Targeted inhibition of gastric cancer progression via a chitosan-RNU11 siRNA nanoparticle delivery system: mechanistic insights and therapeutic potential

Abstract

Background

Gastric cancer (GC) remains a leading cause of cancer-related mortality worldwide. The prognosis for advanced GC is poor, necessitating innovative therapeutic strategies. Small interfering RNA (siRNA) offers a promising avenue for gene-specific cancer treatment. However, effective delivery of siRNA remains a challenge. Chitosan, a natural biopolymer, has demonstrated potential as a nanocarrier due to its biocompatibility and ability to facilitate targeted delivery. This study investigates the therapeutic efficacy of a novel chitosan-RNU11 siRNA drug delivery system in inhibiting GC progression and elucidates its underlying mechanisms.

Methods

Chitosan nanoparticles (CH-NP) loaded with RNU11 siRNA (Si-RNU11 + CH-NP) were synthesized and characterized for size, zeta potential, and siRNA release efficiency. GC cell lines (AGS and HGC27) were treated with various formulations, and functional assays, including EdU proliferation, Transwell invasion/migration, and cell cycle/apoptosis analysis, were conducted. The in vivo efficacy of Si-RNU11 + CH-NP was evaluated in GC xenograft mouse models. Expression of Wnt pathway components was analyzed by Western blot and qRT-PCR.

Results

Si-RNU11 + CH-NP exhibited a uniform size distribution (~ 85 μm) with a zeta potential of + 14 mV, ensuring colloidal stability and efficient cellular uptake. siRNA release exceeded 95% under physiological conditions. In vitro, Si-RNU11 + CH-NP significantly reduced GC cell proliferation, migration, and invasion while inducing apoptosis and G1/G2 cell cycle arrest. Western blotting revealed downregulation of Wnt pathway components, including Wnt1, β-catenin, GSK-3β, c-myc, and CyclinD1. In vivo, Si-RNU11 + CH-NP treatment led to a significant reduction in tumor size and decreased expression of Ki67 and RNU11.

Conclusion

This study demonstrates that the chitosan-RNU11 siRNA drug delivery system effectively inhibits GC progression by targeting the Wnt signaling pathway. The integration of chitosan nanoparticles and RNU11 siRNA provides a novel therapeutic approach for GC, addressing the critical challenge of efficient siRNA delivery and highlighting the potential for clinical application.

Introduction

Gastric cancer (GC) is a malignant neoplasm that originates from the epithelial cells of the gastric mucosa (Smyth et al. 2020), commonly found in people over 50 years old, with a male to female ratio of 2:1. Changes in diet, increased work pressure and infection of Helicobacter pylori have led to a trend of younger patients with GC (Amieva and Peek 2016). GC can manifest in any region of the stomach, with inconspicuous early-stage symptoms. These symptoms often resemble those of gastritis, gastric ulcer, and other chronic gastric diseases, which can easily go unnoticed. The early diagnosis rate of GC in China remains suboptimal, thus indicating a pressing need for improvement. The lymphatic route serves as the primary pathway for metastasis in GC, exhibiting a substantial incidence of lymph node involvement in advanced stages, reaching up to 70%, and early GC can also have lymph node metastasis (Ma et al. 2022). Patients with metastasis have a significantly poor prognosis. At present, the pathogenesis of GC has not been fully elucidated, and it is crucial to further reveal the drugs and genes that have significant therapeutic effects in the development of GC.

Nanotechnology has revolutionized cancer therapeutics, particularly in drug delivery (Jia et al. 2025). Chitosan, a natural polysaccharide derived from chitin, has emerged as a promising biomaterial for nanotechnology applications due to its excellent biocompatibility, biodegradability, and non-toxic properties (Maleki Dana et al. 2021; Zhang and Li 2024). Chitosan-based nanoparticles have shown great potential in cancer therapy as carriers for chemotherapeutic agents and biomolecules. These nanoparticles can encapsulate therapeutic molecules, enhance their stability, and enable targeted delivery to tumor sites, thereby reducing systemic toxicity (Ning et al. 2023). Recent studies have highlighted the anti-tumor potential of chitosan-based systems. For example, Imran H et al. revealed the importance of chitosan-mediated nanoparticles for DOX drug delivery in tumor chemotherapy (Imran et al. 2023). Choukaife H et al. showed that oral administration of chitosan nanoparticles can be used for the treatment of colorectal cancer (Choukaife et al. 2022). Research by Rout SR et al. suggests that chitosan can be used to treat oral mucositis, which is a common complication of cancer treatment (Rout et al. 2023). In addition, Rong L et al. revealed that injectable nanocomposite hydrogel of hyaluronic acid-chitosan derivatives can photothermally chemotherapeutic cancer (Rong et al. 2023). Studies have shown that chitosan, as a key compound in the drug delivery system of GC plays a pivotal role in treatment of GC (Shafabakhsh et al. 2020).

Small interfering RNA (siRNA) has emerged as a revolutionary tool for targeted cancer therapy due to its ability to silence specific oncogenes. RNU11, a small nuclear RNA involved in U12-dependent splicing, plays a critical role in gene expression regulation. Although its role in GC is not well understood, preliminary studies suggest that RNU11 may contribute to tumor progression. For instance, Wang et al. showed that RNU11 may be involved in the regulation of gene expression in bladder cancer (Wang et al. 2021). Relevant investigations have recently revealed that certain small nuclear RNAs may exert a pivotal influence on the progression of GC. Therefore, RNU11, as a small nuclear RNA (Park et al. 2010), may also be associated with the development of GC. However, the effect of chitosan-RNU11 siRNA drug delivery system on the occurrence and development of GC and its mechanism of action are still unknown.

To address these gaps, we developed a novel chitosan-based RNU11 siRNA delivery system to investigate its therapeutic efficacy in GC. Unlike existing studies focusing solely on chitosan nanoparticles or siRNA, our approach integrates these components to achieve a synergistic effect. Moreover, this study elucidates the involvement of the Wnt signaling pathway in mediating the anti-tumor effects of the chitosan-RNU11 siRNA delivery system. Our findings provide a novel perspective on targeting RNU11 to inhibit GC progression and highlight the therapeutic potential of chitosan-based delivery systems. By bridging the gap between chitosan-mediated drug delivery and siRNA therapeutics, this research advances the field of GC treatment and offers a promising avenue for clinical application.

Materials and methods

Sources of animals, cells and reagents

SPF Balb/c mice (5 weeks, n = 40) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. AGS and HGC27 cells were came from ATCC Cell Collection Center. RPMI-1640 medium was purchased from Gibco, Transwell chambers from Corning, and apoptosis detection kit from Thermo Fisher Scientific. Crystal violet and paraformaldehyde were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Antibodies against Wnt1(ab63934), GSK-3β(ab93926), Bax(ab3191), C-caspase(ab32351), Bcl-2(ab16904), β-catenin(ab68183), LGR5(ab75850), c-myc(ab185656) and CycinD1(ab62151) were purchased from Abcam, Inc., and reverse transcription kits were purchased from Takara, Inc., Japan.

Synthesis of nanoparticles

Chitosan nanoparticles (CH-NPs): Chitosan nanoparticles were synthesized using a previously described ionic gelation method (Redhwan et al. 2024). Briefly, 200 mL of 0.5% (w/v) chitosan solution, dissolved in 1% (v/v) acetic acid, was heated to 100 °C under magnetic agitation and reflux. Subsequently, 85 μL of 25 mM HAuCl4 was added dropwise to the chitosan solution and boiled for 1 h under continuous stirring until the solution turned dark red, indicating nanoparticle formation. To remove unreacted materials, the solution was centrifuged at 22,000 ×g for 1 h at 4 °C, and the precipitate was resuspended in deionized water.

Assembly of Si-RNU11 + CH-NP in layers: To load negatively charged siRNA molecules onto NPs as a second layer, siRNA is first dissolved in TPP solution, and then CH-NPs is re-suspended in a 10 mM HEPES buffer with pH = 7. At the same time, siRNA [TPP solution of siRNA (0–640 nM) (0.1% w/v)] with different weight ratios and CH-NPs (1:1–12.5) were mixed and incubated for 1 h under magnetic continuous agitation (400 RPM). Finally, the final chitosan layer is applied by dispersing the nanoparticles in a 0.5% (w/v) chitosan solution that is remixed under continuous agitation for 1 h (CH-NPs). Excess chitosan and unattached siRNA were removed by centrifugation at 22,000 ×g, 4 ℃, 1 h, and the purified particles were re-suspended in RNase-free water.

The characterization of nanoparticles was observed by scanning electron microscope (SEM)

The size and morphology of chitosan nanoparticles were determined by scanning electron microscopy. SEM was used to scan the surface of the nanoparticles, and the defined elements of interest were displayed on the display with different color points, respectively, and correspond to the acquired secondary electron images. The higher the element content of the area, the more bright spots displayed, the higher the brightness.

The absorbance of nanomaterials was analyzed by infrared light

Infrared light is used to analyze the absorbance of nanomaterials. First, qualitative analysis is carried out, and then data processing is carried out. The transmittance in the toolbar is selected and the peak value is marked. The powder of potassium bromide and acetanilide is about 200:1, dried under an infrared lamp, and fully ground in an agate mortar to make it evenly mixed. Take out a small amount of sample evenly spread in a clean mold, and make a transparent sheet on the tablet press. This piece is mounted on the solid sample rack, and the sample rack is placed in the sample pool of the infrared spectrometer, and the absorption spectrum is obtained by wave number scanning.

X-ray photoelectron spectroscopy (XPS) analysis

X-ray photoelectron spectroscopy (XPS) measurements were performed on the K-Alpha instrument (Thermo Scientific, East Grinstead, UK) using a monochromatic X-ray beam (Al Kα) with a spot size of 300 × 300 μm. The spectrometer was equipped with a charge-compensated flood gun that corrects the energy shift caused by the positive charge by fixing the c1s line at 284.4 eV. In the peak fitting process, the Shirley-type background was subtracted from the spectrum, and the peaks were fitted with a symmetric Gaussian function.

Particle size analysis

Particle size analyzer was used observing the particle size and stability distribution. Use the injector to push the sample slowly into the sample pool to ensure uniform sample distribution. The measurement type (particle size or Zeta potential) is selected in the software and the corresponding measurement parameters are set. After the measurement is completed, the particle size distribution and Zeta potential of the particles are recorded and the file saved.

Cell culture, grouping and cell transfection

AGS cells and HGC27 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells were seeded at a density of 5 × 10³ cells/well in 96-well plates for proliferation assays and 2 × 10⁵ cells/well in 6-well plates for functional studies. After reaching logarithmic growth, cells were divided into six groups: si-NC, si-RNU11, CH-NP, si-RNU11 + CH-NP, si-RNU11 + CH-NP + si-LGR5, and si-RNU11 + CH-NP + si-LGR5 + BML284. Transfections were performed using Turbofect, and cells were treated with CH-NPs or 10 μM BML284 for 48 h before analysis.

EdU proliferation assays

After digestion cells in logarithmic growth phase were resuspended in complete medium and seeded in 96-well plates with 100 μl cell suspension in each well. After cultured in cell incubator for 12 h, cells were treated according to the cell grouping in Sect. “Synthesis of nanoparticles”. EdU was diluted to 20 μM with complete medium, and 100 μl of diluted EdU working solution was added to each well of the 96-well plate. After incubation for 2 h, cells were fixed for staining. Finally, the EdU-labeled cells were observed under a fluorescence microscope, and cells were counted.

Transwell invasion and migration assays

Matrigel was diluted at a ratio of 1:6, and then coated in upper chamber of Transwell, 100μL gel solution was added to well, and that were placed in incubator for use. After the cells were treated according to method 2.2, and the cell density was observed to be over 80%, trypsin was added for digestion, and the supernatant was removed by centrifugation. Then the cells were resuspended in RPMI-1640 medium, and 200 μL of counted cell suspension was absorbed and inoculated into the upper chamber of Transwell. RPMI-1640 medium was added to the lower chamber, and after 12 h of culture, the cells were washed twice with PBS and fixed with paraformaldehyde for 30 min. After staining with 0.1% crystal violet for 20 min, and adding PBS buffer solution to wash the cells for 3 times, cells were observed by microscope, and the numbers were recorded and counted in 10 random fields.

Cell scratch healing experiments

AGS cells and HGC27 cells were inoculated into 6-well plates, respectively, and cells were grouped according to method 2.2 and cultured. When the confluence reached about 90%, scratches were made in the cell holes with a 200 μL gun head. After being washed with PBS for 3 times, the supernatant was discarded, and the cells were treated with serum-free medium. After 12 h, complete medium was added for culture, and then the scratch condition of cells at 0 h and 24 h was observed, pictures were taken and the healing rate of cell scratches was calculated.

Apoptosis assays

After transfection and treatment of AGS and HGC27 cells according to method 2.2, the cells were cultured in an incubator, and cells were centrifuged after digestion. The supernatant was discarded and washed with 2 mL PBS. The cells were resuspended with 1 × Binding Buffer, and 100 μL of the above suspension was taken into 5 mL EP tubes, 5 μL FITC Annexin V and 5 μL PI were added. After incubation in a light-proof environment for 15 min, 400 μL of 1 × Binding Buffer was added to tube, and cells were detected on the machine.

Cell cycle assays

After AGS and HGC27 cells were collected from each treatment group, cells were washed with PBS for 3 times, fixed in 70% ice ethanol at 4 °C for 30 min, then fully resuspended in PBS buffer containing propidium iodide (40 μg) and RNaseA (100 μg) and incubated for 30 min. Finally, the cell cycle was detected by flow cytometry.

RT-qPCR

According to the instructions of Invitrogen's Trizol method, total RNA was extracted from each group of cells, and the extracted RNA was reversed into cDNA after concentration determination. Taking GAPDH as internal reference, RT-PCR reaction was performed. The reaction conditions were: 95 °C 3 min, 95 °C 45 s, 55 °C 30 s, 72 °C 45 s, 38 cycles.

Western blotting

When AGS and HGC27 cells were cultured to 80% confluence rate, the cells were washed with PBS and the cell lysate was added. After 5 min of lysis at 4 °C, cells were transferred to EP tubes and centrifuged for 20 min. The supernatant protein concentration was measured by BCA method. The protein samples were electrophoresed, transferred to a membrane, and blocked with milk. The primary antibody was added and incubated at 4 °C.The protein was washed 5 times with TBST, and secondary antibody was incubated for 1 h in absence of light, followed by protein expression detection.

Tumorigenic in nude mice

The nude mice were cultured in a specific environment. They were allowed to drink water and eat freely and adaptively fed for 1 week. After that, they were divided into the following 6 groups: Model, si-RNU11, CH-NP, si-RNU11 + CH-NP, si-RNU11-CH-NP + si-LGR5 and si-RNU11-CH-NP + si-LGR5 + BML284. The density of HGC27 cells in each treatment group was adjusted to 5 × 10⁶ cells/ml and subcutaneously injected. After 30 days, mice were killed for tumor weighing, and tumors were removed for paraffin embedding sections for subsequent experiments. All experimental procedures involving animals were studied and approved by the ethics committee of Shanghai Jiao Tong University School of Medicine, Shanghai, China, following the Institutional Animal Care and Use Committee (IACUC) guidelines.

Immunohistochemistry

The nude mice were killed, and part of the tumor tissue was removed and fixed for 24 h. Subcutaneous tumor tissue was taken to make paraffin specimens of the subcutaneous tumor tissue of nude mice, and paraffin specimens were cut into slices, which were then blocked by endogenous peroxidase, washed with PBS, and incubated at 37 °C for 60 min with primary antibody, and incubated with secondary antibody for 30 min. Horseradish enzymes were added to label streptomycin vitellin and incubated for 20 min, DAB was used for color development, and the slices were sealed after re-dyeing and transparency, and observed under a microscope.

HE staining

After the mice in each group were killed, the subcutaneous tumor tissue were obtained and stored in 10% formaldehyde solution. After 24 h, the tissues were taken out for conventional dehydration, paraffin embedding and sliced (4 mm thickness) and then processed according to the HE staining procedure. Finally, the tumors of mice were observed under an optical microscope (× 200).

Statistical analysis

Statistical analysis was performed using Graphpad prism 7.0 software, and protein expression was processed using ImageJ software. P < 0.05 indicates statistically significant difference.

Results

Expression of RUN11 in clinical tissues and its effect on the proliferation of GC cells

To investigate the role of RNU11 in GC progression, qRT-PCR was performed to quantify RNU11 expression in clinical tissues. The results showed that RNU11 expression was significantly elevated in GC tissues compared to adjacent normal tissues (Fig. 1A), suggesting its potential role in tumor development. To evaluate the functional effects of RNU11 on GC cells, knockdown experiments were conducted using RNU11-specific siRNA transfected into HGC27 cells. Silencing RUN11 significantly inhibited cell proliferation, as observed in EdU assays, reduced cell migration in wound healing assays, and decreased invasion in Transwell experiments (Fig. 1B–D). These findings imply that RNU11 promotes GC cell proliferation and metastasis, making it a promising therapeutic target.

Fig. 1

Expression of RUN11 in clinical tissues and its effect on the proliferation of GC cells. A The expression of RUN11 in clinical tissues was first detected by qRT-PCR. B The effect of RUN11 on the proliferation of gastric cancer cells HGC27 was observed by EDU. C The effect of RUN11 on the migration of HGC27 was observed by cell scratch healing. D The effect of RUN11 on the invasion of HGC27 was observed by Transwell

Synthesis and characterization of nanoparticles

The chitosan-RNU11 siRNA nanoparticles (Si-RNU11 + CH-NP) were synthesized and thoroughly characterized. SEM images revealed uniform, spherical nanoparticles with sizes predominantly in the range of 80–90 μm (Fig. 2A). Based on the histogram in Fig. 2B, we analyzed 40 nanoparticles, distributed as follows: ~ 4 particles in the 60–70 μm range, ~ 8 in the 70–80 μm range, ~ 16 in the 80–90 μm range, ~ 8 in the 90–100 μm range, and ~ 4 in the 100–110 μm range. According to the Fourier transform infrared spectroscopy analysis of Si-RNU11 + CH-NP particles, the size of the particles affects the absorption of light (Fig. 2C). Figure 2D shows the SEM element diagram of the nanoparticles, and the detection results confirm the uniform distribution of all elements (C, N, S, Na, O, and P) in the synthesized nanoparticles. High-resolution XPS spectra of Ti2p nuclear grade of tio2 annealed 600-nh3 samples with different Si-RNU11 + CH-NP loads are shown in Fig. 2E–K. Particle size analyzer was used observing the particle size and stability distribution, the results showed that nanoparticle Si-RNU11 + CH-NP has good stability (Fig. 2L). A particle size analyzer confirmed an average nanoparticle diameter of 85 μm, which is optimal for cellular uptake via endocytosis, a critical process for effective drug delivery (Fig. 2M). Nanoparticles in this size range have been shown to balance penetration into tissues and avoidance of rapid clearance by the reticuloendothelial system, enhancing their therapeutic potential. The zeta potential of Si-RNU11 + CH-NP was measured at + 14 mV (Fig. 2N). This moderately positive zeta potential reflects the surface charge imparted by the chitosan layer and ensures colloidal stability by preventing aggregation. These characteristics collectively contribute to the system’s robust performance in vitro and in vivo. Ultraviolet spectrophotometer was used to measure siRNA release rate. The results show that the synthetic material can achieve a release rate of nearly 100% (Fig. 2O).

Fig. 2

Observation of synthesis and characterization of nanoparticles. A SEM scanning; B, C infrared light analysis; D SEM to observe the surface elements of nanoparticles; E–K X-ray photoelectron spectroscopy (XPS) measurement. L Particle size analyzer was used observing the particle size and stability distribution. M Particle size analyzer was used observing infrared spectroscopy analyzes the intensity and wavelength of particles. N Particle size analyzer was used observing siRNA loading rate. O Ultraviolet spectrophotometer was used to measure siRNA release rate

Effects of the chitosan-RNU11 siRNA drug delivery system on tumor development in GC nude mice

To evaluate the therapeutic efficacy of the chitosan-RNU11 siRNA drug delivery system in vivo, GC xenografts were established in nude mice. According to the results of HE staining, immunohistochemistry and RT-qPCR in Fig. 3, compared to Model group, tumor volume of mice for si-RNU11 and CH-NP groups was smaller, ki67 and RNU11 expressions were decreased. While compared with the si-RNU11 and CH-NP groups, the tumor volume of mice in the RNU11si-RNU11+CH-NP group was significantly smaller, ki67 and RNU11 expressions were significantly decreased. It was suggested that the chitosan-RNU11 siRNA drug delivery system had a better killing effect on GC nude mice. These results demonstrate that the chitosan-RNU11 siRNA system exerts potent anti-tumor effects, outperforming its individual components.

Fig. 3

Effects of chitosan-RNU11 siRNA drug delivery system on the killing effect of GC. A, B Comparison of tumor size in each group; C RT-qPCR was used to detect the expression of RNU11; D HE staining was used to observe the tumor in each group; E The expression of Ki67 was detected by immunohistochemistry

Effects of the chitosan-RNU11 siRNA drug delivery system on malignant development of GC cells

In AGS and HGC27 cells, when compared to NC group, cell viability, cell proliferation, the invasion, migration numbers and cell scratch healing rate for si-RNU11 and CH-NP groups was decreased, but cell apoptosis rate was increased. Besides, the numbers of cell proliferation, invasion, migration and cell scratch healing rate in the si-RNU11+CH-NP group were significantly lower than si-RNU11 and CH-NP groups, however, the apoptosis rate showed an increase. In addition, we can also find from the figure that compared to model group, the Bax and C-caspase proteins expressions of si-RNU11 group was significantly increased, Bcl-2, Wnt1, β-catenin, GSK-3β, c-myc and CyclinD1 proteins showed a decrease, while the downregulation trend of Bax and C-caspase proteins and upregulation trend of Bcl-2, Wnt1, β-catenin, GSK-3β, c-myc and CyclinD1 proteins in the si-RNU11+CH-NP group was more obvious. From the results of cell cycle analysis, we can also find that cell numbers of G1 and G2 phases in si-RNU11+CH-NP group is significantly more than that in the si-RNU11 and CH-NP groups, and the cells were blocked in G1 and G2 phases, as shown in Figs. 4, 5. These results highlight the enhanced efficacy of the combined chitosan and RNU11 siRNA system in suppressing GC cell malignancy.

Fig. 4

Effects of chitosan-RNU11 siRNA drug delivery system on the development of AGS cell. A Cell viability assay; B cell cycle detection; C detection of apoptosis-related proteins; D cell proliferation number detection; E cell scratch healing rate detection; F–G cell invasion and migration number detection; H cell apoptosis rate detection

Fig. 5

Effects of chitosan-RNU11 siRNA drug delivery system on the development of HGC27 cell. A Cell viability assay; B cell cycle detection; C detection of apoptosis-related proteins; D cell proliferation number detection; E cell scratch healing rate detection; F–G cell invasion and migration number detection; H cell apoptosis rate detection

Effects of the chitosan-si-RNU11 drug delivery system on the Wnt pathway

Firstly, we analyzed the effect of chitosan-si-RNU11 drug delivery system on Wnt pathway in AGS and HGC27 cells. Compared to Model group, we found that Wnt1, β-catenin, GSK-3β, c-myc and CyclinD1 expressions of in the si-RNU11-CH-NP + si-LGR5 group were decreased, while Wnt1, β-catenin, GSK-3β, c-myc and CyclinD1 expressions in si-RNU11-CH-NP + si-LGR5 + BML284 group were significantly up-regulated. Then, we analyzed whether the chitosan-si-RNU11 drug delivery system affected the malignant development of AGS and HGC27 cells through Wnt pathway. The results showed that compared to Control and si-NC groups, cell viability and cell numbers in the si-RNU11-CH-NP + si-LGR5 group were decreased, the cell scratch healing rate, number of S phases cells was decreased, the apoptosis rate was increased and number of cells in G1 phases was increased. We can also find that Bax and C-caspase expressions were increased, and protein expressions of Bcl-2, Wnt1, β-catenin, GSK-3β, LGR5, c-myc and CycinD1 were significantly decreased. In addition, compared with the si-RNU11-CH-NP + si-LGR5 group, the si-RNU11-CH-NP + si-LGR5 + BML284 group showed a significantly opposite trend. Importantly, we observed a significantly opposite trend in the si-RNU11-CH-NP + si-LGR5 + BML284 group compared with the si-RNU11-CH-NP + si-LGR5 group (Figs. 6, 7). These results suggested that the chitosan-si-RNU11 drug delivery system affects the malignant development of AGS and HGC27 cells by regulating the Wnt pathway.

Fig. 6

Effects of chitosan-RNU11 siRNA drug delivery system on the development of AGS cell by regulating Wnt pathway. A Cell viability assay; B cell cycle detection; C detection of apoptosis-related proteins; D cell proliferation number detection; E cell scratch healing rate detection; F–G cell invasion and migration number detection; H cell apoptosis rate detection

Fig. 7

Effects of chitosan-RNU11 siRNA drug delivery system on the development of HGC27 cell by regulating Wnt pathway. A Cell viability assay; B cell cycle detection; C detection of apoptosis-related proteins; D cell proliferation number detection; E cell scratch healing rate detection; F–G cell invasion and migration number detection; H cell apoptosis rate detection

Effects of the chitosan-si-RNU11 drug delivery system on GC tumorigenesis in nude mice by regulating Wnt pathway

In our study, we evaluated the tumor condition and compared the tumor size of each group through HE staining. On the one hand, we found that tumor size in si-RNU11-CH-NP+si-LGR5+BML284 group was smaller than that in Model group, but larger than that of the si-RNU11-CH-NP+si-LGR5 group. On the other hand, the expressions of Ki67, RNU11 and LGR5 were detected. And it was found that Ki67, RNU11 and LGR5 expressions in the si-RNU11-CH-NP+si-LGR5+BML284 group were significantly lower than that in Model group, while higher than that in si-RNU11-CH-NP+si-LGR5 group (Fig. 8). Reactivation of the Wnt pathway in the si-RNU11-CH-NP+si-LGR5+BML284 group partially reversed these effects, confirming the central role of Wnt pathway inhibition in the observed therapeutic benefits.

Fig. 8

Effects of chitosan-RNU11 siRNA drug delivery system on the killing effect of GC by regulating Wnt pathway. A, B Comparison of tumor size in each group; C RT-qPCR was used to detect the expression of RNU11; D HE staining was used to observe the tumor in each group; E the expression of Ki67 was detected by immunohistochemistry

Discussion

As a serious malignant tumor, the prognosis of GC is affected by many factors, and many patients are diagnosed at the late stage of cancer (Ning et al. 2023). In addition, when GC metastasizes to distant organs or tissues, such as the liver, lungs, bones, etc., the prognosis is very poor (Obermannová and Lordick 2017). Therefore, early diagnosis, comprehensive treatment, and the overall health management of patients are very important for improving the prognosis of GC patients. The pivotal role of chitosan and small interfering RNA in GC has been demonstrated by numerous studies. Based on this, we conducted an in-depth study on the effect of chitosan-RNU11 small interfering RNA drug delivery system on the occurrence and development of GC. The results showed that chitosan-RNU11 small interfering RNA drug delivery system regulates the malignant development of GC by regulating Wnt pathway.

The role of chitosan and small interfering RNA in the development of GC have been found by many researchers. Babaeenezhad E et al. showed that berberine-loaded chitosan/pectin nanoparticles have therapeutic effects on AGS cells (Babaeenezhad et al. 2024). Jiang Z et al. found that chitosan oligosaccharide coupled with selenium can block the growth of GC (Jiang et al. 2021). In addition, studies have shown that carboxymethyl chitosan–norcantharidin coupling can be used as a new polymer therapeutic agent to exert anti-tumor effects on GC. The above studies have shown that chitosan has a significant anti-tumor effect in GC. Currently, many literatures have reported the role of small interfering RNA in the development of GC. For example, Suo WH et al. showed that small interfering RNA targeting AE1 plays an anti-tumor role in GC (Jiang et al. 2021). Another study showed that survivin targeted small interfering RNA inhibited the development of GC cells (Li et al. 2014). Currently, no studies have revealed the effect of chitosan-RNU11 siRNA drug delivery system on GC development, while our study results found that in AGS and HGC27 cells, the cell proliferation, invasion and migration numbers in si-RNU11 and CH-NP groups were less than that in NC group, the cell scratch healing rate was reduced, and the apoptosis rate was increased. In addition, it was found that the malignant development of AGS and HGC27 cells was significantly reduced, and the cells were blocked in G1 and G2 phases, indicating that the malignant development of AGS and HGC27 cells was significantly inhibited by chitosan and RNU11 siRNA. Our findings revealed a novel GC therapeutic drug system: chitosan-RNU11 siRNA drug delivery system, which is also the innovation of this study. In addition, we found that the glycan-RNU11 siRNA drug delivery system plays a therapeutic role in GC by regulating the Wnt signaling pathway. The Wnt pathway is an important signaling pathway that is essential for processes such as cell proliferation, differentiation, and tissue regeneration. However, aberrant activation of the Wnt pathway is closely associated with a variety of tumors. More and more studies have revealed the relationship between Wnt signaling pathways and tumor development, including GC. Zhu L et al. showed that PROX1 promotes breast cancer metastasis through Wnt/β-catenin pathway (Zhu et al. 2022).Scholars such as MR Rubinstein have found that Fusobacteria promote colorectal cancer by inducing the Wnt regulator annexin A1 (Rubinstein et al. 2019). Guo Q et al. showed that iminodimethylsulfoxide mediates GC migration by Wnt pathway (Guo et al. 2021). Besides, Wang Y et al. demonstrated that aberrant activation for Wnt pathway confers resistance to ferroptosis, which enhances chemotherapy sensitivity in patients with advanced GC (Wang et al. 2022). It was also found that UBE2T inhibitors inhibited GC progression by inhibiting Wnt signaling pathway activation (Yu et al. 2021). Our study revealed that the chitosan-RNU11 siRNA system inhibits GC progression by downregulating the expression of Wnt pathway components, including Wnt1, β-catenin, GSK-3β, c-myc, and CyclinD1. This inhibition promotes apoptosis and blocks the cell cycle, leading to reduced tumor growth both in vitro and in vivo. The mechanistic role of Wnt signaling in mediating the therapeutic effects of the chitosan-RNU11 siRNA system underscores its importance as a target for GC treatment.

Compared to other drug delivery systems, such as liposomes and polymeric nanoparticles, chitosan nanoparticles offer distinct advantages, including high biocompatibility, low toxicity, and the ability to facilitate sustained drug release. While liposomes have been widely used for siRNA delivery, their stability and storage conditions remain a challenge. Polymeric nanoparticles often require complex synthesis processes and may have lower biocompatibility. In contrast, the chitosan-RNU11 siRNA system demonstrated excellent stability (zeta potential of + 14 mV), efficient siRNA release (~ 100%), and superior anti-tumor efficacy. Additionally, the dual functionality of chitosan as both a carrier and a bioactive compound enhances its therapeutic potential.

Despite its promising results, our study has several limitations. First, the findings are based on cell and mouse models, and no clinical validation has been performed. Future research should include clinical trials to confirm the system's safety and efficacy in human patients. Second, while we demonstrated the system’s mechanism of action via the Wnt pathway, additional molecular pathways may also be involved. Comprehensive transcriptomic and proteomic analyses could provide further insights into the system's broader effects. Finally, optimizing the synthesis process for scalability and exploring combination therapies with other chemotherapeutic agents could further enhance the clinical applicability of this system.

In summary, our study presents the chitosan-RNU11 siRNA drug delivery system as a novel and effective therapeutic strategy for GC. By inhibiting the Wnt signaling pathway, this system significantly reduces tumor growth and malignant progression in GC. Our findings offer a foundation for future research and clinical applications in GC treatment, while also highlighting areas for improvement and further exploration.