Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

Summary

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Abstract

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 t_1/2 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.

Introduction

The effectiveness of gene therapy as a therapeutic modality is hampered by the poor targeting capacity of gene therapy vectors^1,2. The lack of proper targeting results in sub-therapeutic levels of transgene expression at the target location and leads to a wide dissemination of vectors to non-target organs³, including those responsible for mounting immune responses against both the vector and encoded therapeutic product^4,5. One potential means to offset the promiscuity of transduction and to promote targeting is to introduce gene vectors at the desired location in a form that precludes their free dissemination via blood and lymph. Typically, such efforts rely on a locally injectable delivery systems comprising of either viral or non-viral vectors admixed with fibrin, collagen or hyaluronic acid hydrogel matrices^6-10 that are capable of transiently sustaining gene vectors at the injection site by physically entrapping them in a polymeric network.

Another generally accepted paradigm for localized gene therapy utilizes immobilization of gene vectors on the surface of implanted prosthetic devices^11,12. Permanent medical implants (endovascular, bronchial, urological and gastrointestinal stents, pacemakers, artificial joints, surgical and gynecological meshes, etc.) are used yearly in tens of millions of patients¹³. While generally effective, these devices are prone to complications that are inadequately controlled for by current medical practices^14-17. Implantable prosthetic devices present a unique opportunity to serve as proxy platforms for localized gene therapy treatment. From the pharmacokinetic standpoint, surface derivatization of medical implants with relatively low input doses of gene vectors results in achieving both high local concentrations of gene vectors on the implant/tissue interface and slowing the kinetics of their elimination from this location. As a consequence of protracted residence and enhanced uptake by the targeted cell population, vector immobilization minimizes spread of the gene vector. Thus the inadvertent inoculation of non-target tissues is reduced.

Surface tethering of gene vectors on implantable biomaterials (also termed as substrate-mediated gene delivery or solid phase gene delivery) has been implemented in cell culture and animal experiments using both specific (antigen-antibody^18-20, avidin-biotin^21,22) and non-specific^23-26 (charge, van der Waals) interactions. The covalent attachment of vectors to the surface of the implanted device has been previously considered as non-functional since excessively strong bonds with the surface preclude vector internalization by target cells. Recently it was demonstrated that this limitation can be overcome through the use of spontaneously hydrolysable cross-linker used as the tethers between the modified metallic surface of the stent and capsid proteins of the adenoviral vector^27,28. Moreover, the vector release rate and time course of transgene expression in vitro and in vivo can be modulated with the use of hydrolysable cross-linkers exhibiting different kinetics of hydrolysis²⁸.

The present paper provides a detailed protocol for the reversible covalent attachment of adenoviral vectors to activated metal surface and introduces a useful experimental setup for studying ensuing transduction events in vitro in cultured smooth muscle and endothelial cells and in vivo in the rat carotid model of stent angioplasty.

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Protocol

1. Preparation of Cy3-labeled Adenovirus for the Release Experiments

1. Suspend 2 x 10¹² particles of Ad_empty (approximately 2 x 10¹¹ infective units) in 650 µl of carbonate/bicarbonate buffer (CBB; pH 9.3).
2. Dissolve the content of 1 vial (0.2 mg) of amine-reactive fluorescent dye (Cy3(NHS)₂) in 1 ml CBB to a final concentration of 0.2 mg/ml.
3. Add 100 µl of the dye solution to virus suspension, vortex for 5 sec and incubate for 1 hr at 28 °C with shaking (100-200 rpm).
4. Equilibrate a 20 ml Sepharose 6B column with PBS. Add 750 µl of Cy3-labeled Ad suspension drop-wise to the center of the gel bed.
5. After viral suspension permeates the resin, add 5 ml of PBS. Discard the eluate.
6. Add 0.5 ml of PBS and collect the eluate in a labeled glass container. Repeat this step 10x collecting a total of ten 0.5 ml fractions.
7. Assay the collected fractions by spectrophotometry (260 and 280 nm) for viral DNA content.
8. Pool the fractions containing more than 10% of total (sum of F1 through F10) optical density (OD) at 260 nm. Note: Typically the fractions F3, F4, F5, and F6 are pooled and mixed.
9. Re-assay the mixed pooled fractions by spectrophotometry (260 and 280 nm) and assay by fluorometry (550_ex/570_em nm) for viral DNA content and Cy3 labeled capsid protein, respectively. Note: A Cy3(NHS)₂ calibration curve covering the 10^-9-10^-13 mol/L range is prepared and fluorimetrically assayed on the same plate as the pooled fractions.
10. Calculate the labeled virus yield and labeling density. Assume that 1.19 x 10¹² virus particles/ml corresponds to 1 OD unit for a 1 cm path length²⁹. Use the formula: Yield = [(OD_260nm/1.19)*10¹²*V/input Ad amount]*100, where, OD_260nm is the optical density of the pooled formulation at 260 nm and V is the volume of the pooled formulation (ml) to calculate Ad recovery yield (%). Use the formula: Labeling Density=C*6.02*10²³/(OD_260nm/1.19)*10¹², where C is Cy3 concentration of the pooled formulation (moles/ml) and OD_260nm is the optical density of the pooled formulation at 260 nm to calculate average Cy3 labeling density . NOTE: A typical recovery of labeled virus is >70% of the input dose. The labeling density is 600-800 fluorophore molecules per single virus particle.
11. Aliquot Cy3-labeled Ad_empty into smaller portions (typically 5 x 10¹¹ particles) suitable for the individual release experiments. NOTE: Current protocol specifies the use of Cy3-labeled Ad solely in the release experiment. All transduction studies are carried out with non-labeled vectors.

2. Activation of Metal Samples

1. Wash the stainless steel mesh disks or stainless steel stents consecutively in isopropanol (5 min x 2) and chloroform (5 min x 2) at 55 °C with shaking (100 rpm). Remove the solvent.
2. Heat the samples for 30 min at 200 °C.
3. Dissolve polyallylamine bisphosphonate with installed latent thiol groups (PABT^27,28,30) in water (1-2% w/v) at 72 °C with shaking (100 rpm). Adjust pH to 4.5-5 with KHCO₃.
4. Incubate metal samples in the PABT solution for 2-4 hr at 72 °C with shaking (100-200 rpm). NOTE: The procedure may be paused at this point. The samples are stable for 3 days at 4 °C.
5. Rinse the samples thrice with double distilled water (DDW).
6. Expose the samples to tris(2-carboxyethyl) phosphine (TCEP; 15 mg/ml in 0.1 M acetic buffer) for 15 min at 28 °C with shaking (100-200 rpm).
7. Rinse 5x in degassed DDW.
8. Expose the samples to 2% polyethyleneimine with installed pyridyldithio groups (PEI_PDT^27,28,30) in degassed DDW in an argon atmosphere for 2 hr at 28 °C with shaking (100-200 rpm). NOTE: The procedure may be paused at this point. The samples are stable for 2 weeks at 4 °C.
9. Rinse the samples three times with DDW.
10. Expose the samples to 10 mg/ml dithiothreitol/DDW to convert pyridyldithio groups to thiols.
11. Rinse 5x again in degassed DDW.

3. Adenovirus Activation and Metal Surface Immobilization

1. Suspend 5 x 10¹¹ particles of either Ad_eGFP or Ad_Luc (approximately 5 x 10¹⁰ infective units) in 487.5-495 µl of carbonate/bicarbonate buffer (CBB; pH 9.3). Note: For the release studies, use 5 x 10¹¹ of Cy3 labeled Ad_empty particles (adjust volume to 487.5-495 µl with CBB).
2. Dissolve HC with varying hydrolysis rates²⁸ [(rapidly (t_1/2 = 5 d), intermediately (t_1/2 = 12 d) and slowly (t_1/2 = 50 d) HC, i.e. RHC, IHC or SHC; Figure 1] or non-hydrolysable cross-linker (NHC), sulfo-LC-SPDP in CBB at 20 mM.
3. Immediately add 5-12.5 µl of cross-linker solution to the virus suspension for a total volume of 500 µl (200-500 µM final concentration of cross-linker), vortex and incubate for 1 hr at 28 °C with shaking (100-200 rpm).
4. Equilibrate a 20 ml Sepharose 6B column with degassed 5 mM EDTA/PBS (EPBS). Add dropwise 500 µl of cross-linker-modified Ad suspension to the center of the resin bed.
5. Add 5 ml of degassed EPBS. Discard the eluate.
6. Add 0.5 ml of degassed EPBS and collect the eluate in a labeled glass container. Repeat this step 10 times collecting a total of ten 0.5-ml fractions.
7. Determine OD of the collected fractions using spectrophotometry at 260 and 280 nm and convert OD to virus titers (1.19 x 10¹²/ml corresponds to 1 OD)²⁹.
8. Pool the fractions containing >10% of the eluted virus (Note: Typically, the fractions F3-F6). Repeat the spectrophotometric titer assay for the pooled suspension.
9. Transfer viral suspension to the vial with the activated metal samples (as per 2.11). Incubate in an argon atmosphere for 1 hr at 28 °C with shaking (100-200 rpm). Note: The Ad-tethered metal samples obtained in this step are further used in the subsequent release and transduction experiments described in the protocol sections 5-7.

4. Quantification of Surface-associated Ad Vector by PCR

1. Prepare meshes formulated with surface-immobilized Ad_eGFP tethered via NHC, SHC, IHC and RHC (n=3 for each type) as described in sections 2 and 3.
2. Use a QIAamp DNA Micro kit to isolate viral DNA. Place the meshes individually into 1.5-ml plastic tubes containing 200 µl of mixture composed of 180 µl of ATL buffer and 20 µl of proteinase K (both from the kit). Add known amount of Ad_eGFP particles (as standards for the calibration curve) into the separate tubes containing the same mixture. Incubate at 56 °C overnight without shaking.
3. Add 200 µl of AL buffer (from the kit), mix by vortexing, add 200 µl of 100% ethanol, and incubate at room temperature for 5 min.
4. Transfer the mixture from each tube onto an individual MinElite column (from the kit) and spin at 8,000 rpm for 1 min. Rinse the mesh-containing tubes with 180 µl of fresh ALT buffer, add onto respective columns and spin at 8,000 rpm for 1 min. Discard the eluates.
5. Add 500 µl of AW1 buffer (from the kit) into the columns and spin at 8,000 rpm for 1 min. Discard the eluates.
6. Add 500 µl of AW2 buffer (from the kit) into the columns and spin at 8,000 rpm for 1 min.
7. Discard the eluates.
8. Spin at 14,000 rpm for an additional 3 min until the columns are completely dry. Discard the eluates.
9. Add 50 µl of MilliQ-grade water into the columns. Spin at 14,000 rpm for 5 min. Collect 50 µl of eluate from each column into individual collection tube (from the kit).
10. Prepare PCR Master Mix solution by combining multiplies of the following (12.5 µl of Power Sybr Green PCR Master Mix, 0.63 µl of 10 µM eGFP sense primer [5’- ACG TAA ACG GCC ACA AGT TC -3’], 0.63 µl of 10 µM eGFP anti-sense primer [5’- AAG TCG TGC TGC TTC ATG TG -3’], 6.3 µl of MilliQ-grade water). Multiply these volumes by the number of planned reactions including “no DNA” controls.
11. Load 5 µl of Ad_eGFP DNA (from step 4.8) and 20 µl of PCR Master Mix into triplicate wells of a MicroAmp Optical 96-well Reaction plate. Seal the plate with MicroAmp Optical Adhesive Film. Spin the plate at 1,000 rpm for 1 min to eliminate air bubbles.
12. Place the plate into the receptacle of a 7500 Real-Time PCR engine. In the main menu of the 7500 System SDS Software (v1.4 or higher) select “Create new experiment”. Click “next”. In the “New experiment wizard” screen choose Sybr Green from a scroll-down menu and click “add”. Click “next” to get a layout of the plate. Highlight all wells to be analyzed, select Sybr Green. Highlight consecutively no DNA control wells, Ad_eGFP DNA standards and unknowns, and mark them using respective designations from the scroll-down menu.
13. Switch to the instrument tab. Select “add dissociation phase”, change the well volume from default 50 µl to 25 µl. Click start.
14. Analyze PCR results using after checking the QC summary for outliers and other irregularities.

5. Release Kinetics of Hydrolysable Cross-linker-tethered Vector Particles from the Model Steel Mesh

1. Wash the mesh samples derivatized with Cy3-labeled Ad via RHC, IHC, SHC and NHC (as per 3.9) in 1% BSA/PBS (1 hr x 3) with shaking (100-200 rpm).
2. Using sterile fine forceps, place the meshes into individual wells of a 96-well plate prefilled with 200 µl of elution buffer (0.1% BSA/0.1% Tween-20/PBS).
3. Take fluorescent images of a central part of each mesh. Record the settings of the microscope and the CCD camera used for image acquisition.
4. Assay the plate fluorimetrically (550_ex/570_em) in well-scan mode with maximum reduction. Use the wells with non-derivatized meshes as a background control.
5. Incubate the plate at 37 °C with shaking (50 rpm).
6. At predetermined times (1-30 days range) aspirate the elution buffer without disturbing the meshes and add 200 µl of fresh elution buffer. Repeat steps 4.3-4.5 after replacing the buffer.

6. Transduction of Cultured Cells by Mesh-immobilized Ad Vectors

1. Wash the Ad_eGFP- or Ad_Luc-derivatized meshes thrice with sterile PBS for 5 min with shaking (100 rpm).
2. Using fine sterile forceps remove the mesh disks one by one and individually place them into the wells of a 96-well plate with the cell type of interest in the log phase of growth.
3. Incubate the cells for 24 hr at 37 °C, in 5% CO₂.
4. Use the respective non-terminal endpoint assay (fluorescent microscopy, fluorometry for eGFP or bioluminescence imaging for luciferase) to determine the extent and the spatial distribution of gene expression in the wells.
5. Optionally, substitute medium for PBS to increase sensitivity of the fluorometry assay (485/535 nm) if low eGFP expression is expected. Note: If a fluorometer is equipped with well-scan capability, read the plate in a well-scan mode to assess the spatial distribution of eGFP-expressing cells in the wells. Exchange PBS for medium after completing fluorometry.
6. Image the transduced cells in the wells with Ad_eGFP-eluting meshes (both underlying and outlying the mesh) using the FITC filter set. Take representative images at 40-200X magnification. Record the exact settings of the fluorescent microscope and the CCD camera used for the acquisition of images.
7. Add 5 µl of luciferin stock in PBS (10 mg/ml) directly to wells with Ad_Luc-eluting meshes to a final concentration of 500 µg/ml and incubate at 37 °C and 5% CO₂ for 10 min prior to bioluminescence imaging. Aspirate media and replace with luciferin-free media after imaging.
8. Repeat endpoint assays at predetermined times (up to 2 weeks) to study the kinetics of reporter gene expression following substrate-mediated gene transfer.

7. Validation of Preserved Transduction Capacity at Delayed Time Points

1. Prepare the Ad_eGFP derivatized meshes using RHC, IHC, SHC and NHC for vector tethering (as per 2.1-2.11 and 3.1-3.9) and individually place the meshes in the wells of a 96-well plate with 60-80% confluent bovine aortic endothelial cells (BAEC).
2. Analyze transduction of cultured BAEC with mesh-immobilized Ad_eGFP at 1 and 2 days post-mesh placement using fluorescence microscopy and fluorometry (as per 6.4-6.5).
3. 48 hr after commencement of transduction wash the mesh-containing wells with PBS twice.
4. Add 200 µl of 0.25% trypsin/EDTA to each well and incubate for 15 min at 37 °C with shaking (100 rpm). Wash 3x with PBS.
5. Use fluorescent microscopy and fluorometry to ascertain complete removal of all eGFP-positive cells associated with the meshes.
6. Seed freshly passaged BAEC into the wells with partially released meshes at a 60-80% initial seeding density (2-2.7 x 10⁴ cells/well).
7. Incubate the plate at 37 °C and 5% CO₂ for predetermined periods of time (1-10 days) prior to assessment of “new” transduction events by fluorescent microscopy and fluorometry.

8. Ad-eluting Stent Deployment in the Rat Carotid Model of Stent Angioplasty

1. All animal procedures described in this protocol conform to Federal regulations on laboratory animal use and were approved by the IACUC of the Children’s Hospital of Philadelphia. To adhere to aseptic surgical conditions all instruments are autoclave sterilized. To assure continuous sterility a bead sterilizer is employed between use of the instruments in up to five consecutive animals.
2. Prepare Ad_Luc-eluting stents according to 2.1-2.11 and 3.1-3.9. Store the virus-derivatized stents in sterile PBS at 4 °C for no longer than 24 hr prior to use.
3. Anesthetize male Sprague-Dawley rats (400-450 g) with IP injection of ketamine (100 mg/kg), and xylazine (5 mg/kg). Determine the depth of anesthesia by paw pinch response and muscle tonus. Apply ophthalmic vet ointment to prevent dryness of cornea and sclera. Note: The inhalation anesthesia with 4% and 2% isoflurane (1 L/min) for inducing and maintaining anesthesia, respectively, is possible but impedes free access to the neck region of the animal and thus is not recommended for an inexperienced user.
4. Reassess depth of anesthesia by toe pinch. Shave and aseptically prep neck and upper chest region. Administer antibiotic (cefazolin; 20 mg/kg; IM), analgesic (meloxicam; 0.5 mg/kg; SC) and saline (10 ml/kg; SC). Catheterize the tail vein with a 24 G catheter and administer heparin (200 IU/kg; IV). Note: A dose of 10 mg/kg (IM) enrofloxacin (Baytril) can be used instead of cefazolin. Avoid use of antibiotics lacking significant activity against Gram-positive bacteria. 10-15 mg/kg carprofen (Rimadyl) may be used instead of meloxicam as a preemptive analgesic. Narcotic analgesics (e.g., morphine, buprenorphine) should be avoided because of their respiration-depressing properties.
5. Perform a midline incision through the skin and neck fascia. Use blunt dissection techniques to isolate the left external carotid artery. Tie-off the external carotid artery at the most distal approachable site. Apply a sliding temporary ligature to the origin of the internal carotid artery.
6. Make a 2-mm arteriotomy incision in the left external carotid artery.
7. Insert a 2-French Fogarty catheter into the common carotid artery through the incision in the external carotid artery. Inflate the tip of catheter with saline and pass 3x from the aortic arch to the carotid bifurcation in order to denude the endothelium.
8. Slide a piece of tubing (1.04 mm OD, 0.99 mm ID) over the Fogarty catheter and into the common carotid artery. Withdraw the Fogarty catheter.
9. Mount and crimp an Ad_Luc-derivatized stent over the balloon of a 1.5 mm diameter angioplasty catheter. Insert the stent through the Teflon tubing and advance it into the mid-section of the common carotid artery. Avoid rubbing the stent against the tube or vessel wall.
10. Deploy the stent at 12 atm for 30 sec and withdraw the angioplasty catheter.
11. Tie-off the external carotid artery proximal to the arteriotomy site and release the temporary ligature on the internal carotid artery.
12. Repair the operative wound in layers with running 4.0 Vicryl suture and staple the skin.
13. Recover the animal on a warming pad until ambulatory and return to its isolated cage. While no signs of pain or discomfort are typically exhibited by the post-operative animals beyond the first 12 hr after the procedure, consider extension of meloxicam  therapy (0.5 mg/kg, SC daily) for 72 hr.

9. Bioluminescence Imaging of Arterial Gene Expression

1. At predetermined time points (1 day – 3 weeks range) after gene-eluting stent deployment in the common carotid artery, anesthetize the rat using isoflurane inhalation anesthesia (2-4% isoflurane in oxygen).
2. Remove the surgical staples, aseptically prepare the site and reopen the operative wound. Using blunt dissection, re-gain access to the left common carotid artery and separate it from the vagus nerve and adjacent connective tissue.
3. Prepare a mixture of 50 mg/ml Luciferin in PBS and 25% Pluronic F-127 in PBS (1:4 v/v) and store it on ice. Note: This formulation presents as a viscous solution at 4 °C and immediately turns to gel upon contact with tissue at 37 °C.
4. Apply 200 µl of a chilled Luciferin/Pluronic mixture directly to the exposed segment of the common carotid artery and verify gel solidity.
5. Place the animal in the supine position in the imaging chamber of the IVIS-Spectrum apparatus and maintain isoflurane anesthesia with a face mask.
6. In the acquisition control panel window (Living Image, version 4.2 or higher) choose the position “B” (6.6 cm camera to object distance) and binning factor “medium” from the dropdown menus entitled “field of view” and “binning”, respectively. Type in “2.5 cm” in the subject height box. Choose “min” as a unit of time in the “exposure time” box, and choose a numerical value of “2”.
7. Three min after application of the Luciferin/Pluronic gel, start image acquisition by clicking the “Acquire” button on the screen. NOTE: Image acquisition time can vary from 1 to 6 min depending on the anticipated signal strength.
8. After acquiring the image, wash the gel off with saline and dab the periarterial space with sterile gauze and cotton applicators.
9. Close the wound with Vicryl suture and staple the skin.
10. Recover the animal and return to its cage. Repeat imaging at later time points to study the time course of arterial expression brought about by the stent-immobilized Ad vectors.
11. Alternatively, perform imaging following a systemic administration of luciferin. Anesthetize and prep the animal as per 9.1-9.2. Catheterize the tail vein with a 24 G catheter and secure catheter with surgical tape.
12. Prepare a solution of luciferin in PBS (50 mg/ml) and inject 1 ml of the solution through the catheter over a 10 sec interval. One min after injection start image acquisition as per 9.7-9.8. Note: A faster injection rate (<10 sec) can provoke seizure activity and respiratory arrest. The animal must be euthanized if seizures or respiratory arrest occur during imaging.
13. Follow step 9.10.

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Results

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 hypothesi...

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Discussion

The presented protocol describes an operational method for substrate mediated gene delivery achieved through reversible attachment of adenoviral vectors to coatless stainless steel surfaces. While developed for the specific purpose of stent-based gene therapy of vascular restenosis, this technique has much broader applications in the areas of biomaterials, biomedical implants and gene therapy.

Although presented studies have solely utilized stainless steel as a prototypical metal substrate, PA...

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Materials

Name	Company	Catalog Number	Comments
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 24 G 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