The study was conducted at Kashi Prefecture Second People’s Hospital (Xinjiang, China) from May, 2023 to March, 2024.

The L-lactic acid (Catalog No. L0165, TCI America, Portland, OR, USA) was employed as the starting material to synthesize lactide oligomers via a dehydration polycondensation methodology. The lactide oligomers were then depolymerized into L-lactide (LLA) under high-temperature vacuum conditions at 50–130 °C. Following crystallization through washing with anhydrous ether and vacuum drying at 35 °C to constant weight, poly LLA (PLLA) was obtained. Using diethyl carbonate (Catalog No. 436131000, Thermo Scientific Chemicals, Waltham, MA, USA) and 1,3-propanediol as raw materials, PTMC oligomers were synthesized through polycondensation under high-temperature vacuum conditions at 180 °C after ethanol removal. The crude product was dissolved in ether and acetone mixture, repeatedly crystallized five times in an ice water bath, washed with anhydrous ether and then vacuum filtered and dried at 25 °C to constant weight, yielding PTMC. Using carboxylic acid as the raw material, the mixture was heated to 140 °C in oil. After dehydration between hydroxy acetic acid molecules, stannous octoate was added under vacuum conditions at 200 °C to yield pale yellow crystals. The product was recrystallized five times using ethyl acetate, washed once with anhydrous ether and vacuum-dried at 35 °C to constant weight, resulting in poly (glycolic acid) (PGA). Under nitrogen protection, PLLA, PTMC, and PGA were melted and mixed uniformly (the mass ratios of PLLA:PTMC:PGA were 1:1:1, 1:1:2 and 1:1:3, respectively), then cooled and solidified in an ice-water bath. The mixture was subjected to vacuum treatment for 3 hrs and reacted at 130 °C for 72 hrs. The copolymer was dissolved in a Dichloromethane solution (Catalog No. D807825, Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China), precipitated with ethanol and vacuum-dried at 60 °C to constant weight, yielding the PLLA-PTMC-PGA composite.

A 5% (w/v) solution of PLLA-PTMC-PGA was dissolved in 1,4-dioxane (Catalog No. D116356, Aladdin Industrial Corporation, Shanghai, China) and stirred for 3 hrs using a magnetic stirrer (Catalog No. 85-2, Shanghai Sile Instrument Co., Ltd., Shanghai, China). Deionized water (prepared using a Milli-Q water purification system, Catalog No. Direct-Q 3, Merck KGaA, Darmstadt, Germany) was then applied and stirring continued for an additional 2 hrs. Metoprolol (Meto, Catalog No. M1013, Sigma-Aldrich, St. Louis, MO, USA) was incorporated and stirred for 1 hr to ensure its uniform distribution within the stent material. The temperature was maintained at approximately 5 °C above the cloud point using a constant temperature water bath (Catalog No. HH-S2, Jintan Medical Instrument Factory, Jintan, China) and then slowly decreased to the gel point for 2 hrs. The clear solution was quickly transferred to liquid nitrogen (Nanjing Special Gas Factory Co., Ltd., Nanjing, China) for 1 hr to freeze rapidly, ensuring stable binding of Meto with the material and preventing separation or drug precipitation.

The resulting Meto-PLGA/PTMC DES was obtained through freeze-drying for 24 hrs using a freeze dryer (Catalog No. FD-1A-50, Beijing Boyikang Experimental Instrument Co., Ltd., Beijing, China). The stent material was placed in 10 mL of Dimethyl Sulfoxide (DMSO, Catalog No. D8371, Sigma-Aldrich, St. Louis, MO, USA) and stirred at 37 °C for 1, 3, 6, 12, 24, 48 and 96 hrs using a thermostatic shaker (Catalog No. THZ-320, Shanghai Jinghong Laboratory Equipment Co., Ltd., Shanghai, China). At each time, 2 mL of release solution was sampled (buffer replenished) and analyzed for Meto release using a UV-1901 ultraviolet Spectrophotometer (Shanghai Lengguang Technology Co., Ltd., Shanghai, China).

The Meto-PLGA/PTMC DES samples were individually dissolved in dichloromethane (Catalog No. D8418, Sigma-Aldrich, St. Louis, MO, USA) to prepare solutions at 10 ng/mL. As 300 µL aliquot of each sample was applied to clean glass slides (Catalog No. 7101, Thermo Fisher Scientific, Waltham, MA, USA), vacuum-dried to constant weight using a vacuum dryer (Catalog No. DZF-6050, Shanghai Jinghong Laboratory Equipment Co., Ltd., Shanghai, China) and then UV-sterilized for 30 min using a UV sterilizer (Catalog No. SW-CJ-2FD, Suzhou Antai Air Technology Co., Ltd., Suzhou, China). The samples were extracted using either physiological saline (Catalog No. P1400, Solarbio Science & Technology Co., Ltd., Beijing, China) or DMEM (Catalog No. 12100046, Thermo Fisher Scientific, Waltham, MA, USA) at a liquid-to-membrane ratio of 1 mL/6 cm² for 72 hrs at 37 °C under sterile conditions in a CO₂ incubator (Catalog No. BB15, Thermo Fisher Scientific, Waltham, MA, USA), filtrated through a 0.22 µm pore filter (Catalog No. SLGP033RB, Merck Millipore, Darmstadt, Germany). Rabbit cardiac fibroblasts (Catalog No. KL-C1025, Shanghai Kanglang Bio-Tech Co., Ltd., Shanghai, China) were cultured under standard conditions and prepared as a cell suspension at 1 $\times$ 10⁴ cells/mL. As 100 µL aliquot of suspension was seeded in a 96-well plate and incubated for 24 hrs. Cell morphology was observed under an inverted microscope (Catalog No. IX73, Olympus, Japan). The rabbit cardiac fibroblasts exhibited a typical spindle-shaped morphology with uniform size, clear cytoplasm and intact cell membranes, consistent with the characteristic phenotype of fibroblasts. Surface marker identification was performed using flow cytometry (Catalog No. FACSCanto II, BD Biosciences, Sussex, NJ, USA). The cells were positive for vimentin ($\ge$95%) and negative for CD31 (2%) and $\alpha$-smooth muscle actin (5%), confirming their identity as cardiac fibroblasts. Mycoplasma detection was conducted using a mycoplasma detection kit (Catalog No. MP0030, Sigma-Aldrich, St. Louis, MO, USA). The result was negative, indicating the absence of mycoplasma contamination in the cell culture. The original medium was then removed and replaced with a 1:1 ratio of medium containing the sample extract. Cells were further incubated for 12, 24, 36, 48 and 96 hrs, with 20 µL of a 5 mg/mL methyl thiazolyl tetrazolium (MTT) reagent (Shanghai Enzyme Linked Bio, China) applied. After a 6 hrs incubation, the supernatant was discarded and 150 µL of DMSO (Sigma-Aldrich, St. Louis, MO, USA) was applied and shaken for 10 min using a microplate shaker (Catalog No. TS-100, BIOSAN, Riga, Latvia). Absorbance at 570 nm was measured via a microplate reader (Catalog No. Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA) and the relative proliferation rate was calculated.

Rabbit blood (Guangzhou Ruite Biotechnology Co. Ltd., China) was anticoagulated and mixed with physiological saline (Catalog No. P1400, Solarbio Science & Technology Co., Ltd., Beijing, China) at a 4:5 ratio. The mixture was centrifuged at 1500 rpm for 10 min using a centrifuge (Catalog No. 5810R, Eppendorf AG, Hamburg, Germany) to collect supernatant. A 10 mL aliquot of the Meto-PLGA/PTMC DES sample extract was incubated at 37 °C for 30 min in a water bath (Catalog No. HH-S4, Jintan Medical Instrument Factory, Jintan, China), mixed with 200 µL of fresh anticoagulated blood and incubated for an additional hour. The mixture was centrifuged at 3000 rpm for 5 min using a centrifuge (Catalog No. 5810R, Eppendorf AG, Hamburg, Germany) and the supernatant’s absorbance was measured at 540 nm employing a microplate reader (Catalog No. Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA).

The hemolytic ratio was calculated using [7]:

$$\text{Hemolytic ratio}={\displaystyle \frac{\left(\text{Sample absorbance}-\text{Saline absorbance}\right)}{\left(\text{Distilled water absorbance}-\text{Saline absorbance}\right)}}\times 100$$

The Meto-PLGA/PTMC DES samples were dissolved in dichloromethane (Catalog No. D8418, Sigma-Aldrich, St. Louis, MO, USA) and evenly coated on the concave center of a glass surface dish (Catalog No. xy085, Shanghai Yubo Biotechnology Co., Ltd., Shanghai, China), then allowed to evaporate at 25 °C. Following this, 200 mL of anticoagulated blood was applied. At 0, 0.25, 0.5, 1 and 2 hrs, the glass surface was gently washed with 100 µL of ultrapure water (prepared using a Milli-Q water purification system, Catalog No. Direct-Q 5, Merck KGaA, Darmstadt, Germany) and absorbance at 540 nm was measured via HBS-1101 microplate reader (Nanjing Detie Experimental Equipment Co., Ltd., Nanjing, China).

A total of 30 adult male New Zealand white rabbits (4–6 kg) were anesthetized for the procedure using 3% sodium pentobarbital (Catalog No. P3761, Sigma-Aldrich, St. Louis, MO, USA) at a dose of 0.05 g/kg, and the animals were purchased from Shanghai Jiagen Biotechnology Co. Ltd., China. A constriction ring (custom-made, Suzhou Weirui Medical Device Technology Co., Ltd., Suzhou, China) was placed at the distal end of the left coronary artery under assisted breathing conditions using a small animal ventilator (Catalog No. SAR-830, CWE Inc., Ardmore, PA, USA). Post-operative disinfection was performed with 75% ethanol (Catalog No. E7023, Sigma-Aldrich, St. Louis, MO, USA) and anti-inflammatory treatment were administered with ceftriaxone sodium (Catalog No. C5793, Sigma-Aldrich, St. Louis, MO, USA) to establish a CAD animal model [14]. Two-dimensional ultrasound was employed using a small animal ultrasound system (Catalog No. Vevo 2100, FUJIFILM Visual Sonics Inc., Toronto, ON, Canada) to visualize the vascular orientation, lumen endoscopy, vessel wall thickness and plaque formation.

The thorax was directly sutured for the Sham group using surgical sutures (Catalog No. 11-0, Ethicon Inc., Somerville, NJ, USA), while animals subjected to modeling and atherosclerotic plaque formation were divided into the Model group and the Stent group (treatment with Meto-PLGA/PTMC DES), with 10 rabbits in each group. Pre-operatively, the Stent group received aspirin (Catalog No. A2093, Sigma-Aldrich, St. Louis, MO, USA) at 25 mg/day and clopidogrel (Catalog No. C3487, Sigma-Aldrich, St. Louis, MO, USA) at 12.5 mg/day. Three days post-operation, angiography was performed under X-ray fluoroscopy using the 1725AX small animal X-ray imaging system (Shanghai Yuyan Scientific Instrument Co. Ltd., Shanghai, China), with a microcatheter (Catalog No. MPC-1.2F, Cook Medical Inc., Bloomington, IN, USA) introduced and the DES positioned at the site of arterial stenosis. Routine intramuscular injections of 4.5 million U penicillin (Catalog No. P3032, Sigma-Aldrich, St. Louis, MO, USA) were administered for 3 days. After recovery, animals were housed in cages (Catalog No. RCC-01, Suzhou Fengshi Laboratory Animal Equipment Co., Ltd., Suzhou, China) and continued a high-fat diet (Catalog No. D12492, Research Diets Inc., New Brunswick, NJ, USA) for 30 days. The 1725AX small animal X-ray imaging system was sourced from Shanghai Yuyan Scientific Instrument Co. Ltd., China.

Post-treatment, 3 mL of venous blood was collected from the ear margin using a 5 mL disposable syringe (Catalog No. 100500, Shanghai Kohama Medical Devices Co., Ltd., Shanghai, China) with a 22G needle (Catalog No. 302220, BD Medical, Franklin Lakes, NJ, USA), anticoagulated with heparin sodium (Catalog No. H3149, Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 10 U/mL and centrifuged at 1200 rpm for 10 min using a centrifuge (Catalog No. 5810R, Eppendorf AG, Hamburg, Germany).The supernatant was utilized to measure serum Interleukin-8 (IL-8) levels via radioimmunoassay using an IL-8 radioimmunoassay kit (Catalog No. RK00012, Shanghai Enzyme Linked Bio, Shanghai, China), while serum Vascular Cell Adhesion Molecule-1 (VCAM-1), Intercellular Adhesion Molecule-1 (ICAM-1) and Tumor Necrosis Factor-Alpha (TNF-$\alpha$) were assessed using a double-antibody sandwich ELISA methodology with VCAM-1 ELISA kit (Catalog No. EK0577, Shanghai Enzyme Linked Bio, Shanghai, China), ICAM-1 ELISA kit (Catalog No. EK0395, Shanghai Enzyme Linked Bio, Shanghai, China) and TNF-$\alpha$ ELISA kit (Catalog No. EK0525, Shanghai Enzyme Linked Bio, Shanghai, China), respectively. The corresponding ELISA kits were sourced from Shanghai Enzyme Linked Bio, China.

Twelve hours after treatment, the animals were anesthetized with 0.05 g/kg of 3% sodium pentobarbital (Sigma-Aldrich, USA). The right common carotid artery and left femoral artery were isolated. A polyethylene (PE-20) catheter was inserted through a right common carotid artery into the left ventricle, with the other end connected to a physiological recorder (Shanghai Renyi Biotechnology Co. China). This setup measured Left Ventricular Systolic Pressure (LVSP), LV end-diastolic pressure (LVEDP) and the maximum rates of pressure increase and decrease ($\pm$dp/dt_max). Arterial systolic blood pressure (BPs) and diastolic BP (BPd) were assessed via catheterization of the left femoral artery.

Twenty-four hours after cardiac function assessment, the animals were euthanized under 3% sodium pentobarbital (Catalog No. P3761, Sigma-Aldrich, St. Louis, MO, USA) anesthesia. Coronary artery tissues were fixed in 4% paraformaldehyde (Catalog No. P6148, Sigma-Aldrich, St. Louis, MO, USA) for 24 hrs. Tissues were rinsed twice with PBS (pH 7.2, Catalog No. P1020, Solarbio Science & Technology Co., Ltd., Beijing, China) , dehydrated through a graded ethanol series (50%: Catalog No. E7023-500ML, Sigma-Aldrich, St. Louis, MO, USA; 70%: Catalog No. 10009218, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; 80%: Custom-prepared, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; 90%: Custom-prepared, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; 100%: Catalog No. 10009218, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and cleared in xylene (Catalog No. X1500, Solarbio Science & Technology Co., Ltd., Beijing, China). They were embedded in paraffin (Catalog No. P1461, Solarbio Science & Technology Co., Ltd., Beijing, China) using a tissue embedding machine (Catalog No. EG1150H, Leica Biosystems, Wetzlar, Germany) and sectioned into 4 µm thick slices with a microtome (Catalog No. RM2235, Leica Biosystems, Wetzlar, Germany).

Staining was conducted according to the instructions of the Hematoxylin-Eosin staining kit (Catalog No. G1120, Shanghai Enzyme Linked Bio, Shanghai, China).The coronary artery tissue morphology was visualized via a CX23 Microscope (Olympus, Japan) equipped with a digital camera (Catalog No. DP27, Olympus, Tokyo, Japan) and image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Rockville, MD, USA) was employed to evaluate intimal thickness, lumen area and stenosis severity.

All data were presented as Mean $\pm$ Standard Deviation and compared via One-way Analysis of Variance with post hoc LSD-t tests, performed using SPSS 22.0 (International Business Machines Corporation, Armonk, NY, USA). A p 0.05 meant statistically significant.

Fig. 1a shows the Fourier Transform Infrared Spectroscopy (FTIR) spectrum of Meto-PLGA/PTMC DES. The spectrum indicated a notable presence of PLLA in Meto-PLGA/PTMC DES, with a prominent C = O peak at 1758/cm and a notable -CH₂ peak at 1431/cm. Fig. 1b depicts that the glass transition temperature (Tg) of Meto-PLGA/PTMC DES is 56.9 °C. Fig. 1c presents the tensile strength is 1831.5 MPa. Fig. 1d illustrates the storage modulus of Meto-PLGA/PTMC DES as a function of temperature, showing a decreasing trend with increasing temperature, with a sharp decline in storage modulus observed above 40 °C. Fig. 1e displays the weight loss versus time curves for Meto-PLGA/PTMC DES during degradation in water and proteinase K. Over time, the weight loss of Meto-PLGA/PTMC DES increased gradually (Fig. 1e), with a higher weight loss rate in proteinase K compared to water (Fig. 1f).

Fig. 1.

Physicochemical properties of Metoprolol-loaded poly (lactic-co-glycolic acid)-poly (trimethylene carbonate)-poly (glycolic acid) drug-eluting stent (Meto-PLGA/PTMC DES). (a) Fourier Transform Infrared Spectroscopy (FTIR) spectrum; (b) Differential Scanning Calorimetry (DSC) curve; (c) Static mechanical properties (the x-axis labels 1, 2, and 3 represent the mass ratios of PLLA:PTMC:PGA as 1:1:1, 1:1:2 and 1:1:3, respectively); (d) Dynamic mechanical properties; (e) Weight loss versus time curve in water and (f) Weight loss versus time curve in proteinase K.

Fig. 2a shows the results of cell proliferation assays for rabbit cardiac fibroblasts cultured in a normal medium and in extracts from Meto-PLGA/PTMC DES. Over time, proliferation activity increased markedly in both conditions, with higher proliferation in the normal medium. The effect of Meto-PLGA/PTMC DES on cell proliferation was minimal, indicating good cytocompatibility and absence of cytotoxicity. The hemolysis rate of Meto-PLGA/PTMC DES was 3.09%, which was below the 5% threshold, indicating that the composite materials meet the hemolysis requirements for medical biomaterials. Fig. 2b presents the dynamic clotting time curve for Meto-PLGA/PTMC DES, which shows a similar gradual decrease as observed in the normal control, indicating comparable clotting behavior.

Fig. 2.

Cell compatibility of Metoprolol-loaded poly (lactic-co-glycolic acid)-poly (trimethylene carbonate)-poly (glycolic acid) drug-eluting stent (Meto-PLGA/PTMC DES) complex. (a) Effect of Meto-PLGA/PTMC DES on the growth of rabbit cardiac fibroblasts and (b) Dynamic clotting time curve of Meto-PLGA/PTMC DES.

Fig. 3 shows the in vitro drug release curve of Meto-PLGA/PTMC DES. Over time, the cumulative drug release rate of the stent gradually increased (Fig. 3a), while the total amount of drug released gradually decreased (Fig. 3b), indicating that the stent enables slow and sustained drug release with a stable release profile.

Fig. 3.

In vitro drug release characteristics of Metoprolol-loaded poly (lactic-co-glycolic acid)-poly (trimethylene carbonate)-poly (glycolic acid) drug-eluting stent (Meto-PLGA/PTMC DES). (a) Cumulative drug release rate and (b) Amount of drug released.