www.sciencetranslationalmedicine.org/cgi/content/full/6/218/218ra6/DC1

Supplementary Materials for

A Blood-Resistant Surgical Glue for Minimally Invasive Repair of Vessels and Heart Defects

Nora Lang, Maria J. Pereira, Yuhan Lee, Ingeborg Friehs, Nikolay V. Vasilyev,

Eric N. Feins, Klemens Ablasser, Eoin D. O’Cearbhaill, Chenjie Xu, Assunta Fabozzo, Robert Padera, Steve Wasserman, Franz Freudenthal, Lino S. Ferreira, Robert Langer,

Jeffrey M. Karp,* Pedro J. del Nido*

*Corresponding author. E-mail: jmkarp@partners.org (J.M.K.); pedro.delnido@cardio.chboston.org (P.J.d.N.)

Published 8 January 2014, Sci. Transl. Med. 6, 218ra6 (2014)

DOI: 10.1126/scitranslmed.3006557

This PDF file includes:

Materials and Methods Fig. S1. Rheologic properties of the PGSA prepolymer. Fig. S2. The adhesive strength of the HLAA to cardiac tissue varies with the degree of acrylation. Fig. S3. UV light transmitted through multiple patch materials. Fig. S4. FTIR evaluation of the HLAA. Fig. S5. Adhesive strength of the HLAA. Fig. S6. Approach for testing the adhesive strength of the HLAA or CA in the presence of flowing blood. Fig. S7 HLAA cytocompatibility. Fig. S8. Cardiac function over the course of the study in the in vivo biocompatibility study and LV wall defect model. Fig. S9. Patch delivery system. Legends for movies S1 and S2

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/6/218/218ra6/DC1)

Movie S1 (.wmv format). Closure of a transmural LV wall defect with an HLAA-coated patch. Movie S2 (.avi format). The HLAA-coated patch resists supraphysiologic, highly dynamic conditions inside the chamber of the heart.

Submitted Manuscript: Confidential template updated: February 28 2012

SUPPLEMENTARY METHODS Chemical and mechanical characterization of the hydrophobic light-activated adhesive

(HLAA)

The degree of acrylation of HLAA networks after purification was evaluated through NMR

(Bruker AVANCE 400 MHz), and calculated as described below. The stiffness and elasticity of

cured HLAA networks (n = 5) were evaluated through a compression cyclical test at a rate of 1

mm/min (eXpert 3600 Biaxial, ADMET) and up to the maximum strain of approximately 15%

for 100 cycles. This maximum strain was selected considering that the global longitudinal strain

of cardiac tissue during systole is ~15%, as reported based on speckle tracking measurements1-2.

The tested samples were cured for 5 s at a light intensity of 0.38 W/cm2 and in the presence of

the UV-transparent borosilicate glass. Samples were 6 mm in diameter and 1 mm in height. The

compressive modulus was calculated as the initial slope (up to 15% of strain) observed.

Rheological properties of the poly(glycerol sebacate acrylate) (PGSA) prepolymer

The rheological properties of the PGSA with an acrylation of 0.5 mol of acryloyl chloride per

mol of glycerol were determined using a using an AR2000 (TA Instruments) stress controlled

rheometer equipped with a 20-mm plate. Oscillatory stress sweep was applied between 0.1 to

1000 Pa at room temperature and at a frequency of 0.1 Hz to evaluate the storage and loss

modulus of the prepolymer. Flow experiments were performed to determine the viscous

properties of the prepolymer at room temperature with a shear rate from 0.01 to 100 s-1 (n = 3).

All measurements were performed at room temperature.

Determination of the degree of acrylation

PGS was acrylated with different mol of acryloyl chloride per mol of glycerol in the prepolymer.

NMR was used to confirm and quantify the degree of acrylation. The degree of acrylation was

determined according to the formula:

Degree of acrylation = δf,g,h × (4/δd),

where δf,g,h represents the average signal resulting from the acrylate protons, and δd the signal

resulting from protons d in the sebacic acid molecule (fig. S2).

PGSU patch synthesis

Poly(glycerol sebacate urethane) (PGSU) patches were prepared as described previously3.

Briefly, PGS prepolymer was solubilized in dimethylformamide (DMF, 10% w/v) and heated to

55ºC in the presence of the catalyst stannous 2-ethyl-hexanoate (0.05% w/v). Hexamethylene

diisocyanate (HDI) was added to the solution in a molar proportion glycerol:HDI of 1:0.5 and

allowed to react for 5 hours. The solution was cast on a Teflon mold and the solvent evaporated.

Prior to in vivo use the patch material was extracted with ethanol and sterilized by autoclave

(121oC, 100 kPa for 15 minutes). HLAA was applied to one PGSU patch surface using a pipette.

Light intensity measurement during UV exposure

The OmniCure S1000 (Lumen Dynamics Inc.) was used for all experiments. A 5-mm light guide

was used for the determination of the light intensity exposed to the tissue. We used a Newport

1918-R optical power meter (Newport), able to measure radiation with a wavelength of 365 nm,

as a light sensor. The power meter was zeroed in ambient light. For light intensity measurements,

the borosilicate glass rod was attached to the light guide. Then, a PGSU patch (diameter = 6 mm,

as used in all experiments) was applied to the light guide and pressed against the detector of the

power meter. The measurements were performed three times and averaged with and without the

addition of a borosilicate glass rod/PGSU patch.

The light source used had an approximate intensity of 0.38 W/cm2 at the tip of the light

guide. Given that UV energy was lost because of the interposed borosilicate and PGSU, the

energy reaching the tissue was approximately 0.12 W/cm2.

Tissue temperature measurement during UV light exposure

Temperature sensors were implanted below the epicardial surface, and the temperature was

monitored during the curing process (3100 Fluoroptic Thermometer, Luxtron). The cardiac tissue

was initially at room temperature (~23ºC) and during UV exposure the maximum temperature

observed was approximately 34ºC.

Determination of UV light transparency of patch materials

The UV light transparency of the PGSU patch and patch materials currently in clinical use

including bovine pericardium (Supple Peri-Guard), porcine small intestine submucosa

(Cormatrix), and polyethylene terephthalate (PET; Dacron) was determined (n = 3 per material).

The same setup used for measuring the light intensity was applied to measure light transparency.

The patch materials were interposed between the light guide/borosilicate rod and the detector of

the power meter, and the total light intensity was measured and compared to the intensity

obtained in the absence of the patch. The quotient was determined.

ATR-FTIR evaluation of the HLAA

ATR-FTIR was performed using a Bruker Alpha spectrophotometer to evaluate the HLAA

chemical composition before and after exposure to UV.

Determination of the HLAA glue layer thickness

The thickness of the HLAA layer applied in the patch materials was controlled through the

amount of material spread over the surface. To determine the average thickness of the layer, the

HLAA was cured on top of the patch and the thickness of the patch, alone or with the glue, was

measured using a caliper. This technique was applied to determine both the thickness of different

glue layers used for adhesion testing, as well as to quantify the glue layer before and after

exposure to blood flow.

Cytocompatibility studies

All materials (80 mg) were incubated in 5ml DMEM media containing 10% FBS (37ºC) for 3

days. 3T3-Fibroblasts were plated in a 96-well plate (5×103 cells per well, n = 5 per experimental

condition). Cells were treated with each extraction media (200 µl per well) and incubated for 24

h at 37ºC and 5% CO2. Cell viability was measured using CCK-8 kit (Dojindo).

Biocompatibility of HLAA and adhesive strength of HLAA- and CA-coated patches in a rat

model

Preoperative echocardiography (Vevo 2100 System, VisualSonics Inc.) was performed. A left

anterior thoracotomy in the 4th intercostal space was performed to gain access to the left

ventricle (LV). After opening the pericardium, a HLAA-coated PGSU patch (diameter = 6 mm)

was attached to the epicardium. While activating the HLAA with UV light, the patch was

pressed against the epicardial surface with the light guide and an interposed borosilicate glass

cylinder. Cyanoacrylate (CA)-coated patches were used as a positive control. At defined survival

time points (7 and 14 days, n = 8 for the HLAA and n = 7 for CA) echocardiography was

performed and animals were euthanized. Hearts were explanted, fixed in 4% paraformaldehyde

(PFA) and hematoxylin and eosin (H&E) and Masson Trichrome (MT) staining was performed.

The degree of necrosis and inflammation was assessed by a blinded pathologist. The following

scale was used for evaluation: 0- negligible, 1-reduced, 2-moderate, and 3- severe.

For determination of the in vivo adhesion strength, HLAA (n = 3) and CA (n = 3) coated

patches were attached to the rat heart epicardium as described above. After 2 days of

implantation, hearts were explanted. The patch with a thin layer of epicardium was immediately

cut from the whole heart and pull-off adhesion testing was performed.

Determination of cardiac function

The fractional shortening (%) is a measure of cardiac function and measures the percent change

in the diameter of the LV between the contracted and relaxed states and was determined by the

following formula:

[(LVEDD – LVESD)/LVEDD] × 100

Therefore, LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were

estimated by means of echocardiography. They were obtained from M-mode images at the

midpapillary level in the parasternal short-axis view.

Determination of the thrombogenic potential of HLAA and PGSU

Circular patches (diameter = 12 mm) of HLAA, PGSU, and glass were incubated with

heparinized porcine blood for 1 h at 37°C on a hematology mixer. The surfaces were rinsed

thoroughly after blood contact with 50 ml of PBS and immersed in 1 ml of 2% Triton X-100

solution for 20 min to lyse surface adherent platelets. The number of deposited platelets on each

sample was then quantified by a lactate dehydrogenase (LDH) assay with an LDH Cytotoxicity

Detection Kit (Promega).

SUPPLEMENTARY FIGURES

Fig. S1. Rheologic properties of the PGSA prepolymer. (A) Viscoelastic properties of the PGSA prepolymer. For angular frequencies where the loss modulus (G’’) is higher than the storage modulus (G’), the HLAA prepolymer behaved as a viscous liquid (n = 3). (B) The PGSA prepolymer presents relatively stable viscosity over different shear rates. Data are means ± SD (n = 3, separate batches).

Fig. S2. The adhesive strength of the HLAA to cardiac tissue varies with the degree of acrylation. (A) NMR spectra and chemical structure of the PGSA prepolymer, confirming the presence of acrylate groups in the prepolymer backbone. PGSA were synthesized with three different degrees of acrylation (0.2, 0.5, and 0.9 mol/mol of glycerol molecules) as determined through NMR. (B) Adhesive strength for three different degrees of acrylation. Data are means ± SD (n = 3–4). P-values determined by one-way ANOVA with Tukey post hoc analysis.

Fig. S3. UV light transmitted through multiple patch materials. Patch materials PGSU, bovine pericardium (BP), porcine small intestine submucosa (SIS), and PET (polyethylene terephthalate) were evaluated. Patch transparency and average adhesion for HLAA-coated patches were measured after 5 seconds of UV light exposure. Data are means ± SD (n = 3 per material).

Fig. S4. FTIR evaluation of the HLAA. (A) Spectrum of the HLAA prior to UV activation. (B) Magnification of the vinyl group stretch peak before and after 5 seconds of HLAA exposure to UV light.

Fig. S5. Adhesive strength of the HLAA. (A to C) The effect of light intensity (A), pre-load (B), and glue layer thickness (C) on HLAA adhesive pull-off strength. Data are means ± SD (n = 4). P-values determined by one-way ANOVA with Tukey post hoc testing.

Fig. S6. Approach for testing the adhesive strength of the HLAA or CA in presence of flowing blood. (A) The test set-up comprised the following steps: HLAA-coated patches were immersed in blood for 5 minutes at 37ºC and the samples were shaken at 500 RPM; the coated patches were then brought in contact with epicardial tissue and the material was activated with UV light; pull-off adhesive strength was measured. (B) Thickness of the HLAA layer before and after exposure to flowing blood. Data are means ± SD (n = 5). P-values determined by an unpaired t-test.

Fig. S7. HLAA cytocompatibility. The cytocompatibility of cured and uncured HLAA was compared with commercially available adhesives including fibrin and CA. Data are means ± SD (n = 5). P-values determined by one-way ANOVA with the Tukey post hoc test.

Fig. S8. Cardiac function over the course of the study in the in vivo biocompatibility study and the LV wall defect model. (A) Cardiac function before and after attachment of HLAA- and CA-coated patches on the rat epicardium for 14 days. CA was used as control group, as detailed in Fig. 3C. Data are means ± SD (n = 3). (B) Cardiac function before and after closure of LV wall defects with HLAA-coated patches or sutures at different postoperative time points. Sutures were used as control group, as detailed in Fig. 3E. Data are means ± SD (n = 3).

Fig. S9. Patch delivery system. (A) Overview of the patch delivery system. Prior to release, the patch is fixed to an angled (90º) frame that favors the contact with the septum during delivery. (B) Location of the nitinol wires. The patch is sutured to the nitinol wires, but not to the patch delivery system, so that once pulled, the release of the patch from the frame is promoted.

SUPPLEMENTARY MOVIES

Movie S1. Closure of a transmural LV wall defect with an HLAA-coated patch. A

representative example of the creation and closure of LV wall defect with a HLAA coated patch

is shown here. After exposure of the heart, a purse string suture is applied and a 2 mm defect is

created. Then, a HLAA coated patch is placed centrally over the defect, pressed onto the heart

and activated with UV light. In case of bleeding at the edges, as shown in this example,

additional HLAA is placed and activated as well to provide a complete leek-proof seal. Finally,

the purse string suture is withdrawn.

Movie S2. The HLAA-coated patch resists supraphysiologic, highly dynamic conditions

inside the chamber of the heart. 2D Echocardiographic image immediately after injection of

epinephrine demonstrating the stability of the attachment of the patch. Left image shows a short

axis view of the heart and the image on the right shows a long axis view.