www.acsabm.

org Article

Tuning of Molecular Interactions between Zein and Tannic Acid to

Modify Sunflower Sporopollenin Exine Capsules: Enhanced Stability
and Targeted Delivery of Bioactive Macromolecules
Ziyu Deng, Shishuai Wang, Yaqiong Pei, Bin Zhou, Jing Li, Xinyao Hou, Bin Li, and Hongshan Liang*
Cite This: ACS Appl. Bio Mater. 2021, 4, 2686−2695 Read Online

ACCESS Metrics & More Article Recommendations *

sı Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: There are multiple obstacles for the storage and

digestion of orally administered bioactive macromolecules. This
study developed a low-cost and sustained-release delivery system
Downloaded via FUDAN UNIV on April 1, 2024 at 11:39:04 (UTC).

(sporopollenin exine capsules with zein/tannic acid modification)

of proteins with excellent storage stability, and at the same time
provided insights into the sustained-release mechanism through
exploring the interaction between zein and tannic acid (TA). β-
Galactosidase (β-Gal) was utilized as a model protein and loaded
into sporopollenin exine capsules (SECs), which were then coated
with the zein/TA system. Under the optimized zein/TA
conditions, the zein/TA system showed better performance than
the zein alone system in the sustained release of β-Gal, with the
residual activity of about 70.26% after 24 h of simulated digestion.
Evaluation of the storage stability demonstrated a β-Gal residual activity of nearly 90% for 28 days at 25 °C. Additionally, FTIR
analysis demonstrated that the stability of the zein/TA system depends on both hydrogen bonding and certain covalent bonding
through the Schiff-base reaction, and the sustained release is regulated by the bonding strength.
KEYWORDS: zein, tannic acid, sunflower sporopollenin exine capsules, controlled release, storage stability, interaction

■ INTRODUCTION
Currently, the oral route has become more popular than the
parenteral route due to its higher patient compliance and lower
cost, which is of great importance for the delivery of
biopharmaceuticals.1,2 However, it is commonly known that
the oral delivery of bioactive macromolecules is a challenge
because of the high susceptibility of these bioactive macro-
molecules to denaturation during storage within the
commercial products and after ingestion into the human
gastrointestinal tract (GIT).3,4 Hence, bioactive macromole-
cules have to be encapsulated into vehicles formulated from
food-grade ingredients with low costs,5 which can provide
protection to the bioactive macromolecules in storage and
facilitate the targeted release in the GIT.6,7 Many kinds of
delivery systems, including micelles, emulsions, liposomes,
polymeric, and nanoparticles, have been developed for
bioactive macromolecule oral delivery.8,9 Among these
systems, there is still a lack of an oral delivery system that
can achieve long-term stability in transportation, storage, and
gastrointestinal tract.
There has been great interest in utilizing SECs for bioactive
macromolecule oral delivery due to their advantages of safety
and low cost.10−15 Compared with these delivery systems,
SECs extracted from natural pollen have resistance to
variations in temperature, pH, ionic strength, and mechanical
stress, as well as the mucosal-adhesion capability to greatly
improve the bioavailability of the delivered macromole-
cules.16−20 However, numerous pores on SECs can lead to
the leakage of the loaded bioactive macromolecules, which is
still a significant challenge.21 Some polymer coatings such as
enteric coating, calcium alginate coating, and carboxymethyl (1
→ 3)-β-D-glucan (CMP) coating have been utilized to address
this limitation. These coatings can protect the encapsulated
macromolecules from the negative effect of the harsh stomach
environment and then release them in the small intestine.22,23
Despite certain progress in the development of coating
approaches, it remains a challenge to develop a cost-effective
coating system that can achieve sustained small intestinal
release of bioactive macromolecules to date.
It is commonly known that the main source of zein is
industrial scraps, and its utilization can not only meet the need
of sustainable industrial production but also be cost-
Received: December 17, 2020
Accepted: February 1, 2021
Published: February 10, 2021

© 2021 American Chemical Society https://dx.doi.org/10.1021/acsabm.0c01623

2686 ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials
Figure 1. (a) Schematic diagram of the development of a new oral vehicle for intestinal protein delivery, including the processes of encapsulation,
coating, release, and storage. (b) Diameter distribution of natural and extracted sunflower pollen grains, and β-Gal-loaded SECs. (c) Confocal
micrographs, SEM, and TEM images of natural, extracted, and (d) β-Gal-loaded SECs.
effective.24,25 As an alcohol-soluble protein, zein possesses
many unique characteristics and can be converted into films
and nanoparticles to be applied in oral delivery systems.26
However, the application of zein is largely hindered by its
susceptibility to variable solubility near the isoelectric
point.27,28 Chemical modification has been used to solve this
problem based on the covalent or noncovalent interaction
between zein and other compounds (polysaccharides, lipids,
and small molecules), which can improve the performance of
zein when used in an oral delivery system.29,30 Previously, we
have fabricated a zein/polysaccharide system as an orally
administered drug carrier based on the noncovalent interaction
between zein and CMP,31 but oral delivery systems with a low
cost and a sustained small intestinal release of bioactive
macromolecules are still not available. Great effort has been
made to utilize proteins and polyphenols to construct delivery
systems due to their interactions. These systems can mediate
drug release on the basis of various factors, including pH,
temperature, and ion nature or concentration.32 More
importantly, these studies have demonstrated that the covalent
protein−polyphenol complex has higher stability than the
noncovalent protein−polyphenol complex.33,34 Therefore, it
can be hypothesized that tannic acid (TA) may help to control
the release of the loaded molecules in the GIT, enhance the
pH-responsiveness, and promote the cost efficiency.
In this study, β-galactosidase (β-Gal), whose oral bioavail-
ability is usually low due to the adverse pH and proteases in
the GIT,35 was employed as the model protein to be loaded in
SECs, and zein cross-linked with TA was used as the secondary
2687
www.acsabm.org Article
coating, with the aim to further enhance the storage stability
and realize a targeted and sustained release in the GIT (Figure
1a). In addition, the zein/TA system was compared with the
zein system in terms of surface morphology, release behavior,
and storage stability. Finally, the interaction between zein and
TA was investigated, and a possible synergistic stabilization
mechanism was elucidated to guide their actual applications.
This study provides important implications for the design of
inexpensive and high-performance oral delivery systems for
bioactive proteins in food and pharmaceutical industries.
■
EXPERIMENTAL SECTION
Materials. Zein was provided by Sigma-Aldrich (Sigma Z3625).
Tannic acid (TA) was provided by Aladdin Chemical Co. β-
Galactosidase (β-Gal) from Escherichia coli (250−600 U/mg, EC
232−864−1) was purchased from Shanghai Yuanye Bio-Technology
Co., Ltd. The optimum temperature for β-Gal was 37 °C and the
optimum pH was 7.3. Natural sunflower pollen grains were the
product of China Carl Science Laboratory. Coumarin-6 was provided
by Invitrogen (USA).
SECs Characterization. SECs Extraction. SECs were obtained
according to the previous procedure.31 After acid hydrolyzation, the
pollen grains were washed with organic reagents.
Microstructure Analysis. Morphological features were observed by
SEM (SU8010, Japan) and TEM (JEM-2100F, JEOL, Japan). A Carl
Zeiss LSM700 confocal laser scanning microscope (CLSM) was used
to observe their morphology. CLSM imaging was conducted with
three laser excitation channels (405, 488, and 561 nm), with three
respective emission filters (440−470, 498−550, 572−620 nm), and
40× or 20× regular objective lens. The particle distribution and
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials
Figure 2. SEM images of SECs coated with zein (a) and zein/TA systems (b); CLSM images of SECs coated with zein or zein/TA systems (c).
particle diameter of the SECs were tested by Malvern MasterSizer
2000 (Malvern, UK).
Analysis the Property of Delivery System. β-Gal Activity. β-
Gal activity was assayed according to our previous work.36 In short, a
chromogenic reaction occurred between β-Gal and o-nitrophenyl β-D-
galactopyranoside (o-NPG). The concentration of free O-NP was
measured by a multilabel microplate reader (Victor X3, PerkinElmer
2030; absorbance at 420).
Encapsulation of β-Gal. β-Gal was loaded into SECs (BLS)
following the descriptions in our previous study.36 In brief, β-Gal and
FITC-β-Gal were encapsulated into SECs by the vacuum loading
technique.
Coating for Oral Administration. First, zein (5%, w/v) was added
into the BLS and vacuumed for 3 h. Zein1, zein2, and zein3
represented the application of 4, 5, and 6 mL of zein, respectively.
Subsequently, the dry samples were put into 5 mL TA solutions (in
0.2 M, pH 7.4, 3-(N-morpholino)-propane-sulfonic acid (MOPS))
with free exposure to air for 6 h. The samples were withdrawn with
the excess TA on the surface being removed by water washing, and
then placed on a desiccator for further characterization. TA1, TA2,
TA3, and TA4 represented the application of 1, 3, 5, and 10 mg/mL of
TA, respectively. The zein was treated under the same conditions
without TA as the control. Coumarin-6 was added to the samples for
labeling the zein following the procedure described above.
In Vitro Release Behavior Assay. In vitro release behavior was
measured using the method reported previously.22 Briefly, the samples
were blended with simulated gastric fluid (SGF, 0.01 M HCl solution,
3.20 mg/mL pepsin) for 2 h and then mixed with simulated intestinal
fluid (SIF, pH 7.4 phosphate buffer, 1.60 mg/mL pancreatin). One
mL of simulated solution was collected at different time intervals and
the enzyme activity was determined.
Storage Stability. Storage stability of the samples was examined for
28 days at 4, 25, and 40 °C, which simulated the refrigerated
conditions, ambient conditions, and hot conditions. Finally, the
residual activity (RA) was calculated with the following equation
Residual activity(%) = (Residual β‐Gal activity/Total β‐Gal activity)
(1)
× 100%
Complex Characterization. Preparation of Complex. The zein
(5%, w/v) was vacuumed for 3 h. Subsequently, the dry samples were
placed in 5 mL TA solutions (in 0.2 M, pH 7.4, MOPS) with free
exposure to air for 6 h. Subsequently, the effect of TA on β-Gal
2688
www.acsabm.org Article
activity was studied. Different TA solutions were mixed with β-Gal
solution and then the enzyme activity was determined.
Microstructure Analysis. The elemental composition of the
complex was analyzed via the X-ray photoelectron spectroscopy
(XPS, VG Multilab 2000, Britain) with Al Kα as the radiation source.
Fourier transform infrared (FT-IR) spectra were acquired on a
Nicolet iS50 FT-IR spectrometer (Thermo Scientific, USA). The
samples were mixed with KBr, ground, and pressed into pellets.
Colorimetry. The color of the complex was determined by a hand-
held colorimeter (Chroma meter CR-400, Konica Minolta, Inc.). The
value of b* represented the color change of blue and yellow.
Test of Total Phenolics. Total polyphenol content was determined
by the Folin−Ciocalteu method using gallic acid as a standard.37
Briefly, the Folin−Ciocalteau reagent (250 μL) and Na2CO3 (10%,
w/v, 50 μL) were added to the TA solution (1 mg/mL, 50 μL),
diluted with water to 1 mL, and incubated at 30 °C for 1 h. The
absorbance of the green color was measured by UV−vis
spectrophotometer (UV-1800, RELY, China) at 760 nm. The
polyphenols bound was calculated as the percentage of TA combined
with zein.
Polyphenols bound (%)
=(Total polyphenol − Free polyphenol/Total polyphenol) × 100%
(2)
Statistical Analysis. All experiments were performed in triplicate
and reported as the mean ± standard deviation. The one-way analysis
of variance (ANOVA) was employed to determine the statistical
difference, and the Fisher LSD was applied as the mean comparison
method to compare the difference between groups at the significance
level of 0.05.
■
RESULTS AND DISCUSSION
Figure 1 presents the processes of encapsulation, coating,
release, and storage. Briefly, the β-Gal was encapsulated into
SECs (BLS) (Figure 1a, Step 1), which were then coated by
zein/TA. After oxidation of TA under mild conditions (with
free exposure to air for 6 h), the complex was obtained for oral
administration (Figure 1a, Step 2), with the aim of further
improving the intestinal release (Figure 1a, Step 3) and storage
stability of β-Gal (Figure 1a, Step 4).
Characterization of the Delivery Systems. SECs
Properties. Sunflower pollen is thick-walled,38 which is not
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials www.acsabm.org Article

Figure 3. In vitro release profiles of β-Gal from zein coated (a) and zein/TA coated (b) SECs. The release profiles were obtained from the
incubation for 2 h in SGF and 22 h in SIF. Data are presented as the average of triplicate measurements with standard deviation (n = 3). SEM
images of zein coated (c) and zein/TA coated SECs incubated for 2 h in SGF (d). SEM images of zein coated (e) and zein/TA coated (f) SECs
incubated for 2 h in SGF and 22 h in SIF.

easily broken under harsh hydrolysis conditions compared with
thin-walled pollen.18,19 Therefore, we chose sunflower pollen
as the study material. First, the characteristics of natural and
extracted sunflower pollen grains were compared. SEM images
(Figure 1c) clearly showed that compared with natural
sunflower pollen grains (NSP), the extracted SECs had the
same surface morphology but obviously more visible pores.
TEM images showed that the internal cavity of NSP contained
pollen constituents, while that of SECs was clean. This is
consistent with the observation from CLSM images, where
strong autofluorescence due to the presence of pollen
constituents was observed for NSP but not for SECs.
Furthermore, the particle diameter was about 41 μm for
2689
NSP but significantly dropped to about 29 μm for SECs, which
could be ascribed to the complete removal of pollen
constituents (Figure 1b). These results suggested that SECs
that have clean hollow cavities were successfully extracted,
which can provide a large space for drug loading.
β-Gal Encapsulation. The RA of BLS was more than 7-fold
that in NSP (Table S1). This was due to the fact that SECs
had more visible pores and larger clean cavity than NSP, which
could provide more channel and space for β-Gal encapsulation.
To determine the localization of the loaded β-Gal in SECs, we
made a comparison between unloaded SECs and FITC-BLS by
CLSM and TEM images (Figure 1d). Empty internal cavities
were observed in the unloaded SECs, whereas the loaded SECs
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials www.acsabm.org Article

Figure 4. Storage stability of zein and zein/TA systems at 4, 25, and 40 °C for 28 days (a). In vitro release profiles of β-Gal from zein coated or
zein/TA coated SECs after storage at 4 °C (b), 25 °C (c), and 40 °C (d) for 28 days. The release profiles were obtained from the incubation for 2
h in SGF and 22 h in SIF. Effects of TA before and after oxidation on the activity of β-Gal (e). Data are presented as the average of triplicate
measurements with standard deviation (n = 3).

emitted strong green fluorescence inside, further confirming
that β-Gal was successfully loaded into SECs, which is in
agreement with the observation from TEM images. Besides,
Figure 1d reveals that the loading process had no influence on
the morphology and structure of SECs, which is significant for
the industrial production of the encapsulation system.39
Zein/TA Coating. Zein/TA coating was used to entrap the
numerous pores on the SEC surface, so as to avoid the leakage
of the loaded β-Gal. The SEM images are shown in Figure
2a,b. Generally, a thicker coating could be formed by the zein/
TA complex to cover the pores on SECs, while zein alone
2690
could only partly cover the pores of SECs, and some visible
pores were still exposed. This effect could be attributed to the
interaction between zein and TA, which would be further
discussed below. In addition, the coating thickness rose along
with increasing TA concentration, possibly because more
intermolecular zones participated in the interaction when the
TA concentration was higher. As observed in Figure 2c, the
SECs (red autofluorescence) were entirely embedded into the
Coumarin-6-zein/TA coating (green fluorescence), which was
in line with the observation from SEM images.
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials
Figure 5. XPS survey spectra of zein (a and b) and zein/TA (c and d); FTIR spectra of zein/TA under different treatments (e and f).
In Vitro Release Behavior. We compared the zein/TA
system with the zein system in terms of the release profile in
the simulated gastrointestinal conditions (SGC). The control
groups were conducted with the uncoated system of BLS and
the zein/TA system without SECs. The in vitro release profiles
in different samples are depicted in Figure 3a and b.
After 24 h of digestion under SGC, the RA of β-Gal was 0%
for the uncoated system of BLS and 46.72% for the zein/TA
system without SECs. For the zein3 system, the RA of β-Gal
remained at 0% steadily under SGC; by contrast, that for the
zein3/TA systems with TA1, TA2, TA3, and TA4 was 54.32%,
70.26%, 45.23%, and 35.96%, respectively, after 24 h of
digestion under SGC. In Figure 3b, after 24 h of digestion
under SGC, the RA of β-Gal in the zein/TA2 system rose with
increasing zein concentration.
According to Figure S1, a burst effect of β-Gal release in
SGF was observed for the uncoated system of BLS due to the
presence of numerous pores on SECs. Similarly, the same burst
effect was also observed in the zein3 system due to the
existence of protease in SGF, which could cause the
inactivation of β-Gal. In comparison, for the zein/TA system,
the interaction between zein and TA would contribute to the
2691
www.acsabm.org Article
stability of the system under SGC. On one hand, the zein/TA
coating could act as a barrier layer to prevent β-Gal from direct
exposure to SGF. For the previous polysaccharide system, the
RA of β-Gal rose dramatically over the first 3 h in SGC,
reaching around 20% and peaking at around 65% at 24 h in
SGC. However, for the zein/TA system, the RA of β-Gal
stayed at about 2% at 3 h in SGC, and reached its peak at
about 70% at 24 h in SGC.22 Hence, the zein/TA system is
superior to the previous polysaccharide systems in sustained
release of β-Gal. This was due to the thick coating and strong
cohesion of the polymer chains, and the β-Gal would be
embedded in the zein/TA layer to avoid immediate release
into the SIF. Besides, the different releasing behaviors between
the zein/TA system with and without SECs indicated that
SECs positively influence the β-Gal release dynamics. More-
over, there is likely a synergetic effect between SECs and the
zein/TA film.
After in vitro release, the microstructure of the samples was
examined by SEM analysis. In the zein/TA system, no pores
were found on SECs after SGF treatment, whereas the zein
system presented some pores on SECs, demonstrating that the
introduction of TA could enhance the antidigestive enzyme
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials
Figure 6. Percentage of polyphenols bound of the samples (a). In vitro release profiles of TA from zein/TA (b,c). The release profiles were
obtained from the incubation for 2 h in SGF and 22 h in SIF. Data are presented as the average of triplicate measurements with standard deviation
(n = 3).
performance of this system (Figure 3c,d). SIF treatment
dissolved the coating of all samples with the exposure of pores
on SECs, resulting in a surface structure similar to that of SECs
without coating (Figure 3e,f). These results are in line with the
results of the releasing behavior, confirming the capability of
this system for targeted delivery and sustained release of β-Gal.
Besides, some studies have shown that different species of
pollen are of different resilience to degradation when exposed
to SGF and SIF.40 These results demonstrate that SECs from
sunflower pollen could be resilient to degradation when
exposed to SGF and SIF.
Storage Stability. Storage stability is of great importance
for the industrial production of delivery systems. The results of
storage stability evaluation are shown in Figure 4a. Generally,
for all samples, the RA of β-Gal decreased with increasing
storage temperature after 28 days of incubation. After
incubation at 4 °C for 28 days, the RA of β-Gal was not
significantly different among samples (p > 0.05). However,
after incubation at 25 °C for 28 days, the RA of β-Gal in the
zein/TA system was higher than that in other systems. The RA
of β-Gal in the zein system was similar to that in the uncoated
system of BLS, while the RA of β-Gal alone as the blank
control was lower. Identical results were obtained under
storage at 40 °C. These results implied that the introduction of
SECs and TA can enhance the storage stability of the delivered
enzyme in this system.
In general, the release behavior of the same samples was
similar after storage at different temperatures. Under SGC, the
RA of β-Gal in the zein/TA system with SECs was consistently
higher than that in the zein/TA system without SECs, and the
difference was gradually enlarged with increasing storage
2692
www.acsabm.org Article
temperature (Figure 4b,c,d), demonstrating that SECs can
improve the thermal stability of β-Gal in the system.
Characterization of Complex Coacervates. Micro-
structure Analysis. To dissect the mechanism for the better
stabilizing effect of the zein/TA system on β-Gal than the zein
system, we studied the interaction between zein and TA. The
zein/TA system provided channels for both the release of β-
Gal under SGC and the outward diffusion of TA. Therefore,
we further examined the effect of TA on β-Gal activity.
Generally, TA showed a less negative effect on β-Gal activity
after oxidation than before oxidation. Increasing TA
concentration enhanced its inhibitory effect on β-Gal activity,
though the effects of TA1 and TA2 on β-Gal activity were
similar (Figure 4e). This result was in agreement with the
result of release behavior, which may explain why the zein3/
TA2 system had the highest RA of β-Gal after 24 h of digestion
under SGC.
XPS analysis was conducted to elucidate the chemical
characteristics of the zein/TA complex (Figure 5a,b,c,d and
Table S2). The survey scan spectrum of zein exhibited
characteristic peaks of C 1s, O 1s, N 1s, and S 2p; and three
peak components of C 1s were located at 284.2, 285.5, and
287.3 eV, which are assigned to CC, CO, and CO or
OCO group, respectively.41 Compared with the spectrum
of zein, that of the zein/TA complex showed significant
increases in the peak intensity of CO. Besides, Table S2
shows that the C/O ratio of zein/TA (2.23) was lower than
that of zein (5.12) because of the rich oxygen in TA,
confirming that TA was bound to the complex surface.
FTIR Analysis. The interaction between TA and zein was
characterized by FT-IR (Figure 5e and f). The spectra of TA
had three common bands, including 1715 cm−1 (carbonyl C
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials
O stretching vibration), 1612 cm−1 (aromatic CC stretching
vibration), and 1533 cm−1 (carbon chain CC stretching
vibration). 42 The spectra of zein had two prominent
characteristic peaks at 1656 cm−1 (amide I, representative of
CO stretching) and 1534 cm−1 (amide II, due to bending of
NH and stretching of CN).32 In the infrared spectra, a
characteristic peak in the range of 3200−3400 cm−1 indicated
the hydrogen bonding. The OH stretching band of the
hydroxyl groups in zein and TA was at 3331 and 3406 cm−1,
respectively, whereas this band in zein/TA shifted to a higher
wavenumber, implying the formation of hydrogen bonds
between the amide groups of glutamine in zein and the
hydroxyl groups in TA.43 Compared with the spectra of zein,
new peaks were observed at around 1216 and 780 cm−1, which
correspond to the CN stretching of aromatic amines and the
−CH bending out-of-plane in the aromatic ring, respectively,
indicating the occurrence of chemical reaction between TA
and zein.5,44 In addition, the spectra of zein/TA showed a
significant decrease in the ratio of amide II stretching/A
stretching peak intensity, which could be attributed to the
consumption of amino groups on zein,45,46 revealing the
formation of covalent bonding between zein and TA.
Meanwhile, the value of b* is shown in Table S3. In the
zein3/TA2 system, the value of b* consistently rose with the
increasing exposure time of the sample in the air. Some studies
have shown that the occurrence of the Schiff base reaction
would cause changes in the color of the sample.47,48 These
results illustrated the occurrence of chemical reactions between
zein and TA, which is consistent with the results of FT-IR
analysis. Therefore, it can be concluded that hydrogen bonds
and covalent bonds cooperatively participate in the binding of
TA with zein. Interestingly, these peaks of the digested sample
resembled those of zein, which can be attributed to the
dissolution of zein/TA coating under SGC.
The stability of the zein/TA system under SGC not only
depended on the hydrogen bonding but also required certain
covalent bonding through the Schiff-base reaction. It is well-
known that the acidity of the solution significantly affects the
strength of the hydrogen bond and Schiff base reaction.
Generally, the hydrogen bond is relatively stable under acidic
conditions, while the Schiff base is relatively stable under
alkaline conditions. 5,42,49 On one hand, under acidic
conditions, the interaction between zein and TA is dominated
by hydrogen bonding, which could help to prevent the
disintegration of the zein/TA system in SGF. On the other
hand, under alkaline conditions, the interaction between zein
and TA is dominated by covalent bonds, which may help to
improve the sustained release performance of the zein/TA
system in SGF. Thus, the interaction between zein and TA in
the zein2/TA3 system may help to achieve a balance between
hydrogen bonding and covalent bonding, contributing to the
optimum release effect under SGC with the highest RA of β-
Gal. This property may make this system applicable in oral
delivery, particularly for the small intestinal release.
Release of TA. Figure 6a shows that the percentage of
bound TA was similar in the zein3/TA systems with different
concentrations of TA, while it showed an upward trend with
increasing concentration of zein in the zein/TA2 systems. The
value of b* is shown in Figure 6a. In the zein3/TA systems, the
value of b* consistently rose with increasing TA concentration.
The same changing trend was found with increasing zein
concentration for the zein/TA2 systems. These results
illustrated the occurrence of chemical reactions between zein
2693
www.acsabm.org Article
and TA, which was consistent with the results of FT-IR
analysis.
To study the dissolution characteristics of the zein/TA
coating under SGC, the release profiles of TA from the zein/
TA coating were examined and shown in Figure 6b and c. After
3 h of digestion under SGC, the release amount of TA for the
zein3/TA1 system reached about 0.30 mg/mL, while that for
the zein3/TA2, zein3/TA3, and zein3/TA4 systems was lower
than 0.26 mg/mL. After 24 h of digestion under SGC, the
release amount of TA for the zein3/TA1 system slightly
increased to 0.39 mg/mL; by contrast, that for the zein3/TA
systems with TA2, TA3, and TA4 was nearly tripled, reaching
0.60 mg/mL, 0.83 mg/mL, and 0.90 mg/mL, respectively. A
similar changing trend was observed with increasing concen-
tration of zein for the zein/TA2 systems. These results revealed
that the zein/TA systems with higher TA and zein
concentrations have a better sustained release effect due to a
lower dissolution rate of the zein/TA systems under SGC,
possibly due to the stronger interaction between zein and TA.
However, due to the extremely low dissolution rates of the
zein3/TA3 and zein3/TA4 systems, the release amount of β-Gal
was particularly low during the monitoring time (Figure 3a).
Therefore, the zein3/TA2 system was confirmed to be the
optimum system for the oral delivery of β-Gal.
■ CONCLUSION
Overall, this study develops a low-cost controlled delivery
system for proteins with excellent storage stability and provides
insights into the sustained-release mechanism by exploring the
interaction between zein and TA. Notably, the proposed zein3/
TA2 system is superior to the zein system in terms of targeted
release with the RA of β-Gal of nearly 70.26% after 24 h of
digestion under SGC, and a high storage stability with β-Gal
RA of about 90% for 28 days at 25 °C. Additionally, the
stability of the zein/TA system under SGC and storage
depends on both hydrogen bonding and covalent bonding
through the Schiff-base reaction, and the bonding strength
could regulate the sustained release of the delivered β-Gal.
This study provides an effective approach for fabricating
efficient delivery systems for bioactive proteins. Future studies
may be focused on the in vivo oral efficacy determination and
sensory evaluation of such delivery systems.
■
ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsabm.0c01623.
Retention rate of β-Gal activity after encapsulation into
SECs (Table S1), Element composition and content on
the surface of zein and zein/TA (Table S2), Value of b*
changed with time exposed to air (Table S3), In vitro
release profiles of β-Gal from zein coated SECs. Release
profiles were obtained from the incubation for 2 h in
SGF and 22 h in SIF (Figure S1). (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Hongshan Liang − College of Food Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China;
Key Laboratory of Environment Correlative Dietology,
Huazhong Agricultural University, Ministry of Education,
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials
Wuhan 430070, China; orcid.org/0000-0003-4338-
7033; Email: lianghongshan@mail.hzau.edu.cn
Authors
Ziyu Deng − College of Food Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China;
Key Laboratory of Environment Correlative Dietology,
Huazhong Agricultural University, Ministry of Education,
Wuhan 430070, China
Shishuai Wang − College of Culinary and Food Engineering,
Wuhan Business University, Wuhan 430056, China
Yaqiong Pei − College of Culinary and Food Engineering,
Wuhan Business University, Wuhan 430056, China
Bin Zhou − Key Laboratory of Fermentation Engineering,
Ministry of Education; National “111” Center for Cellular
Regulation and Molecular Pharmaceutics; Hubei Key
Laboratory of Industrial Microbiology; School of Biological
Engineering and Food, Hubei University of Technology,
Wuhan 430068, China; orcid.org/0000-0002-2839-
4967
Jing Li − College of Food Science and Technology, Huazhong
Agricultural University, Wuhan 430070, China; Key
Laboratory of Environment Correlative Dietology, Huazhong
Agricultural University, Ministry of Education, Wuhan
430070, China; orcid.org/0000-0001-9229-2263
Xinyao Hou − College of Food Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China;
Key Laboratory of Environment Correlative Dietology,
Huazhong Agricultural University, Ministry of Education,
Wuhan 430070, China
Bin Li − College of Food Science and Technology, Huazhong
Agricultural University, Wuhan 430070, China; Key
Laboratory of Environment Correlative Dietology, Huazhong
Agricultural University, Ministry of Education, Wuhan
430070, China; Functional Food Engineering & Technology
Research Center of Hubei Province, Wuhan 430068, China;
orcid.org/0000-0001-7763-4245
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsabm.0c01623
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 31801586), the
Hubei Provincial Natural Science Foundation of China (Grant
No. 2018CFB233), and the Fundamental Research Funds for
the Central Universities (Grant No. 2662018QD016). The
authors greatly thank colleagues of the Key Laboratory of
Environment Correlative Dietology of Huazhong Agricultural
University for offering many conveniences.
■
REFERENCES
(1) Truong-Le, V.; Lovalenti, P. M.; Abdul-Fattah, A. M.
Stabilization challenges and formulation strategies associated with
oral biologic drug delivery systems. Adv. Drug Delivery Rev. 2015, 93,
95−108.
(2) Muheem, A.; Shakeel, F.; Jahangir, M. A.; Anwar, M.; Mallick,
N.; Jain, G. K.; Warsi, M. H.; Ahmad, F. J. A review on the strategies
for oral delivery of proteins and peptides and their clinical
perspectives. Saudi Pharm. J. 2016, 24 (4), 413−428.
2694
www.acsabm.org Article
(3) Moroz, E.; Matoori, S.; Leroux, J.-C. Oral delivery of
macromolecular drugs: Where we are after almost 100 years of
attempts. Adv. Drug Delivery Rev. 2016, 101, 108−121.
(4) Maher, S.; Mrsny, R. J.; Brayden, D. J. Intestinal permeation
enhancers for oral peptide delivery. Adv. Drug Delivery Rev. 2016, 106,
277−319.
(5) Fu, W.; Chen, E.; McClements, D. J.; Cao, Y.; Liu, S.; Li, B.; Li,
Y. Controllable viscoelastic properties of whey protein-based
emulsion gels by combined cross-linking with calcium ions and
cinnamaldehyde. ACS Appl. Bio Mater. 2019, 2 (1), 311−320.
(6) Sikkens, E. C. M.; Cahen, D. L.; Kuipers, E. J.; Bruno, M. J.
Pancreatic enzyme replacement therapy in chronic pancreatitis. Best
Pract. Res. Clin. Gastroenterology 2010, 24 (3), 337−347.
(7) Ismail, R.; Csóka, I. Novel strategies in the oral delivery of
antidiabetic peptide drugs − Insulin, GLP 1 and its analogs. Eur. J.
Pharm. Biopharm. 2017, 115, 257−267.
(8) Liau, J. J.; Hook, S.; Prestidge, C. A.; Barnes, T. J. A lipid based
multi-compartmental system: Liposomes-in-double emulsion for oral
vaccine delivery. Eur. J. Pharm. Biopharm. 2015, 97, 15−21.
(9) Liu, C.; Kou, Y.; Zhang, X.; Cheng, H.; Chen, X.; Mao, S.
Strategies and industrial perspectives to improve oral absorption of
biological macromolecules. Expert Opin. Drug Delivery 2018, 15 (3),
223−233.
(10) Mundargi, R. C.; Potroz, M. G.; Park, S.; Shirahama, H.; Lee, J.
H.; Seo, J.; Cho, N. J. Natural sunflower pollen as a drug delivery
vehicle. Small 2016, 12 (9), 1167−73.
(11) Fan, T. F.; Park, S.; Shi, Q.; Zhang, X.; Liu, Q.; Song, Y.; Chin,
H.; Ibrahim, M. S. B.; Mokrzecka, N.; Yang, Y.; Li, H.; Song, J.;
Suresh, S.; Cho, N. J. Transformation of hard pollen into soft matter.
Nat. Commun. 2020, 11 (1), 1449−1459.
(12) Tan, E.-L.; Potroz, M. G.; Ferracci, G.; Wang, L.; Jackman, J.
A.; Cho, N.-J. Hydrophobic to superhydrophilic tuning of multifunc-
tional sporopollenin for microcapsule and bio-composite applications.
Appl. Mater. Today 2020, 18, 100525−100536.
(13) Zhao, Z.; Hwang, Y.; Yang, Y.; Fan, T.; Song, J.; Suresh, S.;
Cho, N. J. Actuation and locomotion driven by moisture in paper
made with natural pollen. Proc. Natl. Acad. Sci. U. S. A. 2020, 117
(16), 8711−8718.
(14) Fan, T. F.; Hwang, Y.; Ibrahim, M. S.; Ferracci, G.; Cho, N. J.
Influence of Chemical and physical change of pollen microgels on
swelling/de-swelling behavior. Macromol. Rapid Commun. 2020, 41
(21), e2000155−e2000161.
(15) Deng, Z.; Pei, Y.; Wang, S.; Zhou, B.; Hou, X.; Li, J.; Li, B.;
Liang, H. Designable carboxymethylpachymaran/metal ion architec-
ture on sunflower sporopollenin exine capsules as delivery vehicles for
bioactive macromolecules. J. Agric. Food Chem. 2020, 68 (47),
13990−14000.
(16) Lale, S. V.; Gill, H. S. Pollen grains as a novel microcarrier for
oral delivery of proteins. Int. J. Pharm. 2018, 552 (1−2), 352−359.
(17) Diego-Taboada, A.; Maillet, L.; Banoub, J.; Lorch, M.; Rigby, A.
S.; Boa, A. N.; Atkin, S. L.; Mackenzie, G. Protein free microcapsules
obtained from plant spores as a model for drug delivery: ibuprofen
encapsulation, release and taste masking. J. Mater. Chem. B 2013, 1
(5), 707−713.
(18) Prabhakar, A. K.; Potroz, M. G.; Tan, E. L.; Jung, H.; Park, J.
H.; Cho, N. J. Macromolecular microencapsulation using pine pollen:
loading optimization and controlled release with natural materials.
ACS Appl. Mater. Interfaces 2018, 10 (34), 28428−28439.
(19) Park, J. H.; Seo, J.; Jackman, J. A.; Cho, N. J. Inflated
sporopollenin exine capsules obtained from thin-walled pollen. Sci.
Rep. 2016, 6, 28017−28027.
(20) Maric, T.; Nasir, M. Z. M.; Rosli, N. F.; Budanović, M.;
Webster, R. D.; Cho, N. J. Pumera, M. microrobots derived from
variety plant pollen grains for efficient environmental clean up and as
an anti-cancer drug carrier. Adv. Funct. Mater. 2020, 30 (19),
2000112−2000125.
(21) Potroz, M. G.; Mundargi, R. C.; Gillissen, J. J.; Tan, E.-L.;
Meker, S.; Park, J. H.; Jung, H.; Park, S.; Cho, D.; Bang, S.-I.; Cho, N.-
J. Plant-based hollow microcapsules for oral delivery applications:
https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695
ACS Applied Bio Materials www.acsabm.org Article

toward optimized loading and controlled release. Adv. Funct. Mater. (41) Liang, H.; Zhou, B.; Li, J.; Xu, W.; Liu, S.; Li, Y.; Chen, Y.; Li,
2017, 27 (31), 1700270−1700282. B. Supramolecular design of coordination bonding architecture on
(22) Deng, Z.; Pei, Y.; Wang, S.; Zhou, B.; Li, J.; Hou, X.; Li, J.; Li, zein nanoparticles for pH-responsive anticancer drug delivery. Colloids
B.; Liang, H. Carboxymethylpachymaran entrapped plant-based Surf., B 2015, 136, 1224−33.
hollow microcapsules for delivery and stabilization of beta- (42) Zou, Y.; Guo, J.; Yin, S. W.; Wang, J. M.; Yang, X. Q. Pickering
galactosidase. Food Funct. 2019, 10 (8), 4782−4791. emulsion gels prepared by hydrogen-bonded zein/tannic acid
(23) Wei, X. J.; Hu, W. Y.; Hu, T. J. Effects of carboxymethylpachy- complex colloidal particles. J. Agric. Food Chem. 2015, 63 (33),
maran on signal molecules in chicken immunocytes. Int. J. Biol. 7405−14.
Macromol. 2013, 59, 357−62. (43) Wei, X.; Li, J.; Li, B. Multiple steps and critical behaviors of the
(24) Wei, B.; Zhao, Y.; Wei, Y.; Yao, J.; Chen, X.; Shao, Z. binding of tannic acid to wheat starch: Effect of the concentration of
Morphology and properties of a new biodegradable material prepared wheat starch and the mass ratio of tannic acid to wheat starch. Food
from zein and poly (butylene adipate-terephthalate) by reactive Hydrocolloids 2019, 94, 174−182.
blending. ACS omega 2019, 4 (3), 5609−5616. (44) Chen, H.; Hu, X.; Chen, E.; Wu, S.; McClements, D. J.; Liu, S.;
(25) Pirani, S. I.; Arafat, H. A. Solid waste management in the Li, B.; Li, Y. Preparation, characterization, and properties of chitosan
hospitality industry: A review. J. Environ. Manage. 2014, 146, 320− films with cinnamaldehyde nanoemulsions. Food Hydrocolloids 2016,
336. 61, 662−671.
(26) Paliwal, R.; Palakurthi, S. Zein in controlled drug delivery and (45) Chen, H.; McClements, D. J.; Chen, E.; Liu, S.; Li, B.; Li, Y. In
tissue engineering. J. Controlled Release 2014, 189, 108−122. situ interfacial conjugation of chitosan with cinnamaldehyde during
(27) Patel, A. R.; Velikov, K. P. Zein as a source of functional homogenization improves the formation and stability of chitosan-
colloidal nano-and microstructures. Curr. Opin. Colloid Interface Sci. stabilized emulsions. Langmuir 2017, 33 (51), 14608−14617.
2014, 19 (5), 450−458. (46) Balaguer, M. P.; Borne, M.; Chalier, P.; Gontard, N.; Morel, M.
(28) Elzoghby, A.; Freag, M.; Mamdouh, H.; Elkhodairy, K. Zein- H.; Peyron, S.; Gavara, R.; Hernandez-Munoz, P. Retention and
based nanocarriers as potential natural alternatives for drug and gene release of cinnamaldehyde from wheat protein matrices. Biomacro-
delivery: focus on cancer therapy. Curr. Pharm. Des. 2017, 23 (35), molecules 2013, 14 (5), 1493−1502.
5261−5271. (47) Marin, L.; Ailincai, D.; Mares, M.; Paslaru, E.; Cristea, M.; Nica,
(29) Tarhini, M.; Greige-Gerges, H.; Elaissari, A. Protein-based V.; Simionescu, B. C. Imino-chitosan biopolymeric films. Obtaining,
nanoparticles: From preparation to encapsulation of active molecules. self-assembling, surface and antimicrobial properties. Carbohydr.
Int. J. Pharm. 2017, 522 (1−2), 172−197. Polym. 2015, 117, 762−70.
(30) Chuacharoen, T.; Sabliov, C. M. Zein nanoparticles as delivery (48) Marin, L.; Stoica, I.; Mares, M.; Dinu, V.; Simionescu, B. C.;
systems for covalently linked and physically entrapped folic acid. J. Barboiu, M. Antifungal vanillin-imino-chitosan biodynameric films. J.
Nanopart. Res. 2017, 19 (2), 81−93. Mater. Chem. B 2013, 1 (27), 3353−3358.
(31) Deng, Z.; Wang, S.; Zhou, B.; Li, J.; Zhou, P.; Li, B.; Liang, H. (49) Krogsgaard, M.; Andersen, A.; Birkedal, H. Gels and threads:
Carboxymethylpachymaran-zein coated plant microcapsules-based mussel-inspired one-pot route to advanced responsive materials.
beta-galactosidase encapsulation system for long-term effective Chem. Commun. 2014, 50 (87), 13278−13281.
delivery. Food Res. Int. 2020, 128, 108867−108875.
(32) Liu, F.; Ma, C.; McClements, D. J.; Gao, Y. A comparative
study of covalent and non-covalent interactions between zein and
polyphenols in ethanol-water solution. Food Hydrocolloids 2017, 63,
625−634.
(33) Zhu, X.; Chen, Y.; Hu, Y.; Han, Y.; Xu, J.; Zhao, Y.; Chen, X.;
Li, B. Tuning the molecular interactions between gliadin and tannic
acid to prepare pickering stabilizers with improved emulsifying
properties. Food Hydrocolloids 2021, 111, 106179−106188.
(34) Zhou, S.-D.; Lin, Y.-F.; Xu, X.; Meng, L.; Dong, M.-S. Effect of
non-covalent and covalent complexation of (−)-epigallocatechin
gallate with soybean protein isolate on protein structure and in
vitro digestion characteristics. Food Chem. 2020, 309, 125718−
125729.
(35) Zhang, Z.; Zhang, R.; McClements, D. J. Lactase (β-
galactosidase) encapsulation in hydrogel beads with controlled
internal pH microenvironments: Impact of bead characteristics on
enzyme activity. Food Hydrocolloids 2017, 67, 85−93.
(36) Deng, Z.; Li, J.; Pei, Y.; Wan, J.; Li, B.; Liang, H.
Oligosaccharides act as the high efficiency stabilizer for beta-
galactosidase under heat treatment. Int. J. Biol. Macromol. 2019,
137, 69−76.
(37) Zhan, F.; Li, J.; Wang, Y.; Shi, M.; Li, B.; Sheng, F. Bulk, foam,
and interfacial properties of tannic acid/sodium caseinate nano-
complexes. J. Agric. Food Chem. 2018, 66 (26), 6832−6839.
(38) Uddin, M. J.; Liyanage, S.; Abidi, N.; Gill, H. S. Physical and
biochemical characterization of chemically treated pollen shells for
potential use in oral delivery of therapeutics. J. Pharm. Sci. 2018, 107
(12), 3047−3059.
(39) Lam, P.; Gambari, R. Advanced progress of microencapsulation
technologies: In vivo and in vitro models for studying oral and
transdermal drug deliveries. J. Controlled Release 2014, 178, 25−45.
(40) Fan, T. F.; Potroz, M. G.; Tan, E. L.; Ibrahim, M. S.; Miyako,
E.; Cho, N. J. Species-specific biodegradation of sporopollenin-based
microcapsules. Sci. Rep. 2019, 9 (1), 9626−9639.

2695 https://dx.doi.org/10.1021/acsabm.0c01623
ACS Appl. Bio Mater. 2021, 4, 2686−2695