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Original article

Efficacy and safety of a novel nano-porous polymer-free sirolimus-eluting stent in pigs

CHEN, Ming; ZHENG, Bo; WU, Zheng; PENG, Hong-yu; WANG, Xin-gang; ZHANG, bin; HUO, Yong

Author Information
doi: 10.3760/cma.j.issn.0366-6999.20123267

Abstract

The problem of restenosis following percutaneous coronary intervention (PCI) is largely solved with the introduction of drug-eluting stents (DESs).1-3 Most DESs currently in the market use a permanent polymer coating for drug storage and controlled release.4-6 However, it is believed that the polymer causes inflammation and/or a hypersensitive reaction that is responsible for late complications such as stent thrombosis and restenosis.7-10 Under the assumption that a polymer-free DES may prevent these late complications,11-14 several polymer-free DESs have been developed. Researchers developed the Janus stent with Carbofilm, a carbon-based biocompatible coating, which places the drug in reservoirs on the stent’s outer surface, ensuring a targeted release towards the vessel wall.15,16 Peters et al17,18 launched a polymer-free DES which contains micro-meter pores to enable the absorption of different organic substances. The coating solution completely fills the pores and creates a uniform layer after the evaporation of the solvent.

Different from the previous polymer-free DES, our new polymer-free DES utilizes revolutionary nanometer-size pore technology to store and to allow the controlled release of an anti-proliferative drug. The average diameter of the pores is around 400 nm, which allows for easy absorption of drugs and minimizes turbulence due to rough strut surface. The aim of the present study was to evaluate this specially designed sirolimus-eluting stent with a polymer-free nano-porous surface in pigs.

METHODS

DES platform

The polymer-free sirolimus-eluting stents (PFSES) used in the experiments were 3.0 mm × 12.0 mm and made of 316l stainless steel. Numerous pores with a diameter of 100 nm× 1 μm were prepared directly on the stent struts using a mechanical surface treatment. The nano-porous surface of the stent platform allows drug deposition and slow release. The stent surface visualized with a scanning electron microscope is shown in Figure 1 (Hitachi S-4800, Japan). The PartnerTM polymer-coated sirolimus-eluting stents (PCSES) (Lepu Medical, China) used as controls in the experiment were 3.0 mm × 12.0 mm and made of 316L stainless steel with PEMA/PEVA polymer. The sirolimus concentration on the stent was 1.2 μg/mm2. Bare metal stents (BMS) (Lepu Medical) used in this study were 3.0 mm × 12.0 mm and made of 316L stainless steel. The stent platform design and delivery systems of all three types of stents were identical.

Figure 1.
Figure 1.:
Scanning electron microgram of strut surface. Expanded polymer-free sirolimus-eluting stent coated with sirolimus at an original magnification of × 100 (A) and × 50 (B), visually highlighting uniform drug distribution across the entire stent surface. The stent strut surface before (C) and after (D) sirolimus coating at original magnifications of × 10 000 and ×5000, respectively.

To determine the drug concentration on the stent surface after the coating process, PFSES that were coated with sirolimus (n=3) were determined by high performance liquid chromatography (HPLC) analysis.

In vitro and in vivo pharmacokinetics of drug-coated stents were determined by pharmacological release kinetics. Three polymer-free and three polymer-coated stents were submersed in 5 ml phosphate buffered saline (PBS) at 37°C and rotated at 80 r/min. Samples taken at time points were subjected to HPLC-based analysis. The determination of total sirolimus stent dosage was performed by drug elution in 5 ml of methanol for four hours.

To determine sirolimus tissue levels, an additional 10 pigs for each time point were stented in the right coronary artery (RCA), left anterior descending (LAD) and left circumflex (LCX) coronary artery, and euthanized 14 and 28 days after stenting. The vascular segment covered by the stent as well as perivascular tissues adjacent to the stented vascular segment were harvested under a dissecting microscope, snap-frozen in liquid nitrogen (LN2), weighed, and stored at -70°C. Subsequently, the tissue was disaggregated for 10 seconds in 1 ml of ice-cold DPBS (#D8537; Sigma-Aldrich, USA) using a MICCRA RT mixer (#ARTMICCRA D-8, Art-Labortechnik, USA). After three freezethaw cycles, samples were subjected to protein precipitation using an equal volume of a methanol/zinc sulfate solution. Thereafter, cleared supernatants were submitted to an online solid-phase extraction procedure, followed by HPLC analysis.

Porcine coronary stent model

Three days before percutaneous intervention, juvenile Chinese pigs with a body weight of 25 to 30 kg were given 75 mg of clopidogrel and 500 mg of aspirin. Antiplatelet therapy of 75 mg clopidogrel and 250 mg of aspirin a day was maintained throughout the study. A total of 18 anesthetized pigs underwent stent implantation in the LAD, LCX and RCA. A total of 36 coronary stents (12 BMS, 12 PCSES, and 12 PFSES) were placed at a stent-to-vessel ratio of (1.2-1.3):1. Stents in each group were equally distributed in the LAD, LCX and RCA with two stents in each animal. At the time of stenting, the animals received 200 units/kg of heparin intravenously. One month or three months after stenting, angiography and intravascular ultrasound (IVUS) evaluation were performed in each vessel and the pigs were euthanized by pentobarbital. This study was approved by the Institutional Animal Care Committee at Peking University First Hospital.

Quantitative angiographic and IVUS evaluation

Angiograms were recorded before and immediately after the procedure as well as after one month and three months. They were analyzed using the quantitative angiographic analysis software (GE, Germany). The late lumen loss was calculated as the difference in the measured minimal lumen diameter immediately after the procedure, and at one month or at three months.

IVUS (Boston Scientific, USA) evaluation was performed right after the procedure to confirm proper stent apposition as well as at one month and three months after procedure. The luminal area and stent cross-sectional area were measured every 0.5 mm on consecutive IVUS images. Quantitative analysis software was used to calculate the neointimal area and volume. Neointimal area was calculated as the difference between the stent area and lumen area, and neointimal volume was calculated as stent volume minus lumen volume.

Histomorphometric analysis

For histological analysis, the heart was removed and the coronary arteries were rinsed with 250 ml of saline at physiological pressure, followed by perfusion fixation using 10% formaldehyde. Subsequently, stented artery segments were removed and fixed for another 24 hours in 10% formaldehyde. After a dehydration protocol for several days, stented vessels were embedded in methylmethacrylate as previously described. For histomorphometric analysis, 100 μm thick sections were cut with a LEIKA SP1600 microtome, stained with hematoxylin and eosin (HE), and scanned with a Leica DFC300FX microscope. Neointimal formation was evaluated by histomorphometric measurement at the most stenotic site of the vessel. Morphometric analysis was performed by Leica QwinPlus V3.2.1 software (Leica Inc.). For the additional assessment of inflammation, an embedded segment of each implanted stent was HE-stained and analyzed at a thickness of 100 μm. Strut-associated inflammation, stent endothelialization and injury score were assessed as previously reported.5,9,10

Statistical analysis

Significance of variability of the means between the experimental groups was determined by one-way analysis of variance using SPSS 13.0 software (SPSS Inc., USA). Differences among experimental groups were considered to be statistically significant when P <0.05. Unless indicated otherwise, values are given as mean ± standard deviation (SD).

RESULTS

Sirolimus concentration on stent struts

The drug concentration on the stent surface was 2.2 μg/mm2. The surfaces of both an uncoated and a sirolimuscoated PFSES are illustrated in Figure 1.

In vitroandin vivopharmacokinetics

Time-dependent HPLC-based analysis of the sirolimus elution from PFSES and PCSES (n=3 each group) showed sustained sirolimus release over 15 days (Figure 2), with more than two-thirds of the stent-based sirolimus released in PFSES within the first 10 days. Drug-elution during the first 2-5 minutes, a timeframe commonly needed between introduction of the stent into the guiding catheter and stent deployment, was determined to be 30% of the total sirolimus dose located on the PFSES.

Figure 2.
Figure 2.:
Pharmacokinetics of polymer-coated sirolumus-eluting PartnerTM stent and polymer-free sirolimus-eluting stents. The HPLC-based sirolimus release kinetic curve illustrates that more than 2/3 of the sirolimus were released within the first 10 days.

To determine and compare sirolimus levels in the vascular walls of both groups, the entire stented coronary vascular segments were recovered 14 and 28 days after stent deployment (n=10 stents for each time point). Tissue sirolimus levels were (50.9±58.7) ng/mg at 14 days and (11.3±15.7) ng/mg at 28 days in the PFSES group, and (6.9±5.0) ng/mg at 14 days and (2.1±2.2) ng/mg at 28 days in the PCSES group.

Angiographic analysis

All animals survived the experiments without acute or subacute stent thrombosis. At the one month time point, the lumen loss was significantly greater in the BMS group compared to the PCSES and PFSES groups (Figure 3). At three months, the PCSES showed a late catch up phenomenon and the difference in lumen loss was no longer significant between PCSES and BMS groups. PFSES, however, showed a persistent minimal late lumen loss and the difference between PFSES and PCSES became significant; (0.78±0.47) mm vs. (1.45±0.16) mm (P=0.004).

Figure 3.
Figure 3.:
Quantitative coronary angiogram analysis showed a significant reduction in lumen loss in PFSES at 3 months. Lumen loss at 1 month significantly decreased in PFSES as well as in PCSES, compared to BMS. At 3 months, angiogram analysis showed that the lumen loss remained significantly lower only in PFSES. *P <0.05 compared with BMS; †P <0.05 compared with BMS in 3 months.

IVUS analysis

Neointimal area and volume at one month were significantly smaller in the PCSES and PFSES groups, compared to the BMS group (Table 1). At three months, both neointimal area and volume increased in PCSES and the difference between PCSES and BMS was no longer significant. Only PFSES showed a persistent low neointimal area and volume at three months.

Table 1
Table 1:
Intravascular ultrasound assessment after stenting

Histomorphometic assessment

At one month, both the PCSES and PFSES groups showed a significantly smaller neointimal area and a larger lumen area, compared to the BMS group (Table 2). However, at three months, only the PFSES group showed a significantly smaller neointimal area and larger lumen compared to the BMS group (Figure 4).

Table 2
Table 2:
Histomorphometric assessment (mean±SD)
Figure 4.
Figure 4.:
Histomorphometrical assessment showed a significant inhibition of neointimal area in PCSES and PFSES compared with BMS 1 month (*P <0.001) post-implantation and in PFSES compared with BMS 3 months post-implantation (†P=0.01).

At one month, the inflammation score was similar among the three groups. However, the inflammation score became significantly higher in the PCSES group compared to the PFSES group at three months (P=0.006) (Table 3). The injury score was similar among the three groups. At one month, all stent struts were covered by endothelium (Figure 5).

Table 3
Table 3:
Assessment of various parameters after stenting (mean±SD)
Figure 5.
Figure 5.:
Neointimal formation in the porcine coronary artery stent model at 1 month (A, B, C) and at 3 months (D, E, F). Representative histology at 1 month shows minimal neointimal formation in both PCSES and PFSES, compared to BMS. Representative histology at 3 months shows a thin neointima only in PFSES.

DISCUSSION

The introduction of a DESs revolutionized interventional cardiology with reduction of the restenosis rate to less than 10%. However, the currently available DES platforms are coated with a permanent polymer and there is evidence that the polymeric coating may lead to localized hypersensitivity reactions and inflammation, resulting in restenosis and/or late coronary thrombosis. Therefore, the novel concept of a DES without the use of a polymer could be a valuable addendum to existing stent platforms.

Our study demonstrated that both PCSES and PFSES showed a significant reduction in neointimal hyperplasia compared to BMS at one month. However, the inhibitory effect of PFSES was sustained and at three months only the PFSES group showed significant inhibition of neointimal hyperplasia. Histology at three months also shows a significantly lower level of inflammation in the PFSES group compared to the PCSES group. A previous study also demonstrated that polymer free SES showed less inflammation and improved arterial healing in rabbits; however, the inhibition of neointimal hyperplasia was less robust compared to polymer coated SES.19 The stent platforms and sirolimus elution kinetics might have been responsible for this difference.

At three months the PCSES group did not show a sustained inhibitory effect on neointimal hyperplasia. This late catch up phenomenon has been previously reported both in animals and in humans.5,6 Shiode et al20 showed that from six months to three years, stenosis of BMS-treated lesions regressed, but stenosis of SES-treated lesions progressed. Park et al21 also showed that “late catch-up” occurred in both paclitaxel-eluting stents and SES with greater delayed late loss in SES. Carter et al5 compared SES with BMS in the porcine coronary artery stent model. A significant decrease in neointimal area was observed in SES at 30 days, but not at 90-180 days. Notably, the injury score and inflammation score increased progressively between 30 and 180 days post-implantation of SES. Because the drug was almost completely released after 30 days, it is possible that the chronic inflammation was due to continued stimulation of the vessel wall by the polymer, and the chronic inflammation was related to neointimal formation. Noteworthy, this late catch up phenomenon seems to be more significant in the porcine model than in humans. The reason why the inhibitory effect was sustained in PFSES in our study might be explained by a sustained high tissue sirolimus level ((11.3±15.7) ng/mg at 28 days in the PFSES group and (2.1±2.2) ng/mg at 28 days in the PCSES group) and low inflammation score (1.33±0.52 in PFSES and 2.50±0.55 in PCSES, P=0.006) due to absence of the durable polymer.

There are several limitations in this study. The pigs used in this experiment did not have atherosclerosis and stents were deployed in healthy coronary arteries. Therefore, the results should be interpreted with caution. However, this experiment provides preliminary data of a novel stent that warrants further investigation. Second, the follow-up duration was too short. A longer-term experiment is needed to examine outcomes and variables over time.

Acknowledgement: We thank our research staff at the Heart Center of Peking University First Hospital, and the nurses and technologists at the cardiac catheterization laboratories of the Heart Center of Peking University First Hospital.

Figure 6.
Figure 6.:
Neointimal formation in the porcine coronary artery stent model at 1 month (A, B, C) and at 3 months (D, E, F). Representative intravascular ultrasound images at 1 month show minimal neointimal formation in both PCSES and PFSES, compared to BMS. Representative intravascular ultrasound images at 3 months show a thin neointima only in PFSES.

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Keywords:

nano-porous; neointimal hyperplasia; polymer-free; sirolimus; thrombosis

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