Thermal response of a PVCL-HA conjugate

Article abstract of DOI:10.1002/pola.27794

The synthesis and self-assembling of a thermoresponsive conjugate of hyaluronic acid (HA) and poly(N-vinylcaprolactam) (PVCL) is reported. Both polymers were end functionalized: HA via reductive amination, thereby introducing an azide endgroup to the chain end, and PVCL via thioetherification to introduce a propargyl group. The two were coupled with a copper assisted "click" reaction into a bioconjugate composed of HA blocks with the molar mass 3,600 g mol^-1 (1618 saccharide units) and PVCL blocks of 3,500 g mol^-1 (μ25 repeating units). The cloud point temperature measured by transmittance was 50-51 °C in water. The calorimetrically observed phase transition temperature of PVCL in the conjugate increased by 2 °C to 47.7 °C, whereas the enthalpy of the phase transition was unaffected by the conjugation. HA-PVCL conjugate self-assembles in water upon heating into monodisperse, colloidally stable, hollow spherical particles whose size may be tuned with the heating rate of the solution. Slow and fast heating resulted in vesicles with the hydrodynamic radii of 443 or 275 nm, respectively. The heating rate did not, however, affect the cloud point. Salt did not noticeably affect the size of the polymer particles, presumably because of interactions between the HA and PVCL blocks.

Full text of DOI:10.1002/pola.27794

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Thermal Response of a PVCL–HA Conjugate
Jukka Niskanen,1 Mikko Karesoja,1 Vladimir Aseyev,1 Xing-Ping Qiu,2 Franc¸oise M. Winnik,1,2,3
Heikki Tenhu1
1Laboratory of Polymer Chemistry, Department of Chemistry, A.I. Virtasen Aukio 1, P.O. Box 55, 00014 University of Helsinki,
Finland
2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Faculte de Pharmacie et Departement de Chimie, Universite de Montreal, CP 6128 Succursale Centre Ville, Montreal, Quebec,
H3C 3J7, Canada
3International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki,
Tsukuba, Ibaraki, 305-0044, Japan
Correspondence to: H. Tenhu (E-mail: heikki.tenhu@helsinki.fi)
Received 5 July 2015; accepted 19 July 2015; published online 11 August 2015
DOI: 10.1002/pola.27794
ABSTRACT: The synthesis and self-assembling of a thermores-
ponsive conjugate of hyaluronic acid (HA) and poly(N-vinylcap-
rolactam) (PVCL) is reported. Both polymers were end
functionalized: HA via reductive amination, thereby introducing
an azide endgroup to the chain end, and PVCL via thioetherifi-
cation to introduce a propargyl group. The two were coupled
with a copper assisted “click” reaction into a bioconjugate
composed of HA blocks with the molar mass 3,600 g mol21
(1618 saccharide units) and PVCL blocks of 3,500 g mol21 (ꢀ25
repeating units). The cloud point temperature measured by
transmittance was 50–51 8C in water. The calorimetrically
observed phase transition temperature of PVCL in the conju-
gate increased by 2 8C to 47.7 8C, whereas the enthalpy of the
phase transition was unaffected by the conjugation. HA-PVCL
conjugate self-assembles in water upon heating into monodis-
perse, colloidally stable, hollow spherical particles whose size
may be tuned with the heating rate of the solution. Slow and
fast heating resulted in vesicles with the hydrodynamic radii of
443 or 275 nm, respectively. The heating rate did not, however,
affect the cloud point. Salt did not noticeably affect the size of
the polymer particles, presumably because of interactions
C
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between the HA and PVCL blocks.
2015 Wiley Periodicals,
Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 425–436
KEYWORDS: click chemistry; hyaluronic acid; poly(N-vinylcapro-
lactam); thermoresponsive; vesicle
When constructing bioconjugates, polysaccharides may be
functionalized by esterification or etherification of the
hydroxyl or carboxylic acid groups present in the saccharide
units. “Click” reactions, such as copper-assisted alkyneazide
coupling3–5 and thiol-ene chemistry,6,7 have been utilized in
conjugations. Such bioconjugates have brush-like structures
or are cross-linked, however it is possible to prepare linear
diblock copolymers via coupling reactions.8,9 In the present
case, reductive amination is an option that is selective to the
reducing end of the carbohydrate chain. This reaction relies
on the equilibrium between the hemiacetal and free alde-
hyde form of carbohydrates. The free aldehyde reacts with
an added amine and the produced imine is quickly reduced
into a more stable amine by a reducing agent, cyanoborohy-
dride.8,10,11 Oxime formation is another route in preparing
diblock copolymers of polysaccharides, in which the alde-
hyde of the reducing end of the polysaccharide reacts with
an aminooxy group.12,13
INTRODUCTION Hyaluronic acid (HA) is an attractive candi-
date for targeting active substances to certain sites in the
human body. Combination of HA with synthetic, biocompati-
ble, and thermoresponsive poly(N-vinylcaprolactam) (PVCL)
is of interest for applications as drug delivery. HA is ubiqui-
tous in the human body, and its turnover ranges from hours
to several days.1 In the extra cellular matrix, several proteo-
glycans can be found that bind HA,1 and HA plays an impor-
tant role in stabilizing cartilage tissue.1 The binding of HA to
cell surface receptors can initiate endocytosis, catabolism,
cellular proliferation, the binding of cells to other cells, the
formation of specialized cell matrix structures, and can also
influence cell motility.1,2 Targeted conjugates of HA and
hydrophobic drugs, such as paclitaxel, have been prepared,
and it has been shown that these bioconjugates exhibit more
prominent cell toxicity toward cancer cells than the free
drug.2 This is due to over expression of HA receptors in
some cancer cells.
Additional Supporting Information may be found in the online version of this article.
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Currently, HA is primarily produced by fermentation, how-
ever, the molecular weight is usually very high, up to mil-
lions of grams per mole.14 Enzymatic degradation and
fractionation may be used in order to produce shorter and
fairly monodisperse fragments of HA, suitable for prepara-
tion of diblock conjugates.9,11 Thermoresponsive conjugates
of hyaluronic acid have been prepared by conjugating HA
with poly(N-isopropylacrylamide) (PNIPAM),5,7,15–17 poly(2-
ethyl-2-oxazoline),9 and poly(ethylene glycol) methyl ether
methacrylate-co-2-(2-methoxyethoxy) ethyl methacrylate-co-
poly(ethylene glycol) diacrylate (PEGMEMA–MEO2MA–
PEGDA).7 These bioconjugates include brush-like copolymers
with HA as the backbone,5,15–17 cross-linked HA,7 and linear
diblock copolymers.9 Linear diblock copolymers of HA have
also been prepared with poly(ethylene glycol)13,18 and poly(-
lactic acid).19
of the hydrophobic PVAc to hydrophilic PVOH, increased the
TCP from 27 to 36–42 8C.27 Using the same approach, di-
and tri-block copolymers of PVCL and random copolymers
consisting of N-vinylcaprolactam and N-vinylpyrrolidone
have been reported, exhibiting two cloud points in aqueous
solutions.26
In random copolymers of PVCL, with vinylacetate, N-methyl-
N-vinylacetamide, vinyl pivalate, and N-vinylacetamide, the
nature of the comonomer greatly affects the TCP of the copol-
ymer. Vinylacetate and N-methyl-N-vinylacetamide both
increase the TCP, with increasing the fraction of the comono-
mer. Contrary to the more hydrophobic vinyl pivalate and N-
vinylacetamide, which both decrease the TCP with increasing
fraction.30
Self-assembling bioconjugates are materials of interest in
designing vehicles in drug delivery applications. In this
paper, we present the preparation of a diblock bioconjugate
consisting of PVCL and hyaluronic acid. HA, PVCL, and their
conjugate were characterized by size exclusion chromatogra-
phy (SEC), nuclear magnetic resonance (NMR), matrix-
assisted laser desorption/ionizationtime of flight (MALDI-
TOF), and infrared (IR) spectroscopy. The HA block (3,600 g
mol21, 16 to 18 saccharide units) was obtained via enzy-
matic degradation and fractionation of high molecular weight
Poly(N-vinylcaprolactam) is a neutral thermoresponsive
polymer with a cloud point temperature of around 32 8C,
and is regarded as a biocompatible one. Upon the hydroly-
sis of the amide side group, no amines are released, con-
trary to PNIPAM.20 However, both PVCL and PNIPAM have
been found to be well tolerated by cells at concentrations
below 10 mg mL21 and at temperatures below the cloud
points of the polymers. Increasing the temperature to body
temperature increased the cell toxicity of both polymers.21
PVCL exhibits stronger interaction with macrophages both
below and above the cloud point than PNIPAM.22 In
another study, bioconjugate beads of chitosan and PVCL
were found to show a slow and steady release of drug
molecules but also to be nontoxic to human endothelial
cells.23
HA, whereas N-vinylcaprolactam (3,500
g
mol21
,
ꢀ25
repeating units) was prepared by macromolecular design via
interchange of xanthate (MADIX) polymerization. The ther-
moresponsive properties and dynamics of self-assembling of
the bioconjugate were studied in detail by microcalometry
and light scattering.
Several studies have shown that the thermoresponsiveness
of PVCL is affected by diblock formation. It has been shown
that the phase transition of PVCL is shifted to higher tem-
peratures in diblock copolymers with polyethylene oxide
(PEO),24 poly(N-vinyl pyrrolidone); PVP25,26, polyvinyl alco-
EXPERIMENTAL
Materials
Azoisobutyronitrile (Fluka) was recrystallized from methanol.
1,4-dioxane (VWR, analytical grade) and were distilled under
reduced pressure prior use. N-vinylcaprolactam (Aldrich,
98%) was recrystallized from toluene prior use. Deuterated
choloroform (Euriso-top), deuterium oxide (Euriso-top),
dichloromethane (VWR, HPLC > 99.8%), diethyl ether (Sigma-
Aldrich, > 99.8%), diethylene glycol (Sigma-Aldrich, 99%),
dimethylformamide (DMF, Lab-Scan, HPLC > 99.8%), ethanol
(96%, Altia), hyaluronic acid (2 MDa, Soliance, France), hyalu-
ronidase from bovine testes (Sigma-Aldrich, Type I-S, lyophi-
lized powder, 400–1000 units mg21 solid), hydrochloric acid
(VWR, 37%), magnesium sulfate (Alfa Aesar, anhydrous,
99.5%), methanesulfonyl chloride (Fluka, > 98%), methyl-2-
bromopropionate (Aldrich, 98%), pentane (Aldrich, 99%),
potassium ethyl xanthogenate (Aldrich, 96%), propargyl bro-
mide (80 wt % in toluene, Sigma-Aldrich), sodium azide
(Sigma-Aldrich, > 99%), sodium borohydride (Aldrich, > 96%),
sodium carbonate(Sigma-Aldrich, 99.99%), sodium chloride
(Fluka, 99.9%), sodium hydrogencarbonate (JT Baker, 99.7%),
sodium hydroxide (Fluka, > 85%), sodium phosphate (1 H2O,
JT Baker), sodium sulfate (Fluka, anhydrous > 99%), sodium-
hydrogen phosphate (12 H2O, Merck), tetrahydrofuran (THF,
hol (PVOH)27
, or poly(dimethylaminoethylmethacrylate);
PDMAEMA28 as the other block. Double thermoresponsive
diblock copolymers of PVCL have been prepared with
PDMAEMA28 and PNIPAM.29 In the case of PDMAEMA, one
or two phase transitions were observed depending on the
pH of the solution. Lowering the pH of the solution
increased the charge of the PDMAEMA block and thus the
interactions between the PVCL and PDMAEMA were
affected, but more importantly, the phase transitions were
shifted far enough from each other as to be separately visi-
ble. In the case of PVCL-PNIPAM, as is also the case with a
random copolymer of the two, only one transition was
observed.29 Cobalt-mediated polymerization has been used
in preparing block copolymers of PVCL with poly(vinyl ace-
tate); PVAc, which was later hydrolyzed to PVOH.27 The
PVCL-PVAc block copolymers form micelles at room temper-
ature in aqueous solutions, due to their amphiphilic nature,
and show only a slight change in size at elevated tempera-
tures. Whereas the hydrophilic PVCL-PVOH produced large
micelles at temperatures above the TCP of PVCL. Hydrolysis
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HPLC > 99.9%), triethylamine (Fluka, > 98%), and trimethylsi-
lane (Fluka, NMR-grade) were used as received.
thane (5 3 50 mL). The dichloromethane was dried with
Na2SO4. Product 2 (2-(2-azidoethoxy)ethanamine) of 2.42 g
was obtained after evaporation of the dichloromethane. 1H
NMR (500 MHz, CDCl3, TMS, d ppm): 1.60 (N3–CH2–), 3.36
(N–CH2–), 3.50 (O–CH2–), 3.63 (–O–CH2–), 5.28 (–NH2) and
residual solvent peaks from DMF: 2.85 and 7.97.
SYNTHESIS
Enzymatic Degradation of Hyaluronic Acid
The degradation of HA was done as reported by Yang et al.9
In short, 5 g hyaluronic acid (2 MDa) was dissolved in
750 mL saline phosphate buffer (pH 7.2). Hyaluronidase of
160 mg was added and the solution was kept at 37 8C for
30 h. The enzymatic degradation was stopped by heating the
solution to 100 8C for 5 min. A drastic change in viscosity of
the solution was observed during the degradation process.
The HA was then fractionated using ultrafiltration (10 k, 5 k,
and 1 k MWCO). The fraction used was characterized by size
exclusion chromatography (SEC) against pullulan standards
using an aqueous solution of 0.1 M NaNO3 with 3% acetoni-
trile as eluent in order to determine the polydispersity. The
molecular weight of the fraction (Mn 5 3600 g mol21, Sup-
porting Information Fig. 1) was determined by matrix-
assisted laser desorption/ionization (MALDI). The fractions
obtained after dialysis and freeze drying: 500 mg with
Linking of 2-(2-Azidoethoxy)ethanamine to Hyaluronic
Acid (Reaction 2)
103 mg hyaluronic acid (M 5 3,600 g mol21) was dissolved
in 12 mL borate buffer (pH 8.4, 0.1 M with 0.4 M NaCl)
under N2 flow. The 2-(2-azidoethoxy)ethanamine of 40 mg
was added to the solution followed by 50 mg (0.796 mmol)
of sodium cyanoborohydride and the solution was heated to
40 8C. After 9 days the product was purified by dialysis and
the presence of azide was confirmed by IR (Supporting
Information Fig. 3, absorbance band at 2112 cm21). Product
of 100 mg was obtained.
Synthesis of MADIX-CTA, O-ethyl-S-(1-
Methoxycarbonyl)ethyl-Dithiocarbonate
MADIX-CTA,
O-ethyl-S-(1-methoxycarbonyl)ethyl-dithiocar-
M > 10,000 g mol21 and 2.5 g, M > 1,000 g mol21
.
bonate, was synthesized by adapting the synthesis method
reported by Destarac et al.32 Then 20.00 g of (0.12 mol) of
methyl-2-bromopropionate was dissolved in 200 mL ethanol
and cooled in an ice bath. Potassium ethyl xanthogenate
21.20 g (0.13 mol) was added slowly, during 30 min, into
the cold reaction mixture. The reaction was stirred for 20 h
at room temperature, after which it was extracted with
300 mL of pentane-diethylether mixture (2:1). The organic
phase was washed with distilled water and dried with
MgSO4. After evaporation of the solvents, 14.90 g product
was obtained. 1H-NMR (300 MHz, CDCl3, TMS, d ppm): 1.42
(CH2–CH3), 1.56 (CH–CH3), 3.76 (O–CH3), 4.39 (O–CH2–), and
4.62 (–CH–).
2-(2-Azidoethoxy)ethanamine
An amine and azide containing asymmetric linker (2-(2-azi-
doethoxy)ethanamine) was synthesized as reported by
Schwabacher et al.31 Reaction scheme is presented as Sup-
porting Information (Fig. 2). Diethylene glycol (10.03 g,
0.094 mol) was dissolved in 110 mL THF and the solution
was cooled on an ice bath. Methylsulfonylchloride of 18 mL
(0.233 mol) was added to the cold solution. Triethylamine
32.5 mL (0.23 mol), diluted with 30 mL THF, was added
drop wise to the reaction mixture. After 24 h the precipitate
was filtered and the THF evaporated. The product was dis-
solved in a mixture of 80 mL DMF and 60 mL H2O. The pH
of the solution was adjusted to 8 with NaHCO3. Sodium azide
(12.88 g, 0.20 mol) was added and the solution was heated
to 80 8C. After 22 h the solution was extracted with diethy-
lether (6 3 60 mL). The combined ether phases were
washed with brine. The ether phase was dried with Na2SO4
and evaporated. Crude Product 1 (1-azido-2-(2-azidoethoxy)-
ethane) of 12.46 g was obtained. 1H NMR (500 MHz, CDCl3,
TMS, d ppm): 1.80 (N3–CH2–) and 3.41(–O–CH2–) with resid-
ual peaks 2.82 (DMF), 2.91(DMF), 7.95 (DMF), 1.14 (diethyl
ether), 3.36 (diethyl ether), 3.63 (–O–CH2–CH2–O–SO2Me),
and 13.69 (–O–CH2–CH2–O–SO2Me).
Synthesis of PVCL
The synthesis of PVCL was conducted as reported by
Destarac et al.33 N-VCL 5.00 g (0.036 mol), MADIX-CTA
0.052 g (0.25 mmol), and AIBN 0.0082 g (0.050 mmol) were
dissolved in 10 g of dioxane and purged with N2 for 40 min.
The reaction flask was placed in an oil bath at 60 8C. After
22 h the reaction was stopped by quenching it with liquid
nitrogen and product was precipitated twice into hexane.
Polymer of 1.06 g was obtained. The molar mass was deter-
mined by SEC (Mn 5 3,500 g mol21, D 5 1.41).
-
Reduction of the Polymer End Group into a Thiol (PVCL-
SH)
The crude product (5.43 g, 0.034 mol) was dissolved in
40 mL diethylether and 80 mL 1.2 M HCl was added, result-
ing in an emulsion. Triphenylphosphine (5.08 g, 0.019 mol),
dissolved in 40 mL diethylether, was slowly added drop wise
into the emulsion. After 20 h the precipitates were filtered
and the ether was separated. The aqueous layer was washed
with diethyl ether (4 3 60 mL). After traces of ether were
evaporated from the aqueous layer, the pH was adjusted to
11 with sodium hydroxide and the solution was stored at 7
8C. After 24 h the solution was extracted with dichlorome-
PVCL of 319 mg (0.09 mmol) was dissolved in 50 mL THF.
Sodiumborohydride of 77.16 mg (2.03 mmol) was dissolved
in 10 mL water and added to the PVCL solution. After 4 h
the THF was evaporated and the polymer purified by dialysis
against water and freeze dried, yielding 310 mg polymer
(PVCLSH).
Propargyl Functionalized PVCL
PVCL-SH of 150 mg (0.04 mmol) was dissolved in 5 ml THF.
Then 37 mg (0.35 mmol) sodium carbonate was dispersed
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in the solution and 0.37 mL (0.36 mmol) propargyl bromide
solution was added. The reaction was left over night at 40
8C. The polymer was precipitated into hexane twice from
THF and then dialyzed and finally freeze dried. The presence
of the propargyl group was confirmed by 13C NMR (Support-
ing Information Fig. 4.). 1H NMR (500 MHz, D2O, TMS, d
ppm): 1.79, 2.37, 2.55, 3.33, 4.32, and 4.47. 13C NMR (176
MHz, D2O, TMS, d ppm): 23.0, 23.3, 29.0, 29.3, 30.1, 37.6,
42.4, 46.1, 47.2, 68.5 (propargyl), and 176.2.
30 to 1508. LS studies were performed in the temperature
range from 20 to 55 8C. Solutions were filtered through 0.45
R
lm CHROMAFILV Xtra PVDF-45/25 directly into LS cells. Cor-
relation functions of the intensity of scattered light, G2(t), were
recorded simultaneously with the integral time-averaged inten-
sities, Ih ꢁ Iq, where q (5(4pno ko21)sin h/2) is the scattering
vector, no is the refractive index of the medium, and h is the
scattering angle. Methodological aspects of DLS and SLS can be
found elsewhere.35,36 In our LS experiments, bioconjugate solu-
tions in saline water were heated from 20 up to 55 8C either
using equilibrium (EH) or fast heating (FH). LS intensity meas-
ured at 458 scattering angle was a criterion of solution stability
at fixed ambient temperature. The LS intensities at the scatter-
ing angles of 1358, 908, and 458 were recorded at each temper-
ature step simultaneously with the apparent hydrodynamic
radius, Rh. Then the LS intensity and Rh were extrapolated to
zero angle. Contrast between HA and PVCL in water below the
phase separation temperature of PVCL should be similar, which
allows the analysis of LS data with appropriate accuracy. Thus
Click Reaction of HA and PVCL
Propargyl-PVCL (77.0 mg, 0.022 mmol, Mn 5 3,500 g mol21),
HA with azide end group (75.3 mg, 0.025 mmol,
Mn 5 3,600 g mol21) and 25 mg (0.14 mmol) ascorbic acid
were dissolved in 3 mL of borate buffer (pH 8.5 with 0.4 M
NaCl) and 1 mL of water. Copper(I)chloride (24.75 mg, 0.25
mmol) was placed in a separate vial. Both vials were purged
with N2 for 30 min, after which the HA-polymer-solution
was transferred into the vial with the CuCl. After 8 days,
with continuous N2 flow, the product was dialyzed against
water and then ultrafiltrated using a 5000 MWCO mem-
brane. After the filtration the sample was freeze dried. Prod-
uct of 78.8 mg was obtained. SEC of the product gave one
dn/dc of HA is 0.16–0.18 ml g21 37
0.18–0.19 ml g21 24
,
whereas dn/dc of PVCL is
.
NMR spectra were recorded with a 300 MHz UNITYVarian or
500 MHz Bruker Avance III spectrometer. Sample concentra-
tions were 60 mg mL21 in D2O or CDCl3 with tetramethylsi-
lane as the reference.
-
peak with D 1.3. The composition of the conjugate was
determined, using the NMR peaks at 2.03 (3H, HA, 12) and
2.33 (2H, PVCL, P1) ppm, to be 51% HA and 49% PVCL. The
NMR spectra are presented as figures 2 and 3. 1H NMR
(HA1PVCL-Click, 500 MHz, D2O, TMS, d ppm): 1.78 (broad),
2.03, 2.33, 3.34, 3.57, 3.93, 4.32, and 4.48. 13C NMR
(HA1PVCL-Click, 176 MHz, D2O, TMS, d ppm): 22.06, 27.76,
28.95, 33.45, 36.54, 42.24, 47.22, 53.87, 60.04, 67.87, 71.90,
74.99, 79.49, 82.10, 99.90, 103.70, 174.89, and 178.21. 1H
NMR (HA, 500 MHz, D2O, TMS, d ppm): 2.06, 3.39, 3.53,
3.61, 3.75, 3.88, 3.94, 4.49, and 4.59.
MALDI-TOF (matrix-assisted laser desorption/ionization–time
of flight) analysis was conducted with a Bruker Microflex
equipped with 337 nm N2 laser, using 20 kV accelerating volt-
age in pressure 5.0 3 1026 mbar. a-cyano-4-hydroxycinnamic
acid (CHCA) was used as matrix.38 CHCA solution of 10 mg
mL21 was prepared in methanol containing 0.1% TFA. The
samples were prepared by dissolving 1 mg in 100 lL water
and 2 lL was mixed (1:1) with the matrix solution. A blank of
the CHCA matrix was also investigated.
INSTRUMENTS
Size exclusion chromatography (SEC) was used to determine
the molar mass and polydispersity of the polymers. The
apparatus using THF as eluent included the following instru-
ments: Biotech model 2003 degasser, Waters 515 HPLC
pump, Waters 717plus auto sampler, Viscotek 270 dual
detector, Waters 2487 dual k absorbance detector, Waters
2410 refractive index detector, and the OmnisecTM software
from Viscotek. Styragel HR 1, 2, and 4 columns and a flow
rate of 0.8 ml min21 were used in the measurements. PMMA
standards from PSS Polymer Standards Service GmbH were
used for calibration. For aqueous SEC the instrumental set
up was as follows. Water chromatograph equipped with pre-
column (Waters, Ultrahyrdrogel 6 3 40 mm) and three col-
umns (Waters Ultrahydrogel 2000, 250, and 120), HPLC
pump (Waters 515), Autosampler (Waters 717plus), and a
RI-detector (Waters 2410). The HA degradation products
were characterized using an aqueous eluent of 0.1 M NaNO3
with 3% acetonitrile and the diblock copolymer with a 1:1
mixture of dimethylsulfoxide and water.
Calorimetric analyses were conducted with a MicroCal VP
DSC microcalorimeter. Solutions were degased at 5 8C prior
the measurements. The 0.5 ml samples were run from 5 to
90 8C with 60 8C h21, repeating the heating and cooling cycle
three times. The pre-equilibration time was 120 min at 5 8C
before each heating cycle. The polymer concentrations were
corrected by subtracting the amount of hyaluronic acid in
the samples. The measured enthalpy values are given per
repeating N-VCL unit.
The IR spectra were measured with a PerkinElmer Spectrum
One spectrometer.
Light scattering (LS) experiments were conducted using a Broo-
khaven Instruments BI-200SM goniometer, a BIC-TurboCorr
digital pseudo-cross-correlator, and a BI-CrossCorr detector,
including two BIC-DS1 detectors; pseudo-cross-correlation func-
tions of the scattered light intensity were collected with the
self-beating method;34 a Sapphire 488-100 CDRH laser from
Coherent GmbH operating at ko 5 488 nm and the power
adjusting from 10 to 50 mW was used as a light source. Inten-
sity of scattered light was collected in the angular range from
Turbidity measurements: Transmittance as a function of tem-
perature was measured with JASCO J-815 CD-spectrometer
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SCHEME 1 Schematic representation of the procedure for preparing the HA-PVCL block copolymer: (A) End functionalization of
HA with 2-(2-azidoethoxy)ethanamine by reductive amination. (B) End functionalization of PVCL with propargylbromide. (C) “Click”
coupling of HA and PVCL. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
equipped with PTC-423S/15 Peltier-type temperature control
system. The transmittances of the samples were monitored
at wavelength of 488 nm. Sample holder was heated from 10
to 80 8C with varying rates; the reported temperatures are
those of the sample. After heating, the sample holder imme-
diately was cooled back to 10 8C, still monitoring the sample
temperature. Aqueous solutions of the bioconjugate of
0.1 mg/mL were investigated both in pure water and in
saline solution (0.1 M NaCl).
The formation of the conjugate (scheme 1) could be confirmed
from SEC, NMR, and IR data (Supporting Information Fig. 5
and 6). After purification by dialysis and ultrafiltration, the
SEC eluogram of the block copolymer shows no trace of free
PVCL (Fig. 4). However, the presence of PVCL is observed in
the NMR spectra. In the 1H NMR (Fig. 1), the peaks at 2.33
ppm (PVCL –CH2– in the ring) and methyl group from the
hyaluronic acid (2.06 ppm) were used to determine the com-
position of the conjugate. From integration of these peaks, the
composition of the product was estimated to be 49% PVCL
and 51% hyaluronic acid. According to the NMR spectra there
are traces of free hyaluronic acid in the product. The excessive
purification was followed by NMR (Supporting Information Fig.
6), where in the crude product an excess of PVCL was
observed. From the NMR spectra it can be seen how free
homopolymers of PVCL and HA are removed from the product.
The free PVCL was removed by dialysis (3500 MWCO) and
free HA was removed after the dialysis by ultrafiltration (5000
MWCO). It should be noted that during this long purification
RESULTS AND DISCUSSION
Synthesis of the Diblock Copolymer
The molecular weight for the HA was determined by MALDI-
TOF (Mn 5 3,600 g mol21, Supporting Information Fig. 1)
and the molecular mass distribution was determined by SEC
-
(D 5 1.63). Thus, the HA fragment consists of 8 to 9 disac-
charide units. The PVCL block is roughly 25 repeating units
long (Mn 5 3,500 g mol21, D 5 1.41).
-
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FIGURE 1 1H NMR spectra of hyaluronic acid (top), hyaluronic acid–PVCL conjugate (middle) and poly(N-vinylcaprolactam) (PVCL,
bottom).
process, some degradation of the HA might occur. In this case,
it would work in our advantage, since smaller fragments are
removed more readily during the purification process.
and Tmax (Fig. 5). The enthalpies for the phase transition are
similar for both free PVCL (3.8 kJ mol21) and for PVCL
linked to HA (3.7 kJ mol21).
The triazole ring is visible as a very weak signal at 7.14 ppm
(Supporting Information Fig. 6). 13C spectrum was also
recorded to confirm the composition, however, the triazole
ring could not be observed. In the IR spectrum, the charac-
teristic absorption band for azide at 2112 cm21 is dimin-
ished (Supporting Information Fig. 5.) while absorption
bands for both polymers can be observed.
The phase transition temperature of PVCL is dependent on
the molecular weight of the polymer. It has been shown that
Tmax increases from 33.1 to 43.6 8C when the molecular
weight decreases from 1,500,000 g mol21 to 21,000 g
mol21 39
Since the PVCL block is short, the phase transition
.
is expected to occur at a temperature higher than 32 8C, and
indeed, the Tmax for the homopolymer is observed at 45.2 8C
and for the diblock copolymer at 47.7 8C.
Microcalorimetric Observations
Conjugation of PVCL to HA increases its phase separation
temperature, seen calorimetrically as a shift of both Tonset
Hyaluronic acid is known to influence the phase transition of
thermoresponsive polymers. Ohya et al. have studied the
FIGURE 2 1H NMR spectrum of the hyaluronic acid–PVCL conjugate. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
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FIGURE 3 13C NMR spectrum of the hyaluronic acid–PVCL conjugate. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
thermoresponsive behavior of PNIPAM (5,000–84,000
g
8C (26,800 g mol21).5 Both the HA to PNIPAM ratio and the
molecular weight of PNIPAM affect the change in the phase
transition temperatures upon grafting to HA. However, in
both cases the phase transitions for short PNIPAM chains
shifted to higher temperatures.
mol21) grafted to HA (500–1.500 kg mol21) by turbidimetry.
They report that the Tonset of the decrease in transmittance
is shifted in the presence of HA. For pure PNIPAM the cloud
point is observed at 32 8C, whereas in the presence of HA it
is 33.5 to 34.4 8C, depending on the composition of the bio-
conjugates.17 Mortisen et al. have studied the thermorespon-
sive behavior of PNIPAM (10,700–48,200 g mol21) grafted to
HA (1,185 kg mol21). In their differential scanning calorimet-
ric (DSC) measurements they found that the maximum of
the phase transition peak shifts upon grafting. For the short-
est grafts (10,700 g mol21) Tmax increased from 29.2 to 30.2
8C. For PNIPAM grafts with higher molecular weights
(26,800 and 48,200 g mol21) the transition occurred at
lower temperatures, that is Tmax decreased from 30.6 to 29.5
Kirsh et al. found that the cloud point of PVCL shifts to
higher temperatures in the presence of solutes with carbonyl
or amide groups, and that the effect increases upon increas-
ing the amount of the solute. The same was observed in the
presence of glycols.40 On the other hand, Yanul et al.41
observed that the presence of polyethylene oxide (PEO) in
the solution decreases the cloud point of PVCL. Laukkanen
et al. grafted PVCL with alkyl modified PEO. They showed
that with increasing grafting density the phase transition
FIGURE 4 SEC trace of the HA-PVCL conjugate (blue) com-
pared with the traces for HA (red) and PVCL (black). [Color fig-
ure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
FIGURE 5 Micro calorimetric data of the HA-PVCL bioconjugate
and PVCL. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
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FIGURE 6 Turbidity measurements performed on 0.1 mg mL21
HA-PVCL dissolved in aqueous 0.1 M NaCl. Heating (solid lines)
and cooling (dashed lines) rate was varied: 1 (black), 5 (blue),
and 10 (red) 8C min21. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
FIGURE 8 Equilibrium heating: hydrodynamic radius of the bio-
conjugate as function of temperature (extrapolated to 08 angle).
In the inset, the scattering intensity at a 458 angle is presented as
function of temperature. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
temprature of PVCL increases, that is, the Tmax shifted from
34.9 to 38.2 8C. The enthalpy of the phase transition is also
affected by the graft copolymerization, decreasing with
increased the Tc of PVCL.28 Also, the water-soluble poly(N-
vinyl pyrrolidone) has been observed to increase the cloud
point of PVCL in PVCLPVP diblock copolymers.25
increasing PEO content from 32 to 21 J g21 24
When a PVCL-
.
b-PEO block copolymer is attached to the surface of a silica
particle the phase transition shifts to lower temperatures,
that is, from 36.5 to 35.4 8C. In this case the grafting to the
silica surface has a major effect on the enthalpy of transition,
Hou and Wu studied diblock copolymers of PVCL and PNI-
PAM. Only one cloud point was observed, but by using FTIR
measurements, it was possible to resolve two transitions.
The values obtained for Tc with the two different methods
differ noticeably; FTIR gives lower values than the turbidity
measurement. In this case, the PVCL homopolymer and the
PVCL block in the copolymer had different chain lengths.29
increasing it from 1.94 to 5.40 kJ mol21 42
More recently, it
.
was shown that when PVCL was connected to PDMAEMA
either one or two transitions were observed, depending on
the pH. The phase separation was monitored by turbidome-
try and calorimetry. However, in all cases, PDMAEMA
The shifts observed in the present study in the Tonset and Tmax
for the HA-PVCL conjugate are well in line with the reported
observations for PVCL and PNIPAM. Interestingly though, in
this case the HA has no effect on the enthalpy of the transition.
Turbidity Studies
Turbidimetric studies were conducted in aqueous solutions
with and without NaCl (0.1 M) figures 6 and 7. The heating
rate was varied in the measurements, that is, the samples
were heated either 1, 5, or 10 8Cmin21. The polymer concen-
tration cp was 0.1 mg mL21. The solvents and polymer solu-
tions were degassed at 5 8C. Fresh samples were used for
each measurement, to minimize the effect of possible degra-
dation of the HA block at elevated temperatures.
In saline solutions the cloud point (Tcp 5 51 8C, Fig. 6) is not
affected by the heating rate, however, the heating rate has an
overall effect on the transition. The transition is more grad-
ual and is still incomplete at 80 8C with the higher heating
rates (5 and 10 8Cmin21). For the slowest heating/cooling
rate studied (1 8Cmin21), transmittance reaches its lowest
value and the observed hysteresis manifests a slow response
of HA-PVCL. The observation shows that the conjugate has
enough time to self-organize in response to the slow heating,
FIGURE 7 Turbidity measurements performed on 0.1 mg mL21
HA-PVCL (black) and PVCL (red) dissolved in aqueous 0.1 M
NaCl, with heating (solid lines) and cooling (dashed lines) rates
of 1 8C min21. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
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FIGURE 9 Sizes measured at an angle of 458 at 20 8C (left) and after equilibrium heating at 55 8C (right). Top, the size distributions
of HA-PVCL in 0.1 M NaCl. At 20 8C the size of the size measured is 132 nm, whereas upon heating to 55 8C the size increases to
450 nm. Below, the size distributions of HA-PVCL in water. At 20 8C, the hydrodynamic radius measured is 92 to 225 nm, whereas
upon heating to 55 8C, the size increases to 395 nm.
which consequently slows the resolubilization upon cooling.
In aqueous solution without NaCl the cloud point is
observed at practically the same temperatures as for the
saline solution (Supporting Information Figs. 9 and 10) The
hysteresis upon a heatingcooling cycle is, however, smaller in
the salt-free solution.
perature, the intensity of light scattered at 458 angle was
measured to follow the equilibration of the solution. Upon
heating from 20 up to 50 8C, the intensity of scattered light
decreases slightly in accordance with decreasing mean peak
value of the hydrodynamic radius (Rh). The scattering inten-
sity increases strongly between 50 and 51 8C. Thus the Tcp
can be determined to be 51 8C, which is in line with the tur-
bidimetric measurements (Fig. 8).
Light Scattering with Equilibrium Heating (EH)
in 0.1 M NaCl
At 20 8C, HA-PVCL forms aggregate with Rh 5 145–150 nm
(Fig. 9). It is known that high molar mass PVCL may form
loose aggregates at room temperature,24 whereas HA as a
polyelectrolyte swells in water. The existence of loose struc-
tures is supported by the relatively weak scattering by the
polymer solutions. The angular dependence at 20 and 55 8C
are shown in Supporting Information Figure 12.
In the EH procedure, the diblock copolymer solution (0.1 mg
mL21) was heated in 1 8C steps. After each increase in tem-
Both the scattering intensity and the size of the particles,
formed above Tcp (51 8C), increase upon further heating. At
55 8C, Rh 5 443 nm. The dissymmetry (z) in the intensity of
scattered light measured at 45 and 1358 scattering angle,
z 5 I(1358)/I(458), is 10. Assuming a spherical conformation
of the particles, z 5 10 corresponds to a sphere with its
radius of gyration Rg 5 139 nm.43 Such combination of geo-
metrical parameters is not possible for a hard sphere. There-
fore, the angular dependence of the intensity of light
scattered from solutions at 55 8C was analyzed simultane-
ously with Rh measurements. The solution scatters strongly
and primarily forward, which is typical for large particles.
However, the LS intensity is not as high by its absolute value
as one may expect judging by the particle size and
FIGURE 10 Equilibrium heating: Rh versus scattering vector at
55 8C, showing the linear fit in the extrapolation of the Rh to 08
angle (black) and the relaxation rate plotted against the scatter-
ing vector (blue). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
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FIGURE 11 Fast heating: On the left, scattering intensity as function of time, showing the slow reorganization of the forming par-
ticles. Right, size distribution of HA-PVCL in 0.1 M NaCl at 55 8C, measured at 458 angle. The average hydrodynamic radius of the
aggregates is in the order of 280 nm.
thermodynamically poor condition. As a result, at the angles
above 908, light scattered forward and reflected back by sev-
eral quartz surfaces of the goniometer overcomes weak back
scattering and makes data analysis impossible (e.g., Kratky
plot). Distributions of the hydrodynamic radius clearly are
bi- and multi-modal above 1208 scattering angle, whereas
below 908, the distributions are monomodal and narrow. The
second-order cumulants analysis shows that the relaxation
rate decreases linearly with decreasing the scattering vector
below 908 and goes through zero, thus representing transla-
tional diffusion (Fig. 10).
son, it has been reported that a PVCL (330,000 g mol21
,
0.1 mg mL21) has Rh 5 127 nm, Rg 5 54 nm, Rg/Rh 5 0.43,
and the mesoglobules have a density of 650 mg mL21 at 50
8C in aqueous solution.44
Fast Heating, FH in 0.1 M NaCl
In the FH procedure, a polymer solution (0.1 mg mL21) was
first equilibrated at 20 8C and then immersed directly to the
LS goniometer preheated at 55 8C. As follows from our LS,
calorimetric, and turbidity studies, 55 8C is within the transi-
tion range, that is the phase transition of the PVCL block is
not over at this temperature and the formed particles have
freedom to reorganize. This process is slow and the sample
was left to stabilize for 20 h (Fig. 11).
Therefore, Rg was calculated in the angular range of 30 to
908. The second-order polynomial fit to the data in Zimm’s
presentation43 gives Rg 5 478 nm. Rough estimation of the
molar mass of the aggregates (assuming the increment of
refractive index dn/dcp 5 0.18 cm3 g21) suggests packing
density of polymeric material within such particles to be
4 mg mL21. This value is too low for a hard sphere. Taking
into account the weak scattering form the particles, their
low density, and the shape parameter Rg/Rh ꢂ 1.1, our LS
results suggest that the shape of the particles formed at 55
8C upon equilibrium heating is a hollow sphere. For compari-
Angular dependence of the LS intensity was analyzed at 55
8C simultaneously with Rh measurements and Rg was calcu-
lated in the angular range of 30–908 using the Zimm analy-
sis.43] Accordingly at zero scattering angle (Supporting
Information Fig. 10), Rh 5 275 nm, Rg 5 270 nm, packing
density of the particles is 7 mg mL21, and the shape parame-
ter is Rg/Rh ꢂ 1.0. These parameters support a vesicle-like
model for the bioconjugate particles.
One can note that FH procedure yields particles of smaller
size in comparison to EH procedure (Figs. 9 and 11). This
has been observed previously for homopolymers of PVCL,
PNIPAM, and poly(vinyl methyl ether).44 However, kinetics of
the bioconjugate is much slower. Once the particles are
formed, they do not grow with time to adopt the size of the
EH particles. Evidently, the phase-separated PVCL is hin-
dered by tangling HA blocks. Possibly, slow equilibration
originates from reorganization inside the particles.
Light Scattering with Equilibrium Heating (EH) in
Aqueous Solution Without NaCl
In aqueous solutions without salt, the size distributions of
the block copolymers observed at 20 8C are broader than
those in the saline solution (Fig. 9). The size distribution
becomes narrower at 55 8C, however the average radius of
the obtained particles is smaller than in the presence of salt.
FIGURE 12 Equilibrium heating in pure water: hydrodynamic
radius of the bioconjugate as function of temperature (458
angle).
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The size of the aggregates remains fairly stable until around
50 8C, above which the size increased rapidly (Fig. 12). The
size of the block copolymers at 20 8C (ꢀ150 nm) are of the
same order with and without NaCl in the solution. This
shows that the solubility of the block copolymer is affected
to a lesser degree by the presence of salt.
Above the cloud point of the PVCL block, the bioconjugate
self-assembles into colloidally stable particles and the struc-
ture of the particles was investigated by light scattering. The
light scattering studies indicate that at room temperature
the bioconjugate forms loose associates. Above the cloud
point the molecules self-assemble into hollow spherical par-
ticles with a hydrodynamic radius of 443 nm. The heating
rate has an effect on the size of the formed particles. Fast
heating resulted in smaller particles with hydrodynamic
radius of 275 nm. Turbidimetric studies show that the cloud
point of the bioconjugate is unaffected by the heating rate.
The turbidimetric studies also show a hysteresis when heat-
ing and cooling the solutions, this indicating that the phase
separated particles have time to organize during their forma-
tion. This organization process can also be seen in the light
scattering data, when the solution is heated fast to 55 8C,
since the intensity of scattered light takes up to 20 h to
stabilize.
PVCL can complex ionic substances, as has been shown with
surfactants. Cetylpyridinium chloride and sodium dodecyl
sulfate have been shown to bind and affect the size of PVCL
coils or aggregates in aqueous solutions. At low concentra-
tions (< 1:1) the surfactants decrease the size of the aggre-
gates, whereas at higher concentrations (> 1:1) the size was
observed to increase, showing the binding of the surfactants
to the PVCL.45 Similar behavior was observed with PVCL-
PDMAEMA block copolymer, where at room temperature the
polymer was observed to form loose associates, due to inter
molecular interactions between the PDMAEMA and PVCL
blocks.28
The dynamic light scattering studies of HA-PVCL show that,
independent of salt, the polymers form inter molecular loose
associates with broad size distributions, in aqueous solutions
at room temperature (Fig. 9). Upon heating, the interactions
are overcome by hydrophobic interactions during the phase
transition of the PVCL block, resulting in more compact par-
ticles. Knowing that the HA-PVCL polymers interact with
each other sheds further light to the static light scattering
studies, which suggest that the self-assembled particles
obtained at 55 8C are hollow. As the HA-PVCL forms loose
associates at low temperatures, that are later squeezed into
particles; one would expect some of the HA blocks to end up
inside the particles and some to form a corona on the out-
side. The inside of the vesicular aggregate would be signifi-
cantly hydrated by the HA and be less dense than the
collapsed PVCL.
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