Abstract

   Natural compound-based treatments provide innovative ways for
   ulcerative colitis therapy. However, poor targeting and rapid
   degradation curtail its application, which needs to be addressed.
   Inspired by biomacromolecule-based materials, we have developed an
   orally administrated nanoparticle (GBP@HA NPs) using bovine serum
   albumin as a carrier for polyphenol delivery. The system synergizes
   galactosylated bovine serum albumin with two polyphenols,
   epigallocatechin gallate and tannic acid, which is then encased in
   “nanoarmor” of ε-Polylysine and hyaluronic acid to boost its stability
   and targeting. Remarkably, the nanoarmor demonstrated profound
   therapeutic effects in both acute and chronic mouse models of
   ulcerative colitis, mitigating disease symptoms via multiple
   mechanisms, regulating inflammation related factors and exerting a
   modulatory impact on gut microbiota. Further mechanistic investigations
   indicate that GBP@HA NPs may act through several pathways, including
   modulation of Keap1-Nrf2 and NF-κB signaling, as well as
   Caspase-1-dependent pyroptosis. Consequently, this novel armored
   nanotherapy promotes the way for enhanced polyphenol utilization in
   ulcerative colitis treatment research.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-024-02933-3.

   Keywords: Bovine serum albumin, Epigallocatechin gallate, Tannic acid,
   Armored nanotherapy, Ulcerative colitis

Introduction

   Inflammatory bowel disease (IBD) is a chronic and non-specific
   inflammation of the gastrointestinal tract, primarily comprising
   Crohn’s disease (CD) and ulcerative colitis (UC) [[38]1–[39]4]. It is
   characterized by disruption of the epithelial barrier of the colon and
   intestinal homeostasis [[40]5, [41]6]. The two main types of IBD have
   significant differences, with UC being continuous inflammation and CD
   being discontinuous full-thickness inflammation. Research has found
   that the incidence of UC is higher than CD, and the incidence of
   complications and clinical symptoms is also higher than CD. Therefore,
   the severity and complexity of UC are greater than CD. Although
   treatment is challenging, they represent a significant modern medical
   concern due to their severe impact on patient’s quality of life
   [[42]7–[43]9]. As a result, researchers have been trying to find a
   satisfactory cure for IBD.

   The main treatments for IBD include medications such as
   corticosteroids, aminosalicylic acid, and immunosuppressants
   [[44]10–[45]13], these drugs provide relief by scavenging free
   radicals, reducing inflammation, and regulating immune responses
   [[46]14–[47]16]. Nevertheless, these synthetic drugs have extensive
   side effects such as gastrointestinal reactions [[48]17], bone marrow
   suppression [[49]18], infection [[50]10], liver function damage
   [[51]19], etc., which limited their long-term use [[52]20]. So, safer
   and more effective way are a pressing concern.

   Polyphenols, predominantly derived from plants, are the abundant
   natural compounds with phenolic structural [[53]21–[54]23]. Ranking
   second only to sugars, they exhibit extensive physiological activities,
   high biocompatibility, and minimal side effects [[55]24, [56]25]. As
   potent antioxidants, they augment the efficacy of antioxidant vitamins
   and enzymes, inhibit oxidative stress from excessive reactive oxygen
   species, and regulate the intestinal flora [[57]26–[58]31]. Many
   researches indicated that polyphenols can play a therapeutic role in
   diabetes [[59]32], inflammatory diseases like arthritis [[60]33] and
   inflammatory bowel disease [[61]34], neurodegenerative diseases such as
   Parkinson’s disease and Alzheimer’s disease [[62]35], and various
   cancers [[63]36–[64]39]. Our research focuses on two important
   polyphenols, epigallocatechin gallate (EGCG) and tannic acid (TA). EGCG
   is the most abundant polyphenol in green tea, with anti-inflammatory
   and antioxidant properties. Studies have found that it has a
   therapeutic effect in a mouse model of colitis by regulating the
   composition of gut microbiota and reducing levels of inflammatory
   factors to alleviate colitis [[65]27]. TA is a polyphenolic compound
   obtained from schisandra, which has significant antioxidant capacity.
   Current research has found that it also has unique effects in
   anti-inflammatory and anti-tumor aspects, and can alleviate colitis
   [[66]31]. Nevertheless, polyphenols are not without limitations. After
   entering the digestive tract, polyphenols undergo biotransformation due
   to environmental conditions including pH and enzymes [[67]40, [68]41].
   They are affected by different conditions at different stages of
   digestion and undergo degradation in different ways, affecting their
   chemical structure. Meanwhile, the microbiota in the colon can
   decompose phenolic compounds. Therefore, they exhibit stability issues
   and the direct entry of polyphenols into the digestive tract has
   relatively low bioavailability [[69]42, [70]43]. To address these
   problems, developing a polyphenol delivery system becomes a promising
   choice, potentially enhancing the stability and therapeutic efficacy of
   polyphenols in IBD treatment [[71]44–[72]49].

   Oral administration is widely recognized as a convenient and safe
   approach for drug delivery. Our study introduced a nanomedical drug for
   oral administration that contains “nanoarmor” to coat polyphenols for
   the treatment of UC [[73]50, [74]51]. Specifically, galactosylated
   bovine serum albumin is used as a carrier to combine with EGCG and TA
   polyphenols, subsequently enveloping these in layers with ε-Polylysine
   (EPL) and Hyaluronic acid (HA), ultimately forming a “nanoarmor”
   (Scheme [75]1a), which is referred to as GBP@HA NPs. EPL is a cationic,
   biodegradable peptide produced by microorganisms, which has attracted
   increasing attention in oral delivery systems. It can encapsulate
   negatively charged nanoparticles formed by polyphenol and
   galactose-modified bovine serum albumin by electrostatic action and
   enhance their stability [[76]52, [77]53]. HA is the simplest
   glycosaminoglycan, a negatively charged polysaccharide that can
   specifically bind to glycoprotein CD44. CD44 is overexpressed on the
   surface of colonic epithelial cells and macrophages in UC tissue
   [[78]54]. At the same time, the enrichment of positively charged
   proteins is one of the characteristics of inflammatory colon, so the
   outer layer of HA encapsulated nanoparticles with negative surface
   charges can aggregate at the inflammatory site of the colon, enhancing
   the targeting of the system [[79]55]. We hypothesize that this
   nanoarmor could protect EGCG and TA, targeting the colon through the
   gastrointestinal tract. The therapeutic effect of GBP@HA NPs can be
   evaluated in both acute and chronic UC mouse models, and their role in
   alleviating colitis symptoms can be explored through studies of
   multiple mechanisms, and the gut microbiota can be analyzed (Scheme
   [80]1b). The signaling pathways are studied at the molecular biology
   level. In summary, the oral nanoarmor delivery system has the potential
   to exert good therapeutic effects. This system provides new and
   promising methods for the treatment of colitis, and offers more ideas
   for the application of hyaluronic acid and galactose.

Scheme 1.

   [81]Scheme 1
   [82]Open in a new tab

   (a) Schematic illustrations of GBP@HA NPs, (b) Schematic diagram of the
   role of GBP@HA NPs in UC model mice

Materials and methods

Materials

   Hyaluronic acid was purchased from Shanghai yuanye Bio-Technology Co.,
   Ltd, and epigallocatechin gallate was purchased from Shanghai Bidepharm
   Technology Co., LTD. Tannic acid was purchased from Shanghai Aladdin
   Biochemical Technology Co., LTD. Lactonic acid and bovine serum albumin
   were obtained from Tianjin Heowns Biochemical Technology Co., LTD. MDA
   detection kit, SOD detection kit, CAT detection kit and GSH-Px
   detection kit come from Wuhan Servicebio Biotechnology Co., LTD. Caco-2
   cells and RAW 264.7 cells were purchased from Wuhan Prichella
   Biotechnology Co., LTD.

Synthesis and characterization of galactosylated bovine serum albumin (G-BSA)

   5.34 g of lactose acid was dissolved in phosphate buffered saline
   (PBS). After the solution was dissolved at room temperature, the pH was
   adjusted to 5 ∼ 6 with sodium hydroxide solution. Then weigh 5.70 g of
   1-ethyl-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
   (EDC), in the above solution. Then 2.70 g of N-Hydroxysuccinimide (NHS)
   was stirred and activated for 30 min. Adjust the pH with sodium
   hydroxide solution to about 7, add 3.70 g BSA, stir for 2 d. The
   obtained solution was dialyzed with a dialysis bag of 3000 ∼ 7000 Da
   for 2 d. Finally, the solution was freeze-dried to obtain G-BSA and
   stored at 4 ° C for later use. The structure of G-BSA was analyzed by
   infrared spectrometer scanning. The crystal form of G-BSA was analyzed
   by X-ray Diffraction (XRD). Circular dichroism was used to detect the
   changes in the secondary structure of BSA.

Synthesis and characterization of GBP@HA NPs

   Weigh 3.34 g of prepared G-BSA and dissolve in water, weigh 280 mg of
   EGCG and TA respectively and dissolve in 160 mL of water, and add the
   obtained mixed solution of EGCG and TA to G-BSA solution during
   stirring at room temperature. The nanoparticles formed by the
   combination of G-BSA and polyphenol (G-BSA-polyphenol NPs) were
   obtained. Finally, the nanoparticles were washed with water. The above
   G-BSA-polyphenol NPs were dispersed in water and added to EPL aqueous
   solution with a concentration of 10 mg/mL at room temperature. After
   stirring overnight, G-BSA-polyphenol NPs wrapped in EPL (GBP@EPL NPs)
   were obtained, and then washed for three times. Then, GBP@EPL NPs were
   added to HA solution with a concentration of 2 mg/mL at room
   temperature. After stirring overnight, the nanoparticles wrapped in HA
   were obtained. After washing for three times and freeze-drying, the
   final BSA nanoparticles (GBP@HA NPs) were obtained.

   The particle size and polydispersity index (PDI) of GBP@HA NPs were
   measured by dynamic light scattering method and their potential was
   detected. The secondary structure of BSA in GBP@HA NPs was also
   analyzed by circular dichroism. The morphology of GBP@HA NPs was
   observed by transmission electron microscopy, and then GBP@HA NPs were
   incubated in artificial gastric fluid and artificial small intestine
   fluid for 3 h. High performance liquid chromatography (HPLC) was used
   to determine the contents of EGCG and TA in GBP@HA NPs (Supplementary
   Material). Meanwhile, the drug release of GBP@HA NPs were also detected
   by HPLC in vitro.

GBP@HA NPs free radical scavenging experiment

   The scavenging of 2-phenyl-4,4,5,
   5-tetramethylimidazolin-3-oxide-1-oxyl (PTIO), 1,
   1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2’
   -Azino-bis(3-ethylbenzothiazoline 6-sulfonic acid) diammonium salt
   (ABTS) free radicals by different concentrations of GBP@HA NPs was
   investigated. The experimental method can be found in the supplementary
   materials.

MTT assay

   RAW 264.7 macrophages and Caco-2 colon cancer cells were planted in
   96-well plates at the concentration of 1 × 10^4 cells/well and cultured
   at 37℃ and 5% CO[2] for 24 h. The medium was discarded and replaced
   with fresh medium containing different concentrations of GBP@HA NPs,
   and the cells treated with no nanoparticles were used as the control.
   After 24 h incubation, each well was replaced with 200 µL medium
   containing 0.5 mg/mL MTT, and incubated for another 4 h. After the
   supernatant was discarded and 150 µL DMSO was added, the cell viability
   was measured at 492 nm using an enzyme marker after 10 min of shaking.

Cellular oxidative stress damage assay

   Preparing a cell culture medium containing 500 µM hydrogen peroxide
   (H[2]O[2)] and incubate Caco-2 cells for 6 h to construct a cellular
   oxidative stress model. Using cell culture medium without H[2]O[2] as
   the normal cell control group, cells containing H[2]O[2] but without
   GBP@HA NPs were used as the model group, and other experimental groups
   were treated with medium containing 200 µg/mL GBP@HA NPs, medium
   containing the corresponding concentration of EGCG and TA mixture, and
   medium containing G-BSA-polyphenol NPs. After 24 h, the survival of
   cells was detected by MTT assay.

Enzyme-linked immunosorbent assay (ELISA)

   Lipopolysaccharide (LPS) (1 µg/mL) was used to stimulate RAW 264.7
   macrophages for 12 h to form inflammatory cell models. The cytokines
   TNF-α, IL-6, IL-1β and IL-10 in the supernatant of the medium were
   detected by ELISA to investigate the anti-inflammatory effects in
   vitro. MPO, TNF-α, IL-6, IL-1β and IL-10 in the colon of mice were
   analyzed by ELISA kit. The specific steps can be found in the
   supplementary materials.

Reactive oxygen species (ROS) scavenging assay in cells

   After stimulating 264.7 macrophages with LPS (1 µg/mL) for 12 h, medium
   containing 200 µg/mL GBP@HA NPs, medium containing the corresponding
   concentration of EGCG and TA mixture, and medium containing
   G-BSA-polyphenol NPs were added, respectively, and incubated in the
   incubator for 12 h. After the medium was incubated and washed with PBS
   for 3 times, DCFH-DA fluorescent probe (10 µM) was added under the
   condition of avoiding light. The culture medium was incubated for
   30 min and washed with PBS for 3 times. The fluorescence microscope was
   used for observation.

Cell uptake experiments

   RAW 264.7 macrophages stimulated by LPS (1 µg/mL) were used as the
   inflammatory cell model, and the following five groups were set up:
   Normal cells were treated with LPS alone, HA (9–10 W Da, 100 µg/mL)
   treated with LPS, lactose (100 µg/m) treated with LPS, HA and lactose
   pretreated with LPS. Then, rhodamine B (RB) -labeled GBP@HA NPs was
   incubated with the cells for 12 h, the media was discarded, PBS was
   washed 3 times, and the cells were fixed with 4% paraformaldehyde.
   4’,6-diamidino-2-phenylindole (4’, 6-Diamidino-2-phenylindole, DAPI)
   was added and incubated for 30 min, then washed with PBS and observed
   under fluorescence microscope.

Treatment of DSS and TNBS induced acute UC with GBP@HA NPs

   Mice are randomly divided into 7 groups of 6 mice each. The groups were
   as follows: Control group, Model group, 5-ASA group (100 mg/kg),
   Polyphenols group, G-BSA-Polyphenols group, NPs-Low group (GBP@HA NPs
   low-dose group, 50 mg/kg), NPs-High group (GBP@HA NPs high-dose group,
   100 mg/kg). The control group drank drinking water freely, and the
   remaining six groups of mice drank freely 2.5% DSS aqueous solution for
   8 d to complete the induction of UC model. The next day, each dosing
   group began gavage with the corresponding drug for 7 d. In addition,
   the acute UC model was also constructed using 2% TNBS. The treatment of
   other groups was the same as DSS induced acute UC.

Treatment of DSS induced chronic UC with GBP@HA NPs

   DSS induced chronic UC was constructed using a 2% DSS solution. The
   mice were free to drink DSS solution for the first week, water for the
   second week, and then alternating DSS and water for 7 weeks. At the
   same time, each group was given corresponding medication by gavage
   every day. On the 49th day, the mice in each group were killed.

Colitis severity assessment and tissue collection

   During the experiment, the weight, activity, mental condition, stool
   characteristics, blood stool, etc. of each mouse were observed every
   day and recorded. Scoring is based on the Disease Activity Index (DAI).
   DAI = (weight loss fraction + stool trait score + blood in the stool
   fraction)/3. The mice were killed by cervical dislocation, their colon
   was rapidly dissected and removed feces, length measurement was
   performed after treatment, and the thymus and spleen of the mice were
   weighed and recorded at the same time, and their blood cells were
   analyzed.

Analysis of oxidative stress

   Factors and enzymes related to antioxidant effect in the colon of each
   group of mice were detected separately. The mouse colon was accurately
   weighed. Glutathione peroxidase (GSH-Px), malondialdehyde (MDA),
   catalase (CAT) and superoxide dismutase (SOD) were used to detect the
   kits corresponding to glutathione peroxidase (GSH-Px), malondialdehyde
   (MDA), catalase (CAT) and superoxide dismutase (SOD), and the operation
   was performed according to the kit instructions.

Histopathological evaluation

   H&E staining, Masson staining and Alcian blue staining were used to
   evaluate the pathology of tissues. First, 4% paraformaldehyde was used
   to fix the colon tissue, and then embedded with paraffin. The
   conventionally prepared paraffin-embedded tissue is then sliced (5 μm)
   and stained with hematoxylin and eosin (H&E) or Masson kits. In
   addition, H&E or Masson staining of heart, liver, spleen, lung, and
   kidney of normal mice and mice in the NPs-High group were performed
   respectively to further evaluate the biocompatibility of GBP@HA NPs.
   Alcian blue staining was used to observe mucus secretion in the colon
   of mice.

Immunofluorescent staining

   Analyze proteins related to intestinal barrier repair and biomarkers of
   macrophages in the colon of mice in each group using immunofluorescence
   staining technology to study the intestinal barrier repair and
   macrophage polarization regulation of GBP@HA NPs, and analyze proteins
   related to cell apoptosis in the colon of mice in each group. Simply
   put, after antigen repair, paraffin sections are sealed with 3% BSA
   solution, followed by incubation with primary and secondary antibodies,
   and finally stained with DAPI for the nucleus. The primary and
   secondary antibodies used are shown in Table [83]S2 and [84]S3.

In vivo distribution and targeting experiment

   The UC model was constructed by freely drinking 2.5% DSS aqueous
   solution in mice. Cy7 labeled GBP@HA NPs were orally administered on
   the 7th day, and fluorescence in the mice was detected by in vivo
   imaging system (IVIS) at 2 h, 6 h, 12 h, and 24 h. After the mice were
   sacrificed, the heart, liver, spleen, lungs, kidneys and
   gastrointestinal tract were extracted for fluorescence detection.

Real-time quantitative PCR

   On the 8th day of the experiment, the mice were sacrificed to remove
   the colon, extract the total RNA, and measure the expression level of
   the relevant protein mRNA. In simple terms, colonic homogenate is
   obtained by grinding, centrifuged, and then isopropanol is added to
   extract RNA. They are reverse-transcribed, amplified, and mRNA changes
   quantified by the 2^−ΔΔCT method. The primer sequences used are shown
   in Table [85]S4.

Western blotting analysis

   Each group of colon tissues was lysed using RIPA lysis buffer,
   centrifuged, and protein extracted. Quantification was performed using
   BCA assay kit according to the instructions. Detecting proteins in
   colon tissue through western blotting, the specific steps were
   described in the Supplementary Material.

16 S rDNA sequencing

   In order to evaluate the changes in the gut microbial community of each
   group of mice, fecal samples from each group were collected and the
   16 S rDNA gene of the variable region V3-V4 was sequenced and analyzed.

Data analysis

   All data are expressed as mean ± standard deviation (SD). Using
   GraphPad Prism version 8.0 software, one-way analysis of variance
   (ANOVA) was used to analyze significance between three or more groups.
   P < 0.05 was considered a significant difference.

Results and discussion

Synthesis and characterization of GBP@HA NPs

   We prepared and characterized GBP@HA NPs based on our conceptual
   framework (Scheme [86]1a). As shown in Fig. [87]1a, the FT-IR Fig of
   BSA v = 1661 cm^− 1, v = 1537 cm^− 1 and v = 1239 cm^− 1 are attributed
   to the amide (I) peak, amide (II) peak and amide (III) peak,
   respectively. In the spectrum of G-BSA, v = 1655 cm^− 1,
   v = 1542 cm^− 1 and v = 1247 cm^− 1 are amide I, II, III bands,
   respectively, glycosylation can cause the carboxyl group in the
   lactobionic acid molecule and the free amino group in BSA for amide
   reaction, so its amide I, II, III band changes, 1135 cm^− 1 in
   lactobionic acid corresponds to the -OH group expansion vibration in
   the galactosyl group, and the peak wavenumber in G-BSA is 1162 cm^− 1,
   indicating the successful synthesis of G-BSA.

Fig. 1.

   [88]Fig. 1
   [89]Open in a new tab

   Synthesis and characterization of GBP@HA NPs. (a) Infrared spectra of
   G-BSA. (b) XRD spectrum of G-BSA. (c) Potential changes during the
   synthesis of GBP@HA NPs. (d) Circular dichroic spectra of BSA, G-BSA,
   and G-BSA-polyphenol NPs. (e) Particle size distribution of GBP@HA NPs.
   (f) Observe the morphology of GBP@HA NPs through transmission electron
   microscopy (pH = 7) and GBP@HA NPs were incubated in artificial gastric
   juice at pH 1.5 (g) and artificial intestinal juice at pH 6.8 (h) for
   3 h before morphological observation. Release curves of (i) TA and (j)
   EGCG in GBP@HA NPs under different pH conditions (n = 3). The (k) PTIO,
   (l) DPPH, and (m) ABTS free radical clearance rates of GBP@HA NPs. All
   data are presented as mean ± SD

   Then analyze its crystal structure through XRD. XRD spectra
   (Fig. [90]1b) showed that the broad peaks of lactobionic acid and BSA
   showed that both were amorphous, and physical mixing had no effect on
   them, and they were still amorphous, while there were obvious
   diffraction peaks at 31°(2θ) and 45°(2θ) in the spectra of G-BSA,
   indicating that G-BSA was crystalline, further proving the successful
   synthesis of G-BSA.

   Analyze the synthesis process of GBP@HA NPs through potential changes.
   The potential change results of Fig. [91]1c show that the zeta surface
   potential of G-BSA-polyphenol NPs during the synthesis of GBP@HA NPs is
   -30.97 ± 0.29 mV. Because EPL has a positive charge and HA has a
   negative charge, G-BSA-polyphenol NPs are encapsulated layer by layer
   through EPL and HA to form GBP@HA NPs. In this process, GBP@EPL NPs are
   formed after EPL encapsulation, and the potential changes from
   − 30.97 ± 0.29 mV to 21.50 ± 0.53 mV, and then through HA
   encapsulation, it finally becomes − 22.23 ± 3.62 mV.

   The circular dichroism patterns of BSA, G-BSA and G-BSA-polyphenol NPs
   (Fig. [92]1d) were verified and analyzed as shown in Table [93]S1
   (Supplementary Material), when BSA was modified by glycosylation, the
   content of its α-helix structure increased from 32.2%, the content of
   β-fold increased from 30.7 to 34.0%, and the content of β-corner
   decreased from 6.2 to 2.6%, indicating that the content of lactobonic
   acid glycosylation modified BSA The molecular secondary structure was
   affected, confirming the synthesis of G-BSA, but did not cause
   significant changes in the secondary structure. The binding of
   polyphenols to G-BSA further affects the structure of G-BSA, indicating
   that its binding may be related to hydrogen bonding, and cause
   secondary structural changes by affecting the intramolecular hydrogen
   bonding force of G-BSA.

   We also tested the particle size distribution of GBP@EPL NPs and GBP@HA
   NPs, both of which showed a unimodal distribution. The particle size of
   GBP@EPL NPs was 367.57 ± 27.17 nm (Figure [94]S1), with a PDI of 0.207.
   As shown in Fig. [95]1e, the particle size of GBP@HA NPs is
   441.93 ± 17.27 nm with a PDI of 0.268. Both types of nanoparticles have
   good dispersibility and uniform distribution.

Stability and in vitro release profiles of GBP@HA NPs

   Transmission electron microscopy was used to observe the morphology of
   nanoparticles, as shown in Fig. [96]1f-h, and it can be observed that
   the nanoparticles are regular and uniformly spherical, and the particle
   size distribution is about 500 nm, which is consistent with the
   particle size distribution detection results. To evaluate the stability
   of GBP@HA NPs, they were incubated in pepsin-containing artificial
   gastric juice and trypsin-containing artificial small intestinal fluid,
   respectively, and then the morphology of GBP@HA NPs was observed.
   Transmission electron microscopy showed that GBP@HA NPs were still
   relatively complete spherical after 3 h incubation in artificial
   gastric juice and artificial small intestinal fluid, and there is no
   significant change in particle size, so GBP@HA NPs had good stability
   in pepsin and trypsin, which was conducive to their smooth reaching of
   the colon site after oral administration.

   The release curves of GBP@HA NPs under different pH conditions are
   shown in Fig. [97]1i-j, and the cumulative release rates of TA and EGCG
   in GBP@HA NPs within 48 h at pH 1.5 are (30.84 ± 1.24) % and
   (29.96 ± 0.43) %, respectively, indicating that they are more stable
   and less released under acidic conditions. At pH 6.8, the cumulative
   release rates of TA and EGCG increased, (68.84 ± 0.68) % and
   (38.29 ± 1.40) %, respectively, and the cumulative release rates
   reached (91.26 ± 0.42) % and (47.15 ± 0.90) % at pH 7.4, respectively,
   compared with the cumulative release rates at pH 1.5 and pH 7.4.
   Therefore, GBP@HA NPs can effectively resist the acidic environment,
   maintain good stability when administered orally, and carry out a good
   drug release process in the colon.

In vitro free radical scavenging ability of GBP@HA NPs

   PTIO• is commonly used ROS radicals, DPPH• and ABTS•, commonly used
   reactive nitrogen species (RNS) radicals, and the antioxidant capacity
   of GBP@HA NPs was preliminarily tested by free radical scavenging
   experiments. As shown in Fig. [98]1k-m, the scavenging rate of GBP@HA
   NPs in the range of 50 ∼ 500 µg/mL is more than 60% for PTIO radicals,
   more than 70% for DPPH radicals, and more than 98% for ABTS radicals,
   and its clearance gradually increases with the increase of
   concentration, so GBP@HA NPs can effectively remove ROS and RNS,
   indicating that they may have good antioxidant function. At the same
   time, according to the detection results, 200 µg/mL was selected for
   cell experiments.

In vitro and in vivo biocompatibility of GBP@HA NPs

   The safety of a drug is crucial to the application of the drug.
   Therefore, the cytotoxicity of GBP@HA NPs in RAW 264.7 mouse
   macrophages and Caco-2 colorectal cancer cells was first analyzed by
   MTT. As shown in Fig. [99]2a-b, at a concentration of 12.5 ∼ 800 µg/mL,
   the survival rate of RAW 264.7 macrophages and Caco-2 colorectal cancer
   cells was above 80%, and there was no obvious cytotoxicity, which
   proved that GBP@HA NPs had good in vitro biocompatibility. As ideal
   nanoparticles for oral administration, in addition to good therapeutic
   effects, good biocompatibility is also necessary. In this study, the
   heart, liver, spleen, lungs and kidneys of mice continuously given high
   doses of GBP@HA NPs were subjected to histopathological observation by
   H&E staining and Masson staining, and the blood cells of mice were
   analyzed and compared with normal mice.

Fig. 2.

   [100]Fig. 2
   [101]Open in a new tab

   Effect of different concentrations of GBP@HA NPs on cell viability
   after incubation with (a) RAW 264.7 cells and (b) Caco-2 cells for
   24 h, respectively. (n = 3) (c) H&E staining and Masson staining of
   heart, liver, spleen, lung, kidney of mice administered GBP@HA NPs mice
   (Scale bar: 200 μm). (d) Blood cell analysis of mice administered
   GBP@HA NPs (n = 3; ns: No significant difference). All data are
   presented as mean ± SD

   As shown in Fig. [102]2c, cardiomyocytes are complete and neatly
   arranged, liver cells are normal and closely arranged, the structure of
   white pulp and red pulp in spleen tissue is clear and normal, there is
   no alveolar damage in the lungs, no inflammatory cells are produced,
   and the kidney tissue presents normal tubules and glomeruli, all
   without inflammatory reaction. The blood cells of mice were analyzed to
   detect white blood cells (WBC), red blood cells (RBC), platelets (PLT),
   hemoglobin (HGB), granulocytes (GRN), hematocrit (HCT), mean volume of
   red blood cells (MCV), and mean hemoglobin concentration (MCHC) in the
   blood of mice, and the results of Fig. [103]2d showed that there was no
   significant difference between the blood cells in the blood of mice in
   the NPs-High group and those of normal mice. The above results show
   that GBP@HA NPs have no adverse effects on the tissues and blood of
   mice, have good biocompatibility, and can be used as a safe delivery
   system to play a therapeutic role.

Anti-inflammatory, antioxidant and cellular uptake properties of GBP@HA NPs

   Cytokines play an irreplaceable role in the occurrence and development
   of inflammation, in which macrophages are closely related to the
   production of cytokines [[104]56]. The anti-inflammatory effects of
   GBP@HA NPs in vitro were analyzed by investigating the secretion of
   four inflame-related cytokines, TNF-α, IL-1β, IL-6 and IL-10 by RAW
   264.7 macrophages in different treatment groups. As shown in
   Fig. [105]3a, after the stimulation of LPS, the content of
   pro-inflammatory factors TNF-α, IL-1β and IL-6 increased significantly,
   while the content of anti-inflammatory factor IL-10 was significantly
   reduced. After treatment with mixed polyphenols, G-BSA-polyphenol NPs
   and GBP@HA NPs, the pro-inflammatory factors of each group were reduced
   and the anti-inflammatory factors were increased, among which the
   regulatory effect of GBP@HA NPs was the most significant, showing good
   in vitro anti-inflammatory effects.

Fig. 3.

   [106]Fig. 3
   [107]Open in a new tab

   (a) The contents of TNF-α, IL-1β, IL-6 and IL-10 secreted by RAW 264.7
   cells were detected by ELISA (n = 3; ^*P < 0.05; ^**P < 0.01;
   ^***P < 0.001; ^****P < 0.0001). (b) The repair ability of GBP@HA NPs
   for oxidative stress damage caused by H[2]O[2] was detected by MTT
   method in Caco-2 cells. (c) Detection of reactive oxygen species in RAW
   264.7 cells using DCFH-DA fluorescence probe (scale: 50 μm). (d) Uptake
   of GBP@HA NPs by RAW 264.7 cells (blue fluorescence: DAPI-stained
   nucleus; Green fluorescence: Rhodamine B labeled GBP@HA NPs; White
   scale: 50 μm; Green scale: 25 μm). All data are presented as mean ± SD

   We evaluated the ability of GBP@HA NPs to repair oxidative stress
   damage caused by H[2]O[2] using MTT assay. As shown in the
   Fig. [108]3b, H[2]O[2] will cause significant oxidative stress damage
   to cells, leading to a significant decrease in cell survival rate.
   Polyphenols can effectively repair oxidative stress damage and increase
   cell survival rates to be similar to normal cells. The formation of
   GBP@HA NPs has no significant effect on the repair effect of
   polyphenols, and can still significantly improve cell survival rates.

   ROS in cells was detected by DCFH-DA fluorescence probe. Figure [109]3c
   shows that, after LPS stimulation, intracellular fluorescence was
   significantly enhanced and ROS content increased significantly. The
   fluorescence in cells treated with EGCG and TA mixed polyphenols,
   G-BSA-polyphenol NPs and GBP@HA NPs was significantly reduced, which
   proved that the growth of ROS content was inhibited. And We speculate
   that the antioxidant capacity of G-BSA-polyphenol NPs and GBP@HA NPs
   mainly came from natural polyphenols with good antioxidant effect,
   while the formation of G-BSA-polyphenol NPs and GBP@HA NPs can still
   maintain their antioxidant effect.

   The targeting of GBP@HA NPs to macrophages was evaluated in vitro by
   cell uptake experiments. Fluorescence pictures showed that the
   fluorescence in normal macrophages without LPS treatment was weak,
   indicating that fewer GBP@HA NPs were ingested. And the fluorescence
   intensity in LPS-induced inflammatory macrophages was significantly
   enhanced than that of normal macrophages, indicating that the uptake of
   GBP@HA NPs by inflammatory macrophages was significantly increased, and
   GBP@HA NPs had good targeting of inflammatory macrophages
   (Fig. [110]3d). To further explore the targeting of GBP@HA NPs, we
   pretreated inflammatory macrophages with a mixture of lactobionic acid,
   HA, and lactobionic acid and HA, respectively, and then incubated with
   GBP@HA NPs, respectively. As shown in the Fig. [111]3d, the
   pretreatment of lactobionic acid and HA can weaken the fluorescence in
   inflammatory macrophages, and the mixture pretreatment of lactobionic
   acid and HA significantly reduces the fluorescence to be close to that
   of normal cells, which indicates that the good targeting of GBP@HA NPs
   to inflammatory macrophages is related to the galactose group and HA
   contained in GBP@HA NPs. Galactose groups and HA selectively bind to
   macrophages galacto-type lectins (MGL) and CD44 proteins that are
   highly expressed on the surface of inflammatory macrophages,
   respectively, promoting targeted binding of GBP@HA NPs to inflammatory
   macrophages and entry.

GBP@HA NPs alleviate acute UC

   As shown in Fig. [112]4a, after free drinking of 2.5% DSS aqueous
   solution for 8 d, the model group mice had severe diarrhea, and the
   feces were bloody mucus-like, the mobility was significantly reduced,
   and the coat color was visible to the naked eye. It showed that the
   mouse ulcerative colitis model was successfully constructed. In
   Fig. [113]4b, the blood in the stool of mice in each group was observed
   with naked eye, and it was seen that there were obvious traces of blood
   in the stool at the anus of the mice in the model group.

Fig. 4.

   [114]Fig. 4
   [115]Open in a new tab

   Construction of a mouse model of ulcerative colitis and the therapeutic
   effect of GBP@HA NPs on UC. (a) Schematic diagram of experimental time.
   (b) Visual observation of mouse fecal blood situation image. (c)
   Changes in body weight of mice. (d) DAI score of mice. (e) Picture of
   mouse colon. (f) Colon length in mice. (g) Picture of mouse spleen. (h)
   Spleen index of mice. (i-l) The regulatory effects of GBP@HA NPs on
   antioxidant-related enzymes MDA, SOD, CAT and GSH-PX in colon tissues
   of UC mice. (m-r) The regulatory effect of NPs on inflammatory related
   factors MPO, TNF-α, IL-6, IL-1β and IL-10 in colon tissue of UC mice.
   (n = 6; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001). All
   data are presented as mean ± SD

   The weight of mice with colitis decreased significantly, and after
   treatment, the weight of five groups of mice in polyphenol group,
   G-BSA-polyphenol group, NPs-Low group, NPs-High group and 5-ASA group
   all recovered, among which the NPs-High group had the best therapeutic
   effect, indicating that GBP@HA NPs could effectively enhance the
   therapeutic effect of polyphenols, and the therapeutic effect was
   concentration-dependent (Fig. [116]4c). The DAI scores of mice in each
   group also demonstrated this result, with DSS-induced acute UC leading
   to a significant increase in the DAI score of the mice, and a decrease
   in DAI in all treatment groups, as well as the NPs-High group
   (Fig. [117]4d). At the same time, under the treatment effect of each
   group, the blood in the stool caused by colitis in mice was also
   significantly improved (Fig. [118]4b). This result was also confirmed
   in the acute UC model constructed through TNBS (Fig. [119]S2a-c).

   In addition, both DSS and TNBS induced mice developed symptoms of
   colitis, which developed colon congestion and swelling, weight gain,
   and shortened colon length. In addition, as an important component of
   the immune system, the inflammatory response can also affect the weight
   of the thymus and pancreas, so colon length, thymus weight, and spleen
   weight are important indicators of UC severity. As shown in the
   Fig. [120]4e-f and Fig. [121]S2d-e, the colon of the model group mice
   was swollen and the length was significantly shortened, while the high
   dose of GBP@HA NPs had a significant protective effect on the colon,
   and the protective effect was better than that of the polyphenol group
   and the G-BSA-polyphenol group. For spleen changes caused by colitis,
   all groups of mice improved after treatment, and played a good
   alleviation effect on spleen enlargement caused by inflammation, and
   like other indicators, GBP@HA NPs also played a concentration-dependent
   therapeutic effect (Fig. [122]4g-h). Therefore, we found that compared
   with unmodified polyphenols, GBP@HA NPs have a good therapeutic effect
   on DSS-induced colitis, and can effectively improve the therapeutic
   effect of polyphenols, which is closest to that of normal mice.

   In addition, we evaluated the anti-inflammatory and antioxidant effects
   of GBP@HA NPs in mice with acute UC [[123]57, [124]58]. The antioxidant
   capacity of GBP@HA NPs in vivo was analyzed by detecting the levels of
   MDA, SOD, CAT and GSH-Px in the colons of mice in each group. As shown
   in the Fig. [125]4i, DSS-induced colitis led to a significant increase
   in MDA content in the colon of mice, while the MDA content in each
   treatment group decreased compared with it, among which the NPs-Low
   group and NPs-High group were significantly reduced and
   concentration-dependent. After testing, we found that colitis
   significantly caused the reduction of SOD, CAT and GSH-Px antioxidant
   enzymes, and the levels of antioxidant enzymes were significantly
   increased after the treatment of polyphenols, G-BSA-polyphenols,
   low-dose GBP@HA NPs, high-dose GBP@HA NPs and 5-ASA, as shown in the
   Fig. [126]4j-l, the levels of SOD enzymes in the NPs-High group
   increased significantly. The CAT levels of NPs-Low group and NPs-High
   were significantly increased, and the GSH-Px levels of each treatment
   group were significantly increased, so GBP@HA NPs can effectively
   enhance the antioxidant effect of polyphenols to promote the
   improvement of colitis. The experimental results of TNBS induced acute
   UC in mice are consistent with them (Fig. [127]S2f-i).

   To analyze the anti-inflammatory effects of GBP@HA NPs in vivo, we
   examined the inflammation-related factors MPO, TNF-α, IL-6, IL-1β, and
   IL-10 in mouse colons (Fig. [128]4m). MPO is a functional marker and
   activation marker of neutrophils, and the occurrence of inflammation is
   closely related. As shown in the Fig. [129]4n-r, the content of MPO in
   mice with DSS induced acute UC is significantly increased compared with
   normal mice, and after drug treatment, the content of each group shows
   a significant downward trend, and the G-BSA-polyphenol group, NPs-Low
   group, NPs-High group and 5-ASA group are significantly reduced. The
   inflammatory factors TNF-α, IL-6 and IL-1β were significantly increased
   when inflammation occurred in the colon, but showed a significant
   decrease under the treatment effect of each group. And the content of
   anti-inflammatory factor IL-10 in normal mice was higher than that in
   the model group, the contents of the G-BSA-polyphenol group, NPs-Low
   group, NPs-High group and 5-ASA group were significantly higher than
   those in the model group. The detection of inflammatory factors in the
   colon of TNBS induced acute colitis mice further confirms that GBP@HA
   NPs have a regulatory effect on inflammatory factors and can also play
   a role in reducing MPO (Fig. [130]S2j-n). The results of these
   inflammation-related factors showed that GBP@HA NPs had good
   anti-inflammatory effects in vivo and significantly enhanced the
   anti-inflammatory effects of polyphenols.

Histopathological evaluation

   The colons of DSS and TNBS induced acute colitis mice were stained with
   hematoxylin and eosin (H&E) and Masson for histopathological analysis
   (Fig. [131]5 and Fig. [132]S3a). As shown in the Fig. [133]5a, each
   layer of the intestinal wall of the colon of DSS-induced colitis mice
   had different degrees of inflammatory cell infiltration, intestinal
   mucosal defects and crypt structure damage. The pathological injuries
   of mice in the treatment group were improved to varying degrees, among
   which the G-BSA-polyphenol NPs group, NPs-Low group and NPs-High group
   were the closest to the normal colon, and the histopathological scores
   of each group also confirmed this result. At the same time, the
   collagen fibers of the tissue were stained by Masson staining, which
   further confirmed the pathological changes in the colon tissue. As
   shown in the Masson staining image, the colonic tissue structure of
   mice in the model group was destroyed, accompanied by collagen
   deposition and fibrosis, and the treatment of each group had an
   inhibitory effect on colon tissue structure damage, collagen deposition
   and fibrosis, and promoted colon recovery, among which the
   G-BSA-polyphenol NPs group, NPs-Low group and NPs-High group had the
   best treatment effect and was closest to normal tissue.

Fig. 5.

   [134]Fig. 5
   [135]Open in a new tab

   Histopathological observation. (a) H&E staining and Masson staining of
   colon tissue in each group of mice. (b) Alcian blue staining of colon
   tissue in each group of mice. (c) Cy7 labeled GBP@HA NPs were
   administered orally to normal group mice and UC model group mice, and
   whole body imaging was performed using IVIS technology at 4 h, 6 h,
   12 h, and 24 h. (d) The heart, liver, spleen, lungs, and kidneys, as
   well as the digestive tract from the stomach to the colon, were
   collected to detect the fluorescence distribution of each site. All
   data are presented as mean ± SD. (n = 3; ^*P < 0.05; ^**P < 0.01;
   ^***P < 0.001; ^****P < 0.0001. Magnification: 20 ×; Scale: 200 μm)

   In addition, the analysis of colon tissue by alcian blue staining can
   observe the mucus matrix of colon tissue. Intestinal cells secrete
   mucus to cover the epithelial mucosal layer, forming the intestinal
   mucosal barrier, and the mucus contains IgA and antimicrobial peptides
   that work together to defend against the invasion of harmful commensal
   bacteria and pathogens. Figure [136]5b shows that colitis in the model
   group leads to the destruction of colonic tissue, a significant
   reduction in mucus secretion, and damage to the intestinal mucosal
   barrier. Compared with the model group, mucus secretion in colon
   tissues was significantly increased in all treatment groups, and the
   G-BSA-polyphenol NPs group, NPs-Low group and NPs-High group recovered
   to be close to normal mice. Therefore, compared with natural
   polyphenols and 5-ASA, the modified GBP@HA NPs can effectively inhibit
   the pathological damage of the colon caused by colitis and repair the
   intestinal mucosal barrier.

Targeting and biodistribution of GBP@HA NPs in colitis mice

   Next, we analyzed the in vivo distribution of GBP@HA NPs using Cy7
   fluorescent dye. Figure [137]5c shows that significant fluorescence was
   observed in both normal mice and UC model mice after administration,
   and the fluorescence decreased over time. However, the fluorescence in
   normal mice decreased more significantly. According to the fluorescence
   distribution maps in various organs (Fig. [138]5d), during the 24-hour
   distribution process, there was no fluorescence distribution in the
   liver, liver, spleen, lungs, and kidneys, but only in the
   gastrointestinal tract. After 12 h, UC mice showed stronger
   fluorescence in the gastrointestinal tract than normal mice, and there
   was still significant fluorescence in the colon after 24 h, indicating
   that GBP@HA NPs have good colon targeting ability.

Intestinal barrier repair and regulation of macrophage polarization

   The presence of tight junctions between intestinal epithelial cells in
   the intestinal barrier is essential and plays an irreplaceable role in
   maintaining the integrity of the intestinal barrier, and the expression
   of tight junction proteins between intestinal epithelial cells is
   affected when colitis occurs, resulting in changes in the structure and
   function of tight junctions, so the detection of tight junction
   proteins is an important part of evaluating the therapeutic effect of
   colitis. By dual immunofluorescence staining, we analyzed the four
   tight junction proteins ZO-1 and Occludin, Claudin-4, and E-Cadherin,
   respectively. The immunostaining results (Fig. [139]6a-b and Fig.
   [140]S3b) showed that the fluorescence of ZO-1, Occludin, Claudin-4 and
   E-Cadherin in the colon of normal mice was strong, which proved that
   the content of tight junction protein was high and the intestinal
   barrier function was intact. However, the intestinal barrier function
   of mice with colitis was impaired, tight junctions were destroyed, the
   content of related proteins was significantly reduced, and the
   fluorescence intensity was significantly weakened. Through the
   treatment of GBP@HA NPs and 5-ASA, the fluorescence intensity of the
   four proteins was enhanced compared with the model group, that is, the
   content of the four proteins increased, the intestinal barrier function
   was restored. And the fluorescence intensity of the GBP@HA NPs
   treatment group was also stronger than that of the 5-ASA group, which
   was close to that of the normal group, indicating that GBP@HA NPs can
   improve the colon condition of mice and treat colitis by repairing the
   intestinal barrier.

Fig. 6.

   [141]Fig. 6
   [142]Open in a new tab

   Study on repairing effect of intestinal barrier and regulating
   macrophage polarization by GBP@HA NPs. (a) Immunofluorescence staining
   and quantitative analysis of ZO-1 and Occludin in the colon (Blue
   fluorescence: DAPI-stained nucleus; Green fluorescence: ZO-1; Red
   fluorescence: Occludin) (b) Immunofluorescence staining and
   quantitative analysis of Claudin4 and E-Cadherin in the colon (Blue
   fluorescence: DAPI-stained nucleus; Green fluorescence: Claudin4; Red
   fluorescence: E-Cadherin). (c) Immunofluorescence staining and
   quantitative analysis of iNOS and CD206 in the colon (Blue
   fluorescence: DAPI stained nuclei; Green fluorescence: CD206; Red
   fluorescence: iNOS) scale: 400 μm; magnification: 40 ×

   Macrophages, as immune cells that play an important role in the
   intestinal immune barrier, can effectively help maintain and regulate
   intestinal homeostasis [[143]59]. Macrophages in different environments
   can be polarized into M1 (classical activation) and M2 (selective
   activation) two different phenotypes, M1 macrophages have
   pro-inflammatory effects, in the process of colitis occurrence and
   development of M1 macrophages polarization increases, causing a further
   increase in pro-inflammatory cytokines, while M2 has anti-inflammatory
   effects, can promote the secretion of anti-inflammatory factors, and is
   related to the resolution of inflammation [[144]60]. In this study, the
   markers iNOS (inducible nitric oxide synthase) and CD206 of M1 and M2
   macrophages were selected as indicators for the evaluation of
   macrophage polarization, as shown in Fig. [145]6c, iNOS (red
   fluorescence) expression was very little, while CD206 (green
   fluorescence) expression was significantly more than iNOS, indicating
   that macrophages in normal mice were multipolarized to M2 type. In
   contrast, iNOS expression was significantly higher than CD206 in the
   colon tissues of DSS-induced mice with colitis, indicating that colitis
   development is closely related to increased polarization of M1-type
   macrophages. After treatment with GBP@HA NPs and 5-ASA, the expression
   of iNOS in mouse colon tissues decreased, and the expression of CD206
   in the NPs-High group increased significantly compared with the model
   group, indicating that regulating the M1 and M2 polarization of
   macrophages, reducing M1 cells, and increasing M2 cells is another
   important reason why GBP@HA NPs can effectively play a therapeutic
   role.

GBP@HA NPs regulate the intestinal flora

   Important influences of the gut microbiota have been identified in
   maintaining health as well as disease pathogenesis [[146]61, [147]62].
   In this study, the intestinal bacteria in the feces of each group of
   mice were analyzed. First, the richness and diversity of the microbiota
   were analyzed, mainly evaluating the α-diversity index: operational
   taxa (OTUs), Chao1 index and Simpson index. As shown in Fig. [148]7a,
   the OTUs and Chao1 indices of DSS-induced colitis mice were
   significantly lower than those of normal mice, indicating that colitis
   affected intestinal homeostasis and reduced the abundance of intestinal
   flora, while the Simpson index of colitis mice was significantly
   reduced, indicating a decrease in the diversity of intestinal flora in
   mice with colitis. After the treatment of polyphenols and GBP@HA NPs,
   the OTUs, Chao1 index and Simpson index of mice were increased,
   indicating their recovery effect on the abundance and diversity of
   intestinal flora, among which GBP@HA NPs could promote the significant
   increase of OTUs, Chao1 index and Simpson index in mice with colitis,
   indicating its significant role in restoring the abundance and
   diversity of intestinal flora.

Fig. 7.

   [149]Fig. 7
   [150]Open in a new tab

   The role of GBP@HA NPs in regulating gut microbiota. (a) Study the
   effect of GBP@HA NPs on the abundance and diversity of gut microbiota
   through OTUs, chao1 index, and Simpson index. (b) Heat map of gut
   microbiota at the phylum and genus levels. (c) The relative abundance
   of gut microbiota at the genus level. (d) The relative abundance of
   important genera in the gut microbiota. (e) The relative abundance of
   gut microbiota at the gate level. (f) Ratio of Firmicutes/Bacteroidetes
   ratio (f/B). (g) Principal coordinate analysis (PCoA) diagram of
   intestinal microbiota. (n = 6; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001;
   ^****P < 0.0001). All data are presented as mean ± SD

   A detailed analysis of the microbiota was conducted at the phylum and
   genus levels (Fig. [151]7b). The main intestinal bacteria were analyzed
   at the phylum and genus levels, respectively. As shown in Fig. [152]7c,
   tests were performed at the genus level, and the main intestinal
   bacteria genera among them were analyzed. Lactobacillus is the most
   abundant probiotic in the gut, which is conducive to maintaining
   intestinal homeostasis and immune conditions [[153]63]. Akkermansia, a
   normal human intestinal flora, not only plays a metabolic protective
   role in protecting the integrity of intestinal epithelial cells and
   mucus layer, but also plays an anti-inflammatory role in the process of
   inflammation through regulatory T cells, endocannabinoid system and
   non-classical toll-like receptors [[154]64]. Studies have shown that
   enterotoxins produced by the genus Bacteroides are present in the human
   gut during active colitis and are closely associated with colitis
   [[155]65]. Klebsiella has a symbiotic relationship with humans, helping
   to break down food and produce the nutrients and energy the body needs.
   However, when the immune system is damaged or the intestinal barrier is
   damaged, Klebsiella enter parts of the body other than the
   gastrointestinal region, which can cause infection, oxidative stress
   and other adverse effects [[156]66]. As shown in Fig. [157]7d, in the
   intestines of normal mice, Lactobacillus and Akkermania glutinophilus
   were abundant, Klebsiella and Bacteroides were abundant, and the
   opposite was true in colitis mice. The regulatory effect of GBP@HA NPs
   changes the flora abundance, which is close to that of normal mice.
   Figure [158]7e-f shows that at the phylum level, Bacteroidetes,
   Firmicutes, and Proteobacteria have the highest abundance. In colitis
   mice, Bacteroidetes increased while Firmicutes decreased. The
   Firmicutes/Bacteroidetes ratio (F/B), as an important indicator
   reflecting the degree of intestinal microbiota disorder, decreased
   compared to normal mice. After treatment with GBP@HA NPs, the F/B ratio
   of mice significantly increased, confirming the role of GBP@HA NPs in
   regulating intestinal microbiota and inhibiting intestinal disorders.
   The results of principal coordinate analysis (PCoA) (Fig. [159]7g) were
   consistent with it, and the distance between the control group and the
   model group was the farthest, indicating that the difference between
   the two groups was the greatest. GBP@HA NPs treatment could reduce the
   difference between the colitis mice and the control group, that is,
   close to normal mice. Therefore, GBP@HA NPs can effectively regulate
   intestinal flora and restore intestinal homeostasis.

GBP@HA NPs alleviate chronic UC

   Inspired by the therapeutic effect of GBP@HA NPs on acute UC, we
   further explored its therapeutic effect in DSS induced chronic UC mouse
   model (Fig. [160]8a). As shown in the Fig. [161]8b, the weight of
   chronic UC mice induced by DSS was significantly reduced compared to
   normal mice. After 7 weeks of treatment, the polyphenol group,
   G-BSA-polyphenol group, NPs-Low group, NPs-High group, and 5-ASA group
   all showed higher body weight than UC mice, demonstrating a good weight
   recovery effect. Similarly, each treatment group also demonstrated
   therapeutic effects on colon length reduction caused by colitis, with
   the NPs-High group returning to a level close to that of the normal
   group (Fig. [162]8c-d). As shown in the Fig. [163]8e-f, GBP@HA NPs also
   has a good relief effect on spleen enlargement caused by colitis, and
   the spleen volume and index are significantly reduced. Next, the
   inflammation-related factors MPO, TNF-α, IL-6, IL-1β and IL-10 in the
   colon of mice in each group of the chronic UC model were analyzed. As
   expected, GBP@HA NPs can effectively reduce the increase of MPO, TNF-α,
   IL-6, IL-1β caused by chronic UC, and increase the level of
   anti-inflammatory factor IL-10 (Fig. [164]8g-k).

Fig. 8.

   [165]Fig. 8
   [166]Open in a new tab

   The therapeutic effect of GBP@HA NPs on chronic UC. (a) Schematic
   diagram of experimental time. (b) Changes in body weight of mice. (c)
   Picture of the mouse colon. (d) Mouse colon length. (e) Picture of
   mouse spleen. (f) Mouse spleen index. (g-k) The regulatory effect of
   NPs on inflammatory related factors MPO, TNF-α, IL-6, IL-1β and IL-10
   in colon tissue of chronic UC mice. (n = 6; ^*P < 0.05; ^**P < 0.01;
   ^***P < 0.001; ^****P < 0.0001). All data are presented as mean ± SD

   The histopathological studies conducted in chronic UC models have
   further demonstrated the repair effect of GBP@HA NPs on UC-induced
   colon tissue damage. In addition, we conducted histopathological
   studies. The H&E staining results (Fig. [167]9a) indicate that chronic
   UC can also cause severe damage to colon tissue, causing problems such
   as crypt damage and inflammatory cell infiltration. GBP@HA NPs also
   demonstrate good therapeutic effects and effectively repair damaged
   colon tissue. Meanwhile, the Masson staining results (Fig. [168]9a)
   indicate that GBP@HA NPs can play a significant therapeutic role in
   promoting colon recovery in chronic UC induced collagen deposition and
   fibrosis in the colon. Therefore, GBP@HA NPs can also play an excellent
   role in inhibiting colonic pathological damage caused by colitis and
   repairing the intestinal mucosal barrier in chronic UC.

Fig. 9.

   [169]Fig. 9
   [170]Open in a new tab

   Study on repairing effect of intestinal barrier by GBP@HA NPs. (a) H&E
   staining and Masson staining of colon tissue in each group of
   mice. (Magnification: 20 ×) (b) Immunofluorescence staining and
   quantitative analysis of ZO-1 and Occludin in the colon (blue
   fluorescence: DAPI-stained nucleus; Red fluorescence: ZO-1; Green
   fluorescence: Occludin) (c) Immunofluorescence staining and
   quantitative analysis of Claudin4 and E-Cadherin in the colon (blue
   fluorescence: DAPI-stained nucleus; Red fluorescence: Claudin4; Green
   fluorescence: E-Cadherin). Scale: 400 μm; Magnification: 40 ×

   To investigate whether GBP@HA NPs can alleviate colonic intestinal
   barrier damage caused by chronic UC, we detected tight junction protein
   by immunofluorescence staining. As shown in Fig. [171]9b-c, GBP@HA NPs
   showed a good therapeutic effect on the reduction of tight junction
   proteins ZO-1, Occludin, Claudin4 and E-Cadherin caused by chronic UC,
   and the fluorescence of the four tight junction proteins was
   significantly enhanced, indicating a significant increase in their
   content. The damaged intestinal barrier is repaired. We also studied
   the regulatory effect of GBP@HA NPs on macrophage polarization by
   immunofluorescence staining.

The regulatory effect of GBP@HA NPs on metabolites

   We used UPLC-Q-MS technology to detect endogenous metabolites in mouse
   colon tissue, preliminarily screening differential metabolites related
   to GBP@HA NPs alleviating colitis, and conducting a deeper exploration
   of their therapeutic mechanisms. As shown in Fig. [172]10a-d, the blank
   control group and model group were significantly separated, indicating
   a change in the metabolic level of colonic tissue content in mice after
   modeling. There was no aggregation between the model group and the
   GBP@HA NPs group, indicating a good separation. This indicates that the
   metabolic level of colonic content in mice with colitis also changed
   after treatment with GBP@HA NPs. Figure [173]10e shows that colitis
   causes significant changes in metabolites in mice, while treatment with
   GBP@HA NPs reduces the differential metabolites between colitis mice
   and normal mice. Metabolites such as L-tryptophan, phenylacetylglycine,
   biochanin A, hepoxilin A3 and 5-hydroxyindoleacetic acid in the colon
   of model group mice were significantly increased compared to normal
   mice and L-Asparagine, glycitein, epiandrosterone, and L-Aspartic acid
   metabolites were significantly downregulated (Fig. [174]10f). Further
   pathway enrichment analysis of differential metabolites between the
   GBP@HA NPs group and the model group showed that the amino acid
   metabolism pathway is affected, and amino acids not only provide
   energy, but their metabolites can also participate in important
   physiological activities (Fig. [175]10g-h). Cytoplasmic glutamate is
   crucial for maintaining redox balance and avoiding oxidative stress in
   cells by producing glutathione (GSH). We also found that UC development
   is associated with the peroxisome proliferator-activated receptor
   (PPAR) signaling pathway, a ligand-activated receptor in the nuclear
   hormone receptor family that plays an important role in inflammatory
   responses and binds to the NF-κB subunit that regulates the expression
   of inflammatory factors [[176]67]. Therefore, based on its therapeutic
   efficacy and metabolomics research, a deeper exploration of its
   potential mechanisms has been conducted.

Fig. 10.

   [177]Fig. 10
   [178]Open in a new tab

   (a) PLS-DA score plot between the control group and the model group
   under positive ion mode. (b) PLS-DA score plot between the control
   group and the model group under negative ion mode. (c) PLS-DA score
   plot between GBP@HA NPs group and model group in positive ion mode. (d)
   PLS-DA score plot between GBP@HA NPs group and model group in negative
   ion mode. (e) Differential metabolite statistics. Red: increase the
   number; Blue: Decreased number. (f) Z-score heat map of differential
   metabolites. MetaboAnalyst was used to perform KEGG pathway enrichment
   analysis on differential metabolites between the GBP@HA NPs group and
   the model group. (g) Difference enrichment score chart. (h) Scatter
   plot for differential metabolite enrichment analysis

Potential mechanisms by which GBP@HA NPs alleviate colitis

   Studies have shown that oxidative stress caused by oxidation and
   antioxidant imbalance in the body affects the occurrence and
   development of UC [[179]68]. The Keap1-Nrf2 signaling pathway is a key
   pathway in cellular oxidative stress, and the mRNA transcription and
   expression of Keap1 and Nrf2 in the colon tissues of each group of mice
   were analyzed by qPCR and Western blotting [[180]69, [181]70]. As shown
   in the Fig. [182]11a-b, under oxidative stress, the mRNA levels of
   Keap1 and Nrf2 in the colon tissues of colitis mice were significantly
   increased compared with normal mice, and the treatment groups played a
   regulatory role, so that their relative expression levels were
   significantly reduced. Western blotting’s detection of Keap1 and Nrf2
   proteins also confirmed this result, and the regulation of GBP@HA NPs
   was the most obvious, and the NPs-High group decreased to close to the
   normal group (Fig. [183]11c). Therefore, the powerful regulation of
   Keap1-Nrf2 signaling pathway by GBP@HA NPs is an important way to
   alleviate oxidative stress and exert antioxidant effects.

Fig. 11.

   [184]Fig. 11
   [185]Open in a new tab

   Exploration of the role of GBP@HA NPs in regulating signaling pathways.
   (a-b) RT-qPCR measured the relative expression levels of Keap1 and Nrf2
   mRNA in the colon tissues of mice in each group. (c) Western blotting
   measured the expression of Keap1 and Nrf2 in the colon tissues of each
   group of mice. (d-h) RT-qPCR detected the relative expression levels of
   mRNA in NF-κB p65, TNF-α, IL-1β, IL-6 and IL-10 in the colon tissues of
   each group of mice. (i) Western blotting detected the expression of
   NF-κB p65, TNF-α, IL-1β, IL-6 and IL-10 in the colon tissues of mice in
   each group. (j-l) RT-qPCR was used to detect the relative mRNA
   expression levels of NLRP3, Caspase-1 and GSDMD in colon tissues of
   mice in each group. (m) Western blotting detected the expressions of
   NLRP3, Caspase-1 and GSDMD in colon tissues of mice in each group. (n)
   Immunofluorescence staining of NLRP3, Caspase-1, and GSDMD in the colon
   (Blue fluorescence: DAPI stained nuclei; Red fluorescence: NLRP3; Green
   fluorescence: Caspase-1; Pink fluorescence: GSDMD; Scale: 100 μm;
   Magnification: 40 ×). (n = 3; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001;
   ^****P < 0.0001). All data are presented as mean ± SD

   Studies have found that NF-κB controls many genes related to
   inflammation, so the activation of NF-κB signaling pathway is also
   closely related to the inflammatory response in the colon [[186]71].
   The detection of NF-κB mRNA expression level showed that the expression
   level of NF-κB mRNA in colonic cells was significantly increased when
   colitis occurred, which was about 4 times that of normal mice and the
   G-BSA-polyphenol group and NPs-High group were significantly lower than
   the model group. At the same time, the mRNA expression levels of
   pro-inflammatory factors TNF-α, IL-1β, IL-6 and anti-inflammatory
   factor IL-10 were detected, as shown in the Fig. [187]11d-h, which was
   consistent with the detection results of Elisa. Reduced the increased
   mRNA levels of TNF-α, IL-1β, and IL-6 caused by colitis, and increased
   the expression of anti-inflammatory factor IL-10. In addition, Western
   blotting also presented the expression of four inflammation-related
   factors (Fig. [188]11i). As we imagined, under the regulation of GBP@HA
   NPs, TNF-α, IL-1β, IL-6. The expression levels were significantly
   down-regulated, while the expression level of IL-10 was significantly
   increased. These results suggest that GBP@HA NPs may effectively
   inhibit inflammatory response by regulating NF-κB signaling pathway.

   In order to further explore the regulatory role of GBP@HA NPs on the
   cytopyrosis pathway, we analyzed the genes and proteins associated with
   cytopyrosis. First of all, immunofluorescence staining was applied to
   the colon site NLRP3 (red fluorescence), Caspase-1 (green fluorescence)
   and GSDMD (pink fluorescence) preliminary evaluation. As shown in the
   Fig. [189]11n, under normal state the three proteins are less,
   fluorescence intensity is weak, and the DSS-induced colitis mice colon
   three fluorescence in the colon is significantly enhanced, indicating
   that the three important proteins associated with cell pyroptosis are
   significantly increased, while the fluorescence of the GBP@HA NPs
   treatment group is significantly weakened, suggesting a close
   connection to the cytopyrosis pathway. Next, qPCR analysis results
   showed that the mRNA levels of NLRP3, Caspase-1 and GSDMD were
   consistent with the trend shown by fluorescent staining, and the mRNA
   levels of the three proteins were significantly reduced after each
   group of drug administration (Fig. [190]11j-l). The Western blotting
   band further confirmed this result (Fig. [191]11m). GBP@HA NPs exerted
   a powerful inhibitory effect on the increase of NLRP3, Caspase-1 and
   GSDMD expression in colitis, similar to that of normal mice. In
   summary, GBP@HA NPs can downregulate NLRP3, Caspase-1, and GSDMD, thus
   possessing the potential to regulate the cell pyroptosis pathway and
   alleviate colitis.

Discussion

   UC, as an incurable non-specific gastrointestinal inflammation,
   requires safer and more effective treatment methods. We envisioned
   using polyphenols derived from natural plants for treatment, but due to
   their low stability and bioavailability, we developed the GBP@HA NPs
   system to deliver EGCG and TA to the colon through nanoarmor for
   therapeutic effects on UC. Further, by combining galactosylated BSA
   with polyphenols, the stability of polyphenols is improved, and the
   coating of HA further stabilizes the polyphenols to reach the colon.
   GBP@HA NPs have a hyaluronic acid (HA) coating, which is widely used in
   oral drug delivery systems. This coating can protect them from the
   highly acidic gastric environment and prevent degradation by
   proteolytic enzymes such as pepsin and trypsin. Specifically, the
   carboxyl functional groups of HA remain in an undissociated state under
   acidic conditions and can tightly wrap around the surface of the
   contents. At pH 7.4, carboxyl groups are in the form of carboxylate
   ions, which have a repulsive effect on each other, reducing their
   stability and making it easier to release drugs, which is beneficial
   for protecting polyphenols from reaching the colon through the
   gastrointestinal tract.

   Next, in order to investigate the therapeutic effect of GBP@HA NPs, we
   first conducted preliminary validation through cell experiments. The
   Elisa experiment was used to confirm the in vitro anti-inflammatory
   effect of GBP@HA NPs. Meanwhile, we also investigated the in vitro
   antioxidant activity of GBP@HA NPs using hydrogen peroxide and LPS.
   Macrophage uptake experiments have demonstrated the specific binding
   ability of GBP@HA NPs to inflammatory macrophages.

   Furthermore, the in vivo therapeutic effect of GBP@HA NPs was tested in
   a mouse model of UC. The malondialdehyde (MDA) produced by peroxidized
   lipids can reflect the level of lipid peroxidation. As the first line
   of defense of the biological antioxidant system, SOD is the most
   important member of the antioxidant enzyme system. CAT is the most
   important H[2]O[2] scavenging enzyme and plays an important role in the
   reactive oxygen species scavenging system. GSH-Px is an important
   peroxide decomposition enzyme widely present in the body, which can
   reduce or eliminate the damage caused by peroxide to cells. Therefore,
   we detected MDA, SOD, CAT, and GSH-Px antioxidant enzymes in colon
   tissue, demonstrating the in vivo antioxidant effect of GBP@HA NPs. We
   also analyzed the biodistribution of GBP@HA NPs in mice using IVIS,
   providing stronger evidence of its targeting ability.

   In addition, a key feature of IBD is disruption of the intestinal
   barrier, which allows microorganisms and other antigens to translocate
   to the intestinal wall, leading to uncontrolled immune activation
   [[192]72, [193]73]. Abnormal immune response is an important mechanism
   of colitis, and macrophages, as an important cell population of
   immunity, play an important role in the inflammatory response of body
   tissues. Therefore, drug treatment targeting macrophages has great
   potential. The repair of the intestinal barrier and the regulation of
   macrophage polarization are important mechanisms for GBP@HA NPs
   treatment of UC.

   At present, the important role of gut microbiota has attracted the
   attention of many researchers. Studies have shown that dysregulation of
   the intestinal flora is closely related to the occurrence of colitis.
   Bacteria can produce short chain fatty acids, and short-chain fatty
   acids (SCFAs) is usually reduced in the mucosa and feces of UC patients
   [[194]74]. It is an important metabolic product for maintaining
   intestinal homeostasis, protecting the integrity and permeability of
   the intestinal mucosal barrier, promoting intestinal mucus secretion
   balance and the stability of tight junction expression [[195]75].
   Through analysis of the gut microbiota, we discovered the regulatory
   effect of GBP@HA NPs on the gut microbiota, which is also an important
   reason for its ability to treat UC.

   Finally, we explored the potential molecular mechanisms of GBP@HA NPs
   in treating UC. During the research process, based on the intracellular
   antioxidant activity and metabolomics analysis of GBP@HA NPs, it is
   speculated that GBP@HA NPs may exert therapeutic effects by regulating
   oxidative stress pathways. In the existing studies, NF-κB protein
   exists in almost all animal cell types, When cells are subjected to
   various stimuli, NF-κB homo- or heterodimers can be transferred to the
   nucleus and activate target gene transcription. Activated NF-κB will
   induce a large number of pro-inflammatory cytokines and induce
   inflammation. Cell pyroptosis, as an important natural immune response
   of the body, is increasingly being found to be related to UC. Cell
   pyroptosis relies on inflammatory caspase (mainly caspase-1, 4, 5,
   etc.), which mediates the activation of a variety of Caspase, through
   inflammosomes containing NLRP3, resulting in cleavage and
   polymerization of GSDMD, which can result in cell perforation, which in
   turn causes cell death [[196]76–[197]78]. Among them,
   Caspase-1-mediated classical cell pyroptosis cleaves the precursors of
   IL-1β and IL-18 to form active IL-1β and IL-18, and release them
   extracellularly, recruiting inflammatory cells to aggregate and expand
   the inflammatory response [[198]79, [199]80]. Through methods such as
   RT-qPCR and western blotting, we speculate that GBP@HA NPs can treat UC
   by regulating the Keap1-Nrf2 oxidative stress signaling pathway, NF-κB
   inflammatory signaling pathway, and cell pyroptosis signaling pathway.

   In summary, the GBP@HA NPs we constructed can effectively exert the
   role of nanoarmor, target the delivery of polyphenols to the inflamed
   colon to exert therapeutic effects, and alleviate colon symptoms
   through various mechanisms. It also has good biocompatibility and has
   potential in the treatment of UC and the application of polyphenols.

Conclusion

   In general, we have developed a polyphenol-armored nanodelivery system
   (GBP@HA NPs) to deliver EGCG and TA to sites of colonic inflammation.
   In vitro assays showed that GBP@HA NPs had robust anti-inflammatory,
   antioxidant capabilities, macrophage targeting effects, and superior
   biocompatibility. The therapeutic efficacy of GBP@HA NPs has been
   corroborated in both acute and chronic UC mouse models. This nanoarmor
   have been effective in alleviating UC symptoms, repairing damaged colon
   tissue, regulating macrophage polarization, and restoring intestinal
   homeostasis. The underlying mechanisms of action for GBP@HA NPs may
   involve modulation of the Keap1-Nrf2 signaling pathway, inhibition of
   the NF-κB signaling pathway, and regulation of the Caspase-1-dependent
   pyroptosis pathway. Consequently, this research may offer novel
   insights into the therapeutic application of polyphenols for ulcerative
   colitis management.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [200]Supplementary Material 1^ (2.6MB, docx)

Acknowledgements