Journal of Drug Delivery Science and Technology 52 (2019) 670–676

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

pH-sensitive polymeric nanoparticles of quercetin as a potential colon T

cancer-targeted nanomedicine
Suhair Sunoqrot∗, Lujain Abujamous
Department of Pharmacy, Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman, 11733, Jordan

ARTICLE INFO ABSTRACT

Keywords: Quercetin (QCT) is an abundant plant polyphenol with demonstrated efficacy in several diseases including colon
Quercetin cancer. Herein we have developed polymeric nanoparticles (NPs) of QCT based on the pH-sensitive polymer
Eudragit S100 Eudragit® S100 to achieve colon pH-specific drug release following oral administration. NPs were prepared by
Polymeric nanoparticles the nanoprecipitation technique and showed a mean diameter of 66.8 nm and a partially negative surface charge
Colon-targeting
of −5.2 mV. NPs contained on average 2.2% w/w QCT at an encapsulation efficiency of 41.8%. IR spectroscopy
Colon cancer
and differential scanning calorimetry (DSC) both revealed the presence of intermolecular interactions, most
likely H-bonding, between QCT and Eudragit® S100, which likely contributed to drug loading. DSC also indicated
that the drug was present in the NPs in an amorphous state, which was further confirmed by X-ray diffraction. In
vitro release testing showed a delay in drug release in acidic pH, but complete release within 24 h at pH 7.2. A
cytotoxicity assay was performed on CT26 murine colon carcinoma cells, where QCT-loaded NPs displayed
significantly higher potency (IC50 = 0.8 μM) than free QCT (IC50 = 65.1 μM). Our findings present a promising
nanomedicine for colon-targeted delivery of QCT in diseases such as colon cancer.

1. Introduction
Colon cancer is the third-most prevalent type of cancer, accounting
for 1.8 million cases and 862,000 deaths in 2018 alone [1]. The
American Joint Committee on Cancer (AJCC) has classified colon
cancer into five stages; Stage 0, I, II, III, and IV. Stage 0 has a cure rate
of 100% after surgical resection. Stages I – IIC are also treated by sur-
gical resection, with 37–74% 5-year survival rates. Advanced stages
(IIIA – IV) require surgical resection followed by adjuvant che-
motherapy, but patients remain at high risk for metastasis and recur-
rence with a 6% survival rate [2]. Adjuvant chemotherapy is often
administered through the intravenous route, which can result in severe
systemic side effects due to nonspecific distribution of chemother-
apeutic drugs to healthy tissues [3]. On the other hand, the oral route
represents a safer and more logical route of administration for the
treatment of colon-related diseases such as colon cancer. In addition to
the improved patient compliance, this strategy provides direct access to
the disease site, which can enhance drug bioavailability and reduce the
required dose, putting the patient at a much lower risk for developing
chemotherapy-related side effects.
The ability of nanocarriers to guide drugs to specific sites in the
body, enhance their solubility, extend their half-life, and improve their
therapeutic index has revolutionized the treatment protocols for many
diseases especially cancer [4,5]. Among the various classes of nano-
carriers, polymeric NPs are the most versatile, since their controllable
structure provides an opportunity to tailor their properties for various
applications. In particular, NP-based oral formulations shield drugs
from the harsh gastrointestinal environment, and can selectively in-
crease drug concentration inside diseased colon cells, thus elevating
therapeutic efficacy while reducing systemic toxicity [6]. A variety of
polymeric NP-based platforms have been investigated for colon-specific
delivery of therapeutic agents, such as those based on poly(lactide-co-
glycolide) (PLGA) [7,8] and chitosan [9,10]. One of the most efficient
methods for colon-targeted delivery via the oral route is pH-dependent
drug release [6]. To achieve this purpose, the dosage form is coated
with an acid-resistant polymer that can protect it from premature re-
lease in the stomach and upper gastrointestinal tract (GIT). Once the
polymer reaches the large intestine or colon, where the pH rises close to
neutrality, it dissolves allowing the drug to be released [11–14].
Eudragit® polymers are a group of commercially available me-
thacrylic acid copolymers that are typically employed in the pharma-
ceutical industry in modified-release film coatings of tablets and cap-
sules. Among the various types of Eudragit® polymers available,
Eudragit® S100 (Fig. 1a) is the most suitable for colon targeting with a
ratio of methacrylic acid to methyl methacrylate of approximately 1:2
[15]. The carboxyl groups of the methacrylic acid moieties are

∗
Corresponding author. Department of Pharmacy, Faculty of Pharmacy, Al-Zaytoonah University of Jordan, P.O. Box 130, Amman, 11733, Jordan.
E-mail address: suhair.sunoqrot@zuj.edu.jo (S. Sunoqrot).

https://doi.org/10.1016/j.jddst.2019.05.035
Received 5 April 2019; Received in revised form 17 May 2019; Accepted 21 May 2019
Available online 22 May 2019
1773-2247/ © 2019 Elsevier B.V. All rights reserved.
S. Sunoqrot and L. Abujamous Journal of Drug Delivery Science and Technology 52 (2019) 670–676

anti-colon cancer activity, none of the previous reports involved the use
of any type of polymeric NPs to specifically target QCT to the colon
tissue. For these reasons, we chose Eudragit® S100 as a colon pH-sen-
sitive nanocarrier for QCT. Physicochemical characterization of the NPs
was complemented with in vitro release in conditions mimicking GIT
transit, as well as cytotoxicity evaluation in a murine colon cancer
model to further validate the NP formulation as a potential colon-tar-
geted nanomedicine.

2. Materials and methods

2.1. Materials

Quercetin dihydrate was obtained from Acros Organics (Geel,

Belgium). Eudragit® S100 and RS100 were gifts from Evonik Industries
AG (Essen, Germany). Potassium dihydrogen phosphate (KH2PO4) and
absolute ethanol were obtained from Fisher Scientific (Loughborough,
UK). Polysorbate 20 (Tween 20) was provided by Tedia (Ohio, USA).
Sodium acetate, ethyl cellulose (EC), and polyvinyl alcohol (PVA, MW
13,000–23,000, 98% hydrolyzed) were obtained from Sigma-Aldrich
(St Louis, MO, USA). Ultrapure water (specific resistivity ~18.2 MΩ.cm
Fig. 1. Structures of (A) Eudragit® S100 and (B) QCT. (C) Schematic re-
at 25 °C) was prepared using a Milli-Q purification system (EMD
presentation of QCT-loaded NP formation by nanoprecipitation.
Millipore, Billerica, MA, USA).

protonated in acidic media (e.g. stomach) and the side chains are not
2.2. Preparation of QCT-loaded NPs
charged, rendering the polymer insoluble. Upon exposure to neutral or
basic pH values, the carboxyl groups become ionized. This results in
NPs were prepared by the nanoprecipitation method as previously
negative repulsive charges between the carboxylate side groups and an
described [17,33]. Various formulations were prepared using Eudragit®
increase in aqueous solubility, causing the polymer to release its pay-
S100 with or without PVA (0.2% w/v in ultrapure water) as a stabilizer,
load [16]. The versatility of Eudragit® polymers has expanded their
Eudragit® S100 mixed with RS100 (1:1), or EC (1:1). The composition of
application from conventional film coating excipients to NP platforms
each formulation is summarized in Table 1. In a typical procedure,
for various applications including pH-dependent drug release. Pre-
19 mg total polymer and 1 mg QCT were co-dissolved in 2.5 mL ethanol.
paration of NPs from Eudragit® polymers can be achieved by nano-
The polymer/drug solution was then added dropwise to 10 mL ultra-
precipitation, resulting in matrix-type NPs [17].
pure water under stirring at 400 rpm, followed by overnight mixing to
Quercetin (QCT) (Fig. 1b) is the lead compound of flavonoids with
allow ethanol to evaporate. Large aggregates were removed by cen-
demonstrated anti-proliferative activities against different types of
trifugation at 4000 rpm for 5 min, and unentrapped drug was removed
cancer such as colorectal, lung, and breast, through several proposed
by membrane dialysis (MWCO 3500, Spectra/Por 3, Spectrum Labora-
mechanisms that involve free radical scavenging and binding transition
tories Inc., Rancho Dominguez, CA, USA) against deionized water,
metal ions [18,19]. Like many other molecules in its class, QCT is
changing the water every hour for 3 h. Experiments were conducted on
characterized by poor water solubility (log P = 0.35) and extensive
freshly prepared NP dispersions unless otherwise noted. NPs were
metabolism in vivo [20]. Given its promising activity toward colon
lyophilized using a Christ Alpha 1–4 freeze-dryer (Osterode, Germany)
cancer, we set out to develop a novel NP platform for QCT targeted to
without any lyoprotectant.
the colon via the oral route, since it is the most convenient method of
administration and will maximize the drug concentration at the target
site. 2.3. Particle size and zeta potential measurements
QCT has been previously formulated in polymeric NPs [21], mi-
celles [22], and liposomes [23]. Polymer-based carriers in particular Particle size and zeta potential were measured by dynamic light
have been widely explored to improve the oral bioavailability and scattering (DLS) using a Nicomp Nano Z3000 particle size/zeta poten-
stability of bioactive plant flavonoids including QCT [24]. Wegiel et al. tial analyzer (Particle Sizing Systems, Santa Barbara, CA, USA). Two
carried out mid-infrared spectroscopic investigations of QCT-based hundred microliter samples of freshly prepared NPs were diluted with
amorphous solid dispersions in a variety of polymers including Eu- an equal volume of ultrapure water, and each measurement was re-
dragit® E100 (an acid-soluble copolymer which readily dissolves at ported at least three times using different batches of the NPs.
stomach pH) [25]. The study revealed the presence of intermolecular
QCT-polymer interactions with high crystallization-inhibiting and sta- 2.4. Drug loading (DL) and encapsulation efficiency (EE) determination
bilizing potential, making this class of polymers an attractive delivery
platform for QCT. Wu et al. prepared QCT-encapsulated Eudragit® E100 The amount of QCT loaded into the NPs was determined by dis-
NPs for the purpose of increasing its solubility and oral bioavailability solving 1 mg of lyophilized NPs in 1 mL ethanol, followed by measuring
[26], where the NPs were designed to dissolve and release the drug the UV absorbance at 376 nm (UV-1800 Shimadzu spectrophotometer,
immediately in the stomach. Likewise, Tang et al. encapsulated genis- Kyoto, Japan). The amount of QCT loaded was determined based on a
tein, an isoflavone, in Eudragit® E100 NPs to improve its oral bioa- standard curve of QCT absorbance at 376 nm versus concentration in
vailability [27]. On the other hand, Pool et al. reported on the fabri- ethanol (y = 0.0566x + 0.1029, R2 = 0.9881). Each measurement was
cation of QCT NPs from Eudragit® L30-D55 to target the small intestine repeated at least three times using different batches of the NPs. DL and
[28]. QCT has been formulated in Eudragit® S100 but only in the form EE were calculated according to Equations (1) and (2) as follows:
of microspheres for the purpose of treating mustard gas poisoning
DL (w/w%) = (Actual weight of loaded QCT/Weight of QCT-loaded
[29–31]. Enteric-coated PLGA NPs have also been reported for QCT to
NPs) × 100% (1)
improve its intestinal absorption [32]. However, despite its promising

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S. Sunoqrot and L. Abujamous
EE (%) = (Actual weight of loaded QCT/Theoretical weight of QCT
added) × 100% (2)
2.5. Fourier transform (FT)-IR spectroscopy
FT-IR spectra for QCT, Eudragit® S100, and lyophilized drug-loaded
NPs were recorded using a Shimadzu IR Affinity-1 spectrometer (Kyoto,
Japan). All samples were prepared as KBr discs (Acros Organics, Geel,
Belgium).
2.6. Differential scanning calorimetry (DSC) analysis
The thermograms of QCT, Eudragit® S100, drug/polymer physical
mixture, and lyophilized drug-loaded NPs were recorded using a DSC 1
STARe System (Mettler Toledo, Columbus, OH, USA). Approximately
1 mg of each sample was heated in an aluminum pan from 25 to 350 °C
and the scanning rate was conducted at 10 °C/min.
2.7. X-ray diffractometry (XRD)
The X-ray diffraction patterns for QCT, Eudragit® S100, drug/
polymer physical mixture, and lyophilized drug-loaded NPs were ob-
tained using a Shimadzu XRD-6000 apparatus (Kyoto, Japan) with Ni-
filtered Cu Kα radiation. Measurements were performed at a voltage of
40 kV and a current of 25 mA. The scanning angle was set from 2° to 50°
at a scanning rate of 1°/min. Measurements were performed in tripli-
cate.
2.8. In vitro release of QCT from loaded NPs
To mimic the NPs’ transit through the GIT, the release test was
performed in simulated gastric fluid (SGF) at pH 1.2 for the first 2 h,
followed by acetate buffer pH 4.5 for the next 2 h, and then phosphate
buffer pH 7.2 from the 4 h time point up to 24 h. All release media
contained 0.5% w/v Tween 20 to maintain sink conditions [22], and all
were freshly prepared for each experiment. For the release test, 2.5 mL
of freshly prepared QCT-loaded NP dispersion was placed in a dialysis
bag with MWCO 3500 (Spectra/Por 3, Spectrum laboratories Inc.), and
both ends were sealed with clips. The dialysis bag was incubated in
100 mL of SGF in tightly sealed glass beakers. The whole set up was
placed in an automated shaking water bath operating at 37 °C and
100 rpm. At different time intervals (1, 2, 3, 4, 5, 6, 7, 8, and 24 h),
10 mL of release medium was collected and replaced with an equal
volume of fresh medium. After the 2 and 4 h time points, the medium
was completely replaced with acetate buffer pH 4.5 and phosphate
buffer pH 7.2, respectively. The amount of drug released was quantified
by measuring the UV absorbance of withdrawn samples at 376 nm
based on a standard curve of QCT in each medium, and the results were
plotted as percent cumulative amount released versus time. The ex-
periment was repeated three times using different batches of the NPs.
2.9. Drug release mechanism
The Korsmeyer-Peppas model was used to assess the mechanism of
QCT release according to Equation (3) [34,35]:
Mt/M∞ = Ktn (3)
where Mt/M∞ is the fractional cumulative amount of drug released at
time t, K is the release rate constant, and n is the diffusion exponent
indicative of the mechanism of drug release. To determine n, the release
data of ≤60% cumulative release was fitted into the model using
Graphpad Prism 6.0e.
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Journal of Drug Delivery Science and Technology 52 (2019) 670–676
2.10. Cytotoxicity of QCT-loaded NPs against CT26 murine colon cancer
cells
The in vitro cytotoxicity of QCT-loaded NPs was evaluated by an
MTT assay. CT26 murine colon carcinoma cells (ATCC, Manassas, VA,
USA) were cultured in RPMI 1640 medium supplemented with 10%
fetal calf serum, 1% L-glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin, at 37 °C in 5% CO2. For the experiment, cells were seeded
in 96-well plates at a density of 10,000 cells/well (n = 6). After 24 h,
media was removed and cells were treated with free QCT or QCT-
loaded Eudragit® S100 NPs at concentrations ranging from 0.1 to
1000 μM QCT in complete RPMI 1640 for 72 h. Another set of cells was
treated with empty Eudragit® S100 NPs at polymer concentrations
equivalent to QCT-loaded NPs. At the end of the incubation period,
media was removed and replaced with fresh complete RPMI 1640 (100
μL/well) containing 0.5 mg/mL MTT reagent, and cells were incubated
for an additional 3 h. Then, the media was carefully removed, 100 μL
DMSO was added to each well to dissolve the formazan crystals, and
absorbance was read at 570 nm using a microplate reader. Cell viability
was determined relative to untreated controls. IC50 was determined by
fitting the data into a dose-response curve using Graphpad Prism 6.0e.
Statistical analysis was performed using a 2-way ANOVA followed by
Sidak's multiple comparisons test, where p < 0.05 was considered
statistically significant.
3. Results and discussion
Treatment of colon diseases is most efficient by direct delivery of the
drug to the affected area. This selective delivery may also reduce the
required dose of the therapeutic agent [3,6,36,37]. Effective colon
targeting requires the delivery system to protect the drug payload from
being released in the stomach, at the same time exploiting the unique
features of the colon to maximize drug release. In this study, we de-
veloped a polymeric NP formulation for QCT as a potential nanome-
dicine against colon cancer. The NPs were designed for targeted de-
livery to the colon following oral administration by taking advantage of
the pH-dependent dissolution behavior of Eudragit® S100 at the colonic
pH. We hypothesized that the pH-sensitive nature of the polymer would
protect the drug throughout the GIT until it reaches the colon, where it
can be released to exert its therapeutic effect with minimal side effects.
3.1. Preparation and characterization of QCT-loaded Eudragit® S100 NPs
The size and surface charge of the nanocarrier play an important
role in its performance [38,39]. In this study, we prepared QCT-loaded
Eudragit® S100 NPs by the nanoprecipitation technique, and examined
the particle size and surface charge using DLS. Particle size of Eudragit®
NPs has been shown to depend on several factors, such as the pre-
paration method, polymer concentration, the ratio of organic phase:
aqueous phase, and the use of stabilizers [17].
As summarized in Table 1, different compositions were screened to
reach an optimized formulation for QCT. The polymer: drug ratio was
fixed at 19:1 and the ratio of the organic phase: aqueous phase was
Table 1
Compositions of the QCT NP formulations prepared in this study.
Batch Eudragit® Eudragit® EC (mg) QCT (mg) Aqueous Appearance
S100 (mg) RS100 phasea
(mg) (10 mL)
F1 19 - - 1 Water Milky
F2 19 - - 1 0.2% PVA Clear
F3 9.5 9.5 – 1 Water Precipitate
F4 9.5 – 9.5 1 Water Precipitate
a
The organic phase was composed of polymer(s) and QCT dissolved in
2.5 mL ethanol.
S. Sunoqrot and L. Abujamous
Fig. 2. (A) Appearance of the various formulations prepared in this study. (B)
Representative volume-weighted particle size distribution for QCT-loaded
Eudragit® S100 NPs (F1) obtained by DLS.
fixed at 1:4. NP formulations included either Eudragit® S100 alone (F1)
or in combination with Eudragit® RS100 (F3) and EC (F4). The use of a
stabilizer, 0.2% PVA, in the aqueous phase was also examined (F2).
Screening of the NPs was performed based on overall appearance of
each formulation as shown in Fig. 2a. F1 produced a milky colloidal
dispersion that was stable for several days at room temperature. F2
produced a clear solution and no NPs were detected by DLS, most likely
due to solubilization of the polymer and QCT by PVA. F3 (1:1 Eudragit®
S100 and RS100) and F4 (1:1 Eudragit® S100 and EC) both failed due to
the formation of a large amount of precipitate, indicative of in-
compatibility between the polymers. Consequently, F1, composed of
Eudragit® S100 and QCT with no stabilizer, was advanced for further
characterization.
As shown in Table 2, the optimized formulation F1 produced col-
loidal particles with a mean diameter of 66.8 nm. A representative
particle size distribution for F1 is shown in Fig. 2b. The surface charge
of the NPs was on average −5.2 mV. Since the NPs were dispersed in
ultrapure water, the polymer is expected to be in its acidic neutral form,
thus the partially negative surface charge may have resulted from the
presence of sodium lauryl sulfate added by the manufacturer to Eu-
dragit® S100 [15]. We obtained a DL of 2.2% w/w at an EE of 41.8%,
indicating good compatibility between QCT and Eudragit® S100. The
moderate EE indicates that some QCT may have partitioned into the
water phase during NP fabrication.
3.2. FT-IR spectroscopy
FT-IR was employed to better understand the intermolecular inter-
actions between QCT and the polymer. As shown in Fig. 3, QCT ex-
hibited a number of characteristic bands: O–H stretching
Table 2
Characterization of QCT-loaded Eudragit® S100 NPs (F1).
Particle size (nm) Zeta potential (mV) DL (%w/w) EE (%)
66.8 ± 2.3 −5.2 ± 2.4 2.2 ± 0.4 41.8 ± 9.1
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Journal of Drug Delivery Science and Technology 52 (2019) 670–676
Fig. 3. FT-IR spectra of (A) QCT, (B) Eudragit® S100, and (C) QCT-loaded NPs
(F1).
(3200–3500 cm−1); C=O stretching (1660–1680 cm−1); C=C aro-
matic stretching (1612, 1564, and 1522 cm−1); O–H bending
(1385 cm−1); and C–H aromatic bending (825 cm−1). The FT-IR spec-
trum of the polymer showed bands corresponding to O–H stretching
(3400–3700 cm−1), sp3 C–H stretching (2800–3100 cm−1), and C=O
stretching (1600–1800 cm−1). As for QCT-loaded NPs (F1), we noted
broadening and decrease in the intensity of the O–H stretching peak
(3400–3700 cm−1), which could be attributed to the formation of in-
termolecular H-bonding interactions between QCT and Eudragit® S100.
3.3. DSC analysis
DSC analysis was performed to determine the physical nature of the
drug in the final formulation, which would ultimately govern release
characteristics of QCT. It is also a powerful tool for the analysis of
polymer-drug interactions [28]. Such insights are crucial as QCT has
not been previously incorporated in Eudragit® S100 NPs.
As depicted in Fig. 4, QCT used in this study was in a crystalline
state, exhibiting two sharp endothermic peaks at 136.2 and 316.3 °C,
corresponding to dehydration (QCT is supplied as a hydrate) and
melting of QCT, respectively. Eudragit® S100 exhibited two broad en-
dothermic peaks at 94.0 and 228.2 °C, indicating its presence in an
amorphous state. The thermogram of the drug/polymer physical mix-
ture showed thermal transitions similar to those observed in the drug
and polymer individual curves. However, we observed changes in the
location of these peaks and the disappearance of the QCT melting peak
at 316.3 °C. These changes are a strong indicator of the presence of
interactions between QCT and the polymer, which led to changes in the
structural organization of the molecules in the solid state. These in-
teractions are likely the result of H-bonding, as suggested by the
structures of QCT and Eudragit® S100 (Fig. 1). As for QCT-loaded NPs,
the DSC thermogram also showed changes in peak locations and a
disappearance of the QCT melting peak. In addition, we observed a
marked decrease in the intensity of the peak corresponding to QCT
dehydration at 120.8 °C. This result indicates that QCT is dispersed in
the NP matrix in an amorphous state.
3.4. XRD analysis
XRD was employed to further verify the crystalline state of QCT
after loading into Eudragit® S100 NPs. Diffraction patterns were ob-
tained for QCT, Eudragit® S100, drug/polymer physical mixture, and
S. Sunoqrot and L. Abujamous Journal of Drug Delivery Science and Technology 52 (2019) 670–676

Fig. 6. In vitro release profile of QCT-loaded Eudragit® S100 NPs (F1) up to

24 h. The NPs were incubated in SGF (pH 1.2) during the first 2 h, acetate buffer
(pH 4.5) for the next 2 h, and then phosphate buffer (pH 7.2) from the 4 h time
point up to 24 h. Results represent the mean ± SD of percent cumulative
amount released obtained from three independent experiments.

3.5. pH-dependent release of QCT from Eudragit® S100 NPs

Fig. 4. DSC thermograms of (A) QCT, (B) Eudragit® S100, (C) QCT/polymer
Effective colon targeting with pH-sensitive polymers requires the
physical mixture, and (D) QCT-loaded NPs (F1). Changes in peak locations as ability of the delivery system to protect its cargo from the GIT en-
well as the disappearance of the QCT melting peak, are indicative of inter- vironment till it reaches the colon, where the pH will favor drug release
molecular H-bonding interactions between QCT and Eudragit® S100. by promoting polymer dissolution. We verified the ability of QCT-
loaded Eudragit® S100 NPs to effectively achieve this goal by con-
ducting a release test in media with different pH.
QCT-loaded NPs and are presented in Fig. 5. QCT exhibited several
As shown in Fig. 6, no QCT was detected in the release medium
characteristic peaks at 2θ of 10.65°, 12.30°, 15.67°, 24.60°, 26.38°, and
during incubation in SGF (pH 1.2) and acetate buffer (pH 4.5) for the
27.16° (Fig. 5a), indicative of a highly crystalline structure and con-
first 4 h, indicating that Eudragit® S100 can protect QCT against gastric
sistent with DSC results. Eudragit® S100 (Fig. 5b) displayed broad fea-
pH and during small intestinal transit. This is attributed to the proto-
tures, confirming the amorphous nature of the polymer. In the case of
nated state of the methacrylic acid moieties in Eudragit® S100 at low
QCT/polymer physical mixture (Fig. 5c), although most of QCT's
pH, rendering it insoluble and preventing QCT release. As the pH was
characteristic peaks were masked by the polymer incoherent scattering,
increased to 7.2, QCT release was initiated and was almost complete,
the crystalline structure of the drug still persisted. On the other hand,
reaching 91.8%, after 24 h. At this pH, the increased ionization of the
no characteristic peaks appeared in the pattern of QCT-loaded NPs
methacrylic acid moieties in the polymer caused electrostatic repulsion
(Fig. 5d), where the diffraction pattern was identical to that of Eu-
and polymer swelling, leading to the disruption of the NP matrix and
dragit® S100 alone (Fig. 5b). This finding is in agreement with DSC
release of encapsulated QCT. Our results are consistent with previous
analysis and provides strong evidence that QCT is converted to an
reports using similar systems [11,12,28], and strongly indicate that
amorphous state after loading into the NPs.

Fig. 5. XRD patterns of (A) QCT, (B) Eudragit® S100, (C) QCT/polymer physical mixture, and (D) QCT-loaded NPs (F1). QCT's crystalline structure is partially masked
upon mixing with amorphous Eudragit® S100, and completely disappears after loading into the NPs, indicating its conversion to an amorphous state.

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Table 3
Korsmeyer-Peppas release kinetic parameters for the in vitro release of
QCT from Eudragit® S100 NPs (F1).
n K (h−1) R2
9.06 3.33 0.89
Fig. 7. Cell viability of CT26 cells treated with 0.1–1000 μM QCT, QCT-loaded
Eudragit® S100 NPs (F1), or empty Eudragit® S100 NPs up to 72 h. Results are
expressed as mean ± SD (n = 6). QCT and QCT-loaded NPs display dose-
dependent cytotoxicity while empty NPs are nontoxic (IC50 = 1262.0 μM).
QCT-loaded NPs are significantly more potent (IC50 = 0.8 μM) than free QCT
(IC50 = 65.1 μM). ***p < 0.001, ****p < 0.0001 based on 2-way ANOVA
followed by Sidak's multiple comparisons test.
Eudragit® S100 NPs can protect QCT against premature release in acidic
media, but readily release the drug at neutral pH conditions, which
validates their use for colon-specific delivery.
3.6. Mechanism of QCT release from Eudragit® S100 NPs
To better understand the drug release mechanism from the NPs,
release data was fitted into the Korsmeyer-Peppas model. For spherical
particles, a value of n = 0.43 indicates drug release by Fickian diffu-
sion, while n values between 0.43 and 0.85 represent anomalous (non-
Fickian) transport, where drug release is attributed to a combination of
diffusion and polymer chain relaxation. Values of n ≥ 0.85 indicate
supercase II transport, where drug release is governed purely by
polymer relaxation [34,35]. As shown in Table 3, release data de-
monstrated a good fit (R2 = 0.89) with the Korsmeyer-Peppas model,
with an n value of 9.06, confirming that drug release was largely con-
trolled by polymer chain swelling and relaxation upon ionization of the
methacrylate moieties when exposed to neutral pH.
3.7. In vitro cytotoxicity of QCT-loaded Eudragit® S100 NPs
Cytotoxicity of QCT, QCT-loaded NPs, and empty NPs was evaluated
in CT26 murine colon cancer cells after 72 h of incubation. As shown in
Fig. 7, free QCT and QCT-loaded NPs both caused dose-dependent de-
crease in cell viability. However, around 80-fold increase in potency
was observed for QCT-loaded NPs, with an IC50 of 0.8 μM compared to
65.1 μM for free QCT. The lower potency for the free drug may be at-
tributed to its poor water solubility, which could hinder its cellular
uptake. A similar result was reported for rutin-encapsulated Eudragit®
S100 NPs [40]. On the other hand, solubilization of QCT in Eudragit®
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Journal of Drug Delivery Science and Technology 52 (2019) 670–676
S100 NPs enabled the intracellular delivery of a more effective con-
centration of QCT, resulting in a significantly higher cytotoxic effect,
which highlights an additional advantage for the Eudragit® S100-based
QCT NPs. Note that the in vitro release experiment revealed that drug
release from Eudragit® S100 NPs is almost complete within 24 h, sug-
gesting that QCT-loaded NPs are expected to behave similar to free QCT
in the cytotoxicity experiment after 72 h incubation. However, previous
studies have shown that Eudragit® S100 NPs may be internalized by
cells as quickly as after 1 h of incubation [41–43], which supports the
ability of the NPs to shuttle the drug inside the cells early on before they
dissolve in the cell culture medium extracellularly. Furthermore, cells
treated with empty Eudragit® S100 NPs displayed significantly higher
cell viabilities with an IC50 of 1262.0 μM at all concentrations tested,
indicating the biocompatibility of Eudragit® S100 NPs as a drug delivery
vehicle. Interestingly, a recent report by Sankaranarayanan et al. found
that flavonoid metabolites such as 2,4,6-trihydroxy benzoic acid are
more likely to be responsible for the anti-proliferative and cancer-pre-
ventive activity of flavonoids since these compounds are rapidly me-
tabolized in vivo [44]. In light of these findings, it would be noteworthy
to investigate the molecular mechanism of anti-cancer activity of QCT-
loaded NPs compared to the free drug and its metabolites in vitro, and
the possible protective role of Eudragit® NPs on QCT stability in vivo.
4. Conclusions
In this study we report for the first time on an NP formulation of
QCT based on the pH-sensitive polymer Eudragit® S100 for colon-tar-
geted delivery. An optimized formulation composed of QCT loaded in
Eudragit® S100 NPs without any additive was prepared by the nano-
precipitation method, and resulted in good DL and EE values. FT-IR and
DSC analyses indicated the formation of intermolecular H-bonding
between QCT and the polymer, which contributed to their compat-
ibility. DSC and XRD analysis also revealed that QCT was present in the
NPs in an amorphous state. Release tests were conducted in media with
different pH to mimic transit throughout the GIT. Eudragit® S100 NPs
effectively prevented QCT release at low pH (pH 1.2 and 4.5), but re-
leased the drug at neutral pH upon ionization of the carboxylate moi-
eties in the polymer. Fitting the release data into the Korsmeyer-Peppas
model revealed that the release mechanism followed supercase II
transport, where drug release was predominantly dependent on
polymer chain relaxation and swelling. QCT-loaded NPs demonstrated
significantly higher potency compared to free QCT in CT26 colon
cancer cells. Our findings demonstrate that traditional pharmaceutical
excipients can be effectively leveraged to develop a nanomedicine
candidate for QCT to target colon cancer.
Conflicts of interest
There are no known conflicts of interest associated with this pub-
lication.
Acknowledgements
This work was supported by Al-Zaytoonah University of Jordan
(grant no. 15/28/2017–2018). The authors would like to thank Dr.
Eveen Al-Shalabi and Ms. Aya Zuheiri for assistance with FT-IR ana-
lysis.
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