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International Journal of Nanomedicine Volume 14, 2019 - Issue
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Original Research

Nano-scaled MTCA-KKV: for targeting thrombus, releasing pharmacophores, inhibiting thrombosis and dissolving blood clots in vivo

Shurui Zhao1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Ze Li1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Fei Huang2 Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing, People’s Republic of China
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Jianhui Wu1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Lin Gui1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Xiaoyi Zhang1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
,
Yaonan Wang1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Xiaozhen Wang1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
,
Shiqi Peng1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
&
Ming Zhao1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of ChinaCorrespondence[email protected]
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Pages 4817-4831 | Published online: 03 Jul 2019

Figures & data

Figure 1 Preparation of MTCA-KKV. (i) DCC, HOBt, NMM and THF; (ii) H2, Pd/C and CH3OH; (iii) hydrogen chloride in ethyl acetate (4 M); (iv) H2SO4 (5 M), 60ºC; (v) DMF, (Boc)2O and trimethylamine.

Abbreviations: DCC, dicyclohexylcarbodiimide; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine; THF, tetrahydrofuran; (Boc)2O, ditertbutyl dicarbonate; DMF, NN-dimethylformide; MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 1 Preparation of MTCA-KKV. (i) DCC, HOBt, NMM and THF; (ii) H2, Pd/C and CH3OH; (iii) hydrogen chloride in ethyl acetate (4 M); (iv) H2SO4 (5 M), 60ºC; (v) DMF, (Boc)2O and trimethylamine.Abbreviations: DCC, dicyclohexylcarbodiimide; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine; THF, tetrahydrofuran; (Boc)2O, ditertbutyl dicarbonate; DMF, NN-dimethylformide; MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 2 FT-ICR-MS, qCID and NOESY 2D NMR spectra of MTCA-KKV. (A) FT-ICR-MS spectrum of MTCA-KKV in aqueous solution; (B) qCID spectrum of MTCA-KKV in aqueous solution;(C) NOESY 2D NMR spectrum of MTCA-KKV in DMSO-d6;  (D) energy minimized conformation of the dimer of MTCA-KKV.

Abbreviations: MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 2 FT-ICR-MS, qCID and NOESY 2D NMR spectra of MTCA-KKV. (A) FT-ICR-MS spectrum of MTCA-KKV in aqueous solution; (B) qCID spectrum of MTCA-KKV in aqueous solution;(C) NOESY 2D NMR spectrum of MTCA-KKV in DMSO-d6;  (D) energy minimized conformation of the dimer of MTCA-KKV.Abbreviations: MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 3 Tyndall effect, particle size and zeta potential of MTCA-KKV in ultrapure water. (A) Feature of ultrapure water of pH 6.7; (B) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 6.7; (C) Feature of ultrapure water of pH 6.7 with the radiation of 650 nm laser; (D) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 6.7 with the radiation of 650 nm laser; (E) Feature of ultrapure water of pH 2.0; (F) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 2.0; (G) Feature of ultrapure water of pH 2.0 with the radiation of 650 nm laser; (H) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 2.0 with the radiation of 650 nm laser; (I) Seven-days’ size of MTCA-KKV in pH 6.7 and pH 2.0 ultrapure water (0.1 μM); (J) Zeta potential of MTCA-KKV in pH 6.7 ultrapure water (0.1 μM).

Abbreviations: MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 3 Tyndall effect, particle size and zeta potential of MTCA-KKV in ultrapure water. (A) Feature of ultrapure water of pH 6.7; (B) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 6.7; (C) Feature of ultrapure water of pH 6.7 with the radiation of 650 nm laser; (D) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 6.7 with the radiation of 650 nm laser; (E) Feature of ultrapure water of pH 2.0; (F) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 2.0; (G) Feature of ultrapure water of pH 2.0 with the radiation of 650 nm laser; (H) Feature of 0.1 μM solution of MTCA-KKV in ultrapure water of pH 2.0 with the radiation of 650 nm laser; (I) Seven-days’ size of MTCA-KKV in pH 6.7 and pH 2.0 ultrapure water (0.1 μM); (J) Zeta potential of MTCA-KKV in pH 6.7 ultrapure water (0.1 μM).Abbreviations: MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 4 TEM, SEM and AFM images of MTCA-KKV in ultrapure water and plasma of rat. (A and B) TEM image and size distribution of particles of MTCA-KKV in ultrapure water of pH 6.7 (10.0 μM); (C and D) TEM image and size distribution of particles of MTCA-KKV in ultrapure water of pH 6.7 (0.1 μM); (E and F) TEM image and size distribution of particles of MTCA-KKV in ultrapure water of pH 6.7 (1.0 nM); (G and H) SEM image and size distribution of powders lyophilized from a solution of MTCA-KKV in pH 6.7 ultrapure water (10.0 μM); (I and J) SEM image and size distribution of powders lyophilized from a solution of MTCA-KKV in pH 6.7 ultrapure water (0.1 μM); (K and L) SEM image and size distribution of powders lyophilized from a solution of MTCA-KKV in pH 6.7 ultrapure water (1.0 nM); (M) AFM image of rat plasma; (N, O and P) AFM image and size distribution of the particles of MTCA-KKV in rat plasma (10.0 μM, 0.1 μM and 1.0 nM); (Q) MTCA-KKV’s nanoparticle predicted with mesoscale simulation software.

Abbreviations: TEM, transmission electron microscopy; SEM, scanning electron microscopy; AFM, atomic force microscopy; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 4 TEM, SEM and AFM images of MTCA-KKV in ultrapure water and plasma of rat. (A and B) TEM image and size distribution of particles of MTCA-KKV in ultrapure water of pH 6.7 (10.0 μM); (C and D) TEM image and size distribution of particles of MTCA-KKV in ultrapure water of pH 6.7 (0.1 μM); (E and F) TEM image and size distribution of particles of MTCA-KKV in ultrapure water of pH 6.7 (1.0 nM); (G and H) SEM image and size distribution of powders lyophilized from a solution of MTCA-KKV in pH 6.7 ultrapure water (10.0 μM); (I and J) SEM image and size distribution of powders lyophilized from a solution of MTCA-KKV in pH 6.7 ultrapure water (0.1 μM); (K and L) SEM image and size distribution of powders lyophilized from a solution of MTCA-KKV in pH 6.7 ultrapure water (1.0 nM); (M) AFM image of rat plasma; (N, O and P) AFM image and size distribution of the particles of MTCA-KKV in rat plasma (10.0 μM, 0.1 μM and 1.0 nM); (Q) MTCA-KKV’s nanoparticle predicted with mesoscale simulation software.Abbreviations: TEM, transmission electron microscopy; SEM, scanning electron microscopy; AFM, atomic force microscopy; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 5 UV and CD described in vitro interaction between MTCA-KKV and P-selectin or GPIIb/IIIa. (A) P-selectin concentration dependently changes the UV of MTCA-KKV; (B) GPIIb/IIIa concentration dependently changes the UV MTCA-KKV; (C) MTCA-KKV concentration dependently changes the CD of P-selectin; (D) MTCA-KKV concentration dependently changes the CD of GPIIb/IIIa.

Abbreviations: UV, ultra violet spectrum; CD, circular dichroism spectrum; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly- Asp-Val.

Figure 5 UV and CD described in vitro interaction between MTCA-KKV and P-selectin or GPIIb/IIIa. (A) P-selectin concentration dependently changes the UV of MTCA-KKV; (B) GPIIb/IIIa concentration dependently changes the UV MTCA-KKV; (C) MTCA-KKV concentration dependently changes the CD of P-selectin; (D) MTCA-KKV concentration dependently changes the CD of GPIIb/IIIa.Abbreviations: UV, ultra violet spectrum; CD, circular dichroism spectrum; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly- Asp-Val.

Figure 6 AFM feature of rat platelets, erythrocytes and leucocytes with and without MTCA-KKV. (A) AFM feature of rat platelets alone; (B) AFM feature of rat platelets with MTCA-KKV (0.1 nM, in pH 6.7 ultrapure water); (C) AFM feature of rat erythrocytes alone; (D) AFM feature of rat erythrocytes with MTCA-KKV (0.1 nM, in pH 6.7 ultrapure water); (E) AFM feature of rat leucocytes alone; (F) AFM feature of rat leucocytes with MTCA-KKV (0.1 nM, in pH 6.7 ultrapure water).

Abbreviations: AFM, atomic force microscopy; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetra-hydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 6 AFM feature of rat platelets, erythrocytes and leucocytes with and without MTCA-KKV. (A) AFM feature of rat platelets alone; (B) AFM feature of rat platelets with MTCA-KKV (0.1 nM, in pH 6.7 ultrapure water); (C) AFM feature of rat erythrocytes alone; (D) AFM feature of rat erythrocytes with MTCA-KKV (0.1 nM, in pH 6.7 ultrapure water); (E) AFM feature of rat leucocytes alone; (F) AFM feature of rat leucocytes with MTCA-KKV (0.1 nM, in pH 6.7 ultrapure water).Abbreviations: AFM, atomic force microscopy; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetra-hydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 7 In vivo anti-arterial thrombosis and thrombolytic activity, as well as downregulation of the expression of GPIIb/IIIa and P-selectin. (A) Dose-dependently anti-arterial thrombosis activities of MTCA-KKV; (B) Thrombolytic activity of 0.01 μmol/kg MTCA-KKV; (C) GPIIb/IIIa expression of the rats treated with 0.01 μmol/kg MTCA-KKV; (D) P-selectin expression of the rats treated with 0.01 μmol/kg MTCA-KKV; n=12.

Abbreviations: NS, normal saline; MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 7 In vivo anti-arterial thrombosis and thrombolytic activity, as well as downregulation of the expression of GPIIb/IIIa and P-selectin. (A) Dose-dependently anti-arterial thrombosis activities of MTCA-KKV; (B) Thrombolytic activity of 0.01 μmol/kg MTCA-KKV; (C) GPIIb/IIIa expression of the rats treated with 0.01 μmol/kg MTCA-KKV; (D) P-selectin expression of the rats treated with 0.01 μmol/kg MTCA-KKV; n=12.Abbreviations: NS, normal saline; MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 8 (A) ESI(+)-FT-MS spectrum of the extract of arterial thrombus of rats treated with 0.01 μmol/kg MTCA-KKV; (B) ESI(-)-FT-MS spectrum of the extract of arterial thrombus of rats treated with 0.01 μmol/kg of MTCA-KKV.

Abbreviations: MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 8 (A) ESI(+)-FT-MS spectrum of the extract of arterial thrombus of rats treated with 0.01 μmol/kg MTCA-KKV; (B) ESI(-)-FT-MS spectrum of the extract of arterial thrombus of rats treated with 0.01 μmol/kg of MTCA-KKV.Abbreviations: MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 9 (A-C) In the arterial thrombus of the treated rats, MTCA-KKV is degraded via 3 ways to release anti-thrombosis, thrombolytic and thrombus targeting pharmacophores.

Abbreviations: MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 9 (A-C) In the arterial thrombus of the treated rats, MTCA-KKV is degraded via 3 ways to release anti-thrombosis, thrombolytic and thrombus targeting pharmacophores.Abbreviations: MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 10 Graphic conclusion.

Abbreviations: MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Figure 10 Graphic conclusion.Abbreviations: MTCA, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; MTCA-KKV, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val.

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