% relative ester content
100
80
60
40
20
0
0
1
2
3
4
5
6
7
t [d]
Figure S1:
Degradability of HD O (10 mg/mL) at 37°C in aqueous medium at pH 7.4
using 1H NMR spectroscopy to determine the decrease of ester bonds (n=2).
1
A)
100
% relative fluorescence
E
Sp
80
S
O
60
40
20
0
0
1
2
3
4
5
c/p - ratio
B)
% relative fluorescence
100
80
60
ED
BD
40
HD
20
0
0
Figure S2:
1
2
3
c/p - ratio
4
5
DNA binding ability of pseudodendrimers demonstrated by ethidium bromide
(EtBr) exclusion assay performed in HBG. A) Influence of the surface modification unit on
DNA binding ability as demonstrated in a representative experiment with the HD core. B)
Influence of the hydrocarbon spacer length in the pseudodendritic cores on DNA binding
ability as demonstrated in a representative experiment with the ethanolamine surface
modification.
2
RLU / 10 000 cells
1E+7
1E+5
100
% metabolic activity
c/p 0.5
c/p 0.8
c/p 1
c/p 2
c/p 4
A)
80
60
40
20
LPEI
BPEI
B)
c/p 0.5
c/p 0.8
c/p 1
c/p 2
c/p 4
RLU / 5 000 cells
1E+8
1E+6
1E+4
BPEI
LPEI
BPEI
80
60
40
20
LPEI
Figure S3:
LPEI
100
% metabolic acitivity
1E+3
BPEI
In vitro transfection efficiency (left, Luciferase assay) and metabolic activity
(right, MTT assay) of LPEI (22 kDa) and BPEI (25 kDa) in Neuro2a cells (A) and in B16F10
cells (B) in the presence of 10% serum. Polyplexes were formed at indicated c/p –
(conjugate/plasmid, w/w) ratios in HBG at a pDNA concentration of 20µg/mL and remained
for 4h on the cells. 24h after transfection, luciferase expression and metabolic activity of cells
was evaluated. Data were expressed as mean values (± SD) out of three independent
measurements each performed in triplicates. RLU / number of cells represents the measured
light units referred to the number of cells at the time point of cell plating. Two nanograms of
recombinant luciferase correspond to 107 light units.
3
A)
c/p 1
1E+8
c/p 2
1E+8
1E+6
1E+5
1E+4
1E+3
LPEI
N/P 6
BPEI
N/P 6
HD E
HD Sp HD S
c/p 4
c/p 2
100
c/p 4
% metabolic activity
lg RLU / 10 000 cells
c/p 1
80
60
40
20
0
HD O
LPEI
N/P 6
BPEI
N/P 6
HD E
HD Sp HD S
HD O
B)
1E+8
c/p 2
c/p 4
1E+7
1E+6
1E+5
1E+4
1E+3
LPEI
N/P 6
Figure S4:
BPEI
N/P 6
HD E
HD Sp
HD S
HD O
c/p 1
100
% metabolic activity
lg RLU / 5 000 cells
c/p 1
c/p 2
c/p 4
80
60
40
20
0
LPEI
N/P 6
BPEI
N/P 6
HD E
HD Sp
HD S
HD O
In vitro transfection efficiency (left, Luciferase assay) and metabolic activity
(right, MTT assay) of HD E, HD Sp, HD S and HDO. LPEI (22 kDa) and BPEI (25 kDa)
were taken as references at their optimized c/p-ratio of 0.8 (= nitrogen / phosphate ratio (N/P)ratio 6) in Neuro2a cells (A) and in B16F10 cells (B) in the presence of 10% serum.
Polyplexes were formed at indicated c/p – (conjugate/plasmid, w/w) ratios in HBG at a pDNA
concentration of 20µg/mL and remained for 4h on the cells. 24h after transfection, luciferase
expression and metabolic activity of cells was evaluated. Data were expressed as mean values
(± SD) out of three independent measurements each performed in triplicates. RLU / number
of cells represents the measured light units referred to the number of cells at the time point of
cell plating. Two nanograms of recombinant luciferase correspond to 107 light units.
4
A)
c/p 1
c/p 1
c/p 4
c/p 2
% metabolic activity
lg RLU / 10 000 cells
1E+6
1E+5
1E+4
1E+3
LPEI
N/P 6
BPEI
N/P6
BD E
BD Sp BD S
c/p 2
c/p 4
100
1E+7
80
60
40
20
0
BD O
LPEI
N/P6
BPEI
N/P 6
BD E
BD Sp BD S
BD O
B)
c/p 1
c/p 2
1E+7
1E+6
1E+5
1E+4
1E+3
Figure S5:
c/p 1
c/p 4
120
% metabolic activity
lg RLU / 5 000 cells
1E+8
LPEI
N/P 6
BPEI
N/P 6
100
80
60
40
20
0
BD E BD Sp BD S
BD O
c/p 4
c/p 2
LPEI
N/P 6
BPEI
N/P 6
BD E
BD Sp BD S
BD O
In vitro transfection efficiency (left, Luciferase assay) and metabolic activity
(right, MTT assay) of BD E, BD Sp, BD S and BDO. LPEI (22 kDa) and BPEI (25 kDa) were
taken as references at their optimized c/p-ratio of 0.8 (= nitrogen / phosphate ratio (N/P)-ratio
6) in Neuro2a cells (A) and in B16F10 cells (B) in the presence of 10% serum. Polyplexes
were formed at indicated c/p – (conjugate/plasmid, w/w) ratios in HBG at a pDNA
concentration of 20µg/mL and remained for 4h on the cells. 24h after transfection, luciferase
expression and metabolic activity of cells was evaluated. Data were expressed as mean values
(± SD) out of three independent measurements each performed in triplicates. RLU / number
of cells represents the measured light units referred to the number of cells at the time point of
cell plating. Two nanograms of recombinant luciferase correspond to 107 light units.
5
A)
c/p 1
c/p 2
c/p 4
1E+7
1E+6
1E+5
1E+4
1E+3
c/p 1
120
% metabolic activity
lg RLU / 10 000 cells
1E+8
LPEI
N/P 6
BPEI
N/P 6
ED E
ED Sp ED S
100
80
60
40
20
0
ED O
c/p 4
c/p 2
LPEI
N/P 6
BPEI
N/P 6
ED E ED Sp ED S
ED O
B)
c/p 1
c/p 2
c/p 4
1E+7
1E+6
1E+5
1E+4
1E+3
LPEI
N/P6
Figure S6:
BPEI
N/P 6
ED E
ED Sp ED S
ED O
c/p 1
120
% metabolic activity
lg RLU / 5 000 cells
1E+8
c/p 4
c/p 2
100
80
60
40
20
0
LPEI BPEI
N/P=6 N/P 6
ED E
ED Sp ED S
ED O
In vitro transfection efficiency (left, Luciferase assay) and metabolic activity
(right, MTT assay) of ED E, ED Sp, ED S and EDO. LPEI (22 kDa) and BPEI (25 kDa) were
taken as references at their optimized c/p-ratio of 0.8 (= nitrogen / phosphate ratio (N/P)-ratio
6) in Neuro2a cells (A) and in B16F10 cells (B) in the presence of 10% serum. Polyplexes
were formed at indicated c/p – (conjugate/plasmid, w/w) ratios in HBG at a pDNA
concentration of 20µg/mL and remained for 4h on the cells. 24h after transfection, luciferase
expression and metabolic activity of cells was evaluated. Data were expressed as mean values
(± SD) out of three independent measurements each performed in triplicates. RLU / number
of cells represents the measured light units referred to the number of cells at the time point of
cell plating. Two nanograms of recombinant luciferase correspond to 107 light units.
6
1E+10
lg RLU / mg protein
lg RLU / 10 000 cells
A) 1E+8
1E+7
1E+6
1E+5
1E+4
1E+8
1E+6
1E+4
1E+3
LPEI
BPEI
ED
BD
B)
HD
E
lg RLU / mg protein
lg RLU / 5 000 cells
S
BPEI
ED
BD
HD
O
1E+10
1E+8
1E+7
1E+6
1E+5
1E+4
1E+3
LPEI
Sp
LPEI
Figure S7:
BPEI
ED
BD
HD
1E+8
1E+6
1E+4
LPEI
BPEI
ED
BD
HD
In vitro transfection efficiency in Neuro2a cells (A) and B16F10 cells (B) of
ED core, BD core and HD core conjugates in the presence of 10% serum 24h post transfection
(left, luciferase assay correlated to the cell number at the time point of cell plating RLU / cell
number; right, luciferase assay correlated to protein content at the time point when luciferase
was determined RLU / mg protein). All polyplexes of pseudodendrimers were prepared at c/pratio of 2 in HBG. LPEI (22 kDa) and BPEI (25 kDa) were taken as references at their
optimized c/p-ratio of 0.8 (= nitrogen / phosphate ratio (N/P)-ratio 6) All polyplexes were
formed at a pDNA concentration of 20µg/mL and remained for 4h on the cells. 24h after
transfection, luciferase expression and protein content was evaluated. Data were expressed as
mean values (± SD) out of three independent measurements each performed in triplicates.
RLU / number of cells represents the measured light units referred to the number of cells at
the time point of cell plating.
7
1E+8
1E+10
1E+7
lg RLU / mg protein
lg RLU / 10 000 cells
A)
1E+8
1E+6
1E+6
1E+5
1E+4
1E+4
B)
c/p 1 c/p 2
c/p 4
S
HD
TE
T
HD A
PE
HI
HD
O
HD
O
12
HD
00
O
18
00
HD
c/p 8
1E+10
lg RLU / mg protein
lg RLU / 5 000 cells
1E+8
LP
EI
N
BP /P6
EI
N/
P6
HD
E
HD
ED
A
HD
Sp
LP
EI
N
BP /P6
EI
N/
P6
HD
HD E
ED
A
HD
Sp
HD
HD S
TE
T
HD A
PE
H
HD I
O
HD
O
12
HD
00
O
18
00
1E+3
1E+7
1E+8
1E+6
1E+6
1E+5
1E+4
Figure S8:
S
TE
HD TA
PE
HI
HD
HD O
O
1
HD 200
O
18
00
HD
HD
LP
EI
N
BP /P6
EI
N/
P6
HD
E
HD
ED
A
HD
Sp
1E+3
LP
EI
N
BP /P6
EI
N/
P6
HD
E
HD
ED
HD A
Sp
HD
HD S
TE
HD TA
PE
HI
HD
O
HD
O
12
HD
00
O
18
00
1E+4
In vitro transfection efficiency in Neuro2a cells (A) and B16F10 cells (B) of
HD core conjugates in the presence of 10% serum 24h post transfection (left, luciferase assay
correlated to the cell number at the time point of cell plating RLU / cell number; right,
luciferase assay correlated to protein content at the time point when luciferase was determined
RLU / mg protein). All polyplexes of pseudodendrimers were prepared at indicated c/p-ratios
in HBG. LPEI (22 kDa) and BPEI (25 kDa) were taken as references at their optimized c/pratio of 0.8 (= nitrogen / phosphate ratio (N/P)-ratio 6). All polyplexes were formed at a
pDNA concentration of 20µg/mL and remained for 4h on the cells. 24h after transfection,
luciferase expression and protein content was evaluated. Data were expressed as mean values
(± SD) out of three independent measurements each performed in triplicates.
8
A)
diacrylate /
OEI
HD E
surface
amine /
OEI
ester
[%]
yield
[%]
10
3
97
35
HD Sp
11.4
4.1
98
40
HD S
12.5
6.5
96
46
HD O
1.4 *
n.d .
87
40
BD E
8.6
2.1
94
32
BD Sp
8.1
1.8
88
24
BD S
9.6
3.1
94
38
BD O
1.3 *
n.d .
85
25
ED E
8.3
1.5
90
33
ED Sp
6.3
1.4
88
26
ED S
6.3
2
85
30
ED O
0.9 *
n.d .
71
30
B)
D2O
HD S
c
a
b
a´
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
PPM
Table S1 plus representative figure:
A) Composition of pseudodendrimers determined by 1H NMR spectroscopy in D2O.
* = ratio of diacrylate to total OEI (core plus surface OEI). Yields are given as weight of
product/weight of educts (w/w) percent.
B) 1H NMR of HD S as an example for the calculation of the diacrylate to OEI ratio (a/c), the
percentage of residual ester content after synthesis ( a/(a+a´) ) and the surface amine to OEI
ratio (b/c).
9
Vp [mL]
Mp
Mw
Mn
PDI
n.d.
n.d.
n.d.
n.d.
n.d.
HD Sp
11.5
1294
1410
1095
1.3
HD S
11.0
2264
2613
1729
1.5
HD O
10.8
3099
4138
2761
1.5
HD E
n.d.
n.d.
n.d.
n.d.
n.d.
BD Sp
11.3
1610
1989
1243
1.6
BD S
10.9
2850
5178
2163
2.4
BD O
10.8
3179
7905
3049
2.6
ED E
n.d.
n.d.
n.d.
n.d.
BD E
n.d.
ED Sp
11.0
2496
5289
1910
2.7
ED S
10.9
2763
6291
2221
2.9
ED O
10.5
4643
16207
5298
3.1
LPEI
8.3
60058
58499
26854
2.2
BPEI
8.8
35369
34823
9710
3.6
OEI
11.3
1628
1809
1581
1.1
Table S2:
Vp: elution volume at peak maximum; Mp: molecular weight at peak
maximum; Mw: weight average molecular weight; Mn: number average molecular weight;
PDI: polydispersity index; n.d.: not detectable. Molecular weights were measured relative to
Pullulan molecular weight standards which were used for preparing a standard calibration
curve.
10
Pseudodendrimer
mean diameter [nm]
zeta-potential [mV]
HD E
531
(± 62)
2.7
(± 1.5)
HD Sp
144
(± 17)
12.8
(± 3.7)
HD S
167
(± 38)
19.1
(± 3.6)
HD O
162
(± 22)
29.0
(± 2.0)
BD E
247
(±17)
6.8
(± 1.5)
BD Sp
277
(± 54)
18.5
(± 2.1)
BD S
209
(± 35)
24.2
(± 3.6)
BD O
244
(± 18)
25.1
(± 6.0)
ED E
894
(± 78)
- 8.8
(± 5.6)
ED Sp
549
(± 246)
1.8
(± 1.8)
ED S
252
(± 46)
9.9
(± 1.3)
ED O
181
(± 48)
24.0
(± 3.1)
Table S3:
Mean diameters (DLS) of pseudodendrimers at c/p-ratio of 2 at a final DNA
concentration of 10µg/mL were determined 20min after polyplex formation (n=3) in HBG.
For zeta-potential measurements polyplexes were diluted 1:5 to a final DNA concentration of
2µg/mL in 1mM NaCl (n=3).
11