Colloids and Surfaces B: Biointerfaces 224 (2023) 113205

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Colloids and Surfaces B: Biointerfaces

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

Encapsulation of resveratrol within size-controlled nanoliposomes: Impact

on solubility, stability, cellular permeability, and oral bioavailability
Youjin Baek , Eun Woo Jeong , Hyeon Gyu Lee *
Department of Food and Nutrition, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, South Korea

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

Keywords: This study examined the influence of the nanoliposomes (LPs) particle size on the solubility, antioxidant stability,
Particle size in vitro release profile, Caco-2 cellular transport activity, cellular antioxidant activity, and in vivo oral
Nanoliposomes bioavailability of resveratrol (RSV). LPs with sizes of 300, 150, and 75 nm were prepared using the thin-lipid film
Resveratrol
hydration method, followed by ultrasonication for 0, 2, and 10 min, respectively. Formulating small LPs (< 100
Oral administration
Poorly soluble compound
nm) was effective to enhance the solubility, in vitro release profile, cellular permeability, and cellular antioxidant
Stability activity of RSV. A similar pattern was observed for in vivo oral bioavailability. However, the size reduction of
RSV-loaded LPs did not promote the antioxidant stability of RSV, owing to their large surface area used to
interact with harsh environments. This study provides the better understanding of the appropriate particle size
range of LPs to improve their in vitro and in vivo performances of RSV as an effective carrier for oral
administration.

1. Introduction of a hydrophilic core surrounded by a lipid bilayer, are considered po­

Resveratrol (3,5,6′ -trihydroxy-stilbene, RSV), one of the most high­
lighted naturally occurring antioxidants, is a polyphenol phytoalexin
generally abundant in plants such as soybean, peanuts, and grapes [1].
RSV exists as two geometric isomers, trans and cis-RSV; generally,
trans-RSV demonstrates higher therapeutic characteristics than cis-RSV
[2]. Previous studies have reported that RSV has the ability to prevent
obesity, inhibit cancer, and reduce the likelihood of cardiovascular
disease, owing to its high antioxidant activity [3,4].
However, the application of RSV is strictly restricted in the food in­
dustry owing to its degradation by high temperature, alkaline pH, or
ultraviolet light, resulting in the conversion of the trans to cis- form [5].
When administered orally, RSV would demonstrate low intestinal ab­
sorption and low bioavailability due to its poor aqueous solubility
(20–30 μg/mL) [1]. Moreover, the low bioavailability of RSV may be
explained by its conjugation with glucuronic acid and sulfates in the
liver and intestinal epithelial cells after administration, resulting in
rapid elimination [6,7]. Several attempts have been made to overcome
the limitations of RSV associated with solubility, stability, and
bioavailability by incorporating RSV into various lipid-based nano­
carriers, such as nanoliposomes, nanoemulsions, and nanostructured
lipid carriers [8–10]. Among them, nanoliposomes (LPs), which consist
tential lipid-based nanocarriers owing to their capacity to improve the
solubility, stability, and bioavailability of poorly water-soluble com­
pounds [11]. In addition, LPs have received significant attention for
applications in the food industry because of their high biocompatibility
and non-toxicity [12].
Particle size of LPs is one of the main factors influencing in vivo
performances of encapsulated compounds after injection [13]. In the
pharmaceutical industry, the in vivo performance of LPs with different
particle size ranges has been investigated to determine how the particle
size of LPs influences their biodistribution after lateral tail vein, intra­
muscular, and penile vein injection [14–16]. On the other hand, rela­
tively little attention in food industry has been paid to the effects of LPs
with different particle sizes on the properties of phytochemicals found in
food after oral administration. In general, LPs would be transported
across the intestinal mucosa by several mechanisms after oral ingestion;
endocytosis, paracellular uptake, and uptake by microfold cell (M-cell)
[17]. Endocytosis is one of passage mechanisms through the epithelial
cell membrane via transcellular route, which would embed the LPs and
form the membrane vesicles, followed by the release of compounds
within LPs into the cytoplasm [18–20]. Meanwhile, paracellular uptake
is the mechanism by which LPs pass through the intercellular space
between small intestinal epithelial cells controlled by the tight junctions

* Corresponding author.
E-mail address: hyeonlee@hanyang.ac.kr (H.G. Lee).

https://doi.org/10.1016/j.colsurfb.2023.113205
Received 7 December 2022; Received in revised form 2 February 2023; Accepted 10 February 2023
Available online 12 February 2023
0927-7765/© 2023 Elsevier B.V. All rights reserved.
Y. Baek et al.
[19,21]. Finally, M-cells, mainly located in Peyer’s patch, could uptake
LPs via Peyer’s patch and transport LPs directly to the lymphatic systems
[22]. Several previous studies have reported the enhanced cellular up­
take of bioactive compounds via those three mechanisms by nano­
encapsulation within LPs with small particle sizes because LPs with
particle size less than 100 nm have been reported to demonstrate high
cellular transport efficiency [23–26]. However, oral absorption could be
restricted by various physiological obstacles which could be managed by
the particular particle size range of LPs. Therefore, it is crucial to
investigate the overall in vitro and in vivo characteristics of LPs with wide
size ranges, including solubility, antioxidant stability, cellular proper­
ties, and in vivo oral bioavailability, for manufacturing effective orally
administered LPs.
To the best of our knowledge, the impacts of the particle size of LPs
on the in vitro and in vivo performances of RSV after orally administra­
tion, which are of great importance, are not fully investigated. Consid­
ering that the performance of RSV may differ depending on the particle
size of the LPs, this present study aimed to investigate how RSV-loaded
LPs with different particle sizes influence RSV solubility, antioxidant
stability, and in vivo oral bioavailability. RSV-loaded LPs consisting of L-
α-phosphatidylcholine, β-sitosterol, and resveratrol were formulated
using the thin-lipid film hydration method, and their sizes were
controlled by ultrasonication. The physicochemical features of the RSV-
loaded LPs, such as particle size, polydispersity index (PDI), zeta po­
tential (ZP), and entrapment efficiency (EE), were investigated. In
addition, the impact of size-controlled LPs on the characteristics of RSV,
such as solubility, antioxidant stability, in vitro release properties using
simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) models,
cellular transport assay, cellular antioxidant assay using Caco-2 mono­
layers, and in vivo pharmacokinetic assay after oral administration to
Sprague-Dawley (SD) rats were investigated.
2. Results and discussions
2.1. Physical properties of RSV loaded size-controlled LPs
2.1.1. Particle size, PDI, and ZP
The physical properties such as particle size, PDI, and ZP of the RSV-
loaded size-controlled LPs were illustrated in Table 1. RSV-loaded LPs
with ultrasonication times of 0, 2, and 10 min exhibited the particle size
of 318.6 ± 15.9 (LPs 300), 152.6 ± 7.4 (LPs 150), and 73.3 ± 4.9 nm
(LPs 75), respectively. Ultrasonication time longer than 10 min was
considered to have no significant effect on reducing the particle size of
RSV-loaded LPs as predetermined in our preliminary experiments. The
PDI of RSV-loaded LPs 300, 150, and 75 were 0.636 ± 0.070, 0.353 ±
0.075, and 0.244 ± 0.008, respectively. The PDI of LPs less than 0.5
indicates highly narrow particle size distribution, and the PDI higher
than 0.5 illustrates the extensive particle size distribution [27]. Ultra­
sonication times of 2 and 10 min significantly narrowed the PDI of the
LPs compared to LPs without ultrasonication, indicating that the uni­
formity of the LPs can be enhanced by the increasing the ultrasonication
time (p < 0.05) [28]. Thus, the longer the ultrasonication time, the more
sonication energy is delivered to the liposome suspension, leading to a
smaller particle size and a homogenous size distribution [29]. These
results indicated that ultrasonication time (0, 2, and 10 min) can highly
control the particle size and PDI of RSV-loaded LPs, without modifying
the lipid composition.
Table 1
Physical characteristics of resveratrol loaded size-controlled nanoliposomes (LPs).
Sample Ultrasonication time (min) Target size(nm) Measured size (nm)
LPs 300 0 300.0 318.6 ± 15.9a
LPs 150 2 150.0 152.6 ± 7.4b
LPs 75 10 75.0 73.3 ± 4.9c
a-c Different letters in the same column indicate significant differences (p < 0.05)
2
Colloids and Surfaces B: Biointerfaces 224 (2023) 113205
The ZP values of RSV-loaded size-controlled LPs ranged from − 47.7
± 0.7 to − 41.1 ± 5.2 mV. The ZP values in this study exhibited no
significant differences regardless of the ultrasonication time, indicating
that the influence of various ultrasonication times on the surface charge
of RSV-loaded LPs was minimal. In general, a higher ZP value is related
to better LPs stability because the ZP value illustrates the repulsion
between charged LPs, and absolute ZP values greater than 30 mV are
regarded as stable [11]. These results demonstrated that ultrasonication
time did not influence the surface charge of RSV-loaded size-controlled
LPs, leading to electrostatic stability.
2.1.2. EE
The EE of the RSV-loaded LPs 300, 150, and 75 were 80.88 ± 0.05,
80.24 ± 0.01, and 79.82 ± 0.02%, respectively, without significant
differences, as shown in Table 1. This result indicated that the amount of
RSV entrapped within the LPs was not affected by the ultrasonication
time. The high EE of RSV-loaded LPs, regardless of the ultrasonication
time, could be due to the affinity of the core materials for lipid bilayer.
Generally, hydrophobic components, entrapped within the lipid bilayers
of LPs, have a high affinity for lipid bilayers and low affinity for water,
resulting in high EE and low drug leakage [30]. In a previous study, the
authors formulated tacrolimus-loaded LPs with ultrasonication for 0–7
min to investigate the effects of ultrasonication time on the EE of
tacrolimus. The authors affirmed that the EE of tacrolimus was stable at
all ultrasonication times owing to its high affinity for lipid bilayers [31].
Hence, RSV-loaded LPs subjected to 0, 2, and 10 min of ultrasonication
exhibited three distinct particle sizes with high EE, indicating that these
size-controlled LPs successfully encapsulated RSV and could be consid­
ered adequate for further analysis.
2.2. Solubility of RSV-loaded size-controlled LPs
The effects of LPs size on RSV solubility were demonstrated in Fig. 1.
LPs 75, 150, and 300 increased the solubility of RSV by 13.49, 12.96,
and 12.04 times more, respectively, than RSV dissolved in water, sug­
gesting that encapsulation within LPs can significantly enhance the
solubility of hydrophobic compounds (p < 0.05) [11]. In addition, LPs
75 and 300 showed a significant difference in the aqueous solubility of
RSV, indicating that the particle size of the LPs can remarkably influence
Fig. 1. Solubility of free RSV, LPs 75, LPs 150, and LPs 300. a-c Columns with
different letters are significantly different from each other (p < 0.05).
Polydispersity index Zeta potential (mV) Entrapment efficiency (%)
0.636 ± 0.070a − 47.7 ± 0.7a 80.88 ± 0.05a
0.353 ± 0.075b − 41.1 ± 5.2a 80.24 ± 0.01a
0.244 ± 0.008c − 45.2 ± 7.1a 79.82 ± 0.02a
Y. Baek et al.
the solubility of RSV (p < 0.05). This result could be explained by the
fact that the smaller particle size of the LPs can generate a larger surface
area of encapsulated compounds, which can affect surface reactivity,
leading to enhanced aqueous solubility [32]. A previous study reported
that RSV-loaded nanoparticles (NPs) with a particle size of 72 nm
increased RSV aqueous solubility compared to NPs with a particle size of
269 nm owing to their larger contact surface area between RSV and
aqueous media [1]. Thus, this study confirmed that LPs 75, which
exhibited smaller particle sizes, appeared to have a higher aqueous
solubility of RSV than LPs 300.
2.3. Antioxidant stability of RSV-loaded size-controlled LPs
The antioxidant activity of RSV before and after thermal treatment at
90 ℃ for 4 h in its free and encapsulated forms with different particle
sizes was compared in Fig. 2. At 0 h, the DPPH radical scavenging effect
of free RSV was 91.19% and that of RSV-loaded size-controlled LPs was
ranged from 84.71% to 88.62%, indicating that antioxidant activity of
free RSV was higher than that of RSV encapsulated within size-
controlled LPs. Similar findings with the present study have been re­
ported that RSV-loaded LPs and proniosomes exhibited lower DPPH
radical scavenging effect than free RSV [4,33]. These results could be
explained by the location of hydrophobic compounds in LPs as afore­
mentioned [30]. As RSV is not prone to move across the liposomal
membrane due to its high affinity for lipid bilayer, it would be released
from LPs slowly; therefore, it requires more time to interact with the
reaction medium, resulting in lower antioxidant activity than its free
form.
On the other hand, the antioxidant activity of RSV at 4 h showed
entirely different trend. The antioxidant activities of all samples
exhibited a decreasing trend with the increase of thermal treatment
time, indicating that 90 ℃ was high enough to induce the thermal
degradation of RSV and LPs. The antioxidant activity of free RSV was
only 45.94%, while that of RSV-loaded size-controlled LPs was greater
than 56.63% after 4 h of thermal treatment, indicating that LPs could
enhance the thermal stability of bioactive compounds [34]. The differ­
ence in the antioxidant activities depending on the particle size of LPs
was clearly observed; the DPPH radical scavenging effect of LPs 75 was
significantly lower than that of LPs 300 (p < 0.05). Similarly, a previous
study illustrated that NPs with a particle size of 87 nm showed signifi­
cantly lower stability of RSV than those with a particle size of 258 nm
[1]. LPs with smaller particle sizes were highly exposed to the heat
owing to their larger surface area, which provides more sites for RSV
interaction, leading to higher degradation than LPs with larger particle
sizes. Thus, it is tempting to presume that the preparation of LPs with
large particle sizes may efficiently enhance the antioxidant stability of
Fig. 2. DPPH radical scavenging activity of free RSV, LPs 75, LPs 150, LPs 300
at 90 ℃ for 4 h. a-c Means with different letters indicate significant differ­
ences (p < 0.05).
3
Colloids and Surfaces B: Biointerfaces 224 (2023) 113205
core materials which are susceptible to degradation during heat
processing.
2.4. In vitro release profile of RSV-loaded size-controlled LPs
The in vitro release profile of RSV-loaded size-controlled LPs in the
SGF environment followed by that in the SIF environment was demon­
strated to investigate the characteristics of RSV being released in gastro-
intestinal tract (Fig. 3). Free RSV was rapidly released within the first
2 h, and the cumulative amount of RSV reached 81.40% after 12 h.
Meanwhile, the cumulative amounts of RSV released from LPs 75, 150,
and 300 were 52.90%, 45.44%, and 41.32%, respectively, after 12 h,
and LPs 75 released a significantly higher amount of RSV than LPs 150
and 300 (p < 0.05). Within the first 30 min, RSV that was free or
attached to the surface of the bilayer membranes immediately diffused
in the SGF environment, leading to the burst release of RSV [35]. After
30 min, all LPs samples exhibited delayed release of RSV within 2 h,
which could be explained by two different mechanisms. One was pepsin,
which cannot diffuse through bilayer membranes [36]. Another possi­
bility was the aggregation or precipitation of LPs structures due to
electrostatic interactions at low pH, which prevents RSV from being
released [37]. Similar results were obtained by Liu et al., who observed
aggregation of LPs structures as the ZP value of LPs changed to zero at
acidic pH [38].
On the other hand, the release profile of RSV in the SIF environment
demonstrated an entirely different trend owing to the presence of
pancreatin, which degrades the LPs structures primarily composed of
phospholipids. Likewise, previous literature has affirmed that LPs
showed a higher release of curcumin in SIF than in SGF environments
due to the lipid-degrading effect of lipolytic enzymes in pancreatin [39].
Moreover, LPs 75 in this study released a significantly higher amount of
RSV than LPs 150 and 300, indicating that the larger surface area of LPs
75 was more susceptible to the reaction with pancreatin to disrupt the
phospholipid bilayer. Therefore, the release profile of RSV within LPs in
the SGF environment suggests that encapsulating RSV within LPs may
protect RSV from acidic or enzymatic degradation along the way to the
small intestine. In particular, LPs with smaller particle sizes allow for the
release of higher amount of RSV into the SIF environment, which is
sequentially associated with bioavailability.
2.5. Cellular transport activity of RSV-loaded size-controlled LPs
The Papp of free RSV, LPs 75, 150, and 300 across the Caco-2
monolayers was demonstrated in Fig. 4. Compared to free RSV, the
Papp value of RSV increased by 1.53, 1.31, and 1.21 times through
nanoencapsulation within LPs 75, 150, and 300, respectively. The
Fig. 3. In vitro release profile of free RSV, LPs 75, LPs 150, and LPs 300. a-c
Means with different letters are significantly different from each
other (p < 0.05).
Y. Baek et al. Colloids and Surfaces B: Biointerfaces 224 (2023) 113205

Fig. 4. Apparent permeability coefficient (Papp) of free RSV, LPs 75, LPs 150, Fig. 5. Cellular antioxidant activity of free RSV, LPs 75, LPs 150, and LPs 300
and LPs 300 transported across Caco-2 cell monolayers. a-c Means with different in Caco-2 monolayer. a-c Columns with different letters indicates significant
letters are significantly different (p < 0.05). differences (p < 0.05).

results indicated that cellular transport of RSV significantly increased by monolayers than those with particle sizes over 100 nm [21,44]. Be­
nanoencapsulation within the LPs (p < 0.05). In addition, the Papp value sides, reduced particle size leads to an increased surface area between
of LPs was dramatically higher than that of LPs 150 and 300 (p < 0.05). entrapped RSV and 2′ ,7′ -dichlorodihydrofluorescein (DCFH), resulting
In general, free RSV in an aqueous solution with poor solubility is in decreased 2′ ,7′ -dichlorofluorescein (DCF) formation, followed by
limited to reaching the cell surfaces, while RSV-loaded LPs improved the increased antioxidant activity in the Caco-2 monolayer [32]. Therefore,
cellular permeability of RSV owing to their improved solubility [40]. LPs 75, which has a smaller size and higher surface area than other LPs
RSV-loaded LPs 75, which had the smallest particle size and the highest formulations, could have more chances to permeate the Caco-2 mono­
solubility of RSV, demonstrated the highest cellular permeability among layer and interact with DCFH, leading to lower degradation of DCFH to
other LPs formulations. DCF and prevention of free radical formation.
Additionally, nanocarriers can be transported into epithelial cell
layers via two routes. One is the paracellular route, which is controlled
2.7. In vivo bioavailability of RSV loaded size-controlled LPs
by tight junctions between the enterocytes. The transport of nano­
carriers via the paracellular route is limited because the width between
The mean plasma RSV concentrations after the oral administration of
enterocyte cells only increases to 20 nm, even though the tight junction
free RSV and RSV-loaded size-controlled LPs are shown in Fig. 6. The
opens entirely [21,41]. Nanocarriers with a positive charge, such as
plasma concentration of RSV was 0.404 ± 0.249 μg/mL at 1 h,
chitosan nanoparticles or chitosan-coated LPs, have positive effects on
decreased rapidly from 1 to 8 h, and then became undetectable after 8 h.
the opening of tight junction, which has a negative charge due to the
In contrast, the concentration of RSV in plasma was highly elevated by
presence of negatively charged sialic acid groups on the mucus mem­
incorporating within size-controlled LPs. The pharmacokinetic param­
brane [42,43]. In other words, RSV-loaded size-controlled LPs prepared
eters of free RSV, LPs 75, 150, and 300 after oral administration are
in our study may not affect the opening of intercellular tight junctions
demonstrated in Table 2. The Cmax and Tmax values of free RSV were
because of RSV-loaded size-controlled LPs’ negative charge. Another
0.404 ± 0.249 μg/mL and 1.08 h, respectively. In contrast, the Cmax of
mechanism is endocytosis via a transcellular route [19]. Generally,
LPs 75, 150, and 300 were 1.486 ± 0.427, 0.910 ± 0.174, and 0.684
nanocarriers can be easily transported into enterocytes when their
± 0.346 μg/mL, and Tmax values of LPs 75, 150, and 300 were at 2.67,
particle size is 50–100 nm, whereas nanocarriers with a particle size of
2.17, and 1.83 h, respectively. Moreover, the oral bioavailability of RSV
20–500 nm can be transported into M-cells, which can be expressed by a
increased by 12.531, 1.733 and 2.801 times by encapsulation within LPs
co-culture with Raji B cells [44]. This result indicated that modifying LPs
75, compared to that by encapsulation within free RSV, LPs 150, and
to a smaller size could potentially enhance the permeability of bioactive
300, respectively, based on the AUC0–12 values.
materials across the Caco-2 monolayer, leading to an increased chances
In general, free RSV exhibited a peak plasma concentration at
of reaching the bloodstream.

2.6. Cellular antioxidant activity of RSV-loaded size-controlled LPs

As shown in Fig. 5, the cellular antioxidant activity (CAA) unit of free

RSV, LPs 75, 150, and 300 in Caco-2 cell was 20.54 ± 2.63, 41.35
± 2.01, 35.03 ± 1.56, and 30.54 ± 1.69%, respectively, indicating that
the CAA of RSV can be increased significantly by encapsulation within
size-controlled LPs compared to that of free RSV (p < 0.05). Moreover,
among the size-controlled LPs, the CAA value of LPs 75 was significantly
higher than that of LPs 300 (p < 0.05). This result indicated that the
enhanced CAA value of RSV by incorporation into LPs with smaller
particle sizes may be attributed to the improved solubility and perme­
ability of RSV through the Caco-2 monolayer. The increase in CAA with
a decrease in the particle size of LPs may be attributed to two sequential
reasons: the particle size-dependent endocytosis mechanism and
increased surface area of LPs. As aforementioned, nanocarriers with Fig. 6. Mean plasma resveratrol concentration versus time profiles after oral
particle size of 50–100 nm are more prone to endocytosis in Caco-2 administration of free RSV, LPs 75, LPs 150, and LPs 300.

4
Y. Baek et al.
Table 2
Pharmacokinetic parameters, including Cmax, Tmax, AUC0–12, and MRT after oral administration of free RSV, LPs 75, LPs 150, and LPs 300 at 20 mg/kg body weight.
Formulation
Dose mg /kg body weight
Pharmacokinetic parameters
Peak plasma concentration (Cmax) [μg/mL]
Time to reach peak plasma concentration (Tmax) [h]
Area under curve0–12 (AUC0–12) [μg/mL×h]
Mean residence time (MRT) [h]
Data presented as mean ± standard deviation (n = 6). * Values are significantly different from free resveratrol at the level of p < 0.05. ** Values are significantly
different from free resveratrol at the level of p < 0.01. *** Values are significantly different from free resveratrol at the level of p < 0.001.
approximately 0.5–1 h, and the oral bioavailability of RSV has been
reported to be less than 1% [45]. Moreover, as mentioned above, RSV
demonstrates low bioavailability due to highly rapid glucuronic acid
and sulfate conjugation in the intestine and liver [6,7]. Our study
affirmed that encapsulation within LPs could greatly enhance the oral
bioavailability of RSV, mainly owing to the increment of RSV solubility,
stability in the gastrointestinal tract, and absorption properties [11,46].
Among the size-controlled LPs, LPs 75 showed the highest Cmax, Tmax
AUC0–12, and MRT, indicating that controlling the particle size is a
crucial factor in improving the oral bioavailability of RSV. The differ­
ences in RSV oral bioavailability between size-controlled LPs may be
explained by two main mechanisms. One is the increased surface area of
LPs 75, which had more chances of increasing the solubility and
permeation of RSV to the intestinal environment, resulting in increased
oral bioavailability [11,46]. The other is the particle size of the LPs that
can be taken up by lymphatic vessels [22,47]. LPs with particle size
smaller than 10 nm are more prone to escape from the lymphatic vessel,
while nanocarriers with particle sizes ranging from 10 to 100 nm are
appropriate for delivering bioactive compounds to the lymphatic vessel,
thus avoiding first-pass metabolism in the liver [48]. Consequently,
decreased first-pass metabolism of bioactive compounds may lead to
increased bioavailability [49]. A similar result was observed in a pre­
vious study which showed that erythropoietin-loaded LPs with a particle
size of 100 nm were more effectively absorbed in rats after oral
administration than those with a particle size of 200 nm [49]. Therefore,
LPs 75, which exhibited particle sizes smaller than 100 nm, may be a
promising lipid-based nanocarrier to overcome the low oral bioavail­
ability of RSV.
2.8. Conclusion
In this study, encapsulation of RSV in the LPs system was an effective
strategy to overcome the limitations of RSV, including its low solubility,
stability, and bioavailability. Moreover, RSV-loaded LPs were success­
fully formulated in different particle sizes (approximately 75, 150, and
300 nm) with the same concentration of lipid compositions, enabling
the comparison of solubility, antioxidant stability after thermal treat­
ment, cellular permeability, cellular antioxidant activity, and in vivo oral
bioavailability. All in vitro and in vivo performances of RSV showed a
size-dependent manner. The results obviously demonstrated that LPs
with small particle size (75 nm) were positively related to enhancing the
solubility and in vitro release profile due to their large surface area to
interact with the reaction environment compared with LPs with large
particle size (300 nm). Besides, LPs with a particle size of 75 nm
enhanced the cellular permeability and cellular antioxidant activity of
RSV compared with those with 300 nm, indicating that their primary
permeation route may be mediated by endocytosis. This tendency was
consistent with the result of the pharmacokinetic study after oral
administration, indicating that LPs with small particle size are more
effective in improving the bioavailability of RSV. In contrast, when the
particle size of LPs was increased to 300 nm, the antioxidant stability
was improved, which affirmed that a larger particle size is more
acceptable to enhance the stability of RSV than a smaller particle size.
Therefore, small LPs (<100 nm) could enhance the solubility, in vitro
Colloids and Surfaces B: Biointerfaces 224 (2023) 113205
Free RSV LPs 75 LPs 150 LPs 300
20 20 20 20
***
0.404 ± 0.249 1.486 ± 0.427 0.910 ± 0.174* 0.684 ± 0.346
1.08 ± 0.49 2.67 ± 1.03** 2.17 ± 0.98* 1.83 ± 0.41
0.825 ± 0.306 10.340 ± 0.866*** 5.968 ± 1.025*** 3.692 ± 1.918***
1.875 ± 0.378 7.363 ± 2.111*** 6.090 ± 1.530*** 5.779 ± 1.347***
release profile, cellular properties, and in vivo oral availability, while
large LPs (≥300 nm) could increase the thermal stability of RSV. Our
findings may have significant implications for understanding that
appropriate particle size range of LPs for improving the solubility,
antioxidant stability, cellular properties, and in vivo oral bioavailability
of RSV to establish them as an effective delivery system.
CRediT authorship contribution statement
Youjin Baek: Conceptualization, Investigation, Methodology, Data
curation, Visualization, Writing – original draft, Writing – review &
editing. Eun Woo Jeong: Conceptualization, Data curation. Hyeon Gyu
Lee: Conceptualization, Supervision, Project administration, Funding
acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgement
This work was supported by a National Research Foundation of
Korea (NRF) grant funded by the Korean government (MSIT)
(No.2021R1A2C2013460).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.colsurfb.2023.113205.
References
[1] J.H. Chung, J.-S. Lee, H.G. Lee, Resveratrol-loaded chitosan–γ-poly (glutamic acid)
nanoparticles: optimization, solubility, UV stability, and cellular antioxidant
activity, Colloids Surf. B Biointerfaces 186 (2020), 110702, https://doi.org/
10.1016/j.colsurfb.2019.110702.
[2] I. Choi, N. Li, Q. Zhong, Enhancing bioaccessibility of resveratrol by loading in
natural porous starch microparticles, Int. J. Biol. Macromol. 194 (2022) 982–992,
https://doi.org/10.1016/j.ijbiomac.2021.11.157.
[3] E.C. Alexandre, F.B. Calmasini, M.G. de Oliveira, F.H. Silva, C.P.V. da Silva, D.
M. Andre’, F.C. Leonardo, M.A. Delbin, E. Antunes, Chronic treatment with
resveratrol improves overactive bladder in obese mice via antioxidant activity, Eur.
J. Pharmacol. 788 (2016) 29–36, https://doi.org/10.1016/j.ejphar.2016.06.017.
[4] H. Jeong, K.J. Samdani, D.H. Yoo, D.W. Lee, N.H. Kim, I.S. Yoo, J.H. Lee,
Resveratrol cross-linked chitosan loaded with phospholipid for controlled release
and antioxidant activity, Int. J. Biol. Macromol. 93 (2016) 757–766, https://doi.
org/10.1016/j.ijbiomac.2016.09.018.
[5] Š. Zupančič, Z. Lavrič, J. Kristl, Stability and solubility of trans-resveratrol are
strongly influenced by pH and temperature, Eur. J. Pharm. Biopharm. 93 (2015)
196–204, https://doi.org/10.1016/j.ejpb.2015.04.002.
[6] A.Y. Berman, R.A. Motechin, M.Y. Wiesenfeld, M.K. Holz, The therapeutic potential
of resveratrol: a review of clinical trials, Npj Precis. Oncol. 1 (1) (2017) 1–9,
https://doi.org/10.1038/s41698-017-0038-6.
5
Y. Baek et al.
[7] A.R. Neves, M. Lucio, J.L. Lima, S. Resis, Resveratrol in medicinal chemistry: a
critical review of its pharmacokinetics, drug-delivery, and membrane interactions,
Curr. Med. Chem. 19 (11) (2012) 1663–1681, https://doi.org/10.2174/
092986712799945085.
[8] M. Huang, C. Liang, C. Tan, S. Huang, R. Ying, Y. Wang, Z. Wang, Y. Zhang,
Liposome co-encapsulation as a strategy for the delivery of curcumin and
resveratrol, Food Funct. 10 (10) (2019) 6447–6458, https://doi.org/10.1039/
c9fo01338e.
[9] H. Cheng, H. Zhang, D. Li, H. Duan, L. Liang, Impact of oil type on the location,
partition and chemical stability of resveratrol in oil-in-water emulsions stabilized
by whey protein isolate plus gum Arabic, Food Hydrocoll. 109 (2020), 106119,
https://doi.org/10.1016/j.foodhyd.2020.106119.
[10] C. Houacine, D. Adams, K.K. Singh, Impact of liquid lipid on development and
stability of trimyristin nanostructured lipid carriers for oral delivery of resveratrol,
J. Mol. Liq. 316 (2020), 113734, https://doi.org/10.1016/j.molliq.2020.113734.
[11] Y. Zhu, M. Wang, J. Zhang, W. Peng, C.K. Firempong, W. Deng, Q. Wang, S. Wang,
F. Shi, J. Yu, X. Xu, W. Zhang, Improved oral bioavailability of capsaicin via
liposomal nanoformulation: preparation, in vitro drug release and
pharmacokinetics in rats, Arch. Pharm. Res. 38 (2015) 512–521, https://doi.org/
10.1007/s12272-014-0481-7.
[12] B.S. Esposto, P. Jauregi, D.R. Tapia-Blácido, M. Martelli-Tosi, Liposomes vs.
chitosomes: encapsulating food bioactives, Trends Food Sci. Technol. 108 (2021)
40–48, https://doi.org/10.1016/j.tifs.2020.12.003.
[13] M. Leitgeb, Ž. Knez, M. Primožič, Sustainable technologies for liposome
preparation, J. Supercrit. Fluids 165 (2020), 104984, https://doi.org/10.1016/j.
supflu.2020.104984.
[14] Y.-D. Dong, E. Tchung, C. Nowell, S. Kaga, N. Leong, D. Mehta, L.M. Kaminskas, B.
J. Boyd, Microfluidic preparation of drug-loaded PEGylated liposomes, and the
impact of liposome size on tumour retention and penetration, J. Liposome Res. 29
(1) (2019) 1–9, https://doi.org/10.1080/08982104.2017.1391285.
[15] G. Lou, G. Anderluzzi, S. Woods, C.W. Roberts, Y. Perrie, A novel microfluidic-
based approach to formulate size-tuneable large unilamellar cationic liposomes:
formulation, cellular uptake and biodistribution investigations, Eur. J. Pharm.
Biopharm. 143 (2019) 51–60, https://doi.org/10.1016/j.ejpb.2019.08.013.
[16] G. Phan, A. Herbet, S. Cholet, H. Benech, J.R. Deverre, E. Fattal, Pharmacokinetics
of DTPA entrapped in conventional and long-circulating liposomes of different size
for plutonium decorporation, J. Control. Release 110 (1) (2005) 177–188, https://
doi.org/10.1016/j.jconrel.2005.09.029.
[17] S. Haddadzadegan, F. Dorkoosh, A. Bernkop-Schnurch, Oral delivery of therapeutic
peptides and proteins: Technology landscape of lipid-based nanocarriers, Adv.
Drug Deliv. Rev. 182 (2022), 114097, https://doi.org/10.1016/j.
addr.2021.114097.
[18] M.S. de Almeida, E. Susnik, B. Drasler, P. Taladriz-Blanco, A. Petri-Fink, B. Rothen-
Rutishauser, Understanding nanoparticle endocytosis to improve targeting
strategies in nanomedicine, Chem. Soc. Rev. 50 (9) (2021) 5397–5434, https://doi.
org/10.1039/d0cs01127d.
[19] D.J. McClements, Edible lipid nanoparticles: digestion, absorption, and potential
toxicity, Prog. Lipid Res. 52 (4) (2013) 409–423, https://doi.org/10.1016/j.
plipres.2013.04.008.
[20] M.R.I. Shishir, N. Karim, V. Gowd, X. Zheng, W. Chen, Liposomal delivery of
natural product: a promising approach in health research, Trends Food Sci.
Technol. 85 (2019) 177–200, https://doi.org/10.1016/j.tifs.2019.01.013.
[21] E. Roger, F. Lagarce, E. Garcion, J.P. Benoit, Lipid nanocarriers improve paclitaxel
transport throughout human intestinal epithelial cells by using vesicle-mediated
transcytosis, J. Control. Release 140 (2) (2021) 174–181, https://doi.org/10.1016/
j.jconrel.2009.08.010.
[22] P. Pandya, P. Giram, R.P. Bhole, H.I. Chang, S.Y. Raut, Nanocarriers based oral
lymphatic drug targeting: Strategic bioavailability enhancement approaches,
J. Drug Deliv. Sci. Technol. 64 (2021), 102585, https://doi.org/10.1016/j.
jddst.2021.102585.
[23] Y. Sun, J. Chi, X. Ye, S. Wang, J. Liang, P. Yue, H. Xiao, X. Gao, Nanoliposomes as
delivery system for anthocyanins: physicochemical characterization, cellular
uptake, and antioxidant properties, LWT 139 (2021), 110554, https://doi.org/
10.1016/j.lwt.2020.110554.
[24] M. Yang, H. Zhong, R. Guan, X. Liu, W. Zhang, X. Lu, J. Xu, Cellular uptake,
transport mechanism and anti-inflammatory effect of cyanidin-3-glucoside
nanoliposomes in Caco-2/RAW264.7 co-culture model, Front. Nutr. 9 (2022),
995391, https://doi.org/10.3389/fnut.2022.995391.
[25] J. Xian, X. Zhong, H. Gu, X. Wang, J. Li, J. Li, Y. Wu, C. Zhang, J. Zhang, Colonic
delivery of celastrol-loaded layer-by-layer liposomes with pectin/trimethylated
chitosan coating to enhance its anti-ulcerative colitis effects, Pharmaceutics 13
(12) (2021) 2005, https://doi.org/10.3390/pharmaceutics13122005.
[26] R.S. Managuli, J.T.W. Wang, F.M. Faruqu, A. Pandey, S. Jain, K.T. Al-Jamal,
S. Mutalik, Surface engineered nanoliposomal platform for selective lymphatic
uptake of asenapine maleate: in vitro and in vivo studies, Mater. Sci Eng. C: Mater.
Biol. Appl. 109 (2020), 110620, https://doi.org/10.1016/j.msec.2019.110620.
[27] M.M. Seyedabadi, H. Rostami, S.M. Jafari, M. Fathi, Development and
characterization of chitosan-coated nanoliposomes for encapsulation of caffeine,
Food Biosci. 40 (2021), 100857, https://doi.org/10.1016/j.fbio.2020.100857.
Colloids and Surfaces B: Biointerfaces 224 (2023) 113205
[28] J.H. Nam, S.-Y. Kim, H. Seong, Investigation on physicochemical characteristics of
a nanoliposome-based system for dual drug delivery, Nanoscale Res. Lett. 13 (1)
(2018) 1–11, https://doi.org/10.1186/s11671-018-2519-0.
[29] A.K. Sahu, V. Jain, Screening of process variables using Plackett–Burman design in
the fabrication of gedunin-loaded liposomes, Artif. Cells Nanomed. Biotechnol. 45
(5) (2017) 1011–1022, https://doi.org/10.1080/21691401.2016.1200057.
[30] E.A.A. Azim, S.A. Elkheshen, R.M. Hathout, M.A. Fouly, N.M.E. Hoffy, Augmented
in vitro and in vivo profiles of brimonidine tartrate using gelatinized-core liposomes,
Int. J. Nanomed. 17 (2022) 2753, https://doi.org/10.2147/ijn.s370192.
[31] Y. Dai, R. Zhou, L. Liu, Y. Lu, J. Qi, W. Wu, Liposomes containing bile salts as novel
ocular delivery systems for tacrolimus (FK506): in vitro characterization and
improved corneal permeation, Int. J. Nanomed. 8 (2013) 1921, https://doi.org/
10.2147/ijn.s44487.
[32] J.B. Min, E.S. Kim, J.-S. Lee, H.G. Lee, Preparation, characterization, and cellular
uptake of resveratrol-loaded trimethyl chitosan nanoparticles, Food Sci.
Biotechnol. 27 (2) (2018) 441–450, https://doi.org/10.1007/s10068-017-0272-2.
[33] P.A. Shruthi, H.A. Pushpadass, M.E.E. Franklin, S.N. Battula, N.L. Naik,
Resveratrol-loaded proniosomes: Formulation, characterization and fortification,
LWT 134 (2020), 110127, https://doi.org/10.1016/j.lwt.2020.110127.
[34] Y. Liu, D. Liu, L. Zhu, Q. Gan, X. Le, Temperature-dependent structure stability and
in vitro release of chitosan-coated curcumin liposome, Food Res. Int. 74 (2015)
97–105, https://doi.org/10.1016/j.foodres.2015.04.024.
[35] H.J. Je, E.S. Kim, J.-S. Lee, H.G. Lee, Release properties and cellular uptake in
Caco-2 cells of size-controlled chitosan nanoparticles, J. Agric. Food Chem. 65 (50)
(2017) 10899–10906, https://doi.org/10.1021/acs.jafc.7b03627.
[36] S. Hu, M. Niu, F. Hu, Y. Lu, J. Qi, Z. Yin, W. Wu, Integrity and stability of oral
liposomes containing bile salts studied in simulated and ex vivo gastrointestinal
media, Int. J. Pharm. 441 (1–2) (2013) 693–700, https://doi.org/10.1016/j.
ijpharm.2012.10.025.
[37] W. Liu, J. Liu, L.J. Salt, M.J. Ridout, J. Han, P.J. Wilde, Structural stability of
liposome-stabilized oil-in-water pickering emulsions and their fate during in vitro
digestion, Food Funct. 10 (11) (2019) 7262–7274, https://doi.org/10.1039/
c9fo00967a.
[38] W. Liu, J. Liu, W. Liu, T. Li, C. Liu, Improved physical and in vitro digestion stability
of a polyelectrolyte delivery system based on layer-by-layer self-assembly
alginate–chitosan-coated nanoliposomes, J. Agric. Food Chem. 61 (17) (2013)
4133–4144, https://doi.org/10.1021/jf305329n.
[39] M. Hasan, K. Elkhoury, C.J. Kahn, E. Arab-Tehrany, M. Linder, Preparation,
characterization, and release kinetics of chitosan-coated nanoliposomes
encapsulating curcumin in simulated environments, Molecules 24 (10) (2019)
2023, https://doi.org/10.3390/molecules24102023.
[40] J.-J. Ma, Y.-G. Yu, S.-W. Yin, G.-H. Tang, X.-Q. Yang, Cellular uptake and
intracellular antioxidant activity of zein/chitosan nanoparticles incorporated with
quercetin, J. Agric. Food Chem. 66 (48) (2018) 12783–12793, https://doi.org/
10.1021/acs.jafc.8b04571.
[41] J. Sheng, L. Han, J. Qin, G. Ru, R. Li, L. Wu, D. Cui, P. Yang, Y. He, J. Wang, N-
trimethyl chitosan chloride-coated PLGA nanoparticles overcoming multiple
barriers to oral insulin absorption, ACS Appl. Mater. Interfaces 7 (28) (2015)
15430–15441, https://doi.org/10.1021/acsami.5b03555.
[42] H.M. Ezzat, Y.S. Elnaggar, O.Y. Abdallah, Improved oral bioavailability of the
anticancer drug catechin using chitosomes: design, in-vitro appraisal and in-vivo
studies, Int. J. Pharm. 565 (2019) 488–498, https://doi.org/10.1016/j.
ijpharm.2019.05.034.
[43] I. Fiebrig, S.E. Harding, S.S. Davis, Sedimentation analysis of potential interactions
between mucins and a putative bioadhesive polymer, Prog. Coll. Polym. Sci. 94
(1994) 66–73, https://doi.org/10.1007/bfb0115603.
[44] C. Dima, E. Assadpour, S. Dima, S.M. Jafari (Eds.), In Vitro Assays for Evaluating
the Release of Nanoencapsulated Food Ingredients, Vol. 5, Elsevier, Amsterdam,
Netherlands, 2020, https://doi.org/10.1016/b978-0-12-815665-0.00004-7.
[45] A. Amri, J.C. Chaumeil, S. Sfar, C. Charrueau, Administration of resveratrol: what
formulation solutions to bioavailability limitations, J. Control. Release 158 (2)
(2012) 182–193, https://doi.org/10.1016/j.jconrel.2011.09.083.
[46] M. Takahashi, S. Uechi, K. Takara, Y. Asiin, K. Wada, Evaluation of an oral carrier
system in rats: bioavailability and antioxidant properties of liposome-encapsulated
curcumin, J. Agric. Food Chem. 57 (19) (2009) 9141–9146, https://doi.org/
10.1021/jf9013923.
[47] S.G.M. Ong, L.C. Ming, K.S. Lee, K.H. Yuen, Influence of the encapsulation
efficiency and size of liposome on the oral bioavailability of griseofulvin-loaded
liposomes, Pharmaceutics 8 (3) (2016) 25, https://doi.org/10.3390/
pharmaceutics8030025.
[48] R.S. Managuli, S.Y. Raut, M.S. Reddy, S. Mutalik, Targeting the intestinal
lymphatic system: a versatile path for enhanced oral bioavailability of drugs,
Expert Opin. Drug Deliv. 15 (8) (2018) 787–804, https://doi.org/10.1080/
17425247.2018.1503249.
[49] Y. Maitani, M. Hazama, Y. Tojo, N. Shimoda, T. Nagai, Oral administration of
recombinant human erythropoietin in liposomes in rats: influence of lipid
composition and size of liposomes on bioavailability, J. Pharm. Sci. 85 (4) (1996)
440–445, https://doi.org/10.1021/js950477m.