Amphiphilic amylose-g-poly(meth)acrylate copolymers through "click" onto grafting method

Article abstract of DOI:10.1021/bm101143q

Periodate oxidation and subsequent reductive amination with propargylamine was adopted for the controlled functionalization of amylose with alkyne groups, whereas ATRP polymerization was exploited to obtain end-(α)- or end-(ω)-azide functionalized poly(meth)acrylates to be used as "click" reagents in Cu(I) catalyzed azide-alkyne [3 + 2] dipolar cycloaddition. Amylose was effectively grafted with poly(n-butyl acrylate), poly(n-butyl methacrylate), poly(n-hexyl methacrylate), and poly(dimethylaminoethyl methacrylate) with this strategy. Their structure and composition were confirmed by FT-IR, NMR spectroscopies, and thermogravimetric analysis (TGA). Dynamic and static light scattering analyses, as well as TEM microscopy showed that the most amphiphilic among these hybrid graft copolymers self-assembled in water, yielding nanoparticles with ca. 30 nm diameter.

Full text of DOI:10.1021/bm101143q

388
Biomacromolecules 2011, 12, 388–398
Amphiphilic Amylose-g-poly(meth)acrylate Copolymers through
“Click” onto Grafting Method
Monica Bertoldo,*,† Giovanni Zampano,‡,| Federico La Terra,† Valentina Villari,§ and
Valter Castelvetro†,‡
Istituto per i Processi Chimico-Fisici, Consiglio Nazionale delle Ricerche (IPCF-CNR), Area della Ricerca,
Via G. Moruzzi 1, I-56124 Pisa, Italy, Dipartimento di Chimica e Chimica Industriale, Universita` di Pisa, Via
Risorgimento 35- I-56126 Pisa, Italy, and Istituto per i Processi Chimico-Fisici, Consiglio Nazionale delle
Ricerche (IPCF-CNR), Viale Ferdinando Stagno d’Alcontres 37, 98158 Messina, Italy
Received September 23, 2010; Revised Manuscript Received November 25, 2010
Periodate oxidation and subsequent reductive amination with propargylamine was adopted for the controlled
functionalization of amylose with alkyne groups, whereas ATRP polymerization was exploited to obtain end-(R)-
or end-(ω)-azide functionalized poly(meth)acrylates to be used as “click” reagents in Cu(I) catalyzed azide-alkyne
[3 + 2] dipolar cycloaddition. Amylose was effectively grafted with poly(n-butyl acrylate), poly(n-butyl
methacrylate), poly(n-hexyl methacrylate), and poly(dimethylaminoethyl methacrylate) with this strategy. Their
structure and composition were confirmed by FT-IR, NMR spectroscopies, and thermogravimetric analysis (TGA).
Dynamic and static light scattering analyses, as well as TEM microscopy showed that the most amphiphilic
among these hybrid graft copolymers self-assembled in water, yielding nanoparticles with ca. 30 nm diameter.
grafting “from” route is the most commonly used procedure to
obtain graft copolymers with controlled architecture. In this
approach, the growth of copolymer grafts occurs from initiating
sites on the polymer backbone (macroinitiator) by free radical,
ionic, ring-opening, controlled/living radical, or even enzymatic
graft copolymerization.12-14 Conventional free radical polym-
erization does not allow control over grafted chain length and
grafting density,15,16 whereas ring-opening and controlled/living
radical polymerization processes allow predictable molecular
weight of the grafted block copolymer chains to be obtained.
However, controlling the surface density of immobilized initiator
and eventually the density of grafted chains can be problematic,
and the result is scarcely predictable, even by these latter
methods.17
Introduction
Amphiphilic graft copolymers are attractive materials because
they can self-assemble into nanoscale structures in solution, in
bulk, and at interfaces. The micelles formed in solution have
been studied as drug carriers because of their unique charac-
teristics such as core-shell structure, mesoscopic size range,
and prolonged blood circulation.1-4 In addition, amphiphilic
graft copolymers can form complex micro- and nanostructured
rod-like, spheroidal, disk-like, and linelike micelles and vesicles,
the actual shape depending on the interaction parameter between
grafted chains and solvent. Microstructure formation and
aggregate morphology stability also depend on the architecture
parameters such as the position of the first graft point, the
number, average length, and length distribution of branches.5
The present application perspective of amphiphilic graft co-
polymers would be greatly broadened after a better understand-
ing of such processes and correlations. Polysaccharides have
been studied as surface coating materials for their biocompat-
ibility, biodegradability, and mucoadhesive properties; they have
also been considered as candidates for achieving an active
targeting toward tissues and organs. With this purpose, various
drug delivery systems such as liposomes and polymeric micelles
have been decorated with polysaccharides.6,7 In addition,
hydrophobically modified polysaccharides self-assembling into
nanoparticles have been investigated as possible drug delivery
systems8,9 exhibiting high loading efficiency as well as capability
to trap and targeting the release of scarcely soluble and toxic
antitumor drugs.9 Tailor-made graft copolymers can be synthe-
sized using specific polymerization methods mainly based on
grafting “from” and grafting “to” or “onto” approches.10,11 The
In the grafting “onto” approach, a preformed polymer with
reactive end group is coupled to functional groups along the
backbone of a second polymer. When the backbone functional
polymer is a polysaccharide, the nucleophilic character of its
hydroxyl groups is often exploited in condensation and addition
reactions.18 By this way, cellulose-g-polystyrene was prepared
by direct esterification of cellulose with the terminal carboxylic
acid groups of well-defined polystyrene chains.19 Cellulose
substrates have been functionalized with amino groups onto
which poly(isobutyl vinyl ether) and poly(2-methyl-2-oxazoline)
have been grafted.20 Similarly, poly(isobutyl vinyl ether),
poly(ethylene glycol) (PEG), poly(ethyleneimine), polyurethane,
poly(dimethylsiloxane), and polylactide have been grafted to
chitosan21-23 and other polysaccharides.24-27 In most cases,
these procedures are unfortunately not quantitative, resulting
in low grafting densities. Furthermore, removal of the unreacted
polymer may be difficult, a problem that emphasizes the need
of highly efficient, high yield coupling reactions. An attractive
feature of the grafting “onto” approach is that the side chains
are prepared separately. This makes easily accessible such
complex architectures as comb copolymers with different kinds
of side chains along the backbone. With this goal, graft
copolymers from functionalized PEG and guar gum have been
* To whom correspondence should be addressed. Tel: +39 050 3152230.
Fax: +39 050 3152250. E-mail: monica.bertoldo@ipcf.cnr.it.
† Consiglio Nazionale delle Ricerche (IPCF-CNR), Area della Ricerca,
Pisa, Italy.
‡ Universita` di Pisa.
§ Consiglio Nazionale delle Ricerche (IPCF-CNR), Messina, Italy.
| Present address: A. Menarini Industrie Farmaceutiche Riunite Srl, via
Marchetti 7/7 Bis, I-50131 Firenze, Italy.
10.1021/bm101143q
 2011 American Chemical Society
Published on Web 12/30/2010

Amphiphilic Amylose-g-poly(meth)acrylate Copolymers
Biomacromolecules, Vol. 12, No. 2, 2011 389
One FT-IR spectrophotometer interfaced with the software Spectrum
v.3.02 for data acquisition. Samples were analyzed in transmission mode
as cast films on KBr windows or prepared in KBr discs. A liquid KBr
cell 5 × 10-2 mm thick was used for CCl4 solutions in the calculation
recently prepared by Cu(I) catalyzed azide-alkyne [3 +
2]-dipolar cycloaddition (“click”) as the coupling reaction with
grafting chains.28 The modification of galactomannans (Guar
gum, Locust Bean gum, etc.) by “click” grafting “onto” with
poly(oxyalkylenes) is a versatile procedure for achieving various
polysaccharide-based materials.28,29 For this purpose, alkynyl
side groups have been introduced along the polysaccharide
backbone by base-catalyzed etherification with propargyl bro-
mide, targeting a broad range of substitution degrees of the
pyranose ring.30,31 Similarly, hyaluronic acid (HA) functional-
ized with propargylamine has been grafted by “click” coupling
with azido-end-functional poly(N-isopropyl acrylamide) sepa-
rately prepared by controlled radical polymerization.32,33 Finally,
amphiphilic chitosan graft copolymers with mixed side chains
have been synthesized by “clicking” end-functional poly(ε-
caprolactone) and poly(ethylene oxide) onto a polyazidated
chitosan obtained by esterification with 2-bromoisobutyryl
bromide, followed by nucleophilic substitution of the R-bromide
with azide.34 The effectiveness of the “click” reaction has also
been demonstrated in the modification of polysaccharides by
functionalization with small moieties35-40 and cross-linking.33,41
As already mentioned, click reactions have been combined with
different controlled polymerization methods.42 Among them,
atom transfer radical polymerization (ATRP) has been exten-
sively adopted, generating hundreds of research papers.42-46
In this work, amylose, an almost completely linear R-(1,4)-
glucopyranoside, was modified to introduce alkynyl function-
alities along the backbone and then hybridized by graft-clicking
with azido-terminated (meth)acrylic homopolymers that had
been obtained from end-brominated precursors prepared by
ATRP.47 The highly effective and selective “click” reaction was
used to overcome the problems of low yield and selectivity
frequently encountered in grafting “onto” reactions involving
polysaccharides and highly hydrophobic synthetic polymers.
Periodate oxidation and subsequent reductive amination was
adopted for the controlled functionalization of polysaccharides
with alkyne. The combination of periodate oxidation and
reductive amination with either small molecule amines or
polyamine has been previously exploited to obtain sponges to
be used in tissue engineering,48 or polycations for nucleic acid
delivery,49-51 and wastewater treatment.52 Furthermore, graft
and comb copolymers have been synthesized through reductive
amination by exploiting polysaccharide reducing end53-55 or,
in a reverse coupling strategy, amine groups of chitosan.22
Finally, in this work, the self-assembling behavior in water
of the hybrid graft-copolymer was investigated.
1
of the fractional amount of N3-end-functional polymers, dfN3. H and
13C NMR spectra were recorded with either a Varian 200 or a 300
MHz instrument from CDCl3, DMSO-d6, DMF-d8, or D2O solutions.
Sample concentration was ∼30 mg/mL for proton and ∼60 mg/mL
for 13C analyses. At least 128 transients were accumulated for
polysaccharide proton spectra. Thermogravimetric analyses (TGA) were
carried out with a Mettler Toledo TGA/SDTA 851e instrument under
60 mL/min nitrogen flux in the 25-700 °C temperature range at a 10
°C/min heating rate on 20-30 mg samples. Elemental analyses (C, N,
H) were performed on a Carlo Erba 1106 elemental analyzer. Size
exclusion chromatography (SEC) analyses were carried out with a Jasco
instrumental setup consisting of a PU-2089 Plus high pressure pump,
RI 2031 Plus (refractive index) and UV-2077 Plus (UV) detectors, and
column oven. Either two PLgel 5 µm Mixed-D columns (Polymer
Laboratories) or two Ultrahydrogel Linear columns (Waters) mounted
in series with guard column were used for elutions with organic and
aqueous solvents, respectively. The instrument was interfaced with
Borwin 1.21.61 (JMBS development) software for data acquisition and
processing. Sample concentration was in the 3-5 mg/mL range, and
injection volume was 20 µL for all SEC analyses. Poly(butyl acrylate)
(PBA) and all polymethacrylates with the exception of poly(N,N-
dimethylaminoethyl methacrylate) (PDMAEMA) were analyzed using
CHCl3 (1 mL/min, 40 °C) as the eluent and poly(methyl methacrylate)
standards (Polymer Laboratories) for column calibration. A DMF/LiBr
(1 g/L, 0.8 mL/min, 70 °C) eluent solution and polystyrene standards
were used for PDMAEMA, whereas an aqueous NaCl (0.5 M)/NaN3
(1%) eluent solution (0.5 mL/min, 40 °C) and poly(ethylene glycol)/
poly(ethylene oxide) (PEG/PEO) standards (Polymer Laboratories) were
used for the polysaccharides.
Flash chromatography was carried out on a Biotage ZIF-SIM FLASH
instrument equipped with silica gel 60 prepacked cartridges (40 × 150
mm column, particle size 40-63 µm). Gas chromatography-mass
spectrometry (GC-MS) analysis was carried out by using a Hp (Agilent)
GCD series II apparatus with quadrupole analyzer.
We performed transmission electron microscopy (TEM) analysis
using a Philips CM12 instrument operating at 120 keV; images were
recorded with a CCD camera (Gatan 791). We prepared samples by
laying drops of previously sonicated polymer dispersions over graphi-
tized copper grids and removing excess solvent with filter paper,
followed by staining with uranyl acetate (1 M in water) for 10-15
min. Finally, sample grids were washed with deionized water and dried.
Determination of aldehyde groups in oxidized polysaccharide. A
modification of the procedure proposed by Zhao and Heindel was
adopted.56 In brief, the oxidized polysaccharide was dissolved (4.2 g/L)
in water, the pH was adjusted to 4, then an equal amount by volume
of hydroxylamine hydrochloride (2.5 M) at pH 4 was added. The
mixture was stirred for 2 h and then back-titrated with NaOH 0.01 N
to pH 4.
Experimental Part
Materials. Amylose (type III) from potatoes was purchased from
Aldrich. Tetrahydrofuran (THF) was kept on NaOH for 24 h, then
distilled (67 °C) under a nitrogen atmosphere. Sodium borate buffer
(1 M, pH 8.5) was obtained by basification of a boric acid solution.
The monomers (M) butyl acrylate (BA) (50 °C, 100 mmHg), butyl
methacrylate (BMA) (57 °C, 18 mmHg), hexyl methacrylate (HMA)
(87 °C, 18 mmHg), and dimethylaminoethyl methacrylate (DMAEMA)
(82 °C, 18 mmHg) were distilled from CaH2 under a nitrogen
atmosphere. All purified solvents and chemicals were stored at low
temperature (-20 °C) until use. CuBr (Aldrich, 99.999%) was stored
under nitrogen and used without further purification. 1,1,4,7,10,10-
Hexamethyltriethylenetetramine (HMTETA, Sigma-Aldrich, 97%).
N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, Sigma-Ald-
rich, 99%), ethyl-2-bromopropionate (EBP), propargylamine, and all
other chemicals and solvents were used as received.
Determination of the N3-Functionalization Degree (dfN3
) of
End-Functionalized Poly(meth)acrylates. (a) Method by FT-IR spec-
troscopy. Four solutions in CCl4 containing 2-bromo-isobutyric acid
3-azidopropylester in the 3 × 10-4 to 1 × 10-3 M concentration range
and poly(butyl methacrylate) (PBMA) (2.5 g/L), respectively, were cast
onto KBr windows, and their FT-IR spectra were collected after solvent
evaporation. The ratio between the integrals of the absorption bands at
2100 (N3 stretching) and 1066 cm-1 (C-O stretching from PBMA)
plotted against the N3/PBMA mol/g ratio gave a linear plot that was
fitted with a straight line (y ) 0.004 + 1920x; R2 ) 0.99945) used for
calibration. A mathematical deconvolution procedure was adopted to
evaluate the integral of the PBMA band separately from the contribution
of other partially superimposed absorptions. Seven Lorentzian functions
with maxima at 1179.6, 1154.2, 1143.2, 1124.6, 1066.1, 1022.6, and
996.1 cm-1 were used to fit the spectra in the 1215-990 cm-1 range.
The corresponding area ratio in the FT-IR spectrum of the functionalized
Instruments and Analysis Methods. Infrared spectra were recorded
with either a Perkin-Elmer Spectrum GX or a Perkin-Elmer Spectrum

390 Biomacromolecules, Vol. 12, No. 2, 2011
Bertoldo et al.
Table 1. Synthesis of Poly(meth)acrylates through ATRP: Feed Composition and Experimental Results
c
d
M/Ia
M:I:CuBr:CuBr2:ligandb Mn (g mol-1
)
Mn (g mol-1
)
(Mw)/(Mn)
conv. (%)e dfN3 (mol %)f
th
SEC
j
j
j
j
sample
PBA
PBA-N3
PBMA
PBMA-N3-A
PBMA-N3-B
PHMA-N3-A
PHMA-N3-B
PDMAEMA
BA/EBP
45:1:0.15:0:0.15
5800
5800
5700
6150
5700
5350
5450
5030
5030
6350
6350
6100
6600
8500
8200
1.203
1.203
1.30
1.20
1.26
1.25
1.19
1.19
1.34
90
∼100
BMA/EBP
50:1:0.9:0.1:2
50:1:0.9:0.1:2
49:1:1:0:1
80
BMA/BiBAP
BMA/BiBAP
HMA/BiBAP
HMA/BiBAP
DMAEMA/EBP
87
81
74
73
95
59
94
42:1:0.9:0.1:2
41:1:1:0:2
7400
78
32:1:0.9:0.1:2
32:1:0.9:0.1:2
12 6003
15 7503
>95
>95
PDMAEMA-N3 DMAEMA/BiBAP
85
a M ) monomer; I ) initiator; EBP ) ethyl-2-bromopropionate; BiBAP ) bromo-isobutyric acid 3-azidopropylester. b Mole ratio; the ligand was either
HMTETA or PMDETA. c Mnth ) conversion × [M]0/[I]0. d Average molecular weight obtained by SEC analysis; column calibration performed with either PS
or PMMA standards. (See the Experimental Part.) e Conv. (%): monomer conversion. f Degree of end-functionalization of the polymer chains calculated
by 1H NMR. The dfN3 values determined by FTIR for PBA-N3 and PBMA-N3 matched exactly those obtained by 1H NMR. (See the Experimental Part.)
Table 2. Experimental Conditions and Results of Grafting “Onto” Experiments Carried out on Am-PA with N3-Functionalized
Poly(meth)acrylates (Z-N3)
concentration (g/L)
DMSO/DMF
vol/vol
N3/alkyne
(mol/mol)
grafted chains
(wt %)a
grafting yield
(wt %)b
sample
Z-N3
T (°C)
Am-PA
Z-N3
blank
AmBA
AmBMA
AmBMA2
AmHMA
AmDMAE
PBA
PBA-N3
PBMA-N3-A
PBMA-N3-A
PHMA-N3-A
PDMAEMA-N3
7/12
7/12
1/3
0/1
2/7
60
13
13
36
10
27
25
9
9
74
20
70
29
<1
39
52
37
31
80
<3
95
91
63
52
0.33
0.83
0.91
0.66
0.33
40-60c
40-60c
40
60
5/2
40-60c
∼100
a Calculated from TGA as [(synthetic polymer)/(polysaccharide-g-synthetic polymer)] × 100. b [(Synthetic polymer/polysaccharide)TGA/(synthetic polymer/
polysaccharide)feed] × 100. c 60 °C for 5 h (24 h for AmBMA), then 40 °C.
polymer afforded the functionalization degree expressed as mol/g, which
was converted to a mol/mol ratio by using the average Mn value
Samples were sonicated for 1.5 h before analysis. Centrifuged samples
were analyzed after processing at 4400 RCF for 10 min, whereas filtered
samples were processed through 0.45 µm cellulose acetate syringe filters
just before analysis. Cumulant analysis was used to obtain the average
particle size and size distribution from the autocorrelation function.
Preparation of r-N3-Poly(butyl acrylate) by Successive ATRP
and Bromide-Substitution. A mixture of butyl acrylate (20 mL, 139.5
mmol) and PMDETA (97 µL) was degassed by three freeze-pump-thaw
cycles; then, CuBr (66.7 mg, 0.465 mmol) was added under nitrogen.
The catalyst was dissolved at 25-30 °C under stirring; then, the
temperature was raised to 40 °C, and EBP (430 µL) was added to start
the polymerization. The polymerization was carried out for 4 h, then
stopped by exposing the mixture to air and adding THF (20 mL). The
catalyst was removed by eluting with THF through a column packed
with aluminum oxide. The polymer (PBA) was recovered after solvent
and residual monomer evaporation.
j
determined by SEC (Table 1). Similarly, ethyl-2-azidobutyrate (see
Supporting Information for the synthesis details) and PBA were used
to build the calibration plot for calculating dfN3 of PBA-N3. In this
case, the FT-IR spectra were collected from liquid samples, and the
integral of the N3 stretching band at 2110 cm-1 was plotted versus
azide concentration. The linear fit of the experimental values afforded
the following equation: y ) 0.04 + 0.116x (R2 ) 0.9995).
(b) Method by 1H NMR spectroscopy. The functionalization degree,
dfN3, was calculated from the ratio between the integral of the CH2-N3
resonance at ∼3.4 ppm and that of the OCH2 resonance at ∼3.9 ppm
j
due to the polymer side chains. The Mn values determined by SEC
(Table 1) were used to convert dfN3 from mol/g to mol/mol.
Light Scattering Measurements. The dependence of dynamic and
static light scattering on the scattering angle (from 20 to 150°) was
investigated with a homemade apparatus by using a He-Ne laser source
at λ ) 633 nm with a power of 10 mW, linearly polarized orthogonal
to the scattering plane, and a computer-controlled goniometer.57 The
scattered light was collected in a pseudo-cross-correlation mode through
two cooled R943-02 photomultipliers at the same scattering angle,58
and a MALVERN 4700 correlator was used to build up the normalized
intensity autocorrelation function. Amylose-g-PBMA (AmBMA, Table
2) solutions were investigated before and after filtering through 0.45
and 0.2 µm cellulose acetate filters, respectively, the aqueous solutions
obtained after dialysis of the original 2 or 20 g/L DMSO solutions.
The temperature was set at 25 ( 0.01 °C. A reference sample obtained
by dialyzing pure DMSO against deionized water was also measured
for comparison.
PBA (16.2 g) was dissolved in DMF (60 mL) at 30 °C, and NaN3
(220 mg, 3.41 mmol) was added under stirring. After 5 h, the mixture
was diluted with THF and eluted through an aluminum oxide column,
yielding 15.0 g of PBA-N3 after solvent evaporation. FT-IR (KBr
window, cm-1): PBA: 2800-3000 (ν, C-H); 1732 (ν, CdO), 1450
(δa, CH2), 1380 (δa, CH3), 1160 (ν, C-O). PBA-N3: 2800-3000 (ν,
C-H); 2110 (ν, N3), 1732 (ν, CdO), 1450 (δa, CH2), 1380 (δa, CH3),
1
1160 (ν, C-O). H NMR 300 MHz (CDCl3, δ): PBA: 0.96 (t, CH3,
3H), 1.38 (m, CH2, 2H), 1.61 (m, CH2, 2H), 1.05-2.40 (m, CH and
CH2 main chain) 4.05 (t, OCH2, 2H). PBA-N3: 0.96 (t, CH3, 3H), 1.38
(m, CH2, 2H), 1.61 (m, CH2, 2H), 1.05-2.40 (m, CH and CH2 main
chain) 4.05 (t, OCH2, 2H).
Preparation of ω-N3-Poly(methacrylate)s by ATRP. A mixture
of the given methacrylate in THF (1:1 v/v) and HMTETA was degassed
by three freeze-pump-thaw cycles, then CuBr (and eventually CuBr2,
see Table 1) was added under nitrogen. After dissolution of the copper
salt by stirring at 25-30 °C, the temperature was raised to 40 °C, and
the polymerization was started by the addition of either bromo-
isobutyric acid 3-azidopropylester (BiBAP; see the Supporting Informa-
tion for the synthesis details) or EBP as the initiator. We stopped the
polymerization after 3 h by exposing the mixture to air and adding
THF. The catalyst was removed by eluting with THF through an
aluminum oxide column. The polymer was recovered after solvent and
Details of the data analysis procedure can be found in the Supporting
Information.
Comparative dynamic light scattering (DLS) measurements on
amylose-g-PBMA, amylose-g-PBA, and amylose-g-poly(hexyl meth-
acrylate) (AmBMA, AmBA, AmHMA, Table 2) were carried out at
25 ( 0.1 °C with a Brookhaven 90 Plus instrument collecting the
scattered light at 90°. The results of 2 runs of 10 min accumulation
each were averaged. Amylose copolymer dispersions were obtained
from DMSO solutions (20 and 2 g/L), filtered (0.45 µm PTFE), diluted
1:16 with ultrapure water, and dialyzed against water for 2 to 3 days.

Amphiphilic Amylose-g-poly(meth)acrylate Copolymers
Biomacromolecules, Vol. 12, No. 2, 2011 391
Scheme 1. Preparation of Amylose-Based Nanoparticles: Synthesis and Self-Assembling Procedures
residual monomer evaporation, except for the end-functionalized
PBMA-N3-B and PHMA-N3-B (PBMA: butyl methacrylate ho-
mopolymer; PHMA: hexyl methacrylate homopolymer), which were
precipitated in MeOH at -78 °C (acetone/dry CO2 bath), and
PDMAEMAs, precipitated in cyclohexane at 0 °C (ice bath). FT-IR
(KBr window, cm-1): PBMA-N3: 2850-3000 (ν, C-H), 2099 (ν, N3),
1732 (ν, CdO), 1471, 1456 (δa, CH2), 1386 (δa, CH3), 1153 (ν, C-O).
PHMA-N3: 2850-3435 (ν, C-H), 2098 (ν, N3), 1732 (ν, CdO), 1469,
dialysis. The aqueous dispersions were centrifuged, and the collected
solid was washed sequentially with water, acetone, and diethyl ether
(three cycles for each solvent) to remove any unbound polymer. In the
case of AmDMAE, the dialysis was carried out against an acetate buffer
for 3 days, then against water for 7 days to remove catalyst and
unreacted polymer. The collected solids were dried at 40 °C overnight
and then under reduced pressure to constant weight. The adopted
purification procedures allowed quantitative recovery of the grafted
copolymer except for AmHMA, which scantily precipitated under any
experimented conditions. Accordingly, FT-IR analysis of the washing
solvent showed no detectable characteristic polysaccharide stretching band
except for the PHMA derivative. FT-IR (KBr discs, cm-1): AmBA: 3435
(ν, O-H), 2959, 2934 (ν, C-H), 1731 (ν, CdO), 1459 (δa, CH2), 1155
(νa, C-C-O), 1080 (ν,C-C-O), ∼1000 (ν, C-C). AmBM: 3421 (ν,
O-H), 1731 (ν, CdO), 1459 (ν, C-H), 1155 (νa, C-C-O), 1080
(ν,C-C-O), ∼1000 (ν, C-C). AmBMA: 3421 (ν, O-H), 2959, 2934 (ν,
C-H), 1731 (ν, CdO), 1459 (δa, CH2), 1155 (νa, C-C-O), 1080
(ν,C-C-O), ∼1000 (ν, C-C). AmHMA: 3420 (ν, O-H), 2930 (ν, C-H),
1731 (ν, CdO), 1459 (δa, CH2), 1155 (νa, C-C-O), 1080 (ν, C-C-O),
∼1000 (ν, C-C). 1H NMR 300 MHz (DMSO-d6, δ):59 AmBMA2:
0.77-1.07 (CH3), 1.37 (CH2), 1.53 (CH2), 3.38 (H-2, H-4), 3.62 (H-3,H-
5, H-6a,b), 3.88 (OCH2), 4.57 (OH), 5.09 (H-1), 5.38 (OH), 5.49 (OH).
AmBMA (room temperature): 0.79-1.08 (CH3, CH2 main chain), 1.36
(CH2 side chain), 1.55 (CH2 side chain), 3.25 (H-2, H-4), 3.65 (H-3, H-5,
H-6a,b), 3.86 (OCH2), 4.46 (OH), 5.09 (H-1), 5.30 (OH), 5.35 (OH).
AmBMA (60 °C): 0.79-0.91 (CH3, CH2 main chain), 1.37 (CH2), 1.54
(CH2), 3.15 (H-2, H-4), 3.34 (H2O traces), 3.59 (H-5), 3.65 (H-3, H-6a,b),
3.89 (O-CH2), 4.34 (OH), 5.10 (H-1), 5.21 (2OH). AmBMA (80 °C):
0.79-1.08 (CH3, CH2 main chain), 1.39 (CH2), 1.59 (CH2), 3.13 (H-2,
H-4), 3.32 (H2O traces), 3.58 (H-5), 3.63 (H-3, H-6), 3.91 (OCH2), 4.23
(OH), 5.10 (H-1), 5.23 (2OH), 7.86 (H, triazole). AmHMA (room
temperature): 0.74-1.08 (CH3, CH2 main chain), 1.28-1.62 (CH2, 8H),
3.33 (H-2, H-4, H2O traces), 3.62 (H-3, H-5, H-6a,b), 3.88 (OCH2, partly
overlapped), 4.56 (OH), 5.08 (H-1), 5.38 (OH), 5.48 (OH). AmHMA (60
°C): 0.78-0.98 (CH3, CH2 main chain), 1.30-1.85 (CH2, 8H), 3.16 (H-2,
H-4), 3.33 (H2O), 3.64 (H-3, H-5, H-6a,b), 3.91 (OCH2), 4.35 (OH), 5.10
(H-1), 5.21 (2OH). AmBA (room temperature): 0.88 (CH3), 1.3 (CH2),
1.5 (CH2), 1.5-2.2 (CH and CH2 main chain), 3.2-3.4 (H-2, H-4), 3.45
-3.7 (H-3, H-5, H-6a,b), 3.96 (OCH2), 4.8 (OH), 5.1 (H-1), 5.35 (OH),
5.45 (OH), 7.95 (H triazole). AmDMAE (60 °C): 1.65-1.9 (CH and CH2
main chain), 2.2 (NCH3, 6H), 2.5 (CH2N, DMSO-d5-H), 3.25-3.4 (H-2,
H-4), 3.51-3.62 (H-3, H-5, H-6a,b), 3.98 (OCH2), 4.4 (OH), 5.12 (H-1),
5.25 (OH), 8.2 (H, triazole).
1
1456 (δa, CH2), 1380 (δa, CH3), 1149 (ν, C-O). H NMR 300 MHz
(CDCl3, δ): PBMA-N3: 0.85-1.02 (CH3, 6H), 1.20 -1.85 (CH2, 6H),
3.35 (t, CH2-N3), 4.14 (m, H3C-O). PBMA: 0.85-1.02 (CH3, 6H),
1.20-1.85 (CH2), 4.14 (m, H3C-O). PHMA-N3: 0.88-1.01 (CH3, 6H),
1.18-1.89 (CH2, 10H), 3.38 (t, CH2-N3), 4.1 (OCH2). PDMAEMA:
0.85-1.02 (CH3, 6H), 2.25 (H3C-N, 6H), 2.62 (CH2), 4.15 (OCH2).
PDMAEMA-N3. 0.85-1.02 (CH3, 6H), 2.25 (H3C-N, 6H), 2.62 (CH2),
3.4 (CH2-N3), 4.15 (OCH2).
Preparation of Alkynyl-Functionalized Amylose (Am-PA). Amy-
lose (9.07 g, including a 10 wt % of absorbed water and butanol
according to the results of TGA analysis) was dispersed in H2O (∼35
g/L) under stirring at room temperature; then, sodium periodate (0.359
g, 0.04 mol/glucosidic unit) was added, and the solution was stirred in
the dark for 2 h. The oxidized polysaccharide (Aox) was dialyzed
against water (molecular weight cutoff MWCO ) 2000) for 3 days
and then either freeze-dried to constant weight or precipitated in ethanol,
filtered, and dried under reduced pressure. Aox (4 g) was dispersed in
85 mL of aqueous borate buffer (pH 8.5) at room temperature; then,
the temperature was raised to 40 °C before propargylamine (0.18 g)
and NaCNBH3 (0.27 g) were added. After stirring for 24 h, the reaction
mixture was cooled to room temperature and the product (Am-PA)
was filtered and washed with a sequence of water, acetone, and diethyl
1
ether, then dried to constant weight. H NMR 300 MHz (DMSO-d6,
δ): 3.36 (H-2, H-4), 3.55-3.63 (H-3, H-6), 4.56 (OH), 4.85 (H-1 ꢀ),
5.08 (OH), 5.49 (OH). Elemental analysis (wt %): C, 40.45; N, 0.39;
H, 6.26.
Preparation of Graft Copolymers by “Click” Reaction. The
alkynyl-functionalized amylose (Am-PA) and the given N3-end-
functionalized poly(meth)acrylate (Table 2) were separately dissolved
in DMSO or DMF at 40 °C under stirring and deoxygenated by either
purgingwithnitrogenfor2hordegassingthroughthreefreeze-pump-thaw
cycles. DMSO was used as the solvent for amylose, except in the
AmBMA2 synthesis (Table 2) that was performed in DMF. The two
solutions were mixed; then, the catalyst (CuBr/PMDETA, equimolar
to-N3) and freshly prepared aqueous sodium ascorbate (4 M solution,
equimolar to CuBr) were added to start the reaction that was carried
out for 3 days (except AmHMA 6 days) at either 40 or 60 °C (Table
2) under a nitrogen atmosphere. Catalyst removal and solvent replace-
ment were achieved by dialysing (MCWO ) 2000) against water for
3 days. Mixtures rich in DMF were diluted with pure water before
Results and Discussion
A new strategy to prepare self-assembling amphiphilic graft
copolymers based on a grafting “onto” approach was adopted

392 Biomacromolecules, Vol. 12, No. 2, 2011
Bertoldo et al.
Figure 1. FT-IR spectra of N3-end-functionalized polymers (pellets
in KBr): PBA-N3 (solid), PBMA-N3 (dashed-dotted), PHMA-N3 (dotted),
PDMAEM-N3 (dashed). (See Table 1.)
1
Figure 2. H NMR (300 MHz, CDCl3) spectrum of PBMA-N3-B.
Scheme 3. Amylose Functionalization with Alkynyl Groups by a
Two-Step Procedure: Oxidation by Periodate and Successive
Reductive Amination with Propargylamine
Scheme 2. Synthesis of 2-Bromo-isobutyric Acid
3-Azidopropylester (BiBAP)61,62
(Scheme 1). The convergent synthetic design is based on the
combination of two independent reaction routes providing each
a “click” reagent, namely, an alkynyl-functionalized amylose
and an N3-end-functionalized poly(meth)acrylate. These reagents
are then combined under “click” conditions to give graft
copolymers.
Preparation of r-N3- and ω-N3-End-Functionalized
Poly(meth)acrylates by ATRP. The polymerization of butyl
acrylate under ATRP conditions with EBP as the initiator (I)
afforded Br-terminated PBA that was converted to PBA-N3
through a nucleophilic substitution of the terminal bromide with
the azide group of NaN3.60 The N3 stretching band at 2112 cm-1
in the FT-IR spectrum of PBA-N3 (Figure 1) was used to
evaluate the degree of ω-N3 end-functionalization (dfN3, Table
1) which resulted quantitative.
Unlike in poly(acrylate)s, HBr elimination leading to an R-ꢀ-
unsaturated ester chain end is expected to compete effectively
with bromine substitution at the tertiary carbon of end-
functionalized poly(methacrylate)s. To prevent the possible
generation of unreactive chain ends by such side reaction, the
functional ATRP initiator 2-bromo-isobutyric acid 3-azidopro-
pylester (BiBAP) was synthesized (Scheme 2)61,62 and used
instead of EBP in the polymerization of methacrylates, yielding
R-N3-ω-Br-functionalized polymethacrylates. Comparative ex-
periments with EBP and the functional BiBAP initiators led to
comparable monomer conversions (by NMR analysis), molec-
ular weights, and molecular weight distributions (by SEC
analysis) (Table 1). Therefore, the absence of interference of
the azido group in the ATRP reaction was confirmed.
The FT-IR spectra of purified methacrylic polymers obtained
with BIBAP as the initiator present the N3 stretching band at
2099 cm-1 (Figure 1), from which the end-functionalization
degree (dfN3) was determined as 73 and 95 mol % for the
polymers purified by prolonged vacuum drying (sample names
with suffix A, Table 1) and precipitated at low temperature
(suffix B), respectively. The 1H NMR results (Figure 2)
confirmed the dfN3 values obtained by FT-IR. In the case of
PHMA-N3 and PDMAEMA-N3, the dfN3 values were similar
to that of PBMA-N3 after analogous purification process
(samples A and B). The lowest dfN3 value (59 mol %) was
obtained for PHMA-N3, possibly as a result of some photo-
cleavage occurring while drying in vacuum the reaction product
contained in a glass flask in the 40-80 °C temperature range,
without protection from environmental light. In the case of
PDMAEMA-N3 precipitated at 0 °C, the comparatively lower
dfN3 value (85 mol %) is likely to be the result of an
overestimation of the PDMAEMA molecular weight from SEC
with calibration performed using PS standards, in agreement
with the findings of Lowe et al.63
Functionalization of Amylose with Alkynyl Groups. A two-
step procedure was adopted to functionalize amylose with
alkynyl groups: initial oxidation by periodate afforded aldehyde
groups (Aox) which were then modified by reductive amination
with propargylamine (Am-PA) (Scheme 3). Periodate oxidation
quantitatively afforded 8 mol % of aldehyde groups per pyranose
ring (df ) 8.2 mol %, Table 3), as determined by titration with
hydroxylamine hydrochloride. (See the Experimental Part.)
The reaction of Aox with propargylamine in the presence of
NaCNBH3 at pH 8.5 afforded Am-PA. Preferential conversion
of the intermediate imine to amino groups with respect to the
competitive aldehyde reduction to hydroxyl groups is achieved
under alkaline conditions,64 the latter also being effective in
catalyzing the back reaction of hemiacetals to aldehyde, thus
increasing the final amination yield. Indeed, a comparative
reaction performed at pH 5 resulted in degradation of the

Amphiphilic Amylose-g-poly(meth)acrylate Copolymers
Biomacromolecules, Vol. 12, No. 2, 2011 393
Table 3. Functionalization Degree and Average Molecular Weight
by SEC (H2O/NaCl) Analysis of Oxidized (Aox) and
Alkynyl-Functionalized (Am-PA) Amylose
propargylation
j
df
yield
Mn
j
j
sample
(% mol/mol)a
(% mol/mol)b
(g mol-1
)
(Mw)/(Mn)
amylose
Aox
19 800c
9700
13 300
1.6
1.5
1.4
8.2
4.6 ( 0.7
Am-PAd
56 ( 9
a Aldehyde/pyranose ring or alkyne/pyranose ring × 100. b dfalkyne
as suggested by the bimodal profile of the SEC trace. d Average value
from three experiments.
×
100/dfaldehyde.
c Possible overestimation due to the presence of aggregates,
Scheme 4. Synthesis of Amylose-g-PBA Copolymers by “Click”
Grafting onto Reaction
Figure 3. 1H NMR (300 MHz, DMSO-d6, 25 °C) of the AmBA graft
copolymer.
Figure 4. FT-IR of PBA-N3 (dotted), Am-PA (dashed), and AmBA
(solid line). Films cast on KBr disks.
of the graft copolymers, both the CH3 and CH2 proton
resonances from the PBA chains in the 0.5-2.5 ppm range and
those of the amylose protons in the 3-6 ppm range are present
(Figure 3). Accordingly, in the FT-IR spectrum, both the
characteristic CdO stretching band of polyacrylates at 1732
cm-1 and the typical C-C complex stretching band of the
pyranose ring centered at 1022 cm-1 (Figure 4) are present. The
complete disappearance of the N3 stretching band at 2112 cm-1
in the FT-IR spectrum and the appearance of a 1H NMR
resonance at 7.9 ppm due to the triazole ring proton proved the
formation of the amylose-g-PBA copolymer (AmBA) by
azide-alkyne cycloaddition.
When PBA without azide functionality was used (blank, Table
3), only negligible traces of polymer were collected after
purification by subsequent washings with the solvents for the
homopolymers, the residual solid being PBA according to
the NMR and FT-IR spectra. Therefore, the effectiveness of
the purification procedure to remove possible unbound polymers
was confirmed, and the occurrence of any grafting reactions by
a mechanism different from the azide-alkyne cycloaddition
could be excluded. Furthermore, the polysaccharide recovered
from the solvent extracts had a SEC Mn ) 13 900 Da, practically
the same as the unprocessed Am-PA, confirming that no
polysaccharide degradation occurs under the adopted “click”
reaction conditions. Such inertness of polysaccharide chains had
been already observed by some of the authors of this work for
the chitosan azide/alkyne click functionalization in DMF under
oxygen-free conditions.41
polysaccharide chain and no detectable amination. From the
nitrogen content of Am-PA determined by elemental analysis,
a 4.6 ( 0.7 mol % alkynyl functionalization degree (df, Table
3) was determined, corresponding to a 56% aldehyde conver-
sion, slightly lower than the typical 70-80% conversions
reported for the reductive amination of low-molecular-weight
aldehydes.64 The molecular weight of the polymer after func-
tionalization with propargylamine (Am-PA) by reductive ami-
nation was comparable to that of the oxidized precursor (Aox)
j
(Table 3), once considering the Mn increase caused by the grafted
propargylamine.
Preparation of Amylose-g-poly(meth)acrylate Copolymers
by “Click” Reactions. The alkynyl-functionalized amylose (Am-
PA) was used for grafting “onto” reactions with the N3-end-
functionalized poly(butyl acrylate) (PBA-N3) by the “click”
Cu(I)-catalyzed [3 + 2] dipolar cycloaddition (Scheme 4).65 The
reaction was carried out using an equimolar amount of catalyst
with respect to the alkynyl groups in homogeneous phase by
DMSO/DMF mixed solvent (Table 2) because DMSO is a good
solvent for amylose and DMF is a solvent of polymethacrylates
that does not precipitate the polysaccharide. The reaction
temperature was initially set to 60 °C to improve the reagent
1
solubility. The H NMR and FT-IR spectra of the reaction
products obtained under such conditions presented the charac-
teristic features of both the synthetic polymer and the polysac-
charide (Figures 3 and 4). In particular, in the1H NMR spectra
The adopted reaction conditions had been previously opti-
mized in DMF, a single solvent homogeneous phase, by using

394 Biomacromolecules, Vol. 12, No. 2, 2011
Bertoldo et al.
Figure 5. Weight loss rate (first derivative of the TGA weight loss
curve) for the AmBA graft copolymer (solid line), the parent ungrafted
Am-PA (dashed line), and the grafting PBA-N3 (dotted line).
Figure 6. 1H NMR (300 MHz, DMSO-d6/H2O 100/1, 60 °C) of the
AmDMAE copolymer.
was used instead of DMSO/DMF mixed solvent in run
AmBMA2, and the reaction temperature was set to 40 °C (Table
2). As a result, the reaction was carried out in suspension, and
a lower 63 wt % grafting yield was obtained. Similarly, in the
synthesis of AmHMA, an incomplete solubility of the grafting
poly(hexyl methacrylate) resulted in a poor 30 wt % grafting
yield achieved after 6 days of reaction. The above results showed
the importance of having both reactants well-solubilized in the
reaction medium to achieve quantitative grafting.
When the functional PDMAEMA-N3 was used as the grafting
polymer, the result of the TGA analysis performed on the
reaction product AmDMAE indicated quantitative grafting onto
amylose (Table 2), and the 1H NMR spectrum (Figure 6) showed
the presence of the typical resonances from both amylose and
synthetic polymer, both results confirming the effectiveness of
the adopted grafting conditions also when using functional
polymethacrylates. Integration of the resonance peaks gave
values in agreement with the copolymer composition from TGA
data analysis, this being an evidence of the good solubility of
the copolymer in the solvent used for the spectroscopic analysis.
A 4.5 mol % grafted PDMAEMA chains per pyranoside unit
was calculated from the ratio between the resonance integrals
of the triazole proton at 7.95 ppm and of the anomeric proton
of the pyranose ring at 5.11 ppm. Such a grafting density is in
good agreement with the functionalization degree of amylose
by alkynyl groups (Table 3), thus confirming the quantitative
conversion of the alkynyl groups to grafted chains.
Self-Assembling and Nanoparticle Morphology of Grafted
Copolymers. A preliminary investigation on the behavior of the
amphiphilic graft copolymers in water showed their aqueous
solution to be almost completely transparent at concentrations
of amylose-based copolymers <1 g/L. DLS analysis performed
on the aqueous graft copolymers always showed detectable
scattered intensity except in the case of AmDMAE, which was
therefore regarded as fully soluble. The presence of nanoparticles
in all other samples suggested by the light scattering experiments
was confirmed by the TEM observations, showing the presence
of nanoparticles <100 nm (Figure 7). Cumulant analysis of the
DLS data from unfiltered or not centrifuged dispersions of
AmBA, AmBMA, and AmHMA always showed the presence
of both small (<100 nm) and large (g350 nm) nanoparticles
(Table 4). The total scattered intensity decreased after filtration
or centrifugation because of the removal of the larger particle
or particle aggregate families (∆CR, Table 4). In fact, the TEM
micrographs of Figure 7c,d show the presence of aggregates of
spheroidal particles with well-defined size in the 30-40 nm
PBA-N3 and an alkynyl-functionalized branched glucan well-
soluble in DMF. For that purpose, the following parameters had
been varied: (a) Cu(I)/reactive groups ratio, (b) reaction solvent,
and (c) reaction temperature. Quantitative PBA-N3 grafting was
observed to require an equimolar amount of catalyst with respect
to the alkyne groups, only partial grafting being achieved with
a lower catalyst amount. We observed a similar effect by
replacing some of the DMF with water; the lower conversion
being ascribed in this case to the consequent heterogeneity of
the system. Changing the reaction temperature in the 40-60
°C range did not significantly influence the grafting yield. To
understand the quite higher catalyst amount requirement than
that in cycloadditions involving low-molecular-weight species
(0.01 to 0.1 equiv of catalyst),33,36,66 we must consider some
specific characteristics of polysaccharide systems under inves-
tigation. These are the very low concentration of the functional
groups (typically 10-3 to 10-4 mol L-1), the possible capture
of some copper(I) by complexation with the numerous hydroxyl
groups, and the molecular or supramolecular steric hindrance.
The weight loss curve recorded from the TGA analysis of
the AmBA graft copolymer (Figure 5) showed two well-resolved
degradation steps, thus allowing the quantitative evaluation of
its composition (Table 2). For this purpose, the peak integrals
in the weight-loss first derivative curve from the thermograms
of the copolymer and of the grafting parent polymers Am-PA
and PBA-N3 were considered, and the fractional weight loss
attributed to the acrylic chains was calculated accordingly. The
amount of PBA in AmBA was found to be 39 wt %, which
corresponds to an almost quantitative grafting yield (95 wt %
with respect to the amount of PBA-N3 in the reaction feed).
Accordingly, no acrylic homopolymer could be detected in the
washing solvent, the efficiency of the proposed click promoted
grafting “onto” strategy for the preparation of comb like
copolymers being thus confirmed.
The same conditions as those used for the AmBA preparation
were adopted in the grafting of PBMA and PDMAEMA chains
onto amylose (AmBMA and AmDMAE, respectively, Table 3).
In the case of PBMA, the only difference in the synthetic
procedure was the amount of azido-end-functionalized grafting
polymer PBMA-N3, fed in a higher ∼80 mol % with respect to
the alkynyl groups on amylose as compared with the much
smaller ∼0.33 mol % used in the synthesis of AmBA. However,
the grafting yield was again almost quantitative (91 wt %), thus
indicating the possibility of tuning the amount of grafted chains
up to the complete conversion of alkynyl groups on amylose.
In an attempt to simplify the reaction conditions, only DMF

Amphiphilic Amylose-g-poly(meth)acrylate Copolymers
Biomacromolecules, Vol. 12, No. 2, 2011 395
Figure 7. TEM micrographs of nanoparticles from (a,b) filtered and (c,d) unfiltered 1 g/L aqueous dispersions of (a,c) AmBMA and (b,d) AmBA.
Dispersions obtained by solvent exchange dialysis from DMSO solutions at 2 g/L.
Table 4. Average Diameter of Amylose Copolymer Nanoparticles
but cannot prevent coalescence of particles during the prepara-
tion of samples for TEM analysis from relatively concentrated
dispersions.
in Water (concn ) 1 g/L) from DLS Measurement (Dispersions
Obtained by Solvent Exchange Dialysis from DMSO Solutions at
2 g/L)
hydrodynamic
Heterogeneity of both unfiltered and 0.45 µm filtered aqueous
solutions of AmBMA, for instance, is evident by comparing
the respective scattered light intensity profiles. The larger
aggregates contribute to the scattered intensity mainly at small
scattering angle (due to the form factor), and only the filtering
with 0.2 µm pore size significantly decreases such heterogeneity.
(See Figure 8.)
diameter (DH) particles > 350 nm
sample
notea
∆(CR)b
(nm)c
(vol %)
AmBMA
46 ( 6
45 ( 4
31 ( 2
27 ( 3
24 ( 3
30 ( 3
41 ( 4
29 ( 2
26 ( 2
13.0
centrifuged
filtered
21
47
AmBA
13.5
10.0
centrifuged
filtered
37
77
The dependence of dynamic and static light scattering on the
scattering angle θ can add more information on the particle
morphology. In Figure 9 is shown the decay or relaxation rate
Γ, as obtained from the normalized scattered electric field
autocorrelation function for the AmBMA copolymer by a
second-order cumulant expansion. The average translational
diffusion coefficient D, that is, the average value from a
monomodal polydisperse particle size distribution (resulting in
a distribution of decay rates Γ ) DQ2 of monodisperse scatterers
assumed as rigid spheres, where Q is the exchanged wavevector
of the autocorrelation function), can be measured below Q )
20 µm-1, whereas above this value, the onset of the internal
motions regime occurs. The diffusion coefficient does not seem
to depend on the concentration of the aqueous copolymer
solution (data not shown) at the lowest concentration values
(0.1 to 0.01 g/L), indicating that interparticle interactions can
be neglected. Under this condition, the Einstein-Stokes relation
RH ) kBT/(6πηD) (where kB is the Boltzmann’s constant, T is
the absolute temperature, and η is the solvent viscosity) can be
applied, giving an average hydrodynamic radius of RH ) 48 (
2 nm for AmBMA. The crossover at QRH ≈ 1 between
AmHMA
16.0
centrifuged
filtered
26
43
a Centrifuged samples were processed at 4400 RCF for 10 min; filtered
samples were processed through 0.45 µm cellulose acetate syringe filters.
b ∆(CR) ) [(CR) - (CR)T]*100/(CR), where (CR) ) count rate of as
prepared dispersion, (CR)T
) count rate of centrifuged or filtered
dispersion. c Weighed intensity average of the most abundant smaller sized
particle family.
range in the case of AmBMA, whereas undefined morphologies
apparently consisting of much smaller flocculated particles of
irregular shape were observed with the PBA and PHMA
derivatives. To explain such differences, one should take into
account the fact that the two latter polymers have glass-transition
temperatures that are much lower than those of PBMA (PBA
) -51.5 °C; PHMA ) -4.2 °C; PBMA ) +26.0 °C), resulting
in corresponding copolymers with high macromolecular mobility
and tendency toward interparticle coalescence on drying.
Actually, the low molecular weight of the grafted chains, which
is below the entanglement length67 for all investigated copoly-
mers (Table 1), does not favor micelle clustering in solution

396 Biomacromolecules, Vol. 12, No. 2, 2011
Bertoldo et al.
Figure 10. Reciprocal of the excess scattered intensity I(Q) of the
filtered aqueous solution of AmBMA at 0.1 g/cm3 (dispersions
obtained by solvent exchange dialysis from DMSO solutions at 20
g/L). Excess scattered intensity determined from static light scattering
measurements and proportional, at high dilution, to the form factor.
The continuous line is the fit according to the Ornstein-Zernike
equation.
Figure 8. Scattered intensity profile of the AmBMA/water dispersion
at 0.1 g/L before (9) and after the filtering procedure with 0.45 (0)
and 0.2 µm (b) pore size cellulose acetate filters (dispersions obtained
by solvent exchange dialysis from DMSO solutions at 20 g/L). The
scattering intensity wave vector scales with the scattering angle θ as
Q ) (4πn/λ)sin ϑ.
Figure 11. Comparison between the scattered intensity autocorre-
lation function of AmBMA at 0.1 g/L obtained by solvent exchange
dialysis from DMSO solutions at 20 (O) and 2 g/L (b).
Figure 9. Correlation functions decay rate of the filtered aqueous
solution of AmBM2A at 0.1 g/cm3 (dispersions obtained by solvent
exchange dialysis from DMSO solutions at 20 g/L). The straight line
is the linear fit in the Q range below 20 µm-1. In the inset, the
normalized decay rate is reported at different concentration values:
c ) 0.01 (0), 0.1 (b), and 1 g/cm3 (4). The two dashed straight lines
represent the regions in which Γ is proportional to Q2 and Q3,
respectively.
(DH/2 ≈ 16 nm for the filtered AmBMA) is referred to the
singled out family of particles in the smaller size range.
Moreover, the measured particle size was not affected by
dilution, even when starting from 1 g/L aqueous solutions, an
indication of the achievement of more stable particle morphol-
ogy, differently from the case of the dispersions prepared starting
from the 20 g/L DMSO solution.
translational diffusion and internal motions is displayed more
clearly in the inset of Figure 9, in which the results for all
investigated concentrations indicate that the aggregates are not
rigid.
This sample preparation method, that is, solvent exchange
by dialysis starting from 2 g/L DMSO solutions, was thus
adopted to compare the average particle size of samples AmBA,
AmHMA, and AmBMA at 90° scattering angle. At least a
bimodal particle distribution was observed in all samples, with
the smallest particle family listed in Table 4 as the most
abundant both before and after filtration or centrifugation. In
fact, the number fraction of the particles belonging to the largest
families was negligible under all experimental conditions,
although the same did not always hold true for the volume
fraction. The hydrodynamic particle diameter for the most
abundant distribution was centered at ∼30 nm for all three
filtered dispersions of amylose graft copolymers. Therefore, it
must be assumed that the different grafted chains give a similar
contribution to the hydrophobic character of the graft copolymer.
In particular, the slightly smaller average size displayed by
AmHMA suggests a more pronounced hydrophobic contribution
from the PHMA grafted chains, leading to the formation of
The scattered intensity profile can be fitted with the
Ornstein-Zernike equation (Figure 10), describing particles with
nonhomogeneous internal density distribution and correlation
length ꢁ. By taking as the radius of gyration of the aggregate
Rg ) ꢀ3ꢁ, the obtained value for AmBMA was Rg ≈ 95 nm.
Both the form factor of the aggregates, indicated by the
Ornstein-Zernike profile, and the Rg/RH ratio suggest that the
mass of the aggregates is not homogeneously distributed in
the occupied volume. This result is consistent with the formation
of micellar clusters.
When the aqueous dispersions were prepared starting from
less-concentrated DMSO solutions (2 instead of 20 g/L), smaller
values of the average particle size were observed from the
cumulant analysis (Figure 11). In particular, RH was 25 ( 1
nm for AmBMA filtered with a 0.2 µm filter. It is worth pointing
out that the slightly lower average value reported in Table 4

Amphiphilic Amylose-g-poly(meth)acrylate Copolymers
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The results obtained in the grafting “onto” experiments
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Supporting Information Available. Details of the light
scattering data analysis procedure; synthesis and spectroscopy
characterization of 2-bromo-isobutyric acid 3-azidopropylester
(BiBAP) and ethyl-2-azidobutyrate. This material is available
free of charge via the Internet at http://pubs.acs.org.
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