International Journal of Biological Macromolecules 238 (2023) 124133

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Enhanced biocompatibility of silk sericin/caffeic acid nanoparticles by red

blood cell membranes cloaking
Chunru Wang a, Qingyang Lu a, Yingjie Xiang a, Yulan Yin a, Junyao Li a, Yalu Liu b, c, *,
Xiaochen Wu a, **
a
Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
b
Department of Ophthalmology, The Affiliated Xuzhou Municipal Hospital of Xuzhou Medical University, Daxue Road 269, Xuzhou 221002, China
c
Xuzhou First People's Hospital, Daxue Road 269, Xuzhou 221002, China

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

Keywords: Caffeic acid (CA) is an antioxidant phenolic compound that enriched in coffee beans, however, its administration
Caffeic acid often restrains by the instability and low solubility. Nanoparticle encapsulation is an effective approach to
Silk sericin improve the therapeutic activity of CA. For example, silk sericin (SS), a natural biomaterial finds applications in
Red blood cell membranes cloaking
food, cosmetics and biomedical fields, is proved here to be an appropriate encapsulation agent for CA, and a SS/
CA composite nanoparticle has been fabricated. To further improve the biocompatibility of SS/CA, a red blood
cell membranes (RM) cloaking strategy is adopted. The as-formed SS/CA/RM preserves the antioxidant activity
of CA, and shows satisfactory biocompatibility especially under high concentration. Hope this can provide a
potential appropriative strategy to adjust the chemical stability of insoluble drugs and to improve their
biocompatibility.

1. Introduction properties, cross biological barriers, better pharmacokinetic behavior,

Dietary phenolic compounds possess health-promoting properties
due to their various biological effects including high anti-inflammatory
and antioxidant capacity, thus, the consume of foods that are rich in
phenolic compounds may provide therapeutic benefits [1,2]. In this
way, adjuvant phenolic diet therapy is widely accepted during the
comprehensive treatment of cancer, and dietary polyphenols are good
candidates for the study of drug delivery and pharmacological action
[3,4]. However, some of the dietary phenolic compounds are insoluble
in aqueous solution, and showed low bioavailability as well as possible
side effects when delivering as free drugs [5]. Therefore, appropriate
drug delivery strategies are required to reduce their toxic effects and
build high efficacy systems [6]. Nanoscale delivery vehicles (e.g.,
nanoparticle, polymeric micelle, liposome, and colloid) that encapsulate
drugs to overcome water insolubility and improve biological effective­
ness have drawn continuous attention [7,8]. Particularly, nanoparticles
(NPs) show great potential as effective vehicles to deliver drugs, due to
their improvement on drug solubility and stability, specificity to target
site, sensitive to environment stimuli, tailored physicochemical
and lower toxicity effects [9–11]. A variety of metallic and inorganic
NPs (e.g., gold, mesoporous silica, calcium carbonate, magnetic iron
oxide), [12] synthetic polymers (e.g., poly(lactic acid), poly(glycolic
acid), poly(lactic-co-glycolic acid)), [6] and natural materials (e.g.,
collagen, silk fibroin, chitosan, alginate) [13] have been extensively
studied in the preparation of NPs vehicles to encapsulate hydrophobic
drugs, and generally improve the pharmacodynamic and pharmacoki­
netic properties of drugs [14,15]. For example, hydrophobic drug-core
silica-shell NPs were fabricated with high drug encapsulation effi­
ciencies, and exhibited improved efficacy (i.e., in vitro cytotoxic effects
as well as in vivo suppression of tumor growth); [16] zidovudine loaded
polyvinylpyrrolidone/stearic acid-polyethylene glycol NPs exhibited
obvious improvement in cellular internalization, which help to over­
come the shortcomings of free zidovudine (e.g., short half-life in vivo and
frequent administrations); [17] resveratrol loaded sericin NPs could
realize sustained drug release for over 72 h, and increase the potential
therapeutic effect by strongly inhibiting the growth of colorectal
adenocarcinoma [18]. However, even the NPs encapsulated drugs
showed still some inevitable side effects such as inflammatory and

* Corresponding author.
** Corresponding author.
E-mail addresses: liuyalu0407@163.com (Y. Liu), wxcguest@126.com (X. Wu).

https://doi.org/10.1016/j.ijbiomac.2023.124133
Received 21 December 2022; Received in revised form 15 March 2023; Accepted 18 March 2023
Available online 23 March 2023
0141-8130/© 2023 Elsevier B.V. All rights reserved.
C. Wang et al.
immune responses, especially when they were delivered with high
concentrations.
In order to further enhance the biocompatibility and relieve the
possible side effects of NPs-loaded drugs, a cell membrane cloaking
strategy attracts raising attention [19–22]. Cellular functions are
generally regulated by the cell membranes, thus the coating of cell
membranes on the NPs can disguise them as homologous substances,
and empower the NPs with biomimetic characteristics and bio-
interfacing functionalities of the source cells, in this way to improve
the biocompatibility and therapeutic efficacy of the loaded drugs
[23–25]. Clinically, cells (e.g., red blood cells) were collected from the
patients before surgery, lysed to separate the cell membranes, and
coated onto NPs-loaded drugs to acquire composite drugs [26]. For
example, based on the ability of neutrophils to accumulate at inflam­
matory sites to eliminate pathogens, neutrophil membranes were coated
on the NPs-loaded-sparfloxacin to acquire precise targeting ability on
inflammatory sites, the composite showed prolonged circulation time
and controlled-release property, exhibiting large advantages in treating
inflammations; [27] on account of the pathogens clearing ability of
macrophages, macrophage membranes were utilized to coat NPs, and
fabricated a nano-toxoid vaccine that capable to provide protection ef­
fect against bacterial infection; [25] taking advantage of the multiple
tumor antigens on cancer cell membranes, cancer cell membranes were
coated on the surface of poly(lactic-co-glycolic acid)-immunological
adjuvant NPs to form an effective anticancer vaccine, elicit potent
antitumor immunity in vivo, and generate strong antitumor responses
[28]. Additionally, comparing to live cell products, the cell membranes
coated NPs overcome the delivery route and storage instability re­
strictions, providing an alternative strategy for stem cell therapy [29].
In our previous research, silk sericin (SS) as an efficient drug carrier
could load various insoluble drugs to fabricate SS encapsulated drug NPs
(SS/drug NPs) through a facile one-step process [30]. The SS/drug NPs
presented good dispersity in aqueous solution, and exhibited enhanced
antioxidant efficiency, biocompatibility, and in vivo therapeutic effects
comparing to the individual drugs. However, a high concentration (150
mg/kg) of SS/drug NPs presented lower in vivo therapeutic effect than
under low concentrations (50 & 100 mg/kg), probably because the
enhanced toxicity raised from high concentration [30]. Herein, in order
to further increase the biocompatibility of SS/drug NPs, especially under
high concentrations, red blood cell membranes (RM) were used to cloak
SS/drug NPs. Red blood cells are abundant and accessible in animal
bodies, they are appropriate sources for extracting the autologous cell
membranes. Using caffeic acid (CA) as a model drug, the morphology,
size distribution, structure, hemocompatibility and cytocompatibility of
the RM coated SS/CA NPs (SS/CA/RM NPs) were detail studied, espe­
cially their toxicity under high concentrations. Hope this NPs-based cell
membrane cloaking strategy could offer application prospects in
biomedical and biotechnological fields.
2. Experimental
2.1. Materials
Bombyx mori cocoon was collected from Shanxi, China. Caffeic acid
(> 98 %) and 2, 4, 6-tripyridyltriazine were bought from Shanghai
Aladdin Biochemical Technology Co., Ltd. Other reagents were bought
from Sinopharm Chemical Reagent Co., Ltd.
2.2. Preparation of SS/CA/RM NPs
Bombyx mori cocoon was boiled in the aqueous solution of NaHCO3
(0.5 %) for 60 min to extract the SS [30]. Then, CA was added into the
aqueous solution of SS (10 mL, 12 mg/mL) to reach a final concentration
of 12 mg/mL. The mixture was stirred at 30 ◦ C for 10 min, and
neutralized by HCl to get the SS/CA NPs.
Whole blood was collected from male Kunming mice and centrifuged
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International Journal of Biological Macromolecules 238 (2023) 124133
at 3000 r at 4 ◦ C for 6 min, after which the serum was carefully removed
to get the red blood cells. The red blood cells were washed 3 times with
cold PBS. Then, hemolysis was triggered by suspending the red blood
cells in deionized water at 4 ◦ C for 1 h. RM was acquired by centrifuge at
12000 r at 4 ◦ C for 6 min, subsequently washed 3 times with cold water,
and stored at − 20 ◦ C for further usage.
To cloak the RM on SS/CA NPs, 1 mL of SS/CA (24 mg/mL) was
mixed with 0.1 mL of RM (~1 × 107/mm3). The mixture was sonicated
with an ultrasonic cell pulverizer (JY96-IIN, Shanghai Huxi Industry
Co., Ltd) at 3 W until a clear solution (SS/CA/RM) was got.
2.3. Characterization
Transmission electron microscopy (TEM, JEOL JEM-2100F, Japan)
was used to characterize the morphology of the samples. The sample was
prepared by dispersing the SS/CA/RM in H2O, dropping it onto a carbon
support film on a copper grid, removing the excess solution after 1 min,
and leaving to air dry. Granulometric distribution was measured on a
NanoBrook Omni (Brookhaven Instruments, Holtsville, New York). FTIR
spectra were acquired on a Bruker Tensor II Fourier transform infrared
spectrometer.
2.4. Visualization of SS/CA/RM NPs
DiO, a lipophilic tracer, was used to label RM and observed under a
fluorescent microscope (Olympus CKX53, Japan). Briefly, SS/CA/RM
NPs (2 mg/mL, 10 μL) were mixed with DiO (10 μM, 10 μL), incubated at
dark for 30 min, dried, and observed under a fluorescent microscope.
2.5. Intracellular co-localization [21] of SS/CA/RM NPs
To investigate the intracellular location of SS/CA/RM NPs, during
the synthesize process, a fluorescent probe Sudan red III was loaded onto
SS, while another fluorescent probe DiO was labeled on the RM.
NCM460 cells were seeded in 24-well plates, cultured, and incubated
with the dual-fluorophore-labeled SS/CA/RM NPs for 30 min. The cells
were then washed 3 times with PBS and fixed with 4 % para­
formaldehyde solution. Finally, the nuclei were stained with a fluores­
cent probe DAPI for fluorescent imaging and the cells were observed by
a fluorescent microscope.
2.6. Hemolytic evaluation [31] of SS/CA/RM NPs
Different concentrations of SS/CA, SS, CA, and SS/CA/RM NPs (all of
which were dispersed in 0.9 % saline solution) were mixed with anti­
coagulant blood, respectively, and incubated at 37 ◦ C for 1 h. Then the
solutions were centrifuged, and the absorbance of the supernatant at
545 nm was measured on a microplate reader. Negative and positive
controls referred to anticoagulant blood incubated in 0.9 % saline so­
lution and H2O, respectively.
2.7. In vitro antioxidant activity [30] of SS/CA/RM NPs
A ferric reducing antioxidant potential (FRAP) assay was applied to
measure the antioxidant activity of the SS/CA/RM NPs. 1 mM of 2, 4, 6-
tripyridyltriazine (in 40 mM of HCl, 1 mL) and 2 mM of ferric chloride
(in pH 3.6 NaAc, 1 mL) were mixed and denoted as the working fluid.
195 μL of the working fluid was mixed with 5 μL of SS/CA/RM solution,
and the absorption change at 593 nm versus time was immediately
measured on a microplate reader. The antioxidant activity was
expressed as the concentration of the reaction product (Fe2+, mM),
which was calculated by the standard curve of a standard FeSO4
solution.
C. Wang et al.
2.8. In vitro cytotoxicity of SS/CA/RM NPs
Cytotoxicity of the SS/CA/RM and SS/CA was evaluated on human
umbilical vein endothelial cells (HUVEC) and NCM460 cells by a stan­
dard MTT assay, respectively. The SS/CA/RM was dispersed in Dul­
becco's Modified Eagle's Medium (DMEM) and sterilized before usage.
HUVEC cells (4 × 103 cells per well) were seeded in 96-well plates,
cultured at 37 ◦ C for 24 h, and then treated with 100 μL of DMEM
dispersed SS/CA/RM NPs (100 μg/mL and 200 μg/mL) for 24–72 h.
Subsequently, the culture medium was replaced by MTT solution, and
incubated for another 4 h. 150 μL of dimethyl sulfoxide was added
before measuring the optical density at 490 nm. The cell viability results
were compared to the blank control, SS/CA, SS, and CA-treated cells,
respectively.
The morphology of HUVEC after incubating with SS/CA/RM was
measured by an Olympus CKX53 fluorescence microscope (Japan).
HUVEC cells (1 × 105 cells per well) were seeded in 24-well plates,
cultured in the presence of DMEM dispersed SS/CA/RM (100 μg/mL and
200 μg/mL) for 24–72 h, and observed by a fluorescence microscope.
2.9. Antioxidative stress ability [32] of SS/CA/RM NPs in H2O2-
stimulated cells
The SS/CA/RM was dispersed in DMEM and sterilized before use.
NCM460 cells (4 × 103 cells per well) were seeded in 96-well plates,
cultured at 37 ◦ C for 24 h, stimulated with 100 μL of DMEM dispersed
H2O2 (1 mM) for 2 h, and treated with 100 μL of DMEM dispersed SS/
CA/RM NPs (10 μg/mL) for 24 h. Subsequently, the culture medium was
replaced by MTT solution, and the cell viability was measured. The cell
viability results were compared to the blank control, SS/CA, SS, and CA-
treated cells, respectively.
For the characterization of cell morphology, after the stimulation of
H2O2 and treatment of SS/CA/RM, the cells were treated with 2′ , 7′ -
dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min, washed
with PBS for 3 times, and the fluorescence was observed by an Olympus
CKX53 fluorescence microscope (Japan). To measure the fluorescence
intensity in cells semi-quantitatively, the photograph was analyzed by
Image J, and the mean fluorescence intensity was calculated.
Same procedure was carried out on HUVECs.
2.10. Statistical analysis
All the measurements were performed at least in triplicate, and all
the data were shown as mean ± standard deviation. Data were analyzed
using one-way ANOVA and two-way ANOVA. p < 0.05 was considered
significant.
3. Results and discussion
CA, a naturally occurring phenolic compound that exists in fruits,
grains and dietary supplements, has therapeutic potentials including
antioxidant, anticancer, anti-inflammatory, and antiviral effects [9]. It is
a constantly used ingredient in cosmetics, drugs, or dietary supplements
[33]. However, CA has poor solubility and low chemical stability in
aqueous solution, which restricts its further applications [33,34]. The
encapsulation of CA in NPs would favor the improvement of its solubi­
lity, stability, and bioavailability [35]. SS is one of the major proteins
constituting silkworm cocoon, it binds and strengthens the silk fiber, and
usually presents as a by-product of the silk industry. SS has good water
solubility and attractive bioactive properties, has been reported to be
capable of enhancing the bioavailability of natural ingredients in foods,
promoting cellular adhesion and proliferation, suppressing lipid perox­
idation, and inhibiting tyrosinase enzyme activity, thus it is highly
desirable for drug delivery, cell culture, tissue engineering, and cos­
meceutical applications [36–38]. Specifically, due to its biocompati­
bility and biodegradability, SS raises as an attractive drug carrier, the
3
International Journal of Biological Macromolecules 238 (2023) 124133
high chemical reactivity of SS made it possible to modulate the surface
properties to favor drug encapsulation and delivery [13,38]. Addition­
ally, similar to CA, SS can act as a functional food nutrition additive due
to its antioxidant property.
As illustrated in Fig. 1, using CA as a model drug, the SS encapsulated
CA NPs (SS/CA NPs) can be fabricated according to our previous study
[30]. In order to reduce the possible high concentration toxicity, RM was
coated on SS/CA NPs to form a composite NPs (SS/CA/RM NPs) through
a facile procedure. The visualization of RM coating on SS/CA NPs was
achieved by a fluorescent microscope. As shown in Fig. 2A & B, after
labeling with a fluorescent dye (DiO), SS/CA/RM NPs showed distinct
green fluorescence due to the conjugation of DiO with RM, while SS/CA
NPs barely exhibited any fluorescence, indicating the successful coating
of RM. The SS/CA/RM NPs exhibited good dispersibility in aqueous
solution with neither aggregation nor flocculation (Fig. 2C).
To further characterize the microscopic structures, the as-formed SS/
CA/RM NPs were examined under a transmission electron microscope
(TEM). SS/CA NPs were randomly and uniformly dispersed, presenting
clearly a NPs morphology (Fig. 2D). The as-formed SS/CA/RM NPs also
presented a morphology of NPs, which was constructed by the RM and
the SS/CA NPs loading both inside and outside the membranes (Fig. 2E).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed to further verify the existence of RM. As shown in Fig. 2F,
the protein bands of SS/CA/RM NPs were consistent with those of RM,
confirmed the coating of RM. The average diameter of the SS/CA/RM
NPs was measured to be 219.0 nm (PDI = 0.339, Fig. 2G), and the size
could stay almost unchanged for 16 days in aqueous solution (4 ◦ C), with
no visible precipitation was observed. The coating of RM changed the
surface potential of the SS/CA NPs. As shown in Fig. 2H, the Zeta-
potential values of SS/CA/RM NPs were more negative than the SS/
CA NPs under alkaline conditions, which should be resulted from the
coating of RM.
The FT-IR spectra of SS/CA, RM, and SS/CA/RM NPs were shown in
Fig. 2I. SS/CA NPs presented the characteristic absorption peaks of both
SS and CA, including the stretching vibrations of C– – O at 1650 cm− 1, the
deformation vibrations of C–H at 1450 cm− 1, as well as the phenol –OH
vibrations at~3400 cm− 1 [39]. The FTIR spectrum of RM presented
peaks at 3320 cm− 1 and 1650 cm− 1 that belonged to C–H stretching
vibrations and C– – O stretching vibrations, respectively, together with
peaks at 1228 cm− 1 and 1080 cm− 1 that belonged to the P– – O stretching
vibrations and P–O–C stretching vibrations [26]. SS/CA/RM NPs
showed the characteristic absorption peaks of both SS/CA NPs and RM,
confirmed their conjunction.
The antioxidant capacity of SS/CA/RM NPs was measured in Fig. 3
using the kinetics of FRAP. Both SS and CA possessed initial antioxidant
efficiency that mainly due to the functional groups in the backbones.
Although cloaked by RM, the SS/CA/RM NPs preserved the antioxidant
capacity of SS and CA, showing only slightly lower antioxidant effi­
ciency than that of SS/CA NPs, favor for further applications in
biomedical field (Fig. 3A). Moreover, the antioxidant efficiency of the
SS/CA/RM NPs was significantly influenced by their concentration, a
higher concentration led to much increased antioxidant efficiency
(Fig. 3B). The blood compatibility of SS/CA/RM NPs was then investi­
gated by a hemolysis assay, and the results showed no hemolysis
occurred (Fig. 4). No significant hemolysis was presented with the
concentration of SS/CA/RM NPs reaching up to 3.4 mg/mL, demon­
strating that the SS/CA/RM NPs have excellent hemocompatibility.
Cytotoxicity of the SS/CA/RM NPs was assessed on NCM460 cells
and showed in Fig. 5A. After 48 h of co-incubation, SS with a high
concentration of 100 μg/mL hardly affected the viability of NCM460
cells (viability: 94.8 %), showing excellent biocompatibility. However,
free CA with the same concentration (100 μg/mL) only exhibited a low
viability of 69.8 %, significantly different from the control group (p <
0.05). As a dietary antioxidant, the cell viability of CA was directly
proportional to the molecule concentrations. Previous reports have
proved that CA with a concentration of < 0.1 mM (~18 μg/mL) can
C. Wang et al. International Journal of Biological Macromolecules 238 (2023) 124133

Fig. 1. Schematic illustration of the basic constituents of SS/CA/RM NPs. RBC: red blood cells.

Fig. 2. Morphology and physiochemical properties of the SS/CA/RM NPs. (A-B) Representative fluorescence images of (A) SS/CA NPs and (B) SS/CA/RM NPs, both
labeled with DiO dye (green). (C) Image of the SS/CA/RM NPs. (D) TEM image of the SS/CA NPs. (E) TEM image and schematic illustration of the SS/CA/RM NPs. (F)
SDS-PAGE analysis, (G) Diameter and storage stability (inset), (H) Zeta potential, and (I) FTIR spectra of the SS/CA/RM NPs. For SDS-PAGE analysis in (F), RIPA lysis
buffer was used to extract the total proteins from the samples, then the proteins were analyzed via polyacrylamide gel electrophoresis and stained with Kaumas blue,
RBC referred to red blood cells.

Fig. 3. In vitro antioxidant activity of SS/CA/RM NPs on the kinetics of FRAP. (A) With 44 μg/mL of SS/CA and SS/CA/RM NPs, respectively. (B) With different
concentrations of SS/CA/RM NPs.

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biocompatibility.
Due to the fact that cytotoxicity can be sharply influenced by the
concentration of drugs, the cytotoxicity of SS/CA/RM NPs was also
measured under a higher concentration (Fig. 5A). By increasing the
concentration to 200 μg/mL, the cytocompatibility of SS/CA NPs
decreased significantly, a viability of 69.5 % was presented after 48 h of
co-incubation, significantly different from the control group (p < 0.05).
Comparatively, for the cells treated by the SS/CA/RM NPs, a high con­
centration of 200 μg/mL can still retain a viability of 106.9 %, clearly
distinguished from the SS/CA NPs treated group (p < 0.05) and showed
excellent cytocompatibility at high concentration. Similar phenomenon
was shown in the case of HUVEC. As shown in Fig. 5B, SS/CA/RM NPs
exhibited much higher cytocompatibility than SS/CA NPs, especially at
high concentration (200 μg/mL). Besides, high concentration of SS/CA/
RM NPs scarcely affected the morphology of cells (Fig. 5C).
The cell uptake of the SS/CA/RM NPs was verified in Fig. 6A. Sudan
red III, a fluorescent probe, was loaded on SS (defined as S-SS). Another
fluorescent probe, DiO was loaded on RM (defined as D-RM). Then, the
S-SS was coated by D-RM to form dual-fluorophore-labeled NPs, and
incubated with NCM460 cells. As shown in Fig. 6A, around the blue
DAPI-stained nuclei, the red fluorescent signal from Sudan red III (rep­
resenting the SS) and the green signal from DiO (representing the RM)
co-localized, indicating the desirable cell uptake of the SS/CA/RM NPs.
Fig. 4. Hemolysis of SS/CA/RM NPs, A- and A+ referred to the negative (0.9 %
Furthermore, the intracellular ROS elimination property of SS/CA/RM
NaCl solution) and positive (H2O) controls, respectively. ****p < 0.0001
NPs was evaluated on NCM460 cells with the help of a ROS probe DCFH-
compared to the positive control.
DA (Fig. 6B-D). DCFH-DA has no initial fluorescence, it can freely across
the cell membranes and hydrolyzed into DCFH. DCFH stayed inside the
maintain a good cell viability during 48 h of incubation, while con­
cells, and oxidated by intracellular ROS to form fluorescent DCF, whose
centrations larger than 0.2 mM (~36 μg/mL) may cause adverse impact
fluorescence intensity thus indicated the extent of intracellular ROS. As
on the cells [40], consistent with our results. SS/CA NPs preserved the
shown in Fig. 6B & C, almost no fluorescence was observed in cells
cytocompatibility of SS, and showed a high cell viability of 103.9 % after
without H2O2 treatment. After H2O2 stimulation, the intracellular ROS
48 h incubation. The encapsulation of CA by SS largely enhanced the
greatly increased and there appeared intensive fluorescence (**p < 0.01
biocompatibility of CA. The cloaking of RM further enhanced the cyto­
compared to the control group without H2O2 treatment). Both SS and CA
compatibility of SS/CA NPs, and 100 μg/mL of SS/CA/RM NPs produced
can reduce the generation of ROS to some tent, but the fluorescence was
a cell viability of 117.6 % after 48 h co-incubation. The coating of RM
still significantly different from the control group (*p < 0.05). SS/CA
may pretend the SS/CA NPs as homologous substances, and endow the
NPs treated cells showed more obvious decrease in fluorescent intensity
SS/CA NPs with biomimetic characteristics to improve their
due to the higher ROS scavenging ability of SS/CA NPs, which might be

Fig. 5. Cytotoxicity of SS/CA and SS/CA/RM NPs. (A) Cell viability of NCM460 cells incubated with SS/CA and SS/CA/RM NPs for 48 h. (B) Cell viability and (C)
morphology of HUVEC incubated with SS/CA and SS/CA/RM NPs for 24–72 h. *p < 0.05 and **p < 0.005 compared to the control group with the same incubation
time, #p < 0.05 compared to the SS/CA NPs treated group with the same incubation time and same concentration.

5
C. Wang et al. International Journal of Biological Macromolecules 238 (2023) 124133

Fig. 6. (A) Intracellular co-localization of the dual-fluorophore-labeled SS/CA/RM NPs (RM: DiO/green; SS: Sudan red III/red), scale bar: 50 μm. (B) Representative
fluorescence images of total intracellular ROS (DCFH-DA) of NCM460 under different treatments. (C) Quantification of the intracellular ROS level measured by the
fluorescence intensity of DCFH-DA in (B). *p < 0.05 and **p < 0.01 compared to the control group, #p < 0.05 compared to the H2O2 treated group. (D) Viability of
NCM460 cells incubated with SS/CA and SS/CA/RM NPs after different treatments.

raised from the better aqueous dispersibility and stability than free CA.
SS/CA/RM NPs reserved the strong ROS scavenging property of SS/CA
NPs, and significantly decreased the fluorescence intensity. After
coating with RM, the cellular uptake capacity of the SS/CA/RM NPs
should be enhanced, and it was easier for the SS/CA/RM NPs to enter the
cells than the SS/CA NPs. However, the coating of RM also blocked out
some of the antioxidant groups of the SS/CA NPs, which sacrificed the
antioxidant capacity to some tent (which was consistent with the in vitro
antioxidant shown in Fig. 3). Therefore, the ROS scavenging property of
the SS/CA/RM NPs was slightly lower than the SS/CA NPs. Subse­
quently, SS/CA/RM NPs can thus visibly increase the viability of H2O2-
treated cells by protecting them from oxidative stress (Fig. 6D).
Furthermore, in the case of HUVEC, SS/CA/RM NPs can also perfectly
balance the cytotoxicity and antioxidant activity at high concentrations
(Fig. 7), a high concentration of 200 μg/mL still resulted in a cell
viability of 57 % after the cells were stimulated by H2O2. SS/CA/RM NPs
was capable of relieving oxidative stress while retaining high biocom­
patibility, showing great potential in biomedical field.
Fig. 7. Viability of HUVEC incubated with high concentrations of SS/CA and
4. Conclusion SS/CA/RM NPs after H2O2 treatments.

A NPs-based cell membrane cloaking strategy was adopted here to

vivo and in vitro, and showed no hemolysis under a high concentration of
acquire a SS/CA/RM NPs. The SS/CA/RM NPs showed an average
3.4 mg/mL. Moreover, the SS/CA/RM NPs significantly enhanced the
diameter of 219.0 nm, preserved the antioxidant capacity of CA both in
cytocompatibility of the non-cloaked SS/CA NPs, and exhibited

6
C. Wang et al.
excellent cytocompatibility even at a high concentration of 200 μg/mL.
This strategy provided a possible way to adjust the chemical instability
of insoluble drugs and to improve their performances, expecting great
potential in biomedical and biotechnological fields.
CRediT authorship contribution statement
Chunru Wang: Methodology, Formal analysis, Data Curation,
Visualization.
Qingyang Lu: Validation, Data Curation.
Yingjie Xiang: Formal analysis, Visualization.
Yulan Yin: Investigation, Data Curation.
Junyao Li: Resources, Formal analysis.
Yalu Liu: Writing-Review & Editing, Supervision, Funding.
Xiaochen Wu: Investigation, Visualization, Writing-Original Draft,
Funding.
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgements
This work was supported by Xuzhou Municipal Health Commission
Project (XWKYSL20210257), Training of Youth Medical Talents in
Xuzhou First People's Hospital (QMHB2021018), and Talent Foundation
funded by Province and Ministry Co-construction Collaborative Inno­
vation Center of Eco-chemical Engineering (No. STHGYX2206).
References
[1] Z. Cheng, Y. Wang, B. Li, Dietary polyphenols alleviate autoimmune liver disease
by mediating the intestinal microenvironment: challenges and hopes, J. Agric.
Food Chem. 70 (35) (2022) 10708–10737.
[2] O.M. Agunloye, G. Oboh, Caffeic acid and chlorogenic acid: evaluation of
antioxidant effect and inhibition of key enzymes linked with hypertension, J. Food
Biochem. 42 (4) (2018), e12541.
[3] P.Stanely Mainzen Prince, K.Senthil Kumaran, Preventive effects of caffeic acid on
lipids, lipoproteins and glycoproteins in isoproterenol induced myocardial
infarcted rats, Food Res. Int. 45 (1) (2012) 155–160.
[4] D.K. Maurya, T.P.A. Devasagayam, Antioxidant and prooxidant nature of
hydroxycinnamic acid derivatives ferulic and caffeic acids, Food Chem. Toxicol. 48
(12) (2010) 3369–3373.
[5] H.S. Khan, A.F. Alhumaydhi, A.M. Khan, H. Younus, Therapeutic potential of
polyphenols and their nanoformulations in the treatment of colorectal cancer, Anti
Cancer Agents Med. Chem. 21 (16) (2021) 2117–2129.
[6] B. Bahrami, M. Hojjat-Farsangi, H. Mohammadi, E. Anvari, G. Ghalamfarsa,
M. Yousefi, F. Jadidi-Niaragh, Nanoparticles and targeted drug delivery in cancer
therapy, Immunol. Lett. 190 (2017) 64–83.
[7] Y. Guan, H. Chen, Q. Zhong, Nanoencapsulation of caffeic acid phenethyl ester in
sucrose fatty acid esters to improve activities against cancer cells, J. Food Eng. 246
(2019) 125–133.
[8] S.-H. Choi, D.-Y. Lee, S. Kang, M.-K. Lee, J.-H. Lee, S.-H. Lee, H.-L. Lee, H.-Y. Lee,
Y.-I. Jeong, Caffeic acid phenethyl ester-incorporated radio-sensitive nanoparticles
of phenylboronic acid pinacol ester-conjugated hyaluronic acid for application in
radioprotection, Int. J. Mol. Sci. 22 (12) (2021) 6347.
[9] L.E. Aguilar, S.R. Jang, C.H. Park, K.M. Lee, Supramolecular caffeic acid and
bortezomib nanomedicine: prodrug inducing reactive oxygen species and
inhibiting cancer cell survival, Pharmaceutics 12 (11) (2020) 1082.
[10] D. Lombardo, M.A. Kiselev, M.T. Caccamo, Smart nanoparticles for drug delivery
application: development of versatile nanocarrier platforms in biotechnology and
nanomedicine, J. Nanomater. 2019 (2019) 3702518.
[11] M.J. Mitchell, M.M. Billingsley, R.M. Haley, M.E. Wechsler, N.A. Peppas, R. Langer,
Engineering precision nanoparticles for drug delivery, Nat. Rev. Drug Discov. 20
(2) (2021) 101–124.
[12] T. Vangijzegem, D. Stanicki, S. Laurent, Magnetic iron oxide nanoparticles for drug
delivery: applications and characteristics, Expert Opin. Drug Deliv. 16 (1) (2019)
69–78.
[13] A. Jain, S.K. Singh, S.K. Arya, S.C. Kundu, S. Kapoor, Protein nanoparticles:
promising platforms for drug delivery applications, ACS Biomater. Sci. Eng. 4 (12)
(2018) 3939–3961.
[14] W.H.D. Jong, P.J. Borm, Drug delivery and nanoparticles: applications and
hazards, Int. J. Nanomed. 3 (2) (2008) 133–149.
[15] M. Anwar, F. Muhammad, B. Akhtar, Biodegradable nanoparticles as drug delivery
devices, J. Drug Deliv. Sci. Technol. 64 (2021), 102638.
International Journal of Biological Macromolecules 238 (2023) 124133
[16] G. Yang, Y. Liu, H. Wang, R. Wilson, Y. Hui, L. Yu, D. Wibowo, C. Zhang, A.
K. Whittaker, A.P.J. Middelberg, C.-X. Zhao, Bioinspired core–shell nanoparticles
for hydrophobic drug delivery, Angew. Chem. Int. Ed. 58 (40) (2019)
14357–14364.
[17] K.S. Joshy, S. Snigdha, G. Anne, K. Nandakumar, P. Laly, A.T. Sabu, Poly (vinyl
pyrrolidone)-lipid based hybrid nanoparticles for anti viral drug delivery, Chem.
Phys. Lipids 210 (2018) 82–89.
[18] K. Suktham, T. Koobkokkruad, T. Wutikhun, S. Surassmo, Efficiency of resveratrol-
loaded sericin nanoparticles: promising bionanocarriers for drug delivery, Int. J.
Pharm. 537 (1) (2018) 48–56.
[19] L.-L. Li, J.-H. Xu, G.-B. Qi, X. Zhao, F. Yu, H. Wang, Core–shell supramolecular
gelatin nanoparticles for adaptive and “on-demand” antibiotic delivery, ACS Nano
8 (5) (2014) 4975–4983.
[20] X. Chen, J. Li, Bioinspired by cell membranes: functional polymeric materials for
biomedical applications, Mater. Chem. Front 4 (3) (2020) 750–774.
[21] Y. Wang, D. Wang, Y. Zhang, H. Xu, L. Shen, J. Cheng, X. Xu, H. Tan, X. Chen, J. Li,
Tumor microenvironment-adaptive nanoplatform synergistically enhances
cascaded chemodynamic therapy, Bioact. Mater. 22 (2023) 239–253.
[22] Y. Wang, X. Xu, X. Chen, J. Li, Multifunctional biomedical materials derived from
biological membranes, Adv. Mater. 34 (46) (2022) 2107406.
[23] J. Jin, B. Krishnamachary, J.D. Barnett, S. Chatterjee, D. Chang, Y. Mironchik,
F. Wildes, E.M. Jaffee, S. Nimmagadda, Z.M. Bhujwalla, Human cancer cell
membrane-coated biomimetic nanoparticles reduce fibroblast-mediated invasion
and metastasis and induce T-cells, ACS Appl. Mater. Interfaces 11 (8) (2019)
7850–7861.
[24] T. Wu, X. Hou, J. Li, H. Ruan, L. Pei, T. Guo, Z. Wang, T. Ci, S. Ruan, Y. He, Z. He,
N. Feng, Y. Zhang, Microneedle-mediated biomimetic cyclodextrin metal organic
frameworks for active targeting and treatment of hypertrophic scars, ACS Nano 15
(12) (2021) 20087–20104.
[25] X. Wei, D. Ran, A. Campeau, C. Xiao, J. Zhou, D. Dehaini, Y. Jiang, A.V. Kroll,
Q. Zhang, W. Gao, D.J. Gonzalez, R.H. Fang, L. Zhang, Multiantigenic nanotoxoids
for antivirulence vaccination against antibiotic-resistant gram-negative bacteria,
Nano Lett. 19 (7) (2019) 4760–4769.
[26] C. Tao, X. Nie, W. Zhu, J. Iqbal, C. Xu, D.-A. Wang, Autologous cell membrane
coatings on tissue engineering xenografts for suppression and alleviation of acute
host immune responses, Biomaterials 258 (2020), 120310.
[27] K. Wang, Y. Lei, D. Xia, P. Xu, T. Zhu, Z. Jiang, Y. Ma, Neutrophil membranes
coated, antibiotic agent loaded nanoparticles targeting to the lung inflammation,
Colloids Surf. B 188 (2020), 110755.
[28] A.V. Kroll, R.H. Fang, Y. Jiang, J. Zhou, X. Wei, C.L. Yu, J. Gao, B.T. Luk,
D. Dehaini, W. Gao, L. Zhang, Nanoparticulate delivery of cancer cell membrane
elicits multiantigenic antitumor immunity, Adv. Mater. 29 (47) (2017) 1703969.
[29] H. Liang, K. Huang, T. Su, Z. Li, S. Hu, P.-U. Dinh, E.A. Wrona, C. Shao, L. Qiao, A.
C. Vandergriff, M.T. Hensley, J. Cores, T. Allen, H. Zhang, Q. Zeng, J. Xing, D.
O. Freytes, D. Shen, Z. Yu, K. Cheng, Mesenchymal stem cell/red blood cell-
inspired nanoparticle therapy in mice with carbon tetrachloride-induced acute
liver failure, ACS Nano 12 (7) (2018) 6536–6544.
[30] C. Wang, J. Li, X. Han, S. Liu, X. Gao, C. Guo, X. Wu, Silk sericin stabilized
proanthocyanidins for synergetic alleviation of ulcerative colitis, Int. J. Biol.
Macromol. 220 (2022) 1021–1030.
[31] F. Zhang, H. Yang, Y. Yang, H. Wang, X. Li, X. Wu, Stretchable and biocompatible
bovine serum albumin fibrous films supported silver for accelerated bacteria-
infected wound healing, Chem. Eng. J. 417 (2021), 129145.
[32] S. He, L. Wu, H. Sun, D. Wu, C. Wang, X. Ren, Q. Shao, P. York, J. Tong, J. Zhu,
Z. Li, J. Zhang, Antioxidant biodegradable covalent cyclodextrin frameworks as
particulate carriers for inhalation therapy against acute lung injury, ACS Appl.
Mater. Interfaces 14 (34) (2022) 38421–38435.
[33] S.S. Hallan, M. Sguizzato, M. Drechsler, P. Mariani, L. Montesi, R. Cortesi,
S. Björklund, T. Ruzgas, E. Esposito, The potential of caffeic acid lipid
nanoparticulate systems for skin application: in vitro assays to assess delivery and
antioxidant effect, Nanomaterials 11 (1) (2021) 171.
[34] S.S. Hallan, M. Sguizzato, P. Mariani, R. Cortesi, N. Huang, F. Simelière,
N. Marchetti, M. Drechsler, T. Ruzgas, E. Esposito, Design and characterization of
ethosomes for transdermal delivery of caffeic acid, Pharmaceutics 12 (8) (2020)
740.
[35] I.L. Dejeu, L.G. Vicaș, L.L. Vlaia, T. Jurca, M.E. Mureșan, A. Pallag, G.H. Coneac, I.
V. Olariu, A.M. Muț, A.S. Bodea, G.E. Dejeu, O.A. Maghiar, E. Marian, Study for
evaluation of hydrogels after the incorporation of liposomes embedded with caffeic
acid, Pharmaceuticals 15 (2) (2022) 175.
[36] G. Das, H.-S. Shin, E.V.R. Campos, L.F. Fraceto, M. del Pilar Rodriguez-Torres, K.C.
F. Mariano, D.R. de Araujo, F. Fernández-Luqueño, R. Grillo, J.K. Patra, Sericin
based nanoformulations: a comprehensive review on molecular mechanisms of
interaction with organisms to biological applications, J. Nanobiotechnol. 19 (1)
(2021) 30.
[37] R.I. Kunz, R.M.C. Brancalhão, L.D.F.C. Ribeiro, M.R.M. Natali, Silkworm sericin:
properties and biomedical applications, BioMed Res. Int 2016 (2016) 8175701.
[38] L. Lamboni, M. Gauthier, G. Yang, Q. Wang, Silk sericin: a versatile material for
tissue engineering and drug delivery, Biotechnol. Adv. 33 (8) (2015) 1855–1867.
[39] P.O. Kuzema, I.V. Laguta, O.N. Stavinskaya, M.V. Borysenko, V.A. Tertykh, S.
V. Snegir, Preparation and characterization of the composites based on
hydridesilylated nanosilica and caffeic acid, Appl. Nanosci. (2022), https://doi.
org/10.1007/s13204-022-02679-0.
[40] E. Guerriero, A. Sorice, F. Capone, S. Costantini, P. Palladino, M. D'Ischia,
G. Castello, Effects of lipoic acid, caffeic acid and a synthesized lipoyl-caffeic
conjugate on human hepatoma cell lines, Molecules 16 (8) (2011) 6365–6377.