Metal-organic-framework-based sitagliptin-release platform for multieffective radiation-induced intestinal injury targeting therapy and intestinal flora protective capabilities

Abstract

In patients with abdominal or pelvic tumors, radiotherapy can result in radiation-induced intestinal injury (RIII), a potentially severe complication for which there are few effective therapeutic options. Sitagliptin (SI) is an oral hypoglycemic drug that exhibits antiapoptotic, antioxidant, and anti-inflammatory activity, but how it influences RIII-associated outcomes has yet to be established. In this study, a pH-responsive metal-organic framework-based nanoparticle platform was developed for the delivery of SI (SI@ZIF-8@MS NP). These NPs incorporated mPEG-b-PLLA (MS) as an agent capable of resisting the effects of gastric acid, and are capable of releasing Zn²⁺ ions. MS was able to effectively shield these SI@ZIF-8 NPs from rapid degradation when exposed to an acidic environment, enabling the subsequent release of SI and Zn²⁺ within the intestinal fluid. Notably, SI@ZIF-8@MS treatment was able to mitigate radiation-induced intestinal dysbiosis in these mice. restored radiation-induced changes in bacterial composition. In summary, these data demonstrate the ability of SI@ZIF-8@MS to protect against WAI-induced intestinal damage in mice, suggesting that these NPs represent a multimodal targeted therapy that can effectively be used in the prevention or treatment of RIII.

Introduction

While radiotherapy remains a mainstay of treatment efforts for many malignancies, the utility of radiotherapeutic interventions is inevitably constrained by the damage that radiation causes in normal tissues [1]. The small intestine is highly susceptible to the effects of ionizing radiation exposure, which can compromise the integrity of the mucosal barrier that surrounds the intestines, contributing to acute gastrointestinal (GI) symptoms with the potential for life-threatening effects in some instances. There thus remains a pressing need to identify drugs that are capable of effectively promoting intestinal epithelial cell (IEC) regeneration and abrogating the adverse GI toxicity induced by radiation exposure.

The dipeptidyl peptidase IV (DPP-4) inhibitor sitagliptin (SI) is an oral hypoglycemic drug that has been approved by the US Food and Drug Administration (FDA). It functions by inhibiting glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 (GLP-1) degradation leading to the simultaneous enhancement of insulin secretion and the suppression of glucagon secretion [2]. SI has recently been demonstrated to exert beneficial therapeutic effects in a wide range of diseases including acute lung injury, ischemia-reperfusion injury, atherosclerosis, and Alzheimer’s disease [3, 4], benefiting patients through its antiapoptotic, anti-inflammatory, and antioxidant activities. The most recent mechanistic study focused on this drug revealed that sitagliptin is capable of activating NRF2-dependent antioxidant signaling pathways, suppressing inflammation mediated by the NLRP3 inflammasome, and restoring microbial homeostasis in the gut so as to protect against radiation-induced intestinal damage [5]. The severity of radiation-induced intestinal injury (RIII) can be aggravated by the loss of zinc ions (Zn²⁺) and the consequent disruption of appropriate metal ion homeostasis [6], and oral SI monotherapy administration can result in the undesirable degradation of these agents within the acidic gastric environment [7].

Relative to many other metal ions or peptides, Zn²⁺ exhibits greater biodegradability, biocompatibility, and stability while in storage such that they are widely employed in applications aimed at regulating biological processes. While pharmacological Zn²⁺ doses are often employed for the enhancement of intestinal barrier function, achieving controlled Zn²⁺ release remains difficult and necessitates further efforts to improve the performance of associated materials [8]. High Zn²⁺ concentrations can also adversely affect normal tissues and healthy tissues [9]. The design of Zn-loaded biomaterials thus necessitates efforts to carefully balance the bioactivity and biocompatibility of these materials, making this an ongoing challenge with which researchers in the field of tissue engineering continue to grapple.

Metal-organic frameworks (MOFs) are 3-dimensional (3D) porous structures with a cage-like conformation or containing regular rigid pore-like channels. MOFs are produced through the coordination bond-mediated self-assembly of organic ligands and metal ions [9, 10], and they are commonly used in the context of drug delivery and other applications owing to their uniform structure, tunable functions, and other advantageous characteristics [11]. The pH-responsive zeolitic imidazolate framework-8 (ZIF-8) MOFs, which are produced through coordination between Zn²⁺ and dimethylimidazole, are often employed in this setting as they exhibit excellent biocompatibility and can facilitate the controlled delivery and release of cargos of interest. These ZIF-8-based drug delivery platforms tend to rely on pH discrepancies to promote nanocarrier disassembly and cargo release within acidic microenvironmental settings [10]. Advances in ZIF-8-based systems thus represent promising tools for use in the context of RIII repair.

The ability to orally deliver SI is currently constrained by several characteristics of the gastrointestinal (GI) system. For one, SI and other protein drugs can readily be degraded within the GI microenvironment [12], owing to the high levels of acidity within the stomach and the actions of digestive enzymes present within the intestines. These protein-based drugs also tend to exhibit low bioavailability as the permeability of the intestines to these drugs is quite low [10]. These intestinal barriers are comprised of both epithelial cells and a mucus layer. An effective platform for the administration of SI must thus be able to shield it from degradation within the GI tract while also promoting its ability to cross the intestinal epithelium. In the present, a novel nanocomposite carrier was prepared through the embedding of SI@ZIF-8 nanoparticles (NPs) within biodegradable methoxy poly(ethylene glycol)-block-poly(L-lactide) (mPEG-b-PLLA, MS), thereby supporting the oral delivery of this drug. When prepared, MS microspheres present with good resistance against acidic stomach conditions while supporting the intestinal release of associated payloads [13].

This study thus describes the production of pH-responsive SI@ZIF-8@MS NPs, which were encapsulated using MS and care capable of facilitating Zn²⁺ release. MS was capable of successfully preventing SI@ZIF-8 NP degradation in acidic settings while enabling SI and Zn²⁺ release within the intestinal fluid. Further in vivo testing demonstrated the ability of SI@ZIF-8@MS to abrogate intestinal injury in a mouse whole abdominal irradiation (WAI) model, demonstrating the value of this multi-effective targeted approach to treating RIII.

Methods

Ethical overview

Female C57BL/6 mice (6–8 weeks old, 20–22 g) from the Experimental Animal Department of Army Medical University were bred under standard laboratory conditions in the Department of Oncology of Southwest Hospital. The Animal Ethics Committee of Army Medical University approved all animal studies(AMUWEC20226175).

Materials and chemicals

SI was from MedChemExpress (NJ, USA). ZIF-8 and MS-related materials were from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). Reagents used in biological experiments and material characterization assays were from Shiyanjia Lab (www.shiyanjia.com, Hangzhou, China).

SI@ZIF-8@MS synhesis

Synthesis of ZIF-8

Initially, 67.5 mL of Tris solution was used to dissolve 1.98 g of zinc acetate (Solution A), while 90 mL of Tris solution was used to dissolve 11.8 g of dimethyl imidazole (Solution B). These two solutions were then mixed for 2 h with constant stirring, centrifuged, and washed thrice using methanol. Precipitates were subsequently freeze-dried, yielding a white ZIF-8 powder(reactions yields 73.8%).

SI@ZIF-8 preparation

Initially, 67.5 mL of Tris solution was used to dissolve 1.98 g of zinc acetate and 0.56 g of SI (Solution A), while 90 mL of Tris solution was used to dissolve 11.8 g of dimethyl imidazole (Solution B). These two solutions were then mixed for 2 h with constant stirring, centrifuged, and washed thrice using methanol. Precipitates were subsequently freeze-dried, yielding a white ZIF-8 + SI powder(reactions yields 64.7%).

mPEG-b-PLLA synthesis and SI@ZIF-8@MS preparation

Anhydrous methylbenzene was used to dissolve mPEG (3.5 g) and L-lactide (LLA) (21.6 g), with stannous octoate (0.5 M equivalent relative to mPEG) then being added to initiate the ring-opening polymerization of LLA and mPEG(reactions yields 76.0%). An oil/water emulsion strategy was then used for SI@ZIF-8@MS preparation. Briefly, 1 mL of dichloromethane was used to disperse SI@ZIF-8 (2.1 mg) and 10.3 mg of mPEG-b-PLLA, followed by the addition of the solution to 10 mL of aqueous 3% PVA to generate oil/water emulsions via emulsification pretreatment. This mixture was then added to 1.5% PVA with stirring, and rotary evaporation was used to remove the dichloromethane. Microsphere centrifugation (7,000 rpm) and lyophilization were then performed(reactions yields 65.2%). Finally, the drug loading rate test was carried out, which showed that wt% of SI was 23.1% in the final product SI@ZIF-8@MS.

Characterization

SI@ZIF-8@MS characterization was performed through scanning and transmission electron microscopy (SEM and TEM) and through atomic force microscopy (AFM). Dynamic light scattering was employed to evaluate zeta potential and particle sizes. A Nicolet 6700 FTIR spectrometer was utilized to record Fourier transform infrared (FTIR) spectra (4000–600 cm^− 1). Elemental composition analyses were performed through energy dispersive spectroscopy (EDS)/X-ray photoelectron spectroscopy (XPS), while crystal structures were assessed through an X-ray diffraction (XRD) approach. Contact angle (CA) testing was employed to gauge hydrophobicity and hydrophilicity. Study the release of SI in the stomach (pH 1.2 at 37 °C for 2 h) and then, the same material in intestinal conditions (Ringer medium based on a phosphate-buffered saline solution, pH 6.0 at 37 °C for 24 h). SI concentrations were quantified through enzyme-linked immunosorbent assays (ELISAs) using appropriate standards, thereby enabling the quantitative measurement of SI release.

RIII modeling and SI@ZIF-8@MS treatment

All mice were anesthetized through intraperitoneal pentobarbital injection (0.2 mL). WAI modeling was achieved using a linear accelerator that emits 6 MeV X-rays (Elekta Precise, Stockholm, Sweden) through a single dose of radiation at 15 Gy (3 Gy/min, 5 min). All non-abdominal regions were covered using a lead plate (thickness: 1.5 cm). Mice were randomized into control (group A), WAI (group B), WAI + SI (group C), and WAI + SI@ZIF-8@MS (group D) groups. Intestinal experiments were performed by anesthetizing mice using 1.5% sodium pentobarbital. The administration of SI@ZIF-8@MS (30 mg/kg) or PBS control was performed by oral gavage at 1 h prior to WAI or sham irradiation, and was repeated once daily for 3 days post-WAI. A 30 mg/kg dose level and the selected dosing schedule were based on prior reports.

Histological and immunohistochemical (IHC) analyses

After isolation, 4% paraformaldehyde was used to fix the small intestines of experimental mice, after which they were paraffin-embedded, sliced into 4 μm sections, and used for hematoxylin and eosin (H&E), IHC, or periodic acid-Schiff (PAS) staining based on provided instructions. For IHC staining, utilized primary antibodies included anti-Lgr5 (1:2500, Boster, BM4244), anti-Ki67 (1:100, Abcam, ab15580), and anti-γ-H2AX (1:100, CST, #9718). Sections were then imaged via microscopy (Leica, Germany), and two blinded pathologists quantified the numbers of positive cells. A TUNEL assay staining kit (Roche, Germany) was also used as directed and in accordance with prior publications.

Immunofluorescence (IF)

Small intestines were processed using the same approach used for IHC staining, blocking slides using 10% normal donkey serum prior to overnight incubation with anti-γ-H2AX (1:400, CST, #9718) or anti-C-caspase3 (1:200, CST, #9664) at 4 °C, followed by a 1 h incubation with a fluorescent secondary antibody at 37 °C. DAPI (Servicebio, China) was then used for nuclear counterstaining, and slides were imaged with a fluorescent microscope (Leica, Germany).

Analyses of blood glucose and zinc levels

Samples of blood were obtained from the tail vein before administration and at a range of post-administration time points. A glucose meter (ACCUCHEK Performa) was used to quantify blood glucose content. Blood samples were also obtained for pharmacokinetic analyses (n = 3). Blood trace elements were analyzed by centrifuging blood samples and collecting the serum for quantification of Zn²⁺ levels therein.

Quantitative real-time PCR (qRT-PCR)

According to the manufacturer’s protocol, total RNA was extracted from the small intestine tissues using Trizol and then reverse transcribed to cDNA using PrimeScript™ RT reagent, and the qRT-PCR was performed using SYBR Green Real-Time PCR assay kit with the 7900HT RTPCR system.

Analyses of intestinal microbiota composition

After collection, fresh feces were stored in liquid nitrogen pending their use for 16 S rRNA sequencing, which was performed via a standard approach with an Illumina HiSeq platform (Novogene Technology Co., Ltd., China) to evaluate the composition of the intestinal microbiota. Sequencing data were analyzed as in prior reports.

Statistical analyses

GraphPad Prism 9.0^® (CA, USA) was used to analyze all data and to construct figures. Experiments were conducted three times, and the presented results are means ± SD. Student’s t-tests were used for pairwise comparisons, and P < 0.05 was chosen as the threshold for significance.

Results and discussion

SI@ZIF-8@MS overview

An overview of the approach to SI@ZIF-8@MS synthesis, the animal modeling strategy for this study, and the mechanism of action for SI@ZIF-8@MS is presented in Fig. 1. The synthesis of SI@ZIF-8 was achieved with a one-pot approach, yielding a core-shell structure through chemical MS grafting (Fig. 1a). A mouse WAI model was established in which mice were orally treated daily with SI@ZIF-8@MS from 1 day prior to irradiation to day 3 post-irradiation, with samples being collected at 3.5 days post-irradiation (Fig. 1b). Mechanistically, MS functions to shield SI@ZIF-8 NPs against undergoing rapid degradation under acidic conditions, allowing SI and Zn²⁺ to be released into the intestinal fluid (Fig. 1b). The radioprotective effects of SI@ZIF-8 may be associated with the increased Zn²⁺ levels and with reductions in the activity of the NLRP3 inflammasome. Notably, SI@ZIF-8 administration was found to remediate radiation-associated changes in the composition of the GI microflora. Together, the results presented below demonstrate the ability of SI@ZIF-8@MS to protect mice against WAI-induced intestinal injury, underscoring the efficacy of this multi-component targeted approach to RIII treatment.

Fig. 1.

(a) Schematic overview of SI@ZIF-8@MS synthesis. (b) Overview of the WAI-based animal model approach and associated treatment strategy.Schematic overview of the transit of SI@ZIF-8@MS through the stomach and its subsequent dissolution within the intestines, thereby exposing SI@ZIF-8 and allowing it to penetrate the intestinal epithelium

SI@ZIF-8@MS characterization

Here, ZIF-8, SI@ZIF-8, and SI@ZIF-8@MS NPs were synthesized as reported previously. SEM, TEM, and AFM images revealed that the SI@ZIF-8@MS NPs were uniformly spherical with a core-shell structure (Fig. 2a-c). Sheet-like crystals were visible on the surface of ZIF-8 spheres (Fig. 2a, SEM), whereas SI is loaded into the internal pores of ZIF-8 and MS located in the outer surface of ZIF-8. The images in Fig. 2c demonstrate that the prepared material resembles a blooming flower. Zhang et al. previously conducted SEM and TEM analyses of ZIF-8 NPs, revealing them to be uniformly spherical, with ZIF-8-loaded Pd exhibiting a cubic structure [14]. The basic ZIF-8 NPs were approximately 200 nm in size (Fig. 2b), whereas SI@ZIF-8@MS NPs were 200–400 nm in size. The Z-potential values of ZIF-8, SI@ZIF-8, SI@ZIF-8@MS are (23.6 ± 3.8mV), (18.7 ± 2.9mV), and (15.8 ± 2.7mV) respectively, in line with other publications. Hao et al. observed similar NP sizes and Zeta potential values when using ZIF-8 encapsulating materials [15].

Fig. 2.

(a) SEM images of SI@ZIF-8@MS, Scale bar: 2000 nm (left)/ 200 nm (right). (b) Particle sizes (left) and Zeta potential values (right) for ZIF-8, SI@ZIF-8, and SI@ZIF-8@MS. (c)TEM images of SI@ZIF-8@MS, Scale bar: 500 nm (left)/ 100 nm (right). (d) XRD patterns for ZIF-8 (lower) and SI@ZIF-8@MS (upper). (e) FTIR spectra for ZIF-8 (lower) and SI@ZIF-8@MS (upper). (f)AFM images of SI@ZIF-8@MS, Scale bar: 2000 nm (left). (g) XPS patterns for SI@ZIF-8@MS. (h) SI release from SI@ZIF-8 and SI@ZIF-8@MS at pH values of 5.4 and 7.4. (i) EDS of SI@ZIF-8@MS. (j) CA of ZIF-8, SI@ZIF-8, and SI@ZIF-8@MS

XRD patterns for SI@ZIF-8@MS exhibited sizes consistent with those of ZIF-8 (Fig. 2d), while the ZIF-8 and SI@ZIF-8@MS spectra presented with stretching vibration peaks at 2925 and 3138 cm^− 1 respectively corresponding to the C-H bond in the imidazole ring and methyl (Fig. 2e). These data support the successful fabrication of SI@ZIF-8@MS [14, 15]. An XPS analysis of the valence state of SI in SI@ZIF-8@MS was next performed, revealing peaks at 1000–1100,280–300 and 380–400 eV consistent with SI molecules present within the lattice ZIF-8 framework but without any formation of stable chemical bonds (Fig. 2g), a conformation that is compatible with the effective release of this loaded drug [16].

The effective delivery of protein cargo remains challenging and is an important area of research. Individual proteins can be readily degraded. In a prior study focused on radiation dermatitis, the IFI6 protein was successfully delivered through erythrocyte membranes to enable its release in a targeted fashion [17]. To assess the pH-responsive degradation of SI@ZIF-8@MS, we study the release of SI in the stomach (pH 1.2 at 37 °C for 2 h) and then, the same material in intestinal conditions (Ringer medium based on a phosphate-buffered saline solution, pH 6.0 at 37 °C for 24 h), and the methodology is based on the article published by Rojas et al. [18]. In the simulated stomach(pH 1.2 at 37 °C for 2 h), more rapid SI@ZIF-8 structural collapse was observed than that for SI@ZIF-8@MS (Fig. 2h), confirming the resistance of SI@ZIF-8@MS to acid-mediated degradation. Prepared mPEG-b-PLLA (MS) microspheres were able to effectively resist digestive fluid-mediated degradation owing to the mPEG coating surrounding these particles, which shielded the internal components from enzymatic activity. Ma et al. previously demonstrated the pH-responsive nature of ZIF-8, revealing a burst effect for their prepared CLO@ZIF-8 platform within a 12 h incubation at a pH of 3 or 5 that was distinct from the gradual sustained release evident at pH 8 [19]. The use of mPEG shells can thus effectively protect SI@ZIF-8 NPs and SI against acid- and enzyme-mediated degradation.

Finally, the drug loading rate test was carried out, which showed that wt% of SI was 23.1% in the final product SI@ZIF-8@MS.Deepika et al. suggest that the optimized formulation of sitagliptin loaded polymeric nanomicelles (PNM 10) was found to be clear, homogenous nanomiceller solution with round spherical structures [20]. PNM 10 showed drug loading of 38.67 ± 0.23%, entrapment efficiency of 79.67 ± 0.54% and % drug release of 82.34 ± 0.78%.Jeremiah‘s study presents the development and production, by twin-screw melt granulation technology, of a high-dose immediate-release fixed-dose combination (FDC) product of metformin hydrochloride (MET) and sitagliptin phosphate (SIT), with drug loads of 80% w/w and 6% w/w, respectively [21]. EDS analyses revealed that Zn accounted for 16.94% of the material (Fig. 2i), primarily corresponding to ZIF-8, while C and N accounted for 45.09% and 26.96%, respectively, consistent with the organic nature of MS and SI. The synthesis approach used in this study did not impact the hydrophilicity of the main ZIF-8 structure (Fig. 2j), while the overall hydrophobicity of the material remained, potentially benefitting its digestion and intestinal absorption [16, 22].

SI@ZIF-8@MS augments survival and reduces RIII severity

In line with prior findings, WAI resulted in a reduction in intestinal contents and severe congestive and hemorrhagic changes within the small intestines. Hao et al. previously developed a radiation-induced model of damage in mice and detected a significant reduction in survival and weight following irradiation in a manner correlated with radiation dose [23]. SI@ZIF-8@MS administration to mice protected against adverse WAI-associated symptoms, maintaining the intestinal contents in these animals (Fig. 3a). SI was also able to significantly mitigate the WAI-induced shortening of the colon length (Fig. 3a, e). Oral SI@ZIF-8@MS administration was associated with significant reductions in weight loss and survival following murine irradiation (Fig. 3b, c). The DDP-4 inhibitor SI was first designed to treat type 2 diabetes [2]. A growing wealth of evidence, however, suggests that SI also exhibits a diverse array of antioxidant, anti-inflammatory, and anti-apoptotic effects [3]. H&E staining of small intestinal sections revealed the shortening and degeneration of the villi in mice exposed to WAI with corresponding crypt damage, while the administration of SI@ZIF-8@MS was able to significantly increase villus length and crypt integrity in these irradiated animals (Fig. 3d). Goblet cells are vital for appropriate barrier function, and significantly fewer of these cells were evident at 3.5 days post-WAI as detected via PAS staining. However, SI@ZIF-8@MS substantially increased goblet cell counts in the intestines of these irradiated mice (Fig. 3f, g). Zhang et al. employed an MS encapsulation strategy to generate insulin microspheres [22], and they observed robust Rhodamine B fluorescence within the intestines of Ins@MIL100/SDS@MS-treated mice at 1 h following administration, supporting the MOF-NP-incorporated microspheres to deliver insulin to the intestine.

Fig. 3.

SI@ZIF-8@MS protects against intestinal injury in mice subjected to WAI. (a) Macroscopic images of the intestines from the indicated groups. (b, c) Survival analyses and body weight measurements of mice in the indicated groups. (d) H&E staining results for the small intestines from treated mice. (e) Colon length measurements. (f) PAS staining results for the small intestines of mice in each group. (g) PAS staining-based goblet cell quantification (n = 5,*P < 0.05)

Together, these findings support the ability of SI@ZIF-8@MS treatment to protect against WAI-induced damage to the murine intestines, with the efficacy of such treatment being superior to that of SI alone (P < 0.05). The delivery of SI to the intestines in a targeted manner has become an important area of research. For example, George et al. sought to establish an economically feasible approach to achieving sustained SI release using composites [12]. In an effort to overcome the harsh GI microenvironment, these composites were prepared using biodegradable polymers including guar gum, chitosan, and poly(vinyl alcohol), in combination with montmorillonite clay as a nano-filler, using a cross-linker in the form of tetraethyl orthosilicate. In mice in which SI@ZIF-8@MS was orally delivered, greater PAS staining intensity was detected relative to that associated with free SI administration, owing to the ability of SI to be readily degraded within the acidic microenvironment. These data demonstrate that MS-containing SI@ZIF-8 NPs were capable of readily facilitating the delivery of SI to the intestines while also improving the subsequent ability of SI to cross the mucosal epithelium. These findings emphasize the ability of SI to be delivered successfully into the systemic circulation following its oral delivery when utilizing MOF-NP-incorporating microsphere carriers.

SI@ZIF-8@MS treatment abrogates intestinal inflammation, oxidative stress and the apoptotic death of IECs

To evaluate the possible mechanisms whereby SI@ZIF-8@MS protects mice against RIII, TUNEL staining was employed to detect apoptotic death within the murine intestines. This staining revealed that WAI mice presented with more TUNEL-positive cells within their intestinal crypts as compared to controls, while SI@ZIF-8@MS administration markedly reduced the extent of crypt cell TUNEL positivity in these mice (Fig. 4a, b). The regeneration of the intestinal epithelium is dependent on continuous intestinal stem cell (ISC) self-renewal, and these cells express leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), with their activity being vital for the maintenance of GI homeostasis under luminal stress conditions [24]. In line with the above findings, there were fewer Ki67 + cells in the gut of mice from the WAI group relative to controls, while SI@ZIF-8@MS administration effectively abrogated this change in proliferative activity (Fig. 4c, d). Here, a significant reduction in Lgr5-positive cells was observed in WAI group mice as compared to controls, whereas SI@ZIF-8@MS treatment was associated with the restoration of these cell populations (Fig. 4e, f). IHC staining was additionally employed for the detection of Ki67 + cells as a means of clarifying how SI@ZIF-8@MS impacts crypt cell proliferation and differentiation. Together, these findings support the ability of SI@ZIF-8@MS to suppress intestinal inflammation and promote IEC regeneration in mice that have undergone WAI.

Fig. 4.

SI protects IECs against radiation-induced DNA damage and apoptotic death. (a, b) TUNEL staining was employed to evaluate the impact of SI on apoptosis in the context of RIII. (c-f) IHC staining results for Lgr5 + and Ki67 + cells in samples of murine small intestinal tissue. (g) Cumulative Zn²⁺ release profiles. (h) Blood glucose levels over time after oral administration.Representative RT-PCR of NLRP3 (i), Caspase-3 (j) and γ-H2AX(k) mRNA levels in the RIII. (n = 5,*P < 0.05)

SI administration in mice has been shown to reduce the WAI-induced upregulation of pro-inflammatory cytokines including IL-6, TNFα, and IL-1β [5]. Here, SI@ZIF-8@MS treatment enhanced Lgr5 + and Ki67 + cell survival within the murine small intestines, helping to preserve the regenerative functionality of ISCs while maintaining appropriate GI homeostasis. TUNEL-positive cell numbers also declined in irradiated mice that had been treated with SI@ZIF-8@MS, and caspase-3 and γ-H2AX levels were similarly reduced. This provides support for the ability of SI to protect against RIII via the inhibition of inflammatory activity, the induction of more robust ISC regeneration, and the attenuation of radiation-induced DNA damage and apoptotic death in IECs [5, 25].

Serum trace element analyses were also conducted to assess Zn²⁺ release from SI@ZIF-8@MS preparations. A gradual increase in the rate of Zn²⁺ release was observed from 12 to 24 h, peaking around 24 h and then gradually declining thereafter (Fig. 4g), with levels falling by half within ~ 48 h. This suggests that SI@ZIF-8@MS microspheres exhibit a two-stage release process, including the early release of SI@ZIF-8NPs from the microspheres followed by SI and Zn²⁺ release from these NPs. Song et al. previously indicated that in a solution design to simulate the tumor microenvironment, Zn species were released from their prepared ZIF-8/DOX-HA@MIP samples with a corresponding decrease in particle size attributable to the synergistic effects of pH, glutathione, and hyaluronidase, thus exhibiting excellent biodegradability [26].

At pH levels rose to 5.4, SI and Zn²⁺ were gradually liberated from NPs while these release rates grew faster at a pH of 7.4. This pH-dependent release of SI and Zn²⁺ from these NPs may be associated with the uniform mesoporous ZIF-8@MS-based NP structures and the varying sizes of insulin at different pH levels. ZIF-8 may therefore undergo ion-responsive degradation within ionic solutions, altering its initial morphology and limiting its direct biological utilization. Wang et al. found that ZIF-8 can function as a bioactive material compatible with the surface modification of synthetic polymers, suggesting its utility in the context of in situ bone regeneration [27]. In line with prior analyses [4, 12], direct oral free SI administration was not associated with the effective absorption of this drug via the GI tract. Following oral SI@ZIF-8@MS administration (30 mg/kg), WAI model rats exhibited significant declines in blood surfer and this change was more gradual and more pronounced than that associated with oral SI (Fig. 4h). Pharmacokinetic analyses indicated that maximum plasma insulin levels were evident after 24 h, remaining elevated for more than 12 h. The ability of SI@ZIF-8@MS to reduce blood sugar in a prolonged manner thus confirms the utility of this nanoplatform as a tool for the oral delivery of insulin. The more delayed onset of action for oral SI@ZIF-8@MS as compared to oral SI is likely attributable to drug absorption via the GI tract as a result of gradual uptake and transport by IECs whereupon it can enter systemic circulation [12]. Selecting an appropriate timing for administration is thus vital for the practical application of this oral drug delivery system.

The multiprotein NLRP3 inflammasome complex can, when excessively activated, promote the onset and progression of a range of inflammation-associated diseases [28, 29]. Next, NLRP3 mRNA expression were evaluated in the intestines of the established model mice (Fig. 4i). This approach revealed that WAI promoted NLRP3, caspase-3, and γ-H2AX mRNA expression in the intestines of these mice, while SI@ZIF-8@MS administration reduced the upregulation of these factors. SI@ZIF-8@MS treatment was thus capable of suppressing NLRP3 inflammasome activation within IECs in response to irradiation. In a prior study, NLRP3 was shown to play a key role in the pathogenesis of radiation dermatitis [28]. While radiation can significantly increase NLRP3 levels and activity, in this context the application of soy protein hydrogels was sufficient to reduce NLRP3 levels and to thereby minimize cutaneous inflammation. Cleaved caspase 3 (C-caspase3) levels were also analyzed in the intestines of these experimental mice, revealing that there were more C-caspase3 mRNA in the WAI group as compared to the control group, whereas SI was capable of significantly reducing these levels of caspase 3 activity. This is consistent with the ability of SI to prevent small intestinal cell apoptosis in mice subjected to WAI (Fig. 4j. The unrestrained activation of the NLRP3 inflammasome can be spurred by many stimuli, thereby inducing progressive systemic inflammatory activity and initiating adaptive immunity [29, 30]. NLRP3 upregulation can trigger caspase-3-mediated inflammation and induce the upregulation of caspase-3, γ-H2AX, and other downstream inflammatory mediators [31]. NLRP3 inflammasome-mediated pyroptotic cell death has been linked to the exacerbation of RIII and radiation-induced pulmonary injury [30]. In the present study, increases in NLRP3, caspase-3, and γ-H2AX activity were evident in mice subjected to irradiation, while SI@ZIF-8@MS was capable of inhibiting the radiation-induced activation of the NLRP3 inflammasome and suppressing the consequent release of proinflammatory cytokines. SI@ZIF-8@MS may thus be capable of protecting against RIII at least in part owing to its ability to suppress NLRP3 inflammasome activation. γ-H2AX is closely related to the repair of DNA damage, and prior studies have shown that its expression can be suppressed by the application of compounds capable of promoting DNA repair [32]. RT-PCR revealed that radiation-treated mice harbored significantly more γ-H2AX foci as compared to control Groups, whereas SI@ZIF-8@MS treatment attenuated the magnitude of this effect (Fig. 4k). SI thus appears to be capable of reducing the severity of radiation-induced DNA damage. Shentu et al. previously suggested that MCC950-mediated NLRP3 blockade was capable of attenuating the incidence of neuroinflammation and LPS-induced anxiety-like behaviors while reducing γ-H2AX levels [33]. These results demonstrate the ability of SI@ZIF-8@MS to protect RIII against DNA damage induced upon irradiation.

SI@ZIF-8@MS treatment remediates WAI-Induced gut dysbiosis

The composition of the gut microbiome has previously been suggested to impact radioresistance in the host [34, 35]. Hua et al. determined that moderate and high L. casei ATCC334 doses were capable of promoting enhanced mucosal barrier integrity and changes in the structure and metabolic activity of the gut microbiota that were conducive to protecting against RIII [34]. Yang et al. further discussed the fact that, despite being common in the clinic, radiotherapy-based treatment of malignancies in the pelvic and abdominal regions can often result in dysbiosis owing to the disruption of the normal mutualistic relationships that exist between bacteria and their hosts within the GI tract. Accordingly, microbial therapy can serve as a direct approach to reversing radiation-related dysbiosis and thereby protecting against related inflammatory activity in the intestines [35].

Next, the impact of SI@ZIF-8@MS administration on the gut microbiota in WAI model mice was assessed. Pronounced WAI-induced declines in Chao1 and Shannon index values were evident following irradiation (Fig. 5a, b), whereas SI@ZIF-8@MS treatment notably abrogated these changes. Principal coordinate analysis (PCoA) further confirmed the significant changes in gut microbiota composition in response to WAI and SI@ZIF-8@MS treatment in these mice (Fig. 5c). At the genus level, the WAI group exhibited increases in relative Bacteroides, Blautia, Helicobacter, and Lactobacillus abundance with corresponding reductions in Alistipes and Dubosiella abundance, while treatment with SI@ZIF-8@MS largely reversed these irradiation-related changes in bacterial abundance (Fig. 5d, e).

Fig. 5.

The impact of SI@ZIF-8@MS on the composition of the gut microbiota. Feces were collected from different treatment groups for 16 S rRNA sequencing at 3.5 days post-WAI (n = 5). (a, b) Chao1 and Shannon indices. (c) PCoA results based on weighted unifrac distances. (d, e) The relative abundance of the top 10 genera in the analyzed groups. (f) A heat map presenting genus-level differences in relative bacterial abundance. (g, h) KEGG pathway (g) and MetaCyc pathway (h) analyses of SI@ZIF-8@MS group samples

KEGG and MetaCyc pathway analyses were additionally conducted to better understand the possible mechanisms whereby SI@ZIF-8@MS protects against RIII (Fig. 5f and h), revealing that this treatment strategy may favorably alter certain metabolic pathways in the gut microflora. These results highlight a potentially new mechanism through which RIII can interact with gut dysbiosis, providing a foundation for future studies. Zhao et al. previously demonstrated that irradiation resulted in intestinal fibrosis in mice and a corresponding shift in gut microflora composition and beta diversity, with both mouse age and time after irradiation having an impact on these gut microbial bacterial profiles [36]. Long-term supplementation with NMN was sufficient to relieve fibrosis in the intestines while mitigating ionizing radiation-related shifts in the composition of the gut flora. The probiotic species Akkermansia muciniphila and key metabolic pathways including amino acid metabolism and the biosynthesis of other secondary metabolites pathway were also more abundant in irradiated mice following treatment with NMN.

The exposure of the abdomen to radiation can alter the composition of the gut microflora, driving a reduction in bacterial diversity characterized by lower levels of beneficial bacterial species including Lactobacilli and Bifidobacteria [37]. Intestinal dysbacteriosis can further exacerbate the severity of radiation enteritis, weakening intestinal barrier functions and contributing to the expression of higher levels of inflammatory mediators, thus contributing to more pronounced enteritis. Given this relationship, evaluating the composition of the gut microbiome may offer utility as a biomarker for detecting or monitoring radiation enteritis. Treatment strategies including antibiotics, probiotics, and fecal microbiota transplantation all offer potential value as means of restoring the makeup of the gut microbiome, and may thus provide utility as treatments for radiation enteritis [35]. These data suggest that SI may exert its radioprotective benefits at least in part by improving the composition of the gut microflora that was impaired in response to WAI.

Conclusion

Here, the ability of SI@ZIF-8 to protect mice against WAI-induced weight loss and death while improving enterocyte regeneration and preserving intestinal morphological integrity was convincingly demonstrated. SI@ZIF-8 was found to significantly inhibit radiation-induced proinflammatory cytokine production while reducing γ-H2AX expression and apoptotic IEC counts. From a mechanistic perspective, the radioprotective effects of SI@ZIF-8 may be linked to increased Zn²⁺ levels and lower levels of activity of the NLRP3 inflammasome activity. Notably, SI@ZIF-8 was able to significantly remediate radiation-related changes in the composition of the gut microbiome.

Together, these data highlight the promise of SI@ZIF-8@MS as a candidate multi-effective treatment option for RIII that can resist gastric acid-mediated dissolution while enhancing Zn²⁺ levels within the intestines, suppressing inflammation, mitigating oxidative injury, and remediating gastrointestinal dysbiosis.

Author contributions

Dan He: Design and Project administration.ZhiHui Li: Material design and characterization. Min Wang and Dejun Kong: Cell and Animal experiments. Wenyan Guo: Validation, Formal analysis. Xuliang Xia and Dong Li: Writing - review & editing, Supervision. Daijun Zhou: Design and Experiment.

Funding

This study was supported by the Scientific Research Project of China Baoyuan (CBYI202103). The School level Program of Army Medical University (2022XQN26). The Youth Program of Sichuan Natural Science Foundation (2023NSFSC1841). The Program of General Hospital of Western Theater Command of PLA (2021-XZYG-C49/2024-YGJC-A11/2024-YGJS-A04).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The experimental design, experimental process and animal killing methods were reviewed by the Laboratory Animal Welfare and Ethics Committee of Army Medical University, which met the requirements of animal ethics and animal welfare.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.