An opposite pH-responsiveness “gating” strategy: Intelligent sporopollenin exine armor for targeted therapy of colitis

Abstract

Conventional therapy for ulcerative colitis (UC) is often limited by insufficient colonic targeting, short local retention and poor cellular drug uptake at lesion sites. Here, we develop a biomimetic colon-targeted delivery system based on an opposite pH-responsive “gating” strategy. Chrysanthemum sporopollenin (spo) microcapsules with a characteristic spiny architecture serve as the core carrier. Spo exhibits acid-induced contraction and alkali-induced expansion, with germinal apertures opening progressively as pH increases. In contrast, chitosan-butyrate complex (CBC) swells into a gel under acidic conditions but contracts and precipitates in alkaline environments. After drug loading into the spo, surface coating with CBC seals the germinal apertures, constructing an intelligent gate. In gastric fluid, the CBC layer gels and blocks apertures to prevent premature drug release. In intestinal fluid, CBC contracts and precipitates to open the gate; meanwhile, spo expands to further widen germinal apertures and facilitate drug release. The spiny morphology of spo, combined with the mucoadhesive properties of chitosan and active targeting of butyrate, collectively enhances intestinal adhesion and retention, enabling precise colonic drug release and accumulation. Mesalazine is formulated into liposomes to improve aqueous solubility and stability, which enhances cellular uptake and bioavailability, thereby exerting synergistic anti-inflammatory effects with butyric acid at inflamed sites. The chrysanthemum sporopollenin-based gated microcapsules exhibit favorable pH-responsive release, enhanced mucoadhesion and potent synergistic anti-inflammatory activity. This work provides a promising multifunctional targeted delivery strategy for UC therapy and establishes a novel, versatile design concept termed the opposite pH-responsive dual-gating mechanism, which supports the development of oral colon-targeted carriers capable of navigating complex gastrointestinal environments.

Graphical abstract

1. Introduction

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease with rising global incidence, representing a major public health concern [[1], [2], [3]]. A central challenge in current treatment lies in achieving precise delivery and efficient release of therapeutics at colonic lesions [4,5]. Although 5-aminosalicylic acid (5-ASA) is a first-line agent for mild-to-moderate UC [6], its instability and susceptibility to degradation in gastric acid severely limit therapeutic efficacy [7]. To address this limitation, colon-targeted strategies including pH-dependent [8], time-controlled [9] and enzyme-triggered [10] systems have been developed [[11], [12], [13], [14]]. However, their performance remains compromised by interindividual variability in gastrointestinal physiology and the complexity of gut microbiota.

In recent years, natural biomaterials have emerged as promising candidates for constructing intelligent delivery systems. Sporopollenin (spo) microcapsules possess robust mechanical strength, acid resistance and favorable bioadhesion [[15], [16], [17], [18], [19]]. Chitosan (CS) forms gels in gastric acid to protect loaded drugs and enhances mucosal adhesion in the intestine [20]. Butyric acid (BA) functions as an energy source for colon epithelial cells and exerts anti-inflammatory and intestinal barrier-repairing effects [[21], [22], [23]]. This study integrates the advantages of these materials to develop a novel oral colon-targeted delivery system based on an opposite pH-responsive gating strategy.

The system adopts a sophisticated multilayered architecture with synergistic functions for each layer. The innermost core consists of 5-ASA-loaded liposomes (5-ASA@L), which improve encapsulation efficiency and promote intestinal epithelial cell uptake via favorable biomembrane compatibility, laying a foundation for targeted delivery and efficient therapeutic utilization [24]. The second layer is a protective sustained-release carrier composed of spo microcapsules encapsulating 5-ASA@L. As a natural polymeric material, spo exhibits excellent mechanical strength and acid resistance. Under acidic conditions, germinal apertures contract slightly to protect drugs from degradation and premature release in the stomach and small intestine, ensuring safe delivery to the colon. Spo also displays inherent bioadhesion that prolongs intestinal residence, and aperture expansion under neutral conditions enables sustained drug release in the colonic microenvironment. The third layer is an adhesive gating layer formed by a CS-butyrate composite. In gastric acid, CS swells and gels to seal spo germinal apertures and prevent early drug leakage. Upon entry into near-neutral intestinal fluid, the gate opens. Strong mucosal adhesion of CBC synergizes with spo swelling and drug release to prolong formulation residence in the colon, enhancing overall gastrointestinal adhesion and extending the time window for colonic drug accumulation. BA in the composite layer plays dual roles: it serves as the preferred energy source for colonocytes and exerts anti-inflammatory and barrier-repairing effects that synergize with 5-ASA.

In vitro studies evaluate release profiles in simulated gastrointestinal environments. Cell-based models assess adhesiveness, cellular uptake, and biosafety. Comprehensive in vivo evaluations are performed in dextran sodium sulfate (DSS)-induced colitis rats, with measurements of disease activity index, histopathological changes, inflammatory cytokine levels, and drug distribution to verify targeting efficiency, bioavailability, and therapeutic efficacy. This study provides a promising novel delivery strategy for UC therapy and offers a theoretical reference and practical paradigm for multifunctional integration and synergistic design of complex oral delivery systems.

The preparation and proposed in vivo mechanism of the pH-responsive gated oral delivery system based on spiny spo microcapsules were illustrated in Fig. 1. Drugs remain protected within the formulation in acidic gastric fluid. Upon entry into the intestine, spo microcapsules adhere to intestinal villi via their unique spiny structure and natural bioadhesion, prolonging residence and facilitating sustained release. The CBC layer acts as a gatekeeper in gastric fluid to prevent premature release and enhances mucoadhesion in the intestine to extend epithelial contact. Butyrate is incorporated as a targeting moiety; as the preferred energy source for colonocytes, it undergoes active transport and targets inflamed sites through aryl hydrocarbon receptor activation. It also inhibits NLRP3 inflammasome activation, promotes intestinal tight junction repair, and alleviates oxidative stress, thereby synergizing with 5-ASA.

Fig. 1.

Schematic diagram of the preparation process and mechanism of action for spo-CBC-5-ASA@L. Schematic illustration of the preparation and in vivo action mechanism of the oral delivery system spo-CBC-5-ASA@L in response to gastrointestinal pH changes. After oral administration, the formulation remains protected in the stomach where the germinal apertures contract and the CBC gating layer closes. In the intestinal tract, the apertures open, the CBC gate is activated, and drug release is initiated. The spo microcapsules adhere to and embed within the intestinal villi, prolonging residence time to enable sustained drug release. At the inflammatory site, the CBC layer further enhances mucoadhesion and exerts synergistic anti-inflammatory effects.

2. Materials and methods

2.1. Materials

Reagents: 5-ASA (Hubei Xingyinhe Chemical Co., Ltd., Wuhan, China); BA (Beijing GLS Bioengineering Technology Co., Ltd., Beijing, China); CS (Jiangsu Kangting Biotechnology Co., Ltd., Yancheng, China); Acrylic resin (Colorcon, Shanghai, China); Phosphoric acid (Chengdu Kelong Chemical Co., Ltd., Qionglai, China); Ethanol and acetone (Tianjin Fuyu Reagent Factory, Tianjin, China); Hydrochloric acid and sodium hydroxide (Tianjin Hengxing Reagent Factory, Tianjin, China); Chrysanthemum pollen (from the North Tianshan Mountains, Tacheng, Xinjiang, China); Soybean lecithin (Will Pharmaceutical Group Co., Ltd., Nanjing, China); Absolute ethanol, acetone, concentrated hydrochloric acid (China Fuyu Fine Chemical Co., Ltd., Songyuan, China); Methanol (Anhui Shilian Special Solvent Co., Ltd., Anhui, China). Indocyanine green (ICG, self-made); Phosphate-buffered saline (PBS, Tianjin Haoyang Huake Biotechnology Co., Ltd., Tianjin, China).

Animals: The animals used were Sprague Dawley (SD) rats (200–220 g), Male C57BL/6 J mice (18–22 g), and Kunming (KM) mice (18–22 g). All animal procedures were performed under the approval of the SYPU-IACUC (No. SYPU-IACUC-2024-0311-015) and complied with institutional guidelines for animal welfare.

2.2. Preparation of spo

Five grams of chrysanthemum pollen agglomerates were weighed and dispersed in of 50 mL purified water. The mixture was passed through an 80-mesh sieve to remove impurities, followed by vacuum filtration and washing. The collected material was redispersed in 50 mL purified water, and washing was repeated until the filtrate was nearly colorless. The material was sequentially washed with 50% ethanol, 75% ethanol and absolute ethanol, with each step repeated until the filtrate was essentially colorless. The product was dried in an oven at 40 °C for 4 h to yield defatted chrysanthemum pollen. One gram of defatted pollen was suspended in 10 ml 85% (v/v) phosphoric acid in a 25 ml single-neck flask equipped with a condenser. The mixture was stirred under reflux at 50 °C for 12 h. The viscous spo mixture was collected, filtered and washed repeatedly with purified water until neutral. The washed spo was spread in a clean glass dish and dried at 40 °C for 4 h. Dried spo was stored at room temperature.

2.3. Preparation of 5-ASA@L

5-ASA, phospholipid and cholesterol were weighed at a mass ratio of 1:15:1 and dissolved in absolute ethanol. Organic solvent was completely removed using a rotary evaporator at 40 °C to form a uniform lipid film on the inner wall of the flask. Purified water was added, and the mixture was hydrated under stirring at 60 °C for 15 min to obtain a crude liposome suspension. The suspension was probe-sonicated in an ice bath at 300 W with 5 s on and 5 s off cycles for 5 min to produce a milky semitransparent liposome dispersion. Liposomes and free drug were separated by high-speed centrifugation. Mesalazine content was determined by high-performance liquid chromatography (HPLC) to calculate encapsulation efficiency.

2.4. Preparation of spo-CBC-5-ASA@L samples

Prepared 5-ASA@L dispersion was mixed with blank spo microcapsules under stirring. The mixture undergoes vacuum-assisted drug loading in a vacuum drying oven, followed by three washes with purified water. The sample was dried in a vacuum drying chamber at 40 °C for 2 h to obtain drug-loaded spo microcapsules (spo-5-ASA@L). A portion of the spo-5-ASA@L microcapsules was weighed. CS, BA and purified water were weighed at a mass ratio of 1:1:20. CS and BA were dissolved in purified water under magnetic stirring to form a homogeneous solution, which was mixed with spo-5-ASA@L microcapsules. The mixture was reacted under gentle stirring at room temperature for 10 min and was dried in a vacuum drying chamber at 40 °C for 2 h to yield CS-butyrate modified spo microcapsules (spo-CBC-5-ASA@L). All samples were sealed and stored at 4 °C for further use. 5-ASA was extracted from spo-5-ASA and spo-CBC-5-ASA via ultrasonic extraction. Content was determined by HPLC, and drug loading was calculated accordingly.

2.5. Analysis of nitrogen, phosphorus, and sulfur elements

Elemental analysis (vario EL cube, Elementar, Germany) was performed using an elemental analyzer equipped with a thermal conductivity detector. Weighed samples of spo and pollen were wrapped in tin cups and placed in the autosampler. The instrument calculates and reports mass percentages of nitrogen, phosphorus and sulfur based on characteristic peak areas and standard curves.

2.6. TEM observation

Samples were prepared via phosphotungstic acid negative staining. Liposome dispersion was diluted with ultrapure water. A drop of diluted sample was applied to a carbon-coated copper grid and adsorbed for 2 min. Excess liquid was removed with filter paper. A drop of 2% (w/v) phosphotungstic acid aqueous solution at pH 7.0 is added for negative staining for 1 min. The staining solution was removed, and the grids were air-dried. Grids were transferred to the transmission electron microscope (TEM) (Hitachi H-7650, Hitachi Ltd., Tokyo, Japan) sample holder and observed at an accelerating voltage of 80 kV.

2.7. Particle size measurement

Vacuum-dried spo microcapsules were weighed and dispersed in ultrapure water. The suspension was sonicated in an ice bath at 100 W for 3 min to ensure uniform dispersion. An aliquot was transferred to a Malvern particle size analyzer sample cell (MALVERN ZS90, Malvern Instruments Ltd., UK). Measurement temperature was set to 25 °C with 60 s equilibration. Dispersant refractive index was set to 1.330, spo refractive index to 1.48, and absorption to 0.01. Each sample was measured three times with 60 s per run.

2.8. Zeta potential measurement

Sample powder or concentrated dispersion was diluted with ultrapure water to an appropriate concentration and transferred to a folded capillary cell, avoiding air bubbles. The cell was inserted into the Malvern particle size analyzer sample holder. Measurement temperature was set to 25 °C with 120 s equilibration. Each sample was measured three times with 20 submeasurements per run. Average zeta potential and standard deviation are calculated.

2.9. Optical microscopy

A small amount of the spo sample was placed on a clean glass slide. A drop of ultrapure water was added, and a coverslip was gently pressed to form a uniform aqueous monolayer. The slide was observed under an optical microscope (XD-202, Caikang Optical Instrument Co., Ltd., Shanghai, China) with a 100 × oil immersion objective. Digital images were captured for documentation.

2.10. SEM observation

Dried spo powder was dispersed onto a sample stub using conductive double-sided adhesive tape. Loosely adhering particles were removed with a rubber bulb. The sample was sputter-coated with a 10 nm platinum layer using an ion sputter coater. The stub was placed in the scanning electron microscope (SEM) (S-3400 N, HITACHI, Japan) chamber. Representative areas were imaged in secondary electron mode at 5–10 kV accelerating voltage to analyze particle size, shape, surface texture, and multilayer features.

2.11. FTIR and atomic absorption determination

Chemical composition was analyzed via Fourier transform infrared spectroscopy (FTIR) (TENSOR II, Bruker, Germany). Samples were ground and mixed with dried potassium bromide at a mass ratio of 1:100. Mixtures were pressed into translucent pellets using a hydraulic press. Spectra were acquired with a blank potassium bromide pellet for background subtraction. Acquisition parameters include 4000–400 cm⁻¹ wavenumber range, 4 cm⁻¹ resolution, and 32 scans. Successful loading, composite formation, and chemical interactions were assessed via spectral shifts, intensity changes, and new absorption bands. Atomic absorption spectroscopy measures sodium ion concentration changes in sodium hydroxide solution before and after chemical treatment, establishing a quantitative correlation between carboxyl group protonation-deprotonation and swelling rate.

2.12. In vitro release study

Release characteristics were evaluated via dialysis. A precise amount of spo microcapsules equivalent to a specific mesalazine dose was sealed in a dialysis bag with an 8000–14,000 Da molecular weight cutoff. The bag was placed in a dissolution cup containing 200 ml simulated gastric fluid at pH 1.2 and simulated intestinal fluid at pH 6.8 and 7.4. Conditions are maintained at 37 °C and 100 rpm. At predetermined time points, the release medium was removed and replenished with fresh isothermal isovolumetric medium. The collected solution is filtered through a 0.22 µm membrane. Mesalazine content was determined by HPLC (LC-20AT, Shimadzu, Japan), and release curves were plotted and fitted.

2.13. Rheological characterization of CS

Complex viscosity was measured with a rheometer equipped (Kinexus ultra, Malvern, Germany) with a 25 mm parallel-plate geometry. CBC is dissolved in buffer at the specified pH, equilibrated, and deaerated at 37 °C. A sample aliquot was loaded onto the lower plate. The upper plate is lowered to a set gap, and excess material is trimmed. A strain sweep from 0.1% to 100% at 1 Hz determines the linear viscoelastic region. A frequency sweep was performed within the linear viscoelastic region at 1% strain over 0.1–100 rad/s angular frequency. Complex viscosity was recorded as a function of frequency.

Viscoelastic properties were measured with a 40 mm parallel-plate geometry. CBC was dissolved in buffer at the specified pH, equilibrated, and deaerated at 37 °C. A sample aliquot was loaded onto the lower plate. A frequency sweep was performed over 0.1–100 rad/s angular frequency. Storage modulus and loss modulus were recorded as functions of frequency.

2.14. Preparation of fluorescent samples

Indocyanine green (ICG) was dissolved in absolute ethanol to prepare a stock solution. Blank spo microcapsules were added, and the mixture was vortexed. Vacuum-assisted loading was performed in an oven to adsorb ICG into the spo microcapsules. The sample was dried at 37 °C to remove solvent, yielding ICG-loaded spo microcapsules. For coating, spo-ICG was mixed with aqueous CS-butyrate solution. The mixture was gently stirred at 37 °C to allow electrostatic adsorption of the coating onto microcapsule surfaces. Drying at 37 °C yields CS-butyrate-coated samples. Three powder samples including free ICG, spo-ICG and spo-CBC-ICG are suspended in ultrapure water. Fluorescence intensity was measured with a microplate reader, and concentration was calculated using a standard calibration curve.

2.15. Ex vivo intestinal adhesion experiment

Mice were euthanized, and identical intestinal segments were excised and divided into three equal segments. Segments were immediately placed in ice-cold saline. One milliliter of fluorescent sample suspension was injected into one end of each segment. Segments were massaged for 1 min to ensure mucosal contact. Lumen was repeatedly flushed with ice-cold saline. After each flush, segments were imaged using an in vivo imaging system (IVIS Lumina III, PerkinElmer, USA) at appropriate ICG excitation and emission wavelengths. Fluorescence intensity was quantified to monitor signal attenuation during washing.

2.16. In vivo intestinal retention and distribution experiment

Mice were orally administered equivalent doses of fluorescently labeled samples. At predetermined time points post-administration, mice were anesthetized with isoflurane and imaged using a small animal in vivo imaging system. Whole-body fluorescence images track formulation migration and targeted accumulation. Four hours after final imaging, mice were euthanized and dissected. The entire gastrointestinal tract was excised, laid flat, and imaged ex vivo to compare colonic enrichment. Fluorescence intensity was quantified using instrument software.

2.17. Distribution experiment in vivo

Eighteen healthy C57 mice were acclimatized for 3 d and randomly divided into two groups of nine based on body weight: mesalazine solution and CS-butyrate mesalazine spo. Samples were collected at three time points. Drug concentrations in the stomach, small intestine, colon, liver, and plasma were determined by high-performance liquid chromatography coupled with mass spectrometry after oral administration.

2.18. Cytotoxicity assays and in vitro inflammatory cytokine assays

Cytotoxicity assay:​ Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and incubated overnight to allow attachment. The next day, cells were treated with various concentrations of the drug (e.g., 0, 1.6, 8, 40 µM) for 24 h. After treatment, 10 µl of CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated relative to the untreated control group, and experiments were performed in triplicate.

In vitro anti-inflammatory effect:​ Caco-2 cells were pre-treated with LPS (1 µg/ml) for 4 h to induce inflammation, followed by exposure to spo-CBC-5-ASA@L for 12 h. The cell culture supernatants were then collected and centrifuged at 1000 × g for 5 min to remove debris. The levels of TNF-α and IL-1β in the clarified supernatants were measured by ELISA according to the manufacturer’s instructions. All experiments were performed in triplicate.

2.19. In vivo therapeutic efficacy

Sixteen healthy SD rats were acclimatized for 3 days and randomly divided into four groups of four based on body weight: model control, 5-ASA@L solution, spo-5-ASA@L, and spo-CBC-5-ASA@L. From Day 1 to 7, acute colitis was induced with 4% w/v DSS as the sole drinking water. Normal water was restored from days 8 to 10. Starting on Day 1, rats receive daily oral gavage. Treatment groups receive formulations equivalent to 75 mg/kg 5-ASA. Model control receives an equal volume of normal saline. Body weight and fecal characteristics were recorded daily. DAI score was also evaluated daily. Scores were defined as follows: weight loss: 0 (no loss), 1 (1%–5%), 2 (5%–10%), 3 (10%–15%), and 4 (>15%); bleeding: 0 (no blood), 1 (hemoccultpositive), 2 (hemoccult-positive and visual pellet bleeding), and 4 (gross bleeding); stool consistency: 0 (normal), 2 (loose stool), and 4 (diarrhea).

On Day 11, rats were anesthetized and euthanized. Abdominal cavities were opened, gastrointestinal tracts were photographed, and colons were excised. Colon length from the cecum to the anus was measured. Serum inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, and interleukin-1β are quantified.

For histopathological analysis, colon, heart, liver, spleen, lung, and kidney tissues were fixed in 4% paraformaldehyde for 24–48 h. Tissues were dehydrated, cleared, and embedded in paraffin. Serial sections of 4 µm thickness were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin. Sections were dehydrated, cleared, and mounted with resinous medium.

2.20. Statistical analysis

Data were analyzed using GraphPad Prism 9.0. Results were presented as mean ± standard deviation from three independent experiments. Statistical comparisons were performed via one-way or two-way analysis of variance. P < 0.05 indicates statistical significance.

3. Results and discussion

3.1. Preparation and characterization of spo-CBC-5-ASA@L

Sporopollenin (spo), constituting the robust protective exine of pollen grains, exhibits remarkable physicochemical stability, encompassing resistance to acids, alkalis, and enzymatic degradation. Its inherent hollow architecture renders it an exceptional candidate for drug encapsulation. Defatted chrysanthemum spo was successfully obtained through the systematic removal of internal genetic material and surface proteins (Fig. 2A). Raw chrysanthemum pollen presented as large orange-yellow granules, whereas the processed non-activated sporopollenin (nspo) manifested as a finer brownish-yellow powder. Subsequent hydrophilic surface modification yielded spo as a dark brown powder (Fig. 2B). Elemental analysis revealed a substantial reduction in nitrogen, phosphorus, and sulfur contents in the spo relative to raw pollen, corroborating the effective elimination of internal genetic material and proteins (Fig. 2C). Particle size analysis indicated that the size of raw pollen was slightly larger than that of nspo and spo, which was demonstrated that the pectin layer surrounding the pollen also removal (Fig. 2D).

Fig. 2.

Preparation and characterization of spo-CBC-5-ASA@L. (A) Schematic diagram of the spo extraction process from chrysanthemum pollen; (B) Photographs of raw chrysanthemum pollen, nspo and spo; (C) Elemental analysis (N, P, S content) of pollen and spo; (D) Particle size distribution of pollen, nspo and spo; (E) FTIR spectra of pollen, nspo, and spo; (F) Schematic diagram of the preparation process for 5-ASA@L; (G) TEM image and particle size distribution of the prepared 5-ASA@L; (H) Schematic diagram of the preparation process for spo-CBC-5-ASA@L microcapsules; (I) Optical microscopy images and particle size distribution of spo and spo-CBC-5-ASA@L; (J) Particle size distribution of spo, spo-5-ASA@L and spo-CBC-5-ASA@L; (K) Zeta potential of spo-5-ASA@L and spo-CBC-5-ASA@L; (L) Stability of 5-ASA@L content in spo-CBC-5-ASA@L stored at 4 °C over 30 days.

Fourier transform infrared (FTIR) spectroscopy conducted at pH 7 (Fig. 2E) disclosed a broad absorption band within the 3600–3000 cm⁻¹ region, attributable to O–H/N–H stretching vibrations. Pollen exhibited the most intense and broadest band in this region, indicative of abundant proteins, polysaccharides, and bound water containing numerous –OH and –NH groups that constitute an extensive hydrogen-bonding network. The absorption intensity of nspo was markedly attenuated with a concomitant narrowing of the peak, suggesting that preliminary treatment effectively eliminated a substantial proportion of hydrophilic constituents, although residual proteins or polysaccharides may persist. Conversely, spo displayed the weakest and narrowest band, signifying that the rigorous purification protocol successfully removed the majority of proteins, polysaccharides, and other impurities, yielding a sample composed predominantly of spo. Although spo per se contains phenolic hydroxyl groups, their abundance is considerably lower than that of hydrophilic components present in raw pollen.

In the C O stretching region (1740–1710 cm⁻¹) and the aromatic C C skeletal vibration region (∼1600 cm⁻¹), pollen exhibited complex peak profiles, presumably arising from the superposition of signals originating from multiple carbonyl sources. The spectra of both nspo and spo were substantially simplified, confirming the removal of miscellaneous carbonyl-containing compounds and enabling the characteristic carbonyl and aromatic signals of spo to emerge distinctly. Within the fingerprint region (1500–400 cm⁻¹), pollen absorption was complex with numerous fluctuations, consistent with its heterogeneous composition. nspo exhibited reduced spectral complexity, while the spo spectrum was further simplified, displaying clearer and more characteristic peaks. This observation indicates that spo possesses a highly uniform chemical structure representative of purified spo. The FTIR data collectively suggest that the surface chemical functionalities of nspo and spo are distinctly different, implying variations in surface polarity, hydrophilicity/hydrophobicity balance (Fig. 2E).

To enhance the bioavailability of 5-ASA, liposomes were fabricated to facilitate transmembrane absorption. The detailed preparation procedure is illustrated in Fig. 2F. TEM revealed that the resultant liposomes possessed a diameter of approximately 180 nm, the PDI was 0.238,and exhibited a rounded bilayer architecture, confirming successful preparation. The encapsulation efficiency was determined to be 88% (Fig. 2G). The spo-CBC-5-ASA@L system was subsequently constructed as depicted in Fig. 2H Particle size analysis indicated that spo exhibited a distribution centered around 28.8 µm, whereas spo-CBC-5-ASA@L displayed a distribution around 29.2 µm. Optical microscopy revealed no significant morphological alterations between the two samples (Fig. 2I). The unaltered particle size of spo-5-ASA@L compared to pristine spo confirmed that liposomes were successfully loaded into the interior of spo via vacuum-assisted loading. The comparable particle size of the coated spo-CBC-5-ASA@L sample further indicated that the surface coating did not modify the external architecture of spo, thereby preserving its characteristic spiny morphology—a feature critical for intestinal adhesion and retention (Fig. 2J). The drug loading capacities were determined to be approximately 6.8% and 5.6%, respectively.

Zeta potential measurements demonstrated a shift from –6.23 mV for spo-5-ASA@L to +5.91 mV for spo-CBC-5-ASA@L, confirming the successful deposition of CS and butyrate onto the surface through electrostatic adsorption (Fig. 2K). The pollen-derived carrier particles, with a diameter of approximately 30 µm, are classified as a microparticulate formulation. The zeta potential measurements obtained in this study indicated that the uncoated particles possessed a negatively charged surface; following CS coating, the particles acquired a positive surface charge, thereby confirming successful encapsulation. The ζ-potential values (ranging from –6.23 mV to +5.91 mV) suggest limited colloidal stability in aqueous dispersion; consequently, the dried product exhibits favorable stability during storage. The 5-ASA content in spo-CBC-5-ASA@L remained stable over observation periods of 5, 10 and 30 d when stored at 4 °C, indicating acceptable short-term stability (Fig. 2L).

3.2. Investigation of the pH-dependent swelling mechanism of spo

SEM was employed to systematically examine the morphological and dimensional alterations of pollen, nspo and spo under varying pH conditions. As presented in Fig. 3A, all three specimens exhibited increased dimensions with elevating pH. Pollen samples displayed distinct spherical structures at their germinal apertures, which function as natural selective “gates” that protect internal genetic material and permit its release under specific physiological conditions. In marked contrast, the processed nspo and spo retained merely the aperture architecture, with internal contents completely removed, thereby presenting a hollow structural configuration.

Fig. 3.

Investigation of the pH-responsive swelling mechanism of spo. (A) SEM images of pollen, nspo and spo at pH 1 and 7; (B) Statistical analysis of the particle size of pollen, nspo and spo at pH 1, 5, 7 and 10; (C) Statistical analysis of the germinal aperture size of pollen, nspo and spo at pH 1, 5, 7 and 10; (D–F) FTIR spectra of (D) pollen, (E) nspo and (F) spo at pH 1, 5, 7 and 10; (G) Linear relationship plot between the atomic absorption amount of spo and its swelling rate under different concentrations of Na⁺ particles.

Quantitative measurements of sample dimensions and aperture sizes revealed that although all specimens exhibited pH-responsive behavior, pollen demonstrated the most modest degree of contraction and expansion (Fig. 3B), accompanied by minimal aperture size variation. Both nspo and spo exhibited significantly more pronounced dimensional changes compared to pollen. Furthermore, spo demonstrated more marked opening and closing dynamics of the germinal apertures relative to nspo (Fig. 3C).

Comparative analysis of the FTIR spectra of pollen (Fig. 3D), nspo (Fig. 3E) and spo (Fig. 3F) under different pH conditions revealed consistent underlying trends. Under acidic conditions (pH 1 and pH 5), absorption in the 3400 cm⁻¹ region (O–H/N–H stretching) was intensified, presumably attributable to the protonation of carboxyl (–COOH) or phenolic hydroxyl groups. Protonation converts these moieties into their protonated forms, which function as stronger hydrogen-bond donors. Concurrently, the reorganization of intra- and intermolecular hydrogen-bonding networks results in enhanced and broadened O–H stretching absorption. Peak profiles and positions within the 1600–1700 cm⁻¹ region (C O stretching, including aromatic C C vibrations) also exhibited systematic pH-dependent alterations. Notably, in the spectra of processed spo, a relative enhancement of the carboxylic acid C O vibration near 1700 cm⁻¹ was observed under acidic conditions, directly confirming the presence of carboxyl groups and their protonation state. Under acidic pH, carboxyl groups exist predominantly as –COOH, with C O stretching vibrations typically appearing at 1710–1740 cm⁻¹. Under neutral to alkaline conditions, deprotonation to –COO⁻ shifts the asymmetric stretching vibration to approximately 1550–1610 cm⁻¹, accompanied by altered peak morphology.

On the basis of these observations, we propose that pH-induced alterations in the ionization state of functional groups drive the reorganization of intramolecular and intermolecular hydrogen bonds and electrostatic interactions. This molecular-level switching mechanism serves as the fundamental trigger for the macroscopic “acid-contraction, alkali-expansion” behavior. This pH-responsive transformation provides a favorable foundation for drug encapsulation and oral administration aimed at intestinal targeting. To further elucidate the quantitative relationship between the protonation/deprotonation of functional groups and the “acid-induced contraction and base-induced swelling” phenomena, atomic absorption spectrometry was utilized to quantify changes in sodium ion concentration in sodium hydroxide solution before and after contact with spo. Upon interaction with spo powder, sodium ions from the sodium hydroxide solution were transferred to the spo matrix, resulting in a significant decrease in solution sodium ion concentration. This phenomenon quantitatively confirms that the carboxyl groups (-COOH) on the spo surface undergo deprotonation and combine with Na⁺ ions in the solution to form sodium carboxamate (-COONa). Simultaneously, the electrostatic repulsion between the resulting carboxylate ions (-COO⁻) induced swelling of the spo, leading to a significant increase in particle size. As the concentration of sodium hydroxide solution increased, the carboxylate content rose proportionally, and the swelling rate of spo correspondingly increased (Fig. 3G), which is consistent with the results obtained from FTIR spectroscopy. These findings quantitatively elucidate the correlation between the protonation/deprotonation state of the carboxyl groups and the swelling rate. This study further clarifies the pH-responsive expansion and contraction mechanisms, laying the foundation for drug encapsulation, gastrointestinal delivery, and intestinal targeting applications.

3.3. Investigation of the pH-responsive mechanism of CS

The morphological transformations of CS under varying pH conditions were systematically investigated through rheological characterization. Optical microscopy revealed that CS remained soluble under acidic conditions yet precipitated under alkaline conditions. This behavior was visually confirmed using an ICG-containing solution, wherein the acidic CS solution maintained homogeneity, whereas distinct particulate precipitation was observed under alkaline conditions (Fig. 4A).

Fig. 4.

Investigation of the pH-responsive mechanism of CS and in vitro drug release. (A) Optical microscopy images of CS and CBC solutions with/without ICG at pH 1 and 7; (B) Apparent viscosity versus shear rate for CS and CBC solutions at pH 7; (C) Storage modulus (G') and loss modulus (G'') versus angular frequency for CS and CBC solutions at pH 7; (D) Apparent viscosity versus shear rate for CBC at pH 1 and 7; (E) G' and G'' versus angular frequency for CBC at pH 1 and 7; (F-G) SEM image of spo-CBC-5-ASA@L at pH 1 and pH 7; (H) Cumulative drug release profile of spo-5-ASA@L in pH 1.2, 6.8 and 7.4; (I) Cumulative drug release profile of spo-CBC-5-ASA@L in pH 1.2, 6.8 and 7.4; (J) Comparison of total drug release at 4 h for spo-5-ASA@L and spo-CBC-5-ASA@L in pH 1.2, 6.8 and 7.4.

The rheological behavior of CS and the CS–butyrate copolymer (CBC) at pH 7 was comparatively evaluated. As demonstrated in Fig. 4B, the viscosity of CBC was consistently superior to that of CS across the entire frequency range. Additionally, as illustrated in Fig. 4C, both the storage modulus (G′) and loss modulus (G″) were elevated for CBC relative to CS, indicating that the incorporation of BA substantially enhances the stability and viscosity of the CS matrix. We subsequently investigated the complex viscosity together with the storage modulus (G′) and loss modulus (G″) of CBC under different pH conditions, as presented in Fig. 4D and 4E. At pH 1.0, the storage modulus (G′) of CBC consistently exceeded the loss modulus (G″), albeit with a minimal margin between the two parameters. This behavior indicates that the material stores more mechanical energy than it dissipates under applied stress, thereby classifying it as a weak gel possessing certain elastic characteristics. At pH 7.0, the G′ and G″ values were closely matched and considerably lower in magnitude, indicating an extremely fragile material structure—an observation consistent with microscopic examinations.

The surface morphology of the CBC layer in the spo-CBC-5-ASA sample at pH 1 (Fig. 4F) and pH 7 (Fig. 4G) revealed a distinct gel state at pH 1, with uniform deposition onto the surfaces of spo germinal apertures, thereby forming a gate-like architecture. Conversely, at pH 7, no coherent structural layer was observed, leaving the germinal apertures fully exposed and permitting the release of encapsulated contents. These observations compellingly demonstrate that CBC functions as an effective pH-responsive gatekeeper for the spo carrier system.

SEM images of spo-CBC-5-ASA@L provided direct visual confirmation of this mechanistic behavior (Fig. 4F and 4G). At pH 1, the CBC layer formed a continuous gel coating that effectively sealed the spo germinal apertures, whereas at pH 7, the coating disintegrated, leaving the apertures completely exposed. Subsequently, release studies were conducted on spo-5-ASA (Fig. 4H) and spo-CBC-5-ASA (Fig. 4I) samples, evaluating their respective release profiles in simulated gastric fluid and simulated intestinal fluid. The results demonstrated that the spo-5-ASA sample released approximately 20% of the encapsulated drug in simulated gastric fluid, whereas the spo-CBC-5-ASA sample released no >10%. Both spo-5-ASA and spo-CBC-5-ASA samples achieved nearly complete drug release in simulated intestinal fluids (pH 6.8 and 7.4), with marginally elevated release observed at pH 7.4 compared to pH 6.8 (Fig. 4J).

In simulated gastric fluid, the release data were best fitted to the Higuchi model, indicative of a Fickian diffusion mechanism, which effectively suppressed drug burst release and minimized gastric drug loss. In simulated intestinal fluid, the data conformed to a first-order kinetic model, reflecting a synergistic diffusion-swelling mechanism that ensured efficient drug release at the target intestinal site. These findings collectively demonstrate that the CBC layer forms a gel-like structure under gastric acidic conditions, effectively sealing the germinal pores of spo and significantly inhibiting premature drug release in the gastric environment. In contrast, under intestinal fluid conditions, the CBC layer disintegrates, thereby opening the “gate” and facilitating coordinated drug release through spo expansion, resulting in rapid and near-complete drug liberation—constituting a successful validation of the reverse pH-responsive “gate” design concept.

3.4. In vitro and in vivo evaluation of intestinal adhesion, retention, and targeting

Comprehensive in vitro and in vivo experiments were conducted to systematically evaluate the adhesion, retention, and targeting capabilities of the spo-CBC-5-ASA@L delivery system. Initially, an ex vivo intestinal model was established to simulate formulation transit dynamics. ICG was employed to prepare three distinct formulations: an ICG solution (ICG@S), spo-ICG, and spo-CBC-ICG. The fluorescence intensities of these formulations were standardized to equivalent levels prior to ex vivo administration (Fig. 5A). Following repeated saline washes, fluorescence imaging (Fig. 5C) revealed that the spo-CBC-ICG formulation produced the most intense intestinal fluorescence signal, whereas ICG@S generated the weakest signal. This observation suggests that spo-based formulations possess superior intestinal retention characteristics. Histological sections (Fig. 5B) further confirmed that spo adheres readily to the intestinal mucosal surface. Moreover, spo-CBC-ICG exhibited stronger retention than spo-ICG, indicating that the CS coating confers additional mucoadhesive properties (Fig. 5D).

Fig. 5.

In vitro and in vivo evaluation of intestinal adhesion and targeting. (A) Fluorescence images of ICG solution (ICG@S), spo-ICG and spo-CBC-ICG samples adjusted to equal fluorescence intensity for ex vivo studies; (B) Microscopic image of intestinal tissue section showing adhesion of spo particles; (C) Ex vivo fluorescence images of intestinal segments after administration of different samples and saline washing; (D) Quantitative analysis of residual fluorescence intensity in intestinal segments from the ex vivo adhesion experiment; (E) In vivo real-time fluorescence images of mice at different time points after oral administration of ICG@S, spo-ICG and spo-CBC-ICG; (F) Ex vivo fluorescence images of the entire gastrointestinal tract excised 4 h post-administration; (G) Quantitative analysis of fluorescence intensity in the colon region from the ex vivo gastrointestinal tract images; (H) Distribution of the 5-ASA in the stomach, small intestine, colon, liver and blood in mice from the 5-ASA solution group and the spo-CBC-5-ASA@L group at 1, 3 and 6 h post-administration.

The in vivo distribution patterns of the various formulations were evaluated through real-time fluorescence imaging in mice. Mice received oral administration of different fluorescently labeled formulations (standardized for equivalent fluorescence intensity) and were subsequently imaged at 10, 20, 30, 60, 120, 240 and 480 min post-administration. As presented in Fig. 5E, the ICG@S formulation generated a strong signal immediately following administration, which rapidly diminished within 1 h. In contrast, the spo-ICG formulation initially produced a weaker signal; however, the overall abdominal fluorescence intensity progressively increased over the 4 h observation period, eventually surpassing that of the ICG@S group at the 4 h time point, albeit without the formation of distinct focal accumulation sites. The spo-CBC-ICG formulation similarly exhibited a weak initial signal, yet fluorescence rapidly distributed throughout the abdominal cavity. At the 4 h time point, it displayed the highest fluorescence intensity accompanied by concentrated fluorescent foci, indicative of colon-targeted accumulation.

Mice were euthanized 4 h post-dose, and the entire gastrointestinal tract was excised for ex vivo fluorescence imaging (Fig. 5F). In the ICG@S group, the majority of fluorescence had transited to the colon and was largely eliminated. The spo-ICG group exhibited pronounced fluorescence retention along the entire intestinal tract. The spo-CBC-ICG formulation displayed some retention in the small intestine; however, the predominant signal was concentrated in the colon. The overall fluorescence intensity of the spo-CBC-ICG group was significantly higher than that of the spo-ICG group (Fig. 5G). These results collectively demonstrate that the CS coating enhances the mucoadhesive properties of spo, while surface-immobilized butyrate imparts distinct colon-targeting capabilities. To further validate the intestinal targeting properties of spo-CBC-5-ASA, additional in vivo distribution studies were conducted employing different 5-ASA carrier formulations. As shown in Fig. 5H, following administration of spo-CBC-5-ASA@L, the 5-ASA drug was released and primarily distributed in the gastrointestinal tract, with sustained intestinal retention over 6 h, particularly with prolonged retention in the colon. Its metabolite, Ac-5-ASA, exhibited extremely low concentrations in the liver and blood. In contrast, the 5-ASA solution group was degraded by gastric acid and directly entered the bloodstream, making 5-ASA difficult to measure; however, significant amounts of Ac-5-ASA were detected in the liver and plasma, demonstrating that the solution formulation failed to achieve effective retention at the target site., thereby confirming spo-CBC-5-ASA@L intestinal targeting capability.

3.5. In vivo and in vitro therapeutic efficacy and safety evaluation

Cytotoxicity studies (Fig. 6A) demonstrated that the developed formulation exhibits low cytotoxicity profiles. In vitro inflammatory cytokine assays (Fig. 6B) revealed that the spo-CBC-5-ASA formulation effectively suppressed the production of pro-inflammatory mediators TNF-α and IL-1β in cultured cells. The therapeutic efficacy of 5-ASA formulations in colitis treatment was subsequently evaluated in a rat model. The comparative outcomes for the model control, 5-ASA@L solution, spo-5-ASA@L, and spo-CBC-5-ASA@L groups in ameliorating DSS-induced colitis were systematically analyzed (experimental timeline presented in Fig. 6C; necropsy performed on Day 11).

Fig. 6.

In vivo therapeutic efficacy in a DSS-induced colitis rat model. (A) Evaluation of the effects of 5-ASA and different formulations on cell viability; (B) Impact of spo-CBC-5-ASA@L on LPS-induced inflammatory cytokine release in Caco-2 cells; (C) Schematic timeline of the animal efficacy experiment; (D) Macroscopic appearance of the colon from different treatment groups; (E) Photograph of isolated colons from different treatment groups, demonstrating length differences; (F) Statistical analysis of colon length from different treatment groups; (G-I) Statistical analysis of the concentration of (G) IL-1β, (H) TNF-α and (I) IL-6 from different treatment groups; (J) Body weight change curves of rats during the experiment; (K) DAI change curves of rats during the experiment.

Macroscopic examination (Fig. 6D) revealed severe intestinal inflammation in the model control group, characterized by mucosal congestion, edema, and ulcerative lesions. The 5-ASA@L group displayed considerable inflammation, although the severity of symptoms was attenuated relative to the model control. The spo-5-ASA@L group exhibited only mild, localized inflammation confined to the small intestine, without obvious congestion or edema. Remarkably, the intestines of the spo-CBC-5-ASA@L group appeared essentially normal, devoid of inflammatory signs. Colon length was measured as a quantitative indicator of inflammation severity (Fig. 6E). The model control group presented the shortest colons (mean 12.1 cm) and produced no formed feces. The 5-ASA@L group demonstrated marginally longer colons (mean 12.7 cm; not statistically significant) with soft, pelleted feces. The spo-5-ASA@L group exhibited a further increase in colon length (mean 15.5 cm) accompanied by more formed feces. Most notably, the spo-CBC-5-ASA@L group demonstrated the longest colons (mean 16.9 cm), significantly exceeding all other treatment groups, with firm, elliptical feces—indicating pronounced suppression of colitis pathogenesis (Fig. 6F).

Serum inflammatory cytokine levels (TNF-α, IL-6 and IL-1β) are presented in Fig. 6G–6I. Compared with blank plasma, the control group exhibited significantly elevated levels of all cytokines, confirming successful DSS modeling. In contrast to the control group, all three drug administration groups showed a certain degree of reduction in inflammation-related factors, indicating that the drugs possess anti-colitis efficacy. Among these, the spo-CBC-5-ASA@L group demonstrated the most pronounced reduction, demonstrating its superior therapeutic effect against colitis—a finding consistent with the results regarding colon length measurements. Body weight and DAI trajectories were monitored throughout the experimental duration (Fig. 6J and 6K). The model control group experienced progressive weight loss and severe diarrhea during DSS administration, with no recovery observed following restoration of normal drinking water. The 5-ASA@L group similarly lost weight, albeit to a lesser magnitude. The spo-5-ASA@L group initiated weight recovery from Day 6 onward, the severity of diarrhea had decreased, suggestive of a sustained-release therapeutic effect. Most remarkably, the spo-CBC-5-ASA@L group commenced weight regain from Day 4, the severity of diarrhea had also been significantly reduce, achieving complete recovery to baseline body weight by Day 10. This observation suggests that the spo-CBC composite carrier facilitates accelerated transit to the colon, coupled with prolonged local drug release mediated by CS mucoadhesion and spo-based sustained release. Surface-immobilized butyrate may additionally provide nutritional support to damaged epithelial cells and suppress inflammatory mediator production, thereby contributing to the superior anti-inflammatory therapeutic outcome.

Histopathological analysis (H&E staining, Fig. 7A) of the model control group revealed severe inflammatory cell infiltration, mucosal erosion, crypt architectural destruction, and crypt abscess formation. The 5-ASA@L group exhibited extensive inflammatory infiltration but retained a relatively intact mucosal surface. The spo-5-ASA@L group displayed minor inflammatory infiltration with mild mucosal damage; nevertheless, its histopathological score was significantly superior to that of the 5-ASA@L group. The spo-CBC-5-ASA@L group presented healthy-appearing colon tissue devoid of obvious inflammatory pathology, with intact mucosal architecture and uniformly distributed goblet cells. These findings confirm the exceptional therapeutic efficacy of spo-CBC-5-ASA@L, attributable to prolonged intestinal retention and sustained drug release at the lesion site. Histological examinations of other major organs—including the heart, liver, spleen, lungs and kidneys—by H&E staining revealed no significant pathological alterations, thereby indicating a favorable safety profile for the developed formulation (Fig. 7B).

Fig. 7.

In vivo safety evaluation of tissues and organs. (A) H&E-stained colon tissue sections from different treatment groups; (B) H&E-stained heart, liver, spleen, lungs and kidneys tissue sections from different treatment groups.

4. Conclusion

The therapeutic management of ulcerative colitis (UC), a chronic relapsing inflammatory bowel disease, is fundamentally constrained by the imperative to achieve precise drug delivery and prolonged retention at colonic lesion sites. Although 5-ASA remains a first-line clinical therapeutic agent, conventional oral formulations are vulnerable to degradation or premature absorption within the upper gastrointestinal tract. These limitations culminate in subtherapeutic drug concentrations at the target site, diminished bioavailability, and compromised clinical efficacy. Established colon-targeting strategies, encompassing pH-dependent systems utilizing acrylic resins and time-controlled release mechanisms, have been extensively employed to circumvent these challenges. Nevertheless, their therapeutic effectiveness is frequently undermined by reliance upon specific and relatively invariant gastrointestinal parameters—such as pH transition points or intestinal transit times—which exhibit substantial inter-individual variability in UC patients, particularly due to disease-associated dysmotility. Such variability potentially results in mistimed drug release and attenuated targeting precision [11,12]. Enzyme-triggered systems, conversely, are constrained by the inherent complexity and inter-individual variability of gut microbiota composition [13]. Consequently, the development of intelligent delivery systems capable of dynamically adapting to the gastrointestinal microenvironment while incorporating robust, multi-mechanistic targeting and retention strategies represents an urgent unmet clinical need.

Contemporary colon-targeted carrier materials are primarily classified into three categories: natural biomacromolecules, synthetic responsive polymers, and inorganic porous materials. Natural polysaccharides, exemplified by modified pectin and inulin, achieve drug release through degradation by colon-specific microbial enzymes; while exhibiting favorable biocompatibility, these materials generally possess inadequate mechanical stability. Synthetic polymers, predominantly Eudragit S100 and polylactic acid-glycolic acid copolymers, rely upon pH-responsive targeting to the colon and benefit from mature manufacturing processes; however, they typically demonstrate limited intestinal specificity. Inorganic materials, including mesoporous silica and metal-organic frameworks (MOFs), offer tunable pore architectures and elevated drug loading capacities, frequently employing dual enzyme/pH-responsive strategies; notwithstanding, certain representatives of this class exhibit potential cytotoxicity concerns. spo, as an emerging natural pollen wall material, demonstrates exceptional acid-base stability, superior biosafety, and facile surface modifiability. The present study conducted a comprehensive evaluation of its extraction protocol, demonstrating excellent reproducibility and scalability, while optimizing the drug-loading mechanism through stable and repeatable procedures. Furthermore, as a naturally derived product, spo exhibits an outstanding safety profile, rendering it a highly promising carrier material. This investigation established a gate-controlled targeting strategy that triggers drug release specifically within the colonic microenvironment, achieving substantially superior targeting precision and controllability compared to conventional materials—an approach that represents a prominent contemporary research direction in colon-targeted delivery systems.

This investigation successfully developed a novel oral colon-targeted delivery system based on a CS-butyrate copolymer and spo carrier, with comprehensive characterization of its chemical structure and microscopic morphology accomplished through spectroscopic and electron microscopy techniques. The central mechanistic innovation involves a “reverse pH dual-response gating” mechanism: under gastric acidic conditions, the CBC layer undergoes substantial swelling to form a dense gel, while the spo carrier simultaneously experiences slight contraction; these cooperative phenomena collectively affect physical obstruction of drug release channels, achieving potent suppression of premature release (<10%). Upon entry into the weakly alkaline intestinal environment, the CBC layer contracts and precipitates, while the spo carrier expands; these coordinated processes jointly trigger opening of the “gate,” enabling rapid and near-complete drug liberation. This intelligent switching architecture not only facilitates precise pH-responsive drug release but also enhances formulation adhesion and retention within the intestinal mucosa through the viscosity transitions of the CBC layer under neutral conditions and the unique physical architecture of the spo carrier. Additional in vivo distribution studies confirmed that the system efficiently accumulates within the colon, demonstrating excellent active targeting performance. In a DSS-induced colitis rat model, the formulation exhibited remarkable therapeutic efficacy, effectively ameliorating colonic shortening, promoting body weight recovery, and improving histopathological damage indices. These beneficial outcomes are attributed to the precise colonic-targeted release, prolonged local retention, and synergistic anti-inflammatory and epithelial repair effects conferred by the combination of mesalazine and butyrate. Preliminary safety assessments indicated favorable biocompatibility profiles.

In conclusion, this study presents a promising multifunctional delivery strategy for targeted therapy of ulcerative colitis. Future investigations should elucidate the molecular pathways underlying butyrate-mediated active targeting and evaluate the long-term therapeutic efficacy and systemic safety of this platform in chronic disease models. Moreover, the inherent versatility of this spo-based “gated” microcapsule system harbors substantial potential for oral delivery of other challenging therapeutic modalities, including peptides and antibodies, thereby paving a novel avenue for innovative formulation development in the treatment of inflammatory bowel diseases.

CRediT authorship contribution statement

Jun Liu: Writing-review & editing, Writing-original draft, Data curation, Methodology, Investigation. Zifang Ding: Data curation, Investigation. Jianxiong Wei: Data curation, Investigation. Yuanqing Li: Data curation, Investigation. Xiaoqing Wang: Data curation, Investigation. Xiaodan Yan: Data curation, Formal analysis. Dan Li: Data curation, Formal analysis. Tiantian Ye: Data curation, Formal analysis. Shujun Wang: Supervision, Project administration. Jin Sun: Supervision, Project administration.

Conflicts of interest

The authors declare no conflicts of interest.