Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2020.

Supporting Information
for Adv. Mater., DOI: 10.1002/adma.201906128

A Combinatorial Library of Lipid Nanoparticles for RNA

Delivery to Leukocytes
Srinivas Ramishetti, Inbal Hazan-Halevy, Ramesh Palakuri,
Sushmita Chatterjee, Somu Naidu Gonna, Niels Dammes,
Inbar Freilich, Luba Kolik Shmuel, Dganit Danino, and Dan
Peer*
 Supporting information

A Combinatorial Library of Lipid Nanoparticles for RNAi Delivery to Leukocytes

Dr. Srinivas Ramishetti1,†,‡,§, Dr. Inbal Hazan-Halevy1,†,‡,§, Dr. Ramesh Palakuri1,†,‡,§, Dr.

Sushmita Chatterjee†,‡,§, Dr. Somu Naidu Gonna†,‡,§, Dr. Niles Dammes†,‡,§, Inbar Freilichζ,

Dr. Luba Kolik Shmuelζ, Prof. Dganit Daninoζ and Prof. Dan Peer†,‡,§,*

†
Laboratory of Precision NanoMedicine, School of Molecular Cell Biology and

Biotechnology, George S. Wise Faculty of Life Sciences; ‡Department of Materials

§
Sciences and Engineering, Faculty of Engineering; Center for Nanoscience and

Nanotechnology and Cancer Biology Research Center, Tel Aviv University, Tel Aviv

69978, Israel. ζ CryoEM Laboratory of Soft Matter, Faculty of Biotechnology and Food

Engineering, Technion, Haifa, 3200003, Israel

1
These authors contributed equally to this work

*
Corresponding author

Prof. Dan Peer

Laboratory of Precision NanoMedicine, School of Molecular Cell Biology and

Biotechnology, George S. Wise Faculty of Life Science, Tel Aviv University,

Ramat Aviv, Tel Aviv, 6997801

peer@tauex.tau.ac.il
Synthesis of Lipid 1:

BocNHNH2
dryDCM
Na(OAc)3BH

O
O N N
H

1.TFA 2.EDC,DMAP
dry DCM
dryDCM

O
N
N N
H

Lipid 1

The aldehyde (1.5 g, 5.6 mmol) and Boc-hydrazide (0.6 g, 4.5 mmol) were dissolved in
dry CH2Cl2 under argon atmosphere then sodium triacetoxyborohydride (4.7 g, 22.6 mmol)
was added to the reaction mixture and stirred for 24hr at room temperature. The reaction
mixture was quenched with sat. NaHCO3 solution followed by extract with CH2Cl2 (3
times) washing with brine solution and dried over with anhydrous Na2SO4. The solvent
was evaporated and the residue was purified by silica column chromatography (EtOAc:
Hexane (5: 95) yielded 1.3 g of pure compound N,N-dilinoleyl-boc hydrazide. 1H NMR
(400 MHz, CDCl3): δ 5.22-5.45 (8H, m); 3.63 (4H, q, J = 6.93 Hz); 2.77 (4H, t, J = 6.67
Hz); 2.05 (8H, q, J = 6.43 Hz); 1.58 (9H, s); 1.5 (4H, m); 1.2-1.4 (32H, m); 0.89 (6H, t, J
= 6.93 Hz). ESI-MS: m/z 629.6 (M+1)+

N,N-dilinoleyl-boc hydrazide (1.3 g, 2.07 mmol) was dissolved in dry CH2Cl2 under argon
atmosphere. The reaction mixture was cooled to 0°C and trifluoroacetic acid (TFA) (2 mL)
was added drop wise and stirred for 3hr. Upon completion of reaction by TLC analysis, the
reaction mixture was washed with sodium bicarbonate followed by brine solution. The
solvent was evaporated and the crude reaction mixture (0.4 g, 0.76 mmol), N, N-dimethyl
aminobutyric acid (0.19 g, 1.3 mmol) and EDC (0.43 g, 2.26 mmol), DMAP (cat.) were
dissolved in dry CH2Cl2 under argon atmosphere. Then the reaction stirred for 16hr. The
reaction mixture was quenched with sat. NaHCO3 solution followed by extract with CH2Cl2
(3 times) washing with brine solution and dried over with anhydrous Na2SO4. The solvent
was evaporated and the residue was purified by silicagel column chromatography (MeOH:
CHCl3 (2:98) to yield 0.3 g of pure Lipid 1 as colorless liquid. 1H NMR (400 MHz, CDCl3):
δ 5.22-5.45 (8H, m); 2.8 (4H, t, J = 6.93 Hz); 2.6 (3H, m); 2.3-2.5 (4H, m); 2.2 (6H, s); 2.0
(8H, q, J = 6.46 Hz); 1.6-1.8 (8H, m); 1.2-1.5 (32H, m); 0.9 (6H, t, J = 6.87 Hz). ESI-MS:
m/z 642.6 (M+1).+

Synthesis of Lipid 2:

H2N N

OH
EDC, DMAP
N N
dry DCM
O

H
N N N N

O
Lipid 2
N, N-dilinoleyl hydrazide (0.52 g, 1.0 mmol, 1 equiv.), N-methyl piperiazine propionic acid
(0.25 g, 1.5 mmol, 1.5 equiv.), EDC (0.38 g, 2.0 mmol, 2 equiv.) and DMAP (cat.) were
dissolved in dry CH2Cl2 under argon atmosphere and the reaction stirred for 12hr. The
reaction mixture was quenched with sat. NaHCO3 solution followed by extract with CH2Cl2
(3 times) washing with brine solution and dried over with anhydrous Na2SO4. The solvent
was evaporated and the residue was purified by silica gel column chromatography (MeOH:
CHCl3 (4:96) to yield 0.58 g (85%) of pure Lipid 2 as colorless liquid. 1H NMR (400 MHz,
CDCl3): δ 8.7(1H, s), 5.41-5.28 (8H, m); 2.7 (4H, t, J = 6.41 Hz), 2.66 (4H, t, J = 6.41 Hz),
2.59 (4H, t, J = 6.23 Hz), 2.37 (4H, t, J = 5.92 Hz); 2.29 (3H, s); 2.03 (8H, q, J = 6.73 Hz);
1.55-1.40 (4H, m); 1.4-1.2 (32H, m), 0.88 (6H, t, J = 6.97 Hz). ESI-MS: m/z 683.7 (M+1)+;
342.3 (M/2+1)+
Synthesis of Lipid 11 & 3:
O
1. PCC, DCM
H
HO OH
6O 8
2. Decanoic acid, EDC O
1,7-heptane diol DMAP, DCM
H2N
OH

O NaBH(OAc)3
O H
N NH2
HO N
N
NaBH(OAc)3
O
O
EDC, DCM O
DMAP O
H
O N N
N
O
O
O
N N
O
O
O Lipid 3
O
Lipid 11

7-oxoheptyl decanoate:
O
O
OHC

1,7-heptanediol (5.0 g, 37.0 mmol) taken in 100 mL flask and dissolved in dry DCM
followed by portion-wise slowly addition of PCC (8.9 g, 41.6 mmol) over 15 min. Then,
the reaction mixture was stirred at room temperature for 2h. The crude reaction mixture
was filtered through the silica gel pad and washed with CH2Cl2 (2x 50 mL). The organic
solvent dried over with Na2SO4 and solvent removed by under reduced pressure crude
hydroxyl aldehyde used directly without any further purification.

The above prepared crude hydroxyaldehyde (1.3 g, 10 mmol), decanoic acid (2.0 g, 12
mmol) and EDC (2.8 g, 15.0 mmol) taken in to 100 mL flask and dissolved in dry CH2Cl2
and addition of DMAP (cat.) at 0oC. The reaction mixture stirred for 16hr at rt. The reaction
mixture was quenched with sat. NaHCO3 and followed by extract with CH2Cl2 (3 times)
and washed with brine solution and dried over with anhydrous Na2SO4. The crude mixture
was purified by column chromatography gave color less oil with 90% (2.4 g) yield. 1H
NMR (400 MHz, CDCl3): δ 9.76 (1H, t, J = 1.91 Hz); 4.05 (2H, t, J = 6.76 Hz), 2.43 (2H,
dt, J = 7.48, 5.70 Hz); 2.28(2H, t, J = 7.68 Hz);1.78- 1.52 (8H, m); 1.44-1.33 (4H, m);
1.32-1.20 (12H, m), 0.87 (3H, t, J = 6.62 Hz). ESI-MS: m/z 285.8 [M+1]+, 283.8 [M-1]+

(2-(2-(dimethylamino)ethyl)hydrazine-1,1-diyl)bis(heptane-7,1-diyl) bis(decanoate)
(Lipid 3):
O
O
H
N N
N
O
O

The above aldehyde (0.6 g, 2.2 mmol), dimethylaminoethyl hydrazine hydrochloride (0.18
g, 1 mmol) dissolved in dry CH2Cl2 followed by addition of sodium tri acetoxyborohydride
(0.6 g, 3 mmol) was added to the reaction mixture and stirred for 12hr at room temperature.
The reaction mixture was quenched with sat. NaHCO3 and followed by extract with CH2Cl2
(3 times) and washed with brine solution and dried over with anhydrous Na2SO4. The crude
mixture was purified by silica gel column chromatography yielded 73% (0.51 g) pure (2-
(2-(dimethylamino)ethyl)hydrazine-1,1-diyl)bis(heptane-7,1-diyl) bis(decanoate) as pale
liquid. 1H NMR (400 MHz, CDCl3): δ 9.28 (1H, t, J = 5.55 Hz); 4.01 (1H, t, J = 6.60 Hz);
3.70 (2H, t, J = 7.70 Hz); 3.30-3.20 (3H, m); 2.54 (3H, t, J = 7.55 Hz); 2.33-2.21 (6H, m),
2.20 (s, 6H); 1.80-1.70 (4H, m); 1.37-1.17 (24H, m); 0.83 (6H, t, J = 6.90 Hz). ESI-MS:
m/z 640 ([M+1]+).
((2-hydroxyethyl)azanediyl)bis(heptane-7,1-diyl) bis(decanoate):
O
O
HO
N

O
O

The aldehyde (1.0 g, 3.7 mmol), ethanolamine (0.11g, 1.85 mmol) dissolved in dry CH2Cl2
followed by addition of Sodium tri acetoxyborohydride (1.1g, 5.5mmol), the reaction
mixture stirred for 24hr at room temperature. The reaction mixture was quenched with sat.
NaHCO3 and followed by extract with CH2Cl2 (3 times) and washed with brine solution
and dried over with anhydrous Na2SO4. The crude mixture was purified by silica gel
column chromatography yielded 85% (0.91 g) pure ((2-
hydroxyethyl)azanediyl)bis(heptane-7,1-diyl) bis(decanoate) as pale liquid. 1H NMR (400
MHz, CDCl3): δ 4.05 (4H, t, J = 6.98 Hz); 3.60 (2H, t, J = 5.31 Hz); 2.66 (2H, t, J = 5.31
Hz); 2.54 ( 4H, t, J = 7.58 Hz); 2.28 (4H, t, J = 7.58 Hz); 1.68-1.54 (8H, m ); 1.53-1.41
(4H, m); 1.20-1.40 (32H, m), 0.89 (6H, t, J = 6.80 Hz). ESI-MS: m/z 598.6 ([M+1]+).

((2-((4-(dimethylamino)butanoyl)oxy)ethyl)azanediyl)bis(heptane-7,1-diyl)
bis(decanoate) (Lipid 11):
O
O
O
N N
O
O
O

The above alcohol compound (0.4 g, 0.7 mmol) and 4-(dimethylamino)butanoic acid
(0.17g, 1.0 mmol) taken in100mL flask and dissolved in dry CH2Cl2 and EDC (0.27g, 1.4
mmol) was added to the reaction mixture followed by DMAP (cat.) stirred for overnight
for 24hr. The reaction mixture was quenched with sat. NaHCO3 and followed by extract
with CH2Cl2 (3 times) and washed with brine solution and dried over with anhydrous
Na2SO4. The crude mixture was purified by silica gel column chromatography yield 75%
(0.37 g) pure Lipid 11 as colorless oil. 1H NMR (400 MHz, CDCl3): δ 4.11 (2H, t, J = 6.51
Hz); 4.04 (4H, t, J = 6.78 Hz); 2.66 (2H, t, J = 6.51 Hz); 2.43(4H, t, J = 7.73 Hz); 2.33 (
2H, t, J = 7.58 Hz); 2.28 (4H, t, J = 7.58 Hz); 2.21 (6H, s); 1.68-1.54 (6H, m); 1.67-1.52
(8H, m); 1.46-1.36 (4H, m); 1.20-1.40 (32H, m), 0.87 (6H, t, J = 6.80 Hz). ESI-MS: m/z
711 ([M+1]+).
Synthesis of lipid 4:
Cis-2-nonen-1-ol
O
OHC OH
OHC O
O EDC, DMAP (Cat.),
DCM
Boc-NH-NH2,
DCM

H O
O N N

O
O

OH EDC, DMAP, DCM

N
O

H O
N N
N
O
O

Lipid 4
Di((Z)-non-2-en-1-yl) 6,6'-(2-(tert-butoxycarbonyl)hydrazine-1,1-
diyl)dihexanoate:
O

H O
O N N

O
O

The above aldehyde (0.25 g, 1.00 mmol, 2 equiv.) and Boc-hydrazine (0.06 g, 0.5 mmol, 1
equiv.) were dissolved in dry CH2Cl2 under nitrogen atmosphere then sodium tri
acetoxyborohydride (0.63 g, 3.0 mmol, 3 equiv.) was added to the reaction mixture and
stirred for 12h at room temperature. The reaction was quenched with sodium bicarbonate
solution and extract with CH2Cl2 followed by washing with water and brine solution. The
solvent was evaporated and purified by column chromatography yielded 0.21 g (78%) of
pure lipid as colorless liquid. 1H NMR (400 MHz, CDCl3): δ 5.69-5.57 (2H, m); 5.56-5.45
(2H, m); 4.61 (4H, d, J = 7.20 Hz); 2.62 (1H, br s); 2.30 (4H, t, J = 7.59 Hz), 2.08 (4H, q,
J = 7.90, 7.11 Hz);1.70- 1.56 (8H, m); 1.55- 1.40 (12H, m);1.40-1.20 (20H, m); 0.87 (6H,
t, J = 6.75 Hz). ESI-MS: m/z 609 ([M+1]+); 553 ([M-t-Bu]+).

di((Z)-non-2-en-1-yl) 6,6'-(hydrazine-1,1-diyl)dihexanoate:
O

O
H 2N N

The above Boc-protected-hydrazine compound (0.25 g, 0.41 mmol) was dissolved in TFA/
CH2Cl2 (2:8) 10 mL for 2h at room temperature. The reaction was quenched with sodium
bicarbonate solution and extract with CH2Cl2 followed by washing with water and brine
solution and dried over Na2SO4. The solvent was evaporated and without further
purification used for next reaction yielded 0.20 g (99%) of pure hydrazine as pale yellow
liquid. 1H NMR (400 MHz, CDCl3): δ 5.70-5.58 (2H, m); 5.56-5.42 (2H, m); 4.61 (4H, d,
J = 6.92 Hz), 2.44 (4H, t, J = 7.52 Hz); 2.31 (4H, t, J = 7.52 Hz); 2.09 (4H, q, J = 7.52,
7.15 Hz); 1.64 (4H, quint); 1.55 (4H, quint); 1.43-1.20 (18H, m); 0.87 (6H, t, J = 7.15 Hz).
ESI-MS: m/z 509.5 ([M+1]+)
Di((Z)-non-2-en-1-yl) 6,6'-(2-(4-(dimethylamino)butanoyl)hydrazine-1,1-
diyl)dihexanoate (Lipid 4):
O

H O
N N
N
O
O

The above amine (0.20 g, 0.4 mmol, 1 equiv.), N,N-dimethyl aminobutyric acid
hydrochloride (0.10 g, 0.6 mmol, 1.5 equiv.) and EDC (0.15 g, 0.8 mmol, 2 equiv.) and
DMAP (cat.) were dissolved in dry CH2Cl2 under nitrogen atmosphere and the reaction
stirred for 12h. The reaction mixture was quenched with sat. NaHCO3 and followed by
extract with CH2Cl2 (3 times) and washed with brine solution and dried over with
anhydrous Na2SO4. The solvent was evaporated, and the reaction mixture was purified by
column chromatography to yielded 85% (0.20 g) of pure Lipid 4 as pale-yellow liquid.
1
H NMR (400 MHz, CDCl3): δ 5.67-5.57 (4H, m); 5.57-5.44 (4H, m); 4.61 (4H, t, J = 5.52
Hz); 2.66 (2H, t, J = 8.16 Hz ); 2.45 (2H, t, J = 7.47 Hz); 2.40 (2H, t, J = 7.75 Hz); 2.34-
2.20 (4H, m); 2.28 (6H,s); 2.08 (4H, q, J = 7.80 Hz); 2.00-1.90 (4H, m); 1.86-1.76 (2H,
m); 1.70-1.55 (4H, m); 1.54-1.40 (4H, m); 1.38-1.20 (14H, m); 0.87 (6H, t, J = 6.92 Hz).
ESI-MS: m/z 622.6 ([M+1]+).
Synthesis of lipid 5:
O O
H Methyl adipate O OH
N
N NH2 N LAH, THF N
N N
EDC HN
OH
HN
O O
O

LDA, NaH 5
OH OH
7 Iodohexane 7
O O

O
N O
N

HN O
O

Lipid 5

2-hexyldecanoic acid:

COOH

The freshly prepared LDA (9.0 mL, 2M in THF, 18.00 mmol) in THF (30 mL) was slowly
added to a solution of decanoic acid (2.6 g, 15.3mmol) and NaH (60 w/w% mineral oil
suspension, 690 mg, 18.0 mmol) in THF (19 mL) at 0 °C and stirred for 30 min at room
temperature. After addition of n-C6H13I (2.6 mL, 18.00 mmol), the reaction mixture was
stirred for 6 h at 45°C then quenched with 1N HCl at room temperature. The organic layer
was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue
was purified by flash column chromatography to give (3.7 g, 65%) as a colorless liquid. 1H
NMR (400 MHz, CDCl3): δ 2.38-2.28 (1H, m); 1.69-1.53 (2H, m); 1.50-1.40 (2H, m);
1.36-1.20 (20H, m); 0.87 (6H, t, J = 6.87 Hz). ESI-MS: m/z 255 [M-1]+
Dimethyl 6,6'-(1-(2-(dimethylamino)ethyl)hydrazine-1,2-diyl)bis(6-oxohexanoate):
O O
O
N
N

HN
O O
O

Methyl adipate (1.6 g, 10 mmol), dimethylaminoethyl hydrazine (0.88 g, 5 mmol) and EDC
(2.8 g, 15 mmol) dissolved in dry CH2Cl2 followed by addition of triethyl amine. The
reaction mixture was stirred for overnight. The reaction mixture was quenched with sat.
NaHCO3 and followed by extract with CH2Cl2 (3 times) and washed with brine solution
and dried over with anhydrous Na2SO4. The crude mixture was purified by column
chromatography. 1H NMR (400 MHz, CDCl3): δ 3.66 (3H, s); 3.64 (3H, s); 2.26-2.41 (6H,
m); 2.15-2.26 (12H, m); 1.65-1.54 (8H, m). ESI-MS: m/z 388.3 [M+1]+; 410.3 [M+Na]+

6,6'-(1-(2-(dimethylamino)ethyl)hydrazine-1,2-diyl)bis(hexan-1-ol):

OH
N
N
HN
OH

The above compound (0.77 g, 2.0 mmol) was dissolved in dry THF followed by the
addition of excess LAH (10 mL, 2M in THF, 20 mmol). The reaction mixture was refluxed
for 12hr. The reaction mixture was quenched by slow addition of H2O and filtered and
dried over Na2SO4 solvent removed by under reduced pressure purified by silica gel column
chromatography yield 70% (0.4 g) pure 6,6'-(1-(2-(dimethylamino)ethyl)hydrazine-1,2-
diyl)bis(hexan-1-ol). 1H NMR (400 MHz, CDCl3): δ 3.62 (4H, t, J = 6.70 Hz); 2.70 (2H, t,
J = 6.87 Hz ); 2.65 (2H, t, J = 7.10 Hz); 2.50-2.60 (4H, m); 2.44 (2H, t, J = 6.91 Hz); 2.29
(6H, s); 1.65-1.45 (6H, m);1.44-1.25 (8H, m). ESI-MS: m/z 304 ([M+1]+).

(1-(2-(dimethylamino)ethyl)hydrazine-1,2-diyl)bis(hexane-6,1-diyl)
bis(2-hexyldecanoate) (Lipid 5):

O
N O
N

HN O
O

The above diol compound (0.30 g, 1.0 mmol) and 2-hexyl-decanoic acid (0.50 g, 2.2 mmol,
2 equiv.) taken in 50 mL flask and dissolved in dry CH2Cl2 and EDC (0.27g, 1.4 mmol)
was added to the reaction mixture followed by DMAP (cat.) stirred for overnight. The
reaction mixture was quenched with sat. NaHCO3 and followed by extract with CH2Cl2 (3
times) and washed with brine solution and dried over with anhydrous Na2SO4. The crude
mixture was purified by silica gel column chromatography yielded 65% (0.49 g) pure
compound as pale liquid. 1H NMR (400 MHz, CDCl3): δ 3.72 (4H, t, J = 5.65 Hz); 3.20-
3.34 (4H, m); 2.31 (4H, t, J = 6.71 Hz); 2.21 (6H, s); 1.71-1.85 (4H, m); 1.68-1.50 (6H,
m); 1.50-1.36 (6H, m); 1.32-1.17 (50H, m), 0.84 (12H, t, J = 6.91 Hz). ESI-MS: m/z 780
([M+1]+).
Synthesis of Lipid 6:
HO

i) PCC
dry DCM, MS

i) dry DCM
HO NH3 Cl
ii)Na(OAc)3BH

HO N

HyAM-1

O
N EDC, DMAP
dry DCM
OH

O
N
O N

Lipid 6

Linoleic alcohol (3.4 g, 12.6 mmol, 1 equiv.) was dissolved in dry CH2Cl2 under nitrogen
atmosphere and molecular sieves (4A0MS) were added to the reaction mixture. PCC (4.0
g, 19.0 mmol, 1.5 equiv.) was added portion wise to the reaction mixture over a period of
10min. The reaction mixture was stirred for 1.5 hr and after completing the reaction filtered
it through silica gel pad to remove PCC followed by wash with CH2Cl2 (2 times)
evaporating the solvent to give yield 3.0 g of crude linoleic aldehyde.

Linoleic aldehyde (2.64 g, 10.0 mmol, 2 equiv.) and hydroxylamine hydrochloride (0.34 g,
5.0 mmol, 1.0 equiv.) were dissolved in dry CH2Cl2 under nitrogen atmosphere then
sodium triacetoxyborohydride (3.1 g, 15.0 mmol, 3 equiv.) was added to the reaction
mixture and stirred for 16 hr at room temperature. The reaction was quenched with sat.
NaHCO3 solution followed by extract with DCM (3 times) followed by washing with brine
solution and dried over with anhydrous Na2SO4. The solvent was evaporated and the
residue was purified by silica column chromatography (EtOAc: Hexane (5:95) to yield 1.5
g (55%) of pure white color compound N,N-dilinoleyl-hydroxylamine (HyAM-1). 1H
NMR (400 MHz, CDCl3): δ 5.27-5.39 (8H, m); 2.77 (4H, t, J = 6.84 Hz); 2.57-2.68 (4H,
m); 2.05 (8H, q, J = 6.80, 6.87 Hz); 1.50-1.65 (4H, m); 1.22-1.42 (32H, m); 0.89 (6H, t, J
= 6.86 Hz). ESI-MS: m/z 530 [M+1]+

N,N-dilinoleyl-hydroxylamine (HyAM-1) (0.48g, 0.90 mmol, 1 equiv.) N,N-dimethyl

aminobutyric acid hydrochloride (0.30 g, 1.8 mmol, 2 equiv.), EDC (0.34g, 1.8 mmol, 2
equiv.) and DMAP (0.01g, 0.09 mmol, 0.1 equiv.) were dissolved in dry CH2Cl2 under
nitrogen atmosphere. Then the reaction was stirred for overnight. The reaction mixture was
quenched with sat. NaHCO3 solution followed by extract with CH2Cl2 (3 times) washing
with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated
and the residue was purified by silicagel column chromatography (MeOH: CHCl3 (3:97)
to yield 0.6 g (85%) of pure lipid (Lipid 6) as colorless oil. 1H NMR (400 MHz, CDCl3):
δ 5.22-5.45 (8H, m); 2.71-2.87 (8H, m), 2.24-2.36 (4H, m); 2.21 (6H, s); 1.93-2.12 (8H,
m); 1.74-1.81 (2H, m); 1.42-1.58 (4H, m); 1.6-1.8 (8H, m); 1.20-1.40 (32H, m); 0.89 (6H,
t, J = 6.90 Hz). ESI-MS: m/z 643.1 [M]+; 644.1 [M+1]+
Synthesis of lipid 7 & 13:

TBS-Cl, Imidazole
HO COOH TBSO COOH
DCM
10-Hydroxy decanoic acid NaBH4
MeOH
EDC, DMAP 5 5
DCM
5 OH 5 O
O O
TBAF, THF
O O
rt
HO

PCC TBSO
DCM
O O

O O HO
N
OHC
Ethanol amine,
NaBH(OAc)3 O O

Ethanol amine,
NaBH(OAc)3
O O 4-(dimethylamino) EDC, DMAP
butanoic acid DCM
HO N

O O
O O

O
4-(dimethylamino) EDC, DMAP N N
butanoic acid DCM O

O O O O
Lipid 13

O N
N
O
Lipid 7 O O
10-((tert-butyldimethylsilyl)oxy)decanoic acid:

TBSO COOH

To a stirred solution 10-hydroxy decanoic acid (5.0 g, 26.5 mmol, 1 equiv.) and imidazole
(4.32 g, 63.6 mmol, 2.4 equiv.) in dry DMF (50 mL) was added TBS-Cl (4.78 g, 31.8
mmol, 1.2 equiv.) at 0oC portion-wise over a period of 10 min. The reaction mixture was
stirred at room temperature for 8 h. The mixture was then diluted with EtOAc and washed
with water, brine. The organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated. The crude product was purified by column chromatography using ethyl
acetate/ hexane (20:80) to afford TBS protected acid (7.2 g, 3.12 mmol, 90%) as colorless
oil. 1H NMR (400 MHz, CDCl3): δ 3.59 (2H, t, J = 6.61 Hz); 2.34 (2H, t, J = 7.61 Hz);
1.69-1.56 (2H, m);1.55-1.43 (2H, m); 0.89 (9H, s); 0.04 (6H, s).

tridecan-7-ol:
HO

To a stirred solution tridecan-7-one (4.0 g, 20.2 mmol, 1 equiv.) in MeOH (50 mL) was
added NaBH4 (1.15 g, 30.3 mmol, 1.5 equiv.) at 0oC portion-wise over a period of 10 min.
The reaction mixture was stirred at room temperature for 1h. The mixture was
concentrated. The crude product was purified by column chromatography using ethyl
acetate/ hexane (5:95) to afford tridecan-7-ol (3.8 g, 96%) as colorless oil. 1H NMR (400
MHz, CDCl3): δ 3.62-3.52 (1H, m); 1.51-1.35 (8H, m); 1.35-1.20 (12H, m); 0.87 (6H, t, J
= 6.72 Hz).

tridecan-7-yl 10-((tert-butyldimethylsilyl)oxy)decanoate:

O O

TBSO

A mixture of the TBS protected acid (5.0 g, 16.5 mmol, 1 equiv.), tridecan-7-ol (3.3 g, 16.5
mmol, 1 equiv.), EDC (4.75 g, 24.82 mmol, 1.5 equiv.), and 4-(dimethylamino) pyridine
(DMAP) (cat.) was dissolved in CH2Cl2 (75 mL) and stirred overnightat room temperature.
The resulting mixture was quenched with sat. NaHCO3 and extracted with CH2Cl2 (3x 20
mL) and washed by brine solution. The organic layer was then dried over Na2SO4 filtered
and concentrated under reduced pressure. The crude product was purified via flash
chromatography using 5:95 EtOAc/hexanesas to give the TBS ester as colorless oil yield
82 % (6.5 g). 1H NMR (400 MHz, CDCl3): δ 4.86 (1H, quint, J = 6.63 Hz); 3.58 (2H, t, J
= 6.63 Hz); 2.27 (2H, t, J = 7.61 Hz); 1.67-1.56 (2H, m); 1.55-1.43 (6H, m);1.35-1.20
(26H, m); 0.88 (9H, s); 0.87 (6H, t, J = 6.72 Hz); 0.04 (6H, s). ESI-MS: m/z 485.5
([M+1]+); 507.5 ([M+Na]+).
tridecan-7-yl 10-hydroxydecanoate:

O O

HO

To a solution of TBS ester (3.62 g, 7.5 mmol, 1 equiv.) in dry THF (35 mL) was added
TBAF (5.6 mL, 11.2 mmol, 1 equiv.). The mixture was stirred at ambient temperature for
2h.The reaction was quenched by addition of aq. NH4Cl and aqueous layer were extracted
with EtOAc (2 x 20 mL). The combined organic layers were washed with brine, dried with
Na2SO4, filtered and evaporated. The residue was purified by column chromatography to
give alcohol (2.6 g, 92%) colorless oil. 1H NMR (400 MHz, CDCl3): δ 4.85 (1H, quint, J
= 6.75 Hz); 3.62 (2H, q, J = 6.63 Hz); 2.26 (2H, t, J = 7.35 Hz); 1.67-1.40 (8H, m); 1.38-
1.18 (26H, m); 0.86 (6H, t, J = 6.72 Hz). ESI-MS: m/z 393.6 ([M+Na]+).

Tridecan-7-yl 10-oxodecanoate:

O O

OHC

The above alcohol (3.7 g, 10.0 mmol) was dissolved in dry CH2Cl2 under argon and 4A0
molecular sieves then PCC (3.2 g, 15.0 mmol) was added by fractions to the reaction
mixture over a period of 10 min. The reaction mixture was stirred for 2hr at room
temperature and filtered through silica pad to remove PCC followed by evaporating the
solvent to yield 92% (3.3 g) of crude aldehyde. 1H NMR (400 MHz, CDCl3): δ 9.74 (1H,
t, J = 1.69 Hz); 4.85 (1H, quint, J = 6.78 Hz); 2.40 (2H, dt, J = 7.57, 1.89 Hz); 2.26 (2H, t,
J = 7.57 Hz); 1.70-1.54 (4H, m); 1.52-1.40 (4H, m); 1.36-1.13 (24H, m); 0.86 (6H, t, J =
6.72 Hz).

di(tridecan-7-yl) 10,10'-((2-hydroxyethyl)azanediyl)bis(decanoate)
 O O

HO
N

O O

The above aldehyde (0.54 g, 1.46 mmol, 2 equiv.) and Ethanol amine (44.0 mg, 0.73 mmol,
1equiv.) were dissolved in dry CH2Cl2 under nitrogen atmosphere then sodium tri
acetoxyborohydride (0.46 g, 2.10 mmol, 3 equiv.) was added to the reaction mixture and
stirred for 12h at room temperature. The reaction mixture was quenched with sat. NaHCO3
and followed by extract with CH2Cl2 (3 times) and washed with brine solution and dried
over with anhydrous Na2SO4. The solvent was evaporated and purified by column
chromatography yielded 76 % (0.42 g) as colorless liquid. 1H NMR (400 MHz, CDCl3): δ
4.86 (2H, quint, J = 6.78 Hz); 3.59 (2H, t, J = 5.38 Hz); 2.66 (2H, t, J = 5.38 Hz); 2.58-
2.48 (4H, m); 2.27 (4H, t, J = 7.56 Hz); 1.68-1.54 (4H, m); 1.52-1.40 (4H, m); 1.36-1.13
(52H, m); 0.86 (12H, t, J = 6.79 Hz). ESI-MS: m/z 767.1 ([M+1]+).
di(tridecan-7-yl) 10,10'-((2-((4-
(dimethylamino)butanoyl)oxy)ethyl)azanediyl)bis(decanoate)(Lipid 13):
O O
O
N O
N

O O
EA-402

The above alcohol (0.76g, 1.0 mmol, 1 equiv.), N,N-dimethyl aminobutyric acid
hydrochloride (0.25 g, 1.5mmol, 1.5 equiv.) and EDC (0.38 g, 2.0 mmol, 2 equiv.) and
DMAP (cat.) were dissolved in dry CH2Cl2 under nitrogen atmosphere. Then the reaction
stirred for 24h. The reaction mixture was quenched with sat. NaHCO3 and followed by
extract with CH2Cl2 (3 times) and washed with brine solution and dried over with
anhydrous Na2SO4. The solvent was evaporated and the reaction mixture was purified by
column chromatography to yielded 75 % (0.65 g) of pure Lipid 13 as pale yellow liquid.
1
H NMR (400 MHz, CDCl3): δ 4.86 (2H, quint, J = 6.78 Hz); 4.10 (2H, t, J = 6.41 Hz);
2.66 (2H, t, J = 6.41 Hz); 2.42 (4H, t, J = 7.41 Hz); 2.33 (2H, t, J = 7.41 Hz); 2.26 (6H, t,
J = 7.41 Hz); 2.20 (6H, s); 1.77 (4H, quint, J = 7.41 Hz); 1.60 (4H, quint, J = 7.41 Hz);
1.55-1.45 (8H, m); 1.44-1.35 (4H, m); 1.36-1.13 (52H, m); 0.87 (12H, t, J = 6.83 Hz).
ESI-MS: m/z 880.12 ([M+1]+);441.75 ([M/2+1]+).

di(tridecan-7-yl) 10,10'-(hydroxyazanediyl)bis(decanoate):
O O

HO N

O O

The above aldehyde (1.4 g, 4.0 mmol, 2 equiv.) and hydroxylamine hydrochloride (0.13 g,
2.0 mmol, 1 equiv.) were dissolved in dry CH2Cl2 under argon atmosphere, than sodium
triacetoxyborohydride (1.2 g, 6.0 mmol, 3 equiv.) was added to the reaction mixture and
stirred for 12h at room temperature. The reaction mixture was quenched with sat. NaHCO3
and followed by extract with CH2Cl2 (3 times) and washed with brine solution and dried
over with anhydrous Na2SO4. The solvent was evaporated and the residue was purified by
silica column chromatography (EtOAc: Hexane (5:95)to yield 65 % (0.97 g) of pure
colorless oil. 1H NMR (400 MHz, CDCl3): δ 4.86 (2H, quint, J = 6.32 Hz); 2.61(4H, t, J =
7.34 Hz); 2.27 (4H, t, J = 7.78 Hz); 1.70-1.40 (16H, m); 1.38-1.14 (52H, m); 0.88 (12H, t,
J = 6.89 Hz). ESI-MS: m/z 738.6 ([M+1]+).

di(tridecan-7-yl) 10,10'-(((4-
(dimethylamino)butanoyl)oxy)azanediyl)bis(decanoate)(Lipid 7):
O O

O N
N
O
O O

The above hydroxylamine (0.5g, 0.67 mmol, 1 equiv.), N, N-dimethyl aminobutyric acid
hydrochloride (0.16 g, 1.0 mmol, 1.5equiv.), EDC (0.25g, 1.34 mmol, 2 equiv.) and DMAP
(cat.) were dissolved in dry CH2Cl2 under nitrogen atmosphere. Then the reaction was
stirred for 16hr. The reaction mixture was quenched with sat. NaHCO3 and followed by
extract with CH2Cl2 (3 times) and washed with brine solution and dried over with
anhydrous Na2SO4. The solvent was evaporated and the residue was purified by silicagel
column chromatography (MeOH: CHCl3 (3:97) to yield 0.44 g (75%) of pure lipid as
colorless oil. 1H NMR (400 MHz, CDCl3): δ 4.85 (2H, quint, J = 6.32 Hz); 2.78 (4H, t, J
= 7.61 Hz); 2.31 (4H, t, J = 7.32 Hz); 2.26 (4H, t, J = 7.32 Hz); 2.20 (6H, s); 1.80 (2H,
quint, J = 7.73 Hz); 1.59 (4H, quint, J = 7.78 Hz); 1.55-1.40 (12H, m); 1.38-1.18 (52H, m);
0.88 (12H, t, J = 6.89 Hz). ESI-MS: m/z 852.2 ([M+1]+).
Synthesis of Lipid 8:

Linoleic aldehyde(2.64 g, 10.0 mmol, 2 equiv.) and ethanolamine (0.30 g, 5.0 mmol, 1
equiv.) were dissolved in dry CH2Cl2 under nitrogen atmosphere, then sodium tri
acetoxyborohydride (3.1 g, 15.0 mmol, 3 equiv.) was added, and the reaction mixture was
stirred for 16 hr at room temperature. The reaction mixture was quenched with sat.
NaHCO3 solution followed by extract with CH2Cl2 (3 times) washing with brine solution
and dried over with anhydrous Na2SO4.The solvent was evaporated and the residue was
purified by column chromatography (MeOH: CHCl3 (2: 98) to yield 2.3 g (85%) of pure
yellowish color compound EA-1. 1H NMR (400 MHz, CDCl3): δ 5.35-5.46 (8H, m); 3.52
(2H, t, J = 5.38 Hz); 2.77 (4H, t, J = 6.37 Hz), 2.57 (2H, t, J = 5.38 Hz); 2.43-2.49 (4H,
m); 2.05 (8H, q, J = 6.73, 6.75 Hz), 1.43-1.48 (4H, m); 1.32-1.38 (32H, m); 0.89 (6H, t, J
= 6.85 Hz). ESI-MS: m/z 558 [M+1]+

HO Na(OAc)3BH
NH2
dry DCM

N
HO
EA-1

O
EDC, DMAP
N dry DCM
OH

O
N N
O

Lipid 8

N,N-dilinoleyl-aminoethanol (EA-1) (0.55g, 1.0 mmol, 1 equiv.), N,N-dimethyl

aminobutyric acid (0.33g, 2.0 mmol, 2 equiv.), EDC (0.38g, 2.0 mmol, 2 equiv.) and
DMAP (0.01g, 0.01 mmol, 0.1 equiv.) were dissolved in dry CH2Cl2 under nitrogen
atmosphere and reaction mixture was stirred for 24hr. The reaction mixture was quenched
with sat. NaHCO3 and followed by extract with CH2Cl2 (3 times) and washed with brine
solution and dried over with anhydrous Na2SO4. The solvent was evaporated and the
residue was purified by silicagel column chromatography (MeOH: CHCl3 (3: 97) or
EtOAc: CHCl3 (30:70) to yield 0.46 g (70%) of pure Lipid 8 as colorless liquid.
1
H NMR (400 MHz, CDCl3): δ 5.27-5.45 (8H, m); 4.12 (2H, t, J = 6.33 Hz); 2.77 (4H, t, J
= 6.37 Hz); 2.67 (2H, t, J = 6.34 Hz); 2.38-2.49 (4H, m), 2.19-2.38 (4H, m); 2.21 (6H, s);
2.05 (8H, q, J = 6.82, 6.84 Hz); 1.70-1.85 (4H, m); 1.20-1.50 (36H, m), 0.9 (6H, t, J = 6.85
Hz). ESI-MS: m/z 671 ([M]+), 672 ([M+1]+).
Synthesis of lipid 9:
N
HO
EA-1

MsCl, NEt3
dry CH2Cl2

N
MsO
EA-OMs

N NaH
OH dry THF

N N
O

Lipid 9

To a stirred solution of EA-1 (0.55 g, 1.0 mmol, 1 equiv.) and NEt3 (0.28 mL, 2.0 mmol,
2.0 equiv.) in dry CH2Cl2 (10 mL) was added methanesulponyl chloride (Ms-Cl) (4.78 g,
31.8 mmol, 1.2 equiv.) at 0oC portion-wise over a period of 5 min. The reaction mixture
was stirred at room temperature for 6h. The mixture was then diluted with CH2Cl2 and
washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated. The crude EA-OMs product was used for the next reaction without further
purification.

To the solution of NaH (60.0 mg, 60% NaH in oil, 1.5mmol, 1.5 equiv.) and 4-
(dimethylamino)butan-1-ol (0.17 g, 1.5 mmol, 1.5 equiv.) were taken in a 50 mL flask and
dissolved in dry THF at 00C. The reaction mixture was stirred at 00C for 30 min. Then the
EA-OMs compound (0.63 g, 1.0 mmol, 1equiv.) added drop wise to the reaction mixture
warms it to room temperature and stirred it for rt 6 hr. The reaction mixture was quenched
with ice at 00C and added water and extracted with CH2Cl2 (3x20 mL) washed with brine
solution dried over Na2SO4. The crude compound was purified by silica gel column
chromatography to give 0.42g of Lipid 9 (yield 65%) as pure yellow color oil. 1H NMR
(400 MHz, CDCl3): δ 5.44-5.24 (8H, m); 3.80-3.72 (2H, m); 3.69 (2H, t, J = 5.57 Hz); 3.58
(2H, t, J = 5.57 Hz); 3.36 (6H, s); 2.97-2.83 (2H, m); 2.76 (4H, t, J = 6.73Hz); 2.45 (4H, t,
J = 7.30 Hz); 2.04 (8H, q, J = 6.73 Hz); 2.0-1.90 (2H, m); 1.64 (2H, qint, J = 6.73, 5.57
Hz);1.49-1.15 (36H, m); 0.88 (6H, t, J = 6.96 Hz). ESI-MS: m/z 658.0 (M+1)+; 329.5
(M/2+1)+
Synthesis of Lipid 10:

N
OH
EA-1

EDC, DMAP (cat.) O N N

dry DCM, rt, 24 h
65% HO O N N

O
N

Lipid 10

N,N-dilinoleyl-aminoethanol (EA-1) (0.55 g, 1.0 mmol, 1 equiv.), N-methyl

piperizinepropanoicacid(0.25 g, 1.5 mmol, 1.5 equiv.) and EDC (0.38 g, 2.0 mmol, 2
equiv.) and DMAP (0.01 g, 0.1 mmol, 0.1 equiv.) were dissolved in dry CH2Cl2 under
argon atmosphere. Then the reaction was stirred for 24hr. The reaction mixture was
quenched with sat. NaHCO3 and followed by extract with CH2Cl2 (3 times) and washed
with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated
and the reaction mixture was purified by silicagel column chromatography (MeOH: CHCl3
(4: 96) to yielded 0.46 g (65%) of pure Lipid 10 as colorless liquid. 1H NMR (400 MHz,
CDCl3): δ 5.23-5.49 (8H, m); 4.13 (2H, t, J = 6.31 Hz); 2.78 (4H, t, J = 6.36 Hz); 2.63-
2.73 (4H, m); 2.50 ( 2H, t, J = 6.31 Hz); 2.37-2.49 (4H, m); 2.27 (3H, s); 1.95-2.12 (8H, q,
J = 6.78, 6.82 Hz ); 1.20-1.50 (36H, m); 0.89 (6H, t, J = 6.88 Hz ). ESI-MS: m/z 712
([M+1]+).
Synthesis of lipid 12:
3N NaOH
OH IBX
HO OH
O 1,4-dioxane O
O O DMSO
O

cis-3-nonen-1-ol
EDC, DMAP (Cat.),
DCM
O

HO O O
N Ethanol amine
OHC O
NaBH (OAc)3
O
DCM
O

EDC, DMAP (Cat.), OH

N
DCM O

O O
N N
O
O

O
Lipid 12
6-hydroxyhexanoic acid:
HO COOH

𝜖𝜖-caprolactone (2.2 g, 20 mmol) was dissolved in dioxane 6 mL, and 50 ml of a 3M NaOH

solution was added. The mixture was stirred at room temperature overnight. The solution
was washed with ethyl acetate to remove some organic impurities. The aqueous layer was
acidified to pH 3–4 with concentrated HCl 37% and then extracted with ethyl acetate (2 ×
50 mL). The organic layer was washed with saturated NaCl (2x50 mL), dried with Na2SO4,
and filtered. The organic layer was concentrated in vacuum to yield 90% (2.0 g) as colorless
oil. 1H NMR (400 MHz, CDCl3): δ 3.62 (2H, t, J = 6.30 Hz); 2.33 (2H, t, J = 7.45 Hz);
1.63 (2H, quint); 1.55 (2H, q, J = 7.45 Hz); 1.42-1.32 (2H, m).

(Z)-non-3-en-1-yl 6-oxohexanoate:
O

OHC O

IBX (1.0 g, 3.5 mmol, 1.5 equiv.) was added to a solution of the hydroxyl acid (0.60 g, 4.5
mmol) in DMSO (10 mL). The mixture was stirred for 6 h and quenched by addition of
water. The precipitate was removed by filtration. Extraction with ethyl acetate (2x50 mL),
anhydrification with Na2SO4, and removal of the solvent in vacuo gave almost pure oxo
acid yield 0.46 g (78%) as colorless oil. 1H NMR (400 MHz, CDCl3): δ 9.48 (1H, t, J =
1.57 Hz); 2.33 (2H, t, J = 7.45 Hz); 2.20 (2H, t, J = 7.20 Hz); 2.10-1.95 (2H, m); 1.45-1.32
(2H, m).
The above 7-oxoheptanoic acid (0.26 g, 2.00 mmol) and cis-3-nonen-1-ol (0.28 g, 2.0
mmol, 1 equiv.) taken in 50 mL flask and dissolved in dry CH2Cl2 and EDC (0.27g, 1.4
mmol) was added to the reaction mixture followed by DMAP (cat.) stirred for overnight.
The reaction mixture was quenched with sat. NaHCO3 and followed by extract with CH2Cl2
(3 times) and washed with brine solution and dried over with anhydrous Na2SO4. The crude
mixture was purified by silica gel column chromatography yielded 85% (0.44 g) pure
compound as liquid. 1H NMR (400 MHz, CDCl3): δ 9.75 (1H, t, J = 1.69 Hz), 5.55-5.42
(1H, m); 5.37-5.25 (1H, m); 4.05 (2H, t, J = 7.27 Hz); 2.50-2.40 (2H, m); 2.38-2.25 (4H,
m); 2.02 (2H, q, J = 7.27 Hz); 1.70-1.60 (4H, m); 1.40-1.20 (6H, m ); 0.86 (3H, t, J = 6.95
Hz ).ESI-MS: m/z 277 ([M+Na]+).
di((Z)-non-3-en-1-yl) 6,6'-((2-hydroxyethyl)azanediyl)dihexanoate
O O

HO
N

O O

The above aldehyde (1.56 g, 6.08 mmol, 2 equiv.) and ethanol amine (0.18 mL, 3.0 mmol,
1equiv.) were dissolved in dry CH2Cl2 under nitrogen atmosphere then sodium tri
acetoxyborohydride (1.80 g, 9.10 mmol, 3 equiv.) was added to the reaction mixture and
stirred for 12h at room temperature. The reaction was quenched with sodium bicarbonate
solution and extract with CH2Cl2 followed by washing with water and brine solution. The
solvent was evaporated and purified by column chromatography yielded 1.2 g (76%) of
pure lipid as colorless liquid. 1H NMR (400 MHz, CDCl3): δ 5.55-5.43 (2H, m); 5.37-5.27
(2H, m); 4.07 (4H, t, J = 6.87 Hz); 3.56 (2H, t, J = 5.50 Hz); 2.62 (2H, t, J = 5.50Hz); 2.50
(4H, t, J = 7.69 Hz); 2.36 (4H, q, J = 7.42 Hz); 2.29 (4H, t, J = 7.42 Hz); 2.02 (4H, q,
J = 7.42 Hz); 1.62 (4H, quint, J = 7.41, 7.58 Hz); 1.47 (4H, quint, J = 7.41, 7.58 Hz); 1.20-
1.40 (16H, m); 0.87 (6H, t, J = 6.87 Hz). ESI-MS: m/z 538.7 ([M+1]+)
di((Z)-non-3-en-1-yl)6,6'-((2-((4-
dimethylamino)butanoyl)oxy)ethyl)azanediyl)dihexanoate (Lipid 12)
O O

O
N N
O

O O

The above alcohol (0.53 g, 1.0 mmol, 1 equiv.), N,N-dimethyl aminobutyric acid
hydrochloride (0.25g, 1.5 mmol, 1.5 equiv.) and EDC (0.38 g, 2.0 mmol, 2 equiv.) and
DMAP (cat.) were dissolved in dry CH2Cl2 under nitrogen atmosphere. The reaction
mixture was quenched with sat. NaHCO3 and followed by extract with CH2Cl2 (3 times)
and washed with brine solution and dried over with anhydrous Na2SO4. The solvent was
evaporated and the reaction mixture was purified by column chromatography to yielded
72% (0.46 g) of pure Lipid 12 as pale yellow liquid. 1H NMR (400 MHz, CDCl3): δ5.53-
5.39 (2H, m); 5.35-5.22 (2H, m); 4.05 (2H, t, J = 6.39 Hz); 4.01 (4H, t, J = 6.85 Hz); 2.61
(2H, t, J = 6.39Hz); 2.39 (4H, t, J = 7.47 Hz); 2.35-2.22 (12H, m); 2.16 (6H, s); 1.99 (4H,
q, J = 6.39 Hz); 1.73 (2H, quint, J = 7.41, 7.85 Hz); 1.58 (4H, quint, J = 7.41, 7.85 Hz);
1.45-1.34 (4H, m);1.32-1.17 (16H, m); 0.84 (6H, t, J = 6.87 Hz). ESI-MS: m/z 651.8
([M+1]+); 326.6 ([M/2+1]+)
Synthesis of lipid 14
HO
TBDPS-Cl, Imidazole HO
OH OTBDPS
DCM
1,8-Octane diol

EDC, 5
DMAP (cat.) OH
DCM 7
O

HO O
TBDPSO O
O
O
TBAF, THF
rt
PCC, DCM

OHC O

Ethanol amine,
NaBH(OAc)3 DCM

O
O
HO
N
O

OH
N EDC, DMAP (cat.)
O DCM

O
O
O
N N
O O

O
Lipid 14

8-((tert-butyldimethylsilyl)oxy)octan-1-ol:

TBDPSO
OH

To a solution of 1,8-octanediol (2.9 g, 20.0 mmol) in dry CH2Cl2 (100 mL), imidazole (2.99
g, 44.0 mmol) was added and after 10 min tert-butyldiphenylsilylchloride (TBDPS-Cl) (6.0
g, 22.0 mmol). The reaction was stirred at room temperature until TLC analysis showed
the complete conversion (12 h). Excess of solvents was removed under diminished pressure
and water was added to the residue. The aqueous layer was extracted with CH2Cl2 (3 times).
The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The resulting
crude oil residue purified by silica column chromatography EtOAc:Hexane (10:90) yield
90% (7.3g)
as colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.70-7.65 (4H, m); 7.46-7.34 (6H, m); 3.66
(2H, t, J = 6.76 Hz); 3.63 (2H, t, J = 6.83 Hz); 1.56 (4H, quint, J = 7.23 Hz); 1.41- 1.22
(8H, m); 1.05 (9H, s). ESI-MS: m/z 385.6 [M+1]+

8-((tert-butyldimethylsilyl)oxy)octyl 2-hexyldecanoate:
TBDPSO
O

A mixture of the 2-hexyl decanoicacid (0.87 g, 3.1 mmol), silyl protectedoctanol (1.2 g,
3.1 mmol), EDC (4.65 g, 7.8 mmol), and 4-(dimethylamino) pyridine (DMAP) (cat.) was
dissolved in CH2Cl2 (25 mL) and stirred overnightat room temperature. The resulting
mixture was quenched with sat. NaHCO3 and extracted with CH2Cl2 (3x 20 ml) and washed
by brine solution. The organic layer was then dried over Na2SO4, filtered, and concentrated
under reduced pressure. The crude product was purified via flash chromatography using
5:95 EtOAc/hexane as eluent to give the ester as colorless oil (1.6 g, 82 % yield). 1H NMR
(400 MHz, CDCl3): δ 7.70-7.63 (4H, m); 7.46-7.34 (6H, m); 4.06 (2H, t, J = 6.86 Hz); 3.65
(2H, t, J = 6.56 Hz); 2.37-2.26(1H, m); 1.98-1.88 (2H, m); 1.80- 1.70 (2H, m); 1.66- 1.50
(6H, m); 1.49-1.39 (2H, m); 1.39-1.16 (34H, m); 1.05 (9H, s); 0.87 (6H, t, J = 6.83 Hz).
ESI-MS: m/z 645.5 [M+Na]+; 545.5 [M-t-Bu]+
8-hydroxyoctyl 2-hexyldecanoate:
HO
O

A mixture of tetrabutyl ammonium fluoride (TBAF) (4.0 mL, 4.0 mmol, 1.0 M solution in
THF) was added to a solution of silyl ester (1.24 g, 2.0 mmol) in THF (15 mL).The reaction
mixture was stirred atrt for 3h and then quenched with sat. NH4Cl solution and extracted
with of EtOAc (3x30 mL). The organic layer was then washed with brine dried over
Na2SO4 filtered and concentrated under reduced pressure. The crude product was purified
via flash chromatography using 9:1 hexanes/ethyl acetate to give yield 90% (0.64 g)
alcohol as clear colorless oil. 1H NMR (400 MHz, CDCl3): δ 4.06 (2H, t, J = 6.67 Hz); 3.62
(2H, t, J = 6.62 Hz); 2.35-2.25 (1H, m); 1.66-1.48 (6H, m); 1.46- 1.16 (30H, m); 0.86 (6H,
t, J = 6.87 Hz). ESI-MS: m/z 385.4 [M+1]+; 407.4 [M+Na]+
8-oxooctyl 2-hexyldecanoate:

OHC O

8-hydroxyoctyl 2-hexyldecanoate (1.15 g, 3.0 mmol, 1equiv.) taken in 100 mL flask and
dissolved in dry CH2Cl2 followed by portion wise slowly addition of PCC (0.96 g, 4.5
mmol, 1.5 equiv.) over 15 min. Then, the reaction mixture was stirred at room temperature
for 2h. The crude reaction mixture was filtered through the silica gel pad and washed with
CH2Cl2 (2x20 mL). The organic solvent dried over with Na2SO4 and solvent removed by
under reduced pressure crude hydroxyl aldehyde yield 82% (0.93 g) as used directly
without any further purification. 1H NMR (400 MHz, CDCl3): δ 9.75 (1H, t, J = 1.87 Hz );
4.05 (2H, t, J = 6.73 Hz), 2.41 (2H, dt, J = 7.41, 1.87 Hz ); 2.35-2.23 (1H, m); 1.70- 1.50
(8H, m); 1.47-1.30 (2H, m); 1.28-1.16 (24H, m); 0.86 (6H, t, J = 6.87 Hz).

((2-hydroxyethyl)azanediyl)bis(octane-8,1-diyl) bis(2-hexyldecanoate):

O
O
HO
N
O

The above aldehyde (0.76 g, 2.0 mmol, 2 equiv.) and Ethanol amine (0.06 g, 1.0 mmol, 1
equiv.) were dissolved in dry CH2Cl2 under nitrogen atmosphere then sodium tri
acetoxyborohydride (0.63 g, 3.0 mmol, 3 equiv.) was added to the reaction mixture and
stirred for 12h at room temperature. The reaction mixture was quenched with sat. NaHCO3
and followed by extract with CH2Cl2 (3 times) and washed with brine solution and dried
over with anhydrous Na2SO4. The solvent was evaporated and purified by column
chromatography yielded 0.62 g (80%) of pure lipid as colorless liquid. 1H NMR (400 MHz,
CDCl3): δ 4.05 (4H, t, J = 6.67 Hz); 3.54 (2H, t, J = 5.57 Hz); 2.59 (2H, t, J = 5.57 Hz);
2.46 (4H, t, J = 7.65 Hz); 2.34-2.24 (2H, m); 1.68-1.50 (8H, m); 1.48-1.36 (8H, m); 1.36-
1.16 (56H, m); 0.86 (12H, t, J = 6.87 Hz). ESI-MS: m/z 795.9 ([M+1]+).
((2-((4-(dimethylamino)butanoyl)oxy)ethyl)azanediyl)bis(octane-8,1-diyl) bis(2-
hexyldecanoate) (Lipid 14):

O
O
O
N N
O O

The above alcohol (0.79 g, 1.0 mmol, 1 equiv.), N,N-dimethyl aminobutyric acid
hydrochloride (0.25g, 1.5 mmol, 1.5 equiv.) and EDC (0.38 g, 2.0 mmol, 2 equiv.) and
DMAP (cat.) were dissolved in dry CH2Cl2 under nitrogen atmosphere. Then the reaction
stirred for 24h. The reaction mixture was quenched with sat. NaHCO3 and followed by
extract with CH2Cl2 (3 times) and washed with brine solution and dried over with
anhydrous Na2SO4. The solvent was evaporated and the reaction mixture was purified by
column chromatography to yielded 72% (0.65 g) of pure lipid as pale yellow liquid. 1H
NMR (400 MHz, CDCl3): δ 4.11 (2H, t, J = 6.43 Hz); 4.05 (4H, t, J = 6.73 Hz); 2.66 (2H,
t, J = 6.37 Hz); 2.43 (4H, t, J = 7.76 Hz); 2.37-2.25 (6H, m); 2.21 (6H, s); 1.78 (2H, quint,
J = 7.39 Hz); 1.68-1.50 (8H, m); 1.48-1.36 (8H, m); 1.36-1.16 (56H, m); 0.86 (12H, t, J =
6.87 Hz). ESI-MS: m/z 908.3 ([M+1]+).
Figure S1: Transmission electron microscopic (TEM) images of lipids 2, 6, 9, 13 and 14-
based LNPs. Scale bar 200nm.
Figure S2: In vitro safety study of LNPs in multiple myeloma cells; U266 cells were treated
by LNPs containing NC-siRNA at 0.06µM for 72hrs. Cell viability was measured by XTT
assay. Results are average ± SD from triplicate experiments.
Figure S3: Therapeutic study of LNPs in multiple myeloma cells; U266 cells were treated
by LNPs containing siRNA against PLK1 at different doses for 72hrs. Cell viability was
measured by XTT assay. Results are average ± SD from triplicate experiments
*p<0.05, **p<0.001, ***p<0.0001 compared to untreated cells (two-sided student’s T-
test).
Figure S4: Dose-dependent safety study of LNPs in multiple myeloma cells; U266 cells
were treated by LNPs containing siNC at different doses for 72hrs. Cell viability was
measured by XTT assay. Results are average ± SD from triplicate experiments.
Figure S5: PI-AnnexinV-APC analysis of U266 cells after the treatment with MC3, lipid 8
and 10 based-LNPs encapsulated with either siPLK1 or siNC5 at 0.06 µM siRNA
concentration. Cells were collected after 72hr after the treatment, incubated with PI and
annexin V-APC and analyzed by FACS.
Figure S6: Bio-distribution of lipid 8 and 10 based-LNPs in mice 2hr post i.v
administration. Fluorescent images of mouse organs (A) Lipid 10 based-LNPs; (B) Lipid
8 based-LNPs; (C) Corresponding histogram analysis of Cy5 quantity. (n=3, results are
average ± SD, from 2 independent experiments).
Figure S7: Bio-distribution of lipid 2 and 6 based-LNPs in mice 2hr and 24hr post i.v
administration. Fluorescent images of mouse organs and corresponding histogram analysis
of Cy5 quantity (A, B) Lipid 6 based-LNPs; (C, D) Lipid 2 based-LNPs; (n=3, results are
average ± SD, from 2 independent experiments).
Figure S8: Fluorescent intensity of LNPs at different pH values measured by TNS assay.
(n=3, reproduced with two independent experiments).

Figure S9: In vivo CD45 gene silencing efficiency of lipid 2-based tLNPs (A) and lipid 6-
based tLNPs (B) in CD4+ and CD8+ T-lymphocytes (n=4, single experiment).
Figure S10: Stability of LNPs at different serum concentrations. LNPs were incubated with
PBS with or without fetal calf serum (10% or 50%) for 24h in 37 0C, followed by treatment
with or without Triton X-100 (0.5%). The samples then were resolved on agarose gel (2%)
and visualized by the bio-imaging system.

Figure S11: Immune activation study: LNPs administered intravenously into the mice at
1mg/kg siRNA dose. Blood was collected at 2 and 24h post-injection and analyzed for
cytokines expression (n=5).
Figure S12: Gating strategy for the FACS analysis of figure 4D-F.
 WBC RBC HGB Hemat MCV MCH MCHC Neut Lymp Mono Eos Baso Platelt

UT 2.94 9.43 13.9 46.26 49.1 14.74 30.1 13.78 83.62 0.74 1.48 0.18 919
±0.4 ±0.3 ±0.4 ±1.5 ±3.1 ±0.2 ±1.6 ±8.6 ±8.2 ±0.7 ±1.3 ±0.1 ±160
Lipid 2 3.098 9.196 13.42 43.66 47.88 14.64 30.76 18.88 77.28 1.18 2.34 0.14 1012
2h ±0.99 ±1.44 ±1.75 ±5.404 ±4.45 ±0.561 ±2.32 ±2.84 ±4.72 ±1.25 ±3.46 ±0.195 ±265.1
Lipid 2 4.342 9.416 13.94 48.36 51.3 14.78 28.9 13.58 82.48 1.18 2.18 0.14 915
24h ±0.728 ±0.27 ±0.44 ±3.1 ±2.18 ±0.18 ±1.03 ±4.2 ±2.91 ±0.66 ±1.43 ±0.09 ±161.5
Lipid 6 3.816 9.584 14.04 47.96 50.1 14.62 29.28 21.3 71.54 2.38 4.32 0.12 850
2h ±0.875 ±0.27 ±0.42 ±1.63 ±2.74 ±0.14 ±1.7 ±2.63 ±6.1 ±0.9 ±3.8 ±0.13 ±132.9
Lipid 6 5.574 9.858 14.28 48.26 48.98 14.48 29.66 18.86 77.4 1.66 1.54 0.14 953
24h ±1 ±0.28 ±0.32 ±2.8 ±3.36 ±0.19 ±1.72 ±6.66 ±4.77 ±1.67 ±1.43 ±0.134 ±72.42
Lipid 8 4.4 9.92 14.6 48.26 48.56 14.68 30.32 23.98 70.84 2.24 2.68 0.12 1020.2
2h ±0.53 ±0.7 ±1.2 ±5.1 ±3.18 ±0.45 ±1.42 ±4.72 ±5.81 ±3.1 ±2.3 ±0.18 ±0.18
Lipid 8 5.580 9.7 14.4 48.6 50.1 14.82 29.65 12.1 83.67 1.42 2.175 0.2 1096
24h ±1.82 ±0.04 ±0.21 ±1.78 ±1.86 ±0.22 ±0.97 ±1.9 ±2.84 ±0.95 ±0.9 ±0.141 ±112.4
Lipid 10 4.744 10.24 15.28 51.300 50.1 14.92 29.86 47.86 42.7 2.54 6.6 0.1 1012.4
2h ±0.79 ±0.08 ±0.29 ±0.2.3 ±2.2 ±0.18 ±1.155 ±7.213 ±7.87 ±1 ±4.12 ±0.07 ±98.5
Lipid 10 3.56 9.52 14.08 47.92 50.28 14.78 29.4 13.18 82.06 1.36 2.94 0.14 892.4
24h ±0.98 ±0.62 ±0.1 ±4.38 ±0.53 ±0.164 ±0.616 ±3.17 ±5.23 ±0.814 ±2.4 ±0.114 ±393.2
Lipid 13 5.812 9.804 14.56 49.86 50.86 14.82 29.2 40.76 52.74 2.46 3.4 0.24 1006.6
2h ±0.76 ±0.18 ±0.35 ±0.96 ±0.55 ±0.28 ±0.255 ±8.65 ±6.95 ±0.83 ±2.94 ±0.114 ±109
Lipid 13 5.182 9.604 14.22 48.02 50.0 14.78 29.64 15.02 82.54 1.12 1.04 0.1 560.0
24h ±0.712 ±0.49 ±0.62 ±2.92 ±2.32 ±0.268 ±0.96 ±6.63 ±6.78 ±1.55 ±1.42 ±0.141 ±187.25

Table S1: LNPs based on the different lipids (2, 6, 8 and 10) were administered
intravenously to mice at 1mg/kg siRNA dose.
Blood cell count was measured, 2h and 24h post-injection of LNPs.
 Creat Calc Phos Glu Urea Chole TP Alb Glob Biliru Alk SGOT SGPT Na K Chlor
mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl g/dl g/dl g/dl mg/dl Phos IU/L IU/L mmol/L mmol/L mmol/L
IU/L
UT 0.26 9.84 9.88 125.8 42.54 102.8 6.89 3.98 2.91 0.12 147.4 132.4 36.60 156.4 5.78 115.2
±0.04 ±0.19 ±1.37 ±8.2 ±3.07 ±9.34 ±0.24 ±0.28 ±0.07 ±0.02 ±26.91 ±21.87 ±8.73 ±1.52 ±0.48 ±0.4
Lipid 8 0.31 10.35 14.04 99.2 39.72 104.4 7.72 4.50 3.22 0.14 157.8 171.25 73.80 161.6 7.44 118.4
2h ±0.04 ±0.36 ±2.96 ±12.01 ±4.86 ±3.85 ±0.37 ±0.19 ±0.24 ±0.02 ±20.13 ±23.87 ±25.33 ±2.61 ±1.55 ±2.3
Lipid 8 0.25 10.22 8.04 132.0 36.98 105.6 5.90 4.22 1.68 0.12 144.2 155.6 41.20 159.6 6.26 120.0
24h ±0.03 ±0.28 ±1.05 ±16.26 ±8.03 ±4.51 ±0.18 ±0.2 ±0.16 ±0.06 ±15.22 ±15.66 ±11.97 ±1.14 ±0.47 ±1.22
Lipid 10 0.23 10.32 13.40 107.8 37.22 101.4 6.11 4.42 1.69 0.16 129.0 232.0 68.0 164.4 7.12 119.2
2h ±0.13 ±0.58 ±2.73 ±27 ±3 ±14.57 ±0.46 ±0.29 ±0.22 ±0.03 ±22.35 ±41.92 ±15.56 ±2.07 ±1.45 ±6.3
Lipid 10 0.27 10.34 9.46 138.6 32.46 110.8 6.16 4.240 1.92 0.11 132.60 152.40 35.40 161.6 6.94 122.0
24h ±0.03 ±0.28 ±1.51 ±13.11 ±3.4 ±13.81 ±0.23 ±0.11 ±0.27 ±0.02 ±25.33 ±30.42 ±3.78 ±3.78 ±0.47 ±1
Lipid 6 0.29 10.36 10.38 137.6 38.08 96.0 5.94 4.26 1.68 0.09 165.6 136.2 63.20 158.8 6.76 120.0
2h ±0.03 ±0.21 ±0.86 ±14.05 ±3.01 ±7.97 ±0.18 ±0.15 ±0.11 ±0.02 ±17.37 ±46.51 ±38.13 ±1.48 ±0.78 ±1.58
Lipid 6 0.25 10.34 8.18 159.6 46.04 105.8 5.96 4.14 1.82 0.11 170.2 130.8 41.60 160.0 6.48 122.2
24h ±0.02 ±0.14 ±0.31 ±7.5 ±8.42 ±10.13 ±0.13 ±0.18 ±0.14 ±0.03 ±12.11 ±15.39 ±7.4 ±2.35 ±0.37 ±1.92
Lipid 2 0.23 10.03 11.10 127.6 36.84 82.2 5.56 4.02 1.54 0.13 153.4 122.4 45.80 157.4 7.64 120.4
2h ±0.04 ±0.21 ±0.77 ±26.64 ±4.45 ±13.22 ±0.33 ±0.15 ±0.18 ±0.05 ±22.5 ±23.35 ±4.44 ±1.82 ±0.57 ±1.14
Lipid 2 0.26 10.52 8.58 175.2 44.22 104.2 6.06 4.28 1.78 0.09 159.0 178.8 54.80 161.2 6.66 123.2
24h ±0.01 ±0.11 ±1.06 ±14.06 ±8.08 ±8.76 ±0.2 ±0.19 ±0.29 ±0.03 ±14.37 ±38.07 ±6.42 ±3.03 ±0.36 ±3.03
Lipid 13 0.27 10.25 11.06 141.0 42.90 92.80 5.90 4.24 1.66 0.12 174.0 191.8 87.20 159.4 7.70 120.8
2h ±0.02 ±0.3 ±0.43 ±12.39 ±2.18 ±7.92 ±0.15 ±0.09 ±0.09 ±0.04 ±7.75 ±26.83 ±27.23 ±1.34 ±0.16 ±1.64
Lipid 13 0.28 10.44 8.38 155.4 42.70 107.2 5.97 4.06 1.91 0.11 149.8 121.8 37.0 160.2 6.56 121.0
24h ±0.02 ±0.24 ±0.7 ±6.66 ±5.08 ±25.91 ±0.34 ±0.22 ±0.15 ±0.05 ±7.29 ±31.66 ±5.66 ±2.68 ±0.34 ±2.65

Table S2: LNPs based on the different lipids (2, 6, 8 and 10) were administered
intravenously to mice at 1mg/kg siRNA dose. Chemistry parameters were measured in the
serum, 2h and 24h post-injection of LNPs.