BME 272
NCIIA Grant Proposal
Design of a MK2i-loaded PLLA Nanofilm to Combat Intimal
Hyperplasia in Autologous Venous Grafts
Mitchell Weisenberger, James Carrow, Ming Chen, Andrew Schultze
Team GraftWrap
December 6, 2012
Abstract
Intimal hyperplasia, a major cause of autologous graft failure in coronary artery bypass
graft (CABG) surgery, is mediated by the activation of intracellular kinase Mitogen Activated
Protein Kinase Activated Protein Kinase 2 (MK2). MK2 activation leads to smooth muscle cell
(SMC) infiltration into the vessel lumen and subsequent graft occlusion. A cell penetrating
peptide (CPP) conjugated inhibitory sequence (YARAMK2i) has been shown to block MK2 and
reduce downstream inflammatory markers in vitro, yet an adequate delivery method for this
therapeutic to graft SMCs during and following CABG surgery is an unmet challenge.
Separately, PLLA nanofilms have been developed which can load drugs, adhere to wet tissues,
and degrade slowly in vitro. We wish to develop a YARAMK2i-loaded PLLA nanofilm for
direct application to the CABG outer surface to serve as a drug depot for extended delivery of
YARAMK2i and deterrence of intimal hyperplasia. Our primary objectives are to:
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Synthesize YARAMK2i-loaded PLLA nanofilms of various thicknesses and confirm film
thicknesses via atomic force microscopy (AFM).
Conduct drug release and degradation assays to determine release kinetics and
degradation properties of loaded nanofilms respectively, followed by determination of
optimal nanofilm thickness for drug delivery.
Undertake cytotoxicity and ELISA assays to determine biocompatibility and therapeutic
potential of loaded nanofilms in vitro, followed by determination of optimal drug loading
concentration.
Introduction
Coronary Heart Disease and Intimal Hyperplasia:
A primary health concern that has emerged due to recent lifestyle developments is the
occlusion of the blood vessels which nourish the heart, leading to ischemia and mechanical
failure of cardiac tissue. We know it is the leading
cause of death in America, and in 2008 its effects
resulted in one sixth of the total deaths in the U.S. (1).
As of now, standard treatment for a blocked coronary
artery is an autograph from the saphenous vein, which is
used to bypass the blockage and return blood flow. Yet,
Figure 1: Intimal hyperplasia (right) in
almost 20% of these grafts fail in the first year alone (2)
comparison to healthy venous tissue (left)
and thus there is recognizable room for improvement in
entails SMC migration and ECM deposition
the therapy.
into the lumen without distinct organization.
The failure is attributed to the transformation of
vascular smooth muscle cells (VSMCs) in the graft into a pathological state, which causes them
to grow into the lumen of the blood vessel. This leads to the narrowing and even blockage of the
graft itself, a process termed intimal hyperplasia (Fig. 1) (3). Under further investigation, we can
dissect the pathway in which the vessels undergo this physiological change. Within this
pathway, MK2 is an intracellular kinase which phosphorylates heat shock protein 27. From this,
actin dynamics are altered and result in SMC proliferation and migration into the vessel lumen
(Fig. 2). This will be our target of the treatment. During coronary artery bypass surgery,
activation of MK2 leads to smooth muscle cell infiltration into the vessel lumen and subsequent
graft occlusion. Currently MK2i has been used to inhibit the activation of MK2, reducing the
amount of smooth muscle cell infiltration, which in turn reduces occlusion within the vessel
graft. The problem which we look to address is the short timeframe in which the graft is treated
with MK2i. We will do this by integrating MK2i into a nanosheet, which when wrapped around
the graft, will release MK2i over a longer period of time. This would reduce the activation of
MK2 for a longer time than is currently in practice.
Nanofilms for Drug Delivery:
By encapsulating the YARAMK2i in a PLLA
nanofilm, we will have more control of the release
mechanism and limit immediate release of the peptide.
We hope that the release profile will resemble Fickian
diffusion rather than straight dissolution. By tuning our
thickness of the film and drug integration method, we
hope to delay the release of the YARAMK2i for
Figure 2: The molecular mechanism of VSMC
pathogenesis is specifically mediated by
maximal cellular response. Considerations of nanofilm
MK2.
adhesivity will also dictate thickness and loading method. As
coronary artery bypass surgery is
becoming more prevalent in the world, it is necessary to increase the effectiveness of the surgery.
As 20% of grafts fail within the first year due to intimal hyperplasia, there is a profound need to
find a way to combat occlusion of the vessel graft. The YARAMK2i nanosheet we will develop
will decrease the incidence of intimal hyperplasia after coronary artery bypass surgery, and
decrease the likelihood that the graft will fail.
History
YARAMK2i:
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An inhibitory sequence (MK2i) has been developed which binds MK2 and deters the
process of intimal hyperplasia (4), yet an adequate delivery technology is needed.
•
The CPP-conjugated MK2i (YARAMK2i) has been shown to exhibit MK2 and
downstream inflammatory marker inhibition in vitro (5).
•
This YARAMK2i therapeutic has been successfully synthesized and isolated before by
Mitchell Weisenberger (BME) in Vanderbilt’s Advanced Therapeutic Laboratory
directed by Professor Craig Duvall.
•
Professor Craig Duvall has granted his permission to utilize his utilities to synthesize,
characterize, and isolate the YARAMK2i therapeutic for the project.
PLLA Nanofilms:
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PLLA is an FDA approved material for medical applications.
PLLA nanofilms less than 100 nm thick were seen to be stable for more than 3 weeks in
vitro in conditions of a pH of 1 and .5% pepsin, and the sheets also served to facilitate
closing of a gastric lesion in vivo after 7 days (6). This suggests that PLLA nanofilms
could serve as a biomaterial that would slowly degrade over time when wrapped around a
CABG.
PLLA nanofilms less than 100 nm thick were observed to adhere strongly to wet tissue
and organs (6), suggesting that they could adhere well to the outside of a CAGB.
Superparamagnetic iron oxide nanoparticle loaded PLLA nanosheets have been
successfully manufactured with a homogenous distribution of nanoparticles (7),
suggesting the feasibility of YARAMK2i-loaded nanosheets.
PLLA nanosheets of various thicknesses and layers have been synthesized by Jake
Carrow (BME) in Phil Messersmith’s lab in Northwestern’s BME department.
At Northwestern, films were synthesized to carry a wound healing drug with release
profiles measured
Currently at Vanderbilt, nanosheets have been loaded with gold nanoparticles for a laser
stimulation project, but none have been loaded with YARAMK2i presently
Team
Undergraduates:
1. Mitchell Weisenberger (BME): Has synthesized YARAMK2i before in Doctor Craig
Duvall’s lab, and has experience in solid phase peptide synthesis, isolation via high
performance liquid chromatography (HPLC), and identification via liquid
chromatography mass spectroscopy (LCMS). Has worked with cells. Will be
especially focused on the synthesis of YARAMK2i and in vitro analysis of loaded
films.
2. James (Jake) Carrow (BME): Has synthesized multi-layer PLLA nanofilms via spin
coating techniques in Phil Messersmith’s Northwestern University BME lab, and will
be working with such films this year in Doctor Giorgio’s lab. Is proficient in organic
chemistry. Has worked with cells. Will be especially focused on the loading and
synthesis of the PLLA nanofilms and in vitro analysis of loaded films.
3. Ming Cheng (BME): Has worked on peptide synthesis, HPLC, and polymer
chemistry in Doctor Duvall’s lab. Has worked with cells. Will be especially focused
on the synthesis of YARAMK2i and in vitro analysis of loaded films.
4. Andrew Schultze (BME): Has worked with dynamic mechanical testing techniques in
Doctor Nyman’s lab. Will be especially focused on the mechanical application of
films to cell cultures during in vitro analysis, as well as mechanical characterization
of films should the interest arise.
Directors:
1. Doctor Craig Duvall
2. Doctor Hak-Joon Sung
3. Doctor Todd Giorgio
Work Plans and Outcomes
10/1/2012 11/1/2012 12/1/2012 1/1/2013
2/1/2013
3/3/2013
Synthesis of YARAMK2i
YARAMK2i purification and conjugation of FITC
Creation of YARAMK2i-FITC-loaded PLLA nanofilms
Start Date
Characterization of nanofilm thicknesses
Completed
Drug release and nanofilm degredation assays
Remaining
Determination of optimal nanofilm thickness
In vitro culture of loaded films and biological assays
Determination of optimal nanofilm drug loading concentration
Major Outcome: We wish to ultimately construct a YARAMK2i-loaded nanofilm of
optimal thickness and loading concentration which is biocompatible and can cause
inflammatory marker knockdown in vitro. A loaded nanofilm of optimal thickness will be
able to adhere to wet tissue in vitro while still being sufficiently thick enough to display
release kinetics that elicit an optimal therapeutic effect in in vitro ELISA assays. Optimal
loading concentration will be achieved when the loaded film elicits a peak therapeutic effect
in in vitro ELISA inflammatory marker knockdown without causing a significantly greater
cytotoxicity in vitro in comparison to no treatment. Inflammatory marker knockdown in vitro
will be dictated by ELISA assays which display a significant decrease in inflammatory
markers, either IL-6 or TNF-alpha, in comparison to no treatment in an in vitro cell culture of
endothelial cells. The PLLA-loaded nanosheets fit into the lab culture of the Duvall, Sung,
and Giorgio labs in that they are drug delivery platforms that may be loaded with other
compounds for therapeutic treatments.
Sources
(1) Roger, V. L., A. S. Go, et al. (2011). "Heart Disease and Stroke Statistics—2012
Update." Circulation.
(2) Fitzgibbon GM, Kafka HP, Leach AJ, Keon WJ, Hooper GD, Burton JR. Coronary
bypass graft fate and patient outcome: angiographic follow-up of 5,065 grafts related to
survival and reoperation in 1,388 patients during 25 years. J Am Coll Cardiol.
1996;28:616–626
(3) Glagov, S. (1994). "Intimal hyperplasia, vascular modeling, and the restenosis
problem." Circulation 89(6): 2888-2891.
(4) Hayess, K. and R. Benndorf (1997). "Effect of protein kinase inhibitors on activity of
mammalian small heat-shock protein (HSP25) kinase." Biochemical Pharmacology
53(9): 1239-1247.
(5) Brugnano, J. L., B. K. Chan, et al. (2011). "Cell-penetrating peptides can confer
biological function: Regulation of inflammatory cytokines in human monocytes by MK2
inhibitor peptides." Journal of Controlled Release 155(2): 128-133.
(7) Okamura, Y., Kabata, K., Kinoshita, M., Saitoh, D. and Takeoka, S. (2009), FreeStanding Biodegradable Poly(lactic acid) Nanosheet for Sealing Operations in Surgery.
Adv. Mater., 21: 4388–4392. doi: 10.1002/adma.200901035