Biomacromolecules 2005, 6, 2189-2196 2189

pH-Induced Self-Assembly and Capsules of Sodium Alginate

Yi Cao,† Xiaochen Shen,† Ying Chen,† Jian Guo,† Qi Chen,† and Xiqun Jiang*,†,‡
Laboratory of Mesoscopic Chemistry and Department of Polymer Science and Engineering, College of
Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China, and Jiangsu Provincial
Laboratory for Nanotechnology, Nanjing University, Nanjing, 210093, China
Received March 2, 2005; Revised Manuscript Received April 11, 2005

In this investigation, we used a kind of polyelectrolyte, sodium alginate, as a model biomacromolecule to

investigate the aggregation behaviors in aqueous solution after partial protonation of carboxylate groups in
the alginate molecules. It is demonstrated that the alginate assemblies with core-shell structure can be
generated by the partial protonation of carboxylate groups in sodium alginate chains using the protons released
gradually from the reaction of K2S2O8 with water at 70 °C in aqueous solution. The partial cross-linked
alginate assemblies are pH sensitive and can change to hollow structure in the medium with relatively high
pH value. This approach avoids use of block or grafted copolymers as the precursors or any other template
to prepare assemblies and capsules, and provides a functional surface for subsequent chemical reaction at
the surface (e.g., for binding biomolecules and for surface grafting). Such unique assemblies are also expected
to be useful in biomedical fields.

Introduction the shell. To mimic this behavior of the biologic macromol-

ecules, a number of theoretic studies and computer simula-
The synthesis of nanosized polymeric assemblies such as
tions on the aggregation of protein-like copolymers consisting
micelles, nanospheres and nanocapsules in aqueous solution
of uncharged monomer units and charged ones in aqueous
has attracted great interest due to their applications in biologic
medium have been reported.11 Furthermore, a theoretic model
and medical areas such as diagnostic testing, bioseparations,
of two-letter copolymers comprised of two types of monomer
controlled releasing of drugs, and gene therapy.1-3 One
units, H (hydrophobic) and P (hydrophilic or polar), has been
typical approach to construct them in aqueous solution is
proposed.12 These investigations show that the copolymers
mixing oppositely charged polymers,4 polymer/surfactant,5
with a heterogeneous, nonaltering sequence of H and P units
or polymer/small counterion.6 The electrostatic force interac-
along the copolymer chain can form stable, finite aggregates
tion between a pair of oppositely charged polymers, polymer/
with H-core/P-shell structure. A few experimental studies
surfactant, or polymer/small counterion drives the formation
also determined that a random copolymer could aggregate
of polymeric assemblies.
to form unimolecular micelles in aqueous solution.13 Al-
Recently, several groups have reported a novel method to
though advances have been made in understanding aggrega-
prepare pH-sensitive micelles from double-hydrophilic water-
tion behavior of individual protein-like copolymers in
soluble AB diblock or graft copolymers.7,8 These copolymers,
aqueous medium, few experimental studies have investigated
which contain at least one polyelectrolyte block, can dissolve
the multichain aggregation of two-letter copolymers with
molecularly in water in a certain pH range and aggregate
random distribution of two types unit along the backbone
spontaneously to form micelles upon an appropriate change
chains.14
in the pH value. The difference in protonation or deproto-
nation of the polyelectrolyte block in a certain pH range In this paper, we used the polyelectrolyte, sodium alginate,
drives the micellization of the copolymer in aqueous solution as a model biomacromolecule to investigate the aggregation
and determines the pH-responsive behavior of the micelles.9,10 behaviors in aqueous solution. We chose alginate based on
Among these approaches, in most cases, the micelles are the following reasons: (1) alginates are biocompatible
comprised of block or graft copolymers, with the pH- unbranched binary copolymers of (1f4)-linked residues of
sensitive solvent-phobic part forming the core and the â-D-mannuronic (M) and R-L-guluronic (G) acids (the pKa
solvent-philic part forming the corona. On the contrary, in values for them are 3.38 and 3.65, respectively),15 which have
nature, a variety of protein and polypeptide biomacromol- been widely used as a kind of the desired biomaterials in
ecules consisting of hydrophobic, hydrophilic, and/or charged many fields such as cell immobilization, tissue engineering
amino acid residues on the backbone chain can self-assemble and drug delivery;16 (2) the hydrophilic and hydrophobic
to aggregates in aqueous medium with hydrophobic amino units along a molecule chain can be altered by the protonation
acids as the core and hydrophilic or charged amino acids as and deprotonation of carboxyl groups in the backbone chain;
(3) the carboxyl groups existing in alginate can be used for
* To whom correspondence should be addressed. Fax: 86 25 83317761. further chemical modification such as cross-linking reaction.
E-mail: jiangx@nju.edu.cn.
† College of Chemistry and Chemical Engineering, Nanjing University. The dissociated carboxyl groups in alginate chains are
‡ Jiangsu Provincial Laboratory for Nanotechnology, Nanjing University. protonated smoothly by the protons which are released
10.1021/bm0501510 CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/17/2005
2190 Biomacromolecules, Vol. 6, No. 4, 2005 Cao et al.

Scheme 1. Chemic Structure of Sodium Alginate Used, with Chair aqueous solution, the product was dialyzed for 48 h. The
Conformationa size and morphology of the samples were characterized by
dynamic light scattering (DLS), transmission electron mi-
croscopy (TEM), and scanning electron microscopy (SEM).
pH Value Measurement. The pH measurements were
performed with a Delta 320 pH-meter (Mettler Toledo
Instruments Co., Ltd.). Each sample was measured three
times, and the results shown are the mean pH value.
a M, â-D-mannuronate; G, R-L-guluronate. FG ) 0.65 and FGG ) 0.53.
Fluorescence Measurement. Pyrene was chosen as the
gradually upon the thermal decomposition and reaction of fluorescent probe to study the onset of assembly of sodium
potassium persulfate (K2S2O8) in water. By this means, alginate solution. Pyrenes (6.0 × 10-7 molar) were dissolved
alginate molecules can form regular core-shell type ag- in 10 mL of 0.25% (w/w) alginate solutions in the presence
gregates in aqueous solution without the aid of any other of NaCl (0.05 M). The pH value of the solutions was adjusted
homopolymer, copolymer, or surfactant. Such core-shell by HCl and NaOH. Steady-state fluorescent spectra were
structure assemblies can be fixed by cross-linking the measured using a Perkin Elmeter LS50B fluorescence
carboxyl groups in sodium alginate chains at room temper- spectrophotometer in the right-angle optical geometry with
ature. Moreover, the cross-linked assemblies obtained have a bandwidth of 0.5 mm. The excitation wavelength was 390
a pH-dependent structure and can change between a core- nm.
shell structure to a hollow structure depending on the pH Dynamic Light Scattering (DLS) Measurement. The
change of medium. mean sizes of the assemblies were measured by DLS using
a Brookheaven BI9000AT system (Brookheaven Instruments
Corporation, USA). All DLS measurements were done with
Experimental Section a wavelength of 633.0 nm at 25 °C with an angle of detection
Materials. Sodium alginate, purchased from Beijing Stock of 90°. Each sample was measured three times, and the
of China Medicine Company, was refined twice by dissolving results shown are the mean diameter for two replicate
it in distilled water, filtered, precipitated with ethanol, and samples. The zeta potential of the assemblies was obtained
finally dried in a vacuum at 60 °C. Its chemical structure is with Zetaplus (Brookheaven Instruments Corporation, USA).
shown in Scheme 1. The viscous-average molecular weight The results were the average of three runs.
of sodium alginate used was 170 kDa, determined by Transmission Electron Microscopy (TEM). The mor-
viscometric methods.17 The alginate was analyzed by 1H phologies of the samples were observed by TEM (JEOL
NMR spectroscopy at 70 °C using a Bruker DRX-500 (500 JEM-100S, Japan). Samples were placed onto copper grid
MHz) spectrometer.18 The molar fraction of guluronic acid covered with carbon and dried at room temperature. They
residues (FG) and sequence parameters given as the diad were examined without being stained.
(FGG, FMM, FGM, and FMG) was determined by NMR to be Scanning Electron Microscopy (SEM). The morphology
FG ) 0.65, FM ) 0.35, FGG ) 0.53, and FMM ) 0.23. of the samples was examined by SEM (SX-40 scanning
Potassium persulfate (K2S2O8) was recrystallized from deion- microscope, AKA SHI, Japan). The sample was placed onto
ized water. Pyrene was recrystallized two times from the hydrophilic surface of the glass substrate which was
benzene. treated with a solution of H2O2 and H2SO4, washed with
Preparation of Sodium Alginate Assemblies. A total of distilled water, and dried at room temperature.
0.25 g of sodium alginate was dissolved in 100 mL of
deionized water under magnetic stirring, and the temperature Results and Discussion
was raised to 70 °C. The desired amount of K2S2O8, based
on the content of carboxylate groups in alginate, was added Self-Assembly of Alginate Induced by pH. It has been
to the system. With the decomposition of K2S2O8 and the shown that fluorescence technique is a powerful tool for the
reaction of K2S2O8 with water, protons were released investigation of many micellar properties such as micelle
gradually to the solution, and the dissociated carboxyl groups formation, micelle structure, and the kinetics of micelle
of sodium alginate were partially protonated. We sampled formation.22 Using pyrene, a polarity-sensitive molecule, as
the solution at different time points after K2S2O8 was added a fluorescent probe, it is possible to obtain information on
and cooled the samples to 0 °C with ice water. the aggregation of amphiphiles in aqueous solution from a
Cross-Linking of Assemblies and Preparation of Cap- shift of the (0, 0) band from 334 to 337 nm in the excitation
sules. When sodium alginate assemblies were formed in the spectra of pyrene.19 In our case, we chose pyrene as the
aqueous solution, the desired amount of 2,2′-(ethylenedioxy)- fluorescent probe to study the self-assembly of alginate at
bis(ethylamine), as a cross-linker, was added into the aqueous different pH values and to see if the sodium alginate chains
solution in the presence of 1-(3-dimethylaminopropyl)-3- can form the hydrophobic domains after protonation of
ethylcarbodiimide methiodide at room temperature, and the carboxyl groups. Fluorescence excitation spectra of the
cross-linking reaction was allowed to take place for 10 h. pyrene probe dissolved in 0.25% (w/w) alginate aqueous
The cross-linked product was dialyzed against distilled water solutions in the presence of 0.05 M NaCl were measured,
for 72 h to remove the byproducts. Then, the aqueous and the ratio (I337/I334) of the intensity at 337 nm to that at
solutions with different pH values were prepared. In each 334 nm of the solution was estimated at different pH values.
Capsules of Sodium Alginate Biomacromolecules, Vol. 6, No. 4, 2005 2191

Figure 2. pH variation caused by the decomposition and reaction of

Figure 1. Ratios of I337/I334 in pyrene excitation spectra as a function K2S2O8 in water as a function of heating time at different temperatures
of pH in 0.25% (w/w) sodium alginate aqueous solution in the and concentrations.
presence of 0.05 M NaCl.

Figure 1 displays the intensity ratio of I337/I334 of alginate

aqueous solutions as a function of pH value. It can be seen
that, beyond the pH value of 6.6, the I337/I334 ratio is
practically constant around 0.62, indicating that the location
of pyrene probe is in the hydrophilic environment. As the
pH value decreases from 6.6 to 4.1, the I337/I334 ratio increases
substantially and reaches a maximum at pH of 4.1, suggesting
that pyrene is progressively solubilized into the hydrophobic
environment. Below the pH value of 4.1, the I337/I334 ratio
keeps the constant of 1.47, indicating that the pyrene probe
locates primarily in the hydrophobic environment. The
behavior observed indicates that the alginate molecules can
form hydrophilic-hydrophobic aggregates in aqueous solu-
tion depending on the pH of the medium. Here, we postulate
that this pH dependence of “aggregation” as a signature for
self-assembly caused by the partial protonation of dissociated
carboxyl groups in the alginate main chain.
Preparation of Regular Assemblies of Sodium Alginate.
Based on the result of fluorescence measurement, the partial
protonation of dissociated carboxyl groups in the alginate
main chain was selected to prepare alginate assemblies. The
partial protonation of dissociated carboxyl groups in sodium
alginate was achieved by the decomposition of the desired
amount of K2S2O8 in sodium alginate aqueous solution. Since
the thermal decomposition of K2S2O8 will dissociate sym-
metrically into two sulfate free-radicals which can react with
water to produce hydrogen sulfate ions and donate the
protons to aqueous solution,20 alginate can obtain the protons
upon the decomposition of K2S2O8 in aqueous solution.
Figure 2 shows pH variation caused by the decomposition
of K2S2O8 in water as a function of heating time at different
temperatures and concentrations. It can be seen that the pH
Figure 3. Hydrodynamic diameters (Dh) of sodium alginate as-
value of the aqueous solution decreases gradually from a semblies and the solution pH value as a function of heating time. (a)
pH of 7 to a pH of less than 3 in the time period of 400 R ) 0.6, (b) R ) 0.7, (c) the hydrodynamic diameter distribution of
min. As the K2S2O8 concentration in the solution increases assemblies formed at K2S2O8 decomposition time (t) of 140 and 400
from 1.018 to 1.187 g L-1, the final pH value in the given min with R of 0.7.
time period decreases from 2.66 to 2.54 accordingly. At the
same time, the decomposition rate of K2S2O8 increases when temperature. Thus, we use K2S2O8 as a mediate proton-donor-
the temperature of the solution increases from 60 to 70 °C. agent to prepare assemblies of alginate by partial protonation
This result indicates that the protons are gradually released of dissociated carboxyl groups in alginate main chains.
by the decomposition of K2S2O8 into sulfate free-radicals The assemblies of alginate were prepared by dissolving
and the reaction of sulfate free-radicals with water. The total sodium alginate in deionized water at 70 °C followed by
amount of protons released to the solution can be controlled adding the desired amount of K2S2O8. Figure 3 shows the
by the heating time and the K2S2O8 concentration. Also, the average hydrodynamic diameters of alginate assemblies
protonation rate can be adjusted through the decomposition measured by DLS, together with the variation of pH in
2192 Biomacromolecules, Vol. 6, No. 4, 2005 Cao et al.

medium, as a function of heating time. In Figure 3a, the Table 1. Size of Assemblies Prepared at Different Temperature
initial K2S2O8 concentration is 1.018 g/L and the molar ratio, temperature (°C) 〈Dh〉 (nm) polydispersity index (PDI)
R, of the maximum possible amount of protons released upon 60 349 ( 17 0.27
the decomposition and reaction K2S2O8 in water to the total 70 319 ( 6 0.19
carboxylate groups of sodium alginate is 0.6, which means 80 335 ( 14 0.23
that the maximum possible protonation degree of carboxylate 90 380 ( 26 0.30
groups in given sodium alginate solution is 60%. Before the droppinga 412 ( 29 0.31
decomposition of K2S2O8, the pH value of alginate aqueous a The assemblies prepared by direct addition of 0.1 M HCl to sodium

solution is 8.5, and no alginate aggregate was detected by alginate solution at room temperature. (the molar ratio R of HCl and the
carboxylate groups of sodium alginate is 0.6 and the concentration of
DLS. With the decomposition of K2S2O8 at 70 °C, the pH sodium alginate is 0.25% (w/w)).
value of the solution decreases from an initial value of 8.5
to 3.4. Meanwhile, the assemblies occur and the hydrody- about 700 nm. The second peak may be attributed to the
namic diameter of the assemblies decreases substantially aggregation of assemblies which causes the assembly size
from 1070 nm at reaction time of 15 min (pH ) 5.5) to 320 to increase for more than two times. Thus, it is safe to
nm at 400 min (pH ) 3.4). It is apparent that the shrinkage conclude that the increase in assembly size when the pH
in assembly size is attributed to the protonation of the value decreases from 3.4 to 3.2 may result from the
carboxyl groups in alginate chains. In other words, the coalescence of assemblies due to the decrease of charges in
number of dissociated carboxylic groups in alginate chains the shell of assemblies.
decreases with the decomposition of K2S2O8, which makes Table 1 lists the minimum hydrodynamic diameter of
alginate lose its hydrophilicity to some extent. Initially, alginate assemblies prepared by the decomposition and
sodium alginate in aqueous solution would be in an extended, reaction of K2S2O8 in water at various temperatures with R
random coil conformation because of plenty of negative of 0.6. It can be seen that at a temperature of 70 °C the
charges in chains. When some dissociated carboxylic groups minimum assembly size is 319 nm, and the hydrodynamic
in alginate chains are gradually protonated, the occurrence diameter polydispersity index of assemblies (PDI) obtained
of hydrophobicity segments in alginate chains and hydrogen from µ2/Γ2 is 0.19. When the temperature of the system
bonds between protonated carboxylic acid groups drives the decreases to 60 °C or increases to 80 °C, the minimum size
formation of alginate assemblies. With the progressive and PDI of assemblies show a slight increase. However,
decrease of pH, the hydrophobic segments in the alginate when the temperature reaches 90 °C, the size of assemblies
chains increase and the hydrophilic segments decrease, shows a significant increase, only a little smaller than that
resulting in the substantial decrease in the size of the alginate formed by direct addition of HCl to sodium alginate aqueous
assemblies. It has been reported that protonating the alginates solution at room temperature. These phenomena suggest that
with higher content of guluronic acid moieties gives rise to the assembly formation is also related to the protonation rate
alginic gels with a higher aggregate density than the alginates of carboxylate groups in the alginate chains, which deter-
with lower content of guluronic acid moieties because mines the formation rate of hydrophobic segments and
guluronic acids are protonated and form hydrogen bonds assembly behavior of alginate molecules. For example, in
between acid groups more easily.21 the case of the addition of HCl to sodium alginate aqueous
Figure 3b shows the size of alginate assemblies as a solution at room temperature, the protonation rate of car-
function of the decomposition time of K2S2O8 at 70 °C with boxylate groups is fastest, and the assembly size and PDI
the initial K2S2O8 concentration of 1.187 g/L and R of 0.7. also are largest. In addition, the morphology of assemblies
Similar to the behavior observed in Figure 3a, as the pH prepared by direct addition of HCl to sodium alginate
value decreases from 8.5 to 3.4, the assembly size decreases solution at room temperature is comparatively irregular.
from 1000 nm (at 15 min) to 320 nm (at 140 min). However, Thus, it seems that, at an appropriate protonation rate of
when the pH value continues to decrease to 3.2 (at 400 min), carboxylate groups or decomposition temperature, the as-
the assembly size rises from 320 to 540 nm. This can be semblies prepared are smaller in size and more regular in
explained as follows. With the decrease in pH, the charges shape.
at the surface of the assemblies decrease. If the electrostatic In addition, like many other preparation methods of
repulsions are strong enough to keep the assemblies stable, nanosized polymeric assemblies, the concentration of poly-
hydrophobic associations are mostly intra-assembly. If the mer plays an important role to avoid precipitations. In our
electrostatic repulsions among different assemblies are not experiment, we found that the optimum concentration of
strong enough, some of the assemblies may move close and sodium alginate was 0.25%. If the concentration of sodium
aggregate with each other, resulting in the apparent increase alginate was increased from 0.25% (w/w) to 0.5% and the
of assembly size. This is confirmed by Figure 3c, which initial K2S2O8 concentration was 1.018 g/L, the diameter of
shows the hydrodynamic diameter distribution of assemblies resulting assemblies increased greatly from 320 to 754 nm
formed at K2S2O8 decomposition time of 140 and 400 min. after the reaction time reached 400 min at 70 °C. Moreover,
Compared with a unimodal size distribution of assemblies these assemblies precipitated after 24 h of storage at room
at 140 min (pH ) 3.4), the hydrodynamic diameter distribu- temperature. With further increasing the concentration of
tion of assemblies at 400 min (pH ) 3.2) is a bimodal size sodium alginate to 1%, only a gel of alginate was obtained
distribution. One of the peaks occurs at 320 nm with a little after the reaction. However, when we lowered the concentra-
broadening in the peak width and another one appears at tion of sodium alginate to 0.1%, there were no significant
Capsules of Sodium Alginate Biomacromolecules, Vol. 6, No. 4, 2005 2193

Figure 4. Morphologies of the assemblies prepared by the decomposition of K2S2O8 in water, with R of 0.6, at 70 °C for different heating time
(t). (a) TEM image, t ) 15 min, pH ) 5.5; (b) TEM image, magnification of a typical assemblies as seen in (a); (c) TEM image, t ) 80 min, pH
) 4.0; (d) TEM image, t ) 400 min, pH ) 3.4; (e) SEM image of assemblies as shown in (d).

changes in the size of resulting assemblies compared with shell micelles-like assemblies are seldom observed in com-
the case of alginate concentration of 0.25%. mon polymeric micelle system. Based on the ú potential of
It is also interesting that a rather large assembly diameter such assemblies (-54.88 ( 4.46 mV), which is proportional
of around 1000 nm was obtained at a reaction time of 15 to the effective charge at the surface of assemblies, and the
min as shown in Figure 3, panels a and b. Even if the sodium observation by TEM, the swelling shells, shown in Figure
alginate was fully ionized in water, the radius of gyration of 4, panels a and b, should be comprised of the negatively
sodium alginate with weight-average molecular weight 190 charged sodium alginate segments, whereas the cores should
kDa was found to be only 50 nm.22 This means that a single be comprised mainly of the alginic acid segments. Since the
alginate chain could not possibly give an assembly with the core is charge-neutral, it should be somewhat hydrophobic
size of 1000 nm. Therefore, it is reasonable to conclude that compared with the anionic shell. Furthermore, as there is
the observed assembly in our case is a multichain aggregate strong hydrogen bond interactions in alginic acid,21,23 the
and the assembly size shrinks with the increase of protonation hydrogen bonds should be also present in the cores of these
degree in sodium alginate chains. assemblies. Thus, with the increase of the protonation degree
To investigate the morphology of the formed alginate of alginate, the core size should increase, whereas shell
assemblies and the assembly structure, TEM observation was thickness should decrease. This is confirmed by Figure 4c.
carried out. Figure 4 shows the morphology of the assemblies When K2S2O8 has decomposed for 80 min (pH ) 4.0), the
prepared by the decomposition and reaction of K2S2O8 in size of assemblies is found to be 342 ( 41 nm, much smaller
water at 70 °C, with R of 0.6. When the decomposition time than that at pH value of 5.5, whereas the core diameter of
of K2S2O8 is 15 min (pH ) 5.5), the alginate assemblies the assemblies increases to 184 ( 24 nm and the shell
with the core-shell micelle-like structure are observed, as thickness of the assemblies decreases from 322 ( 37 nm
shown in Figure 4a. The diameter of the assemblies is 722 (pH ) 5.5) to 79 ( 8.5 nm thick. The swelling of the
( 80 nm, and the core size and shell thickness are found to assembly shell decreases markedly. Obviously, this can be
be 149 ( 37 and 322 ( 37 nm, respectively. Compared to attributed to a decrease of hydrophilic sodium alginate
the data measured in aqueous solution by DLS, the assembly moieties, and an increase of alginic acid moieties in the
size characterized by TEM is a little smaller due to the dry backbone chains with the decrease of pH value. Meanwhile,
state in TEM observation. At a higher magnification image the ú potential of assemblies decreases from -54.88 ( 4.46
shown in Figure 4b, the core and shell of the alginate to -31 ( 3.62 mV. Therefore, it is safe to conclude that the
assemblies is clearly seen. It is notable that such giant core- core and the shell are comprised of protonated and ionized
2194 Biomacromolecules, Vol. 6, No. 4, 2005 Cao et al.

Table 2. Size of Cross-Linked Micelles with Different

Cross-Linking Degreea
cross-linking degreeb 〈Dh〉 (nm) polydispersity index (PDI)
10% 513 ( 25 0.25
20% 413 ( 21 0.25
30% 359 ( 18 0.19
40% 326 ( 2 0.11
50% 448 ( 6 0.22
60% 560 ( 22 0.27
a The cross-linked samples are dialyzed in the water at pH 3.4 for 2

days to remove all of the impurities. b This refers to the molar ratio of amine
groups in the cross-linker to carboxyl groups of sodium alginate. It reflects
only the maximum possible degree of cross-linking.

alginate segments, respectively. Figure 4d shows the mor-

phology of the assemblies as K2S2O8 has decomposed for
400 min (pH ) 3.4). At this stage, about 60% of the carboxyl
groups in the sodium alginate chain are protonated since
K2S2O8 in the solution is totally decomposed, which can be
proved by the horizontal slope at the curve of pH variation
shown in Figure 3a. It can be seen that the assemblies with Figure 5. (a) TEM image of the cross-linked assemblies at pH )
the diameter of 180 ( 21 nm in the dry state become more 3.4; (b) TEM image of the cross-linked assemblies at pH ) 7.0.
compact in shape and the swollen shell is almost disappeared.
Figure 4e displays the SEM image of assemblies as seen in
Figure 4d at the hydrophilic surface of glass substrate. The
size of assemblies in SEM image is found to be 285 ( 48
nm, which is larger than that in the TEM image, suggesting
the shrinkage of assemblies at the hydrophilic and rough
surface of glass substrate is smaller than that at the
hydrophobic surface of carbon in grid during the drying
process of samples. After close examination, it is also found
that the center region of assemblies is somewhat concave
compared to the edge region of assemblies, indicating the
core of alginate assemblies is comparatively soft. However,
Figure 6. Mean hydrodynamic diameter of cross-linked assemblies
the assemblies prepared at this stage are not stable enough. as a function of pH.
They trend to further assemble and form larger super-
molecular structure after storage for several days in aqueous electrostatic effect between the reacting moieties.25 Thus, in
solution. our case, the cross-linking reaction may mainly take place
Cross-Linked Alginate Assemblies. To improve the at the assembly shell consisting of the negatively charged
stability of the alginate assemblies in aqueous solution, the sodium alginate segments. This is confirmed by the increase
alginate assemblies at minimum assembly size (pH ) 3.4) in ú potential after the cross-linking reaction. As the CMR
were cross-linked by a cross-linker, 2,2′-(ethylenedioxy)bis is below 40%, the cross-linking reaction happens in the intra-
(ethylamine), in the presence of coupling reagent, 1-(3- assemblies, whereas when CMR is above 40%, the cross-
dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDC). linking reaction may take place not only in the intra-
Table 2 displays the size of the assemblies measured by DLS assemblies but also inter-assemblies, resulting in the increase
after cross-linking. Since the cross-linker, 2,2′-(ethylene- in the average size of assemblies.
dioxy)bis (ethylamine), shows the basic property, the addition Figure 5a shows the TEM image of cross-linked alginate
of the cross-linker into alginate assemblies solution caused assemblies at pH 3.4, with the molar ratio of cross-linker to
some increase in assembly size but had not affected the cross- repeat units of alginate being 40%. It can be seen that the
linking reaction in the given system. From Table 2, it can structure of the cross-linked assemblies becomes considerable
be seen that after the cross-linking reaction the assembly size compact and the swollen shell is no longer present. In
decreases initially with the increase in the degree of cross- addition, the shape of cross-linked assemblies is not as round
linking and reaches a minimum value, 326 nm, at the as the non-cross-linked assemblies, suggesting that the cross-
calculated molar ratio of cross-linker to repeat units of linking reaction may not take place homogeneously in the
alginate (denoted as CMR) of 40%. Progressively increasing assemblies.
the amount of cross-linker results in an increase in the An interesting feature of the cross-linked assemblies is
assembly size. It is well-known that EDC is extensively used their response to environmental pH variation. Figure 6
as a coupling reagent in the condensation reaction between displays the size change of cross-linked alginate assemblies
an acid group and amine group and can react with both at the cross-linking degree of 40%. The size was measured
carboxylic acids and carboxylate groups.8,24 However, the by DLS after dialysis against the aqueous solution with
reaction rate with carboxylate groups is faster due to the different pH values. It can be seen that a sudden increase in
Capsules of Sodium Alginate Biomacromolecules, Vol. 6, No. 4, 2005 2195

Figure 7. Schematic representation of pH-induced formation of sodium alginate assemblies, and the transition from the compact structure to
hollow structure.

the assembly diameter between pH ) 4.5 and 8.0 occurs, protonation of carboxylate groups in sodium alginate chains
and the size of cross-linked assemblies expands almost causes the self-assembly of sodium alginate chains and forms
2-fold. In addition, the integration of cross-linked assemblies the assemblies with the core-shell structure. Subsequently,
is still maintained upon pH ) 10.1. This monotonic increase the cross-linking reaction fixes the shape of the assemblies,
in the size with the increase of pH value in the medium most which makes the assemblies yield a hollow capsule structure
likely arises from the swelling of assemblies and the when they are in the medium with relatively high pH value.
electrostatic repulsive inside assemblies. To further inves-
tigate this issue, TEM observation was carried out. Figure Conclusion
5b shows the TEM image of the cross-linked assemblies at
In this investigation, we used a kind of polyelectrolyte,
pH ) 7.0. It is found that the compact structure of cross-
sodium alginate, as a model biomaterial to investigate the
linked assemblies is transformed to hollow capsule structure
aggregation behaviors in aqueous solution after partial
in the medium of pH ) 7.0. A significant contrast between
protonation of carboxylate groups in the alginate molecules.
the center and the shell of the capsules indicates that a cavity
It is demonstrated that the alginate assemblies with core-
in the assembly center is formed, which may result from
shell structure can be generated by the smooth and partial
the increase in assembly size after dialysis in relatively high
protonation of carboxylate groups in sodium alginate chains
pH medium. On the other hand, for non-cross-linked samples,
using the protons released gradually from the decomposition
the size of assemblies also increases with the increase of
and reaction of K2S2O8 in water at 70 °C. The partial cross-
pH value in medium, as shown in Figure 3a and b. However,
linked alginate assemblies are pH sensitive and can change
at high pH (pH > 7), a DLS measurement indicated that
to hollow structure at the medium with relatively high pH
there was almost no assembly in the non-cross-linked system
value. This approach avoids use of block or grafted copoly-
and the disintegration of the assemblies occurred, which are
mers as the precursors or any other template to prepare
agreement with the result in Figure 1. This result indicates
assemblies and capsules, and provides a functional surface
that the cross-linking reaction of alginate assemblies is
for subsequent chemical reaction at the surface (e.g., for
effective. In addition, it is found that the cross-linked sample
binding biomolecules and for surface grafting). Such unique
does not return to the size of the original non-cross-linked
assemblies are also expected to be useful in biomedical fields.
sample at the same pH values by comparison of Figures 3
and 6. Except in the case of pH 3.4 where the diameters of Acknowledgment. This work has been supported by
cross-linked and non-cross-linked assemblies are almost Natural Science Foundation of China (No. 20374026).
equal (326 ( 2.3 nm for cross-linked sample and 322 ( 8.1
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