In-vitro Release of Rapamycin from a Thermosensitive Polymer for the Inhibition of Vascular Smooth Muscle Cell Proliferation

Abstract

Hemodialysis arteriovenous grafts are often plagued by stenosis at the anastomosis, which is due to the proliferation of vascular smooth muscle cells (SMCs). To prevent the stenosis, we have been developing a strategy for the sustained perivascular delivery of an antiproliferative agent, rapamycin, using an injectable biodegradable polymer, ReGel(®). In this study we examined the in-vitro kinetics of rapamycin released from ReGel and its efficacy for inhibiting the proliferation of human and porcine venous and arterial SMCs. To study the release from ReGel, rapamycin was mixed with ReGel and incubated at 37°C in a release medium. The release medium was periodically sampled and assayed for rapamycin concentration by UV. Cellular uptake and release of rapamycin were examined by incubating SMCs with rapamycin for various durations. Intracellular drug was extracted and measured by HPLC. Antiproliferative effects and cytotoxicity of stock rapamycin and that released from ReGel were examined using cell counting and lactate dehydrogenase (LDH)-release assay, respectively. Rapamycin exhibited a sustained-release pattern from ReGel for 52 days. The kinetics of rapamycin transport through the cell membrane was compatible with a passive diffusion mechanism. Rapamycin released from ReGel exhibited antiproliferative activity similar to the free drug. Our results support the concept of sustained delivery of rapamycin using ReGel as a promising strategy to inhibit SMC proliferation for the prevention of hemodialysis arteriovenous graft stenosis.

Figure 1

In-vitro release of rapamycin from ReGel. The release rate is relatively constant over 45 days with no significant initial burst release. Each data point represents the mean ± SD of 3 experiments.

Figure 2

Uptake and release of rapamycin in human venous SMCs. The rate of uptake is faster than the rate of release. In addition, release is extended because rapamycin likely binds within the cell. Each data point represents the mean ± SD of 3 experiments.

Figure 3

Antiproliferative effect and toxicity of rapamycin in (A) human venous SMCs and (B) porcine venous SMCs. The results show that the in-vitro efficacy and therapeutic index is not different between these two cell models. Each data point represents the mean ± SD of 6 experiments.

Figure 4

The impact of rapamycin on venous smooth muscle cells after it was released from ReGel in-vitro. (A) Rapamycin shows a dose-dependent inhibitory activity on the proliferation of human and porcine venous SMCs. (B) The inhibitory activity of rapamycin (10 ng/mL) released from ReGel compared the drug directly from the vial diluted to the same concentration. ReGel does not alter rapamycin’s ability to inhibit SMCs. Samples of the plain culture medium alone were used as a control. Each data point represents the mean ± SD of 6 experiments. The smooth muscle growth medium was supplemented with 10% fetal calf serum, 5 μg/ml insulin, 0.5 ng/ml human recombinant epidermal growth factor (EGF), 2 ng/ml human recombinant fibroblast growth factor (FGF), 50 μg/ml gentamicin and 50 ng/ml amphotericin-B.