Cu(2+)-RGDFRGDS: exploring the mechanism and high efficacy of the nanoparticle in antithrombotic therapy

Abstract

Thrombosis disease has been the leading cause of morbidity and mortality worldwide. In the discovery of antithrombotic agents, three complexes of Cu(2+) and repetitive arginine-glycine-aspartic acid (RGD) sequences, Cu(II)-Arg-Gly-Asp-Ser-Arg-Gly-Asp-Ser (Cu[II]-4a), Cu(II)-Arg-Gly-Asp-Val-Arg-Gly-Asp-Val (Cu[II]-4b), and Cu(II)-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Phe (Cu[II]-4c), were previously reported, of which Cu(II)-4a and Cu(II)-4c possessed the highest in vitro and in vivo activity, respectively. Transmission electron microscopy (TEM) images visualized that Cu(II)-4a and Cu(II)-4c formed nanoaggregates and nanoparticles, respectively. However, the details of the formation of the nanospecies complexes and of the mechanism for inhibiting thrombosis remain to be clarified. For this purpose, this study designed a novel complex of Cu(II) and the RGD octapeptide, Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser (RGDFRGDS), consisting of Arg-Gly-Asp-Phe of Cu(II)-4c and Arg-Gly-Asp-Ser of Cu(II)-4a, to colligate their biological and nanostructural benefits. In contrast with Cu(II)-4a, -4b, and -4c, Cu(II)-RGDFRGDS (Cu(2+)-FS) had high antiplatelet and antithrombotic activities, with the formed nanoparticles having a porous surface. Additionally, this paper evidenced the dimer had the basic structural unit of Cu(2+)-FS in water, theoretically simulated the formation of Cu(2+)-FS nanoparticles, and identified that Cu(2+)-FS activity in decreasing glycoprotein IIb/IIIa, P-selectin, and IL-8 was responsible for the antithrombotic action. Finally, adherence onto the surface and entry into the cytoplasm were considered the steps of a two-step model for the blocking of platelet activation by Cu(2+)-FS nanoparticles. Findings indicated that the antiplatelet aggregation activity of Cu(2+)-FS was 10-52 times higher than that of RGDFRGDS, while the effective dose for antithrombotic action was 5,000 times lower than that of RGDFRGDS.

Keywords: AFM; GPIIb/IIIa; IL-8; SEM; TEM; nanomedicine.

Figure 1

Preparation of RGDFRGDS and Cu²⁺-FS. Notes: (i) NMM, DCC, and HOBt. (ii) HCl/EtOAc. (iii) H₂/Pd/C. (iv) Phenyl methyl ether, CF₃CO₂H/CF₃SO₃H (4/1). (v) CuCl₂. Abbreviations: Cu²⁺-FS, the complexes of Cu²⁺ and RGDFRGDS; DCC, dicyclohexylcarbodiimide; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine; RGDFRGDS, arginine-glycine-aspartic acid-phenylalanine-arginine-glycine-aspartic acid-serine.

Figure 2

UV spectra of RGDFRGDS and Cu²⁺-FS. Abbreviations: RGDFRGDS, Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; Cu²⁺-FS, Cu^2+-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; Abs, absorbance; UV, ultraviolet.

Figure 3

CD spectra of RGDFRGDS (A and B) and Cu²⁺-FS (C and D). Notes: (A) CD spectra of RGDFRGDS in the range 190–400 nm. (B) CD spectra of RGDFRGDS in the range 400–750 nm. (C) CD spectra of Cu²⁺-FS ranging from 190 nm to 400 nm. (D) CD spectra of Cu²⁺-FS ranging from 400 nm to 750 nm. Abbreviations: CD, circular dichroism; RGDFRGDS, Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser.

Figure 4

FT-MS spectrum of Cu²⁺-FS in water, to which the dimer (1,981.57170) is inserted. Notes: The ion peak at 1,981.57170 (inset) equals the mass of two molecules of Cu²⁺-FS plus K (971.3396). Abbreviations: Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; FT-MS, Fourier transform mass spectrometry; Intens, intensity.

Figure 5

Proposed model of Cu²⁺-FS. Notes: (A) Forming the dimer and (B) forming nanoparticles. Abbreviation: Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser.

Figure 6

Nanosizes of Cu²⁺-FS assembled in NS. Notes: (A) Nanoparticle diameters of Cu²⁺-FS in NS during 10 days. (B) Nanoparticle diameters of Cu²⁺-FS in NS during 27 minutes. Nanoparticle diameter is represented as the mean ± SD nm (n=6). Abbreviations: Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; NS, normal saline; SD, standard deviation.

Figure 7

In pH 6.7 aqueous solution, Cu²⁺-FS (0.015 nM) formed nanoparticles having surface pores of 4.2 nm in diameter. Notes: (A) The nanoparticles were 13–57 nm in diameter, with pores on the locally enlarged surface. The scale bar represents 50 nm. (B) The nanoparticles were 26–90 nm in diameter, with pores on the locally enlarged surface. The scale bar represents 100 nm. Abbreviation: Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser.

Figure 8

In pH 7.4 buffer, Cu²⁺-FS formed nanoparticles having pores on their surfaces. Notes: (A) The scale bar represents 50 nm. The nanoparticles (0.015 nM) were 64–244 nm in diameter, with the pores of 3.1 nm on the locally enlarged surface. (B) The scale bar represents 50 nm. The nanoparticles (0.015 nM) were 32–83 nm in diameter, with pores of 3.1 nm on the locally enlarged surface. (C) The scale bar represents 100 nm. The nanoparticles were 21–116 nm in diameter, with pores of 4.2 nm on the locally enlarged surface. (D) The scale bar represents 100 nm. The nanoparticles were 68–99 nm in diameter, with pores of 4.2 nm on the locally enlarged surface. (E) The scale bar represents 100 nm. The nanoparticles (1.5×10² nM) were 15–40 nm in diameter, with pores of 4.5 nm on the locally enlarged surface. (F) The scale bar represents 100 nm. The nanoparticles (1.5×10² nM) were 20–83 nm in diameter, with pores of 4.9 nm on the locally enlarged surface. (G) The scale bar represents 100 nm. The nanoparticles (1.5×10³ nM) were 15–88 nm in diameter, with pores of 5.0 nm on the locally enlarged surface. (H) The scale bar represents 100 nm. The nanoparticles (1.5×10³ nM) were 36–152 nm in diameter, with pores of 5.3 nm on the locally enlarged surface. (I) The scale bar represents 100 nm. The nanoparticles (1.5×10⁶ nM) were 59–135 nm in diameter, with pores of 8.3 nm on the locally enlarged surface. (J) The scale bar represents 100 nm. The nanoparticles (1.5×10⁶ nM) were 26–100 nm in diameter, with pores of 8.7 nm on the locally enlarged surface. (K) The scale bar represents 100 nm. The nanoparticles (1.5×10⁸ nM) were 32–100 nm in diameter, with pores of 9.0 nm on the locally enlarged surface. (L) The scale bar represents 100 nm. The nanoparticles (1.5×10⁸ nM) were 36–112 nm in diameter, with pores of 10.0 nm on the locally enlarged surface. Abbreviation: Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser.

Figure 9

AFM image of Cu²⁺-FS in rat plasma. Notes: The nanoparticles were labeled with a blue arrow. (A) Rat plasma alone, giving no comparable nanoparticles. (B) Rat plasma with Cu²⁺-FS (10^–2 M). The blue arrow indicates the nanoparticles of Cu²⁺-FS in plasma, of 39.3–78.1 nm in height and 234.4 nm in width. (C) Water with Cu²⁺-FS (10⁻² M). The blue arrow indicates the nanoparticles, of 39.6–54.9 nm in height and 234.4–468.8 nm in width. Abbreviations: AFM, atomic force microscopy; Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser.

Figure 10

Interaction of Cu²⁺-FS with rat platelets, GPIIb/IIIa expression, and P-selectin expression. Notes: (A) SEM image of AA-activated rat platelets with 200 nM Cu²⁺-FS, wherein the nanoparticles on the surface of the platelet are indicated by the yellow arrowheads, while the nanoparticles in the process of endocytosis are indicated by the red arrowheads. (B) SEM image of AA-activated rat platelets treated with NS. (C) GPIIb/IIIa levels of AA-activated platelets treated with 0.1–100 nM Cu²⁺-FS. (D) P-selectin levels of AA-activated platelets treated with 0.1 nM Cu²⁺-FS. Abbreviations: AA, arachidonic acid; Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; GP, glycoprotein; NS, normal saline; SD, standard deviation; SEM, scanning electron microscope.

Figure 11

In vitro and in vivo actions. Notes: (A) Antiplatelet aggregation activity of RGDFRGDS in vitro. (B) Antiplatelet aggregation activity of Cu²⁺-FS in vitro. (C) Dose-dependent antithrombotic activity of Cu²⁺-FS (n=12). (D) IL-8 level of the blood of rats treated with 10 nmol/kg Cu²⁺-FS (n=3). Thrombus weight is represented with the mean ± SD mg. Abbreviations: AA, arachidonic acid; ADP, adenosine diphosphate; Cu²⁺-FS, Cu²⁺-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; IC₅₀, half maximal inhibitory concentration; NS, normal saline; PAF, platelet-activating factor; RGDFRGDS, Arg-Gly-Asp-Phe-Arg-Gly-Asp-Ser; SD, standard deviation; TH, thrombin.