Selective light-triggered release of DNA from gold nanorods switches blood clotting on and off

Abstract

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.

Figure 1. TBA and antidote affect coagulation in whole human blood.

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 (t_plasma) 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).

Figure 2. Gold nanoparticles synthesized and loaded for triggered release.

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) D_H (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.

Figure 3. Release from NR- and NB-coronas and their comparison to covalently loaded NRs.

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 t_plasma 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 t_plasma  = 1.0 (gray dotted line). A significant difference (p≤0.05) from baseline t_plasma is indicated with a * (Table S1), d) t_plasma (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) t_plasma (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).

Figure 4. Selective melting of NRs and NBs to switch blood clotting off and on.

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 t_plasma 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 (t_plasma increases to 1.73), and 800 nm+1100 nm (t_plasma 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 t_plasma are indicated with an * (Table S1).