Gene Transfection in High Serum Levels. Case studies with New
Cholesterol based Cationic Gemini Lipids
Santosh K. Misra,a Joydeep Biswas,a Paturu Kondaiahb and Santanu Bhattacharya*,a,c
aDepartment
of Organic Chemistry and bDepartment of Molecular Reproduction, Development and
Genetics, Indian Institute of Science, Bangalore 560 012, India.
cChemical
Biology Unit of JNCASR, Bangalore 560 064, India.
*Corresponding author and also J. C. Bose Fellow, DST, New Delhi, India.
Email: sb@orgchem.iisc.ernet.in
Phone: (91)-80-2293-2664; Fax: (91)-80-2360-0529.
Supporting Information
Contents
Page No.
Elemental analysis results
S2
TEM images of lipoplexes
S3
Variation in Zeta potential
S4
Hydrodynamic diameters of lipid-DOPE coliposomes and lipoplexes
S5
Optimization of lipid:DOPE molar ratio in absence of serum
S6
Optimization of lipid:DOPE molar ratio in presence of serum
S7
Optimization of lipid:DOPE molar ratio in absence of serum
S8
Optimization of lipid:DOPE molar ratio in presence of serum
S9
Flow cytometric scans
S10
Effect of variation in the amount of pEGFP-C3 plasmid DNA
S11
pEGFP-C3 transfection in HEK 293T cells
S12
DNase sensitivity of DNA bound to various lipid formulations
S13
MTT assay of different formulations
S14
BrDU assay
S15
S1
Quantification of GFP Expression
S16
Confocal Images
S17
Abstract Figure
S18
Table S1. Elemental analysis values of new cholesterol based gemini lipids.
Lipid
CholG-D
Formula
Calculated
C66H118Br2N2O4
.
Found
C
H
N
C
H
N
68.13
10.22
2.41
67.96
10.13
2.47
CholHG-D
C68H122Br2N2O6 H2O
65.78
10.07
2.26
65.59
9.89
2.31
CholHG-1ox
C68H122Br2N2O5.2H2O
65.68
10.21
2.25
65.42
10.05
2.29
CholHG-2ox
C70H126Br2N2O6.H2O
66.22
10.16
2.21
66.07
9.97
2.29
CholHG-3ox
C72H130Br2N2O7.2H2O
64.07
10.16
2.08
63.86
10.19
2.17
CholHG-4ox
C74H134Br2N2O8.H2O
65.46
10.1
2.06
65.29
10.15
2.16
S2
A
B
C
Figure S1. Representative negative-stain transmission electron micrographs of aqueous suspensions of
lipoplexes of (A) CholHG-1ox (lipid/DOPE = 1:4 and N/P = 0.5:1); (B) CholHG-3ox (lipid/DOPE = 1:2
and N/P = 0.75:1) and (C) CholHG-D (lipid/DOPE = 1:2 and N/P = 1:1).
S3
Hydrodynamic Radius (nm)
CholHG-1ox:DOPE (1:4)
CholHG-1ox:pEGFP-C3 (0.5:1)
CholHG-2ox:DOPE (1:3)
CholHG-2ox:pEGFP-C3 (0.5:1)
CholHG-3ox:DOPE (1:2)
CholHG-3ox:pEGFP-C3 (0.75:1)
CholHG-4 ox:DOPE (1:1)
CholHG-4 ox:pEGFP-C3 (0.75:1)
CholG-D:DOPE (1:2)
CholG-D:pEGFP-C3 (1:1)
CholHG-D:DOPE (1:2)
CholHG-D:pEGFP-C3 (1:1)
500
400
300
200
100
0
Formulations
Figure S2. Histogram showing the hydrodynamic diameters of lipid-DOPE coliposomes at optimized
lipid/DOPE ratio and lipoplexes at optimized N/P ratio.
S4
25
Zeta potential (mV)
20
(A)
15
CholHG-1ox
CholHG-1ox:DOPE
CholHG-1ox:DOPE:FBS
10
5
0
-5
-10
-15
-20
-25
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
N/P charge ratio
25
Zeta potential (mV)
20
(B)
15
CholHG-3ox
CholHG-3ox:DOPE
CholHG-3ox:DOPE:FBS
10
5
0
-5
-10
-15
-20
-25
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
N/P charge ratio
Figure S3. Variation in the Zeta potential values on inclusion of different percentage of DOPE
and FBS in representative gemini lipid CholHG-1ox and Chol-3ox suspensions. Experiment was
performed using 4 µg of pEGFP-C3/mL of aqueous medium in which (A) CholHG-1ox, CholHG1ox:DOPE (1:4), CholHG-1ox:DOPE:FBS and (B) CholHG-3ox, CholHG-3ox:DOPE (1:4), CholHG3ox:DOPE:FBS were added gradually to vary the N/P charge ratio from 0.125 to 2.
S5
CholHG-1ox (-FBS-FBS)
% GFP cells 100
MFI
80
0
20
20
20
0
0
0
1:
0
Lipid:DOPE molar ratio
Lipid:DOPE molar ratio
CholHG-4ox (-FBS-FBS)
CholHG-3ox (-FBS-FBS)
100
40
20
0
80
80
20
20
20
0
0
0
100
80
0
1:
1:
40
40
20
20
20
0
0
0
1:
0
1:
4
Lipid:DOPE molar ratio
1:
4
60
1:
3
60
MFI
1:
3
20
1:
2
40
1:
1
40
1:
0
60
80
MFI
60
% GFP cells 100
MFI
F
80
% GFP Cells
80
% GFP Cells
CholHG-D (-FBS-FBS)
% GFP Cells 100
MFI
1:
2
E
1:
Lipid:DOPE molar ratio
CholG-D (-FBS-FBS)
100
1:
0
1:
Lipid:DOPE molar ratio
4
40
3
40
2
60
1
60
1:
4
1:
3
40
1:
2
60
1:
1
60
% GFP cells 100
MFI
D
MFI
80
% GFP cells
MFI
80
% GFP Cells
C
1:
0
100
MFI
% GFP Cells
100
1:
4
40
1:
3
40
1:
2
60
1:
1
60
1:
4
1:
3
20
1:
2
40
1:
1
40
1:
0
60
80
MFI
60
% GFP cells 100
MFI
B
80
MFI
% GFP Cells
80
100
1:
1
A
% GFP Cells
100
CholHG-2ox (-FBS-FBS)
Lipid:DOPE molar ratio
Figure S4. Lipid:DOPE molar ratio optimization for achieving highest transfection efficiency while
keeping N/P ratio fixed at 0.5 in absence of serum (-FBS-FBS). Formulations were screened for 5
different ratios from 1:0 to 1:4. (A) CholHG-1ox; (B) CholHG-2ox; (C) CholHG-3ox; (D) CholHG4ox; (E) CholG-D and (F) CholHG-D. Concentration of the DNA = 0.8 g/well. Data are expressed as
number of transfected cells and MFI as obtained from flow cytometry analysis.
S6
CholHG-1ox (-FBS+FBS)
100
0
0
0
0
0
100
% GFP cells 100
MFI
1:
0
0
0
1:
0
1:
4
0
1:
3
20
1:
2
20
1:
1
20
20
1:
3
% GFP Cells
40
40
MFI
40
40
MFI
60
60
1:
2
80
60
60
1:
1
% GFP cells 100
MFI
F
80
80
1:
0
1:
CholHG-D (-FBS+FBS)
CholG-D (-FBS+FBS)
% GFP Cells
1:
Lipid:DOPE molar ratio
Lipid:DOPE molar ratio
80
1:
0
1:
1:
4
1:
3
1:
2
0
4
20
3
20
2
20
1
40
20
1:
1
% GFP Cells
40
40
MFI
60
40
1:
0
80
60
60
E
% GFP Cells 100
MFI
D
80
80
MFI
% GFP Cells
100
% GFP Cells 100
MFI
60
100
1:
CholHG-4ox (-FBS+FBS)
CholHG-3ox (-FBS+FBS)
80
1:
1:
Lipid:DOPE molar ratio
Lipid:DOPE molar ratio
C
4
20
3
20
2
40
1
40
0
60
1:
4
1:
3
1:
2
1:
1
0
1:
0
0
60
1:
50
80
MFI
50
MFI
100
% GFP Cells
80
100
100
% GFP cells 100
MFI
B
1:
4
A
1:
% GFP cells 150
MFI
150
% GFP Cells
CholHG-2ox (-FBS+FBS)
Lipid:DOPE molar ratio
Lipid:DOPE molar ratio
Figure S5. Lipid:DOPE molar ratio optimization for highest transfection efficiency possible while N/P ratio
was 0.5 in presence of serum (-FBS+FBS). Formulations were screened for 5 different ratios from 1:0 to 1:4. (A)
CholHG-1ox; (B) CholHG-2ox; (C) CholHG-3ox; (D) CholHG-4ox; (E) CholG-D and (F) CholHG-D.
Concentration of the DNA = 0.8 g/well. Data are expressed as number of transfected cells and MFI as obtained
from flow cytometry analysis.
S7
CholHG-1ox (-FBS-FBS)
% GFP cells 100
MFI
80
0
0
80
60
60
40
40
20
20
0
0
N/P charge ratio
0
20
20
0
0
0
0.
25
N/P charge ratio
CholG-D (-FBS-FBS)
CholHG-D (-FBS-FBS)
% GFP Cells
100
MFI
20
0
80
20
20
20
0
0
0
0.
0.
0.
0.
12
5
1
N/P charge ratio
1
40
75
40
50
60
25
0
60
0.
0.
25
0.
12
0.
80
% GFP Cells
40
75
40
50
60
0
60
5
80
% GFP Cells 100
MFI
F
MFI
80
100
MFI
% GFP Cells
3
20
3
40
2
40
1
60
N/P charge ratio
E
2
80
60
3
0.
0.
% GFP Cells
20
2
40
1
40
5
60
25
60
% GFP Cells 100
MFI
D
80
MFI
% GFP Cells
80
100
MFI
80
100
1
CholHG-4ox (-FBS-FBS)
% GFP Cells 100
MFI
C
5
N/P charge ratio
CholHG-3ox (-FBS-FBS)
100
0.
25
0.
0.
80
3
20
2
20
1
40
0.
5
40
25
60
% GFP cells 100
MFI
B
MFI
60
MFI
% GFP Cells
80
100
0.
5
A
% GFP Cells
100
CholHG-2ox (-FBS-FBS)
N/P charge ratio
Figure S6. Optimization of N/P charge ratio to achieve highest transfection efficiency at the
optimized lipid: DOPE ratio in absence of serum (-FBS-FBS). Formulations were screened for
different N/P ratios from 0.125 to 3 to obtain maximum transfection efficiency. (A) CholHG-1ox, (B)
CholHG-2ox, (C) CholHG-3ox, (D) CholHG-4ox, (E) CholG-D and (F) CholHG-D. Concentration of
the DNA = 0.8 g/well. Data are expressed as number of transfected cells and MFI as obtained from the
flow cytometric analysis.
S8
CholHG-1ox (-FBS+FBS)
0
50
0
0
0.
25
2
1
N/P charge ratio
N/P charge ratio
CholHG-4ox (-FBS+FBS)
CholHG-3ox (-FBS+FBS)
150
0
0
50
0
0
0.
25
2
1
N/P charge ratio
N/P charge ratio
CholG-D (-FBS+FBS)
CholHG-D (-FBS+FBS)
% GFP Cells 150
MFI
100
50
50
0
0
100
100
50
50
0
0
N/P charge ratio
1
0.
75
0.
12
5
1
0.
75
0.
50
0.
25
0
0.
12
5
% GFP Cells 150
MFI
F
MFI
100
150
% GFP Cells
E
MFI
% GFP Cells
150
0.
50
0.
75
0.
5
0.
25
50
2
50
100
1
50
100
0.
75
100
% GFP cells 150
MFI
D
MFI
100
% GFP Cells
% GFP Cells 150
MFI
C
MFI
% GFP Cells
150
0.
5
0.
75
0.
5
0.
25
50
2
0
100
1
50
100
0.
75
50
% GFP Cells
100
% GFP Cells 150
MFI
B
MFI
100
150
0.
5
% GFP cells 150
MFI
0.
25
0
A
MFI
% GFP Cells
150
CholHG-2ox (-FBS+FBS)
N/P charge ratio
Figure S7. Optimization of the N/P charge ratio to achieve highest transfection efficiency.
Optimized lipid: DOPE ratios were used in serum (-FBS+FBS). Formulations were screened for
different N/P ratios from 0.125 to 3 to obtain maximum transfection efficiency. (A) CholHG-1ox, (B)
CholHG-2ox, (C) CholHG-3ox, (D) CholHG-4ox, (E) CholG-D and (F) CholHG-D. Concentration of
the DNA = 0.8 g/well. Data are expressed as number of transfected cells and MFI as obtained from the
flow cytometry analysis.
S9
S10
A
B
Figure S8. Flow cytometric scans showing comparative green fluorescence intensity due to all negative
control along with our lipoplexes (A) CholHG-1ox/pEGFP-C3 and (B) CholHG-3ox/pEGFP-C3 in 10%
serum condition (-FBS+FBS).
S11
150
% GFP cells 150
MFI
100
100
50
50
0
0
2
1.
6
1.
2
0.
8
0.
4
MFI
% GFP Cells
CholHG-1ox (-FBS+FBS)
DNA amount in g/well
Figure S9. Effect of variation in the amount of pEGFP-C3 plasmid DNA on gene transfection
efficiency. Experiment was performed on CholHG-1ox/DOPE (1:4 mole ratio) formulation at N/P
ratio of 0.5 CholHG-1ox/DNA.
S12
% GFP Cells
MFI
C
200
1
0.
75
ne
fe
ct
e
0
Ef
-3
lH
G
ho
20
N/P charge ratio
C
C
ho
lH
G
-1
ox
0
ox
0
40
5
50
10
60
0.
20
80
0.
25
100
CholHG-1ox
CholHG-3ox
CholHG-1ox/pEGFP-C3
CholHG-3ox/pEGFP-C3
Effectene/pEGFP-C3
100
5
150
30
% Cell Viability
40
120
MFI
% GFP Cells
50
0.
12
B
Formulations
Figure S10. pEGFP-C3 transfection in HEK 293T cells. (A) Comparative FACS histogram of GFP
expression in HEK 293T cell lines after performing CholHG-1ox, CholHG-3ox and Effectene mediated
transfection of pEGFP-C3 with various negative controls; (B) Bar diagram shows slightly better
transfection efficiency of CholHG-1ox formulations compare to Effectene in terms of MFI and (C) Cell
viability bar diagram of different formulations shows considerably high cell viability of HEK 293T cells
in transfection conditions.
S13
A
CholHG-1ox
D/L
DL/Dn
DLF
DLF/Dn
CholHG-3ox
DNA
D/L
DL/Dn
DLF
DLF/Dn
A1
A2
A3
B
CholHG-1ox
DNA
D/L
DL/Dn
DLB DLB/Dn
CholHG-3ox
DNA
D/L
DL/Dn DLB
DLB/Dn
B1
B2
B3
Figure S11. DNase sensitivity of DNA bound to various lipid formulations in presence of 10% FBS.
Experiment was performed with 10µg plasmid DNA per well. Lipid formulations were complexed with
plasmid DNA at N/P ratio 2 for 30 min followed by complexation with FBS/BSA 10% (v/v)/(w/w),
respectively. (A) DNase stability of lipid formulations in presence of 10% FBS. Stability of complexes
after incubation for 2h (A1), 4h (A2), and 6h (A3) at 37 oC using 0.25 unit of DNase I. (B) DNase
stability of lipid formulations in presence of 10% BSA. Stability of complexes after incubation for 2h
(B1), 4h (B2), and 6h (B3) at 37 oC using 0.25 unit of DNase I. Figure shows pure plasmid DNA lane
(DNA), DNA/lipid complex (D/L = 5), DNA/lipid complex incubated with DNaseI(DL/Dn), DNA/lipid
FBS complex (DLF), DNA/lipid FBS complex incubated with DNaseI (DLF/Dn), DNA/lipid BSA
complex (DLB), DNA/lipid BSA complex incubated with DNaseI (DLB/Dn).
S14
% Cell Viability
120
A
CholHG-1ox
CholHG-2ox
CholHG-3ox
CholHG-4ox
CholG-D
CholHG-D
100
80
60
40
20
00
6.
00
3.
25
2.
50
1.
0.
75
0
Lipid concentration (M)
% Cell Viability
120
B
CholHG-1ox/DNA
CholHG-2ox/DNA
CholHG-3ox/DNA
CholHG-4ox/DNA
CholG-D/DNA
CholHG-D/DNA
100
80
60
40
20
2.
00
1.
00
0.
75
0.
50
0.
25
0
N/P charge ratio
% Cell Viability
100
C
pEGFP-C3
Effectene
Effectene/pEGFP-C3
80
60
40
20
0
Formulations
Figure S12. MTT assay of different gemini lipids and their lipoplexes at various charge ratios along
with negative and positive controls, pEGFP-C 3 plasmid and Effectene, respectively. Histograms show
cytotoxicity of (A) liposomal suspensions; (B) lipoplexes (C) DNA alone, Effetene alone and its
complex with DNA. Experiments were performed in 10% FBS using 0.1 µg of pEGFP-C3 plasmid/well
in 96-well plates.
S15
-FBS+FBS (10%)
(A)
% Cell Proliferation
100
Liposome
Lipoplex
80
60
40
20
te
ne
Ef
fe
c
C
ho
l-M
G
-D
ho
lH
C
C
ho
lG
-D
ox
G
-4
ox
ho
lH
C
G
-3
ox
ho
lH
C
G
-2
ho
lH
C
C
ho
lH
G
-1
ox
0
Formulations
+ FBS+FBS(50%)
(B)
% Cell Proliferation
100
Liposome
Lipoplex
80
60
40
20
te
ne
Ef
fe
c
ho
l-M
C
-D
C
ho
lH
G
-D
ho
lG
C
-4
ox
ho
lH
G
-3
ox
C
C
ho
lH
G
-2
ox
G
ho
lH
C
C
ho
lH
G
-1
ox
0
Formulations
Figure S13. BrdU assay of HeLa cells treated with different liposomes and lipoplexes used for
transfection studies. (A) In presence of 10% serum (-FBS+FBS) optimized transfection formulations
did not show any significant reduction in cell proliferations while (B) in presence of 50% serum
(+FBS+FBS), considerable reduction in cell proliferation was noticed. Experiments were performed in
duplicate using 0.8 µg DNA/well in lipoplexes.
S16
ho
Fold Transfection Efficiency
lH
G
C
ho -1o
x
lH
G
C
ho 2o
x
lH
G
C
ho 3o
x
lH
G
-4
ox
C
ho
lG
C
ho -D
lH
G
-D
C
ho
Ef l-M
fe
ct
en
e
(A)
4
3
-FBS+FBS (50%)
+FBS+FBS (50%)
2
1
C
0
Formulations
(B)
(C)
Effectene
Chol-M
CholHG-D
CholG-D
CholHG-4 ox
CholHG-3 ox
CholHG-2 ox
CholHG-1 ox
Cells only
Effectene
Chol-M
CholHG-D
CholG-D
CholHG-4 ox
CholHG-3 ox
CholHG-2 ox
CholHG-1 ox
Cells only
Figure S14. Fold transfection efficiency of pEGFP-C3 transfected HeLa cells. Fluorescence was
observed by fluorescence microscopy and quantified using FACS analysis. (A) Fold transfection
efficiency; (B) FACS histogram obtained upon transfecting pEGFP-C3 in presence of 50 % FBS (FBS+FBS) and (C) 50 % FBS (+FBS+FBS).
S17
Figure S15. Confocal images of pEGFP-C3 transfected HeLa cells nuclear stained with PI. HeLa
cells transfected with (A) CholHG-1ox:DOPE (1:1) in absence of serum (-FBS-FBS); (B) CholHG1ox:DOPE (1:1) in 10% serum (-FBS+FBS); (C) Effectene (1:25) in absence of serum (-FBS-FBS); (D)
Effectene (1:25) in 10% (-FBS+FBS); (E) CholHG-1ox:DOPE (1:1) in 10% serum (-FBS+FBS); (F)
CholHG-1ox:DOPE (1:1) in 50% serum (-FBS+FBS); (G) CholHG-3ox:DOPE (1:1) in 10% serum (FBS+FBS) and (H) CholHG-3ox:DOPE (1:1) in 50% serum (-FBS+FBS).
S18
Confocal Image
GFP
Insert
δ+
H
δ+H
O δH
δO
δ+ H
pEGFP-c3
Plasmid
δ+
+
Hδ
OδH +
δ+H
δO
δ+
δ+ H
FACS Analysis
δO
O δH+
δ
H
δ+
.....
.....
O
O
O
5'
O
δO δ
O δ
O δ
O δ
O δ
H + + H H + +H H + + H H + +H H +
H
δ+ δ+
δ δ
δ δ
δ δ
δ
δ δ
O
O
O
O
P
O
O
O
O
P O
O-
O
-
O
O
O
P O
O
-
O
OO PO
O
O
O P O
O
5'
O
O
O
P O
O-
O
O
-
O
P O
O
O
3'
δ+ H O H δ+ δ+ H O H δ+ δ+H O H δ+ δ+ H O H δ+
δδδδ-
Enhanced Luciferase
Expression
N+
4000
3000
Lipoplex
2000
N+
-(CH2-CH2-O-CH2-CH2)n-
n=1
-CH2-CH2-O
-CH2-CH2-O
1000
0
FB
S]
H
H
H
H
[5
0%
FB
S]
H
C
ho
lH
G
-1
ox
[1
0%
FB
S]
C
ho
lH
Ef
f.
G
-1
ox
[5
0%
FB
S]
H
[1
0%
Ef
f.
Lucifearase activity/g protein
-
O
OP
O
FL1-H
5000
Spine of
Water molecules
H
.
H
δ+ δ+
.....
....
3'
-
.....
....
.
Oδ
H
....
......
....
.
H
.....
....
......
Events
δ+
G-Chol-H-1ox
δ
+
Hδ
H
Formulations
Figure S16. Abstract Figure. Cholesterol based gemini lipid CholHG-1ox possessing -CH2-CH2-OH
at the headgroups and one oxyethylene spacer is at least three times better transfecting agent in vitro
than one of the best-known commercially available transfecting agents, Effectene (Eff.), in presence of
high serum levels (50%).
S19