full papers
M. K. M. Leung et al.
and a 2 μL sample of the particles was taken and analyzed on the 800 nm with a resolution of 169 μm and an intensity of 8. Fluo-
flow cytometer. rescence intensities were quantified using Odyssey 2.1 software.
Degradation of Capsules: The AF488 -labeled crosslinked
Az
(
PEGAlk/PMA ) /(PEG ) capsules were incubated in PBS in the
Alk 2
Alk 3
absence or presence of GSH (5 mM in PBS) at 37 °C with gentle
mixing. At regular time points, the number of capsules and fluo-
rescence intensity of the capsules were quantified using flow
4
cytometry. Triplicate samples, each with ≈5 × 10 capsules, were Supporting Information
measured. Degradation of the capsules was also monitored by
fluorescence microscopy.
Supporting Information is available from the Wiley Online Library
Preparation of in vitro Thrombi and Biofouling Studies: Fresh or from the author.
human blood was centrifuged (186 g, 10 min, no deceleration,
room temperature) for separation of platelet rich plasma (PRP). To
every 1 mL of PRP was added a mixture of adenosine 5′-diphosphate Acknowledgements
(
ADP) (100 μL, 200 mM in water), actin (88 μL) and calcium
chloride (25 μL, 1 M). To form thrombi, aliquots (100 μL) of the PRP This work was supported by the Australian Research Council
mixture were incubated at 37 °C for 12 min. After washing in PBS under the Federation Fellowship (F.C.) and Discovery Project (F.C.)
5
(
with Ca/Mg), each thrombus was incubated with ≈5 × 10 DL800 - schemes. J.-F.L. acknowledges the Fraunhofer Society for financial
Az
labeled capsules (see below for preparation) in PBS (with Ca/Mg) support. Zoya Zarafshani (Fraunhofer Institute for Applied Polymer
at 37 °C for 30 min with gentle mixing. Thrombi were then washed
with PBS (with Ca/Mg) three times to remove unbound capsules.
The fluorescence intensities of capsule-bound thrombi were ana-
lyzed by near infrared imaging.
Research) and Dr. C. E. Hagemeyer (Baker IDI Heart and Diabetes
Institute, Melbourne) are acknowledged for assistance with
polymer synthesis and near infrared scanning measurements,
respectively. The authors thank Dr. H. Lomas and K. Liang
(The University of Melbourne) for TEM and AFM images, respectively.
Dr. R. De Rose (The University of Melbourne) and Dr. C. E. Hagemeyer
and Prof. K. Peter (Baker IDI Heart and Diabetes Institute) are
acknowledged for helpful discussions regarding the preparation of
serum and thrombi, respectively.
Crosslinked capsules of
PMA) /(PVPON/PMA) , PVPON/(PMA/PVPON ) and (PVPON/
Alk 5
PMA(PEGAlk/PMA ) /(PEGAlk/
Alk 2
3
2
PMA
) were prepared as described earlier. DyLight 800 NHS-
Alk 5
Ester (0.2 mg) was dissolved in DMSO (20 μL) and mixed with the
azido-PEG-amine (2 μL) for 4 h to give DL800-PEG-azido (DL800Az).
Without purification, 5 μL of the DL800 suspension together with
Az
−1
−1
sodium ascorbate (4.4 g L , 50 μL) and copper sulfate (1.75 g L ,
0 μL) (all in 50 mM NaOAc, pH 4) were added to each of the cap-
5
sules and labeling was allowed to proceed overnight. After exten-
sive washing, the DL800-labeled capsules were exposed to PBS
for PMA removal as reported earlier to form hollow capsules of
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tungsten lamp, respectively. For AFM and TEM measurements,
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scans were conducted on an Odyssey infrared imager (LI-COR
Biosciences, Lincoln, NE, USA) at an excitation wavelength of
[
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