Abstract
Millions of people worldwide have inflammatory bowel disease (IBD).
Self‐driven micro/nanorobots (MNRs) are efficient in the treatment of
IBD. However, their lack of controllability regarding direction of
motion in the organism and their inability to achieve continuous
navigation limits their further application. In this study,
polydopamine is wrapped around the magnetite surface, loaded with an
anti‐inflammatory drug resveratrol, and wrapped with pH‐responsive
sodium alginate to obtain magnetic MNRs. MNRs can be driven by magnetic
fields to achieve directional movement and targeted transportation. In
addition, MNRs can effectively remove reactive oxygen species from the
inflammation site, repair intestinal damage, inhibit the cellular
pathway of pro‐inflammatory factors, such as MAPK and NF‐κB pathways,
and restore intestinal flora, thereby relieving IBDs. MNRs are safe and
effective for in vivo treatment of IBD and have proven to be a
promising therapeutic platform. This MNRs therapeutic strategy provides
new insights into comprehensive IBD therapy.
Keywords: inflammatory bowel disease, intestinal milieu restoration,
micro/nanorobots, resveratrol, ROS scavenger
__________________________________________________________________
Improved reactive oxygen species scavenging capacities of
functionalized micro/nanorobots (MNRs) are useful for the treatment of
inflammatory bowel disease (IBD). MNRs can break the harmful bacteria
in IBD, thereby eliminating inflammation and restoring the intestinal
barrier function. With its excellent efficacy as an oral drug and
superior biocompatibility, MNRs have great potential for clinical
application in the treatment of IBD.
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1. Introduction
Inflammatory bowel disease (IBD) is a chronic relapsing and remitting
disease commonly categorized into: ulcerative colitis and Crohn's
disease.^[ [34]^1 ^] The prevalence of IBD increased from 1990 to 2017,
with huge implications for global health and the economy.^[ [35]^2 ^]
IBD has a complex pathology characterized by elevated levels of
inflammation, reactive oxygen species (ROS), disruption of the
intestinal barrier, and abnormal intestinal flora.^[ [36]^3 , [37]^4 ,
[38]^5 , [39]^6 ^] This disease environment makes the treatment of IBD
difficult. Currently, IBD is primarily treated with oral medications
such as methotrexate (MTX)^[ [40]^7 ^] and 5‐amino salicylic acid
(5‐ASA),^[ [41]^8 ^] that mitigate the inflammatory response. However,
these drugs have limited effects on IBD. The amide group of MTX is
easily hydrolyzed in acidic environments, making it difficult to
maintain the activity of orally administered MTX.^[ [42]^9 ^]
Intestinal metabolizing enzymes make 5‐ASA ineffective, leading to
treatment failure.^[ [43]^8 ^] These drugs lack targeting mechanisms,
which can easily cause adverse side effects and systemic adverse
reactions.^[ [44]^10 ^] Additionally, imbalances in the gut microflora
continue to induce inflammation in the intestinal area.^[ [45]^11 ^] If
the medication does not change the imbalance in the intestinal flora,
the anti‐inflammatory effect alone will only provide temporary relief.
Resveratrol (Res) is a polyphenol extracted from various natural
species such as grapes, peanuts, and red wine. This substance acts as
an antioxidant and maintains the functional integrity of the mucosal
barrier. As a natural antioxidant, Res reduces ROS induced damage to
mitochondria mainly by scavenging free radicals, reducing lipid
peroxidation and modulating the activity of antioxidant‐related
enzymes.^[ [46]^12 ^] Although Res has the potential to regulate
intestinal flora,^[ [47]^13 ^] its poor water solubility, which results
in low bioavailability, limits its use in organisms.^[ [48]^14 ^] To
overcome this limitation, researchers have developed various
nanocarriers including tetrahedral framework nucleic acids,^[ [49]^12
^] hydrogels,^[ [50]^15 , [51]^16 ^] and polymeric micelles.^[ [52]^17
^] These carriers improve the bioavailability of Res; however, they
cannot actively propel it, especially in oral treatments that rely on
biological fluids for passive transport. Thus, a long period of time is
required for the drug to reach the lesion, which substantially reduces
the timeliness of treatment. Therefore, it is crucial to design a drug
delivery strategy that simultaneously relieves and precisely treats
symptoms.
Micro/nanorobots (MNRs) are intelligent machines that can convert
external energy into kinetic energy, and their driving methods are
mainly categorized as self‐driven or driven by external power.^[
[53]^18 , [54]^19 , [55]^20 , [56]^21 , [57]^22 , [58]^23 ^] They are
widely used for targeted drug delivery because they can actively travel
through organisms to reach lesions that are difficult to reach using
conventional drug delivery methods.^[ [59]^24 ^] Among these,
self‐driven MNRs are often used for gastrointestinal drug delivery
owing to the rich humoral environment in the gastrointestinal tract.^[
[60]^25 , [61]^26 , [62]^27 ^] Although self‐driven MNRs do not require
additional equipment, they lack controllability with the direction of
motion within an organism. Cao et al. reported using mesoporous
manganese oxide (MnO[x])‐based nanomotors driven by H[2]O[2]/ultrasound
in a synergistic manner to cure colon cancer.^[ [63]^28 ^] These
nanomotors do not target certain tissues or cells, nor are they able to
move in a specific direction. Recently, magnetically driven MNRs have
been recognized for their various advantages, such as precise and
wireless three‐dimensional manipulation in physiological environments,
access to small areas, and the use of harmless energy sources. They are
promising tools for the precise delivery of drugs or cells to targeted
areas^[ [64]^29 ^] and have been used to treat other diseases with good
therapeutic results.^[ [65]^30 , [66]^31 , [67]^32 ^] Therefore, we
propose the use of magnetite to prepare MNRs to improve their targeting
ability for IBD treatment. Notably, to the best of our knowledge, this
goal has not been achieved in the field of MNRs for IBD treatment.
Here, we demonstrate that oral drug delivery for intestinal
inflammation therapy can be achieved by safely and effectively using
magnetic MNRs in vivo for colitis treatment. In this study, we prepared
spherical magnetite as the main carrier of the MNRs. Magnetite spheres
were wrapped in polydopamine (PDA) for electrostatic adsorption of Res
and then coated with sodium alginate. They were simultaneously
assembled under a magnetic field to form biocompatible MNRs with
powerful thrust and precise navigation. After oral administration, the
pH‐responsive sodium alginate shell protects the MNRs until they reach
the gut. The negatively charged Fe[3]O[4]@PDA‐Res was then bound to the
inflamed colon via electrostatic interactions (Figure [68]1 ). MNRs
were effective in repairing intestinal damage and restoring the
intestinal environment in a mouse model induced by dextran sulfate
sodium salt (DSS). Moreover, MNRs significantly inhibit ROS and
inflammatory factor levels and ultimately restore the balance of gut
bacteria; MNRs can effectively enhance their bioavailability. Overall,
these MNRs have all the properties that require further development
into a therapeutic tool for IBD. We believe that this study will
provides new insights into the treatment of IBD.
Figure 1.
Figure 1
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Schematic illustration of the preparation of MNRs and mechanisms of its
activity in the treatment of IBDs. MNRs combines with positively
charged intestinal inflammatory areas through electrostatic adsorption.
They are able to remove reactive oxygen species, regulate intestinal
flora disorders, repair damaged intestinal environments and alleviate
intestinal diseases.
2. Results and Discussion
2.1. Synthesis and Characterization of MNR
MNRs were prepared using a convenient method and fully characterized to
determine their nanostructures and modification progress. The
preparation process consisted of four steps (Figure [70] 2A). First,
Fe[3]O[4] nanoparticles with a spherical structure were prepared using
a solvothermal method.^[ [71]^33 ^] The nanoparticles were then
dispersed in an alkaline buffer, followed by the addition of dopamine
to form a PDA layer on their surfaces (Fe[3]O[4]@PDA) via an in situ
polymerization process.^[ [72]^34 ^] Through electrostatic
interactions, the negatively charged drug Res was adsorbed onto the
polydopamine surface. Finally, sodium alginate (SA) was deposited on
the surface to form a shell via a cross‐linking process. SA shells are
biocompatible and pH‐responsive, stabilizing under acidic conditions in
gastric juices and collapsing under neutral intestinal environments,
resulting in on‐demand drug release without premature leakage.
Transmission electron microscope (TEM) and scanning electron microscopy
(SEM) images in Figure [73]2B and Figure [74]S1 (Supporting
Information) depict the morphology of the nanoparticles during each
fabrication step. The synthesized Fe[3]O[4] nanoparticles were
homogeneous spheres, that uniformly formed thin surface layer after
functionalization with PDA and SA. Energy dispersive X‐ray (EDX)
analysis was performed to characterize the elements present in the
resulting nanoparticles. The corresponding results in Figure [75]2C
show that Fe and O are distributed over a very small area, whereas Na
and C cover the entire area, indicating the presence of an SA shell
layer. Additionally, N confirms the successful loading of PDA. The
dynamic light scattering (DLS) results are shown in Figure [76]S2
(Supporting Information). The average sizes of the Fe[3]O[4],
Fe[3]O[4]@PDA, Fe[3]O[4]@PDA‐Res, and Fe[3]O[4]@PDA‐Res@SA
nanoparticles were ≈420, 500, 620, and 676 nm, respectively, indicating
the gradual growth of the shell layer. The Fe[3]O[4]@PDA‐Res@SA
nanoparticles were more stable in gastric fluid, and the SA shell layer
was broken down in the intestinal fluid, resulting in a smaller size
(Figure [77]S3, Supporting Information). Fourier‐transform infrared
(FT‐IR) spectroscopy was used to characterize the surface functional
groups of the nanoparticles at each fabrication stage (Figure [78]2D).
Iron oxide nanoparticles were successfully synthesized, as seen by the
peaks at 539 (Fe‐O stretching vibration), 1618, and 3386 cm^−1
(absorbed water and hydroxy groups) in all curves. The specific
stretching vibration peaks of C = C and benzene ring skeleton at 1605
and 1586 cm^−1 further confirmed the successful loading of Res. The
magnetic properties were investigated using a vibrating sample
magnetometer (VSM) at 300 K (Figure [79]2E). All nanoparticles were
ferromagnetic, as shown by the hysteresis loops in Figure [80]2F. The
gradually increasing proportion of nonmagnetic materials was the main
cause of the saturation magnetization values decreasing from 82, 77,
66, and 53 emu g^−1 for Fe[3]O[4], Fe[3]O[4]@PDA, Fe[3]O[4]@PDA‐Res,
and Fe[3]O[4]@PDA‐Res@SA, respectively. This suggests that
Fe[3]O[4]@PDA‐Res@SA NPs have a strong magnetic attraction under an
external magnetic field and can be used as components of clustered
magnetic MNRs. Notably, the Fe[3]O[4]@PDA‐Res@SA NPs exhibit magneto
thermal properties and can achieve temperature increases under magnetic
fields (Figures [81]S4–S8, Supporting Information). However, the
temperature increase has little effect on the magnetic properties of
the MNRs itself (Figure [82]S9, Supporting Information).
Figure 2.
Figure 2
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Fabrication and characterization of MNRs. A) Schematic of the
fabrication process. B) TEM images of nanoparticles at each fabrication
stage. Scale bar is 150 nm. C) EDX analysis of building block showing
the elemental distribution of Fe, O, C, N and Na in a nanoparticle.
Scale bar is 50 nm. D) FT‐IR spectra of nanoparticles at each
fabrication stage, demonstrating the gradual change of surface
functional groups. E) Magnetic hysteresis loop of nanoparticles at each
fabrication stage. F) Localized magnification of the hysteresis loop
around the origin (x range from −100 Oe to 100 Oe) of E). In (D)–(F),
the black, yellow, blue, and green curves indicate Fe[3]O[4],
Fe[3]O[4]@PDA, Fe[3]O[4]@PDA‐Res, and Fe[3]O[4]@PDA‐Res@SA,
respectively. G) Zeta potential evolution during preparation of
Fe[3]O[4]@PDA‐Res@SA MNRs (n = 3; mean ± SD). H) Drug‐loading rate and
drug encapsulation rate of Fe[3]O[4]@PDA‐Res@SA MNRs (n = 3; mean ±
SD). I) Drug release under different pH conditions Fe[3]O[4]@PDA‐Res@SA
MNRs (n = 3; mean ± SD). J) Simulation of drug release at pH 6.8.
When dispersed in an acidic solution at pH 2.0, the PDA layer exhibited
a strong positive charge with a ζ‐potential of 15.10±0.65 mV
(Figure [84]2G), thereby adsorbing the negatively charged drug Res
through effective electrostatic self‐assembly. At a drug‐to‐carrier
mass ratio of 10, the drug loading and encapsulation rates were ≈40.93%
and 45.02%, respectively (Figure [85]2H; Figures [86]S10 and [87]S11,
Supporting Information). To confirm the pH‐responsive Res release
process from building block, solutions with pH value of 1.2 and 6.8 was
used to simulate gastric and colonic conditions. At pH 1.2, the drug
release rate was 18.56%, whereas upon immersion in pH 6.8 solution,
they collapsed and released their contents at a drug release rate of
88.57% (Figure [88]2I). This suggest that SA has an ideal pH‐dependent
disintegration behavior that protects the encapsulated Res from
premature drug leakage in the harsh acidic gastric environment. In
addition, the negative charge of Fe[3]O[4]@PDA‐Res@SA NPs is expected
to promote their adhesion to positively charged inflamed colonic
epithelial tissues. Finally, the drug release at pH 6.8 was simulated
by software (Figure [89]2J).
2.2. Magnetic Propulsions of Magnetic MNRs
Figure [90]3A,B provide a schematic representation of how the
square‐wave and oscillating magnetic fields drive the behavior of the
functionalized nanoparticles. A square‐wave magnetic field was applied
using a magnetic field with square‐wave signals in the x, y, and z
directions, where the amplitude and frequency of the magnetic field and
the duty cycle of the square‐wave signals were adjustable. The
oscillating magnetic field was created by applying a direct current
signal in the x‐direction and sinusoidal signal in the y‐axis direction
(Movie [91]S1, Supporting Information). By adjusting the applied B(t)
(Figure [92]3C), the MNRs were dynamically reconfigured, transforming
from bulb‐like to dandelion‐like, dandelion‐like back to bulb‐like, and
bulb‐like to ellipsoidal (Movie [93]S2, Supporting Information). This
suggests that the morphology of the MNRs is capable of reversible
transitions. The direction of the MNRs can be controlled by adjusting
B(t). The MNRs can move left‐up, left‐down, down and up in turn. When
frequency is increased from 10 to 100 Hz, the MNRs in square magnetic
field can move up to 34.29 µm s^−1 (Figure [94]3D). The MNRs in
oscillating magnetic field walk slower than those of the square
magnetic field and exhibit a maximum velocity of 22.22 µm s^−1
(Figure [95]3E). The relationship among the motion of the MNRs,
magnetic field strength, and frequency was further explored. The
driving frequency at which the maximum velocity was obtained was
considered the critical value (critical frequency), beyond which the
velocity decreased with increasing frequency. The motion of the MNRs
follows this law when a fixed magnetic field strength of 3, 6, 9, and
12mT were applied to the square‐wave oscillating and oscillating
magnetic field, respectively (Figure [96]S12, Supporting Information).
As the viscosity of the liquid environment increases, the velocity of
the MNRs decreases. In addition, the velocities of the MNRs in
simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were
measured separately (Figure [97]S13, Supporting Information). The
slower velocities in gastrointestinal fluid may be due to the fact that
gastric mucus and intestinal mucus give greater resistance to the
movement of the MNRs. The MNRs could move in a predetermined
rectangular and N‐shaped trajectory by adjusting the direction of B(t)
(Figure [98]3F,G, Figure [99]S14, Supporting Information),
demonstrating their high‐performance motion controllability (Movie
[100]S3 and [101]S4, Supporting Information). We control the MNRs move
the trajectory of the letter “CUG” through a magnetic field
(Figure [102]3H, Movies [103]S5–S7, Supporting Information). Finally,
we compared the performance of our MNR with that of other magnetically
driven MNRs (Table [104]S1, Supporting Information).
Figure 3.
Figure 3
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Motion performance of magnetic MNRs. A, B) Schematic illustration of
the square‐wave magnetic field and oscillating magnetic field. The red
and blue arrows represent the magnetic field and the direction of
motion, respectively. C) MNRs undergo morphological changes and move in
different directions under the control of a magnetic field (f = 5 Hz,
5mT). Scale bar: 2mm. D, E) Average velocity versus frequency in
square‐wave magnetic field and oscillating magnetic field modes (n = 3;
mean ± SD). F) Optical microscopy images of MNRs moving directionally
to follow a rectangular trajectory (f = 5 Hz, 5mT). Scale bar: 2mm. G)
Controllable navigation of MNRs along a rectangular route with a motion
trajectory indicated by the red line (f = 5 Hz, 5mT). (H) Motion
trajectory of the letter “CUG” indicated by the red line (f = 5 Hz,
5mT).
2.3. Anti‐inflammatory Effects In Vitro and Promote Intestinal Epithelial
Barrier Repair
The Fe[3]O[4]@PDA carrier and Res released by Fe[3]O[4]@PDA‐Res entered
the cell for synergistic treatment (Figure [106]4A). Lipopolysaccharide
(LPS) was used to treat RAW264.7 macrophage cells increase the amount
of intracellular ROS produced. Figure [107]4B,C show that IL‐6 and
TNF‐α were significantly increased in LPS group compared with those in
the healthy group, indicating that RAW264.7 cells were at a high level
of inflammation. The Fe[3]O[4]@PDA‐Res treatment showed the best
inhibitory effect on the above three inflammatory factors compared to
that with Fe[3]O[4], Fe[3]O[4]@PDA, and Res. IBD significantly
increased release of several inflammatory factors (e.g., IL‐6 and
TNF‐α) of NF‐κB. These pro‐inflammatory factors further increased ROS
production, forming a vicious cycle of inflammation and ROS production.
As shown in Figure [108]4D,E, Figures [109]S15 and [110]S16 (Supporting
Information), Fe[3]O[4]@PDA‐Res significantly reduced intracellular
ROS. The concentration of the material used in the cellular assay was
determined from the results of the cytotoxicity assay (Figures [111]S17
and [112]S18, Supporting Information). Notably, the SA shell typically
undergoes dissolution in the intestinal fluid; therefore, its
interaction with cells has not been studied. The above evidence
demonstrated that Fe[3]O[4]@PDA‐Res effectively eliminated ROS and
pro‐inflammatory factors to break this vicious circle.
Figure 4.
Figure 4
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In vitro treatment of cells. A) Schematic diagram of Fe[3]O[4]@PDA‐Res
releasing Res to anti‐inflammatory and promote proliferation and
repair. The expression of B) IL‐6 and C) TNF‐α in the RAW264.7 cells
under different treatment (Control, LPS, Fe[3]O[4], Fe[3]O[4]@PDA, Res,
and Fe[3]O[4]@PDA‐Res). D) ROS levels in untreated RAW264.7 cells and
cells treated with four materials (Fe[3]O[4], Fe[3]O[4]@PDA, Res, and
Fe[3]O[4]@PDA‐Res) and stimulated by LPS. E) Representative ROS
staining (green fluorescence) of RAW264.7 cells after various
treatments. Scale bar: 100 µm. F) MMP monitored of RAW264.7 cells by
staining with the JC‐1. Scale bar: 200µm. G) Scratch assay images of
Caco‐2 cells cultivated in the medium supplementary with PBS (Control),
Fe[3]O[4], Fe[3]O[4]@PDA, Res, and Fe[3]O[4]@PDA‐Res. Scale bar:
300 µm. Data in (B–C) are shown as mean ± S.D, (n = 3 per group).
(Statistical significance was performed by one‐way ANOVA test. **p
< 0.01, ***p < 0.001, ****p < 0.0001, and ns: no significance).
Homeostasis of epithelial cells depends on normal mitochondrial
function;^[ [114]^35 ^] however, excessive ROS can damage the
mitochondria and compromise epithelial cell activity.^[ [115]^36 ^]
Thus, the JC‐1 probe was used to measure a change in the mitochondrial
membrane potential (MMP). At higher or lower MMP, the probe forms a
polymer or monomer in the matrix and emits a red or green fluorescent
signal, respectively. The ratio of red to green fluorescence is an
indicator of mitochondrial depolarization and early apoptosis.^[
[116]^37 ^] Figure [117]4F shows that control group cells exhibited a
normal MMP, while RAW264.7, exposed to LPS, exhibited extensive green
fluorescence, which was due to an increasing proportion of JC‐1
monomers and aggregates. Fe[3]O[4]@PDA‐Res completely restored MMP in
the exposed cells. Caco‐2 cell migration is essential in intestinal
barrier repair and determines the therapeutic effect in ulcerative
colitis. In Figure [118]4G and Figure [119]S19A (Supporting
Information), Fe[3]O[4]@PDA‐Res shows a higher migration rate
(88.62±0.41%) than that of Fe[3]O[4] (30.23±1.72%), Fe[3]O[4]@PDA
(59.66±0.79%), and Res (41.20±0.74%) at an interval of 12 h.
2.4. In Vivo Ulcerative Colitis Amelioration
Owing to the excellent biocompatibility and ROS‐scavenging ability of
Fe[3]O[4]@PDA‐Res, its in vivo therapeutic efficacy was further
evaluated in a DSS‐induced BALB/C mouse IBD model (Figure [120]5A).
Enteritis was induced in mice after seven days of feeding with 3.5%
DSS. The mice were randomly divided into seven groups: healthy (blank
control group), enteritis (DSS, negative control group), Fe[3]O[4]
treatment (DSS+Fe[3]O[4], experimental group), Fe[3]O[4]@PDA treatment
(DSS+Fe[3]O[4]@PDA, experimental group), Res treatment (DSS+Res,
experimental group), Fe[3]O[4]@PDA‐Res treatment
(DSS+Fe[3]O[4]@PDA‐Res, experimental group) and Fe[3]O[4]@PDA‐Res@SA
treatment (DSS+Fe[3]O[4]@PDA‐Res@SA, experimental group). All
experimental groups received daily oral administration through gavage,
and therapeutic efficacy was assessed by measuring the disease activity
index (DAI), body weight, and colon length. Compared with the DSS
group, the DSS+Fe[3]O[4]@PDA‐Res@SA group had a lower DAI
(Figure [121]5B), higher body weight (Figure [122]5C), and longer colon
length (Figure [123]5D,E). Compared with the group DSS+Res and healthy
groups, there were fewer differences between the
DSS+Fe[3]O[4]@PDA‐Res@SA and healthy groups. These results showed that
Fe[3]O[4]@PDA‐Res@SA was more effective than Res in treating mice with
DSS‐induced colitis. In the in vivo experiments, mice were orally
administered Fe[3]O[4]@PDA‐Res@SA, and whole bowel fluorescence imaging
10 h later revealed a significant increase in Fe[3]O[4]@PDA‐Res@SA
adhesion and retention in the mouse colon (Figure [124]S19B, Supporting
Information). Hematoxylin and eosin (H&E) staining of colon sections
revealed that mice with colitis displayed significant crypt loss,
histological collapse, substantial immune cell infiltration, and severe
colonic epithelial damage in the inflamed colon. In contrast, the
DSS+Fe[3]O[4]@PDA‐Res@SA group displayed an almost normal histological
microstructure with minimal inflammatory cell infiltration
(Figure [125]5F).
Figure 5.
Figure 5
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In vivo IBD amelioration. A) Schematic a description of the entire
process of creating a model of colitis in mice generated by DSS and
then administering oral therapies. B) DAI score values of the mice over
11 days of the experiment. C) Daily changes in mouse body weight
determined during 11 experimental days. Colon length D) and digital
photographs E) of the excised colons in the indicated groups. Scale
bar: 2 cm. F) H&E‐stained colonic tissue sections from the indicated
groups on day 11 of the experiment. Scale bar: 200 µm. Data in (B–D)
are shown as mean ± S.D, (n = 5 per group). (Statistical significance
was performed by one‐way ANOVA test. **p < 0.01, ***p < 0.001, ****p
< 0.0001, and ns: no significance).
Notably, we compared its efficacy with that of the clinically available
first‐line drugs, DEX (dexamethasone) and 5‐ASA. The results show that
mice treated with DEX and 5‐ASA had DAI indices of 4.4 ± 0.55 and 3.6
± 0.55, respectively, and colon lengths of 7.14 ± 0.22 cm and 7.36
± 0.16 cm (Figure [127]S20, Supporting Information). Therefore, MNRs
showed superior therapeutic efficacy in two of the most important
metrics of IBD (DAI and colon length) compared with that of other
first‐line clinical agents. We used isolated pig intestinal tissues to
mimic the mouse intestinal environment and achieved motility regulation
of MNRs in the pig intestine (Figure [128]S21; Movie [129]S8,
Supporting Information). It can cause morphological changes in and make
full contact with the narrow intestinal wall to improve therapeutic
efficiency (Movie [130]S9 and [131]S10, Supporting Information).
2.5. Anti‐Inflammation Activity In Vivo
Neutrophils express myeloperoxidase (MPO), which regulates IBD by
catalyzing the synthesis of reactive chemicals and hypochlorous acid in
inflamed tissues.^[ [132]^38 ^] Figure [133]6A suggests that although
the MPO levels were elevated in the colon tissue of the DSS group,
Fe[3]O[4]@PDA‐Res@SA treatment significantly reduced their aggregation,
demonstrating that Fe[3]O[4]@PDA‐Res@SA could effectively relieve the
symptoms of IBD. The results presented in Figure [134]6B–D demonstrate
a considerable increase in TNF‐α and IL‐6 in IBD lesions compared with
those in the healthy group. This suggests that the intestinal tissues
of IBD mice were highly inflamed. The Fe[3]O[4]@PDA‐Res@SA treatment
showed the best inhibitory effect on the above three inflammatory
factors compared to that with Fe[3]O[4], Fe[3]O[4]@PDA, Res, and
Fe[3]O[4]@PDA‐Res. MAPK and NF‐κB signaling pathways are activated in
colon tissues of patients with IBD and DSS‐induced colitis mice.^[
[135]^39 ^] Therefore, the potential effects of Fe[3]O[4]@PDA‐Res@SA
intake on the activation of p38 MAPK and NF‐κB p65 signaling pathways
were explored (Figure [136]6E,F). The results showed that NF‐κB p65 and
p38 MAPK signaling pathways were activated in the colon tissues of
DSS‐induced colitis mice. Fe[3]O[4]@PDA‐Res@SA intake reduced the
phosphorylation of NF‐κB p65 and p38 MAPK, thus exhibiting
anti‐inflammatory effects.
Figure 6.
Figure 6
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Anti‐inflammation activity in vivo. A) The amounts of MPO in colon
issue excised from mice exposed to annotated treatments. Expression
levels of inflammatory factors IL‐6 B) and TNF‐α C) in the serum of
mice. D) Immunofluorescent staining images of TNF‐α/IL‐6 inflammatory
factors in colon tissue section from different groups. Scale bar:
800 µm. E) Anti‐inflammatory by inhibition the activation of p38 MAPK
and p65 NF‐κB. F) Western blot analysis showing the differential
expression of p38, p‐p38, p65, and p‐p65 of different treatment groups
in mice (healthy mice, IBD model mice, IBD treated with Fe[3]O[4], IBD
treated with Fe[3]O[4]@PDA, IBD treated with Res, IBD treated with
Fe[3]O[4]@PDA‐Res, and IBD treated with Fe[3]O[4]@PDA‐Res@SA. Data in
(A–C) are shown as mean ± S.D, (n = 3 per group). (Statistical
significance was performed by one‐way ANOVA test. **p < 0.01, ***p
< 0.001, ****p < 0.0001, and ns: no significance).
2.6. Therapeutic Mechanisms of MNRs on IBD
To elucidate the therapeutic mechanism of MNRs in IBD, RNA‐seq was
performed on the mouse colonic tissue. RNA‐seq of colon tissues from
the Robot‐treated, Res‐treated, DSS, and healthy groups showed that
these groups had different transcriptome profiles. After comparing
Robot‐treated colitis animals to DSS mice, the volcano plot revealed
that 319 genes were up‐regulated and 590 genes were downregulated,
indicating a significant change in gene expression (Figure [138]7A).
The Robot group showed higher variability in all, upregulated, and
downregulated genes than the Res group (Figure [139]7B). These results
suggest that the therapy was effective. Principal component analysis
further confirmed this finding (Figure [140]7C). Subsequent
investigation of the normalized heatmap (which shows the top 20
differentially expressed genes) indicated that the colitis animals
treated with the robot were more similar to the healthy group in the
gene profile (Figure [141]7D).
Figure 7.
Figure 7
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Transcriptomic analysis. A)Volcano plot of differentially expressed
genes determined between DSS+Robot and DSS group. B) Volcano plot of
differentially expressed genes determined between DSS+Res and DSS
group. C) The principal components analysis of sample from each group.
D) Heat map of RNA‐seq analysis showing the genes in the healthy, DSS,
DSS+Res, and DSS+Robot groups (fold change in right). E) KEGG pathways
enrichment analysis of ROS‐related signaling pathway in DSS versus
DSS+Robot.
Screened genes were subjected to enrichment and clustering analyses to
clarify their underlying therapeutic processes. Kyoto Encyclopedia of
Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses
(Figures [143]S22 and [144]S23, Supporting Information) were performed
to investigate the biological roles of these DEGs and main enrichment
pathways in more detail. Functional clustering analysis of samples in
ROS‐related signaling pathways was performed using the KEGG database,
with log2 (fold change) as the evaluation criterion. The findings
showed that 10 KEGG pathway categories—MAPK, Ras, Rap1, cancer,
PI3K‐Akt, Melanoma, Neurotrophin, FoxO, Pancreatic cancer, Hepatitis B
signaling pathways—were linked to variations in gene expression between
the DSS and DSS+Robot groups (Figure [145]7E, Figure [146]S24,
Supporting Information). Previous studies have shown that by reducing
ROS levels, and inhibiting NF‐κB and other inflammatory signaling
pathways, robots decrease colon inflammation in mouse models. The
inflammatory signaling pathway regulates the release of inflammatory
factors from cells, and the binding of inflammatory factors to
receptors further activates the inflammatory signaling pathway, forming
a positive feedback loop that exacerbates the inflammatory response.^[
[147]^40 ^] The results of differential gene analysis showed enrichment
in the MAPK signaling pathway, indicating that MNRs significantly
reduced the expression of inflammatory factors, TNF‐α and IL‐6, by
blocking this loop, further enhancing the anti‐inflammatory effect.
Notably, robot treatment altered the PI3K‐Akt signaling pathway,
indicating that it may play a role in regulating intestinal epithelial
barrier repair and intestinal microbiota in IBD treatment.
Short‐chain fatty acids (SCFAs), products of the fermentation of
undigested polysaccharides by anaerobic bacteria in the gut, ameliorate
the inflammatory response by modulating immune cells and enhancing
intestinal barrier integrity.^[ [148]^41 , [149]^42 ^] The development
of colitis in mice significantly reduces the diversity of the
intestinal flora, leading to a decrease in SCFA production and
worsening of the disease.^[ [150]^43 ^] We performed KEGG enrichment
analysis on several key genes involved in SCFA metabolism (e.g.,
acetic, and propionic acid metabolism), suggesting that our therapeutic
regimen could address this issue to some extent (Figure [151]S25,
Supporting Information).
2.7. Restore Gut Microbiota Mechanism of MNRs on IBD
There was a considerable difference in the intestinal flora of IBD
lesions compared with that in healthy intestines. In IBD, harmful
bacteria infiltrate the intestinal wall and excessive ROS disrupt the
integrity of the intestinal mucus and epithelial barrier, triggering an
intense inflammatory response that leads to intestinal epithelial cell
(IEC) apoptosis, creating a vicious cycle between IEC apoptosis and
colonization by dangerous microorganisms (Figure [152]8A).^[ [153]^44 ,
[154]^45 ^] In this regard, ROS act as inflammatory mediators that
activate toll‐like receptors, thereby inducing the release of
inflammatory cytokines.^[ [155]^46 , [156]^47 ^] This process leads to
the release of large amounts of inflammatory cytokines through plasma
membrane rupture, resulting in cell death and accelerating the
progression of inflammation.^[ [157]^48 , [158]^49 ^] Apoptosis
provides key nutrients to the IBD pathogenic flora, leading to
colonization of pathogenic bacteria and resulting in an imbalance of
the intestinal flora.^[ [159]^44 ^] MNRs are able to break this cycle
by scavenging ROS and inhibiting the expression of cellular
inflammatory factor. Compared with the DSS group, the Venn diagram
showed that MNR treatment increased the number of intestinal microbes
at the operational taxonomic unit (OTU) level (Figure [160]8B).
Nonmetric multidimensional scaling (NMDS) analysis of the weighted
UniFrac distance was performed to show the degree of similarity in the
microbial composition between the groups. The Robot‐treated group
showed a different phylogenetic architectural profile compared with
that of the IBD group (Figure [161]8C). Notably, the mice treated with
MNRs had microbiomes more similar to those of healthy control mice. In
addition, nanorobot treatment increased the relative abundance of
beneficial bacteria (norank_f_Muribaculaceae) in IBD lesions, whereas
Res treatment was less effective than MNRs (Figure [162]S26, Supporting
Information). These results suggest that MNRs inhibit harmful bacteria
and increase beneficial bacteria, ultimately restoring the intestinal
flora to a healthy balance.
Figure 8.
Figure 8
[163]Open in a new tab
The effect of MNRs on the gut microbiota of IBD mice. A) Schematic
diagram of MNRs breaking the vicious cycle of intestinal epithelial
cell apoptosis and large‐scale bacterial colonization to induce
inflammation. B) Venn diagram of the intestinal bacterial species
detected in the control, DSS, DSS group exposed to Res, and DSS group
exposed to Robot. C) NMDS analysis of the microbiota composition. D)
Bar chart of the percent of community abundance of gut microbiota in
the healthy group, DSS model group, Res treatment group, and Robot
treatment group. E) Heatmap showing the relative abundance of gut
microbiota composition analysis at the order level in each group. F–I)
Microbial community richness and diversity analyses of normal and
treated mice expressed as ACE F), Chao G), Shannon H) and Simpson I)
indices. Data in (F–I) are shown as mean ± S.D, (n = 4 per group).
(Statistical significance was performed by one‐way ANOVA test. **p
< 0.01, ***p < 0.001, ****p < 0.0001, and ns: no significance).
The relative abundance at the class level is shown in Figure [164]8D.
The gut microbiota of the different groups was mainly composed of
Bacteroidetes, Bacilli, Clostridia, Verrucomicrobiae, and
Saccharimonadia. The top 50 strains with the highest abundance were
further analyzed at the species level. The heatmap presented in
Figure [165]8E provides additional evidence that the microbiota species
composition of the DSS group underwent significant changes, whereas the
microbiota of mice treated with MNRs had a composition similar to that
of healthy mice. The acquired data verified that MNRs could alter the
microbial composition profile of IBD mice from dysbiosis to
homeostasis. Oral MNR treatment improved the community richness
expressed by OTU, ACE index (Figure [166]8F), Chao index
(Figure [167]8G), community diversity Shannon index (Figure [168]8H),
and Simpson index (Figure [169]8I). As shown in the previously
described investigations, MNRs effectively altered the gastrointestinal
milieu by eliminating ROS and altering the gut microbiota.
2.8. Biocompatibility of MNRs
Finally, to confirm that the clinical transformation of MNRs is
feasible, the safety and biocompatibility of the treatment were
assessed. No hazardous components were detected in the MNRs. Even after
treating RAW264.7 cells in vitro for 24 h at escalating MNRs doses
(from 0 to 1280 µg mL^−1), we did not notice a discernible decline in
their viability (Figurse [170]S17 and [171]S18, Supporting
Information). This demonstrated the high degree of cellular
biocompatibility of MNRs. Oral MNRs were collected only from the
gastrointestinal tract, therefore, there was no risk to critical organs
(Figure [172]9A). As anticipated, there were no indications of systemic
toxicity, autoimmunity, or disease in the main organs during MNR
therapy. After oral administration of MNR, liver function (alanine
aminotransferase [ALT], lactic dehydrogenase [LDH]), and renal function
(creatinine [CR]), blood index parameters (red blood cells [RBC], mean
platelets [PLT], and red cell distribution width‐SD [RDW‐SD]) were not
affected (Figure [173]9B–E; Figure [174]S27, Supporting Information).
To assess the potential in vivo safety of magnetothermal therapy, brain
tissues from mice undergoing magnetothermal therapy were collected for
H&E staining. Despite the fact that the abdominal temperature of the
mice rose to nearly 50 °C under alternating magnetic field, no
significant brain damage was detected (Figure [175]S28, Supporting
Information). The therapeutic effect of MNRs minimized damage to other
organs. Its metabolism in organisms has been studied using
pharmacokinetics; it is metabolized and eliminated from the body
relatively quickly (Figure [176]S29, Table [177]S2, Supporting
Information). ICP‐MS was used to evaluate the biosafety and metabolism
of the MNRs. Mice treated with MNRs did not exhibit elevated levels of
metal ions in their heart, liver, spleen, lung, or kidney (Figure
[178]S30, Supporting Information). This evidence suggests that MNRs
have excellent biocompatibility with potential for clinical
translation.
Figure 9.
Figure 9
[179]Open in a new tab
In vivo biosafety assessment. A) H&E‐stained histological sections of
major organs excised from mice after annotated treatments. Scale bar:
200 µm. B) RBC indicators, C) RDW‐SD indicators, D) PLT indicators and
E) ALT indicators of each group. Data in (B–E) are shown as mean ± S.D,
(n = 3 per group). (Statistical significance was performed by one‐way
ANOVA test. **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: no
significance).
3. Discussion
IBD is a chronic and recurrent inflammatory disease of the
gastrointestinal tract that substantially increases the risk of colon
cancer.^[ [180]^50 ^] The pathogenesis of IBD is complex and
clinicopathological studies have shown that intestinal flora dysbiosis
is closely associated with the development of IBD.^[ [181]^51 ^] The
traditional clinical approach to treatment has been primarily through
the administration of anti‐inflammatory drugs to patients with IBD;
however, these drugs have targeting issues that can lead to systemic
side effects and serious complications.^[ [182]^52 ^] Intestinal
inflammation leads to a dramatic increase in ROS level in the
intestinal mucosa and most current research has focused on the topical
delivery of antioxidants to achieve anti‐inflammatory therapy in IBD.^[
[183]^53 , [184]^54 ^] However, restoring intestinal ecological balance
and repairing the epithelium barrier cannot be accomplished by solely
treating inflammation without also considering the dysbiosis of the gut
flora. Therefore, a drug carrier that effectively scavenges ROS and
enables the modulation of the intestinal flora is urgently needed. MNRs
exhibit unique propulsive forces in liquid media and can be targeted by
delivering drugs directly to the disease site.^[ [185]^55 , [186]^56 ,
[187]^57 , [188]^58 , [189]^59 ^] In particular, chemically driven MNRs
are commonly used to treat gastrointestinal disorders and use
biological fluid responses for locomotion.^[ [190]^25 ^] Their speed
and direction cannot be controlled.
In this study, we constructed biocompatible MNRs that can reversibly
change their morphology under the control of a magnetic field and
achieve directed motion. The carboxyl group (‐COOH) of SA is protonated
at low pH in the gastric acidic environment and intermolecular hydrogen
bonding is enhanced to form a dense structure without drug release;
under alkaline conditions, the carboxyl group is deprotonated (–COO–),
the molecule is negatively charged, and electrostatic repulsion leads
to gel dissolution and solubilization. Thus, SA shells exhibit
pH‐responsive behavior and are capable of releasing drugs in the
gastrointestinal environment. However, passive navigation, which relies
on humoral transport, results in slow and poorly targeted drug delivery
and reduces therapeutic efficiency. With the introduction of magnetic
navigation, MNRs achieved controllable speeds and directionality.
Therefore, the integration of the pH responsiveness of SA shells with
magnetic navigation can substantially enhance the delivery efficiency
and therapeutic effect of drug carriers. In addition, the material
synthesis process was reproducible and the structure and properties of
the samples from each batch were consistent, confirming the stability
and reliability of the synthesis method. A magnetic field device for
the modulation of MNRs, which can be combined with clinical imaging
devices (e.g., endoscopes), may enable a wider range of biomedical MNRs
applications for clinical therapy. Overall, MNRs in this study have the
ability to scavenge ROS, repair wounds, and regulate flora, showing
potential for the treatment of various inflammatory diseases, such as
gastrointestinal inflammation, diabetic wounds, and atherosclerosis.
4. Conclusion
In summary, we demonstrated the significant potential of MNRs as an
oral therapy for IBD. Functionalized MNRs exhibit enhanced
ROS‐scavenging capabilities for the diagnosis and treatment of IBD. The
ability of MNRs to efficiently target IBD in vivo has demonstrated a
highly effective therapeutic effect against IBD, which is significantly
better than that of Res. The therapeutic mechanism of MNRs has been
profoundly and systematically revealed, mainly by inhibiting the p38
MAPK and p65 NF‐κB signaling pathway. MNRs can break the harmful
bacteria in IBD, thereby eliminating inflammation and restoring the
intestinal barrier function. Given the advantages of MNR as an oral
therapy, with strong efficacy and superior biocompatibility, they have
substantial potential for clinical translation for IBD treatment.
Furthermore, the viability of Res as a small‐molecule medication for in
vivo transformation is increased by the MNR design, which can influence
the creation of nano‐drug carriers for further small‐molecule
medications.
5. Experimental Section
Physicochemical Characterization Tools and Approaches
TEM images were obtained with a field emission transmission electron
microscope (JEOL JEM‐2100F, Japan). Hydrodynamic diameters and zeta
potentials were measured on zeta potential and particle size analyzer
(Brookhaven 90Plus PALS, USA). Absorption spectra were acquired using
microplate reader measurement (Tecan Spark, Austria). FT‐IR spectra
were obtained using an infrared spectrophotometer (Thermo Fisher
Nicolet iS50, USA). Hysteresis loops were gained with a vibrating
sample magnetometer (LakeShore7404, USA). CLSM images were captured
with a Leica STELLARIS 5 microscope (Leica, Germany). Quantification of
fluorescence intensity of cells using flow cytometry (BD Accuri C6
plus, USA).
Preparation of Fe[3]O[4] Nanoparticles
First, 1.35 g of FeCl[3]∙6H[2]O was dissolved in 40 mL of EG by
magnetic stirring to form an orange‐yellow transparent solution, and
then 3.6 g of NaAc and 1 g of PEG were added. A yellow‐brown turbid
mixture was obtained after 4 h of vigorous stirring. Subsequently, the
mixture was sealed in a 50 mL reactor and heated at 200 °C for 10 h
before cooling naturally to room temperature. The black product was
collected with a permanent magnet and washed three times with deionized
water to remove the residual solvent. Finally, the nanoparticles were
vacuum dried at 60 °C for 12 h and collected for storage.
Preparation of Fe[3]O[4]@PDA Nanoparticles
A smooth PDA layer was encapsulated on the Fe[3]O[4] surface by in situ
polymerization.^[ [191]^60 ^] Dissolve 0.12 g Tris in 100 mL deionized
water and disperse by sonication. The pH of the resulting solution was
adjusted to 8.5 with 1 M hydrochloric acid to form an alkaline buffer
solution. The buffer solution was poured into a 250 mL beaker and 2 mL
of Fe[3]O[4] nanoparticle solution (50 mg mL^−1) was added. Then, the
mixture was stirred for 30 min to completely disperse the magnetite
nanoparticles. Subsequently, 0.02 g of dopamine hydrochloride was added
to the mixture and was mechanically stirred for 4 h at room temperature
to make the polymerization reaction complete. Finally, the mixture was
collected with permanent magnets, washed three times with deionized
water and dried under vacuum at 60 °C for 12 h.
Preparation of Fe[3]O[4]@PDA‐Res Nanoparticles
25 mg of resveratrol powder was weighed and dispersed in 20 mL of
methanol, and ultrasonically dispersed for 5 min. 2.5 mg of
Fe[3]O[4]@PDA was then taken and continued to be ultrasonically
dispersed for 5 min, after which the mixture was shaken away from the
light at 37 °C for 24 h. Finally, the product was collected with a
magnet, washed with methanol for three times, and freeze‐dried to
obtain the resveratrol‐containing magnetite complexes
Fe[3]O[4]@PDA‐Res.
Using UV spectrophotometry, a standard concentration curve of Res was
initially created in order to calculate the drug loading of
Fe[3]O[4]@PDA. 200 µL of supernatant from the drug loading process was
collected, and the UV absorption spectrum of the supernatant was
measured. Using the standard curve, the concentration of Fe[3]O[4]@PDA
in the supernatant was determined. The formulas for drug encapsulation
efficiency and loading efficiency are as follows:
[MATH: Encapsulationefficiency%=WLoaded
ResWTotalRes×100% :MATH]
(1)
[MATH: Loadingefficiency%=WLoaded
ResWFe3O4<
/msub>@PDA−Re
s×100% :MATH]
(2)
Preparation of Fe[3]O[4]@PDA‐Res@SA Nanoparticles
The oil phase was prepared as follows: 57.2 mL of liquid paraffin was
taken in a round‐bottomed flask, heated to 50 °C in a water bath,
1.2 mL of Span‐80 and 0.4 mL of Tween‐80 were added, and the mixture
was mechanically stirred until it was transparent and clarified and
then cooled to 37 °C. The aqueous phase was prepared as follows: 12 mg
of Fe[3]O[4]@PDA‐Res prepared and 20 mg of sodium alginate were
dispersed in 2 mL of deionized water and mechanically stirred for
30 min. The aqueous phase was added dropwise into the oil phase at a
rate of 500 µL min^−1 using a syringe pump, maintaining a temperature
of 37 °C and a rotational speed of 700 rpm during the process. When the
oil phase is fully mixed with the water phase, continue mechanical
stirring for 1 h to make a uniform and stable milky white water‐in‐oil
emulsion. Add 5% calcium chloride solution drop by drop for
crosslinking, mechanical stirring and curing for 2 h, respectively,
washed with isopropyl alcohol and deionized water for 3 times. After
freeze‐drying, the Fe[3]O[4]@PDA‐Res@SA nanoparticles were obtained.
pH‐Responsive Drug Release
In order to simulate the drug release in the gastrointestinal
environment, Fe[3]O[4]@PDA‐Res@SA nanoparticles were immersed in
artificial gastric fluid at pH 1.2 and artificial intestinal fluid at
6.8. The cumulative drug release rate was calculated over time.
Drug Release Simulation
The phenomenon of drug diffusion by MNRs within the intestinal fluid
was computationally simulated using the dilute matter transfer module
of COMSOL Multiphysics software. The concentration of MNRs was maximum
in the center region and zero in the rest of the region at t = 0.
ABTS•+ Scavenging Assay
The antioxidant capacity of the materials was assessed by using the
total antioxidant capacity assay kit (Beyotime, China). The ABTS
solution was mixed 1:1 with the oxidizing agent to configure an ABTS
workhorse solution, which was stored at room temperature and protected
from light for 12 h. The ABTS workhorse solution was diluted with 80%
ethanol until the absorbance was about 1.4. The solution was then
incubated with a range of materials in the dark for 10 min. The mixture
was centrifuged at 8000 rpm for 5 min and the absorbance of the
supernatant at 405 nm was measured using microplate reader.
Magnetothermal Effect
A high‐frequency induction heating device (Shuangping SPG‐10AB‐II,
China) supplied the alternating magnetic field (AMF). The material was
placed in a centrifuge tube at the center of the coil and heated, and
the temperature changes were recorded by an infrared thermographic
camera (Fotric 326, China).
Magnetic Propulsions of MNRs
We built our own experimental setup for the magnetic propulsions of the
MNRs (Figure [192]S31, Supporting Information). The magnetic actuation
experiments were conducted in a customized three‐axis Helmholtz
electromagnetic coil setup fixed on an operating microscope (RWD,
China). At first, the suspension of the Fe[3]O[4]@PDA‐Res@SA NPs was
added into a glass‐bottom tank and placed in the working space of the
electromagnetic coils. Then, square‐wave magnetic field and oscillating
magnetic field were applied by the electromagnetic coils that were
controlled. The magnetic propulsion of the Fe[3]O[4]@PDA‐Res@SA NPs
were observed and recorded through the operating microscope. The speed
of Fe[3]O[4]@PDA‐Res@SA NPs were analyzed using the Video Spot Tracker
V08.01.02 software.
Cell Culture
RAW264.7 cells, Human colon carcinoma cells (Caco‐2) were cultured in
minimum essential medium supplemented with 20% fetal bovine serum, 1%
nonessential amino acid, 1% sodium pyruvate, 1% GlutaMAX, 1%
l‐glutamine, and 1% penicillin/streptomycin. The cells were maintained
at 37 °C with 5% CO[2].
ROS Scavenging In Vitro
RAW264.7 cells were seeded in a six‐well plate and cultured in a CO[2]
incubator at 37 °C until an appropriate cell density was reached.
Fe[3]O[4] (20 µg mL^−1), Fe[3]O[4]@PDA (20 µg mL^−1), Res (20 µg
mL^−1), and Fe[3]O[4]@PDA‐Res (50 µg mL^−1) were added to the RAW264.7
cell culture dishes along with LPS (2 µg mL^−1). The cells were
incubated in the treatment conditions for 3 h.
2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA, 0.5 µg mL^−1)
probe was added and incubated at 37 °C in the CO[2] incubator for
50 min. Subsequently, the cells were washed thrice with a serum‐free
culture medium. The collected cells were resuspended in PBS and
analyzed for intracellular ROS levels using flow cytometry and laser
scanning confocal microscope.
Cytotoxicity Evaluation of MNRs
RAW264.7 cells were seed in 95‐well plates at a density of 2 × 10^5 of
cells per well for 24 h. The cells were then incubated with various
concentrations of Fe[3]O[4], Fe[3]O[4]@PDA, Fe[3]O[4]@PDA‐Res, Res, and
Fe[3]O[4]@PDA‐Res@SA (0, 5, 10, 20, 40, 80, 160, 320, 640, 1280 µg
mL^−1) for 12 or 24 h. Then, the medium was removed and cell viability
was determined by the CCK‐8 method. Briefly, 0.1 m of CCK‐8 solution
was added to each well and incubated at 37 °C for 1 h. Relative cell
viability was finalized by measuring the absorbance of each well at
450 nm using an enzyme marker.
Mitochondrial Membrane Potential Measurements
RAW264.7 cells were seeded in 6‐well plates at a density of 1 × 10^4 of
cells per well. Fe[3]O[4] (20 µg mL^−1), Fe[3]O[4]@PDA (20 µg mL^−1),
Res (20 µg mL^−1), and Fe[3]O[4]@PDA‐Res (50 µg mL^−1) were added to
the RAW264.7 cell culture dishes along with LPS (2 µg mL^−1) for 3 h.
Afterward, the cells were rinsed with PBS and stained in the dark with
JC‐1 (1 µg mL^−1) at 37 °C for 30 min. Microscopy images were captured
using CLSM.
In Vitro Wound Healing Properties
The wound healing properties of different NPs were evaluated using the
scratch method. Caco‐2 cells are a human epithelial cell line used in
the intestinal epithelial barrier model. Cells were inoculated in
6‐well plates at 1 × 10^6 cells per well. After 24 h of incubation, the
monolayers were scored with a pipette tip, rinsed with PBS, and
incubated before and after the addition of Fe[3]O[4] (20 µg mL^−1),
Fe[3]O[4]@PDA (20 µg mL^−1), Res (20 µg mL^−1), and Fe[3]O[4]@PDA‐Res
(50 µg mL^−1) suspension to image the “wound”. The unhealed areas of
the NPs were measured using ImageJ software to evaluate their wound
healing ability.
Enzyme‐Linked Immunosorbent Assay (ELISA) Analysis
To evaluate the levels of inflammatory factors, enzyme‐linked
immunosorbent assay (ELISA) was used to determine the levels of TNF‐α,
IL‐6 and MPO in cell supernatants, mouse serum and mouse colon
homogenates. Mouse intestinal tissue homogenates were prepared at 4 °C.
Each sample was centrifuged at 10 000 × g for 10 min at 4 °C. The
levels of TNF‐α, IL‐6 and MPO enzyme activity in the supernatant were
determined by ELISA.
DSS‐Induced Model of IBD
Female BALB/C mice (20g , 6–8 weeks) were cohoused for 7 days.
Afterward, mice were randomly assigned to the set groups with 5 mice
per group. The mice were normally fed and drinking water was set as the
control group, while the other groups were treated with drinking water
containing 3.5% DSS for 7 days. Then, DSS‐induced mice were orally
administered different treatments (Fe[3]O[4], Fe[3]O[4]@PDA, Res,
Fe[3]O[4]@PDA‐Res, Fe[3]O[4]@PDA‐Res@SA) from day 8 to day 10. During
the experimental period, the body weight change and DAI of the mice
were recorded daily. The DAI scoring criterion were (for a total of
0–10 points): i) weight loss (no change = 0 points, 0–5% = 1 point,
6–10% = 2 points, 11–20% = 3 points, >20% = 4 points); ii) stool
properties (normal = 0 points; soft, intact granular shape = 1 point;
soft, unshaped stool = 2 points; diarrhea = 3 points); and iii)
bleeding (none = 0 points, presence of blood = 2 point, visible
bleeding = 4 points). All groups of mice were sacrificed on day 11 for
subsequent procedures, including excision of the entire colon and major
organs for length measurements, H&E‐stained, western blot analysis and
biosafety estimation. All animal experimental procedures involved in
this study were approved by the Institutional Animal Welfare and Ethics
Committee of Jarvis (Wuhan) biological pharmaceutical Co.,Ltd
(JWS‐20240123‐007).
Microbiome Analysis
A mouse fecal sample was taken and DNA extraction was performed. After
extracting the total DNA of the samples, primers were designed based on
conserved regions and sequencing adapters were added at the end of the
primers. The products were PCR amplified, purified, quantified and
normalized to construct a sequencing library. Sequencing was performed
using Illumina, and the PE reads obtained from sequencing were firstly
spliced according to overlap relationship, while the sequence quality
was quality controlled and filtered.
Biocompatibility and Biodistribution
H&E‐stained colonic tissue sections were used for histological
assessment. The blood and major organs (colon, heart, liver, lung,
kidney and spleen) of the sacrificed mice were collected for
histopathological analysis. Each organ was fixed with paraformaldehyde
and embedded in paraffin. Tissues were stained with H&E and
photographed by microscopy. Routine blood and blood chemistry analyses
to assess systemic toxicity. In addition, ICP of iron ions in various
organs of mice was analyzed three days after oral injection to assess
biological metabolism.
Pharmacokinetics
Mice were weighed, and the Fe[3]O[4]@PDA‐Res@SA nanoparticles were
given by gavage at a dose of 50 mg kg^−1. Blood was collected from the
retro‐orbital venous plexus of mice 0.083, 0.25, 0.5, 1, 2, 4, 8, and
12 h after the administration of the drug, and was placed in 1.5 mL EP
tubes containing EDTA, centrifuged at 5000 r min^−1 for 15 min, plasma
samples were separated, and stored at −80 °C for measurement. The
plasma concentrations at each time point were processed by DAS2.0
software, and the pharmacokinetic parameters of resveratrol were
calculated according to the nonatrial model.
Statistical Analysis
All results are presented as the means ± SD. The significance between
the two groups was analyzed by an unpaired two‐tailed Student's t test.
For multiple comparisons, a one‐way analysis of variance (ANOVA) with
Tukey's post hoc test was used. P values of less than 0.05 were
considered significant. *P < 0.05, **P < 0.01 and ***P < 0.001.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
H.Y. developed the concept for the work. Y.F. and H.Y. conceived the
project and designed the experiments. Y.F. performed the experiments
and characterized the samples, and wrote the draft of the manuscript.
Y.L. performed the numerical simulation. L.L. and M.A. discussed the
results and interpreted the data. Q.Y. proposed amendments. H.Y. wrote
the final manuscript and supervised the research. All authors approved
the final version of the manuscript.
Supporting information
Supporting Information
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Acknowledgements