Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by recurrent diarrhea and abdominal pain1. The etiology and pathogenesis of UC are complex, characterized by dysregulation of immune response, alterations in colonic barrier function, and substantial dysbiosis of gut microbiota2. Conventional therapies such as 5-aminosalicylic acid (5-ASA), thiopurines, and corticosteroids are frequently employed for the management of mild to moderate UC by reducing inflammation, alleviating symptoms, and maintaining remission3,4. Although 5-ASA is considered the first-line therapy for UC, a subset of patients may experience intolerable adverse effects, resulting in treatment discontinuation and an elevated risk of undergoing colectomy5.

Recently, with the deepening of research into the pathogenesis of UC, molecular targeted therapies have gradually emerged. The excessive production of proinflammatory cytokines resulting from extensive infiltration of immune cells represents a pivotal pathological factor in UC, thereby rendering Janus kinases (JAKs) crucial therapeutic targets. Tofacitinib (Tof), an oral pan-JAK inhibitor primarily targeting JAK1 and JAK3, has demonstrated efficacy in inducing remission in active cases of UC6. However, concerns arise due to its extensive systemic distribution, which often leads to suboptimal drug concentrations within the colon, resulting in inadequate anti-inflammatory effects. Additionally, this distribution may have potential off-target consequences such as infections, cancers, and lymphoma7,8.

To address these challenges, oral colon-targeted drug delivery systems (OCDDSs) have been developed with the aim of specifically releasing medication in the colon. This ensures that therapeutic agents are concentrated at the site of disease while minimizing systemic exposure and reducing potential side effects. It is imperative to develop OCDDSs for Tof to enhance its therapeutic efficacy and minimize potential adverse effects, thereby effectively addressing the intricate challenges associated with the treatment of UC9. In our previous study, the valuable polysaccharides, such as chondroitin sulfate (Chs)10, chitosan (Csn)11, and pectin (Pcn)12, have been employed in OCDDSs due to their biodegradability, biocompatibility, and specific targeting capabilities.

In this study, we developed an innovative OCDDSs by desolvating Tofacitinib and encapsulating it within a bovine serum albumin (BSA) core. Chs was then introduced for surface modification, and further cross-linking was achieved through layer-by-layer (LBL) technology using a prebiotic shell composed of Csn and Pcn. The synthesized OCDDSs exhibits excellent pH-dependent and colon-targeting properties, offering promising prospects for clinical applications in UC treatment (Fig. 1).

Fig. 1
figure 1

Schematic illustration of the Tof@BSA-Chs-CP NPs. The Pcn/Csn shell offers pH-dependent release protection in the upper GIT. The pectinase from gut microbiota triggers OCDDSs degradation and further regulates the gut microbiota ecosystem.

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Materials and methods

Materials

Chs was sourced from Solarbio (Beijing, China). Csn (Mw: 140–190 kDa) was obtained from Aladdin (Shanghai, China). 1-Ethyl-(3-Dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (EDCI), N-Hydroxysuccinimide (NHS), Tof, Glutaraldehyde (GA), and Pcn (Mw: 60–100 kDa) with a galactose uronic acid content of ≥ 74% were purchased from Macklin (Shanghai, China). BSA was obtained from Yuanye (Shanghai, China).

Preparation and characterization of Tof@BSA-Chs-CP NPs

Tof@BSA NPs were synthesized via the desolvation method by adding a Tof ethanol solution (3 mg/mL) to a BSA solution (20 mg/mL), followed by 1.6 µL of 50% glutaraldehyde. The mixture was stirred for 2 h, centrifuged, and washed with ddH2O. Next, Tof@BSA-Chs was prepared using EDC/NHS coupling by mixing Tof@BSA NPs with an activated Chs solution (1 mg/mL) for 12 h, then centrifuged and washed. For Tof@BSA-Chs-CP NPs fabrication, a layer-by-layer (LBL) method was used. Csn solution (1% w/v in acetic acid) was gradually added to Tof@BSA-Chs, stirred for 1 h, centrifuged, and washed. Finally, a Pcn solution was added with stirring, washed, and freeze-dried. To track the nanoparticles in experiments, Rhodamine B isothiocyanate (RBITC)-labeled Csn was synthesized based on previous study13.

Characterize the NPs using a transmission electron microscope (TEM, JEM-1200 EX, Tokyo, Japan) at 100 kV. Measure particle size and Zeta potential using dynamic light scattering (DLS) and electrophoretic light scattering (ELS) on a Zetasizer Nano ZS (Malvern Instruments, UK). Analyze chemical interactions via Alpha Fourier-transform infrared (FT-IR) spectroscopy (Bruker, USA) using the KBr press disk method within the 4000 –400 cm− 1 frequency range.

Encapsulation efficiency (EE) and drug loading (LC) capacity

EE and LC were quantified using a high-performance liquid chromatography (HPLC) system with a C18 column (4.6 × 150 mm, 5 μm). The mobile phase comprised a 65:35 volume ratio of phosphoric acid buffer and acetonitrile. The flow rate was set at 1.0 mL/min, and the detection wavelength was 287 nm. A 20 µL sample volume was utilized for the drug analysis. The formulas for EE and LC are as follows:

$${\text{EE}}\:\% \:{\text{ = }}\:\frac{{{\text{Total}}\:{\text{Tof}}\:{\text{weight}}\:{\text{ - }}\:{\text{Free}}\:{\text{Tof}}\:{\text{weight}}\:}}{{{\text{Total}}\:{\text{Tof}}\:{\text{weight}}}}\: \times \:{\text{100}}\:(\% )$$
$${\text{LC}}\:\% \:{\text{ = }}\:\frac{{{\text{Total}}\:{\text{NPs}}\:{\text{weight}}\:{\text{ - }}\:{\text{Free}}\:{\text{Tof}}\:{\text{weight}}}}{{{\text{Total}}\:{\text{NPs}}\:{\text{weight}}}}\: \times \:{\text{100}}\:(\% )$$

In vitro drug release

To evaluate the release rate of NPs in the gastrointestinal tract (GIT), we utilized three distinct pH levels to mimic the environments of the stomach (pH 1.2), small intestine (pH 6.8), and colon (pH 7.4), following the method described in our previous study13. To emphasize the enzyme sensitivity of the NPs, we conducted experiments using solutions containing either a fecal microbiota suspension (50 mg/mL) or a pectinase solution (3750 U/mL). We collected 50 mg fresh fecal of the mice. Then we ground it with 1 ml PBS solution whose pH is 7.4 and we got the bacterial solution with the concentration of 50 mg/mL finally. To determine the in vitro release kinetics of NPs, we precisely weighed 5.0 mg of NPs and placed them into a dialysis bag containing 5 mL of PBS. The sealed bag was then submerged in 200 mL of PBS. At designated intervals (0, 0.5, 1, 2, 3, 4, 6, 8, and 12 h), 5 mL samples were collected from the medium and replaced with an equivalent fresh PBS. Standard curves were established to quantify the concentrations of Tof in the aqueous phase, using a UV-visible spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) at 287 nm. The absorbance of these samples at this wavelength was measured with the same instrument, facilitating the calculation of cumulative drug release. The formulas for cumulative drug release are as follows:

$${\text{The}}\:{\text{cumulative}}\:{\text{drug}}\:{\text{release}}\:\% {\text{ = }}\frac{{{\text{Cumulative}}\:{\text{drug}}\:{\text{release}}\:}}{{{\text{The}}\:{\text{total}}\:{\text{amount}}\:{\text{of}}\:{\text{particle}}\:{\text{loading}}\:\left( {{\text{mg}}} \right)}}\: \times \:{\text{100}}\:\left( \% \right)\:$$

Cell viability and cellular uptake of NPs in Caco-2 cells

Caco-2 cells were cultured in an atmosphere humidified with 5% CO2. The cytotoxicity of NPs was assessed using the Cell Counting Kit-8 (CCK-8) assay. Fluorescence microscopy was used to observe and analyze cellular uptake after specific cultivation periods, utilizing RBITC-labeled NPs. DAPI staining was conducted to detect fluorescence signals of cell nucleus.

Animals and DSS-induced colitis model

Animal protocols were reviewed and approved by the Ethics Committee of the Medical College of Qingdao University (QDU-AEC-2024459), in line with the Guide for ARRIVE to decrease animal numbers and suffering as much as possible. All methods were carried out by relevant regulations and ARRIVE guidelines. C57BL/6J mice (6 weeks old, male, 18–22 g) were obtained from Pengyue Experimental Animal Breeding Co., LTD (Jinan, China). The study was performed on the efficacy of NPs in the treatment of colitis. After a one-week acclimation period, the mice were randomly divided into five groups: (1) NC group: no treatment for two weeks; (2) DSS group: 2.5% DSS for one week and no treatment for additional week; (3) Tof group: 2.5% DSS for one week and 20 mg/kg/day Tof treatment for one week; (4) @BSA-Chs-CP group: 2.5% DSS for one week and 20 mg/kg/day @BSA-Chs-CP treatment for one week; (5) NPs group: 2.5% DSS for one week and NPs (containing 20 mg/kg/day Tof) for one week. Colitis was induced by allowing the mice to drink water containing 2.5% DSS freely for one week.

The body weight of each mouse was recorded daily, and doses for the NPs were adjusted based on body weight changes. The Disease Activity Index (DAI) score was calculated by considering changes in body weight, stool consistency, and the presence of blood in the stool to assess the severity of intestinal inflammation. After anesthetizing the mice, blood samples were collected. The colon was then dissected for length measurement and histological analysis.

Adhesion experiment

After the colitis model was established, we monitored the distribution of RBITC-labeled NPs in the colonic tissues of the NC and DSS-treated mice. This observation occurred over a 12-hour period following oral administration. After the mice were euthanized, frozen sections of the colitis tissues were prepared, stained with DAPI, and examined using a fluorescence microscope.

Histological evaluation

The histopathological changes in colon tissues were analyzed by hematoxylin and eosin (H&E) staining and mucin expression was assessed using alcian blue staining. The detailed methods referred to in our previous study14.

Flow cytometry analysis

Cell suspensions were obtained from the spleen, followed by lysis of red blood cells, and resuspension of cells in PBS post-lysis. Cell counts were conducted before Th17 staining, and subsequently, the cells were stimulated using a PMA mixture for 6 h. Treg cells and Th17 cells were stained according to our previous study15. After staining, the cells were detected using flow cytometry (BD FACSVerseTM, NJ, USA).

Measurement of inflammatory cytokines in serum

Blood samples were centrifuged at 3,000 RPM for 15 min to obtain the serum samples. According to instructions, inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) were measured using ELISA kits (Abclonal, Wuhan, China).

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.3. Comparisons between two groups were made using Student’s t-test, while one-way ANOVA was used for comparisons among multiple groups. Protein-protein interaction (PPI) network analysis and KEGG pathway enrichment analysis were also utilized to explore the relationships and functional pathways. A p-value of < 0.05 was considered statistically significant.

Results

Preparation and characterization of Tof@BSA-Chs-CP NPs

The preparation scheme of Tof@BSA-Chs-CP NPs is shown in Fig. 2A. Briefly, Tof was encapsulated within the BSA core using the desolvation method. Subsequently, it underwent Chs modification and was further crosslinked with Csn and Pcn via LBL technology, resulting in the formation of Tof@BSA-Chs-CP NPs. The morphological characteristics of the Tof@BSA core, Tof@BSA-Csn, and Tof@BSA-Chs-CP NPs were analyzed by TEM (Fig. 2B), which revealed that the Tof@BSA-Chs-CP NPs were roughly spherical, uniformly sized nanoparticles with an average particle size of 300 nm. The particle size and zeta potential were measured by DLS (Fig. 2C, D and Fig. S1A). The Tof@BSA exhibited a size of 183.9 ± 1.2 nm, subsequent to the deposition of Chs in the initial polyelectrolyte layer, there was an increase in particle size to 264.7 ± 2.7 nm. Further augmentation in particle size to 287.6 ± 1.2 nm was observed upon the addition of the second polyelectrolyte layer containing Csn. Finally, with the incorporation of Pcn in the third polyelectrolyte layer, the particle size increased to 308.5 ± 4.8 nm. As shown in Fig. 2D, the zeta potential of NPs was observed to vary in accordance with the preparation protocol, resulting in a final zeta potential of -22.4 ± 0.7 mV for the final NPs. The significant positive and negative shifts in zeta potential confirmed the successful incorporation of Csn and Pcn into Tof@BSA-Chs via LBL electrostatic self-assembly. Furthermore, the PDI for NPs was calculated to be 0.065 ± 0.028, similar to the results observed under the TEM, the PDI results showed that the distribution of NPs was uniform. The extracted Tof from the Tofa-TK-HSA NPs was quantified via HPLC. LE and LC of Tofa in Tof@BSA-Chs-CP were determined as 54.9% and 30.9%, respectively (Fig. S1B). As shown in Fig. 2E and Fig. S2, the FT-IR spectra of Pcn exhibited a broad band at 3100–3500 cm− 1, attributed to O-H stretching, a band at 2941.20 cm− 1 corresponding to C-H stretching, 1746.89 cm− 1 for COOR stretching, 943.04 cm− 1 for O-H out-of-plane bending, and 1627.17 cm− 1 for COO− stretching. Additionally, compared with blank Csn, Chs, and Pcn, the absence of their respective characteristic peaks indicates the successful synthesis of Tof@BSA-Chs-CP NPs, confirming the effective reaction between the amine group in Csn and the carboxyl group in Pcn.

The EE and LE of generated NPs were 49.7% and 30.56%, respectively. As shown in Fig. 2F, Tof has the highest release rate at pH 7.4; only 26.30% of Tof was released at pH 1.2 within 12 h. The strong electrostatic interaction between the carboxyl group of Pcn and the amino group of Csn causes NPs to shrink at low pH, providing Tof in the GIT and the stomach environment. Meanwhile, the release of Tof from NPs mixed with bacterial solution (50 mg/mL) or pectinase (3750 U/mL) exhibited a significant increase within 8 h, reaching about 88.04% and 83.10% (Fig. 2G). The NPs exhibit more excellent responsiveness to enzymes compared to the 58% release amount at 8 h in the blank group. These results indicated that NPs are pH sensitive and responsive to gut microbiota-produced pectinase for controlled release.

Fig. 2
figure 2

The preparation and characterization of Tof@BSA-Chs-CP NPs. (A) Preparation scheme of NPs. (B) TEM images of Tof@BSA core, Tof@BSA-Chs-Csn and Tof@BSA-Chs-CP NPs. Size distribution profiles (C) and Zeta potentials (D) of NPs. (E) FT-IR of Tof@BSA-Chs-CP NPs. (F) Release curves of NPs at different pH conditions. (G) Release curves of NPs with a bacterial solution or pectinase in the simulated digestive system. Experiments were performed in triplicate and repeated three times. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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The uptake efficiency of Tof@BSA-Chs-CP NPsIn vitro and In vivo

To determine the cellular uptake of synthesized OCDDSs in vitro, we synthesize Tof@BSA-Chs-CP NPs tagged with RBITC and co-incubate them with intestinal epithelial cells (Caco-2 cells) for co-localization analysis using blue fluorescence (DAPI) and red fluorescence (RBITC). As shown in Fig. 3A, the uptake efficiency of Caco-2 cells gradually increases over time. The RBITC-labeled NPs efficiently enter into Caco-2 cells and accumulate in significant quantities near the nucleus. These findings indicate that NPs demonstrate outstanding in vitro biocompatibility and capable of being taken up by intestinal epithelial cells. Colitis has the potential to disrupt the gut barrier, and the increased permeability of inflamed tissues allows nanoscale drug delivery systems to remain in the colon longer, leading to selective accumulation of NPs in inflamed tissues. After 12 h treatment, we evaluate the specific attachment and accumulation of RBITC-labeled NPs in both healthy and colitic tissues (Fig. 3B). We found that healthy colon tissues retained significantly fewer NPs at 12 h compared to colitic tissues. This indicates that NPs face the challenge of penetrating the intact structure of healthy intestinal tissues.

Moreover, substantial amounts of RBITC-labeled NPs accumulated and were internalized by epithelial cells, penetrating deeply into the mucosa. Furthermore, biocompatibility is key in evaluating NPs for UC treatment. The CCK-8 assay showed no significant cytotoxicity in Caco-2 cells (Fig. S3). These findings indicate that NPs exhibit excellent uptake efficiency in vivo and in vitro, supporting their efficient and safe use as a drug delivery vehicle. Thus, NPs proved to be an effective delivery system capable of penetrating colitis tissue and facilitating the internalization of Tof into target cells, thereby alleviating DSS-induced colitis.

Fig. 3
figure 3

In vitro Cellular Uptake and Tissue-Specific Uptake Efficiency of Tof@BSA-Chs-CP NPs in Colitis Models. (A) Cellular uptake of NPs by Caco-2 cells. (B) Fluorescence microscopy profile of healthy colonic tissue or colitis tissue. Scale bars represent 50 μm. DAPI for nuclear staining; red, RBITC-labeled NPs.

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Tof@BSA-Chs-CP NPs alleviated DSS-induced colitis in mice

To evaluate the therapeutic effects of NPs on colitis, we established a DSS-induced colitis model (Fig. 4A). Compared to the NC group, DSS treatment significantly reduced the body weight of mice. In contrast NPs treatment reversed this weight loss trend (Fig. 4B). Colon length is a critical indicator of colitis severity, so we measured the colon lengths in different groups (Fig. 4C). The results indicated that NPs could prevent the shortening of the colon induced by DSS. As shown in Fig. 4D, the result showed a significant increase in the DAI score for the DSS group, while the NPs-treated group exhibited a significant reduction, consistent with the changes in body weight. Thus, NPs demonstrated significant efficacy in alleviating DSS-induced colitis and improving the quality of life in mice. Meanwhile, tof and @BSA-Chs-CP nanoparticles alone had no significant therapeutic effect, suggesting that nanoparticles infused with Tof (20 mg/kg/day Tof treatment) require the Pcn/Csn shell to function. To further verify the protective effects of NPs on colon tissue, histological evaluations were conducted using H&E and Alcian blue staining (Fig. 4E). Histological analysis revealed severe inflammatory cell infiltration, gland destruction, goblet cell depletion, and mucosal epithelial necrosis in the DSS group compared to the NC group. NPs treatment markedly alleviated these pathological damages. These findings suggest that NPs can mitigate the histological damage in the colons of DSS-induced colitis mice.

The balance between pro-inflammatory and anti-inflammatory cytokines is crucial for regulating the intestinal inflammatory environment, affecting the progression of UC16. To confirm the anti-inflammatory effects of NPs, we measured the concentrations of pro-inflammatory and anti-inflammatory cytokines in the serum (Fig. 4F-I). The results showed that the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) were significantly elevated in the DSS group. After NPs treatment, the concentrations of these pro-inflammatory cytokines were significantly reduced (P < 0.001), while the levels of the anti-inflammatory cytokine IL-10 were significantly increased (P < 0.001). These results indicate that NPs can significantly decrease the expression of pro-inflammatory cytokines and increase the expression of anti-inflammatory cytokines in DSS-induced colitis mice. This is closely related to the competitive binding of Tof through the ATP binding site of JAK kinase, resulting in the reduction of the production of inflammatory factors. Additionally, the degraded and utilized prebiotic shell bacteria play a role in regulating the body’s inflammatory response. In conclusion, NPs reduce inflammation in colon tissues and alleviate systemic inflammatory responses, significantly improving the symptoms and overall health status in the DSS-induced colitis model.

Fig. 4
figure 4

Tof@BSA-Chs-CP NPs alleviated pathological lesions of DSS-induced colitis. (A) Experimental design showing treatment protocols for each group: NC (no treatment), DSS (DSS-induced colitis without therapy), Tof (free Tof treatment), @BSA-Chs-CP (@BSA-Chs-CP treatment), and NPs (Tof@BSA-Chs-CP NPs treatment). The body weight (B) and DAI scores (D) change during the experiment. (C) The statistical analysis for colon length. (E) The colon sections were stained with H&E and alcian blue. Plotting scale = 100 μm. The concentrations of TNF-α (F), IL-1β (G), IL-6 (H) and IL-10 (I) in serum. n = 6. NC compared with the DSS group, #P < 0.05, ##P < 0.01, ###P < 0.001. NPs compare with DSS group, *P < 0.05, **P < 0.01; ***P < 0.001.

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Tof@BSA-Chs-CP NPs restore Treg/Th17 balance

Th17 and Treg cells are related in differentiation and inhibit each other in function, jointly maintaining the balance of the body’s immune microenvironment17,18. Various autoimmune diseases including UC, are closely related to the disruption of Treg/Th17 balance, which has been observed in both human and mouse models of UC19,20. To investigate the impact of NPs on the balance of Treg and Th17 cells, we conducted a flow cytometric analysis on the spleens of mice treated with oral NPs (Fig. 5A–D). The results demonstrated that NPs increased the number of Treg cells while reducing the number of Th17 cells in the spleens of mice with DSS-induced colitis. As shown in Fig. 5E and F, the result illustrated the relative changes in IL-17 A and IL-10 gene expression, respectively. Our findings indicate that NPs effectively decrease IL-17 A expression and significantly enhance IL-10 expression, contributing to their anti-inflammatory effects. Figure 5G highlights the influence of nanoparticle treatment on the IL-17 and IL-10 signaling pathways using protein-protein interaction (PPI) network analysis. Further enrichment analysis of the KEGG pathway was performed for the above proteins, mainly focusing on the cytokine -cytokine receptor interaction, JAK-STAT signaling pathway and IL-17 signaling pathways, further confirming its anti-inflammatory and immunomodulatory roles. These results suggest that NPs facilitate the rebalancing of the Treg/Th17 cell population and promote immune homeostasis. In conclusion, NPs can restore immune homeostasis in DSS-induced colitis models by rebalancing the Treg/Th17 cell population.

Fig. 5
figure 5

Effect of Tof@BSA-Chs-CP NPs on Treg/Th17 cell balance in colitis tissue. Representative flow cytometry images of Th17 (CD4+) (A) and Treg (CD25+) cells (C) in three groups. Percentage of Th17 cells (B) and Treg cells (D). The relative changes of IL-17 A (E) and IL-10 (F) gene expression. (G) Network map of genes and signaling pathways associated with IL-17 and IL-10. NC compare with the DSS group, #P < 0.05, ##P < 0.01, ###P < 0.001. NPs compare with DSS group, *P < 0.05, **P < 0.01; ***P < 0.001.

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Discussion

The therapeutic approaches for UC are continuously enhanced with an improved understanding of the underlying pathophysiology21. The JAK inhibitor, Tof, widely employed in clinical practice, has gained approval for the treatment of moderately to severely active UC. Although it exhibits rapid onset, high efficacy, and non-immunogenicity, its poor targeting may give rise to various adverse events such as infection, hypercholesterolemia, venous thromboembolism, malignancy, among others22,23,24,25. This study demonstrates the robust biodistribution and accumulation of resulting NPs in colon tissue, rapid cellular uptake, and efficient therapeutic efficacy in a mouse colitis model. The development of this colon-targeted OCDDSs can effectively mitigate adverse events resulting from increased drug concentration, addressing the challenge of low therapeutic target drug concentration in Tof treatment for UC.

OCDDSs provide an effective therapeutic method for targeted treatment of UC with Tof, avoiding its systemic absorption and potential side effects26. However, there are challenges in drug delivery targeting the colon region, including pH changes in the gastrointestinal tract and how to stimulate nanoparticles for drug release27. In this study, LBL technology was used to crosslink the negatively charged Tof@BSA modified by Chs with positively charged Csn and negatively charged Pcn. The strong electrostatic interaction between the carboxyl group of Pcn and the amino group of Csn leads to the contraction of NPs at low pH, so that NPs can smoothly pass through the changes in gastrointestinal pH (Fig. 2F). Furthermore, since both Csn and Pcn as prebiotics lacking relevant degradation enzymes within the human body28, they remain intact without degradation. However, the gut microbes in the colon can secrete relevant degrading enzymes to degrade Pcn and Csn, thereby further releasing Tof encapsulated within nanoparticles (Fig. 2G). As prebiotics, the Csn/Pcn shells can also treat UC by improving the composition of the microbiota, thereby enhancing the therapeutic effect of NPs on UC29,30. Additionally, through the modulation of gut microbiota, the NPs possess the capacity to exert influence over the composition and functionality of microorganisms residing within our physiological systems. This attribute proves particularly advantageous in scenarios where specific bacteria or fungi contribute to disease progression or impede treatment efficacy.

Meanwhile, Th17/Treg cells balance plays a crucial role in the pathogenesis of UC31. Commonly, Th17 cells secrete pro-inflammatory factors to maintain host immunity, while Treg cells secrete anti-inflammatory factors to inhibit the excessive activation of Th17 cells and maintain the balance of immune function31. During the progression of UC, there is a severe imbalance between Th17/Treg cells, which leads to the disorder of the immune system and the persistence of inflammation32. Both flow cytometry analysis and PPI network analysis reveal the NPs treatment effectively restores the dysregulated Treg/Th17 cell population induced by DSS through modulation of IL-17 and IL-10 signaling pathway, thereby exerting potent anti-inflammatory and immune regulatory effects (Fig. 5). Collectively, our synthetic Tof@BSA-Chs-CP NPs present a new and versatile approach for the treatment of UC.

However, despite the promising therapeutic efficacy demonstrated, there are important limitations that should be addressed in future research. A critical aspect that remains to be fully investigated is the biodistribution of tofacitinib in key organs such as the heart, brain, liver, and lungs. Understanding the organ-specific distribution of tofacitinib is crucial for assessing potential off-target effects and systemic exposure. Further studies using advanced imaging techniques and organ-specific biodistribution assays are needed to provide a comprehensive profile of tofacitinib’s pharmacokinetics and evaluate any unintended side effects that may arise from its presence in these vital organs. Additionally, the long-term safety of the NPs, including potential toxicity or accumulation in non-target organs, warrants further exploration.

Conclusion

In this study, based on the current situation of dose-dependent systemic side effects of tofacitinib, we successfully prepared Tof@BSA-Chs-CP NPs, an efficient oral colon targeted drug delivery system. Our results show that the Csn/Pcn shell has pH sensitive properties, can pass through a strong acid environment, and can be degraded by polysaccharide degrading enzymes secreted by the gut microbiota, so that Tof can be released in the colon. Specifically, DAI index in the NPs group decreased by approximately 38.89% compared to the DSS group. In conclusion, this OCDDSs can effectively avoid the side effects of Tof, but also has better therapeutic effect on UC due to its prebiotic shell. Future research should focus on in vivo efficacy and safety evaluations, as well as exploring potential synergies with other therapeutic agents. This system holds promise for advancing the treatment of UC with improved safety and efficacy.