Synthesis and characterization of pH-responsive and folated nanoparticles based on self-assembled brush-like PLGA/PEG/AEMA copolymer with targeted cancer therapy properties: A comprehensive kinetic study

Article abstract of DOI:10.1016/j.ejmech.2012.02.027

In this study, a novel nanocarrier was synthesized based on methacrylated poly(lactic-co-glycolic acid) (mPLGA) as a lipophilic domain, acrylated methoxy poly(ethylene glycol) (aMPEG) as hydrophilic part and N-2-[(tert-butoxycarbonyl) amino] ethyl methacr

Full text of DOI:10.1016/j.ejmech.2012.02.027

European Journal of Medicinal Chemistry 50 (2012) 416e427
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and characterization of pH-responsive and folated nanoparticles based
on self-assembled brush-like PLGA/PEG/AEMA copolymer with targeted cancer
therapy properties: A comprehensive kinetic study
*
Sepideh Khoee , Reza Rahmatolahzadeh
Polymer Chemistry Department, School of Science, University of Tehran, PO Box 14155-6455, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 August 2011
Received in revised form
In this study, a novel nanocarrier was synthesized based on methacrylated poly(lactic-co-glycolic acid)
(
mPLGA) as a lipophilic domain, acrylated methoxy poly(ethylene glycol) (aMPEG) as hydrophilic part
and N-2-[(tert-butoxycarbonyl)amino] ethyl methacrylamide (Boc-AEMA) as pH-responsive segment.
Radical polymerization of the above-mentioned three modified monomers produces amphiphilic brush-
like copolymer. The protecting amine group (Boc) was selectively deprotected and the latter targeted
copolymer was produced through the reaction of this copolymer with activated folic acid. Nano-
precipitation method was used to prepare quercetin-loaded nanoparticles. Dynamic light scattering
2
February 2012
Accepted 14 February 2012
Available online 22 February 2012
Keywords:
Amphiphilic brush-like copolymer
pH sensitivity
Core-shell nanoparticles
Targeting
(
DLS) analysis showed that the produced nanoparticles had nanometric size (<100 nm) and low poly-
dispersity in size at different pHs. Higuchi and KorsmeyerePeppas models were applied to evaluate
release mechanisms and kinetics. Based on in-vitro degradation study, we found that the brush-like
copolymer underwent a rapid weight loss in acidic pH.
Degradation study
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
therapy, a strong attention has been paid to develop drug carriers
with active targeting ability. Moreover, tissue specificity is another
Polymeric micelles have been developed as drug delivery
systems as well as carriers for various contrasting agents in diag-
nostic imaging applications [1e5]. With the amphiphilic structure
of the polymeric micelles, hydrophobic pharmaceutical compounds
are solubilized within the hydrophobic cores, whereas the shell
maintains a hydration barrier that protects the integrity of each
micelle. As a result, polymeric micelles can be used as efficient
containers for reagents with poor solubility and/or low stability in
physiological environments [6e9]. Moreover, the ability to tailor its
numerous end groups offers considerable scope for fine-tuning its
drug loading and targeted drug release properties. Selective drug
targeting to cancer cells is an interesting task [10e13]. This is
especially true for chemotherapeutic cancer treatment because
most anti-cancer drugs cannot distinguish between cancerous and
healthy cells. A well-known approach to accomplish active tumor
targeting is to chemically link specific ligands to the micelle’s outer
layer that can recognize the exact molecular signatures of the
cancer cells. To increase drug delivery efficiency and specific cancer
issue for design of an appropriate carrier. Among various targeting
moieties, such as peptide and antibody [14e17], which can be
recognized and bind to specific receptors that are unique to cancer
cells, the folate receptor is vastly over-expressed in a wide variety of
human tumor cells [18e21]. Drug targeting can also be achieved by
using a polymer that is sensitive to the surrounding temperature or
pH [22e25]. Various stimuli such as ultrasound, light, or micro-
environmental changes in temperature and pH could affect on
stimuli-responsive polymeric micelles. Due to their ability to
release their encapsulated drugs at specific sites where the stimuli
are present, they are known as ‘smart’ delivery systems. The pH-
responsive polymeric micelles and nanoparticles are particularly
useful for application in biological systems because of the
numerous pH gradients that exist in both normal and pathophys-
iological states.
In this work, we describe the synthesis and micellar character-
ization of folated and non-folated amphiphilic brush copolymers
based on methacrylated poly(lactic-co-glycolic acid) (mPLGA) as
the hydrophobic segment which are biodegradable and extensively
used in therapeutic devices. Acrylated methoxy poly(ethylene
glycol) (aMPEG) chains, as hydrophilic domain placed on the
micelle surface can protect the nanocarrier from undesired attacks
*
Corresponding author. Tel.: þ98 21 61113301; fax: þ98 21 6649 5291.
E-mail address: Khoee@Khayam.ut.ac.ir (S. Khoee).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.027

S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
417
in the biological media. Thereby increasing the stability of micelles
2.3.2. Synthesis of PLGA
and prolonging their circulation time in the blood. 2-Aminoethyl
methacrylamide (AEMA), with a primary amine group is expected
to play dual role. A crucial role in offering the pH sensitivity to the
produced nanocarriers and also providing a site to conjugate folic
acid to copolymer and make it suitable for targeted anti-cancer
drug delivery. Since controlling the molar ratio of PLGA:MPE-
G:AEMA can affect physical properties of the final nanoparticles,
the effective ratio of amphiphilic and pH-responsive copolymer
should be tailored via addition copolymerization of the above
modified monomers and hence the drug loading and release level
of the resulting polymeric micelles can be adjusted. The AEMA
groups can become hydrophobic and adhere tightly on the inner
hydrophobic block at neutral pH or change to hydrophilic chains
and create corona layer with PEG chains at acidic pHs. Changing in
AEMA nature affects the drug release profile and to accurately
investigate the drug release mechanism, the release data were
fitted into exponential models such as Higuchi and
KorsmeyerePeppas. The relationship between copolymer compo-
sitions and the rate of copolymer degradation with core-shell
architecture were also evaluated.
Polycondensation reaction of glycolic acid and
D
,
L
-lactic acid was
performed according to the modified procedures described in
reference in the presence of Sb and under nitrogen atmosphere
[27,28]. Briefly, a pre-determined amount of -LA and GA (70/30 of
2 3
O
D,L
molar ratio) was charged into a two-necked round-bottomed flask
equipped with nitrogen inlet and a condenser to transfer the
produced water to the second two-necked round-bottomed flask
which was equipped with nitrogen outlet. The flask was heated in
ꢀ
ꢀ
an oil bath to 120 C for an hour and then 180 C for 5 h to melt the
monomers. Accordingly, 85% of water was removed approximately,
ꢀ
to increase the conversion, the temperature was lowered to 150 C
and reaction media was stirred for another 2 h. The ring-opening
polymerization under nitrogen atmosphere continued for 12 h
with stirring under vacuum at room temperature. After the reaction
was completed, the product was dissolved in dichloromethane and
then re-precipitated from an excess cold diethyl ether three times.
The precipitate was dried under vacuum for 72 h to produce 15.2 g
of PLGA (95.6% yield).
2.3.3. Synthesis of methacrylated PLGA
In a 250 mL three-necked round-bottomed flask, PLGA (4 g,
ꢂ4
8
.0 ꢁ 10 mol) was dissolved in dehydrated dichloromethane
ꢀ
2
. Experimental
(20 mL) and cooled to 4 C for 30 min. Next, triethylamine (0.5 mL,
ꢂ3
3
.73 ꢁ 10 mol) was added dropwise to the cold PLGA solution
2.1. Materials
while stirring and the final solution was stirred for another 15 min.
An excess amount of methacrylic anhydride (1.5 mL,
ꢂ3
Methoxy poly(ethylene glycol) (MPEG) (M
w
¼ 2000) and
D
,
L
-
10.06 ꢁ 10 mol) was added to the reaction vessel, and the solu-
lactic acid and glycolic acid, ethylene diamine (EDA), di-tert-butyl
carbonate were purchased from Fluka Co (Switzerland). Acryloyl
chloride, methacryloyl chloride, methacrylic anhydride, N-hydroxy
succinimide (NHS), dicyclohexylcarbodiimide (DCC) and triethyl-
amine (TEA), trifluoroacetic acid (TFA), folic acid (FA), 2,2-
azobisisobutyronitrile (AIBN) were supplied from SigmaeAldrich
Co (USA). 1,2-dichloromethane, methanol, dimethyl sulfoxide
tion was stirred at room temperature under mild vacuum for 48 h.
The mixture was filtered off to remove any sedimentation and then
precipitated in cold methanol to produce methacrylated PLGA
(3.0 g, 74% yield). The precipitate was dried under vacuum for 72 h.
2.3.4. Synthesis of N-2-[(tert-butoxycarbonyl)-amino] ethyl
methacrylamide (Boc-AEMA)
(
DMSO), 1,4-dioxane, Ethyl ether and acetone were purchased from
Boc-AEMA was synthesized with 55% yield according to the
previously reported procedure [29].
Merck Co (Germany). AIBN was purified by recrystallization from
hexane and acetone, respectively. Other reagents were commer-
cially available and were used as received.
2.4. Synthesis of copolymers
2
.2. Methods
2.4.1. Preparation of brush-like copolymer from acrylated and
methacrylated monomers
1H NMR spectra were recorded in CDCl
solution on a Bruker
Brush-like copolymer was synthesized by free radical copoly-
merization of the three modified monomers. Pre-determined
amount of acrylated PLGA, methacrylated-MPEG, methacrylated-
AEMA-BOC and AIBN (feed molar ratio of monomers to initiator
was 100:6) was dissolved in 15 mL of dehydrated dichloromethane
and placed in a three-necked round-bottom flask equipped with
a magnetic stirrer, condenser and nitrogen inlet and outlet. The
mixture was completely degassed by nitrogen gas for 20 min and
3
(
Germany) DRX 500 (500 MHz) apparatus with the TSM proton
signal for reference. IR measurements were performed using
a Bruker EQUINOX 55 model FT-IR. Dynamic light scattering
experiments were performed with commercially available equip-
ment (Zeta-sizer Nano from Malvern) [United Kingdom] using a 4-
mW HeeNe laser (633 nm wavelength) with a fixed detector angle
of 173 . Size and morphology of the nanoparticles were investi-
gated by scanning electron microscopy (SEM) with Vega-II from
Tescan Co (Brno, Czech Republic). The number average molecular
ꢀ
ꢀ
then stirred under nitrogen blanket for 24 h at 65 C. After poly-
merization, the product was purified by precipitation from petro-
weight (M
n
) and the weight average molecular weight (M
w
) of
leum ether twice to obtain 2.6 g (70.2% yield) of the copolymer.
hybrid copolymer were estimated by gel permeation chromatog-
raphy (GPC) (Agilent 1100) with THF as the mobile phase at 25 C.
ꢀ
2.4.2. Deprotection of t-BOC group from brush copolymer
Poly(styrene) standard samples were used for calibration.
BOC protecting group was removed by TFA. TFA (0.3 mL) was
ꢂ3
added to a solution of copolymer (1.0 g, 0.08 ꢁ 10 mol) in chlo-
roform and ethyl acetate (14 mL, 1:6 mixture). The reaction was
carried out for 1 h at room temperature under magnetic stirring.
The reaction mixture was neutralized with 10% aqueous sodium
bicarbonate solution (2 ꢁ 10 mL) and then washed with aqueous
sodium chloride solution (2 ꢁ 10 mL). The organic layer was dried
2
2
.3. Synthesis of monomers
.3.1. Synthesis of acrylated PEG
Acrylated methyl ether poly(ethylene glycol) (ACMPEG) was
synthesized with 73% yield according to the previously reported
4
over MgSO , filtered and solvent was evaporated under reduced
procedure (Scheme 1 e Step 3) [26].
pressure to yield 0.54 g (55%) of the deprotected copolymer.

418
S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
Scheme 1. Synthesis of methacrylated PLGA (mPLGA), acrylated PEG (aPEG) and methacrylated ethylene diamine (Boc-AEMA).
2
.4.3. Synthesis of folate-copolymer
Folate conjugated copolymer was prepared within a two-step
quercetin were dissolved in acetone (2 mL), and then the obtained
organic solution was added dropwise to distilled water (10 mL)
under stirring at room temperature and then, acetone was thor-
oughly removed under reduced pressure. Finally, the resulting
procedure: (1) carboxylation of folic acid with NHS to yield
folate-NHS; (2) conjugation of copolymer with folate-NHS to
produce folate-copolymer. The carbodiimide-activated folic acid
aqueous dispersion was filtered through 0.2 mm cellulose acetate
can couple with either
tion conditions were set to favor linkage with
a
- or
g
-carboxyl group residue [30]. Reac-
syringe filter to remove aggregated copolymers and non-
incorporated drug crystals and then, freeze-dried to extract fine
nanoparticles from the aqueous medium. Nanoparticles formed
from folated copolymer carrier were prepared as below: folate
conjugated brush copolymer (10 mg) and pre-determined amount
of quercetin were dissolved in DMSO and acetone (3 mL, 1e8), then
the obtained organic solution was added dropwise to distilled
water (10 mL) at room temperature stirringly. Then, acetone was
completely removed under reduced pressure. Finally, the resulting
g
-carboxyl residue. A
ꢂ3
solution of folic acid (1 g, 2.2 ꢁ10 mol) in anhydrous DMSO
(
7
25 mL) was reacted an overnight with NHS (0.9 g,
.81 ꢁ10 mol) in the presence of DCC (0.5 g, 2.4 ꢁ10 mol)
ꢂ3
ꢂ3
under argon at room temperature, and the major by product, 1,3-
dicyclohexyllurea (DCU), was removed by filtration. Subsequently,
the above activated folate solution (3 mL) was added to a solution
ꢂ3
of brush copolymer (0.4 g, 0.03 ꢁ10 mol) in DMSO (5 mL) con-
ꢂ3
taining triethylamine (0.05 mL, 0.35 ꢁ10 mol). The reaction was
aqueous dispersion was filtered through 0.2
mm cellulose acetate
performed at room temperature for 10 h under argon atmosphere.
The final product was centrifuged, filtered and purified by dialysis
against deionized water for 24 h (molecular weight cut-off
syringe filter according to the above procedure.
2
000 Da) and freeze-dried to produce 0.25 g (61% yield) folate
2.6. Nanoparticle yield, drug loading and encapsulation efficiency
conjugated polymer.
The nanoparticle yield was obtained by gravimetric method.
Drug content in the nanoparticles was measured using a dissolu-
tion method. A pre-determined amount of the freeze-dried nano-
particles was dissolved in 5 mL of acetone. The concentration of
quercetin was analyzed with ultraviolet absorption at their
maximum wavelength (362 nm) on a UV spectrophotometer. Three
2.5. Preparation of drug-loaded micelles
Drug-loaded nanoparticles were prepared by nanoprecipitation.
The brush copolymer (10 mg) and pre-determined amount of

S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
419
samples were prepared for each test. The nanoparticle yield,
Table 1
Diffusion exponent and solute release mechanism for spherical shape matrices.
encapsulation efficiency (EE) and drug loadings (DL) were calcu-
lated using the following equations (Eqs. (1)e(3)):
Diffusion exponent (n)
n < 0.43
Overall solute diffusion mechanism
Fickian diffusion
Weight of the nanoparticles
Nanoparticle yieldð%Þ ¼
0
.43 < n < 0.85
Anomalous (non-Fickian) diffusion
Case II transport
Super Case II transport
Total weight of drug and copolymer
0.85 < n < 1
n > 1
ꢁ
100
(1)
2
.9. Statistical analysis
Weight of encapsulated drug
Weight of the nanoparticles
Drug loadingð%Þ ¼
ꢁ 100
(2)
Particle diameters and “in-vitro” release analyses were per-
formed in triplicate. The data are represented as mean ꢃ standard
deviation (SD). Data were analyzed using the T test and one-way
analysis of variance (ANOVA) and considered significantly
different at the level of p < 0.05.
Weight of encapsulated drug
Total weight of drug
Encapsulation efficiencyð%Þ ¼
ꢁ
100
(3)
2.10. Polymer degradation
For degradation study, polymeric nanoparticles were prepared
using nanoprecipitation method. 10 mg (n ¼ 3) of the synthesized
polymer was dissolved in 2 mL of acetone and then the dissolved
polymer was added dropwise to 10 mL of phosphate buffer (pH 7.4
and 5.8) under stirring at room temperature and then, acetone was
thoroughly removed under reduced pressure. Weight loss of water
insoluble amine-modified brush copolymer was measured gravi-
metrically after incubation of polymer nanoparticles in phosphate
buffered saline over 5 weeks. After 4, 12 and 36 days, samples were
freeze-dried and then were carefully washed five times with
distilled water to remove the salt residues. The recovered samples
were dried in a vacuum oven for approximately 3 days at room
temperature until constant masses were obtained. Polymer total
residue was calculated from the following formula:
2
.7. In-vitro drug release studies
The in-vitro release studies of quercetin-loaded nanoparticles
were performed by diffusion technique. The nanoparticle suspen-
sion (10 mL) was placed into a pre-swelled dialysis bag that was
immersed into phosphate buffer saline (PBS) solution (100 mL,
ꢀ
pH ¼ 7.4 and 5.8) at 37 C. At pre-determined time intervals, the
solution (3 mL) was taken out from the release media and replaced
with fresh PBS (3 mL). Then the released drug was measured using
UV spectra at 202 nm.
2.8. Mechanism of drug release
Final weight of a dried sample
The release data were analyzed by model-dependent (curve
Total residue ð%Þ ¼
ꢁ 100
Original weight
fitting) methods. For model-dependent analysis, two theoretical
models describing drug release from polymeric systems were used
according to Higuchi [31] and KorsmeyerePeppas [32]. Higuchi
model describes drug release as a diffusion process based on Fick’s
law according to the following Eq. (4):
The morphology of polymer nanoparticles was characterized
by SEM.
3
. Result and discussion
pffiffi
M
M
t
¼
N
k
t
(4)
3
.1. Design, synthesis and characterization of the brush copolymer
where k is a constant reflecting formulation characteristics, and M
t
The polymer structure containing two types of polymer chains
and M
infinite time, respectively. According to this model, a straight line is
expected for the plot of M /M versus the square root of time if the
N
are cumulative amounts of released drug at time t and
was a brush-like copolymer prepared by macromer copolymeri-
zation. The acrylated macromer of PEG and methacrylated macro-
mer of PLGA, and monomer of EDA was prepared separately
according to Scheme 1. The core of the micelles consisted of
hydrophobic chain of PLGA copolymer to make it biodegradable.
Synthesis of the poly(lactic-co-glycolic acid) macromer has been
presented in Scheme 1, step 1. Sb
catalyst for the esterification reaction. Number (M
(M ) average molecular weights of PLGA were determined by GPC
and were 2280 and 5396 g/mol respectively with PDI of 2.36.
t
N
drug release from the matrix is based on a diffusion mechanism.
The KorsmeyerePeppas model is considered if the drug release
mechanism deviates from Fick’s law and follows an anomalous
behavior described by the following equation:
2
O
3
, as a Lewis acid, acts as the
) and weight
n
M
t
0
n
¼
N
k t
(5)
w
M
where k’ is the kinetic constant and n is an exponent, characterizing
the diffusion mechanism. When pure diffusion obeys the control-
ling release mechanism, n equivalents to 0.5 and Eq. (5) coincide to
Eq. (4). Moreover, Eq. (4) becomes physically realistic for n ¼ 1 since
the drug release follows swelling controlled release or Case II
transport [33]. Both Eqs. (4) and (5) are short time approximations
of complex exact relationships and therefore, their use is confined
for the description of the first 60% of release curve. The n value is
used to characterize different release mechanisms as given in
Table 1 for the spherical shaped matrices.
The secondary hydroxyl group of PLGA reacted efficiently with
methacrylic anhydride in the presence of triethylamine to provide
the methacrylated PLGA (mPLGA) macromer. The structure of this
macromer that contains a double bond group was characterized
with H NMR (Fig. 1a) and FT-IR (their data was not shown)
spectroscopy.
The peak “a” was attributed to the methylene protons of the
glycolic acid segment and peaks “b” and “c” were assigned to the
methyne and pendent methyl protons of lactic acid domain,
respectively.
1

420
S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
Fig. 1. 1H NMR spectra of (a) methacrylated PLGA (mPLGA) macromer, (b) acrylated PEG (aPEG) macromer and (c) methacrylated ethylene diamine (Boc-AEMA) monomer.
In order to make the copolymer more compatible with biolog-
ical systems, we have designed hydrophilic polyethylene glycol-
000 was modified to be incorporated into the copolymer chain
Scheme 1, step 2). Fig. 1b displays the spectrum of acrylated PEG
and peaks “g”, “h”, “i”, and “j” were attributed to the methylenic
PEG groups. The expanded region for “l”, “k” and “m” peaks of the
acrylic group has been demonstrated, which reveals the prepara-
tion of modified PEG macromer.
as AIBN amount (Table 2). Due to ambivalent nature of copolymers,
the new hydrophilic/hydrophobic material has an improved prop-
erties compared with the initial modified monomers. Conse-
quently, the smallest nano-sized formulation has been selected for
drug carrier.
2
(
The structure of resulting brush-like copolymer that contained
1
three modified monomers was determined by H NMR spectros-
copy. The composition of each grafted group in the soluble copol-
ymer was measured by the integral of characteristic peaks of PLGA
(at 5.18 ppm), PEG (at 3.65 ppm) and Boc-AEMA (at 3.39 ppm)
(Fig. 2a). According to this calculation, the average number of each
monomer in the targeted brush-like copolymer was about 1 PLGA
macromer, 2 PEG macromers and 11 Boc-AEMA monomers in the
repeating unit. Therefore the molecular weight of corresponding
copolymer should be around 12 kDa, which is responsible for the
formation of smaller-size nanomicelles.
N-2-[(tert-butoxycarbonyl)-amino] ethyl methacrylamide (Boc-
AEMA) is a pH-sensitive monomer which can be synthesized
successfully by consecutive protection and deprotection of one of
the two amino groups of ethylene diamine. Boc-AEMA has the
necessary features as a carrier for the development of targeted anti-
cancer delivery systems. It also possesses the versatility of facile
modification and functionalization of folate nanoparticles via the
amino groups to achieve dual-targeted nanocarriers. Boc-AEMA
was synthesized from the reaction of methacryloyl chloride with
N-2-[(tert-butoxycarbonyl)-amino] ethyl amine according to the
previously reported procedure (Scheme 1, step 3) [29]. Fig.1c shows
Table 2
1
the H NMR spectra of methacrylated-EDA (Boc-AEMA) monomer.
Chemical composition and physicochemical properties of brush-like copolymers.
Peaks “o”, “p”, “q”, “r”, “s”, “t”, “u” and “n” were assigned to the
AEMA and terminal methyl protons of the Boc-AEMA pendant
group, respectively.
The modified monomers were copolymerized using free radical
copolymerization according to Scheme 2. The brush-like amphi-
philic copolymers with different compositions were synthesized by
varying ratios of the three components in the initial mixture as well
PLGA/PEG/AEMAa AIBNHY (%) Encapsulation efficiency (%) Particle size (nm)
b
2
2
/2/12
/2/12
3
6
3
6
70.1
62.4
57.3
54.0
337
190
159
84
1/2/12
1/2/12
a
Feed molar ratio of monomers.
The molar ratio of initiator to total monomers.
b

Scheme 2. Preparation of amphiphilic brush copolymer via addition copolymerization.
Fig. 2. 1H NMR spectra of (a) t-Boc-protected brush copolymer, (b) deprotected brush copolymer and (c) folate targeted brush copolymer.

422
S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
Scheme 3. Synthetic route for preparation of dual-targeted brush-like copolymer.
Scheme 4. Schematic representation of preparation of the drug-loaded pH-responsive nanoparticles.

S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
423
Fig. 3. SEM images of non-folated nanoparticles at pH 5.8 (a) and pH 7.4 (b).
Here, mono-Boc-protected copolymer was converted into the
corresponding amino-terminated copolymer. Deprotection of t-Boc
group was carried out in trifluoroacetic acid/chloroform and ethyl
acetate solvent mixture (v/v, 0.3/14) for 1 h at room temperature.
Decreasing of pH could make nanoparticles bigger again, so the
nanoparticles have a pH-dependent size as shown in Fig. 4. The
difference in size between DLS and SEM results is due to the fact
that DLS gives the hydrodynamic radius, which strongly depends
on the surface charge. In acidic media, surface charge density
increases and consequently, its mean hydrodynamic size increases.
The zeta potentials of the nanoparticles in different pH have been
measured to determine the stability and the surface charge of the
nanoparticles. The absolute values of zeta potential for nano-
particles at pH 7.4 and 5.8 are ꢂ17.54 and ꢂ9.54 mV respectively.
This means that the absolute zeta potential value decreases with
decreasing pH of the medium and colloidal nanoparticles tend to
aggregate and result in larger particles. As confirmed by Scheme 4
at pH 5.8, the AEMA chains were protonated and became water
soluble. The hydrophobic PLGA chains with hydrophobic drug
molecules associated as the hydrophobic core with the PEG and
protonated AEMA as the corona. After the solution pH rose to 7.4,
the AEMA chains were deprotonated, became hydrophobic and
thus collapsing on the PLGA core as the hydrophobic middle layer.
The PEG chains formed the hydrophilic corona too.
1
Disappearance of the tert-butyl peak at 1.45 ppm of H NMR
spectrum (Fig. 2b) depicts complete removal of the Boc group.
As shown in Scheme 3, deprotected brush-like copolymer was
then coupled with activated folic acid by using of DCC. Folate-NHS
was prepared to activate the folate carboxylic groups for coupling
with terminal amino groups of the AEMA. The reaction between
folate-NHS with the terminal amine in deprotected copolymer led
to the formation of a covalent amide bond. The degree of substi-
1
tution was evaluated to be 91% from H NMR by considering the
integral of the NH
PEG backbone protons (
2
protons of deprotected AEMA (
d
¼ 3.67) to the
d
¼ 3.51) (Fig. 2c).
3.2. Nanoparticle preparation
The nanoparticles were prepared in a single step by nano-
precipitation method. The fabrication of the nanoparticle is shown
in Scheme 4. The brush-like copolymer was firstly dissolved in
acetone and DMSO as organic solvent and then dispersed in water
to produce corresponding micellar nanoparticles.
3.4. Drug loading into the folated and non-folated nanoparticles
Quercetin is a potent anti-cancer drug with poor water solu-
3
.3. Size and morphology of the brush-like copolymer micelles
bility. Quercetin was loaded into the nanoparticles by a nano-
precipitation method. EE of the folated nanoparticles was found to
be higher than that of non-folated ones, which may be due to the
Fig. 3 shows the SEM photographs of non-folated nanoparticles
at different pHs. Shape of the micelles at pH 5.8 was almost
spherical and the sizes were below 100 nm in diameter (Fig. 3a),
whereas at pH 7.4, a morphological change from spherical to bean-
like shape with a huge increase in size was observed. At pH 7.4, the
nanoparticles were neutral but gradually became positively
charged with decreasing pH to 5.8. This suggests that at low pHs,
the AEMA chains become positively charged and correspondingly,
the nanoparticles are arranged individually due to the electrostatic
repulsion. As the surrounding pH increased, AEMA was deproto-
nated and their tendency to get together was increased; this led to
the association of nanoparticles and conversion of their
morphology to bean-like (Fig. 3b).
Fig. 4 shows particle size distribution of folated and non-folated
micelles by DLS. The DLS results revealed that the mean size of
folated nanoparticles was larger than non-folated ones. By adding
folate to the copolymer structure, the diameter of nanoparticles
increased from 75 nm to 84 nm with increasing their dispersity
from 0.081 to 0.193 (Table 3).
Fig. 4. Size of non-folated (a), folated (b) micelles at pH 7.4 and folated (c) micelles at
pH 5.8.

424
S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
Table 3
Particle size, drug loading, and encapsulation efficiency of copolymeric micelles in aqueous solution.
Nanoparticles
Drug/copolymer ratio in feed (%)
Yield (%)
Drug loading (%)
Particle size (nm)
Encapsulation efficiency (%)
Non-folated
Folated
15
15
53
62
12.58
13.7
75
84
54
58
drugefolate interactions. The drug loading into the folated copol-
ymer was as high as 13.7% with an encapsulation efficiency of 58%,
while the EE amount of non-folated copolymer could reach up to
3.5. Release of quercetin from folated and non-folated
nanoparticles
5
4% with 12.58% drug loading. This comparison confirms that the
In-vitro drug release studies were carried out in various pH in
order to evaluate the effect of pH on quercetin-release profile. The
cumulative release of drug from the quercetin-loaded folated and
folated nanoparticles are more effective for encapsulating the
hydrophobic drug, which may be due to the establishment of the
strong interactions between folate and drug functional groups.
The correlation between drugefolate interactions and EE
amount has been recognized by UVeVisible spectrophotometer. As
shown in Fig. 5, free quercetin displays two major absorption bands
in the UV/Vis region. The broad peak with a maximum at 380 nm
ꢀ
non-folated copolymer in pH 5.8 and 7.4 at 37 C has been shown in
Fig. 6. Quercetin release from both copolymers was pH-dependent.
The drug release rate at pH 5.8 was much faster than that of pH 7.4.
The results indicate a conformational change in AEMA chains from
a compacted shape to an expanded one with a decrease in the pH
values. In expanded conformation, drug can diffuse out from the
nanoparticles easier than in compacted form and the release rate of
entrapped drug in the compacted form is reduced by condensed
polymeric layers. Furthermore, drug release from non-folated
nanoparticles in comparison with the folated one at both pH was
slightly slower. The faster drug release of quercetin from the folated
nanoparticles is due to the hydrophilicity of folic acid and its folate
form. This property facilitates the water uptake of nanoparticles
and causes a faster drug release. Hence, it is important to note that,
this kind of copolymer has potential targeting capability as well as
negligible effect on the rate of drug release.
belongs to 10pe due to the contribution of p electrons from eOH
group till carbonyl group (blue line) and a narrower peak with
a maximum at 258 nm corresponds to hydroxyl substitutions on
the benzene rings. Non-folated copolymer doesn’t show any
specific absorption in its UV/Vis spectrum due to the absence of
conjugated chromophore and allowed transitions in copolymer
structure. Folated copolymer shows their maximum absorption at
2
81 nm related to the n / p pe resonance system
* transitions by 6
as shown in Fig. 5 and Scheme 5. The UV spectrum of quercetin-
loaded folated nanoparticles exhibits an approximately 12 nm
hypsochromic shift. This blue shift with an intense increase in the
absorbance is caused by the decrease in conjugated system length
and
transfer complex (CT complex) formation between folate group of
the copolymer and quercetin could convert the forbidden n /
transitions to the allowed
* ones. This conversion leads to
producing an -unsaturated ketone with different chromophores
at position. In contrary, disappearance of the quercetin broad
peak at 380 nm (10 e) after CT complex formation is due to the
p / p
* transitions, respectively. It is observed that charge
3
.6. Release kinetics and mechanism of quercetin-loaded
nanoparticles
p
*
p
/ p
To investigate the kinetics of drug release from folated and non-
folated nanoparticles, the plots of accumulated drug release as
a function of the square root of time at pH 5.8 and 7.4 were plotted
a,b
b
p
(Fig. 7).
hiding of this peak in the folate one according to the low quercetin
concentration in nanoparticles formulation.
Linear relationship is an indicative of diffusion-controlled
mechanism for drug release from the polymeric nanocarrier.
Release profiles from quercetin-loaded nanoparticles at pH 7.4 had
minimum 2 h delay in comparison with similar nanoparticles at pH
5.8 and this was due to the protection of the AEMA-middle layer at
higher pH. All release profiles show two release-stages i) first
release-stage that is characterized by the steeper linear region, and
ii) second release-stage that is characterized by the second linear
region occurring at later time. The slopes of these linear regions for
the polymer samples show that the kinetic of release for the first
stage at different pH are obviously different, but that of the second
stage are completely similar. This means that only the first step of
release is affected by surrounding pH and the second one remains
unaffected. To prove this claim, the release data were analyzed on
the basis of Higuchi equation and KorsmeyerePeppas kinetics. The
release rates, k and n of each model were calculated by linear
regression analysis using Microsoft Excel 2007 software. Coeffi-
2
cients of correlation (r ) were used to evaluate the accuracy of the
2
fit. The r , n and k values have been given in Table 4.
2
Based on the calculations and comparing r values for Higuchi
model, all formulations except the fourth one, gave good fit to the
Higuchi model. According to this model, the drug release from
these nanoparticles may be controlled by nanopore diffusion. All
carriers were best fitted into the KorsmeyerePeppas model and
both nanoparticles showed a Fickian release at second stage of pH
5.8 and pH 7.4. Fickian drug release is identified by a linear
Fig. 5. UVeVis spectra of free quercetin (dd), non-folated copolymer (d d), folated
copolymer (e e e) and quercetin-loaded folated nanoparticles (//) in DMSO.

S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
425
Scheme 5. The structure of quercetin, folated copolymer (left) and folated copolymerequercetin charge transfer complex (right).
dependency of the released drug with the square root of time that is
concentration dependent. The fundamental of diffusion is based on
Fick’s laws, which describes the macroscopic transport of molecules
by a concentration gradient. These nanoparticles showed a non-
Fickian or anomalous release in the first stage of both pHs and
the drug release varies with time t according to the power law.
As, degradation rate depends on the pH of the in-vitro medium,
additional experiments were carried out to investigate the degra-
dation of amine-modified brush-like copolymer in buffer solution
occurring in the core [35]. Fig. 8 shows the weight change and SEM
pictures of in-vitro degradation of folated brush-like copolymer in
pH 7.4 and 5.8. It can be seen that this copolymer has a slow
degradation over 5 weeks period and at both pHs. The reason for
observing this phenomenon may be the decrease in molecular
weight of hydrophobic parts and subsequently, the short polymer
chains may have less chance to be attacked by water molecules
which is required for polymer chain hydrolysis or biodegradation
[36]. For these nanoparticles, the patterns of mass loss were divided
into two phases. During the initial 4 days, the rate of mass loss is
almost similar for both pH media and after that, the rate of mass
loss at pH 5.8 in comparison with pH 7.4 is increased. During 5
(
PBS) at the desired pH values (5.8 and 7.4). In general, two
mechanisms are considered for degradation of polymers: i) bulk
erosion and ii) surface erosion. It is well-known that bulk erosion is
the degradation process of PLGA and acidic conditions catalyze this
kind of degradation [34]. In surface erosion, degradation mecha-
nism proceeds from the outside to the inside with no reaction
Fig. 7. The accumulated quercetin release from folated nanoparticles (at pH 5.8 (A)
ꢀ
Fig. 6. In-vitro release profiles of quercetin from folated nanoparticles (at pH 5.8 (A)
and 7.4 (C)) and non-folated nanoparticles (at pH 5.8 (:) and 7.4 (-)) at 37 C (n ¼ 3).
and 7.4 (C)) and non-folated nanoparticles (at pH 5.8 (:) and 7.4 (-)) at 37 C as
a function of the square root of time (n ¼ 3).
ꢀ

426
S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
Table 4
Statistical values for the release rate modeling.
Entry
Nanoparticles
pH
Stage
Higuchi
KorsmeyerePeppas
Release mechanism
k
r2
k’
r2
n
1
2
3
4
5
6
7
8
Non-folated
5.8
7.4
5.8
7.4
1st
2nd
1st
2nd
1st
2nd
1st
19.765
3.642
11.379
4.33
22.128
4.098
11.447
4.158
0.984
0.984
0.986
0.977
0.991
0.993
0.987
0.994
1.1997
1.6024
0.5523
1.2577
1.2621
1.6194
0.7029
1.3105
0.991
0.997
0.992
0.997
0.999
0.997
0.990
0.998
0.559
Anomalous
Fickian
Anomalous
Fickian
Anomalous
Fickian
Anomalous
Fickian
0.1597
0.8262
0.2814
0.560
0.1671
0.7492
0.263
Folated
2nd
ꢀ
Fig. 8. The residual weight percent and surface morphology of folated nanoparticles incubated in PBS at pH 5.8 (-) and 7.4 (C) at 37 C.
weeks incubation of these nanoparticles under pH 5.8 and 7.4, 3.2
and 1.4% decrease in mass were observed respectively.
the ratio of monomers in the preliminary mixture. Among different
ratios, the most soluble copolymer and smallest nanoparticles were
obtained from PLGA:PEG:AEMA system with 1:2:11 ratio. Folated
copolymers were successfully synthesized by two-step
deprotectionecondensation reactions. Folated and non-folated
copolymers were self-assembled into layered structures, smaller
than 100 nm in diameter with a hydrophobic PLGA in the core with
potent hydrophobic drug loading capability up to 13.7 wt%. The
hydrophobic AEMA block contains amido-amine pendent groups
and hydrolyzes when the surrounding pH is changed from neutral
to acidic. Changing in pH of the medium leads to the change in
nanoparticle size, morphology and characteristics of AEMA from
lipophilic to hydrophilic or vice versa. Shape of the micelles in
acidic pH was spherical and the sizes were below 100 nm in
diameter, whereas a morphological change from spherical to bean-
like shape with a large increase in size was observed in neutral
media by SEM pictures. The pH-sensitive nanoparticles could
deliver the pre-determined encapsulated drug more rapidly at
acidic pH compared to neutral one and hence, they can be highly
efficient carrier for rapid tumoricidal action. Kinetic studies
confirmed that all release profiles showed two release-stages and
the drug release from these nanoparticles might be controlled by
Fick’s laws specifically in acidic pH. It can be concluded that this
kind of nanocarriers, which includes drug targeting with sensing
SEM was carried out to study the surface morphology of incu-
bated nanoparticles. The fresh nanoparticles had smooth spherical
shape, while after 4 days, a dramatic change in nanoparticle shape
was observed. At pH 7, nanoparticles no longer retained their
spherical shape but at pH 5.8, they completely lost their spherical
shape and coalesced to make a smooth film. This can be explained
by the accelerated cleavage of the ester bonds under acidic condi-
tions and their subsequent reaction with amine groups during
degradation process [37]. On the 36th day, a porous surface resulted
for incubated nanoparticles at pH 7.4, which implies bulk or semi-
bulk degradation of nanoparticles, whereas, at pH 5.8 eroded
surface was reconstructed.
4
. Conclusion
Addition copolymerization of methacrylated PLGA, acrylated
PEG and BOC-protected amino functional acrylate was successfully
performed achieving amphiphilic and pH-sensitive brush copoly-
mers. It was further shown that the monomers’ ratio has an
important role on the solubility of copolymers and their nano-
particle size. Molecular weight and hydrophilic to lipophilic
balance (HLB) of copolymers could be easily controlled by adjusting

S. Khoee, R. Rahmatolahzadeh / European Journal of Medicinal Chemistry 50 (2012) 416e427
427
and therapy at the same time, has an excellent potential for the
simultaneous diagnosis and therapy of cancer. Degradation studies
of the copolymer were performed in buffers with varied pH values.
SEM images of degraded copolymeric nanoparticles proved the
degradation and suggested that surface erosion was occurred in
acidic media, while, bulk erosion was observed after incubation of
nanoparticles in PBS buffer at pH 7.4.
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