Food Bioscience 41 (2021) 100923

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Ascorbyl palmitate effects on the stability of curcumin-loaded soybean

phosphatidylcholine liposomes
Junhua Li a, b, Cuihua Chang a, b, Jiali Zhai c, Yanjun Yang a, b, *, Haitao Yu c, **
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China
b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China
c
School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria, 3000, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: The effect of ascorbyl palmitate (AP) on the physical stability of soy phosphatidylcholine (SPC) liposomes and the
Liposome retention rate of curcumin loaded into SPC liposomes enriched with different concentrations of AP was studied.
Soy phosphatidylcholine Results showed that the transmittance, stability coefficient and particle size of liposomes were all influenced by
Ascorbyl palmitate
the addition of AP. The highest stability of AP-stabilized liposomes was achieved when the mass ratio of SPC to
Curcumin
AP was at 50:5 by increasing the surface charge, as indicated by an increase in ζ-potential from − 15.1 mV (pure
liposome) to − 35.4 mV (AP-enriched SPC). Beyond this ratio, excessive AP addition was unfavorable to the
stability of liposomes. Also at this 10% ratio, small angle X-ray scattering and differential scanning calorimetry
results showed the strongest interaction between AP and the SPC liposomes, with the altering of the bilayer
structure and the main phase transition temperature. Atomic force microscopy results showed that the addition
of AP did not obviously change the vesicle microstructure but increased the vesicle height and contact angle.
When curcumin was loaded into AP-enriched liposomes as a model bioactive, the best storage stability was
observed in the liposome formulation with 10% added AP, which correlated well with the physical stability of the
system. These results open up the possibility of AP-modified liposomes being developed as a multifunctional
formulation for encapsulating and delivering nutraceuticals.

1. Introduction (Guldiken et al., 2018; Toniazzo et al., 2014). As a kinetically stable

colloidal system, the physical instability is intrinsically driven by hy­
Liposomes are among the first and most successful delivery systems drophobic interactions, and leads to the flocculation/aggregation of li­
developed for bioactives, nutraceuticals, and drugs (Dutta et al., 2019; posomes during prolonged storage (Zhou et al., 2014).
Jiao et al., 2018). Liposomes are spherical shaped lipid-based vesicles To improve the physical stability of liposomes, particle repulsion
that can be formulated using various preparation methods (Riaz, 1996). forces might be increased either through steric and/or electrostatic
Phospholipids are the most commonly used molecules for liposome mechanisms. The steric repulsion among particles is often increased by
preparation (Akbarzadeh et al., 2013; Shah et al., 2019; Shailesh et al., adding some hydrocolloids or biopolymers such as a gum or lipid
2009). In a single liposome particle, lipid molecules self-assemble into a polyethylene glycol (PEG) derivatives (Caddeo et al., 2018). This steric
spherical structure with a large, empty hydrophilic core and one or mechanism relies on the hydrophilic part of the biopolymers extending
multiple hydrophobic bilayers surrounding the core. The presence of into the aqueous phase and imparting steric hindrance to prevent the
this structure is beneficial to encapsulate various components with particles from coming close to each other. On the other hand, electro­
different polarities, providing substantial opportunities for the encap­ static repulsion refers to increasing the surface charges (usually indi­
sulation of multiple bioactive compounds (Hasan et al., 2014; Lopes cated using the ζ-potential) of the liposome particles to increase
et al., 2019; Marsanasco et al., 2011; Shaker et al., 2017; Vergara & repulsion. Previous studies have shown that increasing the ζ-potential of
Shene, 2019). However, the main limitations of liposome application as colloidal dispersions was effective in improving the physical stability
an effective delivery system come from physical and chemical instability (Gallardo et al., 2005; Gao et al., 2014). A large positive or negative

* Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
** Corresponding author.
E-mail addresses: [email protected] (Y. Yang), [email protected] (H. Yu).

https://doi.org/10.1016/j.fbio.2021.100923
Received 25 March 2020; Received in revised form 25 January 2021; Accepted 11 February 2021
Available online 15 February 2021
2212-4292/© 2021 Elsevier Ltd. All rights reserved.
J. Li et al. Food Bioscience 41 (2021) 100923

ζ-potential of nanoparticles indicates good physical stability of nano­ using the same preparation method.
particle suspensions. An absolute ζ-potential value > 30 mV is generally To investigate the effect of the surface charge state on the stability of
considered to have sufficient repulsive force to obtain better physical liposomes, all samples were prepared separately using 0.15 mol/L NaCl
colloidal stability due to electrostatic repulsion of individual particles (Sinopharm) using the same procedure described above. The curcumin-
(Joseph & Singhvi, 2019). Many factors including material properties, loaded liposomes were prepared with a mass ratio of curcumin to SPC of
presence of additives, and the ionic strength (salt conditions of the bulk 5:50. All the samples were allowed to sit at 4 ◦ C for 24 h before the
environment) can greatly affect the electrostatic repulsion of nano­ measurements.
particle dispersions (Cheng et al., 2019; Cheung & Al-Jamal, 2019;
Zidovska et al., 2009). 2.3. Transmittance and stability coefficient
In this study, the stability of liposomes was improved using elec­
trostatic repulsion after the addition of ascorbyl palmitate (AP). The The transmittance of liposome samples was measured at 600 nm with
amphiphilic structure of AP, a palmitic acid ester of ascorbic acid, may a UV–Vis Cary 60 spectrophotometer (Agilent Technologies, Wilming­
result in electrostatic repulsion of liposomes as its palmitate part in­ ton, DE, USA) using the method of Kawakami et al. (2001). Liposome
teracts with the lipid bilayers and its ascorbic acid group (with a pKa = samples were diluted to 0.05% SPC (w/v) with deionized water before
4.2) ionizes and results in negative charges on the particle surface under the measurement.
neutral pH conditions. AP has been used as an antioxidant in foods, To evaluate the stability coefficient (SC) of liposome dispersions, the
cosmetics and pharmaceuticals, a preservative for various edible oils method of Li et al. (2019) was used. The transmittance of the diluted
(Gosenca et al., 2013; Upadhyay et al., 2017; Upadhyay & Mishra, liposomes (0.05% SPC, w/v) and the supernatant of diluted liposomes
2015), and is therefore safe and biocompatible. In addition, AP is being samples after a mild centrifugation using an Eppendorf centrifuge
studied as an additive to stabilize colloidal systems such as liposomes (5430R, Eppendorf South Pacific Pty. Ltd., Macquarie Park, New South
and emulsions as bioactive carriers (Gopinath et al., 2004; Zariwala Wales, Australia) at 2000×g for 10 min at 25 ◦ C were measured at 600
et al., 2014). nm. The SC was calculated using the following equation:
The aim of this study was to evaluate the effect of AP as an additive in
a liposome formulation to improve the physical stability and bioactive SC =
A2
× 100% (1)
retention ratio of model liposomes made of commonly used soy phos­ A1
phatidylcholine (SPC). The study investigated a range of physicochem­
where A1 = the transmittance of the diluted liposome samples, and A2 =
ical parameters of AP-enriched liposomes, including transmittance,
the transmittance of the supernatant. Measurements of each sample
centrifugal stability coefficient, particle size distribution, ζ-potential,
were carried out in triplicate.
surface tension and membrane micropolarity. In addition, AP-induced
changes of the lipid bilayer structure, the phase transition tempera­
ture, and the surface morphology of the liposomes were characterized 2.4. Z-average particle size, polydispersity index (PDI) and ζ-potential
using small angle X-ray scattering (SAXS), differential scanning calo­ measurements
rimetry (DSC), and atomic force microscopy (AFM), respectively. As a
proof-of-concept study to investigate bioactive-loading applications, the The Z-average particle size, PDI and ζ-potential of the liposome
AP-containing liposomes were used to encapsulate curcumin and samples were determined using a Malvern Zetasizer Nano ZS (Malvern
assessed by examining the curcumin retention ratios in these systems. Instruments Ltd., Malvern, Worcestershire, UK). The Z-average gives an
intensity-based average particle size measurement of a given sample
2. Material and methods based on a cumulative fit to the initial part of the correlation function,
while the PDI is a measure of the breadth of particle size distribution in a
2.1. Materials given polymer sample which is defined as weight average divided by the
number average particle size (or molecular weight). For these mea­
SPC (94.5%) was provided by Lipoid GMBH (Ludwigshafen, Ger­ surements, the scattering angle was set as 173◦ and the refractive index
many). Pyrene (99.0%) and curcumin (curcuminoid content 94.0%, was 1.49 for phospholipids (Tai et al., 2018). ζ-potential is the potential
curcumin 80.0%) were purchased from Sigma Aldrich Co. (St. Louis, difference between the dispersion medium and the stationary layer of
MO, USA). Food grade AP (95.0%) was obtained from Dongguan City fluid attached to the dispersed particle, which is determined using the
Geen Food Technology Co., Ltd. (Dongguan, Guangdong, China). All Smoluchowski relation of the ionic mobility with the surface charge
chemicals were used without further purification. All other chemicals (Kumar & Dixit, 2017; Sze et al., 2003). Diluted liposome samples
were analytical grade bought from Sinopharm Chemical Reagent Co., (0.05% SPC, w/v) measured in the Zetasizer Nano ZS (Malvern In­
Ltd. (Shanghai, China). struments) were the same as the samples used for transmittance. Mea­
surement of each sample was carried out in triplicate at 25 ◦ C after 1 min
2.2. Sample preparation of equilibration in the sample chamber. All calculations were done using
the software within the instrument.
Samples were prepared using the ethanol injection method (Sebaaly
et al., 2016) with modifications described below. Briefly, 50 mg/mL SPC 2.5. Surface tension and membrane micropolarity
and AP (0, 0.1, 1, 5, 10 and 25 mg/mL) were dissolved together in ab­
solute ethanol (Sinopharm) at room temperature (23–25 ◦ C). This so­ The surface tension of the diluted samples (0.05% SPC, w/v) was
lution was dropped into Milli-Q ultrapure water (Millipore Co., Billerica, determined using a Delta-8 high throughput tensiometer (Kibron Inc.,
MA, USA) with a 1.2 mL final volume according to the volume ratio of Helsinki, Finland) at 25 ◦ C. Before the measurement, the tensiometer
1:5. It formed a milky aqueous phase. The final liposomes (SPC: 10 (Kibron) was calibrated with ultra-pure water at = 72.8 mN/m and test
mg/mL and AP: 0, 0.02, 0.2, 1, 2 and 5 mg/mL) were obtained by data should be within the range of 72.8 ± 0.5 mN/m. The metal rods are
removing the superfluous ethanol using evaporation at 40 ◦ C under a automatically cleaned between measurements by exposure to high
reduced pressure (0.08 MPa) using a vacuum oven (Laboratory Equip­ temperature (~1000 ◦ C, as specified by the manufacturer). Samples
ment Pty. Ltd., Marrickville, New South Wales, Australia). The lipo­ were loaded into a 96-well plate and measured in triplicate.
somes prepared at different mass ratio of SPC to AP, 50:0, 50:0.1, 50:1, The membrane micropolarity of the liposome samples was assayed
50:5, 50:10, 50:25 were designated as 0AP, 0.1AP, 1AP, 5AP, 10AP and using fluorescence probe pyrene (Tai et al., 2018). Aliquots of pyrene
25AP, respectively. Free AP control (APC) was prepared with 10 mg/mL (Sigma Aldrich) solution (2 mM in acetone) were added to the diluted

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J. Li et al. Food Bioscience 41 (2021) 100923

liposomes (SPC 0.05%) at a volume ratio of 1:50. This solution was

mixed using an Ohrus vortex mixer (Ohrus Corp., Port Melbourne,
Victoria, Australia) and incubated overnight at 4 ◦ C. The pyrene fluo­
rescence spectra of the samples were collected using an Ensight multi­
mode plate reader (PerkinElmer Inc., Waltham, MA, USA) at the
excitation wavelength of 337 nm and the emission wavelength range of
350–450 nm. Membrane micropolarity was determined as the I1/I3
ratio of the fluorescence intensity of the pyrene monomer peak 1 (~373
nm, I1) and peak 3 (~384 nm, I3). Measurement of each sample was
carried out in triplicate.

2.6. SAXS

SAXS data was collected on a Bruker Microcalix instrument (Bruker

Corp., Karlsruhe, Germany) using 50 W Cu-Kα radiation at 1.54 Å under
vacuum. Samples (10 mg/mL SPC for liposomes) were transferred into
glass capillary tubes and sealed using epoxy glue to prevent sample loss
during the analysis. The exposure time was set to 15 min. The bilayer
structure of the liposomes was determined by the relative position of the
scattering peak corresponding to the scattering vector (q). The repeat
distance (d-spacing) was calculated according to Woolf-Bragg’s equa­
tion (Tang et al., 2007):
2π
d = (2)
q1

where q1 is the scattering vector of the first reflection. Samples were

measured twice and representative patterns are shown in Fig. 4a.

2.7. DSC

The DSC measurements were done on a DSC 2 instrument (Mettler-

Toledo Ltd., Port Melbourne, Victoria, Australia), equipped with a liquid
nitrogen cooling system. To avoid the interference of water, samples
used for DSC measurements were vacuum dried (40 ◦ C, − 0.08 MPa)
overnight. Dried samples (~10 mg) were weighed into 50 μL aluminum
pans and hermetically sealed. The reference sample was a blank
aluminum pan. The sample pan was placed in the calorimeter and
isothermally held at − 50 ◦ C for 5 min, then heated at a rate of 10 ◦ C
Fig. 1. Transmittance (a) and stability coefficient (b) of SPC (0.5 mg/mL) li­
min− 1 to 50 ◦ C. For the AP control sample, the heating endpoint was
posomes at different mass ratios of SPC:AP: 50:0 (0AP), 50:0.1 (0.1AP), 50:1
extended to 150 ◦ C. Two replicates were analyzed for each sample. The
(1AP), 50:5 (5AP), 50:10 (10AP), 50:25 (25AP) and AP control (APC). Data are
peak temperature at the lowest point of the curve (melting peak tem­ shown as mean ± SD. Means with different letters (capital or lowercase) indi­
perature, Tm, ◦ C) was analyzed using STARe software (Mettler-Toledo) cate significant differences (p < 0.05) among the respective sample series.
from the DSC curve.

m2
2.8. AFM Retention ratio = × 100% (3)
m1

Liposome samples were diluted and deposited onto mica disks and where m1 and m2 were the curcumin concentration in liposomes before
dried at room temperature. AFM images of the prepared samples were and after storage. Measurement of each sample was carried out in
acquired in air using a Dimension Icon AFM instrument (Bruker Corp.) triplicate.
with a ScanAsyst-Air tip operating in peak force mode with quantitative
nanomechanical mapping in air. Analyses of images were done using 2.10. Statistical methods
NanoScope Analysis 1.5 (Bruker Corp.) and representative images are
shown in Fig. 5. All measurements were done at least in triplicate except those
specified otherwise, and data were expressed as mean ± standard de­
2.9. Retention ratio of curcumin in liposomes viation (SD). One-way ANOVA (analysis of variance) was done using the
Statistical Package for the Social Sciences (SPSS) software ver. 17.0 (IBM
The storage stability of curcumin-loaded liposomes (10 mg/mL SPC) Corp., Armonk, NY, USA) with the application of Duncan’s test to
was assessed as the retention ratio of curcumin after 1 and 7 days of determine the significant difference among samples at the 95% confi­
storage at 4 ◦ C using the method of Jin et al. (2016). The supernatant of dence level (p < 0.05). To elucidate the inherent link among different
the sample using natural sedimentation was diluted with absolute indicators, correlation analysis (significant correlations at the 95%
ethanol (Sinopharm). The concentration of the curcumin in the super­ confidence level, p < 0.05; highly significant correlations at the 99%
natant was determined using the spectrophotometer at 426 nm. The confidence level, p < 0.01) was determined using Pearson’s correlation
retention ratio (%) of curcumin in liposome suspension was calculated coefficient in bivariate linear correlations using the SPSS software.
using the following equation:

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J. Li et al. Food Bioscience 41 (2021) 100923

Fig. 3. Surface tension (a), pyrene fluorescence spectra (b), and I1/I3 ratio (b,
inset image) of SPC (0.5 mg/mL) liposomes at different mass ratios of SPC:AP:
50:0 (0AP), 50:0.1 (0.1AP), 50:1 (1AP), 50:5 (5AP), 50:10 (10AP), 50:25
(25AP) and AP control (APC). Data are shown as mean ± SD. Means with
different lowercase letters indicate significant differences (p < 0.05)
among samples.

3. Results and discussions

3.1. Transmittance and stability coefficient

The effect of adding different concentrations of AP on the floccula­

tion state of the liposome dispersions with and without 0.15 M NaCl in
the aqueous solution were investigated using the transmission study and
the results are shown in Fig. 1a. Generally, high transmittance indicates
homogeneous colloidal systems with small particle sizes. In the absence
of 0.15 M NaCl, transmittance of liposomes initially increased from 35%
(pure liposomes) with increasing concentrations of AP, reaching a
Fig. 2. Particle size (a), PDI (b) and ζ-potential (c) of SPC (0.5 mg/mL) lipo­ maximum (83%) at the ratio of SPC to AP 50:5 (5AP). However, higher
somes at different mass ratios of SPC:AP: 50:0 (0AP), 50:0.1 (0.1AP), 50:1 concentrations of AP led to a slight decrease of the transmittance. In the
(1AP), 50:5 (5AP), 50:10 (10AP), 50:25 (25AP) and AP control (APC). Data are presence of 0.15 M NaCl, the effect of adding AP on the transmittance of
shown as mean ± SD. Means with different letters (capital or lowercase) indi­ the liposomes followed the same trend. However, the addition of NaCl
cate significant differences (p < 0.05) among the respective sample series. caused a significant decrease in the transmittance of all samples
compared to the samples in pure water with the same composition. This
probably reflected the masking effect of salts on the effective surface
charge and hence the particle size distribution (transmittance) of the

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J. Li et al. Food Bioscience 41 (2021) 100923

stability of the dispersion. The decrease of PDI with increasing AP

concentrations indicated that the size distribution of the liposomes
became smaller and the sample became more homogenous. Sample 5AP
had the smallest Z-average particle size and PDI. The particle size
increased as the AP increased further whereas the PDI continued to
decline. The former could be related to the large particle size of the AP
control dispersions, while the latter might be attributed to the negative
charges that AP provided, which can increase the electrostatic repulsion
among liposomal vesicles, thus inhibiting the vesicle fusion and
decreasing the PDI. As shown in Table S2, there was a positive corre­
lation (p < 0.01) between PDI and ζ-potential values. Additionally, the
incorporation of NaCl into liposomes resulted in an increase in Z-average
particle size and PDI. These results were consistent with the decrease of
transmittance and SC shown in Fig. 1 with the addition of NaCl, con­
firming again the shielding effect of the salts on the particle surface
charge.
The ζ-potential of the liposomes without or with NaCl is shown in
Fig. 2c. Sample 0AP showed the lowest ζ-potential, which was consistent
with previous studies on pure SPC liposomes (Di Sotto et al., 2018). In
comparison, all SPC-AP composite liposomes showed significantly
higher absolute ζ-potential values. The absolute ζ-potential value of 5AP
was almost 2.5 times higher than that of the liposomes without AP. The
ζ-potential reached the maximum with 10AP, and additional AP did not
increase the absolute ζ-potential values, indicating that the excessive
amounts of free AP in the system.
The increased absolute ζ-potential values of liposomes was possibly
due to ionization of the ascorbic acid headgroup of AP at neutral pH
conditions, and hence provided a high energy barrier preventing particle
aggregation and flocculation. A correlation analysis between all the
physical parameters investigated so far is shown in Table S2. These re­
sults indicated that increased negative surface charge contributed
significantly to the decreases in particle size and PDI of liposomes, thus
enhancing the physical stability of liposomal vesicles. The addition of
NaCl significantly decreased the negative charges of liposomes (Fig. 2c).
The Na+ adsorbed on the surface of liposomes may shield charges on the
surface of liposomes, resulting in the decrease of electrostatic repulsion
among liposomes. These results further supported the results of SC
analysis and showed the importance of the electrostatic repulsion for the
stabilization of liposome dispersions. Similar results were obtained in
previous studies which have shown that liposome stability depends on
Fig. 4. SAXS profiles (a), and DSC curves (b) of SPC (0.5 mg/mL) liposomes at
its charge state that can be significantly modulated by the pH and NaCl
different mass ratios of SPC:AP: 50:0 (0AP), 50:0.1 (0.1AP), 50:1 (1AP), 50:5
(5AP), 50:10 (10AP), 50:25 (25AP) and AP control (APC). concentration (Alkhalaf et al., 1989; Wei et al., 2020).

3.3. Surface tension and membrane micropolarity

particles, which was further supported by the ζ-potential study discussed
below. When the AP concentration was >10% SPC, adding NaCl
Fig. 3a shows that the surface tensions of SPC-AP composite lipo­
significantly decreased the transmittance of liposomes (Samples 10AP
somes were significantly lower than the control liposomes without AP.
and 25AP). Free AP suspensions had low transmittance (~20%). This
The surface tension was gradually lowered to 47.8 mN/m (25AP) with
probably reflected the high aggregation and heterogeneous nature of the
increasing AP. The pure AP control had the lowest surface tension,
free AP suspensions.
indicating its amphiphilic nature and strong ability to lower surface
The SC measures the changes of transmittance of the liposome sus­
tension at interfaces. These results indicated that AP can insert into the
pension after centrifugation (Li et al., 2019). A SC value of 1 represents
SPC bilayers and act as a potent co-amphiphile for lowering the surface
the most stable liposomes formulations. Fig. 1b shows that the SC of the
tension. These results were consistent with a previous study which
liposomes with or without 0.15 M NaCl kept increasing until it almost
showed AP interacted with model phospholipid monolayers at air-water
reached 1 with 5AP. The addition of NaCl led to an overall decrease in
interfaces and significantly lowered the surface tension, measured using
SC of the liposomes regardless of the composition, which was consistent
a Langmuir trough (Mottola et al., 2013). As shown in Fig. 2, the
with the transmittance results in Fig. 1a. Further addition of AP did not
incorporation of AP in liposomes was beneficial to form particles with
improve the physical stability of liposomes especially in the presence of
smaller sizes by increasing electrostatic repulsion, thus increasing the
NaCl. The transmittance and SC of the AP-enriched liposomes correlated
adsorption of liposome at air-water interface, subsequently reducing the
well with the particle size and ζ-potential studies discussed below.
surface tension. Table S2 showed that there was a significant positive
correlation (coefficient: 0.823, p < 0.05) between surface tension and
3.2. Z-average particle size, PDI, and ζ-potential PDI. This suggested that lower surface tension could be beneficial to the
formation of homogenous liposomal vesicles. Similar effects have been
Fig. 2a and b shows the particle size and PDI of the AP-enriched li­ reported for the liposomal vesicles sizes and PDI that could be effectively
posomes. Adding AP decreased the average particle size and the het­ decreased after adding some Tween 80 which has a low static surface
erogeneity of the SPC liposomes, which was beneficial for the physical tension (38.3 mN/m) (Ahad et al., 2018; Samaras et al., 2014).

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J. Li et al. Food Bioscience 41 (2021) 100923

Fig. 5. AFM images and height section analysis obtained from selected liposomes at the mass ratios of SPC:AP: 50:0 (0AP) (a), 50:1 (1AP) (b), 50:5 (5AP) (c), 50:10
(10AP) (d) and AP control (APC) (e). The line graphs below the AFM images are the results of the section height analysis profile of the solid line in the corresponding
AFM images. (f): Changes in the average contact angle obtained from three vesicles of different samples.

The pyrene emission spectra and the calculated fluorescence in­ and DSC analysis described below.
tensity ratio of I1/I3 are shown in Fig. 3b. The fluorescence intensity of
the pyrene emission spectra in liposomes with AP was apparently lower
than that in blank liposomes. The control sample APC had low fluores­ 3.4. SAXS and DSC
cence intensity, indicating the presence of a lipophilic group (palmi­
tate), which could be helpful to the formation of a more compact The SAXS data obtained for liposomes containing different AP con­
hydrophobic membrane structure. The micropolarity (I1/I3 ratio) also centrations are shown in Fig. 4a. For 0AP, two scattering peaks char­
decreased when AP was incorporated. These results further confirmed acteristic of the lamellar bilayers with a d-spacing of 6 nm were
that the polarity of micro-environment in liposome bilayers decreased observed, which was consistent with previous studies (Neunert et al.,
with the incorporation of AP. However, the control sample APC has the 2018). There was no shift in the position of the scattering peaks of li­
highest I1/I3 ratio, implying that AP itself could not effectively form a posomes at low ratios of SPC:AP (0.1AP and 1AP), indicating the bilayer
micelle to encapsulate the hydrophobic pyrene probe. structures were not affected by adding small amounts of AP. As the
Based on the above surface tension and fluorescence analysis, it was concentration of AP increased (5AP and 10AP), the first characteristic
concluded that the AP could penetrate into the membrane and its scattering peak shifted to a higher q value corresponding to a d-spacing
palmitate part associates strongly with the hydrophobic domain of the of 5.66 nm, indicating that the incorporation of AP had a compression
phospholipid bilayer. Additional evidence was provided by the SAXS effect on the bilayer.
There was a small sharp peak appearing at a q value of ~0.14 Å− 1 for

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J. Li et al. Food Bioscience 41 (2021) 100923

25AP, which was the same position as the scattering peak of the pure AP saturated fatty acid (due to the palmitate part of the AP molecule),
control. Although the current SAXS pattern did not show a second peak resulting in limited spreading of the liposomes on the mica surface. The
in the pure AP control sample, the first peak at 0.14 Å− 1 was found to be flake morphology of AP control (APC) in Fig. 5e was consistent with a
consistent with a previous study which also studied pure AP in water previous study, which had also shown that a lamellar liquid crystal of AP
using SAXS (Mottola et al., 2013). With this neutral condition at room is easily formed (Benedini et al., 2011).
temperature, pure AP in water showed a lamellar phase. The appearance
of this peak at 0.14 Å− 1 in the AP-enriched liposomes indicated the
presence of excess AP in the bulk environment at this high concentra­ 3.6. Retention ratio of curcumin
tion. The peaks corresponding to the SPC liposomes were restored to the
original q value observed in the pure SPC sample. These results sug­ The storage stability of bioactive-loaded liposomes is an important
gested that there is a maximum loading capacity of the SPC liposomes aspect with regard to applications as functional food products. To assess
for AP, which in the current study was in the range of 5AP and 10AP. the application of the AP-enriched liposomes, curcumin was used as a
Previous results showed that where the maximum insertion of the AP model bioactive or drug and its storage stability was examined. As
into the SPC bilayers is achieved (10AP), the resulting liposomes were shown in Fig. 6a, the retention ratio of curcumin in liposomes after
most homogenous and stable, with the smallest average particle size as storage correlated well with the SC analysis shown in Fig. 1a. These
described in Sections 3.1 and 3.2. results indicated that the curcumin stability in liposomes largely
The heating scan of pure, dehydrated SPC liposomes (Fig. 4b) depended on the physical stability of the liposomes. After 7 days of
showed a broad, unsymmetrical peak at low temperatures (− 48.0 to storage, the retention ratio (23.2%) of curcumin in the liposomes
0.0 ◦ C), suggesting its multicomponent nature containing mainly un­ without AP (0AP) was the lowest in the selected curcumin-loaded li­
saturated phospholipids containing oleic, palmitoleic, linoleic, and posomes; 5AP had the highest retention ratio (52.2%). This was
α-linolenic hydrocarbon chains (Jangle et al., 2013). Furthermore, there consistent with the sample pictures in Fig. 6b where the sediment of
were two endothermic peaks from − 31.4 to − 11.7 ◦ C, corresponding to curcumin-loaded liposomes are shown for 0AP and 10AP. 5AP had the
the pretransition and main gel-to-fluid phase transition temperature of highest retention ratio due to the optimal interaction between AP and
the liposomes, respectively. An early study has also observed the the SPC bilayers.
pre-transition and main transition temperatures for liposomes, which The curcumin in liposomes made of bovine milk or krill phospho­
varied greatly with lipid-lipid interaction (Eklund et al., 1984). Incor­ lipids had a retention ratio >80% (Wu et al., 2020). Another study also
poration of AP into the SPC membrane significantly affected the DSC reported high retention ratio (>80%) of curcumin loaded in egg PC
curves and melting point of the mixtures. Increasing the concentrations
of AP embedded into SPC membranes led to the main phase transition
peak becoming more intense and narrower, suggesting the insertion of
AP into the SPC bilayers. The phase transition temperature of the main
endothermic peak was increased from − 1.7 to 6.1 ◦ C. These results
could also be ascribed to the overall increase in the saturated fatty acid
chain of liposomes with the addition of AP containing the saturated
16-carbon palmitic acid hydrocarbon chain, which increased the crys­
tallinity and saturation of lipid systems (Yang et al., 2018). On the other
hand, the addition of low melting point unsaturated 1,2-dioleoyl-sn-gly­
cero-3-phosphocholine (DOPC) into the high melting point saturated 1,
2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been observed
to induce the occurrence of a gel/fluid phase transition at a lower
temperature (Chen & Santore, 2014; Mills et al., 2009).
Overall, the SAXS and DSC results showed strong interactions be­
tween AP and SPC bilayers of liposomes. This was consistent with pre­
vious studies which have shown strong interactions between AP and
model phospholipid monolayers (Gosenca et al., 2013; Gosenca & Ga
Perlin, 2011). The above results also showed that the strongest inter­
action between SPC bilayers and AP occurred with 5AP, and led to the
best physical stability of the liposomes as indicated by transmittance,
SC, particle size, and PDI analysis.

3.5. AFM

The morphology and size distribution of the AP-enriched liposomes

were examined using AFM. As shown in Fig. 5a–d, all liposome samples
aggregated into spherical caps with different sizes. This was consistent
with the observation from SEM and AFM (Tai et al., 2018). The size of
the aggregated liposomal vesicles was higher than the Z-average particle
size, which was attributed to vesicle fusion when it was dried on the
mica substrate during sample preparation. With the increase in AP
content, the proportion of vesicles with bigger particle sizes seemed to
decrease. The decrease of vesicle fusion can be related to the high ab­
solute ζ-potential value of SPC-AP composite liposomes, which provided Fig. 6. Retention ratio (a) of curcumin in liposomal suspensions after 1 and 7
enough electrostatic repulsion among liposomal vesicles. Additionally, days of storage and represented images of curcumin-loaded liposomes after 7
there was a significant increase in vesicle height and internal contact days storage (b). Data are shown as mean ± SD. Means with different letters
angle from 4.5◦ to 10◦ as the AP content increased. This may be due to (capital or lowercase) indicate significant differences (p < 0.05) among the
the incorporation of AP into SPC increasing the effective proportion of respective sample series.

7
J. Li et al. Food Bioscience 41 (2021) 100923

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