Highly flexible and stable aptamer-caged nanoparticles for control of thrombin activity

Abstract

In this paper, we have demonstrated that the thymine linker length number (T_n, n = 0–60) and stem pair number (P_m, m = 0–16) in the terminal of thrombin binding aptamers (TBAs) have a strong impact on the flexibility and stability of TBA-modified gold nanoparticles (TBA–Au NPs) and thus the binding strength and inhibitory potency toward thrombin. The anticoagulation of TBA–Au NPs increased with an increase in the linker length from T₀ to T₃₀ due to an increase in the flexibility of G-quadruplexes of TBAs on the Au NP surfaces (TBA-T_n–Au NPs). The inhibition of TBA-P_mT₁₅–Au NPs increased with an increase in the P_m from P₀ to P₈ as a result of the increase in the rigidity and the stability of G-quadruplexes of the TBAs on the Au NP surfaces. The best results were observed for multivalent TBA–Au NPs conjugates—TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs—which exhibited ultra-high binding affinity toward thrombin (K_d = 8.86 × 10⁻¹² M) and thus extremely high anticoagulant (inhibitory) potency because of their particularly flexible and stable conformation and multivalency. Compared to the case without inhibitors, their measured thrombin clotting time (TCT) was 296 times longer, whereas for TBA₁₅ alone it was only 3.9 times longer. From the dosage dependence of the TCT delay, we further demonstrated the anticoagulation ability of our TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs was much better than the commercial drugs (argatroban and hirudin). Moreover, the Au NPs modified with TBA with photocleavable (PC) units allow a reversal in the activity of TBA_PC–Au NPs via near-UV light-inducement of TBA release from Au NPs. We believe that our described techniques can be used widely to modify NPs with other anticoagulant DNA or RNA aptamers towards different proteins such as factor IX, activated protein C, and factor VIIa.

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1. Introduction

Aptamers are short, single-stranded oligonucleotides that fold into a specific three-dimensional structure to specifically bind targets, which can range from ions and small organic or inorganic molecules to biomacromolecules and living cells.^1–5 In comparison to antibodies, aptamers are cheaper, easier to synthesize and highly stable.^6–9 Over the past decade, molecular functional aptamers and the integration of aptamers into nanomaterials has become a new interdisciplinary field that aims at providing new hybrid systems for sensors, cancer cell targeting and therapy.^8,10–17 Such multifunctional aptamers exhibit unique optical, electrochemical and magnetic properties, polyvalent interactions, and extraordinary intracellular stability, allowing them to be used as probes, inhibitors or drug delivery systems for target analytes or cells.^18–21

Thrombin plays an indispensable role in the coagulation cascade, converting soluble fibrinogen into insoluble strands of fibrin and catalysing many other coagulation-related reactions, including direct activation of protein C, platelets and feedback activation of pro-cofactors V and VIII.^22–24 Thus, thrombin is a common target for anticoagulants and antithrombotics.^25,26 Anticoagulant drugs that are direct thrombin inhibitors (DTIs), such as heparin, warfarin and bivalirudin, are often used to modulate blood coagulation.^27–31 However, they have side effects such as excessive bleeding, thrombocytopenia, narrow therapeutic windows and highly variable dose–response relationships among individuals.^32–35 As an alternative, aptamers have been shown to be potential hemostasis-factor-specific anticoagulants, with advantages of high affinity, non-immunogenicity, and nontoxicity.^36,37 Two well-known thrombin-binding aptamers (TBAs) possess 15 bases (denoted as TBA₁₅) and 29 bases (denoted as TBA₂₉) that bind to exosite 1 and exosite 2 with dissociation constant (K_d) values of about 100 and 0.5 nM, respectively.^38–40 TBA₁₅, which interacts with fibrinogen-binding exosite 1, exhibits the enzymatic-inhibitory functions required for thrombin-mediated coagulation; however, because of its insufficient binding affinity, large amounts of it are needed to achieve the desired anticoagulant response.

Modifications of aptamer that included 4-thio-2′-deoxyuridine,⁴¹ locked nucleic acid,⁴² unlocked nucleic acid monomers,⁴³ 2′-O-fluoro,⁴⁴ 2′-deoxy-isoguanosine,⁴⁵ 2′-O-methyl-RNA nucleotides,⁴⁶ phosphorothioate internucleoside linkages,⁴⁷ partial inversion polarity,⁴⁸ or changes of loop size,⁴⁹ sequence,⁵⁰ and terminal extension of oligonucleotides (< 8 mer)⁵¹ and poly(ethylene glycol) (PEG)⁵² have been employed to increasing affinity or resistance. Few of these modifications (phosphothioate, 2′-O-methyl, 2′-O-fluoro, etc.), can provide good resistance against enzymatic hydrolysis, while toxicity due to over dose of these non-natural compounds becomes a serious problem. Conjugation of PEG leads to an increase in the stability of aptamer and also facilitates clinical delivery. However, PEG reduces binding affinity between the PEG–aptamers and thrombin molecules.

Recently, several studies have demonstrated that a linear assembly of TBA₁₅, TBA₂₉, and engineering dendritic aptamers improves thrombin inhibition via multivalent interactions.^53–59 Low flexibility of assembly aptamers and low rigidity of G-quadruplex structures of TBAs leads to limiting their inhibitory potencies. Recently, we have reported TBA₂₉-conjugated gold nanoparticles (TBA₂₉–Au NPs) provide a 82-fold improvement in anticoagulant potency via thrombin-mediated coagulation made possible by steric blocking and a high binding affinity toward thrombin in solutions that mimic biological plasma.⁶⁰ We also used TBA–Au NPs with a self-assembled arranged monolayer (SAAM) to enhance the binding affinity of TBAs toward thrombin.⁶¹ More recently, we further incorporated sulfated galactose acid into TBA–Au NPs and demonstrated that it enables effective inhibition of thrombin activity toward fibrinogen.⁶² Although these TBA–Au NPs are ultra-stable in plasma, the low flexibility and rigidity of the G-quadruplex structures of TABs on nanoparticles limits their inhibitory ability.

It has been suggested that the stability and rigidity of aptamers along with the flexibility of assembly aptamers were indispensable for interaction with targeting proteins.^63–65 In this work, we systematically investigated the inhibitory abilities of TBA–Au NPs with various linker lengths and the stem pair numbers of TBAs toward thrombin (Scheme 1a and 1b). We also prepared multivalent Au NP-conjugations of TBA₁₅ and TBA₂₉ on to Au NPs to increase the binding affinity, thereby improving its anticoagulant activity (Scheme 1c). Finally, we demonstrated that Au NPs modified with “caged” TBA with photocleavable (PC) units (2-nitrobenzyl group) allow a reversal in the activity of TBAs–Au NPs via inducement by near-UV light, which breaks a C–O bond releasing a 5′-phosphorylated TBA strand (Scheme 2).

Scheme 1 Schematic representation of the binding and enzymatic inhibition of (a) effect of linker-length with TBA-P₄T_n–Au NPs, (b) effect of stem pair with TBA-P_mT₁₅–Au NPs, and (c) multivalent TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs toward thrombin.

Scheme 2 Schematic representation of reversible inhibitory function of the TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs by irradiation of near-UV light.

2. Experimental

2.1 Materials

Calcium chloride (CaCl₂), magnesium chloride (MgCl₂), potassium chloride (KCl), sodium chloride (NaCl), tris(hydroxymethyl)aminomethane (Tris), trisodium citrate and hydrochloric acid (HCl) were purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). Human α-thrombin (≥ 1000 NIH units/mg proteins), fibrinogen and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). The anticoagulant drugs argatroban and hirudin were purchased from Sigma. The argatroban (100 μM) or hirudin (100 μM) was prepared in biological buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5.0 mM KCl, 1.0 mM MgCl₂, and 1.0 mM CaCl₂). Reagents used for measuring thrombin clotting time (TCT) were purchased from Helena Laboratories (Victoria, Australia). Oligonucleotides and thiol-modified oligonucleotides listed in Table 1 were purchased from Integrated DNA Technologies (Coralville, IA, USA). All other reagents used in this study were purchased from Aldrich (Milwaukee, WI, USA).

Table 1 DNA sequences of TBAs, TBApc and rDNA used in this study

Name	Sequence^a	TBA/Au NP^b
a The underline was used denote the stem pairs. b Number of TBA molecules per Au NP.
TBA15-P0Tn	5′-Tn GGT TGG TGT GGT TGG-3′	—
TBA29-P4Tn	5′-Tn AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′	—
rDNA-Tn	5′-Tn GCT GAC TAC ACG CTG TCA TTA-3′	—
TBA15-P0T0-SH	5′-SH-GGT TGG TGT GGT TGG-3′	120
TBA15-P0T15-SH	5′-SH-T15 GGT TGG TGT GGT TGG-3′	110
TBA15-P0T30-SH	5′-SH-T30 GGT TGG TGT GGT TGG-3′	75
TBA15-P0T45-SH	5′-SH-T45 GGT TGG TGT GGT TGG-3′	66
TBA15-P0T60-SH	5′-SH-T60 GGT TGG TGT GGT TGG-3′	53
TBA29-P4T0-SH	5′-SH-A̲G̲T̲ C̲CG TGG TAG GGC AGG TTG GGG TG̲A̲ C̲T̲-3′	99
TBA29-P4T15-SH	5′-SH-T15 A̲G̲T̲ C̲CG TGG TAG GGC AGG TTG GGG TG̲A̲ C̲T̲-3′	88
TBA29-P4T30-SH	5′-SH-T30 A̲G̲T̲ C̲CG TGG TAG GGC AGG TTG GGG TG̲A̲ C̲T̲-3′	70
TBA29-P4T45-SH	5′-SH-T45 A̲G̲T̲ C̲CG TGG TAG GGC AGG TTG GGG TG̲A̲ C̲T̲-3′	65
TBA29-P4T60-SH	5′-SH-T60 A̲G̲T̲ C̲CG TGG TAG GGC AGG TTG GGG TG̲A̲ C̲T̲-3′	52
TBA15-P4T15-SH	5′-SH-T15 A̲G̲T̲ C̲CG TGG TTG GTG TGG TTG GGG TG̲A̲ C̲T̲ -3′	90
TBA15-P8T15-SH	5′-SH-T15 A̲T̲C̲ T̲A̲G̲ T̲C̲C GTG GTT GGT GTG GTT GGG GTG̲ A̲C̲T̲ A̲G̲A̲ T̲-3′	70
TBA15-P16T15-SH	5′-SH-T15 A̲T̲T̲ C̲T̲A̲ C̲T̲G̲ G̲A̲T̲ A̲G̲T̲ C̲CG TGG TTG GTG TGG TTG GGG TG̲A̲ C̲T̲A̲ T̲C̲C̲ A̲G̲T̲ A̲G̲A̲ A̲T̲-3′	55
TBA29-P0T15-SH	5′-SH-T15 CGT GGT AGG GCA GGT TGG GGT -3′	118
TBA29-P8T15-SH	5′-SH-T15 A̲T̲C̲ T̲A̲G̲ T̲C̲C GTG GTA GGG CAG GTT GGG GTG̲ A̲C̲T̲ A̲G̲A̲ T̲-3′	70
TBA29-P16T15-SH	5′-SH-T15 A̲T̲T̲ C̲T̲A̲ C̲T̲G̲ G̲A̲T̲ A̲G̲T̲ C̲CG TGG TAG GGC AGG TTG GGG TG̲A̲ C̲T̲A̲ T̲C̲C̲ A̲G̲T̲ A̲G̲A̲ A̲T̲-3′	55
TBA15pc-P8T15-SH	5′-SH-pc-T15 A̲T̲C̲ T̲A̲G̲ T̲C̲C GTG GTT GGT GTG GTT GGG GTG̲ A̲C̲T̲ A̲G̲A̲ T̲-3′	60
TBA29pc-P8T15-SH	5′-SH-pc-T15 A̲T̲C̲ T̲A̲G̲ T̲C̲C GTG GTA GGG CAG GTT GGG GTG̲ A̲C̲T̲ A̲G̲A̲ T̲-3′	60
rDNA-T15-SH	5′-SH-T15GCT GAC TAC ACG CTG TCA TTA-3′	105

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2.2 Synthesis of Au NPs

Spherical Au NPs (13.3 nm) were prepared by reduction of AuCl₄⁻ with citrate. Aqueous 1 mM NaAuCl₄ (500 μL) was added all at once into 4 mM trisodium citrate (50 mL) in a round-bottom flask fitted with a reflux condenser.^66,67 The solution was boiled for 8 min whilst stirring (during which it changed color from pale yellow to deep red), cooled to room temperature, and kept at 4 °C until use. The size of the Au NPs were determined by transmission electron microscopy (TEM: H7100, Hitachi High-Technologies Corporation, Tokyo, Japan); the NPs appeared to be nearly monodisperse, with an average size of 13.3 ±0.8 nm. The absorption of the Au NP solution was measured by microplate reader (Synergy 2 Multi-Mode Microplate Reader, Biotek Instruments, Winooski, VT). The particle concentration of Au NPs (15 nM) was determined from Beer's law using an extinction coefficient of ca. 2.0 × 10⁸ M⁻¹ cm⁻¹ at 520 nm and an NP size of 13.3 nm.⁶⁸

2.3 Preparation of TBA–Au NPs

Thiol-modified TBAs were attached to the Au NPs by a modified procedure.⁶⁹ 5′-Thiol-modified oligonucleotides received in the disulfide form HOCH₃(CH₂)₅S-S-3′-oligo reacted directly with the NPs, attaching both the HO(CH₂)₆S and oligo-S units onto the NP surface. Aliquots of aqueous Au NP solutions (15 nM, 990 μL) in 1.5 mL tubes were mixed with thiol-TBA₁₅ (150 μM, 5 μL) and thiol-TBA₂₉ (150 μM, 5 μL), after 2 h it was prepared under salt aging in the presence of 200 mM NaCl, reacted over 16 h. Salt aging resulted in greater numbers of TBA molecules bound to the Au NPs.⁷⁰ The mixtures were centrifuged at a relative centrifugal force (RCF) of 30 000g for 25 min to remove unattached thiol oligonucleotides. The supernatants were removed and the oily precipitates were washed with Tris-HCl (pH 7.4, 5.0 mM, 1.0 mL). For determination of the number of TBA molecules per Au NP, the amount of TBA in the supernatant after centrifugation was measured using OliGreen (OG) reagent.⁷¹ After 3 centrifuge/wash cycles, the colloids (TBA–Au NPs) were resuspended separately in Tris-HCl (pH 7.4, 5.0 mM, 1.0 mL). The ξ (zeta) potentials and hydrodynamic particle sizes of the TBA–Au NPs were measured on a Zetasizer 3000HS analyzer (Malvern Instruments, Malvern, U. K.).

2.4 Measurement of thrombin clotting time

TCT tests were performed to examine the potency of anticoagulation for the TBA–Au NPs by the common pathway of coagulation. Human plasma (200 μL) was pre-incubated at 37 °C with a 2 : 1 mixture of each of the oligonucleotides (100 μL) (TBA, TBA–Au NP or commercial anticoagulant drugs) in biological buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5.0 mM KCl, 1.0 mM MgCl₂, and 1.0 mM CaCl₂] in the presence of BSA (100 μM) for 180 s. Then pre-incubated thrombin (37 °C, 100 μL, 20 nM) was added to initiate the clotting cascade, and time-course light-scattering measurements were performed at 680 nm to the point of saturation. The TCT was taken at the point where the scattering signal was half-way between the lowest and highest points. Measurements were repeated 3 times; each set of experiments was performed using a single batch of plasma.

2.5 Binding constant

The dissociation constant (K_d) of the TBA–Au NPs with thrombin was determined by mixing aliquots of each TBA–Au NP solution (1.0 pM) separately with different concentrations of thrombin (0–10 pM) for 15 min to form thrombin/TBA–Au NP complexes. The solution was centrifuged (RCF 30 000g) for 20 min. Unbound thrombin in the supernatant was diluted with biological buffer and quantified using fibrinogen and 56 nm Au NPs as we described previously.⁷² Fibrinogen was added to a solution of 56 nm Au NPs, causing their ready conjugation into 56 nm Fib–Au NPs via electrostatic and hydrophobic interactions.⁷³ Thrombin was then introduced in the presence of excess fibrinogen, inducing formation of insoluble fibrillar fibrin–Au NP agglutinates via polymerization of unconjugated and conjugated fibrinogen. The mixture was centrifuged and the absorbance of the supernatants was measured at 532 nm. For estimation of the binding properties of TBA–Au NPs, saturation binding data were processed further with the Scatchard equation

N _Thr/[Free-Thr] = N_max/K_d − N_Thr/K_d (1)

where N_Thr is the number of thrombin molecules bound to the TBA–Au NPs at equilibrium, N_max is the apparent maximum number of binding sites, [Free-Thr] is the free thrombin concentration at equilibrium, and K_d is the dissociation constant. The values of K_d and N_max were calculated from the slope and intercept of the linear plot of N_Thr/[Free–Thr] versus N_Thr.

2.6 Reverse of thrombin activity

The reverse of the inhibited reaction was performed by photocleavable TBA_15PC/TBA_29PC-P₈T₁₅ modified Au NPs (TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs) which can reverse thrombin activity via irradiation with near-UV light (365 nm) to cleave the 2-nitrobenzyl PC linker of the terminal-modified TBA.^74,75 TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs were prepared as described for the TBA–Au NPs. Aliquots of 2-fold diluted plasma with physiological buffer (500 μL) containing TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs (2.5 nM) and thrombin (5.0 nM), were subsequently irradiated by 365 nm UV light (FWHM ∼50 nm, 20 mW cm⁻²) for 30 min. Time-course light-scattering measurements for determination of TCT were then performed.

3. Results and discussion

3.1. Effects of linker length of TBA-Tn–Au NPs.

Several DNA bioconjugated Au NPs were prepared through Au–S bonding by separately mixing 15 nM Au NP (diameter: 13.3 ±0.6 nm) solutions with one of the 2.0 μM 5′-thiol-modified oligonucleotides (TBA₁₅, TBA₂₉, and random-sequence control DNA). We performed five linker length (T₀, T₁₅, T₃₀, T₄₅, and T₆₀) to test the effect of the distance between nanoparticle and aptamer toward the inhibitory efficiency. For simplicity, we denoted the Apt–Au NPs having T_n-linker and P_m-stem pair of aptamer molecules on Au NP as TBA-P_mT_n–Au NPs; TBA₂₉-P₄T₁₅–Au NPs represent the TBA₂₉ with 4-stem pair number and 15-mer thymidine on its' terminal. Please note the reported structure of TBA₂₉ has 4 stem pair number (P₄), while TBA₁₅ does not.^38–40 To investigate the effect of linker length and stem pair number of TBA on the inhibition of thrombin, we performed typical TCT tests using mixtures of thrombin (5.0 nM) and 2-fold diluted plasma sample in the presence and absence of the following DNA strands: TBA₁₅-P₀T_n (100 nM), TBA₂₉-P₄T_n (100 nM), TBA₁₅-P₀T₀–Au NPs (0.83 nM), TBA₁₅-P₀T₁₅–Au NPs (0.91 nM), TBA₁₅-P₀T₃₀–Au NPs (1.30 nM), TBA₁₅-P₀T₄₅–Au NPs (1.52 nM), TBA₁₅-P₀T₆₀–Au NPs (1.89 nM), TBA₂₉-P₄T₀–Au NPs (1.00 nM), TBA₂₉-P₄T₁₅–Au NPs (1.14 nM), TBA₂₉-P₄T₃₀–Au NPs (1.43 nM), TBA₂₉-P₄T₄₅–Au NPs (1.54 nM) and TBA₂₉-P₄T₆₀–Au NPs (1.92 nM). TCT is a common test performed in patients suspected of suffering from coagulopathy. The measurement of TCT is a common screening test for factors I, IIa, and XIII of the common pathways.^76–78 The concentration of each of the oligonucleotides was held constant (100 nM) in these experiments. The numbers of TBA₁₅ and TBA₂₉ on each NP are listed in Table 1. We noted the decrease in the number of TBA density on Au NPs with the increase in the linker length from T₀ to T₆₀.

Fig. 1 shows that in the TCT test, the presence of inhibitors prolong the TCT. The inhibition strengths of TBA and TBA–Au NPs toward thrombin were compared based on their TCT delay [(t − t₀)/t₀], where t₀ and t are the TCT in the absence and presence, respectively, of inhibitors; with higher TCT delay representing stronger inhibition efficiency for anticoagulation. Of the two free aptamers, only TBA₁₅ exhibited a significant inhibition effect of thrombin-induced coagulation. This result was expected because TBA₁₅ targets the critical exosite 1, whereas TBA₂₉ does not.^38–40 In controlled experiments, the random DNA (rDNA) and rDNA modified Au NPs (rDNA–Au NPs) exhibit no inhibitory ability (data not shown). The ultra high anticoagulant potency of TBA₂₉-P₄T_n–Au NPs are a result of steric blocking effects and high binding affinity toward thrombin, and were two orders of magnitude higher than that of free TBA₂₉.⁶⁰ The length of the T_n-linker of TBA–Au NPs has a strong impact on their inhibitory ability while having little effect on that of free TBA. The little enhancement of free TBA₁₅ with increasing T_n can be attributed to the increasing electrostatic interaction between thrombin and TBA₁₅.⁵¹ The driving force of TBA and thrombin interaction is thought to be a combination of electrostatic interaction and molecular pattern recognition.^38–40 The anticoagulation of TBA–Au NPs increased with an increase in the linker length from T₀ to T₃₀ and then started to decrease with a further increase in linker length. The increase in linker length will increase the flexibility and the stability of G-quadruplexes of TBAs (avoid formation of stretched, linear TBA structures) on the Au NP surfaces and therefore will improve the anticoagulant potency. Our reasoning of the phenomenon was supported by the fact that there was an increase in the hydrodynamic diameter with increasing linker length of TBA–Au NPs to T₆₀ (Fig. 2a). When the linker length of TBA was longer than T₃₀, there was a lowering of TBA density on Au NPs (Table 1) and possible formation of intermolecular G-quadruplexes of TBA, which resulted in the decrease of anticoagulation activity. The lower TBA density and formation of intermolecular G-quadruplexes were further supported by their electrophoretic mobility (Fig. 2c). The lower TBA density and/or formation of intermolecular G-quadruplexes both resulted in the decrease of surface charge of TBA–Au NPs and thus their decrease in the binding affinity to thrombin.

Fig. 1 The relative thrombin clotting time [(t − t₀)/t₀] in human plasma in the presence of TBA₁₅-P₀T_n, TBA₂₉-P₄T_n, TBA₁₅-P₀T_n–Au NPs or TBA₂₉-P₄T_n–Au NPs. The TCT is taken at the point where the scattering signal is half-way between the lowest and highest points. The TCT for human plasma in the absence and presence of inhibitors is represented by t₀ and t, respectively. Coagulation was initiated by adding plasma to each thrombin sample, and then light scattering was monitored at 680 nm. The control sample contained only thrombin (5.0 nM) and plasma prepared in physiological buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂]. Error bars represent standard deviations from three repeated experiments.

Fig. 2 (a, b) Hydrodynamic diameters and (c, d) gel electrophoresis of various modified TBA-P_mT_n–Au NPs. Lanes in (c): (1) bare Au NPs, (2) BSA capping Au NPs, (3) TBA₁₅-P₀T₀–Au NPs, (4) TBA₁₅-P₀T₁₅–Au NPs, (5) TBA₁₅-P₀T₃₀–Au NPs, (6) TBA₁₅-P₀T₄₅–Au NPs, (7) TBA₁₅-P₀T₆₀–Au NPs, (8) TBA₂₉-P₄T₀–Au NPs, (9) TBA₂₉-P₄T₁₅–Au NPs, (10) TBA₂₉-P₄T₃₀–Au NPs, (11) TBA₂₉-P₄T₄₅–Au NPs, (12) TBA₂₉-P₄T₆₀–Au NPs. Lanes in (d): (1) bare Au NPs, (2) BSA capping Au NPs, (3) TBA₁₅-P₀T₁₅–Au NPs, (4) TBA₁₅-P₄T₁₅–Au NPs, (5) TBA₁₅-P₈T₁₅–Au NPs, (6) TBA₁₅-P₁₆T₁₅–Au NPs, (7) TBA₂₉-P₀T₁₅–Au NPs, (8) TBA₂₉-P₄T₁₅–Au NPs, (9) TBA₂₉-P₈T₁₅–Au NPs, (10) TBA₂₉-P₁₆T₁₅–Au NPs and (11) TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs. (c, d) Gel electrophoresis of various modified Au NPs (50 nM) in a 1.5% agarose gel containing 0.5X TBE buffer with electric field of 20 V cm⁻¹. Other conditions were the same as those described in Fig. 1.

3.2. Stem pair number of TBA-P_mT₁₅–Au NPs.

Fig. 3 displays the TCT delay in the presence of TBA₁₅-P_mT₁₅ (100 nM), TBA₂₉-P_mT₁₅ (100 nM), TBA₁₅-P₀T₁₅–Au NPs (0.91 nM), TBA₁₅-P₄T₁₅–Au NPs (1.11 nM), TBA₁₅-P₈T₁₅–Au NPs (1.43 nM), TBA₁₅-P₁₆T₁₅–Au NPs (1.82 nM), TBA₂₉-P₀T₁₅–Au NPs (0.85 nM), TBA₂₉-P₄T₁₅–Au NPs (1.14 nM), TBA₂₉-P₈T₁₅–Au NPs (1.43 nM) or TBA₂₉-P₁₆T₁₅–Au NPs (1.82 nM). The content of each of the oligonucleotides was held constant (100 nM) in these experiments. TBA₁₅-P_mT₁₅ prolonging the stem pair number will stabilize the G-quadruplexes structure and increase the resistance of nuclease digestion in plasma and therefore increase their inhibitory potency. We noted the inhibition of TBA₁₅-P_mT₁₅–Au NPs and TBA₂₉-P_mT₁₅–Au NPs increased with increase in the stem pair number from P₀ to P₈ and then decreased. As a result of increase in the rigidity and the stability of G-quadruplexes of TBAs on the Au NP surfaces with increasing stem number, the anticoagulant potency increased. In addition, the increase of stem pair number leads to an increase in the distance of G-quadruplexes unit of TBA and thus increases their hydrodynamic diameter (Fig. 2b). The inhibitions of TBA₁₅-P₁₆T₁₅–Au NPs/TBA₂₉-P₁₆T₁₅–Au NPs (TCT delay 2.6/12.3 fold) were lower than TBA₁₅-P₈T₁₅–Au NPs/TBA₂₉-P₈T₁₅–Au NPs (TCT delay 4.2/18.2 fold), probably due to the relatively low TBA density (Table 1 and Fig. 2d) and unexpected intermolecular hybridization or G-quadruplexes. The electrophoretic mobility of TBA-P₁₆T₁₅–Au NPs was unexpectedly faster than TBA-h₈T₁₅–Au NPs.

Fig. 3 The relative thrombin clotting time [(t − t₀)/t₀] in human plasma in the presence of TBA-P_mT₁₅ or TBA-P_mT₁₅–Au NPs. The error bars represent the standard deviation of three repeated measurements. Other conditions were the same as those described in Fig. 1.

3.3. Multivalent of TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs

After optimization of linker length and stem pair number of TBA-P_mT_n–Au NPs, we further prepared multivalent Au NP bioconjugates (TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs) to improve the inhibition of thrombin enzymatic activity. The TCT values for TBA₁₅-P₈T₁₅ (100 nM), TBA₁₅-P₈T₁₅–Au NPs (1.43 nM), TBA₂₉-P₈T₁₅–Au NPs (1.43 nM) and TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs (1.42 nM) are approximately 3.9, 4.2, 18.2 and 296 times longer than that would be in the absence of an inhibitor (Fig. 4). Our result reveals TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs have an ultra strong potency on coagulation delay in this common pathway compared to TBA₁₅ and monovalent TBA–Au NPs. We will emphasize that the TBA₁₅-P₀/TBA₂₉-P₄–Au NPs conjugates were used without modifying the sequence according to the reports,^38–40 leading to only 19.3-times delay in TCT (data not shown). The multivalent interaction between the thrombin and TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs induces interparticle cross-linking. The degree of aggregation of TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs (1.0 nM) tends to increase with increasing thrombin concentration from 10 to 100 nM (ESI, Fig. S1†). Interaction of the TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs with thrombin is strong: K_d = 8.86 × 10⁻¹² M (ESI, Fig. S2†), two orders of magnitude higher than that for free TBA₂₉ and one order of magnitude higher than that for a linear assembly of TBA₁₅ and TBA₂₉ and engineering dendritic aptamer.^{38–40,53–59} In addition, electrostatic interactions and steric blocking between the TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs and thrombin are caused by the high density and negative charge of the aptamer units which cannot be excluded for stronger inhibitions. The stability of nucleic acids toward enzymes and nucleases is an important factor that affects their therapeutic and diagnostic applications. We investigated that the TCT delay for the TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs remains almost constant after 48 h of incubation in the presence of human plasma (ESI, Fig. S3†). High local concentrations of salt in the highly negatively charged TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs are known to cause enhanced resistance to nuclease digestion in plasma.⁷⁹

Fig. 4 (a) Real-time monitoring of the light scattered during the coagulation of mixtures of thrombin, plasma and in the absence (A, control) and presence of inhibitors (B–E). (b) The relative thrombin clotting time [(t − t₀)/t₀] in human plasma in the presence of and in the absence (A, control) and presence of inhibitors (B–E). Other conditions were the same as those described in Fig. 1.

3.4. Comparison with commercial anticoagulant drugs

We further compare our TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs with commercial anticoagulant drugs of direct thrombin inhibitors (argatroban or hirudin) and our previous TBA₂₉-P₄T₁₅–Au NPs in TCT assays (Fig. 5). The dosage dependence of TCT delay indicates the anticoagulation ability of TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs was much better than the two commercial drugs and TBA₂₉-P₄T₁₅–Au NPs. For example, TCT using TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs (0.14 nM; [TBA] = 10 nM) was 961 ±11 s, which is much longer than that in the absence of the inhibitor (20 ±3 s), whereas the TCT time was prolonged to 58 ±4 s, 22± 3 s and 69± 4 s when using argatroban (10 nM), hirudin (10 nM) and TBA₂₉-P₄T₁₅–Au NPs (0.11 nM; [TBA] = 10 nM), respectively.

Fig. 5 Dosage dependence of the delay of TCT for TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs, TBA₂₉-P₄T₁₅–Au NPs, argatroban or hirudin in the human plasma samples. The error bars represent the standard deviation of three repeated measurements. Other conditions were the same as those described in Fig. 1.

3.5. Reverse thrombin activity by irradiation of light

Reversibility of binding between inhibitors and coagulant factors is important for cardiopulmonary bypass.^80–82 Thrombin is also important in the regulation of homeostasis and is associated with many types of cancer.⁸³ Therefore, engineering probes that target thrombin and, moreover, whose function can be precisely regulated by photonic energy, can advance anticoagulation therapy for many diseases. Fig. 6 exhibits that, in the presence of TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs and TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs (both of them are 2.5 nM) inhibit the plasma clotting induced by thrombin (5 nM). The TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs/thrombin complex irradiated with 365 nm UV light (365 nm, 20 mW cm⁻², 30 min) can rapidly reverse the activity of thrombin. TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs with near-UV light allowed the “caged” TBA_15pc/TBA_29pc units to be released from the Au NPs surface. We determined about 80% of the TBA_pc was released in to the solution after irradiation of near-UV light for 30 min. To minimize the damage caused by light to the animal tissue, for real animal system applications, we will use two photon irradiation in our future work.

Fig. 6 Reversible inhibitory function of the TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs (2.5 nM) in plasma by irradiation of near-UV light. (A) thrombin (5 nM) is added to 2-fold diluted plasma immediately in the absence of any inhibitors, (B) 2.5 nM TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs or (C) 2.5 nM TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs incubated with thrombin and plasma, or (D) 2.5 nM TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs incubated with thrombin were irradiated with near-UV light for 30 min and then reacted with plasma. Other conditions were the same as those described in Fig. 1.

4. Conclusions

We have demonstrated that TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs display a high anticoagulant activity toward thrombin due to their highly flexible and stable conformation and multivalent binding. The TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs interact strongly with thrombin, exhibiting a high binding affinity (K_d = 8.86 × 10⁻¹² M) toward thrombin—over 100 times higher than that of monovalent TBA₂₉, 10 000 times higher than that of TBA₁₅, and at least 10 times higher than those that have been reported previously for fusion aptamers, dendritic aptamers, and TBA₂₉–Au NPs.^53–60 The TBA₁₅/TBA₂₉-P₈T₁₅–Au NPs exhibited a 296-fold longer TCT relative to that tested in the absence of any inhibitor, whereas for TBA₁₅ alone, it was only 3.9 times longer. Moreover, the caged TBA_15pc/TBA_29pc-P₈T₁₅–Au NPs allow a reversal of thrombin activity via near-UV light irradiation. Thus, our newly developed light-controllable anticoagulant drug should exhibit high potencies in biomedical applications. We believe that our described techniques can be used widely to modify NPs with other DNA or RNA aptamers toward different proteins or cancer cells.

Acknowledgements

This study was supported by the National Science Council of Taiwan under contract NSC 99-2113-M-019-001-MY2 and the National Health Research Institutes Taiwan under contract NHRI-EX100-10047NI.

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Footnote

† Electronic Supplementary Information (ESI) available: Fig. S1–S3. See DOI: 10.1039/c1ra00344e/

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