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International Journal of Nanomedicine Volume 13, 2018 - Issue
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Original Research

Design of pH-sensitive methotrexate prodrug-targeted curcumin nanoparticles for efficient dual-drug delivery and combination cancer therapy

Jiajiang Xie1 Xiamen Xianyue Hospital, Xiamen, China;2 Research Center of Biomedical Engineering of Xiamen, Key Laboratory of Biomedical Engineering of Fujian Province, Fujian Provincial Key Laboratory for Soft Functional Materials Research, Department of Biomaterials, College of Materials, Xiamen University, Xiamen, China
,
Zhongxiong Fan2 Research Center of Biomedical Engineering of Xiamen, Key Laboratory of Biomedical Engineering of Fujian Province, Fujian Provincial Key Laboratory for Soft Functional Materials Research, Department of Biomaterials, College of Materials, Xiamen University, Xiamen, China
,
Yang Li2 Research Center of Biomedical Engineering of Xiamen, Key Laboratory of Biomedical Engineering of Fujian Province, Fujian Provincial Key Laboratory for Soft Functional Materials Research, Department of Biomaterials, College of Materials, Xiamen University, Xiamen, China
,
Yinying Zhang2 Research Center of Biomedical Engineering of Xiamen, Key Laboratory of Biomedical Engineering of Fujian Province, Fujian Provincial Key Laboratory for Soft Functional Materials Research, Department of Biomaterials, College of Materials, Xiamen University, Xiamen, China
,
Fei Yu3 College of Medicals, Xiamen University, Xiamen, China
,
Guanghao Su4 Children’s Hospital of Soochow University, Suzhou, ChinaCorrespondence[email protected]
,
Liya Xie5 The First Affiliated Hospital of Xiamen University, Xiamen, China
&
Zhenqing Hou2 Research Center of Biomedical Engineering of Xiamen, Key Laboratory of Biomedical Engineering of Fujian Province, Fujian Provincial Key Laboratory for Soft Functional Materials Research, Department of Biomaterials, College of Materials, Xiamen University, Xiamen, ChinaCorrespondence[email protected]
 show all
Pages 1381-1398 | Published online: 09 Mar 2018

Figures & data

Figure 1 (A) Synthetic route of DSPE-PEG-Imine-MTX conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. (B) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). (C) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect.

Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; DHFR, dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.

Figure 1 (A) Synthetic route of DSPE-PEG-Imine-MTX conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. (B) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). (C) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect.Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; DHFR, dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.

Figure 2 Hydrodynamic size distribution, zeta potential, and TEM images of (A) M-CUR, (B) MTX-Amide-M-CUR, and (C) MTX-Imine-M-CUR. (D) XRD spectra of CUR, MTX-Amide-M nanocarriers, MTX-Amide-M nanocarriers/CUR mixture, MTX-Amide-M-CUR nanosystems, MTX-Imine-M nanocarriers, MTX-Imine-M nanocarriers/CUR mixture, and MTX-Imine-M-CUR nanosystems. (E) In vitro release profiles of CUR from M-CUR, MTX-Amide-M-CUR, and MTX-Imine-M-CUR in PBS buffer (pH 7.4 and 5.0) at 37°C. (F) In vitro release profiles of MTX from MTX-Amide-M-CUR and MTX-Imine-M-CUR in PBS buffer (pH 7.4 and 5.0) at 37°C.

Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; PBS, phosphate buffer saline; TEM, transmission electron microscope; XRD, X-ray diffractometer.

Figure 2 Hydrodynamic size distribution, zeta potential, and TEM images of (A) M-CUR, (B) MTX-Amide-M-CUR, and (C) MTX-Imine-M-CUR. (D) XRD spectra of CUR, MTX-Amide-M nanocarriers, MTX-Amide-M nanocarriers/CUR mixture, MTX-Amide-M-CUR nanosystems, MTX-Imine-M nanocarriers, MTX-Imine-M nanocarriers/CUR mixture, and MTX-Imine-M-CUR nanosystems. (E) In vitro release profiles of CUR from M-CUR, MTX-Amide-M-CUR, and MTX-Imine-M-CUR in PBS buffer (pH 7.4 and 5.0) at 37°C. (F) In vitro release profiles of MTX from MTX-Amide-M-CUR and MTX-Imine-M-CUR in PBS buffer (pH 7.4 and 5.0) at 37°C.Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; PBS, phosphate buffer saline; TEM, transmission electron microscope; XRD, X-ray diffractometer.

Figure 3 CLSM images of folate receptor-overexpressing (A) HeLa and (B) MCF-7 cells incubated with free CUR, M-CUR, and MTX-Imine-M-CUR for 0.5 and 2 h.

Notes: DAPI (false-color blue) was used to identify the nucleus. Scale bars are 15 µm.

Abbreviations: CLSM, confocal laser scanning microscopy; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate.

Figure 3 CLSM images of folate receptor-overexpressing (A) HeLa and (B) MCF-7 cells incubated with free CUR, M-CUR, and MTX-Imine-M-CUR for 0.5 and 2 h.Notes: DAPI (false-color blue) was used to identify the nucleus. Scale bars are 15 µm.Abbreviations: CLSM, confocal laser scanning microscopy; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate.

Figure 4 (A) CLSM images, (B) flow cytometry profiles, and (C) mean fluorescence intensity of HeLa cells incubated with free CUR, M-CUR, MTX-Amide-M-CUR (without/with FA pretreatment), or MTX-Imine-M-CUR (without/with FA pretreatment) for 2 h. Error bars indicate SD (n=4). *P<0.05. (D) Subcellular location of MTX-Imine-M-CUR in HeLa cells after incubation for 1 h.

Notes: DAPI (false-color blue) and LysoTracker Red (false-color red) were used to identify the nucleus and lysosomes, respectively. Scale bars are 20 µm.

Abbreviations: CLSM, confocal laser scanning microscopy; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; FA, folic acid; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; NA, no answer.

Figure 4 (A) CLSM images, (B) flow cytometry profiles, and (C) mean fluorescence intensity of HeLa cells incubated with free CUR, M-CUR, MTX-Amide-M-CUR (without/with FA pretreatment), or MTX-Imine-M-CUR (without/with FA pretreatment) for 2 h. Error bars indicate SD (n=4). *P<0.05. (D) Subcellular location of MTX-Imine-M-CUR in HeLa cells after incubation for 1 h.Notes: DAPI (false-color blue) and LysoTracker Red (false-color red) were used to identify the nucleus and lysosomes, respectively. Scale bars are 20 µm.Abbreviations: CLSM, confocal laser scanning microscopy; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; FA, folic acid; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; NA, no answer.

Figure 5 (A) Intracellular drug/nanocarriers distribution of HeLa cells treated with MTX-Imine-M-CUR for 1 and 6 h. DSPE-PEG-Cy5.5 (false-color red) was used to label nanocarriers. (B, C) In vitro cell viability of (B) HeLa cells and (C) MCF-7 cells incubated with free CUR, free CUR/MTX mixture, M-CUR nanosystems, MTX-Amide-M-CUR nanosystems, or MTX-Imine-M-CUR nanosystems for 24 h. (D, E) In vitro cell viability of (D) HeLa and (E) MCF-7 cells incubated with MTX-Imine-M-CUR nanosystems without/with FA pretreatment for 24 h.

Note: Error bars indicate SD (n=4).

Abbreviations: C, cytoplasm; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; FA, folic acid; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; N, nucleus.

Figure 5 (A) Intracellular drug/nanocarriers distribution of HeLa cells treated with MTX-Imine-M-CUR for 1 and 6 h. DSPE-PEG-Cy5.5 (false-color red) was used to label nanocarriers. (B, C) In vitro cell viability of (B) HeLa cells and (C) MCF-7 cells incubated with free CUR, free CUR/MTX mixture, M-CUR nanosystems, MTX-Amide-M-CUR nanosystems, or MTX-Imine-M-CUR nanosystems for 24 h. (D, E) In vitro cell viability of (D) HeLa and (E) MCF-7 cells incubated with MTX-Imine-M-CUR nanosystems without/with FA pretreatment for 24 h.Note: Error bars indicate SD (n=4).Abbreviations: C, cytoplasm; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; FA, folic acid; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; N, nucleus.

Figure 6 In vivo antitumor efficacy of HeLa tumor-bearing nude mice after intravenous injection of 0.9% NaCl, free CUR, free CUR/MTX, M-CUR, MTX-Amide-M-CUR, or MTX-Imine-M-CUR at an equivalent dose of CUR (8 mg/kg).

Notes: (A) Tumor volumes changes, (B) body weight changes, (C) isolated tumor weight, and (D) representative H&E staining histologic images from the tumor tissues. Error bars indicate SD (n=5). *P<0.05.

Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate.

Figure 6 In vivo antitumor efficacy of HeLa tumor-bearing nude mice after intravenous injection of 0.9% NaCl, free CUR, free CUR/MTX, M-CUR, MTX-Amide-M-CUR, or MTX-Imine-M-CUR at an equivalent dose of CUR (8 mg/kg).Notes: (A) Tumor volumes changes, (B) body weight changes, (C) isolated tumor weight, and (D) representative H&E staining histologic images from the tumor tissues. Error bars indicate SD (n=5). *P<0.05.Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate.

Figure S1 (A) 1H NMR, (B) UV–vis absorption, (C) XRD, (D) MALDI-TOF-MS, and (E) GPC spectra of DSPE-PEG-CHO, MTX, and DSPE-PEG-Imine-MTX amphiphilic polymer prodrug.

Abbreviations: 1HNMR, 1H nuclear magnetic resonance; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; DSPE-PEG-CHO, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(aldehyde[polyethylene glycol]-2000); GPC, gel permeation chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MTX, methotrexate; UV–vis, ultraviolet–visible; XRD, X-ray diffractometer.

Figure S1 (A) 1H NMR, (B) UV–vis absorption, (C) XRD, (D) MALDI-TOF-MS, and (E) GPC spectra of DSPE-PEG-CHO, MTX, and DSPE-PEG-Imine-MTX amphiphilic polymer prodrug.Abbreviations: 1HNMR, 1H nuclear magnetic resonance; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; DSPE-PEG-CHO, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(aldehyde[polyethylene glycol]-2000); GPC, gel permeation chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MTX, methotrexate; UV–vis, ultraviolet–visible; XRD, X-ray diffractometer.

Figure S2 In vitro stability of hydrodynamic size distribution of MTX-Imine-M-CUR nanosystems in (A) water and (B) PBS over 72 h. Error bars indicate SD (n=3).

Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; PBS, phosphate buffer saline; PDI, polydispersity index.

Figure S2 In vitro stability of hydrodynamic size distribution of MTX-Imine-M-CUR nanosystems in (A) water and (B) PBS over 72 h. Error bars indicate SD (n=3).Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; PBS, phosphate buffer saline; PDI, polydispersity index.

Figure S3 Blood circulation of MTX-Imine-M-CUR nanosystems. Concentration of CUR in blood at different time points after intravenous injection of MTX-Imine-M-CUR nanosystems.

Notes: The free CUR was used as a control. Error bars indicate SD (n=3).

Abbreviations: CUR, curcumin; MTX, methotrexate.

Figure S3 Blood circulation of MTX-Imine-M-CUR nanosystems. Concentration of CUR in blood at different time points after intravenous injection of MTX-Imine-M-CUR nanosystems.Notes: The free CUR was used as a control. Error bars indicate SD (n=3).Abbreviations: CUR, curcumin; MTX, methotrexate.

Figure S4 Relative DHFR activity in free MTX, MTX-Amide-M, and MTX-Imine-M at a MTX concentration of 0.1 µg/mL (very low concentration).

Notes: Error bars indicate SD (n=3). The enzymatic activity of DHFR in the presence of MTX or MTX conjugates was determined by a DHFR assay kit (Sigma-Aldrich) based on the NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid. The assay was conducted according to the reported literature.Citation1 Free MTX, MTX-Amide-M, and MTX-Imine-M showed a comparable inhibition effect of DHFR enzyme activity.

Abbreviations: DHFR, dihydrofolate reductase; MTX, methotrexate; NADPH, nicotinamide adenine dinucleotide phosphate.

Figure S4 Relative DHFR activity in free MTX, MTX-Amide-M, and MTX-Imine-M at a MTX concentration of 0.1 µg/mL (very low concentration).Notes: Error bars indicate SD (n=3). The enzymatic activity of DHFR in the presence of MTX or MTX conjugates was determined by a DHFR assay kit (Sigma-Aldrich) based on the NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid. The assay was conducted according to the reported literature.Citation1 Free MTX, MTX-Amide-M, and MTX-Imine-M showed a comparable inhibition effect of DHFR enzyme activity.Abbreviations: DHFR, dihydrofolate reductase; MTX, methotrexate; NADPH, nicotinamide adenine dinucleotide phosphate.

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