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

RGDV-modified gemcitabine: a nano-medicine capable of prolonging half-life, overcoming resistance and eliminating bone marrow toxicity of gemcitabine

Wenchao Liu1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Yujia Mao1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School 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, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Yaonan Wang1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Jianhui Wu1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of ChinaORCID Iconhttps://orcid.org/0000-0001-9416-8952
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Shurui Zhao1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China
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Shiqi Peng1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of ChinaORCID Iconhttps://orcid.org/0000-0003-2669-6715
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Ming Zhao1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;2 Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Capital Medical University, Beijing100069, People’s Republic of China;3 Department of Biomaterials, Beijing Laboratory of Biomedical Materials and Key Laboratory of Biomedical Materials of Natural Macromolecules, Beijing University of Chemical Technology, Beijing100026, People’s Republic of ChinaCorrespondence[email protected]
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Pages 7263-7279 | Published online: 06 Sep 2019

Figures & data

Figure 1 HPLC-UV chromatogram, peak area and half-life. (A) After 300 mins incubation, the HPLC-UV chromatogram, peak area and half-life of gemcitabine; (B) After 300 mins incubation in mouse plasma, the HPLC-UV chromatogram, peak area and half-life of Asp(OBzl)-gemcitabine; (C) After 300 mins incubation, the HPLC-UV chromatogram, peak area and half-life of 1,2,3,4-tetrahydroisoquinoline-3-carboxyl-Ile-gemcitabine; (D) After 300 mins incubation, the HPLC-UV chromatogram, peak area and half-life of RGDV-gemcitabine.

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo- lan-2-yl]pyrimidin-2-one.

Figure 1 HPLC-UV chromatogram, peak area and half-life. (A) After 300 mins incubation, the HPLC-UV chromatogram, peak area and half-life of gemcitabine; (B) After 300 mins incubation in mouse plasma, the HPLC-UV chromatogram, peak area and half-life of Asp(OBzl)-gemcitabine; (C) After 300 mins incubation, the HPLC-UV chromatogram, peak area and half-life of 1,2,3,4-tetrahydroisoquinoline-3-carboxyl-Ile-gemcitabine; (D) After 300 mins incubation, the HPLC-UV chromatogram, peak area and half-life of RGDV-gemcitabine.Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo- lan-2-yl]pyrimidin-2-one.

Figure 2 Synthetic route of RGDV-gemcitabine (4)a.

Notes: aReagents: i) DCC, HOBt, NMM and THF; ii) CH3OH and aqueous NaOH (4 M); iii) CH3OH and Pd/C; iv) DCC, HOBt and DMF; v) Hydrochloride in ethyl acetate (4 M).

Abbreviations: DCC, dicyclohexylcarbodiimide; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine; THF, tetrahydrofuran; DMF, N,N-dimethylformamide; RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 2 Synthetic route of RGDV-gemcitabine (4)a.Notes: aReagents: i) DCC, HOBt, NMM and THF; ii) CH3OH and aqueous NaOH (4 M); iii) CH3OH and Pd/C; iv) DCC, HOBt and DMF; v) Hydrochloride in ethyl acetate (4 M).Abbreviations: DCC, dicyclohexylcarbodiimide; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine; THF, tetrahydrofuran; DMF, N,N-dimethylformamide; RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 3 IC50 of gemcitabine and RGDV-gemcitabine against the proliferation of A549 cells. (A) IC50 of gemcitabine alone against the proliferation of A549 cells; (B) IC50 of RGDV-gemcitabine alone against the proliferation of A549 cells; (C) IC50 of gemcitabine plus dipyridamole against the proliferation of A549 cells; (D) IC50 of RGDV-gemcitabine plus dipyridamole against the proliferation of A549 cells, n=3.

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo- lan-2-yl]pyrimidin-2-one.

Figure 3 IC50 of gemcitabine and RGDV-gemcitabine against the proliferation of A549 cells. (A) IC50 of gemcitabine alone against the proliferation of A549 cells; (B) IC50 of RGDV-gemcitabine alone against the proliferation of A549 cells; (C) IC50 of gemcitabine plus dipyridamole against the proliferation of A549 cells; (D) IC50 of RGDV-gemcitabine plus dipyridamole against the proliferation of A549 cells, n=3.Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo- lan-2-yl]pyrimidin-2-one.

Figure 4 (A) FT-MS spectrum gives RGDV-gemcitabine an ion peak of monomer and an ion peak of trimer; (B) qCID spectrum of the trimer gives an ion peak of monomer and an ion peak of dimer; (C) NOESY 2D 1H NMR spectrum gives two cross-peaks that defines the approach manner of RGDV-gemcitabine forming trimer; (D) to fit the NOESY 2D 1H NMR spectrum, the trimer of RGDV-gemcitabine should possess butterfly-like conformation.

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 4 (A) FT-MS spectrum gives RGDV-gemcitabine an ion peak of monomer and an ion peak of trimer; (B) qCID spectrum of the trimer gives an ion peak of monomer and an ion peak of dimer; (C) NOESY 2D 1H NMR spectrum gives two cross-peaks that defines the approach manner of RGDV-gemcitabine forming trimer; (D) to fit the NOESY 2D 1H NMR spectrum, the trimer of RGDV-gemcitabine should possess butterfly-like conformation.Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 5 Faraday-Tyndall effect, particle size and zeta potential of aqueous RGDV-gemcitabine. (A) Without radiation of 650 nm laser, the ultrapure water of pH 6.7 shows no Faraday-Tyndall effect; (B) Without radiation of 650 nm laser, the solution of RGDV-gemcitabine in the ultrapure water of pH 6.7 (0.1 μM) shows no Faraday-Tyndall effect; (C) With radiation of 650 nm laser, the ultrapure water of pH 6.7 shows no Faraday-Tyndall effect; (D) With radiation of 650 nm laser, the solution of RGDV-gemcitabine in the ultrapure water of pH 6.7 (0.1 μM) shows Faraday-Tyndall effect; (E) 7 days’ size of the nano-particles of RGDV-gemcitabine in ultrapure water of pH 6.7 (0.1 μM); (F) On 7th day, the zeta potential of the nano-particles of RGDV-gemcitabine in ultrapure water of pH 6.7 (0. μM).

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 5 Faraday-Tyndall effect, particle size and zeta potential of aqueous RGDV-gemcitabine. (A) Without radiation of 650 nm laser, the ultrapure water of pH 6.7 shows no Faraday-Tyndall effect; (B) Without radiation of 650 nm laser, the solution of RGDV-gemcitabine in the ultrapure water of pH 6.7 (0.1 μM) shows no Faraday-Tyndall effect; (C) With radiation of 650 nm laser, the ultrapure water of pH 6.7 shows no Faraday-Tyndall effect; (D) With radiation of 650 nm laser, the solution of RGDV-gemcitabine in the ultrapure water of pH 6.7 (0.1 μM) shows Faraday-Tyndall effect; (E) 7 days’ size of the nano-particles of RGDV-gemcitabine in ultrapure water of pH 6.7 (0.1 μM); (F) On 7th day, the zeta potential of the nano-particles of RGDV-gemcitabine in ultrapure water of pH 6.7 (0. μM).Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 6 Nano-feature of RGDV-gemcitabine. (A) Nano-feature of the powders lyophilized from a solution of RGDV-gemcitabine in ultrapure water (pH 6.7, 0.01 μM) imaged with SEM; (B) nano-feature of RGDV-gemcitabine in ultrapure water (pH 6.7, 0.01 μM) imaged with TEM; (C) nano-feature of RGDV-gemcitabine in ultrapure water (pH 6.7, 0.01 μM) imaged with AFM (C); (D) nano-feature of mouse serum imaged with AFM; (E) nano-feature of RGDV-gemcitabine in mouse serum (0.01 μM) imaged with AFM.

Abbreviations: SEM, scanning electron microscopy; TEM, transmission electron microscopy; AFM, atomic force microscopy; RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo- lan-2-yl]pyrimidin-2-one.

Figure 6 Nano-feature of RGDV-gemcitabine. (A) Nano-feature of the powders lyophilized from a solution of RGDV-gemcitabine in ultrapure water (pH 6.7, 0.01 μM) imaged with SEM; (B) nano-feature of RGDV-gemcitabine in ultrapure water (pH 6.7, 0.01 μM) imaged with TEM; (C) nano-feature of RGDV-gemcitabine in ultrapure water (pH 6.7, 0.01 μM) imaged with AFM (C); (D) nano-feature of mouse serum imaged with AFM; (E) nano-feature of RGDV-gemcitabine in mouse serum (0.01 μM) imaged with AFM.Abbreviations: SEM, scanning electron microscopy; TEM, transmission electron microscopy; AFM, atomic force microscopy; RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo- lan-2-yl]pyrimidin-2-one.

Figure 7 Mesoscale simulation course of RGDV-gemcitabine forming a sized particle.

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 7 Mesoscale simulation course of RGDV-gemcitabine forming a sized particle.Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 8 Anti-tumor activities of RGDV-gemcitabine. (A) IC50 of gemcitabine and RGDV-gemcitabine inhibiting the proliferation of MCF-7, HCT-8, A549, 95D and HepG2 cells, n=9; (B) tumor weight of gemcitabine and RGDV-gemcitabine treated S180 mice, n=12.

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 8 Anti-tumor activities of RGDV-gemcitabine. (A) IC50 of gemcitabine and RGDV-gemcitabine inhibiting the proliferation of MCF-7, HCT-8, A549, 95D and HepG2 cells, n=9; (B) tumor weight of gemcitabine and RGDV-gemcitabine treated S180 mice, n=12.Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 9 Serum ALT/GPT (A), AST/GOT (B), Cr (C) and BUN (D) of S180 mice treated by 8.4 μmol/kg/day of RGDV-gemcitabine and 84 μmol/kg/day of gemcitabine, n=12.

Abbreviations: ALT/GPT, alanine aminotransferase; AST/GOT, aspartic aminotransferase; Cr, creatinine; BUN, blood urea nitrogen; RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 9 Serum ALT/GPT (A), AST/GOT (B), Cr (C) and BUN (D) of S180 mice treated by 8.4 μmol/kg/day of RGDV-gemcitabine and 84 μmol/kg/day of gemcitabine, n=12.Abbreviations: ALT/GPT, alanine aminotransferase; AST/GOT, aspartic aminotransferase; Cr, creatinine; BUN, blood urea nitrogen; RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 10 Count of red blood cells, white blood cells, platelets and neutrophil of the treated S180 mice. (A) Count of red blood cells; (B) count of white blood cells; (C) count of platelets; (D) count of neutrophil, n=12.

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 10 Count of red blood cells, white blood cells, platelets and neutrophil of the treated S180 mice. (A) Count of red blood cells; (B) count of white blood cells; (C) count of platelets; (D) count of neutrophil, n=12.Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one.

Figure 11 FT(+)-MS spectrum of the extract of the homogenate of tumor tissue and blood of S180 mice treated with 8.4 μmol/kg/day of RGDV-gemcitabine. (A) FT(+)-MS spectrum of the extract of the homogenate of the tumor tissue; (B) locally amplified spectra of Arg-Gly-Asp-Val plus H at 468.2117, Arg-Gly-Asp plus H at 347.1631, Arg-Gly plus H at 232.1424, Arg plus H at 175.1176, Gly plus H at 76.0396, Asp plus H at 134.0443, Val plus H at 118.0868, RGDV-gemcitabine plus H at 691.3013, GDV-gemcitabine plus Na at 557.1737, DV-gemcitabine plus H at 478.1694, V-gemcitabine plus H at 363.2060 and gemcitabine plus H at 264.0771; (C) FT(+)-MS spectrum of the extract of the homogenate of the blood and amplified spectrum of RGDV-gemcitabine plus H at 691.3002.

Abbreviations: RGDV-gemcitabine, 4-[Arg-Gly-Asp-Val-amino-1-[3,3-difluoro-4-hydroxy-5- (hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; GDV-gemcitabine, 4-[Gly-Asp-Val-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; DV-gemcitabine, 4-[Arg-Gly-Asp-Val-amino-1-[3,3-difluoro-4- hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one.

Figure 11 FT(+)-MS spectrum of the extract of the homogenate of tumor tissue and blood of S180 mice treated with 8.4 μmol/kg/day of RGDV-gemcitabine. (A) FT(+)-MS spectrum of the extract of the homogenate of the tumor tissue; (B) locally amplified spectra of Arg-Gly-Asp-Val plus H at 468.2117, Arg-Gly-Asp plus H at 347.1631, Arg-Gly plus H at 232.1424, Arg plus H at 175.1176, Gly plus H at 76.0396, Asp plus H at 134.0443, Val plus H at 118.0868, RGDV-gemcitabine plus H at 691.3013, GDV-gemcitabine plus Na at 557.1737, DV-gemcitabine plus H at 478.1694, V-gemcitabine plus H at 363.2060 and gemcitabine plus H at 264.0771; (C) FT(+)-MS spectrum of the extract of the homogenate of the blood and amplified spectrum of RGDV-gemcitabine plus H at 691.3002.Abbreviations: RGDV-gemcitabine, 4-[Arg-Gly-Asp-Val-amino-1-[3,3-difluoro-4-hydroxy-5- (hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; GDV-gemcitabine, 4-[Gly-Asp-Val-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; DV-gemcitabine, 4-[Arg-Gly-Asp-Val-amino-1-[3,3-difluoro-4- hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one.

Figure 12 In the tumor tissue, RGDV-gemcitabine could release gemcitabine and Arg-Gly-Asp-Val via path A, B, C and D.

Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5- (hydroxylmethyl)oxolan-2-yl]pyri-midin-2-one; GDV-gemcitabine, 4-(Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; DV-gemcitabine, 4-(Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; V-gemcitabine, 4-(Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimi-din-2-one.

Figure 12 In the tumor tissue, RGDV-gemcitabine could release gemcitabine and Arg-Gly-Asp-Val via path A, B, C and D.Abbreviations: RGDV-gemcitabine, 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5- (hydroxylmethyl)oxolan-2-yl]pyri-midin-2-one; GDV-gemcitabine, 4-(Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; DV-gemcitabine, 4-(Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimidin-2-one; V-gemcitabine, 4-(Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylme-thyl)oxolan-2-yl]pyrimi-din-2-one.

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