Amino acid-nucleotide-lipids: Effect of amino acid on the self-assembly properties

Article abstract of DOI:10.1021/la400515m

Hybrid amphiphiles composed of a lipid covalently linked to biomolecules are attracting considerable attention, owing to their unique physicochemical and biological properties. Herein, we have synthesized novel amino acid-nucleotide-lipids (ANLs), presenting phenylalanine and thymidine residues and saturated or unsaturated diacyl glycerol lipid moieties to investigate the effect of the specific aminoacid moieties on both aggregation properties and interactions of ANLs with single strand polyA RNA. Physicochemical studies (DLS, cryo-TEM, and small angle X-ray scattering) indicate that phenylanaline amino acids inserted at the 5′ position of the nucleotide-lipids stabilize multilamellar systems, whereas unilamellar vesicles are formed preferentially in the case of nucleotide-lipids (NLs). Both NLs and ANLs exhibit weak interactions with complementary polyA RNA as revealed by isothermal titration calorimetry investigations. The multilamellar vesicles obtained with ANLs could be used as a versatile carrier, suitable for both hydrophobic and hydrophilic therapeutic molecules.

Full text of DOI:10.1021/la400515m

Article
pubs.acs.org/Langmuir
Amino Acid−Nucleotide−Lipids: Effect of Amino Acid on the Self-
Assembly Properties
Giovanni Tonelli,†,‡,§,∥ Khalid Oumzil,†,‡ Frederic Nallet,§,∥ Cedric Gaillard,⊥ Laurence Navailles,*,§,∥
́
́
́
and Philippe Barthelemy*,†,‡
́
́
†Univ. Bordeaux, ARNA laboratory, F-33000 Bordeaux, France
‡INSERM, U869, ARNA laboratory, F-33000 Bordeaux, France
§Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France
∥CNRS, CRPP, UPR 8641, F-33600 Pessac, France
⊥INRA, UR 1268 Unite
́ ̀
Biopolymeres Interactions Assemblages, F-44300 Nantes, France
S
* Supporting Information
ABSTRACT: Hybrid amphiphiles composed of a lipid covalently linked to biomolecules are attracting considerable attention,
owing to their unique physicochemical and biological properties. Herein, we have synthesized novel amino acid−nucleotide−
lipids (ANLs), presenting phenylalanine and thymidine residues and saturated or unsaturated diacyl glycerol lipid moieties to
investigate the effect of the specific aminoacid moieties on both aggregation properties and interactions of ANLs with single
strand polyA RNA. Physicochemical studies (DLS, cryo-TEM, and small angle X-ray scattering) indicate that phenylanaline
amino acids inserted at the 5′ position of the nucleotide-lipids stabilize multilamellar systems, whereas unilamellar vesicles are
formed preferentially in the case of nucleotide−lipids (NLs). Both NLs and ANLs exhibit weak interactions with complementary
polyA RNA as revealed by isothermal titration calorimetry investigations. The multilamellar vesicles obtained with ANLs could
be used as a versatile carrier, suitable for both hydrophobic and hydrophilic therapeutic molecules.
stable aggregates highly loaded with an anticancer drug, namely
the cisplatin. The resulting nucleolipid-based nanoparticles
were found to be efficient vehicles for the delivery of cisplatin
into different cancer cell lines.
The interactions occurring between the polar heads
themselves or with a partner (nucleic acid), play a determinant
role in the formation of supramolecular assemblies, as
demonstrated by several studies.22−24 Interestingly, additional
moieties can be attached covalently to the nucleoside polar
heads in order to modulate these interactions. Among the
chemical variants of the nucleolipid structure, the incorporation
through 1,3-dipolar cycloaddition of sugar25 and aminoacid26 at
the 5′ positions has been previously described. Here, we report
INTRODUCTION
■
Recent years have witnessed intensive research activities with
the development of hybrid bioinspired amphiphiles based on
the combination of biological units, such as amino acids,1,2
peptides,3−6 sugar,7−11 and nucleic acids,12,13 with lipids. The
combination of a lipid with nucleosides/nucleotides was
initially designed to improve the cellular uptake of nucleoside
drugs14−16 in the treatment of cancers. More recently,
nucleolipids17,18 have been the object of intense investigations,
due to their unique self-organization properties and their
potential applications in the biomedical field. For example,
nucleolipids featuring reduced cytotoxicity have been used for
the transfection of DNA19 and siRNA.20 Likewise, we recently
demonstrated that nucleolipids can be used to control the
precipitation of anticancer drugs for the development of new
drug delivery systems (DDS).21 In this study, it was discovered
that the nucleoside polar heads allow the self-assembly into
Received: February 7, 2013
Revised: March 25, 2013
Published: April 8, 2013
© 2013 American Chemical Society
5547
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
Figure 1. Chemical structures of nucleotide and aminoacid−nucleotide−lipids (NL, up and ANL, down, respectively) used in this study.
followed by oxidation with 110 mL of a solution of I2 (0.02 M in
THF/Pyr/H2O). After 6 h at room temperature, the solvent was
evaporated under high vacuum and the intermediate products were
dissolved in 50 mL of ethyl acetate and then washed with 2 × 25 mL
of a saturated solution of Na2S2O3. The fractions were dried over
anhydrous sodium sulfatek, and the solvent was evaporated. The
resulting product was dissolved in 20 mL of DCM (dichloromethane)
and 15 mL of a solution of trichloroacetic acid (TCA, 3% in DCM)
under argon, after 4 h at room temperature, 20 mL of methanol and 25
mL of sodium hydrogen carbonate solution were added. The aqueous
phase was extracted with 2 × 25 mL of DCM, and then the organic
fractions were collected, dried over anhydrous sodium sulfate, and the
solvent evaporated. The intermediate product was dissolved in 35 mL
of DCM and 25 mL of triethylamine (TEA) and stirred overnight a
room temperature. The solvent was evaporated under high vacuum.
The product was isolated after purification on silica gel (DCM/
methanol/TEA from 98:1:1 to 90:9:1). Yield: 60%.
the synthesis of two new hybrid bioinspired amphiphiles
(Figure 1) having a double functionality based upon the
conjugation of phenylalanine with the 5′ hydroxyl group of
thymidine via an ester function. The base moiety on the polar
head can interact with a nucleic acid, and the amino acid
residue could increase this interaction. In this study, we
demonstrate that the insertion of this specific amino acid into
the nucleolipid structures strongly modifies their self-assembly
properties and promotes the formation of multilamellar
systems. We also show a strong dependence of the strength
and nature of the interaction between bilayers and between a
bilayer and a nucleic acid, according to the presence and the
nature of the amino acid.
MATERIALS AND METHODS
■
1H NMR (300 MHz, CDCl3): δ 0.84−0.91 (m, 6H, CH3, chain),
1.23−1.38 (m, 40H, 2CH2, chain + 9H, 3CH3, TEA) 1.53−1.64 (m,
4H, 2CH2, chain), 1.91 (s, 3H, CH3, base), 1.96−2.04 (m, 8H, chain),
2.24−2.39 (m, 5H, 2CH2, chain + H, 2′ sugar), 2.45−2.57 (m, 1H, 2′
sugar), 3.02−3.14 (m 6H, 3CH2, TEA), 3.77−4.42 (m, 7H, 2CH2,
glycerol + 1H, 4′ 2H, 5′ sugar), 4.85−4.97 (m, 1H, 3′ sugar), 5.17−
5.29 (m, 1H, glycerol), 5.31−5.37 (m, 4H, CHCH chain), 6.17−
6.23 (m, H, 1′ sugar), 7.55 (s, 1H, base). 13C NMR (75 MHz, CDCl3):
δ 8.61 (3C, 3CH3, TEA), 12.62 (1C, CH3, base), 14.15 (2C, CH3,
chain), 22.70 (2C, chain), 24.86 (2C, chain), 27.20−27.23 (4C,
chain), 29.13−29.80 (14C, chain), 31.93 (4C, chain), 34.06−34.24
(2C, chain), 38.98 (1C, 2′ sugar), 45.74 (3C, 3CH2, TEA), 61.93 (1C,
glycerol), 62.59 (1C, glycerol), 70.22 (1C, glycerol), 74.31 (1C,
4′sugar), 84.71 (1C, 3′ sugar), 85.70 (1C, 1′ sugar), 110.93 (1C, base),
129.70 (2C, CHCH, chain), 130.02 (2C, CHCH, chain), 136.16
(1C, base), 150.29 (1C, CO, base), 163.70 (1C, CO, base),
173.09 (1C, ester), 173.52 (1C, ester). 31P NMR (121 MHz, CDCl3):
δ 2.64. ESI-MS: Mw = 925.179; [M − H + 2Na]+ = 969.5
Materials. General Experiments and Analytical Conditions. All
starting materials and solvents were obtained from Aldrich, Bachem,
and Glen Research and were used without further purification. 1,2-
Dioleoyl-sn-glycerol was synthesized as reported.27 All compounds
were characterized using standard analytical and spectroscopic
1
techniques such as H, 13C, and 31P spectroscopy (apparatus Bruker
1
Avance DPX-300, H at 300.13 MHz, 13C at 75.46 MHz, and 31P at
121.49 MHz) and mass spectrometry. The NMR chemical shifts are
reported in parts per million relative to the tetramethylsilane, using the
deuterium signal of the solvent (CDCl3, deuterated dimethylsulfoxide
DMSO-d6 or pyridine-d5) as a heteronuclear reference for 1H. The 1H
NMR coupling constants, J’s, are reported in Hz. High-resolution
electrospray ionization mass spectra (HR ESI-MS) were performed by
the CESAMO (Bordeaux, France) on a QSsat Elite mass spectrometer
(Applied Biosystems). Silica gel 60 (particle size: 45 μm) was used for
flash chromatography (Biotage). Thin layer chromatograms (TLC)
were performed with aluminum plates coated with silica gel 60 F254
(Merck). The revealing solution for TLC is a sulfuric acid solution (95
mL ethanol and 5 mL sulfuric acid)
Thymidine 3′-(1,2-Dimyristoyl-sn-glycero-3-phosphate)-5′-(Phe-
nylalanine-Boc), diC14-dT-Boc-Phe, Compound 2a. Thymidine 3′-
(1,2-Dimyristoyl-sn-glycero-3-phosphate) (0.4g, 1 equiv, 0.49 mmol)
and Boc-Phe-OH (0.26 g, 2 equiv, 0.98 mmol) were dissolved in 10
mL of dry DCM under argon; N,N′-dicyclohexylcarbodiimide (DCC,
0.202 g, 2 equiv, 0.98 mmol) and 4-dimethylaminopyridine (DMAP,
0.03 g, 0.5 equiv, 0.25 mmol) were added, and the reaction mixture
was stirred overnight at room temperature. The dicyclohexylurea was
then filtered, and the solvent was evaporated under high vacuum. The
product was purified on silica gel (DCM/MeOH/TEA from 96:2:2 to
Synthesis. Thymidine 3′-(1,2-Dimyristoyl-sn-glycero-3-phos-
phate), diC14-dT, Compound 1a. The compound 1a was prepared
in the same way as compound 2 in Khiati et al.28
Thymidine 3′-(1,2-Dioleoyl-sn-glycero-3-phosphate), diC18:1-dT,
Compound 1b. 5′-O-(4,4′-Dimethoxytrityl)-2′-deoxythymidine-3′-
[(2-cyano-ethyl)-N,N-diisopropyl)] phosphoramidite (2 g, 1 equiv,
2.69 mmol) and 1,2-dioleoyl-sn-glycerol (2.5 g, 1.5 equiv, 4.0 mmol)
were dissolved in 10 mL of dry THF under argon. A tetrazole solution
in acetonitrile (0.45 M, 8 mL, 1.33 equiv, 3.6 mmol) was added, and
the reaction mixture was stirred overnight at room temperature
5548
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
94:4:2) with an LH20 exclusion column (DCM/MeOH 50:50).Yield:
40%.
71. 81−71.91 (1C, CH, glycerol), 76.99 (1C, 4′ sugar), 83.24 (1C, 3′
sugar), 86.69 (1C, 1′ sugar), 111.68 (1C, base), 128.07−130.58 (5C,
aromatic Phe), 136.63 (1C, aromatic Phe), 136.72 (1C, base), 152.10
(1C, CO base), 165.14 (1C, CO base), 170.76 (1C, ester, AA),
173.65 (1C, ester), 173.81 (1C, ester). 31P NMR (121 MHz, pyridine-
d5): δ 2.03. ESI-HRMS: Mw = 964.172, [M + Na]+ = 986.5477.
Thymidine 3′-(1,2-Dioleoyl-sn-glycero-3-phosphate)-5′-(Phenyl-
alanine), diC18:1-dT-Phe, Compound 3b. Thymidine 3′-(1,2-
Dioleoyl-sn-glycero-3-phosphate)-5′-(Phenylalanine-Boc) (0.288g, 1
equiv, 0.246 mmol) was dissolved in 6 mL of a trifluoracetic (50%)/
dichloromethane (50%) mixture for 4 h. The product was purified
with an LH20 exclusion column (DCM/MeOH 50:50). Yield: 94%.
1H NMR (300 MHz, DMSO-d6): δ 0.82−0.90 (m, 6H, CH3, chain),
1.20−1.36 (m, 40H, 2CH2, chain), 1.46−1.57 (m, 4H, 2CH2, chain),
1.81 (s, 3H, CH3, base), 1.92−2.03 (m, 8H, chain), 2.19−2.42 (m, 6H,
2CH2, chain + 2H, 2′ sugar), 3.83−4.39 (m, 8H, 2CH2, glycerol + 1H,
4′ + 2H, 5′ sugar + H, CH2, Phe), 4.69−4.77 (m, 1H, 3′ sugar), 5.06−
5.16 (m, 1H, glycerol), 5.27−5.38 (m, 4H, CHCH, chain), 6.09−
6.17 (m, H, 1′ sugar), 7.23−7.35 (m, 5H, aromatic, Phe, aromatic),
7.46 (1H, base). 13C NMR (75 MHz, DMSO-d6): δ 12.24 (1C, CH3,
base), 14.02 (2C, CH3, chain), 22.63 (2C, chain), 24.84 (2C, chain),
27.21 (4C, chain), 29.09−29.73 (14C, chain), 31.87 (4C, chain), 33.70
(1C, CH2, Phe), 34.02−34.19 (2C, chain), 36.30 (1C, 2′ sugar), 54.50
(1C, CH, Phe), 62.56 (1C, glycerol), 63.93 (1C, glycerol), 70.28 (1C,
glycerol), 79.99 (1C, 3′ sugar), 81.67 (1C, 1′ sugar), 111.19 (1C,
base), 127.52−130.01 (9C, 2 CHCH, chain + 5, aromatic, Phe),
134.10 (1C, aromatic, Phe), 138.10 (1C, base), 150.29 (1C, CO,
base), 163.84 (1C, CO, base), 167.89 (1C, ester, AA), 173.01 (1C,
ester), 173.31 (1C, ester). 31P NMR (121 MHz, DMSO-d6): δ 1.38.
ESI-HRMS: Mw = 1072.353, [M]+ = 1072.6564.
Preparation of the Samples. Sample solutions are prepared by
weighting the dry compounds and adding the buffer (HEPES 50 mM,
pH 7.2) to obtain the requested concentration (5 mM). The mixture
was stirred overnight at room temperature, followed by several cycles
of heating (50 °C during 1 min), and vigorous mixing (for 5 min) was
performed for 30 min. The homogeneity of the sample is checked
visually, and the dispersions are systematically characterized using
complementary techniques.
Cryo-TEM. Specimens for cryo-TEM observation were prepared
using a cryoplunge cryo-fixation device (Gatan, USA) in which a drop
of the aqueous solution was deposited onto glow-discharged holey-
type carbon-coated grids (Ted Pella Inc., USA). The TEM grid was
then prepared by blotting the drop containing the specimen to a thin
liquid layer, spanning across the holes in the support carbon film. The
liquid film was vitrified by rapidly plunging the grid into liquid ethane
cooled by liquid nitrogen. The vitrified specimens were mounted in a
Gatan 910 specimen holder (Gatan, USA) that was inserted in the
microscope, using a CT-3500-cryotransfer system (Gatan, USA) and
cooled with liquid nitrogen. TEM images were then obtained from
specimens preserved in vitreous ice and suspended across a hole in the
supporting carbon substrate. The samples were observed under low
dose conditions (<10 e−/Å2), at −178 °C, using a JEM-1230 “Cryo”
microscope (Jeol, Japan) operated at 80 kV and equipped with a LaB6
filament. All micrographs were recorded with a Gatan 1.35 K × 1.04 K
× 12 bit ES500 W CCD camera.
1H NMR (300 MHz, CDCl3): δ 0.85−0.91 (m, 6H, CH3, chain),
1.23−1.43 (m, 58H, 20CH2, chain + 9H, 3CH3, TEA + 9H, 3CH3,
Boc), 1.55−1.64 (m, 4H, 2CH2, chain), 1.92 (s, 3H, CH3, base),
1.94−2.04 (m, 3H, 1H, 2′ sugar + 2H, CH2, Phe), 2.24−2.34 (m, 4H,
2CH2, chain), 2.46−2.52 (m, 1H, 2′ sugar), 3.03−3.14 (m, 6H, 3CH2,
TEA), 3.96−4.02 (m, 2H, CH2, glycerol), 4.12−4.19 (m, 1H, CH2,
glycerol), 4.33−4.71 (m, 5H, 1H, CH2, glycerol + 1H 3′ + 1H 4′ + 2H,
5′ sugar), 5.09−5.15 (m, 1H, CH, Phe), 5.22−5.29 (m, 1H, glycerol),
6.25−6.31 (m, H, 1′ sugar), 7.12−7.27 (m, 5H, Phe, aromatic), 8.39
(s, 1H, base). 13C NMR (75 MHz, CDCl3): δ 8.61 (3C, 3CH3, TEA),
12.55 (1C, CH3, base), 14.15 (2C, CH3, chain), 22.72 (2C, chain),
24.88 (2C, chain), 28.26 (3C, 3CH3, Boc), 29.16−29.68 (14C, chain),
31.94 (4C, chain), 34.09−34.29 (2C, chain), 38.41 (1C, 2′ sugar),
45.71 (3C, 3CH2, TEA), 54.77 (1C, CH2, Phe), 62.66 (1C, glycerol),
63.62 (1C, glycerol), 65.07 (1C, CH, Phe), 70.28 (1C, glycerol), 74.98
(1C, 4′ sugar), 80.06 (1C, 3′ sugar), 84.95 (1C, 1′ sugar), 111.38 (1C,
base), 127.17−129.27 (5C, aromatic, Phe), 135.16 (1C, base), 135.84
(1C, aromatic, Phe), 150.02 (1C, CO, base), 154.98 (1C, CO,
Boc), 163.41 (1C, CO, base), 171.84 (1C, ester, AA), 173.09 (1C,
ester), 173.50 (1C, ester). 31P NMR (121 MHz, CDCl3): δ 1.97
Thymidine 3′-(1,2-Dioleoyl-sn-glycero-3-phosphate)-5′-(Phenyl-
alanine-Boc), diC18:1-dT-Boc-Phe, Compound 2b. Thymidine 3′-
(1,2-Dioleoyl-sn-glycero-3-phosphate) (0.3g, 1 equiv, 0.325 mmol)
and Boc-Phe-OH (0.172 g, 2 equiv, 0.65 mmol) were dissolved in 10
mL of dry DCM under argon; DCC (0.134 g, 2 equiv, 0.65 mmol) and
DMAP (0.02 g, 0.5 equiv, 0.163 mmol) were added, and the reaction
mixture was stirred overnight at room temperature. The dicyclohex-
ylurea was then filtered, and the solvent was evaporated under high
vacuum. The product was purified on silica gel (DCM/MeOH/TEA
from 98:1:1 to 95:4:1). Yield: 75%.
1H NMR (300 MHz, CDCl3): δ 0.84−0.91 (m, 6H, CH3 chain),
1.22−1.42 (m, 58H, 20CH2, chain + 9H, 3CH3, TEA + 9H, 3CH3,
Boc), 1.53−1.64 (m, 4H, 2CH2, chain), 1.91 (s, 3H, CH3, base), 1.96−
2.04 (m, 8H, chain), 2.24−2.34 (m, 4H, 2CH2, chain), 2.43−2.52 (m,
1H, 2′ sugar), 3.03−3.13 (m, 6H, 3CH2, TEA), 3.96−4.72 (m, 9H,
2CH2, glycerol +1H, 4′ + 2H, 5′ sugar + 2H, CH2, Phe), 5.09−5.15
(m, 1H, 3′ sugar), 5.21−5.30 (m, 1H, glycerol), 5.30−5.39 (m, 4H,
CHCH chain), 6.25−6.32 (m, H, 1′ sugar), 7.12−7.27 (m, 6H, 5H,
Phe, aromatic + 1H, base). 13C NMR (75 MHz, CDCl3): δ 8.59 (3C,
3CH3, TEA), 12.55 (1C, CH3, base), 14.14 (2C, CH3, chain), 22.69
(2C, chain), 24.86 (2C, chain), 27.23 (4C, chain), 28.26 (3C, 3CH3,
Boc), 29.13−29.76 (14C, chain), 31.91 (4C, chain), 34.06−34.24 (2C,
chain), 38.40 (1C, 2′ sugar), 45.70 (3C, 3CH2, TEA), 54.79 (1C, CH2,
Phe), 62.66 (1C, glycerol), 63.65 (1C, glycerol), 65.0 (1C, CH, Phe),
70.28 (1C, glycerol), 74.98 (1C, 4′ sugar), 80.03 (1C, 3′ sugar), 84.81
(1C, 1′ sugar), 111.41 (1C, base), 127.16−130.01 (9C, 2 CHCH,
chain + 5, aromatic, Phe), 135.13 (1C, base), 135.89 (1C, aromatic,
Phe), 150.21 (1C, CO, base), 154.91 (1C, CO, Boc), 163.64
(1C, CO, base), 171.83 (1C, ester AA), 173.04 (1C, ester), 173.44
(1C, ester). 31P NMR (121 MHz, CDCl3): δ 1.81.
Thymidine 3′-(1,2-Dimyristoyl-sn-glycero-3-phosphate)-5′-(Phe-
nylalanine), diC14-dT-Phe, Compound 3a. Thymidine 3′-(1,2-
Dimyristoyl-sn-glycero-3-phosphate)-5′-(Phenylalanine-Boc) (0.200g,
1 equiv, 0.188 mmol) was dissolved in 6 mL of a trifluoracetic (50%)/
dichloromethane (50%) mixture for 4 h. The product was purified
with an LH20 exclusion column (DCM/MeOH 50:50). Yield: 96%.
1H NMR (300 MHz, pyridine-d5): δ 0.89−0.96 (m, 6H, CH3,
chain) 1.20−1.38 (m, 40H, 2CH2, chain) 1.66−1.77 (m, 4H, 2CH2,
chain), 1.99 (s, 3H, CH3, base), 2.39−2.50 (m, 4H, 2CH2, chain),
2.74−2.81 (m, 2H, 2′ sugar), 3.57−3.63 (m, 2H, CH2, Phe), 4.48−
4.85 (m, 8H, 2CH2, glycerol + 1H, 4′ + 2H, 5′ sugar + H, CH2, Phe),
5.43−5.50 (m, 1H, 3′ sugar), 5.72−5.79 (m, 1H, glycerol), 6.68−6.76
(m, H, 1′ sugar), 7.26−7.56 (m, 6H, 5H, aromatic, Phe, aromatic +
1H, base). 13C NMR (75 MHz, pyridine-d5): δ 13.02 (1C, CH3, base),
14.69 (2C, CH3, chain), 23.38 (2C, chain), 25.79 (2C, chain), 29.80−
30.60 (14C, chain), 32.60 (4C, chain), 34.78−35.01 (2C, chain),
38.44−38.66 (2C, CH2, Phe + 2′ sugar), 56.16 (1C, CH, Phe), 63. 65
(1C, CH2, glycerol), 64.87 (1C, CH2, glycerol), 65.28 (1C, 5′ sugar),
Dynamic Light Scattering (DLS). DLS experiments have been
performed with a Brookhaven Instruments apparatus (BI 9000AT
digital correlator). The signal was detected by a photomultiplier tube,
set at 90° with respect to the incident beam on the sample. The
pinhole was set at 150 μm. The light source was a diode laser (532
nm), linearly polarized in the vertical direction. A 3 min acquisition
time was used for each measurement. The experiments were
performed at 20 °C. The tubes were immersed in a thermostatted
bath of Decalin (decahydronapthalene), necessary for index matching.
The time autocorrelation functions of the scattered light intensity were
collected and analyzed by a cumulant analysis.
X-ray Experiments. Once prepared, samples were carefully
transferred at room temperature into sealed quartz capillaries. High-
flux and high-resolution X-ray experiments were carried out on the
SWING beamline at the synchrotron facility SOLEIL near Paris,
5549
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
a
Scheme 1. Synthesis of Nucleotide-Lipid (NL, diC18:1-dT) and Its Amino Acid Analogue (ANL, Phe-diC18:1-dT).
a
(a) Tetrazole 0.45 M in CH3CN, overnight, RT. (b) I2 0.02 M in THF/Pyr/H2O, 6 h, RT. (c) TCA 3% in DCM, 4 h, RT. (d) TEA, DCM,
overnight, RT, (e) Boc−Phe−OH, DCC, DMAP, DCM, overnight, RT. (f) TFA 50% in DCM, 4 h, RT.
France. With a sample-to-detector distance equal to 1.58 m and a
radiation wavelength of λ =0.103 nm, the scattering wave vectors in
reciprocal space ranged from q = 0.007 nm−1 to q = 7.0 nm−1. The
total resolution of detection was estimated by fitting Gaussian
functions to peaks from silver behenate diffractograms; the value of
Δq was found to be ca. 4.3 × 10−3 nm−1. The beam size at the sample
position was 0.4 × 0.1 mm (H × V). Images were captured by the
Aviex CCD detector of the beamline. Data were radially averaged and
corrected for background scattering by using the software Foxtrot, an
application developed by the SWING beamline staff.
rpm to remove any insoluble particles, if any present. The syringe was
constantly stirred at a rate of 500 rpm, and the measurements were
performed at 25 °C. The first injection was carried out without taking
into account the corresponding observed heat because the first
injection was subject to large errors as a result of the diffusion of
solutions across the syringe tip during the pretitration equilibrium
period. The data analyses were carried out with Origin 7.0 software
(provided by MicroCal), using the standard “one-set-of-sites” model
where a monofunctional ligand interacts with any of the N equivalent
binding sites of a receptor.
A homemade, rotating anode-based setup (MicroMax-007 HF
Rigaku rotating anode) was used to evaluate the scattering profile in
the wide-angle region. The goal of such experiments was to probe the
fluidity of aliphatic chains within the lipid bilayers. The sample-to-
detector distance was 103 mm and, with a radiation wavelength of λ =
0.154 nm, q ranged from 3.5 to 28.0 nm. The detection system is a
mar345 image plate (from Marresearch) coupled to an online scanner.
All above-described experiments were carried out on thermally
equilibrated samples at 25 °C, which was obtained by keeping the
sample holders under water circulation at a controlled temperature.
Isothermal Titration Calorimetry (ITC) Experiments. Titration
experiments were performed with an iTC200 microcalorimeter from
MicroCal Inc. The working cell (205.8 μL) was filled with the desired
nucleolipid (diC14-dT, diC14-dT-Phe) dispersion (5 mM) in a
HEPES buffer (50 mM, pH = 7.2) and the reference cell with water.
The injection syringe was filled with nucleic acid (polyA or polyU with
a degree of polymerization in the range of 50100, purchased from
SIGMA) solution (14.6 mM) in a HEPES buffer (50 mM, pH = 7.2).
The titration schedule consisted of 20 consecutive injections of 2 μL
with an interval of 500 s between each injection. The corresponding
reference blank experiments were also performed, namely, titration of
nucleic acid-free HEPES buffer in the nucleolipid solution and titration
of nucleic acid solution in the nucleolipid-free HEPES buffer. To avoid
the presence of bubbles, all samples were degassed for 10 min, shortly
before starting the measurements and centrifuged for 5 min at 6000
RESULTS AND DISCUSSION
■
Synthesis. To evaluate the impact on the self-assembly
properties of the presence of an amino acid in the amphiphilic
structure, a nucleotide−lipid and its phenylalanine aminoacid
derivate compound were synthesized according to Scheme 1.
We are interested in weak interactions occurring between NL
and nucleic acid partners. Hence, besides the amino group of
the AA, phenylalanine was selected among the 20 AA since it
features an aromatic ring, allowing the formation of π stacking
interactions. The nucleoside-3′-monophosphate bearing 1,2-
diacyl-sn-glycerol as the hydrophobic part was synthesized
following a procedure adapted from Khiati et al.28 Briefly, the
5′-DMT-protected commercial phosphoramidite was coupled
with the 1,2-diacyyl-sn-glycerol in acidic conditions (tetrazole),
and then the resulting phosphite intermediate was treated with
iodine in a THF/pyridine/water mixture to obtain the
cyanoethyl-protected phosphate. The DMT and the cyanoeth-
yl-protecting groups were removed, respectively, under acidic
conditions and basic conditions to obtain the final nucleotide
lipid 1a, 1b (NL, diC14-dT, diC18:1-dT). The aminoacid
derivate compound 3a, 3b was synthesized using a two steps
5550
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
synthetic strategy starting from the corresponding NL 1a, 1b.
The Boc-protected amino acid was allowed to react with the
compound 1a, 1b, in the presence of DCC and DMAP. The
intermediate 2a, 2b was then isolated. The final amino acid−
nucleotide−lipid 3a, 3b (ANL, diC14-dT-Phe, diC18:1-dT-
Phe) was obtained after cleavage of the Boc-protecting group
under acidic conditions. The final compounds were fully
characterized by mass spectroscopy and NMR analysis.
Characterization of the Aggregates. We first studied the
morphology and size of the NLs and ANLs using cryo-
transmission electron microscopy (cryo-TEM), that is partic-
ularly well-adapted for studying such structures.29 Aqueous
samples of lipids at a concentration of either 1 or 5 mg/mL
were prepared in a HEPES buffer (50 mM) at a pH of 7.2. As
observed in Figure 2, the amino acid moiety inserted in the
DLS. The size distribution profile of both NLs and ANLs
were obtained via dynamic light scattering (DLS) measure-
ments. In accordance with cryo-TEM microscopy images, DLS
data (Table 1) show the formation of larger objects in the case
a
Table 1. DLS Data of NLs and ANLs
d (nm)
polydispersity
diC14-dT
46
100
137
653
0.13
0.20
0.31
0.48
diC14-dT-Phe
diC18:1-dT
diC18:1-dT-Phe
a
NLs and ANLs samples were prepared in a HEPES buffer (50 mM,
pH 7.2) at a concentration of 5 mg/mL.
of ANLs (diameter, d, 100 and 653 nm for diC14-dT-Phe and
diC18:1-dT-Phe, respectively) than for NLs (46 and 137 nm
for diC14-dT and diC18:1-dT, respectively).
X-rays. WAXS profiles suggest the fluidity of the bilayers
with a single broad peak observed at about 19 nm−1. This
feature is related to the molecular correlations between water
molecules and, more significantly, any sharp peaks due to a gel-
phase organization of the lipid chains that are characteristically
absent (data not shown). These WAXS profiles clearly indicate
that the bilayer is in the fluid state for all the studied systems.
SAXS data (Figure 3) show two kinds of profiles: in the case of
NLs, a broad peak that corresponds to the form factor of a
vesicular object is observed,30 whereas in the case of diC18:1-
dT-Phe, we observe a characteristic feature that we interpret as
a Bragg peak, overlapped to a broad peak. As we argue later, the
SAXS profile of diC14-dT-Phe is also better described as a
Bragg peak, overlapped to a broad peak, than as a vesicular
form factor profile alone. For both ANLs, the broad peak
corresponds to the form factor of a vesicle. The Bragg peak of
diC18:1-dT-Phe corresponds to the structure factor of a
multilamellar stacking of bilayers.31 We therefore interpret NLs
as unilamellar vesicles and ANLs as multilamellar ones.
Lamellar periodicities and numbers of stacked bilayers can be
roughly estimated by adjusting the ANLs experimental curves
with the Nallet et al. model,32 which also yields a simple
description of the bilayer form factor in all cases. In Figure 3,
the solid lines correspond to the best fits obtained for the
different samples. For NL systems, we use the model form
factor in order to extract the values of the thicknesses of
hydrophilic (δH) and hydrophobic (δT) parts of the bilayer. In
the case of the diC18:1-dT-Phe system, adjusting form and
structure factors simultaneously allows for, in addition, the
extraction of the lamellar periodicity (D) and the number of
stacked bilayers (NB). The set of parameters is reported in
Table 2. In the case of the diC14-dT-Phe system, because there
is not any evidence of a Bragg peak in the SAXS profile and in
agreement with the cryo-TEM observations, we set the lamellar
periodicity value to 25 nm, extracting NB in addition to δH and
δT (see Table 2). It is worth noticing that fitting to the form
factor alone would not have satisfactorily described the diC14-
dT-Phe system.
Figure 2. Cryo-TEM images. NL and ANL samples were prepared in a
HEPES buffer (50 mM, pH 7.2). (a) diC14-dT (1 mg/mL), (b),
diC14-dT-Phe (1 mg/mL), (c) diC18:1-dT (5 mg/mL), and (d)
diC18:1-dT-Phe (1 mg/mL). Scale bars = 200 nm.
polar head changes the morphology of the vesicles observed.
Images collected from both NLs (diC14-dT and diC18:1-dT)
show unilamellar vesicles, whereas the ANLs, diC14-dT-Phe,
and diC18:1-dT-Phe exhibit multilamellar vesicles. This result
indicates that ANLs self-assemble at room temperature into
different objects than NLs. Note that in these conditions, both
oleoyl chains and myristoyl are in a “fluid” state, suggesting that
large vesicles are formed in aqueous solutions. This technique
allows a direct visualization of objects, but the low statistics
does not allow one to accurately determine the structural
parameters such as the vesicle size, for example. However, the
analysis of cryo-TEM images allows estimating the lamellar
periodicity observed for systems with amino acid. In the case of
simple nucleotide lipids (diC14-dT and diC18:1dT), the
estimated sizes of vesicles are lower than for aminoacid
derivatives (diC14-dT-Phe and diC18:1dT-Phe), likely due to
the formation of multilamellar objects. In both systems with
aminoacids, we observe multilamellar vesicles with a variable
number of bilayers up to 6. The average lamellar periodicity for
the diC14-dT-Phe system is estimated at 30 15 nm, while the
system is less swollen in the case of diC18:1-dT-Phe, with a
The thickness of bilayers (2δH + 2δT), the number of
stacked bilayers (NB), and the lamellar periodicity (D) for the
diC18:1-dT-Phe system extracted from the SAXS data appear
compatible with the corresponding information obtained from
the cryo-TEM measurements. In order to explain the fact that
the Bragg peak is observed in one ANL case (diC18:1-dT-Phe)
and not in the other (diC14-dT-Phe), we tentatively argue,
periodicity of about 17
5 nm. The stacking of bilayers
appears less regular in the case of diC14-dT-Phe. In all cases
(ANLs and NLs), the thickness of the bilayer is estimated to be
about 5 nm.
5551
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
□
□
Figure 3. Xray profiles of NLs and ANLs. (a) diC18:1dT ( ) and diC18:1dT-Phe (•), (b) diC14dT ( ) and diC14dT-Phe (•). Samples were
prepared in a HEPES buffer (50 mM, pH 7.2) at a concentration of 5 mg/mL.
identical sites description provided with the instrument. Briefly,
within the usual reaction scheme L + RLk ↔ RLk+1, where a
ligand L binds with the same binding constant K to any k-
coordinated receptor RLk, we consider as ligand L, a monomer
of polyA, and as receptor R, a uni- or multilamellar vesicle built
with nagg-aggregated NL or ANL molecules. The standard law
of mass action and the matter conservation equations of the
model, namely,
Table 2. X-ray Fitting Data for diC14dT, diC14dT-Phe,
diC18:1dT, and diC18:1dT-Phe
δT (nm)
δH (nm)
D (nm)
NB
diC14dT
0.84
0.70
0.84
0.70
1.84
1.40
1.74
1.70
−
25.0
−
3
diC14dT-Phe
diC18:1dT
−
18.5
−
3
diC18:1dT-Phe
comparing Figure 2 panels b and d, that the lamellar stacking is
much more regular for diC18:1-dT-Phe than it is for diC14-dT-
Phe. In addition, the lamellar period being larger for diC14-dT-
Phe, the Bragg peak is expected at a smaller wave vector, that is
to say, in a range where it could be masked by the direct beam
contributions.
Interaction Studies. Isothermal titration calorimetry
(ITC). A further step in this study is to evaluate the possibility
of an interaction between our molecules (NLs and ANLs) and
a nucleic acid (polyA). We have selected the polyA as a nucleic
acid model with the objective to increase the specific
interactions between the thymine moiety of our diC14
molecules and the adenine moiety in the polyA (Watson−
Crick). The ITC experiment allows one to study these
interactions within low quantities of the sample, in volume,
and to quantify the energies involved.
Typical ITC profiles for the titration of polyA into solutions
of diC14 ANL and NL systems are presented in Figure 4. In
both cases, the titration of polyA produces a sequence of
endothermic peaks. An estimation of the parameters of the
interaction is obtained by a nonlinear least-squares fit of the
ITC data (lower row in Figure 4), using as a thermodynamic
model the appropriate adaptation of the standard single set of
Θ
K =
(1 − Θ)[L]
Lt = [L] + NΘRt
where Lt (respectively, Rt) is the total ligand (respectively,
receptor) concentration, [L] the actual ligand concentration, N
the number of binding sites per receptor, and Θ the fraction of
bound receptors, are simply recast in terms of the NL (or
ANL) molecule concentration (mt), using the relation Rt = mt/
nagg. For unilamellar vesicles with outer and inner diameters, d
and d − 2δ, respectively, N is proportional to d2, while nagg is
proportional to d2 + (d − 2δ)2. One therefore obtains
Θ
Lt = [L] +
mt
1 + (1 − 2δ/d)2
as the appropriate form of the equation for matter conservation.
Similarly, since for multilamellar vesicles of outer diameter d
built from NB stacked bilayers with stacking period D, nagg is
now proportional to d2 + (d − 2δ)2 + [d − 2D]2 + [d − 2D −
2δ]2 +...+ [d − 2(NB − 1)D]2 + [d − 2δ]2, the conservation
equation becomes
Θ
Lt = [L] +
2 mt
2
2
2
2
(N − 1)D
(N − 1)D + δ
δ
d
D
d
D + δ
d
⎡
⎤
⎦
⎡
⎤
⎦
⎡
⎤
⎦
⎡
⎤
⎦
⎡
⎤
⎦
B
B
1 + 1 − 2
+ 1 − 2
+ 1 − 2
+ ... + 1 − 2
+ 1 − 2
⎣
⎣
⎣
⎣
⎣
d
d
5552
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
Figure 4. ITC profiles for the titration of polyA into a solution of (a) diC14dT and (b) diC14dT-Phe. Samples were prepared in a HEPES buffer (50
mM, pH 7.2) at a concentration of 5 mg/mL.
It thus appears that the (apparent) stoichiometry of the binding
reaction described by the parameter N in the standard model is,
in our case, smaller than 1. In addition, it is sensitive to the
actual details of the self-assembly process.
The parameters resulting from the above-described analysis
are summarized in Table 3.
stoichiometries for the two studied systems. This calculation
gives N = 0.65−0.80 and N = 0.25−0.4 for the NL and ANL
systems, , respectively, in nice agreement with the fitted values.
It is also interesting to note the second difference, namely, that
the value of ΔH is greater for the system with the amino acid.
Although the binding affinities are rather close, it seems more
difficult to form the interaction in the case of the ANL. In both
systems, the binding is endothermic. Although the energy of a
simple pair binding is expected to be exothermic, the net
measured positive enthalpic penalty can result from a variety of
parallel unbinding(s) involving indifferent, intermolecular
interactions among nucleolipid polar heads, dipole interactions
among polar head group and water molecules, possibly H
bonds, or electrostatic interactions among the polar heads and
ions from the buffer solution. Clarifying these points would
require separating all these different effects, which would prove
extremely difficult to perform. The main conclusion which can
be drawn is thus that the overall mechanism of binding is
definitely an entropy-driven process in both cases. The entropic
gain is commonly associated with the freeing of water
molecules interacting with the polar heads (although the ions
from the buffer could also take part).
Table 3. ITC Three Fitting Parameters for the Titration of
PolyA into a Solution of (a) diC14dT and (b) diC14dT-
a
Phe
N
K (M−1
)
ΔH (J/mol)
ΔS (J/mol/deg)
diC14dT
0.77
0.13
521
313
395.2
53.2
55.3
diC14dT-Phe
2209.8
a
Samples were prepared in a HEPES buffer (50 mM, pH 7.2) at a
concentration of 5 mg/mL. Note that ΔS is not a fitting parameter but
calculated from ΔS =R ln K + ΔH/T.
As already stated, for both systems the interaction is
endothermic and it turns out that the values of the binding
affinity (K) are very close. Two differences can, however, be
noted. The apparent stoichiometry of the process is 0.77 for the
NL system and 0.13 for the ANL system. As a stand-alone
experiment, ITC is usually considered unable to provide
structural information, but we know here, from both X-ray and
cryo-TEM experiments, that with the ANL system, we are in
the presence of multilamellar vesicles when it comes to
unilamellar vesicles in the case of the NL system. It is possible,
knowing the different structural parameters [size of objects (d)
from DLS, lamellar periodicity (D), and number of stacked
bilayers, NB, from X-rays] to calculate the apparent
CONCLUSION
■
We have synthesized and characterized new amino acid−
nucleolipids with phenylalanine and thymidine residues and
saturated or unsaturated diacyl glycerol lipid moieties. Using
different complementary techniques (DLS, cryo-TEM, X-ray
scattering), we have shown that the presence of the
phenylalanine amino acid on the 5′ position of the
nucleotide−lipid stabilizes multilamellar vesicles. Interactions
5553
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
Bolisetty, S.; Mezzenga, R.; Frauenrath, H. Low-Temperature
Preparation of Tailored Carbon Nanostructures in Water. Nano Lett.
2012, 12, 2573−2578.
between bilayers give the possibility to obtain highly swollen
lamellar systems in the case of ANLs, in comparison to NLs or
more conventional systems of phospholipids. The large
thickness of the aqueous layer between bilayers could be used
to insert different hydrophilic molecules for therapeutic
application. ITC experiments indicate that weak, entropically
driven interactions occur between nucleotide−lipids and polyA
RNA. From a biomedical applications viewpoint, the new
amino acid−nucleotide−lipids described in this report provide
multilamellar vesicles suitable for the stabilization of versatile
systems, allowing the encapsulation of both hydrophobic and
hydrophilic therapeutic molecules. At present, the scope is
limited to the particular amino acid phenylalanine and further
research is still needed to establish the structural requirements
for multilayer vesicle formation.
(9) Men
́
dez-Ardoy, A.; Guilloteau, N.; Di Giorgio, C.; Vierling, P.;
ez, F.; Ortiz Mellet, C.; García Fernandez, J. M. β-
Santoyo-Gonzal
́
́
Cyclodextrin-Based Polycationic Amphiphilic “Click” Clusters: Effect
of Structural Modifications in Their DNA Complexing and Delivery
Properties. J. Org. Chem. 2011, 76, 5882−5894.
(10) Clemente, M. J.; Romero, P.; Serrano, J. L.; Fitremann, J.; Oriol,
L. Supramolecular Hydrogels Based on Glycoamphiphiles: Effect of
the Disaccharide Polar Head. Chem. Mater. 2012, 24, 3847−3858.
(11) Nandi, S.; Altenbach, H.-J.; Jakob, B.; Lange, K.; Ihizane, R.;
̈
Schneider, M. P.; Gun, U.; Mayer, A. Amphiphiles Based on d-
̈
Glucose: Efficient Low Molecular Weight Gelators. Org. Lett. 2012, 14,
3826−3829.
(12) McLaughlin, C. K.; Hamblin, G. D.; Sleiman, H. F.
Supramolecular DNA Assembly. Chem. Soc. Rev. 2011, 40, 5647−5656.
́ ́
(13) Patwa, A.; Gissot, A.; Bestel, I.; Barthelemy, P. Hybrid Lipid
ASSOCIATED CONTENT
* Supporting Information
Full synthetic procedures and NMR/MS spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
Oligonucleotide Conjugates: Synthesis, Self-Assemblies and Biomed-
ical Applications. Chem. Soc. Rev. 2011, 40, 5844−5854.
(14) Rosemeyer, H. Nucleolipids: Natural Occurrence, Synthesis,
Molecular Recognition, And Supramolecular Assemblies As Potential
Precursors of Life and Bioorganic Materials. Chem. Biodiversity 2005, 2,
977−1063.
S
AUTHOR INFORMATION
Corresponding Author
*P.B.: e-mail, philippe.barthelemy@inserm.fr. L.N.: e-mail,
navailles@crpp-bordeaux.cnrs.fr.
■
(15) Couvreur, P.; Stella, B.; Reddy, L. H.; Hillaireau, H.; Dubernet,
̂
C.; Desmaele, D.; Lepetre-Mouelhi, S.; Rocco, F.; Dereuddre-Bosquet,
̈
N.; Clayette, P.; Rosilio, V.; Marsaud, V.; Renoir, J.-M.; Cattel, L.
Squalenoyl Nanomedicines as Potential Therapeutics. Nano Lett. 2006,
6, 2544−2548.
Notes
The authors declare no competing financial interest.
(16) Desmaele, D.; Gref, R.; Couvreur, P. Squalenoylation: A
̈
Generic Platform for Nanoparticular Drug Delivery. J. Controlled
Release 2012, 161, 609−618.
ACKNOWLEDGMENTS
This research was supported by the following grants: Reg
■
́
(17) Barthelemy, P. Nucleoside-Based Lipids at Work: From
́
ion
Supramolecular Assemblies to Biological Applications. C. R. Chim.
2009, 12, 171−179.
d’Aquitaine and the Army Research Office (U.S.A.). The
authors thank Pr. Gilles Sigaud for his help in interpreting the
ITC experiments. We are grateful to the scientific team of the
SWING beamline, in particular, to Dr. Javier Perez, for help and
support, and to synchrotron SOLEIL for allocating beam time
to proposal 20120613
(18) Allain, V.; Bourgaux, C.; Couvreur, P. Self-Assembled
Nucleolipids: From Supramolecular Structure to Soft Nucleic Acid
and Drug Delivery Devices. Nucleic Acids Res. 2012, 40, 1891−1903.
(19) Chabaud, P.; Camplo, M.; Payet, D.; Serin, G.; Moreau, L.;
́ ́
Barthelemy, P.; Grinstaff, M. W. Cationic Nucleoside Lipids for Gene
Delivery. Bioconjugate Chem. 2006, 17, 466−472.
REFERENCES
(20) Ceballos, C.; Khiati, S.; Prata, C. A. H.; Zhang, X.-X.; Giorgio,
■
́ ́
S.; Marsal, P.; Grinstaff, M. W.; Barthelemy, P.; Camplo, M. Cationic
(1) Tang, C.; Ulijn, R. V.; Saiani, A. Effect of Glycine Substitution on
Fmoc−Diphenylalanine Self-Assembly and Gelation Properties.
Langmuir 2011, 27, 14438−14449.
Nucleoside Lipids Derived from Universal Bases: A Rational Approach
for siRNA Transfection. Bioconjugate Chem. 2010, 21, 1062−1069.
(21) Khiati, S.; Luvino, D.; Oumzil, K.; Chauffert, B.; Camplo, M.;
(2) Ghosh, A.; Dey, J. pH-Responsive and Thermoreversible
Hydrogels of N-(2-hydroxyalkyl)-l-valine Amphiphiles. Langmuir
2009, 25, 8466−8472.
(3) He, C.; Han, Y.; Fan, Y.; Deng, M.; Wang, Y. Self-Assembly of
Aβ-Based Peptide Amphiphiles with Double Hydrophobic Chains.
Langmuir 2012, 28, 3391−3396.
(4) Ziserman, L.; Lee, H.-Y.; Raghavan, S. R.; Mor, A.; Danino, D.
Unraveling the Mechanism of Nanotube Formation by Chiral Self-
Assembly of Amphiphiles. J. Am. Chem. Soc. 2011, 133, 2511−2517.
(5) Ku, T.-H.; Chien, M.-P.; Thompson, M. P.; Sinkovits, R. S.;
Olson, N. H.; Baker, T. S.; Gianneschi, N. C. Controlling and
Switching the Morphology of Micellar Nanoparticles with Enzymes. J.
Am. Chem. Soc. 2011, 133, 8392−8395.
(6) Dong, H.; Dube, N.; Shu, J. Y.; Seo, J. W.; Mahakian, L. M.;
Ferrara, K. W.; Xu, T. Long-Circulating 15 nm Micelles Based on
Amphiphilic 3-Helix Peptide−PEG Conjugates. ACS Nano 2012, 6,
5320−5329.
́ ́
Barthelemy, P. Nucleoside−Lipid-Based Nanoparticles for Cisplatin
Delivery. ACS Nano 2011, 5, 8649−8655.
(22) Desbat, B.; Arazam, N.; Khiati, S.; Tonelli, G.; Neri, W.;
́ ́
Barthelemy, P.; Navailles, L. Unexpected Bilayer Formation in
Langmuir Films of Nucleolipids. Langmuir 2012, 28, 6816−6825.
(23) Banchelli, M.; Berti, D.; Baglioni, P. Molecular Recognition
Drives Oligonucleotide Binding to Nucleolipid Self-Assemblies. Angew.
Chem., Int. Ed. 2007, 46, 3070−3073.
(24) Milani, S.; Baldelli Bombelli, F.; Berti, D.; Baglioni, P.
Nucleolipoplexes: A New Paradigm for Phospholipid Bilayer−Nucleic
Acid Interactions. J. Am. Chem. Soc. 2007, 129, 11664−11665.
́ ́
(25) Godeau, G.; Bernard, J.; Staedel, C.; Barthelemy, P. Glycosyl−
nucleoside−lipid Based Supramolecular Assembly as a Nanostructured
Material with Nucleic Acid Delivery Capabilities. Chem. Commun.
(Cambridge, U.K.) 2009, 5127−5129.
(26) Yang, H. W.; Yi, J. W.; Bang, E.-K.; Jeon, E. M.; Kim, B. H.
Cationic Nucleolipids As Efficient siRNA Carriers. Org. Biomol. Chem.
2010, 9, 291−296.
(7) Chae, P. S.; Gotfryd, K.; Pacyna, J.; Miercke, L. J. W.; Rasmussen,
S. G. F.; Robbins, R. A.; Rana, R. R.; Loland, C. J.; Kobilka, B.; Stroud,
R.; Byrne, B.; Gether, U.; Gellman, S. H. Tandem Facial Amphiphiles
for Membrane Protein Stabilization. J. Am. Chem. Soc. 2010, 132,
16750−16752.
́
(27) Nyilas, A. A New Protecting Group: 9-Fluorenylmethoxycar-
bonyl (FMOC) in the Synthesis of 1,2-Diacylglycerols. Chem. Phys.
Lipids 1997, 87, 171−174.
(28) Khiati, S.; Pierre, N.; Andriamanarivo, S.; Grinstaff, M. W.;
(8) Szilluweit, R.; Hoheisel, T. N.; Fritzsche, M.; Ketterer, B.;
Fontcuberta i Morral, A.; Demurtas, D.; Laporte, V.; Verel, R.;
́ ́
Arazam, N.; Nallet, F.; Navailles, L.; Barthelemy, P. Anionic
5554
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555

Langmuir
Article
Nucleotide−Lipids for In Vitro DNA Transfection. Bioconjugate Chem.
2009, 20, 1765−1772.
(29) Gaillard, C.; Douliez, J.-P. Cryo-TEM and AFM for the
Characterization of Vesicle-Like Nanoparticle Dispersions and Self-
Assembled Supramolecular Fatty-Acid-Based Structures: A Few
Examples. In Current Microscopy Contributions to Advances in Science
and Technology; Microscopy Series 5; Formatex Research Center:
Badajoz, Spain, 2012; Vol. 2, pp 912−922.
(30) Blaurock, A. E. Evidence of Bilayer Structure and of Membrane
Interactions from X-ray Diffraction Analysis. Biochim. Biophys. Acta,
Rev. Biomembr. 1982, 650, 167−207.
(31) Loudet-Courreges, C.; Nallet, F.; Dufourc, E. J.; Oda, R.
Unprecedented Observation of Days-Long Remnant Orientation of
Phospholipid Bicelles: A Small-Angle X-ray Scattering and Theoretical
Study. Langmuir 2011, 27, 9122−9130.
(32) Nallet, F.; Laversanne, R.; Roux, D. Modelling X-ray or Neutron
Scattering Spectra of Lyotropic Lamellar Phases: Interplay between
Form and Structure Factors. J. Phys. II 1993, 3, 487−502.
5555
dx.doi.org/10.1021/la400515m | Langmuir 2013, 29, 5547−5555