Abstract
Excessive accumulation of reactive oxygen and nitrogen species (RONS)
and dysbiosis of intestinal microbiota are pivotal symptoms for
inflammatory bowel disease (IBD) and its associated complications, such
as intestinal fibrosis. This research introduces a probiotic inulin
hydrogel loaded with polypyrrole (PPy) nanozymes and antifibrotic drug
pirfenidone (PFD) (PPy/PFD@Inulin gel) designed for the concurrent
amelioration of IBD and its fibrotic complication. Upon oral
administration, the inulin gel matrix could extend the gastrointestinal
residence time of PPy nanozymes and PFD, facilitating the efficient
reduction of pro-inflammatory cytokine levels and enhancement of the
intestinal epithelial barrier repair as well as the suppression of
intestinal fibrosis through sustained RONS scavenging, modulation of
gut microbiota and attenuation of the TGF-β/Smad signaling pathway to
inhibit fibroblast proliferation. Notably, the PPy/PFD@Inulin gel
demonstrated significant prophylactic and therapeutic efficacy in acute
and chronic colitis as well as intestinal fibrosis induced by dextran
sodium sulfate (DSS) in mouse models. Thus, the engineered ternary
PPy/PFD@Inulin gel offered a pioneered paradigm for simultaneous
reversal of IBD and its associated complications, such as intestinal
fibrosis, in a single therapeutic regimen.
Subject terms: Ulcerative colitis, Ulcerative colitis, Dysbiosis
__________________________________________________________________
Because of its complex pathophysiology, the management of inflammatory
bowel disease as well as its fibrotic complication has become a global
challenge. Here, the authors show a probiotic inulin hydrogel loaded
with polypyrrole (PPy) nanozymes and antifibrotic drug pirfenidone
(PFD) (PPy/PFD@Inulin gel) designed for the concurrent amelioration of
IBD and its fibrotic complication.
Introduction
Inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and
ulcerative colitis (UC), is a chronic autoimmune disorder that could
affect the entire gastrointestinal tract^[40]1,[41]2. Due to its
intricate pathophysiology, the management of IBD has become a global
challenge^[42]3. Despite the incomplete understanding of its
pathogenesis, it is widely accepted that IBD is associated with
elevated oxidative stress, dysbiosis of intestinal microbiota, and
compromised intestinal barrier function within the gut
microenvironment^[43]4–[44]6. Unfortunately, standard clinical
therapies, such as aminosalicylates, corticosteroids, and
immunosuppressants, etc., offer only transient symptom relief without
addressing the underlying intestinal barrier disruption and microbiota
imbalance^[45]7. Consequently, these inflammatory microenvironments may
progress to more severe conditions such as intestinal fibrosis, a
complication characterized by thickened intestinal walls and reduced
peristalsis, potentially culminating in intestinal
stenosis^[46]8,[47]9. This progression would further lead to
life-threatening intestinal obstruction or colorectal cancer
(CRC)^[48]10,[49]11. Therefore, research aimed at simultaneously
addressing IBD and its fibrotic complication is highly pursued.
Activated macrophages and neutrophils in inflamed intestinal regions
generate excess reactive oxygen and nitrogen species (RONS)^[50]12,
resulting in oxidative protein damage, lipid peroxidation, DNA injury,
and disruption of the intestinal epithelial barrier^[51]13,[52]14.
Recently, nanozymes have garnered attention as promising candidates for
IBD treatment owing to their unique ability to modulate redox
homeostasis in RONS-related inflammation^[53]15. Various nanozymes with
multiple enzymatic activities (including the CeO[2]^[54]16,
NiCo[2]O[4]@PVP^[55]17, and Mn[3]O[4]^[56]18 nanozymes, among others)
have been explored for IBD treatment. However, most reported nanozymes
are composed of metals/metal oxides and exhibit instability as well as
potential toxicity in acidic gastric conditions, raising concerns about
their long-term safety^[57]19,[58]20. In addition, orally administered
nanozymes have a short gastrointestinal residence time, necessitating
repeated administration to achieve therapeutic efficacy. Moreover,
solely targeting oxidative stress may be insufficient to address the
complex pathogenesis of IBD or manage IBD-related complications like
intestinal fibrosis. Increasing evidence highlights the crucial role of
gut microbiota in maintaining intestinal homeostasis and its
dysregulation in IBD^[59]21. Hence, modulating the gut microbiota has
emerged as a popular therapeutic strategy for IBD, either through oral
probiotic administration or by regulating molybdenum cofactors to
inhibit the proliferation of Enterobacteriaceae^[60]22. Nonetheless,
the existing IBD treatments seldom address both IBD and intestinal
fibrosis simultaneously. Therefore, there is an urgent need to design a
biocompatible platform with multifaceted capabilities to regulate
oxidative stress, balance gut microbiota, repair intestinal barriers,
and prolong intestinal retention time of therapeutic agents, enabling
concurrent treatment of IBD and its fibrotic complications.
Hydrogels with a porous three-dimensional matrix have been developed as
multifunctional and biocompatible drug delivery platforms for various
disease treatments^[61]23. Nevertheless, conventional hydrogels
encounter significant challenges in addressing intestinal lesions, such
as poor stability under low pH conditions, insufficient degradation
resistance and complicated synthesis procedures^[62]24. Inulin, a
polysaccharide dietary fiber sourced from chicory roots and Jerusalem
artichokes, is renowned for its probiotic benefits^[63]25,[64]26 and
has demonstrated potential in treating disorders like tumor^[65]25,
non-alcoholic steatohepatitis^[66]27 and IBD^[67]28. This study
introduces an oral-administered ternary inulin gel loaded with
polypyrrole (PPy) nanozymes with excellent ROS scavenging capability
and anti-fibrotic pirfenidone (PFD) drug for the treatment of IBD and
intestinal fibrosis (Fig. [68]1a). The as-prepared PPy/PFD@Inulin gel,
produced through a straightforward heating and cooling process,
exhibited remarkable stability under acidic conditions. Upon oral
administration, the intestinal retention effect of inulin gel would
ensure the sustained release of PPy nanozymes and PFD within the
gastrointestinal tract, thereby extending the therapeutic duration. The
released PPy nanozymes could regulate oxidative stress by eliminating
excessive RONS. On the other hand, the released PFD could inhibit the
proliferation of fibroblasts and down-regulate TGF-β/Smad pathway.
Furthermore, the inulin gel could selectively promote the proliferation
of probiotics, aiding in the restoration of gut microbiota balance in
IBD patients (Fig. [69]1b). Thus, the ternary PPy/PFD@Inulin gel could
perform as a pioneered design for the concurrent treatment of IBD and
associated fibrotic complication.
Fig. 1. Schematic description of the fabrication process of PPy/PFD@Inulin
gel and its mode of action in addressing IBD and intestinal fibrosis.
[70]Fig. 1
[71]Open in a new tab
a Preparation of PPy/PFD@Inulin gel. After oral administration of
PPy/PFD@Inulin gel, the gel undergoes metabolism by the intestinal
microbiota, enhancing the effectiveness of PPy and PFD in the
gastrointestinal tract. b The PPy/PFD@Inulin gel exerts multifaceted
regulation on the mechanisms associated with IBD and intestinal
fibrosis.
Results
Preparation and characterization of ternary PPy/PFD@Inulin gel
Firstly, PPy nanozymes were synthesized via an aqueous dispersion
polymerization technique, achieving a diameter of 60 − 80 nm^[72]29
(Supplementary Fig. [73]1a-c). The surface analysis of PPy nanozymes
revealed a homogeneous distribution of carbon and nitrogen elements,
with no detectable metallic elements, thereby classifying them as
non-metallic nanozymes (Supplementary Fig. [74]1d and e). Supplementary
Fig. [75]2a, b shown that PPy nanozymes has good water dispersibility.
Next, the obtained PPy nanozymes and PFD drug were incorporated into a
heated inulin solution, forming a ternary PPy/PFD@Inulin gel upon
cooling to ambient temperature. As shown in Fig. [76]2a, the gel
exhibited a black coloration attributable to the presence of PPy
nanozymes, and the successful synthesis of PPy/PFD@Inulin gel was also
confirmed by Fourier transform infrared spectroscopy (FTIR) and Zeta
potential characterizations (Supplementary Fig. [77]3a, b). All four
gel variants possessed typical porous hydrogel structures with no
significant morphological differences regardless of the inclusion of
PPy nanozymes and PFD molecules. Satisfyingly, the PPy/PFD@Inulin gel
exhibited outstanding injectability and was capable of traversing
syringes of varying sizes, even with a minimum needle diameter of only
0.4 mm, which is crucial for subsequent oral administration.
Furthermore, the storage modulus (G’) of all hydrogels surpassed the
loss modulus (G”), indicating that the moderate incorporation of PPy
nanozymes and PFD molecules did not adversely affect the hydrogel
performance of inulin while maintaining their excellent shear-thinning
behaviors (Fig. [78]2b and Supplementary Fig. [79]4a–c).
Fig. 2. Synthesis and characterization of PPy/PFD@Inulin gel.
[80]Fig. 2
[81]Open in a new tab
a Four distinct groups of inulin-based gels were depicted in various
aspects: visual appearance, SEM images, and photographs illustrating
their injectability. b Dynamic rheological tests were conducted on
these four groups, measuring both the elastic modulus (G’) and the
viscous modulus (G”). c In vitro degradation kinetics of the
PPy/PFD@Inulin gel were studied under different pH levels and the
presence of Bifidobacterium longum. d The release profiles of PFD were
assessed at varying pH levels at 37 °C. e, f Cy5.5-PPy nanozymes and
Cy5.5-PPy/PFD@Inulin gel were administered orally to mice. The
gastrointestinal tract was observed for (f) 24 h and (e) the mean
fluorescence intensity in the colon was quantified. Data are presented
as mean ± SD (n = 3 biologically independent samples for (c–e)). In a,
the representative SEM images are shown (at least three images were
taken for each sample). In f, images from one representative experiment
of three independent experiments are presented. Statistical analysis
was evaluated with two-tailed Student’s t tests. p > 0.05 (n.s.),
*p < 0.05, **p < 0.01, ***p < 0.001.
The heterogeneity in pH levels across the gastrointestinal tract poses
a substantial challenge for the oral delivery of pharmaceuticals.
Generally, the pH ranges from 1.0-3.0 in the stomach, 6.0-7.5 in the
small intestine, 5.0-7.0 in the proximal colon, and then increases
to7.0-8.0 in the rectum^[82]30. Therefore, four distinct pH solutions
(1.5, 6.0, 7.4 and 8.0) were used to evaluate the stability of orally
administered PPy/PFD@Inulin gel. As illustrated in Supplementary
Fig. [83]5a, b, the diameter of PPy nanozymes exhibited no significant
alterations, indicating their excellent stability across varying pH
levels. Satisfactorily, after soaking the PPy/PFD@Inulin gel in
phosphate-buffered saline (PBS) with different pH values to mimic the
gastrointestinal environment, scanning electron microscopy (SEM)
revealed that gel’s microstructure remained intact, while FTIR and
X-ray diffraction (XRD) analyzes confirmed that the composition and
structure of the gel were unaltered, as depicted in Supplementary
Fig. [84]6a–c. The PPy/PFD@Inulin gel demonstrated greater
susceptibility to disruption under acidic conditions (pH = 1.5) than at
other pH levels. However, the degradation rate remained below 5% after
6 h and below 15% after 12 h (Fig. [85]2c), which were beneficial for
escorting the PPy/PFD@Inulin gel to intestine as gastric emptying
typically occurred within 3-4 h. Although inulin itself cannot be
well-digested or absorbed in the stomach and small intestine, it would
undergo fermentation and degradation by beneficial bacteria in the
colon^[86]31. As shown in Fig. [87]2c and Supplementary Fig. [88]7a–c,
Bifidobacterium longum (BL) could utilize PPy/PFD@Inulin gel to enhance
its growth, thereby increasing lactic acid production. Additionally,
the resulting metabolites created an acidic environment that would
accelerate gel degradation in the presence of BL. Overall, the
PPy/PFD@Inulin gel displayed exceptional stability across different pH
levels, confirming its suitability for oral therapeutic applications in
animal models.
Next, the release kinetics of PFD molecules were further investigated
(Fig. [89]2d and Supplementary Fig. [90]8a, b). The free PFD group
exhibited complete active pharmaceutical ingredient (API) release
within 2 h, whereas the PPy/PFD@Inulin gel demonstrated a prolonged and
complete release within 24 h, indicating that inulin gel could function
effectively as a drug carrier to extend the release duration of PFD
molecules. Previous studies have shown that gel-state polysaccharide
fibers would prolong gastric emptying (owing to thickening), adhere to
the intestinal mucosa, and extend intestinal residence time^[91]32. A
lap-shear adhesion test was conducted to assess the bio-adhesive
properties of PPy/PFD@Inulin gel (Supplementary Fig. [92]9a, b).
Interestingly, the PPy/PFD@Inulin gel adhered to pig skin tissues and
withstood greater deformation compared to PPy nanozymes, attributed to
its gel form. Our study confirmed that oral administration of
Cy5.5-PPy/PFD@Inulin gel resulted in a longer residence time in the
colon than Cy5.5-PPy nanozymes (Fig. [93]2e, f). These findings
validated that the PPy/PFD@Inulin gel could contribute to the sustained
release of PPy nanozymes and PFD in the intestinal tract, thereby
prolonging the therapeutic efficacy.
In vitro and in vivo RONS scavenging and biocompatibility characterization
Elevated RONS levels in inflamed intestinal tracts have become a focal
point in the pathophysiology of IBD. PPy nanozymes, due to the
alternation of π-electron delocalization in single and double
carbon-carbon bonds, can donate electrons or active hydrogen atoms to
neutralize free radicals, thereby bestowing PPy nanozymes with
peroxidase (POD) and superoxide dismutase (SOD) activities^[94]33.
Particularly, at a low concentration of 60 μg/mL, PPy nanozymes
exhibited DPPH and ABTS clearance rates of 59.1 ± 0.4% and 77.7 ± 3.7%,
respectively (Fig. [95]3a, b). In addition, at a treatment
concentration of 40 μg/mL, the clearance rate for O[2]−^- reached 80%.
Employing cyclic voltammetry (CV) to evaluate the H[2]O[2]-scavenging
capacity of PPy nanozymes revealed a reduction peak and increased
current density at −0.6 V when a glassy carbon (GC) electrode coated
with PPy nanozymes was immersed in a PBS solution containing H[2]O[2]
and scanned in the negative direction, indicating H[2]O[2] was reduced
and validating the H[2]O[2] scavenging capacity of PPy nanozymes
(Fig. [96]3c, d)^[97]34. Moreover, the RONS scavenging capacities
remained stable across a range of pH levels from weakly acidic to
alkaline, as well as in simulated gastrointestinal fluid (Fig. [98]3e
and Supplementary Fig. [99]10). Encouraged by the superior RONS
scavenging capacities of PPy nanozymes, the antioxidant activity of
PPy/PFD@Inulin gel was then assessed, showing no significant difference
in antioxidant capacity between PPy nanozymes and PPy/PFD@Inulin gel at
equivalent concentrations (Supplementary Fig. [100]11a–d). Furthermore,
the antioxidant capacity in gel state is largely unaffected by varying
pH values and simulated intestinal and gastric conditions
(Supplementary Fig. [101]12a–c). Hence, the ternary PPy/PFD@Inulin gel
demonstrated stable and outstanding RONS-scavenging properties across
various pH environments.
Fig. 3. In vitro antioxidant effect of PPy/PFD@Inulin gel and cell protection
from ROS-induced damage.
[102]Fig. 3
[103]Open in a new tab
a DPPH, (b) ABTS and (c) O[2]·^- scavenging efficacy. d CVs of the
modified GC electrode with PPy nanozymes as prepared in the presence of
5.0 × 10^−3 M H[2]O[2] in N[2]-saturated 0.01 M PBS (pH 7.4) and bare
GC electrode in PBS (H[2]O[2]). e PPy nanozymes retention activity
after treatment in a simulated gastrointestinal environment was
retained. f Viability of NCM460 cells. g Fluorescent images of
calcein-AM/PI co-stained NCM460 cells and (h) corresponding cell
viability. i NCM460 cells stained with DCFH-DA and (j) the
corresponding mean fluorescence intensity. Data are presented as
mean ± SD (n = 3 biologically independent samples for (a–c, e, f, h,
and j)). Images from one representative experiment of three independent
experiments are presented (g and i). Statistical analysis was performed
using one-way ANOVA. p > 0.05 (n.s.), *p < 0.05, **p < 0.01,
***p < 0.001.
The cytotoxicity assessment of PPy/PFD@Inulin gel was performed by
using human colon mucosal epithelial cells (NCM460) (Fig. [104]3f and
Supplementary Fig. [105]13a and b). Notably, even at elevated
concentrations of 400 μg/mL PPy nanozymes and 1 mg/mL PFD, the cell
viability remained both above 85% after 24 and 48 h of incubation,
emphasizing their favorable biocompatibility. The cytoprotective effect
of PPy nanozymes under H[2]O[2]-induced oxidative stress was then
investigated by using a typical Live/Dead fluorescence assay
(Fig. [106]3 g). Unlike the significant cell mortality seen in the 3 mM
H[2]O[2] treated group, negligible dead cells could be observed in the
group treated with 3 mM H[2]O[2] + 400 μg/mL PPy nanozymes,
highlighting the superior cytoprotective capability of PPy nanozymes,
which was further confirmed by the quantitative cell survival rate
analysis (Fig. [107]3 h and Supplementary Fig. [108]14). Additionally,
a marked reduction in green fluorescence from the oxidation of
2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was observed in the
presence of PPy nanozymes, further demonstrating their eye-catching ROS
scavenging capacity, which also was validated by the H[2]O[2] and
lipopolysaccharide (LPS) stimulation assay (Fig. [109]3i, j and
Supplementary Fig. [110]15a and b).
Biocompatibility and biodistribution of PPy/PFD@Inulin gel
Subsequently, a hemolysis test was conducted, revealing that the
hemolysis rate remained below 5% across all concentrations of PPy
nanozymes, demonstrating excellent hemocompatibility (Supplementary
Fig. [111]16). Motivated by its favorable in vitro biocompatibility,
the in vivo biocompatibility of PPy/PFD@Inulin gel was further
assessed. No significant alterations were detected in hematological or
biochemical parameters after oral administration of the PPy/PFD@Inulin
gel for 4, 7, 15 and 30 days (Supplementary Fig. [112]17a–c), and no
histopathological abnormalities or organ damage were observed
(Supplementary Fig. [113]17d). Furthermore, the biological distribution
of hydrogel components was assessed using Cy5.5-labeled PPy/PFD@Inulin
gel post oral administration. As depicted in Supplementary
Fig. [114]17e, the fluorescent hydrogel predominantly localized in the
intestine, with a peak fluorescence intensity at 8 h and detectable
levels persisting up to 24 h, indicating the extended retention of
PPy/PFD@Inulin gel in the colon, which was beneficial for prolonging
its therapeutic efficacy.
Investigation of the PPy/PFD@Inulin gel effect on intestinal physiology
As depicted in Fig. [115]4a, mice received oral administration of
PPy/PFD@Inulin gel on days 0, 1, 3, 5, and 7, with fecal specimens
collected on day 9. Throughout the 9-day timeframe, the experimental
mice exhibited a reduction in food intake relative to the control
group, attributed to the gastric volume occupation by the hydrogel,
thereby diminishing food consumption (Fig. [116]4b). Body weight
analysis revealed no significant difference between the PPy/PFD@Inulin
gel-treated mice and the normal group (Fig. [117]4c). The capability of
PPy/PFD@Inulin gel to modulate the abundance of gut microbiota in
normal mice was studied using 16S rRNA gene sequencing. Alpha diversity
metrics including observed species, Simpson, and Shannon indices,
indicated an enhancement in both the abundance and diversity of gut
microbiota following PPy/PFD@Inulin gel administration
(Fig. [118]4d–f). The Venn diagram in Fig. [119]4g illustrated the
augmented microbial community richness resulting from the
PPy/PFD@Inulin gel treatment. Principal Coordinate Analysis (PCoA)
based on Bray-Curtis distances also revealed partial overlap yet slight
differentiation between operational taxonomic units (OTUs) from
PPy/PFD@Inulin gel-treated mice and those from the healthy control
group (Fig. [120]4h). Furthermore, at the phylum level, there was a
significant proliferation of Verrucomicrobia in the treated mice, which
was consistent with the genus level heatmap, showing a notable increase
in Akkermansia abundance (Fig. [121]4i, j). This was aligned with
previous research indicating that inulin administration significantly
boosted Akkermansia levels, which were linked to the maintenance of
intestinal barrier function^[122]25,[123]35. In a word, oral
administration of PPy/PFD@Inulin gel can modulate gut microbiota
composition, notably enhancing the abundance of beneficial bacteria
Akkermansia in mice.
Fig. 4. Effect of hydrogel on intestinal physiology.
[124]Fig. 4
[125]Open in a new tab
a Roadmap for experimental design. b The amount of feed loss in 9 days.
c In 9 days, the body weight of mice was recorded. d–f Estimation of
the gut microbial abundance and indices of community α-diversity
determined by the number of observed OTUs (d), Simpson (e), and Shannon
(f). g Venn diagram illustrates the numbers of ASV/OTU in the control
and PPy/PFD@Inulin gel. h Beta diversity is illustrated of the PCoA,
based on Bray-Curtis distances. i Composition analysis of gut
microbiota at the phylum level in different groups. j Heat map of
intestinal microorganisms in mice at genus level. Data are presented as
mean ± SD (n = 5 biologically independent samples for (c–j)).
Statistical analysis was evaluated with two-tailed Student’s t tests.
p > 0.05 (n.s.), *p < 0.05, **p < 0.01, ***p < 0.001.
Prophylactic treatment of DSS-induced ulcerative colitis
Leveraging the superior RONS-scavenging efficacy and excellent
biocompatibility of PPy/PFD@Inulin gel, its prophylactic therapeutic
effects were evaluated in a model of DSS-induced ulcerative colitis.
The optimal dosage for PPy nanozymes was established as 40 mg/kg
(Supplementary Fig. [126]18a-f). PPy/PFD@Inulin gel was then orally
administered on the 1st, 3rd, 5th, and 7th days, with euthanasia on the
9th day (Fig. [127]5a). According to Fig. [128]5b, while all
DSS-treated mice initially experienced weight loss, those in the three
groups receiving PPy nanozymes exhibited weight recovery starting from
day 4. It is noteworthy that PFD demonstrated minimal efficacy in this
IBD model. Ultimately, the body weights of mice treated with PPy@Inulin
gel and PPy/PFD@Inulin gel were most comparable to those of healthy
controls, attributable to the sustained therapeutic effect facilitated
by the inulin hydrogel. As successful induction of UC in mice would
result in increased disease activity index (DAI) scores, the DAI values
of PPy/PFD@Inulin gel treated mice returned to normal, suggesting a
promising prophylactic effect (Supplementary Fig. [129]19). Colon
photographs and length measurements on day 9 revealed that the average
colon length of mice in DSS group was only 6.78 cm, whereas it was
restored to 9.22 cm in the PPy/PFD@Inulin gel-treated group, comparable
to that of normal mice (Fig. [130]5c, d).
Fig. 5. Prophylactic effects of PPy/PFD@Inulin gel on DSS-induced colitis by
oral administration.
[131]Fig. 5
[132]Open in a new tab
a Experimental design of DSS-induced IBD model mice. b Daily changes in
body weight were recorded throughout the experiment. c Colon length of
mice with indicated treatment on day 9. d Digital photos of the colons
of each group of mice on day 9. e H&E staining of representative mouse
colon tissue. f MPO activity in supernatant after homogenization of
mouse colon tissue. g-i Quantitative analysis of pro-inflammatory
factors (TNF-α, IL-1β, and IL-6) in colon tissue. j Immunohistochemical
staining for the expression of TNF-α, IL-1β, and IL-6 in colonic
tissue. Data are presented as mean ± SD (n = 5 biologically independent
samples for (b and c), n = 3 biologically independent samples for
(f–i)). The representative images in e and j are shown from three
independent mice. Statistical analysis was performed using one-way
ANOVA. p > 0.05 (n.s.), *p < 0.05, **p < 0.01, ***p < 0.001.
Unlike the pathological phenomena including reduced goblet cells, crypt
distortion, and extensive inflammatory cell infiltration revealed by
H&E staining of DSS-treated colons, no significant disruption of the
intestinal micro-barrier was observed in the colons post-treatment with
the PPy/PFD@Inulin gel (Fig. [133]5e). Myeloperoxidase (MPO),
predominantly expressed by neutrophils, serves as a crucial mediator of
oxidative stress and is thus frequently utilized as a biomarker for IBD
evaluation^[134]36. As shown in Fig. [135]5f, MPO activity approached
to nearly normal level following the oral administration of
PPy/PFD@Inulin gel, significantly lower than that observed in the
DSS-induced inflammatory mice. Moreover, mice treated with
PPy/PFD@Inulin gel exhibited cytokine expression levels akin to the
normal control group, including TNF-α, IL-1β, and IL-6, in stark
contrast to the DSS-treated group (Fig. [136]5g–i). Immunohistochemical
analysis revealed elevated pro-inflammatory cytokine levels in the
DSS-treated group, which normalized subsequent to PPy/PFD@Inulin gel
administration (Fig. [137]5j). These findings collectively
substantiated the in vivo prophylactic effect of orally administered
PPy/PFD@Inulin gel in mitigating colitis.
Delayed treatment of DSS-induced ulcerative colitis
Encouraged by its promising prophylactic efficacy, the therapeutic
potential of PPy/PFD@Inulin gel in the delayed treatment of acute
colitis was further assessed. As shown in Fig. [138]6a, colitis in mice
was induced by substituting drinking water with 3% DSS solution for the
initial six days, followed by oral gavage on days 7, 9, 11, and 13,
culminating in euthanasia on day 15. During the initial DSS
administration period, the mice experienced a gradual weight loss of
approximately 10% of their original body weights (Fig. [139]6b). Mice
in the three groups treated with PPy nanozymes formulations exhibited
weight recovery post the first dose on day 7. The DAI was recorded
throughout the experiment (Supplementary Fig. [140]20), showing a
reduction in DAI scores for the mice receiving PPy/PFD@Inulin gel,
nearly reaching normal level similar to those observed in the
prophylactic treatment study. The average colon length in mice
subjected to 15 days of DSS treatment was significantly reduced,
measuring only 6.64 ± 0.23 cm. Notably, both the PPy@Inulin gel
(8.4 cm) and PPy/PFD@Inulin gel (8.42 cm) demonstrated substantial
therapeutic benefits, attributed to prolonged retention by the inulin
hydrogel, and with the therapeutic efficacy of PPy nanozymes comparable
to that of clinically used 5-aminosalicylic acid (5-ASA) (Fig. [141]6c,
d). H&E staining of intestial tissues showed that the PPy/PFD@Inulin
gel-treated group exhibited the negligible colonic injury with intact
goblet cells, regular crypt morphology, and an unimpaired intestinal
mucous layer (Fig. [142]6e). Moreover, following PPy/PFD@Inulin gel
treatment, colon tissues exhibited significantly reduced levels of MPO,
TNF-α, IL-1β, and IL-6 compared to the DSS-treated groups, with values
closely resembling those of normal mice (Fig. [143]6f–i).
Fig. 6. PPy/PFD@Inulin gel alleviated DSS-induced colitis in a delayed
treatment mode.
[144]Fig. 6
[145]Open in a new tab
a Experimental design of DSS-induced IBD model mice. b During the
entire experiment, the changes of body weight were recorded every 2
days. c Colon length of mice with indicated treatment on day 15. d
Digital photographs of colon of each group of mice on day 15. e H&E
staining of representative mouse colon tissue. f MPO activity in
supernatant after homogenization of mouse colon tissue. g-i
Quantitative analysis of TNF-α, IL-1β, and IL-6 in colon tissue. Data
are presented as mean ± SD (n = 5 biologically independent samples for
(b and c), n = 3 biologically independent samples for (f–i)). The
representative images in e are shown from three independent mice.
Statistical analysis was performed using one-way ANOVA. p > 0.05
(n.s.), *p < 0.05, **p < 0.01, ***p < 0.001.
PPy/PFD@Inulin gel repairs the intestinal barrier and regulates flora
Next, the underlying reparative mechanism of PPy/PFD@Inulin gel on
compromised colonic epithelial barrier and gut microbiota were
investigated. Damage to intestinal mucosal barrier typically results in
elevated serum FITC-dextran levels post-gavage. By orally administering
FITC-dextran to mice across different experimental groups and
subsequently quantifying serum FITC-dextran using fluorescence imaging
and a microplate reader, the DSS group exhibited significantly higher
fluorescence intensity in the colonic region compared to the control
group (Fig. [146]7a, b). Intervention with PPy/PFD@Inulin gel markedly
decreased colonic permeability to FITC-dextran. Microplate reader
measurements corroborated these findings, indicating that serum
FITC-dextran levels in PPy/PFD@Inulin gel-treated mice approached those
of the control group (Fig. [147]7c). Zonula Occludens-1 (ZO-1) and
occludin proteins are critical components of intercellular tight
junctions and play pivotal roles in maintaining the structure,
function, and barrier integrity of the intestinal epithelium^[148]35.
Western blotting analysis (Supplementary Fig. [149]21a–c) revealed
diminished expression of ZO-1 and occludin in colitis-afflicted mice,
indicative of compromised tight junctions. Notably, PPy/PFD@Inulin gel
treatment significantly upregulated these protein levels.
Immunofluorescence staining and quantitative fluorescence analysis
(Fig. [150]7d and Supplementary Fig. [151]22a, b) further confirmed
reduced ZO-1 and occludin expression in the DSS group, with the gel
effectively enhancing their expression, thereby facilitating intestinal
barrier restoration. Fluorescent in situ hybridization (FISH) imaging
demonstrated high Muc2 protein levels, a major component of intestinal
mucus barrier, in the control group, which effectively shielded
epithelial cells from bacterial invasion (Supplementary Fig. [152]23).
In contrast, the DSS-treated group exhibited significantly increased
bacterial invasion.
Fig. 7. The repair effect of PPy/PFD@Inulin gel on colon epithelium and
intestinal flora regulation.
[153]Fig. 7
[154]Open in a new tab
a, b Fluorescence images and fluorescence quantification detected by
imaging of small animals 2 h after oral administration of 4 kDa
FITC-dextran. c Concentration of FITC-dextran in serum of mice. d
Immunofluorescence staining ZO-1 (green) and occludin (red) proteins,
and the nucleus was stained with DAPI (blue). e, f Estimation of the
gut microbial abundance by the number of OTUs (e) and indices of
community α-diversity determined by Shannon (f). g Composition analysis
of gut microbiota at the phylum level in different experimental groups.
h, i Relative abundance of gut microbes at genus level in mice (h) and
heatmap showing normalized Z-score values of microbial abundance at
genus level (i). Data are presented as mean ± SD (n = 3 biologically
independent samples for (b and c), n = 4 biologically independent
samples for (e–i)). The representative images in d are shown from three
independent mice. Statistical analysis was performed using one-way
ANOVA (b and c) and statistical analysis was evaluated with two-tailed
Student’s t tests (e and f). p > 0.05 (n.s.), *p < 0.05, **p < 0.01,
***p < 0.001.
Furthermore, the efficacy of PPy/PFD@Inulin gel in modulating the gut
microbiota during delayed treatment of DSS-induced colitis was
investigated by analyzing alterations in the intestinal microbiome
through 16S rRNA gene sequencing. Parameters including the observed
species, Shannon and Simpson’s diversity indicated that oral
administration of PPy/PFD@Inulin gel significantly increased the
abundance and diversity of the intestinal microbial community in
DSS-treated mice (Fig. [155]7e, f and Supplementary Fig. [156]24). In
the feces of DSS-treated mice, Firmicutes and Bacteroides were
predominant, indicating a significant imbalance in the flora^[157]37.
In addition, Proteobacteria increased because of their advantage in
utilizing host nitrogen, resulting in an increased abundance of
Enterobacteriaceae in colitis mouse feces^[158]38. Following the
PPy/PFD@Inulin gel treatment, the imbalance of intestinal flora was
significantly improved (Fig. [159]7g). Further analysis at the genus
level, along with the corresponding heat maps (Fig. [160]7h, i), oral
administration of PPy/PFD@Inulin gel substantially increased the
abundance of Akkermansia. Additionally, the populations of Coprococcus
and Oscillospira in the murine gut flora were restored to levels
similar to those in healthy mice. Both bacteria genera contribute to
short-chain fatty acids (SCFAs) production and intestinal
regulation^[161]39,[162]40. Overall, the 16S rRNA sequencing results
indicated that PPy/PFD@Inulin gel treatment could restore the abundance
and diversity of the intestinal microbiota in mice to normal levels by
sustaining the intrinsic regulatory functions by inulin.
Analysis of transcriptome changes after PPy/PFD@Inulin gel treatment using
RNA-seq
To further elucidate the protective efficacy of PPy/PFD@Inulin gel on
DSS-induced colitis in murine models, RNA sequencing (RNA-seq) was
employed for a comprehensive quantitative assessment of the gene
expression landscape in colonic tissues. Initially, the heatmap
depicted in Fig. [163]8a strikingly contrasts the gene expression
profiles between the DSS-treated and PPy/PFD@Inulin gel-treated groups.
Principal Component Analysis (PCA) further clustered the samples,
distinctly segregating the DSS group from both the control and
PPy/PFD@Inulin gel groups (Fig. [164]8b). Volcano plot and Venn
analysis identified 511 differentially expressed genes (DEGs) (228
upregulated, 283 downregulated) between the PPy/PFD@Inulin gel and
DSS-treated groups (Fig. [165]8c and Supplementary Fig. [166]25).
Additionally, 981 DEGs (451 upregulated, 530 downregulated) were
delineated between the control and DSS groups, while 211 DEGs (57
upregulated, 154 downregulated) were found between the control and
PPy/PFD@Inulin gel groups (Supplementary Figs. [167]25 and [168]26a,
b). To ascertain their functional relevance, Gene Ontology (GO)
analysis was conducted on DEGs between the PPy/PFD@Inulin gel and DSS
groups, revealing significant shows the difference of infiltrated
immune cells among control group, DSS group and involvement in
biological signaling pathways such as neutrophil migration, bacteria
response, and inflammatory response (Fig. [169]8d)^[170]41. KEGG
pathway analysis further indicated that these DEGs are implicated in
inflammatory pathways including the IL-17 signaling pathway,
cytokine-cytokine receptor interaction, TNF and JAK/STAT signaling
pathways (Fig. [171]8e)^[172]42. The JAK/STAT pathway plays a crucial
role in modulating both adaptive and innate immune responses,
particularly in IBD^[173]43. The ImmuCC algorithm was employed to
profile the infiltration of 23 types of immune cell types within the
colonic tissues of these three groups. The bar graph in Fig. [174]8f
and heatmap in Fig. [175]8g illustrated the differential abundance of
immune cell infiltrates among the groups. Compared to the healthy
control and PPy/PFD@Inulin gel groups, the DSS group showed elevated
levels of activated dendritic cells, M0 macrophages, and M1 macrophages
in the colonic mucosa.
Fig. 8. RNA sequencing analysis of colon tissues treated with PPy/PFD@Inulin
gel.
[176]Fig. 8
[177]Open in a new tab
a Heat map of the total differential metabolite clustering in the DSS
group and PPy/PFD@Inulin gel group. b PCA of the colon tissue gene
expression values in the Control DSS, and PPy/PFD@Inulin gel. c Volcano
plot of the differential genes in the colon tissues of the Control DSS,
and PPy/PFD@Inulin gel. d GO enrichment analysis of DEGs. BP represents
the “biological process”; CC represents the “cellular component”; MF
represents the “molecular function”. e KEGG pathway enrichment analysis
of DEGs. f The histogram PPy/PFD@Inulin gel group. g Heat map of the
difference of infiltrating immune cells among control group, DSS group
and PPy/PFD@Inulin gel group. (n = 4 biologically independent samples
for (a, b), n = 5 biologically independent samples for (c and d), n = 3
biologically independent samples for (f and g)). Statistical analysis
was evaluated with two-tailed Student’s t tests. p > 0.05 (n.s.),
*p < 0.05, **p < 0.01, ***p < 0.001.
Intestinal fibrosis reversion by oral administration of PPy/PFD@Inulin gel
Intestinal fibrosis, a resultant complication linked to chronic IBD,
manifests as a thickened intestinal wall and reduced peristalsis,
posing a heightened risk of severe intestinal stenosis and potentially
lethal intestinal obstruction. Inspired by the promising therapeutic
efficacy of PPy/PFD@Inulin gel in addressing IBD via RONS scavenging
and microbiota modulation, a chronic colitis model induced through
repeated DSS cycles was established to investigate the capability of
PPy/PFD@Inulin gel for simultaneously reversing IBD and its fibrotic
complication (Fig. [178]9a). As shown in Fig. [179]9b, a lower dose of
1.5% DSS solution had minimal impact on mouse body weight, whereas a
higher dose of 2.5% DSS led to significant body weight loss.
Remarkably, treatment with PPy/PFD@Inulin gel effectively restored the
body weight of mice to normal levels. Upon sacrifice on day 35,
examination of mouse colons revealed distinct differences.
Specifically, DSS-treated mice exhibited shorter and thicker colons
compared to the normal group, with an average colon length of merely
6.52 cm. However, oral administration of PPy/PFD@Inulin gel (7.88 cm)
restored colon length and thickness to levels comparable to the control
group (8.12 cm) (Fig. [180]9c, d). Subsequent examination of
H&E-stained colon tissue cross sections and colonic damage
scores^[181]44 revealed substantial inflammatory cell infiltration in
the DSS-treated group, particularly in the submucosa, in contrast to
the intact crypt structures observed in the normal group (Fig. [182]9e,
f). Quantitative measurements of H&E-stained sections using CaseViewer
software 2.4 showed significantly increased thickness in the muscularis
propria, muscularis mucosa, and intestinal wall of the DSS group
(Fig. [183]9g–i). Masson’s trichrome and immunohistochemical staining
were used to evaluate fibrosis in mice with chronic colitis. The
results illustrated that PPy/PFD@Inulin gel exhibited a protective
effect against fibrous collagen deposition (Fig. [184]9j). Although
single PFD treatment moderately mitigated fibrosis, its efficacy in
alleviating intestinal damage was limited due to persistent chronic
inflammation. Gratifyingly, with the synergistic assistance of RONS
scavenging PPy nanozymes and gut microbiota-modulating inulin hydrogel,
oral administration of PPy/PFD@Inulin gel could not only prevent
intestinal damage induced by RONS but also alleviate intestinal
fibrosis.
Fig. 9. Therapeutic effects of PPy/PFD@Inulin gel on DSS-induced fibrosis
model.
[185]Fig. 9
[186]Open in a new tab
a Experimental design of a mouse fibrosis model induced by DSS. b
Detailed weight changes were recorded every 5 days. c Colon length of
mice on day 35. d Digital photographs of colon on day 35. e H&E
staining of representative mouse colon tissue. f The colonic damage
scores of mice. g Wall thickness of colon in mice with DSS-induced
fibrosis. h Thickness of muscularis propria and (i) thickness of
muscularis mucosa. j Representative images of colon sections stained
with Masson’s trichrome in the eight groups: ECM deposition (blue
area). Representative immunohistochemical staining for α-SMA and TGF-β
in colon sections. Data are presented as mean ± SD (n = 5 biologically
independent samples for (b and c), n = 3 biologically independent
samples for (f), n = 4 biologically independent samples for (g-i)). The
representative images in e and j are shown from three independent mice.
Statistical analysis was performed using one-way ANOVA. p > 0.05
(n.s.), *p < 0.05, **p < 0.01, ***p < 0.001.
Next, a comprehensive analysis of the antifibrotic mechanism was
further performed. One potential antifibrotic mechanism of PFD is its
capacity to attenuate colon fibroblast proliferation without inducing
cytotoxicity. Incorporating 5-ethynyl-2’-deoxyuridine (EdU)
demonstrated that the PPy/PFD@Inulin gel could inhibit the
proliferation of human colon fibroblasts (CCD-18Co) (Fig. [187]10a and
Supplementary Fig. [188]27). The integrity of cells was assessed using
a standard lactate dehydrogenase (LDH) release assay. Negligible
cytotoxic effects were observed with PFD incubation at a concentration
as high as 1 mg/mL during the initial 24 h, with only slight cellular
damage after 48 h incubation (Fig. [189]10b). Subsequently, Live/Dead
fluorescence assays indicated a reduction in cell proliferation
following treatment with gradient concentrations of PFD, which was
accompanied by a decrease in cell size and cytoplasm volume
(Fig. [190]10c and Supplementary Fig. [191]28). Western blot analysis
was then utilized to examine the expression of fibrosis-related
proteins, including α-smooth muscle actin (α-SMA) and TGF-β1, in the
colons of mice with fibrosis. In comparison to the control group, these
proteins were upregulated in the DSS-treated group, whereas treatment
with the PPy/PFD@Inulin gel reduced their expression levels
(Fig. [192]10d–f), indicating that PPy/PFD@Inulin gel blocked the
TGF-β/Smad signaling pathway. Similar to previously reported
anti-fibrotic mechanism of PFD^[193]45, Fig. [194]10g provided an
overview of the potential modulation of TGF-β/Smad pathway by the
PPy/PFD@Inulin gel. Upon TGF-β ligand binding to its receptor, the type
II receptor is activated, which subsequently phosphorylates the type I
receptor. Following the phosphorylation of the type I receptor, Smad3
is specifically targeted and phosphorylated, forming a transcriptional
complex with Smad4 that translocates to the nucleus to regulate the
transcription activity of various genes. Therefore, these data
confirmed that the obtained PPy/PFD@Inulin gel could inhibit the
activated TGF-β/Smad pathway in colitis-related intestinal fibrosis.
Fig. 10. The mechanism of PPy/PFD@Inulin gel for inhibiting intestinal
fibrosis.
[195]Fig. 10
[196]Open in a new tab
a Proliferation of CCD-18Co cells treated with different groups in the
EdU assay. b Relative LDH activity of CCD-18Co cells. c Fluorescent
images of CCD-18Co cells co-stained with calcein-AM/PI dyes. d
Representative Western blot images. e, f Quantitative Western blot
analyzes were performed. g Schematic mechanism of PPy/PFD@Inulin gel
for inhibiting intestinal fibrosis. Data are presented as mean ± SD
(n = 3 biologically independent samples for (b, e, and f)). Images from
one representative experiment of three independent experiments are
presented (a and c). Statistical analysis was performed using one-way
ANOVA. p > 0.05 (n.s.), *p < 0.05, **p < 0.01, ***p < 0.001.
Discussion
IBD is a complex inflammatory disorder modulated by numerous variables.
It is characterized by increased oxidative stress and dysbiosis of the
gut microbiota, leading to the perturbation of intestinal
microenvironment. Consequently, this perturbation instigates recurrent
inflammation, ECM deposition, and thickening of the intestinal wall,
ultimately culminating in intestinal fibrosis, a prevalent complication
of IBD. In this study, a PPy/PFD@Inulin gel was developed to prevent
and alleviate the detrimental effects associated with IBD and
intestinal fibrosis. Notably, the remarkable injectability of
PPy/PFD@Inulin gel endows its rapid delivery to the inflamed areas of
intestines through oral administration. The intestinal retention
properties of PPy/PFD@Inulin gel contributed to the sustained release
of PPy nanozymes and PFD molecules from the intestines, thereby
prolonging their therapeutic efficacy.
The PPy/PFD@Inulin gel exhibited effective RONS clearance along with a
substantial reduction of pro-inflammatory cytokine secretion.
Particularly, PPy/PFD@Inulin gel significantly alleviated the symptoms
of acute DSS-induced IBD symptoms, as demonstrated by rapid body weight
recovery, reduced mucosal injury in colonic tissues, diminished MPO
activity and pro-inflammatory cytokine levels. Moreover, the
PPy/PFD@Inulin gel effectively upregulated the expression of tight
junction proteins in colonic tissues, thereby fortifying the intestinal
barrier. It also modulated the gut microbiota, increasing the
prevalence of beneficial genera such as Coprococcus and Oscillospira,
which contribute to the production of butyrate, a vital SCFAs
instrumental in intestinal barrier repair. In addition, KEGG analysis
revealed that the DEGs between the PPy/PFD@Inulin gel-treated and IBD
groups were predominantly associated with immunoregulatory and
metabolic pathways, such as IL-17 signaling pathway,
cytochrome-cytochrome receptor interactions, TNF and JAK/STAT signaling
pathway. This study showed the therapeutic potential of PPy/PFD@Inulin
gel in regulating intestinal immunity and microbiota composition.
Furthermore, the PPy/PFD@Inulin gel markedly inhibited the
proliferation and viability of colonic fibroblasts and attenuated the
expression of proteins related to the TGF-β1/Smad3 pathway, including
TGF-β1 and α-SMA. Notably, it significantly alleviated chronic colitis
induced by DSS, as reflected by rapid weight recovery, diminished
mucosal damage in the colonic tissue, and decreased intestinal fibrosis
severity.
In conclusion, considering the elevated RONS levels, the disruption of
microbial communities, and related complications in the intestinal
microenvironment associated with gastrointestinal diseases, a ternary
PPy/PFD@Inulin gel was successfully developed to prevent and mitigate
adverse effects related to IBD and intestinal fibrosis. The results of
the above experiments provided compelling evidence of the robust
therapeutic efficacy of PPy/PFD@Inulin gel in both cellular and animal
models. Current research findings indicated that inulin gel exhibited a
certain sustained-release capability. Future optimization of this gel’s
drug-release profile can be achieved by fine-tuning its structure and
components, with the goal of enhancing drug stability and sustained
release for improved therapeutic outcomes. Furthermore, functional
modifications of the components in PPy/PFD@Inulin gel may augment its
targeting specificity to particular intestinal loci, thereby further
boosting therapeutic effectiveness. This multifunctional composite
hydrogel, utilizing prebiotic inulin as a carrier, is safe, reliable,
simple to prepare, scalable for mass production, and capable of
ameliorating the intestinal microenvironment. Such a multifunctional
hydrogel platform could serve as a convenient and valuable modality for
the treatment of a spectrum of other pathologies, presenting novel
strategies for the advancement of biomedical materials.
Methods
Materials
Pyrrole, pirfenidone, Cy5.5 NHS ester, 2,2-Diphenyl-1-picrylhydrazyl,
and 2,2’-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium
salt) were obtained from Aladdin. Inulin from chicory, the EdU Cell
Proliferation Kit, and the Tissue or Cell Total Protein Extraction Kit
were purchased from Sangon Biotechnology Co., Ltd. Total superoxide
dismutase and Reactive Oxygen Species assay kits were obtained from
Beyotime Biotechnology Co., Ltd. Dextran sodium sulfate was obtained
from Meilunbio Co., Ltd.
Synthesis of PPy nanozymes
1.5 g of polyvinyl alcohol was dissolved in 20 mL of deionized water
and stirred at 60 °C until complete dissolution. After cooling to room
temperature, 1.2434 g of ferric chloride hexahydrate was added and
stirred at room temperature for 1 h. Subsequently, 140 μL of pyrrole
was introduced and stirred at 5 °C for 4 h. Upon completion of the
reaction, washed three times with water (15,100 x g/min, 20 min), and
subjected to lyophilization. The resulting PPy nanozymes powder was
obtained by lyophilization.
Synthesis of PPy/PFD@Inulin gel
The PFD was dissolved in a small amount of methanol. Subsequently, 1 mL
of the PPy nanozymes solution and 0.6 g of inulin were added, and the
mixture was stirred to achieve uniformity. The resulting solution was
then placed in a water bath at 80 °C, stirred at 800 rpm for 10 min,
and finally allowed to stand at room temperature for 12 h to obtain
PPy/PFD@Inulin gel. Control samples, including blank inulin gel,
PFD@Inulin gel and PPy@Inulin gel, were also prepared using the same
method.
Characterization of PPy nanozymes and PPy/PFD@Inulin gel
TEM (JEM-1400flash), SEM (Gemini500), and modular compact rheometer
(Anton Paar, H-PTD200) were employed to characterize the PPy nanozymes
and PPy/PFD@Inulin gel. For TEM and SEM sample preparation of PPy
nanozymes, the sample in aqueous solution was deposited on gilder grids
or silicon wafers and allowed to dry naturally. The PPy/PFD@Inulin gel
and control group gels were characterized by SEM after freeze-drying.
The rheological properties of the hydrogels were evaluated using a
25-mm plate pressure-controlled rheometer.
In vitro release of PFD from PPy/PFD@Inulin gel
PFD solution and PPy/PFD@Inulin gel were added to dialysis bags with a
molecular weight cutoff (MWCO) of 1000 Da and placed on an oscillator
at 37 °C with a rotation speed of 100 rpm. Samples were collected at
various time points after adding the corresponding volumes of PBS
buffer. The concentration of PFD was determined by UV−Vis spectrometer
at 310 nm.
In vitro degradation experiment of PPy/PFD@Inulin gel
Before cooling to form the gel, 1 mL of the mixed PPy/PFD@Inulin gel
solution was subpackaged into centrifuge tubes; the mass of the
centrifuge tubes was termed m[0], and the combined mass of the
PPy/PFD@Inulin gel and centrifuge tubes after cooling to form the gel
was termed m[1]. The PPy/PFD@Inulin gel was incubated at 37 °C in
different environments (pH=1.5, 6.0, 7.4, and 8.0, as well as
Bifidobacterium longum solution). The solution above the gel was
discarded at different times, the centrifuge tube was weighed, and the
recorded weight was denoted as m. Subsequently, the percentage quality
loss was calculated. Three samples were collected during each
experimental period.
[MATH:
W%=m−m0<
mrow>m1−m0×<
/mo>100% :MATH]
1
Determination of lactic acid in incubation of PPy/PFD@Inulin gel with BL
bacteria
After co-incubating PPy/PFD@Inulin gel with BL for 2 days, the
supernatant was obtained by 1700 xg/5 min centrifugation and filtered
with 0.22 μm filter membrane. Analytical HPLC with C18 column. In the
HPLC procedure, the mobile phase is composed of two parts: A) dilute
sulfuric acid (95% in volume percentage) and B) acetonitrile (5% in
volume percentage). The flow rate was fixed at 0.5 mL/min, the elution
time was 15 min, and eluent detection was monitored at 210 nm.
Retention of PPy/PFD@Inulin gel in the gastrointestinal system
Preparation of Cy5.5-PPy nanozymes and Cy5.5-PPy/PFD@Inulin gel. For
each milliliter of PPy nanozymes, 8 μg of Cy5.5 was added and placed in
a 37 °C oscillator for 12 h. Subsequently, it was placed into a
dialysis bag (MWCO: 14000 Da) and dialyzed for 24 h to remove excess
Cy5.5. Cy5.5-PPy/PFD@Inulin gel was prepared according to the procedure
described above. The Cy5.5-PPy nanozymes and Cy5.5-PPy/PFD@Inulin gel
prepared were orally administered to mice. Mice were dissected at 0, 2,
4, 8, 12, and 24 h post-administration, and the gastrointestinal tract
from the small intestine to the colon was extracted for analysis. The
fluorescence distribution was photographed using Spectral Instruments
Imaging (SI Imaging AmiX), and the fluorescence intensity was
quantitatively analyzed. The excitation wavelength used was 675 nm, the
emission wavelength was 730 nm, and the exposure time was 10 s.
In vitro RONS scavenging
The capacity of PPy nanozymes to scavenge active nitrogen was analyzed
by detecting DPPH and ABTS free radicals. 0.5 mL PPy nanozymes solution
and 1 mL DPPH or ABTS working solution were incubated in the dark for
30 min, and then the UV absorption at 519 nm or 732 nm was measured.
Where A[0] is the UV absorption value of the DPPH or ABTS working fluid
and water, A[x] is the UV absorption value of the DPPH or ABTS working
fluid and material reaction, and A[1] is the UV absorption value of the
material under the total system.
[MATH:
Scaveng<
mi>ingeffect%=
A0−AX+A1A0
×100% :MATH]
2
The ability of PPy nanozymes to scavenge active oxygen was analyzed by
detecting O[2]·^− and H[2]O[2]. The O[2]·^− scavenging capacity of PPy
nanozymes was assessed using the SOD assay kit. The ability of PPy
nanozymes to scavenge H[2]O[2] was measured using the three-electrode
CV method. Carbon rods and saturated calomel electrodes were used as
counter and reference electrodes, respectively. PPy nanozymes (10 μL,
1 mg/mL) was dripped onto and dried on the GC working electrode. To
evaluate the catalytic activity of PPy nanozymes for the elimination of
H[2]O[2], in the presence of 5.0 × 10^−3 M H[2]O[2] in N[2]-saturated
0.01 M PBS (pH 7.4), the PPy nanozymes-modified GC electrode and the
bare GC electrode in PBS (H[2]O[2]) were used to record the CV curve on
an electrochemical analyzer (CHI760E, China).
Stability of PPy nanozymes and PPy/PFD@Inulin gel at different pHs
Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were used to
regulate the acidity and basicity of the PBS. In this experiment, PBS
with varying pH values (pH=1.5, 6.0, 7.4, and 8.0) was used. The PPy
nanozymes and PPy/PFD@Inulin gel were immersed in PBS at different pH
values for 6 h, and their structural integrity was determined using TEM
or SEM.
Determination of RONS clearance under simulated gastrointestinal conditions
Owing to the varying pH levels in the gastrointestinal tract, HCl was
initially added to the PPy nanozymes solution to achieve a pH of 1.5
and incubated at 37 °C for 4 h. Subsequently, NaOH was added dropwise
to the PPy nanozymes solution to adjust the pH to 8.0 and incubated at
37 °C for 4 h. Following these adjustments, the scavenging ability
against DPPH, ABTS, and O[2]·^− was evaluated using the experimental
steps outlined above.
Cell culture
NCM460 cell lines were obtained from In Cell (San Antonio, TX).
CCD-18Co (CRL-1459) cell lines were obtained from the American Type
Culture Collection (ATCC). NCM460 cells were cultured in 1640 medium
containing 10% FBS, streptomycin (100 g/mL) and penicillin (100 U/mL)
in an incubator at 37 °C. CCD-18Co cells were cultured in DMEM (high
sugar and pyruvate) medium containing 10% FBS, streptomycin (100 g/mL)
and penicillin (100 U/mL) in an incubator at 37 °C.
Cell viability
NCM460 cells were seeded in a 96-well plate at a density of 1 × 10^4
cells/well and cultured in a serum-containing 1640 medium for 24 h. The
experimental materials were prepared in a serum-free medium. Fresh
medium was used for the control group and used to incubate cells for 24
or 48 h. The cells were incubated with 20 μL
3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT)
(5 mg/mL) solution for 4 h, and the supernatant was replaced by
dimethyl sulfoxide (150 μL). Finally, the absorbance was recorded
utilizing a microplate reader (BioTek, ELX800) to calculate cell
viability.
Cellular antioxidation experiment
The protective efficacy of PPy nanozymes against oxidative damage
induced using H[2]O[2] was evaluated by live/dead fluorescence staining
or the MTT assay. NCM460 cells treated with 3 mM H[2]O[2] were
incubated with different concentrations of PPy nanozymes and stained
with calcein-AM and PI dyes. After co-incubating the materials with
1 mM H[2]O[2] for 6 h, the protective efficacy against H[2]O[2]-induced
cellular oxidative damage was quantitatively assessed using the MTT
assay.
NCM460 cells were grown in a 96-well plate at 1 × 10^4 cells/well and
cultured for 24 h. Different PPy nanozymes concentrations were
dispersed in the medium, and 1 mM H[2]O[2] was added. NCM460 cells were
incubated with the above solution at 37 °C for 45 min. Subsequently,
cells were incubated with DCFH-DA at 37 °C for 30 min and photographed
using a fluorescence microscope.
NCM460 cells were grown in a 96-well plate at a density of 1 × 10^4
cells/well and cultured for 24 h. The NCM460 cells were treated with
800 ng/mL LPS for 8 h and then removed. Different concentrations of PPy
nanozymes were dispersed in the medium with cells for 4 h, after which
cells were incubated with DCFH-DA at 37 °C for 30 min and photographed
using a fluorescence microscope.
Animals
Animals were grouped according to different experiments and put in
separate cages. All animals were housed and maintained in pathogen-free
conditions and allowed free access to food and autoclaved water ad
libitum in a 12 h light (8:00 AM-8:00 PM)/dark (8:00 PM-8:00 AM) cycle,
with controlled temperature (25 ± 1 °C) and humidity (40–70%). We
euthanized mice by carbon dioxide suffocation. BALB/c and C57bl/6j mice
(female, 6 weeks old, 18 − 20 g) were purchased from Henan Sikebas
Biotechnology Co., Ltd. All animal experiments were approved by the
Institutional Animal Care and Use Committee of Hefei University of
Technology (No. HFUT20220305003).
In vivo biocompatibility and biodistribution characterization
The BALB/c mice were randomly divided into five groups (n = 5). One
group of healthy mice served as a control, and the other four groups
received oral administration of PPy/PFD@Inulin gel for the first four
days, and euthanasia was performed at various time points (4th, 7th,
15th, and 30th days). Blood and the main organs were collected for
hematology and histological analyzes.
The BALB/c mice were randomly divided into six groups (n = 3), with
each mouse receiving 0.2 mL Cy5.5-PPy/PFD@Inulin gel orally. The mice
were sacrificed at 0, 2, 4, 8, 12, and 24 h. The small-animal imaging
software Spectral Instruments Imaging (SI Imaging AmiX) was used for in
vivo imaging. Following this, the isolated colon segment, heart, liver,
spleen, lung, and kidney, were examined using an imager, and the
biological distribution of the PPy/PFD@Inulin gel in vitro was
evaluated.
Effect of hydrogel on intestinal physiology
The BALB/c mice were randomly divided into 2 groups (n = 8). One group
of healthy mice served as controls, while another group orally received
PPy/PFD@Inulin gel on days 0, 1, 3, 5, and 7. Throughout the 9-day
experiment, daily measurements were taken of food weight consumed by
the mice, as well as their body weights. On the 9th day, the feces of
mice were collected for 16S rRNA sequencing. The NEB Next® Ultra DNA
Library Prep Kit library construction kit was used to construct the
library, and an Agilent 5400 was used for detection and qPCR
quantification of the constructed library. After the library was
qualified, NovaSeq6000 was used for sequencing.
Preventative and delayed treatment of UC by PPy/PFD@Inulin gel
The BALB/c mice were randomly divided into eight groups (n = 8). Before
the experiment began, the animals were domesticated for one week. From
days 0 to 7, the mice received 3% DSS instead of drinking water to
induce colitis. PPy/PFD@Inulin gel was orally administered on the 1st,
3rd, 5th, and 7th days, and the mice were euthanized on the 9th day.
Throughout the 9-day experiment, daily recordings were made for body
weight, fecal morphology, and fecal bleeding. Part of the colon was
stained with H&E. The level of inflammatory factors was assessed by
homogenizing the colon on ice and quantifying the resultant supernatant
using ELISA kits.
In the delayed treatment of UC animal model, the clinically used 5-ASA
was employed as a positive drug for the treatment of IBD. The BALB/c
mice were randomly divided into nine groups (n = 8). The animals were
domesticated for one week before the commencement of the experiment.
From days 0 to 6, the mice received 3% DSS instead of drinking water to
induce colitis. The mice received oral administration of PPy/PFD@Inulin
gel on days 7, 9, 11, and 13 and were euthanized on the 15th day.
Throughout the 15-day experiment, the body weight, fecal morphology and
fecal bleeding were assessed every two days. Part of the colon was
subjected to H&E, immunohistochemical, and immunofluorescence staining.
The levels of inflammatory factors were assessed by homogenizing the
colon on ice, and the supernatant was quantified using commercial ELISA
kits. Fecal samples from nine groups of mice were collected on the 14th
day for 16S rRNA sequencing.
Intestinal permeability test
The BALB/c mice were randomly divided into 3 groups (n = 5). On the
14th day of the delayed treatment model, approximately 2 h after
administering 4 kDa FITC-dextran (Sigma, 10 mg/kg), blood samples were
collected from three mice via retro-orbital bleeding. The blood was
centrifuged at 500 xg/15 min to obtain plasma, which was then measured
for OD values. Subsequently, the colons of the mice were excised and
imaged using a small animal fluorescence imaging system with an
excitation wavelength of 488 nm and an emission wavelength of 520 nm.
Western blotting of ZO-1 and occludin proteins
Proteins were extracted from the colon tissue of delayed treatment
model mice using the Tissue or Cell Total Protein Extraction Kit, and
protein concentration was determined using a BCA assay kit. 7.5% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was
employed to separate an equal amount of colon tissue extract, which was
subsequently transferred to a polyvinylidene fluoride membrane (PVDF,
Millipore). The blocked PVDF membrane was then incubated with the
respective primary antibodies, ZO-1 (Proteintech Group, Inc), and
occludin (ABclonal), at 4 °C overnight. Following that, the membrane
was incubated with the secondary antibody (ABclonal) at room
temperature for 90 min. To visualize protein bands, the membrane was
incubated with horseradish peroxidase (HRP) substrate (Tanon, Shanghai,
China). All protein bands were normalized to β-actin.
Oral administration of PPy/PFD@Inulin gel for the treatment of intestinal
fibrosis
The C57bl/6j mice were randomly divided into eight groups (n = 8).
Prior to the experiment, the animals were domesticated for one week.
The animals were repeatedly exposed to multiple DSS cycles. Briefly,
each cycle comprised a five-day administration of different
concentrations of DSS, with 1.5% DSS administered on days 0 − 5, 2%
administered on days 10 − 15, and 2.5% administered on days 20 − 25.
The mice received drinking water for other days. From day 15, the drug
was administered orally once daily until day 35, after which the mice
were sacrificed. During the 35-day experimental period, the body
weights of the mice were assessed every 5 days. A portion of the colon
underwent H&E and immunohistochemical staining.
Proteins were extracted from the colon tissue of fibrotic mice using
the Tissue or Cell Total Protein Extraction Kit, and protein
concentration was determined using a BCA assay kit. 12% SDS-PAGE was
employed to separate an equal amount of colon tissue extract, which was
subsequently transferred to a PVDF. The blocked PVDF membrane was then
incubated with the respective primary antibodies, α-SMA, and TGF-β1
(ABclonal), at 4 °C overnight. Following that, the membrane was
incubated with the secondary antibody (ABclonal) at room temperature
for 90 min. To visualize protein bands, the membrane was incubated with
HRP substrate. All protein bands were normalized to GAPDH.
CCD-18Co cells were inoculated in a 96-well plate with 1 × 10^4
cells/well and cultured in H-DMEM medium containing serum for 24 h.
Incubated different groups of materials and EdU solution in the well
plate at 37 °C for 8 h. EdU incorporation was measured using an EdU
cell proliferation assay kit. LDH activity in CCD-18Co cells was
detected using the LDH Cytotoxicity Assay Kit. The supernatant of the
culture medium was collected after 24 and 48 h of treatment with 0,
0.25, 0.5, or 1.0 mg/mL PFD and analyzed for LDH.
Statistical Analysis
All results are expressed as the mean ± SD, as indicated. Statistical
analyzes were performed using GraphPad Prism 8. A one-way ANOVA with
Tukey’s post-hoc test was used to evaluate statistically significant
differences in multiple group data (P > 0.05 (n.s.), *P < 0.05,
**P < 0.01, ***P < 0.001). Statistical significance between two groups
was evaluated using the Student’s t-test. (P > 0.05 (n.s.), *P < 0.05,
**P < 0.01, ***P < 0.001) (n.s., not significant).
Reporting summary
Further information on research design is available in the [197]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[198]Supplementary information^ (2.5MB, pdf)
[199]Peer Review File^ (6.2MB, pdf)
[200]Reporting Summary^ (174.1KB, pdf)
Source data
[201]Source Data^ (5.4MB, xlsx)
Acknowledgements