Acetals moiety contained pH-sensitive amphiphilic copolymer self-assembly used for drug carrier

Article abstract of DOI:10.1016/j.polymer.2009.12.034

pH-sensitive nanoparticles were prepared from a novel amphiphilic copolymer poly(2-phenyl-1, 3-dioxan-5-yl methacrylate-co-2-hydroxyethyl acrylate), poly(PDM-co-HEA), which was synthesized from the pH-sensitive hydrophobic monomer 2-phenyl-1, 3-dioxan-5-yl methacrylate (PDM) and the hydrophilic monomer 2-hydroxyethyl acrylate (HEA) with unit ratio (4:6) via radical polymerization. The random amphiphilic polymer could form nanoparticles in aqueous media with sizes of about 167?nm (PDI?=?0.03). The morphology of the nanoparticles was determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). When the nanoparticles solution was adjusted to pH?=?5.5, sizes of the nanoparticles increased from 167?nm to about 800?nm within 24?h, characterized by DLS. The critical aggregation concentration (CAC) of the copolymer was determined to be 5.3?mg/L (1.7?×?10^-7?M). The insoluble Nile Red could be delivered into the Hep3B cells by the nanoparticles and released in cytoplasm determined by fluorescence microscopy.

Full text of DOI:10.1016/j.polymer.2009.12.034

Polymer 51 (2010) 1709–1715
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Acetals moiety contained pH-sensitive amphiphilic copolymer self-assembly used
for drug carrier
*
*
Jushan Lu, Najun Li, Qingfeng Xu, Jianfeng Ge, Jianmei Lu , Xuewei Xia
Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry, Chemical Engineering and Materials Science, Suzhou University, Suzhou, Jiangsu 215123, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
pH-sensitive nanoparticles were prepared from a novel amphiphilic copolymer poly(2-phenyl-1, 3-
dioxan-5-yl methacrylate-co-2-hydroxyethyl acrylate), poly(PDM-co-HEA), which was synthesized from
the pH-sensitive hydrophobic monomer 2-phenyl-1, 3-dioxan-5-yl methacrylate (PDM) and the hydro-
philic monomer 2-hydroxyethyl acrylate (HEA) with unit ratio (4:6) via radical polymerization. The
random amphiphilic polymer could form nanoparticles in aqueous media with sizes of about 167 nm
(PDI ¼ 0.03). The morphology of the nanoparticles was determined by dynamic light scattering (DLS) and
transmission electron microscopy (TEM). When the nanoparticles solution was adjusted to pH ¼ 5.5,
sizes of the nanoparticles increased from 167 nm to about 800 nm within 24 h, characterized by DLS. The
critical aggregation concentration (CAC) of the copolymer was determined to be 5.3 mg/L (1.7 ꢀ 10ꢁ7 M).
The insoluble Nile Red could be delivered into the Hep3B cells by the nanoparticles and released in
cytoplasm determined by fluorescence microscopy.
Received 26 September 2009
Received in revised form
16 December 2009
Accepted 24 December 2009
Available online 11 February 2010
Keywords:
Random amphiphilic copolymer
pH-sensitive nanoparticles
Drug carrier
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
for the purpose of controlled release of drugs therein, it is more
attractive to develop acid responsive drug carriers.
During the past decades, amphiphilic polymers have attracted
considerable attention due to their special self-assembly ability to
form nano-scale aggregates, which have wide applications such as
encapsulation for controlled release of drugs and enzymes, fillers,
pigments, catalysts, adsorption materials and so on [1–4]. Signifi-
cant progress has been made in the design and synthesis of variety
amphiphilic polymers. However, most studies have focused on the
self-assembly of well-defined copolymers such as amphiphilic
block copolymers [5–8]. Only a few reports appeared in literature
did concern the self-assembly of amphiphilic random copolymers
which can be easily prepared and have potential applications in
industry and biomedicine [9–13].
Amphiphilic copolymers responsive to stimuli have emerged as
one of the most promising nano-carrier systems. In aqueous media,
these polymers can self-assemble into various supramolecular
structures and thus provide interiors able to noncovalently
encapsulate guest molecules [14–16]; besides, the release of guest
molecules can be triggered by external stimuli, such as pH [17–22],
glutathione [23], enzyme [24], temperature [25] and so on. Since it
is mildly acidic in tumor and inflammatory tissues as well as in the
intracellular compartments such as endosomes and lysosomes [26],
It is reasonable to insert cleavable linkers into amphiphilic
copolymers to obtain acid-sensitive nanoparticles. So nanoparticles
can be formed through self-assembly of copolymers and destroyed
via cleavage of acid-sensitive bonds in acidic environment. So far,
acid-labile covalent bonds such as hydrazone [27,28], orthoester
[29] and acetal [30,31] were positioned into the main chain, side
chain, or at the terminal of the core-forming blocks. For instance,
Caixia Ding [32] and co-workers prepared acid-sensitive micelles
by conjugating alkyl (C18) to PEG via a novel benzoic-imine linker,
which was assembled into pH-responsive micelles, used for drug
delivery. Heller and co-workers [33] have prepared acid-sensitive
micelles based on block copolymers of poly(ortho ester) and
poly(ethylene glycol) (PEG), in which degradation of the poly(ortho
ester) block under acidic conditions led to the release of loaded
anticancer drugs. PH-sensitive micelle/nanoparticle used for drug
carriers could release the drug molecules under endosomal pH,
achieving high antitumor activity.
Many functional polymers as drug carriers have been prepared
by physical methods from the natural macromolecules, which
usually have much broad size distribution [34,35]. The polymer
microspheres with uniform size are essential for drug delivery
system (DDS) because the distribution of the microspheres in the
body and the interaction with biological cells are greatly affected by
the particle size [36,37]. Additionally, if monodisperse polymer
microspheres are available, the drug release kinetics can be
* Corresponding authors. Tel.: þ86 512 65880367; fax: þ86 512 65882875.
E-mail addresses: lujm@suda.edu.cn (J. Lu), xiaxuewei@suda.edu.cn (X. Xia).
0032-3861/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2009.12.034

1710
J. Lu et al. / Polymer 51 (2010) 1709–1715
manipulated, therefore making it easier to formulate more
sophisticated intelligent DDS.
In this paper, we synthesized a novel amphiphilic pH-sensitive
copolymer poly(PDM-co-HEA), which could self-assemble into
nanoparticles with a rather narrow PDI, and the sizes of nano-
particles increased from nano-scale to almost micro-scale in acidic
environment, which was a very important property for drug
release. Nile Red was employed as hydrophobic and fluorescence
molecules to mimic the drug delivery and release.
Scheme 1. The synthetic route of PDM.
2.3.1. Preparation of 5-hydroxy-1, 3-benzylideneglycerol
2. Experimental section
Benzaldehyde (106 g, 1 mol), glycerol (92 g, 1 mol), 200 mL of
toluene, and 1 g of p-toluenesulfonic acid were placed into
a 500 mL flask that was fitted with an oil–water separator. The
mixture was refluxed with vigorous stirring for 12 h. The conden-
sation product was diluted with half its volume of ether and then
well shaken with 1% solution of potassium carbonate (K2CO3)
(150 mL ꢀ 3) to remove the acid catalyst and any remaining glyc-
erol. The organic layers were combined and dried over anhydrous
magnesium sulfate, filtered and another 250 mL of ether were
added. The mixture was then placed in a refrigerator overnight to
obtain title compound. The crude product was recrystallized from
ether to obtain a product of white, silky needles (yield: 31%); m.p.
2.1. Materials
Benzaldehyde, hydroxyethylacrylate (HEA), glycerol, p-toule-
nesulfonic acid, triethylamine were all purchased from Shanghai
chemical reagent Co. Ltd. as analytical regents and used without
further purification. Phosphotungstic acid and Nile Red were
purchased from Aldrich. Methacryloyl chloride was produced by
Haimen Best Fine Chemical Industry Co. Ltd. and used after redis-
tillation. Tetrahydrofuran (THF), cyclohexanone and N,N-dimethyl
formamide (DMF) were purified by reduced pressure distillation.
AIBN (98%) was recrystallized from ethanol. Other reagents such as
methanol, ether, toluene, potassium carbonate (K2CO3) and
magnesium sulfate were commercially available and used as
received.
80–81 ꢂC. 1H NMR (400 MHz, CDCl3,
d
, ppm): 3.08 (d, J ¼ 11.23 Hz,
1H), 3.65 (d, J ¼ 10.91 Hz, 1H), 4.17 (q, J ¼ 11.87, 11.87, 11.66 Hz, 4H),
5.57 (s, 1H), 7.40 (d, J ¼ 6.82 Hz, 3H), 7.51 (d, J ¼ 6.17 Hz, 2H). Anal.
Calcd for C10H12O3: C, 66.65; H, 6.71. Found: C, 66.25; H, 6.66. TOF-
MS (EI): calcd for C10H12O3: 180.0786, found: 180.0787.
2.2. Instruments and methods
2.3.2. Preparation of 2-phenyl-1, 3-dioxan-5-yl methacrylate
(PDM)
1H NMR spectra were recorded on a Bruker 400 MHz NMR
spectrometer, using CDCl3 or DMSO-d6 as solvent and tetrame-
thylsilane (TMS) as the internal standard at ambient temperature.
Molecular weights (Mn) and polydispersity (Mw/Mn) relative to
PMMA were measured on a gel permeation chromatography (GPC)
utilizing Waters 515 pump and differential refractometer. DMF was
used as a mobile phase at a flow rate of 1.0 mL/min. Conversions for
monomer and polymer were determined by gravimetry. Room
temperature emission and excitation spectra were carried out using
Edinburgh-920 fluorescence spectra photometer. Melting point
determinations were performed on a microscopic (20 ꢀ 10) melting
point apparatus and were uncorrected. TEM observations were
conducted on a Philips CM120 electron microscope at an acceler-
ation voltage of 100 kV. The sample for TEM observations was
prepared by placing a drop of nanoparticle solutions at a concen-
tration of 0.2 mg/mL on copper grids, which were coated with thin
films of Formvar and carbon successively. The size of nanoparticles
was determined using dynamic light scattering (DLS). Measure-
ments were carried out at 25 ꢂC using the Zetasizer Nana-ZS from
Malvern Instruments equipped with a 633 nm He–Ne laser using
back-scattering detection. The single crystal X-ray diffraction data
were recorded on a Rigaku Mercury CCD X-ray diffractometer.
Steady-state fluorescence spectra were recorded with a FLS920
spectrofluorometer (Edinburgh Co., UK) with a slit of 1 nm for both
excitation and emission. The excitation wavelength was 335 nm,
and pyrene was used as the probe. The intensity ratio of the third
band to the first band of the pyrene emission spectrum (I3/I1) was
used to indicate the polarity of the pyrene environment.
5-Hydroxy-1, 3-benzylideneglycerol (23.4 g, 0.1 mol) and trie-
thylamine (10.1 g, 0.1 mol) were dissolved in 100 mL anhydrous
tetrahydrofuran (THF) and cooled to 0 ꢂC in a water–ice bath.
Methacryloyl chloride (10.45 g, 0.1 mol) was added dropwisely.
After the addition, the mixture was reacted at room temperature
for another 12 h. The reaction mixture was filtered, concentrated,
and purified by recrystallized from ethyl alcohol. White crystal was
obtained (yield: 30%); m.p. 66–67 ꢂC. 1H NMR (400 MHz, CDCl3,
d,
ppm): 2.01 (s, 3H), 4.20 (d, J ¼ 12.5 Hz, 2 H), 4.33 (d, J ¼ 12.6 Hz, 2H),
4.77 (s, 1H), 5.58 (s, 1H), 5.65 (s, 1H), 6.29 (s, 1H), 7.38 (d, J ¼ 6.6 Hz,
3H), 7.51 (d, J ¼ 6.3 Hz, 2H). Anal. Calcd for C14H16O4: C, 67.73; H,
6.50. Found: C, 67.10; H, 6.49. TOF-MS (EI): calcd for C14H16O4:
248.1049, found: 248.1046.
2.4. Amphiphilic polymer synthesis
The copolymer poly(PDM-co-HEA) was prepared by reaction
between 2-phenyl-1, 3-dioxan-5-yl methacrylate (PDM) 1 and
Hydroxyethylacrylate (HEA) 2, according to the procedure shown in
Scheme 2.
2.3. Monomer synthesis
The monomer PDM was prepared by reaction of methacryloyl
chloride with glycerol and benzaldehyde (Scheme 1), according to
the modified procedure of previous literature [38].
Scheme 2. The synthetic route of poly(PDM-co-HEA).

J. Lu et al. / Polymer 51 (2010) 1709–1715
1711
Fig. 2. 1H NMR spectrum of polymer 3b in CDCl3.
phosphate buffer, while keeping the salt concentration the same.
The samples were stirred at 37 ꢂC and the fluorescence intensity
was measured at the desired time points.
Fig. 1. The single crystal structure of cis-PDM.
2.4.1. Synthesis of amphiphilic polymer poly(PDM-co-HEA) 3
Polymer 3 was synthesized by copolymerization of 1 and 2 with
different feed ratios. A typical procedure for synthesizing polymer 3
with the feed ratio of 4:6 was described as below. To a solution of
monomer 1 (297.6 mg, 1.2 mmol) and monomer 2 (208.8 mg,
1.8 mmol) in cyclohexanone (2.0 mL), AIBN (4.92 mg, 3.0 mol% to
total amount of monomers) was added. The resulting solution was
degassed by three freeze–evacuate–thaw cycles and heated at 70 ꢂC
for 3.5 h under argon. The mixture was cooled and diluted by THF,
and then poured into a large amount of cooled diethyl ether
(100 mL). The precipitate was filtered and dried under vacuum at
ambient temperature for 24 h to obtain the polymer 3 (yield: 99%).
2.7. Cell culture and fluorescence microscopic characterization
Hep3B cell, a human hepatocellular liver carcinoma cells line,
was purchased from Hu’nan Wanbai Biotechnology Development
Center, Changsha Province, China, and used for the cell uptake
studies. The cells were cultured in complete Dulbecco’s modified
Eagles medium (DMEM, containing 10% Hyclone fetal bovine
serum, 50 units mlꢁ1 penicillin, and 50 units mlꢁ1 streptomycin) at
37 ꢂC and 5% CO2 atmosphere.
The cellular uptake and intracellular release behaviors of Nile
Red loaded 3b micelles were followed with fluorescence micros-
copy (OLYMPUS IX 51) using human hepatocellular liver carcinoma
cells line (Hep3B). After hep3B cells were cultured in a disc to w70%
confluency (w8 ꢀ 104 cells/disc), 100 mL of PB solution of Nile Red
loaded micelles (10 mg Nile Red per disc) was added. After incu-
bation for 0.5 or 2 h, the culture medium was removed and the cells
were rinsed two times with DMEM prior to the fluorescence
assessment.
1H NMR (CDCl3, 400 MHz,
d, ppm): 1.0–1.3 (–CH3); 1.7–2.5 (–CH2–
C(CH3)–COO–) and (–CH2–CH–COO–); 3.5–4.4 (–COO–CH2–CH2–)
and (–O–CH(CH2O–)2–); 4.5–4.65 (–O–CH(CH2O–)2–); 5.4–5.6
((–CH2O)2–CH–ph); 7.3–7.56 (–C5H6).
2.5. Nanoparticle formation
Typically, the nanoparticle solution of the copolymer 3b was
prepared by dropwise addition of 10 mL deionized water to
copolymer 3b solution in THF (2 mg/mL) at room temperature,
followed by stirring overnight to evaporate THF completely. The
final concentration of the copolymer 3b was 0.2 mg/mL.
2.8. In vitro cytotoxicity of drug carrier poly(PDM-co-HEA) 3b
The sulforhodamine B (SRB) assay [39] is used to assess the
cytotoxicity of drug carriers in different concentrations. Well-
growing hepatoma Hep3B cells were placed in 96-well plates
(8 ꢀ 103 cells per well). Six duplicate wells were set up in each
sample. When the cells anchored to the plates, the culture medium
was replaced with the medium containing various concentrations
2.6. Encapsulation and release of Nile Red
Nile Red was loaded into micelles 3b by adding 20 mL of 1 mM
of the drug carrier (0, 7.844, 15.69, 31.38, 62.75, 125.5, 251 mg/mL)
Nile Red in acetone to 0.4 mL of copolymer solution in THF (5 mg/
mL), followed by dropwise addition of 10 mL phosphate buffer
(10 mM, pH ¼ 7.4). To completely evaporate THF and acetone, the
solution was stirred overnight and then vacuumed for 1 h. So the
micelles have a concentration of 0.2 mg/mL and contain 2.0 mM Nile
Red. The solution was divided into three 3 mL samples and the
fluorescence intensity of each was measured as 100% intensity.
Then the samples were adjusted to pH 4.0, 5.0, and 7.4, respectively,
by addition of 75 mL of 0.4 M pH 4.0 and 5.0 acetate buffer or 7.4
Table 1
Results of the Polymerization of poly(PDM-co-HEA).
Run Initial feed Polymer (unit Mn(Mw/Mn)b Conversion [%]c Yield [%]d
ratio (x:y)
ratio [x:y])a
3a
3b
3c
3d
2:8
4:6
6:4
8:2
3a (2:8)
42,200 (2.0) 98
30,500 (2.1) 99
72,000 (3.0) 97
20,500 (2.3) 99
99
98
96
96
3b (4.2:5.8)
3c (6.1:3.9)
3d (7.9:2.1)
a
Determined by 1H NMR (400 MHz) spectrum.
Estimated by estimated by GPC (DMF) using PMMA standard.
Methanol-insoluble parts.
b
c
d
Diethyl ether-insoluble parts.
Fig. 3. TEM image of the nanoparticles formed by amphiphilic copolymer 3b.

1712
J. Lu et al. / Polymer 51 (2010) 1709–1715
Fig. 6. Time dependence of nanoparticle size for copolymer 3b in pH ¼ 5.5 and 7.4
measured by DLS.
Fig. 4. Size distribution profile for nanoparticles of copolymer 3b measured by DLS.
and cultured at 37 ꢂC in a humidified incubator (5% CO2 in air, v/v).
After grown for 24 h, the medium was discarded and 100 mL of 10%
trichloroacetic acid in Hank’s balanced salt solution was added and
held at 4 ꢂC for 1 h. Then, the stationary liquid was poured away;
the cells were washed with deionized water for several times and
polymer 3b, the hydrophilic and hydrophobic content could be
calculated, which was coincided with the feed ratio of the two
monomers (Table 1). The signals assignable to the methenyl in the
acetals were at 5.5 and 4.5 ppm; the chemical shift of 3.4–4.4 ppm
was ascribed to the methylene of acetals and HEA (Fig. 2). So the
hydrophilic and hydrophobic content of the copolymer could be
conveniently tuned through the adjustment of the monomers feed
ratio.
stained with 100
remove of SRB, the cells were washed three times with 0.1% acetic
acid solution. The SRB dye was extracted with 150 L of 10 mmol/L
mL of 0.4% SRB solution for 30 min. Following the
m
tris (hydroxymethyl)-aminomethane solution (pH ¼ 10.5). The
absorbance at 531 nm was measured with a spectrophotometer.
3.3. Nanoparticle formation and behavior of the amphiphilic
copolymer 3b
3. Results and discussion
The micellar characteristics of copolymer 3b in aqueous solution
were investigated using a fluorescence technique, TEM, and DLS
[40]. Fig. 3 shows a TEM image of the copolymer 3b nanoparticles,
and samples were stained with 0.1 wt% phosphotungstic acid
before measurements. It can be seen that the nanoparticles are
spherical in shape with an average diameter of about 170 nm. The
particle size and size distribution of the nanoparticles were inves-
tigated further by DLS (Fig. 4). DLS results show that the nano-
particles have an average diameter of 163 nm, with a narrow size
distribution (PDI ¼ 0.03), which are consistent with the TEM
results. Interestingly, although the copolymer 3b was random
rather than block amphiphilic copolymer, it could be self-
assembled to nanoparticles well.
3.1. Monomer synthesis
According to the synthetic route of Scheme 1, we synthesized
the monomer (PDM) containing an acetal, which was a pH-
responsive moiety. The momomer (PDM) has two configurational
isomers, herein we obtained a single crystal, and the single crystal
structure of PDM was characterized by X-ray diffraction. Finally we
found the configuration of the compound PDM synthesized in our
laboratory was cis-PDM (Fig. 1).
3.2. Design and synthesis of amphiphilic copolymer
Four copolymers were synthesized with different feed ratios 3a–
d (2:8, 4:6, 6:4 and 8:2). According to the 1H NMR spectrum of
The CAC of copolymer 3b was determined by a fluorescence
technique using pyrene as a fluorescence probe [41]. Fig. 5 shows
the intensity ratios (I3/I1) obtained from the fluorescence emission
spectra of pyrene (inset) plotted versus the concentrations of
copolymer 3b (Mn ¼ 30 500, Mw/Mn ¼ 2.1) at pH ¼ 7.4 and 25 ꢂC. I3/
I1 was fluorescent emission intensity ratio of third band (383 nm, I3)
and first band (372 nm, I1) of pyrene in aqueous media containing
amphiphilic polymer 3b. At a certain concentration, the intensity
Fig. 5. Intensity ratios (I3/I1) obtained from the fluorescence emission spectra of pyr-
ene (inset) plotted versus the concentrations of copolymer 3b at pH ¼ 7.4 and 25 ꢂC.
Fig. 7. 1H NMR of copolymer 3b before acidification and after acidification.

J. Lu et al. / Polymer 51 (2010) 1709–1715
1713
Fig. 10. In vitro cell viabilities, measured by the SRB assay, after culture of Hep3B cells
with amphiphilic poly(PDM-co-HEA) as functions of different concentrations (0, 7.844,
15.69, 31.38, 62.75, 125.5, 251 mg/mL).
Fig. 8. Size distribution of nanoparticles in different composition (the molar ratio of
HEA in polymer chain as 20%, 40%, 60% and 80%).
ratio started to increase dramatically. This increase reflects the
incorporation of pyrene into the hydrophobic cores of the nano-
particles. The CAC was determined from the crossover point in the
low concentration range. The CAC of the copolymer 3b was 5.3 mg/
L, which is not only strong evidence to the formation of nano-
particles but also an important parameter in evaluating the stability
of the nanoparticles in the blood post administration, owing to the
low CAC value. The CAC/CMC of polymeric nanoparticles/micelles is
typically of the order of 10ꢁ6–10ꢁ7 M, while that of low molecular
weight surfactant micelles is of the order of 10ꢁ3–10ꢁ4 M [42].
Herein, the CAC of nanoparticle 3b was calculated to be
1.7 ꢀ 10ꢁ7 M (CAC ¼ 5.3 mg/L), which is less prone to dissociation at
low concentrations.
Amphiphilic polymer with a high CAC value seems to be
unsuitable for drug delivery because the nanoparticles may be
dissociated after being administered into the body due to the
dilution effect. Here we have obtained the amphiphilic copolymer
with very low CAC value (CAC ¼ 5.3 mg/L) and this nanoparticle
could be used for potential drug carrier.
consistent to the mildly acidic environment of endosomes and
lysosomes (pH ¼ 5.5–6.5) [32]. Rapid and remarkable swelling of
nanoparticles instead of nanoparticle disruption was observed
according to DLS result (Fig. 6). The nanoparticle size increased
from 167 nm to 255 nm within 10 min, and then reached about
800 nm within 24 h. In contrast, no change of nanoparticle size was
observed over 1 day at pH ¼ 7.4. The reason for this phenomenon
could be ascribed to the cleavage of hydrophobic cyclic acetals
moieties of the polymer. Namely, the acid-sensitive cyclic acetal
functionality could be cleaved under mildly acidic conditions and
transferred into hydrophilic dihydroxypropyl methacrylate unit,
which was highly hydrophilic but not soluble in water, together
with releasing of benzaldehyde. Environmentally responsive poly-
mer capsules ranging in the size from nanometers to microns have
been interesting for the material scientists due to their potential
and practical utilization in controlled release [43].
To further explain the phenomenon of the nanoparticles
swelling, and to make sure that the acetal has been hydrolyzed,
copolymer 3b, which was set in mildly acidic solution for 1 h, was
characterized via 1H NMR. Compared to 1H NMR of copolymer 3b
before acidification (Fig. 7), the specific chemical shifts attributing
to the protons of methenyl group (a) and phenyl group (b) in the
3.4. pH-responsive nanoparticle of amphiphilic copolymer 3b
To determinate whether the nanoparticles of copolymer 3b
were sensitive to acid, one drop of dilute HCl was added to the
nanoparticles, the pH was adjusted to about 5.5, which was
Fig. 11. The pictures of Nile Red in different conditions (a). Nile Red in aqueous
solution; (b). Nile Red in nanoparticles; (c). 15 min after drops of sodium acetate buffer
at pH ¼ 4 in nanoparticles.
Fig. 9. Nile Red fluorescence in micelles 3b as a function of time at different pH at
37 ꢂC.

1714
J. Lu et al. / Polymer 51 (2010) 1709–1715
Fig. 12. Fluorescence microscopy images of Hep3B cells being treated with free Nile Red and Nile Red-loaded 3b nanoparticle with different incubation time at pH ¼ 7.4 (a). a0-free
Nile Red, 0.5 h; (b). b0-Nile Red-loaded 3b nanoparticle, 0.5 h; (c). c0-Nile Red-loaded 3b nanoparticle, 2.0 h.
polymer chain were disappeared after acidification. So it could
conclude that remarkable swelling of nanoparticles under mildly
acidic conditions was due to the hydrolysis of the acetals.
nanoparticles with the size of 167 nm. As for this phenomenon, the
copolymer with hydrophilic ratio of 60% may be the right chemical
composition to self-assemble to nanoparticles used for drug carrier.
So we choose copolymer 3b as potential biomaterials.
3.5. Size distribution of the nanoparticles in different chemical
composition
3.6. Encapsulation and release of Nile Red in vitro
We have prepared the micelles from the amphiphilic copoly-
mers with different chemical composition of PDM and HEA (2:8,
4:6, 6:4 and 8:2). As shown in Fig. 8, the result of DLS shows that the
size of self-aggregates decreased from 510 to 270 nm as the
hydrophilic ratio increased from 20 to 80%, indicating formation of
more dense hydrophobic cores, which is consistent with the papers
reported by Ick Chan Kwon etc before [44,45]. However when the
hydrophilic ratio is 60%, the copolymer can self-assemble to
The encapsulation and release of Nile Red from micelles 3b
were investigated at 37 ꢂC at different pHs (i.e., pH 4.0, 5.0, and
7.4). The release of Nile Red was followed by measurement of
fluorescence and the results showed clearly a pH-dependent
release behavior from micelles 3b, in which the fluorescence of
Nile Red decreased almost 70 and 80% at pH 5.0 and 4.0, respec-
tively, within 6 h, while little change was observed for the sample
at pH 7.4 (Fig. 9).

J. Lu et al. / Polymer 51 (2010) 1709–1715
1715
3.7. In vitro cytotoxicity of the PDM-co-HEA nanoparticles
nanoparticles can be used as a potential smart drug carrier for
cancer therapy.
Hep3B cells were used to assess the cytotoxicity of poly(PDM-
co-HEA). We expected little cytotoxicity because the copolymer
was synthesized from biocompatible and nontoxic monomers. In
fact, the cells cultured after 24 h in the presence of copolymer with
different concentration retained high cell viability (above 85%) in
SRB assays even at high concentrations (350 mg/mL) (Fig. 10). This
result suggests that amphiphilic poly(PDM-co-HEA) may be safe
drug carriers in vivo.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 20876101), Project sup-
ported by the Specialized Research Fund for the Doctoral Program
of Higher Education of China (Grant No. 20070285003) and the
Supporting Project of Jiangsu Province (Industry) (Grant No.
BE2008061).
3.8. Encapsulation of Nile Red by the nanoparticles and in vitro
cellular uptake by Hep3B cells
References
To assess whether the nanoparticles of amphiphilic copolymer
3b could carry and controllably release drugs, Nile Red, as guest
molecules, was encapsulated by the nanoparticles solution of 3b
(0.2 mg/mL, above the CAC). According to Fig. 11, the hydrophobic
Nile Red, insoluble in water (Fig. 11-a), could be solubilized by
amphiphilic copolymers in aqueous media into a homogeneous
state (Fig.11-b). When drops of sodium acetate buffer solution were
added into the solution (pH ¼ 4), encapsulated Nile Red was
deposited from the solution within 15 min (Fig. 11-c). So polymer
3b was a promising candidate for nanoparticle carrier of hydro-
phobic drugs to passively target cancer tissue by EPR effect [46,47]
and controllably release drugs triggered by changing of pH values.
To mimic the in vitro drug controllable delivery and release
behavior of polymer 3b nanoparticles, Nile Red was also used as
guest molecules and Hep3B cell as target cell. By using fluorescence
microscopy, the process of drug (Nile Red) delivery and release with
assistance of nanoparticles could be observed. The result was
shown in Fig. 12-a0 showed that the free Nile Red in water with very
low solubility, so there was little Nile Red in solution interacting
with the cells, resulting in fewer uptakes and almost no fluorescent
image. In contrast, the Nile Red could be solubilized in the nano-
[1] Zhang J, Xu S, Kumacheva E. J Am Chem Soc 2004;126:7908.
[2] Cochran JK. Curr Opin Solid State Mater Sci 1998;3:474.
[3] Gill I, Ballesteros A. J Am Chem Soc 1998;120:8587.
[4] Caruso F. Adv Mater 2001;13:11.
[5] Luo L, Eisenberg A. J Am Chem Soc 2001;123:1012.
[6] Mao J, Ni PH, Mai YY, Yan DY. Langmuir 2007;23:5127.
[7] Zhou JF, Wang L, Wang CL, Chen T, Yu HJ, Yang Q. Polymer 2005;46:11157.
[8] Liu SY, Armes SP. Langmuir 2003;19:4432.
[9] Sun GX, Zhang MZ, He JL, Ni PH. J Polym Sci Part A Polym Chem 2009;47:4670.
[10] Li YB, Deng YH, Tong XL. Macromolecules 2006;39:1108.
[11] Yan LF, Zhang GZ, Liu GM, Ji J, Li W. J Appl Polym Sci 2006;100:3718.
[12] Yu HJ, Wang L, Zhou JF, Dong XC, Jiang GH. J Appl Polym Sci 2007;105:1156.
[13] Yu HJ, Wang L, Zhou JF, Jiang GH, Zhao ZR. J Phys Chem B 2006;110:837.
[14] Discher DE, Eisenberg A. Science 2002;297:967.
[15] Zhou Z, Li Z, Ren Y, Hillmyer MA, Lodge TP. J Am Chem Soc 2003;125:10182.
[16] Neiser MW, Muth S, Kolb U, Harris JR, Okuda J, Schmidt M. Angew Chem Int Ed
2004;43:3192.
[17] Du J, Armes SP. J Am Chem Soc 2005;127:12800.
[18] Chiu HC, Lin YW, Huang YF, Chuang CK, Chern CS. Angew Chem Int Ed
2008;47:1875.
[19] Li Y, Du W, Sun G, Wooley KL. Macromolecules 2008;41:6605.
[20] Yang XY, Chen LT, Huang B, Bai F, Yang XL. Polymer 2009;50:3556.
[21] Chen W, Meng FH, Li F, Ji SJ, Zhong ZY. Biomacromolecules 2009;10:1727.
[22] Auguste D, Furman K, Wong A, Fuller J, Armes SP, Deming T, et al. J Control
Release 2008;130:266.
[23] Takae S, Miyata K, Oba M, Ishii T, Nishiyama N, Itaka K, et al. J Am Chem Soc
2008;130:6001.
particles at the concentration of 20 mg/mL, strong Nile Red fluo-
[24] Thornton PD, Mart RJ, Ulijn RV. Adv Mater 2007;19:1252.
[25] Sundararaman A, Stephan T, Grubbs RB. J Am Chem Soc 2008;130:12264.
[26] Ulbrich K, Subr V. Adv Drug Deliv Rev 2004;56:1023.
[27] Kale A, Torchilin VP. Bioconjug Chem 2007;18:363.
[28] Bae Y, Nishiyama N, Fukushima S, Koyama H, Yasuhiro M, Kataoka K. Bio-
conjug Chem 2005;16:122.
[29] Lin S, Du F, Wang Y, Ji S, Liang D, Yu L, et al. Biomacromolecules 2008;9:109.
[30] Chan Y, Bulmus V, Hadi Zareie M, Byrne FL, Barner L, Kavallaris M. J Control
Release 2006;115:197.
rescence was observed in the cells after 0.5 h incubation with Nile
Red loaded 3b micelles (Fig. 12-b0). It has been reported that
polymeric micelles can be rapidly uptaken by the cells via endo-
cytosis [48]. After a longer incubation time of 2 h, Nile Red fluo-
rescence intensity became stronger, indicating Nile Red escaped
from the micelles (Fig. 12-c0). It has been shown that acetals moiety
could be rapidly cleaved in the intracellular compartment caused in
comparatively acidic condition, such as endosomes and lysosomes
of cells and based on this intriguing phenomenon, intracellular
delivery systems for drugs and DNA have successfully been devel-
oped [21,49].
[31] Murthy N, Thng YX, Schuck S, Xu MC, Frechet JMJ.
2002;124:12398.
J Am Chem Soc
[32] Ding CX, Gu JX, Qu XZ, Yang ZZ. Bioconjug Chem 2009;20:1163.
[33] Toncheva V, Schacht E, Ng SY, Barr J, Heller J. J Drug Target 2003;11:345.
[34] Yun YH, Goetz DJ, Yellen P, Chen WL. Biomaterials 2004;25:147.
[35] Zhou QZ, Wang LY, Ma GH, Su ZG. J Colloid Interface Sci 2007;311:118.
[36] Chattopadhygay P, Shakunov BY, Yim D, Cipolla D, Boyd B, Farr S. Adv Drug
Deliv Rev 2007;59:444.
[37] Shiga G, Muramatsu N, Kondo T. J Pharm Pharmacol 1996;48:891.
[38] Garcı Nuria, Guzma Julio, Riande Evaristo, Garcıa Felix, de la Pen Jose L,
Calle Paloma, et al. Macromolecules 2002;35:2926.
[39] Wang XR, Yue P, Kim YA, Fu HA, Khuri FR, Sun SY. Cancer Res 2008;68:7409.
[40] Klaikherd Akamol, Nagamani Chikkannagari, Thayumanavan* S. J Am Chem
Soc 2009;131:4830.
[41] Zhang H, Ni PH, He JL, Liu CC. Langmuir 2008;24:4647.
[42] Adams ML, Lavasanifar A, Kwon GS. J Pharm Sci 2003;92:1343.
[43] Gao HF, Yang WL, Wang CH, Fu SK. Polymer 2005;46:1087.
[44] Kwon S, Park JH, Chung H, Kwon* IC, Jeong SY. Langmuir 2003;19:10188.
[45] Kim K, Kwon S, Park JH, Chung H, Jeong SY, Kwon* IC. Biomacromolecules
2005;6:1154.
4. Conclusion
We have successfully developed pH-responsive nanoparticles
based on amphiphilic copolymers comprising of a novel acid-labile
cyclic acetal as hydrophobic moiety. The copolymer could undergo
self-association in aqueous media to form its nanoparticles, with
a narrow PDI. The sizes of nanoparticles increased with the pro-
longing of the time in acidic aqueous solution due to the hydrolysis
of the acid-labile acetals. CAC of the copolymer was 5.3 mg/L
(1.7 ꢀ 10ꢁ7 M), which was rather low, and the nanoparticles can
solubilize hydrophobic guest molecules in dilute solution. Hydro-
phobic Nile Red could be solubilized, controllably delivered and
released into liver cancer cell Hep3B. So the present pH-sensitive
[46] Matsumura Y, Maeda H. Cancer Res 1986;46:6387.
[47] Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. J Control Release 2000;65:271.
[48] Savic R, Luo LB, Eisenberg A, Maysinger D. Science 2003;300(5619):615.
[49] Wang BD, Xu CJ, Xie J, Yang ZY, Sun SH. J Am Chem Soc 2008;130:14436.