Submitted to
Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2012.
Supporting Information
for Adv. Healthcare Mater., DOI: 10.1002/adhm.201200478
Arsonium-containing Lipophosphoramides, Poly-functional Nano-carriers
for Simultaneous Antibacterial Action and Eukaryotic Cell Transfection
Tony Le Gall,* Mathieu Berchel, Sophie Le Hir, Aurore Fraix, Jean Yves Salaün, Claude
Férec, Pierre Lehn, Paul-Alain Jaffrès,* and Tristan Montier*
Summary
SI1. Detailed structure of the cationic lipids .............................................................................. 2
SI2. X-ray crystal structure of compound 6 ............................................................................... 3
SI3. Physicochemical characteristics of liposomal solutions ..................................................... 4
SI4. Antibiotic resistance profile of the bacterial strains ........................................................... 5
SI5. Drug resistance study .......................................................................................................... 6
SI6. Stability of a naked DNA under sterile or infected conditions ........................................... 7
SI7. Antibacterial activity and DNA compaction and protection .............................................. 8
SI8. Sequential assay: antibacterial effect then in vitro transfection activity ............................ 9
SI9. Adenylate kinase measurements for monitoring bacterial growth ................................... 10
SI10. Simultaneous assay: antibacterial effect and transfection activity in one pot ................ 11
SI11. References ....................................................................................................................... 12
Abbreviations: AA, antibacterial activity; BGTC, bis(guanidinium)-tren cholesterol; BSV,
Brest synthetic vector; CF, cystic fibrosis; CFU, colony forming unit; CR, charge ratio; Ec,
Escherichia coli; i.p., intraperitoneal; i.v., intravenous; MIC, minimal inhibitory
concentration; MBC, minimal bactericidal concentration; LB, Luria Broth; LFM,
lipofectamine; LX, lipoplex; MRSA, methicillin-resistant Staphylococcus aureus; Pa,
Pseudomonas aeruginosa; pDNA, plasmid DNA; PEI, poly(ethylenimine); RPM, rotation per
minute; RLU, relative light unit; RT, room temperature; Sa, Staphylococcus aureus; TE,
transfection efficiency.
S1
Submitted to
SI1. Detailed structure of the cationic lipids
1 (BSV77)
2 (BSV4)
3 (KLN47)
4 (BSV98)
5 (BSV82)
6 (BSV83)
7 (BSV21)
8 (BSV61)
9 (BSV36)
10 (GLB43)
11 (BSV62)
12 (BSV99)
13 (BSV100)
Figure S1. Chemical structure of the various representative cationic lipids evaluated herein.
S2
Submitted to
SI2. X-ray crystal structure of compound 6
Single crystal Diffraction data were collected at 170 K on an Xcalibur 2 diffractometer
(Oxford Diffraction) using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The
structure was solved by direct methods and successive Fourier difference syntheses and was
refined on F2 by weighted anisotropic full-matrix least-squares methods.[1] All the nonhydrogen atoms were refined anisotropically. All the hydrogen atoms were calculated for both
structures and therefore included as isotropic fixed contributors to Fc. The thermal ellipsoid
drawings were made with the ORTEP program.[2] Data collection and data reduction were
done with the CRYSALIS-CCD and CRYSALIS-RED programs.[3] All other calculations
were performed using standard procedures (embedded with WinGX suite of programs).[4]
A) Unit cell
B) Packing
Figure S2. X-Ray crystal-structure (ORTEP plot – Ellipsoids are represented at the 50%
probability level) of 2-(O,O-diethoxyphosphoramidyl)-ethyltrimethylarsonium iodide
(compound 6) (Data collected at 170 °K, space group P21/n (14); selected data d(Ǻ): P1-O3
1.450(5); P1-O2 1.556(5); P1-O1 1.552 (5); P1-N1 1.611 (5); As1-C6 1.931(5); As1-C8
1.898(5)). A) Asymmetric unit of 6; B) Two units of 6 arranged as a dimer stabilized by
hydrogen bonds (d(Ǻ) N1-H1 0.88; H1…O3 1.96; N1…O3 2.835(6)).
S3
Submitted to
SI3. Physicochemical characteristics of liposomal solutions
Table S1. Size and zeta potential of liposomal solutionsa) prepared with the various
representative cationic lipids evaluated in this study.
Compounds
Mean Particle
Sizes (nm)
Poly Indexb)
Zeta Potential
(mV)
1 (BSV77)
233
0.22
+ 38
2 (BSV4)
224
0.21
+ 41
3 (KLN47)
242
0.36
+ 49
4 (BSV98)
142
nd
+ 35
5 (BSV82)
190
0.49
+ 53
7 (BSV21)
241
0.15
+ 60
8 (BSV61)
179
0.19
+ 40
9 (BSV36)
267
0.36
+ 33
10 (GLB43)
205
0.41
+ 33
11 (BSV62)
145
0.29
+ 40
12 (BSV99)
nd
nd
nd
13 (BSV100)
nd
nd
nd
a)
Liposomal solutions were prepared at 1.5 mM. Of note, at this concentration,
compound 6 does not form any aggregates in water; b) Poly Index, polydispersity index; nd, not determined.
S4
Submitted to
SI4. Antibiotic resistance profile of the bacterial strains
Table S2. Antibiotic resistance profilea) of the bacterial strains.
Mup
Lin
Van
Tei
Fus
Cot
Pef
Pri
Lin
Ery
Dox
Kan
Ami
Tob
Net
Gent
Clav
Amo
Antibioticc)
Oxa
Gram
positive
strainsb)
Sa RN4220
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Sa Newman
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Sa N315
R
R
R
S
S
R
R
R
S
R
R
S
S
S
S
S
S
S
S
Number of antibiotic resistances per strain: Sa RN4220, 0 out of 19; Sa Newman, 0 out of 19; Sa
N315, 8 out of 19.
Tem
Col
Cot
Fos
Cif
Lev
Ami
Net
Tob
Gen
Imi
Cefo
Cefs
Ceft
Aza
Taz
Pip
Clav
Antibioticc)
Tic
Gram
negative
strainsb)
Ec MG1655
R
R
R
S
S
S
R
S
S
S
S
S
S
S
S
S
nd
S
nd
Pa 130709
S
S
S
S
S
S
R
S
R
L
S
R
L
nd
S
R
R
R
R
Pa 240709
R
R
R
R
R
R
R
R
S
R
R
R
R
nd
S
R
R
S
nd
Number of antibiotic resistances per strain: Ec MG1655, 4 out of 17; Pa 130709, 7 out of 18; Pa
240709, 14 out of 17.
a)
as determined with the antibiogram reference method; b) Sa, S. aureus; Ec, E. coli; Pa, P.
aeruginosa; c) Antibiotic abbreviations: Ami, amikacin; Amo, amoxicilline; Aza, aztreonam; Cefo,
cefoperazon; Cefs, cefsulodin; Ceft, ceftazidim; Cif, ciprofloxacin; Clav, amoxicilline+clavulinic acid;
Col, colistin; Cot, cotrimoxazole; Dox, doxicillin; Ery, erythromycin; Fos, fosfomycin; Fus, fusidic acid;
Gent, gentamicin; Imi, imipenem; Kan, kanamycin; Lev, levofloxacin; Lin, lincomycin; Lin, lincomycin;
Mup, mupirocin.; Net, netilmicin; Pef, pefloxacin; Pip, piperacillin; Pri, pristinamycin; Taz,
piperacillin+tazobactam; Tei, teicoplanine; Tem, temocillin.; Tob, tobramycin; Van, vancomycin; Oxa,
oxacilline; Tic, ticarcilline. R, resistant; L, limit; S, sensitive; nd, not determined.
S5
Submitted to
SI5. Drug resistance study
In order to investigate whether or not arsonium-containing cationic lipids might select for
drug-resistant isolates, the MRSA strain N315 was cultivated for serial passages on half-MIC
of 2, MIC values being re-evaluated every 24 h. As a positive control, the antibiotic
norfloxacin (NFX) was evaluated in parallel in the same way.
A
B
MIC (µм)
200
NFX
200
150
150
100
100
50
50
2
0
0
0
1
2
3
4 5 6
Passage
7
8
9 10
0
1
2
N/N
2/2
3
N/N
Passage 0/1
4
2/2
5
6
N/2
2/N
Passage 9/10
Figure S3. Evaluation of the ability of a methicillin resistant S. aureus strain to develop
resistances towards an antibiotic or an arsonium-containing cationic lipid. (A) Minimal
inhibitory concentrations (MIC) were determined while cultivating the strain N315 for 10
passages (one each 24 h) in presence of either Norfloxacin (NFX, N) or 2. Bacteria growing at
one-half of the MIC were used to prepare bacterial dilution (106 to 107 CFU/mL) used for
subsequent passage. (B) As controls, at passage 9 to 10, bacteria previously cultivated in
presence of one drug were exposed to the other one (n=3).
While the susceptibility of bacteria towards 2 did not change after 10 passages, a strong
increase in MIC of NFX was already detected after 3 passages, with a more than 20-fold
increase in MIC measured after 10 days. This supports that arsonium-containing cationic
lipids should not participate in the development of drug-resistant strains.
S6
Submitted to
SI6. Stability of a naked DNA under sterile or infected conditions
Figure S4. Stability of a naked pDNA when diluted in a culture medium either sterile or
inoculated with a bacterial strain. A pDNA was diluted either in water (i), in sterile LB broth
(ii) or in LB broth inoculated with the S. aureus strain RN4220 (iii) and then incubated at 37
°C for 6 h. At 3 time points (H), optical densities at 650 nm were measured (for assessing
bacterial growth) and an aliquot of each condition was analysed by agarose gel
electrophoresis (for assessing DNA integrity). L, 100 bp DNA ladder (Smartladder SF,
Eurogentec); LB, LB broth alone; i, pDNA in water; ii, pDNA in sterile LB broth; iii, pDNA
in inoculated LB broth.
- Lane 2: The lower diffused band visible at the bottom of wells #2, #4, #5, #7, #8, #10, #11,
and #12 should not be confused with DNA. It is likely that it corresponds to some LB broth
components that should interact with ethidium bromide, thereby being visualised on gel.
- Lanes 3, 6 and 9: The clear delineation of distinct bands remaining unchanged at the three
time points considered indicate the stability of pDNA diluted in water, at 37 °C, for at least 6
h (and up to 16 h, not shown).
- Lanes 4, 7 and 10: The dilution of pDNA in sterile LB broth leads to some conformational
changes as the different forms visualised at H+0, H+4, and then H+6 are not the same.
However, clear distinct bands are well observed in each case and no obvious degradation
can be detected there.
- Lanes 5, 8 and 11: For conditions where DNA is diluted in an inoculated LB broth, as
bacteria grow, pDNA conformational changes are first visible at H+4 then a smear with
fragment sizes ranging to 200 up to >1,000 base pairs at H+6. After 16 h at 37 °C, no DNA
is visualised (not shown).
- Lanes 11 and 12: Of note, the intense fluorescence observed in well #11 is related to bacteria
themselves; indeed, such fluorescence was not observed in well #12 which corresponds to
the electrophoresis of the supernatant collected after centrifugation (to pellet bacteria).
S7
Submitted to
SI7. Antibacterial activity and DNA compaction and protection
AA/ Bacterial growth assessments after an overnight incubation at 37 C (H+23)
Incubation with 2-based lipoplexes
1,4
1.4
1,4
1.4
1.2
1,2
1.2
1,2
1.0
1,0
1.0
1,0
OD 650 nm.
OD 650 nm.
Incubation with LFM-based lipoplexes
0.8
0,8
0.6
0,6
0.4
0,4
0.8
0,8
0.6
0,6
0.4
0,4
0.2
0,2
0.2
0,2
0.0
0,0
0.0
0,0
DNA CR½
Conc. (µM):
0
CR1
CR2
CR4
CR6
CR8
9
19
38
56
75
5
DNA CR½
Conc. (µM):
w. bact.
w. bact.
w.o. bact.
w.o. bact.
Legend: DNA, naked (uncomplexed) DNA;
, without bacteria;
0
CR1
CR2
CR4
CR6
CR8
38
75
150
225
300
19
, with bacteria
BB/ DNA condensation and protection within lipoplexes (after incubation)
CR 8.0
CR 6.0
CR 4.0
CR 2.0
CR 0.5
DNA
With bacteria
CR 8.0
CR 6.0
CR 4.0
CR 2.0
CR 1.0
CR 0.5
DNA
Without bacteria
CR 8.0
CR 6.0
CR 4.0
CR 2.0
CR 1.0
CR 0.5
DNA
With bacteria
CR 8.0
CR 6.0
CR 4.0
CR 2.0
CR 1.0
CR 0.5
DNA
Without bacteria
2-based lipoplexes
CR 1.0
LFM-based lipoplexes
- DS
+DS
Figure S5. Lipoplexes prepared with an arsonium-containing lipophosphoramide can
simultaneously kill bacteria and protect DNA against degradation. Lipoplexes were prepared
by mixing DNA with either LFM or 2 in order to form complexes characterized with charge
ratios (CR) ranging from 0.5 up to 8.0 (corresponding cationic lipid concentrations are
indicated in the unit of µM). Thereafter, lipoplexes were diluted in LB broth either sterile or
inoculated with the S. aureus strain RN4220. After an overnight incubation at 37 °C, bacterial
growths were assessed spectroscopically by measuring the absorption at 650 nm and by
spreading an aliquot of each condition onto nutritive non-selective agar plates (A); agarose gel
electrophoresis were conducted to evaluate DNA condensation within lipoplexes, before and
after addition of dextran sulphate (DS) (B). Of note, 3 provided similar results (not shown) as
2.
S8
Submitted to
SI8. Sequential assay: antibacterial effect then in vitro transfection activity
AA/ Lipoplexes +/- bacteria incubated for 24 h at 37 C
With bacteria
CR :
½
1
2
3
BB/ Bactericidal assessments at H+24
Without bacteria
4
5
½
1
2
3
4
5
With bacteria
CR :
1
½ 1
2
3
4
Without bacteria
5 ½ 1
2
3
4
5
1
2
2
3
3
[C] (µM) :
5
11
21
32
42
53
5
11
21
32
42
53
LFM
LFM
[C] (µM) :
1
3
5
8
11
13
1
3
5
8
11
13
CC/ Transfection of human bronchial epithelial cells with mixtures of lipoplexes incubated +/- bacteria
1E+7
TE (RLU mg protein-1).
1E+6
1E+5
1E+4
1E+3
1E+2
1E+1
1E+0
Cells
//
CR½
CR2.0 CR4.0
CL1
1
CR½
CR2.0 CR4.0
CL2
2
CR½
CR2.0 CR4.0
CR½
CL3
3
CR2.0 CR4.0
LFM
Legend: lipoplexes previously incubated with ( ) or without ( ) bacteria for 24 hours at 37 C.
Figure S6. Lipoplexes prepared with an arsonium-containing lipophosphoramide can kill
bacteria while remaining efficient for in vitro transfection of eukaryotic cells. Lipoplexes
were prepared by mixing DNA with either LFM or an arsonium-containing
lipophosphoramide (1, 2 or 3) in order to form complexes characterized with charge ratios
(CR) ranging from 0.5 up to 5.0 (corresponding cationic lipid concentrations [C] are indicated
in the unit of μM). Next, lipoplexes were diluted in DMEM either sterile or inoculated with
the S. aureus strain RN4220. After 24 h at 37 °C, a direct visual inspection of these mixtures
allowed identifying conditions in which a bacterial growth had occurred (A). Bacterial
growths were further confirmed by spreading an aliquot of each condition onto a nutritive
non-selective agar plate and overnight incubation at 37 °C (B). Mixtures of lipoplexes with or
without bacteria were then used for in vitro gene transfection of eukaryotic cells. The
luciferase reporter system was used to evaluate the transfection efficiency measured as RLU
per mg protein. The results shown were obtained by incubating the human airway epithelial
cell line 16HBE with lipoplexes formed at 3 different CR. Of note, similar results were
obtained when using A549 cell line (not shown) (C). Values are mean +/- SD with n = 3.
S9
Submitted to
101
103
100
102
10-1
101
10-2
100
10
0
2
4
6
8
AK activity (RLU)
OD 650 nm
SI9. Adenylate kinase measurements for monitoring bacterial growth
Time (hours)
Figure S7. Comparative evaluation of adenylate kinase (AK) activity and medium turbidity.
LB broth inoculated with the S. aureus strain N315 was incubated for several hours at 37 °C.
At regular time interval, optical density (OD) was determined at 650 nm and a sample was
collected in order to be assayed using the Toxilight kit (Lonza).
S10
1E+5
1E+5
1E+5
1E+5
1E+5
1E+4
1E+4
1E+4
1E+4
1E+4
Submitted to
1E+5
1E+5
1E+4
1E+4
CL2-LX, wo
bacteria
CL2-LX, w
bacteria
0
10
10
1E+5
1E+5
1E+4
504
10
10
bacteria
Cells, w
wo
Cells,
bacteria
10
20
30
40
bacteria
Cells, w Time (hours)
bacteria
Cells, w
wo
Cells,
bacteria
10
20
30
40
bacteria
Cells, w Time (hours)
50
0
10
20
30
40
Time (hours)
1E+4
1E+3
50
0
10
20
30
40
Time (hours)
1E+4
1E+3
50
0
10
20
30
40
Time (hours)
1E+4
1E+3
TE (RLU/µL)
TE (RLU/µL)
LFM-LX, w
CL2-LX,
bacteria
CL2-LX, w
w
bacteria
CL2-LX, wo
bacteria
Cells,
wo
bacteria
LFM-LX,
wo
LFM-LX,
bacteria wo
bacteria
CL2-LX, w
bacteria
bacteria
Cells, wTime
LFM-LX,
w
1E+2
LFM-LX, wo
w
bacteria
LFM-LX,
1E+0
1E+3
1E+2
bacteria
40
bacteria
Cells, wo
50
0 bacteria
10
20
30
40
1E+1
LFM-LX,
Cells, wowTime (hours)
1E+2
bacteria
bacteria
Cells,
w
1E+1
20
1E+2
1E+4
1E+4
80
1E+5
1E+3
1E+1
1E+3
1E+4
60
CL2-LX, wo
Cells, w
bacteria
bacteria
CL2-LX,
wo
1E+0
50
0
10
20
30
40
bacteria
CL2-LX,Timew(hours)
1E+0
501E+1 0
50
bacteria
Cells, wo
bacteria
10
20
30
40
Cells, w Time (hours)
10
30
40
bacteria20
Time (hours)
0
10
20
30
40
Time (hours)
bacteria
CL2-LX, w
bacteria
LFM-LX,
wo
1E+3
LFM-LX, w
bacteria
CL2-LX, wo
bacteria
Cells, wo
bacteria
CL2-LX, w
bacteria
Cells, w
bacteria
7) CR10
LFM-LX, wo
1E+4
1E+3
1E+2
1E+0
50
0
100
10
40
bacteria
CL2-LX,20wo
wo 30
CL2-LX,
bacteriaTime (hours)
AK (% of max.)
AK (% of max.)
1E+0
501E+1 0
TE (RLU/µL)
TE (RLU/µL)
1E+2
1E+4
1E+4
80
1E+5
1E+3
1E+1
1E+3
1E+4
60
6) CR9
LFM-LX, wo
LFM-LX, wo
bacteria
1E+3
1E+1
CL2-LX, w
bacteria
1E+0
1E+3
1E+5
50 1E+5
0
100
10
40
bacteria
CL2-LX,20wo
wo 30
CL2-LX,
bacteriaTime (hours)
TE (RLU/µL)
TETE
(RLU/µL)
(RLU/µL)
TE (RLU/µL)
AK (% of max.)
1E+0
501E+1 0
5) CR8
LFM-LX, wo
LFM-LX, w
CL2-LX,
bacteria
CL2-LX, w
w
bacteria
CL2-LX, wo
bacteria
Cells,
wo
bacteria
LFM-LX,
wo
LFM-LX,
bacteria wo
bacteria
CL2-LX, w
bacteria
bacteria
Cells, wTime
LFM-LX,
w
1E+2
LFM-LX, wo
w
bacteria
LFM-LX,
1E+0
1E+3
1E+2
bacteria
40
bacteria
Cells, wo
50
0 bacteria
10
20
30
40
1E+1
LFM-LX,
Cells, wowTime (hours)
1E+2
bacteria
bacteria
Cells,
w
1E+1
20
TE (RLU/µL)
TETE
(RLU/µL)
(RLU/µL)
TE (RLU/µL)
TE (RLU/µL)
AK (% of max.)
bacteria
Cells, w
wo
Cells,
bacteria
10
20
30
40
bacteria
Cells, w Time (hours)
TE (RLU/µL)
TE (RLU/µL)
1E+4
1E+4
20
30
40
Time (hours)
1E+4
1E+3
3
10
40
1E+0
501E+1 0
1E+3
1E+1
1E+3
1E+4
60
CL2-LX, wo
bacteria
1E+4
0
0
0
0
1E+0
1E+0
Cells, w Time (hours)
10
0
10 20 20 30 30 40 40 5050 0 0 10
10 20 20 30 30 4040 5050 0 0 10
10 20 20 30 30 40 40 5050 0 0 10
10 20 20 30 30 40 40 50
bacteria
bacteria
bacteria
bacteria
1E+0
1E+0
1E+0
1E+0
Time
(hours)
Time
(hours)
Time
(hours)
Time
(hours)
1E+5
1E+5
1E+5
1E+5
Time
(hours)
Time
(hours)
Time
(hours)
Time
(hours)
Time (hours)
20
30
40
Time (hours)
30
1E+5
1E+5
1E+0
LFM-LX, w
CL2-LX,
bacteria
CL2-LX, w
w
bacteria
CL2-LX, wo
bacteria
Cells,
wo
bacteria
LFM-LX,
wo
LFM-LX,
bacteria wo
bacteria
CL2-LX, w
bacteria
bacteria
Cells, wTime
LFM-LX,
w
1E+2
LFM-LX, wo
w
bacteria
LFM-LX,
1E+0
1E+3
1E+2
bacteria
40
bacteria
Cells, wo
50
0 bacteria
10
20
30
40
1E+1
LFM-LX,
Cells, wowTime (hours)
1E+2
bacteria
bacteria
Cells,
w
1E+1
20
1E+2
1E+4
1E+4
80
1E+5
1E+2
1E+5
1E+1
CL2-LX, w
bacteria
1E+0
1E+3
1E+5
50 1E+5
0
100
10
40
bacteria
CL2-LX,20wo
wo 30
CL2-LX,
bacteriaTime (hours)
1E+3
TE (RLU/µL)
1E+0
B
TE (RLU/µL)
10
1E+3
1E+1
1E+3
1E+4
60
CL2-LX, wo
bacteria
TE (RLU/µL)
0
0
Cells,
wo
Cells, w
bacteria
10
20
bacteria
LFM-LX, w
CL2-LX,
bacteria
CL2-LX, w
w
bacteria
CL2-LX, wo
bacteria
Cells,
wo
bacteria
LFM-LX,
wo
LFM-LX,
bacteria wo
bacteria
CL2-LX, w
bacteria
bacteria
Cells, wTime
LFM-LX,
w
1E+2
LFM-LX, wo
w
bacteria
LFM-LX,
1E+0
1E+3
1E+2
bacteria
40
bacteria
Cells, wo
50
0 bacteria
10
20
30
40
1E+1
LFM-LX,
Cells, wowTime (hours)
1E+2
bacteria
bacteria
Cells,
w
1E+1
20
1E+2
1E+4
1E+4
80
1E+5
1E+2
1E+5
1E+4
4) CR7
LFM-LX, wo
1E+0
1E+3
1E+5
50 1E+5
0
100
10
40
bacteria
CL2-LX,20wo
wo 30
CL2-LX,
bacteriaTime (hours)
1E+3
1E+1
CL2-LX, w
bacteria
TE (RLU/µL)
20
LFM-LX, w
CL2-LX, w
bacteria
CL2-LX, w
bacteria
CL2-LX, wo
bacteria
Cells,
wo
bacteria
LFM-LX, wo
wo
LFM-LX,
bacteria
CL2-LX, w
bacteria
bacteria
bacteria
Cells, wTime
LFM-LX,
w
LFM-LX, wo
w
bacteria
LFM-LX,
bacteria
bacteria
bacteria
10
20
30
40
Cells,
LFM-LX,
Cells, wo
wowTime (hours)
bacteria
bacteria
CL2-LX, wo
bacteria
1E+4
3) CR6
LFM-LX, wo
1E+0
1E+3
1E+5
50 1E+5
0
100
CL2-LX, wo
10
40
bacteria
CL2-LX,20wo 30
bacteriaTime (hours)
bacteria
1E+2
1E+5
1E+1
CL2-LX, w
bacteria
1E+4
1E+3
TE (RLU/µL)
TETE
(RLU/µL)
(RLU/µL)
40
CL2-LX, wo
bacteria
TE (RLU/µL)
60
1E+2
1E+5
1E+1
CL2-LX, w
bacteria
AK (% of max.)
TE (RLU/µL)
TE (RLU/µL)
LFM-LX, w
CL2-LX, w
1E+2
bacteria
CL2-LX, w
1E+4
1E+4
bacteria
80
1E+5
bacteria
CL2-LX, wo
Cells, wo
bacteria wo
LFM-LX,
1E+3
LFM-LX,
bacteria wo
1E+3
1E+1
1E+1
1E+3
1E+3
bacteria
1E+4
1E+4
60
60
bacteria
CL2-LX, w
Cells, wTime
bacteria
LFM-LX,
1E+2
LFM-LX, w
w
bacteria
1E+2
1E+0
1E+0
bacteria
1E+3
1E+3
1E+2
1E+2
bacteria
LFM-LX, wo
40
40
20
30
40
50
0 bacteria
10
20
30
40
50
0
Cells,
1E+1
Cells, wo
wo Time (hours)
Time (hours)
1E+2
1E+2
bacteria
LFM-LX, w
1E+1
1E+1
20
20
bacteria
1E+0
Cells, w
1E+1
1E+1 0
bacteria
Cells, wo
0
0
1E+0
1E+0
bacteria
20
30
40
50 0 0 10 10 20 20 30 30 40 40 5050 0 0
1E+0
1E+0
(hours)
1E+5
1E+5
Cells, wTime
5
Time (hours)
Time
(hours)
0 bacteria
10
20
30
40
50
0
1E+4
1E+2
1E+4
AK (% of max.)
10
1E+1
1E+2
TE (RLU/µL)
TETE
(RLU/µL)
(RLU/µL)
1E+2
1E+0
1E+2
1E+3 0
CL2-LX, wo
10
20wo 30
40
bacteria
CL2-LX,
bacteriaTime (hours)
bacteria
CL2-LX, wo
bacteria
2) CR5
LFM-LX, wo
1E+0
1E+3
1E+5
50 1E+5
0
100
TE (RLU/µL)
TE
TE (RLU/µL)
(RLU/µL)
1E+3
1E+1
1E+3
1E+4
1E+4
80801E+5
1E+3
1E+5
0
100
100
of max.)
AK AK
(%
(% of max.)
1E+4
1E+2
1E+4
1E+5
1E+1
CL2-LX, w
bacteria
1)
CR4LFM-LX, wo
1E+0
1E+5
A
1E+5
1E+3
1E+5
µL-1)
TE TE
(RLU
(RLU/µL)
TE (RLU/µL)
TE (RLU/µL)
TE
TE (RLU/µL)
(RLU/µL)
1E+4
CL2-LX, wo
bacteria
1E+2
1E+5
1E+3
TE (RLU/µL)
TETE
(RLU/µL)
(RLU/µL)
1E+1
1E+4
1E+0
1E+1
1E+2
1E+5
1E+3
TE (RLU/µL)
1E+5
1E+3
TE (RLU/µL)
TE (RLU/µL)
SI10. Simultaneous assay: antibacterial effect and transfection activity in one pot
10
40
bacteria
CL2-LX,20wo
wo 30
CL2-LX,
bacteriaTime (hours)
50
LFM-LX, w
CL2-LX,
bacteria
CL2-LX, w
w
bacteria
CL2-LX, wo
bacteria
Cells,
wo
bacteria
LFM-LX,
wo
LFM-LX,
bacteria wo
bacteria
CL2-LX, w
bacteria
bacteria
Cells, wTime
LFM-LX,
w
LFM-LX, wo
w
bacteria
LFM-LX,
bacteria
bacteria
Cells, wo
80
60
40
50
bacteria
LFM-LX,
Cells, wow
bacteria
Cells, w
bacteria
Cells, wo
20
50
0
5050 0
1E+5
Cells, w
bacteria
bacteria
Cells, w
10
30
40
bacteria20
Time (hours)
50
CL2-LX, wo
bacteria
50
1E+4
CL2-LX, w
bacteria
1E+3
LFM-LX, wo
bacteria
10
20
30
40
Time (hours)
80
AK (% of max.)
AK (% of max.)
80
10
60
40
20
30
40
Time (hours)
60
40
1E+1
1E+1
Cells, wo
bacteria
1E+0
50
0
100
1E+0
50
0
100
1E+0
50
0
100
1E+0
50
0
100
1E+0
50
0
100
10
20
30
40
Time (hours)
80
10
20
30
40
Time (hours)
80
60
40
10
20
30
40
Time (hours)
1E+2
80
60
40
40
40
20
20
20
20
0
0
0
0
0
0
20
30
40
Time (hours)
50
0
10
20
30
40
Time (hours)
50
0
10
20
30
40
Time (hours)
50
0
10
20
30
40
Time (hours)
S11
50
0
10
20
30
40
Time (hours)
50
10
20
30
40
Time (hours)
50
10
20
30
40
Time (hours)
50
80
60
20
10
20
30
40
Time (hours)
80
60
20
0
10
AK (% of max.)
0
1E+1
1E+2
AK (% of max.)
1E+0
50
0
100
LFM-LX, w
bacteria
1E+1
1E+2
AK (% of max.)
1E+0
1E+2
1E+1
1E+2
AK (% of max.)
1E+1
100
TETE
(RLU/µL)
(RLU/µL)
1E+2
1E+1
AK (% of max.)
1E+2
(RLU/µL)
TETE
(RLU/µL)
CL2-LX, wo
1E+3
102
CL2-LX,
bacteria wo
bacteria
LFM-LX, wo
1E+3
101
bacteria
bacteria
CL2-LX,
w
LFM-LX, w
1E+4 100
CL2-LX,
LFM-LX,
w 40 50 0 10 20 30 40 50
1E+4
0 10 20 30 40 50 0 10 20 30 401E+2
50 0 10 20 30bacteria
40 50 0 w
10 20 30 40 50 0 10 20 30 40 50 bacteria
0 10 20 30
bacteria Time (hours)
bacteria
Time (hours)
Time (hours)1E+2 Time (hours)
Time (hours)
Time (hours)
Time (hours)
LFM-LX, wo
CL2-LX, wo
Cells, wo
1E+3
CL2-LX,
wo
LFM-LX,
wo
Cells,
wo
bacteria
2-based
lipoplexes,
wo
bacteria
LFM-based
lipoplexes,
wo
bacteria
Untransfected
cells,
wo
bacteria
bacteria
bacteria
1E+1
1E+3
bacteria
bacteria
bacteria
1E+1
CL2-LX,
w
LFM-LX,
w lipoplexes, w bacteria
Cells,
w
2-based lipoplexes,
w bacteria
LFM-based
Untransfected
cells, w bacteria
Cells, w
CL2-LX,
1E+2
LFM-LX, w
bacteria w
bacteria
bacteria
1E+0
bacteria
bacteria
1E+2
bacteria
1E+0
LFM-LX, wo
Cells,
wo
0with10
20
30
40
50
Figure S8.
Lipoplexes
prepared
an
arsonium-containing
lipophosphoramide
can both
LFM-LX,
bacteria wo
0
10
20
30
40
50
Cells, wo
bacteria
Time (hours)
1E+1
bacteria and transfect eukaryotic
inhibit
bacteria
cells
in
one
pot.
During
55
hours,
2-based
lipoplexes
Time
(hours)
bacteria
1E+1
LFM-LX, w
Cells, w
(formed atLFM-LX,
CR ranging
from 4 to 10) were
assayed for their simultaneous ability to (1) inhibit
w
bacteria
Cells, w
bacteria
1E+0
bacteria
the growth
of
the
bacterial
strain
N315
and
to (2) transfect human bronchial epithelial cells
bacteria
Cells, wo
1E+0 0
10
20
30
40
50
Cells,
wo
both0 cultivated
bacteria in EMEM (without any antibiotics). LFM-based lipoplexes were used as
Time (hours)
10
20
30
40
50
controls. bacteria
Antibacterial
Time (hours)activity was monitored by considering the adenylate kinase (AK)
Cells,
w
Cells,
w
bacteria
activity (A)
together
with variations of the medium colour (insets in A). Transfection
bacteria
efficiency
(TE) was quantitated as RLU per µL of supernatant (B). w, with; wo, without.
20
30
40
50
20 (hours)
30
40
50
Time
Time (hours)
60
40
20
0
0
10
20
30
40
Time (hours)
50
0
Cells, w
bacteria
Submitted to
SI11. References
[1]
[2]
[3]
[4]
M. Sheldrick, SHELX97. Programs for crystal structure analysis; Göttingen, Germany, 1997.
C. K. Johnson, ORTEP; Delft, The Netherlands, 1985.
CRYSALIS PRO Software System; Oxford Diffraction Ltd, 2007.
L. J. Farrugia, J. Appl. Crystallogr. 1999, 32, 837.
S12