Design and synthesis of nanoscaled IQCA-TAVV as a delivery system capable of antiplatelet activation, targeting arterial thrombus and releasing IQCA

Abstract

Background: Arterial thrombosis has been associated with a series of pathological conditions, and the discovery of arterial thrombosis inhibitor is of clinical importance.

Methods: By analyzing the pharmacophores of anti-platelet agents, thrombus targeting peptide and anti-thrombotic nano-systems 3S-1,2,3,4-tetrahydroisoquino-line-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val (IQCA-TAVV) was designed and prepared as a nano-scaled arterial thrombosis inhibitor.

Results: In vitro the nanoparticles of IQCA-TAVV were able to adhere onto the surface of activated platelets, attenuate activated platelets to extend pseudopodia and inhibit activated platelets to form aggregators. In vivo IQCA-TAVV targeted arterial thrombus, dose dependently inhibited arterial thrombosis with a 1 nmol/kg of minimal effective dose, and the activity waŝ1670 folds of that of aspirin.

Conclusion: IQCA-TAVV represented the design, preparation and application of nanomedicine capable of adhering on the surface of activated platelets, attenuating platelet activation, targeting arterial thrombus and inhibiting arterial thrombosis.

Keywords: antithrombosis; arterial thrombosis; nanodelivery; thrombus targeting.

Figure 1

The combination of pharmacophores of the antiplatelet agents, thrombus-targeting peptide Arg-Gly-Asp-Val and antithrombotic agents leads to the design of IQCA-TAVV as a thrombus-targeting nanodelivery system capable of antiplatelet activation, targeting arterial thrombus and releasing IQCA. Abbreviations: IQCA, 3S-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; IQCA-TAVV, 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val.

Figure 2

FT-MS spectrum, NOESY 2D NMR spectrum and the dimer of IQCA-TAVV. Notes: (A and B) FT-MS spectrum and qCID spectrum of 1 nM solution of IQCA-TAVV in ultrapure water, and the red box shows the amplified region in the MS spectrum; (C) NOESY 2D NMR spectrum of IQCA-TAVV and red ring–labeled interesting cross-peak; (D) Energy-minimized conformation of IQCA-TAVV’s monomer; (E) Energy-minimized conformation of IQCA-TAVV’s dimer. Abbreviations: FT-MS, Fourier transform mass spectrometry; IQCA-TAVV, 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val; Intens, intensities; NMR, nuclear magnetic resonance; qCID, qualitative contingent influence diagram.

Figure 3

TEM and SEM images, as well as mesoscale simulation software-predicted nanoparticle of IQCA-TAVV. Notes: (A) TEM image of 10 nM IQCA-TAVV in ultrapure water; (B) TEM image of 0.1 nM IQCA-TAVV in ultrapure water; (C) Particle size distribution of TEM image of 0.1 nM IQCA-TAVV in ultrapure water; (D) SEM image of lyophilized powders from 10 nM IQCA-TAVV in ultrapure water; (E) Particle size distribution of TEM image of lyophilized powders from 10 nM IQCA-TAVV in ultrapure water; (F) Mesoscale simulation software-predicted nanoparticle of IQCA-TAVV. Abbreviations: IQCA-TAVV, 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val; TEM, transmission electron microscopy; SEM, scanning electron microscopy.

Figure 4

Tyndall effect, zeta potential and size of IQCA-TAVV in ultrapure water. Notes: (A) Ultrapure water without radiation; (B) ultrapure water with 650 nm laser radiation; (C) IQCA-TAVV in pH 6.7 ultrapure water (1 nM) with 650 nm laser radiation; (D) IQCA-TAVV in pH 1.2 ultrapure water (1 nM) with 650 nm laser radiation; (E) IQCA-TAVV in pH 6.7 ultrapure water (10 nM) with 650 nm laser radiation; (F) IQCA-TAVV in pH 1.2 ultrapure water (10 nM) with 650 nm laser radiation; (G) zeta potential of IQCA-TAVV in ultrapure water (10 nM); (H) particle size of 10 nM solution of IQCA-TAVV in normal saline over 7 days (n=6). Abbreviation: IQCA-TAVV, N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-Ala-Arg-Gly-Asp(Val)-Val.

Figure 5

AFM images of the resting rat platelets and the interaction between nanoparticles of IQCA-TAVV and AA-activated rat platelets. Notes: (A) IC₅₀ of IQCA-TAVV against AA-activated rat platelet aggregation; (B) AFM image of rat plasma without platelets; (C) AFM image of IQCA-TAVV in rat plasma (10 nM) to mirror the feature of the particles in physiologic environment; (D) AFM image of resting platelets without IQCA-TAVV; (E) AFM image of AA-activated platelets without IQCA-TAVV; (F) AFM image of IQCA-TAVV (10 nM)-treated resting platelets; local amplified particle labeled by blue box; (G) AFM image of IQCA-TAVV (100 nM)-treated AA-activated platelets. Besides, the AFM images of normal saline and IQCA-TAVV in normal saline (10 nM) are shown in Figure S5, the height of particle was labeled by red arrow. Abbreviations: IQCA-TAVV, N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-Ala-Arg-Gly-Asp(Val)-Val; AA, arachidonic acid; AFM, atomic force microscopy.

Figure 6

AFM images of the resting rat platelets and the effect of IQCA-TAVV nanoparticles on TH-activated rat platelets. Notes: (A) IC₅₀ of IQCA-TAVV against TH-induced rat platelet aggregation; (B) AFM image of the resting rat platelets; (C) AFM image of TH-activated rat platelets without IQCA-TAVV; (D) AFM image of TH-activated platelets treated with 0.05 nM of IQCA-TAVV; (E) AFM image of TH-activated platelets treated with 0.5 nM of IQCA-TAVV; (F) AFM image of TH-activated platelets treated with 5 nM of IQCA-TAVV. Abbreviations: IQCA-TAVV, N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-Ala-Arg-Gly-Asp(Val)-Val; TH, thrombin; AFM, atomic force microscopy.

Figure 7

SEM images of the action of the nanoparticles of IQCA-TAVV on rat platelets. Notes: (A) SEM images of AA-activated rat platelets treated with normal saline, in which an amplified platelet having a smooth surface is inserted and the extended pseudopodia are labeled with red arrowheads; (B) SEM images of AA-activated rat platelets treated with 200 nM IQCA-TAVV, in which an amplified platelet without pseudopodia and the nanoparticles of IQCA-TAVV on the surface are labeled with blue arrowheads. Abbreviations: SEM, scanning electron microscopy; AA, arachidonic acid; IQCA-TAVV, N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-Ala-Arg-Gly-Asp(Val)-Val.

Figure 8

Anti-arterial thrombosis activities of IQCA-TAVV, n=12. Notes: (A) At 10 nmol/kg dose IQCA-TAVV effectively inhibits the mice forming an arterial thrombus; (B) IQCA-TAVV dose-dependently inhibits the rats forming an arterial thrombus. Abbreviations: IQCA-TAVV, N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-Thr-Ala-Arg-Gly-Asp(Val)-Val; TAVV, Thr-Ala-Arg-Gly-Asp(Val)-Val; NS, normal saline.

Figure 9

ESI(±)-FT-MS spectra of the extract of the organs and thrombus of rats treated with 10 nmol/kg IQCA-TAVV. Notes: (A) ESI(-)-FT-MS spectrum of the thrombus extract; the local amplified inset shows an ion peak at 178.07821, the mass of IQCA subtracting H; (B) ESI(+)-FT-MS spectrum of the thrombus extract; the local amplified inset shows an ion peak at 519.25641, the mass of TARGD plus H; (C) ESI(-)-FT-MS spectrum of the blood extract, the local amplified inset was labeled by red box and shows no ion peak of IQCA; (D–I) The local amplified ESI(-)-FT-MS spectra of the extracts of the brain, heart, lung, liver, spleen and kidney; the local amplified insets show no such ion peak of IQCA. Abbreviations: Intens, intensities; ESI, electrospray ionization; FT-MS, Fourier transform mass spectrometry; IQCA-TAVV, 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val; TAVV, Thr-Ala-Arg-Gly-Asp(Val)-Val.

Figure 10

Effects of IQCA-TAVV on mouse Cr, ALT and AST, n=5. Notes: (A) Serum Cr of ICR mice treated with NS and 1 μmol/kg of IQCA-TAVV; (B) Serum ALT of ICR mice treated with NS and 1 μmol/kg of IQCA-TAVV; (C) Serum AST of ICR mice treated with NS and 1 μmol/kg of IQCA-TAVV. Abbreviations: ALT/GPT, alanine aminotransferase/glutamic pyruvic transaminase; AST/GOT, aspartate aminotransferase/glutamic oxaloacetic transaminase; Cr, creatinine; NS, normal saline; ICR, institute of Cancer Research.

Figure 11

Hypothesized thrombus-specific release of IQCA and Thr-Ala-Arg-Gly-Asp from IQCA-TAVV. Abbreviations: IQCA-TAVV, 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val; IQCA, 3S-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

Scheme 1

Synthetic route to IQCA-TAVV. Notes: (a) HCHO and HCl; (b) (Boc)₂O and NaOH; (c) DCC, HOBt, and NMM; (d) 2N NaOH and methanol; (e) Hydrogen chloride in ethyl acetate (4N); (f) CF₃CO₂H/CF₃SO₃H. Abbreviations: IQCA-TAVV, 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-Thr-Ala-Arg-Gly-Asp(Val)-Val; DCC, dicyclohexylcarbodiimide; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine.