Synthesis of 5-(bromomethyl)-N1,N3-bis(N-Boc-carbamimidoyl)isophtha-
lamide (Compound f): Compound f was synthesized according to the
method shown in Figure 2A.
in Maestro and were converted to 3D structures. A solved nucleic
acid structure (PDB ID: 5zld) required for docking analysis was
nucleic acid were preprocessed with the Protein Preparation Wizard,
Synthesis of dimethyl 5-methylisophthalate (Compound b): Sulfuric
acid (98%, 4 mL) was added dropwise into 5-methy isophthalic acid
(compound a) (10 g, 55.54 mm) in methanol (150 mL) at a temperature
under 30 °C, and the mixture was refluxed at 80 °C for 8 h. The 1H-NMR
spectrum is seen in Figure S1, Supporting Information. The reaction
solution was cooled to room temperature and neutralized with
saturated aqueous sodium bicarbonate to pH ≈8. The resulting solution
and precipitate were extracted with CHCl2 and washed with saturated
aqueous NaCl three times. The organic phase was dried with anhydrous
MgSO4 and removed using rotary evaporation. The product was dried
under vacuum to obtain compound b (10.6 g, 50.94 mm, 91.72%). The
bond orders were assigned, hydrogens were added and the H bond
network was optimized. The structural model of the nucleic acid was
energy-minimized using the OPLS 2005 force field. Molecular docking
was performed using the AutoDock software suite. No restraints were
used during docking. Computational docking was performed by the
Lamarckian Genetic Algorithm and default values.[21]
NMR and 31P NMR Spectra: BGG, adenosine monophosphate (AMP)
and the mixture of BGG and AMP were dissolved in DMSO-d6. The
mixture was mixed thoroughly for 30 s and incubated for 30 min to
1
give the complex. H NMR and 31P NMR spectra were recorded using a
1
structure of compound b was confirmed by H NMR (Varian 400 MHz;
Varian 400 MHz NMR spectrometer (Palo Alto, CA, USA).
Palo Alto, CA, USA) (Figure S2, Supporting Information).
FTIR Spectra: The spectra of BGG, AMP, the physical mixture and
complex of BGG and AMP were obtained by infrared spectroscopy
(Necolet iS5, ThermoFisher, MA, USA). BGG and AMP were dispersed
in KBr separately to observe the FTIR spectra of BGG and AMP. A simple
mix of BGG@ KBr and AMP@ KBr represented the physical mixture
of BGG and AMP. BGG and AMP were mixed thoroughly to obtain
the BGG-AMP complex. The complex was then dispersed in KBr for
scanning by infrared spectroscopy.
Preparation and Characterization of Gene Complexes: The concentrations
of DNA were 80 µg mL−1 for in vitro studies and 1.6 mg mL−1 for in vivo
studies. DNA and all polymers were dissolved in phosphate buffered
saline (PBS). Concentrations of polymers were determined by different
mass ratios of polymers and DNA. An equal volume of polymers and
DNA samples were combined, and the mixture was mixed thoroughly
for 30 s and incubated at room temperature for 30 min. The size
distribution and zeta potential of different polymer/pGL3 complexes
were determined by dynamic light scattering (DLS) (Zetasizer Nano
ZSP, Malvern Instruments, Worcestershire, UK).
Enzymatic Degradation Assay: G5-BGG/pDNA complexes (20 µL) were
made at different mass ratios, then DNase I (4 µL, 5 U/µL) was added
and incubated for 1 h at 37 °C Then EDTA (4 µL, 500 mM) was added and
the mixture was incubated for another 10 min. Finally, heparin Sodium
(12 µL, 100 mg mL−1) was added into the mixture. After including for 2 h,
the DNA protection ability against enzymatic degradation was measured
by gel retardation assay.
Cellular Uptake and Endosome/Lysosome Escape: HEK 293T cells in the
logarithmic growth phase were suspended and seeded on 12-well plates
at a primary cell density of 1 × 105 cells/well. After incubation for 24 h,
the medium in the wells was replaced with fresh culture medium and
cells were treated with 100 µL of different gene complexes containing
4 µg TOTO-3 labeled DNA. The culture medium contained 10% FBS
unless otherwise specified. After 4 h, the mean fluorescence intensity of
cells was measured by flow cytometry (CytoFLEX S, Beckman, CA, USA).
To evaluate the endosome/lysosome escape ability of G5-BGG/DNA,
HEK 293T cells were treated with TOTO-3 labeled gene complexes for
1 h or 4 h. The nuclei were stained with DAPI, whereas lysosomes were
stained with Lyso Tracker. The fluorescence images were observed by
using a confocal laser scanning microscope (CLSM) (LSM 710, Zeiss,
Oberkochen, Germany).
Synthesis of dimethyl 5-(bromomethyl)isophthalate (Compound c):
Compound b (8 g, 38.45 mm) was suspended in CCl4 (200 mL), and
N-Bromosuccinimide (NBS) (7.2 g) and benzoyl peroxide (0.16 g) were
added. After refluxing for 12 h, the reaction solution was filtered under hot
conditions. The raw product, obtained by evaporating to remove CCl4,
was washed with cooled ethanol (50 mL × 3) to afford compound c
(7.2 g, 25.18 mm, 65.48%). The 1H NMR spectrum is shown in Figure S3,
Supporting Information.
Synthesis of 5-(bromonethyl)isophthalic acid (Compound d): Compound c
(6 g, 20.98 mm) was dissolved in acetic acid (100 mL) and aqueous
HBr (40%, 100 mL). After refluxing for 12 h, the mixture was cooled
to room temperature and dropwise added to 1000 mL ice water. The
suspension was filtered to collect the product. The product was dried
under vacuum to afford compound d (3.2 g, 12.41 mm, 59.13%). The
1H NMR spectrum is shown in Figure S4, Supporting Information.
Synthesis of Compound f: Compound d (1 g, 3.88 mm) was dissolved
in CHCl3 (5 mL) and oxalyl chloride (1.2 mL) was added dropwise
under a nitrogen atmosphere at 0 °C. The mixture was stirred for 6 h
to obtain 5-(bromomethyl)isophthaloyl dichloride (compound e) without
additional purification. 1-(tert-Butoxycarbonyl)guanidine was dissolved
in DMF (5 mL) and compound e was added dropwise under nitrogen
at 0 °C. After reacting for 12 h, rotary evaporation was used to remove
CHCl3 in the mixture followed by the addition of 100 mL distilled water.
The resulting solution was extracted with ethyl acetate and washed with
saturated aqueous NaCl three times. The organic phase was dried with
anhydrous Na2SO4 and removed using rotary evaporation. The crude
product was purified by column chromatography (eluent:petroleum
ether/ethyl acetate = 8:1) to collect compound f (276 mg, 0.51 mm,
13.18%). The structure of compound f was verified by 1H NMR
(Figure S5, Supporting Information).
Synthesis of G5-BGG: G5-BGG was synthesized according to Figure 2B.
The PAMAM G5 dendrimer (Figure S6, Supporting Information) and
compound f were dissolved in methanol and DMF at different molar
ratios and stirred for 12 h at 60 °C. Then, 50% trifluoroacetic acid in
CH2Cl2 was added to the reaction and the mixture was stirred for 6 h at
0 °C. The organic phase and trifluoroacetic acid were removed using
rotary evaporation. The remaining solution was dialyzed against distilled
water and G5-BGG was obtained by lyophilization. The modification ratios
were determined by recording 1H NMR spectra. As a negative control, G5
modified with benzyl (G5-BEN) was synthesized through a similar reaction
between G5 and benzyl bromide (Figure 2C), and the modification ratios
In vitro Gene Transfection: HEK 293T and HeLa cells in logarithmic
growth phases were suspended and seeded on 48-well plates at a
primary cell density of 2 × 104 cells/well. After incubation for 24 h, the
medium in the wells was replaced with fresh culture medium and 50 µL
of the different gene complexes containing 2 µg DNA. After incubation
with gene complexes for 4 h (HEK 293T) or 6 h (HeLa), the medium
containing the gene complexes was replaced with fresh culture medium
and the cells were cultured for a further 44 h (HEK 293T) or 42 h (HeLa).
The expression of the EGFP plasmid in HEK 293T and HeLa cells was
directly visualized by fluorescent microscopy (DMI4000B, Leica, Wetzler,
Germany). The expression level of the luciferase plasmid was measured
according to the manufacturer’s protocol (Promega) and the results
are presented as the relative light units per milligram cellular protein
(RLU/mg protein). The apoptosis rates of HeLa cells as determined by
1
were also determined by H NMR. The synthesis of G5-GUA and the
determination of the modification ratios were both performed according
to well-established methods reported previously (Figure 2D).[19h,20] Briefly,
G5 and 1H-pyrazole-1-carboxamidine hydrochloride were dissolved in
distilled water at a molar ratio of 1:5. N,N-Diisopropylethylamine (DIEA)
was added into the mixture and the solution was stirred for 24 h at
25 °C. The reaction solution was then extensively dialyzed against distilled
water and lyophilized to obtain G5-GUA.[20] The modification ratio was
determined by the ninhydrin assay.[19h]
Molecular Docking Studies: The structural models of G5, G5-GUA,
G5-BEN and G5-BGG fragments were generated by the 2D Sketcher
Adv. Funct. Mater. 2020, 2004783
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