Amphiphilic chitosan derivatives-based liposomes: Synthesis, development, and properties as a carrier for sustained release of salidroside

Article abstract of DOI:10.1021/jf4039925

A novel amphiphilic chitosan derivative of N,N-dimethylhexadecyl carboxymethyl chitosan (DCMCs) was synthesized. The structure of DCMCs was confirmed via FT-IR and ¹H NMR, and the critical micelle concentration (CMC) was investigated by fluorescence spectroscopy. The results indicated that DCMCs has hydrophilic carboxyl and hydrophobic methylene groups and the CMC value was 23.00 mg·L^-1. The polymeric liposomes (DCMCs/cholesterol liposomes, DC-Ls) were developed, and its properties were evaluated. The DC-Ls exhibited multilamellar spheres with positive charge (+73.30 mV), narrow size distribution (PDI = 0.277), and good crystal properties. Salidroside was first to encapsulate into DC-Ls. Compared with traditional liposomes (phosphatidylcholine/cholesterol liposome, PC-Ls), DC-Ls showed higher encapsulation efficiency (82.46%) and slower sustained release rate. The in vitro salidroside release from DC-Ls was governed by two distinct stages (i.e., burst release and sustained release) and was dependent on the pH of the release medium. The case II transport and case I Fichian diffusion were the main release mechanisms for the entire release procedure. These results indicated that DC-Ls may be a potential carrier system for the production of functional foods that contain salidroside or other bioactive food ingredients.

Full text of DOI:10.1021/jf4039925

Article
pubs.acs.org/JAFC
Amphiphilic Chitosan Derivatives-Based Liposomes: Synthesis,
Development, and Properties as a Carrier for Sustained Release of
Salidroside
Hailong Peng,†,‡ Wenjian Li, Fangjian Ning, Lihua Yao, Mei Luo, Xuemei Zhu, Qiang Zhao,†
and Hua Xiong*
†
†
§
†,‡
†
,
†
†
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, P.R. China
Department of Chemical Engineering, Nanchang University, Nanchang, Jiangxi 330031, P.R. China
‡
§
School of Life Science, Jiangxi Science & Technology Normal University, Nanchang, Jiangxi 330013, P.R. China
ABSTRACT: A novel amphiphilic chitosan derivative of N,N-dimethylhexadecyl carboxymethyl chitosan (DCMCs) was
1
synthesized. The structure of DCMCs was confirmed via FT-IR and H NMR, and the critical micelle concentration (CMC) was
investigated by fluorescence spectroscopy. The results indicated that DCMCs has hydrophilic carboxyl and hydrophobic
−1
methylene groups and the CMC value was 23.00 mg·L . The polymeric liposomes (DCMCs/cholesterol liposomes, DC-Ls)
were developed, and its properties were evaluated. The DC-Ls exhibited multilamellar spheres with positive charge (+73.30 mV),
narrow size distribution (PDI = 0.277), and good crystal properties. Salidroside was first to encapsulate into DC-Ls. Compared
with traditional liposomes (phosphatidylcholine/cholesterol liposome, PC-Ls), DC-Ls showed higher encapsulation efficiency
(
82.46%) and slower sustained release rate. The in vitro salidroside release from DC-Ls was governed by two distinct stages (i.e.,
burst release and sustained release) and was dependent on the pH of the release medium. The case II transport and case I Fichian
diffusion were the main release mechanisms for the entire release procedure. These results indicated that DC-Ls may be a
potential carrier system for the production of functional foods that contain salidroside or other bioactive food ingredients.
KEYWORDS: amphiphilic chitosan, liposomes, salidroside, sustained release, release mechanism
INTRODUCTION
extensively investigated for its potential using in delivery of
■
14
various nutrients. However, CS is only soluble in aqueous
acidic solutions below pH 6.5 but insoluble in neutral pH and
physiological environments. Therefore, different chemical
agents have been used to improve the CS solubility, and the
formation of micelles has been confirmed. However, only a
small section of the hydrophobic segment can conjugate to the
CS backbone (substitution degree, DS < 10%) because of the
Because of a lipophilic and a hydrophilic group on the same
molecules, polar lipids can be self-assembled and form self-
organized traditional liposomes after interaction with water. As
an active agent’s carrier, traditional liposomes have advantages,
including biocompatible and biodegradable properties and
targeting and protection of entrapped agents. In addition,
traditional liposomes are capable of entrapping both hydro-
philic and hydrophobic agents. Recently, traditional liposomes
have been widely applied in pharmaceutical, cosmetic,
1
2
15
3
limited solubility property. Hence, the loading capacities of
the micelles for hydrophobic bioactive agents are very low.
Thus, CS derivatives were synthesized using the soluble CS
4
5
1
,6,7
nutrition, and food industries.
However, traditional lip-
(
such as carboxymethyl chitosan, CMCs) as a necessary
osomes exhibit notable defects on short circulation half-life in
vitro, instability of the lipid, and a few certain chemical groups
on the liposome surface. Hence, many efforts have been exerted
to design sufficiently stable polymeric liposomes, which may be
a promising carrier with good physical and thermal stability,
excellent solubility, and high encapsulation efficiency. For
promoting the application of polymeric liposomes in the food
industry, choosing the optimal food-grade biopolymers is a
critical factor.
Chitosan (CS) is a natural polymer composed of randomly
distributed β-(1−4)-linked D-glucosamine (deacetylated unit)
and N-acetyl-D-glucosamine (acetylated unit). CS has been
widely used in several areas such as biomedical, pharmaceutical,
and biotechnological fields as well as in the food industry
because of its good properties such as nontoxicity, good
backbone.
Among these CS derivatives, amphiphilic CS derivatives with
hydrophilic and lipophilic blocks have received extensive
15−17
study.
The characteristics of amphiphilic CS derivatives
8
,9
are quite similar to that of polar lipids and surfactant
complexes, which can form micelles via intra- or intermolecular
association between hydrophobic moieties, and this process
primarily minimizes interfacial free energy in contact with
13
aqueous solvents. Evidence has shown that amphiphilic CS
derivatives have been applied to develop liposomes for drugs
8
,9
1
0
and genes delivery because such polymeric liposomes exhibit
higher stability in vivo/vitro and encapsulation efficiency.
Received: September 7, 2013
Revised: December 27, 2013
Accepted: December 29, 2013
Published: December 29, 2013
11−13
biodegradability, and biocompatibility.
food biopolymers for encapsulation and delivery systems, CS
is one of the most popular polymers and has also been
Among various
©
2013 American Chemical Society
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Journal of Agricultural and Food Chemistry
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Figure 1. Schematic illustration of the synthesis process of DCMCs: (1) DA was synthesized using epoxychloropane and N,N-
dimethylhexadecylamine; (2) OCMCs were synthesized using CS and monochloroacetic acid in an alkali environment; (3) DCMCs were then
synthesized using OCMCs and DA in 2-propanol aqueous solution in an alkali environment.
However, to the best of our knowledge, the application of
amphiphilic CS derivatives liposome in the food industry is
rarely reported.
Cholesterol (Chol) and pyrene (with 99.00% purity) were purchased
from Aladdin Chemistry Co., Ltd. (Shanghai, China). All other
chemical agents used were of analytical grade.
Preparation of DCMCs. Figure 1 illustrates the synthesis
procedure of DCMCs. Epoxychloropane (10 mg) and N,N-
dimethylhexadecylamine (4 mg) were added into a flask and were
reacted at 60 °C for 2 h with stirring. The residual epoxychloropane
was then removed via reduced pressure distillation. The hexadecyl
dimethylammonium chloride (DA) production was recrystallized
using acetone and was washed with ether. CS (5 g) was subjected
to swelling by using NaOH solutions (75 mL, 50 wt %) for 24 h in a
flask. Monochloroacetic acid (5 g, 53 mmol) was dissolved in 25 mL of
Salidroside has been widely used as a functional food for the
various pharmacological actions such as antiaging, antioxidative,
18−20
anticancer, and antidepressant activities.
Salidroside was
widely applied in functional food industries in oral solution,
powder, and capsule forms. The application of these forms has
been significantly restricted because of the fluctuation of its
concentration in blood, very high intake frequency, and poor
2
1,22
oral bioavailability.
To overcome these drawbacks, tradi-
2
-propanol and then added dropwise into the flask containing CS for
tional liposomes (phosphatidylcholine/cholesterol liposomes,
2
1,22
reaction of 24 h at room temperature. The pH of the reacted systems
was adjusted to 7 using HCl (2.5 M). The product of O-carboxymethyl
chitosan (OCMCs) was filtered and washed with ethanol. The
production was then dialyzed for 3 days and was subsequently freeze-
dried. OCMCs (5 g) were dissolved in an 2-propanol aqueous solution
(100 mL, 2-propanol/deionized water = 1/3, V/V). In addition, DA
was added slowly. The mixture was trickled with 10 mL of NaOH
solution (42%, w/w) and reacted at 50 °C for 24 h with stirring.
Finally, the solution was dialyzed for 3 days and lyophilized and the
DCMCs were then obtained.
PC-Ls) were developed to encapsulate salidroside.
However, PC-Ls suffer from various disadvantages such as
instability in vivo/vitro environments and leaking of entrapped
salidroside. On the basis of the advantages of amphiphilic CS
derivatives, formulating a polymeric liposome by using
amphiphilic CS derivatives for salidroside encapsulation may
be an effective approach to overcome the above-mentioned
problems.
In this study, the amphiphilic CS derivative (DCMCs) was
synthesized. DCMCs were then used to develop liposomes
with cholesterol (DC-Ls). As a functional food agent,
salidroside was encapsulated first into DC-Ls. The physico-
chemical properties of DCMCs and DC-Ls were studied.
Meanwhile, the encapsulation capacity and in vitro release
properties of DC-Ls were also investigated.
Structure Characterizations of DCMCs. The structure of
1
DCMCs was determined via FT-IR and H NMR. The FT-IR spectra
of CS, OCMCs, and DCMCs were obtained using KBr pellets on a
spectrophotometer (Nicolet 5700, Thermo Electron Corporation,
−1
1
Waltham, MA, USA) in the region of 400−4000 cm . The H NMR
spectra of DCMCs were obtained using a spectrometer operated at
500 MHz (Bruker DPX300, Bruker Corporation, Karlsruhe, Germany)
with D O as the solvent.
2
Substitution Degree (SD) of OCMCs. The SD of OCMCs was
measured via the conductometric method. OCMCs (0.1 g) was
EXPERIMENTAL PROCEDURES
Reagents and Materials. Salidroside (with 98.00% purity) was
purchased from Dingsheng Pharmaceutical Technology Co., Ltd.
Hunan, China). Chitosan (CS) (300 KDa molecular weight, 90%
deacetylation degree) was obtained from Lanji Technology Co., Ltd.
Shanghai, China). Epoxychloropane was purchased from Aladdin
Chemistry Co., Ltd. (Shanghai, China). N,N-Dimethylhexadecylamine
was purchased from Aoke Industrial Co., Ltd. (Shanghai, China).
23
■
dissolved in an HCl solution (100 mL, 0.05 M) with mild stirring. The
pH was adjusted to 2.0−2.2 by adding NaOH solution (0.1 M). The
OCMCs solution was then titrated with NaOH solution (0.1 M) to
pH 11.0−12.0. The typical titration curve was calculated, and the SD
of OCMCs was measured based on the following equation:
(
(
SD = (V − V) × DD/(V − V )
(1)
2
1
3
2
6
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Journal of Agricultural and Food Chemistry
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where SD is the OCMCs substitution degree and DD is the
respectively. The release media was incubated at 37 ± 0.1 °C with
stirring. At specified time intervals, 1 mL of the sample was taken and
the same volume of fresh media was added to maintain a constant
volume. The amount of salidroside release from DC-Ls was estimated
via HPLC. All release tests were performed in triplicate, and the mean
values were reported. The ingredients of simulated fluids were as
deacetylation degree of original CS. V , V , and V are the consumed
1
2
3
volume of NaOH in different linear sections of the titration curve.
Critical Micelle Concentration (CMC) of DCMCs. The CMC of
DCMCs was investigated via fluorescence spectroscopy by using
pyrene as a hydrophobic probe. A known volume of pyrene in acetone
−
6
24
(
6.0 × 10 M, 1 mL) was added to each of a series of 10 mL vials.
follows:
−4
Then 10 mL of various concentration of DCMCs (1.0 × 10 , 1.0 ×
SGF (pH 1.2): NaCl (0.2 g), HCl (7 mL), and pepsin (3.2 g), pH
−
3
−3
−2
−1
−1
1
0 , 2.0 × 10 , 5.0 × 10 , 1.0 × 10 , and 1.0 mg·mL ) were
was adjusted by NaOH to 1.2 ± 0.5.
added into each vial after acetone evaporation was complete. The
SIF (pH 7.4): KH PO (6.8 g), 0.2 N NaOH (190 mL), and
2
4
fluorescence emission spectra were obtained using a fluorometer (F-
pancreatin (10.0 g).
PBS (pH 7.0): 0.2 M KH PO (250 mL), 0.2 M NaOH (145.5
4500, Hitachi, Tokyo, Japan) at an excitation wavelength of 339 nm,
2
4
and the emission wavelength was 360−t450 nm for excitation spectra.
mL), and H O (604.5 mL).
2
Preparation of DC-Ls. The DC-Ls were developed via thin-layer
Release Mechanism of DC-Ls. The first-order, Higuchi, and
Peppas models were used to fit the release data of salidroside from
DC-Ls and were then used to investigate potential release
mechanisms.
9
evaporation method. DCMCs and cholesterol (Chol) (weight ratio
1
/0.81, total lipids 90 mg) were dissolved in ethanol (15 mL) in a
round-bottomed flask. The mixture was dried under reduced pressure
by using an Eyela rotary evaporator (model N-1000; Eyela, Tokyo,
first‐order model: ln(1 − M /M ) = −k
(3)
Japan) at 40 °C under condition of N environment to form a thin
t
∞
t
2
lipid film. The trace solvent was then removed by holding the lipid film
at high vacuum overnight. The thin lipid film was hydrated by using
deionized water (15 mL, with salidroside of 0.5 mg·mL ) and was
then ultrasonicated using an ultrasonic processor (KQ-600B, Kun
Shan Ultrasonic Instruments Co., Ltd., Jiangshu, China) at 25 °C for
1
/2
Higuchi model: Mt/M∞ = kt
Peppas model: ln(M /M ) = n ln t + ln k
(4)
(5)
−1
t
∞
where M /M∞ is the fractional active agents release at time t, k is a
constant incorporating the properties, and n gives an indication of the
release mechanism. The correlation coefficient (R ) is the linear
relationship between salidroside release and time.
3
0 min at 45 W to produce multilamellar liposomes (DC-Ls). For
t
comparison, traditional PC-Ls were also prepared via the same
procedures and conditions except egg yolk phosphatidylcholine (PC)
replaced the DCMCs.
2
For verifying that DC-Ls have the capacity of forming multilamellar
film structure, 25 mg of OA-Fe O was also dissolved in ethanol (15
mL) with DCMCs and Chol, and other procedures were the same as
that in the preparation of DC-Ls. The physical mixtures of DCMCs/
Chol were prepared as follows: DCMCs/Chol (weight ratio 1/0.81)
was mixed with chloroform, and the chloroform was then evaporated
to obtain the dried physical mixtures of DCMCs/Chol.
RESULTS AND DISCUSSION
3
4
■
Synthesis and Structural Characterization of DCMCs.
A novel amphiphilically modified CS molecule with hydrophilic
carboxyl and long hydrophobic methylene chains were
synthesized. Detailed schemes for the DCMCs preparation
1
are shown in Figure 1. FT-IR and H NMR were performed to
Physicochemical Properties of DC-Ls. Transmission electron
microscopy (TEM) images were obtained using JEOL (JEM-2010HR,
Hitachi, Tokyo, Japan) with an operating voltage of 200 kV. X-ray
diffraction (XRD) patterns were recorded using an XRD analyzer (D/
max 2400, Rigaku Corporation, Tokyo, Japan) with a scattering angle
range of 3−45°. DSC was performed using DSC equipment (Perkin-
confirm the structural properties of DCMCs. The FT-IR
spectra of CS, OCMCs, and DCMCs are shown in Figure 2A.
In the FT-IR spectra of CS, the stretching vibrations at 3444.62
and 2927.73 cm− were attributed to the −OH and C−H
groups, respectively. Meanwhile, the stretching vibration of C−
1
−1
Elmer, Waltham, MA, USA) with a heating rate of 10 °C·min . The
average particle size was determined by a dynamic light scattering
Zetasizer (Nano ZS90, Malvern Instruments, Worcester, UK). The ζ
potential was measured using the Nano ZS90 zetasizer (DTS1060,
Malvern Instruments) at 25 °C. The vibrating specimen magneto-
meter (VSM) (model 7407, Lakeshore, Westerville, OH, USA) was
used to examine the magnetic properties of the DC-Ls.
−1 25
O was found at 1092.96 cm . In the FT-IR spectra of
OCMCs, the vibration absorption peaks were observed at
−1
1
575.94 and 1419.22 cm , which indicates that carboxylate
26
groups were successfully conjugated into CS. In the FT-IR
spectra of DCMCs, the characteristic stretching vibration
absorption of long methylene chain was found at 2921.31 and
−1
Encapsulation Efficiency (EE) of DC-Ls. The Sephadex G25
column method was used to determine the salidroside EE in DC-Ls.
A predetermined aliquot of salidroside loaded DC-Ls were first eluted
through the Sephadex G25 column to remove free salidroside. The DC-
Ls was dissolved in chloroform to destroy the liposomes structure. The
trapped salidroside was extracted with deionized water and was then
determined with high performance liquid chromatography (HPLC)
2853.49 cm . Additionally, the appearance of new stretching
vibration absorption at 721.55 cm− corresponds to the
methylene planar rocking vibration. These results showed
that the quaternary ammonium salt group was grafted into
DCMCs. The DCMCs structure was further confirmed by the
1
1
1
H NMR spectra. In the H NMR spectra of DCMCs (Figure
2
B), the signal at δ 4.78, 1.72, 3.67, 3.65, 3.60, and 3.37 ppm
(
1100 series, Agilent Technologies Co., Ltd., Palo Alto, CA, USA) at a
wavelength of 480 nm. The analytical column was a C18 column (5
μm, 4.6 mm × 150 mm, Waters Company, Milford, MA, USA). The
mobile phase was a mixture of methanol/water (20:80, v/v) at a flow
rate of 1.0 mL min , and the injection volume was 20 μL at 25 °C.
The EE of DC-Ls was determined using the equation below. EE test
were performed in triplicate with data reported as the mean values.
were attributed to H-1, H-2, H-3, H-4, H-5, and H-6 of the D-
glucosamine unit of DCMCs, respectively. The new signals at δ
0
.91, 1.32−1.40, 3.25, 3.27, and 3.90 ppm were attributed to H-
−1
e, H-d, H-b, H-a, and H-c, which correspond to the proton
assignment of long-chain alkyl of quaternary ammonium salt.
The results of FT-IR and H NMR confirmed that DCMCs
1
were successfully synthesized, which was consistent with the
previous report of Liang at el.
Substitution Degree (SD) of OCMCs. A typical
conductometric titration curve of OCMCs that consists of
four linear branches is shown Figure 3. The excess HCl was
EE(%) = (salidroside loaded in DC‐Ls/total salidroside added)
27
×
100
(2)
In Vitro Release of DC-Ls. First, 5 mL of salidroside loaded DC-
Ls suspension were placed in dialysis bags and were suspended in 30
mL of simulated gastric fluid (SGF, pH 1.2), phosphate buffer saline
neutralized using the volume of NaOH (0−V
). (V
−V )
1
1
2
(
PBS, pH 7.0), and simulated intestinal fluid (SIF, pH 7.4),
corresponds to the volume of NaOH that reacted with the
6
28
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thereby resulting in the fluorescence emission changes. Figure
4
A shows the fluorescence emission spectra of pyrene in
1
Figure 2. FT-IR spectra (A) of CS, OCMCs and DCMCs, and H
NMR spectra (B) of DCMCs.
Figure 4. Fluorescence spectra of pyrene in different DCMCs
−4
−3
−3
−2
concentrations (1.0 × 10 , 1.0 × 10 , 2.0 × 10 , 5.0 × 10 , 1.0
−
1
−1
×
10 , and 1.0 mg·mL ) (A), and intensity ratio (I372/I383) versus
the DCMCs concentration (B).
−
4
DCMCs solution with concentrations varying from 1.0 × 10
−1
to 1.0 mg·mL . The results indicated that the fluorescence
intensity increased with increasing the concentration of
DCMCs. The pyrene solubility in water is extremely low, and
its dissolved fraction produces negligible fluorescence. When
the concentration is above CMC, the hydrophobic phase
(
micelle core) was formed, which solubilizes pyrene and results
in increased solution fluorescence. Plots of pyrene I (372 nm
1
/
I3 (383 nm) ratio versus logarithm of DCMCs concentration
are shown in Figure. 4B. The scatters are fitted by using a
Figure 3. Conductimetric titration curve of OCMCs.
Boltzmann function, which is expressed as following:
(x+1.88)/Δx
y = 1.29 + 0.52/[1 + e
]
(6)
carboxymethyl groups of OCMCs. (V −V ) represents the
2
3
+
volume added to react with NH of CS. The final volume
where y and x is the pyrene I /I ratio and the DCMCs
3
1
3
(
from V to the end) indicated the excess amount of NaOH in
concentration logarithm, respectively. A common tangent can
be obtained by determining the midpoint, which is obtained by
using the second derivative of y (y″ = 0), and the CMC was
defined as the crossover point of common tangent and flat
3
the solution. The SD of OCMCs was determined according to
eq 1, and the SD value was 70.00%. The results obtained in the
current study was higher than that of the previous study.
Critical Micelle Concentration (CMC) of DCMCs. For
evaluation of the aggregation properties of DCMCs, the probe
fluorescence technique was applied to determine the CMC by
using pyrene as the fluorescence probe. Pyrene has a strong
hydrophobic character with a very low solubility in water and
tends to solubilize into the hydrophobic region of the micelles,
23
28
tangent. The CMC value of DCMCs was determined as
−1
approximately 23.00 mg·L , which was lower than other
similar CMCs-based micelle systems such as deoxycholic acid-
O-carboxymethylated chitosan−folic acid conjugates and
2
9,30
acylated CMCs.
These results indicated that DCMCs are
capable of forming self-organized liposomes easily and are also
6
29
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Journal of Agricultural and Food Chemistry
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capable of maintaining their integrity even upon strong
dilution.
Formulation of DC-Ls. Figure 5 illustrats the preparation
processes of DC-Ls via the thin-layer evaporation method.
Figure 6. TEM images of DC-Ls and DC-Ls@OA-Fe O . DC-Ls
3
4
Figure 5. Schematic illustration of the formation process of DC-Ls.
DCMCs and Chol (or with poorly water-soluble agents) were
dissolved in an organic solvent, which was then completely removed,
and the thin film was obtained. The thin film was hydrated using
deionized water (or with water-soluble agents) and sonicated to obtain
the liposomes. As a soluble agent, salidroside was dissolved in
deionized water and used to hydrate the thin film and thus obtained
the salidroside-load DC-Ls.
exhibit a nearly spherical bubble shape with slight aggregation (A,B),
DC-Ls@OA-Fe O exhibit property of core (OA-Fe O ) and shell
3
4
3
4
(DMCMs films) structure (C,D), and multilamellar films can be seen
on the surface of OA-Fe O (D).
3
4
DCMCs and Chol were dissolved in organic solvents, and a
thin film was obtained after removing the solvent via
evaporation. The DC-Ls solution was obtained after hydration
of thin film by adding an aqueous phase. The cross section of
DC-Ls (Figure 5) showed that the hydrophilic heads of the
DCMCs are oriented toward the water compartment, whereas
the lipophilic tails are directed toward the center of the vesicle.
Thus, the hydrophilic and lipophilic bioactive agents can be
loaded into the water compartment and lipid section of DC-Ls,
respectively.
Figure 7. Magnetic hysteresis loop of OA-Fe O and DC-Ls@OA-
Fe O .
3 4.
3
4
Characterization of DC-Ls. The morphology and bilayer
structure of DC-Ls were observed by TEM (Figure 6). The
results indicated that DC-Ls exhibit a nearly spherical bubble
charge were not captured by the reticuloendothelial cell
systems but were easily absorbed by the negatively charge
with slight aggregation (Figure 6A,B). For verifying OA-Fe O4
3
31
incorporated into DC-Ls, VSM was employed to study the
magnetic properties of the DC-Ls@OA-Fe O . From the
cellular membrane. Consequently, the bioavailability of
bioactive agents can be improved after encapsulation into
DC-Ls.
3
4
magnetic hysteresis loop of OA-Fe O and DC-Ls@OA-
3
4
Fe O (Figure 7), it was found that the saturation magnet-
The XRD of DCMCs, Chol, DC-Ls, and DCMCs/Chol
physical mixture were investigated, and the results are shown in
Figure 9A. CS has the property of the crystal form II at 2θ =
20.5°. However, the crystal peaks of DCMCs at 2θ were sharply
changed with shifting from 20.5° to 21.6°, and a new peak at 2θ
of 5° appeared. These results indicated that DCMCs molecules
have good crystal properties with a billayered lamellar
3
4
ization of OA-Fe O and DC-Ls@OA-Fe O was 66.45 and
3
4
3
4
−1
2
3.45 emμ·g , respectively. These results (Figures 6C and 7)
indicated that OA-Fe O was encapsulated into DC-Ls with
3
4
properties of core (OA-Fe O ) and shell (DCMCs films)
3
4
structure. Meanwhile, the multilamellar film structure was also
found on the surface of OA-Fe O nanoparticles (Figure 6D).
3
4
32
The particle size and ζ potential of DC-Ls were determined
and are shown in Figure 8. The average size of DC-Ls was
structure. The DCMCs peak of 21.6° and all Chol peaks
disappeared for DCMCs/Chol physical mixture, which
indicated that the interaction (i.e., hydrogen bonding and
hydrophobic interactions) between DCMCs and Chol was
32.21 nm with a narrow distribution (PDI = 0.277 < 0.5), and
the ζ potential was approximately +73.30 mV. A previous study
reported that the average size, PDI and ζ potential of the
traditional liposomes (PC-Ls) were 604.00 nm, 0.36, and +7.50
9
occurred. However, the peak at 20.9° appeared for DC-Ls after
hydration, which suggested that DC-Ls can form lamellar-like
structures as lipid.
2
1
mV, respectively. These results indicated that DC-Ls has
better properties than PC-Ls. Evidence has shown that
nanocarriers with a small particle size (<200 nm) and positive
The lipid physical state can change from an ordered gel phase
to a disordered liquid-crystalline phase when temperature is
6
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Journal of Agricultural and Food Chemistry
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changed. This change was defined as phase transition
temperature (T ). The DSC of DCMCs, DC-Ls, Chol, and
m
DCMCs/Chol physical mixture was investigated and is shown
in Figure 9B. The T values of Chol and DCMCs were 44.43
m
and 44.29 °C, respectively. However, the T of DCMCs/Chol
m
physical mixture and DC-Ls decreased and the values were
3
7.50 and 33.61 °C, respectively. The results indicated that the
structure of DCMCs was rearranged and interacted with Chol.
The Chol interaction with DCMCs would increase the rigidity
of the “fluid state” liposomal bilayers of DC-Ls, which results in
33
good thermal stabilization. The XRD and DSC results
suggested that DC-Ls have the same physiochemical properties
as that of PC-Ls.
Encapsulation Efficiency (EE) of DC-Ls. Compared with
DC-Ls, PC-Ls of salidroside loading was also developed and the
EE was only about 54.72%. Previous studies have also reported
that the EE of salidroside loading in PC-Ls was lower than
2
1,22
6
0.00%.
Fortunately, the EE of salidroside in DC-Ls
increased to 82.46%. The high EE (82.46%) may be attributed
to the multilamellar structure of DC-Ls, which favored the
15
formation of a high inner volume. EE depends on the inner
volume of liposomes for water-soluble compounds, thus, a large
inner volume of DC-Ls results in a high EE.
Release of Salidroside from DC-Ls. The release of
salidroside from salidroside solution (control sample), PC-Ls,
and DC-Ls was investigated in PBS solutions (pH 7.0) (Figure
1
0A). The release of salidroside from the control solution
occurred rapidly with a release rate of 86.44% after 6 h. As
expected, the salidroside release from liposomes was slower
than that in the control solution. The release rates were only
about 38.38% and 30.77% for PC-Ls and DC-Ls after 6 h,
Figure 8. Size distribution (A) and ζ potential (B) of DC-Ls.
Figure 10. In vitro release of salidroside from control solution, PC-Ls,
and DC-Ls in PBS solutions (A), and from DC-Ls in SGF, PBS, and
SIF solutions (B), respectively. Data represent the mean ± SD (n = 3).
Figure 9. XRD (A) and DSC (B) spectra of DCMCs, Chol, DCMCs/
Chol physical mixture, and DC-Ls.
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dx.doi.org/10.1021/jf4039925 | J. Agric. Food Chem. 2014, 62, 626−633

Journal of Agricultural and Food Chemistry
Article
2
Table 1. Correlation Coefficients (R ) and Release Exponent (n) of Different Models in SGF, PBS, and SIF Solution,
a
Respectively
SGF solution
PBS solution
SIF solution
stage 2
model
stage 1
stage 2
stage 1
stage 2
stage 1
2
2
2
First model
R
R
R
n
0.939
0977
0.897
0908
0.975
0.984
0.987
0.870
0.941
0.951
0.979
0.259
0.993
0.989
0.995
0.901
0.943
0.933
0.964
0.322
Higuchi model
Peppas model
0.983
0.948
0.767
0.252
a
Stage 1 and 2 is the burst (0−10 h) and sustained release (10−48 h) process, respectively.
respectively, and more interestingly, the release rate was 66.98%
and 82.33% for DC-Ls and PC-Ls after 50 h, respectively.
These results indicated that DC-Ls had a relatively longer
release time than PC-Ls, which may be due to the high molar
mass of DCMCs. Thus, DC-Ls can be used for sustained
release of salidroside.
multilamellar structure with positive charge and narrow size
distribution. Compared with traditional liposomes, DC-Ls have
higher thermal stabilization and encapsulation efficiency. The
release of salidroside from DC-Ls was initially burst release,
followed by sustained release. Furthermore, the case II
transport and case I Fichian diffusion were the main release
mechanisms for the whole release process. In conclusion, DC-
Ls may be a promising carrier system for the production of
functional foods that contain salidroside or other bioactive food
ingredients. However, further in vivo investigation is required
to demonstrate the efficacy and safety of DC-Ls.
To further investigate the effect of gastrointestinal environ-
ment on salidroside release from DC-Ls, the solutions of SGF
(
pH 1.2), PBS (pH 7.0), and SIF (pH 7.4) were used as release
media (Figure 10B). The results suggested that the release rate
of salidroside from DC-Ls decreased with decreasing pH value.
In the SGF solution, the H-bond between −COOH and −OH
caused the aggregation of salidroside in the inner portion of
AUTHOR INFORMATION
■
*
+
DC-Ls. Moreover, the repulsive force between H and the
Corresponding Author
Phone: +86-791-6634810. Fax: +86-791-6634810. E-mail:
huaxiong100@126.com.
positive charge of liposomal surface made DC-Ls more stable.
Consequently, the release rate of salidroside from DC-Ls was
lower in the SGF solution. On the other hand, the positive
charge on the surface of DC-Ls was partially neutralized with
Funding
−
This work was supported by the Planning Subject of “the
Twelfth Five-Year-Plan” National Science and Technology for
the Rural development of China (2013AA102203-05), and the
National Natural Science Foundation of China (31160317,
OH in the SIF solution, which leads to the destruction of DC-
Ls and an improvement in the release rate of salidroside.
Release Mechanism of DC-Ls. Figure 10B shows that two
stages were observed for the release of salidroside from DC-Ls.
The first stage of release was initially rapid and was considered
the burst release, which may be due to the unbound excess of
salidroside on the DC-Ls surfaces. The second stage was a
sustained release process. The burst release of salidroside may
help to reach the effective concentration rapidly in plasma,
whereas the sustained release would maintain the effective
concentration in plasma for a long time. Thus, the
bioavailability and biological half-life time of salidroside can
be improved after loading into DC-Ls.
2
1201098, 2126602, and 81360205)
Notes
The authors declare no competing financial interest.
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