Figure 1.
Chemical structure of 2-(3-[bis(3-aminopropyl)amino]propylamino)-N-ditetradecyl-carbamoylmethylacetamide (DMAPAP).
Figure 1.
Chemical structure of 2-(3-[bis(3-aminopropyl)amino]propylamino)-N-ditetradecyl-carbamoylmethylacetamide (DMAPAP).
Figure 2.
TEM image of γ-Fe2O3 magnetic nanoparticles (MNPs).
Figure 2.
TEM image of γ-Fe2O3 magnetic nanoparticles (MNPs).
Figure 3.
Diagram of ultra-magnetic liposomes (UMLs) or MCLs preparation by reverse phase evaporation method. Phospholipids were dissolved in organic phase (CHCl3 + diethyl ether) (A); then, an aqueous phase containing MNPs was added (B); After 20 minutes of sonication, a water-in-oil emulsion was formed (C); Then, the organic solvent was evaporated, leading to the formation of a gel form (D); At the critical point, some vesicles were broken, and the excess phospholipid in the environment interacted with the residual micelles to complete the lipid bilayer, (E) leading to the formation of liposomes (F).
Figure 3.
Diagram of ultra-magnetic liposomes (UMLs) or MCLs preparation by reverse phase evaporation method. Phospholipids were dissolved in organic phase (CHCl3 + diethyl ether) (A); then, an aqueous phase containing MNPs was added (B); After 20 minutes of sonication, a water-in-oil emulsion was formed (C); Then, the organic solvent was evaporated, leading to the formation of a gel form (D); At the critical point, some vesicles were broken, and the excess phospholipid in the environment interacted with the residual micelles to complete the lipid bilayer, (E) leading to the formation of liposomes (F).
Figure 4.
Diagram of MCLs preparation by post insertion method. Pre-formed UMLs were dispersed in H2O (A); then, a solution of DMAPAP in H2O was added (B); Hydrophobic interaction between the liposome bilayer and the acyl-chain of DMAPAP led to the insertion of DMAPAP into the phospholipid bilayer (C).
Figure 4.
Diagram of MCLs preparation by post insertion method. Pre-formed UMLs were dispersed in H2O (A); then, a solution of DMAPAP in H2O was added (B); Hydrophobic interaction between the liposome bilayer and the acyl-chain of DMAPAP led to the insertion of DMAPAP into the phospholipid bilayer (C).
Figure 5.
Diagram of MCLs preparation by cosolvent sonication method. Cit-MNPs and DMAPAP were mixed at an equal molar ratio in H2O (A); The aggregates (FF-DMAPAP) were collected and washed with ethanol before dispersion in CHCl3 (B); Next, FF-DMAPAP were mixed with other lipids in CHCl3. Then, a second solvent, N-methyl pyrolidone (NMP), was added (C); Due to a long period of sonication, a clear suspension was obtained (D); After the evaporation of CHCl3, addition of H2O, and dialysis, the cationic magnetic liposomes were formed (E).
Figure 5.
Diagram of MCLs preparation by cosolvent sonication method. Cit-MNPs and DMAPAP were mixed at an equal molar ratio in H2O (A); The aggregates (FF-DMAPAP) were collected and washed with ethanol before dispersion in CHCl3 (B); Next, FF-DMAPAP were mixed with other lipids in CHCl3. Then, a second solvent, N-methyl pyrolidone (NMP), was added (C); Due to a long period of sonication, a clear suspension was obtained (D); After the evaporation of CHCl3, addition of H2O, and dialysis, the cationic magnetic liposomes were formed (E).
Figure 6.
TEM images of ultra-magnetic liposomes (UMLs): (A) before and (B) after insertion of 10% or (C) 40% of 2-(3-[bis(3-aminopropyl)amino]propylamino)-N-ditetradecyl- carbamoylmethylacetamide (DMAPAP). 10% or 40% of DMAPAP per total lipid was added to a diluted dispersion of UMLs (50 mM of Fe, 0.36 μmol total lipids). After stirring for 1 h, centrifugation, and magnetic separation, TEM images of the post-inserted UMLs was observed.
Figure 6.
TEM images of ultra-magnetic liposomes (UMLs): (A) before and (B) after insertion of 10% or (C) 40% of 2-(3-[bis(3-aminopropyl)amino]propylamino)-N-ditetradecyl- carbamoylmethylacetamide (DMAPAP). 10% or 40% of DMAPAP per total lipid was added to a diluted dispersion of UMLs (50 mM of Fe, 0.36 μmol total lipids). After stirring for 1 h, centrifugation, and magnetic separation, TEM images of the post-inserted UMLs was observed.
Figure 7.
TEM image of MCLs prepared with: (A) 2.5 mM (REV_MCL_2.5) or (B) 10 mM (REV_MCL_10) of iron.
Figure 7.
TEM image of MCLs prepared with: (A) 2.5 mM (REV_MCL_2.5) or (B) 10 mM (REV_MCL_10) of iron.
Figure 8.
TEM images of cosol_MCL_3 h.
Figure 8.
TEM images of cosol_MCL_3 h.
Figure 9.
TEM images of MCLs prepared by the cosolvent sonication method with various PEG-PE: (A) 14:0 PEG750 PE; (B) 14:0 PEG1000 PE; (C) 14:0 PEG2000 PE.
Figure 9.
TEM images of MCLs prepared by the cosolvent sonication method with various PEG-PE: (A) 14:0 PEG750 PE; (B) 14:0 PEG1000 PE; (C) 14:0 PEG2000 PE.
Figure 10.
Dynamic size and PDI of cosol_MCLs in cell culture medium after incubation at 37 °C. Cosol_MCLs with various PEG-PE (14:0 PEG750-PE, 14:0 PEG1000-PE, 14:0 PEG2000-PE) or without PEG-PE (wo PEG-PE) were diluted 5 times in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (50 U/mL), and streptomycin (50 U/mL), and incubated at 37 °C. Hydrodynamic size was measured by dynamic light scattering after 0 h, 1 h or 3 h incubation. The inserted table presents PDI value of the samples. The data given are averages of 3 different experiments (n = 3); bars, SD.
Figure 10.
Dynamic size and PDI of cosol_MCLs in cell culture medium after incubation at 37 °C. Cosol_MCLs with various PEG-PE (14:0 PEG750-PE, 14:0 PEG1000-PE, 14:0 PEG2000-PE) or without PEG-PE (wo PEG-PE) were diluted 5 times in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (50 U/mL), and streptomycin (50 U/mL), and incubated at 37 °C. Hydrodynamic size was measured by dynamic light scattering after 0 h, 1 h or 3 h incubation. The inserted table presents PDI value of the samples. The data given are averages of 3 different experiments (n = 3); bars, SD.
Figure 11.
(A) TEM and (B) crytoTEM of REV_MCLs.
Figure 11.
(A) TEM and (B) crytoTEM of REV_MCLs.
Figure 12.
(A) TEM and (B) CryoTEM of cosol_MCLs.
Figure 12.
(A) TEM and (B) CryoTEM of cosol_MCLs.
Figure 13.
Attraction of MCL formulations when exposed to a magnet. REV_MCLs and cosol_MCLs were put in 2 vials at a concentration of 5 mM of Fe. Two small NdFeB magnets (0.34 T) were placed next to the vials for 20 min. REV:MCLs by reverse phase evaporation method; cosol: MCLs by the cosolvent sonication method.
Figure 13.
Attraction of MCL formulations when exposed to a magnet. REV_MCLs and cosol_MCLs were put in 2 vials at a concentration of 5 mM of Fe. Two small NdFeB magnets (0.34 T) were placed next to the vials for 20 min. REV:MCLs by reverse phase evaporation method; cosol: MCLs by the cosolvent sonication method.
Figure 14.
T2 weighted MRI at 7 T of: (A) free MNPs; (B) REV_MCLs; and (C) cosol_MCLs at various iron concentrations in water.
Figure 14.
T2 weighted MRI at 7 T of: (A) free MNPs; (B) REV_MCLs; and (C) cosol_MCLs at various iron concentrations in water.
Figure 15.
Transfection efficiency of lipoplexes at RC8 in a CT26 cell line. Cells were seeded at 104 cells/well in a 96-well plate. Luciferase and protein assay were carried out 24 h after the incubation of cells with 100 µL of lipoplexes containing 1 µg of pFAR4-luc in the absence of a magnetic field for 3 h. Transfection efficiency is calculated as RLU/µg protein. The data given are averages of 3 different experiments, each performed in triplicate (n = 9); bars, SD. One-way ANOVA was done with GraphPad Prism software, ** p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001. Inserted figure: (A) RLU, (B) µg protein in 10 µL of cell lysis 24 h after incubation. RLU, relative light unit; RC = nmol DMAPAP/µg pFAR4-luc. Ctrl+ (dark circle): positive control (lipoplexes based on liposome DOPE/DMAPAP/C14PEG1000 49:50:1 mol/mol); REV_MCLs (dark square): lipoplexes based on REV_MCLs; cosol_MCLs (dark triangle): lipoplexes based on cosols MCLs.
Figure 15.
Transfection efficiency of lipoplexes at RC8 in a CT26 cell line. Cells were seeded at 104 cells/well in a 96-well plate. Luciferase and protein assay were carried out 24 h after the incubation of cells with 100 µL of lipoplexes containing 1 µg of pFAR4-luc in the absence of a magnetic field for 3 h. Transfection efficiency is calculated as RLU/µg protein. The data given are averages of 3 different experiments, each performed in triplicate (n = 9); bars, SD. One-way ANOVA was done with GraphPad Prism software, ** p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001. Inserted figure: (A) RLU, (B) µg protein in 10 µL of cell lysis 24 h after incubation. RLU, relative light unit; RC = nmol DMAPAP/µg pFAR4-luc. Ctrl+ (dark circle): positive control (lipoplexes based on liposome DOPE/DMAPAP/C14PEG1000 49:50:1 mol/mol); REV_MCLs (dark square): lipoplexes based on REV_MCLs; cosol_MCLs (dark triangle): lipoplexes based on cosols MCLs.
Figure 16.
Transfection efficiency of lipoplexes based on cosol_MCLs with different PEG-PE in CT26 cell line. Cells were seeded at 104 cells/well in a 96-well plate. Luciferase and protein assay were carried out 24 h after incubation of cells with 100 µL of lipoplexes containing 1 µg of pFAR4-luc in the absence of magnetic field for 3 h. Transfection efficiency is calculated as RLU/µg protein. The data given are averages of 2 different experiments, each performed in triplicates (n = 6); bars, SD. RLU, relative light unit; RC = nmol DMAPAP/µg pFAR4-luc. Ctrl+ (dark circle): positive control (lipoplexes based on liposome DOPE/DMAPAP/C14PEG1000 49:50:1 mol/mol); cosol_MCLs: lipoplexes based on cosols MCLs with 1% of 14:0 PEG1000-PE (dark square) or 14:0PEG2000-PE (clear circle).
Figure 16.
Transfection efficiency of lipoplexes based on cosol_MCLs with different PEG-PE in CT26 cell line. Cells were seeded at 104 cells/well in a 96-well plate. Luciferase and protein assay were carried out 24 h after incubation of cells with 100 µL of lipoplexes containing 1 µg of pFAR4-luc in the absence of magnetic field for 3 h. Transfection efficiency is calculated as RLU/µg protein. The data given are averages of 2 different experiments, each performed in triplicates (n = 6); bars, SD. RLU, relative light unit; RC = nmol DMAPAP/µg pFAR4-luc. Ctrl+ (dark circle): positive control (lipoplexes based on liposome DOPE/DMAPAP/C14PEG1000 49:50:1 mol/mol); cosol_MCLs: lipoplexes based on cosols MCLs with 1% of 14:0 PEG1000-PE (dark square) or 14:0PEG2000-PE (clear circle).
Figure 17.
Effect of magnetic field exposure on the transfection efficiency of lipoplexes (cosol_MCLs/pFAR4-luc, RC8) in a CT26 cell line for different incubation times. Cells were seeded at 104 cells/well in a 96-well plate. Luciferase and protein assay were carried out 24 h after the incubation of cells with 100 µL of lipoplexes containing 1 µg of pFAR4-luc in the absence (clear circle) or presence (dark circle) of a magnetic field for various incubation times. The transfection efficiency is calculated as RLU/ug protein. The data given are averages of triplicates of one experiment (n = 3); bars, SD. Two-way ANOVA was done with GraphPad Prism software, *p ≤ 0.05, ** p ≤ 0.01. RLU, relative light unit; RC = nmol DMAPAP/µg pFAR4-luc.
Figure 17.
Effect of magnetic field exposure on the transfection efficiency of lipoplexes (cosol_MCLs/pFAR4-luc, RC8) in a CT26 cell line for different incubation times. Cells were seeded at 104 cells/well in a 96-well plate. Luciferase and protein assay were carried out 24 h after the incubation of cells with 100 µL of lipoplexes containing 1 µg of pFAR4-luc in the absence (clear circle) or presence (dark circle) of a magnetic field for various incubation times. The transfection efficiency is calculated as RLU/ug protein. The data given are averages of triplicates of one experiment (n = 3); bars, SD. Two-way ANOVA was done with GraphPad Prism software, *p ≤ 0.05, ** p ≤ 0.01. RLU, relative light unit; RC = nmol DMAPAP/µg pFAR4-luc.
Figure 18.
Viability of CT26 (colon cancer) and TIB-75 (hepatocyte) cells after 24 h incubation with naked plasmid DNA (pFAR4-luc), cosol_MCLs, or lipoplexes (RC8). CT26 or TIB75 cells were seeded at 2 × 104 cells/well in a 96 well plate. Alamar blue test was carried out 24 h after incubation of cells with 100 µL of lipoplexes based on cosol_MCLs or cosol_MCLs or naked pFAR4-luc containing various concentration of pFAR4-luc and MNPs. The data given are the averages of 2 different experiments performed in triplicates (n = 6); bars, SD.
Figure 18.
Viability of CT26 (colon cancer) and TIB-75 (hepatocyte) cells after 24 h incubation with naked plasmid DNA (pFAR4-luc), cosol_MCLs, or lipoplexes (RC8). CT26 or TIB75 cells were seeded at 2 × 104 cells/well in a 96 well plate. Alamar blue test was carried out 24 h after incubation of cells with 100 µL of lipoplexes based on cosol_MCLs or cosol_MCLs or naked pFAR4-luc containing various concentration of pFAR4-luc and MNPs. The data given are the averages of 2 different experiments performed in triplicates (n = 6); bars, SD.
Table 1.
Lipid components and iron concentration for magnetic liposome formulations. DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DPPC: 1,2-dipalmitoyl-sn-glycero-3- phosphocholine, DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG-PE: 1,2-dimyristoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)].
Table 1.
Lipid components and iron concentration for magnetic liposome formulations. DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DPPC: 1,2-dipalmitoyl-sn-glycero-3- phosphocholine, DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG-PE: 1,2-dimyristoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)].
Components | MCLs | UMLs |
---|
DOPE (mol%) | 49 | 0 |
DMAPAP (mol%) | 50 | 0 |
14:0 PEG-PE (mol%) | 1 | 0 |
DPPC (mol%) | 0 | 86 |
DSPC (mol%) | 0 | 9 |
18:0 PEG2000-PE (mol%) | 0 | 5 |
Fe (mM) | 2.5–1000 | 1000 |
Table 2.
Characterizations of magnetic cationic liposomes (MCLs) prepared by reverse phase evaporation method using various amounts of magnetic nanoparticles (MNPs) (γ-Fe2O3).
Table 2.
Characterizations of magnetic cationic liposomes (MCLs) prepared by reverse phase evaporation method using various amounts of magnetic nanoparticles (MNPs) (γ-Fe2O3).
Label | Fe Concentration Input (mM) | Z Average (d.nm) | PDI | Zeta Potential (mV) | Encapsulation Efficiency % | Loading Efficiency % |
---|
REV_MCL_2.5 | 2.5 | 126.3 ± 2.6 | 0.188 ± 0.043 | +71.6 ± 4.2 | 70.3 ± 2.1 | 8.3 ± 0.2 |
REV_MCL_5 | 5 | 169.9 ± 24.7 | 0.202 ± 0.012 | +69.1 ± 2.3 | 75.6 ± 2.3 | 16.3 ± 0.4 |
REV_MCL_10 | 10 | 194.1 ± 14.2 | 0.240 ± 0.024 | +68.3 ± 4.0 | 78.8 ± 7.2 | 28.9 ± 1.9 |
REV_MCL_25 | 25 | 371.8 ± 36.7 | 0.388 ± 0.037 | +57.5 ± 5.4 | 35.6 ± 7.2 | 31.3 ± 4.4 |
Table 3.
Characterizations of MCLs by the cosolvent sonication method for different sonication times.
Table 3.
Characterizations of MCLs by the cosolvent sonication method for different sonication times.
Label | Sonication Time (h) | Z Average (d.nm) | PDI | Zeta Potential (mV) | Encapsulation Efficiency % | Loading Efficiency % |
---|
Cosol_MCL_1 h | 1 | 268.0 ± 7.2 | 0.168 ± 0.029 | +37.7 ± 0.7 | 61.0 ± 2.5 | 37.5 ± 1.0 |
Cosol_MCL_3 h | 3 | 215.7 ± 15.3 | 0.158 ± 0.047 | +46.8 ± 7.3 | 67.0 ± 2.7 | 39.7 ± 1.0 |
Cosol_MCL_6 h | 6 | 208.8 ± 24.8 | 0.178 ± 0.052 | +47.1 ± 2.5 | 69.4 ± 3.7 | 40.6 ± 1.3 |
Table 4.
Characterizations of MCLs by the cosolvent sonication method using different PEG-PE.
Table 4.
Characterizations of MCLs by the cosolvent sonication method using different PEG-PE.
PEG-PE | Z Average (d.nm) | PDI | Zeta Potential (mV) |
---|
14:0 PEG750 PE | 175.2 ± 20.2 | 0.143 ± 0.004 | 49.5 ± 1.4 |
14:0 PEG1000 PE | 207.4 ± 11.7 | 0.143 ± 0.022 | 41.7 ± 0.1 |
14:0 PEG2000 PE | 244.4 ± 66.3 | 0.184 ± 0.033 | 44.5 ± 0.8 |
Table 5.
Calculated relaxivities of free MNPs, REV_MCLs, and cosol_MCLs at various concentrations of iron in water at 7 T.
Table 5.
Calculated relaxivities of free MNPs, REV_MCLs, and cosol_MCLs at various concentrations of iron in water at 7 T.
Sample | r1 (s−1.mM−1) | r2 (s−1.mM−1) | r2/r1 | r2* (s−1.mM−1) |
---|
Free MNPs | 2.54 | 159.13 | 62.58 | 178.87 |
REV_MCLs | 0.37 | 172.77 | 461.83 | 279.18 |
Cosol_MCL | 1.07 | 222.59 | 207.99 | 296.74 |
Table 6.
Lipoplexation efficiency calculated from Picogreen assay.
Table 6.
Lipoplexation efficiency calculated from Picogreen assay.
Sample | RC | LE % |
---|
Ctrl+ | 8 | 92.0 ± 3.8 |
REV_MCLs | 8 | 90.5 ± 5.1 |
10 | 94.9 ± 1.8 |
| 12 | 93.9 ± 1.6 |
Cosol_MCLs | 8 | 88.0 ± 3.7 |
10 | 87.8 ± 5.1 |
12 | 89.0 ± 2.4 |