Novel thermo- and pH-responsive hydroxypropyl cellulose- and poly (l-glutamic acid)-based microgels for oral insulin controlled release

Article abstract of DOI:10.1016/j.carbpol.2012.03.095

Novel smart microgel particles made of poly (l-glutamic acid-2-hydroxylethyl methacrylate) (PGH) and hydroxypropyl cellulose-acrylic acid (HPC-AA) have been successfully prepared via emulsion polymerization. The dynamic light scattering measurement reveals that the average hydrodynamic radius 〈R_h〉 and hydrodynamic radius distributions f (R _h) of the microgel particles depend on the temperature and pH value thus the microgel particles exhibit both pH- and temperature-sensitivity. In vitro release study shows that the amount of insulin released from microgels in the gastric juice (at pH 1.2) is significantly less than that in the intestinal fluid (at pH 6.8). These results indicate that the resultant microgels are of potential for use in intelligent oral drug delivery systems.

Full text of DOI:10.1016/j.carbpol.2012.03.095

Carbohydrate Polymers 89 (2012) 1207–1214
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Novel thermo- and pH-responsive hydroxypropyl cellulose- and poly (l-glutamic
acid)-based microgels for oral insulin controlled release
Yunyan Baia,1, Zhe Zhanga,1, Aiping Zhanga, Li Chena,∗, Chaoliang Heb, Xiuli Zhuangb, Xuesi Chenb,∗
a Department of Chemistry, Northeast Normal University, Changchun 130024, PR China
b Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Changchun 130022, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel smart microgel particles made of poly (l-glutamic acid-2-hydroxylethyl methacrylate) (PGH) and
hydroxypropyl cellulose-acrylic acid (HPC-AA) have been successfully prepared via emulsion polymer-
ization. The dynamic light scattering measurement reveals that the average hydrodynamic radius ꢀRhꢁ
and hydrodynamic radius distributions f (Rh) of the microgel particles depend on the temperature and
pH value thus the microgel particles exhibit both pH- and temperature-sensitivity. In vitro release study
shows that the amount of insulin released from microgels in the gastric juice (at pH 1.2) is significantly
less than that in the intestinal fluid (at pH 6.8). These results indicate that the resultant microgels are of
potential for use in intelligent oral drug delivery systems.
Received 9 November 2011
Received in revised form 10 February 2012
Accepted 13 March 2012
Available online 6 April 2012
Keywords:
Thermo-responsive
pH-responsive
Microgel
© 2012 Elsevier Ltd. All rights reserved.
Insulin
Controlled release
1. Introduction
and biocompatible natural polysaccharides as suitable candidates
including starch, dextrans, chitosan.
The design and preparation of “smart” synthetic materials which
are able to respond to external or internal stimuli such as heat,
light, pH, magnetic and electric fields and chemical substances have
attracted a great deal of interest for their potential applications
in tissue engineering, biomedical implants, drug delivery, and bio-
nanotechnology (Li, Luo, & Lam, 2009; Ma, Fan, Liang, & Xu, 2010;
Ma, Liu et al., 2010; Pasparakis & Vamvakaki, 2011; Zhang et al.,
2010). Microgels, as intramolecular cross-linked polymeric parti-
cles of colloidal size, can swell and deswell in response to stimuli
more rapidly than bulk gels, are the most commonly studied types,
especially in the field of controlled release (Heyes & Branka, 2009;
Hoare & Pelton, 2008; Liu, Luo, Guan, & Zhang, 2010; Nur, Pinkrah,
Mitchell, Benée, & Snowden, 2010). Poly (N-isopropylacrylamide)
(PNIPAM), as the first synthesized thermal-responsive polymers
for bio-related applications, exhibits a reversible volume phase
transition around the lower critical solution temperature (LCST,
approximately 32 ◦C) (Chen et al., 2010; Liu, Yu, Tian, Sun, & Fan,
2009). However, the poor biocompatibility of PNIPAM may be a lim-
itation to be used in vivo, especially as drug vehicles (Pelton, 2000).
Therefore, considerable efforts have been made to explore nontoxic
Hydroxypropylcellulose (HPC), as another class of thermal-
responsive polymer, approved by FDA is a derivative of natural
polysaccharide cellulose and widely used in food, drug and
cosmetics areas (Heitfeld, Guo, Yang, & Schaefer, 2008). This
amphiphilic polymer, potentially alternative to PNIPAM, exhibits
a LCST around physiological temperature (about 42 ◦C) due to the
hydrophilic/hydrophobic balance (Lu, Hu, & Gao, 2000; Xia, Tang,
Lu, & Hu, 2003). This kind of phase transition behavior is similar to
that of PNIPAM, which makes it feasible to use HPC as a biodegrad-
able and biocompatible substitute of PNIPAM. Ma et al. studied the
self-assembly properties of HPC-graft-(2-(dimethylamino)ethyl
methacrylate) DMAEMA copolymers in aqueous solutions and
showed that the LCST onset could be precisely tuned (Ma, Fan, et al.,
2010; Ma, Liu et al., 2010).
Very recently, some multi-responsive micro/nanoparticles with
controlled architecture have been designed for biomedical imag-
ing, drug delivery and intracellular biosensing (George & Maria,
2011). Combining thermo-responsive polymer with pH-sensitive
moiety containing pendent carboxylic groups such as acrylic acid
makes it possible to successfully prepare dual stimuli-responsive
microgels which can respond to not only temperature but also pH
stimuli due to changing the ionization–deionization equilibrium
by adjusting the structure and conformation (Dou, Yang, Tao, Li,
& Sun, 2010; Wu et al., 2010). To advantage for the difference
between gastric juice and intestinal juice in vivo, these double-
responsive micro/nanoparticles will be prominent candidates for
∗
Corresponding authors.
E-mail addresses: chenl686@nenu.edu.cn (L. Chen), xschen@ciac.jl.cn (X. Chen).
1
These authors contributed equally to this work.
0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2012.03.095

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Y. Bai et al. / Carbohydrate Polymers 89 (2012) 1207–1214
Scheme 1. Synthetic route of PGA.
protein deliveries, especially on insulin release systems for the
diabetes treatment (Leobandung, Ichikawa, Fukumori, & Peppas,
2002; Schmaljohann, 2006). These “smart” delivery systems may
protect the drugs against a harmful gastric environment (chemical
degradation), encapsulating insulin in particulate carriers can pre-
vent enzymatic degradation, while controlling the drug release and
increasing their absorption in the small intestine.
However, the above mentioned pH-sensitive polymers are not
biodegradable and not suitable for application in vivo. There-
fore, using biodegradable pH-sensitive materials to develop oral
insulin-delivery systems has received considerable attention. Poly
(l-glutamic acid) (PGA) is one of the most widely used pH-
responsive polypeptides in the biomedical area due to their
biocompatibility and biodegradability (Murakami & Aoki, 2006).
The ␥-PGA-based pH-responsive nanoparticles have been success-
fully prepared and used as the delivery of insulin (Sonaje et al.,
2010).
Accordingly, in this article a series of novel pH- and thermal-
responsive microgels composed of HPC-AA and PGH were
synthesized by the emulsion polymerization. The influences of
pH and temperature on the swelling behaviors of the microgels
were investigated. The microgels exhibited special pH-dependent
temperature-sensitivity because of the increase of LCST with pH
value. Based on this property, the intelligent microgels have inter-
esting potential application in insulin controlled release via the
reversible swelling/shrinking transition.
phosphate was dissolved into 500 ml of deionized water, and then
the pH value was adjusted to 6.8 and the solution was diluted to
1000 ml to get artificial intestinal liquid.
Oil phase of emulsion polymerization was prepared as follows.
1.35 g Span-80 and 0.15 g Tween-80 (Span-80/Tween-80 = 9, w/w)
were dispersed into 240 ml of n-hexane and 60 ml of paraffin (n-
hexane/paraffin = 4, v/v) and then the mixture was magnetically
stirred uniformly for later.
2.2. Synthesis of Poly (l-glutamic acid) (PGA)
Firstly, ␥-benzyl-l-glutamate N-carboxyanhydride (BLG-NCA)
was prepared as follows. 10 g of ␥-benzyl-l-glutamate and 6 g of
triphosgene were suspended into 100 ml of dry THF under a flow
of nitrogen. The mixture was stirred at 60 ◦C with nitrogen bubbling
until it turned into a transparent solution. The solution was poured
into 1000 ml of n-hexane to yield white precipitate and the crude
product was recrystallized from the ethyl acetate/n-hexane mixed
solution for three times. The yield was 70%.
Secondly, poly (␥-benzyl-l-glutamate) (PBLG) was synthesized
through the ring-opening polymerization (ROP) of BLG-NCA ini-
tiated by n-hexylamine. A typical experimental procedure was
as follows: BLG-NCA (5 g) was dissolved in 150 ml of anhydrous
dioxane, followed by adding 4.75 ml of 0.1 M n-hexylamine in diox-
ane solution (with a monomer/initiator ratio = 40, mol/mol). After
vigorous stirring for 3 days at room temperature, the reaction
mixture was added to a 10-fold excess of diethyl ether. The precip-
itated polypeptide was washed twice with diethyl ether and dried
overnight under vacuum, yielding PBLG 4.5 g (90%).
Lastly, poly (l-glutamic acid) was prepared by debenzylation
of PBLG using HBr as shown in Scheme 1. Briefly, PBLG (4.5 g)
was dissolved in 45 ml of dichloroacetic acid, followed by addi-
tion of excessive 33 wt% HBr solution in acetic acid (HBr/benzyl
groups = 8, mol/mol). After the solution was stirred at 30 ◦C for 1 h,
it was precipitated into excessive diethyl ether, isolated by filtra-
tion, and repeatedly washed using acetone. Then the product was
dried under vacuum at room temperature for 24 h. The yield was
77%.
2. Experimental
2.1. Materials
Hydroxypropyl cellulose (HPC) (average Mw = 1 × 105) was pur-
chased from Acros Organics and used directly. Acrylic acid (AA) was
obtained from Shanghai Chemical Reagent Co. (China) and distilled
under reduced pressure to remove inhibitors before use. 1-Ethyl-3-
(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl,
Shanghai GL Biochem, China) and 4-dimethylaminopyridine
(DMAP 98% Fluka) were used as received. ␥-Benzyl-l-glutamate
was purchased from Sigma. n-Hexylamine (Aldrich, 99%) was used
as received. Hydroxyethyl methacrylate (HEMA 96% Acros) was dis-
tilled under reduced pressure before use. Ammonium persulfate
(APS) and N,N,Nꢂ,Nꢂ-tetramethylethylenediamine (TEMED) were
obtained from Shanghai Chemical Reagent Co. (China) and used
directly. Trichloromethane and dimethyl sulfoxide (DMSO) were
dried over CaH2, followed by distillation under reduced pressure
prior to use. Distilled water was used throughout.
2.3. Synthesis of PGA-HEMA (PGH)
The synthetic route of PGH was shown in Scheme 2(a). 3.0 g PGA
and 0.3 ml of HEMA (the molar ratio of HEMA and COOH groups
in PGA is 1:10) were dissolved in 90 ml of DMSO. After dissolution,
EDC·HCl (0.9 g) and DMAP (0.06 g) were added as the coupling agent
and catalyst, respectively. The mixture was mixed thoroughly and
then quickly poured into a glass mold. The reaction was allowed to
proceed at room temperature for 72 h. The product was collected
by precipitating in 10-fold diethyl ether.
Artificial gastric juice and artificial intestinal liquid were pre-
pared as follows. 7 ml of hydrochloric acid (37 wt%) and 2.0 g
sodium chloride were dissolved into 1000 ml of deionized water
to prepare artificial gastric juice. 6.8 g potassium dihydrogen

Y. Bai et al. / Carbohydrate Polymers 89 (2012) 1207–1214
1209
Scheme 2. Synthetic route of PGH (a) and HPC-AA (b).
2.4. Synthesis of HPC-AA
2.5. Microgels synthesis
HPC-AA was prepared by esterification reaction of AA in HPC
trichloromethane solution in the presence of DMAP as a cata-
lyst and EDC·HCl as the coupling agent, respectively, as depicted
in Scheme 2(b). Firstly, 5 g HPC was dissolved into 100 ml of
trichloromethane by gentle stirring. Secondly, 0.46 ml of AA (the
molar ratio of AA and hydroxyl groups in HPC was 1:10) was
added into the mixture. Finally, 0.08 g DMAP and 1.3 g EDC·HCl
were subsequently added into the mixture and the reaction was
allowed to proceed for 48 h at room temperature. The crude
product was precipitated in diethyl ether (1000 ml). Then the pre-
cipitate was filtered and dried, followed by dialysis for 7 days
against very frequently changed water. HPC-AA was obtained in
75% yield.
The microgel dispersions were prepared by free-radical emul-
sion polymerization of HPC-AA and PGH, as shown in Scheme 3.
Firstly, HPC-AA and PGH (total amount of 50 mg) at different weight
ratios, e.g. PGH wt% = 30%, 50% and 70%, were dissolved into 5 ml
of phosphate buffer (pH = 7.4, 0.1 M), after dissolution, 5 mg APS
was dissolved into the mixture solution. Secondly, 50 ml of pre-
prepared oil phase was emulsified for 10 min at 4200 rpm on a
homogenizer under ice water bath and then 5 ml of water phase
was added dropwise. After continuous emulsification for another
5 min to form w/o emulsion, 5 ␮l of TEMED as a catalyst was added
to initiate the radical cross-linking reaction for 12 h under room
temperature. Lastly, the resultant was collected by centrifugation,
washed by ethanol, diethyl ether and water and then freeze-dried.
Scheme 3. Synthetic route of microgel.

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Y. Bai et al. / Carbohydrate Polymers 89 (2012) 1207–1214
2.6. Characterization
gastric juice or the artificial intestinal liquid and the microgel solu-
tion was poured into a dialysis bag (cut-off 7000) placed in a
beaker containing 10 ml of the artificial gastric juice and the artifi-
cial intestinal liquid as release medium for 10.5 h, respectively. The
beaker was shaken at 100 rpm at 37 ◦C. At predetermined intervals
1 ml of medium was taken out and replaced with 1 ml of the fresh
solution to keep constant volume. The concentration of the insulin
in the medium was determined by the UV–Vis spectrophotometer
at 280 nm. All the release experiments were carried out in triplicate,
and the averaged results were obtained.
The FT-IR spectra were recorded with a Bruker Vertex 70 Fourier
transform infrared spectrometer using KBr method. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded by a Bruker
300 MHz spectrometer with CDCl3 and D2O as solvent.
2.7. Dynamic light scattering (DLS) measurements
The hydrodynamic radius distributions of the microgels in aque-
ous solution were monitored using DLS performed on a Wyatt QELS
instrument with a vertically polarized He–Ne laser (DAWN EOS,
Wyatt Technology), and 90◦ collecting optics. All samples were
prepared in aqueous solution at a concentration of 0.1 mg/ml and
the measurements were carried out at 25, 35 and 60 ◦C, respec-
tively. Each sample was kept in the thermostat of the apparatus at
various temperatures for 30 min to reach the equilibrium prior to
measurements.
3. Results and discussion
3.1. Preparation of polymers and microgels
PGA was synthesized by ring-opening polymerization of BLG-
NCA using n-hexylamine as the initiator followed by debenzylation
in dichloroacetic acid using 33 wt% HBr of acetic acid solution at
30 ◦C. The degree of polymerization (DP) is 36 calculated from
the intensities of 1H NMR signals at 0.68 ppm of terminal CH3
and 4.14 ppm methine proton (H) ( NHCHCO ) of PGA. And then
PGH was prepared by esterification between PGA and HEMA in
the presence of EDC·HCl and DMAP. The absorption peaks appear-
ing at 5.46 ppm and 5.15 ppm (2H, CH2 CCH3 ) (Fig. 1A) verifies
the successful coupling of HEMA to PGA. The coupling ratio of
HEMA is calculated from the integration ratio between the methene
proton (2H) (CH2 CCH3 ) of HEMA and the methine proton (H)
2.8. Scanning electron microscopy (SEM) measurements
The morphologies of the microgels were investigated by envi-
ronmental scanning electron microscopy (ESEM) on an XL 30 ESEM
FEG Scanning Electron Microscope (Micrion FEI PHILIPS). A drop of
the microgel solution (0.1 mg/ml) was deposited onto a silicon chip
and air-dried. A layer of gold was sputter-coated on the specimen
surface before ESEM measurement.
(
NHCHCO ) of PGA appearing at 5.46 ppm and 4.14 ppm (shown
2.9. Phase transition measurements
in Fig. 1A), respectively, by the following equation:
Ia
Phase transition measurements were performed by a UV-Vis
spectrometer (Shimadzu UV-2401PC) equipped with a temper-
ature controller (Shimadzu S-1700). The concentration of the
microgel solution used for the determination of phase transition
was 0.1 mg/ml. We selected 550 nm as the analyzing wavelength.
The heating rate was 0.5 ◦C/min over the temperature range of
25–60 ◦C. The LCST was determined as the temperature at which
the optical transmittance decreased to 90% of its initial value.
HEMA coupling ratio (mol%) =
× 100
ꢂ
(1)
Ih,h
ꢂ
where Ia and Ih,h are the integral values of the peaks at 5.46 ppm
and 4.14 ppm, respectively. The HEMA coupling ratio of PGH was
8%.
HPC-AA was synthesized by esterification reaction of HPC and
AA in the presence of EDC·HCl and DMAP as the coupling agent
and catalyst, respectively, as depicted in Scheme 2(b). The hydroxyl
groups of HPC reacted with AA to form HPC-AA with active double
bond. 1H NMR measurement was performed to determine whether
the double bond group was introduced into HPC. As presented in
Fig. 1B, HPC-AA exhibits new proton peaks at 6.44 ppm, 5.83 ppm
(2H, CH2 CH ), and 6.10 ppm (1H, CH2 CH ) compared with pure
HPC, which are attributed to the AA moieties introduced into HPC.
From the 1H NMR spectrum of HPC-AA, it is clear that HPC have
been successfully modified with double bond. The coupling ratio of
AA on HPC is calculated from the integral areas of the CH peak at
6.10 ppm from AA, the proton peaks of c, cꢂ over 3.1–4.4 ppm and
the CH3 peaks at 1.2 or 1.4 ppm from HPC by the following equation:
2.10. Insulin load and release
The hydrophilic insulin was loaded into the microgels by a
swelling-diffusion method. Insulin, for the treatment of diabetes,
has poor solubility at neutral pH values while under acidic con-
ditions it is quite soluble (Nolan, Serpe, & Lyon, 2004). According
to this property, the insulin-loaded microgels were prepared as
follows. Firstly, 20 mg insulin was dissolved into 200 ␮l of 0.1 N
HCl to prepare stock solution, and then the solution was diluted
with 99.6 ml of phosphate buffer solution (pH = 7.4) and normalized
with 200 ␮l of 0.1 M NaOH. The final pH value of the solution with
a concentration of 0.2 mg/ml was 7.4. Secondly, 4 mg lyophilized
microgel was immersed into 10 ml of the insulin solution at room
temperature for 24 h to load the insulin into the microgels. Lastly,
after reaching swelling equilibrium the resulting solution was
transferred into centrifuge tubes and centrifuged at 12,000 rpm
at 25 ◦C for 10 min to get rid of unloaded insulin. After collecting
the supernatant in another container, the precipitate was rinsed
with 10 ml of the artificial gastric juice (pH = 1.2) or the artificial
intestinal liquid (pH = 6.8) for 10 min to move the drug adsorbed
on the surface of the microgels. The unloaded insulin solution and
the artificial gastric juice/artificial intestinal liquid used to rinse
the insulin-loaded microgels were collected together. The concen-
tration of insulin was monitored by a UV–Vis spectrophotometer
(Shimadzu UV-2401PC) at a wavelength of 280 nm.
ꢀ
ꢁ
Ib
[Ic,c − (Ia + Ia )]
7
3
AA coupling ratio (mol%) =
∗
∗ 100
(2)
ꢂ
ꢂ
ꢂ
ꢂ
where Ib, Ic,c , Ia and Ia are the integral areas of the peaks at
6.10 ppm, 3.1–4.4 ppm, 1.2 ppm and 1.4 ppm, respectively. The Ic,c
corresponds to all CHs and CH2s on the glucose unit (5CH + 1CH2)
and on the hydroxypropyl substitutes (1CH + 1CH2). Although the
degree of hydroxypropyl substitution is unknown, they can be sub-
tracted from Ic,c because each hydroxypropyl substitute consists of
one CH, one CH2 and one CH3, and the latter gives signals at 1.2 or
1.4 ppm (Ia and Ia , in Fig. 1B). Therefore, Ic,c − (Ia + Ia ) corresponds
to the seven protons on the glucose unit (5CH + 1CH2) and Eq. (2)
stands for the AA coupling ratio on HPC. From it the AA coupling
ratio of HPC is calculated to be 9 mol%.
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
To study the release behavior of insulin from the microgels,
insulin-loaded microgels were dissolved into 1 ml of the artificial
The microgels were prepared by free radical cross-linking
between PGH and HPC-AA using APS as the initiator and TEMED as

Y. Bai et al. / Carbohydrate Polymers 89 (2012) 1207–1214
1211
Fig. 2. FT-IR spectra of HPC, HPC-AA, PGA, PGH and MG 5/5.
where R is the weight percentage of nitrogen in the microgels
determined by elemental analysis and 129*90% + 241*10% = 140 is
the averaged molecular weight of the PGH units, assuming 10 wt%
of HEMA coupling. As shown in Table 1, the PGH contents in the
microgels increase in the order of the feeding ratios.
3.2. pH-triggered swelling of the microgel particles
Fig. 3 illustrates the pH-responsive swelling of the particles in
solution as a function of pH at a constant temperature of 25 ◦C. As
expected, the hydrodynamic radii of the microgel particles increase
with an increase in pH value. This is ascribed to the transformation
of PGA from the hydrophobic protonated form to the hydrophilic
deprotonated form near its pKa. When the pH value is below the
pKa of PGA, the size of microgels remains nearly constant due to
the strong hydrogen bonding between the protonated PGA seg-
ments and HPC segments. When the pH value is above the pKa,
the COOH groups of PGA chains gradually dissociate, which can-
not only weaken the hydrogen bonding between the HPC and PGA
chains, but also induce the Coulombic repulsion among the ion-
ized COO− groups, causing the gradual increase in the size of the
microgels.
3.3. Effects of temperature and pH on the microgels
Fig. 1. 1H NMR spectra of PGH (A) in D2O and HPC-AA (B) in CDCl3.
To investigate the hydrodynamic radius and their distribution
of the obtained microgels, 0.1 mg/ml of the microgel solution at
an accelerator according to the procedures described in Scheme 3.
A series of HPC-AA and PGH with various weight ratios ranging
from 30% to 70% were used to prepare the microgels, which were
marked as MG3/7, MG5/5, and MG7/3, respectively.
The FT-IR spectra of HPC, HPC-AA, PGA, PGH and MG5/5 are
shown in Fig. 2. Comparing the IR spectrum of HPC-AA with that of
HPC, it can be seen clearly that there is a new absorption appearing
at 1720 cm−1 (ꢀCO), which can be assigned to the carbonyl group
to confirm the formation of the acryloyl groups. The character-
istic absorption of the amide group at 1656 cm−1 (amide I) and
1547 cm−1 (amide II) from the PGH and the hydroxyl from HPC at
about 3459 cm−1 (ꢀOH) of the microgels are also observed in the
FT-IR spectrum of MG5/5. These results clearly confirm that the
microgels are prepared successfully.
The contents of PGH (in weight percent) in the microgels are
estimated from the elemental analysis results according to the fol-
lowing equation:
R
R
Fig. 3. Plot of hydrodynamic radius of the microgels as a function of pH value at
PGH wt% =
∗ (129 ∗ 90% + 241 ∗ 10%) =
∗ 140
(3)
25 ◦C.
14
14

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Y. Bai et al. / Carbohydrate Polymers 89 (2012) 1207–1214
Table 1
Feed compositions of microgels.
Samples
Feed compositions
Measured PGH
contenta (wt%)
HPC-AA (mg)
PGH (mg)
APS (mg)
TEMED (␮l)
PB (ml)
Oil (ml)
PGH (wt%)
MG3/7
MG5/5
MG7/3
15
25
35
35
25
15
5
5
5
5
5
5
5
5
5
50
50
50
70
50
30
73.9
50.5
29.8
a
Estimated from the elemental analysis results.
pH 4.92 and 7.38 was tested by DLS, respectively. The measure-
ment was carried out at 25, 35 and 60 ◦C, respectively. The effects
of temperature and pH value on particle size are shown in Fig. 4.
At a given pH value, no matter pH 4.92 or pH 7.38, the maximum
particle size decreases with increasing temperature, obviously due
to the temperature-dependence of the HPC segments. At a given
temperature, the maximum particle size is always smaller at pH
4.92 than at 7.38 due to the pH-dependence of the ionization of
PGA COOHs. As far as particle size distribution is concerned, the
microgels exhibit a relatively narrow size distribution at a lower
pH or at a higher temperature. Under these two conditions, smaller
maximum or average particle size is observed. All results above
indicate that the microgels have an excellent pH- and tempera-
ture sensitivity and can potentially be used to control the release
of drug by changing the temperature and the pH values. SEM was
also used to observe the morphology of the microgels. As shown in
the inset of Fig. 4, the microgel exhibits well-defined and spherical
morphology, consistent with the DLS measurement.
pH-dependence. Fig. 5B illustrates the phase transition behavior
of MG5/5 at pH 4.92, 5.29, and 6.81, respectively. The LCST is 42.8,
43.3 and 45.6 ◦C, respectively. This is because at a higher pH, PGA
is more hydrophilic.
3.5. Insulin load and in vitro release
The above results show that the microgels system composed
of crosslinked HPC-PGH exhibits both pH- and thermo-sensitivity,
and thus, it is expected to protect a hydrophilic drug against the
acidic environment of stomach and release it under the intestine
condition.
Insulin was selected as a model drug to demonstrate the
pH-regulated drug release. The insulin release behavior from
the microgels was investigated in vitro in artificial gastric juice
(pH = 1.2) and artificial intestinal liquid (pH = 6.8) at 37 ◦C, respec-
tively. As shown in Fig. 6A, the release profiles of insulin from
the microgels in artificial intestinal liquid (pH = 6.8) includes three
stages. In the initial stage there is an obvious burst release (ca.
49–54%) in about 25 min. This burst release may be attributed to
the release of insulin molecules located at the microgel surface.
When the dialysis tube is placed into the artificial intestinal liquid,
these surface drugs can be dissolved immediately, which leads to
the burst release. In the second stage (sustained release, ca. 8–10%)
in about 425 min the microgels serve as diffusion barriers and the
drug is mainly released by the swelling-controlled diffusion mech-
anism, so the insulin molecules come out at a relatively slow speed.
In the last stage little insulin is released and there seems to be an
equilibrium between the microgels and the release medium. This
is due to the reduced drug concentration difference between the
microgels and the surrounding medium on one hand, and due to
the interaction between the remaining insulin and the microgels
network on the other hand, which prevents the release of the drugs.
It is also observed in Fig. 6A that the release rate is PGH-content
dependent, i.e., the insulin is released at a rate in the order of MG3/7
>MG5/5 > MG7/3. This is because at pH 6.8, PGA is highly ionized
3.4. Phase transition behavior of microgels
The phase transition behavior of the microgels was character-
ized by monitoring the turbidity of the microgels as a function of
temperature (Zhao et al., 2011). Fig. 5A shows transmittance per-
centage versus temperature of the microgel solutions at pH 6.81.
It is clear that the LCST of the microgels increases with the weight
percentage of PGH. The hydrogen bonding between the HPC chains
and water molecules is a decisive factor to maintain the microgels
in the solution state below LCST. The hydrogen bonds are destroyed
above LCST and the hydrophobic interaction of the polymer chains
becomes predominant, which leads to the collapse and phase sep-
aration. Because PGH itself is hydrophilic, its incorporation into
the microgels would move the transition temperature to a higher
temperature. Moreover, the transition temperature range becomes
broader with the increase of PGH because of its hydrophilic nature.
Because the hydrophilicity of PGA can be modulated by pH
variation, the phase transition behavior is expected to show
Fig. 4. Hydrodynamic radius distributions for MG5/5 in aqueous solution of pH 7.38 and pH 4.92 at 25, 35 and 60 ◦C, respectively. SEM image of MG5/5 is in the inset.

Y. Bai et al. / Carbohydrate Polymers 89 (2012) 1207–1214
1213
Fig. 5. Effect of temperature on the phase transition behavior of the microgels at pH 6.81 (A) and MG5/5 at different pH value (B).
Fig. 6. Cumulative release of insulin from the microgels at pH 6.8 (A) and pH 1.2 (B) as a function of time at 37 ◦C.
and the microgels are in the swollen state. The more the PGH con-
tent, the more swollen the microgels and the fast the insulin release.
Fig. 6B shows the release profiles of insulin from the microgels in
artificial gastric juice (pH = 1.2). Different from Fig. 6A, all samples
show very slow release of insulin. This is because at pH 1.2, all
carboxyl groups in PGA are in COOH state, strong H-bonding and
hydrophobic interaction in the microgels result in shrinkage of the
microgels. In other words, pH can trigger the release of insulin from
the microgels. Specifically, little insulin can be released at pH 1.2
whereas it can be released rapidly at pH 6.8. Therefore, the microgel
system of crosslinked HPC-PGH can serve as an oral delivery system
for insulin.
It is noticed that the microgel composition shows different influ-
ences on release rate in Fig. 6B and A. As stated earlier, the release
rate has the order of MG3/7 > MG5/5 > MG7/3 in Fig. 6A, but in
Fig. 6B, this order is MG3/7 < MG5/5 < MG7/3. This completely oppo-
site influence arises from the great changes of PGA from pH 6.8 to
pH 1.2 as mentioned above, which indicates that the MG3/7 micro-
gels might be the best candidate for oral insulin delivery among the
three formulas examined.
microgels reversibly increased with pH value increasing. The
microgels showed more excellent pH sensitivity and a higher
swelling ratio at a higher pH value. The in vitro release data of insulin
from the microgels demonstrated that the drug release rate at pH
1.2 was much slower than that at pH 6.8 due to the protonation of
the PGA part in an acidic condition. The results showed the pos-
sibility of using such pH- and temperature-sensitive microgels as
intelligent oral drug delivery systems.
Acknowledgements
This research was financially supported by the National Natural
Science Foundation of China (Projects 50903012, 51003103), Jilin
Science and Technology Bureau (International Cooperation Project
20120729), Jilin Human Resources and Social Security Bureau
(201125020).
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