Reducing Neointimal Hyperplasia in Post-Vessel Anastomosis Surgery: A Study on Bivalirudin-Loaded Carbopol 940 Gel.

doi:10.21203/rs.3.rs-3575143/v1

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Research Article

Reducing Neointimal Hyperplasia in Post-Vessel Anastomosis Surgery: A Study on Bivalirudin-Loaded Carbopol 940 Gel.

https://doi.org/10.21203/rs.3.rs-3575143/v1

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Background:

After vascular anastomosis, stenosis at the site of anastomosis is a significant concern as it can lead to severe illness and even death. The primary cause of stenosis in graft anastomoses is the excessive growth of vascular neointima.

Methods:

We used a bivalirudin-loaded carbopol 940 gel, which can effectively inhibit platelet aggregation. Bivalirudin possesses thrombin-inhibitory properties and can suppress platelet activation and aggregation. Bivalirudin was mixed with carbopol 940 gel to achieve slow release. In vivo experiments were conducted on Wistar rats, and longitudinal incisions were made on the abdominal aorta of the rats. Subsequently, the gel was applied around the vascular incision site. The incision was then sutured with polypropylene suture lines.

Results:

The drug-releasing profile showed that approximately 60% of bivalirudin was released in 24 hours, and the remainder was slowly released after 50 hours. WST-1 assay and live/dead staining results obtained from A7r5, L929, and HUVEC revealed that the gel is not cytotoxic. In the in vivo study, the vessel walls in the group untreated with the gel increased by 233.3% in four weeks, whereas it increased by 2.8% in the group treated with the gel (p = 0.039).

Conclusions:

Bivalirudin-loaded carbopol 940 gel can inhibit postoperative neointimal hyperplasia and improve the outcomes of vascular repair surgery.

Carbopol 940 gel

Bivalirudin

vascular anastomosis

intimal hyperplasia

Vascular surgery has become increasingly crucial in modern medicine as it can address various issues related to the circulatory system. Cardiovascular surgery is concerned with the diagnosis and treatment of various conditions affecting the arteries and veins, including peripheral artery disease [1, 2], aneurysms [3–5], carotid artery disease [6], and coronary artery disease [7–9]. Arterial bypass surgery is crucial for restoring blood flow to a blocked artery or bypassing a diseased section of an artery.

Initial injury to the blood vessels may cause the vessel walls to overgrow. This is because inflammatory cells in the vessel walls produce cytokines, which create an environment that promotes the migration and proliferation of vascular smooth muscle cells (VSMCs). The migration and proliferation of VSMCs can lead to hyperplasia in the intimal smooth muscle, which can cause neointimal hyperplasia (NIH) after vascular anastomosis [10, 12], potentially leading to recurrent vascular stenosis [10–14]. This may result in poor blood flow and, in severe cases, complete vessel occlusion. This can lead to severe complications such as stroke in patients with carotid artery stenosis [6] and acute myocardial infarction, which can cause sudden death in patients with coronary artery stenosis [15, 16].

Maintaining the long-term patency of bypass vessels is a significant problem in vascular surgery. Surgical guidelines suggest antiplatelet and anticoagulation medications to prevent postoperative occlusion and stenosis [15–17]. However, these medications may cause bleeding tendencies that can result in gastrointestinal bleeding or intracranial hemorrhage [9, 16]. We aimed to prevent NIH through localized medications using bivalirudin, an antiplatelet agent that blocks the NIH signaling pathway.

Bivalirudin is a synthetic version of the naturally occurring drug hirudin [18, 19]. It primarily acts as an anticoagulant. It functions similarly to antithrombin by directly inhibiting the activity of thrombin [20]. Additionally, it inhibits platelet activation and aggregation, which may reduce the chances of VSMC proliferation and migration [21, 22]. We aimed to prevent NIH by suppressing thrombin and platelet function.

Carbopol 940 gel, a crosslinked polyacrylic acid and synthetic polymer, is often used as an emulsifier in food, cosmetic products, and drug delivery systems. This is a pH-sensitive gel, changing its swelling behavior and rheological properties according to the external pH [23–25]. The gel provides sufficient viscosity to create an area for bivalirudin–platelets interactions, making it an ideal gel medium for our purposes.

Synthesis of bivalirudin-loaded carbopol 940 gel

We dissolved 0.5 g of carbopol 940 powder (Emperor Chemical Co., Taipei, Taiwan) in 50 mL of deionized water to obtain a 1% (w/v) solution. After mixing evenly for 2 h, the solution was placed under ultraviolet (UV) light for 1.5 h in an autoclave. A 5-µM bivalirudin solution (Sigma-Aldrich, Burlington, Massachusetts, USA) solution was filtered using a membrane filer with a pore size of 0.22 µm. After filtering, the two solutions were mixed in a volume ratio of 1:1 to obtain bivalirudin-loaded carbopol 940 gel (C-Biv).

Analysis of bivalirudin-loaded carbopol 940 gel

Drug release profile

Drug-release profiles are used to measure the concentration and rate at which a drug is released from a carrier coated with the drug. In this study, UV–Vis spectrophotometry was used to analyze the release rate of bivalirudin from C-Biv.

C-Biv gels with bivalirudin diluted at the concentrations of 2, 4, 6, 8, and 10 mM were prepared and their absorbance were measured at 264 nm. Deionized water was used as a reference to obtain the calibration curve. Subsequently, 100 µL of 1% carbopol 940 gel and 100 µL of 10^− 2-M bivalirudin aqueous solution were mixed in equal volumes to form a 0.5% carbopol 940 gel containing 5 × 10^− 3-M bivalirudin. The gel was placed at the bottom of a 1.5-mL microcentrifuge tube, and 100 µL of deionized water was added to the top layer. The tube was then placed in an incubator heated to 37°C. The top layer was periodically replaced with 100 µL of fresh deionized water, and the absorbance of the gel was measured at 264 nm. This process was repeated for seven days to obtain the release profile of bivalirudin.

In vitro study

Cells

We used a mouse fibroblast cell line (L929, BCRC, Hsinchu, Taiwan ), rat aortic smooth muscle cell line (A7r5, BCRC, Hsinchu, Taiwan), and human umbilical vein endothelial cells (HUVECs, BCRC, Hsinchu, Taiwan) to conduct in vitro investigations.

L929 cells were cultured in an Eagle's minimal essential medium (Sigma-Aldrich, Burlington, Massachusetts, USA) containing 10% fetal bovine serum ( Gibco, Waltham, Massachusetts, USA) and 1X antibiotic-antimycotic (AA, Wako Pure Chemical, Osaka, Japan) at 37°C and 5% CO₂. The cells were passaged every three days.

A7r5 cells were cultured in a Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, Burlington, Massachusetts, USA) containing 10% FBS and 1X AA at 37°C and 5% CO₂. The cells were changed every two or three days and passaged once a week.

HUVECs were cultured in an endothelial cell growth medium (Sigma-Aldrich, Burlington, Massachusetts, USA) at 37°C and 5% CO₂. The cells were changed every two or three days and passaged once a week. The experiments were conducted after passaging the cells four to six times and thawing them.

Biocompatibility

The biocompatibility of C-Biv was evaluated using the WST-1 assay (Takara Bio, Kusatsu shi, Japan) on L929 cells, A7r5 cells, and HUVECs according to the ISO 10993-5 standard. The unmodified culture medium was used as the control group. The cells treated with extract media containing 0.2-g/mL Al₂O₃ (Sigma-Aldrich, Burlington, Massachusetts, USA) and 0.2-g/mL zinc diethyldithiocarbamate (ZDEC, Sigma-Aldrich, Burlington, Massachusetts, USA) were used as the negative and positive control groups, respectively. The experimental group was added with 0.2-g/mL extract of carbopol 940 encapsulated with and without bivalirudin. The experiment lasted for three days. On the first day, L929 cells, A7r5 cells, and HUVECs were seeded in 96-well plates at a density of 10⁴ cells/well, and the corresponding control- and test-group extract media were placed in an incubator at 37°C and 5% CO₂ for one day. On the second day, the extract media were filtered using a 0.22-µm filter to form a sterile filtrate. Subsequently, the culture medium in the original cell-culture plate was replaced with the extract-medium filtrates and incubated at 37°C and 5% CO₂ for one day. On the third day, the filtrate was replaced with a pre-configured WST-1 reagent and incubated in the dark at 37°C and a 5% CO₂ for 2 h. Absorbance was measured at 450 nm using a multifunctional microplate reader.

Cytotoxicity

Cytotoxicity was evaluated using a live/dead staining kit (Invitrogen, Waltham, Massachusetts, USA) according to the ISO 10993-5 standard. A 0.2-g/mL specimen was added to the culture medium and incubated for 24 h at 37°C to create an extract medium. The cells were then placed in 96-well plates at a density of 1×10⁵ cells per well and incubated for one day at 37°C. The samples and cells were treated for one day with the extract medium instead of the culture medium. On the first day, the cells were treated with the unmodified culture medium and 0.2-g/mL Al₂O₃ to obtain the control group and negative control group, respectively. The cells were treated with 0.2-g/mL of C-Biv was to obtain the experimental group. On the second day, the culture medium in the cell culture plate was replaced with the filtered solutions extracted on the first day, and the cells were cultured for one day. On the third day, a live/dead staining reagent was added to the culture medium at a concentration of 0.5 µL/mL and 2 µL/mL. The positive control group was added with Triton X-100 10 min before the reagent was replaced with the filtered extraction solution, and the cells were co-cultured. Finally, the filtered extraction solution was replaced with the reagent. The cells were stained in an incubator at 37°C and 5% CO₂ for 30 min in the dark, and the cell activity was observed under a fluorescence microscope.

Cell proliferation

The proliferation of A7r5 cells was evaluated using the WST-1 reagent. Cells were seeded in 96-well plates at a density of 1x10⁵ cells/well and incubated for two days at 37°C and 5% CO₂. Subsequently, the original media was replaced with a medium containing 0.5% fetal bovine serum and 4 NIH units/mL thrombin (Sigma-Aldrich, Burlington, Massachusetts, USA). C-Biv was added using the Transwell system. After incubating the cells for two days at 37°C and 5% CO₂, cell growth activity and proliferation were assessed with the WST-1 reagent.

Animal study

The animals were cared for in accordance with the Guidelines for the Care and Use of Laboratory Animals established by the Council of Agriculture Executive Yuan, R.O.C. The animals were obtained from BioLASCO Taiwan Co., Ltd. All procedures were performed with the approval from the Animal Use Review Board of the Chang Gung Memorial Hospital (IACUC number 2020112401).

Six male Wistar rats aged 10–12 weeks and weighing 300–350 g were used. The rats were divided into two groups: the control and experimental groups. Both groups received arterial incisions. The control group (group PR) were treated with a simple PROLENE® suture-line (PR) repair. The wounds of the experimental group (group C-Biv) were treated with C-Biv before primary vessel repair. After four weeks, three rats from each group were euthanized for analysis.

General anesthesia was induced using isoflurane. Prior to surgery, a prophylactic dose of cefazolin sodium (20 mg/kg, Cefa injection; Taiwan Biotech Co. Ltd., Taiwan) was administered. Midline laparotomy was performed to access the inferior vena cava and abdominal aorta. Hematological and biochemical tests were performed on the collected blood samples. Systemic heparin sodium (100 IU/kg; Pine, Huons Co., Ltd., POS, Korea) was administered via the inferior vena cava. Bulldog clamps were applied to the proximal and distal infrarenal aorta. An 8-mm long longitudinal aortotomy was carefully made on the superior surface of the infrarenal abdominal aorta using an Istar knife (961502, 15° Collet). The aortotomy was then sutured using three simple sutures, either with PR (group PR, control, n = 3) or after treatment with 1-mL C-Biv (group C-Biv, experimental, n = 3). Haemostasis was achieved by applying gauze compression for approximately 2 min. The surgical layers were gradually closed. Oral intake was permitted 2 h post-procedure. Additionally, oral pain management with ibuprofen was incorporated into the daily drinking water.

Postoperative care included daily monitoring for surgical site infections and pain assessment. Neomycin ointment was applied twice daily as a part of wound care. After four weeks, the rats were euthanized, and the aortotomy site, including a 5-mm average aorta margin, was excised for further analysis.

Histology analysis

After the aortotomy sites were removed, the aortas were dehydrated in alcohol and embedded in paraffin. Consecutive serial sections with a size of 3 mm and transverse sections of the aorta was obtained using a microtome (Leica RM 2125RT). Five random sections of the aortotomy line were examined. Hematoxylin and eosin (H&E) staining was used to assess endothelialization and overall vascular morphology. We determined the arterial wall thickness (AWT) ratio at the operation site to that at the non-operation location to determine the pace at which the AWT increased.

Immunohistochemical analyses were performed to evaluate arterial wall hyperplasia in different layers. Proliferation of the cells was measured using the proliferating cell nuclear antigen (PCNA, D3H8P, XP® Rabbit mAb #13110, D4K9N, Cell Signaling Technology©, Massachusetts, USA) in terms of the proliferating cell (PC) growth rate (PC thickness at the operation location/PC thickness at the non-operation site). Additionally, the hyperplasia of VSMCs was evaluated via staining using alpha-smooth muscle actin (αSMA, D4K9N, XP® Rabbit mAb #19245, D4K9N, Cell Signaling Technology©, Massachusetts, USA) antibody. The VSMC proliferation rate was determined by dividing the thickness of the VSMCs at the operation site by that at the non-operation location.

The slides were first deparaffinized and rehydrated. The slides were submerged in a 1X citrate unmasking solution and heated in a microwave until boiling began. Subsequently, the temperature was maintained between 95° C and 98°C for 10 min. The slides were washed in dH₂O and incubated in 3% hydrogen peroxide for 10 min. Next, we blocked the slides by treating them with 100–400 L of the recommended blocking solution for 1 h at 25°C to 28°C. PCNA and αSMA were diluted in an antibody diluent (SignalStain®) in the ratios 1:4000 and 1:320, respectively. The primary antibody solutions were applied to the slides, which were then incubated overnight 4°C. The slides were washed three times using a wash buffer. Next, each portion of the slides was treated with SignalStain® Boost Detection Reagent and then incubated in a humidified chamber for 30 min at room temperature. SignalStain® DAB was then applied to each part of the slides and carefully observed. The slides were then dehydrated, rinsed in dH₂O, and counterstained with hematoxylin. Finally, the slides were mounted using coverslips and mounting materials.

The thicknesses of the intima (including the neointima) and media layers at the site of the aortotomy were measured using NIS-Elements software (Nikon Instruments Inc., Tokyo, Japan). These values were compared to the thickness of the layers at the non-operation site on the sections stained with H&E, PCNA, and αSMA. A digital camera connected to a photomicroscope (ECLIPSE Ti; Nikon) was used to capture the images of the stained sections.

Statistical analysis

Statistical graphs were obtained and data analyses were performed using GraphPad 9 for Mac OS (Version 9.4.1 (458), July 18, 2022). The mean and standard deviation of the data were obtained from a minimum of three replicates. Using a single sample t-test, statistical differences across studies were examined using the GraphPad Prism® software. Stars (*) denote statistical differences between groups: one, two, and three stars denote p-values below 0.05, below 0.01, and below 0.001, respectively. A p-value of 0.05 or less was regarded as statistically significant.

Analysis of Bivalirudin-loaded Carbopol 940 gel

Drug releasing profile

Figure 1a shows the standard calibration curve illustrating the relationship between the concentration of bivalirudin and absorbance at 264 nm. The absorbance of the solution varied linearly with bivalirudin concentration. Furthermore, bivalirudin was released from C-Biv in two stages: 63.36 ± 5.72% of bivalirudin was rapidly released within 24 h, after which the release rate decreased with longer release times. After 72 h, 68.27 ± 6.35% of bivalirudin was released (Figure 1b).

In vitro study

Biocompatibility

All the three cell lines exhibited more than 70% cell viability towards C-Biv gel, as required by ISO10993-5, with no apparent cytotoxicity (Figure 2). Carbopol 940 had a mild inhibitory effect on HUVECs, which decreased their cell viability. Nonetheless, HUVECs exhibited a cell viability of 92.68% ± 6.45 with C-Biv.

Cytotoxicity

Except for the positive control group, all groups showed green fluorescence (Calcein AM), indicating the presence of live cells. By contrast, the positive control group exhibited red fluorescence (EthD-1), corresponding to the presence of dead cells (Figure 3).

Cell proliferation

The cell viability of the positive control group co-cultured with A7r5 and 4 NIH units/mL thrombin for 48 h increased by approximately 15%. This indicates that, at this concentration, thrombin promotes the proliferation of smooth muscle cells. When C-Biv was co-cultured with A7r5 cells in a transwell for 48 h, the cell viability decreased to approximately 70%. This showed that C-Biv slightly affected the growth of A7r5 cells; however, it still satisfied the biocompatibility criterion (cell viability over 70%). When 4 NIH units/mL thrombin and C-Biv were added simultaneously and co-cultured with A7r5 cells for 48 h, the cell viability decreased to approximately 55%. This proved that bivalirudin inhibited thrombin by binding to it (Figure 4).

In vivo study

Lab data of animals

Four weeks after surgery, alanine aminotransferase (ALT) and creatinine levels and whole blood analysis results were within a normal range in both PR and C-Biv groups of rats (Table 1). This indicates that C-Biv has good biocompatibility and no hepatorenal or systemic biotoxicity.

Table 1 Biochemistry and whole blood analysis results for PR and C-Biv groups (ATL: Alanine aminotransferase, Cr.: Creatinine, Hb: Hemoglobin, PLT: platelet, WBC: white blood cells)

	ALT(U/L)	Cr.(mg/dL)	WBC(1000/μL)	Hb(g/dL)	PLT(1000/μL)
PR	39 - 42	0.4 - 0.5	7.2 - 11.4	12.2 - 14.4	567 - 1136
C-Biv	35 - 40	0.6 - 0.7	4.7 - 9.5	12.2 - 14.9	929 - 1225

Histology analysis

Hematoxylin and eosin staining

H&E staining of the aortic wall tissue was performed after the rats were sacrificed four weeks after abdominal aortic repair surgery (Figure 5a). The rate of AWT increase in the PR and C-Biv groups were 333.3% and 102.8%, respectively. Compared to the PR group, the C-Biv group exhibited a considerably lower rate of AWT increase (p = 0.0390) (Figure 5d).

PCNA and αSMA immunohistochemistry

As can be observed from Figure 5b, The PR and C-Biv groups exhibited a PC-increase rate of 273.4% and 116.8%, respectively. Compared to the PR group, The C-Biv group exhibited a considerably lower PC-increase rate (p = 0.0069) (Figure 5e).

The rates of VSMC proliferation (VSMC) (Figure 5c) exhibited by the PR and C-Biv groups were 373.8% and 112.8%, respectively. Compared with the PR group, The C-Biv group exhibited considerably lower rates of VSMC proliferation (p = 0.0479) (Figure 5f).

Bivalirudin was selected because it can directly inhibits thrombin and prevent platelet aggregation. Previous in vitro studies have shown that bivalirudin exhibits concentration-dependent inhibition of thrombin, with effective inhibitions observed over the concentration range from 100 nM to 125 mM [18, 20–22, 26]. The concentration of bivalirudin used in our gel was 2.5 µM, which is consistent with the previous study findings.

Carbopol 960 gel is a negatively charged polymer commonly used in daily applications such as cosmetic products and emulsifiers in food and drug delivery systems. Bivalirudin encapsulated in carbopol 940 gel was released slowly, extending the drug action time. Owing to the properties of carbopol 940 gel, the concentration of C-Biv can be controlled to achieve a gel viscosity that avoids vascular occlusion while being easily removable like an aqueous solution. The final product was comfortable for use in an in vivo study.

The drug-release profile of C-Biv revealed that the bivalirudin located on the surface of the hydrogel is released quickly in the early stages, releasing 60% of the drug in the first 24 h (Fig. 1). Subsequently, bivalirudin encapsulated in the inner layer of the hydrogel was released. Therefore, bivalirudin can be rapidly released from C-Biv and quickly bind to thrombin in the blood, inhibiting platelet activation. This helps prevent intimal hyperplasia due to the rapid aggregation and activation of platelets at the wound site after surgery. Additionally, the slow release of bivalirudin encapsulated in the inner layers of C-Biv increases the action time of the drug and maintains the smoothness of blood vessels.

The WST-1 assay and live/dead staining results demonstrated that C-Biv does not have cytotoxic characteristics (Fig. 2–3). C-Biv significantly inhibited thrombin, providing insights into the direct inhibitory role of C-Biv on thrombin. C-Biv did not lose its inhibitory activity even when it was converted into a gel. Moreover, its non- cytotoxic nature adds to its safety for clinical use. The data from the blood samples obtained after the sacrifice of the animals showed that C-Biv not only reduced localized cell proliferation but also imposed no adverse effects on the animals (Fig. 4).

The pathological results from the rat aorta four weeks after the operation showed that the C-Biv Group had reduced NIH at the surgical location. The results of H&E staining and IHC staining with PCNA and αSMA indicated that C-Biv can reduce vascular wall thickening, reduce PCs, and reduce VSMC proliferation (Fig. 5). These results support our hypothesis that c-Biv can inhibit NIH and maintain long term patency at a site of vascular anastomosis.

Bivalirudin was successfully mixed with the Carbopol 940 gel. The drug was released adequately over time. C-Biv demonstrated good biocompatibility in L929, A7r5, and HUVECs. Moreover, it exhibited no cytotoxicity or biological toxicity. AWT, PCNA, and VSMC counts were significantly lower in the C-Biv group than in the PR group after abdominal aortic repair surgery in rats. According to the pathological results of the rat aortas, C-Biv is a promising drug for inhibiting NIH and maintaining a long-term patency for vessel anastomosis.

This study still needs clinical trials to validate C-Biv's efficacy in human patients, optimize the drug release profile for various clinical contexts, assess its long-term effects, and the development of biodegradable gel matrices. Furthermore, evaluating its patient-specific response factors and promoting awareness among healthcare professionals and patients is essential for successful clinical implementation. This comprehensive approach to future research will not only enhance our understanding of C-Biv but also facilitate its transition from the laboratory to clinical practice, ultimately benefiting patients undergoing vascular anastomosis procedures.

1. AA: Antibiotic-antimycotic

2. AWT: Arterial wall thickness

3. C-Biv: Bivalirudin-loaded carbopol 940 gel

4. H&E: Hematoxylin and eosin

5. HUVEC: Human umbilical vein endothelial cells

6. NIH: Neointimal hyperplasia

7. PCNA: Proliferating cell nuclear antigen

8. PC: Proliferating cell

9. VSMC: Vascular smooth muscle cell

Acknowledgments

We would like to extend our gratitude to the Chang Gung Memorial Hospital's Laboratory Animal Center in Keelung for their valuable assistance in conducting the studies and animal care, which contributed significantly to the successful completion of this study. We are extremely grateful to Mr. Hong-Yen Liang from the Chang Gung Hospital Research Department in Keelung for his invaluable help and direction with sample processing, histology, and staining.

Data Avalibility

The data and materials that support the findings of this study are available

from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All animal procedures were approved by the Approval of Animal Use Protocol Chang Gung Memorial Hospital (ICUUCA number: 2019093001).

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

Author contribution

T.-Y.H.: Conceptualization, Data curation, formal analysis, funding acquisition, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing the original draft, review, and editing. W.-C.C.: Data curation, formal analysis, Investigation, Software, Visualization. Y.-J.L.: Project administration, Software, Supervision, Validation, Visualization, Writing, review and editing. P.-Y.C.: Project administration and resources. Y.-C.W.: Project administration and resources. F.-H.L.: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Writing, review, and editing. All authors read and approved the final manuscript.

Funding

This research was funded by research grants from the laboratory of Feng-Huei Lin at the Institute of Biomedical Engineering, College of Medicine, and College of Engineering, National Taiwan University, as well as research funding from Chang Gung Memorial Hospital's Laboratory Animal Center in Keelung. Additionally, personal salary support was provided by Tzu-Yen Huang.

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Reducing Neointimal Hyperplasia in Post-Vessel Anastomosis Surgery: A Study on Bivalirudin-Loaded Carbopol 940 Gel.

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