Design of Polysaccharide-b-Elastin-Like Polypeptide Bioconjugates and Their Thermoresponsive Self-Assembly

Article abstract of DOI:10.1021/acs.biomac.9b01058

The advantageous biological properties of polysaccharides and precise stimuli-responsiveness of elastin-like polypeptides (ELPs) are of great interest for the design of polysaccharide- A nd polypeptide-based amphiphilic block copolymers for biomedical applications. Herein, we report the synthesis and characterization of a series of polysaccharide-block-ELP copolymers, containing two biocompatible and biodegradable blocks coupled via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The resulting bioconjugates are capable of self-assembling into well-defined nanoparticles in aqueous solution upon raising the solution temperature above a specific transition temperature (T_t)- A characteristic of the ELP moiety. To the best of our knowledge, this is the first study where polysaccharides were combined with a stimuli-responsive ELP for the preparation of thermosensitive self-assemblies, providing insight into novel pathways for designing bioinspired stimuli-responsive self-assemblies for biomedical applications.

Full text of DOI:10.1021/acs.biomac.9b01058

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Article
Design of polysaccharide-b-elastin like polypeptide
bioconjugates and their thermoresponsive self-assembly
Ye Xiao, Zoeisha S. Chinoy, Gilles Pecastaings, Katell
Bathany, Elisabeth Garanger, and Sebastien Lecommandoux
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b01058 • Publication Date (Web): 24 Sep 2019
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Design of polysaccharide-b-elastin like polypeptide
bioconjugates and their thermoresponsive self-
assembly
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Ye Xiao,†§ Zoeisha S. Chinoy,†§ Gilles Pecastaings,† Katell Bathany,‡ Elisabeth Garanger,*† and
Sébastien Lecommandoux*†
† Univ. Bordeaux, CNRS, Bordeaux INP, LCPO, UMR 5629, F-33600, Pessac, France
‡ Univ. Bordeaux, CNRS, Bordeaux INP, Chimie et Biologie des Membranes et des Nano-objets
(UMR 5248), Allée Geoffroy Saint Hilaire, F-33600, Pessac, France
§ Authors contributed equally to this work
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ABSTRACT
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The advantageous biological properties of polysaccharides and precise stimuli-responsiveness of
elastin-like polypeptides (ELPs) are of great interest for the design of polysaccharide and
polypeptide-based amphiphilic block copolymers for biomedical applications. Herein, we report
the synthesis and characterization of a series of polysaccharide-block-ELP copolymers,
containing two biocompatible and biodegradable blocks coupled via copper(I)-catalyzed azide-
alkyne cycloaddition (CuAAC). The resulting bioconjugates are capable of self-assembling into
well-defined nanoparticles in aqueous solution upon raising the solution temperature above a
specific transition temperature (Tt) – a characteristic of the ELP moiety. To the best of our
knowledge, this is the first study where polysaccharides were combined with a stimuli-
responsive ELP for the preparation of thermo-sensitive self-assemblies, providing insight into
novel pathways for designing bioinspired stimuli-responsive self-assemblies for biomedical
applications.
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INTRODUCTION
Nanostructured carriers resulting from the self-assembly of amphiphilic block copolymers have
been developed for a broad range of applications, ranging from biomimetics1-3 to
nanomedicines.4,5 Numerous synthetic polymers have been selected as building blocks due to
their biocompatibility and low cytotoxicity, such as poly(ethylene glycol),6,7 poly(L-glutamic
acid),8,9 poly(γ-benzyl-L-glutamate),10-12 poly(2-hydroxyethyl methacrylate),13,14 polylactide,15,16
polycaprolactone,17 and poly(trimethylene carbonate),9,18 and have been approved for biomedical
applications by regulatory agencies. Additionally, biologically derived or inspired polymers, like
polysaccharides19-21 and polypeptides22, show considerable promise as block copolymer
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constructs since they are naturally biodegradable, biocompatible and are potentially
biofunctional. Regarding the biomedical application of block copolymers as carriers, a key
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challenge is to enhance accumulation of the active components at the biological target.23
A
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typical method to achieve this goal is to introduce bioactive targeting moieties on the surface of
nano-objects.24-26 Such surface decorated strategies have led to the design of original bioactive
block copolymers that favor the use of natural targeting functionalities.27-29 In this respect,
bioactive polysaccharides-based (e.g., chitosan,30,31 galactan,32 fucoidan,33 hyaluronan11,34) block
copolymers able to recognize and bind to specific receptors demonstrate particularly promising
interest for potential biomedical applications.35
Recently, stimuli-responsive polymer nanosystems36-41 have been extensively explored due to
their ability of supramolecular assembly or disassembly in response to “internal stimuli”, such as
pH,42,43 reductive-oxidative environment44,45 and enzymes,46 or “external stimuli”, such as
temperature,47,48 light49,50 and magnetic field51. Among them, recombinant elastin-like
polypeptides (ELPs) have gained significant attention as stimuli-responsive protein-like
polymers.52-54 In aqueous solution, ELPs display a lower critical solution temperature (LCST),
similar to synthetic thermo-responsive polymers such as poly(N-isopropyl acrylamide)
PNIPAM,55 also referred to as inverse transition temperature (Tt) that actually corresponds to the
cloud point temperature. Below the Tt, ELPs are water soluble, while upon heating the solution
temperature above the Tt, ELPs aggregate into insoluble coacervates, this phase transition being
fully reversible. Tt can be highly tunable either by playing with molecular parameters such as
varying amino acid residues composition and protein molecular weight, or by controlling
environmental parameters such as concentration, pH or cosolutes.56-58 Post-translational chemical
modification of ELPs, for example, chemoselective modifications of ELPs at the C- or N-
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terminal end,59 or at specific residues such as methionine,60-62 cysteine,63,64 tyrosine,65 or
lysine,66,67 has also been exploited for tuning the Tt of specific ELPs, as well as for facilitating
subsequent conjugation of ELPs with polymers,59,66 dyes,64 or drugs67,68. ELP diblock
copolymers could be obtained by fusing ELP blocks with different Tt, which self-assembled into
micelles by triggering the phase transition of the ELP block with the lowest Tt.69-72 The stimuli-
responsive property of ELPs has also been exploited to obtain well-defined ELP-based
nanomedicines, such as ELP-proteins,73 ELP-drug conjugates,67 and ELP-nucleic acid
complexes.74 Moreover, as a building block of copolymers, combinations of ELPs with other
types of polymer-based blocks, such as poly(γ-benzyl-L-glutamate),75 poly(L-glutamic acid),8
poly(aspartic acid),76 poly(ethylene glycol),59,77 and polyelectrolyte78 has demonstrated to be a
fascinating strategy to obtain functional block copolymers which could self-assemble in aqueous
conditions above a specific and tunable Tt.
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Although polysaccharides and polypeptides-based block copolymers were reported for decades,
benefits from conjugates of polysaccharides with ELPs have not yet been investigated, since a
highly efficient and proper conjugation method is required. We previously synthesized
polysaccharide-b-polypeptide block copolymers via copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) for the preparation of glycoprotein biomimetic vesicles,11,12 and
demonstrated the interest of such design as drug-delivery nanocarriers.79,80 To be able to use
CuAAC “click” chemistry for conjugation, polysaccharides and ELPs need to be functionalized
with either an azide or an alkyne group.81 Polysaccharides can be readily modified at the
reducing end with azide-containing linkers using reductive amination,11,12 or by using
bifunctional oxyamine, oxime or N-methoxyamine linkers82,83. Regarding the introduction of an
alkyne group at the ELP chain end, an alkyne functionalized N-hydroxysuccinimide ester (NHS-
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ester) can be easily prepared and coupled with the N-terminal primary amine group, granting the
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ELP with a functional moiety to conjugate with the polysaccharide.84
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We present here, a modular approach that combines polysaccharide and ELP into block
copolymers capable of self-assembling into nanoparticles in aqueous solution upon heating
above a specific Tt. Both block being composed of biological molecules, make the
polysaccharide-b-ELP bioconjugates biocompatible, biodegradable and possibly
bioactive. The versatile choice of polysaccharide and ELP allow “tailor-made” design of
bioconjugates for a desired propose, such as specific receptor binding and drug loading.
The ELP block can be further modified at methionine residues, allowing subsequent
orthogonal biofunctionalization of bioconjugates with small molecules such as dyes or
inter-chain cross-linking. Three different polysaccharides namely dextran,10,12
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laminarihexaose85,86 and hyaluronic acid,11,25,34 were functionalized with an azide moiety at their
reducing end, while the ELP was modified with an alkyne moiety at the N-terminus by NHS-
coupling chemistry. Three polysaccharide-b-ELP bioconjugates were thus prepared through click
chemistry, isolated and subsequently fully characterized. Their transition temperature behaviors
were studied through turbidity measurements by ultraviolet–visible (UV-Vis) spectroscopy and
their self-assembly properties were subsequently investigated by temperature-varying dynamic
light scattering (DLS) and temperature-controlled liquid atomic force microscopy (Liquid AFM).
To our knowledge, the synthesis and temperature-triggered self-assembly behavior study of
polysaccharide-b-ELPs block copolymers were demonstrated for the first time, providing insight
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into original pathways for designing bioactive thermo-sensitive self-assemblies for biomedical
applications.
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EXPERIMENTAL SECTION
Materials
Acrolein (95%), sodium azide (NaN3, 99.5%), acetic acid (AcOH, 99.8%), methoxylamine
hydrochloride (98%), sodium cyanoborohydride (NaBH3CN, 95%), hydrochloric acid (HCl,
37%), 4-pentynoic acid (97%), N,N'-dicyclohexylcarbodiimide (DCC, 99%), N-
hydroxysuccinimide (NHS, 98%), trimethylamine (TEA, 99%), copper(II) sulfate pentahydrate
(CuSO4, 99%), dichloromethane (DCM, 99.9%), N,N-dimethylformamide (DMF, 99.8%),
dimethyl sulfoxide (DMSO, 99.7%), methanol (MeOH, 99.8%), diethyl ether (99.9%) and
anhydrous magnesium sulfate (MgSO4, 99.5%) were purchased from Sigma-Aldrich. N,N-
Diisopropylethylamine (DIPEA, 99%), sodium acetate (AcONa, 99%) and sodium ascorbate
(NaAsc, 99%) were obtained from Alfa Aesar. Tris(benzyltriazolylmethyl)amine (TBTA, 97%)
were purchased from TCI. Cuprisorb® was purchased from Seachem. Dextran (Dex, T10) was
purchased from pharmacosmos. Laminarihexaose (Lam) was purchased from Megazyme.
Sodium hyaluronate (HA) was purchased from Lifecore Biomedical. Water was purified using
an ELGA PURELAB Classic system. Solvent was purified using PureSolv MD-5 solvent
purification system from Innovative Technology. Dialysis was conducted using a Spectra/Por®6
dialysis membrane.
Synthetic Procedure
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Synthesis of N-(3-azidopropyl)-O-methylhydroxylamine (Azide-linker, 1). Acrolein (1.84 mL,
27.4 mmol) was added to acetic acid (4 mL) at -20 °C, followed by dropwise addition of a
solution of sodium azide (2.38 g, 41.2 mmol) in H2O (10.4 mL). The reaction mixture was let to
stir at -20 °C for 1.5 hour. It was then quenched with a saturated solution of NaHCO3 (80 mL)
and the resulting mixture was extracted with DCM (2 × 100 mL). The combined organic extracts
were washed with sat. aq. NaHCO3 (150 mL), dried over MgSO4, filtered and concentrated in
vacuo to 100 mL. To the solution in DCM, methoxylamine hydrochloride (2.68 g, 31.68 mmol)
and sodium acetate (4.42 g, 54 mmol) were added and the mixture was stirred overnight at r.t.
Sat. aq. NaHCO3 (150 mL) was added to quench the reaction and the resulting mixture was then
extracted with DCM (2 × 100 mL). The combined organic extracts were washed with Sat. aq.
NaHCO3 (150 mL), dried over MgSO4, filtered and concentrated in vacuo to 100 mL. To the
solution in DCM, NaBH3CN (2 g, 32 mmol) was added, followed by dropwise addition of 1M
ethanolic HCl (32 mL, freshly prepared by adding acetyl chloride to ethanol). The reaction
mixture was stirred at r.t for 1.5 hour, concentrated and the resulting white solid suspended in
sat. aq. NaHCO3 (150 mL) and extracted with DCM (2 × 100 mL). The combined organic
extracts were washed with sat. aq. NaHCO3 (150 mL), dried over MgSO4, filtered and
concentrated in vacuo to afford the crude N-(3-azidopropyl)-O-methylhydroxylamine as a yellow
oil. Purification of the crude product by silica gel column chromatography (1-3% MeOH in
DCM) yielded N-(3-azidopropyl)-O-methylhydroxylamine as a colorless oil (Azide-linker, 2.2 g,
62% over 3 steps). 1H NMR (400 MHz, CDCl3): δ 3.55 (s, 3H, CH3O), 3.41 (t, 2H, CH2N3), 3.00
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(t, 2H, NHCH2), 1.83 (p, 2H, CH2CH2CH2). C NMR (101 MHz, CDCl3): δ 62.01 (CH3O),
49.44 (CH2N3), 48.94 (NHCH2), 26.87 (CH2CH2CH2). In agreement with the literature data.82
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Synthesis of Dextran-Azide (Dex-N3, 2). The azide-linker (380 mg, 2.9 mmol) was added to a
solution of dextran (MW=8,000 Da, 1 g, 125 µmol) in acetate buffer (AcOH / AcONa, 2 M, pH
4.6, 4.2 mL), and the reaction mixture was incubated at 40 °C with shaking, for 9 days. The
product was purified by dialysis (dialysis bag MWCO 1 kDa) against pure water for 24 hour
(changing water 3 times per day), followed by lyophilization to obtain the product as a white
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powder (805 mg, 79% yield). H NMR (400 MHz, D2O): δ 4.98 (d, H-1), 4.18 (d,
CHN(OCH3)CH2), 4.04-3.97 (m, H-6), 3.95–3.89 (m, H-5), 3.69–3.80 (br m, H-6’, H-3), 3.61–
3.37 (br m, H-2, H-4, CH2N3), 3.20–3.13 (dt, CHN(OCH3)CH2), 3.01–2.93 (dt,
CHN(OCH3)CH2’), 1.92–1.88 (m, CH2CH2CH2). FT-IR (ATR): 3368, 2906, 2106 (υazide), 1351,
1148, 1164, 1007, 915, 846, 763, 545, 429 cm-1.
Synthesis of Laminarihexaose-Azide (Lam-N3, 3). The azide-linker (900 mg, 6.9 mmol) was
added to a solution of laminarihexaose (500 mg, 0.5 mmol) in acetate buffer ( AcOH / AcONa, 2
M, pH 4.6, 5 mL), and the reaction mixture incubated at 40 °C with shaking, for 8 days
(vortexing 3 times per day). Then the mixture was lyophilized and re-dissolved in water 5 mL,
purified by dialysis (dialysis bag MWCO 100 Da) against pure water for 3 days (changing water
3 times per day and transferring excess dialysis solution to new dialysis bag every 12 hour),
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followed by lyophilization to obtain the product as a white powder (302 mg, 54% yield). H
NMR (400 MHz, D2O): δ 4.81 (d, H-1), 4.23 (m, CHN(OCH3)CH2), 3.97–3.88 (m, H-6), 3.86–
3.68 (br m, H-6’,H-3), 3.67–3.33 (br m, H-2,H-4,H-5, CH2N3), 3.22–3.13 (m,
CHN(OCH3)CH2’), 3.03–2.95 (m, CHN(OCH3)CH2), 1.91 (m, CH2CH2CH2). FT-IR (ATR):
3434, 3151, 2890, 2100 (υazide), 1568, 1403, 1308, 1159, 1072, 1022, 896, 557 cm-1.
Synthesis of Hyaluronan-Azide (HA-N3, 4). The azide-linker (520 mg, 4 mmol) and sodium
cyanoborohydride (65 mg, 1 mmol) were added to a solution of sodium hyaluronate (MW=
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7,000 Da, 1 g, 0.14 mmol) in acetate buffer (AcOH / AcONa, 2 M, pH 5.5, 5 mL), and the
reaction mixture was incubated at 50 °C with shaking, for 5 days. The product was purified by
dialysis (dialysis bag MWCO 1 kDa) against pure water for 24 hour (changing water 3 times per
day), followed by lyophilization to obtain the product as a white powder. (610 mg, 60% yield).
1H NMR (400 MHz, D2O): δ 4.56 (d, GlcNAc H-1), 4.48 (d, GlcUA H-1), 3.99–3.67 (br m,
GlcNAc H-6, H-2, H-3, H-5, GlcUA H-4), 3.66–3.41 (br m, GlcNAc H-4, GlcUA H-3, H-5,
CH2N3), 3.35 (t, GlcUA H-2), 2.94–2.86 (m, CH2N(OCH3)CH2), 2.03 (s, GlcNAc COCH3), 1.87
(m, CH2CH2CH2). FT-IR (ATR): 3323, 2892, 2107 (υazide), 1729, 1642, 1555, 1376, 1315, 1152,
1042, 610 cm-1.
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Synthesis of 4-Pentynoic Acid Succinimidyl Ester (Pentynoic NHS-ester, 5). N, N’-
dicyclohexylcarbodiimide (480 mg, 2.3 mmol) was added to a solution of pentynoic acid (210
mg, 2.14 mmol) in DCM (8mL) and the mixture was stirred at r.t for 5 min and then N-
hydroxysuccinimide (260 mg, 2.3 mmol) was added. The reaction was continuously stirred at r.t
for 3 hour more. Afterwards, the precipitated dicyclohexylurea was filtered over Celite and
washed with cold DCM (2×10mL). The filtrate was collected and DCM was removed in vacuo.
The crude product was re-dissolved in EtOAc (10 mL) and cooled in a refrigerator at 0 °C for 20
min. The precipitate was filtered over Celite, and the filtrate was washed with saturated NaHCO3
(2×50 mL) and brine (2×50 mL), dried over MgSO4, filtered and concentrated in vacuo to give
the crude product as a colorless oil. Purification of the oil by silica gel column chromatography
(petroleum ether/EtOAc = 2:1) yielded pentynoic NHS-ester as a white solid (300 mg, 72%
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yield). H NMR (400 MHz, CDCl3): δ 2.90–2.82 (m, 6H, COCH2CH2CO, OCOCH2CH2), 2.62
(ddd, 2H, CH2C≡CH), 2.05 (t, 1H, C≡CH). 13C NMR (101 MHz, CDCl3): δ 168.89
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(COCH2CH2CO), 167.02 (OCOCH2), 80.84 (CH2C≡CH), 70.04 (CH2C≡CH), 30.31 (OCOCH2),
25.57 (COCH2CH2CO), 14.09 (CH2C≡CH).
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Synthesis of ELP-Alkyne (ELP-Alk, 6). Pentynoic NHS-ester (92 mg, 0.47 mmol) and N,N-
diisopropylethylamine (1.7 mg, 13.2 μmol) were added to a solution of ELP (MW= 17,035 Da,
225 mg, 13.2 μmol) in anhydrous DMSO (18 mL) and the mixture was stirred at r.t for 72h. It
was then diluted with pure water (20 mL). The product was purified by dialysis (dialysis bag
MWCO 15 kDa) against pure water for 48 h (changing water 3 times per day), followed by
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lyophilization to obtain the ELP-Alk as a white powder (210 mg, 93% yield). H NMR (400
MHz, D2O): δ 7.63–7.09 (br, indole H Trp), 4.57 (m, CHα Met), 4.45 (m, CHα Val, Pro), 4.19 (d,
CHα ValXaa), 4.06–3.89 (br m, CH2α Gly, CH2δ Pro), 3.75 (m, CH2’δ Pro), 2.69–2.46 (br m, CH2γ
Met, CH2CH2C≡CH ), 2.33 (m, CH2β Pro), 2.18–1.91 (m, CH2β Met, CH2’β Pro, CH2γ Pro, CHβ
Val, CH3ε Met, CH2C≡CH), 1.05–0.91 (m, CH3γ Val). MALDI-TOF: Calculated Mass = 17,115
Da, Experimental Mass [M+H]+ = 17,120.5 Da.
Synthesis of Dextran-b-ELP (Dex-b-ELP, 7). Copper sulfate (6 mg, 22.8 μmol), sodium
ascorbate (10 mg, 45.6 μmol) and TBTA (12 mg, 22.8 μmol) were added to a solution of ELP-
Alk (65 mg, 3.8 μmol) and Dex-N3 (46 mg, 5.7 μmol) in anhydrous DMSO (8 mL) under argon
atmosphere and the reaction mixture was stirred at r.t for 3 days. It was then diluted with cold
water (20 mL) and cooled in a refrigerator at 4 °C for 20 min. TBTA was precipitated and
removed by centrifugation. Cuprisorb (120 mg) was added to the resulting solution and it was
then incubated at r.t with shaking, for 18 h to remove the copper. The solution containing
cuprisorb was centrifuged and the supernatant was purified by dialysis (dialysis bag 15 kDa)
against pure water for 5 days (changing water 3 times per day), followed by lyophilization to
obtain the product as a white powder (92 mg, 90 % yield). 1H NMR (400 MHz, D2O): δ 7.75 (s,
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triazole H), 7.63–7.09 (br, indole H Trp), 4.99 (d, Dex H-1), 4.55 (m, CHα Met), 4.44 (m, CHα
Val, Pro), 4.17 (d, CHα ValXaa), 4.06–3.87 (br m, CH2α Gly, CH2δ Pro, Dex H-6,H-5), 3.82-3.67
(m, CH2’δ Pro, Dex H-6’,H-3), 3.62–3.49 (Dex H-2,H-4), 2.69–2.48 (br m, CH2γ Met), 2.31 (m,
CH2β Pro), 2.18–1.89 (m, CH2β Met, CH2’β Pro, CH2γ Pro, CHβ Val, CH3ε Met), 1.03–0.88 (m,
CH3γ Val). FT-IR (ATR): 3332, 2929, 1653, 1527, 1443, 1342, 1152, 1106, 1017, 917, 547 cm-1.
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Synthesis of Laminarihexaose-b-ELP (Lam-b-ELP, 8). Copper sulfate (5.5 mg, 22 μmol),
sodium ascorbate (9 mg, 45.4 μmol) were added to a solution of ELP-Alk (62 mg, 3.6 μmol) and
Lam-N3 (20 mg, 18.1 μmol) in anhydrous DMSO (8mL) under argon atmosphere and the
reaction mixture was stirred at r.t for 3 days. It was then diluted with cold water (20 mL), after
which Cuprisorb (110 mg) was added and the resulted solution was incubated at r.t with shaking,
for overnight to remove the copper. The solution containing cuprisorb was centrifuged and the
supernatant was purified by dialysis (dialysis bag MWCO 15 kDa) against pure water for 5 days
(changing water 3 times per day), followed by lyophilization to obtain the product as a white
powder (60 mg, 93% yield). 1H NMR (400 MHz, D2O): δ 7.74 (s, triazole H), 7.60–7.09 (br,
indole H Trp), 4.80 (m, Lam H-1), 4.55 (m, CHα Met), 4.43 (m, CHα Val, Pro), 4.17 (d, CHα
ValXaa), 4.05–3.85 (br m, CH2α Gly, CH2δ Pro, Lam H-6), 3.82-3.66 (m, CH2’δ Pro, Lam H-6’,H-
3), 3.62–3.34 (Lam H-2,H-4, H-5), 2.69–2.48 (br m, CH2γ Met), 2.41–2.24 (m, CH2β Pro), 2.19–
1.88 (m, CH2β Met, CH2’β Pro, CH2γ Pro, CHβ Val, CH3ε Met), 1.05–0.85 (m, CH3γ Val). FT-IR
(ATR): 3322, 2917, 1654, 1522, 1440, 1221, 1105, 1063, 1027, 562 cm-1.
Synthesis of Hyaluronan-b-ELP (HA-b-ELP, 9). HA-N3 was first acidified by adding aq. HCl,
so as to be totally soluble in DMSO. HA-N3 (18mg, 3.5 μmol), copper sulfate (9 mg, 36 μmol),
sodium ascorbate (18 mg, 90 μmol) and TBTA (22 mg, 41 μmol) were added to a solution of
ELP-Alk (60 mg, 3.5 μmol) in anhydrous DMSO (6 mL) under argon atmosphere. The mixture
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was stirred at 40 °C for 4 days, after which the reaction was diluted with cold water (20 mL) and
cooled in a refrigerator at 4 °C for 20 min. TBTA was precipitated and removed by
centrifugation. Cuprisorb (180 mg) was added to the resulted solution and it was then incubated
at r.t with shaking, for overnight to remove the copper. The solution containing cuprisorb was
centrifuged and the supernatant was purified by dialysis (dialysis bag MWCO 15 kDa) against
pure water for 5 days (changing water 3 times per day), followed by lyophilization to obtain the
crude product (53 mg). The crude product was redissolved in deionized water (5.3 mL) and the
solution was then heated to 40 °C and kept for 1 h. The insoluble unreacted ELPs were
centrifuged at 13,000 g for 20 min at 40 °C. The supernatant was lyophilized to give the final
product as a white powder (42 mg, 54 % yield). 1H NMR (400 MHz, D2O): δ 7.75 (s, triazole H),
7.60–7.09 (br, indole H Trp), 4.62–4.37 (br, CHα Met, Val, Pro, GlcUA H-1, GlcNAc H-1), 4.17
(d, CHα ValXaa), 4.04–3.66 (br, CH2α Gly, CH2δ Pro, CH2’δ Pro, GlcUA H-4, GlcNAc H-2, H-3,
H-5, H-6), 3.65–3.42 (GlcUA H-3, H-5), 3.41–3.30 (t, GlcUA H-2), 2.69–2.48 (br m, CH2γ Met),
2.41–2.25 (m, CH2β Pro), 2.20–1.89 (m, CH2β Met, CH2’β Pro CH2γ Pro, CHβ Val, CH3ε Met,
CH3 GlcNAc), 1.06–0.88 (m, CH3γ Val). FT-IR (ATR): 3298, 2964, 1631, 1528, 1440, 1232,
1153, 1044, 541 cm-1.
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Characterization Methods
Nuclear Magnetic Resonance Spectrometry Analysis (NMR)
NMR spectra were acquired in D2O or CDCl3 at 298 K on a Bruker Avance I NMR spectrometer
operating at 400.2 MHz and equipped with a Bruker multinuclear z-gradient direct probe head
capable of producing gradients in the z direction with 53.5 G.cm–1 strength. The relaxation time
was fixed to 3 seconds for homonuclear correlation spectroscopy (COSY) measurements.
Mass Spectrometry Analysis (MS)
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Mass spectrometry analyses were performed on a MALDI TOF/TOF (Ultraflex III, Bruker
Daltonics, Bremen, Germany) equipped with a SmartBeam laser (Nd:YAG, 355 nm). Solutions
of ELPs were prepared as follows: lyophilized ELPs were resuspended in water/acetonitrile (1/1,
v/v) to obtain a final concentration lower than 100 μM. Samples were then mixed with the matrix
solution of sinapinic acid prepared at the concentration of 10 mg/mL in water/acetonitrile (1/1,
v/v). All MALDI-MS measurements were acquired in the linear positive mode and a mixture of
standard proteins was used for external calibration in the suitable mass range (10−20 kDa).
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Size Exclusion Chromatography (SEC)
SEC analysis was performed on a SEC-MALS system with refractive index and laser scattering
detectors (WYATT Technology Optilab rEX and HELEOS-II) using an aqueous buffer (0.1 M
NaNO3, 0.01 M Na2HPO4, 0.02 M NaN3) with a flow rate of 0.6 mL/min at 22 °C. The specific
refractive index increment (dn/dc) of polysaccharide-b-ELP bioconjugates were measured by
means of a differential refractometer (Wyatt Optilab rEX) operating at a wavelength of 658 nm
at 26 °C. A single concentration of each bioconjugate was used to determine the dn/dc
coefficient through the calculation module implemented in the Astra 7.1 software. The measured
dn/dc values of polysaccharide-b-ELP bioconjugates were applied for the calculation of weight
average molecular weight (Mw).
Fourier Transform Infrared Spectroscopy (FT-IR)
FT-IR spectra were recorded using a Bruker Vertex 70 spectrometer with a GladiATR diamond.
Spectra were recorded directly on a powder samples at 400-4000 cm-1 (resolution of 4
wavenumber) range by using attenuated total reflection mode.
Transition Temperature Measurements (Tt)
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Tt were determined by measuring the turbidity at 350 nm between 10 and 80 °C at a 1 °C / min−1
scan rate at different concentrations in DI water. Data were collected on a Cary 100 UV−Vis
spectrophotometer equipped with a multicell thermoelectric temperature controller from Agilent
Technologies (Les Ulis, FR). The Tt is defined as the temperature corresponding to the point
where the maximum standard deviation on the absorbance versus temperature plots.
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Dynamic Light Scattering Measurements (DLS)
Dynamic light scattering measurements were performed on NanoZS instrument (Malvern, U.K.)
at a 90° angle at a constant position in the cuvette (constant scattering volume). The derived
count rate (DCR) was defined as the mean scattered intensity normalized by the attenuation
factor. The DCR was plotted against temperature and the Tt is defined as the temperature
corresponding to the point where the DCR starts increasing on this plot.
Temperature-controlled Liquid Atomic Force Microscopy (Liquid AFM)
Temperature-controlled liquid atomic force microscopy measurements were performed using a
Dimension FastScan Bruker AFM system. The topography images of the bioconjugates were
obtained in Peak Force tapping mode, using a Silicon cantilever (ScanAsyst-Fluid+, Bruker)
with a typical tip radius of 5 nm. The cantilever resonance was 150 kHz and the spring constant
was 0.7 N/m. Substrates were purchased from Agar Scientific. Samples were prepared by drop-
casting a bioconjugates water solution of 50 μM (150 μM for HA-b-ELP) onto a freshly cleaved
mica or HOPG surface, which was directly applied for imaging. AFM imaging process was
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conducted in liquid environment at specific temperature. An external heating stage (Bruker) was
used to achieve the target temperature at the substrate surface.
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RESULTS AND DISCUSSION
Synthesis of polysaccharide-b-ELP block copolymers
The synthetic strategy was based on a coupling reaction between the polysaccharide and ELP
blocks via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) which has previously been
applied for biomacromolecular conjugation with high selectivity and coupling efficiency.87 For
this purpose, the three selected polysaccharides and the ELP needed to be functionalized with
either an azide or an alkyne group. In most cases, polysaccharides can be straightforwardly
modified at their reducing end by azide-containing amines in acidic conditions with excellent
functionalization degrees. Regarding the introduction of an alkyne group at the ELP chain end,
an activated alkyne-functionalized NHS-ester can be exploited and subsequently substituted by
the N-terminal primary amine of the ELP.84 Herein the different polysaccharides were modified
at their reducing end with an azide group using a bifunctional N-methoxyoxyamine linker and
the ELP was modified at the N-terminal end with an alkyne moiety (Scheme 1), allowing the
subsequent coupling of the two blocks by CuAAC as illustrated on Scheme 1C.
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O
1, AcOH, NaN3, H2O,-20oC, 1.5h
O
N
H
O
N3
N
A
B
2, MeONH2HCl, AcONa, r.t, overnight
NHS, DCC
DCM, r.t
HO
O
O
5
O
3, NaBH3CN, HCl(EtOH), r.t, 1.5h
1
O
O
OH
DIPEA, DMSO,
48 h, r.t
ELP
R
Acetate Buffer
Polysaccharide
9
H
N
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O
N
O
O
OH
N3
N
N3
O
S
O
N
O
O
R
or
R
H
N
H
N
H
OH
NH
OH
40
N
N
N
N
O
H
H
H
O
O
O
R'
O
2, 3
4
DMSO
48 h
CuSO4, Sodium
Ascobate, TBTA
C
6
H
N
O
R
OH
7, 8
O
N
N
N
N
O
H
O
O
O
H
H
OH
40
N
N
N
N
OH
N
N
N
H
H
H
O
O
O
R'
O
R
NH
O
S
9
R
: Polysaccharide backbone (2, 7 = dextran; 3, 8 = laminarihexaose and 4, 9 = hyaluronic acid)
R' : -CH(CH3)2 or -(CH2)2SCH3 (Val:Met = 3:1)
Scheme 1. Synthetic strategy of polysaccharide-b-elastin-like polypeptide block copolymers. (A)
Synthesis of the bifunctional linker 1 and of the NHS-alkyne ester 5. (B) Reducing-end
functionalization of polysaccharides and modification of ELP at the N-terminus. (C) Huisgen’s
cycloaddition between the azide-functionalized polysaccharides and the alkyne-functionalized
ELP.
Reducing-end functionalization of polysaccharides
Primary amines have been extensively used for the functionalization of polysaccharides by
reductive amination to form an open-chain monosaccharide at its reducing end.11,12 Recently the
use of functional oxyamine linkers for conjugation to glycans has been demonstrated with highly
efficient coupling.82,83 In comparison to the conventional reductive amination of glycans with
primary amines, the use of oxyamine linkers in acetate buffer without any reducing agent was
able to produce the native closed conformation of the reducing sugar, which is essential for the
recognition of some saccharides to the relevant receptors. In order to explore more on this
methodology for the functionalization of polysaccharides, we first prepared the azide-
functionalized oxyamine linker 1 via a three-step one-pot strategy (Scheme 1A) and
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characterized it by 1D and 2D NMR (Figure S1).82 This azide-linker contains an N-
methoxyamine functional group for conjugation to the reducing end of the polysaccharides and a
chain end azide group for subsequent conjugation to the ELP.82,83 The modification reactions of
Dex and Lam by the oxyamine linker were carried out in acetate buffer at 40 °C at pH 4.6
(Scheme 1B). The 1H, HSQC and COSY NMR spectra in D2O of modified polysaccharides were
recorded and all peaks were assigned (see Figures S2, S3). Full conversion of Dex and Lam were
confirmed by comparing the integral of the peak at 1.84 ppm corresponding to the resonance of
the β-methylene of oxyamine linker with the integral of the peak at 4.1 ppm for Dex and 4.2 ppm
for Lam (anomeric proton of the reducing sugar). The two protons of α-methylene in oxyamine
linker have different resonance frequencies (2.89 ppm and 3.08 ppm) illustrating a
stereochemical non-equivalence of the two protons, which indicated that the reducing sugar
retained its closed conformation with a β-anomeric configuration due to the stereochemical effect
of the methoxy group82, and the proton coupling constant of a β-linked sugar (J = 8.9 Hz).
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Applying the same strategy on the modification of HA by the oxyamine linker, either at pH 4.6
or 5.5 of acetate buffer gave a conversion below 50 %. This may due to the high negative charge
density of HA. Thus conventional reductive amination in a buffer at pH 5.5, which has also
been previously reported for the modification of HA,11 was applied to couple the reducing
end of HA with the oxyamine linker, using sodium cyanoborohydride (NaBH3CN) as a reducing
agent (Figure S4). Also, the integration of the NMR spectra of the starting HA and the
azido-functionalized HA showed the degree of functionalization (Figure S5). As a result
of the reduction, the protons of the α-methylene in the oxyamine linker have stereochemical
equivalence (2.88 ppm), revealing an open chain reducing end of HA (Figure S5). Successful
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azide-functionalization of the three polysaccharides was also assessed by tracking the appearance
of azide vibration signal at 2100 cm-1 in FTIR spectra (Figure S6, S7, S8).
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Alkyne functionalization at the N-terminal end of ELP
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The ELP used in this work was prepared by recombinant DNA and protein engineering
techniques in Escherichia coli as described previously.60 This ELP contains in total 40
pentapeptide repeats with the protein sequence MW[(VPGVG)(VPGMG)(VPGVG)2]10,
corresponding to a molecular weight of 17,035 Da. The ELP-polysaccharide complex reported,
uses lysine-containing ELPs and charged polysaccharides as polyelectrolytes to form the
complex.78 In our strategy, we covalently conjugate the polysaccharide to the ELP to form a
block copolymer, the ELP, having methionine residues, can be further functionalized to provide
diverse possibilities of crosslinking or side-chain modifications.60-62 The MS and NMR spectra
characterization of this “starting” ELP are provided in Figure S9 and S10. In order to introduce
an alkyne group on the ELP, the activated NHS-alkyne ester 5 was synthesized by a coupling
reaction between N-hydroxysuccinimide and 4-pentynoic acid (Scheme 1A). The reaction of the
1
primary amine of ELP with the NHS-alkyne ester 5 was thereafter verified by H NMR and
MALDI-Tof mass spectrometry (Figure S9, S12). Even though the terminal alkyne moiety was
1
not detected by H NMR spectroscopy of the purified chain end-functionalized ELP, the
appearance of peaks for two methylene groups of the alkyne linker at 2.3 ppm was an indication
of successful modification (Figure S12). Full alkyne functionalization of ELP was also
confirmed by the MS shift to 17,119 Da after NHS-coupling chemistry (Figure S9).
Synthesis of polysaccharide-b-ELP copolymers
The emergence of “click chemistry” has revolutionized bioconjugate chemistry by affording
excellent selectivity and using soft reaction conditions amenable to both synthetic polymers and
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fragile biomacromolecules, such as proteins or DNAs.88 A widely used “click reaction” is the
Cu(I)-catalyzed azide-alkyne cycloaddition or Huisgen’s cycloaddition.87 Herein, conjugations of
ELP and polysaccharides were carried out via Huisgen’s cycloaddition in DMSO as common
solvent for the ELP and polysaccharides (Scheme 1C). TBTA, a powerful stabilizing ligand for
copper(I), was used in the conjugation of Dex and HA as they have a much higher molecular
weight than Lam. The mole ratio of polysaccharides to ELP, which are 1.5:1, 5:1 and 1:1,
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for Dex, Lam and HA, respectively. The excess polysaccharides were removed by
extensive dialysis against water and the unreacted ELP was removed by inverse
transition cycling. The success of the click reaction was subsequently monitored by SEC, as
well as by 1H, HSQC and COSY NMR and FTIR spectroscopy.
Figure 1. SEC traces of ELP and polysaccharide-b-ELP bioconjugates in aqueous buffer (0.1 M
NaNO3, 0.01 M Na2HPO4, 0.02 M NaN3) using a RI detector.
The conjugation of polysaccharides to the ELP coincided with a clear increase in the molecular
weight of the different block copolymers as observed in Figure 1 and S13. SEC also highlights
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the low dispersity of the resulting block copolymers (Table 1). All the peaks in 1H NMR spectra
were assigned with the help of corresponding HSQC and COSY NMR, indicating both
polysaccharide block and ELP block (see Figure S14, S15, and S16). All NMR spectra displayed
the characteristic anomeric peaks of polysaccharides at around 5.0 ppm and the triazole proton
peak was found at 7.8 ppm (Figure 2) confirming the desired structures of the block copolymers.
FTIR spectra of the bioconjugates, after the click reaction, were also compared with the ones of
alkyne-functionalized ELP and corresponding azide-functionalized polysaccharides (Figure S6,
S7 and S8). The complete disappearance of the azide band at 2100 cm-1 in the bioconjugates
proved the success of the “click reaction” bioconjugation.
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Polysaccharide
mass fraction
a
Bioconjugates
Mw (kDa)
PDIa Tt in waterb (125 µM)
Dex-b-ELP
Lam-b-ELP
HA-b-ELP
24.9
18.2
24.3
32 %
1.01
1.03
1.02
39 °C
36 °C
48 °C
5 %
30 %
Table 1. Characteristics of the polysaccharide-b-ELP bioconjugates synthesized. aWeight
average molecular weight (Mw) and polydispersity index (PDI) were determined by SEC in
aqueous buffer. bTt was measured by turbidity on UV-Vis.
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Figure 2. 1H and HSQC NMR Spectra of Dex-b-ELP in D2O at 25 °C.
Temperature-triggered self-assembly of polysaccharide-b-ELP copolymers
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Thermo-responsive properties
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The thermo-responsive properties of the different polysaccharide-b-ELP block copolymers were
first studied by measuring the Tt values by turbidity measurement at 350 nm. In contrast with Tt
of ELP (32 °C) at same concentration (Figure 3A), Tt of the bioconjugates shifted to higher
temperature depending on the hydrophilicity of polysaccharides (36 °C, 39 °C and 48 °C for
Lam-b-ELP, Dex-b-ELP and HA-b-ELP, respectively). Consistently, the higher the
hydrophilicity of the polysaccharide, the larger the Tt. One can note that the slight decrease in
absorption of HA-b-ELP at high temperature was most likely due to the formation of large
aggregates, which may sediment (Figure S24). The Tt values of ELP and ELP bioconjugates
were then plotted as functions of concentration (Figure 3B) and the data were fitted using the
empirical law proposed by Chilkoti and coworkers.56 This equation gave satisfactory fits of Tt,
concentration and temperature, enabling accurate prediction of the Tt of ELP and its
bioconjugates at specific concentrations. As the hydrophilicity of the ELP bioconjugates
decreased, the slope of the fit became flatter. This result is consistent with the observations
reported by Chilkoti and coworkers.56
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Figure 3. Turbidity study of ELP and polysaccharide-b-ELP bioconjugates. (A) Absorbance
measured at 125 μM in water as function of temperature. (B) Tt values plotted as function of
concentration in water.
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Self-assembly of polysaccharide-b-ELP block copolymers
In a next step, we were interested in studying the self-assembly properties of polysaccharide-b-
ELP copolymers and their ability to form well-defined micellar structures. The behavior of Dex-
b-ELP (125 μM in water) was first analyzed by DLS. Measuring the total scattering intensity
with temperature allowed the determination of Tt that was found to be around 40 °C.
Consistently with turbidity measurements, this value was comparable and larger than the Tt of
single ELP determined at the same concentration (Figure 4A) due to the conjugation of a
hydrophilic polysaccharide segment. Interestingly, the physical mixture of ELP and Dex did not
influence the Tt of ELP (Figure 4A, S17). As observed in Figure 4A, at low temperature (30 °C)
below Tt of Dex-b-ELP, the scattering intensity is very low, due to the presence of free
copolymer chains in solution and a few aggregates (Figure S23A). The scattering intensity
sharply increased at the transition temperature (around 40 °C) and triggered the self-assembly to
form structures with hydrodynamic diameter (Dh) around 165 nm. Once Dex-b-ELP was heated
above 45 °C, the nanoparticles showed little changes in diameter with a Dh of approximately 290
nm (Figure 4B, S23A), being stable until 60 °C. Repeated heating and cooling on Dex-b-ELP
showed similar Dh and PDI values, illustrating that this temperature-responsive behavior is fully
reversible, which offers a simple method for controlling the transition of ELPs (Figure S18).
Atomic force microscopy in a liquid cell was conducted to investigate the morphology of the
nanostructures formed by Dex-b-ELP below/above Tt. Consistent with the DLS results, very
small objects were observed below Tt at 35 °C (Figure 5A), and spherical-shaped particles were
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observed with an average diameter of approximately 280-300 nm above Tt at 65 °C (Figure 5B)
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and with a particle thickness around 7-10 nm (Figure S25A).
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Figure 4. Dynamic light scattering analysis of the self-assembly of ELP, Dex-b-ELP and
physical mixture of ELP and Dex at 125 μM in water. (A) Scattered intensity as a function of
temperature upon heating. (B) Size distribution in intensity of Dex-b-ELP at selected
temperatures.
Due to the small hydrophilic fraction of Lam-b-ELP, the Tt of Lam-b-ELP was found around 33
°C, which is slightly higher when compared to the Tt of ELP (Figure S19). At low temperature
(30 °C), below Tt of Lam-b-ELP, small objects and a few aggregates were observed with very
low scattering intensity (Figure S23B). The scattering intensity sharply increased at transition
temperature (around 33 °C) and triggered the self-assembly to form structures with Dh around
210 nm (Figure S23B). Unlike Dex-b-ELP, a peak located at 36-38 °C was most likely due to the
formation of unstable big particles, which precipitated thus decreasing the concentration in the
detected area. Repeated heating and cooling of Lam-b-ELP from 25 °C to 36 °C on DLS also
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illustrated this unstable behavior (Figure S20). Nevertheless the temperature-responsive behavior
of Lam-b-ELP is fully reversible. When temperature was heated above 45 °C, the nanoparticles
showed little changes in scattering intensity and displayed diameter with a Dh of approximately
400-500 nm (Figure S20). Liquid AFM even recorded larger diameters (500-900) for the
aggregates of Lam-b-ELP above the Tt (Figure 5D) and particle thickness was found around 3-5
nm (Figure S25B).
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Figure 5. Liquid AFM images of Dex-b-ELP (50 μM in water) on mica substrate at: (A) 35 °C
and (B) 65 °C. Lam-b-ELP (50 μM in water) on HOPG substrate at: (C) 30 °C and (D) 55 °C.
HA-b-ELP (150 μM in water) on mica substrate at: (E) 25 °C and (F) 52 °C. The scale bar
indicates 1 μm.
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Similar to the UV-vis measurements, the Tt of HA-b-ELP on DLS heating ramp was found
around 45 °C. At low temperature (35 °C) below Tt of HA-b-ELP, small objects were observed
with very low scattering intensity (Figure S23C). The scattering intensity sharply increased at
transition temperature and triggered the self-assembly to form structures with Dh around 300 nm
at 48-50 °C (Figure S21, S23C). Once HA-b-ELP was heated above 55 °C, the diameter of
nanoparticles became unstable and separated into two size distributions, which was also
confirmed by liquid AFM (Figure S24). Nanoparticles with an average diameter of
approximately 220-280 nm and a particle thickness of 0.5-1.5 nm were observed by liquid AFM
at 50 °C (Figure 5F, S25C) at the same concentration. The stability of the HA-b-ELP assemblies
at 48 °C was investigated by repeated heating and cooling on DLS. This temperature-responsive
system was again found fully reversible and HA-b-ELP nanoparticles proved relatively stable at
48 °C for 20 minutes with an average diameter around 280 nm on every single heating time
(Figure S22).
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We hypothesized that the polysaccharide-b-ELP block copolymers may behave
similarly to the ELP diblock copolymer, in which self-assembly undergoes a chain
aggregation followed by ELP core dehydration71. However, in the presence of
hydrophilic polysaccharides, molar mass and charge density of the polysaccharide, and
interactions between the polysaccharide and ELP, would contribute to the “unstable”
chain aggregation process. Self-assembly characteristics of polysaccharide-b-ELP block
copolymers were summarized in Table 2.
Bioconjugates Conc.(μM) Tt DLS
DLS
Conc.(μM)
AFM
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T(°C) Size(nm)
T(°C) Size(nm)
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65
30
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/
Dex-b-ELP
Lam-b-ELP
HA-b-ELP
125
125
150
39 °C
33 °C
46 °C
45
300
50
190-340
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45
210
520
/
50
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500-900
/
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Table 2. Self-assembly characteristics of polysaccharide-b-ELP block copolymers.
CONCLUSION
We herein presented a modular approach to combine inert (Dex) and bioactive (Lam and HA)
polysaccharides with a stimuli-responsive elastin-like polypeptide (ELP) into well-defined block
copolymers. Polysaccharides were successfully functionalized with an azide moiety at the
reducing end using a bifunctional N-methoxyoxyamine linker for further conjugation to the ELP.
NHS-ester coupling chemistry was utilized to modify the N-terminal primary amine of ELP with
an alkyne group. Thereafter, smart polysaccharide-b-ELP block copolymers were produced by
CuAAC, and their thermal responsiveness was studied by turbidity measurements. Increasing
temperature above the phase transition of ELP bioconjugates resulted in the formation of
amphiphiles, which self-assembled into well-defined and stable nano-objects. Stable objects with
a size of a few hundreds nanometers have been evidenced, especially when the hydrophilic
polysaccharide segment was large enough. In addition, the self-assembly process was fully
reversible by controlling the temperature. This is the first study that proposes the conjugation of
bioactive polysaccharides with stimuli-responsive ELPs for the preparation of thermo-sensitive
bioactive self-assemblies, providing insight into novel pathways for designing “smart” and
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biologically-active nanocarriers for applications in biomaterial, drug delivery and receptor
recognition.
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ASSOCIATED CONTENT
Supporting Information
Synthesis route of HA-azide; Characterization (NMR, FTIR) of azide-functionalized Dex, Lam,
HA and alkyne-functionalized ELP. MALDI of ELP and ELP-Alk; Characterization (SEC, NMR,
FTIR) of Dex-b-ELP, Lam-b-ELP and HA-b-ELP. DLS heating ramp data of ELP bioconjugates;
Liquid AFM images of HA-b-ELP at 55 °C.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: garanger@enscbp.fr
*E-mail: lecommandoux@enscbp.fr
ORCID
Elisabeth Garanger: 0000-0001-9130-8286
Sébastien Lecommandoux: 0000-0003-0465-8603
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We acknowledge the China Scholarship Council and Université de Bordeaux (UB-CSC 2015)
for the financial support. Campus-B project, CNRS, Univ. Bordeaux, Bordeaux INP and the
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Région Nouvelle Aquitaine are also acknowledged. This work was also supported by the French
National Research Agency (ANR-15-CE07-0002) and the Cancéropole Grand Sud-Ouest
(Emergence 2018-E18). The authors thank Guillaume Goudounet, Delphine Portes and Yunjie
Xia for the bioproduction of ELPs, Amelie Vax and Anne-Laure Wirotius for the help with SEC
and NMR measurements and analysis.
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