Bioprocess Biosyst Eng (2010) 33:457–463
DOI 10.1007/s00449-009-0349-2
ORIGINAL PAPER
Hydrophobic modification of sodium alginate and its application
in drug controlled release
Bolong Yao Æ Caihua Ni Æ Cheng Xiong Æ
Changping Zhu Æ Bo Huang
Received: 14 April 2009 / Accepted: 19 June 2009 / Published online: 4 July 2009
Ó Springer-Verlag 2009
Abstract Sodium alginate was hydrophobically modified
by coupling of polybutyl methacrylate onto the alginate.
The polybutyl methacrylate was previously prepared
through polymerization of butyl methacrylate in the pres-
ence of 2-amino-ethanethiol as a chain transfer agent. The
structure of the product was characterized by Fourier-
transformed infrared spectrometry, nuclear magnetic reso-
nance (1HNMR) and thermogravimetry. The result of
fluorescence analysis showed that the hydrophobicity of the
modified alginate was obviously increased. The modified
alginate conjugate was used for immobilization of bovine
serum albumin in the presence of calcium chloride. In
addition, the release behavior of the drug-loaded alginate in
deionized water and Tris–HCl buffer solution (pH 7.2) was
investigated. It was found that the modified sodium algi-
nate possessed prolonged release behavior compared to
unmodified sodium alginate, and it had potential applica-
tion in controlled release as a drug carrier.
Keywords Sodium alginate
Controlled release

Hydrophobic modification
Polybutyl methacrylate
B. Yao
C. Ni (&)
C. Xiong
School of Chemical and Material Engineering, Jiangnan
University, 214122 Wuxi, China
e-mail: nicaihua2000@163.com
B. Yao
C. Ni
C. Zhu
B. Huang
State Key Laboratory of Hydrology-Water Resources
and Hydraulic Engineering, College of Computer
and Information Engineering, Hohai University,
213200 Nanjing, China
Introduction
Alginate is a linear anionic polysaccharide consisted of two
kinds of hexuronic acid residues including 1, 4-b-D-man-
nuronic acid (M) and a-L-guluronic acid (G) residues which
are arranged in repeating GG, MM blocks or alternating
MG blocks. As a natural biopolymer, alginate has got
increasing biotechnological and biomedical applications in
view of its several advantages such as high biocompatibil-
ity, biodegradability, non-toxicity, non-immunogenicity,
chelating ability, and the possibility of chemical modifica-
tion [1]. In particular, the sodium alginate hydrogels
cross-linked by calcium ions are widely used for the
encapsulation of cells, proteins, oligonucleotides or DNA
and also as scaffolds for cartilage tissue engineering. In
general, biomolecules which do not interact ionically with
the alginate are rapidly released (within a few hours), and
the release profiles are often characterized by a more or less
pronounced burst effect. To reduce the high protein diffu-
sion, it has been proposed to coat the alginate beads, for
example, with polycationic water-soluble polymers such as
poly-L-lysine [2–4], chitosan [5–7], dextrin [8], amino-
poly(oxyethylene) [9] or proteins [10, 11]. Covalent
modification of alginates by hydrophobic materials is an
effective way for the increase of drug loading and the
controlled release. Attempts to incorporate organic com-
pounds into alginate beads to create a hydrophobic gel
matrix have been reported. These includes, for example,
incorporation of long chain alkyl groups [12, 13], esterifi-
cation of alginate by butanol [14], and preparation of algi-
nate derivatives with hydrophobic segments [15]. Grafting
polymerization is a well-established and powerful method
for the development of natural–synthetic polymer hybrid
materials. So far, the grafting of vinyl monomers onto
alginate has been carried out using catalysts such as ceric
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(IV) ions, persulfate, and hydrogen peroxide, etc. However,
those methods will cause ring opening of the alginate,
which results in some problems such as great structural
change of the alginate, low grafting yields, non-controllable
polymerization degree, and cost of catalysts.
In the present study, we have developed a new route
for hydrophobic modification of sodium alginate. Butyl
methacrylate has successfully been grafted onto sodium
alginate through coupling reaction of polybutyl methac-
rylate with alginate. The drug loading and controlled
release behavior of the modified alginate have been
investigated.
Experiments
Materials
Sodium alginate (SA, Mw: 2.1 9 105) and 2-aminoeth-
anethiol hydrochloride (AET, A.R.) were purchased
from Acros. 1-ethyl-[3-(dimethylamino)propyl]-3-ethylcarbo
diimide HCl (EDC) and N-hydroxysuccinimide (NHS,
A.R) were purchased from Shanghai Med Co., Led. Bovine
serum albumin (BSA), initiator (AIBN) and butyl meth-
acrylate (BMA) were A.R. grade and purchased from
National Chemical Groups. Butyl methacrylate (BMA) was
washed successively with an aqueous sodium hydroxide
and distilled water and was then dried over anhydrous
sodium sulfate and distilled under reduced pressure.
Preparation of amino-ended polybutyl methacrylate
(PBMA-NH2)
Monomer BMA, initiator AIBN and chain transfer agent
AET with a molar ratio of 100/1/30 were dissolved in
dimethylformamide (DMF). The reactant solution was
degassed with nitrogen for 30 min. The reactor was sealed
and immersed in a thermostated oil bath at 60 °C for 8-h
polymerization. The polymer produced was precipitated by
methanol and dried in a vacuum at room temperature. The
product was marked as PBMA-NH2.
Preparation of PBMA-grafted alginate (SA-g-PBMA)
Sodium alginate (0.3 g) and PBMA-NH2 (0.6 g) were
dissolved in mixed solvents that contained 30 mL phos-
phate buffer solution (pH = 6.0), DMF (25 mL) and
tetrahydrofuran (THF 5 mL). Then EDC and NHS were
added to the solution to catalyze the reaction between
carboxyl groups of the alginate and amino groups of the
PBMA-NH2. The molar ratio for alginate (with reference to
the carboxyl groups), EDC and NHS was 2:2:1. The mixed
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Bioprocess Biosyst Eng (2010) 33:457–463
Fig. 1 The scheme of preparation of SA-g-PBMA conjugate
solution was continuously stirred for 24 h at room tem-
perature. The polymer solution was dialyzed using dialysis
tubes with cutting off molecular weight of 14,000 for at
least 3 days using distillated water which was replaced
daily, precipitated with THF, extracted with THF using a
soxhlet extractor, and finally dried at room temperature
under a reduced pressure. The synthetic route was showed
in Fig. 1.
Characterization
1
H-NMR was performed on a nuclear magnetic resonance
spectrometer (VARIAN INOVA 400 MHz NMR SYS-
TEM, Varian company, America) at 25 °C. The sample
was dissolved in CDCl3 to a concentration of approxi-
mately 10 mg/ml. Infrared spectrum measurement was
carried out on a FT-IR Analyzer (FTIR, FTLA 2000-104,
America) at a 4 cm-1 resolution using KBr pellets. The
molecular weight was determined by gel permeation
chromatography (GPC, Agillent-1100, America); thermo-
gravimetry analyses (TGA) of SA, SA-g-PBMA and
PBMA-NH2 were carried out on an apparatus (TGA/
SDTA851e, Mettler Toledo, Switzerland) at a heating rate
of 25 °C/min in a nitrogen atmosphere. The morphology
was observed by scanning electron microscope (SEM,
Quanta-200, FEI, Europe).
Fluorescence measurement
Fluorescence measurement was carried out on a Spectro
fluoro photometer (RF5301PC, Japan) using pyrene as a
fluorescence probe. The fluorescence emission spectra
Bioprocess Biosyst Eng (2010) 33:457–463
were recorded in the range of 350–500 nm and the slit
width of 3 nm. A pyrene stocking solution of different
NaCl content was prepared by adding NaCl to a saturated
aqueous pyrene solution. The sample solutions were stored
for more than 24 h to ensure pyrene was completely
entrapped into the hydrophobic microdomains [16].
Preparation of microspheres of alginates containing
BSA
The modified alginate (0.15 g) was dissolved in 10 mL of
water to obtain a 1.5% (w/v) solution, BSA (0.05 g) was
dissolved in the solution, and then the mixed solution was
added dropwise into 50 mL of calcium chloride solution
(0.25 mol/L) through a 22-G needle. The microspheres
were allowed to harden in the calcium chloride solution for
5 min, afterwards they were washed thrice using distillated
water and were dried at room temperature. For comparison,
the sample of BSA loaded on unmodified alginate was
prepared with the same procedure described above.
In vitro release studies
The protein-loaded microspheres were placed in 50 mL of
an aqueous solution containing 0.02 w/v% sodium azide as
a bacteriostatic agent. The vials were equipped on a rotary
shaker at 25 °C with a speed of 100 rpm. At a certain time
interval, 5 mL of the solution was removed for determi-
nation of the BSA concentration on UV (TU-1901 Beijing,
China). The absorbance was monitored at 280 nm [17].
The same amount of fresh solution was added into the
Fig. 2 The 1HNMR spectrum
of PBMA-NH2
459
release system after each removal of the drug-released
solution.
Results and discussion
Synthesis and characterization of PBMA-NH2
The polymerization of butyl methacrylate is easily per-
formed according to free radical polymerization mecha-
nism. The chain transfer agent (AET) in the system has two
functions: (1) controlling the molecule weight of polybutyl
methacrylate through chain transfer action of mercapto
groups of AET; (2) introducing amino groups to the end of
polymer chains which can be used for coupling reaction
with alginate in the next step. Our study has confirmed the
polymer formation of the amino-ended polybutyl methac-
rylate (PBMA-NH2). The molecular weight of the PBMA-
NH2 determined by gel permeation chromatography (GPC)
is 4,980 for Mn and the molecular weight polydispersity
index (PDI) is 1.57. The structure of PBMA-NH2 is further
confirmed by 1H-NMR (Fig. 2). The 1H-NMR spectrum of
PBMA-NH2 indicates that the chemical shift of 2.5 ppm is
attributed to the protons of the methylene which join with
sulfhydryl group, and 2.89 and 2.98 ppm are signals of
methylene that belongs to 2-amino-ethanethiol. In the next
step, PBMA-NH2 will be grafted onto the alginate through
the reaction between amino groups in PBMA-NH2 with
carboxylic groups in SA in the presence of catalysts EDC
and NHS. The product is confirmed by the following
investigation.
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Fig. 3 Infrared spectra of a SA, b SA-g-PBMA, and pure c PBMA-
NH2
FTIR analysis
The FTIR spectra of SA, SA-g-PBMA and PBMA-NH2 are
shown in Fig. 3a, b, c, respectively. There is a wide peak at
3,447 cm-1 in Fig. 3a, b, indicating O–H stretching
vibrations in SA and SA-g-PBMA. In Fig. 3c 2,958 and
2,934 cm-1 are the characteristic peaks of methylene and
methyl groups of PBMA, and the sharp peak 1,734 cm-1
shows the existence of C=O groups in PBMA. However,
these peaks are getting weak in curve b because the related
content of PBMA decreases after being grafted onto SA. In
Fig. 3b the peak 1,629 cm-1 which does not appear in the
curve c is attributed to carboxylate groups (asymmetric
stretching) of SA. Comparing the three curves of Fig. 3, it
is concluded that PBMA-NH2 is successfully grafted onto
the alginate.
Fig. 4 Thermogravimetry analysis of (a) SA, (b) SA-g-PBMA, and
(c) PBMA-NH2
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Thermal analysis
The results of thermogravimetric analyses (TGA) for SA,
SA-g-PBMA and PBMA-NH2 are displayed in Fig. 4a, b,
c, respectively. As showed in Fig. 4a, SA looses weight
slowly from 50 to 230 °C, which may due to the loss of
combined water, and due to the lactonization or trans-
glucosidation of the SA. The SA loses weight rapidly at
230 °C and the weight loss rate is slow down at 271 °C
until the equilibrium at 350 °C. It is explained that SA is
decomposes in this temperature range. When the residue
reaches up to 38.3 w% the weight loss no longer chan-
ges, probably because sodium oxide and sodium car-
bonate have been produced. These compounds cannot be
further decomposed at this temperature. On the curve in
Fig. 4b (SA-g-PBMA), we have observed that the weight
loss behavior is similar to Fig. 4a before 350 °C. After
this temperature, the sample loses weight continuously
until the equilibrium at 510 °C. This is because the
sample SA-g-PBMA contains part of PBMA which
decomposes within this temperature range. For the
polymer PBMA-NH2, it loses weight quickly during the
temperature range of 280–453 °C, and no residue is left
because it is decomposed completely. Additionally, the
grafting yield of PBMA onto SA can be estimated
through calculation based on the TGA curves. In Fig. 4b,
the weight loss within 230–350 °C is attributed to the
decomposition of SA, and the apparent decomposition of
PBMA-NH2 takes place within 350–510 °C. The grafting
yield of SA-g-PBMA is about 20 w% according to
Fig. 4.
Images of solutions and gels of the SA-g-PBMA
To investigate the structural change before and after modi-
fication, digital photograph and scanning electron micro-
scope are employed for the observation of the samples,
respectively. Figure 5a, b are the digital photos of SA and
SA-g-PBMA aqueous solutions, respectively. Apparently
SA solution is almost transparent since SA is very hydro-
philic and is completely dissolved in water. However, when
a hydrophobic segment PBMA is grafted onto the SA, the
product exhibits amphiphilic behavior. Therefore, its aque-
ous solution becomes turbid. Figure 5c is a digital photo of
the microspheric image of the gel SA-g-PBMA cross-linked
by CaCl2, and Fig. 5d is scanning electron microscope of the
network structure inside SA-g-PBMA gel. It is observed in
Fig. 5d that the rough morphology appears inside the
hydrophobically modified SA-g-PBMA gel. This phenom-
enon revealed that the microphase separation occurs in the
gel. It is understood that when hydrophobic blocks of PBMA
are grafted onto hydrophilic alginate blocks, the SA-
g-PBMA demonstrates an amphiphilic structure. PBMA is
Bioprocess Biosyst Eng (2010) 33:457–463 461

Fig. 5 Pictures of: a digital

photo of SA aqueous solution
(1.5 w%); b digital photo of
SA-g-PBMA aqueous solution
(1.5 w%); c digital photo of
SA-g-PBMA microspheres;
d scanning electron microscope
of inside structure of
SA-g-PMMA

insoluble in the aqueous environment, and consequently, it

shows island structure inside the hydrogel.

Hydrophobic change of SA-g-PBMA

Pyrene is one of the effective probes to detect the for-

mation of hydrophobic microdomains by intra- or inter-
molecular associations of amphiphilic copolymers. The
intensity of the first band at 372 nm (I1) increases with
increase of the polarity of solvent, while the intensity of
the third band at 383 nm (I3) is stable in various condi-
tions. Accordingly, I1/I3 value is the indication of micro-
environmental polarity surrounding pyrene molecules.
The decrease in I1/I3 value implies the formation of
hydrophobic microdomains where pyrene is involved. The
fluorescence spectrum of pyrene is determined and the Fig. 6 Pyrene fluorescence of intensity ratio (I1/I3) dependence on
comparison of I1/I3 value for SA and SA-g-PBMA is concentrations of: a SA in 0.15 mol/L NaCl aqueous solution; b SA-
showed in Fig. 6. It is seen that the I1/I3 value (Fig. 6a) g-PBMA in 0.10 mol/L of NaCl aqueous solution; c SA-g-PBMA in
0.15 mol/L of NaCl aqueous solution
remains constant in the whole concentration range for SA
solution, indicating the aqueous nature of the environment
probed. On the contrary, for the hydrophobically modified more in the higher ionic strength solution (0.15 mol/L
SA-g-PBMA, the I1/I3 value decreases significantly with NaCl) than in lower ionic strength solution (0.10 mol/L
increase of the polymer concentration. Obviously the NaCl) (Fig. 6b, c). It is explained that the negative
hydrophobic polymer chain PBMA in the SA-g-PBMA charges of SA are shielded by the oppositely charged ions
causes the polarity decrease of the environment around of the salt solution, which results in the decrease of
pyrene [18]. It is noticed that the ionic strength has also polarity and the increase of hydrophobicity of the poly-
some influence on I1/I3 value. For a SA-g-PBMA solution mer, therefore the I1/I3 value decreases. The higher the
with a certain concentration, the I1/I3 value decreases ionic strength, the more decrease the I1/I3 value.

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Fig. 7 Cumulative BSA release from hydrogels of: (a) SA; and
(b) SA-g-PBMA in distillated water
Controlled release of BSA
The hydrophobically modified alginate is used as a matrix
for loading of BSA, and the controlled release behavior is
investigated. Figure 7 gives the cumulative release results
of BSA from SA-g-PBMA and SA in deionized water. It is
seen from Fig. 7a that the release of BSA from SA expe-
riences a rapid process, the cumulative release reaches up
to 32.3 and 67%, respectively, after 1- and 4-day exposure
to the medium. However, the modified alginate displays
prolonged release behavior, and its cumulative release is
only 22.7 and 32.1%, respectively within the same period
of time. Even after 20 days only about 58% of the BSA is
released from SA-g-PBMA, but the BSA will be com-
pletely released from SA after 20 days.
The release behavior of BSA from SA and SA-g-PBMA
in buffer solution (0.05 mol/L Tris–HCl, ionic strength
0.042) is also investigated, respectively (Fig. 8). We have
also observed the prolonged release behavior from SA-
g-PBMA compared to from SA. For example, at the first
14 h, about 65% of BSA is released from SA, but only
about 45% of BSA is released from SA-g-PBMA. As
explained above, SA-g-PBMA is more hydrophobic than
SA, and the association interaction between SA-g-PBMA
and BSA is stronger since BSA is hydrophobic as well.
This will cause slow release of BSA from the matrix. It is
concluded from Figs. 7 and 8 that the release rate of BSA
from alginate matrix can be controlled through a hydro-
phobic modification to the alginate.
Comparing Figs. 7 and 8, it is found that BSA is
released faster in the Tris–HCl buffer solution than in
deionized water. This is because the release rate is signif-
icantly affected by a diffusion process. For a certain gel,
the average pore size of the gel is bigger in the buffer
solution (pH [ 7.2) than in pure water (pH = 6.8). In an
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Bioprocess Biosyst Eng (2010) 33:457–463
Fig. 8 Cumulative BSA release from hydrogels of: (a) SA; and
(b) SA-g-PBMA in Tris–HCl buffer solution (pH 7.2)
aqueous solution with a higher pH value the carboxyl
groups of SA are ionized, the repulsion among the carboxyl
groups increases which results in increase of the volume
and pore size of the hydrogel. Therefore, it is easier for the
protein to diffuse out from the gel in the buffer solution
than in deionized water, consequently the release rate is
facilitated in the buffer solution.
Conclusion
Alginate can be hydrophobically modified by introduction
of polybutyl methacrylate prepared previously. The modi-
fied alginate demonstrates improved hydrophobicity com-
pared to the original alginate. The product shows prolonged
drug release behavior when it is used as a matrix for BSA
released in deionized water and buffer solution.
Acknowledgment We are grateful for the financial support of the
State Key Laboratory of Hydrology-Water Resources and Hydraulic
Engineering (No: 2007490611).
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