Graphene oxide-loaded rapamycin coating on airway stents inhibits stent-related granulation tissue hyperplasia

Abstract

OBJECTIVES

Our objective was to explore the safety and efficacy of a graphene oxide-loaded rapamycin-coated self-expandable metallic airway stent (GO@RAPA-SEMS) in a rabbit model.

METHODS

The dip coating method was used to develop a GO@RAPA-SEMS and a poly(lactic-co-glycolic)-acid loaded rapamycin-coated self-expandable metallic airway stent (PLGA@RAPA-SEMS). The surface structure was evaluated using a scanning electronic microscope. The in vitro drug-release profiles of the 2 stents were explored and compared. In the animal study, a total of 45 rabbits were randomly divided into 3 groups and underwent 3 kinds of stent placements. Computed tomography was performed to evaluate the degree of stenosis at 1, 2 and 3 months after the stent operation. Five rabbits in each group were sacrificed after the computed tomography scan. The stented trachea and blood were collected for further pathological analysis and laboratory testing.

RESULTS

The in vitro drug-release study revealed that GO@RAPA-SEMS exhibited a sudden release on the first day and maintained a certain release rate on the 14th day. The PLGA@RAPA-SEMS exhibited a longer sustained release time. All 45 rabbits underwent successful stent placement. Pathological results indicated that the granulation tissue thickness in the GO@RAPA-SEMS group was less than that in the PLGA@RAPA-SEMS group. The TUNEL and hypoxia-inducible factor-1α staining results support the fact that the granulation inhibition effect in the GO@RAPA-SEMS group was greater than that in the PLGA@RAPA-SEMS group.

CONCLUSIONS

GO@RAPA-SEMS effectively inhibited stent-related granulation tissue hyperplasia.

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INTRODUCTION

Airway stenosis is a common occurrence in both benign and malignant diseases such as lung cancer, tuberculosis and post-tracheal intubation procedures [1]. This constriction can lead to respiratory difficulties, respiratory failure, obstructive pneumonia and even life-threatening situations. However, only a minority of patients can undergo surgical interventions to eliminate stenosis [2]. Currently, metallic airway stents are widely applied for benign and malignant airway stenoses [3]. The placement of stents can rapidly relieve airway stenosis, alleviating symptoms of respiratory distress immediately [4].

However, stent-related granulation tissue hyperplasia limits the application of metallic stents, which can lead to recurrent stenosis [5]. Post-stenting restenosis is the most common and severe complication following airway stent placement and is the primary reason for stent removal. Previous studies have indicated a restenosis rate ranging from 6.8% to 40% after tracheal stent placement [6]. Therefore, developing a drug-coated airway stent capable of inhibiting mucosal granulation tissue proliferation is imperative for reducing the occurrence of airway restenosis after stent placement and enhancing the long-term efficacy of airway stenting—a pressing clinical challenge that requires prompt resolution.

Research indicates that the hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in wound healing, granulation tissue overgrowth and scar formation [7]. The activation of HIF-1α induces the upregulation of downstream molecules such as vascular endothelial growth factor and transforming growth factor beta 1, stimulating angiogenesis and leading to granulation tissue proliferation [8]. Rapamycin (RAPA) is a macrolide antibiotic that not only has bactericidal effects but also exerts anti-inflammatory effects by reducing the activity of immune cells and altering bacterial cell activity [9]. Sevilla et al. [10] proposed that the anti-inflammatory and immunosuppressive effects of RAPA can inhibit the growth of airway mucosal granulation tissue and local inflammatory reactions.

Studies suggest that RAPA can interrupt the late-phase response to T-lymphocyte activation, inhibit cell progression from the G1 phase to the S phase and disrupt the binding of interleukin-2 to its receptor, directly suppressing the synthesis of proteins closely associated with cell growth and proliferation [11]. Furthermore, as an inhibitor of the mammalian target of rapamycin (mTOR), RAPA can reduce the expression of HIF-1α and its downstream molecules by blocking the PI3K/Akt-mTOR-HIF-1α pathway, effectively inhibiting granulation tissue proliferation [12]. Therefore, theoretically, loading RAPA onto metallic airway stents may suppress local inflammatory reactions and granulation tissue overgrowth, consequently reducing the incidence of airway restenosis after a stent is implanted.

Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable organic polymer commonly used as a coating material for drug-eluting stents to achieve effective drug loading onto the scaffold [13]. Studies have shown that when RAPA-coated tracheal stents were implanted in an animal model of tracheal stenosis, the degree of granulation tissue proliferation and tracheal restenosis was lower than that of plain nickel-titanium alloy stents [14]. However, due to the limited drug-loading capacity of PLGA, the drug content in coated stents is generally low [15].

Graphene is a novel 2-dimensional carbon nanomaterial discovered in recent years that has emerged as a promising material for drug delivery applications. Its oxygenated derivative, graphene oxide (GO), possesses an exceptionally high surface area, allowing for efficient drug loading on both the upper and lower surfaces as well as on the edges [16]. Additionally, GO exhibits excellent biocompatibility, making it an ideal coating material for drug-eluting stents [17].

This study involved the fabrication of an airway stent with a graphene oxide-loaded RAPA coating of a self-expandable metallic airway stent (GO@RAPA-SEMS) and was designed to evaluate the safety and efficiency of GO@RAPA-SEMS in a rabbit model.

MATERIALS AND METHODS

Preparation of drug-coated stents

Uncovered self-expandable metallic stents (SEMSs) with diameters of 8 mm and 20 mm in length were purchased from Nanjing Micro-tech Company, Nanjing, China). The dip-coating method was used to develop the coated stents. The coating solution of PLGA@RAPA-SEMS had a RAPA-to-PLGA mass ratio of 1:10. The GO@RAPA-SEMS coating solution was a mixture of GO and PLGA at a mass ratio of 1:20 and a RAPA-to-mixture solution at a mass ratio of 1:10. A scanning electron microscope (SEM) was used to evaluate the morphology of SEMS, PLGA@RAPA-SEMS and GO@RAPA-SEMS.

In vitro drug release study of drug-eluting stents

A phosphate-buffed saline (PBS) buffer solution was prepared by first creating a 0.1 mol/l NaOH solution. Next, NaH2PO4·2H2O (0.78 g) was added to a 100-ml volumetric flask, 39.5 ml of 0.1 mol/l NaOH solution was added, and the mixture was brought to 100 ml with distilled water to obtain a PBS buffer solution with a pH of 7.4. Subsequently, 0.4 g of sodium dodecyl sulfate was measured in a 100 ml volumetric flask, the prepared PBS buffer solution was added, and the total volume was brought to 100 ml to create the in vitro release medium.

In vitro release experiment

Each set of stents was placed into its respective centrifuge tubes. The volume of the in vitro release medium for the stents was 7 ml. The timing from the moment all the stents were introduced into the fully temperature-controlled oscillating incubator was noted. At regular intervals, forceps were used to extract the stent. The stent was transferred to a new release medium, and the release concentration of the stent was assessed using an ultraviolet–visible spectrophotometer.

Animal study

This study was approved by the ethics committee of the Experimental Animal Center of Zhengzhou University. Forty-five experimentally healthy rabbits with stable vital signs and overall good health were randomly selected. The rabbits were randomly allocated into 3 groups (SEMS, PLGA@RAPA-SEMS and GO@RAPA-SEMS), with each group comprising 15 rabbits. Each group was further divided into 1-month (5 rabbits), 2-month (5 rabbits) and 3-month-old (5 rabbits) subgroups based on the time of euthanasia. A 24-hour light cycle, an ambient temperature of 22–25°C and an air humidity level between 55% and 58% were maintained.

All rabbits were denied water for 6 h prior to the procedure. The stent was placed in the middle trachea under the guidance of fluoroscopy. Multislice computed tomography (MSCT) was performed according to a schedule to assess the degree of tracheal stenosis. Three-dimensional reconstruction of the trachea was performed using postprocessing software, and the inner diameters of the stenotic and normal tracheal lumens were measured on MSCT mediastinal window images to calculate the degree of stenosis. The formula for calculation was as follows: S = [1 − (d1 × d2)/(D1 × D2)] × 100%, where S is the degree of stenosis, d1 is the maximum transverse diameter at the stenotic site, d2 is the longitudinal diameter of the tracheal lumen perpendicular to d1, D1 is the maximum transverse diameter of the trachea and D2 is the longitudinal diameter of the tracheal lumen perpendicular to D1. Five rabbits in each group were sacrificed after CT. The stented trachea and blood were collected for further pathological analysis and laboratory testing.

Alanine aminotransferase and aspartate aminotransferase levels were used to evaluate liver function. The levels of blood-urea nitrogen and creatinine (CR) were used to evaluate renal function. All the indictors were assayed using an automated biochemical analyser (Rayto Life and Analytical Sciences Co., Shenzhen, China).

Tracheal specimens were collected and fixed in 10% neutral buffered formalin for 24 h. After being embedded in paraffin, these samples were sectioned at 5 µm and stained with haematoxylin and eosin for analysis. Tracheal sections were also subjected to Masson's trichrome staining. The thickness of the granulation tissue was analysed using ImageJ software (https://imagej.net/ij/). Additionally, histological sections of tracheal specimens were stained with antibodies targeting HIF-1α and subjected to TUNEL. Measurements were performed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).

Statistical analyses

The results are presented as the mean ± standard deviation for all independent experiments. The data were analysed using two-way ANOVA or Student's t-test, as appropriate, with GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA).

RESULTS

In vitro drug release of drug-coated stents

A morphological analysis of SEMS, PLGA@RAPA-SEMS and GO@RAPA-SEMS was conducted through an SEM. Figure 1A shows that the surface morphology of SEMS reveals a relatively smooth structure without cracks or evident defects, which is favourable for drug coating. Successful drug loading was noticeable in both the PLGA@RAPA-SEMS and GO@RAPA-SEMS groups. High single-layer graphene oxide loading can be seen in the SEM images of GO@RAPA-SEMS compared to those of PLGA@RAPA-SEMS. The high specific surface area of monolayer graphene oxide provides more effective sites for drugs.

The drug release behaviour of PLGA@RAPA-SEMS and GO@RAPA-SEMS was investigated in PBS (pH 7.4). As depicted in Fig. 1B and C, the release profiles of PLGA@RAPA-SEMS and GO@RAPA-SEMS can be divided into two stages: a burst release phase and a sustained release phase. Specifically, both PLGA@RAPA-SEMS and GO@RAPA-SEMS exhibited sudden release on the first day. However, PLGA@RAPA-SEMS essentially entered a plateau phase after the seventh day, indicating that the drug payload of the PLGA-coated stent had been mostly released by the seventh day. In contrast, GO@RAPA-SEMS maintained a certain release rate on the 14th day, not reaching the maximum drug release. Its drug payload significantly surpassed that of PLGA@RAPA-SEMS and exhibited a longer sustained release time. This result provides a theoretical basis for achieving superior long-term efficacy after stent placement.

Multislice computed tomography evaluation after stent placement

All the stents were successfully displaced without pneumothorax or haemoptysis. The MSCT results showed that the stenosis rates at 1, 2 and 3 months poststenting in the SEMS group were 49.15% ± 2.14%, 52.33% ± 2.51% and 58.67% ± 2.52%, respectively. In the PLGA@RAPA-SEMS group, the percentages were 28.33% ± 1.52%, 19.67% ± 1.53% and 44.33% ± 2.08%, respectively. The percentages in the GO@RAPA-SEMS group were 13.33% ±1.53%, 13.66% ± 1.46% and 17.52% ± 2.08%, respectively (Fig. 2). The differences were statistically significant (Fig. 3A).

Drug-coated stents inhibit granulation hyperplasia

The granulation thickness was measured from haematoxylin and eosin-stained slides. As shown in Fig. 4, the SEMS group exhibited the thickest granulation tissue, and the granulation tissue thicknesses of the PLGA@RAPA-SEMS and GO@RAPA-SEMS groups were reduced. Notably, at the third month, the granulation tissue thickness in the GO@RAPA-SEMS group was less than that in the PLGA@RAPA-SEMS group. This result suggests that the long-term efficacy of GO@RAPA-SEMS is superior to that of PLGA@RAPA-SEMS, likely due to the high drug-loading capacity and long-term stable release of GO.

Collagen is a major component of the extracellular matrix, and excessive collagen deposition gradually results in scar formation, leading to tracheal restenosis [18, 19]. The Masson staining results (Fig. 5) showed that the degree of collagen deposition in the SEMS group at 1, 2 and 3 months was 20.32% ± 1.06%, 34.21% ± 0.95% and 49.79% ± 1.55%, respectively; in the PLGA@RAPA-SEMS group, it was 19.70% ± 2.31%, 25.29% ± 0.98% and 46.60% ± 1.08% at 1, 2 and 3 months, respectively. Collagen deposition in the GO@RAPA-SEMS group was 17.54% ± 1.63%, 22.54% ± 1.61% and 22.34% ± 2.01% at 1, 2 and 3 months, respectively. This finding indicates that RAPA-loaded stents can inhibit collagen deposition, and similarly, at the third month, collagen deposition in the GO@RAPA-SEMS group was lower than that in the PLGA@RAPA-SEMS group (Fig. 3B). These differences were statistically significant, suggesting the inferior long-term efficacy of PLGA@RAPA-SEMS.

Finally, we performed TUNEL immunofluorescent staining on tracheal specimens, as shown in Fig. 6. SEMS exhibited lower TUNEL-positive areas, indicating that RAPA can promote apoptosis in proliferative tissues and inhibit granulation tissue proliferation, with statistically significant differences (Fig. 3C). We investigated molecules related to the HIF-1α pathway. Rapamycin is an early-discovered mTOR inhibitor [20], and HIF-1α is an important downstream molecule of mTOR [21]. Importantly, the expression of HIF-1α and its related proteins is closely associated with neovascularization and granulation tissue proliferation. Immunohistochemical staining for HIF-1α (Fig. 7) indicated that the expression of HIF-1α was significantly inhibited in the PLGA@RAPA-SEMS and GO@RAPA-SEMS groups, with the lowest expression level in the GO@RAPA-SEMS group at the third month (Fig. 3D). This further confirms the superiority of GO, because it can load greater amounts of RAPA and continue to exert its effects. Thus, it significantly improves its long-term efficacy after the placement of GO@RAPA-SEMS and reduces the risk of airway restenosis.

Safety evaluation of drug-coated stents

To investigate the safety of SEMS, PLGA@RAPA-SEMS and GO@RAPA-SEMS after airway placement, we assessed liver and kidney function in experimental rabbits by extracting blood specimens. The alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine levels in the 3 groups of experimental rabbits remained within normal ranges. This result indirectly confirms the effectiveness and safety of the 3 airway stents used in the trachea in this study.

DISCUSSION

The placement of an SEMS is an effective method for treating airway stenosis, providing effective support to narrow lumens and rapidly restore airway patency [22]. However, complications associated with stent placement are a major concern in clinical practice and a problem that urgently needs to be addressed [23]. Among these complications, restenosis after the treatment of airway stenosis is the most common and serious complication following airway stent placement, and it is one of the main reasons for stent removal. Because a foreign body continuously dilates the trachea, the stent inevitably stimulates the formation of granulation tissue, leading to restenosis. Under the stimulation of a foreign body, various inflammatory cells interact and ultimately induce the proliferation of fibroblasts and capillaries. Fibroblasts serve as an important source of extracellular matrix and connective tissue in the early stages of inflammation, providing a necessary survival environment for other inflammatory cells. In the late stages of inflammation, they produce collagen, eventually forming scar tissue, leading to restenosis [24].

Compared with silicon stents, SEMSs have the advantages of easy placement and a lower migration rate. Although silicon stents are preferred for benign stenoses, the design and research of drug-eluting metallic stents are still topics of interest. A recent review by Johnson et al. [25] summarized the research on SEMSs as a ‘backbone’ and their application results in animal studies.

In this study, graphene, a newly discovered two-dimensional planar carbon nanomaterial, has high oxygen-containing derivatives, and graphene oxide (GO) has an ultra-high specific surface area that can efficiently load drugs through the top and bottom surfaces and edges. Compared with traditional PLGA, GO has advantages such as a large drug load and stable and persistent release [16]. In addition, GO has good biocompatibility and is an ideal coating material for airway stents. The results of this study indicate that RAPA-loaded PLGA@RAPA-SEMS and GO@RAPA-SEMS can exert their effects through multiple pathways, such as promoting the apoptosis of proliferating tissue, inhibiting the proliferation of granulation tissue, alleviating local inflammation and inhibiting the PI3K/Akt-mTOR-HIF-1α pathway. These effects provide a favourable microenvironment for significantly reducing airway stenosis.

Hypoxia-inducible factor-1α (HIF-1α) is a protective transcription factor in mammalian and human cells that exists in a complex formed by binding with specific coenzymes in a low-oxygen environment [26]. Many studies have shown that HIF-1α plays a crucial role in wound healing, scar formation and granulation tissue proliferation. In recent years, with the advent of drug-eluting stents, drugs can be coated onto polymer-coated stents to achieve sustained release [27]. Scholars both domestically and internationally have prepared paclitaxel, cisplatin and dexamethasone-eluting stents that have been successfully used in animal models and found that these drugs can be released continuously for more than a month, with inhibitory effects on local inflammation and granulation tissue proliferation [28, 29]. Rapamycin is a macrolide antibiotic that has potent bactericidal and anti-inflammatory effects and is widely used in clinical treatment. Rapamycin can block the late-stage response to T-lymphocyte activation, inhibit cells from entering the S phase from the G1 phase and block the binding of interleukin-2 to its receptor, thereby inhibiting the synthesis of proteins closely related to cell growth and proliferation. Additionally, RAPA is a specific mTOR inhibitor that is theoretically able to inhibit the PI3K/Akt-mTOR-HIF-1α pathway and to reduce the expression of HIF-1α and downstream molecules such as vascular endothelial growth factor and transforming growth factor beta 1, thus inhibiting abnormal neovascularization and granulation tissue proliferation [12].

Limitations

This study has several limitations. First, the small sample size may affect the significance of the findings. However, these results can guide further, more detailed research. Moreover, this technique has the potential to be applied in humans who experience granulation tissue hyperplasia. Second, the wound-healing process after stent placement in a normal trachea may differ from that in a trachea with benign or malignant stenosis.

CONCLUSION

GO@RAPA-SEMS can effectively inhibit stent-related granulation tissue hyperplasia better than PLGA@RAPA-SEMS.

FUNDING

This work was supported by the Natural Science Foundation of Henan Province of China (222300420349).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

Author contributions

Zongming Li: Conceptualization; Data curation; Funding acquisition; Investigation; Methodology; Software; Visualization; Writing—original draft. Xin Lu: Data curation; Investigation; Methodology; Visualization. Kunpeng Wu: Data curation; Formal analysis; Investigation; Visualization; Writing—original draft. Jing Wang: Data curation; Investigation; Methodology. Yahua Li: Data curation; Investigation; Methodology. Yifan Li: Formal analysis; Methodology. Kewei Ren: Conceptualization; Funding acquisition; Investigation; Methodology; Supervision; Writing—original draft; Writing—review & editing. Xinwei Han: Conceptualization; Investigation; Supervision; Writing—review & editing.

Reviewer information

The European Journal of Cardio-Thoracic Surgery thanks Noriyoshi Sawabata, Luca Ampollini and the other anonymous reviewers for their contributions to the peer review process of this article.

REFERENCES

ABBREVIATIONS

ABBREVIATIONS
 
• GO

  graphene oxide

 
• GO@RAPA-SEMS

  graphene oxide-loaded rapamycin-coated self-expandable metallic airway stent

 
• HIF-1α

  hypoxia-inducible factor-1α

 
• mTOR

  mammalian target of rapamycin

 
• PBS

  phosphate-buffed saline

 
• PLGA

  poly(lactic-co-glycolic) acid

 
• PLGA@RAPA-SEMS

  poly(lactic-co-glycolic) acid-loaded rapamycin-coated self-expandable metallic airway stent

 
• RAPA

  rapamycin

 
• SEMS

  self-expandable metallic stent

Author notes