Selective Light-Triggered Release of DNA from Gold Nanorods Switches Blood Clotting On and Off

Blood clotting is a precise cascade engineered to form a clot with temporal and spatial control. Current control of blood clotting is achieved predominantly by anticoagulants and thus inherently one-sided. Here we use a pair of nanorods (NRs) to provide a two-way switch for the blood clotting cascade by utilizing their ability to selectively release species on their surface under two different laser excitations. We selectively trigger release of a thrombin binding aptamer from one nanorod, inhibiting blood clotting and resulting in increased clotting time. We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting. Thus, the nanorod pair acts as an on/off switch. One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function. We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.National Science Foundation (U.S.) (Grant DMR #0906838

Effect of Left and Right Coronary Flow Waveforms on Aortic Sinus Hemodynamics and Leaflet Shear Stress: Correlation with Calcification Locations

Coronary flow induces hemodynamic alterations in the aortic sinus region. The objectives of this study are to: (1) investigate the differences among sinus hemodynamics and leaflet wall shear stresses engendered by the left versus right versus non-coronary flow and (2) correlate respective wall shear stresses with leaflet calcification in patients. A left heart simulator flow loop with a tunable coronary circuit provided physiological coronary flow waveforms corresponding to the left coronary cusp case (LCC), right coronary cusp case (RCC), and non-coronary cusp case (NCC). High spatio-temporal resolution particle image velocimetry was conducted to quantify leaflet wall shear stress and sinus vorticity fields and to measure aortic leaflet tip kinematics. Thirty-one patients with severe calcific aortic valve disease were segmented from CT data for the calcific volumes in their respective left, right, and non-coronary cusps. Leaflet tip position during systole shows the RCC has a wider leaflet opening compared to LCC and NCC. Velocity and vorticity fields combined with leaflet position data show that sinus vorticity is diminished (peak ~ 43 s−1) in the LCC while RCC and NCC maintain high vorticity (~ 1200 and ~ 950 s−1 respectively). WSS magnitudes greater than 0.3 Pa show 20 and 81% greater occurrences in the LCC and RCC respectively compared to NCC. Significant differences [X2 (2, n = 31) = 7.31, p = 0.0258] between the calcification levels in each cusp of the patient population. Coronary flow differences between LCC, RCC, and NCC show significant impact on leaflet kinematics and sinus flow hemodynamics. Clinical data correlations of the coronary flow cases indicate the left coronary cusp has a higher likelihood of calcification compared to the right

Gold nanoparticles synthesized and loaded for triggered release.

<p>Absorption spectra of a) NRs, NR-HSA-TBA coronas (LSPR max  = 777 nm), b) NB, NB-HSA-antidote (LSPR max  = 1093 nm). c) TEM image of NRs, scale bar  = 20 nm, d) TEM image of NBs, scale bar  = 100 nm, e) <i>D_H</i> (DLS) of NRs, NR-HS-TBA, NBs, NB-HS-antidote, indicating that a corona contains multiple not a single NR or NB, but multiple ones. f) Zeta potential of NRs, NR-HS-TBA  = −9.8 mV, NBs, NB-HS-antidote  = −10.1 mV g) Quantified DNA payloads of NR-HS-TBA (674±74 TBA/NR), NB-HS-antidote (1307±255 antidote/NB). h) mixture of NR-CTAB + NB-CTAB before (black) and after (red) 800 nm irradiation. i) NR-CTAB + NB-CTAB before (black) and after (red) 1100 nm irradiation.</p

TBA and antidote affect coagulation in whole human blood.

<p>a) Schematic of coronas made from human serum (HS) loaded with NRs and TBA (NR-HS-TBA) + coronas loaded with NBs and antidote (NB-HS-antidote). 800 nm laser irradiation melts the NRs, triggering release of TBA from the coronas, which inhibits thrombin and causes blood coagulation times to increase. Following this, 1100 nm laser irradiation melts the NBs, triggering release of the DNA antidote from the corona. The antidote forms a double-stranded hybrid with TBA, thus restoring thrombin activity and blood coagulation. Fluorescently labeled TBA has a sequence of 5’ GGTTGGTGTGGTTGG-TMR 3’. The fluorescently labeled antidote has the complementary sequence 5’ CCAACCACACCAACC-FAM 3’. Clotting time (<i>t_plasma</i>) for a thrombin test using 10 nM thrombin measured by a coagulometer with b) TBA, for c) 500 nM TBA + varying antidote from [anti]  =  0 to 1000 nM (anti/TBA  =  0 to 2.0).</p

Selective melting of NRs and NBs to switch blood clotting off and on.

<p>NR-HS-TBA and NB-HS-antidote were mixed at a ratio so that the TBA:antidote was 1:1. a) NR-HS-TBA + NB-HS-anti mixture before (black) and after 800 nm irradiation (red), after 800 nm and then 1100 nm irradiation (blue) b) fluorescence of released supernatant before (black) and after 800 nm irradiation (red), and after 800 nm+1100 nm irradiation (blue). c) Normalized <i>t_plasma</i> for samples before irradiation (defined as 1.0, and used to compare the statistical parameters of all samples) of mixture of NR-HS-TBA + NB-HS-antidote with excitation at 800 nm (<i>t_plasma</i> increases to 1.73), and 800 nm+1100 nm (<i>t_plasma</i> restored to 0.88), demonstrating restoration of clotting time. 1100 nm irradiation alone of the mixture NR-HS-TBA + NB-HS-anti, showing no significant increase in clotting time. Irradiation at 800 nm of NR-HS (without TBA) + NB-HS-anti showing no increase in clotting time. To test the effect of the presence of the nanoparticles, HS-NR-TBA+NB-HS-anti were not exposed to any irradiation in blood. Significant differences (p≤0.05) from baseline <i>t_plasma</i> are indicated with an * (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068511#pone.0068511.s005" target="_blank">Table S1</a>).</p

Release from NR- and NB-coronas and their comparison to covalently loaded NRs.

<p>a) Absorption spectrum of NB-HS-TBA before (black) and after (red) 1100 nm irradiation, where [NB-HS-anti]  = 0.3 nM, and released [anti]  = 129±5 nM (430±17 anti released/NB). Inset: fluorescence spectrum of released TBA before (black) and after (red) 1100 nm irradiation. b) Absorption spectrum of NR-HS-TBA before (black) and after (red) 800nm irradiation where [NR-HS-TBA]  = 2.9 nM, and released [TBA]  = 663±23 nM (223±8 DNA released/NR). Inset: fluorescence spectrum of released TBA before (black) and after (red) 800 nm irradiation, c) Effect of the released TBA in blood. Comparing normalized <i>t_plasma</i> from released TBA from the coronas [NR-HS-TBA] = 2.9 nM (red), where released [TBA] = 663±23 nM in a clotting test. Supernatant of NR-HS-TBA with exposed to no irradiation and added to blood is defined as <i>t_plasma</i>  = 1.0 (gray dotted line). A significant difference (p≤0.05) from baseline <i>t_plasma</i> is indicated with a * (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068511#pone.0068511.s005" target="_blank">Table S1</a>), d) <i>t_plasma</i> (normalized) calibration curve of free TBA in a thrombin test (stars). Released TBA from NR-HS-TBA (red circle) and extrapolated equivalent concentration (red dashed line). e) <i>t_plasma</i> (normalized) calibration curve of free thiolated TBA (blue X’s). Released thiolated TBA from NR-thiol-TBA (1460±108 nM, blue square) and extrapolated equivalent concentration (blue dashed line).</p