Modelling drug release from polymer-free coronary stents with microporous surfaces

Traditional coronary drug-eluting stents (DES) are made from metal and are coated with a permanent polymer film containing an anti-proliferative drug. Subsequent to stent deployment in a diseased coronary artery, the drug releases into the artery wall and helps prevent restenosis by inhibiting the proliferation of smooth muscle cells. Although this technology has proven to be remarkably successful, there are ongoing concerns that the presence of a polymer in the artery can lead to deleterious medical complications, such as late stent thrombosis. Polymer-free DES may help overcome such shortcomings. However, the absence of a rate-controlling polymer layer makes optimisation of the drug release profile a particular challenge. The use of microporous stent surfaces to modulate the drug release rate is an approach that has recently shown particularly promising clinical results. In this study, we develop a mathematical model to describe drug release from such stents. In particular, we develop a mathematical model to describe drug release from microporous surfaces. The model predicts a twostage release profile, with a relatively rapid initial release of most of the drug, followed by a slower release of the remaining drug. In the model, the slow release phase is accounted for by an adsorption/desorption mechanism close to the stent surface. The theoretical predictions are compared with experimental release data obtained in our laboratory, and good agreement is found. The valuable insights provided by our model will serve as a useful guide for designing the enhanced polymer-free stents of the future

Analysis of alternative triggers for PI-9 expression.

<p>A) The up-regulation of PI-9 was also found to be triggered by other inflammatory cytokines. HepG2 cells were treated for 16 hours with either IFN-γ at 100 IU/ml, or IL-1β at 50 ng/ml. GAPDH expression was used as a positive control. B) Stimulation of the HepG2 cell line with either IFN-γ (open squares) or IL-1β (open triangles) also inhibited CTL killing compared to the un-stimulated HepG2 cells (closed circle). C) HepG2 cells were left un-infected (Nil), or infected with either a baculovirus expressing a sub-genomic replicon (NS-replicon), or with a control baculovirus expressing LacZ (+Control). PI-9 expression was analysed by RT-PCR. PI-9 was strongly up-regulated only in the cells expressing the sub-genomic replicon. GAPDH expression was used as a positive control.</p

IFN-α reduces hepatocyte sensitivity to CTL cytotoxicity.

<p>Hepatocytes have been described as expressing low to no MHC Class I. As expected, IFN-α treatment increased the levels of MHC Class I on HepG2 (A) and HHL (B); filled curve represents an isotype control, the solid line represents the MCH Class I expression. C) HepG2 cells were stimulated for 16 hours with a serial dilution of IFN-α at 0 IU/ml (closed circles), 10 IU/ml (open diamonds), 100 IU/ml (open squares), and 1000 IU/ml (open inverted triangles), prior to cytotoxicity assay with the CTL line 2. Treatment with IFN-α reduced the HepG2 cells sensitivity to CTL cytotoxicity in a dose dependent manner. D) This phenomenon was also found with the novel human hepatocyte cell lines (HHL). HHL-17 cells were either left untreated (closed circles) or stimulated for 16 hours with 1000 IU/ml (open inverted triangles) prior to co-incubation with the CTL line 1.</p

IFN- treatment did not protect a B-cell line.

<p>Treatment of a BCL with 1000 IU/ml IFN-α (open inverted triangles) prior to the cytotoxicity assay, did not reduce CTLs ability to kill the treated BCL compared to the untreated BCL (closed circles).</p

Expression of PI-9 in liver tissue from patients with chronic hepatitis C.

<p>Liver specimens obtained from diagnostic biopsy were stained for PI-9 expression as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000791#s4" target="_blank">methods</a>. A) Representative negative isotype control. B–D) PI-9 was detected in the majority of hepatocytes. Stronger PI-9 expression, as expected, was seen within the mononuclear infiltrate (highlighted by red arrows), while all the hepatocytes stained positively, albeit at a lower level (the strongest are highlighted by blue arrows).</p

IFN-α treated hepatocytes remain susceptible to FASL-induced apoptosis.

<p>Aii) Induction of apoptosis by FASL was assessed across a concentration range. HepG2 cells, either stimulated with IFN-α (1000 IU/ml) (closed inverted triangles) or left un-stimulated (open diamonds), were incubated with cross-linked rFASL at concentrations ranging from 0.1 pg/ml to 2 µg/ml. Apoptosis was assessed by Annexin V binding and propidium iodine (P.I) incorporation. Ai) Raw FACS data is shown. Bii) HepG2 cells, either un-stimulated (open and closed circles), stimulated with 100 IU/ml (open and closed squares) IFN-α or 1000 IU/ml (open and closed inverted triangles) IFN-α, were incubated with 1 µg/ml (left, open symbols) or 2 µg/ml (right, closed symbols) cross-linked rFASL over a time course of up to 6 hours. Apoptosis was assessed by the cytosolic presence of the activated form of caspase 3. Bi) Raw FACS data.</p

Expression of the granzyme B inhibitor, serine proteinase inhibitor 9 (PI-9).

<p>A) RT-PCR revealed that IFN-α stimulated cells, HepG2 and HHL-9 cells, were able to upregulate PI-9. GAPDH expression was analysed as a positive control. Bi and ii) This was confirmed by FACS analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000791#s4" target="_blank">methods and materials</a>. Upregulation of PI-9 was shown to be dose dependent.</p

Clinical data of HCV patients at time of biopsy.

<p>Clinical data of HCV patients at time of biopsy.</p