How Does Spacer Length of Cationic Gemini Lipids Influence the ...
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How Does Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and Their Relevance in Gene Therapy
Mónica Muñoz-Úbeda,†,‡ Santosh K. Misra,§,‡ Ana L. Barrán-Berdón,† Sougata Datta,§ Clara Aicart-Ramos,Π Pablo Castro-Hartmann,⊥ Paturu Kondaiah,# Elena Junquera,† Santanu Bhattacharya,*,§ and Emilio Aicart*,† †
Grupo de Química Coloidal y Supramolecular, Departamento de Química Física I, and
Π
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
Departments of §Organic Chemistry, and #Molecular Reproduction Development and Genetics, Indian Institute of Science, 560012 Bangalore, India ⊥
Servei de Microscopia, Universitat Autónoma de Barcelona, 08193 Cerdanyola del Vallés, Spain
*Authors to whom the correspondence should be addressed: Santanu Bhattacharya: Fax: +918023600529. e-mail: [email protected] Emilio Aicart: Fax: +34913944135. e-mail: [email protected]
S1
Chemical shifts, δ, in ppm downfield from the internal standard (TMS) and pertinent details of the synthezised gemini lipids. Bis(hexadecyl dimethylammonium)ethane (C16C2C16). 1H-NMR (300 MHz, CDCl3)
δ 0.87 (t, 6H, alkyl chain 2 × CH3), 1.24-1.40 (br m, 52H, alkyl chain 2 × 13 CH2), 1.75 (br s, 4H, alkyl chain 2 × CH2CH2N+), 2.64 (br s, 2H, spacer chain 1 × CH2CH2N+), 3.51 (s, 12H, 2 × 2 N+CH3), 3.68 - 3.72 (m, 4H, alkyl chain 2 × CH2N+), 4.81 (s, 4H, spacer chain 2 × CH2N+). Anal. Calcd. for C38H82N2Br2, 2.0 H2O: C, 59.83; H, 11.36; N, 3.67. Found C, 60.12; H, 11.48; N, 3.43. Bis(hexadecyl dimethylammonium)propane (C16C3C16). 1H NMR (200 MHz, CDCl3)
δ 0.87 (t, 6 H, alkyl chain 2 × CH3), 1.24-1.40 (br m, 52H, alkyl chain 2 × 13 CH2), 1.75 (br s, 4 H, alkyl chain 2 × CH2CH2N+), 2.64 (br s, 2H, spacer chain 1 × 1 CH2CH2N+), 3.36 (s, 12 H, 2 × 2 N+CH3), 3.46 (m, 4H, alkyl chain 2 × CH2N+), 3.77 (m, 4 H, spacer chain 2 × CH2N+). Anal. Calcd. for C39H84N2Br2, 2.0 H2O: C, 60.29; H, 11.42; N, 3.60. Found C, 60.02; H, 11.38; N, 3.33. Bis(hexadecyl dimethylammonium)pentane (C16C5C16). 1H NMR (200 MHz, CDCl3)
δ 0.88 (t, 6 H, alkyl chain 2 × CH3), 1.15-1.45 (br m, 42H, alkyl chain 2 × 10 CH2 and spacer chain CH2), 1.68 (crude t, 16H, alkyl chain 2 × 4 CH2), 2.02-2.20 (br m, 4H, spacer chain 1 × 2 CH2CH2N+), 3.33 (s, 12H, 2 × 2 N+CH3), 3.45 (crude t, 4H, alkyl chain 2 × CH2N+), 3.90 (crude t, 4H, spacer chain 1 × 2 CH2N+). Anal. Calcd. for C41H88N2Br2: C, 64.04; H, 11.54; N, 3.64. Found C, 64.23; H, 11.72; N, 3.47. Bis(hexadecyl dimethylammonium)dodecane (C16C12C16). 1H NMR (200 MHz, CDCl3) δ 0.88 (t, 6H, alkyl chain 2 × CH3), 1.25- 1.38 (s + br m, 68H, alkyl chain 2 × 13 CH2 and spacer chain 1 × 8 CH2), 1.65-1.85 (br m, 8H, spacer chain 1 × 2 CH2CH2N+ and alkyl chain 2 × CH2CH2N+), 3.38 (s, 12H, 2 × 2 N+CH3), 3.44-3.54 (m, 4H, alkyl chain 2 × CH2N+), 3.64-3.74 (m, 4H, spacer chain 1 × 2 CH2N+). Anal. Calcd.
S2
for C48H102N2Br2, 0.5H2O: C, 65.80; H, 11.85; N, 3.20. Found C, 66.09; H, 12.09; N, 3.05.
ζ / mV
40
0
α= α= α= α=
-40
0
4
8
0.2 0.3 0.5 0.8 12
L/D
Figure S-1. Plots of zeta potential, ζ, against the lipoplex composition (L/D) of C16C3C16/DOPE-pDNA at different CL composition, α, in HEPES buffer at 298.15 K, pH = 7.4. Solid line: sigmoidal fit of experimental values. Errors are within ± 5%, [DNA] = 0.05 mg/mL and [L] was varied, depending on the CL composition, to cover a wide L/D range.
S3
(b) 100
(200)
(c)
(1 0 0 ) 100
(D N A ) (2 0 0 )
10
α = 0 .2 5 α = 0 .3 0 α = 0 .4 0
α = 0.20 α = 0.25 α = 0.30 α = 0.40
1
2
3
-1
(f) 100
(300) 10
100
(200) (300)
α = 0.15 α = 0.20 α = 0.40
α = 0.30 α = 0.40
α = 0.15 α = 0.20 α = 0.25 α = 0.30
α = 0.50 α = 0.60 α = 0.80
α = 0.50 α = 0.60 α = 0.80 3
(DNA)
(300)
α = 0.15 α = 0.20 α = 0.25
-1
(100)
I / a.u.
(100) (DNA) (200)
I / a.u.
100
3
q (nm )
-1
q (n m )
(e)
q (nm )
2
1
2
-1
I / a.u.
3
(100) (DNA) (200)
2
α = 0.60 α = 0.80 1
(d)
1
α = 0.30 α = 0.40 α = 0.50
α = 0 .5 0 α = 0 .6 0 α = 0 .8 0
α = 0.50 α = 0.60 α = 0.80 -1
α = 0.15 α = 0.20 α = 0.25
10
α = 0 .1 5 α = 0 .2 0
10
2
(200 ) (300 )
α = 0.15
q (nm )
100
(D NA)
(3 0 0 ) (300)
1
(10 0)
I / a.u.
(DNA)
I / a.u.
(100)
I / a.u.
1000
(a)
3
q (nm )
α = 0.40 α = 0.50 α = 0.60 α = 0.80 1
2
-1
q (nm )
3
Figure S-2. SAXS diffractograms of the C16C3C16/DOPE-pDNA lipoplexes at several CL compositions, α, and effective charge ratios (ρeff): (a) ρeff = 2.2, (b) ρeff = 4.4, (c) ρeff = 6.5, (d) ρeff = 8.7, (e) ρeff = 10.9 and (f) ρeff = 13.1.
S4
1000
(100) (DNA) (200)
(DNA)
I / a.u.
100
(c)
(100) 100
(200)
(100) (DNA) (200)
100
(300)
(300)
10
(300)
α = 0.20
α = 0.25 α = 0.30 α = 0.40
α = 0.50 α = 0.60 α = 0.80 2
α = 0.30 α = 0.40 α = 0.50
α = 0.50 α = 0.60 α = 0.80 1
3
2
3
-1
q (nm )
-1
α = 0.60 α = 0.80 1
2
3 -1
q (nm )
q (nm )
(e)
(DNA)
100
(200)
(100) 100
(DNA)
I / a.u.
(100)
(200)
I / a.u.
(d)
(f) (100)
100
(DNA)
I / a.u.
1
α = 0.15 α = 0.20 α = 0.25
10
α = 0.15 α = 0.20
α = 0.15 α = 0.25 α = 0.30 α = 0.40
I / a.u.
(b)
I / a.u.
(a)
(200)
(300)
(300)
(300)
10
α = 0.15 α = 0.20
10
α = 0.15 α = 0.20 α = 0.25
10
α = 0.15 α = 0.20
α = 0.25 α = 0.30 α = 0.40 α = 0.50
α = 0.30 α = 0.40 α = 0.50
α = 0.25 α = 0.30 α = 0.40 α = 0.50 α = 0.60 α = 0.80
α = 0.60 α = 0.80
α = 0.60 α = 0.80
1
1
2
-1
q (nm )
3
1
2
3
2
-1
q (nm )
3
-1
q (nm )
Figure S-3. SAXS diffractograms of the C16C5C16/DOPE-pDNA lipoplexes at several CL compositions, α, and effective charge ratios, ρeff: (a) ρeff = 2.3, (b) ρeff = 4.7, (c)
ρeff = 7.0, (d) ρeff = 9.3, (e) ρeff = 11.6 and (f) ρeff = 14.0.
S5
(c)
(100)
(100) 100
(DNA)
(200)
(DNA) (200) (300)
10
10
α = 0.15 α = 0.20
10
α = 0.15
α = 0.15
α = 0.25 α = 0.30 α = 0.40
α = 0.20 α = 0.25 α = 0.30
α = 0.20 α = 0.30 α = 0.40
α = 0.40 α = 0.50 α = 0.60
α = 0.50 α = 0.60 α = 0.80
α = 0.50 α = 0.60 α = 0.80
α = 0.80 2
-1
3
1
q (nm )
(d)
2
1
3
-1
2
q (nm )
(e) 100
(DNA) (200)
(f)
(100)
I / a.u.
(100)
(DNA)
100
(200)
(300)
(DNA) (200) (300)
α = 0.15
10
α = 0.15
α = 0.20
α = 0.25
α = 0.20
α = 0.25
α = 0.30 α = 0.40 α = 0.50
α = 0.25 α = 0.40 α = 0.50
α = 0.30 α = 0.40
α = 0.60 α = 0.80
α = 0.50
α = 0.60 α = 0.80
α = 0.60 1
2
3 -1
q (nm )
100
(100)
(300) α = 0.15 α = 0.20
1
3 -1
q (nm )
I / a.u.
1
100
(300)
(300)
1
2
-1
I / a.u.
100
(200)
I / a.u.
(DNA)
1000
(b)
I / a.u.
1000
(100)
I / a.u.
(a)
3
q (nm )
2
3 -1
q (nm )
Figure S-4. SAXS diffractograms of the C16C12C16/DOPE-pDNA lipoplexes at several CL compositions, α, and effective charge ratios, ρeff: (a) ρeff = 1.5, (b) ρeff = 2.9, (c) ρeff = 4.4, (d) ρeff = 5.8, (e) ρeff = 7.3 and (f) ρeff = 8.7.
S6
Table S-1. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C2C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C2C16/DOPE-pDNA α
0.15
ρeff
DOPE rich
2.0
4.0
6.0
8.0
10.0
12.0
q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
0.68 9.24
0.54 11.6
0.61 10.3
0.63 9.97
0.65 9.67
0.20
Main
DOPE rich
Main
0.94 6.68 1.25 5.03 0.94 6.68 1.27 4.95 0.94 6.68 1.28 4.91 0.94 6.68 1.53 4.11 1.06 5.93 1.50 4.19 0.96 6.54 1.48 4.25
0.62 10.13
0.94 6.68 1.31 4.80 0.94 6.68 1.34 4.69 0.95 6.61 1.35 4.65 0.95 6.61 1.33 4.72 0.96 6.54 1.50 4.19 0.95 6.61 1.52 4.13
0.69 9.11
0.57 11.0
0.63 9.97
0.65 9.67
0.66 9.52
0.25 DOPE rich
0.66 9.52
0.65 9.67
0.69 9.11
0.66 9.52
0.60 10.47
0.68 9.24
Main
0.95 6.61 1.34 4.69 0.97 6.48 1.48 4.25 0.97 6.48 1.44 4.36 0.95 6.61 1.44 4.36 0.97 6.48 1.42 4.42 0.97 6.48 1.53 4.11
0.30 DOPE rich
0.67 9.38
0.7 8.98
0.59 10.7
0.63 9.97
0.61 10.30
0.69 9.11
0.40
Main
DOPE rich
Main
0.97 6.48 1.46 4.30 0.97 6.48 1.53 4.11 0.98 6.41 1.53 4.11 0.97 6.48 1.55 4.05 0.97 6.48 1.47 4.27 0.98 6.41 1.62 3.88
0.62 10.13
1.00 6.28 1.48 4.25 0.99 6.35 1.6 3.93 1.00 6.28 1.54 4.08 1.01 6.22 1.56 4.03 1.00 6.28 1.56 4.03 0.98 6.41 1.51 4.16
0.75 8.38
0.59 10.7
0.59 10.7
0.63 9.97
0.65 9.67
0.50 CL rich
DOPE rich
0.63 9.97
0.74 8.49
0.62 10.1
1.33 4.72
0.60 10.5
1.26 4.99
0.65 9.67
0.67 9.38
Main
1.01 6.22 1.54 4.08 1.01 6.22 1.66 3.79 1.01 6.22 1.65 3.81 1.01 6.22 1.53 4.11 1.01 6.22 1.60 3.93 1.00 6.28 1.53 4.11
0.60 CL rich
DOPE rich
0.63 9.97
075 8.38
0.65 9.67
1.31 4.80
0.56 11.2
1.31 4.80
0.66 9.52
0.64 9.82
0.80
Main
CL rich
1.03 6.10 1.59 3.95 1.04 6.04 1.75 3.59 1.02 6.16 1.65 3.81 1.03 6.10 1.58 3.98 1.03 6.10 1.69 3.72 1.01 6.22 1.63 3.85
1.30 4.83
1.22 5.15
1.24 5.07
1.31 4.80
1.32 4.76
Main
1.05 5.98 1.71 3.67 1.04 6.04 1.73 3.63 1.04 6.04 1.68 3.74 1.03 6.10 1.68 3.74 1.04 6.04 1.62 3.88 1.04 6.04 1.6 3.93
Rich gemini
1.23 5.11
1.23 5.11
1.22 5.15
1.31 4.80
1.22 5.15
1.24 5.07
d values in nm; q values in nm-1
S7
Table S-2. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C3C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C3C16/DOPE-pDNA
α 0.15 DOPE rich
ρeff
0.20 Main
0.93 6.76 2.2 1.25 5.03 0.66 0.94 9.52 6.68 4.4 1.27 4.95 0.54 0.94 11.6 6.68 6.5 1.24 5.07 0.63 0.94 9.97 6.68 8.7 1.26 4.99 0.67 0.94 9.38 6.68 10.9 1.31 4.80 0.57 0.95 11.0 6.61 13.1 1.31 4.80 d values in nm; q values in nm-1 q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
DOPE rich
0.65 9.67
0.55 11.4
0.65 9.67
0.68 9.24
0.63 9.97
0.25 Main
0.94 6.68 1.28 4.91 0.94 6.68 1.36 4.62 0.95 6.61 1.31 4.80 0.94 6.68 1.43 4.39 0.97 6.48 1.42 4.42 0.96 6.54 1.36 4.62
DOPE rich
0.63 9.97
0.60 10.5
0.59 10.7
0.74 8.49
0.65 9.67
0.30 Main
0.95 6.61 1.40 4.49 0.95 6.61 1.45 4.33 0.97 6.48 1.39 4.52 0.96 6.54 1.42 4.42 0.97 6.48 1.43 4.39
DOPE rich
0.63 9.97
0.69 9.11
0.77 8.16
0.66 9.52
0.66 9.52
0.40 Main
0.96 6.54 1.49 4.22 0.97 6.48 1.56 4.03 0.97 6.48 1.48 4.25 0.97 6.48 1.48 4.25 0.98 6.41 1.45 4.33
DOPE rich
0.63 9.97
0.69 9.11
0.77 8.16
0.74 8.49
0.68 9.24
0.50 Main
0.98 6.41 1.57 4.00 0.98 6.41 1.50 4.19 0.98 6.41 1.52 4.13 0.98 6.41 1.46 4.30 0.99 6.35 1.51 4.16 0.99 6.35 1.48 4.25
DOPE rich
0.62 10.1
0.77 8.16
0.68 9.24
0.60
0.80
Main
Main
Main
CL rich
1.01 6.22 1.54 4.08 1.00 6.28 1.49 4.22 1.02 6.16 1.60 3.93 1,00 6.28 1.49 4.22 0.98 6.41 1.51 4.16 1.01 6.22 1.52 4.13
1.01 6.22 1.55 4.05 1.02 6.16 1.57 4.00 1.03 6.10 1.63 3.85 1.01 6.22 1.48 4.25 0.98 6.41 1.51 4.16 1.01 6.22 1.5 4.19
1.04 6.04 1.56 4.03 1.03 6.10 1.62 3.88 1.02 6.16 1.65 3.81 1.01 6.22 1.68 3.74 0.99 6.35 1.50 4.19 1.02 6.16 1.69 3.72
1.25 5.03
1.22 5.15
1.23 5.11
S8
Table S-3. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C5C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C5C16/DOPE-pDNA α
0.15
2.3
4.7
7.0
9.3
11.6
14.0
0.20
DOPE rich
ρeff q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
0.59 10.7
0.63 9.97
0.62 10.1
0.62 10.1
0.65 9.67
Main
0.92 6.83 1.29 4.87 0.92 6.83 1.32 4.76 0.92 6.83 1.33 4.72 0.93 6.76 1.31 4.80 0.93 6.76 1.45 4.33 0.94 6.68 1.30 4.83
DOPE rich
0.62 10.1
0.64 9.82
0.25 Main
0.93 6.76 1.30 4.83 0.94 6.68 1.36 4.62 0.94 6.68 1.29 4.87 0.94 6.68 1.37 4.59 0.95 6.61 1.39 4.52 0.95 0.61 1.30 4.83
DOPE rich
0.62 10.1
0.71 8.85
0.30 Main
0.94 6.68 1.32 4.76 0.95 6.61 1.37 4.59 0.95 6.61 1.33 4.72 0.95 6.61 1.36 4.62 0.97 6.48 1.42 4.42 0.96 6.54 1.33 4.72
DOPE rich
0.66 9.52
0.74 8.49
0.40 Main
0.96 6.54 1.49 4.22 0.97 6.48 1.45 4.33 0.96 6.54 1.37 4.59 0.96 6.54 1.37 4.59 0.98 6.41 1.59 3.95 0.97 6.48 1.34 4.69
DOPE rich
0.73 8.61
0.74 8.49
0.50 Main
Main
0.99 6.35 1.53 4.11 1.00 6.28 1.52 4.13 1.00 6.28 1.56 4.03 0.99 6.35 1.49 4.22 1.00 6.28 1.61 3.90 0.99 6.35 1.45 4.33
1.01 6.22 1.57 4.00 1.01 6.22 1.57 4.00 1.00 6.28 1.54 4.08 0.99 6.35 1.56 4.03 1.06 5.93 1.67 3.76 1.01 6.22 1.48 4.25
0.60 CL rich
1.19 5.28
1.21 5.19
1.24 5.07
1.22 5.15
Main
1.01 6.22 1.79 3.51 1.03 6.10 1.74 3.61 1.00 6.28 1.60 3.93 1.00 6.28 1.59 3.95 1.08 5.82 1.51 4.16 1.01 6.22 1.60 3.93
0.80 CL rich
1.20 5.24
1.19 5.28
1.27 4.95
1.22 5.15
1.21 5.19
1.21 5.19
Main
1.02 6.16 1.83 3.43 1.04 6.04 1.75 3.59 1.04 6.04 1.77 3.55 0.99 6.35 1.69 3.72 0.92 6.83 1.78 3.53 1.01 6.22 1.79 3.51
CL rich
1.21 5.19
1.21 5.19
1.20 5.24
1.21 5.19
1.23 5.11
1.22 5.15
d values in nm; q values in nm-1
S9
Table S-4. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C12C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C12C16/DOPE-pDNA α
0.15 DOPE rich
ρeff
0.20 Main
0.94 6.68 1.5 1.25 5.03 0.95 6.61 2.9 1.44 4.36 0.65 0.94 9.67 6.68 4.4 1.33 4.72 0.71 0.94 8.85 6.68 5.8 1.33 4.72 0.67 0.95 9.38 6.61 7.3 1.36 4.62 0.97 6.48 8.7 1.30 4.83 d values in nm; q values in nm-1 q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
DOPE rich
0.68 9.24
0.75 8.38
0.69 9.11
0.65 9.67
0.25 Main
0.96 6.54 1.34 4.69 0.97 6.48 1.53 4.11 0.97 6.48 1.34 4.69 0.96 6.54 1.33 4.72 0.97 6.48 1.45 4.33 1.01 6.22 1.42 4.42
DOPE rich
0.68 9.24
0.73 8.61
0.67 9.38
0.30 Main
0.97 6.48 1.39 4.52 0.98 6.41 1.39 4.52 0.97 6.48 1.34 4.69 0.97 6.48 1.49 4.22 1.03 6.10 1.48 4.25
DOPE rich
0.67 9.38
0.40 Main
0.99 6.35 1.51 4.16 0.99 6.35 1.44 4.36 0.98 6.41 1.40 4.49 0.98 6.41 1.38 4.55 0.98 6.41 1.49 4.22 -
DOPE rich
0.68 9.24
0.50 Main
1.03 6.10 1.69 3.72 1.00 6.28 1.6 3.93 1.03 6.10 1.48 4.25 1.02 6.16 1.42 4.42 1.03 6.10 1.49 4.22 1.02 6.16 1.52 4.13
DOPE rich
0.69 9.11
Main
1.04 6.04 1.57 4.00 1.04 6.04 1.63 3.85 1.04 6.04 1.53 4.11 1.03 6.10 1.47 4.27 1.04 6.04 1.52 4.13 1.04 6.04 1.56 4.03
0.60 CL rich
1.32 4.76
1.23 5.11
Main
1.06 5.93 1.64 3.83 1.05 5.98 1.64 3.83 1.04 6.04 1.64 3.83 1.02 6.16 1.54 4.08 1.04 6.04 1.53 4.11 1.05 5.98 1.57 4.00
0.80 CL rich
1.21 5.19
1.25 5.03
Main
1.08 5.82 1.92 3.27 1.12 5.61 1.93 3.26 0.98 6.41 1.64 3.83 1.04 6.04 1.77 3.55 1.03 6.10 1.62 3.88
CL rich
1.27 4.95
1.21 5.19
1.26 4.99
1.20 5.24
S10
8
d / nm
18
a
d / nm
6
12
0.2
0.4
0.6
0.8
α
6
0.2
0.4
0.6
0.8
α
8
b
d / nm
d / nm
18
6
12 0.2
0.4
0.6
0.8
α
6
0.2
0.4
0.6
0.8
α
c
8
d / nm
d / nm
18
6
12 0.2
0.4
0.6
0.8
α
6
0.2
0.4
0.6
0.8
α
Figure S-5. Plots of the periodic distance of the lamellar structure, d, as a function of CL composition, α, and at several effective charge ratios, ρeff, for C16CnC16/DOPEpDNA lipoplexes. Red symbols, Lα, DOPE rich structure; black symbols, Lα, main structure; and blue symbols, Lα, CL rich structure. The insets are zoom views of the Lα main structures. (a) n = 3, ρeff is: up triangles, 2.2; diamonds, 4.4; down triangles, 6.5; circles, 8.7; squares, 10.9; and lateral triangles, 13.1 (b) n = 5, ρeff is: up triangles, 2.3; diamonds, 4.7; down triangles, 7.0; circles, 9.3; squares, 11.6; and lateral triangles, 14.0; and (c) n = 12, ρeff is: up triangles, 1.5; diamonds, 2.9; down triangles, 4.4; circles, 5.8; squares, 7.3; and lateral triangles, 8.7. S11
8 ρeff = 2.2
a
ρeff = 4.4 ρeff = 6.5 ρeff = 8.7
dpDNA / nm
ρeff = 10.9 ρeff = 13.1
4
0.2
0.4
0.6
0.8
α
8 ρeff = 2.3
b
ρeff = 4.7 ρeff = 7 ρeff = 9.3
dpDNA / nm
ρeff = 11.6 ρeff = 14
4
0.2
0.4
0.6
0.8
α
8 ρeff = 1.5 ρeff = 2.9
c
ρeff = 4.4 ρeff = 5.8
dpDNA / nm
ρeff = 7.3 ρeff = 8.7
4
0.2
0.4
0.6
0.8
α
Figure S-6. Plots of the distance dpDNA as a function of CL composition, α, at several effective charge ratios, ρeff, for C16CnC16/DOPE-pDNA lipoplexes: (a) n = 3; (b) n = 5; and (c) n = 12.
S12
150
150
150
150
% GFP cells MFI
% GFP cells MFI
50
50
0 0.53
ρeff
150
50
50
0 0.1 0.2
2
1.1
100 Lipo2000
0
0 0.07 0.130.26
100
MFI
Lipo2000
% GFP cells
α = 0.25 100
MFI
% GFP cells
α = 0.15 100
150
0.4
200
Lipo2000
50
0
0 2
ρeff
4
100
100
Lipo2000
MFI
50
% GFP cells
α = 0.80 100
MFI
% GFP cells
α = 0.50 100
1
200
% GFP cells MFI
% GFP cells MFI
0.25 0.5
1.8
0.9
ρeff
0
0 0.4 0.7
1.4
2.8
5.7
ρ eff
Figure S-7. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C2C16/DOPE-pDNA in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum condition (-FBS-FBS). Also shown is the transfection by Lipofectamine2000, as a control.
S13
150
α = 0.15
50
0
% GFP cells
50
Lipo2000
50
50
0
0 1.1
0.6
100
MFI
MFI
Lipo2000
α = 0.25
100
100
0.07 0.14 0.3
150 % GFP cells MFI
100
% GFP cells
150
150 % GFP cells MFI
0 0.1 0.2
2
0.5
ρeff
1.0 ρ eff
150
150
150
150 % GFP cells MFI
% GFP cells MFI
α = 0.80
α = 0.50 100
50
50
0
0 1.0
1.9 ρeff
3.8
100
MFI
Lipo2000
% GFP cells
100
MFI
% GFP cells
100
0.2 0.5
1.9
Lipo2000
50
50
0
0 0.4 0.8
1.5
3.1 ρeff
6.1
Figure S-8. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C3C16/DOPE-pDNA lipoplexes in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum (-FBS-FBS). Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control.
S14
150
150 % GFP cells MFI
% GFP cells MFI
α = 0.15 100
50
0
% GFP cells
50
0 0.1 0.2 0.3
0.6
1.2
100 MFI
Lipo2000
Lipo2000
0
2
0.1 0.3
0.5
ρeff
150
α = 0.50
50
0
0 2
4
% GFP cells
50
α = 0.80 100 MFI
Lipo2000
ρeff
150
100
MFI
% GFP cells
100
1
2
150 % GFP cells MFI
100
0.3 0.5
0 1 ρeff
150 % GFP cells MFI
α = 0.25
100
MFI
% GFP cells
100
Lipo2000
50
50
0
0 0.4 0.8 1.6
3.2
6.3
ρeff
Figure S-9. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C5C16/DOPE-pDNA lipoplexes in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum (-FBS-FBS). Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control.
S15
150
α = 0.25
Lipo2000
50
50
0 0.4
0.7 ρeff
1.2
2
150
α = 0.50
150
0
0 2.3
% GFP cells
50
α = 0.80 100
MFI
50
1.2
2
100
MFI
Lipo2000
ρeff
1.2
150
100
0.6
0.6
% GFP cells MFI
100
0.2 0.3
0 0.1 0.2 0.3
ρeff
150 % GFP cells MFI
100 Lipo2000
0
0 0.05 0.1 0.2
100
MFI
% GFP cells
100
MFI
% GFP cells
200 % GFP cells MFI
100
% GFP cells
200
150
α = 0.15
% GFP cells MFI
Lipo2000
50
50
0
0 0.2 0.5
0.9
1.8 ρeff
3.6
Figure S-10. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C12C16/DOPE-pDNA lipoplexes in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum (-FBS-FBS). Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control.
S16
Table S-5. Variation in the Transfection Efficiency of Different Gemini CLs as a Function of Spacer Lengths in Absence (-FBS-FBS) and Presence of Serum (-FBS+FBS)
CL
C16C2C16
C16C3C16
C16C5C16
C16C12C16
-FBS-FBS
-FBS+FBS
-FBS-FBS
-FBS+FBS
-FBS-FBS
-FBS+FBS
-FBS-FBS
-FBS+FBS
% GFP Cells
50
80
40
50
35
12
30
15
MFI
40
275
70
50
90
20
175
25
S17
100
0 ρeff
1
2
0 0.50 ρeff
0 0.50
1 ρeff
2
4
4
CHO cells
% GFP cells
Lipo2000
50
50
0
MFI
0
2
100
H460 cells 50
1
100
MFI
% GFP cells
50
% GFP cells MFI
Lipo2000
0.25
Lipo2000
0.25
4
100 % GFP cells MFI
50
0
0 0.50
100
50
% GFP cells
50
HeLa cells
MFI
% GFP cells
U343 cells
Lipo2000
0.25
100 % GFP cells MFI
MFI
50
100
100 % GFP cells MFI
0 0.25
0.50
1
2
4
ρeff
Figure S-11. Transfection efficiency (% GFP cells and MFI) of C16C2C16/DOPE– pEGFP-C3 plasmid DNA lipoplexes at CL composition α = 0.5 for four cell lines. Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control. Experiments were performed in presence of 10% serum (-FBS+FBS).
S18
200
a
α = 0.15 α = 0.25 α = 0.50
150
α = 0.80
% cell viability
Lipofectamine2000
100
50
0 2.2
4.4
17.4
8.7 ρeff
200 α = 0.15
b
α = 0.25 α = 0.50 α = 0.80
150
% cell viability
Lipofectamine2000
100
50
0 2.3
9.3
4.7
18.6
ρeff
α = 0.15
c
α = 0.25
200
α = 0.50 α = 0.80
% cell viability
Lipofectamine2000
150
100
50
0 1.5
5.8
2.9
11.6
ρeff
Figure S-12. Cytotoxicity assay (MTT) as a function of ρeff, at several CL compositions, α, for C16CnC16/DOPE-pDNA: (a) n = 3; (b) n = 5; and (c) n = 12. Also shown is the cytotoxicity by Lipofectamine2000, as a control.
S19
a
b
20 µm
20 µm
c
d
20 µm
20 µm
Propidium Iodide Green Fluorescence Protein Overlap of PI and GFP
Figure S-13. Confocal Fluorescence Microscopy of C16CnC16/DOPE–pDNA (n = 2, 3, 5 and 12) transfected HEK293T cells, at α = 0.5 and ρeff = 2, in absence of serum (-FBSFBS). Images show the GFP expression in cells after being transfected with (a) C16C2C16/DOPE–pDNA; (b) C16C3C16/DOPE–pDNA; (c) C16C5C16/DOPE–pDNA and (d) C16C12C16/DOPE–pDNA. .
S20
How Does Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and Their Relevance in Gene Therapy
Mónica Muñoz-Úbeda,†,‡ Santosh K. Misra,§,‡ Ana L. Barrán-Berdón,† Sougata Datta,§ Clara Aicart-Ramos,Π Pablo Castro-Hartmann,⊥ Paturu Kondaiah,# Elena Junquera,† Santanu Bhattacharya,*,§ and Emilio Aicart*,† †
Grupo de Química Coloidal y Supramolecular, Departamento de Química Física I, and
Π
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
Departments of §Organic Chemistry, and #Molecular Reproduction Development and Genetics, Indian Institute of Science, 560012 Bangalore, India ⊥
Servei de Microscopia, Universitat Autónoma de Barcelona, 08193 Cerdanyola del Vallés, Spain
*Authors to whom the correspondence should be addressed: Santanu Bhattacharya: Fax: +918023600529. e-mail: [email protected] Emilio Aicart: Fax: +34913944135. e-mail: [email protected]
S1
Chemical shifts, δ, in ppm downfield from the internal standard (TMS) and pertinent details of the synthezised gemini lipids. Bis(hexadecyl dimethylammonium)ethane (C16C2C16). 1H-NMR (300 MHz, CDCl3)
δ 0.87 (t, 6H, alkyl chain 2 × CH3), 1.24-1.40 (br m, 52H, alkyl chain 2 × 13 CH2), 1.75 (br s, 4H, alkyl chain 2 × CH2CH2N+), 2.64 (br s, 2H, spacer chain 1 × CH2CH2N+), 3.51 (s, 12H, 2 × 2 N+CH3), 3.68 - 3.72 (m, 4H, alkyl chain 2 × CH2N+), 4.81 (s, 4H, spacer chain 2 × CH2N+). Anal. Calcd. for C38H82N2Br2, 2.0 H2O: C, 59.83; H, 11.36; N, 3.67. Found C, 60.12; H, 11.48; N, 3.43. Bis(hexadecyl dimethylammonium)propane (C16C3C16). 1H NMR (200 MHz, CDCl3)
δ 0.87 (t, 6 H, alkyl chain 2 × CH3), 1.24-1.40 (br m, 52H, alkyl chain 2 × 13 CH2), 1.75 (br s, 4 H, alkyl chain 2 × CH2CH2N+), 2.64 (br s, 2H, spacer chain 1 × 1 CH2CH2N+), 3.36 (s, 12 H, 2 × 2 N+CH3), 3.46 (m, 4H, alkyl chain 2 × CH2N+), 3.77 (m, 4 H, spacer chain 2 × CH2N+). Anal. Calcd. for C39H84N2Br2, 2.0 H2O: C, 60.29; H, 11.42; N, 3.60. Found C, 60.02; H, 11.38; N, 3.33. Bis(hexadecyl dimethylammonium)pentane (C16C5C16). 1H NMR (200 MHz, CDCl3)
δ 0.88 (t, 6 H, alkyl chain 2 × CH3), 1.15-1.45 (br m, 42H, alkyl chain 2 × 10 CH2 and spacer chain CH2), 1.68 (crude t, 16H, alkyl chain 2 × 4 CH2), 2.02-2.20 (br m, 4H, spacer chain 1 × 2 CH2CH2N+), 3.33 (s, 12H, 2 × 2 N+CH3), 3.45 (crude t, 4H, alkyl chain 2 × CH2N+), 3.90 (crude t, 4H, spacer chain 1 × 2 CH2N+). Anal. Calcd. for C41H88N2Br2: C, 64.04; H, 11.54; N, 3.64. Found C, 64.23; H, 11.72; N, 3.47. Bis(hexadecyl dimethylammonium)dodecane (C16C12C16). 1H NMR (200 MHz, CDCl3) δ 0.88 (t, 6H, alkyl chain 2 × CH3), 1.25- 1.38 (s + br m, 68H, alkyl chain 2 × 13 CH2 and spacer chain 1 × 8 CH2), 1.65-1.85 (br m, 8H, spacer chain 1 × 2 CH2CH2N+ and alkyl chain 2 × CH2CH2N+), 3.38 (s, 12H, 2 × 2 N+CH3), 3.44-3.54 (m, 4H, alkyl chain 2 × CH2N+), 3.64-3.74 (m, 4H, spacer chain 1 × 2 CH2N+). Anal. Calcd.
S2
for C48H102N2Br2, 0.5H2O: C, 65.80; H, 11.85; N, 3.20. Found C, 66.09; H, 12.09; N, 3.05.
ζ / mV
40
0
α= α= α= α=
-40
0
4
8
0.2 0.3 0.5 0.8 12
L/D
Figure S-1. Plots of zeta potential, ζ, against the lipoplex composition (L/D) of C16C3C16/DOPE-pDNA at different CL composition, α, in HEPES buffer at 298.15 K, pH = 7.4. Solid line: sigmoidal fit of experimental values. Errors are within ± 5%, [DNA] = 0.05 mg/mL and [L] was varied, depending on the CL composition, to cover a wide L/D range.
S3
(b) 100
(200)
(c)
(1 0 0 ) 100
(D N A ) (2 0 0 )
10
α = 0 .2 5 α = 0 .3 0 α = 0 .4 0
α = 0.20 α = 0.25 α = 0.30 α = 0.40
1
2
3
-1
(f) 100
(300) 10
100
(200) (300)
α = 0.15 α = 0.20 α = 0.40
α = 0.30 α = 0.40
α = 0.15 α = 0.20 α = 0.25 α = 0.30
α = 0.50 α = 0.60 α = 0.80
α = 0.50 α = 0.60 α = 0.80 3
(DNA)
(300)
α = 0.15 α = 0.20 α = 0.25
-1
(100)
I / a.u.
(100) (DNA) (200)
I / a.u.
100
3
q (nm )
-1
q (n m )
(e)
q (nm )
2
1
2
-1
I / a.u.
3
(100) (DNA) (200)
2
α = 0.60 α = 0.80 1
(d)
1
α = 0.30 α = 0.40 α = 0.50
α = 0 .5 0 α = 0 .6 0 α = 0 .8 0
α = 0.50 α = 0.60 α = 0.80 -1
α = 0.15 α = 0.20 α = 0.25
10
α = 0 .1 5 α = 0 .2 0
10
2
(200 ) (300 )
α = 0.15
q (nm )
100
(D NA)
(3 0 0 ) (300)
1
(10 0)
I / a.u.
(DNA)
I / a.u.
(100)
I / a.u.
1000
(a)
3
q (nm )
α = 0.40 α = 0.50 α = 0.60 α = 0.80 1
2
-1
q (nm )
3
Figure S-2. SAXS diffractograms of the C16C3C16/DOPE-pDNA lipoplexes at several CL compositions, α, and effective charge ratios (ρeff): (a) ρeff = 2.2, (b) ρeff = 4.4, (c) ρeff = 6.5, (d) ρeff = 8.7, (e) ρeff = 10.9 and (f) ρeff = 13.1.
S4
1000
(100) (DNA) (200)
(DNA)
I / a.u.
100
(c)
(100) 100
(200)
(100) (DNA) (200)
100
(300)
(300)
10
(300)
α = 0.20
α = 0.25 α = 0.30 α = 0.40
α = 0.50 α = 0.60 α = 0.80 2
α = 0.30 α = 0.40 α = 0.50
α = 0.50 α = 0.60 α = 0.80 1
3
2
3
-1
q (nm )
-1
α = 0.60 α = 0.80 1
2
3 -1
q (nm )
q (nm )
(e)
(DNA)
100
(200)
(100) 100
(DNA)
I / a.u.
(100)
(200)
I / a.u.
(d)
(f) (100)
100
(DNA)
I / a.u.
1
α = 0.15 α = 0.20 α = 0.25
10
α = 0.15 α = 0.20
α = 0.15 α = 0.25 α = 0.30 α = 0.40
I / a.u.
(b)
I / a.u.
(a)
(200)
(300)
(300)
(300)
10
α = 0.15 α = 0.20
10
α = 0.15 α = 0.20 α = 0.25
10
α = 0.15 α = 0.20
α = 0.25 α = 0.30 α = 0.40 α = 0.50
α = 0.30 α = 0.40 α = 0.50
α = 0.25 α = 0.30 α = 0.40 α = 0.50 α = 0.60 α = 0.80
α = 0.60 α = 0.80
α = 0.60 α = 0.80
1
1
2
-1
q (nm )
3
1
2
3
2
-1
q (nm )
3
-1
q (nm )
Figure S-3. SAXS diffractograms of the C16C5C16/DOPE-pDNA lipoplexes at several CL compositions, α, and effective charge ratios, ρeff: (a) ρeff = 2.3, (b) ρeff = 4.7, (c)
ρeff = 7.0, (d) ρeff = 9.3, (e) ρeff = 11.6 and (f) ρeff = 14.0.
S5
(c)
(100)
(100) 100
(DNA)
(200)
(DNA) (200) (300)
10
10
α = 0.15 α = 0.20
10
α = 0.15
α = 0.15
α = 0.25 α = 0.30 α = 0.40
α = 0.20 α = 0.25 α = 0.30
α = 0.20 α = 0.30 α = 0.40
α = 0.40 α = 0.50 α = 0.60
α = 0.50 α = 0.60 α = 0.80
α = 0.50 α = 0.60 α = 0.80
α = 0.80 2
-1
3
1
q (nm )
(d)
2
1
3
-1
2
q (nm )
(e) 100
(DNA) (200)
(f)
(100)
I / a.u.
(100)
(DNA)
100
(200)
(300)
(DNA) (200) (300)
α = 0.15
10
α = 0.15
α = 0.20
α = 0.25
α = 0.20
α = 0.25
α = 0.30 α = 0.40 α = 0.50
α = 0.25 α = 0.40 α = 0.50
α = 0.30 α = 0.40
α = 0.60 α = 0.80
α = 0.50
α = 0.60 α = 0.80
α = 0.60 1
2
3 -1
q (nm )
100
(100)
(300) α = 0.15 α = 0.20
1
3 -1
q (nm )
I / a.u.
1
100
(300)
(300)
1
2
-1
I / a.u.
100
(200)
I / a.u.
(DNA)
1000
(b)
I / a.u.
1000
(100)
I / a.u.
(a)
3
q (nm )
2
3 -1
q (nm )
Figure S-4. SAXS diffractograms of the C16C12C16/DOPE-pDNA lipoplexes at several CL compositions, α, and effective charge ratios, ρeff: (a) ρeff = 1.5, (b) ρeff = 2.9, (c) ρeff = 4.4, (d) ρeff = 5.8, (e) ρeff = 7.3 and (f) ρeff = 8.7.
S6
Table S-1. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C2C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C2C16/DOPE-pDNA α
0.15
ρeff
DOPE rich
2.0
4.0
6.0
8.0
10.0
12.0
q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
0.68 9.24
0.54 11.6
0.61 10.3
0.63 9.97
0.65 9.67
0.20
Main
DOPE rich
Main
0.94 6.68 1.25 5.03 0.94 6.68 1.27 4.95 0.94 6.68 1.28 4.91 0.94 6.68 1.53 4.11 1.06 5.93 1.50 4.19 0.96 6.54 1.48 4.25
0.62 10.13
0.94 6.68 1.31 4.80 0.94 6.68 1.34 4.69 0.95 6.61 1.35 4.65 0.95 6.61 1.33 4.72 0.96 6.54 1.50 4.19 0.95 6.61 1.52 4.13
0.69 9.11
0.57 11.0
0.63 9.97
0.65 9.67
0.66 9.52
0.25 DOPE rich
0.66 9.52
0.65 9.67
0.69 9.11
0.66 9.52
0.60 10.47
0.68 9.24
Main
0.95 6.61 1.34 4.69 0.97 6.48 1.48 4.25 0.97 6.48 1.44 4.36 0.95 6.61 1.44 4.36 0.97 6.48 1.42 4.42 0.97 6.48 1.53 4.11
0.30 DOPE rich
0.67 9.38
0.7 8.98
0.59 10.7
0.63 9.97
0.61 10.30
0.69 9.11
0.40
Main
DOPE rich
Main
0.97 6.48 1.46 4.30 0.97 6.48 1.53 4.11 0.98 6.41 1.53 4.11 0.97 6.48 1.55 4.05 0.97 6.48 1.47 4.27 0.98 6.41 1.62 3.88
0.62 10.13
1.00 6.28 1.48 4.25 0.99 6.35 1.6 3.93 1.00 6.28 1.54 4.08 1.01 6.22 1.56 4.03 1.00 6.28 1.56 4.03 0.98 6.41 1.51 4.16
0.75 8.38
0.59 10.7
0.59 10.7
0.63 9.97
0.65 9.67
0.50 CL rich
DOPE rich
0.63 9.97
0.74 8.49
0.62 10.1
1.33 4.72
0.60 10.5
1.26 4.99
0.65 9.67
0.67 9.38
Main
1.01 6.22 1.54 4.08 1.01 6.22 1.66 3.79 1.01 6.22 1.65 3.81 1.01 6.22 1.53 4.11 1.01 6.22 1.60 3.93 1.00 6.28 1.53 4.11
0.60 CL rich
DOPE rich
0.63 9.97
075 8.38
0.65 9.67
1.31 4.80
0.56 11.2
1.31 4.80
0.66 9.52
0.64 9.82
0.80
Main
CL rich
1.03 6.10 1.59 3.95 1.04 6.04 1.75 3.59 1.02 6.16 1.65 3.81 1.03 6.10 1.58 3.98 1.03 6.10 1.69 3.72 1.01 6.22 1.63 3.85
1.30 4.83
1.22 5.15
1.24 5.07
1.31 4.80
1.32 4.76
Main
1.05 5.98 1.71 3.67 1.04 6.04 1.73 3.63 1.04 6.04 1.68 3.74 1.03 6.10 1.68 3.74 1.04 6.04 1.62 3.88 1.04 6.04 1.6 3.93
Rich gemini
1.23 5.11
1.23 5.11
1.22 5.15
1.31 4.80
1.22 5.15
1.24 5.07
d values in nm; q values in nm-1
S7
Table S-2. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C3C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C3C16/DOPE-pDNA
α 0.15 DOPE rich
ρeff
0.20 Main
0.93 6.76 2.2 1.25 5.03 0.66 0.94 9.52 6.68 4.4 1.27 4.95 0.54 0.94 11.6 6.68 6.5 1.24 5.07 0.63 0.94 9.97 6.68 8.7 1.26 4.99 0.67 0.94 9.38 6.68 10.9 1.31 4.80 0.57 0.95 11.0 6.61 13.1 1.31 4.80 d values in nm; q values in nm-1 q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
DOPE rich
0.65 9.67
0.55 11.4
0.65 9.67
0.68 9.24
0.63 9.97
0.25 Main
0.94 6.68 1.28 4.91 0.94 6.68 1.36 4.62 0.95 6.61 1.31 4.80 0.94 6.68 1.43 4.39 0.97 6.48 1.42 4.42 0.96 6.54 1.36 4.62
DOPE rich
0.63 9.97
0.60 10.5
0.59 10.7
0.74 8.49
0.65 9.67
0.30 Main
0.95 6.61 1.40 4.49 0.95 6.61 1.45 4.33 0.97 6.48 1.39 4.52 0.96 6.54 1.42 4.42 0.97 6.48 1.43 4.39
DOPE rich
0.63 9.97
0.69 9.11
0.77 8.16
0.66 9.52
0.66 9.52
0.40 Main
0.96 6.54 1.49 4.22 0.97 6.48 1.56 4.03 0.97 6.48 1.48 4.25 0.97 6.48 1.48 4.25 0.98 6.41 1.45 4.33
DOPE rich
0.63 9.97
0.69 9.11
0.77 8.16
0.74 8.49
0.68 9.24
0.50 Main
0.98 6.41 1.57 4.00 0.98 6.41 1.50 4.19 0.98 6.41 1.52 4.13 0.98 6.41 1.46 4.30 0.99 6.35 1.51 4.16 0.99 6.35 1.48 4.25
DOPE rich
0.62 10.1
0.77 8.16
0.68 9.24
0.60
0.80
Main
Main
Main
CL rich
1.01 6.22 1.54 4.08 1.00 6.28 1.49 4.22 1.02 6.16 1.60 3.93 1,00 6.28 1.49 4.22 0.98 6.41 1.51 4.16 1.01 6.22 1.52 4.13
1.01 6.22 1.55 4.05 1.02 6.16 1.57 4.00 1.03 6.10 1.63 3.85 1.01 6.22 1.48 4.25 0.98 6.41 1.51 4.16 1.01 6.22 1.5 4.19
1.04 6.04 1.56 4.03 1.03 6.10 1.62 3.88 1.02 6.16 1.65 3.81 1.01 6.22 1.68 3.74 0.99 6.35 1.50 4.19 1.02 6.16 1.69 3.72
1.25 5.03
1.22 5.15
1.23 5.11
S8
Table S-3. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C5C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C5C16/DOPE-pDNA α
0.15
2.3
4.7
7.0
9.3
11.6
14.0
0.20
DOPE rich
ρeff q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
0.59 10.7
0.63 9.97
0.62 10.1
0.62 10.1
0.65 9.67
Main
0.92 6.83 1.29 4.87 0.92 6.83 1.32 4.76 0.92 6.83 1.33 4.72 0.93 6.76 1.31 4.80 0.93 6.76 1.45 4.33 0.94 6.68 1.30 4.83
DOPE rich
0.62 10.1
0.64 9.82
0.25 Main
0.93 6.76 1.30 4.83 0.94 6.68 1.36 4.62 0.94 6.68 1.29 4.87 0.94 6.68 1.37 4.59 0.95 6.61 1.39 4.52 0.95 0.61 1.30 4.83
DOPE rich
0.62 10.1
0.71 8.85
0.30 Main
0.94 6.68 1.32 4.76 0.95 6.61 1.37 4.59 0.95 6.61 1.33 4.72 0.95 6.61 1.36 4.62 0.97 6.48 1.42 4.42 0.96 6.54 1.33 4.72
DOPE rich
0.66 9.52
0.74 8.49
0.40 Main
0.96 6.54 1.49 4.22 0.97 6.48 1.45 4.33 0.96 6.54 1.37 4.59 0.96 6.54 1.37 4.59 0.98 6.41 1.59 3.95 0.97 6.48 1.34 4.69
DOPE rich
0.73 8.61
0.74 8.49
0.50 Main
Main
0.99 6.35 1.53 4.11 1.00 6.28 1.52 4.13 1.00 6.28 1.56 4.03 0.99 6.35 1.49 4.22 1.00 6.28 1.61 3.90 0.99 6.35 1.45 4.33
1.01 6.22 1.57 4.00 1.01 6.22 1.57 4.00 1.00 6.28 1.54 4.08 0.99 6.35 1.56 4.03 1.06 5.93 1.67 3.76 1.01 6.22 1.48 4.25
0.60 CL rich
1.19 5.28
1.21 5.19
1.24 5.07
1.22 5.15
Main
1.01 6.22 1.79 3.51 1.03 6.10 1.74 3.61 1.00 6.28 1.60 3.93 1.00 6.28 1.59 3.95 1.08 5.82 1.51 4.16 1.01 6.22 1.60 3.93
0.80 CL rich
1.20 5.24
1.19 5.28
1.27 4.95
1.22 5.15
1.21 5.19
1.21 5.19
Main
1.02 6.16 1.83 3.43 1.04 6.04 1.75 3.59 1.04 6.04 1.77 3.55 0.99 6.35 1.69 3.72 0.92 6.83 1.78 3.53 1.01 6.22 1.79 3.51
CL rich
1.21 5.19
1.21 5.19
1.20 5.24
1.21 5.19
1.23 5.11
1.22 5.15
d values in nm; q values in nm-1
S9
Table S-4. Values of q100, d100, qpDNA and dpDNA of the Lα Lamellar Structures Found for C16C12C16/DOPE-pDNA at Several CL Compositions, α, of the Mixed Lipid and at Several Effective Charge Ratios, ρeff C16C12C16/DOPE-pDNA α
0.15 DOPE rich
ρeff
0.20 Main
0.94 6.68 1.5 1.25 5.03 0.95 6.61 2.9 1.44 4.36 0.65 0.94 9.67 6.68 4.4 1.33 4.72 0.71 0.94 8.85 6.68 5.8 1.33 4.72 0.67 0.95 9.38 6.61 7.3 1.36 4.62 0.97 6.48 8.7 1.30 4.83 d values in nm; q values in nm-1 q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA q100 d100 qpDNA dpDNA
DOPE rich
0.68 9.24
0.75 8.38
0.69 9.11
0.65 9.67
0.25 Main
0.96 6.54 1.34 4.69 0.97 6.48 1.53 4.11 0.97 6.48 1.34 4.69 0.96 6.54 1.33 4.72 0.97 6.48 1.45 4.33 1.01 6.22 1.42 4.42
DOPE rich
0.68 9.24
0.73 8.61
0.67 9.38
0.30 Main
0.97 6.48 1.39 4.52 0.98 6.41 1.39 4.52 0.97 6.48 1.34 4.69 0.97 6.48 1.49 4.22 1.03 6.10 1.48 4.25
DOPE rich
0.67 9.38
0.40 Main
0.99 6.35 1.51 4.16 0.99 6.35 1.44 4.36 0.98 6.41 1.40 4.49 0.98 6.41 1.38 4.55 0.98 6.41 1.49 4.22 -
DOPE rich
0.68 9.24
0.50 Main
1.03 6.10 1.69 3.72 1.00 6.28 1.6 3.93 1.03 6.10 1.48 4.25 1.02 6.16 1.42 4.42 1.03 6.10 1.49 4.22 1.02 6.16 1.52 4.13
DOPE rich
0.69 9.11
Main
1.04 6.04 1.57 4.00 1.04 6.04 1.63 3.85 1.04 6.04 1.53 4.11 1.03 6.10 1.47 4.27 1.04 6.04 1.52 4.13 1.04 6.04 1.56 4.03
0.60 CL rich
1.32 4.76
1.23 5.11
Main
1.06 5.93 1.64 3.83 1.05 5.98 1.64 3.83 1.04 6.04 1.64 3.83 1.02 6.16 1.54 4.08 1.04 6.04 1.53 4.11 1.05 5.98 1.57 4.00
0.80 CL rich
1.21 5.19
1.25 5.03
Main
1.08 5.82 1.92 3.27 1.12 5.61 1.93 3.26 0.98 6.41 1.64 3.83 1.04 6.04 1.77 3.55 1.03 6.10 1.62 3.88
CL rich
1.27 4.95
1.21 5.19
1.26 4.99
1.20 5.24
S10
8
d / nm
18
a
d / nm
6
12
0.2
0.4
0.6
0.8
α
6
0.2
0.4
0.6
0.8
α
8
b
d / nm
d / nm
18
6
12 0.2
0.4
0.6
0.8
α
6
0.2
0.4
0.6
0.8
α
c
8
d / nm
d / nm
18
6
12 0.2
0.4
0.6
0.8
α
6
0.2
0.4
0.6
0.8
α
Figure S-5. Plots of the periodic distance of the lamellar structure, d, as a function of CL composition, α, and at several effective charge ratios, ρeff, for C16CnC16/DOPEpDNA lipoplexes. Red symbols, Lα, DOPE rich structure; black symbols, Lα, main structure; and blue symbols, Lα, CL rich structure. The insets are zoom views of the Lα main structures. (a) n = 3, ρeff is: up triangles, 2.2; diamonds, 4.4; down triangles, 6.5; circles, 8.7; squares, 10.9; and lateral triangles, 13.1 (b) n = 5, ρeff is: up triangles, 2.3; diamonds, 4.7; down triangles, 7.0; circles, 9.3; squares, 11.6; and lateral triangles, 14.0; and (c) n = 12, ρeff is: up triangles, 1.5; diamonds, 2.9; down triangles, 4.4; circles, 5.8; squares, 7.3; and lateral triangles, 8.7. S11
8 ρeff = 2.2
a
ρeff = 4.4 ρeff = 6.5 ρeff = 8.7
dpDNA / nm
ρeff = 10.9 ρeff = 13.1
4
0.2
0.4
0.6
0.8
α
8 ρeff = 2.3
b
ρeff = 4.7 ρeff = 7 ρeff = 9.3
dpDNA / nm
ρeff = 11.6 ρeff = 14
4
0.2
0.4
0.6
0.8
α
8 ρeff = 1.5 ρeff = 2.9
c
ρeff = 4.4 ρeff = 5.8
dpDNA / nm
ρeff = 7.3 ρeff = 8.7
4
0.2
0.4
0.6
0.8
α
Figure S-6. Plots of the distance dpDNA as a function of CL composition, α, at several effective charge ratios, ρeff, for C16CnC16/DOPE-pDNA lipoplexes: (a) n = 3; (b) n = 5; and (c) n = 12.
S12
150
150
150
150
% GFP cells MFI
% GFP cells MFI
50
50
0 0.53
ρeff
150
50
50
0 0.1 0.2
2
1.1
100 Lipo2000
0
0 0.07 0.130.26
100
MFI
Lipo2000
% GFP cells
α = 0.25 100
MFI
% GFP cells
α = 0.15 100
150
0.4
200
Lipo2000
50
0
0 2
ρeff
4
100
100
Lipo2000
MFI
50
% GFP cells
α = 0.80 100
MFI
% GFP cells
α = 0.50 100
1
200
% GFP cells MFI
% GFP cells MFI
0.25 0.5
1.8
0.9
ρeff
0
0 0.4 0.7
1.4
2.8
5.7
ρ eff
Figure S-7. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C2C16/DOPE-pDNA in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum condition (-FBS-FBS). Also shown is the transfection by Lipofectamine2000, as a control.
S13
150
α = 0.15
50
0
% GFP cells
50
Lipo2000
50
50
0
0 1.1
0.6
100
MFI
MFI
Lipo2000
α = 0.25
100
100
0.07 0.14 0.3
150 % GFP cells MFI
100
% GFP cells
150
150 % GFP cells MFI
0 0.1 0.2
2
0.5
ρeff
1.0 ρ eff
150
150
150
150 % GFP cells MFI
% GFP cells MFI
α = 0.80
α = 0.50 100
50
50
0
0 1.0
1.9 ρeff
3.8
100
MFI
Lipo2000
% GFP cells
100
MFI
% GFP cells
100
0.2 0.5
1.9
Lipo2000
50
50
0
0 0.4 0.8
1.5
3.1 ρeff
6.1
Figure S-8. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C3C16/DOPE-pDNA lipoplexes in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum (-FBS-FBS). Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control.
S14
150
150 % GFP cells MFI
% GFP cells MFI
α = 0.15 100
50
0
% GFP cells
50
0 0.1 0.2 0.3
0.6
1.2
100 MFI
Lipo2000
Lipo2000
0
2
0.1 0.3
0.5
ρeff
150
α = 0.50
50
0
0 2
4
% GFP cells
50
α = 0.80 100 MFI
Lipo2000
ρeff
150
100
MFI
% GFP cells
100
1
2
150 % GFP cells MFI
100
0.3 0.5
0 1 ρeff
150 % GFP cells MFI
α = 0.25
100
MFI
% GFP cells
100
Lipo2000
50
50
0
0 0.4 0.8 1.6
3.2
6.3
ρeff
Figure S-9. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C5C16/DOPE-pDNA lipoplexes in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum (-FBS-FBS). Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control.
S15
150
α = 0.25
Lipo2000
50
50
0 0.4
0.7 ρeff
1.2
2
150
α = 0.50
150
0
0 2.3
% GFP cells
50
α = 0.80 100
MFI
50
1.2
2
100
MFI
Lipo2000
ρeff
1.2
150
100
0.6
0.6
% GFP cells MFI
100
0.2 0.3
0 0.1 0.2 0.3
ρeff
150 % GFP cells MFI
100 Lipo2000
0
0 0.05 0.1 0.2
100
MFI
% GFP cells
100
MFI
% GFP cells
200 % GFP cells MFI
100
% GFP cells
200
150
α = 0.15
% GFP cells MFI
Lipo2000
50
50
0
0 0.2 0.5
0.9
1.8 ρeff
3.6
Figure S-10. Transfection efficiency (% GFP cells and MFI) of pEGFP-C3 plasmid DNA using C16C12C16/DOPE-pDNA lipoplexes in HEK293T cells against ρeff, at several CL compositions, α, in absence of serum (-FBS-FBS). Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control.
S16
Table S-5. Variation in the Transfection Efficiency of Different Gemini CLs as a Function of Spacer Lengths in Absence (-FBS-FBS) and Presence of Serum (-FBS+FBS)
CL
C16C2C16
C16C3C16
C16C5C16
C16C12C16
-FBS-FBS
-FBS+FBS
-FBS-FBS
-FBS+FBS
-FBS-FBS
-FBS+FBS
-FBS-FBS
-FBS+FBS
% GFP Cells
50
80
40
50
35
12
30
15
MFI
40
275
70
50
90
20
175
25
S17
100
0 ρeff
1
2
0 0.50 ρeff
0 0.50
1 ρeff
2
4
4
CHO cells
% GFP cells
Lipo2000
50
50
0
MFI
0
2
100
H460 cells 50
1
100
MFI
% GFP cells
50
% GFP cells MFI
Lipo2000
0.25
Lipo2000
0.25
4
100 % GFP cells MFI
50
0
0 0.50
100
50
% GFP cells
50
HeLa cells
MFI
% GFP cells
U343 cells
Lipo2000
0.25
100 % GFP cells MFI
MFI
50
100
100 % GFP cells MFI
0 0.25
0.50
1
2
4
ρeff
Figure S-11. Transfection efficiency (% GFP cells and MFI) of C16C2C16/DOPE– pEGFP-C3 plasmid DNA lipoplexes at CL composition α = 0.5 for four cell lines. Also included are the results for Lipofectamine2000–pEGFP-C3 plasmid DNA complexes, as a control. Experiments were performed in presence of 10% serum (-FBS+FBS).
S18
200
a
α = 0.15 α = 0.25 α = 0.50
150
α = 0.80
% cell viability
Lipofectamine2000
100
50
0 2.2
4.4
17.4
8.7 ρeff
200 α = 0.15
b
α = 0.25 α = 0.50 α = 0.80
150
% cell viability
Lipofectamine2000
100
50
0 2.3
9.3
4.7
18.6
ρeff
α = 0.15
c
α = 0.25
200
α = 0.50 α = 0.80
% cell viability
Lipofectamine2000
150
100
50
0 1.5
5.8
2.9
11.6
ρeff
Figure S-12. Cytotoxicity assay (MTT) as a function of ρeff, at several CL compositions, α, for C16CnC16/DOPE-pDNA: (a) n = 3; (b) n = 5; and (c) n = 12. Also shown is the cytotoxicity by Lipofectamine2000, as a control.
S19
a
b
20 µm
20 µm
c
d
20 µm
20 µm
Propidium Iodide Green Fluorescence Protein Overlap of PI and GFP
Figure S-13. Confocal Fluorescence Microscopy of C16CnC16/DOPE–pDNA (n = 2, 3, 5 and 12) transfected HEK293T cells, at α = 0.5 and ρeff = 2, in absence of serum (-FBSFBS). Images show the GFP expression in cells after being transfected with (a) C16C2C16/DOPE–pDNA; (b) C16C3C16/DOPE–pDNA; (c) C16C5C16/DOPE–pDNA and (d) C16C12C16/DOPE–pDNA. .
S20
