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as doi: 10.1002/pi.5987
PEGylated hollow pH-responsive polymeric nanocapsules for controlled
drug delivery
Bakhshali Massoumi1, Mojtaba Abbasian1, Rana Jahanban-Esfahlan2, Sanaz Motamedi1, Hadi
Samadian3, Aram Rezaei3, Hossein Derakhshankhah4, Amir Farnudiyan‐Habibi5,6, and Mehdi
Jaymand∗,3
1. Department of Chemistry, Payame Noor University, Tehran, Iran.
2. Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of
Medical Sciences, Tabriz, Iran.
3. Nano Drug Delivery Research Center, Health Technology Institute, Kermanshah University of Medical
Sciences, Kermanshah, Iran.
4. Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences,
Kermanshah, Iran.
5. Department of Pharmaceutical Biomaterials, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran.
6. Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran.
∗
Correspondence to: Mehdi Jaymand, Nano Drug Delivery Research Center, Health Technology Institute,
Kermanshah University of Medical Sciences, Kermanshah, Iran.
E-mail addresses: m_jaymand@yahoo.com; m.jaymand@gmail.com; mehdi.jaymand@kums.ac.ir
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Abstract
Novel pH-responsive PEGylated hollow nanocapsules (HNCaps) were fabricated through
a combination of distillation–precipitation copolymerization and surface thiol-ene “click”
grafting reaction. For this purpose, silica nanoparticles (SiO2 NPs) were synthesized by
Stöber approach, and then modified using 3-(trimethoxysilyl) propyl methacrylate (MPS).
Afterward, a mixture of triethyleneglycol dimethacrylate (TEGDMA; as crosslinker),
acrylic acid (AA; as a pH-responsive monomer), and MPS-modified SiO2 NPs (as
sacrificial template) were copolymerized using distillation–precipitation approach to afford
SiO2@PAA core-shell NPs. The SiO2 core was etched from the SiO2@PAA using HF
solution, and the obtained PAA HNCaps were grafted with a thiol-end capped
poly(ethylene glycol) (PEG-SH) through thiol-ene “click” reaction to produce PAA-g-PEG
HNCaps. The fabricated HNCaps were loaded with doxorubicin hydrochloride (DOX) as
a model anticancer drug, and their drug loading and encapsulation efficiencies as well as
pH-dependent drug release behavior were investigated. The anticancer activity of the drug-
loaded HNCaps was extensively evaluated using MTT assay against human breast cancer
cells (MCF7). As the cytotoxicity assay results as well as superior physicochemical and
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biological features of fabricated HNCaps, the developed DOX-loaded nanomedicines has
excellent potential for cancer chemotherapy.
Keywords: Hollow nanocapsules, pH-Responsive, PEGylation, Nanomedicine, Cancer
chemotherapy
1. Introduction
At current time, cancer is one of the most important health threats with an increasing
incidence worldwide [1]. The conventional strategies (e.g., radiation therapy) has some
unwanted outcomes, including treat both cancerous and surrounding normal tissues
simultaneously, which led to poor therapeutic effect on tumors and severe toxic side effects
on normal tissues. Given this fact, design and development of personalized medicine to
address traditional cancer therapy issues is pivotal for coming [2-5]. In this context, the
advent of nanotechnology open new opportunities for design and development of de novo
drug delivery systems (DDSs) in the nanometer-sized domain. Since the first use of
nanoparticles (NPs) in the late 1960s by Speiser and his colleagues for biomedicinal
applications, known as nanomedicine, this field progressed significantly [6, 7].
Polymeric hollow nanocapsules (PHNCaps) are one of the valuable advances and
innovative achievements in the field of nanomedicine [8-10]. These type of NPs consist of
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a hollow inner core surrounded by a polymeric shell, have received a great deal of attention
mainly due to their potential biomedical applications, including drug or gene delivery
vehicles, nanoreactors, and protective shells for enzymes [11, 12]. In the case of DDSs,
hollow nanocapsules (HNCaps) exhibit excellent capability for encapsulation of large
quantities of guest molecules (e.g., drug or other bioactive agents) within their inner cavity.
In addition, HNCaps have excellent stability, relatively long circulation in blood, and better
mechanical properties than those of the other DDSs (e.g., liposome, and solid lipid
nanoparticles (SLNs)) [13, 14]. Some synthetic strategies, including layer-by-layer self-
assembly, sol–gel process, template synthesis, crosslinking of micelles, directed self-
assembly approaches, and heterophase polymerization have been applied for the
fabrication of HNCaps [15-17]. In this context, heterophase polymerization approaches
(e.g., emulsion, suspension, and dispersion) are particular of interest due to their
simplicities and efficiencies toward the fabrication of HNCap with well-defined
morphology and shell thickness [17].
On the other hand, the “smart” HNCaps consist of stimuli-responsive polymeric shell have
received increasing attention due to their high flexibility in tuning the permeability. In
DDSs based on “smart” HNCaps, the encapsulated drug can be released owing to response
to external triggers such as pH, temperature, electric field, and ionic strength [17-19].
Among the above mentioned stimuli, the design and development of pH-responsive DDSs
is particular of interest due to simplicity and efficiency of this stimuli in comparison with
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others [20, 21]. In addition, it is worth noting that cancerous tissues have lower pH values
than those of the surrounding normal tissues, which originate from abnormal metabolism
of these tissues [22, 23]. This fact is the key point for the intense interest toward the design
and development of pH-responsive polymeric nanosystems for drug delivery purposes.
In general, pH-responsive polymers processes monomeric units containing basic (e.g.,
ammonium salt) or acidic (e.g., carboxyl) functionalities, which can either accept or release
protons in response to pH changes, accompanying reversible variation in volume, solubility
and equipoise between the extended and collapsed states [20, 21]. In this context,
poly(acrylic acid) (PAA) is the most famous pH-responsive polymer with pendent carboxyl
group in each repeat unit and received a great deal of interest due to its superior
physicochemical as well as biological features [20, 24].
In this contribution, novel pH-responsive PEGylated hollow nanocapsules (HNCaps) were
fabricated through a multistep process. In first stage, the SiO2@PAA core-shell NPs were
synthesized through distillation–precipitation copolymerization of MPS-modified SiO2
NPs, AA, and TEGDMA. Afterward, the SiO2 core was etched from the SiO2@PAA NPs
using HF solution, and the obtained PAA HNCaps were grafted with a PEG-SH through
thiol-ene “click” reaction to produce PAA-g-PEG HNCaps. The fabricated HNCaps were
loaded with DOX as a model anticancer drug, and their drug loading and encapsulation
efficiencies as well as pH-dependent drug release behavior were investigated. The
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anticancer drug delivery performance of PAA-g-PEG HNCaps was evaluated through
MTT assay against MCF7 cell line.
2. Experimental
2.1. Materials
Acrylic acid monomer (Merck, Darmstadt, Germany) was distilled in reduced pressure,
and stored at −20 °C prior to use. The initiator 2,2′-azobisisobutyronitrile (AIBN; Fluka,
Switzerland) was recrystallized from ethanol at 50 °C before use. Tetrahydrofuran (THF)
was purchased from Merck, dried by refluxing over sodium, and distilled under argon prior
to use. Poly(ethylene glycol) monomethylether (Mn=2000 gmol-1), triethyleneglycol
dimethacrylate (TEGDMA), succinic anhydride (SA), N-(3-dimethylamino propyl-N-
ethylcarbodiimide) hydrochloride (EDC•HCl), N-hydroxysuccinimide (NHS), 3-
(trimethoxysilyl) propylmethacrylate (MPS), tetraethyl orthosilicate (TEOS), and
ammonium hydroxide (25% of ammonia) were purchased from Sigma-Aldrich (St. Louis,
MO, USA) and were used as received. Doxorubicin hydrochloride (DOX) was purchased
from Zhejiang Hisun Pharmaceutical Co., Ltd., Taizhou, China. Phosphate buffered saline
(PBS), fetal bovine serum (FBS), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide), and other biological reagents were purchased from Invitrogen
(Carlsbad, CA, USA) and were used as received. All other reagents were bought from
Merck or Sigma-Aldrich and purified according to the standard methods.
2.2. Synthesis of SiO2 NPs
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Silica NPs were synthesized by the hydrolysis of TEOS in ethanol medium in the presence
of ammonium hydroxide as the catalyst. In a typical experiment, ethanol (16 molL−1) was
taken and kept in a sonication bath. After 15 minutes, TEOS (24 mmolL−1) was added
dropwise under sonication. At the end of this stage, ammonium hydroxide (25%, 2
mmolL−1) was added as the catalyst to promote the condensation reaction. Afterward, the
content of the reactor was sonicated for another 2 hours to get a white opalescent
suspension. The synthesized SiO2 NPs were precipitated by centrifugation at 6000 rpm for
about 15 minutes. The white powder was then re-dispersed in ethanol and re-precipitated
by centrifugation several times, in order to remove unreacted reagents. The white powder
obtained was dried under vacuum at room temperature.
2.3. Synthesis of MPS-modified SiO2 NPs
In a typical experiment, SiO2 NPs (1.00 g) was suspended in ethanol (100 mL) through the
sonication for about 20 minutes. The mixture was then transferred into a 250-mL round-
bottomed flask equipped with condenser, and a magnetic stirrer. The silane coupling agent
(MPS; 1.00 mL, 4.2 mmol) was added dropwise to the reaction mixture. The content of the
flask was refluxed overnight at 65 °C. At the end of this time, the MPS-modified SiO2 NPs
were precipitated by centrifugation at 6000 rpm for 15 minutes. The modified NPs were
then re-dispersed in ethanol and re-precipitated by centrifugation several times, in order to
remove unreacted silane coupling agents and byproducts. The product obtained was dried
in a reduced pressure at room temperature.
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2.4. Synthesis of SiO2@PAA core-shell NPs
The SiO2@PAA core-shell NPs were synthesized through distillation–precipitation
copolymerization of AA monomer with MPS-modified SiO2 NPs as template. It should be
pointed out that TEGDMA was used as crosslinker. For this purpose, a 50-mL three-necked
round-bottomed flask equipped with a condenser, gas inlet/outlet, and a magnetic stirrer
was charged with MPS-SiO2 (0.50 g), dried 1,4-dioxene (20 mL), AA monomer (0.35 mL,
5 mmol), and TEGDMA (52 µL, 0.2 mmol). The reaction mixture was stirred for about 1
hours under argon protection in order to obtain a homogeneous suspension. Afterward,
AIBN (20 mg, 0.12 mmol) was added to the flask, and the reaction mixture was refluxed
for about 12 hour at 80 °C. At the end of this time, the content of the flask was cooled using
an ice/water bath in order to stop the copolymerization. The SiO2@PAA NPs were
collected through centrifugation at 6000 rpm for 15 minutes, and washed by
tetrahydrofuran (THF) and ethanol, respectively. The obtained product was dried under
reduced pressure at room temperature.
2.5. Synthesis of PAA HNCaps
The PAA HNCaps were synthesized through HF etching of the SiO2 core from SiO2@PAA
NPs. Briefly, SiO2@PAA NPs (0.50 g) were stirred in a HF solution (50 mL; 20%) at room
temperature for about 24 hours to dissolve the silica core. The mixture was dialyzed in
double distilled water (DDW) for three days using 1000 molecular weight cut-off dialyzed
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bag in order to remove the excess HF and SiF4 byproduct. Finally, the PAA HNCPs were
collected through freeze-drying.
2.6. Synthesis of carboxyl-end capped PEG (PEG-COOH)
The PEG-COOH was synthesized through the reaction between PEG and succinic
anhydride (SA). In a typical experiment, a 50-mL round-bottomed flask equipped with
condenser and a magnetic stirrer was charged with poly(ethylene glycol) monomethylether
(2.50 g , 1.25 mmol), SA (0.15 g, 1.5 mmol), and dried THF (30 mL). The reaction mixture
was refluxed at 60 °C overnight. At the end of this time, the solvent (THF) was evaporated
under reduced pressure, and the obtained crude product was extracted using CH2Cl2/DDW
(50:50 v/v) three times, in order to remove unreacted SA. The organic phase was dried
using Na2SO4 and evaporated under a reduced pressure to afford a PEG-COOH.
2.7. Synthesis of thiol-end capped PEG (PEG-SH)
The PEG-SH was synthesized through the esterification of PEG-COOH using 3-amino-1-
propanethiol hydrochloride in the presence of EDC and NHS. For this purpose, a 25-mL
three-necked round-bottomed flask equipped with condenser, gas inlet/outlet, and a
magnetic stirrer was charged with PEG-COOH (0.85 g , 0.40 mmol), EDC (80 mg, 0.40
mmol), NHS (48 mg, 0.40 mmol), and dried CH2Cl2 (20 mL). The reaction mixture was
stirred for about 4 hours, and then 3-amino-1-propanethiol hydrochloride (50 mg, 0.40
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mmol) was added to the flask. The content of the flask was stirred for about 24 hours at
room temperature. At the end of this time, the crude product was extracted with
CH2Cl2/DDW (50:50 v/v) in order to remove residual reagents or by products. The organic
phase was dried using Na2SO4 and evaporated under reduced pressure to afford a PEG-SH.
2.8. Synthesis of PAA-g-PEG HNCaps
The PAA-g-PEG HNCaps were synthesized through a thiol-ene “click” reaction as follows.
A 25-mL three-necked round-bottomed flask equipped with condenser, gas inlet/outlet, and
a magnetic stirrer was charged with PAA HNCaps (0.30 g), PEG-SH (0.10 g, 0.045 mmol),
and dried 1,4-dioxane (15 mL). The content of the falsk was stirred for about one hour, and
then de-aerated through the bubbling of highly pure argon for some minutes. Afterward,
the initiator (AIBN, 20 mg, 0.12 mmol) was dissolved in 1,4-dioxane (5 mL), de-aerated
using argon gas, and then added to the reaction mixture under an argon protection. The
reaction was allowed to proceed for about 24 hours at 80 °C. At the end of this time, the
synthesized PAA-g-PEG HNCaps was obtained through centrifugation, followed by
washing using THF and ethanol, respectively in order to remove un-grafted PEG chains.
2.9. Preparation of DOX-loaded PAA-g-PEG HNCaps
The PAA-g-PEG/DOX nanomedicine was formulated through the stirring a mixture of
PAA-g-PEG HNCaps (0.30 g) and DOX (20 mg) in DDW (20 mL) for about 48 hours in
the dark at room temperature. At the end of this time, the mixture was filtered using ultra-
filtration method through centrifugal devices (Amicon® Ultra-4 12 kDa, Millipore,
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Temecula, CA, USA) to separate unloaded DOX. The encapsulation (EE) and loading (LE)
efficiencies of the PAA-g-PEG HNCaps were determined through the analyzing of ultra-
filtrate solution for determination of unloaded drug concentration using a UV-vis
spectrophotometer at 480 nm according to following equations.
EE(%)=(− )
× 100
LE(%)=(− )
× 100
In these equations CT is total amount of DOX which taken for loading, CDOX is DOX
concentration in ultra-filtrate solution, and WS is the mass of sample which used for DOX
loading [25].
2.10. In vitro DOX release study
The in vitro release of DOX form the developed PAA-g-PEG/DOX nanomedicine was
evaluated under various pH values (4.0, 5.4, and 7.4) according to our previously reported
procedure [25]. For this purpose, the PAA-g-PEG/DOX nanomedicine was suspended in
PBS sealed in a dialysis membrane bag with a molecular cut-off of 2 kDa, and placed in
PBS (0.01 moll-1, 100 mL, pH=4.0, 5.4, and 7.4) as the release medium. The system was
kept at 37 °C with gentle stirring for predetermined times. Aliquots of release medium (1
mL) were collected at predetermined time intervals, for determination of released DOX
using a UV–vis spectrophotometer at 480 nm. It is worth noting that the aliquots were
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brought back into the reactor to avoid volume change. The drug release values were
calculated according to following equation:
R=M1/M0
In this equation M1 is the cumulative mass of DOX released from the sample at a given
time, and M0 is the total loading amount of DOX in the sample that determined using UV–
vis spectroscopy at 480 nm as described above.
2.11. Cell culture
The MCF7 cells was provided from Iranian National Cell Bank (Pasteur Institute, Tehran,
Iran), and cultivated in RPMI1640 possessing 100 IU penicillin per 100 mg streptomycin,
enriched with 10% (v/v) of FBS. The cells were cultured into flasks and kept in a humidified
incubator at 37 °C and the media refreshed every two days [26].
2.12. In vitro cytotoxicity assay
The well-known MTT assay was applied to evaluate the cytotoxic effects of the developed
PAA-g-PEG/DOX nanomedicine versus free DOX as the reference. In a typical
experiment, the MCF7 cells was trypsinized, harvested and seeded in 96-well plates. After
overnight incubation, the cells were treated with various concentrations (5.0, 10.0, 20.0,
50.0, and 100.0 µgmL-1) of PAA-g-PEG/DOX nanomedicine (based on concentration of
DOX) and free DOX for 24 and 48 hours. After mentioned times, the media was removed
and MTT solution (50 µL) was added to each well followed by 150 µL cultivation medium,
then placed in humidified incubator for another 4 hours. At the end of this time, the
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remaining MTT solution aspirated, the formed formazan crystals were dissolved in
dimethyl sulfoxide (DMSO; 200 µL) containing Sorenson buffer (25 µL) and absorbance
was measured at 570 nm using a spectrophotometric plate reader [26]. The cell viability
(%) was calculated using the following equation. In order to quantification of cytotoxic
effects.
Cell viability (%) = [A570(sample)/A570(control)] × 100
In this equation, A570(sample) and A570(control) represent the absorbances of the sample
and control wells, respectively.
2.13. Statistical analyses
One-way analysis of variance was performed to examine differences in the average values
between groups and the data were analyzed by SPSS 19.0 software (SPSS Inc., Chicago,
IL, USA). Each result is presented as mean±SD. A value of P<0.05 was considered
statistically significant.
2.14. Instrumentation
Fourier transform infrared (FTIR) spectroscopy was performed using a Shimadzu 8101M
FTIR (Kyoto, Japan) at room temperature. The samples were prepared in the pellet form
with potassium bromide (KBr) powder. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on an FT-NMR Bruker spectrometer (Bruker, Ettlingen, Germany)
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with an operating frequency of 400 MHz at 25 °C. Transmission electron microscopy
(TEM) images were obtained using a CM10-TH microscope (Philips, Eindhoven, The
Netherlands) with a 100 kV accelerating voltage. Thermal properties of the synthesized
samples were examined using thermogravimetric analyzer (TGA-PL STA 1640 equipment
(Polymer Laboratories, Shropshire, UK)). The thermogravimetric analysis (TGA)
experiment was conducted under nitrogen atmosphere from room temperature to 700 °C
with heating rate of 10 °Cmin−1. Dynamic light scattering (DLS) experiments were carried
out using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) at room
temperature. Samples were prepared as 0.5% (w/v) solutions in DDW. The powder X-ray
diffraction (XRD) experiment was performed using a Siemens D5000 diffractometer
(Aubrey, Texas, USA), X-ray generator (CuKα radiation with λ=1.5406 Å) in the scan
range from 2 to 80° (2θ) at room temperature. Ultraviolet-visible (UV-vis) spectra were
recorded with a Shimadzu 1650 PC UV-vis spectrophotometer (Kyoto, Japan).
3. Results and discussion
Among the numerous DDSs, polymeric HNCaps have received a great deal of interest due
to their superior features, including high specific surface area, low density, large pore
volume, and mechanical stability that lead to high drug loading capacity and improving
drug stability. In this context, the stimuli-responsive polymeric HNCaps are particular of
interest due to their “smart” and controlled drug release features. According to these facts,
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the aim of this study was to the design and development of novel PEGylated pH-responsive
HNCaps for “smart” chemotherapy of solid tumors (Scheme 1).
(Scheme 1)
3.1. Characterization of SiO2@PAA NPs
The DLS diagram of SiO2 NPs, SiO2@PAA NPs, and PAA HNCaps as well as XRD
pattern of the synthesized SiO2 NPs are shown in Figure 1. According to the DLS results
(Figure 1a) the size distribution of the synthesized SiO2 NPs, SiO2@PAA NPs, and PAA
HNCaps were obtained to be 24, 51, and 40 nm, respectively. In addition, polydispersity
index (PDI) of the SiO2 NPs, SiO2@PAA NPs, and PAA HNCaps were as 0.11, 0.13, and
0.16, respectively that indicate the relatively monodisperse synthesis of all samples.
The XRD pattern of SiO2 NPs (Figure 1b) exhibited a broad diffraction peak approximately
at 23°. This broad diffraction peak related to the small size and incomplete inner structure
of the SiO2 NPs. This demonstrates that the most of the SiO2 NPs are amorphous, but a few
of them are crystalline.
(Figure 1)
The FTIR spectra of the SiO2 NPs, MPS-SiO2 NPs, and SiO2@PAA NPs are shown in
Figure 2. The most important characteristic absorption bands in the FTIR spectrum of the
SiO2 NPs are symmetric and asymmetric stretching vibrations of the Si–O–Si at 791 and
936, and 1067 cm−1, respectively as well as the bending and stretching vibrations of the
surface hydroxyl groups at 1647 and 3360 cm−1, respectively.
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Silane coupling agents have been widely used for the surface modification of inorganic
NPs in order to prevent aggregation as well as further modification through their reactive
functional groups [27]. The appearance of new absorption band at 1722 cm−1 related to
carbonyl group as well as the stretching vibrations of aliphatic C–H at 2950–2800 cm−1
region confirms the successful surface modification of SiO2 NPs using MPS. Furthermore,
after modification of SiO2 NPs using MPS the stretching vibration of hydroxyl groups is
decreased due to coupling of MPS moiety.
The grafting and simultaneous crosslinking of AA monomer onto MPS-SiO2 NPs was
confirmed through the appearance of some characteristic bands, including the stretching
vibration of C–O group at 1196 cm-1, the bending vibration of –CH2 groups at 1467 cm-1,
the stretching vibration of carbonyl group at 1716 cm-1, the stretching vibrations of
aliphatic C–H at 2950 to 2850 cm-1 region, and the hydroxyl stretching vibration (related
to both PAA and SiO2 NPs) as a broad and strong band centered at 3465 cm-1 in the FTIR
spectrum of the SiO2@PAA NPs.
(Figure 2)
3.2. Characterization of PAA-g-PEG HNCaps
The FTIR spectra of the PEG, PEG-SA, PEG-SH, and PAA-g-PEG HNCaps are shown in
Figure 3. The FTIR spectrum of the neat PEG exhibited the stretching vibration of C–O at
1108 cm-1, the bending vibrations of –CH2 groups at 1467 and 1356 cm-1, the stretching
vibrations of aliphatic C–H at 2950 to 2800 cm-1 region, and the stretching vibration of
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hydroxyl or adsorbed water as a broad band centered at 3490 cm-1. The most important
change in the FTIR spectrum of PEG-SA is the appearance of a weak peak at 1732 cm-1
attributable to the carbonyl groups of SA moiety. As seen, the FTIR spectrum of PEG-SH
showed similar absorption bands with minor differences.
The FTIR spectrum of the synthesized PAA-g-PEG HNCaps represent characteristic bands
related to both PAA as well as PEG polymeric chains. The most important absorption bands
are labeled in the FTIR spectrum of the PAA-g-PEG HNCaps.
(Figure 3)
The synthesized PEG-SA, and PEG-SH were further characterized using 1H NMR
spectroscopy as shown in Figure 4. The neat PEG exhibited the chemical resonance of
hydroxyl end group at 2.30–2.60 ppm (a), –OCH3 group at 3.20 ppm (b), and O–CH2
groups of PEG backbone at 3.35–3.85 ppm (c, d). The successful synthesis of PEG-SA was
verified through the appearance of a new chemical shift at 2.85 ppm (e and f) related to –
CH2 groups of SA moiety. Finally, the chemical shifts at 2.35 (j) and 3.60 (k and h) ppm
confirm the synthesis of PEG-SH compound.
(Figure 4)
3.3. TEM observation
Figure 5, shows the TEM images of SiO2@PAA NPs (a) and PAA HNCaps (b). As seen,
the SiO2@PAA NPs exhibited spherical morphology with average sizes distribution of
40±10 nm. In this image, the dark spots attributable to the SiO2 NPs and light regions
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represent PAA matrix, which confirm the core–shell structure for the synthesized NPs. In
contrast, after HF etching of the SiO2 core from SiO2@PAA NPs the most of dark spots
disappeared at the TEM image due to removing of SiO2 core. As seen the PAA HNCaps
have spherical morphology with average size distribution of 30±10 nm.
(Figure 5)
3.4. Thermal property study
The thermal degradation of SiO2 NPs, MPS-SiO2 NPs, SiO2@PAA NPs, PAA HNCaps,
and PAA-g-PEG HNCaps in the course of heating under nitrogen flow are shown in Figure
6. As seen, the SiO2 NPs showed excellent thermal stability with only 8 wt.% weight loss
between 50 to 700 °C. This weight loss is started with evaporation of residual solvent
(ethanol) and adsorbed water (50 to 120 °C) followed by elimination of water molecules
from two unreacted silanol groups (120 to 220 °C), and after which the loss rate slows
down. By considering the TGA curve of MPS-SiO2 NPs, it was found that the weight
percentage of MPS in the modified NPs is about 5 wt.%. The MPS moiety is degrade in
the temperature range of 200 to 250 °C.
The characteristic TGA curve of SiO2@PAA NPs exhibited three-step weight loss
processes as follows. The first step involve the evaporation of residual solvent and adsorbed
water followed by elimination of water molecules from two unreacted silanol groups and
finally the degradation of modifier (MPS) (50 to 250 °C). The second step (250 to 320 °C)
is related to the de-carboxylation and anhydride formation in the PAA segments [28]. The
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final weight loss step (400 to 480 °C) is associated with the degradation of the poly(acrylic
anhydride) backbone, and after which the loss rate slows down. The residue at 700 °C for
this sample is about 54.5 wt.%, which most portion of this residue is related to the SiO2
NPs. The characteristic TGA curve of PAA HNCaps showed similar weight loss processes
as discussed above, and the residue at 700 °C for this sample is about 5.5 wt.%. This
quantity of residue confirmed the successful etching of the SiO2 core from SiO2@PAA
NPs using HF.
The TGA trace of PAA-g-PEG HNCaps also showed three-step weight loss processes. The
decomposition of this sample was started through the evaporation of solvent and adsorbed
water followed by dehydration and degradation of small molecules (e.g., MPS) (50 to 280
°C). The second step is corresponded to de-carboxylation and anhydride formation in the
PAA segments (280 to 380 °C). The final decomposition step is related to the degradation
of the poly(acrylic anhydride) and PEG backbones (380 to 470 °C), and after which the
loss rate slows down. The residue at 700 °C for this sample is about 4 wt.%. As seen, the
thermal stability of PAA HNCaps is improved slightly after grafting of PEG segments.
(Figure 6)
3.4. Investigation of self-assembly behavior
In all pH values the micelles with PAA inner shell and a PEG outer shell is expected.
However, at lower pH values (pH˂4.2) the PAA inner shell shrinkage due to fully
protonation of carboxyl groups (Scheme 2). In detail, at pH 4 the average size of these
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aggregates was found to be 85 nm. In contrast, when the pH value were increased to 5.5 or
7.4 the average sizes of the fabricated PAA-g-PEG HNCaps were increased to 102.2 and
121.5 nm, respectively due to deprotonation of PAA carboxyl groups. This phenomenon
may be originated from disappearance of the hydrogen bonding between the PAA segments
due to ionization of their carboxylic acid groups as well as electronic repulsion between
carboxylate groups (Figure 7).
(Scheme 2)
(Figure 7)
3.5. Stability of the micelles
The stability of a DDS in physiological condition has significantly affect its final
performance. The stability of the fabricated DDS was investigated through DLS
measurements of fresh micelles and incubated micelles for about 3 days in PBS at
physiological condition (pH=7.4 and temperature value of 37 °C). As seen in Figure 8, the
synthesized PAA-g-PEG HNCaps showed excellent stability after 3 days incubation. In
detail, the average hydrodynamic diameters of PAA-g-PEG HNCaps was increased only
23 nm due to slightly aggregation of micelles. This excellent stability of micelles may be
originated from negatively charged of core (PAA) that lead to electronic repulsion as well
as the effect of PEG chains as shell.
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(Figure 8)
3.6. Drug loading and encapsulation efficiencies
As the hollow nature of PAA-g-PEG HNCaps as well as the strong physical interactions
between the HNCaps and DOX (e.g., firm hydrogen bonding and Van der Waals forces), it
is expected that the fabricated nanosystem has high drug loading and encapsulation
efficiencies. The DOX-encapsulation efficiency (EE) and DOX-loading efficiency (LE) of
the synthesized PAA-g-PEG HNCaps were calculated to be 83 and 71%, respectively under
mentioned experimental condition.
3.7. In vitro drug release study
The in vitro drug release behavior of the DOX-loaded PAA-g-PEG HNCaps was
investigated under various pH values (4.0, 5.4, 6.6 and 7.4) at 37 °C. It should be pointed
out that the pHs 4.0, 5.4, and 6.6 are the average pH values of cancer cell lysosomal,
endosomal, and tumor tissue environment, respectively, and the pH 7.4 is the normal
physiological pH in the human body.
As seen in release profiles (Figure 9), drug release from the DOX-loaded PAA-g-PEG
HNCaps is a pH-responsive process where the protonation of carboxyl groups of PAA in
acidic conditions resulted in a faster release of encapsulated cargo. In contrast, at neutral
pH (7.4) the fully deprotonated PAA lead to strong interactions (e.g., firm hydrogen bonds
and Van der Waals forces) between PAA-g-PEG HNCaps and DOX, which delays the
release of loaded drug. According to Figure 9, the conclusion could be drown that the drug
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release values in pHs 4.0, 5.4, 6.6, and 7.4 after 300 hours were 42.1, 29.5, 24.8, and 22%,
respectively. It should be pointed out that in pH 4 the protonation of DOX, which increased
the dissolution of this drug is another reason for the higher drug release value.
It is worth noting that according to acidic feature of tumor microenvironment in
comparison with surrounding normal tissues and based on obtained results, the developed
PAA-g-PEG HNCaps nanomedicines can be considered as de novo drug delivery system
for “smart” chemotherapy of solid tumors.
(Figure 9)
3.8. In vitro cytotoxicity assay
The cytotoxicity effects of PAA-g-PEG HNCaps as well as DOX-loaded HNCaps were
investigated using MMT assay against MCF7 cells and the results obtained were compared
with the cytotoxicity effect of the free DOX as the reference. As seen in Figure 10, the
PAA-g-PEG HNCaps has acceptable biocompatibility during incubation time period of 24
hours, and the cells viability is more than 91% even at high concentration (200 µg mL-1).
In contrast, the free DOX as well as DOX-loaded HNCaps showed cytotoxic effects on
MCF7 cells. In detail, free DOX can kill cancer cells more efficiently than those of the
developed PAA-g-PEG/DOX nanomedicine. This may be originated from the high
availability of DOX in the case of free DOX treatments than those of the DOX-loaded
HNCaps due to slow drug release profile according to Figure 9. Despite, the use of free
DOX is not recommended due to some issues, including rapid drug clearance, poor
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biodistribution, non-ideal physicochemical properties of drugs (e.g., poor solubility), rapid
drug degradation, and systemic toxicity. It is well established that the formulated anticancer
drugs have some advantages, including prolonged release profiles, favorable
biodistribution, higher therapeutic outcomes and minimizing the side effects of the drug
[29]. This theory is confirmed through the treatment of MCF7 cells with samples in the
time period of 48 hours as shown in Figure 11.
The statistical analysis results revealed that although the eradication of plain DOX was
slightly higher compared with PAA-g-PEG/DOX nanomedicine at 24 h (p<0.01) especially
in higher drug concentrations (100 and 200 µgmL-1). In contrast, after 48 h the PAA-g-
PEG/DOX nanomedicine exhibited slightly higher cytotoxicity against MCF7 cells
(p<0.05) especially at 20 and 50 µgmL-1 of DOX.
(Figure 10)
(Figure 11)
4. Conclusions
Novel pH-responsive PEGylated HNCaps was fabricated through a thiol-ene “click”
grafting of a thiol end-capped PEG onto hollow crosslinked PAA-based NPs for “smart”
chemotherapy of solid tumors. The fabricated HNCaps showed excellent drug loading
capacity (~ 71%) as well as “smart” drug release behavior under pH stimuli. In acidic
conditions, the protonation of carboxyl group of the PAA lead to faster release of
encapsulated DOX. In contrast, at neutral pH (7.4) the fully deprotonated PAA lead to
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strong interactions between the PAA-g-PEG HNCaps and DOX, which delays the release
of loaded drug. The biocompatibility of the fabricated PAA-g-PEG HNCaps was
confirmed through MTT assay against MCF7 cells. In addition, the PAA-g-PEG/DOX
nanomedicine exhibited excellent cytotoxic effect against mentioned cancerous cells.
In conclusion, according to acidic feature of tumor microenvironment in comparison with
surrounding normal tissues and based on obtained results from drug loading and pH-
responsive drug release studies as well as cytotoxicity assay, the developed PAA-g-PEG
nanomedicine can be considered as de novo drug delivery nanosystem for smart
chemotherapy of solid tumors.
Acknowledgement
The authors gratefully acknowledge the financial support from Kermanshah University of
Medical Sciences, Kermanshah, Iran (grant number: 980304).
Competing interests
The authors declare that they have no competing interests.
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1
Schemes and Figures
Scheme 1. The overall strategy for the synthesis of PAA-g-PEG HNCaps.
Accepted Article
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2
Figure 1. The DLS diagram of SiO2 NPs, SiO2@PAA NPs, and PAA HNCaps (a), and XRD pattern
of SiO2 NPs (b).
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3
Figure 2. The FTIR spectra of the SiO2 NPs, MPS-SiO2 NPs, and SiO2@PAA NPs.
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4
Figure 3. The FTIR spectra of the PEG, PEG-SA, PEG-SH, and PAA-g-PEG HNCaps.
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5
Figure 4. The 1H NMR spectra of PEG, PEG-SA, and PEG-SH.
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6
Figure 5. The TEM images of SiO2@PAA NPs (a) and PAA HNCaps (b).
Figure 6. The TGA curves of SiO2 NPs, MPS-SiO2 NPs, SiO2@PAA NPs, PAA HNCaps, and PAA-
g-PEG HNCaps.
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7
Scheme 2. The pH responsibility of the fabricated PAA-g-PEG HNCaps.
Figure 7. The DLS diagrams of PAA-g-PEG HNCaps at various pH values in 37 °C.
0
5
10
15
20
25
30
0 50 100 150 200 250
Intencity (%)
Size (d.nm)
pH=4
pH=5.5
pH=7.4
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8
Figure 8. The DLS diagrams of fresh PAA-g-PEG HNCaps micelles and micelles after 3 days
incubation in PBS (pH=7.4 and T=37 °C).
0
5
10
15
20
25
30
0 50 100 150 200 250
Intencity (%)
Size (d.nm)
Fresh
After 3 days
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9
Figure 9. In vitro drug release profiles of DOX-loaded PAA-g-PEG HNCaps at pH values of 7.4, 6.6,
5.4, and 4.0 in 37 °C. [SPSS results: (pH: 7.4/6.6; P<0.05); (pH: 7.4/5.4; P<0.01); (pH: 7.4/4;
P<0.01); (pH: 6.6/5.4; P<0.01); (pH: 6.6/4; P<0.01); and (pH: 5.4/4; P<0.01).
0
20
40
60
0 50 100 150 200 250 300 350
Release (%)
Time (h)
PAA-g-PEG (pH=7.4)
PAA-g-PEG (pH=6.6)
PAA-g-PEG (pH=5.4)
PAA-g-PEG (pH=4)
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10
Figure 10. The cytotoxicity assay results of PAA-g-PEG HNCaps, PAA-g-PEG/DOX, and free DOX
on MCF7 cells in time period of 24 hours.
0
20
40
60
80
100
120
0 10 20 50 100 200
Survival rate (%)
Concentrations (µg mL-1)
PAA-g-PEG HNCaps PAA-g-PEG/DOX Free DOX
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11
Figure 11. The cytotoxicity assay results of PAA-g-PEG HNCaps, PAA-g-PEG/DOX, and free DOX
on MCF7 cells in time period of 48 hours.
0
20
40
60
80
100
120
0 10 20 50 100 200
Survival rate (%)
Concentrations (µg mL-1)
PAA-g-PEG HNCaps PAA-g-PEG/DOX Free DOX
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