All reagents were purchased from Sigma Aldrich UK, unless otherwise stated. For procedures that involved the use of human blood and tissue, informed consent was obtained from healthy volunteers, and the Institutional Review Board (IRB) at the Division of Surgery & Interventional Science at University College London approved the study protocol. All experimental procedures were done in triplicates (n = 3) unless otherwise stated.
POSS-PCU nanocomposite polymer synthesis
Synthesis of POSS-PCU for peptide functionalization has been previously described elsewhere [25]. Briefly, polyhedral oligomeric silsesquioxane (POSS®) (Hybrid Plastics Inc.) was mixed with polycarbonate polyol in a custom-built reaction flask. The mixture was heated and stirred using a mechanical stirrer. 4,4′-methylenebis (phenyl isocyanate) (MDI) and nitrogen gas were introduced into the reaction mixture to form the pre-polymer. Dimethylacetamide (DMAc) was added to the mixture. Chain extension was commenced via addition of ethylenediamine and diethylamine to yield the final product, 18% (w/w) solution of POSS-PCU. Functionalized fumed silica was then incorporated into POSS-PCU using a UIP1000-Exd Ultrasonic Mixer (Hielscher Ultrasonic GmbH).
O-Phthalaldehyde (OPA) fluorescent amine assay
OPA assay was used to detect the presence of primary amines on POSS-PCU that would be functionalized with antibodies. 83 μl of 2-mercaptoethanol and 833 μl of borate buffer (0.05 mol/dm3, pH = 9) were added onto the different test samples. The mixture was transferred to a 96-well plate and left to stand for 2 hours. Thereafter, 34 μl of OPA (10 mg/ml in ethanol) was added. The plate was placed in a Fluoroskan Ascent FL microplate fluorometer/luminometer (Thermo Scientific). A 360 nm excitation filter, and a 460 nm emission filter were selected.
Ultrasonic atomization spray system
The above-mentioned version of POSS-PCU has a high viscosity, and must be diluted for the purposes of using it in the ultrasonic spray atomization system. Briefly, 22 g of tetrahydrofuran (THF) was added to 2 g of POSS-PCU.
The parameters used for the polymer coating were written into the program of the MediCoat DES 1000 Ultrasonic Spray System (Sono-Tek Corporation USA). Polymer solutions were fed into the nozzle, through a syringe, of the ultrasonic atomization spray system. Nitrogen gas (BOC Industrial Gases) pressure was set at 4.5 PSI. Ultrasonic power was set to 0.24 W. Rate of syringe injection was set at 0.1 ml/min. Translational movement of mandrel was set at 2.5 mm/sec, and rotational movement was set at 115 rpm.
Platinum chromium intra-arterial stents (Boston Scientific USA) with diameters of 3.5 mm and lengths of 20 mm were placed on mandrels, in such a way that half of the stent length “overhangs” out from the mandrel, enabling it to be spray coated in both the luminal and abluminal area while the mandrel rotates. Drying gas was utilized during this procedure at 1.0 PSI. The half-coated stent was then placed in a drying oven (Binder GmbH) at 65°C for 3 hours to allow for solvent evaporation. The coating process was repeated for the other half of the stent.
Polymer films (for cell culture) were also fabricated a similar manner, and the rotating mandrel was spray coated with the polymer using identical parameters mentioned above. A vertical incision was made lengthwise on the coated mandrel, and the polymer films were carefully peeled off.
Covalent bonding of anti-CD34 antibodies onto POSS-PCU
The protocol for covalent bonding of peptide motifs on POSS-PCU was previously developed by de Mel in our lab, and in-depth discussion can be found elsewhere [25]. Briefly, 0.008 g of N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), 0.0115 g of N-hydroxysuccinimide (NHS), and 0.05 g of succinic acid were placed in a 50 ml conical tube. To this, 20 ml of phosphate-buffered solution (PBS) was added. The mixture was placed on a roller mixer (Stuart Equipment) for 3 hours, to allow activation.
Circular-cut discs of polymer sheets were placed in a 24-well plate. 500 μl of the above-mentioned EDC-NHS-PBS mixture was pipetted onto each polymer sheet in the wells. The well plate was wrapped in aluminium foil to avoid light, and placed on a Luckham R100 Rotatest Shaker (Richmond Scientific Ltd.) for 3 hours.
500 μl of PBS, and 5 μl of mouse anti-CD34 concentrate (2 μg/ml) (Life Technologies) were pipetted into an eppendorf tube. Equal amounts of the antibody solution were pipetted into 24-well plates. The well plate was wrapped in aluminium foil and placed on a shaker for 30 minutes. It was then transferred to a 4°C fridge and left for 24 hours. After 24 hours, the polymer discs were washed with PBS.
POSS-PCU-coated stents were also covalently-bonded to anti-CD34 antibodies, using a similar protocol as mentioned above.
Fourier transform infrared (FTIR) spectroscopy
Chemical groups were detected using ATR-FTIR with a Jasco FT/IR 4200 spectrometer (JASCO Analytical Instruments). Parameters were set at 20 scans at a 4 cm-1 resolution, with a wavenumber range of 600 cm-1 to 4000 cm-1.
Scanning probe AFM confocal raman spectroscopy
Raman and AFM scanning were performed using NT-MDT NTEGRA Spectra® system with an upright Raman microscope and a universal head. AFM scanning was done in semi-contact mode with commercial rounded cantilevers for large scans, purchased from MicroMasch (R ~ 40 nm, k = 5.7 N/m). Raman scanning was done in backscattering geometry with a Mitutoyo long-working distance objective (100×, 0.7 NA). The excitation source was a 473 nm solid-state Cobolt Blues® laser with power at the sample being 2 mW. Acquisition time per step was 10s and step size was 0.5 microns. Optical images of the area were captured using the same objective.
Water contact angle
A KRÜSS DSA 100 (KRÜSS GmbH) system was used for static water contact angle measurement, using a sessile drop method. Sterile deionized water was used as a solvent, with droplet volume of 3 μl. A highly hydrophobic material, Teflon® (DuPont USA) and a highly hydrophilic material, Acuvue® (Johnson & Johnson) served as controls.
Thromboelastography (TEG)
The effect of polymer material on blood coagulation kinetics was assessed using a TEG® 5000 Thromboelastograph® Hemostasis Analyzer System (Haemonetics Corporation USA). Cuvettes were spray coated with POSS-PCU and further conjugated with anti-CD34 antibodies in the protocol mentioned above. Uncoated cuvettes served as controls. Whole blood was obtained from healthy volunteers and 320 μl of whole blood were pipetted into each cuvette.
Scanning electron microscopy (SEM)
Samples were mounted on an aluminium stub and sputter-coated with gold via vapour deposition using SC500 (EM Scope), and imaged using a Philips 501 SEM (Cambridge, UK).
X-ray photoelectron spectroscopy (XPS)
The analysis of the samples was carried out using a Thermo Scientific Theta Probe XPS recently calibrated in November 2012. Monochromatic Al Kα X-ray (hν = 1486.6 eV) was employed with an incident angle of 30° with respect to surface normal. Photoelectrons were collected at a take-off angle of 50° with respect to surface normal. The analysis area was approximately 400 μm in diameter while the maximum analysis depth lies in the range of 4 - 10 nm. Survey spectra and high-resolution spectra were acquired for surface elemental identification and for chemical state identification, respectively. For chemical state analysis, a spectral deconvolution was performed by a curve-fitting procedure based on a Lorentzian function, and broadened by a Gaussian function.
Endothelial progenitor cell (EPC) and human umbilical vein endothelial cell (HUVEC) culture
Two cell lines were selected for this study: endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs). EPCs were selected as we wanted to find out the capturing efficacy of CD34-POSS-PCU, while HUVECs were used to confirm that CD34-POSS-PCU were able to support the growth and proliferation of endothelial cells. Both cell lines were cultured in the same type of cell culture medium to ensure experimental consistency.
Details of EPC extraction were reported previously by our group [25, 26]. Whole blood was obtained from healthy volunteers after signing an informed consent document. Approximately 20 ml of whole human blood was placed in eight 2.7 ml light blue-capped Vacutainer® citrate tubes (BD USA). 3 ml of Histopaque was added to six 15 ml centrifuge tubes, and 3 ml of whole blood was layered onto the Histopaque. The material at the opaque interface was transferred into 2 clean centrifuge tubes. 10 ml of HBSS (Life Technologies) was added, mixed gently and centrifuged at 250 g for 10 minutes. The supernatant was discarded and the pellet re-suspended in 5 ml HBSS and mixed gently. This centrifugation and supernatant-discarding process was repeated 2 more times. The cells were then re-suspended in 5 ml cell culture medium M199 (Life Technologies) with 10% FBS (Life Technologies), and 1% penicillin and streptavidin (Life Technologies). Cells were counted using Trypan blue exclusion dye, using 10 μl Trypan blue, and 10 μl cell suspension. Cells were seeded at a density of 1.0 × 106 cells per well in a 24-well plate. It was then incubated at 37°C with 5% CO2 for 21 days. Culture medium was replenished every 3 days, and cells were examined under light microscopy every 3 days.
HUVEC extraction was described extensively by our group in a previous report [27, 28]. The protocol was modified to fit our experimental needs in this study. Briefly, human umbilical cords were obtained immediately upon delivery of newborn infants, from the Department of Obstetrics and Gynaecology at the Royal Free London NHS Foundation Trust Hospital, after patients had signed an informed consent document, and approved by the IRB at UCL. Similar to the above-mentioned protocol for EPCs, cells were seeded at a density of 1.0 × 106 cells per well in a 24-well plate. It was then incubated at 37°C with 5% CO2 for 21 days. Culture medium was replenished every 3 days, and cells were visualized every 3 days.
AlamarBlue® assay
AlamarBlue® (Life Technologies) was added to the cell culture medium at a volume to volume concentration of 10%. After 7 days, culture plates were washed with PBS and 1 ml was added to each well. 100 μl was aspirated after 4 hours. Fluorescence was measured using a Fluoroskan Ascent FL microplate fluorometer/luminometer (Thermo Scientific). A 360 nm excitation filter, and a 460 nm emission filter were selected.
Dynamic flow at physiological conditions
A home-built flow circuit was used to mimic physiological flow conditions in the human body. An electromagnetic pump and a variable height fluid reservoir maintained pressure at around 120 mmHg / 80 mmHg, and “heart beat” at 84 beats per minute. An ultrasound Doppler probe was used to monitor and maintain flow conditions using a Light Patient Monitor (Datex Engstrom). Culture medium with peripheral blood cells (1× 106) were used in the flow circuit. Viscosity was approximated to that of blood, by adding 8% low molecular weight Dextran (77000 Da). Anti-CD34 POSS-PCU coated-stents, POSS-PCU-coated stents, POSS-PCU films, anti-CD34 POSS-PCU films, and IgG-POSS-PCU films were placed in a microtubule and subject to physiological flow conditions for 28 days.
Quantum dots for confocal imaging
Quantum dots (QDs) were manufactured in-house by our lab in a study described previously [29], and modified to suit our experimental needs for this study. Briefly, 200 μl of QD (1 mg/ml) was mixed with 200 μl EDC (1 mg/ml) and 200 μl NHS (1 mg/ml) in PBS for 30 minutes at room temperature. 100 μL of anti-x solution (where x denotes the type of antibody used) was added to the mixture and mixed for 1 hour at room temperature. To separate the reagent and unconjugated CdCoTe/MSA/ QDs, 100 kDa Amicon® Ultra-centrifugal filters (Millipore Corporation) with UV monitoring at 280 nm of the retained samples was used. The purified samples were collected and stored at 4°C until further use. The sample was further characterized by NIR fluorescence and FTIR spectroscopy.
Cells were fixed with 2% formaldehyde (PFA) using the following method. Briefly, cells were washed twice with 0.1% PBS-Tween 20 at room temperature. They were then treated with 2% PFA for 20 minutes, and washed 3 times with 0.1% PBS-Tween 20 for 5 minutes per wash. Cells were permeabilized with 0.5% PBS-Tween 20 for 15 minutes. They were then further washed 3 times with 0.1% PBS-Tween 20 for 5 minutes per wash. Cells were blocked and incubated with quantum dots using the following methods. PBS-Tween 20 was removed. Cells were blocked with 500 μl of 1% BSA (in PBS-Tween 20) for 20 minutes at 4°C, and then washed with PBS-Tween 20. 500 μl of 0.25 μg/ml of red QD-Ab (VEGFR2) and green QD –Ab (CD34) to each well and incubated for 2 hours in the dark at 4°C. Cells were then visualized and counted. Samples were washed with PBS-Tween 20 to remove unwanted QDs. 300 μl of diluted DAPI was added to each well and incubated for 5 minutes. They were then washed with PBS-Tween 20.
Images were acquired by a fluorescent microscopy unit (Nikon Eclipse TE 300). The PCM scanning head was mounted on an inverted optical microscope (Nikon Eclipse TE 300), which can operate in fluorescence, reflection and phase contrast modes, and it was fitted with a Plan Fluor dry objective (20×/NA = 0.5). Lasers at 488 nm and 543 nm were the sources, housed in a common module, providing the excitation beams that were delivered to the scanning head through a single-mode optical fiber. Photomultiplier (PMT) tubes were placed within the control unit, and the collected light arrived via high-transmission optical fibers.
Curve fitting and statistical analyses
Curve fitting (least squares method) and statistical analyses were conducted at 95% confidence interval using MATLAB® (MathWorks Inc.). Statistical significance testing was conducted using unpaired Student’s t-test. p-values of less than 0.05 were considered statistically significant.
