Article

Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents. Proc Natl Acad Sci USA

Division of Cardiology, The Children's Hospital of Philadelphia, Department of Chemical, Philadelphia, PA 19104, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2006; 103(1):159-64. DOI: 10.1073/pnas.0502945102
Source: PubMed
ABSTRACT
The clinical use of metallic expandable intravascular stents has resulted in improved therapeutic outcomes for coronary artery disease. However, arterial reobstruction after stenting, in-stent restenosis, remains an important problem. Gene therapy to treat in-stent restenosis by using gene vector delivery from the metallic stent surfaces has never been demonstrated. The present studies investigated the hypothesis that metal-bisphosphonate binding can enable site-specific gene vector delivery from metal surfaces. Polyallylamine bisphosphonate (PAA-BP) was synthesized by using Michael addition methodology. Exposure to aqueous solutions of PAA-BP resulted in the formation of a monomolecular bisphosphonate layer on metal alloy surfaces (steel, nitinol, and cobalt-chromium), as demonstrated by x-ray photoelectron spectroscopy. Surface-bound PAA-BP enabled adenoviral (Ad) tethering due to covalent thiol-binding of either anti-Ad antibody or a recombinant Ad-receptor protein, D1. In arterial smooth muscle cell cultures, alloy samples configured with surface-tethered Ad were demonstrated to achieve site-specific transduction with a reporter gene, (GFP). Rat carotid stent angioplasties using metal stents exposed to aqueous PAA-BP and derivatized with anti-knob antibody or D1 resulted in extensive localized Ad-GFP expression in the arterial wall. In a separate study with a model therapeutic vector, Ad-inducible nitric oxide synthase (iNOS) attached to the bisphosphonate-treated metal stent surface via D1, significant inhibition of restenosis was demonstrated (neointimal/media ratio 1.68 +/- 0.27 and 3.4 +/- 0.35; Ad-iNOS vs. control, P < 0.01). It is concluded that effective gene vector delivery from metallic stent surfaces can be achieved by using this approach.

Full-text

Available from: Ilia Fishbein
Bisphosphonate-mediated gene vector delivery
from the metal surfaces of stents
Ilia Fishbein*, Ivan S. Alferiev*, Origene Nyanguile*, Richard Gaster*, John M. Vohs
, Gordon S. Wong
,
Howard Felderman*, I-Wei Chen
, Hoon Choi
, Robert L. Wilensky
§
, and Robert J. Levy*
*Division of Cardiology, The Children’s Hospital of Philadelphia, Departments of
Chemical and Biomolecular Engineering and
Material Science and
Engineering, and
§
The Cardiovascular Division, Hospital of the University of Pennsylvania, University of Pennsylvania, Philadelphia, PA 19104
Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved November 14, 2005 (received for review April 11, 2005)
The clinical use of metallic expandable intravascular stents has
resulted in improved therapeutic outcomes for coronary artery
disease. However, arterial reobstruction after stenting, in-stent
restenosis, remains an important problem. Gene therapy to treat
in-stent restenosis by using gene vector delivery from the metallic
stent surfaces has never been demonstrated. The present studies
investigated the hypothesis that metal–bisphosphonate binding
can enable site-specific gene vector delivery from metal surfaces.
Polyallylamine bisphosphonate (PAA-BP) was synthesized by using
Michael addition methodology. Exposure to aqueous solutions of
PAA-BP resulted in the formation of a monomolecular bisphospho-
nate layer on metal alloy surfaces (steel, nitinol, and cobalt–
chromium), as demonstrated by x-ray photoelectron spectroscopy.
Surface-bound PAA-BP enabled adenoviral (Ad) tethering due to
covalent thiol-binding of either anti-Ad antibody or a recombinant
Ad-receptor protein, D1. In arterial smooth muscle cell cultures,
alloy samples configured with surface-tethered Ad were demon-
strated to achieve site-specific transduction with a reporter gene,
(GFP). Rat carotid stent angioplasties using metal stents exposed to
aqueous PAA-BP and derivatized with anti-knob antibody or D1
resulted in extensive localized Ad-GFP expression in the arterial
wall. In a separate study with a model therapeutic vector, Ad-
inducible nitric oxide synthase (iNOS) attached to the bisphospho-
nate-treated metal stent surface via D1, significant inhibition of
restenosis was demonstrated (neointimalmedia ratio 1.68 0.27
and 3.4 0.35; Ad-iNOS vs. control, P < 0.01). It is concluded that
effective gene vector delivery from metallic stent surfaces can be
achieved by using this approach.
gene therapy local delivery restenosis
T
he use of balloon expandable met allic stents has resulted
in improved therapeutic outc omes for coronary arter y
disease (1). However, stent angioplast y is c omplicated in many
patients by reobstruction due to the formation of a neointima
in the stented arterial segment, a disease process known as
in-stent restenosis (2). The mechanisms responsible for in-
stent restenosis involve proliferation and migration of medial
smooth muscle cells (SMCs) and an associated increase in
extracellular matrix components (2). The use of poly mer-
c oated dr ug-eluting stents has markedly decreased the inci-
dence of in-stent restenosis observed with unmodified met al
stents (3). However, both experiment al (4) and clin ical (5)
studies indicate a number of concerns about this approach,
because poly mer c oatings on stents cause a more pronounced
inflammator y response than met al surfaces (6), thus delaying
rather than preventing restenosis (7, 8).
Polymer-coated gene-delivery stents have been demonstrated in
animal studies to be effective for both reporter (9–13) and thera-
peutic (14, 15) vector delivery. Nevertheless, their use is problem-
atic because of harmful properties of the polymer coatings (6, 7).
Therefore, the present experiments inve stigated gene delivery
directly from metal surfaces without the use of a polymer coating.
Bisphosphonates are known to demonstrate high-affinity binding to
both mineral and metallic surfaces through phosphonate–metal
coordination (16). It was therefore hypothesized that a metallic
surface could be modified through an aminobisphosphonate expo-
sure, thereby attaching to the metallic surface a derivatizable
polybisphosphonate molecule that could, in turn, be covalently
conjugated with vector-binding agents; this modification could,
hypothetically, enable local gene delivery from metal surfaces
exposed to the aqueous bisphosphonate. The present studies report
(i) the synthesis of a unique water-soluble bisphosphonate, polyal-
lylamine bisphosphonate (PAA-BP); (ii) binding interactions of
aqueous PAA-BP with metal surfaces, thereby enabling the reten-
tion of a PAA-BP molecular monolayer that permits the attach-
ment (via vector-binding molecules) and site-specific delivery of
adenoviral vectors to cells in culture; and (iii) the efficacy of this
approach using a replication-defective adenovirus (Ad) expre ssing
inducible nitric oxide synthase (iNOS) as a model therapeutic gene
for inhibiting in-stent restenosis in experimental animals.
Methods
Materials. Replication-defective type 5 (E1,E3-deleted) adenoviral
vectors were obtained from the Gene Vector Core Facility of the
University of Pennsylvania (Ad-GFP) and from the Gene Therapy
Core Facility of the University of Iowa (Iowa City, IA) (Ad-iNOS).
In both constructs, transgenes were under the control of the human
cytomegalovirus promoter. Stainless steel (316L) foils and meshes
were obtained from Goodfellow (Berwyn, PA) and Electron Mi-
croscopy Sciences (Hatfield, PA), respectively. Nitinol samples,
cobalt–chromium alloy coupons, and 8-mm cobalt–chromium
stents (SVS) were obtained under a material-transfer agreement
from Cordis (Warren, NJ). The anti-knob antibody (IgG) used was
a gift from Selective Genetics (San Diego).
Polymer Synthesis. For the synthesis of PAA-BP, two PAA (PAA)
HCl salts, 15 kDa and 70 kDa, were obtained from Sigma-Aldrich;
PAA relative molecular masses (mass average) were determined
with size-exclusion chromatography by the manufacturer (Sigma-
Aldrich). Vinylidene bisphosphonic acid tetrasodium salt was ob-
tained from Rhodia, (Oldbury, U.K.). PAA base (from either
PAAHCl, 15 kDa, or PAAHCl, 70 kDa, 23.6 mmol of NH
2
in
both) and vinylidene-bisphosphonic acid (20 mmol) were combined
in water, concentrated to a syrup (7.63 g), and heated at 100–110°C
for 5 h. The reaction product was dissolved in water containing an
excess of triethylamine, and pure solid PAA-BP was precipitated
with an excess of HCl. Purified PAA-BP in the free-acid form was
subjected to elemental analysis by using both combustion method-
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Freely available online through the PNAS open access option.
Abbreviations: Ad, adenovirus; i-NOS, inducible isoform of nitric oxide synthase; PAA,
polyallylamine; PAA-BP, PAA bisphosphonate; SMC, smooth muscle cell; SPDP, N-succin-
imidyl 3-(2-pyridyldithio) propionate; XPS, x-ray photoelectron spectroscopy.
To whom correspondence should be addressed at: The Children’s Hospital of Philadelphia,
Abramson Research Center, Suite 702, 3615 Civic Center Boulevard, Philadelphia,
PA 19104-4318. E-mail: levyr@email.chop.edu.
© 2005 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0502945102 PNAS
January 3, 2006
vol. 103
no. 1
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MEDICAL SCIENCES
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ology and proton-induced x-ray emission (Elemental Analysis,
Lexington, KY) to determine composition and
31
P NMR to doc-
ument purity.
Ad Immobilization and Release. For Ad binding, an anti-knob mouse
monoclonal (IgG) antibody (2 mgml) was reduced with 2-mercap-
toethylamine (10 mgml) at 37°C for 30 min and purified by gel
filtration. The human recombinant D1 domain of the Coxsackie-
Ad-receptor protein (CAR) was also used as a binding agent and
was prepared as described in ref. 17, followed by the conjugation of
eluted D1 thioester with cysteine (20 mgml).
Metallic surfaces were exposed to 3% aqueous PAA-BP, pH, 5.5,
at 60°C for 4 h, exhaustively washed, and reacted with N-
succinimidyl 3-(2-pyridyldithio) propionate (SPDP, 20 mgml) for
1 h at 20°C. The higher molecular mass PAA-BP (using 70-kDa
PAA) showed 20% lower metal-binding efficacy than did the lower
molecular mass PAA-BP (using 15-kDa PAA) and thus was not
further inve stigated. PAA-BP-treated SPDP-modified metallic
samples were then reacted with reduced antibody or thiolated D1
for 12 h at 20°C under an argon atmosphere. Ad immobilization was
attained by 3-hour incubations of antibody- or D1-derivatized
metallic specimens in suspensions of 5 10
10
Ad-GFP particles per
ml in 5% BSAPBS. Selected samples of Ad-GFP were rendered
fluore scent by using a Cy3 modification as described in ref. 18
before immobilization. The amount of immobilized Ad was deter-
mined, by using a depletion assay, as the difference between the Cy3
fluore scent signal (540580 nm) elicited from nondepleted and
metal-sample-depleted virus suspensions.
To compare relative rates of Ad dissociation from the anti-
body- and D1-modified surfaces, meshes (n 4 per group)
c onfigured with Cy3-labeled Ad linked via either antibody or D1
tethers were individually ex posed to 350
l of PBS. The incu-
bation was carried out under shaking at 4°C for 1 week, with a
daily change of buf fer. Immediately after virus acquisition and
at the end of the experiment, fluorescence micrographs of the
meshes were taken under standardized settings of the camera.
The digital images were analyzed by using mean luminescence
intensit y of Adobe
PHOTOSHOP-generated histograms for the
quantification of surface-attached Ad.
Surface Analyses. The x-ray photoelectron spectroscopy (XPS)
spectra were collected at room temperature by using an Al K
a
x-ray
source (Vacuum Generators, Hastings, U.K.) and a hemispherical
electron energy analyzer (Leybold, Hanau, Germany). Surface
profiles were visualized by using a Dimension 3100 atomic-force
microscope (Digital Instruments, Santa Barbara, CA). Imaging was
performed in the intermittent noncontact (tapping) mode, by using
oscillating linear Si tips with a resonance frequency range of
300–350 Hz. Each data scan was collected over a 25
m
2
area at a
scanning frequency of 0.50 Hz. Viral surface density, determined by
counting five fields (1
m
2
), was expre ssed as mean SE.
Cell-Culture Experiments. Rat aortic SMCs (A10 cells; American
Type Culture Collection) were cultured to 90% confluence, as
published in ref. 9. Equal amounts of free and either antibody-, or
D1-conjugated mesh-immobilized Ad-GFP were added into wells
of 24-well plates. The GFP expre ssion was assessed after 48 h by
fluore scence microscopy or by fluorimetry (485–535 nm) of cell
lysates. All cell culture experiments were carried out in triplicate.
Rat Carotid Stent Angioplasty Study. Carotid stent angioplasties
were carried out by using 8-mm stents (Cordis), as above, in male
Sprague–Dawley rats (500–550 g, Taconic Farms) assigned to five
experimental groups: (i) Metal stents not treated with PAA-BP
(three rats) were compared with PAA-BP treated (but without Ad)
stents (three rats) in 7-day studies comparing the inflammatory
response in arterial wall between the two groups. (ii) A series (three
rats) of 24-h explants examined the initial arterial-wall distribution
of stent-delivered (anti-knob-antibody-tethered) Cy3-labeled Ad-
GFP. (iii) In 7-day reporter studies to assess the extent of transgene
expre ssion, rats were subjected to stent angioplasty using control
(unmodified) metal stents (three rats) and antibody- (three rats) or
D1-tethered (three rats) Ad-GFP PAA-BP stents. The estimated
adenoviral load on each stent ranged from 2.5 10
9
to 6.3 10
9
particles. (iv) Ad-iNOS delivered for 16 days from stents was
examined in efficacy studies comparing PAA-BP-only modified
controls (n 7), or PAA-BP-modified D1Ad-iNOS (n 5) stents.
(v) Ad-GFP biodistribution after stent delivery by using D1Ad-
GFP (7-day study, n 5) was assessed by PCR (see protocol below).
Morphometrical Methods. GFP expression was assessed by fluore s-
cence microscopy and immunohistochemistry (19) with a primary
monoclonal mouse anti-GFP antibody (Roche). Photomicrographs
of four representative sections of each artery were obtained at 200
magnification by using a Leica DC 500 microscope digital-image
acquisition system. The images were processed in Adobe
PHOTO-
SHOP
to eliminate background and converted into binary (black and
white) images by using the program
IMAGE (Scion, Frederick, MD).
The area of ‘‘black’’ pixels, representing diaminobenzidine staining
of the GFP-positive material, was divided by the total analyzed area
and normalized to a percentage scale. Differences in the inflam-
matory response between PAA-BP-treated and bare-metal stents
were compared by using hematoxylin and eosin staining and
anti-CD68 (Serotec, Oxford, U.K.) immunostaining (20).
For the Ad-iNOS study, stented arterial segments were plastic-
embedded (Technovit 9100, Wehrheim, Germany), sectioned, and
stained by the Verhoff–van Gie sen method. The arterial micro-
graphs were captured as digital images (see above) under 50
magnification, and the areas of lumen, neointima, and media were
calculated by using Scion
IMAGE-generated tracings (see above) of
the respective anatomic arterial compartments.
Biodistribution of GFP Expression by PCR. Ad-GFP stented and
contralateral arteries and the samples of lung, myocardium, spleen,
liver, and kidney were harvested. Phenolchloroform DNA extrac-
tion was performed, and PCR amplification was carried out over 35
cycle s with a PTC-200 PCR engine (MJ Research, Watertown,
MA) by using GFP-specific primers (upstream, 5-GGC TGC TGC
AAA ACA GAT AC-3; downstream, 5-CGG ATC CTC TAG
AGT CGA C-3). Amplified samples were analyzed with agarose-
gel electrophoresis using appropriate standards and positive con-
trols (Ad-GFP-transduced A10 cells) as described in ref. 9.
Statistical Methods. Dat a are expressed as mean SE. The
sign ificance of differences between means of ex perimental
groups was deter mined by using Student t tests.
Results
A water-soluble PAA-BP that can both interact with metal-oxide
surfaces and provide reactive sites for chemical conjugation was
synthesized by a direct Michael addition of PAA to the activated
double bond of vinylidene-bisphosphonic acid (Fig. 1A). The
molecular mass of each allylamine hydrochloride unit (93.56) was
used to calculate the number of reactive units in the 15,000-Da
PAA-HCl polymer used in these studies as 160.3 units per polymer
macromolecule, which were thereby available for bisphosphonate
derivatization. In the initial formulation studied, the extent of
modification with the bisphosphonate groups was calculated based
on elemental analysis of precipitated pure PAA-BP in the free-acid
form, which contained P, 17.7%, H, 6.45%, C, 29.9%, N, 6.18%, and
O, 42.2%; thus, 65% of PAA amine groups were calculated to be
derivatized with bisphosphonate groups in this preparation. Fur-
thermore, based on elemental analysis, the estimated molecular
mass of PAA-BP is 30 kDa, as calculated by using the sums of the
weights of modified (245.1 Da) and nonmodified (57.1 Da) al-
lylamine residue s.
31
P NMR of pure PAA-BP documented a single
160
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Page 2
peak at
16 ppm. In the
13
C NMR spectrum of nonmodified
PAAHCl (Fig. 1B) three distinctive
13
C signals appear at
30
ppm (backbone CH
2
), 34 ppm (backbone CH), and 43 ppm
(pendant CH
2
NH
2
), whereas the
13
C NMR spectrum of PAA-BP
(Fig. 1C) shows three new carbon moieties at 37 ppm (t, 112 Hz,
CH of diphosphonoethyl groups),47 ppm, and 50 ppm (pendant
CH
2
NH of modified links and CH
2
of diphosphonoethyl groups).
Two additional formulations for vector-binding comparisons were
synthesized by using the same PAA preparation described above
but varying the amount of vinylidene-bisphosphonic acid to create
a range of bisphosphonate modifications; elemental analyses dem-
onstrated these two preparations to have 45% and 77% PAA amino
groups modified.
Initial studies used stainle ss steel (316 L) coupons that were
exposed to 3% aqueous PAA-BP (65% modified) and then rinsed
exhaustively with water. XPS confirmed the presence of a PAA-BP
molecular monolayer on the steel surface, demonstrating the
emergence of a characteristic P(2p) signal (Fig. 1D) in the XPS of
the treated sample, which persists after a 30-day incubation under
simulated physiologic conditions (data not shown); a phosphorus
signal is not present in the control 316 L steel (Fig. 1D). Further-
more, the characteristic Fe(2p) peaks of the steel substrate are still
present in the XPS from the PAA-BP-modified sample (Fig. 1E),
indicating that the thickne ss of the PAA-BP coordination layer is
less than the effective XPS sampling depth (5 nm).
The primary amines of surface-bound PAA-BPs (45%, 65%, and
77% bisphosphonate-modified) were further reacted with a bifunc-
tional (amino- and thiol-reactive) crosslinker, SPDP, to introduce
thiol-reactive pyridyldithio groups to 316 L steel surfaces. To
quantitate thiol-reactive functionalities, we reacted PAA-BP
SPDP-treated metal coupon samples with dansyl cysteine. Subse-
quent chemical reduction of the disulfide bond triggers dansyl
release, which was used to quantitate fluorimetrically (355–535 nm)
the thiol-reactive capacity of the surface-associated PAA-BP. All
three candidate formulations were initially compared with 316 L
steel binding studies using the dansyl cysteine assay. The results of
these studies revealed the PAA-BP thiol-reactivity to be 54.5, 29.9,
and 16.0 pmolcm
2
for the 45%, 65%, and 77% BP modifications,
respectively, corresponding to a minimal surface thiol-reactive
density of 1 group per 10 nm
2
. Because the effective immobilization
diameter of an Ig molecule exceeds 400 nm
2
(21), the theoretical
achievable density of thiol-reactive groups is far beyond the mini-
mal requirement for uniform protein surface modification, born out
by the determination of Cy3-Ad attachment to differently treated
steel foils by using
PHOTOSHOP-generated histogram intensities of
surface fluore scence (see Methods) as a relative index of immobi-
lization density. These comparisons demonstrate virtually identical
vector-binding levels for the various PAA-BP formulations (mean
luminescence intensities 26.03 5.93, 26.52 2.29, and 25.19 4.2
for 45%, 65%, and 77% BP modifications, respectively). Thus, the
65% modified PAA-BP was chosen as a lead formulation for
subsequent in vitro and in vivo studies.
Exposure of either PAA-BPanti-knob-antibody-conjugated or
PAA-BPD1-conjugated but not PAA-BPSPDP-modified alloy
samples to an adenoviral suspension resulted in affinity-mediated
surface tethering of the Ads (Fig. 1F). The extent of Ad binding was
quantitated by depletion assays (Table 1). These results revealed
comparable levels of bound vector for the three alloy surfaces
studied, with several-fold greater attachment for D1 (100–150
particles per
m
2
, Table 1) compared with the anti-knob antibody
(35–50 particles per
m
2
, Table 1); no measurable vector was
bound to either bare metal or PAA-BP-treated alloy samples
without the use of either thiolated anti-knob antibody or D1.
PAA-pretreated metallic samples subjected to the SPDP-D1 pro-
tocol described above demonstrated only trace levels of Cy3-vector
binding (data not shown). The dissociation of Cy3-labeled Ad from
the PAA-BP-binding-agents-primed meshe s under sink conditions
demonstrated a 37.4 2.5% and 27.7 7.5% decrease of surface-
associated Ad for the antibody and D1 tethers, respectively (P
0.05) after 1 week, indicating comparable rates of dissociation for
D1 and antibody-mediated vector binding.
Atomic-force microscopy demonstrated multiple groupings of
100-nm-diameter units (Fig. 2 A and B) that repre sent surface-
bound Ads. This surface nanoparticulate pattern was not observed
on steel samples exposed to only PAA-BP (Fig. 2C). Thus, affinity-
mediated tethering of Ad allows for dense packing of the vector on
a metal surface, estimated to be 19 3 and 45 2 viral particles
per
m
2
(see Fig. 2 A and B) for antibody- and D1-primed steel
surfaces, respectively (P 0.001, greater D1-mediated binding).
Fig. 1. The PAA-BP-synthesis reaction scheme (A) is shown with
13
C NMR
spectra of PAA (B) and PAA-BP (C) demonstrating characteristic chemical shifts
(43–50 ppm, re. B vs. C) with peak changes indicating bisphosphonate addition
(C, re. 47- and 50-ppm peaks). XPS to detect phosphorus (D) and iron (E)on
nonmodified and PAA-BP-modified steel surfaces demonstrates the appear-
ance of P(2p) after PAA-BP treatment with persistent Fe(2p) signals. (F)A
schematic representation is shown of the reversible Ad tethering to the
PAA-BP-modified metal surface.
Table 1. PAA-BP-metal coordination bonding enables thiol-based
covalent attachment of Ad-binding agents for tethering Ad:
Ad-binding comparisons of various alloys and D1 vs. antibody
(mean SE)
Metal alloy
Anti-knob antibody-bound Ad
(10
9
particles per cm
2
)
D1-bound Ad
(10
9
particles per cm
2
)
316L steel 4.30 0.34 10.8 0.6*
Nitinol 5.27 0.10 11.6 0.4*
Co–Cr 3.52 1.13 15.1 1.37*
*Greater binding with D1, P 0.001.
Fishbein et al. PNAS
January 3, 2006
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MEDICAL SCIENCES
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These results are lower than the depletion assay data (Table 1),
perhaps due to Ad loss during AFM-related procedures.
Stainless steel meshes configured with PAA-BPantibody-
immobilized GFP-encoding adenoviral vectors were placed in rat
arterial SMC (A10 cells) cultures, resulting in intense highly local-
ized transduction of 68.1 5.7% of cells on the steel surface and
within 50
m of mesh borders (Fig. 3A). However, the same amount
of free virus (2 10
8
particles) caused transduction of only 1.0
0.4% cells throughout the cultures (Fig. 3B). The use of D1 for
tethering resulted in 110-fold-higher levels of GFP expre ssion after
48 h in A10 cell cultures than that achieved with anti-knob antibody
(Fig. 3C).
Initial rat carotid stent angioplasty experiments compared a
series of animals subjected to nonviral carotid stenting with (n 3)
and without (n 3) stent pretreatment with aqueous PAA-BP.
Hematoxylin and eosin staining demonstrated a mild inflammatory
response due to stenting that did not differ between PAA-BP
pretreatment and control bare-metal-stented arteries (results not
shown). Because macrophage s are the predominant cell type in-
volved in the inflammatory process after stenting (22, 23), the
sections were immunostained by using a rat macrophage-specific
anti-CD-68 antibody. Overall, the prevalence of CD-68-positive
cells was higher for the arteries stented with bare metal stents
(45.02 16.83%; Fig. 4A) than PAA-BP-modified stents (33.7
5.03%, Fig. 4B); however, this difference was not statistically
significant (P 0.05).
To examine the robustness of adenoviral attachment to the stent
surface, Cy3-labeled Ad-GFP were immobilized on the PAA-BP
pretreated, antibody-activated stents, and were shown to result in
uniform vector association with stent struts (Fig. 4C). Twenty-four
hours after stent deployment, Cy3-labeled Ads were observed at the
stentartery interface, as verified by en face fluore scence micros-
copy (Fig. 4D). The spatial pattern of the fluore scent signal noted
in stented arterial segments en face corresponds to the shape of
the stent struts (Fig. 4D), implying that the initial distribution of the
virus in the vessel wall is governed by the physical imprint of
the stent’s wire surface. No fluorescent signal was elicited from the
luminal surfaces of carotid arteries subjected to stent angioplasty
with stents treated similarly but excluding the step of Cy3-labeled
Ad exposure (Fig. 4E).
A series of rat carotid stent angioplasty studies with PAA-BP
(Ad-GFP) stents using either anti-knob antibody or D1 as vector-
binding agents was carried out for 7 days. After quenching elastin
autofluore scence with Evans blue, the GFP-positive cells were
consistently demonstrated in the medial, neointimal, and adventitial
layers of arteries treated with PAA-BP stents using either anti-knob
antibody (Fig. 5A)orD1(Fig.5B) as binding agents for Ad-GFP
attachment. The sections of arteries treated with control metal
Fig. 2. Atomic-force microscopy of a PAA-BPanti-knob antibody- (A) and D1- (B) derivatized stainless steel surface exposed to an Ad suspension (5 10
10
particles per ml), demonstrating distribution of viral particles on the PAA-BP-activated surfaces. (C) For comparison, a control is shown consisting of a stainless
steel surface exposed to only PAA-BP. (Scale bar, 1
m and a depth scale of 500 nm is shown.)
Fig. 3. GFP expression in cultured rat arterial SMCs (A10) transduced by the
mesh-antibody-immobilized (A) or free (B) Ad-GFP (2 10
8
Ad particles) with
prominent site-specific expression only with surface-immobilized vector (A).
The multiplicity of infection was 1 for both cultures (magnification, 100, FITC
filter set). (C) Fluorimetric assessment of GFP expression (485510 nm) from
A10 cell cultures transduced by Ad-GFP immobilized on the meshes by using
anti-knob antibody vs. D1 with significantly greater GFP levels with D1 teth-
ering (P 0.001).
Fig. 4. PAA-BP-modified steel stents: Inflammatory response and tissue
distribution of the vector in vivo. Representative DAB-immunohistochemistry
photomicrographs, demonstrating prevalence of CD68-positive macrophages
(indicated by the arrowheads) in arterial sections treated with bare metal (A)
and PAA-BP-modified (B) stents (original magnification, 200). Fluorescent
photomicrograph of a Cy3-Ad-modified stent surface (2.5 10
9
viral particles
per stent) before deployment (C) and its imprint (en face) on the luminal
surface of a rat carotid artery (D) 24 h after stenting. (E) Absence of autofluo-
rescence in a rat carotid artery stented without tethered Ad. (CE) Original
magnification is 200, rhodamine filter set.
162
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Page 4
stents (without bisphosphonate pretreatment) did not demonstrate
fluore scent cells (Fig. 5C). Widespread arterial-wall transduction
was confirmed by anti-GFP immunostaining for both Ad-GFP
tethered by anti-knob antibody (Fig. 5D) and D1 (Fig. 5E). Mor-
phometric evaluation of the representative sections revealed com-
parable extensive transduction with GFP-positive cells in the me-
dial, neointimal, and adventitial compartments of arteries from
both experimental groups (Table 2). GFP-positive immunostaining
(re. false positive) was not observed in arteries treated with control
nonmodified bare-metal stents (Fig. 5F) not exposed to Ads.
GFP-PCR biodistribution studies demonstrated GFP DNA after 35
cycle s of amplification only in the arterial samples underlying the
stents but not in the contralateral carotid arteries, liver, spleen,
myocardium, and lungs (data not shown).
Therapeutic efficacy was demonstrated with PAA-BP-mediated
metal-surface vector binding comparing unmodified metal stents
with the same stents with aqueous PAA-BPD1 exposure by using
Ad-iNOS as a model therapeutic gene vector. The Ad-iNOS
gene-delivery-stent arteries demonstrated a significant therapeutic
effect with diminished in-stent restenosis compared with controls,
as verified by morphometric results quantitating neointimal area,
neointimal-to-medial-area ratios, and difference s in the percent of
luminal stenosis (see Table 3 and Fig. 5 G and H).
Discussion
These results demonstrate successful delivery of a gene vector
f rom a metal surface treated with an aqueous bisphosphonate
solution to enable tethering. The data also show that the aqueous
polybisphosphonate–metal-binding interaction used to attach
Ad vectors occurs with a variety of alloys and thereby enables
c omparable levels of covalent attachment of vector-binding
agents for gene delivery. These studies have also shown that
various vector-binding agents can be used for gene delivery, such
as anti-Ad antibodies, or recombinant proteins, such as D1, a
rec ombinant Ad-receptor fragment. Importantly, this gene-
delivery strategy can be used efficaciously to treat in-stent
restenosis, as demonstrated by the Ad-iNOS results (Table 3).
Catheter delivery of both viral (24) and nonviral (25) gene
therapeutics has been previously investigated for the mitigation of
in-stent restenosis. Adaptation of the stent itself as a platform for
gene delivery has a number of important advantages. First, animal
and clinical studies have consistently shown that mural thrombosis
and arterial SMC proliferation occur predominantly near stent
struts (26). Thus, a relatively small amount of stent-immobilized
gene vectors strategically placed at the interface of tissue and
implant might be sufficient to produce a clinically significant
therapeutic transduction of regional cells. Second, stent-tethered
vectors that are physically shielded from the shearing effect of blood
flow can, hypothetically, persist in tissues. Indeed, our Cy3-labeled
Ad studies (Fig. 4D) revealed the deposition of fluorescent-labeled
virus beneath the struts 24 h after stenting, despite active blood
flow. Thirdly, immobilization of Ad vectors on stents diminishes
distal spread of the vectors to nontarget tissues, as our PCR data
show.
The mechanisms of Ad delivery from metal surfaces in the
present studies, both in vitro and in vivo, must involve dissociation
of the vector from the PAA-BP–binding-agent complex before cell
uptake. The cell culture studies demonstrate a site-specific local-
ization of GFP expression to the region of the PAA-BP-treated
metal mesh (Fig. 3A), whereas the in vivo results show GFP
expre ssion deep in the media and adventitia of the artery (Fig. 5).
This difference is likely because of a number of factors, including
Ad transduction of arterial SMCs in close proximity to the stent
struts, followed by migration of these cells during the early forma-
tion of a neointima and the forceful compression of vector into the
arterial wall, as shown in Fig. 4D. Intact arterial elastic laminae are
considered to be virtually impenetrable for adenoviral particles
(27). However, stenting causes multiple lacerations of the elastic
laminae, thus facilitating egre ss of the vector into the adventitial
compartment. The cellular types within the adventitia (mainly
Fig. 5. PAA-BP-vector binding agent mediated tethering of Ad to steel
surfaces in vivo: reporter (GFP) and Ad-iNOS therapeutic results. Fluorescence
micrographs of rat carotid arteries treated with PAA-BPantibodyAd-GFP
stents (A), PAA-BPD1Ad-GFP stents (B), or control bare stainless steel stents
that show only residual autofluorescence (C). (AC, where
*
indicates lumen)
Original magnification, 200, FITC filter set. Arrowheads point to the GFP-
positive cells. Peroxidase-based (diaminobenzidine, brown color) immunohis-
tochemical detection of GFP expression in the stented carotid arterial seg-
ments harvested 7 days after stenting by using either Ad-GFP stent with
antibody (D) and D1 (E) tethering or a control bare stainless steel stent (F).
(DF) original magnification, 200. Verhoeff–van Giessen-stained represen-
tative stented arterial sections of control bare metal (G) and Ad-iNOSD1-
derivatized (H) cobalt– chromium stents, demonstrating iNOS-mediated inhi-
bition of restenosis. (G and H), original magnification, 200.
Table 2. Arterial GFP expression after stent-based delivery of
Ad-GFP: morphometric results (anti-knob antibody- vs.
D1-tethering for rat carotid stent reporter studies, mean SE)
Ad attachment
strategy Neointima Media Adventitia
PAA-BPantibody
Ad-GFP stents
37.9 20.3 6.8 1.6 19.6 5.6
PAA-BPD1
Ad-GFP stents
23.0 6.4 8.7 4.5 16.4 1.0
Data represent percentage of GFP-positive area relative to the total area of
the respective compartment in microscopic sections.
Table 3. Inhibition of in-stent restenosis with stent-delivery of
Ad-iNOS: morphometric results (Ad-iNOS-D1-tethered vs. bare
metal stents) for rat carotid stent angioplasty studies
(mean SE)
Group
Neointima,
mm
2
Neointima
media
Stenosis,
%
iNOS 0.23 0.02 1.68 0.27 23.1 3.4
Control 0.40 0.04 3.34 0.35 40.7 4.2
Statistical power
(t-test)
P 0.011 P 0.006 P 0.013
Fishbein et al. PNAS
January 3, 2006
vol. 103
no. 1
163
MEDICAL SCIENCES
Page 5
fibroblasts and myofibroblasts) are known to be more susceptible to
adenoviral transduction than medial SMC (28), perhaps explaining
the high extent of transduction in the adventitia observed in our
reporter studies (Table 2). Considering the active role the adventitia
plays in the formation of neointimal lesions (29), the ability of the
stent-delivery approach investigated herein to transduce cells in the
adventitia might be therapeutically relevant. Importantly, the pen-
etration of the vector into the adventitia did not result in the
dissemination of the virus beyond the stented arterial segment, as
verified by the GFP-PCR results.
In addition, the re sults of in vitro studies show a greater Ad-
binding capacity of D1 vs. anti-knob antibody (Fig. 2 and Table 1)
and a significantly higher level of gene expression in cell culture with
D1-tethered Ad (Fig. 3). These difference s may be due to both a
higher Ad binding affinity of immobilized D1 vs. the antibody used
and subsequent facilitated processing of the Ad–D1-receptor com-
plex. The reported dissociation constant (K
d
) (in solution) for the
anti-knob antibody used in our study is 0.31 nM (17); however, this
value might be lower for the immobilized, chemically reduced
molecule (30). Our Ad-release studies, carried out in an acellular
system, demonstrated a somewhat higher dissociation rate for virus
tethered via anti-knob antibody vs. D1-tethered Ad, confirming
tighter binding with the immobilized receptor protein. Further-
more, it has been shown that the initial binding event of Ad to
immobilized dimeric D1 has a much lower affinity (K
d
20 nM)
than an observed secondary binding event (K
d
1 nM), thus
suggesting that conformational changes of covalently attached D1
could actually facilitate more rapid release of Ad than would be
otherwise expected (31). Thus, higher Ad binding with D1 tethering
and facilitated releasetransduction in the presence of cells are not
mutually exclusive.
iNOS was chosen as a model therapeutic gene for these studies
because of its previous efficacious use in delivery-catheter gene-
therapy studies with a pig coronary stent angioplasty model (24)
and the fact that iNOS can inhibit SMC proliferation (32, 33) and
migration (34), platelet activation (35), and extracellular-matrix
production (32). Thus, the therapeutic potential of iNOS is far
broader than any of the current pharmaceuticals used with drug-
eluting stents. Other types of gene vectors could be attached
through binding agents comparable with those used in the present
studies for Ads. To this end, high-affinity receptors for adeno-
associated viruses (36) and retroviruses (37) have been described
and cloned and, thus, could hypothetically be used with PAA-BP as
described herein.
It is concluded that pretreatment of metal alloy surfaces with an
aqueous bisphosphonate solution, PAA-BP, enables vector binding
to bare metal through the formation of a surface-oriented PAA-
BP–metal-coordination complex. This metal–bisphosphonate-
coordination complex has been demonstrated in the present studies
to enable the covalent attachment of vector-binding agents for
therapeutic gene delivery to the arterial wall. Because of the
wide spread use of metallic implants in medicine, these results have
broad implications for a therapeutic approach involving implant-
able medical device s configured with gene-therapy constructs.
We thank Mrs. Jeanne Connolly for her assist ance in preparing the
figures for this article, Ms. Jenn ifer LeBold for manuscript preparation,
and Cordis, Inc. (Warren, NJ) for donating stents through a material-
transfer agreement. This work was supported, in part, by National Heart,
Lung, and Blood Institute Grant HL 72108; a g rant from the Nanotech-
nology Institute; and both the William J. Rashk ind Endowment and
Erin’s Fund of The Children’s Hospital of Philadelphia.
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www.pnas.orgcgidoi10.1073pnas.0502945102 Fishbein et al.
Page 6
  • Source
    • "Briefly, 316L-grade steel foil samples or 304 grade stents (1 mm diameter) were cleaned with isopropanol and nitric acid and exposed to 1% aqueous solution of polyallylamine bisphosphonate with latent thiol groups, PABT, as previously described [22]. We have shown that this treatment creates a stable polymer monolayer on the steel surface due to the formation of coordination bonds between Fe and Cr atoms on the metal surface and bisphosphonic groups of the polymer [23]. After thiol group deprotection by TCEP (12 mg/ml in 0.1 M acetic buffer at 28 C for 10 min) and washing in degassed double distilled water, the PABT-modified surfaces were reacted with 2% polyethyleneimine (PEI) with installed pyridyldithio groups (PDT) for amplification of attachment sites (PEI-PDT [19]) at 28 C for 1 h and washed in double distilled water. "
    [Show abstract] [Hide abstract] ABSTRACT: The effectiveness of endovascular stents is hindered by in-stent restenosis (ISR), a secondary re-obstruction of treated arteries due to unresolved inflammation and activation of smooth muscle cells in the arterial wall. We previously demonstrated that immobilized CD47, a ubiquitously expressed transmembrane protein with an established role in immune evasion, can confer biocompatibility when appended to polymeric surfaces. In present studies, we test the hypothesis that CD47 immobilized onto metallic surfaces of stents can effectively inhibit the inflammatory response thus mitigating ISR. Recombinant CD47 (recCD47) or a peptide sequence corresponding to the Ig domain of CD47 (pepCD47), were attached to the surfaces of both 316L-grade stainless steel foils and stents using bisphosphonate coordination chemistry and thiol-based conjugation reactions to assess the anti-inflammatory properties of CD47-functionalized surfaces. Initial in vitro and ex vivo analysis demonstrated that both recCD47 and pepCD47 significantly reduced inflammatory cell attachment to steel surfaces without impeding on endothelial cell retention and expansion. Using a rat carotid stent model, we showed that pepCD47-functionalized stents prevented fibrin and platelet thrombus deposition, inhibited inflammatory cell attachment, and reduced restenosis by 30%. It is concluded that CD47-modified stent surfaces mitigate platelet and inflammatory cell attachment, thereby disrupting ISR pathophysiology.
    Full-text · Article · Feb 2016 · Biomaterials
  • Source
    • "Despite the encouraging results obtained with the bulk immobilization of gene vectors on polymer stent coatings, several studies have opted instead for a vector-tethering strategy to exclude the need for a polymer coating 123,125,127,182. Fishbein et al. 125 and Wang et al. 182 demonstrated effective gene delivery from BMS surfaces pretreated with polyallylamine bisphosphonate (PAA-BP) and conjugated to anti-pDNA or anti-adenoviral antibodies or recombinant D1 proteins via covalent cross-linking with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) 125,182. "
    [Show abstract] [Hide abstract] ABSTRACT: Percutaneous coronary intervention (PCI) has become the most common revascularization procedure for coronary artery disease. The use of stents has reduced the rate of restenosis by preventing elastic recoil and negative remodeling. However, in-stent restenosis remains one of the major drawbacks of this procedure. Drug-eluting stents (DESs) have proven to be effective in reducing the risk of late restenosis, but the use of currently marketed DESs presents safety concerns, including the non-specificity of therapeutics, incomplete endothelialization leading to late thrombosis, the need for long-term anti-platelet agents, and local hypersensitivity to polymer delivery matrices. In addition, the current DESs lack the capacity for adjustment of the drug dose and release kinetics appropriate to the disease status of the treated vessel. The development of efficacious therapeutic strategies to prevent and inhibit restenosis after PCI is critical for the treatment of coronary artery disease. The administration of drugs using biodegradable polymer nanoparticles as carriers has generated immense interest due to their excellent biocompatibility and ability to facilitate prolonged drug release. Despite the potential benefits of nanoparticles as smart drug delivery and diagnostic systems, much research is still required to evaluate potential toxicity issues related to the chemical properties of nanoparticle materials, as well as to their size and shape. This review describes the molecular mechanism of coronary restenosis, the use of DESs, and progress in nanoparticle drug- or gene-eluting stents for the prevention and treatment of coronary restenosis.
    Preview · Article · Jan 2014 · Theranostics
  • Source
    • "This " coatless " method of vector immobilization distinguishes our approach from other studies employing multi-micron polymer coatings of the stent struts to incorporate gene vectors in the bulk of the polymer. Both, our affinity binding [26] and HC tethering [27] Ad immobilization strategies have demonstrated effective local transduction of vascular tissue and inhibition of restenosis in stented arterial segments using stents configured with Ad vectors encoding inducible nitric oxide synthase (Ad-iNOS). Since the immobilization scheme realized using cleavable (hydrolyzable) cross-linkers[27] allowed for better control of vector loading and release, it was chosen for further development of stent-based gene delivery. "
    [Show abstract] [Hide abstract] ABSTRACT: The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolyzable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37 °C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolyzable cross-linkers with structure-specific release kinetics.
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