PPC (MW 100,000; purity >99%) was purchased from Inner Mongolia Mengxi High Tech Group Co., Ltd. (Ordos, China). Sirolimus (rapamycin) was obtained from Fujian Kerui Pharmaceutical Co., Ltd. (Fuzhou, China). All chemicals were from Jiete'ao Biotechnology Co., Ltd. (Beijing, China). MTT was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Eagle's minimum essential medium (EMEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Various ratios of PPC and sirolimus were used to produce PPC-sirolimus polymers and cell growth experiments, performed by MTT assay as previously described (18), demonstrated that the polymer made with 900 mg PPC and 100 mg sirolimus resulted in the greatest cell inhibitory effect; therefore, this ratio was used in the present study. The polymer solution was as follows: 1,000 mg PPC alone (PPC polymer) or 900 mg PPC plus 100 mg sirolimus (PPC-sirolimus polymer) was dissolved in 10 ml acetonitrile following stirring for 6 h. Electrospinning was performed using the electrospinning apparatus (Model ET2535; Beijing Ucalery Technology and Development Co., Ltd., Beijing, China). The polymer solution was transferred into a syringe attached with a blunt tipped needle. The anode of the power supply was clamped to the needle tip and the cathode was connected to the grounded collector plate (Fig. 1). Electrospinning was performed according to the established protocol (Institute of Polymer Science, Tsinghua University, Beijing, China) (19).

The polymers were coated with silver using the vacuum evaporator (HUS 5GB; Hitachi Ltd., Tokyo, Japan) following an established protocol at the Imaging Center, Shandong University (Jinan, China) (20). The silver coated polymers were analyzed using a scanning electron microscope (H-8010; Hitachi, Ltd., Japan) operated at 15 kV, and images were captured.

The thermal behavior of the PPC-sirolimus polymers was assessed by DSC analysis using the DSC822e differential scanning calorimeter (Mettler Toledo, Greifensee, Switzerland). A total of 4 mg sample (pure sirolimus or PPC-sirolimus polymer) was weighed in a 40 µl aluminum pan and placed in the sample chamber of the calorimeter, and an empty pan was used as the reference. A flow of nitrogen gas was maintained over the sample to eliminate air oxidation of the sample at high temperatures, and to create a reproducible and dry atmosphere. DSC analysis was performed using the STARe software version 8.10 (Mettler Toledo) with the temperature range 20–300°C and the temperature increase being set at a rate of 10°C/min. The endothermic or exothermic peak was automatically recorded.

A PPC-sirolimus polymer of 1×1 cm was placed in a 5-ml test tube and soaked in PBS (pH 7.2). The tube was gently shaken (60 rpm) at 37°C for 4 weeks. Subsequently, the structure of the polymer was examined using SEM.

The efficiency of drug loading and entrapment was determined as follows: 1,000 mg PPC-sirolimus polymer was dissolved in dichloromethane by stirring for 2 h followed by addition of 0.1 M hydrochloric acid. Following mixing by vortex, lower dichloromethane and upper hydrochloric acid layers were formed. The hydrochloric acid layer was collected, centrifuged at 1,000 × g for 5 min at room temperature and the supernatant was collected. The precipitate was mixed with 0.1 M hydrochloric acid containing 0.1% EDTA. When the precipitate was completely dissolved, the solution was added to the previously collected hydrochloric acid supernatant. The sirolimus in the combined solution was measured by high pressure liquid chromatography as described previously (21). The sirolimus loading efficiency (%) was calculated using the following formula: (Total sirolimus amount in the combined solution/polymer weight) × 100. Drug entrapment efficiency was calculated using the following formula: [Total sirolimus amount in the combined solution/total sirolimus amount used for polymer preparation (100 mg)] × 100. A total of three PPC-sirolimus polymer preparations were used to determine the drug loading and entrapment efficiency.

A total of 5 mg PPC-sirolimus polymer was placed in 14 separate 5-ml test tubes. The polymer was immersed in 4 ml PBS/tube and gently shaken (60 rpm) at 37°C. At day 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 following shaking, the polymer was removed and the sirolimus contained in the polymer was isolated and measured as described in the determination of drug loading and entrapment efficiency section. At each time point, the amount of released sirolimus was calculated using the following formula: Total sirolimus in the original 5 mg polymer-total sirolimus in the soaked polymer. The release percentage was calculated using the following formula: (Amount of released sirolimus/total sirolimus in the original 5 mg polymer) × 100.

An MTT assay was performed to examine the anti-cell proliferative effect of the PPC-sirolimus polymer. Rat aortic adventitial fibroblast cells (RAAFCs) were isolated as described previously (22,23). A total of 4 Wistar rats (2 males and 2 females, ~ 7 weeks old, weighing 180–220 g) were used for RAAFC isolation. The animals were purchased from Shandong Experimental Animal Center (Jinan, China), housed in the Shandong University (Jinan, China) Animal Room with temperature at 20±2°C, and fed with standard chow. The use of animals was approved by the Research Ethics Committee of Shandong University in compliance with the Guidelines for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology, China. Cells were cultured in EMEM with 10% FBS, 50 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. For the MTT assay, 5×10³ cells suspended in 100 µl culture medium were seeded into each well of a 96-well plate, into which had previously been added 2 mg of PPC or PPC-sirolimus polymer (each sample in triplicate). Cells seeded in wells with no polymer were taken as the control. Following culturing for 1 and 3 days, the MTT assay was performed as follows: 10 µl MTT solution was added into each well and incubated at 37°C for 4 h, followed by the addition of 50 µl dimethyl sufoxide/well. Following gentle shaking for 10 min, absorbance at a wavelength of 570 nm was measured using a 96-well plate reader (Model 680, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative cell viability (%) of each sample was calculated using the following formula: (Average sample absorbance/average control absorbance) × 100.

Cell growth data are expressed as the mean ± standard error of the mean. The SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Statistical significance was determined using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Electrospinning conditions were optimized and used as follows: The voltage was set at 15 kV; the distance from the needle tip to the collector was 20 cm, and the flow rate of the spinning solution was controlled at 0.2 ml/h by a syringe pump (TJ-3A/W0109-1B; Longer Precision Pump Co., Ltd., Baoding, China) (Fig. 1). SEM analysis demonstrated that the polymer exhibited a regular three-dimensional, grid-intertwined, net-like structure with a smooth surface, and the diameter was ~3 µm (Fig. 2). DSC analysis demonstrated that the crystalline pure sirolimus produced an endothermic peak at ~230°C, the melting point of sirolimus (Fig. 3A). By contrast, the PPC-sirolimus polymer exhibited a glass transition temperature of ~40°C for PPC (Fig. 3B). The in vitro degradation experiment demonstrated that, following soaking in PBS for 4 weeks, the polymer swelled and the regular three-dimensional grid-intertwined structure broke down and fragmented (Fig. 4).

Sirolimus loading and entrapment efficiency were 10.3±3.2 and 95.1±10.6%, respectively. The release of sirolimus was measured for 28 days. As presented in Fig. 5, sirolimus was gradually released from the PPC-sirolimus polymer in a linear-like fashion, particularly for the first 3 weeks. A total of ~90% of the sirolimus entrapped in the polymer was released after 28 days.

An MTT assay was performed in order to evaluate the effect of the PPC-sirolimus polymer on RAAFC growth. The viability of control cells (without the addition of any polymer) was arbitrarily set at 100%. As presented in Fig. 6, following culture for 1 day, the viability of cells treated with PPC and PPC-sirolimus was 92.7±6.7 and 77.2±4.4%, respectively. At day 3, the viability of cells treated with PPC and PPC-sirolimus was 82.9±5.4 and 12.2±2.4%, respectively. Statistical analysis demonstrated that the PPC-sirolimus polymer significantly inhibited the growth of RAAFCs at the two time points, while the PPC polymer did not (Fig. 6).