40, smaller liposomes were prepared with a mean diameter
of 97.4 nm and 95.8 nm, respectively (Fig. 2). Pc functioned as
the structural backbone of liposomes, with chol acting to sta-
bilize the formed liposomes. The optimal pc:chol ratio to make
nano-sized liposome was determined to be 40: 60 and 60:40. The
effect of different concentrations of chitosan on the size of the
liposomes is also shown in Fig. 2. As the concentration of chi-
tosan solution increased, the size of the liposomes increased. The
increasing content of chitosan coated onto the liposome layers
resulted in an increased liposome diameter. High concentrations
of chitosan produced a highly viscous solution that readily coated Fig. 4. Initial mass of VC affects loading efficiency and payload of liposomes: ()
liposomes. loading efficiency; (䊉) payload (data shown are the mean ± S.D., n = 3).
 N. Liu, H.-J. Park / Colloids and Surfaces B: Biointerfaces 76 (2010) 16–19
Fig. 5. Different concentration of chitosan solution affects loading efficiency and
payload of liposomes: (l) loading efficiency; (䊉) payload (data shown are the
mean ± S.D., n = 3).
3.5. Influence of chitosan concentration on loading efficiency and
payload of liposomes
The influence of chitosan concentration on the loading efficiency
and payload of liposomes was investigated by measuring the VC
content in liposomes by HPLC. During the process of preparation of
the liposomes, 0.1%, 0.2%, 0.5% and 1% of chitosan solution were
used, and liposome loading efficiency and payload were deter-
mined. Similar loading efficiencies and payload were evident for
different chitosan concentrations (Fig. 5), indicating that the con-
centration of chitosan solution did not effect these liposome param-
eters, despite the marginal increase in liposome diameter (Fig. 2).
The most favorable explanation is that VC located stably in the
water phase between the lipid layers. During the preparation pro-
cess, chitosan molecules did not destroy the structure of liposomes,
resulting in an invariant VC content of Vitamin C in the liposomes.
3.6. Storage stability of VC in liposomes
Stability of Vitamin C loaded into liposomes was also investi-
gated during storage by HPLC. VC stability in liposomes prepared
with different concentrations of liposome solutions and VC in PBS
is shown in Fig. 6. Compared with the free VC in PBS, liposome-
loaded VC was more stable. After 15 weeks of storage, free VC
Fig. 6. Stability of VC in liposomes prepared with different concentration of chitosan
solutions during storage: () 0% chitosan solution; (䊉) 0.1% chitosan solution; ()
0.2% chitosan solution; () 0.5% chitosan solution; () 1.0% chitosan solution (data
shown are the mean ± S.D., n = 3).
19
was undetectable, while 85% of the encapsulated VC remained.
Chitosan-coated liposomes may have protected VC against oxida-
tion during storage. Also, chitosan can prevent fusion of liposomes
(data not shown), which would act to increase liposome stability.
4. Conclusion
In this paper, pc and chol were used to prepare a new carrier,
chitosan-coated nano-size liposomes, by direct injection. Lipo-
somes were prepared using ethanol as the solvent with different
ratios of pc and chol. With pc:chol ratios of 40:60 and 60:40, smaller
diameter liposomes were generated. Different factors affecting the
loading efficiency and VC payload of the nano-sized liposomes
were investigated. Chitosan-coated nano-sized liposomes prepared
with a pc:chol ratio of 60:40 are particularly promising VC carri-
ers. Liposomes prepared with 100 mg initial mass of VC yielded
the highest loading efficiency. Furthermore, with increasing initial
mass of VC, the liposome payload increased. The chitosan concen-
tration was not influential to the loading efficiency and liposome
payload. Finally, the results indicate that liposomes are a stable
storage system for VC.
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