1. Preparation of Cy3-labeled Adenovirus for the Release Experiments
2. Activation of Metal Samples
3. Adenovirus Activation and Metal Surface Immobilization
4. Quantification of Surface-associated Ad Vector by PCR
5. Release Kinetics of Hydrolysable Cross-linker-tethered Vector Particles from the Model Steel Mesh
6. Transduction of Cultured Cells by Mesh-immobilized Ad Vectors
7. Validation of Preserved Transduction Capacity at Delayed Time Points
8. Ad-eluting Stent Deployment in the Rat Carotid Model of Stent Angioplasty
9. Bioluminescence Imaging of Arterial Gene Expression
Vector Release Experiments
Tethering of adenoviral vectors to the surface of implants, including interventional devices such as endovascular stents, approximates the vector to the disease site, partially obviating the lack of vectors’ physical targeting. However, to be able to achieve therapeutic effects via the transduction of target tissue, the vector must be released from the surface (Figure 2). The use of hydrolysable cross-linkers was hypothesized to allow 1) effective attachment of vectors to polybisphosphonate-modified, thiol-installed metal implants and 2) sustained release mediated by hydrolysis of the cross-linker.
The number of genomic copies of AdeGFP vector associated with the mesh disks by the end of the derivatization procedure is determined by PCR of viral DNA with eGFP-specific primers (Figure 3). Depending on the specific cross-linker used, the amount of bound Ad vector should vary between 3.5 x 109 and 5.7 x 109 particles/mesh. This amount is in fair correspondence with the estimated maximal capacity (2.5 x 109) of a single mesh disk with a total area of 0.25 cm2 to accommodate 100-nm Ad particles in a monolayer arrangement. Since the starting amount of Ad vector at the step of cross-linker modification is 5 x 1011 particles, only ~1% of the modified vector is eventually immobilized on the PABT/PEIPDT-derivatized stainless steel surface.
Both the rate of in-chain ester hydrolysis and the number of links between the vector and the biomaterial are expected to affect the overall kinetics of Ad vector release from the surface.
To facilitate the release experiments, adenoviral particles can be pre-labeled with Cy3 fluorophore (Protocol; section 1) prior to cross-linker modification and surface immobilization (Protocol; section 3). The decrease of surface-associated fluorescence over time assessed concurrently by fluorometry (Figure 4A) and fluorescence microscopy (Figure 4B) can then be used as an indirect method for monitoring vector release into physiological buffer. Ad vectors attached through RHC and IHC linkages should demonstrate significantly faster release in comparison with their SHC- and NHC-attached counterparts. In the given example, a 45% and a 39% loss of surface-associated vector was observed with RHC and IHC-tethered vector by day 30, respectively, while less than 20% of tethered vector was observed to be released in SHC and NHC-immobilized samples (Figure 4A).
Additionally, Ad vectors immobilized by using different concentrations of the same HC and thus presumably having differing numbers of tethers between the vector and metal substrate should have dissimilar kinetics of vector release. Indeed, vector modification using 0.1 mM RHC resulted in significantly faster release rates than observed for vector immobilized with 0.5 mM RHC (Figure 4A). The fluorescence microscopy data (Figure 4B) correlates well with the fluorometry-based quantitative vector release analysis, demonstrating a faster and more profound decrease of surface-associated fluorescence with mesh samples formulated using IHC-, RHC- and especially low concentration RHC-tethered vectors in comparison with their NHC- and SHC-tethered counterparts.
In vitro Transduction with Mesh Immobilized Ad Vectors
By being compatible with both quantitative expression analysis (fluorometry) and spatial observations (fluorescence microscopy), AdeGFP is the preferred reporter vector to monitor transduction in SMC and endothelial cell cultures treated with both free and mesh-immobilized Ad vectors. Chemical modification of Ad capsid with RHC (Figure 5) as well as with other cross-linkers (not shown) at concentrations exceeding 75 µM significantly impairs transduction effectiveness of non-immobilized vector in vitro, most probably due to masking and reconfiguring of fiber knob domains utilized by Ad for binding to the cells through the Coxsackie-Adenovirus receptors (CAR). However, the role of knob-CAR interactions is less vital for the transduction with substrate immobilized vector since the vector is retained in the vicinity of targeted cells by tethering to substrate. In cell culture experiments regardless of the HC type, mesh-associated Ad vectors are consistently capable of spatially restricting transduction events to cells located within 300-500 µm from the mesh borders (Figures 6A and 6C). Typically the peak levels of transgene expression are achieved 3-5 days after the placement of AdeGFP-loaded meshes into SMC (Figures 6A and 6B) or BAEC (Figures 6C and 6D) cultures. At the same vector load, peak eGFP expression levels increase in the following sequence: NHC-, SHC-, IHC- and RHC-tethered vector, reflecting the differences in their respective release kinetics profiles (Figure 4). Furthermore, more durable transgene expression is observed in cells treated with IHC-formulated meshes in comparison to cells treated with the RHC-formulated counterparts (Figure 6D).
The utilized cell culture system presents a convenient set-up for the investigation of delayed transduction events brought about by vector particles released from the metal surface at or later than 48 hr after mesh placement. In this application, after determining eGFP expression by fluorometry and fluorescence microscopy at 48 hr after commencement of transduction, BAEC are trypsinized and aspirated out of the wells without disturbing the meshes. Freshly passaged non-transduced BAEC are then re-plated over the partially released meshes. “New” transduction events caused by the virus particles released from the mesh carrier after the 2 day time point are then assessed by fluorescence microscopy and fluorometry (Figures 7A and 7B). Robust eGFP expression demonstrating the characteristic spatial restriction to a mesh locale is typically observed in these “new” cultures (Figure 7), confirming a well-preserved transduction capacity of the mesh-bound Ad vectors even after 48 hr exposure to complete cell culture medium at 37 °C.
In vivo Studies
To examine potential effects related to the use of different HC on arterial transduction, animals implanted with AdLuc-eluting stents prepared with RHC-, IHC-, SHC- and NHC-modified vector underwent bioluminescent imaging 1 and 8 days after stent deployment (Figure 8). The imaging was carried out following local perivascular administration of luciferin (2 mg) co-formulated with Pluronic gel. This formulation is a viscous fluid when chilled on ice, but undergoes immediate phase transition to gel when brought in contact with tissue at 37 °C. Preliminary experiments (not shown) demonstrated that perivascular delivery of luciferin results in more stable and reproducible luminescence signals in AdLuc-transduced vascular tissue when compared to systemic (intraperitoneal or intravenous) luciferin administration.
If virus tethering to the stent and the stent deployment surgery were technically sound, a well-defined signal corresponding to the stented segment of rat carotid artery is emitted and recorded. One day after stent implantation, animals treated with RHC-formulated AdLuc stents typically exhibit the highest luminescence signal, followed by the rats receiving IHC- and SHC- formulated stents (Figures 8A and 8B). No perceptible signal is observed in the group of rats implanted with stents prepared using NHC-tethered AdLuc, underscoring the importance of unimpeded vector release from the stent for effective transduction of vascular tissue (Figures 8A and 8B). By day 8, the average intensity of luciferase expression with RHC-formulated stents drops several-fold, while it increases 1.4- and 1.8-fold with IHC- and SHC-formulated stents, respectively (Figures 8A and 8B). This finding is consistent with the hypothesis of the more durable vascular transduction with gene-eluting stents exhibiting a slower kinetics of vector release from the stent surface.
Figure 1. Structural formulas of cross-linkers (NHC, SHC, IHC and RHC) used throughout the described studies (modified with permission28).
Figure 2. A scheme representing Ad vector tethering to a PABT/PEIPDT-modified stainless steel surface via a HC and subsequent release of the vector upon cross-linker hydrolysis (modified with permission28).
Figure 3. PCR-based quantification of AdeGFP immobilized via cross-linker tethering on the surface of PABT/PEIPDT-modified stainless steel meshes. The stainless steel meshes were formulated with AdeGFP immobilized via RHC, IHC, SHC, or NHC (n = 3 for each condition). After proteinase K treatment, viral DNA was eluted and purified using MinElute columns. Amplification of viral DNA using eGFP-specific primers was carried out in a 7500 Real-Time PCR engine and was detected with Sybr Green. Data normalization was based on a calibration curve prepared with a known amount of non-immobilized AdeGFP(modified with permission28).
Figure 4. Release kinetics of Ad vector tethered to metal substrates with HC. Stainless steel meshes were derivatized with ~2 x 109 Cy3-labeled Ad particles attached to the PABT/PEIPDT-modified surface after modification with 500 µM NHC, SHC, IHC, RHC and 100 µM RHC (designated as RHC-L). The release of fluorescently labeled Ad-particles was studied at the indicated time points by (A) well-scan fluorometry at 550/570 nm and (B) fluorescent microscopy (rhodamine filter set; original magnification 200X). Fluorometry results are presented as means ± SEM, n = 8-10; p < 0.001 for all comparisons between the NHC and SHC vs IHC, RHC and RHC-L groups were determined by Anova with a post-hoc Tukey test (modified with permission28).
Figure 5. Transduction effectiveness of non-immobilized AdeGFP following modification with RHC. Rat embryonic aorta-derived SMC (A10 line) were transduced at a MOI of 1,000 with either unmodified AdeGFP or vector modified with RHC as indicated. A reporter expression was determined fluorometrically (485/535 nm) 48 hr after transduction (modified with permission27).
Figure 6. Transduction of cultured SMC and BAEC with Ad vector immobilized to stainless steel meshes with HC. Meshes were derivatized with ~2 x 109 AdeGFP particles appended via NHC, SHC, IHC, and RHC. Meshes were then individually placed on top of sub-confluent A10 (A, B) and BAEC (C, D) monolayers. Transduction (expressed as eGFP expression levels) of cells treated with Ad vector-tethered meshes was assessed by fluorescence microscopy (A, C; FITC filter set; original magnification 100X) and well-scan fluorometry (B, D). Fluorometry results are presented as means ± SEM, n = 4 (modified with permission28). Please click here to view a larger version of this figure.
Figure 7. Transduction competence of substrate immobilized Ad vectors at delayed time points. AdeGFP was immobilized on steel meshes through NHC, SHC, IHC or RHC tethers. The meshes were placed on the subconfluent BAEC monolayers. Two days after placement, BAEC were trypsinized and removed without disturbing the meshes. New, non-transduced BAEC were then seeded over the meshes. AdeGFP transduction competency was measured by eGFP expression using fluorescent microscopy (A; FITC filter set; original magnification 100X) and fluorometry (B). Measurements were taken at indicated days, representative images were used and fluorometry results are means ± SEM, n = 4 (modified from with permission28). Please click here to view a larger version of this figure.
Figure 8.Transduction efficiency of stent-immobilized AdLuc in vivo. AdLuc (1.3 x 1010 particles) was tethered to endovascular stents via NHC, SHC, IHC or RHC (n = 3-4 for all groups). Transduction efficiency of AdLuc eluted from stents was measured by bioluminescence imaging (IVIS Spectrum) 1 and 8 days post stent deployment (A), and plotted using a logarithmic scale (B) (modified from with permission28). Please click here to view a larger version of this figure.
316 stainless steel mesh disks | Electon Microscopy Sciences | E200-SS | |
Generic 304-grade stainless steel stents | Laserage | custom order | |
AdeGFP | University of Pennsylvania Vector Core | AD-5-PV0504 | |
AdLuc | University of Pennsylvania Vector Core | AD-5-PV1028 | |
AdEMPTY | University of Pennsylvania Vector Core | A858 | |
Cy3(NHS)2 | GE Healthcare | PA23000 | |
Sepharose 6B | Sigma-Aldrich | 6B100-500ML | |
UV 96-well plates | Costar | 3635 | |
Fluorometry 96-well plates | Costar | 3915 | |
Cell culture 96-well plates | Falcon | 353072 | |
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP ) | Pierce Thermo Scientific | 20490 | |
dithiothreitol (DTT) | Pierce Thermo Scientific | 20290 | |
sulfo-LC-SPDP | Pierce Thermo Scientific | 21650 | |
Spectrophotometer | Molecular Devices | SpectraMax 190 | |
Spectrofluorometer | Molecular Devices | SpectraMax Gemini EM | |
Orbital shaker incubator | VWR | 1575R | |
Horizontal airflow oven | Shel Lab | 1350 FM | |
Centra-CL2 centrifuge | International Equipment Company | 426 | |
Digital vortex mixerer | Fisher Thermo Scientific | 02-215-370 | |
Eclipse TE300 fluorescence microscope | Nikon | TE300 | |
DC 500 CCD camera | Leica | DC-500 | |
7500 Real-Time PCR system | Applied Biosystems | not available | |
IVIS Spectrum bioluminescence station | Perkins-Elmer | not available | |
EDTA dipotassium salt | Sigma-Aldrich | ED2P | |
Bovine serum albumin fraction V (BSA) | Fisher Thermo Scientific | BP1600-100 | |
Tween-20 | Sigma-Aldrich | P1379 | |
Dumont forceps | Fine Science Tools | 11255-20 | |
A10 cell line | ATCC | CRL-1476 | |
Bovine aortic endothelial cells | Lonza | BW-6002 | |
Luciferin, potassium salt | Gold Biotechnology | LUCK-1Ge | |
Pluronic F-127 | Sigma-Aldrich | P2443-250G | |
PBS without calcium and magnesium | Gibco | 14190-136 | |
Fetal bovine serum | Gemini Bio-Products | 100-106 | |
Penicillin/Streptomycin solution | Gibco | 11540-122 | |
DMEM, high glucose | Corning cellgro | 10-013-CV | |
0.25% Trypsin/EDTA | Gibco | 25200-056 | |
QIAamp DNA micro kit | Qiagen | 56304 | |
Power Sybr Green PCR Master Mix | Applied Biosystems | 4367659 | |
MicroAmp Optical 96-well Reaction Plate | Applied Biosystems | N8010560 | |
MicroAmp Optical Adhesive Film | Applied Biosystems | 4360954 | |
Cephazolin | Apotex | not available | |
Loxicom (Meloxicam) | Norbrook | not available | |
Heparin sodium | APP Pharmaceuticals | not available | |
Ketavet (Ketamine) | VEDCO | not available | |
Anased (Xylazine) | Lloid | not available | |
Forane (Isoflurane) | Baxter | not available | |
Curved Moria iris forceps | Fine Science tools | 11370-31 | |
Curved extra-fine Graefe forceps | Fine Science Tools | 11152-10 | |
Dumont #5 forceps | Fine Science Tools | 11252-20 | |
Vannas spring scissors | Fine Science Tools | 15018-10 | |
Fine scissors – ToughCut | Fine Science Tools | 14058-09 | |
Surgical scissors | Fine Science Tools | 14101-14 | |
Vicryl suture (5-0) | Ethicon | J385 | |
Suture thread (4/0 silk) | Fine Science Tools | 18020-40 | |
Michel suture clips | Fine Science Tools | 12040-02 | |
Wound dilator (Lancaster eye specula) | KLS Martin | 34-149-07 | |
Hot bead sterilizer | Fine Science Tools | 18000-45 | |
Michel suture clip applicator | Fine Science Tools | 112028-12 | |
Insyte Autoguard 24G IV catheter | Beckton-Dickinson | 381412 | |
2F Fogarty catheter | Edwards Lifesciences | 120602F | |
Teflon tubing | Vention | 041100BST | |
PTA catheter | NuMed | custom order | |
Gauze pads | Kendall Healthcare | 9024 | |
Cotton applicators | Solon Manufacturing | WOD1003 | |
Saline | Baxter | 281321 | |
10 ml syringe (Luer-Lok) | Beckton-Dickinson | 309604 | |
1 ml syringe (Luer-Lok) | Beckton-Dickinson | 309628 | |
Clippers with #40 blade | Oster | 78005-314 | |
Transpore surgical tape | 3M | MM 15271 | |
Puralube vet ointment | Pharmaderm | not available |
In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.
In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.
In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.