Download Inhibition of Smooth Muscle Cells Proliferation Using Sirolimus

Inhibition of Smooth Muscle Cells Proliferation Using Sirolimus Loaded, Heparin Bound-Polymeric Micelles
Jayesh V Betala,1 Sooneon Bae,1 Eugene M. Langan III, M.D.,2 JeoungSoo Lee, Ph.D.,1 Martine LaBerge, Ph.D. 1
1
Bioengineering Department, Clemson University, Clemson SC, 2 Greenville Health System, Greenville, SC
Statement of Purpose: Drug Eluting Stents (DES) are
significantly popular for reducing in-stent restenosis (ISR)
as compared to bare metal stents.1 However, their long term
efficacy remains a problem, mainly because of late-stent
thrombosis and delayed endothelialization.2 The objective
of this project is to develop a Sirolimus loaded, Heparin
bound polymeric micelles that can be delivered locally,
which can inhibit smooth muscle cells proliferation after
balloon angioplasty. A cationic, amphiphilic polymer, poly
(lactide-co-glycolide) -graft-polyethyleneimine (PLGA-gPEI, PgP) micelle was synthesized in 4D Lab
(Bioengineering, Clemson University). We hypothesized
that Sirolimus, a hydrophobic drug, will encapsulate in the
hydrophobic core and heparin, a negatively charged
molecule, will bind to positively charged PgP. We
evaluated the loading efficiency of Sirolimus and
antiproliferative effects on SMCs after days 3 and 7.
Methods: Sirolimus was loaded using solvent evaporation
method. Briefly, Sirolimus was dissolved in methanol and
various concentration of sirolimus solution (100 µL) was
added to 1 mg/ml PgP, dissolved in DI water. The solution
was stirred at room temperature for 4 hours. The organic
solvent was then evaporated overnight. The non-loaded
drug was removed by filtering through a 0.45 µm syringe
filter. Amount of Sirolimus loaded was analyzed using
HPLC (Symmetry C18, 3.5 µm, Waters). The loading
efficiency was calculated as follows, % loading efficiency=
(the amount of Sirolimus loaded in the PgP/the amount of
Sirolimus added into PgP) X 100. Particle size of PgP was
determined using dynamic light scattering (DLS) before
and after loading. SMCs isolated from rat aorta, between
passage 3 and 5, were seeded at a density of 2×104 and
1×104 cells/ ml for 24 hours, in DMEM with 10% FBS and
1% Ab-Am, for day 3 and day 7, respectively. After 24
hours, SMCs were treated with Sirolimus-loaded PgP (SPgP-50µg/ml, Sirolimus-2.6µg/ml) and Sirolimus alone
(dissolved in DMSO) for 5, 15, 30 and 60 minutes. After
treatment, DMEM containing Sirolimus was removed and
replenished with fresh DMEM with 10% FBS and 1% AbAm. PgP alone and untreated cells were used as control.
SMCs proliferation was evaluated at day 3 and 7 by MTT
assay. Statistical analysis was performed using one-way
ANOVA and Student’s t-test, with p-value <0.05
considered to be significant.
Results: Sirolimus loading efficiency increased with
increasing concentration of Sirolimus added into PgP (1
mg/ml) solution (Fig 1). However, the loading efficiency
was reached maximum, when 300 µg/ml Sirolimus was
added into PgP solution (~23%). Sirolimus solution was
not soluble in water, so it was completely removed after
filtering through syringe filter. PgP increased sirolimus
solubility approximately 25 times than solubility in water.
Figure 1: Sirolimus loading of PgP micelles using solvent
evaporation method. Mean± SE. (n=4)
DLS did not show any significant difference in size before
(130±4 nm) and after loading (125±4 nm).
Sirolimus loaded PgP showed significant inhibition of
SMCs after 5, 15, 30 and 60 minutes-treatment as
compared to sirolimus alone at day 3 and day 7, while no
cytotoxicity was observed from PgP alone. Untreated
SMCs were considered to be at 100 % (Table 1). Anova
indicated significant difference between treated and control
cells with p-value<0.001(n=6).
Figure 2: SMCs proliferation evaluated by MTT assay after day 3
and day 7. * P-value< 0.001 (n=6) compared to untreated control
Conclusion: Sirolimus loaded PgP micelles significantly
inhibited SMCs proliferation up to day 7, as compared to
Sirolimus alone. An anti-proliferative drug, such as
Sirolimus and an anti-thrombotic drug, Heparin, can be
delivered locally at the same time using these micelles.
This biomaterial platform provides promising results for
the local delivery of Sirolimus loaded and Heparin bound
polymeric micelles to inhibit smooth muscle cell
proliferation after balloon angioplasty.
Acknowledgements: NIGMS of the National Institutes of
Health under award number 5P20GM103444-07 and Page
Morton Hunter Endowment.
References: 1) Htay T, Liu MW. Vascular Health and
Risk Management. 2005;1(4):263-276 2) Edoardo C, P.
Gabriel S, and William W. Circulation. 2007; 115:14401455.